[CANCER RESEARCH 35. 397-404, February 1975]

SUMMARY

Ornithine levels rise progressively in the liver of partiallyhepatectomized rats, probably as a consequence of theincreased flow of metabolites through the urea cycle.Ammonia and urea concentrations in the blood and liver ofpartially hepatectomized animals are not significantly different from those of sham-operated rats. However, inregenerating livers, the ability to remove ammonia from theblood is close to its maximal limit. Ammonia overload leadsto the production of large amounts of orotic acid and causesa marked elevation of hepatic ornithine decarboxylaseactivity. Among the pyrimidine precursors dihydrooroticacid injections increase the activity of the enzyme whileorotic acid is without effect. A peak of labeled material thatcorresponds to dihydroorotic acid was identified by partition chromatography of acid-soluble extracts of livers ofpartially hepatectomized rats previously given injections of[14C]bicarbonate. The labeling of dihydroorotic acid from[‘4C]bicarbonate is increased in the liver of rats giveninjections of ornithine. Despite the difficulties involved instudies of ornithine decarboxylase activity in vivo, ourresults suggest that mutual interactions between urea,pyrimidine, and polyamine synthesis take place during liverregeneration.

INTRODUCTION

One of the earliest responses to partial hepatectomy inrats is the elevation of ornithine decarboxylase activity inthe liver remnant (10, 16, 29, 31). This enzyme, whichcatalyzes the synthesis of putrescine (l,4-diaminobutane)from ornithine, is the 1st and probably the rate-limiting stepin polyamine biosynthesis (33, 40). The elevation of putrescine synthesis during liver regeneration coincides in timewith changes in RNA synthesis (10).

The stimuli that are responsible for the elevation of liverornithine decarboxylase activity following partial hepatectomy have not yet been identified. While cycloheximide orpuromycin injections given at any time during the 1st dayfollowing the operation block the activity of the enzyme,actinomycin D inhibits ornithine decarboxylase activity

1 Supported by Grant AM 14706 from the NIH.

Received September 3, 1974: accepted October 25, 1974.

only when injected during the 1st hr after partial hepatectomy (1 1, 30). We suggested that, in addition to increasedsynthesisof the enzyme, posttranscriptional control of theenzyme activity exists in regenerating liver (4, 11).

Although it is possible that amino acids, growth hormone(11), cyclic 3':S'-AMP (I), or a combination ofthese factorsmay be the initial stimulus for the increase in ornithinedecarboxylase activity after partial hepatectomy, it seemsessential to consider the relationships between polyaminesynthesis and other metabolic processes (13). The loss ofliver mass caused by the operation imposes an increasedphysiological demand on the liver remnant that is likely toalter the functioning of key biochemical pathways.

Alterations of the urea cycle that may occur after partialhepatectomy could be reflected in changes in the rate ofpolyamine biosynthesis because ornithine is a key metabolite in the urea cycle and the 1st precursor of the polyaminepathway. In addition the injection of ammonium chloride oracetate into rats is associated with the excretion of largeamounts of orotic acid (20, 32). These observations led us toexplore the hypothesis that possible physiological adaptations in ammonia or amino acid metabolism followingpartial hepatectomy might interact with the processes ofpolyamine and pyrimidine biosynthesis. Our results suggestthat the urea cycle adapting to the metabolic load imposedon the liver remnant (36) might provide precursors for bothpolyamine and pyrimidine synthesisduring liver regeneration.

MATERIALS AND METHODS

Animals. The animals used in these experiments weremale albino rats (The Holtzman Co., Madison, Wis.)weighing 130 to 170 g. They were maintained in a temperature-controlled room with 12-hr dark-light cycles. Allanimals were killed between 9 and I 1 a.m. Initially, all ratsused were starved for 14 hr before the experiments. Wefound, however, that more consistent results could beobtained with rats fed ad libitum, provided that the ratswere maintained in a room with controlled illumination.Unless otherwise indicated, food was not withdrawn untilthe start of the experimental procedures. Hypophysectomized rats were purchased from Charles River BreedingLaboratory, Wilmington, Mass. Rats from the same sup

FEBRUARY 1975 397

Possible Interactions between the Urea Cycle and Synthesis ofPyrimidines and Polyamines in Regenerating Liver'

Nelson Fausto, John T. Brandt, and Leo Kesner

Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912 [N.F., J.T.B.]; and Department of Biochemistry.Downstate Medical Center, State University of New York, Brooklyn, New York 11203 [L.K.]

