Salvage of Circulating Pyrimidine Nucleosides in the Rat1

Home , Pseudouridine, Pyrimidine

[CANCER RESEARCH 41, 3010-3017, August 1981] 0008-5472/81 /0041-OOOOS02.00 Salvage of Circulating Pyrimidine Nucleosides in the Rat1

James D. Moyer, James T. Oliver, and Robert E. Handschumacher2

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 [J. D. M., R. E. H.], and Boehringer Ingelheim Research and Development Center, Ridgefield. Connecticut 06877 [J. T. O.]

ABSTRACT infusions of radiolabeled tracers to measure the salvage of these nucleosides by various tissues. The contribution of cir A new procedure was developed to measure uridine and culating pyrimidine nucleosides to the nucleotide pools has cytidine in plasma. These nucleosides are present in micro- been examined in relation to the requirements of slowly growing molar concentrations in the plasma of rats, mice, and humans. tissues for the maintenance of pools and also to the needs of Inhibitors of pyrimidine synthesis de novo (pyrazofurin or N- rapidly dividing tissue undertaking net synthesis of RNA and phosphonacetyl-L-aspartate) produce only modest decreases DMA. in the concentration of circulating uridine or cytidine in the rat. Since both uridine and cytidine are rapidly cleared from the MATERIALS AND METHODS circulation of the rat, constant infusions of radiolabeled uridine and cytidine were used to establish a steady-state specific Drugs and Chemicals. Succinic anhydride and pyridine were activity of circulating nucleoside without altering the normal purchased from Pierce Chemical Co. (Rockford, III.) . Nucleo sides, nucleotides, heparin, and 2',3',5'-triacetyl uridine were endogenous concentration. These studies permitted an esti mation of the contribution of circulating pyrimidine nucleoside purchased from Sigma Chemical Co. (St. Louis, Mo.). Acetic anhydride, m-aminophenylboronic acid hemisulfate, and 1- to the nucleotide pools of various rat tissues. Most of the uridine entering the circulation (>70%) is catabolized rather ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride than salvaged by formation of nucleotides. Cytidine in the were obtained from Aldrich Chemical Co. (Milwaukee, Wis.). circulation is much more efficiently utilized and is predomi Methanol for HPLC buffers was purchased from Burdick & Jackson Laboratories, Inc., (Muskegon, Mich.). [5-3H]Uridine nantly salvaged. The implication of these results for chemo (27.9 Ci/mmol) and [5-3H]cytidine (27.7 Ci/mmol) were pur therapy based on inhibition of pyrimidine synthesis de novo is discussed. chased from New England Nuclear (Boston, Mass.). HPLC. Chromatography was performed with Altex 110A INTRODUCTION pumps, an Altex 420 microprocessor to generate gradients, and 2 Altex 8-/il flow cell UV detectors monitoring 280 nm and Separate enzymatic pathways exist in mammalian cells to 254 nm with analog output on a dual channel Linear Corp. generate nucleotides by de novo synthesis or by salvage of Model 485 recorder. preformed bases or nucleosides (12,19). Although it has been Anion-Exchange Chromatography of Uridine. This Chro suggested that bone marrow and intestinal mucosa are primar matographie system is a modification of a system described by ily dependent on the salvage pathway to obtain purine nucleo Singhal and Conn (26). The use of the small particle size resin tides (15, 17) and that the liver provides preformed purines has produced greater resolution and sensitivity. A 25- x 0.46- and pyrimidines to other tissues through the circulatory system cm column of HAX-4 anion-exchange resin (Hamilton Co., (15, 16, 23), the extent to which a particular tissue depends Reno, Nev.) was slurry packed, maintained at 50Â°,and eluted on each pathway has not been firmly established. A more with 0.3 M acetic acid:ammonium acetate (pH 9.7) at 1.0 ml/ complete understanding of the balance between alternate path min. Peak height was directly proportional to amount injected ways would help guide the proper chemotherapeutic use of between 0.1 and 5 nmol. The following retention times were PF3 or PALA, inhibitors of the de novo pathway, and p-nitro- observed: cytidine, 2.1 min; adenosine, 4.2 min; thymidine, benzyl-thioinosine (31) or inhibitors of uridine kinase, which 10.8 min; pseudouridine, 17.4 min; deoxyuridine, 17.6 min; may block nucleoside salvage. Quantitating the low levels of uridine, 22 min; PF, 34 min; uracil, 41 min; guanosine, 48 min; circulating pyrimidine nucleosides has been a major difficulty and inosine, 84 min. The Chromatographie efficiency for uridine in studying nucleoside salvage in vivo. Radioimmunoassays was 1200 theoretical plates. Multiple injections of standards (13), microbiological assays (20, 22), and multistep procedures indicated that peak height variation was Â±2% (S.D.). involving thin-layer chromatography and derivatization (8, 27, The resin was regenerated after processing 50 biological 29) have been used previously to measure pyrimidine nucleo samples by 30-min treatment with 50% sulfuric acid at 70Â°. sides. Recently, several HPLC methods have been developed Although human, horse, or mouse plasma samples (pretreated to permit more rapid quantitation of nucleoside levels (10, 14, by borate affinity column separation as described below) gave 25). base line separation in most instances, in 5 of 27 rat plasma In the current study, we have devised a method for quanti extracts, an overlapping peak at 18 min prevented accurate tation of circulating uridine and cytidine and used constant measurement of uridine. Reverse-Phase Chromatography of Nucleosides and 1 Supported by American Cancer Society Grant CH-67T. Bases. A 25- x 0.46-cm Lichrosorb 5-jum particle size column 2 To whom requests for reprints should be addressed. (Altex Co., Berkeley, Calif.) was eluted at 1.0 ml/min with 0.2 3 The abbreviations used are: PF. pyrazofurin; PALA. N-phosphonacetyl-L- M sodium phosphate (pH 6.4) at 25Â°. The following retention aspartate; HPLC, high-performance liquid chromatography. Received January 19, 1981; accepted April 22, 1981. times were observed: AMP, GMP, UMP, CMP, uric acid, uracil,

