The Identification of 2-Dimethylamino-6-hydroxypurine and Its Ribonucleoside in Urine of Normal and Leukemic Subjects

Kay Fink, William S. Adams, Frances W. Davis, et al. Cancer Res 1963;23:1824-1829. Published online December 1, 1963.

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The Identification

of 2-Dimethylamino-6-hydroxypurine

and Its Ribonucleoside in Urine of Normal and Leukemic Subjects*

KAYFINK, WILLIAM ADAMS, RANCES . DAVIS, S. F W ANDMISAENAKATANI
(Departments of Medicine and Biophysics, School of Medicine, University of California at Los Angeles; and Wadsworth Hospital, Veterans Administration, Los Angeles, California') SUMMARY

Urine from normal and leukemic subjects on controlled low-purine diets was frac tionated by ion-exchange column chromatography, and the various effluent peaks exhibiting absorbance at 260 m/i were subjected to two-dimensional filter paper chromatography for further resolution. It appeared from gross examination of the chromatograms that there were a greater number and concentration of blue fluorescent substances in much of the leukemic urine. Two of these compounds have been identi fied as 2-dimethylamino-6-hydroxypurine and its ribonucleoside.

A number of methylated purines have been found in human urine by Weissmann, Bromberg, and Gutman (33). These include 1-methylhypoxanthine, 7-methylguanine, 8-hydroxy-7-methylguanine, 1-methylguanine, and 6-hydroxy-2-methylaminopurine. Their presence has been confirmed by Park et al. (24), who also reported increased excretion of 1-methylhypoxanthine, 7-methylgua nine, and 8-hydroxy-7-methylguanine in a number of leukemic patients. In a continuation of a study of urinary pyrimidines and purines in normal and leukemic urines by a combination of ion-exchange and filter paper chromatography (1), a greater number and concentration of blue fluorescent sub stances were observed in much of the leukemic urine. Two of these compounds have been identi fied as 2-dimethylamino-6-hydroxypurine and its ribonucleoside. Elion et al. (13) have synthesized 2-dimethylamino-6-hydroxypurine, and its biolog ical occurrence as a minor base in RNA has been reported by Smith and Dunn (29).

purine diet for 5 days prior to the collection of a 24-hour urine specimen. Aliquots of the urine were subjected to ion-exchange chromatography by modification of a procedure described earlier (1). The urine was added to a column of Dowex 2-chloride (X8, 200-400 mesh) anin exchange resin, and gradient elution was accomplished by the gradual delivery of a solution of 0.1 M acetic acid and 0.025 Mammonium chloride into a mixing chamber containing a constant volume of solution, which initially consisted of 0.22 Mammonium hy droxide and 0.025 Mammonium chloride. A gradu al fall in pH from 10.4 to about 7.5 was achieved, and the eluate from the column was collected in 2.5-ml. fractions. In most cases a volume of urine equivalent to 6 mg. of creatinine was applied to a column 24 X 0.75 cm., and the volume in the mix ing chamber was 250 ml. Some urine was also fractionated on a larger scale, with 75 per cent of a 24-hour volume, by the following procedure: One-fourth of a total 24-hour collection of urine was reduced in vacuo to approxi MATERIALS AND METHODS mately 50 ml. To this was added an equal volume Normal subjects and leukemic patients of vari of solution #1 (0.6 gm. ammonium formate and ous types were maintained on a controlled low15.0 ml. of concentrated ammonium hydroxide per * This research was supported by Grant CA-02433, United liter), the pH of the mixture was adjusted to 10.4 States Public Health Service, and by Grant P-813 from the with additional ammonium hydroxide, and after American Cancer Society. filtration the material was added to a column (46 X 2.5 cm.) of Dowex 2-formate resin ( X8, 200-400 Received for publication July 27, 1963. 1824

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FINK et al.Urinary 2-Dimethylamino-6-hydroxypurine mesh). The ammonium formate solution jfl was employed as the influent until 40 fractions of 25 ml. each of effluent had been collected. Creatinine and N-methyl-2-pyridone-5-carboxamide were thus removed. At this stage solution #2 (0.6 gm. ammonium formate and 3.8 ml. of formic acid per liter) was started into a mixing chamber containing 2500 ml. of solution #1, thus initiating gradient elution. Pyrimidines consisting primarily of 5-ribosyluracil and uracil were eluted first, fol lowed by the various purines, with uric acid being eluted last. The combined effluents containing the purines (except for uric acid) from three such fractionations on Dowex 2-formate were lyophilized, and ammonium formate was removed by sublimation at 55-60 The residue was taken C. up in 50 ml. of 0.01 N HC1, added to a column (46 X 2.5 cm.) of Dowex 50-H+ (X12, 200-400 mesh) which was in equilibrium with 0.01 N HC1.