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N. Fausto et a!.

potassium oxalate. The tube was kept in ice and thedetermination ofammonia was started not later than 10 mmafter blood withdrawal. The precautions indicated by Ternberg and Hershey (35) for handling of the blood werefollowed carefully. A sample of the blood (0. 1 to 0.2 ml) waspipetted into a flask containing 1.0 ml of saturated sodiumcarbonate. The flask was closed with a rubber stopperperforated by a glass rod. The tip of the rod was dipped intoI N sulfuric acid. The glass flasks were placed in a rotatorapparatus (Scientific Industries Inc., Springfield, Mass.)for 30 mm. After the completion of the microdiffusion, theammonia was determined by the Berthelot procedure usingnitroprusside as a catalyst. The combination of reagentsdescribed by Chaney and Marbach (8) was used for thecolor development. The absorbance of the samples wasmeasured at 625 nm and compared to that of ammoniumsulfate standards processed in a similar manner. A linearrelationship between ammonia content in blood and A wasobtained for samples containing 1 to 8 zg of ammonia. Therecoveries of the microdiffusion step was 85 to 90%. Bloodurea was determined either by urease digestion followed bythe colorimetric procedure described by Chaney and Marbach (8) or with an amino acid analyzer.

Determination of Orotic Acid and Dihydroorotic Acid.Sham-operated or partially hepatectomized rats were giveninjections of 75 @iCi of [‘4C]sodium bicarbonate. Theanimals were killed 60 mm after the injection of theprecursor. The livers were removed and homogenized in0.25 M sucrose. A sample of the homogenate was precipitated with 5% perchloric acid and centrifuged. The supernatant was saved and mixed with 2 more perchloric acidextractions of the pellet. The combined supernatants wereneutralized with potassium hydroxide. After centrifugationthe volumes ofthe supernatants were measured and samplesof the extracts were stored at —20°until used. Orotate anddihydroorotate analyses were performed by the partitionchromatography procedure of Kesner and Muntwyler (20,21). Dihydroorotic acid appears as a peak on the organicacid analyzer in the area of malic acid (21, 22). Theseparation of these 2 substances is often imperfect, makingquantitation difficult. It is possible to cause a samplecontaining a mixture of these 2 substances to react withceric sulfate and sulfuric acid. We have found that variations in concentration of sulfuric acid and ceric sulfate willalter the nature of the oxidation products of malic acid.However, the complete removal of malic acid from thereaction mixture is easily accomplished by the followingprocedure: the fractions containing dihydroorotate obtamed by partition chromatography are dried in a smallbeaker by a stream of air. To the dried residue 300 @tl0. 1 Msulfuric acid and 10 mg solid ceric sulfate are added. Themixture is agitated at room temperature for 30 mm, afterwhich the mixture is filtered or centrifuged. A 200-@.tlaliquotis used for analysis on the organic acid analyzer.

Amino Acid Analysis. The free amino acids in tissuewererun on a Technicon acid analyzer. Frozen tissues werehomogenized in a microblender using 4 ml of 3% sulfosalicylic acid for each 0.5 g of liver. A 0.4-ml aliquot of theextract was analyzed using norleucine as an internal standard.

plier were usedascontrols in experiments involving hypophysectomized rats. Partial hepatectomies were performed bythe method of Higgins and Anderson (15). Animals wereanesthetized with a mixture of ether and oxygen as described by Bucher and Swaffield (6). The operation resultedin the removal of approximately 70% of the liver.Adrenalectomies were performed 7 to 10 days before theexperiments. Adrenalectomized rats were given 1% NaC1solution instead of water.

Materials. DL-[ I -‘4C]Ornithine-HCI (specific activity,12.8 mCi/mmole) and [‘4C]sodium bicarbonate (specificactivity, 50 mCi/mmole) were purchased from New England Nuclear, Boston, Mass. Urease (jack bean meal,URPC, 240 units/mg, lyophilized) was obtained fromWorthington Biochemical Corp., Freehold, N. J. A unit ofactivity corresponds to the liberation of I @imoleof ammonia per mm at 25°. All other chemicals used wereobtained either from Schwarz/Mann, Orangeburg, N. Y.,or from Sigma Chemical Co., St. Louis, Mo.

Ornithine Decarboxylase Activity. Rats were killed bydecapitation. The livers were rapidly removed, weighed, cutinto small pieces, and homogenized in a Potter-Elvehjemhomogenizer in 0.25 M sucrose, 10 m@iTris buffer (pH 7.5),0.5 m@i EDTA, and I m@i mercaptoethanol. The homogenates (20%, w/v) were centrifuged for 60 mm at I 15,000 x gin an International Centrifuge (Model B-60). The cytosolwas collected and used immediately for the enzyme assay.The incubation mixture contained, in a final volume of 1.0ml: 0.2 ml of 0.05 M Tris buffer (pH 7.8), 0.2 zmole of pyridoxal phosphate, and 0.2 to 0.4 ml of the cytosol. After 10mm ofpreincubation at 37°,0.4 ml (0.8,uCi)of[l-'4C]ornithine was added and the flasks were closed with rubber stoppers fitted with a polyethylene center well and a capillarytube. The center well contained 0.25 ml of a CO2-trappingsolution of ethanolamine in ethylene glycol (1:2, v/v). Thesamples were incubated for 30 mm at 37°and the reactionwas stopped by adding 0.5 ml of 5% trichloroacetic acidthrough the capillary tube (4, 10). After 30 mm at roomtemperature, 0. 1 ml of the CO2-trapping solution waspipetted into a vial containing 10 ml of scintillation fluid.The radioactivity was determined in a Nuclear Chicagocounter and all determinations were corrected for countingefficiency. Under the conditions used for determiningornithine decarboxylase activity, addition of dithiothreitolto the incubation mixture slightly inhibited the activity ofthe enzyme. In the presence of high concentrations ofornithine (2 mM) in the assay, 5 m@i dithiothreitol wasnecessaryfor obtaining maximal enzyme activity (17). Theresults obtained with assays using high substrate concentrations and dithiothreitol agreed with those presented here.

