Chromatography and Modification of Nucleosides, Part C: Modification Nucleosides in Cancer and Normal Metabolism : Methods and Applications (Journal of Chromatography Library)

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 45C

chromatography and modification of nucleosides part C: modified nucleosides in cancer and normal metabolism methods and applications

Chromatography and Modification of Nucleosides edited by C. W. Gehrke and K. C. T. Kuo

Part A : Analytical Methods for Major and Modified Nucleosides HPLC, GC, MS, NMR, UV and FT-IR (ISBN 0-444-88540-4) Part B: Biological Roles and Function of Modification (ISBN 0-444-88505-6) Part C: Modified Nucleosides in Cancer and Normal Metabolism Methods and Applications (ISBN 0-444-88598-6) Part D: Comprehensive Database for RNA and DNA Nucleosides Chemical, Biochemical, Physical, Spectral and Sequence (ISBN0-444-8873 1-8)

JOURNAL OF CHROMATOGRAPHY LIBRARY-

volume 45C

chromatography and modification of nucleosides pat?

C: modified nucleosides in cancer and normal metabolism methods and applications

edited by

Charles W. Gehrke and Kenneth C. T. Kuo Department of Biochemistry, University of Missouri-Columbia, and Cancer Research Center, P. 0. Box 1268, Columbia, MO 65205- 1268, U S .A.

ELSEVIER Amsterdam - Oxford

- New York -Tokyo

1990

ELSEVIER SCIENCE PUBLISHERSB.V. Sara Burgerhartstraat25 P.O. Box 21 1, 1000 AE Amsterdam, The Netherlands

Distributors for the United States and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY INC. 655 Avenue of the Americas New York, NY 10010, U.S.A. Library of Congress Catalogingin-PublicationData (Revised for added vol., part C)

Chromatography and modification of nucleosides. ) (Journal of chromatography library ; v. 45AIncludes bibliographical references. Contents: pt. A. Analytical methods for major and modified nucleosides, HPLC. GC, MS, NMR, UV, and FT-IR -- pt. B. Biological roles and function of modification -- pt. C. Modified nucleosides in cancer and normal metabolism. 1. Nucleosides--Analysis. 2. Nucleosides--Metabolism. 3. Nucleosides--Derivatives--Synthesis. 4. Chromatography. I. Gehrke, Charles W. 11. Kuo. Kenneth C. T., 1936111. Series. q ~ m . ~ a a c 4 71989 2547.7i9046 89-25973

.

ISBN 0-444-88598-6

0 Elsevier Science PublishersB.V., 1990 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science Publishers B.V./ Physical Sciences & EngineeringDivision, P.O. Box 330, 1000 AH Amsterdam, The Netherlands. Special regulations for readers in the U S A . -This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the publisher. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made. Although all advertising material is expected to conform to ethical (medical)standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer. This book is printed on acid-free paper Printed in The Netherlands

V

TABLE OF CONTENTS

Special Acknowledgement to Dr. Robert W. Zumwalt - - - - - - - - - - - - - - - - - XIX

Introduction

Early Development of Nucleoside Markers for Cancer T. Phillip Waalkes and Charles W. Gehrke

Chapter 1

Progress and Future Prospects of Modified Nucleosides as Biological Markers of Cancer - - - - - - - - - - - - - C15 Robert W. Zumwalt, T. Phillip Waalkes, Kenneth C. Kuo and Charles W. Gehrke

Chapter 2

Ribonucleosides in Biological Fluids by a HighResolution Quantitative RPLC-UV Method----------- C41 Kenneth C. Kuo, Dat T. Phan, Nathan Williams and Charles W. Gehrke

Chapter 3

Ribonucleosides in Body Fluids: On-Line Chromatographic Cleanup and Analysis by a Column Switching Technique _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _C115 _____ Eckhard Schlimme and Karl Siegfried-Boos

Chapter 4

High-Performance Liquid Chromatography of Free Nucleotides, Nucleosides, and their Bases in Biological Samples Yong-Nam Kim and Phyllis R. Brown

_____________________________

C1

C147

Chapter 5

Isolation and Characterization of Modified Nucleosides from Human Urine - - - - - - - - - - - - - - - - - - C185 Girish B. Chheda, Helen B. Patrzyc, Henry A. Tworek and Shib P. Dutta

Chapter 6

High Performance Liquid Chromatography of Modified Nucleosides in Human Serum--------------------- C231 Edith P. Mitchell, Kenneth KUO, Lisa Evans, Paul Schultz, Richard Madsen, Charles W. Gehrke and John Yarbro

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Chapter 7

Modified Nucleosides in Human Blood Serum as Biochemical Signals for Neoplasia - - - - - - - - - - - - - - - C251 Francesco Salvatore, Lucia Sacchetti, Marcella Savoia, Fabrizio Pane, Tommaso RUSSO, Alfredo Colonna and Filiberto Cimino

Chapter 8

Biochemical Correlations between Pseudouridine Excretion and Neoplasias- - - - - - - - _ -- - - - - - - - - - - - C279 Filiberto Cimino, Franca Esposito, Tommaso Russo and Francesco Salvatore

Chapter 9

High-Performance Liquid Chromatography Analysis of Nucleosides and Bases in Mucosa Tissues and Urine of Gastrointestinal Cancer Patients- - - - - - - - - C293 Katsuyuki Nakano

Chapter 10

Modified Nucleosides as Biochemical Markers of Asbestos Exposure and AIDS- - - - - - - - - - - - - - - - - - - - - C321 Opendra K. Sharma and Alf Fischbein

Chapter 11

RNA Catabolites in Health and Disease- - - - - - - - - - - - C341 Irwin Clark, Win Lin and James W. Mackenzie

Chapter 12

Serum Nucleoside Chromatography for Classification of Lung Cancer Patients and Controls - - - - - - - - - - - - - C367 John E. McEntire, Kenneth C. Kuo, Mark E. Smith, David L. Stalling, Jack W. Richens, Jr., Robert W. Zumwalt, Charles W. Gehrke and Ben W. Papermaster

Chapter 13

Modified Nucleosides and Nucleobases in Urine and Serum as Selective Markers for the Whole-Body Turnover of tRNA, rRNA, and mRNA-Cap - Future Prospects and I m p a c t - - - - - - - - - - - - - - - - - - - - - - - - - - - C389 Gerhard Schoch, Gernot Sander, Heinrich Topp and Gesa Heller-Schoch

__

Combined Subject Index for Parts A, B and C - - - - - - - - - - - - - - - - - - - - - c443 Journal of Chromatography Library (volumes in the series)

- - - - - - - - - c449

VII PREFACE

This volume addresses the role of modified nucleosides as biomarkers in the early detection of cancer and the clinical management of the cancer patient. The introduction by Waalkes and Gehrke describes in some detail the initial concepts, beginning methods used, and applications of the methodology. The modified nucleosides have been thought to be derived predominantly from transfer ribonucleic acid (tRNA) and have been known to be excreted in abnormal amounts in the urine of the patients with various malignant diseases (ref. 1-4) and tumor-bearing animals (ref. 5). Since these modified nucleosides have no salvage pathway, they are excreted in the urine as intact molecules (ref. 6). In 1966, Borek @t al. reported that tRNA methyltransferase activity was higher in cancer tissue than in corresponding normal tissue (ref. 7) and that the concentration of methylated nucleosides in tRNA of cancer tissue was higher than that in corresponding normal tissue (ref. 8). Thus, urinary elevation of modified nucleosides has been suggested to be caused by the increased tRNA turnover in tumor tissue rather than by cell death (ref. 9). The tRNA is a highly complex biomacromolecule and as many as 20% of its approximately 85 bases may be modified. These modifications may be as simple as the introduction of a methyl group or extremely complex, require the action of many enzymes and the modifications occur after the synthesis of the primary sequence of the tRNA (ref. 10). In part A of this series, many contributing scientists have reported that the enzymes involved have been shown to be species specific, base specific, site specific and sequence specific. Some of the modifications require the action of only one enzyme, whereas more complex modifications such as the introduction of a 'Q-base' require the sequential intervention of several enzymes. It has also been shown that the mammalian cell lacks the kinases to convert the modified nucleosides that are released on metabolism into their corresponding nucleoside triphosphates and

VIII

therefore their incorporation into the macromolecule. Consequently, the modified nucleosides are released into the blood and finally excreted in the urine. With this background of investigations and the central role of nucleic acids, involving the important biomacromolecules of tRNA, rRNA, mRNA and DNA in protein synthesis, metabolism, and biological regulation have resulted in intense and continued research on the structure, sequence, and conformation of these macromolecules by scientists representing a wide spectrum of disciplines, from biochemistry to molecular and subcellular biology to clinical research. With new findings many fundamental investigations are continuing on the chemical structure of nucleoside modifications in nucleic acids; the establishment of additional primary sequences and three dimensional conformation of the polymers; and research on the dynamic properties of nucleic acids under physiological conditions, all of which are essential for increasing our understanding of their complex biological and functional relationships of the tRNAs and other nucleic acids. To this end, the uses of chemical, chromatographic, biochemical, genetic, and molecular biological tools and systems, ranging from E. coli, yeast, and cyanobacteria to drosophila, provide the model investigative systems for the researcher. These subjects are discussed in depth by many scientists in their disciplines from molecular biology, microbiology, biochemistry, to chromatographic separation sciences. Please refer to parts A and B of this series on "Chromatography and Modification of Nucleosides" for the latest findings from research on the role and function of nucleic acids. This volume presents a comprehensive treatment of ribonucleoside analysis by reversed-phase high performance liquid chromatography and other chromatographic techniques and application of the methods in many clinical studies. In the early 1970s, the National Cancer Institute (NCI) made the decision to study biochemical materials produced by the body as potential "biological markers" of cancer. Such markers would either indicate the presence of cancer or they would reflect changes in tumor mass and would be useful in following cancer therapy. These markers

IX

might be found in the body fluids, for example, in blood and urine of patients with cancer. Initially, part of the NCI program was devoted to the study of minor components of nucleic acids, known as modified nucleosides. These modified nucleosides appear in body fluids mainly as the result of the destruction of transfer ribonucleic acid (RNA). Since there is no mechanism in the body for re-use of these modified nucleosides, the presence of these compounds in body fluids is a measure of the destruction rate of transfer RNA. Transfer R N A performs critical functions in protein synthesis, and has important roles in regulation, transcription, inhibiting enzyme activity, and protein degradation. Therefore, measurement of the amount of modified nucleosides in body fluids could indicate changes in the body as the result of cancer, and changes in the amount of these compounds might be a measure of the effectiveness of the therapy given the patient. First, however, it was most important to develop new chemical methods for measuring the modified nucleosides as the components are present at only very low levels in body fluids. The conventional techniques of the 1970's of ion-exchange and paper chromatography lacked the speed, sensitivity, resolution, and quantitation for measuring a number of nucleosides simultaneously. These obvious problems in analysis, as well as the cumbersome nature of the methods, made it necessary to develop new chromatographic-analytical techniques. It was also shown by Gehrke and Kuo that the nucleosidekreatinine ratios were remarkably similar in random or "spot" samples as compared to obtaining a total 24 hour urine collection, and therefore the excretion of the nucleosides relative to creatinine is constant, not episodic. Thus, "spot samples" provided data as valid as data from 24 hour collections. Development of methods for the analysis of modified nucleosides has been a major thrust of Dr. Charles W. Gehrke and Kenneth Kuo's research for a number of years. Earlier, gas-liquid chromatography was utilized to search for and measure cancer markers in body fluids, then their research group pioneered high performance liquid chromatographic methods. The development

X

of these methods of measurement has allowed studies with Dr. T.P. Waalkes at the Oncology Center at Johns Hopkins University in Baltimore. Also, these studies revealed that the use of multiple markers may be useful in staging patients, and in detecting minimal, residual, and recurrent cancer. In the late 70s, the development of high performance liquid chromatographic instrumentation, reliable pumps, new detectors and efficient reversed-phase columns offered a new analytical approach, and the development of a highly specific affinity chromatography sample cleanup method for isolation of ribonucleosides, based on the work of Dr. Uziel at Oak Ridge National Labortory (ref. 11) promised to provide a much improved method for studying ribonucleosides in complex matrices such as physiological fluids (ref. 12, 13, 14). At the same time, parallel protocols were being investigated for improved nucleic acid isolation, quantitative enzymatic hydrolysis of RNAs, high resolution preparative RPLC, and affinity chromatography to obtain pure known and unidentified nucleosides for UV absorption spectroscopy and interfaced mass spectrometry investigations for characterization and identification of nucleosides. In addition, nucleoside structure-spectrum relationships, composition, and conformation using the techniques of HPLC-UV, FT-IR, NMR, and MS were developed as well as structure UV-RPLC retention relationships (part A). Nucleotides, nucleosides, and bases are of major importance in biological systems e.g. in the formation and function of nucleic acids. Disorders in purine and pyrimidine metabolism are believed to be involved in diseases such as cancer. Borek's expectation "of finding some unique metabolic products or components of malignant cells circulating in body fluids which can be measured" (ref. 15) was met during the last decade as specific excretion patterns of modified nucleosides in urine were found to be related to distinct metabolic disorders. The Introduction on "Nucleoside Markers for Cancer: and the Chapters that follow in part C, present the advanced experimental approaches and technologies for measuring modified nucleosides in biological fluids. Also, the progress made since 1980 and future

XI prospects of modified nucleosides as biologic markers of cancer are discussed. New findings are presented and conclusions drawn by the leading investigators from many countries on modified nucleosides as "biochemical signals" in cancer and as "selective markers" in metabolism for whole body turnover of tRNA, rRNA, and mRNA. Columbia, Missouri 1989

Charles W. Gehrke Kenneth C.T. Kuo

References: 1.

2. 3.

4.

5.

6.

7. 8.

9.

Waalkes, T.P., Gehrke, C.W., Zumwalt, R.W., d. The urinary excretion of nucleosides of ribonucleic acid by patients with advanced cancer. Cancer 36 (1975) 390-398. Gehrke C.W., Kuo K.C., Waalkes T.P., Borek E. tRNA breakdown products as markers of cancer. Cancer 39 (1979) 1150-1153. Speer J., Gehrke, C.W., Kuo, K.C., Waalkes, T.P., Borek, E., tRNA breakdown products as markers for cancer. Cancer 44 (1979) 21 20-21 23. Fischbein, A., Sharma, O.K., Selikoff, I.J., Borek, E., Urinary excretion of modified nucleosides in patients with malignant mesothelioma. Cancer Res 43 (1983) 2971-2974. M. Shimizu, S . Fujimura, Studies on the abnormal excretion of pyrimidine nucleosides in the urine of Yoshida ascites sarcoma-bearing rats: Increased excretion of deoxycytidine, pseudouridine and cytidine, Biochim Biophys Acta 517 (1978) 277-286. Weissman, SM., Eisen, AZ, Lenrio, M., Pseudouridine metabolism-Ill: studies with isotopically labeled pseudouridine. J. Lab. Clin. Med. 60 (1962) 40-47. Srinivasan, P.R., Borek, E.,tRNA methylases in tumors of animal and human origin. Proc USA 56 (1966)1003-1009. Borek, E., Introduction to symposium. tRNA and tRNA modification in differentiation and neoplasia. Cancer Res 31 (1971) 596-597. Borek, E.,Baliga, B.S., Gehrke, C.W., U.High turnover rate of transfer RNA in tumor tissue. Cancer Res 37 (1977) 33623366.

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10. 11. 12.

13.

14. 15.

Vreken, P., Tavenier, P., Urinary excretion of six modified nucleosides by patients with breast carcinoma. Am. Clin. Biochem. 24 (1987) 598-603. Uziel, M. Smith, L.H., and Taylor, S.A., Modified nucleosides in urine: Selective removal and analysis, Clin. Chem., 22 (1976) 1451-1455. Gehrke, C.W., Zumwalt, R.W., McCune, R.A., and Kuo, K.C., Quantitative high-performance liquid chromatography analysis of modified nucleosides in physiological fluids, tRNA, and DNA, Recent results in Cancer Research 84 (1983) 344-359. Kuo, K.C., Esposito, F., McEntire, J.E., Gehrke, C.W., Nucleoside profiles by HPLC-UV in serum and urine of Controls and cancer patients, in: F. Cimino and F. Salvatore (Eds.) Human Tumor Markers, Walter de Gruyter Berlin and New York, (1987) 519544. Gehrke, C.W., and Kuo, K.C., Ribonucleoside analysis by reversed-phase high performance liquid chromatography, J. Chromatogr. 471 (1989) 3-36. E., Borek, the Morass of tumor markers, Trends Biochem. Sci., 1985; 10: 182-184.

XI11

ERNEST BOREK 1911-1986 A Pioneer in Methylation of Nucleic Acids

XIV

Dedication

ERNEST BOREK 19 11-1986

E r n e s t Borek, p i o n e e r i n n u c l e i c a c i d s m e t h y l a t i o n , was born i n N y i r c s a s z a r i , Hungary, and moved w i t h h i s f a m i l y t o New York C i t y a t t h e age o f 14. He graduated a t C i t y C o l l e g e o f New York C i t y and t o o k h i s Ph.D. i n b i o c h e m i s t r y a t Columbia U n i v e r s i t y . E r n e s t ' s i n t e r e s t s covered many areas o f b i o c h e m i s t r y . He was on t h e f a c u l t y o f t h e Department o f Chemistry o f C i t y U n i v e r s i t y o f New York from 1934-1969 and was a p r o f e s s o r i n t h e Department o f B i o c h e m i s t r y o f Columbia U n i v e r s i t y f r o m 1959-1969, where h i s research e f f o r t s i n c l u d e d such d i v e r s e areas as s t u d i e s on t h e b i o s y n t h e s i s o f c h o l e s t e r o l , t h e d e m o n s t r a t i o n o f a b i o 1o g i c a l l y a c t i v e ir r a d i a t i on p r o d u c t as t h e b a s i s f o r i n d i r e c t i n d u c t i o n o r t h e "Borek-Ryan" E f f e c t , t h e d e m o n s t r a t i o n o f " r e 1 axed c o n t r o l " o v e r RNA s y n t h e s i s i n b a c t e r i a , t h e d i s c o v e r y o f t h e enzymes which m e t h y l a t e n u c l e i c a c i d s a t t h e polymer l e v e l , t h e d i s c o v e r y o f a b e r r a n t t R N A m e t h y l a t i o n i n tumor t i s s u e s , t h e demonstration o f unique t R N A s p e c i e s i n tumor t i s s u e s , and t h e development o f t h e concept o f u r i n a r y a n a l y s i s o f m o d i f i e d nucleosides as b i o l o g i c a l markers f o r cancer. Borek f e l t t h a t a t u r n i n g p o i n t i n h i s c a r e e r was t h e y e a r 1951. By t h e l a t e 1940s, he had become bored w i t h b i o c h e m i s t s ' p r e o c c u p a t i o n w i t h t r a c i n g , atom by atom, t h e o r i g i n s o f v a r i o u s intermediary metabolites. He c o n t i n u e d some r e s e a r c h p e r f u n c t o r i l y , b u t h i s main i n t e r e s t was w r i t i n g a book e n v i s i o n e d as ' B i o c h e m i s t r y f o r t h e M i l l i o n s ' (It t u r n e d o u t t o be, i n those pre-Sputnik days, f o r t h e dozens.). He wrote: "So f a r t h e b i o c h e m i s t s ' e f f o r t s have been d i r e c t e d m a i n l y toward t h e completion o f t h e a t l a s o f m o l e c u l a r anatomy. We must b e g i n t o p a t i e n t l y assemble s t i l l another science, m o l e c u l a r p h y s i o l o g y - t h e s t u d y o f t h e r e l a t i o n between m o l e c u l a r s t r u c t u r e and Chemical Machine, 1952). He was c e l l u l a r f u n c t i o n " (Man. & t

xv ready t o become a m o l e c u l a r b i o l o g i s t b e f o r e t h e t e r m was coined, and he was s t r u c k by a sentence o f S i r F r e d e r i c k Gowland Hopkins, t h e g r e a t Cambridge b i o c h e m i s t and founder o f modern biochemistry: " I n e x p l o r i n g and c u l t i v a t i n g t h e f i e l d s o f n a t u r e t h e c h e m i s t s were b e s t p r o v i d e d w i t h t h e machinery f o r t h i s c u l t i v a t i o n , b u t t h e b i o l o g i s t knew b e s t t h e l a y o f t h e l a n d . " I t was t h e l a t e H e i n r i c h Waelsch who showed Borek a two-sentence a b s t r a c t o f a paper on l y s o g e n i c i n d u c t i o n by Andre' L w o f f , a f t e r " A few days i n t h e l i b r a r y s p e n t r e a d i n g which E r n e s t wrote: L w o f f ' s p r e v i o u s work convinced me t h a t I had my b i o l o g i s t and my phenomenon. " Thus, i n 1951, Borek c o l l a b o r a t e d w i t h L w o f f a t t h e P a s t e u r I n s t i t u t e as a Guggenheim f e l l o w . H i s work on t h e d e p r i v a t i o n o f amino a c i d s i n l y s o g e n i c b a c t e r i a r e s u l t e d in t h e c h a r a c t e r i zaWith h i s t i o n o f an unusual mutant of f s c h e r i c h i a c o l i K12 W6. c o l l e a g u e , t h e l a t e Ann Ryan, he showed t h a t u n l i k e a l l o t h e r amino a c i d auxotrophs, t h e s y n t h e s i s o f RNA was n o t t u r n e d o f f by t h e mutant i n t h e absence o f i t s r e q u i r e d amino a c i d m e t h i o n i n e . T h i s phenomenon was l a t e r c a l l e d r e l a x e d c o n t r o l o f RNA syntheI t was a novel f i n d i n g which was n o t e a s i l y accepted, b u t sis. i t proved t o be a f e r t i l e area o f r e s e a r c h f o r many s c i e n t i s t s . While on a second Guggenheim f e l l o w s h i p , i n t h e course o f o t h e r s t u d i e s o f i r r a d i a t i o n e f f e c t s on t h e i n d u c t i o n o f l y s o g e n i c organisms w i t h Ann Ryan, he d i s c o v e r e d i n 1958 t h a t when U V - i r r a d i a t e d male f. coif which a r e f r e e o f prophage a r e conj u g a t e d w i t h female f. c o l i which c o n t a i n e d prophage, phage f o r m a t i on o r i n d u c t i o n r e s u l t s . T h i s phenomenon o f in d i r e c t ultra-violet i n d u c t i o n , o r Borek-Ryan E f f e c t , was an i m p o r t a n t c o n t r i b u t i o n t o t h e a r e a o f DNA damage and r e p a i r i n b a c t e r i a . C o n t i n u i n g h i s s t u d i e s on r e l a x e d c o n t r o l o f RNA s y n t h e s i s w i t h graduate s t u d e n t Lewis R. Mandel, he showed t h a t t h e RNA w h i c h a c c u m l a t e d d u r i n g m e t h i o n i n e d e p r i v a t i o n was m e t h y l deficient, I n v i v o l a b e l i n g w i t h [3[H]-methyl] methionine demonstrated t h a t t h e m e t h y l groups i n t R N A o r i g i n a t e d f r o m methionine. The f i n d i n g i n 1961 o f l a b e l e d methyl group i n r i b o t h y m i d i n e , an u b i q u i t o u s component o f E . c o l i tRNA, came as a

xv I complete s u r p r i s e . I t had been shown p r e v i o u s l y by A r t h u r Kornberg t h a t t h e methyl group o f thymine i n DNA stems from t h e one carbon pool through t h e f o l a t e pathway. The new pathway f o r t h e s y n t h e s i s o f thymine i n t R N A by t h e d i r e c t t r a n s f e r o f an i n t a c t methyl group l e d him t o e x p l o r e t h e p o s s i b i l i t y t h a t t h e m e t h y l a t e d bases were synthesized a t t h e polymer l e v e l . I n 1962, w i t h graduate s t u d e n t Erwin F l e i s s n e r , he showed, u s i n g methyl d e f i c i e n t t R N A as a s u b s t r a t e , t h a t methyl groups i n t R N A a r e i n t r o d u c e d by t h e enzymes a t t h e macromolecular l e v e l . His subsequent s t u d i e s l e d t o t h e d i s c o v e r y o f DNA m e t h y l a t i n g enzymes as w e l l , and i n t h e l a t e s i x t i e s , Borek and h i s colleagues showed t h a t t h e t R N A m e t h y l a t i n g enzymes a r e a b e r r a n t i n tumor t i s s u e s ; furthermore, t h e y showed t h a t tumors c o n t a i n unique species o f tRNA. Honors f o l l o w e d these d i s c o v e r i e s , and E r n e s t was awarded t h e Medal o f t h e S o c i e t y o f B i o l o g i c a l Chemists o f F i n l a n d i n 1965, as w e l l as r e c e i v i n g t h e Townsend H a r r i s Medal f o r d i s t i n g u i s h e d alumni o f C i t y U n i v e r s i t y o f New York i n 1968. I n 1969 he accepted an appointment as p r o f e s s o r i n t h e D e p a r t m e n t o f M i c r o b i o l ogy, U n i v e r s i t y o f Colorado Heal t h Sciences Center, which he h e l d u n t i l h i s death. There h i s research i n t e r e s t s t u r n e d t o t h e phenomenon o f e l e v a t e d e x c r e t i o n o f m o d i f i e d nucleosides by cancer p a t i e n t s . The e l u c i d a t i o n o f t h e mechanisms o f t h e m e t h y l a t i o n o f t R N A s o l v e d t h e 1ong-standi ng p u z z l e o f t h e o r i g i n o f m e t h y l a t e d p u r i n e s and p y r i m i d i n e s which had o r i g i n a l l y been shown t o be more c o n c e n t r a t e d i n t h e u r i n e o f cancer p a t i e n t s by Alexander Gutman o f Columbia U n i v e r s i t y . These u r i n a r y e x c r e t i o n p r o d u c t s a r e d e r i v e d from t h e breakdown o f tRNA. Borek was i n t i m a t e l y i n v o l v e d i n t h e N a t i o n a l Cancer I n s t i t u t e B i o l o g i c a l Markers Program, t o e l u c i d a t e whether t h e u r i n a r y e x c r e t i o n o f m o d i f i e d n u c l e o s i d e s c o u l d s e r v e as b i o l o g i c a l markers f o r cancer. T. P h i l l i p Waalkes headed t h e B i o l o g i c a l Markers Program a t t h e N C I , and i n 1971-74 c o n t r a c t s were awarded by t h e N C I t o P r o f e s s o r Charles W. Gehrke and h i s g r o u p a t t h e U n i v e r s i t y o f Missouri-Columbia t o develop

XVII

chromatographic methods t o measure m o d i f i e d n u c l e o s i d e s i n u r i n e . D r . Gehrke and h i s g r o u p developed t h e h i g h r e s o l u t i o n q u a n t i t a t i v e HPLC techniques which a1 lowed a c c u r a t e measurement o f a number o f u r i n a r y m o d i f i e d nucleosides. D u r i n g t h e l a s t t e n years o f h i s l i f e , Borek was i n s t r u m e n t a l i n d e v e l o p i n g a program a t AMC Cancer Research Center f o r t h e a n a l y s i s o f t h e e x c r e t i o n o f m o d i f i e d nucleosides as tumor markers and as d e t e r m i n a n t s f o r t h e e f f i c a c y o f cancer therapy, and he c o l l a b o r a t e d e x t e n s i v e l y w i t h P r o f e s s o r Gehrke a t t h e U n i v e r s i t y o f M i s s o u r i i n t h i s research area. H i s a c t i v i t i e s i n cancer research l e d t o h i s appointment as Chairman o f t h e Department o f M o l e c u l a r B i o l o g y a t AMC Cancer Research Center from 1977-1985. He a l s o served as D i r e c t o r o f t h e Colorado Regional Cancer Center i n 1976-77 and was Chairman o f t h e Cancer Centers Support Review Committee o f t h e N a t i o n a l Cancer I n s t i t u t e i n 1978-79. He a p p r e c i a t e d t h e fundamental importance o f n u c l e i c a c i d m o d i f i c a t i o n f o r n u c l e i c a c i d function very e a r l y i n h i s studies, and he was an i n d e f a t i g a b l e advocate f o r t h e concept, n o t e a s i l y discouraged by s k e p t i c i s m . I n 1968 he w r o t e i n an essay f o r t h e M.D. Anderson symposium a t Houston, "It i s n o t so s u r p r i s i n g as i t may a t f i r s t appear t h a t s c i e n t i s t s should be r e l u c t a n t t o A t r u l y new i d e a i s one o f t h e most accept new i d e a s . u n p a l a t a b l e i m p o s i t i o n s a man can i n f l i c t on h i s f e l l o w men. A new i d e a a s s a u l t s t h e v a n i t y o f t h e r e c i p i e n t . If it i s a valid and worthy idea, why d i d he n o t t h i n k o f i t f i r s t ? Obviously t h e r e f o r e , every novel i d e a must be s u b j e c t e d t o c r i t i c a l scrutiny. Moreover, t h e Pooh-Bahs o f science o f t e n w i e l d g r e a t i n f l u e n c e i n d e t e r m i n i n g t h e welcome accorded t o novel ideas, and t h e i r mood i s n o t always r e c e p t i v e . O f t e n t h e y achieve p o s i t i o n s o f eminence n o t on t h e s t r e n g t h o f t h e i r own o r i g i n a l i t y , b u t r a t h e r by developing and e n l a r g i n g t h e i d e a s o f t h e g e n e r a t i o n I t i s n a t u r a l f o r them n o t t o be t o o immediately preceding. encouraging t o u p s t a r t s who s t a r t hacking away a t t h e i d e o l o g i c a l p e d e s t a l s on which t h e y a r e perched. But i t i s j u s t as w e l l t h a t new ideas must s t r u g g l e f o r s u r v i v a l . Only i n t h i s way can t h e

XVIII

few c l e a r i n s i g h t s be s o r t e d o u t from t h e murky f a l s e ones. And i f an i d e a i s sound, i t s u r v i v e s . A f r u i t f u l i d e a v i s - a - v i s i t s opponents i s l i k e s p r o u t i n g grass a g a i n s t a g r a n i t e b l o c k ; i d e a s and grass p r e v a i 1. " He i s E r n e s t Borek was a t a l e n t e d w r i t e r o f s t y l e and w i t . t h e a u t h o r o f f o u r p o p u l a r books f o r t h e n o n - s c i e n t i s t , Man, t h e Code o f Chemical Machine (1952), The Atoms W i t h i n k (1961), &T Life (1965), and The S c u l P t u r e o f L i f e (1973), which d e s c r i b e t h e h i s t o r y and development o f b i o c h e m i s t r y and m o l e c u l a r b i o l o g y . The Atoms W i t h i n Us. has been t r a n s l a t e d i n t o a l l m a j o r languages and r e c e i v e d t h e Thomas A l v a Edison Foundation Award f o r t h e b e s t science book f o r t h e p u b l i c i n 1961. He was t h e c o - e d i t o r w i t h t h e l a t e Jacques Monod of a book o f essays d e d i c a t e d t o D r . Andre' Lwoff, Qf Microbes and L i f e (1972). I n a d d i t i o n t o over 125 s c i e n t i f i c p u b l i c a t i o n s i n r e f e r e e d j o u r n a l s and many reviews, he c o n t r i b u t e d essays t o newspapers and s c i e n t i f i c j o u r n a l s d e s c r i b i n g t h e problems o f o r i g i n a l t h i n k e r s i n science. The honor t h a t he found most t o u c h i n g came i n 1984 when he was awarded an honorary M.D. degree f r o m t h e U n i v e r s i t y o f Szeged, Hungary. H i s f a m i l y had l e f t Hungary because under t h e f a s c i s t regime i n power a t t h e time, he would have been denied t h e o p p o r t u n i t y t o a t t e n d medical school. To be a b l e t o r e t u r n and r e c e i v e t h e degree was v e r y g r a t i f y i n g . He had a z e s t f o r l i f e , e n j o y i n g an e x c i t i n g s k i r u n as much as a s u c c e s s f u l experiment. He was a generous and c a r i n g i n d i v i d u a l w i t h an i n f e c t i o u s enthusiasm. E r n e s t Borek w i l l be remembered w i t h a d m i r a t i o n and a f f e c t i o n as a d i s t i n g u i s h e d s c i e n t i s t , h i s t o r i a n , and a man o f c u l t u r e .

Opendra K. Sharma Dept o f M o l e c u l a r B i o l o g y AMC Cancer Research Center Denver, Colorado

S y l v i a J. K e r r Department o f B i o c h e m i s t r y , B i o p h y s j c s , and Genetics Uni v e r s i t o f Colorado Heal t h Sciences E e n t e r Denver, Colorado

XIX

SPECIAL ACKNOWLEDGEMENT TO DR. ROBERT W. ZUMWALT D r . Robert W. Zumwalt, Research A s s o c i a t e and A n a l y t i c a l Biochemist i n o u r research group o v e r t h e p a s t 20 y e a r s i n t h e Department o f B i o c h e m i s t r y , U n i v e r s i t y o f M i s s o u r i -Col umbi a, and t h e Cancer Research Center, has been an u n t i r i n g r e s o u r c e and consult a n t i n b r i n g i n g t h i s four-volume t r e a t i s e , C h r o m a t o g r a p h y and Modification of Nucleosides, t o a finality. D r . Zumwalt has p l aced h i s t a l e n t s o f g r e a t t e c h n i c a l d e t a i 1 , know1 edge o f chromat o g r a p h i c s , and p a t i e n c e i n a c h i e v i n g c o m p l e t i o n o f t h e s e works. D r . Zumwalt was a c e n t r a l a u t h o r / e d i t o r , w i t h Kenneth Kuo and Charles Gehrke, i n o u r f i r s t three-volume book e n t i t l e d Amino A c i d A n a 7 y s i s b y G a s C h r o m a t o g r a p h y , p u b l i s h e d by CRC Press i n 1987. Those volumes c o n t a i n two m a j o r c h a p t e r s on t h e search f o r amino a c i d s i n l u n a r s o i l and cosmo c h e m i s t r y by Gehrke, Kuo, Zumwalt, and Ponnamperuma. The e d i t o r s extend t h e i r deep a p p r e c i a t i o n t o D r . Zumwalt f o r h i s t e c h n i c a l a b i l i t i e s and e d i t o r i a l e x p e r t i s e i n a c c o m p l i s h i n g the completion o f t h i s t r e a t i s e .

xx EDITORS

The editors, Kenneth C. Kuo and Charles W. Gehrke,in front of the historic col umns . on Francis Quadrangle, University o f Missouri-Columbia. Right: Robert W. Zumwalt.

xx I CHARLES W. GEHRKE C h a r l e s William Gehrke was born i n 1917 i n New York C i t y . He s t u d i e d a t Ohio S t a t e U n i v e r s i t y , r e c e i v i n g a B . A . i n 1939. From 1941 t o 1945, he was p r o f e s s o r and chairman of t h e Department of Chemistry a t Missouri Valley College, Marshal 1 , Missouri , t e a c h i n g chemistry and physics t o s e l e c t e d Navy midshipmen from d e s t r o y e r s , b a t t l e s h i p s and a i r c r a f t c a r r i e r s of World War I 1 i n the South P a c i f i c . These young men r e t u r n e d t o the war t h e a t e r a s deck and flight officers. In 1946, he r e t u r n e d t o Ohio S t a t e U n i v e r s i t y a s i n s t r u c t o r i n a g r i c u l t u r a l biochemistry and received h i s P h . D . degree i n 1947. In 1949 he j o i n e d t h e Col 1 ege o f Agri cul ture a t the U n i v e r s i t y of Mi s s o u r i Columbia, r e t i r i n g i n t h e Fall of 1987 from p o s i t i o n s a s P r o f e s s o r of Biochemistry, Manager of t h e Experiment S t a t i o n Chemical L a b o r a t o r i e s , and D i r e c t o r o f the U n i v e r s i t y I n t e r d i s c i p l i n a r y Chromatography Mass-Spectrometry f a c i l i t y . His d u t i e s a l s o included t h o s e of S t a t e Chemist f o r Missouri F e r t i l i z e r and Limestone Control laws. Dr. Gehrke i s now S c i e n t i f i c Coordinator a t t h e Cancer Research Center i n Col umbi a. P r o f e s s o r Gehrke i s the a u t h o r of over 250 s c i e n t i f i c pub1 ic a t i o n s i n a n a l y t i c a l and biochemistry. His r e s e a r c h i n t e r e s t s i n c l u d e t h e development of q u a n t i t a t i v e , highr e s o l u t i o n gas- and 1 iquid-chromatographic methods f o r amino a c i d s , p u r i n e s , pyrimidines, major and modified n u c l e o s i d e s i n RNA, DNA, and methylated "CAP" s t r u c t u r e s i n mRNA; f a t t y a c i d s ; and b i o l o g i c a l markers i n the d e t e c t i o n of cancer; c h a r a c t e r i z a t i o n and i n t e r a c t i o n of p r o t e i n s , chromatography of b i o l o g i c a l l y important molecules, structural c h a r a c t e r i z a t i o n o f carcinogen-RNA/DNA a d d u c t s , and automation of a n a l y t i c a l methods f o r n i t r o g e n , phosphorus, and p o t a s s i urn i n f e r t i 1 i z e r s . Automated spectrophotometri c methods have been developed f o r l y s i n e , methionine, and c y s t i ne. P r o f e s s o r Gehrke has been an i n v i t e d s c i e n t i s t t o

XXI I l e c t u r e on g a s - l i q u i d chromatography o f amino a c i d s i n Japan, China, and a t many u n i v e r s i t i e s and i n s t i t u t e s i n t h e U n i t e d S t a t e s and Europe. He p a r t i c i p a t e d i n t h e a n a l y s i s o f l u n a r samples r e t u r n e d b y A p o l l o f l i g h t s 11, 12, 14, 15, 16, and 17 f o r amino a c i d s and e x t r a c t a b l e o r g a n i c compounds w i t h P r o f e s s o r Cyri 1 Ponnamperuma, U n i v e r s i t y o f Mary1 and, and w i t h a consortium o f s c i e n t i s t s a t t h e National Aeronautics and Space A d m i n i s t r a t i o n Ames Research C e n t e r , C a l i f o r n i a . I n 1971, he r e c e i v e d t h e annual A s s o c i a t i o n o f O f f i c a1 H a r v e y W. W i l e y Award i n A n a l y t i c a l C h e m i s t s ' (AOAC) A n a l y t i c a l C h e m i s t r y and was r e c i p i e n t o f t h e S e n i o r Facu t y Member Award, UMC C o l l e g e o f A g r i c u l t u r e , i n 1973. In August, 1974, he was i n v i t e d t o t h e S o v i e t Academy o f S c i e n c e s t o make a summary p r e s e n t a t i o n on o r g a n i c s u b s t a n c e s i n l u n a r f i n e s t o t h e O p a r i n I n t e r n a t i o n a l Symposium on t h e "Origin o f Life." I n 1975, he was s e l e c t e d as a member o f t h e American Chemical S o c i e t y C h a r t e r Review B o a r d f o r Chemical A b s t r a c t s . As an i n v i t e d t e a c h e r u n d e r t h e s p o n s o r s h i p o f f i v e C e n t r a l American governments, he t a u g h t c h r o m a t o g r a p h i c a n a l y s i s o f amino a c i d s a t t h e C e n t r a l American Research I n s t i t u t e f o r I n d u s t r y i n Guatemala, 1975. He was e l e c t e d t o Who's Who i n M i s s o u r i E d u c a t i o n and r e c i p i e n t o f t h e F a c u l t y - A l u m n i G o l d Medal Award i n 1975, and was t h e r e c i p i e n t o f t h e p r e s t i g i o u s Kenneth A. Spencer Award f r o m t h e Kansas C i t y S e c t i o n o f t h e American Chemical S o c i e t y f o r m e r i t o r i o u s achievement i n a g r i c u l t u r a l and f o o d chemistry, 1979-80. P r o f e s s o r Gehrke r e c e i v e d t h e T s w e t t "Chromatography Memorial Medal " f r o m t h e S c i e n t i f i c Counci 1 on Chromatography, Academy o f S c i e n c e s o f t h e USSR, Moscow, 1978, and t h e Sigma X i S e n i o r Research Award b y t h e U n i v e r s i t y o f M i s s o u r i - C o l u m b i a C h a p t e r , 1980. I n 1986, he was t h e r e c i p i e n t o f t h e American Chemical S o c i e t y M i d w e s t Award. He was an i n v i t e d s p e a k e r on " M o d i f i e d N u c l e o s i d e s and C a n c e r " i n F r e i b u r g , West Germany, 1982, and gave p r e s e n t a t i o n s as an i n v i t e d s c i e n t i s t t h r o u g h o u t Japan, m a i n l a n d China, Taiwan, P h i l i p p i n e s , and Hong Kong i n 1982

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and 1987. He was s e l e c t e d f o r the Board of D i r e c t o r s and E d i t o r i a l Board o f t h e AOAC, 1979-80; President-Elect of the Association of O f f i c i a l Analytical Chemists I n t e r n a t i o n a l Organization, 1982-83; and was honored by the e l e c t i o n a s the Centennial P r e s i d e n t in 1983-84. He developed " L i b r a r i e s of Instruments" i n t e r d i s c i p l i n a r y research programs on strengthe n i n g research i n American Universities. Dr. Gehrke i s founder and chairman of the Board of D i r e c t o r s , Analytical Biochemistry Laboratories, Inc. , 1968 t o present, a p r i v a t e corporation of 200 s c i e n t i s t s , engineers, b i o l o g i s t s , and chemists s p e c i a l i z i n g i n chromatographic instrumentation, and addressing problems worldwide i n the environment. Over s i x t y masters and doctoral students have received t h e i r advanced degrees i n a n a l y t i c a l biochemistry under the d i r e c t i o n of Professor Gehrke. In a d d i t i o n t o his e x t e n s i v e c o n t r i b u t i o n s t o amino a c i d a n a l y s i s by gas chromatography, Dr. Gehrke and colleagues have pioneered i n the development of s e n s i t i v e , high-resolution, q u a n t i t a t i v e high-performance l i q u i d chromatographic methods f o r over 100 major and modified nucleosides i n RNA, DNA, mRNA, and then applied t h e i r methods i n c o l l a b o r a t i v e research w i t h s c i e n t i s t s i n molecular biology across t h e world. Professor Ernest Borek a t t h e 1982 I n t e r n a t i o n a l Symposium on Cancer Markers, Frei b u r g , West 'Germany, s t a t e d t h a t Professor Gehrke's chromatographic methods a r e being used s u c c e s s f u l l y by more than h a l f o f the s c i e n t i s t s i n attendance a t these meetings. Professor Gehrke, w i t h Dr. Robert Zumwalt and Mr. Kenneth Kuo, i s the s e n i o r a u t h o r / e d i t o r of a three-volume comprehensive t r e a t i s e e n t i t l e d "Amino Acid Analysis by Gas Chromatography, pub1 ished by CRC Press (1987). The volumes include 19 c h a p t e r s contributed by leading s c i e n t i s t s from twel ve n a t i o n s . I n 1 9 8 9 , P r o f e s s o r Gehrke and P r o f e s s o r C y r i 1 Ponnamperuma of the University of Maryland were named cop r i n c i p a l i n v e s t i g a t o r s on a proposal t o place on the moon a chemical l a b o r a t o r y which will be automated, m i n i a t u r i z e d , "

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computer r o b o t i c - o p e r a t e d and w i 11 s u p p o r t NASA programs i n t h e s t u d y o f f i v e aspects o f t h e e x p l o r a t i o n o f space; (a) a s t r o n a u t h e a l t h , (b) c l o s e d environment l i f e s u p p o r t , (c) 1unar resources, (d) exobi o l ogy, and (e) p l a n e t o l ogy. I n 1989, P r o f e s s o r Gehrke and Kenneth Kuo a r e a u t h o r s / e d i t o r s o f t h i s four-volume t r e a t i s e e n t i t l e d "Chromatography and M o d i f i c a t i o n o f Nucleosides," p u b l i s h e d by E l s e v i e r i n t h e J o u r n a l o f Chromatography L i b r a r y s e r i e s . These t h r e e volumes address " A n a l y t i c a l Methods f o r M a j o r and M o d i f i e d Nucleosides", "Biochemical Roles and F u n c t i o n o f M o d i f i c a t i o n " , " M o d i f i e d Nucleosides i n Cancer and Normal Metabo1 ism" , and "Comprehensive Database f o r RNA and DNA Nucl eos ide s "

.

KENNETH C. T. KUO Kenneth C. T. Kuo was born i n 1936 i n China. He s t u d i e d a t Chun-Yen I n s t i t u t e o f Science and Engineering, Taiwan, r e c e i v i n g a B . S . degree i n Chemical E n g i n e e r i n g i n 1960. A f t e r f u l f i l l i n g a m i l i t a r y s e r v i c e o b l i g a t i o n , he e n r o l l e d a t t h e U n i v e r s i t y o f Houston. I n 1963, he j o i n e d t h e Chevron Chemical Company i n Richmond, California, developing p e s t i c i d e r e s i d u e a n a l y t i c a l methods and s t u d y i n g p e s t i c i d e metabolism. R e c o g n i z i n g t h e power o f g a s - l i q u i d chromatography (GLC) and t h e need o f h i g h r e s o l u t i o n , s e n s i t i v i t y , and speed i n t h e a n a l y s i s o f amino a c i d s , he a p p l i e d and was accepted as a member o f t h e r e s e a r c h team under P r o f e s s o r Charles Gehrke a t t h e U n i v e r s i t y o f M i s s o u r i Columbia i n 1968. He developed mixed phase columns f o r h i s t i d i n e , a r g i n i n e , and c y s t i n e , which a l l o w t h e dual column complete q u a n t i t a t i o n o f p r o t e i n amino a c i d s i n 30 minutes by GC. He, along w i t h Drs. Gehrke, S t a l l i n g , and Zumwalt, i n v e n t e d t h e Solvent-Vent Chromatographic System (U.S. P a t e n t No. 3,881,892), which e l i m i n a t e s t h e sample s o l v e n t e f f e c t i n GC a n a l y s i s . T h i s s o l v e n t - v e n t i n g d e v i c e was used i n t h e search f o r amino a c i d s i n t h e r e t u r n e d A p o l l o l u n a r samples o v e r t h e p e r i o d from 1969-1974, t h u s p r o v i d i n g a s e n s i t i v i t y

xxv f a c t o r of 100 g r e a t e r than c l a s s i c a l ion-exchange a n a l y s i s a t t h a t time. He received h i s M . S . degree i n a n a l y t i c a l biochemistry under P r o f e s s o r Gehrke i n 1970. During the l a s t 20 y e a r s , he and Dr. Gehrke have d e d i c a t e d t h e i r research e f f o r t s t o the developments of q u a n t i t a t i v e high r e s o l u t i o n chromatographic methods f o r biochemical and biomedical r e s e a r c h . He p a r t i c i p a t e d i n the NASA Apol 1 o Returned Lunar Sample c o n s o r t i um of s c i e n t i s t s s e a r c h f o r evidence of chemical e v o l u t i o n i n the l u n a r samples from Apollo missions 11 through 17 (1969 t o 1974). He h a s s t u d i e d b i o m a r k e r s f o r c a n c e r , and developed q u a n t i t a t i v e high r e s o l u t i o n chromatographic methods f o r polyamines, protein-bound n e u t r a l s u g a r s , 8-aminoisobutyric a c i d and j3-alanine; and modified r i b o n u c l e o s i d e s i n human u r i n e and serum. In the l a s t f i v e y e a r s , h i s major e f f o r t s have been d i r e c t e d t o the development of a package of methods f o r the complete q u a n t i t a t i v e composition a n a l y s i s of DNA, mRNA, and t R N A by high r e s o l u t i o n HPLC. Through t h e s e methods, more than 70 major and modified r i b o n u c l e o s i d e s , 15 deoxynucleosides, and 9 mRNA cap s t r u c t u r e s can be i d e n t i f i e d and measured i n n u c l e i c a c i d s o r body f l u i d s . He was an i n v i t e d s c i e n t i s t by the Chinese Academy of Science i n 1982 and l e c t u r e d throughout China on t h e chromatography of nucleosides. He has c o n t r i b u t e d t o over f i f t y s c i e n t i f i c pub1 i c a t i o n s i n a n a l y t i c a l chemistry and biochemistry.

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CONTRIBUTORS

KARL-SIEGFRIED BOOS

Karl-Siegfried Boos was born in 1948 in Rastatt/Baden, Federal Republic of Germany. He studied biochemistry at the Technical University of Hannover and graduated with a diploma in biochemistry in 1974. Between 1975 and 1977 he worked on his doctoral thesis under the auspices of professor Eckhard Schlimme and Professor Walther Lamprecht at the Department of Biochemistry, Medical University School, Hannover. Shortly after he received the academic degree Dr. rer. nat. in 1977, he spent one and a half years as an American Muscular Dystrophy Association and German Research Association (DFG) post doctoral fellow at the Department of Biochemistry and Biophysics, Washington State

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University, Pullman, Washington, in the laboratory of Professor Ralph G. Yount. He then rejoined Dr. Eckhard Schlimme at the Laboratory for Biological Chemistry, University of Paderborn, as an assistant. In 1984, he habilitated and received the academic degree 'Privatdozent' from the University of Paderborn. Since 1985 he has been the head of the Laboratory for Biological Chemistry at the University of Paderborn and was appointed Professor of Biological Chemistry in 1987. His research interests are documented in about 50 scientific papers and they focus on the synthesis and properties of modified nucleotides and biomimetic pseudonucleotides as molecular probes in energy transducing biosystems and on the HPLC analysis of marker molecules in biological fluids.

PHYLLIS R. BROWN Phyllis R . Brown was born in 1924 in Providence, Rhode Island. She received her B.S. in chemistry at George Washington University. After an educational hiatus of eighteen years, she returned to school and received her Ph.D. in chemistry in 1968 from Brown University where her graduate advisor was John 0. Edwards. She did postdoctoral work in the Pharmacology section at Brown for three years and stayed on in that section as instructor and then as an assistant professor in research. In 1973 she became an assistant professor in the department of chemistry at the University of Rhode Island where she became an associate professor in 1977 and professor in 1980. Dr. Brown has been a pioneer in the application of HPLC to biomedical research and has made outstanding contributions in the development of HPLC assays for biochemical research and the clinical laboratory. She is best known for her work in developing assays for nucleic acid constituents in biological samples. The HPLC methods she developed in 1970 for the separation of nucleotides in cell extracts are now the standard procedures used by biomedical researchers studying metabolism in normal subjects and patients with various diseases. She developed the highly selective and sensitive enzyme peak shift technique for identification of peaks in chromatograms of biological matrices and was a leader in systematizing methods for identification of biologically important peaks. She was the first to use reversed-phase HPLC (RPLC) methods to determine concentrations of nucleosides and their bases

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in physiological fluids and established a range of normal values for these compounds in blood fluids. Her current interests include the separation of oligonucleotides, mRNAs, neuro and gastrointestinal peptides, fatty acids and triglycerides. In addition she is working on fast, microbore and preparative HPLC and the use of computers in all phases of HPLC. Dr. Brown wrote the first book on biomedical and biochemical applications of HPLC (Academic Press, 1973) and was senior author of the first book devoted entirely to RPLC (Wiley, 1982). Both these books were translated into Japanese. In addition she edited a book on "HPLC in Nucleic Acid Research" (Marcel Dekker, 1984) and with Dr. J. Calvin Giddings and Dr. Eli Grushka edits the "Advances in Chromatography" series published annually by Marcel Dekker. She is on the editoral board of numerous scientific journals and a member of many professional organizations. In 1983 Dr. Brown was a Visiting Professor at Hebrew University in Jerusalem and recently was awarded a Fulbright Fellowship to return to Israel to continue her research there. She

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was awarded the Scholarly Achievement Award for Excellence in Research at the University of Rhode Island in 1985 and also named Woman of the Year by the Business and Professional Women of South County, Rhode Island. In addition she was given the Community Service Award by the Providence Chapter of the National Council of Jewish Women in 1984.

GlRlSH B. CHHEDA

High Mass High Resolution Mass Spectrometer Finnigan MAT-90; Girish B. Chheda (front), Shib P. Dutta, Henry A. Tworek and Helen B. Patrzyc

xxx Girish B. Chheda was born in the state of Gujrat in Western India. He attended high school in Bombay and studied at the University of Bombay receiving his B.Sc. (hons.) in chemistry and B.Sc. Tech. in Pharmaceuticals in 1955 and 1957, respectively. After a short work period at Glaxo Laboratories in Bombay, he came to the United States in 1958. After receiving an M.S. degree in Pharmaceutical Chemistry from the University of Michigan, he attended the State University of New York at Buffalo for his Ph.D. in Medicinal Chemistry. His Ph.D. work on synthesis of tetracycline analogs, under Prof. H. J. Schaeffer, was awarded the Ebert Prize of the American Pharmaceutical Association in 1964. After postdoctoral work with Prof. B. R. Baker, he joined Prof. Ross H. Hall's group as a Senior Research Associate at Roswell Park Memorial Institute in 1964. He became a staff member in 1966, and moved up through the ranks and currently holds the title of Cancer Research Scientist V I in the Department of Biophysics. He also is the director of the mass spectrometry facilities for the institute. He is author and co-author of. over 90 papers in the areas of isolation, characterization, chemistry and biochemistry of modified nucleosides.

FlLlBERTO ClMlNO Filiberto Cimino was born in 1939 in Naples, Italy. He studied at the University of Naples and received his M.D. degree in 1963. After postdoctoral training in the Institute of Biochemistry of the same university, he was associated with the Laboratory of Biochemistry of the National Heart and Lung Institute, NIH, Bethesda, USA (1968-1970). It was in this laboratory, under the guidance of Dr. Earl Stadtman, that he completed his training in enzymology and protein chemistry working on several aspects of the structure and regulation of bacterial glutamine synthetase. Shortly thereafter he was appointed to the University of Naples, moving through the ranks of associate (1970) and full (1975) professor of biochemistry in the Second School of Medicine. At present he holds the position of director of the Institute of Biochemical Sciences at the Second School of Medicine of the University of Naples. Dr. Cimino is author and co-author of over 100 papers in the areas of amino acid metabolism, enzyme regulation, methylation of tRNA, and modified nucleosides as tumor markers. He became interested in HPLC in 1982, and he was the first to apply the

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Some members of the Naples Group: from left to right, Franca Esposito, Francesco Salvatore, Tommaso Russo and Filiberto Cimino

technique to the analysis of pseudouridine and other modified nucleosides in tissue extracts and in the biological fluids of both human and animal experimental systems. His most significant contributions are those dealing with the mechanisms that provoke the increased production of pseudouridine in transformed cells. At present, he is investigating tRNA gene regulation in normal and transformed cells.

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IRWIN CLARK

Irwin Clark was born in Boston, Massachusetts in 1918. He attended Boston Latin School and received an A.B. degree in Biochemical Sciences from Harvard College in 1939. The following year was spent as a graduate student in chemistry at the University

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of Michigan. From 1940 to 1942 he was in the merchant marine. From 1942-1947, he was employed by the Merck Institute for Therapeutic Research. In 1950, he received the Ph.D. degree from the College of Physicians and Surgeons, Columbia University under the direction of Dr. David Rittenberg. From 1950-1951 he was a Senior Fulbright Scholar at the Dunn Nutritional Institute in Cambridge, England. From 1952-1959 he was with the Merck Institute for Therapeutic Research and resigned to go to the College of Physicians and Surgeons, Columbia University as Assistant Professor of Biochemistry rising to Professor of Biochemistry. In 1970 he resigned his position and accepted a position at the University of North Carolina School of Medicine, Chapel Hill, N.C., as Professor of Biochemistry and Surgery and Associate Director of the Orthopedic Research Laboratories. In 1974 he resigned this position to accept a position as Professor of Surgery at UMDNJ-Rutgers Medical School (now Robert Wood Johnson Medical School), a position he still holds. Dr. Clark is the author and co-author of over 70 articles in areas of endocrinological biochemistry, skeletal metabolism, mineral metabolism, analytical metrology, steroids, nucleic acids and cancer. SHIB PRASAD DUTTA Shib Prasad Dutta received his Master of Science and Ph.D. degrees in organic chemistry from the University of Calcutta, India. He has been involved in the synthesis of modified nucleoside derivatives and in the isolation and identification of nucleoside metabolites present in human biological fluids. Presently he is a Cancer Research Scientist II in the Department of Biophysics at the Roswell Park Memorial Institute.

ALF FlSCHBElN Alf Fischbein was born in 1945 in Malmo, Sweden. He is a graduate from the Faculty of Medicine of the University of Lund, Sweden. He came to the Mount Sinai School of Medicine of the City University of New York in 1973 and is currently Associate Professor of Community Medicine (Occupational and Environmental Medicine). His clinical research has focused on health effects associated with various occupational exposures, with emphasis on asbestos related

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disorders, lead induced disease and effects of exposure to halogenated hydrocarbons including PCB's. He has served on expert committees of the U S . Environmental Protection Agency and the International Labour Office in Geneva, Switzerland. His current research focuses on the development of biological markers of exposure and early effects of occupational exposures by incorporating such markers into epidemiological studies. In collaboration with the late Dr. Ernest Borek and his successor Dr.

xxxv Opendra K. Sharma of the AMC Cancer Center and Hospital in Denver, Colorado he has investigated the applicability of using the excretion pattern of modified nucleosides as an indicator of asbestos exposure in order to identify individual members of asbestos exposed populations who are at increased risk for developing occupationally related cancer. He is currently studying the possibility that modified nucleosides can serve as predictive markers for occupational cancer by evaluating the prospective mortality pattern of a large population of asbestos exposed workers.

YONG-NAM KIM

Yong-Nam Kim was born in 1951 in Pusan, Korea. He studied in Seoul at Yonsei University receiving his B.S. degree in Chemistry in 1977, and his M.S. degree in Analytical Chemistry in 1979. After serving as a n instructor at Yonsei University for one year, he joined Kyungnam University in Masan a s a member of the chemistry I n 1981 he went to Northeastern University in Boston, faculty. Massachuetts and worked with Dr. B.L. Karger on reversed-phase high-performance liquid chomatography of proteins. After two years, he moved to the University of Rhode Island in Kingston, Rhode Island and studied high-performance liquid chromatography for biomedical application.. under the direction of Dr. P.R. Brown. In

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graduate school, he was elected to Sigma Xi and was awarded a Perkin-Elmer Graduate Fellowship. He received his Ph.D. degree in Analytical Chemistry in 1987 with a thesis on micellar liquid chromatograph y of n uc le o s ides and bases . Dr. Kim returned to Kyungnam University where he teaches Analytical Chemistry. H e has been writing a textbook on c h r o mat o g r a p h y . H i s research interests include d ev e 1op ni en t of chromatographic methods for biomedical and biological applications. WIN LIN

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Win Lin was born in 1949, Taiwan, Republic of China. She received a B.S. degree from Chung Hsing University, Taiwan, i n 1970. In 1971 she came to the United States and enrolled in the graduate School at the City University of New York and received a Ph.D in Biochemistry. Her thesis work involved the synthesis of fetal and adult hemoglobins. In 1976 her first child was born and in 1979 her second. She joined UMDNJ-Robert Wood Johnson Medical school (formerly Rutgers Medical School) as a Research Specialist and became an Instructor in 1983, a position she currently holds. She is a member of Sigma Xi and the American Association of Cancer Research. She is an expert in the area of liquid chromatography and her present research involves the use of RNA catabolites as markers in health and disease.

JAMES W. MACKENZIE James (“Jim”) William Mackenzie was born in 1925 in Cleveland, Ohio. He obtained his B.S.degree from the University of Michigan in 1948, majoring in English. In 1951 he received his Medical Degree from the University of Michigan Medical School. He completed his internship, a residency in general thoracic surgery at the University Hospital in Ann Arbor, Michigan in 1960. From 19531955 he served in the U.S. Naval Reserve. Upon completing his residency training, he was appointed to the faculty of the University of Michigan from July of 1960 through June of 1962. In 1962 he was appointed Chief of the Section of Thoracic and Cardiovascular Surgery at the University of Missouri, moving through the ranks of Assistant Professor, Associate Professor, Professor and Assistant Chairman of the department of Surgery. In 1969, he left the University of Missouri to assume the position of Professor and Chairman of the Department of Surgery at the College of Medicine and Dentistry of New Jersey (CMDNJ) - Rutgers Medical School (now UMDNJ-Robert Wood Johnson Medical School). From 1971-1975, Dr. Mackenzie was the Dean of CMDNJ-Rutgers Medical School while continuing his position as Professor and Chairman of the Department of Surgery, a position he still holds. Dr. Mackenzie is the author and co-author of over 58 papers, 6 chapters and 28 abstracts in the areas of lung cancer, myocardial infarction, RNA catabolites as cancer markers, bowel ischemia, and molecular biology.

X X X V I II

James W. Mackenzie

JOHN E. McENTlRE John E. McEntire, Ph.D. is Vice President and Technical Director of Tektagen, Inc., a Malvern, PA, company specializing in providing regulatory-driven testing services to the manufacturers of biological pharmaceuticals. As such, he is responsible for the development of methods for protein characterization, quantification of residual DNA, and virology/cell culture services which are performed under strict GMP. Prior to joining Tektagen, McEntire was Vice President for Operations and Product Development at IMBIC Corporation of Columbia, MO and Assistant Director of the Cancer Research Center, also of Columbia, MO. He also held appointments at the University of Missouri. He is a biochemist with experience

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primarily in the production, purification and biochemical He has purified, sequenced, characterization of lymphokines. synthesized and provided materials for clinical trials of macrophage activating factor and holds a patent for purification of this material from lymphoblastoid cells. He has also developed improved assay systems for clinically useful molecules such as monoamine oxidase, macrophage phagocytic function and mycoplasma contamination. He has extensive experience in the purification and analytical characterization of proteins/peptides by HPLC techniques as well as other biochemical analytical HPLC techniques for amino acids, drugs and in protein microsequencing. McEntire received his B.S. in Biology from Texas Christian University, his M.S. in Microbiology and his Ph.D in Biochemistry from the University of Houston. He trained as a postdoctoral fellow at the University of Texas Medical Branch, Galveston.

EDITH MITCHELL Dr. Edith Mitchell is a senior oncologist in the Hematology/Oncology Department. She received her BS (1 969) in Biochemistry from Tennessee State University at Nashville, graduating with distinction and election to Beta Kappa Chi and Alpha Kappa Mu. She completed her MD (1974) from the Medical College of Virginia. She then went to Meharry Medical College for postgraduate studies in Internal Medicine, and later to Georgetown University where she completed a post-doctoral fellowship in Medical Oncology (1981). She was director of Medical Oncology at David Grant United States Air Force Medical Center, Fairfield, California. Since 1985, she has been at the University of MissouriColumbia School of Medicine where she directs tumor immunology research. Current research in her division involves investigations of monoclonal antibodies in diagnostic imaging, development of immunoconjugates for clinical research, clinical evaluation, nuclear science and other tumor markers, and research on markers to monitor cancer. Research discussed in her chapter has been sponsored by the National Institutes of Health, the Cytogen Col., and internal research and developmental funds.

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Edith Mitchell KATSUYUKI NAKANO Katsuyuki Nakano was born in 1943 in Onomichi City, Hiroshima Prefecture, Japan. He studied at the Kanazawa University receiving his B.S. degree in 1965, and his M.S. degree (biophysics) in 1967. During his master course, he studied molecular genetics at the Cancer Institute, School of Medicine, Kanazawa University. He graduated from the training school for ministers of the Perfect Liberty (PL; a religious organization) receiving a PL ministers degree in 1969. He had a position at the PL Medical Data Center and studied in the field of medical information. In 1976, he studied again at the graduate school of the Waseda University in Tokyo, and received his Ph.D. degree (biophysics) in the field of quantum biology

XL I

in 1980. After that time, he returned to the PL Medical Data Center. He studied high-performance liquid chromatography in Prof. P.R. Brown's Laboratory at the University of Rhode Island (URI) as a visiting researcher in 1980. Dr. Nakano is the author of over 30 scientific publications in analytical chemistry, biophysics, and medical information. His present research interests include the development of highperformance liquid chromatographic (HPLC) methods for biochemical markers in the detection of cancer and the diagnosis of genetic diseases, and their application to automated health check-up systems. In the PL Medical Data Center, Dr. Nakano has developed an online computer system and clinical database system of two PL Health Control Centers (Tokyo and Osaka). He participated in a statistical analysis of health examination results, especially in risk factors for cancer and individual normal ranges of clinical test results under the guidance of Dr. Yasaka, Director of the PL Medical Data Center. After he returned from Prof. Brown's laboratory of URI, he studied nucleosides and bases in biological samples of cancer patients in terms of HPLC to find biochemical markers in the detection of cancer.

XLI I He has a membership in the following professional societies: Physical Society of Japan, Biophysical Society of Japan, Japanese Cancer Association, Japan Society of Medical Electronics and Biological Engineering, Japan Society of Clinical Chemistry, and Japan Society of AMHTS. He is currently teaching at the PL Women's Junior College and the PL School of Nursing.

HELEN B. PATRZYC Helen B. Patrzyc received her B.S. degree in Medical Science from the State University of New York at Buffalo and M.S. degree in chemistry from Canisius College. She has been involved in DNA-polynucleotides-solvent interferon induction studies, interactions, and macromolecular chemical modification studies. More recently she has worked toward the development of isolation methodology and in characterization studies of urinary nucleosides. She is a Cancer Research Scientist I at the Roswell Park Memorial Institute in Buffalo, New York.

TOMMASO RUSSO Tommaso Russo was born in Naples on November 26, 1951; he is married with two children. Dr. Russo obtained his degree in medicine and surgery in 1976. From 1976 to 1980 he was assistant physician at the "Cardarelli" hospital in Naples. From 1981 to 1986 he was investigator at the Italian National Research Council (CNR) at the Institute of Biochemical Sciences of the Second School of Medicine and Surgery at the University of Naples. In 1982 he became professor of Special Biochemistry of Organs and Tissues assigned to the School of Specialization in Clinical Biochemistry of the Second School of Medicine at the University of Naples. He is author and coauthor of many scientific publications. He has presented the results of his studies at various international meetings. Dr. Russo's work has focused on the metabolism of tRNA in neoplastic cells and on biochemical signals of neoplastia.

LUCIA SACCHETTI Dr.

Lucia

Sacchetti

is

Associate

Professor

of

Clinical

XLIII Biochemistry at the Second School of Medicine at the University of Naples. She received her Ph.D. degree from the University of Naples in 1972, and between 1974 and 1982, before assuming her present position, she was a member of the Faculty of the Second School of Medicine at the University of Naples. Dr. Sacchetti is a member of the American Association of Clinical Chemistry, the Italian Society of Clinical Biochemistry (SIBioC) and the International Society for Clinical Enzymology. She sits on a committee mandated to investigate the possible areas for the clinical application of isoenzyme determination. She has published a number of scientific articles on both the methodology and the diagnostic relevance of enzyme and isoenzyme evaluation in clinical chemistry. She is also interested in isoenzymes and macroisoenzymes as tumor markers.

FRANCESCO SALVATORE

Some members of the Naples Group: from left to right, Francesco Salvatore, Luciana Sacchetti, Filiberto Cimino, Fabrizio Pane and Tommaso Russo. Unfortunately, Marcella Savoia was not available the day the picture was taken.

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Francesco Salvatore was born in 1934, in Naples, Italy. He studied at the University of Naples, receiving his M.D. in 1956, and a His initial research was Ph.D in biochemistry (Rome, Italy) in 1960. in the field of amino acid metabolism, in particular, nitrogen catabolism and urea synthesis; he also studied the methyl transfer reaction with reference to transfer-RNA post-transcriptional modifications. GERNOT SANDER Gernot Sander was born in 1941 in Gottingen, Germany. He studied biology and chemistry in Gottingen and Tubingen and, in 1968, received his Ph.D. in biochemistry (Dr. rer. nat.) from the University of Gottingen (Dr. H. Matthaei) for work on the binding of transfer RNA to ribosomes. After a postdoctoral stay at Caltech, Pasadena, Calif. (with Dr. J. Bonner) 1969-1971 working on chromatin, he returned to Germany to work with Dr. A. Parmeggiani at the Gesellschaft fur Molekularbiologische Forschung in Braunschweig (1971 -1975) on bacterial elongation factors EF-Tu and EF-G and their interactions with (aa-)tRNA and ribosomes. In 1975 he followed Dr. Parmeggiani to his new Laboratory at the Ecole Polytechnique, Palaiseau, on the outskirts of Paris, France, to continue work on elongation factors, roles of ribosomal proteins in bacterial protein synthesis and the mechanism of action of antibiotics. In 1980, he accepted an appointment as head of a research group in the Laboratory of Dr. H.G. Wittmann at the Max-Plancklnstitut fur Molekulare Genetik, Berlin. Since 1982 he has worked with Dr. G. Schoch as head of a research group (Clinical Molecular Biology) at the Forschungsinstitut fur Kinderernahrung (Research Institute of Child Nutrition, director Dr. med. G. Schoch) in Dortrnund, Germany. Of over 40 original papers published by Dr. Sander as author or co-author, the ones printed up till 1982 mostly dealt with bacterial protein synthesis. Since then, his interest has focused more on RNA turnover in man and other mammals, and on possible links between RNA turnover, protein turnover and energy metabolism.

XLV

Gernot Sander

MARCELLA SAVOIA Marcella Savoia is research assistant at the "lnstituto di Scienze Biochimiche", Section of Clinical Biochemistry, of the Second School of Medicine at the University of Naples. She received a degree in biology from the University of Naples in 1981, and in

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1985 she received a degree in clinical chemistry from the University of Camerino. Marcella Savoia has spent a period of time in the laboratory of Dr. Charles Gehrke where she worked on a project aimed at the analysis of modified nucleosides in body fluids. She has also received training in the laboratory directed by Prof. Morton Schwartz in New York where she investigated several tumor markers and other aspects in clinical biochemistry. Her research interests are centered on the study of serum pseudouridine as a tumor marker, in particular its diagnostic sensitivity and specificity, in patient surveillance.

ECKHARD SCHLIMME Eckhard Schlimme was born in 1937 in Einbeck, Germany. He obtained his diploma degree in Chemistry from the University of Gottingen in 1963. In 1966 he received the degree 'Dr. sc. agr.' from the same University; his scientific adviser was Professor Fritz Scheffer. From 1967 until 1970 he was a research assistant of Professor Friedrich Cramer at the Max-Planck-Institute for Experimental Medicine in Gottingen. During that time he received his 'Dr. rer. nat.' from the Technical University of Braunschweig in 1969. He completed his training in biochemistry from 1971 until 1973 in the Institute for Physiological Chemistry and Clinical Biochemistry (Head: Professor Walther Lamprecht), Medical University School, Hannover. In 1972 he received the academic degree 'Privatdozent' (Habilitation) for Physiological Chemistry in Hannover and was appointed in 1973 to the newly founded University of Paderborn as a professor of organic chemistry (head of the the Laboratory for Biological Chemistry); 1978-1979 he was dean of the Department of Natural Sciences, and he was elected as a 'Prorektor' of the University of Paderborn from 1983 until 1985. In 1985 he was appointed as a Director and Professor to the Federal Dairy Research Centre in Kiel and became the Head of the Institute for Chemistry and Physics. Since 1986 he has been a member of the Medical Faculty of the University of Kiel and became an honorary professor at the University of Paderborn in January 1987. Dr. Schlimme is author and co-author of about 100 papers in the areas of biochemistry and organic chemistry of nucleic acids and related compounds. During the time in Friedrich Cramer's lab he worked together with Dr. Friedrich von der Haar on the structure and function of tRNA. In Hannover, he started working on the topographic

XLVI I

characterization of the mitochondria1 adenine nucleotide carrier by use of synthesized nucleotide analogs developed in Paderborn with Dr. Karl-Siegfried Boos [a hypothesis of the functional mechanism underlying the ATP, ADP transmembrane exchange]. Dr. Schlimme's involvement in liquid chromatography started in 1971 when he pioneered with Dr. Kurt-Wilhelm Stahl and Dr. Fritz Eckstein in separating the disastereomeric forms of adenosine thiophosphate. His present HPLC work is done in close cooperation with Dr. Karl-Siegfried Boos in the field of system integral cleanup and analysis of marker molecules in body fluids. Further interests are related to structural, nutritive and technological aspects of mammal milk constituents.

XLVI I I

GESA HELLER-SCHOCH Gesa Heller-Schoch was born in 1936 in Hamburg, Germany. She studied pharmacy in Freiburg, Germany (1957 to 1960). From 1960 to 1964 she worked as a pharmacist in Munchen and was trained in the arts (painting) at the Kunstakademie, Munchen.

XLIX From 1964 to 1967 she worked with Dr. H. Matthaei at the MaxPlanck-lnstitut fur experimentelle Medizin in Gottingen in deciphering the genetic code. She obtained her Ph.D. (Dr. rer. nat.) from the University of Gottingen (Dr. H. Matthaei) with a dissertation about the non-enzymatic cleavage of aminoacyl-tRNA ester bonds. From 1967 to 1970 she worked as a molecular biologist at the Universitats-Kinderklinik Hamburg, with Dr. R. Neth. She has been working with her husband, G. Schoch, from 1970 - 1981 in Hamburg, and since 1983 at the Forschungsinstitut fur Kinderer-nahrung, Dortmund (director Dr. med. G. Schoch). After early studies on tRNA methylation in tissues and cells of healthy and leukemic donors, she turned to investigate the metabolism of normal and modified nucleosides in humans in normal and malignant growth processes, with special reference to the interrelations of nutrition and RNA turnover. She is the author and co-author of 30 original papers and a monograph.

Gerhard Schoch was born in 1936 in Sarata/Bessarabia. He studied German and French philology and history from 1956 to 1960 in Tubingen and Freiburg, Germany, and Nancy, France. He then studied medicine in Marburg and Tubingen, Germany. From 1966 to 1967 he worked with Dr. H. Matthaei, Max-Planck-lnstitut fur experimentelle Medizin, Gottingen, on the experimental proof of the genetic code in man. This work was the basis for his medical thesis (University of Tubingen, 1967). From 1968 to 1969 he was trained as an intern in different clinical specialties. After receiving a scholarship for biochemical studies from the Deutsche Forschungsgemeinschaft (University of Hamburg, 1970) he acquired his training in pediatrics at the Universitats-Kinderklinik Hamburg-Eppendorf. His special interest in this period was focused on the molecular biology of normal and malignant growth with emphasis on RNA metabolism. In 1976 he o btai ned h is " hab i I it at io n" f o r p ed iat r ics an d cI in ical m o Iec u lar biology. In 1981 he was appointed full professor of pediatrics at the University of Munster, and Director of the Forschungsinstitut fur Kinderernahrung, Dortmund (Research Institute of Child Nutrition). He obtained the following awards: best medical thesis of the

L

University of Tubingen (1 967); Adalbert Czerny-Preis of the Deutsche Gesellschaft fur Kinderheilkunde (1977): Jurgen und Margarete Voss-Preis der Werner Otto Stiftung zu Hamburg (1980).

LI

He published, as author or co-author, 77 original papers in the fields of pediatrics, nutrition and clinical molecular biology, including the monograph by G. Schoch and G. Heller-Schoch: Molekularbiologie und klinische Bedeutung des Stoffwechsels normaler und modifizierter Nucleobasen, Schwabe & Co., BaseVStuttgart 1977. His current interests comprise nutrition in pediatrics with special emphasis on molecular biological aspects of turnover of energy, protein and ribonucleic acids.

OPENDRA K. SHARMA Opendra K. Sharma received a Ph.D. in biochemistry from the University of Lucknow in India. In 1968 he joined the laboratory of Dr. Ernest Borek at Columbia University in New York; in 1969 he

LI I moved with Dr. Borek to the University of Colorado Medical Center in Denver. Dr. Sharma studied regulation of tRNA methylation in normal and tumor tissues. Subsequently he collaborated with Dr. Borek toward the development of modified nucleosides as tumor markers and their application in the new areas of asbestos exposure and AIDS. Dr. Sharma is Chief of the Laboratory of Molecular Biology at AMC Cancer Research Center in Denver. His research interests include mechanisms of regulation, and stability of RNA, regulation of oncogenes by steroid hormones and antiviral agents. HEINRICH TOPP

LIII

Heinrich Topp, born in 1957 in Hagen, Germany, graduated in 1982 in biology from the Ruhr-Universitat in Bochum, majoring in plant physiology, microbiology and immunology. He has since worked at the Forschungsinstitut fur Kinderernahrung (Research Institute of Child Nutrition, director Dr. med. G. Schoch), Dortrnund, contributing important facets to the ongoing work on RNA turnover markers in urine and serum from humans and other mammals, as documented by author and co-authorship in 9 original publications (see chapter by Schoch et af). In 1988 he obtained his Ph.D. (Dr. rer. nat.) from the University of Bochum and has since taken over the direction of part of the activities of the Clinical Molecular Biology research group at the Forschungsinstitut fur Kinderernahrung, Dortmund. HENRY A. TWOREK Henry A. Tworek received the Bachelor of Arts in Psychology and Master of Science in Natural Sciences from the State University of New York at Buffalo. He has completed the Ph.D. requirements for the Department of Physiology, Roswell Park division of SUNY at Buffalo and is expected to receive the Ph.D degree in June 1989. His dissertation and publications are under the guidance of Dr. G.B. Chheda and involve the characterization and quantitation of modified nucleosides and related compounds present in human urine. T. PHILLIP WAALKES

T. Phillip Waalkes received an A.B. degree in 1941; a Ph.D. in Organic Chemistry from The Ohio State University in 1945. During the World War II years he was associated with the Manhattan Engineering District Atom Bomb Project. In 1951 he received the M.D. degree following which he specialized in Medicine and Oncology. For approximately twenty years, he was involved in research and administrative duties at NIH, mostly in the National Cancer Institute, Bethesda, Maryland. For several years he was Associate Director of NCI in charge of Collaborative Programs with responsiblity for NCl's Chemotherapy Program and Cooperative Group Program. For the past fifteen years he has been a Professor of Oncology in the Oncology Center of the John Hopkins Medical School, Baltimore, Maryland. His publications include clinical and

LIV

biochemical areas. In more recent years, his field of research has been in Biological Markers for Cancer, in which he collaborated with and published extensively with Dr. Charles W. Gehrke and his staff at the University of Missouri.

photograph:

T. Phillip Waalkes

LV

CHROMATOGRAPHY AND MODIFICATION OF NUCLEOSIDES PART A:

ANALYTICAL METHODS FOR MAJOR AND MODIFIED NUCLEOSIDES HPLC, GC, MS, NMR, UV, and FT-IR TABLE OF CONTENTS

Introduction and Overview

Charles W. Gehrke and Kenneth C. Kuo

Chapter 1 Ribonucleoside Analysis by Reversed-Phase Liquid Chromatography Charles W. Gehrke and Kenneth C. Kuo Chapter 2 HPLC of Transfer RNAs Using Ionic-Hydrophobic Mixed-Mode Chromatography and HydrophobicInteraction Chromatography Rainer Bischoff and Larry W. McLaughlln Chapter 3 Nucleic Acid Chromatographic Isolation and Sequence Methods Gerard Keith Chapter 4 Affinity Chromatography of Mammalian tRNAs on Immobilized Elongation Factor Tu from Therrnus therrnophilus Mathias Sprinzl and Karl-Heinz Derwenskus Chapter 5 Structural Elucidation of Nucleosides in Nucleic Acids Charles W. Gehrke, Jean A. Desgrbs, Klaus 0. Gerhardt, Paul F. Agris, Gerard Keith, Hanna Sierzputowska-Gracz, Mlchael S. Tempesta and Kenneth C. Kuo Chapter 6 Three-Dimensional Dynamic Structure of Transfer RNA by Nuclear Magnetic Resonance Spectroscopy Paul F. Agris and Hanna Sierzputowska-Gracz Chapter 7 Codon Recognition: Evaluation of the Effects of Modified Bases in the Anticodon Loop of tRNA Using the Temperature-Jump Relaxation Method Henri Grosjean and Claude Houssier Chapter 8 High-Performance Liquid Chromatography of Cap Structures and Nucleoside Composition in mRNAs Kenneth C. Kuo, Chrlstine E. Smith, Zhixian Shi, Paul F. Agris and Charles W. Gehrke Chapter 9 lmmunoassays for Modified Nucleosides of Ribonucleic Acids Barbara S. Vold Chapter 10 Chromatography of Synthetic and Natural Oligonucleotides Heiner Ecksteln and Herbert Schott

PART B:

BIOCHEMICAL ROLES AND FUNCTION OF MODIFICATION TABLE OF CONTENTS

Introduction and Overview

Dieter G. Sbll

Chapter 1

Synthesis and Function of Modified Nucleosides in tRNA Glenn R. Bjbrk and Jiirg Kohl1

Chapter 2

Biosyntheis and Function of Queuine and Queuosine Helga Kersten and Walter Kersten

Chapter 3

Codon Usage and Q-Base Modification in Drosophila Melanogaster E. Kubli

LV I

Chapter 4

Solid Phase lmmunoassay for Determining the lnosine Content in Transfer RNA Edith F. Yamasaki, Altaf A. Wan1 and Ronald W. Trewyn

Chapter 5

Site Directed Replacement of Nucleotides in the Anticodon Loop of tRNA: Application to the Study of lnosine Biosynthesis in Yeast tRNAAla Keith A. Kretz, Ronald W. Trewyn, Gerard Keith and Henri Grosjean

Chapter 6

tRNA and tRNA-Like Molecules: Structural Peculiarities and Biological Recognition Rajlv L. Josh1 and Anne-Llse Haennl

Chapter 7

Mitochondrial-tRNAs Structure, Modified Nucleosides and Codon Reading Patterns Guy Dirhelmer and Robert P. Martin

Chapter 8

The Modified Nucleotides in Ribosomal RNA from Man and Other Eukaryotes B. E. H. Maden

Chapter 9

Modified Uridines in the First Positions of Anticodons of tRNAs and Mechanisms of Codon Recognition Shigeyuki Yokoyama and Talsuo Miyazawa

Chapter 10

Natural Occurring Modified Nucleosides in DNA Melanie Ehrllch and Xlan-Yang Zhang

PART D: A COMPREHENSIVE DATABASE FOR TRNA AND NUCLEOSIDESHPLC, GC, MS, NMR, UV, AND FT-IR Charles W. Gehrke, Jean A. Desgrbs, Mathias Sprlnzl, Klaus 0. Gerhardt, GBrard Keith, Paul Agrls, Susan Dltson, Dat Phan, Hanna Sierputowska-Gracz, Michael S. Tempesta, John A Hayden, Kenneth C.T. Kuo

F.

