Biochemistry

Dr, lJ, Satyan arayana M . S c,.P h.D .,F.l .C .,F.A .C .B . Professor of Biochemistry Siddhartha Medical Colle g e (NTR University of Health Sciences) Vijayawada, 4.P., India

Dr, lJ, Chakrapani M.B .B ,S ., M.S .

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Eiochemistrg First Published : March 1999 Reprinted: 1999 Revised Reprint : August 2000 Reprinted: 2OQO,2001, 2QO2 Second Revised Edition : June 2002 Reprinted: 2003 Revised Reprint : 2004 Revised Reprint : 2005 Third Revised Edition (multicolour) : 2006 Revised Reprint : 2007

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Prefaceto the Third Edition

The responseto the first and the secondeditionsof my book 'Biochemistry'(reprintedseveraltimes in just 6 years)from the studentsand teachersis simply overwhelming.I was flooded with highly appreciative lettersfrom all cornersof India and abroad!This givesme immensesatisfactionand encouragemLnt in this academicventure. I havecorresponded with many biochernistry teachers,inviting their commentsand opinionsfor further improvingthe book. Most of them havebeenkind enoughto offer constructivesuggestions. I also visited severalcollegesand had personalinteractionwith facultymembersand students.Theseexercises, spreadover the past 6 years,have helped me to get direct feedbackon my book, besidesrealisingthe additional requirementsof students. I havegreat pleasurein presentingthe third edition of my book with severalunique/novelfeatures,some high-lightsof which are listedbelow. . A thoroughrevisionand updatingof eachchapterwith latestadvances. Multicolouredillustrationsfor a better understanding of chemicalstructuresand biochemicalreactions. . Increasein the font size of the text for more pleasantand comfortablereading. o Incorporationof a new Sectionon MolecularBiologyand Biotechnology. . Addition of ten new chapters-human genomeproject,gene therapy,bioinformatics,free radicals and antioxidants,tissueproteinsand body fluids,environmentalbiochemistry,genetics,immunologyetc. . An improvedorientationand treatmentof human biochemistryin healthand disease. . Additionof practicalbiochemistryand clinical biochemistrylaboratoryin the appendix. It is true that I representa selectedgroup of individualsauthoringbooks,havingsometime at disposal, besideshard work, determinationand dedication.I considermyselfan eternallearnerand a regularstudent of biochemistry. However,it is beyondmy capabilityto keeptrack of the evergrowing advances in biochemistry due to the exponentialSrowth of the subject.And this makesme nervous,wheneverI think of revising the book. I honestlyadmit that I haveto dependon mature readersfor subsequent editionsof this book. AN INVITATION TO READERS It is not all the time possiblefor me to meetthe readersindividuallyand get their feedback, despitemy ferventwish. Of course,I do write to somepeoplepersonaliyseekingtheir opinions.However,I wish to have the commentsand suggestions of eachoneof the readersof my book.I sincerelyinvite the readersto feelfree and write to me expressing their frank opinions,critical commentsand constructivesuggestions. DT. U. SATYANARAYANA

tr l

I owe a deepdebt of gratitude to my parents,the late Sri U. VenkataSubbaiah,and Smt. Vajramma,for cultivatingin me the habit of earlyrising.The writing of this bookwould neverhavebeenpossiblewithout this healthyhabit. I am gratefulto Dr. B. S. NarasingaRao(formerDirector,NationalInstituteof Nutrition, Hyderabad)for disciplining my professionallife, and to my eldestbrother Dr. U. Gudaru(former Professorof PowerSystems,WalchandCollegeof Engineering,Sangli)for discipliningmy personallife. My elder son, U. Chakrapani(MBBS)deservesa specialplace in this book. He made a significant contribution at everystageof its preparation-writing, verification,proof-readingand what not. I had the rare privilegeof teachingmy son as he happened to be a studentof our college.And a major part of this bookwas written while he waslearningbiochemistry. Thus,he wasthe first personto learnthe subjectof biochemistry from my handwrittenmanuscript.The student-teacherrelation (rather than the father-son)has helpedme in receivinSlconstant feedbackfrom him and restructure the book in a way an undergraduatestudent would expecta biochemistrytextbookto be. Next,I thank Dr. G. PitcheswaraRao(former Professorof Anatomy,SMC,Vijayawada) for his constructive criticism and advice,and Dr. B. Sivakumar(Director,NationalInstitute of Nutrition, Hyderabad)for his helpful sugi5lestions on the microfigures.I am grateful to my nephew,Mr. U. SrinivasaRao,for helping me in drawingsomefigures. Last but not least, I thank my wife Krishna Kumari and my younger son, Amrutpani, without whose cooperationand encouragementthis book could never have been written. The manuscript was carefully nurtured like a new born babyand the book has now becomea full-pledgedmemberof our family. ACKNOWLEDGEMENTS TO THE THIRD EDITION I am indebtedto a largenumberof friends,pen-friends andstudentswho helpedme to reviseand improve the qualityof this book.I haveindividuallyand personallythankedall of them (whonumbera fewhundreds!). I onceagainexpressmy gratitudeto them. I thank my friend and colleague,Mr. M.S.T.JaganMohan, who has helped me with his frequent interactionsto improvethe book,and makeit more student-friendly. I would like to placeon recordmy deep (M.D.)students,Dr. (Mrs.)U.B.VijayaLakshmiand Dr. (Mrs.)Vidya senseof appreciation to my post-graduate DesaiSripad,whoseperiodicalacademicinteractionand feedback havecontributedto the improvementof the biomedicaVclinical aspectsin somechapters.I acknowledge the help of my friend,Dr. P. Ramanujam(Reader in English,AndhraLoyolaCollege,Vijayawada) for his help and encouragement in revisingthe book. I expressmy gratitude to Mr. ArunabhaSen, Director, Books & Allied (P) Ltd. Kolkata, for his wholehearted supportand constantencouragement in revisingthe book in multicolour,and taking all the pains to bring it out to my satisfaction.I thank Mr. ShyamalBhattacharyafor his excellentpage-makingand graphics-workin the book.I am indebtedto Mr. PrasenjitHalderfor the coverdesignof this book. I thank my wife, Krishna Kumari, and my younger son, Amrutpani, for their constant support and encouragement.I am grateful to UppalaAuthor-PublisherInterlinks, Vijayawada,for sponsoringand supportingme to bring out this edition.

DT. U. SAIYANARAYANA Ii i i ]

Biochemistry

The term Biochemistry was introduced by Carl Neuberg in 1903. Biochemistrybroadly dealswith the chemistrv of life and living processes. There is no exaggerationin the statement,'Thescopeof biochemistrg is as uastas lilb itself !' Everyaspectof life-birth, growth, reproduction,agingand death,involvesbiochemistry. For that matter, everymovementof life is packedwith hundredsof biochemicalreactions.Biochemistryis the mostrapidlydeveloping and mostinnovativesubjectin medicine.This becomesevidentfrom the factthat over the years,the major share of Nobel Prizesearmarkedfor Medicineand Physiologyhas gone to researchers engagedir: biochemistry. The discipline of biochemistryservesas a torch light to trace the intricate complexicitiesof biology, that all living besidesunravellingthe chemicalmysteriesof life. Biochemicalresearchhasamplydemonstrated things are closelyrelatedat the molecularlevel.Thus biochemistryis the subjectof unity in the diversified living kingdom. Advancesin biochemistryhavetremendousimpact on human welfare,and havelargelybenefitedmankind and their living styles.Theseincludethe applicationof biochernistryin the laboratoryfor the diagnosisof and the diseases. the products(insulin,interferon,€rowth hormoneetc.)obtainedfrom geneticengiineering, possibleuse of genetherapy in the near future. 0rganization of the Book This texthook,comprising43 chapters,is orgianizedinto serrensecl:ionsin the heirarchicalorder of learninSbiochemistry. . SectionI dealswith the chemicalconstituentsof life-carbohydrates,lipids,proteinsand amino acids, nucleicacidsand enzymes. . SectionII physiologicalchemistryincludesdigestionand ahsorption,plasmaproteins,hemoglobinand prophyrins,and biologicaloxidation. . SectionIII incorporatesall the metabolisms(carbohydrates, lipids,amino acids,nucleotides,minerals) . Section [V covershormones,organ function tests,water,electrolyteand acid-basebalance,tissueproteins and trodi'fluids,and nutrition. . Section V is exclusivelydevotedto molecularbiologyand biotechnology(DNA-replication, recombination, DNAandbiotechnology) ar"lnrepair,transcriptionandtranslation, recombinant regulationof geneexpression, . Section VI gives relevant information on current topics such a^shuman genomeproject, gene therapy, prostaglandins, bioirrtormatics, diabetes,cancer,AIDS etc. . Section VII deals with the basic aspectsfor learning and understandingbiochemistry (bioorganic chenristry', hiophysicalchemistrytools of biochemistry,genetics,immunology). Each chapter in this book is carefully crafted with colour illustrations, headingsand subheadingsto areput facilitatequick understanding. The importantapplications of biochemistryto humanhealthand disease places 'landmarks'. together as biomedical/clinicalconcepts.Icons are used at appropriate to serveas The origins of biochemicalwords, confusablesin biochemistry,practical biochemistryand clinical biochemistrylaboratory,given in the appendixare novel features. The briok is so organizedas to equip the readerswith a comprehensive knowledgeof biochemistry. Ii u]

Gontents

SECTION ONE ChemicalConstituentsof Life 1 > Biomolecules andthecell

3

2 > Carbohydrates

9

3 > Lioids

28

4 > Proteins andamino acids

43

acidsandnucleotides 5 > Nucleic

69

6 > Enzymes

85

7 > Vitamins

176

TWO SECTION PhysiologicalBiochemistry B > Digestion andabsorption

165

9 > Plasma oroteins

182

10 > Hemoglobin andporphyrins

196

11 > Biologicaloxidation

221

SECTION

SECTION FIVE Molecular Biologyand Biotechnology 24 > DNA-replication, recombination andrepair 523 25 > Transcriotion andtranslation 542 26 > Regulation of geneexpression 566 27 b Recombinant DNAandbiotechnology 578

sEcTtcN Current 28 > 29 > 30 F 31 p 32 33 34 35 36

>' > b > l"

37> 38>

THBEE

stx

Topics project Humangenome

619 Genetherapy 625 Bioinformatics 634 'lvletabolism (detoxification) of xenobiotics 638 Prostaglandins andrelated compounds644 Biological membranes andtransport 650 Freeradicals andantioxidants 655 Environmental biochemistry 662 glucose Insulin, homeostasis, mellitus anddiabetes 669 Cancer 58s

Acquired immunodeficiency (AIDS) syndrome

695

241

q3 > Metabolism of carbohydrates

244

*4 > Metabolism of lioids

285

Metabolism of aminoacids

F-, 16 > Int6gration of metabolism

330. 380

17 > Metabolism of nucleotides

387

metabolism 1B > Mineral

403

FOUR SECTION Clinical Biochemistrvand Nutrition 19 > Hormones function tests 20 > Organ 21 > Water, electrolyte and bqlance acid-base > proteins Tissue andbodyfluids 22 23 > Nutrition-

SECTION SEVEN Basicsto Learn Biochemistrv 39 > Introduction to bioorganic chemistry 40 > Overview of biophysical chemistry 41 > Toolsof biochemistrv 42 > lmmunology 43 > Genetics

487

APPENDICES Answers to Self-assessmenl Exercises I Abbreviations usedin thisbook' ll Greek alphabets lll Origins words ol important biochemical lV Common confusables in biochemistry V Practical biochemistry-principles Vl Clinical laboratory biochemistry

502

INEEX

427 453 468

703 708 71 9 732 73 7 745 751 756 tJt

760 764 77 0 77 3

fi

Protuinsand Amino acids

4:

Nucleic acidsand Nucleotides 69

BflomnoXeeutrss aildthsCelll

-l- hu living matter is composed of mainly six hydrogen, oxygenl I elements-carbon, nitrogen, phosphorus and sulfur. These elements togetherconstituteabout 90% of the dry weight of the human body. Severalother functionally important elementsare also found in the cells. These include Ca, K, Na, Cl, Mg, Fe, Cu, Co, l, Zn, F, Mo and Se.

organic compounds.lt is believedthat man may contain about 100,000 different types of molecules although only a few of them have been characterized. Sornpiex

*riomoleeules

The organic compoundssuch as amino acids, nucleotidesand monosaccharidesserve as the monomeric units or building blocks of complex biomolecules-proteins,nucleic acids (DNA and earbon-a unique element of life RNA) and polysaccharides,respectively.The Carbon is the most predominantand versatile important biomolecules(macromolecules) with elementof life. lt possesses a unique propertyto their respective building blocks and major form infinite number of compounds. This is functions are given in Table 1.1. As regards attributed to the ability of carbon to form stable lipids, it may be noted that they are not c ov ale n t b o n d s a n d C -C c h a i n s of unl i mi ted biopolymers in a strict sense, but majority of length. lt is estimated that about 90% of them contain fatty acids. compounds found in living system invariably contain carbon. Structural heirarehy off asn organisnl The macromolecules(proteins,Iipids, nucleic acids and polysaccharides) form supramolecular Life is composed of lifeless chemical assembl i es(e.g. membranes)w hi ch i n tu r n molecules. A single cell of the bacterium, organize into organelles,cells, tissues,organs Escherichiacoli contains about 6.000 different and fi nal l y the w hol e organi sm.

Ghemical

molecules

of li#e

3

B IOC H E MIS TFIY

Major functions

Building block (repeatingunit)

Biomolecule 1. Protein

Amino acids

and Fundamental basisofstructure functions). function anddynamic ofcell(static

acid(DNA) 2. Deoxyribonucleic

Deoxyribonucleotides Ribonucleotides

o.l.! 9199 iFryi{9l1llgt fl_eq_o_sitory

acid(RNA) 3. Ribonucleic

(glucose) 4. Polysaccharide(glycogen) Monosaccharides Fattyacids,glycerol

5. Lipid

Chem *c a !

c o m p o s i ti o n

biosynthesis. required lorprotein Essentially tomeetshortterm formofenergy Storage demands. tomeetlongterm tormofenergy Storage of membranes. components demands; structural

Prokaryotic

o f ma n

The chemical compositionof a normal man, weighing 65 kg, is given in Table 1.2. Water is the solvent of life and contributesto more than 60"h of the weight. This is followed by protein ( m os t lyin mu s c l e )a n d l i p i d (mo s tl yi n adi pose tissue).The carbohydratecontent is rather low which is in the form of glycogen.

The cell is the structuraland functional unit of life. ft may be also regardedas the basic unit of hiological activity.

and eukaryotic

cells

The cel l s of the l i vi ng ki ngdom may be divided into two categories 1. Prokaryotes(Creek : pro - before; karyon nucl eus)l ack a w el l defi nednucl eusand possess relatively simple structure. These include the various bacteria. 2. E ukaryotes(Greek: eu-true; karyonnucleus)possessa well defined nucleusand are more complex in their structure and function. The hi gher organi sms(ani mal sand pl ants)are composedof eukaryoticcells. A comparisonof the characteristicsbetween prokaryotesand eukaryotesis listed in Table 1.3.

The concept of cell originated from the c ont r ibut i o n so f S c h l e i d e na n d S c h w a n n(1838). However, it was only after 1940, the complexitiesof cell structurewere exposed.

Percent(7")

Weight (kg)

Water

6 1 .6

40

Protein

17.0

11

Lipid

1 3 .8

I '|

6 .1

4

Constituent

Carbohydrate Minerals

The human body is composedof about 1014 cells. There are about 250 types of specialized cel{s in- the human body'G.g. erythrocytes, nerve-cells, muscle cells, B cells of pancreas. A n eukaryoti ccel l i s general l y10 to 100 pm in diameter. A diagrammatic representation of a typical rat liver cell is depicted in Fi g.I.t. The pl ant cel l di ffersfrom an ani mal cel l by possessing a rigid cell wall (mostlycomposedof cellulose) and chloroplasts.The latter are the sitesof photosynthesis.

AND THE CELL Chapter 1 : BIOMOLECULES

Eukaryotic cell

Prokaryotic cell

Characteristic

1-10pm) Small(generally

1. Size

2. Cellmembrane 3. Sub-cellular organelles 4, Nucleus metabolism 5. Energy

6. Celldivision 7. Cytoplasm

DNAisfound Notwelldefined; histones areabsent asnucleoid,

of Mitochondria absent, enzymes metabolism bound to energy membrane fission Usually andnomitosis andcytoskeleton 0rganelles absent

T he c e l l c o n s i s tso f w e l l d e fi n e d subcel l ul ar organelles,enveloped by a plasma membrane. tissue By differential centrifugation of homogenate, it is possible to isolate each c ellular o rg a n e l l e i n a re l a ti v e l y p ure form (Refer Chapter 41). The distribution of major enzymes and metabolic pathways in different c ellular o rg a n e l l e s i s g i v e n i n th e chapter on enzymes (Refer Fig.6.6). The subcellular organellesare briefly describedin the following pages.

pm) Large(generally 10-100 plasma flexible Cellisenveloped bya membrane Distinct organelles arefound (e.9.mitochondria, nucleus, lysosomes) Nucleus iswelldefined, surrounded bya membrane: DNAisassociated withhistones Enzymes metabolism ol energy arelocated inmitochondria Mitosis

Contains organelles andcytoskeleton (anetwork oftubules andfilaments)

N ucl eus N ucl eus i s the l argest cel l ul ar organel l e , surrounded bv a doubl e membrane nucl ear envel ope. The outer membrane i s conti nuous w i th the membranesof endopl asmi creti cul um . At certain intervals,the two nuclear membranes have nuclear pores with a diameterof about 90 nm. These pores permit the free passageof the products synthesizedin the nucleus into the surroundi ng cytopl asm.

Mitochondrion

Plasmamembrane Vacuole

Roughendoplasmicreticulum

Ribosomes

Golgi apparatus

Lysosome

Peroxisome Cytoskeleton Cytosol Coatedpits

Ftg. 1.1: Diagrammaticrepresentationof a nt liverell.

B IOC H E MIS TF|Y Nucleus contains DNA, the repository of genetic information. Eukaryotic DNA is associatedwith basic protein (histones)in the ratio of 1 : 1, to form nucleosomes.An assembly of nucleosomesconstitutes chromatin fibres of chromosomes(Creek'.chroma - colour; soma bod y ). T h u s , a s i n g l e h u ma n c h romosome i s c om o o s e do f a b o u t a m i l l i o n n u cl eosomes. The number of chromosomes is a characteristic feature of the species. Humans have 46 chromosomes,compactlypacked in the nucleus. The nucleusof the eukaryoticcell containsa dense bodv known as nucleolus.lt is rich in RNA, p a rti c u l a rl y th e ri b o s o mal R N A w hi ch entersthe cytosol through nuclear pores.

acid cycle, p-oxidation). The matrix enzymes also parlicipate in the synthesisof heme and urea. Mitochondria are the principal producers of ATP in the aerobic cells. ATP, the energy currency,generatedin mitochondriais exported to all parts of the cell to provide energy for the cel l ul arw ork. The mi tochondri almatri x contai nsa ci rcular double stranded DNA (mtDNA), RNA and ribosomes.Thus, the mitochondriaare equipped with an independent protein synthesizing machinery.It is estimatedthat about 10% of the mitochondrial oroteins are produced in the mi tochondri a.

The ground material of the nucleus is often referredto as nucleoplasm. lt is rich in enzymes p o l y me ra s e s and R N A s uc h a s D N A polymerases.To the surpriseof biochemists,the enzy m e s o f g l y c o l y s i s ,c i tri c a ci d cycl e and hexose monophosphateshunt have also been detected in the nucleoplasm.

The structureand functions of mitochondria closely resemble prokaryotic cells. lt is hypothesizedthat mitochondria have evolved from aerobic bacteria.Further,it is believedthat during evolution,the aerobicbacteriadeveloped a symbi oti c rel ati onshi p w i th pri mor dial anaerobiceukaryoticcells that ultimately led to the arrival of aerobic eukaryotes.

Mitochondria

Endoplasmic

The mitochondria (Creek'. mitos - thread; chondros- granule) are the centres for the c ell u l a rre s p i ra ti o n a n d e n e rg ymetabol i sm.They are regarded as the power houses of the cell wit h v a ri a b l es i z e a n d s h a p e .Mi t ochondri aare r od -l i k e o r fi l a me n to u s b o d i e s , usual l v w i th dim e n s i o n s o f 1 .0 x 3 p m. A bout 2,0O0 mitochondria,occupying about 1/5thof the total c ell v o l u me , a re p re s e n ti n a ty p ical cel l .

The network of membrane enclosed spaces that extends throughout the cytoplasm constitutesendoplasmicreticulum (ER).Some of these thread-like structures extend from the nuclear pores to the plasma membrane.

reticulum

A large portion of the ER is studded with ribosomesto give a granularappearancewhich is referred ro as rough endoplasmic reticulum. Ribosomes are the factories of protein During the process of cell biosynthesis. The mitochondriaare comoosedof a double fractionation, rough ER is disruptedto form small membrane system. The outer membrane is It may be noted vesicles known as microsomes. smooth and completely envelopsthe organelle. not occur in the microsomes as such do that The inner membrane is folded to form cristae (Latin- crests) which occupy a larger surface cel l . area. The internal chamber of mitochondria is The smooth endoplasmicreticulum does not referred to as matrix or mitosol. contain ribosomes.lt is involved in the synthesis phospholipids,sterols) The componentsof electron transportchain of lipids (triacylglycerols, of drugs, besidessupplyingCa'?. and metabolism and oxidative phosphorylation (flavoprotein, functi ons. for the cel l ul ar c y t o c h ro m e sb , c 1 , C , a a n d a 3 and coupl i ng factors) are buried in the inner mitochondrial Golgi apparats,r$ membrane.The matrix containsseveralenzvmes concerned with the energy metabolism of E ukaryoti ccel l s contai n a uni que cl ust erof c ar b o h y d ra te sl i,p i d sa n d a m i n o a c i ds(e.g.,ci tri c membrane vesicles known as dictyosomes

AND THE CELL Chapter 1 : BIOMOLECULES whic h, in tu rn , c o n s ti tu teC o l g i a p p a ratus(or Colgi complex).The newly synthesizedproteins are handed over to the Colgi apparatuswhich catalysethe addition of carbohydrates,lipids or sulfatemoietiesto the proteins.Thesechemical modificationsare necessaryfor the transportof proteinsacrossthe plasma membrane.

The pH of the lysosomalmatrix is more acidic (pH < 5) than the cytosol (pH-7) and this facilitatesthe degradationof differentcompounds. The lysosomal enzymes are responsible for maintaining the cellular compounds in a dynamic stafe, by their degradationand recycling. The degradedproducts leave the lysosomes,usually by diffusion, for reutilization by the cell. Certainproteinsand enzymesare enclosedin Sometimes,however, certain residual products, m em br ane v e s i c l e s o f C o l g i a p p a ra t us and rich in lipids and proteins,collectivelyknown as secreted from the cell after the appropriate Iipofuscinaccumulatein the cell. Lipofuscinis signals.The digestiveenzymes of pancreasare the age pigment or wear and tear pigmentwhich or oduc edi n th i s fa s h i o n . has been implicatedin ageingprocess. Colgi a p p a ra tu sa re a l s o i n v o l v e d i n the The digestiveenzymesof cellular compounds membrane synthesis, particularly for the are confinedto the lvsosomesin the best interest f or m at ion o f i n tra c e l l u l a r o rg a n e l l e s (e.g. of the cell. Escapeof theseenzymesinto cytosol peroxisomes,lysosomes). will destroythe functionalmacromolecules of tne cel l and resul t i n many compl i cati ons.The Lysosornes occurrence of several diseases(e.g. arthritis, allergicdisorders) hasbeenpartly Lysosomesare spherical vesicles enveloped musclediseases, by a single membrane.Lysosomesare regarded attributedto the releaseof lysosomalenzymes. as the digestivetract of the cell, since they are ac t iv ely in v o l v e d i n d i g e s ti o n o f cel l ul ar Feroxi somes s ubs t anc e s -n a m e l y p ro te i n s , l i p i d s , carboPeroxisomes,also known as microbodies, are hydratesand nucleic acids. Lysosomalenzymes si ngl e membranecel l ul ar organel l es.They are are categorizedas hydrolases.These include the spherical or oval in shape and contain the following enzymes(with substratein brackets) enzyme catalase.Catalaseprotects the cell from a-C lucosidase(glycogen) the toxic effectsof HrO, by converting it to HrO and Or. Peroxisomesare also involved in tne Cathepsins(proteins) oxidation of long chain fatty acids (> C,s),and L i p a s e s(l i p i d s ) synthesisof plasmalogens and glycolipids.Plants (R N A ) glyoxysomes, contain Ribonucleases a specialized type of

BTOMED|eAL/ CLINICAL COIUCEPTS

A liuing cell is a true representotiueof life with its own organizotion and specialized lunctions. Accumulotion oJ lipofuscin, a pigment rich in lipids and proteins, in the cell has been implicated in ogeing process. Leokageof lysosomalenzymesinto the cell degrodesseuerolfunctional macromolecules and this may leod to certain disorders (e.9. arthritis). rq Zellweger syndrome is a rare diseose characterized by the absence of functional peroxisomes.

E } IOC H E MIS TF| Y per ox is o m e s , w h i c h glyoxylate pathway.

a re i n v o l v e d i n

the

Peroxisome biogenesis disorders (PBDs), are a Broup of rare diseasesinvolving the enzyme activities of peroxisomes. The biochemical abnor m a l i ti e sa s s o c i a te dw i th P BD s i ncl uoe increasedlevels of very long chain fatty acids (C2aand C26) and decreasedconcentrationsof plasmalogens. The most severeform of PBDs is Zellweger syndrome, a condition characterized by the absenceof functional peroxisomes.The v ic t im s o f th i s d i s e a s em a v d i e w i th i n one vear after birth. {iytosol

and cytoskeleton

The cellular matrix is collectively referred to as cytosol. Cytosol is basically a compartment containing several enzymes/ metabolites and saltsin an aqueousgel like medium. More recent studies however, indicate that the cytoplasm actually contains a complex network of protein filaments, spread throughout, that constitutes cytoskeleton.The cytoplasmic filaments are of

three types- microtubules, actin filaments and intermediatefilaments.The filamentswhich are polymers of proteins are responsiblefor the structure,shape and organizationof the cell. IN TE GR A TIOI{ OF C E LLU LA R FU N C TION S The eukaryoticcells perform a wide rangeof complex reactionsfunctionsto maintain tissues, and for the ul ti mate w el l -bei ng of the w hol e organi sm. For thi s purpose, the vari ous intracellularprocesses and biochemicalreactions are tightly controlled and integrated.Division of a cel l i nto tw o daughtercel l s i s good exampl eof the orderly occurrenceof an integratedseriesof cel l ul ar reacti ons. Apoptosisis the programmed cell death or cel l sui ci de. Thi s occurs w hen the cel l has ful fi l l ed i ts bi ol ogi calfuncti ons.A poptosi smay be regardedas a natural cell death and it differs from the cell death caused by injury due to radiation,anoxia etc. Programmedcell death is a highly regulatedprocess.

1. Life is composed ol lifeless chemical molecules. The complex biomolecules, proteins, nucleic ocids (DNA and RNA), polysaccharidesand lipids are formed by the monomeric units amino acids, nucleotides, monosaccharides and fotty acids, respectluely. 2 . The cell is the structurol and functional unit of life. The eukoryotic cell consisfsof well det'inedsubcellulor organelles,enueloped in a plasma membrane. 3. The nucleus contoins DNA, the repository ol genetic int'ormation.DNA, in association with proteins (histones),forms nucleosomeswhich, in turn, make up the chromosomes. The mitochondria qre the centresfor energy metobolism. They are the principal producers of ATP which is exported to all parts of the cell to ptouide energy lor cellular work. 5. Endoplosmic reticulum (ER) ts the network of membrane enclosed spoces that extends throughout the cytoplosm. ER studded with ribosomes, the factorles of protein biosynfhesis, ts relerred to as rough ER. Golgi opparatus sre a cluster of membrane uesiclesto uthich the newlg synthesized proteins are handed ouer for t'urther processing ond export. 6. Lysosomes are the digestiue bodies ol the cell, actiuely involued in the degradotion of cellular compounds. Peroxisomescontoln the enzyme catalose that protects the cell lrom the toxic elfects of HrOr. The cellular ground motrix is referred to as cytosol which, in fact, is composed of a network ot' protein t'ilaments, the cytoskeleton. 7. The eukaryoticcellsperform a wide rangeof complex lunctionsin a well coordinatedand integrated fashion. Apoptosis is the processol programmed cell death or cell suicide.

1^ arbohydratesare the most abundant organic \ - m o l e c u l e s i n n a tu re . T h e y a re pri mari l y composedof the elementscarbon, hydrogen and oxygen.The name carbohydrateliterally means 'hydratesof carbon'. Some of the carbohydrates pos s es sth e e mp i ri c a l fo rmu l a (C .H 2O)nw here n 3 3, satisfyingthat these carbohydratesare in fact carbon hydrates.However,there are several non-carbohydratecompounds (e.g. acetic acid, C2HaO2;lactic acid, C3H6O3)which also appear as hydrates of carbon. Further, some of the genuine carbohydrates (e.g. rhamnohexose, C6H12O5ideoxyribose,C5H16Oa)do not satisfy the generalformula.Hence carbohydrates cannot be always consideredas hydratesof carbon. Carbohydrates may be defined as polyhydroxyaldehydes or ketones or compounds which produce them on hydrolysis. The term ' s ugar ' i s a p p l i e d to c a rb o h y d ra te ssol ubl e i n water and sweet to taste. #-ur*c;tEerEsof earbohydrates Carbohydratesparticipatein a wide range of f unc t io n s

1. They are the most abundantdietarysource of energy (a Cal/S)for all organisms. 2. Carbohydratesare precursors for many organic compounds (fats,amino acids). (asglycoproteinsand glyco3. Carbohydrates lipids) participate in the structure of cell membraneand cel l ul ar functi onssuch as cell growth, adhesionand fertilization. 4. They are structuralcomponentsof many organi sms.Thesei ncl udethe fi ber (cel l ul ose)o f plants,exoskeletonof some insectsand the cell w al l of mi croorgani sms. 5. Carbohydratesalso serve as the storage form of energy(glycogen)to meet the immediate energy demandsof the body. C LA S S IFIC A TION

OF GARBOHYDRATES Carbohydrates are often referred to as saccharides (Greek: sakcharon-sugar). They are broadly classifiedinto three major groupsmonosaccharides, oligosaccharides and polysaccharides.This categorization is based on

t0

B IOC H E MIS TR Y

Monosaccharides(empirical formula)

AIdose

(CgHoOg) Trioses (C+HoO+) Telroses (CsHroOs) Pentoses (CoHrzOo) Hexoses (CzHr+Oz) Heptoses

Glyceraldehyde Erythrose Ribose Glucose Glucoheptose

Ketose

Dihydroxyacetone Erythrulose Ribulose Fructose Sedoheptulose

the number of sugar units. Mono- and oligosaccharides are sweet to taste, crystalline in characterand soluble in water, hence thev are commonly known as sugars.

liberatedon hydrolysis.Basedon the number of monosaccharide units present, the oligosaccharides are further subdivided to disaccharides,trisaccharides etc.

FJtonosaccharides

P ol ysace hari des

(Greek : mono-one)are the Monosaccharides group simplest of carbohydratesand are often referred to as simple sugars. They have the generalformula Cn(H20)n,and they cannot be further hydrolysed. The monosaccharidesare divided into different categories,based on the functionalgroup and the numberof carbonatoms

Polysacchari6ls(Creek:poly-many)are polymers of mondficcharide units with high molecul ar w ei ght (up to a mi l l i on).They are usual l y tasteless(non-sugars)and form colloids with water. The polysaccharidesare of two typeshomopolysaccharides and heteropolysaccharides.

Aldoses : When the functional group in IH monosaccharidesis an aldehyde l-C:oi,

\ ,h"u

are known as aldoses e.g. glyceraldehyde, gluc os e.

is an importantcharacterof Stereoisomerism the monosaccharides. Stereoisomers are Ketoses: When the functionalgroup is a keto compounds that have the same structural \ lt formulae but differ in their spatialconfiguration. \-C:O.l group, they are referred to as ketoses e.g. dihydroxyacetone,fructose.

A carbon is said to be asymmetric when it is to four different atoms or groups. Ihe attached Based on the number of carbon atoms, the of asymmetric carbon atoms (n) number monosaccharides are regarded as trioses (3C), the possible isomers of a given determines tetroses (4C), pentoses (5C), hexoses (6C) and w hi ch i s equal to 2n. C l ucose compound heptoses(7C).Theseterms along with functional 4 contains asymmetric carbons,and thus has 16 groupsare usedwhile naming monosaccharides. tsomers. For instance, glucose is an aldohexose while fructose is a ketohexose(Table 2,1). Glyeeraldehyde T he c om mo n m o n o s a c c h a ri d easn d d i saccha-tfu e ref erqlrt*e cff rb$hyd$'er'&€3 rides of biological importanceare given in the Clyceraldehyde(triose)is the simplestmonoTable 2.2. saccharidewith one asymmetriccarbon atom. lt S S lgos ac c h a ri d e s and has been chosen existsas two stereoisomers (Creek: io representthe the reference carbohydrate Oligosaccharides oligo-few) contain as 2- 1O m ono s a c c h a ri d em o l e c u l e s w h i ch are structureof all other carbohvdrates.

Ghapter 2 : CARBOHYDRATES

11

Trioses incellsasphosphate Glyceraldehyde Found DihydroxyacetoneFound incellsasphosphate Tetroses D-Erythrose

i i i

Glyceraldehyde 3-phosphate isanintermediate inglycolysis ttst -pnosphate isanintermediate inglycolysis

----t - -- - --.. -.. -. --... -- -

i Widespread

Pentoses D-Ribose

asa constituent of I Widespread I RNAandnucleotides

D-Deoxyribose i Asa constituent ofDNA D-Ribulose

during metabolism : Produced

D-Xylose

i i i i

L-Xylulose D-Lyxose

Asa constituent ofglycoproteins anogums ls anintermediate inuronic acidpathway Heart muscle

i --. --. -- --.. ---.. -.. -. --.

Hexoses D-Glucose

D-Galactose D-Mannose D-Fructose

Asa constituent ol polysaccharides (starch, glycogen, cellulose) and (maltose, disaccharides lactose, Alsofoundinfruits sucrose). Asa constituent oflactose (milk sugar) Found inplantpolysaccharides glycoproteins andanimal Fruits andhoney, asa constituent andinulin ofsucrose

Heptoses inolants D-SedoheptuloseFound Disaccharides

Sucrose Lactose

Maltose

Occurrence

Asa constituent ofcanesugarand pineapple beetsugar, Milksugar

Product ofstarch hydrolysis, ingerminating seeds occurs

Forthestructure ofRNAandnucleotide (ATP, coenzymes NAD+, NADP+) Forthestructure ol DNA Itisanimportant metabolite inhexose monophosphate shunt Involved inthefunction ofglycoproteins pentosuria Excreted inurineinessenlial Asa constituent ol lvxollavin ofheartmuscle The'sugar fuel'oflife;excreted inurine in diabetes. Structural unitofcellulose inplants

Converted toglucose, failure leads to galactosemia Forthestructure ofpolysaccharides Itsphosphates areintermediates ofglycolysis

Its7-phosphate isan intermediate in hexose monophosphate shunt,andin photosynthesis Biochemical importance

Most commonly used table sugar supplying calories Exclusive carbohydrate source tobreast fed (lactose infants. Lactase deficiency intolerance) leads todianhea andflatulence Animportant intermediate inlhedigestion of starch

E}IOCHEMISTFIY

12 H-C:O I H-C-OH cH2oH D-Glyceraldehyde H-C:O I H-C-OH I HO-C-H I H-C-OH I H-Q-OH I

cHzoH D-Glucose

H-C:O HO-C-H

to compoundsthat respectivelyrotatethe plane of polarized light to the right or to the left.

cH2oH

An optical isomer may be designated as D(+), D(-), L(+) and L(-) based on its structural L-Glyceraldehyde relation with glyceraldehyde.lt may be noted that the D- and L-configurationsof sugarsare H-C:O I primarily based on structure of the HO-C-H glyceraldehyde,the optical activities however, H-C-OH may be different.

I HO-C-H HO-C-H

cH2oH L-Glucose

Fig.2.1 : DandL- forms of glucose compared with D and L- glyceraldehydes (the reference carbohydrate).

Racemic mixture : lf D- and L-isomersare present in equal concentration,it is known as racemi cmi xtureor D L mi xture.R acemi cmi xture does not exhibit any optical activity, since the dextro- and levorotatorv activities cancel each other. Configuration

D" and L-isomers The D and L isomers are mirror images of each other. The spatial orientation of -H and -OH groups on the carbon atom (Cs for glucose)that is adjacentto the terminal primary alcohol carbon determineswhether the sugar is D- or L-isomer.lf the -OH group is on the right side, the sugar is of D-series,and if on the left side, it belongs to L-series.The structuresof D- and L-glucosebased on the referencemonosaccharide, D- and L-glyceraldehyde (glycerose) are depicted in Fig.2.1.

of D-aldoses

The configuration of possible D-aldoses starting from D-glyceraldehydeis depicted in Fig.2.2. This is a representation of KillianiFischersynthesis,by increasingthe chain length of an aldose, by one carbon at a time. Thus, (4C), startingwith an aldotriose(3C),aldotetroses aldopentoses (5C) and aldohexoses(6C) are formed. Of the 8 aldohexoses,glucose, mannose and galactose are the most familiar. Among these, D -gl ucose i s the onl y al dose monosaccharidethat predominantlyoccurs in nature.

Gonfiguration of D-ketoses It may be noted that the naturallyoccurring Startingfrom dihydroxyacetone(triose),there monosaccharides in the mammalian tissuesare are five keto-sugarswhich are physiologicallr mostlyof D-configuration. The enzymemachinery important.Their structuresare given in Fig,2.3 of cells is specific to metabolise D-series of monosaccharides.

Epimers

fn the medical practice, the term dextrose is used for glucose in solution. This is because of the dextrorotatory nature of glucose.

ff two monosaccharides differ from eacother in their configuration around a singk specificcarbon (other than anomeric) atom. L*ei are referred to as epimers to each orher '.Fig,21 Optlcal activity of sugars For instance, glucose and galactose are efilwl with regard to carbon 4 (Ca-epimers - -^:i 's Optical activity is a characteristicfeature of they differ in the arrangementof -OH g.'ELcr compounds with asymmetric carbon atom. Clucose and mannose are epi-'e--' q drl When a beam of polarized light is passed Ca. regardto carbon 2 (C2-epimers). througha solutionof an optical isomer,it will be rotated either to the right or left. The term The interconversionof epimers e I r::r'e dextrorotatory (+) and levorotatory (-) are used to galactose and vice versai s i- - -^,'- a*

Ghapter 2 : CABBOHYDFATES

13

cHo

I HCOH I cH2oH

'l

Aldotriose

(3c)

t-

Aldotetroses (4c) D-Erythrose

D.Threooe

cHo

cHo

H C OH

H OC H I HCOH

HCOH I HCOH cH2oH D-Ribose

cHo

HCOH

I H C OH I HOCH I HCOH

cH2oH D-Arabinose

cH2oH D-Xylose

cHo

I HOCH Aldo I HOCH I

toses )

HCOH I cH2oH D-Lyxoee

I /\

JT

cHo

tl HCOH HCOH tl HCOH tl HCOH ll cH2oH D-Allose

/\

/\

cHo HOCH HCOH

JT

cHo HCOH

rl

cHo HOCH

HOCH HOCH tl HCOH HCOH HCOH tl HCOH HCOH HCOH ll cH2oH cH2oH cH2oH D-Altrose D-Glucose D-Mannose

*/\+

cHo

cHo

I H C OH I HCOH I HOCH I HCOH

cH2oH D-Gulose

cHo

I HCOH

I

HOCH I HCOH I HOCH

I HOCH

HoCH

noCH

HCOH I

cH2oH D-ldose

cHo H OC H I AldoHOCH hexoses

tl HCOH tt

cHzoH

(6c)

HCOH

cHzoH

D-Galactose D-Talose

Fig.2.2 : ThestructuralrelationshipbetweenD-aldosesshownin Fischerprojection. (TheconfigurationaroundC2(ed) distinguishesthe membersof eachpair).

epimerization, and a group of enzymesnamely-epimerases catalyse this reaction.

The term diastereomersis used to represent the sfereoisomers that are not mirror images of one another.

E nanti o m e rs Enantiomers are a special type of stereoisomers that are mirror images of each other. The two members are designatedas D- and L-sugars.Enantiomersof glucose are depicted in Fig.2.5.

For a better understanding of glucose structure, let us consider the formation of hemiacetals and hemiketals, respectively Majority of the sugarsin the higher animals producedwhen an aldehydeor a ketone reacts (including man) are of D-type (Fig.2.5'1. w i th al cohol .

E } IOC H E MIS TR Y

14

cH2oH cH2oH I

cH2oH

?H2oH

C :O

cH20H I

I

C:O HCOH I I cH2oH cH2oH Dlhydroxyacetone D-Xylulose

HOCH I HCOH

C :O

C :O I HOCH

HCOH

I

HCOH I cH2oH

HCOH I

cH2oH D-Ribulose

D-Fructose

I

C :O I HOCH I HCOH I HCOH I HCOH I

cH2oH

D-Sedoheptulose

Fig.2.3 : Structuresof ketosesof physiologicalimportance.

,H nt-C.1^ + R2-oH l-

Anomers-nrutarotation

Rr-

The a and p cyclic forms of D-glucose are known as anomers. Thev differ from each other Alcohol Aldefry
H-C:O I H-C-OH I HO-C-H I HO- C -H H-C-OH I cH2oH D-Galactose

H-C:O H-C=O I I H-C-OH HO-C-H I I HO-C-H HO-C-H I I H .C -O H H-C-OH I I H-C-OH H-C-OH I I CHzOH cH2oH D-Glucose D-Mannose

H O= C H O_C - H I H-C- Cl-i I H O-C -H I HO-C- Fl I H-C- ii I

OH Fig.2,4: Structuresof epimers (glucose and galactose are Co-epimerswhile glucose and mannose are C2-epimers).

L-Glucose

H I C :O I

f{ c-oH I

H C -C -H i-l- c-oH H -C -OH I t"1-c-H HO D-Glucose

H9.2.5 : Enantiomers(mirrorimages)of glucose.

t5 Ghapter 2 : CARB

1

H -C :O I

H -C -OH I H O-C -H I H -C -OH tc

H -C -OH I cH2oH D-Glucose form) (aldehYde

I

cH20H o'D'Glucose (+ 112.2"\

iHron ftD-Glucose (+ 18.7-)

cH20H (B)

l/A

fil

H6\?H

H6\?H o-D-GlucoPYranose

H OH D-Glucose form,acYclic) (aldehyde

H OH FD-GlucoPYranose

p chai n form. l n aqueoussol uti on'the 17oopen mixture' equilihrium an to 'i, D'glucose forms of more predominant due to its stable forrn Mutarotationdepicted in Fig' 2'6, is summartzeo glucoseare conformation.The cr and p forms of below. a linear through occurs interconvertiblewhich in a" present is such, # Equilibriummixture# B-D-Clucose form. The latter, as cx-D-Clucose 18.7" + + 5 2 .7 " i nsi gni fi cantquanti tY . + 11 2 .2 " Mutarotation of fructose z Frur' mutarotation. ln case or 63o/" exhibits T h e e q u i l i b ri u m m i x tu re c o ntai ns pyranose ring (six-memberqd' p-anomer and 36"/ocl-anomer of glucose with furanose(five-membered)'o' is attained.And fruqt' rotation of -92)2.

(Specificoptical rotation tctl2p0)

I he conv' to levor

':ut" cH20H t-

H-C-OFi

OH HOH cr-D-GlucoPYranose

HOH uranose cr-D-Glucof

Fig.2.7 : Structurcof glucose-pyranose and furanosetorms'

on\ i s kn, anome' i n al kal i r W hen gt. severalhours,

:;r'

chapter 2 : CAFIBoHYDFATES

15

1

H -C = C ) I H-C-OH I

HO-C-H I H-C-OH l5 H-C-OH

I cH2oH

cH2oH D-Glucose (aldehyde form)

cr-D-Glucose (+ 112.2")

cH2oH

20H

H

H OH o-D-Glucopyranose

H OH D-Glucose (aldehydeform,acyclic)

pD-Glucopyranose

Fig. 2.6 : Mutarotation of glucose representing a and p anomers (A) Fischer projections (B) Haworth projections.

forms of D-glucose to an equilibrium mixture. 17oopen chain form. In aqueoussolution,the p Mutarotationdepicted in Fi9.2.6, is summarized form is more predominant due to its stable conformation.The s and p forms of glucoseare below. interconvertiblewhich occurs through a linear s-D-Clucose# Equilibriummircture # p-D-Glucose form. The latter, as such, is present in an + 112.2" + 52.7" + 18.7o i nsi gni fi cantquanti ty. (Specificoptical rotation talf;) Mutarotation of fructose : Fructose also exhibits mutarotation. ln case of fructose, the T h e e q u i l i b ri u m m i x tu re contai ns 63" /" p-anomer and 36h cl-anomerof glucose with pyranose ring (six-membered)is converted to furanose(five-membered) ring,till an equilibrium is attained.And fructose has a specific optical rotation of -92" at equilibrium. The conversion of dextrorotatory (+) sucrose to levorotatory fructose is explained under inversionof sucrose(see later in this chapter). REACTIONS

OF MONOSACCHARIDES

Tautomerization

cr-D-Glucopyranose

c-D-Glucofuranose

Fig.2.7 : Structureof glucose-pyranose and furanose forms.

or enolization

The processof shiftinga hydrogenatom from one carbon atom to another to produce enediols is known as tautomerization. Sugarspossessing anomeric carbon atom undergo tautomerization i n al kal i nesol uti ons. When glucose is kept in alkaline solution for severalhours,it undergoesisomerizationto form

16

BIOCHEMISTFIY H

n-C-ot H-C:O I H- -OH

( HO-(

2H2O+ CueO{-

HO-( R Enediol (common)

t

2Cu(OH)

It may be noted that the reducing property of sugarscannot help for a specificidentificationof any one sugar,since it is a general reaction. 0xida*iern

Depending on the oxidizing agent used, the terminal aldehyde (or keto) or the terminal Fig.2.8 : Formationof a commonenediolfrom alcohol or both the groupsmay be oxidized. For glucose,fructoseand mannose :PI:Is?Iboncolnmonstnf tar:?l,l instance,considerglucose :

:!lo.t|tfr {fr,f,o,F|F|lPffi ,ft

D-fructose and D-mannose. This reactionknown as the Lobry de Bruyn-von Ekenstein transformatiorr-results in the formation of a common intermediate-namely enediol--$or all the three sugars,as depicted in Fig.2.8.

1. Oxidation of aldehyde group (CHO ------> COOH) resultsin the formationof gluconic acid. 2. Oxi dati on of termi nal al cohol grou p (CH2OH ------+COOH) leadsto the production of gl ucuroni caci d.

R edueti on The enediolsare highly reactive,hencesugars When treated with reducing agents such as in alkaline solution are powerful reducing sodium amalgam,the aldehydeor keto group of agents. monosaccharideis reduced to corresponding alcohol, as indicated by the generalformula : ft+r,.luleFr'.lgr!s.l FeF tlsF H The sugarsare classifiedas reducingor nonH-C:O H-C-Ol-t reducing.The reducing property is attributedto I RR the free aldehyde or keto group of anomeric carbon. ln the laboratory, many testsare employed to identify the reducing action of sugars. These incf ude Benedict's test, Fehling's test, Barfoed's tesf etc. The reduction is much more efficient in t he a l k a l i n e me d i u m th a n i n the aci d m ediu m. The enediolforms (explainedabove)or sugars reduce cupric ions (Cu2+)of copper sulphate to cuprous ions (Cu+), which form a yellow precipitate of cuprous hydroxide or a red precipitate of cuprous oxide as shown next.

The important monosaccharidesand their correspondingalcohols are given below. D -Gl ucose D-Galactose------+ D-Dulcitol D-Mannose ------+ D-Mannitol D-Mannitol+ D-Sorbitol D-Fructose --) D-Ribitol D-Ribose -+ Sorbitol and dulcitol when accumulate in tissues in large amounts cause strong osmotic effects feading to swelling of cells, and certain pathologicalconditions.e.g. cataract,peripheral neuropathy,nephropathy.Mannitol is useful to reduce intracranialtension bv forced diuresis.

17

Ghapter 2 : CAFIBOHYDRATES

H-C:O I

H-C--O I H-C-OH I HO - C -H I H-C-OH I H-C-OH

Formation

of esters

The al cohol i c groups of monosaccharides may be esterified by non-enzymatic or enzymatic reactions. Esterificationof carbohydrate with phosphoric acid is a common reaction in metabolism. Glucose 6-phosphate cH20H and glucose 1-phosphateare good examples. Hydrorymethyl furfural ATP donates the phosphate moiety in ester formation.

I

cH2oH D-Glucose

H-C:O

H-C:O I H-C-OH tl l l H-C-OH

I

C----r Conc.HeSoo

I

H-C-OH

CHrou

rH

'1 3H2o

D-Ribose

H-Q I

H-C

L

U

I

H-d---l Furfural

lClycoside bond formation (see below) and mutarotation (discussedalready) may also be referred to, as these are also the characteristic propertiesof monosaccharides.l

GLYCOSIDES

Glycosidesare formed when the hemiacetal or hemiketal hydroxyl group (of anomeric carbon)of a carbohydratereactswith a hydroxyl group of another carbohydrate or a nonDehydration carbohydrate (e.g. methyl alcohol, phenol, When treatedwith concentratedsulfuricacid, glycerol). The bond so formed is known as monosaccharidesundergo dehydrationwith an glycosidic bond and the non-carbohydrate elimination of 3 water molecules.Thus hexoses moiety (when present)is referredto as aglycone. give hydroxymethylfurfural while pentosesgive The monosaccharidesare held together by furfural on dehydration (Fi9.2.9).These furfurals gl ycosi di c bonds to resul t i n di -, ol i go- or c an co n d e n s e w i th p h e n o l i c c ompounds polysaccharides(see later for structures). (a-naphthol)to form coloured products.This is the chemical basis of the popular Molisch test. they are In case of oligo- and polysaccharides, H-C=O by acid, and first hydrolysedto monosaccharides + HrN-NH-CuHu I _ H -C -OH this is followed by dehydration. Fig.2.9 : Dehydration of monosaccharides with concentrated H o. "SO

Osazone

formation

Phenylhydrazine in acetic acid, when boiled with reducing sugars, forms osazones in a reaction summarizedin Fig,2,10. As is evident from the reaction, the first two carbons (Cr and C2) are involved in osazone formation. The sugars that differ in their configuration on these two carbons give the same type of osazones,since the difference is Thus m as k e db y b i n d i n g w i th p h e n y l h y drazi ne. glucose, fructose and mannose give the same osazones. type (needle-shaped) Reducingdisaccharidesalso give osazonesmaltose sunflower-shaped,and lactose powderpuff shaped.

R Glucose

Phenylhydrazine

H-C:N-NH-CoHs I

H-C-OH I R Glucohydrazone l7-H2N-NH-C6H'

I

H-C:N-NH-CoHs I

C:N-NH-CoHs I

R Glucosazone Fig. 2.10 : A summatyof osazonefomation (R rcprcsentsCrto Crof glucose).

t8

B IOC H E MIS TR Y

Naming of glycosidic bond : The nomenclatureof glycosidic bonds is based on the Iinkagesbetween the carbon atoms and the status of the anomeric carbon (o or p). For instance,lactose-which is formed by a bond between C1 of p-galactoseand Ca of glucoseis named as 0(.1-+ 4) glycosidicbond. The other glycosidic bonds are described in the structure of di- an d p o l y s a c c h a ri d e s . Physiologieally

important

glycosides

1 . G lu c o v a n i l l i n (v a n i l l i n -D -g l u c o si de) is a natural substancethat impartsvanilla flavour.

The ami no groups of ami no sugars are sometimes acetylated e.g. N-acetyl D-glucosamrne. N -A cetyl neurami ni c aci d (N A N A ) i s a and pyruvic acid. derivativeof N-acetylmannose It is an important constituentof glycoproteins and glycolipids.The term sialic acid is used to include NANA and its other derivatives. C ertai n anti bi oti cs contai n ami no sugars which may be involved in the antibiotic activity e.g. erythromycin.

5. Deoxysugars: These are the sugarsthat 2. Cardiac glycosides(steroidalglycosides): contain one oxygen less than that present in the Digoxin and digitoxin contain the aglycone parent mol ecul e. The groups -C H OH and steroid and they stimulatemuscle contraction. -C H 2OH become -C H 2 and -C H 3 due to the 3. Streptomycin, an antibiotic used in the absenceof oxygen. D-2-Deoxyriboseis the most important deoxysugar since it is a structural treatmentof tuberculosisis a glycoside. constituentof DNA (in contrastto D-ribose in 4. Ouabain inhibits Na+- K+ ATPase and R N A ). blocks the active transportof Na+. 6. L-Ascorbic acid (vitamin C) : This is a DERIVATIVESOF MONOSACCHARIDESwater-soluble vitamin, the structure of which There are severalderivativesof monosaccha- closely resemblesthat of a monosaccharide. r ides , s o m e o f important

which

a re p h y s i ol ogi cal l y

1. Sugar acids : Oxidation of aldehyde or primaryalcohol group in monosaccharideresults in sugar acids. Cluconic acid is produced from glucose by oxidation of aldehyde (C1 group) whereasglucuronicacid is formed when primary alc ohol g ro u p (C 6 )i s o x i d i z e d . 2. Sugar alcohols (polyols) : They are producedby reductionof aldosesor ketoses.For instance, sorbitol is formed from glucose and mannitol from mannose. 3. Alditols : The monosaccharides, on reduction,yield polyhydroxyalcohols,known as aldit ols . R i b i to l i s a c o n s ti tu e n t of fl avi n coenzymes; glycerol and myo-inositol are componentsof lipids. Xylitol is a sweetenerused in s ugarl e s g sums and candies. 4. Amino sugars : When one or more hydroxyl groups of the monosaccharidesare replaced by amino groups, the products formed are amino sugars e.g. D-glucosamine, D-galactosamine.They are present as constituents of heteropolysaccharides.

The structuresof selected monosaccharide derivativesare depicted in Fig.2.l1.

di sacchari de s A mong the ol i gosacchari des, are the most common (Fig.2,l2). As is evident from the name, a disaccharideconsistsof two monosacchari de uni ts(si mi l aror di ssi mi l ar)hel d together by a glycosidic hond. They are crystalline,water-solubleand sweetto taste.The disaccharidesare of two types '1. Reducingdisaccharideswith free aldehyde or keto group e.g. maltose, lactose. 2. Non-reducing disaccharideswith no free aldehyde or keto group e.g. sucrose,trehalose. Maltose Maltose is composed of two a-D-glucose units held togetherby cl (1 -+ 4) glycosidicbond. The free aldehydegroup presenton C1 of second glucoseanswersthe reducing reactions,besides

Ghapter & : CAFIBOHYDRATES H -C :O I H-C-OH I H O -C -H I H-C-OH

19

cH2oH I

H -C -OH I

I

H-C-OH I COOH D-Glucuronic acid

cH2oH Glycerol

H OH myo-lnositol

H 3C -C --H N

OH H D-2-Deoxyribose

H NHz D-Glucosamine

H OH N-Acetylneuraminic acid

Fiq.2.11 : Structuresol monosaccharidederivatives(selectedexamples).

the osazone formations (sunflower-shaped). and keto) are held togetherand protectedfrom Maltosecan be hydrolysedby dilute acid or the oxidative attacks. enzyme maltase to liberate two molecules of Sucrose is an important source of dietary cr-D-glucose. carbohydrate. lt is sweeter than most other common sugars(exceptfructose)namelyglucose, lactoseand maltose.Sucroseis employed as a sweetening agent in food industry.The intestinal Cellobioseis another disaccharide,identical enzyme-sucrase-hydrolyses sucroseto glucose in structurewith maltose,exceptthat the former and fructose which are absorbed. has p (1 -r 4 ) g l y c o s i d i cl i n k a g e .C e l l obi osei s f or m ed d u ri n g th e h y d ro l y s i so f c e l l ul ose. F-aetsse Suoroee Lactose is more commonlv known as milk ln isomaltose,the glucose units are held togetherby o (1 --+6) glycosidic linkage.

Sucrose(cane sugar)is the sugarof commerce, mostly producedby sugarcane and sugar beets. Sucrose is made up of a-D-glucose and pD-fructose.The two monosaccharides are held togetherby a glycosidicbond (a1 -+ B2),between Cj of c-glucose and C2 of B-fructose.The reducing groups of glucose and fructose are inv olv e di n g l y c o s i d i cb o n d , h e n c e s ucrosei s a non-reducing sugar, and it cannot form osazones.

sugarsi nce i t i s the di sacchari de found i n mi l k. Lactose is composed ol p-D-galactoseand B-Dglucoseheld together by 0 (1 -r a) glycosidic bond. The anomericcarbonof C1 glucoseis free, hence lactose exhibits reducing propertiesand formsosazones(powder-puffor hedgehogshape).

Lactose of milk is the most important carbohydratein the nutritionof young mammals. It is hydrolysedby the intestinalenzyme lactase glucoseand galactose. to produced Sucroseis the major carbohydrate in photosynthesis. lt is transported into the storageorgans of plants (such as roots, tubers lnversion ef suerose and s ee d s ).Su c ro s ei s th e mo s t a b u n dantamong Sucrose,as such is dextrorotatory(+66.5o). t he natu ra l l y o c c u rri n g s u g a rs .l t h as di sti nct But, r,r,hen hydrolysed, sucrose becomes advantagesover other sugarsas a storageand levorotatory(-28.2"). The processof change in transoort form. This is due to the fact that in optical rotation from dextrorotatory (+) to sucrose, both the functional groups (aldehyde levorotatory(-) is referredto as inversion.The

BIOCHEMISTF|Y

(or simply glycans)consistof Polysaccharides repeat units of monosaccharides or their derivatives,held together by glycosidic bonds. They are primarilyconcernedwith two important and storageof energy. functions-structural, Polysaccharides are linear as well as branched polymers. This is in contrast to structureof proteinsand nucleic acids which are only linear polymers. The occurrence of branches in polysaccharidesis due to the fact that glycosidic linkagescan be formed at any one of the hydroxylBroupsof a monosaccharide. H OH Glucose

Fructose Sucrose (a-D-glucosyl (1 --+2) p-D-fructose)

Polysaccharidesare of two types which on hydrolysis 1. Homopolysaccharides yield only a singletype of monosaccharide. They are named based on the nature of the monosaccharideunit. Thus,glucans are polymers of glucose whereas fructosans are polymers of fructose. 2. Heteropofysaccharideson hydrolysisyield a mixture of a few monosaccharidesor their derivatives.

Galactose

Lactose (p-D-galactosyl (1 -+ a) p-D-glucose) $tarch Fig. 2.12 : Structures of disaccharides -maltose, sucrose and lactose.

Starch is the carbohydrate reserve of plants which is the most important dietary source for hi gher ani mal s,i ncl udi ngman. H i gh contentof hydrolysed mixture of sucrose, containing starchis found in cereals,roots,tubers,vegetables gfucose and fructose, is known as invert sugar. etc. Starch is a homopolymer composed of The processof inversion is explained below. D-glucose units held by a-glycosidic bonds. lt is known as glucosan or glucan. Hydrolysis of sucrose by the enzyme sucrase (invertasd or dilute acid liberatesone molecure Starch consists of two polysaccharide each of glucose and fructose. ft is postulatedthat components-water soluble amylose (15-20o/ol sucrose (dextro) is first split into a-D- and a water insoluble amylopectin (80-85%). glucopyranose(+52.5") and p-D-fructofuranose, Chemically, amylose is a long unbranched both being dextrorotatory. However, p-D- chain with 200-1,00OD-glucoseunits held by c fructofuranoseis lessstableand immediatelygets (1 + 4) gl ycosi di cl i nkages.A myl opecti n,on the converted to p-D-fructopyranose which is other hand, is a branchedchain with a (1 --r 6t stronglylevorotatory(-92"). The overall effect is gl ycosi di cbonds at the branchi ngpoi nts and c that dextro sucrose (+66.5") on inversion is (1 -; 4) linkages everywhere else (Fig.2.13). converted to levo form (28.2'\. A myl opecti n mol ecul e contai ni ng a few

21

ChapteF 2 : CARBOHYDFATES

D-Glucose

D-Glucose o-Amylose

+-

6nu vt

(1-* 6) Branch

t2

Main chain Lg

Amylopectin

thousand glucose units looks like a branched saccharideeasily soluble in water. Inulin is not utilized by the body. lt is used for assessing tree (20-30 glucose units per branch). amylase kidney function through measurement of Starches are hydrolysed by (pancreaticor salivary)to liberatedextrins,and glomerular filtration rate (GFR). finally maltoseand glucose units. Amylase acts Gl ycogen specificallyon a (1 -+ 4) glycosidic bonds. Clycogen is the carbohydrate reserve in animals, hence often referredro as animal starch. Dextrins Dextrins are the breakdown products of It is present in high concentration in liver, starch by the enzyme amylase or dilute acids. followed by muscle,brain etc. Clycogenis also Starch is sequentially hydrolysed through found in plants that do not possesschlorophyll (e.9. yeast,fungi). different dextrins and, finally, to maltose and glucose.The various intermediates(identifiedby The structureof glycogen is similar to that of iodine colouration) are soluble starch (blue), amylopectin with more number of branches. amylodextrin (violet), erythrodextrin (red) and Glucoseis the repeatingunit in glycogenjoined achrodextrin (no colour). togetherby u (1 + 4) glycosidic bonds, and a (1 + 6) gl ycosi di c bonds at branchi ng poi nt s I nulin (Fi9.2.1Q.The molecularweight (up to 1 x 108) fnulin is a polymer of fructosei.e., fructosan. and the number of glucose units (up to 25,000) I t oc c u rs i n d a h l i a b u l b s , g a rl i c ,o n i on etc. l t i s vary in glycogendependingon the source from a low m o l e c u l a r w e i g h t (a ro u n d 5 ,000) pol y- which glycogen is obtained.

B IOC H E MIS TR Y

22

decreasing the absorption of glucose and cholesterolfrom the intestine,besidesincreasing the bulk of feces. (For details, Chapter 23) Ghitin Chitin is composed of N-acetyl Dglucosamineunits held together by F (1 -+ a) gl ycosi di cbonds.l t i s a structuralpol ysaccha r ide found in the exoskeleton of some invertebrates e.g. insects,crustaceans.

T

N

T Ot

(B) 9H2OH

y'-O.,

(+

-o--J\,

\J

uqt,

, F--o.

i) - r+

, . / ' o--' \

L-/

CH2oH

,r4-Or

,L^_K. "

\--l

When the polysaccharidesare composed of differenttypes of sugarsor their derivatives,they are referred to as heteropolvsaccharidesor heteroglycans.

X^_

Fiq.2.14: Structureof glycogen(A) Generalstructure (B) Enlargedat a branchpoint.

Cellulose Ce l l u l o s eo c c u rse x c l u s i v e l yi n p l antsand i t i s the most abundant organic substancein plant k ingd o m . l t i s a p re d o m i n a n t c onsti tuentof plant c e l l w a l l . C e l l u l o s e i s to ta l ly absent i n anim a l b o d y . Cellulose is composed of p-D-glucose units linked by 9 0 -+ 4) glycosidic bonds (Fi9.2.1fl. Cellu l o s e c a n n o t b e d i g e s te d b y mammal sincluding man-due to lack of the enzyme that cleavesB-glycosidicbonds (a amylasebreakscr bonds only). Certain ruminantsand herbivorous anim a l sc o n ta i nm i c ro o rg a n i s mi sn the gut w hi ch produce enzymes that can cleave p-glycosidic bonds . H y d ro l y s i s o f c e l l u l o s e yi el ds a disaccharide cellobiose, followed by P-Dglucose. Cellulose, though not digested, has great im porta n c ei n h u ma n n u tri ti o n . l t i s a maj or constituentol fiber, the non-digestablecarbohydrate. The functions of dietary fiber include

MUCOPOLYSACCHARIDES made are heteroglycans Mucopolysaccharides up of repeatingunitsof sugarderivatives,namely amino sugarsand uronic acids. Theseare more known as glycosaminoglycans commonly (GAG). Acetylatedamino groups,besidessulfate and carboxyl groups are generally present in CAC structure. The presence of sulfate and carboxyl groups contributes to acidity of the mol ecul es, maki ng them aci d mucopo ly, sacchari des. are found Some of the mucopolysaccharides in combination with proteins to forrn mucoproteins or mucoids or proteoglycans (Fig.2.l6l. Mucoproteinsmay contain up to 95o, carbohydrate and 5o/" protein.

S-D-Glucose Fig. 2.15 : Structure of cellulose (The repeat:r; -- ' may be several thousands).

CARBOHYDRATES

Hyaluronicacid

23 Mucopolysaccharides are essential components of tissue structure.The extracellularspacesof ti ssue (parti cul arl yconnecti ve ti ssue-carti l age, skin, blood vessels,tendons)consistof collagen and elastinfibersembeddedin a matrixor ground substance. The groundsubstanceis predominantly composedof CAC. The i mportantmucopol ysacchari des i ncl uoe hyal uroni caci d, chondroi ti n4-sul fate,hepari n, dermatansulfateand keratansulfate(Fig.Z.'[1. j 'i '

s:\

-;-s\'' t / : - -sS -\'\Fig. 2.16 : Diagrammaticrepresentationof a prateoglycan complex.

,i r :r :,

|

'.i .

:,\{,ti i i El 'l

H yal uroni caci d i s an i mportantGA C found i n the groundsubstance of synovi alfl ui d of j oi nts and vitreoushumor of eyes. it is also presentas a ground substancei n connecti veti ssues,and forms a gel around the ovum. H yal uroni caci d servesas a l ubri cant and shock absorbanti n j oi nts.

BToMEDtCAt/ CLtft|ICALCO$CEpTS

rlr Glucose is the most important energy source ol carbohgdrates to the mammals (except ruminants). The bulk of dietary carbohydrote(starch)is dlgestedond finally obsorbedas glucose into the body. Ea Dextrose (glucose in solution in dextrorotatory form) is frequently used in medical practice. Rq'CF

Fructose is obundantly found in the semen which is utilized by the sperms for energy. Seueral diseoses are associated with carbohydrate.s e.g., diabetes mellitus, glycogen storage diseoses, galactosemia.

trs Accumulation of sorbitol and dulcitol in the fissues moy cause certoin pathological conditions e.g. cotaract, nephropothy. t-s' Inulin, a polymer of t'ructose,is used fo ossessrenal function by meosuringglomerular filtration rate (GFR). ue The non-digestiblecarbohydrate cellulose plays a signilicant role in human nutriticsn. These include decreasing the intestinal absorption ol glucose and cholesterol, qnd increasingbulk of feces to ouoid eonstipation. rt The mucopolysaccharidehyaluronic acid seruesas a lubricant and shock absorbantin ioints. The enzgme hgaluronidaseof semen degradesthe gel (contains hyaluronic acid) around the ouum. This qllows eft'ectiuepenetration of sperm into the ouum. !3:. [j-

IF

The mucopolysaccharideheparin is an onticoagulant(preuentsblood clotting). The suruiual of Antarctic lish below -2"C is attributed to the antit'reeze glycoproteins. streptomycin is a glycoside employed in the treatment oJ tuberculosis.

B IOC H E MIS TFIY

24 Hy alur o n i c a c i d i s c o mp o s e d o f al ternate unit s of D -g l u c u ro n i c a c i d a n d N-acetyl D- gluc os a mi n e .T h e s e tw o mo l e c u l es form dis ac c ha ri d eu n i ts h e l d to g e th e r b y 0 (t -+ S ) gly c os idi c b o n d (F i 9 ,2 ,1 5 ). H y a l u ro n i c aci d c ont ains a b o u t 2 5 0 -2 5 ,0 0 0 d i s a c c h a ri deuni ts (held by p 1 -+ 4 bonds)with a molecularweight uo t o 4 m i l l i o n . Hyaluronidase is an enzyme that breaks ( B 1 - + 4 l i n k a g e s )h y a l u ro n i c a c i d a n d other CA C. Th i s e n z y m e i s p re s e n t i n hi gh c onc ent r a ti o ni n te s te s ,s e mi n a l fl u i d , and i n certainsnakeand insectvenoms. Hyaluronidase of s em en i s a s s i g n e d a n i m p o rta n t rol e i n f er t iliz at i o n a s th i s e n z y m e c l e a rs the gel ( hy alur on i ca c i d ) a ro u n d th e o v u m a l l ow i ng a better penetration of sperm into the ovum. Hy alur on i d a s eo f b a c te ri a h e l p s th e i r i nvasi on int o t he a n i ma l ti s s u e s . Ghondroitin

D-Glucuronicacid N-Acetylglucosamine Hyaluronic acid

D-Glucuronic acid

N H -C O-C H 3 H N-Acetylgalactosamine 4-sulfate

Chondroitin 4-sulfate

-o-so3

sulfates

Chondroitin 4-sulfate (Greek: chondroscartilage) is a major constituent of various m am m ali a n ti s s u e s (b o n e , c a rti l a g e ,t endons, heart,valves,skin, cornea etc.).Structurally,it is c om par a b l ew i th h y a l u ro n i c a c i d . C h ondroi ti n 4-sulfateconsistsof repeatingdisaccharideunits c om pos e d o f D -g l u c u ro n i ca c i d a n d N-acetyl D-galactosamine4-sulfate(Fig.2.lV.

oH

O-SO;

H

N-Sulfoglucosamine D-Glucuronate-2-sulfate 6-sulfate Heparin

Chondroitin 5-sulfateis also presentin many tissues.As evident from the name, the sulfate group is found on C6 insteadof Ca.

t

Heparin is composed of alternatingunits of N-sulfoD-glucosamine6-sulfateand glucuronate 2-sulfate(Fi9.2.17).

Dermatan sulfate The name dermatan sulfate is derived from the fact that this compound mostly occurs in the s k in. lt i s s tru c tu ra l l yre l a te d to c h o ndroi ti n

-o'r

N H _C O_C H . H N-Acetylgalactosamine 4-sulfate Dermatansulfate

Heparin Heparin is an anticoagulant(preventsblood c lot t ing)t h a t o c c u rsi n b l o o d , l u n g , l i v e r ,ki dney, spleen etc. Heparin helps in the releaseof the enz y m e l i p o p ro te i n l i p a s e w h i c h h el ps i n c lear ingt h e tu rb i d i ty o f l i p e m i c p l a s ma.

NH-SOa

qH2oH

o

H

NH_CO :-

N-Acetylglucosamine 6-sulfate

Keratansulfate Fiq.2.17 : Structuresof common glycosaminogi',-;-: the disaccharidesas repeating units.

25

Ghapter 2 : CAFIBOHYDHATES

Glycosaminoglycan

Composition

Tissuedistribution

Function(s)

Hyaluronic acid

D-Glucuronic acid, Connective tissue, fluid, synovial humor N-acetylglucosaminevitrous

Chondroitin sulfate

D-Glucuronic bone, acid, Cartilage, skin,blood vessel Helps to maintain thestructure N-acetylgalactosamine walls andshapes oftissues 4-sulfate lung, D-Glucuronate 2-sulfate,Blood, liver, kidney, spleen Actsasananticoagulant N-sulfoglucosamine 6-sulfate vessel L-lduronic valves, acid,N-acetyl- Blood heart valves, Maintains theshapes oftissues galactosamine 4-sulfate skin

Heparin

Dermatan sulfate Keratan sulfate

D-Galactose, N-acetyl- Cartilage, cornea, connective glucosamine 6-sulfate tissues

4-sulfate.The only differenceis that there is an inversion in the configuration around C5 of D- glu c u ro n i c a c i d to fo rm L -i d uroni c aci d (Fi9.2.1V.

Serves asa lubricant. and shock Promotes absorber. wound healing

Keeps cornea transparent

and receptors.A selected list of glycoproteins and their major functions is given in Table 2.4.

The carbohydratesfound in glycoproteins include mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine,xylose, Keratan sulfate L-fucoseand N-acetylneuraminicacid (NANA). It is a heterogeneousCAG with a variable NANA is an importantsialic acid (SeeFig.2,l1). sulfate content, besides small amounts of Antifreeze glycoproteins : The Antarctic fish mannose, fructose, sialic acid etc. Keratan live below -2oC, a temperatureat which the sulfateessentiallyconsistsof alternatingunits of D-galactosamine and N-acetylglucosamine 6-sulfate. A summary of the glycosaminoglycans with regardto composition,distributionand functions is given in Table 2.3.

Several proteins are covalently bound to carbohydrateswhich are referred to as glycoproteins. The carbohydrate content of glycoproteinvariesfrom 1o/oto 90o/oby weight, Sometimes the term mucoprotein is used for glycoprotein with carbohydrate concentration more than 4"/o. Clycoproteins are very widely distributed in the cells and perform variety of f unc t io n s .T h e s ei n c l u d e th e i r ro l e a s enzymes, hormones,transportproteins,structuralproteins

Glycoprotein(s)

Collagen proteases, Hydrolases, glycosidases Ceruloplasmin lmmunoglobulins glycoproteins Synovial Thyrotropin, erylhropoietin group Blood substances Fibronectin, laminin Intrinsic factor Fibrinogen

Major function(s)

Structure Enzymes Transport Defense against infection Lubrication Hormones Antigens Cell-cell recognition and adhesion Absorption ofvitamin 8,, Blood clotting

26 blood would {reeze.lt is now known that ihese fish contain antifreeze glycogtrateinwhich lower the freezingpoint of water and interferewith tne crystalformationof ice. Antifreezegiycoproteins c ons isto f 5 0 re p e a ti n gu n i ts o f th e tri pepti de, alanine-alawine-threonine. Each threonine r es idu e i s b o u n d to B-g a l a c to s yl(1 + 3) o( N-acetylgalactosam i ne.

ElIOCHEMISTF|Y

ri#i .f*iCA#

'?

!"r.Ii $: F"r1.*f i { r,.4F.

i4 t: * :il "'.3

The blood group antigens (of erythrocyte membrane) contain carbohydrates as glycoprotei nsor gl ycol i pi ds.N -,A .cetyl gai actosa m ine, gal actose,fucose,si al i c aci d etc. are found in the blood group substances.The carbohydrate content al so pl ays a determi nantrol e i n bl oo d E roup!n8.

X. Carbohydrs,tesare the polyhydroxyaldehydesor ketones,or campounds which produce them on hydrolysis. The term sugor is applied to carbohydratessoluble in water and stDeet to taste. Carbahgdratesqre the major dietary energy sources, besides their inualuement in cell structure and uariousother t'unctions. 2. Carbohydrqtesare broadly c/ossiJiedinta 3 groups-ffionasqccharides,oligosoccharides and ytoiysaccharides. The monosacchsridesare further diuided into dit't'erentcategories bqsed an the presence af t'wnctional groups {oldosesar ketoses)and the number of carbon atoms (trioses, tetroses,pentases, hexosesand heptcses). 3. Glyceraldehyde{triose) is the simplest carbohydrate and is chosen as a reJerenceto write the cont'iguratian of all other rnonasaccharides(D- anc L- forms). It' two rnonosaccharides differ in their structure around o single carbon atom, they ore known as eplmers.Glucoseand galactoseare C4-epimers. 4. D'Glucose is the most predominant naturally occurring aldosdmonosaccharide. Giucoseexisfs cs a and p anemers with dit'Jerentoptical rotations. The interconuersion of a and B anomericforms with changein the optical rotatian is knoun as mutsratation. 5. Manosaccharidespariicipate in seuercl recctions" These include oxidation, reduction. dehydration, asazone formetion etc. Formatian ol esters and glycosides by manosacchqridesis af special significanceln biochemical reactions. 6. Among the oligosacchqrides,disoccharidesare the most common. These include the reducing disaccharidesnamely lactose(rnilk sugar)and maltase (malt sugar)and the non-reducingsucrose(cane sugar). 7. Palysacclwridesare the poiymers ot' monosaccharides or their deriuatiues,held together by glycosidic bonds. Homopalysaccharidessre compased ot' a single manosaccharicle (e.g., starch, glycogen, cellulose, inulin). Heteropolysaccharidescontain a mixture af Jew monasaceharidesor thetr derluatiues(e.g., rnucapolysacaharides). 8. Slorch and glgcogensre the carbohydrate reseruesot' plants and animals respectiuelg. Cellulose,exclusiuelyt'ound in plants, is the structural constituent. Inulin is utilized to ossesskidney tunction bg measuring glomerular t'iltration rate (GFR). 9. Mucopoiysaccharides(glycosominoglycans)are the essential companents o/ tlssue structure. They prouide the mstrix or grownd substanceof extracellular tissue spacesin whtch collagen and elastin fibers are embedded. Hyaluranic ocid, chondroitin 4'sult'ote, heporin, are amang the important glycosaminaglgcdns. 70. Glycoproteins are a group of biochernically important compaunds with a uariable composition of carbohyd.rate(7-900/o),caualently bound to protein. Seueral enzyrnes, hormanes, structura! proteins and cellular receptorsare in fact glycoproteins.

Ghapter 2 : CAFIBOHYDHATES

I.

27

Essayquestions with suitableexamples.Add a note on the functionsof 1. Define and classifycarbohydrates carbohydrates. 2. Describethe structureand functionsof mucopolysaccharides. with specialreferenceto 3. Cive an accountof the structuralconfigurationof monosaccharides, glucose. 4. Discussthe structureand functionsof 3 biochemicallyimportantdisaccharides. and describethe structureof 3 homopolysaccharides. 5. Define polysaccharides

II.

Short notes (fl (c) Osazoneformation,(d) Clycosidicbond, (e) Sugarderivatives, (a) Epimers,(b) Mutarotation, (j) (i) (h) Deoxysugars. Anomers,(g) Enediol, Amino su8ars, Inversionof sucrose,

III.

F ill i n th e b l a n k s 1. N a me a n o n -re d u c i ndgi s a c c hari de 2. The carbohydratethat is taken as a referencefor writing the configurationof others differ in configurationarounda singlecarbonatom, they are known 3. lf two monosaccharides as 4. The s and B cyclic forms of D-glucoseare referredto as moietyfound in glycosidesis known as 5. The non-carbohydrate 6. Cive an exampleof a glycosideantibiotic 7. The glycosidicbondsat the branchingpointsin the structureof starchare employedfor the assessmentof kidneyfunction B . The polysaccharide that servesas a lubricantand shockabsorbantof joints 9. The glycosaminoglycan and glycolipids 10. Name the sialicacid, mostlyfound in the structureof glycoproteins

IV.

Multiple choice questions 11. Riboseand deoxyribosediffer in structurearounda singlecarbon,namely (a) Cr (b) Cz (c) C: (d) Cq. 12. O n e o f th e fo l l o w i n gi s n o t a n al dose (a) Clucose(b) Calactose(c) Mannose(d) Fructose. that servesas an anticoagulant 13. The glycosaminoglycan (a) Heparin(b) Hyaluronicacid (c) Chondroitinsulfate(d) Dermatansulfate. bonds is composedof B-glycosidic 14. The following polysaccharide (a) Starch(b) Clycogen(c) Dextrin (d) Cellulose. 15. The carbonatomsinvolvedin the osazoneformation (a) 'l and 2 (b) 2 and 3 (c) 3 and 4 (d) 5 and 6.

Lirpirdls

The Jat speaks

fl ?"'-oR--c-o1H CH2-H

i fr

:

"\ffith uater, I say, 'Touch me not': T'otlte tongue,I am tasteful;

R3

IY'ithin limits, I am datiful; I am dangerous!" fn excess,

(Creek: lipos-fat) are of Breat 1 . Simple lipids : Estersof fatty acids with I ipids L im por t a n c e to th e b o d y a s th e chi ef al cohol s.Theseare mai nl y of tw o types concentrated storage form of energy, besides (a) Fatsand oils (triacylglycerols) : Theseare t heir r ole i n c e l l u l a rs tru c tu rea n d v a ri o u sother glycerol. with The of fatty acids esters bioc hem ica l fu n c ti o n s . As s u c h . l i o i d s are a between fat and oil is only difference heterogeneous group of compounds ano, physical.Thus, oil is a liquid while fat is therefore, it is rather difficult to define them a solid at room temperature. pr ec is elv . Lipids may be regarded as organic substances relatively insoluble in water, soluble in organic solvents (alcohol, ether etc.), actually or potentially related to fatty acids and utilized by the living cells.

(b) W axes: E stersof fatty aci ds (usual l yl ong chai n) w i th al cohol sother than gl ycerol . These al cohol s may be al i phati c or al i cycl i c.C etylal cohol i s most commonl y found i n w axes.

2. C ompl ex(or compound)l i pi ds: Theseare , ro te ins and Unlik e th e p o l y s a c c h a ri d e s p esters of fatty aci ds w i th al cohol s contai ni ng nuc leic aci d s , l i p i d s a re n o t p o l y me rs .Further, groups such as phosphate, addi ti onal lipids ar e m o s tl y s ma l l mo l e c u l e s . ni trogenous base, carbohydrate,protei n etc They are furtherdi vi ded as fol l ow s Lipids a re b ro a d l y c l a s s i fi e d(mo d i fi edfrom B loor ) into s i mp l e , c o m p l e x , d e ri v e d and m is c ellan e o ul isp i d s ,w h i c h a refu rth e rs u bdi vi ded into differentgroups

28

(a) P hosphol i pi ds:They contai n phosphor,c aci d and frequentl ya ni trogenousbase Thi s i s i n addi ti on to al cohol and fai :. aci ds.

Chapter 3 : LIPIDS

29

(i) Glycerophospholipids : Thesephospho5. Lipids protectthe internalorgans,serveas lipids contain glycerol as the alcohol insulatingmaterialsand give shape and smooth e .9 .,l e c i th i n ,c e p h a l i n . appearanceto the body. (ii) Sphingophospholipids : Sphingosineis the alcohol in this group of phosphol i p i d s e .g ., s p h i n g o myel i n. (b) Glycolipids: These lipids contain a fatty Fatty acids are carboxylic acids with acid, carbohydrateand nitrogenousbase. hydrocarbonside chain. They are the simplest T h e a l c o h o l i s s p h i n g o s i n e,hence they form of l i pi ds. a re a l s o c a l l e d a s g l y c o sphi ngol i pi ds. Clycerol and phosphateare absent e.g., Occurrence cerebrosides,gangliosides. Fattyacids mainly occur in the esterifiedform (c) Lipoproteins: Macromolecularcomplexes as major constituentsof various lipids. They are of lipids with proteins. also present as free (unesterified)fatty acids. (d) Other complex lipids: Sulfolipids,aminoFattyaci ds of ani mal orgi n are much si mpler in l i p i d sa n d l i p o p o l y s a c c h a ri des are among structure in contrast to those of plant origin th e o th e r c o m p l e x l i p i d s . which often contain groupssuch as epoxy, keto, 3. Derived lipids: These are the derivatives hydroxy and cyclopentanerings. obtainedon the hydrolysisof group 1 and group 2lip i d s w h i c h p o s s e s sth e c h a racteri sti csof Even and odd carbon fatty acids lipid s .T h e s ei n c l u d eg l y c e ro la n d o theral cohol s, Most of the fatty acids that occur in natural fatty acids, mono- and diacylglycerols,lipid (fat) lipids are of even carbons (usually 14C-2OC). soluble vitamins, steroid hormones, hydroThis is due to the fact that biosynthesisof fatty carbons and ketone bodies. acids mainly occurswith the sequentialaddition 4. M i s c e l l a n e o u sl i p i d s : T h e se i ncl ude a of 2 carbon units. Palmitic acid (l6C) and large number of compounds possessingthe stearic acid (l$C) are the most common. Among characteristics of lipids €.g., carotenoids, the odd chain fatty acids, propionic acid (3C) squalene,hydrocarbonssuch as pentacosane(in and val eri c aci d (5C ) are w el l know n. bees wax), terpenes etc. N EU T R A T L IPID S: T h e l i p i ds w hi ch are Saturated and unsaturated unchargedare referredto as neutrallipids.These fatty acids are mono-, di-, and triacylglycerols,cholesterol Saturatedfatty acids do not contain double and cholesterylesters. bonds,while unsaturatedfatty acids contain one or more double bonds. Both saturated and Functions of lipids unsaturatedfatty acids almost equally occur in Lipids perform severalimportant functions the natural lipids. Fatty acids with one double and those with 2 or 1. They are the concentratedfuel reserveof bond are monounsaturated, more double bonds are collectivelv known as the body (triacylglycerols). polyunsaturated fafty acids (PIJFA). 2. Lipids are the constituentsof membrane structure and regulate the membrane Nomenclature of fatty acids s n d c hol esterol ). per m e a b i l i ty(p h o s p h o l i p i d a The naming of a fatty acid (systematicname) 3. They serve as a source of fat soluble is based on the hydrocarbonfrom which it is v it am i n s(4 , D , E a n d K ). derived. The saturatedfatty acids end with a 4. L i p i d sa re i mp o rta n ta s c e l l u l ar metabol i c suffix -anoic (e.g., octanoic acid) while the regulators(steroidhormonesand prostaglandins). unsaturatedfatty acids end with a suffix -enoic

30

BIOCHEMISTF|Y

(e.9., octadecanoic acid). In addition to systematic names/ fatty acids have common nameswhich are more widely used (Iable J. l).

Length of hydrocarbon cha:n of fatty acids

Numbering of carbon atoms : lt starts from the carboxylcarbon which is taken as number 1. The carbonsadjacentto this (carboxylC) are 2, 3, 4 and so on or alternatelya, F, T and so on. The terminal carbon containingmethyl group is known omega (or) carbon. Starting from the methyl end, the carbon atoms in a fatty acid are numberedas omega 1, 2, 3 etc. The numbering of carbon atoms in two different ways is given below 7654 3 2 1 cH3 - cH2 - cH2- cH2-cH2 - cH2 - COOH (t)6 01 a2 o)3 ()4 ol5

Common Name

Systematic name

Depending on the length of carbon chains, fatty acids are categorized into 3 groups-short chain with less than 6 carbons; medium chain with 8 to 14 carbons and long cfiain with 16 to 24 carbons. Shorthand representation of latty aclds lnstead of writing the full structures, biochemists employ shorthand notations (by numbers)to representfatty acids. The general rule is that the total number of carbon atomsare written first, followed by the nunrber of double bonds and finally the (first carbon) position of

Abbreviationx

Structure

l. Saturated fattyaclds Aceticacid Propionic acid Butyricacid Valeric acid Caproic acid Caprylic acid Capricacid Lauricacid Myristic acid Palmitic acid Stearic acid Arachidic acid Behenic acid Lignoceric acid

Ethanoic acid n-Propanoic acid n-Butanoic acid n-Pentanoic acid n-Hexanoic acid n-Octanoic acid n-Decanoic acid n-Dodecanoic acid n-Tetradecanoic acid n-Hexadecanoic acid n-Octadecanoic acid n-Eicosanoic acid n-Docosanoic acid n-Tetracosanoic acid

2:0 3:0 4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0

CHsCO0H CHgCHzCOOH CHs(CHz)z0O0H CHo(CHz)gCOOH CHs(CHe)+COOH CHe(CHz)oCOOH CHs(CHz)eC0OH CHs(CHz)roCOOH CHs(CHzhzCOOH CHg(CHz)t+CO0H CHs(CHz)roC0OH CHg(CHz)reCOOH CHs(CHz)zo00OH CH3(CHz)zzCOOH

16:1;9 18:1;9 18: 2;9,12

= CH(CHz)zCOOH CHg(CHz)sCH = CH(CHz)zCOOH CHs(CHz)zCH = CHCHzCH = CH(CHz)zCOOH CHg(CHz)+CH

F .n

ll. Unsaturated fattyacids Palmitoleic acid Oleicacid Linoleic acid** Linolenic acid*x Arachidonic acid

cr1s9-Hexadecenoic acid cls-9-Octadecenoic acid cls,cls-9,12-Octadecadienoic acid Allce9,12,15-0ctadecatrienoic acid Allcls-5,8,11,14-

18: 3;9,12,'15

= CHCHzCH = CHCHzCH CHoCHzCH = CH(CHz)zCO0H = CHCHzCH = CHCHzCH 20:4;5,8,11,14 CHg(CHz)+CH

Elc0:a!tr3e!o!1ci1___

__=9H9'tcl=_cl9F!)49oli

* Totalnunberofcarbonatons,followed posrtion bythenumber andthefirctcarbon otdoublebonds ot thedouble bond(s). ** Essential faw acids.

31

Ghapten 3 : LIPIDS

double bonds, startingfrom the carboxyl end. Thus,saturatedfatty acid, palmitic acid is written as . l 6: 0 , o l e i c a c i d a s 1 8 :1 ;9 , a rachi doni c ac id as 2 0 : 4 ; 5 , 8 , 1 1 , 1 4 . There are other conventionsof representing t he dou b l e b o n d s .Ae i n d i c a te sth a t the doubl e bond is between 9 and 10 of the fatty acid. o 9 representsthe double bond position (9 and 10) from the
H..ar(CHz)zCOOH

H'c'1cHr;rcu, Oleic acid (cls form)

Elaldic acid (frans form) Fig. 3.1 : Cis-trans isomerism in unsaturated fattv acids.

on the posteriorand lateralpartsof limbs,on the back and buttocks,loss of hair and poor wound heal i ng. lsomerism in unsaturated fatiy

aeids

Unsaturated fatty acids exhibit geometric isomerismdepending on the orientation of the groups around the double bond axis.

lf the atoms or acyl groupsare presenton the same side of the double bond, it is a cis configuration. On the other hand, if the groups occur on the opposite side, it is a trans The fatty acids that cannot be synthesizedby configuration. Thus oleic acid is a cis isomer the body and, therefore, should be supplied in while elaidic acid is a trans isomer,as depicted the diet are known as essentialfatty acids (EFA). in Fig.3.1. Cis isomers are less stable than frans Chemically, they are polyunsaturated fatty i somers. Most of the natural l y occurri n g acids, namely linoleic acid (18 : 2; 9, 12) and unsaturatedfatty acids exist as crs isomers. Iinolenic acid (18 : 3; 9, 12, 15). Arachidonic In the cis isomericform, there is a molecular ac id ( 20 :4 ;5 ,8 , 1 1 ,1 4 ) b e c o m e sessenti ali,f bi ndi ng at the doubl e bond. Thus, ol ei c aci d its precursorlinoleic acid is not provided in the exi sts i n an L-shapew hi l e el ai di c aci d i s a diet in sufficientamounts.The structuresof EFA strai ghtchai n. Increasei n the numberof doubl e are given in the Table 3.1. bonds w i l l cause more bends (ki nks) and Biochemical basis for essentiality: Linoleic arachi doni caci d w i th 4 doubl e bondsw i l l have ac id an d l i n o l e n i c a c i d a re e s s e nti al si nce a U-shape. lt is believed that cis isomersof fatty hum an s l a c k th e e n z y me s th a t c a n i ntroduce acids with their characteristic bonds will compactly pack the membranestructure. double bonds beyond carbons 9 to 10. Functions of EFA: Essentialfatty acids are required for the membrane structure and function, transport of cholesterol,formation of lipoproteins,preventionof fatty liver etc. They are also needed for the synthesisof another important group of compounds, namely eicosanoids(Chapter 32.

Hydroxy fatty acids: Some of the fatty acids are hydroxylated.p-Hydroxybutyricacid, one of the ketone bodies produced in metabolism,is a simple example of hydroxy fatty acids. Cerebronic acid and recinoleic acid are long chain hydroxy fatty acids.

Cyclic fatty acids: Fatty acids with cyclic Deficiency of EFA: The deficiency of EFA structuresare rather rare e.g./ chaulmoogric acid results in phrynoderma or toad skin, found in chaulmoogra oil (used in leprosy characterizedby the presenceof horny eruptions treatment)contains cyclopentenylring.

32

B IOC H E MIS TFIY

o

U C H 2 -O -C Fl , A ltl R2-C-O-CH O ttl

cH2-o-c-R3 Triacylglycerol

o

cH2-o-c-R,

Rz-C-o-cH I

cH2oH 1,2-Diacylglycerol

o cH2-o-c -B tHO_CH I cH20H 1-Monoacylglycerol

C H ,_OH O ill R -C -O-C H I cH2oH 2-Monoacylglycerol

Fig. 3.2 : General structures of acylglycerols (For palmitoyl R = CtsHati for stearoyl R = C.rzHssiFor linoleoyl R = qtHsi

Eicosanoids:Thesecompoundsare relatedro of glycerol, are known (Fi5.3.2).Among these, eicosapolyenoicfatty acids and include prosta- triacylglycerols are the most important glandins ,p ro s ta c y c l i n sl e, u k o tri e n e a s n d throm- bi ochemi cal l y. boxanes.They are discussedtogether(Chapter 32). Simpletriacylglycerolscontain the sametype of fatty acid residueat all the three carbonse.g., tristearoylglycerol or tristearin. Mixed triacylglycerols are more common. (formerly They Triacylglycerols contain 2 or 3 different types of fatty acid triglycerides) are glycerol residues. In general,fatty acid attachedto C1 is the esters of with fatty acids. The fats plants saturated,that attached to C2 is unsaturated and oils that are widely distributedin both while that on C3 can be either. Triacylglycerols and anima l s a re c h e mi c a l l y tri a c y l g l ycerol s. T hey ar e i n s o l u b l e i n w a te r a n d n o n -pol ar i n are named according to placement of acyl characterand commonly known as neutral fats. radi calon gl ycerole.9.,' l,3-pal mi toyl2-l i nol eoyl gl ycerol . Fats as stored fuel : Triacylglycerolsare the m os t abu n d a n t g ro u p o f l i p i d s th a t p ri mari l y Triacylglycerols of plants, in general, have function as fuel reservesof animals. The fat higher content of unsaturated fatty acids reserveof normal humans (men 2Oo/o,women compared to that of animals. 25% by weigh$ is sufficientto meet the body's $tereospecific numbering caloric requirementsfor 2-3 months. of gl ycerol Fats primarily occur in adipose tissue: Adipocytes of adipose tissue-predominantly The structureof glycerol gives an impression found in the subcutaneouslayer and in the that carbons1 and 3 are identical.This is not true abdominalcavity-are specializedfor storageof in a 3-dimensionalstructure.In order to represent triacylglycerols.The fat is stored in the form of the carbon atomsof glycerol in an unambiguous globulesdispersedin the entire cytoplasm.And manner, biochemists adopt a stereospecific surprisingly,triacylglycerolsare not the structural numbering(sn) and prefix glycerolwith sn. componentsof biological membranes. Structures of acylglycerols: Monoacylglycerols, diacylglycerols and triacylglycerols, respectivelyconsisting of one, two and three moleculesof fatty acids esterifiedto a molecule

6n,on no-C'.-H 6tr,ot sn-GfcJrol

C*rapter'3 : LIPIDS

33

It should be noted that C1 and C3 are a. tipid peroxidation in vivo: In the living d ifferent. Cells possess enzymes that can cel l s, l i pi ds undergo oxi dati on to produce dis t ingui s h th e s e tw o c a rb o n s. Thus peroxidesand free radicalswhich can damage glycerokinasephosphorylates sn-3 (and not sn-l) the tissue.The free radicalsare believedto cause glycerol to give sn-glycerol3-phosphate. inflammatory diseases, ageing, cancer/ atherosclerosis etc. lt is fortunatethat the cells PROPERTIES OF TRIACYLGTYCEROLS possessantioxidantssuch as vitamin E, urateand superoxide dismutaseto prevent in vivo lipid A few importantpropertiesof triacylglycerols, peroxidation (Chapter 34). which have biochemical relevance, are discussedbelow 1. Hyd ro l y s i s: T ri a c y l g l y c e ro l s undergo stepwiseenzymatic hydrolysisto finally liberate free fatty acids and glycerol. The process of hydrolysis,catalysedby lipasesis importantfor digestionof fat in the gastrointestinal tract and fat mobilization from the adiposetissues.

Tests to check purity of fats and oils Adulterationof fats and oils is increasingday by day. Several tests are employed in the laboratoryto check the purity of fats and oils. Some of them are discussedhereunder

lodine number : lt is defined as the grams 2. Saponification: The hydrolysisof triacyl(number) of iodine absorbedby 100 g of fat or glycerolsby alkali to produceglyceroland soaps oil. lodine number is usefulto know the relative is k nown a s s a o o n i fi c a ti o n . unsaturationof fats, and is directly proportional Triacylglycerol+ 3 NaOH ---------+ to the content of unsaturatedfatty acids. Thus Clycerol + 3 R-COONa(soaps) lower is the iodine number, lessis the degreeof 3. Rancidity: Rancidity is the term used to unsaturati on.The i odi ne numbersof common represent the deterioration of fats and oils oils/fatsare given below. resultingin an unpleasanttaste. Fatscontaining FaUoil lodine number unsaturatedfatty acids are more susceptibleto r anc idit v .

Coconut oil

7-

10

Butter 25- 28 Rancidity occurs when fats and oils are Palm oil 4C 55 exposed to air, moisture, light, bacteria etc. Oliveoil 80- 85 Hydrolytic rancidity occurs due to partial Groundnut oil 85- 100 hydrolysis of triacylglycerols by bacterial Cottonseed oil 100 - 110 enzymes.Oxidative rancidity is due to oxidation Sunflower oil 125 135 of unsaturatedfatty acids. This results in the Linseed oil 175 -200 formation of unpleasant products such as dicarboxylic acids, aldehydes, ketones etc. D etermi nati onof i odi ne number w i l l hel p to Rancid fats and oils are unsuitablefor human know the degree of adulterationof a given oil. c ons um o ti o n . Saponificationnumber : lt is defined as the Antioxidants : The substanceswhich can mg (number) of KOH required to hydrolyse preventthe occurrenceof oxidativerancidityare (saponify) one gram of fat or oiL Saponification known as antioxidants. Trace amounts of number is a measureof the averagemolecular ant iox ida n tss u c h a s to c o p h e ro l s(v i tami n E ), size of the fatty acids present.The value is higher hy dr oqu i n o n e ,g a l l i c a c i d a n d c ,-n a p htholare for fats containing short chain fatty acids. The addedto the commercialpreparationsof fats and saponificationnumbersof a few fats and oils are oils to preventrancidity.Propylgallate,butylated given below hydroxyanisole(BHA) and butylated hydroxyH umanfat : 195-200 toluene (BHT) are the antioxidantsused in food Butter :230-240 preservation. Coconutoil : 250-260

34

E l IOC H E MIS TR Y

(RM) number: lt is definedas Reichert-Meissl the number of ml 0.1 N KOH required to completely neutralize the soluble volatile fatty acids distilledfrom 5 g fat. RM number is useful in testingthe purity of butter since it containsa good concentrationof volatilefatty acids (butyric ac id, c apr oic a c i d a n d c a p ry l i c a c i d ).T h i s i s i n contrast to other fats and oils which have a negligibleamount of volatile fatty acids. Butter has a RM nu m b e r i n th e ra n g e2 5 -3 0 ,w h i l e i t i s les st han I f o r mo s t o th e r e d i b l e o i l s . T h u s any adulteration of hutter can be easily tested by t his s ens it iv eR M n u m b e r. Acid number : lt is defined as the number of mg of KOH required to completely neutralize free fatty acids presentin one gram fat or oil. In normal circumstances, refinedoils should be free from any free fatty acids. Oils, on dec om oos it i o n -d u e to c h e mi c a l o r b a cteri al contamination-yield free fatty acids.Therefore, oils with increasedacid number are unsafefor hum an c ons u m p ti o n .

There are two classesof phospholipids 1. C l ycerophosphol i pi ds(or phosphogl ycerides)that contain glycerol as the alcohol. (or sphi ngomyel i ns) 2. S phi ngophosphol i pi ds that contai n sphi ngosi neas the al cohol . 1. i t\ " .t

.;:i r ,. :

.

,,,.i ., i - l ,

C l ycerophosphol i pi dsare the maj or l i pi ds that occur in biologicalmembranes.They consist of glycerol 3-phosphateesterifiedat its C1 and C 2 w i th fatty aci ds. U sual l y, C 1 contai ns a saturated fatty acid while C2 contains an unsaturatedfatty acid. 1 . Phosphatidic acid : This is the simplest phosphol i pi d. l t does not occur i n good concentration in the tissues. Basically, phosphati di c aci d i s an i ntermedi atei n the phosphol i pi ds. s synthesi sof tri acyl gl yceroland contai ni ng The other gl ycerophosphol i pi ds differentnitrogenousbasesor other groups may be regardedas the derivativesof phosphatidic aci d.

2. Lecithins (phosphatidylcholine)zThese are the mostabundantgroupof phosphol i pi dsi n the cel l membranes.C hemi cal l y,l eci thi n (C reek : These are complex or compound lipids lecithos-egg yolk) is a phosphatidicacid with phosphoric containing acid,in additionto fatty chol i ne as the base. P hosphati dyl chol i nes acids, nitrogenous base and alcohol (Fig.3.3). represent the storage form of hody's choline.

BtoMEDtCAL/ CLtNtCAt CONCEpTS

os Lipids are important to the body as constituentsof membranes,source ol fat soluble (A, D, E and K) uitamins qnd metabolic regulators (steroid hormones and prostaglandlns), e Triacylglycerols (fots) primarily stored in the adipose tissue ore concentrated t'uel reseruesof the body. Fats t'ound in the subcutoneous tissue and around certaln orgons serue os thermal insulators, se The unsaturated fatty acids-linoleic and linolenic acid-
*

35

Chapter 3 : LIPIDS

ol l

g

,11 ill

cH2-o-c-R1

ill

.:1 RI-C-O-CH -l CH2- i- ' - r ' - i' t (1) Phosphatldicacid

tz) Leclthln(phosphatidylcholine)

i

otl I

C H 2 -O -C -R 1

ill

rf R2-C-O-qH CH2-C--l-CH2-CH2-NH2 Ethanolamine C(3) Cephalln (phosphatidylethanolamine)

,E

myalnositol

(4) Phosphatidyllnosltol

o tl cH2-o-c-Rl ? R2-c-o-?H {l

Al tl

,t_ _

C-

.),f,l

(5)Phosphatldylserlne

l-

CH2-.i

Ethanolamine

(6) Plasmalogen(phosphatidalelhanolamine)

r , n- cH2 ? tr. Hc-o-c-R3 ?

cH2-o-c-R1 cH2-, ? I H?-OH R2-C-O-CH ^

.:1

-CHz-CH2-NH2 CH2-t', - i' i---

CH2-r-:- = C-CHz-CH-COO-

o

QH2-O-CF{=CH-Rl

R2-C-O-CH

,: r-.'-CHe

+

R4-C-O-CH2

I ehospnatioytgty."ro, (7)Cardlollpin(diphosphatidylglycerol) lCeramid"

_

(/t'soninoosrne$)> CH3-(CH2)12-CH:CH-CH-?H-NH-C-R

'

,

*.CHg

r_-CHz-CHz-Nf9,Tt (8) Sphlngomyelln

Choline

Fig. 3.3 : Sttucturesof phospholipids.

\'n3

36

BIOCHEMISTF|Y (a) Dipalmitoyl lecithin is an important ph o s p h a ti d y l c h o l i nfo e u n d i n l u n gs,l t i s a surface active agent and prevents the adherence of inner surface of the lungs due to surfacetension. Respiratory distresssyndrome in infants is a disorder characterizedby the absenceof dipalmitoyl lec i th i n .

by an amide linkage to a fatty acid to produce ceramide.The alcohol group of sphingosineis bound to phosphoryl chol i nei n sphi ngomyel i n are important structure(Fig.3.3).Sphingomyelins constituentsof myelin and are found in good quantity in brain and nervoustissues.

(b) Lysolecithinis formed by removal of the fatty acid either at C, or C, of lecithin.

are a group of enzymesthat Phospholipases hydrolysephospholipids.There are four distinct (A r, 42, C and D ), each one of phosphol i pases them specificallyacts on a particularbond. For details, refer lipid metabolism(Chapter l4).

3. Cephafins (phosphatidylethanolamine): Ethanolamineis the nitrogenousbase presentin c ephalin sT, h u s ,l e c i th i na n d c e p h a l i nd i fferw i th regard to the base.

Action

of phospholipases

Functi ons

of phosphol i pi ds

4. Phosphatidylinositol : The steroisomer P hosphol i pi dsconsti tutean i mportantgroup myo-inositolis attachedto phosphatidicacid to of compound lipids that perform a wide variety giv e phos p h a ti d y l i n o s i to l (P Tlh).i s i s a n i mportant of functions me mb ra n e s .T h e a cti on of c om Done n to f c e l l w i th protei ns,phosphol i pi ds 1. In associ ati on (e.9. certain hormones oxytocin, vasopressin)is form the structural components of membranes m ediat edth ro u g h Pl . and regulatemembrane permeability. 5. P ho s p h a ti d y l s e ri n e : T h e a mi n o aci d 2. P hosphol i pi ds (l eci thi n, cephal i n and s er ine is p re s e n ti n th i s g ro u p o f g l y cerophos- cardi ol i pi n)i n the mi tochondri aare responsi bl e pholipids. Phosphatidylthreonine is also found in for maintaining the conformation of electron certaintissues. transportchai n components,and thus cel l ul ar 6. Plasmalogens : When a fatty acid is respiration. attachedby an ether linkageat C1 of glycerol in 3. Phospholipidsparticipatein the absorption gly c e ro p h o s p h o l i p i d s , th e t he resul tant of fat from the intestine. c om poun d i s p l a s m a l o g e n . P h o s phati dal 4. P hosphol i pi ds are essenti al for the et hanola mi n ei s th e m o s t i mo o rta n t w hi ch i s synthesi sof di fferent l i poprotei ns,and thus similar in structureto phosphatidylethanolamine participate in the transport of lipids. but for the ether linkage (in place of ester).An 5. Accumulationof fat in liver (fattyliver)can unsaturatedfatty acid occurs at C1. Choline, inositol and serine may substituteethanolamine be preventedby phospholipids,hence they are regarded as lipotropic factors. t o giv e ot h e r p l a s ma l o g e n s . Z. Cardiolipin : lt is so named as it was first isolated from heart muscle. Structurally, a c ar diolip i n c o n s i s ts o f tw o mo l e c ul es of phos pha ti d i ca c i d h e l d b y a n a d d i ti o n a lgl ycerol through phosphate groups. lt is an important c om pone n t o f i n n e r mi to c h o n d ri a lm e mbrane. Car diolip i n i s th e o n l y p h o s p h o g l y ceri dethat possessesantigenic properties.

6. Arachidonicacid, an unsaturatedfatty acid liberated from phospholipids, serves as a (prostaprecursorfor the synthesisof eicosanoids glandins,prostacyclins,thromboxanesetc.).

7. P hosphol i pi dsparti ci patei n the reverse chol esterol transport and thus hel p i n the removal of cholesterolfrom the body. 8. Phospholipidsact as surfactants(agenL. l ow eri ng surface tensi on). For i nstance S phingomy e l i n s i s an i mportar: di pal mi toyl phosphati dyl chol i ne Sphingosineis an amino alcohol present in fung surfactant. Respiratory distresssyndrome ^ s phingomy e l i n(s s p h i n g o p h o s p h o l i p i ds). They do infantsis associatedwith insufficientproductio^ not containglycerolat all. Sphingosine is attached of this surfactant.

37

Chapter 3 r LIPIDS

Sphingosine

loHlo YIY]

o-cH2

Fig. 3.4 : Structure ot galactosylceramide (R = H). Fot sulfagalactosylceramideR is a sulfatide (R = SOi-).

9. Cephalins,an importantgroup of phospholipids pa rti c i p a tei n b l o o d c l o tti n g .

Ceramide

) 10. P h o s p h o l i p i d s (p h o s p h a ti d y l i nosi tolare inv olv edi n s i g n a tra l n s mi s s i oanc ro s smembranes.

Glucose

I

Galactos f tl N-AcetylN-Acetylgalactosamine neuraminic acid

C lyco Ii p ids (glycosphingol ipids) are i m portant c ons t it u e n tso f c e l l me mb ra n e a n d nervous tissues(particularlythe brain). Cerebrosidesare . h e y contai n a t he s im p l e s tfo rm o f g l y c o l i p i d s T Li poprotei ns are mol ecul ar compl exes of ceramide (sphingosineattachgdto a fatty acid) lipids with proteins. They are the transport and one or more sugars. Galactocerebroside vehi cl esfor l i pi ds i n the ci rcul ati on.There are (galactosylceramide) and glucocerebrosideare five types of lipoproteins, namely chylomicrons, the most importantglycolipids.The structureof very low density lipoproteins (VLDL), low galactosylceramide is given in Fig3.a. lt contains (LDL), high density lipoproteins density cerebronic acid. the fatty acid Iipoproteins (HDL) and free fatty acid-albumin Sulfagalactosylceramideis the sulfatide derived complexes. Their structure, separation, from galactosylceramide. metabolismand diseasesare discussedtogether Gangliosides: Theseare predominantlyfound (Chapter l4). in ganglionsand are the most complex form of glycosphingolipids.They are the derivativesof cerebrosidesand contain one or more molecules of N- ac e ty l n e u ra m i n i a c c i d (N A N A ), the most im oor t a n ts i a l i c a c i d . T h e s tru c tu reo f N A N A i s Steroids are the compounds containing a given in carbohydratechemistry(ReferFig.2.l1\. cycl i c steroi d nucl eus (or ri ng) namel y The most important gangliosidespresent in cyclopentanoperhydrophenanthrene (CPPP). lr t he br a i n a re C M1 , C M2 , C D , a nd C T, consistsof a phenanthrenenucleus (rings A, B (G representsganglioside while M, D and T and C ) to w hi ch a cycl opentaneri ng (D ) i s indicate rnono-, di- or tri- sialic acid residues, attached. The structure and numbering of CPPP are and the number denotes the carbohydrate The to the ceramide). shown sequence attached in Fi9.3.5.The steroid nucleusrepresents ganglioside,CM2 that accumulatesin Tay-Sachs saturatedcarbons, unlessspecificallyshown as diseaseis reoresentednext (outline structure). doubl e bonds. The methyl si de chai ns (19 and

38

BIOCHEMISTF|Y

Structure and occurrence The structure of cholesterol (C27Ha6O)is depicted in Fig.3.5.lt has one hydroxyl group at C3 and a double bond between C5 and C6. An 8 carbon aliphatic side chain is attachedto C17. Cholesterol contains a total of 5 methyl Sroups. Due to the presence of an -OH group, chol esteroli s w eakl y amphi phi l i c.A s a structural component of plasma membranes,cholesterol is an important determinant of membrane permeabilityr,properties. The occurrence of cholesterolis much higher in the membranesof sub-celIu lar organelles. Cholesterolis founi in associationwith fany acids to- form cholestervl esters (esterification occurs at the OH group of C3). Properties and reactions : Cholesterol is an yel l ow i sh crystal l i nesol i d. The crystal s,under the microscope, show a notched (E) appearance.Cholesterol is insoluble in water and sol ubl e i n organi c sol vents such as chloroform, benzene,ether etc. Fig. 3.5 : Sttucturcs of steroids (A, B, C-Perhydroph enanthrene; D-Cyclopentane).

Several reactions given by cholesterol are useful for its qualitative identification and quantitativeestimation.TheseincludeSalkowski's test, Liebermann-Burchardreaction and Zak's test.

18) attachedto carbons10 and 13 are shown as s ingle bo n d s . At c a rb o n 1 7 , s te ro i d s usual l y Functions of cholesterol: Cholesterol is a c ont ain a s i d e c h a i n . poor conductor of heat and electricity,since it T her e a re s e v e ra ls te ro i d si n th e b i ol ogi cal has a high dielectric constant. lt is present in s y s t em .Th e s e i n c l u d e c h o l e s te ro l ,b i l e aci ds, abundance in nervous tissues.lt appears that cholesterolfunctions as an insulatingcover for vitamin D, sex hormones, adrenocortical hor m one s ,s i to s te ro l s ,c a rd i a c g l y c o si desand the transmi ssi onof el ectri cal i mpul ses i n the alk aloid s . l f th e s te ro i d ,c o n ta i n so n e or more nervous tissue. Cholesterol performs several hy dr ox yl g ro u p s i t i s c o m m o n l y k now n as other bi ochemi calfuncti onsw hi ch i ncl ude i ts role in membranestructureand function, in the s t er ol ( m e a n s s o l i d a l c o h o l ). synthesis of bile acids, hormones (sex and CI{OLESTEROL cortical) and vitamin D (for details, Refer Chofesterol, exclusively found in animals, is Chapters 7 and l9). t he m os t a b u n d a n t a n i ma l s te ro l . l t i s w i del y distributedin all cells and is a major component ERGOSTEROL Ergosteroloccurs in plants.lt is also found as of cell membranesand lipoproteins.Cholesterol (Creek: chole-bile) was first isolatedfrom bile. a structuralconstituentof membranesin yeast Cholesterolliterally means 'solid alcohol from and fungi. Ergosterol(Fig.3.5) is an important precursorfor vitamin D. When exposedto light, bile. '

fTi

li

Cfrraptee3 : LIPIDS

39

I

the ring B of ergosterolopens and it is converted to ergocalciferol, a compound containing vitamin D activity.

cH3(cHdn-cooHydrophiic Hydrophobic hydrocarbonchain carboxyl group (tail) (head) (A) Fatty acid

The other sterolspresentin plant cells include stigmasterol and ftsitosterol.

ot l

Hydrophobic tail

A s per defi ni ti on,l i pi ds are i nsol ubl e(hydr ophobi c) i n w ater. Thi s i s pri mari l y due to t he predominant presenceof hydrocarbon groups. However, some of the lipids possesspolar or hydrophi l i cgroupsw hi ch tend to be sol ubl e in water. Molecules which contain both hydrophobicand hydrophilic groups are known as amphipathic (Creek : amphi-both, pathospassi on).

HYdroPhilic head

(B) Phospholipid

Examplesof amphipathic lipids: Among the l i pi ds, fatty aci ds, phosphol i pi ds,sphi ngol i pids, bile salts and cholesterol(to some extent) are amphi pathi ci n nature.

(C) Amphipathic lipid

Aqueous pnase

Fattyacids contain a hydrocarbonchain with a carboxyl (COO-) group at physiologicalpH. The carboxyl group is polar in nature with affinityto water (hydrophilic)while hydrocarbon chain of fatty acid is hydrophobic.

(D)Micelle

OOOOC

ttttl ltttl

Nonpolarphase

oo

Aqueousphase (E)Lipid bilayer

Phospholipidshave a hydrophilichead (phosphate group attachedto choline, ethanolamine, inositol etc.) and a long hydrophobic tail. The generalstructureof an amphipathicIipid may be representedas a polar or hydrophilic head with a non-polar or hydrophobictail (Fig.3.6).

Orientation amphipathic lipids: When the amphipathic lipids are mixed in water (aqueous Aqueousphase phase), the polar groups (heads) orient themsel vestow ards aqueous phase w hi l e t he non-polar (tails) orient towards the opposite directions.This leadsto the formation of mr'celles (Fi9.3.6).Micelle formation, facilitated by bile salts is very important for lipid digestion and absorption (Chapter 8). Me*nhrane

Fig. 3.6 : Summary of amphipathic lipids in the formation of micelle and lipid bilayer.

bilayers

In case of biological membranes,a bilayer of l i pi ds i s formed ori enti ngthe pol ar headsto t he

B IOC H E MIS TR Y

40

BIOMEDICAL/ CLIITIICALCOilCEPTS

The phospholipid4ipalmitoyl lecithin-preuents the adherence of inner surface of the lungs, the absence of which is ossociofed with respiratory disfress syndrome in infants. !ei-

Cepholinsparticipate in blood clotting.

€

The action of certain hormones is mediated through phosphatidylinositol.

g

Phospholipids are important for the synthesis and transport of lipoproteins ond reuerse tronsport ol cholesterol. Cholesterol is essentialfor the synfhesisol bile ocids, hormones(sexand cortical)and uitamin D. Lipoproteins occur in the membrone structure, besidesseruingos o meansol transport uehiclesfor lipids. Lipids are associated with certain disorders----obesityond atherosclerosis.

out er a q u e o u s p h a s e o n e i th e r s i de and the nonpolar tails into the interior (Fig.3.6\. fhe f or m a ti o n o f a l i p i d b i l a y e r i s th e basi s of membranestructure.

combi nati on w i th ti ssue speci fi c anti B ens,ar e used as carriersof drugs to tarBettissues.

Emulsions: These are produced when nonpol ar l i pi ds (e.g. tri acyl gl ycerol s)are mi xed Liposomes: They are produced when amphi- with water. The particlesare larger in size and pat hic l i p i d s i n a q u e o u sm e d i u m a re subj ected stabi l i zed by emul si fyi ng agents (usuallr to sonification. They have intermittent aqueous amphi pathi c l i pi ds), such as bi l e sal ts and phas e s i n th e l i p i d b i l a y e r. L i posomes, i n phosphol i pi ds.

LIPIDS

substances relatiuely inso,luble in water, soluble in organic actually or potentially related to totti-o"iJl'ord or" utilized 2

Lipids are crassifiedinto simpre (fats deriued (fatty acids, steriod n"r_"n"rl ,and.,oirs),c,omprex(phosphoripids, grgcolipids), and miscelloneous(carotenoids). 3' Fatty acidsare the maior constituents of.uarious ripids.saturated and unsaturotedfatty acidsarmostequarsoccurin ri'['i":ur"rrirrated

ii[i:"0"i:':;::i:i

"tt"*i'1io11i, fatty acids(pr'FA) and tinoteni, o,id o," the.s'se,'tiai ioitv o,ia, that needto be

4' Triacylglycerols(simply fats)are the esters glycerol with fatty acids.They are found in adipose tissueand pr:imoriry ,*nrtior.o, 7f ',u"li.r"rr"- oj oli^otr. seuerar tests(iodine number, RM number)are emproged

in tn" ioiorrJo"/iio"i"i'r"ine purityof

fotsand oirs, 5' Phosphotipidsare complex lipids cctntainin.g phosphoric acid. Glycerophospholipids

;;:I:,:Ji:'::;',:i;,:i:;,:ot

andtheseinct-ude'r"'"it"' i-"intin, phosphatravrinoiitot,

6' sphingophospholipids(sp.hingornyelins) contain sphingosineas the alcohol in place of tJi:;fi,!:,ttvcerophosphoti;idsi.ih-osphoripid;";,;;

i;;';";:,

constituents of ptasma

7 cerebrosides are the .simprest form of grycoripidswhich occur in the membranes neruous tissue. Gangriosides of are predominontrg round in ti" t gorstions. Theycontain one or more moteculesof N-acetylneuraminic.";,; ifi;:;r;:,)i." 8. Steroids contain th,ering cyclopentanoperhydrophenanthrene. T importance incrude. r,rr-iir'n,";;;.'I::#r::t i!^i,"Jlfr,,iZ,, hormones-A steroid "nlutt"ii,""i,,tJ'ir,ar, containing'or."or"*or. hydroxyr groupsis known as steror. 9' cholesterar is the most abundqnt animarsterar.It containsone hydroxyrgroup (at cz), a double bond (c51) and an co,rao.nside choin,ti"rn.a b cp. cholesterol-is "igl,t

i,'"ioJ",,,,nesis 2."?;:::::'t:.":"T"#[::;:::;,:x:_i:ao,,,,,",riiJ"i o/ bireacids,

r0

The lipids that oor:::r both hvdrophobic (non -porar)and hydrophiric (porar) groupsare known as amphipathic. rhese in,i"a" iori, o,,;;.;:;;;h;i;,:;:, sphinsoripids and bite ripids on i^poioi constituentsin the biiaeers ';:;r:#::.''athic of the bioroqical

42

B IOC H E MIS TR Y

I . Essayquestions 1. Writean accountof classification of lipidswith suitableexamples. 2. Describethe structureand functionsof phospholipids. 3. Discussthe saturated and unsaturated alongwith theirstructures. fattyacidsof biologicalimportance, 4. Describethe structureof steroids.Add a noteon the functionsof cholesterol. 5. Discussthe biologicalimportance of amphipathiclipids. II. Short notes (a) Structureof triacylglycerols, (b) Clycolipids,(c) Essential fatty acids,(d) Cis-transisomerism, (e) Rancidity,(0 lodine number,(g) Phosphatidylinositol, (h) Sphingomyelins, (i) Steroidnucleus, (j) Micelles. III. Fill in the blanks

1. The lipidsthatfunctionasfuel reservein animals 2. The isomerismassociated with unsaturated fatWacids 3. The cyclic fattyacid employedin the treatmentof leprosy 4. The lipidsthat are not the structuralcomponentsof biologicalmembranes 5. The prefixsn usedto representglycerol,sn standsfor 6. The numberof mg of KOH requiredto hydrolyse1 g fat or oil is knownas The phospholipidthat preventsthe adherenceof innersurfacesof lungs B . The phospholipid that producessecondmessengers in hormonalaction 9. Namethe glycolipidscontainingN-acetylneuraminic acid 10 . The steroids containa cyclic ring knownas IV. Multiple choice questions 11. The nitrogenous basepresentin lecithin (a)Choline(b) Ethanolamine (c)Inositol(d)Serine. 12. The numberof doublebondspresentin arachidonicacid (a)1 (b) 2 (c) 3 @)a. 13. On e o f th e fo l l o w i n gi s a n a mphi pathilci pi d (a)Phospholipids (b) Fattyacid (c) Bilesalts(d)All of the above. 14. Esterification of cholesteroloccursat carbonposition (a)1 (b)2 (c)3 (d)4. 15. Namethe testemployedto checkthe purity of butterthroughthe estimationof volatilefattyacids (a)lodinenumber(b) Reichert-Meissl number(d)Acid number. number(c)Saponification

rl trii,

p rotuint are the most ahundant organic I molecules of the living system. They occur in every part of the cell and constitute about 50'h of the cellular dry weight. Proteinsform the fundamentalbasis of structureand function of life. Origin

of the wotrd 'protein'

The term protein is derived from a Creek word proteiog meaning holding the first place. Berzelius(Swedishchemist)suggestedthe name proteinsto the group of organic compoundsthat are utmost important to life. Mulder (Dutch chemist)in 1838 used the term proteins for the high mo l e c u l a r w e i g h t n i tro g e n -ri chand most abun d a n t s u b s ta n c e sp re s e n t i n a ni mal s and olants .

Structuralfunctions: Certainproteinsperform brick and mortar roles and are primarily responsiblefor structureand strengthof body. Theseinclude collagen and elastinfound in bone matrix, vascular system and other organs and a-keratin presentin epidermaltissues. Dynamic functions : The dynamic functions of proteinsare more diversifiedin nature.These include proteins acting as enzymeq hormones, blood clotting factors, immunoglobulins, membrane receptors,storage proteins, besides thei r functi on i n geneti c control , muscle contraction,respirationetc. Proteinsperforming dynamic functionsare appropriatelyregardedas the working horsesof cell. Elermental

cornposition clf Broteins Proteinsare predominantlyconstitutedbv five major elementsin the following proportion. Functions of proteins Carbon 50 - 55% Proteinsperforma greatvariety of specialized Hydrogen 6 - 7.3% and e s s e n ti afu l n c ti o n si n th e l i v i n g cel l s.These Oxygen 19 - 24% functions may be broadly grouped as static Nitrogen 13 - 19% (structural) and dynamic. S ul fur 0 - 4o/"

43

44

BIOCHEMISTFIY

Besidesthe above, proteins may also contain other elementssuch as P, Fe,Cu, l, Mg, Mn, Zn etc.

The a-carbon atom binds to a side chain representedby R which is differentfor each of the 20 ami no aci dsfound i n protei ns.The ami no The content of nitrogen, an essential acids mostly exist in the ionized form in the component of proteins,on an averageis l6%. biological system(shown above). Estimationof nitrogen in the laboratory(mostly by Kjeldahl's method is also used to find out the Optical isomers of amino acids amount of protein in biologicalfluids and foods. lf a carbon atom is attachedto four different groups, it is asymmetricand thereforeexhibits Proteins are polymers of amano acids opti cal i someri sm.The ami no aci ds (except Proteinson completehydrolysis(with concen- glycine) possessfour distinct groups (R, H, trated HCI for several hours) yield L-cr-amino COO-, NH;) held by c,-carbon.Thus all the ac ids . T h i s i s a c o m m o n p ro p e rty of al l the ami no aci ds (exceptgl yci ne w here R = H ) have proteins.Therefore, proteins are the polymers of optical isomers. Lq"-amino acids. The structuresof L- and D-amino acids are written based on the configuration of L- and STANDARD AMINO ACIDS D-glyceraldehyde as shown in Fig.4.l. The As many as 300 amino acidsoccur in natureproteinsare composedof L-c-amino acids. Of these, only 20-known as standard amino of amino acids acids are repeatedly found in the structure of Glassification proteins, isolated from different forms of lifeThere are different ways of classifyingthe anim al, p l a n t a n d m i c ro b i a l .T h i s i s b e causeof amino acids basedon the structureand chemicat the universalnatureof the geneticcode available nature,nutritionalrequirement, metabolicfateetc. for the incorporation of only 20 amino acids A. Amino acid classification based on the when the proteinsare synthesizedin the cells. structure : A comprehensiveclassificationof The processin turn is controlled by DNA, the ami no aci ds i s based on thei r structureand geneticmaterialof the cell. After the synthesisof chemi calnature.E achami no aci d i s assi gneda proteins,some of the incorporatedamino acids 3 letter or 1 letter symbol. These symbols are undergo modificationsto form their derivatives. commonl y usedto representthe ami no aci ds i n protein structure.The 20 amino acids found in proteinsare divided into seven distinct groups. ln Table 4.1, the different groups of amino A m ino a c i d s a re a g ro u p o f organi c acids,their symbolsand structuresare given.The compounds containing two functional groupssalientfeaturesof differentgroups are described amino and carboxyl. The amino group (-NH2) next is basic while the carboxyl group (-COOH) is ac idic in n a tu re . cHo cHo I I General structure of amino acids H -C -OH oH-c-H I I The amino acidsare termedas cr-aminoacids, cH2oH cH2oH if both the carboxyl and amino groups are D-Glyceraldehyde L-Glyceraldehyde attachedto the same carbon atom, as depicted R R I below H 2N -C -H H -C -N H 2 I I H H cooH cooH I I L-Amino acid D-Amino acid R-C -C O OH R-C-COOI Fig.4.l : D- and L-forms of amino acid based on the NHz NHJ structure ot glyceraldehyde. Exists as ion Generalstructure

Chapter 4 : PFIOTEINS AND AMINO ACIDS

45

Symbol 3 letters I letter

Structure

Special group present

l. Aminoacldswithaliphatic sidechalns

1. Glycine

Gly

2. Alanine

Ala

3. Valine

Val

4. Leucine

Leu

H-CH-COOt+ NHi

Branched chain

t'!r-.rz-QH-coo HgC

Branched chain

*nl

Branched chain

5. lsoleucine

ll. Aminoacidscontainlng hydroxyl(-OH) groups

6. Serine

Ser

7. Threonine Thr

cH2-cH-coooH NHi

Hydroryl

H3C-CH-CH-COO-

Hydroryl

on rrFrt Tyrosine

Tyr

Seeunder aromatic

Hydroxyl Trble

4.1 contd.

nerl

page

46

BIOCHEMISTF|Y

Symbol

Narne

3 letters

Special group present

Structure

I letter

lll. Sulfurcontaining aminoacids 8. Cysteine

Cys

C

cH2-cH-cooSH NHi

Sulfhydryl

cH2-cH-coo-

A rrt I Cystine

Disulfide

I cH2-cH-@ol+ NHi

9. Methionine Met

M

cH2-cH2-cH-@o-

Thioether

b-cH. runt lV. Acidicaminoacidsandtheiramides 10.Aspartic acid Asp

11.AsparagineAsn

po -ooc-cH2-cH- @o

H2N-C-CH2-CH-COO-

6 12.Glutamic acid Glu

p-Carboryl

r+

NHi

Amide

nnl

Y P ct -ooc-cH2-cH2-?H-coo-

yCarboryl

NH; 13.Glutamine Gln

H2N-C- CHz-CH2?H-COO-

o

Amide

NHI

V, Basicaminoacids

14. Lysine

Lys

q e6Y P CH2-CH2-CH>-CHt -CH-@Ol+ l; NHi NHi

e-Amino

NH- CH2- CH2- CH2- CH-@O-

15. Arginine

Arg

?:*t;

NHt

Guanidino

NHz

16. Histidine

His

IINHi

lmidazole

HNN

-rq-cH+-coot.blo

4.1 contd.

nort

pag€

47

AND AMINO ACIDS ehaptee r* r PFIOTEINS

3 letters

Specialgroup present

Structure

Symbol

Name

1 letter

aminoacids Vl. Aromatic Benzene or phenyl

cH2-9H-COO-

Phe 17. Phenylalanine

Nxt 18.Tyrosine

/-\

Tyr

4-'ir----------c 19. Tryptophan Trp

Phenol

'*\:/""-[Xt"o" W

u\_/

''l*r

H2- cH- coolndole

H

Vll. lminoacid

20. Proline

Pynolidine

Pro gH

I

(Note: B groupis shownin red) 1. Amino acids with aliphatic side chains : These are monoamino monocarboxylic acids. This group consists of the most s i m p l e a m i n o a c i d s -g l y c i ne, al ani ne, v a l i n e , l e u c i n e a n d i s o l e u c i ne.The l ast three amino acids (Leu, lle, Val) contain b ra n c h e d a l i p h a ti c s i d e c hai ns, hence thev are referred to as branched chain amino acids. 2. Hydroxyl group containing amino acids : Serine, threonine and tyrosine are h y d ro x y l g ro u p c o n ta i n i n ga mi no aci ds. Tyrosine-being aromatic in nature-is u s u a l l yc o n s i d e re du n d e r a romati cami no acids. 3. Sulfur containing amino acids : Cysteine w i th s u l fh y d ry l g ro u p a n d methi oni ne with thioether group are the two amino acids incorporatedduring the course of

protein synthesis. Cystine, another i mportantsul furcontai ni ngami no aci d , is formed by condensationof two molecules of cysteine. 4. Acidic amino acids and their amides : Aspartic acid and glutamic acids are dicarboxylic monoamino acids while asparagi ne and gl utami ne are th eir resoective amide derivatives. All these four amino acids possessdistinct codons for their incorporationinto proteins. 5. Basicamino acids : The three amino acids l ysi ne, argi ni ne (w i th guani di no group) and histidine (with imidazole ring) are dibasic monocarboxylic acids. They are highly basic in character. 6. Aromatic amino acids : Phenylalanine, tyrosineand tryptophan(with indole ring)

48

BIOGHEMISTF|Y

are aromatic amino acids. Besidesthese, histidine may also be considered under this category. 7. lmino acids: Prolinecontainingpyrrolidine r ing is a u n i q u e a mi n o a c i d . l t h as an imino group (=NH), insteadof an amino group (-NH2) found in other amino acids. Therefore,proline is an a-imino acid. B. Classification of amino acids based on polarity : Amino acids are classified into 4 groups based on their polarity. The polarity in turn reflectsthe functionalrole of amino acids in protein structure. 1. Non-polar amino acids : These amino acids are also referredto as hydrophobic (water hating). They have no charge on t he ' R ' g ro u p . T h e a m i n o a c i d s i n c l uded in this group are - alanine, leucine, isoleucine, valine, methionine, phenylalanine, tryptophanand proline. 2. Polar amino acids with no charge on 'R' group : Theseamino acids,as such, carry no chargeon the 'R'group. They however possess groups such as hydroxyl, sulfhydryl and amide and participate in hydrogen bonding of protein structure. The simple amino acid glycine (where R = H) is also consideredin this category. The amino acids in this group areglycine, serine, threonine, cysteine, glutamine, asparagineand tyrosine.

1. Essentialor indispensableamino acids : The amino acids which cannot be synthesized hy the body and, therefore, need to be supplied through the diet are called essentialamino acids. They are proper Browth and required for maintenanceof the individual. The ten amino acids listed below are essentialfor humans (and also rats) : Arginine, Valine, Histidine, lsoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine,Tryptophan. lThe code A.U HILL, MP., T. T. (first letter of each amino acid) may be memorized to recall essential amino acids. Other useful codes are H. VITTAL, LMP; PH. VILLMA, TT, PW TIM HALL and MATTVILPhLy.I The two amino acids namely arginineand histidine can be synthesizedby adults and not by growing children, hencethese are considered as semi-essential amino acids (remember Ah, to recall). Thus, 8 amino acids are absolutelyessentialwhile 2 are semi-essential. 2. Non-essential or dispensabte amino '10 acids : The body can synthesizeabout amino acids to meet the biological needs, hence they need not be consumed in the diet. These are-glycine, alanine, serine, cysteine, aspartate,asparagine, glutamate, glutamine,tyrosineand proline.

3. Polar amino acids with positive 'R' group : D. Amino acid classification based on their The three amino acids lysine, arginine metabolic fate : The carbon skeleton of amino and histidine are included in this group. acids can serve as a precursor for the synthesis 4. Polar amino acids with negative'R'group : of glucose(glycogenic)or fat (ketogenic)or both. From metabolic view point, amino acids are The dicarboxylic monoamino acidsaspartic acid and glutamic acid are divided into three groups (for details, Refer Chapter lA. consideredin this group. C. Nutritional classification of amino acids : The twenty amino acids (Iable 4.1) are required for the synthesisof variety of proteins, besides other biological functions. However, all these 20 amino acids need not be taken in the diet. Based on the nutritionalrequirements,amino acids are grouped into two classes+ssential and nonessential.

1. Glycogenic amino acids : These amino acids can serve as precursors for the formation of glucose or glycogen. e.g. glycine,methionineetc. alanine,aspartate, 2. Ketogenic amino acids : Fat can be synthesizedfrom these amino acids. Two ami no aci ds l euci ne and l ysi ne are exclusivelyketogenic.

Ghapter 4 : PFIOTE|NS AND AMTNOACTDS 3. Glycogenic and ketogenic amino acids : T he fo u r a m i n o a c i d s i s o l e u c i n e,phenyl alanine, tryptophan, tyrosine are pre_ cursorsfor synthesisof glucoseas well as fat. Selenocysteine

- the 2i st amino

acid

As already stated, 20 amino acids are commonly found in proteins.ln recent years,a 21s tam ino a c i d n a me l ys e l e n o c y s te i nhea sbeen added. lt is found at the active sites of certain enzymes/proteins (selenoproteins). e.g. gluta_ thione peroxidase,glycine reductase,5,-deiodinase,thioredoxin reductase.Selenocysteineis an unus u a l a mi n o a c i d c o n ta i n i n g th e trace elem ents e l e n i u mi n p l a c e o f th e s u l fu r a tom of cysteine.

z-!H-coo-

-cH-coo-

rinJ

NHd Cysteine

Selenocysteine

49 3. Taste: A mi no aci ds may be sw eet (C l y, Ala, Val), tasteless(Leu) or bitter (Arg, lle). Monosodium glutamate (MSC; ajinomoto) is used as a flavoring agent in food industrv,and Chinese foods to increasetaste and flavor. ln some individuals intolerant to MSC, Chinese restaurant syndrome (brief and reversible flu_ like symptoms)is observed. 4. Optical properties: All the amino acids exceptglycine possessoptical isomersdue to the presence of asymmetric carbon atom. Some amino acids also have a second asymmetric carbon e.g. isoleucine,threonine.The structure of L- and D -ami no aci ds i n compari sonw i th glyceraldehydehas been given (SeeFig.4.t). 5. Amino acids as ampholytes: Amino acids contai n both aci di c (-C OOH ) and basi c (-NH2) groups. They can donate a proton or accepta proton, henceamino acids are regarded as ampholytes.

Zwitterion or dipolar ion : The name zwitter Incorporation of selenocysteine into the is derived from the German word which means proteinsduring translationis carried out by the codon namely UCA. lt is interestingto note that hybrid. Zwitter ion (or dipolar ion) is a hybrid UCA is normally a stop codon that terminates molecule containing positiveand negativeionic protein biosynthesis.Another unique feature ts grouPs. that selenocysteineis enzymatically generated The amino acids rarelyexist in a neutralform from serinedirectly on the tRNA (selenocvsteine- with free carboxylic (-COOH) and free amino IRNA), and then incorporatedinto proteins. (-N H 2) groups.In strongl yaci di c pH (l ow pH ), the amino acid is positively charged (cation) Pyrrolysine-the 22nd amino acid? : In tne w hi l e i n strongl y al kal i ne pH (hi gh pH ), i t i s year 2002, some researchers have describedyet negativelycharged(anion).Eachamino acid has another amino acid namely pyrrolysineas the a characteri sti cpH (e.g. l euci ne, pH 6.0) at 22nd amino acid present in protein. The stop which it carries both positive and negative codon UAG can code for pyrrolysine. chargesand existsas zwitterion (Fig.a.Z. IsoelectricpH (symbol pl) is defined as the pH at which a molecule existsas a zwitterion or T he am i n o a c i d s d i ffe r i n th e i r p h ysi co- dipolar ion and carries no net charge. Thus, the c hem ic alpr o p e rti e sw h i c h u l ti m a te l yd e te rmrne mol ecul e i s el ectri cal l y neutral . the characteristics of proteins. The pl value can be calculatedby taking the averagepKa valuescorresponding to the ionizable A. Physical propefiies groups. For instance,leucine has two ionizabre 1. Solubility: Most of the amino acids are groups/and its pl can be calculatedas follows. us uallys olu b l ei n w a te r a n d i n s o l u b l ei n o rgani c solvents. Properties

of amino

acids

2. M elt in g p o i n ts : Ami n o a c i d s g e n eral l y melt at higher temperatures, often above 200.C.

a L

p1=4!9.9 =6.s z

50

BIOCHEMISTFIY H I R -C -C O OH I NHz

groups namely carboxyl (-COOH) group and ami no (-N H 2) group. Reactions due to -COOH

Amino acid

H I R- C- CO O H I, NHi Cation (lowpH)

group

1. A mi no aci ds form sal ts(-C OON a) w i th basesand esters(-COOR') with alcohors. H I R-C-COOI

NHz Anion (hishpH)

2. D ecarboxyl ati on:A mi no aci ds underg o decarboxylationto producecorresponding ami nes. R-CH-COO ----+R-CH2 + CO2

NHa

NHT

+

Thi s reacti onassumessi gni fi cancei n th e l i vi ng cel l s due to the formati onof many biologically important amines. These i ncl ude hi stami ne,tyrami neand y-ami no butyric acid (CABA)from the amino acids histidine, tyrosine and glutamate, respectively.

H I R-C-COOI NHi Zwitterion (isoelectric pH) Fig. 4.2 : Existence of an amino acid as cation, anion and zwitterion.

Leucine exists as cation at pH below 6 and anion at pH above 6. At the isoelectricpH ( pl = 6.0 ), l e u c i n e i s fo u n d a s z w i tteri on.Thus t he pH o f th e m e d i u m d e te rm i n e sthe i oni c nat ur eo f a mi n o a c i d s . F or t h e c a l c u l a ti o no f p l o f a m i n o aci ds w i th more than two ionizablegroups,the pKasfor all the groups have to be taken into account. Titration of amino acids : The existenceof differentionic forms of amino acids can be more easily understood by the titration curves. The graphic representationof leucine titration is depicted in Fi9.4.3.At low pH, leucine existsin a fully protonatedform as cation. As the titration proceedswith NaOH, leucine loses its protons and at isoelectric pH (pl), it becomes a zwitterion. Further titration results in the formation of anionic form of leucine. S om e m o re d e ta i l s o n i s o e l e c tri cpH are discussed under the properties of proteins 1p. 60) .

E, Chemica! properties The general reactions of am t no aci ds are mostly due to the presenceof two f uncti onal

3. Reaction with ammonia: The carboxyl group of dicarboxylic amino acids reacts with NH3 to form amide Aspartic acid + NH, ------;Asparagine Glutamic acid + NH. ------+Clutamine

14 13 12 11

F-C H -C OO-

I NHz

I I pH 7 6 5 3 2 1 0 0.5 -+

1.0

1.5

2.0

Eouivalents of NaOH-=+

Fig, 4.3 : Titrationcurue of an amino acid-leucine (R = (CH),-CH-CH,-; PK, = Dissociationconstant for COOH; pl = lsoelectric pH; pK, = Dissociationconstant for NHI).

-d*-*d,h Ghapter 4 : PFIOTEINS AND AMINO ACIDS Reactionsdue to -NH2 group 4. The amino groups behave as bases and combine with acids (e.g. HCI) to form s a l ts(-N H i C l -).

acidsareverfrp-

nt for proteinstructure and

functions. Selected examples hereunder.

are

given

. Collagen-the most abundant protein in mammals-contains 4-hydroxyproline and 5. Reaction with ninhydrin: The cr-amino 5-hydroxylysine. acids react with ninhydrin to form a pu rp l e , b l u e o r p i n k c o l o u r compl ex . Histones-the proteins found in association (Ruhemann's purple). with DNA-contain many methylated, phosphorylatedor acetylatedamino acids. Amino acid + Ninhydrin---+ Ketoacid + N H r+ C Oz + H y d r i ndanti n Hydrindantin+ NH: + Ninhydrin-----+ e R u h e ma nn' purpl s Ninhydrin reaction is effectivelyused for the quantitativedeterminationof amino acids and proteins. (Nofe : Proline and hydroxyproline give yellow colour with ni n h y d ri n ).

6. Colour reactionsof amino acids : Amino acids can be identifiedby specificcolour reactions (See Table 4.3).

. y-Carboxyglutamic acid is found in certain pl asmaprotei nsi nvol ved i n bl ood cl otti ng. . Cystine is formed by combination of two cysteines. Cystine is also considered as deri ved ami no aci d. B. Non-protein amino acids : These amino acids,although neverfound in proteins,perform several bi ol ogi cal l y i mportant functi ons. They may be either d-or non-cr-amino acids. A selectedlist of theseamino acidsalong with their functions is given in Table 4.2.

7. Transamination: Transfer of an amino group from an amino acid to a keto acid to form a new amino acid is a very important reaction in amino acid metabolism(detailsgiven in Chapter 1fl.

C. D-Amino acids : The vast majority of amino acids isolatedfrom animalsand olantsare of L-category.Certain D-amino acids are also found i n the anti bi oti cs (acti nomyci n-D, val i nomyci n, grami ci di n-S ). D -seri ne and D-aspartate are found in brain tissue. D8. Oxidative deamination: The amino acids Gl utami c aci d and D -al ani ne are present i n undergooxidativedeaminationto liberate bacteri alcel l w al l s. free ammonia (Refer Chapter l5). ]{ON.STANDARD

AMINO

ACIDS

Amino

acids

usefu!

as drugs

Therea certainnon-standardamino acidsthat B es id e s th e 2 0 s ta n d a rd a mi n o aci ds are used as drugs. (described above) present in the protein structure,there are several other amino acids . D -P eni ci l l ami ne (D -di methyl gl yci ne), a whic h ar e b i o l o g i c a l l yi m p o rta n t.T h e sei ncl ude metabol i teof peni ci l l i n, i s empl oyed i n the the amino acid derivativesfound in proteins, chel ati ontherapyof W i l son' sdi sease.Thi s i s non- pr o te i na m i n o a c i d s p e rfo rmi n gs peci al i zed possi bl esi nce D -peni ci l l ami necan effecti vely f unc t ion sa n d th e D -a m i n o a c i d s . chelate copper. A. Amino acid derivatives in proteins : The . N-Acetylcysteineis used in cystic fibrosis, and 20 standard amino acids can be incoroorated chroni c renal i nsuffi ci encv, as i t can functi on into proteins due to the presenceof universal as an anti oxi dant. genetic code. Some of these amino acids undergo specific modification after the protein . Gabapentin (y-aminobutyrate linked to cvclohexane)is used as an anticonvulsant. svnthesisoccurs. These derivativesof amino

r'

52

B IOC H E MIS TR Y

Amino acids

Function(s)

L cr,-Amino acids Ornithine

I I I Arginosuccinic acidl Citrulline

Thyroxine

I I J Triiodothyronine S-Adenosylmethionine Homocysteine Homoserine phenylalanine (DOPA) 3,4-Dihydrory Creatinine Ovothiol Azaserine ll. Non-s,-amino acids p-Alanine p-Aminoisobutyric acid yAminobutyric acid(GABA) &Aminolevulinic acid(ALA) Taurine

Intermediates inthebiosynthesis ofurea.

fromtyrosine. Thyroid hormones derived inbiological system. Methyl donor heart inmethionine metabolism. A riskfactorforcoronary Intermediate diseases metabolisms. Intermediate inthreonine, aspartate andmethionine pigment. serves asa precursor tormelanin A neurotransmitter, lrommuscle andexcreted inurine Derived infertilized eggs, andactsasan Sulfur containing amino acidfound antioxidant Anantibiotic pantothenic A Component ofvitamin acidandcoenzyme metabolism. Endproduct ofpyrimidine produced fromglutamic A neurotransmitter acid (finally ofporphyrin heme) Intermediate inthesynthesis Found inassociation withbileacids. polypeptide chains referredto as subunits.The spatial arrangementof these subunits is known as quaternarystructure.

Proteinsare the polymersof L-cr-aminoacids. lThe structural hierarchy of proteins is The structureof proteinsis rathercomplex which comparablewith the structureof a building. The can be divided into 4 levels of organization amino acids may be consideredas the bricks, (Fig.4.4) : the wall as the primary structure,the twists in a 1. Primary structure: The linear sequenceof wall as the secondarystructure,a full-fledged amino acids forming the backbone of proteins self-containedroom as the tertiary structure.A (polypeptides). bui l di ng w i th si mi l ar and di ssi mi l arrooms w i ll be the quaternarystructurel. 2. Secondary structure: The spatial The term protein is generally used for a arrangement of protein by twisting of the polypeptide containing more than 50 amino poly pep ti d ec h a i n . acids. ln recent years, however, some authors 3. Tertiary structure: The three dimensional have been usi ng' pol ypepti de' even i f the structureof a functional orotein. numberof ami no aci ds i s a few hundreds.They prefer to use protein to an assembly of 4. Quaternary structure : Some of the proteins are composed of two or more polypeptidechains with quaternarystructure.

Chapter 4 : PROTEINS AND AMINO ACIDS

Primary structure

Secondary structure

53

Tertiary structure

Quaternary structure

Fig. 4.4 : Diagrammaticrepresentationof proteinstructure (Note : Thefour subunitsof tuvotypesin quaternarystructure).

PRIMARY STRUCTUREOF PROTEIN

double bond in character.lt generallyexists in trans configuration. Both -C=O and -NH Eachprotein has a unique sequenceof amino groups of peptide bonds are polar and are ac ids w h i c h i s d e te rm i n e d b y th e genes involved in hydrogen bond formation. contained in DNA. The primary structureof a protein is largely responsiblefor its function. A Writing of peptide structures: Conventionally, vast majority of genetic diseasesare due to the peptidechainsare writtenwith the free amino abnormalitiesin the amino acid sequencesof end (N-terminalresidue)at the left, and the free proteins i.e. changes associatedwith primary carboxylend (C-terminalresidue)at the right.The structureof protein. amino acid sequenceis readfrom N-terminalend T he a m i n o a c i d c o mp o s i ti o n o f a protei n to C-terminal end. Incidentally, the protein also startsfrom the N-terminalamino determinesits physicaland chemical properties. biosynthesis aci d. Peptide bond The amino acidsare held togetherin a protein by covalent peptide bonds or linkages.These bonds are rather strong and serve as the cementing material between the individual amino acids (consideredas bricks). Formation of a peptide bond: When the amino group of an amino acid combines with Ihe carboxyl group oI another amino acid, a peptide bond is formed (Fig.a.D. Note that a dipept id ew i l l h a v e tw o a mi n o a c i d s and one peptide (not two) bond. Peptides containing more than 10 amino acids (decapeptide)are referred to as polypeptides. Characteristics of peptide bonds: The peptide bond is rigid and planar with partial

H +l

H3N-C-COO'l

+

Rl Aminoacid 1

+l Fl"N-C-COOR2 Aminoacid2

Hzo HH +l I H3N-C-€O-l'iH-C-COOtl R1

R2 Dipeptide

Fig.4.5 : Fomation of a peptide bond.

B IOC H E MIS TR Y

54 Shorthand to read PePtides: The amino acids in a PePtideor Protein are representedby the 3-letter or one letter abbreviation. This is the chemical shorthandto write proteins.

fruN--glrt"t"te--cysteine-glycine-CooE Glu

_

C

_G

CYs -

GIY

Aminoacidsin a Peptide Onelettersymbols Threelettersymbols

Peptidename - glYcine Glutamyl- cysteinYl Naming of peptides: For naming a peptide Fig.4.6 : lJseof symbolsin representing peptides, the amino acid suffixes and twopeptidebondsis acids amino wrth 3 (Note: Atripeptide (tryptophan), -afe (glycine), -an -ine shown;Free-NHtr is on the teft whilefree-COt is on the right)' Glutamate) are changed to -Yl with the exception of C-terminal amino 2. Degradation of protein or polypeptide acid. Thus a tripeptide composed of an Ninto smaller fragments. terminal glutamate,a cysteineand a C-terminal g l u ta m y l -c y s te i n y l -gl yci ne. gly c in e i s c a l l e d 3. Determinationof the amino acid sequence' ln the Fig.4.6,the naming and representation 1. Determination of amino acid composition of a tripeptideare shown. in a protein : The protein or polypeptide is to liberate the amino Dimensions of a PePtide chain : The completely hydrolysed estimated' The quantitatively dimensions of a fullv extended polypeptide acids which are by acid or either carried out chain are depicted in Fig.4.7.The two adjacent hydrolysismay be hydrolysis' or by enzyme cr-carbonatoms are placed at a distanceof 0.36 alkali treatment however results in enzymes, with Treatment angles and bond nm. The interatomicdistances smaller peptidesrather than amino acids. are also shown in this figure. Pronase is a mixture of non-specific of primary stvueture Deterrnination proteolytic enzymes that causes complete The primary structure comprisesthe identi- hydrolysisof proteins. fication of constituentamino acids with regard Separation and estimation of amino acids: to their quality, quantity and sequence in a The mixture of amino acids liberatedby protein protein structure.A pure sample of a protein or hydrolysis can be determined by chromatoa polypeptide is essentialfor the determination graphic techniques. The reader must refer of primary structurewhich involves 3 stages: Chapter 41 tor the separation and quantitative Knowledge on n f a mi n o a c i d composi ti on. determination of amino acids' 1. D e te rm i n a ti o o

Fig.4.7 : Dimensionsof a futly ertended polypeptide chain' (The-distancebetween two adiacent a-carbon atoms is A'36 nm)'

Chapter 4 : PROTEINSAND AMINO ACIDS

l\

JJ

tFr

OrN{' \-/

-No, Sanger'sreagent i prot"intabetting i Hydrolysis i-+fr"" aminoacids

Edman'greagent i Proteinlabelling Hydrolysis i Hyorolysls ,$ Polypeptide

(-N-terminalM)

R I N-CH-COO-

Dlnltrophenyl(DNP)amlnoacld

II + ldentifled by chromatography

Phenylthlohydantoln (PTH)amino acld v

I

ldontlfledby chromatography

Ftg. 4.8 : Sanger's reagent (llluoro 2,4-dinitrobenzene)and Edman's reagent (Phenyl isothiocyanate)in the determination of amino acid sequence of a protein (AA-Amino acid).

primarystructure of proteinswill be incomplete withouta thoroughunderstanding of chromatography.

(c) Breakdown of polypeptides into fragments: Polypeptidesare degraded into smallerpeptidesby enzymaticor chemicalmethods.

2. Degradationof protein into smallerfragments : Proteinis a large moleculewhich is Enzymaticcleavage: Theproteolyticenzymes sometimes composedof individualpolypeptide such as trypsin, chymotrypsin,pepsin and chains.Separation of polypeptides is essential elastaseexhibit specificity in cleaving the beforedegradation. peptide bonds (Refer Fig.8.V. Among these enzymes/ trypsin is most commonlyused. lt (a) liberation of polypeptides:Treatment hydrolyses the peptidebondscontaininglysine with urea or guanidinehydrochloride or arginine on the carbonyl(-C:O) side of disrupts the non-covalentbonds and peptide linkage. dissociates the protein into polypeptide units.For cleavingthe disulfidelinkages Chemical cleavage: Cyanogen bromide betweenthe polypeptideunits,treatment (CNBr)is commonlyusedto split polypeptides with performicacid is necessary. into smallerfragments. CNBr specificallysplits peptide bonds, the carbonyl side of which is (b) Numberof polypeptides: The numberof contributed by the amino acid methionine. polypeptidechainscan be identifiedby treatmentof protein with dansylchloride. 3. Determinationof amino acid sequence: It specifically bindswith N-terminal amino The polypeptides or their smallerfragments are acidsto form dansylpolypeptides which convenientlyutilizedfor the determination of on hydrolysisyield N-terminaldansyl sequence of aminoacids.Thisis donein a stepaminoacid.Thenumberof dansvlamino wise mannerto finally build up the order of acidsproducedis equalto the numberof amino acids in a protein.Certainreagents are polypeptide chainsin a protein. (Fig,4,A. employedfor sequence determination

55

ElIOCHEMISTRY

Sanger'sreagent: Sangerused 1-fluoro2, ami no aci dsi n a pol ypepti dechai n.B y anal ys ing (FDNB)to determineinsulin the nucleotidesequenceof DNA that codes for 4-dinitrobenzene structure. FDNB specifically binds with protein, it is possibleto translatethe nucleotide N-terminalamino acid to form a dinitrophenyl sequence into amino acid sequence. This (DNP)derivativeof peptide.This on hydrolysis technique,however,fails to identifythe disulfide yields DNP-aminoacid (N-terminal) and free bondsand changesthat occur in the amino acids aminoacidsfrom the restof the peptidechain. after the protein is synthesized(post-translational DNP-amino acid can be identifiedbv chromato- modifications). graphy. Sanger'sreagenthas limited use since the SECONDARYSTRUCTUREOF PROTEIN peptidechain is hydrolysed to aminoacids. The conformation of polypeptide chain by Edman's reagent: Phenyl isothiocyanateis the Edman'sreagent.lt reactswith the Nterminalaminoacid of peptideto form a phenyl thiocarbamyl On treatment with mild derivative. (PTH)-amino acid,phenylthiohydantoin acid,a cyclic compoundis liberated.This can be (Fig.a.A. identifiedby chromatography

twisting or folding is referred to as secondary structure.The amino acids are located close to each other in their sequence. Two types of secondary structures, a-helix and p-sheef, are mai nl y i denti fi ed. lndian scientist Ramachandran made a significant contribution in understanding the spatial arrangementof polypeptidechains.

Edman'sreagenthas an advantagesince a peptidecan be sequentially degradedliberating N-terminalaminoacidsone afteranotherwhich u-l{elix can be identified. This is due to the factthatthe a-Helixis themosfcommonspiralstructure peptideas a whole is not hydrolysedbut only of protein. lt has a rigid arrangementof releases PTH-amino acid. polypeptidechain. a-Helical structurewas Sequenator:This is an automaticmachineto by Pauling and Corey(1951)which is determinethe amino acid sequencein a proposed of the milestonesin the regarded as one polypeptide(with around 100 residues).lt is research. The salientfeaturesof biochemistry basedon the principleof Edman'sdegradation (Fig.a.9\ given below are s-helix (described above).Amino acidsare determined sequentiallyfrom N-terminalend. The PTH1. The a-helix is a tightly packedcoiled amino acid liberatedis identifiedby high- structure with aminoacid sidechainsextending performanceliquid chromatography(HPLC). outwardfrom the centralaxis. Sequenator takesabout 2 hoursto determine 2. The a-helix is stabilized by extensive eachaminoacid. hydrogenbonding.lt is formedbetweenH atom attachedto peptideN, and O atom attachedto Overlapping peptides peptideC. The hydrogenbondsare individually ln the determination of primarystructureof they arestrongenoughto weak but collectively, protein,severalmethods(enzymatic or chemical) helix. stabilize the aresimultaneously employed.Thisresultsin the peptides. formationof overlapping Thisis due to 3. All the peptidebonds,exceptthe firstand the specificactionof differentagentson different last in a polypeptidechain, participatein sitesin the polypeptide.Overlappingpeptides hydrogenbonding. are very usefulin determiningthe amino acid 4. Eachturn of a-helixcontains3.5 amino sequence. acids and travelsa distanceof 0.54 nm. The spacingof eachaminoacid is 0.15 nm. Reverse sequencing technique formed 5. a-Helix is a stableconformation It is the geneticmaterial(chemicallyDNA) with the lowestenergy. which ultimatelydeterminesthe sequenceof spontaneously

Chapten 4 : PR0TEINSAND AMINO ACIDS

57 Clu) or basic(Lys,Arg, His) aminoacidsalso interferewith o-helixstructure. p-Pleated sheet This is the secondtype of structure(hencep after a) proposed by Pauling and Corey. p-Pleated sheets (or simply p-sheets)are composedof two or more segmentsof fully extended peptide chains (Fig,4,10).ln the p-sheets, the hydrogen bonds are formed between the neighbouring segments of polypeptide chain(s). Parallel and anti.parallel

p.sheets

The polypeptidechainsin the p-sheetsmay be arrangedeither in parallel (the same direction)or anti-parallel(oppositedirection). This is illustrated in Fig.4,l0. p-Pleatedsheet may be formed either by separate polypeptide chains (H-bonds are interchain) or a singlepolypeptide chainfolding backon to itself(H-bondsare intrachain).

(A)

Flg.4.9 : Diagrammatic representation of secondary structureof protein-a righthandeda-helix H I (l-lndicate -C-R groupsof aminoacids;

(B) N-Terminal C-terminal

dotted blue lines are hydrogen bonds; Note that only a few hydrogen bonds shown for clarity).

(C) N-Terminal

C-terminal

C-Terminal #

N-terminal

6. The right handedo-helix is more stable thanlefthandedhelix(a righthandedhelixturns in the directionthatthefingersof righthandcurl when its thumbpointsin the directionthe helix rises).

F19.4.10: Structureof B-pleatedsheet(A) Hydrogen bondsbetweenpolypeptidechains(B) Parallelp-sheet (C)AntiparallelB-sheet.(Note : Redctrctesin A

7. Certainaminoacids(particularly proline) disruptthe a-helix.Largenumberof acidic(Asp,

represent emlnoacid skeleton-C-R l.

I

58

B IOC H E MIS TR Y

polypeptideswhich may be identical or "unrelated. Suchproteinsaretermedas oligomers quaternary The individual structure. and possess polypeptidechains are known as monomers, protomers or subunits.A dimer consitsol two polypeptides while a tetramerhasfour.

Ft,.4.11 : Diagrammatic representation of a protein containinga-helixandB-pleatedsheet(blue).

Bonds in quaternary structure: The monomericsubunitsare held togetherby nonconvalent bonds namely hydrogen bonds, and ionic bonds. hydrophobicinteractions

Importanceof oligomericproteins: These proteinsplay a significant role in the regulation and cellularfunction. Occurrence of p-sheets: Many proteins of metabolism containp-pleatedsheets.As such,the cx-helix Examplesof oligomeric proteins: Hemoand p-sheetare commonlyfound in the same globin, lactate aspartate transcarbomylase, protein structure(Fig.4.ll). In the globular dehydrogenase. proteins,p-sheetsform the core structure. Other typesof secondarystructures:Besides Bonds responsible for the cr-and p-structuresdescribed above, the protein structure p-bends and nonrepetitive(less organised Proteinstructureis stabilizedby two typesof structures) secondary structures arealsofoundin and non-covalent. bonds-covalent proteins. 1. Covalentbonds: Thepeptideanddisulfide TERTIARY STRUCTURE OF PROTEIN bondsare the strongbondsin proteinstructure. The three-dimensional arrangement of The formation of peptide bond and its havebeendescribed. protein structure is referred to as tertiary chracteristics structure. lt is a compact structure with Disulfidebonds:A disulfidebond(-5-O is hydrophobic sidechainsheldinteriorwhilethe formedby the sulfhydrylgroups(-SH) of two hydrophilicgroupsare on the surfaceof the cysteine residues, to produce cystine protein molecule.This type of arrangement Gig.a.l2A).The disulfidebondsmay be formed ensures stabilitv of the molecule. in a single polypeptidechain or between polypeptides. Thesebondscontribute to different Bonds of tertiary structure: Besidesthe stability of conformation and the structural hydrogenbonds,disulfidebonds(-S-S), ionic proteins. interactions (electrostatic bonds) and hydrophobicinteractions also contributeto the 2. Non-covalent bonds: There are,mainly, tertiarystructureof proteins. bonds. four typesof non-covalent Domains: The term domain is used to (a) Hydrogenbonds: The hydrogenbonds representthe basic units of protein structure areformedby sharingof hydrogenatoms (tertiary) and function.A polypeptide with 200 between the nitrogen and carbonyl amino acidsnormallyconsistsof two or more oxygen of different peptide bonds domains. (Fig,4.12D. Eachhydrogenbond is weak but collectivelythey are strong.A large OUATERNARYSTRUCTUREOF PROTEIN numberof hydrogenbondssignificantly contribute to the proteinstructure. great proteins A majorityof the arecomposed of single polypeptidechains. Some of the proteins, however, consist of two or more

(b) Hydrophobicbonds: The non-polarside chainsof neutralaminoacidstendto be

AND AMINO ACIDS chapter 4 : PFIOTEINS

59

NH-CH-CO,,'V\,^. gHz

(A)

I S q/sdne (-I,

(d) Van der Waals forces: These are the non-covalent associations between electrically neutral molecules. They are formed by the electrostatic interactions due to permanentor induced dipoles.

i

CHz NH-CH-CO/'.:,,\,,\ (B)

\r,\.//^\,,^\_

C-CH - N ./\./\./\./ ill

?R1 ! 'RaO

/^,,\,,^\- N-;-8,.^\./\./\./ (c)

NH- CH- CO,'^\./\,'\,"\ HC-CHs I lsoleucine CH^ '7 I'

H33 I /,'v'\r,'v,^\NH(D)

Leuclne COl"\r,^\r,^\r,'\.

NH-CH-CO,,'\.,,a..,,\ I Aspanate

t

He

\,4/'\.,/\-

" Lvslna ( HzhI NH-CH- CO,/\./\./

positively of basic

acidic amino acids with charged groups (e.g. -NHj) amino acids (Fi9.4.12D).

Examples of protein structure Structureof humaninsulin: Insulinconsists of two polypeptide chains,A and B (Fig.a.lA, The A chain hasglycineat the N-terminalend and asparagine at the C-terminalend. The B chain has phenylalanine and alanineat the N- andC-terminal ends,respectively. Originally, insulin is synthesized as a singlepolypeptide preproinsulin which undergoes proteolytic processing to give proinsulinand finally insulin. The structuralaspectsof hemoglobinand colfagenare respectivelygiven in Chaptersl0 and 22. Methods to determine protein structure For the determinationof secondaryand tertiaryprotein structures,X-raycrystallography is most commonly used. Nuclear magnetic resonance(NMR)spectraof proteinsprovides structuraland functional informationon the atomsand groupspresentin the proteins.

Flg,4.12: Majorbondsin proteinstructure(A) Dbultide bond (B) Hydrogenbonds(C) Hydrophicbonds

21 closely associatedwith each other in proteins(Fig.a.l2Q.As such, theseare not true bonds. The occurrenceof hydrophobic forces is observed in 1 aqueous environment wherein the moleculesare forcedto staytogether. (c) Electrostaticbonds: These bonds are formed bv interactions between negatively chargedgroups(e.g.COO-)of

ti

SS

ll

19

30 Bchaln

Ftq.4.13: Diagrammatic representation of humaninsulin structure

60

B IOGH E MIS TR Y

Methods for the isolation and purification of proteins

P epsi n-' | .1;C asei n-4.6;H uman al bumi n-4. 7; Urease-S.0;Hemoglobin-6.7 ; Lysozyme-l1 .0.

5. Acidic and basic proteins: Proteins in Severalmethodsare employed to isolateand which the ratio (e Lys + e Ard/(e Clu + e Asp) it purify proteins.Initially,proteinsare fractionated greater than 1 are referred to as basic proteins. by using differentconcentrationsof ammonium sulfate or sodium sulfate. Protein fractionation For acidic proteins,the ratio is lessthan 1 . may also be carried out by ultracentrifugation. 6. Precipitationof proteins: Proteinsexist in Protein separation is achieved by utilizing col l oi dal sol uti on due to hydrati on of pol a r electrophoresis,isoelectricfocussing, immuno- groups(-C OO-, -N H t, -OH ). P rotei nscan be electrophoresis,ion-exchangechromatography, precipitatedby dehydrationor neutralizationof gel-filtration,high performanceliquid chromato- polar groups. graphy(HPLC)etc. The detailsof thesetechniques Precipitationat pl : The proteins in general are describedin Chapter 4l. are least soluble at isoelectric pH. Certain proteins(e.9.casein)get easilyprecipitatedwhen the pH is adjusted to pl (4.6 lor casein). Formati onof curd from mi l k i s a marvel l ous 1. S o l u b i l i ty: Pro te i n s fo rm col l oi dal example of slow precipitationof milk protein, solutionsinsteadof true solutionsin water. This casein at pl. This occurs due to the lactic acid is due to huge size of protein molecules. produced by fermentation of bacteria which 2. Molecular weight : The proteins vary in lowers the pH to the pl of casein. t heir m o l e c u l a r w e i g h ts , w h i c h , i n turn, i s Precipitationby salting out: The processof depend e n t o n th e n u mb e r o f a mi no aci d protein precipitationby the additionalof neutral r es idue s . Ea c h a mi n o a c i d o n a n average salts such as ammonium sulfate or sodium sulfate contributesto a molecularweight of about 1 10. i s know n as sal ti ng out. Thi s phenomenonis Majority of proteinsholypeptides may be explained on the basisof dehydration of protein composed of 40 to 4,000 amino acids with a moleculesby salts.This causesincreasedproteinmolecular weight ranging from 4,000 to protein interaction, resulting in molecular 440,00O.A few proteins with their molecular aggregationand precipitation. weights are listed below : The amount of salt required for protein fns u li n -5 ,7 0 0 My ; o g l o b i n -1 ,7 OO;H emogl obin- preci pi tati ondepends on the si ze (mol ecul ar 64,450; Serum albumin-69,000. weighg of the protein molecule. In general,the 3. S h a p e : T h e re i s a w i d e v a ri a ti on i n the higher is the protein molecularweight, the lower pr ot eins h a p e .l t m a y b e g l o b u l a r(i n sul i n),oval is the salt requiredfor precipitation.Thus, serum globulins are precipitated by half saturation with (albumin)fibrous or elongated(fibrinogen). ammonium sulfatewhile albumin is precipitated 4. lsoelectric pH : lsoelectric pH (pl) as a by full saturation. Salting out procedure is propertyof amino acids has been described.The convenientlyusedfor separating serumalbumins nat ur e o f th e a m i n o a c i d s (p a rti c ul arl ythei r from gl obul i ns. ionizablegroups)determinesthe pl of a protein. The addition of small quantities of neutral T he ac i d i c a mi n o a c i d s (As p , C l u ) and basi c increasesthe solubility of proteins. This salts amino acids (His, Lys,Arg) stronglyinfluencethe process called as nlting rn is due to the pl. At isoelectric pH, the proteins exist as protein-proteininteractionat low salt diminished zwitterions or dipolar ions. They are electrically concentration. neutral (do not migratein the electricfield) with m inim u m s o l u b i l i ty , ma x i mu m p re ci pi tabi l i ty Precipitation by salts of heavy metals : Heavy and least buffering capacity. The isoelectric metal ions like Pb2+, Hg2+, Fe2+, Zn2+t Cd2+ pH(pl) for some proteinsare given here cause precipitation of proteins. These metals P RO P E R T IES

OF P R OT E IN S

51

Ghapter 4 : PFIOTEINS AND AMINO ACIDS being positivelycharged,when added to protein solution(negativelycharged)in alkalinemedium resultsin precipitateformation. Precipitation by anionic or alkaloid reagents: Proteinscan be precipitatedby trichloroacetic c d, ac id, s u l p h o s a l i c y l iac c i d , p h o s p h otungstiaci pic r ic a c i d , ta n n i c a c i d , p h o s p h o mol ybdi caci d etc. By the addition of these acids, the proteins existing as cations are precipitatedby the anionic form of acids to produce proteinsulphosalicylate, protein-tungstate, proteinpicrate etc. Precipitation by organic solvents: Organic solvents such as alcohol are good protein precipitatingagents.They dehydratethe protein molecule by removing the water envelope and cause precipitation. 7. Colour reactions of proteins : The proteins give several colour reactions which are often useful to identify the nature of the amino acids presentin them. Biuret reaction : Biuretis a compoundformed by heating urea to 180"C.

NHe tC :O

biuret test is not clearlv known, lt is believed that the colour is due to the formation of a copper co-ordinated complex, as shown below.

otl

tl

o

The presenceof magnesiumand ammonium ions interfere in the biuret test. This can be overcome by using excessalkali. The colour reactionsgiven by proteinsdue to the presenceof specificamino acids are given in Table 4,3. These reactions are often useful to know the Dresenceor absenceof the said amino aci ds i n the gi ven protei n.

DENATURATION The phenomenon of disorganization of native protein structure is known as denaturation. Denaturation results in the loss of secondary, tertiaryand quaternarystructureof proteins.This i nvol ves a change i n physi cal , chemi cal a nd biological propertiesof protein molecules.

NH C=O NHz Reaction When biuret is treated with dilute copper s ulf a tei n a l k a l i n e m e d i u m, a p u rpl e col our i s obtained. This is the basis of biuret test widely used for identificationof proteinsand peptides. Biuret test is answered by compounds c ont a i n i n gtw o o r mo re C O-N H groups i .e., peptide bonds. All proteins and peptides possessingat least two peptide linkages i.e., tripeptides (with 3 amino acids) give positive biuret test. Histidine is the only amino acid that answersbiuret test.The principle of biuret test is conveniently used to detect the presence of pr ot e i n si n b i o l o g i c a lfl u i d s . T h e m echani smof

Specificgroup or amino acid

1, Biuret reaction

Twopeptide linkages

2. Ninhydrin reaction

a-Amino acids

3. Xanthoproteic reaction

Benzene ringofaromatic amino acids(Phe,Tyr,Trp)

4. Milllons reaction

group(Tyr) Phenolic

5. Hopkins-Cole reaction Indole ring(Trp) 6. Sakaguchi reaction

group(Arg) Guanidino

(Cys) 7, Nitroprussidereaction Sulfhydrylgroups 8. Sulfur test

(Cys) Sulfhydrylgroups

9. Pauly's test

lmidazole ring(His) groups (Tyr) Phenolic

BIOCHEMISTFIY

62

Denaturation .

Nativeprotein

Fiq.4.14 : Denaturationof a protein.

bonds to enzymes. Cooking causes protein denaturation and, therefore, cooked food Heat, violent shaking, (protein) is more easily digested.

Agents of denaturation Physical agents: X-ravs,UV radiation.

8. Denaturation is usually irreversible.For Chemical agents : Acids, alkalies, organic instance,omelet can be preparedfrom an egg solvents(ether, alcohol), salts of heavy metals (protein-albumin) but the reversalis not possible. (Pb, Hg), urea, salicylate. 9. Careful denaturation is sometimesreversible (known as renaturation). HemoSlobin of denaturation Gharacteristics undergoes denaturation in the presence of 1. The native helical structureof protein is salicylate.By removal of salicylate,hemoglobin lost (Fig.4.l4). is renatured. 2. The primary structure of a protein with 10. Denaturedprotein cannot be crystallized. peptide linkages remains intact i.e., peptide Coagulation : The term 'coagulum' refersto a bonds are not hydrolysed. semi-solid viscous precipitate of protein. 3. The protein loses its biological activity. lrreversibledenaturationresults in coagulation. 4. Denatured protein becomes insoluble in Coagulation is optimum and requires lowest t he s olv en ti n w h i c h i t w a s o ri g i n a l l ys o l ubl e. temperature at isoelectric pH. Albumins and globulins (to a lesser extent) are coagulable 5 The viscosity ol denatured protein proteins. Heat coagulation test is commonly (solution) increaseswhile its surface tension used to detect the presence of albumin in urine. decreases. Flocculation: lt is the process of protein 6. Denaturationis associatedwith increasein precipitationat isoelectricpH. The precipitateis ionizable and sulfhydrylgroups of protein. This referredto as flocculum. Casein (milk protein) is due to loss of hydrogenand disulfide bonds. can be easily precipitated when adjusted to 7. Denaturedprotein is more easily digested. isoelectric pH (4.6\ by dilute acetic acid. This is due to increasedexposure of peptide Flocculation is reversible. On application of

63

AND AMINOACIDS PROTEINS heat, flocculum can be converted into an ir r ev er s i b l ema s s ,c o a g u l u m . CLA S S IF IC A T IO N

OF P R OT E INS

Proteinsare classifiedin severalways. Three major typesof classifyingproteinsbasedon their f unc t ion , c h e m i c a l n a tu re a n d sol ubi l i ty properties and nutritional importance are discussedhere.

7. Genetic proteins: Nucleoproteins. 8. Defenseproteins: Snakevenoms, lmmunogl obul i ns. 9. Receptorproteins for hormones,viruses.

Thi s i s a more comprehensi veand popul ar cl assi fi cati onof orotei ns. l t i s based on tne : ! i ti ami no aci d composi ti on,structure,shape and sol ubi l i ty properti es. P rotei ns are broadly Basedon the functionsthey perform,proteins cl assi fi edi nto 3 maj or B roups ar e c las s i fi e di n to th e fo l l o w i n g g ro ups (w i th 1 . Simple proteins: They are composed of ex am pl e s ) only amino acid residues. 1. Structural proteins : Keratin of hair and 2. Conjugated proteins: Besidesthe amino nails ,c o l l a g e no f b o n e . aci ds, these protei ns contai n a non-protein 2. Enzymesor catalyticproteins: Hexokinase, moiety known as prosthetic group or pepsrn. conj ugati nggroup. 3. T ra n s p o rt p ro te i n s : H e mo g l o b i n, serum 3. Derived proteins: Theseare the denatured album in . or degradedproductsof simple and conjugated 4. H o rmo n a l p ro te i n s : In s u l i n, grow th oroterns. nor m on e . The above three classes are further 5. Contractile proteins: Actin, myosin. subdi vi dedi nto di fferentgroups.The summar y of protein classificationis given in the Table4.4. 6. S to ra g ep ro te i n s : O v a l b u mi n ,gl utel i n.

BIOMEDICAL/ CLINICALCONCEPTS

Proteins are the most abundant organic molecules ol life. Theg perform static (structurol) and dynamic functions in the liuing cells. The dynomic t'unctionsof proteins are highly diuersilied such os enzymes, hormones, clotting factors, immunoglobulins, storage proteins and membrane receptors. oe Half of the amino acids (about 70) that occur in proteins haue to be consumed by humans in the diet, hence they qre essentlal. A protein is soid to be complete (or lirst class)protein if oll the essentialamino acids are present in the required proportion by the human body e.g. egg olbumin. Cooking resultsin protein denaturotion exposingmore peptide bondsfor easydigestion. Monosodium glutamate (MSG) ts used os a flauoring agent in loods to increasetaste and flauour. ln some indiuidualsintolerant to MSG, Chinese restaurantsyndrome (brief and reuersiblellu-like symptoms)is obserued.

64

BIOCHEMISTF|Y

Scleroproteins Albumins Globulins Glutelins Prolamines Histones

Gollagens Elastins Keratins

Nucleoproteins Glycoproteins Mucoproteins Lipoproteins Phosphoproteins Chromoproteins Metalloproleins

Coagulated prot€ins

Proteoses Peptones

Proteans Metaproteins

Polypeptides Peptides

Globins Protamines

l. Simpleproteins (a) Globularproteins:Thesearespherical or oval in shape,solublein wateror other solventsand digestible. (i) Albumins: Soluble t n water and dilute salt solutionsand coagulated by heat. e.g. serum albumin, (milk). ovalbumin(e8d,lactalbumin (ii) Globulins:Solublein neutraland dilute salt solutions e.g. serum globulins, vitelline(eggyolk). (iii) Glutelins:Solublein diluteacidsand alkaliesand mostlyfound in plants e.g. glutelin(wheat),oryzenin(rice). (iv) Prolamines:Solublein 7O1"alcohol e.g. gliadin(wheat),zein (maize). (v) Histones: Strongly basic proteins, solublein waterand diluteacidsbut insoluble in diluteammonium hydroxide e.g.thymushistones, histones of codfishsperm. (vi) Globins: Theseare generallyconsideredalongwith histones.However, globinsarenot basicproteinsand are not precipitated by NH/OH. (vii) Protamines:They are stronglybasic and resemblehistonesbut smaller in size and soluble in NH4OH. Protamines are also found in

association with nucleic acids e.g. sperm proteins. (b) Fibrous proteins : These are fiber like in shape,insoluble in water and resistantto digestion,Albuminoids or scleroproteins constitutethe most predominantgroup of fibrous proteins. (i) Collagens are connective tissue proteins lacking tryptophan. Collagens,on boiling with water or di l ute aci ds, yi el d gel ati n w hi ch i s soluble and digestible. (ii) Elastins:These proteinsare found in elastic tissues such as tendons and arteries. (iii) Keratins: These are present in exoskeletalstructurese.g. hair, nails, horns.Human hair keratincontainsas much as 14% cystei ne. 2. Coniugated proteins (a) Nucleoproteins: Nucleic acid (DNA or RNA) is the prostheticBroup e.g. nucleones. histones,nucleoprotami (b) Glycoproteins: The prosthetic group is carbohydrate, which is less than 4"/" of protein, The term mucoprotein is used if the carbohydratecontent is more than 4o/o. e.g. mucin (saliva),ovomucoid(eggwhite).

Chapter 4 : PROTEINSAND AMINO ACIDS

65

(c) Lipoproteins: in (described Fromthe nutritionalpointof Protein found already). proteins prosthetic view, with lipids as the are classified into 3 categories. combination groupe.g.serumlipoproteins, membrane 1. Completeproteins: Theseproteinshave lipoproteins. all the ten essential aminoacidsin the required (d) Phosphoproteins: Phosphoric acid is the proportionby the humanbody to promotegood prosthetic group e.g. casein (milk), growth.e.g. egg albumin,milk casein. vitelline(eggyolk). 2. Partiatlyincompleteproteins:Theseproteins are partiallylackingone or moreessential (e) Chromoproteins: groupis The prosthetic amino acidsand hencecan promotemoderate coloured in nature e.g. hemoglobins, growth. e.g. wheat and rice proteins(limiting cytochromes. Lys,ThO. (0 Metalloproteins : Theseproteins containmetal 3. Incomplete proteins: These proteins ions suchas Fe,Co, Zn, Cu, Mg etc., e.g. (Zn). completelylack one or more essentialamino (Cu),carbonicanhydrase ceruloplasmin acids.Hencethey do not promotegrowthat all 3. Derived proteins : The derived proteins e.g. gelatin(lacksTrp),zein (lacksTrp, Lys). are of two types.The primaryderived are the denaturedor coagulatedor first hydrolysed BIOTOGICALLY IMPORTANT PEPTIDES productsof proteins.The secondary derivedare Severalpeptidesoccur in the living orgathe degraded(due to breakdownof peptide nisms that display a wide spectrumof biobonds)productsof proteins. logicalfunctions. Generally, theterm'peptide'is (a) Primaryderivedproteins appliedwhen the numberof constituent amino acids is less 10. than Some examples of bio(i) Coagulatedproteins: Theseare the peptides logically active functions and their are denatured proteins produced by described here. agentssuch as heat, acids,alkalies etc. e.g.cookedproteins,coagulated 1. Glutathione : lt is a tripeptide composed of albumin(eggwhite). glutathione 3 amino acids.Chemically, is yglutamyl-cysteinyl-glycine. lt is widelydistributed (ii) Proteans: These are the earliest in nature and exists in reducedor oxidized productsof protein hydrolysisby states. enzymes,dilute acids,alkaliesetc. whichareinsoluble in water.e.g.fibrin 2G-SH =+ G=S-S-G formed from fibrinogen. Reduced Oxidized (iii) N,lgf6plsteins : Theseare the second stageproductsof protein hydrolysid obtainedby treatmentwith slightly strongeracidsand alkaliese.g. acid and alkalimetaproteins.

Functions: In a steady state, the cells generallymaintaina ratio of about 100/1 of CSH to G-S-S-C. The reversibleoxidationreductionof glutathione is importantfor manyof its biologicalfunctions.

(b) Secondary derivedproteins: Thesearethe . progressive hydrolyticproductsof protein hydrolysis. These include proteoses, polypeptides peptones, and peptides. r G. Nutritional classification

of proteins

The nutritivevalueof proteinsis determined by the compositionof essentialamino acids

Clutathioneservesas a coenzymefor certain enzymese.B.prostaglandin PCE,synthetase, glyoxylase. lt prevents the oxidation of sulfhydryl (-SH) groups of several proteins to (-S-S-1 groups. disulfide Thisis essential for the protein function, including that of enzymes.

B IOC H E MIS TFIY

66 It is believed that glutathione in association with glutathionereductaseparticipatesin the formationof correctdisulfidebonds in several proteins.

gl utathi oneperoxi dase(a sel eni umcontai ni ng enzyme). 2 GSH+ tjrorl99l!!9!5G

- s - s - G + 2 H2o

2. Thyrotropin releasing hormone (IRFI) : lt Clut at h i o n e (re d u c e d ) p e rfo rm s s p eci al i zed is a tripeptide secretedby hypothalamus.TRH functions in erythrocytes stlmulatespituitary gland to releasethyrotropic ( i) lt ma i n ta i n sR BC m e m b ra n es tru ctureand normone. integrity. 3. Oxytoci n: l t i s a hormone secretedby (ii) lt protects hemoglobin from getting posteri orpi tui tarygl and and contai ns9 ami no Oxytocin causescontraction acids(nonapeptide). oxidized by agentssuch as HzOz. of uterus. Clut ath i o n e i s i n v o l v e d i n th e tra nsoortof 4. Vasopressin(antidiuretic hormone, ADI{) t am ino a c i d s i n th e i n te s ti n e a n d ki dney ADH is also a nonapeptideproducedby posterior y-glutamyl tubules via cycle or Meister cycle pi tui tary gl and. l t sti mul ateski dneys to retai n (Refer Chapter 8). water and thus increasesthe blood pressure. G lut ath i o n e i s i n v o l v e d i n th e d e t oxi cati on 5. A ngi otensi ns:A ngi otensi nI i s a decapepprocess. The toxic substances (organoti de (10 ami no aci ds) w hi ch i s converted to phosphates,nitro compounds)are converted angi otensi nl l (8 ami no aci ds). The l ater has t o m er c a p tu ri ca c i d s . more hypertensive effect. Angiotensin ll also Toxic amounts of peroxidesand free radicals stimulates the release of aldosterone from produced in the cells are scavanged by adrenalgl and.

EIOMEtrICAL / CLIIhIICALCONCEPTE

sa Collagen is the most abundant protein in mammols. lt is rich in hydroxyproline and hydroxylysine. Seuerolbiologicolly important peptides are known in the liuing orgonism.Theseinclude glutathione t'or the maintenonce of RBC structure ond integrity; oxytocin that caases uterus contraction; uosopressin that stimulates retentlon ol water by kidneys; enkephalins that inhibit the senseof poin in the brain. lg

Antibiotics such os actinomycin, gramicidin, bocitracin and tyrocidin are peptide in nature.

6

yCarboxyglutamic acid is an amino acid deriuatiuefound in certain plasma proteins inuoluedin blood clotting. Homocysteine hos been implicated os o risk loctor in the onset ol coronary heart diseoses. Seueral non-protein amino ocids ol biological importonce are known. These include ornithine, citrulline and arginosuccinicacid (intermediotesol urea synthesis),thyroxine and triiodothyronine (hormones),and ftalanine (of coenzymeA). The protein-free liltrote of blood, required tor biochemical inuestigotions(e.g. urea, sugar)can be obtained by using protein precipitating agents such os phosphotungstlc acid ond trichloroaceticacid. of albumin in urine. Heot coagulationtest is mosf commonly employedto detect the pre.sence

PFOTEINS AND AMINOACIDS

67

6. Methionine enkephalin: lt is a pentapeptide found in the brain and has opiate like f unc t ion . l t i n h i b i tsth e s e n s eo f a p a i n.

9. Aspartame: lt is a dipeptide (aspartylphenyl al ani ne methyl ester), produceo by a combi nati on of asparti c aci d and 7. Bradykinin and kallidin : They are nona- phenyl al ani ne.A spartamei s about 200 ti mes and decapeptides,respectively.Both of them act sweeter than sucrose, and is used as a as powerful vasodilators.They are produced l ow -cal ori e arti fi ci al sw eetner i n softdri nk from plasma proteinsby snakevenom enzymes. i ndustry. 10. Gastrointestinal hormones: 8. Peptide antibiotics: Antibiotics such as Castrin, gr amic id i n ,b a c i tra c i nty , ro c i di n a n d a c tinomyci n secretin etc. are the gastrointestinalpeptides w hi ch serveas hormones. are peptide in nature.

I. Protelns are nltrogen containing, most abunddnt organlc mscromoleculeswtdely distributed in animals ond plants. They perform structurol and dynamic lunctions in the organisms. 2. Proteins are polymers composed ol L-a-amino acids. They are 20 in number and classifled into dtlt'erent groups based on their structure, chemical nature, nutritional requirementond metabolicfote. Selenocysteinehss been recently identified as the 27st amlno acld, and is found ln certain protelns. 3. Amino ocidspossesstwo functional groups namely carboxyl(-CooH) qnd amlno (-NH). ln the physiologlcolsystem,they exist as dipolar ions commonly relerred to os zwitteriois. 4. Besidesthe 20 standard omino acidspresent in proteins, there are seuerolnon-stondard amino ocids. These include the omino acid deriuatluesfound in proteins (e.g. hydroxyprollne, hydroxylysine)ond, non-protein amino acids (e.g. ornithine, citrulltne). 5. The structure of protein is dluided into t'our leuels of organizatlon. The primary structure represents the linear sequence of amino ocids. The twisting ond spatial arrangement of polypepttde chdin is the secondary structure. Tertiary structure constltutes the three dimensional structure of a t'unctional protein. The assemblyof slmilar or dtssimilar polypepilde subunils comprlses quaternary structure. 5. The determlnation of primary structure of a protein inuoluesthe knowledgeol quality, quantity and the sequenceof amino acids in the polypeptide. Chemical and enzymatic methods are employed t'or the determinotion of primary structure. 7. The secondary structure of protein mainly conslstsof a-helix anilor ftsheet. a-Helix is stabiltzedby extensive hydrogen bondlng. ftPleated sheet is composedol two or more segmentsof fully extended polypepttde choins. 8. The tertlory and quaternary structures ol protetn are stabiltzed by non-coualent bonds such os hydrogen bonds, hydrophobic interactions, ionic bonds etc. 9' Protelns are classilied lnto three major groups. Simple proteins contain only amino acid resldues (e.g. albumtn). Conjugated proteins contaln a non-protein moiety known os prosthetic group, bestdesthe amtno acids (e,g. glycoprotelns). Dertued protelns are obtained by degradation of simple or conjugoted protelns. 10. In oddition to proteins, seueral peptldes perlorm btologtcolly tmportant functtons. These lnclude glutothlone, oxgtocin and udsopressin.

68

B IOC H E MIS TR Y

I. Essayquestions 1. Describethe classification of amino acids along with their structures. of primary 2. Discussthe organization of proteinstructure.Cive an accountof the determination structureof orotein. 3. Describethe classification of proteinswith suitableexamples. 4. Write an accountof non-standard amino acids. 5. Discussthe importantbiologicallyactivepeptides. II. Short notes (a) Essentialamino acids, (b) Zwitterion,(c) Peptidebond, (d) Edman'sreagent,(e) a-Helix, (fl p-Pleatedsheet,(g) Denaturation, (h) lsoelectricpoint, (i) Clutathione,(j) Quaternarystructure of protein. IIL Fill in the blanks 1. The averagenitrogencontent of proteins 2. Proteinsare the polymersof -. 3. Name the sulfurcontainingessentialamino acid 4. The chargedmoleculewhich is electricallyneutralis known as -. 5. The non -o amino acid presentin coenzymeA -. 6. The bondsformingthe backboneof proteinstructure 7. The amino acid that is completelydestroyedby acid hydrolysisof protein L The numberof peptidebonds presentin a decapeptide -. 9. The chemical name of Sanger'sreagent 10. The phenomenonof disorganization of nativeproteinstructureis known as -. IV.Multiple choice questions 11, T h e i mi n o a c i d fo u n d i n p ro tei nstructure (a )A rg i n i n e(b ) Pro l i n e(c ) H i sti di ne(d) Lysi ne, 12, T h e fo l l o w i n gi s a n o n -p ro te in ami no aci d (a) Ornithine(b) Homocysteine (c) Histamine(d) All of them. 13. The bonds in proteinstructurethat are not brokenon denaturation. (a) Hydrogenbonds(b) Peptidebonds(c) lonic bond (d) Disulfidebonds. 14. Sequenator is an automaticmachineto determineamino acid sequencein a polypeptidechain. The reagentused in sequenatoris (a) Sanger'sreagent(b) CNBr (c) Trypsin(d) Edman'sreaBent, 15. The reactiongiven by two or more peptidelinkagesis (a) Biurettest (b) Ninhydrintest (c) Xanthoproteic reaction(d) Pauley'stest.

nNucXefcAcids andnNucXeotfldes

here are two types of nucleic acids, genes control the protein synthesisthrough the I namely deoxyrihonucleic acid (DNA) and mediation of RNA, as shown below ribonucleicacid (RNA). Primarily,nucleic acids A ----+ RNA-----| Prlteir serve as repositoriesand transmittersof genetic inf or m at i o n . The interrelationship of these three classesof (DNA, RNA and proteins)constitutes biomolecules Brief history the cenfral dogma of molecular biology or more commonly the central dogma of life. DNA w a s d i s c o v e re d i n 1 8 6 9 b y Johann F r iedr ic h M i e s c h e r, a Sw i s s re s e a rcher.The C omponents of nucl ei e aei ds dem ons t ra ti o n th a t D N A c o n ta i n e d geneti c Nucleic acids are the polymers of nucleotides informationwas first made in 1944, by Avery, (polynucleotides)held by 3' and 5' phosphate Macleod and MacCarv. bridges.In other words, nucleic acids are built up by the monomeric units-nucleotides (lt may F unc t ion s o f n u c l e i c a c i d s be recalled that protein is a polymer of amino aci ds). DNA i s th e c h e m i c a l b a s i s o f h e re di ty and nay be regardedas the reservebank of genetic for r r or m at i o n .D N A i s e x c l u s i v e l yre s p o nsi bl e -aintaining the identity of different speciesof Nucleotidesare composedof a nitrogenous c ' ganis m so v e r m i l l i o n so f y e a rs .F u rther,every as oec tof c e l l u l a rfu n c ti o ni s u n d e rth e controlof base,a pentosesugarand a phosphate.Nucleof,\{. The DNA is organized into geneg the tidesperforma wide varietyof functionsin the :urdamental units of genetic information. The livingcells,besides beingthe buildingblocksor

J

69

70

B IOC H E MIS TR Y

the pyrimidinecytosine(C)isfoundin bothDNA and RNA. However,the nucleic acids differ with respectto the secondpyrimidinebase. DNA contains thymine (T) whereas RNA contains uracil (U). As is observed in the Fig,5.2,thymineand uracildifferin structure by (in T) or absence (in U) of a methyl the presence 8roup. Tautomeric fcrms of purines an€i pyriffiidines Flg.5.l : Generalstructureof nitrogenbases (A) Purine(B) Pyrimidine(Th€ pasltionsare nambercd awrding to the intemetif'.'d'lsystern).

The existenceof a molecule in a keto (lactam) and enol (lactim) form is known as The heterocyclicrings of purines tautomerism.

/o \ and pyrimidines with oto LC-/ functional

m onome ri cu n i ts i n th e n u c l e i c a c i d (D N A and groupsexhibittautomerism as simplifiedbelow. RNA) structure. These include their role as OH OH I structural components of some coenzymes of l. ^1, .i - I 'v-r\-C = N B - c om p l e xv i ta m i n s (e .9 . F A D , N A D+ ), i n the Lactamform Lactlmform energy reactions of cells (ATP is the energy currency), and in the control of metabolic o reactions.

STRUCTUREOF NUCLEOTIDES As alreadystated,the nucleotideessentially consistsof nucleobase,sugar and phosphate. Adenlne(A) Thetermnucleoside refersto base+ sugar.Thus, (6-aminopurine) nucleotideis nucleoside + phosphate.

H

Guanlne(G) (2-amino 6-orypurine)

Purines and pyrinnidines The nitrogenous basesfound in nucleotides (and, therefore,nucleic acids) are aromatic heterocyclic compounds.The basesare of two types-purinesand pyrimidines.Their general structures are depictedin Fig.S.l. Purinesare numberedin the anticlockwise directionwhile pyrimidinesare numberedin the clockwise direction.And this is an internationally accepted systemto representthe structureof bases. Major bases in nucleie acids The structures of major purines and pyrimidines foundin nucleicacidsareshownin Fig.5.2.DNA and RNAcontainthe samepurines namelyadenine(A) and guanine(C). Further,

I Cytoslne(C) (2-ory4-aminopyrimidine)

oo H3

HN"\

tI

lll

f*/ Hil

Thymine(T) Uracil(U) (2,A-dioxy-Smethylpyrimidine) (2,4-dioxypyrimidine) Flg.5.2 : Structures of majorpurines(A, G) and pyrimidlnes(C, T, U) foundin nucleicacids.

Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES NHe

t-

NHe

N2-\c

N?c\c

t-

a

lll

lll

i/-

6"'"\*"-"

^

I H

Lactamform

,t\-'

HO

OHH D-2-Deoryribose

D-Ribose Lactim form

Fig. 5.3 : The tautomeric forms of cytosine.

Fig. 5.5 : Structuresof sugarspresentin nucleic acids (riboseis foundin RNAand deoxyribosein DNA;Note the slructuraldifferenceat Cz).

T he p u ri n e -g u a n i n e a n d p y ri m i di nes- Sugars of nucleic acids cytosine,thymine and uracil exhibittautomerism. The five carbon monosaccharides(pentoses) The lactam and lactim forms of cytosine are are found in the nucleic acid structure. RNA representedin Fi9.5.3. contains D-ribose while DNA contains At physiologicalpH, the lactam (keto)tauto- D-deoxyrihose. Ribose and deoxyribosediffer in meric forms are predominantlypresent. structureat C2. Deoxyribosehas one oxygen less Minor basesfound in nucleic acids : Besides at C2 compared to ribose (Fig.s.A. the basesdescribed above, several minor and unusualbases are often found in DNA and RNA. These include 5-methylcytosine, Na-acetylcytosine, N6-methyladenine,N6, N0-dimethyladenine,pseudouraciletc. lt is believedthat the unus ualba s e si n n u c l e i c a c i d s w i l l h e l p i n the recognitionof specific enzymes. Other biologically important bases : The basessuch as hypoxanthine,xanthine and uric acid (Fig.5.4)are present in the free state in the cells. The former two are the intermediatesrn pur ine s y n th e s i sw h i l e u ri c a c i d i s th e end product of purine degradation.

Nomenclattrre

of nueleotides

The addition of a pentose sugar to base produces a nucleoside. lf the sugar is ribose, ribonucleosides are formed. Adenosine, guanosi ne, cyti di ne and uri di ne are the ribonucleosides of A, C, C and U respectively.lf the sugar is a deoxyribose, deoxyribonucleosidesare produced. The term mononucleotide is used when a single phosphate moiety is added to a nucl eosi de. Thus adenosi ne monophosphate (AMP) contains adenine + ribose + phosphate.

Purine basesof plants : Plantscontain certain The principal bases, their respective methylated purines which are of pharmacological interest. These include caffeine (of nucl eosi des and nucl eoti des found i n the coffee), theophylline (of tea) and theobromine structureof nucleic acids are given in Tahle5.1. Note that the prefix 'd' is used to indicate if the (of cocoa). sugar is deoxyribose(e.g. dAMP). The binding components

Hypoxanthine Xanthine Uricacid (6-orypurine) (2,6-diorypurine) (2,6,8-trioxypurine) Fig. 5.4 : Structures of some biologically impoftant purines.

of nucleotide

The atoms i n the puri ne ri ng are .l numbered as to 9 and for pyrimidine as 1 to 6 (SeeFig.S.l).The carbonsof sugarsare represented with (1 an associated prime for differentiation. Thus the pentose carbons are 1' to 5'.

72

BIOCHEMISTFIY

Ribonucleoside

Ribonucleotide (5'-monophosphate)

Abbreviation

(A) Adenine

Adenosine

Adenosine 5'-monophosphate or adenylate

AMP

(G) Guanine

Guanosine

Guanosine 5'-monophosphate or guanylate

GMP

(C) Cytosine

Cytidine

Cytidine Samonophosphate or cytidylate

CMP

(U) Uracil

Uridine

Uridine 5'-monophosphate or uridylate

UMP

Deoxyribonucleoside

Deoxyribonucleotide (5'-monophosphate)

Abbreviation

(A) Adenine

Deoxyadenosine

Deoxyadenosine 5'-monophosphate or deoryadenylate dAMP

(G) Guanine

Deoxyguanosine

Deoryguanosine Slmonophosphate or deoxyguanylate dGMP

(C) Cytosine

Deorycytidine

Deorycytidine 5'-monophosphate or deoxycytidylate

(T) Thymine

Deoxythymidine

Deoxythymidine 5'-monophosphate or deoxythymidylate dTMP

The pentosesare bound to nitrogenousbases by p-N-glycosidicbonds.The Ne of a purine ring binds with C1111of a pentose sugar to form a covalent bond in the purine nucleoside.ln case of pyrimidine nucleosides,the glycosidiclinkage is between Nl of a pyrimidine and C'1 of a pentose. The hydroxyl groups of adenosine are esterified with phosphatesto produce 5'- or 3'-monophosphates.5'-Hydroxyl is the most commonly esterified,hence 5' is usuallyomitted while writing nucleotide names. Thus AMP

dCMP

represents adenosine 5'-monophosphate. However, for adenosine 3'-monophosphate,the abbreviation3'-AMP is used. The structuresof two selected nucleotioes namefy AMP and TMP are depicted in Fig.5.6. H{urr:"freosidedi- and triphosphates Nucleoside monophosphates possess only one phosphatemoiety (AMP,TMP).The addition of second or third phosphatesto the nucleoside resultsin nucleosidediphosphate(e.g. ADP) or triphosphate(e.9. ATP), respectively.

*o o--P-o-H2? o-[

c- P-o-H2g l

AMP

OHH TMP

Fig.5.6 : The structures of adenosine S'-monophosphate(AMP) and thymidineS'-monophosphate(TMP) [*-Addition of second or third phosphate gives adenosine diphosphate (ADP) and adenosine triphosphate (ATP) respectivetyl.

73

Chapter 5 : NUCLEICACIDSAND NUCLEOTIDES

5. Arabinosylcytosine is being used in cancer therapy as it interfereswith DNA replication. 6. The drugs employed in the treatmentof AfDS namely zidovudine or AZT (3-azido 2',3' -dideoxythymid i ne) and d idanosi ne (dideoxyinosine) are sugar modified synthetic nucleotide analogs(For their structureand more details Refer Chapter 3A.

8-Azaguanine Fig. 5.7 : Structures of selected purine and pyimidine analogs.

DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides).lt is composedof monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxyThe anionic properties of nucleotides and cytidylate(dCMP)and deoxythymidylate(dTMP) nucleic acids are due to the negative charges (lt may be noted here that some authors prefer to contributedby phosphategroups. use TMP for deoxythymidylate,since it is found only in DNA). The details of the nucleotide P UR|NE , P YR IM T D T N E structure are given above.

AND NUCLEOTIDEANALOGS It is possible to alter heterocyclic ring or sugar moiety, and produce synthetic analogs of pur in e s , p y ri mi d i n e s , n u c l e o s i des and nucleotides.Some of the syntheticanalogs are highly us e fu li n c l i n i c a l m e d i c i n e .T h e structures of selectedpurine and pyrimidine analogs are given in Fi9.5.7.

Schematic representation of polynucleotides

The monomericdeoxvnucleotidesin DNA are hefd together by 3',5'-phosphodiesterbridges (Fi9.5.81. DNA (or RNA) structure is often representedin a short-handform. The horizontal line indicatesthe carbon chain of sugar with The pharmacologicalapplicationsof certain base attached to C,,. Near the middle of the analogsare listed below horizontalIine is C3, phosphatelinkagewhile at other end of the line is C5, phosphatelinkage the 1. Allopurinol is used in the treatment of (Fig.s.A. hyperuricemia and gout (For details, Refer Chapter l7). Ghargaff's rule of DNA composltion 2. S-Fluorouracil, 6-mercaptopurine, 8-azaErwin Chargaff in late 1940s quantitatively guanine, 3 -d e o x y u ri d i n e ,5 - o r 6 -a zauri di ne, analysed the DNA hydrolysatesfrom different 5- or 6-azacytidineand 5-idouracilare employed species. He observedthat in all the specieshe in the treatmentof cancers.These compounds studied, DNA had equal numbersof adenineand get incorporated into DNA and block cell (A = T) and equal numbersof thymine residues or olif er a ti o n . guanine and cytosine residues(G = C). This is 3. Azathioprine (which gets degraded to known as Chargaff's rule of molar equivalence suppress 6-mercaptopurine) is used to between the purines and pyrimidines in DNA i m munological rejection du ring transplantation. structure.The significanceof Chargaff'srule was not immediatelv for the realised. The double helical is used 4. Arabinosyladenine viral structure of DNA derives its strength from treatment of neurological disease, (discussed Chargaff's rule later). enc eoha l i ti s .

74

B IOC H E MIS TFIY

J

I

DNA structure is considered as a milestone in the era of modern biology. The structure of DNA double helix is comparable to a twisted ladder. The salient features of Watson-Crick model of DNA (now known as B-Df.lA) are described next (Fi9.5.9).

'end

o I

I

,:

I

o I

I

L5' Hzr 2q'

I {K 4

1'

H

uanrne

OH I

n-D-/l-

C 3'end

A

Fig. 5.8 : Structureof a polydeoryribonucleotide segmentheld by phosphodiesterbonds.On the lower part is the representationol shorthad formof oligonucleotides.

Single-strandedDNA, and RNAs which are usually single-stranded, do not obey Chargaff's rule. However, double-strandedRNA which is the genetic material in certain viruses satisfies Chargaff'srule.

A ____T A_ _ _ _

|

u ==::

u

T

----

A

\J

=

=:='J

DNA DOUBLE HELIX The double helical structure of DNA was proposed by lames Watson and Francis Crick in 1953 ( N o b e l Pri z e , 1 9 6 2 ). T h e e l u ci dati onof

Fig.5.9 : (A) Watson-Crick model of DNA helix (B) Complementary base pairing in DNA helix.

Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES 7. The two strands are held together by hydrogen bonds formed by complementary base pairs (Fig.S.|O). The A-T pair has 2 hydrogen bonds w hi l e G-C pai r has 3 hydrog en bonds.The G = C is strongerby about 50% than A = T.

H

(B)

.Z-=.,-N.-,, l' ttt'o

tl

n. .

8. The hydrogen bonds are formed between a puri ne and a pyri mi di ne onl y. l f tw o puri n es face each other, they would not fit into the allowable space. And two pyrimidines would be too far to form hydrogen bonds. The only base arrangement possible in DNA structure, from spatialconsiderationsis A-T, T-A, G-C and

c-c. 9. The complementarybase pairing in DNA helix proves Chargaffs rule. The content of adenine equals to that of thymine (A = T) and guanine equals to that of cytosine (G = C).

Fiq.5.10 : Complementarybase paiing in DNA (A) Thymine pairs with adenine by 2 hydrogen bonds (B) Cytosine pairs with guanine by 3 hydrogen bonds.

1 . Th e D N A i s a ri g h t h a n d e dd o u bl e hel i x. l t consists ol two polydeoxyribonucleotide chains (strands) twisted around each other on a c om m o n a x ts . 2. The two strands are antiparallel, i.e., one strand runs in the 5' to 3' direction while the ot her in 3 ' to 5 ' d i re c ti o n . T h i s i s c o mparabl eto two parallel adjacent roads carrying traffic in opposite direction.

10. The genetic information resideson one of the two strands known as template strand or sense strand. The opposite strand is antisense strand. The double helix has (wide) major grooves and (narrow) minor grooves along the phosphodiesterbackbone.Proteinsinteractwith DNA at these grooves,without disrupting the base pairs and double helix. Sonformations

0f DNA double

helEx

Variation in the conformation of the nucleotides of DNA is associated with conformational variants of DNA. The double helical structure of DNA exists in at least 6 different forms-A to E and Z. Among these, B, A 3. The width (or diameter)of a double helix and Z forms are important (Table 5.2). The is 20 A o (2 n m). B-form of DNA double helix, described bv Watson and Crick (discussedabove),is the most 4. Each turn (pitch) of the helix is 34 A" predominant form under physiological (3.4 nm) with 10 pairs of nucleotides,each pair conditions. Each turn of the B-form has 10 base placed at a distanceof about 3.4 Ao. pai rs spanni nga di stanceof 3.4 nm. The w i dth 5. Each strand of DNA has a hydrophilic of the doubl e hel i x i s 2 nm. deoxyribosephosphatebackbone(3'-5' phosphoThe A-form is also a right-handedhelix. lt diester bonds) on the outside (periphery)of the contai ns11 basepai rsper turn. Therei s a ti l ti ng m olec u l e w h i l e th e h y d ro p h o b i c bases are pairs by 2O" away from the central of the base (core). stackedinside axts. 6. The two polynucleotide chains are not The Z-form (Z-DNA) is a left-handed helix identical but complementaryto each other due and contains '12 base pairs per turn. The to base pairing.

76

BIOCHEMISTFIY

Feature Helixtype

B-DNA

A-DNA

Z.DNA

Right-handedRight-handedLetl-handed Triple-stranded

Helical

(nm) diameter

1.84

per Distance eachcomplete turn(nm)

3.4

3.2

4.5

Riseperbase pair(nm)

0.34

0.29

0.37

Numberol base

pairspercomplete rurn 10 Basepairtilt

Certain antitumor drugs (e.g. cisplatin) produce bent structurein DNA. Such changed structure can take up proteins that damage the DNA.

+19"

l1

-1.2' (variable)

tz

DNA

Triple-strandedDNA formation may occur due to additional hydrogen bonds between the bases.Thus, a thymine can selectivelyform two Hoogsteen hydrogen bonds to the adenine of A-T pair to form I-A-L Likewise, a protonated cytosinecan also form two hydrogenbonds with guanine of C-C pairs that resultsin C-G-C. An outline of Hoogsteentriple helix is depicted in Fig.5.11.

-9"

Triple-helical structure is less stable than double helix. This is due to the fact that the three HelixaxisrotationMajorgrooveThrough base Minorgroove negatively charged backbone strands in triple pairs(variable) helix results in an increased electrostatic repul si on. polynucleotide strands of DNA move in a somewhat 'zig zag' fashion, hence the name Z - DNA . It is believed that transition between different helical forms of DNA plays a significantrole in regulatinggene expression.

Four-stranded

DNA

l

Polynucleotideswith very high contents of guanine can form a novel tetrameric structure

OTHER TYPES OF DNA STRUCTURE It is now recognized that besides double helical structure, DNA also exists in certain unusual structures. lt is believed that such molecular structures are important for recognition of DNA by proteins and enzymes. This is in fact neededfor the DNA to discharge its functions in an appropriate manner. Some selected unusual structures of DNA are brieflv described. Eent

DNA

I n gen e ra l , a d e n i n e b a s e c o n ta i n ing D N A tractsare rigid and straight.Bentconformationof DNA occurs when A-tracts are replaced by other basesor a collapse of the helix into the minor groove of A-tract. Bendingin DNA structurehas also been reported due to photochemical damage or mispairingof bases.

Fig" 5.11 : An outline of Hoogsteen triple helical structure of DNA.

77

chapter 5 : NUCLEICACIDSAND NUCLEOTIDES

C-tetraplexeshave been implicated in the called G-quarfefs. These structures are planar and are connected by Hoogsteen hydrogen recombi nati onof i mmunogl obul i ngenes, and bonds (Fig.S.12A). Antiparallel four-stranded in dimerization of double-strandedgenomic DNA structures, referred to as G-tetraplexes R N A of the human i mmunodefi ci encyvi rus (Hlu . have also been reported Gig.5.12Rl. The ends of eukarvoticchromosomesnamely THE SIZE OF DNA MOLECULE telomeresare rich in guanine, and thereforeform OF LENGTH -UNITS C-tetraplexes.In recent years, telomeres have becomethe targetsfor anticancerchemotherapies. D N A mol ecul es are huge i n si ze. On an average, a pair of B-DNA with a thickness of 0.34 nm has a mol ecul ar w ei ght of 660 daltons. For the measurementof lengths,DNA doublestrandedstructureis considered,and expresssed in the form of base pairs (bp). A kilobase pair (kb) is 103 bp, and a megahasepair (Mb) is 106 bp and a gi gabasepai r (C b) i s 10e bp. The kb, Mb and Cb relations mav be summarized as fol l ow s : 1 kb = 1000 bp 1 Mb = 1000 kb = 1,000,000bp 1 C b = 1000 Mb = 1,000,000,000bp

s',

(B)

/\

f_l

I

?-f ?-? II tt G-G

I

u

G

I

u

I

G _G

G_ G tl

tl tl

tl tl

3'

s',

t -l

3'

The length of DNA varies from species to species,and is usuallyexpressedin termsof base pair composition and contour length. Contour length represents the total lengthof the genomic D N A i n a cel l . S omeexampl esof organi smsw i th bp and contour lengthsare listed. . l , phage vi rus- 4.8 x 104 bp-contour l ength 16.5 mm.

r: I G _G

It may be noted here that the lengthsof RNA mol ecul es (l i ke D N A mol ecul es) cannot be expressedin bp, since most of the RNAs are single-stranded.

s',

Fig.5.12: Four-strandedDNAstructure(A) Parallel G4uartets (B) AntiparallelG-tetraplex.

E. coli 1.5 mm.

4.6 x 106 bp -

contour length

D i pl oi d human cel l (46 chromosomes) 6.0 x 10e bp-contour l ength2 meters. It may be noted that the genomic DNA size is usual l y much l arger the si ze of the cel l or nucl euscontai ni ngi t. For i nstance,i n humans,a 2-meter long DNA is packed compactly in a nucleus of about 1Opm diameter.

78

B IOC H E MIS TtrIY

T he ge n o m i c D N A rn a y e x i s t i n l i near or c ir c ular f o rm s . M o s t D N A s i n b a c teri a exi st as c los e d c i rc l e s . T h i s i n c l u d e s the D N A of bac t e ri a l c h ro mo s o m e s a n d th e extrac hr om os o m a lD N A o f p l a s mi d s .Mi to chondri a an< lc hlor o p l a s ts o f e u k a ry o ti cc e l l s a l s o contai n c ir c ular D N A.

-

Denaturation. R"a"tr-rti*

Chr omo s o m a D l N As i n h i g h e ro rg a n i smsare m os t ly lin e a r. i n d i i z i d u a ih u rn a n c h ro m osomes Two strands c ont ain a s i n q l e D N ,t r-n o l e c u l ew i th vari abl e separated s iz es c om p a c tl y p a c k e d . T h u s th e smal l est c hr om os o mec o n ta i n s3 4 M b w h i l e th e l arsesr Fiq.5.13 : Diagrammaticrepresentationof denaturation and renaturationof DNA. one has 26 3 M b . ffiffiWATUffiAT[ON

MF MN& STffiANffiS

T he t r , v o s tra n d s o f D N A h e l i x a re hel d t oget her b y h y d ro g e n b o n d s . D i s ru pti on of hy c J r ogebno n d s(b y c h a n g ei n p l -1o r i n creasei n iem per at u re ) re s u l ts i n th e s e p a rati on of poly nuc le o ti d es tra n c l sT.h i s p h e n o n re n o n of Joss of helical structure of DNA is kno',vn as denaturatian (Fi9.5.13). The phosp[64;"r*"t bonds ar e n o t b ro k e n b y d e n a tu ra ti o n Loss . of helic al s t ru c tu rec a n b e m e a s u re db y increase in abs orb a n c e a t 2 6 0 n m i i n a sD ectrophotometer).

Melting temperature (Im) is defined as the temperature at w hi ch hal f of the hel i calstructure of D N A i s l ost. S i nce C -C base pai rs are more stable(due to 3 hydrogenbonds)than A-T base pai rs (2 hydrogenbonds),the Tm i s greaterfor D N A s i vi th hi gherC -C content.Thus,the Tm i s 65" C for 35% C -C contentw hi l e i t i s 70' C for 5A % C -C content. Formanri de destabi l i zes hydrogen bonds of base pai rs and, therefore, l ow ersTrn.Thi schemi calcompoundi s effecti vel l , used i n recombi nantD N A experi ments.

EIOMEDICAL/ CLINTCALCONCEPTS

L€ D/VA is the reseruebank of genetic int'ormation,ultimately responsiblet'or the chemical bcsis o/ life and heredity. Gt DNA is organized into genes, the t'undamental units of genetic int'ormation. Genes control protein biosynfhesisthrough the mediation of RNA. u-F Nucleic acids are the polymers of nucleotides.Certoin nucleotidesserue as B-complex uitamin coenzymes(EAD' IVAD+, CoA), carriers ot' high energy intermediates(UDP-glucose, S-adenosylmethionine)and secondmessengersol hormonal action (cAME cGMP). Na Uric ocid is a purine, ond the end product ol purine metabolism, that has been implicated in the disordergout. Certain purine basesfrom plants such os caft'eine(oj coffee), theophylline (of tea) and theobromine (of cocoo) are of pharmacological interest. c'Ji'

Synthetic analogs of bases(5-fluorouracil, 6-mercaptopurine.6-azauridine)are used to inhibit the growth of cancer cells.

ut Certain antitumor drugs (e.g. cisplatin)can producebent DNA structureand damageit.

Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES

79

Renaturation(or reannealing)is the process These 30-nm fibers are further organized into in which the separatedcomplementary DNA loops by anchoringthe fiber at M-rich regions namely scaffold-associated strandscan form a double helix. regions (SARS)to a protein scafold.During the courseof mitosis,the loops are further coiled, the chromosomes condenseand becomevi si bl e.

As alreadv stated.the double-strandedDNA helix in e a c h c h ro m o s o meh a s a l e n g th that i s thousandstimes the diameterof the nucleus.For ins t anc e ,i n h u ma n s , a 2 -me te r l o n g D N A i s packed in a nucleus of about 'l0 pm diameter! T his is m a d e p o s s i b l e b y a c o mpact and m ar v ello u sp a c k a g i n ga, n d o rg a n i z a ti o nof D N A ins ide in c e l l . OrganEzation

of prakaryotFc

Dl{A

I n pr ok a ry o ti cc e l l s ,th e D N A i s o rg a ni zedas a single chromosome in the form of a doublestranded circle. These bacterial chromosomes are packed in the form of nucleoids, by interaction with oroteins and certain cations ( poly am i n e s ).

R N A i s a pol ymer of ri bonucl eoti deshel d together by 3',5'-phosphodiester bridges. A l thoughR N A has certai nsi mi l ari ti es w i th D N A structure,they have specific differences l . P entose: The sugar i n R N A i s ri bose i n contrastto deoxyribosein DNA. 2. P yri mi di ne: R N A contai nsthe pyri mi di ne uraci l i n pl ace of thymi ne (i n D N A ). 3. S i ngl e strand : R N A i s usual l y a si ngl estranded polynucleotide. However, this strand may fol d at certai n pl aces to gi ve a doubl estrandedstructure,if complementarybase pairs are i n cl ose proxi mi ty.

4. Chargaff's rule-not obeyed : Due to the single-strandednature, there is no specific rel ati on betw een puri ne and pyri mi di ne In the eukaryoticcells, the DNA is associated contents.Thus the guani necontenti s not equal with various proteins to form chromatin which to cytosi ne(as i s the case i n D N A ). then gets organized info compact structures namely chromosomes (Fig.SJa\" 5. Susceptibilityto alkali hydrolysis : Alkali can hydrolyseRNA to 2',3'-cyclic diesters.This T he DN A d o u b l e h e l i x i s w ra p p e da roundthe is possible due to the presenceof a hydroxyl core proteinsnamely histoneswhich are basic in group at 2' position. DNA cannot be subjected nature.The core is composedof two molecules to alkali hydrolysisdue to lack of this group. of histones(H2A, H2B, H3 and H4). Each core with two turns of DNA wrapped round it 6. Orcinol colour reaction : RNAs can be (approximatelywith 150 bp) is termed as a hi stol ogi cal l y i denti fi ed by orci nol col our nucleosome, the basic unit of chromatin. reaction due to the presenceof ribose. Nucleosomesare separatedby spacer DNA to which histone H1 is attached (Fig.S.l5). This TYPES OF RNA continuous string of nucleosomes,representing The three major types of RNAs with their beads-on-astringform of chromatin is termed as respecti vecel l ul arcomposi ti onare gi ven bel ow 10 nm fi b e r. T h e l e n g th o f th e DN A i s c ons idera b l yre d u c e db y th e fo rma ti o nof 10 nm 1. MessengerRNA (mRNA) : 5-1O"/" f iber . T h i s 1 0 -n m fi b e r i s fu rth e r c oi l ed to 2. TransferRNA (IRNA) : 10-200/" or oduc e 3 0 -n m fi b e r w h i c h h a s a sol enoi d 3. RibosomalRNA (rRNA) : 50-80% structure with six nucleosomesin everv turn. Srganization

of eukaryotic

DhlA

80

BIOCHEMISTF|Y

Naked DNA doublehelix

Jzn'

'Beads-on-a-string' formof chromatin

I,.",

30-nmchromatin fibrecomposedof nucteosomes

Chromosome in an extendedform (non-condensed loops)

Condensedform ol cnromosome

Metaphase chromosome

,o*'', |

Fig.5.l4 : Organization of eukaryoticDNAstructurein theformof chromatinandchromosomes.

+ I

11nm

lnbmucleosome Fig. 5.15 : Structurcof nucleosomes.

Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES

Type of RNA

Abbreviation

Messenger RNA

81

Funclion(s)

genelicintormation Transfers fromgenesto

Ii99.9_0,.T.99. !9_ 9J. $ltgli'e.p$giLL

...lt_qt:r.qg:l.:.qtE..t lf9l99.t..EllL Transfer RNA

Ribosomal RNA Smallnuclear RNA Smallnucleolar RNA

rRNA snRNA snoBNA

Smallcytoplasmic RNA Transfer-messenger RNA

TMRNA

scRNA

Besidesthe three RNAs referred above, other RNAsare also presentin the cells.Theseinclude heterogeneousnuclear RNA (hnRNA), small nuc lear R N A (s n R N A), s m a l l n u c l e o l ar R N A (snoRNA)and small cytoplasmicRNA (scRNA). The major functionsof these RNAs are given in Table 5.3.

Serves asprecursor formRNA andotherRNAs. Transfers aminoacidto mRNA for protein biosynthesis. Provides structural framework for ribosomes. Involved in mRNAprocessing.

Playsa keyrolein theprocessing of rRNA molecules. Involved in theselection of oroteins forexoort. present Mostly in bacteria. Addsshortpeptide tagst0 proteins to facilitate thedegradation of proteins. incorrectly synthesized nucleotides) which is known as poly (A) tail. This tail may provide stabilityto mRNA, besides preventing it from the attack of 3'-exonucleases. mRNA molecules often contai certain modified bases such as 6-methyladenylatesin the internal structure.

The RNAsare synthesizedfrom DNA, and are Transfer RNA (tRNAl primarily involved in the process of protein Transfer RNA (soluhle RNA) molecule biosynthesis (Chapter 2fl. The RNAs vary in their structureand function. A brief description contains 71-80 nucleotides(mostly 75) with a molecular weight of about 25,000. There are at on the major RNAs is given. least 20 speciesof tRNAs, correspondingto 20 Messenger amino acids present in protein structure. The RNA (mRNAl structure of tRNA (for alanine) was first The mRNA is synthesizedin the nucleus (in elucidatedby Holley. eukaryotes) as heterogeneous nuclear RNA (hnRNA). hnRNA, on processing,liberatesthe The structure of IRNA, depicted in Fig.S.t6, functional mRNA which entersthe cytoplasmto resemblesthat of a clover leaf. IRNA contains participate in protein synthesis.mRNA has high mainly four arms, each arm with a base paired stem. molecular weight with a short half-life. 1. The acceptor arm : This arm is capped The eukaryotic mRNA is capped at the S'-terminal end by 7-methylguanosine w i th a sequenceC C A (5' to 3' ). The ami no aci d triphosphate.lt is believedthat this cap helps to is attached to the acceptor arm. prevent the hydrolysis of mRNA by 5'-exo2. The anticodon arm : This arm, with the nucleases.Further,the cap may be also involved three specific nucleotide bases (anticodon), is in the recognitionof mRNA for proteinsynthesis. responsiblefor the recognitionof triplet codon The 3'-terminal end of mRNA contains of mRNA. The codon and anticodon are a polymer of adenylate residues (20-250 complementaryto each other.

t

82

BIOCHEMISTRY

el

g-Amino acid

I +A r -!,

Acceptorarm

D arm Complementary basepairs

T\rC arm Variablearm Anticodon arm

f,ibosomal

The ribosomes are the factories of protein synthesis. The eukaryotic ribosomes are major nucleoprotein composed of two complexes-60Ssubunit and 40S subunit. The 605 subunit contains 28S rRNA, 55 rRNA and 5.8S rR N A w hi l e the 40S subuni tcontai ns18S rRNA. The function of rRNAs in ribosomesis not clearly known. lt is believed that they play a si gni fi cant rol e i n the bi ndi ng of mR N A to ribosomesand protein synthesis. Other

Fiq.5.16: Structureof transferRNA.

3. The D arm : lt is so named due to the presenceof dihydrouridine.

RNA (rRNAl

RNAs

The various other RNAs and their functions are summarisedin Table 5.3.

CATALYTIG RNAs-RI BOZYMES

In certain instances,the RNA componentof a (RNA irr association with ribonucleoprotein 4. The TYC arm : This arm contains a protein) is catalytically active. Such RNAs are sequence of T, pseudouridine(representedby termed as ribozymes.At leastfive distinctspecies ps i, Y ) an d C . of RNA that act as catalystshave been identified. 5. The variable arm : This arm is the most Three are involved in the self processing v ar iable i n tR N A. Ba s e d o n th i s v a ri abi l i W , reactions of RNAs while the other two tRNAs are classifiedinto 2 categories: are regarded as true catalysts (RNase P and (a) Class I tRNAs : The most predominant rR N A ). (about 75"/") form with 3-5 base pairs Ribonuclease P (RNase P) is a ribozyme len g th " containing protein and RNA component. lt (b) Classll tRNAs : They contain 13-20 base cleaves IRNA precursors to generate mature pai r l o n g a rm. tR N A mol ecul es. Base pairs in tRNA : The structureof IRNA is RNA molecules are known to adapt maintained due to the complementary base tertiarystructurejust like proteins(i.e. enzymes). pairing in the arms. The four arms with their The specific conformation of RNA may be respectivebase pairs are given below responsiblefor its function as biocatalyst. lt The acceptor arm - 7 bp is believed that ribozymes (RNAs) were The TYC arm - 5 bp functioning as catalysts before the occurrence The anticodon arm - 5 bp of protein enzymes, during the course of T h e D a rm evolution. -4 b p

Shapten 5 : NUCLEIC ACIDS AND NUCLEOTIDES

l.

DNA is the chemical basisof heredity organized into genes, the basic units of genetic inJormotion.

2. RNAs ImFNA, fRNA and rRNA) are produced by DNA which in turn carry out protein synfhesis.

3. Nucleic acids are the polymers of nucleotides (polynucleoiides)held by 3' and 5' phosphodiester bridges. A nucleotide essentiolly consists of base + sugdr (nucleoside) and phosphate.

4. Besidesbeing the constituentsof nucleic acid structurc, nucleotidesperform a wide uariety ot' cellulor functians (e.9. energy carriers, metabolic regulators,second messengers etc.)

5. Both DNA ond RNA contain the parines-adenine(A) and guanlne (G)ond the pyrimidinecytosine(C). The secondpgrimidine is thymine (TJin DNA while it is uracil (U) in RNA. The pentose sugar,D-deoxyriboseis lound in DNA while it is D-ribose in RNA.

6. The structure o/ DNA is a double helix (Watson-Crick model) composed ol two antiparollel sfronds ol polydeoxynucleotidestutistedaround each othen The strandsare held together by 2 or 3 hydrogen bonds t'ormed between the basesi.e. A = T: G : C. DNA sfructure satisfiesChargoff's rule that the content of A is equal to T, and that ol G equal to C.

7. Besides the double helical structure, DNA olso exisfs fn certain unusuol structuresbent DNA, triple-strand DNA, four-strand DNA.

8. RNA is usually a single stranded polyribonucleotide. mRNA is capped ot 5'terminol end by 7-methylGTP while at the 3'-terminal end, it contains o poly A toil. mRNA speciliesthe sequenceol amino scids in protein synfhesis.

9. The structure of tRNA resembles that of a clouer leaf with four arms (acceptori anticodon, D-, and T'LC) held by complementargbasepoirs. fRNA deliuersamino acids lor protein synthesis.

10. Certain RNAs that mn function os enzymes are termed os ribozymes. Ribozymes were probablVfunctioningos cofolysfsbefore the occurrenceof protein enzymesduring ewlution.

83

84

BIOCHEMISTFIY

I. Essayquestions 1. Describethe structureof DNA. 2. Name differentRNAsand discusstheir structure. 3. Write an accountof structure,functionand nomenclature of nucleotides. 4. Describethe structureof nitrogenousbasespresentin nucleic acids.Add a note on tautomerism. 5. "The backboneof nucleicacid structureis 3'-5'phosphodiester bridge."-justify. II. Short notes (a) Chargaff'srule, (b) Riboseand deoxyribose,(c) Hydrogenbonds in DNA, (d) Nucleoside, (e) Differentforms of DNA, (f) TransferRNA, (g) Purine basesof plants,(h) Complementarybase (j) hnRNA. pairs,(i) DNA denaturation, III. Fill in the blanks 1 . The fundamentalunit of geneticinformationis known as 2 . DNA controlsproteinsynthesis throughthe mediationof 3 . Nucleic acidsare the polymersof 4 . The pyrimidinepresentin DNA but absentin RNA 5 . Riboseand deoxvribosediffer in their structurearoundcarbonatom 6 . Nucleotideis composedof 7 . The scientistwho observedthat there existsa relationshipbetweenthe contentsof purinesand pyrimidinesin DNA structure(A = T; C = C) B. The basepair G-C is more stableand strongerthan A-T due to 9. Underphysiological condition,the DNA structureis predominantly in the form 10. The acceptorarm of IRNA containsa cappednucleotidesequence IV. Multiple choice questions 1'l. The nitrogenousbasenot presentin DNA structure (a) Adenine(b) Cuanine(c) Cytosine(d) Uracil. 12. The numberof basepairspresentin each turn (pitch)of B-formof DNA helix (a ) e (b ) 1 0 (c ) 1 1 (d ) 1 2 . 13. The backboneof nucleicacid structureis constructedby (a) Peptidebonds(b) Glycosidicbonds(c) Phosphodiester bridges(d) All of them. 14. The following coenzymeis a nucleotide (a) FAD (b) NAD+ (c) CoASH(d) All of them. 15. The nucleotidethat servesas an intermediate for biosyntheticreaction (a) UDP-glucose (b) CDP-acylglycerol (d) AII of them. (c) S-Adenosylmethionin8

The

etrzytnes

spetrr.

:

"We are the catalystsof the liuing worU! Protein in nature, and in d.ctionspecifc, rapid and accurate; Huge in size but with srnall actiae centres; Highly erploited for diseasediagnosisin hb cennel"

In the laboratory,hydrolysisof proteinsby a strong acid at 100'C takes at least a couple of days. The same protein is fully digestedby the enzymes in gastrointestinal tract at body temperature(37'C) within a couple of hours. This remarkable difference in the chemical The student-teacherrelationship may be a ' reactionstaking place in the living system is good example to understand how a catalyst exclusivelydue to enzymes.The very existence works. The studentsoften find it difficult to learn of life is unimaginablewithout the presenceof from a text-book on their own. The teacher enzvmes. explainsthe subjectto the studentsand increases their understandingcapability. lt is no wonder that certain difficult things which the students take daystogetherto understand,and sometimes do not understandat all - are easily learnt under the guidance of the teacher. Here, the teacher Berzeliusin 1836 coined the term catalysis ac t s like a c a ta l y s t i n e n h a n ci ng the (Greek: to di ssol ve).In 1878, K uhne used the understandingability of students.A good teacher word enzyme (Creek: in yeast)to indicate the is always a good catalystin students'life! catalysistaking place in the biological systems. Enzymes may be defined as biocatalysts lsolation of enzyme systemfrom cell-free extract synthesized by living cells. They are protein in of yeast was achieved in 1883 by Buchner. He nature (exception - RNA acting as ribozyme), named the active principle as zymase (later colloidal and thermolahile in character, and found to contain a mixture of enzymes),which specific in their action. could convert sugar to alcohol. ln 1926, James - the catalystsof life. f nzymesare biocatalysts L A catalvsf is defined as a subsfance that increases ihe velocity or rate of a chemical reactionwithout itselfundergoingany change in the overall process.

85

86

BIOCHEMISTF}Y

Sumner first achieved the isolation and crystallizationof the enzyme ureasefrom jack bean and identified it as a protein.

In the early days, the enzymes were given names by their discoverers in an arbitrary manner. For example,the namespepsin,trypsin and chymotrypsinconvey no informationabout the function of the enzyme or the nature of the substrateon which they act. Sometimes,the suffix-asewas added to the substratefor naming the enzymese.g. lipaseacts on lipids; nuclease on nucleic acids; lactaseon lactose.These are known as trivial names of the enzymes which, however, fail to give complete information of

enzyme reaction (type of reaction, cofactor requirementetc.) Enzymesare sometimesconsideredundertwo broad categories : (a) Intracellular enzymesThey are functional within cells where they are synthesized.(b) Extracellular enzymes- These enzymes are active outside the cell; all the digestiveenzymesbelong to this group. (l U B ) U ni on of B i ochemi stry The Internati onal appoi ntedan E nzymeC ommi ssi oni n 1961.Thi s committeemade a thoroughstudy of the existing enzymesand devised some basic principlesfor the classificationand nomenclatureof enzymes. Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six major classes(in that order). the generaltype Eachclasson its own represents of reaction brought about by the enzymes of that class(Table 6.1\.

Enzyme classwith examples*

Reactioncatalysed

1. Oxidoreductases (alcohol Alcohol dehydrogenase : NAD*oxidoreductase E.C.1.1.1.1.), Oxidation------+Reduction A H r+ B -> A + B H , L-andD-amino acidoxidases cytochrome oxidase, 2. Transferases (ATP: D-hexose Hexokinase E.C.2.7.1.1.), Grouptransfer 6-phosphotransferase, phosphorylase A+ B-X A-X+ B------+ transmethylases, transaminases, 3. Hydrolases Lipase(triacylglycerol acylhydrolase E.C.3.1.1.3), choline phosphatases, pepsin, esterase, acidandalkaline urease

Hydrolysis AH+ BOH A- B + H"O------+

4. Lyases (ketose 1-phosphate lyase,E.C.4.1.2.7), Aldolase aldehyde fumarase, histidase

5. lsomerases (D-glyceraldehyde Triosephosphale isomerase 3-phosphate ketoisomerase, E.C.5.3.1.1), retinol isomerase, phosphohexose isomerase 6. Ligases (L-glutamate synthetase ammonia ligase, E.C.6.3.1.2), Glutamine acetyl succinate thiokinase CoAcarboxylase, *For oneenzynein eachclass,systenaticn"r,

Addition ------+Elimination

A- B+i- v- - Ax- Bi lnterconversion of isomers A-> A'

(usually on ATP) dependent Condensation A+B;z-5-+A-B pi ATP ADp+

ttorg *,rh E.C. nunbetis givenin the brackets.

Chapter 6 : ENZYMES

1. Oxidoreductases: Enzymes involved in oxidation-reductionreactions.

made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part).

2. Transferases: Enzymesthat catalysethe transferof functional groups.

Holoenzyme -----+ Apoenzyme + Coenzyme (activeenzyme) (protein part) (non-protein part)

3. Hydrolases : Enzymes that bring about hy dr oly s iso f v a ri o u sc o m p o u n d s .

The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme. The coenzyme can be separatedby dialysisfrom the enzyme while the prostheticgroup cannot be.

4. Lyases : Enzymes specialised in the additionor removalof water, ammonia,COr etc. 5. lsomerases: Enzymesinvolved isornerizationreactions.

the

6. ligases : Enzymescatalysingthe synthetic :eactions (Creek : ligate-to bind) where two nroleculesare joined togetherand ATP is used. lThe word OTHLIL (first letter in each class) rray be memorisedto rememberthe six classes of enzymes in the correct orderl. E ac h c la s s i n tu rn i s s u b d i v i d e di n to manv sub-classeswhich are further divided. A four digit E nz y me C o m m i s s i o n (E C .) n u m ber i s assignedto each enzyme representingthe class ( f ir s tdigit ) ,s u b -c l a s (s s e c o n dd i g i t),s u b -s u bcl ass ( t hir d digit ) a n d th e i n d i v i d u a l e n z y m e (fourth digit ) . E ac h e n z y m e i s g i v e n a s p e c i fi c name indicating the substrate,coenzyme (if any) and the type of the reactioncatalysedby the enzyme. Although the IUB names for the enzymes are specific and unambiguous,they have not been accepted for general use as they are complex and cumbersometo remember. Therefore,the t r iv ial nam e s , a l o n g w i th th e E.C . n u mbersas and when needed, are commonly used and widely accepted.

The word monomeric enzyme is used if it is made up of a si ngl e pol ypepti de e.g. ri bonucl ease,trypsi n. S ome of the enzymesw hi ch possessmore than one polypeptide (subunit) chain are known as oligomeric enzymes e.g. lactate dehydrogenase, aspartate transcarbamoylaseetc. There are certain multienzyme complexes possessingspecific sites to catalyse differentreactionsin a sequence.Only the native i ntactmul ti enzymecompl exi s functi onal l yacti ve and not the individualunits, if they are separated e.g. pyruvatedehydrogenase, fatty acid synthase, prostaglandinsynthaseetc. The enzymesexhibit all the generalpropertiesof proteins(Chapter4). Genetic engineering and modified enzymes Recentadvancesin biotechnologyhave made it possibleto modify the enzymeswith desirable characters-improved catalytic abilities,activities under unusual conditions. This approach is required since enzymes possess enormous potenti alfor thei r use i n medi ci neand i ndustry. Hybrid enzymes : lt is possibleto rearrange genesand produce fusion proteins. e.g. a hybrid enzyme (of gl ucanaseand cel l ul ase)that can more efficiently hydrolyse barley p-glucans in beer manufacture.

Site-directed mutagenesis : Th is is a technique used to produce a specifiedmutation A ll t he e n z y me s a re i n v a ri a b l yp ro tei ns.In at a predeterminedposition in a DNA molecule. recent years, however, a few RNA molecules The result is incorporationof a desired amino have been shown to function as enzvmes.Each acid (of one's choice) in place of the specified enzymehas its own tertiarystructureand specific ami no aci d i n the enzyme.B y thi s approach,i t conformation which is very essential for its i s possi bl eto producean enzymew i th desi rabl e e.g. tissueplasminogenactivator c at aly t ic ac ti v i ty . T h e fu n c ti o n a l u n i t o f the characteristics. enzyme is known as holoenzymewhich is often (used to lyse blood clots in myocardial

88

B IOC H E MIS TF|Y 2" Concentration +

of substrate

Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels.A rectangularhyperbolais obtainedwhen velocity is plotted against the substrate concentration(Fig.6.2).Three distinct phasesof the reactionare observedin the graph (A-linear; B-curve;C-almostunchanged).

I

I II o -9 o o

E N

t!

Fig. 6.1 : Effect of enzyme concentration on enzyme velociy.

infarction) with increased half-life. This is achieved by replacing asparagine(at position 120) by glutamine.

Order of reaction : When the velocity of the reaction is almost proportionalto the substrate concentration(i.e. [S] is lessthan Kn,),the rate of the reaction is said to be first order with respect to substrate.When the ISJ is much greater than Kn', the rate of reaction is independent of substrateconcentration,and the reaction is said to be zero order. Enzyme kinetics and K, value : The enzyme (E)and substrate(S)combine with each other to form an unstableenzyme-substratecomplex (ES) for the formation of product (P).

ln recentyears,it has also become possibleto produce hybrid enzymes by rearrangement of k1. E+ S r, -- rs S r +p genes. Another innovative approach is the ' kproduction of abzymes or catalytic antibodies, Here kl , k2 and k3 represent the velocity the antibody enzymes. constants for the respective reactions, as indicated by arrows.

K' the Michaelis-Mentenconstant(or Brigrs and Haldane's constant),is given by the formula The contact between the enzyme and substrateis the most essentialpre-requisitefor enzyme activity. The important factors that influencethe velocity of the enzyme reactionare discussedhereunder t. Goncentration

of enzyme

K' =

kz + kr

k, The following equation is obtained after sui tabl eal gebrai cmani pul ati on. v=

Vr"* [S] K m+ [S ]

equation(1)

where v = Measuredvelocity, = Maximum velocitv, Vtu" = Substrateconcentration, S = Michaelis- Mentenconstant. K.

As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases (Fig.6.l). In fact, this property of enzyme is made use in determining Let us assumethat the measured velocity(v) the serumenzymesfor the diagnosisof diseases. is equalto f Vrr". Thentheequation(1)maybe By using a known volume of serum,and keeping substitutedas follows all the other factors (substrate,pH, temperature 1v etc.) at the optimum level, the enzyme could be _ v.axlsl assayedin the laboratory. 2.max K . +[ s ]

89

Ghapter 6 : ENZYMES

since V."" is approached asymptotically. By taking the reciprocals of the equation (1), a straightline graphic representationis obtained.

Vr"* -J

TI

I

I 1V.",-F g

1_

Km .. 1

V v-"" -

--+ Substrateconcentration Fig. 6,2 : Effect of substrate concentration on enzyme velocity (A-linear; B-curve; C-almost unchanged).

1-= K m., -,.-i--TiV V..,.

r^1 [".|

-----------i--T [s] v'"- [sJ

---1-

1 IS.|

1 Vr"*

The above equation is similar to y = ax + b. Therefore,a plot of the reciprocalof the velocity I ' I ur. the reciprocalof the substrateconcen\vi

2vmax[S] Vr"*

Km+ [S]

-

K n ' + [S ]

= 2 [S ]

Km

=

[S]

K stands for a constant and m stands for M ic ha e l i s(i n Kn .).

!

K^ or lhe Michaelis-Menten constant is defined as the substrate concentration (expressedin moles/l) to produce half-maximum velocity in an enzyme catalysed reaction. lt indicatesthat half of the enzyme molecules(i.e. 50%) are bound with the substratemolecules when the substrateconcentrationequalsthe K. v alue .

/

The Lineweaver-Burk plot is shown in Fig.6.3. lt is much easier to calculate the K. from the intercept on x-axis which is -(l/Km). Further,the double reciprocal plot is useful in understandingthe effect of various inhibitions (discussedlater). Enzyme reactions with two or more substrates: The above discussionis based on the presumption of a single substrate-enzyme reaction. In fact, a majority of the enzymecatalvsed reactions involve two or more substrates. Even in case of multisubstrate

K. value is a constant and a characteristic feature of a given enzyme (comparable to a t humb i m p re s s i o n o r s i g n a tu r e). l t i s a represe;;rtative for measuringthe strengthof ES complex. A low K^ value indicates a strong affinity between enzyme and substrate, whereas a high K. value reflects a weak affinity between them. For majority of enzymes,the K. values are in the range of 10-s to 10-2 moles. lt may however,be noted that K. is not dependenton the concentrationof enzvme.

1

_1

Lineweaver-Burkdouble reciprocal plot : For the determination of K, value, the substrate saturation curve (Fig.5.2) is not very accurate

_ \

tration l--l-- | givesa straightline. Here the slope \tsl /is K./y'.u* and whose y intercept is 1/y'."*.

Km

tsl

Ftg,6.3 : Lineweaver-Burk doublereciprocalplot,

90

B IOC H E MIS TRY

enzymes, despite the complex mathematical expressions, the fundamentalprinciplesconform to Michaelis-MentenKinetics. 3. Effect

of temperature

Velocityof an enzyme reactionincreaseswith increasein temperatureup to a maximum and then declines. A bell-shaped curve is usually observed (Fig"6.a). Ternperaturecoefficient or Qto is defined as increase in enzyme velocity when the temperatureis increasedby 10"C. For a majority af enzymes, Qlo is 2 between 0"C and 40oC. lncrease in temperature results in higher activation energy of the molecules and more moiecuiar (enzyme and substrate)collision and interaction for the reaction to oroceed faster.

I o q)

E N

UI

The optimum temperature for most of the It is worth noting here that the enzymeshave enzymes is between 40'C-45'C. However, a few been assignedoptimal temperatures basedon the enzymes (e.g. venom phosphokinases,muscle laboratory work. These temperatures,however, adenylatekinase)are activeeven at 100'C. Some may have less relevance and biological plant enzymeslike ureasehave optimum activity significancein the living system. around 60'C. This may be due to very stable structureand conformationof these enzymes. 4. Effect of pH In general,when the enzymesare exposedto lncrease in the hydrogen ion concentration a temperature above 50"C, denaturation leading (pH) considerablyinfluencesthe enzymeactivity to derangementin the native (tertiary)structure a bell-shaped curve is normally obtained and of the protein and active site are seen.Majority (Fig.6.5). Each enzyme has an optimum pH at of the enzymes become inactive at higher w hi ch the vel oci ty i s maxi mum. B el ow and temperature(above 70'C). above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive.

I

Most of the enzymes of higher organisms show optimum activity around neutral pl-l (6-8). Thereare, however,many exceptionslike pepsin (1-2), aci d phosphatase(4-5) and al kaline phosphatase(10-X 1).E nzyrnesfrom fungi and plants are most active in acidic pH (a-6).

o o) o

E N

Hydrogen ions influencethe enzyme activity by al teri ngthe i oni c chargeson the ami no acids (particularly at the active site), substrate,ES complex etc.

ttl

20 30 40 50 60 ("C) Temperature Fig" 6.4 : Ef'fect of Iempenture on enzyme velocity.

5, Effect

of product

concentration

The accumulation of generally decreases the

reaction products enzyme velocity.

Chapter 6 : ENZYMES

For certainenzymes,the productscombine with the active site of enzyme and form a loose c om plex a n d , th u s , i n h i b i t th e e n z y me acti vi ty. ln the living system,this type of inhibition is generally prevented by a quick removal of productsformed. The end product inhibition by feedback mechanismis discussedlater. 6" Effect

Active site

of activators

Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, zn2+, ca2+, co2*, cu2+, Na+, K+ etc" for their optimum aciivity" Rarely,anionsare also needed for enzyme activity e.g. chloride ion (C11 for amylase. Metals function as activators of enz y m ev e l o c i tyth ro u g hv a ri o u sme c h ani smscombining with the substrate, formation of ES-metalcomplex, direct participation in the reaction and bringing a conformationalchange in t he en z y me .

Fig. 6.6 : A diagrammatic representation of an enzyme with active site.

L

Effect

of light and raEliation

Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivatescertain enzyrnes due to the formation of peroxides.e.g. UV rays i nhi bi t sal i varyamyl aseacti vi ty.

Two categoriesof enzymes requiring metals fbr their activity are distinguished . Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchangedeasily with other ions e.g. ATPase(Mg2* and Ca2*)

Enzymes are big in size compared to substrates which are relativelysmaller.Evidently, a smal l porti on of the huge enzyme mol ecul ei s di rectl y i nvol ved i n the substratebi ndi ng and caialysis(Fig.6,6).

Enolase(Mg2*)

The active site (or active centre) of an represents as the small region at wkich enzynte hold " Metalloenzymes : These enzymes tke suhstrate(s) binds and participates in the the metals rather tightly which are not aatalysis. r eadily e x c h a n g e d . e .g .. a l c o h o l d ehydrogenas e ,c a rb o n i c a n h y d ra s e ,a l k a l ine phosphatase, carboxypeptidase and aldolase Salient features of active site c ont ai n z i n c . 1 . The existenceof active site is due to the Phenol oxidase (copper); Pyruvateoxidase (manganese); X ant hi n eo x i d a s e(m o l y b d e n u m ); Cytochromeoxidase (iron and copper). 7. Effect

of time

Under i d e a l a n d o p ti m a l c o n d i ti o n s(l i ke pH , iemperature etc.), the time required for an enzyme reactionis less.Variationsin the time of the reaction are generally related to the alterationsin pH and temperature.

tertiary structure of protein resulting in threedi mensi onalnati ve conformati on. 2. The active site is made up of amino acids (known as catalytic residues)which are far fronr each other i n the l i nearsequenceof ami no aci ds (primary structLrreof protein). For instance,the enzyme lysozyme has 129 amino acids. The activesite is formed by the contributionof amino aci d resi duesnumbered35, 52, 62, 63 and 101. 3. Active sites are regarded as ctrefts or crevicesor pocketsoccupying a small region in a bi g enzyme mol ecul e.

B IOC H E MIS TFIY

92 4. The active site is not rigid in structureand shape. lt is rather flexible to promote the specific substratebinding. 5. Cenerally, the active site possessesa substrate binding sife and a catalytic site. The latter is for the catalysisof the specific reaction. 6. The coenzymes or cofactors on which some enzymesdepend are presentas a part of the catalytic site.

Enzyme-inhibitor complex

7. The substrate(s) binds at the active site by weak noncovalentbonds. 8. E n z y m e sa re s p e c i fi ci n th e i r fu ncti ondue to the existenceof active sites. 9. T h e c o mmo n l y fo u n d a m i n o a ci ds at the active sites are serine, aspartate, histidine, cysteine,lysine,arginine,glutamate,tyrosineetc. Among these amino acids, serine is the most frequentlyfound.

C= Substrate Active site

Non-competitive inhibitor Enzyme-inhibitor comprex

Fig. 6.7 : A diagrammatic representation of (A) Competitive and (B) Non-competitive inhibition.

10. The substratelslbinds the enzyme (E) at the active siteto form enzyme-substrate complex l. Competitive inhibition : The inhibitor (l) (ES).The product(P) is releasedafterthe catalysis which closely resemblesthe real substrate(S) is and t he e n z y m e i s a v a i l a b l efo r re u s e . regarded as a substrateanalogue. The inhibitor competeswith substrateand binds at the active site of the enzyme but does not undergo any catalysis.As long as the competitive inhibitor holds the active site,the enzyme is not available for the substrateto bind. During the reaction,ES and El complexesare formed as shown below Enzyme inhibitor is defined as a substance ES--+E +P whic h bi n d s w i th th e e n z y m e a n d b ri n gsabout a decreasein catalyrtc activity of that enzyme. EI T he inh i b i to r m a y b e o rg a n i c o r i n organi c i n nature. There are three broad categories of The relativeconcentrationof the substrateano enz y m e i n h i b i ti o n inhibitor and their respectiveaffinity with the

il:+:1-i.'

P

1 . Re v e rs i b l ei n h i b i ti o n . 2. I r r e v e rs i b l ei n h i b i ti o n . 3. A ll o s te ri ci n h i b i ti o n . ,' 1 T he i n h i b i i o r b i n d s n o n -c o v a l e ntl y w i th enz y m e a n d th e e n z y me i n h i b i ti o n can be reversed if the inhibitor is removed. The r ev er s ib l ei n h i b i ti o n i s fu rth e r s u b -d i v i dedi nto l. Rev e rs i b l e

i n h i b i ti o n

l. Competitiveinhibition (Fig.6.7A) ll. Non-competitiveinhibition (Fig.6.7B)

enzyme determinesthe degree of competitive i nhi bi ti on.The i nhi bi ti oncoul d be overcomebv a high substrateconcentration.ln competitive inhibition, the K- value increaseswhereas V-u* remains unchanged (Fig.6.A. The enzyme succinatedehydrogenase(SDH) i s a cl assi calexampl eof competi ti vei nhi bi ti on with succinic acid as its substrate. The compounds,namel y,mal oni c aci d, gl utari caci d and oxal i c aci d, have structuralsi mi l ari tyw i th succinicacid and competewith the substratefor bi ndi ng at the acti ve si te of S D H .

93

chaprer 6 : ENZYMES

cooH cH2cooH cH2cooH Succinic acid

I CHr t-

cooH

Malonic acid

enzyme surface. Thi s bi ndi ng i mpai rs the enzyme function. The inhibitor has no structural resemblancewith the substrate.However, there usuallyexistsa strongaffinity for the inhibitor to bind at the secondsite. In fact, the inhibitor does binding. not interferewith the enzyme-substrate But the catalysisis prevented,possiblydue to a distortion in the enzyme conformation.

Methanol is toxic to the body when it is converted to formaldehyde by the enzyme alcohol dehydrogenase (ADH). Ethanol can compete with methanolfor ADH. Thus, ethanol The i nhi bi tor general l y bi nds w i th the can be used in the treatment of methanol enzyme as well as the ES complex. The overall por s o n rn S . rel ati on i n non-competi ti ve i nhi bi ti on is Some more examples of the enzymes with representedbelow substratesand competitive inhibitors(of clinical ) gi ven i n and p h a rma c o l o g i c asl i g n i fi c a n c eare E+sirES-*E+P Table 6.2. ++ I I Antimetabolites : These are the chemical compounds that block the metabolic reactions E I+ S E IS by t h e i r i n h i b i to ry ,a c ti o n ' o n enzymes. Antimetabolitesare usually structuralanalogues For non-competitiveinhibition, the K^ value of substratesand thus are competitive inhibitors (Table 6.2). They are in use for cancer therapy, is unchanged while V^^* is lowered (Fig.5.9). Bout etc. The term antivitamins is used for the Heavy metal ions (Ag+, Pb2+,Hg2+ etc.) can ant im e ta b o l i te sw h i c h b l o c k th e bi ochemi cal non-competitively inhibit the enzymes by ac t io n s o f v i ta m i n s c a u s i n g d e fi ci enci es,e.g. binding with cysteinyl sulfhydryl groups. The s ulph o n i l a mi d ed, i c u ma ro l . general reactionfor Hg2+ is shown below. ll. Non-competitiveinhibition : The inhibitor E -S H + H gt* i ^ E -S . . .H g2++ H + binds at a site other than the active site on the

.lf

tf

I f\/

2'mu

+ II Km

Km,

11 Km

(A)

1

Km'

tsl (B)

Fig.6.8 : Effect of competitive inhibitor (i) on enzyme velocity. (A) Velocity (v) versus substrate (S) plot. (B) Lineweaver-Burk ptot (Red lines with inhibitor; campetitive inhibitor increases K^, unalters V^o).

B IOC H E MIS TR Y

94

Enzyme

Substrate

lnhibitor(s) Allopurinol

Significance of inhibitor(s) of goutto reduce excess Usedin thecontrol production of uricacidfromhypoxanthine.

Xanthine oxidase

Hypoxanthine xanthine

Monoamine oxidase

levels. forelevating catecholamine Ephedrine, Useful Catecholamines (epinephdne, norepinephrine) amphetamine

Dihvdrofolate reductaseDihvdrofolic acid

in thetreatment of leukemia and Aminopterin, Employed amethopterin, othercancers. methotrexate

Acetylcholine esterase Acetylcholine

relaxation, in lormuscle cholineUsedin surgery Succinyl patients. anaesthetised

Dihydropteroate synthase

Paraaminobenzoic acid (PABA)

synthesis of folicacid. bacterial Sulfonilamide Prevents

Vitamin K epoxide

K Vitamin

Dicumarol

Actsas an anticoagulant.

Lovastatin, compactin

biosynthesis Inhibit cholesterol

reductase HMGCoAreductase HMGCoA

Heavv metals also lead to the formation of i rreversi bl e.These i nhi bi tors are usual l v toxr c covalent bonds with carboxyl groups and poisonoussubstances. histidine,often resultingin irreversibleinhibition. lodoacetateis an irreversibleinhibitor of the enzymes like papain and glyceraldehyde inhibition 2. lrreversible lodoacetate combines 3-phosphate dehydrogenase. (-SH) groups at the activesite of The inhibitors bind covalently with the with sulfhydryl enzymes and inactivate them, which is theseenzvmesand makesthem inactive.

7

1/

2

vm ax\

T

Ghapter 6 : ENZYMES

Diisopropyl fluorophosphafe (DFP) is a nerve uridylatewhich inhibitsthe enzyme thymidylate gas developed by the Cermans during Second synthase,and thus nucleotidesynthesis. World War. DFP irreversiblybinds with enzymes containing serine at the active site, e.g. serine 3. A l l osteri c i nhi bi ti on proteases, acetylcholine esterase. The detailsof this type of inhibition are given Many organophosphorus insecticides like under allosteric regulation as a part of the m elat h i o na re to x i c to a n i m a l s(i n c l udi ngman) regulation of enzyme activity in the living as they block the activity of acetylcholine svstem. esterase (essential for nerve conduction), resultingin paralysisof vital body functions. Disulfiram (Antabuse@)is a drug used in the treatment of alcoholism. lt irreversiblvinhibits the enzyme aldehyde dehydrogenase.Alcohol addicts, when treated with disulfiram become sick due to the accumulation of acetaldehyde, leadingto alcohol avoidance.(Nofe ; Alcohol is metabolized by two enzymes. lt is first acted upon by alcohol dehydrogenase to yield acetaldehyde.The enzyme aldehyde dehydrogenaseconvertsacetaldehydeto acetic acid.) T he p e n i c i l l i n a n ti b i o ti c sa c t a s irreversi bl e inhibitors of serine- containing enzymes, and block the bacterialcell wall svnthesis. lrreversibleinhibitors are frequently used to identify amino acid residuesat the active site of the enzymes, and also to understand the mechanismof enzyme action. S nic ide

i n h i b i ti o n

Enzymesare highly specific in their action when comparedwith the chemicalcatalysts.The occurrence of thousands of enzymes in the biological system might be due to the specific nature of enzymes. Three types of enzyme specificityare well-recognised 1. Stereospecificity. 2. Reactionspecificity, 3. Substratespecificity, Specificity is a characteristic property of the active site. 1. Stereospecificity or optical specificity : Stereoisomersare the comoounds which have the same mol ecul arformul a, but di ffer i n the ir structuralconfiguration.

The enzymes act only on one isomer and, S uic i d e i n h i b i ti o n i s a s p e c i a l i zedform of therefore, exhibit stereospecificity. ir r ev ers i b l ei n h i b i ti o n .l n th i s c a s e ,the ori gi nal (the inhibitor structural analogue/competitive e.g. L-amino acid oxidase and D-amino acid inhibitor) is convertedto a more potent form by oxidase act on L- and D-amino acids the sameenzyme that ought to be inhibited.The respectively. so formed inhibitor binds irreversiblywith the Hexokinase acts on D-hexoses; enzyme. This is in contrast to the original Glucokinaseon D-glucose; inhibit o rw h i c h b i n d s re v e rs i b l y . Amylase acts on a-glycosidic linkages; A g o o d e x a m p l e o f s u i c i d e i n hi bi ti on i s allopurinol (used in the treatment of gout, Refer Cellulasecleavesp-glycosidicbonds. Chapter lV. Allopurinol, an inhibitor of Stereospecificity is explained by considering xanthineoxidase,getsconvertedto alloxanthine, three distinct regions of substrate molecule a more effective inhibitor of this enzyme. specifically binding with three complementary The use of certain purine and pyrimidine regionson the surfaceof the enzyme (Fig.5.l0). analog u e si n c a n c e r th e ra p y i s a l s o expl ai ned The class of enzymes belonging to isomerases on t he b a s i s s u i c i d e i n h i b i ti o n . F o r i nstance, do not exhihit stereospecificity, since they are S-fluorouracil gets converted to fluorodeoxy- specializedin the interconversionof isomers.