3010 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Circulating Undine and Cytidine in the Rat and cytosine, all <7.5 min; cytidine, 8.5 min; uridine, 14 min; and chromatographed on the HAX-4 column as described deoxycytidine, 14 min; deoxyuridine, 23 min; thymidine, 35 above, and 1-ml fractions were collected and counted in 10 ml min; and adenine, 30 min. Peak height was directly proportional of Formula 963 (New England Nuclear). to amount injected in the range of 0.1 to 1.0 nmol at a sensitivity Disposition of [5-3H]Uridine and [5-3H]Cytidine in the Rat. of 0.005 absorbance units, full scale. Male Sprague-Dawley rats (250 to 350 g) were anesthetized Analysis of RNA Hydrolysate by AniÃ³n Exchange. Incor with methoxyflurane, and catheters were placed in the jugular poration of [5-3H]uridine into RNA was assayed by separation vein and the carotid artery. [5-3H]Uridine (27.9 Ci/mmol) was of the 2',3'-mononucleotides resulting from alkaline hydrolysis prepared in sterile 0.9% NaCI solution, and 0.1 mCi was on a 25-cm BAX-4 (Benson Co., Reno, Nev.) column at 50Â° injected via the jugular vein in a volume of 0.2 ml. At the with a linear gradient from 0.6 to 1.0 M ammonium acetate, pH indicated times, 100 p.\ of blood from the carotid artery were 8.4, for 35 min during a period of 20 min and subsequent mixed with 400 Â¡i\of ice-cold 0.5 M HCIO4, and the resulting isocratic elution with 1 M buffer at 2 ml/min. extract was processed and chromatographed sequentially on Cation-Exchange Chromatography of Cytidine. A 25- x the borate affinity resin and on BAX-4 anion-exchange resin as 0.46-cm column packed with BPAN6 resin (Benson Co.) was described above. Radioactivity was determined in 10 ml of eluted at 50Â°with a flow of 1.5 ml/min with 0.3 M formic acid Formula 963 at 24% efficiency. adjusted to pH 4.0 with ammonium hydroxide. The following Similar experiments were performed with [5-3H]cytidine. The retention times were observed: void, 2.0 min; uracil, 2.4 min; neutralized HCIO4 extract of blood (50 to 200 pi) was chromat uridine, 2.6 min; guanosine, 5.8 min; adenosine, 15 min; cyti- ographed directly on a 25-cm BPAN6 cation-exchange column dine, 24 min; adenine, 55 min; and cytosine, 57 min. For as described above. Fractions of 3 ml (2 min) were collected, quantitation of unlabeled cytidine in extracts, a better resolution and radioactivity was determined by scintillation spectrometry. from the UV-absorbing components was obtained with a 50- x Infusion of [5-3H]Uridine or [5-3H]Cytidine. Catheters con 0.46-cm column eluted at 60Â°with 0.5 M ammonium formate, taining heparinized (100 units/ml) 0.9% NaCI solution were pH 4.0, at 0.8 ml/min (4000 theoretical plates). Peak height placed in the carotid artery and left jugular vein of a male was proportional to the amount injected over the range of 0.2 Sprague-Dawley rat (250 g) that had been anesthetized with to 2 nmol at a sensitivity of 0.01 absorbance units, full scale. methoxyflurane. The catheters were protected by a spring and Determination of Uridine and Cytidine in Plasma. Plasma attached to swivels to permit sustained infusion in conscious from heparinized blood was mixed with an equal volume of ice- animals. [5-3H]Uridine or [5-3H]cytidine (40pCi/ml) was infused cold 1 M HCIO4 and kept at 4Â°for 20 min. The precipitated via the jugular vein at 2.3 ml/hr. Carotid blood was obtained at protein was removed, and the supernatant was neutralized to 30-min intervals for determination of radiolabeled nucleoside. pH 7 to 9 at 0Â°with 10NKOH. At the end of the 4-hr infusion, tissue samples were frozen in One to 2 ml of the extracts were prechromatographed on 1 liquid N2. Tissue samples were homogenized with 10 volumes ml of borate affinity resin (5). The column was washed with 4 of N HCIO4 at 0Â°, and total acid-soluble uracil and cytosine ml of 0.3 M ammonium acetate, pH 8.8, and eluted with 3 nucleotides were determined after acid hydrolysis as above. portions (1.5, 2, and 2 ml) of 0.1 N formic acid. The last 2 The acid-insoluble fraction was washed 3 times with ice-cold fractions, which contained the nucleosides, were lyophilized 0.3 M HCIO4, and the RNA was hydrolyzed and quantitated by and reconstituted in 0.3 M ammonium acetate buffer for chro- the method of Blobel and Potter (1 ). The resulting oligonucle- matography as described above. The overall recovery of uri otides were further hydrolyzed in 0.5 M KOH at 37Â°overnight dine or cytidine determined by addition of internal standards of to produce a mixture of 2' and 3'-mononucleotides for chro- radiolabeled nucleoside was 92 Â±10% (S.D.) for uridine and matography as described above. 90 Â±6% for cytidine. Acetylation of Uridine Peak. A derivatization procedure was RESULTS performed to confirm the structure of the component identified as uridine. The uridine fraction from HPLC of rat plasma was Distribution and Stability of Uridine and Cytidine in Blood. lyophilized, and pyridine (100 /Â¿I)andacetic anhydride (50 jul) The conversion of uridine to uracil was reported to occur in were added and allowed to react at room temperature for 1 hr dog blood by Tseng ef al. (29). To examine this possibility, to form the triacetyl derivative (2). After removal of acetic whole rat and mouse blood was drawn and assayed for the anhydride and pyridine, the sample was reconstituted in 0.5 ml degradation of uridine using HPLC as described in "Materials of 0.3 M sodium acetate (pH 5.0), adsorbed onto a 25-cm and Methods." In mouse blood incubated at 37Â°, only 6 and Lichrosorb 5-jum particle size C18 reverse-phase column, and 10% of the uridine was converted to uracil after 10 and 20 min, eluted with 0.3 M sodium acetate (pH 5.0,1 ml/min) for 10 min respectively. In rat blood, 10% degradation of added uridine followed by a 15-min gradient to achieve a buffer:methanol mix occurred after 10 min and 18% after 20 min. of 3:1. A uridine standard derivatized as described above was Analysis of rat blood adjusted to hematocrit values ranging converted to 2',3',5'-triacetyl uridine with 87% recovery; the between 0 and 69% and supplemented with 1 /IM [5-3H]uridine uridine component obtained from rat plasma was converted in showed that the plasma concentration was that predicted for a 84% yield. passive distribution into erythrocytes within 1 min at 37Â°.The Stability of [5-3H]Uridine in Blood. Tracer amounts of [5- plasma concentration of radioactivity did not change during a 3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood 30-min incubation at 37Â°; thus, erythrocytes do not concen from BALB/c x DBA/2 F, (hereafter called CD2F,) mice or trate uridine in nucleotide form to a significant extent. This is male Sprague-Dawley rats along with 10 fil of heparin (1000 consistent with the low level of pyrimidine nucleotides in eryth units/ml) and incubated at 37Â°for 10 or 20 min. HCIO4 (0.5 rocytes and with the lack of uridine kinase and phosphorylase M) was added to stop the reaction. The extract was processed activity reported by Tax ef al. (28). Oliver and Paterson (21)