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INTRODUCTION: EARLY DEVELOPMENT OF NUCLEOSIDE MARKERS FOR CANCER

T. Phillip Waalkes and Charles W. Gehrke Johns Hopkins Oncology Center, Johns Hopkins University, Baltimore, Maryland 2 1205 Department of Biochemistry, University of Missouri-Columbia and Cancer Research Center, Columbia, Missouri 6520 1

In the early 197O's, the decision was made to initiate a program within the National Cancer Institute (NCI) in the development and evaluation of biochemical materials as potential biological markers to aid in the clinical management and assessment of cancer patients during the course of their illness. Such components might be found in the body fluids, e.g. blood and urine of patients with neoplastic diseases by appropriate analytical techniques. This decision to establish a biological markers program was based largely on the startling success found in the use of human chorionic gonadotrophin as a marker for following patients with the rare tumor choriocarcinoma. Further impetus to the program's development was also due to the more recent finding of carcinoembryonic antigen (CEA) as a nonspecific but potential biologic marker for a variety of neoplasms. It was determined that this program would have both intramural and extramural components at NCI, the latter to gain essential expertise in the experimental development and utilization of specific advanced analytical methodology. As this program evolved initially, a segment was devoted to the study of minor nucleic acid degradation products, including methylated compounds and pseudouridine, considered to be derived predominantly from transfer ribonucleic acid (tRNA) and identified as excretion products in human urine. The experimental basis for the interest in this area came from two sources. First, ample evidence had indicated that increased tRNA methylase activity in neoplastic cells was a common and consistent finding, and elevated methylated base content in tRNAs of malignant tumors and in urine from tumor-bearing animals had been reported. Much of this early experimental work had been carried out by Dr. Ernest Borek

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and his co-workers in the 1960's. For a summary, the readers are referred to Dr. Borek's review in his introduction to a symposium he chaired entitled "tRNA and tRNA Modifications in Differentiation and Neoplasia" (ref. 1). Of significance, methylation of the bases in tRNA had been found to occur after the macromolecule is formed, and of particular interest, these methylated compounds were not re-incorporated into the tRNA molecule but thought to be excreted intact. Similar evidence had been found for pseudouridine with experimental results to show that it was excreted in urine without further catabolic breakdown. Secondly, preliminary reports had indicated that patients with leukemia may excrete increased amounts of some purine and pyrimidine bases. Similar findings had been reported for pseudouridine to include patients with leukemia, and in addition others with Hodgkin's Disease. For these studies of urinary excretion of nucleobases, conventional techniques of that time of ion-exchange and paper chromatography, with and without pre-precipitation of the purines as silver salts, had been utilized to detect and identify the methylated nucleic acid components. The original semi-quantitative method of Weissman et a/. (ref. 2) or a modification of it has been used for such studies. By this procedure losses could occur, and only the bases were found in urine since the glycosidic bonds were hydrolyzed. Because of these obvious problems in tRNA catabolite determination, as well as the'cumbersome nature of the methods then employed which lacked the necessary qualities for the rapid and precise analysis of large numbers of clinical samples, other analytical techniques were necessary. At this time, at the Oak Ridge National Laboratory under the direction of Dr. Charles Scott, a program had been begun to build the necessary equipment and develop high resolution ionexchange chromatography as an analytical tool and for adaptation to such biological materials as urine. As a consequence, a collaborative effort was started between the Oak Ridge National Laboratory under the direction of Dr. John Mrochek, and the National Cancer Institute under Dr. Waalkes. The approach and the analytic techniques employed to separate and identify specific purine and pyrimidine degradation products of tRNA were reported (ref. 3) and with specific application in a preliminary publication involving urine samples from cancer patients (ref. 4). These

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preliminary investigations were considered of sufficient interest and potential to warrant further development of the analytical techniques employed with adaptation to larger numbers of specimens collected in a sequential fashion to follow the course of patient's disease, to include a larger variety of different clinical cell types of human neoplasms, and to establish the necessary normal control levels. From among many applicants, Professor Charles Gehrke, his laboratory, and staff were selected in 1970 to carry out the additional analytical development studies and the subsequent analyses of urine samples from selected patients, tumor types, and control individuals. Thus began a collaborative effort which continued for several years, and which became the basis for the subsequent development and application of high performance liquid chromatography for the detailed analysis of degradation products of tRNA in a variety of biological samples. This introduction is not intended as a review of modified nucleosides as biological markers of cancer, but rather to describe our experience in the early development and application of RPLC for the measurement of modified nucleosides in physiological fluids. The initial studies conducted by Professor Gehrke involved only three nucleosides, N2, N2-dimethylguanosine, 1m e t h y l i n o s i n e and pseudouridine and utilized gas-liquid chromatography as the analytical method (ref. 5). During these early years of modified nucleoside research, Dr. Ernest Borek served as an interested observer and consultant to the program. As the eventual analytical methodology progressed and settled on HPLC, Dr. Borek became a study participant and conducted a variety of additional related investigations, both basic laboratory and clinical in nature. In early and mid-l970's, two developments led to major advances in the analysis of modified constituents of nucleic acids in physiological fluids. The development of high performance liquid chromatographic instrumentation, reliable pumps, new detectors and efficient reversedphase columns offered a new analytical approach, and the development of a highly specific affinity chromatography sample cleanup method for isolation of ribonucleosides, based on the work of Dr. Uziel at Oak Ridge National Laboratory (ref. 6) promised to provide a much improved method for studying ribonucleosides in complex matrices such as physiological fluids (refs. 10-12).

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The University of Missouri-Columbia (UMC) component of the NCI biological markers program evolved from earlier research at UMC on the gas-liquid chromatographic analysis of nucleobases as their Our GLC studies provided trimethylsilyl derivatives (refs. 7, 8). information on the levels of a limited number of modified nucleosides in urine, however the charcoal column cleanup procedure followed by trimethylsilylation derivatization and GLC analysis of the samples allowed quantitation of only 3-4 modified nucleosides including pseudouridine (ref. 9). At the University of Missouri, we pursued research which focused both on the reversed-phase liquid chromatographic separation of the nucleosides and on the development of boronate gel affinity chromatography for isolation of the nucleosides from urine. The advantages of this combination of boronate gel cleanup and HPLC analysis over the charcoal cleanup-GLC approach were obvious, thus our efforts centered on improving and applying this new methodology in the biological markers program. In 1977, we published the first of a series of comprehensive reports describing the quantitative HPLC analysis of 6 ribonucleosides (Y, ,’A, m’l, m2G, A, and m2,rn2G) in urine (refs. 10-12). These reports described the potential advantages of HPLC with UV detection for nucleoside analysis (ref. lo), and additional research resulted in a rapid method for the analysis of pseudouridine in urine (ref. 11). The chromatographic separation of pseudouridine required less than 8 minutes and was applied to normal subjects and patients with advanced colon cancer. That limited cancer patient study indicated an elevated Ykreatinine ratio was present in the advanced colon cancer patients (ref. 11). Also, in 1978, Gehrke et a/. (ref. 12) reported a comprehensive study and described the use of the affinity gel for nucleoside isolation and presented data on linearity of response, precision, recovery, sensitivity, relative molar responses, stability of the nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. At that point in time, the analytical methods were sufficiently refined to begin to answer questions concerning the excretion of modified nucleosides: for example, the urinary excretion was found to

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be quite constant over a 24 hour period. In our earlier investigations we used only total 24 hour urine collections for the markers study. We soon realized that not only the cost but reliability of a 24 hour urine collection volume presented some serious problems. Thus, at the University of Missouri, we conducted a large scale study comparing collection of "spot Sample" at 8 AM, 10 AM, 3 PM and 8 P M vs. 24 hour collections using urinary creatinine for normalization of the nucleoside concentrations. The data revealed that the nucleoside/creatinine ratios were remarkably similar in random or "spot" samples as compared to 24 hour urine samples. It is apparent, therefore, that the excretion of nucleosides relative to creatinine is constant, not episodic. We also investigated the consistency of day-to-day nucleoside excretion. Thus "spot samples" provided data as valid as data from 24 hour collections, and we were collecting data that suggested that the range of excretion levels of some nucleosides is quite narrow for normal subjects. In addition, variables as the effect of diet, chemotherapeutic drugs, and diseases other than cancer were studied. Our conclusions were that these factors had little or no effect on the levels of urinary nucleoside excretion. From these studies, we established the foundation information on "normal" baseline values for the nucleoside markers program (refs. 12, 13, 18). The 1976 report by Waalkes et a/. (ref. 14) on the elevated excretion of P-aminoisobutyric acid (P-AIBA), a catabolic end product of nucleic acid metabolism, prompted us to develop an automated, rapid, cationexchange method for urinary (3-AIBA and P-alanine (ref. 15). The method was used to establish the normal excretion levels of these two compounds, and to show that essentially all P-AIBA was present in urine in the free state, while most P-alanine was present in a conjugated form which required acidic hydrolysis prior to analysis. During this time, research continued on the application of the analytical data to quantitatively assess the course of disease and response to therapy, with Woo et a/. (ref. 16) applying multiple markers for this task. A 1979 study by Coombes et a/. (ref. 17 ) measured the excretion of four urinary nucleosides by patients with early and advanced cancer to assess their value as tumor-index substances. The summary was that few abnormal excretion levels were found in patients without overt

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metastases and these did not predict early relapse. In those with metastatic cancer, m2,mZG was most frequently elevated (8 of 1 1 patients) and m2,mZG was also described as most likely to have a role in following patients with this disease. However, in early breast cancer, most patients have normal urinary nucleoside excretion patterns, and those with elevated nucleosides are not necessarily the patients who will relapse early (ref. 17). The reversed-phase liquid chromatographic approach to the analysis of major and modified nucleosides in RNAs was also being advanced, (ref. 28) as Gehrke et a/. reported the analysis of the nucleoside composition of RNA hydrolysates, and Gehrke et a/. (ref. 18,19) reported the systematic investigation of the chromatographic parameters affecting the RPLC separation of nucleosides as well as the quantitative enzymatic hydrolysis of microgram amounts of tRNAs (ref. 19). It was shown that nuclease P1 and bacterial alkaline phosphatase were capable of releasing all of the expected nucleosides regardless of the extent of modification. The hydrolysis parameters were systematically investigated and the optimized hydrolysis conditions were established for quantitative hydrolysis of tRNAs. The relationships of parameters which were established as a result of these studies allowed prediction of separation and increased the number of nucleosides which could be resolved in a single analysis to some 18 nucleosides, a number which would later more than double. Also in 1981, Waalkes et a/. (ref. 20 ) reported a preliminary study on multiple biological markers and breast carcinoma. Noting that although no single biological marker for breast cancer exists, the use of multiple biomarkers in combination might be useful as a means of detecting residual or occult tumor not apparent clinically. The results of that study suggested that serial measurement of biological markers has Fotential for indicating the presence of occult disease. In 1982, Dr. Waalkes et a/. of Johns Hopkins and Dr. Gehrke's group at the University of Missouri (refs. 21, 22) reported on the modified nucleosides as markers for patients with small cell carcinoma of the lung. They used a "composite score" approach for expressing urinary nucleoside values, and concluded that a direct

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relationship was found with increasing extent of disease or tumor burden, and that when determined serially the composite score paralleled in general the clinical response categories for individual patients. In the same year, we reported a feasibility study on the development of biological nucleoside markers for ovarian cancer (ref. 23) with the results suggesting that the use of a combination of multiple markers may be useful in staging patients, and in detection of minimal, residual, and recurrent disease. After 1982, our interest broadened to include investigations on RPLC approaches for studying naturally occurring nucleic acid modification, including tRNA, mRNA and DNA as described in earlier chapters of these volumes. A comprehensive paper by Gehrke and Kuo (ref. 26) reported on a new RPLC-UV diode array technology for the simultaneous measurement and identification in complex biological matrices of a large number of nucleosides. We then approached the problem of investigating modified nucleosides in serum as potential biological markers (ref. 27). As discussed by Kuo et a/. (ref. 27), there are several potential advantages of serum over urine as a source of nucleosides for studies of this type. Serum volume is directly related to total surface area of the body, thus allowing direct comparison of data in terms of concentration, rather than normalizing data on the basis of another molecule such as creatinine as is usually required in studies of urine. Additionally, serum nucleosides may be subject to fewer structural alterations than urinary nucleosides, which may account for higher serum levels of some modified nucleosides. Finally, availability of serum samples, ease of collection, and physician preference of serum for clinical chemical analysis favor use of serum over urine and bring serum nucleoside measures a step nearer the goal of a clinical assay. We successfully developed a method for measuring modified nucleosides in serum (ref. 27, 42), with the complete procedure given in detail in Chapter 13 of this volume. This field of study has benefited greatly from research conducted by a number of research investigators, including Bjork and Rasmuson (ref. 29), Brown's group (refs. 30, 31), Cimino et a/. (ref. 32), Clark et a/. (ref. 33), McEntire et a/. (ref. 34), Salvatore et a/. (ref. 354, Borek 8, Sharma e t

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a/. (ref. 36), Schlimme et a/. (ref. 37), Schoch's group (ref. 38), Tamura et a/. (ref. 39), Trewyn et a/. (ref. 40) and others. These investigators have developed analytical methods and conducted investigations of the modified nucleosides in physiological fluids which has greatly advanced knowledge in this area. Only a few years ago, our knowledge of the identities and quantities of modified nucleosides in physiological fluids was extremely limited; however, recent studies have reported on the levels of the array of modified nucleosides in physiological fluids. These studies have helped define whether, and in what circumstances, modified nucleoside levels may serve as biological markers of disease. In these volumes, a number of investigators present their methods for nucleoside analysis and the results of their studies on modified nucleosides as potential biological metabolic markers. Our most recent study has dealt with the concept of cancer patient classification using the array of modified nucleosides found in serum (ref. 34). A broad spectrum of modified nucleosides was quantified by high performance liquid chromatography in serum of 49 male lung cancer patients, 35 patients with other cancers and 48 patients hospitalized for non-neoplastic diseases. Data for 29 modified nucleoside peaks were normalized to an internal standard (m3U) and analyzed by discriminant analysis and stepwise discriminant analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer and 75% of the non-cancer subjects. It also demonstrated 84% sensitivity and 79% specificity when comparing lung cancer to non-cancer subjects, and 80% sensitivity and 55% specificity in comparing lung cancer to other cancers. These data support and expand our previous studies which reported the utility of measuring modified nucleoside levels in serum and show that precise measurement of an array of 29 modified nucleosides in serum by our HPLC-UV method with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring. We have just completed a comprehensive study in collaboration with Drs. Salvatore, Cimino, and their group at the University of Naples. In this study of twelve nucleosides in serum of 83 patients with acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic

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leukemia, and chronic myelogenous leukemia (ref. 41). Only patients without pre-treatment were included in the study. These data have been compared with the serum nucleoside levels in a population of 94 normal individuals. The normal population was selected to include a balanced representation of sex and age distribution. The normal population showed a very narrow level in distribution of the nucleosides and this distribution was independent of age and sex. A very significant elevation (several fold) was observed of most of the modified nucleosides in the leukemic patients over the values of normals. These observations are striking and may indicate that serum modified nucleoside levels and their distribution pattern may be used as important indicators in following the management of the leukemic patient and his prognosis. The chapters that follow present the approaches, investigations, and conclusions of leading investigators from many countries on modified nucleosides as biological markers in cancer and metabolism. REFERENCES: 1. 2. 3.

4.

5.

6.

E. Borek, Introduction to symposium; tRNA and tRNA modification in differentiation and neoplasia, Cancer Res. 31 (1971) 596-597. B. Weissman, P.A. Bromberg, and A.B. Gutman, The purine bases of human urine. I. Separation and identification, J. Biol. Chem. 224 (1 957) 407-422. J.E. Mrochek, S.R. Dinsmore, and T.P. Waalkes, Analytic techniques in the separation and identification of specific purine and pyrimidine degradation products of tRNA: Application to urine samples from cancer patients, J. Natl. Cancer Inst. 53 (1974) 1553-1563. T.P. Waalkes, S . R. Dinsmore, J.E. Mrochek, Urinary excretion by cancer patients of the nucleosides N*$ N2 dimethylguanosine, 1methylinosine, and pseudouridine, J. Natl. Cancer Inst. 51 (1973) 271 -274. T.P. Waalkes, C.W. Gehrke, R.W. Zumwalt, S.Y. Chang, D.B. Lakings, D.C. Tormey, D.L. Ahmann, C.G. Moertel, The urinary excretion of nucleosides of ribonucleic acid by patients with advanced cancer, Cancer 36 (1975) 390-398. M. Uziel, L.H. Smith, and S.A. Taylor, Modified nucleosides in urine: Selective removal and analysis, Clin. Chem. 22 (1976) 145-.

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7. 8.

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C.W. Gehrke and C.D. Ruyle, Gas-liquid chromatographic analysis of nucleic acid components, J. Chromatogr. 38 (1968) 473-491. D.B. Lakings and C.W. Gehrke, Gas-liquid chromatographic analysis for purine and pyrimidine bases in hydrolysates of nucleic acid, Clin. Chem. 18 (1972) 810-813. S. Y. Chang, D. B. Lakings, R.W. Zumwalt, C.W. Gehrke and T.P. Waalkes, Quantitative determination of methylated nucleosides and pseudouridine in urine by gas-liquid chromatography, J. Lab. and Clin. Med. 83 (1974) 816-830. G. E. Davis, R.D. Suits, K.C. Kuo, C.W. Gehrke, T.P. Waalkes, and E. Borek, High performance liquid chromatographic separation and quantitation of nucleosides in urine and some other biological fluids, Clin. Chem. 23 (1977) 1427-1435. K.C. Kuo, C.W. Gehrke, R.A. McCune, T.P. Waalkes, and E. Borek, Rapid, quantitative high performance liquid chromatography of pseudouridine, J. Chromatogr. 145 (1978) 383-392. C.W. Gehrke, K.C. Kuo, G.E. Davis, R.D. Suits, T.P. Waalkes and E.Borek, Quantitative high performance liquid chromatography of nucleosides in biological materials, J. Chromatogr. 150 (1978) 455-476. O.K. Sharma, T.P. Waalkes, C.W. Gehrke and E. Borek, Applications of urinary nucleosides in cancer diagnosis and cancer management, Cancer Detect. and Prevent. 6(1983) 77-85. T.P. Waalkes, C.W. Gehrke, D.B. Lakings, R.W. Zumwalt, K.C. Kuo, S.A. Jacobs and E. Borek, Brief communication: Beta-aminoisobutyric acid in patients with Burkitt's lymphoma, J. Natl. Cancer Inst. 57 (1976) 435-438. K.C. Kuo, T.F. Cole, C.W. Gehrke, T.P. Waalkes and E. Borek, Dualcolumn cation-exchange chromatographic method for 0aminoisobutyric acid and 0-alanine in biological samples, Clin. Chem. 24 (1978) 1373-1380. K.B. Woo, T.P. Waalkes, D.L. Ahmann, D.C. Tormey, C.W. Gehrke, and V.T. Oliverio, A quantitative approach to determining disease response during therapy using multiple biological markers. Application to carcinoma of the breast, Cancer 41 (1978) pp.1871-1882. R.C. Coombes, T.J. Powles, C.W. Gehrke, T.P. Waalkes, and A.M. Neville, Nucleoside excretion in breast cancer: Comparison with other biochemical tumour-index substances, Investigative and Cell Pathology 2 (1979) 11-14. C.W. Gehrke, K.C. Kuo and R.W. Zumwalt, Chromatography of nucleosides, J. Chromatogr. 188 (1980) 129-147.

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19.

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26. 27.

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C.W. Gehrke, K.C. Kuo, R.A. McCune, and K.O. Gerhardt, Quantitative enzymatic hydrolysis of tRNAs: Reversed-phase high performance liquid chromatography of tRNA nucleosides, J. Chromatogr. 230 (1982) 297-308. T.P. Waalkes, M.D. Abeloff, D.S. Ettinger, K.B. Woo, C.W. Gehrke and J.E. Mrochek, Multiple biological markers and breast carcinoma: A Preliminary stuy in the detection of recurrent disease after primary therapy," J. Surg. Oncol. 18 (1981) 9-19. T.P. Waalkes, M.D. Abeloff, D.S. Ettinger, K.B. Woo, C.W. Gehrke, K.C. Kuo and E. Borek, Biological markers and small cell carcinoma of the lung: A clinical evaluation of urinary ribonucleosides, Cancer 50 (1 982) 2457-2464. T.P. Waalkes, M.D. Abeloff, D.S. Ettinger, K.B. Woo, C.W. Gehrke, K.C. Kuo and E. Borek, Modified ribonucleosides as biological markers for patients with small cell carcinoma of the lung, Eur. J. Cancer & Clin. Oncol. 18 (1982) 1267-1274. T.P. Waalkes, M. Rosenshein, J.H. Shaper, D.S. Ettinger, K.B. Woo, J.F. Paone, and C.W. Gehrke, A feasibility study in the development of biological markers for ovarian cancer, J. Surg. Oncol. 2 (1982) 207214. E. Borek, T.P. Waalkes, and C.W. Gehrke, Tumor markers derived from nucleic acid components, Cancer Detect. and Prevent. 6 (1983) 6771. C.W. Gehrke and K.C. Kuo, High resolution quantitative high performance liquid chromatography-UV-spectrometry analysis of nucleosides in tRNA, mRNA, DNA, and physiological fluids, Bull. Mol. Biol. Med. 10 (1985) 119-142. C.W. Gehrke, and K.C. Kuo, Ribonucleoside analysis by reversed-phase high performance liquid chromatography, submitted Nov. 1988 J. Chromatogr. K.C. Kuo, F. Esposito, J.E. McEntire, C.W. Gehrke, Nucleoside profiles by HPLC-UV in serum and urine of controls and cancer patients, in : F. Cimino, G.D. Birkmayer, J.V. Klavins, E. Pimentel, and F. Salvatore (Eds.), Human Tumor Markers, Walter de Gruyter, Berlin-New York, 1987, pp. 519-544. C.W. Gehrke, and K.C. Kuo, High resolution quantitative RP-HPLC-UV of nucleosides in RNA, Human Tumor Markers, 1987 pp. 475-502.

c 12 29.

30.

31.

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36.

T. Rasmuson, G.R. Bjork, L. Damber, S.E. Holm, L. Jacobsson, A. Jeppsson, B. Littbrand, T. Stigbrand, and G. Westman, Evaluation of carcinoembryonic antigen, tissue peptie antigen, placenta alkaline phosphatase and modified nucleosides as biological markers in malignant lymphomas, in: G. Nass (Ed.) Recent Results in Cancer Research, Vol. 84, Springer-Verlag, Berlin-New York, 1983, pp. 331343. R.A. Hartwick, S.P Assenza, and P.R. Brown, Identification and quantitation of nucleosides, bases, and other UV-absorbing compounds in serum using RP-HPLC. I. Chromatographic methods, J. Chromatogr. 186, (1979), pp. 648-658. R.A. Hartwick, A. M. Krstulovic, and P.R. Brown, Identification and quantitation of nucleosides, bases, and other UV-absorbing compounds in serum using RP-HPLC. II. Evaluation of human sera, J. Chromatogr. 186, (1979), pp. 659-676. F. Cimino, T. Russo, A. Colonna, A. Duilio, R. Ammendola, f. Costanzo, A. Oliva, F. Esposito, F. Salvatore, Pseudouridine excretion in experimental neoplasias of retroviral origin, in: F. Cimino, G.D. Birkmayer, J.V. Klavins, E. Pimentel, and F. Salvatore (Eds.), Human Tumor Markers, Walter de Gruyter, Berlin-New York, 1987, pp. 463474. I. Clark, J.W. MacKenzie, J.R. McCoy, and W. Win, Comparison of urinary modified nucleosides and bases in rats with hepatomas and nephroblastomas, in: G. Nass (Ed.), Recent Results in Cancer Research, Vol. 84, Springer-Verlag, Berlin-New York, 1983, pp. 388400. J.E. McEntire, K.C. Kuo, M.E. Smith, D.L. Stalling, J.W. Richens, Jr., R.W. Zumwalt, C.W. Gehrke, and B.W. Papermaster, Classification of lung cancer patients and controls by chromatography of modified nucleosides in serum, Cancer Res., Accepted Nov.1988. F.Salvatore, M. Savoia, T. Russo, L. Sacchetti, and F. Cimino, Pseudouridine in biological fluids of tumor-bearing patients, in: F. Cimino, G.D. Birkmayer, J.V. Klavins, E. Pimentel, and F. Salvatore (Eds.), Human Tumor Markers, Walter de Gruyter, Berlin-New York, 1987, pp. 451-462. O.K. Sharma, F. L. Buschman, E. Borek, D.L. Cohn, K.A. Penley, F.N. Judson, B.S. Dobozin, K.M. Zunich, C.H. Kirkpatrick, Aberrant urinary excretion of modified nucleosides in patients with various manifestations of infection with HTLV-III/LAV, in : F. Cimino, G.D. Birkmayer, J.V. Klavins, E. Pimentel, and F. Salvatore (Eds. ), Human Tumor Markers, Walter de Gruyter, Berlin-New York, 1987, pp.545558.

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37.

38.

39. 40. 41. 42.

E. Schlimme, K.-S. Boos, B. Wilmers, and H.J. Gent, Analysis of ribonucleosides in human body fluids and their possible role as pathobiochemical markers, in: F. Cimino, G.D. Birkmayer, J.V. Klavins, E. Pimentel, and F. Salvatore (Eds.), Human Tumor Markers, Walter de Gruyter, Berlin-New York, 1987, pp. 503-517. G. Sander, J. Wieland, H. Topp, G. Heller-Schoch, N. Erb and G. Schoch, An improved method for the simultaneous analysis of normal and modified urinary nucleosides and nucleobases by high-performance liquid chromatography, Clin. Chim, Acta, 152 (1985), pp. 355-361. S. Tamura, J. Fujii, N. Takaski, H. Toshikazu, H. Kazuyu, Urinary pseudouridine as a tumor marker in patients with small cell lung cancer, Clin. Chim. Acta, 154 (1986), pp. 125-132. R.W. Trewyn and M.R. Grever, Urinary nucleosides in leukemia: laboratory and clinical applications, CRC Critical Rev. Clin. Lab. Sci., 24 (1986), pp. 71-93. F. Salvatore, L. Sacchetti, F. Pane, K.C. Kuo, and C.W. Gehrke, Clinical Evaluation of Serum Nucleosides in Leukemias and Lymphomas. In Preparation. K.C. Kuo, D.T. Phan, N. Williams, and C.W. Gehrke, Ribonucleosides in serum and urine by a high-resolution quantitative RPLC-UV method. Submitted to J. Chromatogr. Biomedical Applications.

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CHAPTER 1 PROGRESS AND FUTURE PROSPECTS OF MODIFIED NUCLEOSIDES AS BIOLOGICAL MARKERS OF CANCER ROBERT w. ZUMWALT~, T. PHILLIP WAALKES~, KENNETH c. K U O ~ ,AND CHARLES W. GEHRKE?

* .4Departmcnt of Biochemistry, University Center, Columbia, MO U.S.A. 65201

of Missouri, and Cancer Rcscarch

*Johns Hopkins University School of Medicine, Baltimore, MD 3Analytical Biochemistry Laboratorics, Inc., Columbia, MO

U.S.A.

U.S.A.

21205

65201

TABLE OF CONTENTS 1.1 Introduction . 1.2 Progress in Modified Nucleoside Studies . 1.3 Prospects: Biomarkers for Leukemias, Lymphomas, and Other Cancers . 1.4 S u m m a r y . 1.5 References.

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1.1 INTRODUCTION The search for biological markers of cancer has mainly focused on three structural categories: hormones, proteins, and nucleic acid components. This chapter will deal with the topic of nucleic acid components as potential biological markers; specifically modified nucleosides. The Introduction to this volume traces the development of reversed-phase HPLC and phenylboronate gel affinity chromatography for the analysis of modified nucleosides in physiological fluids, and the following chapters i n this volume provide detailed accounts of the methods and their applications to various aspects of cancer and normal metabolism research. The term "tumor marker" as coined by Dr. Morton K. Schwartz of the Sloan Kettering Institute refers to some unique metabolic product or

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unusual component of malignant cells which can be measured in body fluids. As emphasized by Borek (ref. 1) and others, the criteria of an effective tumor marker are numerous; it should be specific for malignancy; it should provide a minimum of false-positives and falsenegatives; it should indicate the extensiveness of the malignancy and it should preferably diminish or hopefully disappear after effective therapy. There have been indications for more than 30 years that cancer patients excrete elevated levels of methylated purines and pyrimidines as well as other modified bases and nucleosides (refs. 2, 3). The origin of these compounds was obscure until the discovery of the modification of transfer RNA (ref. 4). The events responsible for the increased excretion of modified nucleosides by cancer patients remain unclear, with increased tRNA turnover, cell death and increased turnover of RNA in the host tissue proposed by various investigators. Investigators have also attempted to elucidate cancer markers which could be utilized to predict which patients are more likely to respond to treatment and which patients have a worse prognosis. Identification of patients with a worse prognosis would perhaps permit the utilization of either more aggressive or innovative forms of treatment for individuals who would not be likely to have a good response to standard approaches (ref. 5). As G. Prodi has pointed out (ref. 6 ) , "a clinical symptom, in this case a tumor marker, is a "sign" that is meaningful only within a previously established theory" (such as for the concept proposed by Borek on altered tRNA metabolism and turnover). Prodi went on to state that "in the field we are now considering, a marker would be an unambiguous "sign" of cancer in the framework of a theory that was previously defined: i.e. a) the specificity of the tumor condition with respect to any other condition in, or occurrence of the b) the link between the considered marker and t h a t organism, and specificity. Only in this way would the data observed "stand for" the tumor - and thus assume the character of the "sign". Unfortunately, such a theory does not yet exist. Cancer research may be defined as an uninterrupted search to define q u a l i t a t i v e l y distinct and specific traits of a tumor cell. What has been defined as

C17 "qualitative" (from Warburg up to the recent immunological studies, and the still more recent oncogene theory) has always been interpreted as "quantitative", that is, as "more or less". The tumor cell is highly mimetic, and it is relatively invulnerable for the same reason as it has such ambiguous markers. Therefore, the history of the study of cancer is also the history of failed markers, or at least markers first held to be absolute, and then relegated to "signs" endowed with a certain ambiguity because they relate to conditions other than cancer. As when we see a cloud on the horizon and do not know whether it is smoke and means fire, or dust and means wind" (ref. 6 ) . At this time, it is generally agreed that presently available biomarkers are ineffective for the primary diagnosis of cancer (ref. 7). Therefore, most recent research on human tumor markers has focused on the surveillance of patients in the post-primary phase of treatment and on comparison of diagnosed cancer patients with non-cancer patients in terms of serum or urine levels of potential biomarkers. This chapter will point to progress and discuss future prospects of modified nucleosides as biological markers of cancer. The Introduction to this volume by Waalkes and Gehrke describes research developments since our involvement in this research, and the other chapters in this volume describe the investigations and provides thorough description, discussions, and reviews of modified nucleosides as biological markers for cancer and normal metabolism. Provided that altered tRNA metabolism is a fundamental aspect of neoplasia, then modified nucleosides resulting from that altered metabolism may indeed be reflective of the neoplastic state.

1.2 PROGRESS IN MODIFIED NUCLEOSIDE STUDIES High performance liquid chromatography with diode array detection (HPLC-UV) has emerged as one of the most popular and powerful techniques for studying the constitutents of nucleic acids, especially in complex samples such as physiological fluids and cell extracts. This chapter will not describe the methodological developments that now permit the accurate measurement of a wide array of major and modified nucleosides in a broad range of sample types; those

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developments are precisely described in other chapters of these volumes, e.g., in Chapter 1, Part A, and Chapter 2, Part C, Gehrke and Kuo describe ribonucleoside analysis by high performance RPLC, and the following chapters in this volume and the references therein provide a thorough description of methodologies for ribonucleoside analysis. Many investigations have been conducted with the general goal of identifying a component of a physiological fluid which would serve as a tumor marker. Concepts, reports, a n d findings which launched investigations of the modified nucleosides as biological markers of cancer have been described in the Introduction and elsewhere in this To add some perspective to the progress of research, a volume. description of the origin and scientific status of the topic of modified nucleosides as biological markers of cancer could be useful. In the period from 1966 to 1972, there were reports that the activities of tRNA-methylating enzymes were elevated in neoplastic cells, that column-chromatographic profiles of tRNAs in neoplastic cells w e r e altered in comparison to their normal counterparts, and preliminary experiments seemed to indicate that bulk tRNA in tumors might be hypermethylated. This information combined with earlier observations that showed elevated excretion of modified nucleobases by cancer patients pointed to the methylated or otherwise modified catabolic products of tRNA as potentially universal biological markers for cancer. W e first published clinical studies of modified nucleosides as potential biological markers of cancer in 1975, using gas-liquid chromatography to measure a very limited n u m b e r of urinary nucleosides (ref. 8-10). That same year, Suits and Gehrke (ref. 11) reported on RPLC analysis of nucleobases and nucleosides, and Gehrke and coworkers followed in 1978 (ref. 12) with the development of the analytical concept which would find widespread acceptance and use in laboratory and clinical research: isolation of nucleosides from complex matrices by phenylboronate gel affinity chromatography followed by their RPLC separation and detection and quantitation by UV absorption. In 1976 Hartwick and Brown had reported on the evaluation of microparticle chemically-bonded reversed-phase column packings for the analysis of nucleosides and their bases (ref. 13). In 1980, Gehrke e t

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al. (ref. 14) further extended the HPLC approach for nucleoside analysis by describing the effects of numerous chromatographic parameters on the separation and quantitation of a number of modified nucleosides. The early studies by Gehrke, Waalkes et al. in the mid-1970s presaged the many research basic and clinical research investigations which would adopt the approach of Gehrke's group for studying modified nucleosides. Numerous research groups in the U.S., Europe, and Japan have studied modified nucleosides and their potential relationships to cancer, and many of these investigations are described or referenced in the chapters of these three volumes. The group headed by F. Salvatore and F. Cimino at the University of Naples has conducted a wide range of studies concerning modified nucleosides and cancer, with much of their work focusing on pseudouridine (ref. 15). Their research on urinary and serum nucleosides has paralleled ours in some ways, and we have obtained very similar results. In addition, we have engaged in collaborative studies with the Naples group, including studies of modified nucleosides in cell cultures and animal model studies (see Chapters 7 and 8, Part C). As described and referenced in the other chapters of this volume, there followed numerous studies on urinary modified nucleoside levels in patients with various cancer types, normal individuals, and patients with diseases other than cancer. Early results from various investigators were mixed, however i t soon became clear that t h e modified nucleosides would not s e r v e as unequivocal universal indicators for the presence or course of all neoplastic disease, but perhaps would function best for specific neoplasias i n following the clinical management of the patient. However, many of the investigations reported i n the literature were encouraging, and in the early and mid-1970s the continuing development of HPLC (pumps, reversed-phase columns, detectors, etc.) offered a much improved analytical approach. The development of a highly specific affinity chromatography method promised to provide a much improved method for isolation of ribonucleosides from the complex matrix of physiological fluids. Since then, progress in

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improving analytical methodologies for accurately measuring modified nucleosides in physiological fluids has been most impressive. The advances in analytical and chromatographic methodology to accurately measure modified nucleosides in physiological fluids has provided investigators with additional research tools to further advance nucleic acid research far beyond the area of biomarkers research (ref. 37). HPLC nucleoside analysis has been developed and extended by Gehrke et aZ. to the quantitative analysis of the complete tRNA molecule, with special emphasis on the extensive and complex modifications present in tRNA (see Chapter 1, Part A), improved and interfaced characterization methods (HPLC-UV, MS, NMR, and FT-IR) for elucidating the structures of unknown modified nucleosides (see Chapter 5 , Part A), developed methods for quantitatively studying the cap structures of messenger RNA (see Chapter 8, Part A), and to a rapid and accurate methodology for studying methylation of DNA (Chapter 10, Part B). 1.3 PROSPECTS: BIOMARKERS FOR LEUKEMIAS, LYMPHOMAS AND OTHER CANCERS. In 1983, Heldman et al. (ref. 16) reported a study of the urinary excretion of modified nucleosides by patients with chronic myelogenous leukemia (CML). They measured urinary modified nucleosides in 15 patients with Philadelphia chromosome-positive CML and determined the correlation with activity of CML. They found that patients in the stable phase of CML had excretion levels one to two times normal, whereas patients in the blastic phase showed elevations up to 12 times normal. The modified nucleosides showing the most significant differences i n excretion between the stable phase and blastic phase were 1-methylinosine, pseudouridine, and N2, N2-dimethylguanosine. Serial nucleoside determinations were made in two patients with CML and found to correlate closely with disease activity. They noted that the degree of elevation and the correlation with disease activity suggest the potential value of quantitation of urinary nucleosides in monitoring patients with CML; in particular, nucleoside excretion may be useful in detecting early blastic transformation.

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Also in 1983, Rasmuson et al. (ref. 17) evaluated urinary pseudouridine as a biologic marker for patients with bronchogenic carcinoma, and reported elevated levels that paralleled clinical stage. Oerlemans and Lange (ref. 18) reported a study of major and modified nucleosides excreted by patients with ovarian cancer. The patients were divided into three groups: benign, borderline, and malignant. They reported that 44% of the measured marker levels of the benign group were in the normal range, whereas 97% of the borderline and malignant groups were outside the normal range. Nielsen and Killman (ref. 19) studied the excretion of pseudouridine and p -aminoisobutyric acid by patients with acute and chronic myeloid leukemia, and compared those levels to healthy control subjects. Pseudouridine excretion was elevated i n over 80% of the patients with untreated AML and CML, and the levels decreased following treatment. Rasmuson and Bjork (ref. 20) studied pseudouridine excretion by 48 patients with malignant lymphomas, and found elevated levels in 50% of patients with histiocytic lymphoma, 33% of patients with lymphocytic lymphoma and 13% with Hodgkin's lymphoma. However, no correlation could be made between level of excretion and clinical stage, and no prognostic value could be attributed to initial excretion levels of pseudouridine. In a later report (ref. 21) Rasmuson and Bjork studied 39 patients with non-Hodgkin's lymphoma before treatment, and found 57% of the patients with highly malignant lymphomas had elevated pseudouridine compared to 28% of patients with low-grade malignancy and 4% for healthy adults. In 1984, Mackenzie et al. (ref. 22) reported their study of RNA catabolites as cancer markers. They found that rats with aflatoxininduced nephroblastomas excreted elevated amounts of urinary modified nucleosides and bases which are catabolites of tRNA. Their study of nucleoside excretion profiles suggested the possiblity for distinguishing between tumors, and their findings indicated that the source of the elevated nucleoside levels may be the host's tissue RNA. Their preliminary studies on humans with lung cancer showed marked elevation of one or more urinary RNA catabolites, and they suggested

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that measurement of urinary RNA catabolites may be useful in the diagnosis, prognosis, and evaluation of therapy in patients with lung cancer. Esposito et al. (ref. 23) evaluated the relationship between increased pseudouridine excretion and retroviral cell transformation. They studied the effect of retrovirus infection and/or tranformation on the rate of pseudouridine excretion by chick embryo fibroblasts. Their results showed that: (a) pseudouridine excretion by chick embryo fibroblasts transformed by Rous sarcoma virus is several times higher than that of normal cells; (b) this increased excretion precedes the appearance of morphological signs of transformation and i t is always present when neosynthesized infectious viral particles are released into the culture medium; and (c) pseudouridine excretion was also increased in cells infected by a mutant of Rous sarcoma virus (RAV-1) which, lacking the src gene, does not transform the cells but replicates normally. In research with Dr. T. Heyman of the Institut Curie (ref. 24), we analyzed modified nucleosides in tRNAs from chicken e m b r y o fibroblasts (CEF), normal and infected with either a wild strain of Rous sarcoma virus, SR-RSV subgroup A (SRA), or a temperature-sensitive transformation mutant (tsNY68). An increased modification in tRNA from SRA-infected CEF cells over normal CEF cells was observed at both the exponential and stationary growth phase. In contrast, n o significant differences were observed in normal CEF tRNA modification levels in relation to the growth phase. We also found that there was a higher increase of modification in tRNA from SRA-infected cells in the stationary phase as compared to that of the exponential phase. Such a difference would be related to the degree of transformation. The tsT mutant (tsNY68) normally replicates but fails to transform cells at high temperature, 42°C. N o increase in the levels of modification was observed in tRNA from tsNY68-infected CEF as compared to tRNA from normal cells, both grown at 42°C. The increase in all detected tRNA modifications (except Q and Y) in transformed cells in comparison to normal cells thus seems to depend on the expression of the src gene (ref. 24).

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Studies of the origin of increased modified nucleoside levels in physiological fluids, such as those of Esposito et al. a n d H e y m a n mentioned above, illustrate the difficulties researchers have encountered in ascribing a source to the nucleoside elevations of patients with cancer. There is still no metabolic mechanism which has been clearly identified as responsible for observed elevated excretion of modified nucleosides by patients with neoplasias. Specifically, in research with Dr. Heyman, we found that the mole percent values of purine and pyrimidine methylated nucleosides in the tRNAs from RSV-infected and transformed CEF cells are 50 to 120% higher than in the tRNAs from non-transformed CEF cells. In addition, the amount of 2'-O-methylated nucleosides and threoninocarbonylated modified adenosine (&A) are only elevated about 10% in RSV-CEF cells over the C E F cells, and the mole percent values of the four major nucleosides found in C E F cells and RSV-CEF cells are essentially identical. This indicates that the only difference between tRNAs from CEF cells and RSV-CEF cells are probably nucleoside modifications. Rasmuson and Bjork (ref. 25) measured pseudouridine excretion in 222 patients with malignant diseases. They found that patients with malignant lymphomas had a 50% frequency of elevated pseudouridine excretion, colorectal carcinomas 25%, bronchogenic carcinomas 3 1%, and in cases of mammary carcinomas 30%. They also reported that excretion increases paralleled increasing clinical stages of the disease. They noted that for patients with bronchogenic carcinoma and possibly malignant lymphomas, elevated pseudouridine excretion is correlated to shorter survival. They concluded that pseudouridine i s a marker of malignancy, and as such it could be used as a complement to clinical stage and to predict the prognosis. Heldman et al. (ref. 26) also studied the differential excretion of modified nucleosides by patients with adult acute leukemia, and reported that the urinary excretion of 1-methylinosine and N2, N2dimethylguanosine may prove to be valuable clinically in following disease activity in patients with acute lymphoblastic leukemia (ALL), and in distinguishing patients with A L L from those with acute myelogenous leukemia (AML).

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Heldman et al. (ref. 27) also studied the relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia (ALL), and demonstrated that excretion of modified nucleosides reflects disease activity in childhood and that the urinary nucleosides may be useful clinical markers for this disease. In 1986, Tamura et al. (ref. 28) studied the urinary excretion of pseudouridine in patients with hepatocellular carcinoma. They reported that the urinary concentration of pseudouridine in the carcinoma patients was significantly higher than in patients with liver cirrhosis or healthy controls. Seventy percent of the 23 patients with hepatocellular carcinoma had urinary pseudouridine levels higher than the mean value for the healthy controls plus 2 standard deviations. When urinary pseudouridine was used in combination with serum alpha-fetoprotein, 83% of the carcinoma patients were positive for marker elevations. Tamura et al. considered urinary pseudouridine and serum alphafetoprotein to serve as complementary markers for diagnosis of hepatocellular carcinoma. Tamura e l al. (ref. 29) also evaluated urinary pseudouridine as a tumor marker in patients with small cell lung cancer. They reported that urinary pseudouridine is not a specific marker for SCLC, but it relates to the tumor burden and reflects the clinical status of patients. They found that in the limited number of cases examined, the positivity rate for urinary pseudouridine concentration was higher than that for CEA, and that when the two markers were combined, the positivity rate is further elevated above that of either single marker. In a study of colorectal cancer patients, Nakano et al. (ref. 30) reported there were no significant differences in the concentrations of pseudouridine, 1 -methylguanosine, N2-methylguanosine, and Nz,N2dimethylguanosine between urine samples taken before and after surgery from eight patients, and that contrary to other reports, no significant differences in modified nucleoside levels were observed between urine samples from colorectal cancer patients and those from normal subjects. Trewyn and Grever (ref. 5) have pointed out that although certain species of tRNA may be hypermethylated in cancer cells (ref. 31), the degree of increased methylation of total tRNA is too low to be consistent

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with the high tRNA methyltransferase activity and capacity. A major question which remains unresolved: Is altered tRNA metabolism a fundamental aspect of neoplasia? Research on tRNA catabolites in urine and serum/plasma has concentrated on HPLC analysis of the modified nucleosides following isolation of the nucleosides by boronate gel affinity chromatography as the nucleosides are generally the major tRNA catabolic excretion products and are easily isolated by the boronate gel. However, immunoassays may be used more widely in the future to quantitate modified nucleosides in biological fluids, especially if the specificity and sensitivity can be achieved. Advancements i n the isolation, identification and measurement of modified nucleosides has been striking, and are now providing greater insights into the value of modified nucleosides as potential tumor markers. Early studies in which urinary modified nucleosides were found highIy elevated led to speculation that tumor tRNA was hypermethylated, and thus the modified nucleosides could be universal tumor markers. Development of these new research tools have brought insight into how modified nucleosides are excreted by healthy adults and children. With healthy adults the normal range of modified nucleoside excretion is very narrow, and it has been shown by Gehrke and Kuo that random urine samples can be utilized instead of 24-hour collections if nucleoside concentrations are expressed relative to creatinine. Adjustments must be made in the case of children, as the creatinine level correlates to body muscle mass. However, the excellent correlation of nucleoside excretion to age allows this adjustment to be made, resulting in a narrow normal excretion range (refs. 1, 32, 33). Trewyn and Grever (ref. 5 ) have provided an excellent review of urinary nucleosides and leukemia. They reviewed the available literature and discuss laboratory analyses, including methods, reference values, and multivariate analyses; clinical studies covering nonmalignant disease and infection, acute leukemia (childhood and adult) and chronic leukemias. They conclude that measurement of urinary nucleoside excretion offers a potential tool for monitoring disease activity in patients with ALL, CML, and perhaps CLL.

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They pointed out that additional work is necessary in following serial determinations of urinary nucleosides at frequent intervals in patients with different types of leukemia in order to assess the true value of these compounds as an accurate monitor of disease activity within the individual patient. They also observed that correlation of the nucleoside excretion pattern with ultimate clinical duration of complete remission is an important aspect that has not been adequately assessed. At this point, the major observations indicate that urinary nucleosides might serve as useful indicators for both prognosis and disease activity, although a significant amount of work remains to be done in order to ensure that these correlations are scientifically valid. Trewyn and Grever further point out that continuing investigation in the area of urinary nucleoside excretion provides a constant stimulus to understanding the biochemical changes which occur at the cellular level in leukemia, and that a clearer understanding of these cellular events may enhance understanding of the leukemogenic process itself. As Clark et al. discuss in Chapter 11, there is still disagreement concerning the source of the elevated RNA catabolites in patients with neoplasms. Although it is usually reported that the source and reason for the increased level of urinary RNA catabolites is increased turnover of tumor tRNA, Clark et al. and others are of the opinion that this increase is derived primarily from increased turnover of RNA in the host tissue (see Chapter 11, this volume). Salvatore et al. have focused especially on pseudouridine in blood serum as a biological marker of cancer, and in Chapter 7 discuss formation of the modified nucleosides, their measurement and normal levels in serum, and results obtained from the analysis of pseudouridine in serum of cancer patients. They report that in all groups of patients affected by tumors there was a definite and significant increase (as compared to normals) in blood pseudouridine levels, with the exception of the less advanced breast cancer groups; pseudouridine elevations were much greater for patients with lymphoma and leukemia: that there was a good correlation between tumor burden and/or spread of the tumor mass with blood pseudouridine levels; and that in the few cases where monitoring of neoplastic disease was correlated with blood

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pseudouridine levels, there was good correlation between the response to therapy and pseudouridine levels. They report this was the case for patients receiving chemotherapy and that underwent surgical treatment. Schoch’s group ha5 done much to clarify the origin of urinary RNA catabolites. They perceived a kind of basic stoichiometry in t h e pattern of the major modified excretion products, and thus began to screen the literature for relevant structural information. They discovered that modified nucleosides were distributed among rRNA, tRNA, mRNA and snRNA in proportions that were calculable. That ultimately enabled them to select specific urinary nucleosides for the whole-body turnover of each of the three miijor species of RNA; 7-methylguanine (i), N2,N2dimethylguanosine (ii), and pseudouridine (iii) are the degradation products from RNA turnover and can be used as markers for the wholebody metabolism of mRNA-cap, tRNA, and rRNA. The relative molar ratios of these molecules in serum is approximately 100:4.7:1.1 (see Chapter 13, this volume). Their approach opened a new way of looking at urinary RNA catabolites, which clearly adds a new dimension to future RNA catabolite investigations. Cimino et a!. report in Chapter 8 that evidence is accumulating where pseudouridine is the most highly and most frequently increased modified nucleoside in neoplastic patients, and that there is a good correlation between serum pseudouridine levels and progression of the neoplastic disease and the response to therapy. In Chapter 8 they describe increased pseudouridine levels in AKR mice and increased pseudouridine excretion by transformed cells. They also discuss enzymes involved in pseudouridine metabolism, modification of tRNAs from neoplastic cells and studies on tRNA primers for reverse transcriptase in tumor of retroviral origin. Still, the molecular basis for elevations of pseudouridine is unclear. After development of reliable methods for measuring modified nucleoside levels in physiological fluids, researchers began comparing urinary nucleoside levels of cancer patients with normal control subjects. Those data were presented in many cases in the form seen in

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Figure 1.2

Correlation of patient survival and urinary nucleoside levels. (From ref. 44 with permission of publisher)

Figure 1.1 as presented by Waalkes et al. (ref. 10). In this figure, the nucleosidelcreatinine ratios of four nucleosides from patients with colon cancer are compared with normal subjects. In many cases elevations were observed for persons with neoplastic disease. In 1982, Waalkes and Gehrke et al. (ref. 44) proposed a "composite score" approach for expressing urinary nucleoside values of patients with small cell carcinoma of the lung. As shown in Figure 1.2, patients with 3 to 5 elevated modified nucleosides had shorter survival times than patients with 0 to 2 nucleosides elevated. When the number

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of nucleosides elevated in concentration increased from 0-2 to 3-5 the survival time dropped from about 24 months to 12 months. A "composite score" approach has considerable merit in monitoring the course of the disease as the patient is receiving treatment. McEntire et al. (ref. 34) reported a study of serum nucleosides in 49 male lung cancer patients, 35 patients with other cancers and 48 patients hospitalized for non-neoplastic diseases. This study provided the most detailed serum nucleoside profiles reported to date, as 29 modified nucleoside peaks were normalized to an internal standard and analyzed by discriminant analysis, stepwise discriminant analysis, and principal components analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer It also demonstrated 84% and 75% of the non-cancer subjects. sensitivity and 79% specificity when comparing lung cancer to noncancer subjects, and 80% sensitivity and 55% specificity i n comparing lung cancer to other cancers. The nucleoside peaks having the greatest influence on the models varied dependent on the subgroups compared, confirming the importance of quantifying a wide array of nucleosides. Using principal components analysis, 65% of the cancer patients and 79% of the non-cancer patients were correctly classified and the modeling power of each of the 29 nucleosides was also determined. These data support and expand previous studies which reported the utility of measuring modified nucleoside levels in serum, and show that precise measurement of an array of 29 modified nucleosides in serum by HPLCUV with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring. Chheda et al. (ref. 35) recently made an evaluation of 5 carbamoylmethyluridine (ncm5U) as an indicator of tumor burden in lung cancer patients and found that the levels of ncm5U were elevated i n the urine and serum of non-small cell lung cancer patients when compared to the levels found in normal subjects (p = < 0.001). Significantly elevated levels of ncm5U were found in the urine of 17 of 18 (98%) of the patients. These investigations support the use of ncm5U as a monitor of tumor load since combined urinary/serum levels reflected advanced malignancy.

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In collaborative programs with Professor F. Salvatore and his research group at the University of Naples Medical school, Dr. Edith Mitchell in the Department of Oncology, University of Missouri Medical School, and Dr. John McEntire of the Cancer Research Center, Columbia, MO, we collected 94 samples of serum from normal healthy donors and 47 serum samples from non-cancer male patients. The normal healthy population consisted of 51 males and 43 females ranging in age from 19 to 84. Thirteen serum modified nucleosides and creatinine were quantified and the data are presented in the following chapter. Briefly, the narrow distribution (RSD, %) of each nucleoside in the 94 samples was essentially the same whether the data were expressed as pmol/ml or as the nucleoside/creatinine ratio. This indicates a stringently controlled metabolic rate of nucleic acids for healthy subjects. There is no age and sex dependency for adults of any of the nucleosides studied. Thirteen serum modified nucleosides in patients with a number of diseases other than cancer (DOTC) were also investigated. This study included 47 males with ages ranging from 27 to 83. The nucleoside values for the DOTC patients were essentially the same as for the n or m a 1s . Sixteen urinary nucleosides and creatinine were measured in 24 hour collections of urine from 18 normal healthy donors (7 males, 11 females, ages 25 to 50). A narrow distribution of each nucleoside was again observed i n the urine of normal healthy subjects as for serum in healthy subjects. In further collaboration with Professor F. Salvatore's group at the University of Naples, serum from pretreatment leukemia and lymphoma patients were collected and analyzed. Brief preliminary results are presented as bar graphs i n the following chapter. Comparisons of the normal serum nucleoside levels to the levels found i n acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, and chronic myeloid leukemia (CML) were made, as was a comparison of the normal serum nucleoside levels to the levels found in Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). We found that the level of modified nucleosides from the patients in all types of leukemia and lymphoma are significantly

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higher than for the normal values, and acute lymphocytic leukemia patients have much higher levels than patients with other types of leukemia and lymphoma. This indicates the excellent diagnostic value of modified nucleosides for leukemia and lymphoma. The preliminary data also shows that the modified nucleoside profiles of some leukemias are different from the other leukemia types. Thus, preliminary studies on serum nucleosides as potential biological markers for small cell lung carcinoma, leukemias and lymphomas were achieved. Some significant correlations were noted between the levels and profiles of serum nucleosides and different neoplasias.

SUMMARY Much research has been conducted with the general goal of identifying a component(s) of a physiological fluid which would serve as a tumor o r cancer marker. The modified nucleosides resulting from altered t R N A metabolism are i m p o r t a n t as potentially useful "biochemical sentinels" in the diagnosis, prognosis, and evaluation of therapy and monitoring the disease activity in cancer. The term "tumor marker" as coined by Dr. Morton K. Schwartz of the Sloan Kettering Institute refers to some unique metabolic product(s) or unique component(s) of malignant cells which can be measured in body fluids. As pointed out by Borek and others, the criteria of an effective tumor marker are numerous; it should be specific for malignancy; it should provide a minimum of false-positives and falsenegatives; it should indicate the extensiveness of the malignancy and it should preferably diminish or hopefully disappear after effective therapy. There have been indications for more than 30 years that cancer patients excrete elevated levels of modified nucleosides. The origin of these compounds was obscure until the discovery of the modification of transfer RNA. However, the events responsible f o r the increased excretion of modified nucleosides by cancer patients remain unclear, with increased tRNA turnover considered as the predominant source, and cell death and increased turnover of RNA in the host tissue also considered by various investigators. 1.4

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Trewyn and Grever have pointed out that although certain species of tRNA may be hypermethylated in cancer cells, the degree of increased methylation of total tRNA is too low to be consistent with the high tRNA methyltransferase activity and capacity. A major question which remains unresolved: Is altered tRNA metabolism a fundamental aspect and/or consequence of neoplasia? Research in our laboratories on tRNA catabolites in urine and serum/plasma has centered on the development of HPLC separation and UV diode array detection of the modified nucleosides following selective isolation of the nucleosides by boronate gel affinity chromatography as the nucleosides are generally t h e major tRNA catabolic excretion However, products and are easily isolated by the boronate gel. immunoassays may be used more widely in the future to quantitate modified nucleosides in biological fluids if the specificity and sensitivity of the method can be improved. Tjaden (ref. 3 6 ) has pointed out that the analytical and preparative separation of specific compounds in complex samples like physiological fluids and cell extracts is of fundamental importance in biomedical research. Nucleotides, nucleosides and their bases are not only the essential constituents of nucleic acids, but also of other structures important for the proper functioning of cells. Since physiological fluid levels of nucleosides are dependent on the metabolic state of cells, nucleoside profiles might be used in monitoring the progression of disease or the therapeutic effects of drugs. Various analytical methods for the determination of these compounds have been developed, but HPLC-UV is one of the most popular techniques in this respect, since it combines the high selectivity of the separation method with sensitivity of detection (ref. 37). With respect to molecular mass and to polarity, a wide array (-30) of modified nucleosides can be measured by HPLC simultaneously, making this technique a powerful analytical tool. In the past few years, advancements i n the isolation, identification and measurement of modified nucleosides has been striking, and are now providing greater insights into the value of modified nucleosides as potential tumor markers. Early studies in which urinary modified nucleosides were found highly elevated led to speculation that tumor

c 34 tRNA was hypermethylated, and thus the modified nucleosides could be universal tumor markers. Our development of these analytical-chromatographic methods (ref. 37) has brought insight into how modified nucleosides are excreted by healthy adults and children. With healthy adults, the normal range of modified nucleoside excretion is very narrow, and we have shown that random urine samples can be utilized instead of 24-hour collections if nucleoside concentrations are expressed relative to creatinine. In normal patients the excretion level of the modified bases was demonstrated as remarkably constant. Adjustments must be make in the case of children, as the creatinine level correlates to body muscle mass. However, the excellent correlation of nucleoside excretion to age allows this adjustment to be made, resulting in a narrow normal excretion range. An area that seems promising for biologic markers is that of Trewyn and Grever have provided an leukemia (ref. 5) research. excellent review of urinary nucleosides and leukemia. They reviewed the available literature, and discuss laboratory analyses, including methods, reference values, and multivariate analyses; clinical studies covering nonmalignant disease and infection, acute leukemia (childhood and adult) and chronic leukemias. They conclude that measurement of urinary nucleoside excretion offers a potential tool for monitoring disease activity in patients with ALL, CML, and perhaps CLL. In addition, they pointed out that further work is necessary in following serial determinations of urinary nucleosides at frequent intervals in patients with different types of leukemia in order to assess the true value of these compounds as an accurate monitor of disease activity within the individual patient. They also observed that correlation of the nucleoside excretion pattern with ultimate clinical duration of complete remission is an important aspect that has not been adequately assessed. At this point, the major observations indicate that urinary and serum nucleosides might serve as useful indicators for both prognosis and disease activity, although a significant amount of serial studies remain to be done in order to ensure that these correlations are scientifically valid.

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Further, continuing investigation in the area of urinary nucleoside excretion provides a constant stimulus to understanding the biochemical changes which occur at the cellular level in leukemia, and that a clearer understanding of these cellular events may enhance understanding of the leukemogenic process itself. During the last two years we have improved and extensively validated the quantitation of ribonucleosides in biological samples (see Chapters 1, and 5, Part A; and Chapter 2 , Part C). This technology represents a significant advancement over t h e methods that we reported earlier (refs. 38, 39). The precision, speed, sensitivity and ruggedness of our methods are well suited for use in clinical research applications. With the described chromatography protocols, twenty known nucleosides in urine or serum and more than ten unidentified nucleosides can be measured in a single 35 minute chromatographic run. The precision and ruggedness of the method was ensured with the introduction of a new internal standard, 3-methyluridine (m3U), which is added to the urine or serum sample before prechromatography Also, the accuracy of the method was improved by treatment. employing a UV diode-array detector and multi-wavelength quantitation protocols. In our laboratory this nucleoside methodology has been applied on approximately 500 human serum samples, and 200 urine samples with consistent satisfactory results. As presented in the following chapter by Kuo and Gehrke, thirteen human serum nucleoside levels and 17 human urinary nucleoside levels were established on analysis of a large number of samples from a normal population. In addition, preliminary studies on serum nucleosides as potential biological markers for small cell carcinoma, leukemias and lymphomas were achieved. Some significant correlations were noted between the levels and profiles of serum nucleosides and different neoplasias. In a number of collaborative investigations (ref. 40-43) we have extended HPLC-UV analysis to the quantitative analysis of the complete tRNA molecule, with special emphasis on the extensive and complex modifications present in tRNA (see Chapter 1, Part A), improved and interfaced characterization methods (HPLC-UV, MS, NMR, and FT-IR) for elucidating the structures of unknown modified nucleosides (see

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Chapter 5, Part A), developed methods for quantitatively studying the cap structures of messenger RNA (see Chapter 8, Part A), and to a rapid and accurate methodology for studying methylation of DNA (see Chapter 10, Part B). In this research we found new modifications in tRNAs, specifically in position 64 of yeast methionine initiator tRNA, in which 0-prib of u r a n o sy 1- ( 1" - 2') -ad e n o sine - 5 " -phosphate is 1inked by a 3 ' ,5 ' phosphodiester bond to G at position 65 (ref. 40). Also, a new major modified nucleoside (C*) has been identified as om5C in canine serum (see Chapter 5 , Part A); the study of antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of schizosaccharomyces pombe has been published (ref. 41), and this technology has been applied to the identification and measurement of polynuclear carcinogen-ribonucleoside adducts in the urine of fish and rat (Chapter 2, Part C). This methodology has also been used to investigate codon discrimination and anticodon structural context (ref. 42), and the finding of 5-carboxymethylaminomethyluridine in the anticodon of yeast mitochondria1 tRNAs recognizing two-codon families ending in a purine (ref. 43). The broad applicability of RPLC-UV real time diode array analysis was demonstrated by the analysis of nucleosides in human plasma, whole blood, and other biological samples. The measurement and simultaneous detection of an array of nucleosides in complex biological matricies has been demonstrated and widely applied. These new nucleoside chromatography research tools will serve to advance biochemical and biomedical investigations, and present new research approaches to further studies in molecular biology.

1 . 5 REFERENCES 1. E. Borek, The morass of tumor markers, Bull. Mol. Biol. Med., 10 (1985) 103-1 17. 2. T.F. Yu, B. Weissman, L. Sharney, S . Kupfer, and A.B. Gutman, On the biosynthesis of uric acid from glycine-N15 in primary and secondary polycythemia, Am. J. Med., 21 (1956) 901. 3. B. Weissman, P.A. Bromberg, and A.B. Gutman, The urine bases of human urine. 11. Semiquantitative estimation and isotope incorporation, J. Biol. Chem., 224 (1957) 423.

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4. L.R. Mandel and E. Borek, The biosynthesis of methylated bases in

ribonucleic acid, Biochemistry, 2 (1963) 555. 5. R.W. Trewyn and M.R. Grever, Urinary nucleosides in leukemia:

6. 7.

8.

9.

10.

11. 12.

13. 14.

IS.

laboratory and clinical applications, CRC Critical Reviews in Clinical Laboratory Sciences, 24 (1986) 71-93. G. Prodi, What does "marker" mean, in : F. Cimino, G. Birkmayer, J. Klavins, E. Pimentel, F. Salvatore (eds.) Human Tumor Markers, Walter de Gruyter, Berlin, New York, 1987, pp. 3-11. J.G.D. Birkmayer, and B. Paletta, New strategies for follow-up of breast cancer patients using CEA, TPA, CA 15-3 and CA 50, in, F. Cimino, G. Birkmayer, J. Klavins, E. Pimentel and F. Salvatore (Eds.), Human Tumor Markers, Walter de Gruyter, Berlin, 1987, pp. 621630. D.C. Tormey, T.P. Waalkes, D.L. Ahman, C.W. Gehrke, R.W. Zumwalt, J. Snyder, and H. Hansen, Biologic markers in breast carcinoma I. Incidences of abnormalities of CEA, HCG, three polyamines, and three minor nucleosides, Cancer 35 (1975) 721-727. T.P. Waalkes, C.W. Gehrke, W.A. Blyer, R.W. Zumwalt, C.L.M. Olwney, K.C. Kuo, D.B. Lakings, and S. Jacobs, Potential biological markers in Burkitt's lymphoma, Cancer Chemotherapy Reports 59 (1975) 721727. T.P. Waalkes, C.W. Gehrke, R.W. Zumwalt, S.Y. Chang, D.B. Lakings, D.C. Tormey, D.L. Ahman, and C.G. Moertel, The urinary excretion of nucleosides of ribonucleic acid by patients with advanced cancer, Cancer, 36 (1975) 390-397. R.D. Suits and C.W. Gehrke, Reversed-phase chromatographic separation of nucleic acid bases from DNA and RNA hydrolysates, 18th West Central States Biochemistry Conference, 1975. C.W. Gehrke, K.C. Kuo, G.E. Davis, R.D. Suits, T.P. Waalkes and E. Borek, Quantitative high-performance liquid chromatography of nucleosides in biological materials, J. Chromatogr. 150 (1978) 455476. R.A. Hartwick and P.B. Brown, Reversed-phase HPLC of nucleosides and bases, J. Chromatogr. 126 (1976) 679-685. C.W. Gehrke, K.C. Kuo, and R.W. Zumwalt, Chromatography of nucleosides, J. Chroinatogr. 188, (1980) 129-147. F. Salvatore, M. Savoia, T. Russo, L. Sachetti, and F. Cimino, Pseudouridine in biological fluids of tumor-bearing patients, in: F. Cimino, G. Birkmayer, J.V. Klavins, E. Pimentel, and F. Salvatore (eds.) Human Tumor Markers, Walter de Gruyter, Berlin, New York, 1987, 451-462.

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16. D. A. Heldman, M. R. Grever, C. E. Speicher, and R. W. Trewyn, Urinary excretion of modified nucleosides in chronic myelogenous leukemia, J. Lab. Clin. Med., 101 (1983) 783-792. 17. T. Rasmuson, G. Bjork, L. Damber, S. Holm, L. Jacobsson, A. Jeppsson, T. Stigbrand, and G. Westman, Tumor markers in bronchogenic carcinoma, Acta Radiolog. Oncology, 22 (1983) 209-214. 18. Oerlemans and F. Lange, Major and modified nucleosides as markers in ovarian cancer: a pilot study, Gynecol. Obstet. Invest., 22 (1986) 212-217. 19. H.R. Nielsen and S. Killman, Urinary excretion of p-aminoisobutyrate and pseudouridine in acute and chronic myeloid leukemia, JNCI, 71 (1983) 887-891. 20. T. Rasmuson and G. Bjork, Pseudouridine: a modified nucleoside as biological marker in malignant lymphomas, Cancer Detect. and Prevent., 6 (1983) 293-296. 21. T. Rasmuson and G. R. Bjork, Pseudouridine: A prognostic marker in non-Hodgkin's lymphomas, Cancer Detect. Prevent., 8 (1985) 287290. 22. J.W. Mackenzie, R.F. Lewis, G.E. Sisler, W. Lin, J. Rogers, I. Clark, Urinary catabolites of ribonucleic acid as cancer markers: a preliminary report of their use in patients with lung cancer, Annals of Thoracic Surg., 38 (1984) 133-139. 23. F. Esposito, T. Russo, R. Ammendola, A. Duilio, F. Salvatore, and F. Cimino, Pseudouridine excretion and transfer RNA primers for reverse transcriptase in tumors of retroviral origin, Cancer Res., 45 (1 985) 6260-6263. 24. T. Heyman, P. Pochart, C.W. Gehrke and K.C. Kuo: Cell transformation by sous sarcoma virus strongly increases tRNA modification, presented at the international conference on tRNA, Vancouver, British Columbia, July, 1989. 25. T. Rasmuson and G.R. Bjork, Excretion of pseudouridine in urine as a tumor marker in malignant diseases, Bull. Mol. Biol. Med. 10 (1985) 143- 154. 26. D.A. Heldman, M.R. Grever, and R.W. Trewyn, Differential excretion of modified nucleosides in adult acute leukemia, Blood 61 (1983) 291-296. 27. D.A. Heldman, M.R. Grever, J.S. Miser, and R.W. Trewyn, Relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia, JNCI 71 (1983) 269-273. 28. S. Tamuro, Y. Amuro, T. Nakano, J. Fuji, T. Yamamoto, T. Hada, and K. Higashino, Urinary excretion of pseudouridine in patients with hepatocellular carcinoma, Cancer, 57 (1986) 1571-1575.

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29. S. Tamura, J. Fujii, T. Nakano,T. Hada, and K. Higashino, Urinary pseudouridine as a tumor marker in patients with small cell lung cancer, Clin. Chem. Acta, 154 (1986) 125-132. 30. K. Nakano, K. Shindo, and T. Yasaka, Reversed-phase highperformance liquid chromatographic investigation of mucosal nucleosides and bases and urinary modified nucleosides of gastrointestinal cancer patients, J. Chromatogr., 343 (1985) 21-33. 3 1 . Y . Kuchino and E. Borek, Tumor-specific phenylalanine tRNA contains two supernumerary methylated bases, Nature (London), 271 (1976) 126. 3 2. G. Schoch, G. Heller-Schoch, Molekularbiologie und Klinische bedeutung des Stoffwechsels normaler und modifizierter Nucleobasen, Helv. Paediatr. Acta Suppl., 38 (1977) 1. 33. J. Speer, C. W. Gehrke, K.C. Kuo, T.P. Waalkes and E. Borek, tRNA breakdown products as markers for cancer, Cancer, 44 (1979) 2120. 34. J.E. McEntire, K.C. Kuo, M.E. Smith, D.L. Stalling, J.W. Jr. Richens, R.W. Zumwalt, C.W. Gehrke, and B.W. Papermaster, Classification of Lung Cancer Patients and Controls by Chromatography of Modified Nucleosides in Serum, Cancer Research, 49 (1989) 10571062. 35. G.B. Chheda, J.G. Antkowiak, H. Takita, A.K. Bhargava, and H.A. Tworek, Evaluation of 5-carbamoylmethyluridine as an indicator of tumor burden in lung cancer patients, International Symposium on the Analysis of Nucleoside, Nucleotide and Oligonucleotide Compounds, Antwerp, Belgium, Sept. 1989. 3 6. U.R. Tjaden, High performance liquid chromatography of nucleosides, nucleotides and oligonucleotides, International Symposium on the Analysis of Nucleoside, Nucleotide and Oligonucleotide Compounds, Antwerp, Belgium, Sept. 1989. 37. C.W. Gehrke and K.C. Kuo, Ribonucleoside analysis by reversedphase high performance liquid chromatography, J. Chromatogr., 471 (1989) 3-36. 38. C.W. Gehrke, K.C. Kuo, G.E. Davis, R.D. Suits, T.P. Waalkes and E. Borek, Quantitative high-perfomance liquid chromatography of nucleosides in biological materials, J. Chromatogr., 150 (1978) 455476. 39. G.E. Davis, R.S. Suits, K.C. Kuo, C.W. Gehrke, T.P. Waalkes, and E. Borek, High performance liquid chromatographic separation and quantitation of nucleosides i n urine and some other biologic fluids, Clin. Chem. 23 (1977). 1427-1435. 40. J. Degrts, G. Keith, K.C. Kuo, C.W. Gehrke, Presence of phosphorylated o-ribosyl-adenosine in T-yr-stem of yeast methionine initiator tRNA, Nucl. Acids Res., 17 (1989) 865-882.

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41. A.M. Grossenbacher, B. Stadelmann, W.D. Heyer, P. Thuriaux, J. Kohli, C. Smith, P.F. Agris, K.C. Kuo and C.W.Gehrke, Antisuppressor mutations and sulfur carrying nucleoside in transfer RNAs of Schizosaccharomyces pombe, J. Biol. Chem. 261 (1986) 1635116355. 4 2 . F. Lustig, T. Boren, Y.S. Guindy P. Elias, T. Samuelson, C.W. Gehrke, K.C. Kuo, and U. Lagerkvist, Codon discrimination and anticodon structural context, submitted to PNAS, Feb., 1989. 43. R.P. Martin, A.P. Sibler, C.W. Gehrke, K.C. Kuo, J.A. McCloskey, G.Dirheimer, 5-Carboxymethylaminomethyluridine is found in the anticodon of yeast mitochondria1 tRNAs recognizing two-codon families ending in a purine, Accepted, Biochemistry, Nov., 1989. 44. T.P. Waalkes, M.D. Abeloff, D.S. Ettinger, K.B. Woo, C.W. Gehrke, K.C. Kuo, and E. Borek, Biological markers and small cell carcinoma of the lung: A clinical evaluation of urinary ribonucleosides, Cancer 50, (1982) 2457-2464.

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CHAPTER 2 RIBONUCLEOSIDES IN BIOLOGICAL FLUIDS BY A HIGHRESOLUTION QUANTITATIVE RPLC-UV METHOD KENNETH C. KUO1, DAT T. PHAG, NATHAN WILLIAMS', and CHARLES W. GEHRKE' ]Department of Biochemistry, University of Missouri and Cancer Research Center, Columbia, MO 65201 (USA) 2Hewlett Packard Corporation, Avondale. PA ( U S A )

TABLE OF CONTENTS 2.1 Introduction 2.2 Experimental . 2.2.1 Chemicals 2.2.2 Ultrafiltration Procedure . 2.2.3 Phenylboronate Gel Column Procedure . 2.2.4 Procedure for Isolation of Urinary Nucleosides . 2.2.5 Procedure for Isolation of Serum Nucleosides . 2.2.6 Preparation of Internal Standard Solutions . 2.2.7 Determination of Adenosine Deaminase Activity in Serum and Urine . 2.2.8 HPLC Instruments and Conditions . 2.2.9 Creatinine Analysis by HPLC-UV . 2.3 Results and Discussion 2.3.1 Chromatography . 2.3.2 Identification of Urine and Serum Ribonucleoside Peaks . . . 2.3.3 Quantitation of Nucleosides 2.3.4 Internal Standard 3-Methyluridine . 2.3.5 Prechromatography Sample Preparation Procedure 2.3.6 Recovery of the Method 2.3.7 Precision of the Method . 2.3.8 Stability of Nucleosides . 2.3.9 Ribonucleotides and Oligoribonucleotides in Normal and Cancer Serum .

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.

. . . . . . . .

C42 c43 c43 c44 c45 c45 c47 c47

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. c49

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C50 C51 . C51 c54 c57 C60 C65 C7 1 c73 c77

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C42 2.3.10

2.4 2.5

Analysis of Creatinine in Urine and Serum by a Modified HPLC Method . . C80 2.3.1 1 A Comparison of Nucleoside Levels in Random and Total 24 Hour Human Urine Collections . . C83 2.3.12 Serum and Urine Ribonucleoside Levels in Normal Populations . . C85 2.3.13 Clearance Values of Nucleosides . C92 2.3.14 Adenosine Deaminase Activity in Serum and Urine . C93 2.3.15 Serum Nucleosides in Canines with Osteosarcoma. . c93 2.3.1 6 Serum Nucleosides in Leukemia and Lymphoma . C98 Patients 2.3.17 Polynuclear Aromatic Hydrocarbon (PAH) Carcinogen, C99 Ribonucleoside Adducts in the Urine of Fish and Rat Summary . C105 References. C107

INTRODUCTION In the late 1970's we developed analytical protocols for the quantitative measurement of nine ribonucleosides in urine (refs. 1-3). The high resolution, speed, and sensitivity of reversed-phase high performance liquid chromatography combined with the selectivity of phenylboronate gel affinity chromatography (refs. 1-4) of this method have been of value to many researchers who have adopted or modified this methodology and used i t in their laboratories and clinical studies on urinary nucleosides as A comprehensive review of potential biological markers (refs. 5-14). research activities in the field of the modified nucleosides as biomarkers is presented in the Introduction on "Nucleoside Markers for Cancer" and the The latest thirteen chapters presented in Part C of this treatise. experimental approaches and technologies of measurement are described in these chapters. Promising results from these studies on the urinary nucleosides (refs. 1550) stimulated interest on investigations of a larger number of ribonucleosides and especially for ribonucleosides in serum (refs. 5 1-64). The concentrations of the modified ribonucleosides in serum are ca. 1002.1

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fold lower (ppb levels) than in urine and the high protein concentration in serum makes serum a most difficult matrix to analyze. Previous serum nucleoside methods were limited either by sensitivity or selectivity which allowed measurement of only a very few of the serum nucleosides in high concentrations ( Y , I, and U). In 1982 we developed a high resolution HPLC-UV method for quantitative measurement of low picomole amounts of nucleosides in tRNAs (ref. 62). However, this technology for the quantitation of total serum nucleosides was not achieved until after the new sample pretreatment protocols were developed (ref. 64). Using this new sample pretreatment and chromatography methodology, we demonstrated that twenty known nucleosides in urine or serum (hU, Y, ncmSU, mlA, I, X, PCNR, mlI, m*G, ac4C m2G, m2m2G, t6A, m6A, mt6A, ms2t6A, C, U, G and A) and ten unidentified nucleosides can be quantified in a single 35 minute chromatographic run. Further, the precision, speed, sensitivity and ruggedness of the methods are well suited for clinical research applications. In this chapter we have described for the investigators fully validated and reliable methodologies for the analysis of nucleosides including pre-chromatography sample preparation techniques of biological samples. Each chromatographic protocol is designed for high resolution, selectivity, and speed of analysis. Comprehensive information is also presented on investigations on the metabolism of ribonucleic acids and their relationships as biologic markers of cancer.

2.2 EXPERIMENTAL 2.2.1 Chemicals The methanol and acetonitrile solvents used were RPLC grade either of B & J Brand from American Scientific Products (McGaw Park, IL) or OmniSolv from EM Chemicals (Cherry Hill, NJ). RPLC water was obtained through a three-step purification process. The first step was reverse osmosis using an RO-Pure apparatus (Model D0640, Barnstead Company, Boston, MA). The second step of purification was accomplished with a Nanopure four cartridge system (Model D1794, Barnstead) composed of one charcoal cartridge for adsorption of organics, two mixed bed ion-exchange cartridges for removal of anions and cations, and one filtration cartridge capable of removing particulates larger than

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0.22 pm. In the third step, the nanopure water was distilled in an all-glass still with teflon tubing connections (Model AG-11, Corning Glass Works, Corning NY). Ammonium phosphate, zinc sulfate, and sodium acetate were purchased from J.T. Baker Chemical Co., (Phillipsburg, NJ). Ammonium hydroxide and phosphoric acid were from Mallinckrodt Co., (St. Louis, MO). The modified ribonucleoside reference standard compounds used were from several sources including Sigma Chemical Co. (St. Louis, MO), Mann Research Labs (New York, NY) and Vega Biochemicals (Tucson, AZ). Nuclease P1 was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Bacterial alkaline phosphatase (BAP) from E. coli Type I11 was purchased from Sigma Chemical Co. , product No. P-4252, ( St. Louis, MO). The bacterial alkaline phosphatase must be pretested for possible contamination of adenosine deaminase. The above enzymes are the only sources that we have tested which are free of adenosine deaminase activity under our hydrolysis protocol. An enzyme blank must also be run for each newly purchased enzyme lot to observe possible RNA and DNA contamination. All of the transfer ribonucleic acids (tRNAs) as listed were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN), unfractionated tRNAs from brewer's yeast (Cat. No. 109 517), unfractionated tRNAs from calf liver (Cat No. 647 576), and unfractionated tRNAs from E. coli MRE 600 RNase negative (Cat. No. 109 541). Amino acid specific tRNAs from E. coli MRE 600, N-formylmethinoine-specific (Cat. No. 109 584), glutamic acid-specific I1 (Cat. No. 109 609), phenylalaninespecific (Cat. No. 109 673), tyrosine-specific (Cat. No.109 703) and valinespecific I (Cat. No. 109 720), and tRNA phenylalanine-specific from brewer's yeast (Cat. No. 109 657). 2.2.2 Ultrafiltration Procedure To one (1.0) ml of serum, 0.50 nanomoles of internal standard 3methyluridine (m3U) in 100 p1 of water was added and mixed well. The sample was then filtered through a micropartition system (MPS-1, with a 25,000-30,000 molecular weight cut off, type YMT membrane; Amicon Co., Danvers, MA). A 30' fixed angle rotor centrifuge (IEC HN SII Centrifuge equipped with model 1 1/80 rotor International Equipment Co., Needham

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MA) at 1500 g was used for centrifugation. After 3 to 4 hours 600 to 700 pl of filtrate should be obtained. If cost is not of major concern, divide the sample into two MPS-I filtration units and centrifuge at 1500 g for 45 to 60 min. In this way 800-900 p1 of filtrate can be recovered. 2.2.3 Phenylboronate Ge1 Column Procedure Bio-Rad Affigel 601, (Cat. No. 153-61-01, from Bio-Rad Laboratories, Richmond, CA 94804), an immobilized boronic acid based gel, is used. The cleanup procedures for urine and serum are described in detail as follows: Cleaning and Conditioning New Affigel Gel 601: The boronate gel (1 g) is placed in water (ca. 25 ml), allowing a contact time of five minutes to permit the gel to swell. The gel is then alternately washed with methanol and water for at least ten cycles. Following this procedure, the gel is washed two times with 25 ml of 0.1 M NaC1, followed with 3 x 25 ml of 0.1 N HCOOH, 3 x 25 ml of 0.25 M CH3COONH4, 3 x 25 ml of 50% CH30H in water, 3 x 25 ml of 0.1 N HCOOH in 50% CH30H in water, 2 x 25 ml 0.1 M NaCI, and then resuspend the gel in 50 ml of 0.1 M NaCI. The gel is now prepared for placement into the cleanup columns. Column Dimensions: The column length is ten cm and 0.3 cm i.d.. The borosilicate glass column is fitted with a 5 ml reservoir and fine tip plugged with glass wool. Packing the Gel Column: a. Pack the gel column to a height of 3 cm with the washed and conditioned gel in 0.1 M NaC1. b. Just prior to sample cleanup, equilibrate the gel column by passing through the column 15 ml of 0.25 M CH3COONH4, pH 8.8. Be sure that all air bubbles have been removed. Air pockets can be removed by gentle stirring of the gel bed with a glass rod. 2.2.4 Procedure for Isolation of Urinarv Nucleosides 1. Aliquot exactly 250 p1 of urine into a 1.5 ml polypropylene microcentrifuge tube. 2. Add 100 pl of 2.5 M CH3COONH4, pH 9.0.

