Chapter 7 Modified Nucleosides in Human Blood Serum as Biochemical Signals for Neoplasia

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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

%.*

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

mature RNAs

multiple

hydrolysis

6

-I'

nucleotides

malor malor nucleosides

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

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

C255

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

'

'

10

30

20

Time ( m i n )

10

20

L

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 =

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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 *ff f

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)

1.00 3.32 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)

3.97 - 7.00 7 . 99 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 t e s t s i n c l i n i c a l biochemistry. However, the use of a s i n g l e 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

80

40

'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

m

60

40

00

100

'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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5.

6. 7.

8. 9. 10. 11.

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19.

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