96

B IOC H E MIS TF| Y glycosidasesacting on glycosidic bonds of carbohydrates,lipasescleavingesterbonds of l i pi ds etc. . Broad specificity : Some enzymes act on closely relatedsubstrateswhich is commonly known as broad substrate specificity, e.g. hexokinaseactson glucose,fructose/mannose and glucosamine and not on galactose. lt i s possi bl e that some structural si mi l arit y among the first four compounds makes them a common substratefor the enzyme hexokinase.

The protein part of the enzyme,on its own, is not always adequateto bring about the catalytic activity. Many enzymes require certain nonprotein small additional factors, collectively referred to as cofactors for catalysis. 2. Reaction specificity : The same substrate The cofactors may be organic or inorganic in can undergo different types of reactions, each nature. catalysed by a separate enzyme and this is referredto as reaction specificity. An amino acid The non-protein, organic, Iow molecular can undergo transamination,oxidative deami- weight and dialysable substance associated with nation, decarboxylation,racemizationetc. The enzyme function is known as coenzyme. enzymes however, are different for each of these The functional enzyme is referred to as reactions (For details, refer Chapter |fl. holoenzyme which is made up of a protein part part and a non-protein 3. Substrate specificity : The substrate (apoenzyme) specificityvariesfrom enzymeto enzyme. lt may (coenzyme); The term prosthetic group is used when a non-protein moiety is tightly bound to be either absolute,relativeor broad. the enzyme which is not easily separableby . Absolute substrate specificity : Certain dialysis. The term activator is referred to the enzymes act only on one substrate e.g. inorganic cofactor (like Ca2+,Mg2+, Mn2+ etc.; glucokinaseacts on glucoseto give glucose6- necessaryto enhance enzyme activity. lt may, phosphate,urease cleaves urea to ammonia however, be noted that some authors make no and carbon dioxide. distinction between the terms cofactor, . Relative substrate specificity : Some enzymes coenzyme and prostheticgroup and use them interchangeably. act on structurallyrelatedsubstances. This, in turn, may be dependenton the specificgroup Coenzymes are second substrates : or a bond present.The action of trypsin is Coenzymes are often regarded as the second a good example for group specificity (Refer substrates or co-substrafes, since thev have Fig.8.7). Trypsin hydrolyses peptide linkage affinitywith the enzyme comparablewith that of inv olvi n g a rg i n i n e o r l y s i n e . C h y motrypsi n the substrates.Coenzymes undergo alterations cleaves peptide bonds attached to aromatic during the enzymatic reactions,which are later amino acids (phenylalanine, tyrosine and regenerated.This is in contrast to the substrate tryptophan). Examples of hond specificity- which is convertedto the product. Fig. 6.10 : Diagrammatic representation of stercospecificity (a', b', Cl-three point attachment of

.l

97

Chapter 6: ENZYMES

Coenzyme (abhreviation)

Derived from vitamin

(TPP) pyrophosphate Thiamine (FMN) Flavin mononucleotide (FAD) Flavin adenine dinucleolide

Thiamine

Aldehyde or keto

Transketolase

Riboflavin

Hydrogen andelectron

L - Aminoacidoxidase

?ieeril

Atom or group transferred

Dependentenzyme (example)

Riboflavin

D - Aminoacidoxidase

Niacin

Lactate dehydrogenase

Lipoicacid Pyridoxine

Aminoor keto

Glucose Sphosphate dehydrogenase Pyruvate dehydrogenase complex Alanine transaminase

Pantothenic acid

Acyl

Thiokinase

Folicacid

Onecarbon (formyl, methenyl etc.)

Formyl transferase

CO,

Pyruvate carborylase

giell.

i1 _ _ 9o$t1ry c9o!a1 gqogn gsv_l !9!n!c9{a1i1 o_e

Methyl/isomerisation Methylmalonyl CoAmutase

* Detakforeachcoenzyne 7on vitanins aregivenin Chapter

Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen,aldehyde, keto, amino, acyl, methyl, carbon dioxide etc. Coenzymesplay a decisive r ole in e n z y me fu n c ti o n .

Table. 6.3, a summary of the vitamin related coenzymesw i th thei r functi onsi s gi ven. Non-vitamin coenzymes: Not all coenzymes are vitamin derivatives.There are some other organic substances, which have no relationwith vitamins but function as coenzymes.They rnay be consi deredas non-vi tami ncoenzymese.g . A TP , C D P , U D P etc. The i mportantnon-vi tamin coenzymesalong with their functions are given in Tahle 6.4.

Coenzymesfrom B-complex vitamins : Most of the coenzymes are the derivativesof water soluble B-complex vitamins. In fact, the biochemicalfunctionsof B-complexvitaminsare exertedthrough their respectivecoenzymes.The Nucleotide coenzymes : Some of the chapteron vitaminsgivesthe detailsof structure and function of the coenzymes(Chapter V. ln coenzymespossessnitrogenousbase,sugar and

Coenzyme

Adenosine triphosphate

Abbreviation

ATP

Biochemical functions

phosphate, Donates monophosphate adenosine andadenosine (AMP)moieties.

diphosphate Cytidine

CDP

Uridine diphosphate

UDP

SAM S- Adenosylmethionine (active methionine) phosphosulfate Phosphoadenosine (active sulfate)

inphospholipid Required synthesis ascarrier ofcholine and ethanolamine. (glucose, galactose), required lor Carrier of monosaccharides synthesis. $ycogen groupinbiosynthetic Donates methyl reactions. Donates sulfate forthesynthesis ofmucopolysaccharides.

9B

BIOCHEMISTF|Y

phosphate. Such coenzymes are, therefore, regarded as nucleotides e.g. NAD+, NADP+, FMN, FAD, coenzyme A, UDPC etc. Coenzymesdo not decide enzyme specificity : A particularcoenzymemay participatein catalytic reactions along with different enzymes. For instance, NAD+ acts as a coenzyme for lactate dehydrogenaseand alcohol dehydrogenase.ln both the enzymaticreactions,NAD+ is involved in hydrogen transfer. The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzvme.

Catalysisis the prime function of enzymes. The nature of catalysis taking place in the biologic a l s y s te m i s s i mi l a r to th a t of nonbiologicalcatalysis.For any chemical reactionto occur, the reactantshave to be in an activated stateor transitionstate. Enzymes lower activation energy : The energy required by the reactantsto undergothe reaction is known as activation energy. The reactants when heated attain the activation energy. The catalyst (or the enzyme in the biological system)reducesthe activationenergy and this causes the reaction to proceed at a lower temperature.Enzymes do not alter the equilibr i u m c o n s ta n ts ,th e y o n l y e n hance the velocity of the reaction. The role of catalystor enzyme is comparable wit h a t u n n e l m a d e i n a mo u n ta i nto reducethe barrier as illustrated in Fig.6.l1. The enzyme lowers energy barrier of reactants, thereby making the reaction go faster. The enzymes reduce the activationenergy of the reactantsin such a way that all the biological systemsoccur at body temperature(below 40"C).

Enzyme.substrate complex formation

+ I

I

II

lLl

B

Fig. 6.11 : Effect of enzyme on activation energy af a reaction (A is the substrate and I is the

E + S$

E S ---+ E + P

A few theorieshave been put forth to explain mechanism of enzyme-substrate complex formation. Lock and key model or Fischer's template

theory

This theory was proposed by a Cerman bi ochemi st,E mi l Fi scher.Thi s i s i n fact the very first model proposed to explain an enzyme cataiysedreaction. According to this model, the structure or conformationof the enzymeis rigid.The substrate fits to the binding site (now active site)just as a key fits into the proper lock or a hand into the properglove.Thusthe activesiteof an enzymeis a rigid and pre-shapedtemplate where only a specificsubstratecan bind. This model does not give any scopefor the flexible natureof enzymes, hencethe model totallyfailsto explainmany facts of enzymaticreactions,the most importantbeing the effect of allostericmodulators.

Induced fit theory The prime requisite for enzyme catalysis is model or Koshland's that the substrate(S) must combine with the Koshland, in 1958, proposed a more enzyme (E) at the active site to form enzymesubstratecomplex (ES)which ultimately results acceptable and realistic model for enzymesubstratecomplex formation.As per this model, in the product formation (P).

Ghapter 6 : ENZYMES

99 MECHANISIII

OF ENZYME GATATYSIS

The formation of an enzyme-substrate compl ex (E S )i s very cruci al for the catal ysi sto occur, and for the product formation. lt is estimated that an enzyme catalysed reaction proceeds106 to 1012 ti mes fasterthan a noncatalysedreaction.The enhancementin the rate of the reaction is mainly due to four processes: 1. A ci d-basecatal ysi s; 2. Substratestrain; 3. C oval entcatal ysi s; 4. Entropy effects.

Fig. 6.12 : Mechanism of enzyme-substrate (ES) complex formdtion (A) Lock and key model (B) Induced fit theory G) Substrate strain theory.

the active site is not rigid and pre-shaped.The essentialfeaturesof the substratebinding site are presentat the nascentactive site.The interaction of the substratewith the enzyme inducesa fit or a conformationchangein the enzyme,resultingin the formation of a strong substratebinding site. Further,due to inducedfit, the appropriateamino acids of the enzymeare repositionedto form the activesiteand bring aboutthe catalysis(Fig.6.12).

t

1 . Acid-base catalysis : Role of acids and basesis quite important in enzymology.At the physi ol ogi calpH , hi sti di nei s the most i mportant amino acid, the protonated form of which functi ons as an aci d and i ts correspondi ng conjugateas a base. The other acids are -OH group of tyrosine, -SH group of cysteine,and e-aminogroup of lysine.The conjugatesof these acids and carboxyl ions (COO-) function as bases. R i bonucl easew hi ch cl eavesphosphodi ester bonds i n a pyri mi di nel oci i n R N A i s a cl assi ca l example of the role of acid and base in the catal ysi s.

2. Substratestrain : lnduction of a strain on the substratefor ESformationis discussedabove. During the courseof strain induction,the energy level of the substrateis raised, leading to a Induced fit model has sufficientexperimental transitionstate. evidence from the X-ray diffraction studies. K os hlan d ' smo d e l a l s o e x p l a i n s th e a cti on of The mechanismof lysozyme (an enzyme of allostericmodulatorsand competitive inhibition tears,that cleavesp-1,4 glycosidicbonds)action on enzymes. is believed to be due to a combination of substratestrain and acid-basecatalysis. Substrate strain theory 3. Covalent catalysis : In the covalent In this model, the substrateis straineddue to catalysis,the negatively charged (nucleophilic) the inducedconformationchangein the enzyme. or posi ti vel y charged (el ectrophi l i c)group i s It is also possiblethat when a substratebinds to present at the active site of the enzyme. This the preformedactive site, the enzyme inducesa group attacks the substrate that results in the strain to the substrate.The strained substrate covalent binding of the substrateto the enzyme. leads to the formation of product. ln the serine proteases(so named due to the ln fact, a combination of the induced fit presence of serine at active site), covalent model with the substratestrain is consideredto catalysisalong with acid-base catalysisoccur, e.g. chymotrypsin,trypsin, thrombin etc. be operativein the enzymatic action.

100

B IOC H E MIS TFIY

4. Entropy effect : Entropy is a term used in t her m o d y n a m i c sl.t i s d e fi n e d a s th e extent of dis or de ri n a s y s te m.T h e e n z y m e sb ri ng about a decreasein the entropy of the reactants.This enables the reactants to come closer to the enzyme and thus increasethe rate of reaction.

3. Endothermic (endergonic) reactions : E nergy i s consumed i n these reacti ons e.g. glucokinase Glucose6-phosphate+ ADP Clucose+ ATP------+

I n t h e a c tu a l c a ta l y s i so f th e e n z y mes,more - acid-basecatalysis, than one of the processes substratestrain, covalent catalysisand entropy ar e s im u l ta n e o u s loy p e ra ti v e .T h i s w i l l hel p the In biological system, regulation of enzyme to attain a transitionstate leadingto substrate(s) activities occurs at different stages in one or the formation of products. more of the fol l ow i ng w ays to achi evecel l ul a r economy. T } I E RM O D YN AMIC S OF 1. A l l osteri cregul ati on ENZYMATIC REACTIOITS 2. Activation of latent enzymes The enzyme catalysed reactions may be 3. Compartmentation of metabolic broadly grouped into three types based on pathways (e n e rg y ) t her m od y n a m i c c o n s i d e ra ti o ns. 4. Control of enzyme synthesis 1 . lsothermic reactions : The energy 5. E nzymedegradati on exchange between reactants and products is g l y c o g e n p h o s p h o ry l a se negligib l ee .g . 6. l soenzymes Cly co g e n+ Pi --+

C l u c o s e 1 -p h osphate

2. Exothermic(exergonic)reactions: Energy is liber a te di n th e s e re a c ti o n se .s . u re ase Urea --+

NH3 + CO2 + energy

i.

& i l ssteri e regul ati on ;rnsl al l o* teri e i nhl hi ti on

Someof the enzymespossessadditionalsites, known as allosteric sites (Greek : allo-other),

EIOMEDICAL/ CLINICAL CONCEPTS

s€ The existenceot' lit'e is unimaginablewithout the presenceol enzymes-the biocotalysts. se Majoritg of the coenzymes (TPP, NAD+, FAD, CoA) are deriued from B-complex uitamins in which t'orm the latter exert their biochemicalJunctions. 0s Competitiue inhibitors of certain enzymesare ot' great biological signit'icance.Allopurinol, emploged in the treatment of gout, inhibits xanthine oxidase to reduce the formation ot' uric acid. The other competitiue inhibitors include aminopterin used in the treatment of cancers,sult'anilamideas antiboctericidalogent and dicumarol as on anticoagulant.. P- The nerue gas(diisopropyl t'luorophosphate), t'irst developed by Germans during Second World Wa4 tnhibits acetylcholine esterqse,the enzyme essentialfor nerve conduction and paralyses the uital body functions. Many organophosphorus insecticides (e.9. melathion) also block the actiuity of acetylcholine esterase. te Penicillin antibiotics irreuersiblyinhibit serine contqining enzymesof bacterial cell wall sunthesis.

101

Ghapter 6: ENZYMES

change in the active site of the enzyme, leading to the inhibition or activation of the catalytic activity (Fig.6.l3). In the concerted model, allostericenzvmes exist in two conformational states-the T (tenseor taut) and the R (relaxed). The T and R statesare i n equi l i bri um. Allostericactivator(or)substrate Allosteric inhibitor Allosteric inhibitors favour T state whereas activators and substratesfavour R state. The substratecan bind only with the R form of the enzyme.The concentrationof enzvme molecule in the R state increasesas more substrateis added, therefore the binding of the substrateto besidesthe active site. Such enzymesare known the allostericenzyme is said to be cooperative. as allosteric enzymes. The allosteric sites are Allostericenzymesgive a sigmoidalcurve (instead unique p l a c e so n th e e n z y me mo l e c ul e. of hyperbola) when the velocity (v) versus Allosteric effectors : Certain substances substrate(S) concentrationare plotted (Fig.5.14). referred to as allosteric modulators (effectorsor The term homotropic effect is used if the modifiers)bind at the allostericsite and regulate substrate influences the substrate binding the enzyme activity. The enzyme activity is through allosteric mechanism, their effect is increasedwhen a positive (+) allostericeffector always positive. Heterotropic effecf is used binds at the allosteric site known as activator when an allostericmodulatoreffectsthe binding site. On the other hand, a negative(-) allosteric of substrate to the enzyme. Heterotropic effector binds at the allosteric site called interactions are either positive or negative. inhibit ors i te a n d i n h i b i tsth e e n z y m e acti vi ty. Selected examples of allosteric enzymes Classesof allosteric enzymes : Enzymesthat responsible for rapid control of biochemical are regulated by allosteric mechanism are pathways are given in Table 6.5. referred to as allosteric enzymes. They are divided into two classesbasedon the influence of allostericeffectoron K, and V.r*. Fig. 6.13 : Diagrammaticrepresentationof an allastericenzyme(A) T-farm;(B] P-foffn; (C] Effect of aftag"eie ffitW1.(D) Etleotaf altoqtide:ac,.4vaiw, . -

l"

. K-class of allosteric enzymes, the effector changes the K. and not the V."". Double reciprocal plots, similar to competitive inhibiti o n a re o b ta i n e d e .g . phosphofructokinase. . V-classof allosteric enzymes,the effectoralters the V.r* and not the Kr. Double reciprocal plots resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase. Conformational changes in allosteric enzymes : Most of the allosteric enzymes are oligom eri c i n n a tu re . T h e s u b u n i ts may be identical or different. The non-covalent reversiblebinding of the effectormolecule at the allosteric site brings about a conformational

Hyperbolic

1 o o E N E

lrJ

Substrate ------+ concentration Fig. 6.14 : Effect of substrate concentration an allos-

ElIOCHEMISTFIY

l02

Allosteric Activator

Enzyme

Metabolicpathway

Inhibitor

Hexokinase

Glycolysis

6-phosphate Glucose

Phosphofructokinase lsocitrate dehydrogenase Pyruvate carborylase 1,6 - bisphosphatase Fructose phosphate I synthetase Carbamoyl Tryptophan oxygenase Acetyl CoAcarboxylase

Glycolysis Krebs cycle Gluconeogenesis Gluconeogenesis Ureacycle Tryptophan metabolism Fattyacidsynthesis

ATP ATP

Feedback

regillation

The process ol inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation. Look at the series of reactionsgiven below A

el

>B

sp

,9 € 9a )C rD rE

A is the initial substrate,B, C, and D are the intermediatesand E is the end product, in a pathway catalysed by four different enzymes (e1, e-2,e3, e4).The very first step (A -+ B by the enzyme e1) is the most effective for regulating the pathway, by the final end product E. This type of control is often called negative feedback regulation since increasedlevels of end product will result in its (er) decreasedsynthesis.This is a real cellular economy to save the cell from the wasteful expenditure of synthesizing a compound which is alreadyavailablewithin the c ell. Feedbackinhibition or end product inhibition is a specialised type of allosteric inhibition necessaryto control metabolic pathways for efficient cellular function.

ADP AMP, NADADP, AcetylCoA

AMP

Palmitale

N- Acetylglutamate L- Tryptophan lsocitrale

Carbamoylphosphate+ Aspartate

Feedback control

Carbamoyl aspartate + Pi I Y Cytidinetriphosphate(CTP)

Carbamoyl phosphateundergoesa sequence of reactionsfor synthesisof the end product, CTP. When CTP accumulates,it allosterically inhibitsthe enzyme aspartatetranscarbamoylase by a feedback mechanism. Feedback regulation or feedback inhibition? Sometimesa distinction is made between these two usages.Feedback regulation representsa phenomenonwhile feedbackinhibition involves the mechanism of regulation. Thus, in a true sense,they are not synonymous.For instance, dietary cholesteroldecreaseshepaticcholesterol biosynthesisthrough feedback regulation.This does not i nvol ve feedback i nhi bi ti on, si nce dietary cholesteroldoes not directly inhibit the regulatory enzyme HMG CoA reductase. However, the activity of gene encoding this enzyme is reduced (repression)by cholesterol.

Aspartate transcarbamoylase (ATCase) is of latent enzymes a good example of an allosteric enzyme 2. Activation Latent enzymes,as such, are inactive.Some inhibited by a feedback mechanism. ATCase catalysesthe very first reaction in pyrimidine enzymes are synthesized as Proenzymes or zymogens which undergo irreversiblecovalent biosynthesis.

103

Ghapter 6 : ENZYMES

There are some enzymeswhich are active in activation by the breakdown of one or more peptidebonds.For instance,proenzymes -namely dephosphorylatedstate and become inactive pepsinogenand plasminogen, when phosphorylatede.g. glycogen synthase, chymotrypsinogen, are respectively convertedto the active enzymes acetyl CoA carboxylase. chymotrypsin,pepsin and plasmin. A few enzymesare activeonly with sulfhydryl Certain enzvmes exist in the active and (-SH) groups, €.8. succinate dehydrogenase, inactive forms which are interconvertible, urease.Substanceslike glutathionebring about depending on the needs of the body. The the stabilityof these enzymes. interconversion is brought about by the E -S H + E -S H reversible covalent modifications, namely E-S-S-E Reduced phosphorylation and dephosphorylation, and Oxidised GS-SG inactive 2G-SH active oxidation and reduction of disulfide bonds. Clycogen phosphorylaseis a muscle enzyme that breaks dow'n glycogen to provide energy. This enzyme is a homodimer (two identical subunits)and existsin two interconvertible forms. Phosphorylase b (dephosphoenzyme)is inactive which is convertedby phosphorylationof serine residuesto active form phosphorylasea. The inactiveenzyme phosphorylase b is producedon dephosphorylationas illustratedbelow.

E

Phosphorylaseb (inactive). .,-_-\ '

P

.-....-.

phosphatase

/'

E

2Pi

Organelle

______----l

P Phosphorylasea z (active) _./

3. Gompartnnentation Thereare certainsubstancesin the body (e.g., fatty acids,glycogen)which are synthesizedand also degraded.Thereis no point for simultaneous occurrenceof both the pathways.Cenerally,the synthetic (anabolic) and hreakdown (catabolic) pathways are operative in different cellular organellesto achieve maximum economy. For instance,enzymes for fatty acid synthesisare found in the cytosol whereas enzymes for fatty acid oxidation are presentin the mitochondria. Dependingon the needsof the body - through the mediationof hormonal and other controlsfatty acids are either synthesizedor oxidized. The intracellularlocation of certain enzymes and metabolic pathways is given in Table 6.6.

Enzym e/metaboIi c pathway

Cytoplasm

peptidases; glycolysis; Aminotransferases; hexose monophosphate shunt; fattyacid purine andpyrimidine catabolism. synthesis;

Mitochondria

Fattyacidoxidation; amino acidoxidation; Krebs cycle; ureasynthesis; electron phosphorylation. transport chainandoxidative

Nucleus

Biosynthesis ofDNAandRNA.

(microsomes) phospholipid Endoplasmic reticulum Protein biosynthesis;triacylglyceroland synthesis; synthesis steroid and P4Eo; esterase. reduction; cytochrome Lysosomes

phosphatases; phospholipases; proteases; Lysozyme; hydrolases; lipases; nucleases.

Golgiapparatus

glucosyF 5'-nucleotidase; Glucose 6-phosphatase; andgalactosyl-transferases.

Peroxisomes

D-amino urateoxidase; acidoxidase; longchainfattyacidoxidation. Catalase;

I

l04 4" Control I I

B IOC H E MIS TR Y

of enzyme

synthesis

Most of the enzymes, particularly the rate limiting ones, are present in very low the amount of the concentration.Nevertheless, enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate Iimiting enzymes have short half-lives. This helps in the efficient regulationof the enzyme levels. There are two types of enzymei-(a) Constitutive enzymes (house-keeping enzymes)-the levels of which are not controlled and remain fairly constant. (b) Adaptive enzymes-their concentrationsincreaseor decreaseas per body needs and are well-regulated.The synthesisof enzymes (proteinsl is regulated by the genes (Refer Chapter 25).

!

{

I

ln general,the key and regulatoryenzymes are most rapidly degraded.lf not needed, they i mmedi atel y di sappear and, as and w hen Though required,they are quickly sysnthesized. not always true, an enzyme with long half-life is usually sluggishin its catalytic activity. 6. lsoenzymes Multiple forms of the same enzyme will also help in the regulationof enzymeactivity, Many of the isoenzymes are tissue-specific.Although isoenzymesof a given enzyme catalysethe same reaction, they differ in K' V.nu* or both. e.g. isoenzvmesof LDH and CPK.

Induction and repression: The term induction is used to represent increased synthesis of enzyme while repressionindicates its decreased Enzymesare never expressedin termsof their synthesis. Induction or repression which (as mg or pg etc.), but are ultimatelydeterminesthe enzyme concentration concentration at the gene level through the mediation of expressedonly as activities. Various methods have been introduced for the estimation of hormonesor other substances. enzyme activities (particularly for the plasma Examplesof enzyme induction : The hormone enzymes). In fact, the activities have been insulin induces the synthesis of glycogen expressedin many ways, like King-Armstrong synthetase, glucokinase, phosphofructokinase units, Somogyi units, Reitman-Frankelunits, and pyruvate kinase. All these enzymes are spectrophotometricunits etc. inv olv e d i n th e u ti l i z a ti o n o f g l ucose. The hormone cortisol inducesthe synthesisof many Katal enzymes e.B. pyruvate carboxylase, tryptophan oxygenaseand tyrosine aminotransferase. In order to mai ntai n uni formi ty i n th e (as units) Examplesof repression: In many instances, expression of enzyme activities substrate can repress the synthesis of enzyme. worldover, the EnzymeCommissionof IUB has Pyruvate carboxylaseis a key enzyme in the suggestedradical changes.A new unit- namely (abbreviatedas kat)-was introduced.One synthesis of glucose from non-carbohydrate katal sourceslike pyruvateand amino acids.lf there is kat denotes the conversion of one mole (mol/sec).Activity may also sufficientglucoseavailable,there is no necessity substrateper second (mkat), microkatals for its synthesis. This is achieved through be expressedas millikatals (pkat) and so on. repression of pyruvate carboxylase hy glucose. 5. Enzyme

degradation

Enzymesare not immortal,since it will create a seriesof problems.There is a lot of variability in the half-livesof individualenzymes.For some, it is in days while for others in hours or in m inut e s ,e .g . L D H a - 5 to 6 d a y s ; L D H I - 8 to 12 hou rs ;a m v l a s e-3 to 5 h o u rs .

International Units (lUf Some workers preferto use standardunits or Sl units (SystemInternational).One Sl unit or InternationalUnit (lU) is defined as the amount of enzyme activity that catalysesthe conversion of one micromol of suhstrate per minute. Sl units and katal are interconvertible.

Chapter 6 : ENZYMES

= 60 pkatal (or) 1 nkatal = 1 .6 7 l U 1lU

Enzyme Laboratory

use of enzyme

units

In the clinical laboratories, however, the units- namelv katal or Sl units-are vet to find a place. Many investigators still use the old units like King-Armstrongunits, Somogyi units etc. while expressingthe enzyme activities. lt is therefore, essential that the units of enzyme activity, along with the normal values, be invariably stated while expressingthe enzymes for comparison.

Ribozymes Ribozymes are a group ol ribonucleic acids that function as biological catalysts,and they are regardedas non-proteinenzymes.

Application

Therapeutic applications Streptokinase/urokinaseToremove blood clots Asparaginase Incancer therapy Paoain Anti-inf lammatory o,-Antitrypsin Totreatemphysema (breathing ditficulty due todistension oflungs) Analytical application reagents(for estimation) Glucose oxidase andoeroxidase Glucose

Urease Cholesterol oxidase Uricase Lipase Luciferase

Urea Cholesterol Uricacid Triacylglycerols Todetect bacterial contamination offoods Alkalinephosphatase/ Intheanalyticaltechnique

lgpp-n9irl eereriq?ee _qL!94

Applications in geneticengineering A lt ma n a n d h i s c o w o rk e rs ,i n 1 983, found Restriction endonucleases Gene lransfer, DNAtinger that ribonucleaseP- an enzyme till then known pnnrng to cleave precursorsof tRNAs to give tRNAsIag DNApolymerase Polymerase chain was functional due to RNA component present reaction in the enzyme and not the protein part of the enzyme. The RNA part isolated from ribonucleaseP exhibiteda true enzyme activityand also obeyed Michaelis-Menten kinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis. RNA moleculesare known to adapt a tertiary structure just as in the case of proteins (i.e. enzymes). The specific conformation of RNA may be responsiblefor its function as biocatalyst. It is believed that ribozymes IRNAs) were functioningas catalystsbeforethe occurrenceof protein enzymesduring evolution.

Industrialapplications

preparation Cheese Production ofhigh fructose syrup Infoodindustry to convert starch toglucose powder Washing

Rennin Glucose isomerase u,-Amylase Proteases

Enzymes as therapeqtic

agents

preparedfrom streptococcus 1. Streptokinase is useful for clearing the blood clots. Streptokinase activatesplasmaplasminogen to plasminwhich, in turn, attacksfibrin to convert into solubleproducts. Plasminogen I

Certain enzymes are useful as therapeutic genetic agents, analytical reagents, in m anip u l a ti o n sa n d fo r i n d u s tri a l a ppl i cati ons (Table 6.V.

Streptokinase J Plasmin

I

Fibrin-l-* (clot)

Solubleproducts

B IOC H E MIS TR Y

106

2. The enzyme asparaginaseis used in the Tumorcellsare dependent treatmentof leukemias. on asparagineof the host's plasma for their the multiplication.By administeringasparaginase, Estimationof enzyme activities in biological host'splasmalevelsof asparagineare drastically (particularly plasma/serum)is of great fluids reduced.This leadsto depressionin the viability cl i ni cal i mportance.E nzymesi n the ci rcul ati on of t um o r c e l l s . are divided into two Eroups- plasma functional and pl asmanon-functi onal . reagents Enzyrmes as analytica! Some enzymes are useful in the clinical laboratory for the measurementof substrates, drugs, and even the activitiesof other enzymes. The biochemicalcompounds(e.g.glucose,urea, uric acid, cholesterol)can be more accurately and specifically estimated by enzymatic procedures compared to the conventional chemical methods. A good example is the estimationof plasmaglucoseby glucoseoxidase and peroxidasemethod. lmmobilized

enzymes

Enzymescan be used as catalytic agents in industrial and medical applications. Some of theseenzymesare immobilized by binding-.them t o a s o l i d , i n s o l u b l e ma tri x w h i c h w i l l not affect the enzyme stability or its catalytic activity. Beaded gels and cyanogen bromide activated sepharose are commonly used for . h e b o u nd enzymes im m ob i l i z a ti o no f e n z y m e s T can be preservedfor long periodswithout lossof activity.

l.

Fl asma

speei fi c

or P l astna

functional enzYrnes Certain enzymesare normally presentin the plasma and they have specific functions to perform. Cenerally, these enzyme activitiesare higher in plasma than in the tissues.They are mostly synthesizedin the liver and enter the ci rcul ati on e.g. l i poprotei n l i pase, pl asmi n , thrombin, choline esterase,ceruloplasminetc. l mpai rment i n l i ver functi on or genet ic disordersoften leadsto a fall in the activitiesof plasma functional enzymes e.g' deficiency of cerul opl asmi ni n W i l son' s di sease. 2, i{on-piasrma specific or plasrna enzymes non-functional

These enzymes are either totally absent or oresent at a low concentration in plasma compared to their levels found in the tissues. The digestive enzymes of the gastrointestinal tract (e.g. amylase, pepsin, trypsin, lipase etc.) present in the plasma are known as secretory Clucoseoxidaseand peroxidase,immobilized enzymes. All the other plasma enzymes and coated on a strip of paper, are used in the associated with metabolism of the cell are clinical laboratoryfor the detectionof glucosein collectivefy referred to as consfitutive enzymes u r ine. acid transaminases, (e.g. lactatedehydrogenase, phosphocreatine phosphatases, and alkaline xidgg9 t Gluconicacid+ Hro, kinase). Glucose Hzoz o-Toluidine (colourless)

Hzo Oxidizedtoluidine (bluecolour)

Estimation of the activities of non-plasma specific enzymes is very important for the diagnosisand prognosisof severaldiseases.

The normal serum level of an enzyme indicatesthe balance between its synthesisand The intensityof the blue colour dependson releasein the routine cell turnover. The raised the concentrationof glucose. Hence, the strip enzyme l evel scoul d be due to cel l ul ardamag e, estimation increasedrate of cell turnover, proliferationof method is usefulfor semi-quantitative cells, increasedsynthesisof enzymesetc. Serum of gluc o s ei n u ri n e .

Chapten 6 : ENZYMES

Serum enzyme (elevated)

l',

107

Disease(most important)

Amylase

Aculepancreatitis

glutamate pyruvate (SGPT) Serum transaminase

(hepatitis) Liverdiseases

glutamate (SGOT) Serum oxaloacetale transaminase

(myocardial Heartattacks infarction)

phosphatase Alkaline

jaundice Rickets, obstructive

Acidphosphatase

gland Cancer ofprostate

(LDH) Lactate dehydrogenase

Heart attacks, liverdiseases

phosphokinase (CPK) Creatine

Myocardial (earlymarker) infarction

Aldolase

Muscular dystrophy

5'-Nucleolidase

Hepatitis

yGlutamyl (GGT) transpeptidase

Alcoholism

enzymes are conveniently used as markers to It may be noted that SCPTis more specificfor det ect th e c e l l u l a r d a m a g e w h i c h ul ti matel y the di agnosi sof l i ver di seases w hi l e S C OT i s for helps in the diagnosis of diseases. heart diseases.This is mainly becauseof their cel l ul ar di stri buti on- S C P T i s a cytosom al A summary of the important enzymes useful enzyme while SCOT is found in cytosol and f or t he d i a g n o s i so f s p e c i fi cd i s e a sesi s gi ven i n mi tochondri a. Table6.8. Detailedinformationon the diagnostic enzymes including referencevalues is provided Alkaline phosphatase (ALP) : lt is elevated in in Table 5.9. A brief account of selected certain bone and liver diseases(normal 3-13 KA diagnosticenzymes is discussed units/dl). ALP is useful for the diagnosis of rickets, hyperparathyroidism, carcinoma of Amylase : The activity of serum amylase is bone, and obstructive jaundice. increasedin acute pancreatitis (normal 80-180 Somogyi units/dl).The peak value is observed Acid phosphatase(ACP) : lt is increased in \\,ithin 8-12 hours after the onset of disease the cancer of prostate gland (normal 0.5-4 KA rvhich returns to normal by 3rd or 4th day. units/dl).The tartaratelabile ACP (normal<1 KA Elevatedactivityof amylaseis also found in urine units/dl)is usefulfor the diagnosisand pi-ognosis of the patients of acute pancreatitis. Serum of prostate cancers i.e. ACP is a good tumor anrylaseis also important for the diagnosisof marker. chronic pancreatitis,acute parotitis(mumps)and Lactatedehydrogenase(LDH): LDH is useful obstructionof pancreaticduct. for the diagnosis of myocardial infarction, Alanine transaminase(ALT/SGPT): SCPT is infective hepatitis, leukemia and muscular elevated in acute hepatitis of viral or toxic dystrophy(serumLDH normal 50-200 lull). LDH or igin ,j a u n d i c ea n d c i rrh o s i so f l i v e r (normal3- has five isoenzymes,the details of which are J,,r lUll). describedlater. Aspartate transaminase (AST/SGOT) : SCOT activity in serum is increased in myocardial iniarction and also in liver diseases(normal - 1- . + it u l l ).

Creatine kinase (CK) . lt is elevated in myocardial infarction (early detection) and muscul ardystrophy(normal10-50 l U l l ). C K has three isoenzymes(describedlater).

o o

Enzymes l. Dig,estive enzYmes Amylase Lipase

ll. Transaminases (ALT) Alanine transaminase or serum glutamate pyruvate (SGPT) transaminase (AST)or Aspartate transaminase serumglutamate oxaloacetate (SGOT) transaminase lll. Phosphatases phosphatase (ALP) Alkaline (pHoptimum 9-10) (ACP) Acidphosphatase (pHoptimum #S) lV. Enrymes of carbohydrate metabolism Aldolase (lCD) lsocitrate dehydrogenase (LDH) Lactate dehydrogenase

V. Miscellaneousenzynes Creatinekinase(GK)or creatine phosphokinase (CPK)

Reference value

Disease(s)in which increased

80-180Somogyi units/dl or 2.5-5.5pKat 0.2-1.5lu/l

(acuteparotitis), in pancreatic duct,severediabetic Aculepancreatitis, mumps obstruction ketoacidosis. Acutepancreatitis, moderate elevation in carcinoma of pancreas.

3-40lu/l or 40-250nKat

(viralor toxic),jaundice, Acutehepatitis cirrhosis of liver.

nKat 4-45lu/l or 5G-320

Myocardial infarction, liverdiseases, livercancer,cirrhosis ot liver.

(related (KA)units/dl Bonediseases activity)-rickets, Pagets'disease, hyperparaIn adults-$13KingArmstrong to higher osteoblastic nKat. thyroidism, ol bone. or 2$-90lU/lor 500-1400 carcinoma jaundice (cholestasis), infective hepatitis, of liver. Inchildren-l Liverdiseases-obstructive cirrhosis 5-30Klr/dl gland(tartarate Prostatic i.e.cancerof prostate liabileACPseryesasa marker 0.5-4KAuniWdl or 2.5-12IU/' carcinoma labile fordiagnosis andfollowup),Pagets' disease. or 10-100nKat.Tanarate ACP-G{.9 KAunits/dl 2-6 tu/l 1-4 tu/l 50-200lu/l or 1-5 pKat

gravis, liverdiseases, myocardial infarction, myasthenia leukemias Muscular dystrophy, (inflammatory toxicor malignant) Liverdiseases pemicious hepatitis, muscular leukemia, Myocardial infarction, acuteintective dystrophy, anaemE.

1150tu/l

(CKusefulfor earlydetection), Myocardial infarction muscular dystrophy, hypothyroidism, alcoholism. Nephrotic syndrome, myocardial infarction jaundice, Hepatitis, obstructive tumors jaundice. infective hepatitis, obstructive Alcoholism, pregnancy. Bacterial infections, collagen diseases, cirriosis,

(ChEl) Cholinesterase 2-10ru/l phosphatase (NTP) 2-15 lu/l S'-Nucleotidase or nucleotide (GGT) yGlutamyl transpeptidase 5-40tu/l (tenooxidase) Ceruloplasmin 2tr50mg/dl

E o o

I m 6

n

Chapter 6 : ENZYMES

Referencevalues

Disease(s)in which decreased

Amylase (ChEll) Pseudocholinesterase

8G-1 80Somogyi units/dlLiverdiseases 10-20tu/dl Viralhepatitis, malnutrition, livercancer, cirrhosis ofliver Ceruloplasmin 20-50mg/dl Wilson's disease (hepatolenticular degeneration) (G6PD) Glucose 6-phosphate dehydrogenase inRBC 121260 tU/1012 RBC Congenital deficiency withhemolytic anemia

y-Glutamyl transpeptidase (GGT) : lt is a sensitivediagnosticmarker for the detection of alcoholism. GGT is also increased in infective hepatitisand obstructivejaundice. Decreased

plasma

enzyme

aetivities

3. An enzyme may be active as monomer or oligomer e.g. glutamatedehydrogenase. 4. In glycoprotein enzymes, differences in carbohydrate content may be responsiblefor isoenzymese.g. alkaline phosphatase.

Sometimes, the plasma activities of the lsoenzymes of lactate enzymesmay be lower than normal which could dehydrogenase (LDHI be due to decreased enzyme synthesis or Among the isoenzymes,LDH has been the congenital deficiency. ln Table 5.10, the most thoroughly investigated. decreasedplasma enzymes in certain disorders are given. LDH whose systematic name is L-lactateNAD+ oxidoreductase(E.C. 1 .'1.1.27)catalyses the interconversion of lactate and pyruvate as shown below The multiple forms of an enzyme catalysing LDfI ? cH3-cH-@oH CH3-e-C OOH the same reaction are isoenzymes or isozymes. They, however, differ in their physical and oH N A D + N A D H+ H + chemical propertieswhich include the structure, Lactic acld Pyruvicacld -,-\---+ electrophoreticand immunological properties, LD H has fi ve di sti nct i soenzymesLD H t, K,n and V.nr" values, pH optimum, relative LDH2, LDH3, LDHa and LDH5. They can be susceptibility to inhibitors .and degree of (celluloseor starch separated by electrophoresis denaturation. gel or agarose gel). LDHI has more positive charge and fastest in electrophoreticmobility Explanation for the w hi l e LD H 5 i s the sl ow est. existence of isoenzymes Strdcture of LDH isoenzymes : LDH is an Many possiblereasonsare offeredto explain (tetrameric)enzyme made up of four oligomeric presence the of isoenzymesin the living systems. polypeptide subunits. Two types of subunits . 1 . lsoenzymes synthesized from different namely M (for muscle) and H (for heart) are genes e.g. malate dehydrogenaseof cytosol is produced by differentgenes.M-subunit is basic differentfrom that found in mitochondria. w hi l e H subuni t i s, aci di c. The i soenzymes 2. O lig o m e ri c e n z y me s c o n s i s ti n gof more contain either one or both the subunits giving than one type of subunitse.g. lactatedehydro- LDHI to LDH'. The characteristicfeatures of genaseand creatine phosphokinase. LDH isoenzymesare given in Table 5.11.

B IOC H E MIS TR Y

110

Isoenzyme

Subunit constitution

H

Principal tissueof origin

Fastest

Percentage of Whether normal serum destroyed by heat (at 60"C) in humans

No

25To

No

35Yo

Fast

Partially

27To

Liverandskeletal muscle

Slow

Yes

8To

muscle andliver Skeletal

Slowest

Yes

5o/o

LDHr

Ha

LDHz

HsM

HeartandRBC

LDH3

HzMz

Brainandkidney

LDHr

HMs

LDHs

Ma

ffi

Electrophoretic mobility

HeartandRBC

@

(c) Fig. 6.15 : Electrophoresis of lactate dehydrogenase with relative proportians of isoenzymes (A) Normal serum (B) Serum from a patient of myocardial infarction (LDH, and LDH2T)(C) Serum from a patient of liver disease (LDH|T)

Significanceof differential catalytic activity : LD H l (H a) i s predomi nantl yfound i n heart muscle and is inhibited by pyruvate- the substrate. Hence, pyruvate is not converted to lactate in cardiac muscle but is converted to acetyl CoA which enterscitric acid cycle. LDH5 (M+) is mostly presentin skeletalmuscle and the i nhi bi ti onof thi s enzymeby pyruvatei s mi ni mal , hence pyruvate is convertedto lactate.Further, LD H 5 has l ow K . (hi ghaffi ni ty)w hi l e LD H l has high Km (low affinity) lor pyruvate. The differentialcatalyticactivitiesof LDHl and LDH5 in heart and skeletal muscle, respectively,are well suited for the aerobic (presenceof oxygen) and anaerobic(absenceof oxygen) conditions, prevai l i ngi n theseti ssues. Diagnosticimportance of LDH : lsoenzymes of LD H have i mmenseval ue i n the di agnosi sof heart and Iiver related disorders (Fi9.6.1fl. ln heal thy i ndi vi dual s, the acti vi ty of LD H 2 i s hi gherthan that of LD H l i n serum.In the caseof myocardial infarction, LDHI is much greater than LD H 2 and thi s happensw i thi n 12 to 24 hours after infarction. Increasedactivity of LDH<

ENZYMES

111

in serum is an indicator of liver diseases.LDH ac t iv it y i n th e R B C i s 8 0 -1 0 0 ti m e s more than t hat in t h e s e ru m .H e n c e fo r e s ti ma ti o nof LD H or it s is o e n z y me ss, e ru m s h o u l d b e to tal l y free f r om hem o l y s i so r e l s e fa l s ep o s i ti v eresul tsw i l l be obt ai n e d .

In heal thy i ndi vi dual s, the i soenzyme C P K 2 (MB ) i s al most undetectabl ei n serum with less than 2'h of total CpK. After the myocardi ali nfarcti on(Ml ), w i thi rr the fi rst 6_.1 g hours,C P K 2i ncreases i n the serumto as hi eh as 209l o(agai nst2oh normal ).C pK ) i soenzvnre,s not el evated i n skel etal mui cl e di sorders. Therefore,estimationof the enzyme CpKz (MB) is the earliest reliable indication of mvoiardial Cr eati n ek i n a s e(C K )o r c re a ti n ep h o s phoki nase infarctian. ( CP K )c at a l y s e th s e i n te r-c o n v e rs i oonf p hosphoc r eat ine( o r c re a ti n ep h o s p h a teto ) c re a ti ne. A s many as si x i soenzymesof al kal i ne phosphatase (A LP )have been i denti fi ed.A Lp i s a monomer, the i soenzymes are due to CP K e x i s ts a s th re e i s o e n z y m es. E ach the difference in the carbohydrate content is oenz y me rs a d i me r composed of two (si al i c aci d resi dues). The most i mportanr s ubunit s -M (mu s c l e )o r B (b ra i n )o r b oth. A LP i soenzymesare cx1 -A Lp, u2-heat l abi l e A LP , o,2-heatstabl e A Lp, pre-B A Lp, y-A Lp Isoenzyme Subunit Tissueof origin etc. cPKl BB Bra i n Increase i n cr2-heat l abi l e A Lp suggests cPK2 MB Heart hepati ti s w hereas pre p-A Lp i ndi cates l tone cPK3 MM S k e l e ta lmuscl e d iseases. Phosphocreatin"+

-9{--------lCreatine ADP ATP

BIOMEDICAL/ CHHIGAL CSNCEPTS

t:;t In the liuing system, the regulation oJ enzgme qctiuities occurs through allosteric inhibition, actiuation of lotent enzymes, compartmentation of metabot-icpathways, control of enzyme synthesis and degrodation. w Feedback(or end product) inhibition is a specialized form oJ allosteric inhibition that controls seuerol metabolic pathways e.g. CTP inhibits aspartote transcorbamoylase; cholesterol inhibits HMG coA reductase. The end priduct inhibition is ufmost important to cellular economysince a compound is synthesized onlg when required. r"-; Certain RNA molecules(ribozymes) function as non-protein enzymes.It is belieuedthat ribozgmes were lunctioning as biocatalystsbefore the orrurr"r4 of protein enzymes during euolution. r'>' Certain enzymesare utilized os therapeutic agents. Streptokinosein used to dissolue blood clots in circulation while asparaginoseis emploged in the treatment of leukemias. r':' Determination of serum enzyme actiuities is of great importance t'or the diagnosisof seueral diseoses(refer Table 6.8). rt'' Lowered body temperature (hypothermia) is accompained by o decrease in enzyme actiuities' This principle is exploited to reduce metobolic demand. during open heart

surgery or transportotion of organs lor transplantation surgery.

lt2

BIOCHEMISTF|Y Creatine phosphokinase (precisefy isoenzyme MB) is the first enzyme to be released into circulationwithin 6-18 hoursafterthe infarction. Therefore,CPKestimationis highly usefulfor the early diagnosisof Ml. This enzyme reachesa peak value within 24-30 hours, and returnsto normal level by the 2nd or 3rd day.

i o E N

ul 0

6 12f8243036

424A*6066724

Hours

5

6

7

I

9 10 11

Days

Fig. 6.16 : Enzyme paftern in myocadial infarction (CPK-Creatine phosphokinase; SGOT-Serum

Fo;t;nritar}iiries sf alcohof, r$e",fraydrogenr.*se Alcohol dehydrogenase (ADH) has two heterodimer isoenzymes. Among the white Americans and Europeans,cx,p1isoenzyme is predominantwhereas in Japaneseand Chinese (Orientals)oB2 is mostly present.The isomerop2 more rapidly convertsalcohol to acetaldehyde.

Aspartate transaminase(AST or SCOT) rises sharplyafter CPK, and reachesa peak within 48 hours of the myocardial infarction. AST takes 4-5 days to return to normal level. Lactatedehydrogenase(LDHl) generally rises from the second day after infarction, attains a peak by the 3rd or 4th day and takes about 10-15 days to reach normal level. Thus, LDH is the fast enzyme to rise and also the last enzyme to return to normal level in Ml.

Cardiac troponins (CT) : Although not enzymes/ the proteins cardiac troponins are hi ghl y useful for the earl y di agnosi sof M l. Among these, troponin I (inhibitory element of actomysin ATPase) and troponin f (fropomysin binding element)are important.Cardiactroponin Accumulation of acetaldehvdeis associated (CTl) | is releasedinto circulation within four with tachycardia (increase in heart rate) and hours after the onset of Ml, reachesa peak value facial flushing among Orientals which is not by 12-24 hours,and remainselevatedfor about commonlv seen in whites. lt is believed that a week. Japaneseand Chinesehave increasedsensitivity The protein myoglobin is also an early marker to alcohol due to the presenceof ap2-isoenzyme for the diagnosisof Ml. Myoglobin is however, of A D H . not commonly used as it is not specific to cardiac diseases.ln the Table 6.12, a summary of the diagnosticmarkersused in Ml is given.

Hnzymes in liver diseases

For the right diagnosisof a particular disease, The following enzymes-when elevated in it is always better to estimate a few (three or more) serum enzymes, instead of a single serum-are useful for the diagnosis of liver enzyme. Examples of enzyme patterns in dysfunction due to viral hepatitis (jaundice), toxic hepatitis,cirrhosisand hepatic necrosis important diseasesare given here. Hnrymes

in nnyoeardial

infarction

1 . A l ani ne transami nase;

2. Aspartatetransaminase; The enzymes- namelycreatinephosphokinase 3. Lactatedehydrogenase; (CPK), aspartatetransaminase(AST) and lactate dehydrogenase(LDH)-are important for the The enzymes that markedly increase in diagnosis of myocardial infarction (Ml). The intrahepaticand extrahepaticcholestasisare : elevationof theseenzvmesin serum in relation to '1. Alkaline phosphatase, 2. 5'-Nucleotidase hours/daysof Ml is given in the Fig.6.l6.

113

Chapter 6 : ENZYMES

Diagnostic marker

Time of peak elevation

Time of return to normal level

Diagnostic importance

4-Ohrs

20-25hrs

Earliest marker, however notcardiac specific.

I Cardiac trooonin

12-24hrs

5-9days

Earlymarker andcardiac specific.

troponin T Cardiac

18-36hrs

5-14days

Relatively earlymarker andcardiac specific. However, inotherdegenerative elevated diseases.

phosphokinase (MB) Creatine

20-30hrs

24-48hrs

Cardiac specific andearlymarker.

(LDHl) Laclate dehydrogenase

48-72 hrs

10-15 days

Relatively latemarker andcardiac specific.

Asparlate transaminase

3048hrs

4-6days

Notcardiac specific,

Serum-y-glutamyltranspeptidaseis useful in the diagnosisof alcoholic liver diseases.

(tartaratelabile) is specific for the detection of prostaticcarcinoma.

lNote : Prostate specific antigen (PSA; mol wL 32 KD), though not an enzyme, is a more probably In themuscular dystrophies, dueto reliable marker for the detection of prostate the increased leakage of enzymes from the cancer. Normal serum concentrationof PSA is damaged cells, serum levels of certain muscle 1-4 n{mll. enzymes are increased.These include creatine A non-specific increase in certain enzymes phosphokinase, aldolase and aspartate like LDH, alkalinephosphatase and transaminase transaminase. Of these,CPK is the most reliable indicator of muscular diseases,followed by may be associatedwith malignancy in any part of the body. aldolas e .

Enzymes in muscle diseases

p-C l ucuroni dase esti mati oni n uri ne i s usefu l for detecting the cancers of urinary bladder, Increase in the serum acid phosphatase pancreasetc.

Enzymes

in cancers

114 B IOC H E MIS TFI Y

r' Enzymesare the protein biocatarystssynthesizedby the

'!ii,iir

cers.They are classified c/osse's---'ox idoreductaies,transferi s"r, nvi 'iuing rZ nu, r, tyases, isomerases r!ri'"r

2' An enzgm" o to::,r!r-:in its action,possessingactiue site, where the substrate binds form enzgme-substrqtecomplex, i"f;;. to the product is t'ormed. 3. Factorslike cont

xtJ,.!J{j,i{T,zT tr;ti,,:";":i",:! riii:{ii%Ti;'i!"Ji,i;:::{:;:::::;:i, n reuersibte (competitiue, and non-competitiue), ,1,i":,I'f*f."iX'J'"i,,J,i2,,:"Y:::!,bv

S Many enzymesrequire prerenceot' non-protein substances .the called cofactors (coenzyntes) Most of tt''' for i*r"v-o-Lre"iriwtiues of B-comptex- uitamins(e.g. NAD+, 'i;;:::7" FAD, 6

The mechanism

"I,:rrr\",a.ctjo2 is e.xproinedbv tock and keymoder (oJFischer),mare recentty induced fit madet(of Koshtand) ,"i ,"triiiJililn ,n.orr. rateof reaction throughacid-base catatysis, ' [l"o;:':ff:r7:;;::,'n" couarentcatarysis

t ,;,!l;i;:,if"i1n]lf,*, thereis a constant regutation of enzymeteuersbroughtaboutbv sm,actiuation ot'proenzgmes, synthesis anddegrad"ri"i.t etc. "*i^Z 9. Estimation of sert

serum o*,u;*-;;,:f i,;:::T:: ,:,,'J",::;i: !:':,,,;"!,:*i;i,f:::;:,?1.'f:",:1j,,:::::;

hepatitis; aspartate,. trarraminas., lorror" dihgdrogenase"(tDH) phosphokinase(CpK) and creatine i,i.rorrrror,, alkaline phosphatase ^ in ^uoroi,"ot rickets and o,,ia

i,n:::i::i::;::;:f

inolin'io''.-"'nprostatic,o,,,no-[;

vstutamv!transpep-

L0' Isoenzymesare the,,murtip,re fotrys of an enzyme catarysingthe same howeue4 dift'er in their phvsicai'rri reoctian which ii"^,car propertie".iD; has five isoenzgmes hasthree'roui""a ciiz;;;'r",u

i:i::ri::

importantin thedragnosis ot'myocardiar

115

Chapter 6 : ENZYMES

I.

Essayquestiosrs and nomenclature. 1. What are enzymes?Describetheir classification 2. Write an accountof the variousfactorsaffectingenzymeactiviti. 3. Describethe mechanismof enzymeaction. Write brieflyon the role of coenzymesin enzymeaction. 4. What are coenzymes? 5. Write an accountof the importanceof serumenzymesin the diagnosisof diseases.

lL Short notes (a) Enzyme specificity,(b) Competitive inhibition, (c) Coenzymes,(d) Allosteric enzymes/ (f) K, value,(g)Serumenzymesin myocardialinfarction,(h) Lactatedehydrogenase, (e)lsoenzymes, (i) Role of metalsin enzymeaction,(j) Active site. lI L F ill i n th e b l a n k s 1. T h e l i te ra lme a n i n go f e n z y m ei s 2. The classof enzymesinvolvedin syntheticreactionsare 3. The non-proteinpart of holoenzyme 4. Enzymeslose the catalytic activity at temperatureabove 70oCdue to 5. Examplesof two enzymescontainingzinc are

and

6. The place at which substratebinds with the enzyme requiresthe coenzyme dehydrogenase 7. The enzymeglucose6-phosphate rs 8. The E.C.numberfor alcohol dehydrogenase activatedby is allosterically 9. Phsophofructokinase 10. The very first enzymeelevatedin serumin myocardialinfarction I V . M u l ti p l e c h o i c e q u e s ti o n s 11. Pepsinis an examplefor the classof enzymesnamely (c) Hydrolases(d) LiSases. (a) Oxidoreductases(b) Transferases 12. The coenzymenot involvedin hydrogentransfer (a) FMN (b) FAD (c) NADP+(d) FH4. 13. In the feedbackregulation,the end productbinds at (a) Active site (b) Allostericsite (c) E-Scomplex(d) None of these. activityin serumis elevatedin 14. y-Clutamyltranspeptidase (a) Pancreatitis (b) Musculardystrophy(c) Myocardialinfarction(d) Alcoholism. 15. In recent years/a non-proteincompound has been identifiedto bring about catalysisin biologicalsystem.The name of the compoundis (a) DNA (b) RNA (c) Lipids(d) Carbohydrates.

Vitamins t, ++ Fat Water soluble soluble

I t i s d i ffi c u l t to d e fi n e v i ta m i ns preci sel y. I vitamins may be regarded as organic compounds required in the diet in small amounts to perform specific biological functions for normal maintenance of optimum growth and health of the organism. The bacterium E.coli does not requireany vitamin, as it can synthesize all of them. lt is believedthat during the course of e v o l u ti o n ,th e a b i l i ty to s y n th esi zevi tami ns was lost. Hence, the higher organismshave to obt a i nth e m fro m d i e t. T h e v i ta m i nsare requi red in s ma l l a m o u n ts , s i n c e th e i r degradati oni s relativelyslow.

the naturalfoods.Funk (1913)i sol atedan act ive pri nci pl e (an ami ne) from ri ce pol i shi ngsand, l ater i n veast, w hi ch coul d cure beri -be r i in pigeons. He coined the term vitamine (Creek : vita-life) to the accessoryfactors with a belief that all of them were amines.lt was later realised that only few of them are amines. The term vitamin, however, is continued without the final letter 'e'.

The usage of A, B and C to vitamins was i ntroduced i n 1915 by McC ol l um and Davis. They first felt there were only two vitaminsfat soluble A and water soluble I (anti-beriberi factor). Soon another water soluble anti-scurvy His to ry a n d n o m e n c l a tu re factor named vitamin C was described.Vitamin I n th e b e g i n n i n g o f 2 0 th c e ntury, i t w as A was later found to possesstwo componentsc lea rl y u n d e rs to o d th a t th e d i ets contai ni ng one that preventsnight blindness(vitaminA) and purified carbohydrate,protein, fat and minerals anotheranti-ricketfactor named as vitamin D. A were not adequateto maintain the growth and fat sol ubl efactorcal l edvi tami nE , i n the abs ence health of experimentalrats, which the natural of which rats failed to reproduceproperly, was foods (such as milk) could do. discovered. Yet another fat soluble vitamin Hopkins coined the term accessoryfactors to concerned with coagulationwas discovered in the unknown and essentialnutrientspresent in mi d 1930s. l t w as named as vi tami n K . In t he

Chapter 7 : VITAMINS

777

sequenceof alphabets it should have been F, but K was preferred to reflect its function (koagulation). As regardsthe water soluble factors,vitamin C was identifiedas a pure substanceand named as ascorbic acid. Vitamin B was found to be a complex mixtureand nomenclaturealso became complex. B1 was.clearly identified as anti-beriberi factor. Many investigators carried out intensiveresearchbetween 192O and 1930 and went on n a m i n g th e m a s th e w a te r sol ubl e v it am ins82 , 8 3 , 8 4 ,8 5 ,8 6 , 8 7 , 8 6 , B g ,8 19, 811 and 812. Some of them were found to be mixturesof alreadyknown vitamins.And for this reason, a few members (numbers!)of the Bcomplex series disappeared from the scene. Exceptfor 81, Bz, Bo and 812, names are more commonly used for other B-complexvitamins.

hematopoietic (folic acid and 812). Most of the water soluble vitamins exert the functions through their respectivecoenzymeswhile only one fat solublevitamin (K) has been identifiedto function as a coenzyme. $ynthesis of vitannims by intestina! bacteria Vitamins, as per the definition, are not synthesizedin the body. However, the bacteria of the gut can produce some of the vitamins, required by man and animals. The bacteria mainly live and synthesizevitamins in the colon region, where the absorptionis relativelypoor. Some of the animals (e.g. rat, deer etc.) eat their own feces, a phenomenon known as coprophagy.

As far as humansare concerned,it is believed that the normal intestinal bacterialsyntfiesig and of vitamins absorption of vitamin K and biotin may be There are about 15 vitamins, essentialfor sufficient to meet the body requirements. For humans. They are classifiedas fat soluble (A, D, other B-complex vitamins, the synthesis and E and K) and water soluble (C and B-group) absorptionare relatively less.Administrationof vitamins as shown in the Table 7.1. The anitibiotics often kills the vitamin synthesizing B - c om plex v i ta m i n s ma y b e s u b -d i v i d ed i nto bacteria present in the gut, hence additional energy-releasing (81, 82, 86, biotin etc.) and consumptionof vitamins is recommended. Glassification

Vitamin A Vitamin D V1amin E Vitamin K

I Vitamin C I

acid(Bn) l-Folic L-Vitamin 8', (cyanocobalamin)

--.._

118 Fa{ soluble

B IOC H E MI STRY vitamins-general

The four vitamins, namely vitamin A, D, E, and K are known as fat or lipid soluble. Their availabilityin the die! absorptionand transport are associatedwith fat. Thev are soluble in fats and oils and also the fat solvents (alcohol, acetoneetc.). Fat soluble vitaminscan be stored in liver and adiposetissue.They are not readily excreted in urine. Excessconsumptionof these vitamins (particularlyA and D) leads to their accumulationand toxic effects. '

Vitamers The term vitamers representsthe chemically similar substances that possess qualitatively similar vitamin activity. Some good examples of vitamersare given below . Retinol,retinal and retinoic acid are vitamers of vitamin A. . Pyridoxine, pyridoxal and pyridoxamine are vitamersof vitamin B..

Alf the fat soluble vitamins are isoprenoid compounds, since they are made up of one or more of five carbon units namely isoprene units ( -C H = C .C H 3 -C H = C H -). F a r sol ubl evi tami ns In the fol l ow i ng pages, the i n dividual perform diverse functions. Vitamin K has a members of the fat soluble and water soluble specific coenzymefunction. vitamins are discussed with regard to the chemistry,biochemicalfunctions,recommended Water sCIluble vitamlels*seneral dietary/dailyallowances(RDA), dietary sources, The water soluble vitamins are a deficiency manifestationsetc.

heterogenousgroup of compounds since they differ chemically from each other. The only common character shared by them is their solubility in water. Most of these vitamins are readily excreted in urine and they are not toxic to the body. Water soluble vitamins are not stored in the body in large quantities (except 812).For this reason,they must be continuously supplied in the diet. Generally, vitamin deficiencies are multiple rather than individual with overlapping symptoms. lt is often difficult to pinpoint the exact biochemical basis for the symptoms. The water soluble vitamins form coenzymes (Refer Table 5.3) that participate in a variety of biochemical reactions,related to either energy generationor hematopoiesis.lt may be due to this reasonthat the deficiencyof vitaminsresults in a number of overlapping symptoms. The common symptomsof the deficiency of one or more vitamins involved in energy metabolism in c l u d e d e rma ti ti s ,g l o s s i ti s(r ed and sw ol l en tongue),cheilitis (ruptureat the cornersof lips), di a rrh e a , m e n ta l c o n fu s i o n , depressi on and malaise.

The fat soluble vitamin A, as such is present only in foods of animal origin. However, its provitamins carotenes are found in plants. It is recorded in the history that Hippocrates (about 500 B .C .) cured ni ght bl i ndness. He prescribedto the patients ox liver (in honey), which is now known to contain high quantity of vi tami n A . Chennistry In the recent years, the term vitamin A is collectively used to representmany structurally related and biologically active molecules (Fig.7.1).The term retinoids is often used to include the natural and synthetic forms of vitamin A. Retinol, retinal and retinoic acirl arc regardedas vitamersof vitamin A.

1. Retinol (vitamin A alcohol) : lt is a primary alcohol containingp-ionone ring. The side chain has two isoprenoid units, four double bonds and D e fi c i e n c yo f v i ta m i n s8 1 , 8 6 and B 12i s more one hydroxylgroup. R eti noli s presenti n anim al closelyassociated with neurologicalmanifestations. tissuesas retinylesterwith long chain fatty acids.

119

Ghapter 7 : VITAMINS

Retinal -C=O I H Retinolc acid

-C=O I OH

p-lonone

Fig. 7.1 : Structuresof vitaminA and relatedcompounds(Redcolour reptesents the substituentgroupsin the respectivecompounds).

2. Retinal (vitamin A aldehyde) : This is an aldehyde form obtained by the oxidation of retinol. Retinal and retinol are interconvertible. Previously, the name retinine was used for r et inal.

As and when needed, vitamin A is released from the liver as free retinol. lt is believed that zinc plays an important role in retinol mobilization. Retinol is transported in the circulation by the plasma retinol binding protein (RBP; mol. wt. 21,000) in association with 3. Retinoic acid (vitamin A acid) : This is pre-albumin.The retinol-RBPcomplex binds to produced by the oxidation of retinal. However, specific receptors on the cell membrane of retinoic acid cannot give rise to the formationof peripheral tissue and enters the cells. Many retinal or retinol. cells of target tissues contain a cellular retinol4. p-Carotene (provitamin A) : This is found binding protein that carries retinol to the in plant foods. lt is cleaved in the intestineto nucl eus and bi nds to the chromati n (D N A ). produce two moles of retinal. ln humans, this It is here that retinol exerts its function in conversion is inefficient, hence p-carotene a manner analogous to that of a steroid possessesabout one-sixth vitamin A activity hormone. compared to that of retinol.

Absorption, transport and mobilization

BIOCHEMICAL FUNCTIONS

Vitamin A is necessary for a variety of functions such as vision, proper growth and Dietary retinyl esters are hydrolysed by differentiatioryreprbduction and maintenanceof pancreaticor intestinalbrush border hydrolases epithelial cells. In recent years, each form of in the intestine,releasingretinol and free fatty vitamin A has been assignedspecific functions acids. Carotenes are hydrolysed by p-carotene (Fig.7.3). l5-1S'-dioxygenaseof intestinal cells to release Vitamin A and vision : The biochemicalfunc2 moles of retinalwhich is reducedto retinol. In tion of vitamin A in the processof vision was first elucidated by Ceorge Wald (Nobel Prize 1968). The events occur in a cyclic process known as Rhodopsin cycle or Wald's visual cycle (Fig.7.4).

BIOCHEMISTFIY

120

lntestinalcell p-Carotene

j Retinal

J

Retina All-fransretinol

II

+ All-transretinal

II

J

Visual cycle (SeeFig.7.a)

Chylomicrons

RBP-t Retinol-RBP

Nuclear receptor

i j

v Specificproteins I I

+ Cell difierentiation

functions transportandbiochemical : summaryof vitaminA absorption, Fig.7.2 (FFA-Freefaw acid; RBP-Retinolbindingprotein)'

121

Chapter 7 : VITAMINS

Dark adaptationtime : When a person shifts from a bri ght l i ght to a di m l i ght (e.9.entry i nto a dim cine theatre), rhodopsin stores are + depleted and vision is impaired. However, Retinol (steroid hormone--{roMh and difterentiation) within a few minutes,known as dark adaptation time, rhodopsin is resynthesizedand vision is improved. Dark adaptationtime is increasedin RetinylPhosphate Retinal (visualcycle) (glycoproteinsynthesis; vitamin A deficient individuals. B-Carotene (antioxidant)

I

I

+ Retinoicacid (steroidhormone-growth and differentiation)

Flg.7.3 : Summaryof thefunctions of vitaminA compounds.

Rods a n d c o n e s

Bleaching of rhodopsin: When exposed to light, the colour of rhodopsinchangesfrom red to yellow, by a process known as bleaching. B l eachi ng occurs i n a few mi l l i secondsand many unstable intermediatesare formed during this orocess. Rhodopsin

-----) Lumirhodopsin Prelumirhodopsin

The retina of the eye possessestwo types of All-trans-retinal+ ll +-- Metartdopsin Opsin +- Metarhodopsin I cells-rods and cones.The human eve has about 10 m illi o n ro d s a n d 5 m i l l i o n c o n e s.The rods Visual cascadeand cGMP : When light strikes are in the peripherywhile conesare at the centre the reti na, a number of bi ochemi cal change s of retina. Rods are involved in dim light vision l eadi ng to membrane hyperpol ari zati onoccur whereas cones are responsiblefor bright light resul ti ngi n the genesi sof nerve i mpul se.The and c ol o u r v i s i o n . An i m a l s -s u c h a s ow l s and hyperpolarizationof the membrane is brought cats for which night vision is more importantabout by a vi sualcascadei nvol vi ngcycl i c C MP . possessmostly rods. When a photon (from ligh$ is absorbed by rhodopsin, metarhodopsinll is produced. The W ald' s v i s u a l c y c l e protein transducin is activated by metarhodopsin Rhodopsin(mol. wt. 35,000) is a conjugated l l . Thi s i nvol vesan exchangeof C TP for C D P on proteinpresentin rods.lt contains11-crsretinaland inactive transducin. The activated transducin This the proteinopsin.The aldehydegroup (of retinal)is activates cyclic GMP phosphodiesterase. linkedto e-aminogroupof lysine(of opsin). T he p ri ma ry e v e n t i n v i s u a l cycl e, on exposureto light, is the isomerizationof 11-cis: retinal to all-trans retinal. This leads to a c onf or ma ti o n a l c h a n g e i n o p s i n whi ch i s responsiblefor the generationof nerve impulse. The all-trans-retinalis immediately isomerized by r et in a l i s o me ra s e(o f re ti n a l e p i thel i um) .to 1 1- c is - re ti n a l .T h i s c o mb i n e s w i th opsi n.:to regeneraterhodopsin and complete the visual cycle (Fig,7.4).However, the conversionof all frans-retinalto 1 1-crs retinal is incomplete. Therefore, most of the all-frans-retinal is transportedto the liver and convertedto all-frans The all-transretinol by alcbhol dehydrogenase. retinol undergoesisomerizationto 1 1-crsretinol whic h i s th e n o x i d i z e d to 1 1 -c i s reti nal to par t ic ip a tei n th e v i s u a l c y c l e .

Light (photon) Nerve

impulse

lsomerase

I

--" ' -.--- | All- i,'ansretinal 11-cl s-reti('nal -------------+

11-cts-retinol +-

l1ferf+All-trane.retinol

Flg.7.4: Wald'sulsualcycle.

122

B IOC H E MIS TRY synthesisand thus are involved in the cell growth and differentiation.

Rhodopsin

lu'no'o"

2. V i tami n A i s essenti alto mai ntai nhe alt hv epi thel i al ti ssue. Thi s i s due to the fact t hat retinol and retinoic acid are requiredto prevent keratin synthesis(responsiblefor horny surface).

Metarhodopsinll

3. Retinylphosphatesynthesizedfrom retinol is necessary for the synthesis of certain glycoproteinq which are required for growth and mucus secretion.

Phosphodiesterase (inactive)

3',5'-cGMP

51GMP

Fig.7.5 : The visual cascade involving cyclic guanosine monophosphate(3" 5' -cGMP).

enzyme degradescyclic CMP ir\;he rod cells (Fi9.7.5).A rapid decreasein cyclic GMP closes the Na+ channels in the membranesof the rod c el l s . T h i s re s u l tsi n h y p e rp o l a r i zati on w hi ch i s an excitatory responsetransmittedthrough the neuron network to the visual cortex of the brain.

4. R eti noland reti noi c aci d are i nvol v ed in the synthesisof transferrin,the iron transport protein. 5. Vitamin A is consideredto be essentialfor the maintenanceof proper immune system to fight againstvarious infections. 6. Cholesterolsynthesisrequiresvitamin A. Mevalonate,an intermediatein the cholesterol biosynthesis,is diverted for the synthesis of coenzyme Q i n vi tami n A defi ci ency. lt is pertinentto note that the discoveryof coenzyme Q w as ori gi nal l y made i n vi tami n A deficient ani mal s.

7. Carotenor'ds(most important p-carotene) function as antioxidants and reduce the risk of C o n e s a re s p e c i a l i z e di n b ri ght and col our cancers initiated by free radicals and strong vision. Visual cycle comparableto that present oxidants.p-Caroteneis found to be beneficialto in ro d s i s a l s o s e e n i n c b n e s .T h e col our vi si on prevent heart attacks.This is also attributedto is governed by colour sensitive pigmentsthe antioxidantproperty. (red), iodopsin (green) and porphyropsin cyanopsin (hlue). All these pigments are retinaldietary Recommended opsin complexes.When bright light strikesthe (RDA) allowance retina, one or more of these pigments are The dai l y requi rement of vi tami n A is ble a c h e d ,d e p e n d i n go n th e p a rt i cul arcol our of as retinol equivalents(RE)rather than expressed light. The pigmentsdissociateto all-trans-retinal Internati onal U ni ts (l U ). and o p s i n ,a s i n th e c a s eo f rh o d opsi n.A nd thi s reactionpasseson a nerve impulseto brain as a 1 retinol equivalent=1 lrg retinol specific colour-red when porphyropsinsplits, =6 Pg P-carotene greenwhen iodopsinsplitsor blue for cyanopsin. = 12 pg othercarote noids Splitting of these three pigments in different = 3.33 l U of vi tamin A proportionsresultsin the perceptionof different activity from retinol colours by the brain.

Golour vision

Other biochemical functions of vitamin

= ' 10 l U of vi tamin A activityfrom p-carotene A

'1. Retinol and retinoic acid function almost like steroid hormones.They regulatethe protein

The RDA of vitamin A for adults is arouno !"Q0Oretiytolequivalents(3,500 lU) for man and 800 retinol equivalents(2,500 lU) for woman.

Chapter 7 : VITAMINS

123

Effect on reproduction : The reproA)tiu" O ne I nt e rn a ti o n aUl n i t (l U ) e q u a l sto 0 .3 mg of r et inol. T h e re q u i re me n ti n c re a s e si n grow i ng system is adversely affected in vitamin A children,pregnantwomen and lactatingmothers. deficiency.Degenerationof germinalepithelium leads to sterility in males.

Dietary sources Animal sourcescontain (preformed)vitamin A. The best sourcesare liver, kidney, egg yolk, milk, cheese,butter.Fish (cod or shark)liver oils are very rich in vitamin A.

Effect on skin and epithelial cells : The skin becomes rough and dry. Keratinization of epithelial cells of gastrointestinal tract, urinary tract and respiratorytract is noticed. This leadsto increasedbacterialinfection.VitaminA deficiencv is associatedwith formationof urinary stones.

Vegetable sources contain the provitamin The plasma level of retinol binding protein is A-carotenes.Yellow and dark green vegetables decreased in vitamin A deficiency. and fruits are good sources of carotenese.g. carrots,spinach,amaranthus,pumpkins,mango, Hypervitaminosis A papaya etc. Excessiveconsumptionof vitamin A leadsto toxicity. The symptoms of hypervitaminosisA Vitamin A deficiency include dermatitis(drying and rednessof skin), The deficiencysymptomsof vitamin A are not enlargement of liver, skeletal decalcification, immediate,sincethe hepaticstorescan meet the tenderness of long bones, loss of weight, body requirements for quite sometime (2-4 irritability,loss of hair, joint pains etc. months). The deficiency manifestations are Total serum vitamin A level (normal 20-50 relatedto the eyes, skin and growth. pgldl) is elevated in hypervitaminosisA. Free Deficiency manifestationsof the eyes : Nrghf retinol or retinol bound to plasmalipoproteinsis blindness (nyctalopia) is one of the earliest actually harmful to the body. lt is now believed symptoms of vitamin A deficiency. The that the vitamin A toxicosis symptoms appear indiv idu a l sh a v e d i ffi c u l ty to s e e i n d i m l i ght only after retinol binding capacity of retinol since the dark adaptation time is increased. binding protein exceeds. Prolonged deficiency irreversibly damages a Higher concentrationof retinol increasesthe num ber o f v i s u a l c e l l s . , synthesisof lysosomalhydrolases.The manifestations of hypervitaminosisA are attributed to Severe deficiency of vitamin A leads to the destructiveaction of hydrolases,particularly xerophthalmia. This is characterized by dryness on the cell membranes. in conjunctivaand cornea,and keratinizationof epithelial cells. In certain areasof conjunctiva, Beneficial effects of 0.carotene white triangularplaquesknown as Bitot's spots Increased consumption of p-carotene is are seen. associatedwith decreased incidence of heart lf xerophthalmiapersisitsfor a long time, attacks,skin and lung cancers.This is attributed corneal ulcerationand degenerationoccur. This to the antioxidant role of p-carotenewhich is resultsin the destructionof cornea,a condition independentof its role as a precursorof vitamin referred to as keratomalacia, causing total A. Ingestionof high doses of p-carotenefor long VitaminA deficiencyblindnessis mostly periods are not toxic like vitamin A. blindness. common in childrenof the developingcountries. Other

deficiency

manifestations

V i tami n D i s a fat sol ubl e vi tami n. l t Effect on growth : Vitamin A deficiency resultsin growth retardationdue to impairment resemblessterolsin structureand functions like a hormone. in skeletalformation.

124

B IOC H E MIS TRY

Absorption,

tlansport

and storage

Vitamin D is absorbedin the small intestine for w hi ch bi l e i s essenti al .Through l ym ph, vi tami n D entersthe ci rcul ati onbound to pl asm a a2-gl obul i n and i s di stri butedthroughout t he bodv. Liverand other tissuesstoresmall amounts of vi tami n D . ME TA B OLIS M A N D FU N C TION S B IOC H E MIC A L V i tami ns D 2 and D 3, as such, are not biologically active. They are metabolized identically in the body and convefted to active forms of vi tami n D . The metabol i sm a nd bi ochemi calfuncti onsof vi tami n D are depi ct ed in Fig.7.8. (Dr) Ergocalclferol Ftg.7.6 : Formationol ergocalciferolfromergosterol. The symptoms of rickets and the benefical effects of sunlight to prevent rickets have been known for centuries.Hess (1924) reportedthat ir r ad i a ti o nw i th u l tra v i o l e t l i g h t i nduced anti rachitic activity in some foods. Vitamin D was is ola te db y An g u s(1 9 3 1 )w h o n a m e di t cal ci ferol . Ghemistry

Synthesis

of 1,25-DHCC

Cholecalciferolis first hydroxylatedat 25th position to 25-hydroxycholecalciferol(25-OH o3) bv a specific hydroxylasepresent in liver. 25-OH D3 is the major storageand circulatory a specific form of vitamin D. Kidney possesses (calciol) enzyme, 25-hydroxycholecalciferol l -hydroxylase which hydroxylates 25-hydroxycholecalciferolat position 1 to produce 1,25(1,2|-DHCC). 1,25 dihydroxycholecalciferol (1,3 groups and 25 D H C C contai ns3 hydroxyl referred Both to as calcitriol. the carbon) hence hydroxylase enzymes (of liver and kidney) requirecytochromePa56,NADPH and molecular oxygen for the hydroxylation process. The synthesis of calcitriol is depicted in Figs.7,7 and 7.8.

Ergocalciferol(vitamin D2) is formed from ergosterol and is present in plants (Fi9.7.6). Chol e c a l c i fe ro(v l i ta m i nD 3 ) i s fo u n d i n ani mal s. Both the sterolsare similar in structureexcept that ergocalciferol has an additional methyl group and a double bond. Ergocalciferol and cholecalciferol are sourcesfor vitamin D activitv and are referred to as provitamins. Regulation of the synthesis of 1125.-DHCC During the course of cholesterolbiosynthesis (Chapter l4), 7-dehydrocholesterol is formed The concentrationof 1,25-DHCC is regulated as an intermediate.On exposure to sunlight, by plasma levels of calcium and phosphate. 7-dehydrocholesterolis converted to choleThey control hydroxylationreaction at position c alc i fe ro l i n th e s k i n (d e rm i s a nd epi dermi s) 1. Low plasma phosphateincreasesthe activity (Fig.2.V Vitamin D is regarded as sun-shine iferol 1-hydroxylase.Low of 25-hydroxycholecalc vitamin. pl asma cal ci um enhances the producti on of The synthesisof vitamin D3 in the skin is parathyroid hormone which in turn activates proportional to the exposure to sunlight. Dark 1-hydroxylase.Thus the action of phosphateis skin pigment lm-qianjn)-adversly influences the di rectw hi l e that of cal ci um i s i ndi recton ki d nev s y nth e s i s ' ocf h o l e c a l c i fe ro l . 1-hydroxyl ase.

125

Ghapter 7 : VITAMINS

Biochemical

functions

Calcitriol (1,25-DHCC)is the biologically active (ormof vitaminD.lt tegulatesthe plasma Ievelsof calciumand phospha,fe. Calcitriolacts at 3 differentlevels(intestine, kidneyand bonel to maintainplasmacalcium(normal9-11 mg/dl). 7-Ilehydrocholestercl (animals) Sunlight 4S k i n

1. Action of calcitriol on the intestine: Calcitriof increlses the intestinal absorptionof calciumand phosphate. In the intestinalcells, calcitriolbindswith a cytosolicreceptorto form a calcitriol-receptor complex.Thiscomplexthen approachesthe nucleusand interactswith a specific DNA leading to the synthesisof a specific calcium binding protein.This protein increases the calciumuptakeby the intestine. The mechanismof action of calcitriolon the targettissue(intestine) is similarto the actionof a steroidhormone. 2. Action of calcitriol on the bone : In the osteoblasts of bone,calcitriolstimulates calcium uptakefor deposition as calciumphosphate, Thus calcitriolis essential for boneformation.Thebone is an important reservoir of calciumandphosphate. Calcitriol along with parathyroidhormone increasesthe mobilizationof calcium and phosphate fromthe bone.Thiscauses in elevation the plasnlacalciumand phosphate levels.

25-Hydroxycholecalciferol (Calcidiol)

II Calcidiol1o-hydroxylase |

(kidney)

J

1,25-Dlhydrorycholecalclferol (1,25DHCCor calcitrlol)

3. Action of calcitriol on the kidney : Calcitriolis also involvedin minimizingthe excretionof calciumand phosphate throughthe kidney, by decreasingtheir excretion'and enhancingreabsorption. The sequenceof eventsthat take place in response to low plasmacalciumconcentration and the actionof calcitriolon intestine,kidney and bone, ultimatelyleadingto the increasein plasma calcium is given in Fi9.7.9. 24,25-Dihydroxycholecalcife rol (24,25-DHCC\ is anothermetaboliteof vitamin D, lt is also synthesized in the kidneyby 24-hydroxylase. The exactfunctionof 24,2S-DHCC is not known.lt is believedthat when' calcitriolconcentration is adequate,24-hydroxylase acts leading to the synthesisof a less importantcompound24, 25-DHCC. In this way, to maintain the homeostasis of calcium,synthesis of 24,25-DHCC is alsoimportant.

4

B IOC H E MIS TFI Y

Skin 7-Dehydrocholesterol

!*ro*-t*a*

lntestine calcitriol(r)

JRecentor(J) Calcitriol

ffi8lrr Bone formation and turnover

Calciumbinding protein Plasma

Ca

Ca2*absorption

Chapter 7 : VITAMINS

127

Plasmacalcium{

In countries with good sunlight (like Indi a), the RDA for vitamin D is 200 lU (or 5mg chol ecal ci ferol ).

J t

Dietary sources

,'i

Cood sourcesof vitamin D include fany fish, fish liver oils, egg yolk etc. Milk is not a good source of vitamin D.

Fig. 7.9 : Summary of the action of calcitiol in elevating plasma calcium.

Y it am in D i s a h o rmo n e not a vitamin-justification

and

Vitamin D can be provided to the bodv in three ways '1. Exposureof skin to sunlight for synthesis of vi tami n D ; 2. Consumptionof natural foods; 3. By irradiating foods (like yeast) that contain precursorsof vitamin D and fortification of foods (milk, butter etc.).

Calc i tri o l (1 ,2 5 -D H C C ) i s n o w consi dered Deficiency symptoms an important calciotropic hormone,. while -:.olecalciferol is the prohormone.The following Vitamin D deficiency is relatively less :"aracteristicfeaturesof vitamin D (comparable common/ si nce thi s vi tami n can be synthesi ze d in the body. However, insufficient exposure .r tn hormone)justify its statusas a hormone. to sunlight and consumption of diet lacking I . Vitamin D3 (cholecalciferol)is synthesized vitamin D results in its deficiency. in the skin by ultra-violetrays of sunlight. Vitamin D deficiency occurs in strict l. T h e b i o l o g i c a l l ya c ti v efo rm o f vi tami n D , vegetar.ians, chronic alcoholics,individualswith : a c it r io l i s p ro d u c e di n th e k i d n e y . Iiver and kidney diseasesor fat malabsorption 3. Calcitriol has target organs-intestine, syndromes.In some people,who cover the entire body (purdah)for religiouscustoms,vitamin D lo.e and kidney, where it specificallyacts. deficiencyis also observed,if the requirementis i. Calcitriol action is similar to steroid not met through diet. lnnnones. lt binds to a receptor in the cytosol Deficiency of vitamin D causes rickets in :-"0 ihe complex acts on DNA to stimulatethe children and osteomalacia in adults. , , - : hes i s o f c a l c i u m b i n d i n g p ro te i n . derived from an old English *ord 'rvr-i.kk"n', J . { c ti n o m y c i n D i n h i b i ts th e a cti on of meaning to twist. Osteomalacia is derived :: :ri:'iol. This supportsthe view that calcitriol from Creek (osteon-bone; malakia-softness). :rets its etfecton DNA leadingto the synthesis Vitamin D is often called as antirachitic vitamin. :- R.\ { (transcription). Ricketsin children is characterizedby bone i Calcitriol synthesisis self-regulatedby a deformi ti esdue to i ncompl ete mi neral i zati on , mechanismi.e., calcitriol decreasesits -:e:3ack resultingin soft and pliable bones and delay in r " " . - S \n th e s i s . teeth formation. The weighrbearing bones are bent to lorm howJegs. In rickets, the plasma Recommended dietary level of calcitriol is decreased and alkaline allowan c e { R D A I phosphatase activity is elevated. Alkaline --t iaill' requirementof vitamin D is 400 phosphataseis concerned with the processof Wenntknal Units or 10 mg of cholecalciferol. bone formation. There is an overproductionof

B IOC H E MIS TR Y

128 alkalinephosphatase relatedto more cellular activitv of the bone. lt is believedto be due to a vain attemptto resultin bone formation.

Hsc.

CHn t-

cH2-(cH2-cH2-cH-cH2)3-H

In case of osteomalacia(adult rickets) HO demineralizationof the bonesoccurs (bones becomesofter),increasingtheir susceptibility to fractures. Rena ! ri c k e ts {renal osteodystrophy)

cHs

(5,7,8-trimethyltocol) cr,-Tocopherol p-Tocopherol (5,8-dimethyltocol) (7,8-dimethyltocol) y-Tocopherol

Fiq.7.10 : Structureof a-tocopherol (Note : The tocopherols differ in the substitution of methyl groups, represented in red).

This is seen in patientswith chronic renal failure. Renalricketsis mainly due to decreased tocopherols (vitamin E vitamers) have been synthesisof calcitriol in kidney. lt can be treated identified-a, p, T, 6 etc. Among these, u-tocopherol is the most active. The tocopherols by ad m i n i s tra ti o no f c a l c i tri o l . are derivatives of 6-hydroxy chromane (tocol) Hypervitaminosis D ring with isoprenoid (3 units) side chain. The Vitamin D is storedmostly in liver and slowly antioxidantpropertyis due to the chromanering. metabolised.Among the vitamins, vitamin D is the mosf toxic in overdoses(10-100times RDA). Toxic effects of hypervitaminosisD include demineralization of bone (resorption) and increasedcalcium absorptionfrom the intestine, Ieading to elevated calcium in plasma (hypercalcemia). Prolonged hypercalcemia is associatedwith depositionof calcium in many soft tissuessuch as kidney and arteries.HypervitaminosisD may lead to formationof stonesin k idne y s (re n a l c a l c u l i ). H i g h c o n sumpti on of vitamin D is associatedwith loss of appetite, nausea,increasedthirst, loss of weight etc.

Absorption,

transport

and storage

Vitamin E is absorbedalong with fat in the small intestine.Bile salts are necessaryfor the absorption.In the liver, it is incorporatedinto lipoproteins (VLDL and LDL) and transported. Vitamin E is stored in adipose tissue, liver and muscl e.The normal pl asmal evel of tocoph er ol is less than 1 mg/dl. Biochemical

functions

Most of the functionsof vitamin E are related to its anfioxidant property. lt prevents the nonenzymaticoxidationsof variouscell components (e.g. unsaturated fatty acids) by molecular oxygen and free radicals such as superoxide Vitamin E (tocopherol)is a naturallyoccurring (Otl and hydrogen peroxide (H2O2). Ttie antioxidant.lt is essentialfor normal reproduction element selenium helps in these functions. in many animals, hence known as anti-sterility V i tami n E i s l i pophi l i c i n characterand is vitamin. Vitamin E is described as a 'vitamin in found in association with lipoproteins, fat searchof a disease.'Thisis due to the lack of any depositsand cellular membranes.It protectsthe s pec i fi cv i ta m i n E d e fi c i e n c yd i s e a sei n humans. polyunsaturated fatty acids (PUFA) from Evansand his associates(1936) isolatedthe peroxidation reactions. Vitamin E acts as a compounds of vitamin E activity and named scavengerand gets itself oxidized (to quinone them as tocopherols(Creek : tokos-child birth; form) by free radicals (R) and sparesPUFA, as shown below pheros-tobear; ol-alcohol).

Clremistry Vitamin E is the name given to a group of tocopherols and tocotrienols. About eight

Chapter 7 : VITAMINS

129

The biochemical functions of vitamin E, related either directly or indirectly to its antioxidantproperty,are given hereunder

Vitamin E and selenium

unsaturatedfatty acids in various tissues and membranes.lt protectsRBC from hemolysisby ox idiz ing a g e n ts(e .g .H 2 O 2 ).

Recommended dietary allowance (RDA)

Theelementseleniumis foundin the enzyme glutathione peroxidase that destroys free 1. Vitamin E is essentialfor the membrane radicals. Thus,Seis alsoinvolvedin antioxidant structure and integrity of the cell, hence it is functionslike vitaminE, and both of them act regarded as a membrane antioxidant. synergistically. To a certainextent,Secan spare the requirement vitaminE, and vice versa. 2, lt prevents the peroxidation of poly-

3. lt is closely associatedwith reproductive functions and prevents sterility. Vitamin E preservesand maintainsgerminal epithelium of gonadsfor proper reproductivefunction. 4, lt increases the synthesis of heme by enhancing the activity of enzymes 6am inolev u l i n i ca c i d (A L A) s y n th a s ea nd A LA dehydratase.

Intakeof vitaminE is directlyrelatedto the consumptionof polyunsaturated fatty acids (PUFA)i.e.,requirement increases with increased intakeof PUFA.A daily consumption of about l0 mg (15 lU) of c-tocopherol for man andI mg (12 lU) for womanis recommended. One mg of a-tocopherolis equal to 1.5 lU. Vitamin E supplemented diet is advisedfor pregnantand lactatingwomen.

5. lt is re q u i re d fo r c e l l u l a r re s p i rati on- Dietary sources through electron transport chain (believed to Many vegetableoils are rich sourcesof stabilizecoenzyme Q).

vitamin E. Wheat germ oil, cotton seed oil, peanutoil, corn oil and sunflower oil are the goodsources of thisvitamin.lt is alsopresentin 7. lt is requiredfor proper storageof creatine meat,milk, butterand eggs.

6. Vitamin E prevents the oxidation of vitamin A and carotenes. in s k elet a lm u s c l e .

Deficiency

symptoms

8. Vitamin E is neededfor optimal absorption of amino acids from the intestine.

The symptomsof vitamin E deficiencyvary from one animal speciesto another.In many 9. lt is involved in propersynthesisof nucleic animals, the deficiency is associatedwith ac ids . sterility, degenerativechanges in muscle, anaemiaand changesin central 10. Vitamin E protects liver from being megaloblastic nervous system. Severesymptomsof vitaminE damaged by toxic compounds such as carbon deficiencyare not seen in humans except tetrachloride. increasedfragility of erythrocytes and minor 11, lt w o rk s i n a s s o c i a ti o w n i th v i ta mi nsA , C neurological symptoms, and p-carotene, to delay the onset of cataract.

12. Vitamin E has been recommendedfor the prevention of chronic diseasessuch as cancer and heart diseases.Clinical trials in this regard are rather disappointing,hence it is no more recommended. However, some clinicians continue to use it particularly in subjects susceptible to heart attacks. lt is believed that vitamin E preventsthe oxidation of LDL. (Note : The oxidized LDL have been implicated to promote heart diseases.)

Toxicity of vitamin E Amongthe fat solublevitamins(A, D, E, K), vitamin E is the leasttoxic. No toxic effecthas been repoftedeven after ingestionof 300 mg/ day for 23 years.

Vitamin K is the only fat soluble vitamin with a specific coenzyme function. lt is required for

130

B IOC H E MIS TRY

the production of blood clotting factors,essentialfor coagulation(in Cerman-Koagulation; hence the nam e K fo r th i s v i ta m i n ). Ghemistry

CH? t-

?r,

C H 2-C H :C -C H 2- (C H 2-C H 2-C H -C H 2) 3- H VitamlnK1 (phylloquinone)

Vitamin K exists in different forms (Fig.7.ll). Vitamin K1 ( phyl l o q u i n o n ei)s p re s e n ti n p l a n ts . V it am i n K2 (me n a q u i n o n e ) i s produced by the intestinalbacteria and a l s o fo u n d i n a n i m a l s .Vi ta m i n K3 (menadione)is a syntheticform. All the three vitamins(K1, K2,K3) are naphthoquinone derivatives. ls opre n o i ds i d e c h a i n i s p re s e n ti n vitamins Kt and K2. The three vitamins are stable to heat. Their activity is, however, lost by oxidizing agents,irradiation,strong ac ids a n d a l k a l i e s .

Absorption, transport and storage Vitamin K is taken in the diet or svnthesized by the intestinal bacteria. Its absorption takes place along with fat (chylomicrons) and is dependenton bile salts.Vitamin K is transported alongw i th L D L a n d i s s to re dm a i n l y i n l i ver and, to a lesserextent, in other tissues. Biochemical

CHs

functions

The functionsof vitamin K are concernedwith blood clotting process.lt brings about the posttranslational (after protein biosynthesis in the cell) modification of certain blood clotting factors.The clotting factors ll (prothrombin), Vil, lX and X are synthesizedas inactive precursors (zymogens)in the liver. Vitamin K acts as a coenzyme for the carboxylation of glutamic acid residuespresentin the proteinsand this reaction is catalysedby a carboxylase(microsomal).lt involves the conversion of glutamate (Clu) to (Gla)and requiresvitamin K, y-carboxyglutamate 02 and COz Gig.7.l\. The formation of y-carboxyglutamate is inhibited by dicumarol, an anticoagulantfound in spoilt sweet clover. Wartarin is a syntheticanaloguethat can inhibit vitamin K action (Fig.7.13).

o

VitaminK2 (menaquinone)

VitamlnK3 (menadione) Fig.7.l1 : Structures ol vitaminK.

Vitamin K is also required for the carboxylation of glutamic acid residues of osteocalcin,a calcium binding protein present in the bone. The mechanismof carboxylationis not fully understood. lt is known that a 2,3-epoxide derivative of vitamin K is formed as. an intermediateduring the course of the reaction. Dicumarol inhibits the enzyme (reductase)that convertsepoxide to active vitamin K. Role of Gla in clotting : The lcarboxyglutamic acid (Cla) residuesof clotting factors are negatively charged (COO-) and they combine with positively charged calcium ions (Ca2+)to form a complex. The mechanism of action has been studied for prothrombin. The prothrombin -Ca complex binds to the phospholipidson the membrane surfaceof the platelets(Fig.7,14. This leads to the increased conversionof prothrombinto thrombin. Recommended dietary (RDA) allowance Strictlyspeaking,there is no RDA for vitamin K, since it can be adequatelysynthesizedin the

131

Ghaprer 7 : VITAMINS

H I Protein,^.,,'\,2\,,rN-CHlll

C/^,/"v'\,,\

cH2o Glu--Jl cHz

VitaminK

coz T

H I PrOtein,^:,,T,2:,,rNl- CH- C,^\,4/'\,,\ lll

cH2o

I CH{-Gla -<4]--- cooH cooH

?

I

cooH

Dicumarol, warfarin

Precursorsof clotting lactors(ll, Vll, lX,X)

Clotting factors (ll, Vll, lX, X)

Fig. 7.12 : Vitamin K dependent carboxylation of the precursors of clotting factors.

gut. lt is however,recommendedthat half of the body requirementis provided in the diet, while the other half is met from the bacterialsynthesis. Accordingly,the suggestedRDA for an adult is 7o-140 ttg/day.

Dietary sources Cabbage, cauliflower, tomatoes, alfa alfa, spinach and other green vegetablesare good sources.lt is also presentin egg yolk, meat, liver, cheeseand dairy products. Deflciency

symptoms

The deficiency of vitamin K is uncommon, since it is presentin the diet in sufficientquantity and/oris adequatelysynthesizedby the intestinal bacteria. However, vitamin K deficiency may occur due to its faulty absorption(lack of bile salts), loss of vitamin into feces (diarrheal

(Protein)-Glu

di seases) and admi ni strati on of anti bi oti cs(ki l l i nS of intestinalflora). Deficiency of vitamin K leads to the lack of acti ve prothrombi ni n the ci rcul ati on.The resul t is that blood coagulation is adverselyaffected. The i ndi vi dualbl eedsprofusel yeven for mi nor injuries. The blood clotting time is increased. Hypervitaminosis

K

Administration of large doses of vitamin K produces hemol yti c anaemi a and j aundi ce, particularlyin infants.The toxic effect is due to increasedbreakdown of RBC. of vitamin

Antagonists

K

The compounds-namelyheparin,bishydroxycoumarin-act as anticoagulants and are antagoniststo vitamin K. The salicylatesand di cumarol are al so antagoni ststo vi tami n K .

(Protein)-Gla

H

-N-CH-Clrl QHzo

Prothrombin

I

.tt

O=C VitaminK (hydroquinone form) \

form 2,3-Epoxide

I

/ neductase \ ,/ Redilcta$e \_ Quinone marol, r{arin

Fiq.7.13 : Summaryof vitaminK cycle in carboxylation reactian.

\

tl tt -oll

\

torm

-ctt

C=O

y-Carboxyglutamate withcalcium complexed

o"".Cd2*

-r=r'#-x

YYYYYYYY AAAAAAAA

Platelet membrane (withphospholipids)

Fiq.7"14: Mechanism of actionof Tcarboxyglutamate

tE

132

B IOC H E MIS TRY

=?-l

are dehydroascorbic acid H owever , acti ve. bi ol ogi cal l y cooH O=Q J. + H2O O=C IvI D-ascorbicacid is inactive.The cooH pl asma O=C I O:C and ti ssues pr eI _Cr H -C -OH domi nantl y contai n ascor bic I I HO-C -H H O-C -H acid in the reduced form. The I tl ratio of ascorbic acid to cH2oH cH2oH cH2oH dehydroascorbicacid in many L-Ascorblcacid D'ahydroL-ascorbic DlketoL€ulonlc Oxalic (reduced ti ssuesi s 15 : 1 . On hydr at ion, form) acld acld (oxidized torm) acid is dehydroascorbic acid Fiq.7.15 : Sttucturesof vitaminC (ascorbicacid) irreversibly converted to 2,3and itsrelatedcompounds. di ketogul oni c aci d w hi ch is inactive. Hvdration reaction is Dicu ma ro li s s tru c tu ra l l re y l a te dto vi tami nK and al most spontaneous,i n al kal i ne or ne ut r al acts as a competitiveinhibitor in the synthesisof solution. lt is for this reason that oxidation of vitamin C is regardedas biological inactivation active prothrombin. (formation of diketogulonic acid). Oxidation of ascorbicacid is rapid in the presenceof copper, Hence vitamin C becomesinactive if the foods Vitamin C is a water solubleversatilevitamin. are preparedin copper vessels. O= C -OH

H-? 6 H-? |

I t p l a y s a n i m p o rta n tro l e i n h u m an heal th and disease. Vitamin C has become the most c on tro v e rs i a vl i ta m i n i n re c e n t years. Thi s i s becauseof the claims and counter-claimson the use of vitamin C in megadoses to cure everything from common cold to cancer.

Biosynthesis

and metabolism

Many ani mal s can synthesi ze asc or bic acid from glucose via uronic acid pathway (Chapter 13). Howevet, mant other primates, guinea pigs and bats cannot synthesizeascorbic acid due to the deficiency of a single enzyme Scurvy has been known to man for centuries. namely L-gulonolactone oxidase. It was the first diseasefound to be associated Vitamin C is rapidly absorbed from the with diet. In the sixteenthcentury about 10,000 lt is not stored in the bodv to a intestine, (scurvy) m a ri n e rsd i e d o f a mi ra c u l o u sdi sease extent. Ascorbic acid is excreted in significant due to lack of fresh vegetablesin their diet. such, or as its metabolitesurine as J ame sL i n d , a s u rg e o no f th e E n gl i shN avy, i n acid and oxalic acid (Fig,7,1fl. diketogulonic 175 3 p u b l i s h e d' T re a ti s eo n S c urvy' .B asedon Lin d ' s o b s e rv a ti o n sth , e R o y a l N avy si nce 1795 functions Biochemical us e d to s u p p l y l i m e o r l e mo n j u i ce to al l the Most of the functionsof vitamin C are related crews. The EnglishNavy used to carry cratesof lemons, hence they were popularly known as to its property to undergo reversibleoxidationreduction i.e., interconversionof ascorbic acid Limeys. and dehydroascorbicacid.

Ghemistry Ascorbic acid is a hexose (6 carbon) derivative and closelv resembles monoin structure(Fig.7.15). saccharides The acidic propertyof vitamin C is due to the enolic hydroxyl groups. lt is a strong reducing agent. L-Ascorbicacid undergoesoxidation to form dehydroascorbicacid and this reactionis reversible.Both ascorbicacid and

1, Collagenformation : Vitamin C plays the role of a coenzyme in hydroxylationof proline and lysine while protocollagenis converted to collagen (i.e. post-translational modification). The hydroxylationreaction is catalysedby lysyl hydroxylase (for lysine) and prolyl hydroxylase (for prol i ne). Thi s reacti on i s dependent on vitamin C, molecularoxygenand a-ketoglutarate (Fig.7.t6).

133

Chapter 7 : VITAMINS

HO

,,'.",,.."z".,,"'.il-cH-8,,"'.,,"..,,".",". I

II

HeC.-

\c-

.CHzl-

Proline

I H2 I a-Ketoglutara,"\ l r,-O, Prolylhydroxylase V Ascorbicacid /|\ ,/l\

+CO2aJt"rg Succinate HO

,..,..,,\-\N-oH-f* HeC-

\n/'

,/

+-Hydroxyproline

Hr''"\

Fig. 7.16 : Ascorbic acid dependent hydroxylation of proline of protocollagen.

5. Tyrosine metabolism: Ascorbic acid is required for the oxidation of p-hydroxy phenylpyruvate(enzyme hydroxylase)to homogenti si caci d i n tyrosi nemetabol i sm. 6. Folic acid metabolism : The active form of the vitamin folic acid is tetrahydrofolate(FHr). Vitamin C is needed for the formation of FHa (enzyme-folic acid reductase). Further, in associationwith FHz, ascorbic acid is involved in the maturation of erythrocytes. 7. Peptide hormone synthesis: Many peptide hormones contain carboxyl terminal amide w hi ch i s deri ved from termi nal gl yci ne. Hydroxylation of glycine is carried out by peptidylglycine hydroxylase which requires vi tami n C . 8. Synthesis of corticosteroid hormones : Adrenal gland possesses high levels of ascorbic acid, particularly in periods of stress. lt is believed that vitamin C is necessaryfor the hydroxylation reactions in the synthesis of corticosteroidhormones.

Hydroxyproline and hydroxylysine are essentialfor the collagen cross-linkingand the 9. Sparing action of other vitamins : strengthof the fiber. In this way, vitamin C is Ascorbic acid is a strong antioxidant. lt spares necessaryfor maintenanceof normal connective vi tami n A , vi tami n E , and some B -compl ex tissueand wound healing process. vitamins from oxidation. 2. Bone formation : Bone tissuespossessan 10. lmmunological function : Vitamin C organic matrix, collagen and the inorganic enhances the synthesi s of i mmunogl obul i ns calcium, phosphateetc. Vitamin C is required (antibodies)and increasesthe phagocyticaction for bone formation. of leucocytes. 3. lron and hemoglobin metabolism: 11. Preventiveaction on cataract: Vitamin C Ascorbic acid enhances iron absorption by reducesthe risk of cataractformation. keeping it in the ferrousform. This is due to the reducing property of vitamin C. lt helps in the 12. Preventive action on chronic diseases: formation of ferritin (storageform of iron) and As an antioxidant, vitamin C reduces the risk mobilization of iron from ferritin. of cancer, cataract, and coronary heart Vitamin C is useful in the reconversionof diseases. methemoglobinto hemoglobin.The degradation dietary of hemoglobinto bile pigmentsrequiresascorbic Recommended (R D A ) al l ow ance ac id. About 60-70 mg vitamin C intake per day will 4. Tryptophan metabolism: Vitamin C is essential for the hydroxylation of tryptophan meet the adult requirement.Additional intakes (enzyme-hydroxylase) to hydroxytryptophanin Q0-4O% increase)are recommendedfor women during pregnancyand lactation. the svnthesisof serotonin.

134

B IOC H E MIS TPY

Slietary sourees Citrus fruits, gooseberry(amla),guava/ green vegetables (cabbage, spinach), tomatoes, po ta to e s(p a rti c u l a rl ys k i n ) a re ri ch i n ascorbi c ac i d . H i g h c o n te n t o f v i ta m i n C i s found i n adre n a lg l a n d a n d g o n a d s .Mi l k i s a poor source of a s c o rb i ca c i d . tsefiaiem*y

symptoms

T h e d e fi c i e n c y o f a s c o rb i c aci d resul ts i n scurvy. This diseaseis characterizedby spongy and sore gums, Ioose teeth, anemia, swollen join ts , fra g i l e b l o o d v e s s e l s, decreased im m u n o c o mp e te n c ed, e l a y e d w ound heal i ng, s lu g g i s hh o rmo n a lfu n c ti o no f a d renalcortexand gonads,haemorrhage,osteoporosisetc. Most of thesesymptomsare relatedto impairmentin the synthesis of collagen and/or the antioxidant pr o p e rtyo f v i ta m i n C . Megadoses of nitannEm C afid it$ r}Gntrobrersy L i n u s P a u l i n g (1 9 7 0 ) fi rs t advocated the

consumpti on of megadosesof ascorbi c acid (even up to 18 g/day, 300 ti mes the dailv requirement!) to prevent and cure common cold. He is remembered as a scientist who suggested ' keepvi tami nC i n gunny bagsand eat in grams.' This generateda lot of controversy' worldover. lt is now clear that megadose of vi tami n C does not oreventcommon col d. But the durati on of col d and the severit y or symptoms are reduced. l t i s bel i eved t hat ascorbic acid promotesleukocytefunction. Megadoses(-a ildaV) of vitamin C are still conti nued i n common col d, w ound healing, trauma etc. A s an anti oxi dant,ascorbi c acid certai nl yprovi dessome heal th benefi ts. A scorbi caci d, as such,has not beenfound t o be toxi c. B ut, dehydroascorbi caci d (oxidized form of ascorbi caci d) i s toxi c. Further,oxalat eis a maj or metabol i teof vi tami n C . Oxal ate has been i mpl i cated i n the formati on of kidner stones.However, there are controversialreports on the megadoses of vi tami nC l eadi ngto ur inar l stones.

BIOMEDIGALI CLINICAL CONGEPTS

It is belieuedthat during the courseof euolution,the ability to synthesizeuitaminsuos lost by the higher orgonisms, hence they should be supplied through the diet. For humans, the normal intestinal bacterial sgnfhesisoJ uitamin K and biotin is olmost sufficient to meet the bodg requirements. Administration ot' antibioticsolten destroysthe uitamin synthesizingbacteria in the gut, henceadditional supplementationot' uitaminsis recommendedduring antibiotic therapy. Vitamin A det'iciencqcousesnight blindness;uifomin D deficiency rickets (in children) or osteomalacia(in adults); uitamin E deficiency minor neurological symptoms;uitamin K deficiency bleeding. Fat soluble uifomins are not readily excreted in urine, hence excesscohsumption leads to their accumulation qnd toxic effects. Vitomin C deliciency cdusesscuruy. The monifestotionsoJ scuruy ore related to the impairment in the synthesisoJ collagen and/or the antioxidant property of uitomin C. ' Megadosesof uitomin C are used in common cold, wound healing, trauma etc. ftCarotene, uitamin E and ascorbic acid serue os entioxidants and reduce the risk oJ heart sttacks and cancers,

Ghapter 7 : VITAMINS

135 Reactive carbon J _

NHz

I )-p ------f----| --T Pyrimidine Methylene Thiazole bridge +l#

O ---l--------

O-

2. cr-Ketoglutaratedehydrogenase ls an enzyme of the citric acid cycle. Thi I nts s enzyme enzvme ts i s comparabl e w i th pyruvate dehydrogenaseand requires TPP.

3. Transketolaseis dependent on TPP. This is an enzyme of the hexose Pyrophosphate monophosphateshunt (HMp shunt). ,

rrv v ' v " v \

4. The branched chain a-keto acid dehydrogenase (decarboxylase) Flg. 7.17: Structures of thiamine andthiamine catalysesthe oxidativedecarboxylation pyrophosphate (TPP)' of branchedchain amino acids (valine, l euci ne and i sol euci ne) to the respectiveketo acids.This enzyme also requires TP P . Thiamine

5. TP P pl ays an i mportant rol e i n the T hiam ine (a n ti -b e ri -b e ri o r a n ti n euri ti c transmissionof nerve impulse. lt is believed that v it am in) is w a te r s o l u b l e . l t h a s a s p eci fi c TPP is required for acetylcholinesynthesisand coenzyme/ thiamine pyrophosphate (TPP)which the i on transl ocati onof neuralti ssue. is mostly associated with carbohydrate m et abolis m . Flecornnrended dietary a!i sw anee { R D A } Chenristry The dai l y requi rementof thi ami ne depends T hiam ine c o n ta i n sa p y ri mi d i n e ri n g and a on the intake of carbohydrate.A dietary supply t hiaz ole r in g h e l d b y a me th y l e n e b ri dge ( F ig. 7. 1V . T h i a mi n e i s th e o n l y n a tural ol 1-1.5 mdday is recommended for adults (about0.5 hrg/1,000C al sof energy).For chi l dren c om pound w i th th i a z o l e ri n g . R D A i s 0.7-1.2 m{day. The requi rement T he alc o h o l (OH ) g ro u p o f th i a mi ne i s marginally increasesin pregnancyand lactation esterfiedwith phosphate(2 moles) to form the (2 mglday),old age and alcoholism. coenzyme, thiamine pyrophosphate (TPP or cocarboxylase). The pyrophosphate moiety is Dietary souree$ donated by ATP and the reaction is catalysed Cereals,pulses,oil seeds,nuts and yeast are by t he e n z y m e th i a mi n e p y ro p h o sphate good sources.Thiamine is mostly concentrated transferase. i n the outer l ayer (bran)of cereal s.P ol i shi ngof rice removesabout 80% of thiamine.Vitamin B, 9it : c hem ic a l fu n e ti e n s is also presentin animal foods like pork, liver, The coenzyme, thiamine pyrophosphateor heart,ki dney,mi l k etc. l n the parboi l ed(boi l i ng cocarboxylaseis intimately connected with the of paddy w i th husk)and mi l l ed ri ce, thi ami nei s energy releasingreactions in the carbohydrate not l ost i n pol i shi ng.S i nce thi ami ne i s a w ater metabolism(FiS.7.lA. soluble vitamin, it is extracted into the water duri ng cooki ng process.S uch w ater shoul d not 1. The enzyme pyruvate dehydrogenase be discarded. catalyses (oxidative decarboxylation) the irreversible conversion of pyruvate to acetyl Deliciency $ymptonns CoA. This reactionis dependenton TPP,besides the other coenzymes (details given in The deficiency of vitamin B1 results in a carbohydratemetabolism,Chapter 13). condition called beri-beri lsinhalese : I cannot

*h

BIOCHEMISTF|Y

136 Glucose ------+ Glucose 6-phosphate

Pyruvate

Oxaloacetate

Ribose5-phosphate

Citrate

(TPP). Fi4.7.18 : Summaryof thereactionsdependenton thiaminepyrophosphate

( s aid tw i c e )1 . Be ri -b e ri i s mo s t l y seen i n popu l a ti o n sc o n s u m i n ge x c l u s i v e l yp ol i shedri ce as staplefood. The early symptomsof thiamine deficiency are loss of appetite (anorexia), weakness, constipation, naLrsea, mental depression, peripheral neuropathy, irritability et c . N u mb n e s si n th e l e g s c o m p l ai ntsof ' pi ns and needlessensations'are reported. Biochemical

changes

in B'' deficiency

3. Thi ami ne defi ci encyl eads to i mpai rment in nerve impulsetransmissiondue to lack of TPP. activity in erythrocytesis 4. The transketolase decreased. MeasuremenLof RBC transketolase activity is a reliable diagnostic test to assess thi ami ne defi ci encv. In adults,two types of beri-beri,namely wet beri-beri and dry beri-beri occur. lnfantile beriberi that differsfrom adult beri-beri is also seen.

1. C a rb o h y d ra te m e ta b o l i s m i s i mpai red. Wet beri-beri : This is characterized by AccumulationoI pyruvate occurs in the tissues edema of legs, face, trunk and serous cavities. which is harmful. Pyruvate concentration in Breathlessness and palpitation are present.The plasmais elevatedand it is also excretedin urine. cal f muscl esare sl i ghtl y sw ol l en. The systolic blood pressure is elevated while diastolic is 2. Normally, pyruvate does not cross the decreased.Fastand bouncing pulse is observed. blood -b ra i n b a rri e r a n d e n te r the brai n. The heart becomesweak and death may occur However, in thiamine deficiency, an alteration due to heart failure. occurs in the blood-brain barrier permittingthe pyruvateto enter the brain directly. lt is believed Dry beri-beri : This is associatedwith neurothat pyruvate accumulation in brain results in Iogical manifestations resulting in peripheral disturbed metabolism that may be responsible neuri ti s. E dema i s not commonl y seen. The weak and walking f or po l y n e u ri ti s . musclesbecomeprogressively

Chapter 7 : VITAMINS

137

becomes difficult. The affected individuals Chemistry depend on support to walk and become Riboflavin contains 5,7-dimethyl isoalloxazine bedridden,and may even die if not treated. (a heterocyclic 3 ring structure) attached to The symptomsof beri-beriare often mixed in D-ribitol by a nitrogen atom. Ribitol is an open which case it is referred ro as mixed beri-beri. chain form of sugar ribose with the aldehyde group (CHO) reducedto alcohol (CH2OH). Infantile beri-beri : This is seen in infants Riboflavin is stable to heat but sensitiveto born to mothers suffering from thiamine deficiency. The breast milk of these mothers light. When exposed to ultra-violet rays of c ont ainslow th i a m i n ec o n te n t.In fa n ti l eb eri -beri sunlight, it is converted to lumiflavin which restlessness, exhibits vellow fluorescence.The substances is characterizedby sleeplessness, v om it ing, co n v u l s i o n sa n d b o u ts o f s c re ami ng namely lactoflavin (from milk), hepatoflavin that resemble abdominal colic. Most of these (from liver) and ovoflavin (from eggs) which symptoms are due to cardiac dilatation. Death were originally thought to be different are structurallyidentical to riboflavin. may occur suddenlydue to cardiac failure. Wernicke-Korsakoff

syndrome

This is a disorder mostlv seen in chronic alcoholics. The body demands of thiamine inc r eas e in a l c o h o l i s m. l n s u ffi c i e n ti n take or im pair ed in te s ti n a la b s o rp ti o no f th i a m i ne w i l l lead to this syndrome.lt is characterizedby loss of memory, apathy and a rhythmical to and fro motion of the eye balls.

Coenzymes

of riboflavin

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the two coenzyme forms of riboflavin. The ribitol (5 carbon)i s l i nkedto a phosphatei n FMN . FA D i s formed from FMN by the transferof an AMP moiety from ATP (Fig,7.19/'. Biochemical

functions

The flavin coenzymes(mostly FAD and to a lesser extent FMN) participate in many redox reactionsresponsiblefor energyproduction.The The enzyme thiaminaseis presentin certain functional unit of both the co€rZln,rs is seafoods.Their inclusion in the diet will destrov isoalloxazinering which servesas an acceptor of t hiam ine by a c l e a v a g ea c ti o n (p y ri mi d i neand two hydrogen atoms (with electrons).FMN or thiazole rings are separated) and lead to FAD undergo identical reversible reactions def ic ienc y .Py ri th i a m i n e a , s tru c tu ra la n a l ogue acceptingtwo hydrogen atoms forming FMNH2 o f th i a mi n e ; i s fo u nd i n or FADH2 and an ant i me ta b o l i te ffi9.7.20). certain plants like ferns. Horsesand cattle often Enzymesthat use flavin coenzymes(FMN or dev elop t hi a m i n e d e fi c i e n c y (fe rn p o i s oni ng) FAD) are called flavoproteins. The coenzymes due to the overconsumptionof the plant fern. (prostheticgroups) often bind rather tightly, to the protein (apoenzyme)either by non-covalent T hiam ine a n ta g o n i s ts bonds (mostly) or covalent bonds in the Pyrithiamine and oxythiamine are the two holoenzyme.Many flavoproteinscontain metal im por t antan ti m e ta b o l i teosf th i a mi n e . atoms(i ron,mol ybdenumetc.)w hi ch are know n as metallofl avoproteins. Thiamine deficiency due to thiaminase and pyrithiamine

The coenzymes,FAD and FMN are associated with certain enzymes involved in carbohydrate, l i pi d, protei n and puri ne metabol i sms,besi des Riboflavinthroughits coenzymes takespart the electrontransportchain. A few examplesare in a variety of cellular oxidation-reduction listed in Table 7,2. Furtherdetails are given in reactions. the respectivechapters,

135

B IOC H E MIS TF\

o

etmfy quirement of tlt is 1.2-1,7 mg. 0.2-0.5 mdday) )r pregnant and ll H_C_OH

,'ees

T

lo

cH2oH L

Mi l k a n d m i l k p ro d u c ts , me at, eggs, liver, kidney are rich sources. Cereals, fruits, vegetablesand fish are moderate sources. Defieienclr

Riboflavin Flavoklnasei

o

symptonrs

Riboflavin deficiency symptoms include cheilosis (fissures at the corners of the mouth), glossitis (tongue smooth and purplish) and dermatitis. Riboflavin deficiency as s uch i s u n c o mmo n .l t i s mo s tl ys e e n along with other vitamin defic ien c i e s . C h ro n i c a l c o h o l i c s a re susceptibleto 82 deficiency. Assay of the enzyme glutathione reductase in erythrocytes will be useful in assessingriboflavin deficiency.

CH, IH-C-OH I H-C-OH I H-C-OH lll

-6

cH2o-P-o-

Flavinmononucleotide(FMN) D svn_thgp.e,

Antimetabolite: Calactoflavin is an a n ti me ta b o l i teo f ri b o fl a v i n .

Niacin or nicotinic acid is also known as pellagra preventive (P.P.) factor oI Coldberg. The coenzymes of n i a c i n (N AD + a n d N AD P+ ) c an be synthesized by the essential am i n o a c i d , try p to p h a n . The disease pellagra (ltalian : rough skin) has been known for centuries. However, its relation to the deficiencyof a dietaryfactorwas first identified by Coldberger. Coldberger and his associates conductedan interestingexperiment

O

o

9Hz I H-C-OH I H-C-OH I

O

H-C-OHO

o-

o-

Flavin adenlne dlnucleotlde (FAD)

Fig. 7,19 : Structuresand biosynthesisof flavin mononucleotide (FMN) and flavin adenine dinucleotide(FADL

139

Ghapter 7 : VITAMINS

I

I H

@

@

Oxidized flavin (FMN or FAD)

Reduced flavin (FMNH2or FADH2)

Fig, 7,20 : Pariicipation of FMN or FAD in oxidation-reduction reactions (R-represents the rest of the structurc of FMN or FAD as depicted in Fiq.7.19).

for this purpose.Twelve convictswere promised pardon if they consumed diet of pellagrous families for one year. The diet consistedof corn meal/ corn starch, rice, sweet potato and pork fat. More than half of the subjects showed symptomsof pellagrain lessthan an year, while no such symptoms were observed in other pr is oner son a re g u l a r d i e t. Ad mi n i s tra ti onof dried meat or liver to the patientscured pellagra (Coldberger, 1928).

nicotine (presentin tobacco leaves).The term ' ni aci n' w as coi ned and more commonl v used for ni coti ni c aci d. Thi s w as done to emohasi ze the role of niacin as a vitamin and avoid the i mpressi onthat ni coti ni c aci d i s an oxi di zed form of nicotine. However, most of the authors use ni aci n and ni coti ni c aci d synonymousl y. GhemistrV amd snrmthesis of coenzymes

Niacin is a pyridine derivative. Structurally,it Much before it was recognizedas a vitamin, nic ot inic ac id w a s w e l l k n o w n a s a c h e m i cal i s pyri di ne3-carboxyl i caci d.The ami deform of compound, produced by the oxidation of ni aci n i s know n as ni aci nami deor ni coti nami de.

Enzyme

FADdependent metabolism l" Carbohydrate (a)Pyruvate complex* dehydrogenase (b)cr-Ketoglutarate dehydrogenase complexx (c)Succinate dehydrogenase ll. Lipidmetabollsm (d)AcylCoAdehydrogenase lll. Proteinmetabolisn (e)Glycine oxidase (f)D-Amino acidoxidase lY. Purinemetabolism (g)Xanthine oxidase

Reaction

Pyruvate ------+AcetylCoA a-Ketoglutarate Succinyl CoA ------+ Succinate -----+Fumarate AcylCoA---+ o, B-Unsaturated acylCoA Glycine -+ Glyorylate + NH3 D-Amino acid-> cl-Keto acid+ NH3 Xanthine ------+Uricacid

FMN dependent L-Amino acidoxidase *

L-Aminoacid---------+a-Keto acid + NHo

componentrequiresFAD Dihydrolipoyl dehydrogenase

.\

B IOC H E MIS TR Y

140 Dietary nicotinamide,niacin and tryptophan ( an es s e n ti a l a mi n o a c i d ) c o n tri b ute to the synthesis of the coenzymes-nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+)" The pathway for the biosynthesisof NAD+ and NADP+ is depicted in Fi9.7.21.Nicotinamide is deam i n a te di n th e b o d v to n i a c i n . N i aci n then undergoesa seriesof reactionsto produce NAD+ and NADP+. Tryptophan produces quinolinate whic h th e n fo rm s n i c o ti n a te m o n onucl eoti de and, ultimately, NAD+ and NADP+. Sixty milligrams of tryptophan is equivalent to I mg of niacin for the synthesis of niacin coenzymes. Phosphoribosylpyrophosphate and ATP, respectively,provide ribosephosphateand AMP moieties for the synthesis of NAD+. Clut am i n ed o n a te sa m i d e g ro u p .l n t he structure of the coenzymes,nitrogenatom of nicotinamide carriesa positivecharge(due to the formationof an extra bond, N is in quaternarystate),hence t he c o e n z y m e s a re N A D + a n d N A D P + . Nicotinamide, liberated on the degradationof NAD+ and NADP+ is mostlv excreted in urine as N -methy Inicoti namide.

Nicotinamide

I

-l

s2

ll

i

N-Methylnicotinamide (excreted in urine)

Nicotinate mononucleotide adenyltransferase

Desamido-NAD*

(Note : Some authors prefer to use NAD/ NADP without a positive charge to represent generalor oxidized form of niacin coenzymes.) Biochemical

------+rryprophan

-NNlacin

Nicotinamide

functions

T he c o e n z v m e s N A D + a n d NA D P + are involved in a variety of oxidation-reduction reactions. They accept hydride ion (hydrogen atom and one electron :H-) and undergo r educ ti o ni n th e p y ri d i n eri n g . T h i s resul tsi n the neutralizationof positive charges.The nitrogen c a rb o n atom of at om a n d th e ' fo u rth nic ot i n a m i d e ri n g p a rti c i p a tei n th e reacti on. W hile o n e a to m o f h y d ro g e n (a s hydri de i on) from the substrate(AH2) is accepted by the coenzyme, the other hydrogen ion (H+) is r eleas e di n to th e s u rro u n d i n gm e d i um. This reaction is reversed when NADH is ox idiz e d to N A D + . N AD P+ a l s o functi ons l i ke NAD+ in the oxidation-reductionreactions. A large number of enzymes (about 40) belonging to the class oxidoreductases are

NADP+

(-O-PO3-)l loH is phosphorylated Fiq.7.21 : Outline of the biosynthesisof nicotinamide nucleotides, NAD" and NADP (NPRT-Nicotinate

ch ap ter 7 : VI TAM I NS

141

H

.Z\>coNH2

coNH2 -+

il

-'--i2

H+

I

Ribose Adenine I @-@-Ribose

@-@-nibose

NAD'(oxidized)

NADH + H+ (reduced)

Ribose Adenine

Overallreaction AH2 + NAD+ HA+ NADH+ H+ Fiq.7.22 : Mechanism ol oxidation and reduction of n icotinami de coenzym e-N AU (Note : Similar mechanism operates for NADP also).

dependenton NAD+ or NADP+.The coenzymes are loosely bound to the enzymes and can be separatedeasily by dialysis.NAD+ and NADP+ par t ic ipate i n a l mo s t a l l th e m e ta bol i sms tcarbohydrate,lipid, proteinetc.).Someenzymes are exclusively dependent on NAD+ whereas some require only NADP+. A few enzymescan use either NAD+ or NADP+. Selectedexamoles of enzymesand the reactionsthey catalyseare given in Table 7.3.

amino acid tryptophancan serve as a precursor for the synthesisof nicotinamidecoenzymes.On an average, i g of a good quality protein containing about 60 mg of tryptophan is equivalent to 1 mg of niacin (conversion ratio 60: 1) for the synthesi s of ni coti nami de coenzymes.Tryptophanhas many other essential and important functions in the body, hence dietary tryptophan cannot totally replace niacin. Increasedconversionof tryptophanto niacin has been reportedin low protein diet and starvation. Tryptophan can replace niacin to an extent of 10o/ofor the synthesisof coenzymes.Therefore, both niacin and tryptophanhave to be invariably provided in the diet. Deficiency

symptonns

N i aci n defi ci encyresul tsi n a condi ti oncal l ed pellagra (ltalian: rough skin). This disease involves skin, gastrointestinaltract and central nervous system.The symptoms of pellagra are commonly referred to as fhree Ds. The disease also progressesin that order dermatitis,diarrhea, dementia, and if not treated may rarely lead to death (4th D).

NADH produced is oxidized in the electron D ermati ti s(i nfl ammati onof ski n) i s usual l y transportchain fo generateATP. NADPH is also found in the areasof the skin exposedto sunlight important for many biosynthetic reactions as it (neck,dorsalpart of feet,ankle and partsof face). donatesreducing equivalents. Diarrheamay be in the form of loosestools,often with blood and mucus. Prolongeddiarrhealeads Rec om m e n d e d d i e ta ry to weight loss. Dementia is associatedwith allowanc e { B D A I degenerationof nervoustissue.The symptomsof dementia include anxiety, irritability, poor T he dail y re q u i re me not f n i a c i nfo r a n adul t i s memory, insomnia(sleeplessness) etc. 15- 20 m g a n d fo r c h i l d re n , a ro u n d 1 0 - 15 mg. Very often, the term niacin equivalents (NE) is Pellagrais mostly seen among people whose us ed while e x p re s s i n gi ts R D A . O n e N E - 1 mg staple diet is corn or maize. Niacin present in niacin or 60 mg of tryptophan. Insteadof mg, mai ze i s unavai l abl eto the bodv as i t i s i n bound t he daily re q u i re me n tsa re k n o w n a s ni aci n form. Further, tryptophan content is low in equivalents.Pregnancyand lactation in women mai ze. im pos e an a d d i ti o n a l m e ta b o l i c b u rd en and increasethe niacin requirement. Therapeuti c uses of ni aei n A dmi ni strati onof ni aci n i n pharmacol ogi cal doses (2-4 g/day, 2OOtimes the RDA) resultsin T he r ic h n a tu ra l s o u rc e so f n i a c i n incl ude a numberof biochemicaleffectsin the body, not liv er , y east, w h o l e g ra i n s , c e re a l s ,p u l ses l i ke relatedto its function as a vitamin. Most of the beans and peanuts. Milk, fish, eggs and effectsare believedto be due to the influenceof veeetablesare moderate sources.The essential ni aci n on cycl i c A MP l evel s. Dietary

source$

142

B IOC H E MISTFI Y

Enzyme

Reaction

NAD+ dependent L Carbohydratemetabolism (a) Glyceraldehyde -----+1, 3-Bisphosphoglycerate 3-phosphate dehydrogenaseGlyceraldehyde 3-phosphate (b) Lactate dehydrogenase (c) Pyruvate dehydrogenase complex (d) a-Ketoglutarate dehydrogenase complex ll. Lipid metabolism (e) B-Hydroxy acylCoAdehydrogenase (f) p-Hydroxybutyrate dehydrogenase I

(g) Alcohol dehydrogenase

Pyruvate -----+Lactate AcetylCoA Pyruvate------+ ----+ Succinyl CoA cr-Ketoglutarate acylCoA acylCoA ------+B-Keto B-Hydroxy p-Hydroxybutyrate ----+ Acetoacetate Alcohol --+ Acetaldehyde

lll. Protein metabolism (h) Branched chainamino acids acidsof branched chainc-ketoaciddehydrogenase cr-Keto (i) Tyramine dehydrogenase

(Leu,lle,Val)-----+Conesponding acylCoAthioesters ----.>p-Hydroxyphenyl acetate Tyramine

NAD+ or NADP+ dependent (a) Glutamate dehydrogenase

+ NH, Glutamate ------+a-Ketoglutarate

(b) lsocitrate dehydrogenase

lsocitrate-----+ Oxalosuccinate

NADP+ dependent (a) Glucose dehydrogenase 6-phosphate

Glucose G-phosphate -----+6-Phosphogluconolactone

(b) Malicenzyme

Malate---+ Pyruvate

NADPH dependent (a) 3-Ketoacyl reductase (b) HMGCoAreductase (c) Squalene epoxidase

enzyme--> 3-Hydroxy acylenzyme 3-Ketoacyl HMGCoA--+ Mevalonale oxide ----+ Squalene Squalene

(e) Phenylalanine hydroxylase

---+ 7o-Hydroxy cholesterol Cholesterol -----+Tyrosine Phenylalanine

(f) Dihydrofolate reductase

Folicacid ---+ Tetrahvdrofolic acid.

(d) Cholesterol 7a-hydrorylase

1. Niacin inhibits lipolysisin the adipose of hyperlipoproteinemia type II b (elevation of tissueand decreases the circulatoryfree fatty LD L and V LD L). acids. Although megadosesof niacin are useful for 2 . T ri a c y l g l y c e ro ls y n th e s isi n the decreased.

the treatmentof hyperlipidemia,there are certain harmful side effectsalso. 'l . Clycogen and fat reservesof skeletaland

3. The serum levels of low density lipoproteins(LDL),very low density lipoproteins cardi acmuscl e are depl eted. (VLDL), triacylglycerol and cholesterol are 2. Thereis a tendencyfor the increasedlevels lowered. Hence niacin is used in the treatment of gl ucoseand uri c aci d i n the ci rcul at ion.

143

Chapter 7 : VITAMINS

3. Prolonged use of n i a c i n re s u l t s i n elevated serum levels of certain enzymes/ suggestingliver damage.

HO

cH2oH

HsC

V it am in 8 6 i s u s e d to c o l l e c ti vel Y represent the three compounds namely pyridoxine, pyridoxal and pyridoxamine (the vitamers of 85,). Chemistry Vitamin 86 comPounds are PYridine derivatives. They differ from each other in the structureof a functional group attached t o 4t h c ar b o n i n th e p y ri d i n eri n g . Py ri d oxi ne is a primaryalcohol, pyridoxalis an aldehyde f or m whi l e p y ri d o x a mi n e i s a n a mi ne (Fig.7.23).Pyridoxamineis mostly presentin plant swhi l e p y ri d o x a la n d p y ri d o x a mi n eare f ound in a n i ma l fo o d s . P y ri d o x i n ec a n be converted to pyridoxal and pyridoxamine, but the latter two cannot form pyridoxine' Symtlresis

Pyridoxal ptto6ph#

o{' coenzYme

The active form of vitamin 86 is the coenzyme pyridoxal phosphate (PLP). PLP from the threecompounds can be synthesized py r idox in e ,p y ri d o x a la n d p y ri d o x a mi n e'86 is excretedin urine as 4-pyridoxic acid. The different forms of 86 and their interrelationshipare depicted in Fig.7.23.

Pyridoxine

tl'PYridoricacid

Fiq.7.23 : Pyridoxine, its derivatives and coenzyme.

1. Transamination: Pyridoxal phosphate is involved in the transamination reaction (by { u n e tE o n s #ic c hem i c a l transaminase)converteingamino acids to keto Pyridoxal phosphate(PLP),the coenzyme of acids. The keto acids enter the citric acid cycle vitamin 86 is found attached to the e-amino and get oxidized to generateenergy.Thus 86 is gr oup of l y s i n e i n th e e n z y m e ' PL P i s cl osel y an energy releasing vitamin. lt integrates associated with the metabolism of amino carbohydrate and amino acid metabolisms acids. The synthesis of certain specialized (Fig.7.24). products such as serotonin, histamine, D uri ng the course of transami nati on,P LP niac in co e n z y me s fro m th e a m i n o aci ds i s with amino acid to form a Schiff base interacts phosphate dependent on pyridoxine' Pyridoxal (Fig.7.25). The amino group is handed over to participates in reactions like transamination, pyridoxamine phosphateand the to form PLP d ecarboxyl ation, deamination, transsulfuration, liberated. is acid keto condensation eIc.

B IOC H E MIS TFIY

144 Glucose

t

impulses,hence it is an inhibitory neurotransmitter. GABA

Glutamate

coz

J

(d) The synthesisof catecholamines(dopamine, norepinephrine and epinephrine) from tyrosine require PLP. Catecholamines are involved in metabolic and neryous regulation.

O:C -H

I -ol4-'rcH2o-@ ttl

H

\

R-C-COOFiq.7.24 : Pyridoxalphosphate (PLP) integratesamino acid and carbohydrate metabolisms (Alanine and aspartate arc the amino acids respectively converted to pyruvate and oxaloacetate, the keto acids).

H3C\i-2

-N: H -I-H H Aminoacid

I H Pyridoral phosphate

2. Decarboxylation : Some of the cr-amino acids undergo decarboxylation to form the respectiveamines.This is carriedout by a group of enzymes called decarboxylases which are depen d e n to n PL P .Ma n y b i o g e n i cami nesw i th important functions are synthesized by PLP decarboxylation. (a) Serotonin (S-hydroxytryptamine,5 HT), producedfrom tryptophanis importantin nerve impulse transmission(neurotransmitter). lt regulates sleep, behaviour, blood pressureetc.

Schifl base

-----+ 5-Hydroxytryptophan Tryptophan

Coz 5-Hydroxytryptamine (b) Histamine is a vasodilator and lowers blood pressure.lt stimulatesgastric HCI secretionand is involved in inflammation and allergic reactions.

R-C-COOtl

o

o-Keto acid

Histamine

Histidine

coz (c) Glutamate on decarboxylation gives yamino butyric acid (CABA). CABA inhibits the transmission of nerve

Fig.7.25 : Formationof Schiff base in transamination'

chapter 7 : VITAMINS

745

Tryptophan Threonine

i

o-Ketobutyrate + NHg

Y

3-Hydrorykynurenine

NAD+ NADP+

FiS. l.?6 | Roteof pyridoxinein tryptophan metabolism(pLp-pyridoxalphosphate).

Tyrosine--+ DOPA

Dopamine___+

coz __+Epinephrine Norepinephrine

7. Serineis synthesizedfrom glycine by plp a . dependentenzyme hydroxymethyltr"nsfer"ru.

9. PLP is neededfor the absorptionof amino acids from the intestine. i 0. Adequateintakeof 86 is usefulto prevent hyperoxaluriaand urinary stone formation. Reconnmended dietary al l ow ance (R D A I

3. Pyridoxal phosphate is required for the synthesisof &amino levulinic acid, the precursor for heme synthesis. Glycine Succinyl CoA

....|Heme 6-Aminolevulinic acid(ALA)

Dietary sources

e xc r et ion of x a n th u re n a te i n i n dic at ionof 86 d e fi c i e n c y .

u ri n e i s a n

5. PLPplaysan importantrole in the metabo_ lism of sulfur containingamino acids(Fig.7.27). Transsulfuration(transferof sulfur) from homo_ cysteine to serine occurs in the synthesis

A ni mal sourcessuch as egg yol k, fi sh, mi l k, meat are rich in 86. Wheat, corn, cabbage, roots and tubers are good vegetablesources. Methionine ---+ Homocysterne

Cystathionine

6. Deaminationof hydroxylgroup containing a m ino ac ids r equi re sp L p . Serine

Pyruvate + NH3

Fiq.7.27 : Role of pyridoxine in the metabolism of sy\ anino acids (pLp-pyridoxat pnospniti).-

BIC]CHEMISTFIY

146 t-,;;;j i,."ian81, $rf ?r!Storrrs

Toxic

Pyridoxine deficiency is associated with neurological symptoms such as depression, i rri ta b i l i tv ,n e rv o u s n e s sa n d mental confusi on. C o n v u l s i o n s a n d p e ri p h e ra l neuropathy are observedin severedeficiency.These symptoms related to the decreased svnthesis of are (s e ro toni n, a mi n e s CABA, biogenic l n chi l dren,86 n o re p i n e p h ri naen d e p i n e p h ri ne). d e fi c i e n c y w i th a d ra s ti c a l l y reduced C A B A p ro d u c ti o nre s u l tsi n c o n v u l s i ons(epi l epsy).

Excessuse of vitamin 86 Q.5 g/day) in the women of premenstrualsyndrome is associated with sensory neuropathy. Some workers have that vitamin 86 more than 200 mg/day suggested may cause neurologicaldamage.

effects

of overdose

vitamiil

B6

Biotin (formerly known as anti-egg white Decrease in hemoglobin levels, associated i nj ury factor,vi tami n 87 or vi tami n H ) i s a sulf ur w i th h y p o c h ro m i cmi c ro c y ti c anaemi a,i s seen contai ni ngB -compl exvi tami n.l t di rectl ypar t iciin 86 deficieny. This is due to a reduction in pates as a coenzyme in the carboxylation heme production. reactions. T h e s y n th e s i so f n i a c i n c o enzymes(N A D + Boas (l 927) observed that rats fed huge and NADP+) from tryptophan is impaired. quantity of raw egg white developeddermatitis Xanthurenic acid, produced in high quantities is manifestations,besidesretardation nervous and excreted in urine, which serves as a reliable growth. however, found that feeding She in index (particularlyafter tryptophanload test)for did not produce any of these egg cooked 86 deficiency. symptoms.lt was later shown that the egg white Dietarydeficiencyof pyridoxineis ratherrare i nj ury i n ratsand chi cksw as due to the p r esence and is mostly observed in women taking oral of an anti-vitamin in egg white. The egg-white contraceptives,alcoholics and infants. injury factor was identified as a glycoproteinavidin and biotin was called as anti-egg white *i *i..r lr-rtirr:ils F,i deficienerr injury factor. Isoniazid(isonicotinicacid hydrazide,INH) is a drug frequently used for the treatment of Shemistry tuherculosis. lt combines with pyridoxal B i oti n i s a heterocycl i c sul fur cont aining phosphateto form inactivehydrazonederivatives monocarboxylicacid. The structureis formed by w h i c h i n h i b i t P L P d e p e ndent enzymes. fusion of imidazole and thiophene rings with a Tuberculosis patients, on long term use of val eri c aci d si de chai n (Fi g.7.2A . Biot in is l w hi ch i s o n i a T i dd, e v e l o p p e ri p h e ra neuropathy coval entl ybound to e-ami nogroup of l ysine t o respondsto B6 therapy. form biocytin in the enzymes.Biocytin nray be The drug penicillamine (B-dimethyl cysteine) regarded as the coenzyme of biotin. is used in the treatment of oatients with rh e u m a to i d a rth ri ti s , Wi l s o n ' s di sease and Site for cystinuria.This drug also reactswith PLPto form i n a c ti v eth i a z o l i d i n ed e ri v a ti ve. Ad mi n i s tra ti o n o f d ru g sn a m el y i soni azi dand p e n i c i l l a mi n es h o u l db e a c c o m pani edby pyri doxine supplementationto avoid 86 deficiency. + ' :-r'lil,:r:r1n€ iEiltaggniSts lsoniazid, deoxypyridoxine and methoxy pyridoxine are the antagonistsof vitamin 86.

Fiq.7.28 : Structure of biotin with binding sites.

Chapter 7 : VITAMINS Biochernical

147

functions

Biotin serves as a carrier of COz in carboxylation reactions. The reaction cataiysed by pyruvate carboxylase, converting pyruvate to oxaloacetatehas been investigatedin detail.This enzyme has biotin bound to the apoenzyme linked to the e-amino group of lysine, forming the activeenzyme (holoenzyme).Biotin-enzyme reactswith CO2 in presenceof ATP (provides energy) to form a carboxybiotin-enzyme c om plex .T h i s h i g h e n e rg yc o m p l e x h a n dsover the CO2 to pyruvate(carboxylationreaction) to produce oxaloacetate(Fig.7.20.

are dependenton biotin. This was later proved to be wrong. There are a few carboxylation reactions which do not require biotin e.g. formationof carbamoylphosphatein urea cycle, incorporationof CO2 in purine synthesis.l Recosnmended dietary (RDAI allowance

A daily intake of about 100-300 mg is recommended for adults. In fact, biotin is normally synthesizedby the intestinalbacteria. However, to what extent the synthesizedbiotin contributes to the body requirements is not As a coenzyme,biotin is involved in various cl earl v know n. metabolic reactions. Metary sources 1 . Gluconeogenesisand citric acid cycle : B i oti ni s w i del y di stri butedi n both ani maland The conversion of pyruvate to oxaloacetate plant foods. The rich sourcesare liver, kidney, by biotin dependent pyruvate carboxylase (describedabove) is essentialfor the synthesisof egg yolk, milk, tomatoes,grains etc. glucose from many non-carbohydratesources. synrptonrs Oxaloacetateso formed is also required for the Deficiency continuousoperation of citric acid cycle. The symptoms of biotin deficiency include 2. tatty acid synthesis : Acetyl CoA is the anemia, loss of appetite, nausea, dermatitis, startingmaterial for the synthesisof fatty acids. The very first step in fatty acid synthesisis a carboxylationreaction. Acetyl CoA

Biot in Acetyl CoAcarboxylase

M alon y l C o A

3. Propionyl CoA is produced in the metabolis m of c e rta i n a mi n o a c i d s (v a l i n e , isol eucine, threonine etc.) and degradation of odd chain fatty acids. lts further metabolism is dependento n b i o ti n . Propionyr coA

Bioti:.--

..-- ) PropionylCoAqafbq,rylase Methylmalonyl CoA

4. ln the metabolismof leucine,the following reaction is dependenton biotin.

0 il

z)

Blotin-Enz

Carboxybiotln-enzyme complex

o tl

cH3-c-cocr Pyruvate U tl *s-c-cH2-c-coo-

CoA B-Metlrylcrotonyl

Oxaloacetate glutaconyl CoA B-Methyl lNote : lt was once believed that all the carboxylationreactionsin the biological system

Fig.7.29: Roleof biotinin thecatuorylationreaction, catalysed by the enzyme pyruvate cafuorylase (Enz-Enzyme).

148

B IOC H E MIS TR Y

glossitisetc. Biotin deficiency may also r es ult i n d e p re s s i o n , h a l l u c i n a ti ons, m us c l e p a i n a n d d e rm a ti ti s , Biotin deficiency is uncomrnon, since it is well distributed in foods and also supplied by the intestinal bacteria. The deficiency may however, be associated with the following two causes. 1. Destructionof intestinalflora due to prolonged use of drugs such as sulfonamides.

Pantothenlcacld

l'^rP l'root +

4'-Phosphopantothonate Cvsteine-.LzATP v

2. H i g h c o n s u mp ti o no f ra w e g g s.The raw egg white contains a glycoproteinavidin, which tightly binds with biotin and blocks its absorption from the intestine.An intakeof about 20 raw eggs per day is needed to produce biotin def ic i e n c y s y m p to m s i n h u mans. Consumption of an occasional raw egg will no t re s u l ti n d e fi c i e n c v .

l)noP

4'-Phosphopantothenyl cysteine

+'-Phosphoiantetheine

lrlr:P t IYPPi +

Dephospho-coenzyme A I

A nt ag o n i s ts

J Coenryme A

De s th i o b i o ti n ,b i o ti n s u l p h o n i c aci d are biotin antagonists. OH ilt

l*-3-"*2-cH2-sH Pantothenic acid (Creek : pantoseverywhere),formerly known as chick anti-dermatitisfactor (or filtrate factor) is widely d i s tri b u te d i n n a tu re . l t' s metabolic role as coenzyme A is also widespread. Chemistry and synthesis of coenzyme A

I oCoenryme A

A of coenzyme Fiq.7.30: (A) Summaryol the synthesis Pantothenic acid consists of two frompantothenicacid (B) Structureof coenzymeA. components,pantoic acid and p-alanine, held togetherby a peptide linkage.Synthesisof coenzymeA from pantothenateoccurs in a series consists of pantothenic acid joined to (thioethanolamine) of reactions (Fi9,7.30). Pantothenate is first p-mercaptoethanolamine at phosphorylatedto which cysteine is added. one end.On the otherside,pantothenic acid is Decarboxylation,followed by addition of AMP heldby a phosphate bridgeto adenylicacid.The moiety and a phosphate(each from ATP) results adenylicacid is made up of adenine,and a in coenzyme A. The structureof coenzyme A phosphate linkedto carbon-3of ribose.

Chapter

149

7 : VITAMINS

o

O ilil| R-C-S-CoA Acyl CoA

tl CHr-C-S-CoA

O

H3C-C-S-CoA CH2-COOH Acetyl CoA SuccinylCoA

Fig.7.31 : Selected examples of compounds bound to coenzymeA.

Biochemical

SuccinylCoA+ Acetoacetate-----+AcetoacetylCoA + Succinate Coenzyme A may be regardedas a coenzyme of metabolic integration, since acetyl CoA is a central molecule for a wide variety of biochemical reactions,as illustratedin Fig.7.32. Succinyl CoA is also involved in many reactions,including the synthesisof porphyrins of heme.

functions

The functionsof pantothenicacid are exerted through coenzyme A or CoA (A for acetylation). Coenzyme A is a central molecule involved in all the metabolisms (carbohydrate,lipid and pr ot ein) . l t p l a y s a u n i q u e ro l e i n i n tegrati ng various metabolic pathways. More than 70 enzymes that depend on coenzyme A are k nown. CoenzymeA has a terminalthiol or sulfhydryl group (-SH) which is the reactive site, hence CoA-SH is also used. Acyl groups (free fatty acids) are linked to coenzyme A by a thioester bond, to give acyl CoA. When bound to acetyl unit, it is called acetyl CoA. With succinate, succinyl CoA is formed. There are many other compounds bound to coenzyme A. Coenzyme A serves as a carrier of activated acetyl or acyl groups (as thiol esters).This is comparable with ATP which is a carrier of activatedphosphorylgroups. A few examples of enzymes involved the participationof coenzymeA are given below. Pyruvate

Besides the various functions through coenzyme A, pantothenic acid itself is a component of fatty acid synthase complex and is involved in the formation of fatty acids. Recommended dietary (R D A I al l ow ance The requirement of pantothenic acid for humansi s not cl earl y know n. A dai l y i ntake of about 5-10 mg is advisedfor adults.

Dietary sources Pantothenicacid is one of the most widely distributedvitaminsfound in plantsand animals. The rich sourcesare egg, liver, meat, yeast,milk etc. Deficiency

symptoms

It is a surpriseto biochemiststhat despitethe involvementof pantothenicacid (ascoenzymeA) in a great number of metabolic reactions, its

Acetyl CoA Pyruvate dehydrogenase

Carbohydrates

Aminoacids

Fafty acids

Cholesterol

Ketone Detoxibodies fication

o-Ketoglutarate c.Ketoglutaratedehydr€€nase Succinyl CoA AcylCoA

Fattyacid ThioHnass

TCA cycle

I

FatV acids

I

I

I

In some of the metabolic reactions, group + + + transferis importantwhich occursIn a coenzyme Energy TriacylVitaminD, Energy glycerolssteroidhormones A bound form. AcetylCoA + Choline-----+Acetylcholine + CoA AcetylCoA + Oxaloacetat€---)

Citrate+ CoA

Fiq.7.32 : An overview of formationand

150 deficiency manifestationshave not been reportedin humans.This may be due to the widespread distribution of this vitamin or the symptomsof pantothenicacid may be s i m i l a r to o th e r v i ta mi n def i c i e n c i e sD . r. C o p a l a n , a w o rl d renowned nutritionist from lndia, linked the burning feet syndrome ( pa i n a n d n u mb n e s s i n th e to es, sleeplessness,fatigue etc.) with pantothenicacid deficiency.

BIOCHEMISTFIY

OH

8.tl-9t-"oo i

cHo

i

9Hz

;l

cooParaamino benzoicacid

Glutamate

Pteroic acid

Pantothenic acid deficiency in ex p e ri m e n ta l a n i m a l s re s u l ts i n anemia, fatty liver, decreased steroid synthesisetc.

Fo l i c a c i d o r fo l a c i n (L a ti n : f oli u m-l e a f) i s a b u n d a n tl y fo u n d in green leafy vegetables. lt is important for one carbon metabolism and is required for the synthesisof certain amino acids, pur i n e sa n d th e p y ri m i d i n e -th y mi ne.

5,6,7,8-Tetrahydrofolic acid

cooFtq.7.33 : Conversionof folic acid to tetrahydrofolicacid (THF).

intestine. The enzyme folate conjugasepresent i n duodenum and j ej unum spl i tsthe gl utam at e Chennistry residues.Only the monoglutamateof folic acid Folic acid consists of three components- is absorbedfrom the intestine.However, inside pt e ri d i n eri n g ,p -a mi n ob e n z o i ca ci d (P A B A )and the cells, tetrahydrofolates are found as glu ta mi ca c i d (1 to 7 re s i d u e s ). F ol i c aci d mostl y polyglutamates(with 5-6 amino acid residues) g l u ta mi c re s i d u e has o n e acid a nd i s know n as derivatives,which appearto be biologicallymost pteroyl-glutamic acid (PGA). potent. As polyglutamate, folic acid is stored to The active form of folic acid is some extent in the liver. The body can store tetrahydrofolate (THF or FHl. lt is synthesized 10-12 mg of fol i c aci d that w i l l usual l yl a st f or from folic acid by the enzyme dihydrofolate 2-3 months. In the ci rcul ati on, N s-m et hyl reductase. The reducing equivalents are tetrahydrofolateis abundantlypresent. provided by 2 moles of NADPH. The hydrogen functions at o m s a re p re s e n ta t p o s i ti o n s5 ,6,7 and 8 of Biochenrical THF (Fig.7.33). (THF or FHa),the coenzyme Tetrahydrofolate of fol i c aci d, i s acti vel y i nvol ved i n the one Absarption, transport and storage carbon metabolism. THF serves as an acceptor Most of the dietary folic acid found as or donor of one carbon units (formyl, methyl polyglutamatewith 3-7 glutamateresidues(held etc.) in a variety of reactions involving amino by peptide bonds) is not absorbed in the aci d and nucl eoti demetabol i sm.

Chapter 7 : VITAMINS

151

The one carbon units bind with THF at liberationof free THF and for its repeateduse in pos it ion y s 6 1 y 1 0 o r o n b o th N s a n d N l o of one carbon metabolism. In Btz deficiency, pteroyl structure. The attachment of formyl conversionof Ns-methylTHF to THF is blocked (-CHO) at position 5 of THF gives Ns-formyl (more details given under vitamin 812). tetrahydrofolatewhich is commonly known as folinic acid or citrovorum factor. The other Reeomnrenried die*ary commonly found one carbon moietiesand their allowance {R$A} binding with THF are given below. The daily requirementof folic acid is around 20O 1tg. In the women, higher intakes are H recommended during pregnancy @0O p{day) and lactation (300 pglday). CH-(Glu)n Dietary

THF-I carbon derivative N5-Formyl THF

s$snrrees

Folic acid is widely distriburedin nature.The rich sources are green leafy vegetables,whole R group(onecarbon unit) grains, cereals, liver, kidney, yeast and eggs. Milk is rather a poor source of folic acid. -cHo

N1o-Formyl THF

-cHo

N5-Formimino THF

-CH=NH

Deficfi*mey

syrynptoms

Folic acid deficiency is probably the most common vitamin defi ciency, observed pri mari Iy in the pregnant women, in both developed -CHs N5- Methyl THF (i ncl udi ng U S A ) and devel opi ng countri es The essentialfunctionsof THF in one carbon (includingIndia).The pregnantwomen, lactating metabofism are summarized in Fig.7.34. women/ women on oral contraceptives,and The interrelationshipbetween the various alcoholics are also susceptible to folate 1- c ar bon T H F d e ri v a ti v e s a l o n g w ith thei r involvement in the synthesis of different Glycine, serine Onecarbon(1C) compounds is given in Fig.l5.32 (Chapter 1fl. histidine etc. donors Many important compounds are synthesizedin I Y one carbon metabolism. N5,N1o-Methenyl THF

N5,Nlo-Methylene THF

:CHz

One carbon (1C) moiety

1. P uri n e s(c a rb o n2 , 8 ) w h i c h a re i ncorporated into DNA and RNA. 2. Pyrimidine nucleotide-deoxythymidylic acid (dTMP),involved in the synthesisof DNA.

1C_THF

3. Clyc i n e ,s e ri n e ,e th a n o l a m i n ea n d chol i ne are produced. 4. N- Fo rmy l me th i o n i n e , th e protein biosynthesisis formed.

i n i ti ator of

Tetrahydrofolate is mostly trapped as Ns-methylTHF in which form it is presentin the circulation. Vitamin Btz is needed for the conversion of Ns-methyl THF to THF, in a reaction wherein homocysteineis convertedto methionine. This step is essential for the

Onecarbonmoiety(1C) acceptedfor the synthesisof

purines Aminoacids Purines Thymidylate Choline (glycine, serine) (2,8 carbons) Fiq.7.34 : An overview of one carbon metabolism (THF-Tetrahydrofolate).

152 deficiency.The folic acid deficiencymay be due to (one or more causes) inadequate dietary intake, absorption, defective use of anti convulsant drugs (phenobarbitone, d i Iantin, phenyltoin), and increaseddemand. In folic acid deficiency,decreasedproduction of purinesand dTMP is observedwhich impairs DNA synthesis. Due to a block in DNA synthesis, the maturation of erythrocytesis slowed down leading to macrocytic RBC. The r api d l y d i v i d i n g c e l l s o f b o n e marrow are seriously affected. The macrocytic anemia (abnormally large RBC) associated with megaloblasticchanges in bone marrow is a characteristicfeature of folate deficiency.

BIOCHEMISTFIY

reductaseand block the formation of THF. The biosynthesisof purines,thymine nucleotidesand hence D N A i s i mpai red. Thi s resul ts i n t he blockage of cell proliferation. Aminopterin and methotrexate are successfully used in the treatment of many cancers, including leukemia. Trimethoprim (a component of the drug septran or bactrim) and pyrimethamine (antimalarialdrug) are structurallyrelatedto folic acid. They inhibit dihydrofolatereductase,and the formation of THF.

S ul fonami des:Fol i c aci d, as such, cannot enter bacterial cells. However, bacteria can synthesizefolic acid from pteridine, PABA ano glutamate.Sulfonamides are structural analogues pregnant Folic acid deficiencyin women may of PABA.They competitivelyinhibit the enzyme cause neural defects in the fetus. Hence high (dihydropteroatesynthase)responsiblefor the doses of folic acid are recommended in incorporationof PABA into pteridineto produce pregnancyto prevent birth defects,. folic acid. For this reason,sulfonamidesare used as antibacterialdrugs. Sulfonamides,have no Folic acid is associatedwith the metabolism effect on human body, since folic acid is not of h i s ti d i n e . F o rmi m i n o g l u ta mate (FIC LU ), synthesizedand supplied through the diet. formed in histidinemetabolismtransfersthe one carbon fragment,formimino group (-CH=NH) to tetrahydrofolateto produce Ns-formimino THF. In case of folic acid deficiency, FIGLU accumulatesand is excreted in urine. Histidine Vitamin 812 is also known as anti-pernicious load test utilizingthe excretionof FIGLU in urine anemia vitamin. lt is a unique vitamin, is used to assessfolic acid deficiencv. synthesizedby only microorganismsand not by animalsand plants.lt was the last vitamin to be F oli c a c i d a n d discovered. hyperhomocystelnemia Elevatedplasma levels of homocysteineare Ghemistry associatedwith increasedrisk of atherosclerosis, V i tami n B tz i s the onl y vi tami n w i th a thrombosis and hypertension. Hyperhomocysteinemia is mostly due to functional folate complex structure. The empirical formula of deficiencycausedby impairmentto form methyl- vi tami n B 12 (cyanocobal ami n)i s C 63H 9sN1a tetrahydrofolate by the enzyme methylene OlaPCo. The structureof vitamin B12consistsof tetrahydrofofate reductase (See Fig.7.39. This a corrin ring with a central cohalt afom. The results in a failure to convert homocysteineto corri n ri ng i s al most si mi l ar to the tetrapy r r ole methionine.Folic acid supplementationreduces ring structure found in other porphyrin hyperhomocysteinemia, and thereby the risk for compoundse.g. heme (w i th Fe) and chl orophyll (with Mg). v ar i o u sh e a l th c o m p l i c a ti o n s . F oli c a c i d a n ta g o n i s ts Aminopterinand amethopterin(alsocalled as methotrexate)are structural analoguesof folic acid. They competitively inhibit dihydrofolate

The corrin ring has four pyrrole units,just like porphyrin. Two of the pyrrole units (A and Dt a are directly bound to each other whereas the other two (B and C) are held by methenebridges The B roups namel y methyl , acetami de and

Ghapter 7 : VITAMINS

153 (a) SlDeoxyadenosyl cobalamin, cyanide is replaced by 5' deoxyadenosineforming an unusal carbon cobalt bond.

AI

lle.

-N

r-{

-N

N-<

CHs CHz

(b) Methyl cobal ami n i n w hi ch cyani de i s replacedby methyl group. Absorption,

transport

and storage

The vi tami n B 12 i s presenti n the di et i n a bound form to proteins.B12 is liberatedby the enzymes (acid hydrolases)in the stomach.The dietary sourceof B12is known as extrinsic factor of Castle.The stomachsecretesa specialprotein called intrinsic factor (lF). lt is a glycoprotein (8-15% carbohydrate) w i th a mol ecul arw ei ght

c Ho[]o Flg. 7.35: Structureof vitamin8,, (cyanocobalamin).

propionamideare the substituents on the pyrrole r ings . V it am i n Btz h a s c o b a l t a to m i n a coordination state of six. Cobalt presentat the centre of the corrin ring is bonded to the four pyrrole nitrogens.Cobalt also holds (below the corrin plane) dimethylbenzimidazole (DMB) c ont aining ri b o s e 5 -p h o s p h a te a n d a m i noisopropanol. A nitrogen atom of dimethylbenz im idazo l ei s l i n k e d to c o b a l t. T h e ami de group of aminoisopropanolbinds with D ring of corrin. The cobalt atom also oossesses a sixth substituentgroup located above the plane of corrin ring (Fig.7.35).The substituentgroup may be one of the following

illeffiylcobalamin

1. Cy ani d e(p re d o m i n a n t) i n c y a n o c o b al ami n

(Brz") 2. Hydroxyl in hydroxycobalamin(B126) 3. Nit r it e i n n i tro c o b a l a m i n(8 1 2 6 ). Thereare two coenzymeforms of vitamin 812 (Fig.7.36).

S'-Deoxyadenosylcobalamin Fiq.7.36 : Coenzyme derivativesof vitamin 8., (Note: Corrin ring represented diagtammatically is identical in all; DMB-Dimethylbenzimidazole).

154

B ]OC H E MIS TRY

DeoxyadenosylBj2

Fig. 7.37: Absorption,transpoftand storageof vitaminBr, (lF-lntrinsicfactor;TC-Transcobalamins (TC-|, TC-ll).

around 50,000. Intrinsic factor is resistantto Methylcobalaminwhich is in excessis taken up proteolyticdigestiveenzymes.lF generallyforms by the liver, convertedto deoxyadenosyl812 and a dimer, binds strongly with 1 or 2 moles of stored in this form. lt is believed that liver can v it a mi n 8 1 2 . T h i s b i n d i n g p ro te c tsvi tami n 812 store about 4-5 mg, an amount sufficientto meet againstits uptake and use by bacteria. the body requirementsof 812 for 4-6 years. The cobalamin-lF complex travels through the gut. The complex binds to specificreceptors on the surfaceof the mucosalcells of the ileum. The binding of the complex and entry of 812 into the mucosal cells is mediated by Ca2* ions. In the mucosal cells, Btz is converted to methyfcobalamin (Fig.7.SV. lt is then transported in the circulation in a bound form to proteins (TC-|, namely transcobalamins TC-ll). Methylcobalaminis mostly bound to TC-l (90%) and to a lesser degree to TC-ll (10%). lt is believed that TC-l acts as a repository of 872, whife TC-ll mediatesthe tissue uptake of 812.

Biochemical

functions

A bout ten enzymes requi ri ng vi tami n B12 have been identified.Most of them are found in bacteria (glutamate mutase, ribonucleotide reductaseetc.). There are only two reactionsin mammal sthat are dependenton vi tami n 8 12. 1. Synthesis of methionine from homocysteine : Vitamin 812, ds methylcobalaminis used in this reaction. This is an important reaction involving Ns-methyl tetrahydrofolate from which tetrahydrofolate is Iiberated (enzyme-homocysteine methyltransferase or

155

C-a ote r 7 : VI TAM I NS

npthionine synthase). This metabolic step ; Enifiesthe interrelationbetween vitamin 812 -'.d folic acid (detailsgiven later)

Odd chain fatty acids

I

Amino acids (Val,Ile, Thr, Met)

I

I

Thymine, uracil

N5.M r:nocystein

tl C-S-CoA

Methionine

H.rC-C-H I

2. lsomerization of methymalonyl CoA to rrccinyl CoA : The degradationof odd chain ;att)' acids, certain amino acids (valine, r = oleuc inee tc .) a n d p y ri m i d i n e s(th y m i ne and -racil) producedirectly or throughthe mediation of propionyl CoA, an important compound nethylmalonyl CoA. This is converted by the enzyme methylmalonyl CoA mutaseto succinyl CoA in the presence of B.tz coenzyme, deoxyadenosyl cobalamin (Fig.7s$. This .eaction involves hydrogen transfer and intramolecularrearrangement. In 812 deficiency, methylmalonylCoA accumulatesand is excreted in ur ine as m e th y l m a l o n i ca c i d .

cooMethylmalonyl CoA

unne

o tl C-S-CoA I QHz I

r^U

Y"i

cooSuccinylCoA

Recommended dietary allowanc e { n O A l A daily intakeof about 3 pg of vitamin B12is adequate to meet the adult requirements.For children, 0.5-1.5 ttg/day is recommended. During pregnancyand lactation,the requirement is 4 1tg/day.

YV

Citricacidcycle

Porphyrins

Fig. 7.38: Roleof vitaminB, in isomerization of methylmalonylCoAto succinylCoA (t-Blockade in 8,, deficiency).

characterized by low hemoglobin levels, decreased number of erythrocytes and Diet ar y s o u rc e s neurologicalmanifestations. One or more of the Foods of animal origin are the only sources following causesare attributedto the occurrence for vitamin B12. The rich sources are liver, of perni ci ousanemi a. k idney ,m ilk , c u rd , e g g s ,fi s h , p o rk a n d c hi cken. 1. Autoimmunedestructionof gastricparietal Curd is a better source than milk, due to the cells that secreteintrinsicfactor. In the absence synthesisoI 812 by Lactobacillus. of l F, vi tami n 812 cannot be absorbed. Vitamin 812 is synthesized only by micro2. Hereditarymalabsorptionof vitamin B1r. organisms (anaerobic bacteria). Plants cannot 3. Partial or total gastrectomy-theseindivisynthesize,hence 812 is never found in plant duals become intrinsicfactor deficient. foods.Animalsobtain 812either by eatingfoods, derivedfrom other animalsor from the intestinal 4. Insufficientproductionof lF and/or gastric bacterialsvnthesis. H C l , occasi onal l yseen i n ol der peopl e. Deileierrcy

syrnptoms

The most important diseaseassociatedwith vitamin 812deficiencyis perniciousanemia.lt is

5. Dietary deficiency of 81, is seen among the strict vegetarians of low socioeconomic group in the developingcountries(lndia,Srilanka etc.).

1'

156

B IOC H E MIS TRY

In vitamin B12 deficiency, increasedfolate From the foregoingdiscussion,it is clear that pernicious anemia is more a disease of the levelsare observedin plasma.The activity of the methyltransferase homocysteine stomachthan due to the deficiencyof vitamin B12. enzyme (methionine synthase)is low in 812 deficiency. Btz deficiency is also associated with As a result, the only major pathway for the neuronal degeneration and demyelination of conversionof Ns-methylTHF to tetrahydrofolate nervous system. The symptoms include is blocked and body THF pool is reduced. paresthesia(numbnessand tingling) of fingers Essentiallv. almost the entire bodv folate and toes. In advancedstages,confusion,loss of becomes trapped as Ns-methyl THF. This is memory and even psychosismay be observed. known as folate trap or methyl frap. ln this The neurologicalsymptomsof perniciousanemia manner, B12 deficiency results in decreased are believed to be due to the accumulationof folate coenzymes that leads to reduced methylmalonyl CoA that interferes in myelin nucleotideand DNA svnthesis. sheathformation in two possibleways. Although the tissuefolate levelsare adequate 1 . The biosynthesis of fatty acids,requiredfor or high, there is a functional folate deficiency myelin formation, is imparied. This is because, due to the lack of THF pool. The outcome is the methylmalonyl CoA acts as a competitive development of megaloblasticanemia. Adminisinhibitor of malonyl CoA in fatty acid synthesis. trati on of the ami no aci d methi oni nehas b een 2. Methylmalonyl CoA cari substitute shown to partially correct the symptomsof 812 malonyl CoA in fatty acid synthesis,resultingin deficiency. This is due to the fact that the a new type of branchedchain fatty acids.These formati on of N 5-methyl TH F i s i nhi bi te d by fatty acids will disrupt the normal membrane S-adenosylmethionine. A combined therapy of structure. vi tami n 812and fol i c aci d i s general l yemployed to treat the patientswith megaloblasticanemia. The excretion of methylmalonic acid (elevated)in urine and estimationof serum 812 level are used to assess812 deficiency.

Treatment V i ta mi n 8 1 2 i s a d m i n i s te re di n therapeuti c dos e s(' 1 0 0 -1 0 0 0p g ) i n tra m u s c u l arl y. Fol i c aci d administrationcan also reverse hematological abnormalities observed in Btz deficiency. However, the neurological symptoms persist. Therefore,a combined supplementationof B12 and folate is employed to treat the patientswith m eg a l o b l a s ti ca n e m i a s .

INTERRELATIONBETWEENFOLIC AC|D AND VITAM|N Br2 -FOLATE TRAP OR METHYL TRAP HYPOTHESIS Th e d e fi c i e n c yo f e i th e rfo l a te or vi tami n 812 r esu l tsi n a s i mi l a rty p e o f a n e m i a.Thi s suggests a probable biochemical interrelation between thesetwo vitamins.There is only one metabolic reaction known, common to folate and vitamin 812 Gig.7.39.

Besidesthe vitamins described above, there are many other compounds presentin foods as accessoryfactors.Earlierworkers have described these factorssometimeor the other, as essential to higher animals. However, their essential natureand requirementin humans has not been established.Although not essentialin the diet, they perform many important functions in the body. Selected examples of such substances which may be regarded as vitamin like compounds are describedhere.

Choline is trimethylhydroxy ethanolamine. H3 +-CH2-CH2OH H3C-

cHs

157

Chapter 7 : VITAMINS

One carbon

/\ /\ Metylcoba-/ lamrn

\

HomocYsteine

Fiq.7.39 : lnterrelationshipbetuveenfolic acid and vitamin B,r.

It can be synthesized in the body (from serine). lt is also available from many dietary sources(e.g. milk, eggs, liver, cerealsetc.). 6 i cchenrica!

functlons

OH 1. Choline, as a component of plrospholipids Llec it hins )is , i n v o l v e d i n m e m b ra n e s tru cture B i ochemi cal functi ons and lipid t r ansp o rt. 1. Inositol is required for the synthesisof 2. Choline preventsthe accumulationof fat (lipositol) which is a consphosphatidylinosifol in liver (as lipotropic factor). lt promotes the tituent of cell membrane. s v nt hes isof ph o s p h o l i p i d sa n d l i p o p ro te i n sand the disposal of triacylglycerols from liver. 3. Due to the presence of three methyl groups (one carbon fragments), choline is actively involved in one carbon metabolism.

2. ft acts as a lipotropic factor (along with choline) and preventsthe accumulationof fat in l i ver.

3. For some hormones, inositol acts as a 4. Choline is a precursorfor the synthesisof second messengerat the membrane level for the ac et y lc holinew h i c h i s re q u i re dfo r tra n s m i ssi on releaseof Ca2+ ions. of ner v e im oul s e . 4. lnositol concentrationin the heart muscle in high, the significanceof which however, is G holine- an e s s e n ti a l n u tri e n t? not known. As such, choline can be synthesizedand 5. Phytin is hexaphosphateof inositol found r eut iliz ed in h u m a n s . T h i s m a y h o w e v e r, be is plants. lt preventsthe absorptionof iron and insufficient to meet the body needs. Some calcium from the intestine. s or k er s label c h o l i n e a s a n e s s e n ti a ld i e t arv rutrient with RDA in the range of 400-500 mglday.

lnositol is hexahydroxy-cyclohexane.lt is also f,non,nas myo-inositolor meso-inositol.

Li poi c aci d (thi octi c aci d) i s a sul fur containingfatty acid (6,8-dithiooctanoicacid). lt exists in an oxidized and reduced form. Lipoic acid is fat as well as water soluble.

B IOC H E MIS TRY

158 Frrg-Crir-cH-(cH2)4cooH

l-lrFl-lr-cH-{cH2)1--{ooH

llll

s-- s

sF

sH

Lipolc acld (reduced)

Llpolc acld (oxidized)

l i poi c aci d) i s gai ni ngi mportance.B ei ngfat and water soluble, it can comfortablyreach various of l i poi c a cid i cati ons The therapeuti appl c ti ssues. are relatedto its antioxidantproperty(regardedas universalantioxidant),some of them are listed

. Lipoic acid is involved in the decarboxylation r eac ti o n sa l o n g w i th o th e r v i ta m i ns (thi ami ne, niac i n , ri b o fl a v i n a n d p a n to th e n i caci d). The conversion of pyruvate to acetyl CoA (by . pyruvate dehydrogenase)and a-ketoglutarateto dehydrogenase) succinylCoA (by cx,-ketoglutarate r equ i re sl i p o i c a c i d . .

R educes the free radi cal s i n brai n t hat otherwise contribute to Alzheimer's disease and mul ti pl e scl erosi s. Li poi c aci d sti mul ates producti on of gl utathi one (GS H ), besi des hel pi ng i n t he recycl eof vi tami nsE and C . and bri ngs do wn R educesi nsul i n resi stance, pl asmal ow densi tyl i poprotei ns.

In recent years, administrationof high doses . May be useful in the preventionof strokeand (100-600 mg/day) of lipoic acid (or dihydromyocardial infarction.

BIoMEDICAL/ GLINICAL CONGEPTS

aer Distinct deliciency conditions of certain B-complex uitamins are known Thiamine Niocin

-

Folic acid -

Beri-furi

Riboflauin -

Cheilosis, glossifis

Pellagra

Pyridoxine -

Peripheral neuropathy

Macroc7tic anemia

Cobalomin -

Pernicious anemia

B-complex uitamin deficiencies are usuolly multiple rather than individuol with ouerlopping symptoms. A combined therapy oJ vitamin Bp and lolic ocid is commonly employed to treat the patients of megaloblosticonemias. Megodosesof niacin are useful in the treatment ol hyperlipidemia. Long term use of isoniazidfor the treatment of tuberculo.siscouses86 deficiency. Folic acid supplementotion reduces eleuated plasmo homocysteine leuel which is associated with atherosclerosisand thrombosis. Sulfonamides serue as antibacterial drugs by inhibiting the incorporation of PABA to produce folic ocid. Aminopterin and amethopterin, the structural analogues of folic acid, are employgd in the treotment of concers. ns' Lipoic ocid is therapeuticolly uset'ul as an antioxidant to preuent stroke, myocordial infarction, etc.

159

Chatrter 7 : VITAMINS

Szent-Cyorgi and his associates (1936) Paraaminobenzoicacid (PABA)is a structural constituentof folic acid. PABA may be regarded observed that flavonoids, isolated from lemon peel (known as citrin) were responsible for as a vitamin in another vitamin (folic acid) maintenanceof normal capillary permeability. The term vitamin P (P for permeability)was used CCIOH to this group of substances.However, they are commonly known as bioflavonoids.

NHz PABA

NHz Sulfonilamide

Bioflavonoidsact as antioxidantsand protect ascorbic acid from being destroyed. lt is suggestedthat this antioxidant property may be responsible for maintenance of capillary permeability.Bioflavonoidshave been used to correct the vascularabnormality in humans.

The deficiencyof PABA was first found to be associatedwith failure of lactation and greying Bioflavonoidsare found in peel and pulp of of black hair in rats. The specific functions of citrus fru its, tobacco leaves and many PABA in humans,except that it is a component vegetables.The requirementof these compounds of folic acid, have not been identified. in humans has not been established. PABA is synthesizedby the bacteria and is essential for their groMh. The sulfa drug ( p -a mi n ob e n z e n es u l fa n i l a m i de) is s ulf onilam ide a structural analogue of PABA. Sulfonilamide competeswith PABA and acts as a bacteriostatic agent. Ingestion of large doses of PABA will compete with the action of drugs and thereforeshould be avoidedduring sulfonilamide therapy (trade name-sulfonamides).

Antivitamins are antagonisticto (oppose and block) the action of vitamins.They usually have structural similaritieswith vitamins.Administration of antivitaminscausesvitamin deficiencies.The common antivitaminsare discussedas antagonists for each vitamin.

160

B IOC H E MIS TR Y

]. Vitamins qre occessorglood factors required in the diet. Theg are classified os t'ot soluble (A, D, E and K) ond water soluble (B-complexand C).

2. Vitamin A is inuolued in uision,proper growth, diJJerentiationand maintenonceoJ epithelial cells. lts deficiencyresu/fsin night blindness.

3 The actiueform ol uitamin D is colcitriol which functions like a steroid hormone and regulatesplasma leuelsol calciumond phosphate.Vitamin D det'iciencyleadsto rickets in children and osteomalaciain adults.

4. Vitamin E is a natural antioxidant necessar7 for normal reproductionin many animals. 5. Vitamin K has a specificcoenzymeJunction.lt catalysesthe carboxylationof glutamic acid residuesin blood clotting factors (Il, Vil, IX and X) and conuerts them to actiue form.

6. Thiamine (Bl), as a cocarboxylase(TPP) is inuolued in energy releasing reactions. Its deficiency leads to beri-beri.

7. The coenzymesof ribot'lauin(FAD and FMN) and niacin (NAD+ and NADP+) take part in a uoriety of oxidation-reductionreactions connected with energg generation. RiboJlauindet'icienc7resultsin cheilosisand glossitis whereasniacin deficiencqleads to pellagro. 8

Pyridoxal phosphate (PLP), the coenzyme of uitamin F6, is mostly associated with amino acid metoboltsm. PLP participates in transomination, decarboxylotion, deaminotionand condensationreactions.

9. Biotin (antiegg white injury factor) participates as o coenzyme in corboxylation reactions of gluconeogenesis,fatty acid sgnthesisetc.

10. CoenzymeA (of pantothenic acid)is inuoluedin the metabolismol carbohgdrates,/ipids and amino acids,and their integration.

11 Tetrohydrofolate(THF} the coenzymeof t'olic acid porticipates in the transt'erof one carbon units (formyl, methgl etc.) in amino acid and nucleotide metabolism. Megaloblasticonemia is coused b9 t'olic acid deficiency.

72. Vitomin Bp has two coenzymes,deoxyadenosylcobalominand methylcobalamin.Bp deficiency results in pernicious anemia. 73. Vitamin C (ascorbicacid) is inuolued in the hydroxylation of proline and lgsine in the formation of collagen. Scuruyis causedby ascorbicacid deficiency. Therapeuticuse oJ megadosesol uitamin C, to cure euerythinglrom common cold to cancer, has become controuersial. 74. Certain uitamin like compounds (choline, inositol, PABA, lipoic acid) participate in many biochemicalreoctions.

161

Chapter 7 : VITAMINS

I. Essayquestions 1. Classifyvitaminsand brieflydiscusstheir functionsand deficiencydisorders. 2. Describethe chemistry,biochemicalfunctions,daily requirements, sourcesand deficiency manifestations of vitamin A. 3. Write an accountof folic acid involvementin one carbon metabolism. 4. Discussthe biochemicalfunctionsof vitaminC. Add a noteon the therapeutic useof megadoses of t his v ita mi n . reactions. 5. Write brieflyabout the coenzymesinvolvedin oxidation-reduction II. Short notes (a) Vitamin D is a hormone-justify, (b) Thiamine pyrophosphate, (c) Coenzymesof niacin, (d) Pyridoxalphosphatein transamination, (e) Folate trap, (f) Tocopherol,(g) Vitamin K in anemra. , ) C h o l i n e ,(j ) Pernrcrous c ar box y lat i o n(h, ) B i o c y ti n (i I I I . F ill in t he b l a n k s 1. The A in coenzymeA standsfor 2. T he v it am i nc o n ta i n i n gi s o a l l o x a z i nrien g 3. The vitaminthat is regardedas a vitamin in searchof a disease I

A nt i- t ube rc u l o s idsru g , i s o n i c o ti n i ca ci d hydrazi de (l N H ) l eads to the defi ci ency of vitamin

). The egg injury factorpresentin raw egg white with the deficiencyof. 6. The 'burningfeetsyndrome'in man is associated The vitaminthat is synthesized by only microorganisms 6 The three Ds in pellagrastandfor, q ,l

and

The fat solublevitamin requiredfor carboxylationreaction F I CLU ( fo rmi m i n o g l u ta mi c a c i d ) i s

excreted i n

uri ne i n

the defi ci ency of

r ita min

r lult iple c h o i c e q u e s ti o n s

-\

\\'hich one of the vitamin A functionsas a steroidhormone '-

ar Retinal(b) Retinol(c) ProvitaminA (d) F-Carotene. T he f uncti o n a l l ya c ti v efo rm o f v i ta m i nD i s (c) Dehydrocholesterol (d) Calcitriol. (b) Ergocalciferol a Cholecalciferol

'j

T^e metaboliteexcretedin urine in thiaminedeficiency

'r

a Pvruvate(b) Glucose(c) Xanthurenicacid (d) FICLU. - ^e c oen z y m ed i re c tl yc o n c e rn e dw i th the synthesiof s bi ogeni cami nes TPP(b) NADP+(c) Biotin (d) Pyridoxalphosphate. used in the treatmentof cancer c acid antagonist(s) \iethotrexate(b) Trimethoprim(c) Sulfonamide(d) All the three.

I

Sl eiotogical Oxidation

Digestion andAbsoqption

The natutal

Joodctu|fs

speah:

"Complex is the ingestedfood, But digested to simltler products, Absarbed by intestinal mwcosal celb, Assimilated and utilized by ail celk."

is requirementof f ood the basic and essential I man for his verv existence.The food we eat consists of carbohydrates, proteins, lipids, v it am ins and mi n e ra l s .T h e b u l k o f th e f ood ingestedis mostly in a complex macromolecular form which cannot, as such, be absorbedby the body. Digestion rb a process involving the hydrolysis of large and complex organic molecules of foodstuffs into smaller and preferably water-soluble molecules which can be easily absorbed by the gastrointestinal tract for utilization by the organism. Digestion of macromoleculesalso promotesthe absorptionof f at s olublev it a mi n sa n d c e rta i nmi n e ra l s .

Organ

Major function(s) Production of salivacontainingc-amylase; partialdigestion of polysac'charides Elaborationof gastricjuice with HCI and proteases; partialdigestion of proteins Releaseof NaHCO3and many enzymes required lor intestinal digestion

Liver

Synthesis of bileacids

Gallbladder

Storageol bile

Smallintestine Finaldigestion of foodstutfs;absorptionol digestedproducts

Cooking of the food, and mastication(in the mouth) significantlyimprove the digestibilityof foodstuffsby the enzymes.

Largeintestine Mostlyabsorption of electrolytes; bacterial utilization of certainnon-digested and/or unabsorbed foods

i f;qi lrsrl*rtlstinal trae t Digestion as well as absorption are complicatedprocessesthat occur in the gastroint es t inalt r ac t (C l T ) i n v o l v i n gm a n y o rg a n s .The

diagrammaticrepresentationof CIT is depicted in Fig.8.l, and the essential organs with their respective major functions are given in Table 8.1. The digestiveorganspossessa large

165

156

BIOCHEMISTFIY saccharides(glucose,fructose).The structuresof carbohydratesare described in Chapter 2. D i gesti cn

Oesophagus

Stomach Gall bladder

Duodenum (- 0.25m)

Pyloric sphincter

+

Pancreas

+- Jejunum (- 2.3m) Small intestine

{- lleum (- 4.6m)

FIg. 8.1 : Diagrammaticrepresentation ot gastrointestinaltract.

reservecapacity. For instance,pancreassecretes enzymes 5-10 fold higher than required for digestionof foods normally ingested. The digestion and absorption of individual proteins,lipids and foods,namely carbohydrates, nucleic acids,is describedhere.The gastrointestinal hormones are discussed under hormones (Chapter l9).

The digestionof carbohydratesoccurs briefly in mouth and largely in the intestine. The polysaccharidesget hydrated during heating which is essentialfor their efficient digestion. The hydrolysisof glycosidicbonds is carriedout by a group of enzymes called glycosidases (Fi9.8.2). These enzymes are specific to the bond, structure and configuration of monosacchari deuni ts. Digestion in the mouth : Carbohydratesare the only nutrientsfor which the digestionbegins in the mouth to a significantextent. During the process of mastication, salivary a-amylase (ptyalin) acts on starch randomly and cleavesc1,4-glycosidic bonds. The products formed i ncl ude cr-l i mi t dextri ns, (contai ni ngabo ut 8 glucoseunits with one or more o-1,6-glycosidic bonds) maltotrioseand maltose. Carbohydrates not digested in the stomach r The enzyme salivary amylase is inactivatedby high acidity(low pH) in the stomach.Consequently, the ongoing degradationof starch is stopped. Digestionin the small intestine : The acidic dietary contents of the stomach, on reaching small intestine,are neutralizedby bicarbonate pancreas. The produced by pancreatic a-amylase acts on starch and continues the digestion process.Amylase specificallyacts on a-l,4-glycosidic bonds and not on q,-1,6-bonds.

H"O----l ' |Glvcosidase

The principal dietary carbohydrates are (starch,glycogen),disaccharides polysaccharides (lactose,sucrose)and, to a minor extent, mono-

Fig. 8.2 : Hydrolysis of a glycosidic bond.

Chapten I : DIGESTION AND ABSOFIPTION

167

a (1-4)

Amylopectin

lo-nmyase

+

(l

F.

(

-)

!somaltose


Fig. 8.3 : Degradation of amylopectin by salivary or pancreatic a-amylase.

The resultant products are disaccharides contrast, Iactase (p-galactosidase) is the rate(maltose, isomaltose) and oligosaccharides l i mi ti ng, and, consequentl y,the uti l i zati on of (Fig.8.3). mi l k sugar(l actose)i s l i mi ted i n humans. The final digestion of di- and oligosaccharides to monosaccharides (Fig.8.4) pr im ar ily oc c u rs a t th e m u c o s a l l ini ng of t he uppe r j e j u n u m. T h i s i s c a rri e d out by oligosaccharidases (e.g. glucoamylase acting on amylose) and disaccharidases (e.g. maltase, sucrase, lactase). The enzyme sucrase is capable of hydrolysing a large quantity of table sugar (sucrose). In

Absorption

of monosaccharides

The principal monosaccharidesproduced by the digestion of carbohydrates are glucose, fructose and galactose. Of these, glucose accounts for nearlv 80o/o of the total monosaccharides.The absorption of sugars mostly takes place in the duodenum and upper jejunum of small intestine.

LowpH stops amylaseactivity

Pancreatico-amylase, a-Glucoamylase-------* lsomaltase,Maltase Lactase Sucrase

Fig. 8.4 : Overviewof digestionof carbohydrates.

Y'

B IOC H E MIS TRY

168 There exists a considerable variation in the absorption o'f different monosaccharides. The relative rates of absorption of important monosaccharides in comparisonwith glucoseare given below Clucose Calactose Fructose Mannose Xylose Arabinose

Capillaries

lntestinalmucosalcell

100 110 43 20 15 9

It is observed that hexoses are more rapidly absorbed than pentoses. Further, among the monosaccharides,galactoseis most e{ficiently absorbed followed by glucoseand fructose.Insulinhas no effect on the absorption of sugars.

Na+-K+ATPase

Flg. 8.5 : Transport of glucose across intestinal epithelium (Note : Transport of amino acids also occurs by a similar

rehydrationfluid contains glucose and sodium. Intestinalabsorptionof sodium is facilitatedby Differentsugarspossessdifferentmechanisms the presenceof glucose. for their absorption.Clucose is transportedinto The mechanismof absorptionof galactoseis the intestinalmucosalcells by a carriermediated similar to that of glucose.The inhibitorphlorizin and energy requiring process(Fig.8.A. blocks the Na+ dependenttransportof glucose Na+ share the same Glucose and transport and galactose. system(symport\ which is referredto as sodiumAbsorption of fructose : Fructose absorption dependent glucose transporter. The concenis relatively simple. lt does not require energy tration of Na+ is higher in the intestinallumen and is independentof Na+ transport.Fructoseis compared to mucosal cells. Na+, therefore, transportedby facilitateddiffusionmediatedby a moves into the cells along its concentration carri er. Insi de the epi thel i al cel l , most of t he gr ad i e n t a n d s i mu l ta n e o u s l y gl ucose i s fructose is converted to glucose. The latter then transported into the intestinal cells. This is entersthe ci rcul ati on. mediatedby the same carrier system.Thus, Na+ Pentosesare absorbed by a processof simple diffusesinto the cell and it drags glucosealong diffusion. gradient with it. The intestinal Na+ is the immediate energy source for glucose transport. carbohydrates T his e n e rg y i s i n d i re c tl ys u p p l i e dby A TP si nce $lon"diEestible (against the concentration the reentry of Na+ The plant foods are rich in fibrous material gradient)into the intestinallumen is an energy- which cannot be digestedeither by the human requiring active process.The enzyme Na+-K+ enzymes or intestinal bacteria. The fibers are ATPase is involved in the transport of Na+ in chemically complex carbohydrates which exchange of K+ against the concentration i ncl ude cel l ul ose,hemi cel l ul ose,pecti ns,l i g nin gradient (for details see Chapter 33). and gums. Fi ber i n nutri ti on i s of spe cial lVleeharnisnrr ei!$ahsorption

Oral rehydration therapy (ORT) : ORT is the most common treatment of diarrhea. The oral

impoftance which is described under nutrition (Chapter 23).

Ghapter 8: DIGESTION AND ABSOFIPTION

169

about 1O"/' of Eskimosof Creenland and 2"/" of North Americans are affected by this disorder. The treatment is to remove sucrose In general,humanspossessan efficientsystem from the diet. of carbohydratedigestionand absorption.Since only the monosaccharidesare absorbed, any The problern of flatulence resultsin defect in the activitiesof disaccharidases Flatulence is characterized by increased the passageof undigesteddisaccharidesinto the intestinal motility, cramps and irritation. This draw water from largeintestine.The disaccharides occurs after ingestion of certain carbohydrates the intestinal mucosa by osmosis and cause is and explained as follows. osmoticdiarrhea.Further,bacterialactionof these The carbohydrates (di-, oligo-, and polyundigestedcarbohydratesleads to flatulence. saccharides) not hydrolysed by a,-amylaseand Disaccharidasesare the intestinal brush intestinal other enzymes cannot be absorbed. borderenzymes.Any alterationin the mucosaof Lactose is not hydrolysed in some individuals the small intestine caused by severe diarrhea, due to the deficiency of lactase. The di-, and malnutrition,intestinaldiseasesor drug therapy oligosaccharides be can degraded by the will lead to a temporary acquired deficiency of (lower part of small present bacteria in ileum The patients with such disorders disaccharidases. intestine)to liberatemonosaccharides. The latter are advised to restrict the consumption of can be metabolized the by bacteria. sucroseand lactose. A bnor m ali tE e s o f digestion carbohydrate

During the course of utilization of monosaccharidesby the intestinalbacteria,the gases such as hydrogen, methane and carbon dioxid*-besides lactate and short chain fatty acids-are released. These compounds cause Lactose intolerance flatulence. Defect in the enzyme lactase(F-galactosidase) The occurrenceof flatulenceafterthe ingestion is the most common disaccharidasedeficiency in humans.lt is estimatedthat more than half of of leguminous seeds (bengal gram, redgram, the world's adult population is affected by beans, peas, soya bean) is very common. They lactoseintolerance.lt is more commonly found conta i n severaI nondigestible oligonccharides by in Africans (blacks) and Asians compared to human intestinalenzymes.Thesecompoundsare Europeans.Surprisingly,according to a recent degraded and utilised by intestinal bacteria estimate, about 90% of the adult Asians are causing flatulence. Raffinose containing lactasedeficient.The mechanismof how lactase galactose,glucose and fructose is a predominant found in leguminousseeds. is lost in adultsis not clear. lt is however,known oligosaccharide that there is a reduced production of lactase rather than an alterationin enzyme activity. Hereditary disorders with deficiency of in infantsand children individualdisaccharidases cause intoleranceof specificdisaccharides.

The treatment of lactose intolerance is quite simple. Elimination of lactose from the diet The proteins subjected to digestion and (severe restriction of milk and dairy products) absorption are obtained from two sourceswill s olv e t h e p ro b l e m. dietary and endogenous. Continuedconsumptionof lactoseby lactose The intake of dietary protein is in the range intolerant individuals causes typical symptoms of 50-100 g/day. About 30-100 e/day of of flatulence (described later). endogenousprotein is derivedform the digestive enzymes and worn out cells of the digestive Sucrase deficiency tract. The digestionand absorptionof proteinsis The deficiencyof the enzyme sucrasecauses very efficient in healthy humans, hence very little intoleranceto dietarysucrose.lt is estimatedthat protein (about 5-10 S/day)is lost through feces.

170 Dietary proteinsare denaturedon cooking and therefore,easily digested. Proteinsare degradedby a classof enzymesnamely hydrolases-which specifically cleave the peptide bonds, hence known as peptidases. They are divided into two groups

B IOC H E MIS TR Y

Peotides Amino acids

i

I CCK,secretin

1 . Endopeptidases(proteases) which attack the internal peptide bonds and releasepeptide fragments,e.g. pepsin, trypsin. 2. Exopeptidaseswhich act on the peptide bonds of terminal amino acids. Exopeptidases are subdivided into carboxypeptidases(act on C-terminal amino acid) and aminopepfidases(act on N- te rm i n a la m i n o a c i d ). The proteolytic enzymes responsiblefor the digestion of proteins are produced by the stomach, the pancreasand the small intestine. Proteinsare not digestedin the mouth due to the absenceof proteasesin saliva. l. Digestion of proteins by gastric secretion

| , rPe,, I

Chymotrypsin Elastase Carboxypeptidases (AandB) Flg, 8.6 : Formationand activationol Wncreatic

Protein digestion begins in the stomach. Gastric juice produced by stomach contains A is the most predominantgastricproteasewhich hydrochloric acid and a protease proenzyme preferentiallycleaves peptide bonds formed by namely pepsinogen. ami no B roupsof phenyl al ani neor tyrosi ne or Hydrochloric acid : The pH of the stomachis l euci ne. < 2 due to the presenceof HCl, secreted by Pepsindigestionof proteinsresultsin peptides parietal(oxyntic)cells of gastricgland. This acid and a few ami no aci ds w hi ch act as sti mul ant s perfornrstwo i mportant f u nctions-denaturation of for the releaseof the hormone cholecystokinin pr ot ei n sa n d k i l l i n g o f c e rta i n mi c roorgani sms. from the duodenum. The denaturedproteinsare more susceptibleto proteasesfor digestion. Rennin : This enzyme, also called chymosin, found in the stomachof infantsand children. is Pepsin: Pepsin(Creek : pepsis-digestion)is i s i nvol ved i n the curdl i ng of mi l k. lt R enni n produced by the serouscells of the stomach as mi l k protei n casei n to cal ci u m converts pepsinogen, the inactive zymogen or paracaseinate which can be effectivelydigested proenzyme. Pepsinogenis converted to active pepsi n. R enni n i s absenti n adul ts. by pepsin either by autocatalysis,brought about by other pepsin molecules or by gastric HCI (pH < 2). Removalof a fragmentof polypeptide ll. Digestion of proteins proteases by pancreatic chain (44 amino acids in case of pig enzyme) makesthe inactiveenzyme active after attaining The proteasesof pancreaticjuice are secreted a proper conformation. as zymogens(proenzymes)and then convertedto Pepsin is an acid-stable endopeptidase activeforms. These processesare initiated by the optimally active at a very low pH (2.0). The releaseof two polypeptide hormones, namely active site of the enzyme containstwo carboxyl cholecystokinin and secretin from the intestine groups,which are maintainedat low pH. Pepsin (Fig.8.6).

Ghapter 8 : DIGESTION AND ABSOFPTION

171

e@@ lll Prot ein. . . CO - Nil

CH - CO - NH- CH - C O - N H . . . . . . . . . . . . C O - N H - C H - C O O -

TI

I

I Enryme

Natureof (ii;

PepsinA

Tyr,Phe,Leu

Carboxypeptidase A

Ala, lle, Leu, Val

Trypsin

Arg,Lys

CarboxypeptidaseB

Arg, LYs

Enryme

ttatureorG)

Chymotrypsin Trp,Tyr,Phe, Leu,Met Elastase Ala,Gly,Ser Fig. 8.7 : Digestion of proteine-Speciticity of enzyme cleavage of peptide bonds. (81 can be from any amino acid)

Releaseand activation of zymogens : The key enzyme for activation of zymogen is enteropeptidase (formerly enterokinase) produced by int es t ina l(mo s tl y d u o d e n a l )mu c o s a l epi thel i al cells. Enteropeptidase cleavesoff a hexapeptide (6 amino acid fragment)from the N-terminalend of trypsinogen to produce trypsin, the active enzyme. Trypsin, in turn, activates other trypsinogen molecules (autocatalysis).Further, trypsin is the common activator of all other pancreatic zymogens to produce the active proteases,namely chymotrypsin, elastaseand (A and B). carboxypeptidases

Zn-proteases.They also possesscertain degree of substrate specificity in their action. For example, carboxypeptidaseB acts on peptide bondsof C OOH -termi nalami no aci d, the ami no group of w hi ch i s contri butedby argi ni ne or lysine (Fig.8.71. The combined action of pancreaticproteases resultsin the formation of free amino acids and smal l pepti des(2-8 ami no aci ds). l l l . D i gesti on rf protei ns by srna!! intestinal enayrnes

The l umi nal surface of i ntesti nalepi thel i a l cel ls contai ns aminopeptidasesand dipeptidases. Aminopeptidaseis a non-specificexopeptidase which repeatedly cleaves N-terminal amino acids one by one to produce free amino acids and smaller peptides.The dipeptidasesact on different dipeptides to liberate amino acids The substrate specificity of pancreatic (Fig.8.8). proteases is depicted in Fi9.8.7. For instance, trypsin cleavesthe peptide bonds, the carbonyl AhsorptFon nf annlno (-CO-) group of which is contributed by acads and di pepti des ar ginineo r l y s i n e . The free amino acids,dipeptidesand to some The amino acid serineis essentialat the active extent tripeptides are absorbed by intestinal centreto bring about the catalysisof all the three epi thel i alcel l s. pancreaticproteases,hence these enzymes are The di- and tripeptides,after being absorbed referred to as serine proteases. are hydrolysed into free amino acids in the Action of carboxypeptidases: The pancreatic cytosol of epithelial cells. The activities of (A and B) are metalloenzymes dipeptidasesare high in these cells. Therefore, carboxypeptidases that are dependent on Zn2+ for their catalytic after a protein meal, only the free amino acids activity, hence they are sometimes called are found in the portal vein. Specffici$ and action of pancreatic proteases : Trypsin, chymotrypsin and elastase are endopeptidases active at neutralpH. Castric HCI is neutralized by pancreatic NaHCO3 in the intestineand this createsfavourablepH for the action of proteases.

B IOC H E MIS TFIY

172

SmallIntestine

Carboxypeptidases Aminopeptidases Dipeptidases

I Fig. 8,8 : Overuiew of digestion of proteins.

The small intestine possessesan efficient systemto absorbfree amino acids.L-Aminoacids are more rapidly absorbedthan D-amino acids. The transport of L-amino acids occurs by an active process(againsta concentrationgradient), in contrastto D-amino acids which takes place by a simple diffusion. Mechanisffi of anrino a$rs*r, gr;ti
aeid

Amino acids are primarily absorbed by a similar mechanism,as describedfor the transport of D-glucose. lt is basically a Na+-dependent active processlinked with the transportof Na+. As the Na+ diffuses along the concentration gradient,the amino acid also entersthe intestinal c ell. B oth N a + a n d a m i n o a c i d ss h a rea common carrier and are transportedtogether.The energy is supplied indirectly by ATP (for details, see absorption of monosaccharidesand Fig.8.5).

ATP are utilized for the transport of a single amino acid by this cycle. For this season,Meister cycle is not a common transport system for amino acid. However, this cycle is operativefor rapid transportof cysteineand glutamine. The y-glutamylcycle appearsto be important for the metabolism of glutathione, since this tripeptideundergoesrapid turnover in the cells. There may be more physiologicalsignificanceof y-glutamylcycle. AbsorptEon of intact and pol ypepE !des.

proteins

A Na+-independentsystem of amino acid transport across intestinal cells has also been identified. The compound cytochalasinI inhibits Na+-independenttransportsystem.

For a short period, immediatelyafterbirth, the small intestine of infants can absorb intact proteinsand polypeptides. The uptakeof proteins occurs by a process known as endocytosisor pinocytosis.The macromoleculesare ingestedby formationof small vesiclesof plasmamembrane followed by their internalization. The direct absorption of intact proteins is very important for the transfer of maternal immunoglobulins (y-globulins)to the offspring.

Another transport system to explain the mechanism of amino acid transfer across membrane in the intestineand kidnev has been put forth. This is known as y-glutamyl cycle or Meister cycle and involves a tripeptide namely glutathione (y-glutamylcysteinylglycine). Three

The intact proteinsand polypeptidesare not absorbed by the adult intestine.However, the in certain macromolecular absorption individuals appears to be responsible for antibody formation that often causes food allergy.

Chapter 8: DIGESTION AND ABSORPTION

773

Abnormalities of protein digestion and amino acid absorption

Minor digestion of liBids in the stomach

Any defect in the pancreaticsecretionimpairs protein and fat digestion.This causesthe loss of undigested protein in the feces along with the abnormal appearanceof lipids. Deficiency of pancreatic secretion may be due to pancreatitis (see later),cystic fibrosisor surgicalremoval of pancreas.

The digestion of lipids is initiated in the stomach, catalysed by acid-stable lipase. This enzyme (alsocalled lingual lipase)is believedto originatefrom the glands at the back of tongue. Stomachcontains a separategastric lipase which can degradefat containingshortchain fatty acids at neutral pH. The digestion of lipids in the stomach of an adult is almost negligible,since lipids are not emulsified and made readv for lipaseaction. Further,the low pH in the stomach is unfavourablefor the action of gastric lipase.

Hartnup's {neutral

disease amino aciduria}

Hartnup is the name of the familv in whom this disease was first discovered. lt is characterized by the inability of intestinal and renal epithelial cells to absorb neutral amino acids. Tryptopftan absorption is most severely affected with a result that typical symptoms of pellagra are observedin the patientsof Hartnup,s disease.This is relatedto the impairmentin the conversionof tryptophanto NAD+ and NADp+, the coenzymesof niacin.

In case of infants, the milk fat (with short chain fatty acids) can be hydrolysedby gastric lipase to some extent. This is because the stomachpH of infantsis closeto neutralitv,ideal for gastric lipase action Emulsification of lipids in the small intestine Emulsification is the phenomenon of dispersionof lipids into smaller dropletsdue to reduction in the surface tension. This is accompaniedby increasein the surfacearea of lipid droplets. Emulsification is essential for effective digestionof lipids, since the enzymes can act only on the surface of lipid droplets. More correctly, lipasesact at the interfacial area between the aqueousand lipid phase.

There is considerablevariation in the daily consumptionof lipids which mostly dependson the economic status and dietary habits. The intake of f ipids is much less (often < 60 {day) in poorer sectionsof the society,particularlyin the The processof emulsificationoccurs bv three less developed countries. In the developed complementarymechanisms countries, an adult ingestsabout 60-150 g of 1 . Detergentaction of bile salts; Iipids per day. Of this, more than 90% is fat (triacylglycerol).The rest of the dietary lipid is 2. Surfactantaction of degradedlipids; 3. Mechanical mixing due to peristalsis. made up of phospholipids, cholesterol, cholesteryl estersand free fattv acids. 1. Bile salts : The terms bile salts and bile acids are often used interchangeably. At Lipids ar e i n s o l u b l eo r s p a ri n g l ys o l ubl e i n physiological pH, the bile acids are mostly aqueous solution. The digestive enzymes, present as anions. Bile salts are the biological however, are presentin aqueous medium. This poses certain problems for the digestion and detergents synthesized from cholesterol in the absorption of lipids. Fortunately,the digestive liver. They are secreted with bile into the duodenum.Bile saltspossesssteroidnucleus,the tract possesses specializedmachineryto side chain of which is attached to either glycine 1 . Increase the surface area of lipids for (glycocholic acid) or taurine (taurocholii acid). digestion; For the synthesisand other detailson bile acids, 2. Solubilize the digested products for refer cholesterol metabolism (Chapter 14. Bile absorption. saltsare the most effectivebiologicalemulsifying

a

BIOCHEMISTF|Y

174

o

otl

cH2-o-c-R1

ill

R2-c-o-?H

?

cH2-o-c-R3

cH2-oH Pan€roariuiDase ? R'-C-O-CH

\2 Hro

Trlacylglycerol

-

+

Crr_o,

2-Monoacylglycerol

RlcooH

RscooH Freefatty acids

Fig. 8.9 : Enzymatic cleavage of dietary fat.

agents.They interactwith lipid particlesand the aqueous duodenal contents and convert them into smaller particles (emulsified droplets). Further,bile salts stabilizethe smaller particles by preverrtingthem from coalescing. 2. Surfactant action of degraded lipids : The initial digestiveproductsof lipids (catalysedby lipase) namely free fatty acids, monoacylglycerols promote emulsification. These compoundsalong with phospholipidsare known as surfactants. They are characterized by possessing polar and non-polar groups. Surfactants get absorbed to the water-lipid interfaces and increase the interfacial area of lipid droplets. Thus the initial action of the enzyme lipasehelps in furtherdigestionof lipids.

(mol. wt. 12,000).lt is also secretedby pancreas as procolipase and converted to active form by trypsin. Colipase binds at the lipid-aqueous interfaceand helpsto anchorand stabilizelipase. Lipid esterase is a less specific enzyme present in pancreatic juice. lt acts on monoacylglycerols,cholesteryl esters, vitamin esters etc. to liberate free fatty acids, The presenceof bile acids is essentialfor the activity of lipid esterase. Degradation

of cholesteryl

esters

A specificenzyme namely pancreaticcholesterol esterase (cholesteryl ester hydrolase) cleavescholesterylestersto produce cholesterol and free fatty acids (Fig.&JA.

3. Besidesthe action of bile saltsand surfactants, the mechanical mixing due to peristalsis Degradation of phospholipids als o h e l p s i n th e e m u l s i fi c a ti o no f l i pi ds. The are enzymes responsiblefor Phospholipases s m alle r l i p i d e m u l s i o n d ro p l e ts are good phospholipids. Pancreaticjuice the hydrolysisof substratesfor digestion. phospholipase cleavesthe A2 which is rich in phospholipids. position of fatty acid at the 2nd DEgestion sf lipids The products are a free fatty acid and a by pancreatic enzyrnes lysophospholipid.Phospholipase42 is secreted The pancreatic enzymes are primarily as a zymogen which is activatedin the intestine responsiblefor the degradationof dietarytriacyl- by the action of trypsin. glycerols,cholesterylestersand phospholipids. An overviewof the digestionof lipids is given in Fig.8.l | . (tatl Degradation of triacylglycerols Pancreaticlipase is the major enzyme that digests dietary fats. This enzyme preferentially cleavesfatty acids (particularlylong chain, above 10 carbons) at position 1 and 3 of triacylgfycerofs. The products are 2-monoacylglycerol (formerfy 2-monoglyceride)and free fatty acids (Fi9.8.9).The activity of pancreatic lipase is inhibited by bile acids which are presentalong with the enzvme in the small intestine. This problem is overcome by a small protein, colipase

Absorption

of lipids

The former and present theories to explain the absorption of lipids are briefly described hereunder 1. tipolytic theory put forth by Verzar : Accordingto this, fats are completelyhydrolysed to glycerol and free fatty acids. The latter are absorbedeither as soapsor in associationwith bile salts.

175

AND ABSORPTION Chapter a : DIGESTION

Cholesteryl esterase

--------T--------+ 'Hzo

o

I

HO

R- C

Cholesterol

Cholesterylester

Fatty acid

Fig. 8.10 : Enzymaticcleavageof cholesterylester.

2. Partition theory proposed by Frazer : This theory statesthat the digestionof triacylglycerols is partial and not complete. The partially digestedtriacylglycerols,in associationwith bile salts,form emulsions.The lipids are taken up by the intestinal mucosal cells. As per this theory, for their entry resynthesis of lipidsis not necessary int o t he c ir c u l a ti o n .

Role mf bile salts

En llnirl &bs#rpt*{}r!

Besidestheir participation in digestion, bile salts are essentialfor absorptionof lipids. Bile salts form mixed micelles with liDids. These mi cel l es are smal l er i n si ze than the l i pi d emul si on dropl ets (uti l i zed for di gesti on, describedabove).The micelles have a disk like shapewith lipids (monoacylglycerol,fatty acids, 3. Bergstromtheory : This is a more recent cholesteroland phospholipids)at the interiorand and comprehensive theory to explain lipid bi l e sal ts at the peri phery. The hydrophi l i c absorption. lt has almost replaced the earlier groups of the lipids are oriented to the outside theories,and is briefly describedhereunder (close to .the aqueous environment) and the The primary productsobtainedfrom the lipid hydrophobicgroupsto the inside.In this fashion, free fatty acids the bile salt micelles exert a solubilizing effect digestionare 2-monoacylglycerol, on the l i oi ds. and free cholesterol.

Stomach

Almost unchanged

Pancrealic lipase i PhospholipaseAz+L Cholesteryl esterase L

Fig. 8.11 : Overview of digestion of lipids.

T ;

BIOCHEMISTFIY

176

CELL INTESTINAL MUCOSAL

7

Phospholipid Fiq.8.12 : Absorption of lipids by intestinal mucosal cell.

pqt*r.:haril}lsan *,S lipEr6 absorption The mixed micelles serve as the major vehicles for the transport of lipids from the intestinal lumen to the membrahe of the intestinal mucosal cells, the site of lipid absorption.The lipid componentspass through the unstirred fluid layer and are absorbed through the plasma membrane by diffusion (Fig.8.l2). Absorption is almost complete for monoacylglycerolsand free fatty acids which are slightly water soluble. However, for water insoluble lipids, the absorption is incomplete. For instance, less than 4OY. of the dietary cholesterolis absorbed.

the intestinalcells, do not undergo any modification. They enter the portal circulation and are transported to the liver in a bound form to al bumi n.

The long chain fatty acids are activated by thiokinase (fatty acyl CoA synthetase) in the intestinal cells. The acyl CoA derivatives so formed combine with 2-monoacylglycerolsto produce triacylglycerols. These reactions are catalysed by a group of enzymes, namely acyftransferases (Fig.8.13). Further, within the intestinal cells, cholesterol is converted to cholesterylester, and phospholipids are regenerated from the absorbed lysophosphoThe micelle formation is also essentialfor the lipids. The newly synthesizedlipids are usually absorption of fat soluble vitamins, particularly different from those consumed in the diet. vitamins A and K. of lipids from The efficiency of lipid absorption is $ecretion i ntesti nal mucosal cel l s the quantity to on the of bile salts dependent solubilizedigestedlipids in the mixed micelles. It may, however, be noted that in the absenceof bile salts,the lipid absorptionoccurs to a minor extent. This is mostly due to the slightly water soluble nature of monoacylglycerolsand free fatty acids. Further,short and medium chain fatty acids are not dependenton micelle formationfor the absorption. Synth:esEs of EiBids isl ttre [ r at e v * i m a l m u c o s a [ c e l l s The fatty acids of short and medium chain length (< 10 carbons),after their absorptioninto

The lipids that are resynthesized(described above) in the intestinalcells are hydrophobic in nature. They are put together as lipid droplets and surrounded by a thin layer consisting of mostly apolipoproteins (At and B-48) and phospholipids.This packageof lipids enveloped in the layer stabilizesthe dropletsand increases their solubility. These particles are known as chylomicrons. Chylomicrons migrate to the plasma membraneof intestinalmucosal cells. They are releasedinto the lymphatic vesselsby exocytosis.

Ghapter I : DIGESTION AND ABSOFPTION

177

Fattyacid ----Z-\-------+

Fattyaryl-CoA

2-Monoacylglycerol

Acytfians{eras6

Cholesterol

Acvttranslerase cholesterol--------------+ TV

cholestervlester

Fatty CoA acyl CoA

Shortchain fattyacids

Portalcirculation

Lymphatic system

t Blood

J Peripheral tissues

Fig. 8,13 : Formation and secretion of chylomicrons by intestinal mucosal cells.

The presenceof chylomicrons (Creek; chylosjuice) gives the lymph a milky appearance, which is observed after a lipid-rich meal. Chylomicronsenter the large body veins via the thoracic duct. Blood from here flows to the heart and then to the peripheral tissues (muscle, adiposetissue)and, finally, to the liver. Adipose tissueand muscle take up a large proportion of dietary lipids from chylomicronsfor storageand transport. lt is believed that this bypass arrangement(passageof chylomicrons through peripheraltissues)protectsthe liver from a lipid overload after a meal.

Abnorrnalities of lipid digestion and absorption The gastrointestinaltract possesses an efficient systemfor digestionand absorptionof lipids. lt can comfortably handle as much as 4 times the normal dai l y i ntakeof l i pi ds. Steatorrhea : lt is a condition characterized by the loss of lipids in the feces.Steatorrheamay be due to 1. A defect in the secretion of bile or pancreaticjuice into the intestine;

B IOC H E MIS TFI Y

178 2. l mp a i rm e n ti n th e l i p i d a b s o rpti onby the int es ti n a cl e l l s .

fat digestion,absorption,and thus obesityare in use. Two approachesare given below

1 . Pancreatic lipase degrades dietary S te a to rrh e ai s c o m m o n l y s e e n i n di sorders as s o c i a te dw i th p a n c re a s ,b i l i a ry obstructi on, triacylglycerolto fatty acids and glycerol which are absorbed. Orlistat is a non-hydrolysable severeliver dysfunctionetc. anal og of tri acyl gl ycerol ,and i s a pow e r f ul #h*e *eii*,rr;i ! ai{q}n* "; inhibitor of pancreaticlipase,hence preventsfat C h o l e s te ro ls to n e fo rm a ti o n i n gal l -bl adder di gesti on,and absorpti on. ( gall s to n e s )i s a fre q u e n th e a l thc ompl i cati on.l t 2. Olestra is a synthetic lipid, produced by s i n mal es esterificationof natural fatty acids with sucrose is f ou n d mo refre q u e n tl yi n fe rrra l ethan often in associationwith obesity.Cholesterolgall (insteadof glycerol).Olestratasteslike a natural stones are formed when liver secretes bile l i pi d. H ow ever, i t cannot be hydrol ysedand ( c on ta i n i n g p h o s p h o l i p i d s , b i l e aci ds etc.), therefore,gets excreted. with respectto cholesterol. supersaturated

SBESITV AruffiFAT ABSORPTIOro Obesity is a major problem in many parts of N ucl ei c aci ds (D N A and R N A ), and t heir t he w o rl d a s th e a v a i l a b i l i tyo f fo o d i s general l y abun d a n ta n d o v e re a ti n gi s c o mmon. Intakeof bases puri nes and pyri mi di nes can be lipid s l a rg e l y c o n tri b u te sto o b e s i ty. l n recent synthesized in the body, and thus they are years, pharmacologicalinterventionsto prevent di etari l ynon-essenti al .

BIOMEDTCAL/ CLINICAT CONCEPTS

Cooking of food significantly improues the digestibility by enzymes. Lactose intolerance due to a delect in the enzyme lactose (ftgalactosidase)is uery common. The treatment oduocated is seuere restriction of lactose (milk and milk products)in the diet. Flatulence, occurring alter ingestion of certain non-digestible oligosaccharides,is characterizedby increosed intestinal motility, cramps and irritation. Direct intestinal absorption of proteins and polypeptides is obserued in the infants, immediately at'ter birth. This is important for the transferot' maternal immunoglobulins (uia breast-feeding)to the offspring. In some odults, macromolecular (protein) absorption by intestine is responsiblefor antiborly formation, olten causingfood allergV. Emulsification ot' lipids is essenfiolfor their et't'ectiuedigestion, since iipcses can act only on the surJaceof lipid droplets. Bile saltsare the mostet'ficientbiolqical emulsifyingagenia. Pharmacological interuentions (e.g. Orlistat, Olestra) to block fat digestion ond/or absorption so os fo preuent obesitg are in recent use. Steotorrhea,characterizedby the loss o/ lipids in t'ecesis commonlg ossociatedwith impaired pancreatic function and biliory obstruction. ag Gosfric ulcers are mainly coused by the bacterium H. pylori. The antibiotics that eliminate this bacterium are effectiue in the treatment. re Acute pancreatitis is coused by autodigestion ol pancreaswhile chronic pancreatius is associatedwith excessiueconsumption ol alcohol.

FS' $5

Chapter a : DIGESTION AND ABSOBPTTON

SmallIntestine Ribonucl€ases

Stomach

Deoxvribonuclebses

Low pH Nucleicacids (DNA,RNA)

DNA,RNA denatured

| I PhosPhodiesterases

+

Nucleotides

I

pi*{ Nucleotidases + Unchanged

Nucleosides (Deoxv)riboseJ

Nucleosidases

+

fruri nes. P yri mrdrni ,,s

Fig. 8.14 : Overview of digestion of nucleic acids.

The digestion of dietary nucleic acids is carried out in the small intestine,primarily by the enzymes of pancreatic juice. Ribonucleases and deoxyribonu cl eases,respectively, hyd ro Iyse RNA and DNA to oligonucleotides(Fig.8.t4). The latterare degradedby phosphodiesterases to form mononucleotides. Nucleotidasesact on nucleotides to liberate phosphate and nucleosides.The nucleosides mav be either directly absorbed or degraded to free bases before absorption. Some of the unabsorbed purines are metabolized by the intestinal bacteria.

Peptic

ulcers

Castric and duodenal ulcers are collectively known as peptic ulcers. Ulceration occurs due to the autodigestionof mucosa by the gastric secretions (pepsin and HCI). In the patients of peptic ulcer, gastric HCI is always presentin the pyloric regions of stomach and the duodenum. Gastic ulcers are mainly caused by the bacterium Helicobacter pylori which lives in the nutrient-rich gastric mucosa, H. pylori i nduces chroni c i nfl ammati on i n the stomach tissues,which gets exposedto acid damage.For this reason, the best mode of treatment for gastric ulcers is the use of antihiotici that eliminate H. pylori.

The dietarypurinesand pyrimidinesare not of much utility for the synthesisof tissue nucleic acids. Further,the purinesafter their absorption Achlorhydria is a less serious disorder are mostlyconvertedto uric acid by the intestinal involving the failure to secretegastric HCl. mucosal cells and excretedin the urine. Pancreatitis

Inflammation of the pancreas is known as pancreatitis.Acute pancreatitisis causedby the autodigestionof pancreas due to the unusual The following are the major abnormalities(of conversionof zymogensinto the active enzymes interestto biochemists)concernedwith digestion by trypsin. ln normal circumstances,this is and absorption of food in the gastrointestinal preventedby trypsin inhibitor. tract. Acute pancreatitis is a lifethreatening Lactose intolerance, deficiency of sucrase, disorder.Measurementof serum amylase(highly Hartnup's diseaseand steatorrheahave already efevated)is used in the diagnosisof pancreatitis. been described.Pepticulcer and pancreatitisare Excessive consumption of alcohol over a long other important disorders associated with peri od i s bl amed as the pri me causeof chroni c pancreatitis. digestivesystem.

B IOC H E MIS TFIY

180

L

Digestion is o process that conuertscomplex foodstufls into simpler ones which can be reodily absorbed b9 the gastrointestinal trsct.

2. Stomach, duodenum and upper part ol small intestine qre the maiot sites of digestion. The small intestine is the prime site for the obsorption of digested foods. 3. The digestion of corbohydrates is initiated in the mouth by saliuary aamylase qnd is completed in the small intestine by pancreotic anmylase, oligosoccharidases ond disaccharidases. 4. Monosacchoridesare the final absorbable products of carbohydrate digestion. Glucose is tronsported into the intestinal mucosol cells by o carrier mediated, No+-dependent energy requiring process. 5. Lactose intolerancedue to a defect in the enzyme loctose(ftgolactosidose)resulting ln the inobitity to hydrolyse lactow (mitk suEar)is the common abnormahtyof corbohydrate digestion' 6. Protein digestion begins in 1he stomach by pepsin, which is oided by gastric HCl. Pancreatic proteoses (trypsin, chymotrypsin and elostase) and intestinal amino' peptidases and dipeptidasescomplete the degradation of proteins to amino ocids and some dipeptides. 7. The intestinol absorption of amino ocids occurs by different transport systems (at least six known). The uptoke ol omino ocids is primarily a No+-dependentenergy requiring process. 8. Digestion of tipids occurs in the small intestine. EmulslJication ol lipids, brought obout by bile solfs, is a prerequisitefor their digestion. Pancreatic lipose aided by a colipase degradestriacylglycerol to 2-monoacylglycerol and free fatty acids. Cholesterol esterase and phospholipases,respectiuely, hydrolyse cholesteryl esters and phospholipids. 9. Lipid obsorption occurs through mixed micelles, formed by bile sslfs in associstion with pioducts ol lipid digestion (primarily 2-monoacylglycerol, cholesterol ond lree fatty acids). In the intestinol mucossl cetls, Iipids ore resynthesized Jrom the obsorbed components and packed as chylomicrons which enter the lymphatic uesselsand then the blood. 10. Dietary nucleic acids (DNA ond RNA) are digested in the small intestine to nucleosides ond/or bases(purines and pyrimidines) which are absorbed.

Ghapter 8: DIGESTION AND ABSOFPTION

181

I. Essayquestions 1. 2. 3. 4. 5.

Write an accountof the digestionand absorptionof lipids. and proteins. Describebrieflythe digestionof carbohydrates Cive an accountof the Na+ dependentintestinaltranspoftof glucoseand amino acids. Describethe role of intestinein the digestionof foodstuffs. tract involved in the digestionof foodstuffs. Write briefly on the enzymesof gastrointestinal

II. Short notes (e) y(a) Mixed micelles,(b) Lactoseintolerance,(c) Salivaryamylase,(d) Disaccharidases, (h) Bilesalts,(i) Synthesis (g)Specificity of proteases, of chylomicrons Clutamylcycle,(fl Zymogens, juice. in the intestinalmucosalcells, (i) Pancreatic III. Fill in the blanks 1. Celluloseis not digestedin humansdue to lackof the enzymethat hydrolyses bonds. with flatulencecausedby ingestionof leguminous associated 2. The mostimportantcarbohydrate seeds 3. Lactoseintoleranceis causedby the deficiencyof the enzyme carbohydrates are collectivelycalled 4. The non-digested 5. GastricHCI is secretedbv 6. Nameof the peptidebelievedto be involvedin the transportof amino acids 7. The disease characterized by impairment in the absorption of neutral amino acids 8. Trypsinhydrolysespeptidebonds,the carbonylgroup of which is contributedby the amino or acids 9. The inhibition of the enzyme pancreaticlipase by bile salts is overcomeby a protein, nam el y 10. The vehiclesfor the transportof lipids from the intestinallumento the membraneof mucosal c ells IV. Multiple choice questions 11. Transport of glucosefrom the lumento the intestinalmucosalcells is coupledwith diffusionof (a) Na+ (b) K+ (c) Cl- (d) HCOI. 12. The key enzymethat convertstrypsinogento trypsinis (a) Secretin(b) Chymotrypsin(c) Elastase(d) Enteropeptidase. are 13. The productsobtainedby the action of pancreaticlipaseon triacylglycerols (a)Clycerol and free fatty acids(b) 1-Acylglyceroland free fatty acids(c) 2-Acylglyceroland free fatty acids (d) 3-Acylglyceroland free fatty acids. in the intestinalmucosalcells from the absorbedlipids are 14. The lipoproteinssynthesized (a) High densitylipoproteins(b) Chylomicrons(c) Low densitylipoproteins(d) Verylow density lipoproteins. 15. Salivaryc-amylasebecomesinactivein the stomachprimarilydue to

Proteins Plasma

p l a s m a i s th e l i q u i d medi um of bl ood Th " | (5 5 -6 0 % ),i n w h i c h th e c e l l componentsnamely erythrocytes, leukocytes, platelets-are suspended.lf blood containing anticoagulants (e.g. heparin, potassiumoxalate) is centrifuged, the plasma separatesout as a supernatantwhile the cells remain at the bottom. The packed cell volume or hematocrit is about 45%.

Separatlon

eit plasma

proteins

The total concentrationof plasma proteins is about 6-8 g/dl. The plasma is a complex mixture of proteins,and severaltechniquesare employed to separatethem. An age-oldtechnique is based on the use of varying concentrations of ammoni um sul fate or sodi um sul fate. By t his method, which is known as salting out process, T h e te rm s e ru m i s a p p l i e d to the l i qui d the plasma proteins can be separated into medium which separates out afterthe blood clots three groups-namel y al bumi n, gl obul ins and (coagulates). Serum does not contain fibrinogen fi bri nogen. and other clotting factors. Thus, the main Electrophoresis: This is the most commonly difference between plasma and serum is the employedanalyticaltechniquefor the separation presence or absence of fibrinogen. of plasma (serum)proteins.The basic principles lFt's6l{,.ti,rrr*+ot $f ood of electrophoresisare described in Chapter 43. T h e to ta l v o l u me o f b l o o d i n an adul t i s Paper or agar gel electrophoresiswith vernol around 4.5 to 5 liters. Blood performs several buffer (pH-8.6) separatesplasma proteins into diversifiedfunctions. These include respiration, 5 distinct bands namely alhumin, a1, a2, B and excretion, acid-base maintenance, water y globulins (Fig.9.l). The concentration of each balance,transportof metabolites,hormonesand one of these fractions can be estimated by a densitometer. drugs, body defenseand coagulation.

182

Chapter 9: PLASMAPROTEINS

183 Stan

2. E xcreti onof al bumi n i nto uri ne i n ki dney damage. 3. Increased production of globulins associated with chronic infections, multiple myelomasetc. Gomponents

Globulins

of plasma

proteins

The important plasma proteins along with their characteristics(based on electrophoretic pattern) and major functions are given in Table 9.1. Some selected plasma proteins are discussedhereunder.

Fig.9.1 : Electrophoresis of plasma proteins-

,i::,mliiftffi Abnormal

lffitl-ii{F ,tH electrophoretic

'j '

pattern

Electrophoresis of serum proteins is conveniently used for the diagnosisof certain diseases

Albumin is the major constituent (600/o)ol plasma proteinswith a concentrationof 3.5-5.0 g/dl. Human albumin has a molecularweight of 69,000, and consists of a single polypeptide chai n of 585 ami no aci ds w i th 17 di sul fi de bonds. Synthesis

of albumin

A l bumi n i s excl usi vel ysynthesi zedby the 1. Multiple myeloma : A sharp and distinct liver. For this reason, measurementof serum M band appearsin the lglobulin fraction. albumin concentrationis conveniently used to decreasedin liver 2. Acute infections : c1- and a2- globulins assessIiver function (synthesis produces diseases). Liver about 12 g albumin per are increased. day which represents25'/. of the total hepatic 3. Nephrotic syndrome : Decreasedalbumin protein synthesis.Albumin has an half-lifeof 20 with sharp and prominent c,2-globulin. days. 4. Primary immune deficiency : Diminished y globulin ba n d . Functions of albumin 5. a1-Antitrypsindeficiency: Diminishedcr1Plasmaalbumin performsosmotic, transport globulin ban d . and nutritive functions Albumin/globulin (A/G) ratio : The albumin 1. Osmotic function : Due to its high concentrationof plasma is 3.5 to 5.0 g/dl while concentration and low molecular weight, that of total globulins is 2.5 to 3.5 g/dl. The albumin contributes to 75-8oo/o of the total normal A/G ratio is 1.2 to 1.5 : 1. The A,/C ratio plasma osmotic pressure (25 mm Hg). Thus, is lowered either due to decreasein albumin or al bumi n pl aysa predomi nantrol e i n mai ntai ni ng increasein globulins, as found in the following blood volume and body fluid distribution. c ondit ions Decreasein plasma albumin level results in a 1. Decreasedsynthesisof albumin by liverusuallyfound in liver diseasesand severeprotein m alnut r it ion .

fall in osmotic pressure,leading to enhanced fluid retention in tissuespaces/causingedema. The edema observed in l<washiorkor,a disorder

184

BIOCHEMISTF|Y

Plasma concentration

Molecular weight

Maior function(s)

Albumin

3.5-5.0g/dl

nutritive transport, andbuffering 69,000 Osmotic,

Prealbumin

2$-30mg/dl

to someextent thyroxine 61,000 Transports

a,-Globulins

0.3.{,5 g/dl

c,-Antitrypsin

< 0.2 g/dl

(HDL) c[1-Lipoproteins

0.2{.3 g/dl

Orosomucoid

< 0.1g/dl

44,000 Bindswithprogesterone

protein (RBP) Retinol binding

3-6 mg/dl

A vitamin 21,000 Transports

globulin (TBG) Thyroxine binding

1-2 mg/dl

58,000 Transportsthyroidhormones

Transcortin or cortisol protein (CBG) binding

3-4 mg/dl

(e.9. of steroid hormones 52,000 Majortransporter cortisol, corticosterone)

oq-Globulins

0.4-0.8g/dl

o9-Macroglobulin

0.2{.3 g/dl

Haptoglobins (Hp1-1;Hp2-1andHp2-21

< 0.3g/dl

and withplasma freehemoglobin 90,000 Binds prevents itsexcretion

Prothrombin

< 0.02g/dl

in bloodcoagulation 63,000 Participates

Ceruloplasmin

< 0.03g/dl

p-Globullns

g/dl 0.6-1.1

p-Lipoproteins (LDL)

0.2{.5 g/dl

Transfenin

0.24.3g/dl

iron 76,000 Transports

Hemopexin

< 0.1g/dl

heme 57,000 Transports

Plasminogen

< 0.05g/dl

yGlobullns

0.8-1.8mg/dl

of trypsin 54,000 Inhibitor Transports cholesterol andphospholipids

activity andantiplasmin 800,000 Antitrypsin

oxidation of Fe2*to Fe$. of copper; 150,000 Transport Transports triacylglycerols andcholesterol

plasmin, involved in fibrinolysis 140,000 Forms functions Antibody

(fmmunoglobulins-lgG, 9.2lordetails) lgA,lgM,lgDandlgE;relerTable Fibdnogen

0.2-0.4g/dl

of protein-energymalnutrition, is attributed to a drastic reduction in plasma albumin level.

in bloodcoagulation 340,000 Participates

prealbumin, retinol binding protein, thyroxine binding protein,transcortinand others as stated in the functionsof plasmaproteinsin Table9.11.

2. Transport functions : Plasma albumin binds to several biochemically important 3. Nutritive functions : Albumin servesas a compounds and transports them in the sourceof amino acidsfor tissueprotein synthesis circulation. These include free fatty acids, to a limited extent, particularly in nutritional bilirubin, steroidhormones,calcium and copper. deprivationof amino acids. 4. Buffering function : Among the plasma [Note : Besidesalbumin, there are several other plasma transport proteins. These include proteins, albumin has the maximum buffering

18s

f ira!"r!;*s"S : PLASMA PFIOTEINS

Emphysema(Greek: emphusan-to inflate) is a term usedto representthe abnormaldistension of lungs by air. At least5% of emphysemacases are due to the deficiency of a1-AT. This is ,'t ; I ::;,-:,! $ Ea;,{,fi ; I f *{: en{:t'* *,}{ o*! *eet:'i:i;,,; associatedwith lung infections(e.g.pneumonia) 1 . A lbum in, b i n d i n gto c e rta i nc o m p o u n d si n and increasein the activity of macrophagesto the plasma, prevents them from crossing the releaseelastasethat damageslung tissues.In the b l ood- br ain ba rri e r e .B . a l b u m i n -b i l i ru b i n normal circumstances, elastase activity is i nhi bi tedby a1-A T. complex, albumin-freefatty acid complex. capacity. However, the buffering action of a lbum in in plas m ai s n o t s i g n i fi c a nct o mp a re dto bicarbonatebuffer system.

(lowered Effectof smoking on crl-AT : The amino acid plasma 2. Hypoalbuminemia methi oni neat posi ti on358 of a1-A T i s i nvol ved a lbum in) is obs e rv e di n ma l n u tri ti o n ,n e p h ro t i c i n bi ndi ng w i th proteases.S moki ng causes sv ndr om eand c i rrh o s i so f l i v e r. oxi dati on of thi s methi oni ne to methi oni ne u ri n e is e x c re te d i n to 3. A lbum in sulfoxide. As a result, a1-AT with methionine (albuminuria) in nephrotic syndrome and in sulfoxide cannot bind and inactivate proteases. certain inflammatoryconditionsof urinary tract. Emphysemais more commonly associatedwith Microalbuminuria (3O-3O0mg/day) is cl i n ically heavy smokingand the situationbecomesworse important for predictingthe future risk of renal in personswith cr1-AT deficiency. diseases(Refer Chapter 36). cl1-Antitrypsindeficiency and liver disease : 4. A lbum in is th e ra p e u ti c a l l yu s e fu l fo r th e Thi s i s due to the accumul ati onof a mutant treatmentof burns and hemorrhage. o1-AT which aggregatesto form polymers.These polymers,in turn-by an unknown mechanismcause liver damage (hepatitis) followed by accumul ati onof col l agen resul ti ng i n fi brosi s Globulins constituteseveralproteinsthat are (ci rrhosi s). separatedinto four distinct bands (cr1,42, p and gr 1-globulins)on electrophoresis(See Fig.9.l). ix ]-11#flc FsffiBrok$Eii C lobulins , in g e n e ra l , a re b i g g e r i n s i ze It i s a hi gh mol ecul ar w ei ght (8,00,000) th an album in. T h e y p e rfo rm a v a ri e ty o f protein and is a major constituentof u,2-fraction. fu nc t ionswhic h in c l u d etra n s p o rta n d i m m u n i ty. cr2-Macroglobulininhibits proteaseactivity and ln Table 9.1, the importantglobulins are given, servesas an anticoagulant.lts concentrationin some of them are discussedhereunder. plasma is elevated in nephrotic syndrome. This r t . 3 ' 1 } 5 { Y ,r

u1-Antitrypsin,more recentlycalled as a-antiproteinase, is a glycoproteinwith 394 amino acids a,rd a molecularweight of 54,000. lt is a major constituent of u1-globulin fraction of plasma :"oteinswith a normalconcentrationof about200 -q, dl. crl-Antitrypsin is a serineproteaseinhibitor. : combines with trypsin, elastase and other :"o:easeenzymesand inhibitstheir activity.

is due to the fact that majority of the low mol ecul ar w ei ght protei ns are l ost i n uri ne (proteinuria)in this disorder. H A P TOGLOB IN H aptogl obi n (H p) i s a pl asma gl ycoprotei n with an approximate molecular weight of 90,000. Hp is an acute phaseprotein since its plasma concentration is increased in several i nflammatoryconditions.

' - :,i r i:11.-.::;:-n{:t+ '

i " v t * 5 U *t,' *

*;c,!yle;;td'i1'rt $ a;S !'*,4p'i r;e6 d# A;r i ge

Haptoglobin binds with the free hemoglobin a. - { nt it r y ps ind e fi c i e n c yh a sb e e n i mp l i c a te d - :,ro diseases,namely, emphysemaand a1-AT (known as extra-corpuscularhemoglobin) that spi l l s i nto the pl asma due to hemol ysi s.The &ficiengy liver disease.

d

186 haptoglobi n-hemoglobin (Hp-Hb) complex (mol. wt. 155,000) cannot pass through glomeruli of k id n e y w h i l e fre e H b (m o l . w t. 65,000) can. Haptoglobin, therefore, prevents the loss of free Hb i n to u ri n e .

B IOC H E MIS TRY

o)

E IE

o

Clinical significance of Hp : Hemolytic E anemia is associatedwith decreased plasma a concentrationof haptoglobin.This is explained as follows. The haltt-life of Hp is about 5 days 45678910fi12 wh i l e th a t o f H p -H b c o mp l e x i s 90 mi n. l n Days ----* hemolytic anemia,free Hb in plasma is elevated leading to increased formation of Hp-Hb Fig.9.2 : The responseof C-reactiveprotein (CRP) in c om p l e x . T h i s c o mp l e x , i n turn, i s rapi dl y response to surgery (The normal acute Phase is depicted cleared from the plasma resulting in decreased by blue line, the development ot infection by red line and the response after treatment by green line). Hp l e v e l s . CERULOPLASMIN

proteins decreases,and they are regarded as Ceruloplasminis a blue colou;'ed,coppernegative acute phase reactants e.g. albumin, c on ta i n i n ga 2 -g l o b u l i nw i th a m o l ecul arw ei ght transferrin. of 150,000.lts plasmaconcentrationis about 30 mg/dl. Ceruloplasminbinds with almost 9O"/"oI C-reactive Frotein (CRPI plasma copper (6 atoms of Cu bind to a CRP is a maior component of acute phase m o l e c u l e ).T h i s b i n d i n g i s ra th e rti ght and, as a proteins.lt is producedin the liver and is present result,copper from ceruloplasminis not readily in the circulation in minute concentration releasedto the tissues.Albumin carrying only (< 1 mgidl). C-reactive protein (C strands for 1)oh of plasma copper is the major supplier of carbohydrateto which it binds on the capsuleof copper to the tissues.Ceruloplasminpossesses pneumococi) is involved in the promotion of oxidase activity, and it is associated with immune system through the activation of Wilson's disease which is discussed under cascade. complement copper metabolism (Chapter 1A. Estimationof CRP in serum is important for TRANSFERRIN the evaluation of acute phase response.The response of CRP to surgery is depicted in Transferrin (T0 is a glycoprotein with a Fig.9.2. ln a normal surgery, serum CRP molecularweight of 76,000. lt is associatedwith p-globulin fraction.Tf is a transporterof iron in increasesand returns to normal level within 7-10 days. lf the recoveryis complicatedby any t he c i rc u l a ti o n . infection, it will be reflectedby the continuous of CRP which requires further elevation ACUTE PI{ASE PROTEINS treatment. Acute phaseresponserefersto a non-specific responseto the stimulus of infection, injury, variousinflammatoryconditions(affectingtissue/ organs),canceretc. This phaseis associatedwith a characteristicpattern of changes in certain plasmaproteins,collectivelyreferredto as acute The higher vertebrates,including man, have phaseproteinse.g. o,1-antitrypsin, ceruloplasmin, evolved a defense system to protect themselves complementproteins,C-reactiveprotein. During against the invasion of foreign substances-a the acute phase, synthesisof certain plasma virus, a bacterium or a protein. The defense

187

Chapter 9 : PLASMA PFIOTEINS lnterchain disulfidebonds

*Hr N

t-"'=\ *iirN

F-',

rN H s 1-

NH-r Fab

S -S S_

Hingeregion

cHo cH2 Fc Intrachain disulfide bonds

coo-

chains(mol. wt. 53,000 to 75,000 each)and two identical light (L\ chains(mol. wt. 23,000 each) hel d together by di sul fi de l i nkagesand noncovalent interactions(Fi9,9.3\.Thus, immunoglobulin is a Y-shaped tetramer (HzLz). Each lmrmunoglobulins-basic concepts heavy chain contains approximately450 amino while each light chain has 212 amino acids lm m unoglo b u l i n s ,a s p e c i a l i s e d g ro u p of The heavy chains of Ig are linked to acids. proteins are mostly associated with y-globulin carbohvdrates, hence i mmunogl obul i ns are fraction (on electrophoresis) of plasma proteins. glycoproteins. Some immunoglobulinshowever,separatealong with P and a-globulins.Therefore,it should be Constant and variable regions : Each chain not ed t hat y-g l o b u l i n a n d i m m u n o g l o bul i n (L or H) of lg has two regions(domains),namely are not synonymous. lmmunoglobulin is a the constant and the variable. The amino functional ferm while y-globulin is a physical termi nal hal f of the l i ght chai n i s the vari abl e term. region (V1)while the carboxyterminal half is the constant region (Cg). As regards heavy chain, S t r uc t ur e o f i s n m u n o g l o b u l i n s approximatelyone-quarterof the amino terminal All the immunog:lobulin (Ig) molecules region is variable(Vx) while the remainingthreeoasigally consist of two identical heavy (H) quartersis constant(Cs,, Csr, Csr). The amino strategies of the body are collectively referredto as immunity, and are briefly described under immunology (Chapter42). lmmunoglobulins(or antibodies)are describedhere.

188

BIOCHEMISTFIY

Type H-Chain L{hains

Molecular formula

Molecular Percentage Serumconc, werght carbohydrate mC/dl

Itaior fundion(s)

responsible for 80f1,500 Mostly immunity humoral

IgG

rorl,

y2r2or y2)r2

IgA

rorl,

(o2rj1aor (oraldr+

-(160,000h-i

IgM

rorl,

p2rj5 or ItzT"zls

- 900,000

12

IgD

rorl,

(Qr2or Qt2)

-180,000

13

1-10 B-cell receptor?

IgE

rorl,

E2K2Ol e2?u2

-190,000

12

0.02-0.05 Humoralsensitivity andhistamine release,

-150,000

acid sequence (with its tertiary structure) of variable regions of light and heavy chains is responsible for the specific binding of im m u n o g l o b u l i n(a n ti b o d y )w i th anti gen.

15G.400 Protects the body surfaces

l mmunogl obul i n

50-200 Humoralimmunity, serves asfirstlineof defense

G (IgGl

IgC is the most abundant (75-80%) class of i mmunogl obul i ns. IgC i s composedof a single Y-shapedunit (monomer).lt can traverseblood Proteolytic cleavage of Ig : An immuno- vesselsreadily.IgG is the only immunoglobulin globulin can be split by the enzyme papain to that can cross the placenta and transfer the their fragments.These are two identical antigen mother's immunity to the developingfetus.IgG binding fragments(Fab) and one crystallizable triggers foreign cell destruction mediated by fragment(Fc).Papaincleavesthe immunoglobin complement system. molecule at the site between Cp,1 and Cp2 Innmunogl obul i n A { fgA } regions which is referred to as hinge region. IgA occurs as a single (monomer)or double unit (dimer)held togetherby J chain. lt is mostly Humans have five classes of immuno- found in the body secretionssuch as saliva,tears, globufins-namely IgG, IgA, IgM, IgD and sweat,milk and the walls of intestine.IgA is the IgE-containing the heavy chainsy, c, p, 6 and most predominantantibody in the colostrum,the E, respectively. The type of heavy chain initial secretion from the mother's breast after a ultimatelydeterminesthe classand the function baby is born. The IgA molecules bind with bacterial antigens present on the body (outer of a given lg. epithelial) surfaces and remove them. In this Two types of light chains-namely kappa (r) way, IgA prcvents the foreign substances from and lambda (l.)-are found in immunoglobulins. enteringthe body cells. They differ in their structurein C1 regions.An immunoglobulin(of any class)containstwo K or l rnmunogl obul i n M (IgM! two l, light chains and never a mixture. The l gM i s the l argesti mmunogl obul i ncomp osed oc c u rre n c e o f r c h a i n s i s m o r e common i n of 5 Y-shapedunits (IgC type) held togetherby hum a n i mmu n o g l o b u l i n sth a n l , chai ns. a J polypeptidechain. Thus IgM is a pentamer. The characteristics of the 5 classesof human Due to its large size, IgM cannot traverseblood vessels,hence it is restrictedto the blood stream. immunoglobulinsare given in Table 9.2.

CLASSESOF IMMUNOGLOBULINS

Ghapter 9 : PLASMA PROTEINS IgM is the first antibody to be produced in responseto an antigen and is the most effective against invading microorganisms. lt may be notedthat IgM can simultaneouslycombine with 5 antigenicsitesdue to its pentamericstructure. I m m unog l o b u l i n

D (Ig D !

IgD is composed of a single Y-shaped unit and is present in a low concentration in the circulation. IgD molecules are present on the surfaceof B cells.Their function, however,is not known for certain. Some workers believe that IgD may function as B-cell receptor.

lmmunoglobulin E (IgEl IgE is a singleY-shapedmonomer.lt is normally presentin minuteconcentrationin blood. IgE levels are elevated in individuals with allergiesas it is associatedwith the body's allergic responses.The IgE moleculestightly bind with mast cells which releasehistamineand causeallergy, Production by m ult ip l e

of ;mmunoglobulins

189 population. Femalesare more susceptiblethan males for this disorder and it usually occurs in the age group 45-60 years. Abnormal lg production : Multiple myeloma is due to the malignancyof a singleclone of plasma cells in the bone marrow. This results in the overproduction of abnormal immunoglobulins, mostly (75%) IgG and in some cases(25"h) IgA or IgM. IgD type multiple myeloma found in younger adults is less common (<2%) but more severe. In patients of multiple myeloma, the synthesi s of normal i mmunogl obul i ns i s diminishedcausing depressedimmunity. Hence recurrentinfectionsare common in thesepatients. Electrophoretic pattern : The plasma of multiple myelomapatientsshowsa characteristic pattern of electrophoresis. There is a sharp and distinct band (M band, for myeloma globulin) between p-and y-globulins.Further,this M band almost replacesthe y-globulin band due to the diminished synthesisof normal y-globulins.

Bencefones proteins : Henry BenceJonesfirst described them in 1847. These are the light (r or l,) of immunoglobulins that are A s alr e a d y d i s c u s s e d ,i mmu n o g l o b ul i nsare chains synthesized in excess.Bence Jonesproteins have composedof light and heavy chains. Each light molecular a weight of 20,000 or 40,000 (for chain is produced by 3 separategenes,namely dimer). In about 2O"/oof the patientsof multiple a variable region (V1) gene/ a constant region myeloma, Bence .fonesproteins are excreted in (Cl) gene and a joining region (J) gene. Each urine which often damagesthe renal tubules. heavy chain is produced by at least 4 different genes-a variable region (Vg) gene, a constant Amyloidosis is characterized by the deposits region (Cg) gene, a joining region U) gene and of light chain fragments in the tissue (liver, diversity region (D) gene. Thus multiple genes kidney, intestine)of multiple myeloma patients. are responsiblefor the synthesisof any one of The presenceof BenceJonesproteinsin urine t he im m u n o g l o b u l i n s . can be detected by specific tests. Antibody diversity : A person is capable of 1 . Electrophoresis of a concentrated urine is generating antibodies to almost an unlimited the best test to detect Bence Jones proteins in range of antigens (more than one billion!). lt ufl ne. should, however, be rememberedthat humans 2. The classical heat test involves the do not contain millions of genes to separately precipitation of Bence Jones proteins when c ode f or i n d i v i d u a li mmu n o g l o b u l i nmol ecul es. slightly acidified urine is heatedto 40-50'C. This The antibodydiversityis achievedby two special precipitate redissolves on further heating of urine processes/ namely comhination of various point. to boiling lt reappears again on cooling structural genes and somatic mutations, urine to about 70oC. MULTIPLE MYELOMA 3. Bradshaw's test involves layering of urine Multiple myeloma, a plasma cell cancer, on concentratedHCI that forms a white ring of constitutesabout\ 1'/" of all cancers affecting the precipitate, if Bence Jones proteins are present. Ee n e s

., B IOC H E MIS TR Y

190

Extrinsic parhway

lntrinsic patnway

I

I

{z

FactorX

bin(ra)

Prothrombin t,lftThro

Fibrinogen(l)

Fibrin (bloodclot)

Fig.9.4: Overuiew of blood cloning with the final common pathway-

The term hemostasis is applied to the sequence of physiological responsesto stop bleeding (loss of blood after an injury). This is carried out by blood clotting.

two of these factors are designatedby a Roman numeral. lt should, however, be noted that the numbers representthe order of their discovery and not the order of their action. The cascadeof blood clotting process is depicted in Fig.9.5 and the salient features are discussedbelow. The active form of a factor is designated by a subscript a. The active clotting factors (with exception of fibrin) are serine proteases.

Blood clotting or coagulation is the body's major defense mechanism against blood loss. A blood clot is formed as a result of a seriesof to fibrin of librinogen different Sonversion reactions involving nearly 20 substances,most of them being glycoproteins, Fibrinogen(factor l) is a soluble glycoprotein synthesized by the liver. that constitutes2-3"/"of plasmaproteins(plasma Blood clotting process involves two concentration0.3 g/dl). Fibrinogenconsistsof 6 polypeptide chains-two A cr, two B p and two 1 independentpathways making the structure(A ct)z (B F)z'lz. 1. The extrinsicpathway is the initial process Fibrinogen undergoes proteolytic cleavage in clotting and involvesthe factorsthat are not presentin the blood (hence the name). catalysed by thrombin to release small fibrinopeptides (A and B), This results in the 2. The intrinsic pathway involves a seriesof formation of fibrin monomers which can stick reactions participated by the factors present in together to form hard clots (Fig.9.6). Clot the blood. formation is further stabilizedby covalentcrossStrictly speaking,the extrinsic and intrinsic l i nki ng betw eengl utami neand l ysi ne resi dues. pathways are not independent,since they are Thi s reacti on cross-l i nksfi bri n cl ots and is coupled together.Further,the final reactionsare catalysedby fibrin stabilizingfactor (Xlll). The identical for both pathways that ultimately lead red colour of the clot is due to the presenceof to the activationof prothrombinto thrombin and red cells entangledin the fibrin cross-links. the conversion of fibrinogen to fibrin clot of prothromhin Conversion lFig.e.4). The blood coagulation factors in human plasma along with their common names and molecularweightsare listed in Table9"3. All but

to thronnbin Prothrombin(ll) is the inactivezymogenform of thrombin (lla). The activationof prothrombin

191

Chapter I : PLASMA PROTEINS

occurson the plateletsand requiresthe presence (lX). The Christmasfactor is also activated by of factorsVa and Xa, besidesphospholipidsand active proconvertin(Vlla). Ca2+ . In the next step, the Staurt factor (X) is activated by Christmas factor (lXa) and this pathwas* The extrinsic reactionrequiresthe presenceof antihemophilic The extrinsic pathway is very rapid and factor (Vllla), Ca2+and phospholipids. occurs in responseto fissueinjury. This pathway conversion of essentiallv involves the proconvertin(Vll) to its activeform (Vlla)and the generation factor Xa. The tissue factor (lll), found to be necessaryto accelerate the action Vlla on a factor X, is presentin lung and brain.

The extrinsic and intrinsic pathways lead to the formation of factor Xa which then participates in the final common pathway to ultimately result in the formation of fibrin clot. Anticoagulants

Severalsubstances,known as anticoagulants, are i n use to i nhi bi t the bl ood cl otti ng.C al ci um The intrinsic pathway is rather slow. lt is essentiallyrequired for certain reactions of involves the participation of a contact system blood coagulation.The substanceswhich bind (wounded surface) and a series of factors to with Ca2+ are very effective as anticoagulants. generate factor Xa. These include oxalate, fluoride, EDTA and citrate. The Hagemanfactor (Xll) is activated(Xlla)on Heparin is an anticoagulantused to maintain exposureto activatingwound surfacecontaining collagen or platelet membranes.The formation normal hemostasis.lt is a heteropolysaccharide of Xlla is acceleratedby kallikrein and HMK. found i n many ti ssuesi ncl udi ngmastcel l s i n the The activated Hageman factor (Xlla) activates endotheliumof blood vessels.Heparincombines factor Xl. The Xla activate the Christmas factor w i th anti thrombi nl l l w hi ch i n turn. i nhi bi tsthe

T he int r ins ic

Factor number

I tl ill IV V vtl vill IX X XI xtl xill

p a th w a y

Common name(s)

Subunit molecular weight

Fibrinogen Prothrombin Tissue factor, thromboplastin (Ca2*) Calcium Proaccelerin, labilelactor (SPCA) conversion accelerator Proconvedin, serumprothrombin globulin (AHG) factor A, antihemophilic Antihemophilic factorB, factor, antihemophilic Christmas (PTC) Plasma thromboplastin component factor Staurt-Prower (PTA) Plasma thromboplastin antecedent Hageman factor LikiLorand factor Fibrin-stabilizing factor(FSF), fibrinoligase, Prekallikrein (HMK) kininogen Highmolecular weight

340,000 720,000 370,000 330,000 50,000 330,000 56,000 56,000 160,000 80,000 320,000 88,000 150,000

theoderof theirdisnveryaN nottheorderof thehaction.FactorVawason@refenedto as factorVl, Note: Thenunbersrepresent hencethereis nQfactorVl.

192

BIOCHEMISTFIY

Prekallikrein

FactorXll Ertrinsic i pathway i

FactorXl Vl la+-,------:

FactorlX FactorVl||----;*-=-+

Factor Vl I i

Factorlll

VlI

FactorX

Thrombin(lla)

lr ilr ' li:!.) n |J;Ll.liilrJV

Fibrinogen(l)

ractoJxttt---.------+

x tt Fibrin (hardclot)

Fig.9,5 : The blood clotting cascade in humans (the active forms of the factors are reprcsented in red with subscript'a').

clotting factors ll, lX, X, Xl, Xll and kallikrein. Streptokinase is a therapeutic fibrinolytic pl asmi nogen. Heparin can be administeredto patientsduring agentwhich activates and after surgery to retard blood clotting. The blood contains another anticoagulantnamely protein C-which is activated by thrombin. Active protein C hydrolyses and inactivatesclotting factorsV and Vlll. Warfarin, a vitamin K antagonist may be consideredas an oral anticoagulanf. This acts by reducingthe synthesisof certain clotting factors ( ll, Vl l , l X a n d X).

FlMnogen

II

ThrombinI l9 Fibrinope

Itr

A andB

Y

\_./------\_/-------\_-/ Fibrinmonorner

i";nlEr"ls*olneii*, The term fibrinolysisrefersto the dissolution or lysis of blood clots. Plasmin is mostly responsiblefor the dissolution of fibrin clots. Plasminogen,synthesizedin the kidney, is the plasmin. Tissue inactive precursor of plasminogen activator (TPA) and urokinase convert plasminogento plasmin.

Y

Flbrinclot representation of Flg.9.6 : Diagrammatic fibrinclot formationfromfibrinogen.

193

frrr:r:,tslr ii : PLASMA PBOTEINS

i :\.l 4 f1 +." +:ri i i

l+.l

r t' , i i.r ir :::i' 1:

:' ,r .:i' :'

and sufferfrom internalbleeding (particularlyin j oi nts and gastroi ntesti nal tract). H emophi l i aA has gained importancedue to the fact that the Royal familiesof Britain are among the affected i ndi vi dual s.

Severalabnormalitiesassociatedwith blood clotting are known. These are due to defectsin clotting factors which may be inherited or ac quir ed.H e mo p h i l i a ,Vo n W i l l e b ra n d ' sd i sease Hemophilia B (Christmasdisease): This is etc., are examples of inherited disorder while to the deficiency of Christmas factor (lX). due is an acquired disease. afibrinogenemia The cl i ni cal symptomsare al mostsi mi l arto that Hemophilia A (classicalhemophilia) : This is found i n hemophi l i aA . a sex-linked disorder transmitted bv females Von Willebrand's disease: This disorder is affecting males. Hemophilia A is the most common clotting abnormalityand is due to the characterizedby failure of plateletsto aggregate deficiency of antihemophilic factor (VIil). The and is due to a defect in the olateletadherence affectedindividualshave prolongedclottingtime factor.

BIoMEDTCAL/ GUNTCALCONCEPTS

rx' Albumin, the most abundant plasma protein, is inuolued in osmotic t'unction, transport of several compounds (fqtty actds, steroid hormones), besides the bulfering action. !ii

Hypoalbuminemia qnd albuminuria are obseruedin nephrotic syndrome. uyAntitrypsin deliciency has been tmplicated in emphysema (abnormal distension ol lungs by qir) which is more commonly associoted with heauy smoking. Haptoglobin preuents the possible loss of free hemoglobin trom the plasma through the kidneys by lorming hoptoglobin-hemoglobin complex.

[.t"

Immunoglobulins (antibodies), a specialized group of plasma globulor proteins, are actiuely inuolued in immunity. lgG and lgM are primarily concerned with humoral immunity while IgE is ossociofed with allergic reactions. Multiple myeloma, a plasma cell cancer diseoseof bone marrow, is characterlzedbg ouerproduction ol abnormal immunoglobulins (mostly lgG). Laborotory diognosis ol multtple myelomo can be made by the presenceof a distinct M band on plasmo/serum electrophoresis,

I i1

Blood clotting or coagulation is the body's major d.efense mechanism against blood loss. Delects in clotting factors cause coagulation abnormalities such as hemophllia A (det'lciency of lactor VIII) qnd Christmos diseose (deficiency of foctor IX).

st

Anticosgulants Inhibit blood clotttng. These include heparin, qxalate, fluoride, EDTA and citrate.

B IOC H E MIS TR Y

194 .a '1F.ftt1iI";F..q'

L

6-8 g/dl. Electrophoresisseporotes The total concentrationof plosma proteins is about a1, &2, p and y globulins' plasma proteins into 5 distinct bqnds, namely albumin,

2 ' A l b u mi n i s th e m a j o rc o n s ti tu ent(60% )oJpl osmoprotei nsw i thaconcentrati onS ' 5to 5 .0 g /d | .Iti s e x c l u s i u e l y s y n thesi zedbgthel i uer,A l bumtnperformsosmoti c,transpor t ond nutritiue functiorts. deficiencv 3. ayAntitrgrpsinis a major constituent ot'.al globul^,!liil',"i^^?:-Antitrvpsin dtsease' liuer his been implicated in emphysemaand a speciJic 4 ' H a p to g l o b i n (H p )b i n d s w i thfreehemogl obi n(H b)thatspi l l si ntothepl asmadueto the glomeruli, hence haptoglobin hemolysis.The Hp-Hb complex connot pass through preuentsthe loss of t'ree hemoglobininto urtne' ceruloplasrnin' C'reoctiue 5. Alterations in the acute phase proteins (e.g' ayantitrypsin, to the stimulus of infection' response protein) are obseruedos o result oJ non-spicit'ii i n j u ry ,i n fl o mma ti o n e tc ' Esti mati onofserumC -reacti ueprotei ni susedforthe euqluotionot' acute phase response' the body ogoinst the forergn 6. Immunoglobulins ore specialized proteins to defend yglobulin t'ractionof plasmaproteins' The. subsfonces.They are mostlg associatedwith choins and two identical heauy ilentical two immunoglofullinsessenfiollg consist ol linkages' Iight chains, held together by disult'ide IgA, lgM., IgD.and lgE-
Ghapter9 : PLASMA PFIOTEINS

t95

[. Essayquestions and major functionsof plasmaproteins. 1. Describethe characteristics along with their functions. 2. Give an accountof differenttypesof immunoglobulins 3. Discussthe cascadeof blood clottingprocess. 4. Describethe structureof differentimmunogloublins. 5. Discussthe role of acute phaseproteinsin healthand disease. II. Slrort notes (a) Electrophoresis of albumin,(c)at-Antitrypsin,(d) Haptoglobin, of plasmaproteins,(b) Functions (0 (e) lmmunoglobulin C, Multiple myeloma, (g) Bencejones proteins, (h) Fibrinogen, (j) Hemophilia. (i) Anticoagulants, III. Fill in the blanks 1. The differencebetweenplasmaand serumis the presenceor absenceof 2. The most commonlyemployedtechniquefor separationof piasmaproteins 3. Haptoglobinbinds and preventsthe excretionof the compound for the productionof immunoglobulins 4. The cells responsible 5. The immunoglobulinthat can crossthe placentaand transferthe mother'simmunityto the developingfetus s a t c a n b i n d w i th mastcel l sand rel easehi stami ne 6. T he i mmu n o g l o b u l i nth proteinsare precipitated when urine is heatedto 7. Bence-Jones 8. The major componentof acutephaseproteinsusedfor the evaluationof acutephaseresponse 9. The extrinsic and intrinsic pathways result in the formation of a common activated factor for the lysisof blood clot 10. The factor mostlyresponsible IV. Multiple choice questions 11. HemophiliaA is due to the deficiencyof clottingfactor (a) X (b) V (c) VIll (d) ll. 12. Plasmaalbumin performsthe following functions (a) Osmotic(b) Transport(c) Nutritive(d) All of them' 13. T he i m m u n o g l o b u l ipnre s e n itn m o stabundantquanti ty (a) lgc (b) lsA (c) IBM (d) leE. 14. Name the immunoglobulininvolvedin body allergicreactions (a) lgA (b) lgE (c) lgD (d) lgM. binds with Ca2+and preventsblood clotting 15. The following anticoagulant (a) Heparin(b) oxalate (c) Proteinc (d) All of them.