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. J. D. Moyer et al. have reported previously that pyrimidine nucleosides rapidly cytidine standard and had an identical 280:254-nm absorb diffuse into erythrocytes without further metabolism. ance ratio. In addition, this component was collected from rat Similar incubations of [5-3H]cytidine in heparinized rat blood plasma and was rechromatographed in 2 additional HPLC and in plasma were formed at 37Â°using 10 or 100 JUMcytidine. systems to confirm its identity. The first sytem consisted of a Less than 10% deamination of cytidine could be observed by 25- x 0.45-cm BPAN6 column at 50Â°eluted with 0.5 M potas cation-exchange HPLC whether in blood or plasma after 30 sium phosphate, pH 3.0, at 1.0 ml/min. In this system, cytidine min, and the concentration of cytidine in plasma from whole and adenosine had retention times of 45 and 42 min, respec blood incubations was that predicted for passive diffusion and tively. The second system consisted of a 25-cm ODS-2 reverse- did not decrease during the incubation. phase column (Whatman, Inc.) that eluted at 50Â°with a 98:2 Determination of Uridine and Cytidine in Plasma. When 3 mixture of 0.005 M sodium heptanesulfonic acid adjusted to ml of blood were processed as described in "Materials and pH 2.2 with phosphoric acid and methanol. At a flow rate of Methods," a totally resolved uridine peak was observed (Chart 1.0 ml/min, cytidine and adenosine had retention times of 28 1). The identity and purity of the UV-absorbing peak were and 48 min, respectively. The collected cytidine fraction of rat established by several lines of evidence. This plasma compo plasma quantitatively cochromatographed with a cytidine nent cochromatographs with uridine in the HAX-4 anion-ex- standard and gave the expected 280:254-nm ratio in both of change systems as described in "Materials and Methods" these systems. without increase of the width at half height and displays the The concentrations of uridine and cytidine observed in rat, same 280:254-nm absorbance ratio as a uridine standard. This mouse, and human plasma, as well as in commercial horse component of rat plasma collected from anion-exchange chro- serum, are given in Table 1. Since the horse serum was not matography shows the correct retention time and 280:254-nm absorbance ratio when rechromatographed on the Lichrosorb 0008 reverse-phase system described in "Materials and Methods." Derivatization of the collected peak from the aniÃ³n exchange with acetic anhydride quantitatively produces a peak which has the retention time and absorbance ratio of 2',3',5'-triacetyl uridine as described in detail in "Materials and Methods." 0006 Cytidine in plasma can also be measured as described in I "Materials and Methods" and is well resolved (Chart 2). The o component identified as cytidine cochromatographed with a