1825

ultraviolet lamp (253.7 m^i), and two spots fre quently observed were characterized by bright blue fluorescence and did not appear to correspond to purines previously reported in urine (24, 33, 34). To obtain these substances in larger quantity with sufficient purity for identification purposes, frac tions from the large-scale procedure containing relatively high concentrations of the compounds in question were streaked along the base line of filter paper and subjected to one-dimensional chromatography. The bands corresponding to the com pounds to be isolated were cut from several papers and were concentrated by a technic previously de scribed (9). The substances thus purified were used for determination of RF values and electrophoretic mobility and were rechromatographed and eluted for determination of ultraviolet absorption spectra with a Beckman DK-2 recording spectrophotometer.

TABLE1 RFVALUES OFURINARYOMPOUND Two METHYLATED C AND GUANINES

VALUES (X100) IX VARIOUS SOLVENT SYSTEMS

COMPOUNDCompound

urine-DimethyIamino-6-hydroxypurine6-Hydroxy-2-methylaminopurineFORMi-Bu505043HAcn-Bu686858HC1i-Pr373747FORMEtAc393929EtAcform440 isolated from

The composition of the various solvents is as follows: FORM <-Bu = <ert-butyl alcohol, methylethylketone, water, formic acid (40:30:15:15), HAc n-Bu = n-butyl alcohol, water, glacial acetic acid (50:25:25), HC1 i-Pr = isopropyl alcohol, water, concentrated HC1 (65:18.4:16.6), FORM EtAc = ethyl acetate, formic acid, water (70:20:10), EtAc form = upper phase from a mixture of ethyl acetate, water, formic acid (60:35:5).

Subsequent elution was carried out stepwise, start ing with 0.01 N HC1, until 100 fractions of 10 ml. each had been collected; then 0.1 N HC1 was em ployed for another 100 fractions of 10 ml. each. At fraction 200, 2 N HC1 was introduced into a mixing chamber containing 2500 ml. of 0.1 N HCI. At fraction 400, 4 N HC1 was started into the same chamber, and at fraction 600, 6 N HC1 was intro duced. Elution was continued until adenine was eluted. This procedure is based on that reported by Wyngaarden (37) for cation exchange chromatography of purines. The ultraviolet absorbancy at 260 m t the of individual fractions collected from the columns was recorded. Portions of fractions comprising various ultraviolet-absorbing peaks were subjected to two-dimensional paper chromatography with isopropyl alcohol, water, and HCI (35), and with n-butyl alcohol, water, and ammonia (20). The chromatograms were examined over a short-wave

RESULTS Of the known compounds initially available for direct comparison, 6-hydroxy-2-methylaminopurine (N2-methylguanine) seemed most closely re lated to one of the substances isolated from urine, as judged by absorption spectral characteristics and Chromatographie properties. However, no dif ficulty was encountered in distinguishing standard 6-hydroxy-2-methylaminopurine from the urinary compound. A sample of 2-dimethylamino-6-hydroxypurine (N2-dimethylguanine) was then ob tained for direct comparison, and the data ob tained left little doubt that the isolated substance was 2-dimethylamino-6-hydroxypurine. In Table 1 RF values for the urinary compound are given in solvents which provided some separa tion of the N2-methylated guanines by the chromatographic methods described earlier (14). A number of other Chromatographie solvents were tested, and, although the isolated compound had