Hemoglobin andPorphyrins

The hemoglobin

sgeahs :

"I arn rhe red of blood,respowible Jbr respirntion; Deliuer A, to tissuesand return CO, n lungs; Influenced byfactorspII, BPG and.Cl- in rnyfunctiens; ahnormalities," Disttn'bedin my dutiesby stru.entral

structure,functionsand abnormalitiesof Th" I hemoglobin, the synthesisand degradation of heme, the porphyrin containing compounds are discussedin this chapter.

Hemoglobin (Hb) is the red blood pigment, Flg. 10.1: Diagrammaticrepresentationof hemoglobin with2u and 2fl chains(Redblocks-Heme). exclusively found in erythrocytes (Creek; erythrose-red; kytos-a hollow vessel). The normal concentrationof Hb in blood in males non-protein part the heme-the is 14 -1 6 B /d l , a n d i n fe ma l e s 1 3-15 B /dl . part-and (prosthetic group). Hemoglobin is a tetrameric Hemoglobin performstwo important biological (Fig.I0.l). protein allosteric functionsconcernedwith respiration Structure of globin : Clobin consistsof four polypeptide chains of two different primary structures(monomericunits).The common form 2. Transportof CO2 and protonsfrom tissues of adult hemoglobin (HbAl) is made up of two to lungs for excretion. a-chai nsand tw o p-chai ns(o.2P ).S omeauth or s consider hemoglobinconsistingof two identical dimers-(ap)1 and (cr0)2.Each a-chain contains Hemoglobin(mol. wt. 64,450)is a conjugated 14' l ami no aci ds w hi l e p-chai n contai ns 146 protein, containing globin-the apoprotein ami no aci ds. Thus H bA , has a total of 574 1. D e l i v e ry o f 0 2 fro m th e l ungs to the t is s ue s .

196

197

AND POFIPHYFIINS Ghapter 1O: HEMOGLOBIN

l-i: ) 1 1 ;'f'-V

a little of it may be present even in adults. Glycosylated hemoglobin (HbA1), formed by covalent binding of glucoseis also found in low concentration.lt is increasedin diabetesmellitus which is successfullyutilized for the prognosisof these patients (details under Diabetes, in Chapter 36). Myogl obi n

Myoglobin (Mb) is monomeric oxygen hemoproteinfound in heart and skeletal binding , N-PM Histidineot (/ muscl e.l t has a si ngl e pol ypepti de(153 ami no ) globin \ ll aci ds)chai nw i th heme moi ety.Myogl obi n(mol . HN*cH, wt. 17,000)structurallyresemblesthe individual subuni ts of hemogl obi n mol ecul e. For thi s FIg. 102 : 9tructureof hene IM-MeW f CH); V-VrnVl reason, the more complex properties of F-,f ff.,t'ffi9-,fl f,fi r-.S',.f'a-,..--'..9',ff fi fidr't. hemoglobin have been convenientlyelucidated ',,,'.(rlS'flalrii,'o,#fi through the study of myoglobin. Myoglobin functions as a reservoir for amino acid residues. The four subunits of hemoglobin are held together by non-covalent oxygen. lt further seryes as oxygen carrier that int er ac t ionspr i m a ri l y h y d ro p h o b i c , i o n i c a nd promotesthe transportof oxygen to the rapidly hydrogen bonds. Each subunit containsa heme respi ri ngmuscl ecel l s. 8r oup. Structure of heme : The characteristic red Functi ons of hemogl obi n colour of hemoglobin (ultimately blood) is due Hemoglobin is largely responsiblefor the to heme. Heme contains a porphyrin molecule transportof 02 from lungsto tissues.lt also helps namely protoporphyrin lX, with iron at its to transportCO2 from the tissuesto the lungs. center. ProtoporphyrinlX consistsof four pyrrole rings to which four methyl, two propionyl and Binding of O" to hemoglobin two vinyl groups are attached(Fig.l0.A. One molecule of hemoglobin (with four Heme is common prostheticgroup presentin hemes)can bind with four moleculesof 02. This cytochromes, in certain enzymes such as is in contrast to myoglobin (with one heme) catalase,tryptophan pyrolase, and chlorophyll w hi ch can bi nd w i th onl y one mol ecul e of (Mg2*). In case of cytochromes,oxidation and oxygen. In other words, each heme moiety can reduction of iron (fe2* rr Fe3+)is essentialfor bind with one 02. their biological function in electron transport c hain. Other

forms

of hemoglobin

Besides the adult hemoglobin (HbAl ) describedabove, other minor hemoglobinsare also found in humans (Tahle l0.l). ln adults a small fraction (< 5%) of hemoglobin, known as HbA2 is present. HbA2 is composed of two a and two 6 (defta) chains. Fetal hemoglobin (HbF) is synthesizedduring the fetal developmentand

TyPe

Composition and symbol

Percentage of total hemoglobin

HbAl

az1z

90%

HbA2

%62 sz^[z

< 3-/o

HbF HbAlc

o2Fr-glucose

< 2o/o < 5'/o

f-

198

BIOCHEMISTFIY

the binding of oxygen to other hemes.Thus the affinity of Hb for the last 02 is about 100 times greaterthan the binding of the first 02 to Hb. This phenomenon is referred to as cooperative bi ndi ng of 02 to H b or si mpl y heme-hem e interaction(Fig.l0.4. On the other hand, release of 02 from one heme facilitatesthe releaseof 02 from others. ln short, there is a communication among heme groups in the hemogl obi nfuncti on.

AI

o -c

=

E so 6

('

U)

s

Transport 50 pO2(mmHg)

100

Fig. 10.3 : Orygen dissociation cuNes of hemoglobin and myoglobin (pOr- Partial prcssure of orygen).

of O" to the tissues

In the lungs,where the concentrationof 02 is high (hencehigh pO2),the hemoglobingetsfully saturated(loaded) with 02. Conversely,at the tissue level, where the 02 concentrationis low (hence low pO2), the oxyhemoglobin releases (unloads)its 02 for cellular respiration.This is often mediated by binding 02 to myoglobin which serves as the immediate reservoir and supplier of 02 to the tissues(Fig.l0.5).

Oxygen dissociation curve : fhe hinding ability of hemoglohin with 02 at different partial pressuresof oxygen (pO2) can be measuredby a graphic representation known as 02 dissociation curve. The curves obtained for hemoglobin and T and R forms of hemoglobin myogfobin are depicted in Fig.l0.3. The four subunits (clzpz)of hemoglobin are It is evident from the graph that myoglobin held together by weak forces. The relative has much higher affinity for 02 than position of these subunits is different in hemoglobin. Hence 02 is bound more tightly oxyhemoglobincomparedto deoxyhemoglobin. with myoglobin than with hemoglobin. Further, pO2 neededfor half saturation(50% binding) of T-form of Hb : The deoxy form of hemoglobin myoglobin is about'l mm Hg comparedto about exists in a T or taut (tense)form. The hydrogen 26 mm Hg for hemoglobin. and ionic bonds limit the movement of monomers. Therefore,the T-form of Hb has low Coo p o ra ti v e b i n d i n g oxygen affinity. $f 0 s to h e mro g i o b i n R-form of Hb : The binding of 02 destabilizes The oxygendissociationcurve for hemoglobin some of the hydrogen and ionic bonds is sigmoidal in shape (Fig.l0.3). This indicates particularlybetweenaB dimers.This resultsin a that the binding of oxygento one heme increases relaxed form or R-form of Hb wherein the

Q2

Increaslngaflinity for 02

Chapter 1O: HEMOGLOBIN AND PORPHYRINS

199

subunits move a little freely. Therefore, the R-form has high oxygen affinity. The existenceof hemoglobin in two forms (T and R) suitably explains the allos t er ic b e h a v i o u r o f h e mo g l o b i n (Fig.t0.a).

\ Transport of CO2 by hemoglobin ln aerobic metabolism, for every m olec uleo f 0 2 u ti l i z e d ,o n e mo l e c u l eo f CO2 is liberated. Hemoglobin actively participatesin the transportof CO2 from the tissuesto the lungs. About 15'/" of CO 2 c ar r ie di n b l o o d d i re c tl yb i n d sw i th Hb. The rest of the tissue COz is transportedas bicarbonate(HCO3). Carbon dioxide moleculesare bound to the uncharged cr-amino acids of hemoglobin to form carbamyl hemoglobinas shown below Hb- NH2+ C O2$

NHCOOO2

CarbamylHb

2

Fe Oxy

Fe Fe

lr " / / NHCOO-

,- TrssuEs Myoglobin1

H b - N H -C O O-+ H +

T he ox yH b c a n b i n d 0 .1 5 mo l e sC O 2 l mole heme, whereasdeoxyHb can bind 0. 40 m olesC O 2 l m o l eh e m e .T h e b i n d i n g of CO2 stabilizesthe T (taut) form of hemoglobin structure, resulting in decreased02 affinity for Hb. Hem og l o b i n a l s o h e l p s i n th e transport of CO2 as bicarbonate, as explained below (Fig.l0.6). As the CO2 entersthe blood from tissues,the enzyme carhonic anhydrase present in erythrocytescatalysesthe formation of carbonic acid (H2CO3).Bicarbonate(HCOJ) and proton (H+) are releasedon dissociationof carbonic acid. Hemoglobin acts as a buffer and immediatelybinds with protons. lt is estimated that for every 2 protons bound to Hb, 4 oxygen molecules are released to the tissues. In the lungs ,bind i n g0 2 to H b re s u l tsi n th e re l easeof protons.The bicarbonateand protons combine to form carbonic acid. The latter is acted upon by carbonic anhydraseto releaseCO2, which is ex haled.

Fig. 10.5 : Diagrammatic representation of

BOHR EFFECT The bi ndi ng of oxygen to hemogl obi n decreases with increasing H+ concentration (lower pH) or when the hemoglobin is exposed to increasedpartialpressureof CO2 (pCOz).This phenomenonis known as Bohr effect.lt is due to a change in the binding affinity of oxygen to hemoglobin. Bohr effect causes a shift in the oxygen dissociation curve to the right (Fig.t0.V. Bohr effect is primarily responsiblefor the releaseof 02 from the oxyhemoglobin to the tissue. This is becauseof increasedpCO2 and decreasedpH in the actively metabolizingcells.

200

BIOCHEMISTFIY

2CO2 + 2H2O

Exhaled

tI

T caroonic anhVdrase J

2CO2+2H2Q

t

2H2COs

2H2CO3

1 2H++ 2HCO!

40z \-_-YJ

LUNGS

Mechanism

of Bohr effect

The Bohr effect may be simplified as follows

pl asma. In order to mai nt ain enters the neutrality, Clerythrocytes and binds with deoxyhemoglobin.The concentration of Cl- is greater in venous bl ood than i n arter ial blood. The four substances namely 2,3-bisphosphoglycerate (describedbelow), CO2, H+ and C l - are col l ecti vel y cal l ed as Thev effectors. allosteric interact with the hemoglobin mol ecul e and faci l i tate t he release of 02 from oxyhemogl obi n. *

EFFECT OF 2,3.B|SPHOSPHO. GLYCERATE ON (,2 AFFINITY OF Hb

HbO2 + H+ -+ Hb H+ + 02

(2,3-BPG;formerly, 2,3-Bisphosphoglycerate is 2,3-diphosphoglycerate)the most abundant lts molar in the erythrocytes. organicphosphate to that is approximately equivalent concentration produced in of hemoglobin. 2,3-BPC is the erythrocytesfrom an intermediate(1,3of glycolysis.This short bisphosphoglycerate) When CO2 binds to hemoglobin, carbamyl pathway, referred to as Rapaport-Leubering metabolism hemoglobinis produced(detailsdescribedunder cycle,is describedin carbohydrate (Chapter l3). transportof CO2). This causesthe removal of Any increase in protons and/or lower pO2 shifts the equilibrium to the right to produce deoxyhemoglobinas happensin the tissues.On the other hand, any increasein pO2 and / or a decreasein H+ shiftsthe equilibrium to the left, whic h o c c u rs i n l u n g s .

protons from the terminal NH2 group and stabilizes the structure of Hb in the T form (deoxyhemoglobin).Therefore, the binding of CO2 promotesthe releaseof oxygen (in tissues). On the other hand, when hemoglobin is oxygenatedin lungs,CO2 is releasedas it binds loosely with R-form of Hb.

N

o

= o

E50 Role of Cl- in oxygen

transport

(u @

Chloride (Cl-) is bound more tightly to deoxyhem o g l o b i n th a n to o x y h e mogl obi n. Thi s facilitatesthe releaseof 02 which is explained as follows Bicarbonate (HCO3) is freely permeable across the erythrocyte membrane. Once produced in the erythrocytes, HCOJ freely movesout and equilibrateswith the surrounding

s

50 pO2(mmHg) Fig. 10.7 : Effect of pH (Bohr effect) on oxygen dissociation curue (pOr-Partial pressure ot Or).

201

AND PORPHYFIINS GhapterlO : HEMOGLOBIN StrippedHb (no 2, 3-BPG)

On oxygenationof hemoglobin, 2,3-BPC is expelledfrom the pocketand the oxyhemoglobin attainsthe R-form of structure.

100 N

o

Normalblood (with2,3-BPG)

= o

{-Blood

E50

of anemic oatient (2,3-BPGT)

6 a

s

Glinical

significance

of 2,3-BPG

S i nce the bi ndi ng of 2,3-B P C w i th hemoglobin is primarily associated with the releaseof 02 to the tissues,this small molecule assumesa lot of biomedical significance.The erythrocytelevels of 2,3-BPC are relatedto tissue demandsof oxygen supply.

1. ln hypoxia : The concentrationof 2,3-BPC in erythrocytesis elevated in chronic hypoxic 50 100 conditions associated with difficulty in O2 pO2(mmHg) suppl y. These i ncl ude adaptati on to hi gh altitude, obstructive pulmonary emphysema Flg. 10.8: Eftectot pH (Bahrettect)on orygen (airflow in the bronchiolesblocked) etc. .,,,,,,tF,F,#tF'I :;PiPffP,f:,Pd,t::,,,,,,:';, !L9,{F-,,,QF,'I:F 0

levels 2. ln anemia : 2,3-BPC are increasedin severeanemia in order to cope up with the oxygendemandsof the body. This is an adaptationto supply as much 02 as possibleto the tissue,despitethe low hemoglobin levels.

Binding of 2,3-BPG to deoxyhemoglobin

2,3-BPC regulates the binding of 02 to hemoglobin.lt specificallybinds to deoxyhemo3. In blood transfusion: Storageof blood in globin (and not to oxyhemoglobin) and acid citrate-dextrosemedium results in the decreasesthe 02 affinity to Hb. The effect of decreasedconcentrationof 2,3-BPC.Such blood 2, 3- B P Con H b ma v b e s u mma ri z e da s fol l ow s when transfusedfails to supply 02 to the tissues 02 i mmedi atel y. ------s + Hb-2,3-BPC HbO2 + 2,3-BPC

oxYHb

Addition ol inosine (hypoxanthine-ribose) to the stored blood preventsthe decreaseof 2,3The reduced affinity of 02 to Hb facilitates BPC. The ribose moiety of inosine gets hexose the releaseof 02 at the partial pressurefound in phosphorylated and enters the gets pathway and finally th e oxygen monophosphate t he t is s ue s .T h i s 2 ,3 -B PC s h i fts (Fi9.10.0. 2.3-BPC. converted to dissociationcurve to the right Mechanism

"","JtTJr:J"o

of action

of 2'3-BPG

One molecule ol 2,3-BPC binds with one molecule (tetramer)of deoxyhemoglobinin the central cavity of the four subunits.This central pocket has positively charged (e.g. histidine, ly s ine) t wo p -g l o b i n c h a i n s . l o n i c b o n ds (sal t bridges) are formed between the positively charged amino acids (of p globins) with the negativelychargedphosphategroupsof 2,3-BPC (Fig.l0.9. The binding of 2,3-BPC stabilizesthe deoxygenated hemoglobin (T-form) by crosslink ing t he p -c h a i n s .

O.t ,0

-c.

o

ttl H-C-O-P-Otl H-C-H OI

oI

O=P-OI

o-

(A)

(B)

Fig. 10.9 : (A) Diagrammatic reprcsentation of binding of 2,3-BPG to deoxyhemoglobin; (B) Structure of 2,3-BPG.

BIOCHEMISTRY

202 4. Fetal hemoglobin (HbF) : The binding of 2,3-BPC to fetal hemoglobin is very weak' Therefore, HbF has higher affinity for 02 comparedto adult hemoglobin(HbA). This may be needed for the transfer of oxygen from the maternal blood to the fetus.

Hemoglobin (Fe2*)

NADH + H+

Hem o g l o b i n (s p e c i fi c a l l y h e me) combi nes Ftg, 10.10: Conuercionof hemoglobinto with different ligands and forms hemoglobin methemoglobin and vice versa' oxyHb derivatives.The normal blood contains and deoxyHb. Besides these, methemoglohin (metHb) and carboxyhemoglobin are the other vomiting and irritability'Adminisbreathlessness, important Hb derivatives' The Hb derivatives 02 through oxygen maskswill help to of tration have characteristic colour and they can be reversethe manifestationsof CO toxicity' detected by absorPtion spectra. Methernoglobin For the biologicalfunction of hemoglobin-to carry oxySen-the iron should remain in the ferrous (Fe2+)state. Hemoglobin (Fe2+)can be oxidized to methemoglobin (Fe3+).In normal however,molecularoxygen does circumstances, not oxidize Hb, it only loosely binds to form

Abnormal hemoglobins are the resultantof p mutations in the genes that code for a or muta nt 400 chai ns of gl obi n' A s many as are them of hemoglobinsare known. About 95% due to alterationin a singleamino acid of globin'

oxyhemoglobin. h e m o gl obi n to of o x i d a ti o n The (metHb) be caused in the may methemoglobin and drugs' radicals free H2O2, living system by to bind (with is unable Fe3+) The methemoglobin the occupies molecule water a to 02. lnstead, metHb. of heme oxygen site in the

Basic

concepts

of globin

synthesis

For a better understanding of abnormal hemoglobins,it is worthwhile to have a basic knowledgeof globin synthesis'The globin genes are organisedinto two gene families or clusters (Fig.l0.tl).

t n n o rma l c i rc u ms ta n c e s ,th e occasi onal 1. o-Gene family : There are two Senes the by oxidation of hemoglobin is corrected coding for a-globin chain presenton each one 'l in present enzyme methemoglobin reductase of chromosome 6. The (-gene, other member erythrocytes(Fig.l 0.1A. of a-gene cluster is also found on chromosome 16 and i s acti ve duri ng the embryo nic Carboxyhemogiobin {COHbl development. (CO) is a toxic compound Carbon monoxide 2. p-Gene family : The synthesisof p-globin in Hb (an industrialpollutant)that can bind with occurs from a single gene located on each one the same manneras 02 binds. However,CO has of chromosome1 1. about 200 times more affinity than 02 for This chromosome also contains four other bind i n g w i th H b . genes. Clinical manifestationsof CO toxicity are One e-gene expressedin the early stagesof observedwhen the COHb concentrationexceeds 20"/". fhe symptoms include headache, nausea, embryonic develoPment.

203

AND PORPHYHINS Ghapter 1O: HEMOGLOBIN

o-Globinlike genes (chromosome16)

Globinchains

azlz

rtt ttl

o262

Hemoglobins

aZFZ

Globinchains

768

p-Globin-likegenes (chromosome 11) Gy Fig. 10.11: Diagrammaticrepresentationof globin genes with the synthesis of globin chains and hemoglobins (("er-Hb Gower 1;a;yr-HbF; arSSHbA; arBr-HbA,).

Two ygenes (Gy and Ay) synthesizey-globin c hainsof f e ta l h e m o g l o b i n(H b F ). One 6-gene producing 6-globin chain found in adults to a minor extent (HbA2). l{emoglobinopathies It is a term used to describe the disorders causedby the synthesisof abnormalhemoglobin molecule or the production of insufficient quantitiesof normal hemoglobinor rarely both. Sickle-cell anemia (HbS) and hemoglobin C disease (HbC) are the classical examples of abnormal hemoglobins. Thalassemias,on the other hand, are causedby decreasedsynthesisof nor m al he mo g l o b i n .

the black population. lt is estimatedthat 1 in 500 newborn black infants in the USA are affectedby sickle-cellanemia. Molecular

basis

of HbS

The structure of hemoglobin (as described already)containstwo cr-andtwo p-globinchains. In case of sickle-cell anemia, the hemoglobin (HbS) has two normal a-globin chains and two abnormal (mutant)p-globin chains. This is due to a differencein a single amino acid. In HbS, glutamate at sixth position of p-chain is replaced by valine (Clu pu -+ Val). S i ckl e-cel l anemi a i s due to a change (mi ssensemutati on) i n the si ngl e nucl eoti de (thymine-+ adenine)of p-globin gene.This error causesthe formation of altered codon (CUC in

S ic k le-c e lal n e m i a(H b S )i s th e m o s t c ommon form of abnormal hemoglobins.lt is so named becausethe erythrocytesof these patients adopt a sickle shape (crescent like) at low oxygen n (Fig.l0.1A. concentratio Occurrence

of the disease

S ic k le-c e l l a n e m i a i s l a rg e l y c o n f i ned to tropical areasof the world. lt primarily occurs in

(A)

(B)

Fig. 10.12: Erythrocytes: (A) Froma normalperson; (B) Froma patientol sickelcell anemia.

BIOCHEMISTFIY

204

-cTc-

-cAc-

DNA

_GAG_

_GUG_

RNA(codon)

t

J *HN-CH-CO,,%

*HN-CH-CO

I CHz tQHz

J

,,t.",..,...*

Amino acid

I CH

/\

HsC CHs

I

coo(-Gtu*)

(-

HemoglobinA

!/6[ * Hemoglobin S

)

6thposirion F-Chain

FIg. 10,13 : Formation of B-chain of hemoglobin in normal and sickle cell anemia (Note : Single base mutation in DNA (T -+ A ) causes replacementof glutamate by valine at 6th positionof B-chain).

place of CAG) which leadsto the incorporation 4. Prematuredeath : Homozygousindividuals glutamate at the sixth of sickle-cell anemia die before they reach of valine instead of p-chain (Fig.l0,l3). position in adulthood(< 20 years). Homozygous and heterozygous HbS : Sicklecell anemia is said to be homozygous,if caused by inheritanceof two mutant genes (one from each parent)that code for p-chains.In case of heterozygousHbS, only one gene (of p-chain) is affected while the other is normal. The erythrocytesof heterozygotescontain both HbS and HbA and the diseaseis referredto as sicklecell trait which is more common in blacks (almost 1 in 10 are affected).The individuals of sickle-cell trait lead a normal life, and do not u s u a l l y s h o w c l i n i c a l s y mptoms. Thi s is in contrast to homozygous sickle-cell anem ta . Abnormalities

associated

with

HbS

Sickle-cell anemia is characterized bv the following abnormalities

of sickling Mechanism in sickle-cell anemia C l utamate i s a pol ar ami no aci d and i t is repl aced by a non-pol ar val i ne i n si ckl e-cell hemoglobin.This causesa marked decreasein the solubility of HbS in deoxygenatedform (Tform). However, solubility of oxygenatedHbS is unaffected. Sticky patches and formation fibres deoxyhemoglobin

of

The substitutionof valine for glutamateresults in a sticky patch on the outer surfaceof p-chains. It is presenton oxy- and deoxyhemoglobinS but absent on HbA. There is a site or receptor complementaryto sticky patch on deoxyHbS.

The sticky patch of one deoxyHbSbinds with 1. Life-long hemolytic anemia : The sickled the receptor of another deoxyHbS and this erythrocytes are fragife and their continuous processcontinuousresultingin the formation of breakdown leads to life-long anemia. long aggregate molecules of deoxyHbS (Fi9.10.1a1. Thus, the polymerizationof deoxypain Tissue and : The cells 2. damage sickled leadsto long fibrous precipitates HbS molecules poor re s u l ti n g i n bl ood bloc k th e c a p i l l a ri e s (Fig.l0.15). These stiff fibres distort the leads to supply to tissues.This extensivedamage or crescent shape into a sickle erythrocytes pain. and inflammationof certaintissuescausing (Fig.l0.lA. The sickled erythrocytesare highly 3. Increased susceptibility to infection : vul nerabl eto l ysi s. Hemolysisand tissuedamage are accompanied ln case of oxyHbS, the complementary by increased susceptibility to infection and receptor is masked,although the sticky patch is diseases.

Ghapten 1O : HEMOGLOBIN AND POFIPHYFINS

205 DeoxyHbS

Fig. 10.14: Diagrammaticrepresentationof stickypatch (Blue)and stic$ patch receptor( > ) in the fomation of long aggregatesof deoxyhemaglobins.

present (Fi9.1O.14).Hence, the molecules of oxyHbS cannot bind among themselvesor with the moleculesof deoxyHbS.

2. More recent studiesindicatethat malarial parasiteincreasesthe acidity of erythrocytes(pH down by 0.4). The lowered pH increasesthe sickling of erythrocytesto about 40% from the Normal deoxyHbA lacks sticky patches but normally occurring 2Y". Therefore,the entry of contains receptors.Absence of sticky patches mal ari al parasi tepromotessi ckl i ng l eadi ng to does not allow the deoxyHbA to participatein lysis of erythrocytes. Furthermore, the the formation of aggregates. concentrationof K+ is low in sickled cells which A s ex pla i n e d a b o v e , s i c k l i n g i s d u e to is unfavourablefor the parasiteto survive. polymerizationof deoxyHbS.Therefore,if HbS Sickle-celltrait appearsto be an adaptation is maintained in the oxygenatedform (or with for the survi val of the i ndi vi dual s i n mal ari am inim um deo x y H b S),s i c k l i n gc a n b e p re v e nted. infested regions. Unfortunately, homozygous i ndi vi dual s,the pati entsof si ckl e-cel lanemi a Sickle-cell trait prov:des (much less frequent than the trait), cannot live resistance to malaria beyond 20 years. T he inc ide n c eo f s i c k l e -c e ldl i s e a s ec o i n ci des wit h t he hig h i n c i d e n c eo f m a l a ri a i n tro pi cal areasof the world (particularlyamong the black Africans). Sickle-celltrait (heterozygousstatewith about 40% HbS) provides resistanceto malaria which is a major causeof death in tropical areas.This is ex plaineda s fo l l o w s 1. M alar ia i s a p a ra s i ti cd i s e a s ec a u s ed by Plasmodium falciparum in Africa. The malarial parasite spends a part of its life cycle in erythrocytes. lncreased lysis of sickled cells ishorter life span of erythrocytes)interruptsthe oarasitecvcle.

Fig. 10.15: Diagrammatic representation a fibreof aggregateddeoxyhemoglobin.

206 Biagnosis

B IOC H E MIS TF| Y

of sickfe.cell

Normal Sickle-cellSickle-cell trait anemia

anemia

@

1 . Sicklingtest : This is a simple microscopic examinationof blood smearpreparedby adding r educ ing ag e n ts s u c h a s s o d i u m d i th ioni te. Sickled erythrocytes can be detected under the mrcroscope. 2. Electrophoresis : When subjected to elec t r ophore s i si n a l k a l i n e m e d i u m (p H 8.6), s ic k le- c ell h e m o g l o b i n (H b S ) mo v e s sl ow l y towards anode (positive electrode) than does adult hemoglobin(HbA).The slow mobility of HbS is due to less negative charge, caused by the absence of glutamate residues that carry negative charge. In case of sickle-cell trait, the fast moving HbA and slow moving HbS are observed. The electrophoresis of hemoglobin obtained from lysed erythrocytes can be routinely usedfor the diagnosisof sicklecell anemia and sickle-celltrait (Fig.t0.16). ifianaEenrent

of sickle.cell

disease

Administration ol sodium cyanate inhibits sickling of erythrocyteg Cyanate increasesthe affinityof 02 to HbS and lowersthe formationof deoxyHbS. However, it causes certain sideeffectslike peripheralnerve damage.

mn II -..Origin.-'.-"-'--'

Fig. 10.16 : Electrophoresis of hemoglobins at pH 8.6 (HbA-Normal adult hemoglobin; H bS-S ickle cell hemoglobin).

H emogl obi n

D

This is causedby the substitutionof glutamine i n pl ace of gl utamatei n the 121st posi ti oi nof B-chain. Severalvariantsof HbD are identified from different places indicated by the suffix. For instance,HbD (Punjab),HbD (LosAngeles). HbD, on electrophoresismoves along with H bS . H emogl obi n

E

Thi s i s the most common abnormal hemoglobinafter HbS. lt is estimatedthat about 1O% of the popul ati on i n S outh-E astA si a (B angl adesh, Thai l and, Myanmar) suffer In patients with severe anemia, repeated from H bE di sease.In Indi a, i t i s preval enti n blood transfusionis required.This may result in West Bengal. HbE is characterized by replacement of glutamate by lysine at 26th iron overload and cirrhosisof liver. posi ti on of p-chai n. The i ndi vi dual s of H bE Replacementof HbS with other forms of (either homozygous or heterozygous)have no hemoglobins has been tried. Fetal hemoglobin cl i ni cal mani festati ons. (HbF)reducessickling.Sickle-celldiseaseawaits gene-replacement therapy! Hem oglobin

G disease

Cooley's hemoglobinemia(HbC) is characterized by substitutionof glutamate by lysine in the sixth positionof p-chain.Due to the presence of lysine, HbC moves more slowly on electrophoresis comparedto HbA and HbS, HbC diseaseoccurs only in blacks.Both homozygous and heterozygousindividualsof HbC diseaseare known. This disease is characterizedby mild hemolytic anemia. No specific therapy is recommended.

Thalassemiasare a group of hereditary hemolyticd isorderscharacterizedby i mpairment/ i mbal ancei n the synthesi sof gl obi n chai ns of H b. Thalassemias (Greek: thalassa-sea)mostly occur i n the regi ons surroundi ng the Mediterranean sea, hence the name. These diseases,however, are also prevalentin Central Africa, India and the Far East.

207

Chapter 1O: HEMOGLOBIN AND POBPHYRINS Molecular

basis

of thalassemias

The basic conceptsin the synthesisof globin chains have been described (See Fig.l0,l41. Hem oglob i nc o n ta i n s2 a a n d 2 p g l o b i n chai ns. T he s y nt h e s i s o f i n d i v i d u a l c h a i n s i s so coordinated that each a-chain has a p-chain partner and they combine to finally give hemoglobin (o"z!). Thalassemias are characterized by a defect in the production of a-or B-globin chain. There is however, no abnor m ali tyi n th e a mi n o a c i d so f th e i n di vi dual c hains . Thalassemiasoccur due to a varietv of molecular defects

number of mi ssi nga-gl obi n genes.The sal i ent featuresof differenta-thalassemias are given in Table 10.2. 1 . Silent carrier state is due to loss of one of the four a-globin genes with no physical manifestations. 2. a-Thalassemiafraif caused by loss of two genes(both from the samegene pair or one from each gene pai r). Mi nor anemi a i s observed. 3. Hemoglobin H disease,due to missingof three genes,is associatedwith moderateanemia. 4. Hydrops fetalis is the most severeform of a-thalassemias due to lack of all the four genes. The fetus usually survivesuntil birth and then di es.

1 . Cene deletion or substitution, 2. Underproductionor instabilityof mRNA, 3. Defect in the initiation of chain synthesis,

B.Thalassemias

4. P r ema tu rec h a i n te rm i n a ti o n .

Decreased synthesis or total lack of the formation of p-glohin chain causes Pu-Thalassemias thalassemias. The production of u-globin chain o,-Thalassemiasare caused by a decreased continuesto be normal, leadingto the formation synthesis or total absence of a-globin chain of of a globin tetramer (o4) that precipitate.This Hb. There are four copies of a-globin Bene,two causes premature death of erythrocytes. There on each one of the chromosome16. Four types are mainly two types of p-thalassemias of cr-thalassemias occur which deoend on the (Fig.t0.17)

Typeof thalassemia Normal Silent canier trait a-Thalassemia (heterozygous form)

J

Hemoglobin H disease Hydrops fetalis

Number of missing genes Nil

Schematic representation of geneson chromosome 16

Clinical symptoms

flfl-gt-El-

Nil

--::giiG fl-g-

Nosymptoms Minor anemia

-.-*i..r-El-,.01.,€F -'.-9i.€l----';.-ql.i-EF -..9i.,r-&Ff--%-,

Mildto moderate anemia mayleadnormal life. Fetaldeath usually occurs at birlh.

BIOCHEMISTF|Y

208

Genesfor p-globin chain

-r----t-------

i:....r:..:::i No-pchain

\_/ 1pC hai n p Thalassemia minor

p Thalassemia malor

Fig. 10.17: Diagrammatictepresentationof gene deletionsin p-thalassemias (each one of the chromosomepair of 11 has one gene for B-globin).

1. p-Thalassemiaminor : This is an heterozygous statewith a defect in only one of the two p-globin gene pairs on chromosome 1 1 . This disorder, also known as p-thalassemia trait, is us uallya s y m p to m a ti cs, i n c e th e i n d i v i dual scan make some amount of p-globin from the affected gene.

N atural l y occurri ng porphyri ns conta in substituent groups replacing the 8 hydrogen atoms of the porphyrin nucleus.

Hans Fischer, the father of porphyrin chemistry, proposed a shorthand model for Accordingly, presentation of porphyrinstructures. each pyrrole ring is representedas a bracket. 2. p-Thalassemia major : This is a Thus porphyrin has 4 closed bracketswith the homozygousstatewith a defect in both the genes I substituentoositions numbered as shown in responsiblefor B-globin synthesis.The infants Fig.l0.t8. born with p-thalassemiamajor are healthy at Type I porphyrins : When the substituent bir t h s i n c ep -g l o b i ni s n o t s y n th e s i z ed duri ngthe groups on the 8 posi ti ons are symmetri cally fetal development. They become severely arrangedthey are known as type I porphyrins, anemic and die within 1-2 years.Frequentblood e.g. uroporphyrin L transfusionis requiredfor thesechildren. This is Type lll porphyrins: They containasymmetric associatedwith iron overloadwhich in turn may Broupsat the 8 positionsand are more common lead to death within 15-20 years. i n the bi ol ogi cal system. Ori gi nal l y, Fi sch er placed them as lX series hence they are more popularlyknown as type lX porphyrins.lt may be observed that the structure of uroporphyrin is Porphyrinsare cyclic compounds composed asymmetric since on ring lV, the order of of 4 pyrrole rings held together by methenyl substituentgroups is reversed(P, A instead of (:CH-) bridges(Fig.l0JA. Metal ions can bind A, P). with nitrogen atoms of pyrrole rings to form The Fischer'sshorthandmodels of important c om ple x e s . H e me i s a n i ro n-contai ni ng porphyri ns(uroporphyriInand l l l ; coproporphy r in porphyrin (See Fi9.10.2)while chlorophyll is a protoporphyrin lX and heme) are lll; I and m agne s i u m -c o n ta i n i n p g o rp h y ri n . Thus heme in Fig.l0.l9. depicted and chlorophyll are the classical examples of metalloporphyrins. in cancer therapy Forphyrins Fresentation and nomenclature The photodynamic propertiesof porphyrins of porphyrins can be used in the treatmentof certain cancers. The structure of porphyrins (c2oH 14N 4) This is carried out by a techniquecalled cancer l l l and l V . phototherapy. Tumors are capable of taking up has four pyrrole rings namely l,

Chapter 1O: HEMOGLOBIN AND POFIPHYFIINS HC_CH

209 AP

l tll Hc"'r-cH H

PA Uroporphyrln I AP

PA Uroporphyrin lll MP

HH Poiphyrln PM Coproporphyrin I MP

PM Coproporphyrin lll 65

Porpt4pin

MV

12

PM Protoporphyrln lX (lll) Fischer's model

MV

Fig. 10.18: Structuresof pynole and porphyrin fllV arepynole rings; 1-Bare substituentpositions; a, F, y, 6 arq n?tlly"lqne(-Cll=) bridgos,l rrore porphyrinsthan normal tissues.The cancer phototherapy is carried out by administering hematoporphyrin (or other related compounds) to the canceroatient.When the tumor is exoosed to an argon laser,the porphyrinsget excited and oroduce cvtotoxic effectson tumor cells.

PM Heme

FIg. 10,19:Fischer'sshorthandmodelsat

210

BIOGHEMISTFIY propionateside chains (P)to vinyl groups (V) and resultsin the formation of protoporphyrinogen.

Heme is the most important porphyrin (c) Protoporphyrinogen oxidase oxidizes c ont ai n i n gc o m p o u n d .l t i s p ri m a ri l ysynthesi zed (-CH2-) groups methylene in the liver and the erythrocyte-producing cells pyrrole rings to methenyl interconnecting of bone marrow (erythroidcells).Heme synthesis groups (= C H -). Thi s l eads to the also occurs to some extent in other tissues. synthesisof protoporphyrinlX. However, mature erythrocytes lacking mitochondria are a notable exception. 5. Synthesis of heme from protoporphyrin Biosynthesisof heme occurs in the following of ferrousiron (Fe2+)into lX : The incorporati,on stages(Fig.l0.20). protoporphyrin IX is catalysedby the enzyme 1. Formation of 8-aminolevulinate : Glycine, ferrochelatase or heme synthetase.This enzyme a non-essentialamino acid and succinyl CoA, an can be inhibited by lead. lt is found that the intermediate in the citric acid cvcle, are the induction of Fe2+ into protoporphyrin lX can starting materials for porphyrin synthesis. occur spontaneouslybut at a slow rate. Glycine combines with succinyl CoA to form 6-aminolevulinate(ALA).This reactioncatalysed Regulation of heme synthesis by a pyridoxal phosphate dependent 6-aminoHeme production in the liver is required for levulinate synthaseoccurs in the mitochondria. the formation of hemoproteins(e.9. cytochrome It is a rate-controllingstep in porphyrinsynthesis. P456 involved in detoxification)while in the 2. Synthesisof porphobilinogen : Two mole- erythroidcells, it is necessaryfor the synthesisof cules of 6-aminolevulinatecondense to form hemoglobin.Two differentmechanismsexist for por pho b i l i n o g e n (PB G ) i n th e c ytosol . Thi s the regulationof heme biosynthesisin the Iiver reactionis catalysedby a Zn-containingenzyme and the erythroid cells. ALA dehydrafase.lt is sensitiveto inhibition by Regulationin the liver : The first committed heavv metals such as lead. catalysedby 6-aminostep in heme biosynthesis, 3. Formation of porphyrin ring : Por- levulinate(ALA) synthase,is regulatory.Heme phyrin synthesis occurs by condensation of or its oxidized product hemin (Fe3+)controlsthis f our m o l e c u l e s o f p o rp h o b i l i n o g e n.The four enzyme activity by three mechanisms pyrrole rings in porphyrin are interconnected 1 . Feedbacki nhi bi ti on by methylene (-CH2) bridges derived from cx,-carbon of glycine. 2. Repressionof ALA synthatase The interaction of two enzymes-namely 3. Inhibition of transport of ALA synthase uroporphyrinogen I synthase and uroporphyfrom cytosolto mitochondria(the site of action). rinogen lll cosynthase-resultsin condensation Effect of drugs on ALA synthaseactivity : The of porphobilinogenfollowed by ring closureand isomerizationto produce uroporphyrinogenlll. activity of ALA synthase is markedly increased by the administrationof a large number of drugs 4. Conversion of uroporphyrinogen lll to phenobarbital, insecti cides, carcinogens etc. e.g. protoporphyrin lX : This is catalysedby a series This is expected since these compounds are of reactions mostly metabolized by a heme containing (a) Uroporphyrinogen decarboxylase decarbo- protein, cytochrome Pa56.On administrationof xylatesall the four acetate(A) side chains drugs, cellular levels of heme are depleted due to form methyl groups (M), to produce to its increasedincorporation into cytochrome coproporphyrinogen. Pa5s.The reducedheme concentrationincreases (b)Coproporphyrinogen oxidase convefts the synthesis(derepression) of ALA synthaseto (oxidative decarboxvlation)two of the meet the cel l ul ardemands.

AND PORPHYRINS Chapter 1O : HEMOGLOBIN

271

cooH l

(CHr) , C-O

c H r-N H , t-

+

cooH

O-Co A Succinyl CoA

Glyclne

Uroporphyrinogen lll I Uroporphyrinogen MP cr-Amino p-ketoadipate

I

synthase CO,+-1 6-Arninolevulinate I

+

cooH

(?r,), I

M

M

P

P

PM Coproporphyrinogenlll

*1

lCo

^- n

8-Amlnolevullnate | CH2NH2 (ALA) COOH

COOH

MV

PM Protoporphyrlnogen lX

II

o,HH,gh1Hl1"'" 2H2o+-1 J OHCO ll CH, l' l- -

MV

COOH ( CHo ) ,

c---c illl

-I

NH,

PM Protoporphyrln lX I Fe2*--.1 FonocheJaiase

I

J

I

I

MV

4 PBG molecules

PM HEME Flg. 10.20 conld. next column

FIg. 10.20: Biosynthesisot heme[A-Acetyl (-

- COC ) ; P-P ropionyl (- CH2- CH2- COC ) ;

B IOC H E MIS TRY

212

SuccinylCoA+ Glycine

|

I D-Aminolevulinate

synthase +I N -F...

\.. HEMOGLOBIN

(ALA) 6-Aminolevulinate ''..

. nl| Uroporphyrinogen

.,.

t -

I

l^ .- ...

u .Jr ,*

.1 AlAdehydratase

Fenp9hetatase

t='... +

Lioht lroporitQn lll#-UroPot 3

| I Lioht

I-

lll Coproporphyrin

alongwithporphyilas Fig. 10.21: Summaryof hemesynthesis (1-Acute intermittentporphyia; 2-Congenitalerythropoieticporphyria;3-Porphyria cutaneatarda;4-Hereditarycoptoporphyia;S-Variegateporphyria;6-Protoporphyria).

Regulation in the erythroid cells : The enzymeALA synthasedoes not appearto control the heme synthesis in the erythroid cells. Uroporphyrinogen synthase and ferrochelatse mostly regulate heme formation in these cells. Further, the cellular uptake of iron also influences heme synthesis.lt is observed that hem e s ti mu l a te sg l o b i n s y n th e s is.Thi s ensures that heme and globin synthesisoccur in the right proportion to finally form hemoglobin.

A l l the know n porphyri asare i nheri te d as autosomal dominant disorders. Howere' congenital erythropoietic porphyria is a' exception, since it is autosomal recessive.The different types of porphyrias are describec (Fig.l0.21, Table 10.3\ l. Acute

intermittent

porphyria

This disorderoccurs due to the deficiencyothe enzyme uroporphyrinogen I synthase. Acute intermittent porphyria is characterized br increased excretion of porphobilinogen and 6-aminolevulinate.The urine gets darkened on exposure to air due to the conversion ol Porphyrias are the metabolic disorders of porphobi l i nogento porphobi l i nand porphvr ir heme synthesis,characterized by the increased The other characteristic features of acut€ intermittentporphyria are as follows porphyrins porphyrin of or excretion precurcors.Porphyriasare either inherited or . lt is usuallyexpressedafterpubertyin human= acquired. They are broadly classifiedinto two . The symptoms i ncl ude abdomi nal paicategories vomi ti ng and cardi ovascul arabnormalit ies 1 . Erythropoietic: Enzymedeficiencyoccurs distrubancesobserved The neuropsychiatric in the erythrocytes. these patients are believed to be due :: reduced activity of tryptophan pyrrola= 2. Hepatic : Enzymedefect lies in the liver.

Chapter 1O : HEMOGLOBIN AND PORPHYRINS

Type of porphyria

213

Enzvme defect

Characteristics

Hepatic porphyria Acuteintermittent

Uroporphyrinogen I synthase

Porphyria cutanea tarda

Uroporphyrinogen decarboxylase Photosensitivity

Hereditary coproporphyria

Corpoporphyrinogen oxidase

pain,photosensitivity, Abdominal neuropsychiatric symptoms

porphyria Variegate

Protoporphyrinogen oxidase

pain,photosensitivity, Abdominal neuropsychiatric symptoms

pain,neuropsychiatric Abdominal symFoms

Erythropoietic porphyria Uroporphyrinogen erythropoietic Congenital lll cosynthase Photosensitivity, increased hemolysis Protoporphyria

Fenochelatase

Photosensitivity

(caused by depleted heme levels), resulting ll, Gongenital erythropoletie in the accumulation of tryptophan and porphyria 5-hydroxytryptamine. This disorderis due to a defect in the enzyme . The symptoms are more severe after u ropo rphy ri nogen I I I cosynthase. Some workers, administrationof drugs (e.g. barbiturates)that however, believe that congenital erythropoeitic induce the synthesisof cytochrome Pouo.This porphyria is caused by an imbalance between is due to the increasedactivityof ALA synthase the activitiesof uroporphyrinogenI synthaseand c aus inga c c u m u l a ti o no f PB C a n d AL A . uroporphyrinogenlll cosynthase.This disease has certain characteristicfeatures . Thesepatientsare not photosensitive sincethe enzymedefectoccursprior to the formationof . lt is a rare congenital disorder caused by uroporphyrinogen. autosomal recessive mode of inheritance. mostly confined to erythropoietictissues. Acute intermittent porphyria is treated by adm inis t r a ti o no f h e m a ti n w h i c h i n h i b i ts the enzyme-AlA synthaseand the accumulationof porphofflinogen. [The disease-acute intermittentporphyriahashis t or i c a il m p o rta n c eK . i n g C e o rg el l l (17601820) r ule d E n g l a n d d u ri n g th e p e ri od of \merican revolution. He was a victim of this disease and possessed the characteristic (suchas red colour urine)and was nranifestations c ons ider e dm a d . T h e d e c i s i o n sta k e n by the deranged King due to acute intermittent porphyria had led to a war followed by American Independence.lt is widely believed that Americanhistorywould have been different, had Ceor g e l l l n o t i n h e ri te d th i s metabol i c d is or der ! l

. The individuals excrete uroporphyrinogenI and coproporphyrinogen I which oxidize respectively to uroporphyrin I and coproporphyrin | (red pigments). . The patients are photosensitive(itching and burning of skin when exposedto visible light) due to the abnormal prophyrins that accumul ate. . Increasedhemolysis is also observed in the individualsaffectedbv this disorder. lll. Forphyria

cutanea

tarda

This is also known as cutaneous hepatic porphyria and is the most common porphyria, usually associatedwith liver damage causedby

r-214

B IOC H E MISTFI Y

alcohol overconsumptionor iron overload. The and feces. Reticulocytes(young RBC) and skir: partial deficiency of the enzyme biopsy exhibit red flourescence. uroporphyrinogen decarboxylase appears to be responsible for the occurrence of porphyria Acquired {toxic} porphyrias cutanea tarda. The other characteristicfeatures The porphyrias,though not inherited,may be in c l u d e acquired due to the toxicity of severaj . lncreasedexcretionof uroporphyrins(l and lll) compounds. Exposure of the body to hearr a n d ra re l y p o rp h o b i l i n o g e n . metal s (e.g. l ead), toxi c compounds ( e. g . Cutaneous photosensitivity is the most hexachlorobenzene) and drugs (e.g.griseofulvin important clinical manifestation of these inhibits many enzymesin heme sylthesis.These include ALA dehydratase, r.r4irporphyrinI oatients. sVnthase and ferrochelatase. . L i v e r e x h i b i ts fl u o re s c e n ce due to hi gh concentrationof accumulatedporphyrins. lV. llereditary

coproporphyria

This disorderis due to a defect in the enzvme coproporphyrinogen oxidase. As a result of this, coproporphyrinogenlll and other intermediates (ALA and PBC) of heme synthesisprior to the blockade are excreted in urine and feces. The victims of hereditary coproporphyria are ph o to s e n s i ti v e . T h e y e x h i b i t the cl i ni cal manifestations observedin the patientsof acute intermittentporphyria. Infusion of hematin is used to control this dis o rd e r. H e ma ti n i n h i b i ts AL A synthaseand thus reducesthe accumulationof various intermediates. V. Variegate

porphyria

The enzyme protoporphyrinogen oxidase is defectivein this disorder.Due to this blockade, protoporphyrin lX required for the ultimate synthesisof heme is not produced. Almost all the (porphobilinogen, intermediates coproporphyrin, uroporphyrin, protoporphyrin etc.) of heme synthesisaccumulatein the body and are excretedin urine and feces.The urine of these patients is coloured and they exhibit photosensitivity. V l. P ro to p o rp h y ri a This disorder, also known as erythropoietic protoporphyria, is caused by a deficiency of the enzyme ferrochelatase.Protoporphyrin lX accumulatesin the tissuesand is excretedinto urine

Erythrocyteshave a life span of 120 days. At the end of this period, they are removed from the circulation. Erythrocytesare taken up and degraded by the macrophages of the reticuloendothelial(RE)systemin the spleenand liver. The hemoglobin is cleaved to the protein part globin and non-proteinheme. About 6 g of hemoglobin per day is broken down, and resynthesizedin an adult man (70 kg). Fate of globin : The globin may be reutilized as such for the formation of hemoglobin or degraded to the i ndi vi dual ami no aci ds. The Iatterundergothei r ow n metabol i sm,i nc luding participationin fresh globin synthesis. Sourcesof heme : lt is estimatedthat abour 80% of the heme that is subjected for degradationcomesfrom the erythrocytesand the rest (2O"/') comes from immature RBC, myoglobin and cytochromes. Heme oxygenase : A complex microsomal enzyme namely heme oxygenase utilizes NADPH and 02 and cleaves the methenyl bridgesbetweenthe two pyrrole rings (A and B) to form biliverdin. Simultaneously,ferrous iron (Fe2+) is oxidized to ferric form (Fe3+) and released. The products of heme oxygenase reaction are biliverdin (a green pigment), Fe3+ and carbon monoxide (CO). Heme promotesthe activity of this enzyme.

l"ireptrrr "l t] : HEMOGLOBIN AND PORPHYFIINS

215

il

Agederythrocytes

I l'

il

I :

Blllrubin

I Bilirubin-albumin complex I

Bilirubin Reutilized or degraded

Otherheme proteins

rl

Glucuronic Glucuronic acid acid I I

o:c

i CH, t-

I ^tu-

V

Bilarubin dlglucuronide (to bile)

INTESTINE/KIDNEY

Microbial enzymes (intestine)

H Biliverdin

Urobilin Tourine

Stercobilin

t

Tofeces

Fig. 10.22: Degradationof heme to bile pigments (No|e : Colours used in structures represent change in the specific reaction only).

""4 B iliv er dinis e x c re te di n b i rd s a n d a mp h i b i a bi l i rubi n i s dai l y producedi n human adul ts.The rvhile in mammals it is further degraded. term bile pigments is used to collectively representbilirubin and its derivatives. Biliverdin reductase : Biliverdin's methenyl bridges(betweenthe pyrrole rings C and D) are Transport of bilirubin to liver : Bilirubin is reduced to methylene group to form bilirubin l i pophi l i c and therefore i nsol ubl e i n aqueous rvellow pigment).This reaction is catalysedby sol uti on.B i l i rubi ni s transportedi n the pl asmai n an NADPH dependent soluble enzyme, a bound (non-covalently) form to albumin. biliverdin reductase(Fi9.10.22).One gram of Albumin has two binding sites for bilirubin-a hemoglobin on degradation finally yields about high affinity site and a low affinity site. 35 mg biliruhin. Approximately 250-350 mg of A pproxi matel y 25 mg of bi l i rubi n can bi nd

Ir

276 tightly to albumin (at high affinity sites)per 100 m l of p l a s m a . T h e re s t o f th e b i l i ru bi n bi nds loosely (at the low affinity sites)which can be easily detached from albumin to enter the tissues. Certain drugs and antibiotics (e.g s ulf ona m i d e ss, a l i c y l a te sc)a n d i s p l a cebi l i rubi n f r om al b u mi n . D u e to th i s , b i l i ru b i n can enter the central nervoussystemand causedamageto neur on s .

E l IOC H E MIS TF\ compound), a small part of which may rt reabsorbedi nto the ci rcul ati on. U robi l i noee can be convertedto urobi l i n (an yel l ow col o-' compound) i n the ki dney and excreted. Tr. characteristic colour of urine is due to urobilin A major part of urobilinogenis convertedc. bacteria to stercobilin which is excreted alon= with feces. The characteristic brown colour oi feces is due to stercobilin.

As the albumin-bilirubin complex enters the rb liv er , bi l i ru b i n d i s s o c i a te sa n d i s ta k en up by sinusoidalsurfaceof the hepatocytesby a carrier mediated active transport. The transport system has a very high capacity and thereforeis not a lim it at io n fo r fu rth e r me ta b o l i s mo f bi l i rubi n. The normal serum total bilirubin concerInside the hepatocytes, bilirubin binds to a tration is in the range of 0.2 to 1.0 mg/dl. a specific intracellularprotein namely ligandin. this, about 0.2-0.6 mg/dl is unconjugatedwhi,e O.2 to 0.4 mg/dl i s conj ugatedbi l i rubi n. Songugatd€ffi #{ hilirubirr is a clinica Jaundice(French: Jaune-yellow) ln the Iiver, bilirubin is conjugatedwith two condition characterizedby yellow colour of the m olec u l e s o f g l u c u ro n a te s u p p l i e d by U D P - white of the eyes (sclerae)and skin. lt is causec glucuronate.This reaction, catalysedby bilirubin by the depositionof bilirubin due to its elevateo glucuronyltransferase (of smooth endoplasmic levels in the serum. The term hyperbilirubinemia reticulum) results in the formation of a water is often used to represent the increasec s oluble b i l i ru b i n d i g l u c u ro n i d e(F i g s.| 0.22and concentrati onof serum bi l i rubi n. (N ofe ; For 10. 23' ) .Wh e n b i l i ru b i n i s i n e x c e s s,bi l i rubi n some more detailson jaundice, referChapter20 monoglucuronidesalso accumulatein the body. T he enz y me b i l i ru b i n g l u c u ro n y l tra nsferase can {i tassi $i eati on E yfi aundi ee be induced by a number of drugs (e.g. Jaundice(alsoknown as icterus)may be more phenobarbital), appropriatelyconsidered as a symptom rather than a disease.lt is rather difficult to classin €xcre'tE41n ,;t hi{tru.e.;','idlti]alirri4e jaundice, since it is frequently caused due to Conjugatedbilirubin is excretedinto the bile multiole factors.For the sake of convenienceto canaliculiagainsta concentrationgradientwhich understand,j aundi ce i s cl assi fi ed i nto three then enters the bile. The transport of hilirubin major types-hemolytic, hepaticand obstructive. diglucuronide is an active, energy-dependent 1 . Hemolyticjaundice: This conditionis assoand rate limiting process. This step is easily with increased hemolysis of erythrocytes ciated s us c epti b l eto a n y i m p a i rme n ti n l i v e r functi on. (e.g. i ncompati bl ebl ood transfusi on,mal ari a . Normally, there is a good coordinationbetween This results in the oversickle-cell anemia). the bilirubin conjugation and its excretion into production of bilirubin beyond the ability of tne bile. T h u s a l m o s t a l l th e b i l i ru b i n (> 9 8% ) that Iiver to conjugate and excrete the same.lt should, enters bile is in the conjugatedform. however be noted that liver possesses a large capacityto conjugateabout 3.0 g of bilirubin per *j .'ite sf lsiBiril!-rFn day agai nstthe normal bi l i rubi n producti onoi Bilirubin glucuronidesare hydrolysedin the 0.3 {day. intestineby specific bacterial enzymes namely In hemol yti c j aundi ce, more bi l i rubi n i s B-glucuronidasesto liberate bilirubin. The latter is then converted to urobilinogen (colourless excreted into the bile leading to the increased

2't7

Chariter 1O: HEMOGLOBINAND POFIPHYBINS

. lncreased activities of alanine transaminase (SGPT) and aspartate transaminase (SCOT) released into circulation due to damage to hepatocytes.

Blood Bilirubin-albumin

. The patients pass pale, clay coloured stools due to the absenceof stercobilinogen. . The affected individuals experience nausea and anorexia (lossof appetite).

I UDPglucuronvl'

zuop-crcun-rl

J

_-,/

3. Obstructive (regurgitation) jaundice : This is due to an obstruction in the bile duct that preventsthe passageof bile into the intestine. The obstructionmay be caused by gall stones, tumors etc. D ue to the bl ockage i n bi l e duct, the conjugated bilirubin from the liver enters the circulation.Obstructiveiaundiceis characterized by . Increased concentration of bi l i rubi n i n serum.

Ftg. 10.23: Summaryof bilirubinmetabolism (UDPG lcUA-U DP-glucuronic acid).

conjugated

. Serum alkaline phosphataseis elevated as it is releasedfrom the cells of the damaged bile duct.

. Dark coloured urine due to elevatedexcretion of bilirubin and clay coloured feces due to absenceof stercobilinogen. formation of urobilinogen and stercobilinogen. Hemolytic jaundice is characterizedby o Fece3 contain excess fat indicating impair. E lev ati o ni n th e s e ru mu n c o n j u g a te dbi l i rubi n. . Increasedexcretionof urobilinogen in urine. . Dark brown colour of feces due to high content of stercobilinogen.

ment in fat digestion and absorption in the absenceof bi l e (speci fi cal l ybi l e sal ts). . The patients experience nausea and gastroi ntesti nalpai n.

2. Hepatic (hepatocellular)jaundice : This JAUNDICE DUE TO type of jaundice is caused by dysfunction of the GENETICDEFECTS Iiver due to damage to the parenchymalcells. Thereare certainhereditaryabnormalitiesthat This may be attributed to viral infection (viral cause jaundice. poisons and toxins (chloroform, hepa!$! carbon tetrachloride,phosphorusetc.) cirrhosis Neonatal.physiologic jaundice of liver, cardiac failure etc. Among these, viral Physiologicaljaundice is not truly a genetic hepatitisis the most common. defect. lt is caused by increased hemolysis Damage to the liver adversely affects the coupled with immature hepatic system for the bilir ubin u p ta k e a n d i ts c o n j u g a ti o n by l i ver uptake, conjugation and secretionof bilirubin. cells. Hepatic jaundice is characterizedby The activity of the enzyme UDP-glucuronyl. Increased levels of conjugated and transferaseis low in the newborn. Further,there unc onj u g a te db i l i ru b i n i n th e s e ru m. i s a l i mi tati oni n the avai l abi l i tyof the substrate . Dark coloured urine due to the excessive U D P -gl ucuroni c aci d for conj ugati on. The net effect is that in some infants the serum ex c r et i o no f b i l i ru b i n a n d u ro b i l i n o gen.

218

B I O C H E M I S TFIY

uncojugatedbilirubin is highly elevated(may go bilirubin gets converted into a non-toxic isomer beyond 25mildl), which can cross the blood- namel y l umi ruhi n. Lumi rubi n can be easilr br a i n b a rri e r.T h i s re s u l tsi n h yperbi l i rubi nemi c excreted by the kidneys in the unconjugated toxic encephalopathyor kernicterusthat causes form (i n contrastto bi l i rubi n w hi ch can not be mental retardation. The drug phenobarbital excreted).S erum bi l i rubi n i s moni tored ever r ' is used in the treatment of neonatal jaundice, 12-24 hours, and phototherapyis continuousl;' as i t c a n i n d u c e b i l i ru b i n metabol i si ng carri ed out ti l l the serum bi l i rubi n be com es enzymes in liver. In some neonates, blood normal (< 1 mg/dl ). transfusionmay be necessaryto prevent brain b damage. P h o to th e ra p y: Bi l i ru b i nc a n absorbbl ue l i ght ( 4 2 0 4 7 0 n m ) ma x i ma l l y . P h o totherapydeal s with the exposureof the jaundiced neonatesto blue light. By a process called photoisomerization, the toxic native unconjugatecl

Thi s i s al so know n as congeni ta l nonhemol yti cj aundi ce. l t i s a rare di sorderand is due to a defect in the hepatic enzyme UDPglucuronyltransferase. Cenerally, the children die within first two years of life.

BIOMEDIGAL 1 CLINICALEONCEPT$

c€ Hemoglobin is primarily responsiblefor the deliuery of 02 lrom lungs to tissueand the transport of CO2 from tissues to lungs. go' lncreased erythrocyte 2,3-BPG leuels in anemia and chronic hypoxia Jocilitate the releaseof more 02 trom the oxyhemoglobin to fhe fissues. s'= Storage of blood couses a decrease in the concentration ot' 2,3-BPG. This can be preuented by the addition of ionosine. lea,Hemoglobin (Fe2+)on oxidation by H2O2, free radicols or drugs, t'orms methemoglobin (Fes+) whtch connot transport 02. *- Corboxyhemoglobinis produced when corbon monoxide, an industrial pollutant, binds to hemoglobin. The clinical manifestations of CO toxicity (> 200/o COHb) include headoche,nausea, breothlessnessand uomiting. av Sickle cell hemoglobin (HbS) couses hemolytic onemia, increased susceptibility to infection and premature death. Howeuer, HbS oft'ersprotection agoinst malaria. ua Thalassemiasare hemolytic disorderscausedby impoirment/ imbalancein the synfhesis of globin choins of Hb. These include a-thalassemia trait, hydrops letalis ond ftthalassemias. s Administration of porphyrins can be used in the treotment certain cancers by phototherapy. r€ Abnormalities in heme synthesiscouse porphyrias which moy be erythropoietic (enzyme defect in RBC) or hepotic (enzyme defect in liuer). Porphyrias ore associatedwith eleuated excretion of porphyrins, neuropsgchiatric disturbances and cardiouqsculor abnormalities. re Jaundice is coused by eleuated serum bilirubin (normal < 0.8 mg/dl) leuels ond is choracterizedby yellow colorotion of white of the eyes, and skin. uq Phototherapy (by exposure to blue light) is used in to control seuerecasesot' neonotal physiologic jaundice.

HEMOGLOBIN AND PORPHYRINS

219 Gilbert's disease: This is not a singledisease but a combi nati onof di sorders.Thesei ncl ude

T his i s a g a i n a ra re h e re d i ta ryd i s o rderand i s 1. A defecti n the uptakeof bi i i rubi n by l i ve r due t o a l e s s s e v e re d e fe c t i n th e bi l i rubi n c onjug a ti o n .l t i s b e l i e v e d th a t h e p ati c U D P - cel l s. gluc ur o n y l tra n s fe rath s ea t c a ta l y s e sthe addi ti on 2. A n i mpai rnrent i n conj ugati on due to of second glucuronyl gror,rpis defective. The reduced acti vi ty of U D P -gl ucuronyl transfera se. s er umb i l i ru b i nc o n c e n tra ti o ni s u s u a l l yl essthan 3. D ecreasedheoati ccl earanceof bi l i rubi n. 20 m g/d l a n d th i s i s l e s sd a n g e ro u st han type l .

7. Hemoglobin (HbA1, mol. wt. 64,450) is a conjugatedprotein containing globin, the apoprotein and the heme, the nonprotein moiety (prostheticgroup). It is a tetrarneric, allostericprotein with 2a and 2B polypeptidechainsheld by non-coualentinteractions. Each subunit containsa heme with iron in the ferrous state, 2. Hemoglobin is responsibleJor the transport of 02 from lungs to the tissues.Each heme (of Hb) can bind with one moleculeof 02 and this is facilitoted by cooperatiuehemeheme interaction. 3. Hemoglobin actiuely participates in the tronspart of CO2 /rom fissues to lungs. Increasedpartial pressure of CO2 fuCO2) accompanied by eleuated H+ decreasesthe hinding ot' 02 to Hb, a phenomenonknown as Bohr effect. CO2, H+ and Cl- are collectiuely 4. The t'our compounds namely 2,3-bisphosphoglycerate, known as qllosteric efJectors.They interact tuith hemoglobin and facilitate the release of 02 lrom oxyHb. 5. Sickle-cell anemia (HbS) is a classicalexomple of abnormal hemoglobins.lt is caused when glutamate at 6th position of ftchain is reploced bg ualine. HbS is characterized by hemolgticanemia, tissuedamage,increasedsusceptibilityto infection and premature death. Sickle-cellanemia, howeueroffers resistanceto rnalaria. 6. Thalassemiasare a group of hereditaryhernolytic disorderscharacterizedby impairment/ imbalance in the sgnthesiso/ glabin (a or p) chain of Hb. Hydrops fetolis, the most seuere form of a-thalassemio is characterized by the death oJ int'ant at birth. ft Thalassemiamajor is another seriousdisorder with seuereanemia and desth of child within 7-2 years. 7. Heme is the most important porphyrin compound,primarily synthesizedin the liuer t'rom the precursors-glycineand succinyl CoA. Heme productioin is regulated by &aminoleuulinatesynthase. Sdorphyriqs are the metabolicdisordersof heme synthesis,characterizedby the increased excretionot' porphyrinsor their precursors.Acute intermittentporphyriaoccursdue to the det'iciencaof the enzyme uroporphgrinogen I synthase and is characterized by increasedexcretion ot' porphobilinogen and &aminoleuulinate.The clinical symptoms include neuropsychiatric disturbancesand cardiouasculsr abnormoI ities. Heme is degradedmainly to bilirubin, an yellow colour bile pigment. In the liuer,it is 9. conjugatedto bilirubin diglucuronide,a more easilgexcretablet'orm into bile. lA. Jaundice is o clinical condition caused by eleuoted serum bilirubin concentrotion (normal <1.0 m7/dl). Jaundice is ol three types'hemolytic(due to increasedhemolysis), hepatic (due to impaired conjugation)and obstructiue(due to obstructionin the bile duct).

220

B IOC H E MIS TFIY

l. E s s a yq u e s ti o n s 1. Describethe structureof hemoglobinand discussoxygentransporr.

U

2. Write an accountof hemoglobinopathies with specialreferenceto sickle-cellanemia. 3. Discussthe biosynthesis of heme.Add a note on the regulationof heme synthesis. 4. What are porphyrias? Describeany three porphyriasin detail. 5. Write an accountof the degradationof heme to bile pigments.Add a note on jaundice. II. Short notes (a)Methemoglobin,(b) Heme-heme interaction,(c) Bohr effect, (d) 2,3-BPC, (e) Sickle cell (g) Acute intermittentporphyria,(h) Heme oxygenase, anemia and malaria, (fl Thalassemias, ( i) B i l i ru b i nd i g l u c u ro n i d e(i, ) C a rb oxyhemogl obrn. lIL Fill in the blanks 1. The total numberof amino acidspresentin adult hemoglobin 2. The oxidationof ferrous(Fe2+)iron to ferric (Fe3+)iron in hemoglobinresultsin the formation of a compoundnamely

3. The enzymethat catalysesthe formationof carbonicacid 4.

Name the compoundthat is increasedin RBCof anemicpatientsto facilitatethe supplyof O, to the tissues

5. Si c k l i n go f R B C i n s i c k l e -c e lal nemi ai s due to pol ymeri zati on of 6. The disorderscharacterizedby decreasedsynthesisor total absenceof globin chains of hemoglobinare collectivelyknown as 7. The intermediate of citric acid cycle that is involvedin heme synthesis B. The enzymedefectin acute intermittentporphyria 9. The enzymethat is regulatedby feedbackinhibitionin heme synthesisis 10. The productformedwhen heme oxygenasecleavesheme IV. Multiple choice questions 11. The characteristic red colour of hemoglobinis due to (a) Heme (b) a-Clobin (c) 0-Globin(d) All of them. 12. The numberof heme groupspresentin myoglobin (a) 1 (b) 2 (c) 3 (d) a. 13. The patientsof sickle-cellanemiaare resistantto (a) Filaria(b) Malaria(c) Diabetes(d) Trypanosomiasis. 14. The compoundthat facilitatesthe releaseof O, from oxyhemoglobin (a) 2, 3-BPC(b) H+ (c) Cl- (d) All of them. 15. Name the amino acid that directlyparticipatesin the synthesis of heme (a) Methionine(b) Aspartate(c) Clycine (d) Tryptophan.

NHz

"/r_i,\. \'^-t_-__l

" ?i"t"'',] r\!

H

l,/')

the hflgh efle1ggl cotnltor,,rrd. ATP cpeoks l "I am thelnery currenry of thecei'lt t

' : r'

Continuousconsamptknand rcgennationis ny thilt Vithout me, all biocbemicalfunrtions rc$ic io'a standsti.ll,; Existenc.e of \tfs is unimaginahl;rwithout my will"t'

u n d e rs ta n d i n go f bi ol ogi cal D uri ng a chemi cal reacti on, heat may be E or a b e tte r I oxidation. it is worthwhile to have a basrc releasedor absorbed.Enthalpy (AH) is a measure knowledgeof bioenergeticsand the role of high- of the change in heat content of the reactants, compared to products. ener gyc o m p o u n d si n b i o l o g i c a lp ro c esses. Entropy (AS) represents a change in the randomness or d isorder of reactants ano products. Entropy attains a maximum as the Bioenergeticsor biochemical thermodynamics reacti onapproachesequi l i bri um.The reacti ons deals with the study of energy changes(transfer of biological systems involve a temporary and ut i l i z a ti o n )i n b i o c h e m i c a l re a c ti ons.The decrease in entropy. reactions are broadly classified as exergonic (energy releasing) and endergonic (energy The relation between the changes of free c ons um i n g ).B i o e n e rg e ti cis c o n c e rn edw i th the energy (AC), enthalpy (AH) and entropy (AS) is initial and final statesof energycomponentof the exoressedas r eac t an tsa n d n o t th e me c h a n i s mo f chemi cal A C = A H -TA S reacuons. T representsthe absolutetemperaturein Kelvin (K = 273+ " C ). The energy actually available to do work lbt iliz ab l e ) i , i n o * n a s fre e e n e rg y .C hangesi n the fiee energy (AG) are valuable in predicting t he f ea s i b i l i ty o f c h e mi c a l re a c ti ons. The reactionscan occur spontaneouslyif they are accompaniedby decreasein free energy.

The term standard free energy representedby AC' (note the superscript') is often used. lt indicates the free energy change when the reactantsor productsare at a concentrationof 1 mol/l at pH 7.0.

221

222

BIOCHEMISTF|Y

change is an additive value. The sum of AC is crucial in determining whether a particular lf free energy change (AG) is representedby a pathwaywill proceedor not. As long as{he sum negative sign, there is a loss of free energy. The of AGs of individual reactionsis negative,the reaction is said to be exergonic, and proceeds pathway can operate.This happensdespitethe spontaneously. On the other hand, a positiveAC fact that some of the individual reactionsmav indicates that energy must be supplied to the have positiveAG. reactants. The reaction cannot proceed spontaneously and is endergonic in character. Negative

and positive

AG

The hydrolysisof ATP is a classicalexample of exergonic reaction ATP + H2O--+

ADP + Pi (AG' =-7.3 Cal/mol)

The reversalof the reaction (ADP + Pi -+ ATP) is endergonicand occurs only when there is a supply of energy of at least 7.3 Cal/mol (AG' is positive). The free energychangebecomeszero (AG = 0) when a reactionis at equilibrium. \ At a constanttemperatureand pressure,AC is dependent on the actual concentration of reactants and products. For the conversion of reactant A to product B (A -+ B), the following mathematicalrelation can be derived Ac = AGo + Rt tn

[B]

where ACo = Standard fr"JXn"rry

Compounds change

R = Gas constant(1.987 Cal/mol) T = Absolutetemperature(273 +'C) In = Natural logarithm [B] = Concentrationof product [A] = Concentration of reactant. AG' is related to equiEibriuarn constant

value for pathways

Biochemicalpathwaysoften involve a series of reactions. For such reactions, free energy

AG'(Cal/mol)

phosphates High+nergy pyruvate Phosphoenol phosphate Carbamoyl AMP Cyclic 1,3-Bisphosphoglycerate Phosphocreatine Acetylphosphate * S-Adenosylmethionine Pyrophosphate AcetylCoA{'* ATP+ADP+Pi

(Kegi

When a reaction A T^ B is at equilibrium (eq), the free energy change is zero. The above equation may be written as [B ]eq' AG=0=AGo+RTln [o] "o' Hence Aco = - RT In *"o. nG is am additive

Certain compounds are encountered in the biological system which, on hydrolysis, yield energy. The term high-energy compounds or energy rich compounds is usually applied to substances which possess sufficient free energy to liberate at least 7 Cal/mol at pH 7.0 (Table l1.l). Certain other compounds which f iberate less than 7.O Cal/mol (lower than ATP hydrolysis to ADP + Pi) are referred to as lowenergy compounds.

phosphates Low-energy ADP+AMP+Pi 1-phosphate Glucose Fructose 6-phosphate 6-phosphate Glucose

_3-qh9s!Et9 _ _ _Gly.J'gl x *'*

conpound Sulfonium Thioestel

-14,8 -12.3 -12.0 -11.8 -10.3 -10.3 -10,0 -8.0 -7.7 -7.3 -6.6 -5.0 -3.8 -3,3 -2.2

223

1 r BIOLOGICALOXIDATION .",.i:?.::r:''i

Bond

Class

Pyrophosphates -c-@-@

o ll

Acylphosphates -c-o-\g

Enolphosphates

-cH -8-o-O

c Thiolesters (thioesters)

-i-O-S-

I -N-P phosphates Guanidio (Phosphagens)

^

energy bonds, since the free energy is Iiberated w hen these bonds are hydrol ysed. Li pmann suggesteduse of the symbol - to represent highExample(s) energy bond. For instance, ATP is written as ATBpyrophosphate A MP -P -P . :r:i;t,ilrr'+p *i q'fa *g{ .iLTF''j: *"rr-!:'-,'r 1,3-Bisphosphoglycerate, . ti r fi :l l i l fu!1{l ;- r ;,- l t..:." s: -r' phosphate, carbamoyl Adenosinetriphosphate(ATP)is a unique and acetylphosphate the most i mportanthi gh-energymol ecul e i n the pyruvate l i vi ng cel l s. l t consi stsof an adeni ne,a ri bose Phosphoenol and a triphosphatemoiety (Fig.ll.l). ATP is a high-energycompound due to the presenceof two phosphoanhydride AcetYl CoA,acYlCoA bonds in the triphosphate unit. ATP serves as the energy currency of the cell as is evident from the ATP-ADP cvcle. Phosphocreatine, phosphoarginine

A ll th e h i g h -e n e rg y c o mp o u nds-w hen hydrolysed-liberate more energy than that of ATP. These include phosphoenol pyruvate, 1,3bisphosphoglycerate, phosphocreatine etc. Most of t he h i g h -e n e rg y c o mp o u n d s contai n phosphategroup (exceptionacetyl CoA) hence they are called high-energy phosphate c om po u n d s . 'ii;:r',.* ;r:i: ':::r:ii' .'

1..

r-..*

:

,:i ' ;r.

A?Fe=et!#i li!,.;1.r4' The hydrolysisof ATP is associatedwith the releaseof large amount of energy. ATP + H2C ------+ADP + Pi + 7.3 Cal. The energy liberated is utilized for various processes like muscle contraction, active transport etc. ATP can also act as a donor of high-energy phosphate to low-energy compounds,to make them energyri ch. On the other hand, ADP can accept high-energy phosphate from the compounds possessing higher free energy content to form ATP.

A TP serves as an i mmedi atel v avai l ab le There are at least 5 groups of high-energy energy currencyof the cel l w hi ch i s constantly c om oo u n d s . e.g. ATP. 1. Pyrophosphates z-

Acyl phosphates e.g. 1,3-bisphosphoglycerate.

3. Enolphosphatese.g. phosphoenolpyruvate. A

l.

Thioesterse.g. acetyl CoA.

e.g. phosphocreatine. 5. Phosphagens Table ll.2 gives some more details on the high- en e rg yc o mp o u n d s , i n c l u d i n g the hi gh- ,energy bonds presentin each category. High-energy bonds : The high-energycompougds possessacid anhydride bonds (mostly phosphoanhydridebonds) which are formed by the condensationof two acidic groupsor related compounds.Thesebonds are referredto as high-

i;'

r-l

U 7 r: i:-O

Triphosphate

OH OH Fig. 11.1: Structureof ATP.

224

B IOC H E MIS TFIY Oxidative

compound. In invertebrates,phosphoargi ni ne (argininephosphate)replacesphosphocreatine.

Oxidation is defined as the loss of electrons and reducti onas the gai n of el ectrons.Thi s may be illustratedby the interconversionof ferrous ion (Fe2+)to ferric ion (Fe3+).

Fig. 11.2: ATP-ADPcyclealongwithsourcesand utilizationof ATP(Notethat -P doesnot existin free form,but is only transferred).

eThe electron lost in the oxidation is accepted by an acceptorwhich is said to be reduced.Thus the oxidation-reduction is a tightly coupled orocess.

being utilized and regenerated. This is representedby ATP-ADPcycle, the fundamental The generalpri nci pl e of oxi dati on-reducti on bas is of en e rg y e x c h a n g e re a c ti o n s i n l i vi ng system (Fig.l 1.2). The turnover of ATP is very i s appl i cabl e to bi ol ogi cal systemsal so. The high. oxi dati onof N A D H to N A D + coupl ed w i th the reduction of FMN to FMNH" is illustrated ATP acts as an energy link between the catabolism (degradation of molecules) and anabolism(synthesis)in the biological system.

Or, ."" O" synthesizedin two ways 1. Oxidative phosphorylation : This is the In the above illustration,there are two redox m ajor s our c eo f A T P i n a e ro b i co rg a n i s ms.l t i s pai rsN A D H /N A D +and FMN /FMN H 2.The redox linked with the mitochondrialelectrontransport (details pairs differ in their tendency to lose or gain chain describedlater). electrons. 2. Substrate level phosphorylation : ATP may be directly synthesizedduring substrate I . ;.'r:, p*rt;+;riv,il AS.,i 'i,i ox idat ion in th e m e ta b o l i s m.T h e h i g h -e nergy compounds such as phosphoenolpyruvate The oxidation-reduction potential or, simply, (intermediatesof redox potential,is a quantitativemeasureof the and 1,3-bisphosphoglycerate gly c oly s isa) n d s u c c i n y lC o A (o f c i tri c a c i d cycl e) tendency of a redox pair to lose or gain can transferhigh-energyphosphateto ultimately electrons.The redox pairs are assignedspecific produce ATP. standard redox potential (Eovolts) at pH 7.0 and 25"C. a-.

- --

(

The redox potentials of some biologically importantredox systemsare given in Table 11.3. Phosphocreatine (creatine phosphate) stored The more negativeredox potential representsa in vertebratemuscle and brain is an energy-rich greatertendency(of reductant)to lose electrons. :7

t:;gl(.,rt,j..

ililll

I

225

BIOLOGICAL OXIDATION

Redoxpair

Eo Volts

Succinate/s-ketoglutarate

- 0.67

zHrl1z

-0.42

NADVNADH

- 0,32

NADP'NADPH

- v.Jz

(enzyme FMN/FMNHz bound)

- 0.30

Lipoate(ox/red)

- 0.29

FAD/FADH2

-0.22

Pyruvate/lactate

-n10

Fumarate/succinate

+ 0.03

Cytochrome b (Fe3+/Fe2*)

+ U .U /

Coenzyme Q (ox/red)

+ 0.10

c1 (Fe3*/Fe2*) Cytochrome

+ 0.23

Cytochrome c (Fe3'/Fe2+)

+ 0.25

a (Fe3+/Fe2+) Cytochrome

+ 0.29

1

ToJHro

Fig. 11.3 : Overview of biological oxidation (ETC-Electron transpott chain).

and FADH2. The latter two reducedcoenzymes passthrough the electron transportchain (ETC) or respiratory chain and, finally, reduce oxygen to water. The passageof electronsthrough the ETC is associatedwith the lossof free energy.A part of this free energy is utilized to generate ATP from ADP and Pi (Fi9.11.3). An overview of the ETC is depicted in Fig.t t.a. :-i

,'

:ir't,'rr,yr;:,

1p.,:,

i',11:r.

,,

i

, !:r

:r,,.:,.:i

,:.

+ 0.82

On the other hand, a more positive redox potential indicates a greater tendency (of oxidant)to accept electrons.The electronsflow from a redox pair with more negative Eg to another redox pair with more positive Es.

The mitochondria are the centres for metabolic oxidative reactions to generate reducedcoenzymes(N A D H and FA D H 2)w hi ch, i n turn, are uti l i zed i n E TCto l i berateenergyi n the form of ATP. For this reason,mitochondrion is appropriately regardedas the power house of the cell.

The redox potential (Eo)is directly relatedto the change in the free energy (AC'). The mi tochondri onconsi stsof fi ve di sti nct parts.These are the outer membrane,the inner membrane,the intermembranespace,the cristae and the matrix (Fig.ll.5). The energy-richcarbohydrates

intermediatesare transferredto coenzymes NAD* and FAD to produce, respectively, NADH

Fig, 11.4: Overuiewof electrcntransportchain (A-Substrate; F,-Flavoprotein; Cyts-Cytochromes).

226

B IOC H E MIS TR Y

ATPsynthase lnner membrane ETCassembly Fig. 11.5: Structureof mitochondriondepictingelectrcntransportchain(ETC)(Fe F,-Protein subunits).

tnner mitochondrialmembrane: The electron transportchain and ATP synthesizingsystemare located on the inner mitochondrial membrane which is a specializedstructure,rich in proteins. It is impermeableto ions (H+, K+, Na+)and small m olecu l e s(AD P,A T P).T h i s me mb ranei s hi ghl y folded to form crisfae.The surface area of inner mitochondrial membrane is greatly increased due to cristae. The inner surface of the inner mitochondrial membrane possessesspecialized particles (that look like lollipops), rhe phosphorylating subunits which are the centres for ATP pr odu c ti o n .

there are certain mobile electron carriersin the respi ratory chai n. These i ncl ude N A D H, coenzyme Q, cytochrome C and oxygen.

Mitochondrial matrix : The interior ground substanceforms the matrix of mitochondria.lt is rich in the enzymes responsible for the citric acid cycle, p-oxidation of fatty acids and . oxidation of amino acids.

There are five distinct carriersthat participate in the electron transport chain (ETC). These carriersare sequentiallyarranged(Fig.|l.V and are responsiblefor the transferof electrons from a given substrateto ultimately combine with proton and oxygen to form water.

The enzyme complexes(l-lV) and the mobile carriersare collectivelyinvolved in the transport of electrons which, ultimately, combine with oxygen to produce water. The largestproportion of the oxygen suppliedto the body is utilized by the mitochondria for the operation of electron transportchain. and reactions Gomponents chain off tftae electron transpcrt

Stluctural organization of respiratory chain

l . N i coti nanni de

The inner mitochondrial membrane can be disruptedinto five distinct respiratoryor enzyme complexes, denoted as complex L Il, ilL IV and V (Fig.l1.6). The complexesl-lV are carriersof electronswhile complex V is responsiblefor ATP synthesis. Besides these enzyme complexes,

Of the two coenzymes NAD+ and NADP+ derived from the vitamin niacin, NAD+ is more activelv involved in the ETC. NAD+ is reduced to NADH + H+ by dehydrogenaseswith the removal of two hydrogen atoms from the substrate (AH2). The substrates include

nucteoti ci es

.-;hi+atey j'l

; BIOLOGICALOXIDATION

Complex ll t,Ailf t; .I Succinate CoQ reductase Fe:i

Complex lll CoQ-cytochromeC reductase -* CYtti + FeS-+ eil c

-0. ._ (-;(-)ajtlJVille .t'

Complexl NADH-CoQ reductase

227

Cyi a - ** Cyt alr

f'el I -. t-tuiNH. NAL IH r t

ADP + Pi

I

ATp

Sirtrltrate Fig. 11"6: Multiprotein complexes in electron transporl chain.

glyceraldehyde-3 phosphate,pyruvate,isocitrate, Succi nate dehydrogenase(succ i nate-coenzyme cr-ketoglutarateand malate. Q reductase)is an enzyme found in the inner mitochondrialmembrane.lt is also a flavoprotein A H 2 + N A D + i -rA+ N AD H + H + with FAD as the coenzyme.Thiscan accept two NADPH + H+ producedby NADP+-dependent hydrogenatoms (2H+ + 2el from succinate. dehydrogenase is not usuallya substratefor ETC. Succinate+ FAD ----+ Fumarate+ FADHT NADPH is more effectivelyutilized for anabolic reactions (e.9. fatty acid synthesis,cholesterol HH8,Iroffi"sulfur preit*En* synthes is). The iron-sulfur (FeS) proteins exist in the oxidizbd (Fe3+)or reduced (Fe2+)state. About [ ! . F & s w * p r* te $ m s half a dozen FeS proteins connected with The enzyme NADH dehydrogenase(NADH- respiratorychain have been identified.However, coenzyme Q reductase)is a flavoprotein with the mechanismof action of iron-sulfurproteins FMN as the prosthetic group. The coenzyme in the ETC is not clearly understood. FMN acceptstwo electronsand a proton to form One FeS participates in the transfer of FMNH2. NADH dehydrogenase is a complex electronsfrom FMN to coenzymeQ. Other FeS enzyme closely associatedwith non-heme iron proteins associated with cytochrome b and proteins(NHl) or iron-sulfurproteins (FeS). cytochrome c1 participate in the transport of NADH + H+ + FMN -----+ NAD+ + FMNH, electrons. Amytal Rotenone Piericidin A

k

J

.t Subslrate --), NALIr

Antimycin A BAL

J

+ f nltrt -Q+ t,;o() -- | Cyt tr -Q+ Cvt c 1 --+ Cytc -+

Cyanide Carbonmonoxide Sodiumazide

i

+

Cyta --) Cyt,r, -90 O" ",[' l*ll nt

;JffiFJ "ffi#3) Fig. 11.7 : Elgctron transpoft chain with sites ot ATP synthesis and inhibitors (BAL-British antitewisite).

228

B IOC H E MIS TFIY

l1l, Goenzgrme Q

F"'* presentin reductionof heme iron Fe2+i cytochromesallows them td function as effective carriersof electronsin ETC.

Coenzyme Q is also known as ubiquinone s inc e i t i s u b i q u i to u s i n l i v i n g s y stem.l t i s a Cytochrome c (mol. wt. 13,000) is a small quinone derivative with a variable isoprenoid protein containing104 amino acids and a heme possess side chain. The mammalian tissues a quinon e w i th 1 0 i s o p re n o i d u n i ts w hi ch i s group. lt is a central member of ETC with an intermediateredox potential.lt is rather loosely known as coenzyme Q1s (CoQro). bound to i nner mi tochondri almembrane and o can be easily extracted.

cHs

Cytochrome a and a3 : The term cytochrome oxidase is frequently used to collectively representcytochrome a and a3 which is the terminal componentof ETC.Cytochromeoxidase o is the only electron carrier, the heme iron of Coe n z y m eQ i s a l i p o p h i l i ce l e c troncarri er.l t which can directly reactwith molecularoxygen. can accept electronsfrom FMNH2 produced in Besides heme (with iron), this oxidase also the ETC by NADH dehydrogenaseor FADH2 contains copper that undergoes oxidationproduced outside ETC (e.g. succinate reduction (Cu2+ Cu+) during the transport ir dehydrogenase, acyl CoA dehydrogenase). of electrons. Coenzyme Q is not found in mycobacteria. ln the final stage of ETC, the transported Vitamin K performssimilarfunction as coenzyme electrons,the free protons and the molecular Q in these organisms. Coenzyme Q has no oxygen combine to produce water. k nown v i ta m i np re c u rs o irn a n i m a l s .l t i s di rectl y synthesized in the body. (Refer cholesterol biosynthesis,Chapter | 4) CH. t" (C H 2 -C H :C -C H2)n-H Ubiquinone(oxidized torm)

V" Sytochromes The cytochromes are conjugated proteins containing heme group. The latter consistsof a porphyrin ring with iron atom. The heme group of cytochromes differ from that found in the structureof hemoglobinand myoglobin.The iron of heme in cytochromesis alternatelyoxidized (Fe3+)and reduced (Fe2+),which is essentialfor the transportof electronsin the ETC.This is in contrast to the heme iron of hemoglobin and myoglobin which remains in the ferrous (Fe2+) state.

The transportof electronsthrough the ETC is linked with the releaseof free energy.The process of synthesizing ATP from ADP and Pi coupled with the electron transport chain is known as oxidative phosphorylation.The complex V (See Fig.Il.6l of the inner mitochondrial membraneis the siteof oxidativephosphorylation. F : O Ratio

The P : O ratio refers to the number of inorganic phosphatemolecules utilized for ATP generationfor every atom of oxygen consumed. Three cytochromeswere initially discovered More appropriately, P : O ratio representsthe from the mammalian mitochondria.Thev were numberof moleculesof ATP synthesizedper pair designatedas cytochromea, b and c depending of electronscarried through ETC. on the type of heme present and the respective The mitochondrialoxidationof NADH with a absorption spectrum. Additional cytochromes P : O ratio of 3 can be represented by the such as c1, b1, b2, a3 etc. were discoveredlater. following equation : The electrons are transported from coenzyme NADH + H+ + * 3A D P + 3P i ------+ Q to cytochromes (in the order) b, c12c2 a ?nd * O, N A D + + 3A TP + 4H 2O a3. The property of reversible oxidation-

Ghapten 11 : BIOLOGICAL OXIDATION P : O ratio of 2 is assignedto the oxidation of MEGHANISM OF OXIDATIVE FADH2. (Nofe : Although yet to be proved PHOSPHORYLATION beyond doubt, some workers suggesta P : O Several hypotheses have been put forth to r at ioof 2. 5 fo r N A D H + H + , a n d 1 .5 fo r FA D H 2, explain the processof oxidativephosphorylation. basedon the proton translocation). The most important among them-namely, chemi cal coupl i ng, and chemi osmoti c-are Sites of oxidative discussedbelow. phosphorylatlon in ETG There are three reactionsin the ETCthat are exergonic to result in the synthesisof 3 ATP molecules(SeeFig.l1.V.fhe three sitesof ATP formation in ETC are 1. Oxidation of FMNH2 by coenzyme Q. 2. Oxidation of cytochromeb by cytochronl€c1. 3. Cytochromeoxidase reaction.

\

Eachone of the above reactionsrepresentsa coupling site tor ATP production. There are only two coupling sites for the oxidation of FADH2 (P : O ratio 2), since the first site is bypassed. Energetics of oxidative phosphorylation

t

Cherniaal

co*rplEng hypothesis

This hypothesis was put forth by Edward S l ater (1953). A ccordi ng to chemi cal coupl i ng hypothesis,during the courseof electrontransfer in respiratorychain, a series of phosphorylated high-energy intermediates are first produced which are utilizedfor the synthesisof ATP.These reactionsare believed to be analogousto the substratelevel phosphorylationthat occurs in glycolysis or citric acid cycle. However, this hypothesislacks experimental evidence, since all attempts,so far, to isolate any one of the high-energy intermediates have not been successfu l.

Ghemiosnnetle hypothesis The transport of electrons from redox pair Thi s mechani sm,ori gi nal l yproposedby P eter NAD+/NADH (Eo = - 0.32) to finally the redox Mi tchel l .(1961), i s now w i del y accepted. l t (Eo= + 0.82) may be simplified pair |OrlflzO explains how the transportof electronsthrough and representedin the following equation the respiratorychain is effectively utilized to produce ATP from ADP + Pi. The concept of ----+ H2o + NAD+ to, * NADu + H* chemiosmotic hypothesis is comparable with The redox potentialdifferencebetweenthese energy stored in a battery separatedby positive t wo r edoxp a i rsi s 1 .1 4 V , w h i c h i s e q u i v al entto and negativecharges. an energy 52 Cal/mol. Proton gradient : The inner mitochondrial Three ATP are svnthesizedin the ETC when membrane,as such, is impermeableto protons NA DH is o x i d i z e d w h i c h e q u a l s to 2 1 .9 C al (H+) and hydroxyl ions (OH-). The transportof (each ATP = 7.3 Cal). electrons through ETC is coupled with the The efficiency of energy conservation is translocation of protons (H+) across the inner mi tochondri almembrane(coupl i ngmembrane) calculatedas from the matrix to the intermembranespace.The 2 1 .9 x 1 0 0 = 4 2 o to . pumping of protons resultsin an electrochemical 52 Therefore,when NADH is oxidized, about or proton gradient. This is due to the 42'/. of energy is trapped in the form of 3 ATP accumul ati onof more H + i ons (l ow pH ) on the and the remaining is lost as heat. The heat outer side of the inner mitochondrialmembrane liberation is not a wasteful process/ since it than the inner side (Fig.l l.A. The proton allows ETC to go pn continuously to Benerate gradient developed due to the electron flow in ATP. Further, this heat is necessaryto maintain the respiratorychain is sufficientto result in the hody temperature. synthesisof ATP from ADP and Pi.

230

B IOC H E MIS TFI Y

mitochondrial membrane Intermembrane space lnner mitochondrial membrane

Fig. 11.8: Outlineof chemiosmotichypothesisfor oxidativephosphorylation.

Enzyme system for ATP synthesis : ATP The protons that accumulate on the synthase,presentin the complex V, utilizes the intermembrane spacere-enterthe mitochondrial proton gradient for the synthesisof ATP. This matrixleadingto the synthesis of ATP. enzyme is also known as ATPasesince it can hydrolyse ATP to ADP and Pi. ATP synthaseis a Rotary ;ft@tor rnodel for ATP complex enzyme and consistsof two functional generation subunits, namely Ft and F0 ffig.l1.9). lts Paul Boyerin 1964 proposed(Nobel Prize, structureis comparablewith 'lollipops'. 1997) that a conformationalchange in the

NAD+ lflr

(alkaline)

gATp

pH gradient

membrane Outermitochondrial Fig. 11.9: Diagrammaticrepresentationof chemiosmotichypothesisfor oxidativephosphorylation (1,lll, lV and V-Respiratotychainamplexes; Fn F,-Proteinsubunitsfor phosphorylation).

231

1,ii: BIOLOGICALOXIDATION

ADP + Pi

lnner mitochondrial membrane

Fig. 11.10 : Structureof mitochondrialATP synthase (FoF,)complex(C units-channelprotein subunits; u, F, and Tare the subunltsof F,-ATPstnthase).

substratesA D P and P i bi nd to B subuni t i n L-conformation. The L site changes to T conformation,and this leads to the synthesisof ATP. The O site changes to L conformation w hi ch bi nds to A D P and P i . The T si te changes to O conformation,and releasesATP. This cycle of conformation changes of B subunits is repeated.And three ATP are generatedfor each revolution (Fig.l l.l l). It may be noted that the ATP releasein O conformationis energy dependent(and not ATP synthesis)and very crucial in rotary motor model for ATP generation. The enzyme ATP synthaseacts as a protondriving motor, and is an example of rotary catalysis. Thus, ATP synthase is the world's smallest molecular motor. ?niicerit*d dEs*pred*yso# u "rii s.iait$roe g*i't**r,pherryiat6tlm

It is estimatedthat about 100 polypeptides are required for oxidative phosphorylation.Of mitochondrial membrane proteins leads to the these, 13 are coded by mi tochondri al D N A synthesisof ATP. The original Boyer hypothesis, (mtD N A ) and synthesi zedi n the mi tochondri a, now considered as rotary motor/engine driving while the restare producedin the cytosol(coded model or binding change model, is widely by nuclear DNA) and transported. mtDNA is acceptedfor the generationATP. The enzymeATP synthaseis FsFl complex (of complex V). The F6 subcomplexis composedof c hannel pr ot ein ' C ' s u b u n i tsto w h i c h F l -ATP synthaseis attached(Fig.l | .10). Fr-ATP synthase consistsof a central y subunit surrounded by alternatingcr and p subunits(o3 and p3). In responseto the proton flux, the 1 subunit physically rotates.This induces conformational changesin t he B 3 s u b u n i tsth a t fi n a l l y l e a dto the releaseof ATP. A c c or dingt o th e b i n d i n gc h a n g em e c h a n i s m, the three B subunits of F1-ATPsynthaseadopt different conformations. One subunit has open (O) conformation, the second has loose (L) conformation while the third one has tight (T) conformation (Fig.l l.l l)

t

B y an unk no w n me c h a n i s m ,p ro to n si n d u ce th e r ot at ionof y s u b u n i t,w h i c h i n tu rn i n d u ces c onf or m at ion c h a n g e s i n B s u b u n i ts . T he

Fig. 11.11: The binding change model (rotary motor/ engine driving model) for ATP synthesisby F.-ATP synthase.

232

B IOC H E MIS TFI Y

maternally inherited since mitochondria from the sperm do not enter the fertilized ovum.

beforethe i nhi bi tor bl ockadesteo and oxi di z eo componentsafter that step.

M it o c h o n d ri a lD N A i s a b o u t 1 0 ti mes more s us c e p ti b l eto m u ta ti o n s th a n n u cl ear D N A . m t DN A mu ta ti o n s a re m o re c o m monl y seen in tissues with h igh rate of oxidative phos p h o ry l a ti o n(e .g . c e n tra l n e rv ous system, s k ele ta la n d h e a rt mu s c l e .l i v e r).

The synthesi sof A TP (phosphoryl ati on)is dependenton el ectrontransport.H ence, al l t he si te-speci fi ci nhi bi torsof E TC al so i nhi bi t ATP formation. Three possiblesitesof action for the inhibitorsof ETC are identified

1. N A D H and coenzyme Q : Fi sh pol s on Leher's hereditary optic neuropathy is an rotenone, barbituatedrug amytal and antibiotic ex am p l efo r mu ta ti o n si n m tD N A. Thi s di soroer piercidin A inhibit this site. is c ha ra c te ri z e b d y l o s so f b i l a te ra lvi si ondue to 2. Between cytochrome b and c1 t Antimycin neuroretinaldegeneration. A -an antibiotic, British antilewisite (BAL)-an I nhibi to rs o f e l e c tro ri antidote used against war-gas-are the two t r ans p o rt e h a i n important inhibitors of the site between cytochromeb and c1. Many site-specific inhibitors of ETC have contributed to the present knowledge of 3. Inhibitorsof cytochromeoxidase: Carbon m it oc h o n d ri a lre s p i ra ti o nSe . l e c te dexampl esof monoxi de, cyani de, hydrogen sul phi de and these inhibitors haven been given in Fig.l 1.7. azi de effecti vel y i nhi bi t cytochrome oxi da se. The inhibitorsbind to one of the cornponentsof Carbon monoxide reacts with reduced form of ETC and block the transportof electrons.This the cytochrome while cyanide and azide react c aus e sth e a c c u mu l a ti o no f re d u c e dcomoonents w i th oxi di zed form.

BIOMEDIEALI CLINIGAL CONCEPTS

ut The most important lunction of food is to supply energy to the liuing cells. This is tinally achieuedthrough biological oxidotion. rx The supply of 02 is uery essentialfor the suruiualof life (exception--anqerobic bacteria). 0g ATR the energy currency of the cell, acts as a |ink between the cotabolism and anabolism in the liuing system. The major production of body's ATP occurs in the mitochondria through oxidatiue phosphorglationcoupled with respiration. us Respiratorychain or electron transport chain (ETC) is blocked by site specilic inhibitors such os rotenone, amytal, antimycin A, BAL, carbon monoxide and cyonide. w Uncoupling of respiration from oxidstiue phosphorylation under naturol conditions ossumes biologicol significance. The brown odipose tissue, rich in electron corriers, brings about oxidation uncoupledfrom phosphorylation. The presenceof actiue brown adipose tissue in some indiuiduals is belieued to protect them from becoming obese. This is becausethe excesscaloriesconsumed by thesepeople are burnt and liberated as heat instead ot' being stored as fat. us Inherited disorders of oxidatiue phosphorylation caused bv the mutations in mitochondrial DNA houe been identified e.g. Leber's hereditary optic neuropathy.

Ghapter 11 : BIOLOGICAL OXIDATION

233

Cyanide poisoning : Cyanide is probably the also believed to act as an uncoupler. This is, most potent inhibitor of ETC.lt binds to Fe3+of however, yet to be proved beyond doubt. c y t oc h ro me o x i d a s e b l o c k i n g m i tochondri al of uncoupling r es p i ra ti o n l e a d i n g to c e l l d e ath. C yani de Significance pois o n i n gc a u s e sd e a th d u e to ti s sue asphyxi a U ncoupl i ng of respi rati on from oxi dat ive (mostlv of central nervoussvstem). phosphoryl ati on under natural condi t ions assumes bi ol ogi cal si gni fi cance. The I NHI BIT OR S OF O X ID A T IVE maintenanceof body temperatureis particularly PHOSPHORYLATION i mportant i n hai rl ess ani mal s, hi berna t ing animalsand the animals adaptedto cold. These Uncouplers ani mal s possessa speci al i zed ti ssue called The mitochondiral transport of electrons is brown adiposetissuein the upper back and neck t ight l y c o u p l e d w i th o x i d a ti v e p hosporyl ati on portions. The mitochondria of brown adipose (ATP synthesis).In other words, oxidation and tissue are rich in electron carriers and are phosphorylationproceed simultaneously.There specialized to carry out an oxidation uncoupled ar e c e rta i n c o mp o u n d sth a t c a n uncoupl e (or from phosphorylation. This causes liberation of delink) the electron transport from oxidative heat when fat is oxidized in the brown adipose phos p h o ry l a ti o n Su . c h c o mp o u n ds, know n as tissue.Brown adiposetissuemay be considered unc o u p l e rs ,i n c re a s eth e p e rme a bi l i tyof i nner as a site of non-shivering thermogenesis. The mitochondrial membrane to protons (H+). The presence of active brown adioose tissue in result is that ATP synthesisdoes not occur. The certain individuals is believed to protect them energy linked with the transportof electronsis from becoming obese. The excess calories dissipated as heat. The uncouplers allow (often consumed by these people are burnt and at accelerated rate) oxidation of substrates (via Iibirated as heat, instead of being stored as fat. NADH or FADH2) without ATP formation. Thermogenin(or uncoupling protein, UCP) is The uncoupler, 2,4-dinitrophenol (DNP), has a natural uncoupl er l ocated i n the i n ner been e x te n s i v e l ys tu d i e d .l t i s a s mal l l i pophi l i c mi tbchondri al membrane of brow n adi pose m ole c u l e .D N P i s a p ro to n -c a rri earnd can easi l y ti ssue. l t acts l i ke an uncoupl er, bl ocks t he dif f u s e th ro u g h th e i n n e r mi tochondri al formation of ATP, and liberatesheat. membrane.In the people seekingto lose weight, lonophores: The term 'ionophores'is usedto DNP was used as a drug. However, this is now collectively representthe lipophilic substances discontinued,as it produces hyperthermiaand that promote the transport of ions across other side effects. In fact, Food and Drug bi ol ogi calmembranes. A dmi n i s tra ti o n(U S A) h a s b a n n e d the use of All the uncouplers(describedabove) are, in DNP . fact, proton ionophores. T h e o th e r u n c o u p l e rsi n c l u d e di ni trocresol , penta c h l o ro p h e n o l , tri fl u o ro c a rbonyl cyani de The antibiotics valinomycin and nigercin act phen y l h y d ra z o n e(F C C P ).T h e l a st compound as ionophoresfor K+ ions. Both thesecompounds (FCCP)is said to be 100 times more effectiveas are al so capabl e of di ssi pati ngproton grad ient an u n c o u p l e r th a n d i n i tro p h enol . W hen acrossthe i nner mi tochondri almembraneand administeredin high doseg the drug aspirin acts i nhi bi t oxi dati vephosphoryl ati on. as an u n c o u o l e r. Other inhibitors of Physiological uncouplers : Certain physiophosphorylation oxidative logical substanceswhich act as uncouplers at higherconcentrationhave been identified.These Oligomycin : This antibiotic prevents the include thermogenin, thyroxine and long chain mi tochondri al oxi dati on as w el l as free fatty acids. The unconjugatedbilirubin is phosphorylation. lt binds with the enzyme ATP

234

B IOC H E MIS TFIY

N AD H+ H *

cH2oH C=O cH2o-

9r,oli

HO-C-H

cH2o-0 Glycerol$phosphate

Dihydroxyacetone phosphate

CH2OH -I i=J

iH,o-@

glycerol Mitochondrial $phosptatedehydrogenase

{

-n O -J Dihydroryacetone FADH2

\

C

H

i- O FAD

Glycerotg.phosphate

Fig. 11.12: Glycerol-phosphateshuftle(reducing equivalents transportedarc shown in Blue).

synthaseand blocks the proton (H+) channels.lt thus prevents the translocation (re-entry) of protons into the mitochondrial matrix. Due to this, protons get accumulated at higher concentration in the intermembrane space. Electrontransport(respiration)ultimately stops, since protons cannot be pumped out against steep proton gradients.

phosphate shuttle and malate-aspartate shuttleare operativeto do this job. They transportthe reducing equivalents from cytosol to mitochondriaand not vice versa. iJ" &{p*cer*{-phosphate

sh$itf s.r

Cytosolicglycerol3-phosphatedehydrogenase oxi di zes N A D H to N A D + . The reduci ng Atractyloside : This is a plant toxin and equivalents are transported through glycerol inhibitsoxidativephosphorylationby an indirect 3-phosphate into the mitochondria. Clycerol mechanism.Adenine nucleotide carrier system 3-phosphate dehydrogenase-present on outer facilitates the transport of ATP and ADP. surface of inner mitochondrial membraneAtractylosideinhibits adenine nucleotidecarrier reduces FAD to FADH2. Dihydroxyacetone and, thus, blocks the adequate supply of ADP, phosphate escapes into the cytosol and thereby preventingphosphorylation. the shuttlingcontinuesas depicted in Fig.l1.12. FADH2 gets oxidized via ETC to generate TRANSPORT OF REDI.|CING 2 ATP.

EOUIVALENTS-SHUTTLE PATHWAYS

$1. &taEate-aspartate

T he inn e r m i to c h o n d ri a l m e m b ra ne i s impermeableto NADH. Therefore,the NADH producedin the cytosolcannot directly enterthe mitochondria. Two pathways-namely glycerol-

ln the cvtosol. oxaloacetate accepts the reducing equivalents (NADH) and beconres malate. Malate then enters mitochondriawhere it is oxidized bv mitochondrialmalate dehydro-

shutt[e

r-:i-,;:f,i:ar't 1 : BIOLOGICALOXIDATION

235

Oxaloacetate

Glutamate

Aminotransferase

Malate

Aspartate

s-Ketoglutarate

CYTOSOL

Fig. 11.13: Malate-aspartate shuttte-

genase.In this reaction,NADH and oxaloacetate ENZVMES INVOLVED IN are regenerated. NADH gets oxidized via BIOLOGICAL OXIDATION electron transport chain and 3 ATP are A l l the enzymes parti ci pati ngi n bi ol ogical produced. This is in contrast to glyceroloxidation belong to the class oxidoreductases. phosphate shuttle where only 2 ATP are These are further grouped into four categories oroduced. 1. Oxi dases In the mitochondria,oxaloacetateparticipates 2. Dehydrogenases in transamination reaction with glutamate to produce aspartate and a-ketoglutarate. The 3. Hydroperoxidases aspartateenters the cytosol and transaminates 4. Oxygenases. with d-ketoglutarateto give oxaloacetateand '1 glutamate.The malate-aspartate shuttleis shown . Oxidases : These enzymes catalyse the in Fi g .| l .l 3 . elimination of hydrogen from the substrates which is accepted by oxygen to form mostly '. ,iitl*r p.Fttfuways and t;ss,ues water, e.g. cytochrome oxidase, tyrosinase, monoamine oxidase (H2O2 formed instead of Liver and heart utilize malate-aspartate

Hzo)'

s hut tl e ,a n d y i e l d 3 A T P p e r m o l e of N A D H . tvlost of the other tissues, however, employ Cytochrome oxidase, the terminal giycerol-phosphateshuttle and liberate 2 ATP componentof electron transportchain, transfers f r om N A D H . electrons (obtained from the oxidation of

236 substrate molecules by dehydrogenases)to the final acceptor,oxygen.

B IOC H E MIS TFIY 4. Oxygenases : This group of enzymes catalysesthe direct incorporationof oxygen into the substratemolecules.

Some flavoproteins containing FAD or FMN also belong to the category of oxidases. . Dioxygenases (true oxygenases) : They are responsiblefor the incorporationof both the €. 8. , L- am i n o a c i d o x i d a s e (F MN ), xanthi ne atoms of oxygen (Or) into the substrate,e.g. ox idas e( FAD ). homogentisateoxidase, L-tryptophan pyrro2. Dehydrogenases: As the name indicates, lase. these enzymes cannot utilize oxygen as oxihydrogenacceptor.They catalysethe reversible . Monooxygenases (mixed function dases) : They catalyse the incorporation of one transfer of hydrogen from one substrate to (+Or) atom of oxygen while the other oxygen another and, thus, bring about oxidationatom is reduced-to HrO. NADPH usually reductionreactions.There are a large number of provi des the reduci ng equi val ents, e.g. enzymes belongingto this group cytochrome Poro monooxySenase system of . NAD+ dependent dehydrogenases, e.g. microsomesis responsiblefor the metabolism alcohol dehydrogenase,glycerol 3-phosphate of many drugs (ami no pyri ne, morphi ne, dehydrogenase. aniline etc.) and biosynthesis of steroid hormones(from cholesterol).The action of Cyt . NADP* dependentdehydrogenases, e.g. HMC Pouois depicted here. CoA reductase,enoyl reductase. . FMN dependentdehydrogenases, e.g. NADH dehydrogenase. . FAD dependent dehydrogenases,e.g. succinate dehydrogenase, acyl CoA dehydroSenase. e The cytochromes : All the cytochromes of electron transportchain (b, c, and c) except the terminalcytochromeoxidase(a+ar)belong t o t his g ro u p .

RH NADPH+ H+

NADP+

Electron transport in prokaryotes

ln contrastto eukaryotes,the prokaryoteslack mitochondria. However, prokaryotespossessa separatesystemfor biologicaloxidation.A set of 3. Hydroperoxidases: Hydrogen peroxide is electron carriers (different from that found in the substrate for these enzymes. There is a mitochondria) and enzymes of oxidative constant production of H2O2 in the reactions phosphoryl ati onare bound to the i nner cel l catalysed by the aerobic dehydrogenases. The membrane in prokaryotes.This arrangementof harmful effects of H2O2 are prevented by oxidative machinery is one of the reasonsto hydroperoxidases, e.g. peroxidaseand catalase. bel i eve that mi tochondri aof hi gher organi sms 2H2O2 ------+2H2O + 02 have descendedfrom prokaryoticcells.

237

ffiii::rpne* $'l : BIOLOGICALOXIDATION

I

Bioenergeticsdeals with the study ol energg changesin biochemicalreactions.Change in free energg6G) is ualuablein predicting the feasibility of o reaction.A negatiueond a positiue AG, respectiuely,represent on exergonic (energy-releasing) and endergonic (energy-consuming)reactions.

2. High-energgcompounds (AG > -7.0 Cal/mol) plag a crucial role in the energg transt'er of biochemical reactions (e.5. ATP, phosphocreatine,phosphoenolpyruuate). ?

ATP is the energy currency of the cell. ATP-ADPcycle octs as a connecting energy link between catabolic and anabolic reactions.

4, Respiratorgchain or electron transport chain (ETC) located in the inner mitochondrial membrane representsthe finol stage of oxidizing the reducing equiualents(NADH and FADHy) deriued from the metabolic intermediqtes to water. tr

ETC is organized into fiue distinct complexes. The complexes I to IV are electron carriers while complex V is responsiblefor ATP production. The components ol ETC are arrangedin the sequence Cyt b ------+Cyt c1 ---s Cyt c ------+Cyt a + ag --+ NAD+ ----+ FMIV ---+ CoQ -------+

02

6. The processof sgnthesizingATP t'rom ADP and Pi coupled with ETC is known os oxiddtiuephosphorylation.NADH oxidotion with q P : O ratio 3 indicatesthat 3 ATP are synthesizedwhile FADH2 oxidation (P : O ratio 2) results in the production ot' 2 ATP.

7. Among the hypotheses put t'orth to explain the mechanism oJ oxidatiue phosphorylalion, the chemiosmotic hgpothesis (ot' Mitchell) is widely accepted. The rotary motor model (of Boger) inuoluing the conlormation changesin the ftsubunits ol ATP synthaseexplains the ATP generation. R NADH produced in the cytosol connot directly enter mitochondris. Glycerol-phosphate

shuttle (generates2 ATP) and malate'asparateshuttle (generates3 ATP) operate to ouercome the diJt'iculty.

9. There are many inhibitors of electron transport choin (rotenone, amytal, antimycin, Cq-ClV, HzS etc.) and oxtdatiue phosphorylation (oligomycin, atractyloside). Uncouplers (e.9. dinitrophenol) are the substancesthat delink ETC Jrom oxidatiue phosphorylation.

10. The enzymesparticipating in biological oxidation belong to the clossoxidoreductoses. There ore fiue groups, namely oxidases,aerobic dehydrogenases,anaerobic dehydrogenases,hydroperoxidasesand oxggenases

B IOC H E MIS TR Y

I. Essayquestions .l . Write an accountof the high-energy compoundsin metabolism 2. Describethe componentsof electrontransportchain and discussthe oxidationof NADH. 3. Defineoxidativephosphorylation. Discusschemiosmotichypothesisin detail. 4. Give an accountof the enzymesinvolvedin biologicaloxidation.

F "i

5. Discussabout the inhibitorsof ETCand oxidativephosphorylation. II. Short notes (a) High-energybonds, (b) Uncouplers,(c) P : O ratio, (d) Redox loops, (e) ATP synthase, (0 Cytochromes, (d Sitesof oxidativephosphorylation, (h) CoenzymeQ, (l) Redoxpotential,(j) ATP as energycurrency. I I I . F ill in th e b l a n k s 1. The relation betweenthe change of free energy (AG), enthalpy (AH) and entropy (AS) is expressedby the equation 2. A negativesign of free energy indicatesthat the reaction is 3. The bonds responsible for a majorityof high-energy compoundsare 4. The storageform of high-energy compoundin invertebrates is 5. A more negativeredox potential representsa greatertendencyto lose 6. The electrontransportchain is locatedin 7. The prostheticgroup presentin cytochromes 8. The component of electron transport chain which can directly react with molecular oxySen 9. The site of ETCinhibitedbv cvanide 10. Superoxideis convertedto H2O2 by the enzyme IV. Multiple choice questions 11. Name the compoundwith the greateststandardfree energy. (a) ATP (b) Phosphocreatine (c) Cyclic AMP (d) Phosphoenolpyruvate. 12. One of the following componentsof ETCpossesses isoprenoidunits (a) CoenzymeQ (b) Cytochrome(c) Cytochromeb (d) Non-hemeiron. 13. T he P: O ra ti ofo r th e o x i d a ti o no f FA D H , i s (a) 1 (b) 2 (c) 3 @) a. 14. lnner mitochondrialmembraneis impermeableto (a) H* (b) K+ (c) OH- (d) All of them. 15. ATP synthaseactivity is associated with the mitochondrialenzymecomplex (a) V (b) lll (c) lV (d) l.

ductionto MetnboXisnn

l_l undreds of reactions simultaneouslytake | | pla c e i n a l i v i n g c e l l , i n a w e l l -organi zed and integratedmanner. The entire spectrum of chemical reactions, occurring in the Iiving system, are collectively referred to as metaholism. A metaholic pathway (or metabolic map) constitutesa series of enzymatic reactions to produce specific products.The term metabolite is applied to a substrateor an intermediateor a product in the metabolic reactions. M et ab o l i s m i s b ro a d l y d i v i d e d into tw o categories(Fig.l2.1).

Energyrich complexmolecules

products Fig. 12.1 : An outline of catabolismand anabolism.

1. Catabolism : The degradative processes several intermediates common to both rne concerned with the breakdown of complex processes. The term amphiholism is also in use m olec ule sto s i mp l e ro n e s , w i th a c o ncomi tant for reactions which are both catabolic and releaseof energy. anabol i c i n nature. 2. Anabolism : The biosynthetic reactions involvigb the formation of complex molecules Gatabolism r r om s r m p rep re c u rs o rs . The very purposeof catabolismis to trap the A clear demarcationbetweencatabolismand energy of the biomoleculesin the form of ATP anabolis m i s ra th e r d i ffi c u l t. s i n c e there are and to generate the substances (precursors)

241

BIOCHEMISTF|Y

242 Polysaccharides

I

II

Stage 1

+ Monosaccharides-

Fattyacids and glycerol

| | Stage2

Proteins

Lipids

J

A tino acids

,-' ---'

(

\lr-'

coz Stage 3

Fig. 12.2: Thethree stagesof catabolism(ETC-Electrontransportchain).

requiredfor the synthesisof complex molecules. few. Theseinclude pyruvate,acetyl CoA and the intermediatesof citric acid cvcle. Besidesthe Catabolism occurs in three stages(Fig.l2.2). availabilityof precursors,the anabolic reactions 1. Conversion of complex molecules into are dependent on the supply of energy (as ATP their building blocks : Polysaccharidesare or C TP ) and reduci ngequi val ents(as N A D P H + lipids to free broken down to monosaccharides, H * ). fatty acids and glycerol,proteinsto amino acids. The anabolic and catabolic pathwaysare not 2. Formation of simple intermediates : The reversibleand operate independently.As such, building b l o c k s p ro d u c e d i n s ta g e (1) are the metabolicpathwaysoccur in specificcellular degraded to simple intermediates such as locations (mitochondria, microsomesetc.) and pyruvate and acetyl CoA. These intermediates are controlled by different regulatorysignals. are not readily identifiable as carbohydrates, The terms-intermediary metabolism and lipids or proteins.A small quantity of energy (as also in use. ATP) is captured in stage2. energy metabolism-are Intermediary metabolism refers to the entire 3. Final oxidation of acetyl CoA : Acetyl CoA range of catabolic and anabolic reactions,not is c om plete l yo x i d i z e dto C O2 , l i b e ra ti n gN A D H involving nucleic acids. Energy metabolism and FADH2 that finally get oxidized to release deals with the metabolic pathways concerned large quantity of energy (as ATP). Krebscycle (ar with the storageand liberationof energy. citric acid cycle) is the common metabolic pat hway in v o l v e d i n th e fi n a l o x i d a ti on of al l energy-richmolecules.This pathwayacceptsthe The biochemicalreactionsare mainlv of four carbon compounds (pyruvate, succinate etc.) types derived from carbohydrates,lipids or proteins. ja.,:r

1 . Oxidation-reduction. ,

-rr,rr

For the synthesisof a largevarietyof complex molecules,the starting materials are relatively

2. Croup transfer. and isomerization. 3. Rearrangement

INTRODUCTION TO METABOLISM 4. Make and break of carbon-carbonbonds. These reactions are catalysed by specific enzymes-more than 2,000 known so far.

';

.:,1.;;' i5

;r ,1,' ,r ,,'j,,",,1.r .,i.

,:i 3:i V

' ,i:tr ,,.i*i- ."i.r r :!

The metabolic reactions do not occur in isolation.They are interdependent and integrated into specific series that constitute metabolic pathways. lt is, therefore, not an easy task to study metabolisms. Fortunately, the basic metaholic pathways in most organisms are essentially identical. For this reason, many organisms can be used to understand metabolisms.

243 employed to measurethe responseof man (or other animals) towards carbohydrate metabolism is a good exampleof the use of whole organism. (b) lsolated organs, tissue slices, whole cells, subcellular organelles,cell-free systems and purified recently components are frequently used to elucidate biochemical reactions and metabolic pathways.

2. Utility of metabolic probes : Two types of metabolic probes are commonly used to trace out biochemicalpathways.Theseare metabolic inhibitors and mutations. In both the cases,there is a specific blockade in a metabolic reaction which helps to understand the pathway. Several methods are employed to elucidate bioc he mi c a l re a c ti o n s a n d th e metabol i c Inhibitorsof electron transportchain have been pathways.These experimentalapproachesmay largely responsibleto elucidatethe sequenceof electron carriers (Chapter 11). The inborn errors be broadly divided into 3 categories of metabolism in higher organisms and the 1. Use of whole organismsor its components. geneti c mani pul ati onsi n the mi croorgani sm s 2. Utility of metabolic probes. have also contributeda lot to the understanding of metabolisrns. 3. Application of isotopes. 3. Application of isotopes : lsotopes are the atoms' w i th the same number of protons but different neutrons. By use of isotopes, the molecules of the living systemcan be labelled 1. Use of whole organismor its components: without altering their chemical properties. (a) Whole organisms: The ultimateaim of Application of isotopes in biochemistry has a biochemist is to know the revolutionizedthe study of metabolisms.More metabolism in the organism as a detailson the utility of isotopesin biochemisuy whole. Clucose tolerance test (CTT), are given elsewhere (Chapter 4l).

The actual methodsemployed may be either in vivo (in the living system)or in vifro (in the test tube) or, more frequently,both.

1

G

The wide range ol chemical reactions occurring in the liuing system are collectiuely known as metabolism. Catabolism is concerned with the degradation of complex molecules to simpler ones coupled with the liberation ol energy (ATil. An the other hand, anabolism deals with the synthetic reactions conuerting simple precursorsto complex molecules, coupled with the consumption of energy (ATp) A metabolic pathwoy constitutesa seriesof ertzymatic reactionsto produce specific products.

2. Seueralmethods are employed to study metabolism.Theseinclude the useol the whole organism or lts components (organ, tissue, cells, organelles etc.), utility ol metabolic probes (inhibitors and mutations) and application of isotopes.

I

ofCarbolrydrates Metabolism

HO- C- H I H- C- OH I HO*C- H I H- C- OH I h-u I cH2 - o H Glucose

t

{ I I

arbohydratesare the major sourceof energy Major pathways f metabol i sm \ . - f o r th e l i v i n g c e l l s . As s u c h , carbohydrates of carbohydrate by s ynthesi zed c o n s ti tu e n ts , ar e t h e fi rs t c e l l u l a r The important pathways of carbohydrate from carbon green plants during photosynthesis metabolismare listed dioxide and water, on absorptionof light. Thus, pathway) : 1. Glycolysis (Embden-Meyerhof light i s th e u l ti m a te s o u rc e o f e nergy for al l lactate. pyruvate and glucose to of oxidation p ro c e s s e s . The biolo g i c a l The monosaccharide glucose is the central 2. Citric acid cycle (Krebs cycle or molecule in carbohydrate metabolism since all tricarboxylicacid cycle) : The oxidationof acetyl the major pathwaysof carbohydratemetabolism CoA to CO2. Krebs cycle is the final common ar e c o n n e c te d w i th i t (F i g .l 3 ,1). C l ucose i s oxidative pathway for carbohydrates,fats or ut iliz e d a s a s o u rc eo f e n e rg y ,i t i s synthesi zed amino acids, through acetYlCoA. from non-carbohydrateprecursorsand stored as 3. Gluconeogenesis : The synthesis of glycogen to releaseglucose as and when the glucose from non-carbohydrateprecursors(e.9. need arises. The other monosaccharides aci ds,gl yceroletc.). ami no important in carbohydrate metabolism are fructose,galactoseand mannose. 4. Glycogenesis: The formation of glycogen The fasting blood glucose level in normal from glucose. individualsis 70-100 mg/dl (+.5-5.5mmol/l) and 5. Glycogenolysis : The breakdown of it is very efficientlymaintainedat this level (for glycogento glucose. details reler Chapter 35). Liver plays a key role 6. Hexose monophosphate shunt (pentose in m o n i to ri n g a n d s ta b i l i z i n g b l ood gl ucose fevels. Thus liver may be appropriately phosphatepathwayor direct oxidativepathway): This pathway is an alternativeto glycolysisand considered as glucostat monitor.

Chapter 13: METABOLISM OF CABBOHYDRATES

OXDATIVE PATHWAYS

Glycolysis

SYNTHETIC PATHWAYS

Othercarbohydrates (galactose, fructose) Glycogenesis

Hexosemonophosphateshunt

Uronicacid pathway

Lipogenesis synthesisof fat) Non-essential aminoacids

Flg. 13.1: Overuiewof glucosemetabolism. (Note : For majoity of the pathways, glucose

TCA cycle for the oxidationof glucose(directlyto carbon dioxide and water).

245

2. Insulin-dependenttransport system : This occurs in muscle and adiposetissue. Glucose transporters : In recent years,at least six glucosetransporters(CLUT-l to CLUT-5 and CLUT-7) in the cell membranes have been identified. They exhibit tissue specificity. For instance, CLUT-I is abundant in erythrocytes whereasGLUT-4 is abundant in skeletalmuscle and adiposetissue. Insulin increasesthe number and oromotes the activitv of CLUT-4 in skeletal muscle and adipose tissue. In type 2 diabetes mellitus, insulin resistanceis observed in these tissues. This is due to the reduction in the quantity of C LU T-4 i n i nsul i n defi ci ency.

Z. Uronic acid pathway : Clucose is convertedto glucuronic acid, pentosesand, in s om eanim a l s to , a s c o rb i ca c i d (n o t i n m a n).Thi s pathway is also an alternativeoxidativepathway for glucose.

Clycolysis is derived from the Creek words (glycose-sweetor sugar;lysis-dissolution).lt is a universal pathway in the living cells. The 8. Galactose metabolism : The pathways complete pathway of glycolysiswas elucidated concerned with the conversionof galactoseto in 1940. This pathway is often referred to as glucoseand the synthesisof lactose. Embden-Meyerhof pathway (E.M, pathway) in 9. Fructose metabolism : The oxidation of honour of the two biochemistswho made a fructose to pyruvate and the relation between major contribution to the knowledge of glycolysis. fructoseand glucose metabolism. 10. Amino sugar and mu.opotyr"ccharide metabolism: The synthesisof amino sugarsand other sugars for the formation of mucopolysaccharidesand glycoproteins. E nt r y of g l u c o s e

i n to c e l l s

Clucoseconcentrationis very low in the cells comparedto plasma (for humans < 100 mg/dl). However, glucose does not enter the cells by simple diffusion.Two specific transportsystems are recognizedfor the entry of glucose into the c ells .l . Insulin-independent transport system of glucose : This is a carrier mediated uptake of glucosewhich is not dependenton the hormone insulin.This is operativein hepatocytes,erythrocltes and brain.

Glycolysis is defined as the sequence of reactions converting glucose (or glycogen) to pyruvate or lactate, with the production of ATP. Salient

features

1. C l ycol ysi stakes pl ace i n al l cel l s of the body. The enzymes of this pathway are present in the closomal fraction of the cell. 2. Clycolysisoccurs in the absenceof oxygen (anaerobic) or in the presence of oxygen (aerobic). Lactate is the end product under anaerobic condition. In the aerobic condition, pyruvate is formed, which is then oxidized to C O2 and H 2O. 3. Clycolysis is a major pathway for ATP synthesisin tissues lacking mitochondria, e.g. erythrocytes,cornea, lens etc.

BIOCHEMISTFIY

246 4. Clycofysis is very essentialfor brainwhich is dependenton glucosefor energy.The glucose in brain has to undergo glycolysis before it is oxidized to CO2 and H2O. 5. Clycolysis(anaerobic)may be summarized by the net reaction Clucose + 2ADP + 2Pi ------+2lactate + 2ATP 6. Clycolysis is a central metabolic pathway with many of its intermediatesproviding branch point to other pathways.Thus, the intermediates of glycolysisare usefulfor the synthesisof amino acids and fat. 7. Reversal of glycolysis along with the alternate arrangements at the irreversible steps, will result in the synthesisof glucose (gluconeogenesis). Reactions

of glycolysis

The sequence of reactions of glycolysis is given in Fig.l3.2. The pathway can be divided into three distinct phases A. Energyinvestmentphaseor priming stage B. Splittingphase C. Energygenerationphase. The sequence of reactions are discussed below. A. Energy investment phase 1 . Glucose is phosphorylatedto glucose hexokinase or 6-phosphate by glucokinase (both are isoenzymes). This is an irreversible reaction, dependent on ATP and Mg2+. The enzyme hexokinaseis presentin almost all the tissues. lt catalyses the phosphorylation of various hexoses (fructose, mannose etc.), has low K,.n for substrates(about 0.1 mM) and is inhibited by glucose 6-phosphate. Clucokinasepresentin liver, catalyses the phosphorylationof only glucose, has high K. for glucose(10 mM) and is not inhibited by glucose 6-phosphate. Due to high affinity (low K.), glucose is utilized by hexokinaseeven at low concentration, whereas glucokinase

acts only at higher levels of glucose i.e., after a meal when blood glucose concentrationis above 100 mg/dl. Glucose i-phosphate is impermeable to the cell membrane. lt is a central molecule with a variety of metabolic fates-glycolysis, glycogenesis,gluconeogenesis and pentose phpsphate pathway. 2. Clucose6-phosphateundergoesisomerization to give fructose 6-phosphatein the presenceof the enzyme phosphohexosd isomeraseand Mg2*. 3. Fructose6-phosphateis phosphorylated by phosphoto fructose1,6-bisphosphate fructokinase(PFK).This is an irreversible and a regulatorystep in glycolysis. B. Splitting phase 4. The six carbon fructose 1,6bisphosphateis split (hence the name glycolysis) to three-carbon two glyceraldehyde 3-phoscompounds, phate and di hydroxyacetonephosphate by the enzyme aldolase(fructose1,6bisphosphatealdolase). 5. The enzyme phosphotrioseisomerase catalysesthe reversibleinterconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Thus, tvvo molecules of glyceraldehyde 3-phosphate are obtained from one mol ecul eof gl ucose. C. Energy generation phase 6. Glyceraldehyde3-phosphatedehydroglyceraldehyde genase converts 3-phosphateto 1,3-bisphosphoglycerate. This stepis importantas it is involvedin the formationof NADH + H+ and a high energy compound 1,3-bisphosphogfycerate. lodoacetate and arsenate inhibit the enzyme glyceraldehyde ln aerobic 3-phosphatedehydrogenase. condition, NADH passesthrough the electron transport chain and 6 ATP (2 x 3 ATP)are synthesizedby oxidative phosphorylation.

*irapEer "?*8: METABOLISMOF CARBOHYDBATES

247

HO-C\

H-c-oH HO - C- H

I

O

H-J-oHI tl

H- C

'

?H'-o-(P F=o cH20H

6nr-or-r Glucose

Phosphotriose isomerase

H-C:O I H -C -OH

cHr-o-@

Dihydroxyacetone phosphate

Glyceraldehye I. 3-phosphate Pi. NAD* N A D H+ H

?

?-o-
H_C_OH

cnr-o-@ 1,3-Bisphosphoglycerate

Glucose G-phosphate

tI Phosphohexose

ADP.I

Fomerase

M1

J cH2oH

ATPI

coo-

?-or ,

Ho-c-H H- C- OH H- - C

H-C-OH

I

O

6Hr-o-@

I

3-Phosphoglycerate

Cur-o-@

I

J

Fructose 6-phosphate

coo-

H-E-o-O t-

Phosohofructokinase

cH2-oH 2-Phosphoglycerate

I

Mg2*l ---,--tsnolase Hro4

?H'-o-O ?-or, Ho-c-H

j

I

coo-

H- C- O H O

J-o-l> v

*-C I

8*,

Jirr-o-@

Phosphoenolpyruvate

Fructose 1,6-bisphosphate .

II

ATP+

J

Fig 13.2 contd,

ADPMg'

next column

Fig 13,2 contd,

next page

E|IOCHEMISTFIY

248

cooI c-oH

tl CHe Pyruvate(enol) I SPontaneous

+

coo-

t

I C :O I CHg

Pyruvate(keto) NADH+ H-\

f

Lacrare dehydrogenas€ NAD++,j

+ coo-

I H- Q - O H I

cHg L-Lactate Fig. 13.2: Thereactionsin thepathwayof glycolysis(Thethree stepscatalysedby hexokinase, phosphofructo4nase andpyruvatekinase, shownin thick linesare irreversible).

10. The enzyme pyruvate kinase catalyses the transferof high energy phosphate from phosphoenol PYruvateto ADR leading to the formation of ATP.This step also is a substrate level phosphorylation. (Pyruvate kinase requiresK+ and either Mg2+ or Mn2*.) This reaction is irreversible.

Gonversion of pyruvate to lactate-signilicance The fate of pyruvate produced in glycolysis dependson the presenceor absenceof oxygen i n the cel l s.U nder anaerobi ccondi ti ons(l ackof Oz), pyruvateis reducedby NADH to lactatein presenceof the enzyme lactate dehydrogenase (competitive inhibitor-oxamate). The NADH utilized in this step is obtainedfrom the reaction catalysed by glyceraldehyde 3-phosphate The formation of lactateallows dehydrogenase. of NAD+ which can be reused regeneration the 3-phosphatedehydrogenase glyceraldehyde by proceeds even in the absence glycolysis so that ATP. supply to of oxygen

The occurrenceof uninterruptedglycolysisis 7. The enzyme phosphoglyceratekinase 1,3-bisphosphoglyceratevery essentialin skeletalmuscle during strenous on acts resulting in the synthesisof ATP and exercise where oxygen supply is very limited. This Glycolysis in the erythrocytes leads to lactate formation of 3-phosphoglycerate. step is a good example of substrate production, since mitochondria-the centresfor Ievel phosphorylation, since ATP is aerobic oxidation-are absent. Brain, retina, synthesizedfrom the substratewithout skin, renal medulla and gastrointestinaltract the involvement of electron transport derive most of their energy from glycolysis. kinase Phosphoglycerate chain. reaction is reversible, a rare example among the kinase reactions. 8. 3-Phosphoglycerate is converted to by phosphoglycerate 2-phosphoglycerate mutase.This is an isomerizationreaction.

Lactic

acidosis

Lactic acid is a three carbon hydroxy acid. Elevationof lactic acid in the circulation(normal plasma 4-1 5 mg/dl) may occur due to its increased production or decreasedutilization. Mild forms of lactic acidosis(not life-threatening) are associatedwith strenuousexercise,shock, respiratory diseases, cancers/ low pyruvate activity,von Gierke'sdiseaseetc. dehydrogenase

9. The high energy compound phosphoenol pyruvate is generated from 2-phosphoglycerateby the enzyme enolase.This enzyme requiresMg2* or Severeforms of lactic acidosisare observed Mn2* and is inhihited by fluoride. For bfood glucose estimation in the due to impairmen/collapse of circulatory system laboratory,fluoride is addedto the blood which is often encountered in myocardial to preventglycolysisby the cells,so that i nfarcti on, pul monary embol i sm, uncontrol l ed hemorrhageand severeshock.This type of lactic blood glucose is correctly estimated.

249

Ghapter 13 : METABOLISMOF CAFIBOHYDHATES

ac ido s i si s d u e to i n a d e q u a tes u p p l yof 02 to the tissueswith a drastic reduction in ATP synthesis ( s inc e th e c e l l s h a v e to s u rv i v e i n anaerobi c c ond i ti o n s )w h i c h ma y e v e n l e a d to death.The term oxygen debf refersto the excessamount of 02 r e q u i re d to re c o v e r. In c l i n i c al practi ce, measurementof plasma lactic acid is useful to know about the oxygen debt, and monitor the patient'srecovery.

w hi ch forms gl ucose 6-phosphate).Thus, in anaerobicglycolysis,3 ATP are produced from gl ycogen. Glycolysis

and shuttle

pathways

In the presenceof mitochondriaand oxygen, the NADH producedin glycolysiscan participate in the shuttle pathways (Refer Chapter 1l) lor the synthesisof ATP. lf the cytosolic NADH uses malate-aspartate shuttle, 3 ATP are generated Production of ATP in glycolysis from each mol ecul eof N A D H . Thi s i s i n contr ast The details of ATP generation in glycolysis to glycerolphosphate shuttle that produces (from glucose)are given in Table 13.1. Under onl y 2 A TP . anaerobic conditions, 2 ATP are synthesized while, under aerobic conditions, 8 or 6 ATP are Gancer and glycolysis synthesized-dependingon the shuttle pathway Cancer cells display increased uptake of that operates. gl ucose, and gl ycol ysi s.A s the tumors grow When the glycolysis occurs from glycogen, rapidly, the blood vesselsare unable to supply one more ATP is generated.This is becauseno adequate oxygen, and thus a condition of ATP is consumed for the activation of glucose hypoxi aexi sts.D ue to thi s, anaerobi cgl ycol ysis (glycogendirectly producesglucose1-phosphate predomi nantl yoccurs to suppl y energy. The

Pathway

Glycolysis

t

Enzyme (method of ATP synthesis)

3-phosphate dehydrogenase Glyceraldehyde phosphorylation) (2 NADH, ETC,oxidative (substrate levelphosphorylation) Phosphoglycerate kinase (substrate kinase levelphosphorylation) Pyruvate TwoATPareconsumed inthereactions catalysed byhexokinase and phosphofructokinase NetATPsynthesisin glycolysisin aerobiccondition

Number of ATP synthesized 6*

2 2 -z

(2 NADH,ETC,oxidative phosphorylation) Pyruvate dehydrogenase Citricacidcycle

(2 NADH,ETC,oxidative phosphorylation) lsocitrate dehydrogenase dehydrogenase a-Ketoglutarate (substrate thiokinase levelphosphorylation) Succinate (2 FADH2, phosphorylation) ETC,oxidative dehydrogenase Succinate (2 NADH,ETC,oxidative phosphorylation) Malatedehydrogenase

TotalATPpermoleof glucoseunderaerobiccondition TotalATPpermoleof glucose underanaerobic condition

z

4 o

38 2

4 6 ATPareproducedif NADHusesmalateshuttle;only4 ATPareproducedif glycerol-phosphate in wh:tchcasetotal shuttleoperates, per nole of glucoseoxidationis 36 and not 38 ATPsynthesized

250

ElIOCHEMISTF|Y

cancer cells get adapted to hypoxic glycolysis throughthe involvementof a transcriptionfactor namely hypoxia-inducible transcription factor (HIF). HIF increasesthe synthesisof glycolytic enzymesand the glucosetransporters.lHowever, the cancercells cannotgrow and survivewithout Fructose proper vascularization.lOneof the modalitiesof 6-phosphate cancer treatmentis to use drugs that can inhibit vascularizationof tumors. \/ lrreversible

steps

in glyeolysis

Most of the reactions of glycolysis are reversible.However,the three stepscatalysedby the enzymes hexokinase (or glucokinase), phosphofructokinase and pyruvate kinase, are irreversible.These three stagesmainly regulate glycolysis. The reversal of glycolysis, with alternate arrangements made at the three irreversible stages, leads to the synthesis.of glucosefrom pyruvate (gluconeogenesis).

\L

cA MP l^ lE'

Fructose 2,6bisphosphate

Fructose2,G-

-t/ bisphosphatase f l@ cAMP

Flg.l3.3 : legutatiollof lru6ose2,Fbisphosphatase.

(activator)for controlling PFK and, ultimately, glycolysis in the liver. F2,6-BP is synthesized from fructose 6-phosphate by the enzyme phosphofructokinase called PFK-2(PFK-1is the glycolytic enzyme). F2,6-BP is hydrolysed by Regulation of glycolysis fructose 2,6-bisphosphatase.The function of The three enzymes namely hexokinase(and synthesisand degradationof F2,6-BPis brought out by a single enzyme (samepolypeptidewith glucokinase),phosphofructokinase and pyruvate kinase, catalysing the irreversible reactions two active sites) which is referred to as bifunctional enzyme (Fig.13.3). In fact, the regulateglycolysis. combined name of phosphofructokinase-2/ Hexokinase is inhibited by glucose fructose2,6-bisphosphatase is usedto referto the 6-phosphate. This enzyme prevents the enzyme that synthesizesand degradesF2,6-BP. accumulation of glucose 6-phosphate due to The activity of PFK-2 and fructose 2,6pr oduc t inh i b i ti o n . Gl u c o k i n a s e , w hi ch is controlled by covalent bisphosphatase specifically phosphorylates glucose, is an modification which, in turn, is regulated by inducible enzyme. The substrate glucose, (cAMP is the second messengerfor pr obably t hr o u g h th e i n v o l v e me n t o f i n s ul i n, cyclic AMP certai n hormones).C ycl i c A MP bri ngs about induc esgluc o k i n a s e . dephosphorylationof the bifunctional enzyme, Phosphofruclokinase (PFK) is the most resultingin inactivationof active site responsible important regulatoryenzyme in glycolysis.This for the synthesisof F2,6-BPbut activationof the enzyme catalyses the rafe limiting committed active site responsible for the hydrolysis of step. PFK is an allostericenzyme regulatedby F2,6-B P . allostericeffectors.ATP, citrateand H+ ions (low Pyruvate kinasealso regulatesglycolysis.This pH) are the most important allostericinhibitors, enzyme is inhibited by ATP and activated by whereas,fructose2,6-bisphosphate, ADP, AMP F1,6-B P . P yruvate ki nase i s acti ve (a) i n and Pi are the allostericactivators. dephosphorylated state and inactive (b) in phosphorylated state. Inactivation of pyruvate Role of fructose Zr6-bisphosphate kinase by phosphorylationis brought about by in glycolysis cAMP-dependentprotein kinase.The hormone(F2,6-BP)is consi- glucagon inhibits hepatic glycolysis by this Fructose2,6-bisphosphate dered to be the most important regulatory factor mechanism(Fig.l3.a).

Ghapten 13 : METABOLISMOF CARBOHYDFATES A

Glucagon -

) cAMir

l@ I

Y

Proteinkinase

Fig. 13.4 : Regulationof pyruvate kinase.

Pasteur

effect

251

and other mammals.Rapaport-Leubering cycle is mainly concerned with the synthesisof 2,3bisphosphoglycerate(2,3-BPG) in the RBC. 1,3B i sphosphogl ycerate(1,3-B P C ) produced in glycolysis is converted to 2,3-BPC by the enzyme 2,3-bisphosphoglycerate mutase (Fig.l3.5).2,3-BPC is hydrolysedto 3-phosphoglycerate by bisphosphoglycerate phosphatase. lNote : There is a difference between the usages-bisphosphate and diphosphate. A bisphosphatehastwo phosphatesheld separately (e.g. 2,3-BPC), in contrastto diphosphate(e.g. ADP) where the phosphatesare linked togethed. It is now believed that bisphosphoglycerate mutase is a bifunctional enzvme with mutase and phosphataseactivities catalysed by two different sites present on the same enzyme.

The inhibition of glycolysis by oxygen (aerobic condition) is known as Pasteureffect. A bout' 15-25o/o of the gl ucose that get s This effect was discovered by Louis Pasteur, convertedto lactatein erythrocytesgoes via 2,3more than a century a1o, while studying BPG synthesis. fermentationby yeast. He observedthat when anaerobic yeast cultures (metabolizing yeast) Signifieanee of 2,3-BFG were exposedto air, the utiliziation of glucose decreasedby nearly sevenfold. 1. Production of 2,3-BPG allows the glycolysis to proceed without the synthesisof In the aerobic condition, the levels of ATP. This is advantageousto erythrocytessince glycolytic intermediates from fructose 1,6glycolysis occurs when the need for ATP is bisphosphateonwardsdecreasewhile the earlier minimal. Rapaport-Leubering cycle is, therefore, intermediatesaccumulate.This clearly indicates regarded as a shunt pathway of glycolysis to that Pasteureffectis due to the inhibition of the dissipate or waste the energy not needed by enzyme phosphofructokinase.The inhibitory erythrocytes. effect of citrate and ATP (produced in the presence of oxygen) on phosphofructokinase 2. 2,3-BPC, however, is not a waste mol ecul ei n R B C . l t combi nesw i th hemoglobin explainsthe Pasteureffect. (Hb) and reduces Hb affinity with oxygen. Grabtree effect Therefore, in the presence of 2,3-BPG, oxyhemoglobin unloads more oxygen to the The phenomenon of inhibition of oxygen fissues. consumptionby the additionof glucoseto tissues hav i n g h i g h a e ro b i c g l y c o l y s i s i s know n as lncreasein erythrocyte2,3-BPC is observed Crabtreeeffect. Basically,this is opposite to that in hypoxic condition, high altitude,fetal tissues, of Pasteur effect. Crabtree effect is due to anemi c condi ti onsetc. i n al l these cases,2, 3increasedcompetitionof glycolysisfor inorganic BPG will enhance the supply of oxygen to the phosp h a te(Pi ) a n d N A D + w h i c h l i mi ts thei r tissues. av ai l a b i l i tyfo r p h o s p h o ry l a ti o n a n d oxi dati on. 3. Glycolysisin the erythrocytesis linkedwith 2,3-BPCproductionand oxygentransport.In the RAFAPORT"LEUBERING CYCLE deficiencyof the enzyme hexokinase,glucoseis This is a supplementarypathway to glycolysis not phosphorylated,hence the synthesisand which is ooerative in the ervthrocvtesof man concentrationof 2,3-BPG are low in RBC. The

B IOC H E MIS TFIY

Glucose

+ t Y

H- C= O I H- C- O H l^

cH2-o-(7

Glyceraldehyde 3-phosphate

known as pyruvate dehydrogenase complex (P D H ),w hi ch i s found onl y i n the mi tochondri a. H i gh acti vi ti esof P D H are found i n cardi ac muscl e and ki dney. The enzyme P D H requi res five cofactors (coenzymes), namely-TPP, l i poami de, FA D , coenzyme A and N A D + (l i poami decontai nsl i poi c aci d l i nkedto e-ami no group of lysine).The overall reactionof PDH is Pyruvate + NAD+ + CoA

Reactions

o

coo-

I H- C- OH

cH2-o-e 3-Phosphoglycerate

+

Y

Pyruvate

FDFI ) Acetyl CoA + C O2+ N A D H + H +

of PDH complex

The sequenceof reactionsbrought about by different enzymes of PDH complex in associationwith the coenzymes is depicted in Fig.l3.6. Pyruvate is decarboxylated to give hydroxyethyl TPP, catalysed by PDH (decarboxylaseactivity). Dihydrolipoyl transacetylasebrings about the formation of acetyl lipoamide (from hydroxethyl-TPP)and then catalyses the transfer of acetyl group to coenzymeA to produceacetyl CoA. The cycle is complete when reduced lipoamide is converted to oxi di zedl i poami deby di hydrol i poyldehydrogenase,transferringthe reducing equivalentsto FAD. FADH2, in turn, transfersthe reducing equivalentsto NAD+ to give NADH + H+, which can pass through the respiratorychain to give 3 ATP (6 ATP from 2 moles of pyruvate formed from glucose)by oxidative phosphorylation.

Fig. 13.5 : Rapaport-Leuberingcycle for the synthesis of 2,s-bisphosphoglycerate (2,3-BPG).

The intermediatesof PDH catalysedreaction are not free but bound with enzyme complex. In mammals, the PDH complex hasan approximate hem oglobin e x h i b i ts h i g h o x y g e n a ffi n i ty i n molecular weight of 9 x |N. lt contains 60 patients.On the other hand, molecules of dihydrolipoyltransacetylase hexokinase-defective and in the patientswith pyruvate kinasedeficiency, about 20-30 molecules each of the other the level of 2,3-BPG in erythrocytesis high, two enzymes (pyruvate dehydrogenase and resultingin low oxygen affinity. dihydrolipoyl dehydrogenase). F or a m o re d e ta i l e dd i s c u s s i o no n 2 ,3 -B P C , A comparabl e enzyme w i th P D H i s reler Chapter 10. a-ketoglutarate dehydrogenase complex of citric acid cycle which catalyses the oxidative CO NV E RS IO N O F decarboxylationof a-ketoglutarateto succinyl PYRUVATE TO AGETYL GoA CoA. Pyruvate is converted to acetyl CoA by Arsenic poisoning : The enzymes PDH and oxidative decarboxylafion. This is an irreversible a-ketoglutaratedehydrogenaseare inhibited by reaction,catalysedby a multienzymecomplex, arsenite.Arsenitebinds to thiol (-SH) groupsof

253

CAFIBOHYDRATES Ghapter 13 : METABOLIqT4_Q|

o

o

C H 3-C -S -Li p-S H

cH3-c-coo\ Pyruvate

OH I

cH3-cH-TPP

Lip<3

NADH+ H'

HydroryethYl-TPP

1+

\erc +

3 ATP

c9!!l?I. (Note : Thereactioninvolvingthe of actionof pyruvatedehydrogenase Fig. 13.6: The mechanism tipoamide,CoASH'FADand NA}')' coenzymes-TPP, five requires conversionol pyruvateto acetytCoA lipoic acid and makes it unavailableto serve as cofactor. Regulation

3. ln patients with inherited deficiency of PDH, lactic acidosis(usuallyafter glucose load) is observed.

of PDH

4. PDH activitv can be inhibited by arsenic for mercuric ions. This is brought about by good and example is a Pyruvatedehydrogenase inhibition' bi ndi ng of thesei ons w i th -S H groupsof l i poi c (acetyl NADH\ CoA, end producf by aci d. regulated is also Besides this, PDH phosphoryIation and dephosphorylation (Fig' l 3'V while it is Metabolic of Pyruvate imPortance PDH is active as a dephosphoenzyme phosphatase PDH inactiveas a phosphoenzyme. Pyruvate is a key metabolite. Besides its activity is promotedby Ca2*,Mg+ and insulin (in conversiori to acetyl CoA (utilized in a wide adipose tissue). lt is of interest to note that range of metabolic reactions-citricacid cycle, calcium released during muscle contraction fatty acid synthesisetc.), pyruvate is a good stimulates PDH (by increasing phosphatase substratefor gluconeogenesis. activity)for energYProduction. PDH kinase (responsibleto form inactive PDH) is promoted by ATP, NADH and acetyl CoA, while it is inhibited by NAD+, CoA and pyruvate.The net resultis that in the presenceof high ener gy s i g n a l s(AT P , N AD H ), th e PD H i s

) Plex! rhosphoenzyme

turned off. Biochemical

imPortance

of PDH

1 . Lackof TPP(dueto deficiencyof thiamine) inhibit s PD H a c ti v i ty re s u l ti n g i n the accumulationof PYruvate. 2. ln t h e th i a mi n e d e fi c i e n t a l c o hol i cs, pyruvateis rapidly convertedto lactate,resulting in lac t ic ac i d o s i s .

PDHkinase f\

.4\ nrp, r.rnoH\ Aceiycon o]r\

\ -f;,11..

Y@pDHphosphatase 't tn

\ytnsulin (adipose ( A. 'pi tissue) eon complex dePhosphoenzyme active /

Fig. 13.7 : Regulation of pyruvate dihydrogenase (PDH) comaex'

254

B IOC H E MIS TR Y Acetyl CoA

T he c it ri c a c i d c y c l e (Kre b s c y cl e or tricarboxylic acid-TCA cycle) is the most important metabolic pathway for the energy supply to the body. About 65-70% of the ATP is synthesized in Krebs cycle. Citric acid cycle essentially involves the oxidation of acetyl CoA to CO2 and H2O. This cycle utilizesabout twothirds of total oxygen consumed by the body. The name TCA cycle is used,since,at the outset of the cycle, tricarboxylic acids (citrate, cisaconitateand isocitrate)participate.

Oxaloacetate

(4c) \ SuccinylCoA

(4c)

Citrate (6c)

f+co,

cr-Ketoglutarate (5c)

Fig. 13.8: An overuiewol Krebscycle.

T$A

egeEe*mm

e*weru*eww

TGA eyeie-*ftfuer eecB€raB metahmlfrc pff{$t}&rffiy

K rebs cycl e basi cal l y i nvol ves the combinationof a two carbon acetyl CoA with a The citric acid cycle is the final common four carbonoxaloacetateto producea six carbon oxidative pathway for carbohydrates,fats and tricarboxvlic acid, citrate. In the reactionsthat am ino ac id s .T h i s c y c l e n o t o n l y s u p p l i e se nergy follow, the two carbonsare oxidized to CO2 and but also provides many intermediatesrequired oxaloacetate is regenerated and recycled. for the synthesisof amino acids, glucose,heme Oxaloacetate is considered to play a catalytic etc. Krebs cycle is the most important role in citric acid cycle. An overview of Krebs central pathway connecting almost all the cycle is depicted in Fig.l3.8. individual metabolic pathways (either directly or indir ec t ly ) . TGA eyeEe.-an @BeE?#S#Ee Krebs cycle is a cyclic process.However, it should not be viewed as a closed circle, since The citric acid cycle was proposedby Hans many compoundsenterthe cycle and leave.TCA Adolf Krebs in 1937, based on the studies of cycle is comparableto a heavy traffic circle in a oxygen consumption in pigeon breast muscle. national highway with many connecting roads. T he c y c le is n a me d i n h i s h o n o u r (N o b e l P ri ze Each intermediate of the cycle connecting f or P hy s iol o g ya n d M e d i c i n e i n 1 9 5 3 .) another pathway is a road! -r [Note : It is of interestto note that the original ReactBoms of citrie a€id eycBe manuscripton TCA cycle submittedby Krebsto the journal 'Nature' was not accepted. He Oxidative decarboxylation of pyruvate to publis hed i t i n a n o th e r j o u rn a l E n z y m ol i gi a. acetyl CoA by pyruvatedehydrogenase complex Krebsusedto carry the rejectionletter(of Nature) i s di scussed above.Thi s step i s a connecti ngl i nk with him, and advisethe researchesnever to De between glycolysis and TCA cycle. A few discouragedby researchpaper rejectionl. authors, however, describe the conversion of pyruvate to acetyl CoA along with citric acid {.}-VsiE+ fr-*aati+er {Eg'P'$;:.1+, cycle. The events of TCA cycle are described hereunder(Fig.l3.9). The enzymes of TCA cycle are located in mitochondrial matrix, in close proximity to the 1. Formation of citrate : Krebscycle proper electron transport chain. This enables the startswith the condensationof acetyl CoA and synthesisof ATP by oxidative phosphorylation oxaloacetate, catalysed by the enzyme citrate wit hout any h i n d ra n c e . synthase. ffirfref hEsfspr-_v

2s5

Ghapter 13 : METABOLISM

(i II

ci-t3-o-cooPyruv#

o ll

C H 3-C -S C oA AcetylCoA

ot l c-cooI NADH+ H*

NADl

cH2-coo-

Oxaloacetate Aconitase nn-

HO-CH-COO_ I

cH2-cooL-Malate

I

c 8t-.ooClsAconitate

t

\,rHzO Aconitasd\

+ cii2__cooI

II

A

fi-tr-lr\Ju

cH-ctf'l-

AAA_

ti -ooc-c-H

Ho-dH-coolsocitrate +

Fumarate

t

FADH,{ o"frtHSlL FAD/\

lsocitrat€ f runo*

dehydlqgenase I / *N A D H +H -

cH2- c00-

cH2-cCIocH2-cOo-

I

cH-coo-

I O:C-COO,,,,, Oxalosuccinate

Fig_1g.g: Thecitricacid (Krebs)cycte.(trreersiblereactionsshownby thickarrows)

256

B IOC H E MIS TR Y

2. and 3. Citrate is isomerized to isocitrate by the enzyme aconitase.This is achieved in a two stage reaction of dehydration followed by hydration through the formation of an i ntermediate-crs-acon itate. 4. and 5. Formation of a-ketoglutarate : The enzyme isocitrate dehydrogenase (lCD) catalysesthe conversion (oxidative decarboxylation)of isocitrateto oxalosuccinateand then to The formationof NADH and the o-ketoglutarate. liberationof CO2 occur at this stage. 6. Conversion of cl-ketoglutarateto succinyl CoA occurs through oxidative decarboxylation, catalysed by o-ketoglutarate dehydrogenase complex. This enzyme is dependent on five cofactors-TPP, lipoamide, NAD+, FAD and CoA. The mechanism of the reaction is analogousto the conversionof pyruvateto acetyl CoA (See Fig.l3.6). At this stage of the TCA cycle, second NADH is produced and the second CO2 is liberated.

Acetyl CoA + 3 NAD+ + FAD + CDP + Pi + 2CO2 + 3NADH + 3H+ + FADH2 + 2H2O ------> GTP + CoA of oxaloacetate

Regeneration

in TGA cycle The TCA cycle basically involves the oxidation of acetyl CoA to COz with simultaneousregenerationof oxaloacetate.As such,there is no net consumptionof oxaloacetate or any other intermediatein the cycle. Requirement

of O" by TGA cycle

There is no direct participation of oxygen in Krebs cycle. However, the cycle operatesonly under aerobic conditions. This is due to the fact that NAD+ and FAD (from NADH and FADH2, respectively)required for the operation of the cycle can be regeneratedin the respiratorychain only in the presenceof 02. Therefore,citric acid cycle is strictly aerobic in contrastto glycolysis 7. Formation of succinate : Succinyl CoA is which operatesin both aerobic and anaerobic convertedto succinateby succinatethiokinase. condi ti ons. reaction is This coupled with the phosphorylation of CDP to CTP. This is a Energetics of citric acid cycle substrate level phosphorylation. GTP is During the processof oxidationof acetylCoA converted to ATP bv the enzvme nucleoside via citric acid cycle, 4 reducingequivalents(3 as diphosphatekinase. NADH and one as FADH2) are produced. CT P + AD P < + AT P + C D P Oxidation of 3 NADH by electron transport 8. Conversion of succinate to fumarate : chain coupled with oxidative phosphorylation Succinate is oxidized by succinate dehydro- resultsin the synthesisof 9 ATP,whereasFADH2 genaseto fumarate.This reaction resultsin the leadsto the formationof 2 ATP.Besides,there is production of FADH2 and not NADH. Thus, a total one substratelevel phosphorylation. 9. Formation of malate : The enzyme of twelve ATP are producedfrom one acetyl CoA. fumarasecatalvsesthe conversionof fumarateto malate with the addition of H2O. Inhibitors of Krebs cycle 10. Conversion of malate to oxaloacetate : Malate is then oxidized to oxaloacetate by malate dehydrogenase.The third and final synthesisof NADH occurs at this stage. The oxaloacetateis regeneratedwhich can combine with another molecule of acetyl CoA, and c ont inuet he c y c l e . Summary

of TCA cycle

The eventsof Krebscycle may be summarized as given in the next column

The important enzymes of TCA cycle inhibited by the respectiveinhibitorsare listed Enzyme

Inhibitor

Aconitase

Fluoroacetate (non-competitive)

o,-Ketoglutarate dehydrogenase

Arsenite (non-competitive)

Succinate dehydrogenase

Malonate (competitive)

257

Ghapter 13: METABOLISM OF CARBOHYDRATES Fluoroacetate-a suicide substrate : The inhibitor fluoroacetate is first activated to fluoroacetvl CoA which then condenseswith oxaloacetate to form fluorocitrate. TCA cycle (enzyme-aconitase) is inhibited by fluorocitrate. The compound fluoroacetate, as such, is a harmlesssubstrate.But it is convertedto a toxic compound (fluorocitrate)by celIu lar metabolism. T his is a s u i c i d ere a c ti o nc o mmi tte db y the cel l , and thus fluoroacetateis regardedas a suicide substrate. Regulation

of citric

acid

cycle

T he c e l l u l a r d e ma n d so f AT P a re cruci al i n controlling the rate of citric acid cycle. The regulationis broughtabout either by enzymesor the levels of ADP. Three enzymes-namely citrate synthase, isocitrate dehydrogenase and a-ketoglutarate dehydrogenase-regu Iate c itric ac id c y c l e .

The most importantsynthetic(anabolic)reactions connectedwith TCA cycle are given (Fig.l3.l0) 1 . Oxaloacetate and o-ketoglutarate,respectively, serve as precursorsfor the synthesisof aspartate and glutamate which, in turn, are requiredfor the synthesisof other non-essential ami no aci ds, puri nesand pyri mi di nes. 2. Succinyl CoA is used for the synthesisof porphyri nsand heme. 3. Mitochondrial citrate is transportedto the cytosol, where it is cleaved to provide acetyl CoA for the biosynthesisof fatty acids, sterols etc. Anaplerosis

or anaplerotic

reactions

The synthetic reactions described above depletethe intermediates of citric acid cycle. The cycle will cease to operate unless the intermediatesdrawn out are replenished. Ihe 1. Citrate synthase is inhibited by ATP, reactions concerned to replenish or to fill up the intermediates of citric acid cycle are called NADH, acetyl CoA and succinyl CoA. anaplerotic reactions or anaplerosis (Creek : fill 2. lsocitrate dehydrogenase is activated by up). l n Fi g.l3.10, the i mportant synthe t ic A DP , a n d i n h i b i te db y A T P a n d N AD H . pathways that draw the intermediates of TCA is inhibited cycle and the anapleroticreactionsto fill them 3. o-Ketoglutaratedehydrogenase by s uc c i n y lC o A a n d N AD H . up are grven. 4. Availability of ADP is very important for The salient featuresof important anaplerotic the citric acid cycle to proceed.This is due to reactionsare described the fact that unlesssufficientlevels of ADP are 1. Pyruvate carboxylase catalyses the available, oxidation (coupled with phosphoconversion of pyruvate to oxaloacetate.This is rylation of ADP to ATP) of NADH and FADH2 an ATP dependentcarboxylationreaction. through electron transport chain stops. The ac c um u l a ti o no f N AD H a n d F AD H 2wi l l l ead to Pyruvate + CO2 + ATP ------+ inhibition of the enzymes(as statedabove) and Oxal oacetate+ A D P + P i als o limi ts th e s u p p l y o f N AD + a n d FA D w hi ch The details of the above reaction are are essentialfor TCA cycle to proceed. describedunder gluconeogenesis.

Amphibolic nature of the citric acid cycle

2. Pyruvateis convertedto malate by NADP+ (malicenzyme). dependentmalatedehydrogenase

The citric acid cycle provides various Pyruvate + CO2+ NADPH+ H+$ intermediates for the synthesis of many Mal ate+ N A D P H + +Hr O compounds needed by the body. Krebscycle is 3. Transaminationis a orocesswherein an both cataholic and anaholic in nature, hence amino acid transfersits amino group to a keto regarded as amphibolic. acid and itself gets converted to a keto acid. The TCA cycle is actively involved in gluco- formation of a-ketoglutarateand oxaloacetate neoge n e s i s ,tra n s a mi n a ti o na n d d eami nati on. occurs by thi s mechani sm.

258

BIOCHEMISTF|Y

Non-essential amino acids,

punnes, pyrimidines

AsDartate

TransaminationAcetyl CoA

Pyruvate

Fattyacids,sterois Citrate.-'....-..-.-f

Citric acld cycle d,-Ketoglutarate

Glutamate

I J Non-essential aminoacids,purines Fig. 13.10 : Major synthetic and anaplerotic pathways of the intetmediates of citric acid cycle.

4. a-Ketoglutaratecan also be synthesized living system,energy is trapped leading to the from glutamate by glutamate dehydrogenase synthesi of s 38 A TP w hi ch i s equi val entto 1,159 action. KJ (1 ATP has high energy bond equivalentto 30.5 KJ).That is, about 48% of the energy in Clutamate + NAD(P)++ H2O <+ glucosecombustionis actuallycapturedfor ATP + NAD(P)H + H+ + NHf cx,-Ketoglutarate generation. Energetics of glucose oxidation When a molecule of glucose (6 carbon) undergoesglycolysis,2 moleculesof pyruvateor lactate (3 carbon) are produced. Pyruvate is oxidatively decarboxylated to acetyl CoA (2 carbon) which enters the citric acid cycle and gets completelyoxidized to CO2 and H2O. The overall process of glucose being completely oxidized to CO2 and H2O via glycolysis and citric acid cycle is as follows C6H1206 + 602 + 38ADP + 38Pi ------+ 6 C O2 + 6 H 2 O + 38A TP

The synthesis of glucose from noncarbohydratecompounds is known as gluconeogenesis. The major substrates/precursors for gluconeogenesis are lactate, pyruvate, glucogenic amino acids,propionate and glycerol. Location

of gluconeogenesis

C l uconeogenesi s occurs mai nl y i n t he The enzlimes of glucose metabolism cytosol, although some precursorsare produced responsible for generating ATP are given in i n the mi tochondri a.C l uconeogenesi smost ly Table 13.1. takes place in liver (about 1 kg glucose When a molecule of glucose is burnt in a synthesized everyday) and, to some extent, in calorimeter,2,780 K) of heat is liberated.In the kidney matrix (aboutone-tenthof liver capacity).

Chrpter'l

3 : METABOLISM OF CAFBOHYDHATES

r ir f ?p * ffi a n c e o f g l u e c n € 0 g e n e s i s Clu c o s e o c c u p i e s a k e y p o si ti on i n the m et a b o l i s m a n d i ts c o n ti n u o u s suppl y i s absolutelyessentialto the body for a variety of f unc t i o n s

259 4. Certainmetabolitesproducedin the tissues accumulate in the blood, e.g. lactate,glycerol, propionate etc. Cluconeogenesis effectively clearsthem from the blood. R eacti ons

of gl uconeogenesH s

C l uconeogenesi s cl osel y resembl es t he reversedpathwayof glycolysis,although it is not the complete reversalof glycolysis.Essentially, 3 (outof 10) reacti onsof gl ycol ysi sare i rreversible. The seven reactions are common for both gl ycol ysi sand gl uconeogenesi(Fi s 9.13.11). The three irreversihle sfeps of glycolysis are 2. C l u c o s e i s th e o n l y s o u rc e t hat suppl i es catalysedby the enzymes, namely hexokinase, energy to the skeletalmuscle, under anaerobic phosphofructokinase and pyruvatekinase.These c ond i ti o n s . three stages-bypassed by alternate enzymes 3. ln fasting even more than a day, specificto gluconeogenesis-arediscussed gluconeogenesismust occur to meet the basal 1. Conversion of pyruvate to phosphoenolrequirementsof the body for glucose and to pyruvate : This takes place in two steps m ain ta i nth e i n te rm e d i a teos f c i tri c aci d cycl e. (Fig.l3.12). Pyruvate carboxylase is a biotinT his i s e s s e n ti afo l r th e s u rv i v a lo f humansand dependent mitochondrial enzyme that converts pyruvateto oxaloacetatein presenceof ATP and ot her a n i ma l s . 1. Bra i n a n d c e n tra l n e rv ous system/ erythrocytes, testes and kidney medulla are depe n d e n to n g l u c o s efo r c o n ti n u o ussuppl y of ener g y .H u m a n b ra i n a l o n e re q u i re sabout 120 g of glucose per day, out of about 160 g needed by the entire body.

BtoMEDtCAt / CLtNtCAt CONCEPTS

ES

G/ycolysisis an important sourceof energy supplyfor brain, retina, skin and renal medulla.

B€

The crucial signilicanceof glycolysisis its obility to generateATP in the absenceof oxygen.

€

Skeletal muscle, during strenous exercise, requires the occurrence ol uninterrupted glycolysis.This is due to the limited supply of oxygen. The cardiac muscle cqnnot suruiuefor long in the obsenceof oxygen sinceit is not well odapted for glycolysisunder anoerobic conditions. Glycolysis in erythrocytes is associoted with 2, 3-bisphosphoglycerate(2,3-BPG)production. In the presenceof 2, S-BPG,oxyhemoglobin unloads more oxygen to the tissues.

tr-g-

The occurrenceof glycolysisis uerg much eleuated in rapidly growing concer cells.

ss Lactic acldosis is olso obserued in patients with deficiency oJ the enzgme pgruuate dehydrogenase.It could also be due to collapse of circulatory systemencountered in myocardial infarction and pulmonary embolism. Citric acid cycle is the final common oxidatiue pathway lor carbohydrates,t'ots and amino ocids.lt utilizes (indirectly)about 2/3 of the total oxygen consumed by the body ond generatesobout U3 of the total energy (ATP). Unlike the other metabolic pathways/cycles,uery few genetic abnormalities of Krebs cycle are known. This may be due to the uital importance ol thts metabolic cycle for the suruiual of liJe

260

B IOC H E MIS TFIY

I

Phosphoenolpyruvate Glucose6phosphate

Oxaloacetate

Glyceraldehyde 3-phosphate I_

. Dihydroxyacetone phosfihate

Oxaloacetate +-@ I 1,3-Bisphosphoglycerate Glycerol3-phosphate

t

3-Phosphoglycerate

I

+i i AD P< I ) Glycerolkinase i ATP_/I i

Malate
s-Ketoglutarate

li

Glycerol

i

2-Phosphoglycerate

I

Fig. l3,ll

contd- next column

Fig. 13.11: The pathway of gluconeogenesis.[The enzymes catalysingineversible steps in glycolysis arc shown in red. The important enzymes participating in gluconeogenesis are shown in shaded green. The substrates for gluconeogenesis are in blue. The numbers represent the entry of glucogenic amino acids : (1) Alanine, glycine, serine, cysteine, threonine and tryptophan; (2) Aspartate and asparagine; (3) Arginine, glutamate, glutamine, histidine, proline; (4) lsoleucine, methionine, valine; (5) Phenylalanine, tyrosine].

CO2. This enzyme regulates gluconeogenesis malate is catalysedby malatedehydrogenase, an and requiresacetyl CoA for its activity. enzyme present in both mitochondria and cvtosol. Oxaloacetate is synthesized in the mitochondrialmatrix. lt has to be transportedto ln the cytosol, phosphoenolpyruvate carboxythe cytosolto be used in gluconeogenesis, where kinase converts oxaloacetate to phosphoenolthe rest of the pathway occurs. Due to pyruvate.CTP or ITP (not ATP) is used in this membrane impermeability,oxaloacetatecannot reaction and the CO2 (fixed by carboxylase)is diffuseout of the mitochondria.lt is convertedto liberated. For the conversion of pyruvate to malate and then transported to the cytosol. phosphoenol pyruvate, 2 ATP equivalentsare Within the cytosol, oxaloacetateis regenerated. utilized. This is in contrastto only one ATP that The reversibleconversion of oxaloacetateand is liberated in glycolysisfor this reaction.

Chapter 13 : METABOLISMOF CARBOHYDRATES

o cH3-c-cooPyruvate ATF co;

tte ase

ADP+ Pi

o -ooc-cH2-c-cooOxaloacetate GTP. GDP

renolpyruvate rxykihise

CO z {

cH2-c-cooPhosphoenolpyruvate Ftg. 13.12: Conversionof pyruvateto phosphoenolpyruvate.

Gluconeogenesis

261 from

amino

acids

The carbon skel eton of gl ucogeni c ami no aci ds(al l exceptl euci neand l ysi ne)resul tsi n the formation of pyruvate or the intermediatesof citric acid cycle (Fig.l3.ll) which, ultimately, result in the synthesisof glucose. Gluconeogenesis

from

gtycerol

Clycerol is liberated mostly in the adipose tissueby the hydrolysis of fats (triacylglycerols). The enzyme glycerokinase(found in liver and kidney, absent in adipose tissue) activates glycerol to glycerol 3-phosphate. The latter is converted to dihydroxyacetone phosphate glycerol 3-phosphate dehydrogenase. by Dihydroxyacetonephosphateis an intermediate in glycolysiswhich can be convenientlyusedfor glucose production. Gluconeogenesis

from

propionate

Oxidation of odd chain fatty acids and the breakdown of some amino acids (methionine, isoleucine)yields a three carbon propionyl CoA. Propionyl CoA carboxylase acts on this in presence of ATP and biotin and converts to methyl malonyl CoA which is then convertedto succinyl CoA in presence of 812 coenzyme (Refer Fig.7.38). Succinyl CoA formed from propionyl CoA entersgluconeogenesis via citric aci d cycl e.

2. Conversion of fructose 1,6-bisphosphate to fructose 6-phosphate : Phosphoenolpyruvate undergoesthe reversalof glycolysisuntil fructose 1, 6- bis p h o s p h a tei s p ro d u c e d . T h e enzyme fructose lr6-bisphosphatase converts fructose 1,6-bisphosphateto fructose 6-phosphate.This enz y m e re q u i re s Mg 2 * i o n s . F ru c t ose 1,6bisphosphatase is absent in smooth muscle and heart muscle. This enzyme is also regulatoryin Gluconeogenesis gluconeogenesi* 3. Conversion of glucose 6-phosphate to gfucose : Glucose 5-phosphatasecatalysesthe conversion of glucose 6-phosphateto glucose. The presenceor absence of this enzyme in a tissuedetermineswhether the tissue is capable of contributingglucoseto the blood or not. lt is mostly presentin liver and kidney but absentin muscletbrain and adiposetissue. The overall summary of gluconeogenesis for the conversion of pyruvate to glucose is shown below 2 Pyruvate+ 4ATP + 2CTP + 2NADH + 2H+ + 6H2O ------+Clucose + 2NAD+ + 4ADP -r 2G DP + 6Pi + 6 H +

from lactate (Cori cyclel Lactateproduced by active skeletalmuscle is a major precursor for gluconeogenesis.Under anaerobic conditions, pyruvate is reduced to lactate by lactatedehydrogenase(LDH) Pyruvate + NADH * H* *\

Lactate+ NAD+

Lactate is a dead end in glycolysis, since it must be reconverted to pyruvate for its further metabolism. The very purpose of lactate production is to regenerate NADH so that glycolysis proceeds uninterrupted in skeletal muscle. Lactate or pyruvate produced in the muscle cannot be utilized for the svnthesisof

262

BIOCHEMISTF|Y

Gl ucose

Glu co se

J Glycogenf Gtucass

1

Glucose6-phosphatei

+

LrvEB Glycogen

I I

Pyruvate

Pyruvate

I

(red)cycle(otherreactions@mmonfor bothcycles). Flg. 13.13: TheCoricycle(blue)andglucose-alanine

glucose due to the absence of the key enzymes of gluconeogenesis(glucose6-phosphataseand fructose 1,6-bisphosphatase). The plasma membraneis freely permeableto lactate. Lactate is carried from the skeletal muscle through blood and handed over to liver, where it is oxidized to pyruvate. Pyruvate, so produced, is converted to glucose by gluconeogenesis,which is then transportedto the skeletalmuscle.

Regulation

of gluconeogenesis

The hormoneglucagonand the availabilityof substratesmainly regulate gluconeogenesis,as discussedhereunder. Influence of glucagon : This is a hormone, secreted by a-cells of the pancreatic islets. Clucagon stimulates gluconeogenesisby two mechani sms

1. Active form of pyruvate kinase is convertedto inactive form through the mediation The cycle involving the syntfiesis of glucose of cyclic AMP, brought abofit by glucagon. in liver from the skeletal muscle lactate and the Decreasedpyruvate kinase results in the reduced reuseof glucosethus synthesizedby the muscle conversion of phosphoenol pyruvate to pyruvate for energy purpose is known as Cori cycle and the former is diverted for the synthesisof (Fig.t3.t A. glucose. 2. Clucagon reduces the concentration of This compound allosfructose2,6-bisphosphate. There is a continuoustransportof amino acids inhibits phosphofructokinase and terically from muscle to liver, which predominantly activates fructose 1,6-bisphosphatase,both oc c ur s du ri n g s ta rv a ti o n .Al a n i n e d o mi nates favour increasedgluconeogenesis. among the transported amino acids. lt is postulated that pyruvate in skeletal muscle Availability of substrates: Among the various undergoestransaminationto produce alanine. substrates, glucogenic amino acids have s. s i s Alanine is transported to liver and used for sti mul ati ngi nfl uenceon gl uconeogenesiThi gluconeogenesis.This cycle is referred to as parti cul arl y i mportant i n a condi ti on l i ke gfucose-alaninecycle (Fig.l3.13). diabetesmellitus(decreasedinsulin level)where Gfle.ee@cie"alanimecycle

'iih*ryter 13 : METABOLISMOF CARBOHYDBATES am ino ac ids a re m o b i l i z e dfro m m u s c l ep r otei n for the purposeof gluconeogenesis. Acetyl CoA promotes gluconeogenesis : During starvation--dueto excessivelipolysis in adipose tissue-acetyl CoA accumulatesin the liver. Acetyl CoA allostericallyactivatespyruvate c ar box y las e re s u l ti n g i n e n h a n c e d g l ucose pr oduc t ion. Alceilrel inhihits

gtu*ofiGesgenesis

Clycogen is the storage form of glucose in animals,as is starchin plants.lt is storedmostly in Iiver (6-8%) and muscle (1-2"/"').Due to more muscle mass,the quantityof glycogenin muscle (250 S) is about three times higher than that in the liver (75 g). Glycogen is storedas granulesin the cytosol, where most of the enzymes of glycogen synthesisand breakdown are present.

Ethanoloxidation in the liver to acetaldehvde by the enzyme alcohol dehydrogenaseutilizes Functians of glycogen NAD+. The excessNADH produced in the liver The prime function of liver glycogen is to interferes with gluconeogenesisas illustrated maintain the blood glucose levels, particularly below. betweenmeals.Liver glycogenstoresincreasein Ethanol+ NAD+--+ Acetaldehyde+ NADH + H+ a well-fed state which are depleted during fasting.Muscle glycogenservesas a fuel reserve Pyruvate + NADH + H+ <-+Lactate + NAD. for the supply of ATP during muscle contraction. Oxaloacetate+ NADH + H+ e+ Malate + NAD+ It is evident from the above reactionsthat Why store glycogen pyruvate and oxaloacetate, the predominant as a fuel re$*rve? substrates for gluconeogenesis, are made unav ailable b y a l c o h o l i n to x i c a ti o n . Thi s happensdue to overconsumptionof NAD+ and excessiveproduction of NADH by alcohol.

As such, fat is the fuel reserve of the bodv. However, fat is not preferred,insteadglycogen is chosen for a routine, and day to day use of energy for the following reasons

Alcohol consumption increasesthe risk of . Glycogen can be rapidly mobilized hypoglycemia(reducedplasma glucose)due to . Clycogen can generateenergy in the absence r educ ed glu c o n e o g e n e s i sT. h i s i s p a rti c ul arl y of oxygen importantin diabetic patientswho are on insulin . B rai n dependson conti nuousgl ucosesuppl y treatment. (which mostly comes from glycogen.) Glueoneogenesis frorn fat? On the other hand, fat mobilization is slow, It is often stated that glucose cannot be synthesizedfrom fat. In a sense, it is certainly true, since the fatty acids (most of them being even chain), on oxidation, produce acetyl CoA which cannot be convertedto pyruvate.Fufther, the two carbons of acetyl CoA disappearas 2 moles of CO2 in TCA cycle. Therefore,even chain fatty acids cannot serve as precursorsfor glucose formation. The prinre reason why animals cannot convert fat to glucose is the absence of glyoxylate cycle (described later).

needs 02 for energy production and cannot produce glucose (to a significantextent).Thus, fat may be consideredas a fixed deposit while glycogen is in the current/savingaccount in a bank!

GTYCOGENESIS The synfhesis of glycogen from glucose is glycogenesis (Fig.|3.14). Glycogenesis takes place in the cytosol and requiresATP and UTp, besidesglucose.

However, the glycerol releasedfrom lipolysis 1. Synthesisof UDP-glucose: The enzymes and the propionateobtained from the oxidation hexoki nase(i n muscl e)and gl ucoki nase(i n l i ver) of odd chain fatty acids are good substratesfor convert glucose to glucose 6-phosphate. gluconeogenesis, as discussedabove. Phosphoglucomutase catalysesthe conversionof

264

BIOCHEMISTFIY

glucose 6-phosphate to glucose 1-phosphate. U ri d i n e d i p h o s p h a te g l u c o se (UD P C ) i s synthesizedfrom glucose 1-phosphateand UTP by UDP-glucosepyrophosphorylase. 2. Requirement of primer to initiate glycogenesis : A small fragment of pre-existing glycogen must act as a 'primel to initiate glycogen synthesis.lt is recently found that in the absence of glycogen primer, a specific pr o te i n -n a m e l y ' g l y c o g e n i n ' -- -< .an accept glucosefrom UDPG. The hydroxyl group of the amino acid tyrosine of glycogenin is the site at which the initial glucose unit is attached.The enzyme glycogen initiator synthasetransfersthe first molecule of glucose to glycogenin. Then glycogeninitselftakesup a few glucoseresidues to form a fragmentof primer which servesas an acceptorfor the rest of the glucose molecules. 3. Glycogen synthesisby glycogen synthase: Clycogen synthase is responsible for the f o rma ti o n o f 1 ,4 -g l y c o s i d i c l i nkages. Thi s enzyme transfersthe glucosefrom UDP-glucose to the non-reducingend of glycogento form cr1, 4 l i n k a g e s . 4. Formation of branches in glycogen : Clycogen synthasecan catalysethe synthesisof a linear unbranched molecule with '1,4 uglycosidic linkages. Glycogen, however, is a branched tree-like structure.The formation of branches is brought about by the action of a branching enzyme, namely glucosyl a-4-6 transferase. (amylo o 1,4 -+ 1,6 transglucosidase).This enzyme transfers a small fragment of five to eight glucose residuesfrom the non-reducing end of glycogen chain (by breaking a-1 ,4 linkages) to another glucose residuewhere it is linked by ct-1,6 bond. This leads to the formation of a new non-reducing end, besidesthe existingone. Clycogen is further elongated and branched, respectively,by the enzymes glycogen synthase and glucosyl 4-6 transferase.

Glucose Al r\l Glucokinase ,l ADP(I + Glucose6-phosphate

I I Phosphoglucomutase J Glucose1-phosphate

UTP. I UDP-glucose \ .,4 pyrophosphorylase P P i <.fI UDP-glucose

(uDP_a) )

l-ot erycJsenin

ff":,[j:H"t"'

Ut

13(UDP--)\l I

Glycogensynthase

13uDP{/J

710

+--

a 1-6-Bond

The overall reactionof the glycogensynthesis for the addition of each glucose residue is (Clucose)n+ Glucose + 2ATP ------> (C l u c o s e )n* + 1 2A D P + P i

Fig. 13.14 : Glycogen synthesis from glucose (glycogenesis).

Ghapter 13 : MEIABOLISMOF CAFIBOHYDBATES

265

Of the two ATP utilized, one is required for the phosphorylationof glucosewhile the other is needed for conversionof UDP to UTP.

GLYCOGENOLYSIS The degradation of stored glycogen in liver and muscle constitutes glycogenolysis. The pathways for the synthesisand degradationof glycogenare not reversible.An independentset of enzymes present in the cytosol carry out glycogenolysis. Clycogen is degraded by breaking d.-'l,4- and a-1,6-9lycosidic bonds (Fig.t3J A. 1 . Action of glycogen phosphorylase: The a1, 4- g l y c o s i d i cb o n d s (fro m th e n on-reduci ng ends) are cleaved sequentiallyby the enzyme glycogen phosphorylase to yield glucose 1-phosphate. This process-called phosphorolysis---<.ontinuesuntil four glucose residues r em a i no n e i th e rs i d e o f b ra n c h i n gpoi nt (a-1,6glycosidic link). The glycogen so formed is known as limit dextrin which cannot be further phosphorylase. Clycogen degraded by phosphorylase possesses a moleculeof pyridoxal phosphate,covalently bound to the enzyme. 2. Action of debranching enzyme : The branches of glycogen are cleaved by two enzymeactivitiespresenton a singlepolypeptide called debranching enzyme, hence it is a bifunctional enzyme. Clycosyl 4 : 4 translerase(oligo rl..-1 ,4 --> 'l ,4 glucan transferase)activity removes a fragment of three or four glucose residues attached at a branch and transfersthem to another chain. Here, one c-1,4-bond is cleaved and the same cr-1,4bond is made, but the placesare different. Amylo cr-1,6-9lucosidasebreaks the o,-1,6 bond at the branch with a single glucoseresidue and releases a free glucose. The remainingmolecule of glycogen is again available for the action of phosphorylaseand debranching enzyme to repeat the reactions stated in 1 and 2. 3. Formation of glucose 6-phosphate and glucose : Through the combined action of glycogen phosphorylase and debranching

Glycogen P i (-)-

Glycogen phosphorylase

F ]1

Gl cose 1phosphate

a

ffi Limit deldrin

I o"nr"n.i,ingenzyme (transferase activity) J ijr

o"br"n.hingemyme I ^.# Glucosesy' (cr1-+6giucosttaCJbciivityy (rree) |

+

| ,rnn"r, actionof PhosPhovlase I

+

Glucose1-phosphate

I I enosptrogtucomutase

J Glycolysis {-

Glucose6-phosphate

I

I GlucoseSphosphatase (in tiver) J GLUCOSE Fig. 13.15 : Glycogen degradation to glucoseglycogenolysis. (The ratio of glucose 1-phosphate to glucose is 8: t).

B IOC H E MIS TR Y

266 enzymet glucose 1-phosphateand free glucose in a ratio of 8 : 1 are produced. Clucose 1-phosphateis convertedto glucose6-phosphate Glucose6phosphate by the enzyme phosphoglucomutase. The fate of glucose 6-phosphatedependson the tissue.The liver, kidney and intestinecontain the enzyme glucose 6-phosphatasethat cleaves glucose6-phosphateto glucose.This enzyme is absent in muscle and brain, hence free glucose cannot be produced from glucose 6-phosphate in these tissues.Therefore, liver is the major glycogen storageorgan to provide glucose into the circulation to be utilised by various tissues. In the peripheraltissues,glucose6-phosphate produced by glycogenolysiswill be used for glycolysis.lt may be noted that though glucose 6-phosphatase is absentin muscle,some amount of free glucose(8-10% of glycogen)is produced in glycogenolysis due to the action. of activity). debranchingenzyme(a-1,6-9lucosidase

ATP II I

(J

I

I

@C I

/

r''1

,/-

Glycogen

U'on"t" Glyic.ogen.syn-th

to I

Glucose6phosphate

l. Allosteric regulation of glycogen metabolism : There are certain metabolites that allostericallyregulatethe activitiesof glycogen and glycogen phosphorylase. The Acid maltase or a-1 ,4-glucosidase is a synthase control is carriedout in such a way that glycogen lysosomal enzyme. This enzyme continuously synthesisis increasedwhen substrateavailability degrades a small quantity of glycogen. The and energy levels are high. On the other hand, significanceof this pathway is not very clear. glycogenbreakdown is enhancedwhen glucose However, it has been observed that the concentrationand energy levels are low. The deficiency of lysosomal enzyme o,-1,4 glycogen metabolism is gluc os idas ere s u l ts i n g l y c o g e n a c c u mu l ati on, allosteric regulation of In a well-fed state, the in Fig.l3.l6. depicted causinga seriousglycogen storagediseasetype is high which glucose of 6-phosphate availability ll ( i. e. P om p e ' sd i s e a s e ). allosterically activates glycogen synthase for more glycogen synthesis.On the other hand, Regerlation of glycogenesis glucose 6-phosphate and ATP allosterically and glycoqenolysis inhibit glycogen phosphorylase.Free glucosein A good coordination and regulation of liver also acts as an allosteric inhibitor of glycogen synthesis and its degradation are glycagen phosphorylase. essential to maintain the blood glucose 2. Hormonal regulation of glycogen metabolevels. Glycogenesis and glycogenolysis are, lisrn : The hormones,through a complex series respectively, controlled by the enzymes of reactions,bring about covalent modification, glycogen synthase and glycogen phosphorylase. namely phosphorylationand dephosphorylation Regulationof theseenzymesis accomplishedby of enzyme proteins which, ultimately control three mechanisms glycogen synthesisor its degradation. 1 . Allosteric regulation cAMP as second messengerfor hormones : 2. Hor m o n a l re g u l a ti o n and The hormones l i ke epi nephri ne 3. lnf l u e n c eo f c a l c i u m . norepinephrine,and glucagon (in liver) activate Degradation of glycogen acid maltase by lysosornal

267

Chapter 13 : METABOLISMOF CAFIBOHYDRATES adenylatecyclaseto increasethe production of breaks cAMP. The enzyme phosphodiesterase down cAMP. The hormone insulin increasesthe phosphodiesterase activity in liver and lowersthe cAMP levels.

ln the Fig.l3.17, the inhibition of glycogen synthesi s brought by epi nephri ne (al so norepinephrine)and glucagonthrough cAMP by converting active glycogen synthase 'a' to i nacti vesynthase' b' , i s gi ven.

Regulation of glycogen synthesis by cAMP : The glycogenesis is regulated by glycogen synthase.This enzyme exists in two formsis not glycogen synthase 'a'-which phosphorylatedand most active, and secondly, glycogen synthase'b' as phosphoryIated i nactive form. Clycogen synthase'a' can be converted to 'b' form (inactive) by phsophorylation. The degreeof phosphorylationis proportionalto the inactive state of enzyme. The process of phosphorylation is catalysed by a cAMPdependent protein kinase. The protein kinase phosphorylates and inactivates glycogen synthaseby converting'a' form to 'b' form. The glycogen synthase'b' can be converted back to synthase'a' by protein phosphatasel.

Regulation of glycogen degradation by cA MP : The hormones l i ke epi nephri ne and glucagon bring about glycogenolysisby their action on glycogen phosphorylase through cAMP as illustrated in Fig.l3.18. Glycogen phosphorylaseexists in two forms, an active 'a' form and i nacti veform' b' .

Glucagon

Adenylate cycrase (inactive)

The cAMP-formed due to hormonal stimulus-activates cAMP dependent protein kinase.This active protein kinasephosphorylates inactive form of glycogen phsophorylase kinase to active form. (The enzyme protein phosphataseremovesphosphateand inactivates phosphorylase kinase). The active phosphorylase kinase phosphorylatesinactive glycogen phosphorylase'b' to active glycogen phospho-

Epinephrine

PLASMA MEMBRANE

5 'A M P

268

B IOC H E MIS TF| Y

Glucagon Epinephrine (liver) (liver,muscle)

Adenylate cyclase (inactive)

PLASMA MEMBRANE

ATP--Q----+

cAMp

II

cAMP-dependent proteinkinase (inactive)

; -

)

ua-ca2*

phosphorylase b (inactive)

rylase'a' which degradesglycogen.The enzyme protein phosphataseI can dephosphorylateand convert active glycogen phosphorylase'a' to inactive 'b' torm.

Th'e synthesis and degradative pathways of metabolism (particularly reactions involving 3. Effect of Ca2+ ions on glycogenolysis : phosphorylationand dephosphorylationutilizing When the muscle contracts, Ca2+ ions are ATP) are well regulatedand subjectedto fine releasedfrom the sarcoplasmicreticulum. Ca2+ tuning to meet the body demands,with minimal bi nds to calmodulin- calci u m modulati ng p rotein wastage of energy and metabolites. Thus, and directly activates phosphorylase kinase glycolysis and gluconeogenesis(breakdown of without the involvement of cAMP-deoendent glucoseto pyruvate,and conversionof pyruvate protein kinase. to glucose), glycogenolysis and glycogenesis The overall effect of hormones on glycogen oDeratein a selectivefashionto suit the cellular metabolism is that an elevated glucagon demands. ff on the other hand, the synthesisand degradative metabolic pathways of a particular or epinephrine level increases glycogen degradation whereas an elevated insulin results substance(say gluconeogenesisand glycolysis in increased glycogen synthesis. refated to glucose) operate to the same extent

Chapter 13 : METABOLISMOF CAFIBOHYDFATES

269

simultaneously, this would resultin futile cycles. enzymes in the glycogen storage disorders is However,futilecycles,consuming energy(ATP) depicted in Fig.l3.19. The biochemical lesions are wasteful metabolic exercises.They are and the characteristicfeaturesof the disorders minimallyoperativedue to a well coordinated are given in Table 13.2. metabolicmachinery. von Gierke's

The metabolic defects concerned with the glycogen synthesis and degradation are collectively referred to as glycogen storage diseases.These disorders are due to defects in the enzymes which may be either generalized (affecting all tissues) or tissue-specific.The inherited disorders are characterized by deposition of normal or abnormal type of glycogen in one or more tissues.A summary of glycogen metabolism along with the defective

Enzvme defecl

disease

(type

ll

The incidence of type I glycogen storage diseaseis 1 per 200,000 persons.lt is transmitted by autosomalrecessivetrait. This disorderresults in various biochemical manifestations. 1. Fasting hypoglycemia : Due to the defect in the enzyme glucose 6-phosphatase,enough free glucose is not releasedfrom the liver into blood. 2. Lactic acidemia : Clucose is not synthesizedfrom lactateproducedin muscleand liver. Lactatelevel in blood increasesand the oH is lowered (acidosis).

Organ(s)involved

Characteristic features

vonGierke's disease Glucose 6-phosphataseLiver,kidney and Gfcogenaccumulates in hepatocytes andrenalcells, (typeI glycogenosis) intesline enhrged liverandkidney, tasting hypoglycemia, lactic goutyarthritis. acidemia; hyperlipidemia; ketosis; Lysosomal cr-1,4glucosidase(acidmaltase)

ll

Pomoe'sdisease

All organs

lll

muscle, Branched Cori'sdisease Amyloa-1,6-glucosidaseLiver, chainglycogen accumulates; liverenlarged; (limitdextrinosis, (debranching enzyme) heart, leucocylesclinical manifestations aresimilar butmilder compared to Forbe's disease) vonGierke's disease.

lV

Anderson's disease Glucosyl 4-6transferase. Mosttissues (amylopectinosis) (branching enzyme)

V

glycogen McArdle's disease Muscle (typeVglycogenosis) phosphorylase

Vl

Her'sdisease

Liverglycogen phosphorylase

Vll

Tarui's disease

PhosphofructokinaseSkeletal muscle, Musclecramosdue to exercise: bloodlactatenot erythrocytes elevated; hemolysis occurs.

Glycogen accumulates in lysosomes in almostall the tissues; heartis mostlyinvolved; enlarged liverand heart,nervous system is alsoafiected; deathoccursat anearlyagedueto heartfailure.

glycogen A rare disease, with onlyfew branches accumulate; cinhosis ol liver,impairment inliverfunction.

muscle Muscle Skeletal glycogen veryhigh,notavailable stores during exercise; subiects cannotperform strenous exercise; sufferfrommuscle cramps; bloodlactate andpyruvate do not increase afterexercise; musclesmay get damaged dueto inadequate energy supply. Liver

Liverenlarged; liverglycogen cannotformglucose (pyruvate andlactatecanbe precursors for glucose); mildhypoglycemia andketosis seen,nota veryserious disease.

Vlll,lX,X andXl havebeenidentified. Theyaredueto defects Rareglycogen disorders in theenzynesconcerned withactivating and deactivating liverphosphorylase.

270

BIOCHEMISTF|Y

Limitdextrin

UDP-glucose

Glucose1phosphate

V (musc!ei VI (liver)

Glycogen (o 1,4and1,6-bonds)

IV

Glycogenunbranched (a 1,4-bonds)

Glucosyl(4-6) transferase

Glucose+ oligosaccharides Fig. 13.19 : Summary of glycogen metabolism with glycogen storage diseases (Red blocks indicate storage disease, l-von Gierke's disease; Il-Pompe's disease; III-Cori's disease; Iv-Anderson's disease; V-ltlc Ardle's disease; W-Her's disease; Wl-Tarui's disease).

3. Hyperlipidemia : There is a blockade in gluconeogenesis. Hence more fat is mobilized to meet energy requirementsof the body. This resultsin increasedplasma free fatty acids and ketone bodies. 4. Hyperuricemia: Glucose6-phosphatethat accumulates is diverted to pentose phosphate pathway, leading to increased synthesisof ribose phosphateswhich increasethe cellular levelsof phosphoribosylpyrophosphateand enhancethe metabolismof purine nucleotidesto uric acid. Elwated plasma levels of uric acid (hyperuricemia)are often associatedwith gouty arthritis (painful joints).

The important featuresof the glycogen storage diseasesare given in Table 13.2.

Hexose monophosphate pathway or HMP shunf is also called pentose phosphatepathway or phosphogluconate pathway. This is an alternative pathway to glycolysis and TCA cycle for the oxidation of glucose. However, HMP shunt i s more anabol i c i n nature, si nce i t i s concernedwith the biosvnthesisof NADPH and pentoses.

Ghapter'13 : METABOLISMOF CAFIBOHYDHATES HMP shunt-a multifunctional

unique pathway

The pathway startswith glucose 6-phosphate. As such, no ATP is directly utilized or produced in HMP pathway. lt is a unique multifunctional pathway,since there are severalinterconvertible substancesproduced which may proceed in differentdirections in the metabolic reactions. Location

GlucoseFphosphab

of the pathway

The enzymesof HMP shunt are locatedin the cytosol. The tissues such as liver, adipose tissue, adrenal gland, erythrocytes, fesfes and lactating mammary gland, are highly active in HMP shunt. Most of these tissues are involved in the biosynthesisof fatty acids and steroidswhich are dependenton the supply of NADPH. Heactions

271

of the pathway

The sequence of reactions of HMP shunt (Fig.l3.2O)is divided into two phases-oxidative and non-oxidative. 1. Oxidative phase : Glucose 6-phosphate dehydrogenase(C6PD) is an NADP-dependent enzyme that converts glucose 6-phosphateto 6-phosphogluconolactone.The latter is then hydrolysedby the gluconolactonehydrolaseto 6-phosphogluconate. The next reactioninvolving the synthesisof NADPH is catalysedby 6-phosphogluconatedehydrogenaseto produce 3 keto 6-phosphogluconate which then undergoes decarboxylationto give ribulose S-phosphate. G6PD regulates HMP shunt : The first reactioncatalysedby C6PD is most regulatoryin HMP shunt. This enzyme catalyses an irreversible reaction. NADPH competitively inhibits G6PD. lt is the ratio of NADPH/NAD+ that ultimatelydeterminesthe flux of this cycle. 2. Non-oxidative phase : The non-oxidative reactionsare concernedwith the interconversion of three, four, five and seven carbon monosaccharides.Ribulose5-phosphateis acted upon by an epimeraseto produce xylulose 5-phosphate while ribose5-phosphateketoisomerase converts ribulose S-phosphateto ribose 5-phosphate.

o ll

lr

cH2o-(7> 6+ttospttogluconolactone

!U L .'

H-C-OH HO-C-H H-C-OH H-C-OH

cH2o-o 6-Phosphogluconate

cH2oH I

C=O I H-C-OH H-C-OH l^ cH2o-€> RibuloseSphoaphale I I I Flg. 13.20 contd.

noxt prgo

B IOC H E MIS TFIY

272 Ribulose S-phosPhate

XyluloseS-phosphate

cH20H

H-C:O

C:O

H-C-OH I H-C-OH

HO- C - H I H-C-OH

cHro-o XyluloseS-phosphate

H2OH

I

H-C-OH l^

cH2o-<9>

Ribose S-phosphate

C:O I

HO-C-H H-C-OH I H - C- O H

| /^\ cH2o-g>

Fructoseo-phosphate cH20H I

Transketolase H - C: O I H-C-OH I

C:O HO - C - H I H- C - O H I

,,r

cH2o-9>

Glyceraldehyde 3-phosphate

H-C-OH I H- C - O H

cHro-o

o{ I Reversar I ulvcolvsis

Sedoheptulose 7-phosphate

+

Fructose6-phosphate Transaldolase

H- C =O H- C - O H I H-C-OH

cHro-e Erythrose4-phosphate

cH20H tG:O HO-C-H I H-C-OH I

H-c-oH lr

cH2o-9> Fructose6-phosPhate shunt.(TPP-Thiaminepyrophosphate) 13.20: The hexosemonophosphate

Ghapter 13 : METABOLISMOF CAFIBOHYDHATES

6 NADP+ 6 NADPH+ 6H+ (6) clucose G-phosphate(OC)I

(6) 6-Phosphogluconolactone (6C)

l-anro

r

v

(6) 6-Phosphogluconate (6C) lr6NADP* u to'*fru*ADPH

(5) Glucose6phosphate (6C)

tI

+

+ 6H+

(6) Ribulose (5C) s-phosphate

I

I (5) Fructose6phosphate (6C)

(2 + 2) Xylulose5-

(2) Ribose5-

(2) Fructose6phosphate (6C)

(2) Glyceraldehyde 3-phosphate(3C)

(2)Sedoheptulose 7-phosphate(7C)

(2) Glyceraldehyde 3-phosphate(3C)

EMhrose4phosphate (4C)

(2) Fructose (6C) 6-phosphate

I of I Reversal elvcolvsis J (1)Fructose (6C) o-phosphate

Fig. 13.21: Overuiewof hexosemonophosphate shuntrepresentingthe numberof molecules(pretixin red) and the number of carbon atoms (suffix in blue). Note that of the i-molecules of glucose 6-phosphate that enter HMP shunt, one molecule is oxidized as S-molecules are finally recovered.

The enzyme transketolase catalyses the transfer of two carbon moiety from xylulose 5-phosphateto ribose 5-phosphateto give a 3-carbon glyceraldehyde 3-phosphate and a 7-carbon sedoheptulose 7-phosphate. Transketolaseis dependenton the coenzyme thiamine (TPP) and pyrophosphate ions. Mg2+ Transaldolasebrings about the transfer o1 a 3-carbon fragment (active dihydroxyacetone) from sedoheptulose7-phosphate to glyceraldehyde 3-phosphateto give fructose6-phosphate and four carbon erythrose 4-phosphate.

Transketolase acts on xylulose 5-phosphateand transfersa 2-carbon fragment (glyceraldehyde) from it to erythrose 4-phosphate to generate fructose 6-phosphate and glyceraldehyde 3-phosphate. Fructose 6-phosphate and glyceraldehyde 3-phosphatecan be further catabolizedthrough gl ycol ysi sand ci tri c aci d cycl e. C l ucose may also be synthesizedfrom thesetwo compounds. An overview of HMP shunt is given in Fig.l 3.21. For the completeoxidationof glucose

274

BIOCHEMISTF|Y

6-phosphate to 6CO2, we have to start with 6 moleculesof glucose6-phosphate.Of these6, 5 moles are regeneratedwith the productionof 'l 2 NA DP H . The overall reaction may be representedas 6 Glucose6-phosphate+ 12 NADP+ + 6H2O - - - - - s6C O 2 + ' 1 2 N A D P H + 1 2 H + + 5 C l ucose 6-phosphate. S ignif E c a n c e

o f H MP s h u n t

HM P s h u n t i s u n i q u e i n g e n e rati ngtw o important products-penfoses and NADPHneededfor the biosyntheticreactionsand other functions.

2 GSH (reduced)\ '*"-.-t / Glutathione peroxidase

H"o(

,/\ \

/'NADP' Glutathione reductase

o-s-s-ev/

/\

(oxidized)

\1rRopH* n-

Glutathione(reduced,GSH) detoxifiesH2O2, peroxidase catalysesthis reaction. NADPH is responsible for the regeneration of reduced glutathionefrom the oxidized one. 4. Microsomal cytochrome P+so system (in liver) brings about the detoxification of drugs and foreign compounds by hydroxylation reacti onsi nvol vi ngN A D P H .

5. Phagocytosisis the engulfmentof foreign parti cl es,i ncl udi ngmi croorgani sms, carri edou t In the HMP shunt,hexosesare convertedinto by white blood cells. The processrequiresthe pentoses, the most important being ribose suppl y of N A D P H . 5-phosphate.This pentoseor its derivativesare 6. Special functions of NADPH in RBC : useful for the synflresis of nucleic acids (RNA NADPH produced in erythrocyteshas special and DNA) and many nucleotidessuch as ATP, functions to Derform. lt maintains the NAD+, FAD and CoA. concentrationof reduced glutathione (reaction S k el e ta l mu s c l e i s c a p a b l e o f s ynthesi zi ng expl ai nedi n 3) w hi ch i s essenti al l yrequi redto pentoses,althoughonly the first few enzymesof preserve the integrity of RBC membrane. HMP shunt are active. lt, therefore,appearsthat NADPH is also necessaryto keep the ferrous (Fe2+)of hemoglobinin the reducedstateso the completepathway of HMP shunt may not be iron that accumul ati onof methemogl obi n(Fe3+ )is required for the synthesisof pentoses. prevented. lnnportance

of pentoses

Innportance

of NADBH

1 . NADPH is required for the reductive biosynthesis of fatty acids and sferoidg hence HM P s h u n t i s m o re a c ti v e i n t he ti ssues c onc er n e dw i th l i p o g e n e s i se, .g . a d i poseti ssue, liv er et c .

Glucose G-phosphate defieiency dehydrogenase G6PD deficiency is an inherited sex-linked trait. Although the deficiency occurs in all the cells of the affectedindividuals,it is more severe in RBC.

2. NADPH is used in the synthesisof certain H MP shunt i s the onl y means of provi di n g amino acids involving the enzyme glutamate NADPH in the erythrocytes.Decreasedactivity dehydrogenase. of C 6P D i mpai rs the synthesi sof N A D P H i n R B C . Thi s resul ts i n the accumul ati on o f 3. T h e re i s a c o n ti n u o u sp ro d u c ti onof H 2C -2 methemoglobin and peroxides in erythrocytes in t he li v i n g c e l l sw h i c h c a n c h e mi c al l ydamage leading to hemolysis. unsaturatedlipids, proteins and DNA. This is, however, prevented to a large extent through Clinical manifestations in C5PD deficiency : antioxidant reactionsinvolving NADPH. Cluta- Most of the patientswith C6PD deficiency do thione mediated reduction ol H2O2 is given in not usual l yexhi bi t cl i ni cal symptoms.S ome o f t he nex t c o l u m n . them, however, develop hemolytic anemia if

Glrapter 13 : METABOLISMOF CAFBOHYDFATES they are administeredoxidant drugs or exposed to a severe infection. The drugs such as pr im a q u i n e (a n ti m a l a ri a l ), a c e tani l i de (antipyretic), sulfamethoxazole (antibiotic) or ingestion of fava beans (favism) produce hemolytic jaundice in these patients. Severe infectionresultsin the generationof free radicals (in macrophages)which can enter RBC and cause hemolysis. G6PD deficiency and resistanceto malaria : It is interestingto note that G6PD deficiency is associatedwith resistanceto malaria (causedby Plasmodium falciparum). This is explained from the fact that the parasitesthat cause malaria are dependent on HMP shunt and reduced glutathione for their optimum growth in RBC. Therefore, G6PD deficiency-which is seen frequently in Africans-protects them from m alar ia,a c o mmo n d i s e a s ei n th i s re g i on. l t i s regardedas an adaptabilityof the people living in malaria-infectedregionsof the world. Wernicke-Korsakoff

syndrome

This is a genetic disorder associatedwith HMP shunt. An alteration in transketolase activity that reducesits affinity (by tenfold or so) with thiamine pyrophosphateis the biochemical lesion. The symptoms of Wernicke-Korsakoff syndrome include mental disorder, loss of memory and partial paralysis.The symptomsare manifestedin alcoholicswhose dietsare vitamindeficient.

275

participate,whereas,in uronic acid pathway,the free sugarsor sugar acids are involved. 1. Formation and importance of UDPglucuronate : Clucose 6-phosphate is first convertedto glucose1-phosphate.UDP-glucose is then synthesizedby the enzyme UDP-glucose pyrophosphorylase. Till this step, the reactions are the same as described in glycogenesis (Fig.l3Jg. UDP-glucose dehydrogenaseoxidizes UDP-glucoseto UDP-glucuronate. UDP-glucuronateis the metabolicallyactive form of gl ucuronate w hi ch i s uti l i zed for conj ugati onw i th many substances l i ke bi l i rubi n, steroid hormones and certain drugs. Several insoluble compounds are converted to soluble ones through conjugationand, further,the drugs are detoxified.UDP-glucuronateis also required for the synthesis of glycosaminoglycansand proteoglycans. 2. Conversion of UDP-glucuronate to L-gulonate : UDP-glucuronateloses its UDP moiety in a hydrolytic reaction and releasesDglucuronatewhich is reduced to L-gulonateby an NADPH-dependentreaction. 3. Synthesis of ascorbic acid in some animals : L-Culonate is the precursor for the synthesisof ascorbic acid (vitamin C) in many animals. The enzyme L-gulonolactoneoxidasewhich converts gulonate to ascorbic acid-is absent in man, other primatesand guinea pigs. Therefore,vitamin C has to be supplementedin the diet for these animals.

In pernicious anemia, erythrocyte trans4. Oxidation of L-gulonate : L-Gulonate is ketolaseactivitv is found to increase. then oxidized to 3-ketogulonate and decarboxylated to a pentose, L-xylulose. L-Xyluloseis convertedto D-xylulosevia xylitol followed by by a reduction(NADPH-dependent) an oxidation (NAD+-dependent) reaction.This is This is an alternativeoxidative pathway for necessary si nce the D -xyl ul ose (and not glucose and is also known as glucuronic acid L-form)-after getting phosphorylated-can enter pathway (Fig.l3.22). lt is concerned with the the hexose monophosphateshunt, for further synthesis of glucuronic acid, pentoses and metabolism. vitamin, ascorbic acid (except in primates and Effect of drugs guinea pigs).Dietary xyluloseentersuronic acid on uronic acid pathway pathway through which it can participate in Administration of drugs (barbital, chloroother metabolisms.In most of the pathwaysof phosphate esters butanol etc.) significantly increasesthe uronic carbohydrate metabolism,

276

BIOCHEMISTF|Y Glucose6-phosphate I Phosphoglucomutase I

acid pathwayto achieve more synthesis of glucuronate from glucose. Certain drugs (aminopyrine, antipyrine) were found to enhance the svnthesis of ascorbic acid in rats. Essential

+

Glucose1-phosphate ri rtr

"

pentosuria

This is a rare geneticdisorderrelated to the deficiencv of an NADPdependent enzyme xylitol dehydrogenase. Due to this enzyme defect, L-xylulose cannot be converted to xylitol. The affectedindividualsexcrete large amounts of L-xylulose in urine. Essential pentosuria is asymptomatic and the individuals suffer from no ill-effects.lt has been reportedthat the administrationof drugsaminopyrineand antipyrine increasesthe excretion, of L-xylulosein pentosuricpatients,

|

uDP-gtucose PYroPhosPhorylase ,i =;-.r( | v )

UDP-glucose 2N A D -\l \ UDPglucose ,i dehydrogenase zN A D Hr H -Y I

+

UDP-glucuronate

Hzo-rl I Ghcuronidase A UDPYI

L-Gulonate

The disaccharidelactose,presentin m ilk a n d mi l k p ro d u c ts ,i s th e p ri n c i pal I dietary source of galactose. Lactase CO"+'1 (p-galactosidase) of intestinal mucosal cells hydrolyseslactoseto galactoseand L-Xylulose glucose. Galactose is also produced tAnpl iN\AU T n ] *+ within the cells from the lysosomal {tn" deftydrogenasq degradation of glycoproteins and NAD 1) glycolipids.As is the case for fructose, galactose entry into the cells is not Xylitol depe n d e n to n i n s u l i n . N A D * \l I The specific enzyme, namely l galactokinase,phosphorylatesgalactose NADH+ H-y'1 v to galactose 1-phosphate.This reacts D-Xylulose with UDP-glucose in an exchange reaction to form UDP-galactose in + Xylulose5-phosphate presence of the enzyme galactose 1-

-+

L-Gulonolactone 'i,,.; L-Gulonolactone oxidase 2) 2-Keto-L-gulonolactone

II

+ L-Ascorbicacid

I phosphate uridyltransferase(Fig.l3.23). + UDP-galactoseis an active donor of Hexose monophosphate shunt galactose many for synthetic Fig. 13.22 : Uronic acid pathway [UDP-uidine diphosphate); reactions involving the formation of (1) Block in essential pentosuria; compounds like lactose, glycosamino(2) Enzyme absent in pimates (including man) and guinea pigsl. glycans,glycoproteins,cerebrosides and

Ghapter 13: METABOLISM OF CAFBOHYDHATES

277

Galactose Aldose reductase Galactitol

Galactose1-phosphate---.r

i r-UDP-glucose\

\/\ Galactos phosphate UDp-hexose 4-ePimerase uridYltransferase ,/\ / / \ Glucose1-phosphatey' UOe-gatactose I I Phosphog mutasl I + Glucose6-phosphate

\+

\ y J Glycolysis Glucose

glycolipids.UDP-galactosecan be converted.to In this UDP-glucoseby UDP hexose4-epimerase. way, galactosecan enter the metabolicpathways of glucose. lt may be noted that galactoseis not an essentialnutrient since UDP-glucosecan be by the enzymeUDPconvertedto UDP-galactose hexose4-epimerase.

DISORDERSOF GALACTOSE METABOLISM Glassical galactosemia

Lactose Glycosaminoglycans (in mammarygland) Glycolipids Glycoproteins

However, with increased levels of galactose (galactosemia), this pathway assumes significance.Galactitol (like sorbitol, discussed later)has been implicatedin the developmentof cataract. galactose 3. The accumulation of 1-phosphateand galactitolin varioustissueslike liver, nervous tissue, lens and kidney leads to i mpai rmenti n thei r functi on. galactose 4. The accumulation of 1-phosphatein liver resultsin the depletion of inorganicphosphate(sequestering of phosphate) for other metabolic functions.

Calactosemiais due to the deficiency of the enzyme galactose 1-phosphate uridyltransferase. lt is a rare congenital disease in 5. The cl i ni cal symptoms of gal actosemi a infants, inherited as an autosomal recessive are-loss of weight (in infants) hepatodisorder.The salientfeaturesof galactosemiaare splenomegaly,jaundice, mental retardationetc. Iisted. In severe cases, cataract, amino aciduria and 1. Calac to s eme ta b o l i s mi s i mp a i re dl eadi ng al bumi nuri aare al so observed. to increased galactose levels in circulation (galactosemia) and urine (galactosuria).

Diagnosis: Earlydetectionof galactosemiais possible (biochemical diagnosis)by measuring the activity of galactose 1-phosphate uridyltransferasein erythrocytes.

2. The accumulatedgalactoseis diverted for the production of galactitol (dulcitol) by the enzyme aldose reductase(the same enzyme that Treatment : The therapy includesthe supply convertsglucoseto sorbitol).Aldose reductaseis of diet deprived of galactose and lactose. presentin lens, nervoustissue,seminal vesicles Galactokinase deficiency : The defect in etc. The conversionof galactoseto galactitol is insignificant in routine galactosemetabolism. the enzyme galactokinase, responsible for

BIOCHEMISTF|Y

278

, i, / x t SorbitolQl{^-Glucose...' AIOOSe ^

Glucose 6-phosphate

ATP Fructokinase ATP (1 ) Fructose1-phosphate AldolaseB

(2)

Triokinase NADH+ H+

Alcohol dehydrogenase

Triosephosphate tsomerase

NAD-

Glycerol

Glycerol kinase

Glycolysis (pyruvate)

Glycerol 3-phosphate . dehvdrooenase ciryceror+DHAP 3-phosphate ..J L.

TriacylglycerolsPhospholipids Fig. 13.24 : Metabolism of fructose (Metabolic defects 1-Fructosuria; 2-Fructose intolerance). (Note: The shaded part representsthe polyol pathway)

phosphorylationof galactose,will also result in is also found in free form in honey and many galactosemia and galactosuria. Here again fruits. ln the body, entry of fructoseinto the cells galactose is shunted to the formation of i s not control l edby the hormonei nsul i n.This is galactitol. Cenerally, galactokinase-deficient in contrastto glucose which is regulatedfor its individuals do not develop hepatic and renal entry into majority of the tissues. complications.Developmentof cataractoccurs Fructoseis mostly phosphorylatedby fructoat a very early age, sometimeswithin an year kinaseto fructose1-phosphate.Fructokinasehas after birth. The treatment is the removal of been identified in liver, kidney and intestine. galactose and lactose from the diet. Hexokinase, which phosphorylates various monosaccharides,can also act on fructose to produce fructose 6-phosphate. However, hexokinase has low affinity (hish K.n) for fructose,hence this is a minor pathway. The major dietary source of fructose is the Fructose1-phosphateis cleaved to glyceral(cane sugar), containing dehyde and dihydroxyacetone phosphate disaccharide sucrose equimolar quantitiesof fructoseand glucose. lt (DHAP) by aldolase B (Fi9.13.24. This is in

METABOLISMOF CAFBOHYDRATES contrast to fructose 6-phosphate which is and split convertedto fructose1, 6-bisphosphate by aldolase A (details in glycolysis-See Fig.l3.2). Clyceraldehydeis phosphorylatedby the enzyme triokinase to glyceraldehyde 3phos phat e w h i c h , a l o n g w i th D H AP , enters gly c oly s isor g l u c o n e o g e n e s i s .

279

Ingestion of large quantities of fructose or many heal th sucrose i s l i nked w i th compl i cati ons. Sslrfu[tef I P$lyc]! pathwfiy

Polyol pathway (so termed since sorbitol is a polyhydroxy sugar) basically involves the The fructoseis more rapidly metabolized(via conversion of glucose to fructose via sorbitol gly c oly s isb) y th e l i v e r th a n g l u c o s e .T h i s i s due (Fig.l3.2a). This pathway is absent in liver. to the fact that the rate limiting reaction in Sorbitol pathway is directly related to glucose glycolysis catalysed by phosphofructokinaseis concentration, and is higher in uncontrolled bypassed.Increaseddietary intake of fructose diabetes. significantly elevates the production of acetyl The enzymealdosereductasereducesglucose CoA and lipogenesis(fatty acid, triacylglycerol and v er y l o w d e n s i ty l i p o p ro te i n s y n t hesi s). to sorbitol (glucitol)in the presenceof NADPH.

BIOMEDICAT/ CLINICAL CONCEPT9

A continuous presence of glucose---+upplied through diet or synthesized in the body (gluconeogenesisFis essential t'or the suruiual of the organism, Alcohol intoxication reducesgluconeogenesis. Human brain consumes about 120 g of glucose per day out of the 760 g needed by the body. lnsufficient supply of glucose to brain may lead to coma and death. Liuer glycogen sen)esas an immediate source for maintaining blood glucose leuels, particularly between the meols. The glycogen sfores in the liuer get depleted after 7218 hours ol fasting. Muscle glycogen is primarily concerned with the supply of hexoses thot undergo g/ycolysis to prouide energg during muscle contraction. by deposition of normol or abnormal type of Glycogen storage diseoses----charocterized glycogen in one or more fissues-result in muscular weakness,or euen death. The occurrence of HMP shunt (NADPH production) tn the RBC is necessaryto rnaintsin the integrity of erythrocyte membrane and to pretlent the occumulation of methemoglobin. Deficiency ol glucose 6-phosphate dehydrogenase results in hemolysis of RBC, cousing hemolgtic anemio. The subjects of G6PD deficiency are, howeuer, resistant to maloria. lJronic acid pathwag is concerned with the production of glucuronic acid (inuolued in detoxification), pentosesand uitamin C. Man is incapoble of synthesizing uitomin C due to the absenceof a single enzyme-L-gulonolactone oxidase. The conuersion of glucose to fructose is impaired in diabetes mellitus, cousing occumulation ol sorbitol. This compound hos been implicated in the deuelopment of cataract, nephropathy, peripheral neutopathy etc. Seuere casesof golactosemiaare associated with the deuelopment of cataract, believed to be due to the accumulation ol galactitol.

280

B IOC H E MIS TR Y

Sorbitol is then oxidized to fructose by sorbitol dehydrogenaseand NAD+. Aldose reductase is absent in liver but found in many tissues like lens and retina of the eye, kidney, placenta, Schwann cells of peripheral neryes,efihrocytes and seminal vesicles. The enzyme sorbitol dehydrogenaseis present in seminal vesicle, spleen and ovaries. Fructose is a preferred carbohydratefor energy needsof sperm cells due to the presenceof sorbitol pathway.

fructose is not converted to fructose 1-phosphate. This is an asymptomaticcondition with excretion of fructose in urine. Treatment involves the restriction of dietary fructose.

2. Hereditary fructose intolerance : This is due to the absence of the enzyme aldolase B. intolerance causes Hereditary fructose .l intracellular accumulation of fructose -phosphate, severe hypoglycemia,vomiting, hepatic failure and jaundice. Fructose 1-phosphate al l osteri cal l yi nhi bi ts l i ver phosphoryl asea nd in Sorbitol pathway blocks glycogenolysisleading to hypoglycemia. diabetes mellitus Early detection and intake of diet free from In uncontrolled diabetes (hyperglycemia), fructose and sucrose, are advised to overcome large amounts of glucose enter the cells which fructose intolerance. are not dependenton insulin. Significantly,the 3. Consumption of high fructose : Fructoseis cells with increasedintracellularglucose levels rapidly converted to fructose 1-phosphateby in diabetes(lens,retina,nerve cells, kidney etc.) The activity of the enzyme aldolase fructokinase. possess high activity of aldose reductase and B is relatively less, and, due to this, fructose sufficient supply of NADPH. This results in a 1-phosphate accumulates in the cell. This leads rapid and efficient conversion of glucose to inorganic of intracellular to the depletion sorbitol. The enzyme sorbitol dehydrogenase, (Pi) levels. The phenomenon of phosphate however, is either low in activity or absent in (like these cells, hence sorbitol is not converted to bi ndi ng of P i to the organi c mol ecul es leads less availabilitv fructose here)-that to the fructose. Sorbitol cannot freely pass through the cell membrane, and accumulate in the cells of Pi for the essentialmetabolic functions-is where it is produced. Sorbitol-due to its known as sequestering of phosphate. Due to the hydrophilic nature-causes strong osmotic decreasedavailability of Pi, which happens in fructose, the liver effectsleadingto swelling of the cells. Some of overconsumption of This includes metabolism is adverselyaffected. the pathological changes asmciated with the lowered synthesisof ATP from ADP and Pi. (like peripheral diabefes cataract formation, period neuropathy,nephropathyetc.) are believed to be High consumptionof fructoseover a long in blood due to the accumulation of sorbitol, as is associatedwith increaseduric acid gout. This is to the excessive leading to due explained above. breakdown of ADP and AMP (accumulateddue It is clearly known that in diabetic animals to lack of Pi) to uric acid. sorbitolcontentof lens,nerve,and glomerulusis elevated. This causes damage to tissues. lt thus appears that majority of the complications associated with diabetes share a common pathogenesis as a consequence of polyol When a hydroxyl group of a sugaris replaced pathway. Certain inhibitors of aldose reductase by an amino group, the resultantcompound is can prevent the accumulation of sorbitol, and an ami no sugar. thus the associatedcomplications.However,this approach is still at the experimental stage. The important amino sugarsare glucosamine, galactosamine, mannosamine, sialic acid etc. They are essential components of glyco1. Essential fructosuria : Due to the saminoglycans,glycolipids (gangliosides)and deficiency of the enzyme hepatic fructokinase, glycoproteins. They are also found in some Defects

in fructose

metabolism

281

Chapter 13 : METABOLISMOF CAFIBOHYDBATES Glucosamine Glucose I I

+

I I

Glutamine

6-flH3i;:,"o5["fJ3fi3," -

Glutamate J Glucosamine 6-phosphate-l

Glucosamine 1-phosphate

II

ncevr con I

I

N-AcetyF

N-Acetyl-

N-Acetyl-

UDP-N-

oligosaccharidesand certain antibiotics. lt is estimated that about 2Oo/" of the glucose is utilized for the synthesisof amino sugars,which mostly occurs in the connective tissue.

UDPglucosamine

Glycosamino-

may result in skeletal deformities, and mental are important retardation.Mucopolysaccharidoses for elucidatingthe role of lysosomesin health and disease.

CAGs are degraded by a sequentialaction of The outline of the pathway for the synthesis of amino sugars is given in Fi9.13.25. Fructose Iysosomal acid hydrolasese.g. exoglycosidases, mucopolyimportant 6-phosphate is the major precursor for sulfatases. Some glucosamine, N-acetylgalactosamine and saccharidosesand the enzyme defectsare listed. acid (NANA).The utilization N-acetylneuraminic Hurler's syndrome (MPS l)-L-lduronidase for the formation of sugars the amino of and Hunter's syndrome (MPS Il)-lduronate sulfatase glycosaminolgycans, glycoproteins gangliosidesis also indicated in this figure. Sanfilippo syndrome (MPS lll)-Four differt enzymes (e.g. heparan sulfamidase) Mucopolysaccharidoses SIy syndrome (MPS Vll)-p-Glucuronidase. The lymsomal storage diseases caused by defects in the glycosaminoglycans(GACs) are The diseases known as mucopolysaccharidoses. name is so given since the original name for (MPS).Theseare GACs was mucopolysaccharides The animals,including man, cannot carry out more than a dozen rare genetic diseases. Mucopolysaccharidosesare characterizedby the the net synthesis of carbohydrate from fat. accumulation of GACs in various tissuesthat However, the planfs and many microorganisms

E|IOCHEMISTF|Y

282 are equipped with the metabolic glyoxylate the machinery-namely into fat convert cycle-fo carbohydrates. This pathway is very significant in the germinating seeds where the stored triacylglycerol (fat) is converted to sugars to meet the energy needs.

Faity acid

I

Y AcetylCoA

Location of the cycle : The glyoxylate cycfe occurs in glyoxysomes,specialized cellular organelles, where fatty acid oxidation is also operative. Reactions of the cycle : The glyoxylate cycle (Fig.13.26) is regarded as an anabolic variant of citric acid cycle. Acetyl CoA produced from fatty condenses with acid oxidation oxaloacetateto give citrate which is then converted to isocitrate. At this stage, isocitrate bypassesthe citric acid cycle and is cleaved by isocitrate lyase to succinate and glyoxylate. Another molecule of acetyl CoA is now utilized to combine with glyoxylate to form malate. This reaction is catalysed by malate synthase and the malate so formed enters citric acid cycle. The glyoxylate cycle is a cyclic pathway that results in the conversion of two 2-carbon fragmentsof acetyl CoA to 4-carbon compound,

Fig. 13.26 : The glyoxylate cycle (enzymes reprcsented in green shade).

succinate. The succinate is converted to oxaloacetate and then to glucose involving the reactions of gluconeogenesis.

Ghapter 13 ; METABOLISMOF CARBOHYDRATES

283

7. Csrbohydrates are the major source ol energy for the ltuing cells. Glucose (normal lasting blood leuel 70-100 m7/dl) is the central molecule in carbohydrate metabolism, actiuely participating in a number of metabolic pathways--glycolysis, gluconeogenesls, glycogenesis, glgcogenolysis, hexose monophosphate shunt, uronic ocid pathway etc. 2. Glucose is oxidized in glycolysis, either in anaerobic (2 ATP formed) or aerobic (8 ATP formed) conditions, resulting in the lormation of 2 moles ol lactate or pyruuate, r*pectioely. 3. Acetyl CoA is produced from pyruuate which is completely oxidized in citric acid cycle, the final common oxidative pathwoy lor all foodstuffs. The complete oxidation of one mole ol glucose generotes 38 ATP. 4. Gluconeogenesis is the synthesis of glucose from noncarbohydrate precursors like amino acids (except leucine and lysine), Iaetate, glgcerol, propionate etc. The reuersal of glycolgsis with olternate arrangements mode ot three irreuersible reactions ol glgcol ysis constitutes gluconeogenesis. 5. Glycogen is the storogeform of glucose. The degradation of glycogen (glgcogenolysis)in musclemeets the immediatefuel requirements,whereasthe liuer glgcogenmaintainsthe blood glucoseleuel. Enzyme defects in synfhesisor degradationol glycogen lead to storage disorders. uon Gierke's diseose (TVpe I) is due to the defect in the enzyme glucose 6-phosphatase. 6. Hexose monophosphate shunt (HMP shunt) is the direct oxidatiue pathway of glucose. HMP shunt ossumes significance since it genercites NADPH and pentoses, respectiuely required for the synthesisot' lipids and nucleic acids. 7. Glucuronate-inuolued in the conjugation of bilirubin, steroid hormones and detoxification of drugs-is synthesizedin uronic acid pathwog. Due to a singleenzyme det'ect (gulonolactone oxidase) in fhis pathwoy, man cannot synthesize ascorbic acid (uitamin C) wheress some onimals can. 8. Goloctosemio is mostly due to the delect in the enzyme galactose 7-phosphate uridyltranst'erase.This re.sultsin the diuersionot' galactoseto produce galactitol which has been implicated in the deuelopment of cataract. 9. Glucose can be converted to Jructose uia sorbitol pathway. In prolonged hgperglgcemia (uncontrolled diabetes), sorbitol accumulates in the fissues, resulting in cataroct, nephropathy, peripherol neuropathy etc. 10. Amino sugors (glucosomine, galactosamine, mannosamine etc.), synthesized from fructose 6-phosphate are essential components of glgcosaminoglycans,glgcolipids and glycoproteins.

BIOGHEMISTRY

284

I. Essayquestions 1. Describebriefly the metabolismof glucose6-phosphate. 2. Cive an account of glycogenmetabolism. 3. Justify that citric acid cycle is the final common metabolic pathway for the oxidation of foodstuffs. 4. Discussthe synthesisof glucosefrom non-carbohydratesources. 5. Describethe hexosemonophosphateshunt and add a note on its significance. II. Short notes (a) Clycogenolysis,(b) UDPC, (c) Calactosemia,(d) Cori cycle, (e) 2, 3- BPG, (0 Clycogen storage diseases,(g) Essentialfructosuria,(h) Conversionof pyruvateto acetyl CoA, (i) Energeticsof TCA cycle, (j) TPP in carbohydratemetabolism. III. Fill in the blanks dehydrogenase or cr-ketoglutarate 1. Name the five vitamins required.by pyruvatedehydrogenase complex 2. Muscle glycogendoes not directly contributeto blood glucosedue to absenceof the enyme 3. Ascorbic acid is not synthesizedin man due to lack of the enzyme 4. The compoundimplicatedin the developmentof cataractin diabeticpatientsis 5. Galactosemiais mostly due to the deficiencyof the enzyme and 6. The two amino acids that are never glucogenicare 7. Substratelevel phosphorylationin citric acid cycle is catalysedby the enzyme 8. The metabolic pathway concerned with the conversion of L-xylulose to D-xylulose is 9. The name of the protein that has been identifiedto serveas a primer for glycogensynthesisis 10. The metabolite among the citric acid cycle intermediatesperforming a catalytic role IV. Multiple choice questions reaction. 11. One of the followingenzymesin glycolysiscatalysesan irreversible (d) (c) All of them. (a) Hexokinase(b) Phosphofructokinase Pyruvatekinase occurs in the tissuenamely. 12. Synthesisof 2, 3-bisphosphoglycerate (a) Liver (b) Kidney (c) Erythrocytes(d) Brain. 13. The hormonethat lowers cAMP concentrationin liver cells is (a) Glucagon(b) Insulin(c) Epinephrine(d) Thyroxine. 14. The number of ATP produced when a molecule of acetyl CoA is oxidized through citric acid cycle ( a ) 1 2 (b ) 2 4 (c ) 3 8 (d ) 1 s . 15. The connectinglink betweenHMP shunt and lipid synthesisis (a) Ribose(b) NADPH (c) Sedoheptulose7-phosphate(d) NADH.

ipids are indispensablefor cell structureand I l-function. Due to their hydrophobicand nonpolar nature, lipids differ from rest of the body compounds and are unique in their action. T r iac yl s l y c e ro l s *tlre body fuei reserve Lipids constitute about "15-20%of the body weight in humans. Triacylglycerols (formerly triglycerides) are the most abundant lipids comprising85-90% of body lipids. Most of the triacyfglycerols(TC; also called neutral fat or depot fat) are stored in the adipose fissue and serve as energy reserve of the body. This is in contrast to carbohydratesand proteins which cannot be storedto a significantextent for energy purposes.Fat also acts as an insulatingmaterial for maintainingthe body temperatureof animals.

1. Triacylglycerols(TC) are highly concentrated form of energy, yielding 9 Cal/g, in contrast to carbohydrates and proteins that produce only 4 Callg. This is becausefatty acids found in TG are in the reduced form. 2. The triacylglycerols are non-polar and hydrophobic in nature, hence sfored in pure form without any association with water (anhydrous form). On the other hand, glycogen and proteins are polar. One gram of glycogen combines with 2 g of water for storage.

For the two reasonsstatedabove, one gram of fat stored in the body yields nearly six times as much energy as one gram of (hydrated) glycogen.In a healthyadult individual(weighing 70 kg), about 10-11 kg of fat is stored (mostly in adipose tissue) which corresponds to a fuel reserveof 100,000 Cals. lf this much of Why slrouHril '$a[ -lre the f***i energy were to be stored as glycogen (insteadof reserve of tfee body? fat), then the weight of the person would Triacylglycerolsare the most predominant increaseby at least55 kg! This explainswhy fat storage form of energy. There are two main has been chosen as a fuel reserve during reasonsfor fat being the fuel reserveof the body evolution.

28s

ElIOCHEMISTFIY

286 Long chain fatty acids (of faQ are the ideal storage fuel reserves of the body. Fats can support the body's energy needsfor long periods of food deprivation. In extreme cases, humans can fast and survive for 60-90 days, and the obese personscan survive even longer (6 months to one year!) without food.

Lipid fraction

Referencevalues(mg/dl)

Totallipid Totalcholesterol LDL-cholesterol HDL-cholesterol VLDL-cholesterol Triglycerides Phospholipids Freefattyacids

400-600 151200 80-150 3H0 20-4;0 7F150 150-200 5-15

Hibernating animals provide good example for utilizing fat reserve as fuel. For instance, bearsgo on hibernationfor about 7 monthsand, during this entire period, the energy is derived from the degradation of fat stores. The rubythroated humming birds fly non-stop between New Englandand West Indies (2,400 km!) at a speed of 40 km/hr for 60 hours! This is possible metabolically.However, later experimentswith only due to the stored fat. isotopestudieshave proved that the body lipids body lipids Other important are continuously being degraded and glycolipidsand cholesterolare resynthesized.As already stated,fat stored in the Phospholipids, major components of cell membranes. adipose tissue is the fuel reserveof the body. Cholesterolis also a precursorfor bile acids and This is in a dynamic state. steroid hormones. Arachidonic acid-an The triacylglycerolstransportedfrom intestine unsaturated fatty acid-is the substrate for the (as chylomicrons)and liver (as VLDL) are stored synthesis of certain intercellular regulators- in the adipose tissue. Besides,they are also prostaglandi ns, th romboxanes,prostacycli ns etc. utilized by muscle, liver, heart etc., as per the needs of the body. An overview of fat Transport of Iipids metabolismis depicted in Fig.l4.l. The insolublelipids are solubilizedin association with proteins to form lipoproteins in which form lipids are transportedin the blood stream. Free lipids are undetectablein blood. ol Synlhesis of Svnthesis from chyld,microns lipoproteins Chylomicrons,very low density lipoproteins dietarylipids (VLDL) (VLDL), low density lipoproteins (LDL), high Small intestine densitylipoproteins(HDL) and albumin-freefatty acids are the differentlipoproteincomplexesthat 'w i t h transport lipids in the blood stream. Details of VLDLwithTG\ / transportedI plasma lipoproteins and their metabolism are J TGtransported discussed later. F las m a

lip i d s

The various fractionsof lipids in the plasma can be estimated by different methods after extractingthem with lipid solvents.The plasma levels of lipids (Table l4.l) are often useful for assessingthe health of the individuals. Dynamic

state

of body

lipids

ft was earlier thought that the lipids are inert storage compounds and are less significant

Triacylglycerols andfattyacidsutilized Muscle,liver,heartetc. Fig. l4.l : Overuiewof fat metabolism.

287

OF LIPIDS chapter'14: METABOLISM

o

tl

cH2-c-o-R1 ? R2-c-o-?H cH2-c-o-R3

Lipotysis + 3H2O

o

Triacylglycerol

Mobilization of tat frcnn adipose tissue Triacylglycerol(TC) is the stored fat in the adipose tissue. The enzyme/ namely hormonesensitive triacylglycerol lipase, removes the fatty acid either from carbon 1 or 3 of the triacylglycerolto form diacylglycerol.The other two fatty acids of TC are cleaved by additional lipases specific for diacylglycerol and monoacylglycerol.The complete degradationof triacylglycerol to glycerol and free acids is known as lipolysis (FigJa.2).

cH2-oH HO-CH cH20H Glycerol

+

RlcooH R2cooH R3COOH Freefatty acids (3)

Fateof glycerol : The adiposetissuelacksthe enzyme glycerol kinase, hence glycerol produced in lipolysis cannot be phosporylated here. lt is transported to liver where it is activatedto glycerol3-phosphate.The lattermay be used for the synthesisof triacylglycerolsand phospholipids.Clycerol 3-phosphatemay also enter glycolysis by getting converted to d i hydroxyacetone phosphate (Fig.14.4).

Fate of free fatty acids : The fatty acids releasedby lipolysisin the adipocytesenter the circulation and are transportedin a bound form to albumin. The free fatty acids enter various tissues and are utilized for the energy. About Regulation of hormone-sensitive 95V' of the energy obtained from fat comes from TG-lipase the oxidation of fatty acids. Certain tissues, T C -l i p a s e i s s o named Hor m o n e -s e n s i ti v e however, cannot oxidize fatty acids, e.g. brain, because its activity is mostly controlled by ervthrocvtes. hormones.Lipaseis presentin an inactiveform 'b' and is activated (phosphorylated)by a cAMP dependent protein kinase to lipase 'a'. Several hormones-such as epinephrine (most effective), nor epine p h ri n e , g l u c a g o n , th y ro x i n e, A C TH The fatty acids in the body are mostly etc.- enhancethe activity of adenylatecyclase oxidized by B-oxidation. p-Oxidation may be . n th e o th er hand, defined as the oxidation of fatty acids on the and, t hus , i n c re a s el i p o l y s i s O insulin decreases cAMP levels and thereby p-carbon atom. fhis results in the sequential inactivateslipase.Caffeinepromoteslipolysisby removal of a two carbon fragment, acetyl CoA. increasingcAMP levelsthrough its inhibition on phosphodiesterase activity.The control of cAMP Fatty acid oxidation mediated lipolysis is illustratedin Fig.|4.3. and tissues -stages As is evident from the foregoing discussion, The p-oxidation of fatty acids involves three increased levels of cAMP promote lipolysis. ln stages contrast, cAMP decreasesfatty acid synthesisby l. Activation of fatty acids occurring in the inhibiting acetyl CoA carboxylase activity cytosol; (discussedlater). lt should be thereforekept in ll. Transportof fatty acids into mitochondria; m ind t ha t l i p o l y s i s a n d l i p o g e n e s i sare not lll. B-Oxidation proper in the mitochondrial simultaneouslyoperative (i.e. futile cycles are matrix. avoided. Refer p. 258).

288

B IOC H E MIS TR Y

Epinephrine Norepinephrine Thyroxine Glucagon, Glucocorticoids TSH,ACTH,GH

Adenylatecyclase

Hormone sensitive TG lipaseb (inactive)

Insulin,niacin,PGE

cAM Ir

lnsulin-o____+l cafieine-e

-

Protein kinase

Phosphatase

I Dhosphodiesterase )l..'' 5 ' AMP

Hormonesensitive TG lipasea (active)

--? Triacylglycerol

I

Diacylglycerol

Freefattyacid

rrn }-+ Monoacylglycerol

I

HFFA Glycerol Flg. 1L? : Controlof liplysis in adiposetissuethroughcyclicAMP (@-Promotingand O-lnhibiting effect; hormone; GH4 rowth hormone;

Fattyacids are oxidized by most of the tissues Il, Transtr*ort o# ailyi C.oS in the body. However, brain, erythrocytes and i nto mi tochondrda adrenal medulla cannot utilize fatty acids for The i nner mi tochondri al membrane i s energy requirement. impermeable to fatty acids. A specialized carnitine carrier system (carnitine shuttle) r. "*";ntEyaeid actffuation operates to transport activated fatty acids from Fatty acids are activated to acyl CoA by cytosol to the mitochondria.This occurs in four thiokinases or acyl CoA synthetases. The steps (Fig.l4.6). reaction occurs in two steps and requiresATP, 1. Acyl group of acyl CoA is transferredto coenzyme A and Mg2+. Fatty acid reacts with (B-hydroxyT-trimethyl aminobutyrate), carnitine ATP to form acyladenylatewhich then combines by carnitine acyltransferase I (present catalysed with coenzyme A to produce acyl CoA the surface inner mitochondrial on outer of (Fig.l4.i't. In the activation, two high energy phosphates are utilized, since ATP is converted membrane). 2. The acyl-carnitineis transportedacrossthe to pyrophosphate (PPi). The enzyme inorganic pyrophosphafase hydrolyses PPi to phosphate membraneto mitochondrialmatrix by a specific (Pi).The immediateeliminationof PPi makesthis carrier protein. reactiontotally irreversible. 3. Carnitineacyl transferasell (found on the Three different thiokinases,to activate long inner surfaceof inner mitochondrialmembrane) c hain ( 1 0 -2 0 c a rb o n ), m e d i u m c h a i n (4-12 convertsacyl-carnitineto acyl CoA. carbon) and short chain (< 4 carbon) fatty acids 4. The carnitine releasedreturns to cvtosol have been identified. for reuse.

Chapter 14 : METABOLISMOF LIPIDS

cH2-oH HO_CH

cH2-oH Glycerol

Glycerokinase

cH2-oH HO_CH

cH2-o-o Glycerol3phcphab

l l l " p" Oxi dati on

proper

Each cycle of p-oxidation, liberating a two carbon unit-acetyl CoA, occurs in a sequenceof four reactions (Fig.l 4.7). 1. Oxidation : Acyl CoA undergoes dehydrogenation by an FAD-dependent flavoenzyme, acyl CoA dehydrogenase. A double bond is formed between o and p carbons (i .e.,2 and 3 carbons). 2. Hydration : Enoyl CoA hydratase brings about the hydration of the double bond to form p-hydroxyacyl CoA. 3. Oxidation : p-Hydroxyacyl CoA dehydrogenasecatalysesthe second oxidation and generates NADH. The product formed is p-ketoacyl CoA. 4. Cleavage : The final reaction in p-oxidation is the liberation of a 2 carbon fragment, acetyl CoA from acyl CoA. This occurs by a thiolytic cleavage catalysed by p-ketoacyl CoA thiolase (or simply thiolase).

Synthesisof triacylglycerols, phospholipids

The new acyl CoA, containing two carbons less than the original, reentersthe p-oxidation cycle. The processcontinues till the fatty acid is completely oxidized.

R-CH2-CH2-GOOFatty acid Fig. 14.4: Fate of glycerol. Pyrophosphatase

It should be noted that the coenzyme A used for activation is different from the one that finally combines with fatty acid in the mitochondria to form acyf CoA. Thus, the cell has two separate pools (cytosolic and mitochondrial) of coenzyme A. Inhibitor of carnitine shuttle : Carnitine acyl transferase I is inhibited by malonyl CoA, a key metabolite involved in fafty acid synthesisthat occurs in cytosol (details given later). In other words, while the fatty acid synthesis is in progress (reflected by high concentration of malonyl CoA), their oxidation does not occur, since carnitine shuttle is impaired.

2P i

R-CH2-CH2-c_AMP Acyladenylate

ll R-CH2-CH2-C-CoA Acyl CoA Fig. 14.5 : Activation of fatty acid to acyl CoA by the enzyme thiokinase.

BIOCHEMISTFIY

290 Olcd

otl

Carnitine

R-C-SCoA AcylCoA

ot l

R-C-SCoA AcylGoA

Carrier protein

Carnitineacyltransferase I

A

/ CoAsH/

/\

o

\ \

n-8-"arnitine Acyl-carnitine

Fig. 14.6: Carnitineshuttlefor

of activated fafty acid (acyl CoA) into mitochondria'

The overall reaction for each cycle of p-oxidation

The standardfree energy of palmitate = 2,34O C al .

Cn Acyl CoA + FAD + NAD+ + H2O + C6-zt Acyl CoA + AcetYl CoA + CoASH + F A DH2 + N AD H + H + .

The energy yield by its oxidation-"|2g (129 x 7.3 Cal) = 940 Cal'

ATP

The efficiency of energy conservationby fatty The schemeof fatty acid oxidation discussed 940 x 100 = 4Oo/o. above correspondsto saturated(no double bond) acid oxidation = 2,340 and even carbon fatty acids. This occurs most predominantlyin biological system. Oxidatisn

of palmitoyl

CoA

The summaryof p-oxidationof palmitoylCoA is shown below Pafmitoyl CoA + 7 CoASH + 7 FAD + 7 NAD+ + 7H2C----> 8 Acetyl CoA + 7 FADH2 + 7 NA DH + 7 H + Palmitoyl CoA undergoes 7 cycles of p-oxidation to yield I acetyl CoA. Acetyl CoA can enter citric acid cycle and get completely oxidized to CO2 and H2O. Energetics

of B-oxidation

The ultimate aim of fatty acid oxidation is to generate energy. The energy obtained from the complete oxidation of palmitic acid (16 carbon) is given in Table 14.2 and Fig.l4.8.

Mechanism

ATP vield

l. p0xidation7 cycles transport byelectron 7 FADH2 [oxidized gives2 ATPI eachFADH, chain(ETC), bYEIC,eachNADH 7 NADH(oxidized 3 ATP) liberates ll. From8 acetylCoA bycitricacid cycle,eachacetyl Oxidized 12ATP CoAprovides CoA Totalenergyfromonemoleof palmitoyl foractivation utilized Energy (tormation CoA) of palmitoyl

14 21

131

-2

129 of palmitate of one molecule Netvieldof oxidation

Chapter 14 : METABOLISMOF LtPtDS

297

o

il R-CH2-CH2-CH2-C-OFatty acid

ott\l-coASH -

Ms"y

Thiokinase I !ro tl R-CH2-CH2-CH2-C-SCoA AMP + PPiy'l

Acyl CoA

|I

tr.

Ca:'nitineti ansportsystem

1a.

I

"yrosol

MrocHoNDRloN

p *o

?

-CH2-CH2-CH2-C-SCoA Acyl CoA

FAD-! (1),1 oerivo'rAd;is€ ^cytc{A Q]..-rnonz+{ oHo W '

ttl -CH2-CH:CH-C-SCoA A2 traneenoyl CoA Hzo._ (2)

OH ttl -CH2-CH-CH2-C-SCoA pHydroxyacyl CoA " IADH+

f"*roxyacyl CoA dehydrogenase

o -cH2-c-cHr-8-s"oo ftKetoacyl CoA

lr

ucAs!-l-l Thiorase\\ (4)l

o+o

-C-SCcn + Cn3-8Acyl CoA (-2C)

Ac€ry

Flg. 14.7 : p-Oxidation of fatty acids : Palmitoyl CoA (16 carbon) undergoes seven cycles to yield I acetyl CoA fl-Activation; ll-Transport; lll-p Oxidation proryr(1) Oxidation, (2) Hydntion, (3) Oxidation and (4) Cleavagel.

Fig. 14.8 : An overview of oxidation of palmitic acid.

SIDS-a disorder due to blockade in j-l.oxidation The sudden infant death syndrome (SIDS) is an unexpecteddeath of healthy infants,usually overnight.The real causeof SIDS is not known. It is now estimatedthat at least 10% of SIDS is due to deficiency of medium chain acyl CoA dehydrogenase. The enzyme defect has a frequencyof 1 in 10,000 births and is, in fact, more prevalent than phenylketonuria. The occurrenceof SIDS is explained as follows Glucose is the principal source of energy, soon after eating or feeding babies. After a few hours, the glucose level and its utilization decreaseand the rate of fatty acid oxidation must simultaneously increase to meet the energy needs. The sudden death in infants is due to a blockade in p-oxidation caused by a deficiency in medium chain acyl CoA dehydrogenase (MCAD). Jamaiean

vomitinE

siekness

This disease is characterized bv severe hypoglycemia,vomiting, convulsions,coma and death. lt is caused by eating unripe ackee fruit which contains an unusual toxic amino acid, hypoglycin A. This inhibits the enzyme acyl

292

BIOGHEMISTF|Y

CoA dehydrogenase and thus p-oxidation of fatty acids is blocked, leading to various complications. Oxldation of odd carbon chain fatty acids The p-oxidation of saturatedfatty acids containing odd number of carbon atoms proceedsin the same manner, as described above for even carbon fatty acids. The only difference is that in the last and final p-oxidation cycle, a three-carbonfragment is left behind (in place of 2 carbon unit for saturated fatty acids). This compound is propionyl CoA which is converted to succinyf CoA as follows (Fig.la.fl

Succinyl CoA

L-MethylmalonylCoA j B,rdeficiency j Methylmalonic acid

TCA cycle

1. PropionylCoA is carboxylatedin the presence of ATP, CO2 and vitamin biofin to D-methylmalonyl CoA. 2. Methylmalonyl CoA racemaseconvertsthe methylmalonyl CoA to L-form. This reaction (D -+ L) is essentialfor the entry of this compound into the metabolic reactionsof the body.

same extent as saturated fatty acids. Therefore, oxidation of unsaturated fatty acids, in general, provides less energy than that of saturated fafty acids.

3. The next enzyme, methylmalonyl CoA mutase, is dependenl on vitamin 812 (deoxyadenosyl cobalamin). lt catalysesthe conversion of methylmalonyl CoA (a branched compound) to succinyl CoA (a straight chain compound), which can enter citric acid cycle.

Most of the reactions involved in the oxidation of unsaturatedfatty acids are the same as found in the p-oxidation of saturated fatty acids. However, the presence of double bonds poses problem for p-oxidation to proceed. This is overcome by two additional enzymes-an isomerase and an epimerase.

Methylmalonie

acidemia

Two types of methylmalonic acidemias are known

F-Oxidation of fatty in peroxisomes

acids

1. Due to deficiency of vitamin 812;

Peroxisomesare organelles present in most eukaryotic cells. The p-oxidation occurs in a modified form in peroxisomes. Acyl CoA (a flavoenzyme) leads to the dehydrogenase In either case, there is an accumulation of formation FADH2, as in p-oxidation. The of methylmalonic acid in body, followed by its reducing from FADH2 are not equivalents increasedexcretion in urine. This causessevere electron transport chain, but transferred to the metabolic acidosis,damagesthe central neryous to This results in the handed over directly 02. systemand retardsthe growth. lt is often fatal in which is by catalase. formation of H2O2, cleaved the early years of life. 2. Due to defect in the enzyme methylmalonyl CoA mutase.

Oxidation

of unsaturated

fatty

acids

Due to the presence of double bonds, the unsaturatedfatty acids are not reduced to the

E-FADH2 + Oz -----) E-FAD + H2O2 Catalase H2O2i::= UrO + !O'

Chapter 14: METABOLISM OF LIPIDS

293

There is no ATP synthesizedin peroxisomal rr-vOxidation of fatty ac;ds p-oxidation of fatty acids, since the reducing This is a minor pathway. lt involves equivalentsdo not passthrough ETC. However, hydroxylation followed by oxidation of or-carbon heat is liberated. present as a methyl group at the other end (at It is now believedthat the peroxisomescarry one end carboxyl group is present)of fatty acid. out the initial oxidation of long chain (C26,C22 This reactionrequirescytochromeP4s0,NADPH etc.) fatty acids which is followed by and 02, besides the enzymes. The overall mitochondrialoxidation. reaction may be representedas follows. Peroxisomal oxidation is induced by high fat diet and administration of hypolipidemic drugs (e.9. clofibrate). Zellweger syndrome : This is a rare disorder characterizedby the absenceof peroxisomesin alnlostall the tissues.As a result,the long chain fatty acids (C26-C36) are not oxidized. They accumulatein tissues,particularlyin brain, liver and kidney. Hence the disorderis also known as cerebroh e pato renal synd ro m e. a"Oxidation

of fatty

acids

p-Oxidationis the most predominantpathway for fatty acid degradation.However, the removal of one carbon unit at a time by the oxidation of cl,-carbon atom of fatty acid is known. a-Oxidation does not involve the binding of fatty acid to coenzymeA and no energy is produced. Refsumts disease is a rare but severe neurologicaldisorder characterizedby cerebral ataxia and peripheral neuropathy.The patients of this diseaseaccumulatelarge quantitiesof an unusual fafty acid, phytanic acid. lt is derived from phytol, a constituentof chlorophyll. Hence it is found mostly in plant foods. However, it is als o pr es e n t i n m i l k l i p i d s a n d a n i mal fats. Phytanicacid cannot undergoB-oxidationdue to the presenceof a methyl group on carbon-3.This fatty acid undergoes initial o-oxidation (to remove c-carbon as carbon dioxide) and this is followed by p-oxidation. Refsum'sdiseaseis caused by a defect in the cr-oxidation due to the deficiency of the enzyme phytanic acid a-oxidase. The result is that phytanic acid cannot be converted to a compound that can be degradedby p-oxidation. The patientsshould not consumedietscontaining chlorophyll (i.e., green leafy vegetables).

cHs-(cH2)n-cooHo-H2c-(6H2)n-coo-

-ooc-16Hry"-cooOxidatian of fatty acids and metabolic urater Fatty acid oxidation (even other forms of aerobic respiration) is accompanied by the production of water, referredto metabolic water. For instance,when one molecule of palmitic acid is oxidized, it releases16 molecules of water. This metabolic water has great significancein some animals. Camel can store Iipids in. its hump which is good source of water, besides energy supply. For this reason, camel can travel in deserts for long periods even without food and water supply. Kangaroo rat is a small animal that is believed to live indefinitely without water. It consumes only oil rich seeds, and the metabolic water produced is adequate to meet its water needs. lt may however, be noted that the use of metabolic water is an adaptation, and is accompanied by reduced output of urine.

The compounds namely acetone, acetoacetate and B-hydroxybutyrate (or 3-hydroxybutyrate)are known as ketone bodies (Fig.la.l0). Only the first two are true ketones while phydroxybutyrate does not possessa keto (C=O) group. Ketone hodies are water-soluble and energy yielding. Acetone, however, is an exception,since it cannot be metabolized.

294

B IOC H E MIS TRY

o

o

cH3-c-cH3

cH3-c-cH2-coo-

Acetone

Acetoacetate

oo

il CH3-C-S-CoA

ll + CH3-C-S-CoA

AcetylCoA

AcetylCoA

---------.---

OH I

I

cH3-cH-cH2-cooftHydrorybutyrate

Col-sHt-J0rc*ottlotase

oi l tlo

Fig. 14.10 : Structures of ketone bodies.

CH3-C-CH2-C-S-CoA Acetoacetyl CoA

Ketogenesis The synthesisof ketone bodies occurs in the Iiver.The enzymes for ketone body synthesisare focated inthe mitochondrial matrix. Acetyl CoA, formed by oxidation of fatty acids, pyruvate or some amino acids, is the precursorfor ketone bodies.Ketogenesis occursthroughthe following reactions(Fig.Ia.l l). 1. Two moles of acetyl CoA condense to form acetoacetylCoA. This reaction is catalysed by thiolase,an enzyme involved in the final step of p-oxidation. Hence, acetoacetatesynthesisis appropriatelyregardedas the reversalof thiolase reaction of fafty acid oxidation.

oHo

ttl -OOC-CH2-C-CH2-C-S-CoA

cHs p-Hydrory-P-methylglutaryl CoA(HMGCoA) I

9l

coAlYase cHr-d-s-coR+-1 HMG AcetylCoA I

+?

-ooc-cH2-c cHs Acetoacetate

2. Acetoacetyl CoA combines with another molecule of acetyl CoA to produce p-hydroxy p-methyl glutaryl CoA (HMC CoA). HMG CoA synthase,catalysing this reaction, regulates the synthesis of ketone bodies. 3. HMG CoA lyase cleaves HMC CoA to produce acetoacetateand acetyl CoA. 4. Acetoacetate can undergo spontaneous decarboxylation to form acetone. 5. Acetoacetate can be reduced by dehydrogeneaseto p-hydroxybutyrate.

a

The carbon skeleton of some amino acids (ketogenic)is degradedto acetoacetateor acetyl CoA and,,therefore, to ketone bodies, e.g. le u c i n e ,l y s i n e ,p h e n y l a l a n i n eetc.

o cH3-c-cH3 Acetone

OH -ooc-cH2-c-cH3 H p-Hydrorybutyrate

sourcesof energy for the peripheral tissuessuch as skeletalmuscle, cardiac muscle/ renal cortex etc. The tissueswhich lack mitochondria (elg. Utillzation of ketone bodies erythrocytes) however, cannot utilize ketone The ketone bodies, being water-soluble,are bodies. The production of ketone bodies and easily transported from the liver to various their utilization become more significantwhen tissues. The two ketone bodies-acetoacetate glucose is in short supply to the tissues, as and p-hydroxybutyrate serve as important observed in starvation, and diahetes mellitus.

