[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 andDevelopment Center, Ridgefield. Connecticut 06877 [J. T. O.]
ABSTRACT
A new procedure was developed to measure uridine andcytidine in plasma. These nucleosides are present in micro-molar concentrations in the plasma of rats, mice, and humans.Inhibitors of pyrimidine synthesis de novo (pyrazofurin or N-phosphonacetyl-L-aspartate) produce only modest decreasesin the concentration of circulating uridine or cytidine in the rat.Since both uridine and cytidine are rapidly cleared from thecirculation of the rat, constant infusions of radiolabeled uridineand cytidine were used to establish a steady-state specificactivity of circulating nucleoside without altering the normalendogenous concentration. These studies permitted an estimation of the contribution of circulating pyrimidine nucleosideto the nucleotide pools of various rat tissues. Most of theuridine entering the circulation (>70%) is catabolized ratherthan salvaged by formation of nucleotides. Cytidine in thecirculation is much more efficiently utilized and is predominantly salvaged. The implication of these results for chemotherapy based on inhibition of pyrimidine synthesis de novo isdiscussed.
INTRODUCTION
Separate enzymatic pathways exist in mammalian cells togenerate nucleotides by de novo synthesis or by salvage ofpreformed bases or nucleosides (12,19). Although it has beensuggested that bone marrow and intestinal mucosa are primarily dependent on the salvage pathway to obtain purine nucleotides (15, 17) and that the liver provides preformed purinesand pyrimidines to other tissues through the circulatory system(15, 16, 23), the extent to which a particular tissue dependson each pathway has not been firmly established. A morecomplete understanding of the balance between alternate pathways would help guide the proper chemotherapeutic use ofPF3 or PALA, inhibitors of the de novo pathway, and p-nitro-
benzyl-thioinosine (31) or inhibitors of uridine kinase, which
may block nucleoside salvage. Quantitating the low levels ofcirculating pyrimidine nucleosides has been a major difficultyin studying nucleoside salvage in vivo. Radioimmunoassays(13), microbiological assays (20, 22), and multistep proceduresinvolving thin-layer chromatography and derivatization (8, 27,
29) have been used previously to measure pyrimidine nucleosides. Recently, several HPLC methods have been developedto permit more rapid quantitation of nucleoside levels (10, 14,25).
In the current study, we have devised a method for quantitation of circulating uridine and cytidine and used constant
1Supported by American Cancer Society Grant CH-67T.2 To whom requests for reprints should be addressed.3 The abbreviations used are: PF. pyrazofurin; PALA. N-phosphonacetyl-L-
aspartate; HPLC, high-performance liquid chromatography.Received January 19, 1981; accepted April 22, 1981.
infusions of radiolabeled tracers to measure the salvage ofthese nucleosides by various tissues. The contribution of circulating pyrimidine nucleosides to the nucleotide pools hasbeen examined in relation to the requirements of slowly growingtissues for the maintenance of pools and also to the needs ofrapidly dividing tissue undertaking net synthesis of RNA andDMA.
MATERIALS AND METHODS
Drugs and Chemicals. Succinic anhydride and pyridine werepurchased from Pierce Chemical Co. (Rockford, III.) . Nucleosides, nucleotides, heparin, and 2',3',5'-triacetyl uridine were
purchased from Sigma Chemical Co. (St. Louis, Mo.). Aceticanhydride, m-aminophenylboronic acid hemisulfate, and 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloridewere obtained from Aldrich Chemical Co. (Milwaukee, Wis.).Methanol for HPLC buffers was purchased from Burdick &Jackson Laboratories, Inc., (Muskegon, Mich.). [5-3H]Uridine(27.9 Ci/mmol) and [5-3H]cytidine (27.7 Ci/mmol) were pur
chased from New England Nuclear (Boston, Mass.).HPLC. Chromatography was performed with Altex 110A
pumps, an Altex 420 microprocessor to generate gradients,and 2 Altex 8-/il flow cell UV detectors monitoring 280 nm and
254 nm with analog output on a dual channel Linear Corp.Model 485 recorder.
Anion-Exchange Chromatography of Uridine. This Chromatographie system is a modification of a system described bySinghal and Conn (26). The use of the small particle size resinhas produced greater resolution and sensitivity. A 25- x 0.46-cm column of HAX-4 anion-exchange resin (Hamilton Co.,Reno, Nev.) was slurry packed, maintained at 50°,and eluted
with 0.3 M acetic acid:ammonium acetate (pH 9.7) at 1.0 ml/min. Peak height was directly proportional to amount injectedbetween 0.1 and 5 nmol. The following retention times wereobserved: cytidine, 2.1 min; adenosine, 4.2 min; thymidine,10.8 min; pseudouridine, 17.4 min; deoxyuridine, 17.6 min;uridine, 22 min; PF, 34 min; uracil, 41 min; guanosine, 48 min;and inosine, 84 min. The Chromatographie efficiency for uridinewas 1200 theoretical plates. Multiple injections of standardsindicated that peak height variation was ±2% (S.D.).
The resin was regenerated after processing 50 biologicalsamples by 30-min treatment with 50% sulfuric acid at 70°.
Although human, horse, or mouse plasma samples (pretreatedby borate affinity column separation as described below) gavebase line separation in most instances, in 5 of 27 rat plasmaextracts, an overlapping peak at 18 min prevented accuratemeasurement of uridine.