0.004 .

0.004

0002

0.003

min Chart 2. Separation of cytidine in rat plasma. An acid-soluble extract of rat plasma was chromatographed on the borateipolystyrene columns. The nucleo- 0.002 side fraction was lyophilized and reconstituted with water, and an aliquot equiv alent to 0.375 ml of plasma was chromatographed on a 50-cm BPAN6 column as described in "Materials and Methods." Cytidine was located by inclusion of a tracer amount of [3H]cytidine. The 280-nm absorbance is shown at a sensitivity of 0.01 absorbance units, full scale, and the cytidine content of the sample was 0.8 nmol.

0.001 Table 1 Concentration of uridine and cytidine in blood plasma Blood was drawn into heparinized tubes by heart puncture or venous puncture from Sprague-Dawley rats, CD2F, mice, or healthy laboratory workers. The horse serum was purchased from Grand Island Biological Co. The blood was immedi ately centrifugea in a tabletop centrifuge at 5Â°.An acid-soluble extract of the plasma was prepared, and uridine or cytidine were determined as described in "Materials and Methods." The values are corrected for the recovery observed on the borate-polystyrÃ¨ne columns and in extraction.

Chart 1. Separation of uridine in rat plasma. Blood was obtained from the (Â¿IM)1.0 OIM)3.3 vena cava of a male Sprague-Dawley rat under pentobarbital anesthesia, and an Â±0.1Â°(22)6 Â±0.2 (8) acid-soluble extract of plasma was prepared and chromatographed on borate: Rat plasma Mouse plasma 2.2 Â±0.1 (7) 1.5 Â±0.2 (5) polystyrene resin. After concentration, a sample equivalent to 0.75 ml of plasma 0.47 Â±0.11 (4) was chromatographed on a 25-cm HAX4 anion-exchange column as described Human plasma 4.9 Â±0.6 <6) in "Materials and Methods." Only the 254-nm absorbance trace is shown; the Horse serumUridine 2.3 Â±0.2 (7)Cytidine <0.3 (4) 280-nm absorbance was also monitored. The location of uridine was documented Mean Â±S.E. by the inclusion of a tracer amount of [3H]uridine. ' Numbers in parentheses, number of samples examined.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Circulating Uridine and Cytidine in the Rat freshly prepared, the value given here may reflect some deg relative to control animals 3 or 24 hr after treatment with PALA radation during shipment or storage at -20Â° but may be of (500 mg/kg) or PF (100 mg/kg). interest to those who use this serum in tissue culture. In all Metabolism of Circulating Uridine. The kineticsof removal cases, the concentrations observed were in the low micromolar of uridine from blood was determined after tracer injection of range. The combined concentration of both nucleosides was [5-3H]uridine (Chart 3). Even after 0.7 min, a large percentage 3.7 to 5.4 fiM, but the ratio of uridine to cytidine varied widely of radioactivity in acid extracts of blood was associated with and may reflect the relative activity of catabolic and anabolic metabolites such as uracil. By 2 min, distribution of total enzymes in the animals. radioactivity was nearly complete, but the concentration of [5- Effect of PALA and PF on Circulating Pyrimidine Nucleo 3H]uridine continues to decrease with a i,/2 of ~3 min. sides. Since salvage of circulating uridine or cytidine might Salvage of Circulating Uridine. Sprague-Dawley rats re replete nucleotide pools of tissues blocked in de novo synthe ceived infusions for 4 hr with tracer amounts of [5-3H]uridine sis, the effect of treatment with PALA and PF on these circu by the jugular vein, and a constant level of labeled uridine was lating pools was examined. Neither PALA nor PF produced achieved within 1 hr in arterial blood even though total circu >40% depletion of cytidine or uridine in Sprague-Dawley rats lating radioactivity increased linearly with time (Chart 4). At an 24 hr after treatment (Table 2). In CD2Fi mice examined in a infusion rate of 0.092 mCi/hr, the radioactivity associated with similar manner, plasma uridine decreased no more than 20% uridine was 4.5 Â±1.3, 8.4 Â±0.3, 9.8 Â±0.5, and 12 Â±0.8 x 103 cpm/ml (mean Â±S.E. for 7 samples taken at 30-min Table 2 intervals) in 4 rats examined. The plasma concentrations of The effect of PALA or PF on pyrimidine nucleoside concentration of rat plasma endogenous uridine in these 4 rats at the conclusion of the Male Sprague-Dawley rats (-300 g) received drugs Â¡.p.;after 24 hr. they were infusion average 0.95 Â¡M,similar to the value obtained for the anesthetized with sodium pentobarbitol, and ~6 ml of blood were drawn and centrifugea in a chilled tube containing 0.05 ml heparin (250 units/ml) at 5Â°.An larger sample of rats examined for Table 1. The specific activity acid-soluble extract was prepared, and the nucleosides were determined as of circulating uridine was an average of 0.024 Ci/mmol for the described in "Materials and Methods." Only the PF effect on cytidine was significant (p < 0.05) by Student's (test. 4 rats, and from this it can be calculated that, in rats, approxi mately 95 /Â¿molofuridine enter and leave the circulation each Treatment0.9% HM3.3 HM0.99 Â±0.3"2.6 day. NaCI solution Â±0.22 In the tissues of these animals, tritiated water was the major PALA (250 mg/kg) Â±0.8 0.71 Â±0.09 metabolite of [5-3H]uridine (Table 3). Ynger ef al. (32) have PF (100 mg/kg)Cytidine 2.3 Â±0.3Uridine 0.61 Â±0.11 " Mean Â± S.E. of 7 (control) or 4 (drug-treated) rats from 2 experiments also reported 3H20 as a metabolite of [5-3H]uridine in the which are combined. mouse. The lower levels of volatile radioactivity in bone marrow and intestine may reflect the washing these tissues receive Metabolism of Circulating 1000 [5-3HJ Uridine A.