Protein was determined by the method of Lowry et a!. (24).Samples containing no cytosol or boiled supernatants wereused as blanks. Enzyme activity is expressed in pmoles of“CO2produced in 30 mm per mg of liver cytosol protein. Ineach assay enzyme activity was determined using 2 differentamounts of cytosol, generally 0.2 and 0.4 ml. A continuouscheck on the linearity of the reaction was thus assured.

Ammonia and Urea Assays. Whole blood used fordetermination of ammonia was collected from the jugularvein and immediately placed in a test tube containing dry

398 CANCER RESEARCH VOL. 35

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TreatmentTime

afteroperation

(hr)Bloodammonia N

(nmoles/ml)Urea

inplasma

(@moles/ml)Ureain liver

(zmoles/g)Sham

operation0I246l29(l00-l59)°

112(94-135)123(108—141)l29(45-l94)1.1

4.32.13.1Partialhepa

tectomy0 0.5I246129

(76-14l)

123(100-159)159(106-200)129(88-159)2.3

3.94.82.6

1.62.0

Interactions between A mmonia, Pyrimidines, and Polyamines

sions showed no alterations in the 1st 6 hr following partialhepatectomy. Other investigators have reported no significant changes in ornithine carbamoyltransferase activity in24-hr regenerating livers (2, 38). Since urea production perliver 24 hr after partial hepatectomy is 60 to 70% higherthan production in sham-operated rats (37), we conclude

Chart I . Blood ammonia after ammonium acetate administration.Sham-operated and partially hepatectomized animals received I mmole ofammonium acetate by stomach tube. Blood was withdrawn from thejugular vein at the times indicated. Free ammonia nitrogen in the blood wasdetermined as described in Table I . A blood sample was taken during theoperations, immediately preceding the administration of ammoniumacetate.

Table I

RESULTS

Ammonia, Urea, and Ornithine Levels after PartialHepatectomy. The loss of liver cell mass caused by partialhepatectomy may produce changes in the levels of substrates involved in the urea cycle. We measured initially theplasma concentrations of ammonia and urea as well as liverurea levels in sham-operated and partially hepatectomizedrats. As presented in Table I, the blood ammonia nitrogenof partially hepatectomized rats does not appear to differsignificantly from that of sham-operated rats in the 1st6 hrfollowing the operations. Urea values in the plasma ofpartially hepatectomized rats increased slightly after theoperation, but a similar increase was found in the shamoperated controls. The concentrations of urea in the liverafter partial hepatectomy are comparable to the concentrations in the sham-operated rats.

Although partially hepatectomized rats remove ammoniafrom the blood efficiently, they are unable to dispose of anyexcess of amino acids or ammonia. When a complete aminoacid mixture or ammonium acetate is given to shamoperated rats, only minimal and transient increases occur infree ammonia in the blood. In contrast, in partiallyhepatectomized animals ammonia levels increase by 6-foldor more after ammonium acetate injection (Chart I).

Table 2 shows the levels of ornithine in the liver ofsham-operated and partially hepatectomized rats. In the 1st2 hr after partial hepatectomy, there is a rapid increase inornithine. The amount of ornithine doubles 2 hr after theoperation and at 18 hr is 4 to 5 times higher than that ofsham-operated rats. The progressive accumulation of ornithine (14, 25) could have been caused by a decrease inornithine carbamoyltransferase activity, as has been demonstrated in hepatomas (38, 39). Measurements ofthe activityof this enzyme in homogenates and mitochondrial suspen

20 40 60time after ammonium acetate administration (mm

A mmonia and urea concentrations after partial hepatectomy

Whole blood was collected from thejugular vein, immediately placed in a test tube containing drypotassium oxalate, and kept on ice. Ammonia determinations were started no later than 10 mm afterblood withdrawal. Microdiffusion of ammonia was carried out for 30 mm at room temperatureusing a rotator apparatus. Recoveries at this step were 85 to 90%. After completion of the diffusion,ammonia was determined by the Berthelot procedure with nitroprusside as a catalyst and thecombination of reagents described by Chancy and Marbach (8). Urea was determined either byurease digestion followed by colorimetric determination or with an amino acid analyzer. The valuesfor each point were obtained from 4 to 7 rats.

a Numbers in parentheses, range of variation.