Reverse-Phase Chromatography of Nucleosides andBases. A 25- x 0.46-cm Lichrosorb 5-jum particle size column(Altex Co., Berkeley, Calif.) was eluted at 1.0 ml/min with 0.2M sodium phosphate (pH 6.4) at 25°. The following retention
times were observed: AMP, GMP, UMP, CMP, uric acid, uracil,
and cytosine, all <7.5 min; cytidine, 8.5 min; uridine, 14 min;deoxycytidine, 14 min; deoxyuridine, 23 min; thymidine, 35min; and adenine, 30 min. Peak height was directly proportionalto amount injected in the range of 0.1 to 1.0 nmol at a sensitivityof 0.005 absorbance units, full scale.
Analysis of RNA Hydrolysate by Anión Exchange. Incorporation of [5-3H]uridine into RNA was assayed by separationof the 2',3'-mononucleotides resulting from alkaline hydrolysison a 25-cm BAX-4 (Benson Co., Reno, Nev.) column at 50°
with a linear gradient from 0.6 to 1.0 M ammonium acetate, pH8.4, for 35 min during a period of 20 min and subsequentisocratic elution with 1 M buffer at 2 ml/min.
Cation-Exchange Chromatography of Cytidine. A 25- x0.46-cm column packed with BPAN6 resin (Benson Co.) waseluted at 50°with a flow of 1.5 ml/min with 0.3 M formic acid
adjusted to pH 4.0 with ammonium hydroxide. The followingretention times were observed: void, 2.0 min; uracil, 2.4 min;uridine, 2.6 min; guanosine, 5.8 min; adenosine, 15 min; cyti-
dine, 24 min; adenine, 55 min; and cytosine, 57 min. Forquantitation of unlabeled cytidine in extracts, a better resolutionfrom the UV-absorbing components was obtained with a 50- x0.46-cm column eluted at 60°with 0.5 M ammonium formate,
pH 4.0, at 0.8 ml/min (4000 theoretical plates). Peak heightwas proportional to the amount injected over the range of 0.2to 2 nmol at a sensitivity of 0.01 absorbance units, full scale.
Determination of Uridine and Cytidine in Plasma. Plasmafrom heparinized blood was mixed with an equal volume of ice-cold 1 M HCIO4 and kept at 4°for 20 min. The precipitated
protein was removed, and the supernatant was neutralized topH 7 to 9 at 0°with 10NKOH.
One to 2 ml of the extracts were prechromatographed on 1ml of borate affinity resin (5). The column was washed with 4ml of 0.3 M ammonium acetate, pH 8.8, and eluted with 3portions (1.5, 2, and 2 ml) of 0.1 N formic acid. The last 2fractions, which contained the nucleosides, were lyophilizedand reconstituted in 0.3 M ammonium acetate buffer for chro-matography as described above. The overall recovery of uridine or cytidine determined by addition of internal standards ofradiolabeled nucleoside was 92 ±10% (S.D.) for uridine and90 ±6% for cytidine.
Acetylation of Uridine Peak. A derivatization procedure wasperformed to confirm the structure of the component identifiedas uridine. The uridine fraction from HPLC of rat plasma waslyophilized, and pyridine (100 /¿I)and acetic anhydride (50 jul)were added and allowed to react at room temperature for 1 hrto form the triacetyl derivative (2). After removal of aceticanhydride and pyridine, the sample was reconstituted in 0.5 mlof 0.3 M sodium acetate (pH 5.0), adsorbed onto a 25-cmLichrosorb 5-jum particle size C18 reverse-phase column, and
eluted with 0.3 M sodium acetate (pH 5.0,1 ml/min) for 10 minfollowed by a 15-min gradient to achieve a buffer:methanol mixof 3:1. A uridine standard derivatized as described above wasconverted to 2',3',5'-triacetyl uridine with 87% recovery; the
uridine component obtained from rat plasma was converted in84% yield.
Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood
from BALB/c x DBA/2 F, (hereafter called CD2F,) mice ormale Sprague-Dawley rats along with 10 fil of heparin (1000units/ml) and incubated at 37°for 10 or 20 min. HCIO4 (0.5
M) was added to stop the reaction. The extract was processed
and chromatographed on the HAX-4 column as describedabove, and 1-ml fractions were collected and counted in 10 ml
of Formula 963 (New England Nuclear).Disposition of [5-3H]Uridine and [5-3H]Cytidine in the Rat.
Male Sprague-Dawley rats (250 to 350 g) were anesthetized
with methoxyflurane, and catheters were placed in the jugularvein and the carotid artery. [5-3H]Uridine (27.9 Ci/mmol) was
prepared in sterile 0.9% NaCI solution, and 0.1 mCi wasinjected via the jugular vein in a volume of 0.2 ml. At theindicated times, 100 p.\ of blood from the carotid artery weremixed with 400 ¡i\of ice-cold 0.5 M HCIO4, and the resulting
extract was processed and chromatographed sequentially onthe borate affinity resin and on BAX-4 anion-exchange resin as
described above. Radioactivity was determined in 10 ml ofFormula 963 at 24% efficiency.
Similar experiments were performed with [5-3H]cytidine. The
neutralized HCIO4 extract of blood (50 to 200 pi) was chromatographed directly on a 25-cm BPAN6 cation-exchange column
as described above. Fractions of 3 ml (2 min) were collected,and radioactivity was determined by scintillation spectrometry.