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3. Exactly add internal standard (m3U), 100 p1 of a 100.0 nmol/ml solution (10.0 nmo1/250 p1 urine). 4. Mix well for a few seconds using a vortex mixer. 5. Centrifuge at 12,000 rpm for three minutes. 6. Transfer the urine sample onto the boronate gel column with a Pasteur pipet and allow free flow, taking care not to disturb the precipitate in the sample tube. 7. Add as rinse 500 p1 of 0.25 M CH3COONH4, pH 8.8, to each sample tube. 8. Mix well for a few seconds using a vortex mixer. 9. Centrifuge at 12,000 rpm for three minutes. 10. Transfer the rinse onto the gel column, taking care not to disturb the precipitate in the sample tube. 11. Wash the gel column with 3 ml of 0.25 M CH3COONH4, pH 8.8, allowing free flow. 12. Wash the gel column with 300 p l of 50% methanol/water (v/v), allowing free flow. 13. Elute the nucleosides with 5.0 ml 0.02 N HCOOH in 50% methanol in water (v/v). Collect in a 10 ml polyethylene tube. 14. Remove the methanol from the eluate using a Speed Vac Concentrator (Savant Instruments Inc., Hicksville, N.Y.) with a water aspirator as vacuum source. When the sample volume is reduced to less than half, essentially all the methanol has been removed. 15. Freeze the sample with the tube in a slanted position. 16. Lyophilize the sample to dryness using the Speed Vac Concentrator with a mechanical vacuum pump and -50°C cold trap. 17. Redissolve the sample in 500 p l of distilled water; vortex for a few seconds. 18. Inject 100 p1 onto HPLC. 19. Prior to column re-use, wash the gel column with 10 ml of 0.02 N formic acid in 50% methanol/water (v/v). This removes strongly adsorbed substances. 20. Wash column with 10 ml 50% methanol/water (v/v). 21. Wash column with 10 ml 0.02 N HCOOH in water.

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22. Store the gel columns in 0.02 N HCOOH each day in water. The gel column can be reused up to fifteen times. If the gel column is not used for several days, store in 0.1 M NaC1. Recovery tests must be conducted to establish quantitation of the cleanup procedure. 2 3 . Prior to use, re-equilibrate the gel column with 15 ml of pH 8.8, 0.25 M CH3COONH4. 2.2.5 Procedure for Isolation of Serum Nucleosides 1. Aliquot exactly 1.0 ml of serum into a 1.5 ml polypropylene centrifuge tube. 2. Add internal standard (m3U), exactly 100 p l of a 5.00 nmol/ml solution (0.500 nmol/l .O ml of serum). 3. Mix well for several seconds using a vortex mixer. 4. See "Ultrafiltration Procedure Section". This step will take approximately four hours and give ca. 600-700 p1 of filtrate from 1.0 ml of serum. 5. Add 250 p1 of 2.5 M CH3COONH4, pH 9.0 to the ultrafiltrate and mix well. 6. Transfer the sample onto a washed, conditioned and preequilibrated boronate gel column. Steps 7 to 16 are the same as for the urinary nucleoside isolation procedure. 17. Redissolve the sample in 200 p1 of distilled water; vortex for a few seconds. 1 8 . Inject 180 p1 of sample onto HPLC. 2.2.6 Preparation of Internal Standard Solutions A 0.500 pmol/ml stock solution of the internal standard (m3U) is made by weighing an accurately known weight (mgs) of m3U (P-L Biochemicals, Milwaukee, WI 53205) and dissolving the m3U (molecular weight = 258.23) in a calculated amount of water (grams) to make the final concentration 0.500 pmol/ml. For example, the weight of m3U is 1.350 mg (5.228 pmol), then 10.45 g of water (by balance) is used to make a concentration of 0.500 pmol/ml. Appropriate dilutions were made from the stock solution to obtain the desired concentrations for the working solutions, 10.0 nmol/nil for

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urine and 0.500 nmol/ml for serum analysis. Each new dilution of working solution should be checked by HPLC for accuracy (compare with the established value of arednmol of m3U). All solutions are maintained frozen at -20°C in small aliquots and thawed out only just prior to use. 2.2.7 Determination of Adenosine Deaminase Activity in Serum and Urine In Serum: 1.0 ml of pooled normal human serum was pipetted into each of six 1.5 ml polypropylene microcentrifuge tubes and the substrate and enzyme were added as follows to study adenosine deaminase activity on A and m l A . 1. a control sample not spiked , no adenosine deaminase added. 2. sample not spiked, add 10 p1 adenosine deaminase (2.5 units). 3. sample spiked with 0.5 nmol adenosine, no adenosine deaminase added. 4. spiked with 0.5 nmol adenosine, add 2.5 units adenosine deaminase. 5. spiked with 0.5 nmol mlA, no adenosine deaminase added. 6. spiked with 0.5 nmol m1A , add 2.5 units adenosine d e am i n a s e . Vortex and centifuge. Incubate over night at 37 "C. Analyze on HPLC.

In Urine: Pipet 250 p1 of urine into each of six 1.5 ml polypropylene microcentrifuge tubes. Add 100 p1 0.1 M KH2PO4 to adjust acidic urine to pH 7.5. Label the centrifuge tubes 1-6 and add substrate and enzyme as follows: 1 A control sample, not spiked with adenosine and no adenosine deaminase added. 2. Sample not spiked, add 1.25 units of adenosine deaminase. 3. Sample spiked with 3 nmol adenosine, no adenosine deaminase added. 4. Sample spiked with 3 nmol adenosine, add 1.25 units adenosine deaminase.

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5. Sample spiked with 3 nmol mlA, no adenosine deaminase added. 6. Sample spiked with 3 nmol m l A , add 1.25 units adenosine deaminase. Vortex and centrifuge. Incubate over night at 37°C. Analyze on HF'LC.

..

2.2.8 HPLC Instruments and Conditions A fully automated LC instrumentation system consisting of a n H P 1090M (Hewlett Packard, Avondale, PA). The HP-1090M system was made up of a DR5 ternary solvent delivery system, variable-volume autoinjector, autosampler, diode-array detector, and heated column compartment. The liquid chromatography workstation is based on an HP model 310 computer supported by Rev. 4.05 operation software; HP-HIL 512 x 400 color monitor with bit-mapped display; and HP-9133H 20 mb Winchester disc drive with 3.5" 710 kb micro floppy disk. A Think-Jet printer and H P 7475A plotter were used for hard copy data presentation. The cooling coil of the heated column compartment was circulated with refrigerated ethylene glycol based antifreeze by a Haake model FJ circulating bath (Saddle Brook, NJ). The cooling bath was positioned inside a small refrigerator and the antifreeze was also circulated through a 10 ft, 114 in. coiled copper tubing which was positioned inside the freezer compartment for additional cooling. Detailed chromatographic conditions are as follows: Column: Supelcosil LC-18s 15 cm x 4.6 mm, with 2.0 x 4.6 mm LC18s-Supelcosil Guard column, (Supelco, Inc., Bellefonte, PA). Flow-rate: 1.O ml/min. Column Temperature: 26 It: 0.5 "C. Elution Buffers: A: 2.50% methanol in 0.010 M NH4H2P04; pH 5.3. B: 20.0% methanol in 0.010 M N a H 2 P 0 4 ; pH 5.3. C: 50.0% acetonitrile in water. The elution gradient is presented in Table 2.1.

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Table 2.1 HPLC Elution Gradient for Separation of Ribonucleosides in Urine and Serum Step No.

1 2

Buffer A

Step Time (min)

Composition,% B C 0.0

100.0 90.0 75.0 40.0 0.0 0.0 0.0 0.0

7.2 4.8 3.0 4.2 7.8 8.0 0.5 4.5 ~

~

~

_

_

_

10.0 25.0

60.0 100.0 60.0 0.0 0.0 _

_

0.0 0.0 0.0 0.0 0.0 40.0 100.0 100.0

Gradient Type

Isocratic Linear Linear Linear Linear Linear Linear Isocratic

_

Equilibrate the column with 100.0% Buffer A for 15 min between runs.

2.2.9 Creatinine Analysis by HPLC-UV

Sample Preparation: 1. Aliquot exactly 100 p1 of serum and/or urine into a 1.5 ml polypropylene microcentrifuge tube. 2. Add 250 p1 of acetonitrile to the sample. 3 . Vortex for 10 seconds, hold at 0 "C for one hour. 4. Centrifuge at 12,000 x g for 2 minutes. For Serum Creatinine Analysis: 5 . Aliquot exactly 100 p1 of the supernatant and transfer to a glass WISP insert. 6. Evaporate to dryness using a Speed Vac concentrator and water aspirator. 7 . Redissolve the sample in 100 p1 of HPLC water and mix well. 8. Inject 50 pl onto HPLC column. For Urine Creatinine Analysis: 5 . Aliquot exactly 20 pl of the supernatant and transfer to a glass WISP insert. 6. Evaporate to dryness using a Speed Vac concentrator and water aspirator.

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7. Redissolve the sample in 200 pl of HPLC water and mix well. 8. Inject 50 p1 onto HPLC column. HPLC Conditions for Creatinine Analysis: Column: Whatman SCX-10, 25 x 0.46 cm with 2 x 4.6 cm Whatman SCX Guard column. Elution Buffer: 0.10 M NH4H2P04, pH 4.8. Flow-rate: 2.0 ml per min, isocratic. Column Temperature: 40 "C. Detection: 254 nm. Using the above conditions on a new column, the retention time for creatinine is between 4.5 and 5.0 min. As the column is used (ca. 100 sample runs), the retention time of the creatinine decreases. In general, the retention time can be restored to 4 to 4.5 min after the column is washed with 70% methanol in water. When the retention time of creatinine decreases to 3 to 3.5 min, the eluant salt concentration is decreased to 0.05 M. A standard creatinine solution is analyzed with each ten samples to re-calibrate the response factor. 2.3 RESULTS AND DISCUSSION 2.3.1 ChromatograDhv The chromatographic conditions that we used for the reversed-phase liquid chromatographic separation of ribonucleosides in urine and serum was essentially the same as the high speed chromatography method for nucleosides in RNA hydrolysates (ref. 66), except the run time was reduced to 35 minutes (steps 7 and 8 in Table 2.1 were used to wash the strongly retained compounds from the column). We did not customize a separation gradient specific for the nucleosides in physiological samples as we wanted to apply the qualitative and quantitative parameters established for RNA hydrolysates directly to these physiological sample analyses. Chromatograms are presented in Figures 2.1-a and 2.1-b from a 254 nm signal of urine and serum nucleosides in a leukemia patient. In these chromatograms twenty-one known nucleosides were identified. The array

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3

Human Serum

4

E

Fig. 2.1 Chromatogram of modified nucleosides in human serum and urine of a leukemia patient. See Experimental for chromatographic details.

of nucleosides in physiological fluids is considerably different from the patterns for tRNAs (ref. 6 6 ) . More than 60 known modified nucleosides were observed in tissue tRNAs but only 15 of these were found in serum and urine. The fate of those missing ribonucleosides is yet unknown. Ten unidentified peaks in serum and urine were classified as unknown ribonucleosides because not only do they possess a ribose moiety but they also have UV spectra similar to the nucleosides. They are either metabolic products of modified nucleosides, minor components of modified nucleosides in tRNAs, or catabolites of other ribose related biomolecules. Since the nucleoside pattern and composition in physiological samples are different from the tRNA hydrolysates a customized HPLC separation protocol for urine and serum nucleosides should be developed. There is a need for an HPLC protocol to increase the separation in two regions of the chromatogram. One region is at the beginning of the run to the guanosine peak. Decreasing the pH of buffer A from 5.3 to 4.5 - 4.0

c53

10

3

Q

E s c4c

0

0

20

10

40

30

50

15

3

< E

0 0

20

10

30

Time (rnin)

Fig. 2.2-a Chromatography of nucleosides in serum (0.20 ml) on a 25 cm x 2.1 mm minibore column. Fig. 2.2-b Chromatography of nucleosides in serum (1.0 ml) on 15 cm x 4.6 mm analytical column. All other chromatographic conditions were the same.

c54

and decreasing the concentration of the methanol to 1% will increase the separation of the y~ peak from the background peaks and improve the separation of m7G, I, T, X, and G peaks. The second region is between the l-methylinosine ( m l I) peak and the 2-methylguanosine (m2G) peak, especially the separation between m2G and 4-acetylcytidine (ac4C) peaks. In serum, the ratio of ac4C and m2G is high and often results in separation insufficient for quantitation. Decreasing the buffer gradient ramp slope between 19 to 24 minutes will improve the separation of the m l I and m l G peaks from their adjacent peaks as well as the separation of ac4C from m2G. Also, the step-time of the gradient step 1 should be correspondingly reduced to maintain the elution efficiency of the late eluting peaks. A major improvement would be the use of minibore (2.1 mm id) or microbore (1.0 mm id) columns. Minibore and microbore columns will greatly improve the sensitivity of the method. Preliminary comparisons of a 15 cm x 4.6 mm analytical column and a 25 cm x 2.1 mm minibore column for serum analysis are shown in Figure 2.2. One ml of serum was injected into the analytical column (Fig. 2-b) and only 0.2 ml of the same serum sample was injected into the minibore column (Fig. 2-a). A fourfold increase in mass sensitivity (peak area or height per unit weight of nucleoside) was observed for the minibore column as compared to the regular analytical column. Still greater increases in sensitivity can be achieved with a 15 cm length minibore column instead of the 25 cm minibore column shown here. With the minibore column, the serum volume needed for the analysis could be reduced to 250 p l or less. This reduction in serum sample volume allows a significant decrease in sample preparation time and simplification of automation of the total analysis process. High quality minibore columns are available from many suppliers and many HPLC instruments are available for the minibore column application. Further, more than a 20-fold increase in sensitivity could be expected from a microbore column as routine gradient applications of microbore HPLC are available. 2.3.2 Identification of Urinarv and Serum Ribonucleoside Peaks Nucleoside peaks from urine and serum were identified by comparing their chromatographic retentions and UV spectra with known reference nucleosides. In addition, some major urinary nucleoside HPLC

c55

Table 2.2 Day-to-Day HPLC Retention Time Reproducibility Chromatography Retention Nucleoside

hU Y

C ncm5~ U m 3 ~ mlA m 5 ~ I G m 3 ~ mlI mcmR~ mlG ac4~ m2G A m2m2G mcm5s2~ t6A m6A mt6A ms2t6A

Mean

3.02 3.21 4.19 4.43 6.03 6.56 7.81 8.61 12.80 14.19 17.21 19.23 19.49 19.84 20.62 20.92 21.68 24.06 25.68 26.16 28.93 30.67 32.42

Time,

of Nucleoside

min

SD 0.035 0.043 0.061 0.070 0.093 0.488 0.273 0.164 0.213 0.203 0.200 0.0467 0.3 12 0.144 (N = 1) 0.125 0.182 0.181 0.243 0.131 0.431 0.0573 0.262

RSD,% 1.16 1.36 1.45 1.33 1.54 0.732 3.50 1.91 1.67 1.43 1.16 0.243 1.60 0.723 0.604 0.838 0.754 1.10 0.500 1.49 0.187 0.807

High Speed Nucleoside Chromatography, a Supelcosil LC-18s 150 x 4.6 mm column was used with 2.0 cm LC-18s guard column. Data were collected from 29 HPLC runs over 3 days.

peaks, such as v, PCNR, mlI, mlG, and m2m2G were collected structure confirmed by mass spectrometry (ref. 67). In routine a combination of retention time, peak shape and the A254tA280 ratio, in general, are sufficient for positive identification (Figures

and their operation, absorption 2.2, 2.3).

C 56

Table 2.3 RPLC-UV Response Factors of Ribonucleosides in Serum pet. Nucleoside

hU Y

C ncms~ U m 3 ~ mlA m 5 ~ m7G I X

G PCNR m 3 ~

Q mlI rncrns~ mlG ac4~ m2G A m2m2G mcm5s2~ t6 A Br8G m2A m6A mt6A ms2t6A

Time(min)

RMR-Rr8G

4.70 5.03 6.55 7.21 9.28 10.21 11.87 13.20 19.60 19.90 20.84 21.77 22.80 27.90 28.31 30.73. 30.98 31.64 32.62 33.14 34.70 38.33 41.04 41.88 42.90 45.20 46.65 48.80 52.70

3.02 3.21 4.19 4.43 6.03 6.56 7.81 8.61 12.80 12.80 13.52 14.19 14.78 17.10 17.95 19.23 19.49 19.84 20.72 20.92 21.68 24.06 25.68 26.16 26.80 28.86 28.93 30.67 32.42

RMR-IIL~Y

254/280

AJuLwuz-

Urine and

MR 254/280/21(

0.37810.505 0.40910.275 0.38 910.561

0.59411.73 0.64210.949 0.61211.94

0.52510.280 0.30510.627 0.67110.244 0.29310.627 0.63010.620 0.68010.250 0.40910.450 0.83710.667 0.879/0.432 0.63610.283 0.52510.531 0.56010.225 0.26610.272 0.83710.695 0.31110.217 0.91810.680 0.781/0.184 0.949/0.811 0.219/1.11 0.41310.629 1.0011.00 0.73310.184 0.61610.713

0.82510.977 29011 14 0.47912.16 1691256 1.0510.842 371199.6 1621256 0.46112.16 0.99012.14 3481253 1.0710.863 3761102 2261184 0.64311.55 1.3212.30 4631272 1.5111.72 4861177 1.0011.00 35211191364 2901217 0.82511.83 3 10191.9 0.88010.776 14711 1 1 0,41810.939 1.3212.40 4621284 0.48910.749 172188.6 1.4412.35 5071278 431175.1 1.2310.635 1.4912.80 5241331 1211453 0.34413.84 0.64912.17 2281257 5531408 1.5713.43 1.1W0.635 406175.1 0.96812.46 3401291

2091206 2261112 2151229

to be re-established to be re-established

HRNC: High Resolution Nucleoside Chromatography; Supelcosil LC-18s 250 x 4.6 mm column with 2.0 cm LC-18s guard column. HSNC: High Speed Nucleoside Chromatography; Supelcosil LC-18s 150 x 4.6 mm column with 2.0 cm LC-18s guard column. R M R - B r 8 G : Relative Molar Response; 8-Bromoguanosine as internal standard. R M R - m 3 U : Relative Molar Response; 3-Methyluridine as internal standard. M R : Molar Response; in units of arealnmol, area is the counts that were obtained from HP-1090M liquid chromatography work station. 254/280/210: 2 5 4 n m l 2 8 0 n m 1 2 1 0 n m . hU: peak area from 210 nm. P C N R : l-Ribosylpyridin-4-one-3-carboxamide

c57

The retention times for 23 nucleosides, which were established in our laboratory, using the experimental protocols presented by Gehrke and Kuo (ref. 62) are given i n Table 2.2. Figure 2.3 shows a 254 nm Thirty-six chromatogram of nucleosides from a pooled urine sample. known and unknown peaks were observed and their corresponding HPLCUV spectra are presented i n Figures 2.4-a and 2.4-b. These spectra were routinely used for additional peak identification and confirmation of the purity of the peaks.

2.3.3 Ouantitntion of Nucleosides The urine and serum nucleosides were quantified by using the dual wavelength (254 and 280 n m ) internal standard method, except for hU for which only 210 nm was used. The relative molar response factors (RMR) using Br*G or m3U as internal standard, and molar response factor (MR) of 27 nucleosides that we obtained are presented in Table 2.3. Molar response factors can only be applied when both an HPLC data system and chromatographic conditions identical to ours are used. The relative molar

I"""""""""'""'""'~"""""'""""~"''

0

10

20

30

40

50

Time (rnin)

Fig. 2.3 HPLC of ribonucleosides in human urine with HPLC-UV spectral identification. Refer to Experimental for chromatographic details.

C58

Fig. 2.4-a HPLC-UV spectra (200 to 350 nm) of ribonucleosides in urine.

c59

I

I

.........

*m m ,

36.1

42.6

I..

.

.

I

Fig. 2.4-b HPLC-UV spectra (200 to 350 nm) of ribonucleosides in urine.

C60

response factors can be used when the same HPLC chromatographic conditions are employed. All of the factors were confirmed using pure known sequenced tRNAs. 2.3.4 Jnternal Standard 3-Methvluridine Due to the multiple sample preparation steps, the internal standard method is essential for the accuracy of quantitation. The internal standard must be added to the sample prior to any sample manipulation. The most desirable internal standard; i) should if possible, be a ribonucleoside or a compound that has the same chemical and physical properties as the ribonucleosides, ii) must not be present in the sample, iii) stable throughout the entire analytical-chromatographic procedure, iv) elute at a position without interference peaks so that it can be integrated accurately. 3-Methyluridine (m3U) clearly met all of the above criteria. It was reported not to occur in tRNAs and only at a very low level in rRNAs. m3U is present at only trace levels in urine and serum from both normal and cancer patients (Tables 2.4 and 2.5). The endogenous m3U in serum and urine is less than 1.5% and 0.91%, respectively, of the m3U added as internal standard to the samples. Thus the presence of endogenous m3U can result in no more than a 1.5% and 0.91% positive bias in the analysis of serum and urine. An unknown nucleoside eluted just before m3U and its concentration varies in different samples. Thus, the HPLC separation must be sufficient to separate this unknown from m3U. Also, 2'-methyluridine (Urn)is a major modified nucleoside in tRNAs, and it is usually present in urine and serum at high concentration. The present HPLC protocol does not separate m3U from Urn. Thus, caution must be taken in using the correct phenylboronate gel isolation steps to ensure a complete removal of Urn. As Urn is not a cis diol it passes through the boronate gel column. The performance of m3U as an internal standard was demonstrated by the quantitative recovery of m3U from the phenylboronate gel columns at physiological concentration (Table 2.6) and quantitative recoveries of spiked yr, mlI, m2m2G, and t6A in serum were obtained based on m3U as the internal standard (Table 2.7). N2lN2-Dimethylguanine (refs. 1-2), 8-bromoguanosine (ref. 69), 6methylisocytosine (2-amino-4-hydroxy-6-methylpyrimidine), Tubercidin (7-deazaadenosine) (refs. 11-13), 5-hydroxymethyluridine (ref. 68),

C61

deoxyadenosine (ref. 2 6 ) , deoxyguanosine (ref. 52) and other nucleoside analogs have been used as internal standards by various investigators. We selected m3U as internal standard to measure ribonucleosides in serum and urine because m3U i s a typical modified pyrimidine and is stable chemically and biochemically. Thus, it is one of the best possible internal standards to ensure the accuracy for the internal standard method.

Table 2.4 Endogenous 3-Methyluridine

(m3U) in Human Serum

Serum

Endogenous nmol/ml

NS-I NS-2 NS-3 cs-I cs-2 cs-3

0.0075 0.006 1 0.0054 0.0074 0.0067 0.0033

Endo./IS,

%

1.5 1.2 1.1 1.4 1.3 0.7

NS = Normal Serum CS = Cancer Serum Endo./IS,% = Percent of endogenous m3U to added m3U; 0.5 nmol/ml of m3U was added.

Table 2.5 Endogenous 3-Methyluridine Urine NU-1 NU-2 NU-3

cu-1

cu-2 cu-3

(m3U) in Human Urine

Endogenous n m o I / ni 1 0.132 0.104 0.364 0.028 0.360 0.028

Endo./IS,

%

0.33 0.26 0.91 0.07 0.90 0.07

NU = Normal Urine CU = Cancer Urine Endo./IS, % = Percent of endogenous m3U to added m3U; 40 nmol/ml of m3U were added.

C62

Secondly, m3U has not been found in tRNAs. It is only found in very low amounts in rRNA. We have examined m3U levels in pooled and individual serum and urine from both normal and cancer patients. Very low amounts of m3U were detected in all the samples. In addition, the amount of m3U does not increase in cancer urine and serum as does the other modified nucleosides (See Tables 2.4 and 2.5). And thirdly, m3U elutes at an open area of the chromatogram and can be integrated accurately. It elutes in the middle of the chromatography run and also in the middle of a gradient ramp, thus the variation between runs can be more readily compensated by relative retention times and relative response factors to m3U. With this internal standard the reliability of peak identification and accuracy of quantitation are enhanced. Over the past two years, the m3U internal standard method has been rigorously validated and applied in our laboratory. More than 500 serum samples and 250 urine samples have been analyzed. The results obtained have been very satisfactory. The performance of this internal standard method was demonstrated by the quantitative recovery of m3U from the phenylboronate gel columns at physiological concentration (Table 2.6). This shows the stability of m3U, and m3U gave essentially an identical recovery as for the other modified nucleosides. Further, the precision of recovery from the boronate affinity column was excellent. Using m3U as the internal standard, quantitative recoveries of four nucleosides mlI, m2m2G, and t6A) in serum were obtained (Table 2.7). This proved that m3U can quantitatively compensate for the loss of endogenous ribonucleosides during ultrafiltration. A slightly lower recovery (85%) of m6A is due to incomplete elution from the gel column. To improve the recovery of m6A, the elution volume of 0.02 N HCOOH in 50% methanol should be increased from 4.5 ml to 5.0 ml. Additional evidence in support of the use of m3U as the internal standard was obtained by comparing physiological nucleoside values that we obtained with corresponding values obtained by other high quality methods. The recent report by John T. Bernert, Jr. et al. (ref. 68) on an in serum and HPLC-UV internal standard method for quantitation of urine provides this comparison. Bernert et al. used 5-hydroxmethyluridine (omsU) as internal standard. The average values that they obtained from 19 normal urine and' serum samples were 2.77 nmol/ml for

(w,

w

w

C63

Table 2.6 Recovery of Standard Reference Ribonucleosides Phenylboronate Gel* Column

Added to

Recovery,% N

Conc.(pmol/ml)

Mean

SD

RSD,%

w

19 20

1150 1150

91.7 91.5

4.46 4.66

4.86 5.09

mlI

19 20

270 270

99.8 104

2.38 2.20

2.38 2.19

m 3 ~

19 20

474 474

95.3 93.9

2.43 2.69

2.54 2.86

m 2m2G

19 20

480 480

2.56 4.30

2.51 4.17

t6A

19 20

967 967

2.46 4.35

2.62 4.42

Nucleoside

102 103 94.0 98.7

1.0 ml of nucleoside standard solution was added to each of the columns. Phenylboronate Gel: Affigel 601 from Bio-Rad Laboratories. Two groups of gel columns were tested approximately 6 months apart. Each group consisted of 19 and 20 columns, respectively.

Table 2.7 Recovery of Added Nucleosides from Human Serum Recovery

%

Run No.

Y

m11

m 2m 2G

t6A

m6A

1

90.5 97.2 97.7 91.5 90.0 94.6 89.8 96.2 93.4 94.0 90.0 96.1

120 99.5 108 96.3 102 99.3 103 101 109 103 108 98.2

91.0 99.7 97.9 91.7 98.0 93.2 99.7 91.3 93.7 102 95.7 92.0

101 104 101 93.9 100 98.5 92.9 101 100 97.5 96.8 95.4

87.2 82.3 85.2 87.7 90.2 89.0 86.9 79.4 88.8 84.2 83.7 79.3

2 3 4 5 6 7 8 9 10 11 12

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Table 2.7 (continued) Mean

SD RSD, %

93.5 2.90 3.10

95.5 3.85 4.03

104 6.46 6.22

98.5 3.31 3.36

85.2 3.65 4.28

Recovery % = [(Cspk - c ) X loo]/ (Cadd) C = determined mean nuclcoside concentration value, in pmol/ml, in pooled normal serum. Cadd = pmol/ml of nuclcoside added to the pooled normal serum. Cspk = determined mcan nucleoside concentration value, in pmol/ml, of nucleoside in spiked pooled normal serum. Pooled normal serum obtained from blood bank. The nucleoside concentration in pmol/ml was dctermined by our nucleoside rncthod:

picomoleslml Y

Mean (N = 5) 1980 SD 67 3.4 RSD, %

m11

m 2 ni 2 G

t6 A

m 6A

73.9 4.0 5.4

24.8 1.1 4.4

39.4 2.1 5.3

<1

---

Table 2.8 Comparison of Human Urine and Serum Pseudouridine Values Bernert's y~ V a l u e s Serum Urine

2.77 nrnol/ml (N=19) 375* mmo1/24 hr (N=19)

Our y~ Values 2.84 nmol/ml (N=94) 331 mmo1/24 hrs. (N=18)

*Calculated from Tablc 1 of rcf. 68. (203.2 x 1.851 = 375 mmo1/24 hrs.)

serum and 375 mmo1/24 hours for urine. These values were in good agreement with the values that we obtained (Table 2.8). These data show that the internal standards n:3U nnd hm5U performed equally well.

C65

2.3.5 Prechromatogaphy Sample Preparation Procedure Two selective isolation protocols, ultrafiltration to remove high molecular weight biopolymers, and phenylboronate gel affinity column chromatography, were used for selective isolation of ribonucleosides from other components of serum and urine. Ultrafiltration is not required for urinary nucleoside analysis. However, the phenylboronate gel isolation step is needed for all physiological fluid samples. Whole blood, plasma, serum, cell culture medium, and other body fluids which contain a high concentration of protein or other high molecular weight biopolymers, require the additional ultrafiltration steps. Enzymatically hydrolyzed tissue RNAs can be directly injected on to the HPLC column without pretreatment. The flow charts of the protocols for cleanup of serum and urine are presented in Figures 2.5 and 2.6 and are detailed stepwise in the Experimental Section. Ultrafiltration of Serum: We have investigated many different physical and chemical deproteinization procedures and found that ultrafiltration gave the lowest and most reproducible background. The direct application of serum onto the phenylboronate gel resulted i n a number of high background peaks. In this case only y~ and Urd can be measured as the serum concentrations of these two nucleosides are more than 100-fold greater than the other nucleosides. Ultrafiltration, with a micropartition system (MPS-1) equipped with YMT membranes produced the lowest and most reproducible background peaks and required less labor than the use of other deproteinization procedures. The disadvantages are the high cost of the filter and a lower absolute recovery of the nucleosides which is due to incomplete filtration. One ml of serum filtered by one MPS-1 filter gave only 0.6 to 0.7 ml of filtrate after centrifugation for 4 hours at 1000 to 2000 x g. Using 0.5 ml of serum per filter usually resulted in the recovery of more than 0.4 ml of filtrate in 45 minutes. This greater recovery of sample in a shorter period of time, however, increased the analytical cost per sample as two filters were needed to obtain the filtrate from one ml of serum. Improved recovery of filtration by washing the protein precipitate resulted i n a considerably lengthened sample preparation time. Since the internal

C66

Fig. 2.5 ISOLATION of RIBONUCLEOSIDES IN HUMAN URINE

250 pl urine 100 pl 2 M NaOAc 1.0 nmole 3-Me-Urdl100 p1 water

Load on washed and conditioned Boronate gel column (Affigel 601) Gel bed: 3 cm x 0.3 cm I

I Wash with: 1 ) 3.0 ml 0.25 M NH40Ac pH 8.8 2 ) 300 ~1 50 % methanol in water

1 I

I

Elute nucleosides with 4.5 ml 0.02 N HCOOH in 50 % methanol in water

I

I Evaporate methanol Lyophilize off water redissolve in 500 pl water

I Inject 100 pl onto HPLC

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Fig. 2.6 ISOLATION of RIBONUCLEOSIDES IN HUMAN SERUM Add to 1.0 ml Serum 0.50 nmoles 3-Me-Urd in 100 p1 water

Filter through Amicon Centricon-10 use micro concentrator with type YMT, 25K-30K cutoff filter.

Collect filtrate, add 250 p1 2.0 M NH40Ac pH 9 solution

Load on washed and conditioned Boronate gel column (Affigel 601) Gel bed: 3 cm x 0.3 cm)

Wash with: 1 ) 3.0 ml 0.25 M NH40Ac 2 ) 300 p.1 50 % methanol in water

C68

standard (m3U) was added before filtration and as there was no selective loss of m3U during the sample preparation, accuracy of the measurement was still maintained. We chose the simplest and most cost-effective procedure; namely, using one ml of serum per filter without additional washing. The recoveries of the five selected nucleosides (w,mlI, m2m2G, t6A, and m6A) from 12 independent determinations, are presented in Table 2.7. These nucleosides were added to pooled normal human serum at 2 to 3-fold their physiological concentrations. The concentrations of the endogenous nucleosides in the pooled serum were determined and are presented as a footnote to the table. Quantitative recovery of all the added nucleosides was obtained, The average recovery of w, mlI, mZmZG, and t6A from 12 independent determinations ranged from 94% to 104%, with a RSD of 3 to 6%. An average recovery of 85 k 4% was obtained for m6A. Phenylboronate Gel Affinity Chromatography Isolation of Ribonucleosides in Urine and Serum: The phenylboronate nucleoside isolation procedure that Uziel and we developed earlier for urine (refs. 14) cannot be directly applied to serum because the resulting background prevents quantifying the low levels (picomol/ml) of serum nucleosides. We have made several improvements in the new protocol to reduce this background. We employed alternate water and methanol washing steps which successfully removed the impurities from the gel. Repeated swelling of the gel in water followed by shrinking of the gel with methanol forced out most of the UV-absorbing materials which were trapped inside the sponge-like gel. The reagent background was reduced by decreasing the size of the gel bed to 210 p1; this decreased the volumes of reagents used for equilibration, washing, and elution. The gel was washed with 50% methanol in water to remove molecules which were retained by the gel matrix through hydrophobic interaction, and 0.02 N HCOOH in 50% methanol was used for the elution of nucleosides. This minimized the cleavage of the amide linkage between the phenylboronate group and the gel matrix which reduced the chromatographic background. The high selectivity of our prechromatography sample preparation procedure is illustrated in Figures 2.7 and 2.8, in which very few nonribonucleoside molecules were observed. A comparison was made of the 254 nm chromatogram and the 210 nm chromatogram for both urine (Figure 2.7) and serum (Figure 2.8). A peak with a very high A2io/A254

C69

I1

SCC Patient 1.0 ml Sample

2 5 4 nrn. 2 0 mAb

~

~

0

"

,

,

"

"

10

"

"

~

"

"

~

"

"

~

20

~

~

30

~

'

~

"

"

~

40

~

~

"

I

"

~

"

"

50

Tirndmin)

Fig. 2.7 Modified nucleosides and UV-absorbing components in human urine.

1"""""""""'""""""""'"'"'"""''''"' 0

10

20

30

40

50

Timdmin)

Fig. 2.8 Modified nucleosides and UV-absorbing components in human serum.

~

C70

ratio most likely is not a ribonucleoside peak. In the urine sample, we observed one large 210 nm absorption peak at 8.8 min and two small absorption peaks at 27 to 28 minutes (between the t6A and m6A peaks). In the serum sample, we observed some large 210 nm absorption peaks before w, peaks at 9.0, 10.1, 10.9, 23.2, and between the ms2t6A and N39.9 peaks. None of these non-nucleoside peaks interfered with the measurement of the nucleoside peaks. In our laboratory, using this cleanup procedure, low chromatographic backgrounds were consistently obtained from hundreds of urine and serum samples. Occasionally we did observe that some of the new gel columns gave a higher background on the first serum sample analyzed. These background peaks appeared between the m l G and m6A peaks with the peak size around 10 to 30 pmol. These background peaks did not interfere with the modified nucleosides of interest such as a d C , m2rnzG and t6A, but care should be taken in assigning the baseline for correct peak integration. For all new gel columns, a pooled serum sample was routinely run on the new columns to verify the column background before use with actual samples. The miniaturization of the gel column to 3 x 0.3 cm greatly increased the speed of analysis. In our laboratory, an analyst can operate a group of 20 gel columns at one time and process 40 samples per day. The exact usable life of the gel column has not been determined. During routine analysis, a pooled control serum and a control urine with known nucleoside values were analyzed with each group of 10 to 20 samples to monitor the performance of the analysis. The columns still performed well after 15 or more urine or serum samples were processed on each column. As a rule, the gel columns were replaced after 15 sample applications or when the columns were stored for a long period of time (several months) for precautionary purposes. The nucleosides exhibit a hydrophobic interaction with the polyacrylic gel matrix, and elute from the Affigel 601 column in the same order as from C-18 reversed-phase chromatography (unpublished data). 8-Bromoguanosine (BrgG) is strongly retained by the Affigel 601 and after 40 column volumes of 0.5 N HCOOH wash, only 60% was recovered. We have not observed the presence of strong hydrophobic nucleosides such as m6m6A, Y, and i6A in most human urine and serum samples. The question of whether they are not in the sample or simply are not eluted from the

C71

Unsplked Serum

1""""'"""""""""""""""""""""""

0

10

20

30

40

60

Fig. 2.9 Recovery of nucleosides added to serum. gel should be investigated. Lowering the pH of the elution reagent (by increasing the concentration of HCOOH from 0.02 N to 0.05 N or 0.1 N) or adding a few % of 1,2-propylenediol to the elution solvent should be tested. Also, experiments should be made to investigate the use of 100 % methanol as the elution solvent. This would eliminate the time consuming lyophilization step which is essential for automation of this method. For an in-depth description of phenylboronate affinity chromatography, and an excellent review of boronate ligands in biochemical separations, see reference (ref. 65).

2.3.6 Recovery of the Method The performance of the phenylboronate gel procedure is presented i n Table 2.6. One nil of an aqueous solution containing five selected nucleosides at concentrations of 1150 to 270 pmol/ml were applied to two groups of 19 and 20 newly prepared gel columns over a time period of six months. Absolute recoveries ranging from 92 to 104 % were obtained. A

C72

Table 2.9 Recovery of Urinary Nucleosides by HPLC Recovery, Nucleoside

w

U

%

Run 1

Run 2

Run 3

Run 4

Ave.

97.1 101.3 105.0 105.8 105.5 97.7 107.8 100.5 99.8 104.5

91.4 101.5 101.5 110.4 105.5 101.9 109.5 93.5 105.8 102.8

94.1 108.9 99.8 112.9 106.2 102.7 108.9 104.0 102.3 107.9

93.7 103.0 115.0 107.2 113.3 107.4 106.9 99.3 106.5 100.2

94.1 103.6 105.3 109.1 107.1 102.5 108.3 99.3 103.6 103.8

~

_

_

_ ~~~~~~~

~

Spike level added as nmoles per 0.25 ml of urine: w 23.5; U 20.5; mlA 1.87; I 1.76; mlI 1.47; m l G 0.905; m2G 1.39; A 0.615; m h 2 G 1.13; and m6A 1.29.

Table 2.10 Recovery of Nucleosides from Human Serum Nucleoside

Std.-

83.4 103.2 96.4 98.2 106.4 104.7 100.9 99.8 103.8 108.1

Std.

-

2(a)

87.3 101.5 94.1 99.4 106.4 106.4 101.9 98.6 104.0 109.3

Serum-1

Serum-2

87.4 106.6 71.7 116.7 102.7 104.6 (b) 24.7 94.5 134.6

81.5 99.5 77.1 92.1 103.6 101.3 95.8 15.4 86.0 123.9

a) 1.0 ml of water spiked with nucleosides processed as a serum sample. Spike level added as nmoles per 1.00 ml of serum: w 4.81; U 4.17; mlA 0.361; I 0.372; mlI 0.306; m'G 0.184; the m2G 0.277; A 0.123; m2m2G 0.226; and m6A 0.253. b) High ac4C peak, which interfered in the integration of the ni2G peak.

c73

slightly lower recovery of Y (92%) was expected as Y has the lowest affinity to the gel and this loss occurred during the wash step of 300 pl of 50% methanol. However, this wash step is essential to reduce the background peaks. Recovery of m3U at 95% may indicate a slight loss of m 3 U on the gel colurnn. Since the loss is only about 5% and the contribution of endogenous m3U is about 1 to 2% (Tables 2.4 - 2.5), the error in measurement should only be about +3%. We chose not to correct the results from the recovery data of the internal standard because a 3% bias is acceptable for quantitation of biological samples. Figure 2.9 presents the chromatograms of a spiked and an unspiked serum sample from a breast cancer patient. Tables 2.9 and 2.10 present the recovery of 10 nucleosides from urine and serum, respectively. The recovery for all the nucleosides from both urine and serum were essentially quantitative. The recovery data i n Table 2.10 were obtained from a nucleoside performance standard and human serum and shows the effects of a serum matrix on the recovery of the nucleosides. No loss of nucleosides was observed in the aqueous performance standards, but ca. 80% of A and 25% of m l A were lost from the two serum samples. The corresponding high recovery of I indicates the conversion of A to I by deaminase in the serum, and the high recovery of m6A indicates the transmethylation of m l A to m6A at alkaline pH. There is no increase in the concentration of mlI. This implies that m*A does not deaminate to form m l I by adenosine deaminase in serum. Also, this is in agreement with our results from the adenosine deaminase study (see section on adenosine deaminase activity in serum and urine). The origin of the high concentration of m l I in body fluids is still an unanswered question.

2.3.7 Precision of the Method The precision of the method is illustrated in Figures 2.10 and 2.11. In Figure 2.10 a normal male urine sample was analyzed independently in triplicate. The chromatogram obtained at 254 nm from each analysis was plotted. There are more than 30 identified and unidentified peaks which all match with respect to peak height and retention time. Figure 2.11 shows the duplicate analysis of serum from a cancer patient with untreated small cell carcinoma of the lung. The excellent reproducibility of

c74

RUN 2 RUN 3

t

r-vr-v----Tl

0

10

1

20

8

7

7

r

I

1

9

...

-7-r7-l-v-

.

.

I

7

60

40

30 TIME (min)

Fig. 2.10 Reproducibility of HPLC analysis of ribonucleosides in human urine from three independent runs.

RUN 1 -

254 nrn. 2 0 mAU

1 ' ' ' ' ' ' 1 ' ' 10 ' ' ' ' ' ' ' ' ' ' ~20' 1

0

~ ' ' ' ' ' ' ' ' ' ' ' ' ' r ' ' ~ ' ' l " ' ' ' " ' ' ' ' '

30

40

50

Tlrnebnin)

Fig. 2.1 1 Reproducibility of HPLC analysis of ribonucleosides in human serum from two independent runs.

c75

the two independent runs for serum analysis was again demonstrated by the exact match of all the peak heights and retention times between the two runs. The non-reproducibility of the peaks at about 10 minutes is due to the fact that the peaks at 9.6 and 10.5 minutes retention are not nucleosides. They are retained by the gel column resulting from adsorption and not covalent bonding. They are present in the final sample due to variations of washing of the gel column in the cleanup step. A pooled serum sample spiked with nucleosides was used as a control sample to monitor the day to day performance over time of the HPLC-UV nucleoside chromatographic method. This control sample was analyzed with each group of ten to twenty samples. Also the pooled serum was spiked with m l I and m 2 m 2 G at levels so that the change of the respective peak height ratios to the m3U (internal standard) could be visually noted during the chromatographic run. In this way by simply observing the chromatogram the analyst will know the performance of the cleanup step. The concentration of 13 nucleosides from each of the 18 control serum samples analyzed over a period of five weeks is presented in Table 2.11. The results are presented in 3 groups. Runs 1 to 6 were obtained in the first week, runs 7 to 12 in the second week, and runs 13 to 18 five weeks after the sample was pooled. The samples were stored at 4 "C between analyses. The relative standard deviations (RSD, %) of the data for the seven nucleosides (w, U, X, m l I , a&, m2m2G, and t6A) ranged from 3.6% to 9.0% and is quite good. It was also observed that the concentration of seven nucleosides (mlA, I, G, PCNR, ac4C mlG and m6A) changed with storage time, indicating the biological and chemical instability of these nucleosides at 4 "C. High RSD, % values were observed for m l A and m6A, which was caused by the conversion of m l A to m6A during sample preparation. Concentrations of I, G, PCNR, and m l G were affected by the storage time at 4°C. The concentration of I decreased with time, and the concentration of G showed a slight increase in the second week and a decrease after five weeks. PCNR showed a slight increase after one week. The increase in concentration of m l G was caused by an increase of the interference peak N20.3, which eluted as a shoulder at the front of the m l G peak.

Table 2.11 Day-to-Day

0

Precision

of

HPLC

G

PCNR

mlI

mlG

ac4C

2 m2G

t6A

m6A

29.4 26.6 74.5 73.1 85.4 69.1

1050 999 932 894 912 792

59.9 59.6 55.1 53.8 59.9 53.3

45.3 40.5 46.8 41.6 42.5 37.2

45.7 54.4 57.0 52.4 45.6 44.9

340 327 329 341 342 299

19.3 25.3 28.4 25.0 21.9 19.2

125 103 155 123 147 117

425 431 450 450 444 398

52.6 62.9 54.5 49.9 50.7 50.0

37.3 35.2 9.7 12.0 11.9 10.6

7920 7870 7610 7880 7680 7830

53.3 30.1 26.4 38.8 25.9 54.0

1010 1020 969 999 999 1020

96.5 96.9 90.0 87.6 98.2 94.1

66.5 66.3 57.2 54.1 67.8 63.5

63.8 67.2 54.4 60.4 58.1 63.2

329 337 342 340 337 326

34.7 35.7 27.2 31.2 30.6 28.7

111 172 128 163 131 138

417 438 419 433 427 435

50.8 49.5 49.7 47.8 55.3 53.2

46.5 35.9 37.5 39.2

3530 3940 3260 3840 3430 3590

7570 7390 7490 7350 7170 7220

68.3 i i i i 81.4

887 527 614 511 529 539

93.3 85.2 87.2 82.4 43.9 77.8

56.8 41.9 46.0 46.5 25.1 44.2

61.5 55.7 55.2 61.7 71.1 57.0

330 356 370 356 349 354

32.2 39.7 39.0 41.6 43.9 38.0

154 103 106 122 119 106

405 421 436 433 409 408

57.4 54.1 51.9 52.7 50.6 54.0

9.2 22.0 28.7 23.9 22.3 12.3

3815 343 9.0

7792 488 6.3

51.3 22.3 43.5

969

89.2 6.69 7.50

51.9 9.64 18.6

56.9 7.40 13.0

338 15.6 4.6

34.9 5.26 15.1

129 21.4 16.6

426 15.5 3.6

52.8 3.58 6.77

27.1 12.6 46.5

u

1 2 3 4 5 6

4140 4540 3960 3660 3700 3580

8650 8430 8420 8500 8470 7580

7 8 9 10 11 12

3770 3620 3920 3720 3740 3900

13 14 15 16 17 18

RSD,%

U Q1

Analysis

X

Y

SD

Nucleoside

I

Run No.

Mean

Serum

m

l

~

55.5

7.5

All values are in units of pmollml of serum. Pooled serum from both normal and cancer patients was divided were obtained within the first week after the serum was pooled 13 through 18 were obtained after the pooled serum was stored Inosine (1) is not stable during storage at 4 "C. Only the values

25.3

39.6

into 1.0 ml aliquots and stored at 4°C until analysis. Runs 1 through 6 , runs 7 through 12 were obtained during the following week, and runs for five weeks. for runs 1 through 12 were used.

c77

Table 2.12 Storage Stability of Human Serum Nucleosides at -20°C After 10 days and 6 Months

-20°C for 10 Davs

-20°C for 6 Mo nths Serum

Nucleoside Y

U mlA I X G

PCNR rnlI

mlG ac4C

m2m * G t6A

O-Day JO-Davs 0-Dav 10-DavS 5020 7680 3.8 220 i 33 24 130 36 350 50 94

4820 7290 3.0 170 i 23 30 160 40 i 49 97

2640 8840 11 360 150 65 36 100 50 240 46 76

O-Dav6-Mos O-Dav

2560 9040 14 100 234 6.9 29 120 57 260 47 74

1010 6600 15 360 86 47 92 41 81 860 170 28

914 6810 12 50 150 11 98 48 100 680 180 26

1630 6040 23 9700 160 130 140 63 130 1100 22 36

D

L-Mos 1610 6170 14 2440 270 40 120 76 130 860 24 37

All values are in units of prnol/ml of serum. i = interference peak. PCNR = l-Ribosylpyridin-4-one-3-carboxamide.

2.3.8 Stabilitv of Nucleosides The storage stability of urinary ribonucleosides was reported earlier. (ref. 21). Eight nucleosides, mlA, PCNR, mlI, m l G , a d C , m2G and m 2 m 2 G are all stable at -20 "C or -70 "C for at least one month. Since one month is more than sufficient for accomplishing the analysis, a longer storage time was not studied. Urine is generally sterile, thus, the concentration of nucleosides are not altered biologically. Serum, however, contains many enzymes, and biological alteration of the nucleosides upon storage is a major concern. From long term precision data (Table 2.11), we observed that the concentration of certain serum nucleosides change over a period of 5 weeks at 4 "C. In another study four serum samples from cancer patients were selected for storage. After establishing their original (0 day) nucleoside concentrations, the samples were stored at -20 "C until the second analysis. Two samples were analyzed 10 days later and the other two were analyzed after 6 months. The results are presented in Table 2.12. w, m l G , m2m2G, and t6A were

w,

c7a

found to be stable at -20 "C for six months. In general, the other nucleosides either increased or decreased to some extent in concentration over time, with some of the changes being inconsistent within the individual samples. PCNR, a&, and m l I concentrations changed less than 20% over the six months. At this time, we do not have the data for serum stored at -70 OC. However, it is strongly recommended that serum samples should be held at -70 "C to minimize alteration of nucleoside concentration as a result of enzymatic action in the serum.

2.3.9 Ribonucleotides a nd Olieoribonucleotides i n Normal and Cancer

Serum The possibility of observing nucleotides or oligoribonucleotides in serum was a concern, especially in serum from cancer patients who have an abnormally high turnover rate of tRNAs and a highly permeable cell wall. Serum samples from both chronic lymphoblastic leukemia and nonHodgkin's lymphoma have very high concentrations of serum nucleosides. A normal serum sample was used for the control and unfractionated calf liver tRNAs were added to another aliquot of the normal serum to ensure that there were no inhibitors of the snake venom phosphodiesterase (SVP) and bacterial alkaline phosphatase (BAP) in the ultrafiltrate of the serum. The corresponding recoveries of major and modified nucleosides from the SVP and BAP hydrolysate of the normal serum filtrate spiked with calf liver tRNAs indicate that serum does not have inhibitors of the enzymes. Results from the two cancer serum samples are shown in Figures 2.12 and 2.13. In each figure, the lower chromatogram shows the free nucleosides in the serum filtrate, and the upper chromatogram is from the same serum filtrate, but incubated with s n a k e venom phosphodiesterase and bacterial alkaline phosphatase at 37 "C for 16 hours. In both hydrolyzed samples only four major ribonucleosides, Cyd, Urd, Guo, and Ado were increased, but the increased levels were low and less than one nanomole per ml. No changes were noted in the concentration of modified nucleosides in the hydrolyzed samples. A decreased concentration of m l A and an increased concentration of m6A after hydrolysis was due to the alkaline pH of the serum filtrate ( c a . pH 9) which converted m l A to m6A. Some unknown peaks which decreased in size after hydrolysis probably are the oligonucleotides, and appeared as

c79 IN'S

LYMPHOMA

Hydrolyzed ( P D A - L B A P, 1 6 h)

Tine Cninl

Fig. 2.12 RNA oligomers and nucleotides in non-Hodgkin's lymphoma patient serum.

CHRONIC

LYMPOBLASTIC

LEUKEMIA

BKG

Hydrolyzed (PDA L BAP. 16 h)

U

Fig. 2.13 RNA oligomers and nucleotides in chronic lymphoblastic leukemia patient serum.

C80

the small amount of major nucleosides in the hydrolyzed samples. We did not observe any significant amount of oligonucleotides or nucleotides in the normal or cancer serum samples. 2.3.10 Analysis of Creatinine in Urine and Serum bv a Modified HPLC

Method From our earlier collaboration with Dr. Waalkes and the late Dr. Borek it became apparent that the urinary nucleoside levels in normal subjects was a function of the muscle mass of the individual. Therefore, we decided to determine the urinary nucleoside levels in random samples and not in 24 h urine collections. The concentration of creatinine in urine is a function of muscle mass. Thus, we related the nucleoside levels to the creatinine level in urine (refs. 69, 70). Most of the studies on urinary nucleosides in cancer marker investigations have used the nucleoside to creatinine ratio.

Table 2.13 Day-to-Day Matrix Dependent Creatinine in Serum

Precision

mgldl

of

HPLC

Analysis

mgldl

Sample

Run 1

Run 2

Sample

Run 1

Run 2

1 2 3 4

8.57 7.26 5.76 7.61 7.81 9.60 8.23 6.08 8.22 9.60

8.09 7.44 5.37 7.68 8.01 9.66 8.54 6.05 7.98 9.64

11 12 13 14 15 16 17 18 19 20

5.20 8.13 4.66 8.4 1 5.56 9.12 6.54 7.84 7.80 7.4 1

5.60 8.33 4.76 8.47 5.72 8.89 6.39 8.04 7.72 7.38

Mean

7.48 mg/dl 0.24 3.2

5 6 7 8 9 10

SD RSD,%

Data obtained from routine serum creatinine analyses over a period of 2 weeks. A duplicate run was made with each set of 20 samples. SD = {[C(X~-X2)12/2P}o~s

of

C81

-

*

URINE

N

Run 2

STD

1.16 mglml I?

*?

w

1.05 mdml

.4 rn N

*

E

c 0)

SERUM STD 6.65 vg/ml 1 -4 0

w

F Fig. 2.14 HPLC-UV chromatographic analysis of creatinine in urine and serum.

C82

Q

T

1""""'""""""""""""""""""'"""''

0

10

20

30

40

so

Time ( m i d

Fig. 2.15 Consistency of urinary nucleoside profiles from three normal males. When we measured the nucleoside levels in serum of non-cancer patients it was noted for a number of cases that very high levels of nucleosides were obtained for those individuals that have kidney malfunction. This observation agreed with those of our collaborators in Naples. Salvatore's group has proposed that a nucleoside/creatinine ratio (defined as nucleoside coefficient) should be used to correct this discrepancy. They reported that a higher correlation to clinical status was obtained when this ratio was used (refs. 71, 72). In our studies, we used the reported creatinine data provided by the clinical laboratories. In general, their data were obtained by an automated Jaffe colorimetric creatinine method. The sensitivity and precision of the Jaffe method for serum creatinine was poor and only one significant figure was given for the measurement. We obtained the same results of imprecision and positive bias in our laboratory when we made the measurements using the colorimetric method. The accuracy and precision of the nucleoside measurements were often compromised by the creatinine method and

C83

data used. Thus, we modified an HPLC ion-exchange-UV method of Chiou et al. (ref. 73). This modified HPLC method gave good separation (Fig. 2.14) of creatinine from background material. In our laboratory a day-today matrix dependent precision was achieved with a RSD,% of 3.2 (Table 2.13). These data were collected over a two week period using results from 20 independent duplicate analyses. The recovery of creatinine added to serum and urine were in the range of 94.9 to 103%. 2.3.11 A Comparison of Nucleoside Levels in Random and Total 24 Hour Human Urine Collections The level of urinary nucleosides from normal male and female populations were separately examined. Random and 24 hour urine collections were obtained from fifteen males and 10 females, ranging in age from 19 to 58 and 20 to 50 years, respectively. The random specimens were caught at 8:00 am, 1O:OO am and 3:OO pm and the 24 hr samples were collected on the next day. The nucleoside concentrations in urine were expressed as nanomoles of nucleoside per micromole of cre at inine .

Table 2.14 A Comparison of Nucleosides from Random and Total 24 Hour Human Urine Collections Ratio of Nucleoside in Random to 24 Hour Samples Nucleoside Y

mlA PCNR mlI

8 AM 1.03 0.98 0.82 0.99

(20.7%) (28.3%) (38.2) (48.4%)

10 AM 1.07 0.86 1.08 0.96

(17.2%) (49.0%) (25.6%) (55.6%)

3 PM 1.00 0.95 0.95 0.95

(19.5%) (16.4%) (35.5%) (34.7%)

24 Hour 1.00 1.00 1.00 1.00

(19.4%) (27.5%) (31.1%) (35.5%)

m2G

1.13 (19.8%)

1.15 (39.0%)

0.97 (34.0%)

1.00 (38.9%)

m2m2G

0.90 (26.3%)

0.90 (31.6%)

0.94 (31.9%)

1.00 (27.5%)

Nucleoside determined as nmol of nucleoside per pmol of creatinine. Value in ( ) = RSD,%.

C84

Table 2.15 Nucleoside levels in Normal Human Serum ~

pmol of Nucleoside per ml of Serum ~~

Nucleoside

Mean

SD

RSD,%

Max.

Min.

Y

2850 5570 93 1560 85 221 64 65 26 144 25 44 15

755 1340 38 2700 44 387 25 21 8.8 54 5.4 9.9 5.0

26.5 24.1 40.7 173 52 1880 39 32 33 38 22 23 33

4994 8712 270 14000 350 1840 232 110

1820 2990 31 29 16 13 19 11 11 18 17 24 6.3

U mlA I

X G

PCNR

mlI mlG ac4~

m2m2G t6A m6A

51

272 41 61 29

N = 94,age range from 19 to 84,51 females and 43 males. PCNR = l-Ribosylpyridin-4-one-3-carboxamide.

Table 2.16 Nucleoside Creatinine Ratios in Normal Human Serum nmol Nucleoside per p m o l Nucleoside Y

U mlA I

X G PCNR

mlI mlG ac4~

m2m2G t6A

Mean

43.7 84.3 1.22 25.2 1.19 3.59 0.92 0.98 0.38 2.14 0.361 0.638

Creatinine

SD

RSD, %

Max

Min

14.4 30.5 0.48 45.4 0.81 6.79 0.35 0.42 0.14 0.87 0.11 0.15

33.0 36.1 39.7 180 67.9 189 38.1 41.5 37.0 40.5 29.5 23.9

111 185 2.81 234 6.2 38.3 2.06 2.19 0.92 4.15 0.93 1.15

22.1 30.8 0.368 0.323 0.253 0.254 0.159 0.171 0.150 0.339 0.186 0.309

C85

Table 2.16 ( c o n t i n u e d ) m6A

0.222

0.069

30.9

0.39

0.091

N = 94, age range from 19 to 84, 51 females and 43 males. PCNR = l-Ribosylpyridin-4-one-3-carboxamide.

Figure 2.15 demonstrates the remarkably constant excretion of urinary nucleosides from three healthy normal subjects. The chromatograms were plotted after normalization of the m2m2G peaks. The only difference noted is for the peak at a retention time of 9.6 minutes, and as pointed out earlier in the serum precision study (Figure 2.11) this peak most likely is not a nucleoside. 2.3.12 Serum and Urine Ribonucleoside Levels in Normal PoDulationS A number of research groups have reported the nucleoside levels in urine and serum from normal populations (refs. 17, 18, 26, 34, 37, 39, 40, 44, 47, 51, 53, 5 8 , and 63). In these reports, only a few modified nucleosides were reported and at times from a relatively small number of individuals. Our new HPLC technology allowed the quantitation of 21 known ribonucleosides, hU, w, C, ncmSU, U, mlA, I, X, G, PCNR, m3U, m l I , m l G , a d C , m2G, m2m2G, mcm5s2U, @A, m6A, mt6A, and ms2t6A in urine and serum. We made a concerted effort using this methodology to establish a reference level of these nucleosides in a normal population of males and females and of different age groups, and from hospitalized patients who have diseases other than cancer. In collaborative programs with Professor F. Salvatore and his research group at the University of Naples Medical school, Dr. Edith Mitchell in the Department of Oncology, University of Missouri Medical School, and Dr. John McEntire of the Cancer Research Center, Columbia, MO, we collected 94 samples of serum from normal healthy donors and 47 serum samples from non-cancer male patients. The normal healthy population consisted of 5 1 males and 43 females ranging in age from 19 to 84. Thirteen serum modified nucleosides and creatinine were quantified and the data are presented in Tables 2.15 and 2.16. Table 2.15 gives the results of the nucleoside levels in pmol/ml of serum and Table 2.16 presents the serum nucleoside/creatinine ratio in units of nmoles

C86

nucleoside/pmole of creatinine. Data are given for 13 serum nucleosides with mean values, RSD, %, and range values. The RSD, % for most of the nucleosides ranged from 22 to 39% for the population. Inosine, xanthosine, and guanosine showed a high RSD, which was expected, and was discussed earlier in this paper as due to the instability of these molecules and The narrow possibility of re-absorption during the excretion process. distribution (RSD, %) of each nucleoside in the 94 samples was essentially the same whether the data were expressed as pmol/ml or as the nucleoside/creatinine ratio. This indicates a stringently controlled metabolic rate of nucleic acids for healthy subjects. A comparison of normal serum nucleoside values by age and sex are presented in bar graph form in Figures 2.16-a, 2.16-b and 2.17-a, 2.17-b. There is no age and sex dependency for any of the nucleosides studied.

Table 2.17 Nucleoside levels in Human Serum from Diseases* Other than Cancer nanomoles/ml Nucleoside Y

U mlA I

X G PCNR mlI mlG ac4~ m2m2G t6A

m6A

Mean 3.52 7.62 0.100 0.243 0.087 0.033 0.044 0.106 0.034 0.237 0.038 0.048 0.017

SD

RSD, %

1.01 2.16 0.069 0.490 0.040 0.023 0.023 0.039 0.019 0.095 0.014 0.014 0.013

28.6 28.3 69.6 20 1 45.5 73.0 52.1 36.7 54.5 39.9 36.0 29.4 74.9

DOTC* = diseases other than cancer. Population (N) of 47 males, age range 27-83. PCNR = l-Ribosylpyridin-4-one-3-carboxamide.

Max 6.14 13.4 0.498 2.35 0.167 0.114 0.161 0.215 0.111 0.598 0.069 0.098 0.064

Min 2.06 4.40 0.005 0.013 0.016 0.0010 0.0091 0.068 0.01 1 0.088 0.014 0.027 0.002

200 AGE 19-29(37) AGE30-39(29) AGE40-49(9) AGE 50-59(9) II] AGE60-84(8)

IAGE

19-29(37) AGE 30-39(29) AGE40-49(9) AGE50-59(9) AGE60-84(9)

5

\ VI

0

k

.-

a 100

1 pseU

Urd NUCLEOSIDE

C

rnlA

x

PCNR m 1 I rnlG ac4C m Z m X t 6 A NUCLEOSIDE

Fig. 2.16-a and 2.16-b. Comparison of serum nucleoside values by age groups. parentheses gives the number of individuals for that age group.

m6A

The number in

0

200

W W

n

Male(51) Female(43)

Male (51) Female(43)

100

0 pseU

Urd Ino NUCLEOSIDE

mlA '

X

'PCNR ' m l I ' mlG 'ac4C m2m2G t 6 A ' m 6 A NUCLEOSI DE

'

Fig. 2.17-a and Fig 2.17-b Comparison of serum nucleoside values of normal males vs females. number in parentheses gives the number of males and females.

The

C89

Table 2.18 Nucleoside Creatinine Ratios for Human Serum from Diseases Other than Cancer nmol of Nucleoside per pmol Nucleoside Y

U mlA I X G PCNR mlI mlG ac4~ m2m2G t6A m6A

Mean 39.0 85.9 1.16 2.99 0.969 0.376 0.484 1.16 0.384 2.68 0.429 0.536 0.193

SD 10.6 30.0 1.01 6.47 0.477 0.327 0.220 0.412 0.231 1.25 0.167 0.158 0.167

RSD, % 27.3 35.0 87.2 216 49.3 86.8 45.6 35.4 60.2 46.9 39.0 29.6 86.5

Creatinine

Max

Min

63.2 193 7.16 32.8 2.22 1.66 1.23 2.58 1S O 7.95 0.790 0.919 0.851

20.6 40.9 0.041 0.178 0.230 0.015 0.069 0.512 0.090 0.939 0.106 0.281 0.023

Population of 47 males, age range from 27-83.

Table 2.19 Nucleoside Levels in Normal Human Urine pmoles per 24 hours Nucleoside hU Y

U mlA

X G PCNR *lI

mlG ac4~ m2G A

Mean 58.6 331 2.08 24.1 0.63 0.88 11.7 21.1 10.6 15.9 6.05 3.45

SD 24.5 112 1.25 8.90 0.43 0.55 4.71 10.8 4.87 6.62 3.00 2.45

RSD, % 41.9 33.8 60.3 37.0 69.0 62.5 40.2 51.0 45.8 41.8 49.5 71.0

Max

Min

123 648 6.08 48.4 1.18 2.15 25.1 52.4 23.9 29.6 11.7 8.33

27.9 186 0.61 7.89 0.06 0.22 6.17 5.62 2.30 1.68 0.47 0.23

c90

Table 2.19 (Continued) A* m2m2G t6A m6A

3.50 16.1 10.6 0.58

1.76 6.44 3.52 0.53

50.2 40.1 33.2 90.4

7.21 35.9 21.9 1.95

1.44 4.83 6.41 0.15

N = 18; 7 males and 11 females, age range from 25 to 53. PCNR = l-Ribosylpyridin-4-one-3-carboxamide. A* = Unknown, probably is a modified A, calculated as A. Creatinine: Mean = 9.77; SD = 3.66; RSD,% = 37.5; Max. = 16.9; Min. 24 hr).

=

3.88 (in mg per

Table 2.20 Nucleoside levels in Normal Human Urine mg per 24 hours

Nucleoside hU Y

U mlA X G

mlI mlG ac4~ m2G A A* m2m2G mcm5s2~ t6A m6A

Mean 14.4 80.8 0.508 6.78 0.178 0.250 5.96 3.15 4.54 1.80 0.922 0.995 5.01 0.808 4.37 0.164

SD

RSD, %

Max

Min

6.02 27.3 0.305 2.50 0.123 0.156 3.05 1.45 1.89 0.893 0.655 0.471 2.00 0.462 1.45 0.148

41.9 33.8 60.3 37.0 69.0 62.5 51.0 45.8 41.8 49.5 71.0 47.2 40.1 52.7 33.2 90.4

30.2 158 1.48 13.6 0.335 0.610 14.8 7.10 8.45 3.48 2.23 1.93 11.2 1.85 9.03 0.548

6.86 45.4 0.148 2.22 0.016 0.062 1.59 0.683 0.480 0.139 0.062 0.385 1S O 0.037 2.64 0.04 1

N = 18; 7 males and 1 1 females, age range from 25 to 53. PCNR = l-Ribosylpyridin-4-one-3-carboxamide. A * = Unknown, probably is a modified A, calculated as A. Creatinine: Mean = 9.77; SD = 3.66; RSD,% = 37.5; Max. = 16.9; Min. (in mg per 24 hr).

=

3.88

c91

T a b l e 2.21 Nucleoside Creatinine Ratios in Normal Human Urine nmol Nucleoside/p mol Creatinine Nucleoside hU Y

U mlA

X G PCNR mlI mlG ac4~ m2G A A* m2m2G mcm5s2~ t6A m6A

Mean

6.13 35.1 0.217 2.51 0.663 0.093 1.25 1.99 1.01 1.56 0.579 0.348 0.356 1.66 0.240 1.13 0.071

SD

1.47 7.17 0.105 0.557 0.062 0.050 0.348 0.534 0.331 0.573 0.208 0.217 0.105 0.350 0.091 0.23 1 0.081

RSD, %

Max

Min

24.0 20.4 48.5 22.2 93.5 53.1

8.18 53.2 0.495 4.14 0.171 0.204 1.97 3.10 1.54 2.86 0.952 0.847 0.603 2.50 0.376 1.74 0.31 1

2.72 24.0 0.087 1.66 0.008 0.031 0.654 0.807 0.331 0.218 0.120 0.034 0.21 1 1.10 0.019 0.675 0.016

28.0

26.8 32.8 36.6 35.9 62.3 29.5 21.1 37.7 20.4 114

N = 18; 7 males and 1 1 females, age range from 25 to 53. PCNR = l-Ribosylpyridin-4-one-3-carboxamide A* = Unknown, probably is a modified A, calculated as A. Creatinine: Mean = 9.77; SD = 3.66; RSD,% = 37.5;Max. = 16.9; Min. (in mg per 24 hours).

=

3.88

Thirteen serum modified nucleosides in patients with a number of diseases other than cancer (DOTC) were also investigated. This study The data are included 47 males with ages ranging from 27 to 83. expressed in units of nmol/ml of serum (Table 2.17) and nmoles of nucleoside per pmol of creatinine (Table 2.18). The nucleoside values for the DOTC patients were essentially the same as for the normals. Sixteen urinary nucleosides and creatinine were measured in 24 hour collections of urine from 18 normal healthy donors (7 males, 11 females, ages 25 to 50). The standard deviation and RSD, % of a population of individuals are prcsented as pmo1/24 hour, mg/24 hour, and nmol of nucleosides per pmole of creatinine in Tables 2.19, 2.20 and 2.21,

c92

respectively. A narrow distribution of each nucleoside was again observed in the urine of normal healthy subjects as for serum in healthy subjects. We have recently reported an estimation and "reference values " of a series of serum and urinary modified nucleosides by HPLC. A comprehensive statistical evaluation was made of the serum nucleosides to estimate the "normality" of the data distribution to determine the effect of sex, age, and ethnic origin (Italian vs. American) groups on this distribution. The values for all of the nucleosides tested showed a normal distribution. A slightly higher serum nucleoside concentration was obtained for the American group as compared to the Italian group. There was also a small sex (male vs. female) difference observed for m l I and m2m2G. 2.3.13 Clearance Values of Nucleosides It is very interesting to note that the observed serum nucleoside profiles are clearly different from the profile of urinary nucleosides, either expressed in concentration units or as a ratio to creatinine. This can only be explained in that the renal excretion for each nucleoside is different. Using the equation, Clearance (ml of serum/ min) = (U/1440)/S U = pmol of nucleoside/24 hour of urine collection S = pmol of nucleoside/ ml of serum we calculated the clearance values for 10 nucleosides and creatinine from 18 healthy subjects (Table 2.22) and 19 untreated leukemia and lymphoma patients (Table 2.23). Similar clearance values for the healthy and cancer population were observed for the respective nucleosides. However, a larger RSD, % of the clearance values was found in the cancer population. This may be due to the larger variation in renal function of the patients. The much lower clearance values of U, G, and X as compared to creatinine indicates the re-absorption of these nucleosides. The lower clearance value for ac4C is caused by the instabliltiy of this compound in urine, thus, a lower than true value of urinary ac4C was obtained for the calculation. It is interesting that PCNR, mlI, mlG, rnZmZG, and t6A gave much higher clearance values than for creatinine. The clearance of mlA is so high, (> 1000 mYmin) that its serum concentration often is too low to be

c93

measured accurately for the calculation. The high clearance for these nucleosides can be explained only in that secretion of these molecules in addition to filtration is taking place. 2.3.14 Adenosine Deaminase Activity in Serum and Urine To assess if adenosine deaminase will cause a change of A to I and m l A to m l I in urine and serum, we spiked serum with m l A and A. Both A and m l A in serum and urine are converted to I and m lI , respectively in the presence of adenosine deaminase. The rate of conversion for m l A to m l I is approximately 20% of the rate of conversion of A to I. 2.3.15 Serum Nucleosides in Canines with Osteosarcoma Figures 2.18 and 2.19 give correlations of the change in serum nucleoside levels in dogs with osteosarcoma to treatment with 1 5 3 s m e t h y 1en ed i ami n e t e tr a m e t h y 1p h o sp h ate ( 53 Sm-EDTMP) . This work was done (1988) with Dr. D. McCaw of the School of Veterinary Medicine, UMC

Table 2.22 Clearance of Nucleosides and Creatinine in Normal Human Subjects Nucleoside Y

U X G PCNR mlI mlG ac4~ m 2rn 2G t6A m6A Creatinine

Mean 82.8 0.268 8.15 17.5 140 207 330 124 496 177 28.5 98.7

SD

RSD.%

Max

Min

24.9 0.24 0.088 17.9 60.6 95.3 132 83.8 154 51.3 24.7 32.5

30.0 87.7 1.08 102 43.4 46.0 40.1 67.7 31.0 29.0 86.9 33.0

138 1.2 8.2 70.9 303 398 613 356 904 321 97.2 144

46.5 0.069 8.1 1.4 58.5 68.0 88.0 4.82 187 106 7.2 476

Population of 7 males and 11 females, age range 25-53. Values are expressed as ml of serum/min.

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Table 2.23 Clearance of Nucleosides and Creatinine in Cancer Patients Nucleoside Y

U X G

PCNR rnlI mlG ac4~

m2rn2G t6A m6A Creatinine

Mean

SD

RSD.9

Max

85.1 0.82 62.0 43.0 131 174 351 76.9 389 163 25.6 109

54.0 1.5 80.3 64.6 94.0 167 3 19 81.4 322 103 52.6 66.0

63.5 180 130 150 71.7 95.8 90.8 106 82.9 63.6 206 60.5

206 6.4 222 287 428 640 1100 319 1179 406 246 276

Min 15.8 0.01 2.7 0.79 22.9 157 6.7 1.1 2.1 43.0 0.07 17.6

There are no data on the sex and age of the above population. Values are expressed as rnl of serumhin.

(ref. 74). Figure 2.18 shows 4 panels on the change in nucleoside levels from a dog that responded to the treatment. Panels A and B show that the levels of eight modified nucleosides, w, m5C, mlI, a&C, @A, mzrnZG, PCNR and N20.3 increased immediately after the treatments (day 1 and day 7), then decreased from day 20 to day 40. Finally the concentration of all 8 nucleosides remained constant and at low level from day 40. Figure 2.19 shows the change in serum nucleoside levels for 3 dogs, one that responded, the second partially responded, and a third dog with progressive disease. The change in serum modified nucleoside levels for eight nucleosides (four nucleosides are shown in this figure) are closely correlated to the clinical prognosis of the dogs under treatment. For the dog that responded the levels of the nucleosides decreased after 20 days and remained at a lower level. For the dog that partially responded the levels of the nucleosides initially decreased (day 2 to day 10 or 20) then all continuously incI;eased until death. For the dog with progressive disease, three of the four nucleosides were continuously increasing until death of the dog. The unknown nucleoside N14.9 level decreased during treatment over time for all 3 dogs. This decrease in the level of N14.9 in all

(')Nucleoside level in percent relative to prelreatmentvalue (day 0).

Fig. 2.18 Four panels on the change in the relative concentration of serum nucleosides for a dog. The nucleoside level is given in percent relative to the pretreatment value (day 0).

c)

ID

m

200

A

Responded

M 7

a,

> W

100

a,

U .r u)

0 W

"1

7

U 3

z

0 0

40

20

60

80

100

-

- I

Days

120

-

=

7

.nn

IW

Ih\

Days

B

Partially Responded

/

h

120

D

N14.9 in All Three Dogs

I

Q

Partially Rospondod

W

> W

7

W

80

U

.r u)

0 W 7

60

V

-a- AC4C

3

z

0

P

4)

80

7 n .. . -0

Days

. . . -

P

4

0

I

8

0

-

I

80

-

a

1

la,

Days

(')Nucleoside level in percent relalive lo prelrealrnenl value (day 0).

Fig. 2.19 Correlation of serum nucleosides in three dogs with osteosarcoma to 153Sm-EDTMP treatment for a dog that responded (panel A), a dog that partially responded (panel B), and a dog with progressive disease (panel C). Panel D gives the response of unidentified nucleoside N14.9 in all three dogs.

c97

three dogs over the course of the treatment is most interesting. The possibility of using N14.9 as a marker for assessing immunological activity should be investigated. Figure 2.20 presents a serum nucleoside chromatogram of a dog with osterosarcoma. A new major modified nucleoside (C*) was identified as 5hydroxylmethylcytidine (om5C) (ref. 75). omSC has only been observed in the serum of dog and cat. The average concentration of om5C in normal canine is 22 nmol/ml which is more that 10 fold higher than the levels of the other serum nucleosides. Also, m5C which was not observed in human serum and urine is present in high concentration in dog serum. In addition, two unknown nucleosides N14.9 and N2c.3, were found in dog serum which are yet to be identified. N14.g has not been observed in human body fluids and the unknown N20.3 is a minor modified nucleoside observed in human serum. This difference in the dog serum nucleoside profile as compared to human indicates a difference in metabolism of RNAs in dog and man.

10 9

OSTEOSARCOMA

0

7

6 3

a E

5 4

3 2

1

0 0

10

20 Time

30

40

50

(min.)

Fig. 2.20 HPLC-UV chromatography of nucleosides in serum of dog with osteosarcoma. For chromatographic details refer to Experimental.

C 98 2.3.1 6 Serum Nucleosides in Leukemia and Lymphoma Patients In collaboration with Professor F. Salvatore's group at the University of Naples, Italy, serum from pretreatment leukemia and lymphoma patients were collected and analyzed. Brief preliminary results are presented as bar graphs. Figure 2.21 shows a comparison of the normal serum nucleoside levels to the levels found in acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, and chronic myeloid leukemia (CML). Figure 2.22 gives a comparison of the normal serum nucleoside levels to the levels found in Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). We found that the level of modified nucleosides from the patients with all types of leukemia and lymphoma are significantly higher than the normal values. Acute lymphocytic leukemia patients have much higher levels than other leukemias and lymphomas. This indicates the excellent diagnostic value of modified nucleosides for leukemia and lymphoma. The preliminary data also show that the modified nucleoside profiles of some leukemias are different from others.

Fig. 2.21 Serum nucleoside levels in leukemia patients. parentheses gives number of subjects in the study.

Number i n

c99

Fig. 2.22 Serum nucleoside levels in lymphoma patients. parentheses gives number of subjects in the study.

Number in

2.3.17 Polynuclear Aromatic Hvdrocarbon Carcinogen-Ribonucleoside Adducts in the Urine of Fish and Rat Monitoring the catabolites of polynuclear aromatic hydrocarbons (PAH) carcinogen-RNA adducts, specifically ribonucleoside adducts (RNadducts) in body fluids as "an indicator" for occupational exposure and environmental contamination has many obvious advantages over the measurement of PAH-DNA adducts. Measuring RN-adducts in body fluids is non-invasive: The samples of urine and blood are easily obtainable while DNA-adducts generally require isolation of DNA from the tissue of the subjects. Secondly, the analytical method for RN-adducts is simpler and faster, thus a much lower cost of analysis than for the DNA-adducts (32P post-labelling 2D-TLC method): Ribonucleoside adducts are the product of RNA metabolism and are present in blood and urine, while

ClOO

DNA-adducts require isolation of the DNA from tissue followed by a hydrolysis of DNA to the nucleotides. Using the methods that we have described in this chapter, RN-adducts can be isolated in high purity and yield from body fluids and then measured by well established techniques such as HPLC-UV, HPLC-fluorescence, GC-MS erc.. Much larger amounts of RN-adducts should be found in urine than in DNA (one adduct per 109 bases) because not only does urine accumulate RN-adducts from RNA turnover over time, but also a higher amount of RNA-adducts should be present initially than for DNA-adducts in the tissue. The enzymatically activated PAHs (epoxides) react directly with the RNAs in the cytoplasm and thus do not need to be transported across the membrane of nuclei as in the reaction with DNA, therefore there should be considerably more RNA-adducts formed with RNA than with DNA. Gerhard Schoch et al. (ref. 7 6 ) calculated that there are about equal amounts of RNA and DNA in most of the eukaryotic cells and that the whole-body turnover of RNA in human adults is about 100 mg/kg body-wt /day. An average adult body weighs about 60 kg, thus the turnover of RNA is about 6 grams per day. With a conservative assumption that the level of PAH-RNA adducts in RNA is the same as for PAH-DNA adducts in DNA, i.e. one adduct per lognucleobases, this would calculate that there should be more than 20 picomol of each RN-adduct excreted into the urine each day. This amount is within the sensitivity of the measurement of modern chromatography-spectrometry techniques. In collaboration with Dr. Mark Smith of the Cancer Research Center, Columbia, Mo., a preliminary experiment was conducted in our laboratory to investigate RN-adducts in the urine of rat and fish that have been exposed to benzoIa1pyrene (BaP). Three female rats, body wt. ca. 200 g each, were injected i.p. with 300 pl dimethylsulfoxide (DMSO) containing 0.30 mg (1.2 pmol) cold BaP and 80 x 106dpm (ca. 0.7 nmol) of 3H-BaP (53 Wmmole). The 3H activity found in the 24 hour urine collected from each of the three rats for three days is presented in Table 2.24. Also, the nonadducted benzo[a]pyrene metabolites were determined in the ethyl acetate extract of the urine, and the 3H activity found is assumed to be the B[a]P metabolites (Table 2.25). Only 6 to 8% of the total 3H activity was found in the first 24 hours of urine collected. The amount of activity in the urine decreased significantly from day 1 to day 3.