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OperationTimeafter

operation (hr)Liver

ornithineconcentration

(@moles/gliver)Sham0

40.680.75Partial

hepatectomy0.5I24180.43

1.61.31.93.0

Hr after injectionBloodammonia

N (nmoles/ml)Ornithine

decarboxylaseactivity (pmolesofCO2/mg of

protein)0.9%

NaCl solution2

46100

931291082

34

4UreaseI

234S6812100

237

258

2003

872747291833

N. Fausto et a!.

for the synthesis of carbamoylphosphate from CO2 andammonia (18, 34). We gave animals injections of variousdosages of N-acetyl-L-glutamate or N-carbamoyl-L-glutamate. Carbamoylglutamate can perform the same functionand is more stable than the natural compound (23).Injections of 50 to 350 zmoles of acetyl- or carbamoylglutamate into adrenalectomized rats led to 20- to 60-foldelevations in liver ornithine decarboxylase activity (Table4). As shown in Table 4 the increase in the activity of theenzyme occurring 5 hr after the injection of N-acetyl-Lglutamate is inhibited by actinomycin injections. Inhibitionof ornithine decarboxylase activity was obtained withactinomycin given either immediately after or 1hr followingthe administration of N-acetyl-L-glutamate.

Orotate Formation and the Effect of Pyrimidines onOrnithine Decarboxylase Activity. Changes in pyrimidinemetabolism, which include a 50% expansion of the endoge

Table 3

Blood ammonia concentrations and liver ornithine decarboxylase activityf ollowingthe injectionof urease

Rats were adrenalectomized 7 days before the experiments and given 1%NaCI solution. The rats were given injections of I unit of urease or 0.9%NaCl solution ( I ml volumes) and killed at the times indicated on the table.Blood for ammonia determinations werecollected from the jugular vein.Ornithine decarboxylase activity was measured in liver cytosol obtained bycentrifugation of liver homogenate for 1 hr at 105,000 x g. For eachdetermination 0.2 and 0.4 ml of cytosol were used (see “Materials andMethods―). The enzyme activity is expressed in pmoles of 14C02 formed in30 mm per mg of cytosol protein. Six rats were used for each determination.

Table 2

Ornithine concentrations in normal and regenerating livers

Sham-operated and partially hepatectomized animals were killedimmediately after or at various times following the operations. A fragmentof the liver was frozen in situ using a clamp previously cooled in liquidnitrogen. The frozen samples were deproteinized with sulfosalicylic acidand the analyses were performed using a Technicon amino acid analyzer.

that the ornithine levels in the liver rise as a consequence ofthe increased flow of metabolites through the urea cycle.The ammonia load per cell in regenerating livers obviouslyis elevated, but the cells of the liver remnant are able toadapt to the metabolic demands.

Urea Cycle Substrates and Ornithine DecarboxylaseActivity. In addition to participation in the urea cycle,ornithine is the substrate for ornithine decarboxylase, the1st enzyme of the polyamine pathway. The increased levelsof ornithine present in the livers of partially hepatectomizedrats might induce or stabilize ornithine decarboxylaseactivity. However, ornithine injections did not increaseornithine decarboxylase activity. Adrenalectomized ratswere given injections of 150 @.imolesof ornithine and werekilled 5 hr later. Ornithine decarboxylase activity wasmeasured in both dialyzed and undialyzed liver cytosol toavoid dilution of the labeled ornithine used as substrate inthe enzyme assay. The enzyme activities of animals giveninjections of 0.9% NaCI solution were 0.5 to 5 pmoles of‘4CO2formed per 30 mm per mg of protein. The ratsreceiving ornithine had activities in the range of 2 to 8pmoles of ‘4CO2.Injections of 100 to 150 @moles ofcitrulline, arginine, argininosuccinate, urea, or glucose intoadrenalectomized rats did not alter hepatic ornithine decarboxylase activity.

Injections of urease into rats stimulate the urea cycle andelevate blood ammonia levels and liver amino acid concentrations (9, 27). A single injection of 1 unit of ureaseproduced a sustained elevation of blood ammonia and a20-fold stimulation of liver ornithine decarboxylase activity(Table 3). The enzyme activity is maximal between 4 and 5hr following urease injection and returns to normal at 8 hr.Neither glucocorticoids nor growth hormone is involved inthis response because the same elevations in enzyme activitywere found in adrenalectomized and hypophysectomizedrats.

Since ornithine decarboxylase activity could be markedlyincreased only by the urea cycle stimulation caused byurease but not by ornithine or the other intermediates, westudied the effects of N-acetyl-L-glutamate on the enzymeactivity. This compound (Chart 2) is the cofactor required

Co2 + GLUTAMINE Co2 ÷ NH3@ I@ETYLGLUTAMATE

CARBAMOVLPHOSPHATE CARBAM@LPH0SPHAT[

ASPARTATEI —ORNITHINE-——-—CITRULLINECARBAMYL I

ASR@,RTATE ______ ARGI UREA@ARGININEa--t@HYDR0OROTATE PUTRESCINE ——‘ SPERMIDINE --- -----‘SPERMINE

0ROTATE—@OROTIDYLATE —*UMP--------.UTP----------.RNA

Chart 2. Urea cycle, pyrimidine. and polyamine pathways.