Infusion of [5-3H]Uridine or [5-3H]Cytidine. Catheters con
taining heparinized (100 units/ml) 0.9% NaCI solution wereplaced in the carotid artery and left jugular vein of a maleSprague-Dawley rat (250 g) that had been anesthetized with
methoxyflurane. The catheters were protected by a spring andattached to swivels to permit sustained infusion in consciousanimals. [5-3H]Uridine or [5-3H]cytidine (40pCi/ml) was infused
via the jugular vein at 2.3 ml/hr. Carotid blood was obtained at30-min intervals for determination of radiolabeled nucleoside.At the end of the 4-hr infusion, tissue samples were frozen in
liquid N2. Tissue samples were homogenized with 10 volumesof N HCIO4 at 0°, and total acid-soluble uracil and cytosine
nucleotides were determined after acid hydrolysis as above.The acid-insoluble fraction was washed 3 times with ice-cold
0.3 M HCIO4, and the RNA was hydrolyzed and quantitated bythe method of Blobel and Potter (1 ). The resulting oligonucle-otides were further hydrolyzed in 0.5 M KOH at 37°overnightto produce a mixture of 2' and 3'-mononucleotides for chro-
matography as described above.
RESULTS
Distribution and Stability of Uridine and Cytidine in Blood.The conversion of uridine to uracil was reported to occur indog blood by Tseng ef al. (29). To examine this possibility,whole rat and mouse blood was drawn and assayed for thedegradation of uridine using HPLC as described in "Materialsand Methods." In mouse blood incubated at 37°, only 6 and
10% of the uridine was converted to uracil after 10 and 20 min,respectively. In rat blood, 10% degradation of added uridineoccurred after 10 min and 18% after 20 min.
Analysis of rat blood adjusted to hematocrit values rangingbetween 0 and 69% and supplemented with 1 /IM [5-3H]uridine
showed that the plasma concentration was that predicted for apassive distribution into erythrocytes within 1 min at 37°.The
plasma concentration of radioactivity did not change during a30-min incubation at 37°; thus, erythrocytes do not concen
trate uridine in nucleotide form to a significant extent. This isconsistent with the low level of pyrimidine nucleotides in erythrocytes and with the lack of uridine kinase and phosphorylaseactivity reported by Tax ef al. (28). Oliver and Paterson (21)
have reported previously that pyrimidine nucleosides rapidlydiffuse into erythrocytes without further metabolism.
Similar incubations of [5-3H]cytidine in heparinized rat bloodand in plasma were formed at 37°using 10 or 100 JUMcytidine.
Less than 10% deamination of cytidine could be observed bycation-exchange HPLC whether in blood or plasma after 30
min, and the concentration of cytidine in plasma from wholeblood incubations was that predicted for passive diffusion anddid not decrease during the incubation.
Determination of Uridine and Cytidine in Plasma. When 3ml of blood were processed as described in "Materials andMethods," a totally resolved uridine peak was observed (Chart
1). The identity and purity of the UV-absorbing peak were
established by several lines of evidence. This plasma component cochromatographs with uridine in the HAX-4 anion-ex-change systems as described in "Materials and Methods"
without increase of the width at half height and displays thesame 280:254-nm absorbance ratio as a uridine standard. Thiscomponent of rat plasma collected from anion-exchange chro-matography shows the correct retention time and 280:254-nmabsorbance ratio when rechromatographed on the Lichrosorbreverse-phase system described in "Materials and Methods."
Derivatization of the collected peak from the anión exchangewith acetic anhydride quantitatively produces a peak which hasthe retention time and absorbance ratio of 2',3',5'-triacetyluridine as described in detail in "Materials and Methods."
Cytidine in plasma can also be measured as described in"Materials and Methods" and is well resolved (Chart 2). The
component identified as cytidine cochromatographed with a
0.004
0.003
0.002
0.001
Chart 1. Separation of uridine in rat plasma. Blood was obtained from thevena cava of a male Sprague-Dawley rat under pentobarbital anesthesia, and anacid-soluble extract of plasma was prepared and chromatographed on borate:polystyrene resin. After concentration, a sample equivalent to 0.75 ml of plasmawas chromatographed on a 25-cm HAX4 anion-exchange column as describedin "Materials and Methods." Only the 254-nm absorbance trace is shown; the
280-nm absorbance was also monitored. The location of uridine was documentedby the inclusion of a tracer amount of [3H]uridine.
cytidine standard and had an identical 280:254-nm absorb
ance ratio. In addition, this component was collected from ratplasma and was rechromatographed in 2 additional HPLCsystems to confirm its identity. The first sytem consisted of a25- x 0.45-cm BPAN6 column at 50°eluted with 0.5 M potas
sium phosphate, pH 3.0, at 1.0 ml/min. In this system, cytidineand adenosine had retention times of 45 and 42 min, respectively. The second system consisted of a 25-cm ODS-2 reverse-phase column (Whatman, Inc.) that eluted at 50°with a 98:2
mixture of 0.005 M sodium heptanesulfonic acid adjusted topH 2.2 with phosphoric acid and methanol. At a flow rate of1.0 ml/min, cytidine and adenosine had retention times of 28and 48 min, respectively. The collected cytidine fraction of ratplasma quantitatively cochromatographed with a cytidinestandard and gave the expected 280:254-nm ratio in both of
these systems.The concentrations of uridine and cytidine observed in rat,
mouse, and human plasma, as well as in commercial horseserum, are given in Table 1. Since the horse serum was not
0008
0006
Io
0.004 .