ClOl Table 2.24 3 8 Activity Found in Urine from Benzo[a]pyrene Treated Rats 3H Rat No.

Activity,

Day 1

Day 2

1

46.0

2

6 1 . 8 ~lo5 (7.8)

3

47.0

x

(dpm)

lo5 (5.8)

105 (5.9)

Day 3

32.8

105 (4.1)

46.9

x lo5 (5.9)

13.0 x lo5 (1.6)

25.8 x 105 (3.3)

9.18 x 105 (1.1)

9.35

105 (1.2)

~

(

) gives the percentage of 3H activity of the total injected excreted per

day.

Table 2.25 Non-adducted

Benzo[a]pyrene Percentage

Metabolites in Rat Urine of

Non-adducted

B[a]P

Metaboliteda)

Rat No.

Day 1

Day 2

Day 3

1

15.4

17.8

15.5

2

21.8

25.8

16.1

3

21.1

22.4

22.4

(a) Percentage of the 3H activity extracted into ethyl acetate phase.

One-half of the 24 hour rat urine collection was evaporated to 2 ml volume and adjusted to p H 9 with 1 N NaOH. Two nmol of internal standard (m3U) were added and the total sample was loaded onto a 3 x 0.6 cm Affigel column. The column was then washed with 10 ml of 0.25 M CH3COONH4, followed with a 2 ml wash of 50% methanol in water. All of the ribonucleosides were eluted from the column in 25 ml of 0.2 M HCOOH in 50% methanol in water. (There was no 3H activity found in the 2nd 25 ml eluate of 0.2 M HCOOH in 50% methanol/H20. This indicates that all of

c102

the RN-adducts were completely eluted in the first 25 ml). The first 25 ml eluate of 0.2 M HCOOH was evaporated to dryness and re-dissolved in 2.0 ml of water. 100 pl of the sample were injected onto the HPLC. The HPLC conditions used were the same as for modified ribonuclesides in biological samples (see Experimental Section) except that elution solvent C was replaced with 60/40 C H 3 C N / H 2 0 and the run time was extended to 60 minutes to elute the BaP-nucleoside adducts. The HPLC eluate was collected in 0.5 ml fractions and 100 p1 aliquots from each fraction were counted for 3H activity. Only 10% (4800 dpm) of the total 3H activity was found in the 0.2 M HCOOH eluate fractions. This calculates as 7 nmol of ribonucleoside-adducts excreted in rat urine in the 1st 24 hours. Thus 35 nmol of RN-adducts per kg body wt. per day were excreted by a rat, or there is one RN-adduct in 105 of the nucleobases. We have synthesized a number of reference ribonucleoside-BaP adducts to assist in the identification of the RN-adducts. The major activated BaP metabolite, Benzo[alpyrene-trans-7,8-dihydrodiol-9,1O-epoxide (BPDE), (Lot No. CSLO85-008-10-20, was purchased from Midwest Research Institute, Kansas City, MO.) and reacted with each of the four major ribonucleosides. 4 011A

131) (IS)

1%

30-

3

a E

PCNR

206

-

24 48 mli (ace R8. 2.23.b

I El-

Fig. 2.23-a. HPLC-UV chromatogram of ribonucleosides in urine from a rat injected i.p with cold and 3H-labelled benzo[a]pyrene. See text for details of chromatography and experimental.

C103 :m-:

r!OU

a: a

82:

-

3

*

BPDE

~

Adenosine Adducts

LC!

-

2c+

32

3 i

36 ?!me

38 Irnin.1

413

42

44

Fig. 2.23-b. Panels A, B, and C show the HPLC-UV chromatograms for synthesized reference BPDE-adducts of Ado, Cyt, Urd, and Guo, respectively. Panels show only the sections of the HPLC chromatograms from 30 to 45 minutes. Panel D is the 3H radiogram between 30 to 45 minutes from Fig. 2.23-a.

The reaction conditions of Jennette et al. (ref. 77) were used with minor modification to prepare BPDE ribonucleoside adducts (BPDE-cytidine, 100 y1 of nucleoside solution (1.0 uridine, -guanosine and -adenosine). mg/ml in water) was reacted with 50 pl of 1.0 mg/ml of BPDE in acetone. After the reaction mixture was incubated at 37 "C for four hours, an additional aliquot of 50 yl of the BPDE solution was added. The reaction was allowed to continue at 37 "C overnight with no pH adjustment. After

C104

extraction of the non-adducted metabolites with ethyl acetate the aqueous layer was analyzed with HPLC-UV using the same chromatography conditions as for the rat urine samples. Figure 2.23-a shows the HPLC-UV chromatogram of the ribonucleosides in rat urine collected on day one from a rat injected with cold and 3H labelled benzo[a]pyrene. The HPLC eluate was collected after the UV detector in 0.5 ml fractions for 3H activity counting. Only the fractions between 30 to 45 min contained any 3H activity and all of our synthesized reference ribonucleoside-BPDE adducts also eluted in this section (30 to 45 min) of the chromatogram. Figure 2.23-b shows a comparison of the HPLC-UV chromatograms from four of our synthesized RN-BPDE adducts to the HPLC-3H activity radiogram of urine from a rat with injected BaP. At least four RN-adducts at about the same level of concentration were observed in the rat urine. The peak at 39.5 min most likely is the N2, BPDE-guanosine adduct. The peak of 37.5 min could be

m3U

/

U

\

I

\

i I6A

?

B

J ri

10

254 nm

-r

20

I

irnc

30 ( r n i r i .

>

40

5u

Fig. 2.24. HPLC-UV chromatogram of ribonucleosides in urine from a channel catfish injected with 3H labelled benzoralpyrene. See text for details of chromatography and experimental.

C105

either the N6, BPDE-adenosine adduct or a BPDE adduct of uridine. Peaks at 32.5 and 34.0 are not nucleoside adducts of BPDE. They are the adducts of other active benzo[a]pyrene metabolites, perhaps from the 4,5-epoxide. In collaboration with Drs. Chris Schmitt and Brian Steadman at the National Fisheries Contaminant Research Center (NFCRC), urine from a benzo[a]pyrene treated (oral injection) channel catfish was collected. The results of HPLC-UV analysis of the urine is presented in Figure 2.24. It is very interesting to note that high levels of the four major nucleosides were found in the urine, and I and t6A were the only two major modified nucleosides excreted. Three adducts were found in the fish urine, the major one eluting at 39 min, probably N2-guanosine-BPDE. The two unknown minor adducts that eluted at 40.0 and 42.5 min have only onetenth of the 3H activity of the 39 min adduct peak, also their retention times do not match with any of the reference RN-BPDE adducts available (see Fig. 2.23-b).

Summary During the last two years we have improved and extensively validated our method for quantitation of ribonucleosides in biological samples. This technology represents a significant advancement over the The precision, speed, methods that we reported earlier (refs. 1, 2). sensitivity and ruggedness of our methods are well suited for use in clinical research applications. With the described chromatography protocols, twenty known nucleosides in urine or serum (hU, Y, ncm5U, mlA, I, X, PCNR, mlI, mlG, ac4C m2G, rn2mzG, t6A, m6A, mt6A, ms2t6A, C, U, G and A), and more than ten unidentified nucleosides can be measured in a single 35 minute chromatographic run. The precision and ruggedness of the method was ensured with the introduction of a new internal standard, 3-methyluridine (m3U), which is added to the urine or serum sample before prechromatography treatment. Also, the accuracy of the method was improved by employing a UV diode-array detector and multiwavelength quantitation protocols. The within-day relative standard deviation (RSD, %) obtained on five nucleosides (y,U, mlI, m2m2G, and t6A) from a pooled human serum is under 5%. Long term (day to day) analytical precision for the five nucleosides (w, U, m11, m2m2G, and t6A) in a pooled serum sample over a period of five weeks (N = 15) gave a RSD in 2.4

C106

the range of 3.6 % to 9.0 %, and the long term recoveries for five representative nucleosides, (w, mlI, m2m2G, t6A and m6A) spiked at physiological levels in human serum, analyzed over a period of three weeks (N = 12) were 94 f 3 %, 104 f 6 %, 96 f 4 %, 99 f 3 % and 85 f 4 %, respectively. In our laboratory this method has been applied to approximately 500 human serum samples and 200 urine samples with consistently satisfactory results. U, mlA, I, X, G, PCNR, Thirteen human serum nucleoside levels mlI, mlG, a&, rnZmZG, t6A, and m6A) and 17 human urinary nucleoside levels (hU, w, U, mlA, I, X, G, PCNR, m*I, mlG, ac4C. m2G, A, m2m2G, ncm5s2U, t6A, and m6A) were established on analysis of a large number of samples from a normal population. In addition, preliminary studies on serum nucleosides as potential biological markers for small cell lung carcinoma, leukemias and lymphomas were achieved. Some significant correlations were noted between the levels and profiles of serum nucleosides and different neoplasias. The broad applicability of this method was demonstrated by the analysis of nucleosides in human plasma, whole blood, and other biological samples. Nucleosides in serum and urine from dog, cat, rat, mouse, monkey, fish and cell culture media have also been successfully measured. This high efficiency chromatography protocol also can be used for the enrichment of PAH and alkylated carcinogen-ribonucleoside adducts in urine and serum, then their measurement by either microbore HPLC with laser-induced fluorescence detection or capillary GC-MS. Recently, we have used this method for the identification and characterization of benzo[alpyrene-ribonucleoside adducts (BaP-RN-adducts) in the urine of fish and rat. High levels of BaP-ribonucleoside-adducts were found in the urine of BaP treated rat and fish. About twenty percent of the BaP inetabolites in rat urine are non-adducted (free), 10% of the metabolites were found in the urine as BaP-RN-adducts, and 70% have not been identified and are probably BaP-protein related adducts. The BaP-RN adducts were found at a level of one .(1) adduct per 105 nucleobases in RNAs. This was calculated from the number of BaP-RN adducts excreted in and the average whole-body turnover for the urine per day (7 nmol) tRNA of 61.3 mg/kg body-wt/day, and for rRNA of 477 mg/kg body-

(w,

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wtlday (ref. 76). Further, we did not take into account the BnP-RN adducts in the feces which is considered to be at much higher levels than in urine. Even with this conservative calculation, the BaP-RNA adducts in the cells are at least 10 to 100 fold higher than the BaP adducts in DNA. These results confirm our initial concepts that there are much higher levels of PAH-RNA adducts than those for DNA. Our findings strongly support that measurement of ribonucleoside adducts can serve as important endpoints in monitoring occupational exposure, environmental contamination, and the roles of RNA-adducts in chemical carcinogenesis.

Ack n owled gm en t We wish to gratefully acknowledge Marion Laboratories of Kansas City, MO, Supelco., Inc. of Bellefonte, Pa., and the University of MissouriColumbia and the State of Missouri for their financial support of a number of research projects reported in this three-volume series. 2.5 1.

2.

3. 4 5.

6.

References C. W. Gehrke, K. C. Kuo, G . E. Davis, R. D. Suits, T. P. Waalkes and E. Borek, Quantitative high-performance liquid chromatography of nucleosides in biological materials, J. Chromatogr., 150, (1978) 455476. G. E. Davis, R.S. Suits, K.C. Kuo, C.W. Gehrke, T.P.Waalkes, and E. Borek, High performance liquid chromatographic separation and quantitation of nucleosides in urine and some other biologic fluids, Clin. Chem. 23, (1977) 1427-1435. K.C. Kuo, C.W. Gehrke, R.A., McCune, T. P. Waalkes, and E. Borek, Rapid, q u a n t i t at i ve high pe rforma nc e liquid chromatogr aphy of pseudouridine, J. Chromatogr. Biomed. Applic. 150, (1978) 455-476. M. Uziel, L.H. Smith and S.A. Taylor, Modified nucleosides in urine: selective removal and analysis, Clin. Chem., 22, (1976) 1451-1455. M. Zakaria, P. R. Brown, M. P. Farnes and B. E. Barker, HPLC analysis of aromatic amino acids, nucleosides, and bases in plasma of acute lymphocytic leukemics on chemotherapy, Clin. Chim. Acta, 126, (1982) 69-80. A.M. Krstulovic, R.A Hartwick, and P.R. Brown. High performance liquid chromatographic determination of serum UV profiles of normal subjects and patients with breast cancer and benign fibrocytic changes, Clin. Chim. Acta 97, (1979) 159-170.

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7.

8.

9.

10. 11.

12.

13.

14

15

16

17.

R.A. Hartwick, A.M. Krstulovic, and P.R. Brown. Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum using reversed-phase high-performance liquid chromatography. 11. Evaluation of human sera, J. Chromatogr. 186, (1979) 659-664. R.C. Simpson and P.R. Brown, High-performance liquid chromatographic profiling of nucleic acid components in physiological samples, J. Chromatogr., 379, (1986) 269-31 1. E. Hagemeier, K. Kemper, K.-S. Boos and E. Schlimme, Development of a chromatographic method for the quantitative determination of minor ribonucleosides in physiological fluids. Characterization and quantitative determination of minor ribonucleosides in physiological fluids, J. Clin. Chem. Clin. Biochem., 22, (1984) 175-184. E. Schlimme, K.S. Boos, E. Hagemeier, K. Kemper and U. Meyer, Direct clean-up and analysis of ribonucleosides in physiological fluids. J. Chromatogr. 378, (1986) 349-360. G. Schoch, J. Thomale, M. Lorenz, M. Suberg and U. Karsten, A new method for the simultaneous analysis of unmodified and modified urinary nucleosides and nucleobases by high-performance liquid chromatography, Clin. Chim. Acta, 108, (1980) 247-257. H. Topp, G. Sander, G. Heller-Schoch and G. Schoch, A high performance liquid chromatography method for the determination of pseudouridine and uric acid in native human urine and ultrafiltered serum, Anal. Biochem., 150, (1985) 353-358. G. Sander, J. Wieland, H. Topp, C. Heller-Schoch, N. Erb and G. Schoch, An improved method for the simultaneous analysis of normal and modified urinary nucleosides and nucleobases by high-performance liquid chromatography, Clin. Chim. Acta, 152, (1985) 355-361. R.A. De Abreu, J.M. van Baal, CHMM de Bruyn, JAJM, Bakkeren, EDAM Schretlen. High-performance liquid chromatographic determination of purine and pyrimidine bases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleosides in biological fluids, J. Chromatogr., 229, (1982) 67-75. T. P. Waalkes, M. D. Abeloff, D. S . Ettinger, K. B. Woo, C. W. Gehrke, K. C. Kuo and E. Borek, Modified ribonucleosides as biological markers for patients with small cell carcinoma of the lung, Eur. J. Cancer Clin, Oncol., 18, (1982) 1267-1274. T. P. Waalkes, M. D. Abeloff, D. S. Ettinger, K. B. Woo, C. W. Gehrke, K. C. Kuo and E. Borek, Biological markers and small cell carcinoma of the lung, Cancer, 50, (1982) 2457-2464. C. W. Gehrke, K. C. Kuo, T. P. Waalkes and E. Borek, Pattern of urinary excretion of modified nucleosides, Cancer Res., 39, (1979) 1150-1153.

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18. J. Speer, C. W. Gehrke, K. C. Kuo, T. P. Waalkes and E. Borek, tRNA breakdown products as markers for cancer, Cancer, 44, (1979) 21202123. 19. E. Borek, 0. K. Sharma and J. I. Brewer, Urinary nucleic acid breakdown products as markers for trophoblastic diseases, Am. J. Obstet. Gynecol., 146, (1983) 906-910. 20. E. Borek, T. P. Waalkes and C. W. Gehrke, Tumor markers derived from nucleic acid components, Cancer Detect. Prev., 6, (1983) 67-71. 21. C. W. Gehrke and K. C. Kuo, Major and modified nucleosides, RNA, and DNA, in : L. J. Marton and P. M. Kabra (Eds.), Liquid Chromatography in Clinical Analysis, Humana Press Inc., Clifton, New Jersey, 1981, pp. 409-443. 22. H.A. Scoble, J.L. Fasching, and P.R. Brown, Chemometrics and liquid chromatography in the study of acute lymphocytic leukemia, Anal. Chim. Acta. 150, (1983) 171-181. 23. J. Thomale, G. Nass, Elevated urinary excretion of RNA catabolites as an early signal of tumor development in mice, Cancer Letter 2, (1982) 149-159. 24. G . Nass, J. Thomale, A. Luz and U. Friedrich, Excretion of modified nucleosides by malignant cells in vivo and in vitro and its clinical relevance. manuscript in preparation. 25. E. Schlimme, K.-S, Boos, B. Wilmers and H.J. Gent, Analysis of ribonucleosides in body fluids and their possible role as pathobiochemical markers, in: F. Cimino, G.D. Birkmayer, E. Pimentel, J.V. Klavins, F. Salvatore (Eds.), Human Tumor Markers, Walter de Gruyter and Co., Berlin (1987) pp. 503-518. 26. R. W. Trewyn, R. Glaser, D. R. Kelly, D. G. Jackson, W. P. Graham I11 and C. E. Speicher, Elevated nucleoside excretion by patients with nasopharyngeal carcinoma. Preliminary diagnostic/prognostic evaluations, Cancer, 49, (1982) 2513-2517. 27. D. A. Heldman, M. R. Grever, J. S. Miser and R. W. Trewyn, Relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia, J. Natl. Cancer Inst., 71, (1983) 269-273. 28. D. A. Heldman, M. R. Grever and R. W. Trewyn, Differential excretion of modified nucleosides in adult acute leukemia, Blood, 61, (1983) 291296. 29. R. W. Trewyn and M. R. Grever, Urinary nucleosides in leukemia: Laboratory and clinical applications, CRC Lab. Sci., 24, (1986) 7 1-93. 30. J.W. Mackenzie, R.J. Lewis, G.E. Sisler, W. Line, J. Rogers and I. Clark, Urinary catabolites of ribonucleic acid as cancer markers: A preliminary report of their use in patients with lung cancer, The Ann. of Thorac. Surg., 38, (1984) 133-139.

CllO 31. I. Clark, J.W. Mackenzie, J.R. McCoy and W. Lin, Comparison of urinary

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modified nucleosides and bases between rats with hepatomas and nephroblastomas, in: G. Nass (Ed,), Recent Results in Cancer Research, Springer-Verlag, Berlin and New York, 84, (1983) 388-400. W. Lin, J. W. Mackenzie and I. Clark, Excretion of RNA catabolites by rats with hepatoma transplants, Cancer Letters, 22, (1984) 187-192. W. Lin, J.W. Mackenzie and I. Clark, Effect of transplanted osteogenic sarcoma on urinary RNA catabolites, Cancer Letters, 35,( 1987) 47-57. T. Rasmuson, G.R. Bjork, L. Damber et al. Tumor markers in bronchogenic carcinoma. An evaluation of carcinoembryonic antigen, tissue polypeptide antigen, placental alkaline phosphatase and pseudouridine. Acta Radiol. Oncol. 22, (1983) 209-214. T. Rasmuson and G.R. Bjork. Pseudouridine: A prognostic marker in non-Hodgkin's lymphoma. Cancer Chemother. Report, 59, (1975) 721727. T. Rasmuson and G. R. Bjork, Excretion of pseudouridine in urine as a tumor marker in malignant diseases, Bull. Mol. Biol. Med., 10, (1985) 143-154. K. Nakano, K. Shindo, T. Yasaka and H. Yamamoto, Reversed-phase liquid chromatographic investigation of nucleosides and bases in mucosa and modified nucleosides in urines from patients with gastrointestinal cancer, J. Chromatogr., 332, (1985) 127-137. K. Nakano, K. Shindo, T. Yasaka and H. Yamamoto, Reversed-phase high-performance chromatographic investigation of mucosal nucleosides and bases and urinary modified nucleosides of gastrointestinal cancer patients, J. Chromatogr., 343, (1985) 21-33. G. Mills, F. C. Schmalstieg and R. M. Goldblum, Urinary excretion of modified purines and nucleosides in immunodeficient children, Biochem. Med., 34, (1985) 37-51. A. Fischbein, 0. K. Sharma, I. J. Selikoff and E. Borek, Urinary excretion of modified nucleosides in patients with malignant mesothelioma, Cancer Res., 45, (1983) 2971-2974. G. Sander, H. Topp, J. Wieland, G. Heller-Schoch and G. Schoch, Possible use of urinary modified RNA metabolites in the measurement of RNA turnover in the human body, Hum. Nutr. Clin. Nutr., 40C, (1986) 103118. H. Topp, G. Sander, G. Heller-Schoch and G. Schoch, A high-performance liquid chromatographic method for the detemination of pseudouridine and uric acid in native human urine and ultrafiltered serum, Anal. Biochem., 150, (1985) 359-368.

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43. H. Topp, G. Sander, G. Heller-Schoch and G.T. Schoch, Determination of 7-methylguanosine, N2,N2-dimethylguanosine and pseudouridine in ultrafiltered serum of healthy adults by high-performance liquid chromatography, Anal. Biochem., 161, (1987) 49-56. 44. S. Tamura. J. Fujii, T. Nakano, T. Hada and K. Higashino, Urinary pseudouridine as a tumor marker in patients with small cell lung cancer, Clin. Chim. Acta, 154, (1986) 125-132. 45. S. Tamura, Y. Amuro, T. Nakano, F. Fujii, Y. Moriwaki, T. Yamamoto, T. Hada, and K. Higashino, Urinary excretion of pseudouridine in patients with hepatocellular carcinoma, Cancer (Phila.) 57, (1986) 1571-1575. 46. P. Vreken and P. Tavenier, Urinary excretion of six modified nucleosides by patients with breast carcinoma, Clin. Biochem., 24, (1987) 598-603. 47. F. Oerlemans and F. Lange, Major and modified nucleosides as markers in ovarian cancer: A Pilot study, Gynecol. Obstet. Invest. 22, (1986), 212-217. 48. J.G. Maessen, G.J. van der Vusse, M. Vork, and G. Kootstra. Nucleotides, nucleosides, and oxypurines in human kidney measured by use of reversed-phase high-performance liquid chromatography, Clin. Chem. 34/6, (1988) 1087-1090. 49. GPJM Gerrits, AAM Haagen, RA De Abreu, LAH Monnens, JM, Gabreels, JM, Grijbels, ALM, Theeuwes and JM, van Baal. Reference values for nucleosides and nucleobases in cerebrospinal fluid of children, Clin. Chem. 34/7, (1988) 1439-1442. 50. L. Buhl, C. Dragsholt , P. Svendsen. et al Urinary hypoxanthine and pseudouridine as indicators of tumor development in mesotheliomatransplanted nude mice, Cancer Res. 45, (1985) 1159-1162. 51. G. Apell, F.L. Buschman and O.K. Sharma, Improved and rapid method for quantitation of modified nucleosides in urine and sera with radialpack cartridge, J. Chromatogr. 374, (1986) 149-154. 52. A. Colonna, T. Russo, F. Cimino and F. Salvatore, Modified nucleosides in biological fluids of cancer patients by high performance liquid chromatography, J. Clin. Chem. Clin. Biochem., 19, (1981) 640. 53. A. Colonna, T. Russo, F. Esposito, F. Salvatore and F. Cimino, Determination of serum pseudouridine and other nucleosides in human blood serum by high-performance liquid chromatography, Anal. biochem., 130, (1983) 19-26. 54. T. Russo, A. Colonna, F. Esposito, F. Salvatore and F. Cimino, Detection and estimation of several modified nucleosides in serum of cancer patients, It. J. Biochem., 31, (1982) 75-78. 55. F. Salvatore, A. Colonna, F. Costanzo, T. Russo, F. Esposito and F. Cimino, Modified nucleosides in body fluids of tumor-bearing patients, Recent Results Cancer Res., 84, (1983) 360-377.

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56. Y. Amouro, H. Nakaoka, S . Shimomura, S . Tanmura. T. Hada and K. Higashino, Rapid high-performacne liquid chromatography for pseudouridine assay in serum and urine, Clin. Chim. Acta, 172, (1988) 117-122. 57. S.Tamura, J. Fujii, TR. Nakano, T. Hada. Serum pseudouridine as a biochemical marker in small cell lung cancer. Cancer Res. 47, (1987) 6138-6141. 58. F. Cimino, F. Costanzo, T. Russo, A. Colonna, F. Esposito and F. Salvatore, Modified nucleosides from transfer ribonucleic acid as tumor markers, in: E. Usdin, R. T. Borchardt, C. R. Creveling (Eds.), The Biochemistry of S-Adenosylmethionine and Related Compounds, MacMillan, London, (1982) 409-412. 59. F. Salvatore, A. Colonna, F. Costanzo, T. Russo, F. Esposito and F. Cimino, Modified nucleosides in body fluids of tumor-bearing patients, Recent Results Cancer Res., 84, (1983) 360-377. 60. F. Salvatore, T. Russo, A. Colonna, L. Cimino, G. Mazzacca and F. Cimino, Pseudouridine determination in blood serum as tumor marker, Cancer Detec. Prev., 6, (1983) 531-536. 61. L. Sacchetti and F. Salvatore, Novel aspects of blood serum nucleosides and isoenzymes as tumor markers, J. Tumor Marker Oncol., 2, (1987) 245-253. 62. K. C. Kuo, F. Esposito , J. E. McEntire and C. W. Gehrke, Nucleoside profiles by HPLC-UV in serum and urine of controls and cancer patients, in: F. Cimino, G. D. Birkmayer, J. B. Klavins, E. Pimentel, F. Salvatore (Eds.) Human Tumor Markers, Walter de Gruyter & Co., Berlin, New York, (1987) 520-542. 63. L. Sacchetti, F. Pane and F. Salvatore, Modified nucleosides in biological fluids: An overview of their role as biochemical signals of human neoplasia, J. Tumor Marker Oncol.,( 1989) 133-147. 64. C.W. Gehrke, K.C. Kuo, R.A. McCune, and P.F. Agris, Quantitative enzymatic hydrolysis of tRNAs: Reversed-phase high-performance liquid chromatography of tRNA nucleosides, J. Chromatogr. Biomed. Applic., 230, (1982) 297-308. 65. S. Fulton and E. Carlson, Boronate Ligands in Biochemical SeparationsApplications, Method. Theory of Matrix Gel PBA Amicon Corporation, Scientific Systems Division 17 Cherry Hill Drive, Danvers, MA 01923. 66. C.W. Gehrke and K. C. Kuo, Ribonucleoside analysis by high performance reversed-phase liquid chromatography, in: C.W. Gehrke and K.C. Kuo (Eds) Chromatography and Modification of Nucleosides, Part A, Elsevier Chromatography Library Series, Amsterdam, 1989, in press. also J. of Chromatogr., 471, (1989), 3-36.

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Davis, Isolation and analysis of major and modified ribonucleosides by high performance liquid chromatogrpahy, Ph.D Dissertation, University of Missouri, Columbia, 1978. 68. J.T. Bernert Jr., C.J. Bell, J. Guntupalli, and W. Harryn Hannon, Pseudouridine is unsuitable as an endogenous renal clearance marker, Clin. Chem. (1988) Vol. 34. No. 6, 1011-1017. 69. E. Borek, Transfer RNA and its by products as tumor markers, in: S. Sell, Cancer Markers, The Humana Press, Clifton, New Jersey, (1980)

445-462. 70. C.W. Gehrke, K.C,.Kuo, T.P. Waalkes, E. Borek, Patterns of urine excretion of modified nucleosides. Cancer Research, 39, (1979) 11501153. 71. M. Savoia, T. Russo, E. Rippa, L. Bucci, F. Mazzeo, F. Cimino and F. Salvatore, Serum pseudouridine, Its evaluation as a biochemical signal of neoplasia, J. Tumor Marker Oncol., 1, (1986) 61-68. 72. F. Salvatore, M. Savoia, T. Russo, L. Sachetti, and F. Cimino, Pseudouridine in biological fluids of tumor-bearing patients, in Human Tumor Markers, F. Cimino et al. (eds) Walter de Gruyter and Co., Berlin, (1 987), 45 1-462. 73. W.L. Chiou, M.A.F. Gadalla, and C.W. Peng, Simple, rapid and micro high-pressure liquid chromatographic determination of endogenous "true" creatinine in plasma, serum, and urine, J. Pharmaceut. Sci, 67,

(1978), 182. 74. D.L. McCaw, K.C. Kuo, C.W. Gehrke, Serum levels of transfer RNA modified nucleosides in normal and tumor bearing dogs. in preparation, Dec. 1989. 75. C.W. Gehrke, J.A. Desgres, G. Keith, P.F. Agris, H. Sierzputowska-Gracz, M. Tempesta and K. C. Kuo, Structural elucidation of unknown nucleosides, in: C.W. Gehrke and K.C. Kuo (Eds) Chromatography and Modification of Nucleosides, Part A, Elsevier Chromatography Library Series, Amsterdam, 1989, in press. 76. G. Schoch, G. Sander, H. Topp and G.H. Schoch, Modified nucleosides and nucleobases in urine and serum as selective markers for the wholebody turnover of tRNA, rRNA and mRNA-cap - Future prospects and impact, in: C.W. Gehrke and K.C. Kuo (Eds) Chromatography and Modification of Nucleosides, Part C, Elsevier Chromatography Library Series, Amsterdam, 1989, in press. 77. K.W. Jennette, A.M. Jeffrey, S.H. Blobstein, F.A. Beland, R.D. Harvey and I.B. Weinstein, Nucleoside adducts from the in vifro reaction of benzo[a]pyrene-7,8-dihydrodiol-9,lO-oxide or benzo[a]pyrene 4,5oxide with nucleic acid. Biochemistry, 5, (1977) 16.

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CHAPTER 3 RIBONUCLEOSIDES I N BODY FLUIDS: ON-LINE CHROMATOGRAPHIC CLEANUP AND ANALYSIS BY A COLUMN SWITCHING TECHNIQUE ECKHARD SCHLIMMEI and KARL-SI EGFRI ED BOOS* ' Z n s t i t u t f u r Chemie und P h y s i k d e r B u n d e s a n s t a l t f u r W i l c h f o r s c h u n g , 2 3 0 0 K i e l , P.O. Box 6 0 6 9 , Germany ( F . R . G . ) 2 L a b o r a t o r i u a f u r B i o l o g i s c h e Chernie d e r U n i v e r s i t a t , 4790 P a d e r b o r n , P.O. Box 1 6 2 1 , Germany ( F . R . G . )

TABLE OF CONTENTS 3.1 Introduction . 3.2 E x p e r i m e n t a l . 3.2.1 Apparatus . . 3.2.2 Reagents . . . . 3.2.3 C r e a t i n i n e D e t e r m i n a t i o n . 3.2.4 Sample P r e p a r a t i o n . . . . 3.2.5 HPLC B u f f e r s . 3.2.6 A n a l y t i c a l P r o c e d u r e 3.3 R e s u l t s and D i s c u s s i o n . . 3.3.1 O f f - L i n e A f f i n i t y - B i o g e l / R P L C Method 3.3.2 On-Li ne HPAC/RPLC Method . 3.3.3 SEC-HPAC/RPLC Method . . . 3.3.4 A p p l i c a t i o n o f t h e SEC-HPAC/RPLC Method 3.4 Summary . . . . . 3.5 F u t u r e P r o s p e c t s . . . 3.6 Acknowledgment . . . . 3.7 R e f e r e n c e s . . . .

........... ... .... . .. . .... . . . ... .. .. ...

. .... .

.. . ..

. .. .. ... ... .. ... ..... ... .... . . .. ... ..... .... . ... ... ..... .. . . . .... . .. . .

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....... . ..... . . . .

. . . .

. ... . . .. . . . . . . .. . . .. . . . . . ...

. .

,

. . . .. . . . . ... ... . ... . . .... ... ... .... . .......

.

.

.

. . . . . .

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INTRODUCTION The d e t e c t i o n o f Val u a b l e d i a g n o s t i c m a r k e r m o l e c u l e s i n h i g h l y complex body f l u i d s such as b l o o d , serum, u r i n e , l y m p h a t i c f l u i d s , l i q u o r and b r e a s t m i l k i s s t i l l a c e n t r a l f i e l d o f p o t e n t i a l i n t e r e s t i n biomedical chemistry. B o r e k ' s e x p e c t a t i o n " o f f i n d i n g some u n i q u e m e t a b o l i c p r o d u c t s o r u n i q u e components o f ma1 i g n a n t c e l l s c i r c u l a t i n g i n body

3.1

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fluids which can be measured" (ref. 1) was met during the last decade as specific excretion patterns of modified ribonuc eosides in urine were found to be related to distinct metabolic d sorders. Since the mid seventies appropriate HPLC methods for the determination of major, modified and hypermodified ribonucleosides, especially from the breakdown of tRNA (ref. 2), have been developed (refs. 3-11). Current HPLC analysis of ribonucleosides in biological fluids, especially in serum, still involves elaborate and manually performed sample processing steps due to the complexity of the sample matrix. Moreover, matrix impurities still present in the parti cul ar sample extracts often interfere with an accurate quantification of trace levels of ribonucleosides. Prior to HPLC analysis, protein-containing samples are commonly processed by deproteinization as, e . g . ultrafiltration or acid-precipitation, and in order to improve selectivity and sensitivity by bonded phase extraction (affinity gel chromatography or reversed-phase cartridge concentration) followed by lyophyl ization (ref. 12). For affinity chromatography, the cis-diol system of ribonucleosides was chosen as a selectivity criterion, as this structural moiety reversibly forms under a1 kal ine conditions a cycl ic di ester with tetrahedral configured boroni c acid (ref. 13). This biospecific affinity ligand was immobilized via its m-aminophenyl derivative to various gel supports, e . g . agarose, cellulose, polyacrylamide, and used for the manual or partially automated (ref. 14) cleanup of ribonucleosides under low pressure conditions (refs. 3, 8, 9, 15-21). After these sample-pretreatment steps, HPLC-analysis of ri bonucleosides is usual ly performed on reversed-phase materials either under i socrati c conditions or in a gradient el uti on mode. A methodological improvement was achieved, when we succeeded with the preparation of a m-aminophenyl boronic acid-substituted silica gel which was suitable for high performance affinity chromatography (HPAC) (ref. 22). On the basis of this affinity material and a column-switching technique, we set up an instrumental ly-connected two-column liquid chromatographic on-line system that can be used for a system-integrated direct cleanup and analysis of ribonucleosides in protein-free fluids (ref. 23-27).

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Surprisingly, only few on-1 ine methods have been reported until now. In the field of nucleic acid research an on-line twostage column chromatography was described recently, which combines reversed-phase and anion-exchange chromatography for the analysis of purine nucleic acid components (ref. 28). More recently, we prepared a new bonded-phase materi a1 (patent pending) which, for the first time, allows the direct appl i cation and subsequent on-1 i ne analysis of protei naceous fluids, such as serum or milk (ref. 29). The precolumn material is a chemically modified, semi-rigid gel and allows a simultaneous performance of two different modes o f high-performance 1 iquid chromatography. Fi rst, by virtue of i ts gel -permeati on properties, macromolecules (e.g. proteins) can be quantitatively separated from the sol Ute (SEC: size-excl usion chromatography). Secondly, by immobilizing a specifically modified phenylboronic acid to the gel support, high performance affinity chromatography (HPAC) of ribonucleosides can be carried out. In cooperation with E. MERCK (Darmstadt, Germany) we finally built a fully automated HPLC analyzer for ribonucleosides, which will be commercially available. This unique device is distinguished by its practicability with respect to routinely quantifying and profiling ribonucleosides in the biological fluids of individuals with different diseases and thus should encourage more extensive research in the biochemical as well as in the clinical field. 3.2 EXPERIMENTAL 3.2.1

Atmaratus A modular HPLC system from Merck-Hi tachi (Darmstadt, Germany)

was used. As shown schematically in Figure 3.1 the SEC-HPAC/RPLC apparatus is composed of a pump Model 655 A-12 (Z), an LC-Controller Model L 5000 (m), a proportioning valve (U), an autosampler Model 655 A-40 (As) for sample introduction, a SEC-HPAC pre-column (40 x 4 mm I.D.; Column l), an end-capped RPLC-column (LiChrospher 100 RP-18 e, 5 pm, Merck, 250 x 4 mm I.D.,; Column 2), an automatic valve switching system Model ELV 7000 (ASS), an UV-spectrophotometer Model 655 A-22 (Uy) , an integrator Model D 2000 (I)and a second pump (p1).

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r-----------

c------

F i g u r e 3.1

O n - l i n e SEC-HPAC/RPLC system setup.

T h i s i n s t r u m e n t a t i o n a l l o w s t h e independent use o f t h e b a s i c g r a d i e n t system i n a d d i t i o n t o t h e m u l t i d i m e n s i o n a l mode. Thus, r e 1 i a b i 1it y o f o v e r a l l system performance can e a s i l y be c o n t r o l l e d by comparing t h e o f f - l i n e (RPLC) a n a l y s i s o f a s t a n d a r d m i x t u r e o f r i bonucl e o s i des w i t h t h e on-1 ine (SEC-HPAC/RPLC) a n a l y s i s o f an a p p r o p r i a t e sample. 3.2.2

Reaaents Adenosine (Ado), c y t i d i n e (Cyd), i n o s i n e ( I n o ) , u r i d i n e (Urd) and guanosi ne (Guo) were purchased from Boehri nger (Mannheim, Germany). N1 -methyl adenosine (m1 Ado), N1 -methyl in o s i ne (m1 I n o ) , N2-methylguanosine (m*Guo), N6-dimethyladenosine (m; Ado), pseudou r i d i ne ($), N3 -methyl u r i d i ne (m3 Urd) , N1 -methyl guanosi ne (ml Guo) ,

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INJECT -

Figure 3.2

HPLC system.

Valve switching positions of the on-line SEC-HPAC/

N6-methyladenosine (m6Ado), N4-acetylcytidine (ac4Cyd), 5-aminoimi dazol e-4-carboxami do-N-ri bofuranosi de (AICAR) were from Si gma (Munchen, Germany) and N2-dimethylguanosine (mSGuo) from P-L Biochemicals Inc. (Milwaukee, Wisc., USA). N6-(carbamoylthreony1)adenosi ne ( t6Ado) and 2-pyri done-5-carboxami do-N-ri bof uranosi de (PCNR) were isolated from urine. For f u r t h e r characterization, PCNR and t6Ado were a l s o chemically synthesized according t o ( r e f s . 30, 31). Structural characterization and i d e n t i f i c a t i o n of the nucleosides were achieved by using post-run U V , mass as well as nmr spectrometric measurements and by comparison with known reference standard compounds. The q u a n t i t a t i v e determinations by UV detection a t 260 nm were carried out according t o the external standard method as described ( r e f . 23). Calibration mixtures of t h e appropriate nucleosides were prepared, based on the above UV data (H20). 3.2.3

Creati n i ne Determi nation Creatinine was measured w i t h the Beckman Creatinine Analyzer 2 (Beckman, Munchen, Germany). Re1 i a b i l i t y of the overall system performance was monitored with Precinorm\ level 2 (Boehringer, Mannheim, Germany) and control serum Desicion\ level 2 (Beckman).

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TABLE 3.1 UV Data o f I n v e s t i g a t e d R i bonucl e o s i d e s

q~ Cyd Urd AICAR m1 Ado Ino Guo PCNR m3Urd Ado m l Ino m1Guo ac4Cyd m2Guo m:Guo m6Ado t6Ado m$Ado

3.2.4

( n m a X 262 = ( n m a x 280 = ( n m a X262 =

( n n a x 307 ( n m a x 257 ( n m a x 248 ( n m a x 253

= = =

=

( n m a X 259 =

( n m a x 262 = ( n m a X260 = ( n m a x 248 =

( n m a X 258 = ( n m a x 247 =

( n m a x 258 = ( n m a x 264 ( n m a x 262 ( n m a x 269 ( n m a x 268

= = =

=

7.9 13.4 10.1 19.7 13.7 12.3 13.6 11.3 9.5 14.9 9.6 9.4 15.2 14.2 12.8 16.6 24.9 18.4

cm2/pmol, cm2 /pmol , cm2/pmol, cm2/pmol, cm2 /pmol , cm2/pmol, cmz/pmol, cm2/pmol, cm2/pmol, cm2/pmol, cm2/pmol, cm2/pmo cm2/pmo cm2/kmo cmz/pmo cm2/pmo cmz/pmol, cm2/pmol,

PH pH pH pH pH pH pH pH pH pH pH

2.0) 2.0) 2.0) 1.0) 1.5) 3.5) 5.5) 5.0) 7.0) 5.5) 1.5)

pH 5.0) PH 1 . 5 )

SamDle P r e o a r a t i o n

Urine

Urine samples a s well a s 24-h urines were c o l l e c t e d , a d j u s t e d w i t h c o n c e n t r a t e d f o r m i c a c i d t o pH 4 and s t o r e d a t - 20°C u n t i l i n v e s t i g a t i o n . P r i o r t o a n a l y s i s , 500 p l o f human urine were membrane-f i 1 t e r e d (Mi 1 1 ox 0.22 pm; Mi 1 1 i p o r e , BUC, France) and an a l i q u o t o f 100 pl a p p l i e d t o the HPLC system. Serum

Serum samples were a d j u s t e d t o pH 4 w i t h c o n c e n t r a t e d f o r m i c a c i d and s t o r e d a t - 20°C u n t i l i n v e s t i g a t i o n . Elilk

Milk samples were a d j u s t e d t o pH 4.6 w i t h c o n c e n t r a t e d f o r m i c a c i d and s t o r e d a t - 20°C u n t i l i n v e s t i g a t i o n . Galactorroea F l u i d

Samples of g a l a c t o r r o e a f 1 u i d ( f i b r o c y s t i c d i s e a s e ) were c l i n i c a l l y prepared by Dr. H.J. Gent, Universittits-Frauenklinik,

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Kiel (Germany), a d j u s t e d t o pH 4 with concentrated formic a c i d and s t o r e d a t - 20°C u n t i l i n v e s t i g a t i o n .

HPLC Buffers The b u f f e r s used were prepared a s needed d i r e c t l y from the a p p r o p r i a t e s a l t s , soni cated and d i s c a r d e d a f t e r f i v e working days. D o u b l e - d i s t i l l e d water and s a l t s from Merck (Darmstadt, Germany), o f the purest grade a v a i l a b l e , were used i n a l l b u f f e r preparations. Column 1 b u f f e r : Used f o r the SEC-HPAC precolumn. 0 . 1 mol/l diammonium hydrogen phosphate, pH 9 . 8 Column 2 buffer: Used f o r the a n a l y t i c a l RPLC column. 0.05 mol/l ammonium formate, pH 3.5. 3.2.5

HPLC-Conditions

Columnll: The SEC-HPAC column (Column 1; 40 x 4 mm I.D.) was f i l l e d wi t h a 1 aboratory-prepared boroni c acid-functional i zed phase m a t e r i a l according t o ( r e f . 29). I t has a binding c a p a c i t y of 0.18 mmol r i b o n u c l e o s i d e per gram d r y weight and t o l e r a t e s pH v a l u e s from 2 t o 12 a s well a s the usual o r g a n i c s o l v e n t s . Column 2: The a n a l y t i c a l column (Column 2; 250 x 4 mm I.D.) c o n t a i n s LiChrospher 100 RP-18 e, 5 p m reversed-phase m a t e r i a l from Merck (Darmstadt, Germany). The temperature of Column 2 was k e p t a t 28" C . Eluents: A. 0.05 mol/l ammonium formate, pH 3.5 B. methanol C. water 0. 0 . 1 mol/l diammonium hydrogen phosphate, pH 9 . 8 Analytical Procedure In p r i n c i p l e , the d e s i r e d g r o u p - s e l e c t i v e p r e f r a c t i o n a t i o n and on-line cleanup of r i b o n u c l e o s i d e s i s c a r r i e d o u t by a simple pH-step e l u t i o n , followed by the a n a l y t i c a l r e s o l u t i o n under

3.2.6

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reversed-phase and gradi ent-el ution c o n d i t i o n s . The o v e r a l l on-line a n a l y s i s c y c l e i s c h a r a c t e r i z e d by f i v e d i s c r e t e steps ( c f . Tables 3 . 2 , 3 . 3 ) : (1) Sample a p p l i c a t i o n (10 - 500 p1 body f l u i d ) v i a the autosampl e r i n valve-position "Load" ( c f . Figure 3 . 2 ) followed by chemoselective binding ( c f . Figures 3 . 3 , 3 . 4 ) as well as enrichment of the ribonucleosides on the a f f i n i t y l i g a n d of t h e precolumn (Column 1, c f . Figure 3 . 1 ) under a l k a l i n e , i . e . b u f f e r D, c o n d i t i o n s ("HPAC-STEP"; c f . Figure 3 . 3 ) . (2) Simultaneous, q u a n t i t a t i v e e l u t i o n o f the r e s i d u a l matrix c o n s t i t u e n t s from the precolumn i n t o the waste ("SEC- STEP"; d u r a t i o n : 4 min. a t a flow r a t e of 0 . 4 ml/min., c f . Table 3 . 2 and Figure 3 . 3 , 3 . 4 ) . (3) Mi croprocessor-control 1ed Val ve-swi t c h i ng i n t o p o s i t i o n " I n j e c t " ( c f . Figure 3 . 2 ) . Q u a n t i t a t i v e , g r o u p - s e l e c t i v e e l u t i o n of the ribonucleosides from the precolumn ( c f . Figure 3 . 3 , 3 . 4 ) by a c i d i f i c a t i o n ( b u f f e r A) of the immobilized cycl i c boronate ester and simul taneous on-1 i ne t r a n s f e r i n a s i n g l e , narrow elution-band t hr ough p o s i t i o n s 2-1-4-3 of t h e valve ( c f . Figure 3 . 2 ) t o the t o p of the series-connected a n a l y t i c a l column ("TRANSFER-STEP"; d u r a t i o n : 3 min. a t a flow r a t e of 1 . 0 ml/min, c f . Table 3 . 2 and Fig. 3 . 3 , 3 . 4 ) . (4) Automated valve-switching i n t o p o s i t i o n "Load"; Analytical s e p a r a t i o n o f the ribonucleosides on Column 2 ( c f . Figure 3 . 1 ) by i n c r e a s i n g t h e amount of methanol ( r e s e r v o i r B, Figure 3 . 1 , Table 3 . 2 ) in the mobile phase ( b u f f e r A; "SEPARATION-STEP"; d u r a t i o n : 33 min, flow rate g r a d i e n t : 1 . 6 - 1 . 0 ml/min, cf. Table 3 . 2 ) . (5) Reconditioning of the t r i hydroxy-boronyl f u n c t i o n a l i t y ( c f . Figure 3 . 3 , 3 . 4 ) f o r a new e x t r a c t i o n c y c l e during the anal y t i c a l run with the i n i t i a l e l u e n t D ("REGENERATION- STEP"; d u r a t i o n : 33 min a t a flow rate of 0 . 4 ml/min, c f . Figure 3.3).

N o t e : As r e t a r d a t i o n of t h e target r i b o n u c l e o s i d e s by the boronic a c i d c a p a c i t y of the s u p p o r t , b u f f e r pH, volume a s well as t h e precolumn dimensions, t h e maximum time and flow r a t e (SEC-HPAC step; TRANSFER-step) which

is l i m i t e d and sample

range of i s accep-

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A

OH

OH

+ HO

=2H20

Figure 3 . 3 : P r i n c i p l e of boronic a c i d based a f f i n i t y chromatography. t a b l e f o r q u a n t i t a t i v e recovery of the compounds of i n t e r e s t has t o be optimized f o r a given instrumental c o n d i t i o n . A1 though t h e binding c h a r a c t e r i s t i c of boronic a c i d with c i s - d i o l s i s h i g h l y s e l e c t i v e , secondary i n t e r a c t i o n s of the bonded phase with o t h e r f u n c t i o n a l groups can cause an u n s p e c i f i c

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kf; / \ 'OH

t IREGENERATION

Figure 3.4

1

SEC

Chromatographic properties o f the SEC-HPAC precol umn.

retention of sample contaminants. These i n t e r a c t i o n s (hydrogen charge t r a n s f e r complexes, ion-exchange phenomena, hydrophobic interaction) a r e diminished by avoiding buffers which contain beta-hydroxyl amines ( e . g . Tricine, T r i s , ethanolamine) or h i g h s a l t concentrations ( > 0.2 mol/l). In order t o keep the elution volume of ribonucleosides from the HPAC column a t a m i n i m u m , the a c i d i c elution buffer should f u l f i l l the following requirements: 1) the pH should be below pH 4 t o minimize unspecific bonded phase-analyte i n t e r a c t i o n s , 2) ionic strength and buffer capacity should lead t o a f a s t a c i d i f i c a t i o n of the HPAC-phase, and 3) i t s h o u l d be f r e e of organic solvents t o ensure enrichment of the compounds of i n t e r e s t on the RP analytical column. bonding,

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TABLE 3.2 Gradient Elution Sequences

Ti me mi n

Flow ml/min

0.0- 4.0 4.1- 7.0 7.1-25.0 25.1-37.0 37.1-40.0

Column 2

Column 1

0.4 1.0 0.4 0.4 0.4

Eluent % A

D

-

100

100

-

-

100 100 100

-

Flow

Pump

Eluent %

Pump

ml/min

A

B

C

1.0 1.0 1.6 1.3 1.0

100 100 99 94 94 50 50

0 0 16 6 50 50

0 0 0 0 0

1 2 1 1 1

2 2 2 2 2

TABLE 3 . 3 Washing Program f o r the Analytical Column

Time mi n

0.0 - 8.0 8.1 - 13.0 13.1 - 18.0 18.1 - 28.0

Column 2

F1 ow

Eluent

[%I

m l /mi n

A

B

C

1.0 1.0 1.0 1.0

0 0 0 100

0 100 0 0

100 0 100 0

Pump

2 2 2 2

During routine analysis a wash-step f o r the analytical column i s recommended a f t e r every ten analyses (cf. Table 3.3).

3.3

RESULTS AND DISCUSSION

3.3.1

Off-1 ine Affini tv-Bioael /RPLC Method Since the middle of the seventies, chromatographic methods

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for the group-specific separation of ri bonucleosides from human urine have been developed mainly by the working groups of Borek, Gehrke and Waalkes (refs. 3, 6, 8, 9, 15). As outlined in the Introduction, boronic acid linked to polymer matrices such as agarose, cellulose or polyacrylamide was used as an affinity ligand for the separation of nucleosides under low pressure conditions. We have prepared a boroni c acid-functi onal i zed polyacrylamide (refs. 18, 24) by coupling Hydrazide-Bio-Gel P2 with succi ni c anhydride and m-ami nobenzene boroni c acid accordi ng to (refs. 3, 15). This affinity gel was used for the prefractionation of ribonucleosides from different biological materials, e . g . urine and deproteinized serum (refs. 23, 24) as well as soil extracts (ref. 32), under 1 ow pressure conditions. The appropriate ri bonucleoside fraction from the affinity-Biogel column was shell frozen, concentrated by lyophilization and separated on a reversed-phase column. The reliability of this off-line mode has been proven (refs. 3, 8, 9, 15, 18, 24) and matrix-dependent as well as matrix-independent recoveries were found to be > 90 %. 3.3.2 On-Line HPAC/RPLC Method A methodological improvement for the group-speci fi c pre-fractionation and enrichment of ribonucleosides from protein-free body fluids were achieved by preparation of a boronic acid-substituted si 1 ica suitable for pH-shift dependent sample pretreatment under The small elution volume of high pressure conditions (ref. 22). this HPAC-col umn a1 1 owed the direct transfer of the ri bonucl eosi de fraction to the analytical RP-column by a columnswitching technique (refs. 23-27, 29). Off-line/on-line comparison:

By comparing the results obtained by the off-line and on-line procedure for the determination of urinary ribonucleosides, a good correlation was found (refs. 23, 24). Figure 3.5 representatively shows this correlation for mlAdo in 25 different human urines (ref. 24). Each value is an average of 3 independent runs. Regression line: y = 1 . 0 0 3 ~+ 0.006; r = 0.963. Generally, both procedures turned out to be appropriate for the quantification of ribonucleosides in protein free or depro-

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LZ

d-

N

:

i2

1 . h

n Q)

S

.CI

I w

w

0

W

0 TI

<

H

E

Fi ure 3 . 5 Of f-1 i ne a f f i n i ty-Bi ogel -RPLC/on-1 i ne HPAC-RPLC corr e g a t i o n (taken from E. Hagemeier, K. Kemper K.-S. Boos a n d . E Schlimme, J . C l i n . Chem. C l i n . Biochem., 2 j (1984) 175, with permission). t e i ni zed b i o l o g i c a l m a t r i c e s . Figure 3.6 i l l u s t r a t e s the c o n s i d e r a b l e advantages of the on-line HPLC mode ( r e f s . 24, 26). The on-line procedure i s d i s t i n g u i s h e d from the o f f - l i n e method by the following f e a t u r e s ( r e f s . 24, 26): (1) Total a n a l y s i s time i s shortened. (2) Laborious and error-prone evaporation and r e d i s s o l ut i o n steps a r e avoided, thus l e a d i n g t o an improvement in analytical precision.

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(3) The analytical procedure can be e a s i l y contro led f o r i t s re1 i a b i l i t y . (4) Sensitive samples can be processed very apid y under mild conditions. (5) Small sample volumes can be d i r e c t l y app ied and analyzed. (6) The apparatus allows easy automation. 3.3.3 SEC-HPAC/RPLC Method By using our newly developed SEC-HPAC/RPLC method we have separated and characterized so f a r the following eighteen ri bonucl eosi des (Figure 3.7) . Figure 3.8 shows an off-line RPLC run of a s y n t h e t i c mixture of eighteen ribonucleosides completed within 33 minutes. Figure 3.9 shows an on-line SEC-HPAC/RPLC run of the same synthetic mixture o f ribonucleosides completed w i t h i n 40 minutes. The comparison of b o t h diagrams i l l u s t r a t e s the good conformity of peak area, peak height, retention times and ribonucleoside pattern array obtained by off- and on-1 i ne runs. Pseudouridi ne, however, i s not quantitatively recovered due t o the r e l a t i v e l y high pHvalue o f 9.8. In Table 3.4 data a r e given f o r the peak area and the approp r i a t e coefficient of variation from 3 independent on-line SEC-HPAC/RPLC runs of the same synthetic mixture of r i bonucleosides (cf. Figure 3.7). Despite the column-switching technique the imprecision f o r quantitation i s very low. To monitor the accuracy of the overall SEC-HPAC/RPLC system the matrix-i ndependent recovery of the aforementioned synthetic mixture o f ribonucleosides was analyzed by three r e p l i c a t e injections (Table 3.4). Table 3.5 i l l u s t r a t e s the precision of the SEC-HPAC/RPLC system w i t h respect t o the analysis of a physiological matrix, e . g . mammal milk o r urine. Data were calculated from 3 independent runs of the same sample. For matrix-dependent recovery the amount of ri bonucl eosides present i n mammal milk or urine was determined. The matrix (milk; urine) was then spi ked with defined amounts of r i bonucl eosi des and analyzed anew ( r e f s . 33, 34). The recovery values a r e given in Table 3.5. Linearity of concentration versus signal p l o t s was v e r i f i e d

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I

off-line

1

Membrane filtration

Application of 1.0ml on affinity gel column

on RP.HPLC

2MpI Serum

ZSOpl Urine

Injection of SON on HPLAC precolumn

Column switching

Figure 3.6 Flow dia ram of the off-line and on-line chromatographic rocedures (ta7c en from E. Hagemeier, K. Kemper, K.-S. Boos and E. fchlimme J. Clin. Chem. Clin. Biochem., 22 (1984) 175, with permission.)

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1

2

3

4

S

8

7

F i g u r e 3.7: Ribonucleosides i n v e s t i g a t e d by on-1 i n e SEC-HPAC/RPLC. Pseudouridine ($ , 1; c y t i d i n e (Cyd), 2; u r i d i n e (Urd), 3; 5ami n o i m i d a z o l e- -carboxamido-N-rj b o f u r a n o s i d e (AICAR) , 4; N1uanosine (Guo), 7; -methyl adenosine m1 Ado), 5; inos1ne ( I n 0 2 - p y r i done-5-car oxami do-N-ri b o f u r a n o s i 6(P?NR) = 1,6-di hydro-6-oxo-l-(R-D-ri b o f u r a n o s y l ) - 3 - p y r i d i n e c a r b o x y l i c amide, 8; N3 -methyl u r i d i ne (m3 Urd , 9; adenosine (Ado), 10; N1 -meth 1 in o s i ne m1 I n o ) 11; N1 -methy guanosi ne Guo) , 12; N4 - a c e t Y c y t i d i ne ac4 Cydj , 13; N2 -meth 1guanosine (m Guo) , 14; N2 -dimethy guanosi ne m; Guo) , 15; N6 -meth Yadenosi ne (m6Ado), 16; N6 -carbamoyl t h r e o n y l N6-dimethyladenosine (m; Ado), 18. adenosine (t6Ado),

d

b

ck

Y

lr;

over t h e c o n c e n t r a t i o n range o f i n t e r e s t f o r t h e f o l l o w i n g r i b o nucleosides: Cyd, Urd, ,?Ado, Ino, Guo, PCNR, m3Urd, Ado, m l I n o , mZGuo, m$Guo, t6Ado. A l l c o r r e l a t i o n c o e f f i c i e n t s f o r l i n e a r r e g r e s s i o n were around 0.99. I n Table 3.6 t h e r e p e a t a b i l i t y f o r t h e r e t e n t i o n times a r e g i v e n from 3 independent on-1 i n e SEC-HPAC/RPLC r u n s o f a s y n t h e t i c m i x t u r e o f r i bonucl e o s i des ( c f . F i g u r e 3.7) The r e p e a t a b i l i t y as w e l l as t h e r e c o v e r y and l i n e a r i t y d a t a o f t h e SEC-HPAC/RPLC a n a l y s i s demonstrate t h a t t h i s method f u l f i l l s t h e p r e c i s i o n as w e l l as accuracy c r i t e r i a r e q u i r e d f o r a

.

C131

e 0 w

S

o 4 0

r

0 n 4

0 c

I

3

I

I

I

I

I

I

10

Figure 3.8 sides.

I

I

I

I

l

15

l

!L i 1

1

I

I

20

I

l

l

,

i

I

25

I

I

I

I

O

30

I

I

Time (min)

O f f - l i n e run of a s y n t h e t i c mixture of ribonucleo-

p r a c t i c a l and r a p i d on-line HPLC system. 3.3.4 Aoolication o f the SEC-HPAC/RPLC Method Urine:

Figure 3.10 shows the on-line SEC-HPAC/RPLC a n a l y s i s of 100 p l of membrane-fi 1 tered normal human u r i n e . The c o n s i s t e n c y of t h e u r i n a r y e x c r e t i o n p a t t e r n of modified r i bonucleosides from normal human subjects was d e s c r i b e d by s e v e r a l groups ( r e f s . 9 , 14, 19, 24, 25, 35). Due t o these f i n d i n g s the a n a l y s i s of u r i n a r y r i b o n u c l e o s i d e s was found t o be useful a s a non-invasive s c r e e n i n g t e s t . Table 3.7 summarizes the

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TABLE 3.4

Matrix-Independent Repeatabi 1i ty o f Peak Area and Recovery o f R i bonucleosi des by the SEC-HPAC/RPLC Method Ri bonucl eo-

cv ,%a

CYd Urd AICAR m’ Ado

2.9 3.6 3.4 2.7 3.9 6.3 3.3 2.6 3.4 5.5 4.1 4.1 3.2 2.9 4.0 3.3 3.9

side

Ino Guo PCNR Urd Ado ml Ino m3

m1 Guo ac4 Cyd m2 Guo m$ Guo m6 Ado t6Ado mq Ado

Recovery,% 94.0 93.0 104.6 104.9 97.9 95.6 102.0 104.7 104.5 105.6 103.6 105.4 103.9 102.6 97.6 99.2 102.8

SD,%b

2.9 1.9 3.0 3.1 3.2 4.0 4.6 2.1 9.1 2.1 1.5 4.5 1.4 6.5 1.8 0.3 3.3

C V : Coefficient of variation; n = 3

SD: S t a n d a r d d e v i a t i o n

excretion Val ues determined for various urinary ri bonucleoside markers of clinical interest. The interindividual means and standard deviations are expressed i n bmol ri bonucl eoside per mmol creatinine. Creatinine proved t o be a reliable basis f o r such comparison (ref. 36), as i t s levels are a function of body mass (ref. 37). Numerous results have been accumulated i n several 1 aboratories (refs. 2, 3, 8, 14, 25, 27, 35, 36, 38-48) d u r i n g the l a s t decade demonstrating t h a t the profile for modified and hypermodified urinary ribonucleosides i s a1 tered i n individuals sufferi n g from cancer diseases. The results indicate t h a t $, m l Ado,

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TABLE 3.5 Matrix-Dependent Repeatability of Peak Area R i bonucleosides by the SEC-HPAC/RPLC Method

and Recovery

Urine Ri bonucl eoside

CV,%a

CY d Urd

5.6 3.6 4.6 3.8 3.5 4.3 4.6 6.0 4.0 3.8 3.9 4.8 4.0 3.5 4.0 2.7 5.9

AICAR Ado Ino Guo PCNR

m1

Urd Ado m3

Ino m1 Guo ac4Cyd m* Guo m; Guo m6Ado t6Ado ml

mi Ado

Recovery,% 98.7 101.3 103.2 100.8 100.4 95.4 109.1 100.4 100.0 102.0 100.9 101.1 99.3 101.2 97.9 99.3 95.5

of

Milk SD,%O

2.7 4.2 3.2 3.4 6.7 14.1 10.4 8.7 3.8 3.8 4.2 3.7 3.0 3.1 3.2 3.2 2.6

Recovery,% 100.9 101.2

SD,%D

2.0 2.2

n.d.c 98.3 100.6 98.4 n,d. n.d. 100.0

1.7 7.7 5.0

3.8

n.d. 101.0 96.2 n.d. n.d. n.d. 103.5 n.d.

1.9 0.6

1.7

aCV: C o e f f i c i e n t o f v a r i a t i o n ; n = 3 bSD: S t a n d a r d d e v i a t i o n Not d e t e c t e d . Cn.d.:

mlIno, m;Guo, PCNR and t6Ado represent marker molecules f o r neop l a s i a s . Most of the data available seem t o suggest a r e l a t i o n s h i p between the urinary excretion level of various modified ribonucleosides as well as the nucleoside excretion p r o f i l e f o r organ-speci f i c neopl a s t i c diseases. Figure 3.11 shows exemplarily the urinary ribonucleoside excretion pattern (100 p1 urine) of a patient suffering from breast carci noma. Serum: Figure 3.12 shows exemplarily an on-1 ine SEC-HPAC/RPLC anal y s i s of 500 p l of human serum from a normal subject.

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TABLE 3.6 Repeatability o f Retention Times by the SEC-HPAC/RPLC Method Ri bonucl eoside

x, min

SDa, min

cv, %b

CYd Urd AICAR m1 Ado Ino Guo PCNR m3 Urd Ado m1 Ino m1 Guo ac4 Cyd m2 Guo mi Guo m6Ado t6 Ado mi Ado

5.77 7.15 8.18 9.17 13.56 14.96 15.84 17.81 20.90 22.99 24.53 25.63 26.30 28.69 29.57 31.34 32.75

0.33 0.06 0.11 0.14 0,34 0.38 0.34 0.34 0.39 0.40 0.40 0.31 0.17 0.05 0.05 0.05 0.05

0.5 0.9 1.3 1.5 2.5 2.6 2.2 1.9 1.8 1.7 1.6 1.2 0.7 0.2 0.2 0.2 0.1

aSD: S t a n d a r d d e v i a t i o n ~ C V :C o e f f i c i e n t o f v a r i a t i o n ; n = 3

Our results obtained prove that the SEC-HPAC/RPLC procedure is suitable for the direct analysis of serum samples without previous deproteinization. This analytical advantage might encourage a more detai 1 ed investigation of serum ri bonucl eosides as additional biochemi cal markers for neopl asi as. The f o l 1 owing ribonucleosides were measured, most of the modified constituents were present only in low concentrations: Pseudouridine, cytidine, uridine, mr Ado, inosine, guanosine, adenosine, m$Guo, t6Ado and m$ Ado. A compari son of uri nary and serum ri bonucl eosi des from normal subjects shows clearly that there are differences in the ri bonucleoside pattern in human serum and the corresponding urine especially concerning the unmodified components adenosine, guanosine, inosine and uridine (ref. 25). In contrast to serum,

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TABLE 3.7 Uri nary Excretion o f Modi f ied Ri bonucl eosi desa

Nucleoside m l Ado PCNR m' Ino m1Guo ac4Cyd m2 Guo m: Guo t6Ado

bmol nucleoside/mmol creatinine X SD 3.69 2.01 1.65 0.96 0.70 0.41 2.07 0.83

0.60 0.46 0.55 0.09 0.11 0.17 0.38 0.16

Number and s e x o f n o r m a l s u b j e c t s : 6 f e m a l e s ( a g e 2 4 - 4 5 ) , (age: 28-49).

3 males

these major ribo-nucleosides are present in urines only in small or at least trace amounts due to reutilization processes. Milk:

Figure 3.13 shows exemplarily an on-1 ine SEC-HPAC/RPLC analysis of 100 p1 of breast milk. Besides the unmodified nucleosides cytidine, uridine, inosine, guanosine and adenosine the following modified ribonucleosides were present: Pseudouridine, AICAR, ml Ado, m;Guo and t6Ado. Only low amounts o f PCNR and the methylated ribonucleosides m3Urd, mlIno and m2Guo were measured (refs. 33, 34). Further investigations are necessary to confirm how the ribonucleoside profile in milk i s influenced by the nursing period post partum and nutritional habits. These interesting questions are currently being studied. Their investigation seems to be worthy o f exploration as to the potential of these minor milk constituents as intrinsic biochemical markers for metabolic disorders, as e.g. fibrocystic disease (galactorroea) (ref. 27). Galactorroea fluid: Figure 3.14 shows exempl ari ly an lysis of 100 pl of galactorroea fluid.

on-1 i ne SEC-HPAC/RPLC ana-

We observed that in most of the cases the ribonucleosides

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I

AA260 nm 0.004

s

W

L

3

Nc

c 0 I

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c y t i d i n e , u r i d i n e , guanosine, adenosine and i n p a r t i c u l a r t6Ado a r e t h e main c o n s t i t u e n t s i n mammary s e c r e t i o n s o f g a l a c t o r r o e a p a t i e n t s whereas l e s s amounts o f t h e m o d i f i e d components pseudou r i d i n e , m3Urd, m l I n o , mlGuo, m2Guo, m:Guo and m6Ado and o n l y t r a c e s o f A I C A R and ml Ado were found. m$Guo/t6Ado r a t i o i n body f l u i d s :

According t o t h e f i n d i n g s o f Schiich and coworkers ( r e f s . 49-51) t h e r e i s a complete r e n a l e x c r e t i o n o f t h e tRNA c a t a b o l i t e s m$Guo and t6Ado. The amount o f each t R N A marker molecules was c a l c u l a t e d t o be 0.51 (m:Guo) and 0.19 (t6Ado) mol p e r mol eucar y o t i c tRNA ( r e f . 51). The u r i n a r y r a t i o o f b o t h n o n r e u t i l i z a b l e

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1 Figure 3.10 On-1 i ne SEC-HPAC/RPLC analysis of ri bonucl eosi des i n normal human urine. ribonucleosides i s 2.7. In urines of healthy subjects the r a t i o of both nonreutilizable t R N A catabolites was estimated t o be 2.4 (ref. 2 7 ) , 3.4 (ref. 49) and 2.5 (ref. 48). In pathological urines of leukemia patients, on the other h a n d , the m$Guo/t6Ado r a t i o i s increased by about twice (ref. 27). In human breast m i l k the r a t i o equals the calculated t R N A value, whereas i n gal actorroea f l u i d s i n contrast t o the corresponding urines w i t h a r a t i o o f 2 . 5 ( r e f . 27) - the r a t i o i s decreased by one order of magnitude t o 0.24, thus showing t h a t other t h a n cell ul a r t R N A breakdown phenomena influence the level of m$Guo and/or t6Ado.

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Figure 3.11 On-1 i ne SEC-HPAC/RPLC analysis of uri nary r i bonucl eosides of a patient suffering from breast cancer. 3.4

SUMMARY

We present an automated, two-column HPLC analysis method f o r the d i r e c t and routine measurement of modified ribonucleosides i n biological f l u i d s . The method combines the s e l e c t i v i t y of h i g h performance a f f i n i t y chromatography (HPAC) on immobi 1 ized boronic acid w i t h the h i g h resolution, speed of analysis and s e n s i t i v e detection of reversed-phase HPLC by use of a column switching technique. By the preparation of a new column material, the bonded phase a f f i n i t y extraction s t e p could be coupled w i t h a simultaneous size-exclusion chromatography (SEC), thus a1 lowing f o r the

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Figure 3.12 On-1 i ne SEC-HPAC/RPLC analysis of ri bonucl eosi des in normal human serum. first time the direct, i . e . on-line analysis of proteinaceous fluids, such as serum or milk. The SEC-HPAC/RPLC technique also can be used for a small scale preparative purpose or trace enrichment for the characterization and identification of ribonucleosides in bi ol ogi cal matri ces. The ri bonucl eosi de-HPLC-analyzer is distinguished by its on-1 i ne sampl e-processi ng mode, its practicability, chemo-selectivity, precision and speed of analysis. 3.5 FUTURE PROSPECTS The described on-1 ine SEC-HPAC/RPLC method combines the chemo-selectivity of high-performance affinity chromatography with

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Figure 3.13 On-line SEC-HPAC/RPLC analysis of ribonucleosides i n breast mi 1 k. the high resolution, precision, accuracy and speed of analysis of reversed-phase HPLC by use of a column switching technique. Due t o these properties the HPLC-analyzer for ri bonucl eosides i n body fluids represents a powerful a n a l y t i c a l method f o r investigations i n the biochemical as well as i n the clinical research f i e l d , as: (1) a method for trace enrichment f o r the structural characterization of ri bonucleosides i n b i o l o g i c a l fluids;

(2) a method for small scale preparation of ribo-

nucleosides;

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F i g u r e 3.14 On-1 ine SEC-HPAC/RPLC anal s i s o f g a l a c t o r r o e a f 1u i d (Column 2 c o n t a i n e d Su e r s p h e r RP-18, 1 5 x 4 mm I.D., i n s t e a d of LiChrospher RP 18, lOOfe, c f . HPLC-Conditions, S e c t i o n 3.2.5).

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(3) a method t o i n v e s t i g a t e d i s o r d e r s i n r i b o n u c l e o s i d e , r i bonucl e o t i d e and/or RNA metabol ism; (4) a n o n - i n v a s i v e s c r e e n i n g t e s t f o r cancer diseases i n humans ; (5) a method t o s t u d y r e n a l r e u t i l i z a t i o n processes;

(6) a method f o r t h e r a p e u t i c d r u g m o n i t o r i n g d u r i n g n u c l e o s i d e chemotherapy.

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3.6 ACKNOWLEDGMENT The former as well as the current Ph.D. s t u d e n t s Dr. E. Hagemeier, Dr. K. Kemper, B. Wilmers and K.-P. Raezke are gratef u l l y acknowledged f o r t h e i r work i n the HPLC project reviewed i n this paper. For f i n a n c i a l support we wish t o t h a n k the 'Forschungskommission' of the U n i v e r s i t y o f Paderborn, and the company E. Merck, Darmstadt. 3.7 1. 2.

3. 4. 5. 6.

7.

8. 9. 10.

11*

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P. Pa ageorgiou, H. Kesarwala, E. Gelfand and V. Roegner-Manisca co, I n c r e a s e d e x c r e t i o n of m o d i f i e d adenine n u c l e o s i d e s by c h i l d r e n w i t h adenosine deaminase d e f i c i e n c y , P e d i a t r . Res., 16 (1982) 362-

Y

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f.

1

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s i d e p r o f i l e s gy HPLC-UV i n serum and u r i n e o f c o n t r o l s and cancer p a t i e n t s , i n : F. Cimino, G.D. Birkmayer, E. Pimentel, J. V. K l a v i n s , F. S a l v a t o r e (Eds.), Proc. 3 r d I n t e r n a t . Conf. on "Human Tumor Markers" W. de G r u y t e r Verlag, Berlin,

(1987) 519-544. 47. M. Savoia, T. Russo, E. Rippa, L. Bucci, F. Mazzeo, F. Cimino and F. S a l v a t o r e Serum s e u d o u r i d i n e : I t s e v a l u a t i o n as a biochemical signa! o f neopyasia, J. Tumor Marker Oncol., 1

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48. ! I

b

A1885Y 355-361

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Topp, G. Sander, G. Heller-Schbch and G. Schbch, A h i g h performance 1 i u i d chromatographic method f o r t h e determlnation o f seu o u r i d i n e and u r i c a c i d i n n a t i v e human u r i n e and u l t r a f i t r a t e d serum, Anal. Biochem., 150 1985 353-358. G. Sander, H. Topp, G. Heller-Schbch and G. Schbcb K a t a b o l i t e n aus Transfer-RNA i m Urin a l s Marker f u Eir tRN Umsatz und Ganzkbrper-Stoffwechsel a k t i v i t a t , ErnahrungsUmschau, 33 (1986) 92.

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CHAPTER 4 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF FREE NUCLEOTIDES, NUCLEOSIDES, AND THEIR BASES I N BIOLOGICAL SAMPLES YONG-NAM KIM1 and PHYLLIS R. BROWN Depar tm e nt o f C h e m i s t r y , U n i v e r s i t y o f Rhode I s l a n d , K i n g s t o n , R l 02881, U . S . A .

TABLE OF CONTENTS 4.1 Introduction . . . . . . . . . . . . . . . . 4.2 Chromatographic Methods 4.2.1 Ion-Exchange Chromatography 4.2.2 Reversed-Phase Liquid Chromatography 4.2.3 Ion-Pair Chromatography 4.2.4 Mixed-Mode Chromatography .... 4.2.5 Micellar Liquid Chromatography 4.3 Applications . . . . 4.3.1 Analysis of Nucleotides 4.3.2 Analysis o f Nucleosides and Bases 4.3.3 Simultaneous Analysis of Nucleotides, Nucl eo-

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4.4 4.5 4.6

... .......... ...... ...... . . . . . . . . . . . . . . . .. . . ....... .. ... sides and Bases . . . . . . . . . . . . . . Future Prospects . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

4.1

INTRODUCTION

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Nucleotides, nucleosides and bases are o f major importance in biological systems; in the formation and function of the nucleic acids, as meditators of hormone actions and as regulators of enzyme reactions and metabolic processes. In addition, disorders in purine and pyrimidine metabolism are believed to be involved in diseases such as neoplastic diseases (refs. 1-3) and hereditary immunodeficiencies (refs. 4-6). In order to separate and quantify the large number o f purines and pyrimidines in biological samples, sensitive and selective rCurrent

address:

Department of

Chemistry,

Kyungnam U n i v e r s i t y ,

Masan 610, Korea

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techniques are required. High-performance 1 iquid chromatography(HPLC) which is a powerful technique meets the requirements for high sensitivity, speed, selectivity, and resolution needed in the analysis of nucleic acid components. Since Cohn (ref. 7) first reported in 1949 on the separation of several nucleic acid components by ion-exchange chromatography, many high-performance 1 iquid chromatographic procedures have been applied to the analysis of nucleotides, nucleosides and bases. These methods include ion-exchange (refs. 8,9), reversed-phase (refs. 10,11), and ionpai r chromatography (refs. 12-14). Since 1970 our 1 aboratory has been involved i n the development of separation techniques for all compounds involved in the purine and pyrimidine metabolic pathways (refs. 10, 15-17, 23, 24, 35-37, 55, 74, 103, 104, 106-108, 110, 111). Recently, we investigated fast, microbore, fast-microbore, and micellar HPLC methods for the analysis o f nucleotides, nucleosides and their bases. We describe here our experience as well as that of others reported in the literature with HPLC methods for the analysis of nucleic acid components in biological samples. The abbreviations for nucleotides, nucleosides and bases used i n this chapter are listed i n Table 4.1. TABLE 4.1 List o f Abbreviations o f Nucleotides, Nucleosides, and Bases

Nucl eo t i des AMP ADP ATP CAMP CMP CDP CTP GMP GDP GTP cGMP IMP I DP ITP NAD B-NAD NADP TMP

Adenosine 5'-monophosphate Adenosine 5'-diphosphate Adenosine 5'-triphosphate Adenosine 3' 5'- c y d i c monophosphate Cytidine 5'-honophosphate Cytidine 5 ' -di phosphate Cyti di ne 5 ' -tri phosphate Guanosine 5'-monophosphate Guanosine 5'-diphosphate Guanosine 5;-triphos hate Guanosi ne 3 , 5 -cycyi c monophosphate Inosine 5'-monophosphate Inosi ne 5 ' -di phosphate Inosine 5'-triphosphate Nicotinamide adenine dinucleotide B-Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide, 3'-phosphate Thymidi ne 5'-monophosphate I

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TDP, dTDP TTP, dTTP UMP UDP UTP XMP XDP XTP

Thymidi ne 5'-diphosphate Thymidine 5'-tri hos hate Uridine 5'-monop osp ate Uridine 5'-diphosphate Uridine 5'-triphosphate Xanthosine 5'-mono hos hate Xanothosine 5'-dipiospiate Xanthosine 5'-triphosphate

it:

Nucl eosi des Ado 6-m-Ado CY d Guo N, -m-Guo 2-m-G N -m-Guo Nf -m-Guo 7 -m-Guo I no dIno l-m- Ino 7-m-Ino Ord Thd, dThd Urd Xao

Adenosine N6 -Methyl adenosi ne Cytidine Guanosi ne l-Meth 1 uanosine N2 -Met y guanos! ne N* -Methylguanosi ne N$ -Dimethylguanosine 7-Methylguanosine Inosine 2'-Deox inosine l-Methyyi nosi ne 7-Methyl i nosi ne Oroti di ne 2'-Deoxythymidine Uri di ne Xanthosine

$9

Bases Ade Caf CYt ThP

EY

Ura Xan

Adenine Caffeine Cytosine Dyphyl 1 i ne H poxanthine TK eobromi ne Theophyll i ne Thymine Uric acid Uraci 1 Xanthine

4.2 CHROMATOGRAPHIC METHODS 4.2.1 Ion-Exchanae Chromatoaraohy In the late 1940s, the first liquid chromatographic separations of nucleic acid components were performed on synthetic resin ion-exchangers (ref. 7). Since that time, substantial improvements in methods (refs. 18,19) and packing materials (refs. 20-22) have greatly decreased the analysis times and increased the efficiency of the separations. Particularly important to the improvement of the separations was the introduction o f microparticulate chemically bonded ion-exchangers (ref. 23). Because nucleotides have negatively charged phosphate groups, anion-exchangers are usually used for the separation of nucleotides (refs. 23-29). Figure 4.1 shows the separation o f 20 nucl eoti de standards on a si 1 ica-based ani on-exchanger (ref. 23). Optimal resolution of the majority o f the nucleotides was obtained

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u s i ng g r a d i e n t e l u t i on. A1 though b o t h c a t i o n - and anion-exchangers were used t o separate n u c l e o t i des, n u c l eosides and bases ( r e f s . 30-34), i o n exchange chromatographic methods a r e n o t as s u i t a b l e f o r t h e s e p a r a t i o n o f nucl e o s i des and bases as reversed-phase 1iq u i d chromatography (RPLC)

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F i g u r e 4.1 S e p a r a t i o n o f mono-, d i - and t r i p h o s p h a t e n u c l e o t i d e s o f adenine, hypoxanthine, xanthine, c y t o s i n e u r a c i l and thymine. Whatman). e l u e n t s Column, P a r t i s i l - 1 0 SAX (25 cm x 4.6 mm I . D . K!H,PO, + 6.50 M KC! low) 0.007 M KH PO, (pH 4.0), ( h i h) 0.25 [pH 4.5), i s o c r a l i c f o r 15. min t\en l i n e a r e g r a d i e n t 0-100% o f h i h - c o n c e n t r a t i o n e l u e n t !n 4g’min.; s e n s i t i v i t y , 0.68 a.u.f.s. (2!4 nm); temperature, ambient; f l o w - r a t e , 1.5 m l h i n . R e p r i n t e d w i t h p e r m i s s i o n from r e f . 23.

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4.2.2

Reversed-Phase L i a u i d ChromatoaraPhv With t h e development o f m i c r o p a r t i c u l a t e c h e m i c a l l y bonded packing m a t e r i a1 s , n u c l eosides and bases c o u l d be r e a d i l y separa t e d ( r e f s . 10,ll). When reversed-phase packings i n which t h e hydrocarbon c h a i n m o i e t i e s a r e c h e m i c a l l y bonded t o t h e m i c r o p a r -

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t i c u l a t e s i l i c a s u p p o r t are used, the separations have the advantages of simplicity, high efficiency, reproducibility, and speed. T h u s , RPLC methods have been extensively used f o r the separation of nucleic acid components e i t h e r with a gradient mode Figure 4 . 2 ( r e f s . 35-41) or w i t h an i s o c r a t i c mode ( r e f s . 42-44). shows the gradient separation o f the major nucleosides and bases and other biologically important compounds on a reversed-phase

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Fi ure 4 . 2 Separation of the major nucleosides and bases and o t er biologically important com ounds. Column, chemically bonded reversed-phase C,, on 10 pm t o t a y l y porous s i l i c a s u p p o r t (30 cm x

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4.6 mm I.D.) (Waters); e l u e n t s , A) 0.02 M KH,PO,.(pH.5.6), B) 60%, s l o p e 0.69%/min. (0-60% methan.01 i n 87 min.); injection volume, 40 ,ul of a s o l u t i o n 1 x 10-5 M i n each s t a n d a r d ; temperature, ambient; flow-rate, 1.5 ml/min. Reprinted with permission from ref. 36.