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MetaboliteAmount injectedOrotateexcreted

in 24 hr(zmoles)0.9%

NaCI solution3mlNoneAmmoniumchloridel000Mmoles7.5Glutamine1000@moles3.0DL-Carbamoylaspartate50

.tmoles12.0

Interactions between A mmonia, Pyrimidines, and Polyamines

Table 4

Effect of urease and cofactors of the urea cycle on hepatic ornithine decarboxylase activity

Rats were adrenalectomized 7 days before the experiments and given 1% sodium chloride.Animals were given injections of various compounds shown on the table and killed S hr later.Ornithine decarboxylase activity is expressed as pmoles of 14CO2 formed in 30 mm per mg of cytosolprotein (see legend of Table 4). Rats received actinomycin at a dosage of 100 @g/l00 g of bodyweight either immediately after or 1 hr after acetylglutamate injections. Urease was inactivated byboiling for I mm. The hypophysectomized rats were not adrenalectomized. Six rats were used foreach determination.

Ornithine decarboxylase activityAmount injected

0.9% NaCI solutionGlucoseUreaseUrease(inactivated)Urease0.9% NaCI solution (hypophysectomized)Urease (hypophysectomized)N-Carbamoyl-L-glutamateN-Carbamoyl-i-glutamateN-Carbamoyl-L-glutamateN-Carbamoyl-i-glutamateN-Acetyl-L-glutamateN-Acetyl-L-glutamate (actinomycin attime0)

!@-Acetyl-i-glutamate(+I hr after)

a Numbers in parentheses, range of activity.

I ml200mg

0.5 unit0.5unitI unitI mlI unit

50 @imoles100 @moIes200 @moles350zmoles100 @moles100 @imoles

3(1-7)―5(2-6)

17 (13-24)2(0-2)

47 (40-48)3(0-6)

43 (38-39)85(46-122)

137 (73-181)160(123-198)209(140-268)120(III-I36)82 (69-100)

100 @moles 40 (2 1-67)

nous UTP and CTP pools, occur in rat livers followingpartial hepatectomy (5-7). As shown in Table 5, injectionsof ammonium chloride, glutamine, or carbamoylaspartateinto fasted intact rats lead to the excretion of large amountsof orotic acid. Thus, ammonia overload appears to reproduce 3 of the early events of liver regeneration: (a) elevationof ornithine concentration in the liver (27); (b) stimulationof pyrimidine biosynthesis (20, 32); and (c) the “induction―of ornithine decarboxylase activity. The next logical step

was to examine the effects of various pyrimidine precursorsand derivatives on the activity of this enzyme (Table 6).Orotic acid, methylorotic acid, and uracil did not alter theactivity of the enzyme. However, further surveysof pyrimidines showed that carbamoylaspartate injections to someextent and dihydroorotic acid injections in particular markedly increase liver ornithine decarboxylase activity. A linearrelationship exists between the dosageofdihydroorotic acidand ornithine decarboxylase activity (Chart 3). The effect ofdihydroorotic acid is not altered by adrenalectomy orhypophysectomy, and the D form is 50 to 70% less activethan the L compound (Table 6).

Labeling of Dihydroorotate in Regenerating Livers. Theeffect ofdihydroorotic acid on putrescine synthesis suggeststhat in rat liver a relationship between ornithine decarboxylase activity and pyrimidine synthesis may exist. This couldexplain some of the events occurring at the early stages ofliver regeneration, provided that dihydroorotic acid synthesis increases after partial hepatectomy. We investigated thelabeling ofdihydroorotic acid in the 1st hr following partialhepatectomy. Partially hepatectomized and sham-operatedrats were given injections of [‘4Cjbicarbonate.The acidsoluble extracts obtained from the liver of these animals

Table 5Conversion of metabolites to orotic acid

Rats fasted for 18 hr were given injections of the metabolites indicatedabove. Urine was collected and pooled. Orotic acid content was determinedby silica gel column chromatography (20, 21).

were analyzed by partition chromatography in silicic acidcolumns, and the peak corresponding to dihydroorotic acidwas further treated by cerium sulfate to eliminate contamination by malic acid (see “Materialsand Methods―). At 0.5hr to 1 hr after partial hepatectomy we detected a distinctpeak of radioactive material corresponding to dihydrooroticacid (Chart 4) that was approximately 5 times higher thanthe peak found in the sham-operated controls. Furtheranalyses of dihydroorotic acid metabolism both in regenerating livers and in Ll210 tumor cells (L. Kesner, unpublished observation) revealed an effect of ornithine ondihydroorotic acid labeling (Chart 5). Rats were given injections of 75 @Ciof [ ‘4C]bicarbonate1hr prior to killing. Agroup of rats received 3 mmoles of ornithine 0.5 hr after theinjection of the label. Partition chromatography of theacid-soluble extracts indicated that the labeling of dihydroorotate is almost 10 times higher in the animals giveninjections -of ornithine, with apparently little change inorotic acid synthesis.