0002
minChart 2. Separation of cytidine in rat plasma. An acid-soluble extract of rat
plasma was chromatographed on the borateipolystyrene columns. The nucleo-side fraction was lyophilized and reconstituted with water, and an aliquot equivalent to 0.375 ml of plasma was chromatographed on a 50-cm BPAN6 column asdescribed in "Materials and Methods." Cytidine was located by inclusion of atracer 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 was0.8 nmol.
Table 1
Concentration of uridine and cytidine in blood plasma
Blood was drawn into heparinized tubes by heart puncture or venous puncturefrom Sprague-Dawley rats, CD2F, mice, or healthy laboratory workers. The horseserum was purchased from Grand Island Biological Co. The blood was immediately centrifugea in a tabletop centrifuge at 5°.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-polystyrène columns and in extraction.
Rat plasmaMouse plasmaHuman plasmaHorse serumUridine
(¿IM)1.0±0.1°(22)6
2.2 ±0.1 (7)4.9 ±0.6 <6)2.3 ±0.2 (7)Cytidine
OIM)3.3
±0.2 (8)1.5 ±0.2 (5)0.47 ±0.11 (4)
<0.3 (4)
Mean ±S.E.' Numbers in parentheses, number of samples examined.
freshly prepared, the value given here may reflect some degradation during shipment or storage at -20° but may be of
interest to those who use this serum in tissue culture. In allcases, the concentrations observed were in the low micromolarrange. The combined concentration of both nucleosides was3.7 to 5.4 fiM, but the ratio of uridine to cytidine varied widelyand may reflect the relative activity of catabolic and anabolicenzymes in the animals.
Effect of PALA and PF on Circulating Pyrimidine Nucleosides. Since salvage of circulating uridine or cytidine mightreplete nucleotide pools of tissues blocked in de novo synthesis, the effect of treatment with PALA and PF on these circulating pools was examined. Neither PALA nor PF produced>40% depletion of cytidine or uridine in Sprague-Dawley rats24 hr after treatment (Table 2). In CD2Fi mice examined in asimilar manner, plasma uridine decreased no more than 20%
Table 2
The effect of PALA or PF on pyrimidine nucleoside concentration of rat plasmaMale Sprague-Dawley rats (-300 g) received drugs ¡.p.;after 24 hr. they were
anesthetized with sodium pentobarbitol, and ~6 ml of blood were drawn andcentrifugea in a chilled tube containing 0.05 ml heparin (250 units/ml) at 5°.An
acid-soluble extract was prepared, and the nucleosides were determined asdescribed in "Materials and Methods." Only the PF effect on cytidine wassignificant (p < 0.05) by Student's (test.
" Mean ± S.E. of 7 (control) or 4 (drug-treated) rats from 2 experiments
which are combined.
Metabolism of Circulating[5-3HJ Uridine
Iff
IO
I2A
Eau
IO
IO
!VV
r\
Totalradioactivity
o
\\
[5-3H] Uridine
2.5 7.5 IO 12.5
mmChart 3. Metabolism of circulating [5- H]uridme after a single injection.
Sprague-Dawley rats (-340 g) received 0.1 mCi of [5-3H]uridine by the jugularvein. Blood samples were prepared and analyzed as described in "Materials andMethods." The concentrations of total radioactivity and radioactive uridine in
blood are shown. Points, mean of results from 3 rats; bars, S.E.
Circulating Uridine and Cytidine in the Rat
relative to control animals 3 or 24 hr after treatment with PALA(500 mg/kg) or PF (100 mg/kg).
Metabolism of Circulating Uridine. The kineticsof removalof uridine from blood was determined after tracer injection of[5-3H]uridine (Chart 3). Even after 0.7 min, a large percentage
of radioactivity in acid extracts of blood was associated withmetabolites such as uracil. By 2 min, distribution of totalradioactivity was nearly complete, but the concentration of [5-3H]uridine continues to decrease with a i,/2 of ~3 min.
Salvage of Circulating Uridine. Sprague-Dawley rats received infusions for 4 hr with tracer amounts of [5-3H]uridine
by the jugular vein, and a constant level of labeled uridine wasachieved within 1 hr in arterial blood even though total circulating radioactivity increased linearly with time (Chart 4). At aninfusion rate of 0.092 mCi/hr, the radioactivity associated withuridine was 4.5 ±1.3, 8.4 ±0.3, 9.8 ±0.5, and 12 ±0.8x 103 cpm/ml (mean ±S.E. for 7 samples taken at 30-min
intervals) in 4 rats examined. The plasma concentrations ofendogenous uridine in these 4 rats at the conclusion of theinfusion average 0.95 ¡M,similar to the value obtained for thelarger sample of rats examined for Table 1. The specific activityof circulating uridine was an average of 0.024 Ci/mmol for the4 rats, and from this it can be calculated that, in rats, approximately 95 /¿molof uridine enter and leave the circulation eachday.
In the tissues of these animals, tritiated water was the majormetabolite of [5-3H]uridine (Table 3). Ynger ef al. (32) havealso reported 3H20 as a metabolite of [5-3H]uridine in the
mouse. The lower levels of volatile radioactivity in bone marrowand intestine may reflect the washing these tissues receive
1000
800
» •?S O 600§ «
I400
200
A.