Figure 4.3 Gradient s e p a r a t i o n of nucleosides and base standard (Perkin-Elmer) 5 cm x mixture. Column, 3 ,urn base-deactivated C pH 5 . 6 ) , B) 9 % 0.02 4.6 mm I.D.); Mobile phase, A) 0.02 M KH,bb, M.KH,PO, (PH 5.6) + 3% methanol, l i n e a r g r a i e n t , 0-100% B i n 3 m i n . ; s e n s i t i v i t y , 0.05 a . u . f . s . ; flow-rate, 1.5 ml/min. Peaks: l = C t , P=Ura, 3=H p, 4=Xan, 5=Urd, 6=Thy, 7=Ade, 8=Ino, 97Guo, l O = f h d , 11=Ado. 280 pmol of each component i n j e c t e d . Unpublished chromatogram. Reprinted with permission of Dr. R.C. Simpson.

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column ( r e f . 36). Using a methanol mobile phase or a wide variety of hydroorganic mobi 1 e phases with reversed-phase packi n g s , these nucleosides and bases could be readily separated i n various types of b i ol o g i cal sampl es . Recent trends i n the design of HPLC columns are towards the reduction i n column length and/or column diameter as well as p a r t i c l e s i z e . The advantages of these smaller columns packed with 3-5 pm p a r t i c l e s i ncl ude reduced sample quantity requirements, solvent consumption, a n d analysis time. In addition greater s e n s i t i v i t y and s e l e c t i v i t y are obtained; thus with these columns, there are lower l i m i t s of detection as well as smaller sample volume requirements. Recently, we investigated the use of f a s t HPLC, microbore HPLC, and fast-microbore HPLC f o r the separation of nucleic acid components ( r e f s . 45-47). In f a s t HPLC, short columns o f 5-10 cm length with a conventional internal diameter (I.D.) of 4.6 mm are used. Decreased analysis time, solvent use, sample requirements, and increased s e n s i t i v i t y were observed (ref. 45). Figure 4.3 shows the gradient separation of nucleosides and bases w i t h f a s t HPLC. The separation of 11 nucleosides and bases was performed i n l e s s than 9 m i n . In microbore HPLC, 25 cm length microbore columns w i t h 2 . 1 mm or 1 mm I.D. allowed the reductions in sample quantity requirements and mobile phase consumption. Figure 4 . 4 shows the gradient separations of 11 nucleosides and bases on three d i f f e r e n t column diameters ( r e f . 4 6 ) . However, microbore HPLC did n o t provide decreased analysis times compared t o conventional columns of the same length with 4.6 mm I.D. This problem was minimized by using shorter microbore columns. Figure 4.5 shows the separation of 11 nucleosides and bases on 5 cm columns with 2 . 1 mm or 1 mm I.D. The combination of f a s t HPLC and microbore HPLC, namely, f a s t microbore HPLC, o f f e r s reductions in analysis times, solvent use, and sample quantity requirements. However, reduced resolution was observed on the 5 cm columns with 1 mm I.D. due t o the extra column band broadening. Therefore, the use of short (5-10 cm) columns with 2 . 1 mm I.D. i s most s u i t a b l e f o r the analysis of nucleic acid components when dealing w i t h limited amounts of biological samples.

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Figure 4.4 Gradient separation of nucleoside and base standard mixture. Column, 10 pm base-deactivated C,, (Perkin-Elmer); mobile phase, A) 0.02 M KH,PO, (pH 5.6), B) 95% 0.02 M + 5% methanol, linear gradient, 0-100% B i n 15 min.; sensitivity, 0.05 25 cm x 4.6 mm column; flow-rate, 1.5 ml/min; 1 nmol of each component cm x 2.1 mm column; flow-rate, 310 pl/min; 400 pmol of each component Incm x 1 mm col-umn; flow-rate, 70 pl/min; 100 pmol of each component injected. Peaks are the same as i n Figure 4.3. Reprinted with permission from ref. 46.

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Figure 4.5 Gradient s e p a r a t i o n of nucleoside and base standard mixture. (A) 5 cm x 2.1 mm column- flow-rate, 310 ul/min; 100 pmol of each component i n j e c t e d , (B) 5 cm x 1 mm column; flowr a t e , 70 pl/min; 50 pmol of each component i n j e c t e d . Other cond i t i o n s and peaks a r e the same a s i n Figure 4.3. Unpublished chromatograms. Reprinted w i t h permission of Dr. R. C . Simpson.

4.2.3

Ion-Pair ChromatoaraPhv For the s e p a r a t i o n of nucleosides and bases, u s u a l l y RPLC has been used e i t h e r w i t h i s o c r a t i c o r g r a d i e n t e l u t i o n . On the o t h e r hand, most of s e p a r a t i o n s of nucleotides have been achieved on anion-exchange columns with g r a d i e n t e l u t i o n . However, g r a d i e n t i on-exchange chromatographic methods r e q u i r e re1 a t i v e l y long a n a l y s i s and r e - e q u i l i b r a t i o n times. Hoffman and Liao ( r e f . 12) reported t h a t the a d d i t i o n t o the mobile phase of ion-pairing reagents, i .e., charged molecules w i t h a hydrophobic moiety, increased the i n t e r a c t i o n of i o n i c compounds with the l i p o p h i l i c s t a t i o n a r y phase. Tetrabutylammonium s a l t was employed a s the cationic-pairing reagent f o r the s e p a r a t i o n of the nucleotides using a g r a d i e n t e l u t i o n of increasing i o n i c s t r e n g t h

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and methanol concentration. Ehrlich and Ehrlich ( r e f . 48) used an anionic-pairing reagent, heptane sulfonate, f o r the i s o c r a t i c separation of six bases normally found i n DNA. These two pairing reagents have been extensively used f o r the separation of nucleic acid components ( r e f s . 49-56). In order t o optimize the conditions f o r the ion-pairing of nucleotides, Perrone and Brown ( r e f . 57) investigated the optimum alkyl chain l ength of the ion-pairing reagent on the retention behavior of 18 nucl eotides. I t was found t h a t t e t r a b u t y l ammoni um phosphate was the most e f f e c t i v e pairing reagent f o r retarding the In addition, they elution of the majority of the nucleotides. studied the e f f e c t s of concentration of the ion-pairing reagent as well as e f f e c t s of the pH of the mobile phase. Pimenov e t a 7 . ( r e f . 58) reported t h a t a simultaneous separation of the major nucleotides, nucleosides and bases could be

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Figure 4.6 Separation of the major nucleotides nucleosides and bases. Column NovaPak C,, l a s t i c c a r t r i d g e 616 cm x 8 mm I.D.) (Waters); mobi'le phase, A) 8.01 M NH,H,PO + .002 M t e t r a b u t y l ammonium hosphate ( H 7.0), B) 85% A + 1 5 % a c e t o n i t r i l e , gradient, B i n 1g m i n . ; temperature, ambient; flow-rate, 2.0 m l / m i n . Reprinted w i t h permission from r e f . 58.

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achieved on a r a d i a l l y compressed C,, column u s i n g an a c e t o n i t r i l e g r a d i e n t w i t h tetrabutylammoniurn phosphate as t h e i o n - p a i r i n g reagent. A r e p r e s e n t a t i v e s e p a r a t i o n i s shown i n F i g u r e 4.6 ( r e f . 58). The m a j o r n u c l e o t i d e s , n u c l e o s i d e s and bases were w e l l separated i n l e s s than 30 min. D i f f e r e n t i o n - p a i r i n g reagents have been used f o r t h e s e p a r a t i o n o f n u c l e i c a c i d components ( r e f s . 14, 55, 59-61). To s e p a r a t e n u c l e o t i d e s , a z w i t t e r i o n p a i r i n g reagent, l l - a m i noundecanoi c acid, was used by Knox and Jurand ( r e f . 14). F l e x i b l e s e p a r a t i o n s were achieved by c o n t r o l l ing w i t h pH t h e quadrupol a r i n t e r a c t i o n s between t h e zwi t t e r i ons and t h e charged n u c l e o t i d e molecule. Kraak e t a 7 . ( r e f . 59) separated n u c l e o s i d e s and bases u s i n g sodium dodecyl s u l f a t e as a p a i r i n g reagent. The s e p a r a t i o n o f 15 n u c l e o s i d e s and bases wa. achieved i s o c r a t i c a l l y i n 6 min. 4.2.4

Mixed-Mode Chromatoaranhv To s e p a r a t e n u c l e o t i d e s , n u c l e o s i d e s and bases s i m u l t a n e o u s l y i s d i f f i c u l t because t h e bases a r e s t r u c t u r a l l y s i m i l a r b u t t h e t h r e e groups a r e v e r y d i f f e r e n t i n p o l a r i t y and charge. F u r t h e r more, i n b i o l o g i c a l samples, t h e r e can be a l a r g e number o f n a t u r a l l y - o c c u r r i n g n u c l e i c a c i d components which can be p r e s e n t i n a l a r g e range o f c o n c e n t r a t i o n s . I n a d d i t i o n endogenous comounds, which can i n t e r f e r e w i t h t h e HPLC a n a l y s i s , can be p r e s e n t . A1 though a r e l a t i v e l y l a r g e number o f n u c l e i c a c i d components were separated on p o l y m e r i c anion-exchange r e s i n s (Aminex s e r i e s ) w i t h g r a d i e n t e l u t i o n ( r e f . 32,33), t h e s e methods have t h e disadvantages o f l o n g a n a l y s i s times (160-225 min) and h i g h o p e r a t i n g Thus, some o f t h e n u c l e i c a c i d components temperature (55-65" C). have been removed d u r i n g sample p r e p a r a t i o n o r d u r i n g t h e HPLC s e p a r a t i o n ( r e f s . 62-70). I n a d d i t i o n , some o f t h e sample p r e p a r a t i o n methods r e q u i r e time-consuming procedures. On t h e o t h e r hand, mu1 t i d i m e n s i o n a l chromatography, i n v o l v i n g column s w i t c h i n g techniques, o f f e r s a combination o f two d i f f e r e n t s e p a r a t i o n modes i n a s i n g l e chromatographic r u n . A1 though van Gennip e t a 7 . ( r e f . 71) used a combination o f weak anion-exchange mode and reversed-phase mode v i a c o l umn s w i t c h i n g techniques, t h e i r method was used o n l y f o r t h e s e p a r a t i o n o f o r o t i c a c i d and o r o t i d i n e f r o m i n t e r f e r i n g substances. Hagerneier e t a 7 . ( r e f s . 72, 73) r e p o r t e d on t h e use o f a b o r o n i c a c i d a f f i n i t y column

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F i g u r e 4.7 Separation o f a standard n u c l e o t i d e , n u c l e o s i d e and The base m i x t u r e o b t a i n e d u s i n g mixed mode chromatograph n u c l e o t i d e s , peaks 1-14, were separated on a P a r t i s i f - 1 0 SAX column (Whatmand A n o n - l i n e a r g r a d i e n t was used w i t h a hos h a t e (pH 5.5) t o 0.5 M KH,PO, [pH g.5) b u f f e r from 0. 65 M KH,PO Flow-rate was 2 ml/min. A t t e r t h e e l u t i o n o f XTP peak, t e c o l u m i was s w i t c h e d o u t o f t h e l i n e and a r a d i a l l y compressed C, column (Waters) was switched i n l i n e . The nucleosides, peaks 15-92, were then separated u s i n a l i n e a r g r a d i e n t e l u t i o n f r o m 0.005 M KH,PO, t o 60% methanol (O-!O% gradient) F l o w - r a t e was 4 ml/min. Peaks: l=CMP, 2=TMP, 3=AMP, 4=GMP, 5=iMP, 6=TDP, 7=CDP, 8=ADP, 9=GDP, 10=TTP, 11=CTP, lP=ATP, 13=GTP, 14=XTP, 15=Ura, 16=Cyt, l!=Hyp, 18=Xan, 19=Xao, EO=Ino, 21=Guo, 22=Ado. R e p r i n t e d w i t h p e r m i s s i o n from r e f . 74.

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coupled t o a reversed-phase c,, column via column switching techniques. However, t h e i r system was used only f o r t h e separation of nucleosides. Our laboratory has u t i l i z e d a combination of anion-exchange mode and reversed-phase mode via col umn switching techniques f o r the separation of a l l three c l a s s e s of nucleic acid components ( r e f . 7 4 ) . This method cal led mixed-mode chromatography provided rapid and s e n s i t i v e separation of a l l t h r e e c l a s s e s of nucleic acid components. Using t h i s method the separation of 22 nucleot i d e s , nucleosides and bases was obtained (ref. 7 4 ) . The mixture i s introduced f i r s t onto the strong anion-exchange column which r e t a i n s nucleotides. The unretained nucleosides and bases a r e then retained by the second column, a reversed-phase C,, column. By u s i n g switching valves, separations of retained compounds on each column can be achieved independently (Figure 4 . 7 ) . Lang and Rizzi ( r e f . 75) used the same columns i n a reverse configuration. Micellar Liauid ChromatoaraPhv In the previous section, ion-pair chromatographic methods were discussed. Those methods required the use of mobile phases containing quaternary ammoni um sal t s or a1 ky! sul fonates or s u l f a t e s . When s u r f a c t a n t s a r e present i n 1 arger concentrations i n the mobile phase, i . e . , above the c r i t i c a l micellar concentration (CMC) , the type of chromatography i s c a l l e d mice1 l a r l i q u i d chromatography. In 1980, Armstrong and Henry ( r e f . 76) reported the separation of phenols and polynuclear hydrocarbons on a reversed-phase column using an aqueous solution of sodium dodecyl s u l f a t e (SDS) micelles. Since then, applications of m i cel l a r l i q u i d chromatography have been demonstrated empl oyi ng SDS or d i f f e r e n t k i n d s of s u r f a c t a n t s ( r e f s . 77-83). Recently, we investigated the separation of nucleic acid components w i t h micellar SDS mobile phases ( r e f s . 84-86). As a r e s u l t of our investigation of the e f f e c t s of an SDS mobile phase w i t h d i f f e r e n t types of s t a t i o n a r y phases, i t was found t h a t the use of a polyvinyl alcohol (PVA) column was most e f f e c t i v e f o r the separation of nucleosides and bases ( r e f . 86). The e 1 u t i on behavior of nucleosides and bases on the PVA column by micellar l i q u i d chromatography i s q u i t e d i f f e r e n t from t h a t on reversedphase columns eluted w i t h hydroorganic mobile phases. In the 4.2.5

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separation of 12 nucleosides and bases on a PVA column by m i c e l l a r l i q u i d chromatography (ref. 84) , the major purines and pyrimidines were we1 1 separated isocratical l y w i t h an SDS mice1 l a r mobi l e phase (Figure 4.8). In contrast t o RPLC where Cyt eluted early i n the chromatogram, Cyt along w i t h the purines w i t h an amino funct i o n a l group i n the 6 position (Ade, Ado) had a longer retention time and a poorer peak shape t h a n other compounds. The elution

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Figure 4.8 and bases.

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I o c r a t i c elution profile of a mixture f nucleosides ( 'lumn, 9 pm Asahipak GS 320H (25 cm ' 7.6 mm I.D. (Asahi Industry); mobile phase, 0.01 M SDS (PH 3.4); flow-rate, ml/min; sample concentration, 2 x 10-5 M each; injection volume, 10 p ! ; sensitivity, 0.01 a.u.f.s.; temperature, ambient. Reprinted w i t h permission from ref. 64.

h

behavior of nucleotides was also investigated under the same conditions (Figure 4.9). All the major nucleotides were eluted i n 5 min. Thus, the separation of nucleosides and bases can be achieved on a PVA column w i t h a SDS micellar mobile phase i n the presence of nucleotides. Reversed-phase col umns w i t h m i cell a r

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m o b i l e phases can a l s o be used f o r t h e s e l e c t i v e a n a l y s i s o f c e r t a i n p u r i nes and p y r i m i d i n e s . One advantage o f m i c e l l a r HPLC w i t h a PVA column o v e r RPLC i s t h a t a broad pH range can be used

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F i g u r e 4.9 I s o c r a t i c e l u t i o n p r o f i l e o f nucleotide standard m i x t u r e . A l l chromatographic c o n d i t i o n s a r e t h e same as i n F i g u r e 4.8. Unpublished chromatogram from t h e l a b o r a t o r y o f D r . P.R. Brown. i n t h e m o b i l e phase. Another advantage o f t h i s t e c h n i q u e i s t h e d i f f e r e n t s e l e c t i v i t i e s obtained. For example, Hyp, Xan and Urd, which have p o o r r e s o l u t i o n w i t h t h e RPLC methods, a r e w e l l separated w i t h t h e m i c e l l a r method. Thus m i c e l l a r l i q u i d chromato-

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graphy can be used i n c o n j u n c t on w i t h RPLC f o r t h e a n a l y s i s o f n u c l e o s i des and bases.

4.3 APPLICATIONS The a n a l y s i s o f n u c l e i c a c i d components i n b i o l o g i c a l mat r i c e s by HPLC i s i m p o r t a n t i n g e n e t i c , biomedical and biochemical research areas. The i d e a l HPLC methods o f f e r h i g h r e s o l u t i o n and s e n s i t i v i t y , good r e p r o d u c i b i 1it y and s h o r t a n a l y s i s t i m e . However, due t o t h e complex n a t u r e o f b i o l o g i c a l samples, i t i s d i f f i c u l t t o balance a l l these f a c t o r s . Therefore, d i f f e r e n t HPLC methods have been developed f o r t h e a n a l y s i s o f s p e c i f i c n u c l eos i d e s and bases and/or n u c l e o t i d e s in v a r i o u s b i o l o g i c a l sampl es. 4.3.1

Analvsis o f Nucleotides Anion-exchange chromatography has been e x t e n s i v e l y used f o r t h e a n a l y s i s o f n u c l e o t i d e s i n a v a r i e t y o f b i o l o g i c a l samples ( r e f s . 25-29, 87-96). F i g u r e 4.10 shows t h e e r y t h r o c y t e nucleot i d e p r o f i l e o f a p a t i e n t w i t h gout and o f a normal s u b j e c t ( r e f . 87). The s e p a r a t i o n was achieved on a m i c r o p a r t i c u l a t e c h e m i c a l l y bonded anion-exchange column w i t h a g r a d i e n t e l u t i o n . A d i s t i n c t i v e i n c r e a s e i n IMP was observed w i t h t h e gout sample. RPLC has a l s o been used f o r t h e a n a l y s i s o f n u c l e o t i d e s i n b i o l o g i c a l samples ( r e f s . 37, 97-102). F o r example, t h e separat i o n o f c y c l i c n u c l e o t i d e s i n r a t - b r a i n e x t r a c t i s shown i n F i g u r e 4.11 ( r e f . 37). The s e p a r a t i o n was c a r r i e d o u t u s i n g a s i l i c a No i n t e r based C,, column w i t h a l i n e a r methanol g r a d i e n t . ferences from o t h e r n a t u r a l l y o c c u r r i n g compounds were observed. I n addition, f o r the analysis o f nucleotides i n biological samples, i o n - p a i r chromatography has been used ( r e f s . 13, 50-53, 158). F i g u r e 4.12 shows t h e s e p a r a t i o n o f p l a t e l e t guanine and adenine n u c l e o t i d e s by i s o c r a t i c e l u t i o n u s i n g a r a d i a l l y compressed C,, column ( r e f . 51). The s e p a r a t i o n was achieved i n l e s s than 9 min. 4.3.2 A n a l v s i s o f Nucleosides and Bases G e n e r a l l y , RPLC has been used f o r t h e a n a l y s i s o f n u c l e o s i d e s and bases i n v a r i o u s b i o l o g i c a l samples ( r e f s . 40, 42, 43, 103111). Our l a b o r a t o r y conducted a comprehensive i n v e s t i g a t i o n o f t h e s e p a r a t i o n o f n u c l e o s i d e s and bases i n serum ( r e f . 35). In t h e chromatogram o f t h e serum p r o f i l e o f a b r e a s t cancer p a t i e n t

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Figure 4.10 Erythrocyte nucleotide profile of a patient w i t h gout (upper) and of a normal subject (lower). Isocratjc f o r 5 m i n . , theallnear gradient i n 35 m i n . ; flow-rate, 2.0 m l / m i n . Other conditions are the same as i n Figure 4.1. Reprinted w i t h permission from ref. 87. w i t h bone metastasis, two methylated nucleosides not found i n normal sera were observed i n the serum from a patient w i t h breast I t was also found t h a t the cancer (Figure 4.13) (ref. 35). concentrations of Ino were elevated i n the plasma from patients w i t h acute lymphocytic leukemia compared t o the concentrations i n

the plasma from the normal subjects (Figure 4.14) (ref. 112). In the leukemic patients, lower Ino levels were f o u n d t o correspond t o a prognosis of remission and very h i g h levels corresponded t o a prognosis of relapse; thus these results suggest t h a t Ino can be used t o predict the prognosis of the disease. In a d d i t i o n , levels of the nucleosides, Guo and Ado as well as the bases, Hyp and Xan

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F i g u r e 4.11 Chromato rams o f A) a r a t - b r a i n e x t r a c t , (B) t h e e x t r a c t c o i n j e c t e d w i t \ cGMP an CAMP, and C) t h e e x t r a c t i n c u bated w i t h d i e s t e r a s e f o r 10 min. Co umn, pBonda ak C (pH 3.7), B) 68% meth: m o b i l e phase, A) 0.02 M KH,PO, (Waters anol, ]:near g r a d i e n t , 0-25% B i n 30 min; s e n s i t J v i t y , 0.04 a.u.f.s: temperature., ambient; f l o w - r a t e , 1.5 ml/min. Peaks: l=cGMP, e=cAMP. R e p r i n t e d w i t h p e r m i s s i o n from r e f . 37.

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were h i g h e r i n t h e leukemic plasma t h a n those found i n normal subjects. When t h e d a t a from t h i s s t u d y and a s t u d y o f HPLC p r o f i l e s i n c h r o n i c l y m p h o c y t i c leukemia was t r e a t e d s t a t i s t i c a l l y , i t was found t h a t serum o r plasma chromatographic p r o f i l e s c o u l d be used p r o v i d e d non-descri p t i ve component ( o r t h e n o i s e component) was minimized. Thus, u s i n g t h e combined techniques o f RPLC and m u l t i v a r i a t e d i s c r i m i n a n t a n a l y s i s , a c u t e leukemias and c h r o n i c leukemias were c l a s s i f i e d and separated f r o m t h e normal p o p u l a t i o n ( F i g u r e 4.15) ( r e f . 113). The c h r o n i c leukemia serum samples and c o n t r o l s were c l a s s i f i e d w i t h a s e n s i t i v i t y o f 93.7% and a s p e c i f i c i t y of 87.5% whereas t h e a c u t e l y m p h o c y t i c leukemias and c o n t r o l s were c a t e g o r i z e d w i t h 100% s e n s i t i v i t y and speci f icity.

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Figure 4.12 Separation of platelet nucleotides by isocratic elution. Column, 10 pm Radial-Pak C, (Waters); mobile phase, 0.005 M tetrabutyl ammoni urn phosphate + 90% acetoni tri 1 e. (pH 7.5) ; injection volume, 10 pl; flow-rate, 6 ml/min. Reprinted with permission from ref. 51. Analyses of nucleosides alone are readily achieved by RPLC.

In addition, analyses of nucleosides in physiological fluids other than serum or plasma (refs. 38, 72, 73), cells (ref. 68), and nucleic acid hydrolysates (refs. 114-117) were reported. For the analysis of the purine or pyrimidine bases i n physiological fluids (ref. 118) or i n DNA hydrolysates (refs. 49, 54, 59, 60), ion pair chomatography is commonly used. Micellar liquid chromatography with a PVA column was recently used for the analysis of nucleosides and bases in biological samples (refs. 84, 85). A n example is the iso-serum by micellar l i q u i d chromatography (Figure 3.16) (ref. 84). Although good separation of all the nucleosides and bases present i n

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Figure 4.13 Serum profile of a breast cancer patient w i t h bone metastasis. Injection volume, 80 p l . Other conditions are the same as i n Figure 3.2. Reprinted w i t h permission from ref. 35. serum i s n o t obtained, this method i s useful f o r the determination of concentrations of Urd and Xan; compounds which are d i f f i c u l t t o q u a n t i f y by RPLC methods. To demonstrate the feasibility of using fast-microbore HPLC, the gradient separation of the low molecular weight UV-absorbing constituents i n urine i s shown i n Figure 4.17 (ref. 47). Decreased analysis time, sol vent use, and sample q u a n t i t y requirements were observed.

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Figure 4.14 Com arison of the concentrations (bmol/l) i n the normal and acute yymphocytic leukemia (ALL) groups. 0 , value f o r patients l e s s than 7 years old; NM, normal mean. The mean and standard deviation are reported f o r each g r o u p . In parentheses are the numbers o f samples i n which the com ound could be quant i f i e d . Reprinted with permission from r e f . f12.

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F i g u r e 4.15 C a l c u l a t e d d i s e a s e i n d e x versus p a t i e n t p r o f i l e number f o r A) t h e a c u t e l e u kemic d a t a and (B) t h e c h r o n i c leukemic data. Symbols below t h e d o t t e d i n e r e p r e s e n t t h e normal p o p u l a t i o n whi 1e t h o s e above r e p r e s e n t t h e p a t h o l o g i c a l popul a t i o n . Disease i n d e x i s t h e dependent v a r i a b l e , y, c a l c u l a t e d from t h e peak areas u s i n g t h e f o l l o w i n g equation: n ( X i k - U i ) b i + bo Yk = 1=1 where X i k i s t h e a u t o s c a l e d peak area measurement, U i t h e mean o f measurement i, and b - and bo t h e r e g r e s s i o n c o e f f i c i e n t s o f t h e e q u a t i o n . R e p r i n t e d w i t h p e r m i s s i o n f r o m r e f . 113.

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F i u r e 4.16 I s o c r a t i c e l u t i o n p r o f i l e o f human serum. I n j e c t i o n vofume, 30 ~ 1 . Other c o n d i t i o n s a r e t h e same as i n F i g u r e 4.8. R e p r i n t e d w i t h p e r m i s s i o n from r e f . 84. 4.3.3

Simultaneous A n a l v s i s o f N u c l e o t i d e s . Nucleosides and Bases When i t i s i m p o r t a n t t o analyze a l l o f t h e p u r i n e and pyrimi d i n e m e t a b o l i t e s i n a s i n g l e chromatographic r u n f o r t h e metabol i c s t u d i e s , t h e method o f F l o r i d i e t a ] . can be used ( r e f . 3 2 ) . For example, s e p a r a t i o n s o f n u c l e o t i d e p o o l s from y e a s t and r a t l i v e r were achieved on a s t r o n g porous anion-exchange r e s i n (Aminex A-14) w i t h a l i n e a r g r a d i e n t . However, l o n g a n a l y s i s t i m e (225 rnin) and h i g h o p e r a t i n g temperature (55" C) were r e q u i r e d .

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Figure 4.17 Gradient se aration of urine. Column, 3 p m baseflow-rate, deactivated C , , (Perkin-Eymer) ( 5 cm x 2 . 1 mm I . D . ) . 310 p l / m i n ; injection volume, 0.5 pl. Other conditions are the same as i n Figure 4.3. Reprinted with permission from r e f . 47. Nissinen ( r e f . 33) developed an HPLC method f o r the separation and quantitation of nucleotides, nucleosides and bases i n fibroblasts. The gradient separation was achieved on an anionexchange column (Aminex A-25) in about 160 min. Figure 4.18 shows elution profiles of cell extracts of normal skin f i b r o b l a s t s and of a patient w i t h hypoxanthine phosphori bosyl transferase (HPRT)

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F i g u r e 4.18 S e p a r a t i o n o f a c i d - s o l u b l e p u r i n e and p r i m i d i n e m e t a b o l i t e s i n f i b r o b l a s t s o f (a) normal s u b j e c t and o f fb) HPRTd e f i c i e n t subject. Column, Aminex A-25 (70 cm x 1.8 mm I . D . (Bio-Rad); m o b i l e phase, A) 0.07 M Na,B,O, + 0.045 M NH4C1 i n 2.5 ethanol (pH 9.15), B) 0.01 M Na,B,O, + 0.50 M NH4C1 (pH 8.80), i s o c r a t i c f o r 15 min. w i t h A, then l i n e a r g r a d i e n t ; i n j e c t i o n temperature 65" C; volume, 50 pl; s e n s i t i v i t y , 0.08 a.u.f.s.; f l o w - r a t e , 0.5 ml/min. R e p r i n t e d w i t h p e r m i s s i o n from r e f . 33.

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deficiency (ref. 33). Again, t h i s method r e q u i r e s l o n g a n a l y s i s t i m e (160 min) and h i g h o p e r a t i n g temperature (65" C) and t h e o p t i m i z a t i o n o f one group o f compounds i s u s u a l l y a t t h e expense

C172

of optimization of another group; thus optimization of r e s o l u t i o n f o r a l l the purines and pyrimidines i s d i f f i c u l t t o o b t a i n . A single-run RPLC method f o r the simultaneous a n a l y s i s of nucleotides, nucleosides and bases was developed by Wynants and van Belle ( r e f . 119). The separation was c a r r i e d o u t i n 40 min using a l i n e a r g r a d i e n t on silica-based C,, columns. This method was applied t o d i f f e r e n t c e l l s and tissues. Figure 4.19 shows a chromatogram obtained from a biopsy of the live r of a fed r a t ( r e f . 119). I t was found t h a t concentrations of NADP and Guo i n l i v e r were d i s t i n c t i v e l y higher than i n h e a r t tissue. Mixed-mode chromatography has been appl ied t o t h e s e p a r a t i o n of human e r y t h r o c y t e s by Lang and Rizzi ( r e f . 75). The s e p a r a t i o n was c a r r i e d o u t by an on-line combination of a s i l i c a - b a s e d C,, column and a subsequent anion-exchange column. Figure 4.20 shows mixed-mode HPLC of ul t r a - c e n t r i fuged lyzed human e r y t h r o c y t e s ( r e f . 7 5 ) . High s e p a r a t i o n power and peak c a p a c i t y were observed; however, the poor base l i n e makes q u a n t i t a t i o n d i f f i c u l t . 4.4

FUTURE PROSPECTS

Although HPLC i s the method of choice f o r the s e p a r a t i o n and q u a n t i t a t i o n of nucleotides, nucleosides and bases i n biological samples, increased r e s o l u t i o n , s e l e c t i v i t y , s e n s i t i v i t y , and speed o f a n a l y s i s w i l l be required i n the f u t u r e f o r biomedical s t u d i e s , biotechnology and the c l i n i c a l l a b o r a t o r y . With recent developments i n column technology, the reduction i n column l e n g t h , column diameter and p a r t i c l e s i z e have improved analyses of nuclei c a c i d c o n s t i t u e n t s by reducing a n a l y s i s time, s o l v e n t use and sample requirements. In a d d i t i o n , because o f the commercial avai 1 abi 1 i t y of low p r i c e c a r t r i d g e columns, these columns w i l l be used extens i v e l y i n the c l i n i c a l laboratory. Improvements i n instrumental systems a r e a l s o needed t o accompany t h e changes in column technology. Instruments must be miniaturized t o accomodate t h e small er col umns. T h u s instruments w i t h very low dead volumes a r e required a s well a s modifications in the d e t e c t o r c e l l design i n o r d e r t o i n c r e a s e s e n s i t i v i t y . An i n j e c t i o n system a l s o must be developed t o handle very small

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volumes of samples. In the f u t u r e , improved data-handling systems w i l l be developed t o handle the l a r g e number o f samples made p o s s i b l e by t h e very f a s t a n a l y s e s . S e p a r a t i o n methods will be aided by the development and production of r i g i d polymeric o r carbon packing m a t e r i a l s which a r e c a p a b l e of r a p i d mass t r a n s f e r and l a r g e peak c a p a c i t y and which a r e a l s o s t a b l e i n s t r o n g a c i d and a l k a l i n e s o l u t i o n s . Because o f t h e pH 1 i m i t a t i o n of s i 1 i ca-based packing materi a1 s , r i g i d polymeric o r carbon packing materi a1 s wi 11 have advantages

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Chromatogram o f u l t r a - c e n t r i fuged l y z e d human e r y t h i n a medium s p i k e d w i t h adenosine ( f i n a l concentraColumn, L i C h r o C a r t RP-18 and i n c u b a t e d f o r 5 m i n . mm I . D . ) (Merck) and MicroPak AX-5 30 cm x 4.0 mm ; m o b i l e phase, A) 100% methanol, Bf 0.75 M KH,PO, 2% a c e t o n i t r i l e , s e c i a l l y p r o rammed. n o n - l i n e a r column s w i t c h i n ; fyow-rate, 1.8 ml/min. Peaks: 7=AdO, 10=IMP, Ql=AMP, 15=ADP, 19=ATP. Reprinted w i t { p e r m i s s i o n from r e f . 75. f o r t h e s e l e c t i v e s e p a r a t i o n o f n u c l e i c a c i d components i n b i o l o g ic a l samples. 4.5

SUMMARY High-performance 1i q u i d chromatographic methods f o r t h e s e p a r a t i o n o f nucl e o t i d e s , nucl eosides and bases a r e described. These methods in c l ude ion-exchange chromatography, reversed-phase l i q u i d chromatography, i o n - p a i r chromatography, mixed-mode chroma-

C175

tography, and m i c e l l a r l i q u i d chromatography. I n addition, the a p p l i c a t i o n s o f s e p a r a t i o n s o f n u c l e o t i d e s o r n u c l e o s i d e s and bases, as w e l l as f o r t h e simultaneous s e p a r a t i o n o f n u c l e o t i d e s , nucl eosides and bases in v a r i o u s t y p e s o f b i o l o g i c a l m a t r i ces a r e discussed.

4.6 REFERENCES 1. K . F i n k and W. S . Adams, Urinat-;cgurines and p y r i m i d i n e s i n normal and leukemic s u b j e c t s , Biochem. Biophys., 126

.

1968) 27-33.

2. !l . S . McFarlane and G.J. Shaw, Observed i n c r e a s e i n m e t h y l a t e d p u r i n e s e x c r e t e d by hamsters b e a r i n g adenovirus-12 induced tumors, Can. J. M i c r o b i o l , 14 (1968) 185-187. 3. C.W. Gehrke, K. C. Kuo, T. P. Waalkes and E. Borek, P a t t e r n s o f u r i n a r y e x c r e t i o n o f m o d i f i e d nucleosides, Cancer Res., 39

.

(1979) 1150-1153. 4. D. A. Carson, J. Kaye and J. E. Seegmiller,

Lymphospecific d e f i c i e n c y and p u r i ne n u c l eoPossible r o l e o f nucleoside USA, 74 (1977) 5677-5681. P u r i n o e n i c immunodeficiency molecuyar mechanisms, Ann.

5.

I n t e r n . Med., 92 (1980) 826-831. 6. R. W. E. Watts, P u r i n e enzymes and immune f u n c t i o n , C l i n . Biochem., 16 (1983) 48-53. 7. W. E. Cohn, The s e p a r a t i o n o f p u r i n e and p y r i m i d i n e bases and o f n u c l e o t i d e s b ion-exchange, Science, 109 (1949) 377-378. 8. C. A . B u r t i s , Txe d e t e r m i n a t i o n of t h e base c o m p o s i t i o n o f

9. 10. 11.

12.

RNA by h i h-pressure cation-exchange chromatography, J. Chromatogr., !l (1970) 183-194. R. P. Singhal and W . E. Cohn, A n a l y t i c a l se a r a t i o n of nucl eosides by ani on-exchange chromatography: n f l uence of pH, so!vents, temperature, c o n c e n t r a t i o n , and f l o w r a t e , Anal. Biochem., 45 (1972) 585-599. R. A. H a r t w i c k and P. R. Brown, E v a l u a t i o n o f m i c r o p a r t i c l e chemical l y bonded reversed-phase p a c k i ngs in t h e h i gh-press u r e 11 u i d chromatographic anal s i s o f n u c l e o s i d e s and t h e i r Chromatogr., 126 (1976f 679-691 bases, F. S . Anierson and R. C. Murphy, I s o c r a t i i s e p a r a t i o n o f some u r i n e n u c l e o t i d e , nucleoside, and base m e t a b o l i t e s from E i o l o g i c a l e x t r a c t s by high-performance 1 i q u i d chromatography, J. Chromatogr., 121 (1976) 251-262. N. E. Hoffman and J. C. Liao, Reversed-phase high-performance l i q u i d chromato r a p h i c s e p a r a t i o n s o f n u c l e o t i d e s i n t h e presence o f soyvophobic i o n s , Anal. Chem., 49 (1977) 2231-

P

!

2234. 13. E. J. J u e n g l i n g

and H. Kammermeier, Rapid assay o f adenine n u c l e o t i d e s o r c r e a t i n e compounds i n e x t r a c t s of c a r d i a c t i ssue by p a i r e d - i on reversed-phase h i h- erformance 1 iq u i d chromatography, Anal. Biochem., 102 (1988) $58-361.

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14. J. H. Knox

and J. Jurand, Z w i t t e r i o n - p a i r chromato raph o f n u c l e o t i d e s and r e l a t e d species, J. Chromatogr., 283 (1881)

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-N. K i m and'P. R . Brown, M i c e l l a r l i q u i d chromatography of a i e n o s i n e i n cacao, J . L i q . Chromatogr., ( i n p r e s s ) . Y.-N. K i m and P. R. Brown, manuscri t i n p r e p a r a t i o n . P. R. Brown, R . A. H a r t w i c k and A. Krstulovic, Analysis o f b l o o d n u c l e o t i d e s , nucleosides, and bases b h i g h p r e s s u r e li u i d chromatography, i n : G. C. Hawk (Ed.), giolo ical/Biome!i c a l Appl ic a t i ons o f L i q u i d Chromatogrphy Marcel Dekker, New York, 1979, p. 295-331. E. J. R i t t e r and L. M. Eruce The q u a n t i t a t i v e d e t e r m i n a t i o n o f deox r i bonucleoside tri hhsphates u s i n high-performance l i q u i d c i r o m a t o raphy, BiocRem. Med., 21 (1879) 16-21 B. F l e i s c h e r , !he nucleotide content o f r a t l i v i r golgi v e s i c l e s , Arch. Biochem. Biophys., 212 (1981) 602-610. E. Harmsen, P. Ph. de Tombe and J. W. de Jong, Simultaneous determi n a t i o n o f m o c a r d i a1 adenine n u c l e o t i des and c r e a t i ne phosphate by hi erformance 1 iq u i d chromatography, J. Chromatogr., 230 (!98$) 131-136. K. Morimoto, K. Ta awa, T: Hayakawa, F. Watanabe and H. Mogami, C e l l u l a r leve! o f p u r i n e compounds i n ischemic g e r b i l brain b h i h-performance l i q u i d chromatography, J. Neurochem., 3g (1912b 833-835. R. W. C u r r i e , Sporns and F. H. Wolfe A method f o r t h e a n a l y s i s o f ATP m e t a b o l i t e s i n b e e f s k e l e t a l muscle by HPLC, J . Food S c i , 47 (1982 1226-1228. E. S. Sharps and R. L. Mc a r l , A h i h-performance liquid chromatographic method t o measure i n c o r p o r a t i o n in t o phosphor 1a t e d metabol it e s i n c u l t u r e d c e l l s, Anal. Biochern.,

R.

8,

88. 89. 90.

K-

91. 92. 93.

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.

i

1

124 (198%) 421-424 94. P. D. Reiss, P. F.'Zuurendonk and R. L. Veech, Measurement o f

tissue purine, y r i m i d i n e , and o t h e r n u c l e o t i d e s by r a d i a l compression h i [-performance 1i q u i d chromatography, Anal. Biochem., 140 (!984) 162-171 95. S.A. Standard, P. Vaux and C:M. Bray, High-performance l i q u i d chromatography o f n u c l e o t i d e s and n u c l e o t i de sugars e x t r a c t e d from wheat embryo and v e g e t a b l e seed, J. Chromatogr., 318

(1985) 433-439. 96. R. Me e r and. K. G. Wa ner, D e t e r m i n a t i o n o f n u c l e o t i d e p o o l s

ii

i n p r a n t t i s s u e by h- erformance l i q u i d chromatography, Anal. Biochem., 148 (198i) $69-276 97. C. Leray, P a t t e r n s . o f p u r i n e n k l e o t i d e s i n e r y t h r o c y t e s , Corn Biochem. P h y s i o l , 648 (1979) 77-82. 98. P. Schweinsberg and T. L. Loo, Simultaneous a n a l y s i s o f ATP, ADP, AMP, and o t h e r u r i n e s i n human e r y t h r o c y t e s b y h i h- erformance l i q u i d c romatography, J . Chrornatogr., 181

b..

(18807 103-107.

.

R

C182

H. V. Hersey, R. A. . L e v i n e K. COY and s. O l i v e l l e , Improved method o f r e s o l v i n n u c i e o t i d e s by r e v e r sed-phase h i h- erformance l i q u i d c romatography, J . Chromatogr., 219 Q19il) 133-139 100. H. Martinez-Valdez, R. M. K o t h a r i , H. V. Hersey and M. W . T a y l o r , Rapid and r e l i a b l e method f o r t h e a n a l y s i s o f nucleot i d e p o o l s by reversed-phase h i h- erformance 1i q u i d chromatograph , J. Chromatogr., 247 (1882p 307-314 101. J. K. Cxristman, Separation o f m a j o r and'minor d e o x y r i b o nucl e o s i des monophosphates by reverse-phase h i h-performance l i q u i d chromato raphy: A s i m p l e method a p p l i c a l e t o q u a n t i t a t i o n o f methy a t e d n u c l e o t i d e s i n DNA, Anal. Biochem., 119

99. M. . W . T a y l o r ,

1

38-48. 6.1982) J.

1

9

Kessler, Reyersed- $ase h i g h - p e r f o r m a n c e 1 i q u i d chromatography o f t h e 2 - and - n u c l e o t i d e monophosphates, J. L i q . Chromatogr. 5 (1982) 111-123. High p e r 103. A. M. K r s t u i o v i c , R. A. H a r t w i c k and P: R..Brown, formance 1i u i d chromatographi c determi n a t i o n o f serum UV p r o f i l e s o? normal sub e c t s and p a t i e n t s w i t h b r e a s t cancer and benign f i b r o c y s t i c cianges, C l i n . Chim. Acta, 97 (1979)

102.

!

159-170. 104. S. P. Assenza and P. R. Brown, Com a r i s o n o f high-performance l i q u i d chromato r a p h i c serum r o f i f e s o f humans and dogs, J. Chromatogr., 187 (1980) 169-176 105. A. McBurney and T. Gibson, Reversed-phase p a r t i t i o n HPLC f o r

d e t e r m i n a t i o n o f plasma p u r i n e s and p y r i m i d i n e s i n s u b ' e c t s w i t h gout and r e n a l f a i l u r e , C l i n . Chim. Acta, 102 (19803 19-

28. 106. A. P. Halfpenny and P. R. Brown,

x

Optimized assa f o r purine n u c l e o s i d e phosphorylase by reversed-phase h i - erformance l i q u i d chromatogra hy, J. Chromatogr., 199 (1981) $75-282 R. Brown, High-performance l i q u i d ckyo107. S. P. Assenza and matographic p r o f i l e s o f low-molecular-weight,.U.V.-absorbing coin ounds i n t h e s e r a o f bea l e s exposed t o c i g a r e t t e smoke, Anaf. Chim. Acta, 123 (1981) 33-40 108. M. Zakaria, P. R. Brown, M. P. F a r i e s and B. E. Barker, HPLC a n a l y s i s o f a r o m a t i c acids, nucleosides, and bases i n plasma o f acute lymphocytic leukemics on chemotherapy, C l i n . Chim. Acta, 126 (1982 69-80. 109. R. P. Agarwal, P. Major and D. W . Kufe, Simple and r a p i d high-performance 1 i q u i d chromatographic method f o r a n a l y s i s 231 o f nucleosides i n b i o l o g i c a l f l u i d s , J. Chromatogr.,

F.

b.

L

(1982 418-424. 110. K. Na ano, S.P. Assenza and P.R.

Brown, Reversed-phase l i q u i d 1ow-mol ecul a r chromatographi c in v e s t i gat1 on o f UV-absorbi n wei h t compounds i n s a l i v a , J. Chromatogr., 833 (1982) 51-60 111. J. S a l l i s , S. C. N i c o l , P. Perrone and P. R. Brown, Higk erformance 1 i q u i d chromato r a p h i c p r o f i l e s o f nucleosides, Eases and t r y p t o p h a n i n t h e pyasma o f t h e tasmanian d e v i l and f o u r o t h e r marsupial species, Comp. Biochem. P h y s i o l . , 79B

8.

112.

391-394. A.a 1984 Za I a r i a , P. R. Brown, M. P. Farnes and B. E. n a l y s i s o f aromatic amino a c i d s ! nucleosides,

Barker, HPLC and bases i n plasma of acute l y m p h o c y t i c leukemics on chemotheraphy, C l i n . Chim. Acta, 126 (1982) 69-80.

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113. H. A. Scoble, M. Z a k a r i a and P. R. Brown,

L i u i d chromator a p h i c p r o f i l e c l a s s i f i c a t i o n o f a c u t e an! chronic leu&emi as, Comput. B i omed. Res , 16 (1983) 300-309. 114. G. E. Davis, C. W. Gehrke, K. C. Kuo and P . F. A g r i s , M a j o r and m o d i f i e d nucleosides i n t R N A h y d r o l y s a t e s by high-performance l i q u i d chromatography, J. Chromatogr., 173 (1979)

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281-298. 115. K.C. Kuo, R.A. McCune, C.W.

Gehrke, R. M i d g e t t - a n d M. Ehrich, Q u a n t i t a t i v e reversed-phase h i g h performance 1i q u i d chromatog r a p h i c d e t e r m i n a t i o n o f m a j o r and m o d i f i e d deox r i b o n u c l e o s i d e s i n DNA, N u c l e i c Acids Res., 8 (1980) 4763-4y76 116. C. W. Heizmann, R. Hobi, G. C. W i n k l e r and.C. C. Kuetgle, The e x t r a DNA o f b r a i n c o r t e x neurons i s q u a l i t a t i v e l y i n d i s t i n u i s h a b l e from o t h e r somatic DNA, Exp. C e l l Res., 135 (1981)

!31-339. 117. T . RUSSO!

F. S a l v a t o r e and F . Cimino, D e t e r m i n a t i o n o f s e u d o u r i d i n e i n t R N A and i n a c i d - s o l u b l e t i s s u e e x t r a c t s by i h- erformance l i q u i d chromatography, J. Chromatogr., 296

I:(1884p 387-393

118. W. V o e l t e r , K.’Zech,

P. A r n o l d and G. Ludwig, D e t e r m i n a t i o n o f s e l e c t e d p y r i m i d i n e s , p u r i n e s and the1 r m e t a b o l i t e s i n serum and u r i n e k.! reversed- hase i o n - p a i r chromatography, J. Chromatogr . , 199 1980) 345-954. van B e l l e , S i n g l e - r u n high-performance 119. J. Wynants and l i q u i d chromatography o f n u c l e o t i d e s , n u c l e o s i d e s , and m a j o r p u r i n e bases and i t s a p p l i c a t i o n t o d i f f e r e n t t i s s u e ext r a c t s , Anal. Biochem., 144 (1985) 258-266.

d.

This Page Intentionally Left Blank

C185

CHAPTER 5 ISOLATION AND CHARACTERIZATION OF MODIFIED NUCLEOSIDES FROM HUMAN URINE GIRISH B. CHHEDA, HELEN B. PATRZYC, HENRY A. TWOREK AND SHIB P. DUTTA Department o f Biophysics, Roswell P a r k Memorial Institute, Buffalo, New Y o r k 14263

TABLE OF CONTENTS

5.1 Introduction . . . . . . . . . . . . . . . . . . . . C186 5.2 Materials and Methods for Column Preparation . . . . C188 5.2 1 Charcoal -Cel i te Column Assembly . . . . . . . . C196 5.2 2 Packing of a Charcoal-Celite Column . . . . . . C196 5.2 3 AGl-X8 Formate Column . . . . . . . . . . . . . C198 5.2 4 DEAE Cell ul ose-Borate Col umn . . . . . . . . . C198 5.3 Ins rumentation Techniques and Experimental ProceC198 dures . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 Preparative HPLC Col urnns Assembly . . . . . . . C198 5.3.2 Semi-preparative and Analytical HPLC . . . . . C199 5.3.3 Instrumentation Methods . . . . . . . . . . . . C199 5.3.4 Chemical Derivatization for GC-MS . . . . . . . C200 5.3.5 Separati on o f Standard Mi xture of Nucl eosi des and Bases on DEAE-Cellulose-Borate Column . . . C200 5.3.6 General Procedure for Isolation of the Unknown Urinary Nucl eosides . . . . . . . . . . . . . . C200 5.3.7 Preparative HPLC of 0.7 M Borate Fraction . . . C202 5.4 Results and Discussion of Methods Used for Isolation and Structure Elucidation . . . . . . . . . . . . . . C203 5.4.1 Purification and Chromatography . . . . . . . . C203 5.4.2 Structure Elucidation Methods . . . . . . . . . C207 5.4.3 U1 traviolet Spectrometry . . . . . . . . . . . C208 5.4.4 NMR Spectrophotometry . . . . . . . . . . . . . C209 5.4.5 Mass Spectrometry . . . . . . . . . . . . . . . c211 5.5 Characterization o f a New Hypermodified Nuc eos de 3- (3-Ami no-3-Carboxypropyl ) uri di ne (acp3 U) . . . . . . C213 5.6 Discussion . . . . . . . . . . . . . . . . . . . . . C218 5.7 Summary . . . . . . . . . . . . . . . . . . . . . . . c222

C186

5.8 Future Prospects and Impact . . 5 . 9 References. . . . . . . . . . . . 5.1

. . . . . . . . . . .

..........

C223 C224

INTRODUCTION

Mammalian urine contains more than 500 suostances with molecular weights of l e s s than one thousand. Many of these compounds are derived from metabolism of nucleic acids, proteins, carbohydrates, l i p i d s , steroids, f a t t y acids, vitamins, cofactors and g r o w t h factors ( r e f . 1 ) . Some of these compounds are present i n extremely small amounts and t h e i r levels may vary w i t h the physical condition of the organism. Our laboratory has been engaged i n t h e i s o l a t i o n a n d c h a r a c t e r i z a t i o n of novel nucl eosides and re1 ated substances present i n microgram amounts i n human urine ( r e f . 2 ) . While many of the urinary nucleosides and bases are derived from the turnover or degradation of nucleic acids such as tRNA, rRNA, mRNA and DNA ( r e f . 3 ) , some of the nucleosides are derived from anabolic nucleotide intermediates and cofactors involved i n biosynthesis of nucleic acids a s well as other macromolecules ( r e f s . 4 , 5 ) . Of more t h a n 65 modified nucleosides known t o be present i n mammalian t R N A ( r e f s . 6 , 7 ) 22 have been isolated from human urine (Table 5.1, Fig. 5 . l a r b , c , d ) . All modified nucleosides, except queuosine ( r e f . 8) and inosine ( r e f . 9 ) , are biosynthesized by alteration of the four common nucleosides present w i t h i n the t R N A precursor polynucleotide chains ( r e f . 10). Because there are no specific enzyme systems t o incorporate the modified nucleosides i n t o the macromolecular nucleic acids, these nucleosides once released in the process of t R N A turnover cannot be r e u t i l i z e d , b u t are e i t h e r metabolized or excreted i n t a c t i n urine ( r e f s . 11,12). Much remains t o be learned about the process of turnover of various t R N A species and about the metabolism of the modified nucleosides released from these tRNAs i n man. As stated above, a second group of modified nucleosides and bases t h a t occur i n urine are the metabolites of the i n t e r mediates involved in the d e - n o v o biosynthesis of purine and p y r i m i d i ne nucl eotides and other biochemical mol ecul e s . Of the 1 arge number of nucl eotides t h a t occur in mammal i an c e l l s , several have been found i n human urine as the corresponding nucleosides such

C187

as oroti di ne 1A. 5-ami noimi dazol e-4-carboxami de ri bonucl eosi de

W, xanthosine U, N6-succinyladenosine 4A and few others (Table 5.2, Fig. 5.2 a,b). These nucleosides in general appear to be utilized very efficiently by mammalian cells and thus one does not find a large excess of these compounds i n normal urine. Elevated levels of modified nucleosides i n urine have been useful as markers for assessing the tumor burden and effectiveness of therapy in neoplastic diseases (refs. 13,14) and may ultimately be utilized as markers for other metabolic disorders. Reported herein is the methodology devel oped i n our 1 aboratory for i sol ation and characterization of small amounts of nucl eosides present in 24 hour collections of human urine. Also summarized herein, is the characterization of a recently isolated new nucleoside, 3-(3-amino-3-carboxypropyl)uridine, 14, from human urine (ref. 15). TABLE 5.1 U r i n a r y Nucleosides Derived from tRNAa

Amounts Nanomol esl micromole of creati ni ne

mg/dayb

-1. l-Methyl adenosi ne

1.76-1.77

6.18-6.21

2. N6-Methyl adenosine

2.85-3.99

10-14

16,17

N-[(9-p-D-Ribofuranosyl9H- uri n-6-yl ) carbamoyl] L-tlreoni ne 0.68-0.78

3.5-4.0

18,19

2'-O-Methyladenosine

0.11-0.36

0.39-1.26

20,21

5. N4-Ace ty 1 cyt id ine

0.20

0.7

22

6. 3-Methylcytidine

0.19

0.6

22,23

No.

3.

4.

Compound

7.

2'-O-Methylcytidine

0.06-0.16

0.2-0.5

8.

N2-Dimethylguanosine

1.20-1.44

4.66-5.60

9.

l-Methyl guanosi ne

0.11-0.16

0.4-0.6

10.

N2-Methylguanosine

0.35-0.41

1.30-1.52

Reference 16

16 24,25,26 16 16,25

C188

Table 5.1 Cont'd.

11. 2'-O-Methylguanosine

0.02-0.07

0.08-0.26

19,21

12.

3-Methyl uridine

0.10

0.32

13.

Pseudouri di ne

22.4-26.7

68.3-81.4

uri dine

A.

0.038-0.16

15

Z'-O-Methyluridine

0.19-0.25

0.6-0.8

31

0.09

0.34c

2

17. 5-Carbamoylmethyl uridine 0.05

0.20

32

B.2-Thi o-5-Carbamoylmethyl-

0.006

0.025

32

19. Thymine riboside

N.Q.

N.Q.~

33,34

a.Inosine

0.05

0.18

34,35

21. l-Methylinosine

1.15-1.18

4.05-4.16

22. 2'-0-Methylinosine

N.Q.

N.Q.~

14. 3- (3-Amino-3-carboxypropyl 15.

14. 5-Carboxymethyluri di ne

uri di ne

009-0.037

aThe u s u a l nucleosides, adenosine, guanosine, uridine are not included i n t h i s table.

27 26,28,29, 30

16,25,26 21

cytidine

and

bThe v a l u e s , m g / d a y , r e p r e s e n t t h e amount p r e s e n t i n 24 h o u r c o l l e c t i o n o f n o r m a l a d u l t human u r i n e , u n l e s s s t a t e d o t h e r w i s e . These v a l u e s were i n most cases d e t e r m i n e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r y on p u r i f i e d m a t e r i a l s . Jn few i n s t a n c e s , f o o t n o t e s h a v e been added t o i n d i c a t e t h e s p e c i f i c c o n d i t i o n u n d e r w h i c h a p a r t i c u l a r compound c a n b e f o u n d . Range v a l u e s I t must b e r e p r e s e n t v a r i a t i o n s f o u n d among d i f f e r e n t s a m p l e s . n o t e d t h a t t h e r e a r e m i n o r v a r i a t i o n s e v e n among n o r m a l s u b j e c t s , t h u s a t t h i s p o i n t t h e v a l u e s mg/day s h o u l d n o t be c o n s i d e r e d absolute. Amounts l i s t e d i n T a b l e 5 . 1 h a v e b e e n t a k e n f r o m a number o f s t u d i e s , i n c l u d i n g o u r own. CAmount p r e s e n t i n l u n g c a r c i n o m a u r i n e . dN.Q.

,

not quantitated.

MATERIALS AND METHODS FOR COLUMN PREPARATION Neutral charcoal (Norit) is purchased from Fisher Scientific Co. Celite 545 is obtained from Johns-Manville Co., DEAE cellulose (DE-23) and AGl-X8 formate (200-400 mesh) anion5.2

1. 1 - Methyladenosine

2. N6- Methylodenosine

3. N-U9-B-D- Ribofuronosyl -9H- purin-6-yl)carbamoyl 3 -L- threonine

- 2-0-Methyladenosine 4.

Fig. 5.l(a).

5. -

N*-Acetylcytidine

Urinary nucleosides derived from t R N A .

6.

3-Methylcytidine

7 2'-0-Methylcytidine

&.

N2- Dimethylguanosine

CH3-NH HO OH

OH

9. I - Methylguanosine -

Fig. 5 . l ( b ) .

OH OH

10. - N2 - Methylquanosine

U r i n a r y nucleosides derived from tRNA.

OH

11.

0-CH,

2'-0-Methylguanosine

0

0

I

""53

OH

OH OH

OH OH

12. 3-Methyluridine

"od ""aHod I

1 3 Pseudouridine

OH

OH

~.3-(3-Amino-3-carboxypropyl)uridine

0 01 5 C H 2-COOH

H

o

d

OH OH

16. -

OH

5-Corborymrthyluridine

Fig. 5.l(c).

OH

17. 5-Corbomoylmrthyluridinr

Urinary nucleosides d e r i v e d from tRNA.

OCH3

15. 2'-0- Methyluridine 0

""0 OH

OH OH

OH

--19.Thymine riboside

20. __

21.

lnosine

I-Methylinosine

""0 OH

OCH,

22. 2'-0-Methylinosine

Fig. 5 . l ( d ) .

Urinary n u c l e o s i d e s d e r i v e d from tRNA.

""33 OH

OH OH

2A. 5-Methyl-2'-

1A. Orotidine -

deoxycytidine

OH OH

OH

3A. 2'- Deoxycytidine

w. N%uccinylodenosine

fp CH -COOH I CHZ -CH,-S OH

OH

5A. S-Adenosylmethionine

Fig. 5.2(a).

OH OH

6A. S-Adenoaylhomocyrteine

Other u r i n a r y nucleosides.

OH OH

?A. 5'- Deoxy-5'-methylthio-

-

adenosine sulfoxide

OH OH I-B-D-Ribofuranosylhypoxanthine

8A.

-

6'

0 II

C- NH2

I H OH

o

OH

OH

9A. 5'- Deoxyinosine

d OH

10A. 5 -Aminoimidarole- 4- carboxamide

OH

OH

E.N'-B-D-Ribofuranosyl pylridin4- one-3- carboxamide

ribonucleoside

0

0 H

o

Hod

d

OH OH

OH OH

12A. Nl-8-D-RibofuranosyIPyridin2- one-5-carboxamide Fig.

5.m). Other urinary

13A. Xanthosine -

nucleosides.

&I OH

14A. 5'-Deoxyxanthosine

C195

TABLE 5.2

Other Urinary Nucl eosides Amounts

No.

Compound

nanomol es/ micromole of creatinine

mg/dayb

Reference

1A. Orotidine

1.42

5. la

36

2A. 5-Me t hy 1 -2 ' -deoxycy t i d i ne

N .Q .

<.01a

37

3A. 2 ' -Deoxy cy t i d i n e

N.Q.

<.01a

37,38

0.21-0.38

1-1.8

4

5A. S-Adenosylmethioni ne

4.79

23.8a

39

M. S-Adenosylhornocysteine

4.79

23. Oa

39

0.036

0. 14a

5,40

8A. 7-fl-D-Ri bofuranosyl hypoxanthi ne

0.12

0.4a

41

9A. 5'-Deoxyinosine

0.35

1. la

5

10A. 5-Ami noimi dazol e-4-carboxami de ri bonucl eoside N.Q.

0.91

16

2-2.5

42

4A.

7A.

11A. -

N6 -Succi nyl adenosine

5'-Deoxy-5'-methyl thioadenosine sul foxi de

N1 -8-D-Ri bofuranosyl pyridi n-4one-3-carboxamide 0.59-0.74

12A. Nl -8-D-Ri bofurano0.87-1.05 13A. Xanthosine

0.13

14A. 5'-Deoxyxanthosine aThese amounts w e r e f o u n d i n t h e specific conditions (see reference). bSee f o o t n o t e b t o T a b l e 5 . 1 .

cN.Q.,

not quantitated.

urines

2.943.54

43

0.45

44

N.Q.c

45

of

subjects

under

C196

exchange resin, a r e obtained from Whatman and BioRad Labs., res p e c t i v e l y . Deuterium oxide (99.96 atom % D) and DMSO-d, (99.5% atom D) a r e purchased from Aldrich Chemical Co. and Merck Isotopes, respectively. Glass d i s t i l l e d a c e t o n i t r i l e and methanol a r e obtained from Burdick & Jackson. Deionized d i s t i l l e d water f o r use i n HPLC i s prepared i n our l a b o r a t o r y . 5 . 2 . 1 Charcoal-Celite Column Assembly. The column used f o r packing the c h a r c o a l - c e l i t e m i x t u r e i s c o n s t r u c t e d from a Fi s c h e r - P o r t e r q u a r t z heavy wall ed gl a s s p i p e with solv-seal j o i n t s , purchased from Lab Crest, Warminster, PA. A column w i t h an i . d . of 3 . 8 cm and a l e n g t h of 50 cm i s adequate f o r processing a 24 hour u r i n e c o l l e c t i o n . The bottom j o i n t of the column i s g l a s s blown and adapted with a t e f l o n stopcock (Fig. 5 . 3 a ) . The t o p of t h e column i s f i t t e d with a s o l v - s e a l j o i n t and modified t o accommodate two stopcocks. The connections a r e secured with a TFE s e a l , Viton O-rings and a compression clamp. A modified heavy duty r e a g e n t b o t t l e s e r v e s a s a r e s e r v o i r . The stopcock a t the t o p of the column is connected by polyethylene tubing t o a r e s e r v o i r . The t o p o f the r e s e r v o i r i s adapted t o connect t o a n i t r o g e n tank o r t o a pump t o p r e s s u r i z e the system f o r s u f f i c i e n t flow r a t e ( 5 ml/min). The system i s made e n t i r e l y from heavy walled g l a s s and t e f l o n components. 5.2.2 Packina of a Charcoal-Celite Column. To 50 g of charcoal and 50 g Celite-545 i s added 75 ml of d i s t i l l e d deionized water and mixed t o a uniform c o n s i s t e n c y of f r e e flowing powder. A one inch l a y e r of g l a s s wool i s placed i n the bottom of the column, and small p o r t i o n s o f c h a r c o a l - c e l i t e mixture a r e packed by means of a plunger (Fig. 5.3b) i n t o the g l a s s column ( 3 . 8 x 50 cm long) t o g i v e l a y e r s of approximately 2.5 cm i n h e i g h t . The t e f l o n plunger head (3.6 x 7.2 cm) contoured t o f i t snugly i n t o the column, i s mounted on a 90 cm long a1 umi num hand1 e. This packi ng procedure provides a col umn of even d e n s i t y and avoids “channeling” of the e l u e n t . In o r d e r t o ensure proper charcoal bed and uniform flow of the column, two l a y e r s (5 cm) of c e l i t e a r e added a t both bottom and t o p o f the charcoal c e l i t e col umn.

2 4 HOUR URlNE COLLECTION

I

b

Adj. t o pH 3

CHARCOAL-CELITE COLUMN

1

1) Column washed w i t h water, then

2 ) E l u t e d w i t h 2N NH,OH

H 30cm

2 N NH,OH-ELUATE

i n 50% ethanol

CONCENTRATED TO 5 0 ML

I

APPLIED TO AG1-XB-FDRMATE COLUMN

I

90cm

Washed w i t h w a t e r ; water wash evaporated t o dryness and r e s i d u e d i s s o l v e d i n 50 ml 0.15 M b o r i c a c i d

DEAE CELLULOSE-BORATE COLUMN

1

1) 0.15 M b o r i c a c i d 2 ) 0.70 M b o r i c a c i d

0.7 M BORIC ACID (NUCLEOSIOE FRACTION) EVAPORATED TO DRYNESS

t

RESIDUE SEPARATED BY PREPARATIVE HPLC

1

PLUNGER

0.1 M NH,OAC

GRADIENT

FRACTIONS PURIFIED FURTHER BY SEMI-PREPARATIVE AND ANALYTICAL HPLC

F i g . 5.3 (a)

. Charcoal -cel it e col umn assembly.(b). Plunger.

Fig. 5.4.

Flow diagram of nucleoside isolation procedure.

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5.2.3 AGl-X8 formate column. Analytical grade A G l - X 8 formate anion-exchange r e s i n , 150 g (200-400 mesh), i s hydrated i n 1.0 N HCOOH f o r one hour a t 22' and then washed with water (-2 l i t e r s ) u n t i l the pH of the wash i s 5.0. The neutral s l u r r y i s packed i n t o a column (3.5 cm x 35 cm) using 5-10 l b s / i n 2 of p r e s s u r e and then washed w i t h 300 ml water before use. A column o f t h i s s i z e i s adequate f o r processing the material obtained from the 2 N e t h a n o l i c ammonia wash of the 24 hour u r i n e from the c h a r c o a l - c e l i t e column. 5.2.4 DEAE c e l l u l o s e - b o r a t e column. F i f t y grams of f i b r o u s DEAE c e l l u l o s e (DE-23) i s s t i r r e d i n t o 500 ml of 0.5 N HC1 and allowed t o hydrate a t room temperature f o r 60 m i n . The s u p e r n a t a n t i s decanted and the c e l l u l o s e i s f i l t e r e d and washed with water on a funnel u n t i l the washings a r e a t pH 4.0. The c e l l u l o s e i s then s t i r r e d i n t o 750 m l of 0.5 N NaOH f o r 30 min a t room temperature. Decanting and water washing steps a r e repeated u n t i l the s u p e r n a t a n t i s a t pH 7. The DEAE c e l l u l o s e i s then allowed t o e q u i l i b r a t e i n 750 ml o f 0.7 M b o r i c acid f o r 16 hours and then washed w i t h water t o n e u t r a l i t y . The c e l l u l o s e i s suspended i n 0.15 M b o r i c a c i d and washed u n t i l the d e s i r e d pH o f the b u f f e r i s a t t a i n e d (pH 4 . 0 ) . The c e l l u l o s e i s then packed i n t o a column (3.5 cm x 50 cm), which under 5 l b s / i n 2 pressure provides a height o f about 30 cm. The packed column i s e q u i l i b r a t e d with 1000 ml of 0.15 M b o r i c acid a t 5 l b s / i n 2 pressure before use. This method of DEAE c e l l ul ose-borate i s p a t t e r n e d a f t e r the procedure of Pi ke and Rottman ( r e f . 46). The material c o n t a i n i n g n e u t r a l and b a s i c components e l u t e d i n the water wash from the A G l - X 8 formate, anion-exchange r e s i n column i s then a p p l i e d t o t h i s column. 5.3

INSTRUMENTATION TECHNIQUES AND EXPERIMENTAL PROCEDURES

5.3.1 PreParative HPLC Columns Assemblv. A reversed-phase Zorbax (Dupont) ODS-C18 column (21.2 mm I.D. x 25 cm length, 8 pm p a r t i c l e s i z e ) was used f o r p r e p a r a t i v e HPLC. [Reversed-phase Dynamax ODS C18 p r e p a r a t i v e column (Rainin), 21.4 mm i . d . x 25 cm l e n g t h , 8 pm p a r t i c l e s i z e , can be s u b s t i t u t e d f o r t h e Zorbax.] Other components o f the p r e p a r a t i v e RP-HPLC system include: Rainin Rabbit HPX h i g h pressure pump;

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Altex UV d e t e c t o r , Model 153, with a p r e p a r a t i v e flow c e l l s e t a t wavelength 254 nm; Rheodyne sample i n j e c t o r f i t t e d with a 5 ml loop, Model 7010; and g r a d i e n t mixing b o t t l e s . 5.3.2 Semi-DreDarative and Analvtical HPLC. Analytical HPLC i s c a r r i e d o u t on an Altex Model 332 g r a d i e n t 1 i q u i d chromatograph, equipped w i t h an A1 t e x model 420 system c o n t r o l l e r programmer, an Altex model 153 a n a l y t i c a l U . V . d e t e c t o r with a 8 pl flow c e l l s e t a t wavelength 254 nm; twin Altex l l O A pumps, Rheodyne sample i n j e c t o r model 7010 and a C-R1A A1 t e x i n t e g r a t o r . An ODs-C18 5 pm a n a l y t i c a l U 1 t r a s p h e r e (Beckman) (0.46 cm I.D. x 25 cm l e n g t h ) column f i t t e d with a 250 p1 loop i n j e c t o r i s used. Reversed-phase U1 t r a s p h e r e (ODs C18) semi-preparative column, used i n o u r s t u d y i s Beckman (1.0 cm I.D. x 25 cm l e n g t h , 5 pm p a r t i c l e s i z e ) f i t t e d with a 2 ml loop. 5 . 3 . 3 Instrumentation Methods. U l t r a v i o l e t s p e c t r a a r e r e c o r d e d on a Cary 219 spectrophotometer which i s zeroed w i t h water using t h e a u t o b a s e l i n e f e a t u r e . Generally the s p e c t r a a r e determined w i t h a scan r a t e of 1 and absorbance range of 0.2 a s f u l l s c a l e . NMR s p e c t r a a r e determined on a Bruker WP200 (200 MHz) spectrometer by u t i 1 i z i n g the Fourier-transform/ q u a d r a t u r e phase d e t e c t i o n mode. Sample temperatures were maintained a t 30°C with the BVT-2000 temperature c o n t r o l l e r of the WP-200. The chemical s h i f t s ( 6 ) were measured i n ppm from i n t e r n a l TSP (sodium 3 - t r i m e t h y l s i lylpropionate-2,2,3,3-d4). The u r i n a r y unknown nucleosides i n the amount of 2-5 A,,, units a r e lyophilized three times from 99.5% D20 and then d i s s o l v e d i n 99.9% D,O f o r d e t e r mining the NMR s p e c t r a . Low r e s o l u t i o n mass s p e c t r a a r e acquired using a Finnigan 4000 GC-MS system with i o n i z i n g energy o f 70 eV, and an ion s o u r c e temperature o f 280°C. The samples ( 1 p g ) i n 1 pl of water o r methanol a r e introduced by d i r e c t probe a f t e r removal of s o l v e n t s e i t h e r i n t h e probe vacuum lock o r o u t s i d e . The probe i s heated b a l l i s t i c a l l y t o a temperature of 300°C and the d a t a a r e processed by an Incos d a t a system. Exact mass v a l u e s a r e determined on a high r e s o l u t i o n instrument such a s Varian MAT 731 o r Finnigan MAT-90 by peak matching ( r e s o l u t i o n 10,000) with per-

c200

fluoroal kane as internal standard. 5.3.4 Chemical derivatization for GC-MS. The unknown polar nucleosidic materials or synthetic standards (0.4-0.8 p g or 0.02-0.04 A t b O units of vacuum dried material) are derivatized by heating i n a mixture o f bis (trimethyl s i lyl) tri fl uoroacetamide (BSTFA) containing 1% trimethylchlorosilane and anhydrous pyridine (l:l, 5 p1) in a sealed melting point capillary tube at 100' for 1.5 hr (ref. 47). The sample is then introduced in the injector end (splitless injection) of a fused silica capillary column (3% SE-30, 30 m long, film thickness, 25 pm), directly interfaced to the mass spectrometer. All mass spectral information was obtained at an ionizing voltage of 70 eV. 5.3.5 SeDaration of standard mixture of nucleosides and bases on DEAE-cel 1 ul ose-borate col umn A 40 ml mixture containing 40 A,,, units of each of the following free bases, Z'-O-modified nucleosides and other nucleosides was prepared: Adenine, 2'-0-methylguanosine, 2'deoxyi nosine, hypoxanthi ne, xanthine, thymine, thymidi ne, uraci 1 , uric acid, 2'-0-methyluridine, adenosine, N 6 - ( A 2 -isopentenyl) adenosine, cytidine, guanosine, uridine, pseudouridine and inosine. The mixture was applied to a DEAE cellulose column prepared and equilibrated with 0.15 M boric acid as described above. The column was eluted first with 1300 ml o f 0.15 M boric acid, followed by 1800 ml of 0.7 M boric acid and then by 800 ml of 1.0 N formic acid. One hundred ml fractions of borate effluent were monitored by UV spectra, and then evaporated to dryness by addition of methanol as described previously (ref. 46). The formic acid eluate was evaporated to dryness separately. The identification of the compounds i n each eluate was carried out by comparison of HPLC mobilities and U V spectra of the fractions with the individual standards. The compounds were eluted in the following order in each eluant. 5.3.6 General Drocedu re for isolation of the unknown urinarv nucl eosi des (Fi a. 5.41 . Urine from a patient or a normal subject is collected for 24 hours in a glass j u g containing 50 ml of toluene. Before pro-

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TABLE 5.3

Compounds E l u t e d i n Order o f I n c r e a s i n g Volume ~

0.15 M b o r i c a c i d a;IAl

v o l ume

1. Thymine

0.7 M b o r i c a c i d [;h&aAl

v o l ume

1.0 M f o r m i c a c i d total ml)

Boo

volume

9 . Xanthine

16. Guanosine

2. U r a c i l

10. C y t i d i n e

17. U r i c a c i d

3. Thymidine

11. Pseudouri d i ne

4. 2'-O-Methyl u r i d i ne

12. N6 -(A? -isopenteny1)adenosine

5. 2'-Deoxyinosine

13. U r i d i n e

6. 2'-0-Methylguanosi ne

14. Adenosine

7. Adenine

~~

15. I n o s i n e

8. Hvooxanthine

c e s s i n g t h e u r i n e , t h e t o l u e n e l a y e r i s separated and t h e u r i n e (1200-1400 m l ) i s a d j u s t e d t o pH 3, a l l o w e d t o s t a n d a t 4'C f o r 2 hours and f i l t e r e d . The f i l t e r e d u r i n e i s passed t h r o u g h a column (3.8 x 50 cm) o f c h a r c o a l - c e l i t e (50 g each) prepared as d e s c r i b e d above and processed s t e p w i s e as o u t l i n e d i n F i g . 5.4. The column i s washed w i t h water (-3 l i t e r s ) u n t i l t h e washings a r e c h l o r i d e - f r e e , then t h e column-bound m a t e r i a l i s e l u t e d w i t h (The ammonia 2 N NH40H i n 50% aqueous ethanol (-3 l i t e r s ) . e l u a t e c o n t a i n s about 60% o f t h e A 2 6 0 u n i t s o r i g i n a l l y a p p l i e d t o t h e column.) The e l u a t e which g e n e r a l l y c o n t a i n s 5-6 g o f r e s i due i s c o n c e n t r a t e d t o a small volume (50 m l ) and a p p l i e d t o a column (3.5 cm x 35 cm) o f 150 g o f AGl-X8 f o r m a t e r e s i n prepared as d e s c r i b e d above. The column i s e l u t e d w i t h about 2.5 l i t e r s o f w a t e r a t a f l o w r a t e o f 5 ml/min; t h e UV absorbance f a l l s below 0.2 a t 260 nm a t t h i s p o i n t . The n e u t r a l and b a s i c components e l u t e d w i t h water a r e evaporated t o near dryness f o r t h e [The a c i d i c components chromatography on DEAE c e l l u l o s e - b o r a t e . r e t a i n e d on t h e column a r e removed by e l u t i o n w i t h 3.0 N f o r m i c

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acid.] The water wash r e s i d u e (20,000 A 2 6 0 u n i t s ) i s d i s s o l v e d i n 50 m of 0.15 M b o r i c a c i d and then a p p l i e d t o a column of DEAE ce l u l o s e - b o r a t e e q u i l i b r a t e d with 0.15 M b o r i c a c i d a s described above. The column i s then e l u t e d with 1500 ml 0.15 M b o r i c a c i d a t 2 ml/min flow r a t e and monitored a t 260 nm. When t h e UV absorbance of t h e e l u a t e f a l l s below 0 . 3 A,,, u n i t s per ml, the column i s e l u t e d with 0.7 M b o r i c a c i d (-1700 m l ) . The p r o f i l e of t h i s e l u t i o n i s shown i n Fig. 5 . 5 . The column i s then washed with 1.0 N formic a c i d t o remove the remaining m a t e r i a l . Both 0.15 M and 0.7 M b o r a t e e f f l u e n t s a r e f l a s h evaporated s e p a r a t e l y t o near dryness by repeated a d d i t i o n of methanol and then f r a c t i o n a t e d by HPLC. 5.3.7 P r e u a r a t i v e HPLC of 0.7 M b o r a t e f r a c t i o n . The 0.7 M b o r a t e ( c i s - d i o l ) f r a c t i o n eluted from the DEAE c e l l u l o s e - b o r a t e column (-2500 A 2 6 0 u n i t s ) i s d i s s o l v e d i n 15 ml water and c e n t r i f u g e d a t 3500 rpm f o r 20 min. The s u p e r n a t a n t i s f r a c t i o n a t e d on a Zorbax p r e p a r a t i v e HPLC column i n three equal p o r t i o n s . The e l u t i o n i s c a r r i e d o u t by a 0 . 1 M ammonium a c e t a t e b u f f e r (pH 6.8) i n methanol g r a d i e n t of 0-25% i n 60 min with a flow r a t e of 8 ml/min a t 22°C monitored a t 254 nm. A 100% methanol wash removes t h e remaining col umn-bound m a t e r i a l . A t o t a l of a s many a s 35 peaks may be c o l l e c t e d from the g r a d i e n t e l u t i o n (Fig. 5.6) and f r a c t i o n s with a d e s i r e d r e t e n t i o n time a r e pooled from t h e t h r e e i n j e c t i o n s . Each f r a c t i o n i s concent r a t e d t o 2 ml and p u r i f i e d on an U l t r a s p h e r e (semi-preparative) ODS C,, reversed-phase column. The c o n d i t i o n s used f o r d e s a l t i n g and p u r i f i c a t i o n a r e predetermined on an a n a l y t i c a l column f o r each f r a c t i o n . For example, c o n d i t i o n s e s t a b l i s h e d f o r d e s a l t i n g and p u r i f i c a t i o n of 5’-deoxyinosine SA a r e : e l u t i o n w i t h water f o r 30 min followed by a 0-25% water-methanol g r a d i e n t f o r 1 hour with a flow r a t e of 4 ml/min ( r e f . 5 ) . The n u c l e o s i d e acp3U 14 was p u r i f i e d i s o c r a t i c a l l y with water on t h e U l t r a s p h e r e column ( r e f . 15). The p u r i f i e d peaks, i f homogenous, a r e then utilized f o r structural elucidation studies.

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RESULTS AND DISCUSSION OF METHODS USED FOR ISOLATION AND STRUCTURE ELUCIDATION. 5.4 1 Purification and Chromatoaraphv On an average, a 24 hour urine collection (1200-1400 m l ) from a 70 kg human adult contains about 50-60 g of solid residue, of which a b o u t 30 g i s urea, 12 g i s sodium chloride, 6 g other electrolytes a n d the remai nder i s composed of creati nine, uric acid, amino acids, purines, pyrimidines, nucleosides, pigments, proteins, sugars, steroids and other substances ( r e f . 1). In order t o i s o l a t e microgram quantities of nucleosides and other re1 ated heterocycl i c substances, the 24 hour col 1 ection of urine needs t o be desalted and cleaned up. On a small scale (5 t o 20 m l urine), removal of s a l t s can be achieved through Sephadex G-25 or Sephadex LH-20; however, these methods when scaled up f o r a 24 hr urine collection, become t o o cumbersome t o use. In our laboratory desalting and clean-up i s achieved r e l a t i v e l y e a s i l y and inexpensively on a charcoal-celite column. A majority of the nucl eosides and other heterocycl i c materi a1 s bind t o charcoal . These are then eluted off the column by 2 N ammonium hydroxide in 50% ethanol. In s p i t e of some losses (20%) of standard UVabsorbing materi a1 s on charcoal , t h i s method of uri ne cl ean-up a n d desalting i s quite effective and reproducible. Celite i s added t o charcoal t o provide proper coarseness f o r packing and t o maintain an adequate flow r a t e . The desalted material obtained from 24 hour urine (5-6 g ) i s passed t h r o u gh a column of anion-exchange resin, AGl-X8 formate. This retains the acidic substances such a s organic acids, f a t t y acids, uric acid and some amino acids a n d phenolic compounds, while neutral and basic compounds including many of the purine and pyrimidine nucleosides pass through the column. About 40% of the total material applied t o the column i s eluted in the water wash. The ribosides and other material contained in the aqueous wash are fractionated further on a column of DEAE cellulose impregnated with boric acid ( r e f . 46) ( F i g . 5.5). The electropositive diethylaminoethyl moiety of DEAE cellulose forms a ligand with boric acid which i n t e r a c t s with planar cis-diol components leading t o the formation of borate complexes ( r e f s . 48,49). T h u s , the cis-diol compounds are bound t o the modified 5.4

c)

N

0 P

4.0

0.4

2oc

Volume (mL)

Fig. 5.5.

F r a c t i o n a t i o n of water wash from A G l - X 8 formate column on DE-23 (DEAE-cellulose) b o r a t e column ( s i z e , 3.5 cm x 35 cm) e l u t e d with 0.15 M H 3 B O 3 , 0.7 M H3BO3 and 1 N HCOOH, flow r a t e 2 ml/min; temperature 22oC.