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OrnithinedecarboxylaseAmount

injectedactivity0.9%

NaCl solutionI ml3 ( I-7)―Aspartatel50@zmoles32(10-72)CarbamoylaspartateI

50 @imoles32 (2 1-89)L-Dihydrooroticacid75 @moles65(50-73)D-DihydroOroticacid75 Mmoles20 ( I2-27)i-Dihydrooroticacid75 @moles57(20-108)(hypophysectomized)D-DihydrOorotic

acid75 @moles22(10-36)(hypophysectomized)Orotic

acidl50MmolesS(3-6)Methylorotic acid1 50 @moles2 ( I -5)

N. Fausto et a!.

metabolism of the urea cycle and has also been demonstrated experimentally in rat liver slices (3). Alternatively,regenerating livers could clear ammonia from the bloodthrough the synthesis of glutamate, which might stimulatethe 1st step in pyrimidine biosynthesis (18, 34). Some of thepyrimidine precursors formed by either or both of theseprocesses may participate in the “induction―of ornithinedecarboxylase activity during liver regeneration.

The labeling of dihydroorotic acid from bicarbonate isapproximately 5 times higher in regenerating livers than insham-operated controls. In addition, a distinct peak corresponding to dihydroorotic acid was detected in acid-solubleextracts of the liver of rats given injections of ornithine. Wedo not know at this time the nature or the significance ofthis effect of ornithine on dihydroorotic acid metabolism.Dihydroorotate oxidation to orotic acid is the only step ofthe pyrimidine pathway that takes place in mitochondria(19). Ornithine carbamoyltransferase (28) and most of thesteps of urea biosynthesis also take place in this organelle.Shifts in the intracellular distribution ofdihydroorotate andornithine would have to be examined for their possibleeffects on urea, orotic acid, and polyamine synthesis. Thedetection of a distinct peak of dihydroorotic acid shortlyafter partial hepatectomy is also of interest because inEhrlich ascites cells most enzymes involved in de novosynthesis of pyrimidines appear to form a complex, and nointermediates between bicarbonate and UMP accumulateunder normal circumstances (18).

Our results indicate that ornithine levels in the liver riserapidly after partial hepatectomy. These results are inagreement with the findings of Ferris and Clark (14) andOrd and Stocken (25), who reported changes in amino acidpools in liver and blood following partial hepatectomy.a Numbers in parentheses. range of activity.

Chart 3. Effects of dihydroorotic acidon ornith me decarboxylase activity.Adrenalectomized rats were given i.p. injections of the doses of dihydroorotic acidshown on the abscissa and killed 45 hr later.Ornithine decarboxylase (ODC) activity wasmeasured in the cytosol after centrifugationof the liver homogenates for I hr at 105.000x g. Enzyme activity is expressed in pmolesof “'CO2released in 30 mm per mg ofcytosol protein. Each point is the averagevalue from 3 animals.

>

@mc@esof dihydrooro@cacid injected

402 CANCER RESEARCH VOL. 35

DISCUSSION

The flow of metabolites through the urea cycle isaugmented after partial hepatectomy, and the ability toremove ammonia from the blood is close to its maximallimit. Under these conditions carbamoylphosphate notutilized in citrulline synthesis would be channeled to thepyrimidine pathway leading to the synthesis of orotic acid(20, 32). Cross-over of carbamoylphosphate from the ureato the pyrimidine pathway occurs in inborn errors of

Table 6

Effect of various pyrimidine precursors and derivatives on hepaticornithine decarboxylase activity

Rats were adrenalectomized 7 days before the experiments andmaintained on 1% NaCI solution. Ornithine decarboxylase activity isexpressed as pmoles of “'CO2formed in 30 mm per mg of cytosol protein(see legend of Table 4). Hypophysectomized rats were not adrenalectomized. Six rats were used for each determination.

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10

Interactions between A mmonia, Pyrimidines, and Polyamines

15 20 25

.@

0frac@,onnumber fract@,nnumber

Chart 5. Effect of ornithine on dihydroorotic acid labeling in regenerating livers. Partially hepatectomized rats were given injections of either0.9% NaCl solution or 3 mmoles ofornithine 18 hr after the operation. Allanimals received 50 @Ciof [‘4C]sodium bicarbonate 30 mm after 0.9%NaCI solution or ornithine injection and were killed 30 mm after theinjection of the labeled precursor. The livers were frozen in situ and theacid-soluble extracts were analyzed by partition chromatography in silicicacid columns. Ordinate, radioactivity present in each fraction. —, ratsgiven injections of ornithine; ----, rats given injections of 0.9% NaCIsolution. The location of the appropriate markers is shown.

considerable range of variation in enzyme activity existsboth under basal conditions and after stimulation. None ofthe compounds that stimulated ornithine decarboxylaseactivity in vivo appears to have an effect when tested incrude cell extracts in vitro. Within these limitations, ourresults point to some interrelationships between polyamineand pyrimidine synthesis and the functioning of the ureacycle after partial hepatectomy.