METABOLISMOF L|PIDS

295

During prolonged starvation, ketone bodies intermediate in citric acid cycle. Thiophoraseis are the major fuel saurce for the brain and other absent in liver, hence ketone bodies are not pafts of central nervous system. lt should be utilized by the liver.Thiolasecleavesacetoacetyl noted that the ability of the brain to utilize fatty CoA to two moles of acetyl CoA (Fig.l4.l2). acids for energy is very limited. The ketone The summary of ketone body synthesrs, bodies can meet 5O-7Oo/o of the brain's energy utilization and excretion is depicted in needs.This is an adaptationfor the survival of Fig.t4.13. t he or ga n i s m d u ri n g th e p e ri o d s of food .i ' . i :.r ..i ta deprivation. r I n r i t,,i :::, - ;' !gg" ::r :u;+ bfg$!* :g, Reactions of ketone bodies : B-Hydroxybrutyrate is first converted to acetoacetate (reversal of synthesis) and metabolized. Acetoacetate is activated to acetoacetyl CoA by a mitochondrial enzyme thiophorase (succinylCoA acetoacetate CoA transferase). The coenzyme A is donated by succinyl CoA, an

l n normal i ndi vi dual s,there i s a constant production of ketone bodies by liver and their utilization by extrahepatictissues.The concentration of ketone bodies in blood is maintained around 1 ng/dl. Their excretion in urine is very low and undetectableby routine tests(Rothera,s fes0.

BrgntEDrcAL/ cLtnitcf,LcotucEFni

An adult human body contains qbout 10-11 kg of t'at reseruecorrespondingto about 100,000 Cal. This can meet the energg requirements for seueral weeks of t'ood depriuationin man. The sudden infant death syndrome (SIDS)----on unexpectedouernight death oJ healthy infants-is ottributed to a blockade in ftoxidation of Jatty acids,causedby a deftciency of medium chain acyl CoA dehydrogenase(MCAD). Jamaican uomiting sicknessis due to consumptionof unripe ackee fruit contoining hypoglycin A which blocks ftoxidation. Methylmalonic acidemia occurseither due to a deficiencyol the uitamin 812 or o det'ect in an enzyme methyl malonyl CoA mutase. This disorder retards growth and damages central neruoussystem. Zellwegersyndrome is causedby the absenceof peroxisomesin fissues;as a result, the Iong chain fatty acids cannot be oxidized. Refsum's disesse is due to a defect in a-oxidation of lattg acids. The patients are oduisednot to consumediets containingchlorophyll. Ketosis is commonly associated with uncontrolled diabetes mellitus and starvation. Diabetes ketoacidosisis dangerous-moy result in coma or euen death. Starvation, howeue4 is noi accompaniedby ketoacidosis. Insulin promotes fatty acid synfhesisby stimulating the conuersionol pyruuate to ocetyl CaA. The lack of the ability ol the organismsfo introduce double bonds in fatty acidsbeyond C9 and Crc makes linoleic and linolenic acids essentialto mammals.

296

B IOC H E MIS TRY

OH cH3-cH-cH2-coop-Hydrorybutyrate NAD oxybutyrate lrogenase NADH + H*

otl

cH3-c-cH2-cooAcetoacetate

cH2-cooCH2-CO-SCoA

Thiophorase

SuccinylCoA

cH2-coocH2-cooSucclnate

oi l rlo

Diabetes mellitus : Diabetes mellitus is associatedwith insulin deficiency.This resultsin impaired carbohydrate metabolism and increased lipolysis, both of them ultimately leading to the accumulationof acetyl CoA and its conversion to ketone bodies. In severe diabetes,the ketone body concentration in blood plasma may reach 100 mgldl and the urinary excretion may be as high as 500 m{day. of ketogenesis

Regulation

CH3-C-CH2-C-SCoA Acetoac€tyl CoA

I 'o^tt\|nr,rhr" I oYo il tl CH3-C-SCoA + CH3-C-SCoA Acetyl GoA

productionof acetyl CoA which cannot be fully handled by citric acid cycle. Furthermore,TCA cycle is impaired due to deficiency of oxaloacetate, since most of it is diverted for glucose synthesis to meet the essential requirements (often unsuccessful)for tissues like brain. The result is an accumulationof acetyl CoA and its diversion for overproduction of ketone bodies.

Acetyl CoA

Fig. 14.12 : Metabolism (utilization) of ketone bodies to acetyl CoA.

When the rate of synthesisof ketone bodies rate of exceeds the utilization, their concentrationin blood increases,this is known as ketonemia. Ketonemia is predominantly due to incresed production of ketone bodies rather than the deficiency in their utilization.The term ketonuria represents the excretion of ketone bodies in urine. The overall pictureof ketonemia and ketonuria is commonly referredto as kefosis Smell of acetone in breath is a common feature in ketosis. Ketosis is most commonly associated with starvationand severeuncontrolleddiabetes m e l l i tu s . Starvation : Starvation is accompanied by increased degradation of fafty acids (from the fuel reserve triacylglycerol) to meet the energy needs of the bodv. This causes an over-

The ketone body formation (particularly overproduction)occurs primarily due to nonavailabilityof carbohydratesto the tissues.This is an outcome of excessive utilization of fatty acids to meet the energy requirementsof the ceffs. The hormone glucagon stimulates ketogenesis whereas insulin inhibits. The increasedratio of glucagon/insulinin diabetes mellituspromotesketonebody formation.This is due to disturbancescausedin carbohydrateand lipid metabolisms in diabetes, as discussed elsewhere (Chapter 35). Ketogenic

and antiketogenic

substances The dietary compounds are divided into two categoriesdepending on whether they promote ketone body formation (ketogenic) or inhibit (antiketogenic). The ketogenicsubstancesinclude fatty acids and certainamino acids(leucine,lysine,tyrosine etc.). The antiketogenicsubstancesare glucose, glycerol and glucogenic amino acids (".9. I glycine, alanine, serine,glutamateetc.) Ketoacidosis Both acetoacetateand p-hydroxybutyrateare strong acids. Increasein their concentrationin blood would causeacidosis.The carboxyl group

Chapter 14: MEIABOLISMOF LIPIDS

297 BLOOD Fattyacids Ketonebodies

Kldneys

Ketone bodies -

zCO2 Ketonebodies (in urine)

has a pKuaround 4. Therefore,the ketone bodies in the blood dissociate and release H+ ions which lower the pH. Further, the volume of plasma in the body is reduced due to dehydrationcausedby the excretionof glucose and ketone bodies. Diabetic ketoacidosis is dangerous-may resultin coma, and even death, if not treated. Ketosis due to starvation is not usually accompaniedby ketoacidosis.

Acetone (exhaled)

the cytosomal fraction of the cell. Acetyl CoA is the source of carbon atoms while NADPH provides the reducing equivalehts and ATP suppliesenergy for fatty acid formation. The fatty acid synthesismay be learnt in 3 stages l.' Productionof acetyl CoA and NADPH ll. Conversionof acetyl CoA to malonyl CoA lll. Reactionsof fatty acid synthasecomplex.

Treatment of ketoacidosis : Rapid treatment l. Production of acetyl CoA of diabetic ketoacidosisis required to correct the ANd NADPH metabolic abnormalities and the associated Acetyl CoA and NADPH are the prerequisites water and electrolyteimbalance.Administration of insulin is necessaryto stimulate uptake of for fafty acid synthesis.Acetyl CoA is produced glucoseby tissuesand inhibition of ketogenesis. in the mitochondriaby the oxidationof pyruvate and fatty acids, degradation of carbon skeleton of certain amino acids, and from ketone bodies. Mitochondria, however, are not permeable to acetyl CoA. An alternate or a bypass The dietary carbohydratesand amino acids, arrangement is made for the transfer of acetyl when consumed in excess,can be converted to CoA to cytosol. Acetyl CoA condenses with fatty acids and stored as triacylglycerols.De oxaloacetate in mitochondria to form citrate. novo (new) synthesis of fatty acids occurs Citrate is freely transportedto cytosol where it is predominantly in liver, kidney, adipose tissue cleaved by citrate lyase to liberate acetyl CoA and factating mammary glands. The enzyme and oxaloacetate.Oxaloacetate in the cytosol is machinery for fatty acid production is located in convertedto malate (Fig.la.l4.

298

B IOC H E MIS TR Y

Glucose i

Pyruvate

Fig. 14.14 : Transfer oI acetyl CoA from mitochondria to cytosol (HMP shunt-Hexose monophosphate shunt; @-also known as malate dehydrogenase).

Malic enzyme converts malate to pyruvate. lll. $teaetions of fiatty aeld NADPH and CO2 are generatedin this reaction. synthase cor$pleJ( Both of them are utilized for fatty acid synthesis. The remainingreactionsof fatty acid synthesis Advantages of coupled transport of acetyl are catalysed by a multifunctional enzyme CoA and NADPH : The transport of acetyl CoA known as fatty acid synthase (FAS) complex.ln from mitochondriato cytosol is coupled with the eukaryotic cells, including man, the fatty acid cytosomal production of NADPH and COz synthaseexists as a dimer with two identical the activitiesof whic h i s h i g h l y a d v a n ta g e o u sto th e cel l for units. Each monomer possesses seven different enzymes and an acyl carrier optimum synthesisof fatty acids. ll. Forruration of nnalony! GoA Acetyl CoA is carboxylatedto malonyl CoA by the enzyrne acetyl CoA carboxylase Gig.la.lfl. This is an ATP-dependentreaction and requires biotin for COz fixation. The mechanismof action of acetyl CoA carboxylase is similar tci that of pyruvatecarboxylase(Refer Chapter 7, Fi9.7.29).Acetyl CoA carboxylase is a regulatory enzyme in fatty acid synthesis (detailsgiven later).

n i

Cl'i3 C SCon Acetyl CoA coo -.---^\l I AcetvlCoA A'r-J cab
+o

Biotin i

-OOC-CH2 -C-'CoA MalonylCoA Fig. 14.15 : Conversion of acetyl CoA to matonyl CoA.

Ghapter 14 : METABOLISM OF LIPIDS

299 p-Hydroryacyl-AGP

Plsc' q9-st

AcetvlCoA A.CP transacylase

Fafty acid synthase

sH c

Irans a2-EnoylAGP

il

S_{, CH. Acetyl-S-ACP I Transferof

tocyste J -OOC-CH2-C-SCoA MalonylCoA

Acetyl S-enzyme

Acyl-AGP(butyryl-AoP)

MalonvlCoA-ACP trarisacylase

o

c)

I Transferofcarbon ohaintromAOFtoCys | Y

,dyi;s-c-cH2 -cH2-cH3 )(

qry.SH Acylmalonylenzyme (3) . | p-retoacyuce

Acyl-S-enryme

svnthase

A*a+1

6G\sH ^

)(Y \i q97-s-.-cH2- c cH3 p-KetoacyFACP N AD P *

-l 1,r,, B-KetoacyFACP reductase , )"

NADP-sJ

s-c-(cH2)1 3- cH;'--cHa ,ro-,]

pahitoyl

Jthioesterase

s to llr

s-c-cH2-cH -cH3 p-HydroxyacyFACP Fig. {4.16

contd.

n€xt column

Fig. 14.16: Biosynthesisof long chainlatty acid-palmitate.(CyE-Cysteine; ACP-Acylcarrierprotein; Thepathwayrepeats7 timesto produceWhrtaE; the firsttwo carbonsat the methylend are directlyfromacetylCoA,the rest of the carbonscomeframnalonyl CoA). protei n (ACP) bound to 4'-phosphopantethei ne. F at t y a c i d s y n th a s efu n c ti o n s a s a si ngl e uni t catalysingall the seven reactions.Dissociation of the synthasecomplex results in loss of the enzyme activities. In the lower organisms (prokaryotes),the fatty acid synthesis is carried out by a mu l ti e n z y mec o m p l e x i n associ ati on

with a separateacyl carrier protein. This is in contrast to eukaryotes where ACP is a part of fatty acid synthase. The sequence of reactions of the extramitochondrialsynthesisof fatty acids (palmitate) is depicted in Fig.14.15.and described in the next page.

300

BIOCHEMISTF|Y

1. The two carbon fragment of acetyl CoA is B Acetyl CoA + 7 ATP + 14 NADPH + 14 H+ transferred to ACP of fatty acid synthase, ---+ Palmitate+ 8 CoA + 7 ADP + 7 Pi + 6HrO catalysed by the enzyme, acetyl CoA-ACP transacylase.The acetyl unit is then transferred Fatty acid synthase complex from ACP to cysteine residue of the enzyme. The diagrammaticrepresentation of the model Thus ACP site falls vacant. for fatty acid synthase (FAS) multienzyme 2. The enzyme malonyl CoA-ACP complex is depicted in Fi9.14.17. This model is transacylase transfers malonate from malonyl tentative and is largely based on the work of W aki l . CoA to bind to ACP. 3. The acetyl unit attached to cysteine is transferredto malonyl group (bound to ACP). The mafonyl moiety losesCO2which was added by acetyl CoA carboxylase.Thus, CO2 is never incorporatedinto fatty acid carbon chain. The decarboxylationis accompaniedby loss of free energy which allows the reaction to proceed forward. This reaction is catalyzed by p-ketoacyl ACP synthase. 4. p-Ketoacyl ACP reductase reduces ketoacyl group to hydroxyacyl group. The reducing equivalentsare supplied by NADPH.

Fatty acid synthase is a dimer composed of two identical subunits(monomers),each with a molecular weight of 240,000. Each subunit contains the activities of 7 enzymesof FAS and -SH group. an ACP *;11't4'-phosphopantetheine The two subunitslie in antiparallel(head+o-tail) orientation. The -SH group of phosphopantetheineof one subunit is in close proximity to the -SH of cysteine residue(of the enzyme ketoacyl synthase)of the other subunit.

Each monomer of FAS contains all the enzymeactivitiesof fatty acid synthesis.But only the dimer is functionally active. This is because 5. B-Hydroxyacyl ACP undergoesdehydration. the functional unit consists of half of each A moleculeof water is eliminatedand a double subunit interactingwith the complementaryhalf bond is introduced between a and p carbons. of the other. Thus, the FAS structure has both 6. A second NADPH-dependentreduction, functi onal di vi si on and subuni t di vi sion catafysed by enoyl-ACP reductase occurs to (Fig.|4.177.The two functional subunitsof FAS produce acyl-ACP.The four-carbon unit attached independently operate and synthesizetwo fatty to ACP is butyryl group. aci ds si mul taneousl y. The carbon chain attached to ACP is transferredto cysteine residue and the reactions 2-6 are repeated6 more times. Each time, the fatty acid chain is lengthenedby a two-carbon unit (obtainedfrom malonyl CoA). At the end of 7 cycles, the fatty acid synthesisis complete and a 16-carbon fully saturatedfatty acid-namely palmitate-bound to ACP is produced.

Functional significance of FAS complex The organizationof different enzymes of a metabolic pathway into a single multienzyme functional unit has distinct advantages for cel l ul arfuncti on

1. The FAS complex offers great efficiency 7. The enzyme palmitoyl thioesterase that is free from interference of other cellular separatespalmitate from fatty acid synthase.This reactions for the synthesisof fatty acids. completesthe synthesisof palmitate. 2. Since the entire processof the metabolic' pathway is confined to the complex, there are Sumnnary of palrmitate synthesis no permeability barriers for the various Of the 16 carbons presentin palmitate,only intermediates. two come from acetyl CoA directly. The remaining '14 are from malonyl CoA which, in turn, is produced by acetyl CoA. The overall reaction of palmitatesynthesisis summarized

3. The multienzynrepolypeptidecomplex is coded by a single gene. Thus, there is a good coordination in the synthesisof all enzymes of the FAS complex.

fhanter 14: tulfl{tlgllsM OF LIPIDS

301

Kotoacyl Acetyl Malonyl synthase transacylase transacylase

KetoacYl Dehvdratase EnoYl reductase reductase

anp

4,-Phosphopantetheine

I SH

SH

I

cys

AcP

Ketoacvr reductase

5191l.

reductaSe

oehydrarase

Fig. 14'17 : Fatty acid synthase multienzyme comptex (ACP-Acyl canier protein; FAS has two identical subunits which organize into two functional subunits to simuianeously'synthe,size two fatty acids).

Reg*lE*tlorr s'f fatty

acid

synthesis

Insulin stimulatestissue uptake of glucose, and conversion of pyruvate to acetyl CoA. This also facilitates fatty acid formation.

Fatty acid production is controlled by enzymes, metabolites,end products, hormones and dietary manipulations. Some of the Dietary regulation : Consumption of high important regulatorymechanismsare discussed carbqhydrate or fat-free diet increases the hereunder. synthesis of acetyl CoA carboxylase and fatty acid synthase, which promote fatty acid Acetyl CoA carboxylase : This enzyme formation. On the other hand, fastingor high fat controls a committed step in fatty acid synthesis. diet decreasesfatty acid production by reducing Acetyl CoA carboxylase exists as an inactive the synthesisof these two enzymes. protomer (monomer) or an active polymer. Citrate promotes polymer formation, hence Availabilityof NADPH : The reducingequivaincreasesfatty acid synthesis.On the other hand, lents for fatty acid synthesis are provided by palmitoyl CoA and malonyl CoA cause NADPH which come either from citrate (acetyl depolymerizationof the enzyme and, therefore, CoA) transportor hexosemonophosphateshunt. inhibit fatty acid synthesis. About 50-607oof required NADPH is obtained Irom HMP shunf, which significantlyinfluences Hormonal influence : Hormones regulate fatty acid synthesis. acetyl CoA carboxylase by a separate mechanism-phosphorylation (inactive form) F-}*satLsration of fatty aend ehains and dephosphorylation (active form) of the enzyme. Clucagon, epinephrine and norepineA microsomal enzyme system called fafty acyl phrine inactivate the enzyme by cAMp- CoA desaturaseis responsiblefor the formation dependentphosphorylation.Insulin,on the other of unsaturatedfatty acids. This reaction also hand, dephosphorylates and activates the involves flavin-dependent cytochrome bs enzyme. Thus, insulin promotes fatty acid reductase, NADH and molecular O2. The synthesis while glucagon inhibits. monounsaturated fatty acids-namely oleic

BIOCHEMISTF|Y

302

1.

Majortissues

2.

site Subcellular Precursor/substrate Endproduct

3. 4. c.

Intermediates to arebound requirement Coenzyme

Fatty acid synthesis

p-Oxidation

Liver,adipose tissue Cytosol AcetylCoA Palmitate protein Acylcarrier

liver Muscle, Mitochondria

(supplying NADPH reducing equivalents) 7. Carbon unitsadded/degradedMalonylCoA -----+cytosol) Citrate(mitochondria syslem L Transport LongchainacylCoA(inhibits 9. lnhibitor acetylCoAcarboxylase) increased Afterrichcarbohydrate diet 10. Thepathway 11. Hormonal statusthatpromotes Highratioof insuliniglucagon Multifunctional complex 12. Status enzyme of enzyme(s) 6,

AcylCoA AcetylCoA A Coenzyme FADandNAD*(getreduced) AcetylCoA (cytosol -----+mitochondria) Carnitine Malonyl CoA(inhibits l) carnitine acyltransferase ln starvation Lowratioof insulin/glucagon Individual enzymes

acid and palmitoleic acid-are, respectively, catalysedby fatty acid synthase.A specific group of enzymes, namely elongases,bring about fatty synthesizedfrom stearateand palmitate. acid chain elongation. Mammals lack the ability to introducedouble The mitochondrialchain elongation.isalmost bonds in fatty acids beyond carbons 9 and 10. Henc e,l i n o l e i ca c i d (1 8 ;2 ;9 , 1 2 ) a n d l i nol eni c a reversal of p-oxidation of fatty acids. Acetyl ac id ( 18 :3 i 9 , 1 2 , 1 5 ) a re e s s e n ti afo l r man i n CoA molecules are successivelyadded to fatty acid to lengthen the chain. The reducing t he diet . H o w e v e r,a ra c h i d o n i ca c i d (2 0 :4;5,8, '11,'14)can be synthesizedfrom linoleic acid by equivalentsare derived from NADPH. desaturationand chain elongation.Arachidonic between fatty acid acid is the precursorfor eicosanoids(prostaglan- Comparison oxidation and synthesis dins and thromboxanes),a group of compounds with diversified functions, discussedelsewhere The synthesisof fatty acidsand their oxidation (Chapter 34. are tvvo distinct and independent pathways. A comparison between these two metabolic SYNTHESIS OF LONG CHAIN pathways in given in Table 14,3.

FATTY ACIDS FROM PALMITATE Palmitate is the end product of the reactions of fatty acid synthase system that occurs in cytosof. Further, chain elongation can take place either in mitochondria or in endoplasmic (microsomes), reticulum separate by mechanisms.The microsomal chain elongation is more predominant and involves successive additionsof malonyl CoA with the participation of NADPH. These reactionsare similar to that

Triacylglycerol(TG)synthesismostlyoccursin Iiver and adipose fissue, and to a lesserextent in other tissues.Fatty acids and glycerol must be activated prior to the synthesis of triacylglycerols. Conversion of fatty acids to acyl CoA by thiokinaseis alreadydescribed(SeeFig.|4.5).

Chapter'14: METABOLISM OF LIPIDS Synthesis

of glyeerol

3.phosphate

Two mechanisms are involved for the synthesisof glycerol 3-phosphate 1 . In the liver,glycerolis activatedby glycerol kinase.This enzyme is absentin adiposetissue. 2. In both liver and adipose tissue,glucose servesas a precursorfor glycerol 3-phosphate. Dihydroxyacetonephosphate(DHAP) produced in glycolysisis reduced by glycerol 3-phosphate dehydrogenaseto glycerol 3-phosphate. Addition

of aeyl groups

303 1. Formation of lecithin and cephalin ; Cholineand ethanolamine firstget phosphorylated and then combinewith CTPto form, respectively, CDP-cholineand CDP-ethanolamine (Fig.ta.t9l. Phosphatidylcholine(lecithin) is synthesized w hen C D P -chol i necombi nesw i th 1,2-di ac ylglycerol. Phosphatidylethanolamine(cephalin) is produced when CDP-ethanolaminereactswith 1,2-diacylglycerol.Phosphatidyl ethanolamine can be converted to phosphatidyl choline on methylation.

to forrn TG

Choline and ethanolamine, used for phospholipid synthesis,are mostly derived from G lycerol3-phosphateacyltransferase catalyses preexisting the phospholipids. Thus, the the transfer of an acyl group to produce phospholipid synthesis startingwith choline or lysophosphatidicacid. DHAP can also accept ethanofamine is regarded as salvagepathway. acyl group, ultimatelyresultingin the formation of lysophosphatidicacid. Another acyl group is 2. Synthesisof phosphatidylserine : Phosphaadded to lysophosphatidic acid to form tidyl ethanolamine can exchange its phosphatidic acid (1,2-diacylglycerol phosphate). ethanolaminegroup with free serineto produce The enzyme phosphatasecleaves off phosphate phosphatidylserine.The latter, on decarboxyof phosphatidicacid to produce diacylglycerol. lation, gives the former. lncorporationof anotheracyl groupfinally results 3. Formation of phosphatidylinositol : CDPin synthesisof triacylglycerol(Fig.l4.1A. diacylglycerolproducedfrom phosphatidicacid The three fatty acids found in triacylglycerol combines with inositol to form phosphatidyl are not of the same type. A saturatedfatty acid inositol (Pl). This phospholipidcontains arachiis usually presenton carbon 1, an unsaturated donic acid on carbon 2 of glycerol which serves fatty acid is found on carbon 2, and carbon 3 as, a substrate for prostaglandin synthesis. mav have either. Further,PI is important for signal transmission across membranes. The intermediates phosphatidic of TC synthesis acid and diacylglycerol are also utilized for 4. Synthesis of phosphatidyl glycerol and phospholipidsynthesis(describedlater). cardiolipin : CDP-diacylglycerolcombines with glycerol 3-phosphate to form phosphatidyl glycerol 3-phosphate, which then forms phosphatidylglycerol. The latter combines with another molecule phosphatidylglycerolto of P h o s p h o l i p i d sa re a s p e c i a l i zedgroup of fi nal l y produce cardi ol i pi n (Fi g.ta . I 9) . lipids performing a variety of functions. These Cardiolipin phospholipid possessing is the only include the membrane structureand functions, properties. antigenic involvement in blood clotting, and supply of arachidonic acid for the synthesis of 5. Formation of plasmalogens : These are prostaglandins(for details Refer Chapter 32). phospholipidswith fatty acid at carbon 1 bound by an ether linkage insteadof ester linkage.An Synthesis of phospholipids important plasmalogen, 1-alkenyl 2-acetyl Phospholipids are synthesized from glycerol3-phosphocholine, causesblood platelet phosphatidicacid and 1,2-diacylglycerol,inter- aggregation and is referred to as plateletmediates in the production of triacylglycerols activating factor (PAF). The ourline of the Gig.l4.l8'1. Phospholipidsynthesisoccurs in the pathway for the synthesisof plasmalogensis s m oo th e n d o p l a s mi cre ti c u l u m . depicted in Fi9.14.20.

304

B IOC H E MIS TFIY

clucose

CH2-OH H O -C -H li

i

cH2-oH

i

Glycerol t: \ Glycerol k'1 kinase

; Glycolysis i :

++

u'tffil

cH2-oH H O-C -H

cH2-o-

--7 NAD-

Glycerol3-phosphate

C H 2 -O-C -R 1

foR :'

NADPH+H+

C H 2-O-C -R 1

H O-C -H

C :O 1-AcylDHAPreductase

CH2-O-,

Err-O-

Lysophosphatidic acid

,''

1-AcylDHAP

o ?

Phosohatase

Hzo

Phosphatidic acid

cH2-o-c-Rl

R 2-C -O-C -H cH2-oH

Pi

R.-C-SCoA

Acyltransferase

YY

PHOSPHOLlPIDS

o cH2-o-c-R1 ? R2-C-O-C-H ? cH2-o-c-R3 Triacylglycerol Fig. 14.18 : Synthesis of triacylglycerol.

ffihaBter 1al : METABOLISMOF L|PIDS

305

Glycerol3-phosphate (or dihydroryacetone phosphate) For details see Fig. 14.18

Phosphatidic acid

Cholin-(Ethanolamine)

ATP\l )

(1)

ADP+/l + Phosphocircli;re (Phosphoethanolamine)

vo

g

o

cnr-o-8-n,

ill R2-C-O-C-H

cH2-oH 1,2-Dlacylglycerol

?

cur-o-d-n,

R2-C-O-C-H

cHr-o-o-9

CDP-choline (CDP-ethanolamine)

Cytidine

+o

Ch o lin e ( Eth a n o la m in e )

Pl'iosphaticlylcholine (Phosphatidylethanolamine) '

CO

Inositol pfrosphatidyl lnosltol Phospharidyt- ________________ phosphatidyt '

phosphatidylglycerol I l/-qh.ogphati-

[_rGlycerol -dylglvcerol J ._. . Cardiotipin (Diphosphatidylglycerot)

Fig. 14,19: Biosynthesis of phospholipids [Theenzymesare numbered-(t) Chotinekinase,(2) phosphochotine cytidyltransferase' (3) Phosphatidate phosphohydrotase, (4) Phosphochotine aiacytgtycerotiansterasi, (s) cTpPhosphatidatecytidyltransferase, (6) CDP-Diacylgtycerotinositotiransferasel.

306

BIOGHEMISTRY

6. Synthesis of sphingomyelins : These are phospho- Dihydroryacetone lipids containing a complex lrnosfinate amino alcohol, sphingosine, instead of glycerol. Palmitoyl CoA and serinecombineand undergoa sequenceof reactions 1-Alkylglycerol to producesphingosine which is 3-phosphate ,/ then acylated to produce ceramide. Sphingomyelin is

synthesized when ."r"ria" combines with CDp-chotine ffig.la.2l).

I

|,-R,(CHc\c-oH f l\n,cooH 1_AlkyldihYydroryacetone - phosphaie

\

NADP- NADPH+ H*

t'o., ----+ n,r;!-fl1t11f,3:toh"," +

;'

1-Alkvl2-acvlslvcerol

CDp-ethanotamine,/,

Degradation of hospholinids

cwp<-__/

ospholipidsare degraded

byphosphoripases whichcreave phosphodiester the (Fig.14.22).

1-Acvldihvdroxvacetone '''ii'rii,!b-ri,itJ*'"""

( '|'

CDp_chotine

\

rcMp \

'F t-+'1ili3;?'JJfly,i;"J"' .-l'#:tJr?€fiy]fl'J3fl?,1"

bonds

PhospholipaseA1 specifically cleaves the fatty acid at Cr pos it ion phospholipids of r es ult ing i n l y s o p h o s p h o l i p i d . The latter can be further acted by lysophospholipase, phospholipase B to remove the tu.ond aryl group at C2 position.

I l r-A cyl C oA t J'*CoA 1-Alkenyl 2-acetylglycerol (PAF) 3-phosphocholine

Fig. 14.20: Summaryof the biosynthesis of plasmalogens Phospholipase 42 hydrolyses (PAF-Plateletactivationtacto0' the fatty acid at Ci position of phospholipids.Snakevenom and bee venom are rich sourcesof phospholipase 42. Role of LCAT in lecithin metabolisrn T his enz y me i s fo u n d i n m a n y ti s s u es and Lecithin-cholesterol acyltransferase (LCAT) pancreatic juice. Phospholipase42 acts on phosphatidyl inositol to liberate arachidonic is a plasma enzyme, synthesizedin the liver. acid, the substrate for the synthesis of LCAT activity is associated with apo A1 of HDL. This enzyme esterifies cholesterol by prostaglandins. transferringacyl group from the secondposition PhospholipaseC specifically cleavesthe bond of l eci thi n phosphate and between glycerol of phos pholi p i d s . T h i s e n z y me i s p re sent i n lysosomesof hepatocytes. Lecithin + Cholesterol 't"* , PhospholipaseD hydrolysesand removesthe nitrogenousbase from phospholipids.

Lysolecithin+ Cholesterolester

The above reaction is responsiblefor the The degradedproductsof phospholipidsenter reverse cholesterol transport mediated by the metabolic pool and are utilized for various HDL (more details given under lipoprotein purposes. metabolism).

Chapter 14 : METABOLISM OF LIPIDS Degradation

307

of sph;ngornyelins

The enzyme sphingomyelinase of lysosomes hydrolyses sphingomyelins to ceramide and phosphoryIch oline (Fig. | 4.23). Ceramide formed can be further degradedto sphingosineand free fattv acid. Niemann-Pick disease : lt is an inherited disorder due to a defect in the enzyme sphingomyelinase.This causes accumulation of

Sphingosine CoA

Ceramide UDP-ga UP

ucose

Galactocerebroside Glucocerebroside

l-pRps Y I + Galactocerebroside 3-sulfate (sulfatide) Fig. 14.24 : Biosynthesis of cerebrosides and sulfatides (PAPS-Phosphoadenosyl phosphosu lfate).

+ Sphingosine CoA SH Ceramide choline

sphi ngomyel i ns i n l i ver and spl een,resul ti ngi n the enlargement of these organs. Victims of Niemann-Pickdiseasesufferfrom severemental retardation, and death may occur in early chi l dhood.

Farber's disease : A defect in the enzyme ceramidase results in Farber's disease. This Sphingomyelin disorderis characterizedby skeletaldeformation, Flg. 14,21 : An outlineof the synthesisof sphingomyelin. subcutaneousnodules, dermatitis and mental retardation.lt is fatal in earlv life" Phospholipase 41 Pho

eAz

I

t?

c H 2 -o Y C -R 1 I

o

o-c-H I

CH2-O6P aiTBase

r----

Phospholipase C

a- |

PhosPholiPase D

Fig. 14.22 : Degradation of phospholipids by phospholipases. Sphingomyelinase I

Ceramide

Y

Phosphorylcholine

(sohinOosinelFFA ) Ceramidase

Fig. 14.23 : Site of action of sphingomyelinase and ceramidase on sphingomyelin.

Clycolipids are derivatives of ceramide (sphingosine bound to fatty acid), hence they are more appropriately known as glycosphingolipids. The si mpl est form of gl ycosphi ngol i pi dsare cerebrosides containing ceramide bound to monosaccharides.Galactocerebroside(Cal-Cer) (Clu-Cer)are the common and glucocerebroside glycosphingolipids. Calactocerebroside is a major component of membrane lipids in the nervous tissue(high in myelin sheath).Clucocerebroside is an intermediate in the synthesis and degradati onof compl exgl ycosphi ngol i pi ds. Synthesis

of eerebrosides

The outline of the synthesisof cerebrosides and sulfatideis given in Fi9.14.24.

308

ElIOCHEMISTRY

Galactocerebroside (Gal-Cer)

Krabbe's disease

p-Galactosidase Galactose Farber's Falty dicaaca

Glucocerebroside (Glc-Cer)

aClO

Ceramide

SPhingosine

Niemann-Pick disease

Sphingomyelin (choline-P-Cer) Fig. 14.25: Degradationof cerebrosidesand sphingomyelinswithmetabolicdisorders.

Metabolic

dlsorders

of eerebr$sid*s

The degradationof cerebrosidesalong with the associated inborn errors is depicted in Fig.l4.25. Gaucher's disease: This is due to a defect in the enzyme B-glucosidase.As a result, tissue glucocerebroside levelsincrease.This disorderis commonly associatedwith enlargementof liver and spleen, osteoporosis,pigmentationof skin, anemia, mental retardation etc. Sometimes, Caucher'sdiseaseis fatal. Krabbe's disease : Defect in the enzyme p-galactosidaseresults in the accumulation of galactocerebrosides. A total absenceof myelin in the nervoustissue is a common feature.Severe mental retardation, convulsions, blindness, deafnessetc. are seen. Krabbe's disease is fatal in early life.

causes of gangliosides Defectin the degradation gangliosidosis, diseaseetc. Tay-Sach's Sptrinognlipidoses representing IysosomaI Lipi d storaged iseases, Theyare disorders. are inherited storagedefects, of complex the accumulation by characterized lipids. The term sphingolipidosesis often used to collectivelyrefer to the genetic disordersthat lead to the accumulationof any one of the (glycosphingolipids and sphingosphingolipids myelins).Some examplesof sphiogolipidoses (lipid storagediseases)with important features in Tahle14.4. are summarized

Niemann-Pick disease and Farber's disease is found exclusivelyin animals, Cholesterol connected with sphingomyleinmetabolism are hence it is often called as animal sterol. already described. They are also depicted in in an adult Thetotalbodycontentof cholesterol Fig.14.25.

man weighing7O kg is about 140 I i.e., Gangliosidesare complex glycosphingolipids around 2 dke body weight. Cholesterolis both mostlyfound in ganglioncells.They contain one amphipathicin nature,since it possesses in regions the or more molecules of N-acetylneuraminicacid hydrophilicand hydrophobic (NANA) bound ceramide oligosaccharides. structure.

Chapter 14: METABOLISM OF LIPIDS

Missing/defeclive enzyme

Disease

309

Major storage compound

Symptoms Enlargement of liver,spleen, mental retardation. joints. Painful anddefomed

Niemann-Pick disease

Sphingomyelinase

Sphingomyelins

Fa6e/sdisease

Ceramide

Gaucher's disease

Ceramidase p-Glucosidase

Krabbe's disease

p-Galactosidase

GalactocerebrosidesAbsence of myelin formation, liver andspleen enlargement, mental retardation.

Tay-Sachs disease

A Hexosaminidase

Ganglioside GM,

Blindness, mental retardation, death within2-3years.

Fabry's disease

c-Galactosidase

Ceramide trihexoside

Renal failure, skinrash,painin lowerextremities.

Glucocerebroside

iltumctions of cherl:, r.lt,:; . l Cholesterol is essentialto life, as it performsa number of importantfunctions 1. lt is a structural component of cell membrane.

Enlargement of liverandspleen, osteoporosis, mental retardation.

reducing equivalentsare supplied by NADPH while AIP provides energy. For the production of one mole of cholesterol,18 moles of acetyl CoA, 36 moles of ATP and 16 moles of NADPH are required.

By administering acetate with 14C isotope 2. Cholesterol is the precursor [or the label .either on the methyl (-CH3) group or synthesisof all other steroidsin the body. These carboxyl (-COO) group, the origin of carbon include steroid hormones, vitamin D and bile atoms in the entire molecule of cholesterolhas acids. been established.The sources of carbon atoms and the key intermediates of cholesterol 3. lt is an essentialingredientin the structure formation are depicted in Fig.14.26, and the of lipoproteinsin which form the lipids in the detailed reactions are given in Fi9.14.27. body are transported. 4. Fatty acids are transported to liver as cholesteryl estersfor oxidation.

The synthesisof cholesterolmay be learnt in 5 stages 1. Synthesisof HMG CoA

CHOLESTEROL

BIOSYNTHESIS

2. Formation of mevalonate (6C) About 1 g of cholesterol is synthesized per 3. Productionof isoprenoidunits (5C) day in adults.Almost all the tissuesof the body 4. Synthesisof squalene(30C) participate in cholesterol biosynthesis. The (5O"/o), fargest contribution is made by liver 5. Conversion of squalene to cholesterol intestine ('15"/o'),skin, adrenal cortex, repro- (27C). ductive tissueetc. 1. Synthesis of p-hydroxy p-methylglutaryl The enzymes involved in cholesterolsynthesis CoA (HMG CoA) : Two moles of acetyl CoA are found in the cytosol and microsomal condense to form acetoacetyl CoA. Another fractions of the cell. Acetate of acetyl CoA molecule of acetyl CoA is then added to produce providesall the carbon atoms in cholesterol.The HMG CoA. Thesereactionsare similar to that of

310

B IOC H E MIS TR Y cholesterolbiosynthesis.This enzyme is present in endoplasmic reticulum and catalyses the reduction of HMC CoA to mevalonate. The reduci ngequi val entsare suppl i edby N A D PH.

H3C-C-OO-

(acetate)

CH3contributes to carbonsat positions 1 ,3 ,5 ,7 ,9 , 1 3 ,1 5 ,1 7 ,1 8 ,19,21, 22,24,26and27 COO-contributes to the remaininocarbonatoms (B)

Acetyl CoA(2C) i Y

HMG CoA (6C) i \7

Mevalonate(6C) i

+ lsoprenoidunits (5C;building blocks) i 6 units i condense + (30C) Squalene Y

Lanosterol(30C)

3. Productionof isoprenoidunits : In a threestep reactioncatalysedby kinases,mevalonateis 3-phospho 5-pyrophosphoconverted to mevalonate which on decarboxylation forms isopentenyl pyrophosphate (lPP). The latter isomerizesto dimethylallylpyrophosphate(DPP). Both IPPand DPP are 5-carbonisoprenoidunits. 4. Synthesis of squalene : IPP and DPP condense to produce a 1O-carbon geranyl pyrophosphate(GPP).Another molecule of IPP condenses with CPP to form a 1S-carbon farnesyl pyrophosphate (FPP). Two units of farnesyl pyrophosphateunite and get reduced to produce a 30-carbon squalene. 5. Conversion of squalene to cholesterol : Squalene undergoes hydroxylation and cyclization utilizing 02 and NADPH and gets converted to lanosterol. The formation of cholesterolfrom lanosterolis a multistepprocess with a seriesof about 19 enzymatic reactions. The following are the most important reactions . Reducingthe carbon atoms from 3O to 27. . Removal of two methyl groups from Co and one methyl group from Cro. . Shift of double bond from Cs to Cs.

Y

Cholesterol(27C) Fig. 14.fr : Outlineof cholesterolbiosynthesis(A) Derivationof carbanatomsfroma@tate,

. Reduction in the double bond between Cro and Crr.

present

The enzymes (about 19?) i nvol ved i n t he conversionof lanosterolto cholesterolare associ ated w i th endopl asmi c reti cul um. 14Desmethyllanosterol,zymosterol,cholestadienol ketone body synthesis. However, the two and desmosterolare among the intermediatesin pathways are distinct, since ketone bodies are the cholesterol biosynthesis. Ihe penultimate produced in mitochondria while cholesterol product is 7-dehydrocholesterol which, on synthesisoccurs in cytosol.Thus,there exist fwo reduction, finally yields cholesterol. pools of HMG CoA in the cell. Further, two Cholesterolbiosynthesisis now believed to isoenzymesof HMG CoA synthaseare known. be a part of a major metabolic pathway The cytosomalenzyme is involved in cholesterol concerned with the synthesisof several other synthesiswhereasthe mitochondrialHMC CoA isoprenoid compounds. These include synthaseparticipatesin ketone body formation. ubiquinone (coenzyme of electron transport Q 2. Formation of mevalonate : HMG CoA chain) and dolichol (found in glycoprotein).Both reductase is the rate limiting enzyme in of them are derived from farnesylpyrophosphate. (B) Key intermediates with the carbon atoms.

Cirapter 14 : METABOLISMOF LIPIDS

311 S-Pyrophosphomevalonate

H.C-C-S-CoA

o

2AcetylCoA

Kinase

II

CoA.SHl Thiolase

J

-oo\

o

t{t

'.oe )\2"!'

cHz cHz 'o-O-@

HoC-C-CH2-C-S-CoA

o

3-PhosphoS-pyrophosphomevalonate

AcetoacetylCoA

CH. t" -OOC-CH2-C-CH2-C-S-CoA

,//

CIr

\./

lsopentenylpyrophosphate(5C)

+ 9Hs .Cr \ , 4 \ -Ctlz R R cHz cH -1 o-g-€>

,.OH

,trz

\,,, \ cHz oH

cHz

\,/

.ctlz

\ 6fi, dn, 'o-O-O

OH GoA(HMGCoA) B-Hydroxy B-methylglutaryl

-ooc. ).1

.c.

Dirnethylallylpyrophosphate(5C)

Mevalonate (6C)

cls-Prenyl transferase

9Hs 9Hs ,c. .c.Hz.Q QHz.

.(.\(\(,v[-eGeranylpyrophosphate(10C)

S-Phosphomevalonate

cls-Prenyl transferase

t{t

-ooc. \,.'

.roH

'pa

\,/'

g!12 \

cHz cHz 'o-O-e

S-Fyrophosphomevalonate F,g, 14,27 contd.

next column

-zA-1p\ '\'/ \'/ Farnesylpyrophosphate (15C) F,9.14.27

contd. trext tEg.

312

B IOC H E MIS TR Y (15C) 2 Farnesylpyrophosphate NADPH+ Hl 2L

2!

Mg- , Mn-

Squalene synthase

NADPPP i

R egui ati on

of chol esterol

synthesi s

Cholesterolbiosynthesisis controlled by the rate limiting enzyme HMG CoA reductaset at the beginningof the pathway (Fig.la.28). HMC CoA reductase is found in association with endoplasmic reticulum, and is subjected to different metabolic controls. 1. Feedback control : The end product cholesterol controls its own synthesis by a feedback mechani sm.Increasei n the cel l ular concentration of cholesterol reduces the synthesisof the enzyme HMG CoA reductase. This is achieved by decreasing the transcription of the gene responsiblefor the production of HMC CoA reductase.Feedbackregulation has been investigatedwith regardto LDl-cholesterol taken up by the cells, and the same mechanism is believed to operate whenever cellular cholesterollevel is elevated.

W"

NADPH+ H* NADP'

Oz

Epoxidase Hydtoxylase Cyclase

Hzo

2COz NADPH,Oz

A seriesot (about19) reactions

NADP'

Fig. 14.27 : Biosynthesis of cholesterol.

2. Hormonal regulation : The enzyme HMG CoA reductase exists in two interconvertible forms. The dephosphorylatedform of HMC CoA reductase is more active while the phosphorylated form is lessactive.The hormones exerttheir influencethroughcAMP by a seriesof reactionswhich are comparablewith the control of the enzyme glycogensynthase.The net effect is that glucagon and glucocorticoids favour the formation of inactive HMC CoA reductase (phosphorylated form) and, thus, decrease chofesterolsynthesis.On the other hand, insulin and thyroxine increasecholesterolproduction by enhancing the formation of active HMC CoA form). reductase(dephosphorylated 3. Inhibition by drugs : The drugs compactin and lovastatin (mevinolin)are fungal products. They are used to decreasethe serum cholesterol level in patients with hypercholesterolemia. Compactin and lovastatin are competitive inhibitors of the enzyme HMG CoA reductase and, therefore, reduce cholesterol synthesis. About 50 to 60"/" decreasein serum cholesterol level has been reported by a combined use of these two drugs. 4. HMG CoA reductaseactivity is inhibited by bile acids. Fastingalso reducesthe activity of thi s enzvme.

Ghapter'14: METABOLISM OF LIPIDS

mRNA

t -C Cholesterol

I

>lTranscription

I

DNA Fig. 14.28: Regulationof cholesterolbiosynthesis by HMGCoAreductase(&-Prcmoting effect;Q-lnhibitory effect). F.

DEGRADATIONOF CHOLESTEROL The steroid nucleus (ring structure) of the cholesterol cannot be degraded to CO2 and H2O. Cholesterol (50%) is converted to bile acids (excretedin feces),servesas a precursor for the synthesisof steroidhormones,vitamin D, coprostanoland cholestanol.The latter two are the fecal sterols,besidescholesterol. l. Synthesis

function as surfactants. In the bile, the conjugated bile acids exist as sodium and potassiumsaltswhich are known as bile salts. Cholesterol

of bile acids

The bile acids possess24 carbon atoms,2 or 3 hydroxyl groups in the steroid nucleus and a s ide c h a i n e n d i n g i n c a rb o x y l Bro up. The bi l e ac ids a re a m p h i p a th i c i n n a tu re si nce they possessboth polar and non-polar groups.They serve as emulsifyingagentsin the intestineand ac t iv e l y p a rti c i p a te i n th e d i g e sti on and absorptionof lipids. The synthesisof primarybile acidstakesplace in the liver and involves a series of reactions (Fi9.14.29).The step catalysed by 7 a-hydroxylase is inhibited by bile acids and this is the rafe Iimiting reaction. Cholic acid and chenodeox y c ho l i c a c i d a re th e p ri ma ryb i l e a ci dsand the f or m er i s fo u n d i n th e l a rg e s at mo u n t i n bi l e. On conjugationwith glycine or taurine, conjugated bile acids (glycocholic acid, taurocholic acid etc.) are formed which are more efficient in their

7-Hydrorycholesteror seterv ' ^rra.-"e .-:/ep. g.uuu"=t 'i

GIY

Cholifacid ri ne

Chenodeorycholic acid

_.9Jy-"9:,,x .Ta.uro-. .* chol i c aci d'

chol i c aci cl '

I I lntestinal I bacteria + Deoxycholic acid**

Tauro-or glycochenodeoxycholicx acid I Intestinal bacteria I LithochJic acid**

Fig. 14.29 : Outline ot bile acid synthesis(*-Primary bile acids, **-Secondary bile acids).

BIOCHEMISTFIY

314 In the intestine,a portion of primarybile acids undergoesdeconjugationand dehydroxylationto form secondary bile acids (deoxycholic acid and lithocholic acid). These reactionsare catalysed by bacterialenzymes in the intestine.

(27C) Cholesterol

I

+ (21C) Pregnenolone

I

+

Enterohepatic circulation : The conjugated bile sa l tss y n th e s i z e di n th e l i v e r a ccumul atei n (21C) Progesterone gall bladder. From there they are secretedinto the small intestine where they serve as emulsifying agents for the digestion and (1BC) (21C) Estradiol absorption of fats and fat soluble vitamins. A (21C) Aldosterone Cortisol large portion of the bile nlts (primary and Flg. 14.30: Outlineof steroidhormonesynthesis secondary) are reabsorbed and returned to the from eholesterol(Numbersin the brackets liver through portal vein. Thus the bile saltsare reDresentthe number of carbon atoms). recycledand reusedseveraltimes in a day. This is known as enterohepaticcirculation.About 15(d) Androgens (e.9. testosterone) 30 g of bile saltsare secretedinto the intestine each day and reabsorbed.However, a small (e) Estrogens(e.9. estradiol). portion of about 0.5 g/day is lost in the feces.An A brief outline of steroid hormonal synthesis equal amount (0.5 g/day) is synthesizedin liver given in Fig.l4.30 and more details are is to reolace the lost bile salts.fhe fecal excretion of bile salfs is the only route for the removal of discussedunder 'Hormones' (Chapter 19). cholesterol from the hody. l l l . S ynthesi s of vi tami n D Cholelithiasis: Bile salts and phospholipids an intermediatein the 7-Dehydrocholesterol, are responsiblefor keepingthe cholesterolin bile in a s o l u b l e s ta te . D u e to th e i r defi ci ency synthesisof cholesterol,is converted to chole(particularly bile salts), cholesterol crystals calciferol (vitamin O3) bV ultravioletrays in the precipitatein the gall bladder often resultingin ski n. cholelithiasis-cholesterol gall stone disease. A brief summary of prominent sources and Cholelithiasismay be due to defectiveabsorption the major pathwaysfor utilization of cholesterol of bile salts from the intestine, impairment in with the liver as the central metabolic organ is liver function, obstructionof biliary tract etc. depicted in Fi9.14.31. The patientsof cholelithiasisrespond to the administrationof bile acid chenodeoxy cholic Trarrsport of cholesterol acid, commonly known as chenodiol. lt is Cholesterol is present in the plasma believed that a slow but gradual dissolutionof lipoproteinsin two forms gall stonesoccurs due to chenodiol. For severe 1. About 70-75% of it is in an esterifiedform casesof cholelithiasis,surgical removal of gall with long chain fatty acids. bladder is the only remedy. 2. A bout 25-30% as free chol esterol .This form of cholesterolreadily exchangesbetween different lipoproteins and also with the cell is the precursor for the synthesis membranes. Cholesterol

trl" $ynthesis of steroid hormones from cholesterol

of all the five classes of steroidhormones Role of ICAT : High density lipoproteins (a) Clucocorticoids (e.9.cortisol) (HDL) and the enzyme lecithin-cholesterol (b) Mineralocorticoids (e.9.aldosterone) acyltransferase (LCAT) are responsible for the (e.g.progesterone) (c) Progestins transportand eliminationof cholesterolfrom the

3t5

Chapter 14 : METABOLISM OF LIPIDS

Dietarycholesterol body. LCAT is a plasma enzyme, (500 mg/day) synthesizedby the liver. lt catalyses Bile salts and bile acids the transfer of fattv acid from the CHOLESTEROL (250 mg/day) Cholesterol POOL second position of phosphatidyl synthesis in ( 1 0 0 0m g ) c holin e (l e c i th i n ) to th e h y d ro x y l liver (500 mgiday) Cholesterollost in bile group of cholesterol (Fig.Ia32). (500 mg/day) HDL-cholesterolis the real substrate for LCAT and this reaction is freely is reversible. LCAT activity extrahepatic associatedwith apo-A1 of HDL. tissues(variable) The cholesterol(cholesteryl)ester Majorsourcesof liver l a rt o f H D L . In th i s f or m sa n i n te g ra p Majorroutesof cholesterol cholesterol utilization manner, the cholesterol from the peripheraltissuesis trapped in HDL, Fig. 14.31Summaryof majorsourcesof livercholesterol and its by a reactioncatalysedby LCAT and utilization(valuesgivenin bracketsare variable). then transported to liver for degradation and excretion. This m ec ha n i s m i s c o m m o n l y k n o w n as revefse Carr and Dructor method and, more recently, cholesterol oxidase method. HDL- cholesterol cho Ie sterol t ran sport. can be determined after precipitatingLDL and V LD L by pol yethyl ene gl ycol (P E C ). V LDL Flasma eholesterol.l/5th cholesterol is equivalent to of plasma bionnedical innportance (TC) triacylglycerol in a fasting state. LDLI n h e a l th y i n d i v i d u a l s , th e to tal pl asma cholesterol can be calculated from Friedewald cholesterolis in the range of 150-200 mg/dl. ln formula given below. the new born, it is lessthan 100 mgldl and rises LDl-cholesterol= Total cholesterol- (HDLto about 150 mg/dl within an year. The women have relatively lower plasma cholesterol cholesterol+ TCl5). which is attributed to the hormones-esfrogens. The above formula is not valid if TC Cholesterollevel increaseswith increasingage concentrationis above a)O mg/dl. ( in wo me n p a rti c u l a rl ya fte r m e n o pause),and is about In adults,the normal LDL-cholesterol also in pregnancy. 80-150 mgi dl w hi l e H D L-chol esteroli s around Plasmacholesterolis associatedwith different 30-60 mg/dl. Elevation in plasma HDLlipopro te i nfra c ti o n s(L D L , VL D L a n d H D L). cholesterol is beneficial to the body, since it Total cholesterolcan be estimatedby many protects the body from atherosclerosis and methods such as Libermann-Burchardreaction, coronarv heart diseases(CHD). On the other hand, increasein LDl-cholesterolis harmful to the body as it may lead to variouscomplications, .\f Phosphatidvrch" choresterol i ncl udi ngC H D .

I{YPERCHOLESTEROLEM IA Lecithincholesterol acyltransferase(LCAT)

/ Lysophosphatiavt"notin"

\

a no,"r," rotester

Fig. 14.32 : Reaction catalysedby LCAT.

Increasein plasma cholesterol(> 200 mg/dl) concentrationis known as hypercholesterolemia and is observedin many disorders 1. Diabetes mellitus : Due to increased cholesterol synthesissince the availability of acetyl CoA is increased.

B IOC H E MIS TFIY

I 316

/

tyl d.rsm due,E u"_

:

T h ts IS i n tthe he HD DL L

Due to an

,r€ -t i or cholesterol iev heoaFetion be\ 31.;n "ou;{", [::f;''; I Ev-, -.t

(described earlier) and its excretion from the body. The oi l s w i th ri ch P U FA content i ncl u de cottonseedoil, soya bean oil, sunfloweroil, corn oi l , fi sh oi l s etc. Ghee and coconut oi l are poor sourcesof PUFA. 2. Dietary cholesterol: Cholesterolis found

pl ant food s. 3. oltlj?jtome : Increase in plasma onl v i n ani mal foods and not i n

Dietary cholesterol influence on plasma oSstruc$ofation is the characteristic is minimal. However, avoidance of cholesterol l1rou$hrotic syndrome. Cholesterol foods is advocatedto be on the cholesterol-rich z u e to i n c re a s ei n p l a s mal i poprotei n safe side. n th i s d i s o rd e r. ,crcholesterolemia is associated with .iosclerosis and coronary heart disease. is characterizedby depositionof .nerosclerosis c holes te ryel s te rsa n d o th e r l i p i d s i n the i nti ma of the arterial walls often leading to hardening of coronary arteriesand cerebral blood vessels. A positive correlation between raised plasma lipids w i th a th e ro s c l e ro s ios n o n e ' hand and coronarv heart diseaseon the other has been established.More specifically,LDL-cholesterolis positivelycorrelated,whereasHDL-cholesterolis negatively correlated with cardiovascular diseases.

3. Plant sterols : Certain plant sterols and their esters(e.g.sitostanolesters)reduceplasma cholesterol levels. They inhibit the intestinal absorptionof dietary cholesterol. 4. Dietary fiber : Fiber presentin vegetables decreasesthe cholesterol absorption from the i ntestine. 5. Avoiding high carbohydrate diet : Diets rich in carbohydrates (particularly sucrose) should be avoided to control hypercholesterolemia.

6. lmpact of lifestyles: Elevationin plasma Bad cholesterol and good cholesterol : cholesterolis obseved in people with smoking, Cholesterolis a natural metaboliteperforminga abdominal obesity,lack of exercise,stress,high wide range of functions (membrane structure, blood pressure,consumption of soft water etc. precursorfor steroid hormones,bile acids).The Therefore,adequatechangesin the lifestyleswill usagesgood and bad to cholesterol,although bri ng dow n pl asmachol esterol . inappropriate,are still in use. The cholesterolin Z. Moderate alcohol cosumption : The high concentration,presentin LDL,is considered beneficial effectsof moderatealcohol intakeare had due to its involvement in altherosclerosis masked by the ill effectsof chronic alcoholism. and related complications.Thus, LDL may be particularly beneficial due to its wine is Red regardedas lethally dangerouslipoprotein. On low alcohol content. antioxidants, besides the other hand, HDL cholesterol is good since its high concentrationcounteractsatherogenesis. 8. Use of drugs : Drugs such as lovastatin HDL m a y b e c o n s i d e re da s h i g h l y desi rabl e which inhibit HMG CoA reductaseand decrease fipoprotein. cholesterolsynthesisare used. Statinscurrently in use include atorvastatin, simvastatin and pravastatin. Certain drugs-cholestyramine and col esti pol -bi nd w i th bi l e aci ds an d Several measuresare advocated to lower the Thi s helps decreasethei r i ntesti nalreabsorpti on. plasma cholesterollevel in the conversion of more cholesterol to bile 1. Consumptionof PUFA : Dietary intake of acids and its excretion through feces. Clofibrate polyunsaturatedfatty acids (PUFA) reduces the increases the activity of lipoprotein lipase plasma cholesterol level. PUFA will help in and reduces the pl asma chol esterol an d transoort of cholesterol bv LCAT mechanism triacylglycerols. Gontrol

of hypercholesterolemia

316

B IOC H E MIS TFIY

2. Hypothyroidism (myxoedema) : This is believed to be due to decrease in the HDL receptors on hepatocytes. 3. Obstructive jaundice : Due to an obstruction in the excretion of cholesterol t hr ough b i l e . 4. Nephrotic syndrome : Increasein plasma globulin concentration is the characteristic feature of nephrotic syndrome. Cholesterol elevationis due to increasein plasmalipoprotein fractionsin this disorder. Hypercholesterolemia is associated with atherosclerosis and coronary heart disease. Atherosclerosis is characterizedby depositionof cholesterylestersand other lipids in the intima of the arterial walls often leading to hardening of coronary arteriesand cerebral blood vessels. A positive correlation between raised plasma lipids with atherosclerosison one h4nd and coronary heart diseaseon the other has been established.More specifically,LDL-cholesterolis positivelycorrelated,whereasH DL-cholesteroli s negatively correlated with cardiovascular diseases.

(describedearlier) and its excretion from the body. The oi l s w i th ri ch P U FA content i ncl ude cottonseedoil, soya bean oil, sunfloweroil, corn oi l , fi sh oi l s etc. C hee and coconutoi l are poor sourcesof PUFA. 2. Dietary cholesterol: Cholesterolis found only in animal foods and not in plant foods. Dietary cholesterol influence on plasma cholesterol is minimal. However, avoidance of cholesterol-richfoods is advocatedto be on the safe side. 3. Plant sterols : Certain plant sterols and their esters(e.g.sitostanolesters)reduceplasma chol esterol l evel s. They i nhi bi t the i ntesti na l absorptionof dietary cholesterol. 4. Dietary fiber : Fiber presentin vegetables decreasesthe cholesterol absorption from the i ntesti ne. 5. Avoiding high carbohydrate diet : Diets rich in carbohydrates (particularly sucrose) should be avoided to control hvpercholesterol emi a.

6. lmpact of lifestyles: Elevationin plasma Bad cholesterol and good cholesterol : cholesterolis obseved in people with smoking, Cholesterolis a natural metaboliteperforminga abdominal obesity,Iack of exercise,stress,high wide range of functions (membrane structure, blood pressure,consumption of soft water etc. precursorfor steroid hormones,bile acids).The Therefore,adequatechangesin the lifestyleswill usagesgood and bad to cholesterol,although bring down plasma cholesterol. inappropriate,are still in use. The cholesterolin 7. Moderate alcohol cosumption : The high concentration,presentin LDL, is considered beneficial effectsof moderatealcohol intake are bad due to its involvement in altherosclerosis masked by the ill effectsof chronic alcoholism. and r ela te d c o m p l i c a ti o n s T . h u s , L D L may be particularly beneficial due to its Red wine is regardedas lethally dangerousIipoprotein. On low alcohol content. antioxidants, besides the other hand, HDL cholesterol is good since its high concentrationcounteractsatherogenesis. 8. Use of drugs : Drugs such as lovastatin HDL m a y b e c o n s i d e re da s h i g h l y desi rabl e w hi ch i nhi bi t H MC C oA reductaseand decrease fipoprotein. cholesterolsynthesisare used. Statinscurrently in use include atorvastatin, simvastatin and pravastatin. Certain drugs-cholestyramine and col esti pol -bi nd w i th bi l e aci ds and Severalmeasuresare advocatedto lower the Thi s hel ps decreasethei r i ntesti nalreabsorpti on. plasma cholesterollevel of more cholesterol to bile in the conversion feces. 1. Consumptionof PUFA : Dietary intake of acids and its excretion through Clofibrate pofyunsaturatedfatty acids (PUFA) reduces the increases the activity of lipoprotein lipase plasma cholesterol level. PUFA will help in and reduces the plasma cholesterol and transport of cholesterol by LCAT mechanism triacylglycerols. Gontrol

of hypercholesterolemia

F Ghapter 14 : METABOLISM OF LIPIDS

317 1. Chylomicrons : They are synthesizedin the intestine and transport exogenous (dietary) triacylglycerol to various tissues. They consist of highest(99"/")quantityof lipid and lowest (1%) concentration of protein. The chylomicrons are the leastin densityand the largestin size,among the lipoproteins.

Shell (coat)

Fig. 14.33: A generalstructurcof lipoprcteincomplex. (Note : For the sakeof clarity,only a part of the shell and coreare filled with the constituents).

2. Yery low density lipoproteins (VtDt) : They are produced in liver and intestineand are responsiblefor the transport of endogenously synthesizedtriacylglycerols. 3. Low density lipoproteins(LDt) : They are formed from VLDL in the blood circulation.Thev transportcholesterolfrom liver to other tissues.

a. High densitylipoproteins(HDt) : They are mostly synthesized in liver. Three different Hypoeholesterolemia fractionsof HDL (1, 2 and 3) can be identified A decrease in the plasma cholesterol, by ultracentrifugation.HDL particles transport although less common, is also observed. cholesterol from peripheral tissues to liver p e rn i c i o u s Hy per t hy ro i d i s m , anemi a, (reversecholesteroltransport). m alabs or p ti o ns y n d ro me , h e mo l y ti c j a undi ce 5. Freefatty acids-albumin : Freefatty acids etc., are some of the disordersassociatedwith in the circulation are in a bound form to hypocholesterolemia. al bumi n. E ach mol ecul e of al bumi n can hol d

(-) Cathode

Lipoproteins are molecular complexes that consist of lipids and proteins (conjugated proteins).They function as transportvehiclesfor lipids in bl o o d p l a s ma .L i p o p ro te i n sd e l i ver the lipid components (cholesterol, triacylglycerol etc.) to various tissuesfor utilization. Structure

Origin Chylomicrons

LDL(p-lipoprotein)

of lipoproteins

A lipoprotein basically consistsof a neutral lipid core (with triacylglyceroland/orcholesteryl ester) surrounded by a coat shell of phospholipids, apoproteins and cholesterol The polar portions (amphiphilic)of ffigJa3\. phospholipidsand cholesterolare exposed on the surfaceof lipoproteinsso that lipoprotein is s oluble in a q u e o u ss o l u ti o n . G las s if ic a ti o n

Mobility

VLDL(pre-plipoprotein)

HDL(cr-lipoprotein)

o f l i p o p ro te i n s

Five major classes of lipoproteins are ident if ied i n h u ma n p l a s ma , b a s e d o n thei r separation by electrophoresis(Fig.l 4.34).

(+) Anode

Fig. 14.34: Electrophoresis of plasma(serum) Iipoptoteins.

318

B IOC H E MIS TR Y

about 20-30 moleculesof free fatty acids. This lipoprotein cannot be separated by electrophoresis. Apolipoproteins

(apoproteins!

The protein cornponentsof lipoproteinsare known as apolipoproteins o(, simply, apoproteins. They perform the following functions

Chylomicrons(nascent)are synthesizedin the small intestine during the course of fat absorption. They contain apoprotein Ba6 and mostly triacylglycerols.Apo 846 name is given since this apoproteincontains 48'h of protein coded by apo B gene (apo B1s6is found in LDL and VLDL). Chylomicrons are produced when nascentparticlescombine with apo C ll and apo E, derived from HDL.

The liver synthesizes nascent VLDL contai ni ngapo 8166 w hi ch are ri ch i n tri acyl 2. Recognize the cell membrane surface gl ycerol s and chol esterol . C i rcul ati ng H D L receptors. donatesapo C ll and apo E to convert nascent 3. Activate enzymes involved in lipoprotein V LD L to V LD L. metabolism. Role of lipoprotein lipase : The enzyme The comparative characteristic features of l i poprotei nl i pasei s presenti n the capi l l aryw al l s different lipoproteins with regard to electro- of adipose tissue, cardiac and skeletal muscle, phoretic patterns, size, composition etc. are besidesother tissues.lt hydrolysesa portion of given in Tahle 14.5. triacylglycerols present in chylomicrons and VLDL to liberate free fatty acids and glycerol. Metabolism of lipoprotein$ Lipoproteinlipase is activatedby apo C ll. -a general view Uptake of chylomicron remnants by liver : A generalpictureof lipoproteinmetabolismis As the triacylglycerols of chylomicrons and depicted in Fig.|4.35. VLDL are degraded,they losethe apo C ll which 1 . Act as structuralcomponentsof lipoproteins.

Characteristic

Chylomicrons

VLDL

Electrophoretic mobility

Origin

Pre-p

po

Density

<0.96

0.96-1.006

1.006-1.063 1.063-1.21

(nm) Diameter

100-1,000

30-90

2o-25

10-20

Apoproteins

AI,AII B4s

B1oo, Cl, Cll cilt, E

B,oo

Al,All,cl, cll, clll,D,E

2

10

20

40

98

90

80

60

88

55

12

12

(freeandester) Cholesterol

4

24

59

40

Phospholipids

I

20

28

47

(%,approximaie) Composition Protein Lipid(total) (%) Lipidcomponents Triacylglycerol

1 1 __Jgr_flttv_1ci!s__ ___l_ (VLDL : Vetylowdensity liryrcteins; LDL:Lowdensity lipoproteins: HDL:Highdensity lipoproteins).

1

Ghapter 14 : METABOLISMOF LIPIDS

319

tissues Extraheoatic Fig. 14.35 : Summary of metabolism of lipoproteins (Apoproteins-A, B#, 8ffi, Cll and E; TG-Triacylglycerol; C-Cholesterol; P-Phospholipid; VLDL-Very low density lipoprotein; IDL-lntermediate density lipoprotein; LDL-Low density lipoprotein; HDL-High density Iipoprotein).

is returnedto HDL. The chylomicron remnants apo E is returned to HDL. LDL contains high are taken up by receptors present on the cholesterol (free and esterified) and less triacylglycerol. hepatocytesof liver. Cholesterol ester transfer protein (CETP) : CETPis synthesizedin the liver, and it facilitates During the course of VLDL metabolism, the exchange of components between different intermediatedensity lipoprotein (lDL) is formed lipoproteins.CETPcan transfercholesterolesters which lose apo-Eand get convertedto LDL. The from HDL to VLDL or LDL, in exchangefor TC. Conversion

cf tJtr-S[- 1".;"LF{.

320

B IOC H E MIS TR Y

LDL receptors and supply of cholesterol t o t is s ue s The most important function of LDL is to supply cholesterol to the extrahepatic tissues. The LDL particles bind to the specific pits receptor (identified as glycoprotein) on the cell membrane.The shapeof the pit is stabilized by a protein called clathrin. Apo Bles is responsiblefor the recognition of LDL receotorsites.

hol esterol (C )

Bileacidsand cholesterol (in bile)

Deficiency of LDL receptors : A defect in LDL receptorsresultsin the elevationof plasrnaLDL, hence plasma cholesterol. However, plasma triacylglycerol concentration remains normal. Deficiency of LDL receptors is observed in type IIa hyperbetalipoproteinemia. This disorder is associated with a very high risk of atherosclerosis(particu Iarly of coronary artery).

High density lipoproteinsare synthesizedin t he liv er a s d i s c o i d a l p a rti c l e s -n a s c entH D L. They contain free cholesteroland phospholipids (mostlylecithin)and apoproteins(A, Cll, E etc.). Role of LCAT in HDI metabolism : The plasma enzyme lecithin-cholesterol acyttransferase(LCAT) catalysesthe esterification of free cholesterol(by fatty acid of lecithin)present in the extrahepatictissuesand transfersto the HDL. Apoprotein A promotes the activity of LCAT. HDL also accepts free cholesterol from other lipoproteins in circulation and cell membraneof peripheraltissues(Fig.|4.56). Any free cholesterol taken up by HDL undergoes LCAT-catalysed esterification. Due to the addition of cholesterol,HDL particles become s pher ic al .

receptor-mediated endocytosis.ln the liver, the cholesterylesters are degraded to cholesterol. The latteris utilizedfor the synthesisof bile acids and lipoproteins or excreted into bile (as cholesterol). Functionsof HD[ 1. Transport of cholesterol (as ester) from peripheraltissueto liver for its degradationand excretion (scavengeraction). 2. HDL servesas a reservoirof apoproteins. They accept apoproteins(Cll and E) and donate the sameto other lipoproteins-chylomicrons and VLDL (SeeFig.IaSD. 3. The apoprotein Cll of HDL servesas an activatorof lipoprotein lipase.

Inherited disorders of lipoproteins are encounteredi n some i ndi vi dual s resul ti ng i n primary hyper- or hypolipoproteinemias. These are due to genetic defects in lipoprotein metabolism and transport. The secondary acquired lipoprotein disordersare due to some other diseases(e.g. diabetesmellitus, nephrotic syndrome,atherosclerosis, hypothyrodismetc.), resulting in abnormal lipoprotein pattern The HDL particles, with cholesteryl ester which often resemblesthe primary inherited trapped inside, enter the hepatocytes by a condi ti on.

321

Chapter 14: METABOLISM OF LIPIDS

HyperlipoproeinemiaType

Inueased plasma lipoprotein(s)

Increasedplasma Probablemetabolic lipid (nost) defect

Chylomicrons

Triacylglycerols Deficiency of lipoproteinMayincrease

Riskof atherosclerosis

Suggested trcatment Lowlat diel

lipase

Deficienry of LDL receptors Triacylglycerols Overproduction of andcholesterol apo-B in apo-E TriarylglycerolsAbnormality andcholesterol Triacylglycerols Overproduction of TG Gholesterol

lla

ill

IDL

IV

VLDL

andVLDL Triacylglycerols Chylomicrons

Veryhigh(mostly in Lowcholesterol fat artery) diet:cholestvramine coronary

Veryhigh(mostly in Lowfat and low peripheral vessels)caloricdiet;clofibrate Mayor maynol Increase -00-

Lowfal and low caloricdiet;niacin

-do-

5. Type lV : This is due to overproductionof endogenous triacylglycerolswith a concomitant Elevationin one or more of the lipoprotein rise in VLDL. Type lV disorder is usually fractions constitutes hyperlipoproteinemias. associatedwith obesity, alcoholism, diabetes These disorders may be either primary or mel l i tusetc. secondary.Some authors use hyperlipidemiasor dyslipidemiasinsteadof hyperlipoproteinemias. 6. Type V : Both chylomicronsand VLDL are Frederickson's classification of hyperliporoelevated.This is mostly a secondarycondition, tei nemias-based on the electrophoretic patterns due to disorderssuch as obesity, diabetesand of plasma lipoproteins-is widely accepted to excessivealcohol consumptionetc. understand these disorders. lt is given in Hypo!ipoproteinemias Table 14.6 and briefly discussedhereunder. '1. Type | : This is due to familial lipoprotein A l though l ow l evel s of pl asma l i pi ds (not Iipase deficiency. The enzyme defect causes HDL!) within the normal rangemay be beneficial increase in plasma chylomicron and triacyl- to the body, very low lipid levels are glycerol levels. undesirable. These are commonly associated with certain abnormalities hyperbeta2. Type lla : This is also known as Hyperlipoproteinemias

lipoproteinemiaand is causedby a defect in LDL receptors. Secondary type lla hyperlipoproteinemia is observedin associationwith diabetes mellitus, hypothyroidism, nephrotic syndrome etc. This disorder is characterized bv hypercholesterolem ia.

1. Familialhypobetalipoproteinemia : lt is an i nheri ted di sorder probabl y due to an impairmentin the synthesisof apoproteinB. The plasma LDL concentration in the affected i ndi vi dual s i s betw een 10 to 50% of normal val ues. Thi s di sorder i s harml ess, and,the 3. Type llb : Both LDL and VLDL increase individualshave healthy and long life. along with elevation in plasma cholesteroland 2. Abetalipoproteinemia : This is a rare t r iac y lgly c e ro l T . h i s i s b e l i e v e d to b e due to disorder due to a defect in the synthesisof overproduction of apo B. apoprotein B. lt is characterized by a total 4. Type lll : This is commonly known as broad beta disease and characterized bv the appearanceof a broad p-band correspondingto intermediate density lipoprotein (lDL) on electrophoresis.

absence of B-lipoprotein (LDL) in plasma. Triacylglycerolsare not found in plasma, but they accumulate in liver and intestine.Serum cholesterollevel is low. Abetalipoproteinemiais associated with decreased absorotion of fat

322

B IOC H E MIS TR Y

and f at - s o l u b l ev i ta m i n s .l m p a i rme n ti n physi cal ffig.|aJV. In the normal liver, Kupffer cells growth and mental retardation are commonly contain lipids in the form of droplets. In fatty observed. liver, dropletsof triacylglycerolsare found in the entire cytoplasm of hepatic cells. This causes Familial alpha-lipoprotein deficiency (Tangier in metabolicfunctionsof liver. Fatty impairment disease): The plasma HDL particlesare almost l i ver i s associ atedw i th fi broti c changes and absent. Due to this, the reverse transport of Fatty l i ver may occur due to tw o mai n ci rrhosi s, cholesterol is severely affected leading to the causes. accumulationof cholesterylestersin tissues.An absence of apoprotein C ll-which activates 1. Increasedsynthesi sof tri acyl gl ycerol s lipopr o te i n l i p a s e -i s a l s o fo u n d . T h e pl asma 2. l mpai rmenti n l i poprotei nsynthesi s. triacylglycerollevels are elevated.The affected individualsare at an increasedrisk for atheros1. Increased triacylglycerol synthesis ; c ler os i s . Mobilization of free fatty acids from adipose ti ssueand thei r i nfl ux i nto l i ver i s much hi gher than thei r uti l i zati on. Thi s l eads to the overproducti on of tri acyl gl ycerol sand their T he n o rm a l c o n c e n tra ti o no f l i p i d (mostl y accumulation in liver. Diabetes mellitus, phos ph o l i p i d i)n l i v e r i s a ro u n d 5 7 o .L i ver i s not starvation, alcoholism and high fat diet are a storage organ fo r fa t, u n l i k e a d i p ose ti ssue. associatedwith increasedmobilization of fatty However, in c e rta i n c o n d i ti o n s, l i pi dsacids that often cause fatty liver. Alcohol also es pec ia l l y th e tr i acy lgly cerolc- ac cu m u I ate inhibits fatty acid oxidation and, thus, promotes ex c es si v e l yi n liver, resulting in fatty liver fat synthesi sand i ts deposi ti on.

EIOMEI'ICAL/ CLINICAT COHCEPTS

Niemann-Pick disease,caused by a delect in the enzgme sphingomyelinase, results in the accumulation of sphingomyelinsin liuer and spleen, About a dozen glycolipid storoge diseosesare known. These include Gaucher'sdisease and Krabbe's diseose. Hypercholesterolemia is ossocioted with atherosclerosis and coronary heart diseases. Consumption of polyunsaturated fottg ocids and liber decreoses cholesterol in circulation. Drugs---+uchqs louastatin, cholestgramine, compactin and clofibratereduce plasma cholesterol. Cholelithiasis, a cholesterol gall stone disease,is coused by o defect in the absorption of bile salts from the intestine or biliary trqct obstruction. oi' High density lipoproteinsln association with lecithin-cholesterol acyltrans' ferase (LCAT)----areresponsible lor the transport and elimination of cholesterol from the body. Hyperlipoproteinemiasare a group ot' disorderscausedby the eleuation of one or more of plasma lipoprotein fractions. Excessiueaccumulation ot' triocylglycerols couses fatty liuer which can often be preuented by the consumption of lipotropic t'actors(choline, betaine, methionine).

323

Chapt er 14 : M ET A BOL IS O M F L IPID S

Diabetes Siarvation Alcohol Free fatty acids

Cholesterolt

Apo B

(free+ester) |

Protein synrnests

?1[?S#I

Carbon tetrachloride

Nascenl VLDL

II + VLDL

2. lmpaired synthesisof lipoproteins : The synthesisof very low density lipoproteins(VLDL) actively takes place in liver. VLDL formation requiresphospholipidsand apoprotein B. Fatty liver caused by impaired lipoprotein synthesis may be due to : . a defect in phospholipidsynthesis; . a block in apoproteinformation;

o a failure in the formation/secretionof lipoprotein. Among the three causes,fatty liver due to i mpai rmenti n phosphol i pi dsynthesi shas been studied in some detail.This is usuallyassociated with the dietary deficiency of lipotropic factors such as choline, betaine, inositol etc. (more details given later). Deficiency of essentialfatty

324

B IOC H E MIS TFIY

acids leads to a decreased formation of D efi ci ency of vi tami n E i s associ atedwit h phos p h o l i p i d s .F u rth e r,e x c e s s i v econsumpti on fatty liver. Seleniumacts as a protectiveagent in of cholesterolcompeteswith essentialfatty acids such a condi ti on. and i mp a i rsp h o s p h o l i p i ds y n th e si s. Endocrine factors : Certain hormones like Ce rta i nc h e mi c a l s(e .g .p u ro m y ci n,ethi oni ne, A C TH , i nsul i n, thyroi d hormones, adrenoc ar bo n te tra c h l o ri d e , c h l o ro form, l ead, corti coi dspromotedeposi ti onof fat i n l i ver . phos p h o ru se tc .) th a t i n h i b i t p ro t ei n synthesi s c aus efa tty l i v e r.T h i s i s d u e to a b l o ckadei n the s y nt h e s i so f a p o p ro te i n B re q u i r ed for V LD L or od u c ti o n . These are the substancesthe deficiency of Li p o p ro te i n s y n th e s i s a n d th e i r secreti on which causesfaf (triacylglycerol)to accumulate r equ i reA T P. D e c re a s ei n th e a v a i l a bi l i tyof A TP , in liver. This may happen despitethe fatty acid s om e ti me sfo u n d i n p y ri d o x i n ea n d pantotheni c synthesi sand uptakeby l i ver bei ng normal . ac id d e fi c i e n c y ,i m p a i rs l i p o p ro tei nformati on. T he a c ti o n o f e th i o n i n e i n th e d e vel oomentof fatty liver is believedto be due to a reductionin t he a v a i l a b i l i tyo f A T P. E th i o n i n ec o mpetesw i th These include choline, betaine, methionine r net h i o n i n ea n d tra p s th e a v a i l a b l e adenosi ne and i nosi tol .Fol i c aci d, vi tami n 812,gl yci neand ( as a d e n o s y l e th i o n i n e )-th e re b reduci y ng A TP serine also serve as lipotropic factors to some lev el s . extent.

EIOMEDICALI CHNICiAL COHCEPTS

s$ Obesity is an abnormal increasein body weight due to excessiuet'at deposition(>250/o). Ouereating, Iack of exercise and genetic predisposition play a significont role in the deuelopment oJ obesity. w Some indiuiduals with octiue brown adipose fissue do not become obese despite ouereating,since whateuerthey eat is liberated as heat due to uncoupling of oxidation and phosphorylationin the mitochondrio. us A protein namely leptin, produced by the adipose fissue, hqs been identified in mice. Injection of leptin to obesemice causedreduction in body t'at, increasedmetabolic rote ond increasedinsulin concentration, besidesreducedJood intake. Leptin has also been detectedin humans. n* Anorexia neruosais a psychiatricdisorder ossociatedwith total loss o/ appetite-mostly found in females in the age group 70-30 years. ut Atherosclerosisis characterizedby hardening of arteries due to the accumulation of Iipids ond other compounds. The probable cduses of atherosclerosis include hyperlipoproteinemias,diabetes mellitus, obesity, high consumption of soturated fat, lack of exerciseond sfress. sq Atherosclerosis and coronary heart disease are directly correlated with plasma cholesterol and LDL, inuersely with HDL. Eleuation of plasma lipoprotein o suggesfs increosed risk of CHD. rs' Alcoholism is ossociotedwith t'atty liue4 hyperlipidemia and atherosclerosis.

Ghapter 14 : METABOLISM OF LIPIDS A c t ion

o f l i p o tro p i c

fa c to rs

Cholin e a n d i n o s i to l a re c o m o o n ents of phos phol i p i d sa n d , h e n c e , re q u i re d for thei r synthesis. The other lipotropicfactorsare directly or indirectly concerned with transmethylation r eac t ions a n d , u l ti m a te l y , th e s y n t hesi s of choline. Severe protein deficiency (e.g. kwashiorkor)causesfatty liver. This is due to a defect in the synthesisof choline as a result of ins uf f ic ie nat m i n o a c i d (p a rti c u l a rl ym e t hi oni ne) s upply . I n o th e r w o rd s th e n o n -a v a i l abi l i tyof methyl groups may lead to fatty liver (Fig.|4.37). Gholine

deficiency

and {atty

tiver

Severalexplanationsare offeredto understand c holine de fi c i e n c yc a u s i n gfa tty l i v e r :

32s BMt (kg/m2l= l"'tnt'ut'(m)21 lheisht

Obesity is categorizedinto three grades . Crade I obesity or overweight - BMI 25-30 ke/m2 . Grade l l or cl i ni cal obesi ty- B MI > 30 kg/m2 . Crade lll or morbid obesity- BMI > 40 kg/m2 Obesity is associated with many health complications e.g. type ll diabetes, CHD, hypertension, stroke, arthritis, gall bladder disease.Hence, treatmentof obesity assumesa lot of significance in the prevention of these d iseases.

ln recent years, the ratio between waist and hip sizes(for men < 0.9 and for women < 0.85) 1. Dec re a s e d phospholipid synthesi s is considered as more effective than BMI, $ig.ta37) particularly with regard to the risk of heart 2. lmpaired formation of lipoprotein mem- diseases.The lower is the waist to hip ratio the orane l ow er the ri sk for heal th compl i cati ons.ano therefore better is the health. 3. Red u c e d s y n th e s i so f c a rn i ti n e due to insufficientsupply of methyl groups Geneti cs, ohesi tv and l epti n 4. lmpairment in fatty acid oxidation. There is strong evidence to suggest that obesity has genetic basis.Thus, a child born to two obese people has about 25% chances of being obese. One gene namely ob gene, Obesity is an abnormal increasein the booy expressedin adipocytes(of white adiposetissue) weight due to excessivefat deposition. producing a protein called leptin (mol. wt. I6,000 dal tons), i s cl osel y associ atedw i th Nut r it iona l b a s i s obesity. Men and women are consideredas obese if t heir weig h t d u e to fa t (i n a d i p o s e ti ssue), respectively,exceeds more than 20"/" and 25oh of body weight. Obesity is basicallya disorderof ex c es s c a l o ri e i n ta k e , i n s i m p l e l a n guageovereating. lt has to be rememberedthat every 7 caloriesof excessconsumption leads to 1 g fat depos it a n d i n c re a s e i n b o d y wei ght. Overeating-coupled with lack of physical exercise-contribute to obesity. B ody r na s s i n d e x

tBMl l

Leptin is regarded as a body weight regulatory hormone. lt binds to a specific receptorin the brain and functionsas a lipostat. When the fat stores in the adipose tissue are adequate,l epti n l evel sare hi gh. Thi s si gnal sto restrict the feeding behaviour and limit fat deposi ti on. Further, l epti n sti mul atesl i pol ysi s and i nhi bi ts l i pogenesi s. A ny geneti c defect i n leptin or its receptor will lead to extreme overeating and massive obesity. Treatment of such obese i ndi vi dual s w i th l epti n has been shown to reverseobesity.

Clinical obesity is representedby body mass D uri ng starvati on,l epti n l evel s fal l w hi ch index . B M I i s c a l c u l a te d a s th e w e i ght (i n promote feeding, and fat production and its k ilogr am s)d i v i d e d b y th e h e i g h t (i n m e t ers2). deposi ti on.

326 Oh'c,rsrt:fand adEpsse tissue There are two types of adipose tissues t. White adipose tissue : The fat is mostly storedand this tissueis metabolicallylessactive. 2. Brown adipose tissue : The stored fat is relativelylessbut the tissueis metabolicallyvery active. Brown adipose tissue possesses high proportionof mitochondriaand cytochromesbut low activity of ATP synthase.This is an active centre for the oxidation of fatty acids and glucose and is responsiblefor the diet-induced thermogenesis. The peculiarity of mitochondria of brown adipose tissue is that the oxidation and phosphorylationare not coupled. Mitochondrial oxidation produces more heat and less AIP. A specific protein-namely therhogenin-has been isolated in the inner membrane of these mitochondria.Thermogeninfunctions like an uncoupler and dissipatesthe energy in the form of heat, and thus blocks the formation of ATP. Brown adipose tissue is mostly found in hib e rn a ti n ga n i m a l s ,a n d th e a n i m al sexposedto cold, besides the newborn. In adult humans, though not a prominent tissue, it is located in the thoracic region. lt is significantto note that brown adipose fissue is almost absent in obese percons.Some individualsare fortunateto have active brown adipose tissue. They eat and liberateit as heat with the resultthat thev do not become obese.

CACHEXIA

B IOC H E MIS TRY

is aplenty.And some membersin thesefamilies mav be even obese!Anorexia nervosais more a psychiatricdisease.

XANTHOMATOSIS The depositionof yellow-orangecolour lipids in liver, spleen and flat bones of the skull is known as xanthomatosis (Creek: xanthosyellow). This is usually associatedwith severe hyperlipidemiaand hypercholesterolemia.

Atherosclerosis(Creek athere-mush) is a complex diseasecharacterizedby thickening or hardening of arteries due to the accumulation of lipids (particularly cholesterol, free, and esterified)collagen,fibroustissue,proteoglycans, calcium depositsetc. in the inner arterial wall. Atherosclerosisis a progressivedisorder that narrows and ultimately blocks the arteries. lnfarction is the term used to indicate the stoppageof blood flow resultingin the death of affected tissue. Coronary arteries--the arteries supplying blood to heart-are the most commonly affected leading to myocardial infarction or heart attacks. and coronary The incidenceof atherosclerosis heart diseasesare higher in developedcountries (e.g.U S A , U .K .)than i n the devel opi ngcount r ies (lndia, Africa etc.). Causes of atherosclerosis and CHD : The and the risk for developmentof atherosclerosis the coronary heart disease (CHD) is directly correlatedwith plasmacholesteroland LDL. On the other hand, pl asma H D L i s i nver sely correlatedwith CHD.

This is exactly opposite of what is seen in obesity.Cachexiais characterizedby a failure to maintain normal lipid stores in the body. It B[sorcfers $h*e lttay Gause involveshigher rate of fat mobilizationthan the atherosclerosis deposition.In extremecases,the adiposetissue Certain diseasesare associatedwith atherosmay totally disappear. cl erosi s. These i ncl ude di abetes mellit us, Anorexia nervosa is a total loss of appetite. hyperlipoproteinemias, nephrotic syndrome, This is mostly seen in females in the age group hypothyroidism etc. Many other factors like 10-30 years.Surprisingly,majorityof the affected obesity, high consumption of saturated {at, individualsare from wealthv familieswhere food excessivesmoking, lack of physical exercise,

f,IhAPtEr 1TT: METABOLISM OF LIPIDS

327

hypertension, stress etc., are the probable Alcohol (ethanol or ethyl alcohol) is readily causesof atherosclerosis. absorbed by the stomach and intestine. Consequently, less than 2"/' of the alcohol h$e0$tieci"e fuettm*en $Sf;)L and GHD consumed is excretedthrough lungs, urine and sweat. The increasedlevels of plasma HDL (good cholesterol)are correlatedwith a low incidence Alcohol gets oxidized in the liver by alcohol of cardiovasculardisorders.Women have higher dehydrogenaseto acetaldehyde. HDL and are less prone to heart diseases compared to men. This is attributed to estrogens Alcohol dehvdrooenase in women. Strenuous physical exercise, cH3-cH2-oH -CH3CHO moderate alcohol intake, consumption of Alcohol NAD+ NADH+ H+ Acetaldehyde unsaturatedfatty acids (vegetableand fish oils), reduction in body weight-all tend to increase B esi desA D H , mi crosomalethanol oxi di zi ng HDL levelsand reducethe risk of cardiovascular system (MEOS) is also involved in the diseases.Some more details on cholesteroland metabolismof alcohol. Aldehyde, produced by atherosclerosis are given under hyper- the action of eitherADH or MEOS,is responsible cholesterolemia. for the manifestations of alcohol The enzyme aldehyde dehydrogenaseconverts aldehyde to l" ipopr ot e i n a a * d e H D acetic acid which then entersKrebscycle in the Lipopr o te i na (L p -a ) i s a l m o s t i d e n t i cal i n form of acetyl CoA. structure to LDL. Lp-a contains an additional Aldehyde apopr ot ein , a p o -a . L p -a i n h i b i ts fi b ri nol ysi s. dehvdrooenase ---7=-----+ cH3-cHo cHscooH Recent studies have shown that elevation of AcetaldehydeNAD* NADH+ H+ Aceticacid lipoprotein-ain the plasma (>30 mgldl) suggests increasedrisk of CHD. lt is hypothesizedthat Since the activity of aldehydedehydrogenase elevated Lp-a reduces the breakdown of blood is less than that of alcohol dehydrogenase, clots and triggersheart attacks. acetaldehyde accumulates leading to various complications.Disulfiram, a drug used for the lhmtlsxidamts amd atherosclerosis treatment of alcoholism, inhihits aldehyde Antioxidants, in general, decrease the dehydrogenase. oxidationof LDL. There is some evidence,based changes in alcoholisrn on t he epi d e mi o l o g i c a ls tu d i e sth a t ta ki ng of Biochemical antioxidants(vitamins E and C or p-carotene) The metabolism of alcohol (by both reducesthe risk of atherosclerosis, and thereby dehydrogenases)involves the consumption of CHD. However, more researchis neededin this NAD+, and consequentlya high NADH/NAD+ dir ec t ion. ratio.This is mostly responsiblefor the metabolic alterationsobservedin alcoholism.Someof them are listed. 1. High concentrationof NADH favoursthe conversionof pyruvateto lactatewhich may lead to lactic acidosis.

W alk er h a s ri g h tl y s a i d ' a l c o h o l c a n be a food, a drug or a poison dependingon the dose.' In small quantities,alcohol relievestension and 2. Hypoglycemia due to reduced gluconeoanxiety. Unfortunately,consumptionof alcohol genesis is observed. This happens as a result s eldom en d s w i th s ma l l d o s e s , h e n ce the of decreased availability of pyruvate and beneficial effects are over-shadowed by the oxaloacetate(the latter gets converted to malate harmful effects. by hi sh N A D H ).

B IOC H E MIS TFI Y

328 3. C i tri c a c i d c y c l e i s i mp a i re d si nce the availabilitv of oxaloacetate and NAD+ is reduced. As a result, acetyl CoA accumulates which gets diverted towards ketogenesis, cholesterologenesis,and fatty acid synthesis. Accumulation of fats leads to fatty liver and hy pe rl i p i d e mi a . 4. Increasedconcentrationof serum uric acid due to its reduced excretion is observed in alc oh o l i s m .T h i s i s d u e to l a c ti c a ci dosi s.

2.

3.

4.

5.

6.

7.

the 5. Acetaldehyde interferes with overall an with functioningof neurotransmitters, effect of neurologicaldepression. 6. Acetaldehyde causes headache, nausea/ tachvcardia,reduced blood pressureetc. E ffects

sf * i xrur' si c,a* * cl ttaE i snt

C hroni c al cohol i sm i s associ ated wit h cirrhosis of liver, neurodegenerativechanges, cardiomyopathy,diuresis,impotenceetc'

tissue. Tiiacylglycercls(TG) are the highly concentratedform ot' energy, stored in adipose os tronsported are which acids to TG hydrolyses lipase fatty t'ree Hor^ii.-r"nsitiue albumin-FFA complexes. where Fotty acidsare octivated (acil CoA) and transported by carnitine to mitchondria of one oxidotion Complete energy. to liberate (mostly by ftoxidation) they get oxidized ATP 129 palmitate liberates mole ond Excessiue utilization ol fatty acids occurs in uncontrotled diabetes mellitus (in namely liuer), bodies ketone of starwtion. This resulrs in the ouerproduction acetone, acetoacetic acid ond fthydroxy butyric ocid. The lqst tttro ketone bodies serue as energy source t'or peripheral tissues. Fatty acid biosynthesis occursfrom acetyl coA in the cytosol through the inuoluement (ACP). The reducing ol a multienzyme complex associated with ocyl corrier protein shunt' HMP by mostly e.quiualents(NADPH + H+) are supplied Synthesis of triacylglycerols and phosphotipids (PL) occurs lrom glycerol 3-phosphate. ond dthydroxgo"etJi" phosphate *ith th" addition of acyl CoA, and actiuated nitrogenous bases (for PL). CoA, Cholesterol is synthesized lrom acetyl CoA in a seriesoJ reactions inuolving HMG crs serues Cholesterol the intermediates. as squalene ond units isoprenoid meualonote, a precursort'or bile acids, steroid hormones and uitomin D' Lipoproteins are the transport uehiclesfor lipids in the plasma. Lipoprotein disorders qie issocioted with abnormalities in their plasma leuels. Eleuation in LDL and WDLin association with cholesterol and TG-poses o serious heatth problem with increased

rtsk of atherosclerosisand CHD. be due 8. Excessiueaccumulotion of triocylglycerols in liuer causeslatty liuer, which may The (VLDL) synthesis' lipoprotein in impairment ir production of TG to increased lipotropic called subsfonces certoin oJ det'iciency the with associoted mostly latter is lactors (e.g, choline, betaine, methionine etc.) to fat)' 9. Obesity is an obnormal increase in body weight (with more than 250/o due fissues adipose brown active oJ lack obesity, ol Among the many causatiue Jactors (which burn fat ond liberate heat) in these indiuiduols is gaining importance. to the, 70. Atherosclerosisis o complex disease choracterized by thickening ol arteries due and LDL with correlated directly are occumulation of lipids. Atherosclero.sisond CHD inuersely with HDL o

Ghapter 14: METABOLISM OF LIPIDS

329

I. Essayquestions 1. Describethe functionsand metabolismof phospholipids. 2. Cive an account of cholesterolbiosynthesis. Add a note on the significanceof plasma cholesterolestimation. 3. Describein detail the extramitochondrial synthesis of fatty acids. 4. Write aboutthe types,characteristics and metabolismof lipoproteins. Add a noteon lipoprotein disorders. 5. Give an accountof fatty acid oxidation. II. Short notes (a) Carnitine, (b) LCAT,(c) Fatty liver, (d) Ketone bodies, (e) Lipotropic factors, (fl Acyl carrier protein,(g) Degradationof cholesterol,(h) HDL, (i) Lipoproteinlipase,(j) Brown adiposetissue. III. Fill in the blanks The most predominantlipid componentof chylomicrons 2. Cholesterolsynthesisis controlledby feedbackinhibitionof the enzyme 3. A compound possessinghydrophobicand hydrophilic groups in its structureis known as 4. Niemann-Pickdiseaseis due to a defectin the enzvme 5. The lipoproteininvolvedin the reversecholesteroltransportis 6. The total number of ATP produced by the oxidation of a molecule of palmitic acid is 1

7. The long chainfattyacids(C26-C35)are not oxidizeddue to the absenceof peroxisomes. This disorderis known as 8. Acetyl CoA from the mitochondria is transportedinto the cytosol after iG conversion to 9. Plasmalipoproteinthat is inverselycorrelatedwith coronaryheartdiseaseis 10. The fatty acid that is commonlyfound in the C2 of triacylglycerols is IV. Multiple choice questions 11. The following substance(s) is (are)ketogenic (a) Fattyacids(b) Leucine(c) Lysine(d) All of them. 12. The lipoproteinpossessing the highestquantityof phospholipid ( a ) H D L (b ) L D L (c ) V L D L (d ) C hvl omi crons. 13. Hypercholesterolemia is observedin the disorder(s) (a) Hypothyroidism(b) Diabetesmellitus(c) Nephroticsyndrome(d) All of them. 14. The two final productsin the B-oxidationof odd chain fafty acidsare (a) Acetyl CoA and malonyl CoA (b) Acetyl CoA and acetyl CoA (c) Acetyl CoA and propionyl CoA (d) Acetyl CoA and succinylCoA. 15. Hormonesensitivelipaseactivityis inhibitedby the hormone (a) Epinephrine(b) Insulin(c) Thyroxine (d) Clucocorticoids.

Aefrdl$ Nflmtmhollfismm mf,Ammirmo

irir'1rrcrirclrs o-Ketogruta.at€

TFsessrsine eeids sglech :

TEnsamination N ] \+ctut"."t" II i.eit' :r r l.

I Deamina + NHg

+

,J;cr

roteins are the most abundant organic E compounds and constitute a major part of the body dry weight (10-12 kg in adults).They perform a wide variety of static (structural)and dynamic (enzymes,hormones, clotting factors, receptorsetc.) functions.About half of the body protein(predominantlycollagen)is presentin the supportivetissue(skeletonand connective)while t he ot h e r h a l f i s i n tra c e l l u l a r.

mel ani n,serotoni n,creati neetc.).C ertai nami no acids may directly act as neurotransmitters (e.g. glycine aspartate, glutamate). Protein metabolism is more appropriately learnt as metabolism of amino acids.

Proteins are nitrogen-containing macroAn adult has about 100 g of free amino acids molecufes consisting of L-a-amino acids as the repeatingunits. Of the 20 amino acids found in which representthe amino acid pool of the proteins, half can be synthesizedby the body body. The ami no aci d pool may be an (non-essential) while the resthaveto be provided oversimplificationof the facts, since there is no singlecompartment-rather,severalcompartin t he d i e t (e s s e n ti aal m i n o a c i d s ). ments exrst. The proteins on degradation (proteolysis) r eleas ei n d i v i d u a la mi n o a c i d s .A m i no aci ds are Glutamate and glutamine together constitute not just the structuralcomponentsof proteins. about 50"/", and essentialamino acids about E ac h o n e o f th e 2 0 n a tu ra l l yo c c u rri ng ami no 10"h of the body pool (100 g). The concentration acids undergoes its own metabolism and of i ntracel l ul ar ami no aci dsi s al w ayshi ghertha n performsspecific functions.Some of the amino the extracel l ul ar ami no aci ds.A mi no aci dsenter acids also serveas precursorsfor the synthesisof the cells against a concentration gradient by m any b i o l o g i c a l l y i m p o rta n t c o mpounds (e.g. active transoort.

330

33r

OF AMINO ACIDS Ghamcer1$i: METABOLISM The amino acid pool of the body is maintained by the sourcesthat contribute (input) and the metabolic pathwaysthat utilize (output) the amino acids(Fi9.15.1). 6" &.:titrr{;{+s t.",i&i,ztirtt.rr {"r,ihid$xee!

Protein breakdown Protein synthesis (350-400 g/day) (300-400 g/day)

Synthesis of non-protein compounds (30g/day;creatine, porphyrins, phospholipids, purines, pyrimidines etc.)

Dietaryprotein (40-100g/day)

Turnover of body il,oily protein, intake of dietary arrrrlC aCid pool i 100;) protein and the synthesisof non- ess e n ti a a l mino acids contribute to the bodv Synthesisof non- Proteinloss from essentialamino body (30-50 g/day) am ino a c i d p o o l . acids (variable)

J

Carbohydrates, fat

Energy(10-15%of body'sdailyrequirement)

(a) Frotein turnover : Utilization Sources The protein present in the body is in a dynamic state. It is estimated that about Urine 300-400 g of protein per Fig. 15.1 : Overview of body's amino acid pool-sources and utilization. day is constantlydegraded and s y n th e s i z e d w h i c h represents body protein There is no storage form of amino acids as is turnover. There is a wide variation in the proteins. the case for carbohydrates(glycogen)and lipids For instance,the turnoverof individual plasma proteins and digestive enzymes are (triacylglycerols).The 6xcess intake of amino rapidly degraded,their half-livesbeing in hours acids are metabolized-oxidized to provide or days. The structuralproteins (e.8. collagen) energy/ convertedto glucose or fat. The amino have long half-lives,often in months and years. groupsare lost as urea and excreted.The protein consumpti on i n devel oped countri es i s much Control of protein turnover : The turnover of higherthan the recommendeddietaryallowance the protein is influenced by many factors. A (i.e. lgikg body weighVday).The daily protein smafl polypeptide called ubiquitin (mol. wt. intakeby an adult in most countriesis 40-100 g. 8,500) tags with the proteins and facilitates Protein is digested by proteolytic enzymes to degradation.Certain proteins with amino acid amino acids which are absorbedin the intestine sequenceproline, glutamine(one letter code E), and enter the body pool of amino acids. serineand threonine(PESTsequence)are rapidly (c) Synthesis of non-essential amino acids : degraded. Ten out of the 20 naturally occurring amino (b) Dietary protein : There is a regularlossof acids can be synthesizedby the body which nitrogen from the body due to degradation of contributeto the amino acid pool. am ino a c i d s . In h e a l th y a d u l ts , i t i s esti mated that about 30-50 g of protein is lost everyday dfr"*lttlieatiour of amino acfids from the body. This amount of protein (30-50 g/ trorn rrody pool day) must, therefore, be supplied daily in the (a) Most of the body proteins (300-400 g/day) diet fo maintain nitrogen balance.The purpose of dieta ry p ro te i n i s to s u p p l y a m i no aci ds degraded are synthesizedfrom the amino acid (particularlythe essentialones) for the synthesis pool. These include enzymes, hormones, immunoproteins,contractileproteinsetc. of proteinsand other nitrogen compounds.

J

BIOCHEMISTFIY

332

Dietary Body protein protein

(b) Many important nitrogenouscompounds (porphyrins, purines, pyrimidines, etc.) are produced from the amino acids. About 30 g of protein is daily utilized for this purpose.

Synthesis of N-compounds

synthesis

(c) Generally,about 1O-15o/o of body energy requirementsare met from the amino acids.

aza-Ketoglutarate

(d) The amino acids are converted to carbohvdrates and fats. This becomes predominantwhen the proteinconsumptionis in excessof the body requirements.

Ketoacids

J--')l +++

The amino acids undergo certain common reactions like transamination followed by deamination for the liberation of ammonia.fhe am in o g ro u p o f th e a m i n o a c i d s i s' uti l i zed for the formation ol urea which is an excretoryend product of protein metabolism. The carbon skeletonof the amino acids is first convertedto keto acids (by transamination)which meet one or more of the following fates. 1. Utilized to generateenergy. 2. Used for the synthesisof glucose. 3. Diverted for the formation of fat or ketone bodies.

Urea

I t---.

Energy GlucoseFat

Non'essential aminoacids

Fig. 15.2 : An overuiewof amino acid metabolism.

Salient

features

of transamination

require pyridoxal phos1. All transaminases (PLP), phate a coenzyme derived from vitamin B 6. exist for each pair 2. Specifictransaminases of amino and keto acids. However, only twonamely, aspartate transaminase and alanine transaminase-make a significant contribution for transamination.

4. lnvolved in the productionof non-essential 3. There is no free NH3 liberated,only the am in o a c i d s . transferof amino group occurs. A generalpictureof amino acid metabolismis depicted in Fi9,15,2.

4. Transaminationis reversihle(Fig.l5.3).

The detailsof generaland specific metabolic reactionsof amino acids are described in the following pages.

COO-

R1-

R1-C-COO-

o

Amino acid-l

Keto acld-l

TRANSAMINATION fhe tansfer of an amino (- NH2) group from an amino acid to a keto acid is known as transamination. This process involves the interconversionof a pair of amino acids and a pair of keto acids, catalysed by a group of enzymes called transaminases (recently, aminotransferased.

R2-C-COO-

R2-

OO-

o Keto acid-ll

Amino acid-ll

Fig. 15.3 : Transaminationreaction. L

333

OF AMINO ACIDS Chapter'1 5: METABOLISM 5. T ra n s a mi n a ti o ni s v e ry importantfor the redistribution amino of B ro u p s a n d production of non'essential amino acids, as per the requirement of the cell. lt catabolism involves both (degradation)and anabolism (synthesis) of amino acids. 6. Transamination diverts t he e x c e s s a mi n o a c i d s towards energy generation. acids a mi n o 7. Th e undergo transamination to finally concentratenitrogen in glutamate. Glutamate is the only amino acid that undergoes oxidative deamination to a significant extent to liberate free NH3 for urea synthesis.

(A)

-o-E

Pyridoxalphosphate

Tnl

ll

c-cooI QHz I CH, t-

CHc tCHc t-

coo-

coo-

cr-Ketoglutarate

Glutamate

n ..cH-cooi

9. T ra n s a m i n a ti o ni s n o t restrictedto c-amino BrouPS only . F o r i n s ta n c e , 6 -a m i n o is gr oup of o rn i th i n e transaminated.

of Mechanism transamination T r a n s a m i n a ti o no c c u rs i n two stages (FigJ5.a)

Pyridoxamine phosphate

H-C-COO-

8. Al l a mi n o a c i d s e x c e p t ly s ine ,th re o n i n e ,p ro l i n e a n d hydroxyproline participate in t r ans a m i n a ti o n .

10. S e ru m tra n s a mi n a s e s are important for diagnostic prognostic purposes. and (Refer Chapter 6).

cH2-o-E

t,-*\Q-,

IFr"*l

cH2-o-E

I

(C H r) a I

NHz Enryme-PLP Schiffbase

Amino acid PLP-Schiff base

Fig. 15.4 : Mechanism of transamination-(A) lnvolvement ol pyridoxal phosphate (PLP) in the transfer of amino group, (B) Formation ol enzyme' PLP-Schiff base and amino acid-PLP-Schiff base. Note that when the amino acid binds, enzyme separates.

All the transaminases require pyridoxal phosphate(PLP),a derivativeof vitamin 85. The al dehydegroup of P LP i s l i nked w i th e-amino group of lysine residue,at the active site of the p yri doxami ne enzyme forming a Schiff base (imine linkage). g ro u p of 2. T h e a m i n o to When an amino acid (substrate)comes in keto acid phosphateis then transferredto a with with the enzyme, it displaceslysine and contact producea new amino acid and the enzyme new a S chi ffbase l i nkagei s formed.The amino PLP is regenerated.

1 . Transfer of the amino Sroup to the coenzyme pyridoxal phosphate (bound to the coenzyme)to form pyridoxaminephosphate.

334

BIOCHEIVIISTFIY

acid-PLP-Schiff base tightly binds with the enzyme by noncooI covalent forces. Snell and CHc tproposed Braustein a Ping c Pong Bi Bi mechanism H_C HI inv olv ing a s e ri e s o f t(a l d i m i n e s cooint er m ediat e s and k et im ines ) in tra n s a m i n a ti o n L-Glutamate reaction.

coocooI

H2r:

I CHc tCHr tCHe + NHi tC:{:

coo([-Iminoglutarate

c'Ketoglutarate

Fig. 15.5 : Oxidation ol glutamate by glutanate dehydrogenase (GDH).

DEAMINATIOH The removal af amino groupfrom the amino occursthroughthe formationof an intermediate, ac ids as NH3 i s d e a m i n a ti o n .T ra n s a mi n ati on o-im inogiutarate (Fig.IS.trt. (discussedabove) involvesonly the shufflingof Glutamatedehydrogenasecatalysedreaction am ino gr oups a mo n g th e a mi n o a c i d s . O n the i s i mportantas i t reversi bl yl i nks up gl utamate ot her hand, de a m i n a ti o nre s u l tsi n th e l i b e ra ti on metabolism with TCA cycle through cr-ketoof am m onia f o r u re a s y n th e s i sSi . m u l ta n e ousl y, gl utarate.GD H i s i nvol vedi n both catabol i cand the carbon skeletonof amino acids is converted anabol i creacti ons. to keto acids. Deamination may be either Regulation of GDH activity : Clutamate oxidative or non-oxidative. dehydrogenasei s a zi nc contai ni ng mi toAlthoughtransaminationand deaminationare chondri al enzyme. l t i s a compl ex enzyme separatelydiscussed,they occur simultaneously, consi sti ngof si x i denti caluni tsw i th a mol ecul ar often involving glutamate as the central w ei ght of 56,000 each. C D H i s control l ed by m olec ule.F or th i s re a s o n ,s o me a u th o rsu s e the allosteric regulation. GTP and ATP inhihitierm transdeaminafion while describing the whereas GDP and ADP activafe-glutamate reactions of transaminationand deamination. dehydrogenase.Steroid and thyroid hormones par t ic ular lyin v o l v i n gg l u ta m a te . i nhi bi t GD H . !. Oxidative

dearnination

Oxidative deamination is the liberation of free ammonia from the amino group of amino acids coupled with oxidation. This takes place m os t ly in liv e r a n d k i d n e y . T h e p u rp o se of oxidativedeaminationis to provide NH3 for urea synthesis and c-keto acids for a variety of reactions,including energy generation.

After ingestion of a protein-rich meal, liver glutamate level is elevated. lt is converted to with liberationof NH3. Further, u,-ketoglutarate w hen the cel l ul ar energy l evel s are l ow , the degradationof glutamateis increasedto provide a-ketoglutarate which enters TCA cycle to liberateenergy.

Oxidative deamination by amino acid oxidases: L-A mi noaci d oxi daseand D -ami noaci d Role of glutamate dehydrogenase : In the oxidase are flavoproteins,possessingFMN and crocessof transamination,the amino groups of the FAD, respectively. They act on rnost amino acids are transferredto a-ketocorrespondi ngami no aci ds (L or D ) to produce glutarateto produce glutamate. Thus, glutamate o-keto acids and NH3. In this reaction,oxygen is serves as a'collection centre' for amino groups reducedlo H2O2,which is later decomposedby in t he biolo g i c a l s y s te m . G l u ta m a te ra p i dl y catalase(Fig.l5.6). undergoesoxidative deamination,catalysedby glutamate dehydrogenase (CDH) to liberate The activity of L-amino acid oxidase is much am m onia.T hi s e n z y me i s u n i q u e i n th a t i t can low while that of D-amina acid oxidase is high utilize either NAD+ or NADP+ as a coenzvme. i n ti ssues(mostl yl i ver and ki dney).L-A mi noaci d Conversion of glutamate to cr-ketoglutarate oxidasedoes not act on glycine and dicarboxylic

chapter'n5 : METABOLTSM OF AMTNOACTDS

L-Aminoacio$acid

oxidlgact-Keto

335 undergo deami nati on coupl ed desulfhydrationto give keto acids.

acid+NH.

rvi t h

/\ Desulfhvdrases Cysteine-----\----------+ Pyruvate NH.+ HrS (c) Deamination of histidine : The enzyme hi sti daseacts on hi sti di neto l i berateN H 3 by a non-oxidativedeaminationorocess. Histidase Histidine ---------\------+

Fig. 15.6 : Oxidative deamination ol amino acids.

Urocanate

It

NH.

acids. This enzyme,due to its very low activity, does not appear to play any significantrole in the amino acid metabolism. Fate of D-amino acids : D-Amino acids are found in plants and microorganisms.They are, however,not presentin the mammalianproteins. B ut D- a m i n oa c i d sa re re g u l a rl yta k e n i n the di et and metabolized by the body. D-Amino acid oxidase converts them to the respectivea-keto acidsby oxidativedeamination.The cr-ketoacids so produced undergo transamination to be convertedto L-amino acids which participatein variousmetabolisms.Ketoacids may be oxidized to Benerateenergy or serve as precursorsfor gluc os ea n d fa t s y n th e s i sT. h u s , D -ami no aci d oxidase is important as it initiatesthe first step for the conversionof unnaturalD-amino acidsto L-amino acids in the body (Fig.l5.7).

A mmoni a i s constantl ybei ng l i beratedi n the metabolism of amino acids (mostly) and other ni trogenouscompounds. A t the physi ol ogi c al pH, ammonia exisfs as ammonium (NIfi ion. B" For*nati+g1 a;1imrurinar, mqm The production of NH3 occurs from the ami no aci ds (transami nati on and deami nati o n) , bi ogeni c ami nes, ami no group of puri nes and pyrimidines and by the action of intestinal bacteria (urease)on urea. EE"YfansS.r,cr?;r;E'S F:ti.i,si*tj€: +{ F!:i Despitea regularand constantproductionof NH3 from various tissues,its concentrationin

&$" Fdmm- mx $c$mt frwe n$eli:qir;ls$ilga+{ $€}fll D-Aminoacids

Some of the amino acids can be deaminated to liberate NH3 without undergoingoxidation (a) Amino acid dehydrases: Serine,threonine and ho mo s e ri n ea re th e h y d ro x y a m i no aci ds. They undergo non-oxidative deamination catalysed by PLP-dependent dehydrases (dehydratases). Serine .

In fgonlne- | Homoserine

Dehydratase

L-Amino

Resoective .'-Keloaclos

\

a,-Ketoacids

NH.

s-Keto acid

Transaminases

Enerov

t"

Glucose,Fat

(b)Amino acid desulfhydrases : The sulfur aminoacids,namelycysteine andhomocysteine,

Fig. 15.7 : Metabolic fate of D-amino aads

B IOC H E MIS TR Y

336

t he c ir c u l a ti o ni s s u rp ri s i n g l yl o w (n o r mal plasma 10-20 me/dl). This is mostly because the body has an efficient mechanism for NH3 transport and its immediate utilization for urea synthesis. The transport of ammonia between Glutamine various tissues and the liver mostly Glutamate glutamine or occurs in the form of alanineand not as free ammonia.Alanine is important for NH3 transport from muscle to liver by glucose-alaninecycle Fig. 15.8 : Synthesisof glutamine and its (Refer Fig.l3.l3). conversion to glutamate. (Note : The reactions are independent and irreversible). Role o f g l u ta mi n e : Gl u ta m i n e i s a storehouse of NH3. lt is present at the highestconcentration(8 mgldl in adults) (a) Ammoniotelic : The aquatic animals in bloo d a mo n g th e a mi n o a c i d s . C l utami ne servesas a storageand transportform of NH3. lts disposeoff NH3 into the surroundingwater. synthesis mostly occurs in liver, brain and (b) Uricotelic : Ammonia is convertedmostly muscle. Ammonia is removed from the brain to uric acid e.g. reptilesand birds. pr edom i n a n tl ya s g l u ta m i n e .C l u ta mi n ei s freel y diffusible in tissues,hence easily transported. (c) Ureotelic : The mammals including man convert NH3 to urea. Urea is a non-toxic and (a mitochondrial Glutamine synthetase sol ubl e compound, hence easi l yexcreted. enzyme) is responsible for the synthesis of

glut am i n e fro m g l u ta ma tea n d a mmoni a. Thi s reaction is unidirectionaland requiresATP and Mg2+ ions. Clutamine can be deaminatedby hydrolysis to release ammonia by glutaminase(Fig.l5.A an enzyme mostlyfound in kidney and intestinal c ells . llfl" Funetions

of ammonia

Ammonia is not just a waste product of nitrogen metabolism.lt is involved (directly or via glutamine) for the synthesis of many compounds in the body. These include nonessential amino acids, purines, pyrimidines, am ino s u g a rs ,a s p a ra g i n ee tc . Ammo ni um i ons (NHa*) are very importantto maintain acid-base balance of the body. 7'i

[!il:*esal

of ammonia

rs{-?oxieity

of amrnonia

E ven a margi nal el evati on i n the bl ood ammonia concentration is harmful to the brain. Ammonia, when it accumulates in the body, results in slurring of speech and blurring of the vision and causestremors. lt may lead to coma and, finally, death, if not corrected. Hyperammonemia: Elevationin blood NH3 l evel may be geneti cor acqui red.l mpai rmenti n urea synthesisdue to a defect in any one of the five enzymes is described in urea synthesis. All these disorders lead to hyperammonemia and cause mental retardation. The acquired hyperammonemia may be due to hepatitis, alcoholism etc. where the urea synthesis becomesdefective,hence NH3 accumulates. Explanation for NH3 toxicity : The reaction catalysedby glutamatedehydrogenaseprobably explainsthe toxic affectsof NH3 in brain

T he o rg a n i s ms , d u ri n g th e c ourse of evolution, have developeddifferentmechanisms + r* *o"* NADPH for the disposalof ammonia from the body. The animals in this regardare of three differenttypes o,-Ketoglutarate + NH. Clutamate

Ghapter 15: METABOLISM OF AMINOACIDS A c c um u l a ti o no f N H 3 s h i ftsth e e q u i l i bri um to the right with more glutamate formation, hence more utilization of a-ketoglutarate.oKetoglutarateis a key intermediate in TCA cycle and its depleted levels impair the TCA cycle. The net result is that production of energy (ATP) by the brain is reduced.The toxic effectsof NHs on brain are, therefore, due to impairment in ATP formation. Trappingand eliminationof ammonia : When the plasma level of ammonia is highly elevated, intravenousadministrationof sodium benzoate and phenyllactateis done. These compounds can respectively condense with glycine and glutamate to form water soluble products that can be easily excreted. By this way, ammonia can be trapped and removed from the body. In some instances of toxic hyperammonemia, hemodialysismay become necessary.

337

O

Ornithine

Citrulline

\-o.'"n",. (n'-uHa) /

J Arginine

Arginosuccinate

NHi ions with COz to form carbamoyl phosphate.This step consumestwo ATP and is Urea is the end product of protein irreversible, and rate-limiting. CPS I requires Nmetabolism (amino acid metabolism). The acetylglutamafefor its activity. Another enzyme, nitrogenof amino acids, convertedto ammonia carbamoyl phosphate synthase ll (CPS ll)(as describedabove), is toxic to the body. lt is i nvol ved i n pyri mi di ne synthesi s-i s presenti n convertedto urea and detoxified.As such, urea cytosol. lt acceptsamino group from glutamine accountsfor 80-90% of the nitrogencontaining and does not require N-acetylglutamatefor its substancesexcreted in urine. activity. Urea is synthesized in liver and transported to kidneys for excretion in urine. Urea cycle is the frrsf metabolic cyclethat was elucidated by Hans Krebsand Kurt Henseleit(1932), hence it is known as Krebs-Henseleit cyde. The individual reactions,however,were describedin more detail later on by Ratnerand Cohen.

2. Formation of citrulline : Citrulline is synthesized from carbamoyl phosphate and ornithine by ornithine transcarbamoylase. Ornithine is regeneratedand used in urea cycle. Therefore, its role is comparable to that of oxaloacetatein citric acid cycle. Ornithine and ci trul l i neare basi cami no aci ds.(Thevare never found in protein structuredue to lack of codons). (-NH) groups, one Urea has two amino producedin this reactionis transported Citrulline derived from NHj and the other from aspartate. to cytosol by a transporter system. Carbon atom is suppliedby CO2. Urea synthesis is a five-step cyclic process,with five distinct 3. Synthesis of arginosuccinate : Arginoenzymes. The first two enzymes are present in succinate synthase condenses citrulline with mitochondria while the rest are localized in aspartate to produce arginosuccinate. The cytosol. The details of urea cycle are described second amino group of urea is incorporatedin (Figs.l5.9 and l5JA. this reaction. This step requires ATP which is 1. Synthesis of carbamoyl phosphate : Carbamoyl phosphate synthase | (CPS l) of mitochondria catalvses the condensation of

cleaved to AMP and pyrophosphate(PPi).The latter is immediatelybroken down to inorganic phosphate(Pi).

BIOCHEMISTFIY (b) Many important nitrogenouscompounds (porphyrins, purines, pyrimidines, etc.) are produced from the amino acids. About 30 g ' protein is daily utilized for this purpose. 1*

( c ) C e n e ra l l y ,a b o u t 1 0 -1 5 % o f b o requirementsare met from the arr'

ld2 v6 '

C=O I HN I \{l QHZ CHe tCHr tH C _N He

qJ gJ

\

I

cooCihrlllne

Arginocod succinate I synthase - C H o t-

-c-NHl t.l

l$H; ill

coo-

coo-

Aspartate

9Hz

c-NH-C-H

HN I

Arginosuconase

coo-

CHr tCHe tCHr

coo-

I H -C ll

c-H I

coo-

Fumamte group is Fig. 15.10: Reactionsof urea cycle (NAG-N-acetylglutamate; in the formation of urea, one amino derived from free ammonium ion while the other is from aspaftate; carbon is obtained from COr;

the restof theenzymesarecytosomal).

B IOC H E MIS TFIY

ffio irtl

l-l,N-c-o-P-O-l

o-

Carbamoyl PhosPhate

transOrnithine carbamoYlasery

f*a ?" ?*, 9Hz

MITOCHONDRION

I

H-?-NHi coo Ornithine

Arginocoosuccinate I synthase/L. CH, IH-C-NHi

coo-

Aspartate

Arginosucclnase

I CHe tCHe t-

coo-

I H-c c-H I

coo-

Fumarate Fig,|5'10:Reactionsofureacycle(NAG-N.acety|gtutamate;inthefo|mationofurea,oneaminogroupts derivedfromfreeu,,o,i,^ionwhiletheotherisfromaspartate;carbonisobtainedfromCo,;

* mitachondrialenzymes,the restof theenzymesare cytosomal)'

*h;rpter nIt: METABOLISM OF AMINO ACIDS

339

4. Cleavage of arginosuccinate : Arginosuccinase cleaves arginosuccinate to give ar gininea n d fu ma ra teA . rg i n i n ei s th e immedi ate precursor for urea. Fumarate liberated here provides a connecting link with TCA cycle, gluconeogenesis etc. 5. Forrnationof urea : Arginase is the fifth and final enzyme that cleavesarginine to yield urea and ornithine. Ornithine, so regenerated, enters mitochondria for its reuse in the urea cycle. Arginaseis activatedby Co2* and Mn2+. O r nit hin e a n d l y s i n e c o mp e te w i th argi ni ne (competitiveinhibition).Arginaseis mostlyfound in the liver, while the rest of the enzymes(four) of urea cycle are also present in other tissues. For this reason,arginine synthesismay occur to varying degrees in many tissues.But only the liv er c an u l ti m a te l yp ro d u c e u re a .

Fig. 15,11: Formationand degradationof N-acetylglutamate.

the synthesis of carbamoyl phosphate. The remainingfour enzymesof urea cycle are mostly controlled bv the concentration of their respectivesubstrates. Disposai

Swey+rFf;r+ir$'di$n and erflergetles The urea cycle is irreversibleand consumes4 ATP. Two ATP are utilized for the synthesisof carbamoyl phosphate.One ATP is convertedto AMP and PPi to produce arginosuccinatewhich equals to 2 ATP. Hence 4 ATP are actually consumed. NH4+ + CO2 + Aspartate + 3ATP -----+ Urea + F um a ra te+ 2 AD P + 2 P i + AMP + P P i ffi+-"gi.rff aii,i*fr\ rlt: urea

f

e-v*$rl

of urea

Urea produced in the liver freely diffusesand is transported in blood to kidneys, and excreted. A small amount of urea enters the intestine where it is broken down to CO2 and NH3 by the bacterialenzyme urease.This ammonia is either lost in the feces or absorbedinto the blood. ln renal failure, the blood urea level is elevated (uremia),resultingin diffusionof more urea into intestine and its breakdown to NHs. Hyperammonemia (increased blood NH3) is commonly seen in patientsof kidney failure. For these patients,oral administrationof antibiotics (neomycin)to kill intestinalbacteria is advised.

The first reaction catalysed by carbamoyl phosphate synthase t (CPS l) is rateJimiting reactionor committedstep in urea synthesis.CPS I is allostericallyactivatedby N-acetylglutamate trntegration between (NAC). lt is synthesizedfrom glutamate and urea cycle and TGA eycle acetyl CoA by synthase and degraded by a Urea cycle is linked with TCA cycle in three hydrolase(Fig.l5.1 l). different ways (Fig.15.12).This is regarded as The rate of urea synthesisin liver is correlated bicyclic integration between the two cycles. with the concentration of N-acetylglutamate. 1. The production of fumarate in urea cycle High concentrationsof arginine increaseNAC. is the most importantintegratingpoint with TCA The consumption of a protein-rich meal cycle. Fumarateis convertedto malate and then increasesthe level of NAG in liver, leading to to oxaloacetate in TCA cvcle. Oxaloacetate enhanced urea synthesis. undergoestransaminationto produce aspartate Carbamoyl phosphate synthase I and which entersurea cycle. Here, it combineswith glutamate dehydrogenaseare localized in the citrulline to produce arginosuccinate.Oxalomitochondria.They coordinatewith each other acetate is an important metabolite which can in t he f o rma ti o no f N H 1 , a n d i ts u ti l i zati onfor combine with acetyl CoA to form citrate and get

B IOC H E MIS TRY

340

N02

Carbamoyl phosphate

\ /\

/x

Fig. 15.12: lnterrelationbetuveenureaand tricarboxylicacid (TCA)cycle(Depictedin blue colour).

finally oxidized. Oxaloacetatecan also serve as i ntake margi nal l y i ncreasesbl ood urea l e vel, a precursor for the synthesis of glucose however this is well within normal range.About (gluconeogenesis). 15-30 g of urea (7-15 g nitrogen)is excretedin uri ne per day. 2. AfP (2) are generatedin the TCA cycle while AIP (4) are utilized for urea synthesis. Blood urea estimation is widelv used as a screening testfor the evaluationol kidney (renal) 3. Citric acid cycle is an importantmetabolic is estimated in the laboratory either function. lt pathway for the complete oxidation of various or diacetyl monoxime (DAM) urease method by metabolitesto CO2 and H2O. fhe CO2liberated procedure. in blood urea may be Elevation in TCA cycle (in the mitochondria) can be into three categories. broadly classified ut ili z e d i n u re a c v c l e . 1. Pre-renal : This is associated with increased protein breakdown, leading to a Metabolic defectsassociatedwith each of the negative nitrogen balance, as observed after five enzymes of urea cycle have been reported major surgery,prolongedfever, diabetic coma, (Table l5.l). All the disorders invariably lead thyrotoxicosis etc. In leukemia and bleeding to a b u i l d -u p in blood ammoni a disordersalso, blood urea is elevated. (hyperammonemia), leading to toxicity. Other metabolites of urea cycle also accumulate which, however, depends on the specific The clinical symptoms enzyme defect. Enzyme involved Defect associatedwith defect in urea cycle enzymes include vomiting, lethargy,irritability,ataxiaand phosphate I synthase typeI Carbamoyl Hyperammonemia mental retardation. transcarbamoylase typell Ornithine Hyperammonemia Metabolic

Blood

disorders

urea-clinical

of urea cycle

importance

In healthy people, the normal blood urea concentration is 10-a0 mg/dl. Higher protein

Citrullinemia aciduria Arginosuccinic Hyperargininemia

Arginosuccinate synthase Arginosuccinase Arginase

Chapter 15 : METABOLTSM OF AMTNOACTDS

341

2. Renal : In renal disorderslike acufe glomerulonephritis, chronic nephritis, nephrosclerosis, polycystickidney, blood urea is increased. 3. Post-renal: Whenever there is an ohstruction in the urinary tract (e.g. tumors, stones, enlargementof prostategland etc.),blood urea is elevated.This is due to increasedreabsorptionof urea from the renal tubules. T he t erm ' u re mi a ' i s u s e d to i n di care increasedblood urea levelsdue to renal failure. Azotemia reflects a condition with elevation in blood urea,/orother nitrogen metaboliteswhich may or may not be associated with renal diseases. Non.protein

nitrogen

Specialized product(s) Glycine

glutathione, Creatine, heme,

purines, conjugated bileacids. Thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, melanin. NAD*,NADP+ (coenzymes of niacin), serotonin, melatonin.

Tyrosine

rrypdtr;;

Activemethionine, creatine,

Cysteine

......9f i19.?.1fl 9:.p9r.)/..11t9t : ................

(NpN)

Arginine As is obvious from the name, the term NpN Lysine refers to all the nitrogen-containingsubstances ciJifi;i;' other than proteins. These include urea (most abundant), creatinine, creatine, uric acid, peptides,amino acids etc. In healthy persons, NPN concentrationin blood is 20-4O mg/dl. The molecularweight of urea is 60 and about half of it (28) is contributedby the two nitrogen atoms. Thus, if blood urea concentrationis 60 mg, then about half of it-28 mg-is hlood urea nitrogen (BU N). Therefore, BUN

= -l- NPN

NP N

= 2 BU N

In some countries, estimationsof BUN or NPN are used rather than blood urea for assessingkidney function.

ln the preceding pages,the general aspectsof amino acid metabolismhave been discussed.A summary of the biologically important or specialized products obtained from or contributed by the amino acids is given in the Table 15.2.The metabolismof individual amrno acids with special emphasison the specialized products is described next.

Glutathione, taurine, coenzyme A, active sulfate. Histamine Creatine, nitricoxide Carnitine acid,glutathione, tAminobutyric ycarboryglutamate.

.ly.l r:r.Lv..ni9.' H:.1T.'.l.9. lt9if: pyrimidines Purines, Phosphatidylserine, sphingomyelins, choline.

Coenzyme A

Clycine (Cly, C) is a non-essential,optically inactive and glycogenic (precursor for glucose) ami no aci d. l t i s i ndi spensabl for e chi cks. The outline of glycine metabolism is depicted in Fig.l5.l3. Glycine is actively involved in the synthesisof many specializedproducts (heme, purines, creatine etc.) in the body, besides its incorporation into proteins, synthesisof serine and glucose and participation in one-carbon metabolism. C l yci ne i s the most abundant ami no aci d normally excreted into urine (0.5-l .0 g,/g creatinine). Glycine

in proteins

Glycine is one among the commonestamino acids found in protein structure.Beingsmall and non-polar, glycine is mostly present in the

342

B IOGH E Mi S TFI Y

Oxalate Glucose NHs

Purines

G L Y

c I I

(C+,Cs, N7 atoms) Glutathione Conjugation (bileacids,detoxification)

N E

Formate

Heme Creatine

Synthesis

of speeialized

products

1. Formation of purine ring : The entire molecule of glycine is utilized for the formation of positions4 and 5 of carbon and position 7 of nitrogen of purines. +u nt- nu

- t- r \n-

Glycine

J

One-carbon pool Fig. 15.13 : Overview of glycine metabolism.

2. Synthesisof glutathione : Clutathione is a tripeptide (y-glutamyl-cysteinyl-glycine)and requires three amino acids for its formation

(Fig.|s.1s). 3. Conjugation reactions : As a conjugating interior structureof protein. Collagen contains glycine performstwo importantfunctions agent, (about glycine. very high 307o)content of S y mth e s i s o f g l y c i n e Clycine is synthesizedfrom serine by the enzyme serine hydroxymethyltransferasewhich is dependenton tetrahydrofolate(THF).Clycine can also be obtained from threonine,catalysed by th re o n i n e a l d o l a s e . Gl y c i n e synthasecan convert a one-carbon unit (N5, N1o-methylene T H F ), C O 2 a n d N H 3 to g l y c i n e . fl}e*gradation of glyeine Glycine undergoesoxidative deaminationby glycine synthaseto liberateNH4+,CO2 and onecarbonfragmentas N5, N10-methylene THF. This providesa major route for glycine breakdown in m a mma l s .C l y c i n e s y n th a s ei s a mul ti enzyme complex and requiresPLP, NAD+ and THF for its activity. This reaction is reversible and, therefore, glycine can be generated from onecarbon unit (methylenefragmentof THF). Glycine is reversiblyconverted to serine by THF dependent serine hydroxymethyl transferase.Pyruvate produced from serine by serine dehydratase,serves as a precursor for glucose.Serineis degradedto glyoxylatewhich undergoestransaminationto give back glycine. Glyoxylate is also converted to oxalate, an excretory product and formate which entersonecarbon pool (Fig.l 5.14).

(a) The bi l e aci ds-chol i c cholic chenodeoxy conjugatedwith glycine.

aci d and acid-are

Cholic acid + Clvcine-----) C l ycochol icacid Chenodeoxycholicacid + Clycine ------+ Clycochenodeoxycholic acid (b) Clycine is important for detoxification of benzoic acid (commonly used as a food preservative)to hippuric acid.

Benzoic acid

Hippuricacid

4. Synthesisof heme : Clycine condenses with succinvl CoA to form 6-amino levulinate which servesas a precursorfor heme synthesis (details given in porphyrin metabolismChapter 10). Succinyl CoA+ Glycine

ALA synthase levulinate(ALA)

5. Biosynthesis of creatine : Creatine is presentin the tissues(muscle,brain, blood etc.) as the high energy compound, phosphocreatine and as free creatine. Three amino acidsglycine, arginine and methionine-are required for creatine formation (Fig.l5.l6). The first

Chapter 15 : METABOLISMOF AMINO ACIDS

H3N-CH2-COO-

343 Threoninealdolase

Threonine

Ns, Nto-Methylene THF

Glycinesynthase

cH2-oH

NADH + H'

+H3N-CH2-COOSerine

N5, Nlo-Methylene

Co2 + NH|

Serine dehydratase

THF Transamination

+H3N-CH2-CH2-OH Ethanolamine

NH"J

J

O=CH-COO

HO-CH2-COOGlycocolate

L?, cooI cooOxalate

Lo" Formate

f"'

N 1o-Formyl TH F Fig. 15.14 : Metabolism of glycine (THF-Tetrahydrofolate; PLP-Pyridoxal phosphate; w -Block in primary hyperoxaluria).

reaction occurs in the kidney. lt involves the transfer of guanidino group of arginine to glycine, catalysed by arginine-glycine transamidinase to produce guanidoacetate (glycocyamine). S-Adenosylmethionine(active methionine) donates methyl group to glycocyamineto produce creatine.This reaction occurs in liver. Creatine is reversiblv phosphorylated to phosphocreatine (creatine phosphate)by creatine kinase. lt is stored in muscle as high energy phosphate. Cr eat i n i n ei s a n a n h y d ri d eo f c re a ti ne.l t i s formed by spontaneouscyclizationof creatineor creatine phosphate. Creatinine is excreted in unne.

Glutamate+ Cysteine

Arr--.-\l ADp + pi+/

I )

l.clutamyl cysteinesynthaso

|

+

y-Glutamylcysteine Gl yci ne ATP

Glutathione synthase

Glutathione(y-Glu-Cys-Gly) Fig. 15.15 : Outline of glutathione synthesis.

344

B IOC H E MIS TRY

H 3 N-C H 2-C OOGlycine Arginine-glycine transamidinase

+

H Guanidoacetate S-Adenosylmethionine(r

ranidoacetate thyltransferase

S-Adenc homocys'

Estimationof serum creatinine (along with blood urea) is used as a diagnostictest to assess kidney function. Serum creatin i ne concentration is not influencedby endogenousand exogenous factors,as is the case with urea. Hence, some workers consider serum creatinine as a more reliable indicator of renal function. The amount of creatinineexcretedis proportional to total creatinephosphatecontent of the body and, in turn, the muscle mass.The daily excretion of creatinine is usually constant. Creatinine coefficient is defined as the mg of creatinine and creatine (put together)excreted per kg body weight per day. For a normal adult man, the val ue i s 24-26 mg, w hi l e for a w om an, it is 20-22 mg. lncreased output of creatine in urine is referredto as creatinuria.Creatinuriais observed i n muscul ar dystrophy, di abetes mel lit us, hyperthyroidism,starvationetc.

, NH,

HrN-i:

Metabolic N -C H 2-C OO-

N-CH2 t-

cHs

Creatini

coo-

II

Fig. 15.16 : Metabolismof creatine.

Creatine and creatinine-clinical importance : The normal concentrations of creatineand creatinine in human serum and urine are as follows Serum Creatine Creatinine-

0.2-0.6 mg/dl 0.6-1 mg/dl

Urine Creatine Creatinine-

0-50 mgiday 1-2 {day

disorders

of glycine

1. Gl yci nuri a: Thi s i s a rare di sorder.S er um glycine concentrationis normal, but very high amount of it (normalO.5-1{day) is excretedin uri ne. l t i s bel i evedthat gl yci nuri a i s due t o a defective renal reabsorption Glycinuria is characterized by increased tendency for the formation of oxalate renal stones. However, urinary oxalate level is normal in these patients. 2. Primary hyperoxaluria : This disorder is characterized by increased urinary oxalate resultingin oxalatestones.Depositionof oxalate (oxalosis) in various tissues is observed. The urinary oxalate is of endogenousorigin and not due to dietary consumptionof oxalate. Primary hyperoxafuria is due to a defect in glycine transaminase coupled with impairment in glyoxalateoxidation to formate. It is now known that primary hyperoxaluriais mainly due to a defect in protein targeting (i.e. defect in transport of protein from one compartment to another). As a result, the enzyme glycine transaminase is found in mitochondriainsteadof its normal distributionin peroxisomes.

-fli-.,r:.i.rr:'r5' METABOLISM OF AMINO ACIDS

345

ln vitamin 86 deficiency, urinary oxalate is elevatedwhich can be correctedby 86 supplem ent ati o n .H o w e v e r, 8 6 a d m i n i s trati onhas no effect on endogenoushyperoxaluria.

compou nds-epinephrine, norepinephrine, dopamine (catecholami nes), thyroid hormonesand the pi gmentmel ani n(Fi 9.15.11.D uri ngth e course of degradati on, phenyl al ani ne and tyrosineare convertedto metaboliteswhich can serve as precursorsfor the synthesisof glucose and fat. H ence, these ami no aci ds are bot h glucogenic and ketogenic. Biochemists attach P he n y l a l a n i n e(P h e , F ) a n d ty ro s ine(Tyr, Y ) speci al si gni fi cance to phenyl al ani ne and are structurally related aromatic amino acids. tyrosine metabolismfor two reasons-synthesis P henyl a l a n i n ei s a n e s s e n ti aal m i n o aci d w hi l e of bi ol ogi cal l y i mportant compounds and th e t y r os in e i s n o n -e s s e n ti a l . B e si des i ts metabolic disordersdue to enzyme defects. , e o n l y functi on of inc or p o ra ti o ni n to p ro te i n s th t:t*'f-lt r;lr:l,.1li p h e n y I a I a mi n e pheny l a l a n i n ei s i ts c o n v e rs i o nto tyrosi ne.For :rl.l"rvi,i.r t* tyr,i:lsin* reason, this ingestionof tyrosinecan reducethe diet ar y re q u i re me n t o f p h e n y l a l a ni ne. Thi s U nder normalci rcumstances, the degradation phenomenon is referredto as 'sparing action' of of phenyl al ani nemostl yoccursthroughtyrosi n e. tyrosine on phenylalanine. P henyl al ani nei s hydroxyl atedat para-posi tion T he p re d o m i n a n t m e ta b o l i s m of phenyl - by phenyl al ani ne hydroxyl ase to produce alanine o c c u rs th ro u g h ty ro s i n e . Tyrosi ne i s tyrosi ne (p-hydroxyphenyl al ani ne).Thi s i s a n incorporatedinto proteinsand is involved in the i rreversi bl e reacti on and reoui res tn e s y nt he s i so f a v a ri e ty o f b i o l o g i c a l l yi mportant participationof a specific coenzyme biopterin

BIOMEDICAL/ CLInIICALCONCEPTS

ts Abaut 300400 human bodg.

g of protein per dag is constantly degraded and sgnthesizedin the

The amino acids are mainly utilized for protein biosynfhesis,production of specialized products (creatine,porphyrin, amines, purines, pyrimidines) and generation of energy. Glutqmqte is the collection centre t'or the omino groups in the biological systemwhile glutomine is the storehouseof NHg. Free IVH3 con be liberoted predominantly from glutamote. Ammonia accumulation in blood is toxic to brain cousing slurring of speech, blurring of uision,tremors and euendeath. Mammals conuert iVH3 to urea, o non-toxicexcretory product. Metabolic det'ectsin urea cycle enzymes result in hyperammonemia. Dietarg consumption of a protein rich meal increq.ses the leuel ol N-acetylglutamatein Iiuer which enhancesurea production. Primory hyperoxaluria---smetabolic disorder due to a delect in the enzyme glgcine transaminase-is characterizedby eleuated urinary oxalate and the Jormotion of oxalate stanes. Blood urea estimation is commonly used to ossessrenol function. Eleuation of blood urea leuel (normol 1040 mg/dl) is associatedwith seueral disorderswhich may be prerenal (diabeticcoma), renal (ocuteglomerulonephritis)and post-renal(tumors or stones in the urinary tract), Estimation of serum creotinine (normal < 1 mg/dl) is considered to be a more reliable indicator for the eualuation of ktdney function.

BIOCHEMISTFIY

346

P H E N Y L A L A N I N E

Melanins (skin,hair,eye)

NADPH. Due to a defect in phenylalanine hydroxylase,the conversion of phenylalanine to tyrosine is blocked resulting in the disorder phenylketonuria (PKU).

T Y R

Dopamine(CNS)

DEGRADATIONOF TYROSINE (PHENYLALANINEI

I N E

Norepinephrine, epinephrine (adrenalmedulla)

The metabolismof phenylalanineand tyrosine is considered together. The sequence of the reactions in the degradation of these amino acids, dbpicted in Fig.I5.l9, is described hereunder

o s

Thyroxine,T3 (thyroid gland)

Ftg. 15.17: Overuiewof phenylalanineand tyrosine netabolism(CNffientn!. npvoul systeq; .n,,,.' .,..t;:.:,ll.Tg1l . ,,11t.,,..t::t' , :,1.,....,. ,;.,., .',..,.,.t:'l ( c on ta i n i n gp te ri d i n eri n g ) w h i c h i s structural l y related to folate. The active form of biopterin is tetrahydrobiopterin (Ha-biopterin). In the phenylalaninehydroxylasereaction,tetrahydrobiopterin is oxidized to dihydrobiopterin (H2-biopterin). Tetrahydrobiopterin is then regeneratedby an NADPH-dependentdihydrobiopterin reductase(Fig,l5J A.

1 . As phenylalanineis convertedto tyrosine (details in Fig.l5.18), a single pathway is responsiblefor the degradation of both these ami no aci ds,w hi ch occurs mostl y i n l i ver. 2. Tyrosinefirst undergoestransaminationto give p-hydroxyphenylpyruvate. This reaction is catalysed by tyrosine transaminase (PLP dependent).

hydroxylase(or 3. p-Hydroxyphenylpyruvate dioxygenase)is a copper-containingenzyme. lt catalysesoxidative decarboxylationas well as hydroxylationof the phenyl ring of p-hydroxyphenylpyruvateto produce homogentisate.This The enzyme phenylalanine hydroxylase is reaction involves a shift in hydroxyl group present in the liver. In the conversion of from para position to meta position, and phenylalanineto tyrosine,the reaction involves incorporates a new hydroxyl group at para the incorporation of one atom of molecular position. This step in tyrosine metabolism oxygen (O2) into the para position of requiresascorbic acid. phe n y l a l a n i n ew h i l e th e o th e r a tom of 02 i s 4. Homogentisate oxidase (iron metalloreduced to form water. lt is the tetrahydrobiopterinthat supplies the reducing protein) cleaves the benzene ring of equivalents which, in turn, are provided by homogentisate to form 4-maleylacetoacetate.

/,----i\

( \.rr-gr-coo\_./ lrnl Phenylalanine

l-\

\tscH2-qH-coo-

\-,i

l|l-rl

HO<' Dihydrobiopterin

Tyrosine

Ghapten 15: METABOLISM OF AMINOACIDS

347

Phenylalanine

'f,?H$}ffit' (See Fig. 15.18)

*o{

//

\\

\_./ }cH2-cH-cooNHI

4-Maleylacetoacetate

I

Tyrosine

I

J H-C-COOtl H-C-C-CH2-C-CH2-COOi l -i l -

oo

4-Maleylacetoacetate (rewritten) p-Hydroxyphenylpyruvate Maleylacetoacetate isomefas€ Ar

t-?'ffiy;tri:[{8tJ#ff" Dehydroas- t

corbate + H2

-(]0c-c-H il H -C -C + C H 2-C -C H 2-C OOil

oo

tl

4-Fumarylacetoacetate

-ooc-c-H O2

H -C -C OOFumarate /, TCAcycle+/ '-f Glucose

H3C-C-CH2-COO-

o

Acetoacetate Fat

Fig, 15.{9 contd. noxt eolumn

Fig. 15.19 : Tyrosinemetabolism-degradative pathway [aKG-a-Ketoglutarate; Glu-Glutamate; The circled numbers indicate metabolic defects (1) Phenylketonuria; (2) Tyrosinemia type Il; (3) Neonatal tyrosinemia; (4) Alkaptonuia; (5) and (6) Tyrosinosis (tyrosinemia, type l)1.

Molecular oxygen is requiredfor this reactionto break the aromatic ring.

Fumarate is an intermediate of citric acid cycle and can also serve as precursor for gluconeogenesis. Acetoacetateis a ketone body 5. Maleylacetoacetate undergoes isomerifrom which fat can be synthesized.Phenylzation to form 4-fumaryl acetoacetateand this alanine and tyrosine are/ therefore, both reaction is catalysed by maleylacetoacetate gl ucogeni cand ketogeni c. rsomerase. The i nborn errors of phenyl al ani ne and 6. Fumaryl acetoacetase(fumaryl acetoacetate hydrolase)brings about the hydrolysis of tyrosine metabolismare indicated in Fig.l5.l9. fumaryl acetoacetate to Iiberate fumarate and Detailed informationon these disordersis given later. acetoacetate.

'g

BIOCHEMISTFIY

348 Synthesis

t't

of melanin

/|

M e l a n i n(C re e k : me l a n -b l a ck) i s the pigmentof skin, hair and eye.The s y n th e s i s o f m e l a n i n o c c u rs i n melanosomes present in melanocytes, the pigment-producingcells. Tyrosineis the precursorfor melanin an d o n l y o n e e n z y me r n a m el y copper-containing tyrosinase (a oxygenase), is involved in its formation. Tyrosinase hydroxylates tyrosine to form 3,4-dihydroxyphenylalanine (DOPA) (Fig.l5.20). DOPA can act as a cofactor for tyrosinase.The next reaction is also catalysed by tyrosinase in which DOPA is convertedto dopaquinone. It is believed that the subsequent reactions occur couple of spontaneously, forming leucodopachromefollowed by 5,6-dihydroxy5, ind o l e . T h e o x i d a ti o n o f 6-dihydroxyindole to indole 5, 6-quinone is catalysedby tyrosinase, and DOPA servesas a cofactor.This reaction, inhibited by tyrosine regulatesthe synthesisof melanin. Melanochromes are formed from indole quinone, which on polymerization are converted to hlack me l a n i n . pathway from Another i s a l s o i d e n ti fi ed. do p a q u i n o n e with dopaCysteine condenses quinone and in the next series of reactionsresultsthe synthesisof red me l a n i n s .T h e s tru c tu reo f me l ani n pigmentsis not clearly known. Melanin-the colour pigment : T h e s k i n c o l o u r o f th e i n d i v i d u al i s by the relative determined concentrations of black and red me l a n i n s T . h i s , i n tu rn , i s d e p e n dent on many factors, both genetic and en v i ro n me n ta l .T h e s e i n c l u d e the activity of tyrosinase, the density of melanocytes, availability of tvrosineetc.

,'ycnz-QH-coo|I lnl

Ho\-"'Tyrooine

\l) Tyrosinase(Cu2)

H (l

Y

cH2-?H-cooNHi (DOPA) 3,'t-Dihydroryphenylalanine

I ry,o"tn"r"

)

"JcH2-?H-cooNHi Dopaquinone |

(cYtttin'

*oo"ne'l'nott" .-

)olymers D I

H 5,6-Dihydroxyindole ot-.j I -Tyrosinase

Hro4

oY\F------,r I

I

&'\7\N,/

ll

Mehnin porymbrs

tsLAcK

i

.'|Metanochrome

I H Indole 5,6-quinone Fig. 15.20 : Metabolism of tyrosine-biosynthesis of melanin (Defect in tyrosinase causes albinism).

Chapter 15 ; METABOLISM OF AMINO ACIDS Thyroglobulin

The presenceof moles on the body represents a localized severe hyperpigmentation due to hyperactivityof melanocytes.On the other hand, localized absence or degeneration of melanocytesresultsin white patcheson the skin commonly known as leucoderma. Albinismis an inborn error with generalized lack of melanin synthesis(detailsdescribedlater).

I

t"

+a

,"

-l-

OH Active iodine

Biosynthesis

Hzoz

Thyroglobulin

Thyroglobutin

ll

QHz

oH

QHz

oH

/\

Monoiodotyrosine /

\

349

Diiodotyrosine

of thyroid

hornnones

Thyroid hormones-fhyroxine (tetraiodothyroni ne) and tri i odothyroni ne-are synthesized from the tyrosine residues of the protein thyroglobulin and activated iodine (Fig.tS.2t). lodination of tyrosine ring occurs to produce mono- and diiodotyrosine from which triiodothyronine (Tl) and thyroxine (T+) are synthesized. The proteinthyroglobulinundergoes proteolytic breakdown to release the free hormones,T3 and Ta. B i osynthesi s

of catechol ami nes

The name catechol refers to the dihydroxylated phenyl ring The amine derivatives of catechol are called catecholamines.

OH

f-Pr.teorYsis*H3N-cH-coo-

Catechol

'H3N-CH-COO-

o

o

OH (T3) Trilodothyronine

OH (Ta) Thyroxine

Fig. 15.21 : Metabolismof tyrosinesynthesis of thyroid hormones.

Tyrosine is the precursorfor the synthesisof catecholamines, namely dopamine, norepinephrine (noradrenaline) and epinephrine (adrenal i ne). The conversionof tyrosineto catecholamines occurs in adrenal medulla and central nervous system involving the following reactions (Fig.t5.2). Tyrosine is hydroxylated to 3,4-dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase. This enzyme catalysesthe rate limiting reaction and requires tetrahydrobiopterinas coenzyme (like phenylalaninehydroxylase).In contrastto this enzyme, tyrosinasepresent in melanocytes converts tyrosine to DOPA. Hence, two

BIOGHEMISTF|Y

350

l-\

HO<'

\FCH2-CH-COO

\-,/

rlnl Tyrosine

H,-Bior Hr-Biotr

H

Iyrosine 'dtol
CH,-CH-COO-l

NHA (DOPA) Dihydroryphenylalanine ,romaticamlnoacid decarboxylase

rl

)H 2 -C H2-N H l Dopamine

different enzyme systems exist to tyrosineto DOPA.

decarboxyDOPA undergoesPLP-dependent l ati on to gi ve dopami ne w hi ch, i n turn, is produce norepinephrine. hydroxylated to Methylation of norepinephrineby S-adenosylmethi oni ne gi ves epi nephri ne.The di ffer ence i s only betw eenepi nephri neand norepi nephri ne a methyl group (rememberthat norcpinephrine has no methyl group). There existstissuespecificityin the formation In adrenalmedul l a,synt hesis of catechol ami nes. hormones, norepi nephri ne and of the epi nephri ne i s promi nent. N orepi nephr ineis produced in certain areas of the brain while dopami ne i s predomi nantl y synthesi zed in substantianigra and coeruleusof brain. Functions of catecholamines : Norepinephrineand epinephrineregulatecarbohydrate and l i pi d metabol i sms.They sti mul ate t he degradation of triacylglycerol and glycogen. They cause an increasein the blood pressure. D opami ne and norepi nephri ne serv e as in the brain and autonomous neurotransmitters nervoussystem. Dopamine

Norepinephrine S-Adenosyl methionine S-Ader homoc

H

Phenyleftanolamin€ N.melhylirarJsferase

C H -C H2-N -C H 3 II

OH H Epinephrine Fig. 15.22 : Metabolism of tyrosine-synthesis of catecholamines (dopamine, norepinephrine, epinephrine; PLP-pyridoxal phosphate).

convert

and Parkinson's

dlsease

Parkinson'sdiseaseis a common disorder in many elderly people, with about 1% of the population above 60 years being affected.lt is characterized by muscular rigidity, tremors, expressionless face, lethargy, involuntary movements etc. Biochemical basis : The exact biochemical cause of this disorder has not been identified. Parkinson's disease is, however, Iinked with a decreasedproduction of dopamine. The disease is due to degenerationof certain parts of the brain (substantianigra and locus coeruleus), l eadi ng to the i mpai rment i n the synthesisof dopami ne. Treatment: Dopaminecannot enterthe brain, hence its administration is of no use. DOPA (levodopaor L-dopa)is used in the treatmentof P arki nson' sdi sease. In the brai n, D OPA is decarboxylatedto dopaminewhich alleviatesthe

Chapterl5:

METABOLISM OF AMINOACIDS

351

symptoms of this disorder. Unfortunately, Phenylalanine hydroxylase dopamine synthesis occurs in various other Phenylalanine Tyrosine tissuesand resultsin side-effects such as nausea, I vomiting, hypretension etc. Administration of I Trans J dopa analogs-that inhibit dopa decarboxylase Phenylpyruvate (in various tissues)but not enter brain (due to blood-brain barrier)-are found to be effective. NADH + H+ Carbidopaand lmethyl-dopa (dopaanalogs)are NADH+ Hl NAD* administered along with dopa for the treatment of Parkinson'sdisease. Phenylacetate Phenyllactate

DISORDERSOF TYROSINE (PHENYLALANTNEIMETABOLTSM Several enzyme defects in phenylalanine/ tyrosine degradation leading to metabolic disordersare known. ln Fig.l5.19, the deficient enzymes and the respectiveinborn errors are depicted and they are discussedhere under. Fheny!ketonuria

lr-Glutamine I

J*"'o

Phenylacetylglutamine

Clinical/biochemical manifestationsof PKU : The di sturbed metabol i smof phenyl al ani nePhenylketonuria(PKU) is the most common resulting in the increased concentration of m et abolicd i s o rd e r i n a mi n o a c i d m e t abol i sm. phenylalanine and its metabolitesin the bodyT he inc ide n c eo f PK U i s 1 i n 1 0 ,0 0 0 b i rths.l t i s due to the deficiency of the hepatic enzyme, causes many cl i ni cal and bi ochemi cal phenylalanine hydroxylase, caused by an manisfestations. 'l . Effectson central nervous autosomal recessivegene. In recent years, a system : Mental variant of PKU-due to a defect in retardation,failure to walk or talk, failure of dihydrobiopterinreductase(relativelyless)-has growth, peizuresand tremor are the characteristic been reported.This enzyme deficiency impairs findings in PKU. lf untreated,the patientsshow the synthesisof tetrahydrobiopterinrequired for very low lQ (below 50). The biochemical basis the action of phenylalanine hydroxylase (See of mental retardation in PKU is not well Fig.l\.l8). The net outcome in PKU is that understood. There are, however, many phenylalanineis not convertedto tyrosine. explanationsoffered Phenylalanine metabolism in PKU : . A ccumul ati on of phenyl al ani ne i n brai n P heny lk e to n u ripari m a ri l yc a u s e sth e a c cumul aimpairsthe transportand metabolismof other t ion of ph e n y l a l a n i n ei n ti s s u e sa n d b l o od, and aromatic amino acids (tryptophan and resultsin its increasedexcretionin urine. Due to tyrosine). disturbancesin the routine metabolism,phenylr The synthesis of serotonin (an excitatory alanine is diverted to alternate pathways neurotransmitter) from tryptophan is (Fig.l5.23), resultingin the excessiveproduction insufficient. This is due to the competition of of phenylpyruvate,phenylacetate,phenyllactate phenylalanine and its metabolites with and phenylglutamine.AII these metabolitesare tryptophan that impairs the synthesis of excretedin urine in high concentrationin PKU. serotonin. Phenylacetategives the urine a mousey odour. . Defect in myelin formation is observed in PKU The name phenylketonuriais coined due to patients. the fact that the metabolite phenylpyruvate is a 2. Effect on pigmentation : Melanin is the keto acid (C6H5CH2-CO-COO-) excreted in pigmentsynthesizedfrom tyrosineby tyrosinase. ur ine in h i g h a mo u n ts .

352

B IOC H E MIS TR Y

A c c um u l a ti o n o f p h e n y l a l a n i n ec o m peti ti vel y phenylacetate,N-acetyltyrosine-and tyramine inhibits ty ro s i n a s e a n d i mp a i rs mel ani n are observed. formation. The result is hypopigmentation that Tyrosinemiatype ll is characterizedby skin causeslight skin colour, fair hair, blue eyes etc. (dermatitis)and eye lesionsand, rarely, mental Diagnosisof PKU : PKU is mostly detected by retardation.A disturbedself-coordinationis seen screeningthe newborn babiesfor the increased in these patients. plasmalevelsof phenylalanine(PKU, 20-65 mg/ dl; normal 1-2mg/dl).This is usually carried out Neonatal tyrosinemia by Guthrie fest, which is a bacterial (Bacillus The absenceof the enzyme p-hydroxyphenylsubtilis)bioassayfor phenylalanine.The test is pyruvate dioxygenase causes neonatal usually performed after the baby is fed with tyrosinemia.This is mostlya temporarycondition breastmilk for a couple of days. All the babies and usually responds to ascorbic acid. lt is born in USA are screenedfor PKU by testing explained that the substrateinhibition of the elevatedlevelsof phenylalanine.Phenylpyruvate enzyme is overcomeby the presenceof ascorbic in urine can be detected by ferric chloride test aci d. (a green colour is obtained). This test is not specific, since many other compounds give a (B l ack uri ne di seasef A l kaptonuri a false positivetest. Alkaptonuriahas great historicalimportance. Treatment of PKU : The maintenance of It was first describedby Lusitanusin 1649 and plasma phenylalanine concentration within the characterizedin 1859. Garrod conceived the normal range is a challenging task in the idea of inborn errors of metabolism from his treatmentof PKU. This is done by selectingfoods observationon alkaptonuria.The prevalanceof with low phenylalaninecontent and/or feeding this autosomalrecessivedisorderis 1 in 25,000. synthetic amino acid preparations, low in phenylalanine.Dietary intake of phenylalanine Enzyme defect : The defective enzyme in should be adjustedby measuringplasma levels. alkaptonuriais homogentisateoxidasein tyrosine Early diagnosis(in the first couple of months of metabolism (See Fig.l5.l9). Homogentisate baby's life) and treatment for 4-5 years can accumulatesin tissuesand blood, and is excreted prevent the damage to brain. However, the into urine. Homogentisate,on standing, gets restrictionto protein diet should be continued oxidized to the correspondingquinones,which for many more years in life. Since the amino polymerizeto give black or brown colour. For acid tyrosine cannot be synthesized in PKU this reason, the urine of alkaptonuric patients patients, it becomes essential and should be resembles coke in colour. provided in the diet in sufficientquantity. Biochemical manifestations : Homogentisate In some seriously affected PKU patients, gets oxidized by polyphenol oxidase to benzotreatment includes administration of 5-hydro- quinone acetate which undergoes polyxytryptophan and dopa to restore the synthesis merization to produce a pigment called alkapton (Fig.l5.24. Alkapton deposition occurs in of serotoninand catecholamines. connective tissue, bones and various organs Tyrosinemia type ll (nose,ear etc.) resultingin a condition known as ochronosis. Many alkaptonuric patients suffer This disorder-also known as Richnerfrom arthritisand this is believed to be due to Hanhart syndrome, is due to a defect in the the depositionof pigmentalkapton(in the joints), enzyme tyrosine transaminase.The result is a produced from homogentisate. blockade in the routine degradativepathway of tyrosine.Accumulationand excretionof tyrosine Diagnosis: Change in colour of the urine on and its metabolites-namely p-hydroxyphenyl- standingto brown or dark has been the simple pyruvate, p-hydroxyphenyllactate, phydroxy- traditional method to identify alkaptonuria.The

353

Ghapter 15 : METABOLISM OF AMINO ACIDS Homogentisate

oxidase I eotyprrenot

+

Benzoquinoneacetate

I Povmerizatior"

J

Alkapton

I

+

About 150 genes have been identified in mice. The mel ani n synthesi scan be i nfl uencedby a variety of factors.Many possiblecauses(rather explanations)for albinism have been identified 1 . Deficiencyor lack of the enzymetyrosinase. 2. Decreasein melanosomesof melanocytes. 3. l mpai rmenti n mel ani n pol ymeri zati on.

Bindsto tissues

4. Lack of protein matrix in melanosomes. ,|Fiigi lF.?., iiQptlvq$:io!!.gf!lqn!, a(lgeqtg,lpall$ptan,.

(tyrosine) 5. Limitationof substrate availability.

6. Presenceof inhibitorsof tyrosinase. urine gives a positive test with ferric chloride The most common cause of al bi ni sm i s a and silver nitrate. This is due to the strong reducing activity of homogentisate.Benedict's defect in tyrosinase,the enzyme most responsible test-employed for the detectionof glucoseand for the synthesisof melanin (SeeFig.l5.20). other reducing sugars-is also positive with Clinical manifestations: The most important homogentisate. function of melanin is the protectionof the body Treatment : Alkaptonuria is nof a dangerous from sun radi ati on.Lack of mel ani n i n al bi nos disorder and, therefore, does not require any makes them sensitive to sunlight. Increased specific treatment. However, consumption of susceptibility to skin cancer (carcinoma) is protein diet with relatively low phenylalanine observed. Photophobia (intolerance to light) is content is recommended. associatedwith lack of pigment in the eyes. However, there is no impairmentin the eyesight Tyrosinosis or tyrosinemia type I of al bi nos. This is due to the deficiencyof the enzymes fumarylacetoacetate hydroxylase and/or maleylacetoacetateisomerase.Tyrosinosisis a rare but serious disorder. lt causes liver failure, rickets, renal tubular dysfunction and polyneuropathy. Tvrosine,its metabolitesand many other amino acids are excreted in urine.

H yp*:p i E ltreffi t€ht i orl In some individuals,a reduced svnthesisof melanin (insteadof total lack) is often observed. Hypopigmentation disorders may be either diffuse or localized.

A good example of diffuse hypopigmentation is oculocutaneous albinism which is mostly due In acute tyrosinosis, the infant exhibits to mutations in the tyrosinasegene. The degree diar r hea, v o mi ti n g , a n d ' c a b b a g e -l i k e'odor. hypopigmentation of depends on the type and Death may even occur due to liver failure within genes. severity of mutated one year. For the treatment, diets low in t y r os ine, p h e n y l a l a n i n e a n d me th i o n i ne are Vitiligo and leukoderma are the important recommended. amongthe localizedhypopigmentation disorders. Vitiligo is an acquired progressivediseasewith A lbinis m loss of pigmentationaround mouth, nose, eyes Albinism (Greek:albino-white) is an inborn and ni ppl es. Leukodermai s comparabl ew i th error, due to the lack of synthesisof the pigment vi ti l i go, but l ack of pi gmentati onusual l ybegi ns melanin. lt is an autosomal recessivedisorder with hands and then spreads. with a frequency of 1 in 20,000. C reyi ng of hai r i s due to l ack of mel ani n Biochemical basis : The colour of skin ano synthesiswhich usually occurs as a result of of melanocytes from the hair roots. hair is controlled by a large number of genes. disappearance

B IOGH E MIS TRY

354

Glucose Fat lndoleacetic acid

T R Y P T

o

NAD1NADP* (coenzymesof niacin)

P H A N

5-Hydroryindoleaceticacid

metabolism. Fig. 15.25: Overuiewof tryptophan

I

-l

Tryptophan (Trp, W) was the first to be identified as an essenfialamino acid. lt contains an in d o l e ri n g a n d c h e m i c a l l y i t i s s-ami no B - ind o l e p ro p i o n i c a c i d . T ry p to p [an i s both glucogenic and ketogenic in nature. lt is a precursor for the synthesis of impoftant compounds, namely NAD+ and NADP+ (coenzymesof niacin), serotonin and melatonin (Fig.l5.25). The metabolismof tryptophanis divided into | . Kynureni ne (kynureni ne-anthrani Iate) pathway; ll. Serotoninpathway.

Kynureninase,a pyridoxal phosphate(PLP)dependent enzyme acts on the 3-hydroxykynurenineand splits off alanine.Tryptophanis glucogenic,since alanine is a good precursorfor gl ucose. The enzyme kynureni nasei s v er y sensitiveto vitamin 86 deficiency. Due to the reactionis blockedand lack of PLP,kynureninase 3-hydroxykynurenine is diverted to form xanthu renate. EIevated excretion of xanthurenate serves as an indication of 86 deficiency. Administrationof isoniazid, an antituberculosis drug-induces B6 deficiency and results in xanthurenateexcretion in urine. Defects in the activity of kynureninase(in 86 deficiency)cause reduced synthesisof NAD+ and NADP+ from tryptophan.The symptomsof pellagra-observed in 86 deficiency-are explainedon this basis. The enzyme kynureni ne hydroxyl ase is inhibited by estrogen,hence women, in general, are more susceptibleto Pellagra. 3-Hydroxyanthranilate is cleaved by an oxidase (Fe2+dependent)to form an unstable intermediate, 2-amino 3-carboxy muconate semialdehyde.This compound has three fates. 'I . lt undergoes spontaneouscyclization to form quinolinatefor NAD+ synthesis.

2. Picolinatecarboxylasedecarboxylatesthe which cyclizesto producepicolinate. intermediate I. Kynurenln,e pathway This enzyme competes with the formation of pi col i nat e This pathwaymostlyoccurs in liver leadingto qui nol i nate. H i gh acti vi ty of oxidation of tryptophan and the synthesisof carboxylasein some animals (e.g. cat) deprives from tryptophan.In other them of NAD+ synthesis NAD+ and NADP+ (Fig.l5.26). is exclusivelydependenton niacin for words, cat Tryptophan pyrrolase or oxygenasecleaves its coenzymes(NAD+, NADP+),since tryptophan t he f i v e -m e m b e re dri n g o f th e i n d ol e nucl eusto serveas a precursor. cannot produceformylkynureni ne. Tryptophanpyrrolase is a me ta l l o p ro te i n c o n ta i n i n ga n i ron porphyri n 3. The intermediate undergoes decarboxyr ing. l t i s a s u b s tra tei n d u c i b l e e nzyme and i s lation, catalysedby amino carboxysemialdehyde controlled by feedback regulation (by NADPH decarboxylase to produce 2-aminomuconate and other niacin derivatives). Tryptophan semialdehydethat entersglutaratepathway.The pyrrolase activity is also elevated by cortico- semialdehydeis convertedto 2-aminomuconate The aminomuconate,in a steroids. Formamidase hydrolyses formyl- by a dehydrogenase. reactions involving reduction, kvnurenine and Iiberatesformate which enters series of the one carbon pool. Kynurenineformed in this deamination,decarboxylationetc., is converted reaction is a branch point with different fates. to glutaryl CoA and finally to acety CoA. The In the prominentpathway,kynurenineundergoes latteris eithercompletelyoxidized via TCA cycle NADPH-dependent hydroxylation to give or converted to fat (hence tryptophan is 3- hy d ro x y k y n u re n i n e . ketogenic).

Ghapter'!5: METABOLISM OF AMINOACIDS

355

H 2 -C H - C OO-

NFrA I H Tryptophan

c-cH2-cH-cooNH5

c-cH lll

HO N-Formylkynurenine

KYnurenate< _ at'n[

Picolinate

o H2_cH_coo-

Alanine

entnranitateY V,["u,"n,r,"'

Spontaneous

Decarboxylase

Hzo

n t -

z\

ttl O:C H

ll

-ooi^NH2

02, NADPH

Kynurenine hydroxylase

2-Aminomuconate semialdehyde

H2O,NADP

I

NAD\I Atdehyde . I dehydroNADH+ H-rJ Senase

coo-

a-\

ttl - ooc rl -ooi-NHz Ribos+--F' Nicotinate mononucleotide

2-Aminomuconate

i

I

+ NAD+

+ Glutaryl CoA

I

I

+ Fig, 15.26 contd.

next column

NADP+

+

Acetyl CoA

Fig. 15.26 : Metabolism of tryptophan-kynurenine pathway (PLP-Pyridoxal phosphate; QP4T-Quinolinate phosphoribosyl transferase; PRPP-Phosphoribosyl pyrophosphate).

356

B IOC H E MIS TFI Y

NAD+ Pathway : Tryptophan is not a 2. l t i s cl osel y i nvol ved i n the regul at ionof precursor for the synthesis of free niacin. cerebralactivity (excitation). Q u i n o l i n a te u n d e rg o e sd e c a rb o xyl ati onand i s 3. Serotonin controls the behavioural converted to nicotinate mononucleotideby the enz y me q u i n o l i n a tep h o s p h o ri b osyltransferase patterns, sleep, blood pressure and body (QPRT).The synthesisof NAD+ and NADP+from temperature. nic o ti n a tem o n o n u c l e o ti d ei s s i mi l arto that from 4. Serotonin evokes the release of peptide nia c i n a s d e s c ri b e di n th e c h a o teron vi tami ns hormonesfrom gastrointestinal tract. (Refer Fig.7.2l).

:-l

5. lt is also necessaryfor the motility of GIT Conversion of tryptophan to indole acetate : (peri stal si s). T r y p to p h a n u n d e rg o e s d e a m i nati on and decarboxylationto produce indolepyruvateand Serotonin and brain : The brain itself tryptamine,respectively.Both these compounds synthesi zes 5H T w hi ch i s i n a bound form. The are converted to indoleacetate (Fig.15.2V and outside serotonincannot enter the brain due to excreted in urine. bl ood-brai n barri er. P ri mari l y, serotoni n is a stimulator (excitation) of hrain activity, hence ll. $ e * ' .ri i : 'l rr:,:a;abt its deficiencycausesdepression.Serotoninlevel Serotonin or S-hydroxytryptamine (5HT) is a is decreasedin psychosispatients. neurotransmitter,synthesizedfrom tryptophan. Effect of drugs on serotonin : The drug, No rm a l l y , a b o u t 1 % o f th e tryptophan i s (isopropyl isonicotinyl hydrazine) iproniazid convertedto serotonin.The production of 5HT i nhi bi ts monoami ne oxi dase (MA O) and occurs in the target tissues. elevatesserotoninlevels,therefore,this drug is a Synthesisof serotonin : In mammals, the psychi c sti mul ant. On the other h and, largestamount of serotoninis synthesizedin the reserpine increases the degradation of int e s ti n a lc e l l s . T h e fo rma ti o n of serotoni n i s serotonin, hence acts as a depressantdrug. production comparable with the of Lysergic acid diethylamide (LSD) competes catecholamines. Tryptophanis first hydroxylated with serotonin and, therefore, acts as a at 5th carbon by tryptophan hydroxylase.This depressant. enzyme requires tetrahydrobiopterin as a cofactor.5-Hydroxytryptophanis decarboxylated Malignant carcinoid syndrome : Serotoninis by aromatic amino acid decarboxylase(PLP- produced by argentaffincells of gastrointestinal dependent)to give serotonin(Fig.l5.27). tract. When these cells undergo uncontrolled growth, they develop into a tumor called Plateletscontain high concentrationof 5HT, malignant carcinoid or argentaffinomas.The t he s i g n i fi c a n c eo f w h i c h i s not cl ear. A s patients exhibit symptoms like respiratory such, plateletsdo not carry out the synthesisof distress,sweating,hypertensionetc. s er o to n i n . N ormal l y about 1% of the tryptophan is Degradation of serotonin : Monoamine oxidase (MAO) degrades serotonin to 5- utilized for serotonin synthesis. In case of (5HlA) which is excreted carci noi d syndrome,very hi gh amount (u p t o hydroxyindoleacetate 60%) of tryptophan is diverted for serotonin in u ri n e . production.This disturbsthe normal tryptophan Functions of serotonin : Serotonin is a neurometabol i smand i mpai rsthe synthesi sof NAD+ transmitter and performs a variety of functions. and N A D P + . H ence, the pati entsof carc inoid 1 . Serotonin is a powerful vasoconstrictor syndromedevelop symptomsof pellagra (niacin and results in smooth muscle contraction in deficiency). Further, negative nitrogen balance br o n c h i o l e sa n d a rte ri o l e s . is also observed.

357

Ghapten 15 : METABOLISM

Ho'

cH2-cH-coo$lt'lWl}ai;" nnt Ho-BioPterin

H2-BioPterin

-

HH H

t-

| +

urine

Serotonin

s-HYdrorYir acetate

,n-

4-\fac+2-cH2ll ll I

c- cu.

\-\*/ H NAcetylserotonin S-AdenosYlmethionine S-AdenosY homocyste

cetvlserotontn lthYitransferase

H3c-o €-\--TcH2-cH2-

V\--! H Melatonin synthesis : Metabolismof tryptophan-serotoninand melatonin Fia. -1PLP-Pyridoxal ' '"' '-15.27 phosphate;MAO-Mono:ryin: oxllase\

c-cH3

358

B IOC H E MIS TRY

Diagnosis : The excretion of S-hydroxy indole acetate in urine is tremendously elevated (upto 500m9/day against normal <5 mg/day) in carcinoid syndrome.The estimationof 5 HIA in ur ine i s u s e dfo r th e d i a g n o s i so f th is di sorder.l n general,urine concentrationof 5 HIA above 25 mg/dayshould be viewed with caution as it may be suggestiveof carcinoid syndrome.Sufficient precautionshould,however,be taken for sample c olle c ti o n .D u ri n gth e c o u rs eo f u ri ne col l ecti on, the patients should not ingest certain foods (banana,tomato etc.) that increaseurine 5 HlA.

acids and their elevated urinary excretion. Increasedurinaryoutput of indoleaceticacid and indolepyruvicacid is also observed.

Pellagralike symptoms are common in these patients.There is an impairmentin the synthesis of NAD+ and serotoninfrom tryptophan.Some authors(earlier)attributedHartnup'sdiseaseto a defect in the enzyme tryptophanpyrrolase.This, however, does not appear to be true. Hartnup's disease is now believed to be due to an impairment in the absorption and/or transport of tryptophan and orher neutral amino acids from the intestine,renal tubules and, probably M ela to n i n brain. Some more details on Hartnup's disease Melatonin is a hormone, mostly synthesized are given under digestion and absorption by the pineal gland. Serotonin-produced from (Chaptur A. tryptophan-is acted upon by serotonin N-acetylase(the rate limiting enzyme), to give N-acetylserotonin. The latter undergoes methylation, S-adenosylmethionine' being the methyl group donor to produce melatonin or N-acetyl 5-methoxyserotonin(Fi9.15.2V. fhe ami no aci dsare methioThe sul fur-contai ni ng synthesisand secretionof melatoninfrom pineal nine, cysteine and cystine. Among these, only gland is controlled by light. methionine is essential.lt serves as a precursor for the synthesisof cysteineand cystine which Cysteinecan spare are, therefore, non-essential. 1. Melatonin is involved in circadian the requirement of methionine in the diet. rhythms or diurnal variations (24 hr cyclic Cysteineand cystineare interconvertible. Cystine process)of the body. lt plays a significantrole in i s found excl usi vel yi n protei ns.Methi oni nea nd sleep and wake process. cysteine,besidesbeing present in proteins,are involved in many importantmetabolic reactions 2. Me l a to n i n i n h i b i ts th e p roducti on of (Fi9.15.25).Methionine is also required for the melanocyte stimulating hormone (MSH) and i ni ti ati on of protei n bi osynthesi s.The sulf ur adrenocorticotropichormone (ACTH). contai ni ngami no aci ds are al mostan excl usive 3. lt has some inhibitory effect on ovarian dietary source of sulfur to the body. functions. Functions

o# melatonin

4. Melatonin also transmitter function. l{art*rup's

disease

performs a

neuro-

ME TA B OLIS M

OF ME TH ION IN E

Methi oni ne (or sul fur ami no ac ids) metabolismmay be divided into three parts.

1 .Utilization of methionine for transmethvThis disorderwas first describedin the family of Hartnup,hencethe name-Hartnup's disease. lation reactions. It is a hereditary disorder of tryptophan 2.C onversi onof methi oni neto cystei nea nd m et a b o l i s m. T h e c l i n i c a l s y m p t oms i ncl ude cystine. dermatitis, ataxia, mental retardation etc. Hartnup'sdiseaseis characterizedby low plasma 3.Degradationof cysteineand its conversion Ievels of tryptophan and other neutral amino to specializedproducts.

Ehapterl5:

METABOLISM OF AMINO ACIDS

M ;a t{ I

lnitiationof protein

biosynthesis

I

r'{ H

c

Y S T E

I

t{ E

t

359

enzymesinvolved in the transferof methyl group are collectively known as methyltransferases. Transmethylation S-Adenosylmethionine transfersthe methyl group to an acceptor and gets itself converted to Cystathionine S-adenosylhomocysteine. The lossof free energy in this reaction makes the methyl transfer Polyamines essentiallyirreversible.S-Adenosylhomocysteine is hydrolysedto homocysteineand adenosine. Homocysteine can be remethylated to methionineby Ns-methyltetrahydrofolate. In this Glutathione manner/ methionine can be regeneratedfor reuse. lt should be noted that there is no net Taurine svnthesis of methionine in the S-adenosvlCoenzymeA methionine cycle (homocysteine,the precussor for methionine has to be derived from Active sulfate methi oni ne).H ence, methi oni nei s an essenti al ami no aci d.

Proteins{Fig. 15.28 : Overview of the metabolismof sulfur amino acids.

S-Adenosylmethionine(carbon fragment) is also involved in the synthesisof polyamines (spermidine, spermine). The most important transmethylation reactions are listed in Tahle 15.3.

TransrmethylatE*n fhe transfer of methyl group (-CH3) from active methionine to an acceptor is known as Methioninehas to be activated transmethylation. to S-adenosylmethionine (SAM) or active methionineto donate the methyl group. Synthesis

S-Adenosylmethionine S-Adenosylhomocysteine

of S"adenosylmethionFme

occurs The synthesisof S-adenosylmethionine by the transfer of an adenosyl group from ATP Methyl group acceptor Methylatedproduct to suffur atom of methionine (Fig.15.29).This Guanidoacetate Creatine reaction is catalysedby methionineS-adenosylNorepinephrine Epinephrine transferase.The activation of methionine is Epinephrine Metanephrine unique as the sulfur becomesa sulfonium atom Ethanolamine Choline (SAM is a sulfoniumcompound) by the addition of a third carbon. This reaction is also unusual Nicotinamide N-Methylnicotinamide since all the three phosphates of ATP are Acetylserotonin Melatonin (PPi)and inorganic eliminatedas pyrophosphates Phosphatidylethanolamine Phosphatidylcholine phosphates(Pi). Three high energy phosphates Serine Choline 8 ATn arc consumed in the formation of Carnosine Anserine SAM. IRNAbases Methylated IRNAbases FclmetEssrs sf S-adecrosylmethEsn&rse is highly reactivedue S-Adenosylmethionine positive presence charge. The the of a to

Protein-amino Prolein-mehylated aminoacids acid (histidine, residues lysine, (methylhistidine, methyllysine,methylarginine etc.) arginine etc,)

BIOCHEMISTF|Y

360

coon-c-runl tCHr t-

?r,

Methionineadenosyltransferase

cooI

Hzo ATP 2Pi+ Pi

N

CHc t2

-

HsC-

H3C-S Meffrionine

N \

tH-C-NHi

9Hz

OH

OH

9Adenosylmefiionine

cooH_C_NHI t"

CHc tCHc tSH Homocysteine

coo-

product

t-

Adenosinehomocysteinase

,/\-\

Adenosine

Hzo

-?-rra

?,, ?"

S-Adenosine

$Adenosylhomocysteine Fig. 15.29: S-Adenosylmethioninecycle-synthesis, utilizationand regenerution.

and deaminatescystathionineto cysteine and s-ketobutvrate. The sum of the reactions 1 . Transmethylation is of great biological catalysed by cystathionine synthase and significance since many compounds become cystathioninase is a good example of functionally active only after methylation. transsulfuration (transfer of sulfur from one 2. Pro te i n(a m i n o a c i d re s i d u es)methyl ati on compound to another).lt should be noted that helps to control protein turnover. In general, only the sulfur atom of cysteine comes from protects the proteins from homocystei ne(ori gi nal l ymethi oni ne)w hi l e t he methylation rest of the molecule is from serine. immediate degradation. Significance

of transmethylation

3. In p l a n ts , S -a d e n o s y l me thi oni ne i s the H omocystei ne and heart attacks precursorfor the synthesisof a plant hormone, Homocysteine is an intermediate in the ethylene, which regulates plant growth and methionine cysteine from synthesis of dev e l o p me n ta n d i s i n v o l v e d i n th e ri peni ngof (Fig.l5.30), Elevation in plasma homocysteine fru its. (normal < 15 pmol /l ) has been i mpl i cated in althoughthe mechanism coronaryarterydiseases, Synthesis of cysteine is not known. lt is believed that homocysteine Homocvsteineformed from methionine is a reacts with collagen to produce reactive free precursor for the synthesis of cysteine radicals,besidesinterferingwith collagen cross (Fig.l5.30). Homocysteinecondenseswith serine l i nks. H omocystei nei s al so i nvol ved i n t he to form cystathionine.This reaction is catalysed aggregationof LDL particles.All this leadsto an by a PlP-dependentcystathioninesynthase.The increased tendency for atherogenesis, and (PLP-dependent) enzyme cystathioninase cleaves consequentlyheart complications.

Chapter 15 : METABOLISM OF AMTNOACTDS Meffiionine i*

.| -ooc-cH-cHr-cHr-SH I

NHd Homocysteine

1l !i

-ooc-cH-cH2-cH2-s - cH2-cH- cooNHa NHi Gystathionine

351 The enzyme cysteine dioxygenaseoxidizes cystei neto cystei nesul fi natew hi ch, on further oxidation, is convertedto cysteicacid. The latter undergoesdecarboxylationto produce taurine which conjugateswith bile acids. Cysteic acid can also be degraded to pyruvate, which is glycogenic(Fig.l5.31). Cysteine sulfinate cleaves off alanine to produce sulfitewhich is convertedto sulfateand excreted in urine. Some amount of sulfate condenseswith ATP to form active sulfate or 3'-phosphoadenosine S'-phosphosulfafe (PAPS). Active sulfate(PAPS)is utilized for the synthesis of mucopolysaccharides (sulfation), besides being used in detoxification.Sulfate is also a structuralcomponent of some protei ns, l i pi ds etc. Cysteinecan be degradedby desulfhydrase to liberatesulfur (as H2S),ammonia and pyruvate.

h -OOC-fr-CH2-CH3+ HS-CH2-CH- COOo NHI o-Ketobutyrate Cysteine** : + noC-S-fr-CH2-CH2-COO-

Cysteine is a component of tripeptide gl utathi one (synthesi s descri bed i n gl yci ne metabol i sm). Inborn errors of sulfur ami no aci d metabol i sm

Cystinuria (cystine-lysinuria): Cystinuria is one of the most common inherited diseaseswith SuccinylGoA a frequencyof 1 i n 7,000.l t i s pri mari l ycharac terized by increasedexcretionof cystine (25-40 Fig. 15.30 : Synthesisof cysteine from methionine ti mes normal ).E l evati oni n the uri naryoutput of (x-For reactions of methionineconversion to homocysteinesee Fig. 15.29)(x*-ln cysteine synthesis, l ysi ne,argi ni neand orni thi nei s al soobserved.A only sulfur is obtained from homocysteine, the rest specific carrier system exists in kidney tubules of the molecule is lrom serind. for the reabsorpti onof ami no aci ds, namel y cystei ne, orni thi ne, argi ni ne and l ysi ne (rememberC OA L to recal l ). In cysti nuri a,thi s S upp l e me n ta ti o no f d i e t w i th fo l i c aci d, carrier systembecomes defective leading to the v it am in 8 1 2a n d v i ta mi n 8 6 h a v es o mebenefi ci al excreti onof al l thesefour ami no aci ds i n uri ne . affectsin lowering plasma homocysteinelevels (Refer Chapter fi. C ysti nei s rel ati vel yi nsol ubl eand i ncreasei n its concentration leads to precipitation and Degradation of cysteine formationof cystinestonesin kidney and urinary tract. C ysti nuri a i s usual l y i denti fi ed i n the Cystine and cysteine are interconvertibleby laboratory by cyanide nitroprussidetest. The an NAD+-dependentcystine reductase.Cysteine treatmentincludesrestrictedingestionof dietary on decarboxylation produces mercaptocysti neand hi gh i ntakeof fl ui ds. ethanolamine which is involved in the biosynthesisof coenzyme A from the vitamin Cystinosis (cystine storage disease) : Cystine pant ot h e n i ca c i d . crystalsare depositedin many tissuesand organs

o

362

B IOC H E MIS TR Y ami no aci duri a.The affectedpati entsdi e usually within 'l 0 years, mostly due to renal failure. A l though the underl yi ng metabol i c defect in cystinosis is not clearly known, some authors attributethis to the defect in the enzyme cystine reductase. This is, however, not accepted by others.

9H2-S-S-CH2 *H.N-cH Hc-NHi "t l

coo-

coo-

Cystine ,f NADL ' I \ Cvstine redlaase NADH+ ***-J

+

Homocystinurias : Homocystinurias are a group of metabolic disorderscharacterizedby the accumul ati on and i ncreased uri n ar y excretion of homocysteine and S-adenosylmethionine.Plasmaconcentrationof methionine is increased.

?H2-sr-l

H-C-NHi

coo-

Cysteine

tro" c

cH2-so;

CHo t" C=O

coo-

coo-

tHC-NHi

I

Hzc Mercaptoethanolamine

Cysteinesulfinate

I

Pyruvate

rPantoi thenate CoenzymeA

Homocystinuria type I has been more thoroughly investigated.lt is due to a defect in the enzyme cystathionine synthase. Accumulation of homocystetne results in various complications-thrombosis, osteoporosis and, very often, mental retardation. Further, the deficiency of cystathionine is associatedwith damageto endothel i alcel l sw hi ch mi ght l ead t o atherosclerosis.Two forms of type I homocystinurias are known, one of them can be corrected with vitamin B6 supplementation (86 responsive) while the other does not respond to 86. The treatment includes consumption of di et l ow i n methi oni neand hi gh i n cysti ne. The patients of homocystinuria have high levels of homocysteine, and usually die of myocardial infarction, stroke, or pulmonary embol i sm. are associatedwith The other homocvstinurias enzyme defects (as stated below) in the conversion of homocysteineto methionine by remethylation.

Fig. 15.31 : Metabolism of cysteine and cystine.

of reticuloendothelial system throughout the body. These include spleen,lymph nodes, liver, kidney, bone marrow etc. A defect in the Iysosomal function is said to be the primary cause of this disorder. In fact, cystine accumulatesin the lysosomesof varioustissues. lmpairment in renal function is commonly seen in cystinosis.lt is characterizedby generalized

Homocystinuriall : 51s-51O-y"thylene THF reductase. Homocystinuria lll : 5s-511o-yuthyl THFThis is mostly homocysteinemethyltransferase. due to impairment in the synthesisof methylcobal ami n. HomocystinurlalV : Ns-Methyl THF homocysteine methyl transferase.This is primarily due to a defect in the intestinalabsorptionof vi tami n 812.

e?iapter'15 : METABOLISM OF AMINOACIDS

363 2. Histidine contributesformimino fragment to produce Ns-formiminoTHF.

3. When serine is convertedto glycine, Ns, A m ino a c i d m e ta b o l i s m i s p a rti cul arl y THF is formed. This is the most important for the transferor exchange of one- N1O-methylene carbon units. The following one-carbon predominantentry of one carbon units into one fragments are encountered in the biological carbon pool. reactions,which constituteone-carbonpool 4. Choline and betaine contribute to the (-c H 3 ) Methyl formation of Ns-methvl THF. (-c H 2 o H ) Hydroxymethyl The differentderivativesof THF carryingone(:CHr) Methylene carbon units are interconvertible,and this is (-C H :) Methenyl metabol i cal l ysi gni fi cantfor the conti nui ty of (-C H = O) Formyl one- carbon pool (Fig.f5.32). (-C H = N H ) F or m im i n o fr8. ffitf;Eiu;st&eatqpf *ffic"s€nrhorr lNote : lt may be statedhere that CO2 is also mqEfr€lttes a one-carbon unit. Carbon dioxide is involved (carboxylation)in many biochemical reactionsf One-carbonfragmentsfrom THF are used for which are dependent on biotin. For instance, the synthesisof a wide variety of compounds. conversion of pyruvate to oxaloacetate in These i ncl ude puri nes,formyl methi oni neIR N A gluconeogenesis. Most of the authors,however, (required for initiation of protein synthesis), ignore CO2 as one-carbonunit and do not even glycine, pyrimidine nucleotide(thymidylate)etc. c ons ider it w o rth me n ti o n i n g .T h i s w o u l d be unf air t o CO2 !l l l l " ffi el e cai m.+ thi eni rre amd B -2 En opre".c..*;"l.prprlsitet,ahc! tsrn (THF) Tetrahydrofolate is a versatile coenzyme that actively participates in oneMethyl (-CH3) group is an important onecarbon metabolism.With regard to the transfer carbon unit. The role of active methionine as of methyl groups from S-adenosylmethionine, methyl dohor in transmethylationreactions is v it am in 812 i s a l s o i n v o l v e db e s i d e sT H F . already described.After the releaseof methyl group, methionineis convertedto homocysteine. For the regeneration of methionine, free homocysteineand Ns-methyl THF are required and this reaction is dependent on methyl cobal ami n(vi tami n812).The one-carbon pool, under the control of THF, is linked with methionine metabolism (transmethylation) The one-carbon metabolism is rather through vi tami n B 12.H ence vi tami n B 12 i s al so complex, involving many reactions.For the sake involved in one-carbonmetabolism. of better understanding, it is divided into generationand utilization of one-carbon units, and t he r ole o f me th i o n i n ea n d v i ta mi n 8 12. The one-carbon unit covalently binds with T HF at oos i ti o n 5 s o r;1 ' l 0 o r o n b o th N s and N10 of pteroyl structureof folate. The details of differentone-carbonunits binding with THF and the structuresof THF derivativesare given under vitamin-folic acid (Chapter 7).

l, Generation

of one.carbon

un;ts

Valine, Ieucine and isoleucine are the M any c om p o u n d s(p a rti c u l a rl ya mi n o a ci ds) branchedchain and essentialamino acids.These act as donors of one-carbonfragments three ami no aci ds i ni ti al l y undergoa common 1. The formate released from glycine and pathway and then diverge to result in different tryptophan metabolism combines with THF to end products. Based on the products obtained form Nlo-formyl THF. from the carbon skeleton, the branched

364

B IOC H E MIS TRY

-* FTGLU

Purines(C6)

Ns-Formimino THF-------+

Majorproducts

Majorsources

Homocvsteine

r u.\

\

Methionine

/

s-Ad"noryl-y'

methionine Transmethylation (for the synthesis of creatine,epinephrine, choline,melatoninetc.) Fig. 15.32 : Summary of one-carbon metabolism (THF-Tetrahydrofolate; +CHr-Active methyl group).

c ha i n a m i n o a c i d s a re e i th e r g l ycogeni c or ketogenic

coenzymes-TPP, lipoamide, FAD, coenzymeA its action. cr-Keto acid and NAD+-for oxidative decarboxydehydrogenase catalyses glycogenic Va l i n e to the correspondingacyl lation of the keto acids Leucine ketogenic is a regulatory enzyme thioesters. This CoA glycogenicand ketogenic. lso l e u c i n e i n the catabol i sm of branched chai n am ino The first three metabolic reactions are aci ds. c om m o n to th e b ra n c h e d c h a i n ami no aci ds 3. Dehydrogenation: The dehydrogenationis (Fig.15.33). similar to that in fatty acid oxidation. FAD is the 1. Transamination: The three amino acids coenzyme and there is an incorporation of a undergoa reversibletransaminationto form their double bond. lt is now believed that there are resoectiveketo acids. two enzymesresponsiblefor dehydrogenation. 2. Oxidative decarboxylation : s-Keto acid After the initial three common reactions,the deh y d ro g e n a s ei s a c o m p l e x mi tochondri al metabol i sm of branched chai n ami no a cids enzyme. lt is comparablein function to pyruvate diverges and takes independent routes. In a dehydrogenase complex and employs 5 seriesof reactionsthat follow, valine is converted

Chapterl5:

365

OF AMINO ACIDS METABOLISM Valine, leucine, isoleucine

propionyl CoA, a to precursor for glucose. Leucine produces acetyl CoA and acetoacetate, the substrates lor fatty acid s y nt hes is . l s o l e u c i n e i s degraded to propionyl CoA and acetylCoA. Thus,valine is gly c oge n i ca n d l e u c i n e i s k et ogenicw h i l e i s o l e u c i n ei s glycogenic and both ketogenic.

chain I Branched I aminoacid I transarninase

+

Correspondingo-keto acids (cr,-ketoisovalerate, c-ketoisocaproate, a-keto B-methylvalerate NAD+,CoASH NADH

coe Corresponding o, p-unsaturated acylCoA (isobutyryl thioesters CoA,isovaleryl CoA, a,-methylbutyryl CoA)

Metabolic defects of branched chain am ino ac i d s 1 . Maple syrup urine disease: This is a metabolic disorder of branched chain amino acids. The urine of the affected individuals smells like maple syrup or sugar-hence the burnt nam e.

MethylacrylylCoA

PropionylCoA

I I

+ Methylmalonyl CoA

TriglylCoA

CoA B-Methylcrotonyl

+

HMG CoA

MethylacetoacetylCoA

I

19Acetvl CoA

Y

A^ ^ +^ ^ ^ ^ +^ +^ n u g L vq u g ta t9

i |

ii

.:ri, i

Enzyme defect : Maple Glucose Fat {-'--'--'----' syrup urine diseaseis due to a defect in the enzvme branched chain a-keto acid dehydrogenase. This causes a blockadein the conversion of a-keto acids to the respectiveacyl CoA thioesters.The plasma and Diagnosisand treatment : An early diagnosis urine concentrationsof branched amino acids by enzyme analysis-preferablywithin the first and their keto acids are highly elevated. This week of life-is ideal. Estimation of urinary disease is afso known as branched chain branchedami no aci ds and keto aci ds w i l l al so ketonuria. hel p i n di agnosi s. The treatment is to feed a diet with low (or no) content of branched amino acids. The A c c um u l a ti o no f b ra n c h e dc h a i n a mi n o aci ds pl asma l evel s of branchedami no aci ds shoul d causes an impairment in transport and be constantly monitored for adjusting their function of other amino acids. dietary intake. Protein biosvnthesisis reduced. 2. lntermittent branched chain ketonuria : B r anc he d c h a i n a mi n o a c i d s c o mp eti ti vel y This is a lessseverevariantform of maple syrup inhibit glutamatedehydrogenase. urine disease.The enzyme defect is the sameThe disease results in acidosis, lethargy, u-keto acid dehydrogenase. As such, there is an convulsions, mental retardation,coma and, impairment and no total blockade in the conversion of a-keto acids to their respective finally, death within one year after birth. Biochemical complications and symptoms :

.

. . .

366

I

ElIOCHEMISTFIY

acyl CoA thioesters. The symptoms are not as Histidinemia: The frequencyof histidinemiais severeas in maple syrup urine disease.Careful I in 20,000. lt is due to a defect in the enzyme diet planning is adequate to ovecome this histidase.Histidinemiais characterizedby elevated plasmahistidinelevelsand increasedexcretionof disorder. imidazolepyruvateand histidinein urine. Most of 3. lsovaleric acidemia : This is a specific the patientsof histidinemiaare mentallyretarded inborn error of leucine metabolism.lt is due to and have defect in speech. No treatmentwill a defect in the enzyme isovaleryl CoA improvethe conditionof the patients. dehydrogenase. The conversion of isovaleryl CoA to methylcrotonyl CoA is impaired. The Fro{ine excretion of isovalerateis high in urine. The Proline is oxidized to pyrroline S-carboxylate affected individuals exhibit a 'cheesy' odor in which undergoesa non-enzymaticconversionto the breath and body fluids. The symptoms glutamate 5-semialdehyde. The latter is include acidosisand mild mental retardation. converted to glutamate and then transaminated 4. Hypervalinemia : This inborn error is to cr-ketoglutarate.The five carbons of proline characterizedby increasedplasmaconcentration are converted to cr-ketoglutarate. of v al i n e w h i l e l e u c i n e a n d i s o l e uci ne l evel s Hyperprolinemia type | : lt is due to a defect remain normal. The transaminationof valine in the enzyme proline oxidase (proline alone is selectivelyimpaired. dehydrogenase). Another metabolic disorder-hyperproli nemia type ll-associated with hydroxyproline metabolism is also reported. The metabolism of histidine, proline and ArgglgriEse arginine is consideredtogether,as all the three Arginine is cleaved by arginaseto liberate are converted to glutamate and metabolized urea and produceornithine.Ornithine undergoes (Fig.ts.s4). transamination of 6-amino group to form glutamatey-semialdehydewhich is convertedto HEstEe!6m.r* glutamate. Hyperargininemia is an inborn error The metabolismof histidine is important for in arginine metabolism due to a defect in the the generation of one-carbon unit, namely enzyme arginase. formimino group. The enzyme histidaseacts on Nitric oxide (NO) : Arginine is the substrate histidineto split off ammonia. Urocanateformed for the productionof nitric oxide (NO), a wonder in this reaction is acted upon by urocanaseto molecule with a wide range of functions. The produce 4-imidazole 5-propionate. lmidazole enzyme nitric oxide synthase (three isoenzymes ring of the product is cleaved by a hydrolaseto known) cleavesthe nitrogenfrom the guanidino give N-formiminoglutamate (FIGLU). Tetra- group of arginine to form NO. This reaction hydrofolate(THF)takes up the formimino group requires NADPH, FMN, FAD, heme and to form Ns-formimino THF, and glutamate is tetrahydrobiopterin. NO has a very short half-life liberated. Deficiency of folate blocks this (about 5 seconds). reaction and causes elevated excretion of FIGLU in ur i n e . H i s ti d i n e l o a d i n g te s t i s commonl y C=O employed to assessfolate deficiency. I NO synthase HN HN --lz=-\-+ Histidine, on decarboxylation, gives the I (9Ht)' corresponding amine-histamine. Histamine Q2 NO H-C_NHI regulates HCI secretion by gastric mucosa. H-C-NHi lproductionof histaminecausesasthma Excessive coocooCltrulline Arginine and allergic reactions.

ilr; ?-*r'

(tnds.

Tr,

367

Ghapten i 5 r METABOLISMOF AMINO ACIDS

-ooc-9H-cH2-cH2-

c-c-cHz-tH-cooNH; ,'i-^-iln

Nnl

?-|r, NHi

Hlstldine ,

Arginhre

I Histidase

.. I Arginase utea?l

NH;+-J

Y

HC:C-CH=CH-COO-

-ooc-9H-cH2-cH2-

ll

t.j\._/,NH

Nttl

t..i Urocanate

2 I

Omithine Pyrrolines-carboxylate _r._

II

xro-.'| urocanase

J

c-c-cH2-cH2-coo-

"t"l l'.,1-.,.-""/,NH H

{-lmldazoleS-propionate lmldazole .tzv\ I propionate I hydrolase J

----- Non-enzYmatit -\ \ -oo Glutamate5semialdehyde

NAD(P)H+

-ooc-cH-cH2-cH2-coo-N H N-Formlminoglutamate

Fig. 15.34 : Metabolismof histidine,proline, arginine, glutamate and glutamine (THF-Tetrahyd rofolate; a-KG--s- Ketoglutarate; Glu4l utamate).

of The occurrenceof high concentrations citrullinein humanbrain has been known for severalyears.Only recentlyit is realizedthatthe citrullineis formed during the courseof NO synthesis [Note : Nitric oxide (NO) shouldnot with nitrousoxide(NOz)-laughing be confused gas-usedas an anestheticl.

Functionsof NO : The role of nitric oxide as a therapeuticdrug (in the form of nitroglycerine and amyl nitrate) for the treatment of angina pectorishas been known since 1867. However, it is only recentlythat in vivo productionand the biological importanceof NO are recognized.ln fact, the endothelium derived releasingfactor

BIOCHEMISTFIY

368 (EDRF)which causessmooth muscle relaxation is none other than a gas, NO. Nitric oxide acts as a mediator for several biological functions. 1. NO functions as a vasodilatorand causes relaxationof smooth muscles. 2. lt is a key molecule in the regulation of blood flow and the blood pressure(inhibitors of NO synthesismarkedly raise blood pressure). 3. NO acts as an inhibitor of platelet aggregationand adhesion. 4. lt functions as a messengermolecule of the nervous system (neurotransmittef). 5. NO mediatesthe bactericidal actions of macrophages. 6. It is involved in the erecfion of. penis. -l

Mechanismof action : Nitric oxide promotes the synthesisof cGMP. lt is believed that some of the actions of NO are mediated through cGMP and protein kinase C. Agmatine : lt is a derivative of arginine produced in the brain. Agmatine possesses antihypertensiveproperties.

ie

LySli

a,-Ketoglutarate\

I I

Saccharopine dehYdrogenase

+ Saccharopine I Saccharopine Glutamate +1 dehydrogenase

+ cr-AminoadiPate 6-semialdehYde

I Semialdehyde

I dehydrogenase I

+

d-Aminoadipate I Aminotransferase

+

0-Ketoadipate I Dehydrogenase

J

CoA Glutaryl I

I D"hydrog"n""", I Decarborylase + Crotonyl CoA

Acetyl CoA Fig. 15.35: Summary of lysine metabolism.

serves as a precursor for the synthesis of carnitine,a compound involved in the transport of fatty acids to mitochondria for oxidation. lt should be noted that free lysine is not methylated, hence it will not be a substratefor carnitine formation.

Lysine is an essential amino acid. Cereal proteins are deficient in lysine. lt does not participatein transaminationreactions.Some of the lysine residues in protein structure are present as hydroxylysine, methyllysine or Synthesisof carnitinefrom trimethyllysineis a acetyllysine.Thesederivativescan be hydrolysed 4-step reaction involving oxidation, splitting off to liberatefree lysine. gl yci ne resi due,dehydrogenati onand, fi nal ly, Lysine is a ketogenic amino acid. The oxidation (Fig.l5.36). summary of lysine metabolism is depicted in Fig.l5.35. Eilt';r.;u+rte!$*Ennrportance of carnitine Synttlresls

of carnitine

Carnitine plays a key role in the fafty acid oxidation (Chapter 15).

Some of the lysine residuesin proteins are Human requirementsof carnitine are usually found in methylated form. The methyl groups (SAM). with the endogeneousbiosynthesisand the met Such are obtainedfrom activemethionine (by proteolysis)will dietarysupply.Cood sourcesof carnitineinclude proteins on degradation release the methyllysines.The trimethyllysine meat, fish, poultry and dairy products.

Ghapterl5:

METABOLTSM OF AMTNOACTDS e-N-Trimethyllysine 02r

o-KC Succinate

retfryllysine diorygenase

nn

e-N-trimethyilysine B-Hydroxy

Gtvcine -

txine-: l ymethyl_ ferase

y-Butyrobetaine aldehyde

369 immediate precursor for glutamate formation. Clutamate-besides being converted ro glutamine-is involved in the synthesis of certain specializedproducts (Fig..lS.Sl. 1. Gl utathi one i s a tri pepti dethat contai ns glutamate. lts formation is described unoer gl yci ne metabol i sm. 2. N-Acetylglutarnate is an allosterrc regulatorof carbamoylphosphatesynthasel, the first enzyme in urea synthesis. 3. Clutamate is presentin Ihe clotting factors (ll, Vll, lX, X) as y-carboxyglutamate" and is i nvol ved i n coagul ati on.

NAD lrogenase NADH+ H+ y-Butyrobetaine CABA undergoestransaminationfollowed by oxidation to form succinatewhich enters TCA cycle (Fig.tS.JB).The reactionsinvolvingthe fate of GABA constitutea bypassroute for glutamate L-Carnitine Fig" 15.36: Synthesisof carnitine

Proteins

G L U T A M A T E

NHs Glucose

Some researchfindings suggestthat carnitine supplementation has some beneficial effects in the treatment of myocardial dysfunctions, AIDS, etc.

Histidine Proline Arginine

y-Aminobutvric acid

(cABA)

Glutathione N-Acetylglutamate y-Carboxyglutamate (clottingfactors)

( G

Purines (Ns,Ns) Clutamate and glutamine are non-essential L U glycogenic amino acids. Both of them play a Proteins Pyrimidines(N3) T pr edom inantr ol e i n th e a mi n o a c i d m e ta b o l i s m, A NHe and are directly involved in the final transferof M t" Aminosugars + I amino group for urea synthesis. Urea N Detoxification E T he am ino a c i d s -h i s ti d i n e , p ro l i n e a nd arginine-are converted in their metabolismro glutamate (See Fig.l S"S4). a-Ketoglutarate-an Fig" lS.SV : Overviewof glutamate and glutamine metabolism. intermediate in TCA cycle-serves as an

B IOC H E MIS TFI Y

370

900Glutamate decarboxvlase

CHo

COOI

----------------

vrl2

Y"2

+l-

H_C _ N H ; {;()CIGlutamate

I

cor

?",

HrC-NH; yAminobutyricacld (GABA)

Clutamine is the donor of nitrogenatoms for puri ne and pyri mi di nesynthesi s.l t i s the chief source of ammoni a i n ki dneys. The NHr production is elevated in acidosis to maintain acid-base balance" Clutamine also takes part in conjugation reactions.

+

0,-Ketoglutarate

cooI CHe tCHa t-

coo-

Succinate semialdehyde dehydrogenase

\ { r'H2 NADH+ H* NAD* I"

c -H

o

;Succinate

Succinate semialdehyde

Ftg, 15.38: Metabolismof y-aminobutyric acid (PLP-Pyridoxalphosphate). to enter TCA cycle, which is known as GABA shunt.

Both these amino acids are non-essentialand from formed Aspartate is glycogenic. oxaloacetate (an intermediate in TCA cycle) by transamination.Aspartatetransaminase(AST) is an importantenzyme for the interconversionof glutamateand aspartate. Glutamate

Oxaloacetate

cr'-Ketoglutarate

Aspartate

The diagnostic importance of the enzyme AST has already been described (Chapter 6). Aspartate has certain important functions (Fig.|s.39).

Functionsof GABA : lt is one of the major 1. l t donates one ami no group for t he n th e brai n. GA B A synthesisof urea (the other amino group in urea inhib i to ryn e u ro tra n s mi tte irs regulatesthe activity of neuronsby discouraging directly comes from ammonia). t he t r a n s m i s s i osni g n a l s l.t i s b e l i e v edthat C A B A 2. Aspartateforms a connectinglink between open s c h l o ri d e c h a n n e l s a n d i ncreasesthe urea cycle and TCA cycle (via oxaloacetate). permeabilityof post-synapticmembranes.Thus CABA functions as an inhibitory neurotransmifter. Decreased CABA levels will c aus ec o n v u l s i o n s . Vitarnin 86 deficiency and GABA : CABA synthesis requires pyridoxal phosphate, a coenzyme of vitamin 86. In 86 deficiency, the production of GABA is reduced. The result is neuronal hyperexcitability,causingconvulsions.

(Ns, C+, C5 and C6)

$! r , , ' i ;' ," .;,'' Cl u ta m i n e i s a v e rs a ti l e ami no aci d. Ammonia is temporarily stored in the form of glut a m i n e . G l u ta m i n e i s fre e l y d i ffusi bl e and, hence, easily transported. The synthesis and degradation of glutamine are described (See Fig.t s.8).

Fig. 15.39 : Overview of aspartate and asparaginemetabolism.

371 ATP

COO-

\

Asparagine ADP + Pi

synthetas._,O

n-C-ruu; 4-**jffir-C-*' fim; r o 9H,

?-*r.

cH^ l'

cooAspartate

COO-

co-ildl-{2 *

alanine-pyruvate shuttle for carrying nitrogen to be reutilizedor convertedto urea. The ami no aci d p-al ani nei s a consti tuentof the vi tami n pantotheni c aci d, and thus the coenzyme A.

Asparagin*" m)O Asparagine

Fig. 15.40 : Synthesis of asparagine and its conversion to asparlate (Note : The reactions are independent and irreversible).

Serine is a non-essential glycogenic amino acid. As describedin glycine metabolism,serine glycine are interconvertible.Serinecan be 3. ft is utilized for the synthesisof purines and (N1 and NH2 at 6th position) and pyrimidines synthesizedfrom the intermediatesof glycolysis (3-phosphoglycerate). The metabolicreactionsof (N3, Ca C5 and C6 atoms). serine are describedhereunder(FigJ|.al) 4. Malate-aspartateshuttle is important for 1. S eri ne parti ci pates i n transami nati on the transferof reducingequivalents(NADH) from reactions. lt undergoes deamination to form the cytosol to mitochondria(Refer Fig.ll.13). pyruvate. Asparagineis synthesizedfrom aspartateby a synthetase in an irreversible ATP-dependent cooc reaction. Asparaginasehydrolyses asparagine Serinedehvdratase I I H-? and liberates ammonia (Fig.l5.a0). These PLP\------+ ?:o NHs reactionsare comparableto glutaminesynthesis CHs C Pyruvab and its breakdown. Serine 2. S eri ne i s i nvol ved i n one-carbon metabolism.lt donatesmethylene(-CH2) moiety to tetrahydrofolate(THF). The non-essentiaf amino acid alanine performstwo importantfunctions in the body 1 . Incorporationinto the structureof proteins;

COO-

Serinehydroxymethyl- COO(PLP) transferase | ---7--\---------+ H2C-NHi / Gtyeine \ THF Cljr-THF

Proteins

One carbon metabolism Sphingomyelins

H-

2. P ar t ici p a ti o ni n tra n s a mi n a ti o na n d N H 3 transport. As alreadydiscussed,ammonia is toxic to the body, hence it cannot be transported in free f or m . Clut ama te a n d g l u ta m i n e s h o u l d er the m ajor bur de n o f a m m o n i a tra n s p o rt.Al a n ine i s also important in this regard. In the peripheral tissues(most predominantly-muscle), pyruvate produced in glycolysisgets convertedto alanine (by transamination)and is transportedto liver. from alaninein liver Pyruvatecan be regenerated and the pyruvate so produced serves as a precursorfor glucose.Amino group is diverted for transaminationor urea formation.This is an

Glucose Glycine Alanine Cysteine

I + Cystine

Phosphatidylserine Selenocysteine Ethanolamine

+

Choline

Fig. 15.41 : Overuiewof serine metabolism.

372

BIOCHEMISTFIY

Threonine undergoes deamination (by 3. On decarboxylation (PLP-dependent) serineforms ethanolaminewhich is the precursor threonine dehydratase)to s-ketobutyratewhich is convertedto propionyl CoA. Threoninecan be f or ch o l i n e s y n th e s i s . cleaved to glycine and acetaldehydeby serine coohydroxymethyltransferase. Dehydrogenation H C -N H I H,C -N H I followed by decarboxylationof threonineresults t" in aminoacetonewhich mav be converted to cH2oH cH2oH pyruvate or lactate. Ethanolamine Serine 4. Serine is utilized for the synthesis of cysteine(SeeFig.I5.30).lt may be noted that the entire cysteinemolecule is derived from serine exceptthe sulfurthat comesfrom homocysteine. 5. Serine is involved in the formation of selenocysteine,the 21st amino acid found in certain proteins.

t'

The metabolic reactionsof individual amino acids are describedabove. After the removal of amino groups/ the carbon skeleton of amino 6. Serinedirectly participatesin the synthesis acids is converted to intermediatesof TCA cycle of p h o s p h o l i p i d -p h o s p h a ti d ysl e ri ne (detai l s or their precursors.The carbon skeletonfinally describedin lipid metabolism,Chapter l4). has one or more of the following fates 7. Serineis also involved in the svnthesisof s phi n g o my e l i nas n d c e p h a l i n s . 8. In the structureof proteins,serine (-OH group) servesas a carrier of phosphatewhich is involved in the regulation of many enzyme activities.

1. Oxidation via TCA cycle to produce energy (about 10-15% of body needs). 2. Synthesisof glucose. 3. Formationof lipids-fatty acids and ketone bodi es. 4. Synthesisof non-essentialamino acids.

Threonine is an essentialhydroxy amino acid. It is glycogenic and does not participate in transaminationreactions.Threonine is often a carrier of phosphate group in the protein structure.The outline of threoninemetabolismis depicted in Fi9.15.42.

The carbon skeletonsof the 20 standard(or more) ami no aci ds (or the ami no aci ds of proteins)are degradedto one of the following a-ketoglutarate, seven products-pyruvate, succinyl CoA, fumarate, oxaloacetate, acetyl CoA and acetoacetate. Some authors use the term amphibolic (Creek:amphiboles-u ncertain) intermediatesto these compounds due to their mul ti pl e metabol i cfuncti ons. The amino acids are classified into two groups/ based on the nature of the metabolic end productsof carbon skeleton.

Proteins o-Ketobutyrate

J

PropionylCoA

1. Glycogenic (glucogenic) amino acids : These are the amino acids whose carbon skeletonis finally degradedto pyruvateor one of the intermediatesof TCA cycle (c,-ketoglutarate, These succinylCoA, fumarateand oxaloacetate). intermediates serve as good substrates for gluconeogenesisleading to the formation of glucoseor glycogen.

Chapter 15 : METABOLISM OF AMINOACIDS

373

Asparagine Aspafiate---|

Tryptophan Oxaloacetate

f Phenvtataninel .Krebs ryiosinE- l------+Fumarate cycle lsoleucineI \ Methionine

I Threonine I Valine l

SuccinylCoA

*'---_--/

cr-Ketoglutarate{-

Giutamate

t

lilutamire

Flg. 15.43: Summaryof theproductsfonnedfromcahon skeletonof aminoacids (colourindication,Blueflucogenic; Greenshade-glucogenicand ketogenic;Red-ketogenic).

2. Ketogenicamino acids : The amino acids whose carbon skeletonis metabolizedto acetvl CoA or acetoacetate can be converted to fat (i.e., fatty acids or ketone bodies).Acetoacetate is a ketone body (besides acetone and p-hydroxybutyrate). Someof the amino acids are both glycogenic and ketogenic since they serve as precursorsfor glucose as well as fat. The classificationof amino acids (glycogenic, ketogenic, or both) is given in Table 15.4. The various products obtained from the carbon skeleton of amino acids and their connection with the citric acid cycle is depicted in Fig.t 5.43.

Glycngeniq Q;iucagentc)

Alanine Arginine* Aspartate

Tyrosine

Cysteine

Tryptophan*

Glutamine Glutamate Glycine Histidinex Methionine*

. Pyruvate : Alanine, cysteine, glycine, hydroxyproline,serine and threonine.

Serine Threoninex

glutamate,

. Succinyl CoA : lsoleucine, methionine, threonineand valine.

Ketogenic

Phenylalaninex Leucinex lsoleucine* Lysinex

The details on the formation of amphibolic intermediatesby the degradationof amino acids are given in the metabolismof respectiveamino acids. They are summarizedhereunder

. c-Ketoglutarate : Clutamine, ar ginine,hi s ti d i n ea n d p ro l i n e .

Glycogenicand ketogenic

Hydroxyproline Proline

Valinex * Essential aninoacids;(Helpful tipsto recall--ketogenic anino acidsstal withletter'L'; PITTfor glyn- andketogenic amino acids;rcstol the20 aminoacidsareonlyglycogenh).

BIOCHEMISTFIY

374

non-essential in the diet, as they can be in human body. This is carried out synthesized . Oxaloacetate : Asparagine and aspartate. by the biosynthesisof carbon skeleton,followed . Acetyl CoA and acetoacetate: Phenylalanine, by the addition of amino group via ty ro s i n e ,try p to p h a n ,i s o l e u c i ne,l euci ne and transamination.In the Table 15.5,the sourcesof ly s i n e . carbon skeletonfor the synthesisof non-essential L e u c i n ea n d l y s i n ea re o n l y k etogeni c,si nce ami no aci ds are gi ven. they produce acetoacetateor acetyl CoA. a n d tyrosi ne. . Fu ma ra te: Ph e n y l a l a n i n e

Of the 20 amino acids,about half of them are

Severalinheriteddisordersare associatedwith amino acid metabolism. The details of these

BIoMEDICAI/ CLINICALCONCEPTS

Melonin-the pigment oJ, skin, hair qnd eyes-is produced t'rom tyrosine. Lack of melanin synfhesis (mostly due to a deficiency of tyrosinase)couses albinism' Porkinson's disease----acommon disorder of the elderly-is linked with decreosed synthesisot' dopamine. It is charocterizedby muscular rigiditg, tremors, lethorgy etc. Phenylketonuria, due to a defect in the enzyme phenylalanine hydroxylose, is choracterizedby failure of growth, seizuresand mentol retardation (low lQ). Alkaptonuria couses the occumulation of homogentisate which undergoes oxidation followed by polymerization to produce the pigment alkapton. Deposition ol olkapton in fissues (connective fissue, bones) cousesochronosis u.rhichis associotedwith arthritis. Serotonin, an excitatory neurotransmitter, is synthesized lrom tryptophan. Psychic stimulant drugs (iproniazid) eleuate serotonin leuels while depressant drugs (LSD) decrease. Malignant carcinoid syndrome, a tumor of argentaffin cells ot' gastrointestinal tract, is choracterized by tremendously increased production of serotonin. This disorder can be diagnosed by the eleuated leuels of S-hydroxyindoleocetotein urine. w- Melotonin, produced from serotonin, is inuolued in circadion rhythms or diurnol uariations,i.e., maintenance of body's biological clock. (f Homocysteine has been implicated os o risk factor in the onset of coronary heort diseoses. rg

Histidine loading test, characterized by eleuoted excretion ol N-t'ormiminoglutamate (FIGLU) is commonly employed to ossessthe deficiency of the uitamin, folic acid. Nitric oxide (NO), synthesizedfrom arginine, is inuoluedin seueralbiologicol tunctionsuasodilation, platelet aggregotion, neurotronsmission ond bactericidal action.

re yAminobutyric acid (GABA), produced Jrom glutomate, is an inhibitory neurotrans' mitter. Low leuels of GABA result in conuulsions. The carbon skeleton ol amino acids may be conuerted to glucose (glycogenic) or fot (ketogenic), besides being responsible for the synthesis of non-essential amino acids. Polyamines (spermine, putrescine) are inuolued in the sgnthesis ol DNA, RNA proteins and, thus, they are essentialfor cell growth ond differentiotion.

and

G hapt er 1 5 ' M ET A BOL IS MOF A M IN OA C ID S

375 Biosynthesis

Amino acid Glycine Alanine serine ff[:|d I ttl,:HiJt:.. | GtutamineI Proline ./ Cysteine Tyrosine

Source(s)of carbon skeleton Serine pyruvate 3.phosphogtycerat,

,ntermediares of Xrebs cycle (sultur Serine donated bymethionine) Phenylalanine

Ornithine and S-adenosylmethionine are the precursorsfor polyamine synthesis.lt should, however, be noted that only the four-carbon moiety of SAM (not the methyl group) is involved in polyamine formation. Ornithine decarboxylaseacts on ornithine to split off Co2 and produce putrescine(Fig.lS. a). The enzyme o rnithine decarboxylat" itt the shortest halflife (about 10 minutes) among the known mammalian enzymes. lt regulates polyamine synthesis. The activity of this enzyme is increased by hormones like corticosteroids, testosteroneand growth hormone.

putrescine is converted to spermidine and then spermine with the involvement of SAM. is first decarboxylatedto metabolic disorders are described in the S-Adenosylmethionine respective amino acids. Tahle 15.6 gives u give decarboxylatedSAM. SAM decarboxylaseis summarv of the inborn errors of amino acid a rare example of an enzyme that does not require pyridoxal phosphateas coenzyme. An metabolism. amino acid residue bound to pyruvate is believed to function as a cofactor. The propylamino group of decarboxylatedSAM is transferred to putrescine to give spermidine. In general, the decarboxylation of amino Synthesis of spermine requires one more acids or their derivativesresultsin the formation molecule of decarboxylated SAM and this of amines. reaction is catalysedby sperminesynthase. 2

R-

-css6trg@€r.(|+R-cH2-NH2

Aminoacid

ci''

Amine

A summary of the biogenic amines derived from different amino acids and their major functions are given in Table 15.7.

DegradatEcncptpstyanrfimes The enzyme polyamine oxidase (of liver peroxisomes)oxidizes spermine to spermidine and then to putrescine. Spermidine and putrescineare excretedin urine in a conjugated form, as acetvlatedderivatives.Some amount of putrescineis also oxidized to NH3 and CO2.

Functi ons of pol yami nes Polyamines (Creek: poly-many) possess r1 . h| : Polvamines are basic in natureano possess ' multfple amlno Eroups. rutrescrnet spermrne _r-, ., .,. mul ti pl eposi ti vecharges. H encethey are readi l y , ., , . , . ,, .-:: ano spermrorneate rne ororoSrcailyrmponanr r , .' _ associatedwith nucleic acids (DNA and RNA). por, y am r n e s .)p e rm rn e a n o s p e rmro rnew ere originally detected in human semen (sperms), 2. They are involved in the synfhesisof DNA, hence they are so named. RNA and proteins.

B IOC H E MIS TRY

376

Disorder

Metabolic defect (enzyme/other)

l. Defects in urcasynthesis 15.1 -ReferTable ll. Phenylalanine andtyrosine 1. Phenylketonuria

hydroxylase Phenylalanine

2. Tyrosinemia typell

transaminase Tyrosine p-Hydrory phenylpyruvate dioxygenase

3. Neonataltyrosinemia 4. Alkaptonuria (tyrosinemia 5. Tyrosinosis typel)

Homogentisate oxidase

6. Albinism

Tyrosinase

hydrolase isomerase acetoacetate or fumaryl Maleyl acetoacetate

lll. Sulfuraminoacids(methionine, cysteine andcystine) 7. Cystinuria 8. Cystinosis

Defect in renalreabsomtion (defect function) in lysosomal in cystine utilization lmpairment

typeI 9. Homocystinuria

synthetase Cystathionine

10. Homocyslinuria typell

Ns,N1o-Methylene THFreductase

11. Homocystinuria typelll

THF-homocysteine methyltransferase N5-Methyl

12. Cystathionuria

Cystathioninase

lV. Glycine 13. Glycinuria

Delectin renalreabsorption

14. Primary hyperoxaluria

transaminase Glycine

V. Tryptophan 15, Haftnup's disease

intestinal absorption Defective

Vl. Branched chainaminoacids(valine, leucine andisoleucine) 16. Maple syrupurinedisease

Branched chains-ketoaciddehydrogenase

(lesssevere) 17. lntermittent branched chainketonuriaVariant of theaboveenzyme 18. Hypervalinemia

Valine transaminase

19. lsovaleric acidemia

lsovaleryl CoAdehydrogenase

Vll. Histidine 20. Histidinemia

Histidase

Vlll. Proline 21. Hyperprolinemia typeI

Proline oxidase

Ghapter 15: METABOLISM OF AMINO ACIDS

377

+ H 3 N -C H -(C H 2 ) 3-N H i

-

coo-

CooOrnithine

I

S-Adenosylmethionine(SAM)

Omithine vwz?lI decarboxvlase

J

I

COz?]SAM decarborylase

+ H 3 N -C H 2 -(C H 2 )3-N H l

J

Putrescine CH3-S-Adenosine Methylthioadenosine

DecarborylatedSAM

+H3N-CH2-CH2- CH2-NH-CH2- (CHz)s-NHd Spermidine

CH3-S-Adenosine ' Spermine synthase Methylthioadenosine *H3N-CH2-CH2-CH2-NH-CH2-(CH2)3-NH-CH2- CH2- CH2- NHi Spermine Fig. 15.44 : Biosynthesis of polyamines-putrescine,

spermidine and spermine.

3. They are essential for cell growth and proliferation. Amino acid

Amine

Function(s)

4. Some enzymes are inhibited polyamines,e.g. protein kinase.

by

5. Thev are believed to be involved in the stabilizationof the membranestructure(cell and cel l ul arorganel l es). PhenylalanineDopamine

Tryptophan Tryptamine Serotonin Melatonin Cysteine Taurine

Forthesynthesis of nore- Glinieal importance and polyamines pinephrine andepinephrine (increases Vasoconstrictor The excretion of polyamines is found to be bloodpressure) elevated in almost all types of cancers, Elevates bloodpressure e.g. l eukemi as;carci noma of l ungs, bl adder, cerebral activity kidney etc. Diagnostically, putrescine is an Stimulates rhythms Circadian ideal marker for cell proliferation whereas of bileacid Constituent spermidineis suitablefor the assessment of cell (taurocholic acid) destruction.

BIOCHEMISTFIY

378

7 ' T h e b o d y p ro te i n s a re i n a d y nami cstate(degradati onandsynfhesi s)andtherei san purpose' actiue amino acid pool (100 S) maintained t'or this to liberate smmonia for the 2. The amino acids undergo tronsamination and deamination synthesisaf ureo, the end product ol protein metabolism' proteins, omino acids participate in 3. Besides being presentas structural components of compounds' the formation of seaeral biotogically important heme, purines, glutothione etc' 4. Glycineis inuoluedin the synthesisof creatine, is a precufsor for the production of. 5. Phenylalanine is hydroxylated to tgrosine, uhich (dopa^ire, epinephrine and norepinephrine) skin pigment (melanin),'catecholomines (73 ond Ta)' ond thgroid hormones qnd IVADP+, the coenzymes of niacin, serotonin 6' Tryptophan is converted fo NAD+ h neurotransmitter)and melotonin' .1

group (transmethylotion) t'or the The actiue methionine (SAM) is a donar of methyl choline, methylcatosineetc')' synthesisof mony biological compounds(epinephiini,

methylene (Jormyl, .t'ormimino, Mang amino acids contribute to one-carbon t'ragments control of the under is mostly etc.) lor participation in one-carbon metabolism-which tetrahydrofolote. either in the synthesis of glucose 9. The carbon skeleton of amino ocids is inuolued (glycogenic) or fat (ketogenic), or both-glucose ond fat'

6.

in amino acid metabolism haue lo. Many inborn errors(mostly due to enzyme defects) (de7"ct-phenylolaninehvdroxylose)' been identified. These initude phenylketonur:io a l b i n i s m(d e fe c t.ty ro s i n ase),mapl esyrupuri nedi sease(defect_a-ketoaci ddehydr o. genase) etc.

Ghapter 15 : METABOLISMOF AMINO ACIDS

379

I. Essayquestions 1. Describethe reactionsin the synthesis of urea. 2. Cive an accountof the formationof specializedproductsfrom glycine. 3. Discussthe metabolismof phenylalanine and tyrosine. 4. Describethe fate of carbonskeletonof amino acids. 5. Write brieflyon variousinborn errorsof amino acid metabolism. II. Short notes (a)Amino acid pool, (b) Transmethylation, (c) Transamination, (d) Deamination,(e) Ammonia toxicity, (0 One-carbonmetabolism,(g) Albinism, (h) Serotonin,(i) Glutamateand glutamine,

(j) Polyamines. III. Fill in the blanks 1. Thecoenzyme thatparticipates in transamination reactions is 2. The most importantenzymeinvolvedin oxidativedeaminationis 3. N-Acetylglutamateis requiredfor the activationof the enzyme 4. Primaryhyperoxaluriais due to a defectin the enzyme 5. The cofactorrequiredfor the conversionof phenylalanine to tyrosineis 6. Parkinson's diseaseis linkedwith decreasedsynthesis of 7. The metaboliteexcreted(elevated)in alkaptonuriais 8. The diseasein which very high amountof tryptophan(nearly60%) is convertedto serotonin ts 9. The mammalianenzymewith the shortesthalf-life(aboutl0 minutes)is 10. The branchedchain amino acid that is only ketogenicis IV. Multiple choice questions 11. The synthesis of urea occursin (a) Kidney(b) Liver (c) Muscle(d) Brain. 12. The amino acid requiredfor the formationof glutathione (a) Clycine (b) Cysteine(c) Glutamate(d) All of them. 13. In the synthesis of cysteine,the carbonskeletonis providedby (a) Serine(b) Methionine(c) Clutamate(d) Alanine. 14. The amino acids are said to be ketogenicwhen the carbon skeletonis finally degradedto (a) SuccinylCoA (b) Fumarate(c) Acetyl CoA (d) Pyruvate. 15. The amino acid that does not participatein transamination (a) Lysine(b) Glutamate(c) Alanine (d) Tryptophan.

The energg metabolism

sgcths

t

"Carbohydrate,fat, protein m.etabolismsintegrate; Cells, tissuesand organs coordinate| Tb meet the body\ fuel deman*; The essentialrequisitefor existence,"

etabolism is a continuous process,with thousands of reactions. simultaneouslv Energysupply oc c urri n g i n th e l i v i n g c e l l . H ow ever, (variable) biochemistsprefer to presentmetabolismin the form of reactionsand metabolic pathways.This is done for the sake of convenience in presentationand understanding.

Oz CO2 + H2O

Energydemands (variable)

fn the preceeding three chapters (13-15), we Fig. 16.1: A summaryof body'senergysupplyand have learnt the metabolism of carbohydrates, (Note : ATP seruesas the energycurrency), lipids a n d a mi n o a c i d s .W e s h a l l n ow consi der demanda the organism as a whole and integrate the metabolismwith particular referenceto energy The humans possessenormous capacity for demandsof the body. food consumption.lt is estimatedthat one can consume as much as 100 times his/her basal ii;31€i"9tr$ *i,prm*rue{anrcl surp:3rly requirements! Obesity, a disorder ol overnutrition mostly prevalentin affluentsocieties,is The organisms possess variable energy primarily a consequenceof overconsumption. dem a n d s h , e n c eth e s u p p l y(i n p u t)i s al soequal l y variable. The consumed metabolic fuel may be ilattegx+a{imm*rf rxt*ljor imd}'t,i*futli$* burnt (oxidized to CO2 and H2O) or stored to pa*hways €)f e$ergy .rmetalsoHisatt meet the energy requirementsas per the body needs. AIP serves as lhe energy currency of the An overview of the interrelationshipbetween cell in this process(Fig.|5.l). the important metabolic pathways, concerned

380

Ghapter 16 : INTEGRATION OF METABOLISM

Glycogen

aminoacids

381 Triacylglycerols

'-

FADH;

Fig. 16.2 : An overview of integrationof metabolicpathways of energy metabolism (HMP shunt-Hexose monophosphate shunt).

with fuef metabolism depicted in Fig.l6.2, is brieflv describedhere. For detailed information on these metabolic pathways,the reader must refer the resoectivechaoters.

different fuel sources (carbohydrates,lipids, amino acids). Acetyl CoA enters citric acid (Krebs)cycle and gets oxidized to CO2. Thus, citric acid cycle is the final common metabolic pathway for the oxidation of all foodstuffs.Most 1. Glycolysis: The degradationof glucoseto pyruvate (lactate under anaerobic condition) of the energy is trapped in the form of NADH generates8 ATP. Pyruvateis converted to acetyl and FA D H 2. CoA. 5. Oxidative phosphorylation : The NADH FADH2, produced in different metabolic and 2. Fafty acid oxidation : Fatty acids undergo pathways, are finally oxidized in the electron sequential degradation with a release of (ETC).The ETC is coupled with transport chain 2-carbon fragment, namely acetyl CoA. The phosphorylation oxidative to generateATP. energy is trapped in the form of NADH and F A DH2. 3. Degradation of amino acids : Amino acids, particularly when consumed in excess than requiredfor protein synthesis,are degraded and utilized to meet the fuel demands of the body. The glucogenicamino acids can serve as precursorsfor the synthesisof glucose via the formation of pyruvate or intermediatesof citric acid cycle. The ketogenicamino acids are the precursorsfor acetyl CoA. 4. Citric acid cycle : Acetyl CoA is the key and common metabolite, produced from

6. Hexose monophosphate shunt : This pathway is primarily concerned with the liberationof NADPH and ribose sugar.NADPH is utilized for the biosynthesis of several compounds, including fatty acids. Ribose is an essentialcomponent of nucleotidesand nucleic acids (Nofe .'DNA containsdeoxyribose). 7. Gluconeogenesis : The synthesis of glucose from non-carbohydrate sources constitutesgluconeogenesis. Severalcompounds (e.g. pyruvate,glycerol, amino acids) can serve as precursorsfor gluconeogenesis.

382

BIOCHEMISTFIY

3. Protein metabolism : Increaseddegradation 8. Glycogen metabolism : Glycogen is the storage form of glucose, mostly found in liver of amino acids and protein synthesis. and muscle. lt is degraded(glycogenolysis) and synthesized (glycogenesis) by independent Adipose tissue pathways. Glycogen effectively serves as a fuel Adipose tissue is regarded as the energy reserve to meet body needs, for a brief period storage tissue. As much as 15 kg. of (between meals). triacylglycerol (equivalent to 135,000 Cal) is stored in a normal adult man. The major pathways Regulation of rnetabolic metabolic functions of adipose tissue in an The metabolic pathways, in general, are absorptive state are listed here. controlled by four different mechanisms 1. Carbohydrate metabolism : The uptake of glucoseis increased.This follows an increasein 1. The availabilityof substrates glycolysisand hexosemonophosphateshunt. 2. Covalent modificationof enzymes 2. Lipid metabolism : The synthesisof fatty 3. Allosteric regulation acids and triacylglycerols is increased. The 4. Regulationof enzyme synthesis. degradationof triacylglycerolsis inhibited. The details of these regulatory processesare discussed under the individual metabolic Skeletal mussE{r pathways, in the respectivechapters. The metabolism of skeletal muscle is rather variable depending on its needs. For instance, the resting muscle of the body utilizes about 3O"/oof body's oxygen consumption.However, during strenuousexercise,this may be as high as 90%. The important metabolic functions of skeletalmuscle in an absorptivestateare Iisted. The various tissuesand organs of the body 1. Carbohydrate metabolism : The uptake of work in a well coordinatedmanner to meet its glucose is higher, and glycogen synthesis is metabolic demands. The major organs along increased. with their most importantmetabolicfunctions,in a well-fed absorptive state (usually 2-4 hours 2. tipid metabolism : Fatty acids taken up after food consumotion),are described. from the circulation are also important fuel sourcesfor the skeletalmuscle. Liver 3. Protein metabolism : Incorporation of The liver is specializedto serveas the body's ami no aci ds i nto protei nsi s hi gher. central metabolic clearing house. lt processes and distributesthe nutrientsto different tissues Brain for utilization.After a meal, the liver takesup the The human brain constitutesabout 2"/oof the carbohydrates,lipids and most of the amino body's weight. But it utilizes as much as 20o/oof acids, processesthem and routesto other tissues. the oxygen consumed by the body. Being a vital The major metabolic functions of liver, in an organ, special priority is given to the metabolic absorptive state, are listed needsof the brain. 1. Carbohydrate metabolism : Increased 1. Carbohydrate metabolism : In an glycolysis, glycogenesis and hexose monoabsorptivestate,glucose is the only fuel source phosphateshunt and decreasedgluconeogenesis. to the brai n. A bout 120 g of gl ucosei s uti lized 2. tipid metabolism : Increasedsynthesisof per day by an adult brain. This constitutesabout fatty acids and triacylglycerols. 6O"h of the glucose consumed by the body at

Chapten 16 : INTEGFATION OF MFI-ABOLISM

383

Organ/Tissue

Energy compound(s) preferably utilized

Energy compound(s) exported

Liver Adipose tissue Skeletal muscle

Aminoacids,glucose, fattyacids Fattyacids Fattyacids Glucose (instarvation) Glucose, ketone bodies

Glucose, fattyacids,ketone bodies Fattyacids,glycerol, None Lactate None

Brain

rest. lt is estimated that about 50% of the energy consumed by brain is utilized by plasma membrane Na+-K+-ATPase to maintain membrane potential required for nerve impulse t r ans m is s i o n .

reserve of the body. The survival time of an individual on starvationis mostly dependenton his/her fat stores. And for this reason, obese i ndi vi dual s can survi ve l onger than l ean i ndi vi dual sw i thout consumi ngfood.

2. tipid metabolism : The free fatty acids cannot crossthe blood-brainbarrier,hence their contributionfor the supply of energyto the brain is insignificant.Further, in a fed state, ketone bodiesare almost negligibleas fuel sourceto the brain. However, brain predominantly depends on ketone bodies during prolonged starvation (detailsgiven later).

Protein is basically a structural constituent, mostly presentin the muscle. However, during starvation, protein can also meet the fuel demandsof the body. lt is estimatedthat about 1ftrd of the body's protein can be utilized towardsenergy needswithout compromisingthe vital functions.

Starvation is associated with a decrease in insulin level and an increasein glucagon. The The metabolic interrelationshipamong the metabolic changes during starvation are major tissuesin an absorptivestateare given in discussed with referenceto the major organs/ Fig.|6.3. The fuel sources that are preferably tissues. utilized by the major organsand the compounds exported from them are listed in Table t6.l . LEverin

starvatton

Starvationmay be due to food scarcity or the desire to rapidly lose weight or certain clinical conditions(e.9.surgery,burns etc.).Starvationis a metabolic stress which imposes certain metabolic compulsions on the organism. The metabolism is reorganized to meet the new demandsof starvation. Clucose is the fuel of choice for brain and muscle. Unfortunately,the carbohydratereserve of the body is so low that it cannot meet the energy requirementseven for a day. The fuel stores (or energy reserves)of a 7O kg normal man are given in Table 16.2. Triacylglycerol (fat) of adipose tissue is the predominant energy

1. Carbohydratemetabolism : An important function of liver is to act as a blood glucose buffering organ. The action of Iiver is to suit the metabolic needsof the body. During starvation, increased gluconeogenesis and elevated glycogen degradation furnish glucose to the needy tissues(mostly brain).

Energy source (main storage tissue)

Triacylglycerol (adipose tissue) (muscle) Protein (muscle, Glycogen liver)

Weight

(kt)

Energy equivalent (in Cal)

15

135,000

A

24,000 800

0.2

384

BIOCHEMISTFIY

Glucose---+ HMP shunt

evruu"te----|AcetylCoA---+Fattyacids

$1ff,, "_:___l tl YI *,".

Amino acids

r |Y

Urea

Proteins

Aminoacids AcetylCoA{-

I

Pyruvate

1

proteins

Glucose

,( KrebsJ

|

,-

Fig. 16.3 : Metabolic interrelationship among the maior tissues in a well fed state (HMP shunt-Hexose monophosphate shunt).

EIEME$GALI CLINICATGONtrEPTs

Biochemists,for their conuenience, Iearn body chemical processesin terms of indiuidual metabolic reactions and pathways, although thousands of reactions simultaneously occur in a liuing cell. The metabolic pathwaysin uariousfissuesand organs are well coordinated to meet the demands of the body. Liver is oppropriatelg regardedas the bodg's 'central metabolic clearing house' while adipose fissuesconstitute the energy (Jat)storehouse. Broin is o uital metabolic organ thot consumesabout 200/ool body's oxygen, although it constitutes only ?/o of body weight. The metobolismin sforuofion is reorgonizedto meet the body's changed demands and metabolic compul sions. Under normol circumstances,glucose is the only fuel source to brain. Howeuer;during staruation, the brain slowly gets adopted to use ketone bodies for energy needs.

r<

Chapter 16 ; INTEGHATION OF METABOLTSM

38s

Glucosa

Fattyacids \.+Glycerol

Glycerol, Amino acids

Fattyacids

+

Aminoacids AcetylCoA

II

Ketonebodies

Ketonebodies \

Proteins

r,il i1

H i$

ff

2. tipid metabolism : Fattyacid oxidation is ,rt,r$!y:r:rsetissue in stervafimn increased with an elevated synthesisof ketone 1. Carbohydrate metabolism : Clucose bodies. This is due to the fact that TCA (Krebs) uptake and its metabolismare lowered. cycle cannot cope up with the excessproduction of acetyl CoA, hence the lafter is diverted for 2. Lipid metabolism : The degradation ketone body synthesis. of triacylglycerol is elevated, leading to an Ketone bodies (primarily p-hydroxybutyrate) increasedreleaseof fatty acids from the adipose effectively serve as fuel source for the peripheral tissue which serve as fuel source for various tissues.The brain slowly adapts itself to use tissues (brain is an exception). The glycerol ketone bodies. Thus, after a 3-day fast, about liberated in lipolysis servesas a precursor for 1ftrd of the brain's fuel demands are met by glucosesynthesisby liver. The synthesisof fatty ketone bodies, while, after 40 days, starvation, acids and triacylglycerolsis totally stopped in they countribute to about 7O,'/"of energy needs. adipose tissue.

386

E | IOC H E MIS TFIY

As alreadystated,glucoseis the preferredfuel source by brai n. D uri ng the fi rst 2 w eeks of starvation,the brain is mostly dependent on gl ucose,suppl i edby l i ver gl uconeogenesiThi s. s, 2. Lipid metabolism : Both fatty acids and i n turn, i s dependenton the ami no aci dsrel eased k et oneb o d i e sa re u ti l i z e db y th e mu scl eas fuel from the muscle protein degradation.Starvation source. However, on prolonged starvation beyond 3 w eeks general l yresul tsi n a marke d beyond 3 weeks, the muscie adapts to increasein plasma ketone bodies. By this time, ex c lus i v e l y u ti l i z e fa tty a c i d s . T hi s further the brain adapts itself to depend on ketone inc r eas e sth e l e v e l o f k e to n e b o d i es i n the bodies for the energy needs. c ir c ula ti o n . The metabol i c i nterrel ati onshi pamong the 3. Protein metabolism : During the early major organs in starvation are depicted in period of starvation, muscle proteins are Fig.|6.4. The biochemical changes that occur degradedto liberatethe amino acids which are during starvation are such that an adequate ef f ec t iv e l y u ti l i z e d b y th e l i v e r fo r gl ucose supply of fuel molecules is maintained to s y nt he s i s (g l u c o n e o g e n e s i s ).O n prol ongeo various fissues to meet the energy demands. starvation, however, protein breakdown is This is a naturaladaotationfor the survivalof the reduced. organrsm. 1 . Carbohydrate metabolism : Clucose upt ak e a n d i ts m e ta b o l i s m a re v ery much deoressed.

1. The metabolism of carbohydrates,/ipids and proteins is integrated to meet the energg and metobolic demandsof the organism.The metabolic pathways---glycolysis, latty acid oxidation, citric acid cycle and oxidotiue phosphorylation--aredirectly concerned with the generotion ol ATP. Gluconeogenesis,glycogenmetqbolism, hexosemonophosphate shunt ond amino acid degrodation are olso ossociotedwith energy metabolism,

2. The organ{tissues, urifh their respectiuespecializations,coordinate with eoch other to meet the metqbolic demands of the organism os o whale. Liuer is specializedto serue as the body's central metabolic clearing house.It processesand distributesthe nutrients to different tissuesfor their utilization. Adipose fissue is primarily a storage organ ol fat. The major bulk of the bady protein is located in the muscle tissue.

3. Brain is o specializedorgan which, in the normal situotion, is exclusiuelydependent on the supply of glucose (120 Sldeil t'or its fuel needs.

4. Staruation is a metabolic stress, as it imposescertain metabolic compulsionson the organism. The stored fat ol adipose fissue ond the muscle protein are degraded and utilized to meet the body's fuel demonds. Brain gradually adapts itself to use ketone bodies (instead ol glucose)for its energy requirements. Staruation is, fhus, qssociofed with metobolic reorganizationfor the suruiualof the organism.

Nfletffillfl smofNuelieotfl dles

ucleotidesconsistof a nitrogenousbase,a \f | \ pentose and a phosphate. The pentose s ugaris D- r ib o s ei n ri b o n u c l e o ti d eosf R N A w hi l e in deoxyribonucleotides(deoxynucleotides)of DNA, the sugaris 2-deoxy D-ribose.Nucleotides par t ic ipat e i n a l m o s t a l l th e b i o c h e mi cal processes/either directly or indirectly.They are t he s t r uc t ur acl o mp o n e n tso f n u c l e i ca c i d s(D N A , RNA), coenzymes, and are involved in tne regulationof severalmetabolic reactions.

Aspariaie--'N .,,,t

.J

. i

t

Y

Glutamine

Fig. 17.1 : The sources of individuat atoms in purine ring. (Note : Same colours are used in the syntheticpathway Fig. lZ.2).

M any c omp o u n d s c o n tri b u te to th e p u ri ne ring of the nucleotides(Fig.t7.l). 1. N1 of purine is derived from amino group of aspartate.

n T.

C4, C5 and N7 are contributedby glycine.

5. C6 directly comes from COr.

It should be rememberedthat purine bases are not synthesizedas such, but they are formed 2. C2 and Cs arise from formate of N10- as ribonucleotides. The purines are built upon a pre-existing ribose S-phosphate. Liver is the formyl THF. maj or si te for puri ne nucl eoti de synthesi s. 3. N3 and N9 are obtainedfrom amide group Erythrocytes, polymorphonuclearleukocytesand of glut am ine . brai n cannot produce puri nes.

388

BIOCHEMISTF|Y

m-gg-o-=_ |l

Formylglycinamide ribosyl S-phosphate

H)

Kn

H \-Y

Glutam +ATt

OH

OH OH

Glutame + ADP

cl-D-Ribose-S-phosphate

orr-l o"t*']

PRPPsYnthetase

+

EO-qn2-O.l./

\l

H)

KH

u \.]_j^/ II

r,\-iEl-/^\-td

PRpp glutamyl amidotrahsfera-se

1../ \H

\cH

- it l

Hrcl

H N :C --

H

OH OH S-Phosphoribosylo-pyrophosphate

E-o-gH4.o-\

t'1 ,N

O -N H I Ribose5-P Formylglycinamidine ribosyl-s-phosphate I

ATP\l ) Synthetase ADP+ PiYl + H 2O +

NHz

\ | H)

HtsrH OH OH p-S-Phosphoribosylamine

I Ribose5-P S-Amlnoimidazole ribosyl-S-phosphate

I

aor--l Carborytase I I

I ,, ;;,.:i!:r+ ATp_.rl I Synthetaee A D P+ P i a ' l

o+ tl

+

CH2 f.iHZ

;-; -/-/

E-o-cHz-o.NH D/ \l f\H H)

H \-Jn tt

OH OH Glycinamiderlbosyl S-phosphate

Ribose5-P 5-Aminolmidazolecarborylate rlbosyl s-phosphate Aspartate +Al

rrmyltransferase

ADP+I

-ooc | HCl\_

H f.l

t,, --'\cH ,.1 ::r..

CH,

r-

il

co(

O

NH I Ribose5-p Formylglycinamide ribosylS-phosphate Fag. l?.2

contd.

next column

n

li -51 ir,

.PH

/

IRibose5-P

S-Aminoimidazole 4-succinylcarboxamide ribosyl 5-phosphate Fag. 17.2 contd.

next page

389

Ghapter 17 : METABOLISMOF NUCLEOTIDES 5-Aminoimidazole 4-succinylcarboxamide ribosyl 5-phosphate

ll

Ribose5-P S-Aminoimidazole 4-carboramide ribosyl S-phosphate

1. Ribose 5-phosphate, produced in the hexose monophosphateshunt of carbohydrate metabolism is the starting material for purine nucleotidesynthesis.lt reactswith ATP to form phosphoribosylpyrophosphate(PRPP). 2. Clutamine transfersits amide nitrogen to PRPP to replace pyrophosphateand produce 5-phosphoribosylamine. The enzyme PRPP glutamyl amidotransferase is controlled by feedback inhibition of nucleotides (lMP, AMP and GMP). This reaction is the 'committed step' i n puri ne nucl eoti debi osynthesi s. 3. Phosphoribosylaminereacts with glycine in the presence of ATP to form glycinamide ribosyl 5-phosphate or glycinamide ribotide

(cAR). 4. N10-Formyl tetrahydrofolatedonates the formyl group and the product formed is formylglycinamide ribosyl 5-phosphate.

tl

5. Clutamine transfers the second amido amino group to produce formylglycinamidine ribosyl 5-phosphate. 5-Formaminoimidazole 4-carboxamide ribosyl 5-phosphate

II u n)

o

Cyclohydrolase

+

tl

Ribose5-P lnoaane monophosphate

6. The imidazole ring of the purine is closed in an ATP dependentreactionto yield 5-aminoimidazole ribosyl S-phosphate. 7. Incorporation of COz (carboxylation) occurs to yield aminoimidazole carboxylate ribosyl 5-phosphate. This reaction does not require the vitamin biotin and/or ATP which is the case with most of the carboxvlation reactions. 8. Aspartatecondenseswith the product in reaction 7 to form aminoimidazole 4-succinvl carboxamideribosyl S-phosphate.

Fig. 17.2 : The metabolicpathway for the synthesisof inosine monophosphate,the parent purine nucleotide (PRPP-Phosphori bosyl pytophosph ate; PPi-Pyrophosphate).

9. Adenosuccinatelyase cleaves off fumarate and only the amino group of aspartateis retained to yi el d ami noi mi dazol e4-carboxami deri bosyl 5-phosphate.

The pathway for the synthesis of inosine monophosphafe (lMP or inosinic acid), the 'parent' purine nucleotide is given in Fig.l7.2. The reactionsare brieflv described in the next c olum n.

10. N1O-Formyltetrahydrofolatedonates a one-carbon moiety to produce formaminoimidazole 4-carboxamide ribosyl 5-phosphate. With this reaction, all the carbon and nitrogen atoms of purine ring are contributed by the resoectivesources.

BIOCHEMISTFIY

390 1'l . The final reaction cycloby catalysed hy dr olas e l e a d s to ri n g c los ur ew i th a n e l i mi n a ti o n of water molecule. The productobtainedis inosine monophosphate(lMP), the parent purine nucleotide f r om wh i c h o th e r p u ri n e nucleotidescan be synthesized. lnhibit o rs o f purine synthesis F olic a c i d (T H F ) i s essentialfor the synthesis purine nucleotides of ( r eac t io n s 4 a n d 1 0 ). the Sulfonamides are paraof analogs structural acid am inobe n z o i c (PABA).These sulfa drugs can be used to inhibit the synthesis of folic acid by This microorganisms. the reduces indirectlv p u ri n e s a nd, s y nt hes i so f acids nucleic therefore,the ( DNA R N A). and no have Sulfonamides h u m a ns, inf luenc e o n i s n ot s inc e f o l i c a c i d is and svnthesized s upplie dth ro u g h d i e t.

o tl

Hr.r/-YN\

|il) \rlT,

Ribose5-P (lMP) InosinemonophosPhate Asoartate+ GTP

NAD: GDP + Pi

Adenylsuccinate synthetase

IMP dehydrogenase

r HrO NADH + H*

o

rcoc-cH2-?H-cooNH I

ru---YNr\

|il) \*tT,

Ribose5-P Adenylsuccinate

XanthosinemonophosPhate (XMP) Glutamine

+ ATP+ HrO

I Fumarate4

Adenylsuccinase Glutamate + AMP + PPi

I + NHz

o N.

H2 AdencinemonophcPhab (AMP)

\\ /) N' I

Ribose5-P GuanosinemonoPhosPhate (GMP)

Fig. 17.3 : Synthesisof AMP and GMP from inosine monophosphate.

The structural analogs of folic acid (e.9. methotrexate) are widely used to control cancer. They inhibit the synthesisof purine nucleotides ( r eac t io n4 a n d 1 0 ) a n d , th u s ,n u c l e i ca ci ds.B oth these reactionsare concernedwith the transferof one-carbon moiety (formyl group). These inhibitorsalso affectthe proliferationof normally growing cells. This causes many side-effects inc ludin ga n e mi a ,b a l d n e s ss, c a l y s k i n etc.

(Fig.l7.3). Aspartatecondenseswith IMP in the presence of CTP to produce adenylsuccinate which, on cleavage,forms AMP. For the synthesisof CMP, IMP undergoes NAD+ dependent dehydrogenation to form xanthosine monophosphate(XMP). Glutamine then transfersamide nitrogento XMP to produce C MP .

6-Mercaptopuri nei s an i nhi bi tor of the synthesis of AMP and GMP. lt acts on enzyme adenylsuccinase (of AMP the pathway) and IMP dehydrogenase(of GMP immediate ts the lnosine monophosphate precursorfor the formationof AMP and GMP pathway).

$yrrthesis of AMF and GMP from IMP

Shapt*rr 17 : METABOLTSM OF NUCLEOTTDES Nucleoside monophosphate (AMP,cMP)

ATP\l

I

,l NMPkinase ADPT-i Y

Nucleosidediphosphate (ADP,cDP) I

the metabolic reactions.This is achievedby the transferof phosphategroup from ATp, catalvsed by nucl eosi demonophosphate(N Mp) ki nase s and nucl eosi de di phosphate (N D p) ki nase s (Fig 17.4). Salvage

ATP\l

J ADP*- I

391

NDpkinase

+

Nucleotidetriphosphate (ATE GTP) Fig" 17.4 : Conversionof nucteosidemonophosphates to di- and triphosphates(NMp-Nucleoside monophosphate; NDP-Nucleosjde diphosphate).

E**'B0?wayfor p*rfine*s

The free puri nes (adeni ne, guani ne and hypoxanthine)are formed in the normal turnover of nucl ei c aci ds (parti cul arl yR N A ), and arso obtained from the dietary sources.The purines can directly converted to the corresponding _be nucleotides, and this process is known as 'salvage pathway' (Fig.l 7.fl.

Adenine phosphoribosyltransferasecatalyses the formation of AMp from adenine. H ypoxanthi ne-guani nephosphori bosyl trans_ Forrnatiem of p*erime n*86;Ee#side ferase (HCPRT) converts guanine ano riipFros;phaBes em# trfrea[ao*pfuote* hypoxanthine, respectively,to CMp and lMp. T he nu c l e o s i d emo n o p h o s p h a te (A s Mp ano Phosphoribosyl pyrophosphate (pRpp) is rne CMP) have to be convertedto the corresponding donor of ribose 5-phosphate in the salvaee di- and triphosphatesto participatein most of pathway.

AdeninephosphoribosylIransterase

Adenine 4p1p Ribose5-P

oo H^r/^\yN\ "'l' II tt l-

Hlr\*A^rl Guanine

\ /

Hypoxanthine-guanine * ^, pnosphoribosyltransferase+

ffi

,n,-/---N., t') tf

\

rr*\*Ar) GMP

Ribose5-p

Fig' 17-5 : Salvagepathways of purine nucteotide synthesis(PRPP-phosphoribosyt pyrophosphate; PPi-lnorganic pyrophosphate; AMP-Adenosine monophosphate: GMp-Guanosine monophosphate;

IMP-lnosinemonophosphate;* Detieiencyof HGpRT causesLesch-Nyhan "Vni[oiJ".

BIOCHEMISTF|Y

392

O-o-O-o-H2c

//'o\Base

O-o-O-o-t,? Ribonucreotidereducrase

ry-juffi OH OH

Ribonucleoside Thioredoxin diphosphate (2SH, reduced)

Thioredoxin (S-S-, oxidized)

(ADP,GDP, CDEUDP)

r,'o-

Base

fi;). OHH Ribonucleoside diphosphate (ADP,GDP,

cDe UDP)

NADP*

NADPH+ H*

Fig. 17.6 : Formation of deoxyribonucleotides frcm ribonucleotides.

The salvagepathway is particularlyimportant moiety (Fig.t7.6).This reactionis catalysedby a in certain tissuessuch as erythrocytesand brain mul ti subuni t (tw o B 1 and tw o 82 subuni t s) where de novo (a new) synthesis of purine enzvme, rihonucleotide reductase. nucleotidesis not operative. Supply of reducing equivalents : The enzyme A defect in the enzyme HGPRTcausesLesch' ribonucleotide reductase itself provides the hydrogen atoms needed for reduction from its Nyhan syndrome (details given later). sul fhydrylB roups.The reduci ngequi val ents,i n Regula ti o n o f p u ri n e turn, are supplied by thioredoxin, a monomeric biosynthesis protein with two cysteineresidues. nucleotide T he o u ri n e n u c l e o ti d e s y n th e s i s i s w el l coordinatedto meet the cellular demands.The intracellular concentration of PRPP regulates purine synthesisto a large extent. This, in turn, is dependent on the availability of ribose S-phosphateand the enzyme PRPPsynthetase.

NADPH-dependent thioredoxin reductase converts the oxidized thioredoxin to reduced form which can be recycled again and again' Thioredoxin thus serves as a protein cofactor in an enzymatic reaction.

Regulationof deoxyribonucleotide synthesis: is controlled Deoxyribonucleotides PRPPglutamyl amidotransferase are mostlyrequiredfor the bv a feedback mechanismby purine nucleotides. synthesisof DNA. The activity of the enzyme That is, if AMP and CMP are available in ribonucleotidereductasemaintainsthe adequate adequate amounts to meet the cellular supply of deoxyribonucleotides. requirements,their synthesisis turned off at the Ribonucleotide reductase is a complex amidotransferase reaction. enzyme with multiple sites (active site and Another importantstageof regulationis in the allosteric sites) that control the formation of conversion of IMP to AMP and CMP. AMP deoxvribonucleotides. inhibits adenylsuccinatesynthetasewhile CMP inhibits IMP dehydrogenase.Thus, AMP and CMP control their respectivesynthesisfrom IMP by a feedback mechanism. Conv e rs i o n o f ri b o n u c l e o ti d e s to deoxyribonucleotides

The end product of purine metabolism in T he s y n th e s i s o f p u ri n e a n d pyri mi di ne humans is uric acid. fhe sequence of reactions ribo- in purine nucleotide degradation is given in deoxyribonucleotides occurs from nucleotidesby a reduction at the Cr of ribose Fig.17.7.

ffihapter "T7 ; METABOLTSM OF NUCLEOTTDES

AMp

,I H2u-..1 .. Nucleotidase ^.t .{ J Y |

393

AMp deaminase

H,o

Hzor

ilt.