XK)
in
I 234
HourChart 4. Concentration of total radioactivity and [5-3H]uridine in arterial blood
during infusion. A male Sprague-Dawley rat (325 g) was given an infusion via thejugular vein at a rate of 0.092 mCi/hr with [5-3H]uridine (28.5 Ci/mmol). and
blood concentrations were determined by a 2-step Chromatographie procedureas described in "Materials and Methods." This result is typical of that obtained
in 4 rats examined in this manner. The average blood concentration of [5-3H]uridine obtained for all 4 is given in the text.
Sprague-Dawley rats (~325 g) received infusions via the jugular vein with [5-3H]urldine at 0.092 mCi/hr as describedIn "Materials and Methods." After 4 hr of infusion, the rats were anesthetized with sodium pentobarbital and rapidlydissected. 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%.
Table 4Acid-soluble pyrimidine nucleotide and RNA pyrimidine content of rat tissues
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-sates were prepared as described in "Materials and Methods." The RNA pyrim
idine content is calculated from a 45% by weight pyrimidine nucleotide contentof rat RNA.
during the workup. Assuming equal distribution throughoutbody water, 3H2O comprises ~70% of the infused dose at 4 hr.
Much of the nonvolatile radioactivity found in tissues is notassociated with nucleotides and probably represents intermediate degradation products such as/?-alanine and /S-ureidopro-
pionate. These are ultimately catabolized further, and, at theconclusion of a 24-hr infusion, 85 to 99% of acid-solubleradioactivity in every tissue examined is present as 3H2O.
The incorporation of radioactivity into uracil nucleotides perg of tissue was much greater than into cytosine nucleotides, asmight be expected, since cytosine nucleotide pools are smaller(Table 4), and additional steps are required to convert uracil tocytosine nucleotides. The ratio of radioactivity from uridine inuracil compared to cytosine nucleotides ranged from 3:1 to 7:1 in RNA, lower than in the acid-soluble pools. This is areflection of the smaller cytosine nucleotide pools and cytosine-rich composition of rat liver RNA (cytosine:uracil = 1.6). Since
in the liver substantial amounts of radioactivity released byalkaline hydrolysis were not 2',3'-monophosphate, the incor
poration of label into the RNA was based on the results fromthe chromatography of the hydrolysate and detection in theuridylate and cytidylate peaks.
Under these steady-state conditions of infusion, differences
in the utilization of circulating uridine among tissues werestriking (Table 3). Bone marrow, spleen, kidney, and lung wereheavily labeled, while the intestine and thymus incorporatedmoderate amounts, and the liver and skeletal muscle had
minimal activity. In general, the degree of incorporation intoRNA was a reflection of the incorporation into acid-soluble
nucleotides. In the bone marrow and intestine, both of whichcontain a significant proportion of dividing cells, the amount oflabel in RNA equaled that in acid-soluble pools after 4 hr of
infusion, whereas, in all other tissues, the fraction of radioactivity incorporated into RNA was much smaller.
Metabolism of Circulating [5-3H]Cytidine. As a preliminaryto infusion of [5-3H]cytidine, the clearance of a single bolus i.v.injection of [5-3H]cytidine was examined (Chart 5). In contrast
to uridine, clearance is slower and produces much smalleramounts of catabolites. Both total radioactivity and [5-3H]cyti-
dine continue to decrease throughout the 60-min course of theexperiment. Although the turnover of cytidine (ft ~20 min)appeared slower than that of uridine (fs ~3 min), it was suffi
ciently rapid to attain a steady state in subsequent infusionexperiments.
Salvage of Circulating Cytidine. To estimate the degree ofsalvage of endogenous cytidine from the circulation, a steady-
state specific activity was established by constant infusion of[5-3H]cytidine. The concentrations of total radioactivity and
tritiated cytidine in blood obtained by this procedure are shownin Chart 6. After 30 min, the concentration of [5-3H]cytidine
was somewhat lower than at subsequent times, but between 1and 4 hr, the concentration of [5-3H]cytidine was nearly con
stant, while the blood concentration of total radioactivity increased about 2-fold. In 3 experiments, the steady-state bloodconcentrations of [5-3H]cytidine were 91 ±6 x 103, 120 ±7X 103, and 120 ± 7 x 103 cpm/ml (mean ± S.E., n = 7
samples) at an infusion rate of 0.092 mCi/hr. This radioactivitycan be considered a tracer amount since it represents a concentration of 5 x 10~9 M [5-3H]cytidine, and plasma concen
tration averaged 3.3 /¿M.Thus, the specific activity was about0.04 Ci/mmol, and based on steady-state calculations, 50¿imolof cytidine transverse the circulation each day. The 10-fold-higher blood concentration of [5-3H]cytidine obtained incomparison with that achieved on infusion of [5-3H]uridine is
consistent with the slower clearance of cytidine seen in theexperiments using a single injection.
Substantial salvage of endogenous cytidine by conversion tonucleotides and incorporation into RNA was seen after 4 hr(Table 5). In all tissues except muscle, the nucleotides andRNA comprised >65% of the total radioactivity in the tissue.
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
samples (100 /il) were drawn from the carotid artery at the indicated times, andtotal radioactivity and [5-3H]cytidine were measured in the acid-soluble extractas described in "Materials and Methods." Data from 3 experiments are com
bined. Points, mean for the 3 rats; bars, S.E.