It is not yet known whether the changes in RNAmetabolism and polyamine synthesis occurring in regeneration are triggered by a common stimulus or by independentmechanisms. Whatever the case may be, polyamine andRNA metabolism in regenerating rat liver might be linkedat various levels: (a) by the effect of pyrimidines onornithine decarboxylase activity, the 1st and rate-limitingstep of the polyamine pathway; (b) by an effect of ornithineon dihydroorotate metabolism; and (c) by a possible effectof polyamines on RNA transcription (12).

REFERENCES

I. Beck, W. T., Bellantone, R. A., and Canellakis, E. S. The In VivoStimulation of Rat Liver Ornithine Decarboxylase Activity by Dibutyryl Cyclic Adenosine 3'-5'-Monophosphate, Theophylline and Dexamethasone. Biochem. Biophys. Res. Commun.. 48: 1649-1655, 1973.

2. Bhide, S. V. Comparative Study of Metabolic Profiles of PrimaryHepatoma. Regenerating Liver, and Liver in Newborn Swiss Mice. J.NatI. Cancer Inst., 47: 797-800, 1971.

3. Bourget, P. A., Natale, P. J., and Tremblay. G. C. Pyrimidine

Chart 4. Dihydroorotic acid labeling in regenerating livers. PartialLyhepatectomized and sham-operated rats were given injections of 75 zCi of[‘4C]sodiumbicarbonate. Thirts' mm after the injection the liver was frozenin situ using a clamp previously chilled in liquid nitrogen. The liver sampleswere deproteinized with perchloric acid and neutralized with potassiumhydroxide. The acid-soluble extracts were analyzed by partition chromatography in silicic acid columns as described by Kesner and Muntwyler(20, 21). Unlabeled compounds were used as markers. The fractionscorresponding to the dihydroorotate peak were collected and treated withcerium sulfate to eliminate contamination by malic acid. After this

treatment the samples were rechromatographed and the fractions corresponding to dihydroorotic acid were collected and counted in a liquid

scintillation counter.

However, it does not appear that the elevated levels ofornithine in regenerating livers cause the increase in ornithine decarboxylase activity after partial hepatectomy.Ornithine, citrulline, argininosuccinate, arginine, and ureainjections do not alter hepatic ornithine decarboxylaseactivity. This activity is markedly elevated after urease,acetylglutamate, or carbamoylglutamate injections, all ofwhich may lead to increases in ammonia, glutamine, orcarbamoylphosphate.

Orotic acid, methylorotic acid, uracil, and dihydrouracilinjections have no effect on liver ornithine decarboxylaseactivity. In contrast, dihydroorotic acid in dosages above 20jzmoles causes a very large increase in ornithine decarboxylase activity, which is proportional to the amount of thecompound injected. The same effect of L-dihydroorotic acidon liver ornithine decarboxylase activity was obtained inintact, adrenalectomized, or hypophysectomizedanimals.

In an attempt to circumvent the difficulties involved within vivo studies of ornithine decarboxylase activity (3 1), mostof the metabolites used were tested for their effect on theactivity of the enzyme in adrenalectomized and hypophysectomized rats. Although it has been possible to exclude theparticipation of adrenal and growth hormones, it is extremely difficult to ascertain if other hormones (glucagon,insulin) are released under these conditions (26). In ourexperiments there was an obvious effect of various compounds on liver ornithine decarboxylase activity, but a

FEBRUARY 1975 403

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N. Fausto et al.

Biosynthesis in Rat Liver: Studies on the Source of Carbamoylphosphate. Biochem. Biophys. Res. Commun., 45: 1109-1114, 1971.

4. Brandt, J. T., Pierce, D. A., and Fausto, N. Ornithine DecarboxylaseActivity and Polyamine Synthesis During Kidney Hypertrophy.Biochim. Biophys. Acta, 279: 184-193, 1972.

5. Bresnick, E. Early Changes in Pyrimidine Biosynthesis after Partial

Hepatectomy. J. Biol. Chem., 240: 2550-2556, 1965.6. Bucher, N. L. R., and Swaffield, M. N. Nucleotide Pools and (6-―C)

Orotic Acid Incorporation in Early Regenerating Liver. Biochim.Biophys. Acta, 129: 445-459, 1966.

7. Bucher, N. L. R., and Swaffield, M. N. Ribonucleic Acid Synthesis inRelation to Precursor Pools in Regenerating Rat Liver. Biochim.Biophys. Acta, 174: 491 -502, 1969.

8. Chancy, A. L., and Marbach, E. P. Modified Reagents for Determination of Urea and Ammonia. Clin. Chem., 8: 130-132, 1962.

9. Clifford, A. J., Prior, R. L., and Visek, W. J. Depletion of ReducedPyridine Nucleotides in Liver and Blood with Ammonia. Am. J.Physiol., 217: 1269-1272, 1969.