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1963

of the leukemic urines with the small column of Dowex 2 resin, subsequent chromatography re vealed the presence of 2-dimethylamino-6-hydroxypurine in the effluent adenine peak, and occa sionally it also extended into the adjacent hypoxanthine peak. Comparable aliquots of the effluent peaks obtained from normal and leukemic urines were used for paper chromatography, and only once was the dimethyl-guanine derivative observed in the normals (seven cases), whereas it was easily detected in two-thirds of the leukemic specimens (30 cases of a variety of types including acute and chronic granulocytic, lymphocytic, and myelocytic). The compound was routinely detected chroTABLE2 in the effluents from the Dowex ELECTROPHORETIC MOBILITIES URINARY OF COMPOUND matographically 50 columns used in the large-scale procedure with ANDTwo METHYLATED GUANINES both normal and leukemic urine; but it was not confined to a single peak, and a quantitative esti MOBILITIES (CM.)* mation of its level has not as yet been undertaken. With a visual inspection of the chromatograms, a COMPOUNDCompound HBoratepH CitratepH M rough classification of the concentration could be 3.04 made on the basis of spot size and intensity, and 9.2000 it appeared that the acute leukemias and chronic urine2 isolated from cathodal4 .5 lymphocytic leukemias were associated with an -hydroxypurine6-Hydroxy-2-methylaminopurine0.02 -Dimethy lamino-6 cathodal3.8 .5 cathodal0.01 increased excretion of this compound in compari son with what was found in normals and chronic * 4 hours, 800 volts, Whatman #1 filter paper. granulocytic leukemias. TABLE 3

the same RF values as 2-dimethylamino-6-hydroxypurine, separation from the 2-monomethyl-substituted guanine on the same chromatogram was not obvious. The color of the urinary compound on chromatograms observed over an ultraviolet lamp was variable, depending on what Chromato graphie solvents were used and how soon the chromatogram was examined after removal from the solvent. It varied from a dark absorbing spot in ammoniacal and neutral solvents to an intense bright blue fluorescent substance in the isopropyl alcohol: H2O:HC1 solvent. With the latter solvent

ULTRAVIOLET ABSORPTION CHARACTERISTICS OFAUTHENTIC 2-DiMETHYLAMiNO-6HYDROXYPURINE AND THE COMPOUND ISOLATED FROM URINE

l>w256,
COMPOUND2-Dimethylaniino-6-hydroxvpurine

li^roa245,

Compound isolated from urinepH * Inflection.

285* 255, 285*Xmn233234pH

279* 245, 279*Xn,in268270

6-hydroxy-2-methylaminopurine exhibited con siderably less fluorescence, and the color was dark er. In side-by-side Chromatographie comparisons of the isolated compound, synthetic 2-dimethylamino-6-hydroxypurine, and 6-hydroxy-2-methylaminopurine in a variety of solvents, the isolated compound routinely exhibited the same gradations in color as the authentic 2-dimethyl-substituted guanine. In Table 2 the electrophoretic mobilities of the compounds are given, and in Table 3 ultraviolet absorption spectra are presented. Elion, Lange, and Hitchings (13) and Smith and Dunn (29) have reported previously spectral characteristics for 2dimethylamino-6-hydroxypurine. After fractionation of the purines in a number

The ribosyl derivative of 2-dimethylamino-6hydroxypurine was isolated from the urine of a normal subject. It was noted on paper chromato grams prepared from an effluent fraction of the purines obtained from chromatography on Dowex 2-formate resin, and a sufficient quantity was iso lated by paper Chromatographie technics to permit identification. Its Rp value in sec-butyl alcohol saturated with water was 0.35, but it remained at the origin when borate was added (8). The marked ly lower RP value in the presence of borate was consistent with a ribonucleoside structure. The Chromatographie and electrophoretic properties (migration toward the anode in 0.01 M borate buffer, pH 9.2) of the urinary compound agreed with those reported by Smith and Dunn for 2-di--