Iff 800

Â» â€¢? !V S O 600 Total Â§ Â« radioactivity

V 400 IO r\ I I 2 200 A

E o ua

IO \ X \ K) in

[5-3H] Uridine I 234 IO 2.5 7.5 IO 12.5 Hour Chart 4. Concentration of total radioactivity and [5-3H]uridine in arterial blood mm during infusion. A male Sprague-Dawley rat (325 g) was given an infusion via the Chart 3. Metabolism of circulating [5- H]uridme after a single injection. jugular vein at a rate of 0.092 mCi/hr with [5-3H]uridine (28.5 Ci/mmol). and Sprague-Dawley rats (-340 g) received 0.1 mCi of [5-3H]uridine by the jugular blood concentrations were determined by a 2-step Chromatographie procedure vein. Blood samples were prepared and analyzed as described in "Materials and as described in "Materials and Methods." This result is typical of that obtained Methods." The concentrations of total radioactivity and radioactive uridine in in 4 rats examined in this manner. The average blood concentration of [5- blood are shown. Points, mean of results from 3 rats; bars, S.E. 3H]uridine obtained for all 4 is given in the text.

AUGUST 1981 3013

Table 3 Salvage of infused [5-3H]uridine Sprague-Dawley rats (~325 g) received infusions via the jugular vein with [5-3H]urldine at 0.092 mCi/hr as described In "Materials and Methods." After 4 hr of infusion, the rats were anesthetized with sodium pentobarbital and rapidly dissected. Radioactivity in the acid-soluble and RNA fractions was measured as described In "Materials and Methods." The skeletal muscle was a portion of the abdominal wall. The mean circulating [5-3H]uridlne concentrations obtained in these rats are given in the text. The efficiency of scintillation counting was 37%. Acid soluble 10~3}Uracil(cpm/g x

nucleo-TissueBone nucleo-tides15121710125265Â± 10~3)Â±

marrowIntestineKidneyLiverLungMuscleSpleenThymusTotal4704801500130011007501500650Â±Â±Â±Â±Â±Â±-Â±66a56751306039160130tides160Â±84 4Â± 40Â± 38Â± Â±310 2Â± 40Â± 10Â± Â±44 4Â± 80Â± 16Â± Â±220 3Â± 120Â± 2Â± Â±10500 2Â± 50Â± 10Â± 40Â± Â±44 8Volatile210310790730660670680540Â±50Â± 10Â± Â±21224545010014Cytosme 300RNA(cpm/g1605752867211013X2 Mean Â±S.E. from 4 rats.