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c e l l u l o s e m a t r i x a t 0.15 M b o r i c a c i d , while a s t r o n g e r b o r i c a c i d s o l u t i o n (0.7 M) a p p a r e n t l y d i s r u p t s t h i s i n t e r a c t i o n leading t o the r e l e a s e of c i s - d i o l s , such a s s u g a r s and n u c l e o s i d e s from t h e DEAE c e l l ulose-borate m a t r i x . To t e s t the a b i l i t y of t h e DEAE c e l l u l o s e impregnated w i t h b o r i c a c i d , a s t a n d a r d mixture c o n t a i n i n g 6 common bases and 11 nucleosides was chromatographed on t h i s column ( s e e Methods section). All compounds behaved p r e d i c t a b l y except f o r u r i c a c i d , xanthine and guanosine. Xanthine i s e l u t e d i n t h e nucleoside f r a c t i o n , i . e . , i n the f i r s t 400 ml o f the 0.7 M H,BO, f r a c t i o n r a t h e r than i n t h e 0.15 M b o r i c a c i d f r a c t i o n . We have on occasion observed a few hydroxypurine bases such a s 6,8dihydroxypurine ( r e f . 5 0 ) , e l u t i n g i n the 0.7 M H,BO, f r a c t i o n . The a c i d i c n a t u r e and i n s o l u b i l i t y of u r i c a c i d and guanosine r e q u i r e d formic a c i d f o r t h e i r e l u t i o n . A t y p i c a l e l u t i o n p a t t e r n o f the DEAE c e l l u l o s e - b o r a t e column f o r the d e s a l t e d and cleaned-up u r i n e i s shown i n Fig. 5.5. A t y p i c a l chromatogram of t h e 0.7 M H,BO, eluate obltained from normal human u r i n e and f r a c t i o n a t e d on a p r e p a r a t i v e HPLC column i s shown i n Fig. 5.6 and a number of peaks a r e l a b e l e d with the n u c l e o s i d e s which have unequivocally been i d e n t i f i e d i n t h o s e peaks. I t should be noted t h a t none of the peaks i s o l a t e d from the p r e p a r a t i v e HPLC column a r e homogenous. P u r i f i c a t i o n of these compounds t o homogeneity o f t e n n e c e s s i t a t e s a t l e a s t two a d d i t i o n a l passes through a semi-preparative o r a n a l y t i c a l column, using d i f f e r e n t s o l v e n t s o r i n o r g a n i c b u f f e r systems. Complete id e n t i f i c a t i o n i s achieved through UV , NMR and G U M S comparison with an a u t h e n t i c s t a n d a r d . C o - i n j e c t i o n s of the p u r i f i e d u r i n a r y m a t e r i a l with a known s t a n d a r d on an a n a l y t i c a l HPLC with s e v e r a l b u f f e r systems i s u t i l i z e d t o e s t a b l i s h the i d e n t i t y of t h e u r i n a r y m e t a b o l i t e . Often the n u c l e o s i d e l a b e l e d on the peaks i s one o f t h e t h r e e t o f o u r compounds p r e s e n t i n t h a t peak, e.g. peaks c o n t a i n i n g H X 7 R 8A ( r e f . 4 1 ) , dms50A 7A ( r e f s . 5,40) and 5'-deoxyinosine ( r e f . 5) were a l l p u r i f i e d f u r t h e r b e f o r e i d e n t i f i c a t i o n was attempted. From the p r o f i l e i t i s obvious t h a t while 19 p r e v i o u s l y c h a r a c t e r i z e d n u c l e o s i d e s and r e l a t e d compounds have been i d e n t i f i e d i n t h i s u r i n a r y f r a c t i o n , a t l e a s t twenty more remain t o be c h a r a c t e r i z e d . In the u r i n e of

c)

N

-*

0

-

Q1

m$G

4-PCNR

I

ncm'~, acp3U

m'G, dI

1

I

I

10

F i g . 5.6.

I

I

i0Time ( M i d

20 30 40 50 RP-HPLC of 0.7 M H3BO3 f r a c t i o n from a normal human u r i n e . Column Zorbax ODS C,, pm) 21.2 mm x 25 cm. G r a d i e n t 0 - 25% methanol i n 0 . 1 M ammonium a c e t a t e b u f f e r o v e r 60 min, pH 6.8, f l o w r a t e 8 ml/min; AUFS 0.64; temperature 22°C.

(8

A b b r e v i a t i o n s used f o r t h e comoounds i n f i a . 5.6 are: Pseudouridine ($), n e o p t e r i n (Np), 5-carboxymethyl u r i d i ne cm5 u , 3- 3-ami no-3-carboxypropyl ) u r i d i ne ( acp3 U) , 5c y t id i ne carbamoylm%il u r i d i ne (ncm5 U) 5-ami nd-l:(,!!-D-ri kofuranos 1 ) - i m i dazol e-4-carboxami de ( A l C A R ) b i o t e r i n (Bp) , 5-carbamoylmethyl-2-thiouridine(ncm5SzU~ i n o s i n e (I) 7-8-D-ribofuranos y l y p o x a n t h i ne (Hx7 R) , N1 -fl-D-ri b o f u r a n o s y l p y r i d i n-4-one-5-carboxamide [4-PCNR), 3-methylu r i d i n e (m3U), l - m e t h y l i n o s i n e (ma1 I),adenosine (A), 5 ' - d e o x y i n o s i n e ( d I ) , l-methylguanosine (ml G), 5'-deoxy-5-methyl t h i o a d e n o s i n e s u l f o x i d e (dms50A) N2-dimethylguanosine (mZG),

I:

A d d i t i o n a l a b b r e v i a t i o n s used are: "(9-8-D-ri ( t 6 A ) N6-A2-isopentenyladenosine ( I P A ) .

bofuranosyl-9H-purin-6yl)carbamoyl]-L-threonine,

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c a n c e r p a t i e n t s , nucl e o s i d i c drugs o r re1 a t e d compounds administered t o the p a t i e n t s may be e x c r e t e d and e l u t e d with the unknown r i b o s i d e compounds. Drugs such a s a c y c l o v i r and o t h e r nucleoside analogs o r t h e i r m e t a b o l i t e s can i n t e r f e r e and deserve a t t e n t i o n during q u a l i t a t i v e o r q u a n t i t a t i v e s t u d i e s . Most of t h e unknowns were i s o l a t e d i n 5.0 A,,, units or less. Exhaustive s t u d y of t h e 0.15 M H,BO, f r a c t i o n (non-cis-diol p o r t i o n ) using HPLC p u r i f i c a t i o n followed by GC-MS i s underway i n our l a b o r a t o r y . In a d d i t i o n t o t h e unusual bases such as a number of modified u r a c i l d e r i v a t i v e s ( r e f s . 51,52) t h i s f r a c t i o n a l s o c o n t a i n s 2'-O-methylnucleosides ( r e f s . 2 1 , 3 1 ) , 2'deoxynucleosides and some sugar and base modified n u c l e o s i d e s . The a c i d i c n u c l e o s i d e s such a s N-[ (9-B-D-ri bofuranosyl-9H-puri n-6yl)carbamoyl]-L-threonine, 3, ( P A ) (18) and N6-succinyl adenos i n e 4A ( r e f . 4) a r e e l u t e d i n the 3 N HCOOH wash from the A G l - X 8 formate col umn. 5.4.2 S t r u c t u r e Elucidation Methods. Because of the avai 1 abi 1 i t y of a 1 imi ted q u a n t i t y (50-500 pg) of t h e u r i n a r y n u c l e o s i d i c unknowns, i d e n t i f i c a t i o n i s g e n e r a l l y c a r r i e d o u t by physicochemical methods such a s UV, NMR and mass spectrometry. I t i s very important t h a t the m a t e r i a l be p u r i f i e d t o homogeneity before any c h a r a c t e r i z a t i o n i s attempted by these methods. I t i s a l s o important t o be aware of the t y p e s of i m p u r i t i e s t h a t could e x i s t i n a s o - c a l l e d p u r i f i e d nucleos i d i c sample, s i n c e t h i s information could be h e l p f u l i n i n t e r p r e t i n g the UV, NMR and mass s p e c t r a l d a t a . Although i n i t i a l p u r i f i c a t i o n i s u s u a l l y achieved by low p r e s s u r e column chromatography, t h i n 1 ayer chromatography o r paper chromatography, u l t i m a t e l y t h e substances have t o be p u r i f i e d by HPLC t o remove remaining t r a c e s of impuri t i e s . The materi a1 s a r e g e n e r a l l y s u b j e c t e d f i r s t t o the non-destructive techniques of UV and NMR spectrometry and t h e compounds a r e o f t e n recovered a f t e r these procedures. Mass spectrometry, a1 though a d e s t r u c t i v e technique, i s a l s o c a r r i e d o u t i n the i n i t i a l s t a g e s of ident i f i c a t i o n s i n c e i t consumes only a microgram o r l e s s of the material. Subsequently, the unknowns a r e s u b j e c t e d t o proper chemical r e a c t i o n s o r microchemical t e s t s depending upon t h e a v a i l a b i l i t y of the m a t e r i a l and upon the information o b t a i n e d from UV, NMR and mass s p e c t r a l d a t a .

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5.4.3 Ultraviolet SoectroPhotometrv. After comparing t h e u l t r a v i o l e t s p e c t r a of the homogeneous substances w i t h the spectra of the known U V absorbing compounds present i n urine, the urinary substances a r e assigned the s t a t u s o f "Unknowns". In instances of s i m i l a r i t y , the unknowns are cochromatographed with the known substances t o confirm o r exclude their identity. Whereas the amount needed f o r UV spectra by conventional instrumentation i s about 0.1 A 2 6 0 u n i t s (2 pg), diode array detectors, however, can provide good spectra on 0.005 A,,, u n i t s (100 ng) of the material. In the case of nucleosides, protonation of the heteroaromati c bases in aqueous aci di c media or deprotonati on in a1 kal i ne media produces c a t i oni c and ani oni c species, respectively. These species cause c h a r a c t e r i s t i c s h i f t s in the U V absorption maxima and minima often providing informat i o n about the e l e c t r o n i c s t r u c t u r e of the compound. Even t h o u g h t h e r e a r e no simple r u l e s or c o r r e l a t i o n s f o r multisubstituted heteroaromatic nucleosides t o predict accurately the hmax of the nucleoside, nonetheless, the U V spectrophotometry provides useful and complementary information on an u n k n o w n when proper model compounds a r e available. By comparing t h e UV spectrum o f the pure unknown compound w i t h t h a t of the model nucleoside compound, i t i s frequently possible t o obtain information about the main chromophore as well as the position of s u b s t i t u t i o n i n the chromophore. For example, uridine e x h i b i t s h m a x a t 262 nm a t pH 11, while thyrnidine a t t h a t pH absorbs a t 265 nm which i s c h a r a c t e r i s t i c of 5-substituted uridines. By inspection of the UV spectral p r o f i l e of two recently i s o l a t e d urinary unknowns, 5carboxymethyluridine E (Xmax 266, pH 11 ( r e f . 2) and 5carbamoylrnethyluridine &! ( r e f . 32) (Xmax 265, pH ll), i t was p o s s i b l e t o s u g g e s t t h a t these were uridine d e r i v a t i v e s substituted a t the 5-posi tion of u r a c i l . The UV spectrum of a model compound N1-methylpyridin-4-one-3-carboxamide was useful in suggesting the s t r u c t u r e of the Nl-b-D-ri bofuranosyl pyridin-4one-3-carboxamide U. Similarly by comparing t h e spectra o f 7methylhypoxanthine (hmax 262, pH 11) the ribose moiety in H x ~ R , 8A ( r e f . 41) was assigned a t the 7-position of hypoxanthine r a t h e r t h a n the usual 9 position i n the nucleosides.

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S u b s t i t u t i o n o f 0 - a l k y l groups i n p l a c e o f hydrogen i n t h e r i b o s e m o i e t y does n o t cause s i g n i f i c a n t s h i f t s i n t h e s p e c t r a . The s i m i l a r i t y o f UV s p e c t r a o f t h e 2 ' - 0 - m e t h y l u r i d i n e Is t o those o f u r i d i n e was u s e f u l i n p r o v i d i n g a l e a d f o r t h e s t r u c t u r e of t h e former n u c l e o s i d e ( r e f . 31). The UV s p e c t r a o f t h e new n u c l e o s i d e 5'-deoxy-5'-methyl t h i o a d e n o s i n e s u l f o x i d e ( max 259, a t n e u t r a l pH) were s i m i l a r t o t h o s e o f adenosine ( r e f s . 5,40) s u g g e s t i n g t h a t t h i s n u c l e o s i d e was v e r y s i m i l a r t o adenosine and may be s u b s t i t u t e d i n t h e r i b o s e p o r t i o n o f t h e molecule. S i m i l a r l y i n t h e i d e n t i f i c a t i o n o f 5 ' - d e o x y i n o s i n e UV s p e c t r a l d a t a i n d i c a t e d t h a t t h e u r i n a r y compound was perhaps an i n o s i n e d e r i v a t i v e ( r e f . 5 ) . T h i s i n f o r m a t i o n on t h e p o s i t i o n o f s u b s t i t u t i o n cannot be e a s i l y o b t a i n e d by mass s p e c t r o m e t r y . Thus, UV spectrophotometry serves as a v e r y u s e f u l and complementary t o o l i n r e s o l v i n g t h e s t r u c t u r e s o f unknown n u c l e o s i d e s . However, i t should be noted t h a t t h e t y p e o f sugar, i t s a b s o l u t e c o n f i g u r a t i o n , t h e c o n f i g u r a t i o n o f t h e sugar base l i n k a g e and s u b s t i t u e n t s on sugars need t o be i n v e s t i g a t e d by NMR o r by o t h e r techniques,

a,

5.4.4 NMR suectrometrv. NMR s p e c t r o m e t r y u s i n g ' H ( r e f . 53) and I 3 C ( r e f . 54) n u c l e i has served as a v e r s a t i l e and complementary t e c h n i q u e f o r s t r u c t u r a l e l u c i d a t i o n o f a number o f s i m p l e as w e l l as complex m o d i f i e d n u c l e o s i d e s ( r e f . 55). P r o t o n magnetic resonance (PMR) s p e c t r a o f a l a r g e number o f common h e t e r o c y c l i c bases, nucleos i d e s and n u c l e o t i d e s have been s t u d i e d ( r e f . 53) and b y measuri n g t h e chemical s h i f t s and c o u p l i n g c o n s t a n t s (J v a l u e s ) , t h e p o s i t i o n and t h e n a t u r e o f t h e m o d i f i c a t i o n s i n t h e unknown modi f i e d n u c l e o s i d e can be determined. By comparing t h e PMR specw i t h t h e spectrum trum determined i n a s o l v e n t l i k e DMSO-d, determined i n t h e presence o f D,O, one can i d e n t i f y t h e exchanga b l e p r o t o n s i n t h e groups such as OH, SH, NH2, and COOH i n t h e molecule. From a s t u d y o f t h e PMR spectrum a f t e r d e u t e r a t i o n a t H-8 o f m o d i f i e d p u r i n e bases, one can c o r r e c t l y a s s i g n t h e p o s i t i o n o f H-2 and H-8 p r o t o n s i n t h e NMR spectrum o f some o f t h e p u r i n e n u c l e o s i d e s ( r e f s . 56,57). I t i s often possible t o a s s i g n t h e anomeric c o n f i g u r a t i o n i n t h e unknown n u c l e o s i d e on t h e b a s i s o f t h e chemical s h i f t and c o u p l i n g c o n s t a n t ( J , , , , , ) o f

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t h e C - 1 ' p r o t o n ( r e f . 58). Furthermore, by i n t e g r a t i n g t h e area under each peak i n t h e PMR spectrum i t i s o f t e n p o s s i b l e t o det e r m i n e t h e number o f each t y p e o f p r o t o n w i t h r e s p e c t t o t h e s i n g l e p r o t o n resonance used as a s t a n d a r d i n t h e molecule. I n t h e study on m o d i f i e d n u c l e o s i d e s i s o l a t e d from human u r i n e , we have used PMR spectrometry r a t h e r e x t e n s i v e l y f o r s t r u c t u r a l assignments. For example, i n 5 - c a r b o x y m e t h y l u r i d i n e 16 t h e absence o f a p r o t o n a t t h e C-5 p o s i t i o n o f t h e u r a c i l m o i e t y a l o n g w i t h a s i n g l e t peak f o r t h e C-6 p r o t o n a t 7.73 pprn suggested t h e p o s i t i o n o f t h e carboxymethyl s i d e c h a i n a t t h e 5p o s i t i o n o f u r a c i l ( r e f . 2). Comparison o f t h e PMR spectrum o f 1 6 w i t h t h a t o f u r i d i n e i n d i c a t e d t h a t t h e r i b o s e i n 16 was n o t s u b s t i t u t e d ( r e f . 2). I n t h e PMR spectrum o f N1-p-Dribofuranosylpyridin-4-one-3-carboxamide U, t h e presence o f t h r e e a r o m a t i c p r o t o n s ( d o u b l e t a t 9.33, a p a i r o f d o u b l e t s a t 8.62 and 8.54 and another d o u b l e t a t 7.24 ppm) suggested t h a t t h i s compound was a p y r i d i n e d e r i v a t i v e ( r e f . 42). From t h e s t u d y o f t h e s p l i t t i n g p a t t e r n ( J Z , 6 = 3 Hz, J6,2 = 3 Hz, J6,5 = 8 Hz and J5,6 = 8 H z ) , i t was p o s s i b l e t o a s s i g n a s t r u c t u r e o f 3 , 4 - d i s u b s t i t u t e d p y r i d i n e r i b o s i d e U t o t h i s compound ( r e f . 42). I n t h e case o f 7-p-D-ribofuranosylhypoxanthine the presence o f two s i n g l e t s a t 8.22 and 8.54 ppm i n t h e a r o m a t i c r e g i o n o f t h e NMR spectrum, i n d i c a t e d a p u r i n e m o i e t y such as hypoxanthine ( r e f . 41). For t h e sugar m o d i f i e d nucleoside, 2'-0m e t h y l u r i d i n e fi, t h e presence o f two d o u b l e t s a t 8.38 and 6.38 ppm and o t h e r resonances i n t h e NMR spectrum i n d i c a t e d t h e base p o r t i o n o f t h e n u c l e o s i d e t o be an u n s u b s t i t u t e d u r a c i l ( r e f . 31). I n many o f t h e m o d i f i e d u r i n a r y n u c l e o s i d e s c h a r a c t e r i z e d by us, t h e anomeric c o n f i g u r a t i o n was i n i t i a l l y assigned on t h e b a s i s o f NMR s p e c t r a and f i n a l l y c o n f i r m e d by comparison o f t h e s p e c t r a l d a t a w i t h those o f t h e a u t h e n t i c m a t e r i a l . The 1 3 C NMR s p e c t r a o f n a t u r a l l y o c c u r r i n g p u r i n e and p y r i m i d i n e nucleosides p r o v i d e excel l e n t s e p a r a t i o n between t h e peaks observed f o r t h e carbon atoms o f t h e sugar and aglycon ( r e f . 59). The use o f 1 3 C NMR spectrometry, i n i t i a l l y hampered by v e r y low n a t u r a l abundance (1.1%) o f 1 3 C , i s now a p p l i c a b l e through t h e use o f i n t e r n u c l e a r double resonance, and through t h e s i g n a l enhancement by t h e use o f t h e n u c l e a r Overhauser e f f e c t

a,

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and by the use of the computer averaged transients (ref. 54). The 2D NMR spectra using COSY and NOESY methods of analysis can be of help in establishing conformations of these molecules (ref. 60). 5.4.5 Mass soectrometrv.

The key feature of mass spectrometry, in contrast to other analytical techniques, is that it provides vital and complementary information such as the molecular weight, fragmentation pattern and elemental composition on a microgram of the unknown biological material. With the advent of soft ionization techniques such as fast atom bombardment (FAB) (refs. 61,62), thermospray LC-MS (ref. 63), and with the availability of MS/MS (ref. 64), mass spectrometry in the past few years has reached a state of unprecedented versati 1 i ty in sol vi ng compl i cated structural and analytical problems deal ing with compounds of biological interest. Mass Spectrometry either alone, or in conjunction with GC, is one of the most commonly used techniques for the detection, characterization and quantitation of modified nucl eosi des present in bi ol ogi cal matrices . McCloskey and his associates have made a major contribution in the mass spectrometry of common nucleosides (ref. 65) and i n the structure elucidation of complex modified nucleosides such as Q isolated from tRNA (ref. 5 5 ) . Mass spectrometry of nucleic acid components giving interpretation of the mass spectra of some of the modified bases and nucleosides has been reviewed by Hignite (ref. 66). One of the most important pieces of information needed for the structure elucidation of any unknown compound is its molecular weight and molecular formula. For a hitherto unknown modified nucleoside, this information usually is provided through low and high resolution mass spectrometry. This can be obtained directly from the mass spectra of the material as such or after suitable derivatization. The two most common techniques of derivatization used in the area of nucleosides are trimethylsilylation (ref. 47) and permethylation (ref. 67). The trimethyl si lyl ati on procedure described by McCl oskey and his associates (ref. 47) generally provides volatile and heat stable derivatives in good yield. Through trimethylsilylation of nucleosides with deuterated reagents, it is possible to estimate

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t h e number o f a c t i v e hydrogens i n t h e m o l e c u l e and peak assignments a r e o f t e n c l a r i f i e d . Knowledge o f t h e number o f s i l i c o n atoms i n t h e d e r i v a t i z e d molecules a l l o w s t h e r e d u c t i o n o f t h e number o f p o s s i b l e m o l e c u l a r formul as d e r i v e d from t h e computer data. Most o f t h e e a r l i e r s t r u c t u r a l s t u d i e s on n u c l e o s i d e s were conducted a f t e r d e r i v a t i z a t i o n , a c o n s t r a i n t imposed by t h e p o l a r i t y and 1ow v o l a t i 1 it y o f many n u c l e o s i des. T h i s , however, can now be circumvented w i t h t h e use o f t h e FAB i o n i z a t i o n o f t h e p o l a r n u c l e o s i d e s ( r e f s . 68,69). Since t h i s method i s reproduci b l e and r e l a t i v e l y easy t o adapt, an i n c r e a s i n g number o f s t u d i e s i n c l u d i n g DNA adducts a r e b e i n g conducted t h r o u g h t h i s and A t times during the r e l a t e d i o n i z a t i o n procedures ( r e f . 70). process o f d e r i v a t i z a t i o n , unforeseen changes such as r e a r r a n g e ments and degradation c o u l d occur, w h i l e w i t h FAB, one i s i o n i z i n g t h e substance i t s e l f a t -35"C, thus p r e v e n t i n g t h i s p o s s i b l e c o m p l i c a t i o n . D e r i v a t i z a t i o n o f n u c l e o s i d e s f o l l o w e d by GC-MS a n a l y s i s , however, p r o v i d e s on-1 i n e p u r i f i c a t i o n o f t h e nucleosides, l e a d i n g t o t h e s p e c t r a f r e e o f t h e i o n s due t o i m p u r i t i e s ( r e f . 71). I n addition, electron i o n i z a t i o n o f d e r i v a t i z e d nucleosides g e n e r a l l y p r o v i d e s mass s p e c t r a having g r e a t e r s t r u c t u r a l d e t a i l than i n t h e case o f s p e c t r a f r o m FAB. The f o l l o w i n g examples o f s t r u c t u r a l s t u d i e s on m o d i f i e d u r i n a r y nucleosides i11u s t r a t e t h e u t i 1it y o f mass s p e c t r o m e t r y i n t h e s t r u c t u r e e l u c i d a t i o n s t u d i e s . The mass spectrum of a TMS d e r i v a t i v e o f t h e n u c l e o s i d e N l - f l - D - r i b o f u r a n o s y l pyridin-4-one-3carboxamide 11A i n d i c a t e d a m o l e c u l a r w e i g h t o f 558 ( r e f . 42). s i l y l group r e s u l t e d i n a An experiment u s i n g l a b e l e d Si(CD,), m o l e c u l a r w e i g h t o f 594, i n d i c a t i n g a mass s h i f t o f 36 amu. These s t u d i e s i n d i c a t e d t h e presence o f f o u r TMS groups i n t h e d e r i v a t i z e d molecule t h u s e s t a b l i s h i n g t h e m o l e c u l a r w e i g h t o f t h e f r e e n u c l e o s i d e as 270. A s t r u c t u r a l e l u c i d a t i o n s t u d y on 5 ' - d e o x y i n o s i n e $lA u s i n g mass spectrometry gave a m o l e c u l a r w e i g h t o f 252 and i o n s i n d i c a t i n g t h e presence o f unmodified hypoxanthine and a deoxyribose i n t h e molecule ( r e f . 5 ) . T h i s i n f o r m a t i o n l e d t o a comparison of o t h e r deoxyribofuranosylhypoxanthines w i t h t h e unknown i n terms o f t h e i r s p e c t r o s c o p i c and chromatographic prop e r t i e s . From these data, i t was p o s s i b l e t o a r r i v e a t t h e iden-

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t i f i c a t i o n of the compound as 5'-deoxyinosine N. Similarly, f o r 7-p-D-ri bofuranosyl hypoxanthine 8A (M? 268), comparison o f i t s mass spectrum w i t h t h a t of inosine indicated t h a t the two compounds were very similar b u t n o t identical ( r e f . 4 1 ) . The molecular weights were identical as was the general fragmentation pattern, b u t the r e l a t i v e i n t e n s i t i e s of a number of peaks were d i f f e r e n t , suggesting p o s s i b l y a d i f f e r e n t p o i n t of attachment between the sugar and the hypoxanthine moiety in the unknown ( r e f . 41). The UV spectral data suggested the compound t o be 7isomer of inosine, which was confirmed by comparison w i t h the For 2'-0-methyluridine Is, also, the mass authentic sample. spectrum provided the molecular i o n (m/z 258) and revealed the presence of a uracil moiety through the ions a t m/z 112, 113 and 2'-0-methyl-ribose (m/z 147) i n the molecule ( r e f . 31). With the help o f UV, NMR and mass spectra, i t i s often possible t o suggest a tentative structure f o r an unknown nucleoside. The final step i n the structure elucidation process i s the confirmation of the t e n t a t i v e structure of the unknown by comparing i t s physi cochemi cal properties and chromatographic mobilities w i t h those of the authentic material. To i l l u s t r a t e these techniques and the approach used i n structure elucidation of microgram amounts of nucleosides, discussed below i s the characterization of a recently isolated new nucleoside, 3-(3ami no-3-carboxypropyl ) uri di ne 14 (acp3 U) from human uri ne ( r e f . 15). 5.5 CHARACTERIZATION OF A NEW HYPERMODIFIED NUCLEOSIDE 3-(3AMINO-3-CARBOXYPROPYL) URIDINE (acp3 U) This material, designated as nucleoside X, was isolated from a 24 hour collection of urine by the procedures described in the methods section in this chapter. I t was further fractionated from the 0.7 M borate fraction by HPLC ( F i g . 5 . 6 ) . The peak a t the retention time in the range of 18 minutes was purified f u r t h e r t o s e p a r a t e t h i s compound from another modified nucleoside, ncm5U ( r e f . 32) t o give 160 p g of acp3U in a 24 hour urine sample. The u l t r a v i o l e t spectra of the purified urinary nucleoside X exhibited maxima a t 263 nm in neutral, acidic and a1 kal ine media

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(Table 5.4) with almost no change i n the molecular e x t i n c t i o n c o e f f i c i e n t s . These d a t a were s u g g e s t i v e o f a u r a c i l n u c l e o s i d e s u b s t i t u t e d a t the 3 - p o s i t i o n . These maxima coupled w i t h t h e s i m i l a r molecular e x t i n c t i o n c o e f f i c i e n t s a t d i f f e r e n t pH's further suggested t h a t t h e r e were no i o n i z a b l e p r o t o n s on t h e TABLE 5.4 Corn a r i s o n o f the U l t r a v i o l e t Absorption S p e c t r a o f New Urinary Nucyeoside X and Authentic acp3U

Urinary nucleoside X

acp3~ authentic

PH

?I max

Xmin

1.0

263

235

2.56

6.0

263

233

2.56

11.0

263

236

2.50

1.0

263

235

2.56

6.0

263

235

2.56

11.0

263

235

2.56

60 lA2 80

u r a c i l nitrogen and t h a t t h e compound may be an N-3 s u b s t i t u t e d uridine. Retention time of t h e u r i n a r y n u c l e o s i d e X on RP-HPLC and d i f f e r e n c e s i n N M R r e s o n a n c e s c l e a r l y e x c l u d e d 3methyluridine a s a s t r u c t u r e f o r this unknown. I n v e s t i g a t i o n of o t h e r known modified nucleosides ( r e f . 72) suggested t h a t t h i s u r i n a r y substance could be the o t h e r 3-substi tuted u r i d i n e , 3-(3ami no-3-carboxypropyl ) uri d i ne ( acp3 U) o c c u r r i ng i n t R N A ( r e f . 73). The NMR spectrum of the unknown n u c l e o s i d e i n D20 revealed a p a i r of d o u b l e t s centered a t 7.89 and 5.97 ppm s u g g e s t i n g t h a t these could be assigned t o the C6-H and C,-H protons o f the u r i d i n e moiety (Table 5 . 5 ) . A doublet c e n t e r e d a t 5.94 ppm and mult i p l e peaks i n the 3.6-4.5 ppm region were c o n s i s t e n t with t h e s p l i t t i n g p a t t e r n s o f the anomeric proton and the o t h e r r i b o s e protons r e s p e c t i v e l y . In a d d i t i o n , the unresolved peaks c e n t e r e d

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a t 2.25 ppm c o u l d be assigned t o CH, o f >CH-CH,-CH, side chain a t t a c h e d t o a p y r i m i d i n e moiety. The NMR spectrum o f a u t h e n t i c acp3U i n D,O r e v e a l e d 3 s e t s o f d o u b l e t s a t 7.89, 5.98 and 5.95 ppm and a m u l t i p l e t c e n t e r e d a t 2.23 ppm (Table 5.5).. These resonances agreed w e l l w i t h those p r e s e n t i n t h e NMR spectrum o f the u r i n a r y nucleoside X. Mass s p e c t r o m e t r y s t u d i e s gave t h e m o l e c u l a r w e i g h t as w e l l as f u r t h e r i n s i g h t i n t o t h e s t r u c t u r e o f t h e unknown u r i n a r y n u c l e o s i d e X . The TMS d e r i v a t i v e o f t h i s u r i n a r y n u c l e o s i d e gave a m o l e c u l a r i o n a t m/z 633 w i t h an M-15 i o n a t m/z 618 ( F i g u r e 5.7a). Based on t h e mass s p e c t r a o b t a i n e d by McCloskey and h i s a s s o c i a t e s ( r e f . 73) on 3-(3-amino-3-carboxypropyl)uridine, it appeared t h a t t h i s was a spectrum o f t h e t e t r a t r i m e t h y l s i l y l d e r i v a t i v e (TMS), o f acp3U. The measured e x a c t mass o f t h e M-15 i o n was 618.2543 ( c a l c d . 618.2519) s u g g e s t i n g a m o l e c u l a r compos i t i on o f C,, H, N, 0, Si, f o r t h e s i l y l a t e d compound and C, H, N, 0, f o r the f r e e nucleoside. The i o n s a t masses 516 and 461 a r e unique t o t h i s u r i n a r y n u c l e o s i d e and a r i s e from t h e m o d i f i e d base, w h i l e t h e i o n s a t m/z 349, 259, 243 and 217 correspond t o t h e unmodified r i b o s e ( r e f s . 71,47). High r e s o l u t i o n mass measurement o f t h e i o n a t 516.2378 showed t h a t i t r e p r e s e n t e d t h e c h a r a c t e r i s t i c l o s s o f C0,TMS ( c a l c d . 516.2381) which f u r t h e r corresponding s u p p o r t s t h e m o l e c u l a r composition o f C, 3 H, 9N30,, t o a m o l e c u l a r w e i g h t o f 345 f o r t h e u n d e r i v a t i z e d n u c l e o s i d e . The mass spectrum o f t h e t e t r a TMS d e r i v a t i v e o f t h e a u t h e n t i c sample were i d e n t i c a l t o t h a t o f t h e u r i n a r y n u c l e o s i d e ( F i g u r e 5.7b). I n a d d i t i o n t o t h e t e t r a TMS d e r i v a t i v e , a penta TMS d e r i v a t i v e was a l s o formed f r o m b o t h t h e unknown X and t h e s y n t h e t i c acp3U. The l a t t e r d e r i v a t i v e gave a m o l e c u l a r i o n a t m/z 705 and M-15 i o n a t m/z 690. The r e t e n t i o n times on RP-HPLC o f t h i s n a t u r a l l y - o c c u r r i n g n u c l e o s i d e and t h a t o f t h e a u t h e n t i c acp3U were v e r y s i m i l a r i n f o u r s o l v e n t systems as shown i n Table 5.6. On c o i n j e c t i o n w i t h a u t h e n t i c acp3U, t h e m i x t u r e e l u t e d as a s i n g l e d i s c r e t e homogenous peak, t h u s f u r t h e r s u p p o r t i n g t h e i d e n t i t y o f t h e unknown. Complete agreement i n UV, NMR and GC/MS s p e c t r a l d a t a and HPLC and GC r e t e n t i o n times w i t h those o f t h e a u t h e n t i c acp3U u n e q u i v o c a l l y i d e n t i f i e d t h e unknown u r i n a r y n u c l e o s i d e as 3-(3ami no-3-carboxypropyl ) u r i d i ne ( acp3 U)

,

,

.

618.2543

d K

Fig. 5.7.

Mass spectra o f trimethylsilyl derivatives (TMS,) o f a)new urinary nucleoside X, b)authentic acp3U 14.

TABLE 5 . 5

Chemical Shifts ( ) and J Values (Hz) for Urinary Nucleoside X Compared with Authentic acp3U in D,O

Urinary nucleoside ( X )

7.89 (8.0)

5.97 (8.1)

5.94(4.3)

2.25 (m)

Authentic acp3U

7.89 (8.1)

5.98(8.0)

5.95 (4.2)

2.23 (m)

(m)

=

mu1 t i p l e t

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TABLE 5.6

Corn arison o f RP-HPLC Retention Time (Min) Nucyeoside and the Authentic acp3U

of

the

Urinary

Sol vents*

Uri nary Nucl eosi de ( X )

9.15

19.40

11.83

11.81

Authentic acp3U Mixture of X and authentic acp3U

9.07

19.74

12.06

11.78

9.28

19.36

12.01

11.76

*The following RP-HPLC solvent systems w e r e used for coinjection s t u d i e s : A ) i s o c r a t i c , 5% m e t h a n o l i n w a t e r , f l o w r a t e 3 m l / m i n ; B) i s o c r a t i c , 0.1 M a m m o n i u m a c e t a t e b u f f e r , p H 5.3, f l o w r a t e , 3 m l / m i n ; C) i s o c r a t i c , 0.1 M a m m o n i u m a c e t a t e b u f f e r , p H 3.8, flow r a t e , 3 m l / m i n ; D ) g r a d i e n t e l u t i o n w i t h 0-15% m e t h a n o l in w a t e r in 15 m i n , f l o w r a t e , 3 ml/min.

DISCUSSION While the majority of the modified nucleosides i n urine l i s t ed i n Table 5 . 1 are derived from transfer ribonucleic acids, some compounds such as 2'-O-methylnucleosides, pseudouridine, and N6-methyladenosine may well be contributed i n part, from the turnover of ribosomal and messenger RNA ( r e f . 74). The modified nucleosides such as t 6 A are exclusively present i n t R N A , and since these nucl eosi des are made a t a post-transcri p t i onal 1 eve1 i t can be concluded t h at urinary t 6 A 3 originates from turnover of human t R N A (re fs . 75,12). Adenosine and guanosine as well as the corresponding deoxynucl eosi des are converted t o uric acid; however, the methylated adenosines and methylated guanosines do not appear t o metabolize t o methylated uric acids. Some of the modified nucleosides of t R N A may not be detected i n urine because major portions are being metabolized t o several different compounds and 5.6

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the unchanged material excreted in urine therefore decreases to microgram amounts. Nucleosides such as Y have not been detected in urine because their levels after metabolism may be very low, whi 1 e nucl eoside Q or its metabol i tes may be primarily reuti 1 ized and not be excreted in urine in detectable amounts. For example, studies on the metabolism of N6-(A2-isopentenyl) adenosine (IPA) indicate that major portions of this compound are catabolized to non-UV absorbing materials, while less than 5% is excreted as UVabsorbing metabolites (ref. 76). Our studies on t6A, another anticodon-adjacent modified nucleoside of tRNA, indicate that 72% of this nucleoside is excreted unchanged in urine (ref. 77). This explains the failures to detect some nucleosides such as IPA in human urine as compared to the easy detectability of t6A in all urine samples. Our studies on the metabolic fate of 1methyl inosine in rat indicate that this anticodon-adjacent nucleoside is partially catabolized to l-methylhypoxanthine while 27% is excreted as the unchanged nucleoside (ref. 78). Similar studies on l-methyladenosine 1 in rat indicate that 55% of this nucleoside is excreted unchanged, while 32% is converted to 1methylhypoxanthine and 7% is metabolized t o l-methylinosine (ref. 79). From these studies it is suggested that l-methyl adenosine does not contribute significantly to urinary N6 -methyl adenosi ne 2 through i n v i v o rearrange-ment (ref. 79). It is clear that one cannot easily predict the metabolic fate of the modified nucleosides with different structures. Each of these modified nucl eosides undergoes catabol ism to a varying degree before being excreted i n urine. Fourteen other urinary nucleosides listed in Table 5.2 are derived from sources other than RNA. Small amounts of 5-methyl2'-deoxycytidine (ref. 37), Z'-deoxycytidine 3A and 2'deoxyuridine (ref. 38) present in urine are derived from DNA. Urinary thymidine glycols produced from DNA damage and repair have been correlated to the aging process (ref. 80). Nucleosides such as orotidine 1A (ref. 36), N6-succinyladenosine 4A (ref. 4), 5-ami noimidazole-4-carboxami de ri bonucl eosi de 10A (ref. 16) and xanthosine 13A (ref. 44) are derived from the intermediates that occur in the d e n o v o biosynthesis of purine and pyrimidine nucl eoti des. Incl uded in this group are the nucl eosi des derived

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from NAD and NADP c o f a c t o r s such as p y r i d i n e d e r i v a t i v e N1 -8-Dr i b o f uranosyl p y r i d i n-4-one-3-carboxami de, 11A ( r e f . 42) and it s 2 - o n e - i somer , N1 - 8 - D - r i b o f u r a n o s y l p y r i d i n-2-one-5-carboxami de, 12A ( r e f . 43). Recently t h e presence o f f o u r unusual n u c l e o s i d e s i n human u r i n e has been r e p o r t e d . These a r e 5 ' - d e o x y i n o s i n e 9 ( r e f . 5), 5'-deoxyxanthosine 14A ( r e f . 45) , 5'-deoxy-5'-methyl t h i o a d e n o s i n e s u l f o x i d e ZA ( r e f s . 5,40) and 7-B-D-ri b o f u r a n o s y l hypoxanthine 8A ( r e f . 41). I n terms o f o r i g i n , these n u c l e o s i d e s pose a challenge, s i n c e t h e y a r e n o t known t o occur as such i n any b i o l o g i c a l systems. One p o s s i b l e source o f 5 ' - d e o x y i n o s i n e i n mammal s appears t o be 5 '-deoxyadenosi ne which is 1ib e r a t e d from coenzyme-vi tami n B, through non-enzymati c r e d u c t i ve cleavage o f t h e 1a b i 1e cobal t-carbon bond. R e c e n t l y i t has been r e c o g n i z e d t h a t 5'-deoxyadenosine, which i s n o t deaminated by adenosine deaminase, i s an e x c e l l e n t s u b s t r a t e f o r t h e enzyme 5'-deoxy-5'methyl t h i o a d e n o s i n e (MTA) phosphorylase ( r e f . 81). When cleaved by MTA phosphorylase, 5'-deoxyadenosine g i v e s r i s e t o adenine and 5'-deoxyribose-l-phosphate. The l a t t e r i n t h e presence o f hypox a n t h i ne and p u r i n e n u c l eoside phosphoryl ase coul d g i v e r i se t o 5 ' - d e o x y i n o s i n e as has been found i n c u l t u r e d c e l l s ( r e f . 82). Schram and h i s a s s o c i a t e s a r e i n v e s t i g a t i n g t h e o r i g i n o f 5'-deoxyxanthosine ( r e f . 45) i n u r i n e . The n u c l e o s i d e , 7 - 8 - D - r i b o f u r a n o s y l hypoxanthine 8A i s i s o m e r i c w i t h i n o s i n e 20 and l i k e 5 ' - d e o x y i n o s i n e 9 has n o t been d e t e c t e d i n normal u r i n e . T h i s new m a t e r i a l c o u l d a r i s e from t h e c o n t a i n i n g 7-8-Dmetabolism o f an analog o f v i t a m i n B,,, r i bofuranosyl hypoxanthine. Since CML p a t i e n t s a r e known t o have because o f t h e abnormal metabolism ( r e f . 83) o f v i t a m i n B,, e l e v a t e d l e v e l s o f t r a n s c o b a l ami ne-I b i n d i n g p r o t e i n s i n t h e i r plasma, i t appears t h a t t h i s substance may be p r e s e n t o n l y i n p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e disease. I t i s n o t unreasonable t o s p e c u l a t e t h a t t h e abnormal metabolism o f v i t a m i n B,, i n CML p a t i e n t s may be r e s p o n s i b l e f o r t h e e x c r e t i o n o f t h e s e u r i n a r y nucleosides, &A and 9A. The s t r u c t u r e o f t h e n u c l e o s i d e , 5'-deoxy-5'-methylthioadenosine s u l f o x i d e B, suggests t h a t i t most p r o b a b l y o r i g i n a t e s from o x i d a t i o n o f 5 ' -deoxy-5 ' -methyl t h i o a d e n o s i ne

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(MTA) by l i v e r microsomes o r by t h e a c t i o n o f i n v i v o p e r o x i d e o r superoxide ( r e f . 40). MTA i n t u r n i s produced as a by-product from S-adenosylmethionine (SAM) d u r i n g t h e b i o s y n t h e s i s o f t h e p o l yami nes , spermi ne and spermi d i ne ( r e f . 84) Although i n d i v i d u a l l y n e i t h e r t h e macromolecules such as carcinoembryonic a n t i g e n (CEA) , n o r t h e small molecules such as m o d i f i e d n u c l e o s i d e s c o u l d q u a l i f y as r e l i a b l e q u a l i t a t i v e tumor markers, however, i n combination w i t h o t h e r substances, s e v e r a l m o d i f i e d n u c l e o s i d e s c o u l d be considered as u s e f u l i n d i c a t o r s o f tumor burden as w e l l as f o r m o n i t o r i n g therapy. For example, i n c o n j u n c t i o n w i t h CEA and HCG, Nz -dimethyl guanosi ne has served as a q u a n t i t a t i v e i n d i c a t o r o f tumor a c t i v i t y i n b r e a s t carcinoma. I n 57% o f t h e s u b j e c t s w i t h m e t a s t a s i s , N2-dimethylguanosine 8 was e l e v a t e d w h i l e one o f t h e t h r e e i n d i c a t o r s was e l e v a t e d i n 97% o f t h e p a t i e n t s ( r e f . 85). L e v e l s o f p s e u d o u r i d i n e 13 and 7 a m i n o b u t y r i c a c i d i n u r i n e a l s o have been v a l u a b l e i n assessing t h e t r e a t m e n t o f b l a d d e r cancer and i n h e l p i n g t o p r e d i c t r e c u r r e n c e i n these p a t i e n t s ( r e f s . 86,87). Research groups o f Waalkes, Gehrke and Borek ( r e f . 88) showed t h a t 85% o f t h e c o l o n carcinoma p a t i e n t s e x c r e t e d e l e v a t e d l e v e l s o f t h e n u c l e o s i d e s N2-dimethylguanosine, l - m e t h y l i n o s i n e and p s e u d o u r i d i n e . Pseudouri d i ne 1eve1 s have c o r r e l a t e d we1 1 w i t h response i n chroni c myelogenous leukemia and i n o t h e r cancers, i.e. t h e l e v e l s f a l l t o normal w i t h t h e r e m i s s i o n o f t h e disease ( r e f s . 89,90). P r e l i m i n a r y s t u d i e s u s i n g a novel nucleoside, N 6 - s u c c i n y l adenosine (N6-SAR) as t h e marker i n d i c a t e t h a t u r i n a r y l e v e l s o f t h i s n u c l e o s i d e a r e e l e v a t e d i n p r o s t a t e and m e t a s t a t i c l u n g Speer e t a ) . ( r e f . 13) have shown t h a t o u t carcinoma ( r e f . 4). o f t h e seven n u c l e o s i d e s t h a t were measured i n t h e u r i n e o f 27 p a t i e n t s w i t h 13 d i f f e r e n t malignancies, one o r more substances was always e l e v a t e d . Thomale and Nass's experiments w i t h 3methyl chol anthrene-i nduced tumors in mice a1 so in d i c a t e t h e e l e v a t i o n o f m o d i f i e d n u c l e o s i d e s i n t h e tumor b e a r i n g animals as compared t o normal mice ( r e f . 91). The above s t u d i e s i n d i c a t e t h a t t h e u r i n a r y m o d i f i e d n u c l e o s i d e s can be u s e f u l as markers f o r assessing t h e tumor burden as w e l l as f o r d e t e r m i n i n g t h e e f f e c t i v e n e s s o f t h e r a p y . The success i n t h e search f o r s p e c i f i c tumor markers may y e t l i e

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i n i n v e s t i g a t i n g t h e minor, l e s s abundant, novel u r i n a r y m e t a b o l i t e s which may be p r e s e n t i n nanogram amounts. One o f these c o u l d t u r n o u t t o be a d i a g n o s t i c r a t h e r t h a n a p r o g n o s t i c i n d i c a t o r o f tumor burden. Both RNA and DNA o f t e n undergo damage and c a t a b o l i s m under t h e i n f l u e n c e o f chemotherapy and r a d i a t i o n t h e r a p y . Identific a t i o n o f t h e c h e m i c a l l y a l t e r e d n u c l e o s i d e s and bases may shed some l i g h t on t h e mechanism o f a c t i o n o f t h e new a n t i t u m o r agents, w h i l e q u a n t i t a t i o n o f these p r o d u c t s c o u l d s e r v e as a measure t o determine t h e end p o i n t f o r t h e therapy. Another aspect t h a t concerns t h e u r i n a r y m e t a b o l i t e s i s t h e need t o e v a l u a t e some o f t h e unusual substances f o r t h e i r mutagenic and c a r c i nogeni c a c t i v i t y . The compounds such as pyridones and t h e i r d e r i v a t i v e s ( r e f . 42) appear t o have a p o t e n t i a l o f r e a c t i n g w i t h macromolecules such as DNA and t h u s need t o be e v a l u a t e d f o r t h e i r m u t a g e n i c i t y . The m o d i f i e d base 6-amino-5-N-formylaminouracil and i t s d e r i v a t i v e s (51,52) c o n s t i t u t e an i m p o r t a n t group o f u r i n a r y m e t a b o l i t e s . The i n v e s t i g a t i o n s t o determine t h e o r i g i n o f these u r a c i l d e r i v a t i v e s may y e t uncover new enzymes i n v o l v e d i n t h e metabolism of m e t h y l a t e d xanthines. For example, h y d r a t i o n o f t h e N7,C8 double bond o f m e t h y l a t e d x a n t h i n e s t o g i v e m e t h y l a t e d d i h y d r o u r i c a c i d suggests t h e presence o f a s p e c i f i c enzyme. S i g n i f i c a n t p o r t i o n s (>35%) o f m e t h y l a t e d x a n t h i n e s undergo metabolism by t h i s pathway. It i s o f interest t o note t h a t the r e c e n t l y c h a r a c t e r i z e d novel n u c l e o s i d e s 5 ’ - d e o x y i n o s i n e (9A) and may l e a d t o some new 7 - 4 - D - r i bofuranosylhypoxanthine metabol i c pathways i n man i n v o l v i n g v i t a m i n B,

(a)

5.7

.

SUMMARY T h i s c h a p t e r d e a l s w i t h t h e procedures f o r i s o l a t i o n and t h e methods f o r c h a r a c t e r i z a t i o n o f n u c l e o s i des and r e 1 a t e d substances p r e s e n t i n human u r i n e . The i s o l a t i o n procedures a r e designed f o r s e p a r a t i n g a small amount (-50 pg) o f a n u c l e o s i d e m a t e r i a l from a l a r g e volume (-1500 m l ) o f u r i n e . U r i n e i s f i r s t d e s a l t e d and cleaned up on a c h a r c o a l - c e l i t e column. The d e s a l t e d m a t e r i a l i s passed through an Agl-X8 f o r m a t e i o n

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exchange column t o remove t h e a c i d i c components p r e s e n t in urine. The m a t e r i a l s e l u t e d i n water a r e separated i n t o c i s - d i o l and non-ci s-diol substances on a DEAE-cel 1 ul ose-borate col umn. The cis-diol p o r t i o n c o n t a i n i n g t h e n u c l e o s i d i c material i s f r a c t i o n a t e d by p r e p a r a t i v e HPLC and t h e r e s u l t i n g f r a c t i o n s a r e separated and p u r i f i e d t o homogeneity by a n a l y t i c a l HPLC. For s t r u c t u r a l assignment t h e homogenous materi a1 s a r e s u b j e c t e d t o u l t r a v i o l e t s p e c t r a l a n a l y s i s , h i g h r e s o l u t i o n mass spectrometry In o r d e r t o e s t a b l i s h the i d e n t i t y , t h e and NMR spectrometry. u r i n a r y material i s compared w i t h several candidate a u t h e n t i c o r s y n t h e t i c samples i n UV, NMR and mass s p e c t r a l and chromatographic c h a r a c t e r i s t i c s . Using t h i s methodology, c h a r a c t e r i z a t i o n of a new nucleoside 3-(3-ami no-3-carboxypropyl ) uri dine, i s discussed a s an example Included i n this r e p o r t i s t h e l i s t of t h e u r i n a r y nucleosides the amount i s o l a t e d from human urine and their chemica structures. Also discussed i s the o r i g i n , metabolic f a t e s i g n i f i c a n c e and f u t u r e prospects of the u r i n a r y nucleosides. 5.8

FUTURE PROSPECTS AND IMPACT

Of more than 115 modified nucleosides t h a t a r e known t o be present a s p a r t s of macromolecules and small e r bi omol ecul es, 35 have been i s o l a t e d and c h a r a c t e r i z e d from human u r i n e . Our labo r a t o r y has i s o l a t e d 20 new compounds from human urine, some of t h e s e may turn o u t t o be nucleosides o r their m e t a b o l i t e s derived from RNA o r DNA or t h e i r anabolic i n t e r m e d i a t e s . With the improvement i n HPLC methodology f o r i s o l a t i o n and p u r i f i c a t i o n of nucleosides and with the i n c r e a s e i n s e n s i t i v i t y of the d e t e c t i o n methods many a d d i t i o n a l modi f i e d nucl e o s i d e s and t h e i r metabolites w i l l be i d e n t i f i e d i n human urine. Particularly, mass spectrometry through s e l e c t e d ion monitoring and through t h e use of LC/MS and the MS/MS techniques, w i l l prove t o be very useful i n d e t e c t i o n , i d e n t i f i c a t i o n and q u a n t i t a t i o n of u r i n a r y substances. In a d d i t i o n t o mass spectrometry, radioimmunoassay methods w i l l be very useful i n d e t e c t i o n and q u a n t i t a t i o n of these nucleosides a t a femtomole l e v e l . The more abundantly excreted nucleosides such a s pseudouridine and N2-dimethylguanosine have been shown t o be reasonably good i n d i c a t o r s of

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tumor burden and f o r e f f e c t i v e n e s s o f therapy. The u t i l i t y o f these nucleosides may be enhanced by measuring them i n conj u n c t i o n w i t h t h e growth f a c t o r s such as j3-TGF o r o t h e r substances. U r i n a r y nucleosides o f f e r a non-invasive method t o s t u d y a number o f aspects o f human b i o c h e m i s t r y i n c l u d i n g n u c l e i c a c i d damage and r e p a i r . Measurement o f u r i n a r y n u c l e o s i d e s such as pseudouridine may u l t i m a t e l y serve as a means t o determine human tRNA t u r n o v e r . M o d i f i e d nucleosides such as 5 ' - d e o x y i n o s i n e and 7-j3-D-ribofuranosyl hypoxanthine may p r o v i d e v a l u a b l e i n f o r m a t i o n on a n a b o l i c pathways as w e l l as enzyme d e f i c i e n c i e s . Thymine g l y c o l s i n u r i n e have been suggested t o be a measure o f DNA damage ( r e f . 80) and r e p a i r which i n t u r n i s r e l a t e d t o t h e a g i n g process i n man. I n t h e near f u t u r e a number of o t h e r damaged bases and n u c l e i c a c i d adducts may be found i n t h e u r i n e s o f p a t i e n t s r e c e i v i n g chemotherapy and r a d i a t i o n t h e r a p y . On t h e whole, t h e r e i s a p r o m i s i n g f u t u r e i n terms o f a p p l i c a t i o n o f u r i n a r y nucleosides f o r a number o f c l i n i c a l and biochemical s t u d i e s as t h e d e t e c t i o n c a p a b i l i t i e s improve. 5.9 REFERENCES 1. J.F. Van Pilsum, 1 s t e d i t i o n , i n : Altman and D i t t m e r (Eds.), Metabolism, Fed. o f Am. SOC. E x p t l . B i o l . Sect. I X , 1968, 2.

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81. T.M. Savarese, G.W. Crabtree, and E. Parks, J r . , thioadenosine phosphorylase-I. S u b s t r a t e activi:i-Moefth{'I deox adenosine w i t h the enz me from Sarcoma 180 c e l l s , B i octem. Pharmacol , 30 (19gl) 189-199 Plagemann, and R.M. Wohl hueter, 5'-Deoxyadenosine 82. P.G.W. metabolism i n v a r i o u s mammalian c e l l l i n e s , Biochem. Pharmac. , 32 (1983) 1433-1440. 83. M.F. Beard W.R. Pitney,. and E.H. Sanneman, Serum conceni n p a t i e n t s s u f f e r i n g from leukemia, t r a t i o n s of' vitamin B Blood, 9 (1954) 789-7d4 and H.G. Williams-Ashman, On the r o l e of S84. A . E . Peg adenosy1-f:methionine i n the b i o s y n t h e s i s of spermidine by r a t p r o s t r a t e , J . Biol. Chem., 244 (1969) 682-693. 85. D.C. Tormey, T.P. Waalkes, D. Ahmann, C.W. Gehrke, R.W. Zumwalt, J . Snyder, and H. Hansen, Biolo i c a l markers i n b r e a s t carcinoma, Cancer, 35 (1975) 1095-1108 86. H . R . Nielsen, K. Nyhqlm, and K . E . S j o l i n , Relationship between urinary j-ami no1 sobutyri c a c i d and t r a n s f e r RNA turnover i n cancer p a t i e n t s , Cancer Res., 34 (1974) 3428-3432. 87. C.C. I r v i n g , Biochemically d e t e c t a b l e tumor markers i n urine of bladder cancer p a t i e n t s , Cancer Res., 37 1977) 2872-2874. 88. T . P . Waalkes, C.W. Gehrke, R.W. Zumwalt, . Y . Chang D . B . Lakings, D.C. Tormey, D . L . Ahmann, and C.G. Moertef, The u r i n a r y e x c r e t i on of nucl eosi des of r i bonucl ei c a c i d by a t i e n t s with advanced cancer, Cancer, 36 (1975) 390-398. S a l v a t o r e , A . Cojonna, F. Costanzo, T. Russo,. E . Esposito, 89. and F. Cimino, Modified nucleosides !n body f l u i d s of tumorbearing p a t i e n t s , Recent Results i n Cancer Research, 84 1983) 360-377. 90. Rasmuson, G . R . Bjork, L. Damber,. S . E . Holm, L. J a c o b s o n , A. Jeppsson, B. Littbrand, T . Stigbrand, and G. Westman, Eva1 uation of carcinoembryonic a n t i g e n , t i s s u e polypeptide antigen, placental a1 k a l i n e phosphatase, and modified nucleos i d e s a s biological markers i n malignant 1 mphomas, Recent Results i n Cancer Research, 84 (1983) 331-34J. 91. J . Thomale, and G . Nass, Elevated urinary e x c r e t i o n o f RNA c a t a b o l i t e s a s an e a r l y signal of tumor development i n mice, Can. Lett., 15 (1982) 149-159.

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CHAPTER 6 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF MODIFIED NUCLEOSIDES I N HUMAN SERUM EDITH P . MITCHELLa , KENNETH KUOb , LISA EVANS”, PAUL SCHULTZ” , RICHARD MADSENa, CHARLES W . G E H R K E b , AND JOHN YARBROa a S c h o o l o f M e d i c i n e , U n i v e r s i t y of M i s s o u r i - C o l u m b i a , C o l u m b i a , 65201 USA b D e p a r t m e n t o f B i o c h e m i s t r y , U n i v e r s i t y of M i s s o u r i - C o l u m b i a , C o l u m b i a , MO 65201 USA

MO

TABLE OF CONTENTS

6.4 6.5 6.6

Introduction. . . . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . . 6.2.1 Selection of Normal Subjects . . . . . . . . 6 . 2 . 2 Blood Collection a n d Handling. . . . . . . . 6.2.3 S t a t i s t i c a l Considerations . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Effect of Storage Conditions . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . .

6.1

INTRODUCTION

6.1 6.2

6.3

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Methylated purines and pyrimidines and other modified nucleosides, derived predominately from t r a n s f e r ribonucleic acid (tRNA), have been shown t o be excreted i n abnormal amounts in the urine of patients w i t h cancer ( r e f s . 1-9). By contrast, excretion of modified nucleosides by normal adults i s r e l a t i v e ly low ( r e f . 1 0 ) . These excretory products are predominately minor components o f t R N A which originate from the degradation of macromolecules ( r e f . 11). Evidence indicates t h a t methylation of t R N A occurs only a f t e r synthesis of the i n t a c t molecule. Since no kinases have been found t h a t will reincorporate the monomer units into t R N A , the modified bases and nucleosides are excreted fol 1 owing metabol i c degradation of t R N A mol ecul es (ref. 12).

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A d d i t i o n a l s t u d i e s show t h a t p s e u d o u r i d i n e i s n o t c a t a b o l i z e d b u t e x c r e t e d i n u r i n e as t h e i n t a c t molecule ( r e f s . 13, 14). E f f o r t s have been made t o use m e t h y l a t e d n u c l e o s i d e s as biochemical markers f o r n e o p l a s t i c diseases. E l e v a t e d concent r a t i ons have been suggested as p o s s i b l e markers f o r 1eukemi a ( r e f s . 4, 9), lymphoma ( r e f s . 15, 16), mesothelioma ( r e f s . 6, 17), cancers o f t h e l u n g ( r e f s . 18, 19, 20, 21, 22, 23), ovary ( r e f s . 24, 25), b r e a s t ( r e f s . 26, 27), l i v e r ( r e f . 28), nasopharynx ( r e f . 4), g a s t r o i n t e s t i n a l t r a c t ( r e f s . 29, 30), and Hodgkin's disease. I n addition, urinary levels o f modified n u c l e o s i d e s have been suggested as u s e f u l f o r m o n i t o r i n g progress o f disease and response t o therapy ( r e f s . 31, 32). Furthermore, abnormal l e v e l s o f these compounds have been d e t e c t e d i n hamsters w i t h adenovirus-12 induced tumors ( r e f . 33), r a t s w i t h t h y m i c lymphoma ( r e f . 34), mice w i t h mammary carcinoma, and l a b o r a t o r y animals s u b j e c t e d t o i r r a d i a t i o n ( r e f . 35). A1 though m o d i f i e d nucleosides and bases have been s t u d i e d e x t e n s i v e l y i n u r i n e , p r o f i l e s i n serum have n o t , m a i n l y due t o t h e l a c k o f adequately s e n s i t i v e a n a l y t i c a l methods. H a r t w i c k e t a 7 . ( r e f . 36) noted changes i n sera o f p a t i e n t s w i t h cancer u s i n g HPLC; however, e l u t i o n i n t e r f e r e n c e s d i d n o t a l l o w i d e n t i f i c a t i o n o r q u a n t i t a t i o n o f t h e m a j o r i t y o f t h e compounds. The development o f a r a p i d method f o r a n a l y s i s o f u r i n a r y n u c l e o s i d e s u t i l i z i n g reversed-phase h i g h performance l i q u i d chromatography f o l l o w i n g c o n c e n t r a t i o n by a boronate g e l ( r e f . 37) tremendously improved t h e accuracy o f a n a l y s i s . F u r t h e r m o d i f i c a t i o n s o f t h i s method were developed by Gehrke and Kuo and a l l o w e d t h e i s o l a t i o n and q u a n t i t a t i o n o f serum nucleosides ( r e f s . 38-40). W i t h improvement i n a n a l y t i c a l methods t h a t a1 l o w i d e n t i f i c a t i o n and q u a n t i t a t i o n o f m o d i f i e d n u c l e o s i d e s i n serum, i t was f e l t t h a t serum a n a l y s i s would o f f e r d i s t i n c t advantages compared t o u r i n e , such as e a s i e r and more r e l i a b l e sample c o l l e c t i o n and d i r e c t measurements o f t h e c o n c e n t r a t i o n o f t h e nucleosides p e r u n i t volume w i t h o u t r e l a t i o n s h i p t o c r e a t i n i n e and o t h e r metabol it e s . I n t h i s study t h e c o n c e n t r a t i o n s o f t e n m o d i f i e d serum nucleosides were determined i n t h e s e r a o f 37 normal s u b j e c t s (20 ma1 es, 17 females) u t i 1iz i ng reversed-phase h i g h performance

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l i q u i d chromatography ( r e f s . 37-40, 49) In a d d i t i o n , s e r a from p a t i e n t s w i t h several malignant d i s e a s e s were analyzed and the r e s u l t s compared t o normal c o n t r o l s . 6.2

MATERIALS AND METHODS

Our previous publications have described i n d e t a i l the instrumentation, reagents, nucleoside standard compounds, chromatographic columns, HPLC b u f f e r s and phenylboronate a f f i n i t y chromatography f o r nucleoside i s o l a t i o n s ( r e f s . 38-40, 49). Included i n these references a r e comprehensive discussions of i nstrument operation, preparation of chromatographically pure water f o r b u f f e r and sample preparation, p u r i t y of r e a g e n t s , preparation of t h e phenylboronate columns and t h e i r use f o r i s o l a t i o n of nucleosides from urine and serum, and the use of ul t r a f i l t r a t i o n t o prepare serum samples f o r the phenyl boronate i sol a t i o n of nucleosi des. The reader i s r e f e r r e d t o r e f e r e n c e s 38-40.

6.2.1

Selection of normal s u b j e c t s Donors were s e l e c t e d from normal volunteers w i t h no manifestation of disease. Sera was obtained from 20 men and 1 7 women who ranged i n age from 25-70 y e a r s (mean, 45 y e a r s ) . A1 1 were i n good health and none was taking any medication a t t h e time of the s t u d y . Additionally serum samples were obtained from randomly s e l e c t e d p a t i e n t s w i t h a v a r i e t y of ma1 ignant d i s e a s e s and evaluated. 6.2.2

B1 ood Col 1 e c t i on and Hand1 i n q

S e r a were c o l l e c t e d by s u b c u b i t a l venipuncture i n t o v a c u t a i n e r tubes. After c l o t formation, the tubes were centrifuged a t room temperature f o r 5-10 minutes. Sera were then t r a n s f e r r e d t o polyethylene sample v i a l s and s t o r e d a t -2OOC. Five serum samples were evaluated p r i o r t o f r e e z i n g and again a f t e r thawing t o e v a l u a t e the e f f e c t s of f r e e z i n g on a n a l y s i s . 6.2.3

S t a t i sti cal considerations

Separate uni vari a t e analyses of the 10 v a r i a b l e s u s i n g nonparametric s t a t i s t i c s was c a r r i e d out using the Wilcoxon's Rank Sum t e st i n analyzing the d a t a .

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6.3

RESULTS

Representative chromatograms used f o r i d e n t i f i c a t i o n and quantification of nucleosides are given in F i g , 6.1. Figure 6.1 is a standard chromatogram showing the separation and resolution of reference nucleosides using the chromatographic conditions described. Figure 6.2 i s a chromatogram of a normal human serum and i s representative of the HPLC method used in this report. Repeat runs demonstrate the accuracy and precision of the technique. Sera from t o t a l of thirty-seven volunteers with no evidence of systemic disease were analyzed for ten nucl eosides. The mean age was 45 and the range was 25-70. The graphs i n F i g . 6.3 represent the median levels measured in a l l samples. The bars indicate the standard deviation observed f o r each nucleoside measured. Graphs of the levels observed in the serum samples of twenty males and the seventeen females tested are given i n Fig. 6.4. No significant difference was observed between the values obtained from males and females. ( ~ ~ 0 . 1 5 ) The graphs i n Fig. 6 . 5 compare the levels observed w i t h respect t o age. Eighteen were above age 35 and 17 were between No difference i n values were observed with respect t o 25-35. age. To show changes i n the serum nucleoside values observed with ma1 ignant diseases, chromatograms of sera obtained from patients with cancer are shown. Figure 6.6 represents the chromatogram obtained from a forty-seven year old female with acute myelomonocytic leukemia. Elevation of several nucleosides i s apparent as evidenced by an increase in the s i z e of the peaks. The bar graph in Figure 6.7 represents a comparison of nucleoside levels in t h i s patient t o normal values. Elevated levels of a l l nucleosides measured were demonstrated. Figure 6.8 represents the chromatogram from a f i f t y - f i v e year o l d male with large c e l l carcinoma of the l u n g . The bar graph in Figure 6.9 compares levels observed in t h i s patient t o normal controls. Significant elevations of most nucleosides were found. These chromatograms were typical of the values obtained in patients w i t h malignant diseases.

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6.3.1

Effect of Storaae Conditions No significant difference was observed in values obtained from serum samples analyzed immediately after collection and those frozen for 1,3, and 6 months. Therefore, whole sera may be frozen u p to six months without no substantial changes in nucleoside levels. 6.4 DISCUSSION

The urinary levels of modified serum nucleosides derived predominantly from tRNA have been proposed as useful biochemical markers of malignant diseases (refs. 1-9). An evaluation of carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), and placental alkaline phosphatase (PLAP) in serum and pseudouridine i n urine were analyzed in 37 patients with col orectal cancer. The incidence of a1 1 four markers increased with advancing stages of disease (ref. 8). The urinary excretion of ,¶-aminobutyrate (B-AIB) and pseudouridine were investigated in 26 patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The excretion of ,¶-AIB correlated to the leukocyte count in CML, while that of pseudouridine correlated positively with the blast count i n AML (ref. 42) An elevation of pseudouridine was observed in patients with ma1 ignant lymphomas; however, no correlation between the level of excretion and the clinical stage was found (ref. 43). Significantly higher concentrations of l-methyl i nosi ne, N2, N2 -dimethyl guanosi ne, 1methylguanosine, and pseudouridine were found in the urine of patients with acute lymphoblastic leukemia at initial diagnosis or in relapse when compared to the concentrations found in normal controls and patients in remission (ref. 44). The urinary excretion of seven nucleosides were measured in patients with chronic myelogenous leukemia and were highest in patients whose disease was in the blastic phase, with the most significant d i f f e r e n c e s n o t e d in t h e l e v e l s o f l-methylinosine, pseudouridine, and N2, N2-dimethylguanosine (ref. 9). In a study of nucleosides in small cell carcinoma, the urinary concentrations of pseudouri di ne l-methyl adenosi ne, 1methylinosine, N2-methylguanosine, and N2 , N2-dimethyguanosine correlated to the stage of disease, with elevated values in 40%

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of patients w i t h limited disease and 81% i n patients w i t h extensive disease. Additionally, elevated urinary excretion of one o r more nucleosides has been demonstrated i n patients w i t h breast carcinoma (ref. 45) , hepatocell u l a r carcinoma (ref. 7 ) , and myel omatosi s (ref. 4 6 ) . Studies of urinary nucleosides i n animals revealed similar findings. Tumor-bearing mice were f o u n d t o excrete increased amounts of nucleic a c i d catabol i t e s , compared t o normal controls. Furthermore, the excretion rate of l-methyl adenosine, pseudouridi ne, and N4 -acetyl cytidine increased prior t o tumor diagnosis by other methods. Untreated control mice showed no alteration i n the excretion value of any modified nucleoside bases determined ( r e f . 4 7 ) . Level s of hypoxanthi ne and pseudouridi ne increased i n mice w i t h transplanted mesothel iomas and decreased f o l l owing growth cessation by chemotherapy (ref. 48). There are several potential advantages of a n a l y z i n g serum nucleosides rather t h a n urinary nucleosides (refs. 37-38). These include direct comparison of d a t a in terms of concentration, rather t h a n normalizing on the basis of another molecule as i s required i n urine studies. Also, serum nucleosides may be subject t o fewer structural a1 terations t h a n u r i n a r y nucleosides. A d d i t i o n a l ly, ease of col lection and physician preferences f o r serum values for other analytes favor serum. On the other hand, urine i s a much less complex matrix and contains higher levels of most nucleosides. The development of methods t h a t separate and remove proteins and o t h e r substances has allowed identification and q u a n t i t a t i o n of nucleosides i n serum. Using this technology i t i s now possible t o i d e n t i f y and quantitate more t h a n 65 modified nucl eosides. Precise analyses provi de reproduci bl e Val ues and superior resol u t i o n . Prior t o the evaluation of this method i n m a l i g n a n t diseases i t was necessary t o establish normal serum nucleoside levels. E v a l u a t i o n of sera obtained from 37 normal a d u l t donors provided standard Val ues o f ten nucleosides. These preliminary studies i n d i c a t e d t h a t normal p r o f i 1 es are reproducible and consi stent w i t h i n a narrow range. No significant differences were noted i n observed values w i t h regard t o sex or age. Elevated levels of

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some nucleosides were demonstrated i n patients w i t h a variety of m a l i g n a n t diseases. I t i s not clear i f i n d i v i d u a l patterns of serum nucleoside elevations occur i n specific cancers or i f the levels correlate w i t h extent of tumor burden, other staging and prognosti c indicators, or response t o changes associated w i t h therapy. The narrow ranges f o r serum levels of modified nucleosides obtained i n normal controls and the degree of elevation observed i n patients w i t h ma1 i g n a n t diseases suggest the potential value as biochemical markers of cancer. 6.5

SUMMARY

Methylated purines a n d pyrimidines derived from the degradation of transfer ribonucleic acid have been shown t o be excreted i n abnormal amounts i n the urine of patients w i t h cancer. Recent techno1 ogy has a1 1 owed the i sol a t i on and quantification of modified nucleosides i n serum using reversedphase high-performance 1 i q u i d chromatography. Serum levels of 10 modified nucleosides were measured i n 37 normal healthy adults t o establish normal values and t o correlate serum levels w i t h age and sex. Patients w i t h several malignancies were studied t o determine nucl eoside 1 eve1 s associated w i t h these diseases. Levels of modified nucleosides i n normal individuals were consistently reproducible and showed no significant v a r i a t i o n among males vs. females or w i t h age. Patients w i t h m a l i g n a n t diseases showed consistently elevated levels, w i t h highest elevations f o u n d i n patients w i t h more advanced disease. The evidence of no significant differences i n the mean levels of serum modified nucleosides w i t h age or sex i n normal adults, and elevations i n patients w i t h ma1 ignancies, demonstrate the potential value of modified nucleosides as cancer biomarkers.

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6.6 1.

2. 3.

REFERENCES Waalkes T P , Gehrke CW, Zumwalt RW,Chang S Y , Lakings DB, Tormey DC, Ahmann DL, and Moertel CG. The u r i n a r y e x c r e t i o n o f n u c l e o s i d e s of r i b o n u c l e i c a c i d by p a t i e n t s with advanced cancer. Cancer P h i l a . ) (36) 390-398, 1975. Gehrke CW, Kuo Waaikes TP, and Borek E. P a t t e r n of u r i n a r y e x c r e t i o n of modified n u c l e o s i d e s . Cancer Res. (39)

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44) 2120-8123, 1979 ! ~ ~ ~ ~ Glaser ~ o ~ R;W Kell , DR, Jackson DG, Graham WP, and a t i e n t s with S p e i c h e r CE. Elevated nucreoside e x c r e t i o n b nasopharyngeal carcinoma. Cancer (Phi 1 a . ) [4f) 2513-2517, 1982. 5. Borek E , Sharma OK, and Waalkes TP. New a p p l i c a t i o n of u r i n a r y nucl e o s i de markers, Recent R e s u l t s Cancer Res. (84) 301-316, 1983. 6. Fischbein A Sharma OK, S e l i k o f f I J , and Borek E. Urinary e x c r e t i o n o f modified n u c l e o s i d e s i n a t i e n t s with malignant mesothel ioma. Cancer Res. \43) 2971-5974, 1983 7 . Tamura S , Amuro Y , Nakano F u j i i F, Moriwaki Y , Yamamoto T , Hada T , and Higashin; K. Uri n a y e x c r e t i o n of pseudouridi ne i n p a t i e n t s with hepatocel ul a r carcinoma. Cancer ( P h i l a . ) (57) 1571-1575, 1986. 8. Rasmuson T , BJOrk G R , Damber L , Holm SE, Jakobsson D , Jeppsson A , Stigbrand T , and Westman G. Tumor markers i n An e v a l u a t i o n of carcinoembryonic bronchogenic carcinoma. a n t i g e n , t i s s u e polypeptide a n t i g e n , p l a c e n t a l a1 k a l i n e hosphatase and pseudouridine. Acta Radiol. Oncol. (22) 509-213, 1983. Urinary 9. Heldman DA, Grever MR, S p e i c h e r C E , Trewyn RW. e x c r e t i o n of modified n u c l e o s i d e s i n c h r o n i c myelogenous leukemia. J . Lab Med (101) , 783-792, 1983. 10. Gehrke CW, Kuo KC, Waalkes T P , Borek E. P a t t e r n s o f u r i n a r y e x c r e t i o n of n u c l e o s i d e s . Cancer Res (44), 1150-1153, 1979. Ori i n of u r i n a r y 11. Mandel L , S r i n i v a s o n PR, and Borek E. meth l a t e d p u r i n e s . Nature (London) (209), 58l-588, 1966 Atypical t r a n s f e r RNA's and t h e i r 12. Bore{ E , and Kerr SJ. o r i g i n i n n e o p l a s t i c c e l l s . Advances Cancer Res. (15), 163190, 1972. 13. Wei ssman SM, Ei sen AZ, Lenri ne M. Pseudouridi ne metabol i sm 111. S t u d i e s with i s o t o p i c a l l y l a b e l e d pseudourine. J . Lab C l i n Med (660), 40-47, 1962. Lack of c a t a b o l i s m o f 4-rib0 14. Dlugajcz k A, E i l e r J J . syluraci! i n man. Nature (London) (212), 611-612, 1966. 15. Waalkes T P , Gehrke CW, Bleyer WA, Zumwalt RW, Olweny CLM, Kuo KC, Lakings DB, and Jakobs SA. Potential biologic markers i n B u r k i t t ' s lymphoma. Cancer Chemomother. Rep. (59) 721-727, 1975. Pseudouridine: A r o g n o s t i c 16. Rasmuson T , and Bjbrk G R . marker i n Non-Hodgkin's lymphoma. Cancer Detect. brev. (8), 287-90, 1985. T r a n s f e r RNA 17. Fishbein A , Sharma OK Solomon S, e t a l . breakdown products i n the urine o f a s b e s t o s workers. Cancer Detect. Prev. (1) , 247-252, 1984. 4.

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Waalkes TP, Abeloff MD, Ettinger DS, Woo KB, Gehrke CW, and Borek E. Biological markers and small c e l l carcinoma of the l u n g : a c l i n i c a l evaluation of u r i n a r y ribonucleosides. Cancer ( P h i l a ) , (50) 2457-2464, 1982. 19. Tamura S , F u j i i J , Nakano T , Hada T , and Higashino K,. Urinary seudouridine a s a .tumor .marker i n p a t i e n t s w i t h small c e 1 l u n g cancer. C l i n . C h i m . Acta. (154) 125-132, 1986. 20. Tamura S, Fu’ioka H , Nakano T e t a l . Serum pseudouridine a s a biochemica marker i n smal! c e l l l u n g cancer. Cancer Res (471, 6138-6141, 1987. Carcinoembryonic 21. Waa kes, G E , Abeloff MD, Woo KB, e t a l . a n t i en f o r monitoring p a t i e n t s w i t h small c e l l carcinoma of the Yun . Cancer Res. (40), 4420-4427, 1980. 22. Carney \ N , Ihde DC, Cohen MH, Marangos PJ, B u n n PA, Minna JD, and Gazdar AF. Serum neuron s p e c i f i c enolase: a marker f o r d i s e a s e e x t e n t and response t o therapy of small c e l l l u n g cancer. Lancet, 1 583-585, 1982. , Forbes JT, Hainworth JD, Welch RW, 23. Johnson D H , Marangos Hande KR, and Greco FA. P o t e n t i a l u t i l i t y . of serum neurons p e c i f i c enolase l e v e l s i n small c e l l carcinoma of the l u n g . Cancer Res., (44) 5409-5414, 1984. 24. Waalkes TP, Rosenshein NB, Sha er JH, Ettinger DS,. Woo.KB e t a l . A f e a s i b i l i t y s t u d y i n !he development of b i o l o ica! markers f o r ovarian cancer. J: Surg Onc (21), 207-214, Q982 25. Oerlemans. F, and. Lange F. Major and modified nucleosides a; markers i n ovarian cancer: A p i l o t s t u d y . Gyneol Obstet Invest (22), 212-17, 1986. 26. Krstulovic AM, Hartwick RA, and Brown PR. Hi h performance l i q u i d chromatographic determination of serum V p r o f i l e s - o f normal s u b j e c t s and p a t i e n t s w i t h b r e a s t cancer and benign f i b r o c y s t i c changes. C l i n . C h i m . Acta (97), 159-170, 1979. 27. Tormey DC, Waalkes T P , Simon RM: Biological markers i n b r e a s t carcinoma 11. C l i n i c a l c o r r e l a t i o n s w i t h human chorionic gonadotropin. Cancer (39) 2391-2396, 1977. 28. Higashino K , Tamura S , F i y l o k a t t , Amuroy e t a l . Urinary pseudouridine concentration i n p a t i e n t s w i t h he a t o c e l l u l a r carcinoma and l i v e r c i r r h o s i s . In T. Oda and KO h d a (eds New Trends i n Hepatology, pp. 268-274. Tokyo: Medical Tos 6 Co., L t d , 1986. 29, Nakano K, Shindo K, Yasaka T , Yamado A , Reversed-phase h i g h performance 1 i q u i d chromatographic i n v e s t i g a t i o n of mucosal nucl eosi des and bases and u r i n a r y modi f i ed nucl e o s i d e s of a s t r o i n t e s t i n a l cancer p a t i e n t s . J . Chromatogr. (343), 21!3, 1985. 30. Nakano K , Shindo K , Yasaka T, Yamamoto A: Reversed-phase 1 i q u i d . chromatographic i n v e s t i g a t i o n of nucleosi.des and bases i n mucosa and modified nucleosides i n urine from p a t i e n t s w i t h g a s t r o i n t e s t i n a l cancer. J . Chromatogr. (332) 127-137, 1985. 31. Miller J , Erb N , Heller-SchBch G , Loren H , e t a l . M u l t i v a r i a t e a n a l y s i s of u r i n a r y RNA c a t a b o l i t e s i n ma1 ignancies: Cross-sectional and lon i t u d i n a l studies. Recent Results Cancer Res (84), 317-30 1883 32. Heldman DA, Grever MR, Trewyn RW. D i f # e r e n t i a l e x c r e t i o n of modified nucleosides i n a d u l t a c u t e leukemia. Blood (61), 18.

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291-6,

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33. 34.

35. 36.

37. 38.

39.

40.