Pir

+

o

Ribose Adenosine

lno Pi_. Ribose a_ l-phosphate n

--,\ Hry

tl

Lr

\-N/

Hypoxr H 2O + O2:,

H2o2y'

n

I o

u ,,, H

Xanthine

Xanthine oxidase

o

Uric acid

Fig. 17.7: Degradation of purine nucteotides to uricacid(AMp_Aa"nii*iffiipnrr", lMp-lnosine monophosphate; GMp_Guanosnemonophasphate).

Guanine

BIOCHEMISTFIY

394 Uric acid

I un""r"

J

Allantoin I I Allantoinase

+

Allantoicacid I Glvoxvlate+_,,1Allantoicase

+

Urea I I Urease

Most animals (other than primates)however, oxidize uric acid by the enzyme uricase to al l antoi n, w here the puri ne ri ng i s cl eaved. Allantoin is then convertedto allantoic acid and excreted in some fishes (Fig.|7.8). Further degradation of allantoic acid may occur to produce urea (i n amphi bi ans,most fi shes and some mol l uscs) and, l ater, to ammoni a (i n marine invertebrates).

J

Ammonia Fig. 17.8 : Degradation of utic acid in animals other than man.

Hyperuricemia

and gout

1. The nucleotide monophosphates(AMP, U ri c aci d i s the end product of puri ne IMP and CMP) are convertedto their respective metabolism in normal humans. The nuc leos i d e fo rm s (a d e n o s i n e , i n o si ne and concentrationof uric acid in the serum of adults guanosine) by the action of nucleotidase. is in the range of 3-7 m{dl. In women, it is sl i ghtl yl ow er (by about 1 mg) than i n men. The 2. fhe amino group, either from AMP or daily excretionof uric acid is about 500-700 mg. adenosine,can be removed to produce IMP or Hyperuricemia refers to an elevation in the inosine, respectively. serum uric acid concentration.This is sometimes 3. Inosine and guanosine are/ respectively, associatedwith increased uric acid excretion convertedto hypoxanthineand guanine (purine (uri cosuri a). bases) by purine nucleoside phosphorylase. Gout is a metabolic disease associatedwith Adenosine is not degraded by this enzyme, of uric acid. At the physiological overproduction hence it has to be convertedto inosine. pH , uri c aci d i s found i n a more sol ubl eform as 4. Cuanine undergoes deamination by sodium urate. ln severe hyperuricemia,crystals guanase to form xanthine. of sodium urate get depositedin the soft tissues, parti cul arl y i n the j oi nts. S uch deposi ts are 5. Xanthine oxidase is an important enzyme known as tophi. This causes commonly that converts hypoxanthine to xanthine, and i n the j oi nts resul ti ngi n a pai nful i nfl ammati on x ant hine to u ri c a c i d . T h i s e n z v me contarns gouty arthritis. Sodium urate and/or uric acid F A D, m o l y b d e n u ma n d i ro n , a n d i s e xcl usi vel y precipitatein kidneysand uretersthat f ound in l i v e r a n d s ma l l i n te s ti n e . Xanthrne may also in renal damage and stone formation. results oxidase liberatesH2O2 which is harmful to the tissues.CatalasecleavesH2O2 to H2O and 02. Historically, gout was found to be often associated with high living, over-eating and Uric acid (2,6,8-trioxypurine)is the final alcohol consumption In the previous centuries, excretory product of purine metabolism in alcohol was contaminatedwith lead during its humans. Uric acid can serve as an important manufacture and storage. Lead poisoning leads antioxidant by getting itself converted (nonto kidney damage and decreased uric acid enzymatically)to allantoin.lt is believedthat the excretion causing gout. antioxidant role of ascorbic acid in primates is replacedby uric acid, since these animals have fhe prevalence of gout is about 3 per 1,000 persons,mostlyaffectingmales.Post-menopausal lost the ability to synthesizeascorbic acid.

*harpten'l 7 ; METABOLISM OF NUCLEOTIDES women, however,are as susceptibleas men for this disease.Cout is of two types-primary and secondary.

395

GlYcogen.

Grucose

1. P r ima ry g o u t : l t i s a n i n b o rn error of metabolism due to overproduction of uric acid. This is mostly relatedto increasedsynthesisof purine nucleotides.The following are the imoortant metabolic defects (enzymes)associatedwith primarygout (Fig.t 7.e) . PRPP synthetase : ln normal circumstances,PRPP synthetase is under feedback control by purine nuc leot i d e s (A D P a n d C D P ). However, variant forms of PRPP synthetase-which are not subjected to feedback regulation-have been detected.This leadsto the increased productionof purines.

Ribose5-phosphate

II I PRPPsvnthetaset

I

+

PRPP

PRPP qlutamvlamidotrinsferdseI

PRPP glutamylamidotransferase : The lack of feedbackcontrol of this enz y m e b y p u ri n e n u c l e o ti d e sa l s o leads to their elevatedsynthesis. HGPRT deficiency : This is an enzyme of purine salvagepathway, and its defect causes Lesch-Nyhan syndrome.This disorderis associated with increased synthesis of pur ine n u c l e o ti d e s b y a tw o -fo l d mechanism. Firstly, decreased ut iliz at i o no f p u ri n e s (h y p o x a n th i n e and guanine) by salvage pathway, r es ult in g i n th e a c c u mu l a ti o na n d div er s io n o f P R P P fo r p u ri n e nucleotides.Secondlv,the defect in salvagepathway leads to decreased lev els o f IMP a n d C M P c a u s i n g im pair m e n ti n th e ti g h tl y c o n tro l l e d feedback regulation of their production. Glucose 5-phosphatasedeficiency : ln type I glycogen storage disease (von Cierke's),glucose 6-phosphate cannot be converted to glucose due t o th e d e fi c i e n c y o f g l u c o s e 6-phosphatase.This leads to the

Glutamine

5-Phosphoribosylamine

Ii ITPPFJ Hypoxanthine r lno.in" ro*ophosphate unom

Guanine

"-""'

|

"rl

\'

) GMP

AMp<-

Adenine

uypoxlntrine

iI 7 |

Xanthineoxidase

I

+

lanthine

\

II

oxidase | Xanthine

I

+

Uric acid Fig. 17.9 : Summary of possible enzyme alterationscausing gout ( I -t ncreased enzyme activily; ; -Decreased enzyme activity; GSH-Reduced glutathione; G-S-S-G-Oxidized glutathione; PRPP-Phosphoribosyl pyrophosphate; HGPRT-Hypoxanthineguanine phosphoribosyltran sferase).

396

B IOC H E MIS TFIY

increasedutilization of glucose 6-phosphate by hexosemonophosphateshunt (HMP shunt), resulting in elevated levels of ribose 5- phos p h a te a n d PR PPa n d , u l ti ma tel y,puri ne overproduction.von Gierke's diseaseis also increased activitv of associated with glycolysis.Due to this, lactic acid accumulates in the body which interfereswith the uric acid excretionthrough renal tubules. . Elevation of glutathione reductase : Increased glutathionereductasegeneratesmore NADP+ whic h i s u ti l i z e d b y H MP s h u n t.T h i s causes increased ribose S-phosphate and PRPP synthesis.

N N H

Allopurinol

H Alloxanthine

Fig. 17.10: Structuresof hypoxanthine and itsstructuralanaloos.

Among the five enzymesdescribed,the first three are directly involved in purine synthesis. oxidase.This type of inhibition is referredto as T he r ema i n i n g tw o i n d i re c tl y re g u l a te puri ne suicide inhibition (For more details, Refer production.This is a good exampleto show how Chapter 6). an abnormality in one metabolic pathway Inhi bi ti onof xanthi neoxi daseby al l opuri nol influences the other. leads to the accumulationof hypoxanthineand 2. Secondarygout : Secondaryhyperuricemia xanthine. These two compounds are more is due to various diseasescausing increased soluble than uric acid, hence easily excreted. synthesisor decreasedexcretion of uric acid. Besidesthe drug therapy,restrictionin dietary Increaseddegradationof nucleic acids (hence intake of purines and alcohol is advised. more uric acid formation)is observedin various Consumption of plenty of water will also be cancers(leukemias,polycythemia, lymphomas, useful. etc.) psoriasisand increased tissue breakdown (trauma,starvationetc.). The anti-inflammatory drug colchicine is used for the treatmentof gouty arthritis.Other antiThe disordersassociatedwith impairment in inflammatory drugs-such as phenylbutazone, renal function cause accumulationof uric acid i ndomethaci n, oxyphenbut azoneI corticosteroidswhich may lead to gout. are also useful. Uric acid

pool in gout

FseudoEout By administrationof uric acid isotope (N1s), of pseudogoutare The clinical manifestations the miscible uric acid pool can be calculated.lt gout. But this disorderis causedby the is around 1,200 mg in normal subjects.Uric acid similarto pool is tremendouslyincreasedto 3,000 mg. or deposition of calcium pyrophosphate crystals in joints. Further/serum uric acid concentrationis even more/ in patientssufferingfrom gout. normal in pseudogout. Treatment of gout Lesch-Nyhan syndrome The drug of choice for the treatment of primary gout is allopurinol. This is a structural Thi s di sorder i s due to the defi ci ency of analog of hypoxanthine that competitively h ypo xa nthi n e-guan i ne ph ospho r ibosy ltran sferase inhibits the enzyme xanthine oxidase. Further, (HCPRT),an enzyme of purine salvagepathway allopur in o l i s o x i d i z e d to a l l o x a n thi ne by (SeeFig.l7.fl. lt was first describedin 1964 by xanthine oxidase (Fig.l7.l0). Alloxanthine, in Mi chael Lesch(a medi cal student)and W i l l i am turn, is a more effective inhibitor of xanthine L. Nyhan (his teacher).

1j;fi;iirjje{c..1 ,; : METABOLISM

397

OF NUCLEOTIDES

Lesch-Nyhan syndrome is a sexJinked metabolic disorder since the structural gene for HCP RT i s l o c a te d o n th e X-c h r omosome. It affectsonly the males and is characterizedby excessive uric acid production (often Bouty arthritis), and neurological abnormalifies such as mental retardation,aggressivebehavior,learning dis abili tye tc . T h e p a ti e n tso f th i s d i s orderhave an ir r es i s ti b l eu rg e to b i te th e i r fi n g e r sand Ii ps, of t en c a u s i n gs e l f-m u ti l a ti o n .

hel ps to decreaseuri c aci d producti on,has no affecton the neurologicalmanifestations in these oatrents.

Tw o di fferent i mmunodefi ci encydi sorde r s associ ated w i th the degradati on of puri n e nucleosidesare identified.The enzyme defects are adenosine deaminase and purine nucleoside phosphorylase, involved in uric acid synthesis T he o v e ro ro d u c ti o no f u ri c a c i d i n Lescn(SeeFig.l7.7). Ny han s y n d ro me i s e x p l a i n e d . H C P R T def ic ien c yre s u l tsi n th e a c c u m u l a ti o nof P R P P The defi ci encyof adenosi nedeami nase(A D A) and de c re a s e i n C M P a n d l M P, ul ti matel y causes severe combined immunodeficiency leadingto i n c re a s e ds y n th e s i sa n d d e gradati on (S C ID ) i nvol vi ng T-cel l and usual l y B -cell of pur in e s (m o re d e ta i l s g i v e n u n d er pri mary dysfuncti on.l t i s expl ai nedthat A D A defi ci ency gout ) . resul tsi n the accumul ati onof dA TP w hi ch i s an i nhi bi tor of ri bonucl eoti de reductase and , T he b i o c h e m i c a l b a s i s fo r th e n e urol ogi cal therefore,D N A synthesi sand cel l repl i cati on. symptomsobservedin Lesch-Nyhansyndromeis not c lea rl y u n d e rs to o dT . h i s m a y b e rel atedto The defi ci encyof puri ne nucl eoti dephospho t he dep e n d e n c eo f b ra i n o n th e s a l v a gepathw ay ryl ase i s associ atedw i th i mpai rmentof T-cell lor de n o v o s y n th e s i o s f p u ri n en u c l e oti des.U ri c function but has no effect on B-cell function. ac id is n o t to x i c to th e b ra i n ,s i n c ep a ti entsw i th U ri c aci d synthesi si s decreasedand the ti ssue s ev er e h y p e ru ri c e m i a(n o t re l a te d to H C P R T l evel sof puri ne nucl eosi des and nucl eoti desare def ic ien c y ) d o n o t e x h i b i t a n y n e urol ogi cal hi gher. l t i s bel i eved that dGTP i nhi bi ts the s y m pt o ms . F u rth e r, a l l o p u ri n o l tre a tment that devel opmentof normal T-cel l s.

8|ail'tE3|cAl,/ SUfiltCAL COilECEp?S

Folic acid is essentiol t'or the synthesis oJ purine nucleotides. Folic acid analogs (methotrexate)are employed to control concer. The saluage pathwag, inuoluing the direct conuersion of purines to corresponding nucleotides,is important in fissues-brain and erythrocytes. Gout is the disorder associatedwith the ouerproduction of uric cicid, the end product of purine metobolism.Allopurinol is the drug ol choicet'or the treatmentof gout. Lesch-Nghansyndrome is causedby a delect in the enzyme hypoxanthine-guaninephosphoribosyltransferase. The patients haueon irresistibleurge to bite their lingers and lips. A defect in the enzyme adenosine deaminose (ADA) results in seuere combined immunodeficiency(SCID) inuoluing both T-cell and B-cell dgsfunction. A girl suflering from SCID wos cured by transferring ADA gene (in 1990) and that was the first attempt for gene therapy in modern medicine. Orotic aciduria, a metabolic delect in pyrimidine biosynthesis,is chorocterized by anaemia and retorded growth, besidesthe excretion of orotic acid in urine.

BIOCHEMISTF|Y

398

details). Prokaryoteshave only one carbamoyl phosphate synthetasewhich is responsiblefor s argi ni neand pyri mi di ne s. the bi osynthesiof Aspartate

Fig. 17.11: Sources of individualatoms in pyrimidine ing (Note: Same colours are used in the syntheticpathway, Fig. 17.12).

Carbamoyl phosphate condenses with aspartate to form carbamoyl aspartate. This aspartate by is catalysed reaction Dihydroorotasecatalysesthe transcarbamoylase. pyri mi di neri ng cl osurew i th a l ossof H 2O.

The three enzymes-CPS ll, aspartatetranscarbamoylase and dihydroorotase are the domains (functionalunits) of the same protein. Hypouricemia This is a good example ol a multifunctional Decreased uric acid levels in the serum enzyme. ( < 2 m g /d l ) re p re s e n t h y p o u ri c emi a. Thi s i s The next step in pyrimidine synthesisis an mostly associatedwith a rare genetic defect in the enzyme xanthine oxidase. lt leads to the NAD+ dependent dehydrogenation,leading to xanthine and the formation of orotate. increased excretion of hypoxanthine.Xanthinuriafrequentlycausesthe Ribose 5-phosphateis now added to orotate formationof xanthinestonesin the urinarvtract. to produce orotidine monophosphate(OMP). This reaction is catalysedby orotate phosphoan enzyme comparablewith ribosyltransferase, H C P R T i n i ts functi on. OMP under goes decarboxylation to uridine mono-phosphate (U MP ). T h e s y n th e s i so f p y ri mi d i n e s i s a much simpler process compared to that of purines. Aspartate, glutamine (amide group) and CO2 contribute to atoms in the formation of py r im i d i n e ri n g (F i 9 .1 7 .1 1 ).Py ri mi di ne ri ng i s first synthesizedand then attached to ribose 5- ph o s p h a te .T h i s i s i n c o n tra st to puri ne nuc le o ti d es y n th e s i sw h e re i n p u ri n e ri ng i s bui l t upon a pre-existing ribose 5-phosphate.The pathway of pyrimidine synthesisis depicted in Fig.|7.12, and the salientfeaturesare described below .

and OMP Orotate phosphoribosyltransferase protein.A a single of decarboxylaseare domains orotic causes defect in this bifunctional enzyme aciduria (details given later). By an ATP-dependentkinase reaction, UMP is convertedto UDP which servesas a precursor for the synthesisof dUMP, dTMP, UTP and CTP. Ribonucleotide reductase converts UDP to dUDP by a thioredoxin-dependentreaction. Thymidylatesynthetasecatalysesthe transferof a methyl Broup from N5, N1O-methylene tetrahydrofolate to produce deoxythymidine monophosphate(dTMP).

Clutaminetransfersits amido nitrogento CO2 to produce carbamoyl phosphate.This reaction UDP undergoes an ATP-dependentkinase is ATP-dependentand is catalysedby cytosomal enzyme carbamoyl phosphate synthetase ll reactionto produce UTP. Cytidine triphosphate (CTP) is synthesizedfrom UTP by amination. (cPS il). CTP synthetaseis the enzyme and glutamine CPS ll is activated bv ATP and PRPP and providesthe nitrogen. inhibited by UTP. Carbamoyl phosphate synthetase| (CPS l) is a mitochondrial enzyme synthesis Regulatiom of pyrimidine which synthesizescarbamoyl phosphate from ln bacteria, aspartate transcarbamoylase ammonia and CO2 and, in turn urea (Refer (ATCase) catalyses a committed step in more protein metabolism, Chapter 15, for

Ghaprer'87 : METABOLISM OF NUCLEOTTDES CO2 + Glutamine hate

2ATP + H2O

399 PRPp\l Orotate ) phosphoribosylPPil lransrerase

2ADP+ Pi

/'

NHc tO=C I

llt

o = " -, , , , -t '

O:@

Carbamoyl phosphate

I

Aspartate pi< transcarbamoylase

+

'_

:.J,.:..,:T

Ribose5-P Orotidinemonophosphate(OMP)

I

CO2+-1OMPdecarboxylase

-,

+

Ijil' a l

' :'

H-rN 1, O:C

'f ,'i

llN-

.i.. .. - i,.: .' r._..,: . i_,r

Carbamoyl aspartatre

HNI I

O:C \ -

IDihydroorotase

H,O+4 -J

:"r I

HN"'

o :C\

I

,..ii

Dihydroorotate NrAn-

'r^v\ NADH +

Dihydroorotate dehYdrogenase

"**-r'{+

uN/ n- ^

|

.,

li' Orotate Fis' 17,12 contd. n6xt column

Fig. 17.12 : Metabolicpathway for the synthesisof pyrimidine nucteotides.

py r im idi n e b i o s y n th e s i s .A T C a s e i s a good example of an enzyme controlled by feedhack mechanism by the end product CT P . I n c e rta i n b a c te ri a , U T P a l s o i nhi bi ts ATCase. ATP, however, stimulates ATCase activity.

Carbamoyl phosphate synthetasett (CPS il) is the regulatoryenzymeof pyrimidine synthesisin animals. lt is activated by PRPPand ATp and i nhi bi tedby U D P and U TP .OMp decarboxyl ase , i nhi bi ted by U MP and C MP , al so control s pyri mi di neformati on.

400

BIOCHEMISTFIY

phosphoribosyl and orotate transferase OMP decarhoxylase of pyrimidine synthesis (Fig.l7.l2). Both these enzyme activities are T he p y ri mi d i n e n u c l e o ti d e su n d e rgo si mi l ar present on a single protein as domains reactions(dephosphorylation,deamination and (bifunctional enzyme). cleavageof glycosidic bond) like that of purine nucleotidesto liberatethe nitrogenousbasesFeeding diet rich in uridine and/or cytidine is cytosine,uracil and thymine. The basesare then an effective treatment for orotic aciduria. These degradedto highly soluble products-p-alanine compounds provide (through phosphorylation) and B-aminoisobutyrate. These are the amino pyri mi di ne nucl eoti desrequi red for D N A and acids which undergo transaminationand other R N A synthesi s. B esi des thi s, U TP i nhi bi ts reactions to finally produce acetyl CoA and carbamoyl phosphatesynthetaseIl and blocks s uc c inylC o A . svnthesisof orotic acid. Oegradation of pyrirnieline nucleotides

Reye's syndrome : This is considered as a secondaryorotic aciduria. lt is believed that a T he p y ri mi d i n e s(l i k e p u ri n e s )c a n a l so serve (of urea defect in ornithine transcarbamoylase as precursors in the salvage pathway to be cycle) causes the accumulation of carbamoyl converted to the respective nucleotides. This phosphate.This is then divertedfor the increased pyrimidine reaction is catalysed by synthesisand excretion of orotic acid. phosphoribosyltransferase which utilizes.PRPP as the source of ribose 5-phosphate. Biosynthesls of nucleotide coenzymes Disorders of pyrimidine metabolism S elv age

p a th w a y

Orotic aciduria : This is a rare metabolic The nucleotidecoenzymesFMN, FAD, NAD+ disordercharacterizedby the excretionof orotic NADP+and coenzymeA are synthesized from the acid in urine, severe anemia and retarded B-complexvitamins.Their formationis described growth. lt is due to the deficiencyof the enzymes under the sectionon vitamins(Chapter71.

METABOLISM OF NUCLEOTIDES

401

1. Nucleotides participate in a wide uarietg of reactions in the liuing cells--synthesisol DNA and RNA; as constituents ot' mang coenzymes;in the regulation of metabolic reactions etc.

2. Purine nucleotides are synthesized in a series of reactions stqrting from ribose

S-phosphate.Glycine,glutamine, aspartate,t'ormateand COt contribute to the svnthesis of purine ring.

3. Purine nucleotidescan also be synthesizedt'rom lree purines by a saluagepathway. The deJect in the enzyme HGPRT couses Lesch-Nghansyndrome. Deoxyribonucleotidesare formed t'rom ribonucleotidesby a reductian processcatalysed by ribonucleotidereductase.Thioredoxinls the protein cot'actorrequiredt'or this reaction.

( Purine nucleotidesare degrodedto uric acid, the excretoryproduct in humans.lJric acid seruesos a naturql antioxidont in the liuing system.

6. Uric acid in many animal species(other than primates)is conuertedto more soluble forms such as allantoin, allantoic acid etc., and excreted. Gouf is a metabolic diseoseassociatedwith ouerproductionot' uric acid. This oJten leads to the accumulation of sodium urate crystals in the joints, causing painfitl gouty arthritis. Allopurinol, an inhibitor ol xanthine oxidase, is the drug used t'or the treatment of gout. 8. Pyrimidine nucleotides are synthesizedt'rom the precursors aspartate, glutamine and CO2, besidesribose S-phosphate.

9. Orotic aciduria is a defect in pyrimidine sgnfhesiscaused by the deficiency oJ orotate phosphoribosyltransJerase and OMP decarboxylase.Diet rich in uridine and/or cytidine is an effectiue treatment for orotic aciduria. 70. Pyrimidines are degradedto amino acids, nomely ftalanine and ftaminoisobutyrote which are then metabolized.

402

B IOC H E MIS TR Y

I. Essayquestions 1. Describethe catabolismof purine nucleotidesand the associated metabolicdisorders. 2. Write an accountof the biosynthesis of inosinemonophosphate 3. Discussthe synthesisand degradationof pyrimidines. 4 . D e s c ri b eth e ro l e o f P R P Pi n puri neand pyri mi di nesynthesi s. Add a note on Lesch5. Write an accountof salvagepathway in purine nucleotidesynthesis. Nyhan syndrome. II. Short notes (e) lmmuno(a) Cout, (b) PRPB(c) Synthesis (d) Functionsof nucleotides, of deoxyribonucleotides, (f) (g) phosphate synthetase deficiencydiseases in purinemetabolism, Orotic aciduria, Carbamoyl ll, (h) HCPRI (l) Degradationof uric acid in differentanimals,(j) Regulationof purine synthesis

(k) Inhibitors of purinesynthesis. III. Fill in the blanks are 1. Theaminoacidsrequired for the synthesis of purinesandpyrimidines 2 . T h e e n z y m ex a n th i n eo x i d asei s i nhi bi tedby 3. Tophiare mostlymade up of 4. Hypouricemiais due to the deficiencyof the enzyme 5. The disorderin which the patientshave an irresistibleurge to bite their fingersand lips is 6. The cofactorrequiredby the enzymeribonucleotidereductaseis 7. The 'parent'nucleotidesynthesized in the biosynthesis of purinesis B. Xa n th i n eo x i d a s ec o n v e rtsal l oouri nolto 9. The amino acid that contributesto the synthesisof more than half of the pyrimidinering

10. Theregulatory in animalsis enzymein the pyrimidine biosynthesis IV. Multiple choice questions 11. Namethe enzymeassociated with hyperuricemia (a) PRPPsynthetase (b) HCPRT(c) Clucose6-phosphatase (d) All of them. disease 12. An enzymeof purine metabolismassociated with immunodeficiency (a) Adenosinedeaminase(b) Xanthineoxidase(c) PRPPsynthetase (d) HCPRT. 1 3 . O ro ti c a c i d u ri ac a n b e tre a tedby a di et ri ch i n (a )A d e n i n e(b ) C u a n i n e(c ) U ri di ne(d) A ny one of them. 1 4 . T h e e n d p ro d u c to f p u ri n emetabol i smi n humansi s (a ) X a n th i n e(b ) U ri c a c i d (c) U rea (d) A l l antoi n. 15. The nitrogenatomsin the purine ring are obtainedfrom (a) Clycine (b) Clutamine(c) Aspartate(d) All of them.

Tlrc ninetal,

caJ,oiam speohr !

,

"I am the most abundant mineral; Calcify and stengthen bones,teeth,..... Coagulate binod end contract musclz; Regalated by calcitriol, PTH and calcitonin"'z

mineral (inorganic)elements constitute * i l i sa!l i * ;rtr* i : Th" I only a small proportion of the body weight. The mi neral s are cl assi fi ed as pri nci pal There is a wide variation in their bodv content. elementsand trace elements. For instance,calcium constitutesabout 2oh of body weig h t w h i l e c o b a l t a b o u t 0 .0 0 0 0 4% . The seven principal elements (macrominerals) constitute 60-80% of the body,s inorganic material. These are calcium, G ener aE fu n e ti c n s phosphorus, magnesium, sodium, potassium, Mineralsperformseveralvital functionswhich chloride and sulfur. are absolutely essentialfor the very existenceof The pri nci pal el ements are requi red i n the organism. These include calcification of greaterthan 100 mg/day. amounts bone, b l o o d c o a g u l a ti o n , n e u ro m uscul ar ir r it abilit y ,a c i d -b a s ee q u i l i b ri u m,fl u i d bal ance The (microminerals) are required in amounts and osmotic regulation. lessthan 100 mg/day. They are subdividedinto three categories Certain minerals are integral componentsof biologic a l l y i m p o rta n t c o m p o u n d s such as 1. Essentialtrace elements : lron, copper, hem oglob i n(F e ),th y ro x i n e(l ), i n s u l i n ( Zn) and iodine, manganese/zinc, molybdenum, cobalt, v it am in 81 2 (C o ). S u l fu r i s p re s e n ti n th i ami ne, fl uori ne,sel eni umand chromi um. biot in, lip o i c a c i d a n d c o e n z y me A . S everal 2. Possiblyessentialtrace elements: Nickel, mineralsparticipateas cofactorsfor enzymes in vanadi um,cadmi um and bari um. m et abolism(e .9 . Mg , M n , C u , Z n , K). S ome elements are essential constituentsof certain 3. Non-essentialtrace elements: Aluminium. enzymes(e.9. Co, Mo, Se). lead, mercury, boron, silver, bismuth etc.

403

404

Element Calcium

B IOC H E MIS TR Y

Major functions

Deficiency Recommended disease/symptoms dietary allowance

Constituent of bones and Rickets: osteomalacia. teeth:muscle contraction.osleooorosis nervetransmission

Phosphorus Constituent of bonesand

Rickets,

Major sources

0.8-1.0g/d

Milkandmilkproducts, leafy vegetables, beans

0.8-1.0g/d

Milk,cereals,leafyvegetables

teeth;in the lormation ot osteomalacia highenergyphosphates, nucleic acids,nucleotide

coenzymes. Magnesium

Constituent of bonesand Neuromuscular weakness, 300-350mg/d for enzymes irritation teeth;cofactor

vegetables, fruits, Cereals, milk

e.g.kinases.

Sodium

Chiefcationof extracellularAlmost unknown on normal fluids:acid-base balance. diet osmoticpressure; nerve function andmuscle

Potassium

Chiefcationof intracellular Muscular mental }.4 S/d weakness, fluids:acid-base balance: confusion osmoticpressure; muscle function

Chlorine

Regulation of acid-base formation balance; ol HCI

Sulfut

Constituent of sulfur Almostunknown containing amino acids, (thiamine, vitamins certain biotin) andothercompounds (heparin, chondroitin sulfate).

Almostunknown on normal 5-10 g/d diet

A summary of the major characteristicsof pr inc ipal e l e me n ts a n d tra c e e l e ments i s respectivelygiven in Tables l8.l and 18.2. The individual elementsare describednext.

Calcium is the rnost abundant among the minerals in the body. The total content of c alc ium in a n a d u l t m a n i s a b o u t 1 to 1 . 5 kg. A s much as 99o/oof it is presentin the bones and teeth.A smallfraction('l"h)ol the calcium,found outside the skeletal tissue, performs a wide variety of functions. B ioc hem i e a l

5-10 g/d

f u rn c ti o n s

1. Development of bones and teeth : Calc ium , a l o n g w i th p h o s p h a te ,i s re qui redfor

Tablesalt,saltaddedfoods

Fruits, nuts,vegetables

Tablesalt

aminoacids Sulturcontaining

the formation (of hydroxyapafite) and physical strengthof skeletaltissue.Bone is regardedas a mineralizedconnectivetissue.Boneswhich are in a dynamic state serve as reservoir of Ca. Osteoblastsare responsiblefor bone formation resul ti n demi neral i zati on. w hi l e osteocl asts 2. Muscle contraction z Ca2+ interacts with troponin C to trigger muscle contraction. Calcium also activates ATPase, increasesthe interactionbetween actin and myosin. 3. Blood coagulation : Several reactions in the cascade of blood clotting process are dependenton Ca2+(factorlV). 4. Nerve transmission: Ca2* is necessaryfor of nerve i mpul se. the transmi ssi on 5. Membrane integrity and permeability : Ca2* influences the membrane structure and transportof water and severalions acrossit.

Ghapter 18 : MINEHALMETABOLTSM

Element

Copper

Major functions

Deficiency disease,/symptoms

Recommended dietary allowance

Major sources

Constituent of heme Hypochromic, microcytic e.g.hemoglobin, myoglobin, anemia cytochromes; involvedin 0, transport andbiological oxidalion.

10-15mg/d

Organ meats(liver, heart), leafyvegetables, ironcookware

Constituent of enzymes Anemia,Menke'sdisease e.g.cytochrome C oxidase, catalase, tyrosinase; in iron transporl.

2-3 mg/d

Organ mealscereals, leafy vegetables

Constituent of thyroxine and triiodothvronine Manganese Cofactor forenzymes pyruvate e.g.arginase, glycoprotein carborylase; svnthesis.

t#

405

c;i;il;;;;;;;;

goiter,myxedema 150-200pg/d Cretinism,

lodizedsalt,seafoods

Almostunknown

2-9 mg/d

Cereals,leafyvegetables

poor Growth retardation,

10-15mg/d

Meat,lish,milk

e.g.alcoholdehydrogenase, wound healing, hypogonadism carbonicanhydrase, lactatedehydrogenase.

MolybdenumConstituent ol enzymes Almostunknown 75-250 ltgld e.g.xanthine oxidase Constituent (asin ot vitamin B1r, Pernicious anemia 5-8 pg/d required lortheformation vitamin B,, deficiency) of efihrocytes Fluorine Helpsin theproper formation Dental caries, osteoporosis 24 ngld ol bones andteeth Selenium Involvedin antioxidant Muscular degeneration, 50-200pg/d functionalongwithvitaminE; cardiomyopathy

Vegetables Foodsof animalorigin

Drinking water Organ meats, seafoods

constituent of glutathione

Chromium

peroxidase andselenocysteine Promotes insulin function glucose lmpaired tolerance 1G-100 pg/d (asglucose tolerance factor)

6. Activation of enzymes : Ca2+ is needed for the direct activation of enzymes such as lipase (pancreatic), ATPase and succinate dehydrogenase.

yeast, Brewer's meat,whole grarns

mediationof Ca2+(insteadof cAMP).Calcium is regarded as a second messenger lor such hormonal acti on e.g. epi nephri ne i n l i ver glycogenolysis. Calcium serves as a third messengerfor some hormones e.g. antidiuretic hormone (ADH) acts through cAMP, and then ca2+ .

7. Calmodulin mediated action of Ca2+ : Calm odulin (mo l . w t. 1 7 ,0 0 0 ) i s a c a l ci um binding regulatory protein. Ca-calmodulin complex activates certain enzymes e.g. 9. Release of hormones : The release of adenylate cyclase, Ca2+ dependent protein certain hormones(insulin,PTH, calcitonin)from kinases. the endocrine glands is facilitatedby Ca2+. 8. Calcium as intracellular messenger : Certain hormonesexert their action through the

10. Secretory processes : Ca2+ regulates microfilament and microtubule mediated

406

BIOGHEMISTFIY lonizedCa (biologicallyactive)

processessuch as endocytosis,exocytosisand c ell m ot ilit y . 11. Conta c t i n h i b i ti o n : C a l c i u m i s b e l i eved to be involved in cell to cell contact and adhes ionof c e l l s i n a ti s s u e(R e fe rp . 6 9 2 al so). T he c ell t o c e l l c o m m u n i c a ti o nm a v a l s o r equi re c a2*.

Ca comolexedwith citrate,phosphate, bicarbonate

12. Action on heart : Ca2* acts on myocardium and prolongs systole. Protein-bound non-diffusible Ca

#i*r't*r:;g ;requinennents Adult men and women W om en d u ri n g pregnancy/lactation and post-menopause Childr en (1 -1 8 y rs .) Infants(<1 year) *il#ir,I;r,irriill;

B es ts ou rc e s Good sources -

Afo,*r.:,rFr,; *,.*l,*

800 mg/day

Fig. 18.1 : Different forms of circulating calciurn.

Factors

1.5 g/day

* * m-* ,* * n# #a absorpti on

1 . Phytatesand oxalatesform insolublesalts

o.8-1.2 g/day and interferewith Ca absorption. 2. High content of dietary phosphateresults i n the formati onof i nsol ubl ecal ci um phosphate and preventsCa uptake.The dietary ratio of Ca and P-between 1 : 2 and 2 : 1-is ideal for M i l k a n d m i l k o roducts opti mum C a absorpti onby i ntesti nalcel l s. Beans,leafyvegetables, 3. The free fatty acids react with Ca to form fish, cabbage,egg yolk. i nsol ubl e cal ci um soaps. Thi s i s parti cul arl y observedwhen the fat absorptionis impaired.

- 300-500 mg/day

The absorptionof calcium mostly occurs in the duodenum by an energy dependent active process.lt is influencedby severalfactors. Fa*tur:** C*rsffiTstirESGa absorption 1 . Vitamin D (through its active form c alc it r iol) i n d u c e s th e s y n th e s i s o f c a l ci um binding pr o te i n i n th e i n te s ti n a e l p i th e l i alcel l s and promotesCa absorption.

4. A l kal i ne condi ti on (hi sh unfavourablefor Ca absorption.

pH )

is

5. High contentof dietaryfiber interfereswith C a absorpti on. Plasma

*&5{::i#iE*?

Most of the blood Ca is presentin the plasma since the blood cells contain very little of it. The normal concentrationof plasma or serum Ca is 9-11 mg/dl (4.5-5.5 mEq/h. About half of this (5 mg/dl) is in the ionized form which is 2. Parathvroid hormone enhances Ca functionallythe most active(Fi9.18.1).At least'l absorption through the increased synthesisof mg/dl serum Ca is found in associationwith c alc it r iol. citrate and/or phosphate.About 40% of serum 3. Acidity (low pH) is more favourablefor Ca Ca (4-Smg/dl) is bound to proteins, mostly absorption. al bumi nand, to a l esserextent,gl obul i n.l oni zed and citrate (or phosphate)bound Ca is diffusible 4. Lactosepromotescalcium uptake by intesfrom blood to the tissueswhile protein bound Ca t inal c ells . i s non-di ffusi bl e. In the usual l aboratory 5. T he a mi n o a c i d s l y s i n e a n d a r gi ni ne determinationof serumCa, all the threefractions facilitate Ca absorption. are measuredtogether.

407

Chapter 18 : MINERALMETABOLISM

lntes

r tn

I

lcitriol€-Vitamin

D

Bone Ca

Fig. 182 : Overuiewof calciumhomeostasis (PTH-Parathyroidhonnone).

FACTORS REGULATING PLASMA Ga LEVEL

degradedto proPTH and, finally, to active PTH. The rate of formation (by degradationof proPTH) and the secretion of PTH are promoted by low Ca2+ concentration.Thus, the releaseof PTH from parathyroid glands is under the negative feedback regulationof serum Ca2+. Mechanism of action of PTH : PTH binds to a membrane receptor protein on the target cell and activates adenylate cyclase to liberate cA MP . Thi s, i n turn, i ncreases i ntracel l ul ar calcium that promotes the phosphorylationof proteins(by kinases)which, finally brings about the biological actions. PTH has 3 independent tissues-bone, kidneysand intestine-to exert its action. The prime function of PTH is to elevate serum calcium level.

Action on the bone : PTH causes decalcification or demineralization of bone, a process carried out by osteoclasts. This is broughtout by.PTH stimulatedincreasedactivity of the enzymes pyrophosphatase and collagenase. These enzymes result in bone resorption.Demineralizationultimately leads to an increasein the blood Ca level. The action of G alc it r iol PTH on bone is quantitativelyvery significantto maintain Ca homeostasis.lt must, however, be The physiologicallyactiveform of vitamin D is noted that this is being done at the expenseof a hor m one ,n a me l yc a l c i tri o lo r 1 ,2 5 -d i hydroxyloss of Ca from bone, particularlyin dietary Ca cholecalciferolfi,25 DHCC). The synthesisof defi ci encv. calcitriol and its wide range of biochemical As already stated, calcium is almost exclusivelypresentin blood plasma (or serum). The hormone s-calcitriol, parathyroid hormone (PTH) and calcitonin are the major factors that regufate the plasma calcium (homeostasisof Ca; Fig.l8.2) within a narrow range (9-11 mg/dl).

actionsare describedunderVitamins(Chapter7).

Action on the kidney z PTH increases the Ca reabsorption by kidney tubules. This is the most Calcitriol induces the synthesisof a specific rapid action of PTH to elevate blood Ca levels. c alc ium bi n d i n g p ro te i n i n th e i n te s ti nalcel l s. quantitatively, However, this is less important This protein increasesthe intestinalabsorptionof compared to the action of PTH on bone. calcium as well as phosphate.Thus blood Ca promotes production PTH the of calcitriol level is increasedby calcitriol (the activevitamin (1,25 D H C C ) i n the ki dney by sti mul ati ng D). Furthermore, calcitriol stimulates calcium 1-hydroxylation of 25-hydroxycholecalciferol. uptake by osteoblasts of bone and promotes calcification or mineralization (deposition of Action on the intestine : The action of PTH calcium phosphate)and remodelling. on the intestine is indirect. lt increasesthe intestinal absorption of Ca by promoting the hormone Parathyroid synthesisof calcitriol. Parathyroidhormone (PTH) is secretedby two Galcitonin pairs of parathyroid glands that are closely Calcitonin is a peptide containing 32 amino associated with thyroid glands. Parathyroid hor m one ( mo l . w t. 9 5 ,0 0 0 ) i s a s i n g l e chai n acids. lt is secreted by parafollicular cells of poly pept id e ,c o n ta i n i n g 8 4 a mi n o a c i d s. l t i s thyroid gland. The action of CT on calcium riglna//y synthesized as preproPTH wh)ch )s metabd)sm )s antagon)silc to tbat ol PTH. Tbus,

408

BIOCHEMISTF|Y

calcitonin promotes calcification by increasing the activity of osteoblasts.Further, calcitonin decreasesbone resorption and increasesthe excretion of Ca into urine. CT, therefore,has a decreasing influence on blood calcium. lmportance

of Ca : P ratio

The ratio of plasma Ca : P is important for calcificationof bones.The product of Ca x P (in mg/dl) in children is around 50 and in adults around40. This product is lessthan 30 in rickets. Excretion

of calciurn

Calcium is excreted partly through the kidneys and mostly through the intestine.The r en a l th re s h o l d fo r s e ru m C a i s 10 mgl dl . Calcium gets excreted into urine beyond this concentration.Ingestionof excessprotein causes inc re a s e dc a l c i u m e x c re ti o n i n ' uri ne. Thi s i s m a i n l y d u e to a n i n c re a s ei n th e aci di tyof uri ne as a result of high protein diet.

renal losses) and increase in alkaline phosphatase activity are also found in hyperparathyroidism.Elevation in the urinary excretion of Ca and P, often resulting in the formation of urinary calculi, is also observedin these patients. The determinationof ionized serum calcium (elevated to 6-9mg/dl) is more useful for the diagnosis of hyperparathyroidism.lt has been observed that some of the patients may have normal levels of total calcium in the serum but differ with regardto ionized calcium. The symptoms of hypercalcemia include lethargy, muscle weakness, loss of appetite, constipation, nausea, increased myocardial contractilityand susceptibilityto fractures. H ypocal cemi a

Hypocalcemia is a more serious and life threatening condition. lt is characterized by a fall in the serum Ca to below 7 m{dl, Excretionof Ca into the feces is a continuous causing tetany. The symptomsof tetany include pr o c e s s a n d th i s i s i n c re a s e d i n vi tami n D neuromuscul ar i rri tabi l i ty, spasms and deficiency. convul si ons. Galcium

i n th e te e th

The teeth calcium is not subjected to regulation as observedfor bone calcium. Thus the adult teeth, once formed, do not undergo decalcification to meet the body needs of calcium. However, proper calcificationof teeth is i mp o rta n ti n th e g ro w i n g c h i l dren.

DISEASE STATES T h e b l o o d C a l e v e l i s m a i n tai nedw i thi n a narrow range by the homeostaticcontrol, most predominantlyby PTH. Hence abnormalitiesin Ca metabolism are mainly associated with alterationsin PTH.

Hypocalcemia is mostly due to hypoparathyroidism. This may happen after an accidental surgicalremovalof parathyroidglandsor due to an autoi mmunedi sease. Rickets Ricketsis a disorderof defectivecalcification of bones. This may be due to a low levels of vitamin D in the body or due to a dietary both. The deficiencv of Ca and P-or concentrationof serum Ca and P may be low or normaf. An increase in the activity of alkaline phosphatase is a characteristic feature of rickets. Renal rickets

Hypercalcemia El e v a ti o ni n s e ru m C a l e v e l (normal 9-1 1 mg/dl) is hypercalcemia. Hypercalcemia is associated with hyperparathyroidism caused by increased activity of parathyroid glands. Decreasein serum phosphate(due to increased

Renal rickets is associatedwith damage to renalti ssue,causi ngi mpai rmenti n the synt hesis of calcitriol. lt does not respondto vitamin D in ordinary doses, therefore, some workers regard this as vitamin D resistant rickets. Renal rickets can be treated bv administrationof calcitriol.

Ghapter 18 : MINEBALMETABOLISM Osteoporosis Osteoporosis is characterized by demineraIization of bone resulting in the progressiveloss of bone mass. Occurrence : The elderly people (over 60 yr.) of both sexes are at risk for osteoporosis. However, it more predominantlyoccurs in the post-menopausal women. Osteoporosisresultsin fractures which are a major cause frequent bone of disability among the elderly. lt is estimated that more than 50% of the fracturesin USA are due to this disorder. Osteoporosis may be regardedas a silent thief. Etiology : The etiology of osteoporosisis largely unknown, but it is believed that several causativefactorsmay contributeto it. The ability to produce calcitriol from vitamin D is decreased with dB€, particularly in the postmenopausal women. lmmobilized or sedentary individuals tend to decrease bone mass while those on regular exercise tend to increasebone mass.Deficiency of sex hormones (in women) has been implicated in the developmentof osteoporosis.

409 the bones and teeth. About 10"/o of body P is found in musclesand blood in associationwith proteins, carbohydrates and lipids. The remai ni ng10% i s w i del y di stri butedi n vari ous chemi calcompounds. B i ochemi cal functi ons .l . Phosphorus is essentialfor the development of bones and teeth. 2. lt playsa centralrole for the formationand utilizationof high-energyphosphatecompounds e.g. ATP, GTP, creatine phosphateetc. 3. Phosphorusis requiredfor the formationof phosphol i pi ds, phosphoprotei nsand nucl ei c aci ds (D N A and R N A ). 4. lt is an essentialcomponent of several nucleotide coenzymes e.g. NAD+, NADP+, pyridoxal phosphate,ADP, AMP. 5. Severalproteinsand enzymesare activated by phosphorylation. 6. Phosphatebuffer system is important for the maintenanceof pH in the blood (around7.4) as w el l as i n the cel l s.

Treatment : Estrogen administration along with calcium supplementation(in combination Dietary requirements with vitamin D) to postmenopausalwomen reduces the risk of fractures. Higher dietary The recommendeddietary allowance (RDA) intake of Ca (about 1.5 glday) is recommended of phosphateis basedon the intake of calcium. for elderly people. The ratio of Ca : P of | : I is recommended(i.e. 800 mg/day)for an adult. For infants,however, Osteopetrosis the rati o i s around 2 :1, w hi ch i s basedon the (marble bone diseasef rati o found i n human mi l k. C al ci um and phosphate are distributed in the majority of Osteopetrosis is characterized by increased naturalfoods in 1 : 1 ratio. Therefore,adequate bone density.This is primarily due to inabilityto intake of Ca generally takes care of the P resorb bone. This disorder is mostly ohserved in requi remental so. association with renal tubular acidosis (due to a defect in the enzyme carbonic anhydrase)and Sources cerebralcalcification. Milk, cereals,leafy vegetables,meat, eggs. Absorption Phosphateabsorptionoccurs from jejunum An adult body containsabout 1 kg phosphate and it is found in every cell of the body. Most of 1 . Calcitriol promotes phosphate uptake it (about807o)occurs in combinationwith Ca in al ong w i th cal ci um.

410

BIOCHEMISTF|Y

2. A b s o rp ti o no f p h o s p h o ru sa n d cal ci um i s optimum when the dietary Ca : P is between 1: 2and2 :1 . The adult body contains about 20 g 3. Acidity favours while phytate decreases magnesium,70'/. of which is found in bones in phosphateuptake by intestinalcells. combinationwith calcium and phosphorus.The remaining 307o occurs in the soft tissuesand Serum phosphate bodv fluids. The phosphate level of the whole blood is around a0 mg/dl while serum containsabout 3a m{dl. This is becausethe RBC and WBC have very high content of phosphate. The serum phosphatemay exist as free ions (40'h) or in a complex form (50%) with cations such as Ca2*, Mg2*, Na+, K+. About 10o/oof serum phosphate is bound to proteins. lt is interesting to note that the fasting serum phosphate levels are higher than the postprandial. This is attributed to the fact that following the ingestion of carbohydrate (glucose),the phosphatefrom the serumis drawn by the cells for metabolism (phosphorylation reactions). Exeretion About 500 mg phosphateis excretedin urine per day. The renal threshold is 2 mg/dl. The reabsorptionof phosphate by renal tubules is inhibit e db y P T H .

Biochemical

functions

1. Magnesium is required for the formation of bones and teeth. 2. Mg2* serves as a cofactor for several enzymes requiring ATP e.g. hexokinase, glucokinase, phosphofructokinase, adenylate cVclase. 3. Mg2* is necessary for proper neuromuscular function. Low Mg2+ levels lead to neuromuscularirritabilitv. Dietary

requirements

Adult man

350 mglday

Adult woman -

300 mg/day

Sources Cereals, nuts, beans, vegetables (cabbage, cauliflower),meat, milk, fruits. Absorption

Magnesiumis absorbedby the intestinalcells through a specificcarrier system.About 50% of 1. Serum phosphate level is increased in the dietary Mg is normally absorbed. hypoparathyroidism and decreasedin hyperpara- Consumption of large amounts of calcium, t hy r oidi s m . phosphate and al cohol di mi ni shes Mg 2. ln severerenal diseases/serum phosphate absorption.PTH increasesMg absorption. content is elevatedcausing acidosis. Serum Mg 3. Vitamin D deficientricketsis characterized Normal serumconcentrationof Mg is 2-3 m{ by decreasedserum phosphate(t-Z mg/dl). dl. lt is present in the ionized form (60%), in 4. Renalricketsis associatedwith low serum combinationwith other ions (10%)and bound to phosphate levels and increased alkaline proteins(30%). phosphataseactivity.

Disease states

5. In diabetes mellitus, serum content of organic phosphate is lower while that of inorganic phosphateis higher.

Disease states 1 . Magnesi um defi ci ency causes neuromuscular irritation. weaknessand convulsions.

4tl

Chapter 18: MINEBAL MEIABOLISM These symptomsare similar to that observedin tetany (Ca deficiency)which are relieved only by MS. Malnutrition,alcoholismand cirrhosisof Iiver may lead to Mg deficiency. 2. Low levels of Mg may be observed in uremia, ricketsand abnormal pregnancy.

Absorption Sodium is readily absorbedin the gastrointestinal tract and, therefore,very little of it (< 2%) is normally found in feces. However, in diarrhea, large quantitiesof sodium is lost in feces.

Plasma sodium In the plasma (serum), the normal concentration of sodium is 135-145 mEqfi. Sodium is the chief cation of the extracellular Sodium is an extracellular cation, therefore, the fluid. About 50'/' of body sodium is present in blood cells contain much less (35 mEq/l). The the bones,4Oo/o in the extracellularfluid and the mineralocorticoids,secretedby adrenal cortex, i nfl uence sodi um metabol i sm.A decrease in (1 0 % ) i n th e s o ft ti s s u e s . r em ain i n g plasma sodium and an increase in its urinary ' excretion are observed in adrenocortical functions Biochemical insufficiency. 1. ln a s s o c i a ti o n w i th c h l ori de and bicarbonate, sodium regulatesthe body's acidExcretion base balance. Kidney is the major route of sodium excretion 2. Sodium is requiredfor the maintenanceof from the body. As much as 800 g Na/day is osmotic pressureand fluid balance. filtered by the glomeruli, 99Yo of this is 3. lt is necessaryfor the normal muscle reabsorbed by the renal tubules by an active irritability and cell permeability. process. This is controlled by aldosterone. 4. Sodium is involved in the intestinal Extreme sweating also causes considerable absorption of glucose, galactose and amino amount of sodium loss from the body. There is, ac ids . however, individual variation in sodium loss 5. lt is necessary for initiating and through sweat. maintainingheart beat.

Dietary requirements For normal individuals, the requirement of s odium i s a b o u t 5 -1 0 g /d a y w h i c h i s mai nl y consumed as NaCl. For personswith a family history of hypertension,the daily NaCl intake should be less than 5 g. For patients of hypertension, around 'l g/day is recommended. It may be noted that 10 g of NaCl contains4 g of s od i u m . T h e d a i l y c o n s u m p ti on of N a i s generallyhigherthan requireddue to its flavour. Souvces The common salt (NaCl) used in the cooking m edium i s th e ma j o r s o u rc e o f s o di um. The ingestedfoods also contribute to sodium. The good sourcesof sodium include bread, whole grains, leafy vegetables,nuts, eggs and milk.

Disease states 1 . Hyponatremia : This is a condition in which the serum sodium level falls below the normal. Hyponatremia may occur due to diarrhea, vomiting, chronic renal diseases, adrenocortical insufficiency (Addison's disease). Administrationof salt free fluids to patientsmay also cause hyponatremia. This is due to overhydration. Decreased serum sodium concentration is also observed in edema which occurs in cirrhosis or congestive heart failure. The manifestationsof hyponatremiainclude reduced blood pressureand circulatoryfailure. 2. Hypernatremia : This condition is characterized by an elevation in the serum sodium level. The symptomsinclude increasein

412

BIOGHEMISTFIY

blood volume and blood pressure.lt may occur due to hyperactivity of adrenal cortex (Cushing's syndrome), prolonged administration of cortisone, ACTH and/or sex hormones. Loss of water from the body causing dehydration, as it occurs in diabetes insipidus, results in hypernatremia. Rapid administration of s odium s a l ts a l s o i n c re a s e s s e ru m sodi um concentration. lt may be noted that in pregnancy, steroid and placental hormones cause sodium and water retention in the body, leading to edema. In edema, along with water, sodium concentration in the body is also elevated. Administration of diuretic drugs increases the urinary output of water along with sodium. fn the patients ol hypertension and congestive cardiac failure salt (Na+) restriclion is advocated

Sources Banana, orange, pineapple, potato/ beans, chicken, and liver. Tender coconut water is a rich source of potassium.

Absorption The absorptionof K+ from the gastrointestinal tract is very efficient(90%) and very little is lost through feces. However, in subjects with diarrhea, a good proportion of K+ is lost in the feces.

Plasma potassium The plasma (serum) concentration of potassium is 3.4-5.O mEqI.The whole blood contains much higher level of K+ (50 mEdD, since it is predominantlyan intracellularcation. Care should, therefore, be taken to avoid hemolysisof RBCfor the estimationof serum K+.

Excretion

Potassiumis the principal intracellular cation. I t is equ a l l y i m p o rta n ti n th e e x tra c e ll ul arfl ui d for specific functions. Biochemical

functions

1. Potassiummaintains intracellular osmotic Pressure. 2. lt is required for the regulation of acidbase balance and water balance in the cells.

Potassiumis mainly excreted through urine. The maintenance of body acid-base balance influences K+ excretion. Aldosterone increases excretionof potassium.

Disease states Serum potassiumconcentrationis maintained within a narrow range. Either high or low concentrationsare dangerous since potassium effects the contractility of heart muscle.

Hypokalemia : Decreasein the concentration 3. The enzyme pyruvatekinase(of glycolysis) of serum potassium is observed due to overactivity of adrenal cortex (Cushing's is dependenton K+ for optimal activity. syndrome), prolonged cortisone therapy, 4. Potassiumis requiredfor the transmission intravenous administration of K+-free fluids, of nerve impulse. treatment of diabetic coma with insulin, 5. AdequateintracellularconcentrationK+ is prolongeddiarrhea and vomiting. necessaryfor proper biosynthesisof proteins by The symptoms of hypokalemia include ribosomes. irritability, muscular weakness, tachycardia, 6. ExtracellularK+ influencescardiac muscle cardiomegalyand cardiac arrest.Changesin the ECG are observed (flattenedwaves with inverted activity. T wave).

Dietary requirements About 3-4 g/day.

Hyperkalemia : Increasein the concentration of serum potassiumis observedin renal failure,

413

Ghapter 18 : MINEHALMETABOLISM adrenocortical i nsufficiency (Addison's disease), diabetic coma, severedehydration,intravenous administrationof fluids with excessivepotassium salts.

contains higher level of Cl- (125 mEq/l).This is due to the fact that protein content is low in CSF and, therefore,Cl- is higher in order to maintain D onnan membraneeoui l i bri um.

The manifestationsof hyperkalemiainclude Excretion depression of central nervous system, mental confusion,numbness,bradycardiawith reduced There exists a parallel relationshipbetween heartsoundsand, finally, cardiacarrest.Changes excretion of chloride and sodium. The renal in ECG are also observed (elevated T wave). thresholdfor Cl- is about 110 mEq/|.

Disease states 1. Hypochloremia: A reductionin the serum Cl- level may occur due to vomiting, diarrhea, Chlorine is a constituentof sodium chloride. respiratory alkalosis, Addison's disease and Hence, the metabolismof chlorine and sodium excessivesweating. are intimately related. 2. Hyperchloremia : An increase in serum Cl- concentration may be due to dehydration, B ioc henr i c a l fu n c ti o n s respiratoryacidosisand Cushing'ssyndrome. 1. Chloride is involved in the regulationof acid-baseequilibrium, fluid balance and osmotic pressure.These functions are carried out by the interactionof chloride with Na+ and K+. 2. Chloride is necessaryfor the formation of HCI in the gastricjuice.

Sulfur of the body is mostly present in the organic form. Methionine, cysteineand cystine 3. Chlorideshift involvesthe activeparticipa- are the three sulfur-containing amino acids tion of Cl-. present in the proteins. Cenerally, proteins contain about 17o sulfur by weight. 4. The enzyme salivary amylase is activated by chloride. Biochemical functions Dietary

requirements

1. Sulfur-containingamino acids are very The daily requirementof chloride as NaCl is essential for the structural conformation and 5-10 g. Adequate intake of sodium will satisfy biological functions of proteins (enzymes, hormones,structuralproteinsetc.).The disulfide the chloride requirementof the body. linkages(-S-S-) and sulfhydryl groups (-SH) are largely responsiblefor this. Sources Common salt as cooking medium, whole grains, leafy vegetables,eggs and milk.

2. The vitaminsthiamine, biotin, lipoic acid, and coenzyme A of pantothenic acid contain sulfur.

Absorption

3. Heparin, chondroitin sulfate,glutathione, In normal circumstances,chloride is almost taurocholicacid are some other importantsulfurcontaining compounds. totally absorbed in the gastrointestinaltract. phosphosulfate(PAPS) 4. Phosphoadenosine is the active sulfate utilized for several reactions The normal plasmaconcentrationof chloride e.g. synthesis of glycosaminoglycans,detoxiis 95-105 mEq/|. Cerebrospinal fluid (CSF) fication mechanism. P las m a

c h l o ri d e

414

BIOCHEMISTF|Y

4. lron is associatedwith effective immunoaci d 5. T he s u l fu r-c o n ta i n i n g a mi n o (as of the body. is actively competence methionine S-adenosylmethionine) reactions. involved in transmethylation

Dietary requirements SSetary requirements acrd sources

-

Adult man

10 mg/day

There is no specific dietary requirementfor - 18 mg/day Menstruatingwoman sulfur. Adequate intake of sulfur-containing Pregnantand lactatingwoman - 4O m{day essentialamino acid methionine will meet the body needs. Food proteins rich in methionine Sources and cysteine are the sources of sulfur. Rich sources Organ meats (liver, heart, kidney). Excretion Leafy vegetables, pulses, Cood sources The sulfur from different compounds is cereals,fish, apples,dried oxidized in the liver to sulfateand excreted in fruits, molasses. urine. The urine contains inorganic sulfate ($OV"),organic or conjugated or ethereal sulfate Milk, wheat, polished Poor sources ( 10%) an d u n o x i d i z e d s u l fu r (1 O% ). The ri ce. unoxidized sulfur is in the form of sulfurcontaining amino acids, thiocyanatesetc. transport and storage Absorption, lron is mainly absorbed in the stomach and duodenum. ln normal people, ahout 10o/" of dietary iron is usually absorbed. However, in The total content of iron in an adult body is iron deficient (anemic)individualsand growing 3-5 g. About 70% of this occurs in the children, a much higher proportion of dietary erythrocytes of blood as a constituent of iron is absorbed to meet the increased bodv hemoglobin.At least5"h ol body iron is present demands. in myoglobin of muscle. Heme is the most lron is mostly found in the foods in ferric form predominant iron-containingsubstance.lt is a (Fe3+),bound to proteinsor organicacids. In the proteins/enzymes of several constituent acid medium provided by gastric HCl, the Fe3+ (hemoprotei nslhemoglobi n, myoglobin, is released from foods. Reducing substances cytochromes, xanthine oxidase, catalase, such as ascorbic acid (vitamin C) and cysteine tryptophan pyrrolase,peroxidase.Certain other convert ferric iron (Fe3+)to ferrousform (Fe2+). proteins contain non-heme iron e.g. transferrin, lron in the fenous form is soluhle and readily ferritin, hemosiderin. absorbed. Bf,ociremical

functions

1 . lron mainly exertsits functionsthroughthe compounds in which it is present.Hemoglobin and myoglobin are required for the transport of 02 and CO2. 2. Cytochromes and certain non-heme proteins are necessary lor electron transport chain and oxidative phosphorylation.

Factors

affecting

Fe absorptEen

1. Acidity, ascorbic acid promote iron absorption.

and

cysteine

2. In iron deficiencyanemia,Fe absorptionis increasedto 2-10 times that of normal. 3. Small peptides and amino acids favour iron uptake.

4. Phytate (found in cereals) and oxalate 3. Peroxidase, the lysosomal enzyme, is requiredfor phagocytosisand killing of bacteria (found in leafy vegetables) interfere with Fe absorption. by neutrophils.

415

Ghapten 18 : MINERALMETABOLISM

Bonemarrow(Hb) Muscle(Mb) Othertissues (Cyts& NHI) Fig. 18.3: lron absorptionand transpott(GlT4astrointestinaltract; llb-Hemqlobin; Mb4tyoglobin; Cyts4ytochromes; NH[-Non-hemekon).

5. A diet with high phosphate content Storageof iron : lron is stored in liver, spleen decreasesFe absorption while low phosphate and bone marrow in the form of ferritin. ln the promotes. mucosal cells, ferritin is the temporary storage form of iron. A molecule of apoferritin(mol. wt. 6. lmpaired absorptionof iron is observedin 500,000) can combine with 4,000 atomsof iron. malabsorption syndromes such as steatorrhea. The maximum iron content of ferritin on weight 7. In patients with partial or total surgical basis is around 25o/o. removal of stomach and/or intestine, iron Hemosiderin is another iron storage protein absorptionis severelyimpaired. which can hold about 35"h of iron by weight. lron in the mucosal cells : The iron (Fe2+) Hemosiderinaccumulatesin the body (spleen, entering the mucosal cells by absorption is liver) when the supply of iron is in excess of oxidized to ferric form (Fe3+)by the enzyme body demands. ferroxidase.Fe3+then combines with apoferritin to form ferritin which is the temporary storage rflAri€:*\tu{*ff Stfh tftF,'tf{} form of iron. From the mucosal cells, iron may lfOn ES d enter the blood stream (which mainly depends lron metabolism is unique as it operates on the body needs) or lost when the cells are in a closed system. lt is very efficiently desquamated. utilized and reutilized by the body. Further, iron Transport of Fe in the plasma : The iron liberated from the ferritin of mucosal cells enters the plasma in ferrous state. Here, it is oxidized to ferric form by a copper-containing protein, ceruloplasmin which possesses ferroxidase activity. Another cuproprotein ferroxidasell also helps for the conversion of Fe2+to Fe3+.

lossesfrom the body are minimal (< 1 mg/day) which may occur through bile, sweat, hair foss etc. lron is not excreted into urine. Thus, iron differs from the vitamins or other organic and inorganic substanceswhich are either inactivated or excreted during the course of metabolic function. Hence, iron is appropriately regarded as a one-way substance.

Ferric iron then binds with a specific ironprotein, namely transferrin or binding lron entry into the body is controlled at the siderophilin (a glycoprotein with mol. wt. level, dependingon the body needs. with absorption can bind 90,000).Eachtransferrinmolecule periodical loss in menstruatint blood Thus the (Fe3+). plasma The iron ferric of atoms two iron Increased its requirements. increases women bind can mgldl) (concentration 250 transferrin pregnancy, in with 4O0mg of iron/dl plasma. This is known as demands are also observed lactation, and in growing children. total iron binding capacity (flRC) of plasma'

416 *uer Y i e v # s $ i rn n ne*ta:l!:*liss, r A general overview of iron metabolism is depicted in FigJ8.a. lt shows the distribution of iron in the body and its efficient reutilization. lt may be noted that about 1-2 mg of iron is absorbed per day to replace the loss.

BIOCHEMISTRY

Bi:ciy srcir:*e

1,000mg Fe

,' FoodFe Absorption , l-a,orou (1-2mg/day)-l 4mg

Dis*a:+el *iair*s 1. lron deficiency anemia : This is the most prevalent nutritional disorder worldover, including the well developed countries (e.g. USA). Several factors mav contribute to iron deficiencyanemia.Theseinclude inadequate intake or defective absorptionof iron, chronic blood loss, repeated pregnancies and hookworm infections.

t

/

/

J for synthesis Utilization (20mg/daY)

Release by degradation (20 mg/day)

Fig. 18.4 : A general overview of iron metabolism.

to a high intake of iron from their staple diet corn and their habit of cooking foods in iron pots.

Strict vegetariansare more prone for iron deficieny anemia.This is due to the presenceof inhibitors of iron absorption in the vegetarian 3. Hemochromatosis : This is a rare disease foods, besidesthe relatively low content of iron. in which iron is direclly deposited in the tissues (liver,spleen,pancreasand skin). Hemosiderosis lron deficiency anemia mostly occurs in is sometimesaccompaniedby hemochromatosis. growing children, adolescentgirls, pregnantand Bronzed-pigmentationof the skin, cirrhosis of lactating women. lt is characterized by liver, pancreaticfibrosis are the manifestationsof microcytic hypochromic anemia with reduced this disorder. Hemochromatosis causes a bfood hemoglobin levels (<12 B/dl). The other condition known as bronze diabetes. manifestations include apathy(dull and inactive), sluggish metabolic activities, retarded growth and loss of appetite. 2. Hemosiderosis: This is a less common disorder and is due to excessiveiron in the body. It is commonly observed in subjects receiving repeated blood transfusionsover the years, e.g. patients of hemolytic anemia, hemophilia. As already stated, iron is a one-way compound, once it enters the body, it cannot escape. Excessive iron is deposited as ferritin and hem os i d e ri n .

The body contains about 100 mg copper distributed in different organs. lt is involved in several importantfunctions. tsioelremieai

f rnmctioms

1. Copper is an essential constituent of several enzymes. These include cytochrome oxidase, catalase, tyrosinase, superoxide Hemosiderosisis commonly observedamong dismutase,monoamine oxidase, ascorbic acid the Bantutribe in SouthAfrica. This is aftributed oxidase, ALA synthase, phenol oxidase and

Chapter 1a : MINEHALMETABOLISM

417

uricase.Due to its presencein a wide variety of Plasma copper enzymes,copper is involved in many metabolic The copper concentrationof plasma is about reactions. 100-200 mg,/dl. Most of this (95%) is tightly 2. Copper is necessaryfor the synthesisof bound to ceruloplasminwhile a small fraction hemoglobin(Cu is a constituentof ALA synthase, (5% ) i s l oosel y hel d to al bumi n. N orma l concentration of serum ceruloplasmin is 25-50 neededfor heme synthesis). mg/dl. lt contains about 0.34% copper (6-8 3. Lysyloxidase(a copper-containing enzyme) atomsof Cu per molecule,half in Cu2+stateand is required for the conversionof certain lysine the other half in Cu+ state). residuesof collagenand elastinto allysinewhich are necessaryfor cross-linkingthese structural Disease states proteins. 1. Copper deficiency : Severedeficiency of 4. Ceruloplasminservesas ferroxidaseand is copper causes demineralization of bones, involved in the conversionof iron from Fe2* to demyelination of neural tissue,anemia, fragility Fe3+ in which form iron (transferrin) is of arteries, myocardial fibrosis, hypopigtransportedin plasma. mentationof skin, greying of hair. 5. Copper is necessaryfor the synthesis of m elani n a n d p h o s p h o l i p i d s .

2. Menke's disease: This disorderis due to a defect in the intestinalabsorptionof copper. lt is 6. Developmentof bone and nervoussystem possible that copper may be trapped by metallothionein in the intestinalcells. The symptoms ( m y elin )re q u i re sC u . of Menke's diseaseinclude decreasedcopper in 7. Certain copper-containingnon-enzymatic pl asma and uri ne, anemi a and depi gmentati on proteins have been identified, although their of hai r. functions are not clearly known. These include (hepatolenticular 3. Wilson's disease hepatocuprein(storageform in liver), cerebrodegeneration) : lt is a rare disorder of abnormal (i n (i n c upr ein b ra i n ) a n d h e mo c u p re i n R B C ). copper metabolismand is characterizedby the 8. Hemocyanin,a copper protein complex in fol lowing manifestations. invertebrates, functions like hemoglobin for 02 . Copper is depositedin abnormal amounts in transport. l i ver and l enti cul arnucl eusof brai n.Thi s ma y lead to hepatic cirrhosisand brain necrosis. Dietary requirements

Adults

2-3 m{day

fnfantsand children -

0.5-2 mg/day

Sources Liver, kidney, meat, egg yolk, cereals, nuts and green leafy vegetables. Milk is a poor source. Absorption

. Low levels of copper and ceruloplasmin in plasma with increasedexcretionof copper in uri ne. . Copper deposition in kidney causes renal damage.This leads to increasedexcretion of amino acids, glucose, peptides and hemogl obi ni n uri ne. . Intestinalabsorptionof copper is very high, about 4-6 times higher than normal.

Probable causes of Wilson's disease : The About l Oh ol dietary copper is absorbed, following explanationsare offeredto understand mainfy in the duodenum. Metallothionein is a the etiology of this disease. transport protein that facilitates copper 1. A fai l ureto synthesi ze absorption. Phytate, zinc and molybdenum cerul opl asmi n or an impairmentin the binding capacityof copper to decreasecopper uptake.

418

E|IOCHEMISTFIY

this protein or both. As a resultof this, copper is free in the plasmawhich easilyentersthe tissues (liver, brain, kidney),binds with the proteinsand gets deposited.The albumin bound copper is either normal or increased.

About 80% of body's iodine is stored in the (a iodothyroglohulin form as organic glycoprotein)in the thyroid gland. This protein diiodotyrosine and contains thyroxine, triiodothyroninein different proportions.

Excretion of iodine mostly occurs through 2. Sorne workers suggest that a reduced intestinalexcretionof copper may be responsible kidney. lt is also excreted through saliva, bile, for the occurrenceof Wilson's disease. ski n, and mi l k (i n l actati ngw omen). Treatment : Administrationof penicillamine, Plassna iodine a na tu ra l l yo c c u rri n gc o p p e r c h e lati ngagent,i s The normal concentrationof plasmaiodine is used for the treatmentof Wilson's disease. a-1O mg/dl. Most of this is present as protein bound iodine (PBI) and representsthe iodine contained in the circulating thyroid hormones. PBI level decreases in hypothyroidism and The total body containsabout 20 mg iodine, increases in hyperthyroidism. RBC do not most of it (80%) being present in the thyroid contai n i odi ne. gland. Muscle, salivaryglands and ovaries also c on ta i n s o m e a mo u n t o f i o d i n e .

Disease

BiochennieaB fugrctlons

The disordersof iodine metabolism-simple goiter and toxic goiter-are discussed in detail under thyroid hormones(Chapter l9).

The onlv known function of iodine is its requirement for the synthesis of thyroid (T+) and hormones namely, thyroxine triiodothyronine (Tr). These hormones are involved in several biochemical functions (Chapter 19). Functionally, T3 is more active than Ta.

Dietary requirememts Adults Pregnantwomen

states

The total body contentof manganeseis about 15 mg. The l i ver and ki dney are ri ch i n M n. W i thi n the cel l s, Mn i s mai nl y found i n t he nucl ei i n associ ati onw i th nucl ei c aci ds.

100-150pilday 2OO1t{day

B i ochemi cal

and excretion

3. Mn is necessary for the synthesis of mucopolysaccharides and glycoproteins.

funeti ons

1 . Mn serves as a cofactor for several enzymes. These include arginase, pyruvate Sources isocitrate dehydrogenase, carboxylase, Seafoods,drinking water, vegetables,fruits superoxi de di smutase (mi tochondri al ) and (grown on seaboard).High altitudesare deficient peptidase. in iodine content in water as well as soil. Plant 2. Mn is requiredfor the formation of bone, and animal foods of these areas, therefore, proper reproductionand normal functioning of contain lesseramount of iodine. In theseregions, nervous system. iodine is added to drinking water or to table salt. Absorption,

storage

lodine as iodide is mainly absorbedfrom the . o rma l l y ,a b o u t 30% of di etary s m a l l i n te s ti n e N iodi n e i s ta k e n u p b y th e i n te s ti nalcel l s. l odi ne absorptionalso occurs through skin and lungs.

4. H emogl obi nsynthesi si nvol vesMn. 5. Mn i nhi bi tsl i pi d peroxi dati on. for cholesterolbiosynthesis. 6. Mn is necessary

Ghapter 18 : MINEHALMETABOLISM

Dietary requirements The exact requirementof Mn is not known. About 2-9 m{day is recommendedfor an adult.

Sources

419 3. The storageand secretionof insulin from the B-cellsof pancreasrequire Zn. 4. Zn is necessaryto maintain the normal levels of vitamin A in serum. Zn promotesthe synthesisof retinol binding protein.

Cereals,nuts, leafy vegetablesand fruits. Tea is a rich source of Mn.

5. l t i s requi red for w ound heal i ng. Zn enhances cel l grow th and di vi si on, besi d es stabiIi zi ng bi omembranes.

Ahsorption

6. Gusten, a zinc containing protein of the saliva, is important for taste sensation.

About 3-4o/o of dietary Mn is normally absorbedin the small intestine.lron inhibits Mn absorption.

7. Zn is essentialfor proper reproduction.

Dietary requ;rernents Serum

Mn

Zi nc requi rementfor an adul t i s 10-15 mil Manganesein the serum is bound to a specific day. lt is increased(by about 50%) in pregnancy carrier protein-fransmagnanin (a p-globulin). and lactation. The normal blood contains about 5-20 m{dl. Sources

Disease states M n d e fi c i e n c yi n a n i m a l sc a u s e s 1. Retardedgrowth, bone deformitiesand, in severedeficiency,sterility.

Meat, fish, eggs, milk, beans,nuts.

Absorptiom

Zi nc i s absorbedmai nl v i n the duodenum.Zn from the animal sourcesis better absorbedthan 2. Accumulation of fat in liver. the vegetablesources.Zn absorptionappearsto 3. lncreased activity of serum alkaline be dependent on a transport protein-metallothionein. Phytate, calcium, copper and iron phosphatase,and interferewhile small peptidesand amino acids 4. Diminished activity of p-cells of pancreas promote Zn absorption. ( low in s u l i n ). Serum

The total content of zinc in an adult body is about 2 g. Prostategland is very rich in Zn (100 mglg).Zinc is mainly an intracellularelement.

Zn

The concentrationof Zn in serum is about 100 mg/dl. Erythrocytescontain higher content ol Zn (1.5 mg/dl )w hi ch i s found i n associ ation with the enzyme carbonic anhydrase.

Disease states

1. Zinc deficiency is associatedwith growth retardation,poor wound healing,anernia,lossof 1 . Zn is an essentialcomponent of several appetite, loss of taste sensation, impaired enzymes e.g. carbonic anhydrase, alcohol spermatogenesis etc. lt is reported that Zn dehydrogenase,alkaline phosphatase,carboxydeficiencyin pregnantanimalscausescongenital peptidase,superoxidedismutase(cytosolic). malformationsof the fetus.Deficiencyof Zn may 2. Zinc may be regarded as an antioxidant result in depression, dementia and other since the enzyme superoxide dismutase (Zn psychiatric disorders. The neuropsychiatric containing)protectsthe body againstfree radical manifestationsof chronic alcoholism may be damage. partly due to zinc deficiency. Biochemieal

functions

420

BIOCHEMISTRY

Acrodermatitis enteropathica is a rare inherited metabolic diseaseof zinc deficiency caused by a defect in the absorption of Zn from the intestine.

Fluoride is mostly found in bones and teeth. The beneficial effects of fluoride in trace 2. Zinc toxicity is often observedin welders amounts are overshadowedby its harmful effects due to inhalation of zinc oxide fumes. The caused by excessconsumption. manifestationsof Zn toxicity include nausea, gastriculcer, pancreatitis,anemia and excessive B i ochemi cal functi ons salivation. 1. Ffuoride prevents the development of dental caries. lt forms a protective layer of acid resistantfluoroapatite with hydroxyapatiteof the enamel and preventsthe tooth decay by bacterial acids. Further, fluoride inhibits the bacterial enzymesand reducesthe production of acids. Molybdenum is a constituentof the enzymes xanthine oxidase, aldehyde oxidase and sulfite 2. Fluoride is necessary tor the proper oxidase. Nitrite reductase(containingMo) is a developmentof bones. plant enzyme, required for nitrogenfixation. 3. lt inhibitsthe activitiesof certainenzymes. The requirements of Mo are not clearly Sodium fluoride inhibits enolase (of glycolysis) known. However, it is widely distributed in while fluoroacetateinhibits aconitase(of citric the natural foods. Dietary Mo is effectively acid cycle). (60%-70%)absorbedby the small intestine. and sources Dietary requirements Some workers have reported that Mo An intake of lessthan 2 ppm of fluoride will decreasesthe mobilization and utilization of meet the daily requirements.Drinking water is copper in the body. the main source. Molyhdenosis is a rare disorder caused by excessiveconsumptionof Mo. lts manifestations Disease states inc lud e i mp a i rm e n t i n g ro w th , d i arrhea and 1. Dental caries: lt is clearly establishedthat anemia. Intestinal absorption of copper is water containing less than 0.5 ppm of drinking dim inis h e d . fluoride is associatedwith the developmentof dental cari esi n chi l dren.

Cobalt is only important as a constituentof vitamin 812. Cobalt content of vitamin 812 is about 4oh by weight. The functions of cobalt are the same as that of vitamin 812 (Chapter 7). Administration of cobalt stimulates the productionof the hormoneerythropoietin,which promoteserythropoiesis. Prolongedadministrationof cobalt is toxic as it results in polycythemia (increased RBC in blood).

2. Fluorosis: Excessiveintake of fluoride is harmful to the body. An intake above 2 ppm (parti cul arl y > 5 ppm) i n chi l dren causes mottling of enamel and discolorationof teeth. The teeth are weak and become rough with characteristicbrown or yellow patcheson their surface. These manifestationsare collectively referredto as dental fluorosis. An intake of fluoride above 20 ppm is toxic and causespathologicalchanges in the bones. Hypercalcification,increasingthe density of the bones of limbs, pelvis and spine, is a characteristicfeature.Eventhe ligamentsof spine

,. i:iI::'.':!?

M I NERAL M ETABO LI SM

421

and collagenof bonesget calcified.Neurological with fluoride toothpastes. There is some disturbancesare also commonly observed.The rethinking on these aspects due to the toxic manifestations describedhere constituteskeletal effectsof excessfluorioe. fluorosis.ln the advancedstages,the individuals ar e c r ippled a n d c a n n o t p e rfo rm th e i r dai l y r out inewor k d u e to s ti ffj o i n ts .T h i s c o n d i tionof advanced fluorosis is referredto as genu valgum. S el eni um w as ori gi nal l y i denti fi ed as an The fluoride contentof water in some partsof el ement that causestoxi ci ty to ani mal s (al kal i A ndhr a P r ad e s h P , u n j a ba n d K a rn a ta k ai s qui te disease)in some parts of USA, containing large high. F luor o s i si s p re v a l e n t i n th e s e re g i ons, amounts of Se in the soil. Laterwork, however, c aus ing c on c e rn to g o v e rn m e n t a n d h eal th has show n that S e i n smal l er amounts i s of f ic ials . biologically important. 3. Fluoridationof water and use of fluoride tooth-pastes : In order to prevent the dental c ar iesin c hild re n ,s o me a d v a n c e dc o u n tri esl i ke 1. Selenium, along with vitamin E, prevents USA have startedfluoridation of water. Further, the development of hepatic necrosis and the consumermarketstill recentlywere flooded muscul ardystrophy.

EIOMEtrICAL I CLINIGALCONCEFTS

@ Serum calcium leuel is increased (normal 9-11 mg/dl) in hyperparathyroidism. This condition is olso associotedwith eleuated urinary excretion of Ca and P, ot'ten leading to stone formation. w Tetany, coused by a drastic reduction in serum Ca, is characterizedby neuromuscular irritability and conuulsions. !s Rickefs is due to defectiuecalcilication oJ bones. This may be causedby deficiency ot' Ca and P or uitamin D or both. @ Osteoporosisis the bone disorder of the elderly, characterized by deminerolization resulting in a progressiue loss of bone moss. /t is the major causeol bone fractures and disability in the old people. @ Decreasedlevels of serum Na (hyponatremio) is obserued in diarrhea and uomiting, besides Addison's disease, while increased serum Na (hypernatremia) is found in Cushing's syndrome. vs lron deliciency anemia is the most preualent nutritional disorder worldouer.Il is mosf commonly obseruedin pregnant and lactating women. re Wilson's disease is due to an abnormal copper metabolism. It is characterized by abnormal deposition of copper in liuer and brain, besidesthe low leuels of plosma copper ond ceruloplasmin. s

Endemic goitre, due to dietory iodine deficiency, is uery common. consumption of iodized salt is aduocatedto ouercome this problem.

r€ F/uorosis is caused by on excessiueintake of fluoride. The manilestations include mottling of enomel and discolorationof teeth. In the aduancedstages,hgpercalcification oJ limb bones and ligomentsol spine get calcified, ultimately crippling the indiuidual.

422

BIOCHEMISTFIY

2 . S e i s i n v o l v e d i n ma i n tai ni ngstructural are associatedwith increased risk of cardiointegrityof biological membranes. vasculardisease,and various cancers. 3. Se as selenocysteine is an essential component of the enzyme glutathione peroxidase. This enzyme protects the cells againstthe damagecausedby H2O2. lt appears from recentstudiesthat selenocysteine is directly incorporated during protein biosynthesis. Therefore, selenocysteineis considered as a s e p a ra te(2 1 s t)a m i n o a c i d .

Toxficity Selenosisis the toxicity due to very excessive intake of Se. The manifestationsof selenosis i ncl ude w ei ght l oss, emoti onal di stu r bances, diarrhea,hair loss and garlic odor in breath.The compound dimethyl selenide is responsible for the garlic odor.

4. Se preventslipid peroxidationand protects t h e c e l l s a g a i n s t th e fre e ra di cal s, i ncl udi ng superoxide(Ol). 5 . S e p ro te c ts a n i ma l s from carci nogeni c The total human body contains about 6 mg chemicals. However, the precise role of Se in chromium. The Cr content of blood is about 20 humanswith regardto cancer preventionis not m{dl. Cr performs several biochemical clearly identified. functions. 6. Se binds with certain heavy metals (Hg, Cd) and protects the body from their toxic effects. 7. A selenium containing enzyme S'-deiodinase converts thyroxine (T4) to triiodot h y ro n i n ei n th e th y ro i d g l a n d.

1. In associ ati onw i th i nsul i n, C r p r om ot es the utilization of glucose.Cr is a component of a protein namely chromodulin which facilitates the bi ndi ng of i nsul i n to cel l receptorsit es. 2. Cr lowersthe total serumcholesterollevel.

3. lt is involved in lipoprotein metabolism. Cr decreasesserum low density lipoproteins purine Thioredoxin reductase, 8. involved in (LDL) and increaseshigh density lipoproteins nucleotide metabolism,is also a selenoprotein. (HDL) and, thus, promoteshealth. ffiequ;r'e;t-:+c'*ds mffid st?€dFe*,,4;

A daily intake of 50-200 mg of Se has been recommendedfor adults.The good sourcesof Se are organ meats (liver, kidney) and sea foods. ffi frse.iilir* s:;;,sii tg:i

4. lt is believed that Cr participatesin the transportof amino acids into the cells (heartand l i ver). The dietary requirementof Cr is not known. lt is estimatedthat an adult man consumesabout 10 to 100 mg/day. The good sources of Cr include brewer's yeast, grains/ cereals/cheese and meat.

Deficiency : Se deficiencyin animalsleadsto muscular dystrophy, pancreatic fibrosis and reproductive disorders. In humans, Keshan C hromi um defi ci encycausesdi sturb ancesin disease,an endemic cardiomyopathy(in China) carbohydrate, lipid and protein metabolisms. is attributedto the deficiency of Se. Epidemio- Excessiveintake of Cr resultsin toxicity, leading logical studies reveal that low serum Se levels to liver and kidney damage.

1 .., , . . . i,M I NERAL M ETABO LI SM

423

\' The minerals or inorganic elements are required for normal growth and maintenance of the bodg. They are classifiedas principal elements ond t)are elements. There are seuenprincipal erements-ca, n Ms, Na, K, cr and s. The trace erementsincrudeFe Cu, I, Zn, Mn, Mo, Co, F, Se and Cr. 2' Calcium is requiredfor the deuelopmentoJ bones and teeth, muscle contraction, blood coagulation, nerue transmissionetc. Absorptionof Ca Jrom'the duodenumis promoted by uitamin D, PTH and acidity while It is inhibited by-phytate, oxalote, acids and fiber' The normql leuel of serum Ca (s-11 mg/iD is'cont'rolled t'ree fatty by an interplay o! PTH, calcitriol and calcitonin. 3. serum ca leuel is. eleuated in hgperparathgroidism and diminished in hypopara_ thgroidism' Hypocalcemia cousestetany, the symptomsof which include neuromusc,lar irritability, spasmand conuulsions. 4. Phosphorus, besides being essential t'or the deuelopment of bones and teeth, is a constituent of high energyphosphatecompounds(Arp, GTp) ind nucleotiie-;;;""v:;", (NAD+, NADP+). 5' Sodium, potassiumand chlorine are inuoluedin the regulation ol acid-baseequilibrium, Jluid balance ond osmotic pressurein the body. sodlum is the principal extracellular cation (serum leuel 135-745 mEfl), while potassiumis the chief intracellular cation (serum leuel 3.5-5.0 mEq,4), 6' Iron is mainly required t'or 02 transport and cellular respiration. Absarptionof iron is promoted bg ascorbicacid, cysteine, acidity and small peptid.es u.)hileit is inhibtted bv ) phytate, oxalate and high phosphote. 7 ' Iron (Fe3+)is transported in the plasmq in o bound t'orm to transferrin.It is stored as ferritin in liuer, spleen and bone marrow. Iron deficiency anemia couse$ microcatic hypochromic onemia- Excessiueconsumptionot' iron resu/is in hemosidero.sis which is due to the tissue deposition of hemosiderin. 8' Copper is on essenfioI constituentof seueralenzymes(e.g catalase,cytochromesxidase, tyrosinase) Ceruloplasmin ts o copper containingprotein required ior the tronrpori-of iron (Fes+)in the plasma wilsonis disease is,an qbnormolity in copper metabolism, characterizedby the deposition of copper in riuer, brain and. kidney. 9' lodine is important as a component of thyroid hormones(Ta and Tg) white cobaltis s constituent of uitamin 812' Zinc is necessary t'or the storage and secretionot' insulin and maintenanceof normar uitamin A leuelsin serum, b.jdes beinga ,o^plr.nr'o.. seueralenzymes (e.g. carbonic anhydrase, alcohol dehydrogenase). 10 Fluorine in trace amounts (<2 ppm) preuents dentql caries while its higher intake Ieads to t'luorosis.Selenium is ossignedan antioxidant role as it protectsthe cellsJrom free, radicals' Chromium promotes the utilization oJ glucose snd red.uces serum cnotesterot.

BIOCHEMISTFIY

424

I. Essayquestions 1. Write brieflyon the trace elementsand their metabolismin the body. sourcesand absorptionof calcium. 2. Discussthe biochemicalfunctions,dietaryrequirements, 3. Write an essayon the iron metabolismin the body. 4. Describethe metabolismof copper,zinc and manganese. 5. Write on the biochemicalimportanceand diseasestatesof fluorineand selenium. II. Short notes (a) Homeostasisof calcium, (b) Osteoporosis,(c) Phosphorus,(d) Sodium and chlorine, (h) Wilson'sdisease,(i) Iodine, (f) FactorsaffectingFe absorption,(g) Hemosiderosis, (e) Potassium, (j) Magnesium. III. Fill in the blanks of serumcalcium 1 . The normal concentration 2 . The vitamin derivedhormonethat regulatescalcium homeostasis 3 . The inorganic element found in the structure of majority of high-energycompounds 4. Severalkinaseenzymesrequirethe mineralcofactor fluid 5. The principalcation of extracellular of serumpotassium 6. The normal concentration 7. lron is transportedin the plasmain a bound form to a protein to ferric iron 8. The coppercontainingproteininvolvedfor the conversionof ferrousiron (Fe2+) (Fe3+)in the plasma

9. The zinc containingprotein in the salivainvolvedin tastesensation 10. The elementinvolvedin the protectionof cells againstthe damageof H2O2 and other free radicals IV. Multiple choice questions is(are)involvedin the regulationof plasmacalcium level 11. The following substance(s) (a) Calcitriol(b) Parathyroidhormone(c) Calcitonin(d) All of them. ami no aci d 1 2 . T h e fo l l o w i n gi s a s u l fu rc o ntai ni ngessenti al (a) Methionine(b) Cysteine(c) Cystine(d) All of them. 1 3 . l ro n i n th e m u c o s a cl e l l sb indsw i th the protei n (d) Hemosiderin. (b) Ferritin(c) Ceruloplasmin (a) Transferrin 1 4 . T h e fo l l o w i n ge l e me n ti s i n vol vedi n w ound heal i ng (a ) C a l c i u m(b ) S o d i u m(c ) Zi nc (d) Magnesi um. 1 5 . Pick up elementthat preventsthe developmentof dentalcaries (d) S odi um. (a ) F l u o ri n e(b ) C a l c i u m(c ) P hosphorus

pfll tissue proteinsand Body Fluids

t:lt ;i.,r

l i v i n g b o d y p o s s e s s e sa re markabl e T h" $ communication system to coordinate its biologic a l fu n c ti o n s .T h i s i s a c h i e v ed kry tw o Hormones may be classifiedin many rt'ays distinctly organizedfunctional systems. basedon their characteristics and fr,rnctions. lwo iypes of classification are discussed herc 1. The nervoussystemcoordinatesthe body f unc t ion s th ro u g h th e tra n s mi s s i o nof el ectro*. ff+;xsee,1 iDsrri':t-'' r;!rq:et,r;ec*F *eataxre c hem ic a l i mo u l s e s . The hormonescan be categorizedinto three 2. The endocrine systemacts throrrgh a wide groupsconsi deri ngthei r chemi cal nai rrrr:, range of chemical rnessengers known as 1. P rotei n or pepti de hormonese.g. i nsui i n, hor m on e s . gl ucagon,anti di rrreti chormone,e* ,,{ oci ri . Hormones are conventionally defined as 2. Steroid horrnones e.g. glucocorticoids, organic substances,pradwced in snnall"arnounts mineralocorticoids,sex hormc,n*s. hy specific fissues (endocrine glands), secrefed 3. Amino acid derivativese.g. epinephrine, into the hlood stream ta control the norepinephrine,thyroxine (TJ, triiodoll')rir+irine metaholic and hiolagical activities in the target (T' )' cells" l-lormones may be regarded as the in {nessengers involved the chemical !i, Sase*l +n Atr!,e+ {".ls?ffB?i:fir"E;,-f:?}'E {.r$ transmissionof information frorn one tissue to "t:i{f:':i;;{?9t anot her a n d fro m c e l l to c e l i . T he maj or Flormones are ciassified into two broad endoc r i n eo rg a n si n h u m a n b o d y a re d e pi ctedi n groups (l and ll) based on the Lrc.atinnr:f the Fig"n e.t).

427

428

B IOC H E MIS TRY

Pinealgland Pituitarygland

Thyroidgland

Pancreas Adrenalgland

Testis(male)

4t I

stimulate the release of certain molecules, namely the second messengers which, in turn, perform the biochemical functions. Thus, hormones themselves are the first messenSers. Croup ll hormonesare subdividedinto three categoriesbased on the chemical nature of the second messengers. (a) The second messenger is cAMP e.g. ACTH, FSH, LH, PTH, glucagon, cal ci toni n. (b) The second messenger is phosphatidylinositolkalcium e.g. TRH, GnRH, gastrin, CCK. (c) The second messenger is unknown e.g. growth hormone, insulin, oxytocin, prolactin. The pri nci palhuman hormones,thei r cl a ssif ication based on the mechanismof action, and major functions are given in Tahle 19.1. of action Mechanism group I hormones

of

Thesehormonesare l i pophi l i c i n natureand can easily pass across the plasma membrane. They act through the intracellular receptors located either in the cytosol or the nucleus. The hormone-receptor complex binds to specific regions on the DNA called hormone Fig. 19.1: Diagrammaticrepresentationof majorendocrineglands. responsiveelement (HRE)and causesincreased expression of specific genes (Fig.19.2. lI is believed that the interaction of hormone receptorsto which they bind and the signals receptorcomplex with HRE promotes initiation used to mediate their action. and, to a lesser extent, elongation and The of RNA synthesis(transcription). termination 1. Group I hormones: Thesehormonesbind production of specific is the ultimate outcome to intracellular receptors to form receptor(the hormone complexes intracellular proteins (translation)in responseto hormonal m es s e n g e rsth ) ro u g h w h i c h th e i r bi ochemi cal action. functions are mediated. Croup I hormones are lipophilic in natureand are mostlyderivativesof Mechanism of action of (exception-T, cholesterol To). group ll hormones and e.g. estrogens, androgens, glucocorticoids, These hormones are consideredas the first c alc i tri o l . messengers.They exert their action through 2. Group ll hormones: Thesehormonesbind mediatorymolecules,collectivelycalled second to cell surface(plasmamembrane)receptorsand messengers.

429

Chapter 19 : HOFIMONES

Hormone(s)

Origin

Major Function(s)

RECEPTORS THATBINDTOINTRACELLULAR GtoupL HORMONES cortex Female Ovaries andadrenal sexual characteristics, menstrual Estrogens cycle. Progestins Ovaries andplacenta Involved in menstrual maintenance cycleand of pregnancy. Testes andadrenal cortexMalesexual Androgens characteristics, spermatogenesis. Affectmetabolisms, Adrenalcortex suppress immune Glucocorticoids system, Adrenalcortex Maintenance Mineralocorticoids of saltandwaterbalance. (1,25-DHCC) Kidney(finalform) Promotes Calcitriol absorption of Ca2*fromintestine, kidneyandbone. general Promote Thyroid hormones Thyroid metabolic rate. O3,TJ THATBINDTOCELLSURFACE RECEPTORS Groupll. HORMONES is cAMP A. Thesecondmessenger (ACTH)Anterior pituitary Adrenocorticotropic hormone Stimulates therelease of adrenocorticosteroids. (FSH) Anterior pituitary hormone ln females, Follicle stimulating slimulates ovulation andestrogen synthesis. promotes Inmales, spermatogenesis. (LH) pituitary hormone Anterior Stimulates Luteinizing synthesis of estrogens and progesterone and causes ovulation. Promotes androgen synthesis bytestes. progesterone Anteriorpituitary Stimulates release fromplacenta. Chorionicgonadotropin(hCG) pituitary (T,,To). hormone Promotes Thyroid stimulaling therelease of thyroid hormones [ISH) Anterior p-Endorphinsandenkephalins (painrelievers). Natural Anteriorpituitary endogenous analgesics pituitary (stored)Promotes (ADH) Posterior Antidiuretic hormone waterreabsorption by kidneys. glucose glycogenolysis Pancreas Increases blood level,stimulates Glucagon andlipolysis. (PTH) promotes Parathyroid Increases Parathyroid hormone serum calcium, frombone. Ca2*release Thyroid Lowers serum calcium. Decreases Cd+uptake byboneandkidney. Calcitonin medulla Increases glycogenAdrenal heartrateandbloodpressure. Epinephrine Promotes olysis in liverandmuscle andlipolysis inadipose tissue. Adrenal medulla Stimulates lipolysis in adipose Norepinephrine tissue. B. Thesecondme3senger is phosphatldyl InositoUcalcium Hypothalamus Thyrotropin-releasinghormone(tRH) (GnRH)Hypothalamus hormone Gonadotropin+eleasing Gastrin Stomach (CCK) Intestine Cholecystokinin ls unknown/unsettled C.Thesecondmessenger pituitary (GH) Anterior hormone Growth

Promotes TSHrelease. Stimulates release of FSHandLH. gaskicHCIandpepsinogen Stimulates secretion. Stimulates contraction ofgallbladder andsecretion ol pancreatic enzymes.

groMhof thebody(bones Promotes andorgans).

(PRL) Prolactin

pituitary Anterior

Oxytocin

pituitary (stored)Stimulates Posterior uterine contraction andmilkejection.

glands GroMhof mammary andlactation.

Insulin

Pancreas

(hypoglycemic promotes protein Lowers bloodglucose efiect), synthesis andlipogenesis.

(insulinlike Somatomedins growthfactors,IGF-|,IGF-ll)

Liver

GroMhrelated functions of GHaremediated. growth Stimulates ol cartilage.

E}IOCHEMISTF|Y

430

Cy c l i c AMP (c A M P, c y c l i c adenosine3',5'-monophosphate) is a u b i q u i to u s n u c l e o ti d e . l t consistsof adenine,riboseand a phosphate (linked by 3',5' linkage).cAMP acts as a second messenger for a majority of polypeptide hormones. enzvme The membrane-bound adenylatecyclaseconvertsATP to cyclic AMP. cAMP is hydrolysed to S'-AMP by phosphodiesterase (Fig.1e.3). Adenylate

cyclase

system A seriesof eventsoccur at the membrane level that influence the activity of adenylatecyclase leadingto the synthesisof cAMP. This process is mediated by G-proteins, so designateddue to t heir a b i l i ty to b i n d to g u a n i n e nucleotides.

MR N A

MRNA

Action ttf cAMP-a general view produced, cAMP Once performs its role as a second Fig. 19.2 : Mechanism of action of steroid hormones (H-Hormone; R-Receptor; HR-Hormone-receptor complex). m es se n g e r i n e l i c i ti n g b i o (Fig.l9.a). chemical responses cAMP activatesprotein kinaseA (A standsfor cAMP). This enzyme is a heterote- protein that ultimately causesthe biochemical tramerconsistingof 2 regulatorysubunits(R)and response. 2 catalyticsubunits(C). It should, however, be remembered that cAMP binds to inactive protein kinase and cAMP does not act on all protein kinases.For instance, on protein kinase C (the second causesthe dissociationof R and C subunits. messengeris diacylglycerol). 4cAMP + R2C2----; R2(4cAMP) + 2C (inactive) (active) ( in a ctive ) Dephosphorylation of proteins : A group of The active subunit (C) catalysesphosphory- enzymes called protein phosphataseshydrolyse lation of proteins(transferof phosphategroup to and remove the phosphate group added to serineand threonineresidues).lt is the phospho- proteins.

ehapter 19 : HOFIMONES

431 hormonal action. In responseto the stimuli of central nervous system,hypothalamusliberates certain releasing factors or hormones. These factors stimulate or inhibit the release of correspondingtropic hormonesfrom the anterior pituitary. Tropic hormones stimulate the target endocrine tissuesto secretethe hormonesthey synthesize. The relationship between hypothalamus and pituitary with endocrine glands is illustratedin Fig.19.6.In general, the hormonal system is under feedback control. For instance,adrenocorticotropichormone (ACTH) inhibits the release of corticotropin releasing hormone (C R H ).

3',5'-Cyclicadenosine monophosphate (cAMP) I

H^n_-- | \ I Phosphodiesterase

J

5'-AM;T

Hypothalamusproducesat leastsix releasing factorsor hormones.

1. Thyrotropin-releasinghormone (TRH) : lt is a tripeptideconsistingof glutamatederivative (pyroglutamate),histidine and proline. TRH stimulatesanterior pituitary to releasethyroidDegradation of cAMP : cAMP undergoes stimulatinghormone(TSHor thyrotropin)which, rapid hydrolysis, catalysed by the enzyme in turn, stimulates the release of thyroid phosphodiesterase to 5' AMP which is inactive. hormones(T3 and Ta). Hence, the effect of cAMP will be shortlived if the hormone stimulating adenylate cyclase is 2. Corticotropin-releasinghormone (CRH) : removed. Caffeine and theophylline It stimulates anterior pituitary to release (methylxanthine derivatives) can inhibit adrenocorticotropichormone (ACTH) which in phosphodiesterases and increasethe intracellular turn, acts on adrenal cortex to liberate levels of cAMP. adrenocorticosteroids. CRH contains 41 amino aci ds. Fig. 19.3 : Synthesisand degradationof oAMP.

3. Gonadotropin-releasing hormone(GnRH): It is a decapeptide.CnRH stimulatesanterior pituitary to release gonadotropins, namely l utei ni zi nghormone(LH ) and fol l i cl e sti mul ati ng The pituitary gland or hypophysis(weighing hormone(FS H ). about 1 g) is located below the hypothalamus 4. Growth hormone-releasing hormone of the brain. lt consistsof two distinct parts(GRH) with 44 amino acids stimulates the the anteriorpituitary (adenohypophysis) and the releaseof growth hormone(CH or somatotropin) posteriorpitutitary(neurohypophysis) connected which promotesgrowth. by pars intermedia (Fig.l9.fl. The latter is 5. Growth hormone release-inhibiting almost absent in humans, although found in (GR IH ) hormone : l t 14 ami no aci ds contai ns lower organisms. and is also known as somatostafin.CRIH inhibits Hypothalamus is a specialized center in the the releaseof growth hormonefrom the anterior brain that functions as a masfer coordinator of pituitary.

432

BIOCHEMISTFIY

tl

Hormone

Ii I

l1 I i1

!l I

I

j

ri

Fig. 19.4 : Overuiew of synthesis and action of oAMP (RzCe--cAMPdependent protein kinase A; R2-Regulatory subunits; Cr-Catalytic subunits; C-Active catalytic unit of R2C).

6. Prolactin release-inhibiting hormone (PRIH) : lt is believedto be a dopamine and/or a small peptide that inhibits the release of prolactin (PRL)from anterior pituitary.

hormones that influence-either directly or indirectly-a variety of biochemicalprocessesin are the body. The hormonesof adenohypophysis broadly classifiedinto three categories. l. The growth hormone-prolactingroup. ll. The glycoproteinhormones.

Anteriorpituitaryor adenohypophysis is truly the masterendocrineorgan. lt producesseveral

The pro-opiomelanocortin peptide familv.

433

Chapter 19 : HOFIMONES

Grow th Hypothalamus

hormone

(GH I

The growth hormone (or somatotropin) is produced by somatotropes,a special group of aci dophi l i ccel l s of anteri orpi tui tary.

Regulationof GH release: Two hypothalamic Anteriorpituitary (adenohypophysis) factors play a prominent role in the releaseof growth hormones.Theseare the growth hormoneParsintermedia releasinghormone(CRH) that stimulatesand the (intermediate lobe) growth hormone release-inhibiting hormone (GRlH,somatostatin) that inhibits.This, in turn, is pituitary Posterior regulated by feedback a mechanism. (neurohypophysis) Ftg. 19.5: A diagrammaticviewof pituitarygland.

l. T he gr owth gr oup

h o rm o n e -p ro l a c ti n

Crowth hormone production is influencedby many factors such as sleep, stress(pain, cold, surgery),exercise,food intakeetc. lt is observed that the largestincreasein the productionof CH occurs afterthe onset of sleep.This supportsthe adage /'lf you don't sleep, you won't grow."

Crowth hormone (CH), prolactin (PRL)and Biochemical functions of GH : Crowth chorionic somatomammotropin(CS; placental hormone promotesgrowth, and also influences lactogen) are protein hormones with many the normal metabolisms(protein, carbohydrate, striking similaritiesin their structure. l i pi d and mi neral )i n the body.

f

Hypothalamus

ll

I

l[ -

Feedback

G nRH

T SH

ACTH

LH

Adrenal coltex

Ovaryffestes

I +

Th\irord

I

J

GRH,GRIH PRIH

J

FSH

GH

Prolactin

IJ

Oxytocin

tl

++

L' r ' T, Adrenocor Estrogens Androge ' + t ic os ier oids Proqestins I

tl

++ Tarqettissues--+ Liver,muscle, heartetc.

il

JJ Reproductive organs

Bone

f Mammary I (--waCi-l I

I urerinel

gland | [ reabsorption I I contractionI

Flg. 19.6 : Hormonal heirarchy relationships between hypothalamus and pituitary with other endocrine glands ITRH-Thyrotropin releasing hormone; CRH-Corticotropin releasing hormone; GnRH-Gonadotropin releasing hormone; GRH-Grovvth hormone releasing hormone; G4lH4rowth hormone release inhibiting hormone; TSH-Thyroid stimulating hormone; ACTH-Adrenocorticotropic hormone; LH-Luteinizing hormone; FSH-Folticle stimulating hormone; GH-Growth hormone; ADH-Antidiuretic hormone; TgTriiodothyronine; T4-Tetraiodothyronine (thyroxine)1.

434

B IOC H E MIS TFIY

1. Effectson growth : As is obvious from the name, CH is essential for the growth. The growth-related effects of GH are mediated through insulin like growth factor I (lCF-l)which is also known as somatomedin C (formerly sulfationfactor), produced by liver.

P rol acti n Prolactin (PRL) is also called lactogenic hormone,luteotropichormone,mammotropinor luteotropin.

Biochemical functions of PRt : Prolactin is pri mari l y concerned w i th the i ni ti ati on and 2. Effects on protein metabolism : Crowth maintenance of lactation in mammals. PRL hormone has an anabolic effect on protein increasesthe levelsof severalenzymesinvolved metabolism. lt promotes the uptake of amino in carbohydrate and lipid metabolism. PRL acids into the tissuesand increasesthe orotein promotes HMP shunt, increases lipid synthesis.The overall effect of GH is a positive biosynthesisand stimulateslactoseproductionin nitrogen balancethat leads to increasein body mammarygl ands. weight. Prolactinpromotesthe growth of corpus luteum (hence also known as luteotropichormone)and 3. Effects on carbohydrate metabolism : the production of progesterone. stimulates Growth hormone is antagonisticto insulin and causeshyperglycemia.CH increasesgluconeoglycoprotein hormones genes is ,d e c re a s e sg l u c o s e u ti l i z a ti o n, i mpai rs ll. The glycolysis and reduces the tissue uptake of The following four hormonesare glycoprotein glucose. in nature and possess certain structural similarities,despitetheir functional diversity. 4. Effects on lipid metabolism : Crowth 1. Thyroi d sti mul ati nghormone (TS H ) hormonepromoteslipolysisin the adiposetissue and increasesthe circulatory levels of free fatty 2. Fol l i cl esti mul ati nghormone (FS H ) acids and their oxidation. lt increases 3. Lutei ni zi nghormone (LH ) ketogenesis,particularlyin diabetes. 4. H uman chori oni cgonadotropi n(hC G). 5. Effects on mineral metabolism : Growth hormone promotesbone mineralizationand its growth, as clearly observed in the growing c hildr en . Abnorrnalities

of Gl{ production

Deficiency of GH : lmpairment in the secretionof growth hormone in the growing age causesdwarfism. The other deficiency metabolic effects are not that serious in nature. production Overproductionof GH : Excessive of CH causes gigantism in children and acromegaly in adults. This usually occurs in the acidophil tumor of pituitary gland. Gigantism is characterizedby increasedgrowth of long bones and this is observedbeforethe epiphysealplates close. Acromegaly occurs after epiphyseal closure and is characterizedby increasein the size of hands, facial changes (enlarged nose, protruding jaw), excessive hair, thickening of skin etc.

The last three hormones (2-4) are collectively referred to as gonadotropins due to their involvement in the function of gonads. The hormone hCC is produced by human placenta and not by pituitary. However, due to its structuralresemblancewith other hormones,it is also consideredhere. 1. Thyroid stimulatinghormone (TSH) : TSH is a dimer (a0) glycoprotein with a molecular weight of about 30,000. Regulationof TSH production : The releaseof TSH from anterior pituitary is controlled by a feedbackmechanism.This involvesthe hormones of thyroid gland (T3 and Ta) and thyrotropinreleasinghormone (TRH)of hypothalamus. Functions of TSH : The biochemical effectsof TSH on thyroid gland are briefly discussedhere. TSH binds with plasmamembranereceptorsand stimulatesadenylatecyclasewith a consequent increase in cAMP level. TSH, through the mediation of cAMP, exerts the following effects.

Clra pte n 19 : HO RM O NES

435

o Promotesthe uptake of iodide (iodide pump) from the circulation by thyroid gland.

P t5 The prc-opE omel anoeortan {PGM*} peptide farr.rily

. E nhanc esth e c o n v e rs i o n o f i o d i d e (l-) to active iodide (l*), a process known as organification.

This family consists of the hormonesadrenocorticotropic (ACTtil, hormone lipotropin (tPffi and melanocyte stimulating hormone (MSFI) and several (about 24) neuromodul ators such as endorphi ns ano enkephali ns.

. lncreasesthe proteolysisof thyroglobulin to releaseT, and To into the circulation.

TSH increases the synthesis of proteins, The synthesis of POMC family. is very nuc leic ac id s a n d p h o s p h o l i p i d s i n th yroi d interesting. All the members of POMC are gland. produced from a single gene of the anterior Gonadotropins : The follicle-stimulating and intermediate lobes of pituitary. lt is hormone (FSH), luteinizing hormone (LF] and fascinating that a single polypeptide-prohuman chorionic gonadotropin hcq are opiomelanocortin-is the precursor (approxicommonly known as gonadotropins.All three matel y 285 ami no aci ds)that contai nsmul ti pl e are glycoproteins. hormones. The name pro-opiomelano-cortin is derived since it is a prohormone to opioids, The releaseof FSH and LH from the anterior pituitary is controlled by gonadotropin-releasing melanocyte-stimulating hormone and corticotropin. hor m one ( Cn R H 1o f h y p o th a l a m u s . Products of POMC : The pituitary 2. Biochemicalfunctionsof FSH : In femares, FSH stimulatesfollicular growth, increasesthe multihormone precursor is synthesizedas prePOMC weight of the ovaries and enhances the proopiomelanocortin from which is formed. The POMC consists of 3 peptide production of estrogens. Sroups. ln males, FSH stimulates testosterone 1. ACTH that can give rise to o-MSH and production, required f or spermatogenesis. corticotropin like intermediate Iobe peptide FSH also promotes growth of seminiferous (C LIP ). tubu les. 2. p-Li potropi n (B -LP H ) that can produce i 3. Biochemicalfunctions of LH : Luteinizing hormone stimulates the production of y-LPH,p-MSH and p-endorphin.The latteryields progesterone from corpus luteum cells in females y- and cr-endorphins. and testosteronefrom Leydig cells in males. LH 3. An N-terminal peptide that forms y-MSH. and FSH are collectively responsiblefor the The products obtained from POMC are development and maintenance of secondary depicted in Fig.20.7. These products undergo sexual charactersin males. many modi fi cati ons such as gl ycosyl ati on, 4. Human chorionic gonadotropin (hCC) : acetylation etc. hCC is a g l y c o p ro te i n (m o l . w t. 1 0 0 ,000), produced by syncytiotrophoblast cells of 1 . Adrenocorticotropic hormone (ACTH) : placenta.The structureof hCC closely resembles A C TH i s a pol ypepti dew i th 39 ami no aci dsano t hat of LH. a mol ecul arw ei ght of 4,5o0. Thi s hormone i s primarily concerned with the growth and T he lev el s o f h C C i n p l a s m a a n d uri ne functions of adrenal cortex. increase almost immediatelv after tne implantationof fertilizedovum. The detectionof Regulation of ACTH production : The release hCG in urine is conveniently used for the early of ACTH from the anteriorpituitary is under the detection (within a week after missing the regul ati on of hypothal ami c hormone, namel y menstrual cycle) of pregnancy. corticotropin releasinghormone (CRH).

BIOCHEMISTFIY

436 ACTH

Biochemical functions of ACTH promotes the . ACTH conversion of cholesterol to pregnenolonein the adrenal cortex.

1-39

CLIP

o-MSH

Frt--l

1LPH

l=1o-or-l lZfti--l

p'Endorphin (31A'As)

t1o4-1s4 I (15A.As) yEndorphin

. lt enhancesRNA and protein synthesisand thus promotes adrenocorticalgrowth. . ACTH increaseslipolysis by activating lipase of adipose t is s u e .

F-LPH(s3A.As)

(14A.As) o-Endorphin Enkephalin

E (POMC)family Fig. 19.7: Themembersof thepro-opiomelanocoftin deived fromPOMCcleavage.(Numbersin blocksrepresentaminoacids in sequence;ln the bracketsarc the numberof aminoacids-AAs; (ACTH-Adrenocorticottopic n; hormone; LPH-Lipotropi hormone; CLlP4orticotropin like MSH-Melanocyte-stimulating intermediate lobepe ptide).

Overproduction of ACTH : Cush i n g ' ss y n d ro me i s c a u s e d by an excessiveproduction of ACTH which may be due to a tumor. This syndrome is characterized by hyperpigmentation and increased production of The associatedsymptoms adrenocorticosteroids. include negative nitrogen balance, impaired glucosetolerance,hypertension,edema, muscle atrophy etc.

2. p-Lipotropin (p-tPH) : p-LPH is derived from POMC and contains 93 carboxy terminal amino acids. This polypeptide consists of y-LPH and p-endorphin from which p-MSH and y-endorphin are, respectively, formed. y-Endorphincan be converted to c,-endorphin and then to enkephalins (Fi9.19.V. p-LPH is found only in the pituitary and not in other tissuessince it is rapidly degraded.

is derived from POMC (Fig,l9.7). p-Lipotropin has 31 amino acids while its modified products have 15 and 14 ami no acids, a and y-endorphi ns respectively.Methionine enkephalin (Tyr-GlyCly-Phe-Mefl and leucine enkephalin(Tyr-GlyCly-Phe-leu) are the two important pentapeptide derivativesof B-endorphin. Biochemical actions : Endorphins and enkephalins are peptide neurotransmittersthat produce opiate-like effects on the central nervous system,hence they are also known as opioid-peptides.They bind to the same receptors as the morphine opiates and are believed to control the endogenous pain perception. Endorphins and enkephalins are more potent (20-30 times)than morphine in their function as anal gesi cs.

The biochemical functions of p-LPH, as such, are limited. lt promotes lipolysis and increasesthe mobilization of fatty acids. The most importantfunction of p-LPH is its precursor It is believed that the pain relref through role for the formation of B-endorphin and acupuncture and placebos is mediated through enk e p h a l i n s . opioid peptides. Endorphins and enkephalins : These are the natural analgesics that control pain and emotions. They were discovered after an unexpected finding of opiate receptors in the hum a n b ra i n .

3. Melanocyte-stimulating hormone (MSH) : Three types of MSH (cr, 0 and T) are present in the precursor POMC molecule. In humans, y MS H i s i mportantw hi l e i n some ani mal sa and p are functional. The activity of y-MSH is Synthesis : Endorphins and enkephalins containedin the moleculey-LPH or its precursor are produced from p-endorphinwhich, in turn, F-LPH(Fig.te.7).

Chapter 19 : HORMONES 19 (A)Cys-Tyr-

437

lle-Gln -Asn - Cys- Pro- Leu- Gly e_e__

19 (B)Cys-Tyr-

Phe-Gln -Asn-Cys-

Pro-Arg -Gly

s_ _ s_ _ Fig, 19.8: Structuresof (A) Humanoxytocinand (B) Humanantidiuretichormone(ADH).

A nti di ureti c

hormone

(A D H )

The releaseof ADH (also called vasopressin) is mostly controlled by osmoreceptors(of hypothalamus) and baroreceptors(of heart). Any increasein the osmolarity of plasma stimulates ADH secretion. Biochemical functions : ADH is primarily concernedwith the regulationof water balance in the body. lt stimulateskidneysto retain water and, thus, increasesthe blood pressure.

The functions of MSH has been clearly establishedin some animals.MSH promotesthe synthesis of skin pigmentmelanin(melanogenesis) In the absence of ADH, the urine output and dispersesmelanin granulesthat ultimately would be around 20 l/dav. ADH acts on the leadsto darkeningof the skin. In humans,MSH distal convolutedtubules of kidneysand causes does not appear to play any role in melanin water reabsorptionwith a result that the urine output is around 0.5-1.5 l/day. svnthesis. Mechanism of action : ADH stimulates adenylatecyclasecausing production of cAMP. Water reabsorption is promoted by cAMP. Inhibitors of adenylate cyclase (e.g. calcium) inhibit the activity of ADH. This supports the view that ADH action is mostly mediated through cAMP.

Two hormones namely oxytocin and antidiuretic hormone (ADH, vasopressin) are produced by the posterior pituitary gland (neurohypophysis). Both Diabetes insipidus : This disorder is characof them are nonapeptides(9 amino acids). Their structures terized by the excretion of large volumes of dilute urine (polyuria). lt may be due to are depicted in Fig.l9.8. insufficient levels of ADH or a defect in the receptors of target cells. Oxytocin The release of oxytocin from posterior pituitary gland is causedby the neural impulses of nipp l e s ti m u l a ti o n . T h e o th er sti mul i responsiblefor oxytocin releaseinclude vaginal and uterine distention.

Thyroid gland (weighsabout 30 g in adults)is located on either side of the trachea below the Biochemical functions larynx. lt produces two principal hormones (Fig.l9.9)-thyroxine (T+; 3,5,3',5'-tetraiodo1. Effect on uterus : Oxytocin causes the (Tl)thyronine) and 3,5,3'-triiodothyronine contractionof pregnantuterus(smoothmuscles) which regulate the metabolic rate of the body. and induces labor. Thyroidgland also secretescalcitonin,a hormone 2. Etled on milk ejection : ln mammals, concernedwith calcium homeostasis(discussed oxytocin causes contraction of myoepithelial under calcium metabolism,Chapter 181. cells (look like smooth muscle cells) of breast. This stimulatesthe squeezingeffect,causingmilk Biosynthesis of thyroid hormones ejection from the breast. lodine is essentialfor the synthesisof thyroid 3. Oxytocin synthesizedin the ovary appears hormones. More than half of the body's total to inhibit the synthesis of steroids. iodine content is found in the thyroid gland.

438

B IOC H E MIS TR Y

(thyroxine, 3, 5, 3', s'-Tetraiodothyronine T4)

(T.) 3,5,3'-Triiodothyronine

\"*,-9*-cooH I

NHz

(reverseTr, rTs) 3, 3', S'-Triiodothyronine Flg. 19,9: Structuresof thyroidhormones (ReferFig. 15.21for their biosynthesis).

Uptake of iodide : The uptake of iodide by the thyroid gland occurs againsta concentration gr ad i e n t(a b o u t2 0 : 1 ). l t i s a n e n e rgyrequi ri ng processand is linked to the ATPasedependent Na+ -K+ p u m p . l o d i d e u p ta k e i s pri mari l y controlled by TSH. Antithyroid agents such as thiocyanate and perchlorate inhibit iodide transport. Formation of active iodine : The conversion of iodide (l-) to active iodine (l+) is an essential step for its incorporationinto thyroid hormones. T hy r o i d i s th e o n l y ti s s u eth a t c a n oxi di ze l - to a higher valence state l+. This reaction requires HzOz and is catalysed by the enzyme thyroperoxidase(mol. wt. 60,000). An NADPH dependentsystemsuppliesHzOz.

Thyroglobulin and synthesis of T3 and Ta : Thyrogl obul i n (mol . w t. 660,000) i s a glycoproteinand precursorfor the synthesisof T3 and Ta. Thyrogl obul i ncontai ns about 1 40 tyrosine residueswhich can serve as substrates for iodine for the formationof thyroid hormones. Tyrosine(of thyroglobulin)is first iodinatedat position 3 to form monoiodotyrosine(MlT) and then at position 5 to form diiodotyrosine(DlT). Two moleculesof DIT couple to form thyroxine (Ta).One molecule of MlT, when coupled with one molecule of DlT, triiodothyronine (T3) is produced. The mechani smof coupl i ng i s not well understood.The details of synthesisof T3 and Ta are given under tyrosine metabolism (Chapter Ifl. A diagrammatic representationis depicted in Fig.l9.l0. As the process of iodination is completed, each molecule of thyroglobulin contains about 6-8 moleculesof thyroxine (Tl). The ratio of T3 to Ta i n thyrogl obul i ni s usual l yaround 1 : 10. Storage and reieese thyroid hormones

of

Thyrogl obul i ncontai ni ngTa and T3 can be storedfor severalmonths in the thyroid gland. lt is estimatedthat the storedthyroid hormonescan meet the body requirementfor 1-3 months. Thyroglobulin is digested by lysosomal proteolytic enzymes in the thyroid gland. The (9O%) and free hormones thyroxine (10% ) rel eased i nto t he are tri i odothyroni ne process TSH. MIT and blood, a stimulated by gland produced in the thyroid undergo DIT deiodinationby the enzyme deiodinaseand the i odi ne thus l i beratedcan be reuti l i zed. Transport

arf ?o erid T"

Two specific binding proteins-thyroxine bi ndi ng gl obul i n (TB C ) and thyroxi ne bi nding prealbumin (TBPA)-are responsible for the transportof thyroid hormones.Both T4 and T3 are more predominantlybound to TBC. A small promotes TSH the oxidation of iodide to fractionof free hormonesare biologicallyactive. ac t iv e i o d i n e w h i l e th e a n ti th yroi d drugs Ta has a half-lifeo[ 4-7 dayswhile T3 has about (thiourea,thiouracil, methinazole)inhibit. one day.

439

Gha pte r'1 9: HO FI M O NES

correlatedto thyroid hormonesand this, in turn, w i th A TP uti l i zati on.Obesi tyi n some i ndi vi dual s is attributedto a decreasedenergy utilization and heat producti on due to di mi ni shed N a+ -K + ATPaseactivitv. 2. Effect on protein synthesis : Thyroid hormonesact like steroidhormonesin promoting protein synthesisby acting at the transcriptional level (activateDNA to produce RNA). Thyroid hormones,thus,functi on as anabol i chormones and causepositivenitrogenbalanceand promote growth and development.

J

coupring

J

Totargettissues

3. Influence on carbohydrate metabolism : Thyroid hormonespromote intestinalabsorption of glucose and its utilization. These hormones increase gluconeogenesisand glycogenolysis, with an overall effect of enhancing blood glucose level (hyperglycemia). 4. Effect on lipid metabolism : Lipid turnover and utilization are stimulated by thyroid hormones. Hypothyroidism is associatedwith elevatedplasmacholesterollevelswhich can be reversedby thyroid hormone administration. Regulation

of Ta and T4 synthesis

The synthesis of thyroid hormones is controlledby feedbackregulation(Fig.l9.1l.f 3 appearsto be more actively involved than Ta in the regulationprocess.The productionof thyroid stimulating hormone (TSH) by pituitary, and thyrotropin releasing hormone (TRH) by hypothal amus are i nhi bi tedby T3 and, to a l esser Biochemical functions of degree, by T+. The increasedsynthesisof TSH thyroid horrmones and TRH occurs in response to decreased circulatory levels of T3 and Ta. As already Triiodothyronine(I:) is about four times more discussed, the body has sufficient stores of active in its biological functions fllan thyroxine (Ia). The following are the biochemicalfunctions hormones to last for several weeks. Hence it takessome monthsto observethyroid functional attributedto thyroid hormones (T3 and Ta). deficiency. 1. Influence on the metabolic rate : Thyroid hormones stimulate the metabolic activities and IHetabolic fate of T" and To increasesthe oxygenconsumptionin most of the Thyroid hormones undergo deiodination in tissues of the body (exception-brain, lungs, the peripheraltissues.The iodine liberatedmay testesand retina). be reutilized by the thyroid. T3 and T4 may get Na+-K+ATP pump : This is an energydepen- conjugatedwith glucuronicacid or sulfatein the dent processwhich consumesa major share of Iiver and excreted through bile. Thyroid cellular ATP. Na+-K+ATPaseactivitv is directlv hormonesare also subiectedto deaminationto Fig. 19.10: Biosynthesis of thyroidhormonesdiagrammaticrepresentation[Note : ReferFig. 15.21for synthesiswithstructures;Tgb-Thyroglobul in; I+-Active iodine; TgTriiodothyronine ; To-Thyroxine;MlT-MonoiodoUrosine; DIT-DiiodoUrosine; A. As-Aminoacidsl.

440

BIOCHEMISTRY

produce tetraiodothyroaceticacid (from Ta)and triiodothyroaceticacid (from T3) which may then undergo conjugationand excretion. Abnormalities function

of thyroid

A m o n g th e e n d o c ri n e g l a nds, thyroid is the most susceptible for hypo- or hyperfunction. Three abnormalities associated with thyroid functions are known.

I

Goiter : Any abnormal increase in the size of the thyroid gland is known as goiter. Enlargementof thyroid gland is mostly to compensatethe decreasedsynthesis of thvroid hormones and is associated with elevated TSH. Co i te r i s p ri m a ri l y d u e to a fa i l ure in the autoregulationof T3 and Ta synthesis.This may be caused by deficiency or excessof iodide. Metabolic

Protein

Carbohydrate Utilization

Maintenance

Goitrogenic substances rateT synthesisT metabolisml of lipidst of H2O,electrolyte Datance (goitrogens) : These are the substances that interfere with the and functionsof thyroid Fig. 19.11: Regulation of synthesis ulating hormone; hormones-an overview(TRH-Thyrotropin-stim production of thyroid hormones. TSH-Thyroidstimulatinghormone ; ; TgTriiodothyronine These include thiocyanates,nitrates 4nhibitory etfect). ettect; T4-Thyroxine; S-Promoting e and perchloratesand the drugs such as th i o u re ath , i o u ra c i lth , i o c a rb a m i de etc. Certainplant foods-cabbage, cauliflowerand irritability, anxiety, rapid heart rate, loss of turnip-contain goitrogenic factors (mostly thio- weight despite increased appetite, weakness, cyanates). diarrhea,sweating,sensitivityto heat and often protrusionof eyeballs(exopthalmos). Simple endemic goiter : This is due to iodine Hyperthyroidism is caused by Grave's disease deficiency in the diet. lt is mostly found in the geographicalregionsaway from sea coastwhere (particularlyin the developedcountries)or due the water and soil are low in iodine content. to increasedintake of thyroid hormones.Grave's Consumption of iodized salt is advocated to disease is due to elevated thyroid stimulating overcome the problem of endemic goiter. In IgC alsoknown as long acting thyroid stimulator certain cases,administrationof thyroid hormone (LATS) which activates TSH and, thereby, increasesthyroid hormonal production. is a l s o e mp l o y e d . Hyperthyroidism : This is also known as thyrotoxicosis and is associated with overproduction of thyroid hormones. Hyperthyroidismis characterizedby increased metabolic rate (higher BMn nervousness,

Thyrotoxicosisis diagnosedby scanningand/ or estimationof f 3,Tt (both elevated)and TSH (decreased)in plasma. The treatment includes administration of antithyroid drugs. In severe cases,thyroid gland is surgicallyremoved.

441

C:h ao ter 1 9: HO FI M O NES

Hvpothyroidism : This is due to an in the function of thyroid gland that -cairment :.en causesdecreasedcirculatory levels of T3 a-.c T.1.Disordersof pituitary or hypothalamus : so contribute to hypothyroidism.Women are -€ie susceptiblethan men. Hypothyroidismis :-aracterized by reduced BMR, slow heart rate, . r eiqht ga i n , s l u g g i s hb e h a v i o u r,c o n sti pati on, =^sitivity to cold, dry skin etc. H;,pothyroidismin children is associatedwith : - r s ic al a n d me n ta l re ta rd a ti o n ,c o l l ecti vel y (-o\\'n as cretinism.Early diagnosisand proper reatment are essential.Hypothyroidismin adult :auses myxoedema, characterized by bagginess -.der the eyes, puffinessof face, slowness in : ^r s ic al a n d m e n ta l a c ti v i ti e s .

Zona glomerulosa Zona fasciculata Zona reticularis Medulla

Flg. 19.12: Adrenal gland with zones (3) and medulla.

Thyroid activity and serum cholesterol

Serum cholesterol level is increased in hypothyroidism and decreasedi n hyperthyroidism. Unfortunately, cholesterolestimationwill be of no of thyroidfunction.This is Thyroid hormonaladministrationis employed value in the assessment due to the fact that serum cholesterollevel is :. treat hypothyroidism. elevated in many other disorders (diabetes, obstructivejaundice, nephrotic syndrome etc.). Laboratory diagnosis of However,cholesterolestimationmay be utilized t hy r oid fu n c ti o n for monitoringthyroid therapy. Measurementof basal metabolic rate (BMR) r.as once used to reflect thyroid activity. The o f s e ru m p ro te i n b o u n d i o d i ne (P B l ), --epresenting t im at ion the circulating thyroid hormones, The adrenalglandsare two small organs(each u,asemployed for a long time to assessthyroid -unction.The normal serum PBI concentrationis weighingabout 10 g), locatedabovethe kidneys. Eachadrenalconsistsof two distinct tissues-an 3-8 vsfiOO ml. outer cortex (with 3 zones) and inner medulla Hypothyroidismis associatedwith decreased (Fig.|9.12). P B I and h y p e rth y ro i d i s m w i th i n c re a s edP B l . As many as 50 steroid hormones (namely In recent years, more sensitiveand reliable adrenocorticosteroids),produced by adrenal :ests have been developed to assess thyroid cortex, have been identified. However, only a activity. The concentration of free T3 and T4, few of them possessbiological activity. and TSH are measured(by RIA or ELISA)and Ad renoco rticosteroids are c Iassif ied i nto th ree :'leir serum normal concentrationsare groups according to their dominant biological action. However, there is some overlap in their Freetriiodothyronine(T:) - 8o-22o n{dl functi ons. Freethyroxine (To) - 0.8-2.4 ng/dl 1 . Glucocorticoids : These are 21-carbon Totalthyroxine (T+) - 5-12 1tg/dl steroids,produced mostly l:y zona fasciculata. They affect glucose (hence the name), amino T hy r oi ds ti mu l a ti n g acid and fat metabolism in a manner that is hormone (TSH) - <10 pU/ml opposite to the action of insulin. Cortisol (also Radioactive iodine uptake (RAIU) and known as hydrocortisone)is the most important of thyroid gland are also used for glucocorticoid in humans. Corticosterone is 'canning predominantlyfound in rats. c r as nos is .

BIOCHEMISTFIY

442 2. Mineralocorticoids : These are also 21carbon containing steroids produced by zona glomerulosa.They regulatewater and electrolyte bafance. Aldosterone is the most prominent mineralocorticoid. 3. Androgensand estrogens: The innermost adrenal cortex zona reticularis produces small quantities of androgens (19-carbon) and estrogens(18-carbon).Thesehormonesaffecting sexual development and functions are mostly producedby gonads.Dehydroepiandrosteronea precursor for androgens-is synthesized in adrenal cortex. Synthesis

of adrenocorticosteroids

Cholesterol elimination of pregnenolone. precursor for hormones.

undergoes cleavage with an a 6-carbon fragment to form Pregnenolone is the common the synthesis of all steroid

Conversionof cholesterolto pregnenoloneis catalysedby cytochromePa5ssidechain cleavage enzyme. This reaction is promoted by ACTH. The enzymes-hydroxylases, dehydrogenases/ lyases associated with isomerases and m i to c h o n d ri a o r e n d o p l a s mi c reti cul um-are responsiblefor the synthesisof steroidhormones. The metabolicpathwayfor the formationof major is given in Fig.l9.13. adrenocorticosteroids functions Biochemical adrenocorticosteroids

of

1 . Glucocorticoid hormones : The impoftant gfucocorticoids are-cortisol, cortisone and corticosterone. They bring about several biochemical functions in the body. (a) Effects on carbohydrate metabolism ; Clucocorticoidspromote the synthesisof glucose(gluconeogenesis). This is brought about by increasing the substrates (p a rti c u l a rl ya m i n o a c i d s)and enhanci ng the synthesis of phosphoenolpyruvate carboxykinase,the rate limiting enzyme in gluconeogenesis. The overall influence of glucocorticoids on carbohvdratemetabolismis to increase

glucose concentration. The blood bi ol ogi cal acti ons of gl ucocort icoids general l yopposethat of i nsul i n. (b) Effects on lipid metabolism : Clucocorticoids increasethe circulating free fatty aci ds.Thi s i s causedbv tw o mechanism s. (i) Increasedbreakdownof storagetriacylgl ycerol(l i pol ysi s)i n adi poseti ssue. (ii) Reducedutilizationof plasmafree fatty acids for the synthesisof triacylglycerol s. (c) Effects on protein and nucleic acid metabolism : Clucocortiocoids exhibit both catabolic and anabolic effects on protei n and nucl ei c aci d metabolism . promote transcriPtion (RNA They synthesis) and protein biosynthesis in liver. These anabolic effects of glucocorticoids are caused bY the stimulationof specific genes. C l ucocorti coi ds (parti cul arl y at high concentration)cause catabolic effects in extrahepatictissues(e.g. muscle, adipose tissue,bone etc.).This resultsin enhanced degradationof proteins. (d) Effects on water and electrolyte metabolism : The influenceof glucocorticoids on water metabolismis mediatedthrough antidiuretic hormone (ADH). Deficiency of glucocorticoids causes increased production of ADH. ADH decreases glomerular filtration rate causing water retentionin the body. (e) Effects on the immune system : Clucocorti coi ds(parti cul arl ycorti sol ),i n high doses, suppress the host immune response.The steroid hormones act at different levels-damaging lymphocytes, anti bodY sY nt hesis, i mpai rment of suppressionof inflammatoryresponseetc. (0 Other physiological effects of glucocorticoids : Glucocorticoidsare involved in severalphysiologicalfunctions. (i) Stimulatethe fight and flight response (to face sudden emergencies) of catecholamines.

Chapter 19 : HORMONES

443

HO Pregnenolone

Dehydrogenase/ Isomerase

17a'Hydroxylase

CH; tC:O

17'Hydroxypregnenolone

cHo lC:O

progesterone

17-Hydroxyprogesterone

I

C17-c26 t-yase J

c2lHvaroxvtaseJ

cH2oH

+;

" aY^)* a-(?

c2,Hydroxyrase .,1

J\)\) Dehydroepiandrosterone

')',,.-

11-Deoxycorticosterone

Corticosterone

Fig. 19,13 : Biosynthesis of major adrenocoriicosteroids.

444

B IOC H E MIS TR Y (ii) Increasethe production of gastric HCI and pepsinogen. ( ii i ) l n h i b i t th e b o n e fo rma ti o n ,hence the subjectsare at a risk for osteoporosis.

Mechanism of action of glucocorticoids : Clucocorticoidsbind to specificreceptorson the target cells and bring about the action. These hormones mostly act at the transcriptionlevel and control the protein synthesis.

lossof weight, lossof appetite(anorexia),muscle weakness,impaired cardiac function, low blood pressure,decreasedNa+ and increasedK+ level in serum, increasedsusceptibilityto stressetc. Cushing's syndrome : Hyperfunction of adrenal cortex may be due to long term pharmacological use of steroids or tumor of adrenal cortex or tumor of pituitary. Cushing's syndrome is characterized by hyperglycemia (due to increased gluconeogenesis),fatigue, muscle wasting, edema, osteoporosis,negative nitrogen balance, hypertension,moon-faceetc.

2. Mineralocorticoid hormones : The most active and potent mineralocorticoid is aldosterone.lt promotes Na+ reabsorptionat the of adrenocortical distal convoluted tubules of kidney. Na+ Assessment retention is accompanied by corresponding functi on ex c r et i o no f K+ , H + a n d N H f i o n s . The adrenocorticalfunction can be assessed Regulation of aldosterone synthesis : The by measuringplasmacortisol (5-15 pgldl at 9.00 etc. production of aldosterone is regu.lated by AM), plasma ACTH, urinary 17-ketosteroids different mechanisms. These include reninangiotensin,potassium,sodium and ACTH. aldosterone action : Mechanism of Aldosteroneacts like other steroid hormones.lt binds with specificreceptorson the targettissue and promotestranscriptionand translation.

Adrenal medulla is an extension of sympathetic nervous system. lt produces two Metabolism of adrenocorticosteroids : The important hormones-epinephrine (formerly steroid hormones are metabolized in the liver adrenaline) and norepinephrine (formerly and excreted in urine as conjugates of noradrenaline). Both these hormones are glucuronidesor sulfates. catecholaminessince they are amine derivatives of catecholnucleus(dihydroxylatedphenyl ring). The urine contains mainly two steroidsEpinephrine is a methyl derivative ot 1 7-hydroxysteroids a nd 1Z-ketosteroids-derived is another norepi nephri ne. D opami ne from the metabolism of glucocorticoids and catecholamine, produced as an intermediate mineralocorticoids. Androgens synthesized epi nephri ne. synthesi s of duri ng the by gonads also contribute to the formation of N orepi nephri neand dopami ne are i mportant 17-ketosteroids. neurotransmittersin the brain and autonomic Urinary 17-ketosteroidsestimated in the nervous system. The structures of the three laboratory are expressed in terms of catecholaminesare given in Fi9.19.14. dehydroepiandrosterone and their normal Synthesis of catecholamines excretion is in the range of O.2-2.Om{day. The amino acid tyrosine is the precursorfor the synthesisof catecholamines.The pathway is described under tyrosine metabolism (Chapter lmoairment in 15, Fig.l5.2). Catecholaminesare produced in Addison's disease : adrenocortical function results in Addison's responseto fight, fright and flight. Theseinclude disease. This disorder is characterized bv the emergencies Iike shock, cold, fatigue, decreasedblood glucose level (hypoglycemia), emotional conditions like anger etc.

Abnormalities function

of adrenocortical

Ghapter 19 : HOFIMONES

445

Biochemical functions of catecholamines cH2-cH2-NH2

Catecholamines cause diversified biochemical effects on the body. The ultimate goal of their action is to mobilize energy resourcesand preparethe individuafs to meet emergencies (e.9. shock, cold, low blood glucoseetc.).

l,lorcplnephdne Epinephrine 1. Effects on carbohydrate Fig. 19.14 : Catecholamines (dopamine,norepinephrineand metabolism : Epinephrine and epinephrine) produced by adrenal medulla norepinephrine in general (ReferFig. 15.22for biosynthesis). increase the degradation of (glycogenolysis), glycogen synthesis of glucose (gluconeogenesis)and transferase(COMT) and monoamine oxidase (MAO), found in many tissues act on decreaseglycogenformation (glycogenesis). The overall effect of catecholaminesis to catecholamines. The metabolic products elevate blood glucose levels and make it metanephrine and vanillylmandelic acid (VMA) availablefor the brain and other tissuesto meet are excreted in urine. the emergencies. 2. Effects on lipid metabolism : Both epinep h ri n e a n d n o re p i n e p h ri n ee nhance the breakdown of triacylglycerols (lipolysis) in adipose tissue.This causesincreasein the free fatty acids in the circulationwhich are effectively utilized by the heart and muscle as fuel source.

Abnormalities catecholamine

of production

Pheochromocytomas : These are the tumors of adrenal medul l a. The di agnosi s o f pheochromocytomais possibleonly when there is an excessiveproduction of epinephrine and norepinephrine that causesseverehypertension. The metabolic effects of catecholamines are ln the individuals affectedby this disorder,the mostly related to the increase in adenylate cyclaseactivity causingelevationin cyclic AMP ratio of norepinephrine to epinephrine is levels (refercarbohydrateand lipid metabolisms increased. The measurement of urinary VMA (normal <8 mg/day) is helpful in the diagnosisof for more details). pheochromocytomas. 3. Effects on physiological functions : In general, catecholamines (most predominantly epinephrine) increase cardiac output, blood pressureand oxygen consumption.They cause smooth muscle relaxation in bronchi, gastroThe gonads (testes in males, ovaries in intestinaltract and the blood vesselssupplying females) perform closely relateddual functions. skeletal muscle. On the other hand, catecholamines stimulate smooth muscle contraction of the blood vesselssupplying skin and kidney. Plateletaggregationis inhibited by catecholamines. Metabolism

of catecholamines

Catecholaminesare rapidly inactivated and metabolized.The enzymes---catechol-O methyl-

1. Synthesizesex hormones; 2. Producegerm cells. The steroidsex hormonesare resoonsiblefor growth, development, maintenance and regulationof reproductivesystem.Sex hormones are essentiallyrequired for the developmentof germ cel l s.

446

BIOCHEMISTF|Y

The sex hormonesare categorizedinto three

Sexual differentiationand secondarv sexual characteristics.

Sroups 1. Androgens or male sex hormones which are C-19 steroids.

o

Spermatogenesis.

a

Male pattern of aggressivebehavior.

2. Estrogensor female sex hormoneswhich are C-18 steroids.Ring A of steroid nucleus is phen o l i ci n n a tu rea n d i s d e v o i d o f C -19 methyl

2. Biochemical functions : Many specific biochemicaleffectsof androgensthat ultimately influence the physiological functions stated 8roup. above are identified.Androgensare anabolic in 3. Progesteroneis a C-21 steroid produced nature. during the luteal phase of menstrualcycle and . Effects on protein metabolism : Androgens als o d u ri n g p re g n a n c y . promote RNA synthesis (transcription)and protein synthesis (translation). Androgens ANDROGENS cause positive nitrogen balance and increase The male sex hormones or androgens are the muscle mass. producedby the Leydigcells of the testesand to a minor extent by the adrenalglands in both the . Effectson carbohydrate and fat metabolisms : Androgens increase glycolysis fatty acid sexes.Ovaries also produce small amounts of synthesisand citric acid cycle. anoro8ens. . Effects on mineral metabolism : Androgens promote mineral depositionand bone growth before the closure of epiphysealcartilage. Cholesterolis the precursorfor the synthesis of androgens. lt is first converted to pregnenolonewhich then forms androstenedione ESTROGENS by two pathways--either through progesterone predominantly ovarian Estrogens are (Fig.l9.1 51. or through 1 Z-hydroxypregnenolone hormones, systhesized by the follicles and Testosteroneis produced from androstenedione. corpus luteum of ovary. These hormones are The productionof androgensis under the control responsiblefor maintenanceof menstrualcycle of LH a n d F SH . and reproductiveprocessin women. Active form of androgen : The primary product of testes is testosterone.However, the Syrathesis of estrogens active hormone in many tissues is not Estrogensynthesisoccurs from the precursor testosterone but its metabolite dihydro(Fi9.19.1fl. Estrogensare produced chofesterol testosterone (DHT). Testosterone,on reduction (formationof aromatic ring) of by aromatization by the enzyrne 5 s-reductase,forms DHT. This The ovary producesestradiol(Ez)and androgens. conversion mostly occurs in the peripheral estrone(Er) while the placentasynthesizes these tissues.Someworkers considertestosteroneas a (E3). The hormones and estriol two steroid prohormone and dihydrotestosterone, the more synthesisof estrogensis under the control of LH potent form as the hormone. and FS H . Biosynthesis

of androgens

Physiological and biochemical Physiological and biochemical functions of androgens estrogens funetEorrs of 'l . Sex-related physiological functions : The 1. Sex-related physiological functions : The androgens, primarily DHT and testosterone, estrogensare primarily concernedwith inf lu e n c e: . Growth, development and maintenance of male reproductiveorgans.

. Growth, development and maintenance of female reproductiveorgans.

shepren'1 9 : HoFIMoNES

447 Cholesterol

II

J

Pregnenolone

Progesterone )sterone

I

+

17-a,-Hydroxylydroxyprogeslerone slerone

17-d-Hydroryprogesterone

+

Dehydroepiandrosterone

I ,-

Androstenedione

5 d-Dihydrotestosterone (DHT)

Fig. 19.15 : Biosynthesisof steroid sex hormones from cholesterol (Note : Male and female sex hormones are given togethe\.

. Maintenanceof menstrualcycles. . Developmentof female sexualcharacteristics.

women have relatively more fat (about 5%) than men.

. Hypocholesterolemic effect : Estrogenslower the plasmatotal cholesterol.The LDL fraction of lipoproteins is decreasedwhile the HDL . Lipogenic effect : Estrogensincrease lipofraction is increased. This explains the genesisin adiposetissueand, for this reason, low incidenceof atherosclerosis and coronary 2. Biochemical functions : Estrogensare involved in many metabolic functions.

448

B IOC H E MIS TRY

heart diseasesin the women during reproductive age.

women after menopause,due to deficiencyof activity is low. the transhydrogenase estrogens, Thi s resul ts i n the di versi on of N A DPH towards I ipogenesis-causingobesity.

in generalpromote Anabolic effect : Estrogens transcriptionand translation.The synthesisof m a n y p ro te i n s i n l i v e r i s e l evated e.B . P R OGE S TE R ON E t r a n s fe rri nc, e ru l o p l a s m i n . Progesteroneis synthesizedand secretedby E f f e c t o n b o n e g ro w th : E s trogensl i ke corpus luteum and placenta. Progesterone,as androgens promote calcification and bone such, is an intermediate in the formation gr o w th . l t i s b e l i e v e d th a t d e cal ci fi cati on of steroid hormones from cholesterol (See of bone in the postmenopausal women Fig.20.l3). LH controls the production of leading to osteoporosisis due to lack of progesterone. estrogens. fJi**i:c-:;vr*cai C{rrrs:tnons off Effect on transhydrogenase : Transhydrof {:}n + *-.$ t*'"-;t &+ genaseis an enzyme activatedby estrogen.lt h"r tl is c a p a b l eo f tra n s fe rri n re g d u c i ngequi val ents is essentiallyrequiredfor the 1. Progesterone from NADPH to NAD+. The NADH so formed i mpl antati onof ferti l i zedovum and mai ntena nce c an b e o x i d i z e d . l t i s e x o l a i n e dthat i n the of pregnancy.

BIOMEDICAL/ CLINICAL CONCEPTS

Ef |rg

Growth hormone deficiency cousesdwort'ism while its excessiueproduction results in gigantism (in children) or acromegaly (in adults). Identification of hCG in urine is emploged for the early detection of pregnancy. Cushingt syndrome is due to ouerproduction of ACTH that results in the increosed synfhesis of adrenocorticosteroids. The symptoms of this syndrome include hypertension, edema and negative nitrogen balance. Endorphins ond enkephalinsare the natural poin-killersin the broin. It is belieuedthat the pain relief through acupunctureand placebosis mediated through thesecompounds. Deficiencq of ADH cousesdiabetes insipidus, a disorder charscterizedby excretion of large uolumes of dilute urine (polyuria). Thyroid hormones directly int'IuenceNo* - K* ATP pump which consumesa major share of cellular ATP. Obesity in some indiuiduals is attributed to decreased energy utilization (heat production) due to diminished No* - K+ ATPase actiuity. Catecholaminesare produced in responseto t'ight, fright ond flight. The ultimate goal ot' catecholaminet'unctionis to mobilize energy resourcesond prepore the indiuidual to meet emergenciessuch os shock, cold, fotigue, anger etc. Pheochromocytomasare the tumors oJ adrenal medulla, characterized bg excessiue production ot' epinephrine and norepinephrine, associatedwith seuere hypertension. Sex hormones are primarily responsiblet'or growth, deuelopment, maintenance and regulotion of reproductiuesystem. The low incidence of atherosclerosisond coronary heart diseasein the women during reproductiueage is due to estrogens.

Ghapter 19 : HORMONES

449 2. Luteal phase : After the ovulation occurs, the ruptured follicles form corpus luteum and start producing progesteronednd estradiol.The predominanthormone of luteal phase is progesterone which prepares the endometrium of uterus for implantation of the fertilized ovum. LH maintainsthe corpus for a few days. ln the absence of implantation, the corpus luteum regresses and sheds endometrium causing menstruation.And another new cycle begins. The luteal phaseis always fixed, with "14+ 2

c) o o o

E o I

1

days in length. The observedvariations in the length of menstrualcycle are due to changesin the follicular phase. In case of implantationof the ferti l i zed ovum, human chori oni c Fig. 19.16 : Hormonal pattern in women during gonadotropin(hCC) is produced by the cells of mestrual cycle (FSH-Follicle stimulating hormone; implanted early embryo. hCC stimulatescorpus LH-Lutein izing hormone). luteum to synthesize progesterone. This continues till the plancenta starts making high 2. lt promotesthe growth of glandulartissue quantitiesof progesterone. in uterus and mammary gland. Menopause 3. Progesteroneincreasesthe body temperaThe menstrual cycl es w hi ch begi n i n the ture by 0.5-1 .5 Fo.The exact mechanismof this women after puberty,continuetill the age of 45thermogenic effect is not clearly known. The years. 5O The cycles ceasearound this age which measurementof temperaturewas used as an coincides with the loss of ovarianfunction. The indicator for ovulation. progesteroneand estrogenlevelsare very low in thesewomen. However,the concentrationof LH and FSH are elevated due to lack of feedback inhibition by estrogens. The occurrenceof menstrualcycle is a good Post-menopausalwomen are susceptibleto example of coordination among the hormonal two complications associatedwith insufficient functions. In humans, the menstrual cycle is levels of sex hormones. under the control of FSH, LH, estrogensand 1. Atrophy of secondarysex tissues: Mainly progesterone. The cycle normallyvariesbetween the epithelialtissueof vagina and lower urinary 25 and 35 days in length, with a mean of 28 tract. days. The menstrualcycle can be divided into 2. Osteoporosis: Decreaseddensity of bones two phases-follicular phase and luteal phase and increasedsusceptibilityto fractures. (Fig.t9.t6). 048 Menstruation

12 14 16 20 24 -----+Days J Ovulation

1. F ollic ula r p h a s e : F o l l i c u l a r s ti m u l a ti ng hormone (FSH) causes the development and maturation of ovarian follicles. As the follicle enlarges,estradiolprogessivelyrisesand reaches its peak value 24 hours before LH and FSHattain their respectivemaximum levels. LH surge or peak initiatesovulation-release of ovum from the rupturedfollicles.The levelsof progesteroneare l ow dur ing f olli c u l a r p h a s e

The digestion and absorption of nutrients (Chapter 8) is a complicated processwhich is regulatedby the autonomicnervoussystem.This occurs in associationwith peptide hormonesof gastrointestinal tract (ClT).

450

B IOC H E MIS TR Y

CIT hormones show certain structural T he s p e c i a l i z e d c e l l s l i n i n g th e C IT are responsiblefor the productionof GIT hormones. relations and may be considered under two Hence CIT may be considered as the largest fami l i es. mass of cells that secrete hormones. A large (i) Gastrin family : Some of the C-terminal number of CIT hormones have been identified. ami no aci ds are i denti cal . Thi s familv However, only four GIT hormones have been i ncl udesgastri nand C C K . well characterised. (ii) Secretin family : Secretin, CIP and 1. G a s tri n: T h i s h o rmo n ec o n ta i ns17 ami no glucagon are structurally related, hence acids and is produced by gastric mucosa. lt may be consideredunder this family. stimulates the secretion of gastric HCI and Besides the hormones described above, pepsinogen(proenzymeof pepsin).The release severalother hormones(in hundreds!)from the of gastrin is stimulated by vagus nerve of have been identified.These hormones are stomachand partiallydigestedproteins.HCI and CIT known as candidate hormones, since their c er t ai no th e r h o rmo n e si n h i b i t g a s tri nrel ease. often biological functions are yet to be precisely 2. Secretin: lt is a 27-aminoacid containing identified. The candidate hormones include polypeptide and resemblesglucagon in many vasoactive intestinal peptide (VIP), motilin, ways. Secretinis synthesizedby the mucosa of enteroglucagon, substance P, neurotensin, the upper small intestine. lt is released in somatostatin and enkephalins. responseto the presenceof HCI in chyme in the of action duodenumr,r,hichis passedon from the stomach. Mechanism Secretinstimulatespancreaticcells to produce of GIT hormones bicarbonate(HCOI) in order to neutralizeHCl. Many of the CIT hormoneshave receptorsites 3. Cholecystokinin (CCK) : lt contains 33 amino acids and is produced by the upper part of small intestine. The secretion of CCK is stimulatedby the products of protein and lipid digestion,namely peptides,amino acids, monoor diacylglycerols,fatty acids and glycerol. Cholecystokininstimulatesthe contractionof gall bladder and increasesthe flow of bile into duodenum. lt also promotes the secretion of digestive enzymes and HCOj from pancreas4. Gastric inhibitory peptide (GlP) : lt . c ont ain s 4 3 a mi n o a c i d s a n d i s p roduced by duodenal mucosa. The release of CIP is stimulatedby the presenceof glucosein the gut. The most important function of CIP is to stimulatethe releaseof insulin from pancreas. This is evident from the fact that the plasma insulin level is elevatedmuch beforethe increase in blood glucose. CIP also inhibits gastric HCI secretion,gastricmotility and its emptying.

specific for their action. At least two distinct mechanismshave been identifiedthrough which these hormonesact. 1. Productionof cAMP throughthe activation of adenylatecyclasee.B. secretin,VIP etc. 2. S ti mul ati onof i ntracel l ul arC a2+ usua lly metabolism of mediated through the phosphati dyl i nosi tol e.g. gastri n, C C K . Both these mechanismsultimately influence the enzyme secretions/otherbiological effects. Other

hormones

Besidesthe hormonesdiscussedabove, there are a few other important hormoneswhich are not referred to in this chapter. Insulin and glucagon are describedunder diabetesmellitus (Chapter 36) while parathyroid hormone and calcitonin are discussed under calcium metabolism (Chapter 18) These hormones are not given here to avoid repetition.

Gha pte r {9:

HO RM O NES

457

1. Hormones are the organic substances,produced in minute quantitiesby specit'icfissues (endocrineglonds)and secretedinto the blood stream to control the biological actiuities in the target cells. They may be regardedas the chemiccl massengersinuoluedin the regulation and coordination oJ body t'unctions. 2' Hormones are classit'iedbased on their chemical nature or mechanism ot' action. chemically, they may be proteins or peptides (insulin, oxgtocin), sferoids (glucocorticoids,sex hormones)and amino acid deriuatiues(epinephrine, thyroxine). By uirtue of the t'unction, group I hormonesbind to the intracellular receptors(estrageni, calcitriol), while group II hormones (ACTH, LH) bind to the cell surt'acereceptori and act through the second messengers. 3. Cyclic AMP (cAMP) is an intracellular second messengerfor a majority o! patypeptide hormones. Membrane bound adenglate cyclase enzyme, through the meiiatiin'ot' G proteins, is responsiblefor the synfhesisof cAMP cAMP acts through protein lcinoses that phosphorglate specit'icproteins which, in turn, cause the ultimate biochemicsl response.Phosphatidylinositol/calciumsystemalso functionsas a secondmessenger for certain hormones (TRH, gastrin). 4. Hypothalamus is the master coordinator of hormonal action as it liberates certain releasing t'actors or hormones (TRH, CRI1, GRH, GRIH) that stimulate or inhibit the corresponding trophic hormones t'rom the anterior pituitary. 5. Anterior pituitorg gland is the master endocrine organ that producesseueral hormones which influenceeither directly or indirectly(through the mediationol other endocrine organs)a uariety ol biochemical processesin the body. For instance, growth hormone is directlg inuolued in growth promoting process while TSH, FSH and ACTH, respectiuelyinfluence thyroid gland, gonadsand adrenol cortex to synthesizehormones. 6. Thyroid glond produces two principal hormones-thyroxine (Tq) and triiodothyronine (T3)-whrch are primarily concernedwith the regulation of the metabolic actiuityot' the body. Goiter is a disorder causedby enlargement of thgroid gland.and is matnly due to iodine deficiencyin the diet. 7. Adrenal cortex synthesizesglucocorticoids(e.g. cortiso!) that inlluence glucose,amino acid and fat metabolism, and mineralocorticoids(e.g. aldosterone) that regulate water and electrolyte balance. Androgens and estrogens(sex hormones)in small quantities are also sgnthesizedby the adrensl cortex. 8. Adrenal medulla produces two importont hormones----epinephrine and,norepinephrine (catecholamines). They inJluencediuersifiedbiochemical functionswith an ultimate goal to mobilize energy resourcesand prepare the indiuidual to meet emergencies(shock, anger,fatigue etc.) 9

The steroid sex hormones, primarily androgensin malesand estrogensin females,are respectiuelysynthesizedby the testesand ouaries. These hormonesare responsible for growth, deuelopment,mointenanceand regulationot' reproductiuesgstemin either sex.

70. Seuerol gastrointestinal hormones (e.g. gastrin, secretin) haue been tdentified thot are closely inuolued in the regulation of digestion and absorption of t'oodstufJs.

452

BIOCHEMISTF|Y

I. [ssay questions in hormonalaction. 1. Describethe role of secondmessengers 2. Write an accountof the anteriorpituitaryhormones. 3. Discussin detail the synthesisand biochemicalfunctionsof thyroid hormones. 4. Describethe hormonesof adrenalcortex with special referenceto glucocorticoids. 5. Write brieflyon the synthesisand biochemicalfunctionsof sex hormones. IL Short notes (a) 'C'-Proteins, (b) Inositol triphosphate,(c) Hypothalamic hormones, (d) ACTH, (e) Coiter, (fl Epinephrine, (g) Cortisol,(h) Gastrin,(i) ADH, U) Aldosterone. III. Fill in the blanks 1. The enzymethat catalyses the formationof cAMP from ATP is 2. The inorganicion that can act as a secondmessenger for certainhormonesis 3. The endocrineorgan responsiblefor the synthesisof trophic hormonesis 4. The compounds that produce opiate-like effects on the central neryous system 5. The enzyme that convertsiodide (l-) to active iodine (l+) 6. The most predominantmineralocorticoid by adrenalcortex synthesized 7. The major urinary excretoryproduct of catecholamines B. The male sex hormone, testosterone,is converted to a more active form, namely 9. The precursorfor the synthesisof steroid hormones 10. The gastrointestinalhormone that increasesthe flow of bile from the gall bladder IV. Multiple choice questions 11. lmpairmentin the synthesis factorfor the disorder of dopamineby the brain is a majorcausative (a) Parkinson'sdisease(b) Addison'sdisease(c) Cushing'ssyndrome(d) Coiter. 12. One of the following hormonesis an amino acid derivative (a) Epinephrine (b) Norepinephrine (c) Thyroxine(d) All of them. 13. The most activemineralocorticoid hormoners (d) Corticosterone. (a) Cortisol (b) Aldosterone(c) 't1-Deoxycorticosterone 14. Name the hormone,predominantlyproduced in responseto fight, fright and flight (a) Thyroxine(b) Aldosterone(c) Epinephrine (d) ADH. 15. The hormoneessentiallyrequiredfor the implantationof fertilizedovum and maintenanceof pregnancy (a) Progesterone (b) Estrogen(c) Cortisol (d) Prolactin.

&g* F-unctionTssts

The liaet

speahs:

"MasterorganI nrn,for the bofu'sntetabolh?n! Darnageto rny cellscausesmalfunction; Raisingserumbilintbin and certain enzlmesmarhedly, Tbsted in lab.for myfunctionalmeasarmtent,l'

perform its f ach organ of the body has to L biochemicalfunctionsto keep the body, as a whole, in a healthy state.This is possibleonly when the cells of the organ are intact in structure and f unc t io n . An y a b n o rma l i ty i n th e ti ssue, causedby exogenousor endogenousfactors,will s er ious lyim p a i r th e o rg a n fu n c ti o n w h i ch, i n turn, influencesthe health of the organism. B as ed on th e fu n c ti o n a l c a p a b i l i ti e so f the organs, specific biochemical investigations have been developed in the laboratory, to assesstheir f unc t ion. I n th i s c h a p te r, th e b i o c h e mi cal investigationsto assessthe functioning of liver, kidney,stomachand pancreasare discussed. The teststo evaluatethe function of endocrineorgans are discussedelsewhere(Chapter l9).

Liver performsseveraldiversifiedfunctions.lt is the central organ of body's metabolism.

F*rnctions

nf liver

1 . Metabolic functions : Liver actively participates in carbohydrate, lipid, protein, mi neral and vi tami n metabol i sms. 2. Excretory functions : Bile pigments,bile saltsand cholesterolare excretedin the bile into intestine. 3. Protective functions and detoxification : Kupffer cells of liver perform phagocytosisto el i mi nate forei gn compounds. A mmoni a i s detoxified to urea. Liver is responsiblefor the metabolismof xenobiotics(detoxification). 4. Hematological functions : Liver participates in the formation of blood (particularlyin the embryo),synthesisof plasma proteins (including blood clotting factors) and destructionof erythrocytes. 5. Storagefunctions : Clycogen, vitaminsA, D and 812 and trace element iron are stored in l i ver.

4s3

BIOCHEMISTFIY

454 The above list is an oversimplificationand inconclusivewith regardto the role of liver in the body.

Tests to assess liver function The liver function tests (LFT) are the to assessthe capacity biochemicalinvestigations of the liver to carry out any of the functions it performs. LFT will help to detect the abnormalitiesand the extent of liver damage. Two importantfacts should be borne in mind while c a rry i n go u t L F T . 1. Liver is a large-size factory of safety. Therefore, it can perform many of its functions almost normally, despitethe damage.

li

2. Selectionof the right test is important in LFT. This is due to the fact that since liver participatesin severalfunctions,the function that is measuredin LFT may not be the one that is adversely affected. ./ The major liver function tests may be classifiedas follows 1. Tests based on excretory functionMeasurement of bile pigments, bile salts, bromosulphthalein.

Hepatic function

Hemecatabolism Enzymes

Protein synthesis catabolism Protein

Common plana,/serum marker(s) for impaired function

tBitirubin fAlaninetransaminase transaminase tAsoartate tlGlutamyltranspeptidase JAlbumin time lProthrombin

tUrea

tAmmonia TCholesterol tTriglycerides tHalf-lives of drugs Drugmetabolism Bileacidmetabolism tBileacids

Lipidmetabolism

functionsare listed in Table 20.1. The most importantmarkersnamely,bilirubin,enzymes, time anddrugmetabolism albumin,prothrombin to jaundiceandotherliver with specialreference diseases are described. BILIRUBIN

2. Tests based on serum enzymes derived andis theexcretory Bilirubinis a bile pigment, from liver-Determination of transaminases, product lt is conjugated of hemedegradation. end y-glutamyl5'-nucleotidase, alkaline phosphatase, and in the liverto form bilirubindiglucuronide, transpeptidase. bilirubin 3. Tests based on metabolic capacityCalactosetolerance,antipyrineclearance. 4. Tests based on synthetic functionsProthrombintime, serum albumin.

excreted in bile. The details of (Chapterl0). elsewhere metabolism arediscussed Serum bilirubin

of serumbilirubin The normalconcentration Of this, the is in the range of l!!.OAg/dL 5. Tests based on detoxification-Hippuric conjugated bilirubfi=--TffiJ_ucuronide75'/"; acid synthesis. 25%)is about0.2-0.4m{dl, monoglucuronide This above list, although inconclusive, while the unconjugated bilirubin is 0.2-0.6 contains the most important biochemical mgldl. investigationsto assessLFT. Among these, the commonly used tests are described in the following pages.

Ecterus index

This is a simple test to measurethe yellow colow of serum due to bilirubin. This test is hfiarkers of Etver Sunction rathercrudeand almostoutdated.However,it is of neonatal The important liver functions and the often usefulfor a rapid assessment markersfor the impaired jaurtdice. common plasma,/serum

Chapter 2O: ORGANFUNCTION TESTS van den Bergh

reaction

:

This is a specific reaction to identify the increasein serum bilirubin (abovethe reference level). Normal serum gives a negativevan den Bergh reaction. Mechanism of the reaction : van den Bergh reagent is a mixture of equal volumes of s ulf anilicac i d (i n d i l u te H C I) a n d s o d i u m n i tri te. The principle of the reaction is that diazotised sulfanilicacid (in the above mixture)reactswith bilirub,into form a purple coloured azobilirubin. Direct and indirect reactions : Bilirubin as such is insoluble in water while the conjugated bilirubin is soluble.van den Berghreagentreacts with conjugated hilirubin and gives a purple c olour im m e d i a te l y (n o rm a l l y w i th i n 30 seconds).This is referred to as a direct positive van den Bergh reaction. Addition of methanol (or alcohol) dissolvesthe unconjugated biliruhin which then gives the van den Bergh reaction ( nor m allywith i n 3 0 mi n u te s )p o s i ti v ea n d thi s i s referred to as indirect positive. lf the serum contains both unconjugated and conjugated bilir ubinin h i g h c o n c e n tra ti o nth , e p u rp l ec ol our is produced immediately(direct positive)which is further intensifiedby the addition of alcohol (indirectpositive).This type of reactionis known as biphasic. van den Bergh reaction and jaundice : This r eac t ion is h i g h l y u s e fu l i n u n d e rs ta n d ingthe natureof jaundice.This is due to the fact that the type of jaundice is characterizedby increased serum concentrationof unconjugatedbilirubin (hemolytic),conjugatedbilirubin (obstructive)or both of them (hepatic). Therefore, the response of van den Bergh reaction can differentiate the jaundice as follows Indirect positive -

Hemolytic jaundice

Direct positive -

Obstructivejaundice

B iphas ic

H e p a ti cj a u n d i c e .

Bilirubin

-

in urine

455 B i l i rubi n i n uri ne can be detectedbv Fouchet' s test or Cmelin's test.

Bromosulphthalein (BSP| test Bromosulphthaleinis a dye used fo assessthe excretory function of liver. lt is a non-toxic compound and almost exclusivelyexcreted by the liver (through bile). BSP is administered intravenously (5 mg/kg body weight) and its serum concentrationis measuredat 45 min and at 2 hrs. In normal individuals,lessthan 5.h of the dye is retained at the end of 45 min. Anv impairmentin liver function causesan increased retention of the dye. This test is quite sensitive to assess liver abnormality with particular reference to excretory function. SERUM

EIIIZYilES

DERIVED FROM LIVER Liver cells contain several enzvmes which may be released into the circulation in liver damage. Measurementof selected enzymes in serum is often used to assessthe liver function. It must, however,be noted that there is no single enzyme that is absolutelyspecificto liver alone. Despite this fact, serum enzymes provide valuable information for LFT. Some of these enzymesare discussedhereunder.

Transaminases or aminotransferases The activitiesof two enzymes-namely serum glutamatepyruvatetransaminase (SGPT;recently called as alanine transaminase--AlT) and serum glutamate oxaloacetate transaminase (SGOT; recentfy k nown as aspartate t ra nsami n ase- AST) -are widely used to assessthe liver function. ALT is a cytoplasmicenzymewhile AST is found in both cytoplasmand mitochondria.The activity of these enzymes is low in normal serum (ALT 5-aO lU/l; AST 5-45 lUll). Serum Alf and AST are increasedin liver damage.However,alanine transaminaseis more sensitiveand reliable for the assessment of LFT.

The conjugatedbilirubin, being water soluble, Estimation of serum transaminasescannot is excreted in urine. This is in contrast to identify the causes(etiology)of hepaticdamage. unconjugated bilirubin which is not excreted. Further,they do not have much prognosticvalue.

456 A lk al i n e

B IOC H E MIS TRY

Other

p h o s p h a ta s e

Alkaline phosphatase(ALP)is mainly derived f r om b o n e a n d l i v e r (th e c e l l s l i n i ng the bi l e c ana l i c u l i ).A ri s e i n s e ru mAL P (n o rmal3-13 K A units/dl),usuallyassociatedwith elevatedserum bilir u b i n i s a n i n d i c a to r o f b i l i a r y obstructi on (obstructive/posthepatic jaundice). ALP is also elevatedin cirrhosisof liver and hepatictumors.

enzymes

isocitrate dehydrogenase and Serum (LDH+and isoenzymesof lactatedehydrogenase LDH5) are also useful in LFT. H nzyme combi nati ons

Very often, a combination of serum enzyme estimations(insteadof a singleone) is usedfor a Liver is not the sole source of alkaline better understanding of liver functions. For phosphatase.Therefore,its measurementhas to instance, a large increase in transaminases be carefully viewed (along with others) before (particularlyALT) relativeto a small increasein ar r iv i n ga t a n y c o n c l u s i o n .T h e l i ver and bone al kal i ne phosphatasei ndi cates hepatocellular isoenzymes of ALP can be separated by damage.On the other hand, a small increasein transaminasesand a large increaseof alkaline electrophoresis. phosphataseshows biliary obstruction. tra n s p e p ti d a s e G lu ta my l i

i

I ,l'

'll

T h i s i s a m i c ro s o ma l e n zyme w i del y dis t r i b u te d i n b o d y ti s s u e s , i n cl udi ng l i ver. (CCT) Measurementof y-glutamyltranspeptidase activity providesa sensitiveindex to assesliver abnormality.The activity of this enzyme almost par a l l e l s th a t o f tra n s a mi n a s esi n hepati c damage.Serum CCT is highly elevated(normal 5-4O lU/D in biliary obstruction and alcoholism. F ur t h e r,s e v e ra ld ru g s (e .g . p h e n ytoi n) i nduce (liver synthesis)and increase this enzyme in c ir c u l a ti o n .

Jaundice(French : jaune-yellow) is characterized by yellow coloration of sclera (of eyes) and skin. This is due to the elevated serum bilirubin level, usually beyond 2 mg/dl (normal < 1 mg/dl).

The metabolismof heme to produce bilirubin and its conjugated derivatives and the types of jaundice have already been described. The reader must refer this (Chapter 10) now. The 5' - Nu c l e o ti d a s e and the relatedparameters biochemical,changes diagnosis of the three types of for the differential The serum activity of S'-nucleotidase(normal jaundice (hemolytic,obstructiveand hepatic)are 2-15 U/l) is elevatedin hepatobiliarydiseaseand given in Table 20.2. this parallelsALP. The advantagewith 5'-nucleotidase is that it is not altered in bone diseae(as ln the Fig.20.l, the normal and abnormal is the case with ALP). bi l i rubi n metabol i sm(al ongw i th the assoc iat ed

Parameter

Hemolytic jaundice (preheptic jaundice)

Serum bilirubin

t Unconjugated bilirubin

positive vandenBerghreaction Indirect

Obstructive jaundice (posthepatic iaundice)

Hepatic jaundice (l ntrahepatic j aund ice)

1 bilirubin Conjugated

BothT

Dkectoositive

Biphasic T ALTandASTtT, ALPmarginal

Serum enzymes

ALT,ASTandALP-+

t ALPtt, ALTandASTmarginal

Bilirubin in urine

Notexcreted

Excreted +0 r J

Ghapter2O: ORGANFUNCTION TESTS

(A)

457 (B)

ErythrocytesO

C

I I + Blood

Liver

Unconjugated bilirubin (complexed withalbumin)

tConjugated bilirubinT

Cdnjugatedbilirubin

Conjugatedbilirubin (withglucuronate)

Kidney

Fi9.20.1 : Normal and abnormal bilirubin metabolism (A) Normal bilirubin metabolism (B) Alterationsin bitirubin metabolism along with enzymes in three types of jaundice (Note : Colours indicate major changes; Red--changesin hemolytic iaundice; Green-ohanges in hepatic jaundice; Blue-changes in'obstructive jaundice; Dotted tinesindicate minot pathways;ALT-Alanine transaminase;AST-Aspaftate transaminase;ALP-Atkaline phosphatase).

enzyme changes) are depicted. The major Fig,2l.2. lt may be noted that the transaminase changes in the 3 types of jaundice are listed activities(more predominantlyALT) are elevated below much before the bilirubin startsincreasing. Hemolytic jaundice : Elevated serum 'tolerance unc onjugat edb i l i ru b i n , a n d i n c re a s e du ri n ary Galactose excretionof urobilinogen. Calactoseis a monosaccharide, almostexclusively metabolized by the liver. The liver jaundice Obstructive : Elevated serum conjugatedbilirubin and increasedactivitiesof function can be assessedby measuring the e P ),a l a n i n etra n s a m i nase utilization of galactose. This is referred to alk alinephos p h a ta s(AL galactosetolerance test. The subject is given (ALT)and aspartatetransaminase(AST). intravenousadministrationof galactose(about Hepatic jaundice : Elevated serum 300 mg&g body weight). Blood is drawn at 10 unconjugated and conjugated bilirubin, and minute intervals for the next 2 hours and increasedactivitiesof ALT and AST. galactoseestimated.In the normal individuals, The pattern of rise in the serum alanine the half-life of galactoseis about 10-15 minutes. transaminase, aspartate transaminase' and This is markedly elevated in hepatocellular bilir ubin in ac u te v i ra l h e p a ti ti si s d e p i c te d i n damage (infectivehepatitis,cirrhosis).

458

BIOCHEMISTFIY prothrombintime which is prolonged in patients with liver damage, compared to normal. The half-livesof clotting factors are relativelyshort (5-72 hrs.), therefore, changes in prothrombin time occur quickly. Hence, this test is useful to assessacute as well as chronic liver damages; besi desi ts hel p i n the prognosi s.

o (E o

E N

IJJ

+

II II II I

345

10

15

20

Vitamin K is required for the synthesisof blood clottingfactorsIl, Vll, lX and X. Therefore, vitamin K deficiency can also cause prolonged prothrombin time which must be ruled out, before draw i ng concl usi ons on the l i ver functi ons. Thi s i s done by measuring prothrombintime beforeand afteradministration of vitamin K.

Weeks Ftg.20.2: Pattemof ise in serumenrymesand bilirubin in viral hepatitis.

Serum albumin

I

il

Albumin is solely synthesizedby the liver. lt has a half-life of about 20-25 days, therefore, it is a good marker to assesschronic (and not acute) liver damage. Low serum albumin is commonly observedin patientswith severeliver damage. lt must, however, be noted that the serum afbumin concentration is also decreased due to other factors such as malnutrition. Functional impairment of liver is frequently associatedwith increasedsynthesisof globulins. Cirrhosis of the liver causes a reversal of albumin/globulin ratio (A/C ratio). Serum electrophoresis of proteins reveals increased albumin and decreasedy-globulinconcentration. This, however, may not have much diagnostic importancesince severaldiseasesare associated with altered electrophoretic pattern of serum proteins.

Hippuric

acid

synthesis

The liver is the major site for the metabolism of xenobiotics (detoxification).Measurementof hippuric acid synthesis is an ideal test for assessing the detoxification function of liver. H i ppuri c aci d i s produced i n the l i ver w hen benzoi c aci d combi nesw i th gl yci ne. About 6 g of sodium benzoate(dissolvedin about 250 ml water), is orally given to the subject,aftera light breakfast(usually2 hrs later) and afteremptyingthe bladder.Urine collections are made for the next 4 hoursand the amount of estimated. hippuric acid excreted is Theoretically,6 g of sodium benzoate should yi el d 7.5 g of hi ppuri c aci d. In the heal thy persons, about 60% of sodium benzoate (equi val entto 4.5 g hi ppuri caci d) i s excretedin urine. A reduction in hippuric acid excretion (particularly <3 g) indicates hepatic damage.

Ghoice of liver functions tests

The choice of biochemical tests to measure liver functions mostly depends on the purpose of the investigation.The clinical history of the Prothrombin time subject is often a guiding factor in this regard.A si ngl e test i n i sol ati on may have a l i ttle The Iiver synthesizes allthe factorsconcerned diagnosticvalue. with blood clotting. A decrease in the concentrationof plas-maclotting factorsis found Frequently, a combination of laboratory in the impairmentof liver function. This can be investigations are employed in LFT. These assessed in the laboratory by measuring i ncl ude serum bi l i rubi n (conj ugated an d

459

Chapter 2O: OBGAN FUNCTIONTESTS unconjugated),alanine transaminase, aspartate transphosam inas e, alk a l i n e phatase, 'lglutamyl transpeptidase proteins and ( album in,glob u l i n s ).

Proximal convoluted tubule Bowman'scapsule Glomerulus Distalconvoluted tubule

The kidnevs are the vital organsof thq body, performing the following major functions.

Collectingduct

1. Maintenance of homeostasis: The kidneysare largely responsiblefor the regulation of water, electrolyte and acid-base balance in the bodv.

Loop of Henle

2. Excretion of metabolic waste products : The formation of urine The end products of protein and nucleic acid Nephron is the functionalunit of kidney. Each metabolismare eliminatedfrom the body. These kidney is composed of approximately one include urea, creatinine, creatine, uric acid, million nephrons.The structureof a nephron,as sulfateand phosphate. depicted in Fig.20.3, consists of a Bowman's 3. Retention of substancesvital to body : The capsule (with blood capillaries), proximal kidnevs reabsorband retain several substances convoluted tubule (PCT),loop of Henle, distal of biochemical importance in the body e.g. convoluted tubule (DCT) and collecting tubule. gluc os e,am ino a c i d s e tc .

The blood supply to kidneys is relatively large. About 1200 ml of blood (650 ml plasma) 4. Hormonal functions : The kidneys also passes through the kidneys,every minute. From function as endocrine organs by producing 120-125 ml is filtered per minute by this, about hormones. the kidneys and this is referred to as glomerular . Erythropoietin,a peptide hormone,stimulates filtration rafe (GFR). With a normal GFR hemoglobin synthesis and formation of (12O-125ml/min), the glomerularfiltrateformed erythrocytes. in an adult is about 175-180 litres per day, out \ of which onlv 1 .5 litres is excreted as urine. (calcitriol). 1,25-Dihydroxycholecalciferol Thus, more than 99% of the glomerularfiltrate is t he bioc hem i c a l l ya c ti v efo rm o f v i ta m i n Dreabsorbed by the kidneys. is finally produced in the kidney. lt regulates \process calcium absorptionfrom the gut. The of urine formation basically involves two steps-glomerular filtration and . Renin, a proteolytic enzyme liberated by tubular reabsorption. kidney, stimulates the formation of angio1. Clomerular filtration : This is a passive tensin ll which, in turn, leads to aldosterone production.Angiotensinll and aldosteroneare process that results in the formation of the hormones involved in the regulation of ultrafiltrate of blood. All the (unbound) constituentsof plasma,with a molecularweight electrolytebalance.

460

B IOC H E MISTRY

laborato'ryto assesskidney (renal) function. lt must, however, be rememberedthat about twothi rds of the renal ti ssue must be func t ionally damagedto show any abnormalityby thesetests. 2. Tubular reabsorption : The renal tubules The kidney fgnction tests may be divided into (PCT, DCT and collecting tubules) retain water lour Sroups. and most of the soluble constituentsof the 1. Glomerular function tests : All tne glomerular filtrate by reabsorption.This may occur either by passiveor active process.The cl earance tests (i nul i n, creati ni ne, urea) ar e excreted urine has an entirely different i ncl uded i n thi s B roup. composition compared to glomerular filtrate 2. Tubular function tests : Urine concenf r o m w h i c h i t i s d e ri v e d. The normal tration or dilution test, urine acidficationtest. composition of urine is given elsewhere(Refer 3. Analysis of blood/serum : Estimationof inside backcover). bl ood urea, serum creati ni ne, protein and electrolyte are often useful to assess renal f te n a i th re s h o l d s u b s ta n ces functi on. There are certain substancesin the blood 4. Urine examination: Simpleroutineexamiw h o s e e x c re ti o ni n u ri n e i s d e pendenton thei r nation of urine for volume, pH, specificgravity, concentration.Such substances are referredto as osmolality and presence of certain abnormal renal threshold substances. At the normal constituents (proteins, blood, ketone bodies, concentrationin the blood, they are completely gl ucoseetc.) al so hel ps,of courseto a lim it ed reabsorbedby the kidneys,with a resultthat their degree,to assesskidney functioning. ex c re ti o ni n u ri n e i s a l mo s t n e gl i gi bl e. Someof the imoortantrenal function testsare The renal threshold of a substanceis defineo di scussedi n the fol l ow i ng pages. as its concentration in blood (or plasma)beyond which it is excreted into urine. The renal CLEARANGETESTS threshold for glucose is 180 mg/dl; for ketone bodies 3 mg/dl; for calcium 10 mg/dl and for The clearancetests,measuringthe glomerular bicarbonate30 mEq/|.While calculatingthe renal filtration rate (GFR) are the most useful in thresholdof a particularcompound,it is assumed assesSing the renal function. The excretionof a that both the kidneysare optimally functioning, substancecan be expressedquantitatively by w i th o u t a n y a b n o rm a l i ty .Bu t th i s i s not al w ays using the concept of clearance. true-in which casethe renalthresholdis altereo. C l earance, i n general , i s defi ned as t he For instance, renal glycosuria is associatedwith volume of plasma that would be completely reduced threshold for glucose due to its cleared of a substance per minute. In other d i m i n i s h e dtu b u l a r re a b s o rp ti o n. words, clearance of a substance refers to the The term tuhular maximum (Im) is used to milliliters of plasma which contains the amount indicatethe maximum capacityof the kidneysto of that substance excreted by kidney per absorb a particular substance. For instance, minute. Clearance(C), expressedas ml/minute, t u b u l a r m a x i m u m fo r g l u c o se (TmC ) i s 350 can be cal cul atedby usi ng the formul a m g /m i n . UxV C-

less than about 70,000, are passed into the filtrate. Therefore, the glomerular filtrate is al m o s ts i m i l a r i n c o m p o s i ti o nto pl asma.

Te s ts to a s s e s s

re n a l functi on

In view of the important and sensitive functions the kidney performs (described already), it is essentialthat the abnormalities (renal damages),if any, must be detectedat the earliest. Several tests are employed in the

where U = Concentration of the substance rn uri ne. V = Volume of urine in ml excreted per minute. = P Concentration of the substance in olasma.

467

Chapter 2O: OBGAN FUNCTIONTESTS

Care should be taken to expressthe concentrations of plasma and urine in the same units (mmol/l or mg/dl). The clearance of a given substance is determined by its mode of excretion. The maximum rate at which the plasma can be cleared of any substanceis equal to the CFR. This can be easily calculatedby measuringthe clearanceof a plasmacompound which is freely f ilt er ed by th e g l o me ru l u s a n d i s n e i ther absorbed nor secretedin the tubule. Inulin (a plant carbohydrate,composedof fructoseunits) and s lCr - E D T A s a ti s fy th i s c ri te ri a . In u l i n i s intravenouslyadministeredto measure GFR. In practice, however, measurement of clearancefor the substancesalready present in the blood is preferred. The two compounds, namefy creatinine and urea, are commonly employed for this purpose.Creatinineclearance (-'145 ml/min) is marginallyhigherthan the CFR as it is secretedbv the tubules. On the other hand, urea clearance(-75 ml/min) is less than the CFR, since it is partially reabsorbedby the t ubules . Diodrast (diiodopyridoneacetic acid) is used as a contrastmedium to take urinarytract X-rays. Diodrast and para amino hippuric acid (PAH) are peculiar substancesas they are entirely excreted by a single passage of blood through the kidneys.lt is partly filteredby the glomerulus and mostly excretedby the tubules. PAH has a clearance of about 70O ml/min (or 1,20O ml, if expressedas blood). Thus clearance of PAH represents the renal plasma flow. Greatinine

clearance

test

Creatinine is an excretory product derived from creatine phosphate (largely present in muscle). The excretion of creatinine is rather constant and is not influenced by body metabolismor dietary factors.As alreadystated, c r eat inineis fi l te re d b y th e g l o m e ru l ia n d o nl y marginallysecretedby the tubules.The value of creatinineclearance is close to CFR, hence its measurementis a sensitiveand good approach to assess the renal glomerular function. Creatinine clearance mav be defined as the

volume (ml) of plasma that would be completely cleared of creatinine per minute. Procedure : ln the traditional method, creatinine content ol a 24 hr urine collection and the plasmaconcentrationin this period are estimated.The creatinineclearance(C) can be calculatedas follows : f=

UxV

P = where U Urine concentrationof creatinine V = U ri ne output i n ml l mi n (24 hr uri ne volume divided by 24 x 6O) P = Plasmaconcentrationof creatinine. As alreadystated,creatinineconcentrationin urine and plasma should be expressedin the same units (mgldl or mmol/l). Modified procedure : Insteadof a 24 hr urine collection, the procedure is modified to collect urine for t hr, after giving water. The volume of urine is recorded.Creatininecontentsin plasma and urine are estimated. The creatinine clearancecan be calculatedby usingthe formula referred above. Reference values : The normal range of creatinine clearance is around 120-145 ml/min. These values are slightly lower in women. In recentyears,creatinineclearanceis expressedin terms of bodv surtace area. Diagnostic importance : A decrease in creati ni ne cl earance val ue (< 75o/" normal ) servesas sensitiveindicatorof a decreasedCFR, due to renal damage.This test is useful for an early detectionof impairmentin kidney function, are seen. often beforethe clinical manifestations [!rea

clearance

test

Urea is the end product of protein metabolism.After beingfilteredby the glomeruli, it is partially reabsorbedby the renal tubules. Hence, urea clearanceis lessthan the CFR and, further, it is influencedby the protein content of the diet. For these reasons,urca clearanceis not as sensitiveas creatinine clearancefor assessing renal function. Despite this fact, several laboratoriestraditionally use this test.

462

BIOGHEMISTFIY

Urea clearanceis defined as the volume (ml) of plasma that would be completely cleared of urea per minute. It is calculatedby the formula

UxV where C. = Maximum urea clearance U = Urea concentration in (mg/ml)

uflne

V = Urine excretedoer minute in ml P = Urea concentration in olasma (mg/ml). T he a b o v e c a l c u l a ti o n i s a p p l i cabl e i f the output of urine is more than 2 ml per minute. This is referred to as maximum urea clearance and the normal value is around 75 ml/min.

I

i i

20 40 60 80 100 120 140 160 GFR(ml/min)

Standard urea clearance : lt is observed that the urea clearancedrasticallychangeswhen the v olum e o f u ri n e i s l e s s th a n 2 ml /m i n. Thi s i s known as standard urea clearance (C) and the nor m a l v a l u e i s a ro u n d 5 4 m l /mi n. l t i s calculatedby a modified formula

Osmolality and specific gravity : The osmol al i tv of uri ne i s vari abl e. In normal i ndi vi dual s, i t may range from 500-1,2 00 mi l l i osmol es/kg. The pl asmaosmol al i tyi s aroun d 3-0j milljosmoled_ks. The normal ratio of the Diagnostic importance : A urea clearance osmolality between urine and plasma is around value below 75'/' of the normal is viewed 2-4. lt is found that the urine (without any seriously, since it is an indicator of renal proteinor high molecularweight substance) with damage. Blood urea level as such is found to an osmolality of 800 mosm/kg has a specific increase only when the clearance falls below gravity of 1.020. Therefore, measurement of 50% normal. As already stated, creatinine uri ne osmol al i tyw i l l al so hel p to assesstubul a r clearanceis a better indicator of renal function. functi on.

Urine concentration test

Analysis

This is a test to assessthe renal tubular function. lt is a simple test and involves the accuratemeasurementof specific gravity which dependson the concentrationof solutesin urine. A specificgravity of 1.020 in the early morning ur ine s a m p l e i s c o n s i d e re dto b e n o r mal .

Estimation of serum creatinine and blood urea are often used to assessthe overall kidnev function, although these tests are less sensitive than the clearance tests. Serum creatinine is a better indicator than urea in this regard. The diagnostic importance of urea and creatinine estimationsare discussedelsewhere(Refer Chapter 1fl.

Several measures are employed to concentrate urine and measure the specific gravity. These include overnight water deprivation and administration of antidiuretic hormone.lf the specificgravityof urine is above 1.020 for at leastone of the samplescollected, the tubular function is consideredto be normal.

of blood

(or serumf

The relationship between CFR and serl.rm creatinine levefs is depicted in Fi9.20.4. lt is observedthat the CFR must fall to about 5O'/"ol its normal value before a significantincreasein serum creatinine occurs. Therefore. a normal

463

Chapter 2O : OBGAN FUNCTIONTESTS serumcreatininelevel does not necessarilymean that all is well with the kidney. lt is estimated that a loss of 50"h of the functions of nephrons leads. to (approximate) doubling of serum creatinineconcentration.

The routine urine examinationis undoubtedly a guiding factor for renal function. The volume of urine excreted, its pH, specific gravity, osmolality, the concentration of abnormal constituents(such as proteins, ketone bodies, glucose and blood) may help to have some preliminaryknowledgeof kidney function. More informationon urine laboratorytestsis given in the appendix. of renal function

CO2+ H2O

I ca,ooni,

U r ine ex am in a ti o n

Ghoice

Parietalcell

anhvdrase J ATP H2CO3

HCOa cr -

tests Fiq.20.5 : Mechanism of HCI secretion

of kidney function In general,the assessment (U-rcpresentsK activatedATPase). starts with the routine urine examination, followed by serum creatinine and/or blood urea estimations and, finally, the specific tests to A unique enzyme-namely K+ activated m eas ur et he t u b u l a r a n d g l o me ru l a rfu n c ti ons ATPase-present in the parietal cells is (clearancetests). connectedwith the mechanismof HCI secretion (Fig.2O.S). The processinvolvesan exchangeof H+ ions (of the parietalcells)for K+ ions (of the l umen).Thi s i s coupl edw i th the consumpti onof The stomach is a major organ of digestion energy/ suppl i ed by A TP . The H + are continuouslygeneratedin the parietal cells by and performsthe following functions the di ssoci ati onof carboni caci d w hi ch, i n turn, 1. Stomach is a reservoir of ingested is produced from CO2. The bicarbonate ions foodstuffs. (HCOj), Iiberated from the carbonic acid 2. lt has a g re a t c h u rn i n g a b i l i ty w h i ch (H2CO3) dissociation, enter the blood in promotesdigestion. exchangefor Cl- ions. The latter diffuse into the gastric lumen to form HCl. Castrin-a peptide proteases 3. Stomach elaborates HCI and (pepsin)which are responsiblefor the initiation hormoneof gastrointestinal tract-stimu latesHCI secretion. of digestiveprocess. Following a meal, there is a slight elevation 4. The products obtained in the stomach (peptides,amino acids) stimulatethe releaseof in the plasma bicarhonate concentration which is linked to the gastric HCI secretion.This is pancreaticjuice and bile. referred to as alkaline tide. of gastric HCI Secrefion

TESTS TO ASSESS

The parietal (oxyntic) cells of gastric glands GASTRIC FUITIGTION pr oduc eHCl. T h e p H i n th e g a s tri cl u m e n i s a s There are several tests for gastric function low as 0.8 (againstthe blood pH 7.Q. Therefore, the protons are transported against the evaluation, some of the important ones are bri efl v di scussed. concentrationgradient by an active process.

464

B IOC H E MIS TR Y

residual juice. The gastric juice elaboratedfor the next one hour is collectedand pooled which This is rather old and not used these days. representsthe basal secretion. Pentagastrin(5 Fractionaltest meal involves the collection of mg/kg body weigh0 is now given to stimulate stomachcontentsby Ryle's tube in fasting.This gastricsecretion.The gastricjuice is collectedat is f ol l o w e d b y a g a s tri cs ti m u l a ti o ngi , vi ng a test 'l 5 minute intervalsfor one hour. This represents meal (rice gruel, black coffee etc.) The stomach the maxi mum secreti on. contentsare aspiratedby Ryle'stube at different Eachsampleof the gastricsecretioncollected t im e p e ri o d s (u s u a l l ye v e ry 1 5 m i n for 2 hrs.) The samples are analysed for free and total is measuredfor acidity by titrating the samples acidity in the laboratory.The resultsare normally w i th N /10 N aOH to pH 7.4. The end poi nt m ay representedby a graph. be detectedby an indicator(phenolred) or a pH meter. ln this case,the test meal in the form of 100 ml of 7"h alcohol is administered.The response to alcohol test meal is more rapid, and the test t im e c a n b e re d u c e d to 1 1/z hour. C l ear specimenscan be collectedby this test, and the free acidity levelsare relativelyhighercompared to FTM.

Basal acid output (BAO) refers to the acid output (mi l l i mol per hour) under the basal condi ti onsi .e. basal secreti on. Maximal acid output (MAO) represents the aci d output (mi l l i mol per hour) after the gast r ic sti mul ati on by pentagastri n i .e. maxi mum secretion. In normal i ndi vi dual s,the B A O i s 4-10 mm ol/ hr w hi l e the MA O i s 20-50 mmol /hr.

Pentagastrinis a synthetic peptide which stimulates the gastric secretion in a manner similar to the natural gastrin.The test procedure adapted is as follows

Histamine is a powerful stimulant of gastric secretion.The basalgastricsecretionis collected The stomachcontentsare aspiratedby Ryle's for one hour. Histamine (0.04 mg/kg body tube in a fastingcondition. This is referredto as weight) is administeredsubcutaneouslyand the

BIOMEBICAL/ELINICALCONCEFTS

The impairment in the functions ol any organ in the body will aduerselyinfluence the health of the organism. Orgon function tests are the lqborotory tools to biochemically eualuotethe working of a giuen orgon. Acute uiral hepatitis is ossociofedwith eleuatedalonine tronsaminase(predominontly), asportotetransaminaseand bilirubin. Increose in serum yglutamyl transpeptidase is obserued in biliory obstruction ond alcoholism. ts A combinotion of laboratory investigations-instead of a single one---are commonly employed in ossessingorgon function. Kidney function can be accurately ossessedbg clearance tests, meosuringglomerular t'iltration rate. A reduction in clearance rellects renal domoge. w Zollinger-Ellison syndrome, o tumor of gastrin secreting cells of the pancreas, is associatedwith increased gastric HCI production.

465

Chapter 2O : ORGANFUNCTIONTESTS gastric contents are aspirated for the next one hour (at 15 minute intervals).The acid content is measuredin all these samples.

lnsulin test meal

Abnormalities

of gastric

function

Increased gastric HCI secretion is found in Zollinger-Ellisonsyndrome (a tumor of gastrin secreting cells of the pancreas), chronic duodenal ulcer, gastric cell hyperplasia, excessivehistamineproduction etc.

This is also known as Hollander's tesf. lt is mainly done to assess the completenessof A decrease in gastric HCI is observed in vagotomy (vagal resection).Insulin (0.1 uniVkg gastritis,gastriccarcinoma,perniciousanemiaetc. body weight) is administered intravenously, which causes hypoglycemia (blood glucose about 40 mg/dl), usually within 30 minutes, in normal persons. lf the vagotomy operation is successful, insulin administration does not cause any increase in the acid output, compared to the basal level. This test has to be carefully perfomed, since hypoglycemia is dangerous.

PANCREATIG

FUNCTION

TESTS

The pancreas is a specialized organ with exocrineand endocrinefunctions.The endocrine functionsare discussedunder the topic diabetes meflitus (Chapter 35).

The exocrine functions involve the synthesis of pancreaticjuice containing severalenzymes Tubeless gastric analysis (for the digestion of foodstuffs)and bicarbonate. major enzymes of pancreatic juice are The In the traditional methods of gastric analysis, trypsin, chymotrypsin, elastase, carboxya tube is invariably passedinto the stomach to peptidase, amylaseand lipase. collect the gastric juice. This causes Pancreatic enzymes in serum : Serum inconvenience to the subject. Recently, some tests involving tubeless gastric analysis amylase and lipase measuremenfs are commonly the pancreaticfunction. Both have been developed. Such tests, however, are employedto'assess these enzyme activities are elevated in acute mostly useful for preliminaryscreening. pancreatitis, obstruction in the intestine and/or The principle of tubeless gastric analysis pancreatic duct. involves administration of a cation exchange resin that gets quantitatively exchanged with the THYROID FUNCTION TESTS H+ ions of the gastric juice. The resin is then Thyroid gland produces two principal excreted into urine which can be estimated for (T+) and triiodothyronine hormones-thyroxine an indirect measure of gastric acidity regulate which the metabolic rate of the (concentrationof H+ ions). The laboratory tests employed for the body. Diagnex blue containing azure-A-resin is diagnosisof thyroid function are describedin the employed in the tubelessgastricanalysis. Chapter 19 on hormones.

466

B IOC H E MIS TR Y

$ :tr s

1. Specific laboratory biochemical inuestigotionsare emploged fo ossessthe functioning of the orgons such os liuer, kidney, stomach and pancreas.

2. The liuer t'unction con be eualuatedby the testsbasedon its excretoryfunction (serum bilirubin), serum enzymes(transaminases), metabolic capability(galoctosetolerance test) and syntheticfunctions (prothrombintime)

3. Serum bilirubin (normal < lmg/dl) is deriuedfrom heme degrodation.lt is mostly (750/o) found in the conjugatedt'orm. uan den Bergh reaction is a specilic test to identit'y the increased serum bilirubin. Conjugated bilirubin giuesa direct positiue test while the unconjugatedbilirubin giuesan indirect positiuetest.

4. The serum enzymes-namely alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatose(ALP) and y glutamyltranspeptidase(GGT)-are frequently used lor LFT. Increase in the actiuitiesof these enzymes indicates an impairment in Iiuer function.

5. Jaundice is due to eleuatedserum bilirubin leuel (>2 mg/dl). The three types of jaundice (hemolytic,obstructiueand hepotic)can be diflerentiollg diognosedb9 biochemicoltests. Thus, unconjugated bilirubin (indirect positiue) is increosed in hemolytic jaundice, conjugated bilirubin (direct positiue) in obstructiuejaundice qnd both ol them (biphasic) are increasedin hepatic jaundice.

6. lmpaired galactose tolerance test, diminished serum olbumin concentration and prolonged prothrombin time are o/so ossociof ed with liuer molfunction.

7. The renal (kidneil t'unction is usuol/y ossessedby eualuatingeither the glomerular (clearoncetests)or tubular function (urine concentration test). This is often guided by blood onalysis(for ureo, creatinine) and/or urine examination.

8. The clearanceis defined as the uolume of the plasma that u)ould be completely cleared of o substanceper minute. Inulin clearancerepresentsglomerular filtrotion rate (GFR). Creotinine clearanceand urea clearance tests are ot'ten used to ossessrenol function. A decreosein their clearance is an indication of renal damoge.

9. Impoirment in renol function is often associatedwith eleuotedconcentration of blood urea, serum creatinine, decreasein osmolality and specific grauitg of urine (by urine concentrotion tesil.

10. The tests to eualuate gastric function include troctional test meql, pentagastrin stimulation test, augmented histamine test and tubelessgostric onolysis. Gastric HCI secretion is eleuoted in chronic duodenal ulcer and gastric hyperplosia. Gastritis and pernicious anemio ore associatedwith decreased gastric HCI. Pancreatic function is ossessedby serum omylaseond lipase.Both of them are eleuatedin acute pancreatitis.

Ghapten 2O : OFIGANFUNCTIONTESTS

467

I. Essayquestions employedto assessliver function. 1. Write brieflyon the differentlaboratoryinvestigations 2. Discussthe biochemicalparameters for the differentialdiagnosisof jaundice. 3. Cive an accountof the serumenzymesderivedfrom liver and their importancein LFT. 4. Describethe renal function tests. 5. Discussthe different laboratoryinvestigationsto evaluategastricfunction. II. Short notes (a)Serumbilirubin,(b) van den Berghreaction,(c) Calactosetolerancetest,(d) Prothrombintime as LFT, (e) Renal threshold substances,(0 Clomerular filtration rate, (g) Creatinineclearance, (h) Standardurea clearance,(i) Urine concentrationtest, (j) Gastricfunction tests. III. Fill in the blanks 1. Bilirubinis the excretoryend productof 2. The laboratoryreactionmostcommonlyemployedto detectthe elevationof serumbilirubinis 3. The serumenzymemost predominantlyelevatedin viral hepatitisis by an increasein the serumenzyme 4. Obstructivejaundiceis characterized 5. The excretoryfunctionof liver can be evaluatedby usinga dye 6. The renalthresholdfor glucoseis 7. The exogenoussubstanceused to measureglomerularfiltrcationrate (GFR)is 8. Standardureaclearanceis calculatedwhen the volumeof urineoutputis lessthan 9. Name the stomachtube usedto aspirategastricjuice 10. Name the syntheticpeptideusedto stimulategastricsecretionfor evaluationof gastricfunction IV. Multiple choice questions 11. In hemolyticjaundice,van den Berghreactionis (a) Indirectpositive(b) Direct positive(c) Biphasis(d) None of these. 12. The serumenzymeelevatedin alcoholiccirrhosisof liver is (c) Alcohol dehydrogenase (a) Alaninetransaminase (b) Aspartatetransaminase (d) y-Glutamyl transpeptidase. 13. B ilir ub i ni s n o t e x c re te di n u ri n e i n (a) Obstructivejaundice(b) Hepaticjaundice(c) Hemolyticjaundice(d) All three. 14. Urea clearanceis lessthan CFR becauseit rs (a) Partiallysecretedby the renaltubules(b) Partiallyreabsorbed by the tubules(c) Only filtered by glomeruli(d) None of these. 15. The serumenzymeusedto evaluatepancreaticfunction is (a) Alkaline phosphatase(b) Amylase (c) Aspartatetransaminase(d) Lactatedehydrogenase.

Watero Electro

The seiil-bsse hsmeostssis speeks : - oK^ + loq -

IHco;1 lunn tr rwv"l

I

"We wtei.utainthe bloodpH nt 7.4! Regulatedby buffe,rs,lwtgs arzdkiineys; fit*eased./tj,rirogeaion causetacitlos,ii;

., .

h.l,rirogen ion leadsto alhal,nsis." Decrensed

organismpossesses tremendouscapacity Jhe I t o s u rv i v e a g a i n s t o d d s a n d mai ntai n homeostasis. This is particularlytrue with regard to water, electrolvteand acid-basestatusof the body. These three are interrelated,hence they are consideredtogetherfor the discussionin this chapter. Kidney actively participates in the regulation of water, electrolyte and acid-base balance. The general functions of kidney have already been described (Chapter 20).

2. Water directly participatesas a reactantin severalmetabolic reactions. 3. lt serves as a vehicle for transport of solutes. 4. Water is closely associated with the regulationof body temperature. D i stri buti on

of w ater

Water is the major body constituent.An adult human containsabout 60% water (men 55-70%, women 45-60%).The women and obese individuals have relativelylesswater which is due to the higher content of storedfat in an anhydrous Water is the solvent of life. Undoubtedly, form. water is more important than any other single A 70 kg normal man containsabout 42 litres compound to life. lt is involved in severalbody of w ater. Thi s i s di stri buted i n i ntracel l ular f unc t io n s . (i nsi dethe cel l s 281)and extracel l ul ar(outsi de the cells 141)compartments,respectivelyknown F unc t i o n s o f w a te r as intracellular fluid (lCF) and extracellular fluid 1. Water provides the aqueous medium to (ECD.The ECF is further divided into interstitial the organismwhich is essentialfor the various fl ui d (10.51)and pl asma(3.51).The di stri bution biochemical reactionsto occur. of water in man is given in Table 21.1.

468

{:iraster- 21 : WATER. ELECTHOLYTE AND ACID-BASEBALANCE

469

of water from the body-urine, skin, lungs and feces. 'Compartment

% Body weight

Volume (l)

Total

60

42

Intracellular fluid(lCF)

40

28

Extracellular fluid(ECF)

20

14

lnterstitial fluid

15

10,5

c

3,5

Plasma

Urine : This is the major route for water loss from the body. In a healthy individual,the urine output is about 'l-2 l/day. Water loss through kidneys although highly variable, is well regulated to meet the body demands-to get rid of water or to retain. lt should, however, be rememberedthat man cannot completely shut down urine production, despitethere being no water intake. This is due to the fact that some amount of water (about 500 ml/day) is essential as the medium to eliminate the waste products from the body.

Hormonal regulation of urine production : lt The body possessestremendous capacity to is indeedsurprisingto know that about 180 litres regulate its water content. In a healthy of water is filteredby the glomeruli into the renal indiv idual, t h i s i s a c h i e v e d b y b a l a n c i ng the tubules everyday. However, most of this is reabsorbedand only 1-2 litres is excreted as daily water intake and water output. urine. Water excretion by the kidney is tightly controlled by vasopressin also known as bllater intake antidiuretic hormone (ADH) of the posterior Water is supplied to the body by exogenous pituitary gland. The secretion of ADH is and endogenous sources. regulatedby the osmotic pressureof plasma.An Exogenous water : Ingested water and increasein osmolalitv promotesADH secretion beverages, water content of solid foods- that leads.to an increasedwater reabsorption constitute the exogenoussource of water. Water from the renal tubules(lessurine output).On the intake is highly variable which may range from other hand, a decreasein osmolality suppresses 0.5-5 litres. lt largely depends on the social ADH secretion that results in reduced water habits and climate. In general,people living in reabsorptionfrom the renal tubules (more urine hot climate drink more water. Ingestionof water output). Plasmaosmolality is largely dependent is mainly controlled by a thirst centre located in on the sodium concentration, hence sodium the hypothalamus.lncrease in the osmolality of indirectly controls the amount of water in the plasma causes increased water intake by bodv. stimulatingthirst centre. Diabetes insipidusis a disorder characterized the deficiency of ADH which results in an by Endogenous water : The metabolic water produced within the body is the endogenous increased loss of water lrom the body. water. This water (300-350 ml/day) is derived Skin : Loss of water (450 ml/day) occurs from the oxidation of foodstuffs. lt is estimated through the body surfaceby perspiration.This is that 1 g each of carbohydrate, protein and fat, an unregulated process by the body which respectively,yield 0.5 ml, 0.4 ml and 1.1 ml of mostly depends on the atmospherictemperature water. On an average/about 125 ml of water is and humidity. The loss is more in hot generatedfor 1,000 Cal consumedby the body. climate. Fever causes increased water loss through the skin. lt is estimatedthat for every W at er out p u t 1oC rise in body temperature, about 15"/" Water losses from the body are variable. increase is observed in the loss of water (through There are four distinct routesfor the elimination ski n).

470 Drinking water andbeverages Foodstuffs (1,500ml) (700 ml)

BIOCHEMISTF|Y Metabolic water

Water intake (2,s00ml)

Urine Lungs Feces Skin (1,500ml) (450ml) (400ml) (150mD

Water output (2,500ml)

instance, NaCl does not exist as such, but it exists as cation (Na+) and anion (Cl-). The concentration of electrolytes are expressedas milliequivalents (mEdl) rather than mi | | igrams. A gram equivalent weight of a compound is defined as its weight in gramsthat can combine or displace 1 g of hydrogen. One gram equivalent weight is equivalent to 1,000 mi l l i equi val ents. The following formula is employedto convert the concentration mgll to mEq/|.

Fig.21.1 : Water balanee in the body, representedby

mEdl = Lungs : During respiration/some amount of water (about 400 ml/day) is lost through the expiredair. The latteris saturatedwith water and expelled from the body. In hot climates and/or when the person is suffering from fever, the water loss through lungs is increased.

I

I

mg per litre x Valency Atomic weight

cortlpos;tion Electrolyte ol body fluids

Electrolytesare well distributed in the body fluids in order to maintain the osmotic equi l i bri umand w ater bal ance.A compari sonof electrolytespresentin extracellular(plasma)and The loss of water by perspiration(via skin) i ntracel l ul ar (muscl e) fl ui ds i s gi ven in and respiration(via lungs)is collectivelyreferred Table 2l .2. The total concentration of cations and anions in each body compartment(ECFor to as insensible water loss. ICF) is equal to maintain electrical neutrality. Feces : Most of the water entering the There is a marked difference in the concengastrointestinal tract is reabsorbed by the intestine.About 150 ml/day is lost through feces tration of electrolytes (cations and anions) in a healthy individual. Fecal loss of water is betweenthe extracellularand intracellularfluids. Na+ is the principal extracellular cation while tremendouslyincreasedin diarrhea. K+ is the intracellular cation. This difference in A summaryof the water intake and output in the concentrationis essentialfor the cell survival the body is depicted in Fig.2l.l,lt may be noted which is maintained by Na+ - K+ pump (for that water balance of the body is regulated details, Refer Chapter 33). As regards anions, predominantlyby controlling the urine output. Cl- and HCOI predominantly occur in This happensafter an obligatory water loss via extracel l ul arfl ui ds, w hi l e H P O;, protei nsand skin, lungs and feces. organicacidsare found in the intracellularfluids. The abnormalities associated with water balance-dehydration and overhydration-will Osrnolarity and osmolalaty be described, following a discussion on of body fluids electrolvte balance.

the concenTherearetwo waysof expressing trationof moleculeswith regardto the osmotic pressure.

Electrolytes are the compoundswhich readily dissociate in solution and exlbf as ions i.e. positively and negativelycharged particles.For

1. Osmolarity: The number of moles(or per literof solution. millimoles) 2. Osmolality: The number of moles (or millimoles)per kg of solvent.

Ghapter 21 : WATER, ELECTFIOLYTE AND ACID-BASEBALANCE

Extracellular fluid (plasma)

Cations

Intracellular fluid (muscle)

Anions 142

Na+

ct-

K+

5

Ca2*

5

HCO; HPO14-

Mgz+

3

soiProteins

471

Cations

Anions

103

K+

150

27

Na*

10

2

Mg'*

40

1

Ca2r

2

16

Organic acids

6

140

HPO42-

Hcot clsoot Proteins Organic acids

10 2 40 5 202

tcc

lf the solventis pure water, there is almost no not be valid in severe hyperproteinemiaand difference between osmolarity and osmolality. l i pemi a. However, for biological fluids (containing molecules such as proteins),the osmolality is Osmolality of EGF and ICF more commonly used. This is about 6% greater Movement of water across the biological than osmolarity. membranes is dependent on the osmotic pressure differences between the intracellular Osmolality of plasma fluid (lCF) and extracellular fluid (ECF).In a Osmolalityis a measureof the soluteparticles healthy state, the osmotic pressure of ECF, pr es entin t h e fl u i d me d i u m . T h e o s m o l a l i tyof mainly due to Na+ ions, is equal to the osmotic plasma is in the rangeof 285-295 milliosmoles/ pressureof. ICF which is predominantlydue to kgffable 21.3).Sodiumand its associatedanions make the largestcontribution (-90%) to plasma osmolality.Osmolality is generallymeasuredby osmometer. For practical purposes, plasmaosmolality can be computed from the concentrations(mn'rol/l) of Na+, K+, urea and glucose as follows 2( Na+)+ 2 (K+ )+ U re a + C l u c o s e The factor 2 is used for Na+ and K+ ions to account for the associatedanion concentration (assumingcompleteionizationof the molecules). S inc e plas m a N a + i s th e m o s t p re d o m i nant contributorto osmolality,the above calculation is further simplified as follows P las m aos mo l a l i ty= 2 x Pl a s ma N a * (mmol/kg) (mmol/l) The above calculation holds good only if plasmaconcentrationof glucoseand urea are in the normal range.This calculation,however,will

Constituent (solute)

Osmolality (mosm/kg)

Sodium Associated anions Potassium Associated anions Calcium Associated anions Magnesium Associated anions Urea Glucose Protein Total

135 135 3.5 3.5 1.5 1.5 1.0 1.0 b.u

5.0 1.0

472

B IOC H E MIS TFI Y

K+ ions.As such,there is no net passageof water m olec u l e s i n o r o u t o f th e c e l l s . due to thi s os m o ti c e q u i l i b ri u m . Regulation

of electrolyte

balance

zA\ ,-'(/ Ansioterisin ',

oj:"ff'

Electrolyteand water balance are regulated togetherand the kidneysplay a predominantrole in this regard.The regulationis mostly achieved through the hormones aldosterone,ADH and renin-angiotensin. AngiotensinI

Aldosterone : lt is a mineralocorticoid produced by adrenal cortex. Aldosterone increasesNa+ reabsorptionby the renal tubules at the expenseof K+ and H+ ions. The net effect is the retentionof Na+ in the bodv.

Aldosterone

\ \nin Angiotensinogen

Fig.2l.2: Hormonalregulationof Antidiuretic hormone (ADH) : An increasein Na' balanceby the kidney. the plasma osmolality (mostly due to Na+) stimulateshypothalamusto releaseADH. ADH effectively increaseswater reabsorption by renal these ions (Na+ and Cl-). Therefore,the amount t ubule s . of N a+ i n the E C F ul ti matel v determi nesit s Renin-angiotensin : The secretion of vol ume. aldosterone is controlled by renin-angiotensin system.Decreasein the blood pressure(due to a Dietary intake and fall in ECFvolume) is sensedby juxtaglomerular electrolyte balance apparatusof the nephron which secreterenin. C eneral l y, the consumpti on of a w ellRenin acts on angiotensinogento produce bal anceddi et suppl i esthe body requi rementof angiotensin l. The latter is then converted to electrolytes.Humans do not possessthe ability angiotensin ll which stimulatesthe releaseof to distinguishbetweenthe salt hunger and water aldosterone. hunger.Thirst,however,may regulateelectrolyte The relation between renin, angiotensinand intakealso. In hot climates,the lossof electrolyte aldosteronein the regulationof Na+ balance is is usuallyhigher.Sometimesit may be necessary depicted in Fig.2l.2. Aldosterone and ADH to supplementdrinking water with electrolytes. coordinate with each other to maintain the nor ma l fl u i d a n d e l e c tro l y teb a l a n ce. Dehydration Atrial natriuretic peptide : This is a Dehydration is a condition characterizedby polypeptide hormone secreted by the right water depletion in the body. lt may be due to atrium of the heart. Atrial natriuretic peptide insufficient intake or excessivewater loss or increases the urinary Na+ excretion. The both. Dehydrationis generallyclassifiedinto two significanceof this hormone, however, is not types. c lear . 1. Due to loss of water alone. Na* concentration

and ECF

It is important to realise that Na+ and its anion s (ma i n l y C l -) a re c o n fi ned to the extracellularfluid. And the retentionof water in the ECFis directlv relatedto the osmoticeffectof

2. Due to electrolytes.

deprivation of

water

and

Causes of dehydration : Dehydration may occur as a resultof diarrhea,vomiting, excessive sweating, fluid loss in burns, adrenocortical

AND ACID-BASEBALANCE Ghapter 21 : WATEB, ELECTFIOLYTE

473

dysfunction,kidney diseases(e.9. renal insuffi- lumen. Theseions collectivelyraisethe osmotic ciency), deficiency of ADH (diabetesinsipidus) pressureand suck the water into lumen. This results in diarrhea with a heavy loss of water etc. (5-10 liters/day). lf not treated in time, the Characteristicfeatures of dehydration : There victims of cholera will die due to dehydration are three degrees of dehydration-mild, and loss of dissolved salts. Thus, cholera and moderate and severe. other forms of severe diarrhea are the major The salient featuresof dehydrationare given ki l l ers of young chi l dren i n many devel opi n g hereunder countries. 1. The volume of the extracellular fluid (e.g. Oral rehydration therapy (ORT) is commonly plasma)is decreasedwith a concomitantrise in used to treat cholera and other diarrheal electrolyteconcentrationand osmotic pressure. diseases. 2. Water is drawn from the intracellularfluid Overhydration t hat r es u l ts i n s h ru n k e n c e l l s a n d di sturbed metabolisme.g. increasedprotein breakdown. Overhydrationor water intoxicationis caused by excessive retention of water in the body. This 3. ADH secretion is increased.This causes may occur due to excessive intake of large increased water retention in the body and vol umes of sal t free fl ui ds, renal fai l ure, consequentlyurine volume is very low. overproduction of ADH etc. Overhydration is 4. Plasma protein and blood urea concentra- observed after major trauma or operation, lung tions are increased. infectionsetc. 5. Water depletion is often accompaniedby a loss of electrolytes from the body (Na+, K+ etc.).

Water intoxicationis associatedwith dilution of ECF and ICF with a decreasein osmolalitv. The cl i ni cal symptoms i ncl ude headache, lethargy and convulsions. The treatment 6. The principal clinical symptomsof severe advocated is stoppage of water intake and dehydration include increasedpulse rate, low administrationof hypertonicsaline. blood pressure,sunkeneyeballs,decreasedskin turgor, lethargy,confusion and coma.

Water tank model

Treatment : The treatment of choice for dehydration is intake of plenty of water. ln the subjectswho cannot take orally, water should be administered intravenously in an isotonic solution (usually5% glucose).lf the dehydration is accompaniedby loss of electrolytes,the same should be administeredby oral or intravenous routes. This has to be done by carefully monitoringthe water and electrolytestatusof the body. Osmotic imbalance in cholera

and dehydlation

The distribution of body water (in the ECF and ICF),dehydrationand overhydrationcan be better understood by a water tank model (Fi9.21.3).The tank has an inlet and outlet, respectively, representing the water intake (mostlyoral) and water output (mainly urine) by the body. Dehydrationis causedwhen the water output exceeds the intake. On the other hand, overhydration is due to more water intake and less output.

Metabolism of electrolytes -r Lnotera rs transmrfleo tnrougn water ano The body distribution,dietaryintake,intestinal foods, contaminated by the bacterium Vihrio produces which absorption cholerae.This bacterium a toxin and biochemical functions of stimulatesthe intestinalcells to secretevarious individual electrolytesare discussedunder the ions (Cf-, Na+, K+, HCOI etc.) into the intestinal section mineral metabolism (Chapter 18). The

B IOC H E MIS TR Y

474 lnlet

t--l tl tl tl \./

O a

Normal

Overhydration Fig. 21.3 : Water tank model representingbody fluid compaftments (N-Normal level; ECF-Extracellular fluid; ICF-lntracellular fluid)-

electrolytedisorders,particularlyhypernatremia the vol ati l e aci ds l i ke carboni c aci d (most and hyponatremia(of sodium);hyperkalemiaand predominent, about 20,000 mEq/day) or nonhypokalemia(of potassium)mustalso be referred. volatile acids (about 80 mEq/day)such as lactic acid, sulfuricacid, phosphoricacid etc. Carbonic acid is formed from the metabolicproduct CO2; lactic acid is producedin anaerobicmetabolism; sulfuric acid is generated from proteins (sulfur phosphori c aci d i s The normal pH of the blood is maintained in contai ni ng ami no aci ds); phosphates (e.9. from organic derived the narrow range of 7,35-7.45, i.e. slightly phosphol i pi ds). aci ds add up H + i ons A l l these r u i d i s rather alk aline .T h e p H o f i n tra c e l l u l a fl proteins rich in animal A diet to blood. the variable. Thus, for erythrocytesthe pH is 7.2, the body that produclion by acid results in more while for skeletalmuscle,it may be as low as 6.0. ultimately leadsto the excretionof urine which Maintenance of blood pH is an important is profoundly acidic. homeostaticmechanismof the body. In normal the regulationis so effectivethat Production circumstances, of bases by the body the blood pH variesvery little. Changesin blood The formation of basic compounds in the pH will a l te rth e i n tra c e l l u l apr H w h i ch, i n turn, body, in the normal circumstances,is negligible. influence the metabolism e.g. distortion in Some amount of bicarbonateis generatedfrom protein structure, enzyme activity etc. lt is the organicacids such as lactateand citrate.The estimatedthat the blood pH compatibleto life is ammoni a produced i n the ami no aci d 6.8-7.8. (For a good understandingof acid-base metabolism is converted to urea, hence its balance, adequate knowledge on acids, bases, contribution as a base in the body is pH and buffers is essential.The reader,therefore, insignificant.A vegetariandiet has a tendency must first refer Chapter 40 tor this purpose. for a net production of bases.This is due to the These basic aspects are not discussed here to fact that vegetariandiet producessaltsof organic avoid repetition). acids such as sodium lactatewhich can utilize H+ ions produced in the body. For this reason,a Production of acids by the body vegetarian diet has an alkalizing effect on the The metabolismof the body is accompanied body. fhis is reflectedby the excretion of neutral by an overall productionof acids.Theseinclude or slightly alkaline urine by these subjects.

Ghapten 21 : WATER, ELECTROLYTE AND ACIIBASE BALANCE

475

By takingthe reciprocals (for and logarithms logs,multiplication becomesaddition). The body has developedthree lines of defense to regulate the body's acid-base balance and maintain the blood pH (around 7.4). L Blood buffers ll. Respiratorymechanism

roe4 = roe** brl:gl Ka lH+t loq 1 = oK^ -K

......(3)

[H2co3J

The equation3 may now be written as

lll. Re n a l me c h a n i s m .

[Hco;l

l. Blood buffers

pH = pK " + l og-.

...... (4)

[H2co3l A buffer may be defined as a solution of a The above equation is valid for any buffer weak acid (HA) and its salt (BA) with a strong base. The buffer resiststhe change in pH by the pair. The general equation referred to as addition of acid or alkali and the buffering Henderson-Hasselbalch equation for any buffer capacity is dependent on the absolute is written as concentrationof salt and acid. lt should be borne in mind that the buffer cannot remove H+ ions Is"t"l p H = p K "+ lo g f . $ . . . . . . .(5) from the body. lt temporarily acts as a shock absorbantto reduce the free H+ ions. The H+ Incid] ions have to be ultimately eliminated by the It is evidentfrom this equationthatthe pH is renal mechanism(describedlater). dependenton ratio of the concentrationof the baseto acid (HCO3and H2CO3in equation4).

The blood contains 3 buffer systems.

Blood pH and the ratio of HCO! to H2CO3 : The plasma bicarbonate(HCO]) 2. Phosphatebuffer conceritration is around24 mmol/ (range22-26 3. Protein buffer. mmol/l).Carbonicacid is a solutionof CO2 in water. lts concentration is givenby the product 1. Bicarbonate buffer system : Sodium bi(arterial pcoz partial of pressureof CO2 = 49 (NaHCO3 carbonateand carbonicacid - H2CO3) is the most predominant huffer system of the mm Hg) and the solubilityconstantof CO2 plasma. (0.03). extracellular fluid, particularly 1. Bicarbonatebuffer

the Carbonic acid dissociatesinto hydrogen and bicarbonateions.

H2co3r^ H* + HCof By the law of mass action, at equilibrium (1) (Ka = Dissociationconstantof H2CO3). The equation may be rewritten as follows

I- corl . = K"H''--'i ["..| I r

[nco;l WeknowthatpH = loc;fi.

(2) ......

ThusH2CO3= 40 x 0.03 = 1.2 mmol/|. The Henderson-Hasselbalch equation for bicarbonatebuffer is [Hco. ]

pH= pK"+ log+ - :+ . [H2co3l Substituting the values(bloodpH = 7.4; pK^ = for H2CO3 6.1; HCOg= 24 mmol/l;H2COJ= 1.2 mmol/ll, in the aboveequation 7.4 = 6.'l * loe 24 - 1.2 = 6.1 + log 20 = 6.1 + 1.3 = 7.4

476 lt is evident that at a blood pH 7.4, the ratio of bicarbonate to carbonic acid is 20 : 1. Thus, the bicarbonateconcentration is much higher (20 times)than carbonic acid in the blood. This is referredto as alkali reserveand is responsible for the effective buffering of H+ ions, generated in the body. In normal circumstances,the concentrationof bicarbonateand carbonic acid determines the pH of blood. Further, the bicarbonate buffer system servesas an index to understand the disturbancesin the acid-base balance of the body.

B IOC H E MIS TR Y are described elsewhere (Chapter 10, Refer Fig.l0.6). The large volumes of CO2 produced by the cel l ul ar metabol i c acti vi ty endangerthe aci dbase equi l i bri um of the body. B ut i n normal circumstances, all of this CO2 is eliminated from the body in the expired air via the lungs, as summarizedbelow. Carbonicanhydrase

H2CO3

C Or+ H rO.

The rate of respiration(or the rate of removal of CO2) is controlled by a respiratorycentre, 2. Phosphate buffer system : Sodium locatedin the medullaof the brain.This centreis dihydrogen phosphateand disodium hydrogen highly sensitiveto changesin the pH of blood. phosphate(NaH2POa- Na2HPOa)constitutethe Any decreasein blood pH causeshyperventilation phosphate buffer. lt is mostly an intracellular to blow off CO2, thereby reducing the H2CO3 bufferand is of lessimportancein plasmadue to concentration.Simultaneously,the H+ ions are its low concentration.With a pK of 6.8 (closeto el i mi natedas H 20. blood pH 7.4), the phosphatebuffer would have Respiratorycontrol of blood pH is rapid but been more effective,had it been presentin high only a short term regulatory process/ since concentration. lt is estimated that the ratio of hyperventilation cannot proceed for long. baseto acid for phosphatebuffer is 4 compared to 20 for bicarbonate buffer. Hemoglobin as a buffer : Hemoglobin of is also important in the respiratory erythrocytes 3. Protein buffer system : The plasma pH. At the tissuelevel, hemoglobin regulation of proteinsand hemoglobintogetherconstitutethe binds to H+ ions and helps to transportCO2 as protein buffer systemof the blood. The buffering HCOt with a minimum change in pH (referred capacity of proteins is dependenton the pK of isohydric transport). ln the lungs, as to as ioniz ableg ro u p so f a m i n o a c i d s .T h e i m i dazol e with H+ ions are hemoglobin combines 02, group of histidine(pK = 6.7) is the most effective with HCOt to form removed which combine contributor of protein buffers. The plasma lafter to release H2CO3. The dissociates CO2 to proteins account for about 2oh of the total (Refer Fig.l0.6). be exhaled bufferingcapacity of the plasma. Hemoglobin of RBC is also an important buffer. lt mainly buffersthe fixed acids, besides being involved in the transportof gases(Oz and CO2). More details on hemoglobin are given underrespiratorymechanismfor regulationof pH. ll. Respiratory mechanism for pH regulation Respiratory system provides a rapid m ec hanis mfo r th e m a i n te n a n c eo f a ci d-base balance. This is achieved by regulating the concentrationof carbonic acid (H2CO3)in the blood i.e. the denominator in the bicarbonate buffer system. The details of CO2 transport and the role of hemoglobin in this process

Generationof HCO3 by RBC : Due to lack of aerobic metabolic pathways,RBC produce very little CO2. The plasmaCO2 diffusesinto the RBC along the concentration gradient where it combines with water to form H2CO3. This reaction is catalysedby carbonic anhydrase(also called carbonate dehydratase).In the RBC, H2CO3 dissociatesto produce H+ and HCOt . The H+ ions are trapped and buffered by hemoglobin. As the concentration of HCOf increasesin the RBC, it diffuses into plasma along with the concentration gradient, in exchange for Cl- ions, to maintain electrical phenomenon, referred neutrality. This to as chloride shift, helps to generate HCOI (Fig.2t.Q.

477

AND ACID-BASEBALANCE Chapter 21 : WATER, ELECTBOLYTE

the renal regulationof pH which occurs by the fol l ow i ng mechani sms.

Erythrocy'te

1. E xcreti onof H + i ons 2. Reabsorptionof bicarbonate

CO2+ H2O

3. Excretionof titratableacid

l"o +

4. E xcreti onof ammoni um i ons.

H2C03

t HHb HCo!+ n.-1 Hb

1. Excretionof H+ ions : Kidney is the only route through which the H+ can be eliminated from the body. H+ excretion occurs in the proxi malconvol utedtubul es(renaltubul arcel l s) and is coupled with the regeneration of HCOj. The process depicted in Fig.2l .5, occurs as follows.

Carbonic anhydrasecatalysesthe production of carbonic acid (H2CO3)from CO2 and H2O in the renal tubular cell. H2CO3then dissociatesto H+ and HCO!. The H+ ions are secretedinto the lll. Renal mechanism for pH tubul ar l umen i n exchangefor N a+ . The N a+ i n regulation associationwith HCOI is reabsorbedinto the This is an effective mechanism to The role of kidneys in the maintenanceof blood. el i mi nate aci ds (H + ) from the body w i th a acid-base balance of the body (blood pH) is generation of HCO3. The latter simultaneous highly significant.The renal mechanismtries to adds up to the alkali reserveof the body. The H+ provide a permanent solution to the acid-base with combines a non-carbonate base and is disturbances.This is in contrastto the temporary in excreted urine. buffering system and a short term respiratory mechanism,describedabove. 2. Reabsorptionof bicarbonate: This mechanism is primarily responsibleto conserve the The kidneys regulate the blood pH by blood HCO3, with a simultaneousexcretion of maintainingthe alkali reserve,besidesexcreting H+ ions. The normal urine is almost free from as or reabsorbingthe acidic or basic substances, HCOI. This is explained as follows (Fi5.21.5). the situationdemands. Fig. 21.4 : Generation of bicarbonate by the erythrocyte (CA-Carbon ic anhydrase; Hb-Hemoglobi n).

Urine pH normally lower than blood pH : T he pH of u ri n e i s n o rma l l ya c i d i c (-6 .0 ) . Thi s clearly indicates that the kidneys have contributedto the acidificationof urine, when it is formed from the blood plasma (pH 7.4). In other words, the H+ ions generatedin the body in the normal circumstances,are eliminated by ac idif ied ur i n e . H e n c e th e p H o f u ri ne i s nor m ally ac i d i c (-6 .0 ), w h i l e th a t o f b l o od i s alk aline ( 7. 4 ). U ri n e p H , h o w e v e r, i s v a ri abl e and may range between 4.5-9.5, dependingon the concentrationof H+ ions. Carbonic anhydrase and renal regulation of pH : T he en z y mec a rb o n i ca n h y d ra s e(i n hi bi ted by acetazolamide) is of central importance in

Blood

Renaltubularcell

Na-

N aHC03

Tubularlumen

HCOJ+ H*

1

H2C03

l"o CO2+ H2O

H*+ B-

II

+ HB ;

Fig. 21.5 : Renal regulationof blood pH-Excretion of ll ions (CA-Carbonic anhydrase).

I

478

B IOC H E MIS TR Y

Renaltubularcell

Tubularlumen

HCOf + H*

1 I H2C03

H2C03

1"u H2O+ CO2

J CO2+ H2O

l"u

FIg.2l.6 : Renalreglulationof btoodpH4eabsorption t t" :::|:::qL:0 E ilt!t!4Ftfeli..,i,:::: .4d,/i9i!ryF!+r, ,'..;, Bicarbonatefreely diffusesfrom the plasma into the tubular lumen. Here HCO3 combines with H+, secreted by tubular cells, to form H2CO3. H2CO3 is then cleaved by carbonic anhydrase(of tubular cell membrane)to form CO2 and H2O. As the CO2 concentrationbuilds up in the lumen, it diffusesinto the tubular cells along the concentrationgradient.In the tubular cell, CO2 again combines with H2O to form H2CO3 which then dissociatesinto H+ and HCOI. The H+ is secreted into the lumen in exchangefor Na+. The HCO3 is reabsorbedinto plasma in associationwith Na+. Reabsorptionof HCOI is a cyclic processwith the net excretion of H+ or generation of new HCOt. This is because the H+ is derived from water. This

Blood

Renaltubular cell

Na*

Na*

mechanism helps to maintain the steady state and will not be effectivefor the elimination of H+ or generationof new HCOJ. 3. Excretion of titratable acid : Titratable aciditv is a measureof acid excreted into urine by the kidney. This can be estimatedby titrating urine back to the normal pH of blood (7.4). ln quantitative terms, titratable acidity refers to the numberof mi l l i l i tersof N /l 0 N aOH requi redto titrate 1 liter of urine to pH 7.4. Titratableacidity reflectsthe H+ ions excreted into urine which resultedin a fall of pH from 7.4 (that of blood). The excreted H+ ions are actually buffered in the urine by phosphate buffer as depicted in Fi9.21.7,and briefly describedhereunder. As alreadydiscussed,H+ ion is secretedinto the tubul ar l umen i n exchangefor N a+ i on. Thi s Na+ is obtained from the base, disodium hydrogen phosphate (Na2HPOa).The latter in turn combines with H+ to produce the acid, sodium dihydrogen phosphate (NaH2POa), in which form the major quantity of titratableacid in urine is present.As the tubular fluid moves down the renal tubules/more and more H+ ions are added, resultingin the acidificationof urine. This causesa fall in the pH of urine to as low as 4.5. A ny further fal l i n the pH w i l l cause depletion of Na+ ions. 4. Excretion of ammonium ions : This is another mechanismto buffer H+ ions secreted i nto the tubul arfl ui d. The H + i on combi nesw i th

Tubularlumen Na2HPOa

HCO3

pH 7.4

Na*+f,+ NaHPOZ

HCOI + H*

I

HzCQ

1'o

CO2+ H2O

NaHzPO+ +

Excreted

Fiq.21.7 : Renal regulationof blood pH-Excretion of titratableacid by phosphate buffer mechanism (CA-Carbonic anhydrase).

pH4.5

479

Ghapter 21 : WATEFI, ELECTFOLYTE AND ACID-BASEBALANCE

Renal tubular cell

Tubularlumen

Glutamine

I Iutaminase

f-*tt,

-+ NH3

aerobic metabolismmay be exhaled via lungs, or converted to HCOt by erythrocytes and kidneys to add up to the alkali reserveof the body. Buffers

of intracellular

fluids

Glutamate

Na' HCO;

HCO!+ H"

T

NH;

H2COg

l"o

CO2+ H2O

The regulation of pH within the cells is as important as that discussed above for the extracellularfluid. The H+ ions generatedin the cells are exchangedfor Na+ and K+ ions. This is particularly observed in skeletal muscle which reducesthe potentialdangerof H+ accumulation i n the cel l s.

Excreted

Fiq.21.8 : Benal regulationof blood pH-Excretion of ammonium ions (CA-Carbonic anhydrase).

The body has developed an efficient system for the maintenance of acid-base equilibrium with a result that the pH of blood is almost NH3 to form ammonium ion (NH;). The renal constant (7.4. The blood pH compatible to life tubular cells deamidateglutamine to glutamate is 6.8-7.8, beyond which life cannot exist. and NH3. This reaction is catalysed by the enz y m eglut a mi n a s eT. h e N H 3 , l i b e ra te di n thi s For a betterunderstandingof the disordersof reaction,diffusesinto the tubular lumen where it acid-base balance, the Henderson-Hasselbalch combines with H+ to form NHi Gig.2l.A. equation must be frequentlyconsulted. Ammonium ions cannot diffuseback into tubular cells and, therefore,are excreted into urine. lHco-] NHf is a major urine acid. lt is estimatedthat about half to two-thirds of body acid load is eliminated in the form of NHf, ions. For this reason, renal regulation via NHf, excretion is very effective to eliminate large quantities of acids produced in the body. This mechanism becomespredominantparticularlyin acidosis. central Garbon dioxide-the molecule of pH regulation

pH=pK"+rosfi,c;t

Lungs (CO, exhaled)

As is observedfrom the foregoingdiscussion, CO2 is of central importance in the acid-base balanceof the body. lt hasthe ability to combine Erythrocytes Kidneys with H2O to from H2CO3 which can dissociate (CO,transported, gene1HCO3 to HCOJ and H+. A summaryof the interaction HCOJgenerated) H+lost) 161sfl, between the lungs, erythrocytesand kidneys in Fig.21.9 : Carbon dioxide-the central molecule of handling CO2 to maintain pH of the blood is blood pH regulation. depicted in Fig.2l.9. The CO2 generated by

I

BIOCHEMISTF|Y

480 It is evident from the above equation that the blood pH (H+ ion concentration)is dependent on the relative concentration (ratio) of bicarbonate(HCOI) and carbonic 26ii (H2CO3). The acid-basedisordersare mainly classifiedas 1. Acidosis-a decline in blood pH (a\ Metabolic acidosis-due to a decrease in bicarbonate. (b) Respiratory acidosis-due increasein carbonic acid.

to

an

2. Alkalosis-a rise in blood pH (a\ Metabolic alkalosis-Aue increasein bicarbonate. (b) Respiratory alkalosis-due decreasein carbonic acid.

to to

Metabolic acidosis

mellitus Diabetes (ketoacidosis)

asthma Severe Pneumonia anest Cardiac

failure Renal

in airways Obstruction

Lacticacidosis

Chestdeformities

diarrhea Severe acidosis Renaltubular

of Depression center(by respiratory drugse.g.opiates)

an a

Respiratory acidosis

Metabolic alkalosis

Respiratory alkalosis

vomiting Severe

Hyperventilation

Hypokalemia

Anemia Highaltitude

administration lntravenous of bicarbonate

poisoning Salicylate

The four acid-base disorders referred above are primarily due to alterations in either bicarbonateor carbonicacid. lt may be observed that the metabolic acid-basebalance disorders are causedby a direct alterationin bicarbonate concentrationwhile the respiratorydisturbances are due to a change in carbonic acid level (i.e. CO2). This type of classification is more theoretical. ln the actual clinical situations, mixed type of disordersare common.

makes every attempt to restorethe pH to normal level (7.4). This is referredto as compensation which may be partialor full. Sometimesthe acidbase disordersmay remain uncompensated.

alkalemia, terms acidemia and The respectively,refer to an increaseor a decreasein tH*] ion concentration in blood. They are, however, not commonly used.

The principal acid-basedisturbances,along with the blood concentration of HCO3 and H2CO3, in acute and compensated states are given in the Table. 21.5.

For the acute metabolic disorders (due to changesin HCO!, respiratorycompensationsets in and regulatesthe H2CO3 (i.e. CO2) by hyperThe most important clinical causes/disease or hypoventilation. As regards acute respiratory statesthat result in acid-basedisordersare listed disorders (due to changes in H2CO3), the in Table 21.4. Metabolic acidosis could occur renal compensation occurs to maintain the due to diabetes mellitus (ketoacidosis),lactic HCOJ level, by increasing or decreasing its acidosis,renal failure etc. Respiratoryacidosisis excretion. common in severe asthma and cardiac arrest. fn the lable 2l ,6, a summary of the acid-base Vomiting and hypokalemia may result in primary changes and metabolic alkalosiswhile hyperventilationand disorders with severeanemia may lead to respiratoryalkalosis. compensatorymechanismsis given.

Glinical causes of acid-base disorders

Gompensation of acid-base disorders To counter the acid-base disturbances,the body gears up its homeostaticmechanism and

Anion

gap

For a better understanding of acid-base disorders,adequateknowledge of anion gap is essential.The total concentrationof cations and

Chapter 21 : WATEFI,ELECTROLYTE AND ACID-BASEBALANCE

48r

anions (expressedas mEq/l)is equal in the body disordersare often associatedwith alterations in f luids . T his is re q u i re d to ma i n ta i n e l e c t ri cal the anion gap. neutrality. Metabolic acidosis The commonly measuredelectrolytesin the The primary defect in metabolicacidosisis a plasmaare Na+, K+, Cl- and HCOJ. Na+ and K+ reduction in bicarbonate concentrafion which together constitute about 95% of the plasma leads to a fall in blood pH. The bicarbonate cations. Cl- and HCO3 are the major anions, concentration may be decreased due to its contributingto about 80"h of the plasmaanions. utilization in buffering H+ ions, loss in urine or The remaining 20"/. of plasma anions (not gastrointestinal tract or failure to be regenerated. normally measured in the laboratory) include proteins, phosphate,sulfate, urate and organic The most important cause of metabolic ac ids . acidosis is due to an excessiveproduction of Anion gap is defined as the difference between the total concentration of measured cations(Na+ and K+)and that of measuredanion (Cl- and HCOj). The anion gap (A-) in fact representsthe unmeasured anions in the plasma which may be calculated as follows, by substituting the normal concentration of efectrolytes (mEq/l). Na*+

K*

=

Cl-

+

HCO, + A-

organicacidswhich combine with NaHCOj and deplete the alkali reserve. NaHCO3 + Organic acids -----+ Na saltsof organic acids + CO2 Metabolic acidosis is commonly seen in severe uncontrolled diabetes mellitus which is associated with excessive production of acetoaceticacid and p-hydroxybutyric acid (both are organic acids).

Anion gap and metabofic acidosis : Increased production and accumulation of organic acids t5 nEql A- causes an elevation in the anion gap. This type T he anion g a p i n a h e a l th y i n d i v i d u a l i s of picture is seen in metabolic acidosis around 15 mEq/l (range8-18 mEo/l).Acid-base associatedwith diabetes (ketoacidosis). 136+ { - 10 0 + 2 5 + A -

Disorder

Blood pH

lHCo;l

IH2CO3]

J J

-)

-)

1 t

Metabolic acidosis Acute (byt ventilation) Compensated

\or-)

Respiratory acidosis Acute (HCOIretained by kidney) Compensated

\0r-+

I

Metabolic alkalosis Acute (byJ ventilation) Compensated

t

t 1

--)

V o( --)

Hespiratory alkalosis Acute by kidney) Compensated fHCO; excretion

1 v or --+

-) J

J

v

A

v

t I

482

BIOCHEMISTRY

Primary change

Disorder

Metabolic acidosis

Decreased olasma bicarbonate

ptasma Metabolic alkalosis Increased oicii6oni6 pCO, Respiratory acidosis Increased

Respiratoryalkalosis DecreasedpCO,

Compensation of metabolic acidosis : The acute metabolicacidosisis usuallycompensated hy hyperventilation of lungs. This leads to an increased elimination of CO" from the bodv (hence H2CO3U. but respiratorycompensation is only short-lived.Renal compensationsets in within 3-4 days and the H+ ions are excretedas NHi io n s . Respiratory

acidosis

Compensatory mechanism

Timescalefor compensation

Hyperventilation (decrease in pCOr)

to hours Minutes

Htd6llhtio;

Minutes to hours

(increase in pCOJ Elevation in plasma increase in bicarbonate; of renalreabsorption bicarbonate in plasma Reduction degease bicarbonate: in renalreabsorption of bicarbonate

Days

Days

sodium bicarbonate for therapeutic purposes (e.g. control ol gastric acidity). Cushing's of aldosterone)causes syndrome(hypersecretion increased retention of Na+ and loss of K+ from the body. Metabolic alkalosis is commonly low K+ concentration associated with (hypokalemia).In severeK+ deficiency,H+ ions are retained inside the cells to replace missing K+ ions. In the renal tubular cells, H+ ions are exchanged (instead of K+) with the reabsorbed Na+. Paradoxically,the patient excretes acid urine despitealkalosis.

The primary defect in respiratoryacidosis is due to a retention of CO2 (H2CO3|. There may be severalcausesfor respiratoryacidosiswhich The respiratory mechanism initiates the include depression of the respiratory centre compensation by hypoventilation to retain CO2 (overdose of drugs), pulmonary disorders (hence H2CO3T).This is slowly taken over by (bronchopneumonia) and breathingair with high renal mechanism which excretes more HCO3 content of CO2. and retains H+. The renal mechanismcomesfor the rescueto compensaterespiratoryacidosis.More HCO3 is Respiratory alkalosis generatedand retained by the kidneys which The primary abnormality in respiratory adds up to the alkali reserveof the body. The alkalosis is a decreasein H2CO3 concentration. excretion of titratable acidity and NHf is prolonged to This may occur due elevated in urine. hyperventilationresultingin increasedexhalation of COz by the lungs. Hyperventilation is M et ab o l i c a l k a l o s i s observed in conditions such as hysteria, primary The abnormality in metabolic hypoxia, raised intracranialpressure,excessive artificial ventilation and the action of certain afkalosis is an increase in HCO3 concentration. This may occur due to excessive vomiting drugs (salicylate) that stimulate respiratory (resultingin lossof H+) or an excessiveintakeof centre.

WATER. ELECTROLYTE AND ACID-BASEBALANCE The renal mechanismtries to compensateby inc r eas ingt he u ri n a rye x c re ti o no f H C O 3 . : r,!:rl j:i,i; ;iqi - if?;E..s *,* gl i * ti.*.'d + dtt

483

pl asmaK + )or hypokal emi a(l ow pl asmaK + )can be Iife-threatening. The relevanceof potassium bal ance i n certai n aci d-base di sorders i s di scussedbri efl y.

Sometimes,the patientmay have two or more Potassiumand diabetic ketoacidosis : Tne ac id- bas edis t u rb a n c eosc c u rri n gs i mu l ta n e o usl y. hormone i nsul i n i ncreasesK + uptake by cel l s y In such instances,both HCO3 and H2CO3 are (parti cul arlfrom skel etalmuscl e).The pati entof (of severeuncontrolleddiabetes(i.e. with metabolic alt er ed.I n gen e ra l ,i f th e b i o c h e mi c a ld a ta blood gas analysis)cannot be explained by a aci dosi s)i s usual l y w i th hypokal emi a.W hen specific acid-basedisorder, it is assumedthat a such a pati ent i s gi ven i nsul i n, i t sti mul atesK + m ix ed dis t ur b a n c ei s o c c u rri n g .M a n y a ti mes, entry into cells.The resultis that plasmaK+ level compensatory mechanisms may lead to mixed is further depleted. Hypokalemia affects heart functi oni ng,and i s l i fe threateni ng. acid-base disorders. Potassium and alkalosis : Low plasma concentrationof K+ (hypokalemia)leads to an increasedexcretion of hydrogen ions, and thus P las m a po ta s s i u m c o n c e n tra ti o n (n o r mal may cause metabol i c al kal osi s. C onversel y, 3.5-5.0 mEq/l) is very importantas it affectsthe metabolic alkalosisis associatedwith increased contractility of the heart. Hyperkalemia (high renal excretion of K+. :i n;ifjl""S,*lt*#'d*aerders j,ilr',r l:l rli +slmlia P@ffi *SE{.C""t.$

B|oMEDTCAL/ CLIIU|CALCONCEPTS

s

Existence ot' life is unimaginable in the absenceof water.

w Kdneys play a predominant role in the regulationof water;electrolyteand acid-basebalance. we Electrolyte and water balanceregulotion occurs through the inuoluementof hormonesaldosterone,ADH and renin-ongiotensin. sE Seueredehydration is characterizedby low blood pressure, sunken egeballs,Iethargy, confusion and coma. re' Sodium is the principal extracellular cation while K+ is intracellular. The maintenance of the differentiol concentration of these electrolytesis essentialfor the suruiualoJ liJe which is brought about by No+-K+ pump. ss The body metabolism is accompaniedby the production of acidssuch os carbonic acid, sult'uric ocid, phosphoric acid etc. w

Vegetariandiet hason alkalizingefiect on the body. This is ottributed to the formation of organicocidssuch os sodium lactate which can depleteH+ ions by combining with them.

s€ The blood pH is maintained by blood buffers, respiratory and renal mechanisms. *q Carbon dioxide is the central molecule of acid-baseregulation. w

Disturbancesin acid-baseregulotion result in acidosis(decreasedblood pH) or alkalosis (raised blood pH).

w

Uncontrolled diabetesmellitus is associatedwith metabolic ocidosis,commonly referred to as ketoacidosis(due to the ouerproduction of ketone bodies)

BIOCHEMISTFIY

484 In view of the importancediscussedabove, the measurementof plasma K+ concentration assumessignificancein the acid-basedisorders. In cases of these disorders associated with hypokalemia, potassiumsupplementation(with carefull monitoring of plasma K+) needs to be considered.

of blood gas is an important The measurement investigationin the laboratoryservice.In certain conditions associatedwith respiratory failure and/or acid-base disorders, blood gas (COz and 02) measurementassumessignificance.Based on the results obtained and the severity of the condition, oxygen treatment or artificial ventilation is carried out. For blood gas analysis,a sample of arterial blood collected from (most commonly) radial artery in the forearm, or (lesscommonly) from the femoral artery in the leg is used. The

biochemical profile measured include POz, pCOz, and pH (H+ ion concentration).The concentration of bicarbonate is calculated by equation. In fact, using Henderson-Hasselbalch the blood gas analysers employed in the hospitals are designed to perform the various calculations automatically and give the final results. The reference ranges of blood gas analysisare given in Table 21 .7.

Parameter

Concentration/value

tH-1

35-43mmoUl

pH

7.3y7.45

PC0z

4.5-6.0kPa

Poz

10.5-13.5 kPa

Bicarbonatex

2_ryqTq9r4

xBianbonate is nfulatd fronpHaN {'Orvafues. con@ntration

chapter 21 : WATEFI,ELECTROLYTE AND ACID-BASEBALANCE

485

1. Water is the soluent of life and constitutes about 600/oof the total body weight, distributedin intracellularand extracellularJluids. The daily water intake (by drinking, from loodstuffs and metabolic water) and output (/oss uio urine, skin, lungs and t'eces) maintain the body balonceof water.

2. Electrolytesare distributedin the intracellularond extracellularfluids to maintoin the osmotic equilibrium and water balance,No+ is the principal extracellularcation while K+ is the intracellularcation As regardsonions, CI- and HCO1 predominantlyoccur in the extracellulor t'luidswhile HPO42-, proteins and organic acidsare present in the intracellularfluids.

3. The osmolality of plasma is about 285 milliosmoles/kg,which is predominontly contributed by No+ and its ossociatedonions. Thus, Jor practical purposes, plosma osmololity can be calculated from Na+ concentration (2 x No+ in mmol/l).

4. Water and electrolyte balance are usually regulated together and this is under the antidiuretichormone and renin. control of hormones---aldosterone,

5. Dehydration of the body may be due to insufficient woter intake or its excessiue/oss or both. Depletion of water in the ICF causes disturbance in metabolism. The manifestationsof seueredehydration include increasedpulse rate, Iow blood pressure, sunken eyebolls,decreasedskin turgor, lethorgy and coma. The normal pH of blood is maintained in the narrow range of 7.35-7.45. The metabolismot' the body is accompaniedby an ouerall production of acids. The body has deuelopedthree lines of det'ense(blood buffers, respiratory and renal mechanisms)to regulatethe qcid-basebalanceand maintain the blood pH.

7. Among the blood buffers, bicarbonatebut't'er(with a rotio ol HCOS to H2CO3 as 20 : 1) is the most important in regulating blood pH. Phosphate and protein buft'er systems also contribute in this regard. The respiratory system regulates the concentration of carbonicacid by controlling the elimination of CO2 uia lungs.

8. The renal (kidney) mechanism regulotes blood pH by excreting H+ and NHy' ions besidesthe reabsorptionof HCO7. The pH of urine is normally acidic which indicates thot the kidneyshaue contributed to the acidificationof urine.

9. The ocid-basedisordersare classit'iedos ocidosis(metabolicor respiratory)and alkalosis (metabolicor respiratory),respectiuely,due to a rise or foll in blood pH. The metabolic are associotedwith olterotionsin HCOS concentrationwhile the respiratory disturbonces disorders ore due to changes in H2CO3 ft.e. CO2).

10. Blood gasmeasurementincludesthe parameterspO2, pCO2, pH and bicorbonate,and it is uery important to eualuateand treat acid-basedisorders.

B IOC H E MIS TR Y

486

I. Essayquestions 1. Describethe role of kidney in the regulationof blood pH. 2. Cive an accountof the water distributionand its balancein the body. fluids.Discussthe and intracellular 3. Camparethe compositionof electrolytes in the extracellular regulationof electrolytebalance. 4. Describethe role of blood buffersin the acid-basebalance. mechanisms. 5. Classifyacid-basedisordersand discussthem with compensatory II. Short notes (a)Dehydration,(b) Vasopressin and water balance,(c) Osmolalityof plasma,(d) Acids produced in the body, (e) Henderson-Hasselbalch equation, (fl Bicarbonatebuffer, (g) Excretionof H+ by kidney,(h) Titratableacidity,(i) Metabolicacidosis,(j) Anion gap. III. Fill in the blanks 1. The hormonecontrollingwater excretionvia kidneysis 2. The principalcation of extracellular fluid is 3. The normal osmolalitvof plasmais 4. Na+ reabsorption by renal tubulesis increasedby the hormone 5. The most predominantvolatileacid generatedin the body is 6. The most importantbuffer systemregulatingblood pH is 7. At a normal blood pH 7.4, the ratio of bicarbonateto carbonicacid is 8. The body acid load is predominantlyeliminatedin the form of 9. The primary defect in metabolicacidosisis a reduction in the plasma concentrationof of 10. The respiratory alkalosisis primarilyassociated with a decreasein the plasmaconcentration IV. Multiple choice questions 11. The metabolic(endogenous) water is derivedby the oxidationof (a) Carbohydrate(b) Protein(c) Fatsd) All of them. 12. The most predominantanion in the extracellular fluids (a) Cl- (b) HCof (c) HPoi- (d) Protein. 13. The only routethroughwhich H+ ions are eliminatedfrom the body (a) Lungs (b) Stomach (c) Kidneys (d) None of them. 1 4 . N a meth e a m i n oa c i dfro m whi ch ammoni ai s deri vedi n the renaltubul arcel l sw hi ch i s fi nallv excretedas NHf, (a) Asparagine(b) Clutamine(c) Clutamate(d) Aspartate. (in the laboratory) plasmaanion concentration and is 15. The anion gap refersto the unmeasured representedby (a) Proteinsand organicacids (b) Phosphate and sulfate(c) Urate (d) All of them.

The ptotein,

collagen;,, speaks t

l

"I am the vnostabundant protei/t in marnmab; Triple helical in structure, with distinct types; Predominantly cortposedof glycine and pralinl; Gry-X-Y-G!-X-Y

Gy

X-Y

I giue strength, support and shapteto tissues,"

-l-hu body possesses a vast number of proteins I designed with specific structuresto perform specialized functions.A selectedfew of the most importantproteinsthat are intimatelyconnected with the tissuestructureand functionsare briefly des c r ibedin th i s c h a p te r.In a d d i ti o n ,th e b ody f luids ar e als o d i s c u s s e d .

of the total body protein. Collagen is the predominant component of the connective tissue,although its distribution varies in different tissues.For instance,collagenforms 90% of the organic matrix of bones, 85"/t of tendons, 707o of skin, and 4oh of liver. Fu;lc t itins t,i i:+:;rl;i,Eeii

1 . B ei ng a maj or component of the connective tissue, collagen gives strength, support and shape to the fissues. The tensile The connective tissue or extracellular matrix strengthof collagenfiber is impressive.To break (ECM) refers to the complex material a collagen fiber of 1 mm in diameter,a load of surroundingthe mammaliancells in tissues.The 104O kg is needed! However, in diseasedstates major protein components of ECM include with altered collagen structure, the tensile c ollagen,elas ti n , fi b ri l fi n , fi b ro n e c ti n , l a m i ni n strengthis reduced. and proteoglycans.Besidesthese proteins, the 2. Collagen contributesto proper alignment structural proteins namely keratins are also which in turn helps in cell proliferation, of cells, described. and their differentiationto different tissuesand orS ans. COLLAGEN Collagen is th e m o s t a b u n d a n t p ro te i n i n nammals, comprising approximately one-third

3. Collagen(that is exposedin blood vessels) contributesto thrombus formation.

487

I

488 Types

B IOC H E MIS TRY of eoltagen

Collagen is not a single homogeneous protein,but a group of structurallyrelated an d g e n e ti c a l l y d i s ti n c t p ro tei ns. In humans, at least 19 different types of 300nm collagens, composed of 30 distinct polypeptidechains (encodedby separate genes),have been identified.The typesof collagen are numbered (by Roman numerals)as l, ll...XlX. The differenttypes of collagen are suited to perform specialized functions in tissues. For instance,collagenstype I and type ll are Gly-X-Y-Gly-X-Y-Gly-X-Y respectivelyfound in skin and bone. Stnueture

of collagen

Collagen molecule

i1.4 nm Triplehelix <-j

o-charn (potypeptide) A m i n oa c i qs e qu e n ce

Fig. 22.1 : A diagrammatic representation of the structure of collagen and fibril (X and Y represent amino acids other than glycine)

In principle, all types of collagen are triple helical structures. The triple helix may occur throughoutthe molecule,or only a part of t he mo l e c u l e .

t,

Fibril

Type I mature collagen, containing about 1000 amino acids (for each polypeptidechain) possesses triple helical structurethroughoutthe molecule. lt is composed of three similar polypeptidechainstwisted around each other to form a rod like molecule oI 1.4 nm diameter, and about 300 nm length (Fig.22.l). The amino ac i d c o mp o s i ti o n o f c o l l a g e n i s uni que. Approximately 1/{d of the amino acids are contributed by glycine i.e. every third amino acid in collagen is glycine. Hence, the repetitive amino acid sequenceof collagen is represented by (GIy-X-\, where X and Y represent other amino acids.Thus, collagenmay be regardedas a polymer of glycine-ledtripeptide.Among the other amino acids, proline and hydroxyproline are present in large quantities (about 100 residues each). These two amino acids confer rigidity to the collagen molecule.

occursby a quarterstaggeredalignmenti.e. each tri pl e hel i xof col l ageni s di spl acedl ongi tu dinally from its neighbour by about one-quarterof its length (Fig.22.l). The strength of the collagen fibers is contributed by the covalent cross links formed between lysine and hydroxylysineresidues.The degree of collagen cross-linkingincreaseswith age. Thus, i n ol der peopl e, the collagen contai ni ng ti ssues (e.g.ski n, bl ood ve ssels) become less elastic and more stiff, contributing to health complications. Eiosynthesis

of collagem

Collagen synthesisoccurs in fibroblasts,ano the cells related to them e.g. osteoblastsin bones, chondroblastsin cartilage,odontoblasts in teeth.

Collagenis synthesizedon the ribosomesin a precursor form namely preprocollagen. This contains a signal peptide which directs the protein to reach the endoplasmicreticulum(ER). The triple helical structure of collagen is In the ER, the signal peptide is cleaved to form stabilizedby an extensivenetwork of hydrogen procollagen. The latter undergoesextensivepostbonds, covalent cross-links, electrostatic and translational modifications (hydroxylation and hydrophobic interactions,and van der Waals glycosylation) and disulfide bonds formation. forces. The procollagenso formed is secretedinto tne Th e tri p l e h e l i c a l mo l e c u l e s of col l agen extracellular medium, and subjected to the assembleand form elongatedfibrils,and then rod action of aminoproteinase and carboxylike fibers in the tissues.The fibril formation proteinaseto remove the terminal amino acios.

AND BODYFLUIDS Chapter 22 : TISSUEPROTEINS This is followed by a spontaneousassemblyof t he polyp e p ti d ec h a i n s(w i th a b o u t 1 0 00 ami no acids in each) to form triple helical structuresof c ollagen .

489

responsiblefor the extensibility and elasticity of ti ssues.E l asti ni s found i n l arge quanti ti esi n Iungs,arterialblood vessels,elasticligmentsetc.

