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The Metabolism of Ring-Labeled Orotic Acid in Man

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The Metabolism of Ring-Labeled Orotic Acid in Man THE METABOLISM OF RING-LABELED OROTIC ACID IN MAN S. M. Weissman, … , M. Lewis, M. Karon J Clin Invest. 1962;41(7):1546-1552. https://doi.org/10.1172/JCI104611. Research Article Find the latest version: https://jci.me/104611/pdf Journal of Clinical Investigation Vol. 41, No. 7, 1962 THE METABOLISM OF RING-LABELED OROTIC ACID IN MAN By S. M. WEISSMAN, A. Z. EISEN, H. FALLON, M. LEWIS AND M. KARON (From the Metabolism Service, Dermatology and Medicine Branches, National Cancer Institute, Bethesda, Md.) (Submitted for publication January 19, 1962; accepted March 29, 1962) Considerable knowledge concerning purine me- Total plasma and urine radioactivity was determined by tabolism has been obtained from chemical and first digesting the plasma or urine with an equal vol of isotopic studies of urinary uric acid. Correspond- 5 N KOH for 2 hours at 70° C, decolorizing with 1 to 2 drops of hydrogen peroxide, and counting 0.2 ml of this ing information in man is lacking for the closely digest in 20 ml of the toluene-methanol solution (8). In related field of pyrimidine metabolism. The avail- all cases counting efficiency was determined by the addi- ability of ring-labeled orotic acid as a specific tion of internal standards. pyrimidine precursor (1), together with the pres- Alternatively, radioactivity of urine was determined by ence of pseudouridine (5-ribosyluracil) and uracil, plating serial dilutions of each urine specimen on stainless steel cups, and extrapolating the count rates to infinite as specific end products of pyrimidine metabolism thinness. After the first 8 days, the count rate in total in man, in conveniently assayable amounts in urine was generally too low to be conveniently determined human urine (2-4), makes it possible to conduct directly. Extrapolations of total urine radioactivity were certain kinetic studies. In the present investiga- based on the assumption that total urine radioactivity had tion the metabolism of ring-labeled orotic acid and a constant ratio to urinary pseudouridine activity, since such parallelism was observed in days 3 through 8. and the time course of isotope incorporation into uri- urinary pseudouridine constituted about one-third of total nary *pseudouridine and uracil were studied in urine radioactivity during this period. order to develop methods of measurement and Timed expiratory air samples were collected in rubber to obtain values for the whole-body rate of py- balloons. The carbon dioxide was trapped by bubbling rimidine synthesis as well as for the turnover rate the air through 0.5 N sodium hydroxide. Barium hy- droxide was added, barium carbonate collected by filtra- of certain ribonucleic acid fractions. tion, dried overnight at 1100 C, weighed, pulverized, and a weighed aliquot counted in a liquid scintillation counter METHODS AND EXPERIMENTAL PLAN after suspension in a thixotropic gel (9). Relative effi- ciency of this counting method had been previously deter- Uracil and pseudouridine were isolated from 15-minute mined in this laboratory (9). samples of urine by a previously described procedure Patients employed in this study were selected because employing column chromatography in a mixed borate- of the presence of an inoperable but slowly progressing chloride gradient (4). Specimens were concentrated and malignancy and normal hepatic and renal function. None desalted by absorption on alkaline DOW-1 formate and of the subjects lost any weight during the first 6 weeks elution with 0.1 N formic acid, when necessary for paper of the study. All subjects were maintained on a purine- chromatography or counting of low specific activity sam- free, 70-g protein, isocaloric diet for 5 days before and ples (5). Descending paper chromatography was per- at least 12 days after the orotic acid infusion. The selec- formed by standard techniques on Whatman no. 1 paper tion of this diet was made to simplify the urinary chro- with isopropanol:acetic acid:water (60:30:10) as the matogram and to limit the exogenous supply of pseudo- solvent (4). Serial 2 cm-strips were cut from the paper uridine and other pyrimidines. and eluted with water, and the radioactivity in the eluates Urine and expired-air samples were collected at pro- determined by counting at infinite thinness on stainless gressively increasing intervals during the first 2 days, steel cups in a low-background (less than 2 cpm) gas- daily thereafter for about 1.5 to 2 weeks, and once or flow counter. Pseudouridine and uracil specimens were twice weekly thereafter when possible, as indicated in counted in a liquid scintillation counter, after dissolving the graphs (Figures 1-4). in a dioxane-containing solvent (6). Appropriate stand- Patients I, II, and III received an intravenous