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FINK et al.Urinary

%-Dimethylamino-6-hydroxypurine

1827

methylamino-6-hydroxypurine ribonucleoside, and the ultraviolet absorption spectra also corresponded to the ones shown by these investigators (29). Ribose and 2-dimethylamino-6-hydroxypurine were detected on chromatograms after hydrolysis of the urinary compound in l N HC1 at 100 . for C 1 hour. The sugar component was visualized on chromatograms by the aniline hydrogen phthalate reagent (25), and the RF values agreed with those reported for ribose (14) ; the purine base obtained by hydrolysis was identified as 2-dimethylamino6-hydroxypurine on the basis of its absorption spectra and also by direct Chromatographie com parison with an authentic standard, with the solvents listed in Table 1. A careful identification of the nucleoside has been made in urine from only one subject, but a blue fluorescent spot in the proper Chromatographie position for the compound has been observed in urine from a number of patients. DISCUSSION The list of methylated purines found as minor constituents of ribonucleic acids has continued to expand since the initial report of Littlefield and Dunn (19). It now includes 1-methylguanine (2, 29), 2-dimethylamino-6-hydroxypurine (29), 6-hydroxy-2-methylaminopurine (2, 29), 7-methylguanine (12), 2-methyladenine (18, 19), 6-methylaminopurine (2, 18, 19), 6-dimethylaminopurine (18, 19), and 1-methyladenine (11, 12). These methylated purines occur in the sRNA (10, 5) involved in the transfer of amino acids, but they are not incorporated directly into the RNA (30). The methyl groups originate from methionine (6, 22, 23), and the methylation is carried out at the level of the pre-formed polynucleotide structure by a soluble enzyme system, RNA methylase (15). Further purification of the system has demon strated that the transmethylation is performed by S-adenosylmethionine and that specific enzymes may be involved for the particular base methylated (16, 17). The function of these minor bases in the sRNA has not yet been determined, and evidence by Starr (31) indicates that the methylation is not essential for the amino acid acceptor role of the sRNA. The question as to a possible coding func tion of the methylated bases was raised by Can toni et al. (7) in a recent investigation in which 2-dimethylamino-6-hydroxypurine was found to be the only methylated base in the serine sRNA of yeast. It seems probable that 2-dimethylamino-6-hydroxypurine, as well as some of the urinary meth ylated purines observed by Weissmann et al. (33) namely, 7-methylguanine, 1-methylguanine, 6-

hydroxy-2-methylaminopurine, 1-methylhypoxanthine, and 8-hydroxy-7-methylguaninerepresent metabolic end-products of the sRNA. All but the last two are known components of sRNA, and it is not unlikely that 1-methylhypoxanthine may be formed biologically from 1-methyladenine (or its corresponding nucleoside or nucleotide). If 7methylguanine is a precursor of 8-hydroxy-7-methylguanine, an oxidase other than xanthine oxidase would presumably be required, since it has been demonstrated that 7-methylguanine is refractory to xanthine oxidase (34, 36). Other mechanisms may be mentioned which might contribute to the urinary level of the meth ylated purines, but available information is not adequate to assess them critically. First, it is con ceivable that in some abnormal states an excessive methylation of nucleic acid might occur, with a resultant increase in the level of the methylated purines excreted. The excessive methylation might be accomplished by donors other than S-adenosyl methionine, since Magee and Farber (21) have reported methylation of liver RNA and DNA after intraperitoneal administration of a C14-labeled car cinogen, dimethylnitrosamine. Second, methyla tion of purines other than in nucleic acid linkage may occur. The early high specific activity of uri nary 7-methylguanine and 8-hydroxy-7-methylguanine observed after administration of labeled glycine to some individuals with gout, polycythemia vera, and myeloid metaplasia (37, 38) could be consistent with some non-nucleic acid purine methylations. Axelrod and Daly (3) obtained ex tracts of rabbit tissues which could methylate adenine to 3-methyladenine, but the latter com pound could not be detected in DNA or RNA, and it or the corresponding hypoxanthine derivative has not been identified in urine. Remy reported an S-adenosylmethionine transmethylase of E. coli that could N-methylate some unnatural synthetic 2-amino-substituted purines, but it exhibited no appreciable activity toward guanine (26,27). How ever, in a footnote to a later report concerning a mammalian system for S-methylation of 6-thiosubstituted purines, he stated that E. coli extracts containing an active 2-aminopurine transmethyl ase system synthesized 6-hydroxy-2-methylaminopurine in the presence of S-adenosylmethionine and guanosine-8-C14; thus, the nucleoside or nu cleotide derivative may be the natural methyl acceptor, rather than guanine (28). Third, an en hanced level of urinary methylated purines may reflect a lowered metabolism of these compounds, such as demethylation or cleavage of the ring structure. Townsend and Robins (32) have recent ly demonstrated that methylation at position 7