Elastin is synthesizedas tropoelastinwhich post-translational modifications undergoes Abnormalities associated (formation of hydroxyproline, and no wit h eo l l a g e n hydroxylysine).Compared to collagen, elastin T he b i o s y n th e s i so f c o l l a g e n i s a compl ex structure is simple-no triple helix, no repeat process,involving at least30 genes(in humans), sequenceof (Gly-X-Y)n. and about 8 post-translationalmodifications. Expectedly,many inherited diseasesdue to gene Abnormalities associated mutations,linked with collagen formation have with elastin been identified.A few of them are listed below. . Williams syndromeis a geneticdiseasedue to . Ehlers-Danlossyndrome-a group of inherited impairment in elastin synthesis. The disorders characterizedby hyperextensibility connective tissue and central nervous system of skin, and abnormal tissuefragility. are affected. . Alport syndrome-due to a defect in the . Decreased synthesisof elastin is found in formation of type lV collagen fibres found in aging of skin and pulmonary emphysema. the basementmembrane of renal glomeruli. The patients exhibit hematuria and renal FIB R ILLIN diseases. Fibrillin is a structural component of . Osteogenesis imperfecta-characterized by myofibrils found in various tissues. abnormal fragility of bones due to decreased formation of collagen. . Epidermolysisbullosa-due to alteration in the structure of type Vll collagen. The victims exhibit skin breaksand blistersformationeven f or a m i n o r tra u ma .

Marfan syndrome is a genetic disorder due to a mutati on i n the gene for fi bri l l i n. l t i s characterizedby hyperextensibility of joints and skeletal.system. Consequently,the patients of Marfan syndromeare tall, and have long digits. These patients may also have cardiovascular complications. Some researchersbelieve that Abraham Lincoln was a victim of Marfan svndrome.

Scurvy : This is a disease due to the deficiency of vitamin C (ascorbicacid). Although not a genetic disease, scurvy is related to the improper formation of collagen, hence referred here (vitamin C is needed for the postFIB R ON E C TIN translationalmodificationsof collagen). Scurvy is characterized by bfeeding of gums, poor Fibronectin, a glycoprotein, is closely wound healing and subcutaneoushemorrhages. involved in the interaction of cells with lathyrism : lt is a diseaseof hone deformities extracellular matrix. lt actively participates in caused by the consumption of Kesari dal cell adhesion and cell migration. In general, (Lathyrussativa)in some parts of lndia. The toxic tumor cells are deficient in fibronectin which c om poun d n a me l y p -o x a l y l a mi n o al ani ne resultsin the lack of adhesionamong the tumor (BOAA), found in kesaridal, interfereswith the cells that may often lead to metastasis. cross-linkingof lysine amino acids in collagen. B O A A is fo u n d to i n h i b i t e n z y m e l y s y l oxi dase. LA MIN IN

The basal lamina of glomerular membrane(of renal cel l s)contai nsl ami ni n. In fact, l ami ni n i s Elastin is another important (besides one of the first extracellularproteinssynthesized collagens)connectivetissueprotein. lt is mainly during embryogenesis.lt is actively involved in E LA S T I N

490

B IOC H E MIS TR Y

neuronal growth and nerve regeneration. ln the patients of Alzheimer's disease, high concentrationsof laminin are found.

S

I

S

tl

KERATINS

S

I s

tttl

SSSS

tttr

SSSS

tttl

Keratins proteins arestructural foundin hair, s k in, nail s , h o rn s e tc . T h e 3 p o l y p e p ti desof keratin form a-helical structure and are held togetherby disulfide bonds. The toughnessand strength of keratin are directly related to the num ber o f d i s u l fi d e b o n d s . T h u s , th e harder keratin possessesmore disulfide bonds. The mechanical strengthof the hair is attributedto dis ulf ideb o n d s . Hair waving

J."0,"*on llllr l

SH

SH

SI-I

SH

SH

SH

SH

SH

SH

SH

SFI

SH

rrtttl

(curling)

When the hair is exposedto moist heat, the a-helices of a-keratin can be stretched. This results in the formation of p-conformatioh from cx,-helices.On cooling, the hair structure is reverted back to o-conformation. This property of u- and p-conformations of keratin is exploited in hair wa v i n g o r c u rl i n g . The hair to be curled is first bent to appropriateshape.By applyinga reducingagent, the disulfide bonds (of cystine)are convertedto sulfhydyl groups (cysteine).This results in the uncoiling of a-helical structure.After some time, Fiq.22.2 : A diagrammaticrepresentationof hair waving th reducing agent is removed,and an oxidizing with suitable altentions in keratin structure f- S S agent is added. This allows the formation of conesponds to disulfide bonds of cystine; - SH some new disulfide bonds between cysteine indicates sutfhydryl groups of cysteine) residues(Fig.22.21. The hair is now washed and cooled. The desiredcurls are formed on the hair due to new disulfidebonds and alteredo-helical geneticdisordersnamely mucopolysaccharidoses structureof keratin. lt may however, be noted (Chapter l3). that a permanentcurling of hair is not possible. The new hair that grows will be the native or iginal ha i r o n l y (w i th o u t c u rl s ). The proteins that are involved in the PROTEOGLYCANS movement of body organs (e.g. muscle, heart, proteins. lt is Proteoglycans are conjugated proteins lung) are regardedas contractile to understand the basic structureof containing glycosaminoglycans (CACs). Several worthwhile proteins. learning the contractile proteoglycanswith variations in core proteins muscle before and CACs are known e.g. syndecan,betaglycan, aggrecan,fibromodulin.For more informationon STRUCTUREOF MUSCLE the structure and functions of proteoglycans Muscleis the single largestfissueof the Refer Chapter 2. CAGs, the components human bodv. Muscle constitutesabout 20% of of proteoglycans, are affected in a group of body mass at birth, 4O"/oin young adults and

AND BODY FLUIDS Chapter 2P : TISSUEPFIOTEINS

497

(A)

Musclefibre

H band

Zline

1- , pt {

(B)

I band

A band

I band

I

Z band

Zband ;-5--; t'--'-' 2300nm -"'--''' (Sarcomere)

:.-..-- 15OO -i n^ (Sarcomere)

Exrendedform

Contracted

Fig. 22.3 : (A) Structureof myofibrilof a straitedmuscle(B)Anangementof filamentsof myofibrilin ertendedand (Note : The length of sarcomere is reduced from

3oo/oin found smooth. striated striated.

1m to

aged adults.Three types of musclesare membrane,the sarcol emma.The muscl e fi bre in vertebrates-skeletal,cardiac and cells are long which may extendthe entire length The skeletal and cardiac muscles are of the muscle. The intracellular fluid of fibre cells while the smooth muscles are non- is the sarcoplasm(i.e. cytoplasm)into which the myofibrils are embedded. The sarcoplasmis rich The structureof striatedmuscle is represented in glycogen, ATP, creatine phosphate,and the in Fig.22.3. lt is composed of bundles of enzymes of glycolysis. m ult inuc leat e dmu s c l e fi b re c e l l s . E a c h c e l l i s W hen the myofi bri l i s exami ned under surroundedby an electrically excitable plasma electron microscope, alternating dark bands

I

B IOGH E MIS TR Y

492

a) nn..OOOOqOOOO -

oQ oouo o o Xo-n"o U c-actin O

Assembledthin fliament

Fig. 22.4 : A diagrammaticrepresentation of the thin filamentof a sarcomere.

( anis o tro p i c o r A b a n d s ) a n d li ght bands (isotropic or I bands) are observed. The less dense central region of A band is referred to as H ban d (o r H l i n e ).A n a rro w a n d d enseZ l i ne bisectsthe I band. The regionof the musclefibre between Wvo Z lines is termed as sarcomere. S ar c o me rei s th e fu n c ti o n a lu n i t o f muscl e.

More than 20oh of the muscle mass is composedof proteins.This is largelycontributed by structuralproteinsnamely actin, myosin, and the actin cross-linkingproteins,tropomyosinand troponi n. Muscl e al so contai nsother protei n smyoglobin, collagen,enzymesetc.

ln the electron microscopy, it is further observed that the myofibrils are composed of ACTIN t hic k a n d th i n l o n g i tu d i n a fi l l a m e n ts.The thi ck filaments contain the protein myosin, and are Actin is a major constituentof thin filaments confined to A band. The thin filamentslie in the of sarcomere.lt existsin two forms- monomeric I band, and can extend into A band (but not into G-actin (i.e. globular actin) and polymeric H line).Thesethin filamentscontain the proteins F-actin (i.e. f lament actin). C-actin constitutes actin, tropomyosinand troponin. about 25"/" of the muscle proteinsby weight. In presenceof Mg2+ ions, C-actin polymerizes the During the course of muscle contraction, the (non-coval entl y)to form an i nsol ubl e double thick and thin filaments slide over each other (sliding filament model of muscle contraction). hel i cal F-acti n w i th a thi ckness of 6-7 n m (Fig.22.4). Consequently,the H bandsand I bandsshorten. However, there is no change in the length of Tropomyosin and troponin : These tlvo are thick and thin filaments.The lengthof sarcomere cross-l i nki ngprotei nsfound i n associ ati onw it h which is around 2300 nm in an extendedform actin. Although,minor in termsof mass,they are of m y o fi b ri l i s re d u c e d to 1 5 0 0 nm i n a important in terms of their function. contracted form (Fi9.22.38). Tropomyosin,composedof two chains,attaches

493

Ghapter 22 : TISSUEPROTEINSAND BODY FLUIDS

to F-actin in the grooves (Fig.22.a). Troponin consistsof three polypeptidechains- troponin T (TpT binding to fropomyosin),tropinin I (TpI that inhibits F-actin mvosin interaction) and troponin C (TpC, calcium binding polypeptide). T pC is c o mp a ra b l eto c a l mo d u l i n . MYOSINS Myosinsare actuallya family of proteinswith about 1 5 me mb e rs . T h e m y o s i n that i s pr edom i n a n tl yp re s e n ti n mu s c l e i s m yosi n l l .

Light

and heavy

meromyosins

When myosin is digested with trypsin, two fragmentsnamely light meromyosin(LMM) and heavy meromyosin(HMM) are produced. Light meromyosinrepresents the a-helical fibresof the tail of myosin, and cannot bind to F-actin. Heavy meromyosincontains the fibrous and gl obul ar porti ons of myosi n. H MM i nhi bi t s ATPaseactivitv and binds to F.actin.

On digestion by papain, heavy meromyosin yields two sub-fragments S-1 and S-2 (HMM S-1, In terms of quantity, myosin constitutes HMM S-2).HMM S-2 fragmentis fiber-like,does approximately 55% of muscle protein, and is not bind to F-actinand has no ATPaseactivity. found in thick filaments.Myosin is composedof On the other hand, HMM S-1 is globule-like, six polypeptidechains(hexamer).lt containsone binds to L-chains,and possesses ATPaseactivity. pair of heavy (H) chains, and two pairs of light ( L) c hain s . MUSCLE CONTRACTION Limited digestionof myosin with trypsin and papain has helped to understandits structureand function (Fig.22.5).

A n outl i ne of the reacti onsi nvol vi ngmuscl e contraction is depicted in Fi9.22.6, and briefly described in the next page.

494

B IOC H E MIS TR Y

Sources nf ATF fcr fr!trscie coritrai: lt€r$ ATP is a constantsourceof energyfor muscle contraction and relaxation cycle. ATP can be generatedfrom the following ways. . By substratelevel phosphorylationof glycolysis usi ng gl ucoseor gl ycogen. . By oxidative phosphorylation. . From creatine phosphate. OTH E R P R OTE IN S Fiq.22.6 : Major biochemical events occurring during a cycle of muscle contraction and relaxation (The numbers 1-5 represent the steps in muscle contraction; The zig zag rounds indicate high energy states).

OF MU S C LE

There are a large number of other proteins that are involved in the structureand functions of muscl e. These i ncl ude ti ti n, nebul i n, dystrophin,calcineurinand desmin. Titin is the largest protein known. The gene coding for dystrophin is the largestgene (2,300 bp).

1. Dur i n g th e re l a x a ti o n p h a s e o f muscl e Museul ar dystrcphl + contraction,the S-1 head of myosin hydrolyses Muscular dystrophyis a hereditarydiseasein A T P t o A D P a n d P i . T h i s re s u l tsi n th e fo r mati on which musclesprogressivelydeteriorate.This is high energy ADP-P| myosin complex. causedby mutationsin the gene (locatedon X2. On contraction,the musclegetsstimulated chromosome)coding for the protein dystrophin. (through the participation of actin, Ca2*, troponin,tropomyosinetc.) to finally form actin- P R OTE IN MIS FOLD IN G m y os in- A D P -Pic o mp l e x . AND DISEASES 3. The next step is the power sfroke which drives movementof actin filamentsover myosin filaments.This is followed by the releaseof ADP and P i, an d a c o n fo rma ti o nc h a n g e i n m yosi n. The actin-myosincomplex is in a low energy state. 4. A fresh molecule of ATP now binds to form actin-myosinATP complex. 5. Actin is released,as myosin-ATPhas low affinity for actin. This step is crucial for relaxationwhich is dependenton the binding of ATP to actin-myosincomplex. A fresh cycle of muscle contraction ano relaxationnow commenceswith the hydrolysis of ATP and the formation of ADP-Pi-myosin complex. lt has to be noted that it is ultimately the ATP that is the immediatesource of energy for muscle contraction-

The processof proteinfolding is complex and has been briefly described in Chapter 25. Sometimes,improperly folded proteins may be formed (either spontaneous or by gene mutati ons). S uch mi sfol ded protei ns usual l y get degraded within the cell. However, as the individuals d1e, the misfolded proteins accumulate and cause a number of diseases. Prion diseasesand amyloidosis,two groups of diseasesdue to protein misfolding are briefly d i scussed. Fri*n

,-diar.*#s*ti

The term prion represents proteinous infectious agents. Prion proteins ern are the altered forms of normal proteins. However, no differencesin the primary structure(i.e. amino acid sequence) and post-translationalmodifications are observed.

49s

Chapter 22 : TISSUEPFOTEINSAND BODY FLUIDS Certain changes in three-dimensional structureare seen in prion proteins.The major alteration is the replacement of a-helices by p-sheets in PrP. This confers resistance to proteolytic digestion of prion proteins. PrP are highly infectious, and can act as template to convert non-infectiousproteins (with cl-helices) to infectious forms (Fig.22.V. This process c ont inu e s i n a n e x p o n e n ti a l m a nner to accumulatea large number of prion proteinsin tissues.

cr-Helixof a protein (non-infectious)

lnfectiousprion (withp-sheets)

Prion proteins are implicated as causative agentsin the following diseases. . Transmissible spongiform encephalopathies (TSEs) and Creutzfeldt lacob disease in hum a n s . . Scrapie in sheep . Bovine spongiformencephalopathy(popularly known as mad cow disease)in cattle. Kuru is an interesting prion disease. lt was first reportedin PapauNew Guinea in the tribal people who practice cannibalism (they eat the brains of the dead people). As of now, there is no treatment for prion diseases.Transmissiblespongiform encephalopathiesare invariablyfatal in humans. Arnyloidosis

prions Twomolecules of infectious (withp-sheets)

II

J

The term amyloids is used to refer to the Thesetwo moleculesseparateandconvert proteinsto altered proteins (with B-sheefs)that accumulate anothertwo non-infectious infectiousprions in the body, particularly in the nervous system. Amyloids are formed by protein misfolding or due to gene mutations.They are not infectious agents as prion proteins. However, as the age advances,amyloids accumulate,and they have been implicated in many degeneratingdiseases. A total of at least 15 different proteins are Fig. 22.7 : A model for the formation of infectious prions inv olv ed i n a m v l o i d o s i s . (Redthick linesrepresentp-sheetsin protein). Afzheimer's disease is a neurodegerative disorder, affecting about 5-'l 0'h of the people above 60 years of age. lt is characterized by m em or y l o s s , c o n fu s i o n , h a l l u ci nati ons, personalitychanges with abnormal behaviour. the patient may enter As the diseaseprogresses, a vegetative state, and may die after 10 years after the onset of the diseasemanifestations.The

accumulation of amyloids (in the form of amyloid plaque) has been clearly demonstrated in the patientsof Alzheimer'sdisease. A specificprotein,namely p-amyloidwhich is prone for self aggregation is believed to be the causative agent of Alzheimer's disease,

B IOC H E MIS TFIY

496

Constituent

Water Totalsolids(gidl)

Buffalo

Human

Cow

87.6

87.2 12.8

83,5 16.5

87.0

4.4

5.4

4.6

U.U

o.c

a(

3.3

4.3

3.7

12.4

(g/dl) Carbohydrates (gidl) Lipids (g/dl) Proteins

7.5 3.8 1.1

(mg/dl) Calcium (mg/dl) Magnesium (mg/dl) Phosphorus (mg/dl) Sodium (mgidl) Potassium

35 2.2 16 15 cc

150 13 100

160 10 100

60 140

60 130

13.0

175 8 70 50 85

p-Amyloid is formed from a conformational carbohydrates,lipids, proteins, minerals and transformation of a-helix. Apolipoprotein E vi tami ns. promotes the conformational change of in nnilk Garbohydrates a-amyloid to p-amyloid. Milk containsthe disaccharidelactosewhich imparts sweetness.Human milk has a higher concentration of lactose (7'5%) compared to milk of other species.Thus, human milk is sweet milk, are the body of fluids The specialized enough for the babies to relish. Milk sugar aqueous fl u i d , fl u i d , a mn i o ti c (lactose)servestwo major functions' c er ebr o s p i n a l perspective, In a broader and tears. humor, sweat 1 . lt providesgalactose,a structuralunit for blood, plasma and serum are also biological the growing infant. fluids.Their biochemicalimportanceis discussed 2. In the intestine, it gets metabolized to elsewhere (Hemoglobin, Chapter 10; Plasma acid which eliminatesharmful bacteria' lactic enzymes, Diagnostic 9; proteins, Chapter 2l). Chapter balance, Chapter 6; Acid-base Li pi ds !n mi l k Ur ine is a n e x c re to ryb i o l o g i c a lfl u i d . The l i pi ds i n the mi l k are di spersedas smal l gl obul es. Mi l k fat i s mai nl y composed of M ilk i s s e c re te db y ma mma ry g l ands. l t i s triacylglycerols.Mono- and diacylglycerolsare almost a complete natural food. Milk is the only also present in trace quantities.The fatty acids food for the offspringsof mammalson their birth. found i n mi l k (i .e. i n TG) are mostl ymedi um or short chai n, and saturatede.g. pal mi ti c aci d, myristicacid, stearicacid, lauric acid and butyric OF M'LK COMPOSIT'ON acid. Oleic acid, an unsaturatedfatty acid, is The major constituentsof milk in different also present. species-human, cow, buffalo and goat are given in Table 22.1. Water is the major P rotei ns i n mi l k constituent,with a concentrationin the rangeof The major milk proteins are casein (about 83-87o/o, depending on the species. The of r em aini n g 1 3 -1 7 % i s m a d e u p o f sol i ds- 80% ) and l actal bumi n.S mal l concentrati ons M I LK

Chapter 22 : TISSUEPROTEINSAND BODY FLUIDS

497

enzymes (proteases,lipase, xanthine oxidase, spaces surroundingthe brain and spinal cord. lysozyme)and immunoglobulinsare also found. The major functions of CSF are listed. Milk casein (a phosphoprotein)is almost a . CSF servesas a hvdraulic shock absorber.lt complete protein (next to egg albumin), can diffuse the force from a hard blow to the c ont aining a l l th e e s s e n ti a la m i n o a c i d s. l t i s skull that might otherwisecausesevereinjury. present in milk in the form of aggregatescalled r lt helps in the regulation of intracranial micefles. The white colour of milk is due to the pressure. dispersion of calcium caseinate micelles. o lt is believed that CSF influencesthe hunger Whey proteins : lf milk is acidified, casein sensationand eating behaviours. gets precipitated at isoelectric point (pH 4.2). The supernatantfluid contains whey proteins Collection of GSF (2O% of milk proteins). These include lactalbumin,lactoglobulinand variousenzymes. Cerebrospinalfluid is usually collected by a spinal puncture for the purposeof biochemical Minerals in milk analysis. The puncture is performed in the M ilk is r i c h i n c a l c i u m , ma g n e s i u m,phos- lumbar region, betweenthe third and fourth, or phorus, sodium, potassium and chlorine. between the fourth and fifth lumbar vertebrae. However, milk is a poor source of iron and coPper.

The sterile lumbar puncture (spinal tap) is carried out in a side lying (lateral)position with headfixed into the chestand knees.This position V it am ins i n mi l k helps to increasethe space between the lumbar vertebrae so that the needle can be insertedwith Both fat soluble and water soluble vitamins position of the patient with head ease. A sitting are found in good concentration in milk. flexed to chest can also be used for lumbar However mifk is deficient in vitamin C. puncture.., Galorific

value

of milk

Gomposition of GSF in health and Due to variabilityin the nutrientcomposition disease (carbohydrates,fats and proteins), the calorific The normal compositionof cerebrospinalfluid value of milk from different speciesvaries. Thus, given is in the Table 22.2. From the diagnostic human milk can provide about 70 Cal/l 00 ml, point of view, the total cell count of lymphocytes while for buffalomilk, it is around95 Cal/100ml. (Reference: 0-5 x 106/1),protein concentration (1545 mg/dl) and glucoseconcentration(45-85 GEREBROSPTNAL FLUID (CSF) mg/dl) are important. Cerebrospinal fluid is a clear, colourless In the lable 22.3, Ihe major alterationsin the liquid formed within the cavities (ventricles)of brain and around the spinal cord. CSForiginates CSF in the diseasestatesare given. The total cell in the choroid plexus (as an ultrafiltrate of count and protein content are increased while plasma)and returnsto blood through arachnoid glucose concentration is reduced in tuberculosis villi. About 500 ml of CSF is formed everyday. meningitis. In case of brain tumors, there is no However, at any given time, there is about change in total cell count while the protein 120-150 ml CSF in the system.Further,CSF is concentrationmay be marginally increased. completely replacedabout three times a day. The colour and appearance of CSF is sometimes a guiding factor in the disease Functions of CSF diagnosis. For instance, CSF is opalescent As the brain has no lymphatic system,CSF and slightly yellow coloured in tuberculosis drains into the ventricularsystemand movesinto meni ngi ti s.

BIOCHEMISTFIY

498

Functions of amniotic fluid a a

D escr ip ti otdco n cent r ation

Parameter

Volume 9f150 ml Appearance Clearandcolourless gravity 1.006-1.008 Specific 28f290 mOsrn/kg Osmolality Totalcellcount(lymphocytes) G-5x 105/l pH 7.T7.4 Protein 15-45mg/dl l/G ratio(albumin/globulin) 8: 1 45-85mg/dl Glucose 118-130 mEq/l Chloride 2.1-2.7nEq/. Calcium 145-155mEq/l Sodium Potassium 2.0-3.5 mEq/l

A M NI O T IC

F L U ID

A m n i o ti c fl u i d i s a l i q u i d p ro d u ced by the membranesand the fetus. lt surroundsthe fetus throughout pregnancy.The volume of amniotic fluid increaseswith the gestationalage. Thus, the volume increasesfrom 30 ml (at 2 weeks of gestation)to 350 ml (at 20 weeks),and thereafter t o 500-' 1 0 0 0m l . A m n i o ti c fl u i d i s a lmostcl ear with some desquamatedfetal cells and a little lipid.

Colour and aPPeafance

Normal

Clearandcolourless

It provides physical protectionto the fetus. Amniotic fluid is a medium for the exchange of various chemicals.

importance Diagnostic fluid amniotic

of

The term amniocentesis is used for the processby which amniotic fluid is collectedfor analysis.The diagnosticimportanceof amniotic fluid is given below. Assessmentof fetal maturity : Fetal maturity can be assessedby cytological staining of fat cells, and estimationof creatinineconcentration (> 1 .6 mg/dl indicatesfetal maturity). Lung maturity : The fetal lung maturity is evaluated by measuring lecithin+phingomyelin (L/S) ratio. A L/S ratio of 2 :1 or more indicates lung maturity. lf L/S ratio is lessthan 1.2 : 1, it is better to delay the induced delivery until the lung has become more mature. Diagnosis of congenital disorders : Amniotic fluid analysisis usefulfor the prenataldiagnosis of congenital disorders.Some of the important ones are listed. . Chromosomal disorders such as Down's syndorme. . Metabolic disorderse.g. cystic fibrosis. . S ex-l i nkeddi sorderse.g. hemophi l i a. . Enzymedefectse.g. Tay-Sachsdisease.

Glucose

Total cell count

0-5 x 106/l

Tuberculosis meningitis Opalescent andslightly lncreased yellow

15-45mg/dl

4$€5 mg/dl

Increased

low Relatively

Bacterial meningitis

Opalescent andturbid

decreased increasedMarkedly increasedMarkedly Markedly

Braintumour

Clearandcolourless

Nochange

lncreased

Low

RBCandWBC present

lncreased

normal Almost

hemorrhage Slightly bloodcolour Subarachnoid

Chapter €f, : TISSUEPROTEINSAND BODY FLUIDS ,dssessment of hemolytic diseases: Estimation of bilir ub i ni n a mn i o ti cfl u i d i s u s e fu lto eval uate the severityof hemolytic diseases.

499

AQUEOIIS HUruOR

A queous humor i s the fl ui d that fi l l s the anteri or chamber of the eye. Thi s fl ui d Measurement of a-fetoprotein : Increased i s responsi bl efor mai ntai ni ngthe i ntraocul ar levels of cr-fetoprotein(normal 154O 1t{ml tension. Aqueous humor, secreted by the during gestation;at 40 weeks < 1.0 pg/ml) are ciliary body, enters the anterior chamber. associatedwith neuraltube defects,fetal distress, B l ockade i n the fl ow of aqueous humor Turner syndrome. Elevated a-fetoprotein may causes glaucoma due to increasedintraocular pressure. also indicate a possibledeath of the fetus.

atoMEDICAL,/CLilUIGALGO|UCEtrTS

lmproper formation of collogen is associatedwith certain genetic diseasese.g. EhlersDanlos sgndrome(obnormal tissuefragilitg), osteogenesisimperlecto(abnormallragility of bones). Defectiueformation of collagen is obseruedin scuruy,causedby uitamin C deficiency. This resuhs in bleeding of gums and poor wound healing. Hair wauing(curling) through artificial means is possible with suitable alterationsin the structure of keratins. Muscular dystrophy occurs due a mutation in the gene coding lor the protein dystrophin. Protein misfolding results in prion diseoses(e.g. mad cow disease)and amyloidosis (Alzheimer's disease). Biochemical onolysis of cerebrospinal Jluid is uselul t'or the diognosis of certoin -tuberculosis meningifis (increosed protein and decreased glucose diseoses concentrations). Amniotic fluid is analysedfo ossessfetal maturitg (creatinineconcentration> 7.6 mg/ dl), Iung maturity (lecithin-sphingomyelin ratio > 2 : 1) ond for the prenatal diognosis of congenital disorders(e.9. hemophilia, Down's syndrome).

500

B IOC H E MIS TFI Y

1. The maJor proteins of connectiue tissue are collagen, elastin, fibrillin, Iaminin and proteoglgcans.Among these, collagen is the most abundant, constituting one-third of the total body proteins.

2. Type I mature collagen is a triple helicol structure i.e. contoinsthree polypeptlde chains each with about 1000 omino acids. The repetitiue amino acid sequenceof collagen is (Glv-X-Y)". Glycine constitutes about 7/3 rd of the qmino acids while X and Y represent other amino acids.

3. Kerotins are structural proteins t'ound in hair; skin, nails and horns. The strength oJ the keratins is directly related to the number ol disulfide bonds.

4. Muscle is the single largest tissue of the human body (3040o/o of body weight). lt is composed of t'ibre cells into which myofibrils are embedded. Each myot'ibril contains alternoting A and I bands. Sarcomere is the functional unit of muscle.

5. Actin, myosin, tropomyosin qnd troponin ore the major controctile proteins t'ound in muscles.The muscle contraction and relaxation occur due to the actiue inuoluementof these proteins. ATP is the immediate source ot' energy lor muscle contractlon.

6. Proper folding of proteins is essentialt'or thetr structure. Mislolding ol proteins results in certsin diseosese.g. mad cow disease,Alzheimer's disease.

7. The specialized fluids of the body include milk, cerebrospinalfluid, amniotic t'luid, aqueous humor, sweat and tears.

8. Milk is almost a complete t'ood with uariousnutrients---carbohydrotes,Iipids, proteins, uitamins and minerals. Howeue4 milk is deflcient in uitamin C, iron and copper.

9. Cerebrospinal t'luid is an ultrat'iltrate oJ plosma. ln the disease,tuberculosis meningitis, the total cell count and protein concentrationare increesed,while glucoseconcentration is decreased tn CSF

10. Amniotic fluid is a liquid produced by the t'etus. lts biochemicalonolysis is importont for the diagnostic purpose-ossessment of |etal maturity, diognosis of congenital diseqses.

Ghapten 22 : TISSUEPHOTEINSAND BODY FLUIDS

501

I. Essayquestions 1. Cive an account of the structureand functionsof collagen.Add a note on the abnormalities associated with collagen. 2. Describethe muscleproteins,and musclecontraction. 3. Discussthe proteinmisfoldingand variousdiseasesrelatedto it. 4. Give an accountof the compositionof milk. 5. Describethe functionsand compositionof cerebrospinalfluid. Add a note on the alterationsin CSF in diseasedstates. II. Short notes (a) Biosynthesis of collagen,(b) Collagenand scurvy,(c) Elastin,(d) Light and heavy meromyosins, (e) Priondiseases, (fl Amyloidosis, (g) Hair waving,(h) Vitaminsand mineralsin milk, (i) Collection of CSF,(j) Amniotic fluid. III. Fill in the blanks 1. The most abundantproteinin mammals 2. The amino acid that contributesto one-thirdof the total numberof amino acids in collagen 3. The toxic compound that interfereswith the cross-linkingof lysine in collagen,causing lathyrism 4. Marfan syndromeis a geneticdisorderdue to a mutationof the gene coding for 5. Name the carbohydratesassociatedwith the structureof proteoglycans 6. The region of the muscle fibre betweentwo Z lines is termed as 7. Name the major proteinfound in the structureof thin filamentsof sarcomere 8. The white colour of milk is due to the dispersionof 9. Name the vitamin deficientin milk 10. The fetal lung maturityis evaluatedby measuring

ratio.

IV. Multiple choice questions 11. The numberof polypeptidechainspresentin collagen (a) 1 (b) 2 (c) 3 d\ 4. 12. The functionalunit of muscle (a) Fibrecell (b) Myofibril (c) H band d) Sarcomere. 13. The immediatesourceof energyfor musclecontraction (a) ATP (b) Creatinephosphate(c) CTP d) Phosphoenolpyruvate. 14. O n e o f th e fo l l o w i n gmi n e ra l si s l acki ngi n mi l k (a) Calcium(b) Sodium(c) lron d) Potassrum. 15. One of the following biochemicalparameters is increasedin tuberculosis meningitis (a) Clucose(b) Protein(c) Sodiumd) Chloride.

T|rc nattitlon

spea,hs t

"Sorne eat to liae, And some liae to eat!

Ml,function is Tocaterforall," '

hether a man eats for living or lives for e a ti n g , fo o d i s h i s p ri me concern. Nutrition may be defined as the utilization of food by living organr'srns.Biochemists have largely contributedto the scienceof nutrition.

nutrition, undernutrition and areas-ideal overnutrition ldeal nutrition is the concern of everyone.U ndernutri ti oni s the pri me con cer n of devel opi ngcountri esw hi l e overnutri ti o nis a seriousconcern of developedcountries.

N u tri ti o n s i g n i fi c a n tl y p ro motes man' s development,his healthand welfare.The subject nutrition,perhaps,is the most controversial.This is due to the fact that nutrition is concernedwith food, and everyone feels competent enough to t alk l i k e a n e x p e rt o n n u tri ti o n . Further,hi gh public awarenessand the controversialreports by scientistsalso contributeto the controversy.

A sound knowledgeof chemistryand metabolipids, proteins, lism of foodstuffs(carbohydrates, prereq uisit e vi tami nsand mi neral s)i s an essenti al of nutrition.The reader for a betterunderstanding must, therefore,first refer these chapters.The principlesof nutrition with special referenceto energy demands, carbohydrates,fats, proteins, recommended dietary/daily allowances (RDA), bal anced di et and nutri ti onal di sorders ar e Methodology in nutrition : Most of the i n the fol l ow i ng pages. di scussed ex is ti n g k n o w l e d g e o n n u tri ti o n i s ori gi nal l y der i v e d fro m a n i m a l e x p e ri m e n tati on.Thi s i s despite the fact that there may exist several differences in the biochemical composition between man and animals! For instance,some ani ma l sc a n s y n th e s i z e a s c o rb i caci d w hi l e man Food is the fuel source of the body. The cannot do so. ingestedfood undergoesmetabolismto liberate Study of human nutrition : The study of energy required for the vital activities of the nut ri ti o n m a y b e l o g i c a l l y d i v i ded i nto three bodv.

502

Otraster 23 : NUTFI|TION

ffivtr CrHrydrate h Pr*il Abdd

503

Energy value (Cal/g) In bomb calorimeter In the body

4.1 9.4 5.4 7.1

It must be noted that the nutrients,namely vitamins and minerals,have no calorific value, although they are involved in severalimportant body functions, including the generation of energy from carbohydrates,fats and proteins.

4 I

Respiratory quotient of foodstuffs

4

The respiratoryquotient (R. Q.l is the ratio of the volume of CO2 produced to the volume of 02 utilized in the oxidation of foodstuffs.

'7

Energy content of foods The calorific value (energycontent)of a food ;s calculatedfrom the heat releasedby the total combustion of food in a calorimeter.

Carbohydrates : The carbohydrates are completely oxidized and their R. Q. is close to 1, as representedbelow for glucose.

C6H12O6+ 6C2 ---+ 6COz + 6HzO Unit of heat : Calorie is the unit of heat. One caforie representsthe amount of heat required to = =t=,. R.Q. forcarbohydrate + z rilr the temperature ol one gram of water by loC i. e. f r om 1 5 o to 1 6 " C ).A c a l o ri ei s to o smal l a Fats : Fats have relatively lower R.Q. since unit. Therefore,it is more convenientlyexpressed they have a low oxygen content. For this reason, as kilocalories (1,000 times calorie) which is fats require more 02 for oxidation. The R.Q. represented by kcal or simply Cal (with capital 'C'). for the oxidation of the fat, tristearin is given The joule is also a unit of energy used in below. some countries. The relationship between 2 C57HrgO6+ 16 3O2------+114 CO2+ 110 H2O caloriesand joules (J) is (-(-l tu2 1 Cal (1 kcal) = 4.128 Kl = 114 = 0.7. R. Q. for fat = o" 163 The joule is /ess commonly used by Proteins : The chemical nature of proteins is nutritionists. highly variable, and this cannot 6e represented Calorie value of foods : The energy values of by any specific formula. By indirect the three principal foodstuffs-carbohydrate, fat measurements, the R.Q. of protein is found to be and protein-measured in a bomb calorimeter around 0.8. and in the body are given in the Table23.1.The Mixed diet : The R. Q. of the diet consumed carbohydratesand fats are completely oxidized (to CO2 and H2O) in the body; hence their fuel is dependent of the relative composition of values,measuredin the bomb calorimeteror in carbohydrates,fats and proteins.For a normally the body, are almost the same. Proteins, ingesteddiet, it is around 0.8. however, are not completely burnt in the body as they are convefted to products such as urea/ creatinineand ammonia, and excreted.Due to this reason,calorific value of protein in the body is lessthan that obtained in a bomb calorimeter. Man consumes energy to meet the fuel The energy values of carbohydrateg fafs and demands of the three ongoing processess in the proteins(when utilized in the body) respectively, body. are 4, 9 and 4 Cal/9. 1 . Basal metabolic rate Alcohol is a recent addition to the calorie 2. Specificdynamic action 1ZCal/e)contribution,as it is a significantdietary 3. Physicalactivity. component for some people.

B IOC H E MIS TFIY

504 Besidesthe above three, additional energy supply is neededduring growth, pregnancyand lactation.

BASAL METABOLIC RATE Basal metabolism or basal metabolic rate (BMR) is defined as the minimum amount of energy required by the body to maintain life af complete physical and mental rest in the postabsorptivestate(i.e. 12 hoursafterthe lastmeal). ft may be noted Ihat resting metabolic rate (RMR) is in recent use for BMR. Under the basal conditions, although the body appears to be at total rest, several functions within the body continuously occur. These include working of heart and other nerve impulse, organs, conduction of reabsorption by renal tubules, gastrointestinal motility and ion transport across membranes (Na+-K+ pump consumes about 50% of basal energy).

Measurement of BMR

For the calculationof body surfacearea, the si mpl eformul adevi sedby D u B oi sand D u B ois i s used. A = H0 72sx W0.42sx 71.84 where A = Surfacearea in cm2 H = H ei ght i n cm W = Weight in kg. To convertthe surfacearea into squaremeters (m2),di vi de the above val ue (cm2) by 10,000 . Nomogramsof body surfacearea (directlyin m2) from heightsand weightsare readilyavailablein literature. Normaf values of BMR : For an adult man 35-38 Cal/sq. m/hr; for an adult woman 32-35 Cal/sq.m/hr. A BMR value between -157o and +20o/ois consideredas normal. Some authors continue to representBMR as Cal/day. For an adult man BMR is around 1,600 Cal/day, while for an adult woman around 1,400 Cal/day.This is particularlyimportantfor easily calculatingenergy requirementsper day.

Preparationof the subject : For the Factors measurementof BMR the subject should be awake, at complete physicaland mental rest, in a post-absorptive state and in a comfortable s ur r oun d i n g(a t 2 5 ' C ). Measurement: The BMR is determinedeither by the apparatus of Benedict and Roth (closed circuit device) or by the Douglas bag method (open circuit device). The former is more frequently used.

affecting

BMR

1. Surfacearea : The BMR is directly proportional to the surfacearea. Surfacearea is related to weight and height. 2. Sex : Men have marginally higher (about 5% ) B MR than w omen. Thi s i s due to the hi gher proportion of lean muscle mass in men. 3. Age : ln infantsand growing children,with l ean muscl emass,the B MR i s hi gher.In adul ts, BMR decreases at the rate of about 2o/o per decade of life.

By Benedict-Rothmethod, the volume of 02 consumed (recordedon a graph paper) by the subject for a period of 2-6 minutes under basal 4. Physical activity : BMR is increased in conditions is determined.Let this be A liters for persons(notably athletes)with regular exercise. 6 minutes.The standardcalorific value for one This is mostly due to increasein body surface liter 02 consumed is 4.825 Cal. area. Heat produced in 6 min = 4.825 x A

5. Hormones: Thyroid hormones(T3and Ta) have a stimulatoryeffect on the metabolismof Hea t p ro d u c e di n o n e h o u r = 4 .8 25A x 10 the body and, therefore, BMR. Thus, BMR is Units of BMR : BMR is expressedas Calories raised in hyperthyroidism and reduced in per square meter of body surface area per hour hypothyroidism. In fact, the measurement of BMR was earlier used to assessthvroid function. i.e. Callsq.m/hr.

Ghapter 23 : NUTRITION

505

The other hormones such as epinephrine, However, when 25 g protein is utilized by the cortisol, growth hormone and sex hormones body, 130 Cal of heat is liberated.The extra 30 increaseBMR. Cal is the SDA of protein.Likewise,consumption of 100 C al of fat resul tsi n 113 C al and 100 C a l 6. Environment: In cold climates,the BMR is of carbohydratein 105 Cal, when metabolized higher compared to warm climates. in the body. SDA for protein, fat and Z. Starvation : During the periods oJ carbo)>yltale a/e 32%t /3% and f%, starvation, the energy intake has an inverse respectively. Thus, proteins possess the highest relation with BMR, a decreaseup to 50% has SDA while carbohydrates have the lowest. been reported.This may be an adaptation by the SDA for mixed diet : For a mixed diet, the body. SDA is not an additive value of differentfoods 8. Fever : Fevercausesan increasein BMR. but it is much less. The presenceof fats and An elevation by more than 1O'/' in BMR is carbohydratesreducesthe SDA of proteins.Fats observedfor every 1'C rise in body temperature. are most efficient in reducingSDA of foodstuffs. For a regularlyconsumedmixed diet, the SDA is 9. Diseasestates : BMR is elevated in various around l0o/". infections, leukemias, polycythemia, cardiac Significance of SDA : For the utilization of failure, hypertensionetc. In Addison's disease (adrenal insufficiency), BMR is marginally foods by the body, certain amount of energy is consumedfrom the body stores.This is actually lowered. an expenditureby the body for the utilizationof 10. Racialvariations: The BMR of Eskimosis foodstuffs.lt is the highestfor proteins (30o/') and m uc h hig h e r.T h e BMR o f O ri e n ta lw o men l i vi ng lowest for carbohydrates(5%) and for a mixed in USA is about 10% lessthan the averageBMR diet around 10%. lt is, therefore,essentialthat of American women. an additional 10% caloriesshould be added to the total energy needs (of the body) towards Significance of BMR S D A . A nd the di et shoul d be pl anned, BMR is important to calculate the calorie accordingly. (SDA is quite comparable to the requirement of an individual and planning of handling charges levied by a bank for an diefs. Determinationof BMR is useful for the outstationcheque). function. In assessment of thyroid The higher SDA for protein indicatesthat it is hypothyroidism, BMR is lowered (by about not a good source of energy. Fat is the best -40o/o) while in hyperthyroidismit is elevated source of energy due to its lowering effect on (by about +70%). Starvationand certaindisease SDA. However, excessiveutilization of fat leads c ondit io n s a l s o i n fl u e n c e B M R ( descri bed to ketosis. above). Mechanism of SDA : The exact cause of SDA is not known. lt is generallybelievedthat SDA of SPECIFIC DYNAMIC ACTION foods is due to the energyrequiredfor digestion, The phenomenon of the extra heat absorption,transport,metabolismand storageof production by the body, over and above the foods in the body. cafculatedcaloric value, when a given food is Intravenousadministrationof amino acids or metabolized by the body, is known as specific the oral ingestion of proteins gives the same dynamic action (SDA). lt is also known as SDA. This shows that the SDA of proteinsis not calorigenic action or thermogenic action or due to their digestion and absorption. thermic action (effec0 of food. HepatectomyabolishesSDA, thereby indicating SDA for different foods : For a food that SDA is closelyconnectedwith the metabolic containing25 g of protein, the heat production functions of liver. The SDA of proteins is f r om t he c a l o ri c v a l u e i s 1 0 0 C a l (2 5 x 4 C al ). primarily to meet the energy requirementsfor

505

B IOC H E MIS TRY their physical activity and the requirementof enerSy.

Physical activity

(quietly) Sitting (quietly) Standing g/eating/reading Writin Cardriving Typing Household work(dishwashing) (slow) Wdking Sexual intercourse (slow) Cycling (moderate) Running Swimming Walking upstairs

Energy requirement (Cal164

25 30 30 60 75 80 130 140 150 500 600 800

Light work 3040% of BMR (teachers,office workers, doctors) Moderatework (housewives,students)

-

4O-5O"hof BMR

Heavy work 50-60% of BMR (agriculturallabourers,miners)

Veryheavywork

-

60-100%of BMR

(construction workers, pullers) rickshaw Energy requirements of man

As already stated, the three factors-basal metabolic rate, specific dynamic action and physical activity-determine the energy needed by the body. In an individual with light work, about 50% of the calories are spent towards BMR, about 30% for physical activity and about l0% to take care of the SDA.

The daily requirement of energy is rather deamination,synthesisof urea, biosynthesisof variable which depends on the BMR (in turn proteins,synthesisof triacylglycerol (from carbon dependson age,sex,body size etc.)and physical skeleton of amino acids). lt has been activity. As per some rough calculation,caloric demonstrated that certain amino acids requirementsof adults per day (Cal/day)are in (phenylalanine, glycine and alanine)increasethe the following ranges. SDA. lt is a common experience that Light work 2,200-2,500 consumptionof a protein rich diet makesus feel warm and comfortablein cold weather. This is Moderatework 2,500-2,900 due to the high SDA of proteins. Heavy work 2,900-3,500 The SDA of carbohydratesis attributed to the Very heavy work 3,500-4,000 energy expenditurefor the conversion of glucose to glycogen. As regards fat, the SDA may be due to its storage,mobilization and oxidation.

PHYSICAL ACTIVITY OF THE BODY The physical activity of the individual is highly variable. The amount of energy needed for this depends mainly on the duration and intensityof muscularactivity.The expenditureof energy for the various physical activities has been cafculated (Table 23.2\.

Dietary carbohydratesare the chief source of energy. They contribute to 60-70% of total caloric requirement of the body. lncidentally, carbohydraterich foods cost less.

Carbohydratesare the most abundantdietary constituents, despite the fact that they are not essential nutrients to the body. From the For the sake of convenience,the individuals nutritional point of view, carbohydrates are are grouped into four categorieswith regard to grouped into 2 categories.

507

Ghapter 23 : NUTBITION

6. Synthesis of pentoses : Pentoses (e.g. ribose)are the constituentsof severalcompounds in the body e.g. nucleic acids (DNA, RNA), (NAD+, FAD). These pentosesare coenzymes 2. Carbohydratesnot utilized (not di8ested) produced in carbohydrate metabolism. by the body-cellulose, hemicellulose,pectin, gums etc. 7. Synthesis of non-essential amino acids : The intermediatesof carbohydrate metabolism, Among the carbohydrates utilized by the mainly the keto acids (e.g. pyruvic acid), serve body, starch is the most abundant. The as precursorsfor the synthesisof non-essential consumption of starch has distinct advantages ami no aci ds. due to its bland taste, satiety value and slow

1. Carbohydrates utilized by the bodystarch, glycogen, sucrose/ lactose, glucose, fructose etc.

digestion and absorption. Sucrose (the table can be consumedto sugar),due to its sweetness, a limited extent. Excessive intake of sucrose causes dental caries, and an increase in plasma lipid levels is associatedwith many health complications.

8. Synthesis of fat : Excessconsumption of carbohydratesleadsto the formation of fat which is stored in the adipose tissue.

9. lmportance of non-digestible carbohydrates : These are the carbohydrates not utilized by the body. Yet, they are important since they improve bowel motility, prevent Functions of carbohydrates constipation, lower cholesterol absorption and 1. Major sourcesof energy: Carbohydrates improve glucose tolerance (details discussed are the principalsourceof energy,supplying later).

of the body. 60-80%of the caloricrequirements

2. Proteinsparingaction : Proteinsperforma specializedfunction of body building and growth.The wastefulexpenditureof proteinsto meet the energyneedsof the body should be cometo the rescueand curtailed.Carbohydrates proteins for caloric from being misused sparethe purpose.

Glycemic

index

Thereare variationsin the increaseand fall of blood glucose levels after the ingestion of different carbohydratecontaining foods. These quantitative differencesare assayedby glycemic index which measuresthe fime course of postprandial glucose concentrations from a graph. 3. Absoluterequirementby brain : The brain Glycemic index may be defined as the area and other parts of central nervoussystemare under the blood glucosecurve afterthe ingestion dependenton glucosefor energy.Prolonged of a food compared with the area under the hypoglycemiamay lead to irreversiblebrain blood glucose curve after taking the same amount of carbohydrate as glucose. lt is damage. expressedas percentage.

4. Requiredfor the oxidationof fat : Acetyl CoA is the product formed in fatty acid Area under the blood glucosecurve after oxidation.Foritsfurtheroxidationvia citricacid ingestionof test meal cycle,acetylCoA combineswith oxaloacetate, x 100 the latter is predominantlyderived from Glycemic irdo<= Areaunderthe curveafter lt may thereforebe stated'Faf carbohydrates. ingestion of glucose burns in a fuel of carbohydrate'. Excess A graphic representationof high and low utilizationof fats coupledwith deficiencyof glycemic indices is depicted in Fig.2?.l. leadsto ketosis. carbohydrates The glycemic index of a complex 5. Energy supply for muscle work : The (i.e. starch) is lower than a refined carbohydrate glycogen acid to lactic muscle is brokendown (glycolysis)to provide energy for muscle carbohydrate(i.e. glucose).This is explainedon the basis of slow digestion and absorption of contraction.

508

BIOCHEMISTFIY High glycemic index

Time(in minutes)after ingestionof food F19.23.1 : The glycemic index curue after the

complex carbohydrates.Further, the glycemic index of carbohydrateis usually lower when it is combinedwith protein,fat or fiber. The glycemic index of some selected foods is given in Table 23,3. The food item like ice cream has relatively lower glycemic index. This may be explainedon the basis of high fat content which lowers the glucose absorption.

The complex carbohydrates that are nof digestedby the human enzymesare collectively referred to as dietary fiber, These include cellulose, hemicellulose, pectin, Iignin, gums and mucilage. lt may, however, be noted that some of the fibers are digestibleby the enzymes of intestinalbacteria (e.g. pectins,gums). For a long time, fiber was regarded as nutritional waste. And now nutritionists attach a lot of importanceto the role of fiber in human health. The most important beneficial and the adverse effectsof dietary fiber are briefly described. Beneficial

effects

of fiber

1 . Prevents constipation : Fiber helps to maintain the normal motility of gastrointestinal tract (ClT) and preventsconstipation. 2. Eliminatesbacterial toxins : Fiber adsorbs large quantities of water and also the toxic compoundsproduced by intestinalbacteriathat lead to increased fecal mass and its easier expul si on.

3. Decreases GIT cancers : The lower The nutritionalimportanceof glycemic index tract (e.g. incidence of cancersof gastrointestinal is controversial.This is due to the fact that the and rectum) in vegetarians compared to colon foods with low glycemic index need not be non-vegetarians is attributed to dietary fiber. good for health. However, low glycemic index foods usually have higher satiety value (creatinga sense of stomachfulness), and thus m ay be h e l p fu l i n l i m i ti n g th e c a l ori c i ntake. Nutritionists are of the opinion that foods with high fiber content and low glycemic index Glycemic index Food item (e.9. whole grains,fruits, vegetables)should be preferredfor consumption. 100 Glucose Sources

of carbohydrates

Carbohydrates are abundant in several naturally occurring foods. These include table sugar(99oh),cereals(60-80%), pulses(50-60%), roots and tubers (20-40%) and bread (5p-60%).

Requirement of carbohydrates In a well balanceddiet, at least 40% of the caloric needs of the bodv should be met from carbohydrates.

Canots

90-95

Honey

80-90

rice Bread, potato Banana,

7H0 60-70

Sweetpotato

5H0

apples Oranges,

40-45

lcecream, milk

35-40

Fructose

2o-25

Soybeans

15-20

Chapter 23 : NUTFI|TION

509

4. lmproves glucose tolerance : Fiber 15-50% of the body energy requirements. improvesglucosetoleranceby the body. This is Phospholipids and cholesterol (from animal mainly done by a diminished rate of glucose sources) are also important in nutrition. The absorption from the intestine. nutritional and biochemical functions of fat, 5. Reduces plasma cholesterol level : Fiber phospholipidsand cholesterolhave alreadybeen decreasesthe absorption of dietary cholesterol discussed in detail and the reader must from the intestine.Further,fiber binds with the invariably refer them now (Chapters 3 and l4). bile salts and reduces their enterohepatic functions of lipids circulation.This causesincreaseddegradationof Major nutritional cholesterolto bile saltsand its disposalfrom the Dietary lipids have two major nutritive body. functions. 6. Satiety value : Dietary fiber significantly 1. S uppl y tri acyl gl ycerol s that normal l y adds to the weight of the foodstuff ingestedand constituteabout 90% of dietary lipids which is a gives a sensationof stomachfullness. Therefore, concentrated source of fuel to the body, is without satiety achieved the consumptionof 2. Provideessentialfatty acidsand fat soluble excesscalories. vitamins (A, D, E and K). Adverse affects of fiber Some of the food fads went to the extent of ESSENTIAL FATTY ACIDS ingestinghuge quantitiesof rice bran to achieve The unsaturatedfatty acids which the body all the benefits of fiber. This led to several cannot synthesize and, therefore, must be complications.In general,the harmfuleffectsare consumed in the diet are referred to as essential mostly observed in people consuming large fatty acids (EFA). quantities of dietary fiber. The fatty acids-linoleic and linolenic acid1. Digestion and absorption of protein are cannot be synthesizedby humans. In a strict adversely affected. sense, only these two are essential fatty acids. 2. The intestinal absorption ol certain Arachidonic acid can be synthesized from minerals (e.g. Ca, P, Mg) is decreased. l i nol ei c aci d i n some ani mal speci es,i ncl udi ng man. However, the conversion efficiency of 3. Intestinal bacteria ferment some fibers, Iinoleic acid to arachidonic acid is not clearly causing flatulence and often discomfort. known in man. And for this reason, some nutritionists recommend that it is better to Sources of dietary fiber include some amount of arachidonic acid also Fruits, Ieafy vegetahles, vegetables, whole in the diet. wheat legumes,rice bran etc. are rich sourcesof fiber. The ideal way to increasefiber intake is to Functions of EFA reduce intake of refined carbohydrates,besides 1. Essential fatty acids are the structural eating vegetables,fresh fruits and whole grains. components of biological membranes. In general,vegetariansconsumemore fiber than non-vegetarians.An average daily intake of 2. Participatein the transportand utilization about 30 g fiber is recommended. of cholesterol. 3. Preventfat accumulationin the liver. 4. Required prostaglandins.

for

the

synthesis

of

5. Maintain proper growth and reproduction Triacylglycerols (fats and oils) are the concentrateddietary sourceof fuel, contributing of the organisms.

sl0 Deficiency

BIOCHEMISTFIY

of EFA

Essentialfatty acid deficiency is associated with several complications. These include impairment in growth and reproduction, increased BMR and high turnover of phospholipids.The EFAdeficiency in humans is characterized by a scaly dermatitis on the posterior and lateral parts of limbs and buttocks. This condition is referred to as phrynoderma or toad skin. Poor wound healing and hair loss is also observedin EFA deficiencv. EFA content

of foods

The essential fatty acids, more frequently called polyunsaturatedfatty acids (PUFA), are predominantly present in vegetable oils and fish oils. The rich vegetable sources include sunfloweroil, cofton seedoil, corn oil, soyabean oil et c . The fat of animal origin (exception-fish), contain less PUFA e.g. butter, fat of meat, pork and c hi c k e n .

Dietary intake of EFA

It is an accepted fact that reduction in serum cholesterollevel lowers the risk of heart diseases.

REOUIREMENTOF DIETARY FAT Consumption of dietary fats and oils is consideredin termsof their contributiontowards the energy needs of the body. There is a wide variation in fat intake. lt is much higher (up to 5O"/" of daily calories) in affluent societies compared to the poorer sections of the people (about 15"/oof calories). The recommended fat intake is around 2O-3O"/"of the daily calorie requirement,containingabout 50% of PUFA.

Proteinshave been traditionally regardedas 'body-building foodt . However, 10-15% of the total body energy is derived from proteins.As far as possible, carbohydratesspare proteins and make the latter available for body-building process.The functionscarriedout by proteinsin a living cell are innumerable,a few of them are listed hereunder.

Nutritionistsrecommendthat at least30% of the dietary fat should contain PUFA. Very high intake of PUFA (i.e. totally replacing saturated Funetions of proteins fatty acids)may not be advisable.This is due to 1. Proteinsare the fundamentalbasisof cell the fact that excessPUFA, unlessaccompanied (vitamin E, is by antioxidants carotenes), believed structureand its function. to be injurious to the cells due to the 2. All the enzymes, several hormones, overproduction of free radicals. immunoglobulins, transport carriers etc., are proteins.

CHOLESTEROLIN NUTRITION

It is proved beyond doubt that the elevated serum cholesterol (> 200 mg/dl) increasesthe risk of atherosclerosisand coronary heart diseases(For details, Refer Chapter 14). But the role of dietary cholesterolin this regard is still controversial.Cholesterolsynthesiscontinuously occurs in the body which is under a feedback regulation.Somenutritionistsbelievethat dietary cholesterolmay not have much influenceon the body levels while others recommend to avoid the consumptionof cholesterolrich foods (e.g. egg yolk) for a better health.

3. Proteinsare involved in the maintenance of osmotic pressure,clotting of blood, muscle contraction etc. 4. During starvation,proteins (amino acids) serveas the major suppliersof energy.lt may be noted that the structural proteins themselves serveas 'storageproteins' to meet the emergency energy needsof the body. This is in contrastto lipids and carbohydrates which have the respective storage forms triacylglycerols (in adipose tissue) and glycogen (in liver and muscl e).

Chapter 23 : NUTFIffION Essential

alnino

511 Body protein

acids

The nutritional importance of proteins is basedon the contentof essentialamino acids. The details of the essentialamino acids are described under chemistry of proteins (Chapter 4). There are ten essential amino acids-arginine, valine, histidine, isoleucine. I euc ine, l y s i n e , me th i o n i n e , p h e n y l a l ani ne, tryptophanand threonine(code to recall-AV HILL MP TT). Of thesetwo-namely arginine and histidine-are semi-essential. The requirementof 8 essentialamino acids per kg body weight per day is given in Table 23.4. Cysteineand tyrosinecan, respectively,sparethe requirementof methionineand phenylalanine.

Anabolism

Catabolism [l :l reti on as i ;r'ea* N l j i

Skin

(Nit;og;en out: U)

(s)

Fiq.23.2 : Overuiewof nitrogen balance (At equilibrium N input = N output; For positive N balance, N input > N output; for negative N balance N inDut < N outpuil.

Thus, an individual is said to be in a nitrogen balance if the intake and output of nitrogenare the same (Fig. 23.2). There are two other NITROGEN BALANCE situations-a positive and a negative nitrogen Dietary protein is almost an exclusivesource balance. of nitrogen to the body. Therefore,the term nitroPositive nitrogen balance : This is a state in gen bafancetruly represents the protein (160/"of which the nitrogen intake is higher than the which is nitrogen)utilization and its loss from output. Some amount of nitrogen is retained in the body. the body causing a net increase in the body Nitrogenbalanceis determinedby comparing protein. Positive nitrogen balance is observed in the intake of nitrogen (chiefly by proteins)and growing children, pregnant women or during the excretion of nitrogen (mostly undigested recoveryafter seriousillness. protein in feces;urea and ammonia in urine).A Negative nitrogen balance : This is a situation normal healthyadult is in a nitrogenequilibrium in which lhe nitrogen output is higher than the since the daily dietary intake (l) is equal to the input. fhe result is that some amount of nitrogen loss through urine (U), feces(F) and sweat (S). is lost from the body depletingthe body protein. Prolongednegative nitrogen balance may even lead to death. This is sometimesobserved in children suffering from kwashiorkor or marasmus.

I= U + F + S

Amino acid

Valine lsoleucine Leucine Lysine Methionine* Phenylalaninex Tryptophan Threonine

Requirement (n g/kg body weight/d ay)

14 12 16 12 10 16 3 8

* Cysteineand tyrosinecan,respectively, spare(partly)the requircnent ofmethionine andphenylalanine.

Negativenitrogen balance may occur due to inadequatedietary intake of protein (deficiency of even a single essential amino acid) or destruction of tissuesor serious illness. In all these cases,the body adapts itself and increases the breakdown of tissue proteins causing loss of nitrogen from the body. Other factors influencing nitrogen balance Besides the major factors discussed above (growth, pregnancy, protein deficiency, injury, illness)several other factors influence nitrosen balance.

E}IOCHEMISTFIY

512

Hormones: GroMh hormoneand insulinpro- fecesand urine samples.Biologicalvalue can be mote positivenitrogenbalancewhile corticoste- calculatedby the following formula roids result in negativenitrogen balance. (N absorbed- N lostin metabolism) x100 BV = Disease states : Cancer and uncontrolled N absorbed diabetescause negativenitrogen balance.

ASSESSMENTOF NUTRITIVE VALUE OF PROTEINS

-u \

tr"-(F"-r.)l-(u gy- Ln \ n ,Lrl r ,n -c,f ,atgg In-(Fn -Fc)

where In = Nitrogen ingested Knowledge on the quantity of dietary protein alone is not sufficientto evaluatethe nutritional Fn = Nitrogenin feces(on protein diet) importance of proteins. This is in contrast to Fc = Nitrogenin feces(on protein-freediet) dietary carbohydratesand lipids. The quality of Un = Nitrogen in urine (on protein diet) the proteinswhich dependson the composition Uc = Nitrogenin urine (on protein-freediet) of essential amino acids is more important. Several laboratory methods are in use to assess For the calculation of BV of proteins, the nutritive value of proteins. Of these, four experiments can be done even in human methods-protein efficiency ratio, biological subjects.The BV for different protein sources is v alue, ne t p ro te i n u ti l i z a ti o n a n d c hemi cal given in Table 23.5. score-are discussedbriefly. The biological value provides a reasonably good index for the nutritive value of proteins. Protein effieiency ratio (PER| But unfortunatelythis method has an inherent This test consistsof feedingweaning (21 day drawback. lt cannot take into account the ofd) albino ratswith a'l}"/o test protein diet and nitrogenthat might be lost during the digestion recordingthe gain in body weight for a period of process.For instance,if the ingestednitrogen is 4 weeks. PER is representedby gain in the 100 mg, absorbedis 10 mg and retainedis 8 mg, weight of rats per gram protein ingested. the BV 8/'l0x 100 = 80. This figure is erroneous, since the major part of the protein (90 mg) did Cain in bodyweight(g) . ,r* _ not enter the body at all for utilization. Proteinin8ested (g) The PERfor egg protein is 4.5; for milk protein 3.0; for rice protein 2.2. B iologic a l

v a l u e (BV l

The biological value of protein is defined as the percentage of absorbed nitrogen retained by the body. UU _

Nitrogenretained Nitrogenabsorbed

N et protei n

(N P U I

NPU is a better nutritional index than biological value, since it takes into account the digestibilityfactor. The experimentalprocedure for NPU is similar to that of BV. Net protein utilization can be calculatedas N P U=

,..,OO

For the measurementof BV, the experimental animals,namelyweaning albino ratsare chosen. They are first fed with a protein-free diet for 10 days. Then they are kept on a lOoh protein diet to be testedfor BV. Urine and feces are collected for both the periods i.e. protein-free diet and protein diet. Nitrogen is estimatedin the diet,

uti l i zati on

Chemical

Nitrogenretained Nitrogeningested

x100

seote

This is based on the chemical analysis of the protein for the composition of essential amino acids which is then compared with a reference protein (usuallyegg protein).The chemicalscore is defined as the ratio between the quantitity of the most limiting essentialamino acid in the test

Ghapter 23 : NUTRITION

513

proteinto the quantityof the sameamino acid in the egg protein, expressedas percentage. Chemical score mgof the limitingaminoacid/g testprotein mg of the sameamino acid /g egg protein

x 100

The chemical score of egg protein/ for any one of the essentialamino acids, is taken as I00 and the rest of the proteins are compared.

different foods, simultaneously.This helps to overcome the deficiency of certain essential amino acids in one food by being supplemented from the others. This phenomenon is referred to as mutual supplementation. For instance, an Indian diet with cereals(wheat,rice) is taken al ong w i th pul ses(dal ).The l i mi tati onof l ysi ne and threonine in cereal proteinsis overcome by their supplementation from dal proteins. Simultaneously, the limitation of sulfurcontai ni ng ami no aci ds i n dal i s al so compensatedby the cereals which are rich in them.

fn the lable 23.5, the four methods employed ( P E R,B V , N P U a n d c h e mi c a l s c o re ) for the assessmentof nutritive value of proteins are compared with regard to the different sourcesof The nutritive value of protein of a particular dietary proteins. Although there are certain provides food can be enhanced by appropriate variations, anyone of these methods combination with other foods. Due to the sufficient information on the nutritive value of proteins. consumption of mixed diets, dietary deficiency of essential amino acids is most uncommon. Mutua! supplementation of proteins. Further,the principle of mixed diet takescare to As is observedfrom the Table23.5, the suppl y adequatequanti ti esof essenti alami no animal proteins are superior in their nutritive acidsto the people subsistingon pure vegetarian value compared to the proteins of vegetable diets. lt has to be rememberedthat the effect of origin. Further,some of the essentialamino acids mutual supplementation in proteins is best (or at leaston the are limiting in certain foods. For instance,rice observedwith the same meal and wheat proteins are limiting in lysine and same day). threonine while the protein of Bengal gram is lim it ed in s u l fu r-c o n ta i n i n g a mi n o aci ds (methionineand cystine). It is fortunatethat humans (worldover)have the habit of consuming a mixed diet, with

Sourceof protein

PER

NPU

Requirement of proteins The requirementof protein is dependenton its nutritive value, caloric intake and physiological states (growth, pregnancy,

Chemical score

Limiting amino acid(s)

Egg 4.5 100 94 90 Nil oc Mitk 3,0 84 S-Containing amino acids 7n Fish OU 3.0 85 Tryplophan Meat 75 76 70 2.7 S-Containing amino acids Rice 2.2 60 60 68 Lysine, threonine Wheat 1.5 47 42 58 Lysine, threonine gram Bengal 1 .7 47 45 58 S-Containing amino acids Redgram 1 .5 46 45 57 S-Containing amino acids 1 .7 cc 45 44 Groundnut Lysine, threonine, S-amino acids cc 2 .1 65 55 Soyabean S-Containing amino acids protein PER-Protein efficiency ratio;BV-Biological value;NPU-Net utilization; S-Sultur.

514

BIOCHEMISTFIY

lactation)of the individual.For an adult, 0.8-1.0 g protein/kg body weight/day is adequate. The requirementshould be nearlydouble for growing children, pregnantand lactatingwomen.

RDA an for adult man

The detailsof RDA for each of the nutrientsin relation to age, sex and physiologicalstatus is describedin the respectivechapters.For a quick recapitulation, the RDA of macronutrients Dietary sou;ces of proteins (carbohydrate, fat and protein) and selected The protein content of foods is variable, micronutrients (vitamins and minerals) for an cereafs have 6-12%; pulses 18-22%; meat 18- adult man weighing 70 kg are given in 25"h, egg 1O-14%; milk 3-4% and leafy Table 23.6. vegetables'l-2"/o.ln general, the animal proteins are superior than vegetable proteins as the dietary source.

After discussingthe nutritionalaspectsof dietary ingredientsand their RDA, it t s worthwhile to formulatea diet for man. includingmetabolism, The nutritionalaspects requirebiochemicalfunctions,dietarysorlrces, ments and associateddisordersfor vitamins (ChapterV and for minerals(Chapterl8) have alreadybeendiscussed in much detail. Nutrient(s)

The recommended dietary/daily allowances (RDA) represents the quantities of the nutrients to be provided in the diet daily for maintaining good health and physical efficiency of the body. It must be remembered that RDA is not the minimum amount to just meet the body needs, but allowance is given for a safe margin. Factors

affecting

RDA

1. Sex : The RDA for men is aboul 20% higher than that lor women. lron is an exception as the requirement is greater in menstruating women. Additional requirements(20-30%above normal) are needed for pregnant and lactating wom e n . 2. Age: ln general,the nutrient requirement is much higher in the growing age.For instance, the protein requirementfor a growing child is about 2 g/kg body wVday compared to 1 g/kg body wt/day for adults.

Carbohydrates Fats Proteins Essentialfatty acids Vitamin A Vitamin D Vitamin E K Vitamin Ascorbic acid Thiamine RiboflavinI Niacin Pyridoxine Folicacid Cobalamin Calcium Phosphorus lron

RDA

400 g 7og 56 g* 4g 1,000pgxx 5 Pgx:Fx 1o pg 7o pg 60 mg 1.5mg 2mg 20 mg 2mg 150pg 2 trg 800 mg 800 mg 10 mg

xx Retinol * 0.8gkgbodyweight/day, quivalentq *** 1ss6s1&alciftrol

515

Chapter 23 : NUTFI|TION calanced diet or prudent diet is defined as the diet which contains different types of foods, possessingthe nutrients-carbohydrates, fats, proteins,vitaminsand minerals-in a proportion to meet the requirements of the body. A balanceddiet invariablysuppliesa little more of eac h nut r ientth a n th e m i n i m u m re q u i re m e ntto rvithstand the short duration of leannessand keep the body in a state of good health.

fats and oils, sugar and groundnuts.Meat, fish and eggsare presentin the non-vegetarian diets. ln case of vegetarians,an additional intake of mi l k and pul sesi s recommended. The nutri ti onal composition of the most commonly consumed Indi an foods gi ven i n the A ppendi x V l l . The nutri ti onal aspects of mi l k are gi ven i n the Chapter 22.

T he bas ic c o mo o s i ti o no f b a l a n c e d d i et i s highly variable, as it differs from country to country, dependingon the availabilityof foods. Socialand cultural habits,besidesthe economic status, age, sex and physical activity of the indiv iduallar g e l yi n fl u e n c eth e i n ta k eo f d i et.

eountries

Balanced

diet

in developed

Some people in developed countries (e.9. U.S.A) consume excessivequantitiesof certain nutrients.lt is recommendedthat such people have to reduce the intake of total calories,total fat, saturated fattv acids, cholesterol, refineo sugars and sal t. The U .S . C overnment recommendsa daily intake of lessthan 30'/" fat againstthe present 40-50% towards calories.

The Nutrition Expert Croup, constituted by the Indian Council of Medical Researchhas recommendedthe compositionof balanceddiets f or I ndians T . h i s i s d o n e ta k i n g i n to a c c o u ntthe c om m only a v a i l a b l e fo o d s i n In d i a . The composition of balanced diet (vegetarianand for an adult man and an adult non-vegetarian), woman are, respectively, shown is Table 23,7 and Table 23.8.

W hi l e the peopl e of devel opi ng countri es suffer from undernutrition,overnutrition is the The lndian balanced diet is composed of major concernof the developedcountries.Some cereals(rice, wheat, jowar), pulses,vegetables, of the important nutritional diseases are roots and tubers,fruits, milk and milk products, discussedhereunder.

Sedentarywork Vegetarian Non-vegetarian iVegetarian

@ 400 Cereals 70 Pulses lealyvegetables 100 Green 74 vegetables Other Roots 75 andtubers Fruits 30 Mitk 200 Fatsandoils 35 Meat andfish Eggs Sugar andjaggery ;; : Groundnuts

@

i(9@

400

i

cc

100 75 /c

30 100 40 30 30 30

475

i8 0 i tzs i7 5 i 100

is o i zoo

i* i

i...

i+ o

'..

Non-vegetarian

475 65 125 75 100 30 100 40 30 30 40

@

@

650 80 125 100 100 30 200 50

650

:" rCU

* Formulations (RDA) (1989) allowances of thelndianCouncil otMediulBesearch ontherecommended dietary based @aW)

bc tzt

100 100 30 100 50 30 30 55 50

516

BIOCHEMISTRY

Protein-energy malnutrition

manifestationsof kwashiorkor include stunted growth, edema (particularlyon legs and hands), Protein-energymalnutrition (PEM)-somediarrhea,discolorationof hair and skin, anemia, times calfed protein-calorie malnutrition apathy and moonface.

(PCMI-is the mostcommonnutritionaldisorder of the developingcountries.PEM is widely Biochemical manifestations : Kwashiorkor is prevalentin the infantsand pre-school children. associated with a decreased plasma alhumin Kwashiorkorand marasmusare the two extreme concentration (<2 g/dl against normal 3-4.5 formsof protein-energy malnutrition. {dl),lafty liver, deficiencyof K+ due to diarrhea.

Edema occurs due to lack of adequateplasma proteins to maintain water distribution between blood and tissues. Disturbances in the The term kwashiorkorwas introducedby metabolism of carbohydrate,protein and fat are CicelyWilliams(1933)to a nutritional disease also observed. Several vitamin deficiencies affectingthe people of Gold Coast (modern occur. Plasma retinol binding protein (RBP) is Chane)in Africa. Kwashiorkorliterallymeans reduced. The immunological responseof the sicknessof the deposedchild i.e. a diseasethe child to infection is very low.

Kwashiorkor

child getswhen the next baby is born.

Treatment : Ingestionol protein-rich foods or

Occurrence and causes : Kwashiorkoris the dietary combinations to provide about 3-4 g predominantly found in childrenbetween1-5 of protein/kg body weight/day will control yearsof age.Thisis primarilydue to insufficient kwashiorkor.The treatmentcan be monitoredby intakeof proteins, asthe dietof a weaningchild measuring plasma albumin concentration, mainlyconsists of carbohydrates. disappearanceof edema and gain in body

Clinical symptoms : The major clinical weight.

Additional allowances during Prcgrnncy Lactation @@

Cereals

300

3s0

350

Pulses

45

70

cc

i 125 Green vegetables

125

125

125

vegetables i 75 Other

75

75

Roots andtubers 50

50

Fruits

30

Mitk

30

i

475

475 cc

75

i70 i 125 i '100

100

75

75

I

100

30

30

igo I

100

125

100

200

100

Fatsandoils 30 Sugar andjaggery 30

35

35

40

ioo

45

30

30

30

i40

40

Meatandfish

30

30

30

Eggs

30

30

i"' i... i40

40

Groundnuts !

...

200

25

30

200

i

50

100

lzin 10

30

* Formulatbns (ROA) (1989) based on therectmmended dietary allowanccs ot ilrelndian council ol MediulFesrrrrch

517

Chapter 23 : NUTBITION

CIinical/b iochemical parameter

Kwashiorkor

Marasmus

Ageof onset

(1-5yr) Pre-school children

infants(<1 yr) Weaned

cause Mainnutritional

Lowproteinintake

Lowcalorieintake

Bodyweight

6G-80%of normal

Lessthan600/o of normal

Growth

Mildretardation

retardation Severe

Oedema

Present

Absent

Facialappearance

Moonface

Likeoldman'sface

Abdomen

Protruding

Shrunken

Dermatitis

Dryandatrophic

Muscles

wasting Undergo

Weakandatrophic

fat Subcutaneous

Present

Absent

Vitamin deficiencies

Present

Present

albumin Serum

0.5-2 g/dl

2-3 gtdl

Serum cortisol

Normal or decreased

Increased

Fasting bloodglucose

Decreased

Decreased

SerumK+

Decreased

Normal

Marasmus

resulting in a codition called cachexia.This is mainly due to the loss of body proteins as a Marasmusliterally means'to waste'.lt mainly result of hypermetabolism, particu larly i ncreased oc c ur s in c h i l d re n u n d e r o n e y e a r age. basal metabolic rate. Further, increased Marasmus is predominantly due to the oxidation of metabolic fuels leading to deficiency of calories. This is usually observed thermogenesisis also observed in cancer and in children given watery gruels (of cereals)to A ID S . supplementthe mother's breastmilk. The symptoms of marasmusinclude growth Nutritional anemias retardation,musclewasting(emaciation),anemia Anemia is lower characterized by and weakness.A marasmicchild does not show (reference concentration hemoglobin 14-1 6 of edema or decreased concentration of plasma with reduced to a ability transport oxygen. albumin, This is a major differenceto distinguish e/dl) Nutritional anemiasare classifiedbased on the marasmusfrom kwashiorkor. size of erythrocytes. ln the Table 23.9, a comparison between . Microcytic anemia-most common/ with kwashiorkorand marasmusis given. reduced RBC size. Occurs due to the deficiency of iron, copper and pyridoxine. Signs comparable to marasmus in

advanced cancer and AIDS The patientsof certain chronic diseaseslike cancer and AIDS are fequently undernourished,

. Macrocytic anemia-RBC are large and immature. Mostly due to the deficiency of fol i c aci d and vi tami n B rr.

518

B IOC H E MIS TFI Y

. Normocytic anemia-Size of the RBC is no rma l , b u t th e i r q u a n ti ty i n bl ood i s protein-energy low. Mostly found in m a l n u tri ti o n .

The nutri ti onal status of an i ndi vi du al is i mportant i n cl i ni cal practi ce. The di et ar y requirementsof nutrientsare variable, and are mostly relatedto age and sex, and physiological status.Some examplesof listed

. Infants and young children have increased There are several other nutritional disorders needsof protei n,i ron and cal ci um. which have been discussedelesewhere.These include obesity, body mass index and . D uri ngteenage,hi gh cal ci um and magne sium are recommended. (Chapter 14); vitamin deficiency atherosclerosis disorders-xerophthalmia, rickets, beri-beri, . In pregnancy,and lactation,the requirements of i ron, cal ci um, magnesi um,fol i c aci d and pellagra,scurvyand perniciousanemia(Chapter vitamin Bu and 8.,, are increased. V; goiter and other disorders of minerals (Chapter 18). The biochemical ramificationsof . Elderly people have to take more of vitamins starvation are discussed along with the B , and 812, fol i c aci d and vi tami n D , and integrationof metabolism(Chapter l6). mi neral schromi um.zi nc etc.

BIOMEDICAL/ GLINICAL CONGEPTS

lg Mosf of the informotion on human nutrition is bosed on the reseorchcarried out in experimental animals. The body at total rest (physicaland mental) requiresenergy to meet the basal require' ments suchos working of heart, conduction of nerue impulse, membrane transport etc. Corbohydrotes are the most obundont dietary constituents despite the fact that they are not essential nutrients to the body. Adequote intake of dietarg fiber preuents constipation, eliminotes bacterial foxins, reduces GIT concers, improues glucose tolerance and reduces plasma cholesterol. In general, uegetable oils ore good sourcesfor essentiallatty acids while animal protelns are superior for the supplg ot' essential omino acids. The biologicol uolue (BV) of protein represents the percentage of absorbed nitrogen retained in the body. The BV for egg protein is 94 while that for rice is 68. The recommendeddietary ollowance (RDA) of nutrients depends on the sex and age, besidespregnancy and lactqtion in the women. The habit of consuming mixed diet by man is largely responsibleto enhance the nutritiue uolue of foods, besidespreuenting seueral nutritional deficiencies (e.9. omino acids). Kwashiorkor and marasmus,the two extreme forms of protein-energymolnutrition in infants and pre-schoolchildren, are highly preualent in deueloplng countries.

i.Jh6pss* fii3 ; NUTFIITION

519

I n gene ra l , i l l n e s s a n d me ta b o l i c stress inc r eas et he n u tri ti o n a ld e m a n d s .F o r i n s t ances, liver and kidney diseasesreduce the formation Drug Risk of nutrient deficiencies of active vitamin D (calcitriol),and storageand Bu,vitaminB,r,folicacid ut iliz at iono f v i ta m i n s -fo l i c a c i d , v i ta m in B 12 OralcontraceptivesVitamin and v it am in D . Diuretics Potassium, zinc Sruq amd nutrient

iffitesa*tielns

Many drugs are known to lead to potential nutrientdeficiencies(Table23.10). For instance, or al c ont r ac e p ti v em s a y re s u l ti n v i ta m i n 8 6 ,812 and f olic ac i d d e fi c i e n c i e s .

Anticonvulsants Folicacid,vitamin D,vitamin K lsoniazid Vitamin Bu Corlicosteroids Vitamin D,calcium, potassium, zinc Alcohol

Thiamine, vitamin Bu,folicacid

1. The calorific ualues ol carbohydrates,/ots and proteins respectiuelysre 4, 9 and 4 Cal/g. These three nutrients (macronutrients)supplg energy to the body to meet the requirementsof basal metabolic rate, specific dynamic action and physical actiuity.

2. Basal metabolic rate (BMR) representsthe minimum amount of energy required by the body to maintain lit'eot completephgsicolond mental rest, in the post-absorptiue state. The normal BMR for an adult man is 35-38 CaVm2 bodg surfacqhr.

3 . Specific dynamic oction (SDA) is the extra heat produced by the body ouer qnd aboue the calculated calorific ualue of foodstuff. It is higher for proteins (300/o),lower for carbohgdrates(50/o),and lor a mixed diet, it is around 70V0.

4. Carbohydratesore the major source of body fuel supplying about 40-700/oof bodV calories. The non-digested carbohydrates (cellulose, pectin) are ret'erred to as fiber. Adequate intake ol fiber preoentsconstipation, improuesglucose toleranceand reduces plasma cholesterol.

5. Lipids are the concentrated source of energy. They also prouide essentialt'atty acids (linoleic and linolenic ocids)and t'at-solubleuitamins (A, D, E and K). Proteins are the bodg building t'oods that supplg essentialamino acids, besidesmeeting the body energg requirementpartly (70-150/o).

7. Seueral methods are employed fo ossessthe nutritiue ualue of proteins, These include protein efficiencyrotio, biologicalualue, net protein utilization snd chemicalscore.

8. The recommendeddietary ollowance (RDA) representsthe quantities ol nutrients to be prouided daily in the diet lor maintaining good health ond physicaleft'iciency.The RDA for protein is lgkg body weight/day.

9. A bolanced diet is the diet which contains dit'ferent tgpes of foods with the nutrients, namely carbohydrates,Jats, proteins, uitamins and minerals, in o proportion to meet the body requirements.

10. Protein-energymalnutrition (PEM) is the most common nutritionol disorder in the deuelopingcountries.Kwashiorkoris primarily due to inadequateprotein intake while merasmusis mainlg causedby colorie defiaency.

l

520

B IOC H E MIS TR Y

I. Essay questions 1. 2. 3. 4. 5.

Define BMR. Discussthe factorsaffectingBMR. Describethe different methodsemployed for the nutritional evaluationof proteins. Definea balanceddiet. Formulatea diet for a medicalstudent. Discussthe protein-energymalnutritionwith special referenceto kwashiorkor. Cive an accountof the recommended dietaryallowance(RDA)for macro-and micronutrients.

II. Short notes (a) Essentialamino acids, (b) Mutual supplementationof proteins, (c) Caloric value of foods, (d) Specificdynamicaction,(e) Energyrequirements of man, (0 Fiberin nutrition,(g) Kwashiorkor, (h) Limitingamino acids,(i) Nitrogenbalqnce,(j) Biologicalvalue of proteins. III. Fill in the blanks 1. One calorieof energyis equivalentto

Joules(KJ).

2. The endocrineorgan most predominantlyassociatedwith BMR is 3. The non-digestiblecarbohydratesare collectively known as 4. The major sourceof energyto the body is suppliedby 5. The nutritional assessmentmethod used to know the most limiting essentialamino acid in relationto a standardprotein is 6. The daily normal requirementof proteinin an adult is 7. The percentageof absorbednitrogen retainedin the body represents 8. The proteinsof Bengalgram are limiting in the amino acids 9. The nutrientrequiredin greateramountsin menstruatingwomen comparedto men is -. 10. The biochemicalparameteroften usedas an indexfor monitoringthe recoveryfrom kwashiorkor ts IV. Multiple choice questions 11. The specificdynamic action (SDA) is the greatestfor the following foodstuff (a) Protein(b) Carbohydrate(c) Fat (d) Vitamins. 12. The referenceproteinfor the calculationof chemicalscore (a) Meat protein (b) Fish protein (c) Milk protein (d) EeSprotein. 13. T h e e s s e n ti aal m i n o a c i d l i m i ti ngi n ri ce (a) Methionine (b) Tryptophan(c) Lysine(d) Histidine. 14. A continuoussupply of energyto the body is necessaryto meet the requirementsof (a) Basalmetabolicrate (b) Specificdynamicaction (c) Physicalactivity(d) All of them. 15. One of the following is the most importantessentialfatty acid in the diet (a) Linoleicacid (b) Arachidonicacid (c) Oleic acid (d) Palmiticacid.

DITA-Replicationu andRepair Recombtnationu

eoxy ri b o n u c l e i c a c i d (D N A) i s a !l macromolecule that carries genetic lJ informationfrom generationto generation.lt is responsibleto preservethe identityof the species over millions of years.DNA may be regardedas a reserve bank of genetic information or a m em or y b a n k .

Transcri pti on._-. _ Transl ati on

DllA---

RNA#Protein

llepiication

Fiq.24.1 : The central dogma of life.

A s ing l ema mma l i a nfe ta l c e l l c o n ta i nsonl y a f ew pic o g ra m s(1 0 -1 2g ) o f D N A. l t i s surpri si ng The central dogma of life that this little quantityof DNA storesinformation fhe biological information flows from DNA that will determinethe differentiationand everv to RNA, and from there to proteins. This is the f unc t ion o f a n a d u l t a n i ma l " central dogma ol lifu (Fig.24.l). lt is ultimately the DNA that controlsevery function of the cell Why did DNA evolve as genetic through protein synthesis. rnaterial? As the carrierof genetic information,DNA in RNA mo l e c u l e s i,n p ri n c i p l e ,c a n p erformthe a cell must be duplicated (replicated), c ellularfu n c ti o n sth a t a re c a rri e do u t by D N A . maintainedand passeddown accuratelyto the In fact, many virusescontain RNA as the genetic daughter cells. Three distinct processes are material.Chemicallv, DNA is more stablethan designed for this purpose. The 'three Rt of RNA . He n c e , d u ri n g th e c o u rs e o f evol uti on, DNA-replication,recombination,and repair, are DNA is p re fe rre da s a mo re s u i ta b l emol ecul e dealt with in this chaoter. There are certain for long-term repositoryof genetic information. common featuresbetween the three Rs.

523

524 a a

B IOC H E MIS TR Y

They act on the same substrate(DNA). They are primarily concernedwith the making and breaking of phosphodiesterbonds (the backbone of DNA structure). Enzymes used in the three processes are mostly sim i lar/comparable.

DNA is the genetic material.When the cell divides, the daughtercells receive an identical copy of geneticinformationfrom the parentcell. Replication is a processin which DNA copies itself to produce identical daughter molecules of DNA. Replication is carried out with high fidelity which is essentialfor the survivalof the species.Synthesisof a new DNA molecLle is a complex processinvolving a seriesof steps. The salient features of replication in prokaryotesare described first. This is followed by some recent information on the eukaryotic replication.

DaughterDNA

ParentDNA

DaughterDNA

Fig. 24.2 : DNAreplication-semiconservative.

REPLICATIONIN PROKARYOTES

These sites mostly consist of a short sequenceof A-T base pairs. A specific protein called dna A (20-50 monomers)binds with the site of origin for replication.This causesthe double-stranded DNA to separate.

Replication is semiconservative

R epl i cati on

The parent DNA hastwo strandscomplementary to each other. Both the strands undergo simultaneous replication to produce two daughter molecules. Each one of the newly synthesizedDNA has one-half of the parental DNA (one strand from original) and one-half of new DNA (Fig.2a.). This type of replication is known as semiconservative since half of the original DNA is conserved in the daughter DNA. The first experimental evidence for the semiconservative DNA replicationwas provided by Meselsonand Stahl (1958).

The two complementary strands of DNA separate at the site of replication to form a bubbl e.Mul ti pl e repl i cati onbubbl esare formed in eukaryoticDNA molecules,which is essential for a rapid replication process (Fi9,2a.9.

Initiation

of replication

The initiation of DNA synthesisoccurs at a site called origin of replication. In case of prokaryotes,there is a single site whereas in eukaryotes,there are multiple sites of origin.

bubbl es

R N A pri mer For the synthesis of new DNA, a short fragment of RNA (about 5-50 nucleotides, variable with species)is required as a primer. The enzyme primase (a specific RNA polymerase)in associationwith single-stranded bi ndi ng protei ns forms a compl ex cal l ed primosome, and produces RNA primers. A constant synthesisand supply of RNA primers should occur on the laggingstrandof DNA. This is in contrast to the leading strand which has al most a si ngl e R N A pri mer.

AND REPAIFI RECOMBINATION, Ghapter 24 : DNA-FIEPLICATION,

525

proteins.They possessno enzyme activity. SSB proteins bind only to single-strandedDNA (separatedby helicases), keep the two strands separateand provide the template for new DNA synthesis.lt is believed that SSB proteins also protect the single-stranded DNA degradationby nucleases.

j

Fiq.24.3 : Schematicrepresentation of multiple replicationbubblesin DNAreplication.

is semidiscontinuous DNA synthesis and bidirectional The replication of DNA occurs in 5' to 3' direction,simultaneously,on both the strandsof DNA. On one strand, lhe leading (continuous or DNA synthesis is forward) strand-the continuous. On the other strand, the lagging (discontinuous strand-the or retrograde) synthesis of DNA is discontinuous. Short pieces of DNA (15-250 nucleotides)are produced on the laggingstrand.

DNA synthesis catalysed by DNA polymerase lll The synthesisof a new DNA strand,catalysed by DNA polymerase lll, occurs in 5'->3' di recti on. Thi s i s anti paral l el to the parent template DNA strand. The presenceof all the four deoxyribonucleosidetriphosphates(dATP, dCTP, dCTP and dTTP) is an essential prerequisitefor replicationto take place. The synthesis of two new DNA strands, simultaneously,takes place in the opposite direction---oneis in a direction (5'-r3') towards the replicationfork which is continuous,the other in a direction (5'-+3') away from the replication fork which is discontinuous(Fig.24.4).

The incoming deoxyribonucleotides are added one after another,to 3' end of the growing DNA chain (Fig.2a.S).A molecule of pyrophosphate(PPi)is removed with the addition of each nucleotide.The template DNA strand (the ln the replicationbubble, the DNA synthesis parent) determines the base sequence of the occurs in both the directions(bidirectional)from newly synthesizedcomplementaryDNA. the point of origin. Replication

fork and DNA synthesis

The separation of the two strands of parent DNA results in the formation of a replication fork. The active synthesisof DNA occurs in this region. The replication fork moves along the parent DNA as the daughterDNA moleculesare synthesized. DNA helicases: These enzymes bind to both the DNA strands at the replication fork. Helic as es mo v e a l o n g th e D N A h e l i x and separate the strands. Their function is comparabfe with a zip opener. Helicases are dependenton ATP for energy supply.

Polarity

problem

The DNA strand (leading strand) with its 3'-end (3'-OH) orientedtowards the fork can be elongated by sequential addition of new nucleotides. The other DNA strand (lagging strand)with S'-end presentssome problem, as there is no DNA polymeraseenzyme (in any organism) that can catalyse the addition of nucl eoti des to the 5' end (i .e.3' -+ 5' di recti on)of the growing chain. This problem however is solved by synthesizingthis strand as a seriesof small fragments.These pieces are made in the normal 5'-)3' direction, and later joined together.

Okazaki pieces : The small fragments of the Single-strandedDNA binding (SSB)proteins : Theseare also known as DNA helix-destabilizing discontinuously synthesized DNA are called

526

BIOCHEMISTRY

Okazaki pieces. These are produced on the lagging strand of the parent DNA. Okazaki pieces are later joined to form a continuousstrandof DNA. DNA polymerase I and DNA ligaseare responsible for this process (details given later).

Proof.reading function of DNA polymerase lll

;-dnaA

s'

s' NatlveDNA p-dna A protein

., G u. Replicationbubble

6'

-c

5' Fidelity of replicationis the most important for the very existence of an organism. Besides its 5'-)3' directed catalytic Leading function, DNA polymerase strand llf afso has a proof-reading activity. lt checks the incoming nucleotides and allows only the correctly (i.e. matched bases complementary bases) to be added to the growing Newly DNA strand. Further,DNA synthesized DNA polymerase edits its (if mistakes any) and removes the wrongly placed nucleotidebases. Replacement

protein

39flt' AN

'S,ij'ltn ,

J

--

-

3', Originof replication

of

RNA primer by DNA The synthesis of new DNA strandcontinuestill it Lagging 3' strand I is in close proximity to RNA primer. Now the DNA polymerase I comes into picture. lt removesthe RNA primer and takes its position. DNA polymerase

Repllcationfork

(5'+3' direction)of a I catalyses the synthesis the DNA synthesizedby DNA polymeraselll and fragment of DNA that replacesRNA primer the small fragments of DNA produced by DNA (Fig.24.6).

polymerasel. This process-nick sealing-requires

The enzyme DNA ligase catalyses the energy, provided by the breakdown of ATP to formationof a phosphodiester linkagebetween A MP and P P i .

527

AND BEPAIR RECOMBINATION, Ghapter 24: DNA-REPLICATION,

..lt'..1'..f'..f '$'..f'.'f" ttttttl

ZcTGAACTGAT

iltililtilltillllllrtl lll ll lll ll ll

E EGACUTGACTA

RNAprimer

lll

ll

lll

ll

Gro

Fiq.24.5 : DNA replication with a growing complementarystrand.

Another enzyme-DNA polymerase ll-has been isolated. lt participates in the DNA repair Process. Supercoils

and DNA topoisomerases

As the double helix of DNA separatesfrom one side and replicationproceeds,supercoilsare formed at the other side. The formation of supercoils can be better understood by comparing DNA helix with two twisted ropes tied at one end. Hold the ropes at the tied end in a f ix ed p o s i ti o n .An d l e t y o u r fri e n d p ul l the ropes apart from the other side. The formation of supercoilsis clearly observed. The problem of supercoilsthat comes in the way of DNA replicationis solved by a group of enzymes called DNA topoisomerases.Type I DNA topoisomerasecuts the single DNA strand (nucleaseactivity) to overcome the problem of supercoils and then resealsthe strand (ligase activity). Type ll DNA topoisomerase (also known as DNA gyrase) cuts both strands and reseals them to overcome the problem of s uper c oils .

REPLICATIONIN EUKARYOTES Replication of DNA in eukaryotes closely resemblesthat of prokaryotes.Certaindifferences, however, exist. Multiple origins of replication is a characteristicfeatureof eukaryoticcel[. Further,

at leastfive distinct DNA polymerases are known in eukaryotes. Greeklettersare usedto number theseenzymes. 1. DNA polymerasea is responsible for the synthesis of RNAprimerfor boththe leadingand laggingstrandsof DNA. 2. DNA polymerasep is involved in the repairof DNA. lts functionis comparable with DNA polymerase I found in prokaryotes. 3. DNA polymeraseI participatesin the replication of mitochondrial DNA. 4. DNA polymerase6 is responsible for the replication on the leadingstrandof DNA. lt also possesses proof-reading activity. 3'--4

5'

3',

5' Newlysynthesized DNA - 3' DNAtemplate A polymerase I

-s',

3',

Nicksealed by DNAligase 5'DaughterDNA

Ji-

3' ParentDNA Fig. 24.6 : OveNiew of the action of DNA

528

BIOCHEMISTF|Y

5. DNA polymerase e is involved in DNA catalyses the assembly of proliferating cell synthesison the laggingstrandand proof-reading nuclear antigen (PCNA) molecules. The DNA polymerase6 binds to the sliding clamp and function. elongates the Okazaki fragmentto a final length The differences in the DNA replication 150-200 bp. By this elongation, the of about between bacteria and human cells, attributed to replication complex approachesthe RNA primer the enzymesf are successfully used in previous of the Okazaki fragment. antibacterialtherapyto target pathogen(bacterial) replicationand sparethe host (human)cells.

The RNA primer removal is carried out by a pair of enzymes namely RNase H and flap PROCESS OF BEPL'CAT'ON endonuclease| (FENI).This gap createdby RNA IN EUKABYOTES removal is filled by continued elongationof the The replication on the leading (continuous) new Okazaki fragment (carried out by strand of DNA is rather simple, involving DNA polymerase6, describedabove).The small nick polymerase 6 and a sliding clamp called that remains is finally sealedby DNA ligase. proliferating cell nuclear antigen (PCNA). PCNA EukaryoticDNA is tightly bound to histones is so named as it was first detected as an antigen (basic proteins) to form nucleosomeswhich, in in the nuclei of replicatingcells. PCNA forms a turn, organize into chromosomes.During the ring around DNA to which DNA polymerase6 course of replication, the chromosomes are binds.Formationof this ring also requiresanother relaxedand the nucleosomesget loosened.The factor namely replication factor C (RFC). DNA strands separatefor replication, and the fhe replication on the l"ggiry (discontinuous) parental histones associate with one of the strand in eukaryotes is more complex when parental strands. As the synthesisof new DNA compared to prokaryotesor even the leading strand proceeds, histones are also produced strand of eukaryotes. This is depicted in simultaneously,on the parentstrand.At the end Fig.24,7, and briefly described hereunder. of replication,of the two daughterchromosomal The parental strandsof DNA are separatedby DNAs formed,one containsthe parentalhistones the enzyme helicase. A single-strandedDNA while the other has the newlv svnthesized binding protein called replication protein A histones. (RPAI binds to the exposed single-stranded template.This strandhas been opened up by the ,N']|B'TORS OF DNA REPLICATION replication fork (a previously formed Okazaki Bacteria contain a specific type Il fragmentwith an RNA primer is also shown in topoisomerasenamely gynse. This enzyme cuts Fig.2a.$. and resealsthe circular DNA (of bacteria),and thus overcomes the problem of supercoils. primase The enzyme forms a complex with gyrase is inhibited by the antibiotics Bacterial DNA polymerasea which initiatesthe synthesis novobiocin and nalidixic acid. ciprofloxacin, of Okazaki fragments.The primase activity of pol a-primasecomplex is capable of producing These are widely used as antibacterialagents 1O-bpRNA primer. The enzyme activity is then since they can effectivelyblock the replication of DNA and multiplication of cells. These switched from primase to DNA polymerasec which elongatesthe primer by the addition of antibacterial agents have almost no effect on 20-30 deoxyribonucleotides. Thus, by the action human enzymes. of pol c,-primase complex, a short stretch of Certain compounds that inhibit human DNA attached to RNA is formed. And now the topoisomerasesare used as anticancer agents complex dissociatesfrom the DNA. e.g. adriamycin, etoposide, doxorubicin. The The next step is the binding of replication nucleotideanalogsthat inhibit DNA replication factor C (RFC) to the elongated primer (short are also used as anticancer drugs e.g. RNA-DNA). RFC servesas a clamp loader, and 6-mercaptopuri ne, 5-fluorouracil.

AND REPAIFI RECOMBINATION, Chapter 24: DNA-REPLICATION,

DNApolymerase cr- RNAorimer primasecomplex

Fig.24.7 : An outline of DNA replicationon the lagging strand in eukaryotes(RPA-Replicationprotein A;

529

530

B IOGH E MIS TR Y

CELL GYCLE AND DNA REPI.ICATION

Detectionof

o"s,tfl"o

The cell cycle consistsof four distinct phases in higher organisms-mitotic, G1, S and C2 Detectionof phases(Fi9.24.A. When the cell is not growing, incomplete it exists in a dormant or undividing phase (Co). replication Cj phase is characterized by active protein svnthesis. Replication of DNA occurs only once in S-phaseand the chromosomesget doubled i.e. diploid genome gets converted into tetraploid. The entire process of new DNA synthesis takes place in about 8-1 0 hours, and a large number of DNA polymerases(500-1,000) are simultaneouslyinvolved in this process. lt is believed that methylation of DNA servesas a m ar k e rto i n h i b i t re p l i c a ti o n .

... ''

Detectionof mproper spindle

'G o

DNA Damaged detected

Gancer and cell cycle

Cancer representsan excessivedivision of cells. ln cancer, a large quantity of cells are in The C2 phaseis characterizedby enlargement mitosis and most of them in S-phase. of cytoplasm and this is followed by the Majority of the drugs used for cancer therapy actual cell division that occurs in the mitotic phase. are designedto block DNA replicationor inhibit the enzymes that particlpate in replication (directly or indirectly). Methotrexafe (inhibits Gyclins and cell cycle dihydrofolate reductase) and S-fluorouracil Cyclinsarea groupof proteins thatareclosely (inhibitsthymidylate synthase)block nucleotide associatedwith the transition of one phaseof synthesis.

cell cycle to another,hencethey are so named. fn recent years, topoisomerase inhibitors are The most importantcyclins are cyclin A, B, used. They block the unwinding of being D and E. The concentrations of cyclinsincrease parental DNA strands and prevent replication. or decreaseduring the courseof cell cycle. Thesecyclinsact on cyclin-dependent kinases (CDKs)that phosphorylatecertain substances TELOMERES AND TELOMERASE essentialfor the transition of one cycle to There are certaindifficultiesin the replication another.

of linear DNAs (or chromosomes)of eukaryotic cells. The leading strand of DNA can be Gell cycle check points completely synthesized to the very end of its As depicted in Fi9.24.8, there occurs a template. This is not possible wih the lagging continuousmonitoringof the cell cycle with strand, since the removal of the primer respect to DNA replication, chromosome RNA leaves a small gap which cannot be segregation and integrity.lf any damageto DNA filled (Fi9.24,9A). Consequently, the daughter is detectedeitherin G1 or C2 phaseof the cycle, chromosomes will have shortened DNA or if thereis a formationof defective spindle(i.e. molecules.This becomessignificantafterseveral incompletechromosomal segregation), the cell cell replication of cycles involving cycle will not progressuntil appropriately chromosomes.The result is that over a period of corrected.lf it is not possibleto repair the time, the chromosomes may lose certain damage done, the cells undergo apoptosis essential genes and the cell dies. This is (programmed cell death). however, avoided to a large extent.

531

AND FEPAIFI Ghapter 24 : DNA-FIEPLICATION, FIECOMBINATION,

3' ParentalDNAstrand Telomeres are the special struclures that (A) s' s'. 5' LaggingDNAstrand prevent the continuous loss of DNA at the end RNA primer I of the chromosomes during the course of i--Okazakifragment--f replication. Thus, they protect the ends of [ removed the chromosomes/ and are also responsible ", l> prevent from fusing with the chromosomes to 5' -3' LTelomere each other. Telomeres are many repeat 3S' present at the of six nucleotides sequences ends of eukaryotic chromosomes. Human (B) s'-TTAGGGTTAGGG 3' telomerescontain thousandsof repeat TTAGGG f sequencestwhich can be up to a length of ParentalDNAstrand 1500 bp. Newlysynthesized (withtelomeres) 1 incomplete lagging I Bindingto strand telomerase Role of telomerase f 5,-TTAGGGTTAGGG 3' Telomeres are maintained by the enzyme telomerase, also called as telomere terminal transferase.Telomeraseis an unusual enzyme as it is composedof both protein and RNA. In case I Extension of 3'end (NewDNAsynthesis) of humans, the RNA component is 450 | .t' nucleotidesin length,and at the 5'-terminaland s/-TTAGGGTTAGGG G 3' it containsthe sequence5'-CUAACCCUAAC-3'. 3';CAAUCCCMUC-1 \./ It may be noted that the central region of this s', 3',r the telomere to sequence is compfomentary I Translocation repeatsequence5'-TTACCC-3'.The telomerase Y for used as a template RNA sequence can be G 3' 5,-TTAGGGTTAGGG extension of telomeres (Fig.2a.9B). 3',(CAAUCCCAAUC)

The telomeraseRNA base pairs to the end of the DNA molecule with telomeresand extends to a small distance. Then translocation of telomeraseoccurs and a freshextensionof DNA takes place. This processof DNA synthesisand translocationis repeatedseveraltimes until the chromosome gets sufficiently extended. The extension process gets completed through the participation of DNA polymerase and primase complex and sealing of the new DNA formed. It may be noted here that as such the telomeresdo not encode proteins. Hence, when extended by telomerase, they need not have to remain the same length, and some shortening will not pose any problem. During the courseof repeated cell cycles, there occurs progressive shortening of telomeres, and this has to be prevented,which is appropriately carried out hy telomerase.

3', I lNewDNAsynthesis fExtension)

I o andprimase

CompletedDNAstrands Fiq.24.9 : Replicationof DNA with telomeres

s',

532

BIOCHEMISTFIY

TELOMERE IN SENESCENCE AND CANCEB

A B C D E FGH IJK LK N OP ?

Telomerase is highly activein the early

abcdefghijklmnop

embryo, and after birth it is active in the reproductiveand stem cells. Stem cells divide continuously throughout the lifetime of an organism to produce new cells. These cells in turn are responsibleto tissuesand organs in the functional state e.g. hematopoietic stem cells of bone marrow.

ABCDEFGHljklmnoP ?

Many biologists link the process of telomere shortening with cell senescence(i.e. cell death). This is mainly based on the-observationsmade in the in vitro rnamrhalian cell cultures. questionthis relation However,some researchers between telomere shortening and senescence. Cancerous cells are able to divide continuously. There is a strong evidence to sg8Bestthat the absenceof senescencein cancer cells is linked to the activation of the enzyme telomerase.Thus, telomere length is maintained throughoutmultiple cell divisions.lt is however, not clear whether telomerase activation is a cause or an effect of cancer. There is however, evidence to suggestthat telomeraseactivation is in fact the cause of certain cancers e.g. dyskeratosiscongenita due to a mutation in the gene responsiblefor the RNA component of telomerase.

abcdefghiJKLMNOP

with Chromosomes DNAfrombothparents

paternal and (Fig.24JA.

maternal chromosomal pairs

2. Non-homologous recomhhration : This is regarded as illegitimate redmbination and does not require any special homologoussequences. Transposition is a good example of nonhomologous recombination. Random integration of outside genes into mammalian chromosomes is another example.

HOilOLOGOUS RECOMBINATION

The enzyme telomerase is an attractive target It is a known fact that the chromosomes are for cancer chemotherapy. The drugs have been not passed on intact from generation to designed to inactivate telomerase, and generation. Instead,they are inherited from both consequentlyinduce senescencein the cancer the parents.This is possibledue to homologous cells. This in turn prevents the rapid cell recombination. Three models have been out proliferation. forth to explain homologousrecombinations. .

H ol l i day model

.

Meselson-Radding model

Recombination basically involves the . Double-strandbreak model. exchange of genetic information. There are mainlytwo typesof recombinations. Holliday model 1. Homologous recombination : This is also calfed as general recombination, and occurs between identical or nearly identical chromosomes(DNA sequences).The best example is the recombinationbetween the

Holliday model (proposed by Holliday in 1964) is the simplest among the homologous recombination models. lt is depicted in Fig.24.1l, and briefly explained in the next pa8e.

Ghapter 24: DNA-REPLICATION, HECOMBINATION, AND BEPAIR

ab Two homologousDNAmolecules with single-strandbreaks I Y

CrossDNAstrands AYB

ao Heteroduplex sealed by DNA ligase

lcrtz

533

The two homologous chromosomes come closer, get properly aligned, and form singlestrand breaks.This resultsin two aligned DNA duplexes.Now the strandsof each duplex partly unwind and invade in the oppositedirection to form a two strands cross between the DNA mol ecul es. There occurs si mul taneousunw i ndi ng and rewinding of the duplexes in such a way that there is no net change in the amount of base pairing,but the positionof crossovermoves.This phenomenon referred to as branch migration, results in the formation of heteroduplex DNA. The enzyme DNA ligasesealsthe nick. The two DNA duplexes (4 strands of DNA), .ioined by a single crossoverpoint can rotateto create a fourstanded Holliday juncfion. Now the DNA moleculesare subjectedto synlnetrical cuts in either of the two directions,and the cut ends3re resealedby ligase.

IA

The DNA exchange is determined by the direction of the cuts, which could be horizontal or vertical. tf the corss strands are cut hori zontal l y (cut 1), the fl anki ng genes (or markers,. i.e. AB/ab) remain intact, and no recombinationoccurs.On the other hand, if the parental strands are cut vertically (cut 2), the flanking genesget exchanged(i.e. Ab/aB)due to recombination.

a'b

NOI{.HOMOLOGOUS

RECOMBINATION Hollidayintermediate(moleculerotated)

The recombination process without any special homologous sequences of DNA is regardedas non-homologousrecombination.

..::9'"W,{.:r," """;P $

hxa

-J-+d

r . . . . . . '- . . . . - r

u

Recombineddaughter DNA strands Fiq.24.11 : Holliday model fot homologous recombination (Note : Heteroduplex regions are shown in dotted boxes).

Transposition

Transposi ti on pri mari l y i nvol ves the movement of specific pieces of DNA in the genome.The mobile segmentsof DNA are called transposons or transposahle elemenfs. They were first discoveredby BarbaraMcClintock (in 1950) i n mai ze, and thei r si gni fi cancew as ignored for about two decadesby other workers. Transposonsare mobile and can move almost to any place in the target chromosome. There are two modes of transoosition. One that

E}IOCHEMISTF|Y

534 Transposon

Transcription

accu mu fation of transposons. Shorf interspersed elements (SlNfs) are repeats of DNA sequences which are present in about 500,000 copies per hapfoid human genome e.g. Alu seguences. Long interspersed elements (LlNEs) are also repeated DNA sequencesand are present in about 50,000 copies in the human Benomee.g. L1 elements.

A-*nUn

Some of the diseasescaused by mutations are due to insertion of transposonsinto genes.

Transposon copy (retrotransposon)

l

Being the carrier of genetic information,the cellular DNA must be replicated (duplicated), maintained, and passeddown to the daughter cells accurately. In general, the accuracy of involves an RNA intermediate,and the other replicationis extremelyhigh. However,there do which does not involve RNA intermediate. occur replication errors. lt is estimated that Retrotransposition : Transposition involving approximatelyone error is frthoducedper billion RNA intermediate represents retrotransposition base pairs during each ipl5l'of replication.The By the normal process of cells do possesthe capabilityto repair damages Gig.2a.|A. transcription, a copy of RNA formed from a done to DNA to a large extent. transposon(also called as retrotransposon).Then by the enzyme reverse transcriptase, DNA is Gonsequences of DNA damage copied from the RNA. The newly formed DNA Despite an efficient repair system for the which is a copy of the transposongets integrated DNA, replicationerrorsdo accumulate damaged into the genome. This integration may occur that ultimately result in mutations.The human randomly on the same chromosome or/ on a 1014 nucleatedcells, each with body possesses different chromosome. As a result of the retro3 x 10e base pairs of DNA. lt is estimatedthat transposition, there are now two copies of the about 1016cel l di vi si onsoccur i n a l i feti me.lf transposon,at different points on the genome. 10-10 mutations per base pair per cell generation DNA transposition : Some transposons are escaperepair,this resultsin about one mutation capable of direct transpositionof DNA to DNA. per 106 base pairs in genome. This may occur either by replicativetransposition or conservative transposition (Fig.24.13). Both the mechanismsrequireenzymesthat are mostly coded by the geneswithin the transposons. DNA transposition is less common than retrotranspositionin caseof eukaryotes.However, in caseof prokaryotes,DNA transposonsare more important than RNA transposons. Significance

of transposition

It is now widely acceptedthat a large fraction of the human genome has resulteddue to the

Conservative

-

l: Adiagrammatic representationof DNA

535

AND REPAIR Chapter Ztl : DNA-FIEPLICATION, BECOMBINATION, Besidesthe possibleerrors in replication,the DNA is constantly subjected to attack by both phy s ic al an d c h e m i c a l a g e n ts .T h e s e i ncl ude radiation, free radicals, chemicals etc., which also result in mutations.

Category

Types

Slngle-base alteratlon Deamination (C-+U;A-+hypoxanthine)

It is fortunate that a great majority of the mutations probably occur in the DNA that does not encode proteins,and consequentlywill not have any serious impact on the organism. T his is not, h o w e v e r, a l l th e ti me tru e , si nce m ut at ionsd o o c c u r i n th e c o d i n g re g ions of DNA als o. T h e re a re s i tu a ti o n si n w h i c h the TWo-base alteration c hange in a s i n g l e b a s e p a i r i n th e human genome can cause a seriousdiseasee.g. sickleChainbreaks c ell anem ia .

Depurination Basealkylation lnsertion or deletion of nucleotides of baseanalogue Incorporation pyrimidine UV lightinduced (T-T) dimeralteration

freeradicalformation Oxidative lonizing radiation

TYPES OF DNA DAMAGES

Cross-linkage

T he dam a g e s d o n e to D N A b y p h ysi cal , chemical and environmental agents may be broadly classified into four categories with different Wpes (Table 24.1). The DNA damage.mayoccur due to singlebase alterations(e.g. depuri nation, deami nation), two-base alterations (e.g. pyrimidine dimer) chain breaks (e.g. ionizing radiation) and crosslinkages (e.g. between bases).Some selected DNA damagesare briefly described.

Between basesin thesameor opposite strands Between theDNAandprotein molecules

Ultraviolet radiationsresult in the formation of covalent links between adjacent pyrimidines along the DNA strand to form pyrimidine dimers. DNA chain breaks can be caused by i oni zi ng radi ati ons(e.9.X -rays).

MUTATIONS

The occurrenceof spontaneousdeamination The genetic macromolecule DNA is highly basesin aqueoussolution at37"C is well known. stable with regard to its base composition and Cytosine gets deaminatedto form uracil while sequence.However, DNA is not totally exempt adenine forms hypoxanthine. from gradual change. depurination,due to cleavageof Spontaneous Mutation refers to a change in the DNA glycosyl bonds (that connect purines to the structure of a gene. The substances(chemicals) backbone) also occurs. lt is estimated that which can induce mutations are collectivelv 2000-10,000 purines may be lost per known as mutagens. mammalian cell in 24 hours. The depurinated The changesthat occur in DNA on mutation sites are called as abasic sifes. Originally, they reflected in replication, transcription and are were detected in purines, and called apurinic translation. sites (AP sifes) which represent lack of purine. Now, the term AP sites is generally used to Types of mutations represent any hase lacking in DNA. Mutations are mainly of two major typesThe production of reactive oxygen species is point mutations,frameshiftmutations (Fig,2a,l4). often associated with alteration of bases e.g. 1. Point mutations : The replacementof one formation of 8-hydroxy guanine. Free radical formation and bxidative damage to DNA base pair by another resultsin point mutation. They are of two sub-types. increaseswith advancementof age.

535

B IOC H E MIS TFIY

(a) Transitions: In this case, a purine (or a pyrimidine) is replaced by another.

(A)

-9-9-4-9llr lll' l

(b) Transversions : These are characterizeo by replacement of a purine by a -G-C p y ri m i d i n eo r v i c e v e rs a .

-9-q

2. Frameshiftmutations : Theseoccur when one or more basepairsare insertedin or deleted -G-C from the DNA, respectively,causing insertionor deletion mutations. (B)

rransition-9-9-G-9C-

-G-C-C-C-

-P-

-9-9-'--9rransversion

-C-

-G-C-A-C-

ttt- ttl

ffionselqu*rli:{is

-c-G-

of pc*nt il}{.lEiitirlns

-f-G-

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T h e c h a n g e i n a s i n g l e b a s e sequence i n -c-c -cpoint mu ta ti o nma y c a u s eo n e o f the fol l ow i ng (Fi9.24.1fl. -G-C-T-C-

19

1. Silent mutation : The codon (of mRNA) containing the changed base may code for the same amino acid. For instance,UCA codes for serine and change in the third base (UCU) still codesfor serine.This is due to degeneracyof the genetic code. Therefore,there are no detectable effects in silent mutation.

-*N-i-i-i-

rr

2. Missense mutation : In this case, the changed base may code for a different amino acid. For example, UCA codes for serine while ACA codes for threonine. The mistaken (or

r <."\

G-C-G-

Flg. 24.14: An illustration ol mutations(A)-Point (81-Framesh ift mutations. mutations;

missense) amino acid may be acceptable, partially acceptable or unacceptable with regard to the function of protein molecule. Sickle-cell anemia is a classical examole of missense mutation.

A few mtcrogroms (7O-12S) of DNA tn a letal cell stores the genetic tnlormation thot wlll determlne the dtfferentlqtlon and euery lunction of on adult onimal. This is the maruel of moleculor biology. Topolsomerase inhlbitors (e.g. adriamycin, etoposide) are useful to preuent DNA replicatlon, and thus uncontrolled cell proltleration in cancen These compounds block the unwlndlng ol DNA stronds, The progressiue shorfen lng of telomeres (DNA sequences ot chromosomal ends) is preuented by telomerase. Thts enzyme ls an sttroctiue target t'or cancer therapy. ffi

Mutations may sometlmes result ln serlous dlseoses.e.g, sickle-cell onemla, cancen

!iai'

Xeroderma plgmentosum Is a rare dlseasechorocterlzed by photosenstttutty ond rlsk lor skfn concer: Thls ls due to a defect tn the nucleotlde exclslon repalr of the damaged DNA (caused by UV rays). Heredttary nonpolyposis colon concer ls a common tnhertted cancer, ond is due to a laulty mlsmatch repalr ol delectlue DNA.

AND REPAIR FECOMBINATION, Chapter 24 : DNA-REPLICATION, UCU (codonfor Ser)

tI

Silent mutation

I UCA

537

in the reading of codons, translationcontinues' The result is that the protein synthesizedwill have several altered amino acids and/or prematurelyterminatedProtein.

Mutations and cancer Mutations are permanentalterationsin DNA structure,which have been implicated in the etiopathogenesisof cancer. REPAIR OF DNA

of pointmutations Fig.24.15:An itlustration (represented bYa codonot nRNA).

3. Nonsense mutation : Sometimes,the codon with the alteredbase may becomea termination(or nonsenselcodon. For instance, change in the second base of serinecodon (uCN may resultin UAA. The alteredcodon actsas a stopsignaland causesterminationof at that Point. proteinsynthesis, Gonsequences of frameshift mutations

As already stated,damage to DNA caused by replicationerrorsor mutationsmay have serious consequences,The cell possessesan inbuilt system to repair the damaged DNA. This may be achieved by four distinct mechanisms (Table 24.2). 1. B aseexci si on-reP ai r 2. Nucleotide excision-rePair 3. Mismatch rePair 4. Double-strandbreak rePair.

Base excision.repair

The basescytosine,adenineand guaninecan The insertionor deletion of a base in a to respectively depurination spontaneous gene resultsin an alteredreadingframeof the undergo These xanthine. and Lnrun (hence the name frameshift)'The form uracil,hypoxanthine DNA, normal the not exist in machineryof mRNA (containingcodons)does alteredbasesdo carried This is that a basewas missingor a new and thereforeneedto be removed. not recognize repair(Fi9.24.16). basewasadded.Sincethereareno punctuations out by baseexcision

Mechanism

Baaeexclslon.rePalr

basedueto to a single Damage or bY alteration sDontaneous means' or radiation chemical

of thobaseby N-glycosylase; Removal replacement' sugarremoval, abasic

exclslon'rePalr Nucleotlde

of DNAbY to a segment Damage or radiation chemical spontaneous, means.

(- 30 nt of theDNAtragment Removal andreplacement. length)

rePair Mlsmatch

errors dusto coPyhg Damage (1-5baseunPaired looPs).

of thestrand(byexonuclease Removal andreplacement' digestlon)

breakrePah Doublestnnd

andligetion. alignment radiations,unwinding, byionizing caused Damage otc. chemotherapy freeradicals,

ll

538

BIOCHEMISTRY TCCT

Il l

AGGA NormalDNA

I

+

TCUT

llll

AGGA DefectiveDNA

I

.l UracilDNA U+/l glYcosvlase

+ TCXT

llll

(exinuclease) damagedpart.An excisionnuclease (upstream and cuts the DNA on either side downstream)of the damaged DNA. This defectivepiece is degraded.The gap createdby the nucleotideexcisionis filled up by DNA polymerase which gets ligatedby DNA ligase (Fig.z4.tV. Xeroderma pigmentosum (XP) is a rare autosomal recessive disease. The affected patientsare photosensitiveand susceptibleto thatXP is due skincancers.lt is now recognized to a defectin the nucleotideexcisionrepairof the damagedDNA. ,

AGGA

II Endonucleases

J

AGGA

Mismatch

repair

defects Despitehigh accuracyin replication, do occurwhenthe DNA is copied.Forinstance, cytosine (i,nsteadol thymine) could be incorporatedoppositeto adenine.Mismatch

I

polymerase IDNA IDNAligase + TCCT

Itl

AGGA

A defective DNA in which cytosine is deaminatedto uracil is acted upon by the enzymeuracil DNA glycosylase. This resultsin the removalof the defectivebase uracil. An endonuclease cutsthe backboneof DNA strand near the defectand removesa few bases.The gap so createdis filled up by the actionof repair DNA polymerase and DNA ligase. Nucleotide

excision.repair

The DNA damagedue to ultravioletlight, ionizing radiation and other environmental factors often results in the modification of certainbases,strandbreaks,cross-linkages etc. Nucleotideexcision-repair is ideallysuitedfor such large-scaledefects in DNA. After the identificationof the defectivepieceof the DNA, the DNA doublehelixis unwoundto exposethe

Defectrecognition andunwinding

Cuttingat two sites to removedefective oligonucleotide

Degradation of defectlveDNA Resynthesis and religation

Ghapter 24: DNA-REPLICATION, RECOMBINATION, AND REPAIR repair correctsa single mismatch base pair e.g. C to A, instead of T to A.

?".

The template strand of the DNA exists in a methylatedform, while the newly synthesized strand is not methylated.This differenceallows the recognitionof the new strands.The enzyme CATC endonuclease cuts the strand at an adjacent methylated CATC sequence Gig.2aJA. This is followed by an exonuclease digestion of the defective strand, and thus its removal.A new DNA strand is now synthesized to replace the damaged one.

CHa

I Singlestrand cutbVGATCendonuclease I v CHa t"

Jr*onr.,""." I DNApolymerase I

+ CHn t-

bteak

9Ht I

Hereditary colon cancer (HNPCC) is one of the most common inherited cancers.This cancer is now linked with faulfv mismatch repair of defective DNA. Doubfe"sttand

539

CHe

repair

Double-strand breaks (DSBs) in DNA are dangerous.They result in geneticrecombination which may lead to chromosomaltranslocation, broken chromosomes,and finally celI death. DSBs can be repaired by homologous r ec om bin a ti o no r n o n -h o mo l o g o u se n d j oi ni ng. Homologous recombination occurs in yeasts whife in mammals,non-homologousand joining dominates.

s',

CHe t-

s'. 3',

Fig. 24,18: A diagrammatic representation of mismatchreoairof DNA.

altered. Defects in the genes encoding proteins i nvol vedi n nucl eoti de-exci si on repai r.mi smatcn DEFECTSIN DNA REPAIR repai r and recombi nati onalrepai r are l i nked to AND CANCER human cancers.For i nstance,as al readyreferred Cancer develops when certain genes above, HNPCC is due to a defect in mismatcn that regulate normal cell division fail or are reDatr.

l

rt

B IOC H E MIS TR Y

540

7. The central dogma of llfe reuolues around the flow of information from DNA fo RNA, and from there to proteins. 2. Repltcation is o process in which DNA copies itself to produce identicol daughter moleculesol DNA. DNA replication is semiconserustiue,bidirectional and occurs by the formation of bubbles and forks. 3. DNA syntheis is catalysed by the enzyme DNA polymerase III. This enzyme possesses proof-reoding acttuity and edtts the mistokes that might occur during nueleotide incorporatton. 4. Repftcatton {n eukoryotes (particularly on the )agging strand) is more complex and inuolues seuerql foctors e,g. replication protein A, replication factor C, flap endonucleose. 5. Telomeres (repeat TTAGGG sequences)ore the special structures that preuent the continuous loss o/ DNA ot the end ol the chromosome during the course oJ replication. 6. Recombination inuolues the dxchange of genetic information through the exchange ol DNA. Transposition ret'ers to the mouement of speciJic pieces o/ DNA (called fronsposons)in the genome. '7. Domage fo DNA may be due to single bose alterotion, two-bosealterqtion, chain breaks qnd cross linkages.The cells possessan inbuilt system to repair the damaged DNA

I. Essayquestions 1. 2. 3. 4. 5.

D e s c ri b eth e re p l i c a ti o no f D N A . Cive an accountof recombinationof DNA. Discussdifferenttypesof DNA damages,and the repairmechanisms. of mutations. What are mutations?Describedifferenttypes,and consequences and cancer' Cive an accountof telomeresand their role in senescence

II. Short notes (e) Inhibitorsof (a) Replication fork, (b) Okazakipieces,(c) RNA primer,(d) DNAtopoisomerases, (h) (g) Holliday model of DNA recombination, Transposition, DNA replication,(0 Telomerase, (i) Frameshift mutations,(j) Missensemutation,(k) Mismatchrepair,(l) Xerodermapigmentosum. I I L F i l l i n th e b l a n k s 1. DNA strandsfor replicationprocessare separatedby the enzyme 2. The small fragmentsof DNA producedduring replicationare called functionis carriedout by the enzyme 3. Duringthe courseof DNA replication,the proof-reading

HECOMBINATION, AND FEPAIH Ghapter 24: DNA-FIEPUCAT|ON,

541

4. The problemof supercoilsin DNA replicationis overcomeby a group of enzymes,namely 5. The proteinsthat are associatedwith the transition of one phase of cell cvcle to another 6. Name the DNA sequencethat preventsthe continuousloss of DNA at the end of the chromosomeduring the course of replication 7. The mobile segmentsof DNA are called 8. Any change in the DNA sequenceof a gene is commonly referredto as 9. Sickle-cellanemiais a good exampleof

mutation.

10. One common example of inherited cancer with fautty mismatch repair of defective DNA

IV. Multiple choice questions 11. The chemical natureof the primer requiredfor the synthesisof DNA (a) DNA (b) Histone(c) RNA (d) hnRNA. 12. The enzymeresponsiblefor the synthesisof RNA primer in eukaryotes (a) DNA polymeraseo (b) DNA polymerase0 (c) DNA polymerasey (d) Topisomerases. 13. The repeatsequenceof nucleotidesin telomeres (a) TTCCCA (b) TTACCG (c) CCCATT (d) TTCACG. 14. The DNA damagecausedby deaminationis an exampleof (a) Single-basealteration(b) Two-basealteration(c) Chain breaks(d) Crosslinkage. 15. The mutation involving the replacementof one purine by another (a) Frameshiftmutation (b) Transition(c) Transversion(d) None of the above.

I

Transcription andTranslation TIrc genctic

aodc speahs t

"Tipkt haseseqaence RNA,I am; of messenger in character; Uniatrsal,Eeqific,non-oaefkry** degqnerata Faithfullyworhunderthedictansof DNA : Tbexecute orders tn! master's for prowinqntllesis,"

-f. h" conventionalconceptof centraldogma of I lif e whic h i n e s s e n c ei s " D N A ma k e s R N A makes protein" is an oversimplification of m olec ular b i o l o g y . W i th th e a d v a n c e sin cel l biology an d ra p i d d e v e l o p me n ts i n bi oinformatics, the terms genome, transcriptome and proteome are in current use to represent t he c ent r a l d o g ma o f mo l e c u l a r bi ol ogy 1Fig.25.l). Some information on the new c onc ept san d te rmi n o l o g yi s g i v e n h e re u nder.

rr'i, ilil'*5 i:5i !SjTL]1'll:

I

Conventional concept (pre-bioinformatics era)

GENOME

ITranslation lpl+orr'.or,nL-] concept Current (bioinformatics era)

Fiq,25.1: Thecentraldogmaof life (or molecular biology)representedin the formof conventional The total DNA (genetic information) and currentconcepts. contained in an organism or a cell is regardedas the genome.Thus, the genome is the storehouse of biologi c a l i n fo rma ti o n . l t i n c l u d es the structuralmotifsand completeproteinstructures. c hr om os om e si n th e n u c l e u s a n d th e D N A i n Comparative genomics involves the study of m it oc hond ri a a , n d c h l o ro p l a s ts . comparativegene function and phylogeny. Genomics : The study of the structureand function of genome is genomics. The term functional genomics is used to represent the gene expressionand relationshipof genes with gene products. Structural genomics refersto the

TRANSCBIPTOME The RNA copies of the active protein coding genes represent transcriptome. Thus, transcri ptomei s the i ni ti al product of gene

542

543

AND THANSLATION Ghapten 25 : TFANSCRIPTION expression which proteins.

directs the synthesis of

Transcriptomics: The study of transcriptome that involvesall the RNA moleculesmade by a cell, tissueor an organism is transcriptomics.

q

PROTEOIIE The cell's repertoi re (reposito rylstorehou se) of proteins with their nature and biological functions is regarded as proteome. Thus, proteome representsthe entire range of proteins and t hei r b i o l o g i c a lfu n c ti o n si n a c e l l .

Fig. 25.2 : RNApolymerase of E. coli.

Proteomics : The study of the proteome. Metabolomics : The use of genome sequence analysisfor determiningthe capability of a cell, tissue or an organism to synthesize small molecules(metabolites)is metabolomics. Whether the central dogma of life is representedin the conventionalor more recent form, replication, transcription and translation are the key or core processesthat ultimately control life. Reolication of DNA has been described in Chapter 24, while transcriptionand translationare discussedin this chapter.

some selectedregionsof DNA. For certain other regions of DNA, there may not be any transcription at all. The exact reason for the selectivetranscriptionis not known. This may be due to some i nbui l t si gnal s i n the D N A mol ecul e. The productformed in transcriptionis referred to as primary transcript. Most often, the primary RNA transcripts are inactive. They undergo certain alterations(splicing,terminal additions, base modifications etc.) commonly known as post-transcriptional modifications, to produce functionally active RNA molecules.

There exist certain differences in the transcriptionbetweenprokaryotesand eukaryotes. Transcriptionis a processin which ribo- The RNA synthesisin prokaryotesis given in some nucleic acid (RNA) is synthesizedfrom DNA. detail. This is followed by a brief discussionon The word gene refersto the functional unit of eukaryotictranscription.

the DNA that can be transcribed.Thus, the geneticinformationstoredin DNA is expressed TRANSCRIPTION IN PROKARYOTES throughRNA. For this purpose,one of the two A single enzyme-DNA dependent RNA strandsof DNA servesas a template(non-coding strand or sensestrand)and produces working polymerase or simply RNA polymeraseTheotherDNA strand synthesizesall the RNAs in prokaryotes.RNA copiesof RNAmolecules. which does not participatein transcriptionis polymeraseof E. coli is a complex holoenzyme strand (mol wt. 465 kDa) with five polypeptide referredto as codingstrandor antisense (frequentlyreferredto as coding strandsince subunits-2c, 1p and 1p' and one sigma(s)factor with the exceptionof T for U, primarymRNA (Fi9.25,2).The enzyme without sigma factor is containscodonswith the samebasesequence). referred to as core enzyme (ozpp'). An overview of RNA synthesisis depicted in Fig,25.3. Transcription involves three different Theentiremoleculeof DNA is not expressed stages-initiation, elongation and termination only for (Fig.25.4. in transcription. RNAsare synthesized

Transeription

is selective

452

BIOCHEMISTRY

I. Essayquestions 1. Describethe role of second messengers in hormonal action. 2. Write an account of the anterior pituitary hormones. 3. Discussin detail the synthesisand biochemicalfunctionsof thyroid hormones. 4. Describethe hormonesof adrenalcortex with special referenceto glucocorticoids. 5. Write briefly on the synthesisand biochemicalfunctionsof sex hormones. II. Short notes (a)'C'-Proteins, (b) Inositol triphosphate,(c) Hypothalamic hormones, (d) ACTH, (e) Goiter, (fl Epinephrine,(g) Cortisol, (h) Castrin, (i) ADH, (j) Aldosterone. III. Fill in the blanks 1. The enzyme that catalysesthe formation of cAMP from ATP is 2. The inorganicion that can act as a secondmessenger for certainhormonesis 3. The endocrineorgan responsiblefor the synthesisof trophic hormonesis 4. The compounds that produce opiate-like effects on the central nervous system are 5. The enzyme that convertsiodide (l-) to active iodine (l+) 6. The most predominantmineralocorticoidsynthesizedby adrenalcortex 7. The major urinary excretoryproduct of catecholamines 8. The male sex hormone, testosterone, is converted to a more active form, namely 9. The precursorfor the synthesisof steroid hormones 10. The gastrointestinalhormone that increasesthe flow of bile from the gall bladder IV. Multiple choice questions 11. lmpairmentin the synthesis of dopamineby the brain is a majorcausative factorfor the disorder (a) Parkinson'sdisease(b) Addison'sdisease(c) Cushing,ssyndrome(d) Coiter. 12. One of the following hormonesis an amino acid derivative (a) Epinephrine(b) Norepinephrine (c) Thyroxine(d) All of them. 13. The most activemineralocorticoid hormoneis (a) Cortisol (b) Aldosterone(c) 11-Deoxycorticosterone (d) Corticosterone. 14. Name the hormone,predominantlyproducedin responseto fight, fright and flight (a) Thyroxine (b) Aldosterone(c) Epinephrine(d) ADH. 15. The hormoneessentiallyrequiredfor the implantationof fertilizedovum and maintenanceof pregnancy (a) Progesterone (b) Estrogen(c) Cortisol (d) Prolactin.

544

BIOCHEMISTRY

and, further,this enzyme for RNA polymerase possess activity. endo-or exonuclease doesnot The bindingof the enzymeRNA polymerase (proof-reading function Due to lack of the latter to DNA is the prerequisite for the transcription to hasno abilityto repair activity),RNApolymerase start.The specificregionon the DNA wherethe This is in the mistakesin the RNA synthesized. enzyme binds is known as promoter region. which is carriedout contrastto DNA replication There are two base sequenceson the coding with high fidelity.lt is, however,fortunatethat DNA strandwhich the sigma factor of RNA mistakesin RNA synthesis are lessdangerous, polymerasecan recognizefor initiation of to the daughter are not transmitted since they transcription(Fig.2|.A. cells. 1. Pribnowbox (TATAbox) : Thisconsistsof of DNA unwinds Thedoublehelicalstructure 6 nucleotidebases(TATAAT), locatedon the left goes otr, resulting in the transcription as side about 10 basesaway (upstream) from the supercoils.The problem of supercoils is startingpoint of transcription. (more details in overcomeby topoisomerases 2. The '-35' sequence: This is the second Chapter 24). recognition sitein the promoterregionof DNA. It containsa basesequence TTCACA,which is Termination locatedabout 35 bases(upstream,hence-35) The process of transcription stops by away on the left side from the site of terminationsignals.Two typesof terminationare transcriptionstart. identified. Initiation

termination: A specific 1. Rho(p) dependent protein,namedp factor,binds to the growing As the holoenzyme, RNA polymerase RNA (andnot to RNA polymerase) or weaklyto recognizes the promoterregion,the sigmafactor DNA, and in the bound stateit acts as ATPase is releasedand transcriptionproceeds.RNA is and releases RNA. and terminates transcription synthesizedfrom 5' end to 3' end (5'-+3') The p factor is also responsiblefor the antiparallel to the DNA template. RNA from DNA. of RNA polymerase dissociation polymerase utilizesribonucleotide triphosphates 2. Rho (p) independenttermination : The (ATP,CTP, CTP and UTP)for the formationof in this caseis broughtaboutby the termination RNA.Forthe additionof eachnucleotide to the RNA. hairpinsof newly synthesized formation ol growing chain, a pyrophosphatemoiety is presence palindromes. of This occurs due to the released. A palindromeis a word that readsalikeforward The sequenceof nucleotidebasesin the and backwarde.g. madam,rotor.The presence mRNA is complementary to the templateDNA of palindromes in the basesequenceof DNA strand.lt is however,identicalto that of coding template(samewhen readin oppositedirection) strandexceptthat RNAcontainsU in placeof T in thetermination regionis known,As a resultof in DNA (Fig.2s.6). RNA folds to form this, the newly synthesized RNA polymerase differs from DNA hairpins(due to complementary base pairing) polymerase in two aspects. No primeris required that causeterminationof transcription. Elongation

Tranecrlptlon unlt

If o a N

DNA template

3 -I]

za

C) ll T

z 3' 5',

z o -1 fl

z a

h> -.1

d

z

9l

s 9l

546

BIOCHEMISTF|Y

-35 Sequence 5' -TTGAC

Coding strand

Template a, strand

Startof transcription Fiq.25.5: Promoterregionsof DNAin prokaryotes. 3' Codingstrand 5' Templatestrand RNA----------------+s'..--..A U G C A U G G C A........3', Fig. 25.6 : nanscription-Complementary

TRANSGRIPTION

IN EUKARYOTES

RNA synthesisin eukaryotesis a much more complicated process than the transcription describedabove for prokaryotes.As such, all the details of eukaryotic transcription (particularly about termination)are not clearly known. The salientfeaturesof availableinformationare given hereunder. RNA po l y n te ra s e s The nuclei of eukaryotic cells possessthree distinct RNA polymerases(Fi9.25.7).

base pair relationship.

2. RNA polymerase ll synthesizes the precursorsfor mR N A s and smal l nucl earR N A s. 3. RNA polymerase lll participates in the formation of tRNAs and small ribosomal RNAs. Besidesthe three RNA polymerasesfound in the nucleus, there also exists a mitochondrial RNA polymerase in eukaryotes. The latter resembles prokaryotic RNA polymerase in structureand function. Promoter

sites

ln eukaryotes,a sequenceof DNA bases1. RNA polymeraseI is responsiblefor the which is almost identical to pribnow box of synthesisof precursorsfor the large ribosomal prokaryotes-is identified (Fig.25.A. This RNA s . sequence,known as Hognessbox (or TATA box),

s',

3',

3',

5'

I I +

I I RNApolymerase ll RNApolymerase II I

RNApolymerase I

+

e.

r\

r-

I

+

q'l tl

{ a-1 )t-r<

CJ

C)

I/

Ribosomal RNAs

Messenger RNA

Transfer RNA

Fig. 25.7 : An oveNiew of transciption in eukaryotes.

DNA

Chapter 25 : THANSCRIPTION AND TRANSLATTON

547

CMT box Non-coding strand Coding strand -70 bases

is located on the left about 25 nucleotides away (upstream) from the starting site of mRNA synthesis. There also exists another site of recognition between 70 and 80 nucleotides upstream from the start of transcription.This second site is referred to as CAAT box. One of these two sites (or sometimes both) helps RNA polymerase ll to recognize the requisite sequenceon DNA for transcription.

Initiation of transcription

-25 bases

^'

Codingregionof gene J

Heterogeneous RNA (hnRNA)

nuclear

The primary mRNA transcript produced by RNApolymerase ll in eukaryotes is oftenreferred to as heterogeneous nuclearRNA(hnRNA). This is then processed to producemRNA neededfor proteinsynthesis. POST.TRAl{SCRIPTIONAL MODIFICATIONS

The RNAsproducedduringtranscription are primarytranscripts. called They undergo many initiation of transcription in eukaryotes are alterations-terminal base additions, base complex, and broadly involve three stages. modifications, splicing etc., which are 1. Chromatin containing the promoter collectively referredto as post-transcriptional sequence made accessibleto the transcription modifications. Thisprocessis requiredto convert machinery. the RNAs into the active forms. A group of enzymes/namelyribonucleases, are responsible 2. Binding of transcription factors (TFs) to for the processing of tRNAs and rRNAsof both DNA sequencesin the promoter region. prokaryotesand eukaryotes.

The moleculareventsrequiredfor the

3. Stimulationof transcriptionby enhancers. A farge number of transcription factors interact with eukaryotic promoter regions. In humans, about six transcription factors have been identified(TFllD,TFllA, TFllB,TFllF,TFllE, TFIIH).lt is postulatedthat the TFs bind to each other, and in turn to the enzyme RNA polymerase.

Enhancercan increasegene expressionby about100fold.Thisis madepossibleby binding of enhancersto transcriptionfactors to forri activators.ltis believedthatthe chromatinforms a loop that allowsthe promoterand enhance' to be close together in space to facilitate transcription.

The prokaryoticmRNA synthesizedin transcriptionis almostsimilarto thefunctionalmRNA. In contrast,eukaryoticmRNA (i.e. hnRNA) undergoes extens ive post-transcri ptionalchanges. An outline of the post-transcriptional modificationsis given in Fig.21.g,and some highlightsare described. Messenger RNA tn: primarytranscriptof mRNAis the hnRNA . in eukaryotes,which is subjected to many changesbeforefunctionalmRNA is produced' l. The 5, capping: The 5, end of mRNA is cappedwith z-methylguanosine by an unusual

BIOCHEMISTFIY

hnRNA(preRNA)

End

Chemical modifications

E Cap

Poly(A)tail

Introns removed

Cutpieces

New chemical groups added

Flg. 25.9: An outlineof post-transcriptional nuclearRNA). modificationsof RNA(hnRNA-Heterogeneous

5'-+5' triphosphate linkage. S-AdenosylA diagrammatic representation of the m et h i o n i n ei s th e d o n o r o f me th yl group. Thi s relationship between eukaryotic chromosomal cap is requiredfor translation,besidesstabilizing DNA and mRNA is depicted in Fig,25,ll. the structureof mRNA. l';?tEremt rnHf*As produceei 2. Poly-A tail : A large number of eukaryotic [:y al ternate spl i ci ng mRNAs possessan adenine nucleotidechain at Alternatepatternsof hnRNA splicing result in t he 3 ' -e n d . T h i s p o l y -A ta i l , a s such, i s not produced during transcription.lt is later added different mRNA molecules which can produce to stabilizemRNA. However, poly-A chain gets reduced as the mRNA enterscvtosol. 3. Introns and their removal : Intronsare tne int erv e n i n g n u c l e o ti d e s e q u e n ces i n mR N A which do not code for proteins. On the other hand, exons of mRNA possess genetic code and are responsible for protein synthesis. The splicingand excisionof introns is illustratedin Fi9.25.10.The removalof intronsis promoted by small nuclear ribonucleoprotein particles(snRNPs).snRNPs(pronounced as snurps) in turn, are formed by the as s o c i a ti o no f s m a l l n u c l e a r R N A (snR N A )w i th pr ote i n s . The term spliceosome is used to represent t he s n R N P a s s o c i a ti o nw i th h n R N A at the ex on -i n tro nj u n c ti o n . Post-transcriptional modifications of mRNA occur in the nucleus. The mature RNA then enters the cytosol to perform its function (translation).

Exon1

Intron

Exon2

(r

Exon 1

Exon2

,i

+ 1,/ Excised intron

Flg.25.10: Formationof matureRNAfromeukaryotic mFNA (SnRNPs-Small nuclear ribonucleoprotelnpaft icles).

Ghapter 25 : TFANSCFIPTION AND TBANSLATION

1 .5x 1 o Bbp

1. 5x 106bp

549

conversion of CAA codon in mRNA (of apoprotein B gene) to UAA by the enzyme cytidine deaminase.As a result,originatingfrom the same gene, the liver synthesizesa 100-kDa protein (apoB 100) while the intestinal cells synthesize 48-kDa protein (apoB 48). This happensdue to formationof a terminationcodon (UAA) from CAA in RNA editing.

Transfer RNA

Onegene(with8 exonsand 7 introns)I

2.5 x 104 bp

I

+

8x1o3nt Primarytranscript

I + mRNA

2x103nt

Fig. 25.11: A diagrammatic representation of the relationshipbetweeneukaryoticchromosomalDNAand mRNA(bp-Basepair; nt-Nucleotides).

different proteins. Alternate splicing results in mRNA heterogeneity.In fact, the processingof hnRNA molecules becomes a site for the regulationof gene expression. Faultysplicingcan causediseases: Splicingof hnRNA has to be performedwith precision to produce functional mRNA. Faulty splicing may resultin diseases. A good example is one type of p-thalassemiain humans. This is due to a mutationthat resultsin a nucleotidechangeat an exon-intronjunction. The result is a diminished or lack of synthesisof p-chain of hemoglobin, and consequentlythe diseasep-thalassemia. mRNA

editing

T he s equenc e i n th e D N A d e te rm i n e sth e co ding s equence i n mR N A , a n d fi n a l l y th e amino acid sequencein the protein. However, in recent years, changes in the coding information by editing of mRNA have been reported.lt is estimatedthat about 0.01% of the mRNAs undergoesediting. One example is the

All the tRNAs of prokaryotesand eukaryotes undergopost-transcriptional modification.These include trimming, convertingthe existing bases into unusual ones, and addition of CCA nucleotidesto 3' terminal end of tRNAs. Ribosonral

RNA

The preribosomalRNAsoriginallysynthesized are converted to ribosomal RNAs by a seriesof post-transcriptional changes. Inhibitors

of transcription

The synthesisof RNA is inhibited bv certain anti bi oti csand toxi ns. Actinomycin D : This is also known as dactinomycin. lt is synthesizedby Streptomyces. Actinomycin D binds with DNA templatestrand and blocks the movement of RNA polymerase. This was the very first antibiotic used for the treatmentof tumors. Rifampin : lt is an antibiotic widely used for the treatment of tuberculosis and leprosy. Rifampinbinds with the p-subunitof prokaryotic RNA polymeraseand inhibits its activity. a-Amanitin : lt is a toxin produced by mushroom,Amanita phalloides.This mushroom i s del i ci ous i n taste but poi sonousdue to the toxin o-amanitin which tightly binds with RNA polymerase ll of eukaryotes and inhibits transcri pti on.

CELLULAR RNA CONTENTS A typi cal bacteri um normal l y contai ns 0.05-0.10pg of RNA which contributesto about 6Toof the total weight. A mammaliancell, being larger in size, contains20-30 pg RNA, and this

550

B IOC H E MIS TRY

Fig, 25.12: A diagrammaticreprcsentationof RNAcontentof a cell (Note; FN,4srepressntedin black are found in all organisms; RNAsin colour and exclusivelypresent in eukaryotesonly; hnRNA-Heterogeneous nuclear RNA; rt snRNA-Small nuclear RNA: snoRNA-Small

represents only 1% of the . cell weight. Transcriptome,representingthe RNA derived from protein coding genes actually constitutes only 4o/",while the remaining 96oh is the noncoding RNA (F8.25.1A. The different noncoding RNAs are ribosomal RNA, transferRNA, s m a l l n u c l e a r R N A, s ma l l n u c l eol ar R N A and small cytoplasmic RNA. The functions of different RNAs are described in Chapter 2 (Refer Table 2.3\.

protein.The mRNA can be utilized as a template for the synthesis of double-stranded complementary DNA (cDNA) by using the enzyme This cDNA can be used as reversetranscriptase. a probe to identify the sequence of DNA in 8enes.

The genetic information stored in DNA is passedon to RNA (through transcription),and ultimatelyexpressedin the languageof proteins. The biosynthesis of a protein or a polypeptide in a living cell is referredto as translation.The term Some of the viruses-known as rctrcvirusestranslationis used to representthe biochemical possess RNA as the genetic material. These translation of four-letter language information virusescause cancers in animals, hence known from nucleic acids (DNA and then RNA) to 20 as oncogenic. They are actually found in the letter language of proteins. The sequence of transformedcells of the tumors. The enzyme RNA dependent DNA polymerase -or simply reverse transcriptaseis responsible for the formation ol DNA from RNA (Fig,25.13).This DNA is complementary (cDNA) to viral RNA and can be transmittedinto host DNA. Synthesisof cDNA from mRNA : As already described, the DNA expresses the genetic informationin the form of RNA. And the mRNA determines the amino acid sequence in a

s',-

5'Viral RNA

Primer

s', c

5' R NA 3' D NA of RNAvirus. Fiq.25.13: Reversetranscription

Chapter 25 : THANSCRIPTION AND TRANSLATION amino acids in the protein synthesized is determinedby the nucleotidebase sequenceof m RNA. Variability

of cells

in translation

There are wide variations in the cells with respectto the quality and quantity of proteins synthesized.This largely depends on the need and abifity of the cells. Erythrocy4es(red blood c ells ) la c k th e m a c h i n e ry fo r tra n sl ati on,and therefore cannot synthesize proteins.

551

The codons AUG-and, sometimes, GUGare the chain initiating codons.

Other characteristics of genetic code The genetic code is universal,specific, nonoverlappingand degenerate. 1. Universality : The same codons are used to code for the sameamino acids in all the living organisms. Thus, the genetic code has been conservedduring the courseof evolution.Hence genetic code is appropriately regarded as universal.There are, however, a few exceptions. For instance,AUA is the codon for methionine in mitochondria.The same codon (AUA) codes for isoleucine in cytoplasm. With some exceptionsnoted, the genetic code is universal.

I n g e n e ra l ,th e g ro w i n g a n d d i v i di ng cel l s produce larger quantities of proteins. Some of the cells continuously synthesizeproteins for export. For instance,liver cells produce albumin and blood clotting factors for export into the blood f o r c i rc u l a ti o n .T h e n o rma l Ii v e r cel l s are very rich in the protein biosyntheticmachinery, 2. Specificity : A particular codon always and thus the liver may be regarded asthe protein codes for the same amino acid, hence the factory in the human body. genetic code is highly specific or unambiguous e.g. UGG is the codon for tryptophan.

GENETIC CODE The fhree nucleotide (triplet) base sequences in nRNA that act as code words for amino acids in protein constitutethe geneticcode or simply codons.The genetic code may be regardedas a dictionary of nucleotidebases(A, C, C and U ) that determinesthe seouenceof amino acids in oroteins. The codons are composed of the four nucleotide bases,namely the purines-adenine ( A ) and g u a n i n e (C ), a n d th e p y r i mi di nescytosine (C) and uracil (U). These four bases produce 64 different combinations(43) of three base codons, as depicted in Table 25.1. The nucleotidesequenceof the codon on mRNA is wriften from the S'-end to 3' end. Sixty one codons code for the 20 amino acids found in protein. The three codons UAA, UAG and UCA do not code for amino acids. They act as stop signalsin protein synthesis.Thesethree codons are collectively known as termination codons or non-sensecodons. The codons UAC, UAA and UCA are often referred to, respectively, as amber, ochre and opal codons.

3. Non-overlapping: The geneticcode is read from a fixed point as a continuousbasesequence. It is non-overlapping, commalessand without any punctuations.For instance,UUUCUUACACGC is read as UUU/CUU/ACA/GCG. Addition or deletionof one or two baseswill radicallychange the messagesequencein mRNA. And the protein synthesizedfrom such mRNA will be totally different. This is encountered in frameshift mutations which cause an alteration in the readingframe of mRNA. 4. Degenerate: Most of the amino acids have more than one codon. The codon is degenerate or redundant, since there are 6l codons available to code for only 20 amino acids. For instance,glycine has four codons. The codons that designatethe same amino acid are called synonyms. Most of the synonyms differ only in the third (3' end) base of the codon. The Wobble hypothesis explains codon degeneracy(describedlater). Godosr.antEcodon

recognition

The codon of the mRNA is recognizedby the anticodon of IRNA (Fig.25.14). They pair with

BIOCHEMISTRY each other in antiparalleldirection (5'+ 3' of mRNA with 3' -) 5' of IRNA). The usual conventional complementary base paiiing (A=U, C=C) occurs between the first two bases of codon and the last two bases of anticodon. The third base of the codon is rather lenient or flexible with regardto the complementarybase. The anticodon region of IRNA consists of seven nucleotides and it recognizes the three letter codon in mRNA.

complementary base in the anticodon (5'-base). Wobbling is attributed to the difference in the spatial arrangement of the 5'-end of the anticodon.The possiblepairingof 51end baseof anticodon (of IRNA) with the 3'-end base of codon (mRNA) is given

Anticodon Codon

cA_ U_ G_

Wobble hypothesis putforthby Crick,is the Wobblehypothesis, phenomenon in which a single IRNA can recognize more than one codon. This is due to the fact that the third base (3'-base)in the codon often fails to recognizethe specific

Gr

basepairing [ ] Conventional base G orA Non-conventional 1 pairing U or C J (coloured)

Wobble hypothesisexplainsthe degeneracyof the geneticcode, i.e. existenceof multiple codons for a single amino acid. Although there are 61 codonsfor amino acids,the number of tRNAs is far less(around40) which is due to wobbling.

Second base (middle one)

ucc

.I UAU lrry uAc_l

UGU I lcvs uccI

c

UCA

UAA Stop

UGA Stop

A

UCG

UAG Stop

UGG Trp

G

CGU

U

UCU

ccu ccc ccA

cGc

Pro

CGA

ccG i ACU ACA

c

Arg

A . . . . . . , , , i . , . . . , , -, . . . --. . . -. . . --------

AAU

i

i Acc

U

c G G iG

i-"---""""-"--'---"'

i i

fhird base 3'end

Thr

i t99

AAC]*. AAA

a:

AGUI lSer

AGcl

i i

i

U

c

MG ]',

GCU GCC GCA

Ala

GGC Glv GGA

c A

GCG *AuG ,r*r, as initiating as nonsense uAA,UAGandt)GAcalted residue in prcteinsyntresis; Mon, besi(tes Ming lot neffiionine fortermination of proteinspthesis. cofuns,are responsible

553

AND TRANSLATION Ghapter 25 : TFANSCRIPTION fMet 3'e nd A-

fhe protein synthesis which involves the translation of nucleotide base sequence of mRNA into the languageof amino acid sequence may be divided into the following sfagesfor the convenienceof understanding. l. Requirementof the components Anticodon

ll. Activation of amino acids lll. Protein synthesisproper lV. Chaperonesand protein folding

ililltl

5'

AUG M 3' m RNA

tI

Codon Fig. 25.14 : Complementary binding of codon (of nRNA) and anticodon (ot iRNA).

Mutations and genetic code Mutations result in the change of nucleotide sequencesin the DNA, and consequentlyin the RNA. The different types of mutations are described in Chapter 24. The ultimate effect of mutations is on the translation through the alterationsin codons.Some of the mutationsare harmful. The occurrence of the disease sickle-cell anemia due to a single base alteration (CTC+ CAC in DNA, and CAG + GUC in RNA) is a classicalexampleof the seriousness of mutations.The result is that glutamateat the 6th positionof B-chainof hemoglobinis replacedby valine. This happens since the altered codon GUG of mRNA codes for valine instead of glutamate(coded by CAC in normal people). Frameshift mutations are caused by deletion or insertion of nucleotides in the DNA that generatealteredmRNAs.As the readingframe of mRNA is continuous, the codons are read in continuation,and amino acids are added. This results in proteins that may contain several altered amino acids, or sometimesthe protein synthesismay be terminatedprematurely.

V. Post-translational modifications. I. R E QU IR E ME N T GOMPONENTS

OF TI{E

The protein synthesismay be consideredas a biochemicalfactory operatingon the ribosomes. As a factory is dependent on the supply of raw materials to give a final product, the protein synthesisalso requiresmany components. 1. Amino acids : Proteins are polymers of amino acids. Of the 20 amino acids found in protein structure, half of them (10) can be synthesized by man. About lO essentialamino acids have to be provided through the diet. Protein synthesiscan occur only when all the amino acids neededfor a particularprotein are available. lf there is a deficiency in the dietary supply of any one of the essentialamino acids, the translationstops. lt is, therefore,necessary that a regular dietary supply of essentialamino acids, in sufficientquantities,is maintained,as it is a prerequisitefor protein synthesis. As regards prokaryotes, there is no requirementof amino acids, since all the 20 are synthesizedfrom the inorganic components. 2. Ribosomes: The functionally active ribosomes are the centres or factories for protein synthesis.Ribosomesmay also be considered as workbenchesof translation.Ribosomesare huge complex structures(70S for prokaryotesand 80S for eukaryotes)of proteinsand ribosomalRNAs. Eachribosomeconsistsof two subunits-one big and one small. The functional ribosomehas two

5s4

B IOC H E MIS TR Y

protein synthesized Completely

Fig. 25.15: A polyribosomein proteinsynthesis.

sites-A site and P site. Eachsite covers both the subunits.A siteis for binding of aminoacyltRNA and P sife is for binding peptidyl IRNA, during the courseof translation.Some authorsconsider A site as acceptor site, and P site as donor site. In caseof eukaryotes,there is anothersite called exif site or Esife. Thus, eukaryotescontain three sites (A, P and E) on the ribosomes.' The ribosomes are located in the cytosomal fractionof the cell. They are found in association with rough endoplasmicreticulum (RER)to form clusters RER-ribosomes, where the protein synthesis occurs. The term polyribosome (polysome) is used when several ribosomes s im ulta n e o u s l ytra n s l a te o n a s i n gl e mR N A (Fig.2s.rA. 3. Messenger RNA (mRNA) : The specific informationrequiredfor the synthesisof a given protein is presenton the mRNA. The DNA has passedon the genetic information in the form of codons to mRNA to translate into a protein sequence. 4. Transfer RNAs (tRNAs) : They carry the amino acids,and hand them over to the growing pept id e c h a i n . T h e a mi n o a c i d i s coval entl y bound to IRNA at the 3'-end. Each IRNA has a three nucleotide base sequence-the anticodon, which is responsibleto recognize the codon (complementary bases) of mRNA for protein svnthesis. In man, there are about 50 different tRNAs whereasin bacteriaaround 40 tRNAs are found. Some amino acids (particularly those with multiple codons) have more than one IRNA.

5. Energy sources z Both ATP and GTP are required for the supply of energy in protein synthesis.Some of the reactions involve the breakdown of ATP or CTP, respectively,to AMP and GMP with the liberationof pyrophosphate. Each one of these reactionsconsumestwo high energy phosphates(equivalentto 2 ATP). 6. Proteinfactors : The processof translation involves a number of protein factors.Theseare neededfor initiation,elongationand termination of protein synthesis. The protein factors are more complex in eukaryotes compared to prokaryotes.

il. ACTTVATTONOF AMr{O ACTDS Amino acids are activated and attached to tRNAs in a two step reaction. A group of enzymes-namely aminoacyltnNn synthetasesare required for this process.These enzymes are highly specific for the amino acid and the correspondingtRNA. The amino acid is first attachedto the enzyme utilizing ATP to form enzyme-AMP-aminoacid complex. The amino acid is then transferredto the 3' end of the IRNA to form aminoacvl IRNA (Fig.2s.t6). III. PROTEIN

SYNTI{ESIS

PROPER

The protein or polypeptide synthesisoccurs on the ribosomes (rather polyribosomes).The nRNA is read in the 5'-+3' direction and the polypeptide synthesis proceeds from N-terminal end to C-terminal end. Translation is directional and col l i nearw i th mR N A .

Ghapter 25 : THANSCRIPTION AND TFANSLATION

JJJ

Aminoacid

-AMP-Aminoacid

tRNA Fiq.25.16 : Formationof aminocacyl IRNA (AA-Amino acid; E-Enzyme).

The prokaryotic mRNAs are polycistronic, protein biosynthesis in eukaryotes is better s inc e a s ingl e mR N A h a s m a n y c o d i n g re gi ons understoodnow. that code for different polypeptides.In contrast, Translation in eukaryotes is briefly descrihed eukaryotic mRNA is monocistronic, since it here, along with some relevdnt features of codes for a single polypeptide. prokaryotic protein biosynthesis. Translation In caseof prokaryotes,translationcommences proper is divided into three stages-initiation, beforethe transcriptionof the gene is completed. el ongati on and termi nati on (as i t i s done for T hus , s im ult a n e o u tra s n s c ri p ti o na n d tra n s l ati on transcription). are possible.This is not so in case of eukaryotic or ganis m s s i n c e tra n s c ri p ti o n o c c u rs i n the ,NITIATION OF TRANSLAT'ON nucleus whereas translationtakes place in the cytosol. Further,the primary transcript(hnRNA) The initiation of translationin eukaryotesis formed from DNA has to undergo several complex, involving at least ten eukaryotic modificationsto generatefunctional mRNA. initiation factors (etFs).Some of the elFs contain Protein synthesisis comparativelysimple in multiple (3-8)subunits.The processof translation case of prokaryotes compared to eukaryotes. initiation can be divided into four steps Further, many steps in eukaryotic translation (Fig.25.1V. were not understoodfor ouite sometime. For 1 . Ribosomaldissociation. these reasons,majority of the textbooksearlier 2. Formationof 43S preinitiationcomplex. used to describe translation in prokaryotesin detail, and give most important and relevant 3. Formationof 48S initiation complex. informationfor eukaryotictranslation.With the 4. Formationof 80S initiation complex. advancesin molecular biology, the processof

BIOGHEMISTRY

Met

Met

48SinitiationcomPlex

['iTl+[-gl +P i

80S initiation comPlex

AND TFANSLATION Ghapter 25 : THANSCRIPTION Ribosomal

dissociation

The 80S ribosomedissociates to form 40S and 605 subunits.Two initiating factors namely elF3 and elF-lA bind to the newly formed 40S subunit,and therebyblock its reassociation with 605 subunit.For this reason,some workersname elF-3 as anti-association factor.

Formation

JJ/

of 8OS initiation

complex

48S initiationcomplex binds to 605 ribosomal subunit to form 80S initiation complex. The binding involvesthe hydrolysisof CTP (boundto elF-2).This step is facilitatedby the involvement of el F-5.

As the 80S complex is formed, the initiation factors bound to 48S initiation complex are Formation of 43S preinitiation released,and recycled. The activation of elF-2 complex requi res el F-2B (al so cal l ed as guani ne A ternary complex containingmet-tRNArand nucleotide exchange factor) and CTP. The elF-2 bound to CTP attachesto 40S ribosomal activatedelF:2 (i.e. bound to CTP) requireselFsubunit to form 43S preinitiationcomplex. The 2C to form the ternary complex. pr es enc e o f e l F -3 a n d e l F -1 A s ta b i li zesthi s Regulatidnof initiation complex (Nofe .' Met-tRNA is specifically involved in binding to the initiation condon The elF-4F, a complex formed by the AUGs; hence the superscript'is used in met- assembly of three initiation factors controls t RNA l. initiation, and thus the translationprocess.elF4E, a component of elF-4F is primarily Formation ol 48S initiation complex responsiblefor the recognition of mRNA cap. And this step is the rate-limitingin translation. T he bi n d i n g o f mR N A to 4 3 S p re i ni ti ati on elF-2 which is involved in the formation of complex resultsin the formationof 48S initiation complex through the intermediate43S initiation 43S preinitiationcomplex also controls protein complex. This, however, involves certain biosynthesisto some extent. interactions between some of the elFs and activationof mRNA. Initiation of translation elF-4F complex is formed by the association of elF-4C, elF-4A with elF-4E. The so formed elF-4F(referredto as cap binding protein)binds to the cap of mRNA. Then elF-4A and elF-48 bind to mRNA and reduce its complex structure. This mRNA is then transferredto 43S complex. For the appropriate association of 43S preinitiationcomplex with mRNA, energy has to be supplied by ATP. Recognition of initiation codon : The ribosomal initiation complex scans the mRNA for the identification of appropriate initiation codon. S'-AUG is the initiation codon and its recognitionis facilitatedby a specific sequence of nucleotides surrounding it. This marker sequencefor the identificationof AUC is called as Kozak consensus sequences. In case of prokaryotes the recognition sequence of initiation codon is.referredto as Shine-Dalgarno sequence.

in prokaryotes The formation of translation initiation complex in prokaryotes is less complicated compared to eukaryotes. The 30S ribosomal subunit is bound to initiation factor 3 (lF-3)and attached to ternary complex of lF-2, formyl metIRNA and CTP. Another initiationfactor namely lF-l also participates in the formation of preinitiation complex. The recognition of i ni ti ati on codon A U G i s done through S hi neDalgarnosequence.A 50S ribosomeunit is now bound with the 30S unit to produce 70S initiation complex in prokaryotes.

ELONGATION OF TRANSLATTON Ribosomeselongatethe polypeptidechain by a sequenti aladdi ti onof ami no aci ds.The ami no acid sequenceis determinedby the order of the codons in the specific mRNA. Elongation, a cyclic process involving certain elongation

558

BIOCHEMISTRY

factors(EFs),may be dividedinto threesteps In case of prokaryotes,the elongation factors (Fig.2s.tA. are different, and they are EF-Tu,EF-Ts(in place 1. Binding of aminoacyl I-RNA to A-site.

of of EF-1a)and EF-G (insteadof EF-2).

2. Peptide bond formation.

Incorporation of amino acids

3. Translocation. Binding A-site

of aminoacyl-tRNA

It is estimated thataboutsixaminoacidsper to

second are incorporated during the course of elongation of translation in eukaryotes. In case of prokaryotes,as many as 20 amino acids can be incorporated per second. Thus the processof protein/polypeptide synthesis in translation occurs with great speed and accuracy.

The 8OS initiation complex contains mettRNAi in the P-site,and the A-site is free. Another aminoacyl-tRNA is placed in the A-site. This requiresproper codon recognitionon the mRNA and the involvement of elongation factor 1a TEBil,,INATTONOF TBANSLATTON (EF-la)and supply of energy by GTP. As the Terminationis a simple processwhen aminoacyl-tRNAis placed in the A-site, EF-1d, compared to initiation and elongation. After and CDP are recycled to bring another severalcyclesof elongation,incorporatingamino aminoacyl-tRNA. acids and the formation of the specific protein/ polypeptide molecule, one of the stop or Peptide bond formation termination signals (UAA, UAG and UCA) The enzyme peptidyltransferase catalyses the terminates the growing polypeptide. The formation of peptide bond (Fi9.25.19). The terminationcodons which act as stop signalsdo activity of this enzyme lies on 28S RNA of 605 not have specific tRNAs to bind. As the ribosomal subunit. lt is therefore the rRNA (and termination codon occupies the ribosomal not protein) referred to as ribozyme that .A-site, the releasefactor namely eRF recognizes catalyses the peptide bond formation. As the the stop signal.eRF-CTPcomplex, in association amino acid in the aminoacyl-tRNA is already with the enzyme peptidyltransferase,cleavesthe activated, no additional energy is required for peptide bond between the polypeptide and the IRNA occupying P-site.ln this reaction,a water peptide bond formation. molecule, instead of an amino acid is added. The net result of peptide bond formation is This hydrolysis releasesthe protein and tRNA the attachment of the growing peptide chain to from the P-site.The 80S ribosome dissociatesto the IRNA in the A-site. form 40S and 605 subunitswhich are recycled. The mRNA is also released.

Translocation

As the peptide bond formation occurs, the ribosome moves to the next codon of the mRNA (towards 3'-end). This process called translocation,basically involves the movement of growing peptide chain from A-site to P-site. TranslocationrequiresEF-2 and GTP. GTP gets hydrolysedand suppliesenergyto move mRNA. EF-2 and CTP complex recycles for translocation ln recent years, another site namely exit site (E-site) has been identified in eukaryotes. The deacylated IRNA moves into the E-site, from where it leavesthe ribosome.

,N']TBITOBS OF PROTEIN

svtTttEsrs Translationis a complex processand it has become a favourite target for inhibition by antibiotics. Antibiotics are the substances produced by bacteriaor fungi which inhibit the growth of other organisms. Majority of the antibiotics interfere with the bacterial protein synthesisand are harmlessto higher organisms. This is due to the fact that the process of translationsufficiently d iffersbetweenprokaryotes and eukaryotes.The action of a few important antibioticson translationis describednext.

AND TBANSLATION 25: TBANSCRIPTION

Met

I

AAr

I AAz

l-

AAr I

Met 4Az

Peptidyltransferase

@ ,z- PePtidebond

f$et I l

AAr I AAz

i

Translocation

AAr I'

I

Ain @

mRNA Hg. E.l8

con|;d. nort Golumn

Peptide synthesized

BIOCHEMISTFIY

560

Peptidyltransferase

3'mRNA

Ribosome Flg. 25.19: Formationof peptidebondin translation(P-site - PeptidylIRNAsite;A-site- AminoacylIRNAsite).

Streptomycin : Initiation of protein synthesis IV. GI{APERONES AND is inhibitedby streptomycin.lt causesmisreading PROTEIN FOLDING of mRNA and interfereswith the normal pairing The three dimensional conformation of between codons and anticodons. proteins is important for their biological Tetracycline : lt inhibits the binding of functions. Some of the proteins can aminoacyl IRNA to the ribosomal complex. In spontaneouslygeneratethe correct functionally fact, tetracycline can also block eukaryotic active conformation e.g. denatured pancreatic protein synthesis. This, however, does not ribonuclease. However, a vast majority happen since eukaryotic cell membrane is not of proteins can attain correct conformation, permeableto this drug. only through the assistance of certain Puromycin: This has a structuralresemblance proteins referredto as chaperones.Chaperones to aminoacylIRNA. Puromycinentersthe A site are heat shock proteins (originally discovered and gets incorporated into the growing peptide in response to heat shock). They facilitate the interactions on the favour chain and causes its release. This antibiotic and prevents protein synthesis in both prokaryotes polypeptide surfaces to finally give the protein. a of conformation specific and eukaryotes. to reversibly bind Chaperones can Chloramphenicol : lt acts as a competitive hydrophobic regions of unfolded proteins and inhibitor of the enzyme peptidyltransferase and folding intermediates. They can stabilize thus interfereswith elongationof peptide chain. prevent of formation intermediates, Erythromycin : lt inhibits translocation by incorrect intermediates, and also prevent binding with 50S subunit of bacterialribosome. undesirableinteractionswith other proteins.All Diphtheria toxin : lt preventstranslocation in these activities of chaperoneshelp the protein to eukaryotic protein synthesis by inactivating attain compact and biologically active elongation factor eEF2. conformation.

Ghapten 2s : TFANSCRIPTION AND THANSLATION Types

of chaperones

Chaperonesare categorizedinto two major Sroups 1. Hs p 7 0 s y s te m : T h i s ma i n l y c o nsi stsof Hsp70 (70-kDa fieat shock protein) and Hsp40 ( 40- k Da H rp ). T h e s e p ro te i n s c a n bi nd (p ro te i n a ) n d hel p i n indiv idua l l yto th e s u b s tra te the correct formation of protein folding. 2. Chaperonin system : This is a large oligomericassemblywhich forms a structureinto which the folded proteins are inserted. The c haper on i ns y s te mm a i n l y h a s H s p 6 0a n d H spl 0 i. e. 60 k D a H s p a n d 1 0 k D a H s p . C h a p eroni ns are requiredat a later part of the protein folding process/ and often work in association with Hsp70 system. P r ot ein

m i s fo l d i n g

and diseases

The failure of a protein to fold properly generally leads to its rapid degradation.Cystic fibrosis (CF) is a common autosomal recessrve disease.Some casesof CF with mutationsthat result in altered protein (cystic fibrosis transmembrane conductance regulator or in short CFTR)have been reported.Mutated CFTR cannot fold properly, besidesnot being able to

561

get glycosylatedor transported.Therefore,CFTR gets degraded. C ertai nneurol ogi caldi seasesw hi ch are due to cel l ul ar accumul ati on of aggregatesof mi sfol ded protei ns or thei r parti al l y degraded products have been identified.The term prions (proteinous infectious agents) is used to col l ecti vel yrepresentthem. P ri onsexhi bi t the characteri sti cs of vi ral or microbial pathogensand have been implicated i n many di seases.e.g. mad cow di sease, Creutzfeldt-Jacob disease,Alzheimer's disease, Huntington'sdisease(Refer Chapter 2). V. POST.TRANSLATIONAL MOD IFIC A TION S OF P R OTE IN S The proteinssynthesizedin translationare, as such, not functi onal .Many changestake pl ace in the polypeptidesafter the initiation of their synthesisor, most frequently, after the protein synthesis is completed. These modifications i ncl ude protei n fol di ng (descri bed al ready), tri mmi ng by proteol yti c degradati on, i ntei n spl i ci ng and coval ent changes w hi ch are post-translational col l ecti vel y know n as modifications (Fig.25.20).

BIOM=DIGAL,/ CUilICilL DOISGEPIE

re Faulty splicing oJ hnRNA may result in certq.in diseaese.g. ftthalassemia. Inhibitors of transcription are used os therapeutic agents. Thus, actinomgcin D was the first antibiotic used in the treatment ot' tumors. Rifampin is employed to treat tuberculosisand leprosy. i9

Retrouiruses(RNA ts the genetic material) are oncogenic i.e. cause cancers.

l,9

Seueralantibiotics selectiuelyblock bacterial translation, and thus inhibit their growth e.g. streptomycin,tetracqcline,puromycin. Protein mislolding often results in the t'ormation of prions (proteinous inlectious agents)which houe been implicated in mang diseosese.g. mad cow disese,Alzheimer's diseose.

t:t Lebers' hereditary optic neuropathy is caused by mutation in mtDNA in males. The uictims become blind due fo loss of central uision os a result of neuroretinol degeneration.

BIOGHEMISTF|Y

562

Polypeptide

Intein Chemical(covalent) modifications splicing

removed intein

Ftq,25.20: An outlineof post-translational modifications of proteins.

group of enzymescalledprotein kinasescatalyse phosphorylation are while proteinphosphatases Many proteins are synthesized as the (removalof responsiblefor dephosphorylation precursorswhich are much bigger in size than phosphate group).Many enzymesthat undergo the functional proteins. Some portions of phosphorylation areknown or dephosphorylation precursormoleculesare removed by proteolysis (e.g.glycogensynthase). in metabolisms Proteolytic

degradation

to liberate active proteins. This process is commonly referred to as trimming. The formation of insulin from preproinsulin, conversion of zymogens (inactive digestive enzymes e.g. trypsinogen)to the active enzymes are some examplesof trimming.

2. Hydroxylation: During the formationof collagen,the aminoacidsprolineand lysineare convertedto hydroxyprolineand respectively This hydroxylation occursin the hydroxylysine. reticulumand requiresvitaminC. endoplasmic

3. Glycosylation: The aftachmentof carbohydratemoiety is essentialfor someproteinsto Inteinsareinterveningsequences in certain perform their functions. The complex moiety is attachedto the amino proteins. These are comparable to introns in carbohydrate mRNAs. lnteins have to be removed, and exteins acids, serine and threonine(O-linked)or to (N-linked), of leadingto the synthesis ligated in the appropriateorder for the protein to asparagine glycoproteins. become active.

lntein splicing

Govalent modifications

Vitamin K dependent carboxylation of glutamicacid residues in certainclottingfactors modification. is alsoa post-translational

The proteins synthesizedin translation are subjectedto many covalent changes.By these fn the lable 25.2, selectedexamplesof postmodificationsin the amino acids, the proteins translationalmodificationof proteinsthrough may be converted to active form or inactive their aminoacidsare given. form. Selected examples of covalent modificationsare describedbelow. 1 . Phosphorylation : The hydroxyl group containing amino acids of proteins, namely serine, threonine and tyrosine are subjectedto phosphorylation. The phosphorylationmay either increaseor decreasethe activityof the proteins.A

The eukaryoticproteins(tensof thousands) are distributed between the cytosol, plasma membraneand a numberof cellularorganelles

s63

Ghapter 25: TBANSCRIPTION AND TBANSLATION

Certain glycoproteins are targeted to reach lysosomes, as the lysosomal proteins can recognize the glycosidic compounds e.g. N-acetylglucosami ne phosphate. Amino acid

Amino-terminal aminoacid Carbory terminal

Post-translational modification(s)

For the transport of secretory proteins, a speciaf mechanism is operative.A signatrpeptide containing 'l 5-35 amino acids, located at the Glycosylation, acetylation, myristoylation, formylation. amino terminal end of the secretory proteins Methylation, ADP-ribosylation facilitates the transport.

3.TIe_.99iq.................. Arginine Aspartic acid (-SH) Cysteine

Glutamic acid Histidine Lysine Methionine Phenylalanine Proline Serine Threonine

Tryptophan Tyrosine

Methylation Phosphorylation, hydroxylation (-S-S-) tormation, Cystine formation, selenocysteine glycosylation. ycarboxylation. Methylation, phosphorylation, Methylation, Acetylation, methylation, hydrorylation, biotinylation. formation. Sulfoxide hydrorylation, Glycosylation, glycosylation. Hydrorylation, glycosylation. Phosphorylation, Phosphorylation, methylalion glycosylation. Hydrorylatron. phosphorylation, Hydrorylation, iodnation. sulfonylation,

Protein

targeting

to mitochondria

Most of the proteins of mitochondria are synthesizedin the cytosol, and their transport to mitochondria is a complex process.Majority of the proteins are synthesizedas larger preproteins with N-terminal presequencesfor the entry of these proteins into mitochondria. fhe fiansport of unfolded proteins is often facilitated by chaperones. One protein namely mitochondrial matrix targetingsignaf involved in protein targetinghas been identified. This protein can recognize mitochondrial receptor and transport certain proteinsfrom cytosolto mitochondria.This is an energy-dependentprocess.

Protdin targeting to other organelles

Specificsignalsfor the transportof proteinsto organelles such as nuclei and peroxisomeshave (nucleus,mitochondria, reticulum endoplasmic identified. been places, perform

etc.). At the appropriate their functions.

they

The smaller proteinscan easily passthrough

The proteins,synthesized in translation,have nuclear pores. However, for larger proteins, to reach their destination to exhibit their nuclear localization signals are needed to biological activities. This is carried out by a facilitate their entrv into nucleus. process called protein targeting or protein sorting or protein localization. The protefns move from one compartment to another by multiplemechanisms. The protein transportfrom the endoplasmic reticulum through the Golgi apparatus,and beyondusescarriervesicles.lt may be, however, notedthat onfy the correctly folded proteinsare recognizedas the cargo for transport. Protein targeting and post-translationalmodifications manner. occur in a well coordinated

The mitochondrial DNA (mtDNA) has structural and functional resemblanceswith prokaryotic DNA. This fact supports the view that mitochondria are derivativesof prokaryotes. mtDNA is circular in nature and containsabout 16,000 nucleotidebases.

564

B IOC H E MIS TR Y

A vast majority of structuraland functional proteinsof the mitochondriaare synthesizedin t he c v to s o l .u n d e rth e i n fl u e n c eo f n u cl earD N A . However, certain proteins(around 13), most of them being the componentsof electrontransport c hain , a re s y n th e s i z e di n th e m i to c h ondri a(e.9. c y t oc h ro m eb o f c o m p l e x l l l , tw o subuni tsof ATP synthase).Transcriptiontakes place in the m it oc h o n d ri al e a d i n gto th e s y n th e s i of s mR N A s, tRNAs and rRNAs.Two typesof rRNA and about 22 speciesof IRNA have been so far identified. Transcriptionis followed by translationresulting in pr o te i n s y n th e s i s . T he mi to c h o n d ri ao f th e s p e rm cel l do not ent er th e o v u m d u ri n g fe rti l i z a ti on,therefore,

from the mother. mtDNA is inherited to Mitochondrial DNA is subjected high rate of mutati ons(about 10 ti mes more than nucl e ar DNA) that causesinherited defectsin oxidative phosphorylation.The best known among them are certain mitochondrial myopathies and Leber'shereditaryoptic neuropathy.The latter is mostly found in males and is characterizedby bl i ndnessdue to l ossof centralvi si on as a result of neuroretinal degeneration. Leber's hereditary optic neuropathy is a consequence of single basemutati oni n mtD N A . D ue to thi s,the amino aci d hi sti di ne, i n pl ace of argi ni ne, is incorporatedinto the enzyme NADH coenzyme Q reductase.

1. Tronscription is the processin which RNA is synthesizedfrom DNA, urhich is carried out in 3 stoges-initiation,elongationand termination.

2. In case of prokaryotes, a single enzyme synthesizesall the RNAs. ln eukaryotes,RNA polymerase l, Il and III respectiuelgcatalyse the lormotion o/ rRNAs, mRNAs ond fRNAs.

3. The primary mRNA transcript(i.e. hnRNA) undergoespost-transcriptionalmodifications e.g. base modifications,splicing etc.

4. Reuersetranscription is the proce.sso/ synfhesizing DNA t'rom RNA by the enzyme reuersetranscriptase. 6

Biosynlhesisof a protein or a polypeptide is known os tronslation. The amino ocid sequenceof a protein is determined by the triplet nucleosidebosesequencesof mRNA, arranged os codons.

6. The genetic code (codons)-
7. Ribosomesore the t'actoriesof protein biosgnfhesis.Translation inuoluesactiuation of omino acids,protein synfhesisproper (initiation,elongotionand termination),protein lolding and post-translational modit'ications. 8. The post-translationalmodifications include proteolytic degradation, intein splicing and couolent modit'icotions (phosphorylation, hydroxylation, glycosylation etc.). These modificotionsare required to moke the proteins biologicallyactiue.

9. The proteins synthesizedin translation reach the destinotion to exhibit their biological actiuitg. This is csrried out by a processcalled protein targeting or protein sorting.

10. The mitochondriopossessindependentDNA with the machineryfor transcriptionand translation. Howeuer,only a few proteins (around 73) are actually synthesizedin the mitochondria.

Ghapter 25: TRANSCFIIPTEN AND TRANSLATION

565

I. Essay questions 1. Give an account of transcription.Compare the RNA synthesisbetween prokaryotesand eukaryotes. (translation). 2. Describeproteinbiosynthesis 3. Discussthe inhibitorsof transcriptionand translation. 4. Cive an account of post-transcriptional and post-translational modifications. 5. What is geneticcode? Describethe characteristics of geneticcode. Add a note on the effects of mutationson genetic code. II. Short notes (a) Cenome,(b) Heterogeneous (d) Introns nuclearRNA (hnRNA),(c) EukaryoticRNA polymerases, and exons, (e) Reversetranscription,(0 Wobble hypothesis,(g) Anticodon, (h) Shine-Dalgarno (j) Chaperones,(k) Proteintargeting. sequence,(i) Peptidyltransferase, III. Fill in the blanks 1. The total DNA (Beneticinformation)contained in an organism (or a cell) is referredto as

2. Th e p ri m a ry tra n s c ri p tp ro d u cedbyR N A po| ymerasel | i seukaryotes-. 3. The interveningnucleotidesequencesin mRNA that do not code for proteins 4. The synthesisof complementary DNA (cDNA) from mRNA is catalysedby the enzyme 5. A singleIRNA is capableof recognizingmore than one codon, and this phenomenonis referred to as 6. The factoriesfor protein biosynthesisare 7. The enzymepeptidyltransferase calalysesthe formationof peptidebond during translation.The chemicalnatureof this enzymeis 8. The proteins that facilitate the formation of specific conformation of proteins are 9. The common term used for the diseasesdue to misfoldingof proteins 10. The processof delivery of proteins in a cell to the site their biological activity is IV. Multiple choice questions 11. The codon(s)that terminate(s)protein biosynthesis (a) UAA (b) UAG (c) UCA (d) All of them. 12. The nitrogenousbasethat is never found in the geneticcode (a) Adenine(b) Cuanine(c) Thymine(d) Cytosine. 13. The total DNA (geneticinformation)contained in a living cell (or organism)is regardedas (a) Cenome (b) Transciptome(c) Proteome(d) Cene. 14. The enzyme responsiblefor the synthesisof mRNAs in eukaryoticcells (a) RNA polymerase| (b) RNA polymerasell (c) RNA polymerasellt (d) RNA polymerasea,. 15. MitochondrialDNA is inheritedfrom (a) Mother only (b) Fatheronly (c) Both of them (d) Eithermother or father.

-+]1b1]lt--+L

'-,t-

-+b - ]+-

*.- t}

the gcnes speah t

- -'- -il--Ir*ir

"Functional units of DNA, u)eare; Uhimate for all cellular actiaities; 'lailored to expressllt per thsue demands; Mystery of our molecularactionsawait unfolding."

th e c h e mi c a l v e h i c l e of heredi ty,i s n N A, l-,/ 6er1pe5si of functional units, namely genes. The term genome refers to the total genetic information contained in a cell. The bacterium Escherichiacoli contains about 4,400 genes present on a single chromosome. The genome of humans is more complex, with 23 pa i rs o f (d i p l o i d ) c h ro mo s o mes contai ni ng 6 b i l l i o n (6 x ' l 0 e ) b a s e p a i r s of D N A , w i th an estimated 30,00040,000 genes. At any given time, only a fraction of the genome is expressed.

GENE BEGULATION-GENERAL The regulation of the expression of genes rs growth, for the absolutely essential development, differentiation and the very existenceof an organism.There are two types of gene regulation-positive and negative. 1. Positive regulation : The gene regulation is said to be positive when its expression is increased by a regulatory element (positive regulator).

2. Negative regulation : A decreasein the gene expression due to the presence t.,f a The living cells possessa remarkableproperty regulatoryelement(negativeregulator)is referred to adapt to changes'in the environment by to as negativeregulation. regulating the gene expression. For instance, It may be noted here that double negative insulin is synthesizedby specialized cells of pancreasand not by cells of other organs (say effect on gene regulation results in a positive k i d n e y ,l i v e r),a l th o u g hth e n u c l ei of al l the cel l s phenomenon. of the body contain the insulin genes.Molecular .;.'l{",y;,:,; t gir.:;,iri:,ir{i ji n {i dgtt* ;le":I *pflt* S e nes regulatorymechanismsfacilitate the expression of insulin gene in pancreas,while preventingits The genesare generallyconsideredunder two exp re s s i o ni n o th e r c e l l s . categories.

566

Ghapter 26 : FIEGULATION OF GENEEXPFIESSION products 1 . Constitutive genes : The (proteins)of these genesare required all the time in a cell. Therefore,the constitutivegenes (or housekeeping genes) are expressed at more or less constant rate in almost all the cells and, further, they are not subjected to regulation e.g. the enzymesof citric acid cycle. 2. Inducible genes: The concentrationof the proteins synthesized by inducible genes is regulated hy various molecular signals. An inducer increasesthe expressionof these genes while a repressor decreases,e.g. tryptophan pyrrolaseof liver is induced by tryptophan. One cistroh.oh€

subunit

concept

The chemical product of a gene expressionis a protein which may be an enzyme. lt was originally believed that each gene codes for a specificenzyme, leadingto the popular concept, one gene-one enzyme. This however, is not necessarilyvalid due to the fact that several enzymes (or proteins) are composed of two or more nonidenticalsubunits(polypeptidechains).

567

on the regulation of lactosemetabolism in E. coli. Thisis popularlyknownas lac operon. LACTOSE (LACI OPERON Structure of lac operon The lac operon (Fig.26,1) consists of a regulatorygene (l; I for inhibition), operator gene (O) and three structuralgenes (2, Y, A). Besides these genes, there is a promoter site (P), next to the operator gene, where the enzyme RNA pofymerase binds. The structural genes Z, Y and A respectively, code for the enzymes p-galactosidase, galactoside permease and galactosideacetylase.p-Calactosidasehydrolyses lactose (p-galactoside)to galactose and glucose while permeaseis responsiblefor the transportof lactose into the cell. The function of acetylase (coded by A gene) remains a mystery.

The structural genes Z, Y and A transcribe into a single large mRNA with 3 independent translationunits for the synthesisof 3 distinct enzymes.An mRNA coding for more than one protein is known as polycistronic mRNA. The cistron is the smallest unit of genetic Prokaryoticorganismscontain a large number of expression.lt is the fragmentof DNA coding for polycistronicmRNAs. the subunit of a protein molecule. The original concept of one gene-one enzyme is replaced by Repression of |ac operon one cistron-one suhunit. The regulatory gene (l) is constitutive.lt is expressed at a constant rate leading to the synthesisof lac repressor.Lac repressoris a gene expression tetrameric (4 subunits) regulatory protein (total Elucidation of the regulation of gene mol. wt. 150,000) which specifically binds to expressionin prokaryoteshas largely helped to the operatorgene (O). This preventsthe binding understand the principles of the flow of of the enzyme RNA polymeraseto the promoter informationfrom genesto mRNA to synthesize site (P), thereby blocking the transcription of specific proteins. Some important features of structuraf genes (2, Y and A). This is what prokaryoticgene expressionare describedfirst. happens in the absenceof lactose in E. coli. The This is followed by a brief account of eukaryotic repressor molecule acts as a negative regulator of gene expression. gene expression. Models

for the study

of

Derepression of lac operon In the presenceof lactose (inducer) in the medium, a small amount of it can enter the The operon is the coordinatedunit of genetic E. coli cells.The repressormoleculeshave a high expressionin bacteria.The concept of operon affinity for lactose. The lactose molecules bind was introducedby Jacob and Monod in 1961 and induce a conformational change in the (NobefPrize1965),basedon theirobservationsrepressor.The result is that the repressor gets

568

BIOCHEMISTFIY Regulatory gene

Promoter Operator gene site

(A)

I

P

(B)

I

P

o

genes Structural

z

A

z

A

CAP-cAMP

(c)

mRNA Polycistronic

p-GalactosidasePermease Acetylase

Inactive repressor

inactivatedand, therefore,cannot bind to the galactosidepermeaseand galactosideacetylase. operator gene (O). The RNA polymeraseattaches Lactose acts by inactivating the repressor to the DNA at the promotersite and transcription molecules, hence this process is known as of lac operon. proceeds, leading to the formation of derepression (for polycistronicmRNA genesZ, Y and A) and, Gratuitous inducers : There are certain finally, the 3 enzymes.Thus, lactoseinducesthe structuralanalogsof lactosewhich can induce synthesisof the three enzymes p-galactosidase,the lac operon but are not the substrates for the

Ghapter 26 : FIEGULATION OF GENEEXPRESSION enzyme p-galactosidase.Such substancesare known as gratuitous inducers. lsopropylthiogalactoside(IPTC) is a gratuitous inducer, extensivelyused for the study of lac operon. The catabolite gene activator protein : The cells of E. coli utilize glucose in preferenceto lactose;when both of them are present in the medium. After the depletion of glucose in the medium, utilization of lactose starts. This indicatesthat glucose somehow interfereswith the inductionof lac operon. This is explainedas follows. The attachment of RNA polymeraseto the promoter site requires the presence of a cataholite gene activator protein (CAP) bound to cyclic AMP (Fig.26.A. The presence of glucoselowers the intracellularconcentrationof cAMP by inactivating the enzyme adenylyl cyclase responsiblefor the synthesisof cAMP. Due to the diminished levels of cAMP, the formation of CAP-cAMP is low. Therefore, the binding of RNA polymeraseto DNA (due to the absenceof CAP-cAMP)and the transcriptionare almost negligible in the presence of glucose. Thus, glucose interferes with the expression of Iac operon by depleting cAMP levels.Addition of exogenous cAMP is found to initiate the transcription of many inducible operons, including lac operon. It is now clear that the presenceof CAP-cAMP is essential for the transcription of structural genesof lac operon. Thus, CAP-cAMPacts as a positive regulatorfor the gene expression.lt is, therefore,evidentthat lac operon is subjectedto both positive (by repressor,describedabove) and negativeregulation.

559 Glucose

cAMP (o)

(19) CAP-CAMP

I lacoperon I Polycistronic mRNA Fiq.26.2 : Control of lac opercn by catabolitegene activator protein (CAP) and the role of glucose.

The tryptophan operon of E. coli is depicted in Fi9,26,3.This operon containsfive structural genes (trpF, trpD, trpC, trpB, trpA), and the regulatory elements-primary promoter (frpfl, operator (trpO), attenuator (trpa), secondary internaf promoter (TrpP2),and terminator (trfr). The five structuralgenesof tryptophan operon code for three enzymes (two enzymes contain two differentsubunits)requiredfor the synthesis of tryptophanfrom chlorismate. The tryptophan repressoris always turned on, unless it is repressedby a specific molecule called corepressor. Thus lactose operon (described already) is inducible, whereas tryptophan operon is repressihle. The tryptophan operon is said to be derepressed when it is actively transcribed.

Tryptophan operon regulation

Tryptophanis an aromaticamino acid, and is by a repressor required for the synthesisof all proteins that contain tryptophan. lf tryptophan is not present Tryptophan acts as a corepressorto shut down in the medium in adequate quantity, the the synthesis of enzymes from tryptophan bacterialcell has to make it, as it is requiredfor operon. This is brought out in association with a the growth of the bacteria. specific protein, namely tryptophan repressor.

570

B IOC H E MIS TRY STRUCTURAL GENES REGULATORY f/PI ELEMENTS

I

II

+

Anthranilate synthase(CoI)

Rnthlanilate synthase(Cotr)

Anthranilatesynthetase (Col+iou)

phosohoribosyl Tryptophan Tryptophan lranilate synthetasep synthetase a nerase, \ ) ---r/ e glycerol te synthetase I t\ .r t\

l\ | \

T'v?P!!11

sYnthetase

chrorismatelJ+Anthranilatef tH:R["J,',:?:rl- ] cdRP{ } nae Glutamine

PRPP

7l-rryptophan

POLYPEPTIDES

ENZYME COMPLEXES

CATALYSED REACTIONS

L-Serine

Fig. 26,3: Tryptophanoperonin E.coli[regulatoryelementsare promoter(trpP),operator(trpo), '(trpa), secondaryinternalpromoter(trpP) and terminator(trpt);Col, Coll-ComponentI and componentII;

Tryptophanrepressor, a homodimer(contains brought out by binding of tryptophan at an two identicalsubunits) bindswith two molecules allostericsite on anthranilatesynthetase. of tryptophan,and then bindsto the frp operator to turn off the transcription.lt is of interestto Attenuator as the second control notethat tryptophanrepressor also regulatesthe site for tryptophan operon transcription of the gene(trpR)responsible for its Attenuator gene (frpa) of tryptophan operon own synthesis. lies upstream of trpE gene. Attenuation is the second level of regulation of tryptophan operon.

Two polycistronicmRNAsare producedfrom The attenuator region provides RNA polymerase tryptophanoperon-one derivedfrom all the five which regulatestranscription.In the presenceof structuralgenes,and the otherobtainedfrom the prematurely tryptophan, transcription is last threegenes. terminated at the end of attenuator region.

Besidesacting as a corepressorto regulate However, in the absence of tryptophan, the tryptophanoperon,tryptophancan inhibit the attenuator region has no effect on transcription. activity of the enzymeanthranilatesynthetase. Therefore, the polycistronic mRNA of the five This is referredto as feedbackinhibition, and is structural genes can be synthesized.

Chapter 26 : REGULATION OF GENEEXPHESSION

E ac hc e l l o f th e h i g h e ro rg a n i s mc o n tai nsthe entire genome. As in prokaryotes, gene expressionin eukaryotesis regulatedto provide the appropriate responseto biological needs. T his m ay o c c u r i n th e fo l l o w i n g w a y s

571

Genes

Chromosome number

phosphatase Alkaline 1 Apolipoprotein B 2 . Expressionof certain genes (housekeeping Transferrin 3 genes)in most of the cells. Alcohol dehydrogenase 4 r Activation of selectedgenes upon demand. HMGCoAreductase c . Permanentinactivationof severalgenesin all Steroid 21-hydrorylase b but a few types. Arginase In caseof prokaryoticcells, most of the DNA Carbonic anhydrase 8 is organized into genes which can be o |nterteron In transcribed. contrast, in mammals, very little of the total DNA is organized into genes and Parathyroid hormone 11 their associated regulatory sequences. The Glyceraldehyde 3-phosphate dehydrogenase 12 function of the bulk of the extra DNA is not Adenosine deaminase 13 k nown. c,-Antitrypsin 14 Eukaryoticgene expressionand its regulation Cytochrome P*o 15 are highly complex. Some of the important Hemoglobin aspectsare briefly described. c-chain 16 Growth hormone 17 CI{ROMATIN SRUGTURE Prealbumin 18 AND GENE EXPRESSION phosphokinase (Mchain) Creatine 19 The DNA in higher organismsis extensively Adenosine deaminase 20 folded and packed to form protein-DNA Superoxide dismutase 21 complex called chromatin. The structural organizationof DNA in the form of chromatin (i,chain) lmmunoglobulin 22 plays an important role in eukaryotic gene expression. In fact, chromatin structure provides an additional level of control of gene expressron.

Glucose O-phosphate dehydrogenase Steroid sulfatase

X Y

A selected list of genes (representedby the Acetylation or deacetylationof histones is an products)along with the respectivechromosomes important factor in determining the gene on which they are located is given in Table 26.1; expression. In general, acetylation of histones In general, the genes that are transcribed leads to activation of gene expression while within a particularcell are less condensedand deacetylation reversesthe effect. more open in structure.This is in contrast to genesthat are not transcribedwhich form highly condensedchromatin.

Acetylation predominantly occurs on the l ysi ne resi duesi n the ami no termi nal ends of histones.This modification in histonesreduces the positivechargesof terminal ends (tails),and Histone aeetylation and deacetylation decreasestheir binding affinity to negatively Eukaryotic DNA segments are wrapped charged DNA. Consequently, nucleosome around histone proteins to form nucleosome. structureis disruptedto allow transcription.

BIOCHEMISTF|Y

572

fn the if lustration given in the Fi9.26.5, gene I is activated by a combination of activators 1, 2 and 3. Cene ll is more effectivelyactivatedby Cytosine in the sequence CG of DNA gets the combined action of 1, 3 and 4. Activator 4 methylated to form S'-methylcytosine.A major is not in direct contactwith DNA, but it forms a portion of CG sequences(about 2O%) in human bridge between activators 1 and 3, and activates DNA exists in methylated form. In general, gene ll. As regardsgene lll, it gets inactivatedby methylation leads to loss of transcriptional a combinationof 1, 5 and 3. In this case,protein activity, and thus inactivation of genes. fhis 5 interfereswith the binding of protein 2 with occurs due to binding of methylcytosinebinding the DNA, and inactivatesthe gene. proteins to methylated DNA. As a result, methylated DNA is not exposed and bound to MOTIFS IN PROTEINS transcription factors. lt is interestingto note that AND GENE EXPRESSION methylationof DNA correlateswith deacetylation of histones.This provides a double means for A motif literally means a dominant element. genes. of repression Certainmotifs in proteinsmediatethe binding of proteins (transcription factors) to The activation and normal expression of regulatory

Methylation of DNA and inactivation of genes

genes, and gene inactivation by methyfation are depicted in Fig.26.!.

DNA

ENHANCERSAND TISSUE.SPECIFIC GENE EXPRESSION Enhancers(or activators)are DNA elements that facifitate or enhance gene expression.The enhancers provide binding sites for specific proteins that regulate transcription. They facilitatebinding of the transcriptioncomplex to promoter regions. Some of the enhancerspossessthe ability to promotetranscriptionin a tiss,ue-specific manner. For instance,gene expressionin lymphoid cells for the production immunoglobulins (lg) is promoted by the enhancer associatedwith lg genesbetweenJ and C regions.

Geneactivationand expression

JDNA

methylation

Transgenicanimalsare frequentlyusedfor the expression.The available study of tissue-specific evidencefrom various studiesindicatesthat the gene expression is largely tissue-specific mediated through the involvement of enhancers,

OF DNA ELEMENTS COMBIT{ATION AND PROTEINSIN GEI{E EXPRESSION

+ Geneinactivationand no expression Gene expression in mammals is a complicatedprocesswith severalenvironmental Fig.26.4: Methylationof DNA and inactivationof genes stimuli on a single gene. The ultimate response (A) Gene activation in the absence of DNA methylation (B) Gene inactivation due to methylation of the gene which may be positiveor negativeis brought out by the associationof DNA elements and proteins.

OF GENEEXPRESSION Ghapter 26 : REGULATION

Gene activated

I

Gene activated

E

A great majority of specific protein-DNA interactions are brought out by four unique moti fs-hel i x-turn-hel i x (H TH ), zi nc fi nger , l euci nezi pper, hel i x-l oop-hel i x(H LH ). These amino acid motifs bind with high affinity to the specific site and low affinity to other partsof DNA. The motif-DNA interactions are maintainedby hydrogenbonds and van der Waals forces. H el i x-turn.hel i x

J

Gene inactivated Fig, 26.5 : A diagrammatic representation of the associationof DNA elements and proteins in gene regulation.A, B and C representgenes I, ll and lll ( 1... 5 represent proteins).

573

moti f

The helix-turn-helix(HTl{\ molif is about 20 amino acids which representsa small part of a large protein. HTH is the domain part of the protein which specifically interacts with the DNA (Fi9.25.6A).Examplesof helix-turn-helix motif proteins include lactose repressor,and cyclic AMP cataboliteactivatorprotein (CAP)of E. coli, and several developmentallyimportant transcriptionfactors in mammals.

Zi nc fi nger moti f DNA. The specificcontrol of transcriptionoccurs Sometime ago, it was recognized that the by the binding of regulatoryproteinswith high transcriptionfactor TFlllA requires zinc for its affinity to the correct regionsof DNA.

BIOMEDICAI./ CLINICAL CONCEPTS

| = Regulation of gene expression to adapt to the changes in the enuironment is o remarkable property of liuing cells e.g. sgnfhesis of insulin by ftcells ot' pancreas ond nowhere else. ',' The growth, deuelopment and dit'ferentiation of an organism inuolues complex mechanisms which ultimatelg depend on gene regulation, t;

The house-keeping genes or constitutiue genes ore expressed at almost a constant rate in the cells, and they are not usually subjected to regulations e.g. enzymes of Krebs cycle.

-tt The malignant cells deuelop drug resistance to long term administration ot' methotrexate. This occurs b9 amplification of the genes coding for dihgdrofolate reductase. ':

The human body has the capability to produce around 70 billion antigen-specific immunoglobulins. This is achieued by a process called gene rearrangement.

'=' Knowledge on the gene expression and its regulation helps in the understanding and control of seoerol diseases, including concer.

B IOC H E MIS TR Y

574

The zinc fingersbind to the major groove of D N A , and l i e on the face of the D N A ' This binding makesa contact with 5 bp of DNA' The steroid hormone receptor transcription factors use zinc finger motifs to bind to DNA. Leueine

.t!eliT:!Yr.t:l,eIiI

(B)Zinc finger(CYs-CYs)

zipper

mot:f

The basic regions of leucine zipper (bZlfl protei nsare ri ch i s the ami no aci d l euci ne.Ther e occurs a periodic repeat of leucine residuesat every seventh position. This type of repeat structure allows two identical monomers or heterodimersto zip together and form a dimeric complex.This protein-protein complex associates and interacts with DNA (Fig.26.6Q. Cood examples of leucine zipper proteins are the enhancerbi ndi ng protei ns(E B P )-fosand j un ' 1{el ix-!oop-helix

motif

Tw o amphi pathi c(l i teral l ymeansa feel i ngof closeness)cr-helical segmentsof proteins can form hel i x-l oop-hel i xmoti f and bi nd to D NA' The di meri cform of the protei nactual l ybi ndst o DNA (Fig.26.6D).

The imoortant features of eukaryotic gene expressionalong with the regulatoryaspectsare described in the preceeding paBes. Besides transcri pti on, eukaryoti c cel l s al so emp loy varietv of other mechanismsto regulate gene expression.The most important ones are listed below, and briefly describednext' 1 . Cene amPlification 2. Cene rearrangement (D) Helix-loop-helix Fig. 26.6 : A diagrammatic representation of common

activity. On analysis,it was revealedthat each T F fllA c o n ta i n s z i n c i o n s a s a repeatl ng coordinatedcomplex.This complex is formed by the closely spaced amino acids cysteine and cysteine,followed by a histidine-histidine pair. ln some instances, His-His is replaced by a second Cys-Cys pan Gig.26.6R).

of R N A 3. P rocessi ng 4. AlternatemRNA sPlicing of mRNAfrom nucleusto cytoplasm 5. Transport 6. Deeradationof mRNA. Gene affiplificatiotl of a gen e is In thi s mechani sm,the expressi on i ncreased several fol d. Thi s i s commonly observed during the developmental stages of eukaryotic organisms.For instance,in fruit fly

OF GENEEXPFESSION Chapter 26 : FIEGULATION

J /U

estimated that the human body can produce about 10 bi l l i on (1010)anti bodi esi n responseto anti gensti mul ati ons. The mol ecul armechani sm of this antibody diversitywas not understoodfor long. lt is now explained on the basis of gene rearrangement or transposition of genes or somatic recombination of DNA.

Fig. 26.7 : A diagrammatic representation of gene amplification (the genes are depicted in colour shade and colour).

The structure of a typical immunoglobulin moleculeconsistsof two light (L) and two heavy (H ) chai ns. E ach one of these chai ns (L or H) contains an N-terminal variable (V) and C-terminal constant (C) regions (Refer Fig.9.3'1. The V regi ons of i mmunogl obul i ns ar e responsiblefor the recognitionof antigens.The phenomenon of gene rearrangementcan be understoodfrom the mechanismof the synthesis of l i ght chai nsof i mmunogl obul i ns(Fi 9.26.9.

(Drosophila),the amplificationof genes coding for egg shell proteins is observed during the course of oogenesis.The amplification of the Each light chain can be synthesizedby three gene (DNA) can be observed under electron distinct DNA segments,namelythe variable(V1), microscope(Fig.z5.V. the joining (J1) and the constant (C1). The The occurrenceof gene amplificationhasalso mammal i an genome contai ns about 500 V'been reported in humans. Methotrexate is an segments,6 J, segments and 20 C,_ segments. ant ic an c e r d ru g w h i c h i n h i b i ts th e enzyme During the course of differentiation of dihydrofolate reductase. The malignant cells B-lymphocytes,one V,_segment(out of the 500) long term develop drug resistance to is brought closer to J1 and C, segments.This administrationof methotrexateby amplifyingthe occurson the samechromosome.For the sakeof genescoding for dihydrofolatereductase. illustration,100thVL, 3td J,.and 1OthCL segments are rearrangedin Fig.26.8.The rearrangedDNA Gene rearrangment (with VL, J1and C, fragments)is then transcribed The body possessesan enormous capacity to to produce a single mRNA for the synthesisof a synthesize a wide range of antibodies. lt is specific light chain of the antibody. By

v (s00)

J (6)

c (20)

o-{+-.-li'^J-^-^-ll

f{{+{{-ll

orisinar or.rR

lrFrl

ReanangedDNA

{{{-l{-^-

Primary transcript

mRNA

JI

Protein (lightchainof lg)

Fig, 26.8 : A diagrammaticrepresentationof gene rearrangementtor the synthesisof light chain of immunoglobulin.

576

B IOC H E MIS TFI Y

innum e ra b l e c o m b i n a ti o n so f V 1 , J1 and C .. segments/ the body's immune system can a n ti g e n speci fi c B ene ra te m i l l i o n s o f im m u n o g l o b u l i nm o l e c u l e s . The formation of heavy (H) chains of im m u n o g l o b u l i n sa l s o o c c u rs b y re arrangement of 4 distinct genes-variable (VH),diversity(D), joinin g (J H )a n d c o n s ta n t(C s ). P r oc e s s i n E

structure

AU richregion

Fig. 26.9: A diagrammaticrepresentationof a typical sequences) eukaryoticnRNA.(NCS-Non-coding

of RNA

T he R N A s y n th e s i z e d i n transcri pti on unde rg o e sm o d i fi c a ti o n sre s u l ti n gi n a functi onal RNA . T h e c h a n g e si n c l u d e i n tro n -exonspl i ci ng, polyadenylationetc. (Chapter 2fl. Alternate

cap

ffiRf{A

splicing

Eukaryoticcells are capable of carrying out alternate mRNA processing to control gene expression.Different mRNAs can be produced by alternate splicing which code for different proteins (for more details, Refer Chapter 25). Degr a d a ti c n

o f mR N A

The expression of genes is indirectly influenced by the stability of mRNA. Certain

hormonesregulatethe synthesisand degradation of some mRNAs.For instance,estradiolprolongs the half-life of vitellogenin mRNA from a few hours to about 200 hours. It appearsthat the ends of mRNA molecules determi ne the stabi l i ty of mR N A . A typi cal eukaryotic mRNA has 5'-non-codingsequences (5' -N C S ),a codi ng regi onand a 3' -N C S .A l l t he mRNAs are capped at the 5' end, and most of them have a polyadenylate sequence at the 3' end (Fig.26.0. The 5' cap and poly (A) tail protect the mRNA against the attack by exonuclease. Further, stem-loop structures in N C S regi ons,and A U ri ch regi onsi n the 3' N CS al so provi de stabi l i tyto mR N A .

1. DNA, the chemical uehicle of heredity, is composed of genes. The regulation of gene

3. 4.

5. 6.

expressionis absolutelyessentialfor the growth, deuelopmentand dilJerentiationof an organism. A positiue regulation increasesgene expressionwhile a negatiue regulation decreases. The operon is the coordinated.unit ot' gene expression.The lac operon o/ E. coli consists of regulatorygenesand structural genes. The lac repressorbinds to the DNA and halts the process of transcription of structural genes. Howeuer, the presence of lactose inactiuatesthe repressor(derepression)Ieading to the expressiono/ structural genes. Trgptophon operon is regulated by a repressor. Tryptophan repressor binds to tryptophan, snd then to trp operator gene to turn ot'f the transcription. Eukaryotic gene expression and its regulation are highly complex. Acetglation of histones leods to gene expressionwhile deacetylation reuersesthe ett'ect.ln general, methylation of DNA results in the inactiuation ot' genes. The protein-DNA interactions, brought out by motit's (helix-turn-helix, zinc finger, leucine zipper, helixloop-helix), are inuolued in the control ot' gene expression. Eukaryotic cells haue deuelopedseuerolmechanismsto regulategene expression.These include gene amplification, gene rearrangement, and processing, transport and degradation ol DNA.

chapter 25 : FIEGULATION OF GENEEXPRESSION

s77

I. Essay questions 1. Describelactose(lac)operon. 2. Write briefly on the gene expressionand its regulationin eukaryotes. II. Short notes (a) One cistron-onesubunitconcept/(b) Catabolitegeneactivatorprotein,(c) Cene inactivationoy DNA methylation,(d) Zinc finger motil (e) Cene amplification. III. Fill in the blanks 1. The numberof genesfound in human genome 2. The genesresponsible for the productionof proteinsthat are requiredall the time in a cell are regardedas 3. The earlierconceptof one gene-oneenzymeis replacedby 4. The chromatinin higherorganismsis chemicallycomposedof IV. Multiple choice questions 5. The structural'Z' geneof lactose(lac)operon is responsiblefor the synthesisof the enzyme(s) (a) 0-Calactosidase (b) Permease(c) Acetylase(d) All of them. 6. Methylationof DNA resultsin (a)Activationof genes(b) Inactivationof genes(c) No effecton genes(d) Inactivationof protein motifs. 7. The productionof a wide rangeof immunoglobulins is explainedon the basisof (a) Cene amplification(b) Cene rearrangement,(c) Alternate mRNA splicing (d) mRNA degradation. a 8. The specificcontrol of transcriptioninvolvesthe following motif(s) (a) Helix-turn-helix (b) Zinc finger (c) Leucinezipper (d) All of them.

RecombinantDNA

*f-h"

term biotechnologyrepresentsa fusion or I an alliance between biology and technology. Frankly speaking, biotechnology is a newly discovereddiscipline for age-old practicese.g. preparationof wine, beer, curd, bread. These natural processes are regarded as old or traditional biotechnology.

.

Genetic modifications

. New genetics. Brief history of recombinant DNA technology

The presentday DNA technologyhas its roots in the experiments performed by Boyer and I rnu new or modern biotechnology embraces Cohen in 1973. In their experiments, they all the genetic manipulations, cell fusion successfullyrecombinedtwo plasmids(pSC 101 techniques,and improvementsmade in the old and pS C 102) and cl oned the new pl asmi d i n biotechnologicalprocesses.The biotechnology E.coli.ln the laterexperimentsthe genesof a frog with particularreferenceto recombinantDNA in could be successfully and expressed transplanted, human health and diseaseis brieflv describedin in E.coli.This madethe real beginningof modern this chapter. rDNA technology and laid foundationsfor the G enet i c e n g i n e e ri n gp ri m a ri l y i n v o l ves the present day molecular hiotechnology. manipulation of genetic material (DNA) ro Some biotechnologistswho admire Boyerachieve the desired goal in a pre-determined Cohen experimentsdivide the subject into two way. Some other terms are also in common use chronologicalcategories. to describegenetic engineering. 1. 88C-biotechnology Eefore Eoyer and o Gene manipulation C ohen. . Recomhinant DNA (IDNA) technology 2. ABC-biotechnology After Eoyer and c Cene cloning (molecular cloning) Cohen.

578

,l" l

Chapter 27 : BECOMBINANTDNA AND BIOTECHNOLOGY

1. Ceneration of DNA fragments and selection of the desired piece of DNA (e.g. a human gene).

tf \-/

579

2. Insertionof the selectedDNA into a cloning vector (e.g. a plasmid) to create a recombinant I Restriction DNA or chimeric DNA (Chimera is a monster in Greek mythologythat has a lion's head, a goat's Jendonuclease body and a serpent's tail. This may be comparabl eto N arasi mhai n l ndi an mythol ogy ) .

PlasmidDNA

DonorDNA I nestriction endonuclease I

+ o"r,,ffi0,"""

I

v

Cut plasmidDNA

3. Introduction of the recombinant vecrors into host cells (e.g. bacteria). 4. Mul ti pl i cati on and sel ecti on of cl ones contai ni ngthe recombi nantmol ecul es. 5. Expressionof the gene to produce the desired product. RecombinantDNA technology with special referenceto the following aspectsis described

DNA Recombinant Y

'"i3!i33'13'" /'-\r \

(/------l\ a (l/h l r\-/

\____)az

. Molecular tools of genetic engineering. . Host cells-thefactoriesof cloning. . Vectors-thecloning vehicles. r Methods of gene transfer. . Gene cloning strategies.

MOTEGT'LARTOOLS OF ; .",Yli',[f'3iXiJ.",Gffi${ETIG ENGINEERIruG

Fig.27.1: Thebasicprincipleof recombinantDNA technology.

RecombinantDNA technologyis a vast field. T he bas i c p ri n c i p l e sa n d te c h n i q u e sof rD N A technology along with the most important applicationsare briefly describedin this chapter.

The term geneticengineermay be appropriate for an individual who is involved in genetic manipulations. The genetic engineer's toolkit or molecular tools namely the enzymes most commonl y used i n recombi nant D N A experimentsare brieflv described. Restriction endonucleasesDNA cutting enzymes

Restrictionendonucleases are one of the most important groups of enzymes for the manipulation of DNA. These are the hacterial enzymes that can cut/split DNA (from any BASIG PRINCIPLES source) at specific sites. They were first OF rDNA TECHNOLOGY discoveredin E.coli restrictingthe replicationof There are many diverse and complex bacteriophages,by cutting the viral DNA (The techniques involved in gene manipulation. host E.coli DNA is protected from cleavage by However, the basic principles of recombinant addition of methyl groups).Thus, the enzymes DNA technology are reasonably simple, and that restrict the viral replication are known as broadly involve the following stages(Fig.27.1). restriction enzymesor restrictionendonucleases.

B IOC H E MIS TR Y

580 Nomenclature : Restrictionendonucleasesare named by a standardprocedure,with particular reference to the bacteria from which they are isolated.The first letter(in italics)of the enzymes indicatesthe genus name, followed by the first two letters (also in italics) of the species, then comes the strain of the organism and finally a Romannumeralindicatingthe order of discovery. A couple of examplesare given below.

specifically recognize DNA with a particular sequenceof 4-8 nucleotidesand cleave. Cleavage patterns : Majority of restriction endonucleases(particularlytype ll) cut DNA at defined sites within recognition sequence. A selected list of enzymes/ recognition sequences,and their productsformed is given in Table 27,1.

The cut DNA fragments by restriction endonucleases may have mostly sticky ends (cohesive ends) or hlunt ends, as given in Table 27,1. DNA fragments with sticky ends are particularly useful for recombinant DNA experiments.This is becausethe single-stranded Recognitionsequences: Recognitionsequence sticky DNA ends can easily pair with any sticky is the site where the DNA is cut by a restriction other DNA fragmenthaving complementary ends. endonuclease. Restriction endonucleasescan

EcoRI is from Escherichia(D coli (co), strain Ry13 (R), and first endonuclease (t) to be discovered. HindlII is from Haemophilus (t{l influenzae (in), strain Rd (d) and, the third endonucleases(llt) to be discovered.

Products

Enzyme (source)

Recognition sequence

EcoRl (Escherichiacoli)

s',...... GXA-A-T-T-C......3', ...5' ,'.....g-1-1-4-ngG..

A-A-T-T-C G

BanHl (Bacillus amyIoliquetaciens\

. ...3', 5',......Gh-A-T-C-C 3'""'C-C-T-A- ""'5'

G-A-T-C-C..... G......

Haelll (HaemophiIus aegyptius)

.3' 5'.....G_GXGC.... .....5' . .C-C.IG-G 3'.

......G ....'.H-T-A-G

*c-c G-G

te-c GC

Hindlll (Haemophilus influenzae)

s'......AXA-G-C-T-T..,..3'

Noil (Nocardiaotitidi$

5'"""G -G@""'3' ......s', 31.....H-Cfr4

....'5' 31....'T-T-C-G-A.YA

A-G-GT-T....'. A..... ......4 ......T-T-C-G-A

6 arewithstickyends\' arcwithbluntendswhilefor tln rest,theproducts (lVote: Scrssorc indiutethesitesof cleavage.Theproducg

Ghapter 27 : FIECOMBINANT DNA AND BIOTECHNOLOGY

581

'l\1{l\"1" .I\'TI"I1"

Many enzymes are used in the recombinant DNA technology/genetic engineering.A selected list of theseenzymesand the reactionscatalysed by them is given in Table 27.2.

H OS T C E LLS THE FACTORIES Fig. 27.2 : Action of DNA ligase in the formation ot p hosphodie ster bond (B-b ase).

DNA ligases-DNA

joining

enzymes

OF CLONING

The hosts are the living systems or cells in which the carrierof recombinantDNA molecule or vector can he propagated. There are different types of host cells-prokaryotic(bacteria) and eukaryoti c (fungi , ani mal s and pl ants). S ome examples of host cells used in genetic engineeringare given in Table 27.3.

The cut DNA fragmentsare covalentlyjoined together by DNA ligases.These enzymes were originally isolatedfrom viruses.They also occur Host cells, besides effectively incorporating in E.coli and eukaryoticcells. DNA ligasesacti- the vector's genetic material, must be vely participatein cellular DNA repair process. conveniently cultivated in the laboratory to The action of DNA ligases is absolutely collect the products. In general, microorganisms requiredto permanentlyhold DNA pieces.This are preferred as host cells, since they multiply is so since the hydrogenbonds formed between faster compared to cells of higher organisms the complementarybases(of DNA strands)are (pl antsor ani mal s). not strong enough to hold the strandstogether. Prokaryotic hosts DNA Iigasejoins (seals)the DNA fragmentsby Escherichia coli :The bacterium, Escherichia forming a phosphodiester bond between the phosphate group of S'-carbon of one coli was the first organism used in the DNA deoxyribose with the hydroxyl group of technologyexperimentsand continuesto be fhe host of choice bv manv workers. 3'-carbon of another deoxyribose (Fi9.27.A.

Enzyme

phosphatase Alkaline Bal31nuclease DNAligase I DNApolymerase DNase I Exonuclease lll l, exonuclease kinase Polynucleotide Restriction enzymes Reverse transcriptase RNase A RNase H Iag DNApolymerase Sl nuclease Terminal transferase

Use/reaction

groups phosphate fromS'-ends Removes of double/single-stranded DNA(orRNA). shortening of DNA. Fortheprogressive phosphodiester linkages DNAsegments. byforming between JoinsDNAmolecules to a DNAtemplate. DNAcomplementary Synthesizes nicksin DNA. Produces single-stranded from3'-end of DNA. Removes nucleotides fromS'-end of DNA. Removes nucleotides phosphate fromATPto S'-OHendsof DNAor RNA, Transfers DNAwitha specific recognition site. Cutdouble-stranded DNAfromRNA. Synthesizes RNA(andnotDNA), Cleaves anddigests theRNAstrand of RNA-DNA heteroduplex. anddigests Cleaves chainreaction Usedin polymerase DNAandRNA, Degrades single-stranded to the of DNAor RNA.Useful in homopolymer tailing. Addsnucleotides 3'-ends

582

ElIOCHEMISTRY

VECTORS V E H IC LE S

Group

Examples

Prokaryotic Bacbrta

Escheichia coli Bacillussubtilis Streptonyces sp

Eukaryotic Fungi

THE CLONING

Vectors are the DNA molecules,which can carry a foreign DNA fragment to be cloned. They are self-replicatingin an appropriatehost cell. The most important vectors are plasmids, artificial bacteriophages, cosmids and chromosome vectors. Plasmid

Saccharonyces cerevisiae Pfasmids are extrachromosomal, doubleAspergillus nidulans stranded, circular, self-replicating DNA moleInsect cells cules. Almost all the bacteria have plasmids Oocytes containinga low copy number (1-4 per cell) or Mammalian cells a hi gh copy number (10-100per cel l ). The size Wholeorganisms of the plasmidsvariesfrom 1 to 500 kb. Usually, Protoplasts plasmidscontributeto about 0.5 to 5.0% of the lntactcells total DNA of bacteria (Note : A few bacteria Wholeplants

Animals

Plants

contain linear plasmids e.g. Streptomyces sp, Borella burgdorferi).

The major drawback however, is that E.coli (or even other prokaryotic organisms)cannof perform post-translational modifications.

Nomenclature of plasmids : lt is a common practice to designateplasmid by a lower case p, followed by the first letter(s) of researcher(s) Bacillus subtilis : Bacillus subtilis is a rod names and the numerical number given by the shaped non-pathogenicbacterium. lt has been workers. Thus, pBR322 is a plasmid discovered used as a host in industryfor the production of by Bolivar and Rodriguezwho designatedit as enzymes, antibiotics, insecticides etc. Some 322.Someplasmidsare given namesof the places workers consider B.subtilis as an alternative to where they are discoverede.g. pUC is plasmid from University of California. E.coli. Eukaryotic

hosts

Eukaryoticorganismsare preferredto produce human proteinssince these hostswith complex structure (with distinct organelles) are more suitable to synthesize complex proteins. The most commonly usedeukaryotic organism is the yeast, Saccharomyces cerevisiae.

pBR322- the most common plasmid vector : pBR322ol E.coli is the most popularand widely usedplasmidvector, and is appropriatelyregarded as the parent or grand parent of several other vectors.

pBR322has a DNA sequenceof 4,361 bp. lt for ampicillin (Ampr)and carriesgenesresistance tetracycline (Telr) that serve as markers for the Mammalian cells .' Despite the practical identificationof clones carrying plasmids.The difficulties to work with and high cost factor, plasmid has unique recognition sites for the mammalian cells (such as mouse cells) are also action of restriction endonucleasessuch as employed as hosts.The advantageis that certain EcoRl, Hindlll, BamHl, Sall and Pstll (Fig.27.3). complex proteinswhich cannot be synthesized by bacteria can be produced by mammalian Other plasmid cloning vectors : The other cells e.g. tissue plasminogenactivator. This is plasmids employed as cloning vectors include mainly becausethe mammaliancells possessthe pU C 19 (2,686 bp, w i th ampi ci l l i n resist ance machinery to modify the protein to the active gene), and deri vati ves of pB R 322-p8 R325, pB R 328and pB R 329. form (post-translational modifications).

Chapter a7 : RECOMBINANT DNA AND BIOTECHNOLOGY

Artificial

Hindlll

583 chromosome

ueetors

Human artificial chromosome (HAC) : Developedin 1997 (by H. Willard), human artificiaf chromosome is a synthetically produced vector DNA, possessing the characteristicsof human chromosome.HAC Sa1 may be considered as a self-replicating microchromosomewith a size ranging from 'l/ 10th to 1/5th of a human chromosome. The advantagewith HAC is that it can carry human genes that are too long. Further, HAC can carry genes to be introduced into the cells in gene therapy.

Originof replication

Yeast artificial chromosomes (YACs) : Introducedin'1987 (by M. Olson),yeastartificial chromosome(YAC) is a syntheticDNA that can accept )a6te hagtments ol ,/are)g,n DNA (particularly human DNA). lt is thus possib/e to clone large DNA pieces by using YAC.

Fiq.27.3 : Genetic map of plasmid cloning vector pBB322.

B ac r er ioph a g e s

Bacterial artificial chromosomes(BACs) : The construction of BACs is based on one F-plasmidwhich is largerthan the other plasmids used as cloning vectors. BACs can accept DNA inserts of around 300 kb.

Bacteriophages or simply phages are the viruses that replicate within the hacteria. ln case of certain phages, their DNA gets incorporated into the bacterial chromosome and remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes.The advantage with phagesis that they can take up larger DNA segments than plasmids. Hence phage vectors are preferred for working with genomes of human cells. The most commonly used phages are bacteriophagel, (phage l,) and bacteriophage M 13). ' phage

Among the several factors, the size of the foreign DNA is very important in the choice of vectors. The efficiency of this process is often crucial for determiningthe successof cloning. The sizesof DNA insertthat can be acceptedby different vectors is shown in Table 27.4.

GosmFds

I}fETHODS

Choiee

of wee*or

I

Cosmids are the vectors characteristicsof both plasmid phage 1,. Cosmids can b , , t. t , a oy aoornSa rragmenroTpnageA cos site,to plasmids.A foreign kb) can be inserted into The recombinant DNA so fc , , , , pacKeo as pnages anq InJecr( Once inside the host cell, cosmir just like plasmids and repficate. Tho advantagewith cosmids is that ttey can carri Iarger fragments of foreign DNA compared to plasmids.

OF GENE TRAilSFER

l

I a foreign DNA (i'e' the gene)into importanttask in biotechnology' of this processis often crucial for e success of cloning' The most ployed gene transfer methods' lrmation, conjugation, electrolP'ofection, and direct transferof y described' Yransforrmat$on Transformation is the method of introducing foreign DNA into bacterialcells (e.9.E.colr).The

I

BIOCHEMISTF|Y

584

Vedor

I

Host

Foreign insert DNA size

E. coli Phage 7r E. ali Cosmid lv E. @li Plasmid ailifical (PAC) chromosome Bacterial arlificial E. coli (BAC) chromosome Yeastchromosome S. cerevisiae

F25 kb 35-45kb 100-300 kb 10H00 kb 2012000kb

uptakeof plasmidDNA by E.coliis carriedout heat in ice-coldCaCl2(0-5"C),and a subsequent shock (37-45oCfor about 90 sec). By this technique, the transformationfrequency,wh ich refersto the fraction of cell population that can be transferred, is reasonably good e.g. one cell per 1000 (10-3)cells. approximately Gonjugation Conjugation is a natural microbial recombination prccess.During conjugation,two live bacteria(a donorand a recipient)cometogether, join by cytoplasmicbridgesand transfersinglestrandedDNA (fromdonor to recipient).Inside the recipientcell, the new DNA may integrate (ratherrare)or may remain with the chromosome (as plasmids). with is the case free The naturalphenomenonof conjugationis exploitedfor genetransfer.This is achievedby plasmid-insert DNA from one cell to transferring another. In general, the plasmids lack conjugativefunctionsand therefore,they are not as such capable of transferringDNA to the recipientcells. However,some plasmidswith conjugative propertiescan be prepared and used. Electroporation

is a techniqueinvolving electric field'mediated membranepe rmeabiIizafion. EIectric shocks can DNA also inducecellularuptakeof exogenous (believedto be via the poresformedby electric pulses) from the suspending solution. is a simpleand rapidtechnique Electroporation for introducinggenesinto the cellsfrom various (microorganisms, plantsand animals). organisms Liposome-mediated

gene transfer

which arecircularlipid molecules, Liposomes nucleic can carry interior that havean aqueous acids.Severaltechniqueshave been developed TheliposomeDNA in liposomes. to encapsulate to as gene is referred transfer mediated Iipofection. On treatment of DNA fragment with liposomes,the DNA pieces get encapsulated canadhereto Theseliposomes insideliposomes. cell membranesand fuse with them to transfer DNA fragments.Thus,the DNA entersthe cell and thento the nucleus.The positivelycharged liposomesvery efficientlycomplex with DNA, bind to cellsand transferDNA rapidly. Direct transfer

of DNA

It is possibleto directlytransferthe DNA into the cefl nucleus.Microiniection and particle bombardmenfarethe two techniquescommonly usedfor this purpose. GENE CLONING STRATEGIES cells, A clone refersto a grouPof organisms, moleculesor other objects, arisingfrom a single individual, Clone and colony are almost synonymous. Gene cloning strategies in relation to recombinantDNA technologybroadlyinvolve the following aspects(Fi5.27.q. o Cenerationof desiredDNA fragments. . Insertionof these fragmentsinto a cloning vector.

r Introductionof the vectorsinto host cells. is basedon the principlethat Electroporation of the recipientcellsfor or screening high voltageelectric pulsescan induce cell r Selection molecules. DNA plasmamembranes to fuse.Thus,electroporation the recombinant

585

FECOMBINANT DNA AND BIOTECHNOLOGY

| I

+ |

useful if the gene sequence is short and the (Restrictionendonucleasedigestion, compl etesequenceof ami no aci ds i s know n. PCR,chemical synthesis) cDNAsynthesis,

(Ligationof bluntends or cohesiveends, homoOolVmer tailing,linkermolecules)

I

+ @

There are several techni oues used i n recombi nant D N A technol ogy or gene (Transformation, transduction) mani pul ati on.The mostfrequentl yusedmethods lransfection, | I are l i sted.

J

r/OA/ CH SCfiEEI.//NG (Hybridization, PCR,immunochemical methods,protein-proteininteractions, f unctionalcomplementation) Fig. 27.4 : An overuiew of cloning strategies in recombinant DNA technology.

r:L$${ING FBOfoI GENOMIG *€ m RNA ?

JTTI*A

a

lsolation and ourification of nucleic acids.

a

N ucl ei c aci d bl otti ngtechni ques.

a

D N A sequenci ng.

a

Methods of gene transfer(describedalready).

a

P ol ymerasechai n reacti on.

. P roducti on of (Chapter 4l).

monocl onal

anti bodi es

. Constructionof gene library. protein

. Site-directed mutagenesis and enS rneenng.

DNA represents the completeBeneticmaterial of an organismwhich is referredto as genome. Theoreticallyspeaking, cloning from genomic DNA is s u p p o s e dto b e i d e a l . B u t th e D N A containsnon-codingsequences(introns),control "egionsand repetitivesequences. Thiscomplicates :ne cloning strategies,hence DNA as a source naterial is not preferred, by many workers. ilowever,if the objectiveof cloning is to elucidate then genomicDNA .re controlof geneexpression, ^as t o be in v a ri a b l yu s e d i n c l o n i n g .

A l most al l the experi mentsdeal i ngw i th gene manipulationsrequire pure forms of either DNA or RNA, or sometimeseven both. Hence there is a need for the rel i abl ei sol ati onof nucl ei caci ds from the cel l s.The puri fi cati onof nucl ei c aci ds broadly involvesthree stages.

The use of mRNA in cloning is preferredfor : ' r e f ollowin g re a s o n s .

1. B reaki ngor openi ngof the cel l sto expose nucl ei c aci ds.

. mRNA represents the actual inf or m at i o nb e i n g e x p re s s e d .

2. S eoarati onof nucl ei c aci ds from other cel l ul arcomponents.

genetic

, Selection and isolation mRNA are easv.

IS @ LA TIOH A N D P U R IFIC A TIOhI OF N U C LE IG A C ID S

3. Recoveryof nucleic acids in a pure form.

. As introns are removed during processing, The basi c pri nci pl es and procedures for mRNA reflects the coding sequence of the nucl ei c aci d puri fi cati onare bri efl y descri beo. 8ene. . T he s y nt h e s i so f re c o m b i n a n p t ro te i ni s much eas ierwit h mR N A c l o n i n g . B es idest h e d i re c t u s e o f g e n o mi c D N A or ^-R\A, it is possibleto synthesizeDNA in the :-Doratory(by polymerasechain reaction),and - s e it in c lo n i n g e x p e ri m e n tsT. h i s a p p roachi s

P U N IF' E A TION

OF C E LLU LA B

DNA

The first step for DNA purificationis to open the cel l s and rel easeD N A . The method shoul d be gentle to preservethe native DNA. Due to vari abi l i ty i n cel l structure,the approachesto break the cells are also different.

"*-*.
rlil|fli|ul 1 r|rr;;

585

B IOC H E MIS TFI Y

Lysis .sf cells

formati onof an i nsol ubl ecomol ex w i th nu cleic aci ds.Thi s compl ex,i n the form of a prec ipit at e Bacterial cells : The bacterial cells (e.g. E. is collected after centrifugationand suspended coli) can be lysedby a combinationof enzymatic i n a hi gh-sal tsol uti onto rel easenucl ei c a cids. and chemical treatments.The enzyme lysozyme By treatmentwith RNase,RNA is degraded.Pure and the chemical ethvlenediaminetetraacetate DNA can be isolatedby ethanol precipitation. (EDTA)are usedfor this purpose.This is followed by the addition of detergentssuch as sodium The second technique is based on the dodecyl sulfate(SDS). pri nci pl e of ti ght bi ndi ng betw een D N A and si l i ca parti cl esi n the presenceof a dena t ur ing A n i ma l c e l l s : A n i ma l c e l l s, parti cul arl y agent such as guani di ni um thi ocyanate .The c ul tu re d a n i ma l c e l l s , c a n b e e asi l y opened by isolationof DNA can be achievedby the direct direct treatmentof cells with detergents(SDS). addi ti on of si l i ca parti cl es and guani d inium Pl a n t c e l l s : P l a n tc e l l s w i th s trongcel l w al l s thiocyanateto the cellular extract,followed by require harshtreatmentto break open. The cells centrifugation.Alternately,a column chromatoare frozen and then ground in a morter and graphy contai ni ng si l i ca can be used, and pestle.This is an effectiveway of breaking the through thi s the extract and guani d inium c el l u l o s ew a l l s . thiocyanateare passed.The DNA binds to the si l i ca parti cl es i n the col umn w hi ch ca n be f.-l!I ;i:iit+ds t* p.';*r[:i=*ft iir,i, recovereo. There are two differentapproachesto purify DN A fro m th e c e l l u l a re x tra c ts .

PARlFlGAfrOtr @F mRNA

A mong the R N A s, mR N A i s frequent ly 1. Purificationof DNA by removing cellular components : This involves the degradationor required in a pure form for geneticexperiments. complete removalof all the cellular components After the cells are disrupted on lysis by ot h e r th a n D N A . T h i s a p p ro a c hi s sui tabl ei f the different techniques (desciibed above), the c el l sd o n o t c o n ta i nl a rg eq u a n ti t i esof l i pi dsand cellularextractis deproteinisedby treatmentwith carbohydrates. phenol or phenol/chloroform mixtures. On The cellular extract is centrifugedat a low centrifugation,the nucleic acids get concentrated speed to remove the debris (e.g. pieces of cell i n the upper aqueousphasew hi ch may the n be wall) that forms a pellet at the bottom of the precipitatedby using isopropanolor ethanol. tube. The supernatantis collected and treated The puri fi cati onof mR N A can be achi evedby with phenol to precipitate proteins at the affinity chromatography using oligo (dlinterface between the organic and aqueous cellulose (Fi5.27.D. This is based on the lay e rs . T h e a q u e o u s l a y e r, contai ni ng the pri nci pl ethat ol i go (dT)-cel l ul ose can specif ically dissolvednucleic acids, is collected and treated bind to the poly (A) tails of eukaryotic mRNA. wit h th e e n z y m eri b o n u c l e a s (R e N ase). The R N A Thus, by this approach, it is possibleto isolate is d e g ra d e dw h i l e th e D N A re mai nsi ntact.Thi s mR N A from D N A , rR N A and IR N A . DNA can be precipitatedby adding ethanol and isolated after centrifugation,and suspendedin A s the nucl ei caci d sol uti oni s passedthr ough an appropriatebuffer. an affinity chromotographic column, the ol i go(dT)bi nds to pol y(A ) tai l s of mR N A. By 2. Direct purification of DNA : In this w ashi ngthe col umn w i th hi gh-sal tbuffer,DNA, approach, the DNA itse\i \s se\ective\y removed rRNA and IRNA can be eluted, while the mRNA from the cellular extract and isolated.There are i s ti ghtl y bound. Thi s mR N A can be then e lut ed two ways for direct purificationof DNA. by washing with low-salt buffer. The mRNA is ln 6ne method, the addition of a detergent precipitated with ethanol and collected by cetyltrimethyl ammonium (CTAB) results in the centrifugation (Fi9.27.5).

587

DNA AND BIOTECHNOLOGY Chapter e7 r RECOMBINANT

AT AT AT AT AT AT

Cellulosebead with oligo (dT)

immobilization of sample nucleic acids on solid support (nitrocelluloseor nylon membranes). The blotted nucleic acids are then used as targets in the hybridizationexperimentsfor their specific detection.An outline of the nucleic acid blotting technique is depicted in Fi9.27.5. Types

Columnwith oligo(dT)-cellulose

of blotting

techniques

The most comonly used blotting techniques are listed below . Southern blotting (for DNA) . Northern blotting (for RNA) . Dot blotting (DNA/RNA) The Southern blotting is named after the scientistEd Southern(1975) who developed it. The other narnes Northern blotting and Western blotting are laboratoryjargons which are now accepted. Western blotting involves the transfer of protein blots and their identification by using specific antibodies.

High-saltwash

I

+

A diagrammatic representationof a typical blotting apparatus is depicted in Fig.27.7.

SOUTHEBN BLOTTTNG Southernblottingtechniqueis the first nucleic acid blotting procedure developed in 1975 by

lmmobilization of nucleic acids

Southern blot(DNA) Northernblot(RNA) Dot-blot(DNA/RNA)

Low-saltwash Fiq.27.5 : Purification of nRNA by affinity chromatography with oligo(dT)-cellulose.

NUCI.EIC ACID BLOTTING TECHNIOUES Blotting techniques are very widely used anafytical tools for the specific identification of desired DNA or RNA fragmenfs from thousands of molecules. Blotting refers to the process of

Fig, 27.6 : An outline of the nucleic acid blotting techniques.

BIOCHEMISTF|Y

588

. Forensically applied to detect minute quantitiesof DNA (to identify parenthood, thieves,rapistsetc.).

GenomicDNA

, lf f if f i ,-t

,'

t

t

It." t-

\

I

Southern.lt is depicted in Fi9.27.8,and briefly described.

-

-:

,

t

\ \

t-a a

'-.

-.

-l

DNAfragments

The genomicDNA isolatedfroln cellytissues is digestedwith one or more restrictionenzymes. This mixtureis loadedinto a well in an agarose or polyacrylamidegel and then subiectedto DNA, being negativelycharged electrophoresis. migratestowardsthe anode (positivelycharged electrode);the smaller DNA fragmentsmove faster. The separatedDNA moleculesare denatured by exposureto a mild alkali and transferredto nitrocelluloseor nylon paper.This resultsin an exact replicaof the patternof DNA fragmentson the gel. The DNA can be annealedto the paper or on exposureto heat(80'C).The nitrocellulose nylon paper is then exposed to labeled cDNA probes. These probes hybridize with complementaryDNA moleculeson the paper. The paperafterthoroughwashingis exposed This to X-ray film to develop autoradiograph. to the DNA revealsspecificbandscorresponding fragmentsrecognized by cDNA probe. Applications

of Southern

Nitrocellulose (or nylonmembrane)

I oltnprooe

+

blotting

Southern bloting technique is extremely specificand sensitive,although it is a simple are listed. technique.Someof the applications . lt is an invaluablemethodin gene analysis. . lmportantfor the confirmationof DNA cloning results.

Autoradiograph

DNA AND BIOTECHNOLOGY ' -RECOMBINANT

589

. Highly u s e fu l fo r th e d e te rmi n a t i on of DOT.BLOfTING restriction fragment length polymorphism Dot-blottingis a modificationof Southernand (RFLP) with pathologicalconditions. associated Northernblottingtechniquesdescribedabove. ln this approach, the nucleic acids (DNA or RNA) NORTHERNBLOTT'NG are directly spotted onto the filters, and not Nor t her n b l o tti n g i s th e te c h n i q u e for the subjectedto electrophoresis.The hybridization specific identification of RNA molecules. The procedure i s the same as i n ori gi nal bl otti ng procedure adopted is almost similar to that techni ques. describedfor Southernblotting and is depicted Dot-blotting technique is particularly useful in Fig.27.9. RNA molecules are subjected to i n obtai ni ngquanti tati vedata for the eval uati on electrophoresis, followed by blot transfer, of gene expression. hy br idiz at i o na n d a u to ra d i o g ra p h y . RNA mo l e c u l e s d o n o t e a s i l v b i nd to Western bEotting nit r oc ellulo s ep a p e r o r n y l o n m e m b ra n e s.B l otWestern blotting involves the identification of transferof RNA moleculesis carriedout by using proteins. lt is very useful to understand the a chemically reactive paper prepared by nucl ei c aci d functi ons,parti cul arl yduri ng the to create diazotizationof aminobenzyloxymethyl courseof gene mani pul ati ons. diazobenzyloxymethyl(DBM) paper. The RNA The techniqueof Westernblottinginvolvesthe can covalently bind to DBM paper. transferof electrophoresedprotein bands from Northern blotting is theoretically, a good pol yacryl ami degel to nyl on or ni trocel l ul ose t ec hniquef o r d e te rmi n i n gth e n u m b e r o f genes membrane.These proteins can be detected by ( t hr oughm R N A ) p re s e n to n a g i v e n D N A . B ut specificprotein-ligandinteractions. Antibodiesor t his is not r e a l l yp ra c ti c a b l es i n c ee a c h g e n emay lectins are commonly used for this purpose. give riseto tvvoor more RNA transcripts.Another drawback is the presenceof exons and introns. A uteradi o#raFhy Autoradiographyis the processof localization and recording of a radiolabel within a solid specimen,with the productionof an image in a photographic emulsion. These emulsions are composedof silver halide crystalssuspendedin gel ati n.

rRNA bands

When a p-particleor a T-rayfrom a radiolabel passesthrough the emul si ons,si l ver i ons are convertedto metallic silveratoms.This resultsin the devel opmentof a vi si bl e i mage w hi ch can be easily detected. .4#elf.feaffons @rea{Jf#r# dioryaphy As already described, autoradiography is closely associatedwith blotting techniquesfor the detectionof DNA, RNA and proteins. D N A S E QU E N C IN G

F19.27.9: An outlineof Northemblotting.

Determination of nucleotide sequence in a DNA molecule is the basic and fundamental

BIOGHEMISTF|Y

608

rDNA product lnsulin hormone Growth cr,-lnterferon Hepatitis B vaccine plasminogen Tissue ac'livator Factor Vlll DNase Erylhropoietin

Applications/uses

Trade name(s)

Humulin Protropin/Humatrope lnlronA B Recombinax HB/Engerix Activase Kogenate/Recombinate Pulmozyme Epgen/Procrit

medical and commercial importance. An approval,by the concernedauthorities, for using recombinant insulin for the treatment of diabetes mellitus was given in 1982. And in 1986, Eli Lilly companyreceivedapprovalfo market human insulln under the trade name Humulin.

Plasmid Translorm into

E.cott

Techniquefor production of recombinant insulin: Theorginaltechnique(described briefly above) of insulin synthesisin E. coli has undergoneseveralchanges,for improvingthe yield.e.g.additionof signalpeptide,synthesis of A and B chainsseparatelyetc. The procedureemployedfor the synthesisof two insulin chains A and B is illustratedin Fig.27.28. The genesfor insulinA chain and B chainare separately insertedto the plasmidsof two differentE. coli cultures.The /ac operon system(consistingof inducergene, promoter gene, operatorgene and structuralgeneZ for p-galactosidase) is used for expressionof both the genes.The presenceof lactosein the culture mediuminducesthe synthesis of insulinA and B chains in separatecultures.The so formed insulin chains can be isolated,purified and joined togetherto give a full-fledgedhuman insulin.

Diabeles Pituitary dwarfism Hdrycellleukemia Hepatitis B Myocardial infarction Hemophilia fibrosis Cystic withkidney damage Severe anemia

r-'

-^--A.-

I

I +

-

A chain

.-^--^.-' B chain

Humaninsulin Ftq.2728 : Theproductionof recombinantinsulinin

RecombinantDNA technology in recent years, has become a boon to produce new generationvaccines.By this approach,someof

E. coli (l-lnducer gene, P-Promoter gene,

chapar

27 : BECOMB'NANT DNA AND B'OTECHNOLOGY

609

the limitations(low yield, high cost, side effects) of traditional vaccine production could be overcome. In addition, several new strategies, involving gene manipulation are being tried to create novel recombinantvaccines. The list of diseasesfor which recombinant vaccines are developed or being developed is given in Tahle 22.7. lt may be stated here that due to very stringentregulatoryrequirementsto use in humans, the new Beneration vaccines are first tried in animals, and it may take some more years before most of them are approved for use in humans.

llepatitis B vaccine -the first symthetic vaccine

Disease

Pathogenic organism

Viral diseases Acuteinfantib gastroenteritis Acuterespiratory diseases AIDS pox Chicken

Rotavirus lnfluenza A andB viruses Human immunodeficiency virus Varicella-zoster virus

Encephalitis

encephalitis virus Japanese ulcers Genital Herpes simplex virustyp+2 Hemorrhagic fever Dengue virus Liverdamage Hepatitis A virus Liverdamage Hepatitis B virus andlower Yellow fevervirus Upper respiratory tractlesions

ln 1987, the recombinantvaccinefor hepatitis B (i.e. HBsA{ became the first synthetic vaccine for public use. lt was marketed by trade names Recombivax and Engerix-B. Hepatitis B vaccine is safe to use, very effective and produces no allergic reactions. For these reasons, this recombinant vaccine has been in use since Bacterial diseases 1987. The individuals must be administered Vibiocholerae Cholera three doses over a period of six months. lmmunization against hepatitis B is strongly Diarrhea E. co| recommendedto anyone coming in contact with Dysentery strain Shigella the health blood or body secretions. All Niesseiagonorroheae Gonorfiea professionals-physicianst surgeonst medical leprae Mycobacteriun Leprcsy laboratory technicians, nurses/ dentists, besides get p/ice ofrcers, /r7efigrhters etc., mast l/e/aela nezlrVzllUis iweningrts vaccinated against hepatitis B. pneunoniae Streptomccus

Pneumonia fever Rheumatic

Hepatitis B vaccine in India tndiais thefourthcountry(afterUSA,France

Tetanus

groupA Streptocomus Clostidiuntetani

and Belgium) in the world to develop an indigenoushepatitisB vaccine. lt was launched in 1997, and is now being used.

Tuberculosis

Mycobaderiu m tube rculosis

Typhoid

Salnonella typhi groupB Streptocorcus

tract Urogenital infection Parasiticdiseases Filariasis

Genetic immunization by using DNA vaccines is a novel approach that came into being in 1990. The immune resPonseof the bdy is stimulated hy a DNA molecule. A DNA

Wuchereria bancrofti

Plasmodium sp volvulus Onchocerca mansoni Schistosona Schistosomiasis sp sickness Trypanosona Sleeping Mafaria Riverblindness

603

DNA AND BIOTECHNOLOGY Chapter 27 : RECOMBINANT Applications

of DNA fingerprinting

The amount of DNA required for DNA fingerprint is remarkably small. fhe minute quantities of DNA from blood strains, body ffuids, hair fiber or skin fragments are enough. Polymerase chain reaction is used to amplify this DNA for use in fingerprinting.DNA profiling has wide range of applications-most of them related to medical forensics. Some important ones are listed below. . ldentificationof criminals,rapists,thievesetc. . Settlementof paternitydisputes. o Use in immigrationtest casesand disputes.

R1

R2

Bs

DNA 1 I Restriction endonuclease I

+

4 fragments

Br

Fs DNA 2

I nesrriction endonuclease J 3 fragments

Flg. 27.22 : An outlineof the restrictiontragmentlength I n gene ra l , th e fi n g e rp ri n ti n gte c h n ique i s polymorphism (RFLP) (81, R2 Rr represent carriedout by collectingthe DNA from a suspect (or a person in a paternity or immigration dispute)and matching it with that of a reference sample (from the victim of a crime, or a close person's chromosomesand have no apparent r elat iv ein a c i v i l c a s e ), functi on. DNA i'ARKERS IN DISEASE DIAGNOSIS AND FINGERPRINTING The DNA markers are highly useful for genetic mapping of genomes. There are four types of DNA sequenceswhich can be used as markers.

A DNA molecule can be cut into different fragments by a group of enzymes called restrictionendonuc leases(SeeTable 2 7. l). These fragments are called polymorphisms (literally means mdny forms).

An outline of RFLPis depicted in Fi9.27.22. The DNA molecule 'l has three restrictionsites 1. Restrictionfragmentlengthpolymorphisms (R1 R2, R3), and when cleaved by restriction , (RFLPs,pronouncedas rif-lips). endonucleasesforms 4 fragments.Let us now 2. Minisatellitesor variable number tandem consider DNA 2 with an inherited mutation (or a genetic change) that has altered some base repeats(VNIRs, pronouncedas vinters). pairs.As a result,the site (Rz)for the recognition 3. Microsatellitesor simple tandem repeats by restrictionendonucleaseis lost. This DNA (SIRs). molecule2 when cut by restrictionendonuclease forms only 3 fragments(insteadof 4 in DNA 1). 4. Single nucleotide polymorphisms (SNPq pr onounc eda s s n i p s ). As is evident from the above description,a The general aspects of the above DNA stretch of DNA exists in fragments of various markersare describedalong with their utility in Iengths (polymorphisms), derived by the action of restriction enzymes, hence the name diseasediagnosisand DNA fingerprinting. restrictionfragment length polymorphisms.

BESTR,CTTONFNAGMENT LENGTH POLY lttO RPrlrSMS (RF LPsI

RFLPs in the diagnosis

A RFLP representsa stretch of DNA that seryesas a markerfor mapping a specifiedgene. RFLPs are located randomly throughout a

lf the RFLP lies within or even close to the locus of a gene that causesa particulardisease, it is possibleto trace the defectivegene by the

of diseases

602

E|IOCHEMISTF|Y p-Globin gene

Mstll

Pro

ccr 5

{ Glu Glu GAG GAGt-

Baseseouence in normalgene F-Amino acidnumber

CCT GTG G A GF B a s e sequencei n sickle-cellgene Pro Val Glu (p-globingene)

a person's cells (usually white blood cells) is stored. As and when a DNA probe for the detection of a specific diseaseis available,the storedDNA can be usedfor the diagnosisor risk assessment of the said genetic disease. In fact, some institutions have established gene banks. They store the DNA samples of the interested customers at a fee (one firm was charging $200) for a specified period (say around 20-25 years). For the risk assessmentof any disease, it is advisable to have the DNAs from close relatives of at least 2-3 generations. D N A FIN GE R P R IN TIN G DNA PROFILING

OR

DNA fingerprinting is the presentday genetic detective in the practice of modern medical forensics. The underlying principles of DNA DNA fragments in and around B-globin gene, fingerprintingare briefly described. followed by the electrophoretic pattern of the The structure of each person's genome is DNA fragmentsformed. The change in the base The only exception being monozygotic unique. p-globin from A to T in the gene destroys identical twins (twins developed from a (CCTGAGG) the recognition site for Mstll (Fig.27,21). Consequently, the DNA fragments single fertilized ovum). The unique nature formed from a sickle-cell anemia patient for of genome structure provides a good opporp-globin gene differ from that of a normal tunity for the specific identification of an person.Thus, sickle-cellanemiacan be detected i ndi vi dual . by digesting mutant and normal p-globin It may be remembered here that in the genes by restriction enzyme and performing traditional fingerprint technique, the individual a hybridization with a cloned p-globin DNA is identified by preparing an ink impressionof probe. the skin folds at the tip of the person'sfinger. This is based on the fact that the nature of these GENE BANKS-A NOVEL CONCEPT skin folds is geneticallydetermined,and thus the As the search continues by scientistsfor the fi ngerpri nt i s uni que for an i ndi vi dual. ln identification of more and more genes contrast, the DNA fingerprint is an analysis of responsiblefor variousdiseases,the enlightened the nitrogenous hase sequence in the DNA of public (particularlyin the developedcountries), an individual. is very keen to enjoy the fruits of this research outcome. As of now, DNA probes are available History and terminology for the detection a limited number of diseases. The ori gi nal D N A fi ngerpri nti ngtechnique Researchers continueto develop DNA probesfor was developed by Alec Jaffreys in 1985. a large number of genetically predisposed Although the DNA fingerprintingis commonly disorders, used, a more general term DNA profiling is Gene banks are the centres for the storage of preferred.This is due to the fact that a wide individual's DNAs for future use to diagnose range of tests can be carried out by DNA diseases.For this purpose,the DNA isolatedfrom sequencingwith improved technology.

I

604

BIOCHEMISTFIY

H

(A)

-DNAwith

DNAprobe

restriction(R)sites One band

Twobands Nylonmembrane

(B)

Oneband I

Suspected site

JR2

R1

R3 \tl

+r\

V

-E

PCRprimers DNAwithrestrictionmap

Twobands

(RFLP) Fiq.27.23: Twocommonmethodsusedfor scoringrestrictionfrcgmentlengthpolymorphism

anal y s i so f R F L Pi n D N A . T h e p e rson' scel l ul ar DNA is isolated and treated with restriction enzymes.The DNA fragmentsso obtained are separatedby electrophoresis.The RFLPpatterns of the disease suspected individuals can be compared with that of normal people (preferably with the relatives in the same family). By this approach,it is possibleto determinewhetherthe individual has the marker RFLPand the disease gene. With 957o certainity, RFLPs can detect single gene-based diseases.

2. Polymerase chain reaction : RFLPs can also be scored by PCR. For this purpose, PCR primers that can anneal on either side of the suspected restriction site are used. After ampl i fi cati onby P C R , the D N A mol ecul esar e treated with restriction enzyme and then analysed by agarosegel electrophoresis.lf the restrictionsite is absentonly one band is seen, while iwo bands are found if the site is found (Fig.27.238).

Applications of RFIPs : The approach by Methods of RFLPscoring : Two methods are RFLP is very powerful and has helped many in common use for the detection of RFLPs genesto be mapped on the chromosomes.e.g. (Fig.27.23). sickle-cell anemia (chromosome 1 I ), cystic fibrosis (chromosome7), Huntington's desease 1. Southern hybridization : The DNA is (chromosome4), retinoblastoma(chromosome digested with appropriate restriction enzyme, 13), A l zhei mer' sdi sease(chromosome21). and separatedby agarose gel electrophoresis. The so obtained DNA fragments are transferred to a nylon membrane.A DNA probe that spans VAN'ABLE NUTilBEN TANDEM BEPEATS (VNTBS) the suspectedrestrictionsite is now added, and the hybridized bands are detected by VNTRs, also known as minisatellites,like autoradiograph.lf the restrictionsite is absent, RFLPs,are DNA fragmentsof different length. then only a single restriction fragment is The main differenceis that RFLPsdevelop from detected.lf the site is present,then two fragments random mutations at the site of restriction are detected (Fi9.27.23A). enzyme activity while VNTRs are formed due to

BIOCHEMISTRY

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l_./

\J

Plasmids

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( 3,

6

5

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Fig'27'17:"*"::,;;',fr :::i?if",i!i"?{,JiJi,ii'ii";;;;;;^i^i;#;'"""'

Chapter 27 : RECOMBINANT DNA AND BIOTECHNOLOGY

597

accomplishedby isolatingthe completegenome ,e nt ir e DNA f r o m a c e l l ) w h i c h i s c u t i n to fragments,and cloned in suitablevectors.Then the specific clone carrying the desired (target) DNA can be identified, isolated and characterized.In this manner,a library of genes or clones (appropriately considered as gene Dnnh ior the entire genome of a speciescan be constructed.

Screening by DNA hybridization : The target sequencein a DNA can be determinedwith a DNA probe (Fig.27.lA. To start with, the double-strandedDNA of interest is converted into single strands by heat or alkali (denaturation).The two DNA strands are kept apart by binding to solid matrix such as nitrocellulose or nylon membrane. Now, the single strands of DNA probe (100-1,000 bp) labeled with radioisotope are added. Establishing a gene library Hybridization(i.e.,basepairing)occurs between for humans the complementarynucleotidesequencesof the T he hum an c e l l u l a rD N A (th e e n ti reg e n o me) target DNA and the probe. For a stable base may be subjected to digestion by restriction pairing, at least 80% of the bases in the two (target DNA and the probe) should be endonucleases (e.9., EcoRI). The fragments strands The hybridized DNA can be detected matching. formed on an average are of about 4 kb size. by autoradiography. (i. e. , 4000 nit ro g e n e s b a s e s ). Ea c h h u man chromosome,containingapproximately100,000 (Note ; DNA prohe or gene probe represents kb can be cut into about 25,000 DNA fragments. a segmentof DNA that is tagged with a label As the humans have 23 different chromosomes (i.e. isotope) so as to detect a complementary (24 in man), there are a total of 575,000 base sequence with sample DNA after fragmentsof 4 kb length formed. Among these hybridization) 575,000 DNA fragmentsis the DNA or gene of interest(say insulin gene). Now is the selectionof a vector and cloning process.E.coli,a harmlessbacteriumto humans is most commonly used. The plasmids from E. coli are isolated. They are digested by the same restriction enzyme as was used for cutting human genome to form open plasmids. The human chromosomaf DNA fragmentsand open plasmids are joined to produce recombined plasmids.Theseplasmidscontain differentDNA fragmentsof humans.The recombinedplasmids are insertedinto E. coli and the cells multiply (Fig.27.lV. The E coli cells possessall the human DNA in fragments.lt must, however be remembered that each E. coli cell contains different DNA fragmenfs. All the E. coli cells put together collectively represent genomic library (containingabout 575,000 DNA fragments).

Screening strategies

SITE.DIRECTEDMUTAGENESIS AND PROTEINENGINEERING

Modificationsin the DNA sequenceof a gene are ideal to create a protein with desired properties. Site-directed mutagenesis rb the technique for generating amino acid coding changes in the DNA l6ene). By this approach specific (site-directed) change (mutagenesis) can be made in the base (or bases) of the gene to produce a desired enzyme. The net site-directed mutagenesis is result in incorporationof a desired amino acid (of one's choice) in place of a specific amino acid in a protein or a polypeptide. By employing this technique,enzymesthat are more efficient and more sui tabl e than the natural l y occurri ng counterparts can be created for industrial applications. But it must be rememberedthat site-directedmutagenesisis a trial and error method that may or may not result in a better protein.

Once a DNA library is created, the clones (i.e., the cell lines) must be screened for A couple of proteins developed by siteidentificationof specific clones. The screening and proteinengineeringare techniquesare mostly basedon the sequenceof directedmutagenesis the clone or the structurefunction of its product. given next.

DNA AND BIOTECHNOLOGY Ghapter 27 : RECOMBINANT (A)

@@-@-o-rrE

s91

with ddA. In a similar fashion, for the other 3 reaction tubes, DNA synthesis stops as the respectivedideoxynucleotidesare incorporated. The synthesisof new DNA fragments in the four tubes is depicted in Fig.27.12.