Infusion of [5-3HJ Cytidine
&oX
IOO
400 r
300
200
= 100
igT5o
1 i [s-3H] Cytidine
I
hoursChart 6. Infusion of [5-3H]cytidine. Sprague-Dawley rats (~250 g) were given
infusions of [5-3H]cytidine via the jugular vein at a rate of 0.092 mCi/hr, andarterial blood radioactivity (•)and [5-3H]cytidine (O) were determined as described in "Materials and Methods." Points, mean from 3 animals examined on
2 days; bars, S.E.
The remaining acid-soluble radioactivity was found in the void
volume of the Chromatographie system used for analysis ofUMP and CMP. More radioactivity was present in cytosinenucleotides than in uracil nucleotides except in the liver, although both pools showed incorporation of tritium. Most notable is the >7-fold-greater labeling of RNA by cytidine comparedto uridine. The more efficient incorporation of [5-3H]cytidine
into RNA recommends it for use in estimating RNA synthesis invivo.
In tissues other than the kidney and liver, the radioactivitypresent in RNA exceeded that in acid-soluble nucleotides. This
suggests that these pools turn over in less than 4 hr. Calcula
tions from the data of Tables 4 and 5 reveal that, in no case,however, does the specific activity of the cytosine nucleotidepool exceed 0.008 Ci/mmol. Since the specific activity ofcirculating cytidine during the infusion was 0.045 Ci/mmol, nomore than 18% of the cytosine nucleotide pool in any tissuewas derived from circulation during the 4-hr course of theinfusion. In bone marrow, spleen, and thymus, comparison ofthe specific activity of the cytosine nucleotide pool to that ofthe circulating cytidine reveals that approximately 7% of thispool was derived from the circulation in 4 hr.
From the specific activity of circulating uridine and cytidineduring the infusions and the incorporation of radioactivity intoacid-soluble nucleotides and nucleic acids, it is possible to
estimate the contribution of circulating pyrimidine nucleosidesto the nucleotide pool and RNA of each tissue (Table 6). Sincethe precise pyrimidine nucleotide requirement of a tissue hasnever been established, direct calculation of the percentage ofcontribution of circulating pyrimidines to the total requirementis not possible. However, the relationship of this amount to theturnover of RNA pyrimidines in a nondividing tissue or theminimum amount required to double RNA and acid-soluble
pyrimidine nucleotide pools in a rapidly growing tumor can beestimated. In the liver, it has been reported that the pyrimidinesof RNA turn over with a f} of ~5 days (1 ). Since this tissue
contains 7 mg of RNA per g and since 45% of this is pyrimidineribonucleotide by weight, the daily requirement would be 1.2jumol/g/day or about 3 times the calculated daily contributionfrom circulating uridine and cytidine.
Table 5Salvage of infused [5-3H]cytidine
Sprague-Dawley rats (~250 g) were given infusions via the jugular vein with[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 describedin "Materials and Methods." The musice was a portion of the abdominal wall.
The mean blood levels of radiolabeled cytidine obtained in these 3 rats are shownin Chart 6.
Table 6Contribution of circulating pyrimidine nucleosides to the nucleotide pools and
RNA pyrimidines of rat tissues
The radioactivity in pyrimidine nucleotides and RNA was converted to/imol byuse of the specific activity of circulating uridine and cytidine obtained in theinfusion.
Blood flow has been measured in a number of experimentaltumors of rats by Gullino and Grantham (7), who found thatthese tumors are much less well vascularized than liver. At anaverage blood flow of 10 ml/hr/g, only 1 /¿molof pyrimidineper g could be supplied per day assuming 100% clearanceand conversion to nucleotide. We calculate that this is far lessthan the estimated 10 /imol/g/day required for those tumorsto grow with the doubling times (<2 days) observed (7).
DISCUSSION
Enzymes and transport systems for utilization of extracellularnucleosides have been demonstrated in numerous cells andtissues. However, very few studies have examined the dynamics of salvage of nucleic acid precursors in vivo (11, 15, 17).The current study examines the concentration of circulatingpyrimidine nucleosides available for salvage, the turnover ofthese nucleosides, and their conversion to nucleotides byselected tissues of the rat.
The analytical method described here, together with therigorous confirmation of the identity of the nucleosides measured, has established that low micromolar concentrations ofuridine and cytidine are available for salvage from the plasma(Table 1). The uridine concentrations reported here are similarto those reported earlier for sheep plasma (2 /tw, Ref. 8) anddog plasma (0.4 ¡J.M,Ref. 29) as determined by a multistepseparation by thin-layer chromatography. The uridine concen
tration found in human plasma is slightly higher than thatreported for human serum by Hartwick ef al. (3.2 /IM, Ref. 10).One earlier report had suggested a much higher concentrationof uridine in rat plasma (32 /IM, Ref. 25), perhaps because ofincomplete resolution from another UV-absorbing component.