infusion ards were counted in the same systems. of 50 ,uc of 6-C'4-, 2-C14-, and 2-C"-orotic acid (2 to 4 ,uc A fraction of urine containing orotic acid was separated per ,umole), respectively, over a period of 15 minutes. by DOW-1 formate column chromatography, by proce- Patient IV received 35 ,uc of 6-C14-orotic acid intrave- dures described elsewhere (7). This fraction was puri- nously over 90 minutes. Patient I had frequent plasma fied on paper chromatograms with methyl acetate:water: samples drawn over the first 3 hours for determination formic acid (65:35:5). The orotic acid was then eluted 4of radioactivity, in order to obtain an estimate of minimal with water and its specific activity determined. Irenal clearance rates for orotic acid. Simultaneous en- 1546 METABOLISM OF RING-LABELED OROTIC ACID IN MAN 1547 PATIENT I W9, AGE 50, COLON CARCINOMA WITH METASTASES 15C 120)o - 90)of 60 30 .- .- - Al - .A. ^ I- 1% en ^^ nNAa fn v 2 1 6 4 6 8 10 12 14 16 18 21 22 24 26 28 HOURS DAYS TIME FIG. 1. dogenous creatinine clearance was measured by standard RESULTS methods. Rat liver was examined for acid-soluble pseudouridine All radioactivity emerging from the borate col- compounds. Twelve g of liver was homogenized and umns in the uracil area migrated as uracil when treated with 0.5 N cold perchloric acid, centrifuged, and chromatographed on paper. After the first 2 the supernate neutralized with 2 N KOH. It was then hours, all radioactivity in the pseudouridine area treated with sufficient rattlesnake venom and bacterial alkaline phosphatase in 0.05 M Tris buffer, pH 8.0, to of the column chromatogram behaved like pseudo- convert nucleotide derivatives to nucleosides. The extract uridine on paper. The earliest urine specimens was processed by column chromatography in the same contained a radioactive compound, generally not manner as the urine samples. The eluates from the associated with detectable amounts of ultraviolet- uridine and pseudouridine areas of the chromatogram absorbing material, that emerged just ahead of were desalted and carefully examined for pseudouridine by paper chromatography. This was done also with the pseudouridine during column chromatography. liver of a rat previously given a total of 200 jtc of 2-C"- In order to get accurate pseudouridine specific orotic acid in six intraperitoneal doses over 24 hours. activities on these specimens, it was necessary, Both radioactive and ultraviolet-absorbing materials were prior to assay, to purify pseudouridine further by sought for and neither was found. The sensitivity of this approach is such that acid-soluble pseudouridine deriva- paper chromatography. tives corresponding to 1 part in 500 of the acid-soluble As shown in Figure 5, when the orotic acid was uridine derivatives would have been readily detected. administered over 15 minutes, the first few urine A pseudouridine standard was prepared from com- specimens had an excess of radioactivity, presum- mercial yeast ribonucleic acid by the methods of Cohn ably owing to spillage of orotic acid. In Patient I (10). All other chemicals, rattlesnake venom, and Escherichia coli alkaline phosphatase were commercial all detectable radioactivity had disappeared from products. the orotic acid-containing fraction of the appro- 1548 S. M. WEISSMAN, A. Z. EISEN, H. FALLON, M. LEWIS AND M. KARON PATIENT 1I W9,33, ADENOCARCINOMA STOMACH WITH METASTASES 800 400,2~ 200L 0 TIME FIG. 2. PATIENT m Wao.< 53, ADENOCARCINOMA COLON WITH METASTASES 4.8 3.6 01.24 I 1.L 1 II 22 24 26 28 30 32 34 HOURS TIl ME DAYS FIG. 3. METABOLISM OF RING-LABELED OROTIC ACID IN MAN 1549 priate column chromatogram by the end of 3 hours after administration of the isotope. Patient IV, who was given the orotic acid infusion more slowly (over a 90-minute period), did not show an early excess of radioactivity in her urine. The * PAT/ENTZf similarity of the general form of the specific activ- z ity curves in this and the other studies suggests 0 that the chemical amount of orotic acid given to OL PATIENT 39 I!Z WY, z ADENOCARCINOMA, 1ISITE UNKNOWN WITH METASTASES TO LUNGS 0 -J .05 1 2 3 4 5 6 HOUR S "L FIG. 5. TOTAL COUNTS EXCRETED IN URINE PER 15- MINUTE INTERVAL AFTER THE ADMINISTRATION OF ISO- TOPICALLY-LABELED OROTIC ACID. Observed counts not corrected for the 48 per cent efficiency of the counting instrument. rates for orotic acid. The recovered orotic acid from the urine in period U 1 (Table I) had a specific activity of 1,401,000 cpm per jumole, whereas the administered orotic acid had a specific TIME activity of 1,962,000 cpm per ptmole. Approx- FIG. 4. imately 1.3 pmoles (0.2 mg) of orotic acid was FIGS. 1-4. TIME COURSE OF SPECIFIC ACTIVITY OF URI- presumably produced endogenously and excreted NARY PSEUDOURIDINE AND URACIL AND OF RESPIRATORY during this 30-minute period. This would cor- CO2 AFTER THE ADMINISTRATION OF ISOTOPICALLY-LABELED OROTIC ACID.
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