(B)

(M-o-rzQ

Fiq.27.11: Structureof (A) dideoxynucleotide

The DNA pieces are denatured to yield free strandswith radiolabel.The samplesfrom each tube are separated by polyacrylamide gel electrophoresis. This separation technique resolvesDNA pieces, different in size even by a single nucleotide.The shortestDNA will be the fastestmoving on the electrophoresis.

The sequenceof bases in a DNA fragment is determined by identifying the electrophoretic incoming nucleosidetriphosphateis attachedby (radiolabeled)bands by autoradiography.In the ;1s5'-phosphate group to the 3lhydroxyl group Fig.27.13,the sequenceof the newly synthesized of the last nucleotideof the growing chain (Refer DNA fragment that is complementary to the Chapter 24) when a dideoxynucleotide is original DNA piece is shown. lt is conventional incorporatedto the growing chain, no further to read the bands from bottom to top in 5'to 3' replication occurs. This is because direction. By noting the order of the bands first dideoxynucleotide,lacking a 3'-hydroxyl group, C, secondC, third T and so on, the sequenceof cannot form a phosphodiesterbond and thus the the DNA can be determined accurately.As many DNA synthesisterminates. as 350 base sequencesof a DNA fragmentcan process Sequencing method : The of be clearly identified by using autoradiographs. sequencingDNA by dideoxynucleotidemethod Modifications of dideoxvnucleotide method : is briefly described.A single-strandedDNA to be Replacementof 32P-radioiabelby 33P or 3sS sequencedis chosen as a template. lt is attached improves the sharpness of autoradiographic to a primer (a short length of DNA images. DNA polymeraseof the thermophilic oligonucleotide) complementary to a small bacterium, Thermus aquaticus (in place of section of the template. The 3'-hydroxyl group of Kfenowfragmentof E. coliDNA polymerasel) or the primer initiatesthe new DNA synthesis. a modified form of phage T7 DNA polymerase improvesthe technique. DNA synthesisis carried out in four reaction (sequenase) tubes. Each tube contains the primed DNA, Klenow subunit (the larger fragment of AUjOMATED DNA SEOUENC'NG DNA polymerase of E. coli), four dideoxyDNA sequencingin the recentyearsis carried ribonucleotides (ddATP, ddCTP, ddCTP or out by an automated DNA sequencer. In this ddTTP). lt is necessaryto radiolabel (with 32fl technique,flourescenttagsare attached to chainthe primer or one of the deoxyribonucleotides. terminating nucleotides (dideoxynucleotides). As the new DNA synthesisis completed, each This tag gets incorporated into the DNA one of the tubes contains fragments of DNA of molecules, while terminating new strand varying length bound to primer. Let us consider synthesis. Four different fluorescent dyes are the first reaction tube with dideoxyadenosine used to identify chain-terminatingreactionsin a (ddATP). In this tube, DNA synthesisterminates sequencing gel. The DNA bands are separated whenever the growing chain incorporates ddA by electrophoresis and detected by their (complementaryto dT on the template strand). fluorescence.Recently, four dyes that exhibit Therefore, this tube will contain a series of strong absorption in laser are in use for different length DNA fragments, each ending automated sequencing,

592

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3'=-i

DNA* primer dn ruewrysynthesized

Prlmerwith nucleotide extended

Rgacllon tub€ wlth dldeorynucleotlde ddATP

ddCTP

ddGTP

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Primer+ 1

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Primer+ 6

Primer-CGTAGddC

Primer+ 2

Primer{ddG

Primer+ 5

Primer-CGTAddG

Primer+ 3

PrimertGddT

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ddTTP

ddGTP

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s',

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Chapter 27 : FIECOMBINANT DNA AND BIOTECHNOLOGY

593

Advantagesof automated sequencing : lt is a 'apid and accuratetechnique.Automated DNA sequencer can accurately sequence up to 100,000nucleotidesper day. The cost works out to be not more than $0.2 per nucleotide. Automated DNA sequencing has been successfullyused in the human genome project.

DHA CHiPS (MTCBOARBAYS) DNA chips or DNA microarraysare recent developmentsfor DNA sequencingas result of advancesmade in automationand miniarization. A large number of DNA probes,each one with different sequence,are immobilized at defined positionson the solid surface,made up of either nylon or glass.The probes can be short DNA molecufes such as cDNAs or synthetic oligonucleotides. For the preparation of high density arrays, of igonucleotidesare synthesizedin situ on the surface of glass or silicon. This results in an oligonucleotide cfiip rather than a DNA chip.

Hybridizing signals

Technique of DNA sequencing A DNA chip carrying an array of different oligonucleotides can be used for DNA sequencing. For this purpose, a fluorescently labeled DNA test molecule, whose sequenceis to be determined, is applied to the chip. Hybridization occurs between the complementary sequences of the test DNA molecule and oligonucleotidesof the chip. The positions of these hybridizing oligonucleotides can be determined by confocal microscopy. Each hybridizing oligonucleotide representsan 8nucleotidesequencethat is presentin the DNA probe. The sequenceof the test DNA molecule can be deduced from the overlaps between the sequencesof the hybridizing oligonucleotides (Fig.27.tt0. Applications

of DNA chips

There have been many successeswith this relativelynew technologyof DNA chips. Some of them are listed. . ldentification of genes responsible for the development of nervous systems.

+ AGTCCCT'^ 6TCCCTTG\ Hybridizing TcccrTcc\ligonucleotides (8) CCCTTGGC' - - - DNAsequence - - -AGTCCCTTGGC

genes Detection of i nflammatorydiseases.

responsible

for

Construction of microarraysfor every gene in the genome of E. coli, and almost all the genes of the yeast Saccharomyces cerevisiae. Expressionof several genes in prokaryoteshas been identified. Detection and screeningof single nucleotide polymorphisms(SNPs). Rapid detection of microorganisms for environmentalmonitoring.

599

Chapter 87 : RECOMBINANT DNA AND BIOTECHNOLOGY

SourceDNA(double-stranded) I

I Denaturation binding I membrane * DNAs Single-stranded

-*

The basic principles underlying the DNA diagnosticsystems,and their use in the diagnosis of certain pathogenic and genetic diseasesare described.Besidesthese,the variousapproaches for DNA fingerprinting (or DNA profiling) are also discussed.

F* i(x)K

METHODS

HybridDNAs H9.27.18 : Screeningby DNAhybridization ( { indicates radioisotope label in the DNA probe)

Tissue

plasminogen

actiyator

the specific organism. Thus, the presence of a disease-causing pathogen can be detected by identifing a gene or a set of genes of the organism. Likewise an inherited genetic defect can be diagnosed by identifying the alterations in the gene. In the modern laboratory diagnostics,DNA analysisis a very usefuland a sensitivetool.

(tFA|

Tissueplasminogenactivatoris therapeutically used to lvse the blood clots that cause myocardialinfarction.Due to its shorterhalf-life (around 5 minutes),tPA has to be repeatedly administered. By replacing asparagine residue (at position 12O) with glutamine, the half-life of tPA can he substantially increased.This is due to the fact that glutamine is less glycosylated than asparagine and this makes a difference in the half-life of tPA.

OF DNA ASSAY

The specific identification of the DNA sequence is absolutely essential in the Iaboratory diagnostics.This can be achieved by employing the following principles/tools. Nucleie

acid

hybridization

Hybridization of nucleic acids (particularly D N A ) i s the basi s for rel i abl e D N A anal vsi s. Hybridization is based on the principle that a single-strandedDNA molecule recognizesand specifically binds to a complementary DNA strandamid a mixtureof other DNA strands.This is comparable to a specific key and lock relationship. The general procedure adopted for nucleic acid hybridization has been described(Seep. 597 and Fig.27.l8). Some more information is given below (Fi9,27,19.

Hir udin

The single-stranded target DNA is bound to a membrane support. Now the DNA probe (singleHirudin is a protein secretedby leech salivary gland, and is a s tro n gth ro m b i n i n h i b i to r (i.e., acts as an anticoagulant). By replacing ACGTTAGCA asparagine (at 47 position) with lysine, the potency of hirudin can he increased severalfold.

A C GTTA GC A --f-

DNA, being the geneticmaterialof the living organisms, contains the information that contributesto various characteristicfeaturesof

||r||l

TGCAATCGTComplementarypairing

Fig. 27.19 : Hybidization of target DNA with DNAprobe (with tadioactive isotope label).

ftWr

DNA AND BIOTECHNOLOGY 27 : RECOMBINANT

605

Use of RFLPs and VNTRs in genetic fingerpfinting Restriction enzyme

DNA2

RFLPscausedby variationsin the number of VNTRs between two restriction sites can be detected (Fi9.27.2fl. The DNAs from three individualswith differentVNTRs are cut by the specific restriction endonuclease. The DNA fragments are separatedby electrophoresis,and identified after hybridization with a probe complementary to a specific sequence on the fragments.

(A) dirierent number of base sequencesbetween two points of a DNA molecule. In general, VNTRs are made up of tandem repeats of short base requences(10-100 base pairs).The number of elementsin a given region may vary, hencethey are known as variable number tandem repeats.

1

An individual's genome has many different VNTRs and RFLPs which are unique to the individual. The pattern of VNTRs and RFLPs forms the basis of DNA fingerprinting or DNA profiling. lnthe Fig.27.24,twodifferentDNA molecules \vith different number of copies (bands) of VNTRs are shown. When these molecules are subjected to restriction endonucleaseaction (at two sites R1 and R2), the VNTR sequencesare released, and they can be detected due to variability in repeatsequencecopies.Thesecan be used in mapping of genomes,besidestheir utility in DNA fingerprinting. VNTRs are useful for the detection of certain genetic diseasesassociatedwith alterationsin the degree ol repetition of microsatellites e. g. Hunt ingt on ' sc h o re a i s a d i s o rd e r w h i ch is found when the VNTRs exceed 40 repeat units. limitations of VNTRs : The major drawback of VNTRs is that they are not evenly distributed throughout the genome. VNTRs tend to be localized in the telomeric regions,at the ends of the chromosomes.

(B)

Fig. 27.25 : Use of restriction fragment length polymorphisms(RFLPS)caused by variable number tandem repeats (VNTRS)in genetic fingerprinting (A) An illustration of DNA structurc from three

individuals(B) Hybridizedpatternof DNAfragment witha probecomplementaryto the sequenceshownin greencircles(1, 2 and 3 representthe individuals;

606

BIOCHEMISTF|Y DNAI

---.---.---..-----..GGCGAGAGAGAG (5 repeatingunitsof GA) DNA2

(10repeating unitsof GA) Fiq.27.26 : Two alleles of DNA molecules representing

MTCBOSATELLITES (SIMPLE TANDEM BEPEATST

An oligonucleotideis a short single-stranded DNA molecule, synthesizedin the laboratory with a length not usually exceeding 50 nucleotides.Under appropriateconditions,this nucleotidesequencewill hybridizewith a target DNA strandif both have completelybasepaired structure.Even a single mismatch in base pair will not allow the hybridizationto occur.

DNA chip technology is most commonly used to screen SNPshybridization with oligonucleotide(Seep. 593). CURRENT TECHNOLOGY OF DNA FINGERPRINTING ln the forensicanalysisof DNA, the original

Microsatellites areshortrepeatunits(1G-30 techniques and VNTRsare now basedon RFLPs

copies) usually composed of dinucleotide or tetranucleotide units. These simple tandem repeats (STRs) are more popular than minisatellites(VNTRs)as DNA markersfor two reasons. 'l . Microsatellites are evenly distributed throughoutthe genome.

(shorttandem largelyreplacedby microsatellites repeats).The basic principle involves the by polymerase amplificationof microsatellites chain reactionfollowedby their detection. It is now possibleto generatea DNA profile by automatedDNA detectionsystem (comparable equipment). to the DNA sequencing

2. PCR can be effectivelvand conveniently used to identify the length of polymorphism. (A) Two variants(alleles)of DNA moleculeswith -oAGCTGTCGAT5 and 10 repeatingunits of a dimer nucleotides (CA) are depicted in Fig.27.26. -oAGCTCTCGATBy use of PCR, the region surroundingthe microsatellitesis amplified, separatedby agarose gel electrophoresisand identified.

(B) _S N P Y

-oAGC

S"VGIE NUCLEOT'DE POLYIilOEPHTShIS (SNPs) SNPs representthe positions in the genome where some individuals have one nucleotide (e.e.C) while others have a differentnucleotide (e.S. C). There are large numbers of SNPs in genomes.lt is estimatedthat the human genome containsat least 3 million SNPs.Some of these SNPs may give rise to RFLPs. SNPsare highly usefulas DNA markerssince there is no need for gel electrophoresis and this savesa lot of time and labour. The detectionof SNPs is based on the oligonucleotide hybridizationanalysis(Fig.27.2V.

-

rCG41||il lllll GTCGACAGCTA -

Target DNA oligonucleotide

LMatched base Completeandstable hybridization of basepairs r-SNP Y

-

oAGCTGTCGAT -

TargetDNA

oligonucleotide GTCGAGAGCTA .f. LMismatched base notformed Hybridization dueto mismatchbasepair F19.27.27 : (A) An illustration of single nucleotide polymorphism (SNP) (B) Oligonucleotide

\'\

500

BIOCHEMISTRY

stranded and labeled with a detector substance) D]{A IN THE DIAGNOSIS is added. Under appropriate conditions OF INFECTIOUSDISEASES (temperature, ionic strength), the DNA probe The use of DNA analysis(by employing DNA pairs with the complementarytarget DNA. The probes) is a novel and revolutionary approach unbound DNA probe is removed. Sequenceof for specifically identifying the disease-causing nucleotidesin the target DNA can be identified pathogenic organisms.This is in contrastto the from the known sequenceof DNA probe. traditional methods of disease diagnosis by There are two types of DNA hybridization- detection of enzymes, antibodies etc., besides radioactive and non-radioactive respectively the microscopic examination of pathogens. using DNA probes labeled with isotopes and Although at presentnot in widespreaduse, DNA non-isotopesas detectors. analysis may soon take over the traditional diagnostic tests in the years to come. Diagnosis of selected diseases by genetically engineered THE DNA CH'P.MICBOABNAY techniques or DNA probes or direct DNA OF GENE PBOBES analysis is briefly described. The DNA chip or Genechip contains thousands of DNA probes (4000,000 or even Tuberculosis more) arrangedon a small glassslide of the size Tuberculosis is caused by the bacterium of a postagestamp. By this recent and advanced Mycobacterium tuberculosis. The commonly approach, thousandsof target DNA molecules used diagnostic tests for this disease are very can be scannedsimultaneously. slow, and sometimes may take several weeks. This is because M. tuberculosrsmultiplies very Technique for use of DNA chip slowfy (takes about 24 hrs. to double; E. coli The unknown DNA molecules are cut into takes just 20 minutes to double). fragments by restriction endonucleases. A novel diagnostictest for tuberculosiswas Fluorescentmarkersare attachedto these DNA developed by genetic engineering, and is fragments. They are allowed to react with the ilfustrated in Fi9.27.20. A gene trom firefly, probesof the DNA chip. Target DNA fragments encoding the enzyme luciferase is introduced with complementary sequencesbind to DNA into the bacteriophage specific for M. probes. The remaining DNA fragments are tuberculosis. The bacteriophage is a bacterial washed away. The target DNA pieces can be virus, frequently referredto as luciferasereporter identified by their fluorescence emission by phage or mycophage.The geneticallyengineered passing a laser beam. A computer is used to phage is added to the culture of M. tuberculosis. record the pattern of fluoresence emission and The phage attachesto the bacterial cell wall, DNA identification. penetratesinside,and insertsits gene (alongwith T h e te c h n i q u e o f e m p l o y i ng D N A chi ps i s Iuciferase gene) into the M. tuberculosis very rapid, besidesbeing sensitiveand specific chromosome.The enzyme luciferaseis produced for the identification of several DNA fragments by the bacterium.When luciferin and ATP are simultaneously.Scientistsare trying to develop added to the culture medium, luciferasecleaves Cenechipsfor the entiregenomeof an organism. luciferin.This reactionis accompaniedby a flash of light which can be detectedby a luminometer. Applications of DNA chip This diagnostic test is quite sensitive for the The presence of mutations in a DNA confirmationof tuberculosis. sequencecan be convenientlyidentified.In fact, Cenechipprobe arrayhas been successfully used for the detection of mutations in the p53 and BRCA I genes. Both these genes are involved in cancer (Seep. 593 also).

The flash of light is specific for the identification of M. tuberculosis in the culture. For other bacteria, the genetically engineered phage cannot attach and enter in, hence no flash of light would be detected.

DNA AND BIOTECHNOLOGY Gtrapter 27 : FIECOMBINANT

601

DNA IN THE DIAGNOSISOF GENETIC DISEASES Mlubacterium tuberculosis

Traditional laboratorytests for the diagnosis of genetic diseases are mostly based on the estimation of metabolitesand/or enzymes. Thi s i s usual l y done after the onset of Viral genome symptoms. Luciferase gene

The laboratorytests based on DNA analysis can specificallydiagnosethe inherited diseases at the genetic level. DNA-basedtestsare useful to discover, well in advance, whether the individualsor their offspringsare at risk for any genetic disease.Further,such testscan also be employedfor the prenataldiagnosisof hereditary disorders, besides identifying the carriers of genetic diseases.

Although not in routine use in the laboratory service,methods have been developedor being developed for the analysis of DNA in the diagnosis of several genetic diseases.These i ncl ude si ckl e-cel l anemi a, cysti c fi brosi s, llalaria Duchenne's muscular dystrophy, Huntington's Malaria, mainly caused by Plasmodium disease,fragileX syndrome,Alzheimer'sdisease, falciparum, and P. vivax, affectsabout one-third of certain cancers (e.g. breast cancer, colon the world's population. The commonly used cancer), type ll diabetes, obesity, Parkinson's laboratorytestsfor the diagnosisof malariainclude diseaseand baldness. microscopicexaminationof blood smears,and detectionof antibodiesin the circulation.While Sickle-cell anemia the former is time consumingand frequentlygives S i ckl e-cel l anemi a i s a geneti c di sease false-negative tests,the latter cannot distinguish characterizedby the irregular sickle (crescent between the past and presentinfections. like) shape of the erythrocytes.Biochemically, diagnostic test for A specific DNA this disease results in severe anemia ano identification of the current infection of P. progressivedamageto major organsin the body falciparum has been developed. This is carried (heart,brain, lungs,joints). out by using a DNA probe that can bind and hybridize with a DNA fragment of P. falciparum S i ckl e-cel lanemi a occurs due to a si ngl e genome, and not with other species of ami no aci d change i n the B -chai n of Plasmodium. lt is reported that this DNA probe hemogl obi n. S peci fi cal l y, the ami no aci d can detect as little as 1 ng of P. falciparum in glutamate at the 6th position of B-chain is blood or 10 pg o f i ts p u ri fi e d D N A. replaced hy valine. At the molecular level, si ckl e-cel lanemi a i s due to a si ngl e-nucl eoti de Acquired immunodeficiency change (A -+ 7) i n the p-gl obi n gene of codi ng syndrome (AlDSl (or antisense)strand. In the normal p-globin DNA probes,with radioisotopelabel, for HIV gene the DNA sequence is CCTGACCAG, DNA are now available.By using PCRand DNA w hi l e i n si ckl e-cel l anemi a, the sequence i s probes, AIDS can he specifically diagnosed in CCTGTCCAG.This single-basemutation can be detected by using restrictionenzyme Mstll to cut the lahoratory. by usinga 'tuberculosis

RECOMBINANT DNA AND BIOTECHNOLOGY

- ^e adv en to f re c o m b i n a n tD N A te c h n o l ogy - - ' : ied a ne w c h a p te rfo r th e p ro d u c ti o nof a

Disorder

r : r ange o f th e ra p e u ti ca g e n ts i n s u ffi ci ent - - : - ir t ies f o r h u ma n u s e . T h e c o m m e rci al ., : citation of recombinant DNA (rDNA) iechnology began in late 1970s by a few : : ec hnolog i c acl o m p a n i e sto p ro d u c ep ro t ei ns. -:'e are at least 400 different proteins being : ' - luc ed ( by D N A te c h n o l o g yw ) h i c h m a y serve , , : r er apeut i ca g e n tsfo r h u m a n s .A s e l e c te dl i st - :rme important human proteinsproduced by ' - - r m binant D N A te c h n o l o g yp o te n ti a lfo r the - : aim ent of h u ma n d i s o rd e rs i s g i v e n i n Table 27.5. As of now, only a selectedfew of ^enr (around30) have been approvedfor human -se and the most important among these are . , , en in T abl e2 7 .6 . -:'1iit"$ru F.nggpffif,&ffi€"#g$ Diabet es m e l l i tu s i s c h a ra c te ri z e d by icreased blood glucose concentration (hyper. r c em ia) whic h o c c u rs d u e to i n s u ffi c i e nror ' r ef f ic ientins u l i n . In th e e a rl y y e a rs , i n s u l i n rsolatedand purified from the pancreasesof pigs and cows was used for the treatment of severe diabetics. This often resulted in allergies. Rec om binantD N A te c h n o l o g y h a s b e c o me a boon to diabetic patients.

Attemps to produce insulin by recombinant DNA technologystartedin late 1970s.The basic technique consisted of inserting human insulin gene and the promoter gene of lac operon on to the plasmidsof E. coli. By this method human i n s ulin was pr o d u c e d . l t w a s i n J u l y 1 9 8 0, seventeenhuman volunteerswere, for the first ti m e, adm inis t e re d re c o m b i n a n t i n s u l i n fo r treatmentof diabetesat Cuy's Hospital,London. And in f ac t , i n s ul i n w a s th e fi rs t e v e r phar m ac eut ic a p l ro d u c t o f re c o mb i n a n t D N A te c hnology h u m a n s. a d mi n i s te re d to R ec om binantins u l i nw o rk e d w e l l , a n d th i s g a v e hope to scientiststhat DNA technologycould be successfullyemployed to produce substancesof

607

Recombinantprotein(s)

Anemia

Gihma. AiGroscierosi; Delivery " ' Biodii;iotJ' Burns C;iie/

Emphysema Femaleinfertility Freeradicaldamage

lTilTHryI

Growthdefects

Hearlattacks

rgrypr'itigl Hemophilia B Hepatitis B Hypoalbuminemia lmmunedisorders Kidneydisorders LouGehrig's disease

growth Growlhhormone, hormonereleasing f actor, somatomedin-C

Prourokinase Faclor Vlll

Firdlx Heptiiii" a ;;;i;; Serum albumin Interleukins, growth B-cell factors Etyiilp;letin' Brain-derived neurotropic

s.clerosis) factor JlTyllgpllg lglgfgl Muttro! 991ej9i" 1ii19rt9191s 1u,B;ti

Nsrye grs$l 9eleqs . lsryg B9l9l o_'ieqneleqiq 9_elsilslll Pain Rheumatic disease

Ulcers Viralinfections

Endorphins and enkephalins Adrenocorticotropic hormone

Urogastrone (a, F,y) Interferons

1- tr

596 . Nested PCR . Inverse PCR

B IOC H E MIS TRY

18 i n fol l i cul ar l ymphoma) i nvol vi ng kn own genes are identified by PCR.

PCR in sex determination of embryos : Sex of human and live stock embryos fertilized in vitro, Reversetranscription PCR (RT-PCR) can be determinedby PCR,by using primersand AsymmetricPCR DNA probes specific for sex chromosomes. Real-timequantitativePCR Further,this technique is also useful to detect sexlinked disordersin fertilized embryos. Random amplified polymorphic DNA (RAPD)

. Anchored PCR . o . .

. Amplified fragment length polymorphism (AFLP)

PGR in DNA sequencing

. Rapid amplificationof cDNA ends (RACE).

As the PCR technique is much simpler and quicker to amplify the DNA, it is conveniently used for sequencing.For this purpose, singlestrandsof DNA are required.

APPLICAT'ONS OF PCR

The advent of PCRhad, and continuesto have tremendousimpact on molecular biology. The PGR in comparative studies applicationsof PCR are too many to be listed of genomes here. Some of them are selectively and very The differences in the genomes of two briefly described.Other applicationsof PCRare can be measuredby PCRwith random organisms discussedat appropriateplaces. primers. The products are separated by electrophoresisfor comparative identification. PGR in clinical diagnosis Two genomesfrom closely relatedorganismsare The specificityand sensitivityof PCRis highly expectedto yield more similar bands. useful for the diagnosisof various diseasesin PCR is very important in the study of humans. These include diagnosis of inherited hiology, more specifical ly referred evolutionary disorders (genetic diseases), viral diseases, to as phylogenetics. As a technique which can bacterial diseasesetc. amplify even minute quantitiesof DNA from any Prenatal diagnosis of inherited diseases : source (hair, mummified tissues,bone, or any PCR is employed in the prenatal diagnosisof fossilizedmaterial),PCR has revolutionizedthe inh e ri te d d i s e a s e sb y u s i n g c hori oni c vi l l us studies in palaentology and archaelogy. The samples or cells from amniocentesis.Thus, movie 'Jurassic Park', has created public diseaseslike sickle-cell anemia, p-thalassemia awarenessof the potential applications of PCR! and phenylketonuriacan be detectedby PCR in these samples. PGR in forensic medicine Diagnosis of retroviral infections : PCR from cDNA is a valuable tool for diagnosis and monitoring of retroviral infections, e.g., HIV infection.

A single molecule of DNA from any source (blood strains,hair, semenetc.) of an individual is adequatefor amplificationby PCR.Thus, PCR is very importantfor identificationof criminals.

Diagnosisof bacterial infections : PCR is used for the detection of bacterial infections e.g., tuberculosis by Mycobacterium tuberculosis.

The reader may refer DNA finger printing technique describedlater in this chapter.

GE N E LIB R A R IE S Diagnosisof cancers: Severalvirally-induced cancers(e.g.,cervical cancer causedby human The collection of DNA fragments (specifically papilloma virus) can be detected by PCR. genes)from a particular speciesrepresents gene Further, some cancers which occur due to libraries. The creation or construction of chromosomaltranslocation(chromosome14 and gene libraries (broadly genomic libraries) is

594 The future of DNA chips The majorlimitationof DNA chipsat present is the unavailability of completegenomearrays higher eukaryotes, includinghumans.lt is for within the next few years such expectedthat will Thiswill help the DNA chips be available. biotechnologiststo capture the functional of the genomein action for higher snapshots organisms.

BIOGHEMISTFIY

that can withstandat a 4. A DNA polymerase temperatureup to 95oC(i.e.,thermostable). The actual technique of PCR involves repeatedcyclesfor amplificationof targetDNA. Eachcycle hasthree stages. 1. Denaturation: On raisingthe temperature to about 95oCfor about one minute,the DNA getsdenaturedand the two strandsseparate.

2. Renaturation or annealing : As the temperatureof the mixutre is slowly cooled to (Dl{A AlrtPLrFtCATrONl about 55oC,the primersbase pair with the complementaryregionsflanking target DNA The polymerasechain reaction(PCR)is a or strands.This processis called renaturation laboratory (in vitro\ technique for generating of primerensures High concentration annealing. Iarge quantitiesof a specifiedDNA. Obviously, annealingbetweeneach DNA strandand the PCR is a cell-free amplification technique for primer ratherthan the two strandsof. DNA. multipleidenticalcopies(billions) synthesizing of any DNA o{ interest.Developedin 1984 by : The initiationof DNA synthesis 3. Synthesis Karry Mullis (Nobel Prize, "1993),PCR is now occurs at 3'-hydroxylend of each primer. The consideredas a basic tool for the molecular primers are extendedby joining the bases biologist. As is a photocopier a basic complementaryto DNA strands,The synthetic in an office,so is the PCRmachine process in PCR is quite comparableto the requirement in a molecularbiologylaboratory! DNA replicationof the leading strand (Refer Chapter24). However, the temperaturehas to Principle of PCR be keptoptimalas requiredby the enzymeDNA polymerase.For Iaq DNA polymerase,the The double-strandedDNA of interest is optimumtemperatureis around75'C (for E. coli denatured to separate into two individual DNA polymerase,it is around 37"C). The strands.Eachstrandis then allowedto hybridize reaction can be stopped by raising the with a primer(renaturation). Theprimer-template (to about 95'C). temperature duplex is usedfor DNA synthesis(the enzymeDNA polymerase). These three stepsThe 3 stagesof PCRin relationto temperature denaturation, renaturation and synthesis are and time are depictedin Fi9.27.15.Eachcycle repeatedagain and again to generatemultiple of PCRtakesabout3-5 minutes.ln the normal formsof targetDNA. practice,the PCRis carriedout in an automated machine. Technique of PGR As is evidentfrom the Fi9.27.16(cyclel), the The essential requirements for PCRare listed new DNA strandjoinedto eachprimeris beyond below the sequencethat is complementaryto the secondprimer.Thesenew strandsare referredto 1. A targetDNA (100-35,000bp in length). as long templates,and they will be used in the (synthetic primers of secondcycle. 2. Two oligonucleotides 17-30 nucleotides length) that are Forthe secondcycleof PCR,the DNA strands complementaryto regionsflanking the target (original+ newlysynthesized longtemplate) are DNA. denatured,annealedwith primersand subjected (dATP,dCTP, to DNA synthesis. At the end of secondround, 3. Four deoxyribonucleotides dGTP,dTTP). long templates, and short templates (DNA POTYIIERASE CHAIN REACTION

ChAPTET27 : FIECOMBINANT DNA AND BIOTECHNOLOGY

Denaturation (1 min)

Variations

of PCR

The basic technique of PCR has been described. Being a verqatiletechnique, PCR is modified as per the specific demands of the situation.Some of the variantsof PCRare listed

90 e- 80 5

t

!u

i7n

a'" E

,0)

60

Y

2345 Time(minutes)

E 1

strandswith primer sequenceat one end, and sequence complementary to the other end primer) are formed. I n t he t hir d c y c l e o f PC R ,th e o ri g i n a l D NA strandsalong with long and short templatesare the starting materials. The technique of denaturation/ renaturation and synthesis are repeated.This procedure is repeatedagain and again for each cycle. lt is estimatedthat at the end of 32nd cycle of PCR, about a million-fold target DNA is synthesized.The short templates possessingpreciselythe target DNA as doublestrandedmoleculesaccumulate. of DNA polyanerase

ln t he or igi n a l te c h n i q u e o f P C R , Kl e n ow fragmentof E. coli DNA polymerasewas used. This enzyme, gets denatured at higher temperature, therefore, fresh enzyme had to be added for each cycle. A breakthroughoccurred (Lawyer1989)with the introductionof Iaq DNA polymerase from thermophilic bacterium, Thermus aquaticus. The lag DNA polymerase is heat resistant, hence it is not necessary to freshly add this enzyme for each cycle of PCR.

f

orunsvntnesis -

Fig. 27.15 : The three stages in each cycle ot PCR in relation to temDerature and time (Each cycle takes approximately &5 minutes).

Sources

595

Original strand

Original strand

t-

Long template

C Y L E 2

I L

Longtemplate

U

rtE 3

Fiq.27.16 : The polymerase chain reaction (PCR)

representingthe initialthreecycles (, -' indicateprimers).

BIOGHEMISTF|Y

590 requirementin biotechnology.DNA sequencing is important to understandthe functions of genes, and basis of inherited disorders.Further, DNA cloning and gene nianipulation invariably require knowledge of accurate nucleotide sequence.

DNA Double-stranded

J into4 tubes Distributed

IIilAXAM

AND GI'.BEBT

TEC'IN'QUE

The first DNA sequencingtechnique, using chemical reagents, was developed by Maxam and Gilbert (1977). This method is briefly describedbelow (Fi9.27.10).

.//

\\ tr r t)

A strandof sourceDNA is labeledat one end with 32P. The two strands of DNA are then separated.The labeled DNA is distributedinto four samples(in separatetubes).Each sample is subjected to treatment with a chemical that specifically destroysone (C, C) or two bases (A + G, T + C) in the DNA. Thus,the DNA strands are partiallydigestedin four samplesat sitesC, A + C, T + C and C. This resultsin the formation of a seriesof labeledfragmentsof varyinglengths. The actual lengthof the fragmentdependson the site at which the base is destroyed from the labeled end. Thus for instance,if there are C residuesat positions4, 7, and 10 away from the labeled end, then the treatment of DNA that specificallydestroysC will give labeledpiecesof length 3, 6 and 9 bases. The labeled DNA fragmentsobtained in the four tubes are subjected to electrophoresisside by side and they are detected by autoradiograph.The sequenceof the basesin the DNA can be constructedfrom the bandson the electrophoresis.

e

A +G

(Specificbasesdestroyedandfragmentsformed) separated I Fragments by electrophoresis I

+ G

A +G

T +C

C

T

c A G

c G T

c A T A

Shorter Bandson autoradiograph

DIDEOXYNUCLEOT'DE METHAD Currently, the preferred technique for determiningnucleotidesequencein DNA is the one developed hy Sanger (1980). This is an enzymatic procedure commonly referred to as chain the dideoxynucleotide method or termination method (Note : FredrickSangerwon Nobel prize twice, once for determining the structureof protein, insulin; the secondtime for s e q u e n c i n gth e n u c l e o ti d e si n a n R N A vi rus).

strand ATACTGCGACT Sequenced strand TATGACGCTGA Complementrary

at both the 2' and 3' carbonsof the sugar (Fig.27.11). This is in contrastto the natural deoxyribonucleotidethat possessesat 3' hydroxylgroupon the sugar.

Terminationrole of dideoxynucleotide: ln A dideoxynucleotide is a laboratory-made group normal processof DNA replication,an the chemical molecule that lacks a hydroxyl

B IOC H E MIS TRY

610 v ac c i n ec o n s i s tso f a g e n ee n c o d ingan anti 8eni c pr o te i n , i n s e rte d o n to a p l a s mi d, and then inco rp o rte di n to th e c e l l s i n a ta rgetani mal .The pla s m i d c a rry i n g D N A v a c ci ne normal l y c on ta i n s a p ro m o te r s i te , c l o n i ng si te for the DN A v a c c i n e g e n e , o ri g i n o f repl i cati on, a s el e c ta b l ema rk e r s e q u e n c e (e .9. a gene for am p i c i l l i nre s i s ta n c ea)n d a te rm inatorsequence ( a p o l y -A ta i l ).

N asal spray i nj ecti on Intramuscul ar Intravenousi nj ecti on Intradermali nj ecti on C ene gun or bi ol i sti c del i very (i nvolves pressuredel i veryof D N A -coatedgol d be ads) .

D N A v a c c i n e -o l a s mi d sc a n b e admi ni stered A n i l l ustrati on of a D N A vacci ne and t o th e a n i m a l sb y o n e o f th e fo l low i ng del i very the mechani sm of i ts acti on i n developing methods.

trLIruEAL GONCEPTS EIOF/IEDICAL,l

!it

iig

Biotechnologgis o newly discouereddiscipline t'or oge'old practices(e.g. preparation of curd, wine, beer), with special emphasison genetic manipulations. the characteristics Humon ortit'icialchromosome(HAC) is o syntheticuectorlpossessing of human chromosome.HAC is capable of carrying large-sizedhuman genes that may be useful in gene theropy. Southern blotting technique (that specit'ically detects DNA) is employed for the identit'icationof thieues,ropists,ond settlementof parenthood.

t. Polymerose chain reaction is useful Jor the diognosis ot' inherited diseases,tn DNA sequencing,and in forensic medicine. By employing site-directed mutagenesis,it is possible to produce more efficient ond more suitableenzVmesfor theropeuticond industrial purposes. The analysisol genetic material DNA (gene/genes)is employed lor the diognosisof certain diseoses,and in medical Jorensicse.g. AIDS, sickle-cellanemia, certain cancers, DNA fingerprinting. The pharmaceuticol products of rDNA technology haue reuolutionizedthe treatment of certain diseosese.g. diabetes, asthma, atherosclerosis,heart attacks, hemophilio. Recombinant uaccinefor hepatitis B is the lirst synthetic uaccine.lt is effectiue,sat'e and producesno allergic reactions. is o nouelconcept.lt hasbeenshown that Geneticimmunizationby using DNA uaccine.s the immune response(humoralond cellular)of the body csn be stimulatedby a DNA molecule. Transgenicmice thot serueas animal models t'or human diseoseshaue been deueloped. These include human mouse (model t'or immune system), Alzheimer's mouse, oncomouse (model lor cancer), prostate mouse, knockout mice (fo, allergg, tronsplantationetc.). Transgeniconimals serueas bioreactorst'or the production ot' therapeuticallyimportont proteins e.g. interferon, Iactot'errin,urokinose. t-..-

Certain pet animals (cats, dogs)are being cloned by some companies.

3FlatrGer 27 : RECOMBINANT DNA AND BIOTECHNOLOGY

r.r*Lnrt\ is given in Fig.27.29. The plasmid ili4.cne carrying the DNA (gene)for antigenic :r:tein enters the nucleus of the inoculated ?rrE€t cell of the host. This DNA produces #"1\{ and in turn the specific antigenic ;rEfein- The antigen can act directly for m eloping hum o ra li mmu n i tyo r a s fra g me n tsi n I ity class as*sociation with major histocompatabi \ r HC) m olec u l e s fo r d e v e l o p i n g c e l l u l ar 'TrmunrW.

Humoral immunity \s the antigen molecules bind to Blrmphocytes, they trigger the production of antibodies which can destroy the pathogens. Some of the B-lymphocytesbecome memory cells that can protect the host against future rnfections.

6tl

lmportance of transgenic animals-general Transgenesis has now becomea powerfultool for studying the gene expression and developmental processesin higher organisms, besides the improvement in their genetic characteristics. Transgenicanimalsserveas good models for understandingthe human diseases. Further,severalproteinsproduced by transgenic ani mal s are i moortant for medi cal and pharmaceuti cal appl i cati ons. Thus, the transgenic farm animals are a part of the lucrative world-wide biotechnology industry, with great benefitsto mankind.

TRANSGEruAG NfiTCH ANF TffiHEffi&PPLflGAYE@NS

Mouse, although not close to humans in its biology, has been and continuesto be the most Cellular immunity exploited animal model in transgenesis The proteinfragmentsof the antigenbound to experiments.The common featurebetweenman \{HC molecules can activate the cytotoxic and mouse is that both are mammals.Transgenic T-lvmphocytes.They are capable of destroying mice are extensivelyused as animal models for infected pathogenic cells. Some of understandi nghuman di seases,and for the $€ tfre activated T-lymphocytes become memory productionof therapeuticagents.Adequatecare, cells which can kill the future infecting however,must be exercisedbeforeextrapolating cathogens. data of transgenicmice to humans.

With the advent of modern biotechnology,it is now possibleto carry out manipulationsat the geneticlevel to get the desiredcharacteristics in animafs. Transgenesisrefers to the phenomenon ol introduction of exogeneous DNA into the tenome to create and maintain a stable lrcritable character. The foreign DNA that is introducedis called transgene.And the animal whose genome is alteredby adding one or more transgenes is said to be transgenic. The transgenes behavelike other Benespresentin the animals' genome, and are passed on to the o r t s pr ings . T h u s , tra n s g e n i c a n i m a l s are genetically engineered or genetically modified organisms (GMOs) with a new heritable character.

Mouse models for several human diseases (cancers, muscular dystrophy, arthritis, A l zhei mer' s di sease, hypertensi on, al l ergy, coronary heart disease, endocrine diseases, neurodegenerative disorders etc.) have been develooed. A selected few of them are l i sted. The human mouse,the transgenicmouse that di spl ayshuman i mmune system. The Alzheimer's mouse to understand the pathologicalbasis of Alzheimer'sdisease. The oncomouse,the animal model for cancer. The prostate mousetthe transgenic mouse to understand prostate cancer. The knockout mice, (developedby eliminating specific genes) for certain diseases e.g. SCID mouse/ knockout mouse for transpl antati on.

o\ l9

YY YY

Memory B-lvmphocvte (protecis ag'airistf ufure infection)

Antibodies Antigen

HUMORALIMMUNITY CELLULAR IMMUNITY

Fragments of antigen

Kill pathogenic cells T-lymphocyte boundto antigen

Cellular Nucleus DNA lnoculated cell

Activatedcytotoxic T-lymphocytes \-)

MemorycytotoxicT-lympfocyte. againstfutureinfection) @rotect6

m

d o

I m 6 -l ll

Cfiapter 27 : RECOMBTNANT DNA AND BTOTECHNOLOGY A}IIHAL

613

BIOREACTORS

Transgenesis is wonderfully utilized for r,roductionof proteins for pharmaceuticaland -'edical use. In fact, any protein synthesizedin :re human body can be made in the transgenic a:rirnals, provided that the genes are correctly c'rogrammed.The advantage with transgenic animals is to produce scarcehuman proteinsin huge quantities.Thus, the animals'servingas for production of biologially faories inprtant products are referred to as inimal t*reaclors or sometimespharm animals. Some transgenicanimals that serve as bioreactorsare listed . Transgenic cow for the lactoferrin and interferons.

(ae) \ \_-.//

Mammary glandcell

Ovumwithnucleus

I

I riu"oay.

I nutrient Jdeprivation

J--@

production of

. Transgenic Boat to synthesize tissue plasminogen activator,and antithrombin fll.

Dormant totipotentcell

Enucleatedovum

. Transgenic mouse for the production o! im m unog l o b u l i n sa, n d u ro k i n a s e . . Transgenicpig to produce hemoglobin.

//-\)

r ( a) t

\\7

Dolly, the first ever mammal clone was developedby Wilmut and Campbell in 1997. lt is a sheep (female lamb) with a mother and no hther. The technique primarily involves nuclear transfer and the phenomenon of totipotency. The character of a cell to develop into different cells, tissues,organs,and finally an organismis referred to as totipotency or pluripotency. Totipotency is the basic character of embryonic cells. As the embryo develops, the cells specializeto finally give the whole organism.As such, the cells of an adult lack totipotencv. Totipotency was induced into the adult cells for xeloping Do//y. -n: cloning of sheep for producing Dolly, ilu-s:ated in Fig.27.30, is briefly described here. *ire rammary gland cells from a donor ewe m,ere isolated. They were subjected to total n-rb.ientdeprivation (starvation)for five days. Bv : s process,the mammary cells abandon their

(mammary cer.,I"YffSfif;ii ovum enverope) I n vitro J embryoculture

Embryo

Fig. 27.30 : The cloning of sheep for developing Dolly.

614 normal growth cycle, enter a dormant stage and regaintotipotencycharacter.An ovum (eggcell) was taken from another ewe, and its nucleus was removed to form an enucleated ovum. The dormantmammarygland cell and the enucleated ovum were fused by pulse electricity. The mammary cell outer membrane was broken, allowing the ovum to envelopethe nucleus.The fused cell, as it had gained totipotency, can multiply and develop into an embryo. This embryo was then implanted into another ewe which served as a surrogatefostermother. Five months later, Dolly was born.

BIOCHEMISTRY

2. Strong proponents who consider that the biotechnology will provide untold benefits to society. They argue that for centuriesthe society has safely used the products and processesof biotechnology. 3. A neutral group of people who have a balancedapproach to biotechnology. This group believes that research on biotechnology (with regulatory systems),and extending its fruits to the society should be pursued with a cautious approach. BENEFITS

OF BIOTECHNOLOGY

As reported by Wilmut and Campbell, they The fruits of biotechnology are beneficial to fused277 ovum cells, achieved'13 pregnancies, the fiefds of healthcare, agriculture, food and of theseonly one pregnancyresultedin live production, manufaclure of industrial enzymes birth of the offspring-Dolly. and appropriateenvironmentalmanagement. It is a fact that modern technologyin various forms is woven tightly into the fabric of our lives. Our day-to-day life is inseparable from Someof the companiesinvolved in transgenic technology.lmagine life about 1-2 centuriesago experimentshave startedcloning pet animalslike where there was no electricity,no runningwater, cats and dogs. Little Nicky was the frrst pet cat sewage in the streets,unpredictable food supply that was cloned at a cost of $50,00 by an and an expected life span of less than 40 years. Americancompany (in Dec. 2OO4).More cloned U ndoubtedly, technology has largely contributed cats and dogs will be made available to to the present day world we live in. Many interestedparties (who can afford) in due course. pepople considerbiotechnologyas a technology that will improve the quality of life in every Some people who own pet animals are country, besidesmaintaining living standardsat interestedto continue the same pets which is a reasonablyhigher level. possible through cloning. There is some opposition to this approach as the cloned ELSI OF BIOTECI{NOLOGY animals are less healthy, and have shorter life Why so much uproar and negativity to span, besidesthe high cost factor. biotechnology?This is mainly becausethe major part of the modern biotechnology deals with genetic manipulations.These unnatural genetic manipulations,as many people fear, may lead to Advances in biotechnology, and their unknown consequences. applications are most frequently associatedwith controversies. Based on their perception to biotechnology,the people may be grouped into three broad categories.

ELSfis the short form to representthe ethical, Iegal and social implications of biotechnology. ELSI broadly covers the relationship between biotechnology and society with particular referenceto ethical and legal aspects.

1. Strong opponents who oppose the new technology,as it will give riseto problems,issues Risks and ethics of biotechnology and concernshumans have never faced before. They consider biotechnology as an unnatural The modern biotechnologydealswith genetic manipulativetechnology. manipulations of viruses, bacteria, plants,

\,

:-;!*ien

27 : RECOMBINANT DNA AND BIOTECHNOLOGY

. - - : 5, f is h a n d b i rd s . In tro d u c ti o no f fo re i gn : = - = . int o v a ri o u s o rg a n i s m sra i s e s c o n c e rns : ,- -: the safety, ethics and unforeseen : : - ; equenc es.So meo f th e p o p u l a rp h ra s e su sed - --= w h i l e re fe rri n gto e x p e ri me n tson - r edia = . -- - oinant D N A te c h n o l o g ya re Ii s te d .

615

gui del i nes for research deal i ng w i th D N A manipulation were very stringentin the earlier years.

So far, risk assessment studieshave failed to demonstrateany hazardouspropertiesacquired by host cells/organismsdue to transfer of . ' , ' anipulat io no f l i fe DNA. Thus, the fears of genetic manipulations . : ar ing Cod may he unfounded to a large extent. Consequently,there has been some relaxationin . ' , 1a: t - m ade e v o l u ti o n the regulatoryguidelinesfor recombinantDNA -re g e n eti c m ajo r a p p re h e n s i o n o f research. = - . r eer ing is th a t th ro u g h re c o mb i n a n tD N A It is now widely acceptedthat biotechnology i, : €r im ent s , u n i q u e m i c ro o rg a n i s m os r v i ru ses : ' . r ' er inadv e rte n tl yo, r s o m e ti me sd e l i b e ra tel y i s certai nl ybenefi ci alto humans.B ut i t shoul d -:- the purposeof war) may be developedthat not cause problems of safety to people and , - d c aus e e p i d e mi c s a n d e n v i ro n m e ntal environment, and create unacceptable social, -:::sirophes. Due to these fears, the regulatory moral and ethical issues.

I. Recombinant DNA (rDNA) technology is primarily concerned with the manipulation ol genetic material (DNA) to achieuethe desired goal in a pre-determined way. 2. The procedure t'or rDNA technology inuoluesmolecular tools (enzymese.g. restriction endonucleases), hosf cells (E. coli, S. cerevisiae),uectors(plasmids,bacteriophages), gene tronst'er(transformation,electroporation)snd the strqtegiesof gene cloning. 3. Blotting techniquesare employedt'or the identification of desired DNA (southern blot), RNA (Northern blot), and protein (Western blot). 4. Polymerasechain reaction is on in vitro technique for generoting large quantities oJ a specified DNA i.e. cell-t'reeamplit'ication 5 Gene librories or genomic libraries representsthe collection of DNA t'ragments(i.e. genes)from a genome of a particular species. 6. Site-directedmutogenesisis fhe techniquelor generating amino acid coding changesin the DNA (gene)to produce a desired protein/enzgme. 7. Analysis ol DNA (i.e. detection ot' gendgenes)can be used as a diagnostic systemt'or the detection of many pathogenic and genetic diseosese.g. tuberculosis,maloria, AIDS, sickle-cellanemia, certain concers. 8 DNA fingerprinting or DNA prot'iling is the present day genetic detectiuein the practice ol modern medical forensics. Four types of DNA markers are used in DNA fingerprinting-RFlFs, VNTRs, STRs, ond SNPs. 9. Many pharmaceuticalcompoundsoJ health importance (for diseasetreatment)are being produced by rDNA technologye.g. insulin, growth hormone, intert'erons,erythropoietin, hepatitis B uaccine. I0. Transgenicanimals can be deuelopedby introducing a t'oreign DNA (transgene).These animols are genetically modified or engineered with new heritable characterse.g. oncomouse,knockout mouse,prostate mouse.

h

ri 3 3 F

BIOCHEMISTF|Y

616

[. Essayquestions 1. 2. 3. 4. 5.

Describethe basic principlesunderlyingthe recombinantDNA technology. Give an accountof the nucleicacid blottingtechniques.Add a note on their importance. Describethe polymerasechain reactionalong with its applications. Write brieflyon the utility of DNA in diseasediagnosisand medicalforensics. productsof DNA technology. Cive an accountof the pharmaceutical

III. Short notes (c) Methodsof genetransfer,(d) Purification (a) Restriction (b) Plasmids, of nucleic endonucleases, acids,(e) Westernblotting,(fl DNA sequencing,(d DNA chips (h) Cene libraries,(i) Restriction (j) Recombinant vaccines. fragmentlengthpolymorphisms, III. F i l l i n th e b l a n k s 1. The most commonlyusedprokaryotichost in rDNA technologyis 2. Northernblottingtechniqueis usedfor the detectionof 3. Namethe blottingtechniquein which nucleicacids(DNA or RNA)are directlyblottedonto the filterswithout electrophoresis 4. The bacterialsourceof the enzyme laq DNA polymerase,that is widely used in polymerase chain reaction 5. The collection of DNA fragmentsfrom the genome of a particular species represents 6. The techniquefor generatingamino acid coding changesin the DNA (gene)is regardedas 7. The trade name for insulinproducedby rDNA technology 8. The first syntheticveccinedevelopedby rDNA technology to representhumans 9. The mostcommonlyusedanimal model in transgenesis 10. Name the first ever mammmalthat has been cloned IV . Mu l ti p l e c h o i c e q u e s ti ons nicks in DNA 11. One of the following enzymeproducessingle-stranded (a) DNA ligase(b) DNA polymerase(c) DNaseI (d) Sl nuclease. 12. Westernblottingis the techniquefor the identificationof (a) DNA (b) RNA (c) Carbohydrates (d) Proteins. 13. The DNA markersused in the diagnosisof diseases and DNA fingerprinting (c) Single (b) Minisatellites (a) Restriction and microsatellites, fragmentlengthpolymorphisms, (d) Any one of the above. nucleotidepolymorphisms, productof recombinantDNA technologyapprovedfor human use 14. The first pharmaceutical (a) Insulin(b) Growth hormone(c) Interferon(d) HypatitisB vaccine. of 15. Cenetic immunizationinvolvesthe administration (a) Antigens(b) Antibodies(c) DNA (d) RNA.

Biological Membranes and Transport Free Radicalsand Antioxidants

Uancer Acquired lmmunodefic Syndrome (AIDS)

617

Hum

oject

involvingresearchgroupsfrom six countriesUSA, UK, France,Germany,Japanand China, and severalindividuallaboratories and a large number of scientistsand techniciansfrom variousdisciplines.This collaborativeventure was named as International Human Genome Seguencing Consortium UHGSA and was THE BIRTH AND ACTIVITY OF headedby Francis Collins.A totalexpenditure of HUMAN GENOME PROJECT $3 billion,and a time periodof 10-15 yearsfor The human genomeproject WGn was the completionof HCP was expected.A second conceived in 1984, and officially begun in humangenomeprojectwas set up by a private earnestin October 1990. The primary objective company- CeleraGenomics, of MarylandUSA of HCP was to determine the nucleotide in 1998.Thisteamwas led by CraigVenter. he most important features of a DNA molecule are the nucleotide sequences,and the identificationof genes and their activities. Since 1920, scientistshave been working to determinethe sequencesof pieces of DNA.

sequence of the entire human nuclear genome. In addition, HGP was also entrustedto elucidate the genomesof severalother model organisms e.g. Escherichia coli, Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (roundworm), Mus musculus (mouse). James Watson (who elucidatedDNA structure)was the first Director of HCP .

Announcement of the draft sequence of human genome

The date25th lune200Owill be remembered as one of the mostimportantdatesin the history of scienceor evenmankind.lt was on this day, FrancisCollinsand CraigVenter,the leadersof the two humangenomeprojects,in the presence fn 1997, United States established the of the President of U.S.,jointly announcedthe National Human Genome Research Institute workingdraftsof humangenomesequence. The (NHCRT).The HCP was an internationalventure detailedresultsof the teamswere laterpublished 619

EiIOCHEMISTFIY

620

Gene

I

-

l'r, n cene tinkagemap Gene

fragments Restriction

4. Physical map : This is the ultimate map of the chromosome with highest resolution base sequence. Physical map depicts the location of the active genes and the number of bases between the active genes.

Restriction fragment A$}trROAC}IES map

FffN #5' !* OME 9E A U E N C IN G

l

| | | I | | | | | | | | | | | | | | | I | | | i I | | i i1l I I I lil I I I i r ii Physicalmap Base sequence

Fig. 28.1 : Different types of genome maps.

A list of different methods used for mapping of human genomes is given in Table 28. l. These techniquesare also useful for the detection of in humans. normal and disease.genes

For elucidating human genome, different approacheswere used by the two HCP groups. in February2001 in scientific journals Nature IHGSC predominantly employed map first and (IHGSC)and Science(CeleraGenomics). sequencelater approach.The principal method heirarchical shotgun sequencing. This was The human genome project results attracted involves fragmentationof the Eenome technique worldwide attention. This achievement was fragments (100-200 kb), inserting small into hailed with many descriptionsin the.media. them into vectors (mostly bacterial artificial o The mysteryof life unravelled. chromosomes,BACs) and cloning. The cloned fragmentscould be sequenced. . The library of life. . The periodic table of life. . The Holy grail of human genetics. M A P P IN G

OF T H E H U MA N

GE N f} FdE

The most important objective of human genome project was to constructa seriesof maps for each chromosome.ln Fi9.28.1, an outline of the different types of maps is given.

Celera Genomics used whole genome shotgun approach. This bypassesthe mapping step and savestime. Further,Celera Sroup was lucky to have high-throughput sequenators and powerful computer prcgrammes that helped for the early completion of human genome sequence. Whose

genome

was

sequenced?

One of the intriguing questions of human genome project is whose Senome is being 1. Cytogenetic map : This is a map of the sequencedand how will it relateto the 6 billion chromosomein which the active genesrespond or so populationwith variationsin world? There to a chemical dye and display themselvesas is no simple answer to this question' However, bands on the chromosome. looking from the positive side, it does not matter 2. Gene linkage map : A chromosomemap whose genome is sequenced, since the in which the active genes are identified by phenotypic differences between individuals are locating closely associatedmarker genes. The due to variationsin just 0.1% of the total genome most commonly used DNA markers are sequences.Thereforemany individual genomes restriction fragment length polymorphism can be used as source materialfor sequencing. (RFLP), variable number tandems repeats Much of the human Benome work was (VNTRs) and short tandem repeats (STRs). performedon the materialsuppliedby the Centre VNTRs are also called as minisatelfites while for Human Polymorphismin Paris,France.This STRs are microsatellites. institute had collected cell lines from sixty 3. Restriction fragment map : This consistsof different French families, each spanning three the random DNA fragments that have been generations.Thus, the material supplied from Pariswas used for human genome sequencing. sequenced.

.r t

621

Chapter 2a : HUMAN GENOMEPROJECT H U MA N GE N OME S E QU E N C E R E S U LTS S U MMA R IS E D

Method

Comments

DNAsequencing

Physicalmapof DNAcan be identified withhighest resolution.

Useof probes

RFLPS, To identify STSand SNPs.

genome intolarge hybridmappingFragment Radiation pieces markers andlocate somatic andgenes. Requires cellhybrids. Tolocalize a geneon Fluorescence rh srlu (FISH) chromosome. hybridization Applicable to anypartol DNA taggedsite Sequence (STS)mapping if somesequence sequence is available. information A variant of STSmapping; sequence Expressed genesareactually expressed tag (EST)mapping mapped andlocated. gel Fortheseoaration and Pulsed{ield (PFGE) isolation of largeDNA electrophoresis fragments. of To isolateDNAfragments in vectors Cloning phages, (plasmids, lengths. variable YACs, BACs) cosmids, Toamplifygenefragments Polymerase chain reaction(PCR)

walking Chromosome

iildffi;ffiidpins

forcloning of Uselul DNAfragments overlapping (restricted to about200kb).

DNA ffi 6;uiil6 his; for fragments andcircularized

;i.yrodilii; :';'#iilft1t#ill; D;Gt'"

abnormalities

the by cloning be identified genese.g.Duchenne affected muscular dystrophy.

Databases

facilitate Existing dalabases geneidentification by comoarison of DNAand protein sequences.

The information on the human genome projects is too vast, and only some highlights can be given (Iable 28.2). Some of them are briefly described.

. Thedraftrepresents about90%of the entirehuman genome. pafts lt is believed thatmostof theimportant havebeenidentified. r Theremaining 10%ofthegenome sequences areatthe (i.e.telomeres) veryendsof chromosomes andaround thecentromeres. . Human genome iscomposed of3200Mb(or3.2Gb)i.e. 3.2billion basepairs(3,200,000,000). . Approximately 1.1to 1.5%of the genome codesfor oroteins. . Approximately 24o/o ol thetotalgenome is composed of (exons), introns thatsplitthecoding regions andappear as repeating sequences withnospecific functions. . Thenumber genesis in therangeof of protein coding 30,000-40,000. r An average geneconsists of 3000bases,the sizes geneis the largest however varygreatly. Dystrophin genewith2.4million known human bases. . Chromosome (the 1 largest human chromosome) contains the highest number whilethe Y of genes(2968), chromosome hasthelowest. Chromosomes alsodifferin theirGCcontent andnumber of transoosable elements. . Genesand DNAsequences associated with many diseases suchas breastcancer, musclediseases, havebeenidentified. deafness andblindness . About100codingregions appear to havebeencopied (retroand movedby BNA-basedtransposition transposons). . Repeated sequences constitute about 50%ofthehuman genome. o A vastmajority (- 57"/")hasno known of thegenome functions. r Betweenthe humans,the DNAdiffersonlyby 0.2o/o or onein 500bases.

. Morethan3 million polymorphisms singlenucleotide

(SNPs)havebeenidentified. (RFLP-Restrbtion STS-Sequence . Human tngnentlenglhpolymorphisn; DNAis about987oidentical to thatof chimpanzees. polynorphism; YAC-Yeast nucleotide hgged site;SNP-.Slngle o genes About 200 to that foundin bacteria. are close aftilicialchronosome) BAC-Bacterial artiticial chromosone:

622

B IOC H E MIS TFI Y

,^ ^5 \ t E t^ , - r g

! d^ ,l - i u qEfrt

flana

, ^ ;u t F^i!t-d. rt d rcnaa<

1200llir

Fiq.28.2 : An overuiewof the organizationof human genome (LlNEs-Longinterspersednuclear elements; SINEs-Short interspersed nuclear elements; LTR-Long terminal repeats).

Most of the genome is id e n ti fi e d

sequence

About 90% of the human genome has been s equ e n c e d .l t i s c o m p o s e do f 3 .2 bi l l i on base pair s (3 2 0 0 Mb o r 3 .2 C b ). l f wri tten i n the format of a telephone book, the base sequence of h u ma n g e n o m e w o u l d fi l l about 2OO telephonebooksof 1000 pageseach. Someother interesting analogshidelights of genome arc given in Table 28.3. lndividual differencesin genomes: lt has to be r e me mb e re dth a t e v e ry i n d i v i dual , except iden ti c a l tw i n s , h a v e th e i r o w n versi ons of genome sequences. The differences between individualsare largely due to single nucleotide polymo rphisms (SNPs).SN Ps representpositions in t he g e n o mew h e re s o me i n d i v i d u al shave one nucleotide(i.e. an A), and othershave a different nuc le o ti d e (i .e . a C ). T h e frequency of occurrenceof SNPs is estimatedto be one per 1000 b a s e p a i rs . A b o u t 3 mi l l i on S N P s are believed to be oresentand at least half of them have been identified. O r ga n i z a ti o n

of human

g e n ome

A n o u tl i n e o f th e o rg a n i z a ti o no f the human genome is given in Fig.28.2.Of the 3200 Mb,

.

in humangenome wouldfillabout Thebasesequence 200telephone booksof 1000pageseach.

.

lf the genomeis recitedat the rateof onebaseper to secondfor 24 hoursa day,it wouldtakea century recitethe bookof life. ll a typisttypesat the rateof 60 wordsper minute (i.e.360letters) for 8 hoursa day,he/shewouldtake around50 yearsto typehumangenome. lf theDNAsequence is typedin lines10cmcontaining the humangenome 60 nucleotide basesandprinted, (froma singlecell)wouldstretcha distance sequence of 5000km. lf theDNAin theentirehumanbodyis putendto end, it wouldreachto the sun and backover600 times (Note: Thehumanbodycontains 100trillion cells;the between lengthof DNAin a cellis 6 feet;thedistance thesunandearthis 93 millionmiles).

a

for humangenomeprojectwas Thetotalexpenditure hasto be billion. The magnitude ofthishugeamount $3 at a non-stop rateof appreciated. lf onestartscounting a dollarper second,it wouldtakeabout90 yearsto comolete.

623

:-aot er 28: HU M AN G E N OMEPF OJ EC T

T

1

End of biological information

Start of biologicalinformation

Fig. 28.3 : A diagrammatic representation of a typical structure of an average human gene.

-,nll' a small fraction (48 Mb) representsthe :ctual genes,while the restis due to gene-related and intergenic r€quences(introns,pseudogenes) f,\A (long interspersednuclear elements,short nterspread nuclear elements, microsatellites, DNA transposons etc.). Intergenic DNA representsthe parts of the genome that lie betlveen the genes which have no known function. This is appropriatelyregardedas junk D NA . Genes

present

in human

genome

The two genome projects differ in their estimates of the total number of genes in humans.Their figuresare in the rangeof 30,00040,000 genes.The main reasonfor this variation is that it is rather difficult to specifically recognizethe DNA sequenceswhich are genes and which are not.

Human

genes

encoding

proteins

It i s now cl ear that onl v 1.1-1.5% of the human genome codes for proteins. Thus, this figure 1 .1-15% representsexons of genome. As already described,a huge portion of the genome is composedof introns, and intergenic sequences(j unk D N A ). The major categoriesof the proteinsencoded by human genes are listed in Table 28.4. The functions of at least 40'h ol these oroteins are not known. BENEFITS/APPTICATIONS OF I{ U MA N GE N OME S E QU E N C IN G It is expectedthat the sequencingof human genome/ and the genomes of other organisms w i l l dramati cal l ychangeour understandi ng and perceptionsof biology and medicine. Some of the benefitsof human genome project are given.

Before the results of the HCP were announced,the best guessof human geneswas . ldentification of human Benes and their in the range of 80,000-100,000. This estimate functions. was basedon the fact that the numberof proteins i n hum an c ells is 8 0 ,0 0 0 -10 0 ,0 0 0 , a n d th u s s o many genesexpected.The fact that the number '.de.s.@t^ of genesis much lower than the proteinssuggests that the RNA edifing (RNA processing) is v.s" widespread,so that a singlemRNA may code for *os 17.s"k more than one orotein.

d..:;""t''o""*

A diagrammatic representationof a typical structureof an averagehuman gene is given in Fi9.28.3.lt has exons and introns. A broad categorizationof human genecatalog in the form of a pie chart is depicted in Fig.28.4. About 17.5"/oof the genes participate in the g ener albioc hem i c a fu l n c ti o n so f th e c e l l s ,2 3 % i n t he m aint ena n c eo f g e n o me , 2 1 % i n s i g n al tra ns duc t ion wh i l e th e re ma i n i n g 3 8 % a r e involved in the productionof structuralproteins, transportproteins,immunoglobinsetc.

=.^!o,9"

\

o<

Do

Fig. 28.4 : A pie chart showing a broad categorization of the human gene catalog (About 13000genes whose functions are not known are not included),

ll

B IOC H E MISTFI Y

624 U n d e rs ta n d i n go f p o l y g e ni c di sorders cancer, hypertension,diabetes. lmprovementsin gene therapy

lmproved diagnosisof diseases Developmentof pharmacogenomics Cenetic basis of psychiatricdisorders Understandingof complex social trait lmproved knowledge on mutations i ng of developmentalbiology Betteru nderstand

Category of proteins

Percentage Actual number of genes

functions 41.0% Unknown Nucleic acidenzymes 7.5o/o Transcription factors 6.0% Receptors 5.0% 4.07o Hydrolases proteins Regulatory 3.2To (G-proteins, cellcycle regulators etc.) Protooncogenes 2.91o proteins of 2.8o/o Structural cytoskeleton Kinases 2.87o

12,809 2,308 1,850 1,543 1,227 988

902 876 868

(Note: Thistableis basedon the roughdraftof humangenome reportedby CeleraGenomics.Thepercentages are derivedlron a totalof 26,383genes)

'l

Comparativegenomics Developmentof biotechnology .-; {'{ii-;

;+r'.lu;r**Ufu€AfCSfr},l*f!r'i.

The researchon human B enomesw ill m ake very sensitivedata availablethat will affect the personaland pri vate l i ves of i ndi vi du als.For instance,once it is known that a personcarries genes for an incurable disease,what would be the strategyof an insurancecompany?How will the society treat him/her?There is a possibility that individuals with suhstandard genome sequences may be discriminated. Human genome results may also promote racial discriminationcategorizingthe people with good and bad genome sequences.Considering the gravity of ethics related to a human genome, about 3% of the HCP budgetwas earmarkedfor ethical research.

Human Genome Project is an internotional uenture inuoluing seueralloboratories,and o large number of scientists and technicians t'rom vorious disciplines.

2. About 900/oof the human genome hos been sequenced.lt is composedol 3.2 billion base patrs.

3. The total number of genes in the humans is in the ronge ol 30,00040,000. 4. About 7.7-7.50/oot' the human genome codeslor proteins while the remoining portion is regorded as junk DNA (composedof introns ond intergenic sequences). 5. Human genome sequencing has wide range ol applications4etter understanding ol genetic diseoses,improuements in gene therapy, deuelopment of pharmacogenomics, and oduancementof biotechnology.

igl,,lg "l-lfnmruutpV

. .-:_ 1r,-;r: ;i Ffrrl . T,+i Ffi i t!,rri r dv anc es in b i o c h e mi s try a n d m o l e c u l ar biology have helped to understand the There are two approachesto achieve gene genet ic bas is o f i n h e ri te d d i s e a s e s .l t w a s a therapy. to replacethe defective dream of the researchers genes with good ones, and cure the genetic 1 . Somatic cell gene therapy : fhe nond isorders. reproductive (non-sex) cells of an organism are Cene therapy is the process of inserting referredto as somaticcells.Theseare the cells of genes into cells to treat diseases.The newly an organi smother than spermor egg cel l s,e.g., i nt r oduc ed gen e s w i l l e n c o d e p ro te i n s a nd bone marrow cel l s, bl ood cel l s, ski n cel l s, correct the deficienciesthat occur in genetic intestinalcells. At present,all the researchon d is eas esT. hus , g e n e th e ra p y p ri m a ri l y i n v o l ves gene therapy is directed to correct the genetic genetic manipulations in animals or humans fo defectsin somaticcells. In essence,somaticcell correct a disease,and keep the organism in good gene therapy involves the insertion of a fully healt h.T he initi a l e x p e ri m e n tso n Be n eth e ra py functional and expressible gene into a target a r e c ar r iedout i n a n i m a l s ,a n d th e n i n h u ma ns. somatic cell to correct a genetic disease Obviously, the goal of the researchersis to permanentl y.

benef itt he m an k i n d a n d i m p ro v e th e i r h e a l th. An overview of gene therapy strategiesis depicted in Fi9,29.1. ln gene augmentation therapy, a DNA is inserted into the Benome to replacethe missinggene product.In caseof gene inhibition therapy, the antisense gene inhibits th e ex pr es s iono f th e d o mi n a n t g e n e .

2. Germ cell gene therapy : The reproductive (sex) cells of an organism constitute germ cel l l i ne. Gene therapy i nvol vi ng the introductionof DNA into germ cells is passedon to the successivegenerations.For safety,ethical and technical reasons,germ cell gene therapy is not being attempted at present.

625

]!

' ii' lllir r ' r l !

626

ElIOCHEMISTFIY The genetic alterationsin somatic cells are not carried to the next generations.Therefore, somatic cell gene therapy is preferred and extensivelystudiedwith an ultimateobjectiveof correctinghuman diseases. A large numberof geneticdisordersand other diseasesare currently at various stagesof gene therapy trials. A selectedlist of some important ones is given in Table 29.1. There are two types of gene therapies. Inhibitoryaction

Fig. 29.1 : Overview of two major gene therapy strategies (A) Gene augmentation therapy (B) Gene inhibition therapy.

l. Ex vivo gene therapy : This involves the transfer of genes in cultured cells (e.9., bone marrow cells) which are then reintroducedinto the oatient.

Disease (SCID) immunodeficiency Severecombined Cysticfibrosis Familial hypercholesterolemia Emphysema Hemophilia B Thalassemia Sickle-cell anemia Lesch-Nyhan syndrome Gauche/s disease Peripheral arterydisease Fanconi anemia Melanoma Melanoma, renalcancer (braintumor), AIDS,ovarian Glioblastoma cancer Headandneckcancer Breastcancer AIDS

Gene therapy

(ADA). Adenosine deaminase (CFIR). regulator fibrosis transmembrane Cystic (LDL) lipoprotein receptor. Lowdensity o.,-Antitrypsin Factor lX o- or B-Globin 9-Globin (HGPRT). phosphoribosyltransferase Hypoxanthine-guanine Glucocerebrosidase growth (VEGF) factor Vascular endothelial Fanconi anemia C (TNF) Tumor necrosis factor (lL-2) Interleukin-2 (herpes kinase virus) Thymidine simplex 053

resistance I Multidrug rev andenv

Colorectal cancer, melanoma, renalcancer Duchenne muscular dystrophy

(HLA-87) Histocompatability locusantigen-B,

Shortstature*

Growthhormone

Diabetes*

(GLUT-2), glucokinase transporter-2, Glucose

Phenylketonuria*

Phenylalanine hydroxylase

Citrullinemia*

Arginosuccinate synthetase

* Mostlyconfinedto animalexpetinents

Dystrophin

Chapter 29 : GENE THERAPY

627

ll. In vivo gene therapy zThe direct delivery of genes into the cells of a particulartissueis referredto as in vivo gene t her apy .

The ex vivo gene therapy can be applied t o on l y s e l e c te dti s s u e s(e .g ., oone m ar r o w ) w h o s e c e l l s c a n b e cultured in the laboratory. The technique of ex vivo gene t her apy inv o l v e s th e fo l l o w i n g s te p s (Fi9.29.2). 1. lsolate cells with eenetic defect from a patient.

p80d (COCz

nO /v

OO vvA

,rf)

tO 6

\

generrc oelecl

-^^^L,)

Genetically transformed cells selected Fig. 29.2 : The procedure for ex vivo gene therapy.

V'RUSES

2. Cr ow t h e c e l l s i n c u l tu re .

The vectors frequently used in gene therapy are viruses,particularlyretroviruses.RNA is the 3. Introducethe therapeuticgene to correct genetic materialin retroviruses. As the retrovirus gene defect. enters the host cell, it synthesizesDNA from 4. S elec t th e g e n e ti c a l l y c o rre c te d c el l s RNA (by reversetranscription). The so formed (stabletransformants) and grow. viral DNA (referred to as provirus) gets 5. T r ans p l a n t th e mo d i fi e d c e l l s to the i ncorporatedi nto the D N A of the host cel l . The provi ruses are normal l y harml ess. H ow ever, patient. there i s a tremendousri sk, si nce some of the The procedure basically involves the use of retrovi rusescan convert normal cel l s i nto the patient's own cells for culture and genetic cancerous ones. Therefore, it is absolutery correction, and then their return back to the essenti alto ensurethat such a thi ng does not patient. This technique is therefore, not happen. as s oc iat ed w i th a d v e rs e i m m u n o l o gi cal responsesafter transplanting the cells. Ex vivo HUMAN ARTIFICIAL CHROMOSOME gene therapy is efficient only if the therapeutic The human arti fi ci al chromosome (H A C ) gene (remedialgene) is stably incorporatedand i s a syntheti cchromosomethat can reol i cate continuouslyexpressed.This can be achievedby w i th other chromosomes, besi des encodi ng use of vectors. a human protei n. A s al ready di scussed above, use of retroviruses as vectors in VECTORS IN GENE THERAPY gene therapy is associatedwith a heavy risk. The carrier particles or molecules used to Thi s probl em can be overcomei f H A C i s useo. deliver genes to somatic cells are referred to as S ome success has been achi eved i n thi s vectors.The important vectors employed in ex di recti on. vivo gene therapy are listed below and briefly describednext. BONE MARROW CELLS . Viruses . Hum an ar t ifi c i a lc h ro mo s o m e r B one m ar r o w c e l l s .

Bone marrow contains totipotent emhryonic stem (ES) cells. These cells are capable of dividing and differentiating into various cell types (e.g., red blood cells, platelets, macro-

B IOC H E MIS TR Y

628 phages, osteoclasts, B- and T-lymphocytes). For this reason,bone marrow transplantationis t he m o s t w i d e l y u s e d te c h n i q u e for several genetic diseases.And there is every reason to believethat the geneticdisordersthat respondto bone marrow transplantation are likely to respondto ex vivo gene therapyalso e.g. sicklec ell an e mi a ,SC ID ,th a l a s s e m i a .

has been depicted in Fi9.29.2. The same procedurewith suitable modificationscan also be applied for other gene therapies. A pl asmi d vector beari nga provi ral D N A is selected.A part of the proviral DNA is replaced by the A D A gene and a gene (C 418) codi ngfor anti bi oti c resi stance,and then cl oned. The gene w i l l hel p to sel ectt he anti bi oti cresi stance desi redcl onesw i th A D A gene. A diagrammatic representation of the treatmentof ADA deficientpatient is depicted in Fig.29.3.

'

r ** r-:*i ;qi.r \

;-=t.)i,A-r ;4 it,t

l1'i i.;..i ; :'j ?

rJ -:-; ..}';l1i;'ts-f* {i H * E :':j: '.1:I [.i"] i-' : T he ffrs t a n d th e mo s t p u b l i ci sed human gene therapy was carried out to correct the deficiency of the enzyme adenosinedeaminase ( A DA ). T h i s w a s d o n e o n Se ptember 14, 1990 by a team of workers led by Blaese and Andersonat the National Instituteof Health, USA (Thegirl's name is Ashanti,4 yearsold then). ii:;,;,

, r.!,,.,

a,,",,,,:i,-t.,'s,;'.'

+:3"

: i

';

. 1

'"1

'r

Circulating lymphocytesare removed from a patient suffering from ADA deficiency. These cells are transfectedwith ADA gene by exposing carryi ngthe sai d gene. to bi l l i onsof retrovi ruses The genetically-modifiedlymphocytesare Erown i n cul turesto confi rm the expressi onof A DA gene and returned to the patient. These l ymphocytes persi st i n the ci rcul ati on a nd the abi l i ty of the A D A . C onsequentl y, synthesi ze pati ent to produce anti bodi es i s i ncreased. However, there is a limitation.The lymphocytes have a short life span (just live for a few months), hence the transfusionshave to be carried out freouentl v.

S C ID i s ra re i n h e ri te d i m m u ne di sorder associated with T-lymphocytes, and (to a lesserextent) B-lymphocytesdysfunction.About 5O'h of SCID patientshave a defect in the gene (located on chromosome 20, and has 32,000 bas e p a i rs a n d 1 2 e x o n s ) th a t e ncodes for In 1995, ADA gene was transferred into aden o s i n ed e a mi n a s eIn . th e d e fi c i encyof A D A , the stem cel l s,obtai nedfrom the umbi l i calco r d deoxy a d e n o s i n ea n d i ts m e ta b o l i tes(pri mari l y bl ood, at the ti me of baby' sdel i very.Four da ys 5'-triphosphate) accumulateand after birth, the infant receivedthe modified cells deoxyadenosine destroy T-lymphocytes. T-Lymphocytes are back. B y thi s w ay, a permanentpopul ati onof es s en ti a l fo r b o d y ' s i m m u n i ty. B esi des A D A gene produci ngcel l s w as establ i shed . participating directly in body's defense, they promote the function of B-lymphocytes to produce antibodies.Thus, the patientsof SCID (lackingADA) suffer from infectious diseasesand die at an young age. Previously,the children rhe direct delivery of the therapeutic gene sufferingfrom SCIDwere treatedwith conjugated (DNA) into the target cellsof a particulartissue bovine ADA, or by bone marrow transplantation. of a patient constitutes in vivo gene therapy (Fig.29.Q. Many tissues are the potential for thi s approach.Thesei ncl udel i ver , candi dates muscl e,ski n,spl een,l ung,brai n and bl ood cells. The generalschemeof gene therapy adopted Cene deliverycan be carriedout by viral or nonfor introducing a defective gene in the patient viral vector svstems.The successof in vivo gene

Ghapter2g

629

: GENE THEHAPY

Vector DNA

Human ADA'gene Retrovirus containingADA+ gene

with Lymphocytes viralDNAandADA+gene Childwith SCID (ADA- gene)

\

r

___._ _______--. _=InfuselYmPhocYtes withADA-gene into expresston patient

Cellculturesto verifyexpression of ADA- transgene

Fig.2g.g : Treatmentof adenosinedeaminase (ADA) deficientpatient by somatic ex vivo gene therapy (SCtD-Severe combined immunodeficiency)'

therapy mostly depends on oarameters

the following

. The efficiency of the uptake of the remedial (therapeutic)gene by the target cells. . lntracellulardegradationof the gene and its uptake by nucleus.

retroviruses, adenoviruses, adeno-associated virusesand herpessimPlexvirus'

GENE DELIVERYBY NON'VIRAL SYSTEMS

There are certai n l i mi tati ons,i n usi ng vi ral vectors in gene therapy. In addition to the . The expressioncapability of the gene. prohibitive cost of maintaining the viruses,the viral proteins often induce inflammatory GENE DELIVERY BY VIRUSES responses in the host. Therefore, there is a by researchers to find Many viral vector systems have been continuous search systems. vector viral developed for gene delivery' These include alternativesto

B IOC H E MIS TR Y

630

Target tissue

,...-fplr.

treatment strategies (surgery, chemotherapy, radiationtherapy).Cene therapy is the latestand a new approach for cancer treatment. Some of the developments are briefly described hereunder. Turnor

necrosis

factor

gene therapy

Tumor necrosis factor (TNF) is a protein TNF provides producedby human macrophages. defenseagainstcancer cells. This is brought out by enhanci ng the cancer-fi ghti ngabi l i ty of tumor- infiltrating lymphocytes (TI Ls), a speciaI type of i mmune cel l s.

Therapeutic gene

The tumor-infiltrating lymphocytes were transformed with a TNF gene (along with a Patienl neomycin resistant gene) and used for the representation of invivo gene treatment of malignant melanoma (a cancer of Fi,.29.4: Diagrammatic thempy.(p-Promotergenespecificfor therapeutiegene) mel ani nproduci ngcel l s,usual l yoccursi n ski n ) . TNF as such is highly toxic, and fortunatelyno toxic side effectswere detected in the melanoma The non-viralgene delivery systemsare listed patients injected with genetically altered TlLs . Pure DNA construcfs that can be directly with TNF gene.Someimprovementin the cancer patientswas observed. introduced into targettissues. .

Lipoplexes,lipid-DNA complexes that have DNA s u rro u n d e db y l i p i d l a y e rs .

S ui cE de gene therapy

.

Human artificial chromosome which carry large DNA (one or more genes).

The gene encoding the enzyme thymidine kinaseis often referredto as suicide gene, and is used for the treatmentof certain cancers.

can

Thymi di ne ki nase (TK ) phosphoryl ates nucleosidesto form nucleotideswhich are used for the synthesi sof D N A duri ng cel l di vi si on. The drug ganciclovir (GCV) bears a close of leading cause death Cancer is the structural resemblanceto certain nucleosides despite the intensive throughout the world,

GEI{E THERAPY STRATEGIESFOR CANCER

BIOMEtrItrAL/ GLINIGATCONcETT]E

n9

Theoreticallg,gene theropy is the permanent solution Jor genetic diseases.

09

A large number ot' genetic disordersand other diseosesare at uariousstoges of gene therapg trials e.g. sickle-cellanemia, cystic t'ibrosis,AIDS, cancer. Ganciclouir(o drug with structural resemblanceto thymidine) has been used (suicide gene theropy) lor the treatment ot' broin tumors, olthough with limited success. Despite extensiuereseorchond triols, as ol now, no diseasehas been permanently cured by gene theropy. Howeuer, a breakthrough may come at anytime.

ehapter ?9 : GENE THEHAPY

631

---'

DNAsynthesis

ENucleotide

. Inhibits , DNA ' potymerase

-

DNAsynthesis blocked

I

+

Cancer cells die F19.29.5 : The action of ganciclovir mediated by thymidine kinase to inhibit the grov,tthof cancer cells.

(thymidine). By mistake, TK phosphorylates ganciclovir to form triphosphate-GCV,a false and uns ui ta b l en u c l e o ti d efo r D N A s ynthesi s. Triphosphate-GCV inhibits DNA polymerase (Fi9.29.5).The resultis that the elongationof the DNA molecule abruptly stops at a point containing the false nucleotide (of ganciclovir). Further,the triphospate-Ccv can enter and kill t he neighb o u ri n gc a n c e r c e l l s , a p h e n omenon referred to as bystander effect. The ultimate r es ult is t h a t th e c a n c e r c e l l s c a n n o t mul ti pl y, and thereforedie. Thus,the drug ganciclovircan be used to kill the cancer cells.

Some workers have tried to reolace the damaged ps3 gene by a normal gene by employing adenovirusvector systems.There are some encouraging results in the patients with l i ver cancer. The antisensetherapyfor cancer is discussed as a part of antigeneand antisensetherapy.

In general, gene therapy is carried out by introducing a therapeuticgene to produce the defective or the lacking protein. But there are certain disorders (cancer, viral and parasitic infections, inflammatory diseases)which result in an overproductionof certain normal proteins. It is possibleto treat these diseasesby blocking transcriptionusing a single-stranded nucleotide Gene replacement therapy sequence (anti gene ol i gonucl eoti de) that hybridizes with the specific gene, and this is A gene named f3 codes for a protein with a called antigene therapy. Antisense therapy refers m olec ularw e i g h t o f 5 3 k i l o d a l to n s(h e n cep53). to the inhibition of translation by using a singlep53 is consideredto be a tumor-suppressorgenel strandednucleotide (antisenseoligonucleotide). s inc et he pr o te i ni t e n c o d e sb i n d sw i th D N A and Further, i t i s al so possi bl e to i nhi bi t both inhibit s r ep l i c a ti o n .T h e tu m o r c e l l s o f several transcriptionand translationby blocking (with tissues(breast,brain, lung, skin, bladder, colon, oligonucleotides) the transcription factor bone) were found to have altered genesof ps3 responsbilefor the specific gene expression. (mutated p53), synthesizing different proteins Nucleic acid therapy refers to the use of DNA from the original. Thesealtered proteinscannot inhibit DN A re p l i c a ti o n .l t i s b e l i e v e dthat the or RNA molecules for therapeutic purposes, as damagedps3 gene may be a causativefactor in statedabove. The naturallyoccurring sequences of DNA and RNA (with suitable modifications) tumor development. Canciclovir is frequently referred to as a prodrug and this type of approach is called prodrug activation gene therapy. Ganciclovir has been usedfor treatmentof brain tumors (e.9., glioblas t o m a a , c a n c e r o f g l i a l c e l l s i n brai n), alt houghw i th a l i m i te d s u c c e s s .

J

632

BIOCHEM]STRY

(A) Fs.pill

broad senseto reflect antigenetherapy as well as antisense therapy, discussed in the previous paragraph).

Transcription

I 12 Primarytranscript

Antisense RNA

mRNA-antisense RNAcomplex 12

I

+

No translation

(B)

ANTISENSE TIIERAPY FOR CANCER Oncogenesare the genes responsiblefor the causation of cancer. The dominantly acting oncogenes can be targeted in antisense technology by using antisense transgenesor oligonucleotides.Antisenseoligonucleotidesare usedfor the treatmentof myeloid leukemiain as earl y as 1991. AntisenseRNA moleculesare more frequently used in cancer therapy. This approach is effective only if the antisenseoligonucleotide (antisense mRNA) specifically binds to the target mRNA, and blocks protein biosynthesis (translation). This can be achieved in two ways, as illustrated in Fig.29.6. The anti sensecD N A can be cl oned and transfected into cells. Antisense mRNA is synthesizedby transcription.This can readily bi nd w i th the speci fi c mR N A and bl o ck translation (Fig.29,6A).The mRNA is actually formed by a gene containingexons and introns through transcription,followed by processing.

Antisense RNA

12

mRNA-antisense RNAcomplex

II

J

No translation Fig. 29.6: lnhibitionof translationby antisenseRNA (A) TheclonedAS aDNAintroducedintocells to ptoduce antisenseRNA(B) AntisenseRNA

The other way to block translation is to directly introduce antisenseRNA into the cells. This hybridizes with target mRNA and blocks translation (Fig,29.68). The antisensemRNA therapywas tried for the treatment of a brain tumor namely malignant glioma and the cancer of prostategland. In case mal i gnantgl i oma,the protei ni nsul i n-l i kegroM h factor | (lCF-l)is overproduced,while in prostate cancer,insulin-likegrowth factor I receptor(lCFlR) protein is more synthesized.For both these cancers,the respectiveantisensecDNAs can be used to synthesizeantisensemRNA molecules. These in turn, are used to block translation,as briefly described above, and illustrated in Fig.29.6.

or the syntheticones can be employed in nucleic acid therapy. Theoretically, there is a vast potentialfor use of nucleic acids as therapeutic agents.But most of the work that is being carried THE FUTURE OF GENE THERAPY out relates to the use of RNA in antisense therapy. Some of these are described below Theoreticalfy, gene therapy is the permanent (Note : Some authorsuse antisensetherapy in a solution for genetic diseases. But it is not as

533

Srao ter 29: G ENE THEFAPY

; -rple as it appears since gene therapy has ! e\ er al inb u i l t c o mp l e x i c i ti e s .C e n e t herapy :,roadly involves isolation of a specific gene, -laking its copies, inserting them into target :lssuecellsto make the desiredprotein.The story does not end here. lt is absolutelyessentialto ensurethat the gene is harmlessto the patient and it is appropriatelyexpressed(too much or too little will be no good). Another concern in genetherapyis the body's immune systemwhich reactsto the foreign proteins produced by the new 8enes.

involving various gene therapies.Unfortunately, the gene therapistsare unable to categorically claim that gene therapy has permanentlycured any one of these patients!Some people in the media (leading news papers and magazines) have openly questionedwhether it is worth to continue researchon gene therapy!!

It may be true that as of now, gene therapy due to severallimitations,has not progressed the way it should, despite intensiveresearch.But a breakthrough may come anytime, and of course, this is only possiblewith persistentresearch.And The public, in general, have exaggerated a day may come (it might take some years)when expectationson gene therapy. The researchers, almost every diseasewill have a gene therapy, at least for the present, are unable to satisfy as one of the treatment modalities.And gene fhem. As per the records,by 1999 about 1000 therapy will revolutionize the practice of A m er ic ans h a d u n d e rg o n e c l i n i c a l trai l s medi ci ne!

7. Gene therapy is the processof inserting genes into cells to treat diseases.Somatic cell gene theropy, inuoluing the insertion ol an expressiblegene into somotic cells, is the prelerred approach. 2, Ex vivo gene therapy inuolues the transt'er of genes in cultured cells which are then reintroduced into the patient. The direct delivery of genes into the cells of o porticular fissue is regardedos in vivo gene therapg. 3. Gene therapy uros success/ully carried out in a patient of seuere combined immunodeficiency(cousedby the deliciency of the enzyme adenosine deaminose). 4. Antigene therapy inuoluesblocking of transcription (by antigene oligonucleotide)while in antisense theropy, translation is inhibited (bg antisense oligonucleotide). These opproachesare in the experimental sfogesJor the therapy ol cancer ond AIDS. 5. Although os of now, gene therapy hos not offered any permanent cure to ang human patients, a breakthrough may come anytime. And gene therapy mag reuolutionize the practice of medicine.

-r,

Ul

Bioinformatics

ioinformatics is the combination (or Q l) marriagel) of biology and information technology. Basically, bioinformatics is a recentlydevelopedscienceusing informationto understandbiological phenomenon. lt broadly involves the computationaltools and methods used to manage/ analyse and manipulate volumes and volumes of biological data.

managementand analysisof the data pertaining to DNA, RNA and protein sequences.As the biological data is being produced at an unprecedented rate, their management and interpretationinvariably requiresbioinformatics. Thus, bi oi nformati csnow i ncl udesmany other types of biological data. Some of the most imoortant ones are listed below

Bioinformaticsmay also be regardedas a part of the computational biology. The latter is concerned with the application of quantitative anal y ti c a lte c h n i q u e si n mo d e l i n g and sol vi ng problems in the biological systems. B ioin fo rma ti c si s a n i n te rd i s c i p l i nary approach requiring advanced knowledge of computer science,mathematicsand statisticalmethodsfor the understandingof biological phenomena at t he m o l e c u l a rl e v e l .

. Gene expressionprofiles

i{istory and relevtrnee of bioinformatics in

The term bioinformatics was first introduced 1 9 9 0 s . Ori g i n a l l y , i t d e a l t w i th the

. Protein structure o Protein interactions . Microarrays(DNA chips) . Functi onalanal ysi sof bi omol ecul es . D rug desi gni ng. B i oi nformati csi s l argel y (not excl usi vely)a discipline.Computersare in fact computer-based very essential to handle large volumes of biological data, their storageand retrieval. We have to accept the fact that there is no computer on earth (however advanced)which

634

635

BIOINFORMATICS

1. Creation of databases: This involves the i- ; - : ' e i n fo rm a ti o na, n d p e rfo rmth e fu ncti ons : : J i v i n g c e l l . T h u s a h i g h l y compl ex organizing, storage and management of the ' - ' : ' - : r ion te c h n o l o g yl i e s ri g h tw i th i n the cel l s biologicaldata sets.The databasesare accessible . - or g a n i s m. T h i s p ri ma ri l y i n c l u des the to researchers to know the existing information - . : - ) ll s g e n e s a n d th e i r d i c ta te s for the and submit new entries.e.g. protein sequence - . : - s r s b i o l o g i c a lp ro c e s s e a s n d b e h avi our. data bank for molecularstructure.Databases will be of no use until analysed. : : *, . i :# V ER &GE 2. Development of algorithms and statistics : I : -;.'-r.t{+:#ffiilfiA,TAGS This involves the develooment of tools and :- : nformaticscovers many specializedand . : : - c ed a re a so f b i o l o g y . Functionafgenomics : ldentification of genes . - : - - : r re s p e c ti v efu n c ti o n s .

resourcesto determine the relationshipamong the membersof large data sets e.g. comparison of protein sequence data with the already existing protein sequences.

3. Analysis of data and interpretation : The appropriateuse of components 1 and 2 (given above) to analyse the data and interpret the C'wnparativegenomics : For understanding resul tsi n a bi ol ogi cal l ymeani ngfulmanner.Thi s *. : . - : m e s o f d i ffe re n ts p e c i e so f o rg a ni sms. i ncl udes D N A , R N A and protei n sequences, protein structure,gene expressionprofiles, and D\{ microarrays : These are designed to bi ochemi calpathw ays. - , : : ; - ' : ih e l e v e l so f g e n ee x p re s s i o n i n di fferent - - : : r a ri o u s s ta g e so f d e v e l o p m e n tand i n BISII{FORTlf,ATEGS ltructural genomics : Predictionsrelated to :- - :- :^s of proteins.

)i-^^^^ut5cd5c5.

A l \| tr TH E IN TE R ITIE T r , ( edic a l i n fo rma ti c s : T h i s i n v o l ves the The internet is an intemational computer - . - : . e' nen t o f b i o m e d i c a l d a ta w i th speci al network. A computer network involves a group . - : ' - - : e to b i o m o l e c u l e s ,i n v i tro a s savsand of computers that can communi cate (usual l y - : . ir ia l s . over a telephone system) and exchange data between users. j,. "::.j,.j:.,jTg$8F S {#{ FrtF{9b3M.d,T'ff gS :'., It is the internet protocol (lP) that determines :;ormatics how the packets of information are addressed I comprises three components ,

-

-

BtoMEDtCAt / CL|NTCALCONCEpTS

Bioint'ormatics hos largelg benet'ited biologicol and medical sciences,porticulorly related to molecular biology and biotechnology.Some applicationsore listed : . Sequencingof macromolecules(proteins, DNA, RNA) . Human genome sequencing . Molecular modelling of biomolecules . Handling of uast biological data . Designing of drugs for the treatment of dtseases . Deuelopment of models for the lunctioning of cells, fissuesand organs As such, there is no field of biological science that is not benefited by bioinformatics.

ffi

^*

B IOC H E MIS TR Y

636

and routed over the network. To access the (hyperfext transferprotocol) that can identify the internet, a computer must have the correct protocol for communicationover www. hardware (modem/network card), appropriate software and permission for access to network. BIOLOGICAL DATABASES For this purpose, one has to subscribe to an The collection of the biological data on a internet service provider (ISP). computer which can be manipulatedto appear World wide web (www) : www involvesthe in varying arrangements and subsetsis regarded exchangeof informationover the internetusing as a database.The biological informationcan be a programmecalled browser. The most widely stored in differentdatabases.Each databasehas used browsers are Internet exolorer and its own website with unique navigationtools. Netscapenavigator. The biological databases are, in general, www works on the basisof Uniform resource publicly accessible. Selected examples of Iocator (URL) which is a document with a biological databases are briefly described unique address.URLs takes the format nttp.// (Table 30.1\.

Database(s)

Salient features

Primary nucleotidesequencedatabases GenBank (www.ncbi.nih.goviGeneBanU)

maintained bythe databases nucleotide sequence Provides (NCBI), USA. Information National Centerfor Biotechnology

databases Other nucleotidesequence UniGene (www.ncbi.nih. gov/UniGene/) Biology Genome (www.ncbi.nlm.nih.gov/Genomes/)

intheformof clusters, of GenBank Thenucleotide sequences genes areavailable. representing genomes is available. thecompleted about Theinformation

Protein sequencedatabase SWISS-PROT (www.expasy.ch/sport)

itsdomains of a protein, of thestructure thedescription Provides post-translational variants etc.lt hashigh modifications, structure, levelof andminimal withotherdatabases levelof integration redundancv.

Protein sequencemotif databases PROSITE

(www.expasy.ch/prosite/)

lt alsohas families anddomains. onprotein information Provides functions. patterns profiles and biological for sequences and

Macromolecular databases PDB (www.rcsb.org/pdb)

(3'D)structures tor3-dimensional database Thisis theprimary (determined X+ay andNMR by macromolecules ol biological studies).

Other databases KEGG (www. genome.ad.ip/kegg4

(KEGG) is a of Genes andGenomes Encyclopedia TheKyoto information on biomolecules withlatestcomouterised database pathways, provides details oninformation KEGG andcellbiology. linkswithgenes. andtheconnecting interacting molecules

637

Ghapter 3O : BIoINFoFIMATICS Nucleotide sequence databases The nucleotide sequencedata submitted by the scientistsand genome sequencinggroups is at t he d a ta b a s e s n a me l y C e n Ba n k, E MB L (EuropeanMolecular Biology Laboratory)and DDBJ(DNA Data Bankof Japan).There is a good coordination between these three databasesas t hey ar e sy n c h ro n i z e do n d a i l y b a s i s . Besides the primary nucleotide databases (referredabove), there are some other databases also to provide informationon genes,Benomes and ongoing researchprojects.

A P P LIC A TION S

OF B IOIN FOR MA TIC S

The advent of bioinformatics has revol uti oni zedthe advancementsi n bi ol ogi cal science.And biotechnologyis largely benefited by bioinformatics. The best example is the sequenci ngof human genome i n a record ti me w hi ch w oul d not have been possi bl ew i thout bioinformatics.A selectedlist of applicationsof bioinformaticsis given below. . S equencemappi ng of bi omol ecul es(D N A , R N A , protei ns).

ldentification of nucleotide sequences of ' Protein sequence databases functi onalgenes. Protein sequence databases are usually . Finding of sitesthat can be cut by restriction prepared from the existing literature and/or in enzymes. consultation with the experts. In fact, these databases represent the translated DNA o Predictionof functional gene products. databases. r To trace the evolutionarytrees of genes. of databases Molecular structure T he t h re e d i me n s i o n a l (3 -D ) s tru cturesof macromolecules are determined by X-ray c r y s t allog ra p hayn d n u c l e a rm a g n e ti cresonance ( NM R) . P D B a n d S C OP a re th e pri mary databases of 3-D structures of biological m olec ule s .

. For the prediction of 3-dimensionalstructure of proteins. . Mol ecul ar model l i ngof bi omol ecul es. . Designingof drugs for medical treatment. . H andl i ng of vast bi ol ogi cal data w hi ch otherwise is not possible.

. Development of models for the functioning Other databases vari ouscel l s,ti ssuesand organs. KEGG database is an important one that The above list of applicationshowever, may provides informationon the current knowledge of m olec u l a r b i o l o g y a n d c e l l b i o l o gy w i th be treatedas incomplete,since at presentthere special referenceto information on metabolic is no field in biological sciencesthat does not pathways,interactingmoleculesand genes. i nvol ve bi oinformati cs.

1.. Bioinformatics(a computer-baseddiscipline)representson alliance betweenbiology and information technology, 2. The storage,managementand interpretation of uast biological data inuarioblyrequires bioinformatics. 3. Bioinformatics comprises three components-creationof dota base, deuelopment of algorithms and statistics,and onolysis of dota ond interpretation. 4. Biological dotabases,containing the biologicol information, are publicly accessiblee.g. GenBank (www,ncbi.nih.gou/GeneBonk). 5. Bioinformatics has reuolutionizedthe aduancementsot' biological ond medical sciences e.g. sequencingof human genome.

etabolllsm ofXenobiotflas

The detoxilicdtioln

ll

+I

f Oxidation I Reduaion I Hydrclysis tl r | 'Y

I I I

ConjugationConju(

'"J

J

',

"l dealuith the nntabolisinof {ontCo compounds; ': lryr{rolysis Throaghoxidaiion,reduction, and mnju.gation, . xenabiotics inn nkble 7b conaen fonls;, , . For rheirffictiue elbn,ination from thehodg.",

an is continuously exposed to several f o re i g n c o mp o u n d s s u c h a s drugs, pollutants,food additives,cosmetics,pesticides etc. Certain unwantedcompoundsare produced in the large intestineby the bacteriawhich enter t he c ir c u l a ti o n . T h e s e i n c l u d e i n dol e from tryptophan, cadaverine from lysine, tyramine f r om t yro s i n e ,p h e n o lfro m p h e n y l a l a ni ne etc. In the normal metabolism of the body, certain was t e c o mp o u n d s(e .g .b i l i ru b i n )a re formed.A vast majority of the foreign compounds or the produced in the body, are unwantedsubstances, toxic and, therefore, they should be quickly eliminatedfrom the body. The term detoxication or detoxificafion refers to fhe series of biochemical reactions occurring in the body to convert the foreign (often toxic) compounds to non-toxic or less toxic, and more easily excretable forms. Detoxif ication-a

sgegrhs't

nrisnonner?

Detoxification is rather misleading, since sometimes a detoxified oroduct is more toxic

than the original substance(e.g. procarcinogens to carcinogens).lt appearsthat the body tries to get rid of a foreign substance by converting it into a more soluble (often polar), and easi l y excretabl e compound, and thi s may be sometimes associated with increaseo toxicity (e.g. conversionof methanol to formaldehyde). In recent years/ the term detoxification is replaced by biotransformation or metabolism of xenobiotics (Creek : xenos-strange, foreign) or simply metabolism of foreign compounds. Site of detoxification The detoxification reactionsare carried out mainly in the liver which is equipped with the enzyme machi nery. K i dney and other organ s may sometimes be involved. The products formed by detoxificationare mostly excretedby the kidneys,lessfrequentlyexcretedvia fecesor expi red ai r.

638

539

Chapter 31 : METABOLISMOF XENOBIOTICS [DETOXIFICATION] XENOBIOTICS

I

I PHASEI

Oxidation Reduction Hydrolysis

Acetic acid

C6H'CH2OH -_) Benzylalcohol

C6H5COOH Benzoic acid

C6HsCHO

I + Conjugation Conjugation tl

tl

J+ Excreted

----------)cH3cooH

Ethanol

Aldehydes : Aldehydes are oxidized produce the correspondingacids.

I

PHASEII

c2HsoH

Excreied

Fiq.31.1 : Phase I and phase ll reactions in the metabolism of xenobiotics.

B enzoi c aci d

C.CI3CHO --) Chloral

CCI3COOH Trichloroacetic acid

Amines and their derivatives : Alipahtic aminesare convertedto the correspondingacids, l i berati ngurea w hi l e aromati cami no aci ds are oxidized to phenols. RCH2NH2 ---r Aliphatic amine C6H5NH2 -----) Aniline

The metabolism of xenobiotics may be divided into two phases which may occur togetheror separately(Fig.3l.l).

----------)C6H5COOH

B enzal dehyde

R-COOH + NH2{O-NH2 Aliphatic acid Urea HO{6H4-NH2 p-Aminophenol

Aromatic hydrocarbons : Benzene may be oxidized to mono, di- and trihydroxy phenolsas shown below

Phase | : The reactions of Phase I are oxidation, reduction and hydrolysis. Phasell : Theseare the conjugationreactions, inv olv ing c o mp o u n d ss u c h a s g l u c u ro n i caci d, am ino ac i d s (g l y c i n e ), g l u ta th i o n e , s ul fate, acetateand methyl group. Generally, detoxification of a compound involvesphaseI as well as phasell reactions.For instance,oxidation followed by conjugation is the most frequent processin the metabolismof xenobiotics.

Benzene

Oxidation A large number of foreign substancesare detoxifiedby oxidation.These include alcohols, to sul furi caci d. aldehydes,amines, aromatic hydrocarbonsand s ulf ur c om p o u n d s . l n g e n e ra l , a l i phati c Drugs : Meprobamateis a tranquilizer. lt is compounds are more easily oxidized than oxidized to hydroxymeprobamateand excreted aromatic ones. i n uri ne.

sutrur compo""or, 3:;:T:'"[lT:H:::

Alcohols : Aliphatic and aromatic alcohols Peso undergo oxidation to form the corresponding Role of cytochrom€ ac ids . Most of the oxidation reactions of detoxificationare catalysedby monooxygenase CH3OH ---------) HCOOH or cytochrome P+so.This enzyme, also called Formicacid Methanol

-1

640

BIOCHEMISTF|Y

mixd ftnction oxidase,is associatedwith the llydrolysis microsomes.The usage P+so refers to the The hydrolysisof the bonds such as ester, absorptionpeak (at 450 nm), exhibitedby the glycoside and amide is important in the enzymewhen exposedto carbon monoxide. metabolismof xenobiotics.Severalcompounds Most of the reactions of cytochrome P45s undergo hydrolysis during the course of involve the addition of a hydroxyl group to their detoxification.These include aspirin, aliphaticor aromaticcompoundswhich may be acetanilide, atropine diisopropylfluorophosphate, representedas and procaine. RH+ O, + NADPH--) Salient

features

ROH+ HrO + NADP+

of cytochrome

P.5o

1. Multiple forms of cytochromeP45sare believedto exist, rangingfrom 20 to 200. At least6 specieshavebeenisolatedand workedin detail. 2. They are all hemoproteins,containing heme as the prostheticgroup.

cooH 'ococH3 Asplrln (acetylsalicylicacid) Acetanilide

{.o

liro (qFho)2PoF

3. CytochromeP45sspeciesare found in the Diisopropyl fluoro phosphate highestconcentration in the microsomes of liver. Hzo In the adrenalgland,theyoccur in mitochondria. AtropineS+fropic

4. The mechanismof action of cytochrome Pa56is complexand is dependent on NADPH.

Aniline+ Aceticacid

Hzo Procaine-!}

5. Thephospholipid-phosphatidylcholine is a constituentof cytochromePcsosystemwhich is Goniugation for the action of this enzyme. necessary

(qFbo)2Po(oH)+ HF Dialkylphosphate

acid+ Tropine p-Aminobenzoic acid+ Diethylaminoethanol

Severalxenobioticsundergodetoxificationby 6. CytochromeP45eis an inducibleenzyme. Itssynthesis is increasedby the administration of conjugationto producelesstoxic and/or more easilyexcretablecompounds.Conjugationis the drugssuch as phenobarbitol. processin which a foreign compound combines 7. A distinctspeciesnamelycytochromePaa6 with a substanceprducd in the hody. The pro(with absorptionpeak at 448 nm) has been cessof conjugationmay occur eitherdirectlyor studied. lt is specific for the metabolismof after the phaseI reactions.At least8 different polycyclic aromatic hydrocarbons,hence it conjugatingagentshave been identifiedin the is also known as aromatic hydrocarbon body. These are glucuronic acid, glycine, hydroxylase. cysteine (of glutathione),glutamine,methyl group,sulfate,aceticacid and thiosulfate. Beduction Glucuronic acid : Conjugation with A few examplesof detoxificationby reduction glucuronic acid is the mostcommon.Theactive are given. form of glucuronic acid is UDP-glucuronic c5H2oH(NOz)r-> C6H2OH(NO2)2NH2acid produced in the uronic acid pathway Picramicacid Picricacid (Chapter l3). The microsomalenzymesUDPCCI3.CH(OH)2 ------) CCI3CH2OH participatei n glucuronide glucuronyltransferases Trichloroethanol Chloral formation.A generalreactionof glucuronide csHsNO2 + C6H5NH2 conjugationis shown below (X-OH represents Nitrobenzene Aminobenzene xenobiotic).

(OETOXIFICATION) chapter 31 : MEIABOLISMOF XENOBIOTICS

X-oH + UDp-gtucuronic acio

641

R-X

u?P-glYcuro, nyltransf€rase-

+ GSH

YI

HXJGSH

XO-glucuronide+ UDP

transfar,ase

+

R-SG

Certain drugs (e.g. barbiturates) when administeredinduce glucuronyltransferase and this increasesthe glucuronideformation.

I rGlutamylGlutamate +-,1 transPePtidase + Cysteinylglycine

Clucuronic acid conjugationmay occur with compounds containing hydroxyl, carbonyl, sulfhydrylor amino groups.A few examplesof glucuronideconjugationare given here.

I CystainyF GlVcine{ OlVcinase R -S -C H 2-C H -C OOH NHz R-Cystelne AceiyCoA1l

) N-Aceryltransferase CoASH{/l v

R -S -C H 2-C H -C OOH Phenyl glucuronlde UDPBenzoicacid + Glucuronic+ acid UDP. Bilirubin+ Glucuronic--J acid

Flg. 91.2 : Role of glutathione in conjugation to fom mercapturic acid (R-X-A xenobiotlc; A$lrctutiltime).

Benzoyl r I lne glucuronide

Bilirubin-diglucuronide

Glycine : Many aromatic carboxylic acids (e.g. benzoic acid, phenylacetic acid) are conjugatedwith glycine.Hippuric acid is formed when glycine is conjugated with benzyl CoA. The excretion of hippuric acid (Greek : hipposhorse)was first reportedin 1829 in the urine of cows and horses. COS-CoA + H2N-CH2-COOH BenzoylCoA

N H -C OC I-13 Mercapturicacid

GlYclne

Phenylacetic acid+ Glycine-* Cholicacid+ Glycine-+

Glycocholic acid

Glutathione : The tripeptide glutathione (Clu-Cys-Gly),is the active conjugatingagent.A wide rangeof organic compoundssuch as alkyl or aryl halides, alkenes, nitro compounds and epoxides get conjugated with cysteine of glutathione. The formation of mercapturic acid is depicted in Fig.3l.2. The glutamate and glycine of glutathioneare removedand an acetyl group is added to the cysteineresidue. Glutamine : Phenylaceticacid is conjugated with glutamine to form phenylacetylglutamine. Conjugation with glutamine is, however, relativelyless important. Methyl group : The methyl group (-CH3) of S-adenosylmethionine is frequently used to methylatecertain xenobiotics.This is catalysed by the enzyme methyltransferase. S-Adenosylmethionine + X-OH

Hlppurlcacld (benzoylglycine)

phenylaceturic acid

Methyltransterase

+ XO-CH3 S-Adenosylhomocysteine

-tdl[

642

BIOGHEMISTF|Y

Thiosulfate : The highly toxic cyanides are Sulfate : The active form of sulfatelf ate (PAPAconjugated with thiosulfateto form less toxic 3'-phosphoadenosi ne 5-phosphosu participates in conjugation reactions and the thiocyanate. enzvme sulfotransferaseis involved in this Cyanide+ Thiocyanate + process. Several aliphatic and aromatic -+ Sodiumthiosulfate Sodiumsulfate compounds undergo sulfation. Detoxification by drugs : lt may be surprising to know that some drugs are administeredto + FAir detoxify foreign substances.The toxic effects of Phospho- certain metals such as arsenic, mercury and adenosylPhenyl phosphate cadminum could be overcome by administering sulfate BAL (British antilewisite).This compound was Acetic acid : Acetyl CoA is the active form of developed during the World War ll and was acetic acid that takes part in conjugating used as a detoxifying agent for certain war r e a c ti o n s . D ru g s s u c h a s s u l fani l ami de are poisons.The mechanismof action of BAL is not converted to acetyl derivatives. clearly known. lt is believed that BAL readily + AcetylCoA-+ combines with metals and gets easily excreted Sulfanilamide Acetylsulfanilamide + CoASH i nto uri ne. Sulfotransferase --------------if

.

Knowledge of the metabolism ol xenobiotics is essenfial for the understanding of toxicology, pharmacology and drug addiction. The bodV possessesthe capabilitg to get rid of the foreign subsfoncesby conuerting them into more easily excretobleforms. Detoxit'ication is not necessarily associatedwith the conuersion ol toxic into non-toxic compounds. For instance,methonol is metabolized to a more toxic formaldehyde. Detoxification primarily occurs in the liuer through one or more of the reactions, namely oxidation, reduction, hydrolysis and conjugation. British antilewisite (BAL), o compound deuelopedduring Second World Wsn urosused to detoxify certain uror poisons.

Crapter 31 : METABOLISMOF XENOBIOTICS IDETOXIFICATION]

r.

Detoxiflcation deals with the series ol blochemicol reactions occurring in the body to conuert the foreign (often toxic) compounds to non-toxic or less toxic and more easily excretoble forms. Liuer is the major site of detoxification. ln recent years, the term detoxiftcation is replaced by biotransformation or metabolism of xenoblotics.

2. Detoxification may be diuided into phase I (oxidation, reduction, hydrolysis) and phase II reactions (conjugotion). Oxidation is o major processof detoxification, inuoluing the microsomal enzyme cytochrome P4gg which is an inducible, NADPH dependent hemoprotein. 3. Conjugation is o process in which a loreign compound combines with a substance produced in the body. The process of conjugation may occur either directly or after phase I reoctions. At least 8 diflerent conjugottng agents haue been identified in the body--glucuronic acid, glycine, cystelne, glutomine, methyl group, suffate, acetic acid and thiosulfate.

643

Tltc p:osla1'landtryrFif

al' \---.,..

,

s

"Twentycarbonca?tpoundsafrcwe! Syntbesized. from arachidoniiacid.; infunctiou Act aslacalhoflnones Wid"b usedastherupcuticagan*.4

prostaglandins and their relatedcompoundsI prostacyclins (PGl), thromboxanes (TXA), leukotrienes(LT) and lipoxins are collectively known as eicosaniods, since they all contain 20 carbons (Creek : eikosi-twenty). Eicosanoidsare considered as locally acting hormones with a wide range of biochemical functions.

,'

. ''

phosphate buffer (PCF, F for fosfat, in Swedish). All other prostaglandinsdiscovered later were denoted by a letter-PCA, PCH etc. S tructure

o{ prostagl andi ns

derivatives of a Prostaglandins are hypothetical 2O-carbon fatty acid namely prostanoic acid hence known as prostanoids. History : Prostaglandins(PCs) were first This has a cyclopentanering (formed by carbon discoveredin human semenby Ulf von Euler(of atoms 8 to 12) and two side chains, with differ Sweden)in 1930. Thesecompoundswere found carboxylgroup on one side. Prostaglandins to stimulate uterine contraction and reduce in their structuredue to substituentgroup and blood pressure.von Euler presumed that they double bond on cyclopentanering. The different were synthesizedby prostategland and hence prostaglandinsare given in Fig.32.l. named them as prostaglandins.lt was later The structures of the most important realized that PCs and other eicosanoids are prostaglandins(PGF2and PGF2o),prostacyclins synthesizedin almost all the tissues(exception(PCl2), thromboxanes(TXA2) and leukotrienes erythrocytes). By then, however, the name (LTA+)along with arachidonicacid are depicted prostaglandinswas accepted worldwide, and in Fi9.32.2.A subscript numeral indicatesthe hence continued. number of double bonds in the two side chains. The prostaglandinsE and F were first isolated A subscriptc-denotes that the hydroxyl group at from the biological fluids. They were so named Ce of the ring and the carboxyl group are on the due t o th e i r s o l u b i l i ty i n e th e r (PC E ) and same side of the ring.

644

Chapter 32 r PFIOSTAGIANDINS AND RELATEDCOMPOUNDS

645

Proetaglandln Substltuentgroup

13

t5

17

PGE

Ketogroupat Ce -OH groupat C11

PGF

-OH groupat Ce andat C11

Structurc

19

Prostanolcacld Proetaglandin Substltuentgroup

Struc'tur€ o il

DoublebondCro- Crr KetogroupCe

/-a \rl \---r\

o tl

PGB

DoublebondCe- Cre KetogroupCe

/-a'

(.Fz t l o

PGC

DoublebondCr - Crz KetogroupCe

PGG

PGH

(l \ttr' OH I

PGD

-OH groupat Ce Ketogroupat C11

Hg.3al

(l

H o

PGI

Attachment of orygen betweenC6and Ce lorminganothers-carbon ring

contd. n rt colurwl

Hg.9l2.l : Prostagladinswith suhtituent grcupsaN stnlriures (l{ota t Prmtanoicacidis the parentnudeustor all the PGs;B, reptaaentsC, to Crof prastanoicacid,exceptin PGI whereR, is C, to C; B, representsC,rto Crot prostanoicacid).

Synthesis of prostaglandins

2. Oxidationand cyclizationof arachidonic acid to PGG2which is then converted to PCH2 Arachidonicacid (5,8,11,14-eicosatetraenoic by a reducedglutathionedependentperoxidase. acid) is the precursor for most of the 3. PGH2servesas the immediateprecursor prostaglandins of in humans.The biosynthesis for the synthesisof a numberof prostaglandins, PCs was describedby scene Bergstromand prostacyclins including and thromboxanes. Bengt Samuelsson(1960). lt occurs in the reticulumin the followingstages, The above pathway is known as cyclic endoplasmic as depictedin Fi9,32.3. pathway of arachidonic acid. ln the linear pathwayof arachidonicacid, leukotrienes and 1. Release of arachidonic acid from (detailsgivenlater). lipoxins are synthesized phosphophospholipids by membranebound Cyclooxygenase-asuicide enzyme : lt is lipaseA2-this reactionoccursdue to a specific stimuli by hormonessuch as epinephrineor interestingto note that prostaglandinsynthesis can be partly controlledby suicidalactivity of bradykinin.

ltiF

B IOC H E MIS TRY

646

cortisol) prevent the formation of arachidonic acid by inhibitingthe enzyme phospholipase42.

Arachidonic acid

Many non-steroidal anti-inflammatory drugs prostaglandins, inhibit the synthesis of prostacyclinsand thromboxanes.They do so by blocking the action of the enzyme cyclo' oxygenase. Aspirin inhibits PG synthesis: Aspirin (acetyl salicylic acid) has been used since nineteenth century as an antipyretic (fever-reducing)and anal gesi c(pai n rel i evi ng).The mechanism of action of aspirin however, was not known for a Iong peri od. l t w as onl y i n 1971, John Vane discoveredthat aspirin inhibits the synthesisof PC from arachidonic acid. Aspirin irreversibly inhibits the enzyme cyclooxygenase. Other antiinflammatorydrugs, such as indomethacin and phenylbutazoneact as reversibleinhibitors of the enzyme cyclooxygenase.

PGI2

OH TXAq

LTA4

Ft1.32.2: The structuresof arachidonicacid, common

Degradation of prostaglandins : Almost all the eicosanoidsare metabolized rapidly. The lung and liver are the major sites of PC degradation. Two enzymes, namely 15-crhydroxy PG dehydrogenase and 13-PC reductase,convert hydroxyl group at C15 to keto group and then to C 13 and C tq di hydr oderivative. actions Biochemical of prostaglandins Prostaglandinsact as local hormones in their function. They, however, differ from the true hormones in many ways. Prostaglandinsare produced in almost all the tissuesin contrastto hormonal synthesiswhich occurs in specialized glands.PGsare not storedand they are degraded to inactive products at the site of their production. Further, PCs are produced in very small amounts and have low half-lives.

Prostaglandinsare involved in a variety of the enzyme cyclooxygenase.This enzyme is biologicalfunctions.The actionsof PCs differ in capableof undergoingself-catalysed destruction different tissues. Sometimes,PGs bring about to switch off PG synthesis. opposing actions in the same tissue. lnhibition of PG synthesis : A number of Overproduction of PGs results in many structurally unrelated compounds can inhibit symptoms which include pain, fever, nausea, prostaglandin synthesis. Corticosteroids (e.g. vomi ti ng and i nfl ammati on.

arij : PFIOSTAGLANDINSAND FELATED COMPOUNDS ,:.i1;i-,7'

Prostaglandinsmediate the regulation of blood pressure, inflammatory response, blood clotting,reproductivefunctions, responseto pain, fever etc.

647

Phospholipids (membranebound) codicosteroicrs

42 sPholiPase Lysophospholipid

$Liporygenase _

1. Regulation of blood pressure : The prostaglandins ( P CE , P G A a n d P C l 2 ) a re 5-HPETE v as odilat or in fu n c ti o n . T h i s II resultsin increasedblood flow (LT) and decreased peripheral Leukotrienes resistarrceto lower the blood J pressure.PGs serveas agentsin (LX) the treatmentof hypertension. Lipoxins

+

. Arachidonicacid

Cyclooxygenase Aspirin Phenylbutazone lndomethacin lbuprofen

2. Inflammation : The pr os t aglandi n s PC El and PCE2 induce the symptoms (redness, inflammation of s welling, ede m a e tc .) d u e to ar t er iolarv as o d i l a ti o nT. h i s l e d to the belief that PCs are natural mediators of inflammatory reactions of Fig. 32.3 : Overuiewof biosynthesisof prostaglandinsand related r heum at oidar th ri ti s (i n v o l v i n g compounds (5-HPETE-5 -Hydroxyperoxycicosatetraenoic acid; joint s ) , (s k i n ), ps o ri a s i s PG- Prostaglandins; PGlr- Prostacyclin I; TXA hromboxane A). junctivitis (eyes) etc. con "-T Corticosteroidsare frequentlv used to treat these inflammatorvreactions,since secretion. PGs are used for the treatment of gastric ulcers. However, PCs stimulate t hey inhibit pr o s ta g l a n d i n s y n th e s i s . pancreaticsecretionand increasethe motility of have wide3. Reproduction: Prostaglandins intestinewhich often causesdiarrhea. spreadapplicationsin the field of reproduction. 6. Influence immune on system i PGE2 and PGF2 are used for the medical termination of pregnancy and induction of MacrophagessecretePCE which decreasesthe Iabor. Prostaglandins are administeredto cattle immunologicalfunctionsof B-andT-lymphocytes. to induce estrus and achieve better rate of 7. Etfects on respiratory function : PCE is a fertilization. bronchodilatorwhereasPGFacts as a constrictor 4. Pain and fever : lt is believed that of bronchial smooth muscles. Thus, PCE and pyrogens (fever producing agents) promote PCF oppose the actions of each other in the prostaglandin biosynthesis leading to the lungs. PCEI and PCE2are used in the treatment formation of PCE2in the hypothalamus,the site of asthma. of regulation of body temperature. PCE2 along 8. Influence on renal functions : FCE with histamine and bradykinin cause pain. increasesglomerular filtration rate (GFR) and . p i ri na n d o ther promotesurine output. Excretionof Na+ and K+ M igr aineis als o d u e to PGE 2 As non-steroidaldrugs inhibit PG synthesisand thus is also increasedbv PCE. control fever and relieve pain. 9. Effects on metabolism : Prostaglandins 5. Regulation of gastric secretion : In influence certain metabolic reactions,probably gener al, pr os ta g l a n d i n s(P C E ) i n h i b i t g a s tri c through the mediation of cAMP. PCE decreases

BIOGHEMISTFIY

648 lipolysis, increases glycogen formation and promotescalcium mobilization from the bone. 10. Platelet aggregationand thrombosis : The prostaglandins, namely prostacyclins (PGI2), inhihit platelet aggregation. On the other hand, thromboxanes (TXA2) and prostaglandin E2 promote plateletaggregationand blood clotting that might lead to thrombosis.PGl2, produced by e n d o th e l i a l c e l l s l i n i n g th e bl ood vessel s, preventsthe adherenceof plateletsto the blood vessels.TXA2 is releasedby the platelets and is responsiblefor their spontaneousaggregation when the plateletscome in contact with foreign th ro mbi n. Thus, s ur fa c e , c o l l a g e n o r prostacyclinsand thromboxanesare antagonists in their action. In the overall effect PCl2 acts as a vasodilator,while TXA2 is a vasoconstrictor. The balance between PCI2 and TXA2 is important in the regulation of hemostasisand t hr o mb o s i s . Mechanism

0f aeti$n

of *r&,*

The mechanismof action of prostaglandinsis not known for certain.They bind to the specific c ell u l a rre c e p to rsa n d b ri n g a b o u t thei r acti on at the molecular level. lt is believedthat PGs may act through the mediation of cyclic nucleotides. PCE increases cAMP levels whereas PGF elevates cGMP.

Bromrednr;ad dipp$acattotts of $'{is As described above, prostaglandinsperform diversifiedfunctions.And for this reason,PCs (or other derivatives) are the most exploited in therapeutic applications.They are used in the treatment of gastric ulcers, hypertension, thrombosis, asthma etc. Prostaglandinsare also employed in the medical termination of pregnancy, prevention of conception, induction of labor etc. Inhibitors of prostaglandin synthesis (e.9. aspi ri n, i buprofen) are uti l i zed i n contro lling fever, pain, migraine, inflammationetc.

Leukotrienesare synthesizedby leucocytes, mast cells, lung, heart, spleen etc., by lipoxygenasepathway of arachidonic acid. The synthesisof different leukotrienes(A4, 84, C4, Da and Ea) through the intermediate,5-hydroperoxyeicosatetraenoic acid (5-HPETE) is depicted in Fig.32.4. A naphyl axi s i s a vi ol ent and fatal al ler gic reaction.lt is now known that leukotrienes(C4, Da and Ea)are the components of slow-reacting substancesof anaphylaxis (SRS-A),released after i mmunol ogi calchal l enge.S R S -A i s 100-1 , 000

BIOMEDICAL/ CLINIGAL CONCEPTS

ss Prostoglandins, synfhesized in almost all the tissues (exception--erythrocates)of the body, oct as local hormones. PGs perform diuersified biochemical functions. These include lowering of blood pressure, inhibition oJ gastric HCI secretion, decreose in immunologicol response and induction ol labon Ouerproduction of PGs couses symptoms such os poin, fevet; uomiting, nausea, inflammation etc. Aspirin/ibuprofen/corticosteroid administration inhibits PG synthesis and relieues these symptoms. ue Platelet aggregotion that moy leod to fhrombosis is promoted by thromboxanes and prostoglandins E1 and inhibited by prostacyclins. re Leukotrienes are implicated in hypersensitioity (allergy) and asthma. ss Consumption ot' lish foods containing the unsaturated eicosapentaenoicocid is oduocated to preuent heart attacks.

t'atty

acrd nomely

r::' .i# :PBOSTAGLANDINS AND FELAIEDCOMPOUNDS Arachidonicacid

I s-Lipoxygenase |

J 5.HPETE

IHH,O

LTA.hydrolase

LeukotrieneBo (LT84)

+

LeukotrieneAo (LTA.)

Gtutathione--rlu,r,",n,on" ) S-transferase

J

Leukotriene C4(LTC4) | ^lGlutamylGlutamate +-zl transferase

+

LeukotrieneD4 (LTD4)

I

. I DioePtidase ulyclne+/l

J LeukotrieneE4 (LTE4) Fiq.32.4 : Synthesis of leukotrienes from arachidonic acid (5-HPETE-S-Hydroperorycicosatetraenoic acid).

649

adhesi on of w hi te bl ood cel l s and rel easeof lysosomalenzymes. Lipoxins are involved in vasoactive, and immunoregulatoryfunctions. There is a strong evidence to support that lipoxins act as counterregul atory compounds of i mmune response. Ei,i^:f."r:r{'r rslarine lipids en relaticn 'i:i lsfi'rl:" i-Ts ,*nd heart dlseases E ski mosof C reenl andhave a l ow i nci dence of coronary heart diseases,despitethe fact that they consume high quantitiesof fat and cholesterol . Thi s i s due to the hi gh i ntake of mari ne lipids containing unsaturatedfatty acids (UFA). The most predomi nantU FA i n the fi sh foods consumedby E ski mosi s 5, 8, 11, 14,' l Z-ei cosapentaenoicacid (EPA).EPAis the precursorfor l eukotri enes-Sseri es w hi ch are much l ow er i n thei r acti vi ty than the l eukotri ene-4seri es, produced from arachi doni c aci d. Further, eicosapentaenoic acid inhibits the formation thromboxanes (TXA). As already describeo, TXA2 promotes platelet aggregation and thrombosis.

times more potent than histamine or prostaglandinsin its action as a stimulant of The di et ri ch i n mari ne l i pi ds (w i th E P A ) aller gicr eac t io n sL. e u k o tri e n easre i mp l i c a te di n decreases plasma cholesterol and triacylasthma,inflammatoryreactions,hypersensitivity glycerols. These factors, along with reduced (allergy)and heart attacks. synthesisof TXA2 are believedto be responsible Leukotrienescause contraction of smooth for the low incidence of heart attacks in muscles, bronchoconstriction,vasoconstriction, E ski mos.

1. Prostaglandins(PGs)and related compounds prostacyclins(PGI), thromboxqnes(TXA) ond leukotrienes(LT) are collectiuelyknown as eicosanoids.They are the deriuatiuesof a hypotheticol 20 carbon fattg acid, namely prostanoic acid. Prostaglandins are synthesizedfrom arochidonic acid, releosedfrom the membrane bound phospholipids. Corticosteroidsand ospirin inhibit PG synthesis.

2. Prostoglandinsocf os local hormones and ore inuolued in a wide range of biochemical functions. In generol, PGs are inuolued in the lowering of blood pressure,induction of inflammation, medical termination ol pregnancy, induction of laboa inhibition of gastricHCI secretion, decreasein immunological responseand increasein glomerular t'iltration rate. Thromboxanes(TXA2) and prostaglandinEl promote while prostocyclins (PGI) inhibit platelet aggregotion.

,i

Transport The plosrrro nnem,btane spco&s ; "I earmarh the cell territory; For protection fr*

hostile enaironmeftt;

fugttlate solate import a,nd export' By passiueor actiue transport""

h " p l a s m a m e m b ra n e i s an envel ope I s u rro u n d i n g th e c e l l \R e fe r Fi g.l .l ). l t separatesand protectsthe cell from the external hostile environment.Besidesbeing a protective barrier,plasmamembraneprovidesa connecting system between the cell and its environment. T he s u b c e l l u l a r o rg a n e l l e s s u c h as nucl eus, mitochondria,lysosomesare also surroundedby m emb ra n e s . T

$trsscture,

of r*terit&'ra$:s :'

A lipid bilayer model originally proposed for membrane structure i n 1935 by D avson and D ani el l e has been modi fi ed.

Fluid mosaic model, proposed by Singer and Nicolson,is a more recentand acceptablemodel membrane structure. The biological for usual l yhavea thi cknessof 5-8 n m . A membranes membrane i s essenti al l ycomposedof a lipid Che m i c a l c o rmp c s i tro n bilayer.The hydrophobic(nonpolar)regionsof the lipids face each other at the core of the bilayer T h e m e m b ra n e s a re c o mp o sed of l i pi ds, while the hydrophilic(polar)regionsfaceoutward. proteins and carbohydrates.The actual compoClobular proteinsare irregularlyembeddedin the sition differs from tissue to tissue. Among the l i pi d bi l ayer (Fi 9.33.1).Membraneprotei n sar e lipids, amphipathic lipids (containing hydrocategorizedinto two Broups. pho b i c a n d h y d ro p h i l i c g ro u p s ) namel y phospholipids, glycolipids and cholesterol, are found 1. Extrinsic (peripheral)membrane proteins in t h e a n i m a l me mb ra n e s . are loosely held to the surfaceof the membrane and they can be easilyseparatede.g. cytochrome Ma n v a n i ma l c e l l me mb ra nes have thi ck c of mitochondria. coating of complex polysaccharidesreferredto as glycocalyx. The oligosaccharides of 2. Intrinsic (integral)membraneproteinsare glycocalyx interactwith collagenof intercellular ti ghtl y bound to the l i pi d bi l ayer and they can matrix in the tissues. be separatedonly by the use of detergentsor

650

551

*.][rafrter33 : BIOLOGICAL MEMBHANESAND THANSPOFIT or ganic s olv e n ts e .g . h o rmo n e receptors,cytochrome P45g. T he m em br a n e i s a s y m m e tri c due to the irregulardistributionof p r ot eins . T he l i p i d a n d p ro te i n s ubunit sof t he m e m b ra n eg i v e a n a ppear anc of e m o s a i co r a c e ra m i c t ile. Unlik ea f ix e d c e ra mi cti l e , th e mernbrane freely changes, hence the structureof the membrane is consideredas fluid mosaic. Tvamsport

protein

Fig.33,l : The fluid mosaic model of membrane structure.

&crqlss Fsnembrames

The biological membranes are relatively impermeable.The membrane,therefore,forms a barrierfor the free passageof compoundsacross it. At leastthree distinct mechanismshave been identified lor the transoort of solutes (metabolites)through the membrane(Fi9.33.A. I . Passivediffusion 2. Facilitateddiffusion 3. Active transoort; 1. Fassivediffusion : This is a simple process which dependson the concentrationgradientof a oarticular substance across the membrane. Fassageof water and gasesthrough membrane occurs by passivediffusion. This processdoes not require energy.

2. Facilitated diffusion : This is somewhat comparable with diffusion since the solute moves along the concentrationgradient (from higher to lower concentration)and no energy is needed. B ut the most i mportantdi sti ngui shi ng featureis that facilitateddiffusionoccursthrough the mediation of carrier or transport proteins. Specific carrier proteins for the transport of gl ucose,gal actose,l euci ne, phenyl al ani neetc. have been isolatedand characterized. Mechanism of facilitated diffusion : A pingpong model is put forth to explainthe occurrence of facilitated diffusion (Fig.33.3).According to this mechanism,a transport(carrier)proteinexists in two conformations.In the pong conformation, it is exposed to the side with high solute

tl

Concentration gradient

Membrane

Passive diffusion

Facilitated diffusion

_-=r-tl

Active transport

Passivetransport Fig. 33,2 : Mechanism of transport across biological membrane

(Note : Transportmoleculeare representedin blue; the carrierprateinsin red).

lll

652

B IOC H E MIS TR Y

Pong

Ftq.33.3 : A diagrammaticrepresentation ot 'ping-pong'modelfor facilitateddiffusion.

c onc en tra ti o nT. h i s a l l o w s th e b i n d i ng of sol ute to specificsiteson the carrierprotein.The protein then undergoesa conformationalchange (ping state) to expose to the side with low solute concentration where the solute molecule is released.Hormonesregulatefacilitateddiffusion. For instance,insulin increasesglucosetransport in m u s c l e a n d a d i p o s e ti s s u e ; a mi no aci d transportin liver and other tissues.

intracellularATP. The Na+-K+pump, depicted in Fi9.33.4,is summarized. 3 Na+ (in) + 2K+ (out) + ATP -----+ 3Na* (out) + 2K * (i n) + A D P + Pi A ma.ior portion of the cellular ATP (up to 7o" h,i n nervecel l s)i s i n fact uti l i zedby N a+ -K+ pump to maintain the requisite cytosolic Na+ and K + l evel s.Ouabai n (pronouncedas W ah biiin) inhibits Na+-K+ ATPase. Ouabain is a steroid derivative extracted from the seedsof an African shrub. lt is a poison used to tip the hunting arrows by the tribals in Africa.

3. Active transport : Active transportoccurs against a concentration gradient and this is dependent on the supply of metabolic energy (AIP). Active transportis also a carriermediated process like facilitated diffusion. The most Na+-cotransportsystem : The amino acids important primary active transport systems are and sugarsare transportedinto the cells by a ion- pu m p s (th ro u g hth e i n v o l v e mentof pump Na+-cotransport system.This processessentially ATPasesor ion transportingATPases). consistsof the passageof glucose(or amino acid) into the cell with a simultaneousmovement of Na+-K+pump : The cells have a high intraNa+. ATP is required to pump out the cellular K+ concentrationand a low Na+ conceni ntracel l ul ar N a+ through the medi ati on of tration.This is essentiallyneededfor the survival Na+-K+ATPase.More detailson the cotransport of t he ce l l s . H i g h c e l l u l a r K+ i s re q ui redfor the system are given under digestionand absorption optimal glycolysis(pyruvatekinase is dependent (Chaptur A. on K+)and for protein biosynthesis.Further,Na+ and K+ gradientsacrossplasma membranesare needed for the transmissionof nerve impulse. T he N a + -K+ p u m p i s re s p o n s i bl efor the m aint e n a n c e o f h i g h K+ a n d l ow N a+ c onc en tra ti o nisn th e c e l l s .T h i s i s b roughtabout by an integralplasmamembraneprotein,namely the enzyme Na+-K+ATPase(mol. wt. 250,000). It consistsof two cx,and two p subunitswhich may be representedas (a0)2. Na+-K+ ATPase pum ps 3 N a + i o n s fro m i n s i d eth e c e ll to outsi de and bri n g s 2 K + i o n s fro m th e o u tsi de to the ins ide w i th a c o n c o m i ta n t h v drol vsi s of

Outside

Membrane

Inside Fig. 33.4: Diagrammaticrepresentationof N*-IC pump(Note: Redcolourblock representsthe enzymeNa.-K- ATPase).

653

MEMBRANESAND TRANSPORT f;hapter 33 : BIOLOGICAL

Membrane

Antiport

Symport

----TCotransport Fig. 33.5: Diagrammaticreprcsentationof transportsystems.

Transport systems The transport systemsmay be divided into 3 categories(Fi9.33.5). 1. Uniport system : This involves the movement of a single molecule through the membrane e.g. transport of glucose to the erythrocytes.

Cotransport system : In cotransport, the transport of a substance through the membrane is coupled to the spontaneous movement of another substance. The symport and antiport systems referred to above are good examples of cotransport system.

Proton pump in the stomach : This is an antiport transport system of gastric parietal cells. lt is brought out by the enzyme H+-K+ ATPase to maintain highly acidic (pH = l ) condi ti ons i n the l umen of the antiports two 3. Antiport system : The simultaneoustrans- stomach. Proton pump (2H+) protons cytoplasmic and two extracellular port of two different molecules in the opposite (2K+) potassium ions for a molecule of ATP direction e.g exchangeof Cl- and HCO3 in the hydrolysed. The ions chloride secreted by Clerythrocytes. Uniport, symport and antiport protons gastric channels combine with to form systems are considered as secondary active H C I. transport systems.

2. Symport system : The simultaneous transportof two differentmoleculesin the same directione.g. transportof Na+ and glucoseto the intestinalmucosal cells from the gut.

EIOMEDICAL/ CLINICAL CONCEPTS

r:-.t;pi6lsgi6,ql membranes are relatiuelg impermeable protectiue barriers that provide a connecting link between the cell (or its organelle) and its enuironment. t* The cells must contain high K+ and low Na+ concentrationsfor their suruiual.No+-K+ pump, which consumes a major portion of the cellular metabolic energy (ATP), is responsiblefor this. r.r Ouabain inhibits No+-K+ ATPose (No+-K+ pump). lt is extracted from the seeds of an African shrub and used os poison to tip the hunting arrou)s by the tribals. ,r-+:Disturbancesis osmosis ore ossociatedwith diarrhea, edema, inflammation o/ fissues etc.

/

654

B IOC H E MIS TFI Y

Fass*ve tre*ras;*ert of water"osr-ms"+is O s mo s i s i s th e o h e n o m e non of movement of water from low osmotic pressure(dilute solution)to high osmotic pressure (concentrated solution) across biological membranes.The movementof water in the body occursthroughosmosis, and this process does nat require energy (ATP). Certain medical and health complicationsare due to disturbancesin osmosis. e.g. edema, diarrhea, cholera, inflammation of tissues.The reader mav refer Chapter 40 lor more information on osmosis,water and electrolyteimbalance in cholera,/diarrhea. Trams;pmr* m$ ersm*roms3e:;eriq': The transport of macromolecules such as proteins, polysaccharidesand polynucleotides across the membranes is equally important. This is brought about by two independent mechanisms namely endocytosis-intake of macromolecules by the cells and exocytosisreleaseof macromoleculesfrom the cells to the outside.

Exocytosis

Fig. 33.6 : Diagrammatic representation of endocytosis and exocytosis (Note : The red coloured pafticles indicate the transport material).

form coated vesicleswhich contain an unusual protein called clathrin. The process of endocytosisis depicted in Fi9.33.6.The uptake of l ow densi ty l i poprotei n(LD L) mol eculesby the cells is a good example of endocytosis.

Exocytosis: The releaseof macromoleculesto the outside of the cells mostly occurs via the participation of Golgi apparatus. The Endocytosis : lt is estimated that macromoleculesare transportedto the plasma approximately 2% of the exterior surface of membranein vesiclesand let out (Fi9.33.6).The plasma membrane possesses characteristic secretionof hormones (e.9. insulin, parathyroid coated pifs. These pits can be internalizedto hormone) usually occurs by exocytosis.

7. The biological membranesare the barriers that protect the cell and the subcellulor organellesfrom the hostile enuironment. The membranesare primarily composedof a Iipid bilayer onto which the globular proteins are irregularly embeddedto form a fluid mossic model. 2. Transportof moleculesthrough membranesoccurseither by passiuediffusion, facilitated diflusion or actiue transport. Actiue transport occurs against a concentration gradient which is dependent on the supply of metabolic energy (ATP). Na+-K+ pump is responsiblefor the maintenanceof high K+ and /ourNo+ concentrotionsinside the cells, an essentialrequisite for the suruiual of cells. 3. The transport systemsare diuided into 3 categories-uniport, symport and ontiport 4. The transport of macromoleculestakesplace by endocytosis(ingestionby the cells)and exocytosis (release Jrom the cells).

Ttlr- Jtce rdaicols

speaks t,

"V(eexistas indepatdent moledar specics; Generatedby cellukr metabolismand enuironntentalc_fferts; Impliratel in the causationof seueraldiseau; OH- (Hydrorylradical)

Destroyedhy antioxidants to plrtett cells/tissaesi body" "

-l-h"

r,r,.. (Molecular orygen)

supply of oxygen is absolutelyessential I for the existence of higher organisms.As the saying goes too much of even the best is bad. Very high concentrationsof 02 are found to be toxic, and can damagetissues.The present day concept of oxygen toxicity is due to the involvement of oxygen free radicals or reactive oxygen species (ROS). In fact, the generation of reactive metabolites of 02 is an integral part of o ur daily lif e. A free radical is defined as a molecule or a mofecular species that contains one or more unpaired electrons, and is capable of independent existence. Types

of free

radicals

reY

O, (Superoxide) h€,2H+ J H rO, (Hydrogen peroxide)

l. e-,H' Hzo+1 +

OH- (Hydroxylradicat) h e-'H*

+

,-r,r' (W ater) Besidesthe above (O2, H2O2, OH-), the other free radicals and reactive oxygen species of biological importance include singlet oxygen (1O2), hydroperoxy radical (HOO-), lipid peroxide radical (ROO-), nitric oxide (NO-) and peroxynitrite (ONOO-).

Oxygen is required in many metabolic reactions,particularlyfor the releaseof energy. During these processes, molecular O2 is The common characteristic features of free completely reduced, and converted to water. radicalsare listed However, if the reduction of 02 is incomplete, a . Highly reactive seriesof reactiveradicalsare formed, as shown in the next column. o Very short half-life

655

il

I

1

656 o

Can generatenew radicalsby chain reaction

a

Cause damage to biomolecules, cells and tissues

Free radicals and reactive oxygen species (ROS)-not synonymous : By definition, a free radical containsone or more unpairedelectrons. e.g. Or, OH-, ROO-. There are certain nonradical derivatives of 02 which do not contain unpaired electrons e.g. H2O2, 102. The term reactive oxygen speciesis used in a broad sense to collectively representfree radicals, and nonfree radicals (which are extremely reactive) of the biological systems. However, most authors do not make a clear distinction between free radicals and ROS, and use them interchangebly.

SOURCES AND GEI{ERATION OF FREE RADICALS

il

BIOCHEMISTF|Y

I Cellularmetabolism lromtherespiratory chain . Leakage of electrons (ETC). enzymes . Production of HrO,or O, byoxidase (e.9.xanthine NADPH oxidase). oxidase, . Dueto chainreactions of membrane lipid peroxidation. generation . Peroxisomal of O, andHrOr. . During thesynthesis of prostaglandins. . Production of nitricoxidefromarginine. (asa padof . Duringthecourseof phagocytosis bactedcidal action). . In theoxidation of hemeto bilepigments. . Asa resultof auteoxidation e.g.metalions flavin ascorbic acid,glutathione, [Fd*,Cu2*]; coenzymes.

The major sources responsiblefor the II Environmental effects generationof free radicalsmay be considered . As a resultof drugmelabolism e.g.paracetamol, undertwo categories halothane, cy{ochrome P* rehtedreactions. l. Due to normal biological processes(or . Dueto damages caused by ionizing radiations (e.9.X-rays) ontissues. cellularmetabolism). . Photolysis of O, by light. ll. Due to environmentaleffects. . Photoexcitation of organic molecules It is estimatedthat about 1-4o/oof the 02 . Cigarette contains freeradicals, smoke andtrace taken up by the body is convertedto free metals thatgenerate OFI-. radicals. A summary of the sources for promoting . Alcohol, lipidperoxidation. generationof free radicals is given in the Tahle34.1, and a couple of the processes are brieflydescribed. latter,in turn, can removeH-atomfrom another (LOOH). PUFA(LH)to form lipid hydroperoxide Lipid peroxidation L- + 02 -----+ LOOFree radical-induced peroxidation of membrane lipidsoccursin threestages-initiation, LOO- + LH -----+ LOOH + Lpropagation and termination The hydroperoxidesare capable of further Initiation phase : This step involves the stimulatinglipid peroxidation as they can form removal of hydrogen atom (H) from alkoxy (LO-) and peroxyl(LOO-) radicals. polyunsaturatedfatty acids (LH), caused by Fe',Cu hydroxylradical 2LOOH LO- + LO2 + H20 ' LH + OH- -----+ L- + H2O LOOH -----+ LO- + LOO- + aldehydes Propagation phase : Under aerobic Termination phase : Lipid peroxidation (L-) proceeds conditions,the fatty acid radical as a chain reactionuntil the available takes up oxygen to form peroxy radical (LOO-). The PUFAgetsoxidized.

-

Chapter 34 : FFIEEFADICALSAND ANTTOXTDANTS

657

*lalondialdehyde (MDAI as a marker for lipid peroxidation ,Vost of the products of lipid peroxidation are u ns t able e .g . c a rb o n y l s , es t er s , alka n e s , a l k e n e s , 2 alk enal,2,4 -a l k a d i e n a lMD , A. O f t hes e , m a l o n d i a l d e h y d e r - CHO - CH 2 -C H O -) i s th e most extensivelystudied, and is us ed a s a b i o c h e m i c a l Flg.34.l : Generationof free ndicats by macrophages and respiratoryburst. marker for the assessmentof lipid per ox i d a ti o n .M D A a n d other aldehydes react with thiobarbituric acid H A R MFU L E FFE C TS and produce red-coloured products namery OF FREE RADICALS thiobarbituric acid reactive substances(TBARS) which can be measuredcolorimetrically. Free radicals and biomolecules The estimationof serumMDA is often usedto Free radicals are highly reactive, and are assessoxidative stress,and free radical damage capable of damaging almost all types of to the body. biomolecules (proteins, lipids, carbohydrates, nucleic acids).The fact is that free radicalsbeget Eamages cau$ed free radicals i.e. generate free radicals from by lipid peroxidatian normal compoundsw hi ch conti nuesas a chai n The productsof lipid peroxidationare highly reaction. destructive.They damage the membranes,cells Proteins : Free radicals cause oxidation of and even tissues.Lipid peroxidation has been sulfhydryl groups, and modification of certain implicatedin many diseases(Seeharmful effects (e.g.methionine,cysteine,histidine, amino acids of free radicals). tryptophan,tyrosine).ROS may damageproteins by fragmentation,cross-linkingand aggregation. Generation of ROS hy macrophages The net result is that free radicals mav often During the course of phagocytosis, resultin the lossof biologicalactivityof proteins. i nflammatorycells, particularly the macrophages producesuperoxide(Oz),by a reactioncatalysed lipids : Polyunsaturatedfatty acids (pUFA) by NADPH oxidase (Fig3a.t). This 02 radical are highly susceptible to damage by free gets converted to HzOz, and then to radicals. Details have been given under lipid hypochlorous acid (HCIO). The superoxide peroxidation. radical along with hypochlorous ions brings Carbohydrates : At physiological pH, about bactericidal action. This truly represents oxidation of monosaccharides (e.g.glucose)can the beneficial affects of the free radicals produce H2O2 and oxoaldehydes.lt appearsthat generatedby the body. the linkage of carbohydrates to proteins A large amount of 02 is consumed by (glycation)increasesthe susceptibilityof proteins macrophagesduring their bactericidalfunction, to the attack by free radicals. This character a phenomenon referredto as respiratory burst. lt assumessignificancein diabetesmellitus where is estimatedthat about 10'/" of the 02 taken up protein glycation is associatedwith manv health by macrophagesis utilized for the generationof compl i cati ons e.g. di abeti c mi croangi opathy, free radicals. diabetic nephropathy.

558

BIOCHEMISTFIY

Cataract : Increasedexposure to oxidative Nucleic acids : Freeradicalsmay causeDNA strand breaks, fragmentation of bases and stresscontributesto cataractformation,which is deoxyribose.Such damagesmay be associated mostly relatedto aging. with cytotoxicity and mutations. Male infertility : Free radicalsare known to reduce sperm motility and viability, and thus Free radicals and diseases may contribute to male infertility. As discussedabove, free radicalsare harmful Aging process : Free radicals are closely to biomolecules,and in turn cells and tissues. Free radicals have been implicated in the associatedwith the various biochemical and morphol ogi calchangesthat occur duri ng normal causationand progressof severaldiseases. agi ng. Cardiovascular diseases(CHD) : Oxidized low density lipoproteins (LDL), formed by the Other diseases: Freeradicalsplay a key role action of free radicals, promote atherosclerosis in Parkinson's disease, Alzheimer's disease, and CHD. mul ti pl e scl erosi s, l i ver ci rrhosi s, muscul ar toxemia of pregnancy etc. dystrophy, Cancer : Free radicals can damage DNA, and cause mutagenicity and cytotoxicity, and thus play a key role in carcinogenesis.lt is believed ANTAOXIDAI{TS IN t hat RO Sc a n i n d u c emu ta ti o n sa, n d i n h i bi t D N A BIOLOGIGAT SYSTEM repair process,that resultsin the inactivationof To mitigate the harmful/damaging effects of certain tumor suppressor genes leading to free radicals,the aerobic cells have developed cancer. Further, free radicals promote anti oxi dantdefensemechani sms.A bi ol ogi cal bioc hem i c a l a n d mo l e c u l a r c h a n g e sfo r rapi d antioxidant may be defined as a suhstance growth of tumor cells. (presentin low concentrationscompared to an Inflammatory diseases; Rheumatoid arthritis oxidizabfe substrate) that significantly delays or is a chronic inflammatory disease. The free inhibits oxidation of a substrafe. Antioxidants radicals produced by neutrophils are the may be considered as the scavengers of free predominant causativeagents.The occurrence radicals. of other inflammatory disorders-chronic The production of free radicals and their glom er ulo n e p h ri tiasn d u l c e ra ti v ec o l i ti s i s al so neutralizationby antioxidantsis a normal bodily due to the damages caused by ROS on the process.There are different ways of classifying extracellular components (e.g. collagen, antioxidants. hy alur on i ca c i d ). Respiratory diseases : Direct exposure of l" Ant$oxidants in relation to lungs to 100% oxygen for a long period (more Nipid peroxBdation than 24 hrs) is known to destroy endothelium 1. Preventive antioxidantsthat will block the and cause lung edema.This is mediatedby free production of free radicalse.g. catalase, initial r adic als . R OS a re a l s o re s p o n s i b l efo r adul t glutath ione peroxidase. (ARDS), respiratorydistresssyndrome a disorder pulmonary characterizedby edema. 2. Chain breaking antioxidants that inhibit propagativephase of lipid peroxidatione.g. the Cigarette smoke, as such, contains free superoxide dismutase,vitamin E, uric acid. radicals,and further it promotesthe generation of more free radicals.The damagescaused to ll. Antioxidants according to lungs in the smokersare due to ROS. their Eacation Diabetes : Destructionof islets of pancreas due to the accumulationof free radicalsis one of 1. Plasma antioxidants e.g. p-carotene, the causes for the pathogenesisof insulin- ascorbi caci d, bi l i rubi n,uri c aci d,cerul opl asmi n, dependentdiabetesmellitus. transferri n.

FREERADICALSAND ANTIOXIDANTS Cell membrane antioxidants e.g. d_toco_ _

:

ul

,- Intracellular antioxidanfs e.g. superoxide l ;nr.ltase,catalase,glutathioneperoxidase. I irtic::'{E#mt"{t#ffi##*flditrg E* rn:+;r maf*str# effi# ffi#Xie{a '

Enzymatic antioxidants e.g. superoxide : ^'rutase, catalase, glutathione peroxidase, -tathione reductase. N o n -enzy mati c antioxidants Nutrient antioxidants e.g. carotenoids (B-carotenel,a-tocopherol, ascorbic acid, s elen i u m .

659

(A) O, +or +2H+ (B) 2F42O2

Superoxidedismutase

catalase

H2Q2+02 > 2H2O+ 02

(c) Glutathione reductase

/

\*ooPH

+ H+

Fig. 34.2 : The antioxidant enzyme system (G- SHreduced glutathione; GS- gG-oxidized glutathione).

cr-Tocopherolcan directly act on oxyradicals (b) Metabolic antioxidants e.g. glutathione, (e.9.02, OH-, singletoxygen),and thus servesas c er ul o p l a s m i n , a l b u m i n , b i l i rubi n, an importantchain breakingantioxidant. transferrin, ferritin, uric acid Ascorbic acid (vitamin C) : lt is a vitamin that participatesin many normal metabolic reactions of the body. Ascorbicacid is an importantwaterThe antioxidant enzymes are truly the sol ubl eanti oxi danti n bi ol ogi calfl ui ds. V i tami n scavangersof free radicals.The major reactions C efficientlyscavangesfree radicals,and inhibits of these enzymes are depicted in Fig.34.2, some Iipid peroxidation. lt also promotes the regeneration of cr-tocopherol (from o,highlight sare g i v e n b e l o w . tocopheroxyl produced radical during Superoxidedismutase: lt convertssuperoxide scavengingof ROS). (O2) to hydrogen peroxide and 02 ffig3a.2A). Carotenoids : These are the natural This is the first line of defenseto protect cells compounds w i th l i pophi l i c properti es.A bout from the injurious effectsof superoxide. 500 different carotenoidshave been identified, Catalase: Hydrogen peroxide, produced by among them B-carotene is the most important. lt superoxidedismutase,is metabolisedby catalase can act as an antioxidant under low partial ffig3a.2R). pressureof C'-2.p-Caroteneusually functions in association with vitamins C and E. Lycopene,a Glutathione peroxidase: lt detoxifies H2O2 fat soluble pigment is a carotenoid. lt is (G-S H ) t o H2O , whi l e re d u c e d g l u ta th i o n e is responsible for (GS-SG). convertedto oxidized glutathione colour of certain fruits and The reduced glutathionecan be regeneratedby the vegetables (e.g. tomato). Lycopene possesses enzyme glutathione reductaseutilizing NADPH antioxidant propefty. Lutein and zeaxanthin are GigJa.2Q. The hexosemonophosphateshunt is also carotenoid pigmentsthat impart yellow or green colour to fruits and vegetables.These the major source of NADPH. pigmentscan also serve as antioxidants. F{utriesrt antis:cidaerts Selenium : lt is an essentialtrace element, Tocopherols (vitamin E) : Vitamin E is fat and is proved to be a significant antioxidant. soluble, and among the tocopherols, cr-toco- Selenium works with vitamin E in fighting free pherol is biologically the most active. lt is an radicals.lt is also requiredfor the function of an ant iox idantp re s e n t i n a l l c e l l u l a r me mb ranes, important antioxidant enzyme, namely and protects against lipid peroxidation. glutathione peroxidase. The amt$*,w&&nm9*ffieyms* 6:ywk*wn

'**il

B IOC H E MIS TR Y

660 c r - Li p o i ca c i d : l t i s v i ta mi n -l i k ecompound, produced in the body, besidesthe supply from plant an d a n i m a l s o u rc e s a. -L i p o i c a ci d pl ays a key role in recyclingother importantantioxidants such as ascorbic acid, cx,-tocopheroland glutathione. Besides the above, there are many other important nutrient antioxidants,some of them are listed below . Coen z y m eQ ro o f u b i q u i n o n efa m il y

E Vitamin (tocopherols)

vegetable oils Unprocessed (cottonseedoil,peanutoil, oil)wholegrains, sunflower legumes leafyvegetables,

Vitamin C

grapes) fruits(oranges, Citrus guava, green gooseberry (amla), (cabbage, spinach), vegetables melons cauliflower, green fruitsandvegeCanots, turnip, tables, spinach, apricots. Tomatoes, andtheirproducts pink papaya, (tomato sauce),

(ascorbic acid)

. Proanthocyanidins of grape seeds . Catechinsof green tea . Cur c u mi n o i d so f tu rm e ri c r Q uerc e ti no f o n i o n s ln the lable 34.2, some important nutrient antioxidants and their dietary sources are given. Consumption of a variety of nutrient antioxidants is important, since each antioxidant targets certain types of damaging free r adic al s .

.. .. .919Y9:.Y.?!91T.9191,....

greenleafy Eggyolk,fruits, Leutein andzeaxanthin vegetables, corn,greenpeas, Selenium

Seafoods,meats,organmeats, wholegrains

a-Lipoicacid

iedm;;i;iiv;;;t;;st oidiil i6d;i'#ni;6ei; 'eaii chicken.

Coenzyme Q,o

Metsbm&6* xmtisxida*x{s Glutathione : Reduced glutathione (CSH) play s a k e y ro l e i n th e b i o l o g i c a l a nti oxi dant enzyme system(SeeFi9.34.2O.CSH and H2O2 are the twin substrates for glutathione peroxidase.The reduced glutathione(GSH)gets regenerated from the oxidized glutathione (GS-SC) throughthe participationof glutathione reductaseand NADPH. lt is sugestedthat the ability to synthesize GSH decreasesas age

Dietarv Source

Antioxidant

tea redwine,green Onions, pomegranates walnuts, Berries, lemon. fruits(oranges), Citrus

EtgifiEtrleAl 1 ct-lHlcAt corucEFTs

Free radicals haue been implicated in the causation and progressof seueral diseasese.g. atherosclerosisand CHD, canceri respiratory diseases,aging. The estimation of serum malondialdehyde is ot'ten used fo qssessoxidatiue sfressond free radical damage to the body. The respiratory burst oJ macrophages, occompanied by the generation ot' ROS (HFz and HCIO), brings about bactericidal action, ond is beneliciol to the body. Dietary consumption ot' a variety of nutrient antioxidants (uitamins C and E, ft carotene, Iycopenes,Se, ftlipoic acid) is desirable sinceeoch antioxidont targets certain types of damaging free radicals.

Chapter 34: FREEHADICALSAND ANTIOXIDANTS

661

advances,and this has been implicatedin certain . Transferrin binds to iron and prevents ironcatalysed free radical formation. diseasese.g. cataract. There are many more metabolic antioxidants . Albumin can scavange the free radicals formed on its surface. of biological importance.A selectedfew of them are listed below Bilirubin protectsthe albumin bound free fatty . Uric acid, a powerful scavengerof singlet acids from peroxidation. oxygen (rOr) and OH- radicals. Haptoglobin binds to free hemoglobin and prevents the acceleration of lipid peroxi. Ceruloplasmin inhibits iron and copper dependentlipid peroxidation. dati on.

1. Free radicals are the moleculesor molecular speciescontaining one or more unpoired electrons with independent existence.e.g. Ot HzO2, OH-, 1O2.

2. ROS ore constantly formed during the normal cellular metabolism, (e.g. Iipid peroxidotion) and due to uariousenuironmental influences (e.g. ionizing radiotions).

3. Free radicals are highly reactiue and are capable of damaging almost all types of biomolecules(proteins, lipids, corbohydrates,nucleic acids),ond haue been implicated in the cousation of many diseasese.g. cardiouasculardiseoses,cance\ inflammatory diseases.

4. To mitigate the harmJul eft'ects oJ t'ree radicals, the aerobic cells houe developed antioxidont defense mechanisms-enzgmatic antioxidants (superoxide dismutase, catalase)and non-enzymotic antioxidants (glutathione, Se, a-tocopherol, ftcarotene).

Biochemistty

(ai r, normal temperature (despite cold and heat f nv i ro n me n t c o n s ti tu te sth e n o n -l i vi ng and Lwate r, l a n d ,e n e rg ye tc .)a s w e l l as the l i vi ng surroundi ngs)for opti mal physi ol ogi cal functi ons. ( biolo g i c aal n d s o c i a l )s y s te mss u rroundi ngman. bi ochemi cal E nv iro n m e n tabl i o c h e mi s tryp ri ma ri l ydeal sw i th t he me ta b o l i c (b i o c h e m i c a l ) re sponses and EXPOSURETO COLD adaptationsin man (or other organisms)due to Short-termexposureto cold causesshivering the environmentalfactors. (mainly due to skeletalmuscle)to produce extra A h e a l th y e n v i ro n me n t i s re qui red for a heat. Heat is generatedby the hydrolysisof ATP' healthylife which is however,not really possible or pr a c ti c a b l e .T h i s i s m a i n l y b e causeof the tw,+. g,,:t:t;j,i+'r e"t:94 r:li:*s;ie (c l i m a ti c ) and c h a nges at m o s p h e ri c Chronic exposure to cold results in nonp o l l u ti o n . env ir o n m e n ta l shi veri ng phase w hi ch i s characteri zed by Environmental biochemistry is a very vast severalmetabolic adaPtations. subject. The basic concepts regarding the Energy metabolism : Heat generation by a atmospheric changes and environmental processcalled chemical thermogenesisoccurs oollu ti o n o n h u ma n sa re d e a l t w i th here. in non-shiveringphase.The foodstuffsundergo oxidation to generateheat at the expenseof growth and other anabolic processes. Elevation in BMR, and increased intake oi foods are observed. T h e c l i m a ti c c h a n g e si n c l u d e col d, heat etc. The body makes every effort to maintain its

. Lipid metabolism : Storedfat (triacylglycerol) i n the adi poseti ssue i s mobi l i zed to supply

662

BIOCHEMISTRY ENVIHONMENTAL free fatty acidsfor oxidationand productionof energy. Brown edipose tissue, particularly in neonat al lif e , s i g n i fi c a n tl y c o n tri b u te s to t her m ogenes i s . . Hormonal changes : Thyroxine, a hormone closely associatedwith energy metabolism,is elevated.Further,corticosteroidsare increased on exoosureto cold.

EXPOSURETO HEAT There is a continuous generationof heat by the body due to th e o n g o i n g b i o c h e mi cal processes,referred to as metabolic heat. This heat has to be exchangedwith the environment to maintain a constant body temperature.On exposureto heat in surroundings,as happensin sLrmmer, the body is subjected to an uncomfortablesituation(sincetemperatureof the s ur r oundingsis m u c h h i g h e r th a n th a t o f th e body). However, heat is still lost from the body throughsweatingand evaporation.Normally,the body (thermoregulation)gets acclimatized to highert em per a tu rew i th i n 3 -5 d a y s .

t

663 The term pollutant refers to a substance w hi ch i ncreasesi n quanti ty due to human activity and adversely affects the environment (e.9.carbon monoxi de,sul fur di oxi de, l ead).A substancewhich is not present in nature but rel eased duri ng human acti vi ty i s the contaminant (e.g. methyl isocyanate, DDT, malathion).A contaminanthowever, is regarded as a pollutant when it exertsdetrimentaleffects. E nvi ronmentalpol l uti on may be consi deredi n different ways-industrial pollution; agricultural pollution;pollution due to gaseouswastes,liquid wastesand solid wastes.Environmentalpollution with reference to air, water and foodstuffs is briefly discussed. A IR P OLLU TION

The maj or componentsof ai r i ncl udeni trogen (78.1'/.), oxygen (20.93%) and carbon dioxide (0.03%),along with water vapour and suspended particles.The rapid growth of industriescoupled w i th changi ng l i festyl esof man (urbani zati on, smoking, use of motor vehicles etc.) largely contri buteto ai r pol l uti on.The maj or chemi cal constituentsof air pollution are sulfur dioxide, Heat stroke : lt is characterizedby the failure oxides of carbon (CO2 and CO), oxides of of the heat regulatorysystem(thermoregulation) nitrogen, hydrocarbons and particulates.The of the body. The manifestationsof heat stroke biochemical affects of air pollution are i n c lude high bo d y te mp e ra tu re ,c o n v u l s i o ns, described. partial (some times total) loss of consciousness. In extreme cases, heat stroke may cause $ulfur elFoxrs!+; irreversibledamage to brain. The treatmentfor Sulfur dioxide (SO2) is the most dangerous the heat stroke involves rapid cooling of the pollutantgas to man. lndustrialactivitiessuch as body. burni ng of coal and oi l emi t l arge quanti ti esof The milder form of heat stroke is referredto soz. as heat syncope. Although the body temperature S ul fur di oxi de pol l uti on pri mari l y affects i s not r ais edm u c h i n th i s c o n d i ti o n ,th e b l o o d respiratory system in man. lrritation of the pressure falls and the person may collapse respiratory tract and increasingairway resistance suddenly. Heat syncope is easily reversible. (breathingdifficulty) are observed. Lung tissue may get damaged due to acidic pH. Further, di pal mi tyl l eci thi n, the phosphol i pi dacti ng as the lung surfactant,gets affected. Continuous exposure to SO2 (> 1 ppm) for several days Environmentalpollution may be regardedas causesbronchi ti sand i n some i ndi vi dual sl ung the addition of extraneous(foreign)materialsto cancer.AtmosphericSO2when dissolvedin rain air, water or land which adverselyaffects the water becomesvery acidic (acid rain) damaging q ualit y of lif e. P o l l u ti o n ma y b e c a u s e d by soil, plantsand vegetables.Exposureof plantsto phy s ic al,c hem ic a lo r b i o l o g i c a lp ro c e s s e s . SO2 destroys leaves.

664 earbon

BIOCHEMISTFIY

rnonoxide

N i trogendi oxi de (i n the form of H N O3) along with SO2 (as H2SOa)contributes to acid rain.

Carbon monoxide (CO) is mostly produced by incomplete combustion of fuel or carbonH ydrocarbons c ont a i n i n g c o m p o u n d s .Au to mo bi l es,ai rcrafts, Many hydrocarbonspollutingthe environment r ail e n g i n e s a n d b u rn i n g o f c o al i n factori es affect human life. The aromatic hydrocarbons c ont ri b u teto C O o o l l u ti o n . causei rri tati onto i nj uri es. C a rb o nm o n o x i d ec o m b i n e sw i th hemogl obi n to form carboxyhemoglobin (Refer Chapter 10). P arti cul ates This causesa drastic reduction in the supply of The solid dust particles suspended in the 02 to tissues.At a CO concentrationaround l ppm , i mp a i rm e n t i n m e n ta l p e rf ormanceand atmosphereconstitute particulates.The sources visual perception take place. With a further of particulatesare grinding, spraying, erosion, increasein CO level, headache,dizzinessand smoki ngetc. los s o f c o n s c i o u s n e s so c c u r. D eath may be inevitablein personsexposedto above 750 ppm of CO. Car h o n

disxide

The particulateshave ill-affectson humans. Theseinclude interferencein respiratoryfunction (coughing,sneezing)and toxicity causedby the absorpti on parti cul atechemi cal s. Further , t he dust particles carry microorganismsand other infectiveagentsto spreaddiseases.

C a rb o n d i o x i d e (C Oz ), c o n s tituti ngonl y a fraction (0.03%)of the atmosphericgases,plays a s ig n i fi c a n ro t l e i n c o n tro l l i n gth e cl i mate.Thi s Ozsne layer is done by trapping the heat radiationfrom the Ozone is formed from atmosphericoxygen earth's surface.Without the presenceof CO2, duri ng hi gh energy radi ati ons of el ectr ical the earth would be as cold as moon! discharges.This ozone forms a layer above the (15-35 km). lt absorbs harmful Carbon dioxide is often referred to as earth's surface radiations of sun which would ultraviolet greenhouse Bas. The term greenhouse and mutations, cause skin diseases effect refers to an elevation in CO2 near earth's otherwise of earth. increasing the temperature surface that traps sunlight and increases besides atmospheric temperature. Deforestation, bur n i n g o f c o a l , o i l s e tc ., e l e v a teatmospheri c CO2 resulting in greenhouseeffect. Hence the global propagandafor increased plantation of trees!

In recentyears,a decreasein the ozone layer i s observeddue to chemi calpol l uti on i n the air . Nitrogen oxides (releasedfrom enginesof aeroplanes)and chlorofluorocarbons(usedin refrigerators and air conditioners)deplete the ozone laver.

F o rtu n a te l yma , rg i n avl a ri a ti o n si n atmospheri c CO2 are tolerated by the cells. The body gets r:dATElt POLLUTIOH adapted to prolonged exposure to higher concentrations of CO2 (evenupto 1%) with minor Water is the most predominantconstituentof alterationsin electrolytebalance. living matter. The very existence of life is uni magi nabl ew i thout w ater. fltlEtrogen dioxide As such, pure water does not exist in nature. N i tro g e nd i o x i d e(N O 2 )l i k e c a rb onmonoxi de The avai l abl e w ater contai ns di ssol vedgases, (CO), combines with hemoglobin and reduces mi neral s and some suspended particles. the supply of 02 to the tissues.NO2 is more Pollution of water occurs due to waste disposal harmfulto human healththan CO. lt is fortunate from i ndustri es,agri cul tureand muni ci palit ies. that the atmosphericconcentrationof NO2 is The pol l utants may be organi c, i norganic, relativelvlower. sediments,radioactive,thermal etc., in nature.

." .JIi . ENVIFIONMENTALBIOCHEMISTHY

66s

i r?G4$Sdf P0&I'{,t7-ArVfS

rodenticides.Basedon their structure,pesticides are classifiedas follows. The organicpollutantsinclude agentscarrying (a) Chlorinated hydrocarbons : e.g. aldrin, water borne diseases,oxygen demandingwastes di el dri n,endri n,di chl orodi phenyl tri chl oroand or ganicc h e mi c a l s . ethane(D D T). ! ' f ; r ler . bor n e d i s e a s e a g e n ts (b) Organophosphates : e.g. malathion, Severalpathogenicorganismsfind their entry di azi non. into water and cause diseases.The water borne (c) Carbamatese.g. baygon, carbaryl (sevin) disease include typhoid, paratyphoid, cholera, (d) Chforophenoxy e.g. 2,4-dichlorophenoxy am oebias is g , i a rd i a s i sa n d i n fe c ti o u sh e p ati ti s. acetic acid. Thesediseasescan be preventedby disinfection techniquesemployed for the treatmentof water. The useof pesticideshas helped in controlling certain diseases (malaria, typhus), besides 'l}'clr_err du'rmanding waste$ boosting food production. However, pesticides Sewage, and wastes from industries and pollute water and cause several health agricultureprovide good nutrientsfor algae. As compl i cati ons to humans, besi des damagi ng the algae grow utilizing the wastes, oxygen acquati cl i fe. depletion occurs. This phenomenon of water Dichloro-diphenyltrichloroethane(DDT) is a deoxygenation is technically referred to as widely used pesticide to control cotton and eutrophication. As a consequence of peanut pests, besides malaria. However, eut r ophic at i o nfi, s h a n d o th e r a c q u a ti ca ni mal s conti nuoususeof D D T l eads i ts to accumul ati on die (due to lack of O2), causingfoul smell. in foods causing ill effects (hence banned in countri esl i ke U S A ). some #r gar *i* c h e m Ec a l s DDT, being fat soluble, accumulates in the T he or ga n i c c h e m i c a l p o l l u ta n ts o f w ater adipose fissue and is not excreted. Thus, its include pesticides and several synthetic concentration in the body goes on increasing. compounds (detergents, paints, plastics, DDT causes nervous irritability,muscletwitching pharmaceuticals,food additivesetc.) and convul si ons. F es t ic ides A l dri n and di al dri n are al so fat sol ubl e and Pesticides is a broad term used for their effectson humansare comoarablewith that herbicides, fungicides insecticides, and of DDT.

BIOMEDICAL/CLINICALCONGEPTS

The body makeseuergeflort to malntqin its normal temperature,despite cold and heat surroundings,t'or optimal physiologicaland biochemical functions. Failure of heat regulatory system (thermoregulation) Ieads to heot stroke chorocterized by high body temperature, conuulsions etc. Sulfur dioxide (SOz)is the most dangerouslndustriol pollutant gas to man. lt primarilg affects

the respirotory

system, and

mov

result

in bronchifis,

and euen lung

cancen

Corbon monoxide (CO) combines uifh hemoglobin to form carboxyHb. This reduces 02 supply to fissues. rs Pollution ol water with pathogenic organisms couses many diseases e.g. typhoid, cholera, omoebiasis. r* Lead toxicity at'fectscentrol neruous system-learning disabilities, mental retqrdation etc.

]

666

Organophosphates and carbamates are powerful neurotoxic agents. They prevent the t r ans m i s s i o n o f n e rv e i m p u l s e b y i n hi bi ti ngthe enzyme cholinesterase.

BIOCHEMISTFIY

consumi ngfi sh contai ni ng methyl mercury, a s i ndustri alpol l utant). i -3 4 4

: r: -

rrr

r!

rruoFcAntc pof[urAryrs

The outbreak of cadmium toxicity was reported in Japan in the form o( itai itai or ouch Hea v y me ta l s (l e a d , me rc u ry , cadmi um, disease. Cadmium poisoning causes fragile alum in i u m ,a rs e n i ce tc .)a re th e mo st dangerous bones, anemia, bone marrow disorders and am ong th e i n o rg a n i cp o l l u ta n ts . ki dney damage. B i ochemi cal l y, cadmi um replaceszinc and adversely influencesseveral ilean€j metabolic reactions. Lead is the most common inorganicpollutant found in water, air, foods and soils.The sources of lead p o l l u ti o ni n c l u d ep e tro l ,g a s o l i ne,pai nts, cigarettes,news papers, lead pipes and xerox copies. The plasma concentrationof > 25 1tg/dl in adu l ts a n d > 1 0 p g l d l i n c h i l d ren resul tsi n toxic manifestations. The principal targetof lead toxicity is central nervous system. In the growing children, Pb c aus esl e a rn i n gd i s a b i l i ti e sb, e h a v i o uralchanges (hyperexcitability)and mental retardation. In adult s, c o n fu s i o n , i rri ta b i l i ty ,a b d o mi nal col i c and severe anemia are associatedwith lead toxicity. Lead inhibits several enzymes, particularly, 6-aminolevulinate (ALA) synthase, ALA dehydrataseand ferrochelatase of heme synthesis (Refer Chapter 10 also). This results in severe anemia.There has been an increasingawareness worldover on the toxic manifestationsof lead. This has lead to the supply of unleaded petrol in most countries. flfler*rlryt M erc u ry i s a c o m m o n i n d u s tri al (pl asti c, paints,electricalapparatus,fungicides)pollutant. Acute mercuric poisoning causes gastritis, v om it in g a n d p u l mo n a ry e d e m a . C hroni c manifestations of Hg include emotionalchanges, loss of memory and other neuropsychiatric dis t ur b a n c e sl n. a d d i ti o n ,d e p o s i ti o nof mercuri c salts mav cause renal failure. O r g a n i c me rc u ri c p o i s o n i n g i s commonl y referred to as rninamata disease (as it first oc c ur re d i n M i n a ma ta , J a p a n i n 1953-60 by

+!(irYI,ftr:;;,ir;* The sourcesof al umi ni um i ncl ude cooki ng vessels,building materials,food additives and cosmetics.Aluminium toxicity is associatedwith Alzheimer'sdisease,anemia and osteomalacia. r'rirtrd.

fi : i'

A rseni c, commonl y found i n many insecticidesand fungicides,is toxic to the body. Arsenic binds with-SH groups of several enzymesand i nhi bi tsbi ochemi calreacti onse. g. pyruvatedehydrogenase. Further,arseniccauses coagulation of proteins and blockage of ATP generation(functionsas an uncoupler). N OIS E P OI.LU TIGN The unw anted sound i s noi se, w hi ch i s a major urban environmentalpollutant. Man can tol eratenoi se upto 100 deci bel s(speaki nB - 60 decibels;telephonebell 70 decibels;motor cycle 110 deci bel s; rockets 170 deci bel s).A noi se above 150 deci bel si s uncomfortabl e. The affects of noise pollution include headache,increasedblood pressure,irritability, neuromuscul artensi on, confusi on, di sturbed vi si on and di gesti on,depressi onand l oss o f heari ng. RADIGAGTIVE

POTLI'TilOI$

The pollution due to radioactivesubstancesis the most dangerousto human life. The health hazards of radi oacti ve ool l uti on i ncl ude gene mutatrbns, cancer, destruction of living cells etc.

I:-It::.!" i;'i , ENVIRONMENTALBIOCHEMISTRY

TOXiE COMPOUNDS lid FGOE}STUFFS

667 Anti-vitamins : Avidin of raw egg is a good example of anti-vitaminof biotin.

The foodstuffsconsumedby humans contain g r uf f t* T.t' rr F;;"':gl*rflf *ur a irit r; erf ri*i.,ol.d severaltoxic compounds which may be either The foodstuffsmay get polluted with several normally presentor enter foodstuffsduring the toxi c chemi cal s w hi ch mi ght occur duri ng course of cultivation, processingor storage. cultivation, processingor storage. :',.1 ril t':;-'lti! {i:: 4i;,ri::: 1,r",f t:,,,;,tie;*qgffq-:

Neurotoxins : Kesari dal (Lathyrussativus) is pulse grown in some partsof Madhya Pradesh, a Bihar and Uttar Prodesh.Excessive consumption of kesari dal causes paralysis of lower limbs referred to as lathyrism. This is due to a p-oxalylaminoalanine neurotoxin namely (BOAA). BOAA damagesupper motor neurons, and inhibits the enzyme lysyl oxidase (reduces c ollagen c ro s s -l i n k i n g ).C o o k i n g o f k e s ari dal 2-3 times and removal of the supernatantwater will elim ina teth e to x i n .

Cultivation : Pesticidesand other unnatural chemicals used during cultivation do find an entry into the foodstuffs.lt is fortunate that most of these chemicals can be removed by peeling the outer layersof vegetablesand fruits, besides repeatedwashings. Processing: Defectsin freezing,and packing provide a suitableenvironmentfor the growth of severalorganismswhich releasetoxic products e.g. milk contaminationby Salmonella.

Several food additives are in use for preservationand enchancingflavour. Not all of Proteaseinhibitors : Certain legumes (soya them are safee,g. ani l i nedyes usedas col ouri ng bean, peanut) contain inhibitors of protease agents are carcinogenic; sweetening agent enzymesparticularlytrypsin. Normally, protease cyclamate may cause bladder cancer. inhibitors are destroyedby cooking. However, Storage : Contamination of stored foods partial cooking does not totally destroythem. In occurs mostly due to fungal infections. s uc h a c as e , p ro te a s e i n h i b i to rs c a n inhi bi t Aflatoxins are produced by Aspergillus favus digestionand proteins. when ground nuts or coconuts are stored in Goitrogens : These compounds prevent moist conditions.Aflatoxinsare heoatotoxicano upt ak eand u ti l i z a ti o no f i o d i n e b y th y ro i dgl and. carcinogenic. Goitrogens are found in cabbage and turnips (thioglycosides),mustard and rape seed oils ( t hioc y ana te s ),g ro u n d n u ts a n d a l m onds ( poly phenoi cl g l y c o s i d e s ). Biogenic amines : Bananas and cheese c ont ain bio g e n i c a m i n e s n a m e l y h i s t ami ne, tryptamine,tyramine serotoninand epinephrine. In normal metabolism, they are degraded by m onoam ine o x i d a s e (M AO). H o w e v er, i n personstaking MAO-inhibitors, the foodstuffs with amines may cause hypertension.

The group of chemi cal sthat causecancer i n man and animals are collectively referredto as carcinogens(Refer Chapter 3V. Environmental pol l uti on i s undoubtedl y associ ated w i th increasedrisk of cancer.The topic 'cance/ may be considered as a part of environmental bi ochemi stryfor l earni ngpurpose.

568

B IOC H E MIS TR Y

1. Enuironmentol biochemistry deals with the biochemical responsesand odaptations in man (ond other organisms)due to enuironmental foctors. 2. The atmospheric (climottc) changes like cold and heat inJluence the body. Seuerol metabolic adaptotions occur to ouercome the aduerseoffects. 3. The major chemical constituentsof oir pollution include SO2, CO, Ca2 ond oxides of nitrogen. Among these, sulfur dioxide is the most dongerous.

4. Water pollution occurs mainly due to r.oostedisposal lrom industries,agriculture and municipalities. The pollutants may be organic (pathogenic organisms, pesticides),or inorganic (leod, mercurg).

5. The Joodstuffsconsumed by humans may contain seueraltoxic compounds. These may be normally present (e.g. BOAA causing lothyrtsm)or enter the loodstulls during the course of cultiuation (e.9. pesticides),or storage (e.g. aflatoxins).

andDiabetes Mellitus

iabetes mellitus is the third leading cause ll lJ of death (after heart diseaseand cancer) in many developedcountries.lt affectsabout 2 to 3% of the generalpopulation.The complications of diabetes affect the eye, kidney and nervous system.Diabetesis a major cause of blindness, renal failure, amputation, heart attacks and stroke.(The term diabetes,whenever used, refers to diabetes mellitus. lt should, however, be noted that diabetes insipidus is another disorder characterized by large volumes of urine excretion due to antidiuretic hormone deficiency).

An important feature of diabetes is that the body cells are starved of glucose despite its very high concentrationaround i.e. scarcityin plenty. For a comprehensiveunderstandingof diabetes,the relevanthormones,namely insulin and glucagon, homeostasisof blood glucose, besidesthe biochemicalaspectsof diabetes,are di scussedi n thi s chaoter.

lnsufin is a polypeptide hormone produced by the B-cells of islets of Langerhans of Diabet es me l l i tu s i s a c l i n i c a l c o ndi ti on pancreas. lt has profound influence on the characterized by increasedblood glucose level metabolism of carbohydrate,fat and protein. (hyperglycemia)due to insufficient or inefficient Insul i ni s consi deredas anabol i chormone,as i t synthesis of glycogen, ( inc om pet e n t)i n s u l i n .l n o th e r w o rd s , i n sul i n i s promotes the triacylglycerols proteins. and This hormone has produced either not in sufficient quantity or inefficientin its action on the targettissues.As a been implicated in the developmentof diabetes consequence,the blood glucoselevel is elevated mel l i tus. whic h s pillso v e r i n to u ri n e i n d i a b e te smel l i tus Insul i noccupi esa speci alpl ace i n the hi story (Creek : diabetes-a siphon or running through; of bi ochemi stryas w el l as medi ci ne.Insul i nw as mellitus-sweet). the first hormone to be isolated, purified anq

669

670

BIOCHEMISTF|Y

synthesized;first hormone to be sequenced;first hormone to be produced by recombinantDNA technology.

Structure of insulin contains Humaninsulin(mol.wt. 5,7341 5l amino acids, arranged in two polypeptide c hains .T h e c h a i n A h a s 2 1 a mi n o a c i dsw hi l e B has 30 amino acids. Both are held together by two interchaindisulfide bridges,connectingA7 to 87 and A2s to 819. In addition, there is an intrachaindisulfide link in chain A betweenthe am ino a c i d s 6 a n d 1 1 .

Preproinsulin

Biosynthesis of insulin of theislets Insulinis produced by thep-cells The genefor this of Langerhans of pancreas. protein synthesisis locatedon chromosome1 1. The synthesisof insulin involvestwo precursors, nam ely p re p ro i n s u l i nw i th 1 0 8 a m i no aci ds ( m ol. wt. 1 1 ,5 0 0 )a n d p ro i n s u l i nw i th 86 ami no acids (mol. wt. 9,000). They are sequentially degraded (Fig.36.l) to form the active hormone insulin and a connecting peptide (C-peptide). Insulinand C-peptideare producedin equimolar concentration. C-peptide has no biological activity, however its estimation in the plasma serves as a useful index for the endogenous pr oduc ti o no f i n s u l i n . I n t h e p -c e l l s , i n s u l i n (a n d a l s o p roi nsul i n) combines with zinc to form complexes.In this form, insulin is stored in the granules of the cytosol which is releasedin responseto various stimuli (discussedbelow) by exocytosis.

Regulation of insulin secretion About40-50unitsof insulinis secreted daily Thenormalinsulinconcenby humanpancreas. trationin plasmais 20-30pUlml. The important factorsthat influencethe releaseof insulinfrom the p-cellsof pancreas arediscussed hereunder.

B-chain Ineulin C-peptide

meal).A risein bloodglucoselevelis a signal for insulinsecretion. 1. Factors stimulating insulin secretion : These include glucose, amino acids and . Amino acids inducethe secretionof insulin. gastrointestinal hormones. the ingestion observed.after Thisis particularly mealthat causestransientrise of protein-rich . Glucoseis the most importantstimulusfor Among in plasmaamino acid concentration. insulin release.The effect is more predothe amino acids, arginineand leucine are minantwhen glucoseis administered orally (eitherdirect or througha carbohydrate-rich potentstimulators of insulinrelease.

C:'apter sE : INSULIN,GLUCOSEHOMEOSTASIS, AND DIABETESMELLITUS

*letaholism

Net effect

Crbotrydrate metabolism ' Glycolysis

677

Effect on important enzyme(s)

Increased

Z Gluconeogenesis

Decreased

3. Glycogenesis 4. Glycogenolysis 5. HMP shunt

Increased Decreased Increased

Glucokinase 1 Phosphofructokinase t Pyruvate kinase t carboxylase J Srruvate pyruvate Phosphoenol carboxykinase J Glucose o-phosphatase J Glycogen synthetase t phosphorylase Glycogen J Glucose 6-phosphate dehydrogenase'f

Increased Decreased Decreased

Acetyl CoAcarboxylase t Hormone sensitive lioase J HMGCoAsynthetase J

Increased Decreased

RNApolymerase t Transaminases J Deaminases J

Li*l metabolism 6. Lipogenesis 7. Lipolysis 8. Ketogenesis Protein metabolism 9. Protein synthesis 10. Protein degradation

. Gastrointestinal hormones (secretin, gastrin, pancreozymin) enhance the secretion of insulin. The GIT hormonesare releasedafter the ingestionof food. 2. Factors inhibiting insulin secretion : Epinephrine is the most potent inhibitor of insulin release. In emergency situations like stress, extreme exerciseand trauma, the nervous system stimulates adrenal medulla to release epinephrine. Epinephrine suppresses insulin release and promotes energy metabolism by mobilizingenergy-yieldingcompounds-glucose from liver and fatty acids from adiposetissue. Degradation

of insulin

F#etabolic

cffects

of insulin

lnsulin plays a key role in the regulationof carbohydrate, lipid and protein metabolisms (Table 35.1). lnsulin exerts anabolic and anticatabolicinfluenceson the body metabolism. 1 . Effectson carbohydrate metabolism : In a normal individual, about half of the ingested glucose is utilized to meet the energy demands of the body (mainly through glycolysis).The other half is converted to fat (- 40%) and glycogen (- 10%). This relation is severely i mpai redi n i nsul i ndefi ci ency.Insul i ni nfl uences glucosemetabolismin many ways. The net effect is that insulin lowers blood glucose level (hypoglycemic promoting effect) by its utilization and storage and by inhibiting its production.

ln the plasma,insulin has a normal halfJifeof 4-S minutes. This short half-life permits rapid metabolic changes in accordance to the alt er at ion si n th e c i rc u l a ti n g l e v e l s o f i nsul i n. . Effect on glucose uptake by tissues z Insulin is This is advantageous for the therapeutic required for the uptake o{ glucose by muscle (skeletal, cardiac and smooth), adipose tissue, purposes.A proteaseenzymef namely insulinase (mainly found in liver and kidney), degrades leukocytesand mammary glands.Surprisingly, ins ufin. about 80% of glucose uptake in the body is

672

B IOC H E MIS TR Y

utilization of acetyl CoA for oxidation (Krebs not d e p e n d e n to n i n s u l i n .T i s s u e si nto w hi ch glucosecan freely enter include brain, kidney, cycle) and lipogenesis. Therefore, the availability of acetyl CoA for ketogenesis,in erythrocytes,retina, nerve, blood vesselsand the normal circumstances,is very low intestinal mucosa. As regards liver, glucose entry into hepatocytes does not require 3. Effectson protein metabolism : Insulin is ins u l i n . H o w e v e r, i n s u l i n s ti mu l a tesgl ucose an anabolic hormone. lt stimulatesthe entry of ut ili z a ti o n i n l i v e r a n d , th u s , i ndi rectl y amino acids into the cells, enhances protein promotes its uptake. synthesisand reducesprotein degradation. . Effect on glucoseutilization : Insulin increases Besides the metabolic effects described glycolysis in muscle and liver. The activation above, insulin promotes cell growth and as well as the quantities of certain key repl i cati on.Thi s i s medi ated through certa in enz y m e s o f g l y c o l y s i s ,n a me l y g l ucoki nase factors such as epidermal growth factor (EGF), (not hexokinase) phosphofructokinaseand platelet derived growth factor (PDGF) and py r u v a te k i n a s e a re i n c re a s e d b y i nsul i n. prostagland ins. Clycogen production is enhanced by insulin by increasing the activity of glycogen Mechani sm sf aeti on of i nsul i n synthetase. lt is now recognized that insulin binds to . Effect on glucose production : Insulin plasma membrane receptorspresenton specific decreasesgluconeogenesisby suppressingthe target tissues,such as muscle and adipose. the enzymes pyruvate carboxylase,phosphoenol in a series of reactions ultimately This results pyruvate carboxykinase and glucose 6to the biological action. Three distinct leading pho s p h a ta s e .In s u l i n a l s o i n h i b i ts gl ycoi nsul i n acti on are know n. On e of mechani sms genolysisby inactivatingthe enzymeglycogen induction of transmembrane with the concerned phosphorylase. (si gnal transducti on),second w i th the si gnal s 2. Effectson lipid metabolism : The net effect glucosetransportacrossthe membraneand the of ins u l i n o n l i p i d m e ta b o l i s mi s to reducethe third with induction of enzyme synthesis. release of fatty acids from the stored fat and 1. Insulin receptor mediated signal trans' decrease the production of ketone bodies. duction Among the tissues,adipose tissue is the most sensitiveto the action of insulin. Insulin receptor : lt is a tetramerconsistingof . Effect on lipogenesis : Insulin favours the synthesis of triacylglycerols from glucose by providing more glycerol 3-phosphate (from gly c o l y s i s )a n d N AD PH (fro m HMP shunt). Insulin increasesthe activity of acetyl CoA carboxylase, a key enzyme in fatty acid synthesis. . E f f e c t o n l i p o l y s i s : l n s u l i n d e creasesthe activity of hormone-sensitivelipase and thus reduces the releaseof fany acids from stored fat in adiposetissue.The mobilizationof fatty ac id s fro m l i v e r i s a l s o d e c re a s e dbv i nsul i n. I n t h i s w a y , i n s u l i n k e e p sth e c i rc ul ati ngfree fatty acids under a constantcheck.

4 subunits of two types and is designatedas a2p2. The subunitsare in the glycosylatedform. They are held togetherby disulfidelinkages.The . w t. 135,000)i s extracel l ul ar and cx-subuni(mol t i t contai ns i nsul i n bi ndi ng si te. The p-subunit (mol. wt. 95,000) is a transmembraneprotein w hi ch i s acti vatedby i nsul i n.The cytopl asmic domain of p-subunithas tyrosinekinaseactivity. The insulin receptoris synthesizedas a single polypeptide and cleaved to a and p subunits The i nsul i n recep t or w hi ch are then assembl ed. has a hal f-l i feof 6-12 hours. There are abo ut 20,000 receptorsper cell in mammals.

Signaltransduction : As the hormone insulin . Effect on ketogenesis : Insulin reduces binds to the receptor,a conformationalchange ketogenesisby decreasingthe activity of HMG of i nsul i n receptor . i s i nduced i n the cr-subuni ts CoA synthetase.Further,insulin promotesthe Thi s resul tsi n the generati onof si gnal sw hi ch

Ghapter 36 : INSULIN,GLUCOSEHOMEOSTASIS, AND DIABETESMELLTTUS

acti vi ty of i ntracel l ul ar p-subuni t of i nsul i n receptor.This causesthe autophosphorylation of tyrosineresidueson B-subunit.lt is believedthat receptor tyrosine kinase also phosphorylates insulin receptor substrate(lRS). The phosphorylated lRS, in turn, promotesactivationof other protein kinasesand phosphatases, finally leading to biological action (Fig.36.2).

It,i-

Cytoplasm

Cytoplasm

673

\,rr_-u, ,l

2. Insulin-mediated glucose transport : The bi ndi ngof i nsul i nto i nsul i nreceptorssi gnal sthe transl ocati on of vesi cl es contai ni ng gl ucose transporters from intracellular pool to the plasma membrane. The vesicles fuse with rne membrane recruiting the glucose transporters. The glucosetransportersare responsiblefor the i nsul i n-medi ated uptakeof gl ucoseby the cel l s. As the insulin level falls,the glucosetransporters move away from the membrane to the intracellular pool for storage and recycle (Fig.s6.3).

3. Insulin mediated enzyme synthesis : Insulin promotesthe synthesisof enzymessuch as gl ucoki nase, phosphofructoki nase and pyruvate kinase. This is brought about by are transducedto p-subunits.The net effect is i ncreased transcri pti on (mR N A synthesi s), that insulinbindingactivatestyrosinekinase followed by translation(protein synthesis). Flg. 36.2 : lnsulin receptormediatedsignal tnnsduction (IRS-l nsulin receptorsubstrate).

Flg. 36.3: lnsulinmediatedglucosetrunsport.

.-t

674

Clucagon, secretedby a-cells of the pancreas, opposesthe actionsof insulin.lt is a polypeptide hormonecomposedof 29 amino acids (mol. wt. 3, 500) i n a s i n g l e c h a i n . C l u c a g o n i s actual l y synthesizedas proglucagon (mol. wt. 9,000) which on sequentialdegradationreleasesactive glucagon. Unlike insulin, the amino acid sequence of glucagon is the same in all mammalian species(so far studied).Clucagon has a short half-life in plasma i.e. about 5 minutes. f, ++qu[eti tr ! af q! Nle;tg€'re li€rcrr!.fidi*l

B IOC H E MIS TR Y

Glucose is carbohydrate curuency of the body. A n adul t human body contai nsabout 1B g free glucose. This amount is just sufficient to meet the basalenergy requirementsof the body for one hour! The liver has about 100 g stored glycogen. Besidesthis, it is capable of produci ngabout 125-150mg gl ucose/mi nute or 180-220 {24 hrs.

Expressionof glucose concentration : In most developed countries,plasma glucose(insteadof The secretion of glucagon is stimulated by glucose) blood is estimatedand expressedas Sl Iow blood glucose concentration, amino acids (mmol/l). units This is not however so, in protein derived from dietary and low levels of developing countries for practical reasons. lt epinep h ri n e . In c re a s e d b l o o d g l u cose l evel may plasma concentration be noted that the markedly inhibits glucagon secretion. of glucose is slightly higher (about 15%) than blood glucose. Further, a glucose ftiletahe;iit: ei'fe:lts oi qgir.l*;:r';nn concentrationof 180 mg/dl (plasma or blood) Clucagon influencescarbohydrate,lipid and corresponds to 10 mmol/|. In this book, protein metabolisms.In general, the effects of expressionof blood glucose as mg/dl is more this hormone oppose that of insulin. frequently used. 1. Effects on carbohydrate metabolism : A heal thyi ndi vi duali s capabl eof mai ntai ni n g Glucagon is the most potent hormone that the blood glucoseconcentrationwithin a narrow enhancesthe blood glucoselevel(hyperglycemic). range. The fasting blood glucose level in a postPrimarily, glucagon acts on liver to cause absorptive state is 70-100 mddl (plasmaglucose increasedsynthesisof glucose(gluconeogenesis) 80-120 me/dl ). Fol l ow i ng the i ngesti on of a and enhanced degradation of glycogen carbohydrate meal, blood glucose may rise to (glycogenolysis). The actions of glucagon are '120-'l40mg/dl. The fastingblood glucosevalue mediatedthrough cyclic AMP (Chapter t3). i s comparati vel y l ow er i n rumi nant ani mals 2. Effects on lipid metabolism : Clucagon (sheep 30-aOmg/dl; cattle 50-60 mg/dl), while it promotes fatty acid oxidation resulting in is higher in birds (250-300 mg,/dl). energy production and ketone body synthesis (ketogenesis). The term hyperglycemia refers to an increase in the blood glucose above the normal level. 3. Effectson protein metabolism : Glucagon Hypoglycemia represents a decreased blood increasesthe amino acid uptake by liver which, glucose concentration.Excretionof glucose in in turn, promotes gluconeogenesis. Thus, urine is known as glycosuria. The concentration glucagon lowers plasma amino acids. of blood glucoseis dependenton the quantityof glucosethat entersthe circulation from various Meehanisrn of ;.icti*"rri #[ g{e.E$fil{g}f, sources (dietary carbohydrates,glycogenolysis, Clucagon binds to the specific receptorson gluconeogenesisetc.) and the amount that is the plasma membrane and acts through the utilized for different metabolic purposes mediationof cyclic AMP, the secondmessenger. (glycolysis,glycogenesis,fat synthesisetc.) as The details are given elsewhere(Chapter l9). illustratedin Fig.35.4.

Ghapter 35 : INSULIN,GLUCOSEHOMEOSTASIS, AND DIABETESMELLTTUS Dietarycarbohydrates (starch,sucrose,glucose) \ \ Digestionandabsorption \ Glycogenolysis \ in muscle Glucosein liver

Horrrlonal requlatior,'

l

+ BI-OODGLUCOSE Fasting70-100mg/dl

675

Glycolysis and TCAcycle Glucose-+ COr,HrO Glycogenesis in liverandkidney Synthesisof other monosaccharides and aminosugars

Post-prandial 120-140mg/dl

Glycogenolysis in liver

I + Excretedinto urine(>'180mg/dl bloodglucose)

HMPshuntfor pentoses andNADPH Synthesisof fat i J t;l ,z ai i J n , j f DioL-r(lgi-rcr)srl

Sourcesof blood glucose

Sources of bloo
8 roo o

2. Gluconeogenesis : The degradationof ar€ glycogenin muscleresultsin the formation 5 of lactate.Breakdownof fat in adiposetissue oo will producefree glyceroland propionate. 0 Lactate,glycerol,propionateand someamino Mid- Breakfast Lunch Dinner Midnight acids are good precursors for glucose night synthesis (gluconeogenesis) that actively Fig. 36.5 : Sources of blood glucose during occursin liverand kidney.Cluconeogenesis a normal day (24 hours). continuously addsglucoseto the blood.Cori cycle is responsible for the conversionof musclelactateto glucosein liver. liver (weighingabout 1.5 kg) can provideonly g of blood glucosefrom glycogen,that 40-50 3. Glycogenolysis : Degradationof glycogen can last only for a few hoursto meet the body freeglucose.Thisis in contrast in liverproduces requ irements. where glucoseis not to muscleglycogenolysis formed in sufficientamountdue to lack of the enzyme glucose i-phosphatase.However, the contributionof liver glycogenolysis to blood glucoseis ratherlimitedand can meetonly the short intervalsof emergency.This is due to the \\m\ted presence of g\lcogen \n \\rrer. frn adu\t

ln the Fi9,36.5,the sourcesof blood glucose during a normal d^y Qq hours) are given. Clucoseis primarilyderivedfrom glycogenolysis (of hepatic glycogen) between the meals. Cluconeogenesis becomesa predominantsource o\ g\ucose\n \ate nrght \aher dep\etron oi hep atrc

:l

rit

I

i'

676

BIOCHEMISTF|Y Bloodglucose(mg/dl) 40

50

60

70

80

90

100 110 120 130 't40 150 160 170 180 190 200

T Fasting (<100mg/dl)

Renaltirreshold

Post-prandial (<130mg/dl)

J To urine

Hypoglycemiceffect

Hyperglycemiceffect

lnsulin

Glucagon

Glucoseuptaket 1 Glycolysis

t Gluconeogenesis t Glycogenolysis

Glycogenesis t

Epinephrlne

HMPshuntt

t Glycogenolysis Thyrorlne t Gluconeogenesis Glucocorticoids T Gluconeogenesis 1 Glucoseutilization (extrahepatic) Growthhormoneand ACTH GlucoseuotakeJ Glucoseutilization J

Lipidsynthesis t J Gluconeogenesis Glycogenolysis J

Flg. 36.6 : Hormonalregulationol blood glucose,

glycogen). During day time, gluconeogenesis Kidneyplaysa specialrole in the homeostasis may be more or less active, depending on the of blood glucose. Clucose is continuously frequencyof consumptionof snacks,coffee,tea, filtered by the glomeruli, reabsorbedand fruit juices etc. returnedto the blood.lf the levelof glucosein

bfood is above 160-180 m{dl, glucose This value is excretedin urine (glycosuria). (160-180 mg/dl) is referred to as renal Certain tissueslike brain, erythrocytes,renal thresholdfor glucose.The maximumability of m edu l l a a n d b o n e ma rro w a re excl usi vel y the renal tubules to reabsorbglucose per dependent on glucose for their energy needs. minute is known as tubular maximum for When the body is at total resf, about two-thirds (TmC). The value for TmG is 350 glucose of the blood glucoseis utilized by the brain.The mg/minute. r em a i n i n go n e -th i rdb y R BC Ut iliz a ti o n

of blood glucose

a n d s k el etalmuscl e. A regularsupply of glucose,by whatevermeans it may be, is absolutely required to keep the Role of hormones in blood glucose homeostasis br ain fu n c ti o n a l l yi n ta c t. The differentmetabolic pathways(glycolysis, glycogenesis, HMP shuntetc.)responsiblefor the utilizationof blood glucoseare alreadydiscussed (Chapter 13). The synthesis of fat from acetyl CoA a n d g l y c e ro l i s d e s c ri b e d i n l i pi d metabolism(Chapter 14).

Hormonesplay a significantrole in the regulation of blood glucose concentration (Fi9s.35.6and 36.4. Primarily, insulin lowers blood glucose level (hypoglycemic)while the rest of the hormonesopposethe actions of insulin (hyperglycemia).

Growth hormone

Glucocorticoids

Hyperglycemic

130

160

|90

220

Bloodglucoseconcentration (mg/dl)

Fig. 35-7 : A cartoon of tug of war illustrating hormonal action on btood glucose regutation. g\

\ \

678 I ns u l i n : l n s u l i n i s p ro d u c e db y B-cel l sof the islets of Langerhans in response to hyperglycemia (elevated blood glucose level). Some amino acids, free fatty acids, ketone bodies,drugssuch as tolbutamidealso causethe s ec r e ti o no f i n s u l i n . I ns u l i n i s b a s i c a l l ya h y p o g l y c e mi chormone that lowers in hlood glucose level through v ar io u s me a n s . l t i s a n a n ti -di abetogeni c hor m o n e .F o r d e ta i l so f i n s u l i na c ti onon gl ucose homeostasisrefer metabolic effects of insulin ( c ar b o h y d ra te m e ta b o l i s m)i n th i s chapter.

B IOC H E MIS TFY lln Fi9.36.7,regulationof blood glucoselevel by hormonesis depictedas a game of tug of rvar w i th el ephant(representi ng i nsul i n)on one side and the other ani mal s(as rest of the hormones) on the opposi tesi de.Thi s i s j ust an i l l ustrati on( a cartoon)for a qui ck understandi ngof gl uc ose homeostasisl .

When the blood glucoseconcentrationfalls to less than 45 mg/dl, the symptoms of Glucagon: Clucagon is synthesizedby a-cells include hypoglycemia appear.The manifestations of the islets of Langerhansof the pancreas. headache, anxiety, confusion, sweating, slurred Hy po g l y c e mi a (l o w b l o o d g l u cose l evel ) if not speech, seizures and coma, and, corrected, s t im u l a te si ts p ro d u c ti o n .G l u c a g on i s basi cal l y involved in elevating blood glucose death. All these symptoms are directly and glucose concentration.lt enhancesgluconeogenesis and indirectly related to the deprivation of (particularly nervous supply to the central system gly c o g e n o l y s i s . the brai n)due to a fal l i n bl ood gl ucosel ev el. Epinephrine : This hormone is secreted by The mammal i anbody has devel opeda well adr en a lme d u l l a .l t a c tsb o th o n mu scl eand l i ver t o b ri n g a b o u t g l y c o g e n o l y s i sb y i ncreasi ng regulaiedsystemfor an efficient maintenanceof phosp h o ry l a s ea c ti v i ty . T h e e n d product i s blood glucose concentration (details already gluc o s ei n l i v e r a n d l a c ta tei n mu scl e.The net described). Hypoglycemia, therefore, is not outcome is that epinephrine increasesblood commonly observed.The following three types of hypoglycemiaare encounteredby physicians. glucose level. 1 . Post-prandialhypoglycemia: This is also Thyroxine : lt is a hormone of thyroid gland. reactivehypoglycemiaand is observedin called I t ele v a te sb l o o d g l u c o s e l e v e l b y sti mul ati ng subjects with an elevated insulin secretion hepa ti cg l y c o g e n o l y s iasn d g l u c o neogenesi s. fol l ow i ng a meal . Thi s causes transient Glucocorticoids : These hormones are hypogl ycemi a and i s associ ated w i th m ild produced by adrenal cortex. Clucocorticoids symptoms. The patient is advised to eat s t im u l a te p ro te i n me ta b o l i s m a nd i ncrease frequently ratherthan the 3 usual meals. gluc o n e o g e n e s i s(i n c re a s e th e a cti vi ti es of enzymes-glucose 2. Fastinghypoglycemia: Low blood glucose 6-phosphatase and f r uc to s e 1 ,6 -b i s p h o s p h a ta s e ).The gl ucose concentration in fasting is not very common. ut iliz a ti o n b y e x tra h e p a ti cti s s u e si s i nhi bi ted However, fasting hypoglycemia is observed in by glucocorticoids. The overall effect of patients with pancreatic B-cell tumor and glucocorticoids is to elevate blood glucose hepatocellular damage. concentration. 3. Hypoglycemiadue to alcohol intake : In Growth hormone and adrenocorticotropic some individualswho are starvedor engagedin hormone (ACTH) : The anterior pituitary gland prolonged exercise, alcohol consumption may secretesgrowth hormoneand ACTH. The uptake cause hypogl ycemi a. Thi s i s due to t he of glucose by certain tissues (muscle, adipose accumul ati onof N A D H (duri ng the course of tissue etc.) is decreasedby growth hormone. alcohol metabolismby alcohol dehydrogenase) A CT H d e c re a s e sg l u c o s e u ti l i z a ti on. The net which diverts the pyruvate and oxaloacetate effect of both these hormones is hyperglycemic. (substrates of gluconeogenesis) to form,

AND DIABETESMELLITUS INSULIN.GLUCOSEHOMEOSTASIS.

679

respectively,lactateand malate.The net effect is lhat gluconeogenesis is reduced due to alcohol consumption.

common, accounting for 80 to 9Oo/"of the di abeti c popul ati on. N ID D M occurs i n adul ts (usuallyabove 35 years)and is less severethan ID D M. The causati vefactorsof N ID D M i ncl ude 4. Hypoglycemia due to insulin overdose : geneti c and envi ronmental . N ID D M more T he m os t c o m m o n c o m p l i c a ti o n o f i n sul i n commonl y occurs i n obese i ndi vi dual s.Overt her apy in di a b e ti c p a ti e n ts i s h y p o g l y c e mi a. eati ng coupl ed w i th underacti vi tyl eadi ng to This is particularlyobservedin patientswho are obesity is associatedwith the development of on int ens iv etre a tme n tre g ,i m e . NIDDM. Obesityacts as a diabetogenicfactor in geneti cal l ypredi sposed i ndi vi dual sby i ncreasi ng the resi stance to the acti onof i nsul i n.Thi s i s oue to a decreasein insulin receptorson the insulin responsive(target)cells. The patientsof NIDDM may have either normal or even increased Diabetesmellitus is a metabolic dr'sease, more insulin levels. lt is suggestedthat over-eating appr opr iat el ya d i s o rd e ro f fu e l me ta b o l i s m l.t i s causes i ncreased i nsul i n oroducti on but mainly characterized by hyperglycemia that decreasedsynthesisof insulin receptors.This is leads to severallong term complications. based on the fact that weight reduction by diet Diabet esm e l l i tu s i s b ro a d l y d i v i d e d i n to 2 control alone is often sufficient to correct gr oups , nam e l y i n s u l i n -d e p e n d e n td i a b etes N ID D M. m ellit us ( I DD M) a n d n o n -i n s u l i n d e p e n dent The comoarisonbetween IDDM and NIDDM is diabet esm elli tu s(N ID D M). T h i s c l a s s i fi c a ti on is given in Tahle 36.2. m ainly bas edo n th e re q u i re me n ot f i n s u l i n for treatment.

GLUCOSETOLERANCETEST (GTT!

The diagnosisof diabetescan be made on the basisof individual'sresponseto the oral glucose IDDM, also known as type I diabetesor (less load, commonly referred to as oral glucose frequently) juvenile onset diabetes, mainly tolerancetest (OCTT). oc c ur sin c hild h o o d(p a rti c u l a rl yb e tw e e n ' l2 -15 yrs age).IDDM accountsfor about 1Oto 2oohof lf'resara'tEog? ,.;i*;i,ia' 5;ubi*et fgr G?T t he k nown d i a b e ti c s . T h i s d i s e a s e i s The person shoul d have been taki ng characterized by almost total deficiency of carbohydrate-rich diet for at least3 days prior to ins ulindue t o d e s tru c ti o no f B -c e l l so f p a n c r eas. the test. A l l drugs know n to i nfl uence The b-cell destructionmay be causedby drugs, carbohydrate metabolism should be v ir us esor aut o i mmu n i ty .D u e to c e rta i ng e neti c discontinued (for at least 2 days). The subject v ar iat ion,t he B -c e l l sa re re c o g n i z e da s n o n -sel f should avoid strenuousexerciseon the previous and they are destroyed by immune mediated day of the test. Helshe should be in an overnight injury. Usually,the symptomsof diabetesappear (at least 10 h) fasting sfafe. During the course when 80-90% of the p-cells have been of CTT, the person should be comfortably destroyed. The pancreas ultimately fails to seated and shoul d refrai n from smoki ne s ec r et eins ulin i n re s p o n s eto g l u c o s ei n g e sti on. and exercise. T he pat ient so f ID D M re q u i rei n s u l i nth e ra p y. ;,lrtrE;,rr.r.,sjduff€f iiq':

il": T'

Clucose tolerance test should be conducted NIDDM, also called type II diabetesor (less preferabl yi n the morni ng (i deal9 to 11 A M). A frequently) adult-onset diabetes, is the most fasti ng bl ood sampl e i s draw n and uri ne

j

580

B IOC H E MIS TRY

Character

General Prevalence Ageatonset Bodyweight predisposition Genetic Biochemical Defect Plasma insulin Autoantibodies Ketosis Acute complications Clinical Duration olsymptoms Diabetic at complications diagnosis drugs Oralhypoglycemic Administration ofinsulin

Insulin-dependent diabetesmellitus (IDDM)

Non-insulin dependent diabetes mellitus (N IDDM)

population 10-20% ofdiabetic (<20 yrs) Usually childhood Normal orlow Mildormoderate

population 80-90o/o ofdiabetic Predominantly inadults(>30yrs) Obese Verystrong

Insulin dueto deficiency destruction ofB-cells Decreased orabsent Frequently found Verycommon Ketoacidosis

lmpairmenl intheproduction of insulin byp-cells and/or resistance oftargetcellsto insulin Normal orincreased Rare Rare Hyperosmolar coma

Weeks

Months toyears

Rare Notuseful fortreatment Always required

Found in 10-20% cases Suitable fortreatment notnecessary Usually

c olle c te d . T h e s u b j e c t i s g i v e n 7 5 g gl ucose 250 (13.8) orally, dissolvedin about 300 ml of water, to be dr un k i n a b o u t 5 mi n u te s . B l o od and uri ne samplesare collected at 30 minute intervalsfor 200 S (11.1) at least2 hours.All blood samplesare subjected E t o gl u c o s ee s ti ma ti o nw h i l e u ri n e sampl esare E qualitativelytestedfor glucose. E') 150 E (8.3) o Interpretation of GTT o

lmpaired glucose tolerance

o

The graphic representation of the CTT results E (E 100 is depicted in Fig,36.8. The fasting plasma E (5.5) o (E glucoselevel is 75-110 mgldl in normal persons. (L On oral glucose load, the concentration inc r e a s e sa n d th e p e a k v a l u e (140 mg/dl ) i s 50 (2.7) reached in less than an hour which returns to normal by 2 hours. Clucose is not detected in any o f th e u ri n e s a m p l e s . n

In individuals with impaired glucose tolerance,the fasting(110-126mg/dl) as well as 2 ho u r (1 4 O-2 O0mg /d l ) p l a s m a g l ucose l evel s are elevated.Thesesubjectsslowly developfrank

1

1+2

Hours

Flg. 36.8 : Oral glucose tolerancetest.

I

,''iicrcr:*ffi

: INSULIN, GLUCOSEHOMEOSTASIS,AND DIABETESMELLITUS

Condition

681

Plasmaglucose concentration as mmoll @A/d|

Fasting

2 hoursafter gruc0se

Normal

Impaired glucose tolerance

Diabetes

< 6 .1

< t.u

>7.0

(< 1 1 0 )

(<126)

(>126)

<7.8 (<140)

< 11.1

> 11,1 (>200)

(<2oo)

diabetes at an estimated rate of 2% per 6. Corticosteroid stressedGTT is employed vear. Dietarv restriction and exercise are to detect latent diabetes. advocatedfor the treatmentof impaired glucose Gl yeosuri a t oler anc e. T he W HO c ri te ri a to r th e d i a g n o s is of diabetesby OCTT is presentedin Table 36.3. A person is said to be suffering from diabetes mellitus if his/her fastingplasma glucoseexceeds 7.O mmol/l O26 mA/dD and, at 2 hrs. 11.1 mmol/l (200 mg/dll. CIther rele\tant

aspect$

@#&TT

Renal glycosuria : Renal glycosuria is a benign condition due to a reduced renal threshold for glucose. lt is unrelated to diabetes 1.7se/ke). and, therefore, should not be mistaken as 2. I n c as e o f p re g n a n tw o m e n , 1 0 0 g o ral diabetes.Further,it is not accompaniedby the glucoseis recommended.Further,the diagnostic classicalsymptomsof diabetes. criteriafor diabetesin pregnancyshould be more A l i mentarygl ycosuri a: l n certai ni ndi vi dual s, stringentthan WHO recommendations. bl ood gl ucose l evel ri ses rapi dl y after meal s 3. ln the mini GTT carried out in some resul ti ng i n i ts spi l l over i nto uri ne. Thi s laboratories, fastingand 2 hrs. sample(insteadof condition is referred to as alimentary (lag l/2 hr. intervals)of blood and urine are collected. storage) glycosuria. lt is observed in some normal people, and in patients of hepatic 4. T he CTT i s ra th e r u n p h y s i o l o g i c a l .To di seases,hyperthyroi di sm and pepti c ul cer. ev aluat et he gl u c o s eh a n d l i n go f th e b o d y u n der phy s iologic alc o n d i ti o n s fa , s ti n gb l o o d s a mpl ei s ltletabolic changes in diabetes drawn, the subject is allowed to take heavy breakfast,blood samplesare collectedat t hour Diabetes mellitus is associatedwith several and 2 hrs (post-prandiaL-meaning after food). metabolic alterations. Most important among Ur ine s am ple sa re a l s o c o l l e c te d .T h i s ty p e of them are hyperglycemia,ketoacidosisand hypert es t is c om mo n l y e mp l o y e d i n e s ta b l i shed triglyceridemia (Fig.36.9). diabetic patientsfor monitoring the control. 1. Hyperglycemia: Elevationof blood glucose 5. F or ind i v i d u a l s w i th s u s p e c te d mal - concentrati oni s the hal l mark of uncontrol l ed absorption, intravenous GTT is carried out. di abetes. H ypergl ycemi ai s pri mari l y due to l. F or c on d u c ti n g C T T i n c h i l d re n , oral gluc os eis giv e n o n th e b a s i so f w e i g h t (1.5 to

I

The commonestcauseof glucoseexcretionin urine (glycosuria)is diabetesmellitus.Therefore, gl ycosuri a i s the fi rst l i ne screeni ngtest for di abetes.N ormal l y,gl ucosedoes not appear i n uri ne unti l the pl asma gl ucose concentrati on exceeds renal threshold (180 mg/dl) As age advances,renal threshol dfor gl ucosei ncreases margi nal l y.

682

B IOC H E MIS TFI Y

Hepaticketone bodiest

HYPERGLYCEMIA

KETOACIDOSIS

IIYPERTRIGLYCERIDEMIA

Fig. 36.9 : Major metabolic alterations in diabetes mellitus.

reduced glucose uptake by tissues and its inc r e a s e dp ro d u c ti o n v i a g l u c o n e ogenesiand s glycogenolysis.When the blood glucose level goes beyond the renal threshold, glucose is excretedinto urine (glycosuria).

IUlanagement

3. Hypertriglyceridemia: Conversionof fatty acids to triacylglycerolsand the secretion of V LDL a n d c h y l o m i c ro n si s c o m p a r ati vel yhi gher in diabetics.Further,the activity of the enzyme lipop ro te i n l i p a s e i s l o w i n d i a b eti c pati ents. Consequently,the plasmalevelsof VLDL, chylomicrans and triacylglycerols are increased. Hypercholesterolemia is also frequently seen in diabe ti c s .

advised to consume low calories (i.e. low carbohydrate and fat), high protein and fiber rich diet. Carbohydratesshould be taken in the form of starchesand complex sugars.As far as possible,refinedsugars(sucrose,glucose)should be avoi ded. Fat i ntake shoul d be drasti cally reduced so as to meet the nutritional requirementsof unsaturatedfatty acids. Dief control and exercisewill help to a large extent obese N ID D M oati ents.

of dEabetes

D i et, exerci se, drug and, fi nal l y, i ns ulin are the management opti ons i n di abet ics. Approximately,50ohof the new casesof diabetes can be adequatelycontrolled by diet alone, 202. Ketoacidosis: Increasedmobilization of 30% need oral hypogl ycemi cdrugs w hi l e t he fatty acids results in overproductionof ketone remai ni ng20-30% requi rei nsul i n. bodies which often leads to ketoacidosis. Dietary management: A diabetic patient is

$-ong term

effects;

ef ddabetes

Hypoglycemicdrugs : The oral hypoglycemic drugs are broadly of two categories-sulfonylureas Hy p e rg l y c e mi a i s d i re c tl y o r i ndi rectl y and The latter are less commonly biguanides. as s o c i a te d w i th s e v e ra l c o mpl i cati ons. used these days due to side effects. These include atherosclerosis, retinopathy, nephropathy and neuropathy. The biochemical Sulfonylureas such as acetohexamide, tolbutabas is o f th e s e c o m p l i c a ti o n s i s not cl earl y mi de and gl i bencl ami deare frequentl y used, understood.lt is believed that at least some of They promote the secretion of endogenous them are related to microvascular changes i nsul i nand thus hel p i n reduci ngbl ood gl uc ose caused by glycation of proteins. l evel .

ri.r,INSUL|N,GLUCOSE HOMEOSTASIS, AND DTABETES MELLTTUS Management with insulin : Two types .' ins u l i n p re p a ra ti o n s a re c o m merci al l y a.ailable-short acting and long acting. The r . or t acti n g i n s u l i n sa re u n mo d i fi e d and thei r ac t ion la s tsfo r a b o u t 6 h o u rs .T h e l o ng actrng rsulins are modified ones (suchas adsorptionto crotamine) and act for several hours, which dependson the type of preparation. T he a d v e n t o f g e n e ti ce n g i n e e ri n gi s a boon io diabe ti c p a ti e n ts s i n c e b u l k q u a nti ti es of insulin can be produced in the laboratory.

Diagnostic importance of HbA1" : The rate of synthesisof HbA,. is directly related to the exposure of RBC to glucose. Thus, the concentrationof HbA1. servesas an indicatron of the blood glucose concentration over a period, approximating to the half-life of R B C (hemogl obi n) i .e. 6-8 w eeks. A cl ose correlation between the blood glucose and HbAt c concentrations have been observed w hen si mul taneousl ymoni tored for several months. N ormal l y, H bA l c concentrati on i s about 3-57' of the total hemogl obi n. l n di abeti c pati ents,H bA tc i s el evated(to as hi gh as 15% ). Determinationof HbAl . is used for monitoring of diabetes control. HbAlc reflects the mean blood glucose level over 2 months period prior to its measurement.

'r:!;r;.',ftir: i3d in gifrees i"$ri siil*?ll i,r: {$ntf

683

,nl

For a diabetic patient who is on treatment (drug or insulin therapy), periodical assessfnenf of the efficacy of the treatment is essential. Ur ine gl u c o s e d e te c ti o n a n d b l o o d gl ucose estimationsare traditionallyfollowed in severar In the routi necl i ni cal practi ce,i f the H bA l c laboratories.In recent years, more reliable and long- t er m b i o c h e mi c a l i n d i c e s o f di abeti c concentration is less than 7o/o, the diabetic patient is considered to be in good control. c ont r ol a re i n u s e . Glycated hemoglobin : Clycated or glycosylated hemoglobin refers to the glucose derived products of normal adult hemoglobin (HbA). Clycation is a post-translational,nonenzymatic addition of sugar residue to amino acids of proteins. Among the glycated hemoglobins,the most abundantform is Hb\c. HbAtc is produced by the condensation of gluc os ew i th N -te rmi n a lv a l i n e o f e a c h B -chai n of HbA .

Fructosamine: BesidesHbA16,severalother proteins in the blood are glycated. Glycated serum proteins (fructosamine) can also be measuredin diabetics.As albumin is the most abundant pl asma protei n, gl ycated al bumi n largely contributes to plasma fructosamine measurements.A l bumi n has shorter hal f-l i fe than H b. Thus, gl ycated al bumi n represents glucose status over 3 weeks prior to its determination.

BtoMEDtCAL/ CL|N|CAL CONCEPTS

ta- Diabetes alt'ects about 2'30/oof the population and is a major causeof blindness, renal failure, heart attack and stroke. r::' The hormone insulin has been implicated in the deuelopment of diabetes. t:;- Diabetic ketoacidosis is t'requently encountered in seuere uncontrolled diabetics. The management includes administration of insulin, fluids and potassium. w+ The hypoglgcemic drugs commonly used in diabetic patients include tolbutamid,e. gl ibenclamide and acetohexamide. t." Measurementof glycated hemoglobin (HbAil seruesos a marker t'or diabetic control.

)

684

B IOC H E MIS TFIY

M ic ro a l b u m i n u ri a : Mi c ro a l b u mi nuri a i s defined as the excretion of 30-300 mg of albumin in urine per day. lt may be noted that m ic r oa l b u mi n u ri a re p re s e n tsa n i ntermedi ary stagebetweennormal albumin excretion(2.5-30 m g/ d) a n d ma c ro a l b u mi n u ri (> a 3 0 0 mg/d).The small increase in albumin excretion predicts impairment in renal function in diabetic

pati ents.Mi croal bumi nuri aservesas a si gnalof early reversiblerenal damage. Serum lipids : Determinationof serum lipids (total cholesterol,HDL, triglycerides)servesas an index for overall metaboliccontrol in diabetic pati ents. H ence, serum l i pi ds shoul d b e frequently measured.

7. Diabetes mellitus is a common metabolic disorder,charocterizedby insuft'icientor inefficient insulin. 2. lnsulin is a polypeptide hormone, secretedby the ftcells ol pancreas.It hos a prot'ound influence on carbohydrate,t'at and protein metabolisms. Insulin lowers blood glucose concentration (hypoglycemiceffect). 3. Glucagon,secretedby the a-cellsot' pancreas,in general opposesthe actions of insulin, The net et't'ectof glucogon is to increaseblood glucoseconcentration (hyperglycemicet'fect). 4. ln a healthgperson, the blood glucoseleuel(fasting 70-100 mg/dl) is maintainedby a well coordinatedhormonal action regulating the sourcesthot contribute to glucase (gluconeogenesis, glycogenolysis),and the utilizotion pathways (glycolysis,glycogenesis, lipogenesis).lnsulin is hypoglycemic while other hormones (glucagon, epinephrine, thyroxine, glucocorticoids)are hyperglycemrc. 5. ln hypoglycemia(blood glucose <45 mS/dU,there is depriuation of glucose supply to brain resulting in symptomssuch as headache,confusion,anxiety and seizures. 6. Diabetes mellitus is broadly classit'iedinto 2 categories-insulin dependent diabetes mellitus (IDDM) and non-insulindependentdiabetesmellitus (NIDDM). 7. The laboratory diagnosisol diabetes is t'requentlycorried out by orol glucosetolerance test (GTT). As per WHO criteria, a person is soid to be suffering Jrom diabetes if his/ her fasting blood glucose exceeds 126 mg/dl, and 2 hrs. alter oral glucose load goes beyond 200 mg/dl. 8. Diabetes is associatedwith seueral metabolic derangementssuch as ketoacidosisond hypertriglyceridemia, besides hyperglycemia. The chronic complications oJ diabetes include atherosclerosis, retinopathy,nephropathyand neuropathg. 9. Diet, exercise,drug and insulin are the optionslor diabetic control. lt is estimated that about halJ of the new diabetic patients can be adequatelycontrolled by diet and exercrce. l0.Esfimofion of glycoted hemoglobin (HbArc), plosma t'ructosamine,microalbumin in urine, and serum lipids serueas biochemicalindices to monitor diabeticcontrol.

the proliferation 2. Malignanttumors or cancers: They are I n the normalcircumstances, proliferation I of bodv cellsis understrictcontrol.The cells characterized by uncontrolled and differentiate, divide and die in a sequential spreadof cells to variouspartsof the body, a manner in a healthy organism.Cancer is process referred to as mefastasis.Malignant characterized by loss of control of cellular tumorsare invariablylife-threatening e.g. lung growth and developmentleadingto excessive cancer/leukemia. proliferation and spread of cells. Cancer is About 100 differenttypesof humancancers derivedfrom a Latinword meaningcrab. lt is have been recognized.Cancersarising from presumedthat the word canceroriginatedfrom epithelial cells are referred to as carcinomas the characterof cancerouscells which can while that lrom connectivetissuesare known as migrateand adhereand causepain (likea crab) sarcomas.Methodsfor the early detectionand to any part of the body. treatment of cancers have been developed. Neoplasia literally means new growth. However,little is knownaboutthe biochemical Uncontrolledgrowthof cells resultsin tumors(a basisof cancer. word originally used to representswelling). Oncology(Greek:oncos-tumor) deafswith the Incidence studyof turnors. Canceris the secondlargestkiller disease(the first being coronary heart disease) in the The tumors are of two types. developedcountries.lt is estimatedthat cancer 1. Benigntumors : They usuallygrow by accountsfor more than 2oo/oof the deathsin expansion in a layerof United States.Basedon the current rate of and remainencapsulated connectivetissue.Normallybenigntumorsare incidence,it is believedthat one in every 3 not life-threatening e.g.moles,warts.Thesetypes persons will developcancerat sometimeduring of benigntumorsare not considered as cancers. his life. 685

B IOC H E MIS TR Y

686

Althoughhumansof all agesdevelopcancer, chemically non-reactive procarcinogen is with advancement of converted to an ultimate carcinogen by a series the incidenceincreases age. More than 70"/oof the new cancer cases of reactions. occur in personsover 60 years.Surprisingly, The carcinogens can covalently bind to canceris a leadingcauseof deathin childrenin purines, pyrimidines and phosphodiesterbonds the age group3-13 years,half of them die due of DNA, often causingunrepairabledamage.The to leukemia. frequently cause

carcinogens chemical mutations(a change in the nucleotidesequence of DNA) which may finally lead to the developmentof cancer,hencethey are regarded as mutagens.

In general, aremultifactorial in origin. cancers The causative agentsincludephysical,chemical, genetic and environmentalfactorc.A survey in USA has shown that about 90"/" of all cancer deaths are due to avoidablefactors such as tobacco,pollution,occupation, alcoholanddiet. Most of the cancersare causedby chemical radiationenergyand viruses. These carcinogens, agentsmay damageDNA or interferewith its replicationor repair. Ghemical carcinogens

Ames assay: This is a laboratory test to check the carcinogenecity of chemicals. Ames assay employs the use of a special mutant strain of bacterium, namely Salmonella typhimurium (His-).This organismcannot synthesizehistidine; hence the same should be supplied in the medium for its growth. Addition of chemical carcinogenscausesmutations(reversemutation) restoringthe ability of the bacteriato synthesize histidine lHis+). By detecting the strain ol Salmonella(His+)in the colonies of agar plates, the chemical mutagenscan be identified. The Ames assay can detect aboul 9oo/o of the chemical carcinogens.This test is regardedas a Animal screening procedure. preliminary experiments are conducted for the final assessmentof carcinogenecity.

It is estimatedthat almost 80% of the human cancersare causedby chemicalcarcinogensin nature.The chemicalsmay be organic (e.g. dimethylbenzanthracene, benzo (a) pyrene, dimethyl nitrosamine)or inorganic (arsenic, Promoters of carcinogenesis : Some of the cadmium)in nature.Entryof the chemicalsinto the body may occur by one of the following chemicals on their own are not carcinogenic. Certain substancesknown as promoting agents mechanisms. make them carcinogenic. The application of benzo- (a)pyreneto the skin, as such, does not 2. Diet e.g. aflatoxinB producedby fungus cause tumor development. However, if this is (AspergiIIus fIavus\ contami nation of foodstuffs, followed by the applicationof croton oil, tumors will develop. ln this case, benzo(a)pyreneis the particularly peanuts. initiating agent while croton oil acts as a 3. Drugs-certaintherapeuticdrugscan be promoter. Several promoting agent or carcinogenic e.g. diethylstibesterol. compounds that act as promoting agents in 4. Life stylee.g. cigarettesmoking. variousorgansof the body have been identified. These i ncl ude sacchari nand phenobarbi tal .

1. Occupatione.g. asbestos, benzene.

Mechanismof action : Althougha few of the majorityof chemicalsare directlycarcinogenic, prior them require metabolismto become carcinogenic. The enzymessuchas cytochrome P+so responsiblefor the metabolism of (Chapter3ll areinvolvedin dealing xenobiotics In general,a with the chemicalcarcinogens.

Radiation energy Ultraviolet rays, X-raysand lrays have been proved to be mutagenic in nature causing cancers. These rays damage DNA which is the basic mechanismto explain the carcinogenicity

687

Ghapter 37 : CANCEFI

CIass Dl{Aviruses Adenovirus Herpesvirus Papovirus RilAvlrusee typeB Retrovirus Retrovirus typeC

Memberc

12and18 Adenovirus Epstein-Ban virus,herpes simolex virus Papilloma virus,polyoma virus

1. Cancersare transmittedfrom mother to daughter cells.lh otherwords,cancercellsbeget cancercells. 2. Chromosomal abnormalities are observed in manytumorcells. 3. Damageto DNA causedby mutations often resultsin carcinogenesis. 4. Laboratoryexperimentshave provedthat purifiedoncogenes can transformnormalcells into cancercells.

Mammary tumorvirusofmouse Leukemia, sarcoma.

of radiationenergy.For instance,exposureto UV rays resultsin the formationof pyrimidine dimers in DNA while X-ravs cause the production of free radicals. This type of molecular damages are responsiblefor the carcinogeniceffectsof radiations. Carcinogenie viruses

Canceris causedby a geneticchangein a single cell resulting in its uncontrolled multiplication. Thus,tumorsare monoclonal. Two types of regulatorygenes-oncogenesand antioncogenesare involved in the development of cancer (carcinogenesis). In recentyears,a third categoryof genesthat controlthe cell death or apoptosisare also believedto be involvedin carcinogenesis.

The involvement of virusesin the etiologyof cancerwas first reportedby Rousin 1911. He demonstratedthat the cell-free filtrates from 0ncogenes (tumorsof connective certainchickensarcomas The genescapableof causingcancer are tissues)promote new sarcomasin chickens. known as oncogenes(Greek: oncos-tumor or Unfortunately, this epoch-making discoveryof mass). Oncogenes wereoriginallydiscovered in Rous was ignoredfor severalyears.This is tumor causing viruses. Theseviral oncogenes evidentfrom the fact that Rouswas awardedthe Nobel Prize in 1966 at the age of 85 for his werefoundto be closelysimilarto certaingenes presentin the normal host cells which are discovery in 1911! referredto as protooncogenes. Now, about 40 The presenceof viral particlesand the viral and cellular protooncogenes have been enzyme reverse transcriptase,besides the identified.Protooncogenes encode for growthoccurrenceof base sequencein the DNA of regulating proteins. The activation of malignant cells,complementary to tumorviruses protooncogenes to oncogenesis an important indicatethe involvementof virusesin cancer. stepin the causationof cancer. The virusesinvolved in the developmentof In the Table 37.2, a selected list of cancer,commonlyknown as oncogenicviruses, may containeitherDNA or RNA.A selectedlist oncoproteins, protooncogenes and the associated humancancersis given. of tumor virusesis given in Table37.1. DF{A-the ultlnrate earcinogenesis

in

Activation of protooncogenes

to oncogenes

DNA is the ultimatecriticalmacromolecule Thereare severalmechanisms for converting in carcinogenesis. This fact is supportedby the protooncogenes to oncogenes,someof the severalevidences. importantonesare describednext.

688

BIOCHEMISTRY

Oncoproteins Growthfactore growlh (PDGF) Platelet factor derived growlh (EGF) Epidermal factor Growthfactorleceptors

proteins SignaFtransduclng proleins GTP-binding Non+eceptor tyrosine kinase

Protooncogene

Associated human cancer(s)

hst-1

Osteosarcoma andbladder ofstomach, breast Cancers

erffi, erFB, erf-lB,,

Lungcancer Stomach cancer Breast cancer

HS

andcolon Leukemias, cancers oflung,pancreas Leukemia

slb

abl

1. Viral insertioninto chromosome : When (geneticmaterialRNA)infect certainretroviruses cells, a complementaryDNA (cDNA).is made from their RNA by the enzyme reverse transcriptase.The cDNA so produced gets insertedinto the host genome (Fig.37.l).The integrateddouble-stranded cDNA is referredto as provirus.This pro-viralDNA takesover the control of the transcription of cellular chromosomalDNA and transformsthe cells. Activationof protooncogene myc to oncogene by viral insertion ultimately causing carcinogenesisis well known (e.g. avian leukemia).

(Fi9.37.2).This results in the activationof inactive myc gene leading to the increased synthesis of certainproteinswhich makethe cell malignant. 3. Gene amplification: Severalfoldamplifiare observed cationsof certainDNA sequences of anticancer in some cancers.Administration (an inhibitorof the enzyme drugsmethotrexate is associated with gene dihydrofolatereductase) amplification.The drug becomesinactivedue to geneamplificationresultingin a severalfold (about 400) increase in the activity of reductase. dihydrofolate

is 4. Pointmutation: The rasprotooncogene SomeDNA virusesalsoget insertedinto the the bestexampleof activationby pointmutation host chromosome and activate the proto(changein a single base in the DNA). The oncoSenes. mutated ras oncogene produces a protein 2. Chromosomaltranslocation: Someof the tumorsexhibitchromosomal abnormalities. This myc is due to the rearrangement of geneticmaterial (DNA) by chromosomaltranslocationi.e. splittingoff a small fragmentof chromosome which is joined to another chromosome. Chromosomaltranslocationusually resultsin overexpressionof protooncogenes. Burkitt's lymphoma, a cancer of human B-lymphocytes,is a good example of chromosomaltranslocation.In this case, a fragmentfrom chromosomeB is split off and joinedto chromosome 14 containingmyc gene

Activated T Provirus myc

Chapter 37 : CANCEFI

689 A selected list of polypeptide growth factors, their sources and rnajor functions is given in Table 37.3. The cell proliferation is stimulated by grovvth factors. In general, a growth factor binds to a protein receptoron the plasma membrane.This binding activates cytoplasmic protein kinases leading to the phosphorylationof intracellular target proteins.The phosphorylatedproteins,in turn, act as intracellularmessengers to stimulate cel l di vi si on, the mechani smof w hi ch i s not clearly known. Transforming growth factor (TCF-d) is a protein synthesizedand requiredfor the growth of epithelial cells. TCF-a is produced in high concentration in individuals suffering from psoriasis,a diseasecharacterizedby excessive proliferationof epidermal cells.

Growth factor receptors : Some oncogenes encoding growth factor receptors have been identified. Overexpression and/or structural alterations in growth factor receptors are with carcinogenesis. For instance,the (CTPase) which differsin structureby a single associated overexpression of gene erb-9, encoding ECFamino acid. This alterationdiminishesthe receptor is observed in lung cancer. key involvedin Fig. 37,2 : Diagrammaticrepresentationof reciprocal translocationoccurring in Burkitt's lymphoma.

activityof CTPase,a enzyme the control of cell growth (detailsdescribedlater).

The presenceof ras mutdtionsis detectedin severalhumantumors-9}%" of pancreatic,50o/o of colon and 30% of lung. However, ras mutationshave not been detectedin the breast cancer.

GTP-binding proteins : These are a group of signal transducing proteins. Cuanosine triphosphate (CTP)-binding proteins are found in about 30%" of human cancers. The mutation of ras protooncogeneis the single-mostdominant cause of many human tumors.

The involvementof ras protein (productof ras gene) w i th a mol ecul arw ei ght 2' 1,00O(P 21)i n cell multiplication is illustrated in Fi9.37.3. The Oncogenes encode for certain proteins, inactiveras is in a bound statewith GDP. When namely oncoproteins. These proteins are the the cells are stimulated by growth factors, ras and alteredversionsof their normalcounterparts P21gets activatedby exchangingGDP for CTP. are involved in the transformationand This exchange processis catalysedby guanine multiplication of cells.Someof the productsof nucleotidereleasingfactor (CRF).The active ras oncogenes are discussed below. P21 stimulates regulatorssuch as cytoplasmic Growth factors : Several growth factors kinases,ultimatelycausingDNA replicationand stimulating the proliferation of normalcellsare cell division. In normal cells, the activity of ras known.Theyregulate celldivisionby transmitting P21 is shortlived.The CTPaseactivity, which is acrossthe plasmamembraneto the an integralpart (intrinsic)of ras P21,hydrolyses the message interior of the cell (transmembrane signal CTP to CDP, reverting ras 21 to the original transduction). lt is believedthat growth factors state,There are certain proteins,namely CTPase play a key role in carcinogenesis. activatingproteins (GAP), which acceleratethe

Mechanism of action of oncogenes

690

BIOCHEMISTFIY

Growth factor

Source(s)

growth Epidermal factor(EGF) growth Platelet derived factor(PDGF)

gto*th g[_9i:s) ._lg$lgtqlg ig:lgt growth Transforming factor-B tIGF-p) Erythropoietin growth Nerve factor(NGF)

gland, growlh Salivary libroblasts Stimulates ofepidermal andepithelialcells growth Platelels ol mesenchymal cells, Stimulates promotes wound healing Similar to EGF placenta Inhibitory (sometimes Platelets, kidney, stimulatory) effect oncultured tumor cells Kidney Stimulates development erythropoietic cells gland growth Salivary Stimulates the ofsensory and neurons sympathetic Eoithelialcell

(lGF-l Insulin likegrowth lactors andIGF-ll, respectively known assomatomedins CandA)

Serum

Tumor necrosis factor(TNF-a)

Monocytes Monocytes, leukocytes Lymphocyles (mainly T-helper cells).

.ltleleylllrl(hl (lL-2) Interleukin-2

Major function(s)

incorDoration ofsulfates into Stimulates cartilage; exerts insulinlike action on cells certain Necrosis oftumorcells Stimulates synthesis oflL-2. growth Stimulates andmaturation ofT-cells

hydrolysisof CTP of ras Pv. Thus, in normal cells, the activity ol ras P21 is well regulated. Point mutations in ras gene result in the production of altered ras P21, lacking CTPase activity. This leads to the occurrenceof ras P21 in a permanently activated state, causing uncontrol l edmul ti pl i cati onof cel l s.

o

Btockirr mulateciras

GDP

v

G TP Activated ras P>t

J-

Activation (cytoplasmic kinases)

I DNAsynthesis

andcellmultiplication Fig. 37.3 : Model for the mechanism of action of ras P^ protein (GRF-Guanine nucleotide releasing factor; GAP-GTPase activating proteins).

Non-receptor tyrosine kinases : These proteins are found on the interior of the inner plasma membrane. They phosphorylate the cellular targetproteins(involvedin cell division) in responseto externalgrowth stimuli. Mutations in the protooncogenes(e.9. abl) encoding nonreceptor tyrosine kinases increase the kinase activity and, in turn, phosphorylationof target protei nscausi ngunl i mi tedcel l mul ti pl i cati on. Antioncogcnes A special category of genes, namely cancer suppressor genes (e.g. pFs gene) ot, more commonly, antioncogenes,have been identified. The products of these genes apply breaks and regulate cell proliferation. The loss of these

Chapter 37 : CANCEFI

Oncogenic v,ruses

Environmental tactors(physical and chemical) I

\

J ONCOGENEACTIVATION

.)/ .//

(+ ./l

691

CARCJNOGENESIS

Fig. 37.4 : A simplified hypothesis for the development of cancer.

The biochemical indicators employed to detect the presenceof cancersare collectively referred to as tumor markers. These are the abnormally produced molecules ol lamor ce,//s such as surface antigens, cytoplasmic protdns. enzymes and hormones. Tumor markers can be measuredin serum (or plasma). In theory, the tumor markers must ideally be useful for screeningthe population to detect cancers. In practice,however,this has not been totallv true. As such,the tumor markerssupportthe diagnosis of cancers,besidesbeing useful for monitoring the response to therapy and for the earlv detectionof recurrence.

suppressorgenes removes the growth control of cells and is believed to be a key factor in the A host of tumor markershave been described development of several tumors, e.g. retinoblastoma, one type of breast cancer, and the list is evergrowing.However,only a few of them have proved to be clinically useful. A carcinoma of lung, Wilms' kidney tumor. selectedlist of tumor markersand the associated With the rapid advancesin the field of genetic cancers are given in Table 57.4. engineering, introducing antioncogenes to a A couple of the most commonly used tumor normal chromosome to correct the altered growth rate of cells may soon become a reality. markersare discussedhereunder. 1. Carcinoembryonicantigen (CEA)z This is Genes that regulate apoptosls a complex glycoprotein,normally produced by A new category of genes that regulate the embryonic tissueof liver, gut and pancreas. programmed cell death (apoptosis) have been The presenceof CEA in serum is detected in discovered.These genes are also important in severalcancers(colon,pancreas,stomach,lung). In about 67'/. of the patients with colorectal the developmentof tumors. cancert CEA can be identified. Unfortunatery, The gene, namely bcl-2, causes B-cell serum CEA is also detected in several other lymphoma by preventing programmed cell disorders such as alcoholic cirrhosis (70o/o), death. lt is believedthat overexpression of bcl-2 emphysema(57%) and diabetesmellitus (38"/.). allows other mutationsof protooncogenesthat, Due to this, CEA lacks specificity for cancer ultimately, leads to cancer. detection. However, in established cancer patients (particularlyof colon and breast),the Unified hypothesis serum level of CEA is a usefulindicatorto detect of careinogenesis the burden of tumor mass, besides monitoring The multifactorial origin of cancer can oe the treatment. suitably explained by oncogenes.The physical and chemical agents,virusesand mutationsall lead to the activation of oncogenes causing carcinogenesis. The antioncogenes and the genes regulatingapoptosisare intimately involved in development of cancer. A simplification of a unified hypothesisof carcinogenesisis depicted in Fi9.37.4.

2. Alpha-fetoprotein(AFP): tt is chemicallya glycoprotein,normally synthesizedby yolk sac in early fetal life. Elevation in serum levels of AFP mainly indicatesthe cancers of liver ano germ cells of testis and, to some extent, carcinomasof lung, pancreasand colon. As is the case with CEA, alpha-fetoproteinis not specific for the detection of cancers. Elevated

692

BIOCHEMISTRY

Tumormarker

Associatedcancer(s)

antigens Oncofetal (CEA) antigen Carcinoembryonic (AFP) Alpha tetoprotein antigen-1 25(CA-1 25) Cancer

lung,pancreas andbreast ofcolon, stomach, Cancers ofliverandgermcellsoftestis Cancer Ovarian cancer

Hormones gonadotropin (hCG) Human chorionic Calcitonin Catecholamines andtheir (mainly metabolites vanillyl mandelic acid)

Choriocarcinoma thyroid Carcinoma ofmedullary and Pheochromocytoma neuroblastoma

Enzymes Prostatic acidphosphatase Neuron specific enolase

Prostate cancer Neuroblastoma

proteins Specilic (PSA) Prostate specific antigen lmmunoglobulins

Prostate cancer Multiple myeloma

levelsof AFP are observedin cirrhosis,hepatitis and pregnancy.However/measurement of serum AFP provides a sensitiveindex for tumor therapy and detection of recurrence.

The morphological and biochemical changes in the growingtumorcellsare brieflydescribed here.Theseobservations are mostlybasedon the in vitro culturestudies.Knowledgeon the in the biochemicalprofileof tumor alterations cellsguidesin the selectionof chemotherapy of cancers.

Further,they cannot they form monolayers. moveawayfrom eachother.The cancercells form multilayersdue to loss of contact As a result,the cancer inhibition(Fig.37.5). in anypart cellsfreelymoveandgetdeposited of the body, a property referred to as metastasis. Lossof anchoragedependence: The cancer cells can grow without attachmentto the This is in contrastto the normalcells surface. which firmly adhereto the surface. Alteration in permeabilityproperties : The tumor cells have altered permeabilityand transport.

1. Generaland morphologicalchanges Shapeof cells : The tumor cells are much rounderin shapecomparedto normalcells. Alterationsin cell structures: Thecytoskeletal structureof the tumor cells with regardto actinfilamentsis different. . Lossof contactinhibition: The normalcells are characterized bv contact inhibition i.e.

(B) Flg. 37,5 : Growthcells in culture (A) Normal cells formingmonolayer(exhibitingcontact inhibition);(B) Cancer cells forming multilayers(loss of contact lnhibition).

Chapter 37 : CANCER

693

2. Biochemicalchanges Increased replication and transcription : The synthesisof DNA and RNA is increasedin c anc erc ell s . Increased glycolysis : The fast growing tumor cells are characterizedby elevationin aerobic and anaer o b i c g l y c o l y s i s d u e to i n c re ased ener gydem a n d so f m u l ti p l y i n gc e l l s .

known. lt is believed that the morphological changesi n tumor cel l s,l ossof contacti nhi bi ti on, lossof anchoragedependenceand alterationsin the structure of certain macromolecules are among the important factors responsible for metastasis.

. Reduced requirement of growth factors : The tumor cells require much less quantities of growth factors. Despite this fact, there is an increased production of growth factors by t hes ec ells .

Chemotherapy,employing certain anticancer drugs, is widely used in the treatmentof cancer. ln the lable 37.5, a selected list of the most commonly used drugs,and their mode of action of anticancerdrugs is . Synthesisof fetal proteins : During fetal life, is given. The effectiveness certain genes are active, leading to the inverselyproportionalto the size of the tumor synthesisof specificproteins.Thesegenesare i .e. the number of cancer cel l s. The maj or suppressedin adult cells. However,the tumor limitation of cancer chemotherapy is that the cells synthesize the fetal proteins e.g. rapi dl y di vi di ng normal cel l s (of hematopoi eti c system, gastrointestinaltract, hair follicles) are carcinoembryonicantigen, alfa fetoprotein. also affected.Thus,the use of anticancerdrugs is . Alterations in the structure of molecules : associatedwith toxic manifestations. Changesin the structureof glycoproteinsand glycolipids are observed. For the treatmentof solid tumors,surgeryand radiotherapy are very effective. M et as t as is Metastasisrefersto the spreadof cancer cells from the primary site of origin to other tissuesof the body where they get depositedand grow as secondarytumors. Metastasisis the major cause ln recent years, certain precautionary of cancer related morbidity and mortality. The measuresare advocatedto prevent or reduce the biochemical basis of metastasisis not clearly occurrence of cancer. The most imoortant

B|oMEDICAL/ CHNTCALCONGEPTS

6 r€

s€

About 800/oof the human cancers are caused by chemical carcinogens. The products ol oncogenes(growth factors, GTP-binding proteins) have been implicated in the deuelopment ol cancer. Antioncogenes apply breaks and regulate the cell proltferation. The physical and chemlcal agents, uiruses and mutatlons result in the actiuqtion of oncogenes causing carctnogenests, The abnormal products of tumor cells, referred to as tumor markers (CEA, AFe PSA) ore useful far the dlognosis and prognosis of cancer. Anttconcer drugs (e.9. methotrexate, clsplatin) are commonly used tn the treotment of cancer. Antloxldants (ultamlns E and C, ftcarotene, Se) decreqse the risk of carclnogenesisond hence their increosed consumption ls adwcated.

694

B IOC H E MIS TR Y

Anticancerdrug

Chemical naturc

Methotrexate

Folicacidanalogue

6-Mercaptopurine

Mode of action

(inhibits the Blocks theformatin oftetrahydrofolate THFisrequired for enzyme dihydrofolate reductase). nucleotide synthesis. Inhibits theformation ofAMPfromlMP.

6-Thioguanine

Mitomycin C

Antibiotic

Actinomycin D Vinblastine andvincrisline

Antibiotic

Cisplatin

Platinum compound

Alkaloids

Blocks reaction. lhymidylate synthase Results intheformation ofcrossbridges between DNA pairs. base Blocks transcriotion (ofcelldivision) lnhibit spindle movement andinterfere withcytoskeleton formation Results intheformation of intrastrand DNAadducts.

among them, from the biochemicalperspective, system, and promote detoxification of various are the antioxidants namelv vitamin E, carcrnoSens. p-carotene,vitamin C and selenium. In general, most of the vegetablesand fruits The antioxidants prevent the formation or are rich in antioxidants. Their increased detoxify the existing free radicals (free radicals consumption'is advocated to prevent cancer. are known to promote carcinogenesis).In (For more dethils on free radicals and addit io n ,a n ti o x i d a n tss ti m u l a teb o d y' s i mmune antioxidants, Refer Chapter 34).

1.

Cancer is characterizedby uncontrolledcellular growth and deuelopment,Ieading to excessiueproliferotion ond spread of cells. Cancer is the second largest killer diseose (next to heart disease)in the deueloped world.

2.

Regulatorygenes-namely oncogenes, antioncogenes and genescontrollingcell deathare inuolued in the deuelopment ot' cancer. Actiuatlon ol oncogenesis a /undomental step in corcinogenesis. This may occur by insertion of uiral DNA into host chromosome, translocationof chromosomes,gene amplilication ond point mutation.

3. The products of actiuated oncogenessuch os growth t'actors,growth factor receptors, GTP-binding proteins, non-receptor tyrosine kinoses haue all been implicoted in the deuelopment of cancer.

4.

Tumor markers of cancers include carcinoembryonicantigen (CEA), alpha fetoprotein (AFP), cancer antigen-721 and prostote specilic antigen (PSA). They are mainly uselul to support diognosis, monitor therapy and detect recurrence.

5.

There are seueral morphologicol and biochemical chonges in the tumor cells which distinguish them from the normal cells. The cancer cells ore chorqcterizedb9 loss oJ contact inhibition, altered membrane transport, increosed DNA ond RNA synthesis, increased glycolysis,alteration in the structure of certain molecules etc.

syndrome(AIDS) Epidemiology A cquiredimmunodeficiency first reported in 1981 in'homosexual Awas AIDS was first describedin USA and this men. AIDS is a retroviraldiseasecaused by country hasthe majorityof reportedcases.The human immunodeficiencyvirus (HlV). The prevalenceof AIDS has been reported from diseaseis characterizedby immunosuppression, almostevery country.The numberof people secondary neoplasma and neurological living with HfV worldwide is estimatedto be manifestations. AIDS is invariably fatal since around40 million by the end of the year2005. there is no cure. In the USA, it is the fourth (lndia alone has about 5 million persons). At leadingcauseof deathin men betweenthe ages least5 milliondeathsoccurredin 2005,due to 15 to 55 years. AIDS.AIDS is truely a globaldiseasewith an No other disease has attracted as much alarmingincreasein almosteverycountry. publicand aftention asAIDSby thegovernments, Transmission of HIV : Transmission of AIDS scientists. AIDShasstimulated an unprecedentedessentiallyrequires the exchangeof body fluids amountof biomedicalresearchwhich led to a (semen, vaginal secretions, blood, milk) majorunderstanding of thisdeadfydiseasewithin containingthe virusor virus-infected celfs.There a short periodof time.So rapidis the researchon are three major routes of HIV transmissionAIDS(particularly relatingto molecularbiofogy), sexualcontact,parenteralinoculation,and from any review is destinedto be out of date by the infectedmothersto their newborns. time it is published! The distributionof riskfactorsfor AIDStransThe isolationof human immunodeficiencymissionare as follows. virus (HIV) from lymphocytesof AIDS patients (homosexuals) - 60"/" wasindependently achievedby Gallo(USA)and Sexbetweenmen Sex betweenmen and women Montagnier(France) in 1984. -15% 695

696 Intravenous drug abusers

B IOC H E MIS TR Y

- 15"/"

Transfusionof blood and blood products-

6%

All others

4o/o

-

predominant methods of The HIV transmission(about 75o/") are through anal or vaginal intercourse. The risk for the transmission is m uc h h i g h e r w i th a n a l th a n wi th vagi nal intercourse.The practice of 'needle sharing' is mainly responsiblefor the transmissionof HIV in drug abusers.PediatricAIDS is mostlycausedby vertical transmission(mother to infant). It should, however,be noted that HIV cannot be transmitted by casual personal contact in the household or work place. Further, the transmissionof AIDS from an infectedindividual to health personnel attending on him is extremelv rAre.

The lipid membrane of the virus is studded with two glycoproteins Bprzo and gpot. The Virology of HIV surface antigeir 8p126 is very important for the AIDS is causedby a retrovirus,namely human viral infection and the detection of AIDS. immunodeficiency virus (HlY), belonging to Genome and gene products of HIV : The HIV lentivirus family. Retrovirusescontain RNA as genome contains 3 structural Benes-gag,pol and the geneticmaterial.On entry into the host cell, they transcribeDNA which is a complementary env that, respectively, code for core proteins, copy of RNA. The DNA, in turn is used, as a reverse transcriptase and envelop proteins. On either side of the HIV genome are long terminal template to produce new viral RNA copies. repeat (LTR)genes which control transcription. Two different forms of HlV, namely HIV-I Besides the structural genes/ HIV contains and HIV-2 have been isolated from AIDS several regulatory genes including vif, vprt tat, patients.HIV-1 is more common, being found in rev, vpu and nef (Fi9.38.4. These genes control AIDS patients of USA, Canada, Europe and the synthesisand assembly of infectious viral Central Africa while HIV-2 is mainly found in proteins. In fact, the regulatorygenesof HIV play West Africa. Both the virusesare almost similar a key role in the developmentof AIDS. except they differ in certain immunological properties. HIV-1 is describedin some detail. Structureof HIV : The viruse is sphericalwith a diameterof about 110 nm. lt containsa core, surroundedby a lipid envelop derived from the host pfasma membrane (Fig.3fl.l). The core of the HIV has two strandsof genomic RNA and four core proteins, PZq,PtB, reversetranScriptase (poolpsr)and endonuclease(p32).Note that the namingof the proteinsis basedon the molecular weight. For instance,a protein with a molecular weight of 24,0OOis designated as p2,4.

lmmunological abnormalities in AIDS

As is evident from the name AIDS, (or immunosuppression) is immunodeficiency primarily AIDS the haflmark of this disease. affectsthe cell-mediatedimmunesystemwhich protectsthe body from intracellularparasites protozoaand mycobacteria. This suchasviruses, (cluster is caused by a reduction in CD+ antigen4) cells of T-lymphocytes, determinant besidesimpairmentin the functionsof surviving CDa cells.

SYNDHOME(AIDS) IMMUNODEFICIENCY Chapter 3a: ACGIUIRED CDa cells may be regardedas 'naster cells of cell mediated rnmunity.They producecytokines, rracrophage chemotactic factors, remopoietic growth factors, and others involved in the bodv im m unit y .

Core proteins transcriDtase

Entryof HIV and lysisof CDacells: Thevirus HIV bindsto the entersthe CDaT-lymphocytes. specificreceptorson CDa cells by using its surface membrane glycoprotein (gprzo). Followingthe entry into the hostcells,RNA of HIV is transcribed into DNA by theviralenzyme reverse transcriptase.The viral DNA gets incorporated into the host genomicDNA. The virusmay remainlockedin the hostgenomefor monthsor yearsand this is consideredas the latent period. The viral DNA may undergo replication , and translation, respectively, producingviral R'NAand viral proteins.The resultin new viruses. lattertwo, on assembly, virusesleavethe host The newly synthesized cells by forming buds on plasmamembrane. Extensive viral buddingis associated with lysis The new viral and deathof CDa cells(Fi9.38.3). particlesinfectother host cells and repeatthe wholeprocess, ultimatelyresulting in a profound loss of CDa cells from the blood. Most of the immunodeficiency symptoms of AIDS are associatedwith the reduction in CDa cells. Other immunological

697

Envelope glycoproteins

Fiq.38.2: Genomeof HlV.

CDncell

J

abnormalities

The viral membraneprotein gp12s binds with normal T-helper cells and kills them. AIDS patientsalso display abnormalitiesin (humoral antibodyproductionby B-lymphocytes immunity). Abnormalitiesof central nervous system : HIV also infectsthe cells of central nervous system. lt is believed that HIV infected monocytesenterthe brain and causedamage, the mechanism of which remainsobscure. Consequences of immunodeficiency: The various clinical symptoms (fever, diarrhea, multiple weightloss,neurological complications, opportunisticinfections,generalizedlymphadenopathy,secondaryneoplasma etc.)of AIDS

Extensiveviral multiplication

Lysisof CDocells Fig. 38.3 : lmmunological abnomalities in CD4 cells on HIV infection.