In addition, the cytidine concentration was 10 /¿M,rather higherthan that found in this study. The reason for this discrepancyis not clear, although different extraction procedures wereused, and the rats examined in Ref. 25 were female. It ispossible that nutritional factors may influence the circulatinglevels, particularly since Tseng ef a/. (29) have shown that theconcentration of uridine rises severalfold in dogs fed 2 poundsof meat. Obviously, meat would be a food high in nucleic acidcompared to commercial animal chow. The influence of diet isunclear and requires further study. On the other hand, a recentstudy by Sonoda and Masamiti (27) indicates very poor utilization of 14C-labeled RNA fed to mice except by the intestinal
mucosa.The kinetics of uridine clearance after i.v. injection (Charts
3 and 5) confirms an earlier report (4), which showed anextremely rapid clearance of a single injection of this nucleo-
side but a slower clearance of cytidine in the rat. The currentinfusion experiments using an infusion of radiolabeled uridineor cytidine to steady-state levels reveal that approximately 140/iimol of pyrimidine nucleosides enter the circulation of a 300-g rat in a day. Earlier studies by Tseng era/. (29) using differenttechniques had indicated that a similar rapid flux of uridine andcytidine occurred in the circulation of the dog. The currentinfusion experiments have also permitted measurement of nu-cleoside salvage from the circulation.
In addition to providing an estimate of the daily contributionof blood-borne pyrimidine nucleosides to tissue nucleotide
pools (Table 6), the data of Tables 3 and 5 allow severalqualitative statements, (a) Greater than 70% of the uridine that
enters the plasma pool in the rat is catabolized rather thansalvaged. (£>)Although cytidine turnover is less rapid, thecirculating cytidine is more efficiently converted to tissue nucleotides. This fact, together with the differences in nucleotidepool sizes (Table 4), accounts for the observation of Hammar-
sten ef a/. (9) that cytidine was much more effective in labelingRNA in vivo than was uridine. The difference in salvage ofcytidine compared to uridine by the rat may not be seen inother species, since the rat is notably deficient in cytidinedeaminase but has somewhat higher uridine phosphorylaseactivity relative to other mammals (3, 24).
The failure of PF or PALA to substantially deplete plasmaconcentrations suggests that the source is not very sensitive tothese inhibitors of pyrimidine synthesis de novo. Recently, adetermination of circulating uridine concentrations in patientstreated with PALA was reported to be slightly higher than thatreported here, but relatively little change was observed after a5-day course of therapy (14), consistent with the inability of
PALA to decrease circulating levels in mice or rats given toxicdoses of this inhibitor of de novo synthesis.
The source of circulating nucleotides is currently not known.The diet, intestinal flora, or ' 'feeder' ' organs are all possibilities.
Possibly, constant leakage from acid-soluble nucleotide pools
in a wide range of tissues occurs at a rate influenced by theconcentration of monophosphate and the relative activities ofphosphorylating (kinase) and dephosphorylating enzymes. Recently cultured cells have been shown to excrete small amountsof uridine, cytidine, and pseudouridine into the medium, generating micromolar concentrations, particularly in the plateauphase (30). Ongoing studies in this laboratory are directedtoward understanding the factors controlling the concentrations and flux of circulating pyrimidine nucleosides, with particular focus on the essentially complete clearance of uridine inthe portal vein by the liver and its replacement by uridinederived from de novo synthesis (5).
Although only a small fraction of the pyrimidine nucleotidepools in tissues is derived from the circulation, during chemotherapy with inhibitors of pyrimidine synthesis de novo, thismay change markedly. The failure of PALA or PF to greatlydeplete circulating levels in treated animals suggests that salvage should be considered as an ongoing process duringtherapy. Further studies on salvage by tumors both sensitiveand resistant to PALA and PF are required for a more completeunderstanding of the role of nucleoside salvage. Similar studiesextending this work to the salvage of thymidine and deoxycy-tidine would be essential in experiments with the several chem-
otherapeutic drugs that inhibit the production of the corresponding deoxynucleotides.
REFERENCES
1. Blobel, G.. and Potter, V. R. Distribution of radioactivity between the acid-soluble pool and the pools of RNA in the nuclear, nonsedimentable andrlbosome fractions of rat liver after a single injection of labeled orotic acid.Biochim. Biophys. Acta, Õ66:48-57. 1968.
2. Boutagy, J., and Harvey. D. J. Determination of cytosine arabinoside inhuman plasma by gas chromatography with a nitrogen-sensitive detectorand by gas chromatography-mass spectrometry. J. Chromatogr., 146: 283-
296, 1978.3. Camiener, G. W., and Smith, C. G. Studies on the enzymatic deamination of
cytosine arabinoside. Biochem. Pharmacol., 14: 1405-1416, 1965.4. Dahnke, H., and Mosebach, K. In-vivo-Untersuchungen zur Metabolisierung
der Pyrimidin-Nucleoside. Hoppe-Seyler's Z. Physiol. Chem., 356. 1565-
5. Gasser, T., Moyer, J. D., and Handschumacher, R. E. A unique mode ofplasma regulation: the complete single pass exchange of circulating uridinein rat liver. Science (Wash. D. C.). 211: in press, 1981.
6. Gehrke, C. W., Kuo, K. C., Davis, G. E., Suits, R. D., Waalkes, T. P., andBorek, E. Quantitative high-performance liquid chromatography of nucleo-sides in biological material. J. Chromatogr., 150: 455-476, 1978.
7. Guilmu. P. M., and Grantham, F. H. Studies on the exchange of fluidsbetween host and tumor. J. Nati. Cancer Inst., 27. 1465-1484, 1961.
8. Gurpide, E., Tseng, J., Escarcena, L., Farming, M., Gibson, C., and Fehr, P.Fetomaternal production and transfer of progesterone and uridine in sheep.Am. J. Obstet. Gynecol., 113: 21-32, 1972.