Table 4 minimal activity. In general, the degree of incorporation into Acid-soluble pyrimidine nucleotide and RNA pyrimidine content of rat tissues RNA was a reflection of the incorporation into acid-soluble The tissues from the Sprague-Dawley rats given infusions of [5-3H]uridine or [5-3HJcytidine were homogenized, and acid-soluble extracts and RNA hydroly- nucleotides. In the bone marrow and intestine, both of which sates were prepared as described in "Materials and Methods." The RNA pyrim contain a significant proportion of dividing cells, the amount of idine content is calculated from a 45% by weight pyrimidine nucleotide content label in RNA equaled that in acid-soluble pools after 4 hr of of rat RNA. infusion, whereas, in all other tissues, the fraction of radioac nucleo- nucleotides pyrimidine tivity incorporated into RNA was much smaller. TissueBone tides(/imol/g)0.11 (/Â¿mol/g)0.50 (nmol/g)7.86.45.410.03.81.89.17.1Metabolism of Circulating [5-3H]Cytidine. As a preliminary Â±0.005a0.13 marrowIntestineKidneyLiverLungMuscleSpleenThymusCytosineÂ±0.040.78 to infusion of [5-3H]cytidine, the clearance of a single bolus i.v. Â±0.0060.12 Â±0.020.75 Â±0.00070.12 Â±0.021.4 injection of [5-3H]cytidine was examined (Chart 5). In contrast Â±0.010.12 Â±0.10.52 to uridine, clearance is slower and produces much smaller Â±0.0070.05 Â±0.040.21 amounts of catabolites. Both total radioactivity and [5-3H]cyti- Â±0.0010.20 Â±0.050.82 Â±0.010.30 Â±0.040.70 dine continue to decrease throughout the 60-min course of the Â±0.05Uracil Â±0.05RNA experiment. Although the turnover of cytidine (ft ~20 min) Mean Â±S.E. for tissues from 7 rats. appeared slower than that of uridine (fs ~3 min), it was suffi ciently rapid to attain a steady state in subsequent infusion during the workup. Assuming equal distribution throughout experiments. body water, 3H2O comprises ~70% of the infused dose at 4 hr. Salvage of Circulating Cytidine. To estimate the degree of Much of the nonvolatile radioactivity found in tissues is not salvage of endogenous cytidine from the circulation, a steady- associated with nucleotides and probably represents interme state specific activity was established by constant infusion of diate degradation products such as/?-alanine and /S-ureidopro- [5-3H]cytidine. The concentrations of total radioactivity and pionate. These are ultimately catabolized further, and, at the tritiated cytidine in blood obtained by this procedure are shown conclusion of a 24-hr infusion, 85 to 99% of acid-soluble in Chart 6. After 30 min, the concentration of [5-3H]cytidine radioactivity in every tissue examined is present as 3H2O. was somewhat lower than at subsequent times, but between 1 The incorporation of radioactivity into uracil nucleotides per and 4 hr, the concentration of [5-3H]cytidine was nearly con g of tissue was much greater than into cytosine nucleotides, as stant, while the blood concentration of total radioactivity in might be expected, since cytosine nucleotide pools are smaller creased about 2-fold. In 3 experiments, the steady-state blood (Table 4), and additional steps are required to convert uracil to concentrations of [5-3H]cytidine were 91 Â±6 x 103, 120 Â±7 cytosine nucleotides. The ratio of radioactivity from uridine in X 103, and 120 Â± 7 x 103 cpm/ml (mean Â± S.E., n = 7 uracil compared to cytosine nucleotides ranged from 3:1 to 7: samples) at an infusion rate of 0.092 mCi/hr. This radioactivity 1 in RNA, lower than in the acid-soluble pools. This is a can be considered a tracer amount since it represents a con reflection of the smaller cytosine nucleotide pools and cytosine- centration of 5 x 10~9 M [5-3H]cytidine, and plasma concen rich composition of rat liver RNA (cytosine:uracil = 1.6). Since tration averaged 3.3 /Â¿M.Thus, the specific activity was about in the liver substantial amounts of radioactivity released by 0.04 Ci/mmol, and based on steady-state calculations, 50 alkaline hydrolysis were not 2',3'-monophosphate, the incor Â¿imolof cytidine transverse the circulation each day. The 10- poration of label into the RNA was based on the results from fold-higher blood concentration of [5-3H]cytidine obtained in comparison with that achieved on infusion of [5-3H]uridine is the chromatography of the hydrolysate and detection in the uridylate and cytidylate peaks. consistent with the slower clearance of cytidine seen in the Under these steady-state conditions of infusion, differences experiments using a single injection. in the utilization of circulating uridine among tissues were Substantial salvage of endogenous cytidine by conversion to striking (Table 3). Bone marrow, spleen, kidney, and lung were nucleotides and incorporation into RNA was seen after 4 hr heavily labeled, while the intestine and thymus incorporated (Table 5). In all tissues except muscle, the nucleotides and moderate amounts, and the liver and skeletal muscle had RNA comprised >65% of the total radioactivity in the tissue.