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renders guanosine and xanthosine very susceptible to ring opening between C-8 and N-9 under mild conditions of temperature and pH. Thus, if 7methylguanosine monophosphate from the sRNA were biologically degraded to 7-methylguanosine, it is possible that a portion of it might undergo cleavage of the imidazole ring yielding a 5-formylaminopyrimidine derivative. Demethylation of 6-methylaminopurine has been demonstrated with a rat liver homogenate (100,000 X g supernatant fraction), which yielded hypoxanthine and uric acid as two of the products (27), but biological demethylation of the naturally occurring methyl ated guanines has not been shown, and they are either refractory to xanthine oxidase or acted on at an exceedingly slow rate (34, 36). The increased urinary excretion of some of the methylated purines in many of the leukemic pa tients is of considerable interest, but the signifi cance of that observation awaits further investiga tion of the biochemical mechanisms involved.
ACKNOWLEDGMENTS We gratefully acknowledge our appreciation to Dr. Ger trude Elion for a gift of 2-dimethylamino-6-hydroxypurine and to Dr. J. D. Smith and Dr. George B. Brown for samples of 6hydroxy-2-methyIaminopurine. REFERENCES ADAMS, . S.; DAVIS,F.; and NAKATANI, Furine and W M. Pyrimidine Excretion in Normal and Leukemic Subjects. Am. J. Med., 28:726-34, 1960. ADLER,M.; WEISSMANN, and GUTMAN, B. Occur B.; A. rence of Methylated Purine Bases in Yeast Ribonucleic Acid. J. Biol. Chem., 230:717-23, 1958. AXELROD, ., and DALY,J. The Enzymic Conversion of J Adenine to 3-Methyladenine. Biochim. Biophys. Acta, 61:855-56, 1962. BERGMANN, KWIETNY,H.; LEVIN, G.; and ENGEL F.; BERG,H. Studies on the Enzymic Oxidation of Aminopurines. Biochim. Biophys. Acta, 37:433^11, 1960. BERGQUIST, L., and MATHEWS, E. F. Occurrence and P. R. Distribution of Methylated Purines in the RNA of Subcellular Fractions. Biochem. J., 86:305-13, 1962. BISWAS, . A. ; EDMONDS, ; and ABRAMS, The MethylB M. R. ation of the Purines of Soluble Ribonucleic Acid with Methyl-labeled Methionine. Biochem. Biophys. Res. Comm., 6:146-49, 1961. CANTONI, L.; RICHARDS, and TANAKA, A Coding G. H.; K. Function for the Methylated Bases in S-RNA? Fed. Proc., 22:230, 1963. CLINE,R. E.; FINK, R. M.; and FINK, K. Synthesis of 5Substituted Pyrimidines via Formaldehyde Addition. J. Am. Chem. Soc., 81:2521-27, 1959. DAVIS,F.; DOBBS,C. A.; and ADAMS, S. Elution Con W. centration of Paper Chromatogram Spots. Anal. Chem., 34:175-76, 1962. DUNN,D. B. Additional Components in Ribonucleic Acid of Rat-liver Fractions. Biochim. Biophys. Acta, 34:286-88, 1959. . The Occurrence of 1-Methyladenine in Ribonu cleic Acid. Ibid., 46:198-200, 1961.

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33. WEISSMANN, BROMBERG, A.; and GTMAN, B. B.; P. A. The Purine Bases of Human Urine. I. Separation and Iden tification. J. Biol. Chem., 224:407-22, 1957. 34. WEISSMANN, and GTMAN, B. The Identification of B., A. 6-Succinoaminopurine and of 8-Hydroxy-7-methylguanine s ormal Human Urinary Constituents. J. Biol. Chem., N 229:239-50, 1957. 35. WYATT, . R. The Purine and Pyrimidine Composition of G Deoxypentose Nucleic Acids. Biochem. J., 48:584-90, 1951.

36. WYNGAARDEN, B. 2,6-Diaminopurine as Substrate and J. Inhibitor of Xanthine Oxidase. J. Biol. Chem., 224:45361, 1957. 37. WYNGAARDEN,B.; BLAIR,A. E.; and HILLEY,L. On the J. Mechanism of Overproduction of Uric Acid in Patients with Primary Gout. J. Clin. Invest., 37:579-90, 1958. 38. Ytf, T. F.; WEISSMANN, SHARNEY, KUPFER,S.; and B.; L.; GTMAN, On the Biosynthesis of Uric Acid from GlyB. cine-N15in Primary and Secondary Polycythemia. Am. J. Med., 21:901-17, 1956.

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