9. Hammarsten, E., Reichard, P., and Saluste, E. Pyrimidine nucleosides asprecursors of pyrimidines in polynucleotides. J. Biol. Chem.. 783. 105-109,
1950.10. Hartwick, R. A., Krstulovic, A. M., and Brown, P. R. Identification and
quantitation of nucleosides, bases, and other UV-absorbing compounds inserum, using reversed-phase high-performance liquid chromatography. II.Evaluation of Human Sera. J. Chromatogr.. 786. 659-676, 1979.
11. Henderson, J. F., and LePage, G. A. Utilization of host purines by transplanted tumors. Cancer Res., 79. 67-71, 1959.
12. Henderson. J. F., and Paterson, A. R. Nucleotide Metabolism. New York:Academic Press, Inc.. 1973.
13. Hughes, W. L., Christine, M., and Sfollar, B. D. A radioimmunoassay formeasurement of serum thymidine. Anal. Biochem., 55. 468-479, 1973.
14. Karle, J. M., Anderson, L. W., Erlichman, C., and Cysyk, R. L. Serum uridinelevels in patients receiving W-(phosphonacetylM.-aspartate. Cancer Res.,40: 2938-2940, 1980.
15. Lajtha, L. G., and Vane, J. R. Dependence of bone marrow cells on the liverfor purine supply. Nature (Lond.), 782. 191-192, 1958.
16. Levine, R. L., Hoogenraad, N. J., and Kretchmer, N. A review: biologicaland clinical aspects of pyrimidine metabolism. Pediatr. Res., 8. 724-734,
1974.17. Mackinnon, A. M., and Deller, D. J. Purine nucleotide biosynthesis in
gastrointestinal mucosa. Biochim. Biophys. Acta, 3Õ9. 1-9. 1973.18. Munro, H. N., and Fleck, A. The determination of nucleic acids. In: D. Glick
(ed.), Methods of Biochemical Analysis, Vol. 14. pp. 113-176. New York:Interscience, 1966.
19. Murray, A. W., Elliott, D. C.. and Atkinson, M. R. Nucleotide biosynthesis
Circulating Uridine and Cytidine in the Rat
from preformed purines in mammalian cells: regulatory mechanisms andbiological significance. Prog. Nucleic Acid Res. Mol. Biol., 70. 87-119,1970.
20. Nottebrock, H., and Then, R. Thymidine concentrations in serum and urineof different animal species and man. Biochem. Pharmacol., 26. 2175-2179.1977.
21. Oliver, J. M.. and Paterson, A. R. P. Nucleoside transport. I.A mediatedprocess in human erythrocytes. Can. J. Biochem., 49: 262-270, 1971.
22. Parry, T. E. Serum uracil levels in acute childhood leukemia. Br. J. Haema-tol., 39. 241-248, 1978.
23. Pritchard. J. B., O'Connor, N., Oliver, J. M., and Berlin, R. D. Uptake and
supply of purine compounds by the liver. Am. J. Physiol., 229. 967-972,
1975.24. Reichard, P., and Skold, O. Enzymes of uracil metabolism in the Ehrlich
ascites tumour and mammalian liver. Biochim. Biophys. Acta, 28: 376-385,1958.
25. Rustum, Y. M. High-pressure liquid chromatography. I. Quantitative sepa
ration of purine and pyrimidine nucleosides and bases. Anal. Biochem., 90:289-299, 1978.
26. Singhal, R. P., and Conn. W E. Analytical separation of nucleosides byanion-exchange chromatography. Anal. Biochem., 45: 585-599, 1972.
27. Sonoda, T., and Masamiti, T. Metabolic fate of pyrimidines and purines indietary nucleic acids ingested by mice. Biochim. Biophys. Acta, 527. 55-66. 1978.
28. Tax, W., Peters, G., and Veerkamp. J. Pyrimidine metabolism in lymphocytesand erythrocytes of man, horse, and cattle. Int. J. Biochem., JO: 7-10,1979.
29. Tseng, J., Barelkovski, J., and Gurpide, E. Rates of formation of blood-borne uridine and cytidine in dogs. Am. J. Physiol., 227. 869-876, 1971.
30. Uziel, M., and Selkirk, J. K. Pyrimidine nucleoside, pseudouridine, andmodified nucleoside excretion by growing and resting fibroblasts. J. CellPhysiol., 99. 217-222, 1979.
31. Warnick, C. T., Muzik, M., and Paterson. A. R. P. Interference with nucleoside transport in mouse lymphoma cells proliferating in culture. Cancer Res.32. 2017-2022, 1972.
32. Ynger, T., Petersen, I., and Lewan, L. Metabolism of [5-3H]uridine in mouseand specificity for labeling of liver ribonucleic acid. Int. J. Biochem., 8. 395-401, 1977.
![Page 1: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/1.jpg)
![Page 2: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/2.jpg)
![Page 3: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/3.jpg)
![Page 4: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/4.jpg)
![Page 5: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/5.jpg)
![Page 6: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/6.jpg)
![Page 7: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/7.jpg)
![Page 8: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/8.jpg)
![Page 9: Salvage of Circulating Pyrimidine Nucleosides in the Rat1 · Stability of [5-3H]Uridine in Blood. Tracer amounts of [5-3H]uridine (3.6 X 107 cpm/ml; 1.3 pmol) were added to blood](https://reader036.documents.pub/reader036/viewer/2022071116/5ffc87423757ea188c1819e1/html5/thumbnails/9.jpg)