3014 CANCER RESEARCH VOL. 41

Metabolism of Circulating Cytidine tions from the data of Tables 4 and 5 reveal that, in no case, however, does the specific activity of the cytosine nucleotide pool exceed 0.008 Ci/mmol. Since the specific activity of circulating cytidine during the infusion was 0.045 Ci/mmol, no more than 18% of the cytosine nucleotide pool in any tissue 10' was derived from circulation during the 4-hr course of the infusion. In bone marrow, spleen, and thymus, comparison of the specific activity of the cytosine nucleotide pool to that of the circulating cytidine reveals that approximately 7% of this pool was derived from the circulation in 4 hr. Total Radioactivity From the specific activity of circulating uridine and cytidine during the infusions and the incorporation of radioactivity into ,o acid-soluble nucleotides and nucleic acids, it is possible to a u estimate the contribution of circulating pyrimidine nucleosides to the nucleotide pool and RNA of each tissue (Table 6). Since the precise pyrimidine nucleotide requirement of a tissue has never been established, direct calculation of the percentage of contribution of circulating pyrimidines to the total requirement is not possible. However, the relationship of this amount to the turnover of RNA pyrimidines in a nondividing tissue or the IO 10 20 30 40 50 60 minimum amount required to double RNA and acid-soluble min pyrimidine nucleotide pools in a rapidly growing tumor can be Chart 5. Metabolism of circulating cytidine. Sprague-Dawley rats (~250 g) received 0.15 mCi of [5-3H]cytidine by bolus injection into the jugular vein. Blood estimated. In the liver, it has been reported that the pyrimidines of RNA turn over with a f} of ~5 days (1 ). Since this tissue samples (100 /il) were drawn from the carotid artery at the indicated times, and total radioactivity and [5-3H]cytidine were measured in the acid-soluble extract contains 7 mg of RNA per g and since 45% of this is pyrimidine as described in "Materials and Methods." Data from 3 experiments are com ribonucleotide by weight, the daily requirement would be 1.2 bined. Points, mean for the 3 rats; bars, S.E. jumol/g/day or about 3 times the calculated daily contribution Infusion of [5-3HJ Cytidine from circulating uridine and cytidine.

Table 5 Salvage of infused [5-3H]cytidine Sprague-Dawley rats (~250 g) were given infusions via the jugular vein with 400 r [5-3H]cytidine at 0.092 mCi/hr as described in "Materials and Methods." At 4 hr, the animal was anesthetized with sodium pentobarbitol and quickly dissected. Radioactivity in the acid-soluble and RNA fractions was measured as described 300 in "Materials and Methods." The musice was a portion of the abdominal wall. & o The mean blood levels of radiolabeled cytidine obtained in these 3 rats are shown X in Chart 6. IO Acid soluble O 200 10~3TissueBone cpm/g x

1 i [s-3H] Cytidine nu- nu- (cpm/g x 10'3)2000 = 100 cleotides65 cleotides290 Â± 60a marrow Â± 21 Â±30 Â±200 gi T5 Intestine 780 Â± 20 100 Â± 17 240 Â±15 1000 Â±100 o Kidney 2700 Â±400 610 Â±100 750 Â±80 1100 Â±150 I Liver 1800 Â±300 570 Â±220 450 Â±90 1300 Â±400 Lung 800 Â± 60 130 Â± 12 240 Â±26 510Â± 50 hours Muscle Chart 6. Infusion of [5-3H]cytidine. Sprague-Dawley rats (~250 g) were given 330 Â± 30 50 50 55 Â± 6 infusions of [5-3H]cytidine via the jugular vein at a rate of 0.092 mCi/hr, and Spleen 1200 Â± 30 120 Â± 19 470 Â±30 1200 Â±110 ThymusTotal670 730 Â± 30Uracil arterial blood radioactivity (â€¢)and [5-3H]cytidine (O) were determined as de 35 Â± 9Cytosine260 Â± 9RNA 530 Â± 37 scribed in "Materials and Methods." Points, mean from 3 animals examined on ' Mean Â±S.E. from 3 rats. 2 days; bars, S.E. Table 6 The remaining acid-soluble radioactivity was found in the void Contribution of circulating pyrimidine nucleosides to the nucleotide pools and RNA pyrimidines of rat tissues volume of the Chromatographie system used for analysis of The radioactivity in pyrimidine nucleotides and RNA was converted to/imol by UMP and CMP. More radioactivity was present in cytosine use of the specific activity of circulating uridine and cytidine obtained in the nucleotides than in uracil nucleotides except in the liver, al infusion. though both pools showed incorporation of tritium. Most nota TissueBone (Â¿imol/g/day)0.13a0.060.170.030.130.010.230.03Cytidine(/imol/g/day)0.370.220.410.370.140.020.290.14 ble is the >7-fold-greater labeling of RNA by cytidine compared marrowIntestineKidneyLiverLungMuscleSpleenThymusUridine to uridine. The more efficient incorporation of [5-3H]cytidine into RNA recommends it for use in estimating RNA synthesis in vivo. In tissues other than the kidney and liver, the radioactivity present in RNA exceeded that in acid-soluble nucleotides. This

suggests that these pools turn over in less than 4 hr. Calcula Mean for 4 uridine or 3 cytidine infusions.