McFarland ES and Shaw GJ. Observed i n c r e a s e i n methylated u r i di nes e x c r e t e d by hamsters b e a r i n adenovi rus-12 induced tumors. Can J Microbiol (14), 185-187, 1968 Shimizu M, and Fujimura S. Studies on the abiormal e x c r e t i o n of pyrimidine nucleosides i n the u r i n e of Yoshida A s c i t e s sarcoma-bearing r a t s . Increased e x c r e t i o n o f d e o x y c y t i d i n e , s e u d o u r i d i n e , and c y t i d i n e . Biochem Biophys Acta (517), 877-286, 1978. Russo T , Colonna A , S a l v a t o r e F, Cimino L , e t a l . Serum pseudouridine a s a biochemical marker i n the development of AKR mouse 1 mphoma. Cancer Res (44), 2567-2570, 1984. K r s t u l o i c , and Brown PR. I d e n t i f i c a t i o n and Hartwick quanti t a t i o n of n u c l e o s i d e s , bases and 0the.r UV-absorbing compounds i n serum u s i n g reversed-phase h i gh-performance I1 Evaluation of human s e r a , J . l i q u i d chromato r a h Chromatogr . 18g) %5&664, i979 Gehrke CW, iumwalt RW, McCunl, RA, Kuo KC, Q u a n t i t a t i v e high-performance 1 i h u i d chromatograpic a n a l y s i s o f modified n u c l e o s i d e s i n ph s i o l o g i c a l f l u i d s , t R N A and DNA, Recent R e s u l t s i n Cancer i e s e a r c h (84), 344-359, 1983 Kuo KC, Esposito F, McEntire J , and Gehrke dW. Nucleoside p r o f i l e s by HPLC UV i n serum and u r i n e o f c o n t r o l s and cancer a t i e n t s i n F. Cimino, GZ Birkmayer, J , Klavins E . , Pimentef, F. S a f v a t o r e , (eds) Human Tumor Markers, B e r l i n W. Germany: Walter de Gruyter, 1987. Kuo KC, Phan DT, Williams N , and Gehrke CW. Ribonucleosides i n serum and u r i n e by a hi h r e s o l u t i o n q u a n t i t a t i v e RPLC-UV method i n : CW Gehrke and \C Kuo ( e d s . ) , Chromatograph and m o d i f i c a t i o n of n u c l e o s i d e s , E l s e v i e r S c i e n t i f i c P u b l i s i e r s , Amsterdam, 1989. u a n t i t a t i v e RPGehrke CW, and Kuo KC, High r e s o l u t i o n HPLC-UV of nucleosides i n RNA, DNA, and mRN\, i n F. Cimino, G Birkmayer, J Klavins, E Pimentel, and F S a l v a t o r e ( e d s . ; Human Tumor Markers, Walter de Gruyter, B e r l i n ; dew Yor , 1987 475-502. Mitcheyy. E P , Gehrke CW, e t a l . Proc Amer Assoc Cancer Res, (78 347 1987. Urinary e x c r e t i o n of betaNie sen HR, and Killman SA. am1 no1 sobut r a t e and pseudouri d i ne in a c u t e and chroni c myeloid leuiemic. JNCI (5) 887-891, 1983 P s e u d o u h d i n e : A modified Rasmuson T, and Bjork GR. i c a l markers i n malignant lymphomas. n u c l e o s i d e as Cancer Detect 293-6, 1983. JS, and Trewyn R W . R e l a t i o n s h i p Heldman DA, of u r i n a r y e x c r e t i o n of modi f ied nucl e o s i des t o d i s e a s e s t a t u s i n childhood a c u t e lymphoblastic leukemia, JNCI ( 7 1 ) , 269-73, 1983. Vold BS, Kraus L E , Rimer VG, and Coombes RC. Use o f a monoclonal antibody t o d e t e c t e l e v a t e d 1-evels of a modified n u c l e o s i d e , .N-[g-(b-D;ri bofuranosyl) purin-6-yJ carbamo 1 -Lt h r e o n i n e , i n the urine of b r e a s t cancer p a t i e n t s . ancer Res. 1986 Jun; 46(6):3164-7. Urinary Sorenson SH, Brown DA, Coo er EH, e t a l . pseudouridine e x c r e t i o n i n mye omatosis. Br J Cancer ( 5 2 ) , 863-6, 1985.

RJ,

I

41. 42. 43. 44. 45.

1

z

46.

f

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47. Thomale J, Nass G.

Elevated urinary excretion o f RNA catabolites as an earl si nal o f tumor development i n mice. Cancer Letter 2, 149-58 1882 Urinary 48. B u h l L, Dragsholt 6, Svendsen P . e t a 7 . hypoxanthi ne and pseudouri d i ne as indicators o f tumor develo ment i n mesothel ioma-transplanted nude mice. Cancer Res. 4E 1159-62, 1985. 49. Matthew; DE, and Farewell UT. Using and Understanding Medical Statistics, Karger, Base1 (Switzerland), Second Edition, 1988.

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CHAPTER 7 MODIFIED NUCLEOSIDES I N HUMAN CHEMICAL SIGNALS FOR NEOPLASIA

BLOOD SERUM AS BIO-

FRANCESCO SALVATORE, LUCIA SACCHETTI, MARCELLA S A V O I A , F A B R I Z I O PANE, TOMMASO RUSSO, ALFRED0 COLONNAI and FILIBERTO C I M I N O Dipartimento di Biochimica e Biotecnologie Mediche, I I Facolta di M e d i c i n a e C h i r o r g i a , U n i v e r s i t a d i N a p o l i , V i a S. P a n s i n i 5 , 80131 N a p o l i , I t a l y . 'Istitoto d i Oncologia Sperimentale e Clinica, Facolta di Medicina e Chirurgia, Universita di Reggio Calabria, Via Tommaso Campanella, 88100 C a t a n z a r o , I t a l y

TABLE OF CONTENTS 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . 7.2 Formation o f M o d i f i e d Nucleosides i n C e l l Metabolism . 7.3 E s t i m a t i o n Methods and Normal Reference I n t e r v a l s o f Blood Serum M o d i f i e d Nucleosides 7.4 Blood Serum M o d i f i e d Nucleosides i n P a t i e n t s A f f e c t e d by Neoplasias, I n c l u d i n g Leukemia and Lymphoma . . . . 7.5 D i a g n o s t i c Performance o f M o d i f i e d Nucleosides . . . . 7.6 Concluding Remarks . . . . . . . . . . . . . . . . . . 7.7 Summary . . . . . . . . . . . . . . . . . . . . . . . 7.8 Acknowledgements . . . . . . . . . . . . . . . . . . . 7.9 References . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . .

7.1

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INTRODUCTION T h i s b r i e f r e v i e w w i l l c o n c e n t r a t e on a l l t h e f i n d i n g s t h a t have appeared t o - d a t e concerning t h e a n a l y s i s o f t h e s o - c a l l e d "minor", o r "odd", n u c l e o s i d e s i n b l o o d serum, w i t h s p e c i a l r e f e r e n c e t o p s e u d o u r i d i ne ($) M i n o r n u c l e o s i d e s have a t t r a c t e d a g r e a t deal o f a t t e n t i o n over t h e l a s t decade because t h e i r conc e n t r a t i o n i n body f l u i d s has been shown t o be h i g h l y s u g g e s t i v e o f t h e presence o f n e o p l a s i a s ( r e f s . 1-8). However, d a t a on t h e e s t i m a t i o n o f t h e s e compounds i n human b l o o d serum was slow i n coming, and i n t e r e s t was a t f i r s t focused on m i n o r n u c l e o s i d e s e x c r e t e d i n u r i n e ( r e f s . 9-17). The f i n d i n g s c o n c e r n i n g t h e u r i n a r y e x c r e t i o n o f m i n o r n u c l e o s i d e s ( r e f s . 18-21), and t h o s e

.

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d e s c r i b i n g experimental systems aimed a t s t u d y i n g t h e biochemical mechanisms u n d e r l y i n g t h e p r o d u c t i o n o f p s e u d o u r i d i n e i n tumor c e l l s and i n c u l t u r e media o f t r a n s f o r m e d c e l l s ( r e f . 22) a r e reviewed i n o t h e r c h a p t e r s o f t h i s volume. T h i s r e v i e w w i l l , t h e r e f o r e , deal w i t h : i) t h e f o r m a t i o n o f m o d i f i e d n u c l e o s i d e s d u r i n g t h e process o f c e l l metabolism; ii) t h e methods f o r t h e e s t i m a t i o n o f p s e u d o u r i d i n e and o t h e r m o d i f i e d nucl eosides i n human b l o o d serum and t h e a n a l y s i s o f r e f e r e n c e i n t e r v a l s i n normal s u b j e c t s ; iii) t h e d e t e r m i n a t i o n o f b l o o d serum pseudouridine and o t h e r m o d i f i e d n u c l e o s i d e s i n p a t i e n t s a f f e c t e d by s e v e r a l n e o p l a s i a s a t d i f f e r e n t stages, i n c l u d i n g leukemia and lymphoma; i v ) t h e d i a g n o s t i c performance o f these biochemical s i g n a l s f o r t h e assessment o f t h e n e o p l a s t i c s t a t u s . FORMATION OF MODIFIED NUCLEOSIDES I N CELL METABOLISM The f o u r m a j o r nucleosides, i . e . , adenosine, guanosine, c y t i d i n e and u r i d i n e , a r e formed d u r i n g c e l l m e t a b o l i c processes a f t e r t h e d e g r a d a t i o n o f r i b o n u c l e i c a c i d (RNA), t h r o u g h t h e a c t i o n o f h y d r o l y t i c enzymes t h a t produce 5 ' - n u c l e o t i d e s by cleavage o f t h e phosphodiester bond, a f t e r which phosphatase detaches t h e phosphoric group. These n u c l eosides may undergo f u r t h e r c a t a b o l i s m t o u r i c a c i d ( d e r i v i n g from adenosine and guanosine) and t o fl-alanine ( d e r i v i n g f r o m c y t i d i n e and u r i d i n e ) . Whereas, i n man, u r i c a c i d i s an end p r o d u c t and i s e x c r e t e d i n u r i n e , 8 - a l a n i n e i s n o t produced from c y t o s i n e and u r a c i l degradat i o n a l o n e and consequently t h e t u r n o v e r o f c y t o s i n e o r u r a c i l n u c l e o t i d e s and n u c l e o s i d e s cannot be e s t i m a t e d from t h e end p r o d u c t o f t h i s pathway. Furthermore, t h e m a j o r n u c l e o s i d e s , i . e . , u r i d i n e , c y t i d i n e , adenosine and guanosine, a r e s p e c i f i c a l l y phosphoryl a t e d t o n u c l e o s i d e monophosphates, and t h e n t o d i - and t r i p h o s p h a t e s , t h e r e b y c o n s t i t u t i n g t h e s o - c a l l e d " s a l v a g e pathways" f o r p u r i n e and p y r i m i d i n e m o i e t y r e u t i l i z a t i o n . Thus, t h e m a j o r n u c l e o s i d e s cannot be taken as i n d i c a t o r s o f t h e metabolism of t h e compounds from which t h e y d e r i v e d u r i n g c e l l metabolism (see r e f . 8 ) . M i n o r nucleosides, on t h e o t h e r hand, a r e formed a t t h e p o s t t r a n s c r i p t i o n a l l e v e l by chemical m o d i f i c a t i o n s o f m a j o r nucleos i d e s w i t h i n t h e RNA molecule, and t h e y a r e then r e l e a s e d d u r i n g c e l l metabolism i n a manner analogous t o t h e m a j o r n u c l e o s i d e s . 7.2

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However, these minor nucleosides are n o t reutilized i n cell metabolism nor are they further degraded. They are excreted unchanged i n the urine a n d may thus be considered a f a i t h f u l index of the turnover of the RNA of the cell t h a t contains them. The different metabolic routes of the major and minor nucleosides are shown i n Figure 7 . 1 which i s taken from a recent paper by two of the present authors (ref. 2 3 ) . RNA transcripts

modifying enzymes

(tRNA, rRNA, snRNA)

nucleases

uric acid

%.*

-

multiple

%

uric acid 8-alanine, etc. (end-product s)

mature RNAs

hydrolysis

6

nucleotides

malor malor nucleosides

minor nucleosides (end-products, not reut ili r e d 1

I

-I' J

Figure 7 . 1 Schematic pathways of the formation of major and minor nucleosides i n cell metabolism (taken from reference 23, w i t h the permission of the pub1 isher). Various groups have analyzed the composition of minor nucleosides i n a number of RNAs (refs. 24-27) and details are provided elsewhere i n this t r e a t i s e ( r e f . 28). However, i n summary, i t may be concluded t h a t the major sources of modified nucleosides, i n the cells of higher animals, are transfer RNA ( t R N A ) , ribosomal RNA (rRNA) and small nuclear RNA (snRNA) ( r e f . 2 9 ) . In a typical situation where cell proliferation i s enhanced, f o r example during the course o f cell transformation or cancer formation and evolution, nucleic acids are metabolized a t an increased rate, thus producing an i ncrease of pseudouri d i ne and other minor nucleosides (ref. 2 ) . These products are released from the cell and accumulate i n the body fluids, particularly blood and urine, which are the most accessible ones i n clinical biochemi s t r y ; hence the interest f o r the estimation o f these compounds i n biological fluids of patients affected by various types of tumors.

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7.3

ESTIMATION METHODS AND NORMAL REFERENCE INTERVALS OF BLOOD SERUM MODIFIED NUCLEOSIDES The measurement o f m o d i f i e d r i b o n u c l e o s i d e s i n body f l u i d s stems from t h e p i o n e e r i n g s t u d i e s o f Borek ( r e f s . 1, 2) on t h e c o r r e l a t i o n between a b e r r a n t p r o d u c t i o n and e x c r e t i o n o f these compounds from tumor c e l l s . However, whereas t h e e s t i m a t i o n o f m o d i f i e d nucleosides i n t h e u r i n e o f normal and n e o p l a s t i c subj e c t s was a c t i v e l y pursued (see r e f s . 3, 6, 9, 13, 30, 31), t h e i r e s t i m a t i o n i n b l o o d was more r e s t r i c t e d p r o b a b l y because o f t h e low l e v e l s o f m o d i f i e d nucleosides i n b l o o d as opposed t o u r i n e . The f i r s t method o f measurement o f m o d i f i e d n u c l e o s i d e s i n b l o o d was a radioimmunoassay devised by L e v i n e e t a l . ( r e f . 32). The procedure was based on e x t r a c t i o n o f n u c l e o s i d e s f r o m s e r a u s i n g m e t h y l a l - e t h a n o l , and d e r i v a t i z a t i o n o f r i b o n u c l e o s i d e s t o t h e i r 6-aminocaproate d e r i v a t i v e s ( t h e hapten s t r u c t u r e o f t h e immunogen). The d e r i v a t i v e s r e s u l t e d i n a g r e a t e r s e n s i t i v i t y o f t h e radioimmunoassay. Indeed, sensi t i v i ' t y was i n c r e a s e d b y about 1,000 times f o r p s e u d o u r i d i n e and 1Q t i m e s f o r N2 ,N2-dimethylguanosine. The normal r e f e r e n c e values o b t a i n e d w i t h t h i s method a r e l o w e r than a l l t h e o t h e r methods t h a t have been used s i n c e . However, no r e c o v e r y d a t a were presented f o r t h e method t o g e t h e r w i t h t h e procedure f o r n u c l e o s i d e p u r i f i c a t i o n . Thus, i t cannot be excluded t h a t t h e r e was some loss and t h a t t h e l e v e l o f n u c l e o s i d e s was underestimated (see d i s c u s s i o n i n Colonna e t a l . , r e f . 33). To o u r knowledge, besides t h e paper by L e v i n e and co-workers ( r e f . 3 2 ) , t h e r e has been no o t h e r paper concerning t h e e s t i m a t i o n o f b l o o d serum pseudouridi ne o r o t h e r nucl eosides u s i n g r a d i oimmunoassay. The reasons f o r t h i s a r e unknown; however, d a t a from o t h e r l a b o r a t o r i e s (personal communication) i n d i c a t e t h a t i t i s d i f f i c u l t t o produce a n t i bodies a g a i n s t p s e u d o u r i d i n e and t h i s i s supported a l s o by o u r own experience. A m a j o r advance i n t h e d e t e r m i n a t i o n o f p s e u d o u r i d i n e and o t h e r nucleosides i n b l o o d serum came w i t h t h e p u b l i c a t i o n by Colonna e t a 7 . ( r e f . 33) w i t h personal communication i n p u t from t h e l a b o r a t o r i e s o f Gehrke and Kuo. With t h i s procedure HPLC i s used t o separate pseudouridine and o t h e r m a j o r nucleosides, so making i t p o s s i b l e t o determine t h e i r e x a c t l e v e l s i n blood. The method r e q u i r e s four steps: i)d e p r o t e i n i z a t i o n o f serum samples

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by acetonitrile; i i ) purification of nucleosides and concentration of the samples by a f f i n i t y chromatography on phenyl-boronate columns; i i i ) lyophilization of samples; and i v ) nucleoside separation on reversed-phase HPLC. HPLC separation profiles of nucleosides i n serum of normal and lymphoma-bearing subjects are reported i n Figure 7.2. From the profiles i t can be easily seen

t h a t pseudouridine, being well resolved from other nucleosides, and from other interfering substances, can be determined w i t h h i g h specificity and sensitivity. As shown i n Figure 7 . 2 the level of t h i s nucleoside i s increased i n the serum of tumor-bearing patients. These results w i l l be discussed i n a later section. I

A

U

I

I 0

'

'

L

10

30

20

Time ( m i n )

10

20

30

Time ( m i n )

Figure 7 . 2 HPLC separation profiles o f nucleosides present i n serum of normal (A) and 1 mphoma-bearing (B) patients. # = pseudouridine; U = uridine; = inosine; G = guanosine; dG ( I . S . ) = deoxyguanosine, internal standard. (Taken from reference 34, w i t h the permission o f the publisher).

f

The method i s very reliable i n t h a t i t gives a good linear response curve w i t h pseudouri d i ne concentration; and 1 ow concentrations, down t o 25 pmol for each sample, were analyzed w i t h h i g h precision. The linearity o f HPLC estimation for added pseudouridine ( r e f . 33) i s shown i n Figure 7.3, and precision and recovery analyses for pseudouridi ne determi nation i n blood serum

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are shown i n Table 7.1. The precision i s good; indeed, we o b t a i n e d a CV of imprecision r a n g i n g between 3.44% and 4.78%. Recovery was complete (99-103%) except i n the presence of amounts of pseudouridine (14 nmol/ml) well above the highest levels amenable t o HPLC analysis; even i n these cases recovery was h i g h l y satisfactory. The limitations of Colonna's method are t h a t : i ) among the minor nucleosides i t estimates pseudouridine only; i i ) i t requires pretreatment of the b i o l o g i c a l samples w i t h boronate gel a f f i n i t y chromatography; and i i i ) i t involves a laborious l y o p h i l i z a t i o n step. This method has been used mainly by the Naples research group and i t has always given reproducible and reliable results. Schlimme's group (ref. 35) has described a method based on an on-1 i ne mu1 t i col umn HPLC for selective cl ean-up and analysis o f major and minor ribonucleosides i n body fluids i n c l u d i n g serum. This method has the advantage of reducing t h e t o t a l time o f the analysis as i t lends i t s e l f t o automation, and i t avoids the lyophi 1 i z a t i o n step. However, the method has been used excl usively i n Dr. Schlimme's l a b o r a t o r y , and the preparation of the column i s

FSEUDOURIMNE (nmol injected)

Figure 7.3 Linearity of seudouridine estimation by HPLC. from reference 33, w i t h t!e permission of the publisher).

(Taken

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TABLE 7.1

Precision and Recovery Analysis f o r Pseudouridine Determination i n Blood Serum Pseudouridine (nmol /ml ) Added

Founda

SD

1 5 10

5.60 6.59 10.77 13.94

0.26 0.31 0.50 0.48

Re cove ry

(%I ~~

4.78 4.67 4.64 3.44

99 103 83

4 pooled serum; relative standard deviation percentage (RSDX) c a l c u l a t e d a s SD x 1 0 0 / n m o 7 f o u n d . ( T a k e n f r o m r e f . 33, w i t h t h e p e r m i s s i o n o f t h e publisher).

lengthy and requires s k i l l t h a t i s not e a s i l y found i n routine cl i n i cal 1aboratori es . T h i s on-1 i ne cl ean-up analysi s procedure has been mainly used f o r urinary nucleosides, while results on serum nucleosides a r e s t i l l preliminary ( r e f s . 35, 36). In 1980 Schbch et a l . ( r e f . 37) described a l i q u i d chromatographic method f o r the simultaneous analysis of nucleobases and nucleosides i n the same sample. W i t h the method i t i s a l s o poss i b l e t o determine systematically t h e r a t i o o f nucleosides t o nucleobases i n biological f l u i d s , the r a t i o being an important byproduct of the procedure. More recently, the same group ( r e f . 38) published an HPLC method for the determination of pseudouridine i n human urine and u l t r a f i l tered serum. W i t h t h i s new method, pseudouridine and u r i c acid a r e separated on a cation-exchange r e s i n , so t h a t diluted native urine and deproteinized serum can be d i r e c t l y analyzed: untreated samples of biological f l u i d a r e injected d i r e c t l y i n t o the HPLC columns. The method permits the continuous and r e l i a b l e analysis of a large number o f samples, as i s typical i n c l i n i c a l l a b o r a t o r i e s . Recovery of t h e substances i s approximately 96%. However, neither the method described by Schlimme e t a l . (ref. 36) nor t h a t of Topp et a7. ( r e f . 38) has ever been used f o r the estimation of minor nucleosides on a large number of human serum samples.

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In 1986, Apell e t a ] . ( r e f . 39) published a method for the estimation of modified nucleosides i n urine and s e r a . The procedure e n t a i l s the use of a C-18 radial-pack c a r t r i d g e and i s based e s s e n t i a l l y on the methods devised by Gehrke and Kuo ( r e f . 30) and Uziel and co-workers ( r e f . 40) which include phenylboronate a f f i n i t y chromatography before HPLC separation. Ape1 1 e t a ] . quantitated seven modified nucleosides, including pseudouridine, i n blood serum and analyzed samples of male and female subjects. Their data coincided w i t h those obtained by Colonna e t a l . ( r e f . 3 3 ) and Gehrke e t a l . ( r e f . 3 0 ) . Apell e t a 7 . used a radial compression column w i t h a l a r g e r diameter which required a four-fold increase i n volumetric eluent flowrate, thus r e s u l t i n g i n a d i l u t i o n of the eluted nucleosides and a lowered s e n s i t i v i t y of analysis. A real breakthrough i n the f i e l d of the estimation of serum nucleosides has come from recent work conducted i n Prof. Gehrke's laboratory ( r e f s . 41 and 42). The procedure i s an improvement of t h e i r previous method. The new method i s very reproducible, w i t h a CV of imprecision ranging from 1.25% t o 7.29%, depending on the modified nucleoside estimated. The method also shows very good recovery f o r nucleosides i n serum (from 94.1% t o 109.3%). More than 12 modified nucl eosides have been determined w i t h this method and i t i s easy t o compare urine and blood samples from the same patient. Preliminary data ( r e f s . 41 and 42) had already shown a constant concentration over 24 hours f o r some of the nucl eosides. The r e l a t i v e amount of nucleosides i n serum and urine were a t a similar level w i t h the exception of m l A , mlI, m l G and m 2 G , which were two t o s i x times higher i n urine as compared t o serum. Also, serum nucleoside l e v e l s of samples obtained a t 8:OO am and 8:OO pm from the same subjects on the same day were e s s e n t i a l l y the same except f o r inosine. The 8 pm serum inosine levels f o r a l l of the 4 subjects was approximately ten times higher than f o r the 8:OO am samples. A higher ATP metabolic r a t e d u r i n g the daytime may account f o r t h i s large difference. The technique showed excellent precision and recovery, i t can be used f o r ribonucleosides i n urine and serum and can be applied t o the c l i n i c a l s i t u a t i o n . In addition, the method can be combined w i t h chemometric analysis t o allow quantitation of a large number of nucleosides and pattern

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r e c o g n i t i o n o f n u c l e o s i d e s i n serum and u r i n e . W i t h i n t h e framework o f an ongoing m u l t i c e n t r i c study, which i n c l u d e s o u r l a b o r a t o r y i n Naples, a s e r i e s o f i n v e s t i g a t i o n s i s b e i n g conducted t o assess t h e v a l i d i t y o f t h i s methodology and i t s a p p l i c a t i o n i n t h e f i e l d o f tumor markers. F i g u r e 7.4 shows a t y p i c a l p a t t e r n o b t a i n e d by Gehrke and Kuo ( r e f . 41) w i t h t h i s new method i n serum f r o m a normal s u b j e c t and from a c h r o n i c l y m p h a t i c leukemia p a t i e n t . I n t h e l a t t e r case, 15 known d i f f e r e n t n u c l e o s i d e s have been separated and q u a n t i t a t i v e l y analyzed, p l u s s e v e r a l u n i d e n t i f i e d compounds. The peak l a b e l l e d PCNR i n F i g u r e 7.4 has r e c e n t l y been i d e n t i f i e d as 4pyridone-3-carboxamide-N1 -ri b o f u r a n o s i d e b y D r . Gordon M i 11s ( r e f . 5 6 ) a t t h e U n i v e r s i t y of Texas Medical Branch o f Galveston, r a t h e r than t h e 2-5 isomer. D e s p i t e s e v e r a l attempts aimed a t e x p l o i t i n g t h e v a r i o u s procedures f o r t h e e s t i m a t i o n o f m a j o r and m i n o r n u c l e o s i d e s i n serum, few c l i n i c a l i n v e s t i g a t i o n s have been conducted i n t h i s f i e l d , t h e l a r g e s t p o p u l a t i o n s t u d i e d b e i n g t h e one from o u r l a b o r a t o r y , which i s d e s c r i b e d i n S e c t i o n 7.4.

1.0 ml Pooled

I\

,. 0

,

, ,

. ~, , , , . . . . , . . . . 10

20

...

..

Normal Serum

~-t~-----~r~---

30

40

60

T h e (mln)

F i g u r e 7.4 Comparison o f serum n u c l e o s i d e s of c h r o n i c l y m p h a t i c leukemia (CLL) p a t i e n t s vs. normals. yj = pseudouridine; U =

C260

uridine; m1A = N1-methyladenosine, I = inosine; PCNR = 4-p ridone3ycarboxami de-Nl -ri bofuranoside; m3 U (IS) = N3 -methyfurldine (internal standard); mlI = l-meth linosine; ac4C = c tidine; A = adenosine; m2G = N2, NJ-dimethylguanosine; Nti!C%iI tireonyl adenosi ne; m6 A = Nt -meth 1 adenosine; mt6A = N6 -methyl -N6 threonyladenosi?e; ms2 t6A = &methyl thio-N6-threonyladenosine. The numbers indicate the retention time of unidentified compounds. (Taken from ref. 41, with the permission of the publisher). Before reporting the data that have been collected so far, we shall briefly review the normal reference intervals of minor and major nucleosides i n blood serum. The data refer to different methodologies, most of which entail the use of HPLC. Table 7.2a for minor or modified nucleosides, and Table 7.2b for major nucleosides, contain most of the analytical data obtained in this field. Data on human urine are more abundant, both for reference normal subjects and for subjects affected by various diseases including neoplasias, with respect to those on blood serum. From the data of Table 7.2a and b it emerges that the level of pseudouridine is about two orders of magnitude greater than that of all the other modified nucleosides; among the major nucleosides, uridine is the most abundant and adenosine the least. Gehrke and Kuo (ref. 42) have observed that the level of cytidine in serum of normal subjects were all less than the detection limit of the method ( < 5 pmol/ml). The fate of cytidine is obscure, is it totally deaminated to Urd? The low level of adenosine is due to the presence of high adenosine deaminase activity in blood serum (see ref. 33). Data concerning inosine are thus reported in Table 7.2b. A comparative analysis of serum pseudouridine levels indicates that practically all the data collected with the various procedures agree within quite narrow limits. A lower value of 1.72 nmols/ml was obtained with the radioimmunoassay procedure and the reasons for this have been mentioned earlier in this section. The quantitation and precision of the method of Gehrke and Kuo are quite good with little degradation of the nucleosides It has been (refer to Chapters One and Five of Volume I). observed that ac4C is unstable and some breakdown occurs in all of the methods. Also, the m l I values agreed quite closely with the data of others (refer to 36,39). In general, the data are in good

TABLE 7.2a Modified Nucleosides in Blood Serum of Normal Subjects

Nucl eosi des

Serum concentration (mean f SD) (nmol /ml ) 2.83 f 0.51 2.64 f 0.56 3.14 f 1.11 1.72 f 0.77** 2.48 f 0.13 2.29 f 0.48 2.76 f 0.46

Pseudouri di ne

($1

4-pyridone-3-carboxamide-

N1 -ri bofuranoside

69.5 f 20

(PCNR) N1-methylguanosi ne (m’ G ) l-methylinosine

(m’ 1) c

N

Authors and References

48 73 37 57 20 30 10

Apell e t a7. Apell e t al. Mitchell e t a l . Levine e t a l . Russo e t a]. Savoia e t a]. Topp e t a1 .

37

39.7 f 16.1 45.4 f 20.4 29.3 f 11.0

48 73 37

42.1 f 9.8 45.8 f 19.9 54.5 f 15.6 50

48 73 37 5

Mitchell

et

a/.(*)

Apell

a7. al.

ref.39 ref.39 al.[*)

et

Apell e t Mitchell Apell e t Apell e t Mitchell Schlimme

et

a].

a7.

ref.39 fref.391

et a7.(*)

e t a7.(ref.36)

0 N

m

c)

N

m N

TABLE 7.2a (Continued) Modified Nucleosides in Blood Serum of Normal Subjects

N2 -methylguanosi ne (m2GI N4 -acetyl cyti di ne (ac4 C)

21.0 f 19.5 f

10.2 5.72

48 73

Apell Apell

71.6 f 63.5 f 165 f

18.4 18.8 52

48 73 37

Apell Apell

Apell e t a 7 . Apell e t a l . Mitchell e t a 7 . Levine e t a ] . Topp e t a 7 . Apell e t a l . ref.39 Apell e t a 7 . ref.39 Mitchell e t a ] . / * ) Schlirnme e t a 7 . ref.36) Mitchell e t a 7 . [ * )

N2, N2-dimethylguanosine (m:G 1

46.5 f 47.0 T 26.9 f 11.6 f

21.5

19.2 5.5 6.0** 3.7

48 73 37 44 9

N6 -threonyl adenosine (PA)

71.9 f 59.3 f 48.9 f

21.7 19.6 10.2

48 73 37

30 19.7

5 37

N1 -methyladenosine (m’ A ) (*)vo7.

100

f

85.8 T

e t a7. et al.

ref.39 tref.391

e t a7. ref.39 et a ] . ref.39 Mitchell e t a 7 . [ * )

I I I , C h a p t e r 6 o f t h i s s e r i e s ; * * v a 7 u e s o b t a i n e d by

RIA analysis

TABLE 7.2b M a j o r Nucleosides and I n o s i n e i n B l o o d Serum of Normal S u b j e c t s Nucl eosides Uridine (U)

Guanosi ne (GI

Serum C o n c e n t r a t i o n " (mean k SD o r min-max,

5.05 3.17 1.90 2.54 3.66

1.22 1.11 8.40 f 0.63 f 1.24** f f

0.88 f 0.51

0.67 0.22 0.26 0.45

** 0.27 0.08 f 0.17** f

0.57

Adenosine (A)

0.11

I n o s i ne (1)

5.62 f 2.87 0.35 2.20**

0.23

0.56**

* 0.07 *

2.57 4.35 1.22 f 0.72 0.60 f 0.45**

)

N

37 31 13 20 15 31 20 5 13 37 6 5 31 6 37 5 15

Authors and References M i t c h e l l e t al. H a r t w i c k e t al. K a r l e e t al. Russo e t a ] . Z a k a r i a e t al.

r e f . 34

H a r t w i c k e t al. RUSSO e t al. Schlimme e t al. Z a k a r i a et a7. M i t c h e l l e t al. Pfadenhauer and Sun-de Tong Schlimme e t al. H a r t w i c k e t al. ( r e f . 4 5 ) Pfadenhauer and Sun-de Tong r e f . 48) M i t c h e l l e t al. r e f . * ) Schlimme e t al. l r e f . 3 6 1 Z a k a r i a e t al. ref.47

a T h e v a l u e s a r e e x p r e s s e d in n m o l / m l ; * * v a l u e s o b t a i n e d o n p l a s m a s a m p l e s . *Volume III, Chapter 6 of this series.

C264

agreement except for the ac4C values. These l a t t e r are a b o u t twof o l d greater t h a n other reported values. This i s due t o Gehrke and Kuo's method h a v i n g a higher recovery w i t h less breakdown of this nucleoside w h i c h i s sensitive t o pH changes and the incomplete separation of a trace of m 2 G . The large range f o r inosine values shown by diffe en t investigators i s due t o time of sample collection d u r i n g the d aY as shown by Kuo and Gehrke (refs. 41 and 42), and the i n s t a b i i t y o f inosine d u r i n g storage of serum a t -2OOC (ref 42). A f i n a l comment t o this section i s t h a t more d a t a must be col lected t o o b t a i n reference normal interval ranges for these analytes i n b l o o d serum before reliable compar sons can be made among d a t a o b t a i n e d i n various 1 aboratori es. 7.4

BLOOD SERUM MODIFIED NUCLEOSIDES I N PATIENTS AFFECTED BY NEOPLASIAS, INCLUDING LEUKEMIA AND LYMPHOMA

The complexity of the methods available f o r the estimation of modi f i ed nucl eosi des i n body f 1 u i ds has hampered thei r w i despread use i n clinical biochemistry laboratories. Over the l a s t few years there has been a spate of reports on the evaluation of pseudouridine and other modified nucleosides i n urine (refs. 13-17). In almost a l l these studies, w h i c h are reviewed i n other chapters of this book (refs. 18-21), good correlations have been found between the presence and amount of modified nucleosides i n urine and the s t a t e of the neoplastic disease evaluated on the basis of the spread and burden i n various types of tumors. The v a r i a b i l i t y o f analytes excreted i n urine, i s due t o such factors as dilution, difficulty i n o b t a i n i n g an accurate t o t a l 24 hour urine collection, etc. The expense of sample collection i s also a factor. This has prompted us t o use blood serum as the body f l u i d of choice t o evaluate the levels of modified nucleosides derived from cell catabolism. W i t h t h e method described i n the previous section (ref. 33) we have evaluated a series of groups of patients affected by various types of heoplasia for their content of blood pseudouridine (see Table 7 . 3 ) . The other minor nucleosides were n o t estimated because their low serum content ( f i f t y t o one-hundred times lower t h a n pseudouridine) was d i f f i c u l t t o quantitate by the

C265

methodology then avai 1 able. Table 7.3 shows a l l the results o b t a i n e d concerning the estimation of blood serum pseudouridine, expressed either as nmols/ml or as pseudouridine i n d e x . A series of considerations can be drawn from this collection of d a t a : In a l l groups of patients affected by tumors there was a definite and significant increase (as compared t o d a t a obtained i n normal subjects, see ref. 43) i n blood pseudouridine levels, w i t h the exception of the less advanced breast cancer groups. The pseudouridine increases were gradually higher g o i n g from the less advanced t o the advanced tumor-bearing patients and they increased even further i n patients affected by leukemia and lymphoma (refs. 43, 49, 50, see also 51). I t may be concluded t h a t the correl a t i o n between tumor burden and/or spread of the tumor mass i s well correlated w i t h pseudouri d i ne b l ood 1 eve1 s . The pseudouridine index, w h i c h was devised t o a v o i d the i nterference of renal diseases t h a t enhance blood pseudouridine independent of tumor presence, i s i n principle, more directly related t o tumor cell metabolism (refs. 43, 50, 51). Pseudouridine increases i n lymphoma and leukemia patients were much greater t h a n those present i n other tumors (ref. 50, 51). This may be related t o the peculiarity of t h e biochemical pathogenesis of these diseases, as i s discussed i n Chapter 8, Volume I 1 1 o f this book (ref. 2 2 ) . In a few cases where monitoring of neoplastic disease was studied, also through pseudouridine blood levels, there was a good correlation between the response t o therapy and these levels (ref. 4 9 ) . This was true b o t h i n patients receiving chemotherapy and i n those w h o underwent surgical treatment. d a t a concerning the estimation o f blood serum modified nucleosides besides o u r own, are presented by the f o l lowing. The most recent results are given i n Chapter 2 , Volume 111, by Gehrke and Kuo who present d a t a on an a r r a y o f nucleosides by RPLC-UV i n b i o l o g i c a l f l u i d s (Chapter 2 ) , i n Chapter 6 Mitchell e t a l . give t h e levels of modified nucleosides i n b l o o d Additional

C266

serum, and i n Chapter 12 McEntire e t a l . present their studies on classification o f l u n g cancer and controls by chromatography of Modified Nucleosides i n serum. Hartwick e t a ] . (ref. 45) found i n breast cancer patients an increase of l-methylinosine and N 2 methylguanosine i n 45.5% and 22.7% of patients, respectively. Thus, a l t h o u g h an exact q u a n t i t a t i o n was n o t performed, HPLC showed i n several patients affected by various types o f neoplasias and other diseases, serum nucleoside profiles t h a t were signif i c a n t l y different from those obtained i n normal subjects. In earlier studies, Levine e t a 7 . (ref. 32) used the radioimmunoassay technique t o determine blood serum levels of modified nucleosides. I t was found t h a t breast cancer patients and acute leukemia patients had much higher serum levels of pseudouridine and N2 ,N2-dimethylguanosine t h a n normal subjects. Other tumors, whose nature was not indicated i n the paper, d i d n o t show such an increase. As has been mentioned i n Section 7.3 of this chapter, Gehrke's group has initiated an extensive investigation using serum of cancer patients and controls t h a t has started t o produce some interesting results on a variety of different cancer types and on a t least 15 modified nucleosides besides pseudouridine. The preliminary quantitative results (Gehrke, personal communication) are reported i n Table 7 . 4 and i n Chapters 2 , 6, and 12 of Volume 111.

DIAGNOSTIC PERFORMANCE OF MODIFIED NUCLEOSIDES The most promising results on pseudouridine levels i n tumor patients have been described i n the previous section. S a v o i a e t a 7 . (ref. 43) have collected a series of d a t a on blood sera of patients affected by several non-neoplastic diseases (see Table 7 . 5 ) . The groups of diseases t h a t have been studied are: diabetes, vascular and respiratory diseases, acute hepatitis and a group of miscellaneous disorders. These studies have b r o u g h t a b o u t a more careful eval u a t i o n of the diagnostic speci f i c i t y of pseudouri d i ne as a tumor marker. In a l l the diseases except one, pseudouridine blood levels were comparable t o those reported for normal subjects. The except i o n was renal failure, which showed values a b o u t 4 times higher than normal. This alerted us t o the possibility t h a t renal failure 7.5

TABLE B1ood Index Types

7.3 Serum Pseudouri d i n e C o n c e n t r a t i o n (nmol /ml ) and Pseudouri d i ne (mol$/mol C r e a t i n i n e x 103) i n P a t i e n t s A f f e c t e d by D i f f e r e n t o f Cancer

Type o f Cancer

N

M i s c e l 1aneousa M i s c e l 1aneousa Breastb Col on-Rectal G a s t r i cb M i s c e l 1aneousb M i s c e l 1aneousb Leukemias and 1ymp h oma s

12 10 24 18 6 16 12 72

Stage

or Type Advanced Less advanced Less advanced Less advanced Less advanced Less advanced Advanced Different types

$ (nmol /ml ; mean f S.D.)

6.40 3.66 2.34 2.93 2.63 3.61 4.89 8.20

f k

f

*f *ff

3.42 1.41 0.55 0.46 0.68 1.60 1.29 14.0

$ index (mean f S.D.)

36.94 40.24 40.53 46.19 53.53 60.88

k

10.3

f 11.2

*f

12.6 11.6 f 9.9 f 45.6

aData from r e f e r e n c e 49; D d a t a from r e f e r e n c e 4 3 ; C d a t a from r e f e r e n c e 5 0 .

0 N 01 U

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a f f e c t e d p s e u d o u r i d i n e b l o o d l e v e l s . We attempted t o overcome t h i s o b s t a c l e by expressing p s e u d o u r i d i ne Val ues a f t e r normal iz i ng them t o c r e a t i n i n e b l o o d serum values. I n f a c t , a f t e r t h i s normali z a t i o n , t h e parameter, which we c a l l e d " p s e u d o u r i d i n e i n d e x " , almost e q u a l l e d t h e r e f e r e n c e v a l u e f o r normal s u b j e c t s (see Table 7.5). Furthermore, t h e p s e u d o u r i d i n e i n d e x values o b t a i n e d i n a l l t h e non n e o p l a s t i c d i s o r d e r s l i s t e d i n Table 7.5, were h i g h e r t h a n those o f normal s u b j e c t s ; t h i s i n d i c a t e s t h a t t h e p s e u d o u r i d i ne i n d e x i s a more s e n s i t i v e t e s t t h a n p s e u d o u r i d i n e c o n c e n t r a t i o n measured i n nmol / m l , The e v a l u a t i o n o f t h e d i a g n o s t i c sensi t i v i t y and t h e diagnost i c s p e c i f i c i t y ( r e f . 52) a t v a r i o u s c u t - o f f v a l u e s produces r e c e i v i n g o p e r a t i n g c h a r a c t e r i s t i c (ROC) curves t h a t maximize t h e e v a l u a t i o n o f d i a g n o s t i c e f f i c a c y ( r e f s . 53, 54). A s e r i e s o f ROC curves a r e presented i n F i g u r e s 7.5 and 7.6 ( r e f . 55, and unpublished r e s u l t s from o u r group). The b e s t c u t - o f f v a l u e f o r a marker on each curve i s t h e one c l o s e s t t o t h e l e f t - u p p e r c o r n e r o f t h e f i g u r e ( t h e 100% p o i n t ) . I n f a c t , a t t h i s p o i n t t h e r e i s t h e maximum combination s c o r e f o r b o t h d i a g n o s t i c s p e c i f i c i t y ( t h e p e r c e n t o f t r u e n e g a t i v e f o r t h e t e s t o v e r t h e t o t a l number o f reference s u b j e c t s ) , and d i a g n o s t i c s e n s i t i v i t y ( t h e p e r c e n t o f t r u e p o s i t i v e f o r t h e t e s t over t h e t o t a l number o f diseased s u b j e c t s ) , t h u s maximizing d i a g n o s t i c e f f i c a c y . Paramount i n t h e e v a l u a t i o n o f t h e d i a g n o s t i c s p e c i f i c i t y i s t h a t t h e e v a l u a t i o n be performed toward d i f f e r e n t groups o f p a t i e n t s and n o t o n l y toward normal s u b j e c t s . I n f a c t , d i a g n o s t i c speci f i c i t y increases w i t h t h e a b i 1 it y t o d i s c r i m i n a t e among diseases t h a t a r e c l o s e t o ( e . g . , t h a t a f f e c t t h e same organ, e t c . ) t h o s e f o r which t h e biochemical s i g n a l i s used. The ROC curves i n F i g u r e 7.5 (panel A) show t h a t t h e d i a g n o s t i c e f f i c a c y of t h e two serum pseudouridine parameters i s v e r y good when t h e s i g n a l s a r e used f o r a l l n e o p l a s i a p a t i e n t s versus normal subj e c t s . The d i a g n o s t i c e f f i c a c y decreases when t h e two parameters a r e used f o r n e o p l a s i a p a t i e n t s versus normal s u b j e c t s p l u s nonn e o p l a s i a p a t i e n t s ( F i g u r e 7.5, panel B ) . When t h e d i a g n o s t i c e f f i c a c y of t h e two parameters was measured i n leukemia and lymphoma p a t i e n t s versus normal s u b j e c t s ( F i g . 7.6, panel A) b o t h s i g n a l s were h i g h l y i n d i c a t i v e . When t h e s i g n a l s were used f o r leukemia and lymphoma p a t i e n t s versus normal s u b j e c t s p l u s non-

TABLE 7.4 Modified Nucleosides in Normal Human Serum and Neoplastic Subjects*

Nucl eo-

sides

IIa

PCNRb m 1 Gb m1 Ib ac4 Cb m2 Gb

ti Ab

Normal n=37 (mean f S . D . ) 3.14 69.5 29.3 54.5 165 26.9 48:9

f f f f f f f

Colon Ca. n=3 (min-max)

3.32 1.00 89.0 20 11.0 36.0 16.0 117 52 186 5.5 50.0 10.2 72.7

- 5 . 20 - 260 - 66. 0 - 206 - 464 - 79. 0

-

91

NH L n=4 (mi n-max) 2.86 111 48.0 107 211 41.0 72.7

SCLC n=3 (mi n-max)

CLL n=3 (mi n-max)

- 7.00 7 . 99 3.97 10.64 31.37 - 142 8 5 . 0 - 117 130 - 905 - 79.0 5 8 . 0 - 103 114 - 193 - 379 - 355 125 408 - 789 - 514 211 - 808 348 - 1276 - 108 44.0 - 154 154 - 283 - 165 8 9 . 3 - 256 285 - 64 1

NHL: n o n - H o d g k i n ' s l y m p h o m a ; S C L C : s m a l l - c e l l lung c a r c i n o m a ; C L L : ghronic lymphatic leukemia. aConcentration expressed as nmol/ml; concentration expressed as pmol/ml; for nucleoside abbreviations, see Table 7 . 2 a . *Unpublished data from Gehrke and co-workers, University o f Missouri-Columbia.

C270

neoplastic diseases ( F i g . 7.6, panel B ) , the diagnostic efficacy was s t i l l q u i t e good (ref.55, and unpublished r e s u l t s from our group), Furthermore, ROC curves a l s o a1 low a comparison t o be made between the two markers. In f a c t , i n F i g . 7.5, panel A , i t i s c l e a r l y shown t h a t the index has a g r e a t e r diagnostic efficacy, thereby a1 1 owing a b e t t e r cl i n i cal decision. 7 . 6 CONCLUDING REMARKS

Although data on the estimation of pseudouridine and other modified nucleosides i n blood serum a r e not y e t as numerous as those on more widely used biochemical signals of disease, they encourage the notion t h a t these compounds a r e potential markers i n the management of the neoplastic s t a t u s . Methodological d i f f i c u l t i e s have hampered the exploitation of these s i g n a l s as routine However, the use of a s i n g l e t e s t s i n c l i n i c a l biochemistry. column t o p u r i f y and separate each sample, and the f a c t t h a t more than twelve minor nucleosides may be estimated i n a s i n g l e HPLC run, w i t h an enhanced discriminating capability, w i l l c e r t a i n l y increase the diagnostic performance of the estimation of blood nucleosides. I t is not d i f f i c u l t t o foresee t h a t i n the near f u t u r e much e f f o r t w i l l be devoted t o the search f o r c o r r e l a t i o n s between disease s t a t u s , p a r t i c u l a r l y neoplasia, and the quali-quantitative pattern of modified nucleosides i n blood serum, established by HPLC and fol 1 owed by chemometri cs. 7.7 SUMMARY This review summarizes most of the available data on the estimation of modified nucleosides i n the blood serum of p a t i e n t s affected by various types of tumors. While other chapters o f this

volume concentrate mainly on the urinary excretion of modified nucleosides as potential biochemical indicators of cancer, our e f f o r t s have been directed toward investigating the use of blood serum modified nucleosides t o signal and monitor cancer. We f i r s t b r i e f l y review data on the formation of modified nucleosides. We then describe the original method devised by us for the estimation of pseudouridine i n blood; a f t e r which we i l l u s t r a t e the r e s u l t s obtained w i t h this method. Other procedures

C271

36.9

0 : 3.25

b: 30.0 c: 25.0 d: 20.0

f : 2.50 g: 2 . 0 0 h: 1.25

8:

-z

1

'

t 2

20

-c

3

20

80

40

80

'V,, .

a: 55.0, b: 36.8 c: 30.0

20

d: 20.0

#I

,

100

~,

0,: 5.50

f : 3.25 g: 2.50 h: 1.25

e 20

40

60

80

100-DIAGNOSTIC SPECIFICITY

100 (K)

Figure 7.5 Receiver operating characteristic (ROC) curves obtained by plottin diagnostic s e n s i t i v i t y in per cent vs. the values o f the comp ement t o 100 of diagnostic specificity, for each of the cut-off values, chosen (for the sake of c l a r i t y , only a selection of values i s provided) among the two t h a t give maximum sensitivitfy or maximum s p e c i f i c i t . The groups of patients utilized o r panel A are the normar subjects (N=30) vs. patients affected by neoplastic diseases (N=76). The g r o u p o f patients utilized f o r panel B are normal Subjects p l u s patients affected by non-neoplastic diseases (N=112) vs. neoplastic patients (N=76). The usually taken cut-off values, equal t o reference normal value p l u s two standard deviations, are indicated by large solid c i r cl es.

B

0 N U

N

TABLE 7.5

Blood Serum Pseudouridine Levels i n Normal S u b j e c t s and i n S u b j e c t s Affected by Non-Neopl a s t i c Diseases Pseudouridine Concentration #nmol /ml

o f serum

S.D.

(mean value) Normal Subjects (n=76)

R

Diabetes (n=6 Vascular and es i r a t o r y Diseases ( n r l 6 r Acute Hepatitis (n=20) Other Diseases (n=40) Renal Fai 1ure (n=lO)

$index

S.D.

(mean value)

2.44

0.53

24.24

6.33

2.32 2.95

0.71 1.03

26.77 36.92

4.11 11.26

2.37 2.63 9.99

0.94 0.97 4.61

33.41 34.05 28.81

10.90 13.12 11.14

( T a k e n f r o m r e f e r e n c e s 43 a n d 5 1 , w i t h t h e p e r m i s s i o n o f t h e p u b l i s h e r ) .

C273

a: 36.9 b: 30.0 C: 25.0 d: 20.0

z

3.25 f : 2.75 g: 2.50 h: 1 . 3 0

8:

c

*

m

20

w

60

40

00

100

m

'fJ CONC. a: 55.0 b: 36.9 C: 30.0 d: 20.0

20

20

60

40

8 : 5.00

t : 3.25 g: 2.50 h: 1.30

00

100

100-DIAGNOSTIC SPECIFICITY (X)

Figure 7.6 Receiver operating characteristic (ROC) curves obtained by p l o t t i n diagnostic sensitivity ip per cept .vs. the values of the com fement t o 100 of diagnostic s ecifi-city for each of the cut-of values, chosen (for the sake o c l a r i t y , bnly a selection of values i s provided) among the two t h a t give maximum sensitivity or maximum specificity. Thg groups of patients

P

P

C274

Figure 7.6 (continued) u t i l i z e d f o r panel A a r e the normal s u b j e c t s (N=30) vs. leukemia and lymphoma p a t i e n t s (N=87). The group of p a t i e n t s u t i l i z e d f o r panel B a r e normal Subjects p l u s p a t i e n t s a f f e c t e d by non-neoplastic d i s e a s e s (N=112) vs. leukemia and lymphoma p a t i e n t s (N=87). The u s u a l l y taken c u t - o f f v a l u e s , equal t o r e f e r e n c e normal .value plus two standard d e v i a t i o n s , a r e i ndi c a t e d by 1 arge s o l i d c i r c l es . f o r the determination of modified nucleosides i n blood serum a r e described t o g e t h e r with the r e s u l t s obtained i n s e l e c t e d groups of human d i s o r d e r s . T h u s a l l the d a t a a v a i l a b l e on blood serum e s t i m a t i o n a r e c o l l e c t e d i n t h i s review and i n c h a p t e r s 1, 2 , 6 , and 12 of Volume 111. Most of the d a t a i n our review concerns pseudouridine, the most abundant and most widely p r e s e n t modified n u c l e o s i d e . Very r e c e n t d a t a , obtained using an improved methodology devised by Dr. Gehrke's group a t the U n i v e r s i t y o f MissouriColumbia, a r e b r i e f l y mentioned. In our view t h i s new methodology, coupled with chemometric measurements, w i l l lead t o a very e x t e n s i v e s e t of s t u d i e s t h a t cannot f a i l t o i n c r e a s e the vocabulary of biochemical s i g n a l s i n the f i e l d of tumor marker oncology. 7.8 ACKNOWLEDGMENTS The experimental work performed i n the a u t h o r s ' 1 aboratory was supported by research grants-in-aid from the "Mini s t e r o del l a Pubblica I s t r u z i o n e " Rome, I t a l y , and from the "Consiglio Nazional e d e l l e Ri cerche (CNR) , Progetto Final i z z a t o Oncol ogi a " Rome, Italy. 7.9 REFERENCES 1. E. Borek, I n t r o d u c t i o n t o symposium: t R N A and t R N A modific a t i o n i n d i f f e r e n t i a t i o n and n e o p l a s i a , Cancer Res., 31 11971) 596-597. 2. Borek, B. S . Baliga, C . W. Gehrke, K.C. Kuo, S. Belman, W. Troll and T . P . Waalkes, High turnover r a t e of t r a n s f e r RNA i n tumor t i s s u e , Cancer Res., 37 (1977) 3362-3366. 3. G. E. Davis R. D. S u i t s , K.C. Kuo, C.W. Gehrke, T . P . Waalkes and E. Borek, High-performance 1 i q u i d chromatographic separat i o n and q u a n t i t a t i o n of nucleosides i n u r i n e and some o t h e r b i o l o g i c a l f l u i d s , C l i n . Chem., 23 1977 1427-1435. 4. J. Speer, C. W . Gehrke, K . C. Kuo, 4 . P . haalkes and E. Borek, t R N A breakdown products a s markers f o r c a n c e r , Cancer, 44 (1979) 2120-2123.

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i

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617-654. 27. D. H. Gauss 28. 29. 30. 31. 32. 33. 34.

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75-78.

35.

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A. b

il

1

9

45.

E

46. 47. 48.

C278

49. 50.

51.

52. 53. 54. 55 *

F. Salvatore, T. Russo, A. Colonna, L. Cimino G. Mazzacca and F. Cimino, Pseudouridine d e t e r m i n a t i o n i n b l o o d serum as tumor marker, Cancer Detec. Prev., 6 (1983 531-536. L. S a c c h e t t i , M. Savoia, G..Fortunato, F, ane, A. Camera, B. R o t o l i and F. S a l v a t o r e , Biochemical s i g n a l s i n m a l i g n a n t lymphomas and 1eukemi as: 1eve1 s o f 1a c t a t e deh drogenase isoenzymes and pseudouridine i n serum, Q u i m i c a J i n i c a , 5 1986) 239. Salvatore, M. Savoia, T. Russo L. S a c c h e t t i and F. Cimino, Pqeudouridine i n b i o l o i c a i f l u i d s o f tumor b e a r i n g a t i e n t s , i n : F. Cimino, G. D. irkmayer, J. V . K l a v i n s , E. h m e n t e l F. S a l v a t o r e (Eds.), Human Tumor Markers, W a l t e r de G r u y t e r 81 Co., Berlin-New York, 1987, pp. 451-462. R.S. Galen and S.R. Gambino, Beyond n o r m a l i t y : t h e p r e d i c t i v e v a l u e and e f f i c i e n c y o f medical diagnoses, John W i l e y & Sons, New York, N.Y. 1975, D. A . Turner, An i n t u i t i v e approach t o r e c e i v e r o e r a t i n g c h a r a c t e r i s t i c c u r v e a n a l y s i s , J . Nucl Med., 19 (1978) 213220. E . A. Robertson and M. H. Zweig, Use o f - r e c e i v e r o p e r a t i n g c h a r a c t e r i s t i c curves t o e v a l u a t e t h e c l i n i c a l performance o f anal t i c a l systems, C l i n . Chem., 22 (1981) 1569-1574. M. g a v o i a F. Pane, G. Fortunato,.F. S a l v a t o r e and L. Sacc h e t t i , The use o f r e c e i v e r o p e r a t i n c h a r a c t e r i s t i c (ROC) curves a n a l y s i s i n t h e e v a l u a t i o n o f t e d i a g n o s t i c e f f i c i e n c o f serum s e u d o u r i d i n e as a tumor marker, I t a l J . B i o cxem., 37 (19g8) 119-127 G. C. M i l l s , N. J . Davi; and K. L. Lertratanangkoon I s o l a t i on and I d e n t i f i c a t i o n o f 1-Ri b o s y l Pyri done Nuc! e o s i des from Human Urine, Nucleosides and N u c l e o t i d e s , 8 (2) 1989.

b

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CHAPTER 8 BIOCHEMICAL CORRELATIONS BETWEEN PSEUDOURIDINE EXCRET I O N AND NEOPLASIAS* F . CIMINO, F. ESPOSITO, T . RUSSO and F. SALVATORE

Istituto di Scienze Biochimiche, I I Facolta di Nedicina e Chirurgia, Universita di Napoli, via S. Pansini 5 - 80131 Napoli, Italy TABLE OF CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . Increased Pseudouridine Levels i n AKR Mice . . . . . . Increased Pseudouridine Excretion by Transformed Cel'ls Enzymes Involved in Pseudouridine Metabolism: Studies on Their Activity in Neoplastic Sources . . . . 8.5 Are the tRNAs from Neoplastic Cells "Hypermodified"? . 8.6 Studies on t R N A Primers f o r Reverse Transcriptase i n Tumor of Retroviral Origin. . . . . . . . . . . . . . . 8.7 Concluding Remarks . . . . . . . . . . . . . . . . . . 8.8 References . . . . . . . . . . . . . . . . . . . . . . 8.1 8.2 8.3 8.4

8.1

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INTRODUCTION

Modified nucleosides derive from the catabolism of r i b o Basically they can be considered final products of RNA catabolism as there i s no salvage mechanism whereby they can be r e u t i l i z e d , and there i s no operative degradation pathway by which they are transformed t o u r i c a c i d or j3-alanine a s occurs f o r the major nucleosides. Among the d i f f e r e n t RNA c l a s s e s , t R N A i s the molecule in w h i c h the modified nucleosides are the most widely represented. I n f a c t , more t h a n 50 positions a l o n g i t s primary s t r u c t u r e can be occupied by these modifications ( r e f . 1) a n d u p t o 80 d i f f e r e n t ribonucleosides have been identified i n t h i s nucleic a c i d species ( r e f . 2 ) . Pseudouridine ($), with an average of 3-4 residues per RNA molecule ( r e f . 3 ) , i s the most a b u n d a n t modified nucleoside i n tRNA. However, t R N A i s n o t the only $-containing RNA species:

nuc e i c acids.

* Thi s work was search C ounci l,

s uppor ted by Rome, S p e c i a l

g r a n t s of t h e I t a l i a n P r o j e c t "Oncologia".

N a t i o n a l

Re-

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rRNAs and snRNAs also show variable amounts of this modified nucleoside, even t h o u g h i t s representation i n t R N A (normalized t o 100 nucleotides) i s much higher than t h a t found i n other molecules (5 residues/100 nucleotides i n t R N A versus 0.6 t o 2.5 i n rRNAs and snRNAs). The only exception i s U, snRNA which contains up t o 6.3 $ residues/100 nucleotides. However, the concentration of this RNA species i n the c e l l i s much lower than t h a t of r R N A and t R N A so t h a t i t cannot be considered the main source of $ excretion. Compared t o the other modified nucleosides, l a r g e r amounts of $ a r e found i n the serum and urine of normal subjects; this is not related s o l e l y t o i t s representation i n t R N A , b u t a l s o t o the elevated chemical stabi 1 i t y of the unusual C; -C, glycosidic bond between the sugar and the base (ref. 4 ) , which i s d i f f e r e n t from the normal C; -N; present i n uridine. Pseudouridine biosynthesis, i n both eucaryotic and procaryotic c e l l s , i s performed by enzymes t h a t catalyze the pseudouridylation o f uridine residues, which a r e present i n RNA precursors, d u r i n g the maturation process, s p e c i f i c a l l y d u r i n g an early event of t R N A processing ( r e f . 5). Basicall y , $ biosynthesis occurs by way of tRNA-psynthase a c t i v i t y t h a t cleaves the N'-glycosidic bond of a s p e c i f i c uridine residue and forms the C,-C; glycosidic bond c h a r a c t e r i s t i c of $. Another enzymatic a c t i v i t y t h a t y i e l d s $MP from uracil and ribose-5P has been f o u n d i n prokaryotes and few other organisms ( r e f . 6 ) , b u t i t has not yet been extensively characterized. Modified nucleosides have been proposed as biochemical tumor markers of d i f f e r e n t k i n d s of tumors: i n f a c t , they a r e present i n very h i g h concentrations i n the serum and urine of cancer patients ( r e f s . 7-17). Evidence i s accumulating t h a t $ i s the most h i g h l y and most frequently increased modified nucleoside i n neoplastic p a t i e n t s . Furthermore, t h e r e i s a good correlation between $ serum levels and progression of the neoplastic disease and the response t o therapy ( r e f s . 10, 18). Recently, a s p e c i f i c relationship between $ overexcretion and tumors of r e t r o v i r a l origin has been found by our group ( r e f . 19, 20). Our data, described below, support the hypothesis t h a t , indeed, $ can be considered a tumor marker of viral-induced neopl asi as. Some possible biochemical mechani sms re1 ated t o $ overexcretion i n d i f f e r e n t tumors a r e a l s o discussed.

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8.2

INCREASED PSEUDOURIDINE LEVELS I N AKR MICE Our p r e v i o u s s t u d i e s have demonstrated i n c r e a s e d $ l e v e l s i n serum o f cancer p a t i e n t s ( r e f s . 10, 18, 21). I n addition, i n s t u d i e s on $ values i n d i f f e r e n t k i n d s o f tumors, we found a good c o r r e l a t i o n between $ l e v e l s and s t a g e and spread o f t h e d i s e a s e We ( r e f . 18, 22; see a l s o F. S a l v a t o r e e t a 7 . i n t h i s volume). a l s o i n v e s t i g a t e d t h e biochemical mechanisms u n d e r l y i n g $ o v e r p r o d u c t i o n by n e o p l a s t i c c e l l s i n tumor-beari ng animal s . The AKR mice spontaneously develop a t h y m i c lymphoma (100% i n female mice) s h a r i n g a n a l o g i e s w i t h a c u t e l y m p h o b l a s t i c leukemia ( r e f . 23). With t h i s system i t i s p o s s i b l e t o s t u d y $ e x c r e t i o n d u r i n g t h r e e d i f f e r e n t stages o f tumor development: t h e " p r e l e u k e m i c " stage, i n which no tumor c e l l s a r e d e t e c t e d and a recombinant r e t r o v i r u s , b e l i e v e d t o be t h e e t i o l o g i c a l agents o f t h e disease, i s generated ( r e f . 24); a second stage, i n which tumor c e l l s a r e p r e s e n t , b u t o n l y i n t h e thymus; a t h i r d s t a g e i n which tumor c e l l s spread t o lymphoid o r non-lymphoid organs. Our r e s u l t s ( r e f . 19) show t h a t $ serum l e v e l s a r e h i g h e r i n h e a l t h y females mice t h a n i n h e a l t h y males. Moreover, females show a h i g h e r i n c i d e n c e o f lymphoma; $ serum l e v e l s a r e s i g n i f i c a n t l y and r e p r o d u c i b l y h i g h e r i n lymphomatous mice (AKR s t r a i n ) than i n normal ones (BALB/c s t r a i n ) w i t h a d i r e c t c o r r e l a t i o n between serum values and t h e tumor burden ( t h e h i g h e s t l e v e l s a r e found i n t h e most d i s s e m i n a t e d tumors). Moreover, serum $ i n c r e a s e s d u r i n g t h e "preneopl a s t i c s t a g e " i n t h e absence o f any c l i n i c a l and h i s t o p a t h o l o g i c a l s i g n o f t h e disease. These d a t a i n d i c a t e t h a t , i n t h i s system, 3 f u l f i l l s such general c r i t e r i a o f tumor markers as: i)i n c r e a s e d l e v e l s i n animals w i t h cancer, s t r i c t l y c o r r e l a t e d w i t h t h e s t a g e o f t h e tumor, and ii) i n c r e a s e d l e v e l s i n t h e absence o f any c l i n i c a l , and h i s t o p a t h o l o g i c a l s i g n o f t h e disease. T h i s i n d i c a t e s t h a t $ serum determi n a t i on can be a p r e c l in i c a l marker o f lymphoma development i n AKR mice. I n t e r e s t i n g l y , $ serum l e v e l s a r e w i t h i n t h e normal values i n b l o o d serum o f mice t r a n s p l a n t e d w i t h c e l l s from chemical l y - i n d u c e d lymphoma (MOPC-460) which suggests t h a t t h e recombinant r e t r o v i r u s i s i n v o l v e d i n t h e genesis of t h i s phenomenon. Obviously, t h e p o s s i b i l i t y o f u s i n g serum $ as an i n d i c a t o r o f speci f i c neopl a s i as i s a v e r y n t e r e s t i ng h y p o t h e s i s t h a t deserves t o be f u r t h e r i n v e s t i g a t e d .

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8.3

INCREASED PSEUDOURIDINE EXCRETION BY TRANSFORMED CELLS Our r e s u l t s d o n ' t show whether i n c r e a s e d 11, serum l e v e l s i n t h e AKR system i s due t o t h e appearance and/or p r o l i f e r a t i o n o f n e o p l a s t i c f o c i , o r i f t h e y a r e a consequence o f biochemical a1 t e r a t i o n s c o r r e l a t e d t o t h e v i r a l i n f e c t i o n . By u s i n g c u l t u r e d c h i c k embryo f i b r o b l a s t s (CEF), we i n v e s t i g a t e d t h e r e l a t i o n s h i p between increased 11, c o n c e n t r a t i o n s and oncogenic v i r u s e s ( r e f . 20). I n t h e s e s t u d i e s CEF were i n f e c t e d w i t h a r e t r o v i r u s , Rous sarcoma v i r u s (RSV) and/or w i t h a mutant o f t h e same v i r u s (RAV-1) d e l e t e d o f t h e t r a n s f o r m i n g oncogene 5 r c ( r e f . 25). This l a t t e r v i r u s cannot cause t r a n s f o r m a t i o n i n CEF, and hence t h e c e l l phenotype i s i d e n t i c a l t o a normal c e l l , even though v i r u s r e p l i c a t i o n occurs i n these c e l l s . F i g u r e 8.1 shows t h e l e v e l s o f @ e x c r e t e d i n t h e c u l t u r e medium o f these c e l l s , a f t e r t h e v i r a l i n f e c t i o n . I t was found t h a t : i)@ c o n c e n t r a t i o n s were d r a m a t i c a l l y h i g h e r i n t h e c u l t u r e medium o f RSV-transformed c e l l s t h a n i n t h a t o f t h e normal ( u n i n f e c t e d ) c e l l s (0.15 p m o l e s / c e l l x 103, 60 and 72 h r s a f t e r t h e seeding f o r CEF versus 0.75 t o 1 p m o l / c e l l x l o 3 , a t t h e same t i m e s a f t e r v i r a l i n f e c t i o n f o r CEF-RSV); ii) comparable increases were observed i n CEF i n f e c t e d w i t h t h e transformation-defective v i r u s (RAV-1) , which demonstrates a d i r e c t r e l a t i o n s h i p between 11, i n c r e a s e and t h e metabolism o f t h e oncogenic v i r u s e s , r a t h e r t h a n an involvement o f t h e transformat i o n process i t s e l f ; iii)t h e i n c r e a s e o f t h e n u c l e o s i d e i s always p r e s e n t when neosynthesized v i r a l p a r t i c l e s a r e d e t e c t e d i n t h e c u l t u r e medium and precedes by many hours t h e appearance o f t h e transformed phenotype i n CEF-RSV. These d a t a c o n f i r m t h e p r e v i o u s r e s u l t s o b t a i n e d i n humans and i n t h e AKR m i c e system, and demons t r a t e t h a t the virus-cell interaction i s responsible f o r the i n c r e a s e d 11, p r o d u c t i o n , r u l i n g o u t t h e p o s s i b i l i t y t h a t enhanced 11, l e v e l s a r e a consequence o f t h e t r a n s f o r m a t i o n process, as suggested by RAV-1 s t u d i e s . Furthermore, t h e h i g h l e v e l s o f 11, found i n stages preceding t h e morphological changes o f t h e transformat i o n f o r CEF-RSV, a r e y e t another i n d i c a t i o n t h a t 11, i s a p r o m i s i n g c a n d i d a t e f o r a biochemical marker o f neopl a s i as.

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8.4 ENZYMES

INVOLVED IN PSEUDOURIDINE METABOLISM: STUDIES ON THEIR ACTIVITY IN NEOPLASTIC SOURCES Many data indicate an increased activity of tRNA modifying enzymes, such as tRNA methyl transferases, in tumor cells (refs. 10, 26, 27). To determine if the increased $ levels could be caused by an enhanced activity of its synthesizing enzyme, the biosynthesis of this nucleoside in normal and tumorous AKR thymuses, as well as in CEF and CEF-RSV was studied (ref. 28). The availability of a bacterial mutant that lacks the enzyme synthesizing the $ residue in the anticodon region (ref. 29) allowed us to use, as a substrate, a hypopseudouridylated tRNA, purified from this organism. These studies revealed an enhanced tRNA-$-

TIME (hrs)

Figure 8.1 Pseudouridine concentration i n culture medium of normal ( 0 ) , RSV-i nfected ( A ) , or RAV-1-1 nfected ( various times after seeding of secondary cultures. Close arrows, CEF at presence of infectious virus in the medium (RSV or RAV-1); open arrows, presence of morphological signs of transformation (for RSV-i nfected cell s ) . See ref. 20 for experimental conditions. (Taken from ref. 20 with the permission of the publisher).

d

synthase activity both in AKR thymocytes and in CEF. Specifically, the extent of pseudouridylation in extracts from lymphomatous tissues was two-fold higher than that of the normal source. Furthermore, the studies demonstrated that this enzyme, both from normal and tumorous counterparts, modifies sites other than those present in the anticodon loop. In fact, new $ were found when the wild-type E . c o 7 i tRNA was used as substrate. Similarly,

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t h e r e was enhanced enzymatic a c t i v i t y o f tRNA-$-synthase i n tumor sources compared t o t h e i r normal c o u n t e r p a r t s ( r e f . 28). 8.5

ARE THE tRNAS FROM NEOPLASTIC CELLS "HYPERMODIFIED"? The d i r e c t consequence o f t h e enzymatic s t u d i e s d e s c r i b e d above would be t h a t t h e tRNAs p u r i f i e d f r o m t r a n s f o r m e d c e l l s a r e To v e r i f y h y p e r m o d i f i e d compared t o those o f t h e normal c e l l s . t h i s h y p o t h e s i s t h e tRNAs from experimental systems (normal and lymphomatous thymuses i n t h e AKR system and from CEF and CEF-RSV) have been analyzed. B r i e f l y , t R N A was p u r i f i e d as d e s c r i b e d e l sewhere ( r e f . 30), enzymati c a l l y h y d r o l y z e d t o n u c l eosides and t h e l a t t e r compounds were separated by an HPLC method d e s c r i b e d by Gehrke ( r e f . 31). Surprisingly, the quantitative analysis o f $ r e s i d u e s p r e s e n t i n these tRNAs shows no d i f f e r e n c e s i n $ c o n t e n t between normal and lymphomatous thymus (3.79 and 3.98 moles o f $ p e r 100 moles o f m a j o r nucleosides, r e s p e c t i v e l y ) ( r e f . 30). S i m i l a r r e s u l t s were o b t a i n e d i n t R N A p u r i f i e d f r o m CEF and CEFRSV ( r e f . 32). F i g u r e 8.2 d e p i c t s an HPLC p r o f i l e o f t h e nucleos i d e s p r e s e n t i n CEF tRNA. I d e n t i c a l HPLC p a t t e r n s were o b t a i n e d On t h e o t h e r f o r t R N A p u r i f i e d from CEF-RSV ( d a t a n o t shown). hand, t h e c o n c e n t r a t i o n o f $ p r e s e n t i n a c i d s o l u b l e e x t r a c t s from lymphomatous thymuses was almost t w o - f o l d h i g h e r t h a n t h a t found i n t h e e x t r a c t s from normal t i s s u e s ( r e f . 30). A p o s s i b l e explana t i o n f o r t h i s apparent paradox ( i . e . enhanced tRNA-$-synthase a c t i v i t y i n n e o p l a s t i c c e l l s and l a c k o f h y p e r m o d i f i c a t i o n s i n t h e tRNAs s u b s t r a t e s e x t r a c t e d from t h e same sources) c o u l d be an i n c r e a s e d t u r n o v e r r a t e o f some RNA s p e c i e s i n neopl a s t i c c e l l s . T h i s h y p o t h e s i s would t h u s be i n agreement w i t h p r e v i o u s s t u d i e s i n d i c a t i n g an i n c r e a s e d t R N A t u r n o v e r i n mice w i t h b l a d d e r c a r cinoma when compared t o normal mice ( r e f . 33). 8.6

STUDIES ON tRNA PRIMERS FOR REVERSE TRANSCRIPTASE I N TUMOR OF RETROVIRAL ORIGIN The i n c r e a s e d $ l e v e l s i n t h e serum o f lymphomatous mice (AKR s t r a i n ) and i n t h e c u l t u r e medium o f RSV/RAV-1 i n f e c t e d f i b r o b l a s t s , as w e l l as t h e absence o f h y p e r p s e u d o u r i d y l a t e d b u l k tRNAs p u r i f i e d from t h e r e l a t i v e sources, suggested t h a t t h i s $ i n c r e a s e c o u l d be a t t r i b u t a b l e t o an i n c r e a s e d b i o s y n t h e s i s and/or catabo-

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lism of specific RNA species, possibly those a c t i n g as primers of the reverse transcriptase. This enzyme, required for retrovirus replication, s t a r t s the viral DNA synthesis from a "primer" identified as a t R N A molecule of cellular o r i g i n (refs. 34, 35). Incidentally, the tRNAs f u n c t i o n i n g as primers of reverse transcriptase of MuLV and RSV, the t R N A P r o and T r p respectively, contain an extra $ residue i n t h e i r sequence (refs. 34, 35). These observations prompted us 'to assay the concentrations of nonacyl ated pro1 i ne and tryptophane-accepti ng tRNAs. The results indicated i n Table 8.1 show that, indeed, the metabolism of these specific t R N A species i s altered. In particular, proline amino a c i d acceptance on AKR thymus was 4-fold higher compared t o the normal tissue. Simi 1 a r l y , the tryptophane acceptance in CEF-RSV or CEF-RAV-1 was two-fold higher t h a n t h a t found i n CEF.

Figure 8.2 HPLC profile of the nucleosides present i n t R N A purified from chick embryo fibroblasts ( C E F ) . Chromatographic conditions: 25pg of t R N A , purified from C E F (ref. 30) were

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h y d r o l y z e d as d e s c r i b e d ( r e f . 312 Column: Supelco LC 18 DB; 8%, 12% methanol i n 0.01 M Tern e r a t u r e : 27°C. E l u e n t : 2. %, NH4R PO , pH 5.3, 30% a c e t o n i t r i l e i n t h e same b u f f e r ; D e t e c t o r : UV a i 254 nm; Flow r a t e : 1 ml/minute. Abbreviations: $ = pseudouridine; C = c y t i d i n e , U = u r i d i n e ; mlA = 1-methyladenosine; m5C = 5 - m e t h y l c y t i d i n e ; Cm = 2 ' - O - m e t h y l c y t i d i n e ; m7G = 7methylguanosine; I = i n o s i n e ; T = 5 - m e t h y l u r i d i n e ; G = guanosine; dG = deoxyguanosine; Um = 2 ' - 0 - m e t h y l u r i d i n e ; dT = thymidine; m1I = 1-methylinosine; Gm = 2'-O-methylguanosine; m1 G = 1-methylguanosi ne; ac4 C = N4 - a c e t y l c y t i d i ne; m Z G = N2 -methyl guanosi ne; A = adenosine; dA = deoxyadenosine; m$G = N 2 , N2 - dimethylguanosine; Am = 2'-O-methyladenosine; mcm5s2U = 2-thio-5-carboxymethyluridine methyl e s t e r ; Br8G = 8-bromoguanosine; t 6 A = N6 -(N-threonyl c a r bonyl) adenosine; m6A = 6-methyladenosine; i 6 A = N6-(A2-isopen-

t e n y l ) adenosine. These samples were prepared i n t h e l a b o r a t o r y of t h e a u t h o r s and analyzed i n t h e l a b o r a t o r y o f D r . C.W. Gehrke a t t h e U n i v e r s i t y o f M i s s o u r i , Columbia. TABLE 8.1 Amino Acid Acceptance o f tRNA P u r i f i e d f r o m Normal and Lymphomatous AKR Thymus and f r o m Normal and RSV-Transformed CEF pmol o f charged tRNA/A,,,

u n i t s o f t o t a l tRNAa

AKR

Norma1 t RNAP r o t RNAT r p t RNAT y r

8.75 29.65 6.15

Lymphomat ou s 34.30 24.75 5.10

CEF

Normal

RS V

RAV - 1

105.00 21.45 5.25

74.20 33.30 5.70

96.20 44.80 7.80

a C h a r g e d tRNA amount was c a l c u l a t e d on t h e b a s i s o f a m i n o a c i d specific activity. S e e r e f . 20 f o r e x p e r i m e n t a l c o n d i t i o n s . ( T a k e n f r o m r e f . 20 w i t h t h e p e r m i s s i o n o f t h e p u b l i s h e r ) .

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chromatography of the aminoacyl ated tRNAs excluded the presence of "new" t R N A P r o and T r p species i n neoplastic c e l l s ( r e f . 20), as has been described i n other systems ( r e f . 36). These r es ul t s , together w i t h the other experimental data described, demonstrated t h a t the h i g h levels of $ found i n neoplastic systems i s d i r e c t l y or indirectly related t o a retrovirus t h a t i s involved i n the genesis of the disease. The molecular cloning of the genes coding f o r these t R N A s w i l l permit the study of the regulation of t h e i r expression i n transformed c e l l s . Three genes coding f o r mouse t R N A P r o have a1 ready been is01 ated from a mouse genomic 1 i brary ( r e f . 37) : two of these genes code f o r the t R N A primers of reverse transcriptase of MuLV; these genes are clustered t o other t R N A genes as described i n other systems (ref. 3 8 ) . The t h i r d one i s unable t o f o l d i n the cloverleaf s t ru c t u re because of several mutations and deletions i n the matured t ran s cri p t , b u t i t shows normal transcripTwo t R N A T r p genes have been isolated from a tional act i vit y . chicken genomi c 1 i brary ( u n p u b l i shed results) and t h e i r structural characterization i s i n progress. RPC-5

8.7 CONCLUDING REMARKS On the basis of the experimental results obtained so f a r the observed increase of $ biosynthesis i n neoplastic c e l l s appears t o be a complex phenomenon. In summary, we have demonstrated that: 1) $ concentrations are higher i n the serum of lymphomatous AKR mice and i n the culture medium of RSV transformed fibroblasts than i n normal counterparts; 2) this increase i s evident before the appearance of any morphological s i g n of transformation i n b o t h systems; 3) i t correlates very well w i t h the tumor burden as observed i n neoplast i c mice a t d i fferen t stages o f tumor development; 4 ) a clear correl ation has been found between $ serum 1 eve1 s and therapeutic treatment ( w i t h normalization of $ concentrations i n cases of c l i n i c remission); 5) $ increase i s not present "aspecifically" i n a l l k i n d s of tumors; i n fa c t normal serum levels were found i n mice carrying a chemically induced lymphoma; 6) these increased concentrations seem t o be independent on the transformation process i t s e l f : i n f a c t h i g h levels are found i n the culture

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medium of fibroblasts infected w i t h a transformation-defective mutant of RSV where an active viral replication i s present, a l t h o u g h the cells are phenotypically normal. All these experimental d a t a led us t o an i n t r i g u i n g hypothesis: could enhanced $ levels be a consequence of increased metabo1 ism of tRNAs primers of reverse transcriptase of retroviruses? Basically, a retrovirus i s involved i n the genesis of the disease i n b o t h the experimental systems used, and the phenomenon i s absent from chemically induced lymphomas. Furthermore, some steps i n the virus-cell interaction must be responsible f o r or related t o this phenomenon. In fact, elevated $ concentrations have been found i n RAV-1 infected cells (Figure 8.1), where no tumor i s evident and only viral replication is present, and i n AKR mice i n the "preneoplastic" period before morphological signs of the disease are evident, b u t i n the presence of active v i r a l replicat i o n . During rep1 i c a t i o n of the retroviruses, reverse transcri ptase triggers viral DNA synthesis from a primer, w h i c h i s known t o be a t R N A molecule of cellular o r i g i n (refs. 34, 35). I t seems conceivable t h a t i n particular circumstances there are some mechanisms t h a t lead t o an increased synthesis of these p a r t i c u l a r t R N A species and thereby reverse transcriptase i s able t o enhance v i r a l DNA synthesis which i s specifically required a t t h a t moment. In favour of our hypothesis is the fact t h a t b o t h the tRNAs primers of the reverse transcriptases of MuLV and RSV ( t R N A P r o and tRNATrp, respectively) contain an extra $ residue i n the IV l o o p instead of the normal ribothymidine. As shown i n Table 8.1 the amino a c i d acceptance of t R N A purified from normal and neoplastic systems i s 4-fold higher for proline i n lymphomatous mice compared t o normal mice, whereas t R N A T r p and t R N A T y (tested as controls) appeared unchanged i n the two counterparts. Similarly, i n CEF a E-fold increase of the specific t R N A primer ( t R N A T r p ) was observed i n RSV/RAV-I infected fibroblasts, whereas t R N A p r o and tRNATyr, used as control s , remained unchanged. The significant increase i n the concentrations o f t h e two specific tRNAs primers confirms t h a t there i s a correlation between $ overproduction and increased turnover of speci f i c t R N A molecules f u n c t i o n i n g as primers of the retroviral polymerase. A specific mechanism of transcriptional a c t i v a t i o n of particular

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t R N A s p e c i e s coul d a1 so be specul ated. The o b s e r v a t i o n s d e s c r i bed i n t h i s a r t i c l e a r e d i f f i c u l t t o interpret, p a r t i c u l a r l y i n the l i g h t o f recent findings i n the human n e o p l a s i a s . I n l a t t e r t h e e t i o l o g i c a l involvement of r e t r o v i ruses has n o t r e c e i v e d any d i r e c t d e m o n s t r a t i on. However, i t cannot be excluded t h a t r e t r o v i r a l i n f e c t i o n can be a s s o c i a t e d t o the neoplastic transformation o f the c e l l o r t h a t the e t i o l o g i c a l mechanism of these diseases ( e . g . oncogene a c t i v a t i o n ) induces s e v e r a l phenomena observed i n r e t r o v i r a l i n f e c t i o n . We a r e now c o n d u c t i n g experiments t o s t u d y t h e r e g u l a t i o n o f t h e expression o f t h e genes coding f o r t h e t R N A p r i m e r s o f t h e r e v e r s e t r a n s c r i p t a s e i n t h e normal and i n t h e n e o p l a s t i c c e l l .

8.8 1.