698

BIOCHEMISTF|Y Acute phase

Crisis phase

Chronic phase

1,200 E

8 1,100 E 1,000 E

=g

eoo

8.

800

;f

700

1 :512 1 :256

Clinicallatency --1

1 :128 o

J--

1 :64 Opportunistic diseases 1 :32

[]- eoo

.g E o '5

E o

soo

*tr 5 *o o o

400

1:16 E o

300 2oo 100

1:8

(! 6

E

1i 4

1:2 0 2

4

6 8 1 0 1 ,2 Weeks-4

1294

s

67 I 91011121314 Years.l

FIg. 38.4: Graphicrepresentationof a typicalcourseof HIV infection.

are directly or indirectly related to the immunosuppression causedby HlV. Due to the deficiencyin the immune system,the body of AIDS patient is freely exposedto all sorts of infections(viral,bacterial,fungal). Natural course of AIDS Threedistinctphasesof HIV interaction with the immunesystemof infectedbody havebeen identified.Theseare the early,acutephase;the intermediate,chronic phase;the final, crisis phase(Fi9.38.4).

hence this phase is also referred to as seropositiveperiod. 3. Crisis phase : A failure in the defense system of the ,body, caused by immunothe crisisphase. suppression by HlV, represents -tremendously The plasma level of virus i5 increased.CD+ T-lymphocyteconcentration drasticallyfalls.A patientwith lower than 200 CDa T-lymphocytes/plblood is consideredto have developed AIDS. Crisis phase is characterizedby opportunisticinfectionsand In Western the relatedclinical manifestations. countries, a cancer-Kaposis sarcoma-is with AIDS. associated

1. Acute phase: This represents the initial body response to HIV infection. lt is characterizedby high rate of production of In general,AIDS patientsdie between5-10 viruseswhich are lodgedin the lymphoidtissues years after HIV infection. Treatment ffidy, and the antiviralimmuneresponse of the body. however,prolongthe life. This periodmay lastfor about8-12 weeks. 2. Chronic phase: During this periodthat may last for 5 to 10 yearsor even more, the body tr:iesto contain the virus. The immune systemis largelyintact.The personobviously appearsnormal,althoughhdsheis the carrierof HIV which can be transmittedto others. Antibodiesto HIV are found in the circulation,

Laboratory

diagnosis

of AIDS

The following laboratorytestsare employed to diagnosethe HIV infection. 1. The detection of antibodies in the immunocirculationby ELISA(enzyme-linked sorbantassay).

SYNDHOME(AlDSl Ghapter 38 : ACGIUIRED IMMUNODEFICIENCY

3'-Azldo2',3'dlod€oxythymldlne(AZT)

699

2',3'-Dldeorylnoslne(DDl)

Fi,.38.5: Structureof anti-AIDSdrugs

2. Westernblot technique,a more specific to the T-lymphocytessince cellular DNA test for the HIV antibodies,is employedfor pofymerasehas low affinity for AZT. However, AZT is found to be toxic to the bone marrow of ELISApositivecases. confirmation the patientsdevelopanemia. PCRcan cells,therefore, 3. A morerecentand sophisticated be used to detect the presenceof the HIV Themechanism is of actionof dideoxyinosine genomein the peripheralblood lymphocytes. almostsimilarto that of AZf . Drugs for the treatment

of AIDS

Althoughthere is no cure for AIDS, use of certain drugs can prolong the life of AIDS patients. Zidovudine or AZT (3'-azido 2', 3'-dideoxythymidine),a structuralanalog of was the first drug used and deoxythymidine continuesto be the drug of choice for the treatmentof Al DS. Didanosine(dideoxyionosine, DDt) is anotherdrug employedto treat AIDS. The structuresof AZf and DDI are shown in Fig.38.s.

Vaccine against AIDS -a failure so far

HIV exhibitsgeneticheterogenecity with a resultthatseveralspecies of virusmay be found in the sameAIDSpatient.Theprincipalcausefor the geneticvariationis the lack of proof-reading activityby the enzymereversetranscriptase. This leads to very frequent alterations in the DNA base sequence synthesized from viral RNA which, in turn, influencesthe amino acid sequenceof proteins.Thus,the proteinproducts Mechanismof action : AZT is takenup by the of HIV are highly variablein the amino acid fymphocytes and convertedto AZt triphosphate composition and, therefore, the antigenic which inhibits the enzyme HIV reverse properties.For this reason, it has not been AZT triphosphatecompeteswith possibleto develop a vaccine againstAIDS. transcriptase. dTTPfor the synthesisof DNA from viral RNA. However,there have been some encouraging which raisefresh Further,AZI is addedto the growingDNA chain animaland in vitroexperiments is halted.Thisdrugis not toxic hopesfor a vaccinein the nearfuture. andthe synthesis

B IOC H E MIS TR Y

700

BIOMEDICAL/ CLINIGAL GOIUCEFfS

u1? AIDS is a global diseasewith an alarming increosein the incidenceof occurrence.By the year 2005, more than 40 million people were globally ot't'ectedby AIDS. Lg

Homosexuality (predominantly in men) qnd intrauenous drug abuse ore the major foctors in the risk of AIDS fronsmission.

G+- The patientsol AIDS are destined to die (within 5-70 gearsaJter infection), since there is no cure. Howeue4 administration of certain drugs (AZT, DDI) prolongs life. E-' The clinicol manifestations ol AIDS are directly or indirectlg related to immunosuppression (mostly due to reduced CDa cells). AIDS patients are lreely exposed to all sorts ol infections (uiral, bacterial, fungal).

1. A/DS is a retoruiral disease coused by human immunodet'iciency uirus (HIV)' It is charocterized by immunosuppression, secondary neoplasms and neurological manifestations. Tronsmission of HIV occurs by sexual contact (more in male homosexuqls),parental inoculation (intrauenous drug abusers) and from infected mothers to their newborns.

2.

HIV enters CDa Tlymphocytes where its genetic material RNA is transcribedinto DNA by the enzyme reuerse transcriptose.The virol DNA gets incorporated into the host genomeultimately leodingto the multiplicationof the uirusand the destructionof CD4 cells. This is the root causeof immunosuppressionleading to opportunistic inlections in A/DS.

chronic ond crisis. A potient 3. The natural course ol AIDS has 3 distinct phases----acute, with lower than 200 CDa Tlymphocytedltl is consideredto haue deueloped AIDS. The sensitiuelaborotory testsfor AIDS detection are-ELISA, Western blot technique and, recently PCR.

4.

There is no cure t'or AIDS. The potients generally die within 5-10 years aJter HIV infection. Administration of drugs (zidouudineand didanosine),howeuer,prolongs the life oJ AIDS patients. These drugs inhibit the uiral enzyme reuerse tronscriptaseand halt the multiplication of the uirus. due to the uoriations The attempts to produce uoccinefor AIDS haue been unsuccesst'ul in the genome (and, thereJore,the protein products)ol the HlV.

Introduction to Bior Chemistry

,,

Overview of Biophysical

chemistry

lli ,,,ii:,

-a

lS

fools of Biochemistry

Bioorganic Chemistry

I s life comes from previous life, it was vitamins are the most common organic A believed for a longthatthecarboncompounds compoundsof life. Their chemistryhas been (hence in Section| (Chapters1-V. the nameorganic)arosefrom discussed of organisms force thury. This is referred to as vital fife onfy. that urea Comnnon funetiona! groups FriedrichWohler(1825)firstdiscovered (NH2-CO-NH2),the organiccompound,could in bEochemistry be preparedby heating ammonium cyanate Most of the physicaland chemicalproperties (NH4NCO), Thereafter, thousands of organiccompoundsare determinedby their in the laboratory. of organiccompoundshavebeen functionalgroups.Biomolecules and thousands possess certain synthesized outsidethe livingsystem. functional groups which are their reactive Organic chemistry broadly deals with the centres.The common functional groups of chemistry of carhon compounds, regardlessof importancein biomoleculesare presentedin their origin. Biochemistry, however, is Table39.1. concerned with the carbon chemistry of life Gomrmon ring structures of organicchemistry only.Thegeneralprinciples in biochemistry provide strongfoundationsfor understanding Thereare manyhomocyclicand heterocyclic exclusively However,biochemistry biochemistry. rings, commonlyencountered in biomolecules. that occur in the living dealswith the reactions given A list is in Fig.S9.l. selected of them systemin aqueousmedium. Homocyclicrings : Phenylring derivedfrom Most commoR organie eonnpounds benzene is found in several biomolecules found in living systenr (phenylalanine, tyrosine, catecholamines). The organic compounds,namely carbo- Phenanthreneand cyclopentaneform the hydrates,lipids, proteins,nucleic acids and backboneof steroids(cholesterol, aldosterone).

703

704

E}IOCHEMISTRY

Functional group Name Group

Hydroryl

General structural Type of formula compound

o

Aldehyde

-c-H

tl R _ C _H

Keto

o -c-

tl R1-C-R2

il

o Carboxyl

Sulfhydryl

Ester

tl

Aldehyde

glucose Glyceraldehyde,

Ketone

Fructose, sedoheptulose

o

o ll

-c-oH

R-C-OH

acid Carboxylic

Aceticacid,palmitic acid

-NHz

R-NH2

Aminoacid

Alanine, serine

H I -N-

H I R_N-

lminoacid

Proline, hydroryproline

_ SH

R _ SH

-o-

R1-O-R2

o

o

tl

-c-o-R1

o

Amido

Glycerol, ethanol

R_OH

o

Examplesof biomolecule(s)

-d--
ll R2- C - O - R 1

coenzyme A Cysteine, Ether

Thromboxane A2

Cholesterol ester

o

n.-8-r.r1l'

N-Acetylglucosamine

CoenzymeQ is an exampleof benzoquinone and indole are examples of fused heterowhile vitaminK is a naphthoquinone. cyclic rings. Heterocyclicrings: Furanis the ring structure found in pentoses. Pyrroleis the basicunit of porphyrins (heme) found in many biomolecules while pyrrolidineis the ringpresentin the amino acid, proline.Thiophenering is a part of the The compounds possessing identical vitaminbiotin.Theaminoacidhistidinecontains molecular formulae but different structures imidazole. are referredto as isomers.The phenomenon Pyranstructureis found in hexoses. Pyridine of existenceof isomersis called isomerism nucleusis a part of the vitamins-niacin and (Creek : isos-equal; meros-parts). lsomers (cytosine, pyridoxine.Pyrimidines thymine)and differ from each other in physicaland chemical purines(adenine,guanine)are the constituentsproperties. Isomerismis partly responsiblefor of nucleotidesand nucleic acids. lndole ring the existence of a large number of organic is found in the amino acid tryptophan.Purine molecules.

705

TO BIOORGANICCHEMISTRY Ghapter 39 : INTRODUCTION

(A)

Naphthalene

Phenol

B€nzene

o Naphthoqulnon€

Anthracens

Phenanthrene

o-Naphthol

Gyclopentane

(B)

Furan

I H Pyrrole

Thlophene

Pyran

Pyrldlne

Pyrlmldlne

i H lmldazole

Fig.39.1 : Commonring structuresfoundin biomolecules(A) Homocyclicrings (B) Heterocyclicrings.

Considerthe molecularformulaJ2H5O. There isomerism)or difference in the position of are two importantisomersof this-ethyl alcohol functionaf Broups (position isomerism) or (C2HsOH) and diethyl ether (CH3OCH3) difference in both molecular chains and functionalgroups(functional isomerism). depictednext. HHHH lll H-C- -H

Structural isomerism, as such, is more H-C-O-C-H common in general organic molecules. lll Tautomerism,a type of structuralisomerism, HH H occurs due to the migrationof an atomor group Ethylalcohol Dl€thyleth3r positionto the other e.g. purinesand from one lsomerism is broadly divided into two cate(Chapter5). pyrimidines gories-structuralisomerismand stereoisomerism. Structural

isomerism

Stereoisomerism

(Greek: stereos-spaceoccuStereoisomerism The difference in the arrangementof the framework) pying)is, perhaps,more relevantand important atomsin the molecule(i.e.molecular is responsiblefor structuralisomerism.This to biomolecules. The differential space of atomsor groupsin molecules mav be due to variationin carbonchainskhain arrangement

I I

i l

I

t I

I

706

BIOCHEMISTRY

gives rise to stereoisomerism. Thus, stereoisomershave the samestructuralformula but differ in their spatialarrangement. Stereoisomerismis of two types-geornetric isomerism and optical isomerism. Geometricalisomerism: This is also called cis-transisomerismand is exhibitedby certain possessing molecules doublebonds.Ceometrical isomerismis due to restrictionof freedomof rotationof groupsarounda carbon-carbon double bond (C:C). Maleicacid and fumaricacid are classicalexamplesof cis-transisomerism. H-C-COOH llll H-C-COOH Malelcacld(cis)

H-C-COOH HOOC-C-H Fumarlcacld(trans)

Asymmetric F19.39.2: Asymmetricand symmetricobjects.

B B I I A-C-D D-C_A I I E E Minor

When similargroupslie on the sameside,it is called cis isomer(Latin: cis-on the same side).On the other hand,when similargroups The numberof possibleopticalisomersof a lie on the oppositesides,it is referredto as trans moleculedependsupon the specificnumberof isomer (Latin ; trans-across).As is observed chiralcarbon(n). lt is givenby 2n. from the above structure,maleic acid is a crs What is optical activity? form while fumaricacid is a transform. Geometricisomerismis also observedin sterolsand porphyrins.crs-fransisomersdiffer in physicaland chemicalproperties.

in all directions. Theordinarylightpropagates However,on passingordinarylight througha Nicol prism,the planeof polarizedlightvibrates in one direction only (Fig.39.31.

Optical isomerism : Optical isomers or enantiomersoccur due to the presenceof an Certainorganiccompounds(opticalisomers) asymmetriccarbon (a chiral carbon).Optical which are said to exhibit optical activity rotate isomersdiffer from each other in their optical the planeof polarizedlighteitherto the left or to activityto rotatethe planeof polarizedlight. the right. What is an asymmetric

carbon?

The term levorotatory (indicated by 1 or (-) sign)is usedfor the substances which rotate the plane of polarizedlight to the left. On the other hand, the term dextrorotatory(indicated rotating by d or (+) sign)is usedfor substances the planeof polarizedlight to tight (Fi9.39.31.

An object is said to be symmetricalif it can be dividedinto equalhalvese.g.a ball.Objects which cannotbe divided into equal halvesare asymmetric, e.g. hand (Fi9.39.2). An asymmetric objectcannotcoincidewith itsmirrorimage.For The term racemicmixture representsequal instance,left hand is the mirror imageof right handand thesetwo can neverbe superimposed.concentrationof d and I forms which cannot In contrast,a symmetricalobject like a ball rotatethe planeof polarizedlight. superimposes its image. Gonfiguration of chiral moleeules A carbonis saidto be chiral(Greek:hand)or of chiral While representing the configuration asymmetricwhen it is attachedto four different groups.Theirmirrorimagesdo not superimposemofecules,the configurationof glyceraldehyde is taken as a referenceshndard. with eachother.

Ghapter 39 : INTFIODUCTION T0 BIOORGANICCHEMISTFIY

9Ho

H-C-OH cH2oH

707

gHo HO-C-H cH2oH

DGlyccraldehyde L-Glyccraldehyde Ordinarylightwaves NicolPrism It must,however,be remembered that vibrating in all directions D- and L- do not representthe direction of the rotationof planeof polarizedlight.

Plane of polarizedlight vibratingin one direction

Existence of chiral biomolecules As you know, you can never come acrossanybodywho is yourmirrorimage. Thesameis truewith biomolecules. Only one type of molecules(D or L) arefound in the living system.Thus,the naturally occurringaminoacidsareof L-typewhile the carbohydratesare of D-type.

Planerotatedto the left(levorotatory)

Plane rotated to the right (dextrorotatory)

-l-h" general laws and principlesof chemistry Structure of water I and physics are applicable to biochemistry The H2O molecule existsin a bent Seometry. as well. lt is, therefore,worthwhile to have a i s 104.5" and the The bond angl e of H -O-H brief understanding of some of the basic O-H bond has a distance of 0.958Ao. There chemicaland physicalprinciplesthat have direct existselectrical polarity in H2O due to electrorelevanceto life. negativity(the power of an atom in a molecule It must, however, be remembered that this to attract electrons)difference between H and O. chapterdealswith quite unrelatedtopics to each This results in the polarization of a positive chargeon H and a negative chargeon O. Thus other. H2O molecule,althoughcarryingno net charge, possesses an electrical dipole. The polar character of water has tremendous biological significance. Water is the most abundantfluid on earth. lt is justifiably regarded as the solvent of life. As much as 70'h of a typical cell is composed of wat er . T h e u n i q u e p h y s i c a l a n d chemi cal properties of water have profound biological importance. The structures of biomolecules ( pr ot ei n s , n u c l e i c a c i d s , l i p i d s a nd carbohydrates)are maintaineddue to their interaction with water, which forms an aqueous environment.This is essentialfor sustaininglife.

Hydrogen bonds between H2O molecules : The presence of electrical dipoles on HzO molecules is responsible for their attraction. Hydrogen bonds are formed due to polarity different with atoms two between the transient in H2O, Thus, electronegativities. negative charge on the O atom of one H2O molecule and the transient positive charge on the H atom of another H2O molecule attract each other to form a hydrogen bond. The water

708

Grrfer

4O : OVEBVIEWOF BIOPHYSICALCHEMISTFY 6-

709

Acid

Base

HC\

H+ + C\-

H2CO3

H + + H C OJ

H2PO4

H+ + HPO,

NHi

H+ + NH3

Hzo cH3cooH

H+ + OH-

HA

H+ + CH3COOH++A(general)

(general)

It is evident that an acid dissociatesto form proton and base. On the other hand, the base combines with proton to form acid. The molecules are interlinked with each other by difference between an acid and its corresponding profuse hydrogen bonding. The energy of base (more commonly referred to as conjugate each hydrogen bond is very small comparedto base) is the presenceor absenceof a proton. In that of a covalent bond. But the collective general,a strong acid has a weak base while a strength of H-bonds is due to their large weak acid has a strong base. For instance,strong numbers. Hydrogen bonds are important for acid HCI has weak base Cl-, weak acid HCN the three-dimensionalstructures of biomole- has a strong base CN-. cu l es . Alkalies : The metallic hydroxides such as NaOH and KOH are commonly referred to as Water expands on freezing : Water is one of alkalies.Thesecompoundsdo not directly satisfy the very few substances that expands on the criteriaof bases.However,they dissociateto freezing. Thus, ice has a density of 0.92 g/ml, form metallic ion and OH- ion. The latter,being while water at OoChas density of 1.0 g/ml. For a base, accepts H+ ions. this reason,ice floats on water. And this property Ampholytes : The substances which can is essentialto maintain water equilibrium in the function both as acids and basesare referred to environment,and to sustainlife. as ampholytes. Water is the best example for (lmaginethat water contractedon cooling and ampholytes. becomesdenser.In such a case, ice would sink Dissociation of water to the bottom of seasand lakes and would never Water is a weak electrolvte and dissociatesas get exposed to sun rays. Thus, frozen water would permanentlyremain as ice. lf this were to follows. happen,earth would have a permanentice age!)

H zO#H + + OH The proton reacts with another molecule of water to form hydronium ion (HtO*), H+ + H2O l^

HrO*

For the sake of convenience, the presenceof According to Lowry and Bronsted,an acid is proton as H3O+ is ignored. defined as a substance that gives off protons By applying the law of mass action for the while base is a substancethat acceptsprotons. Thus, an acid is a proton (H+) donor and a base dissociationof water. is a proton acceptor. A few examples of acids and their correspondingbasesare given in the next column.

*

710

BIOCHEMISTRY

Here K is a constant; the concentrations are expressed in molarity. Since the degree of dissociationis very small, the concentrationof undissociated[HzO] may be taken as constant. Ko = [H+] [OH-] K, is the dissociationconstantfor water. lts v a l u e i s 1 0 -1 4a t 2 5 o C . tH+l tOH-l = le-14 In a neutral solution lH*l = [OH-] = 10-7 Hydrogen

ion concentration

(pH)

pH

FIuid juice Pancreatic (orwholeblood) Bloodplasma fluid Cerebrospinal Tears lnterstitial fluid Human milk Saliva fluid(cytosol) Intracellular juice Gastric Urine

7.5 7.357.2 7.2 7.2 7.2 6.4 6.5 1.5 5,0 -

8.0 7.45 7.4 7.4 7.4 7.4 7.0 6,9 3.0 7.5

The acidic or basic nature of a solution is measuredby H+ ion concentration. The strength of H+ ions in the biologicalfluids is exceedingly low. For this reason,the conventionalunits such as moles/l or g/l are not commonly used to The pH of a given solution can be easily expressH+ ion concentration. altered by the addition of acids or bases.Buffers are defined as the solutions which resist change Sorenson(1909) introduced the term pH to in pH by the addition of small amounts of acids express H+ ion concentration. pH is defined as or bases.A buffer usually consistsof a weak acid the negative logarithm of H+ ion concentration. and its salt (e.g. acetic acid and sodium acetate) pH=_loglH+I or a weak base and its salt (e.9. ammonium hydroxide and ammonium chloride). Several The pH is a narrow scale, ranging from 0 to bufferscan be prepared in the laboratory. Nature 14 which correspondsto 1 M solution to 10-14 has provided many buffers in the living system. M solution of [H+] concentration. As explained under dissociation of water, pure water has an equal concentrationof H+ and OH- ions i.e. 10-7 M each. Thus,pure water has a pH 7 which is neutral.Solutionswith pH less than 7 are said to be acidic while those with pH greater than 7 are alkaline. lt must be rememberedthat the term acidic or alkaline are not absolutebut only relative.Thus, a solution with pH 3.0 is more acidic when comparedwith a solution of pH 4.5. A rise in H+ concentration decreasespH while a fall in H+ concentrationincreasespH. The reverseis true for OH- concentration.The pH o f a s o l u ti o nc o n ta i n i n g1 N [H + ] i s 0 w hi l e t h a t c o n ta i n i n g1 N [O H -] i s 1 4. The pH of important biological fluids is presented in Table 40.1.

Mechanism

of buffel

action

Let us consider the buffer pair of acetic acid and sodium acetate.Acetic acid, being a weak acid, feebly ionizes.On the other hand, sodium acetate ionizes to a large extent. C H 3C OOH #C H TC OOC H 3C ooN a#cH rcoO-

+ H+ + N a+

When an acid (say HCI) is added, the acetate ions of the buffer bind with H+ ions (of HCI) to form acetic acid which is weakly ionizing. Therefore,the pH change due to acid is resisted by the buffer. H+ + CH3COO- --+

CH3COOH

When a base (say NaOH) is added the H+ ions of the buffer (acetic acid) combine with OH- ions to form water, which is weakly

trhapter 4O : OVEBVIEWOF BIOPHYSICALCHEMISTBY

71t

orissociated.Thus, the pH change due to base addition is also prevented by the buffer.

solutioncan be preparedby dissolving1 mole of solute in 1,000 g of solvent.

OH- + H+ ---------+H2O

Normality : Molarity is based on molecular weight while normality is based on equivalent weight. One gram equivalent weight of an element or compound representsits capacity to combine or repface 1 mole of hydrogen. ln general, the gram equivalent weight of an elernent or a compound is equal to its molecular weight divided by the total positive valence of the constituent ions. Thus, for NaOH and KOH, the molecular and equivalent weights are the same,while, for H2SOa,equivalentweight is half the of molecular weight. The term milliequivalent per liter (mEq/l) is used for smaller concentrations.

Buffering capacity :The efficiency of a buffer t maintaining a constant pH on the addition of acid or base is referred to as buffering capacity. ft mostly depends on the concentration of the auiier components. The maximum buffering capacity is usually achieved by keeping the same concentrationof the salt as well as the acid. For a comprehensivediscussion on blood buffers, refer Chapter 21.

Solutions may be regarded as mixtures of substances. In general,a solution is composedof two parts-solute and solvent. The substance Thomas Graham (1861), regarded as the that is dissolvedis solute and the medium that colloidal chemistry', divided dissolvesthe solute is referredto as solvent.The 'father of substances into two classes-crystalloids and particle size of a solute in solution is < 1 nm. colloids. The relativeconcentrationsof substancesin a Crystalloids are the substanceswhich in solution can be measuredby severalways. solution can freely pass (diffuse) through Per cent concentration : This representsparts parchment membrane e.g. sugar, urea, NaCl. per 100. The most frequentlyused is weight per Colloids (Creek : glue-like),on other hand, are volume (w/v) e.g. 9o/" saline (9 g/1O0 ml the substancesthat are retained by parchment solution). For expressingsmaller concentration, membranee.g. gum/ gelatin,albumin.The above mg (10-39), pg (10-6 g), ng (10-e g) and pg classificationof Graham is no longer tenable, (10- t z t , ar e us e d . since any substancecan be converted into a Parts per million (ppm) : This refers to the col l oi d by sui tabl emeans.For i nstance,sodi um number of oarts of a substancein one million chloride in benzeneforms a colloid. parts of the solution. Thus 10 ppm chlorine means 10 pg of chlorine in 1 g of water. Molarity (M) : lt is defined as the number of moles of solute per liter solution NaCl has a molecular weight of 58.5. To get one molar (1 M) or one mole solution of NaCl, one gram m olec ular weig h t (5 8 .5 g ) o f i t s h o u l d b e dissolvedin the solvent(HzO) to make to a final total volume of 1 liter. For smaller concentrations,millimole and micromole are used.

Colloidal state : As such, there are no group of substancesas colloids, rather,substancescan exist in the form of colloidal sfafe or colloidal system. Colloidal state is characterized by the particle size of 1 to 100 nm. When the particle si ze i s < 1 nm, i t i s i n true sol uti on. For the particle sizes >100 nm, the matter exists as a visible precipitate.Thus, the colloidal stateis an intermediate between true solution and precipitate.

Mofality : lt representsthe number of moles of solute per 11000 g of mlvenf. One molal

Phasesof colloids : Colloidal state is heterogeneous with two phases.

772

B IOC H E MIS TF| Y

1. Dispersed phase (internal phase) which constitutesthe colloidal particles. 2. Dispersionmedium (externalphase)which r ef er s to th e m e d i u m i n w h i c h th e col l oi dal particlesare suspended. CLASSIFICATION

separationof charged colloids can be achieved by the analytical technique-electrophoresis (Refer Chapter 4l). 4. Osmotic pressure : Since the colloidal particlesare larger in size, their contribution to osmotic pressureis relativelyless.

OF COLLOIDS

5. Non-dialysable nature : The colloidal particles, being larger in size, cannot pass through a membrane(cellophaneor parchment). The membrane, however, allows dispersion medium and smaller particlesto escapethrough 1. tyophobic (Creek: solvent-haring) : These the pores.This processis referredto as dialysis colloids do not have anv attraction towards and is useful for the separationof colloids. dispersion medium. When water is used as dispersionmedium, the colloids are referredto 6. Donnan membrane equilibrium : The as hydrophobic. presenceof non-diffusiblecolloidal particles(e.g. protein) in the biological systemsinfluencesthe 2. tyophilic (Creek : solvent-loving): These concentration of diffusible ions across the colloids have distinct affinity towards dispersion membrane. This is an important phenomenon, medium. The term hydrophilic is used for the the details of which are given on page 714. c olloid s w h e n w a te r i s th e d i s p e rs i onmedi um. Based on the affinity of dispersionmedium with dispersedphase, colloids are classifieoas lyophobic and lyophilic colloids.

The terms gel and sol are, respectively,used t o jelly -l i k ea n d s o l u ti o n -l i k ec o l l o i d s. E mul si ons are the colloids formed by two immiscible liquids (e .g .o i l + w a te r).F re q u e n tl y,emul si ons can be stabilized by using agents known as emulsifiers. For instance,the protein casein acts as an e m u l s i fi e rfo r mi l k . Micelles are the aggregates of colloidal particles.Soap(sodiumpalmitate)in water is the classicalexample for the micelles formation. Properties

of colloids

1. Brownian movement: The continuousand haphazard motion of the colloidal particles is known as Brownian movement.

Biological

importance

of colloids

1. Biological fluids as colloids : These i ncl ude bl ood, mi l k and cerebrospi nal fl ui d. 2. Biological compounds as colloidal particles : The complex molecules of life, the hi gh mol ecul arw ei ght protei ns,compl ex l i pi d s and polysaccharides exist in colloidal state. 3. Blood coagulation : When blood clotting occurs, the sol is convertedfinally into the geL 4. Fat digestion and absorption : The formation of emulsions, facilitated bv tne emulsifying agents bile salts, promotes fat digestionand absorptionin the intestinaltract.

5. Formationof urine : The filtrationof urine 2. Optical properties : When light is passed i s basedon the pri nci pl eof di al ysi s. through a colloidal solution, it gets scattered. This phenomenon is referredto as Tyndal effect. 3. Electrical properties : The colloidal particlescarry electricalcharges,either positive or negative.The electricalchargemay be due to ionizationof the colloidal particlesor adsorption of the ions from the medium, or both. The stability and precipitation of colloids is determinedby the ionic chargesthey carry. The

The mol ecul es i n l i qui ds or gases are i n continuous motion. Diffusion may be regarded as the movement of solute molecules from a higher concentration to a lower concentration. Diffusion is more rapid in gasesthan in liquids.

Ghapten 4O : OVERVIEWOF BIOPHYSICAL CHEMISTFIY

713

Thesmallerparticles diffusefasterthanthe larger weight, in general, exhibit greater osmotic ones.The greaterthe temperature, the higheris pressure. Further,for ionizablecompounds, the the rateof diffusion. totalosmoticpressure is equivalent to the sumof the individualpressures exertedby eachion. For Diffusionoccursin truesolutions aswell as in instance, one molarsolutionof NaCl will exert colloidalsolutions. double the osmotic pressureof one molar solutionof glucose. Thisis because NaClionizes Applications of diffusion to Na+ and Cl- while glucoseis non-ionizable. 1. Exchange of 02 and CO2 in lungsand in The solutionsthat exert the same osmotic tissuesoccursthroughdiffusion. pressureare said to be isoosmofic.The term 2. Certainnutrients areabsorbed by diffusion isotonicis usedwhen a cell is in direct contact in the gastrointestinal tract e.g. pentoses, with an isoosmotic solution(0.9%NaCl)which minerals,watersolublevitamins. doesnot changethe cell volumeand,thus,the A solutionwith relatively 3. Passageof the waste productsnamely cell toneis maintained. greater pressure osmotic is referred to as ammonia,in the renal tubulesoccursdue to hypertonic. On the other hand, a solutionwith diffusion. refatively fower pressure is hypotonic. The term oncotic pressureis commonlyused to representthe osmotic pressureof colloidal (e.g.albumin,globulin). substances Osmosis (Greek : push) refers to the movement of mlvent (most frequently water) through a semipermeable memhrane. The flow of solventoccurs from a solution of low concentration to a solution of high concentration, when both are separated by a semipermeablemembrane.ln a strict sense,the semipermeable membrane is expected to be permeableto the solvent and not to the solute.

Osmotic

pressure

Units of osmotic pressurez Osmole is the unit of osmotic pressure.One osmole is the numberof moleculesin grammolecularweight of undissociated solute.One gram molecular weight of glucose (180 g) is one osmole. However,one gram molecularweight of NaCl (5B.5g) is equivalentto 2 osmoles,sinceNaCl ionizesto give two particles(Na+,Cl-). Osmotic pressureof biological fluids is y expressed as milliosmoles. The frequentf osmotic pressure of plasma is 280-300 milliosmoles/|.

Osmotic pressuremay be defined as the excesspressurethat must be applied to a Applications of osmosis solutionto preventthe passageof solventinto the solution,when both are separatedby a 1. Fluid balanceand blood volume : The semipermeable membrane. fluid balanceof the differentcompartments of the body is maintained due to osmosis.Further, Osmosis is a colligative property i.e. a osmosis significantly contributes to the regulation characterwhich dependson the number of of blood volumeand urineexcretion. soluteparticlesand not their nature.Osmotic pressure is directly proportional to the 2. Redblood cellsand fragility : When RBC (number)of the solutemolecules are suspendedin an isotonic (O.9lo NaCl) concentration (e.g. solution,the cell volume remainsunchanged or ions.Low molecularweightsubstances NaCl, glucose)will have more number of and they are intact. In hypertonicsolution(say moleculescomparedto high molecularweight 1.5% NaCl), water flows out of RBC and the (albumin,globulin)for unit mass. cytoplasmshrinks,a phenomenonreferredto as substances Therefore,the substances with low molecular crenation,

714

BIOGHEMISTF|Y

On the other hand, when the RBC are kept in hypotonic solution (say O.4o/oNaCl), the cells bulge due to entry of water which often causes rupture of plasma membrane of RBC (hemolysis). Osmotic fragility testlor RBC is employed in Iaboratories for diagnostic purposes. For a normal human blood, RBC begin to hemolysein 0.45% NaCl and the hemolysis is almost compfete in 0.33% NaCl. lncreased fragility of RBC is observed in hemolytic jaundice while it is decreasedin certain anemias.

Initial Na+

Prcl-

Na+

cltl

I At equilibrium Fiq.40,2 : Diagrammaticrepresentationof

3. Transfusion : lsotonic solutions of NaCl (O.9%)or glucose(5%) or a suitablecombination of these tvvo are commonly used in transfusion in hospitals for the treatment of dehydration, burns etc. 4. Action of purgatives: The mechanismof action of purgatives is mainly due to osmotic phenomenon. For instance, epson (MgSOa 7H2O ) o r C l a u b e r' s (N a 2 S O a 1 0 H 2O) sal ts withdraw water from the body, besides preventingthe intestinalwater absorption. 5. Osmotic diuresis: The high blood glucose concentrationcausesosmotic diuresis resulting in the loss of water, electrolytesand glucose in the urine. This is the basisof polyuria observed in diabetesmellitus. Diuresiscan be produced by administering compounds (e.g. mannitol) which are filtered but not reabsorbedbv renal t ubules .

When membraneis freely permeableto ions (say Na+, Cl-) and if the concentrationof ions on both the sides is different,the ions freely diffuse to attai n equal concentrati on.Gi bbs-D onnan observed that the presence of a non-diffusible ion on one side of the membrane alters the diffusion of diffusible ions.

In the molecule sodium proteinate(Na+Pr-), the protein (Pr) ion is non-diffusablethroughthe membrane. Let us consider two sides of a compartmentseparatedby a membrane.Initially, sodi um protei natei s on si de I w hi l e sodi um chloride is on side ll (Fig.40.2).Diffusible ions (Na+,Cl-) can freely passthroughthe membrane. On si de l , N a+ i ons w i l l bal ancethe i ncomi ng 6. Edema due to hypoalbuminemia : C l - i ons besi desP r i ons, w hi l e on si de l l N a+ Disorderssuch as kwashiorkorand glomeruloions have to balance only Cl- ions. Therefore, nephritis are associatedwith lowered plasma the concentrationof Na+ on side I is greaterthan albumin concentration and edema. Edema is on side ll, However, from the thermodynamical caused by reduced oncotic pressureof plasma, poi nt of vi ew , at equi l i bri um,the concentrati on leading to the accumulationof excessfluid in of Na+ Cl- on both the sidesshould be the same. tissuespaces. Thus N a+C l - (l ) = N a+ C l - (l l ) 7. Cerebral edema : Hypertonic solutionsof > N a+ (l l ) N a+ (l ) Since salts (NaCl, MgSOa) are in use to reduce the < cl- (il) cl- (r) volume of the brain or the oressure of c er ebr o s p i n afll u i d . Consequently,the concentrationof Cl- ions should be greater on side ll. Further,the total 8. lrrigation of wounds : lsotonic solutions are used for washing wounds. The pain concentrationof ions on side I is higher than on experienced by the direct addition of salt or si de l l . sugar to wounds is due to osmotic removal of water.

The salient features of Donnan membrane equi l i bri umare l i sted next.

Ghapter 4O : O\EFIVIEWOF BIOPHYSICAL CHEMISTRY

1. The presenceof a non-diffusiblet o n influencesthe concentrationof diffusible ions acrossthe membrane. 2. The concentrationof oppositely charged ions (Na+), is greater on the side of the membrane containing non-diffusible ions

(Pr). 3. The concentration of similarly charged ions (Cl-) is higher on the side of the membrane not containing non-diffusibleions (Pr).

715

Liquidor fluid hasa tendencyto flow which is referredto as fluidity.The term viscositymay be defined as the infernal resistanceoffered by a liquid or a gas to flow. The property of viscosityis due to frictionalforcesbetweenthe layerswhile their movementoccurs.Viscosity may be appropriatelyregardedas the internal frictionof a liquid.

Liquidsvary widely as regardstheir viscosity. For instance, etherhasvery low viscositywhile 4. The net concentration of total ions honey and bloodare highlyviscous.Amongthe will be greater on the side of the membrane containing non-diffusibleions. This leads to a several factors that contribute to viscosity, differencein the osmotic pressureon either side densityof the liquid,concentration of dissolved substances and their molecularweightand the of the membrane. molecularinteractions are important.Increase in temperature decreases viscositywhile increase Applications of Donnan in pressure increases viscosityto someextent. membrane equilibrium Viscosityof colloidal solutions,particularly 1. Differencein the ionic concentrationsof Iyophilic colloids,is generally higherthantrue biologicalfluids : The lymph and interstitial solutions. fluids have lower concentrationof inorganic Units of viscosity: The unit of viscosityis of cations(Na+,K+) and higherconcentration anions (Cl-) compared to plasma. This is poise, after the scientistPoiseuille,who first studiedtheflow of liquids.A poise attributedto the higherprotein(Pr) contentin systematically representsdynes/otf. the plasma. 2. Membrane hydrolysis : The relative Applications of viscosity strengthof H+ and OH- ions and, therefore, 1. Viscosityof blood : Bloodis about4 times the acidic or alkaline nature on either side moreviscousthanwater.The viscosityof blood is influenced by the presence of of a membrane, is mainly attributedto suspended blood cellsand ions.Thisphenomenon is referred non-diffusible colloidal plasma proteins.As the blood flows Donnanmembrane to as membranehydrolysis. through capillariesthe viscositydecreases to equilibriumexplainsthe greaterconcentration facilitate free flow blood. Blood viscosity is of o( H+ ions in the gastric juice. increasedin polycythemia(elevationof RBC), A 3. Lower pH in RBC : The hemoglobinof while it is reducedin anemiaand nephritis. cardiacwork load. RBC is negativelycharged and, therefore, moreviscousbloodincreases causesthe accumulationof positivelycharged When dehydrationoccurs,the viscosityof the ' ions including H+. Therefore,the pH of blood increases. RBCis slightlylower(7.25)thanthat of plasma 2. Viscositychangein muscle: Excitationof (7.4). the muscleis associated with increasein the 4. Osmoticimbalance: Donnanmembrane viscosityof the musclefibres.This delaysthe muscle. equilibrium-whichresultsin the differentialchangein the tensionof the contracting 3. Vitreous body : This is an amorphous of distributionof ions in differentcompartments the body-partly explainsthe osmoticpressure viscousbodylocatedin the posterior chamberof theeye.lt is rich in albuminandhyaluronic acid. differences.

BIOCHEMISTF|Y

776

FIg.40.3: Surtacetensionof a liquid.

4. S y n o v i a fl l u i d : l t c o n ta i n sh y a l u r oni caci d whic h im p a rts v i s c o s i ty a n d h e l p s i n the lubr ic at i n gfu n c ti o n o f j o i n ts .

A moleculein the interiorof a liquid is by othermoleculesin all directions. In attracted contrast,a moleculeon the surfaceis attracted and not upwards only downwardsand sideways ffig.aO3).Due to this,the surfacelayerbehaves like a stretchedfilm. Surtacetensionis the force with which the molecules on the surtace are held together. lt is expressedas dynes/cm. Surface tension decreaseswith increase in temperature. Due to the phenomenon of surfacetension, any liquid occupiesthe minimum possible volume.

Sulfurpowder, saltsin urineof jaundicepatients. when sprinkled on the surface of urine possessing bile salts,sinks.This is in contrastto a normalurinewheresulfurpowderfloats.Hay's test is basedon the principlethat bile saltsin urinelowersurfacetensionwhich is responsible for sulfurto sink. 3. Surfactantsand lung function : The low surfacetensionof the alveoli keepsthem apart and allows an efficientexchangeof gasesin predominantly lungs.In fact, certainsurfactants, dipalmitoylphosphatidylcholine (dipalmitoyl for maintaininglow lecithin)are responsible surface tension in the alveoli. Surfactant deficiency causesrespiratory distresssyndrome in the infants. 4. Surfacetensionand adsorption: Adsorption, being a surfacephenomenon,is closely relatedto surfacetension.Due to the coupled the formationof action of thesetwo processes, complexesof proteinsand lipidsoccursin the biologicalsystems. 5. lipoprotein complex membranes: The structureof plasmamembraneis composedof namely surfacetension reducingsubstances, proteins. of absorption Thisfacilitates lipidsand thesecompounds.

Accordingto the principleof Cibbs-Thomson, Adsorption is a surface phenomenon lt is the the compounds which lowerthe surfacetension get concentratedat the surface(or interface) orocess of accumulation of a substance layer while those compoundswhich increase (adsorbate)on the surface of another substance surfacetensionget distributedin the interior (adsorbent).Adsorption differs from absorption, portion of the liquid. ln general, organic as the latter involves the diffusion into the (proteins,lipids)decreasewhereas interior of the material. substances inorganic substances(NaCl, KCI) increase The capacityof an adsorbentdependson the t surfacetension. surface area. Therefore,porous substancesserve as betteradsorbentse.g. charcoal,alumina,silica gel. Adsorption is a dynamic and reversible 1. Digestionand absorptionof fat : Bile salts process which decreases with rise in reduce the surface tension. They act as temperature.

Applications

of surface tension

detergentsand cause emulsificationof fat, of adsorption thereby allowing the formation of minute Applications particlesfor effectivedigestionand absorption. 1. Formation of enzyme-substratecomplex : 2. Hay's sulfur test : This is a common For the catalysisto occur in biological system, laboratorytestemployedfor the detectionof bile formation of enzyme-substrate complex is a

CHEMISTRY OF BIOPHYSICAL Ghapten 4O : OVEFIVIEW

prerequisite.This happensby adsorptionof substrateon the enzyme.

777

1. o-Rays-an o particlepossessing 2 protons i .e. hel i um nucl ei .

2. p-Rays-due to the emission of electrons. 2. Action of drugsand poisons: On adsorption at the cell surface,drugsand poisonsexert 3. y-Rays-due to emission of high energy their action. photons. 3. Adsorption in analytical biochemistry: The radiationsemmittedby radioactivenuclei The principleof adsorptionis widely employed are characteristicof the isotope. For instance, in the chromatographytechnique for the 3H , 14C , and 32P al l emi t p-parti cl esi n the separationand purification of compounds respecti veenergi esof 0.018, 0.155 and 1.71 (enzymes, immunoglobul ins). MeV. The B and 7 emitting radioisotopes are employed in biochemical research. These isotopes are produced in nuclear reactors. The simple chemicals so produced are then lsotopes have revolutionized biochemistry converted to radiolabelled biochemicals by when they became available to investigators chemicaf or enzymatic synthesis. soon after Second World War. lsotopes are Units of radioactivity z Curie (Ci) is the basic defined as the elements with same atomic unit of radioactivedecay. lt is defined as the number but different atomic weights. They amount of radioactivity equivalent to 1 g of possessthe same number of protonsbut differ in radium i.e. 2.22 x '1O12disintegrationsper the neutronsin their nuclei. Therefore,isotopes mi nute (dpm). Mi l l i curi e (mC i ) and mi crocuri e (Creek : iso-equal; tope-place) occupy the (pCi), respectively,correspondingto 2.2 x 1Og same place in the periodic table. The chemical and 2.2 x 106 dpm, are more commonly used. properties of different isotopes of a particular Half-lives of isotopes : The unstable element are identical. radioisotopesundergo decay. The radioactivity lsotopes are of two types-sfable and gets reducedto half of the original within a fixed unstable.The latter are more commonly referred time. This representsthe half-life which is to as radioactive isotopes and they are of characteristicfor a given isotope. particular i nterestto biochemists.Conventionally Some of the commonly used radioactive while representingisotopes,the atomic weight is written on upper left side of the elementsymbol. isotopes in biochemical research with their characteristicsare given in Tahle 40.2.

Stable isotopes They are naturallyoccurring and do not emit radiations (non-radioactive) e.g. deuterium (heavy hydrogen) 2H; 13C; 1s51' 186. 51"51" isotopes can be identified and quantitated by mass spectrometry ot nuclear magnetic resonance(NMR). They are lessfrequentlyused in biochemical investigations. Radioactive

isotopes

The atomic nucleusof radioactiveisotopesis unstable and, therefore, undergoes decay. The radioactive decay gives rise to one of the f ollowing 3 io n i z i n g ra d i a ti o n s .

Measurement of radioactivity of isotopes Severaltechniquesare in usefor the detection of radioactivity of the isotopes. The most commonly employed in biochemical research are-Geiger counters, Iiquid scintillation counter and autoradiography. Geiger counters are almostoutdated.Liouid scintillationcounters are now widelv used. ln the liquid scintillationcounter,the sample is dissolved or suspended in a solution containing one or two fluorescent organic compounds (fluors).The fluors emit a pulse of

ElIOGHEMISTRY

718

Isotope 3H 14C

Radiation

p B

22Na 32P 35S 6Ca seFe

p p p

131 |

12.2years years 5,700 2.5years 14.5days 87days 164days

F'T

45days 5.25years

v F'v

60days

6oCo 1251

Half-life

8.1days

are widely used in establishing Radioisotopes the precursor-productrelationships in metabolisms and understanding of the complex metabolic pathways. A few important applicationof radioisotopes are 1. By the useof isotopetracers,the metabolic origin of complex molecules such as heme, cholesterol,purines and phospholipidscan be determined.As early as 1945, it was established that nitrogen atom of heme was derived from glycine.This was done by feedingratswith (1sN) gl yci ne and detecti ng(1sN )heme. 2. The precursor-product relationship in several metabolic pathways has been investigatedby radioisotopes.e.g. Krebs cycle, p-oxidation of fatty acids, urea cycle, fatty acid synthesis.

light when struck by radiation. The light, 3. Radioisotopesare conveniently used in proportional to the radiation energy, can be the study of metabolic pools (e.g. amino acid detected.The advantagewith liquid scintillation pool) and metabolic turnovers (e.g. protein counter is that it can discriminatethe particlesof turnover). different energies. Thus, two or more isotopes 4. C ertai n endocri ne and i mmunol ogi cal can be simultaneouslydetected. studiesalso depend on the use of radioisotopes In autoradiography, the radiations are e.g. radioimmunoassay. detectedby its blackeningof photographicfilm. are employed in elucidating 5. Radioisotopes T his t e c h n i q u e i s c o mmo n l y u s e d for the metabolism. drug detection of radioactive substancesseparatedin polyacrylamidegel electrophoresis(PACE). in Radioisotopes diagnosis and treairnerrt ct Applleatiolrs

radioisotopes in bioeher*rEstryr Radioactive isotopes have become indispensable tools of biochemistry.They can be conveniently used as tracers in biochemical researchsince the chemical propertiesof different isotopesof a particular element are identical. Therefore,the living cells cannot distinguishthe radioactive isotope from a normal atom.

are used in the scanning Certainradioisotopes bone (eoSr)ano of organs-thyroid gland (1311), ki dney (1311hi ppuran). Radioactivity has been employed in the treatment of cancers. This is based on the principle that radiations produce ionizations w hi ch damage nucl ei c aci ds. Thus, th e uncontrolledproliferationof cells is restricted.

Vlorhingon thepinciplesof adsorption, ?/trt;tiln, ion-achange; I an a heybiochanical toolin kboratoryexperimentation."

p iochemistryis an experimentalratherthan a Lltheoretical science.The understandingand developmentof conceptsin biochemistryare a result of continuous experimentation and evidenceobtained therein. lt is no exaggeration to state that the foundations for the present(and the future,of course!)knowledgeof biochemistry are based on the laboratory tools employed for biochemical experimentation. Thus, the development of sensitive and sophisticated analytical techniques has tremendously contributedto our understanding of biochemistry.

!

Methods of DNA assav

a

DNA fingerprintingor DNA profiling.

Chromatographyis one of the most usefuland popular tools of biochemistry.lt is an analytical technique dealing with the separation of closely related compounds from a mixture. These include proteins, peptides,amino acids, lipids, carbohydrates,vitamins and drugs.

A detailed discussion on the tools of F,i($ttrrit:aF For'$Fective biochemistryis beyond the scope of this book. The credit lor the discovery of The basic principles of some of the commonly employedtools are describedin this chapter.The chromatographygoes to the Russian botanist reader must, however, refer Chapter 27, for the Mikhail Tswett.lt was in 1906, Tswettdescribed following techniques related to molecular the separationof plant leaf pigmentsin solution by passingthrougha column of solid adsorbents. biology,andrecombinantDNA technology He coined the term chromatography(Creek : . lsolation and purificationof nucleic acids chroma--colour; graphein-to write), since the . Nucleic acid blotting techniques technique dealt with the separationof colour . DNA sequencing compounds (pigments). Coincidently, the term Tswettmeanscolour in Russian!Truly speaking, . Polvmerasechain reaction

719

720

B IOC H E MIS TR Y

chromatographyis a misnomer, since it is no longer limited to the separation of coloured c om po u n d s . Principles and classification Chromatography usually consists o[ a mobile phase and ---+ Paperchromatography a stationary phase.The mobile I phase refers to the mixture of VY Singledimensional Twodimensional substances(to be separated), dis s ov e di n a l i q u i d o r a g a s . Ascending lThe stationary phase is a L Descending porous solid matrix through Thin layerchromatography which the sample contained in the mobile phase percolates. Gas-liquidchromatography The interaction between the Fi,.41.1 : lmportanttypesof chromatography mobile and stationary phases resultsin the separationof the compounds from the mixture. These interactionsinclude the (spotted)at one end, usually -2 cm physicochemicalprinciples such as adsorption, partition, ion-exchange, molecular sieving and above, a strip of filter paper (Whatman No. 1 or 3). The paper is dried and affinity. dipped into a solvent mixture The interactionbetweenstationaryphaseand consistingof butanol, acetic acid and mobile phase is often employed in the water in 4 : 1 : 5 ratio (for the sepaclassification chromatography e.g. partition, ration of amino acids). The aqueous adsorption, ion-exchange. Further, the classicomponentof the solventsystembinds fication of chromatographyis also based either to the paper and forms a stationary on the natureof the stationaryphase(paper,thin phase. The organic component that layer, column), or on the nature of both mobile migrates on the paper is the mobile and stationary phases (gas-liquid chromatophase. When the migration of graphy). A summary of the different methods the solvent is upwards, it is referred (classes) of chromatography is given in to as ascending chromatography. ln Fig.at J. the chromatography, descending 1. Partition chromatography : The molecules solvent moves downwards (Fig.al.2. As the solventflows, it takesalong with of a miKure get partitionedbetweenthe stationary The rate of phas e a n d mo b i l e p h a s e d e p e n d i n g on thei r it the unknown substances. migrationof the moleculesdependson relative affinity to each one of the phases. the relativesolubilitiesin the stationary (a) Paper chromatography : This phase (aqueous) and mobile phase technique is commonly used for the (organic). separation of amino acids, sugars, After a sufficient migration of the sugarderivativesand peptides.In paper chromatography, a few drops oI solvent front, the paper (chromatogram) is removed, dried and developed for solution containing a mixture of the compoundsto be separatedis applied the identificationof the specific spots.

Chapter 41 : TOOLSOF BIOCHEMTSTRY

721

Mobileohase

Paperstrip

Mobileohase

Fig. 41.2 : Paper chromatography-ascending and descending types.

Ninh y d ri n , w h i c h fo rms p u rpl e complex with c'-amino acids/ is frequentlyused as a colouring reagent. The chemical nature of the individual spots can be identified by running known standardswith the unknown mixture. The migration of a substance is frequentlyexpressedas Ri value (ratio of fonts) -'

Distancetravelledby the substance Distancetravelled bv solventfront

The R1 value of each substance, characteristicof a given solventsystem and paper, often helps for the identificationof unknown. Sometimes, it is rather difficult to separate a complex mixture of substancesby a single run with one solvent system. ln such a case, a second run is carried out by a different solvent system, in a direction perpendicularto the first run. This is referred to as two dimensional chromatography which enhances the separation of a mixture into the individual comDonents. (b) Thin layer chromatography (TtC) : The principle of TLC is the same as described for paper chromatography (partition).ln place of a paper,an inert

substance, such as cel l ul ose, i s employed as supporting material. C el l ul osei s spreadas a thi n l ayer on glass or plastic plates.The chromatographic separation is comparatively rapi d i n TLC . In case of adsorption thin layer chromatography, adsorbents such as acti vatedsi l i cagel , al umi na,ki esel guhr are used. (c) Gas-liquid chromatography (GLC) : This is the method of choice lor the separation of volatile subsfancesor the volatile derivatives of certain nonvol ati l e substances. l n C LC , the stationary phase is an inert solid material (diatomaceous earth or powdered firebrick), impregnated with a non-vol ati l e l i qui d (si l i con or polyethyleneglycol). This is packed in a narrow column and maintained at high temperature (around 200"C). A mixture of volatile material is injected i nto the col umn al ong w i th the mobi l e phase, which is an inert gas (argon, helium or nitrogen).The separationof the volatile mixture is based on the partition of the components between the mobile phase (gas)and stationary phase (l i qui d), hence the name gasliquid chromatography.The separated compounds can be i denti fi ed and

722

BIOCHEMISTRY Recorder

Amplifier

Sarnple Detector

J

T

+ - - ]ll

tl

Fiq.41.3 : Diagrammaticrepresentationof gasJiquid chromatography(GLC).

powder and calcium hydroxyapatiteare packed into a column in a glasstube. This servesas the stationaryphase.The samplemixture in a solvent i s l oaded on thi s col umn. The i ndi vi du al Cas-liquidchromatographyis sensitive, componentsget differentiallyadsorbedon to the rapid and reliable.lt is frequentlyused adsorbent.The elution is carried out by a buffer for the quantitative estimation of system (mobi l e phase). The i ndi vi du al b i o l o g i c a l ma te ri a l s s u c h as l i pi ds, compoundscome out of the column at different drugs and vitamins. rates which may be separatelycollected and 2. Adsorption column chromatography : The identified (Fig.4l.4). For instance,amino acids adsorbentssuch as silica gel, alumina, charcoal can be identified by ninhydrin calorimetric quantitated by a detector (Fig.4l.3). The detectorworks on the principlesof ionization or thermal conductivity.

Buffer

J

Elution

Chapter 4{ : TOOLSOF B|OCHEM|STFY

723 Sample

J lon exchange cotumn

Amino acid separation

method. An automatedcolumn chromatography pH. This plinciple is exploited in the separation apparatus-fraction collector-is frequently of molecules in ion-exchangechromatography. used nowadays. A mixtureof amino acids(proteinhydrolysate) 3. lon-exchange chromatography : lon- or proteins can be conveniently separatedby exchange chromatography involves the ion-exchange chromatography.The amino acid separationof molecules on the basis of their mixture (at pH around 3.0) is passedthrough a electrical charges. lon-exchange resins-cation cation exchangeand the individual amino acids exchangers and anion exchangers-are used for can be eluted by using buffersof different pH. this purpose. An anion exchanger (R+A-) The various fractions eluted, containing exchanges its anion (A-) with another anion individual amino acids,are allowed to reactwith (B-) in solution. ninhydrin reagent to form coloured comolex. This is continuously monitored for qualitative R+A- + B-__) R+B_+ A_ and quantitative identificationof amino acids. Similarly, a cation exchanger (H+R-) The amino acid analyser, first developed by exchangesits cation (H+) with another cation Moore and Stein, is based on this principle (C+) in solution.

Gig.a|.5).

H+R -+ C + + C + R -+ H + Thus, in ion-exchangechromatography,ions in solution are reversibly replaced by ionexchange resins. The binding abilities of ions bearing positive or negativechargesare highly pH dependent,since the net charge varieswith

Several types of ion exchangers are commerci al l y avai l abl e. These i ncl ude pol ystyreneresins(anion exchangeresin, Dowex 1; cation exchangeresin, Dowex 5O),DEAE(diethyl aminoethyl) cellulose, CM (carboxy methyl) cellulose, DEAE-sephadex and CM-sephadex.

BIOCHEMISTRY

724

Small

molecule

Large molecule

4. Gel fittration chromatography : ln gel filtration chromatography, the separation of molecules is based on their size, shape and molecularweight. This techniqueis also referred to as molecular sieve or molecular exclusion chromatography. The apparatus consists of a column packed with spongelike gel beads contain i ng (usually cross-linked polysaccharides) the pores.The gels serveas molecularsieves,,for molecules bigger and smaller of separation

molecular fishhook to selectivelypick up the proteinspass desiredproteinwhilethe remaining protein, desired The column' the through using by eluted be can ligand, the by captured reagents some Alternately, molecules. freeligand canalso interaction thatcanbreakprotein-ligand seParation' the for be employed Affinity chromatographyis useful for the vitamins,nucleicacids, of enzymes, purification drugs,hormonereceptors,antibodiesetc'

ffig.a|.6).

6. High performanceliquid chromatography (HPtC) : In general, the chromatographic techniquesare slow and time consuming'The can be greatlyimprovedby applying separation psi in the rangeof 5,000-10,000 pressure high (poundsper squareinch),hencethis technique is also referred (less frequently)to as high HPLCrequires liquid chromatography' pressure and materials resin non-compressible of ih" uru column the of eluants The columns. metal strong are detectedby methodssuchas UV absorption and fluorescence.

The solution mixture containingmoleculesof differentsizes(sayproteins)is appliedto column and eluted with a buffer. The larger molecules cannot pass through the pores of gel and, therefore, move faster' On the other hand, the The movementof charged particles(ions)in smaller molecules enter the gel beads and are selecting an electricfield resultingin their migration left behind which come out slowly. By electrode is the gel beadsof different porosity, the molecules towards the oppositely charged can be separated.The commercially available gelsinc lu d eS e p h a d e x(C -1 0 ,G-2 5 , C -l 00), B i ogel (P-10, P-30, P-100) and sepharose(68, 48, 28). The gel-filtrationchromatographycan be used for an approximatedeterminationof molecular weights. This is done by using a calibrated column with substancesof known molecular weight. 5. Affinity chromatography : The principle of affinity chromatographyis basedon the property of specificand non-covalentbinding of proteins to other molecules, referred to as ligands' For instance,enzymes bind specificallyto ligands such as substratesor cofactors. The technique involves the use of ligands covalentlyattachedto an inert and porousmatrix in a c olu mn . T h e i m m o b i l i z e d l i g a n ds act as

@

Negative ions

Clupter 41 : TOOLSOF BIOCHEMISTFY Moleculeswith a net <mwn as electrophoresis. positive charge (cations) move towards the negativecathode while those with net negative charge(anions)migratetowards positiveanode. Electrophoresisis a widely used analytical technique for the separation of biological moleculessuch as plasma proteins,lipoproteins a nd im m unoglo b u l i n s . The rate of migration of ions in an electric field depends on several factors that include shape,size, net chargeand solvationof the ions, viscosity of the solution and magnitude of the current employed.

Different types of electrophoresis Among the electrophoretictechniques,zone (paper,gel), isoelectricfocussing electrophoresis are important and and immunoelectrophoresis commonly employed in the laboratory. The original moving boundary electrophoresis, developed by Tiselius (1933), is less frequently used thesedays. In this technique,the U-tube is filled with protein solution overlaid by a buffer solution.As the proteinsmove in solutionduring they form boundarieswhich can electrophoresis, be identified by refractive index.

ami do bl ack or bromophenol bl ue are employed. The serum proteins are separated into five distinct bands-albumin, d.t-, d2-, p- and y-globulins (Refer Fig.g.l). For the electrophoretic pattern of serum lipoproteins, reler Fig.l 4.34. (b) Gel electrophoresis: This technique involves the separationof molecules based on their size, in addition to the electrical charge. The movement of large molecules is slow in gel (this is in contrastto gel electrophoresis filtration). The resolution is much higher in this technique. Thus, serum proteins can be separatedto about 15 bands, instead of 5 bands on paper electrophoresis. The gels commonly used in gel electrophoresis are agarose and polyacrylamide, sodium dodecyl sulfate (SDS).Polyacrylamideis employed for the determinationof molecularweights of proteins in a popularly known electrophoresistechnique known as SDS-PACE.

1. Zone electrophoresis: A simple and modi2. lsoelectric focussing : This technique is fied method of moving boundaryelectrophoresis primarily based on the immobilization of the An inert supporting molecules is the zone electrophoresis. pH at isoelectric during material such as paper or gel are used. electrophoresis.Stable pH gradientsare set up (a) Paper electrophoresis : ln this (usually in a gel) covering the pH range to t ec hniq u e ,th e s a m p l e i s a p p l i e d o n a include the isoelectricpoints of the components strip of filter paper wetted with desired in a mixture. As the electrophoresisoccurs, the buffer solution. The ends of the strip molecules (say proteins) migrate to positions are dipped into the buffer reservoirsin corresponding to their isoelectric points, get which the electrodes are placed. The immobilized and form sharp stationarybonds. efectric current is applied allowing the The gel blocks can be stained and identified. By moleculesto migratefor sufficienttime. isoelectric focussing, serum proteins can be The paper is removed, dried and separated to as many as 40 bands. lsoelectric stained with a dye that specifically focussing can be conveniently used for the colours the substancesto be detected. purificationof proteins. The coloured spots can be identified 3. lmmunoelectrophoresis: This technique by comparing with a set of standards involves combination of the principles of run simultaneously. electrophoresisand immunological reactions. For the separationof serum proteins, lmmunoelectrophoresis is useful for the Whatman No. 1 filter paper,veronalor analysis of complex mixtures of antigens and tris buffer at pH 8.6 and the stains antibodies.

I j

il i

726

BIOCHEMISTFIY

ooooo

a

a -

Elechophoretically separatedproteins

Precipitin arc

Fig. 41.8 : Diagrammatictepresentationof immunoelectrophoresis.

The complex proteins of biological samples (say human serum) are subjected to electrophoresis. The antibody (antihuman immune serum from rabbit or horse) is then applied in a trough parallelto the electrophoretic separation.The antibodies diffuse and, when they come in contactwith antigens,precipitation occurs/ resulting in the formation of precipitin bands which can be identified(Fig.a|,8).

depends on the concentrationof the absorbing molecules).And accordingto Lambert'slaw, the transmitted light decreasesexponentially with increase in the thickness of the absorbing molecules (i.e. the amount of light absorbedis dependenton the thicknessof the medium). By combining the two laws (Beer-Lambert law), the following mathematicalderivationcan be obtained I = Ior.r where I = Intensityof the transmittedlight Io = Intensityof the incident light

Photometry broadly deals with the study of the phenomenon of light absorption by molecules in solution. The specificity of a compound to absorb light at a particular wavelength (monochromatic lighfl is exploited in the laboratory for quantitative measurements. From the biochemist'sperspective,photometry forms an important laboratory tool for accurate estimationof a wide variety of compounds in biological samples. Colorimeter and spectrophotometer are the laboratory instruments used for this purpose.They work on the principles discussedbelow.

e = Molar extinction coefficient (characteristicof the substancebeing investigated) c = Concentration of the absorbing substance(moles/lor g/dl) t = Thickness of medium w hi ch l i ght passes.

through

When the thicknessof the absorbingmedium is kept constant(i.e. Lambert'slaw), the intensity of the transmitted light depends only on concentrationof the absorbingmaterial.In other words, the Beer's law is operative.

The ratio of transmitted light (l) to that of When a light at a particular wavelength is incident light (I0) is referred as to transmittance passedthrough a solution (incident light), some (r). amount of it is absorbedand, therefore,the light t=f that comes out (transmittedlight) is diminished. Io The nature of light absorption in a solution is governed by Beer-Lambert law. Absorhance (A) or optical density (OD) is very commonly used in laboratories. The relation Beer's law states that the amount of between absorbance and transmittance is transmittedlight decreasesexponentiallywith an expressedby the following equation. increase in the concentration of absorbing material (i.e. the amount of light absorbed A =2 -logroT=2-log%f

e hapter l[1 : TOOLSOF BIOCHEM|STF|Y

727

Fl _.*F;;l__* @ ____+ mil ____* F;;;l___* F;;l Fig. 41.9 : Diagrammatic representation of the components in a colorimeter.

Golorimeter Colorimeter (or photoelectriccolorimeter)is the instrument used for the measurement of coloured substances. This instrument is operative in the visible range (400-800 nm) of the electromagnetic spectrum of light. The working of colorimeter is based on the principle of Beer-Lambert law (discussed above). The colorimeter, in general consists of light source, filter sample holder and detector with display (meter or digital). A filament lamp usually serves as a Iight source.The filters allow the passage of a small range of wave length as incident light. The sample holder is a special glass cuvette with a fixed thickness. The photoelectric selenium cells are the most common detectors used in colorimeter. The diagrammatic representationof. a colorimeter is depicted in Fig.4t.9. $pectrophotorneter The spectrophotometerprimarily differs from colorimeter by covering the ultraviolet region (200-400 nm) of the electromagneticspectrum. Further, the spectrophotometer is more sophisticatedwith several additional devices that ultimately increase the sensitivity of its operation severalfold when compared to a colorimeter.A preciselyselectedwavelength ( s ay 23 4 n m o r 6 1 0 n m) i n b o th ul traviolet and visible range can be used for measurements.In place of glass cuvettes (in colorimeter), quartz cells are used in a spectrophotometer.

When certain compounds are subjected to light of a particular wavelength, some of the molecules get excited. These molecules,while they returnto ground state,emit light in the form of fluorescence which is proportional to the concentration of the compound. This is the principle in the operation of the instrument fluorometer.

Flame photometry primarily deals with the quantitative measurement of electrolyfes such as sodium, potassiumand lithium. The instrument, namely flame photometer, works on the fol l ow i ng pri nci pl e.A s a sol uti oni n ai r i s fi nal ly sprayed over a burner, it dissociatesto give neutral atoms. Some of these atoms get excited and move to a higher energy state. When the excited atoms fall back to the ground state,they emit light of a characteristicwavelengthwhich can be measured.The intensityof emissionlight is proportional to the concentration of the electrolytebeing estimated.

The ultracentrifuge was developed by a Swedish biochemist Svedberg (1923). The principle is based on the generation of centrifugal force to as high as 600,000 g (earth's gravity C - 9.81 m/s2) that allows the sedimentationof particles or macromolecules. Ultracentrifugationis an indispensabletool for The spectrophotometer has similar basic the isolation of subcellular organelles, proteins components as described for a colorimeter and nucl ei c aci ds. l n addi ti on,thi s techni quei s (Fig.4l.9),and its operationis also basedon the also employed in the determinationof molecular Beer-Lambertlaw (alreadydiscussed). weights of macromolecules.

B IOC H E MIS TR Y

728

where v = Migration (sedimentation)of the molecule (0= Rotation of the centrifuge rotor in radiansAec f= Distancein cm from the centre of rotor

J 7 0 0 S X1 0 mi n

I Supernatant Nuclear fraction

J'u ,o o o sxl smi n

The sedimentationcoefficienthas the units of seconds.lt was usuallyexpressedin units of 1013s (since several biological macromolecules occur in this range),which is designatedas one Svedberg unit. For instance, the sedimentation coefficientof hemoglobin is 4 x 10-13 s or 45; ribonucleaseis 2 x 10-13s or 25. Conventionally, the subcellularorganellesare often referredto by their S value e.g. 70S ribosome.

lsolation of subcellular organelles by centrifugation to disruptionby The cells are subjected sonication or osmotic shock or by use of homogenizer.This is usually carried out in an isotonic (0.25 M) sucrose.The advantagewith sucrose medium is that it does not cause the organellesto swell. The subcellularparticlescan be separated by differential centrifugation. The most commonly employed laboratory method separates subcellular organelles into 3 major fractions-nuclear, mitochondrial and microsomal(Fig.alJA.

FIg. 41.10 : Separation of subcellular

The rate at which the sedimentationoccurs in primarily dependson the size ultracentrifugation and shape of the particles or macromolecules (i.e. on the molecularweight). lt is expressedin terms of sedimentation coefficient(s) and is given by the formula. S= -

a2r

When the homogenateis centrifugedatTOOg for about 10 mi n, the nucl earfracti on(i ncl udes plasma membrane) gets sedimented. On centrifugingthe supernatant(l) at 15,000 g for about 5 min mitochondrialfraction(that includes lysosomes, peroxisomes) is pelleted. Further centrifugationof the supernatant(ll) at 100,000 g for about 60 min separatesmicrosomalfraction (that includes ribosomes and endoplasmic reticulum). The supernatant(lll) then obtained correspondsto the cytosol. The purity (or contamination) of the subcellularfractionationcan be checkedby the use of marker enzymes. DNA polymerase is the marker enzyme for nucleus, while glutamate dehydrogenaseand glucose 6-phosphataseare the markers for mitochondria and ribosomes, respectively.Hexokinaseis the marker enzyme for cvtosol.

Ghapter 41 : TOOLSOF BIOCHEMISTRY

729 antigen and the same quantities of antibody and labelled antigen.

(RlA)was developedin The labelled antigen-antibody (Ag+-Ab) Radioimmunoassay 1959 by Solomon,Bensonand RosalynYalow complex is separated by precipitation. The for the estimationof insulinin humanserum. radioactivity of 1311 present is Ag+-Ab is Thistechniquehasrevolutionized the estimation determined. of severalcompoundsin biologicalfluids that are found in exceedinglylow concentrations Applications (nanogram or picogram). RIAis a highlysensitive RIA is no more limited to estimating of and specificanalyticaltool. hormones proteins

and that exhibit antigenic properties. By the use of haptens (small Principfe molecules such as dinitrophenol, which, by Radioimmunoassay combinesthe principles themselves,are not antigenic),severalsubstances of radioactivity of isotopesand immunological can be made antigenic to elicit specific antibody reactionsof antigen and antibody, hence the responses. ln this way, a wide variety of compounds have been brought under the net of name. RIA estimation. These include peptides, steroid The principle of RIA is primarily basedon the hormones, vitamins, drugs, antibiotics, nucleic competitionbetweenthe labelledand unlabelled acids, structural proteins and hormone receptor antigensto bind with antibody to form antigenproteins. antibody complexes (either labelled or Radioimmunoassay unlabel l e d ). T h e u n l a b e l l e d a n ti g e n i s the has tremendous substance(say insulin) to be determined.The application in the diagnosis of hormonal antibody to it is produced by injecting the disorders,cancersand therapeuticmonitoringof antigen to a goat or a rabbit. The specific drugs, besides being useful in biomedical antibody (Ab) is then subjected to react with research. unlabelled antigen in the presence of excess amounts of isotopically labelled (1311)antigen (Ag+) with known radioactivity. There occurs a competition between the antigens (Ag+ and Ag) to bind the antibody. Certainly, the labelled Ag+ will have an upper hand due to its excess Enzyme-linked immunosorbant assay (El.lSA) presence. is a non-isotopic immunoassay.An enzyme is used as a label in ELISAin place of radioactive Ag*+Ab -----t Ag*Ab isotopeemployed in RlA. ELISA is as sensitive Ag as or even more sensitivethan RlA. ln addition, there is no risk of radiation hazards (as is the case with RIA) in ELISA. Ag-Ab Principle As the concentrationof unlabelled antigen (Ag) increasesthe amount of labelled antigenantibody complex (Ag+-Ab)decreases.Thus, the concentration of Ag+-Ab is inversely related to the concentration of unlabelled Ag i.e. the substance to be determined. This relation is almost linear.A standardcurve can be drawn by using different concentrations of unlabelled

ELISA is based on the immunochemical principles of antigen-antibody reaction. The stages of ELISA, depicted in Fig.41.11, are summarized. 1. The antibody against the protein to be determined is fixed on an inert solid such as polystyrene.

730

B IOC H E MIS TR Y

2. The biologicalsamplecontainingthe protein to be estimatedis applied on the antibody coated surface. 3. The protein antibody complex is then reactedwith a second protein specific antibodyto which an enzyme is covalently link ed. T h e s e e n z y m e s m u s t b e e asi l y assayableand producepreferablycoloured products.Peroxidase, amylaseand alkaline phosphataseare commonly used.

Il -4.

4. After washingthe unbound antibody linked enzyme, the enzyme bound to the second antibody complex is assayed.

Il t

t (

-

Enzyme second antibody

5. The enzyme activity is determined by its action on a substrate to form a product (usuallycoloured).This is related to the concentration of the protein being estimated. The principle for the use of the enzyme peroxidasein ELISAis illustratednext. Peroxlrtage Hz O z # H 2 O+ O (substrate)

/- ----------Diaminobenzidine (colourless)

(nascentorygen)

Fiq.41.11 : Diagrammaticrepresentationof enzymelinked immunosorbant assay (ELISA).

-y Oxidized diamt6oben:idine

have the desired properties but are found with many other antibodieswhich undoubtedly are not required.A simple,convenientand desirable Applications method for the largescale productionof specific antibodiesremaineda dream for immunologists ELfSAis widely used for the determination of for a long period. ln 1975, Ceorge Kohler and small quantitiesof proteins(hormones,antigens, Cesar Milstein (Nobel Prize 1984) made this antibodies)and other biological substances. The dream a reality. They created hybrid cells that most commonly used pregnancy test for the will make unlimitedquantitiesof antibodieswith detection of human chorionic gonadotropin which are termed as (hCC) in urine is based on ELISA.By this test, defined specificities, monoclonal antihodies (McAb). This discovery, pregnancy can be detected within few days after often referred to as hybridoma technology, has conception. ELISA is also been used for the revolutionizedmethodsfor antibody production. diagnosisof AIDS. Principle This is based on the fusion between myeloma cells (malignant plasma cells) and spleen cells Conventional methods adopted in the from a suitably immunized animal. Spleencells laboratoryfor the production of antiseraagainst die in a shortperiod underordinarytissueculture antigenslead to the formation of heterogeneous condi ti ons w hi l e myel oma cel l s are adopted antibodies.Among these antibodiesa few may to grow permanently in culture. Mutants of

731

Chapter 41 : TOOLSOF BIOCHEMISTRY

lmmunizedanimal

Mvdomaline

SPleen---y,.cells

myeloma cells lack the enzyme hypoxanthine (azaquinine guanine phosphoribosyltransferase resistant) or thymidine kinase (bromodeoxyuridine resistant).Thesemutantscannot grow in a medi umcontai ni ngami nopteri n,suppl emented w i th hypoxanthi ne and thymi di ne (H A T medium). Hybrids betweenthe mutant myeloma cel l s and spl een cel l s can be sel ected and cul turedi n H A T medi um. From the grow i ng hybri ds, i ndi vi dual clones can be chosen that secretethe desired antibodies (monoclonal origin). The selected cl ones l i ke ordi nary myel oma cel l s can be mai ntai ned i ndefi ni tel y. In short, the hybridoma technology for the production of monocl onal anti bodi es i nvol ves the fol l ow i ng steps. 1. l mmuni zati onof appropri ateani mal sw i th antigen (need not be pure) under study. 2. Fusionof suitabledrug resistantmyeloma cells with plasmacells,obtainedfrom the spleen of the i mmuni zedani mal .

I

Recloning l{------)

I

fch"".6;l

l*------+ | clonesSelectI | | variants I + Freeze

Propagationof selected

of Tumours

3. S el ecti onand cl oni ng of the hybri d cel l s that grow in culture and produce antibody molecule5of desiredclassand specificityagainst the antigen of interest. H ybri doma technol ogy can make avai l abl e hi ghl y speci fi canti bodi esi n abundantamounts. The clones once developed are far cheaper than the tradi ti onal l y empl oyed ani mal s (horses, rabbits) for producing antibodies. The clones developed from the hybrids will also ensure constancy of the quality of the product and will also avoid the batch to batch variation inherent in the conventional methods. Applications antibodies

Fig" 41.12 : Basic protocol for the derivationof

of monoclonal

The antibodies produced by hybridoma technologyhavebeenwidely usedfor a varietyof purposes.These include the early detection of pregnancy, detection and treatment of cancer, diagnosis of leprosy and treatment of autoi mmunedi seases.

I

t l I

i

li

ll

{

'. 1

The imrrumologg

sgeahs t

systemdth,e body; "I representthed.efense Mainly composed of B-lymphocytes arcdT-lymphocytes; Designedn eliminateinuadingmimobesand moles;

mmunology deals with the study of immunity Fi rst E i ne of defense I I and immune systemsof vertebrates.lmmunity The skin is the largestorgan in the human (immunis literally means exempt/ree from body, constitutingabout 15"h of the adult body burden) broadly involves the resistance w ei ght.The ski n provi desmechani calbarri erto shown, and protection offered by the host preventthe entry of microorganismsand viruses. organism against the infectious diseases. The The aci di c (pH 3-5) envi ronmenton the ski n immune systemconsistsof a complex networkof surface inhibits the growth of certain c ells and mo l e c u l e s , a n d th e i r i n te r acti ons. Further,the sw eat contai nsan mi croorgani sms. I t is s p e c i fi c a l l y d e s i g n e d to e l i mi nate enzyme lysozymethat can destroybacterialcell infectious organisms from the body. This is w al l . possible since the organism is capable of dis t inguish i n gth e s e l f fro m n o n -s el f, and Second line of defense elim inat en o n -s e l f. Despite the physical barriers, the microlm m uni ty i s b ro a d l yd i v i d e d i n to tw o typesorganismsdo enter the body. The body defends innate (non-specific)immunity and adaptive or itself and eliminates the invading organisms by ac quir ed( s p e c i fi c )i m m u n i ty . non-specific mechanismssuch as sneezing and secretionsof the mucus. In addition, the body I NNA T E IM M U N IT Y also tries to kill the pathogensby phagocytosis I nnat e i mmu n i ty i s n o n -s p e c i fic, and (i nvol vi ng macrophages and compl ement represents the inherentcapabilityof the organism system).The inflammatory responseand fever to offer resistanceagainstdiseases.lt consistsof responseof the body also form a part of innate defensivebarriers. i mmuni ty.

732

The immune systemrepresentsthe third and most potent defense mechanism of the body. Acquired (adaptive or specific) immunity is capable of specifically recognizing and eliminating the invading microorganisms and roreignmolecules(antigens). In contrastto innate immunity, the acquired immunity displaysfour d istinct characteristics

nodes Thymus Lymphnodes Spleen Peyerspatches nodes vessels

. Antigen specificity ' Recognitiondiversity . lmmunological memory . Discriminationbetween self and non-self. The body possesstremendous capability to specifically identify various antigens(antigen is a foreign substance, usually a protein or a carbohydrate that elicits immune response). Exposureto an antigenleadsto the development of immunologicalmemory. As a result,a second encounter of the body to the same antigen results in a heightenedstate of immune response.The immune system recognizes and responds to foreign antigensas it is capableof distinguishing self and non-self. Autoimmune diseases are causeddue to a failure to discriminateself and non-selfantigens.

Fig. 42.1 : A diagrammaticrepresentationof humanlymphaticsystem.

Secondary

lymphoid

organs

Theseare the sitesfor the initiation of immune response. e.g. spleen, tonsils, Iymph nodes, appendix, Peyerspatchesin the gut. Secondary lymphoidorgansprovidethe microenvironment for interaction between antigens and mature lymphocytes.

CELLS OF THE IMMUNE SYSTEM

ORGANIZATIONOF IMMUNE SYSTEM

Two typesof lymphocytesnamely B-cellsand T-cells are critical for the immune system. In addition, several accessory cells and effector The immune systemconsistsof severalorgans cells also participate. distributed throughout the body (Fig. a2,t). These lymphoid organs are categorized as B.lymphocytes primary and secondary. The site of development and maturation of B-cells occurs in bursa fabricius in birds, and These organs provide appropriate micro- bone marrow in mammals.During the courseof environmentfor the developmentand maturation immune response.B-cells mature into plasma of antigen-sensitive lymphocytes(a type of white cells and secreteantibodies(immunoglobulins). bfood cells). The thymus (situated above the The B-cells possess the capability to heart) and bone marrow are the central specificallyrecognizeeach antigenand produce or primary lymphoid organs. T-lymphocyte antibodies (i.e. immunoglobulins) against it. maturation occurs in the thymus while B-lymphocytesare intimately associated with B-lymphocytematurationtakesplace in the bone humoral immunity. Immunoglobulins are marrow. described in Chapter g.

Primary lymphoid organs

734

B IOC H E MIS TR Y

function of antibodies in defending the body from the invading antigens.The complementary The maturation of T-cells occurs in the factors are heat labile and get inactivated if lhymus, hence the name. The T-cellscan identify heated at 56"C for about 30 minutes. The viruses and microorganismsfrom the antigens complementsystemhelps the body immunity in displayed on their surfaces.There are at least 4 ways four different types of T-cells. 1. Complement fixation : The complement . Inducer T-cellsthat mediate the development system binds to the foreign invading cells and of T-cells in the thymus. causeslysis of the cell membranes. . Cytotoxic T-cells (T), capable of recognizing 2. Opsonization : The process of promoting and killing the infectedor abnormal cells. the phagocytosisof foreign cells is referredto as . Helper T-cells (Ti that initiate immune opsonization. responses. 3. lnflammatory reaction : The complement . SuppressorT-cellsmediate the suppressionof system stimulates local inflammatory reaction rmmune response. and attracts phagocytic cells. T-lymphocytesare responsiblefor the cell4. Clearanceof antigen-antibodycomplexes : mediated immunity. The complementsystempromotesthe clearance

T.lymphocytes

MAJOR HISTOCOI'PATIBILITV COMPLEX

of antigen-antibodycomplexesfrom the body. Nomenclature of complement system : The complement proteins are designatedby the letter 'C', followed by a component number-C'r, Cz, C3 etc.

The major histocompatibilitycomplex (MHA representsa special group of proteins, presenton the cell surfaces of TJymphocfies. MHC is Types of reaction : The complement system involvedin the recognitionof antigenson T-cells. brings about two sets of reactions : It may be noted here that the B-cell receptors 1. Antibody dependentclassicalpathway. (antibodies) can recognizeantigenson their own, 2. Antibody independentalternativepathway' while T-cells can do so through the mediation of M H C . Eachone of the pathwaysconsistsof a series In humans,the MHC proteinsare encodedby of reactions converting inactive precursorsto a cluster of genes located on chromosome 6 (it active products by serine proteases which is on chromosome17 for mice).The major histo- resemblesblood coagulation. compatibility complex in humans is referredto as human leukocyte antigen (HtA). Three classes of MHC molecules (chemically glycoproteins) ar e k n o w n i n h u m a n . C l a s s I m o l ecul es are found on almost all the nucleated cells of the body. Classll moleculesare associatedonly with leukocytes involved in cell-mediated immune molecules are the response. Class lll secretedproteins possessingimmune functions' e.g. complement components (Cz, C/, tumor necrosis factor.

The complementsystemis composedof about

20 plasma proteins that 'complement' the

The immune response refers to the series of reactionscarried out by the immune system in the body againstthe foreign invader.When an infection takes place or when an antigen enters the body, it is trapped by the macrophagesin lymphoid organs.The phagocyticcells which are guardingthe body by constantpatrollingengulf and digest the foreign substance.However, the partiallydigestedantigen(i.e. processedantigen) with antigenic epitopes attachesto lymphocytes. T-helpercells (TH)play a key role the immune response(Fig. a2,2).This is brought out through

Chapter 42 : IMMUNOLOGY

735

Class I MHC

Antigenfragment T-cellreceptor TH cell

Ts cell

Cytokines are a group of proteins that bring about communication between different cell fypes involved in immunity. They are low molecular weight glycoproteins and are produced by lymphoid and non-lymphoid cells during the course of immune response. Cytokinesmay be regardedas solublemessenger molecules of immune system. They can act as short messengersbetween the cells or long range messengers by circulating in the blood and affectingcells at far off sites.The latter function is comparable to that of hormones. The term interleukin (IL) is frequently used to representcytokines.There are more than a dozen producedby di fferent i nterl euki ns(l L-I......1112), cells with wide range of functions. The main function (directlyor indirectly)of cytokinesis to amplify immune responses and inflammatory responses.

I

I + Gell-mediated immunity

Therapeutic

uses of *gtckines

It is now possible to produce cytokines in vltro. Some of the cytokines have potential applications in the practice of medicine. For instance,lL-2 is used in cancer immunotherapy, and in the treatment of immunodeficiency diseases. lL-2 induces the proliferation and differentiation of T-and B-cells, besides increasingthe cytotoxiccapacityof naturalkiller the participationof antigenpresentingcell (APC), cel l s. usuallya macrophage.Receptorsof Ts cell bind to class ll MHC-antigen complex displayed on A group of cytokinesnamely interferonscan the surfaceof APC, APC secretesinterleukin-1, combat vi ral i nfecti on by i nhi bi ti ng thei r which activatesthe Tn cell. This activated Tn replication. cell activelygrows and dividesto produceclones of T" cells.All the T" cells possessreceptorsthat are specificfor the MHC-antigencomplex. This facilitatestriggeringof immune responsein an exponential manner. The TH cells secrete interleukin-2which promotesthe prolifirationof The prime function of immune system is to cytotoxic T cells (Tc cells) to attack the protect the host againstthe invading pathogens. infected cells through cell-mediatedimmunity. Further, interleukin-2 also activates B-cells to The body tries its best to overcome various pr oduc e i mmu n o g l o b u l i n s w h i c h perform strategiesof infectious agents (bacteria,viruses), and provides immunity. hum or al im m u n i ty .

736

BIOCHEMISTF|Y

Someof the importantimmunologicalaspects In case of humans, maj ori ty of organ in human health and disease are briefly transplantations are allografts (between two described, individuals). The term xenograft is used if tissues/organsare transferredfrom one species AUTOIMIIUNE DISEASES to another e.g. from pig to man. In general,the immune systemis self-tolerant i.e. not responsive to cells or proteins of self. Sometimes, for various reasons, the immune system fails to discriminate between self and non-self. As a consequence,the cells or tissues of the body are attacked.This phenomenon is referred to as autoimmunity and the diseasesare regarded as autoimmune diseases. The antibodies produced to self molecules are regarded as autoantibodies. Some examples of autoimmunediseasesare listed. . Insulin-dependentdiabetes (pancreaticp-cell autoreactiveT-cellsand antibodies). . Rheumatoid arthritis (antibodies against proteinspresentin joints). o Myasthenia gravis (acetylcholine receptor autoantibodies).

Organ transplantation is associated with immunological complications, and tissue rejection.This is becausethe host body responds to the transplantedtissuein a similar way as if it were an invading foreign organism. Major histocompatibilitycomplex (MHC) is primarily involved in allograftrejection.This is due to the fact that MHC proteins are unique to each individual, and the immune system responds promptly to foreign MHCs. Organ transplantation between c losely related family members is preferred,since their MHCs are also likely to be closely related.And major immunologicalcomplicationscan be averted.

GANCERS

Crowth of tumors is often associatedwith the formation of novel antigens. These tumor . Autoimmune hemolytic anemia (erythrocyte antigens (also referred to as oncofetal antigens autoantibodies). e.g. c-fetoprotein) are recognized as non-self by the immune systems. However, tumors have Mechanism of autoimmunity : lt is widely developedseveralmechanismsto evadeimmune acceptedthat autoimmunitygenerallyoccurs as responses. a consequence of body's rcsponse against bacterial, viral or any foreign antigen. Some of AIDS the epitopes of foreign antigens are similar Acquired immunodeficiency syndrome (homologous)to epitopes presenton certain host (AIDS), caused by human immunodeficiency proteins.This resultsin crossreactionof antigens virus, is characterizedby immunosuppression, and antibodieswhich mav lead to autoimmune neurological secondary neoplasma and diseases. manifestations.AIDS primarily affectsthe cellmediated immune system which protects the ORGAN TRANSPLANTATION body from intracellularparasitessuch as viruses, The phenomenon of transferof cells, tissues and bacteria. Most of the immunodeficiency or organs from one site to another (in the same symptoms of AIDS are associated with a organism, autograft or from another organism reduction in CDa (clusterdeterminantantigen4) allograft) is regarded as organ transplantation. cel l s.

enetics is the study of heredity. lt is BRIEF HISTORYAND appropriately regarded as the sciencethat DEVELOPMENTOF GENETICS explains the similaritiesand differencesamong the related organisms. The blood theory of inheritance in humans For many centuries, it was customary to ex plain inh e ri ta n c ei n h u ma n s th ro u g h bl ood theory. People used to believe that the children receivedblood from their parents,and it was the union of blood that led to the blending of characteristics.That is how the terms 'blood relations','blood will tell', and 'blood is thicker than water' came into existence.They are still used, despite the fact that blood is no more involved in inheritance.With the advances in genetics,the more appropriateterms should be as follows .

Gene relations in place of blood relations.

.

Genes will tell instead of blood will tell.

C eneti cs i s rel ati vel yyoung, not even 150 years. The blood theory of inheritance was questionedin 1850s, basedon the fact that the semencontainedno blood. Thus, blood was not being transferredto the offspring.Then the big questionwas what was the hereditarysubstance. Mendel's experiments : lt was in 1866, an Austrian monk named Gregor Johann Mendel, for the first time reported the fundamental laws of inheritance. He conducted se",eral experiments on the breeding patterns of pea plants. Mendel put forth the theory of transmissible factors which states that inheritance is controlled by certain factors passed from parents to offsprings.His results w ere publ i shedi n 1866 i n an obscurej ournal Proceedingsof the Societyof Natural Sciences. For about 35 years,the observationsmade oy Mendel went unnoticed, and were almost

737

738

B IOGH E MIS TFIY

forgotten. Two European botanists (Correns and . Both read maganizesfrom back to front. Hugo d e Vri e s ) i n 1 9 0 0 , i n d e p e n dentl yand . Both stored rubber bands on their wrists. simultaneously rediscovered the theories of . Both liked to sneezein a room of strangers. Mendel. The year 1900 is important as it marks BesidesOskar and Jack, many other studies the beginningthe modern era of genetics. conducted on identical twins point out the The origin of the word gene : In the early importanceof geneson the inheritedcharacters years of twentieth century, it was believed that relatedto personalityand mannerisms. the Mendel's inheritancefactorsare very closely related to chromosomes (literally coloured BASIE PRENGIPLES GF bodies) of the cells. lt was in 1920s, the term }IEREEITV IN }IUMANS gene (derived from a Creek word gennan genetic how The understanding of meaningto produce)was introducedby Willard Johannsen.Thus, gene replaced the earlier terms characteristics are passed on from one generationto the next is basedon the principles inheritance factor or inheritance unit. developedby Mendel. Chemical basis of heredity : There was a As we know now, the human genome is controversyfor quite sometimeon the chemical into a diploid (2n) set of 46 organized basis of inheritance.There were two groupsThey exist as 22 pairs of chromosomes. protein the supportersand DNA supporters.lt and one pair of sex chromosomes autosomes presented was in 1944, Averyand his associates (XX/XY). During the course of meiosis, the c onv inc i n ge v i d e n c eth a t th e c h e m i cal basi sof number becomeshapl oi d(n).Thus , chromosome her edit yl i e s i n D N A, a n d n o t i n p rotei n.Thus, gametes- sperm and female male and haploid DNA was finally identified as the genetic formed. are On fertilization oocyte respectively, material. lts structurewas elucidated in 1 952 bv statusis by the sperm, the diploid of the oocyte Watson and Crick. restored.This becomes possible as the zygote receivesone member of each chromosomepair lmportance of genes in from the father, and the other from the mother. inheritance-studie$ on twins regards the sex chromosomes,the maleshave As Monozygotic or identical fwins contain the while the femaleshave XX. The sex of X and Y, same geneticmaterial- DNA or genes.Studies the child is determinedby the father. conducted on identical twins make startling revelationswith regardto inheritance.One such Monogenic and Folygenic traits study is describedhere. The genetictraitsor charactersare controlled Oskar Stohr and Jack Yufe were identical by si ngl egenesor mul ti pl e genes.The change s twins separatedat birth. Oskar was taken to in genesare associatedwith genetic diseases. Cermany where he was brought up by his Monogenic disorders : These are the single grandmotheras a Christian.Jack was raised by gene disease traits due to alterations in the his father in lsrael as a Jew. The two brothers gene e.g. si ckl e-cel l anemi a, were reunited at the age of 47. Despite the correspondi nB phenylketonuria. Inheritance of monogenic different environmental influences, their follows the Mendelian pattern behavioural patterns and personalities were disordersusually of inheritance. r em ar k a b l ys i mi l a r Polygenic disonders ; The genetic traits . Both men had moustaches,wore two pocket conferred by more than on gene (i.e multiple shirts,and wire-rimmed glasses. genes),and the disordersassociatedwith them . Both loved spicy foods and tended to fall are very important e.g. height, weight, skin asleep in front of television. colours,academic performance,blood pressure, length of life. . Both flushed the toilet before using. aggressiveness,

I

Chapter 43 : GENETICS

739

(A) Autosomal dominant PARENTS

CHILDREN Genotyperatio-l : 1 Aa to aa Phenotype-50%affected -50% normal

Male (d\ Genotype-Aa Phenotype-Affected male Female (Q) Genotype-aa Phenotype-Normalfemale

(B)Autosomalrecessive PARENTS Male (d) Genotype-Bb PhenotypeCarriermale Femate(9) Genotype-Bb Phenotype -Carrierfemale

CHILDREN Genotyperatio-1 :2: 1 BB/Bb/Bb/bb Phenotype-25%affecied -257o normal -507o caniers

.B R'

\b

(C)X-chromosome(sex chromosomeFlinkedinheritance PARENTS

CHILDREN

Mate(d) Genotype -XY Phenotype-Normal male Fenale(9\ Genotype-X"X Phenotype - Carrierfemale

Genotyperatio-1: 1 : 1 : 1 X)VXY/X"XX"Y Phenotype - 50o/"of malesaffecteci

J

y'x

XX

\"

XY

X"X

d'

Fiq.43.1 : Patterns of inheritance-autosomaldominant,autosomal recessiveano X-linked (Note : Genotype refers to the description of genetic composition, while phenotype

representsthe obseruabtecharacterdisplayedby an organism).

PATTEBNS OF TN']ERITANGE The hereditv is transmitted from parent to offspring as individual characterscontrolled by genes. The genes are linearly distributed on chromosomesat fixed positionscalled loci. A gene may have different forms referred to as alleles. Usually one allele is transferredfrom the father, and the other from the mother. The alfele is regarded as dominanf if the trait is exhibiteddue to its presence.On the other hand, the allele is said to be recessive if its effect is masked by a dominant allele. The individuals are said to be homozygous if both the alleles are the same.When the allelesare differentthey are said to be heterozygous.

The pafternof inheritanceof monogenic traits may occur in the following ways (Fig. aZ.l). 1. A utosomaldomi nant 2. Autosomal recessive 3. Sex-linked. 1. Autosomal dominant inheritance : A normal allele may be designatedas a while an autosomal domi nant di sease al l el e as A (Fig. 43.1A). The male with Aa genotype is an affectedone while the female with aa is normal. Half of the genes from the affected male will carry the diseaseallele. On mating, the male and female gametes are mixed in different combinations. The result is that half of the

740

BIOCHEMISTRY

I nh er ited p attern/di sease

Estimated incidence

Salient features

Autosomaldominant Familial hypercholesterolemia Huntington's disease Familial retinoblastoma Breastcancergenes (BRACI and2) p-Thalassemia

12000 1 800

Highriskforheartdiseases Nervous disorders, dementia Tumors of retina Highriskforbreast andovarian cancers

1 : 2500(inpeople of Meditenanean descent)

A blood disorder; theblood appears to beblue instead of red

1 500 1 5000 I

Autosomalrecessive 100(inAfricans)

Sickle-cell anemia

lifethreatening anemia; confers Severe resistance to malaria Cystic fibrosis 2500(inCaucasians) Defective iontransport; severe lunginfections andearlydeath(before theyreach30years) Phenylketonuria 1 2000 Mental retardation dueto braindamage u, -Antitrypsin deficiency s000 Damage to lungsandliver Tay-Sachs disease 1 3000(inAshkenazi disorder; blindness andparalysis Jews)Nervous (only Severe combined immunodeficiency Rare Highly defective immune system; earlydeath 100cases (SCID) disease reported worldwide) a I

Sex-linked Colour blindness (I/B) Hemophilia Duchenne muscular dystrophy

1 : 50males 1 : 10,000 males 1 : 7000males

Unable to distinguish colours Defective bloodclotting Muscle wastage

Notknown

to opticnerves, mayleadto blindness Damage

Mitochondrial Leberhereditary opticneuropathy

children will be heterozygous(Aa) and have the disease. Example of autosomal dominant inherited diseases are familial hypercholesterolemia,B-thalassemia,breast cancer 8enes.

3. Sex (X)-linked inheritance : In the Fig. 43.1C, sex-linked pattern of inheritanceis depicted. A normal male (XY) and a carrier female (XcY)will produce children wherein, half of the male children are affectedwhile no female child is affected.This is due to the fact that the 2. Autosomal recessiveinheritance : ln this male children possessonly one X chromosome, case,the normal allele is designatedas I while and there is no dominantalleleto mark its effects the disease-causing one is a (Fig. 43.18). The (as is the case with females).Colour blindness gametesof carrier male and carrierfemale (both and hemophilia are good examplesof X-linked with genotype Bb) get mixed. For these diseases. heterozygouscarrier parents,there is one fourth chance of having an affected child. Cystic A selected list of genetic disorders fibrosis,sickle-cellanemia and phenylketonuria (monogenic traits) due to autosomal and sexare some good examplesof autosomalrecessive l i nked i nheri tance i n humans i s gi ven i n disorders. Tahle 43.1.

Ghapter 43 : GENETICS

741

Eugenicsis a highly controversialsubjectdue to social, ethical, and political reasons. The The pattern of inheritance and monogenic proponentsof eugenicsargue that people with traitsalong with some of the associateddisorders desirable and good traits (good blood) should are described above (Table 43.1). Besides reproduce while those with undersirable gene mutations, chromosomal abnormalities (bad blood) should not. The advocates characters (aberrations)also result in genetic diseases. of eugenics, however, do not force any policy, Aneuploidy : The presence of abnormal but they try to convince the people to perform number of chromosomes within the cells is their duty voluntarily.The object of eugenicsis referred to as aneuploidy. The most common to limit the production of people who are unfit aneupfoid condition is trisomy in which three to live in the society. copies of a particular chromosome are present in a cell instead of the normal two e.g. trisomy-2| Eugenics in Nazi Germany causing Down's syndrome; trisomy-18 that results in Edward's syndrome. These are the Cermany developed its own eugenic examplesof autosomalaneuploidy. programmeduri ng 1930s. A l aw on eugeni c 'l ln case of sex-linked aneuploidy, the sex sterilizationwas passed in 933. In a span of chromosomesoccur as three copies.e.g. pheno- three years/ compulsory sterilization was typically male causingKlinefelter'ssyndromehas done on about 250,000 people, who allegedly XXY; trisomy-X is phenotypicallya female with suffered_from hereditary disabilities, feeble mi ndedness, epi l epsy,schi zophreni a, bl i ndness, XXX. physical deformaties, and drug or alcohol addiction. EUGENICS G E NE T I C

DI SE AS ES IN H U M AN S

The German Covernment committed many is a scienceof improvinghuman Eugenics race based on genetics. lmproving the traits of atrocities in the name of racial purity. Other p lant s and a n i ma l s th ro u g h b re e d i ng countries,however do not support this kind of programmes has been in practice for centuries. eugenics.

, t ,'l'1"' l, i\itlll{ s l' jlt lt ) lt ltjqr it ) Answers to Seff-assessrnentExerct'ses

V45

Abbreviations used in this Eook

751

Greek Alphabets (Cornrnoniyused as synrbols) 756, Oniglnrsof lrnportant BiochennlcalWords

757

Cornrnon Confusahles in Eiochernistry

V6*

Fractical Eiochennistry-Principles

764

'':tt:;:t'i::

Clinical Eioc

743

Lgboratory

CHAPTER 2 Answers to fll and fV 1. Sucrose, 2. Clyceraldehyde, 3. Epimers, 4. Anomers, 5. Aglycone, 6. Streptomycin, 7. a-l ,6-Clycosidicbond, 8. I nu l i n , 9. Hy a l u ro n i ca c i d , .l 0. N-Acetylneuraminic acid,

8. Phospharidylinositol, 9. Cangliosides, 10. Cyclopentanoperhydrophenanthrene, 11. a, 12. d, 13. d, 14. c, 1s. b.

CHAPTER4 Answers to lll and tV

1' t . b,

1. 16yo,

12. d,

2. L-a-Aminoacids,

13. a,

3. Methi oni ne,

14. d,

4. Zwitterion,

15. a.

, 5.

CHAPTER 3 Answers to lll and lV 1. Triacylglycerolds, 2. Ceometricisomerism(cis_trans isomerism), 3. Chaulmoogricacid, 4. Triacylglycerols,

B-Alanine,

6. Peptidebonds, 7. Tryptophan, 8.9, 9. 1-Fluro2,4-dinitrobenzene (FDNB), 10. D enaturati on, 11. b,

5. Stereospecific number,

12. d, " t3.b,

6. Saponification number,

" t4.d,

lmitoyl lecithin,

15. a.

BIOCHEMISTRY

746 CHAPTER Answers

CHAPTEP

5

to lll and lV

Answerc

7

to lll and lV

1. Cen e ,

1. Acetylation,

2. RNA,

2. Riboflavin,

3. Nucleotides,

3. VitaminE (tocopherol),

4. Thymine,

4. Pyridoxine(Br),

5. 2,

5. A vi di n,

6. Base+ sugar+ phosphate,

acid, 6. Pantothenic

7. Erwin Chargaff,

7. C obal ami n(B ' r),

8. 3 Hydrogenbonds (in place of 2 in A-T),

8. Dermatitis,diarrheaand dementia,

9. B-Form,

9. Vitamin K,

10. CCA(5'to 3'),

10. Fol i caci d,

11. d,

11. b,

12. b,

12. d,

13. c ,

13. a,

14. d,

14. d,

15. d.

15. a.

CHAPTER 8

CHAPTER 6 Answers to lll and lV

Answere to lll and lV

1. In yeast,

1. p-Clycosidicbonds,

2. Ligases,

2. Raffinose,

3. Coenzyme,

3. Lactase(p-galactosidase),

4. Denaturation,

4. Fiber,

5. Alcohol dehydrogenase, carbonic anhydrase,

5. Parietal(oxyntic)cells,

6. Active site,

6. Clutathione,

7. NADP+,

7. Hartnup'sdisease,

8. E . C .' .t.' 1 .1 .1 ,

8. Arginine,lysine,

9. AMP/ADB

9. C ol i pase

(CPK), 10. Creatinephosphokinase

10. Mixed micelles.

11. c ,

11. a,

12. d,

12. d,

13. b,

13. c,

14. d,

't4. b,

1 s.b.

15. a.

ANSWERS TO SELF-ASSESSMENTEXERCISES CHAPTEH Answers

747

9

to lll and lV

CHAPTER {1 Answers

to lll and lV

1. F i b ri n o g e n ,

1. AC = AH - TAS (I = Absolutetemperature),

2. Electrophoresis,

2. Exergonicor spontaneous,

3. He mo g l o b i n ,

3. Phosphoanhydride bonds,

4. B-Lymphocytes,

4. Phosphoarginine,

5. lgG ,

5. Electrons,

6. lgE,

5. lnner mitochondrialmembrane,

7. 4A-50"C,

7. Heme (porphyrinwith iron),

8. C-reactiveprotein,

8. Cytochromeoxidase(cyt a + a3),

9. Staurtfactor (Xa),

9.C ytochromea+ a3/

10. P l a s mi n ,

10. Superoxidedismutase,

11. c ,

11. d,

' t 2. d,

12. a,

13. a,

13. b,

14. b,

14. d,

15. b.

15. a.

CHAPTER10 Answerc to lll and lV 1. 574 , 2. Methemoglobin, 3. Carbonic anhydrase, 4. 2,3 -Bisphosphoglycerate, 5. Deoxyhemoglobin, 6. Thalassemias, 7. Succinyl CoA, 8. Uroporphyrinogen synthaseI, 9. 6 -Aminolevulinatesynthase, 10. Biliverdin, '|1. a, 12. a, 13. b, 14. d, 15. c .

GHAPTEB13 Answirs to lll and lV 1. Thiamine,riboflavin,lipoic acid, niacin, pantothenicacid, 2. Absenceof glucose6-phosphatase, 3. L-Gulonolactone oxidase, 4. Sorbitol, 5. Calactose1-phosphateuridyltransferase, 6. Leucineand lysine, 7. Succinatethiokinase, 8. Uronic acid pathway, 9. Clycogenin, 10. Oxaloacetate, 11. d, 12. c, 't3. b, 't4. a, 15. b.

748

B IOC H E MIS TF| Y CHAPTER

Answers

14

3. Sodiumurate, 4. Xanthineoxidase,

to Ill and lV

1. Triacylglycerols, 2. HMC CoA reductase, 3. Ampipathic, 4. Sphingomyelinase, 5. HDL , 6. " 129 A T P, 7. Zellweger syndrome, 8. Citrate,

5. Lesch-Nyhan syndrome, 6. Thioredoxin, 7. lnosinemonophosphate, 8. Alloxanthine, 9. Aspartate, 10. Carbamoylphosphatesynthetase ll, 11. d, ,t2. a, 13. c,

9. HDL , 10. Unsaturatedfatty acid, ' tI . d,

14. b, 1s. d.

12. a,

CHAPTER18

13. d, ' 14.c ,

Answers to lll and lV 1. 9-11 mg/dl. (a.5-s.5 mEq./|.),

15. b.

2. C al ci tri ol ,

CHAPTER15 Answers to lll and lV 1. Pyridoxalphosphate,

3. Phosphorus, 4. Magnesium, 5. S odi um,

2. Clutamatedehydrogenase,

6. 3.s-5.0 mEq/l, 7. Transferrin,

3. Carbamoylphosphatesynthasel,

8. Ceruloplasmin,

4. Clycine transaminase,

9. Custen,

5. Tetrahydrobiopterin,

10. S el eni um, ' t1. d,

6. Dopamine, 7. Homogentisate, 8. Malignantcarcinoidsyndrome, 9. Ornithinedecarboxylase, 10. Leu c i n e , 11. b,

12. a, ' t3. b, "14.c, 15. a.

GHAPTER19

12. d,

Answers to lll and lV

13. a, ' t 4. c ,

1. Adenylatecyclase,

15. a.

2. Caz*,

CHAPTER17 Answers to lll and lV

3. Anterior pituitary, 4. E ndorphi ns and enkephal i ns, 5. Thyroperoxidase,

1. Glutamineand aspartate,

6. Aldosterone,

2. A llop u ri n o l ,

7. V ani l l ylmandel i caci d (V MA ),

749

ANSWERS TO SELF-ASSESSMENTEXERCISES (DHT), 8. Dihydrotestosterone

12. a,

9. Cholesterol, 10. Cholecystokinin(CCK),

13. c, 't4. b,

1" .1 a,

15. d.

12. d,

CHAPTEB22

13. b,

Answers to lll and lV

14. c ,

1. C ol l agen,

15. a.

2. C l yci ne,

CHAPTER 20 Answers to lll and lV

3. B -oxal ylami noal ami ne, 4. Fi bri l l i n,

1. Hem e,

5. Clycosaminoglycans,

2. van den Bergh reaction,

6. Sarcomere,

3. Alaninetransaminase,

7. Actin,

4. Alkalinephosphatase,

8. Calciumcaseinate,

(BSP), 5. Bromosulphthalein

9. Lecithin/Sphingomyel in,

6. 180 mg/dl,

10. V i tami nC ,

7. I nulin, 9. Ryle'stube,

11. c, ' t2. d, "13.a,

10. Pentagastrin,

14. c,

8. 2m \ / m i n ,

1s.b.

11. a, ' 12.d,

CHAPTEB23

13. c ,

Answers to lll and lV "1. 4.128,

14. b, 1s . b.

2. Thyroidgland,

CHAPTER 21 Answers to lll and lV

3. Fiber, 4. Carbohydrates,

1. Antidiuretichormone(ADH),

5. Chemicalscore,

2. Na+,

6. 1ilke body weighVday,

3. 285-295 milliosmoles/kg,

7. Biologicalvalue (BV)of protein,

4. Aldosterone,

8. S ul furcontai ni ngami no aci ds,

5. Carbonicacid (HrCOr),

9. l ron,

6. Bicarbonatebuffer

10. P l asmaal bumi n,

7. 20 : 1,

11. a,

B. Ammonium ion (NHf),

12. d,

9. Bicarbonals(HCO3),

13. c,

10. Carbonicacid (HrCOr)or CO,

14. d,

11. d,

15. a.

750

BIOCHEMISTRY CHAPTER

Answers

24

to lll and lV

1. DNA h e l i c a s e ,

13. a, 14. b, 15. a.

2. Okazaki pieces,

CHAPTER

3. DNA polymeraselll, 4. DNA topoisomerases,

Answers

26

to lll and lV

5. Cyclins,

1. 30,000--40,000,

6. Telomere,

2. Constitutivegenes,

7. Transposons or transposableelements,

3. One cistron-onesubunit concept,

8. Mutation,

4. Protein-DNAcomplex,

9. Missense,

5.a,

10. Hereditarynonpolyposis colon cancer,

6.b,

't'1. c, ' 12.a,

7. a, 8. d.

13. b, CHAPTER

14. a,

1s.b.

Answers

27

to lll and lV

1. Escherichiacoli,

CHAPTER25 Answers to lll and lV

2. RNA, 3. Dot-blofting,

1. G enom e ,

4. Thermusaquaticus,

2. hnRN A ,

5. Cenomic library/DNAlibrary,

3. Introns,

6. Site-directedmutagenesis,

4. Reversetranscriptase,

7. H umul i n,

5. Wobble hypothesis,

8. HepatitisB vaccine,

6. Ribosomes,

9. Mouse,

7. r RNA ,

10. Sheep(Dolly),

B. Chaperones,

11. c,

9. Prion diseases,

12. d,

10. Proteintargeting,

13. d,

11. d,

14. a,

12. c ,

15. c.

I I I

A Ab ACP ACTH Acyl CoA ADA ADH ADH ADP AFP AFLP Ag NG A I DS ALA A LP ALT AMP APC Apo-A AP sites AST AT ATCase ATP BAL BAO BHA BHT 6MR BOAA bp BP

adenine, adenosine BPC bisphosphoglycerate (2,3-BpG, antibody 1,3-B P G) acyl carrierprotein BSP bromosulphthalein adrenocorticotropic hormone BUN blood ureanitrogen falty acid derivativeof coenzymeA BV biological value adenosinedeaminase c cytosine,cytidine alcohol dehydrogenase CA carbonicanhydrase antidiuretic hormone Cal calorie adenosinediphosphate Cam cal modul i n o-fetoprotein cAMP 3',5'-cyclic adenosine amplifiedfragmentlength monophosphate(cyclicAMp) polymorphism CAp cataboliteactivatorprotein antigen CBG corticosteroidbi ndi ng globulin a(bumin/globulin(ratio) CCK cholecystokinin acquiredimmunodeficiency syndrome cDNA complementaryDNA d-aminolevulinicacid CDo clusterdeterminant antigen4 alkalinephosphatase CDp cytidinediphosphate afaninetransaminase CEA carcinoembryon ic antigen adenosinemonophosphate CF cystic fibrosis antigenpresentingcell CFTR cystic fi brosis transmembrane apoprotein-A regulator apurinic sites cGMP 3',5'-cyclic guanosinemonophosaspartatetransaminase phate cr,-antitrypsin CH constantheavv chain aspartatetranscarbamoylase CHD coronaryheartdisease adenosinetriphosphate C hE choli nesterase British antilewisite cht ch{orophylf basalacid output CL constantlight chain butylatedhydroxyanisole C LIP corticotropin like i ntermediatelobe peptide butylatedhydroxytoluene 6asafmeta6o(fcrate CMP cytidine monophosphate p-oxalylaminoalanine cNs centrafneryoussystem basepair CoAoTCoASHcoenzymeA blood pressure C OH b carboxyhemoglobin 751

/

I I

i

I

I ! I

I I

* t

; I I

752 COMT CoQ CPK(CK)

catechol-o-methyltransferase coenzymeQ (ubiquinone) (creatine creatinephosphokinase kinase) CP P P cyclopentanoperhydrophenanth rene CPS carbamoylphosphatesynthase CRH corticotropinreleasinghormone CS chorionic somatomammotropin CSF cerebrospinal fluid CT calcitonin CTP cytidinetriphosphate dA deoxyadenosine dADP deoxyadenosine diphosphate DC diacylglycerol DAM diacetylmonoxime dAMP deoxyadenosine monophosphate dATP deoxyadenosi ne triphosphate dCMP deoxycytidi ne monophosphate DCT distalconvolutedtubule DEAE diethylaminoethylamine DF Por DI FP diidopropylfluorophosphate dCM P deoxyguanosi ne monophosphate DHA P d ihydroxyacetonephosphate DHCC dihydroxycholecalciferol (1, 25DHCC;24,2i-DHCC) DHE A dehydroepiandrosterone DHF dihydrofolate DHT dihydrotestosterone DIT diiodotyrosine dl deciliter DM B dimethyl benzimidazole DMS dimethylsulfate DNA deoxyribonucleic acid DNase deoxyribonuclease DNP 2, 4-dinitrophenol DOPA dihydroxyphenylalanine DPC diphosphoglycerate DPP d imethylallyl pyrophosphate dTMP deoxythymidine monophosphate Eo redoxpotential EC enzymecommtssron ECF extracellular fluid E DRF endothelium-derived releasingfactor EDTA ethylened iami ne tetraacetate EF elongationfactor EFA essentialfatty acids elFs eukaryoticinitiationfactors E CF epidermalgrowth factor

B IOC H E MIS TR Y ELISA EM ER ES EScells E-site ETC FA Fab FAD FADH2 FAS F 1,6-B P F 2,6-B P Fc FD N B FFA FC F FH o FIC LU fMet FMN FMN H 2 F l -P F 6-P Fp FS H FTM C a o

AC CABA CAC Cal-Cer GAR GD H CDP GFR CGT (GT) CH CHRH GIP GIT Cla C LC Clu-Cer

Glv CLUT

enzyme-linked immunosorbentassay Embden-Meyerhof endoplasmic reticulum enzyme-substrate complex embryonicstemcells existsite electrontransportchain fafty acid antigenbindingfragment flavinadeninedinucleotide reduced FAD fatty acid synthase fructose1, 6-bisphosphate fructose2, 6-bisphosphate crystalline fragment 1-fluoro2, 4-dinitrobenzene free fatty acid Jlbroblastgrowth factor tetrahydrofolate formiminoglutamic acid N-formylmethionine flavinmononucleotide reduced FMN fructose1-phosphate fructose6-phosphate flavoprotein follicle stimulatinghormone fractionaltest meal guanine,guanosine gram free energychange y-aminobutyric acid glycosaminoglycans galactocerebroside glycinamideribotide glutamatedehydrogenase guanosine diphosphate glomerularfi ltrationrate y-glutamyltranspeptidase growth hormone growth hormonereleasinghormone gastricinhibitory peptide gastrointestinal tract y-carboxyglutamate gasliquid chromatography glucocerebroside glycine glucosetransporters

Appendix | : ABBREVIATIONSUSED lN THIS BOOK CN CM P CnRH G RH G RI H c 6-P C 6- P D GPP GSH CSSC CTP CTT AH HAC Hb HbAl HbA r c HbF Hbo2 HBsAg HbS

hcc HDL HCP RT HIAA HI F HI V HLA HLH HMC CoA HM P HNPCC hnRNA Hp HP LC HRE Hsp 5HT HTH I CD I DDM I DL I DP IF

glucose-nitrogen(ratio) guanosinemonophosphate gonadotropin'releasinghormone growth hormonereleasinghormone growth hormonerelease-inh ibiting hormone glucose6-phosphate glucose6-phosphatedehydrogenase geranylpyrophosphate glutathione (reducedform) glutathione(oxidizedform) guanosine triphosphate glucosetolerancetest changein enthalpy humanartificialchromosome hemoglobin adulthemoglobin glycosylatedhemoglobin fetal hemoglobin oxyhemoglobin hepatitisB surfaceantigen sickle-cel I hemoglobi n humanchorionicgonadotropin h igh densitylipoproteins guanine hypoxanthine phosphoribosyltransferase hydroxyindoleaceticacid Hypoxiainducibletranscription factor humanimmunodeficiencv virus human leukocyteantigen helix-loop-helix p-hydroxyp-methylglutarylCoA hexosemonophosphate hereditarynonpolyposiscolon cancer heterogeneous nuclearRNA haptoglobin high performance liquid chromatography hormoneresponsiveelement heatshockprotein 5-hydroxytryptamine helix-turn-helix isocitratedehydrogenase insulindependentdiabetesmellitus intermediatedensityIi poproteins inosinediphosphate initiationfactor

Ig lgc IGF IL IMP IN H InsP,(lPr) InsP,(lPr) IP P IR ITP IU IV K KA K^ Kbp KD Keq c-KC Ki Km KJ LATS LCAT LD H LD L LFT LH LIN E S LP H LT Lp-a LS D M MAO MAO Mb Mbo2 MCAD MD H mEq m8 MH C MI MIT mol

753 i mmunogl obul i n i mmunogl obul iCn insulin-likegrowthfactor interleukins inosinemonophosphate isonicotinicacid hydrazide (isoniazid) inositol1, 4-bisphosphate inositol 1, 4, S-triphosphate isopentenylpyrophosphate infrared inosinetriphosphate international unit intravenous dissociation constant KingArmstrong dissociation constantof acid kilo basepair kilodalton equilibriumconstant cx,-ketoglutarate inhibitionconstant Michaelisconstant ki l oj oul e longactingthyroidstimulator lecithin cholesterolacyltransferase lactatedehydrogenase low densitylipoproteins liver functiontests luteinizinghormone Long interspesed elements Iipotrophichormone(lipotropin) leukotrienes lipoprotein-a lysergicacid diethylamide molar maximalacid output monoamineoxidase myoglobin oxymyoglobin mediumchainacyl CoA dehydrogenase malatedehydrogenase mi l l i equi val ents mi l l i gram Iity complex major histocompatibi mvocardialinfarction monoiodotyrosine mole(s)

BIOCHEMISTRY

754 MM

mol. wt. M RNA MSH MTDNA MW NAD+ NA DH NADP+ NADPH NAG NANA NDP NE NEFA ng NCF NI DDM NM P NM R NP N NP U OAA

ob OD OMP Osm PABA PAF PACE PAH PAPS PBG PBI PCM PCNA PCoz PCR PCT P DCF P DH PEG PEM PEP PER PEST

millimolar molecularweight RNA messenger melanocytestimulating hormone mitochondrialDNA molecularweight nicotinamideadeninedinucleotide reducedNAD* nicotinamideadeninedinucleotide phosphate reducedNADP+ N-acetylglutamate ic acid N-acetylneuramin nucleosidediphosphate niacinequivalents non esterifiedfatty acid nanogram(10-e g1 nerve growth factor non-insuIi n dependentd iabetes m e l l i tu s nucleosidemonophsphate nuclearmagneticresonance non-proteinnitrogen net proteinutiIization oxaloacetate obese optical density orotidine monophosphate osmoles paraaminobenzoicacid platelet-activati ng factor polyacrylamide gel electrophoresis paraaminohippurate lfate phosphoadenosi ne phosphosu porphobilinogen proteinbound iodine protein-caloriemalnutrition proliferatingcelI nuclearantigen partialpresenceof CO, polymerasechain reaction proximalconvolutedtubule plateletderivedgrowth factor pyruvatedehydrogenase glycol polyethylene protein-energy malnutrition phosphoenolpyruvate proteinefficiencyratio proline,glutamine,serine,threonine

PFK PC PGA pH PI Pi p' P IF PlP2 pK" PKU PL P LP Poz POMC PPi ppm P R IH PRL PRPP PT PTH PTH PUFA QPRT RACE RAIU RAPD ras RBC RBP RDA rD N A RE RER RF Rf RFC RFLP R-form RIA RMR RNA RNAP RNase

phosphofructokinase prostaglandins pteroylglutamicacid negativelog of (H+) phosphatidylinositol inorganicphosphate isoelectricpH prolactininhibitoryfactor i nositol4, S-bisphosphate negativelog of Ka phenylketonuria phospholipid pyridoxalphosphate partialpressureof O, pro-opiomelanocortin i norganic pyrophosphate partsper million nhibiting prolactinrelease-i hormone prolactin 5-phosphoribosyl1-pyrophosphate prothrombintime p3rathyroidhormone phenylthiohydantoin polyunsaturated fattYacids quinolinate phosphoribosyltransferase of cDNA ends rapidamplification radioactiveiodine uptake randomamplifiedpolymorPhicDNA rat sarcoma red blood cells retinolbindingprotein recommendeddietary(dailY) allowance recombinantDNA retinolequivalents reticulum roughendoplasmic releasingfactor ratio of fronts replicationfactor C restrictionfragmentlength polymorphism relaxedform radioimmunoassay restingmetabolicrate ribonucleicacid RNA polymerase ribonuclease

'/

Appendix | : ABBFIB/IATIONSUSED lN THIS BOOK R 5-P RPA RQ rRNA RSV KT rT: SAM S CI D SDA

sf SCOT SCPT S HB C S I DS S I NE s sn SNPs s nRNA s nRNP sRNA SRS STRs T T T3 T4 TBG TBPA TCA TF T-form TC Tgb

riboseS-phosphate replicationproteinA respiratoryquotient ribosomalRNA rousesarcomavirus reversetranscriptase reverseT, S-adenosylmethionine severecombinedimmunodeficiencv specificdynamicaction Svedbergfloatation serumglutamateoxaloacetate transaminase serumglutamatepyruvate transaminase sexhormonebindingglobulin suddeninfantdeathsyndrome shortinterspersed elements stereospecific number singlenucleotidepolymorph isms smallnuclearRNA smalI nuclearribonucleoprotein solubleRNA slow reactingsubstance simpletandemrepeats thymine,thymidine thymus(T-lymphocyte) 3,5,3'-triiodothyronine i ne 3,5,3',5'-tetraiodothyron (thyroxine) thyroxinebindingglobulin thyroxinebindingprealbumin tricarboxylicacid tissuefactor taut or tenseform triacylglycerol thyroglobulin

TCF TH F TIB C TLC Tm TMP TN F tPA TPP TRH tRNA TSH TX pm UBG UCP UDP UDPC pl pM U MP UTP UV VH V IP VL V LD L VMA V.", VNTRs WBC X MP XP Xvl YAC

/r, transforminggrowth factor tetrahydrofolate total iron bindingcapacity thin layerchromatography tubularmaximum thymidi ne monophosphate tumor necrosisfactor tissueplasminogenactivator th iaminepyrophosphate thyrotropinreleasinghormone transferRNA thyroidstimulatinghormone thromboxane micrometer(10-66) urobilinogen uncouplingprotein uridinediphosphate glucose uridinediphosphate (10-6 microliter l) micromoles(10-6M) uridinemonophosphate uridinetriphosphate ultraviolet variableheavychain vasoactiveintestinalpeptide vari abl el i ghtchai n very low densitylipoproteins vanillylmandelicacid velocitymaximum variablenumbertandemrepeats white blood cells xanthosi ne monophosphate xerodermapigmentosum xylose yeastartificialchromosome

t

ondixII : C'r

a

Alphabet C[

Alpha Beta

p

G a mma

^'l

Delta

6

Ep s i l o n

r

Zera

n e

Eta Theta KapPa L a mb d a

l.

Mu

p F

Xi

L

Pi Rho

p

Sigma

o

Psi

a x v

Omega

o

Ph i chi

756

Apesrdtx III : odginsoflmportant Btoehemieatr Words

Acid (Latin)acidus-sour

Biology (Greek)bios-life; logos-discourse

Acidosis (Latin) acidus-sou r; osis-condition

Bovine (Latin)bovinus-pertainingto cow or ox

Afbinism(Greeb albino-white

Calorie (tatin) calor-heat

Alkali (Arabic)al-qite-ashesof saltwort

Cancer (Latin) crab

Aflergy (Creek)allos-other;ergon-work

Carbohydrate carbo (Latinl-coal; hydor (Creek)water

Alloseric (GreeHallo-the other

Caries (latin)Jecay

Amentia (Latin) amentis-mentaldeficiency Amnesia (Greeb a-not; mnesis-memory

Casein (Latin) caseus-cheese

Amphipathic (Greek)amphi-both; pathos-feeling

Catabolism(G reek)kata-down; ballein-to throw

Amphiphilic(Greek)amphi-both;philic-love

Catalysis(G reek)kata-down; lysis-degradation

Anaerobe (Greek)a-nou aer-air; bios-life

Cathepsin(Creek)to digest

Anapferotic (Greek)ana-up; plerotikos-tofill

Cephalins(Greek)kephale-head

Androgen (G reek)aner-man; genesis-production

Cheilitis(Creek)cheilos-lip;itis-inflammation

Anemia (Greek)a-not; haima-blood

Cheifosis(Greek)cheilos-lip;osis-condition

Anorexia (Greek)a-not; orexis-appetite

Chirafity (Creek)cheir-hand

(Greek)chloros-palegreen;phyllonAnticoagufant anti (Creeklagainst; coagulare Chforophyll leaf (Latin)-to curdle (Greek)chole-bile; lithos-stone;asisAntimetabolite (Greek) anti-against; metabole- Cholelithiasis condition cnanSe (Greek)chole-bile;sterol-solidalcohol Arterioscferosis arteria (Latin)-arterv; sclerosis Cholesterol (Creek)hardening. Chromatography (Greek) c hroma-colour; graphein -to write Arthritis (Greek) arthron-jo i nt; itis-infIammation (Greek)chroma-colour;soma-body Atherosclerosis (G reek)athere-porridge;sclerosis- Chromosome hardening

Chyle (Greek) chylos-ju ice

Beri-beri(Singhalesellcannot (saidtwice)

Chyfuria (Greek)chylos-juice;auron-urine

Biochemistry (Greek)bios-life; chymos-juice

Chyme (Greek)chymos-juice 757

758 Cirrhosis (Greek) kirrhos--orange-tawny;osiscondition Cis (Latin) same side Coagulation(Greek)coagulare-tocurdle Collagen (Greek) kolla-glue; genesthai-to be produced

BIOCHEMISTF|Y Hepatitis (C reek)hepar-liver; itis-inflammation Hormone (Greek) hormain-to excite Hydrophilic (C reek)hydro-water; phi Iic-l iving Hydrophobic (G reek)hydro-water; phobic-hating

Colloid (Greek)kolla-glue; eidos-form

Hyperglycemia(Creek)hyper-above;glycos-sweet; haima-blood

Consanguinity(Latin)con-with; sanguis-blood

Hypertonic (C reek)hyper-above;tonos-tension

Creatine (Greek) kreas-flesh

Hypogfycemia (C reek) hypo-below; glycos-sweet; haima-blood

Cristae (latin) crests Cutaneous(lafin) cutis-skin

Hypotonic (Creek)hypo-below; tonos-tension

Cytofogy (G reek)kytos-cell; logos-discourse

lcterus (Creek)ikteros-jaundice

Cytoplasm (G reek)kytos-cell; plassein-to mould

lmmunity (Latin) immunis-exempt from public burden

Dermatitis (Greek)derma-skin; itis-inflammation Diabetesmellitus (G reek)d iabetes-running th rough (or a siphon);mellitus-sweet

lnflammation (Latin) inflammare-to set on fire In situ (Latin)in the correct position

Eicosanoids(Greek)eikosi-twenty

In vitro (Latin) in a test tube

Embolism(Greek)embolos-to plug

In vivo (Latin)in the living tissue

Emphysema(Creek)emphysan-toinflate Enkephalin(Creek)in the brain

f somerism(Gre*) iso-equal;mesoFpart

Enthalpy(Greek)to warm within Entropy (Creek)in turning

lsotope (Creek)iso--equal;topos-place

Enzyme (Greek) in yeast Erythrocyte (C reek)erythros-red; kytos-cell Eukaryotes(Creek)eu-true;karyon-nucleus Ferrous (Latin) ferrum-iron Folate (Latin) fol ium-leaf Gafactose (Greek)gala-milk Gastritis (C reek)gaster-belly; itis-inflammation Gene (Greek)genesis-descent Genome (Creek)genos-birth Globin (latin) globus-ball Globulin (Latin) globulus-little ball Glossitis(Creek)glossa-tongue;itis-inflammation Glycofysis (C reek)glycorsweet; lysis-dissolution Goitre (Lafinlgultur-throat Gonadotrophin (Creek) gona-generation;trophenourishment Hemoglobin haima (GreekFblood;globus (LatinF ball

lsotonic (Greek)iso--equal;tonos-tension faundice (French/jaune-yellow Keratin (Creek) keras-horn Kwashiorkor (Ca-African) sicknessof the deposed chi l d lactafbumin (Greek)lac-milk; albumin-white lecithin (Greek) lekithos-egg yolk Lipids (Creek) lipos-fat Lactosuria lac (LatinFmilk; ovron (GreekFurine Leukocytes(Ceek/ leukos-white;kytos-<ell Leukoderma(Creek)leukos-white;derma-skin Ligase (Greek) ligate-to bind Mafaria (ltalian)bad air (Latin) Mafnutrition nourishment

malus-bad;

nutrire-

Marasmus (CreeH to waste Melanin (Greek)melan-black Menopause(Greek)men-month; pausis-stopping Metabolism (Creek)metabole-change

Appendix Ill : OR|GINSOF |MPORTANTBTOCHEMTCAL WORDS Mitochondria granufe

(Creek) mitos-thread; chondros-

Mitosis (Greek) mitos-thread; osis-condition

759

Porphyrin (Greek)porphyra-purplecolour Post-prandial (tafrn)-after food

Prokaryotes (Greek) pro-before; karyon-nucleus Monosaccharide (Greek)-mono-one; saccharinProteins (Greek) proteiorholding first place sugar Rickets (Old English)wrickken-to twist Myeloma (Creek)myelos-marrow;oma-tumor Serum (Latinlwhey Nephritis (Gre-ck)nephrorkidney; itiri nflammation Sphingosine(Greek)sphingein-robind tight Neurosis(Greek)neuron-nerye;osis--condition Steatorrhea (Greek)stear-fat; rheein-to flow Oedema or edema (Greek)oidema-swelling Stereoisomerism(Greek)stero-space Ofigosaccharides (Greek) oligo-few; saccharonSterol (Creekl steros-solid;ol- alcohol suSar Thafassemia(Greeb thalassa-sea Osmosis (Greeklpush Osteomalacia(Creek)osteon-bone;malakia-softness Oxyntic (Greek)oxynein-to make acid

Thermodynamics (G reek)therme-heat; dynamicr power

Oxytocin (Creek)-rapid birth

Thermogenesis (Greek) therme-heat; genesisproduction

Pafindrome(Greeklto run back again

Thrombosis(Greek)thrombos-cloUosis-
Pantothenic acid (Greek) pantos-everywhere

Thyfakoid (Greek)thylakora sac or pouch

Pathogenesis (Creek) pathos-disease; genesisproducing

Tocopherol (Greek) tokos-child birth; pheros-to bear; ol-alcohol

Pellagra (/talianfrough skin

Trans (Lafin) across

Pepsin(Creek)pepsis-digestion

Tumor (Latin)swelling

Phagocytosis (Greek) phagein-to eat; kytos-<ell; osis-condition

Mtamin (coined inappropriatelyin 19O6) (Latin) vita-life; amine

Phobia (Greek)phobos-fear

Xanthoma (Greek)xanthos-yellow

Polysaccharide (Greek) poly-many; saccharin- Xenobiotics (Creek) xenos-strange suSar Zwitterion (Cerman) zwitter-hybrid.

Appendix N: Common in Biochemistry Confusables

Acetone;acetate- Acetone is a ketone;acetateis a carboxylic acid. Acetyl CoA; acyl CoA - Acetyl CoA is a specific compound containing acetate bound to coenzyme A; acyl CoA is a generalterm used to refer to any fatty acid (acyl group) bound to coenzymeA.

heme;bile saltsare the sodiumand potassium saltsof bile acids (glycocholate,taurocholate) produced by cholesterol. Biliverdin; bilirubin - Both are bile pigments. Biliverdin is produced from heme in the reticuloendothelial cells; bilirubin is formed by reductionof biliverdin.

Albumin; albinism- Albumin is a serum protein; Biotin; biocytin - Biotin is a B-complexvitamin; albinism is a genetic disease in tysosine biocytin refersto the covalentlybound biotin metabolism. to enzymes(throughe-aminogroup of lysine). Amino; imino - Amino group (-NHr) is found in B-Lymphocytes;T-lymphocytes- B-lymphocytes majorityof amino acids;imino group (:NH) (antibodies) produceimmunoglobulins and are is presentin a few amino acids like proline involvedin humoralimmunity;T-lymphocytes and hydroxyproline. for cellular immunity. are responsible Anabolism; catabolism- Anabolism refersto the Bisphosphate; diphosphate- Bisphosphate hastwo biosynthetic reactions involving the formation phosphates held separately e.g. 2,3-BPC; of complex molecules from simpler ones; diphosphate has two phosphates linked catabolismis concernedwith the degradation togethere.g. ADP. of complex moleculesto simpler ones with a Calcitriol; calcitonin- Calcitriol (1,25-DHCC)is concomitant releaseof energy. the physiologicallyactive form of vitamin D; Anomersl epimers - Anomers refer to two calcitoninis a peptidehormone,synthesized stereoisomersof a sugar that differ in by thyroid gland. configurationaround a single carbonyl atom; epimers are two stereoisomersthat differ in Calorimetry;colorimetry- Calorimetrydealswith the measurement of heat production by configurationaround one asymmetriccarbon organism;colorimetry is concernedwith the of a sugarpossessing two or more asymmetric measurement carbon atoms. of colour compounds. Apoenzyme;coenzyme- Apoenzymeis the protein Carboxyl; carbonyl - These two are functional part of the functional enzyme (holoenzyme); groups found in organic substances;carboxyl coenzyme is the non-protein organic part group -Coou; carbonyl-8-. associatedwith enzyme activity. Bile pigments;bile salts- Bile pigments(biliverdin, Carnitine; creatine; creatinine Carnitine bilirubin) are the breakdown products of transportsactivatedfatty acids (acyl CoA) from 760

Appendix lV : @MMON CONFUSABLESlN BIOCHEMISTFY

761

Genel genome- A gene refersto the DNA fragment of a chromosome that codes for a single polypeptide; all the genes of a cell or an organismare collectively known as genome. Choline; cholic acid - Choline is a trimethyl Glu; Gla - Glu is the code for glutamic acid; Cla is the code for ycarboxy glutamic acid. quaternary base and is a constituent of acetylcholine;cholic acid is an importantbile Glucuronic acid; gluconic acid - Both are derived acid. from glucose; oxidation of C, results in glucuronic acid while oxidation of C, yields Chyle; chyme - Chyle refersto lymph with milky gluconic acid. Clucuronic acid is producedin appearancedue to chylomicrons; chyme is uronic acid pathway;gluconic acid is formed the partiallydigestedfood in the stomachthat in hexosemonophosphateshunt. passesto deodenum. Glutaric acid; glutamic acid - Glutaric acid is a Configuration;conformation- Configurationis the dicarboxylic acid; glutamic acid (c-amino geometric relationship between a given set of glutaric acid) is an amino acid. atoms (e.g. L- and D-amino acids). Conformation is the special relationship of Glycogen;glycogenin- Glycogenis a storageform every atom in a molecule (e.9. secondary of carbohydrate(polysaccharide) in the animal structureof protein). body; glycogenin is a protein which servesas a primer for the initiation of glycogensynthesis. Cysteine;cystine- Both are sulfi.rrcontainingnonGlycoproteins; mucoproteins Both are essential amino acids. Cysteine contains conjugated proteins containing carbohydrate sulfhydryl (-SH) group; cystine is formed by as the prostheticgroup. The term glycoprotein condensationof two cysteine residues and is used if the carbohydrate content is <4o/o; containsa disulfide(-S-S-) group. mucoprotein contains >47" carbohydrate. Dextrins; dextrans; dextrose - The first two are polysaccharides composedof glucose.Dextrins Hydrophilic; hydrophobic - Hydrophilic refersto affinity to water; hydrphobic means hatred are the breakdownproductsof starch;dextrans towards water. are gels produced by bacteriafrom glucose. Dextroseis glucose in solution (dextrorotatory) Insulin;inuiin- Insulinis a peptidehormone;inulin used in medical practice. is a polysaccharidecomposed of fructose. Diabetes mellitus; diabetes insipidus - Diabetes In vivo; in vitro - In vivo refers to within the cell mellitusis primarilyan impairmentin glucose or organism;in vitro means in the test tube. metabolism due to the deficiency of, or lsoniazid; iproniazid lsoniazid is an antiinefficient insulin; diabetes insipidus is tuberculosis drug; iproniazid is an anticharacterizedby excretion of large volumes of depressantdrug. urine (polyuria),caused by the deficiency of Lactam;lactim - Thesetermsare usedto represent antidiuretichormone(ADH). tautomerism.Lactam indicatesthe existence Endocytosis;exocytosis- Endocytosisis the intake of a molecule in keto form; lactim represents of macromoleculesby the cells; exocytosis a moleculein enol form. refers to the releaseof macromoleculesfrom lactase- Lactoseis a disaccharide;lactase Lactose; the cells to the outside. is an enzyme that cleaves lactose to glucose Epinephrine;norepinephrine- Both are catechoand gulactose. lamines synthesized from tyrosine. Linoleicacid; linolenicacid - Both are 18 carbon Epinephrine is methylated while norepineunsaturatedfatty acids. Linoleic acid has two phrine does not contain a methyl group. double bonds;linolenic acid hasthree double Exons; introns - Exons are the DNA sequences bonds. coding for proteins;intronsare the intervening Lipoproteins;lipotropic factors - Lipoproteinsare DNA sequencesthat do not code for proteins. molecularcomplexescomposd of lipids and proteins; lipotropic factors are the substances GABA; PABA - y-Aminobutyricacid (CABA) is a (e.g.choline, betaine),the deficiencyof which p-ami nobenzoicacid (PABA) neurotransmitter; is a vitamin. causesaccumulationof fat in liver. cytosol to mitochondria; creatine is mostly found in the muscle as creatinephosphate,a high energy compound; creatinine is the anhydrideof creatine.

762

BIOCHEMISTF|Y

held by an ether linkage in phosphatidal B-Lipoprotein;B-lipotropin - p-Lipoproteinrefers ethanolamine. to the low density lipoproteins;p-lipotropin is a peptide hormone derived from proPhytic acid; phytanic acid - Phytic acid is formed opiomelanocortin(POMC) peptide. by the addition of six phosphatemoleculesto Lyases;ligases - Lyases are the enzymes that inositol, it is an inhibitor of the intestinal catalyse the addition or removal of water, absorptionof calcium and iron; phytanicacid ammonia, CO, etc.; ligases catalyse the is an unusalfatty acid derived from phytol, a syntheticreactionswhere two moleculesare constituentof chlorophyll. joined together. are the cells Prokaryotes;eukaryotes- Prokaryotes Malate; malonate; mevalonate - Malate is an that lack a well defined nucleus;eukaryotes intermediatein the citric acid cycle; malonate possessa well-definednucleus. is a competitive inhibitor of the enzyme protamines- Bothare simpleproteins. succinate dehydrogenase;mevalonate is an Prolamines; are solublein alcohol;protamines Prolamines intermediatein cholesterolbiosynthesis. are basic protein soluble in NHTOH. Melanin; melatonin- Melanin is the pigmentof skin and hair; melatonin is a hormone Pyridine;pyrirnidine;pteridine- All the three are heterocyclic rings containing nitrogen, as synthesizedby pineal gland. depicted below. Maltose; maltase - Maltose is a disaccharide; maltaseis an enzyme that cleavesnaltose to two moleculesof glucose. Methvl, methenyl; methylene- All the three are one-carbonfragmentsas shown in brackets, methyl (-CHr); methenyl(-CH:;' methylene ( - c H 2 -). Molarity; molality - Molarity is defined as the numberof molesof a soluteper liter solution; molality representsthe number of moles of a solute per 1,000 g of solvent. Nicotinic acid; nicotine - Nicotinic acid is a B-complexvitamin; nicotine is an alkaloid present in tobacco leaves.

Pyridine ring is found in niacin and pyridoxine;pyrimidineis presentin thiamine (vitamin Br), thymine, cytosine and uracil; folic acid containspteridinering. Pyridoxine;pyridoxal- Pyridoxineis the primary alcohol form of vitamin Bu; pyridoxal is the aldehydeform of 8..

(recommendeddietary/daily Nucleoside; nucleotide- A nucleosideis composed RDA; SDA - RDA the quantities of represents allowance) of a nitrogen base and a sugar; nucleotide provided in the diet daily for to be nutrients containsone or more phosphategroupsbound good health and physical of maintenance to nucleoside. efficiency; specific dynamic action (SDA) is Osmolarity; osmolality - Osmolarity represenG the extra heat producedby the body over and osmotic pressure exerted by the number of above the caloric value of foodstuffs. moles (milli moles) per liter solution; osmolality refers to the osmotic pressure Renin;Rennin- Reninis synthesizedby the kidneys and is involved in vasoconstrictioncausing exertedby the number of moles (milli moles) hypertension;rennin is an enzyme found in per kg solvent. gastric juice responsiblefor coagulation of Palmitate; palmitoleate - Both are even chain mi l k. (16-carbon)fatty acids. Palmitateis a saturated fatty acid; palmitoleateis a monounsaturated Ribosomes;ribozymes- Ribosomesare the sitesof protein biosynthesis;ribozymes refer to the fatty acid. RNA moleculeswhich function as enzymes. Phosphatidylethanolamine;phosphatidalethanolamine Both are phospholipids. In Retinol; retinal - Retinol is the alcohol form of vitamin A; retinal is the aldehyde form phosphatidylethanolamine,the fafty acid is obtained by the oxidation of retinol. bound by an ester linkage.The fafty acid is

Aspendix lV : COMMON CONFUSABLEStN B|OCHEM|STFIY :c{eroproteins;selenoproteins- Scleroproteins are a group of fibrous proteins; selenoproteins contain the amino acid selenocysteine.

763

structure.

:eiotonin; melatonin - Serotonin is a neuro- Thiokinase; thiolase * Thiokinase activates fatty transmitter synthesized from tryptophan; acidsto acyl CoA; Thiolasecatalysesthe final melatonin is a hormone derived from reaction in p-oxidationto liberateacetvl CoA serotoninin the pinealgland. from acyl CoA. >omatotropin; somatostatin; somatomedin Iranscription; translation - Transcription refers Somatotropinis the other name for growth to the synthesisof RNA from DNA; translation hormone (GH); growth hormone release involves the protein synthesis from the inhibiting hormone (CRIH) is also called RNA. somatostatin; somatomedin refers to the insulin-likegrowth factor -l (lCF-l), produced Uric acid; uronic acid - Uric acid is the by liver in responseto GH action. end product of purine metabolism; uronic acids are formed by the oxidationof aldehyde Sucroselsucrase- Sucrose is a disaccharide; group of monosaccharides(e.g. glucuronic sucraseis an enzyme that cleavessucroseto acid). glucoseand fructose. Synthase; synthetase - Both the enzymes are concerned with biosynthetic reactions. organisms(e.9. reptiles)convert NH, to uric Synthasedoes not require ATp; synthetaseis acid. dependent on ATP for energy supply. (Nofe : This distinctionbetweensynthaseand Vitamin A; coenzyme A Vitamin A is {at soluble synthetasehowever,is not maintainedstrictly vitamin; coenzyme A is derived from water by most authors). solublevitamin,pantothenicacid.

a I

QUAtTTAT|VE

EXPERIMENTS

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Severallaboratoryqualitativeexperimentsare performedto indentifythe compoundsof biochemical importance (carbohydrates,proteins/ami no acids, non-protein nitrogenoussubstances)and to detect the abnormalconstituents of urine.The principlesof the reactionspertainingto the mostwidely employed qualitativetestsare describedhere.

The carbohydratesused in the laboratory for the qualitativetests include glucose and fructose (monosaccharides), sucrose, lactose and maltose (disaccharides) and starch (polysaccharide). The principles of the reactions of carbohydratesare Srven: 1. Molischtest l lt is a generaltestfor the detection of carbohydrafes.The strong H2SOa hydrolyses carbohydrates(poly- and disaccharides) to liberate m onos ac ch a ri d e s .T h e m o n o s a c c h a ri desget dehydrated to form furfural (from pentoses)or hydroxy methylfurfural (from hexoses)which condensewith o-naphtholto form a violet coloured complex. 2. lodine test : Polysaccharides combine with iodine to form a coloured complex. Thus, sfarch gives blue colour while dextrins give red colour with iodine. 3. Benedict's test : Thisis a testfor the identification of r educ i n g s u g a rs , w h i c h fo rm e n edi ol s (predominantlyunder alkaline conditions).The enediolformsof sugarsreducecupric ions (Cu2+)of copper sulfateto cuprous ions (Cu+)which form a yellow precipitateof cuprous hydroxide or a red precipitateof cuprousoxide. 4. Earfoed'stest : The principle of this test is the same as that of Benedict's test except that the 74

reductionis carriedout in mild acidicmedium.Since acidic medium is not favourablefor reduction,only give this strongreducingsugars(monosaccharides) test positive. Thus, Barfoed's test serves as a key reaction to distinguish monosaccharidesIorm disaccharides. 5. Seliwanoff'stest : This is a specific test for ketohexoses.Concentrated hydrochloric acid to form furfural derivatives dehydratesketohexoses which condensewith resorcinolto give a cherryred complex. 6. Foufger'stest : This is also a testfor ketohexoses. The furfural derivativesformed from ketohexoses condensewith urea in the presenceof stannous chlorideto give a blue colour. are converted 7. Rapidfurfural lest: Ketohexoses to furfural derivativesby HCI which form a purple colour complex with o-naphthol. in aceticacid, B. Osazonetest : Phenylhydrazine when boiled with reducingsugarsforms osazones. The first two carbons(Cr and Cz) are involved in thi s reacti on. The sugars that di ffer i n thei r configurationon thesetwo carbonsgive the same type of osazones,sincethe differenceis markedby bi ndi ng w i th phenyl hydrazi ne.Thus, gl ucose, fructoseand mannosegive the same type (needle shaped) of osazones. However, the osazones of reducing disaccharidesdiffer - maltose gives sunfl ow er-shapedw hi l e l actose pow der-puff shaped. 9. Sucrosehydrolysistest : Sucroseis a nonreducing sugar,hence it does not give Benedict's and Barfoed'stests.Sucrosecan be hydrolysedby concentratedHCl, to be converted to glucose which and fructose (reducingmonosaccharides) answer the reducing reactions.However, after sucrose hydrol ysi s, the medi um has to be made alkaline (by adding Na2CO3)for effective reductionprocess.

Appendix V : PBACTICALBIOCHEMISTRY-PRINCIPLES

765

precipitating agents.They dehydrate the protein moleculeby removingthat water envelopeand cause precipitation. The proteinsemployedin the laboratoryfor the qualitativetestsinclude albumin, globulins,casein, gelatin and peptones.The principle of the most c om m on r e a c ti o n s o f p ro te i n s /a m i n o aci ds performedin the laboratoryare given hereunder. A. PRECIPITATION

REACTIONS

Proteinsexist in colloidal solution due to hydrationof polar groups(-{OO-, -NHj, -OH). T hey c an b e p re c i p i ta te d b y d e h y d ra t i on or neutralizationof polar groups.Severalmethodsare in useto achieveprotein precipitation. 1. Precipitationby neutral salts : The processof proteinprecipitationby the additionof neutralsalts such as ammonium sulfateor sodium sulfateis referredto as salting out. This phenomenonis explained on the basis of dehydrationof protein moleculesby salts.This causesincreasedproteinproteininteraction,resultingin molecularaggregation and precipitation. T he am o u n t o f s a l t re q u i re d fo r protei n precipitationdependson the size(molecularweighg of the proteinmolecule.In general,the higheris the protein molecular weight, the lower is the salt requiredfor precipitation.Thus,serumglobulinsare precipitatedby half saturationwith ammoniumsulfate while albumin is precipitatedby full saturation. 2. Precipitationby saltsof heavy metals: Heavy metal ions fike Pb2+,Hg2+,Fe2+,Znz+, Cd2+ cause pr ec ipit at io no f p ro te i n s . T h e s e m e ta l s bei ng positivelycharged,when addedto protein solution (negativelycharged)in alkalinemedium result in precipitateformation.

B. COLOURREACTIONS The proteinsgive severalcolour reactionswhich are often useful to identify the nature of the amino acids presentin them as shown in the table. Colour reactionsof proteins/aminoacids Reaction

Specific group or amino acid

1. Biuret reaction 2. Ninhyddn reac'tion 3. Xanhoproteic reac'tion 4. Millons reaction 5. Hopkins-Cole reaclion 6. Sakaguchi reaction 7. Nitroprusside reaction 8. Sulfurtest 9. Paufstest 10. FolirGolcalteau's test

Twopeplide linkags c-Amino acids Benzene ilngofaromatic amino acids(Phe,Tyr,Trp) groupfiyr) Phenolic lndolering(Ip) group(Arg) Guanldino groups (Cla) Sullhydryl (Cys) Sulftydrylgrougs lmidazole ring(His) groups Phenolic [Iyr)

1. Biuret reactions: Biuretis a compoundformed by heating urea to 180'C. When biuret is treatd with dilute copper sulfate in alkaline medium, a purple colour is obtained.This is the basisof biuret test used for identification of proteinsand peptides. Biurettestis answeredby compoundscontaining two or more CO-NH groups i.e., pptide funds. All proteins and peptides possessingatleast two peptidelinkagesi.e.,tripeptides(with 3 aminoacids) give positivebiuret test.The principle of biuret test is convenientlyusedto detectthe presenceof proteins in biologicalfluids.The mechanismof biurettest is not clearly known. lt is believedthat the colour is due to the formation of a copper co-ordinated complex.

3. Precipitationby anionic or alkaloid reagents: Proteinscan be precipitatedby trichloroaceticacid, sulphosalicylic acid, phosphotungstic acid, picric acid,tannic acid, phosphomolybdic acid etc. By the additionof theseacids,the proteinsexistingascations are precipitatedby the anionic form of acids to produceprotein-suIphosalicylate,protein-tungstate, 2. Ninhydrin reaction : The cr-aminoacids react protein-picrateetc. with ninhydrinto form a purple,blue or pink colour The anionic reagen8such as phosphotungstic complex (Ruhemann'spurple). acid and trichloroaceticacid are used to prepare Amino acid + Ninhydrin--------+ protein-freefiltrate of blood needed for several Keto acid + NH3 + CO2 + Hydrindantin estimationsre.g..urea,sugar)in the laboratory. Hydrindantin+ NH3 + Ninhydrin----------) 4 P r ec ip i ta ti o nb r o rg a n i cs o l v e n ts: O rgani c purple Ruhemann's s olv ent s s u c h a s a l c o h o l a re g o o d p rotei n

766 3. Xanthoproteicreaction r Xanthoproteicreaction is due to nitration oI aromatic amino acids (tryptophan,tyrosineand phenylalanine)on treatment with strongnitric acid at high temperature. 4. Millon's test : This test is given by the amino acid tyrosine,or any other compound containing hydroxyphenylring. A red colour or precipitateis obtained in this reaction due to the formation of mercurycomplex of nitrophenolderivative. 5" Hopkins'Colereaction: This reactionis specific for the indole ring of tryptophan lt combineswith formaldehydein the presenceof the oxidizingagent (sulfuricacid with mercuricsulfate)to form a violet or purple colouredcompound.

BIOCHEMISTRY identified by a colour change in phenophthalein indicator(pink colour in alkalinemedium). 3. Benedict'suric acid test : Uric acid being a to strongreducingagent,reducesphosphotungstate tungstenblue in alkalinemedium. 4. Murexidetest: Uric acidis oxidizedby nitric acid to give purpuricacid (reddishyellow).This in turn combines with ammonia to form purple red colourammoniumpurpurate(murexide). 5. faffe'stest r Creatininereactswith picric acid in alkalinemediumto form orangeredcolourcomplex.

6. S ak agu c h ire a c ti o n : Arg i n i n e , c o n tai ni ng Urine is the mostimportantexcretoryfluid from guanidinogroup,reactswith a-naphtholand alkaline the body. Someof the diseasesare associatedwith hypobromiteto form a red colour complex. an excretionof abnormalconstituentsin urine. The 7. Sulfur test : This is a test specific for sulfur identificationof suchcompoundsin urine is of great containingaminoacidsnamelycydeineand cystine, diagnosticimportance. but not methionine.When cysteineand cystineare boiled with sodium hydroxide,organic sulfur is Associated disorde4s) Urine abnormal convertedto inorganicsodium sulfide.This reacts constituent with lead acetateto form a black precipitateof lead (glomerulonephritis) Kidney damage Albumin sulfide. Methionine does not give this test, since Damage to kidneys or urinarytract. Hemoglobin sulfur of methionineis not split by alkali. glycosuria. renal Diabetes mellitus, Glucose B. Pauly/stest : This reactionis specificfor histidine Diabetes mellitus, starvation. Ketonebodies (imidazolering).Diazotisedsulfanilicacid reactswith jaundice Obstructive Bilesalts imidazole ring in alkaline medium to form a red jaundiceandhepatic Obstructive Bilepigments colouredcomplex. jaundice. 9. Molisch test : This is a specific test for the detectionof carbohydrafes.The proteinscontaining acid te*t: Proteinsgetprecipitated 1. Sulfosalicylic carbohydrates(e.9., glycoproteins)give this test by sul fosal i cyl i c aci d by formi ng protei npositive.Albumin containscarbohydratebound to sulfosalicylate. it, henceanswersMolisch test. 2. Heat coagulationtest : This is a test for the detectionof albumin and/or globulinsin urine. Heat coagul ati ontest i s based on the pri nci pl e of denaturationof proteins,followed by coagulation. (Note : Smallamountsof dilute aceticacid are added to dissolvethe phosphatesand sulfatesthat The non-proteinnitrogenous(NPN)substances get precipitatedon heating.) of biochemicalimportanceinclude urea, uric acid 3. Benzidinetest : Thistestdetectsthe presenceof and creatinine. bl ood. H emogl obi n (acts l i ke peroxi dase) 1. Sodiumhypobromitetest : This is a testfor the decomposeshydrogenperoxideto liberatenascent detectionof urea. Sodiumhypobromitedecomposes oxygen(O-) which oxidisesbenzidineto a greenor ureato liberatenitrogen.The lattercan be identified blue colouredcomplex. by brisk efferyescence. (Note : Pus cells of urine possessperoxidase 2. Specificureasetest : The enzymeurease(source- activitywhich interferesin benzidinetest.This can horse gram) specificallyacts on urea to liberate be eliminatedby boilingthe urine prior to the testto ammonium carbonate (alkali). The latter can be inactivatethe enzyme).

Appendix V r PRACTICAL BIOCHEMISTRY-PRINCIPLES 4. Benedict'stegt : This is a semiquantitative test for the detectionof urine reducingsugars(primarily glucose).Benedict'stest is basedon the principleof reducingpropertyof sugars(describedin detailunder reactionsof carbohydrates).Colour of the precipitate formedindicatesthe approximateamountof glucose presentin urine.Thus,greenturbidity= traces;Breen precipitate= O.So/oi yellow precipitate= 1"h; orange precipitate = 1.5o/" brick red precipitate - 2%. ( Not e: B e n e d i c t' s te s t i s n o t s p e ci fi c to glucose,since it can be answeredby any reducing substance). 5. Glucoseoxidasetest : This is a strip testfor the specific detection of glucose.The enzyme glucose oxidase oxidizes glucose to liberate hydrogen peroxide which in turn is converted to nascent oxygen(O-) by peroxidaseenzyme.The compound O-diansidinecombineswith nascentoxygento form a coloured(yellow to red) complex. in alkalinemedium 6. Rothera/s test : Nitroprusside reacts with keto group of ketone bodies (acetone and acetoacetate) to form a purple ring. This test is not given by p-hydroxybutyrate. 7. Hay's test : This test is basedon the surface tension lowering property ol bile salts (sodium glycocholate and sodium taurocholate).Sulfur powdersprinkledon the surfaceof urine containing bile saltssinksto the bottom. B. Petternkofer's test : This test is employed for the detection of bile salts. The furfural derivatives(by reacting sugar with concentrated HzSO+)condensewith bile saltsto form a purple ring. 9. G m eli n ' s te s t : N i tri c a c i d o x i d i z es the bile pigm e n t b i l i ru b i n to b i l i v e rd i n (g r een)or bilicyanin (blue). Gmelin's test gives a play of colours and is used for the identificationof bile pigments. 10. Fouchet'stest : This test is also employedfor the detection o( bile pigments. Bile pigmentsare adsorbed on barium sulfate. Fouchet's reagents (containingferric chloride in trichloroaceticacid) oxidizesbilirubinto biliverdin(green) and bilicyanin (blue).

767

QUANTTTATTVE EXPERTMENTS aaaaaaooaoaooaoooaoaoaaaoa

Quantitativeexperiments,dealing with the determi nati on of concentrati ons of several biologicallyimportantcompoundsand the assayof many enzymes/are of great significancein the laboratorypractice.Very often,the ultimatediagnosis and prognosisof a large number of diseasesare guidedby the quantitative biochemicalinvestigations. The pri nci pl es i nvol ved i n some of the quantitativeexperiments,commonly employed in the biochemistrylaboratoryby an undergraduate student are briefly describedhere. 1. Blood glucose estimation Thequantitative determination of blood (plasma/ serum)glucoseis of greatimportancein the diagnosis and monitoringof diabetesmellitug. 0

FolinWu methodzAlkalinecopper(cupric ions) is reducedby glucosewhen boiled with protein free blood filtrateto cuprous oxide. The cuprous oxide in turn reacts with phosphomolybdicacid to form blue coloured oxides of molybdenum. The intensityof the colour can be measuredin a colorimeter at a wavelength680 nm. lFolin Wu method is ratherold and is not specific for glucose determination,since other substances(e,g., fructose, lactose, glutathione)also bring about reduction. Consequently the blood glucoselevelwhen estimatedby Folin Wu method is higher i.e., normalfastingis 8O-120mg/dl against true glucose50-100mg/dll

(ii) O-Toluidine method: Glucose combines w i th O-tol ui di ne w hen boi l ed i n aci d mediumto form a greencolouredcomplex which can be measuredin a colorimeterat a w avel ength 630 nm. (Thi s method determinesglucosealone). (iii) Glucose oxidase-peroxidase(GOD-POD) method: Thi s i s an enzvmati c determinationof blood glucose.Clucose gets oxi di zed by gl ucose oxi dase to

768

B IOC H E MIS TF| Y gluconicacid and hydrogenperoxide.The enzyme peroxidaseconverts hydrogen peroxideto water and oxygen.The oxygen in turn reactswith 4-aminophenzonein the presenceof phenol to form a pink coloured complex, the intensityof which can be measuredat 530 nm.

2. Blood urea estimation

Biuret method: Peptidebonds(-CO-NH) of proteinsreactwith cupric ions in alkalinemedium to form a violet colour complexwhich is measured at a wavelength530 nm. Thismethodis suitablefor total serumproteinswith estimation. Bromocresol green (BCG) dye method : This techniqueis employedfor the estimationof serum albumin.BCC dve reactswith albuminto form an intenseblue-green colouredcomplexwhich can be measuredat 628 nm.

Determinationof blood urea (referencerange 10-a0mg/dl)is importantfor the evaluationof kidney (renal)function.Elevationof blood ureais associated 5. Estimationof serum bilirubin with pre-renal(diabeticcoma,thyrotoxicosis), renal The total bilirubin concentrationin serum is (acute glomerulonephritis,polycystic kidney) and 0.2-1 m{dl (conjugated- 0.6 mgldl; unconjugated post-renal(obstructionin the urinary tract, due to me/dl ). E l evati on i n serum bi l i rubi n O.4 tumors,stones)conditions. concentrationis observedin jaundice.Unconjugated Diacetyl monoxime(DAM) method: Ureawhen bi l i rubi n i s i ncreasedi n hemol yti c j aundi ce, heated with diacetyl monoxime forms a yellow conjugatedbilirubin in obstructive jaundice,while colouredcomplexof dioximederivatives which can both of them are increasedin hepaticjaundice. be measuredat 520 nm. van den Bergh reaction : Serum bilirubin 3. Serum creatinine estimation estimationis basedon van den Berghreaction.The Estimationof serumcreatinine(referencerange principleof the reactionis that diazotisedsulfanilic (formedby mixingequalvolumesof sulfanilic 0.5-1.5 mg/dl) is usedas a diagnostictest to assess acid in acid HCI and sodiumnitrite)reactswith bilirubin kidney function.Serumcreatinineis not influenced to form a purplecolouredazobilirubinwhich can by endogenousand exogenousfactors,as is the case be measured at 540 nm. wit h ur e a . H e n c e , s o m e w o rk e rs consi der serumcreatinineas a more reliableindicatorof renal function. Alkaline picrate methodt This method is based on Jaffe'sreaction.Creatininereactswith alkaline picrate to form creatinine picrate, an orange red coloured complex, which can be measuredin a colorimeterat 530 nm. ( Note : U ri n a ry c re a ti n i n e c a n al so be determinedby employingthe sameprinciple given above). 4. Determination of serum proteins

6. Estimation of serum cholesterol Serumcholesterolconcentration(referencerange diabetes 150-225mg/dl)is elevatedin atherosclerosis, jaundiceand hypothyroidism. mellitus,obstructive Decreasedlevelsare observedin hyperthyroidism. Acetic anhydride method: Serum cholesterol of glacial reactswith aceticanhydridein the presence aceticacid and concentratedH2SOato form a green colouredcomplex.Intensityof thiscolouris measured at 560 nm. 7. tstimation of serum uric acid

The normalconcentration of totalserumproteins is in the range 6-8 g/dl (albumin 3.5-5.0 g/dl; globulin s2 .5 -3 .5{ d l ; N C ra ti o i s 1 .2 to 1.5 : 1). The A/G ratio is loweredeitherdue to a decreasein albuminor an increasein globulins.

U ri c aci d i s the end product of puri ne metabolism.lts concentrationin serumis increased (referencerange- men 4-8 mg/dl; women 3-6 mg/ dl) in gout.

Serumalbumin concentrationis decreasedin liver diseases,severe protein malnutrition,and excretionof albuminin urine(dueto renaldamage). Serumglobulin concentrationis elevatedin chronic infectionsand multiplemyeloma.

Henry-Caraway's method : Uric acid in the protei n-free f i l trate w hen treated w i th phosphotungstic acid in the presenceof sodium carbonate(alkalinesolution)givesa blue coloured complexwhich can be measuredat 660 nm.

Appendix V : PRACTICALBIOCHEMISTHY-PRINCIPLES

769

The keto acid (pyruvateor oxaloacetate),formed i n the above reacti on, w hen treated w i th Serumcalcium level is elevated(referencerange 2, 4-dinitrophenylhydrazine forms dinitrophenyl and decreased hydroazone(brown colour) in alkaline medium 9-11 mg/dl) in hyperparathyroidism in hypothyroidism. which can be measuredat 505 nm. B. Estimation of serum calcium

O-Cresolphthalein complexone method : 11. Determination of serum alkaline Calcium reactswith the dye, O-cresolphthalein phosphatase complexone(CPC)in alkaline solution to form a complex which can be measuredat a wavelength The activity of the enzyme serum alkaline phosphatase(normal range 3-13 KA Units/dl) is 660 nm. elevatedin ricketsand obstructivejaundice. 9. Estimationof serum phosphorus (inorganic) Principle of assay: Alkaline phosphatase Serumphosphate(referencerange 3-a.5 mddl) hydrolysesdisodium phenylphosphateliberating is increasedin hypoparathyroidism,and decreased phenol. On treatment with -amino antipyrine in and renal rickets. in hyperparathyroidism alkalinemedium,phenolgivesferricyanide(reddish Forthe determinationof serumphosphate,serum colour) which can be measuredat 520 nm. proteinsare precipitatedby trichloroaceticacid.The protein-freefiltrate containing inorganic phosphate is reacted with molybdic acid reagent to form phosphomolybdate. The latter in turn is reducedto molybdenum blue by treatmentwith l-amino 2naphthol-4sulfonic acid (ANSA).The intensityof the blue colour is measuredat 689 nm. 10. Determination of SGPT and SGOT (SGPT; Serumglutamatepyruvatetransaminase alanine t ra n s a m i n a s e )a n d s e ru m g lutamate ox aloac e ta te tra n s a m i n a s e(S G OT ; a spartate transaminase) aretwo importantdiagnosticenzymes. SGPT activity (referencerange 5-40 IUA) is more specificallyincreasedin Iiver diseases(hepatic jaundice). SGOT activity is elevated (reference range 5-45 lUlL) in heart diseases(myocardial infarction). Principleof assay:SGPTcatalysesthe following reaction L-Alanine + c-ketoglutarate-----+ L-glutamate+ pyruvate SGOT bringsabout the following reaction L-Asparticacid + cr-ketoglutarate -----+ L-glutamate+ oxaloacetate

12. Determination of serum amylase Serumamylaseactivity is increased(reference range80-180 SomogyiUnits/dl)in acutepancreatitis. Principle of assay:Amylase acts on starchand hydrolysesto dextrinsand maltose.Starchforms blue coloured complex with iodine, a decreasein the colour (measuredat 670 nm) is proportionalto the activity of amylase. i3. Analysis of cerebrospinalfluid Cerebrospinal fluid (CSF)is the aqueousmedium surroundingthe brain and spinal cord. From the biochemicalperspective, estimationof proteinsand glucose in CSF is important. lncreasein protein (reference range 15-a0 mg/dl) and decrease in glucose (referencerange 50-75 mg/dl) in the cerebrospinalfluid are observedin tuberculosis meningitis. CSFprotein estimation: Sulfosalicylicacid (in sodium sulfate solution) precipitatesCSF proteins and the turbidity is measuredat 680 nm. aSFgluco* estimationz Any one of the standard methodsemployed for the determinationof blood glucose (alreadydescribed)can be used for CSF glucoseestimation.

The ultimate application of the biochemistry subject is for the health and welfare of mankind. Clinic al bio c h e mi s try(a l s o k n o w n a s cl i ni cal chemistryor chemicalpathology) is the laboratory serviceabsolutelyessentialfor medicalpractice.The carriedout resultsof the biochemicalinvestigations in a clinical chemistrylaboratorywill help the (diagnosis) and cliniciansto determinethe diseases from the for follow-up of the treatment/recovery hold Biochemicalinvestigations illness(prognosis). the key for the diagnosisand prognosisof diabetes m ellit us ,ja u n d i c e , m y o c a rd i a li n fa rc ti o n ,gout, pancreatitis,rickets,cancers,acid-baseimbalance etc. Successfulmedical practiceis unimaginable wit hout t h e s e rv i c e o f c l i n i c a l b i o c h e mi strv laboratory. fhe biological fluids employed in the clinical biochemistry laboratory include blood, urine, cerebrospinalfluid and pleuralfluid. Among these, blood (directlyor in the form of plasmaor serum)is in the clinical frequentlyusedfor the investigations biochemistrylaboratory.

CHOICE OF BLOOD SPECIMENS Biochemicalinvestigationscan be performed blood,plasma, on 4 typesof blood specimens-whole serum and red blood cells. The selectionof the specimendependson the parameterto be estimated. Whole blood (usuallymixed with an anticoagulant) i s used for the esti mati on of hemogl obi n, pH, glucose,urea,non-protein carboxyhemoglobin, pyruvate, ammoniaetc. (Note : for lactate, nitrogen, plasmaispreferedin recentyears). glucosedetermination, Plasma,obtained by centrifugingthe whole blood collectedwith an anticoagulant,is employed glucose,bicarbonate, for the parameters-fibrinogen, acid etc. ascorbic chloride, Serum is the supernatantfluid that can be collectedafter centrifugingthe clotted blood. lt is the most frequentlyused specimenin the clinical biochemistrylaboratory.The parametersestimated i n serum i ncl ude protei ns (al bumi n/S l obul i ns), creati ni ne, bi l i rubi n, chol esterol , uri c aci d, electroylets(Na+,K+,Cl-), enzymes(ALT,AST,LDH, CK, ALP,ACP,amylase,Iipase)and vitamins. R ed bl ood cel l s are empl oyed for the determinationof abnormal hemoglobins,glucose pyruvatekinaseetc. 6-phosphatedehydrogenase,

Venous blood is most commonly used for a lt can be majority of biochemicalinvestigations. drawn from any prominentvein (usuallyfrom a vein on the front of the elbow). Capillary blood (<0.2 ml) obtainedfrom a fingeror thumb, is less f requentlyemployed. Arterialblood (usuaIly d rawn under loc a l a n e s th e s i ai)s u s e d fo r b l o od gas determinations.

ANTICOAGULANTS

Certain biochemicaltests require unclotted blood. Anticoagulantsare employedfor collecting such specimens. Heparirr: Heparin(inhibitsthe conversionof prothrombinto thrombin)is an ideal anticoagulant, si nce i t does not cause any change i n bl ood composition. However, other anticoagulantsare for bloodcollection: Useof sterile preferedto heparin, due to the cost factor. Precauiicns ( pr ef er abl yd i s p o s a b l e )n e e d l e s a n d s yri nges, Potassiilm or sodiumoxalate: Thesecompounds cleaningof patientsskin,blood collectionin clean precipitatecalcium and inhibit blood coagulation. and dry vials/tubesare some of the important Beingmoresoluble,potassium oxalate(5-10mg per precautions. 5 ml blood) is prefered. 77l|

I

Appendix Vl r CLINICAL BIOCHEMISTRYLABORATOFIY

771

3. Dynamicfunction tests: Thesetestsare designed Potasiumoxalateand sodium tluoride : These to measurethe body'sresponseto externalstimulus anticoagulants are employedfor collectingblood to estimateglucose. Furthersodium fluoride inhibits e.9., oral glucosetolerancetest (to assessglucose glycolysisand preservesblood glucoseconcentration. homeostasis) : bromosulphthalein test(to assessliver function). potassium oxalate : A oxalate and Ammonium mixtureof thesetwo compoundsin the ratio 3 : 2 is used for blood collection to carry out certain hematologicaltests. Enthylenediaminetetraceticacid (EDTA) : lt chelateswith calciumand blockscoagulation.EDTA is employed to collect blood for hematological examinations.

4 Screeningtests : These tests are commonly employedto identifythe inbornerrorsof metabolism, and to check the entry of toxic agents(pesticides, lead, mercury)into the body.

HEMOLYSIS

The term emergencyfesfs is frequently used in the clinical laboratory.lt refersto the teststo be performedimmediatelyto help the clinician for propertreatmentof the patiente.g., blood glucose, urea, serumelectrolytes.

the cellular The ruptureor lysisof RBC,releasing c ons t it u e n ts i n te rfe re s w i th th e l aboratory careshouldbe taken investigations. Therefore,.utrnost to avoid hemolysiswhen plasmaor serumare used for biochemicaltests.Use of dry syringes,needles and containers,allowing slow flow of blood into syringeare amongthe importantprecautionsto avoid hemolysis.

5. Metabolic work-up tests : The programmed intensiveinvestigationscarried out to identify the endocrinological disorderscome underthis category.

Urine,containingthe metabolicwasteproducts of the body in water is the most important excretory fluid. For biochemicalinvestigations, urine can be Plasmaor serumshould be separatedwithin 2 hoursafterblood collection.lt is idealand advisable colfectedasa singlespecimenor for 24 hours.Single to analyseblood,plasmaor serum,immediatelyafter speci mensof uri ne, normal l y col l ected i n the the specimencollection.This however,may not be morning,are usefulfor qualitativetestse.9., sugar, alwayspossible.In sucha case,the samples(usually proteins.Twenty four hour urine collections(done plasmalserum) can be storedat 4oC until analysed. between8 AM to 8 AM) areemployedfor quantitative estimation of certain urinary constituentse.9., For enzymeanalysis,the sampleare preservedat proteins,hormones,metabolites. 20"c.

PRESERVATION OF BLOOD SPECIiIEITS

Preservatives for urine : For the collection of 24 hr urine samples,preservatives have to be used or else urine undergoeschangesdue to bacterial action.Hydrochloricacid,toluene,light petroleum, thymol, (on formalin etc., are among the common blood/ The biochemical investigations used. plasma/serum) carriedout in the clinicalbiochemistry preservatives laboratory may be grouped into different types. 1. Diseretionaryor on-off tests r Most common clinicalbiochemistry teststhat aredesignedto answer specific questions.e.9., does the patient have CSFis a fluid of the nervoussystem.lt is formed inc r eas e d b l o o d u re a /g l u c o s ec o n c entrati on? by a processof selectivedialysisof plasmaby the Normally,thesetesbareusefulto supportthe diagnosis. choroid plexusesof the ventriclesof the brain. The 2. Biochemicalprofiles : Thesetestsare basedon total volumeCSFis 100-200ml. the fact that more usefulinformationon the patients Collection of CSF ; CSF is collected by diseasestatuscan be obtained by analysingmore constituentsratherthan one e.g., plasmaelectrolytes puncturingthe interspacebetweenthe 3rd and the (Na+,(+, Cf-, bicarbonate,urea); liven function tests 5th lumbar vertebrae,under asepticconditionsand (serumbilirubin,ALT,AST). local anesthesia.

772

BIOCHEMISTRY

Biochemical investigations on CSF : Protein, laboratory the resultsshould u"ry *itftin a narrow gluc os e a n d c h l o ri d e e s ti ma ti o n s are range. usually performed in the clinical biochemistry Externalquality control deals with the analysis laboratory. of a sample received from outside, usually from a nationalor regionalquality coneol centre.The resultsobtained are then compared. Q uality c o n tro l i n c l i n i c a l b i o c h emi stry laboratory refers to the reliability of investigative service.Any error in the laboratory will jeopardize the lives of patients.lt is thereforeutmost important that the laboratoryerrorsare identifiedand rectified. Quality control comprises of four interrelated factors namely precision, accuracy, specificity and sensitivity. Precisionrefersto the reproducibilityof the result when the same sample is analysed on different occasions(replicate measurements)by the same person.For instance,the precision is good, if the blood glucose level is 78, 80 and 82 mg/dl on replicates. Accuracy meansthe closenessof the estimated resultto the true value e.9., if true blood urea level is 50 mg/df,"the laboratoryreporting45 mg/dl is more accuratethan the one reporting 35 mg/dl. Specificity refersto the ability of the analytical method to specifically determine a particular parametere.g.,glucosecan be specificallyestimated by enzymatic glucoseoxidase method. Sensitivitydealswith the ability of a particular method to detect small amounts of the measured constituent. METHODS OF OUALITY COilTROL Intemal quality control refersto the analysisof the same pooled sample on different days in a

The heavywork load in the clinicalbiochemistry laboratoryhas leadto the discoveryof autoanalysers. These modern equipment are useful to analyse hundredsof samplesin a shorttime. Singlechannel based and multi-channelmachines(autoanalysers) on the principles of either continuous or discrete analysisare availableon the market.

A s al ready stated, cl i ni cal bi ochemi stry laboratoryis a service-orientedestablishmentfor the benefit of patient health care. The reader may refer toofs of biochemistry Ghapter 4ll and principlesof practicaf biochemistry (Appendix-rll for a brief knowledge on the principles of some of the equipment used and the laboratory investigations employed. The details on the biochemistry of health and diseasestatesin relationto the normal and abnormal biochemical data are described in the text of this book. For ready reference, the most common referencebiochemicalvaluesaregivenon the inside of back cover.

t'

Index

A

il

# # rft '* s, ,&, .d f l

it

{-site, 558 {basic sites,535 Abetalipoproteinemia, 521 Abnormalhemoglobins, 202 Abzymes,S8 Acetaldehyde,327 Acetanilide,640 Acetohexamide,682 Acetazolamide,477 Acetic acid, 640, 642 Acetoacetate,293 Acetone,293 Acetylcholine, 149 Acetylcholineesterase,94 AcetylCoA, 149, 242, 252,287, 297, 309, 381, 642 AcetylCoA carboxylase, 147,298 N-Acetylcysteine, 51 N-Acetyfglucosamine,24, 28'l N-Acetylglutam ate, 337, 369 N-Acetylneuraminic acid, 6, 18, 281 Achlorhydria,179 Achrodextrin,21 Acid(s),474, 709 Acid-basebalance,474 Acid-basecatalysis, 99 Acid-basedisorders,474 Acidemia,480 {cidic amino acids,47 \cid maltase,266 \c d number,34 \c cc=rs -lB0 {. c a.osphatase, 1O7,

Aconitase,256 Acquired porphyria,2"1 4 Acrodermaticisenteropathica,463 Acromegaly,434 ACTH (adrenocorticotropic hormone),435, 678 Actin, 4O4,492 Actinomycin D, 549 Active iodine, 438 Active methionine (seeS-adenosylmethionine) Active site, 91 Active transport,652 Acute glomerulonephritis, 341 Acute intermittentporphyria,212 Acute pancreatitis,1O7,179 Acute phaseprotiens,186 Acute porphyria,214 Acyladenylate, 288 Acyl carrierprotein,298 Acyl CoA, 149,288 Acyl CoA dehydrogenase, 289 Acyl CoA synthetase,288 Acylglycerols,32 Adaptiveenzymes,104 Addison'sdisease,411, 444 Adeni n e ,7 0 , 3 9 1 Adenohypophysis, 431 Adenosine,72,394 Adenosinedeaminase, 397 Adenosinedeaminasedeficiency, 39 7 , 6 2 8 Adenosinediphosphate,72 Adenosinemonophosphate, 72, 390 Adenosinetriphosphate, 72, 223, 231, 249, 290, 430 S-Adenosylhomocysteine, 359 773

S-Adenosylmethionine, 97, 343, 359, 375, 64'l Adenylatecyclase,267,268, 430 Adrenal cortical hormones,441 Adrenal cortex, 441 Adrenaline(seeepinephrine) Adrenal medulla,444 Adrenocorticosteroids, 441 Adrenocorticotropichormone (seeACTH) Adsorption,7'16,722 Aerobic dehydrogenases, 235 Aerobic glycolysis,245 Affinity chromatography,586, 724 Aflatoxins,667 .l A/C ratio, 83, 458 Agarosegel electrophoresis, 588 A g e i n g ,7 , 6 5 8 Aglycone, 17 Agmatine,368 AIDS,595 Air pollution,663 Alanine,45, 371 p-Alanine,52, 371, 40O Alaninetransaminase (alanineaminotransferase), I 08, 1 1 2 , 1 4 4 , 4 5 5 ,4 s 7 ALA synthase, 210 Albinism,353, 549 A l b u m i n ,1 8 3 , 4 5 8 Albumin/globulin ratio, 185, 458 .185 Albuminuria, Alcohol, 179, 263, 327, 394, 639, 679 Alcohol dehydrogenase, 112, 327 Alcoholism,'1O7,179, 322, 328 Alcoholtest meal,464 Alditols,18

BIOCHEMISTF|Y

774 Aldohexoses,10 246, 278 Aldolase,'1O7, AldolaseB, 278, 280 Aldopentoses,13 Aldose,10, 12 Aldosterone,314, 41'', 442, 472 Aldotetroses,13 Aldotriose,13 681 Alimentaryglycosuria, Alkalemia,480 1'l'1, Afkalinephosphatase,'107, 't't2, 127, 408, 4s6 Alkalies,709 Alkalinetide, 463 Alkali reserve,476 Alkalo ids , 6l Alkalosis,480 Alkapton, 352 352 Alkaptonuria, Allantoicacid,394 Allantoin,394 Allergy,189 Allogralt, zzo Allo pu rinol,94, 396 Allostericenzymes,101 Allostericeffectors,101, 200 Allostericregulation,95, 1OO,266 Allostericsite(s),101 Alloxanthine,396 Alpha fetoprotein,691 Alport syndrome,489 534 Alu sequences, Alzheimer'sdisease,495, 561 cl-Amanitin,549 Ames assay,686 152 Amethopterin, Amino acid(s) absorption,171 activation,554 as drugs,51 biosynthesis,324 chemical reactions,50, 6l classification,44 decarboxylation,96, 375 48 essential, glycogenic,48,372 inbornerrors,374,376 ketogenic,48,373 metabolism,330 n on - pr ot ein, 51 non-standard,5l optical isomers,44 properties,49

protein biosynthesis,553 standard,44 structures,44 titration, 50 Amino acid pool, 330 D-Amino acid oxidase,334 L-Aminoacid oxidase,334 Aminoacyl IRNA, 554 Aminobenzene,640 p-Aminobenzoic acid, 150, 159, 390 1-Aminobutyrate,1 44, 369 p-Aminohippuric acid, 461 p-Aminoisobutyricacid, 400 a-Amino p-ketoadipate,211 &Amino levulinicacid, 145,21O p-Aminophenol, 639 Aminopeptidase,170 Aminopterin,94, 152 Amino sugars,''8, 28O Aminotransferases (seetransaminases) Ammonia,335 Ammoniumions,335, 478 Ammonium sulfate,60 Ammoniotelic,336 Amnioticfluid, 498 AMP (seeadenosine monophosphate) Amphibolism,241 Amphipathiclipids,39, 650 Amphipathicpathways,257 Ampholytes,709 Amylase,2'1, 1O7, 166, '179 Amylodextrin,21 Amyloidosis,189, 495 Amyloid proteins,495 Amylose,20 Amylopectin,20 Amytol, 227 Anabolism,242 236 Anaerobicdehydrogenases, Anaerobic glycolysis, 249 Anaphylaxis,648 Anaplerosis, 257 Anderson'sdisease,269 Androgens,446 Anemia, 2O1, 416 Aneuploidy,741 Angiotensin,66 472 Angiotensinogen, Aniline,639

Animal starch,21 Anion gap, 480 Annealing,594 Anomers,l4 Anorexia nervosa,326 Anterior pituitary,432 Antibodies,187, 729 Antibodies,monoclonal,730 Antibody enzymes,88 Anticoagulants,191 Anticodon,552 Antidiuretic hormone,437, 469, 472, 669 Anti egg-whiteinjury factor, 146 Antifreezeglycoproteins,25 Antigen,729, 733 Antigentherapy,631 Anti-inflammatorydrugs, 396 Antimetabolites,92 AntimycinA, 227, 232 Antioncogenes,690 Antioxidant(s),33, 128, 132, 274, 394, s10, 658 Antioxidantenzvmes,659 Antioxidantsystem,659 Antioxidantvitamins,659 anemiavitamin, Antipernicious 't 5 2 Antiport system,653 Antiproteinase,185 Antisensetherapy,631 Antisterilityvitamin, 128 Antithyroiddrugs,438 183, 185 c,-Antitrypsin, 93, 159 Antivitamins, Apoenzyme,ST Apoferritin,415 318 Apolipoproteins(apoproteins), Apoptosis,8, 691 Apurinicsites,535 Aqueoushumor, 499 Arabinosvladenine,73 Arabinosylcytosine,73 Arachidonicacid, 31, 5O9,645 356 Argentaffinomas, Arginase,339 Arginine,48, 336, 339, 339 Arginosuccinase, 337 Arginosuccinate, aciduria,340 Arginosuccinic Aromaticaminoaciddecarboxvlase, 350 Aromaticamino acids,47,345

INDEX Arsenate,246 Arsenite,256 Arthritis,7, 352, 394, 647 Ascorbicacid, 18, '132,275, 4'14 Asparaginase,'106, 371 Asparagine, 46,370 Aspartame,67 (aspartate Aspartatetransaminase aminotransferase), 107, 112, 't44, 455 Aspartatetranscarbamoylase, 102, 399 Asparticacid (aspartate), 46, 337, 370 Aspirin, 640, 646 Asymmetriccarbon, 44, 706 Atherosclerosis, 316, 326 ATP (seeadenosinetriphosphate) ATP-ADP cycle, 223 ATPase,230 ATP synthase,230 Atractyloside,234 Atrial natriureticpeplides,472 Atropine,640 Augmentationhistaminetest, 464 Autoantibodies,736 Autograft,736 Autoimmunediseases, 736 Autoimmunity,736 AutomatedDNA sequencer,591 Autoradiography,589, 7'lB Autosomaldominant inheritance,739 Autosomalrecessiveinheritance, 740 Autosomes,739 Avidin, 146, 667 8-Azaguanine, 73 Azathioprine,73 Azotemia,341 Azure-A-resin,465

Bacteriophages, 583 Bad cholesterol,316 Balanceddiet, 514 Barbiturates, 641 Barfoed'stest, 16 Basalacid output, 464

775 Basalmetabolicrate, 504 Basalmetabolism, 504 Bas e( s ) ,4 7 4 , 7 O 9 Baseexcisionrepair,537 Basicamino acids,47 Beer-Lambert law, 726 proteins,189 Bence-Jones Benedict'stest, 16 Benedict-Rothapparatus,504 Bent DNA, 76 Benzaldehyde,639 Benzene,639 Benz oi ca c i d , 3 4 2 , 6 3 9 Benzylalcohol,639 Bergstromtheory, 175 Beri-beri,136 Bet aine , 3 2 4 Bicarbonatebutfer,475 Bifunctionalenzyme(s),25O, 265, 398 Biguanides, 682 Bile, 173 Bile acids,173, 313 Bile pigments,214, 454 Bile safts,173, 313 Bilirubin,215, 454t 641 Bilirubindiglucuronide, 216, 641 Bilirubin glucuronyltransferase, 2"16 Bilirubinmetabolism, 217 Biliv er d i n2, 1 4 Biliverdinreductase, 215 Bindingchangemodel,231 Biocatalyst,85 Biocytin,146 Bioenergetics, 221 Bioflavonoids, 159 Biogenicamines,375, 667 Bioinformatics,634 Biologicaldatabases,636 Biologicaloxidation,221 Biologicalvalueof proteins,512 Biomembranes, 650 Biopterin,345, 356 Biotin,146, 259, 292, 298 Biotransformation, 638 glycerate, 247 1,3-Bisphospho 2,3-Bisphosphoglycerate, 2OO,251 Bisphosphoglycerate mutase,251 Biuretreaction,61 Bitot'sspots, 123 Blood buffers,475 Blood clotting,190

Blood gas analysis,484 Bloodgroup antigens,26 Blood pH, 474 Blood urea nitrogen,341 Blottingtechniques,5BZ B-Lymphocytes, 733 Body fluids,496 BMR (seebasal metabolicrate), Body massindex, 325 Bohr effect, 199 Bone marrow cells Bovinespongiform encephalopathy, 495 Bowman'scapsule,459 Bradshawstest, 189 Bradykinin,6T Branchedchain amino acids,45, 363 Branchedchain a,-ketoacid dehydrogenase, 135, 365 Branchedchain ketonuria,365 Brig's-Haldane's constant/88 Britishantilewisile, 227, 642 Broad beta disease,321 Bromosulphthalein test, 455 Bronzediabetes,416 Brown adiposetissue,233, 326 Brownian movement,712 Buffering capacity, 7'l'l Bufters,475,710 Buffersystemsof blood, 475 Burkitt'slymphoma,688 Burningfeet syndrome,150 Butyric acid, 30

CAAT box, 547 Cachexia,326 Cadaverine,638 Cadmium,403 Caffeine,71, C a l c i d i o l ,1 2 5 Calciol,125 Calcitonin,407 Calcitriol,125, 407, 459 Calcium,125, 268, 404 Calciumbindingprotein,125 Calmodulin,268, 4O5

776 Caloric requirements(seeenergy requirements) Calorie,503 Calorificvalue of foodstuffs,503 Calorigenicaction,505 cAMP (seecyclic AMP) Cancer,539, 685 antioncogenes, 690 carcinogenesis,hypothesis, 691 chemicalcarcinogens, 686 environmentalfacto,rs,686 etiology,685 free radicals,658 glycolysis, 249 incidence,685 malignant,685 metastasis, 685, 693 molecularbasis,682 oncogenes,687 phototherapy,208 suppressorgenes,690 tumor markers,691 viruses,687 Candidatehormones,458 Carbamoylaspartate,102 Carbamoylphosphatesynthetasel, 33 7 Carbamoylphosphatesynthetasell, 39 8 Carbidopa,351 Carbohydrate(s), chemistry,9 classification, 9 definition,9 digestionand absorption,166 functions,9, 507 metabolism,244 nutritionalimportance,506 Carbondioxide in pH regulation, 479 Carbondioxidetransport,199 Carbonicacid, 199, 476 Carbonicanhydrase, 199, 476 Carbon monoxide,2O2, 227, 232 Carbontetrachloride,323 Carboxybiotin, 147 y-Carboxyglutamate, 130 Carboxyhemoglobin, 2O2 Carboxylation, 147, 389 Carboxypeptidase, 171 Carcinoembryonic antigen,691 Carcinogenesis, 686, 691 Carcinogenicviruses,687 Carcinogens,686

B IOC H E MIS TF| Y Cardiacglycosides, 18 Cardiactroponins,112 Cardiolipin,36, 303 Cardiomegaly, 412 Carnitine,288, 368 288 Carnitineacyltransferase, p-Carotene,119, "123,659 p-Carotenedioxygenase,1l 9 Carotenoids, 122, 659 Casein,60, 65 Catabolism, 241 Catabolitegene activatorprotein, 56 9 Catalase,236, 292, 394, 659 Catalyst,85 CatalyticRNAs,105 Catalyticsite, 92 Cataract,277,658 Catechins, 660 Cat ec h o l3, 4 9 , 4 4 5 , 6 3 9 Catecholamine(sl, 144, 349, 445 445 Catechol-O-methyltransferase, Cathepsin,7 CDa cells,697 CDP (seecytidine diphosphate) CDP-choline, 303 CDP-ethanolamine, 303 CeleraCenomics,619 Cell cycle,530 enzymedistribution,103 structure,3 membrane,550 Cell cycle and cancer,530 Cell-mediated immunity,734 Cellobiose,19, 22 Cellulose,22, 5OB Centraldogma of life, 69, 523 Centrifugation,728 Cephalin,36, 303 Ceramide,36 Cerebrafedema,714 Cerebrocuprein, 417 Cerebrohepatorenal syndrome,293 Cerebronicacid, 37 Cerebrosides, 37, 307 Cerebrospinalfluid, 497 Ceruloplasmin, 109, 186, 415, 41 7 cCMP (seecyclic CMP) Chain isomerism, 705 Chaperones,560 Chaperonin,561

Chargaff'srule, 73, 79 acid, 31 Chaulmoogric Chemicalcarcinogens, 686 Chemical coupling, 229 427 Chemical messengers, Chemicalscore,512 C h e i l o s i s1, 3 8 Chemiosmotichypothesis,229 Chenodeoxycholic acid, 313 ChimericDNA, 579 Chiral,706 Chitin, 22 Chloral,639 Chloramphenicol, 560 Chloride shift,476 Chlorine,413 Chlorophyll,'152,293 Cholecalciferol, 124 17O,45O Cholecystokinin, 314 Cholelithiasis, Cholera, 473 Cholesterol, absorption,176 biomedicalimportance,315 biosynthesis, 309 degradation,3l3 effect of drugs,316 functions,309 nutrition,510 ' structure,38 transport,314 Cholesterolesterase,174 Cholesterolestertransferprotein, 319 Cholesterolstones,178 Cholesteryleste\ 174 Cholestyramine, 316 C h o l i ca c i d , 3 1 3 Choline,34, '156,324 24 Chondroitinsulphates, Christmas disease,193 Christmasfactor, 191 Chromanering, 128 Chromatin,79 720 Chromatography, Chromium, 422 Chromoproteins,65 recombination, 532 Chromosomal Chromosomaltranslocation,688 Chromosomes,6, 79, 528, 688 176, 317 Chylomicrons, 170 Chymotrypsin, "l70 Chymotrypsinogen,

INDEX Circadianrhythms,358 Cirrhosis,322, 458 Cistron,567 Citrate, 254 Citratesynthase,254 Cihic acid cycle, 254, 381 Citrovorumfactor, 151 Citrulline,337, 366 Citrullinemia, 340 Clathrin,32O, 654 Clearancetests,460 Clone,584 Cloning,584 Clofibrate,293, 316 Clotting of blood, 190 Clottingfactors,130, 19O, 369 Clottingtime, 131 CMP (seecytidine monophosphate) Coagulatedproteins, 65 Coagulation,62 Coagulation factors, 191 Coatedpits, 654 Cobafamin,152, 292, 420 cobalt, 152, 420 Cocarboxylase,135 Coding strand,543 Cod on, 55l CoenzymeA, 148 Coenzyme Q, 228 Coenzymes,87,96 Coenzymesof B-complexvitamins, 97 Colchicine.396 Colestipol,316 Colipase,'174 Collagen,64,132, 487 Collagenase, 407 Colloids,711 Colorimeter,726 Colourvision,122 Committedstep,250, 389, 398 Common cold,'i,34 Compactin,312 Competitiveinhibition, 92 Competitiveinhibitors,94 Complementarybases,75 ComplementaryDNA, 550, 5BB Compfementsystem,734 Computationalbiology, 634 Congenitalerythropoietic porphyria,213 Conjugated proteins, 64

777 Conjugation,313, 342, 640 Connectivetissueproteins,407 Constitutiveenzymes,104 Constitutivegenes,567 Contact inhibition, 406, 692 Contaminant,663 Contractileproteins,63, 49O Copper,91, 416 Copper coordinatedcomplex, 61 Coprophagy, 117 Coproporphyrin,208 Coproporphyrinogen, 209 Cori cycle,261 Cori's disease,269 Coronaryheart diseases,3'16, 326, 649 Corpus luteum, 449 Corrin ring, 152 Corticosteroids, 442, 647 Corticosterone,442 Corticotropinreleasinghormone, 431 Cortisol,314, 441 Cortisone,442 Cosmids,583 C-peptide,670 Cosubstrate,96 Cotransportsystems,653 Covalentbonds,58 Covalentmodifications,562 Crabtree effect, 251 Creatine,342 Creatinephosphate,224, 344 Creatinephosphokinase,'1O7, 111, Creatinine,344 Creatinineclearancetest, 461 Creatininecoefficient,344 Creatinuria,344 C-reactiveprotein, 186 Crenation,713 Cretinism,441 Creutzfeldt-Jacob disease,495 Criggler-Najjarsyndrome,2l I Cristae,6, 226 Curcuminoids,650 Curie,717 Cushing'ssyndrome,412, 444 Cutaneoushepatic porphyria,2'13 Cyanide,227, 233,642 Cyanidepoisoning,233 Cyanocobalamin, 153

Cyanogenbromide, 55 Cyclic AMP,250 Cyclic GMP, '121 ,266, 287, 43O Cyclins,530 Cyclooxygenasepathway, 645 Cyclopentanoperhydrophenanthrene, 37 Cystathionine,145, 361 C y s t e i n e4, 6 , 3 6 0 , 6 4 1 Cystic fibrosis,626 Cystine,46, 58,374 Cystine-lysinuria, 361 Cystine reductase,361 Cystinestoragedisease,361 Cystinuria,361 Cystinosis,361 Cytidine,72 Cytidine diphosphate,92 Cytidine monophosphate,22 Cytidine triphosphate,1O2, 3O3., 398 CytochalasinB, 172 Cytochromes, 228, 4'14 Cytochromeoxidase,228 CytochromeP$o,21O, 274, 293, 639 Cytogenetic map, 620 Cytokines,735 Cvtosine,70 Cytoskeleton,I Cytosol,I

D Dansyl chloride, 55 Dark adaptation,121 Davsonand Danielle model, 650 DEAECellulose.723 Deamination,145, 334 Debranchingenzyme,265 Decarboxyfation,1 44, 375 Degeneracy(of geneticcode), 551 Dehydratase,335 Dehydration, 472 Dehydrationin cholera,473 Dehydroascorbicacid, 132 7-Dehydrocholesterol, 124, 310 Dehydroepiandrosterone, 443 Dehydrogenases, 235

B IOC H E MIS TF| Y

778 439 Deiodinase, Denaturation, 61, 78, 90 Dental caries,420 Dental fluorosis,420 72 Deoxyadenosine, 153, 5-Deoxyadenosylcobalamin, 15 5 Deoxyadenylate(dAMP),73 Deoxvcholicacid, 313 Deoxycytidylate(dCMP),73 Deoxyguanylate(dGMP),73 Deoxyhemoglobin,2O1, 204 179 Deoxyribonucleases, Deoxyribonucleic acid (seeDNA) 71 Deoxyribonucleosides, 72, 387, Deoxyribonucleotides, 392 Deoxvribose,71 Deoxysugars,18 Deoxythymidylatephosphate,73 567 Derepression, Dermatansulfate,24 Dermatitis,141 Derivedlipids,29 Derived proteins,65 140 Desamido-NAD", Desaturase, 301 274,342, 454, 638 Detoxification, De ute rium,7 17 De xtrin s,2 l Dextrorotatory,10, 706 Dextrose,12 Diabetesinsipidus,437, 469, 669 Diabetesmellitus blood glucose,669,674 classification,679 hemoglobinArc, 683 insulin-dependent, 679 management,682 metabolicacidosis,481 metabolicchanges,681 non-insulindependent,579 sorbitol pathway,279 Diabetic ketoacidosis,481, 682 Diacetyl monoxime, 340 Diacylglycerol, 303 Diagnexblue, 465 Diagnosticenzymes,106 Diaminobenzidine,730 Diastereomers, 13 Dicarboxylicmonoamino acids,47 Dicumarol,94,'l30 Dictyosomes,6

Didanosine,699 Dideoxynucleotide,590 Difiusion,712 Digestionand absorption of carbohydrates,166 of lipids,1 7 3 of nucleicacids,178 of proteins,169 Dihydrobiopterin,346, 350 Dihydrofolatereductase,"15O,1 52, 575 Dihydroorotase,399 Dihydroorotate,399 Dihydrotestosterone, 446 Dihydroxyacetonephosphate,246 1,25-Dihydroxycholecalciferol, 124, 407 24,25-Dihydroxycholecalc iferol, 125 Diisopropylfluorophosphate, 95, 640 Dimethylallylpyrophosphate, 310 Dimethylselenide,422 Dinitrocresol,233 2,4-Dinitrophenol, 233 Diketo L-gulonicacid, 135 Diodrast,461 Dioxygenases, 236 Dipalmitoyllecithin,36, 7'16 Dipeptidases, 171 glycerol,303 Diphosphatidyl Dipolar ions, 49, 60 Diptheriatoxin, 560 Disaccharidases, 167 Disaccharides, 10, 18 Dissociationconstant,710 Disulfidebonds,58 Disuffiram,95, 327 Diurnalvariations,358 DNA amplification, 594 chips,593 composition,73 damageand repair,534 finger printing, 602 helicase,525 hybridization, 599 isolationand purification,585 libraries,597 ligas e, 5 2 6 methylation,572 polymerase lll, 525 polymerases,527 probes,597

profiling,602 recombination, 532 repair, 537 sequencing,589 structure,73 527 topoisomerases, Wp e s , 7 6 vaccines,510 dna A, 524 D o l i c h o l s3 , 10 Domains,58, 398 Donnanmembraneequilibrium, 7 '1 2 ,7 1 4 DOPA (dihydroxyphenylalanine), 349 Dopamine,349 Dopaquinone,34S Dot-blotting,589 Double helix, 74 Double reciprocalplot, 89 Double-strandbreak repair,539 Douglasbag method, 504 Down's syndrome,741 Du Bois and Du Boisformula,504 Duchennemusculardystrophy, 626 D u l c i t o l ," 1 6 ,2 7 7 Duodenafulcer,179 321 Dyslipidemias, Dystrophin,494

E ECoRl,580 Edman'sreagent,56 Edward'ssyndrome,741 syndrome,489 Ehlers-Danlos Eicosanoids,32, 644 Eicosapentanoic acid, 649 Elaidicacid, 31 Efastins, 64,689 Efectrolyte balance, 47O Electrontransportchain, components,226 inhibitors,232 in prokaryotes,236 organization, 225 228 oxiditivephosphorylation, sitesof ATP synthesis,227 724 Electrophoresis,

779

rtMlur Itr riirr[E.€€El--<

205

illt !iltifljfinr; lra(elns, :fir srurm -::

trogroteins.

182 317

9{G:

584 rifilrrrucnrrrmmr"mr:r rods, 59 iillNlrrnrmmmlc r$.Ji$*ir:.relinked j*.rnuj' orbant assay),729 iactors, 557 rilliirrmnfaarr:r pathway, 245 irffi-\t6'erhof 'l E:'qp'r.secta, 85 ?murs;cationof liPids,173 3n-rsons, 40, 712 i-a-oomers, 13 iloocytosis,172,654 i"dopeptidases,170 :-rdoplasmicreticulum,6 436 Endorphins, derivedreleasing Endothelium factot,367 End productinhibition,102 En ediols15 , Energymetabolism,242 Energyrequirements,506 En ginedr iv ingm odel,231 547, 572 Enhancers, 67, 436, 45O Enkephalin, 248 Enolase, Enolization,15 450 Enteroglucagon, circulation,314 Enterohepatic 171 Enteropeptidase, 171 Enterokinase, Enthalpy , 221 Entropy, 221 Environmentalbiochemistry662 Enzyme(s), active site, 91 adaptive,104 affinity,89 106 analyticalreagents, 86 classification, clinical(diagnostic) importance,166 commission(EC),87 compartmentation,103 constitutive,104 effect of activators,91 effect of pH, 90 effect of substrate,88 effect of temperature,90 86 extracellular, im m obiliz ed,106 104 inductionand repression,

inhibition,allosteric,95, 100 inhibition,competitive,92 94 inhibition,irreversible, inhibition, non-competitive,93 inhibition, reversible,92 S6 intracellular, kinetics,88 K. value,88, 92 mechanismof action,98 monomeric,87 oligomeric,87 specificity,95 substratecomplex, 98 therapeuticagents,105 units,104 Vmax,88, 92 Epidermalgrowth factor,688 bullosa,489 Epidermolysis 12 Epimerases, E p i m e r s1, 2 144, 267, 287, 349, Epinephrine, 444, 678 Epoxide,131 virus,687 Epstein-Barr 124 Ergocalciferol, Ergosterol,3B n, 2 1 Erythrodextri 560 Erythromycin, Erythropoieticprotoporphyria,2 14 Erythropoietin,420, 459 Erythrose,13 amino acids,48, 511 Essential fatty acids,31, 509 Essential Essentialfructosuria,280 pentosuria, 276 Essential 314, 447 Estradiol, Eslriol, 447 314, 442, 446 Estrogens/ E t h a n o l2, 6 3 , 3 2 7 , 6 3 9 36, 303 Ethanolamine, E t h e r e asl u l p h a t e , 4 1 4 Ethicsand human genome,624 Ethylene,360 Eugenics,74l 4, 546 Eukaryotes, Eutrophication,665 Exergonicrcaction,221 Exitsite,558 Exocytosis,654 Exons,548 170 Exopeptidases, enzymes,86 Extracellular fluid, 468 Extracellular

fular matrix, 487 Extracef Extrinsicfactor of Castle,'l 53 Extrinsicpathway (of blood 191 coagulation), Ex vivo gene therapy, 627

F Fab fragment,187 Facilitated diffusion,651 FAD, '.t37,139, 225, 252, 335 626 Familialhypercholesterolemia, Farber'sdisease,307 310 Farnesylpyrophosphate, Fat(s) chemistry,32 digestionand absoprtion,174 nutrition,509 , 18 F a t s o l u b l ev i t a m i n s 1 Fattyacid(s) activation,288 deficiency,3l elongation,302 31 essential/ 31 isomerism, 29 nomenclature, oxidation,287, 3B'l peroxidation, 33, '128,656 polyunsaturated, 31, 128, 316, 509 saturated,29 s y n t h e s i s1,4 7 , 2 9 7 29,292 unsaturated, Fattyacid synthase,298 Fatty liver, 322 Favism,275 Fc fragment,187 Feedbackinhibition,102, 389, 399 Feedbackregulation,1O2,3'12, 392 Fehling'stest,16 F e r r i t i n4, 1 5 Ferrochelatase,2l0 415 Ferroxidase, 201 Fetalhemoglobin,'197, Fetal lung maturity,498 Fetalmaturity,498 691 0,-Fetoprotein, Fiber,22, 'l68, 316, 508

BIOCHEMISTF|Y

780 Fibrillin 48 , 9 Fibrin ,1 90 Fibrinmonomer,190 190 Fibrinogen, 192, 327 Fibrinolysis, Fibronectin,489 190 Fibrinopeptides, Fibrousproteins,64 Fischerprojections,13 Fischer'sshort hand models,209 Fischer'stemplatetheory,98 Flame photomete\ 727 l69 Flatulence, Flavinadeninedinucleotide (seeFAD) (seeFMN) Flavinmononucleotide 227 Ffa vo pro tein s,' 137, 62 Flocculation, Fluid mosaicmodel,650 421 Fluoridation, Fluoride,248, 42O Fluorine,420 , 257, 420 Fluoroacetate, Fluorometer,T2T 420 Fluorosis, 73 5-Fluorouracil, FMN, 13 7, ',13 9, 227,334 Fola cin ,15 3 Folateconjugase,150 + Folatetrap, 156 Folic acid (folate),150 Folin icacid, 1 51 Folliclestimulatinghormone,434, 449 Follicular phase,449 Food allergy,'172 Forbe'sdisease,269 638 Formaldehyde, Formiminoglutamicacid, 152, 366 557 N-Formylmethionine, Fouchetstest, 455 DNA, 77 Four-siranded Fractional test meal,464 Fragilitytest,713 Frameshift mutations,536, 553 classification, 321 Frederickson's Free energy,221 Freeradicals,66, 422,5'10,655, 68 7 formula,315 Friedewald 278,280 Fructokinase,

Fructosamine, 683 Fructosan,20 Fructose14, 21, 168, 278 26"1 Fructose1,6-bisphosphatase, 246, Fructose1,5-bisphosphate, 261 250 Fructose2,6-bisphosphate, Fructose6-phosphate,246 Fructoseintolerance,280 Fructosuria,essential,280 FSH (seefollicle stimulating hormone) Fucose,26 256 Fumarase, Fumaric acid, 7O6 Fumarate,256, 339 Functionalisomerism,705 Furanose, 14 Furtural,"l7 Futilecycles,268

G Cabapentin,51 C-proteins,430 C-quartets, 77 C-tetraplex,77 CABA shunt,370 Calactitol, 277 307 Calactocerebroside, Calactoflavin, 138 Calactokinase,277 Calactosamine,280 Calactose,'12,276 277 Calactosemia, Calactose1-phosphate uridyltransferase, 277 Calactosetolerancelest, 457 167, 276, 3O8, B-Calactosidase, 568 Calactosuria,277 Calf stones,178, 314 Canciclovir,630 Cangfiosides,37, 3O8 721 Cas-liquidchromatography, Castric function tests,463 Castric HCl, '17O,463 Gastric inhibitory polypeptide,450 Castricjuice, 170 Castriclipase,173

Gastriculcers,179 Castrin, 45O, 463 hormones,67, Castrointestinal 450, 672 tract, 165 Castrointestinal Caucher'sdisease,308 Ceiger counters,717 Celatin,65 725 Cef efectrophoresis, 724 Cel-filtrationchromatography, Cene(s) amplification,574, 688 chip, 600 constitutive,567 571 expression, inducible,567 library, 597 mutations,535 575 rearrangement, regulationof expression,566 therapy,625 625 Cene augmentation, Cene augmentationtheraPy,625 Cene cloning,578 Cene deliveryby viruses,629 Cene family,202 Gene inhibitiontherapy,625 Cene libraries,596 Cene linkagemap, 620 Cene therapy,625 Geneticcode, 551 578, 585 Ceneticengineering, Ceneticimmunization,609 Cenetics,737 Cenome, 542, 619 Cenomic library,596 Cenu valgum,421 31, 706 Ceometricisomerism, 311 Ceranylpyrophosphate, Cerm cell gene therapy,625 principle,716 Cibbs-Thomson Cigantism,434 Cilbert'sdisease,219 682 Clibenclamide, Clobin(s),64, 196, 202 Clobularprotein,64 Clobulins,64, 185 Clomerularfiltration'ate, 459 Clomerulus,459 Clossitis,138 Clucagon,262, 267, 288, 312, 674, 678 C l u c a n ,2 0

781

INDEX 312, 44'1,678 Clucocorticoids, Cfucogenicamino acids,48, 261, 3 72 246 Clucokinase, 147, 258, 38"1, Cluconeogenesis, 675 Clu c onicac id, 16 280 Clucosamine, Clucosan,20 Clucose absorption,168 alaninecycle,262 blood level,244, 674 homeostasis, 674 insulinsecretion,670 244 metabolism, monitor-liver,244 structure,13 tolerancelest,679 uptake by tissues,671 261, 266, Clucose6-phosphatase, 269, 395 246, 266, Clucose6-phosphate, 2 71 Clucose6-phosphate '109,271, 274 dehydrogenase, 245 Clucose transporters, p-Clucosidase, 308 Clucosuria(seeglycosuria) Clucuronicacid, 16, 275, 640 216 B-Glucuronidase, Clu c ov anillin,1B 274, Clutamatedehydrogenase, 334 46, Clutamicacid (glutamate), .l s 0, 333, 365, 369, 479 Glutaminase, 336, 369, 479 Clutamine,336, 369, 398, 479, 641 336 Clutaminesynthetase, '-Clutamyl cycle,66, 172 -aClutamyltranspeptidase, 107, 1 13, 454 I -:a.ic acid,92 - --:: ' ione, 65, 172, 342, 369, 274, 422, - --:-- :-e peroxidase, e d u cta se , 1 3 8 , 2 7 4 ,

:

_-i

-

: '=-

-emnolnhin\

10, 44, 7O7 Glyceraldehyde, Clycemicindex,507 3-phosphate C lyceraldehyde 246, 252 dehydrogenase, Clycerol,32, 259, 287 287 Clycerokinase, shuttle,234 C lycerol-phosphate 29, 34 Clycerophospholipids, Clycine,45, 210, 341, 641 Clycinuria, 344 Clycineoxidase,343 Glycocalyx,650 Clycocholicacid, 173, 313, 641 342 Clycinesynthase, Clycogen,21, 263, 382 C lycogenesis,263 Clycogenin,264 Clycogenicamino acids,48 (seeglucogenicamino acids) 265, 675 Clycogenolysis, 103, Clycogenphosphorylase, 145,265 Glycogenstoragediseases,269 Clycogen synthase,264 Clycolipids,29, 37, 3O7 Clycolysis,245, 381 Clycoproteinhormones,434 Clycoproteins,25, 64, -190 22, 281 Clycosaminoglycans, 166 Clycosidases, Clycosides,17 Clycosidicbonds,17 Clycosphingolipids, 37 Clycosuria,674, 681 hemoglobin,197, Clycosylated 683 562 Clycosylation, Clyoxylatecycle, 28l Clyoxysomes,7, 282 Cmelin'stest,455 Coiter, 418, 440 Coitrogens,44O, 667 6 Colgi apparatus, Conadalhormones,445 Conadotropinreleasinghormone, 431 434 Conadotropins, Cood cholesterol, 316 Cramicidin,67 Cratuitousinducers,568 Cout, 394, 396 Couty arthritis,270, 394

Crave'sdisease,440 CroMh factors,689 Crowth hormone, 433, 678 Crowth hormone releasing hormone,431 Cuanidoacetate, 343 Cuanine,70, 391 C u a n o s i n e7, 2 , 3 9 3 (CDP),391 diphosphate, (CMP),390 monophosphate (CTP),391 triphosphate L-Culonate,275 L-Culonolactone, 276 L-Cufonolactone oxidase,132, 275 Custen,419 Cuthrietest,352 Cyrase,528

H Hagemenfactor, 191 Hair waving,490 Half-maximalvelocity,89 Hapten,729 Haptoglobin,185 Hartnup'sdisease,173, 358 HAT medium,731 Haworth prolections,15 Hay's test, 716 Heat shockprotein,560 Heat stroke,663 Heat syncope,663 Heavymeromyosin, 493 Helicobacter pylori, 179 o-Helix, 56 Helix-loop-helix motil, 574 Helixturn-helixmotif, 573 Hematocrit,182 Heme, 197, 21O,2'14,342, 414 Heme oxygenase, 214 Heme synthase, 210 Hemicellulose, 508 Hemin,210 416 Hemochromatosis, H e m o c u p r e i n4,l 7 H e m o c y a n i n4, 1 7 Hemoglobin(sl abnormal,202 as bufter,476

782 Hemoglobin(s) contd. biochemicalfunctions,197 CO, transport,199 derivatives,202 glycated,683 O, transport,198 oxygendissociation curve,200 structure,196 T and R forms,198 types, 197 HemoglobinA1c,197 HemoglobinC, 206 HemoglobinD, 206 HemoglobinE, 206 HemoglobinH disease,207 Hemoglobinopath ies,203 He molysis, 27 5, 71 4 Hemolyticjaundice,216, 457 He mop hilia 19 , 3 Hemoproteins, 414 Hemosiderin, 4l 5 Hemosiderosis, 416 Hemostasis, 190 Henderson-Hasselbach equation, 4 75 Heparin,24, 19"1 Hepaticjaundice,217, 457 Hepatitis,107 HepatitisB, 609 HepatitisB vaccine, 609 Hepatocuprein, 417 Hepatoflavin, 137 Hepatolenticular degeneration, 417 Heptose,11 Hereditarycoproporphyria, 214 Hereditaryfructoseintolerance, 280 Heredity,738 Her'sdisease,269 Heterocyclicrings, 704 Heteroduplex DNA, 533 Heterogeneous nuclearRNA, 81, 54 7 Heteropol,vsaccharides, 10, 20, 22 Hexosemonophosphate shunt, 270, 381 Hexokina se24 , 6,2 50 Hexoses,11 HCPRTase, 391,396 High densitylipoproteins (seeFIDL) High-energy bonds,223 High-energy compounds,222

B IOC H E MIS TF| Y phosphates, High-energy 223 High performance liquid chromatography, 624 Hippuric acid, 342, 458 Hir udin,599 Histamine,144, 366, 377, 464 Histaminestimulationtesl,464 Histidase, 335, 366 Histidine,46, 366 Histidinemia, 366 Histoneacetylation,571 Histones,64, 79 HIV (humanimmunodeficiency virus),695 HM C CoA, 29 4 , 3 'l O '102,312 HMC CoA reductase, Hognessbox,546 Hollander'stest,465 Hollidaym ode l ,5 3 2 Holoenzyme,87 Homeostasis of plasmacalcium, 407 Homeostasisof blood glucose,674 Homocyclic rings,7O3 Homocysteine, 154, 360 Homocysteine methyltransferase, 156 Homocystinuria(s), 362 Homogentisate, 346, 352 Homogentisate oxidase,352 Homologousrecombination, 532 Homopolysaccharides, 10, 20 Homoserine,52, '145 Hoogsteenhydrogenbonds, 76 Hopkins-Cole test,61 Hormone(s),427 adrenalcortex,441 adrenalmedulla,444 anterior pituitary,432 c las s if ic a t i o n , 4 2 T gastrointestinal,449 gonads,445 hypothalamic, 431 mechanismof action,428 ovarian,446 pancreatic, 670, 674 posteriorpituitary,437 receptors,428 secondmessengers, 430 thyroid, 437 v it am inD, 1 2 7 Hormone sensitivelipase,287 Hos t c ells in cl o n i n g ,5 8 1 Housekeepinggenes,567

583 Human artificialchromosome, 4 Human body composition, Human chorionicgonadotropin, 435 Human genomeproject,619 Human leukocyteantigen,734 Humoralimmunity,733 H u m u l i n ,6 0 8 Hurler'ssyndrome,281 Hunterssyndrome,281 Hyaluronicacid,23 Hyaluronidase, 24 Hybrid enzymes,87 Hybridomatechnology,730 Hydrochloricacid,'170 Hydrogenbonds, 58, 75 Hydrogenion concentration,710 Hydrogenperoxide,236,394, 655 H y d r o l a s e s7,, 8 7 , 1 7 O Hydrolysis,640 236 Hydroperoxidases, acid, Hydroperoxyeicosatetraenoic 648 Hydrophobicbonds,58 Hydropstelalis,207 Hydroxyapatite,4O4, 42O p-Hydroxy bulyrate,294 25-Hydroxycholecalciferol, 125 12B 6-Hydroxychromane, 153 Hydroxycobalamin, S-Hydroxyindole acetate,358 Hydroxylysine, 132 p-Hydroxy B-methyglutaryl CoA, (seeHMC CoA) Hydroxyproline, 132 17-Hydroxysteroids, 444 (seeserotonin) 5-Hydroxytryptamine Hyperammonemia, 336 Hyperargininemia, 366 ipoproteinemia,320 Hyperbetal Hyperbilirubinemic toxic 2 18 encephalopathy, Hypercalcemia, 408 Hypercholesterolemia, 315 413 Hyperchloremia, Hyperglycemia, 669, 674, 681 Hyperhomocysteinemia, 152 412 Hyperkalemia, 269, 321 Hyperlipidemia, 142, 321 Hyperlipoproteinemias, Hypernatremia, 411 Hyperoxaluria,344 408 Hyperparathyroidism,

783

TNOEK Hyperprolinemia,366 Hyperproteinemia, 471 Hypertonicsolution,713 Hyperthyroidism, 440 t lvpertriglyceri demia, 682 - ' cerurlcemia, 27O,394 - .:€'! alrnemia,366 - . ae^.rtaminosis A, 123 - . cen rtaminosis D, 128 -r ' coalbum inem ia, 7' 14 ir pobetalipoproteinemia,321 rlr pocalcemia, 408 Hr pocholesterolemia, 3'l7 Hrp oc hlor em ia, 413 Hypoglycemia, 269, 465, 674, 678 HypoglycinA, 291 Hypo k alem ia, 412 Hypolipoproteinemias, 321 Hyponatremia, 411 Hypoparathyroidism, 408 Hypopigmentation, 353 Hypothyroidism, 316, 441 Hypotonicsolution,713 Hypouricemia, 398 Hypoxanthine, 71, 39'l Hypoxanthi ne-guani ne phosphoribosyltransferase (see HCPRTase) Hypoxia,201 Hypoxia-inducible transcription factor, 250

lnduced fit theory,9E Inducibleenzymes,104 Induciblegenes,567 lnfantile beri-beri,137 Initiationcodons,551 Initiationfactor(s) of DNA synthesis,524 of proteinsynthesis, 555 of transcription,544 Inorganicpollutants,666 l n o s i n e2 , 01,393 Inosinemonophosphate, 389 ln o s i t o f ,1 9 , 3 5 , 1 5 7 Insecticides, 210 Insulin blood glucoseregulation,676 deficiency,679 glucosetransport,673 history,669 mechanismof action,672 metabolic elfects,67'l receptors,672 recombinant, 607 resistance,679 structure,59,670 synthesis,670 Insulinase,6Tl Insulinlike growthfactor,434 Insulintest meal,465 lnterferon,609 IntergenicDNA, 623 lnterleukin,735 Intermediary metabolism, 242 International Union of Biochemistry,86 International Human Cenome Consortium,619 Sequencing Intermediatedensity lipoproteins, 52 |

lcterusindex,454 IDDM (insulindependentdiabetes mellitus),679 lmidazolegroup,46, 146 Imino acids,48 lmmunesystem,733 lmmunity,732 lmmunodeficiency,697 lmmunoelectrophoresis, 725 Immunoglobuli ns , 186, 575 lmmunology,732 lmmunosuppression, 698 IMP (seeinosinemonophosphate) fnbornerrorsof amino acids,376 lnd oler ing,47, 354

Internet,635 lntracellulartluid, 468 Intracellular enzymes,86 Intrinsicfactor,153 Intrinsicpathway,190 Introns,548 lnulin, 21, 461 Inulin clearance, 461 lnvertase,20 Invert sugar,20 lodide,438 In vivo gene therapy, 628 lo d i n e ,4 1 8 , 4 3 8 lodine number,33 lodoacetate,246

lodothyroglobu Iin, 41I lon-exchangechromatography, 723 lonophores, 233 lproniazid,356 lron, "133,4"14 lron-sulfurproteins,227 lsletsof Langerhans, 669 lsoalloxazine, 137 lsocitrate,256 lsocitratedehydrogenase, 256 lsoelectricfocussing,725 lsoelectricpH, 49, 60, 725 fsoenzymes, 109,112 lsohydrictransport,476 lsofeucine,45, 363 lsomaltose, 19 lsomerases, 87 lsomerism,704 lsomers,704 lsoniazid(seeisonicotinicacid hydrazide) lsonicotinicacid hydrazide,146 pyrophosphate, lsopentenyl 311 lsoprene,118 lsoprenoid(s), 118, 228, 31O lsotonicsolution,713 lsotopes,243,717 lsovalericacidemia,366 Isozymes(see isoenzymes)

J chain, 188 J-polypeptide Jacoband Monod operon concept, )o/

291 Jamaicanvomitingsickness, Jaundice,216, 456 Joule,503 J u n kD N A , 6 2 3 Juvenileonsetdiabetes,679

l( K" (dissociation constanto. aclc 475 K - ( M i c h a e l i sc o n s t a l . : 5 c 9 :

784 (dissociationconstantof HrO), 709 Kallid in,6 7 Kallikrein19 , 1 Katal,104 Keratansulfate,25 Keratin,64,490 Keratinization, 123 Keratomalacia, 123 Kernicterus, 218 Keshandisease,422 Keto acid(s),332 a-Keto acid dehydrogenase, 365 Ketoacidosis,296, 481, 682 Ketogenesis, 294, 672 Ketogenicamino acids, 48, 373 d-Ketoglutarate,256, 333, 373 cr-Ketbgl utaratedehydrogenase, 1 35 ,25 2, 2 56 Ketonebodies, 293,385, 481 Ketonemia,296 Ketonuria,296 Ketoses, 10, 12 Ketosis,296 17-Ketosteroids, 444 p-Ketothiolase, 294 Kidneyfunction,340, 344, 459 Kidney functionstests,459 Kidneyhormones,459 Kidney threshold(see rena thresholdsubstances) Killiani-Fischer synthesis, I2 Kjeldahlsmethod,44 Koshlands model,98 Krabbe'sdisease,308 Krebscycle, 254, 291, 372, 38'l Krebs-Henseleil cycle, 337 Kupffercells, 322 Kuru,495 Kwashiorkor, 183, 516 Kynureninase,'l 45,354 Kynurenine, 354 Kynureninepathway,354 (

L Lac operon, 567 Lactam form, 70 Lactase,"167,'169,276

B IOC H E MIS TF| Y Lactate (lactic acid), 248, 259 Lactatedehydrogenase, 109, 248 Lacticacidemia,269 Lactic acidosis,248 Lactim form, 70 Lactoflavin,l3T Lactose,19,276, 567 Lactoseintolerance,169 Lactosesynthase,277 Laggingstrand,525 Laminin,489 Lanosterol, 312 Lathyrism,489, 667 Lauricacid, 30 LCAT(lecithin-cholesterol acyltransferase), 314, 32O LDL (low densitylipoproteins), 129, 317 Lead, 394, 4O3, 666 Leadingstrand,525 Leber'shereditaryoptic myopathy, 232 Lecithin,34, 3O3 Lesch-Nyhan syndrome,391, 395 Leuc ine,45, 3 6 3 , 3 7 3 Leucine zipper, motif, 574 Leucodopachrome, 348 Leukemia,396 Leukoderma,353 Leukotrienes, 648 Leuteinizing hormone,435 Levodopa,350 Levorotatory,10,706 Liebermann-Burchard reaction,38 Ligandin,216 Ligands,724 Ligases,87 Lightchains,187 Light meromyosin, 493 Lignin,508 Liki Lorandfactor, 191 Limeys,| 32 Limit dexkin, 265 plot, 89 Lineweaver-Burk Linoleicac id, 3 1 , 5 0 8 Linolenicac id , 3 1 , 5 0 8 Lipase,173 Lipid(s), amphipathic,39 classification, 28 definition,28 dietary requirements,510

digestionand absorption,173 dynamic state,286 functions,29 membranecomposition,650 metabolism, 285 nutritional importance,509 peroxidation,33 plasmacomposition,286 transport,286 Lipid bilayer,650 Lipid esterase,174 Lipid storagediseases,309 Lipofection,584 Lipofuscin,7 Lipogenesis, 245,672 Lipoicacid, "157,660 Lipolysis,287, 672 Lipoproteins, 38, 65, 317 atherosclerosis, 320 characteristics, 3 17 classification, 317 coronary heart disease,327 electrophoresis, 317 metabolism,3 18 structure,317 Lipoprotein-a(Lp-a),327 Lipoproteinlipase,318 Liposomes,40 Lipotropicfacto(s), 157, 324 p-Lipotropin, 436 Lipoxygenase, 647 Leptin,325 Lipostet,325 Lipoxins,649 Liquidscintillationcounter,717 L i t h o c h o l i ca c i d , 3 1 3 Liver function markers,454 Liver function tests,453 Lobry de bruyn-vonEkenstein transformation, 16 Local hormones,646 Lock and key theory,98 Loop of Henle,459 Long interspersed nuclear elements,622 Lovastatin,3-12,3-16 Low-energyphosphates,222 L u m i r h o d o p s i n1,2 1 L u m i f l a v i n1, 3 7 L u m i r u b i n2, 1 8 Lungsurfactant, 716 Luteal phase,449 Lutein,659

785

INDEX alj Luteinizingutse. Lyases,8i Lycopene,66t1 B-Lymphocrtes733 Lymphoma &rrkitt's. 588 L yo ph ilic:,12 Lyophobic.712 Lysine,46, 368 Lysolecithin,36, 306 Lysosomalstoragediseases,281 Lysosomes,7 Lysozyme,91 Lysyl hydroxylase,132

M Macroglobulins, 105 Macrocyticanemia,152 Macronutrients,403 Macrophages, 214 Mad cow disease,495 Ma gn esium , 410 ity complex, Major histocompatibil 734 Malaria,2O5,275 Malate, 256, 260 Malate-aspartate shuftle,234 260, 298 Mafate dehydrogenase, Maleic acid, 706 Malic enzyme,298 ,Valignantcarcinoidsyndrome, 35 6 vtalnutition,458, 516 \taloflate, 256 ualordialdehvde, 657 v.aorc acid. 93 tra€r'rr Ca\ 289 297 ttatia!€

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--t

rrmrrc fie i:'t

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i4;

Maxam and Cilbert technique,590 Maximal acid output, 464 M-band,189 McArdle'sdisease,269 Megaloblastic anemia,156 Meistercycle,66,'172 Melanin,348, 353 Melanochrome,348 Melanocytes,353 Malanocytestimulatinghormone, 358 Melanosomes,353 Melatonin,358 Melting temperature,78 Membraneantioxidant,659 715 Membranehydrolysis, Membranes,650 Menadione,130 130 Menaquinone, Mendel'sexperiments,737 417 Menke'sdisease, Menopause,449 Menstruafcycle, 499 Meprobamate,639 6-Mercaptopurine,73, 390 Mercapturicacid, 641 Mercury 666 Meselson-Stahl experiment,524 Messenger RNA, 81, 547,554 purification,586 Metabolic acidosis,481 Metabolic alkalosis,482 Metabolic heat, 653 Metabolic integration,380 Metabolic pathways,definition, 24"1 Metabolic probes,243 Metabolic watet, 293, 469 Metabolism,definition, 241 Metabolismin starvation,383 Metabolomics,543 Metalloenzymes, 91 Metalloflavoproteins, 137 Metalloproteins,65 Metanephrine, 445 .\letallothionein,417, 4'19 \letaproteins,65 \tetarhodopsin,121 \tetastasis,685, 693 rtethanol, 539 ttethemoglobin, 2O2,274

Methionine,46, 156, 358 Methionineenkephalin,67 Methioninesulfoxide,185 Methotrexate,152, 39O, 575 Methylation(seetransmethylation) 153 Methylcobalamin, Methylcytosine,71 Methyldopa,351 Methylfolatetrap, 156 Methyl malonicacid, 155 Methyl malonylCoA, 155, 292 N-Methylnicotinamide,140 Ns-MethylTHF, 151, 364 Methyltransferases, 359 Mevalonate,310 Micelles,39, 175 Michaelis-Mentenconstant,88, 92 Microafbuminuria, 684 Microarrays,600 Microbodies,7 Microcytichypochromicanemia, 416 Microminerals, 403 Microsatellites,620 Microsomes,6 Microtubules, 8 Migraine,647 M i l l i e q u i v a l e n t 7s 1 ,1 Millons reaction,61 Milk composition,496 Mineralocorticosteroids, 442 Mineral metabolism,403 Minisatellites, 620 Mismatch repair,538 Missensemutations,536 Mitochondria,6, 225, 563 MitochondrialDNA, 563 Mitochondrialmyopathies,564 Mixed acid-basedisorders,483 Mixed function oxidase,640 Molaliry 711 Molarity,711 Molecularexclusion chromatography,724 Molecularmotor,231 Mofecufarsieve,724 Molisch lest,17 Molybdenosis, 420 Molybdenum,420 Monoacylglycerol,32 Monoamineoxidase,356, 445

786 Monochromaticli8'ht,726 Monoclonalantibodies,731 Monogenicdisorders,738 Monomers,58 Monooxygenases, 236 10, 15, 18 Monosaccharides, Monosodiumglutamate,49 Morphine,436 Motilin, 450 mRNA (seemessengerRNA) mRNA editing,549 Mucoids,22 Mucopolysacch arides,22 Mucopolysaccharidoses, 281 Mucoproteins,22 Multienzymecomplexes,87, 252, 300 Multifunctional enzyme,398 Multiple myeloma,183, 189 Muscleproteins,492 Muscle structure,490 Muscularcontraction,4O4, 493 Musculardystrophy,494 Mutagens,535 Mutarotation,14 Mutation(s),535 Myelomacells,730 M ye lin,35 1 Myocardialinfarction,1O7,"l'12 Myofibril,491 Myoglobin,197, 414 myo-lnositol,35 Myosin,493 Myristicacid, 30 Myxoedema,316t 441

N NAD+, 1 40 , 25 3,290, 354 NADP+,14 O,27 1, 354 NADPH, 142, 274, 297, 334 NarK* ATPase,168, 653 Na'-K*pump, 470, 5o4,652 Naphthoquinones, l30 NationalHuman Genome Researchlnstitute,619 jaundice, Neonatal-physiologic 2 "1 7

B IOC H E MIS TF| Y Neonataltyrosinemia,352 Nephron,459 Nephroticsyndrome,183, 185, 3'16 Nerve gas, 95 Nerve growth factor,690 Net proteinutilization,512 Neurodegenerative diseases,495 Neurohypophysis, 431 Neuroretinaldegeneration,232 Neurotensin, 450 Neurotoxins,667 Neurotransmitter, 356, 37O Niac in,138, 3 5 4 Niacin equivalents, 141 Nicotine,139 Nicotinamide,|40 Nicotinamideadeninedinucleotide (seeNAD+) Nicotinamideadeninedinucleotide phosphate.(see NADP+) Nicotinamidenucleotides, 140, 226 Nicotinicacid (seeniacin) NIDDM (non-insulin dependent diabetesmellitus),679 Niemann-Pick disease,307 Nigercin,233 Night blindness,t 23 Ninhydrinreagent,51 Nitric oxide, 366 Nitric oxide synthase, 366 Nitrocellulose, 589 Nitrocobalamin, 153 Nitrogenbalance,511 Nitrogendioxide, 604 Nitrogenequilibrium,511 Noisepollution,666 Non-competitive inhibition,93 Non-essential amino acids,48 Nonhem eion , 4 1 4 Non-homologous recombination, 533 Non-oxidative deamination, 335 Non-proteinnitrogen,341 Nonsensemutations,537 (seenorepinephrine) Noradrenaline Norepinephrine , 349, 445 Nor m alit y , 7 1 1 Northern blot technique,589 Nucleicacids,69 (seeDNA and RNA)

Nucleolus,6 Nucleoplasm, 6 Nucleoproteins,64 Nucleosidases, 179 Nucleosides, 72 Nucleosome(s), 6, 79 107, 179, 393, 456 Nucleotidase, Nucleotideexcisionrepair,538 Nucleotides,69, 274, 387 Nucleus,5 659 Nutrientantioxidants, Nutrition,502 Nutritionalanemias,517 Nutritionaldisorders,515 Nutritionalimportanceof 506 carbohydrates, lipids,509 proteins,510 23 Nyctalopia(nightblindness),1

o Obesity,325 Obesity and fat absorption,178 Obesity and leptin, 325 Ob gene,325 jaundice,217, 316, Obstructive 457 352 Ochronosis, Oculocutaneous albinism,353 Olestra,178 Okazakipieces,525 Oleic acid, 30 Oligomer,58 Of igomeric enzymes,ST Oligomycin,233 chip, 593 Oligonucleotide 593 Oligonucleotides, Of igosaccharid asest167 10, 169 Oligosaccharides, OMP (orotidinemonophosphate), 399 398 OMP decarboxylase, Oncogenes,687 Oncogenicviruses,55O,687 Oncoproteins,689 Oncotic pressure,713 One-carbonmetabolism,150, 157, 363

tl

787

INDEX One-carbonunits,151, 363, 390 Operon, 567 Opioid pePtides,436 Opsin,1 21 Optical activitY'12 Optical densitY,726 Optical isomerism,706 192 Oral anticoagulant, Oral rehydrationtherapy,168, 473 Orcinol,79 Organ function tests,453 Organicpollutants,665 comPounds,95 OrganophosPhorus Orlistat, 178 Ornithine,336 375 Ornithine decarboxylase, 337 Ornithine transcarbamoYlase, Orotate phosphoribosyltransferase, 399 Orotic acid, 399 Orotic aciduria,400 Osazones,'17 Osmolality, 470 Osmolarity,470 Osmo le,7 13 Osmosis,713 Osmoticdiuresis,714 713 Osmoticpressure, Osteocalcin,130 Osteoblasts,407 Osteoclasts,407 imperfecta,489 Osteogenesis 127 Osteomalacia, 409 Osteopetrosis, 688 Osteosarcoma, 4O9, 449 Osteoporosis, Ou ab ain ,18 Overhydration,473 Oxalic acid (oxalate),93, 132, 344 Oxaloacetate,256 Oxalosis,344 Oxalosuccinate,256 p-Oxalylaminoalanine,667 Oxidases,235 Oxidoreductases,87 Oxidation, 224, 639 o-Oxidation,293 p-Oxidation,287 co-Oxidation,293 Oxidative deamination,334

252, Oxidative decarboxYlation, 364 228, Oxidative PhosPhorYlation' 381 87, 140, 235 Oxidoreductases, Oxygenases,236 curve, 198 Oxygendissociation 198 Oxyhemoglobin, Oxyntic cells,170 Oxythiamine,137 Oxytocin,66, 437 Ozone layer, 664

276 essential, Pentosuria, stimulationtest,464 Pentagastrin Pepsin,170 170 Pepsinogen, Pepticulcers,179 53, 65 Peptide(s), Peptideantibiolics,67 Peptidebond, 53 Peptidylsite (seeP-site) 558 Peptidyltransferase, Peptones,64 136 PeripheralneuroPathY, 174 Peristalsis, anemia,155 Pernicious

P P-site,554 PABA(seep-amino benzoic acid) Paget'sdisease,108 Palindromes,544 Palmiticacid (palmitate),30, 29O, 300 Palmitoleicacid, 30 PalmitoylCoA, 290 Pancreaticfunction tests,465 lipase,174 Pancreatic Pancreatitis,179 Pantoicacid, 149 acid,149 Pantothenic Papain,187 Paperchromatography,720 725 Paperelectrophoresis, PAPS(seephosphoadenosyt phosphosulfate) Parathyroidhormones,124, 407 Par ie t acl e l l s , 1 2 0 disease,350 Parkinson's 720 PartitionchromatograPhY, Passive diffusion,651 Pasteureffect,251 Paulystest, 61 chain PCR(seepolymerase reaction) Pectins,168, 508 Pellagra,138, 141 Pellagrapreventive(PP)factor, 138 famine,51, 146 Penicif Pentosephosphatepathway (see hexosemonophosphateshunt) ?€.-tc€s 11 271

Peroxidase,659 656 Peroxidation, Proxisomebiogenesisdisorders,8 7 Peroxisomes, Pesticides,665 pH,90, 71o Phagocytosis,274 Phenanthrenering, 37 210, 640 Phenobarbitol, Phenol,64.1 P h e n y l a l a n i n4e7, , 3 4 5 346, hydroxylase, Phenylalanine 351, 17 Phenylhydrazine, Phenyl isothiocyanate(seeEdman's reagent) a, 347, 351 Phenylketonuri 445 Pheochromocytoma, P h l o r i z i n1 , 68 Phosphatebuffer,476 acid, 34, 305 Phosphatidic 34, 303 Phosphatidylcholine, 36, 303 Phosphatidylethanolamine, glycerol,303 Phosphatidyl 36, 303 Phosphatidylinositol, 36, 303 Phosphatidylserine, ne phosphosulfate, Phosphoadenosi 362 (seecreatine Phosphocreatine phosphate) bonds, 74 Phosphodiester Phosphoenolpyruvate,247, 259 Phosphoenolpyruvate 260 carboxykinase, 247 Phosphofructokinase, 265 Phosphoglucomutase, Phospholipase(s),36

788

B IOC H E MIS TFIY I

Phospholipids, 28, 34 Phosphoprotein(s), 65 pyrophosphate, Phosphoribosyl 389, 391 PhosphoruS, 409 Phosphorylation, oxidative,224, 228 substratelevel,224 Photoisomerization, 218 Photometry,T26 Photophobia, 353 Phototherapy,2 18 Phrynoderma, 31, 510 Phylogenetics, 596 Phytanicacid,293 Phylloquinone, 130 Phytanicacid a-oxidase,293 Phytate,406,414 Phytol,293 Picric acid, 640 Piercidin A, 227 Pinealgland,358 Pign-pongmodel, 652 Pinocytosis,l 72 Pituitaryhormones,431 pKa (dissociationconstantof acid), 475 PKU (seephenylketonuria) Plasmacell cancer,189 Plasmalogens, 36, 303 Plasmaproteins,182 Plasmatransportproteins,184 Plasmids, 582 Pla smin1, 92 Plasminogen, 192 Platelet-activating factor,303 Plateletaggregation,648 Plateletderived growth factor,690 p-Pleatedsheet,57 P : O ratio,228 Porntmutations,535 Polaramino acids,48 Pollutant,665 Polyacrylamidegel electrophoresis, 7"18 Polyamines, 375 Poly A tail, 548 Polycistronic, 555, 567 Polycythemia, 396 Polyethyleneglycol, Polygenicdisorders,738

Polyglutamate, 150 Polymerasechain reaction,534 Polynucleotides, 73 Polyol pathway, 279 Polypeptides,52 Polyribosomes, 554 Polysaccharides, 1O, 2O, Polyunsaturated fafty acids, 31, 128 , 3 16 , 5 0 9 Polysomes,554 Polyuria,437 Pompe'sdisease,269 Por phobili n o g e n , 2 l 0 Porphyriacutanaetarda,213 Porphyrias,212 Porphyrins,208 Positionisomerism, 705 Post-transcriptional modifications, J+/

Post-translational modifications, 130, 132, 561 Potassium,412, 483 Potassiumin acid-basedisorders, 481 Prealbumin,184 Pregnenolone, 314,446 Preproinsulin,59, 670 Pribnowbox,544 Primaryhyperoxaluria, 344 Primarystructureof protein, 54 Primase,524 Primosome,524 Prion diseases,494 Prions, 494 Proanthocyanidins, 660 Procarcinogens, 686 Proenzymes, 102, 17'l Progesterone, 314, 448 Prohormone,127, 435, 670 Proinsulin,591, 670 Prokaryotes, 4, 543, 557, 563 Prolactin,434 Prolamines,64 Profine, 47, 366 Prolineoxidase,366 Prolyl hydroxylase,132 Pronase,54 Pro-opiomelanocortin, 435 Proparathyroidhormone,407 Propionicacid, 258 Pr opionyC l oA,155,292

PropionylCoA carboxylase,292 Prostacyclins, 644, 648 Prostaglandins, 302, 644 Prostanoic acid, 644 Prostanoids, 644 Prostatecance\ '107, 692 Prostatespecificantigen,113 Prostheticgroup, 63, 87 Protamines,64 Proteaseinhibitors,667 Proteases,170 Protein(s), biologicalvalue,512 biosynthesis, 553 classification,63, 65 denaturation,61 digestionand absorption,I69 functions,43 metabolism, 330 misfolding,494 mutualsupplementation, 513 nitrogenbalance,511 nutritionalimportance,510 nutritivevalue assessment, 512 properties,60 requirements, 513 structure,52, 59 Proteinbuffer,476 ProteinC, 192 Protein-boundiodine, 441 Protein-calorie malnukition,516 Proteinefficiencyratio, 512 Protein-energy malnutrition,516 Proteinengineering,597 Proteinkinase(s),562 ProteinkinaseC, 430 Proteinmisfolding,494 Proteinsorting,563 Proteinsparingaction, 507 Proteintargeting,562 185 Proteinuria, Proteoglycans, 22 Proteolysis,349 Proteolyticdegradation,562 Proteome,542 Proteomics,542 Prothrombin, 130, 190, 458 Prothrombintime, 458 Protomer,58 Protongradient,229 Protooncogenes,687 Protoporphyria,214

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a Qro, 90 Quaternarystructureof protein, 52, 68 Quercetin,660 Q u i n o l ,6 3 9 Quinolinate,354 Quinolinate phosphoribosyltransferase, 356

t.a tin, 166 r.iA (seepolyunsaturated fatty acids) 714 Purgatives, Purines(s), biosynthesis,387 degradation,392 disorders,394 nucfeotideanalo1s,T3 salvagepathway,391 structures/70 tautomericforms, 70 Puromycin,560 Putrescine,375 Pyranose,14 Pyridoxal,143 Pyridoxalkinase,143 Pyridoxalphosphate,143 143 Pyridoxamine, Pyridoxine,143 Pyrimidine(s), 398 biosynthesis, degradation,400 disorders,400 nucleotideanalogs,73 salvagepathway,400 structure,71 tautomericforms, 70 Pi rithi am ine,137 hrophosphate,inorganic,288 Prroohosphatase,289 Pr'rcle ll8 h'.O,rSr-.e h'--\a:e .\--"f h-.".e h--r't l:-

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R Racemicmixture,12 717 Radioactivity, Radioactiveisotopes,717 Radioactivepollution, 656 729 Radioimmunoassay, Ra f f i n o s e , 1 6 9 Rancidity,33 cycle, 200, Rapaport-Leubering 251 Reactiveoxygen species/656 RecombinantDNA technology, 578 insulin,607 Recombinant vaccines,688 Recombinant Recommendeddietary (daily) allowance,5l4 Redox potential,224 Reducingequivalents,234 Reducingsugars,16 Reduction,224 Refsum'sdisease,293 Regulationof, citric acid cycle, 257 fafty acid synthesis,301 gene expression/566 261 gluconeogenesis, glycogenesis, 266 glycogenolysis, 266 210 heme synthesis, 296 ketogenesis, purinesynthesis, 392 pyrimidinesynthesis, 398 urea cycle, 339 Releasefactors,558 number,34 ReichertMeissl

Renalfunction tests,459 46O, 681 Renalglycosuria, Renalpfasmaflow, 459 Renalregulationol pH, 477 Renalrickets,128,4O8 460 Renalthresholdsubstances, Renaturationof DNA, 79 Renaturationof protein,62 Renin, 459, 472 Rennin,170 472 Renin-angiotensin, Replication, in eukaryotes,527 in prokaryotes,524 bubbles,524 Replication Replicationfactor C, 528 Replicationfork, 526 RepficationproleinA, 528 566 Repression, Repressorprotein, 566 356 Reserpine, Respiratoryacidosis,482 Respiratoryalkalosis,482 Respiratoryburst, 657 Respiratorychain (seeelectron transportchain) Respiratorydistresssyndrome,36 Respiratoryregulationof pH, 476 Repiratoryquotient,503 Restingmetabolicrate, 504 579 Restrictionendonucleases, Restrictionfragmentlength polymorphisms, 603 Restrictionfragmentmap, 620 Retina,121 R e t i n a l1 , 19 Retinoicacid, 119 Retinol1 , 19 Retinolbindingprotein,119 534 Retrotransposition, 550, 688 Retroviruses, 550, 688 Reversetranscriptase, Reversetranscription,550 Reversetriidothyronine,438 Rf value,721 Reye'ssyndrome,400 9 Rhamnohexose, Rheumatoidarthritis,647 Rhodopsin,12l Rhodopsincycle,121 Rho factor, 544 R i b i t o l ,1 3 7 R i b o f l a v i n1, 3 7

BIOCHEMISTF|Y

790 Ribonuclease P, 105 179 Ribonucleases, Ribonucleicacid (seeRNA) 71 Ribonucleosides, reductase, 392 Ribonucleotide 72, 79, 387 Ribonucfeotides, Ribose,71 272, 387 RiboseS-phosphate, RibosomalRNA, 79, 82 Ribosomes, 553, 557 '105,558 Ribozymes, Ribu lose,11 , ' 14 Ribuloses-phosphate, 271 Richner-Hanhart syndrome,352 Rickets,127, 408 Rifampin,549 RNA, 79, 523, s42, s89 RNA editing,549 RNA polymerase, 543, 546 RNA primer,524 RNA viruses,550, 688 Ro ds,12 1 Rotarymotor model, 230 Rotenone, 227 Rothera'stest, 295 Roussarcomavirus 687 purple,51 Ruhemann's Ryle'stube, 646

s Sakagauchireaction,61 Salicylic acid, 640 Salivaryamylase,l66 Salkowski'stest, 3B Saltingin, 60 Saltingout, 60, 182 Salvagepathways,303, 391, 400 Sanfilipposyndrome,281 Sanger'sreagent,56 Saponification, 33 Saponificationnumber,33 Schinb ase,143, 333 Scleroproteins, 64 Scurvy,'l 34 Secondarymessengers, 428, 43O Secondarystructureof protein, 56 Secretin,'17O,450 Sedimentationcoefficien! 728 14 Sedoheptulose, Sedoheptulose7 -phosphale,273

N.

Selenium,49, 128, 421, 659 48, 422 Sefenocysteine, Selenoproteins, 48 Selenosis,422 Sequenator,56 Serine,45, 361,37'l Serineproteases, 95, 17'l Serotonin,144,356 Serotoninpathway.354 Severecombined immunodeficiency,397 738 Sex chromosomes, Sex hormones,445 740 Sex-linkedinheritance, SCOT (seeaspartatetransaminase) SCPT(seealanine transaminase) Shine-Dalgarno sequence,557 Short interspersed nuclear elements,622 Short (simple)tandem repeats, 603, 620 Sl units,104 Sialicaci d , 1 8 Sickle-cellanemia,2O3.601 Sickle-cellhemoglobin,203 Sickle-cellttait, 204 Sigmafactor,543 Silentmutations,536 Simpletandem repeats,603 Singlenucleotidepolymorphisms, 606, 622 Single-stranded DNA binding proteins,525 Site-directedmutagenesis, 597 Sitosterol,39 Small nuclear ribonucleoorotein particles,548 SmallnuclearRNA. 81, 550 Snakevenom, 306 Sodium ,4 1 1 Sodiumfluoride. 284, 42"1 SolubleRNA, 81 Solutions,711 Somaticcell gene therapy,625 SomatomedinC, 434 Somatostatin, 433 Somatotropin,433 Sorbitol,16 Southernblot technique,587 Specificdynamic action, 505 Specificoptical rotation,14 Spectrophotometer, 726 Spermidine, 375 Spermine,375 Sphingolipidoses, 308

308 Sphingolipids, 308 Sphingomyelinase, 36 Sphingomyelins, 36 Sphingosine, Splicingof introns,548 Squalene,310 Standardlree energy,221 Starch,20 Starvation,383 Stearicacid, 30 Steatorrhoea,117 216 Stercobilin, 10, 705 Stereoisomerism, 10, 705 Stereoisomers, Stereospecificiry95 Steroid(s), 37 37 Steroidderivative(s), Steroidhormones,38, 314 Sterols,38 39 Stigmasterol, .l05, 193 Streptokinase, Streptomycin,18, 560 Subcellularotganelles,727 Substratelevel phosphorylation, 224 Substratestraintheory 99 Subunitvaccines,608 Succinate(succinicacid),93, 255 93, 227, Succinatedehydrogenase, 255 SuccinylCoA, 149, 21O,255, 292 67 Sucrase,l Sucrose,19 Suddeninfant death syndrome,291 Sugars,10 Suicideenzyme,645 Suicideinhibition,95 Suicidesubstrate,257 Sulfate,362, 4'13, 642 Sulfatides,37 Sulfhydrylgroups,46, 59,659 Sulfite,362 94,'153, 159, 390, Sulfonamides, 642 682 Sulfonylureas, Sulfur,413 Sulfur amino acids, 46, 357 Sulfurdioxide,663 Sun-shinevitamin, 124 655 Superoxide, Superoxidedismutase,659 Surfacetension,716 716 Surfactants, Svedbergunits,728 Symportsystem,653 Synovialfluid, 716

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r; '*':r-re coefficient,90 ,,rmrr;:n= Strand/543 r{'.*-,::ton/ rr ::anscription/ 544 558 :r translation, l.{r nationcodons,55.1 , 558 -e- ary structureof protein, 58 -,j5osterone/ 446 :*any, 4O8 ?tracycline,560 ctrahydrobiopterin,346, 357 Tetrahydrofolate, 150, 363, 389 Tetramer,58 Tetroses,11 Thalassemia(s), 206 Theobromine, 71 Theophylline, 71 Thermodynamics, 221 Thermogenesis, 327 Thermogenic action,505 Thermogenin, 327 Thiamine,'l 35 Thiaminepyrophosphate, 135, )q)

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Th iazoler ing, 135 Thin layerchromatography, 721 Thiobarbituric acid reactive substances, 657 Thiocyanate,642 Thioethergroup,46 Th iokinas e, 177, 288 Thiolase,289, 296, 311 Thiophenering,146 Thiophorase, 296 -riore dox in, 392 -^ io s ulf at e, 642

Thiouracil,43S Thiourea,438 T h r e eD s , 1 4 1 l n r e o n t n e4/ 5 , 5 / 2 Thresholdsubstances, 460 Thirstcerrtre,469 .l Thrombin, 90 Thromboplastin 1 ,9 1 Thrombosis, 648 Thromboxanes, 644, 648 I n y m r n e /, / u Thyroglobulin,349, 438 T h y r o i df u n c t i o nt e s t s , 4 4,14 6 5 Thyroidhormones,349, 431r Thyroidstimulatinghormone,434, 439 Thyrotropinreleasinghormone, 431 Thyroxine(T4\,233, 349, 418, 438, 504 T h y r o x i n eb i n d i n gg l o b u l i n ,4 3 8 Tissuefactor,191 Tissueplasminogen activator/599 Tissue-specific geneexpression/ 572 Titratableacid, 478 T-Lymphocytes, 737 TMP (thymidinemonophosphate), 72 T o a ds k i n ,3 1 , 5 1 0 Tocopherols, 128, 659 Tocotrienols,128 Tolbutamide, 682 Tophi,394 Topoisomerases, 527 Totaliron bindingcapacity,415 Traceelements,405 Tracertechnique,718 Transaminases, 86, "107,332 Transamination, 143, 332 Transcobalamin, I54 Transcription,542 Transcriptome, 542 Transcriptomics,543 Transdeamination, 334 Transducin, 122 Transferases, 87 .l Transferrin, 86 TransferRNA, 81, 554 Transformation, 583 Transforminggrowth factor,690 Transgene, 6-l1 Transgenesis, 6 11 Transitions,536 Transketolase, 135, 272

Translation,550 Transmethylation,35g Transmissible spongiiorm encephalopathies,4g5 Transport acrossmembranes, 651 Transportproteins,184 Transportsystems,653 Transposable elements,533 Transposition,533 Transposition of genes,533 Transposons, 533 Transsulfuration, 360 Transversions, 536 Triacylglycerol lipase,287 T r i a c y l g l y c e r o l3s2, , 1 74 , 2 8 7 (seetriacylglycerors.r Triglycerides Triiodothyronine, 349, 439 Trimethoprim, 152 .l Trioses, 1 Tripeptide,65, 342 Triple-stranded DNA, 76 r f l p t e tc o o o n s /5 5 I Tropomyosin, 492 Troponins,112, 492 Trisaccharides, 10 frisomy, 741 Tritium(:H), 717 tRNA (seetransferRNA) Trypsin,17'l Trypsininhibitor,179 Trypsinogen,170 Tryptamine,377 Tryptophan,47, 354 Tryptophanoperon, 569 Tryptophanpyrrolase,354 Tryptophanrepressor,569 Tubeless gastricanalysis,465 Tuberculosis, 146, 601 Tuberculosis meningitis, 492 Tubularmaximum(Tm),460 Tumorcells,692 Tumormarkers,691 Tumorsuppressor genes,631 (seecancersuppressor gene: tyramtnetJ/ / Tyrosinase,34B Tyrosine, 46,345 Tyrosinehydroxylase3,:T y r o s i n et r a n s a m i n a slej Tyrosinemia type ll 3-;: T y r o s i n o s {t i sy r o s i ^ e -: . : : 353

I

792

BIOCHEMISTF|Y

U Ubiquinone(CoQ),228 Ub iqu itin,3 31 UDB 39 8 276 UDP-galactose, UDP-glucose,263 275 UDP-glucuronate, 215 UDP-glucuronyltransferase, Uftracentrifugation, 727 UMP,7 2 Uncouplers, 233 Uncoupling protein,233 5'l5 Undernutrition, Uniform resource locator, 635 Uniport systems,653 Uracil,70 Ure a,3 37 , 34O Urea clearancetest, 461 Urea cycle,337 Urease,90 Uremia,341 Ureotelic,336 Uric acid, 71, 394 Uric acid pool, 396 Uricase,394 Uricotelic,336 Uridin e,7 2, 398 Uridylicacid (seeUMP) Urinarystones,134 Urine formation,459 Urine production, hormonalregulation,469 Urob ilin,216 Urobilinogen,215 Urocanate,335 Uronic acid pathway,275 Uroporphyrin,209 Uroporphyrinogen,211

ffi Vmax,BB Valeric atid,

Valine,45, 363 Valinomycin,233 van den Bergh reaction,455 Van der Waals forces, 59 Vanillyl mandelic acid, 445

Variablenumber tandem repeats, 604, 620 Variegate porphyria, 2'l 4 Vasoactiveintestinalpeptide,450 Vasopressin, 66, 437 Vectors,582 Viral RNA, 688 Viscosity,715 Visualcvcle, 121 Vital force theory 703 Vitamers,118,'143 Vit am i nA , 1 1 8 Mtamin B, (seethiamine) Vitamin B, (seeriboflavin) Vitamin 86 (seepyridoxine) Vitamin8.,, (seecobalamin) Vitamin C (seeascorbicacid) Vit am i nD , 1 2 3 VitaminE, 128 VitaminE and selenium,129 VitaminK, 129 VitaminB 159 r/itamins,116 classification,117 definition,116 fat soluble-general, 118 nomenclature, 116 synthesis,117 water soluble-general, 118 Vitellin, 65 Vitiligo,353 Mtreousbody, 7'15 VLDL (very low density lipoproteins), 315 von Cierke'sdisease,269 Von Willenbrand'sdisease,193

Water tank model, 473 Watson and Crick model,74 Waxes,28 syndrome,137 Wernicke-Korsakoff Westernblot technique,589 Whey proteins,497 White adiposetissue,326 Williams syndrome,489 Wilson'sdisease,417 Wobble hypothesis,552 World wide web, 636

x X a n t h i n e7, 1 , 3 9 3 Xanthineoxidase,393 Xanthinestone, 398 398 Xanthinuria, Xanthoproteicreaction,61 Xanthurenate, 145,354 X-chromosome, 397, 74O Xeaxanthin,659 Xenobiotics,638 Xenograft,736 Xerodermapigmentosum,538 Xerophthalmia, 123 xylitol, 273 273 Xylitol dehydrogenase, Xylose,11 Xylulose,11

z w Wafd's visual cvcle, 129 131

709 46 endogenous,469 exogenous,469 functions,468 intake,469 output, 469 structure,709 Water pollution, 664

Zak's test, 38 Zein, 65 Zellwegersyndrome,8 Zidovudine,699 Zinc,4"19 Zinc finger motif, 573 Zona fasciculata,441 Zona gfomerulosa,441 Zona reticularis,441 syndrome,465 Zollinger-Ellison Zn-proteases,171 725 Zone efectrophoresis, Zymogens,102,170, 562 Zymosterol,3l0 Zwitterion, 49, 60

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