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Blood flow has been measured in a number of experimental enters the plasma pool in the rat is catabolized rather than tumors of rats by Gullino and Grantham (7), who found that salvaged. (Â£>)Although cytidine turnover is less rapid, the these tumors are much less well vascularized than liver. At an circulating cytidine is more efficiently converted to tissue nu average blood flow of 10 ml/hr/g, only 1 /Â¿molof pyrimidine cleotides. This fact, together with the differences in nucleotide per g could be supplied per day assuming 100% clearance pool sizes (Table 4), accounts for the observation of Hammar- and conversion to nucleotide. We calculate that this is far less sten ef a/. (9) that cytidine was much more effective in labeling than the estimated 10 /imol/g/day required for those tumors RNA in vivo than was uridine. The difference in salvage of to grow with the doubling times (<2 days) observed (7). cytidine compared to uridine by the rat may not be seen in other species, since the rat is notably deficient in cytidine DISCUSSION deaminase but has somewhat higher uridine phosphorylase activity relative to other mammals (3, 24). Enzymes and transport systems for utilization of extracellular The failure of PF or PALA to substantially deplete plasma nucleosides have been demonstrated in numerous cells and concentrations suggests that the source is not very sensitive to tissues. However, very few studies have examined the dynam these inhibitors of pyrimidine synthesis de novo. Recently, a ics of salvage of nucleic acid precursors in vivo (11, 15, 17). determination of circulating uridine concentrations in patients The current study examines the concentration of circulating treated with PALA was reported to be slightly higher than that pyrimidine nucleosides available for salvage, the turnover of reported here, but relatively little change was observed after a these nucleosides, and their conversion to nucleotides by 5-day course of therapy (14), consistent with the inability of selected tissues of the rat. PALA to decrease circulating levels in mice or rats given toxic The analytical method described here, together with the doses of this inhibitor of de novo synthesis. rigorous confirmation of the identity of the nucleosides mea The source of circulating nucleotides is currently not known. sured, has established that low micromolar concentrations of The diet, intestinal flora, or ' 'feeder' ' organs are all possibilities. uridine and cytidine are available for salvage from the plasma Possibly, constant leakage from acid-soluble nucleotide pools (Table 1). The uridine concentrations reported here are similar in a wide range of tissues occurs at a rate influenced by the to those reported earlier for sheep plasma (2 /tw, Ref. 8) and concentration of monophosphate and the relative activities of dog plasma (0.4 Â¡J.M,Ref. 29) as determined by a multistep phosphorylating (kinase) and dephosphorylating enzymes. Re separation by thin-layer chromatography. The uridine concen cently cultured cells have been shown to excrete small amounts tration found in human plasma is slightly higher than that of uridine, cytidine, and pseudouridine into the medium, gen reported for human serum by Hartwick ef al. (3.2 /IM, Ref. 10). erating micromolar concentrations, particularly in the plateau One earlier report had suggested a much higher concentration phase (30). Ongoing studies in this laboratory are directed of uridine in rat plasma (32 /IM, Ref. 25), perhaps because of toward understanding the factors controlling the concentra incomplete resolution from another UV-absorbing component. tions and flux of circulating pyrimidine nucleosides, with partic In addition, the cytidine concentration was 10 /Â¿M,ratherhigher ular focus on the essentially complete clearance of uridine in than that found in this study. The reason for this discrepancy the portal vein by the liver and its replacement by uridine is not clear, although different extraction procedures were derived from de novo synthesis (5). used, and the rats examined in Ref. 25 were female. It is Although only a small fraction of the pyrimidine nucleotide possible that nutritional factors may influence the circulating pools in tissues is derived from the circulation, during chemo levels, particularly since Tseng ef a/. (29) have shown that the therapy with inhibitors of pyrimidine synthesis de novo, this concentration of uridine rises severalfold in dogs fed 2 pounds may change markedly. The failure of PALA or PF to greatly of meat. Obviously, meat would be a food high in nucleic acid deplete circulating levels in treated animals suggests that sal compared to commercial animal chow. The influence of diet is vage should be considered as an ongoing process during unclear and requires further study. On the other hand, a recent therapy. Further studies on salvage by tumors both sensitive study by Sonoda and Masamiti (27) indicates very poor utili zation of 14C-labeled RNA fed to mice except by the intestinal and resistant to PALA and PF are required for a more complete understanding of the role of nucleoside salvage. Similar studies mucosa. extending this work to the salvage of thymidine and deoxycy- The kinetics of uridine clearance after i.v. injection (Charts tidine would be essential in experiments with the several chem- 3 and 5) confirms an earlier report (4), which showed an otherapeutic drugs that inhibit the production of the corre extremely rapid clearance of a single injection of this nucleo- sponding deoxynucleotides. side but a slower clearance of cytidine in the rat. The current infusion experiments using an infusion of radiolabeled uridine or cytidine to steady-state levels reveal that approximately 140 REFERENCES

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James D. Moyer, James T. Oliver and Robert E. Handschumacher

Cancer Res 1981;41:3010-3017.

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