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L. Buhl, C. Dra s h o l t , P. Svendsen, E. Hage and M. R. Buhl, U r i n a r y hypoxant ine and pseudouri d i ne as i n d i c a t o r s o f tumor development i n mesothel ioma t r a n s p l a n t e d nude mice, Cancer Res., 45 (1985) 1159-1162. E: Schlimme, K.S, Boos, B. Wilmers and H.J. Gent, Anal s i s o f r i b o n u c l e o s i d e s i n human body f l u i d s and t h e i r o s s i b e r o l e as athobiochemical markers, T h i r d I n t e r n a t i o n a y Conference on luman Tumor Markers, Lacco Ameno d ' I s c h i a , Naples, I t a l y ,

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Sa vatore, T. Russo, A. Colonna! L. Cimino G. Mazzacca and F. Cimino, Pseudouridine d e t e r m i n a t i o n i n b l o o d serum as tumor marker, Cancer Det. and Prev. , 6 1983) 531-536. 19. T. Russo, A. Colonna, F. S a l v a t o r e , F. dimino S. B r i d g e s and C. Gurgo, Serum s e u d o u r i d i n e as a b i o c h e m i c i l marker i n t h e develo ment o f A R mouse lymphoma, Cancer Res., 44 (1984)

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F. Es o s i t o , T. Russo, R. Ammendola, A. D u i l i o , F. S a l v a t o r e and Cimino, Pseudouridine e x c r e t i o n and t r a n s f e r RNA p r i m e r s f o r r e v e r s e t r a n s c r i p t a s e i n tumors o f r e t r o v i r a l o r i i n , Cancer Res. , 45 1985) 6260-6263. F. t i m i n o , F. Costanzo Russo, A. Colonna, F. E s p o s i t o and F. S a l v a t o r e , M o d i f i e i nucleosides from t r a n s f e r r i b o n u c l e i c a c i d as tumor markers, i n : E. Usdin, R. T. B o r c h a r d t , and C.R. C r e v e l i n g (Eds.) , B i o c h e m i s t r y o f S-Adenosyl-Methionine and r e 1 a t e d compound , MacMi 1 1an London , (1982) 409-412. M. Savoia T. Russo, E. Rippa, L. Bucci, F. Mazzeo, F: Cimino and F. Salvatore, Serum pseudouridine: i t s e v a l u a t i o n as a biochemical s i g n a l o f neoplasia, J. o f Tumor Marker Oncology,

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CHAPTER 9 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF NUCLEOSIDES AND BASES I N MUCOSA TISSUES AND URINE OF GASTROINTESTINAL CANCER PATIENTS KATSUYUKI NAKANO PL Wedical Data Center. Japan

1 Kamiyamacho.

T o n d a b a y a s h i . Osaka 584.

TABLE OF CONTENTS 9.1 I n t r o d u c t i o n 9.2 M a t e r i a l s and Methods 9.2.1 Apparatus 9.2.2 Reagents . . . . . . . . . . . . . . . . . . . 9.2.3 Buffers 9.2.4 Chromatographic C o n d i t i o n s 9.2.5 Boronate Gel A f f i n i t y Chromatography 9.2.6 Sample C o l l e c t i o n . . . . . . . . . . . . . . 9.2.7 Sample P r e p a r a t i o n . . . . . . . . . . . . . . 9.2.8 Peak I d e n t i f i c a t i o n s . . . . . . . . . . . . . 9.2.9 Biochemicals . . . . . . . . . . . . . . . . . 9.3 R e s u l t s and D i s c u s s i o n . . . . . . . . . . . . . . . . 9.3.1 Chromatography o f Standard Compounds . . . . . 9.3.2 Chromatograms o f E x t r a c t s o f Mucosa and Muscle 9.3.3 Nucleosides i n E x t r a c t s o f Mucosa 9.3.4 Compound L e v e l s i n Mucosa f r o m Cancer P a t i e n t s 9.3.5 U r i n a r y Nucl eosi des A n a l y s i s 9.3.6 Q u a n t i t a t i o n o f U r i n a r y M o d i f i e d Nucleosides 9.4 F u t u r e Prospects and Impact 9.4.1 A p p l i c a t i o n o f Tumor Markers t o P e r i o d i c a l Tests 9.4.2 HPLC A n a l y s i s i n N u c l e i c A c i d Research . . . . 9.5 Summary 9.6 Acknowledgments 9.7 References

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INTRODUCTION The analysis of nucleotides, nucleosides and bases in biological samples is of great importance for the understanding of the metabol ism of nucl eic acids, re1 ated disorders and re1 ated pathological states. During the 1 ast decade, the development of high-performance 1 iquid chromatography (HPLC) has made it possible to determine accurately and with high resolution the naturally occurring nucleotides, nucleosides, bases, and their metabolites i n very small samples of biological fluids (for review, see refs. 1-3). The determinations of these compounds in serum or plasma (refs, 4-11), urine (refs. 12-14), saliva (ref. 15), cerebrospinal fluids (ref. 16), and blood cells (refs. 8, 10) have been performed using several HPLC methods. Some o f these HPLC techniques have been applied to the determination of nucleic acid metabolites i n plasma and serum of patients with neoplastic diseases (refs. 4, 9, 11). Nucleotides in tissue samples have also been studied using several modes of HPLC in order to examine physiological states in the liver (refs. 17-20), heart (refs. 17, 21-27), brain (refs. 17, 28, 29), granulation tissue (ref. 30), HeLa cells (ref. 31) and tissue perfusates (refs. 32, 33). However, less information is available on the level o f nucleosides, bases, *and their metabolites i n tissues, because the major metabolites of nucleic acid catabolism i n tissues examined so far were nucleotides and coenzymes. Very recently, the measurement of adenosine, inosine and hypoxanthine in human term placenta with reversed-phase HPLC (RPLC) has been reported (ref. 34). Until our studies were published (refs. 35, 36), the endogenous compounds in acid extracts of gastrointestinal (GI) mucosa have never been studied by the HPLC method. The investigation of mucosal compounds is of primary importance because most GI adenocarcinomas arise originally from the mucosal tissue of stomach or intestine. Therefore, differences in the profiles of ultraviolet (UV)-absorbi ng compounds in normal and neopl astic in mucosa could provide valuable information on the nucleic acid metabolism in mucosa of patients with malignant cancer. On the other hand, the urinary modified nucleosides derived 9.1

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from t h e enzymatic d e g r a d a t i o n o f t r a n s f e r RNA have been i n v e s t i g a t e d f o r t h e i r c l i n i c a l s i g n i f i c a n c e as biochemical markers i n cancer d e t e c t i o n ( r e f s . 37-40). I n r e c e n t s t u d i e s , t h e s e nucleos i d e s have been p r e f r a c t i o n a t e d u s i n g a b o r o n a t e a f f i n i t y g e l column, and then determined by RPLC techniques f o r u r i n e samples ( r e f s . 41-44) and b l o o d samples ( r e f s . 44, 45). Recently, an on1 i n e mu1 t i - d i m e n s i o n a l high-performance a f f i n i t y chromatography (HPAC) -RPLC method has been developed u s i n g a b o r o n i c s i 1 ica p r e column connected d i r e c t l y t o a reversed-phase c o l umn by a c o l umn s w i t c h i n g t e c h n i q u e ( r e f s . 46, 47). I n most o f t h e p r e v i o u s i n v e s t i g a t i o n s , t h e e l e v a t i o n s o f t h e u r i n a r y n u c l e o s i d e l e v e l s i n cancer p a t i e n t s have been s t u d i e d u s i n g t h e comparative d a t a f o r normal s u b j e c t s and cancer pat i e n t s . However, t h e change i n n u c l e o s i d e l e v e l s i n u r i n e t a k e n b e f o r e and a f t e r s u r g e r y on t h e same p a t i e n t have n o t been exami ned Recently, we used t h e reversed-phase mode o f HPLC t o examine t h e UV-absorbing compounds i n perch1 o r i c a c i d (PCA) e x t r a c t s o f t h e normal p o r t i o n and t h e n e o p l a s t i c p o r t i o n o f mucosa, r e s e c t e d s u r g i c a l l y f r o m p a t i e n t s w i t h GI cancer. We a l s o s t u d i e d t h e change i n m o d i f i e d n u c l e o s i d e l e v e l s i n u r i n e samples c o l l e c t e d b e f o r e and a f t e r s u r g i c a l o p e r a t i o n on t h e i d e n t i c a l p a t i e n t s w i t h c o l o r e c t a l cancer.

.

9.2 MATERIALS AND METHODS 9.2.1 A t n a r a t u s High-performance 1iquid-chromatographi c equipment ( H i t a c h i 638-30; H i t a c h i , Tokyo, Japan) w i t h a mu1 ti-wave1 e n g t h UV-moni t o r ( H i t a c h i 635-M) was used. Peak areas and r e t e n t i o n t i m e s were measured a t 260 nm w i t h a Chromatopack C-R2A (Shimadzu, Kyoto, Japan). Another r e c o r d e r was used t o m o n i t o r t h e 280-nm s i g n a l and t o c a l c u l a t e peak h e i g h t r a t i o s (280/260 nm). UV-absorption s p e c t r a o f f r a c t i o n a t e d HPLC peaks were measured w i t h a s p e c t r o photometer ( H i t a c h i 200-10). 9.2.2

Reaaents Potassium dihydrogen phosphate, potassium hydroxide, methanol (HPLC grade), ammonium a c e t a t e , f o r m i c a c i d and p e r c h l o r i c a c i d were o b t a i n e d from t h e Wako Pure Chemical I n d u s t r i e s (Osaka, Japan).

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9.2.3

Buffers A 0.02 M potassium dihydrogen phosphate b u f f e r (pH unadjusted; pH 4.53) was used f o r HPLC a n a l y s i s . And a 0.25 M ammonium a c e t a t e b u f f e r , pH 8.8 was used f o r boronate gel a f f i n i t y chromatography. 9.2.4

ChromatoaraPhic Conditions Nucleosides and bases i n b i o l o g i c a l samples were s e p a r a t e d on commercially a v a i l a b l e columns (250 x 4.6 mm I.D.) packed w i t h 5pm Devel o s i 1 ODS-5 (Nomura Chemical s , Nagoya, Japan) . A precol umn (50 x 4.0 mm I.D.; Chemuco, Osaka, Japan) packed with 15-30 pm Develosil ODS (Nomura Chemicals) was used t o p r o t e c t the a n a l y t i cal column. A f i l t e r ( 1-pm mesh s i z e ; Nomura Chemicals) was f i t t e d between the i n j e c t o r and pre-column. For t h e s e p a r a t i o n , l i n e a r g r a d i e n t e l u t i o n from 0.02 M potassium di hydrogen phosphate, pH 4.53 t o 40% methanol -water (3:2, v/v) i n 35 min. was used. The flow-rate was 1.2 ml/min. and the column temperature was ambient. All e l u e n t s were degassed by purging with helium. The i n j e c t i o n volume was 100 p1 unless s t a t e d otherwise. UV d e t e c t i o n was a t 260 and 280 nm (0.16 a.u.f.s.). Boronate Gel Affini t v ChromatoaraDhv The boronate a f f i n i t y gel column technique was used t o i s o l a t e nucleosides i n 1 ml of p e r c h l o r i c a c i d e x t r a c t s o f mucosa homogenate and 0.5 ml of u r i n e samples. The i s o l a t i o n procedure was a s l i g h t modification of the method developed by Gehrke e t . a l . ( r e f . 41). The boronate g e l , Affi-gel 601 (Bio-Rad Labs., Richmond, CA), was packed i n the p l a s t i c column (60 x 9 mm I.D.) t o a h e i g h t of 13 mm (bed volume 0.83 ml), and e q u i l i b r a t e d with 0.25 M ammonium a c e t a t e b u f f e r , pH 8.8. A l-ml volume of the mucosa e x t r a c t s o r 0.5 ml of u r i n e samples (samples were a d j u s t e d t o pH 9.5 with 2.5 M ammonium a c e t a t e ) was loaded on t o p of the column. The column was washed with 8 ml of 0.25 M ammonium a c e t a t e b u f f e r , pH 8.8. Nucleosides were e l u t e d w i t h 4 ml of 0.2 M formic a c i d . The e l u a t e was evaporated under reduced p r e s s u r e and r e d i s s o l v e d in 0.5 ml o f t h e s t a r t i n g b u f f e r o f HPLC a n a l y s i s . 9.2.5

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9.2.6

Samole Collection Specimens of normal and n e o p l a s t i c mucosa of colorectum and

stomach were obtained through s u r g i c a l operation on eig h t p a t i e n t s (four male, f o u r female; age, 52-72) w i t h malignant c o l o r e c t a l cancer (colon, 1; sigmoid colon, 3; rectum, 4 ) , and f o u r p a t i e n t s (three maleh one female; age, 41-81) w i t h malignant g a s t r i c cancer, r e s p e c t i v e l y . The h i s t o p a t h o l o g i c a l s t a g e s (ref. 48) of these cases were c l a s s i f i e d a s follows: s t a g e 11, 3; s t a g e 111, 3; s t a g e IV, 2 f o r c o l o r e c t a l cancer, and s t a g e 111, 3; s t a g e IV, 1 f o r g a s t r i c cancer. I n the preliminary experiments, normal and n e o p l a s t i c mucosa from another three p a t i e n t s w i t h malignant c o l o r e c t a l cancer (colon, 1; rectum, 2) were examined. Mucosa specimens resected from tissues a f t e r s u r g i c a l o p e r a t i o n were immediately frozen i n a deep-freezer a t -70°C u n t i l used. Urine samples one day before and one week a f t e r s u r g i c a l operation f o r eight p a t i e n t s ( t h r e e male, f i v e female; age, 38-76) w i t h malignant c o l o r e c t a l cancer (colon, 1; rectum, 7 ) were c o l l e c t e d i n the morning a f t e r a 12-h f a s t i n g period. The histopathological s t a g e s of these cases were a s follows: s t a g e 11, 2; s t a g e 111, 3; s t a g e IV, 1; recurrence, 2. Urine samples from s i x t e e n normal s u b j e c t s (eight male, eight female; age, 35-72) were obtained from the PL Osaka Health Control Center. 9.2.7 Samol e Preoarati on Each mucosa specimen (1.25 g wet weight) was chopped o f f from the normal portion and the n e o p l a s t i c portion of mucosa of t h e same p a t i e n t using a s c i s s o r . The chopped mucosa were immediately minced w i t h a s c i s s o r t o f a c i l i t a t e the following homogenization. After t h e a d d i t i o n of 3 ml of cold water, the minced mucosa samples were homogenized u s i n g a pre-chi 11 ed micro Wari ng b l ender ( N K Micronizer; Nihon S e i k i , Tokyo, Japan). After a s t a i n l e s s s t e e l blender vessel had been washed w i t h 1 m l of cold water, 5 m l of cold PCA (5%, w/v) were added t o the homogenized mucosa and the mixture was vortexed vigorously f o r about 3 m i n . After standing i n i c e about 30 m i n . , the mixture was c e n t r i f u g e d a t 1500 g f o r about 10 m i n . and the s u p e r n a t a n t was a d j u s t e d t o approximately pH 5 w i t h 10 M potassium hydroxide. The pH-adjusted sample was centrifuged t o remove precipi t a b l e p e r c h l o r a t e and the s u p e r n a t a n t f l u i d s were s t o r e d a t -20°C u n t i l HPLC a n a l y s i s .

C298

I n t h e p r e l i m i n a r y experiments, homogenized mucosa were f r a c t i o n a t e d u s i n g t h e d i f f e r e n t i a l c e n t r i f u g a t i o n method. The perch1 o r i c a c i d s o l u b l e f r a c t i o n s o f 105,000 g s u p e r n a t a n t were used as t h e sample f o r HPLC a n a l y s i s . U r i n e samples were s t o r e d a t -20°C and c e n t r i f u g e d a t l o w speed t o remove p r e c i p i tab1 e compounds b e f o r e use. Peak I d e n t i f i c a t i ons The UV-absorbing compounds i n t h e p e r c h l o r i c a c i d e x t r a c t s o f G I mucosa and i n t h e p r e f r a c t i o n a t e d u r i n e s were i d e n t i f i e d on t h e b a s i s o f t h e r e t e n t i o n times, simultaneous i n j e c t i o n o f standards, peak-height r a t i o s , UV-absorption s p e c t r a o f f r a c t i o n a t e d HPLC peaks, and t h e enzymatic peak s h i f t , as developed by Brown and coworkers ( r e f s . 4, 49, 50). Moreover, t h e n u c l e o s i d e s were conf i r m e d by t h e HPLC s e p a r a t i o n o f samples p r e f r a c t i o n a t e d on t h e boronate a f f i n i t y g e l column. 9.2.8

9.2.9

B i ochemi c a l s The f o l l o w i n g standard compounds used i n o u r experiments were creatinine ( C r t ; SIGpurchased from Sigma ( S t . Louis, MO): C4255), p s e u d o u r i d i n e ($; P0509), u r i c a c i d (U.A.; U2625), hypoxa n t h i n e (Hyp; H9377), x a n t h i n e (Xan; X0125), u r i d i n e (Urd; U3750), M5001) , 7-methylguanosine (m7Guo; l - m e t h y l a d e n o s i ne (ml Ado; M0627), in o s i ne (Ino; 14125), guanosi ne (Guo; G6752), 4 - t h i o u r i d i ne (S4 Urd; T4509), l-methyl in o s i n e (ml Ino; M8257), l - m e t h y l guanosi ne (ml Guo; M6254) , and N2 -methyl guanosine (m2 Guo; M4004), and adenosine (Ado; A9251). The f o l l o w i n g enzymes were a l s o from Sigma: u r i c a s e (U3250) and x a n t h i n e o x i d a s e (X1875). N2 ,N2d i m e t h y l guanosi ne (m: Guo; Vega-10452) was purchased from Vega Biochemical s (Tucson, AZ) and u r a c i 1 (Ura; WAKO-214-00061) from Wako (Osaka, Japan). RESULTS AND DISCUSSION ChromatoaraDhv o f Standard ComDounds The chromatogram i n F i g u r e 9 . 1 ( r e f . 35) shows t h e s e p a r a t i o n o f 0.7-3.5 nmol o f each standard o f s i x t e e n n u c l e o s i d e s , bases, and t h e i r metabol it e s i n terms o f t h e reversed-phase HPLC condit i o n s d e s c r i b e d under M a t e r i a l s and Methods. The chromatographic c o n d i t i o n s a r e s l i g h t m o d i f i c a t i o n s o f t h o s e developed by H a r t w i c k

9.3

9.3.1

C299 e t . a 7 . (ref. 51) improving the s e p a r a t i o n a t the f r o n t p a r t of a chromatogram of the serum p r o f i l e ( r ef . 4 ) . The r e t e n t i o n time of u r i c acid was delayed by lowering the pH o f the mobile phase compared w i t h the serum p r o f i l e . 9.3.2 Chromatoarams of E x t r a c t s of Mucosa and Muscle The UV-absorbing compounds a r e e x t r a c t e d from the normal and n e o p l a s t i c p o r t i o n s of mucosa of i d e n t i c a l p a t i e n t s w i t h GI cancer u s i n g p e r c h l o r i c acid. Figure 9.2 (ref. 35) shows the chromatograms f o r p e r c h l o r i c acid e x t r a c t s of normal and n e o p l a s t i c mucosa of rectum, resected s u r g i c a l l y from a r e c t a l cancer p a t i e n t . The chromatograms i n Figure 9.3 (ref. 36) show the UV-absorbing compounds i n the normal and n e o p l a s t i c p o r t i o n s of mucosa, and of normal muscle of sigmoid colon from a p a t i e n t w i t h sigmoid colon cancer. These chromatograms a r e t y p i c a l of the p r o f i l e s observed f o r most GI mucosa. The peaks present i n the m a j o r i t y of GI mucosa samples a r e numbered.

U L

m L

=3

3

'

0

1

I

I

10

I

I

20 Time (rnin)

I

I

1

30

Figure 9 . 1 HPLC separat-ion of a standard s o l u t i o n of nucleosides, bases and the1 r metaboli tes.. Column: Develosi 1 ODs-5 (5-pm, 250 x 4.6 mm I.D., Nomura Chemicals). Eluents: A, 0.02 M potassium dihydrogen phosphate, pH 4.53; B , methanol-water ( 3 : 2 , v / v ) ;

C300

t o 40% B i n 35 min.; i g u r e 9 . 1 c o n t ' d . ) l i n e a r g r a d i e n t from 1.2 ml/min. Detector: UV a t 260 nm 0.16 a . u . f . s . ) . ITemFlow-rate. e r a t u r e : ambient. I n ' e c t i o n volume: 70 c l o i a s o l u t i o n 5 x A

10- P m o l / l i n each t h i o u r i d i n e and 1 x 35).

s t a n d a r d except 2.5 x 10m o l / l o f 4m o l / l o f N2-methylguanosine. (From r e f .

Based on a l l t h e d a t a from t h e i d e n t i f i c a t i o n techniques, t h e endogenous compounds p r e s e n t i n G I mucosa were id e n t i f i ed as u r a c i l , u r i c a c i d , hypoxanthine, x a n t h i n e , u r i d i n e , i n o s i n e , and guanosine. Peak 1 i n F i g u r e s 9.2 and 9.3 were c o n f i r m e d as u r a c i l from t h e c h a r a c t e r i s t i c change (bathochromic s h i f t ) o f t h e UVa b s o r p t i o n spectrum a t a l k a l i n e pH as shown i n F i g u r e 9.4 ( r e f . 36). The m a j o r i t y o f these peaks corresponded t o components found i n serum ( r e f . 4) and s a l i v a ( r e f . 15). Compared w i t h t h e p r o f i l e s o f serum and s a l i v a , t h e HPLC p r o f i l e s o f G I mucosa and muscle showed r e l a t i v e l y h i g h l e v e l s o f hypoxanthine, xanthine, and e s p e c i a l l y u r a c i l , and a low l e v e l o f u r i c a c i d as seen i n F i g u r e s 9.2 and 9.3. These f i g u r e s a1 so compare t h e chromatograms o f normal mucosa A conand those o f n e o p l a s t i c mucosa from t h e same p a t i e n t s . s i d e r a b l e i n c r e a s e i n s e v e r a l components was found i n n e o p l a s t i c mucosa as d e s c r i b e d i n d e t a i l l a t e r . Peak 2 i n F i g u r e 9 . 3 ~ r e p r e s e n t s u r i c a c i d and n u c l e o t i d e s . From t h e HPLC a n a l y s i s o b t a i n e d a f t e r u s i n g t h e b o r o n a t e a f f i n i t y gel column, i t was found t h a t muscle samples c o n t a i n e d r e l a t i v e l y 1arge amounts o f n u c l e o t i des. 9.3.3

Nucleosides i n E x t r a c t s o f Mucosa The n u c l e o s i d e s i n p e r c h l o r i c a c i d e x t r a c t s o f G I mucosa were p r e f r a c t i o n a t e d u s i n g t h e boronate g e l a f f i n i t y chromatographic procedure d e s c r i b e d under M a t e r i a1 s and Methods. F i g u r e 9.5 ( r e f . 35) shows HPLC s e p a r a t i o n o f n u c l e o s i d e f r a c t i o n s o b t a i n e d from t h e same mucosal specimen as d e s c r i b e d i n F i g u r e 9.2. The m a j o r peaks were i d e n t i f i e d as u r i d i n e , i n o s i n e , and guanosine. Pseudouridine and adenosine, which were c o n f i r m e d i n serum nucleosides ( r e f . 45), were n o t observed i n d e t e c t a b l k amounts i n p e r c h l o r i c a c i d e x t r a c t s o f mucosa. An unknown peak w i t h r e t e n t i o n t i m e of about 7.5 min. was suggested t o c o n t a i n n u c l e o t i d e s

C301 6

3

1 12

I

I

1

I

20 Time (min)

I

3

5

I

I

6

I

I

10 20 Time ( m i n )

0

1

30

10

1

I

1

I

0

I

I

I

30

3"

Chromatograms of perchl o r i c acid e x t r a c t s o f normal mucosa a) and neoplastic mucosa (b) of the rectum from a p a t i e n t wFigure i t h ma ignant rectal cancer. Injection volume: 100 1 , corresponding t o 13.9 m of wet mucosa. For chromatograp i c condit i o n s , see Figure 8.1. Peaks: 1 = u r a c i l ; 2 = u r i c acid; 3 = hypoxanthine; 4 = xanthine; 5 = uridine; 6 = inosine; 7 = guanos i n e . (From r e f . 35).

f:

such as ATP and ADP from the retention behavior. 9.3.4

Comoound Levels in Mucosa from Cancer Patients The concentrati ons o f endogenous compounds i n perchl o r i c aci d e x t r a c t s o f GI mucosa were determined by the HPLC method described

C302 3

I ,

0

I

I

I

10

20

30

20

30

1 345

0

10

3

6

(C)

Figure 9.3 Chromatograms of p e r c h l o r i c a c i d e x t r a c t s of normal mucosa ( a ) , n e o p l a s t i c mucosa (b) and normal muscle (c) of the siqmoid colon from a p a t i e n t with malignant sigmoid colon cancer. I n j e c t i o n volume: 100 p l . For chromatogra h i c c o n d i t i o n s , see Figure 9.1, and f o r peaks, s e e Figure 9.2. (From r e f . 36).

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200

250

300

350

Wavelength (nm)

Figure 9.4 UV absorption s p e c t r a of peak 1 ( u r a c i l ) i n Figure 9.2 and 9.3. (From r e f . 36).

above. Table 9 . 1 ( r e f . 35) shows the l e v e l s of these compounds i n normal and n e o p l a s t i c mucosa from the same p a t i e n t s w i t h col o r e c t a l cancer. These compound 1 eve1 s were i 1 1 u s t r a t e d in Figure 9.6 ( r e f . 36). A s i g n i f i c a n t e l e v a t i o n of u r a c i l was found i n n e o p l a s t i c c o l o r e c t a l mucosa (adenocarcinoma) from e i g h t p a t i e n t s w i t h c o l o r e c t a l cancer ( P < 0.01, s t a t i s t i c a l l y s i g n i f i c a n t with the paired t t e s t ) . The mean l e v e l o f u r a c i l i n n e o p l a s t i c c o l o r e c t a l mucosa was 2.7-fold higher than i n normal mucosa a s seen i n Table The e l e v a t i o n of the u r a c i l l e v e l i n n e o p l a s t i c mucosa t o 9.1. the same e x t e n t was a l s o observed i n another three p a t i e n t s with c o l o r e c t a l cancer examined i n the prel imi nary experiments.

C304

-E 0

W

N

>

2

v

I '

W V

K

m L

n

0 v)

n

4:

0

10

20

30

Time (min) 5

6

-E 0

W

N

3 > v

(u

U

c m

1?,

n L

0 v)

n

4:

L

1

0

10

20

30

Time (min)

F i g u r e 9.5 Chromatograms o f n u c l e o s i d e s i n p e r c h l o r i c a c i d e x t r a c t s o f normal mucosa (a) and n e o p l a s t i c mucosa (b) o f t h e rectum from t h e same p a t i e n t as i n F i g u r e 9.2. I n j e c t i o n volume: 100 p l , corresponding t o 27.8 mg o f wet mucosa. F o r chromator a p h i c c o n d i t i o n s , see F i g u r e 9.1 and f o r peaks, see F i g u r e 9.2. From r e f . 35).

9

I n t h e n e o p l a s t i c mucosa o f colorectum, t h e l e v e l s o f hypoxa n t h i n e and u r i d i n e were a l s o s i g n i f i c a n t l y h i g h e r t h a n those i n normal mucosa (P < 0.05, w i t h t h e p a i r e d t t e s t ) . However, an i n c r e a s e i n t h e u r a c i l l e v e l i n n e o p l a s t i c mucosa was found i n o n l y one o u t o f f o u r p a t i e n t s w i t h g a s t r i c cancer, as shown i n Table 9.2 ( r e f . 35) and F i g u r e 9.7 ( r e f . 36). I n the n e o p l a s t i c mucosa o f stomach, t h e l e v e l o f i n o s i n e was s i g -

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TABLE 9.1

Comparison of Perch1 o r i c Acid Extract Compound Levels Between Normal and Neoplastic Mucosa from Col orectal Cancer P a t i e n t s (from ref. 35)

I

Pati entsa (cancer)

Mucosa

Level o f compounds (nmol g wet weight of mucosa UraD U.A. Hypc Xan UrdC Ino Guo

#38 F , 66)

152 757

221 583

760 1188

100 205

474 371

564 771

333 357

400 715

428 1216 152 2045

716 1212

374 787

306 483

969 1684

818 285 708 409

303 923

187 584

393 817

712 1276

269 399

210 207

671 66.1 515 23.1

155 557

790 512

1010 1969

297 679

229 584

584 549

84.6 102 490 408

727 1976

333 822

213 601

531 10.9 512 65.2

(rectum)

Normal Neopl a s t i c Normal Neoplastic Normal Neopl a s t i c Norma 1 Neopl a s t i c Normal Neoplastic Norma 1 Neopl a s t i c Normal Neoplastic Normal Neoplastic

315 598

904 1236

705 596

275 288

296 548

Mean

Normal

(colon)

#lOO(F, 72) (si gmoi d ) #106(M, 52) (si gmoi d ) #111(M,

66)

(sigmoid)

#101(F, 56)

(rectum)

#105(F, 63)

( r e c t um)

#107(M, 49)

(rectum)

#108(M, 65)

(S.D.)

287 448

703 151 1141 199

167 143

11.9 10.5

153 155

579 563

58.2 48.5

346 657

231 45.2 409 67.4 33.9 97.2

46.0 62.8

29.8 44.1

522 233 420 37.8 221 375 858 (124) (206) (207) (232) (66.9) (191) (20.0)

Neoplastic 587

520 52.4 472 1518 739 388 (186) (189) (467) (311) (203) (214) (27.3)

a s e x ( F , f e m a l e ; M , m a l e ) and a g e a r e g i v e n i n p a r e n t h e s e s . b l %s i g n i f i c a n t w i t h t h e p a i r e d t t e s t . c5% s i g n i f i c a n t w i t h t h e p a i r e d t t e s t .

n i f i c a n t l y higher than t h a t i n normal mucosa according t o the paired t t e s t ( P < 0.05). In 1954, Horrigan ( r e f . 52) reported an increased excretion of urinary uraci 1 i n p a t i e n t s w i t h chronic myel ocyti c 1eukemi a (approximately twice the normal excretion). This observation was confirmed i n p a t i e n t s w i t h chronic and acute leukemia by Adams e t .

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Ura

U.A.

Ino

Guo

2000

1500 5 wl

0 V

3

E '+-

0

c,

r

m

'5 1000 3 c, W

3

cn 1 7

E 500

0

Figure 9.6 The levels of compounds i n perchloric acid extracts of normal .and neoplastic mucosa from colorectum, resected surgically from eight patients w i t h colorectal cancer. The broken lines are the mean levels. The d o t s represent males, the crosses females. Abbreviations used: Ura = uracil; U.A. = uric acid; Hyp = hypoxanthive; Xan = xanthine; Urd = uridine; Ino = inosine; Guo = guanosine. (From ref. 36). a l . (ref. 53) and i n a child w i t h a malignant tumor,of the b r a i n by Berglund e t . a 7 . ( r e f . 54).

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TABLE 9.2

Comparison of Perchlori c Acid E x t r a c t Compound Levels Between Normal and Neoplastic Mucosa from G a s t r i c Cancer P a t i e n t s (from ref. 35) Pati e n t s a (cancer)

Mucosa

Level of compounds (nmol of mucosa UraD U.A.

Hyp

Xan

Urd

InoD Guo

#102(M, 41)

(stomach)

Normal Neoplastic

169 139

349 368

1095 647

#104(M, 58)

(stomach)

Normal Neoplastic

544 420

1643 625

630 1940

1311 379 424 434

100 579

10.5 77.9

#109(F, 63)

Normal Neoplastic

325 319

352 934 7 1 3 776

697 418 164 344

255 948

27.9 5.81

(stomach)

81)

Normal Neoplastic

315 662

470 599

869 1474

Mean (S.D.)

Normal

338 (155

704 (629

882 (193

Neoplastic

385 416 1209 363 280 813 63.9 (218) (257) (608) (226) (145) (368) (47.8)

(stomach)

#llO(M,

251 248 512 209 94.8 457

882 656

393 249

66.3 52.0

299 16.4 1268 120

785 360 292 30.3 (439)(76.1)(170)(25.1)

a s e x ( F , f e m a l e ; M , m a l e ) and a g e a r e g i v e n i n p a r e n t h e s e s . b5% s i g n i f i c a n t w i t h t h e p a i r e d t t e s t .

In the salvage pathway of pyrimidine metabolism, d i hydroura c i l dehydrogenase (DHUDH, E.C. 1.3.1.2) i s a r a t e - l i m i t i n g enzyme of the degradation system of u r i d i n e and thymidine. A d e c r e a s e i n DHUDH a c t i v i t y has been observed i n r a t hepatoma ( r e f . 55), embryonic l i v e r ( r e f . 55) and human leukemia ( r e f . 56). The e l e v a t e d level of u r a c i l i n n e o p l a s t i c mucosa of c o l o r e c t a l cancer p a t i e n t s observed i n this study may be r e l a t e d t o the d e c r e a s e i n DHUDH a c t i v i t y . The a c t i v i t y of xanthine oxidase (E.C. 1.2.3.2), the r a t e 1 imi t i n g enzyme of i n o s i n e monophosphate (IMP) catabolism, has been observed t o decrease i n hepatoma and i n o t h e r tumors ( r e f . 55). The increased level of hypoxanthine and xanthine i n neop l a s t i c mucosa from colorectum we observed may be caused by a

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decrease i n this enzyme a c t i v i t y . 9.3.5 Urinary. Nucleosides Analysis Urinary nucleosides were i s o l a t e d by u s i n g a boronate a f f i n i t y gel column. They were separated and q u a n t i f i e d by the HPLC

U.A

Ura

2000

-

1500

-

1000

-

Hyp

Urd

Xan

Ino

Guo

a

m 0 U S

E

lt

0

c,

L:

:.

a,

3

c,

% m \ 7

E=

500-$

0 V

i

-

\ u

V .r

u

.r

Y

U .r

U

V

.r

U

U .r

CI

Figure 9.7 The levels of compounds i n p e r c h l o r i c a c i d e x t r a c t s o f normal and n e o p l a s t i c mucosa from the stomach, resected s u r g i c a l l y from f o u r p a t i e n t s w i t h a s t r i c cancer. For symbols and abbrevit i o n s , see Figure 9.6. !From r e f . 36).

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procedure described. Samples equivalent t o 200 ,ul of urine were used f o r each HPLC analysis. Urine samples were collected before and a f t e r surgery from each colorectal cancer p a t i e n t and analyzed w i t h the HPLC as shown i n Figure 9.8 ( r e f . 35). In many instances, the l e v e l s o f the urinary nucleosides i n the pre- and postoperative samples showed considerable differences (maximum tenfold). 1 (a)

2

n

6

1.:

1 I1

I

0

I

1

I

10

I

20

I

I

I

30

T i m e (min)

1 n

2

T i m e (min)

Figure 9.8

Chromatograms of nucleosides in urine taken before (a)

and a f t e r surgical operation (b) from a p a t i e n t with malignant rectal cancer. Injection volume: 200 p l , equivalent t o the same

volume of urine. For- .chromatographic conditions, see Figure 9.1. Peaks: 1 = pseudouridine; 2 = uridine + 1-methyladenosine; 3 = 1methylinosine + an unknown compound; 4 = 1-methyl uanosine; 5 = N2-methylguanosine; 6 = N2 ,N*-dimethylguanosine. (from r e f . 3 5 ) .

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Based on the data from the i d e n t i f i c a t i o n techniques and the r e s u l t s reported by Gehrke e t . a l . ( r e f s . 41, 42), t h e nucleosides present in the majority of urine samples were i d e n t i f i e d as pseudouridine, 1-methylguanosine, N2-methylguanosine and N2, N 2 dimethylguanosine. The peak with the retention time of 13 min. (peak 2) contained 1-methyladenosine + uridine and the peak w i t h the retention time of 23.5 min. (peak 3) was i d e n t i f i e d as 1methyl inosi ne together with an unknown compound. The recovery f o r the boronate a f f i n i t y gel column has determined by processing 0.5 m l of a standard mixture on the a f f i n i t y column. The average recoveries of two runs were 93.9% f o r pseudouridine, 105.0% f o r uridine, 98.5% f o r inosine, 93.3% f o r guanosine, 96.7% f o r 1-methyl inosine, 93.8% f o r 1-methylguanosine, 97.2% f o r N2-methylguanosine, 85.0% f o r adenosine and 97.3% f o r N 2 ,N2-dimethylguanosine. The recoveries of the nucleoside standards were s l i g h t l y lower than those reported by Gehrke e t . a 7 . ( r e f . 41). 9.3.6

Ouantitation of Urinary Modified Nucleosides The concentration of modified nucleosides i n urine samples taken before and a f t e r surgery from the eight p a t i e n t s w i t h col orectal cancer and from sixteen normal subjects was determined by the HPLC method. The urinary nucleoside l e v e l s were then converted on the basis of the urinary c r e a t i n i n e level of each sampl e . Figure 9.9 ( r e f . 36) and Table 9.3 ( r e f . 35) show a comp a r i son of urinary nucl eoside 1 eve1 s pre- and post-operation f o r cancer p a t i e n t s and normal subjects. Contrary t o previous reports (refs. 37-40), the present r e s u l t s did not show an elevation of modified nucleosides in urine from p a t i e n t s with colorectal cancer. The low creatinine l e v e l s in post-operative p a t i e n t s seems t o cause the r e l a t i v e l y h i g h nucleosides/creatinine l e v e l s i n these patients. However, the difference of c r e a t i n i n e l e v e l s may merely indicate t h a t the change in urinary compound l e v e l s i s caused by changes i n the physiological condition of the p a t i e n t s between pre- and post-operation. Several investigators have reported an elevation of the modified nucleoside levels i n urine from cancer p a t i e n t s ( r e f s .

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37-40). Recently, Tamura e t . a l . (ref. 57) reported t h a t the urinary pseudouridine level of patients w i t h l u n g cancer was significantly higher t h a n those of controls. However, our results for the same Japanese showed no elevation of modified nucleosides i n the pre-operative urine of colorectal cancer patients compared w i t h the post-operative urine and normal urine. mZGuo 4 60

23

a,

c

.r

.r

S

S

.r

.r

+-‘ ro

c,

40

V

L W V

7

7

L 0

E 2

E

a-

7 \

\

7

7

0

0

E c

E

=

20

1

0 0

0

W

a >

.r

Q

0

> .r

r

c,

o

L

W

a J a

, - n o m o c, I

,

z

z

EO &L v Ol a

a

L a J W a

-

r o o

P

r o c

r o L

r o L

rrrL

o

I

m

n

z

L a J W Q

~

o

n

I

o

L a J W P

7

r o o

a

o

I

E & O% OE L& O% OE L& O Z O L a

a

a

z

n

a

Figure 9.9 The leve1.s of uri.nary modified nucleosides pre- and post-operation for eight patients w i t h colorectal cancer, a n d sixteen normal subjects. For symbols, see Figure 9.6.. Abbreviations used: $ = pseudouridine; m 1 G u o = 1-methylguanosine; m2Guo = N2 -methyl guanosi ne; ;Guo = N Z ,N2 -dimethyl guanosi ne. (From ref. 36).

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The mean 1eve1 of u r i nary pseudouri d i n e / c r e a t i nine i n normal subjects i n the present study (see Table 9.3) was s l i g h t l y higher than t h o s e reported by Speer e t . a 7 . ( r e f . 39) and Tamura e t . a 7 . ( r e f . 57), b u t lower than those r e p o r t e d by Evans e t . a 7 . ( r e f . 13). These d i f f e r e n c e s might be caused b y - d i f f e r e n c e s i n physiol o g i c a l environments including aging and n u t r i t i o n , and problems of q u a l i t y control i n microanalysis. In f a c t , Tritsch e t . a 7 . ( r e f . 58) reported t h a t the pseudouridine/creatinine l e v e l of o l d e r p a t i e n t s (60-90 y e a r s ) was almost t w i c e higher than t h a t of 20-55-year-01 d normal s. TABLE 9.3 Urinary Modi f i ed Nucl e o s i d e Level s o f Col o r e c t a l Cancer P a t i e n t s (Pre- and Post-Operation) and Normal S u b j e c t s (from ref. 35.) Uri vary modified nucleosidesa

Pseudouridine 1-Methylguanosine N2 -Methyl guanosi ne N2 ,N* -Dimethyl guanosi ne Urinary c r e a t i n i n e b

Col o r e c t a l cancer p a t i e n t s (n=8)

Normal (n=16)

Pre-operative

Post-operati ve

33.9 2 5.17 1.21 2 0.411 1.06 2 0.258

43.9 2 9.36 1.49 2 0.704 1.38 2 0.586

35.6 2 5.00 1.07 2 0.325 0.805 2 0.193

2.20 2 0.399 105.1 2 89.6

2.70 2 0.764 60.9 2 53.4

2.09 2 0.315 101.2 2 44.7

aunits: nmo7/pmo7 c r e a t i n i n e . Mean S.D. were calcu7ated from t h e d a t a o f e i q h t p a t i e n t s w i t h c o 7 o r e c t a 7 c a n c e r and s i x t e e n normal s u b j e c t ; . ’ b C r e a t i n i n e l e v e l ( u n i t s : mg/d7) was a n a l y s e d by t h e J a f f e method using a G r i n e r s e l e c t i v e analyser II C ( G r i n e r , Langental, S w i t zerland)

.

Very r e c e n t l y HPLC methods t o measure modified blood serum have been developed by Kuo e t . a 7 . Colonna e t . a 7 . ( r e f . 45). Their r e s u l t s s t r o n g l y modified nucleoside e s t i m a t i o n i n serum should be biochemical marker of the n e o p l a s t i c d i s e a s e ( r e f s .

nucleosides i n ( r e f . 44) and

syggested t h a t e x p l o i t e d as a 38, 44).

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9.4

9.4.1

FUTURE PROSPECTS AND IMPACT

ADDlication of Tumor Markers t o Periodical Tests In the past, many potential tumor markers have been studied i n an attempt t o i d e n t i f y the presence of e a r l y cancer o r cancer risk. Unfortunately, most of these markers have not been found t o be of value i n f u r t h e r ' t r i a l s . A t present the following markers have mainly been examined as oncofetal proteins, cancer-associated potential tumor markers: antigens, t i s s u e associated antigens, ectopic hormones, isozymes, tumor growth f a c t o r s and metabolic products. Among metabolic products, polyamines and modified nucleosides a r e closely related t o the elevated r a t e of neoplastic c e l l p r o l i f e r a t i o n , thus having l e s s s p e c i f i c i t y f o r a s p e c i f i c organ. These markers a r e r a t h e r useful as c l i n i c a l t e s t s f o r cancer screening i n health check-up system. However, t h e s e n s i t i v i t y of cancer detection w i t h the markers i s not s o h i g h , e s p e c i a l l y f o r the e a r l y detection of cancer. Moreover, the amount of t e s t - t o - t e s t fluctuation i n the assay needs t o be considered. A variety of nontumor-related f a c t o r s , including technical variation, therapy i t s e l f , and benign inflammatory o r other t r a n s i e n t diseases, could cause t r a n s i e n t fluctuations o r increases i n the assay. For almost a l l tests, therefore, i t i s important t o obtain repeated specimens over a period of time and t o observe p e r s i s t e n t or progressive increase i n marker 1 eve1 s. In 1970 and 1971, we established the P L Health Control Center i n Tokyo and Osaka, respectively, and have developed t h e periodical health check-up system supported by c l i n i c a l t e s t s twice a year f o r subjects w i t h membership ( r e f . 59). Using these periodical t e s t data stored i n the database system, we have s t r e s s e d the importance of subject-specific normal range instead of conventiona l , v i z . populational normal range f o r the e a r l y detection of adult diseases showing actual cases ( r e f . 60, 61). We a l s o showed t h a t risk f a c t o r s f o r cancer were calculated u s i n g these periodical t e s t data, and would be u t i l i z e d t o predict the potential cancer p a t i e n t s ( r e f . 62). In our center, several cancer markers such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) were already added

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i n t o screening t e s t menu. I f the analytical technique and clinical value of modified nucleosides, especially i n serum o r plasma, would be established, the subject-specific normal ranges f o r the markers m i g h t show t h e i r effectiveness only i n the periodical t e s t system described. 9.4.2 HPLC Analysis i n Nucleic Acid Research As reviewed i n the book edited by Brown ( r e f . 2 ) , HPLC techniques have been applied t o the research o f nucleic acids and t h e i r metabolites covering wide s c i e n t i f i c f i e l d s . The technique i s a l s o playing a v i t a l r o l e i n the s t u d i e s of the diseases related t o the altered l e v e l s of nucleic acid metabolites and related t o the s p e c i f i c enzymatic disorder of purine and pyrimidine metabolism, and neoplastic diseases ( r e f . 63). In t h e near future, other disorders re1 ated t o purine and pyrimidine metabolism should be found w i t h the help of HPLC. I t has been known t h a t neoplastic diseases o r i g i n a t e i n the activation of human oncogenes due t o a s p e c i f i c r e t r o v i r u s , point mutation i n a s p e c i f i c oncogene, amplification of a s p e c i f i c oncogene and rearrangement of s p e c i f i c chromosomes including a special oncogene. In addition, several genetic diseases have been d i r e c t l y diagnosed w i t h the r e s t r i c t i o n fragment length polymorphism (RFLP) on electrophorogram due t o the mutation of a s p e c i f i c gene, and by the d i r e c t analysis of the mutation w i t h synthetic DNA probes. Although these abnormalities of DNA have h i t h e r t o been analyzed w i t h electrophoresis, HPLC could be u t i l i z e d t o analyze the r e s t r i c t i o n fragments of DNA including the oncogene and the s p e c i f i c gene related t o genetic diseases. HPLC w i l l become a major tool not only i n biochemistry, i n c l i n i c a l chemistry, i n physiology, and i n pharmacology b u t a l s o i n pathological studies of d i sease processes and i n genetic engi neeri n g f o r oncogene and DNA diagnosis. 9.5

SUMMARY

Reversed-phase h i gh-performance 1 i q u i d chromatography (HPLC) has been used t o identify nucleosides, bases and their metabolites i n perchloric acid e x t r a c t s of g a s t r o i n t e s t i n a l mucosa, and t o determine the level of these nucleic acid metabolites. By comparing the l e v e l s of these compounds i n the normal portion w i t h

C315

t h e l e v e l s i n t h e n e o p l a s t i c p o r t i o n o f mucosa, r e s e c t e d s u r g i c a l l y from t h e same p a t i e n t s w i t h m a l i g n a n t cancer, i t was found t h a t u r a c i l was s i g n i f i c a n t l y e l e v a t e d i n t h e n e o p l a s t i c c o l o r e c t a l mucosa (adenocarcinoma) o f e i g h t p a t i e n t s w i t h c o l o r e c t a l cancer (P < 0.01, s t a t i s t i c a l l y s i g n i f i c a n t w i t h t h e p a i r e d t t e s t ) . The mean l e v e l qf u r a c i l i n n e o p l a s t i c c o l o r e c t a l mucosa was 2 . 7 - f o l d h i g h e r than t h a t i n normal mucosa. However, i n n e o p l a s t i c g a s t r i c mucosa, o n l y one o u t o f f o u r p a t i e n t s w i t h g a s t r i c cancer showed e l e v a t e d u r a c i 1 . I n neopl a s t i c mucosa, t h e 1eve1 s o f h b o x a n t h i ne and u r i d i n e f o r c o l o r e c t a l cancer, and i n o s i n e f o r g a s t r i c cancer, were a l s o s i g n i f i c a n t l y h i g h e r than those i n normal mucosa (P < 0.05, w i t h t h e p a i r e d t t e s t ) . The u r i n a r y m o d i f i e d n u c l e o s i d e s were p r e f r a c t i o n a t e d w i t h a boronate a f f i n i t y g e l column, and t h e i r l e v e l s were determined by t h e same HPLC method. The c o n c e n t r a t i o n s o f pseudouridine, 1methylguanosine, N2-methylguanosine, and N2, N2 -dimethylguanosine i n u r i n e samples taken b e f o r e and a f t e r s u r g e r y from t h e e i g h t p a t i e n t s w i t h ma1 i g n a n t c o l o r e c t a l cancer were determined t o compare t h e d i f f e r e n c e s between b o t h p h y s i o l o g i c a l s t a t e s . C o n t r a r y t o o t h e r r e p o r t s , no s i g n i f i c a n t d i f f e r e n c e s i n t h e l e v e l s o f m o d i f i e d n u c l e o s i d e s were observed i n p r e - and p o s t o p e r a t i v e u r i n e s from p a t i e n t s w i t h c o l o r e c t a l cancer and normal u r i nes. 9.6

ACKNOWLEDGMENTS The a u t h o r acknowledges D r . P.R. Brown (The U n i v e r s i t y o f Rhode I s l a n d ) f o r h e r k i n d and h e l p f u l guidance i n HPLC t e c h niques, D r . K. Kiyoshima and M r . H. Murao (PL Osaka H e a l t h C o n t r o l Center) f o r t h e measurement o f c r e a t i n i n e and t h e s u p p l y o f some samples, D i r e c t o r S. Oda and M r . K. Imaizumi (PL B o t a n i c a l I n s t i t u t e ) f o r t h e use o f equipment and f o r advice, and D r . T. Yasaka ( D i r e c t o r , PL Medical Data Center) and D r . K. Shindo and D r . H. Yamamoto (Osaka U n i v e r s i t y , Medical School) as t h e co-workers o f p a r t o f t h i s study. T h i s s t u d y was supported by a g r a n t from P a t r i a r c h T a k a h i t o M i k i and t h e " P e r f e c t L i b e r t y " O r g a n i z a t i o n .

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9.7 REFERENCES 1. M. Z a k a r i a and P. R. Brown, 2. 3. 4.

5.

High-performance li u i d column chromatograph o f n u c l e o t i d e s , n u c l e o s i d e s an! bases, J . Chromatogr., $26 (1981) 267-290 P.R. Brown (Ed.), HPLC i n N u c l e i c A c i d Research: Methods and A p p l i c a t i o n s , Marcel Dekker, New York, 1984. K. Nakano, HPLC a n a l y s i s o f ox p u r i n e s and r e l a t e d compounds, J . Cazes and P. R. Brown i n : J . C. Giddings, E. Grushta Vol.'25, Marcel Dekker, New York, Eds. , Adv. Chromatogr., 1985 , pp. 245-277. 1.A. a r t w i c k , A.M. K r s t u l o v i c and P.R. Brown, I d e n t i f i c a t i o n and q u a n t i t a t i o n o f nucleosides, bases and o t h e r UV-absorb1 ng compounds in serum, us1 ng reversedTphase h i gh-performance I1 E v a l u a t i o n o f human sera, J . l i q u i d chromatogra h Chromatogr., 186 (1878) 654-676 W . E . Wung and S.B. Howell I Simuitaneous l i q u i d chromatography o f 5 - f l o u r o u r a c i l , u r i d i n e , h poxanthine, x a n t h i n e , u r i c a c i d a1 l o p u r i n o l , and o x i p u r i n o i n plasma, C1 i n . Chem., 26

b

Y

h198b) 1704-1708.

.

A. T a y l o r P. J . Dady and K. R. H a r r a , Q u a n t i t a t i v e h i g h erformance !i u i d chromatograph o f nuc eosides and bases i n Ruman plasma, Chromatogr., 183 (1980) 421-431 7. A. McBurney an4 T. Gibson, Reversed-phase p a r t i t i o n HPLC f o r d e t e r m i n a t i o n o f 1asma p u r i n e s and p y r i m i d i n e s i n sub ' e c t s w i t h gout and rena f a i l u r e , C l i n . Chim. Acta, 102 (19803 19-

6.

!

3

!

28.

8. R. J . Simmonds and R . A. Harkness,

High- erformance l i q u i d chromato r a p h i c methods f o r base and nuc e o s i d e a n a l y s i s i n e x t r a c e l y u l a r f l u i d s and i n c e l l s , J . Chromatogr., 226 (1981)

!

369-381. 9. M. Zakaria, P. R. Brown, M. P. Farnes and B: E. Barker,

HPLC a n a l y s i s o f a r o m a t i c amino acids, nucleosides, and bases i n l a m a o f acute lym h o c y t i c leukemics on chemotheraphy, C l i n . !him. Acta, 126 (19g2 69-80 B a l t k s a t and C. Gonnet, Hypoxanthine 10, R. Boulieu, C. Bory and x a n t h i n e l e v e l s determined by high-performance li u i d chromatogra hy i n plasma, e r y t h r o c y t e , and u r i n e samples ?o,m h e a l t h y suEjects. The problem o f .hy o x a n t h i n e l e v e l evol u t i o n as a f u n c t i o n o f time, Anal. Bioclem., 129 (1983) 398-

b.

404. 11. A. M. K r s t u l o v i c , R.A. H a r t w i c k and P.R.

Brown, High-performavce li u i d chromatographic d e t e r m i n a t i o n o f serum UV prof i l e s o normal S u b j e c t s and p a t i e n t s w i t h b r e a s t cancer and benign f i b r o c y s t i c changes, C l i n . Chim. Acta, 97 (1979) 159-

?

12 *

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J . F. Kuttesch, F.C. Schmalstieg and J.A. Nelson, A n a l y s i s o f

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a

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C319

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g'":

Y

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6 d

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-.A

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34u.

G . L. T r i t s c h , J. M. Luch, J. T. Evans and A. M i t t e l m a n , Age dependence o f human u r i n a r y pseudouri d i ne e x c r e t i o n , B i ochem. Med., 22 (1979) 387-390; T. Yasaka, K. Nakano and T. Mizunuma, C l i n i c a l Data Evalua t i o n and t h e PL H e a l t h C o n t r o l I n f o r m a t i o n System, Computer Pro rams i n Biomed., 8 (1978) 51-70. T. ?asaka and K. Nakano, Data-Base designs and a p p l i c a t i o n s o f s u b j e c t - s p e c i f i c ph s i o l o g i c a l f l u c t u a t i o n s i n t h e PL h e a l t h c o n t r o l system, f n f o r m a t . Med., C o l l . I R I A Toulouse, 1976 I R I A , Domaine de Voluceau - Rocquencourt, trance, pp. 737-f60. K. Nakano, T. Atobe, Y. H i r a k i and T. Yasaka, E s t i m a t i o n o f s u b j e c t - s p e c i f i c normal ranges based. on some s t a t i s t i c a l models o f an i n d i v i d u a l ' s p h y s i o l o g i c a l v a r i a t i o n s , Med. Inform., 6 1981) 195-205. K. Nakano, Yasaka, M. S h i n b o r i and M. Tamura, R i s k f a c t o r s A mu1 t i v a r i a t e a n a l y s i s of f i v e - y e a r s ' f o r g a s t r i c cancer: o b s e r v a t i o n i n t h e PL h e a l t h c o n t r o l system and i t s a p p l i c a t i o n s , Med. Inform., 6 (1981) 115-130. M. Zakaria, N u c l e i c a c i d c o n s t i t u e n t s i n d i s e a s e processes i n : P. R. Brown (Ed.), HPLC i n N u c l e i c A c i d Research, Marcel Dekker, New York, 1984, pp. 365-388.

f.

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CHAPTER 10 MODIFIED NUCLEOSIDES AS BIOCHEMICAL MARKERS OF ASBESTOS EXPOSURE AND AIDS* OPENDRA K. WARMAl, P h . D . and ALF FISCHBEINZ, M . D . ' L a b o r a t o r y o f M o l e c u l a r B i o l o g y , AMC C a n c e r R e s e a r c h C e n t e r , 1600 P i e r c e S t r e e t , Denver, Colorado

2 D i v i s i o n o f E n v i r o n m e n t a l and O c c u p a t i o n a l W e d i c i n e , Mount S i n a i S c h o o l o f M e d i c i n e o f t h e C i t y U n i v e r s i t y o f New Y o r k , One G u s t a v e L . Levy P l a c e , New Y o r k , New Y o r k

TABLE OF CONTENTS

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . C321 10.2 Results and Discussion . . . . . . . . . . . . . . . . . C324 10.2.1 Asbestos Exposure and Modified Nucleosides . . . . C324 10.2.2 Urinary Excretion of Modified Nucleosides in Patients With Various Manifestations of Infection With H I V . . . . . . . . . . . . . . . . . . C331 10.3 References . . . . . . . . . . . . . . . . . . . . . . C337 10.1 INTRODUCTION Cancer p a t i e n t s and tumor-beari ng animals excrete in t h e i r urine increased amounts of modified purines and pyrimidines ( r e f s . 1-6). These modified nucleosides, synthesized a t the macromolecular level ( r e f . 7 ) , are primarily constituents of t R N A and t o a l e s s e r extent of other RNAs. When RNA i s catabolized, most of these modified nucleosides cannot be r e u t i l i z e d ; consequently they a r e excreted. Pioneering s t u d i e s o f Borek e t a l . ( r e f . 8) have suggested t h a t excretion of elevated amounts of modified nucl eosides in tumor-beari ng animal s resul t s from increased t R N A turnover r a t h e r than from c e l l death. The molecular mechanisms of elevated excretion a r e unclear. Extracts of neopl a s t i c t i s s u e s have aberrant t R N A methyltransferases ( r e f . 7 ) and i t has been suggested t h a t the high turnover of t R N A i s due t o rapid degradation of aberrantly modified tRNAs ( r e f s . 7 , 8 ) . * T h i s work was supported by USPHS Grants HL-32432, HD-20612 OH-02122, NIEHS Center Grant E S 00928, CDC Grant OH-02122 and a g i f t t o t h e AMC Cancer Research Center from G e r a l d M . Q u i a t .

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More recent studies indicate t h a t catabolism of other RNA species and altered RNA metabolism of host tissue (refs. 5, 9 ) may also contribute t o elevated urinary levels of modified nwleosides. The urinary excretion of modified nucleosides and j-AIBA (degradation product of thymine) expressed re1 a t i ve t o creati n i ne i s remarkably constant for normal 18-60 year o l d subjects (refs. 1, 10). Infants and children excrete lower levels o f modified nucleosides compared t o adults (ref. 2 ) . Patients suffering from non-cancerous diseases do n o t excrete significantly elevated levels of modified nucleosides (ref. 11), however, elevated excretion i s observed i n some patients w i t h acute hepatitis (ref. l l ) , g o u t and psoriasis (ref. 12). Children w i t h acute infections do n o t excrete significantly elevated levels of modified nucleosides when compared t o healthy children (ref. 13). S l i g h t elevations i n u r i n a r y modified nucleoside excretion from subjects w i t h bacterial pneumonia and significant elevations from some subjects w i t h urinary tract infections have been noticed ( r e f . 143. Therefore, urinary excretion of modified nucleosides should be interpreted w i t h caution i n subjects w i t h syndromes t h a t interfere w i t h the creatinine o u t p u t . Tables 10.1 and 10.2 show modified nucleoside content i n urine and serum from healthy adults (refs 10,48). Work from our laboratory i n collaboration w i t h Drs. Brewer, Gehrke, and Waalkes (ref. 1) and others (refs. 3-6) have shown t h a t 1 eve1 s of urinary modified nucl eosi de excretion correlate w i t h stage of disease and response t o therapy. Elevated levels of urinary modified nucleosides i n cancer patients return t o normal levels soon after effective therapy. Elevated levels of modified urinary nucleosides and bases i n animals precede the appearance of tumor. Thomale and Nass (ref. 5) have studied the excretion of various breakdown products of t R N A by mice treated w i t h a carcinogen, 3-methylcholanthrene. The tumor developed i n s i t u i s palpable a f t e r sixteen weeks. Death usually occurs around the twenty-third week. Modified nucleosides of 24 hr urine samples were determined from the i n i t i a t i o n of the experiment u n t i l demise of the animals. By the seventh week, when the tumor was n o t diagnosable, excretion o f the nucleosides was elevated. In the sixteenth week, levels of the various nucleosides may be elevated a s much as 2- t o 4-fold above those of

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TABLE 10.1 M o d i f i e d Nucl e o s i des creatinine) N $ m1A

PCNR mlI m lG ac4C m2G mG; t6A B-A1 BA

49 42 39 49 22 32 47 46 20 36

Male

and

p-AIBA

Content

X

S.D.

N

23.3 1.70 1.08 1.36 0.88 0.52 0.39 1.27 0.58 6.14

2.70 0.32 0.24 0.21 0.16 0.08 0.09 0.17 0.10 3.59

54 38 37 47 15 23 41 46 11 120*

in

Urine

Femgl e

(nmol / w o l

X

S.D.

27.0 1.94 1.10 1.34 0.84 0.56 0.39 1.30 0.73 5.8

5.21 0.42 0.38 0.39 0.29 0.12 0.15 0.41 0.11 4.1

N , number o f subjects; X , mean; , standard d e v i a t i o n . These values are very s i m i l i a r t o those reported e a r l i e r ( r e f . 10 and 48) except t h a t t h e values for t 6 A have been corrected by a f a c t o r o f 0.55 due t o e a r l i e r discrepancy w i t h the standard. These corrected values are very s i m i l a r t o those determined by radioimmunoassay ( r e f . 53). We a r e g r a t e f u l t o Dr. Barbara Vold and Dr. Eckhard Schlimme f o g p r o v i d i n g authentic t 6 A . Males 18-59 years and females 18-60 years o l d . From Kuo e t a l . ( r e f . 51).

untreated control s. The p r e c i s e mol e c u l a r mechanism which g i v e s r i s e t o t h i s l a r g e e x c r e t i o n has n o t been a s c e r t a i n e d . We e x p l o r e d whether s i m i l a r e a r l y changes i n abnormal excret i o n o f m o d i f i e d n u c l e o s i d e s a r e e v i d e n t i n humans b e f o r e c l i n i c a l m a n i f e s t a t i o n s o f cancer become apparent. T h i s may be u s e f u l f o r d e t e c t i n g p r e c l in i c a l b i ochemi c a l changes p r e d i c t i v e o f f u t u r e neoplastic manifestations. We d e s c r i b e here r e s u l t s o f o u r ongoing s t u d i e s on asbestos-exposed workers who a r e a t h i g h n e o p l a s t i c r i s k and s u b j e c t s i n f e c t e d w i t h H I V , A I D S v i r u s . The a b b r e v i a t i o n s used a r e : Pseudouridine ($), l - m e t h y l adenosine (m1A) ; 2 - p y r i done-5-carboxami de-Nl-ri b o f u r a n o s i de (PCNR, a d e g r a d a t i o n p r o d u c t o f N A D t ) ; l-methyl i n o s i n e (mlI); l-methylguanosi ne (m1G) ; N2-methyl guanosi ne (mZG) ; N2N2-dimethylguanosine (m;G) ; N4-acetyl c y t i d i ne (ac4C) ; N6-threonyl adenosi ne ( t 6 A ) and p a m i n o i s o b u t y r i c a c i d (p-AIBA)

'

.

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10.2 RESULTS AND DISCUSSION 10.2.1 Asbestos EXDOSUre and M o d i f i e d Nucleosides Asbestos-associ a t e d diseases a r e o f g r e a t concern i n *nany countries. The c u r r e n t i n c r e a s e i n t h e i n c i d e n c e o f t h e s e diseases n o t e d i n t h e U n i t e d S t a t e s appears t o be r e l a t e d t o t h e l a r g e number of i n d i v i d u a l s who o v e r t h e p a s t t h r e e o r f o u r decades have been employed i n work environments i n which t h e r e was p o t e n t i a l f o r exposure t o hazardous l e v e l s o f a i r b o r n e asbestos I t has been e s t i m a t e d t h a t , s i n c e t h e 1940's, more t h a n fibers. 25 m i 11 i o n persons have experienced exposure t o asbestos a t t h e i r places o f work ( r e f . 15). A l o n g p e r i o d o f l a t e n c y between t h e onset of exposure and t h e c l i n i c a l m a n i f e s t a t i o n o f d i s e a s e i s one o f t h e c h a r a c t e r i s t i c s o f asbestos-associ a t e d d i s o r d e r s ( r e f . 16). T h i s a p p l i e s p a r t i c u l a r l y t o t h e m a l i g n a n t diseases, such as l u n g cancer, p l e u r a l and p e r i t o n e a l mesothel ioma, which have been s t r o n g l y a s s o c i a t e d w i t h asbestos i n e p i d e m i o l o g i c a l s t u d i e s ( r e f . 17). I n general, t h e d i a g n o s i s o f a s b e s t o s - r e l a t e d cancer i s made a t a stage when t h e disease i s f a r advanced, and t h e t h e r a p e u t i c p o s s i b i l i t i e s are very l i m i t e d . Because o f t h i s s i t u a t i o n and because o f t h e p u b l i c h e a l t h problem t h a t asbestos-i nduced diseases pose i n s o c i e t y , t h e r e i s an u r g e n t need t o develop new methods t o i d e n t i f y i n d i v i d u a l s who may be a t h i g h r i s k o f developing t h e s e types o f o c c u p a t i o n a l cancers, and t o i n c r e a s e t h e p o t e n t i a l f o r e a r l y d i a g n o s i s and more e f f e c t i v e t r e a t m e n t . The term "asbestos" i s used t o d e s c r i b e a group o f n a t u r a l l y o c c u r r i n g f i b r o u s s i 1 ic a t e s and a r e c h a r a c t e r i z e d by g r e a t t e n s i 1e s t r e n g t h and r e s i s t a n c e t o chemicals and heat. The d e f i n i t i o n o f asbestos i s l i m i t e d here t o t h e f i b r o u s m i n e r a l s o f t h e s e r p e n t i n e and amphibole s e r i e s A s b e s t i f o r m m i n e r a l s can be c a t e g o r i z e d i n t o two m a j o r subd i v i s i o n s , namely c h r y s o t i l e , which belongs t o t h e s e r p e n t i n e s e r i e s and t h e amphiboles, which i n c l u d e c r o c i d o l i t e , a c t i n o l i t e t r e m o l i t e , amosite and a n t h o p h y l l i t e . The c r y s t a l l i n e s t r u c t u r e s of b o t h c h r y s o t i l e and amphiboles have been c l a r i f i e d . C h r y s o t i l e c o n s i s t s o f a l a y e r of magnesium oxide-hydroxide octahedra bonded t o a layer o f s i l i c o n dioxide tetrahedra. The s h e e t l i k e l a y e r tends t o r o l l i t s e l f i n t o a h o l l o w t u b e w i t h t h e magnesium hydrox-

.

TABLE 10.2 Modified Nucleosides and B-AIBA Content in Serum

pmol/ml N $

m1I ml G ac4C m2G m:G t6A 8-AIBA

37 21 18 29 27 19 10 20

X

2810 41.5 41.0 71.6 19.8 47.1 38.2 2140

Ma1 e nmollpmol of creati nine -

U

523 9.80 17.1 18.9 7.59 20.0 12.6 960

X

40.3 0.61 0.61 1.04 0.29 0.73 0.59 32.4

pmol /ml U

8.07 0.16 0.27 0.27 0.12 0.40 0.18 14.7

N

48 35 16 36 33 29 19 27

T

2520 42.8 56.9 61.6 18.9 46.9 32.5 1680

Femal e nmollpmol o f creatinine -

U

538 17.2 18.4 20.1 5.72 21.4 10.5 684

X

46.3 0.76 1.15 1.11 0.35 0.91 0.62 30.7

U

12.5 0.29 0.55 0.35 0.13 0.50 0.23 12.5

D e t a i l s a r e same as f o r Table 9 . 1 . Males 19-56 years and females 21-52 years o l d . C r e a t i n i n e i n serum (males 13.8 nmol/ml) was determined by HPLC ( r e f . 52). 70.8 2 11.1; females 56.5

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ide on the outer surface. In contrast, amphiboles consist of doubl e chains of 1 inked si 1 i con-oxygen tetrahedra posi t i oned para1 1el t o the vertical crystal 1 ographi c axis and bound 1a t e r a l l y by m e t a l l i c ions. The chemical composition of the two asbestos types a l s o d i f f e r i n t h a t chrysotile contains l e s s s i l i c a and iron oxides than the amphiboles, b u t has a higher content of magnesium than the amphiboles ( r e f . 18). Asbestos-contai n i ng materi a1 s have been used extensively i n industry since i t s commercial introduction i n the l a t e 19th Century. I t i s estimated t h a t some 3,000 types of products contain some form of asbestos. One of i t s most important uses has been as a component i n heat and f r o s t insulation, and asbestos i s therefore often encountered i n s h i p b u i l d i n g and r e p a i r , construct i o n , power production, chemical manufacturing and i n the manufact u r i n g of automobile brakes ( r e f . 19). Adverse health e f f e c t s of inhaled asbestos f i b e r s began t o appear among occupational ly-exposed groups a t the e a r l y p a r t of the century. I t was subsequently shown t h a t inhaled asbestos f i b e r s can be retained i n the l u n g t i s s u e , where they can be i d e n t i f i e d e i t h e r by l i g h t microscopy, phase contrast microscopy, and electron microscopy, depending upon f i b e r s i z e and s t r u c t u r e ( r e f s . 20-22) . Asbestosis, i . e . , i n t e r s t i t i a l pulmonary f i b r o s i s , was the f i r s t disease related t o exposure t o airborne a s b e s t o s . ' I t i s s t i l l the most common manifestation of asbestos-induced e f f e c t s among occupational ly-exposed individuals. There i s usually a 1 atency period of approximately ten years before radiographic abnormalities occur. These are characterized radiographically by l i n e a r and r e t i c u l a r opacities affecting the middle and lower l u n g f i e l d s . I t i s also known t h a t a group of conditions of the pleura such a s thickening and c a l c i f i c a t i o n can be induced by asbestos ( r e f . 23).\ The f i r s t indications t h a t asbestos exposure m i g h t r e s u l t i n cancer appeared i n the 1930's, when a few cases of, 1 ung cancer I t was were reported i n persons w i t h asbestosis ( r e f . 2 4 ) . subsequently shown, i n epidemiological s t u d i e s , t h a t populations of individuals w i t h a h i s t o r y of occupational exposure t o asbestos

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have h i g h m o r t a l i t y r a t e s o f l u n g cancer ( r e f . 25-27). I t was a l s o c l a r i f i e d t h a t asbestos seems t o e x e r t i t s e f f e c t s y n e r g i s t i c a l l y w i t h tobacco smoke, and t h a t asbestos i n s u l a t i o n workers who smoke c i g a r e t t e s r u n t h e r i s k o f an e x t r a o r d i n a r i l y h i g h m o r t a l i t y r a t e i n l u n g cancer ( r e f . 28, 29). However, t h e r e i s some e v i dence, based. upon a small number o f d a t a p o i n t s , t h a t asbestos may i n c r e a s e t h e r i s k o f l u n g cancer even i n nonsmoking i n d i v i d u a l s . I t was r e c e n t l y observed t h a t t h e m o r t a l i t y o f l u n g cancer decreased s i g n i f i c a n t l y among asbestos workers who d i s c o n t i n u e c i g a r e t t e smoking as compared t o t h o s e who c o n t i n u e t h i s h a b i t ( r e f . 30). Although l u n g cancer may be t h e most common neoplasm among asbestos workers, m a l i g n a n t mesothel ioma o f t h e p l e u r a and p e r i toneum i s t h e most a s b e s t o s - s p e c i f i c m a l i g n a n t d i s e a s e ( r e f . 31). An e x t r e m e l y r a r e tumor i n t h e general p o p u l a t i o n , i t accounts f o r a p p r o x i m a t e l y 8% o f a l l deaths o f asbestos i n s u l a t i o n workers, a c c o r d i n g t o some s t u d i e s ( r e f . 17). Survival time i s usually l e s s t h a n a y e a r a f t e r d i a g n o s i s w i t h c u r r e n t l y a v a i l a b l e diagnosI t i s o f i n t e r e s t t o note t i c methods and t r e a t m e n t m o d a l i t i e s . t h a t c i g a r e t t e smoking does n o t appear t o be r e l a t e d t o t h e r i s k o f devel o p i ng mesothel ioma ( r e f . 29) A c l e a r dose response r e l a t i o n s h i p , which seems t o e x i s t f o r asbestosis, has n o t been i d e n t i f i e d w i t h t h e same degree o f c e r t a i n t y f o r e i t h e r l u n g cancer o r mesothel ioma. I n a d d i t i o n t o l u n g cancer and mesothelioma, o t h e r neoplasms have a1 so been a s s o c i a t e d w i t h o c c u p a t i o n a l exposure t o asbestos. These i n c l u d e c o l on-rectum cancer, esophageal and stomach cancer, as w e l l as cancer o f t h e oropharynx and l a r y n x ( r e f s . 17, 32, 33). I t should be emphasized, however, t h a t t h e degree o f evidence f o r t h e a s s o c i a t i o n between t h e l a t t e r groups o f cancers and asbestosexposure i s l e s s s t r o n g than f o r l u n g cancer and mesothelioma ( r e f . 34). As m e n t i o n e d , a s b e s t o s - r e l a t e d diseases, including the asbestos-associated cancers, usual l y become c l i n i c a l l y mani f e s t o n l y a f t e r a l o n g t i m e l a p s e f r o m onset o f exposure. For asbestosis, t h i s i s u s u a l l y a decade o r so, b u t f o r t h e asbestosr e l a t e d n e o p l a s t i c diseases, two t o f o u r decades i s u s u a l l y t h e r u l e , D u r i n g t h a t l a t e n c y p e r i o d , t h e i n d i v i d u a l may e i t h e r have

.

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radiographic evidence of asbestosis or may be entirely without symptoms or signs of exposure. I n either case, the exposed individual does not manifest any sign or symptom that would make possible the identification of the individual's high neoplastic risk. The protracted period of latency poses a difficult problem for early detection and, especially, for successful therapy. The information obtained in retrospective prospective epidemiological investigations of asbestos insulation workers provides evidence for the serious consequences that may occur when there has been a delay in establishing a cause-effect relationship, and when the biological and biochemical events during the 1 atency period remain poorly understood. Because of this situation, there is an urgent need for developing new diagnostic methods which could assist i n the identification of pathophysiological changes at a stage when early intervention might be of added advantage. We report observations of the urinary excretion patterns of modified nucleosides in patients with malignant mesothel ioma as a possible approach for the early detection of this disease. The first pilot study addressed the question whether patients w.ith asbestos-associated ma1 ignant mesothel ioma excrete elevated levels of modified nucleosides i n their urine. Eight patients with mesothelioma were studied and the results showed that several nucleosides were elevated, with $ elevated in all patients, Table 10.3 (ref. 35). Subsequently, similar results were obtained from additional seven patients with ma1 ignant mesothel ioma (Unpublished observation). These results indicate that mesothelioma produces elevated excretion of nucleosides and that this may be an additional diagnostic tool . No fa1 se-negative resul ts were observed i n this small group of patients with mesothelioma and the laboratory investigators were able to identify all as "cancers" on the basis of the quantitation of nucleosides. A recent stu y has shown increased excretion of $ and hypoxanthine in nude mice transplanted with mesothelioma (ref. 49). Excretion of $ s related to the growth of mesothelioma and an increased excretion commences at a time when the tumor is just measurable. The potentially useful predictive value of study ng nucleo-

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side excretion patterns i s reflected i n the i n i t i a l p i l o t study. Ten of 13 workers whose onset of asbestos exposure preceded the examination date by 30 t o 40 years had elevated levels of nucleosides. Elevated excretion of pb was the p r i n c i p a l a b n o r ma l i t y . This i s of interest since this gr oup of workers constitutes a population a t p a r t i c u l a r l y h i g h risk of neoplastic disease development (ref. 17). Follow-up information t o date on 3 of the 10 workers gave evidence of cancer. One developed lperi toneal mesothe1 i oma. A second study was therefore undertaken t o investigate whether apparently well asbestos insulation workers w i t h l o n g histories of asbestos exposure (30 years or longer), b u t w i t h o u t current evidence of cancer, would show unusual urinary excretion patterns of modified nucleosides. A group of 47 male individuals TABLE 10.3

Elevated Modified Nucleosides i n Patients w i t h Mesothelioma Patient 1 2 3 4 5 6 7 8 Normal

Sex

Marker Levels (nmol/fimol creatini ne) $

M M M M M

49.2 41.1 114.5 38.6 32.9 M 38.4 M 48.3 M 36.6 M 22.4

mlA

1.80 1.84 4.85 2.97 NRa 3.23 2.78 2.22 1.77

PCNR

2.81 1.74 3.75 1.90 1.97 1.90 1.02 1.56 0.87

m'I

mlG

mzG

m:G

B-AIBA

2.30 2.85 5.50 2.16 1.69 1.94 1.63 1.31 1.15

0.87 1.20 3.76 NR 1.44 2.15 1.30 0.79 1.06

0.54 0.44 1.46 0.53 0.73 0.73 0.40 0.68 0.35

2.19 2.26 4.32 1.96 1.72 2.24 1.44 1.30 1.2

6.0 4.0 21.0 30.0 3.0 4.0 9.4 4.0 4.27

(k2.10)b(*0.29) (i0.25) (i0.27) F

26.7

1.76

1.05

1.18

(e4.5) (k0.48) (+0.26)(e0.39) aNR, N o t R e s o l v e d hean S.D.

(k0.07) (20.15) (k1.93)

1.07 0.41

1.44

5.8

(k0. 12) (k0.38) (k4.11)

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was examined. They had been selected from a population of close to 2,000 workers from whom we have collected urine samples in the course of cl i ni cal examinations. The results of this feasi bi 1 i ty study showed that asbestos insulation workers exhibit significantly higher levels of seven of the investigated nucleosides when compared to .b control group. Twenty-seven (57%) of the asbestos workers had three or more nucleosides elevated, indicating a highly abnormal excretion profile (ref. 36). ' An increasing severity of radiographic a1 terati ons was associated with a greater frequency of elevated nucleosides, especially with mrA, mlI, mlG, and m$G. Duration since onset o f exposure was directly related to $, mlI, and m$G. We have also examined d
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Because of the possi bi 1i t y of re1 a t i ng .the nucl e o s i d e l e v e l s t o e x t e n s i v e c l i n i c a l and l a b o r a t o r y information a v a i l a b l e on this high r i s k population, i t i s a n t i c i p a t e d t h a t important information w i l l be obtained about determinants of nucleoside l e v e l s and m u l t i p l e f a c t o r i n t e r a c t i o n s which may g i v e c l u e s t o p r e v e n t i v e measures ( r e f . 38). 10.2.2 Urinary Excretion of Modified Nucleosides i n P a t i e n t s w i t h Various Manifestations o f I n f e c t i o n with HIV The d i s e a s e , Acquired Immune Deficiency Syndrome (AIDS) has r e c e n t l y approached epidemic proportions i n many p a r t s of the world. AIDS i s c h a r a c t e r i z e d by severe immune depression and s u s c e p t i b i l i t y t o neoplasms and o p p o r t u n i s t i c i n f e c t i o n s . Recent evidence s t r o n g l y i m p l i c a t e s a lymphotropic r e t r o v i r u s , HIV, a s the primary e t i o l o g i c agent of AIDS ( r e f . 39-42). During a f i v e y e a r period, 5-19% of i n d i v i d u a l s with HIV a n t i b o d i e s developed AIDS and there was a l a t e n c y period o f 1-5 y e a r s between i n f e c t i o n and development of d i s e a s e . Pathognomonic immunological o r biochemical a b e r r a t i o n s t h a t d e l i n e a t e t h i s syndrome a r e not known. We have observed e a r l ier e l evated 1eve1 s o f modi f i ed nucleosides and j-AIBA i n u r i n e from p a t i e n t s w i t h AIDS and i n a male homosexual population from New York C i t y ( r e f . 43). A goal of o u r s t u d y i s t o explore whether i t i s p o s s i b l e t o i d e n t i f y immunological o r biochemical abnormal i t i e s i n persons with HIV i n f e c t i o n s t h a t may i d e n t i f y s u b j e c t s with p r e d i s p o s i t i o n f o r development of AIDS. Conceivably, such a s t u d y might allow selection of subjects f o r early therapeutic intervention. The s u b j e c t s (20-52 y e a r s o l d ) f a l l i n t o f o u r c a t e g o r i e s : 1) T h i r t y - f i v e h e a l t h y homosexual men (HHS), none of whom a r e c h r o n i c c a r r i e r s of h e p a t i t i s B s u r f a c e a n t i g e n ; a n a l y s i s of s t o r e d frozen serum by an enzyme-linked immunosorbent assay (ELISA) f o r HIV antibody divided t h i s group r e t r o s p e c t i v e l y i n t o two subcategori e s : 20 h e a l t h y homosexual men who were s e r o n e g a t i v e (HHS-) f o r antibody t o the HIV (ELISA r a t i o l e s s than 3.00) a t the time of the immunol ogi c'al and c l i n i cal eval u a t i ons and nucl e o s i de d e t e r minations and 15 heal t h y homosexual men who were , s e r o p o s i t i v e (HHS+) f o r antibody t o the HIV (ELISA r a t i o g r e a t e r than o r equal t o 5.00) a t the time of e v a l u a t i o n s ; 2) 38 asymptomatic homosexual

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men w i t h Chronic lymphadenopathy syndrome (CLS), defined a s d i s c r e t e lymph nodes i n two o r more non-inguinal s i t e s of three o r more months duration ( r e f . 44); none of t h e s e p a t i e n t s had symptoms o r f i n d i n g s of fevers, n i g h t sweats, w e i g h t l o s s , f a t i g u e , d i a r r h e a , Herpes z o s t e r , Herpes simplex o r i d i o p a t h i c thrombocytopenia; 3) 14 s u b j e c t s w i t h AIDS r e l a t e d complex (ARC) defined a s a syndrome of i n t e r m i t t e n t o r continuous fevers >38.50 f o r > one month, watery d i a r r h e a of two weeks d u r a t i o n f o r which no o t h e r e t i o l o g i e s were e s t a b l i s h e d , unexplained weight l o s s of >15 pounds o r >lo% of the body