10. Fausto, N. Studies on Ornithine Decarboxylase Activity in Normal

and Regenerating Livers. Biochim. Biophys. Acta, 190: 193-201, 1969.I I. Fausto, N. The Control of Ornithine Decarboxylase Activity During

Liver Regeneration. Biochim. Biophys. Acta, 238: I 16-128, 1971.12. Fausto, N. RNA Metabolism in Isolated Perfused Normal and

Regenerating Livers: Polyamine Effects. Biochim. Biophys. Acta, 281:543-553, 1972.

13. Fausto, N., Brandt, J. T., and Kesner, L. Interrelationships Betweenthe Urea Cycle. Pyrimidine and Polyamine Synthesis during LiverRegeneration.In: R. Lesch (ed), Liver Regeneration after Experimental Injury. Intercontinental Book Publishers. in press.

14. Ferris, G. M., and Clark, J. B. Early Changes in Plasma and HepaticFree Amino Acids in Partially Hepatectomized Rats. Biochim.Biophys. Acta, 273: 73-79, 1972.

15. Higgins. G. M., and Anderson, R. M. Experimental Pathology ofLiver. I . Restoration of the Liver of White Rats following PartialSurgical Removal. Arch. Pathol., 12: 186-202, l931.

16. Höltta,E.. and Jänne,J. Ornithine DecarboxylaseActivity and theAccumulation of Putrescine at Early Stages of Liver Regeneration.Federation European Biochem. Soc. Letters, 23: 117- 121, 1972.

17. Jänne, J., and Williams-Ashman, H. G. On the Purification ofi-Ornithine Decarboxylase from Rat Prostate and Effects of ThiolCompounds on the Enzyme.J. Biol.Chem., 246: 1725-1732,1971.

18. Jones, M. E. Regulationof Uridylic Acid Biosynthesisin EukaryoticCells. Current Topics Cellular Regulation, 6: 227-265, 1972.

19. Kennedy,J. Distribution,SubcellularLocalizationand Product Inhibition of Dihydroorotate Oxidation in the Rat. Arch. Biochem. Biophys.. 157: 369-373, 1973.

20. Kesner, L. The Effect of Ammonia Administration on Orotic AcidExcretion in Rats. J. Biol. Chem., 240: l722-l724, 1965.

21. Kesner, L., and Muntwyler, E. Separation of Citric Acid Cycle andRelated Compoundsby Partition ColumnChromatography.MethodsEnzymol../3:415-425,1968.

22. Kesner, L., Yao, T. T., and Dell, R. B. Determination of TotalOrganic Acids in Urine by Extraction with Organic Solvents. Clin.Chem., 19. 593-596. 1973.

23. Kim, S., Paik, W. K., and Cohen. P. P. Ammonia Intoxication inRats: Protection by N-Carbamoyl-i-Glutamate plus Arginine. Proc.NatI. Acad. Sci. U.S.. 69: 3530-3533, 1972.

24. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall, R. J.Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.,193:265-275,1951.

25. Ord, M., and Stocken, L. A. Uptake ofAmino Acids and Nucleic AcidPrecursors by Regenerating Rat Liver. Biochem. J.. 129: 175 181,1972.

26. Panko, W. B., and Kenney, F. T. Hormonal Stimulation of HepaticOrnithine Decarboxylase. Biochem. Biophys. Res. Commun., 43:346-350,1971.

27. Prior, R. L., Clifford, A. J.. and Visek. W. J. Tissue Amino AcidConcentrations in Rats during Acute Ammonia Intoxication. Am. J.Physiol.. 219: 1680 1683, 1970.

28. Raijman. L. Citrulline Synthesis in Rat Tissues and Liver Content ofCarbamoyl Phosphate and Ornithine. Biochem. J.. 138: 225-232.1974.

29. Russell, D. H., and Snyder, S. H. Amine Synthesis in RapidlyGrowing Tissues: Ornithine Decarboxylase Activity in RegeneratingRat Liver, Chick Embryo and Various Tumors. Proc. NatI. Acad. Sci.U.S.,60: 1420-1427,1968.

30. Russell, D. H., and Snyder. S. H. Amine Synthesis in RegeneratingRat Liver: Extremely Rapid Turnover of Ornithine Decarboxylase.Mol. Pharmacol., 5. 253, 262, 1969.

31. Schrock, T. R., Oakman, N. J., and Bucher, N. L. R. OrnithineDecarboxylase Activity in Relation to Growth of Rat Liver. Effects of

Partial Hepatectomy, Hypertonic Infusions, Celite Injection or OtherStressful Procedures. Biochim. Biophys. Acta 204: 564 577. 1970.

32. Statter, M., Russell, A., Abzug-Horowitz. S.. and Pinson, A. Abnormal Orotic Acid Metabolism Associated with Acute Hyperammonaemia in the Rat. Biochem. Med., 9. 1 18, 1974.

33. Tabor, H., and Tabor, C. W. Biosynthesis and Metabolism of1,4-Diaminobutane, Spermidine, Spermine, and Related Amines.Advan. Enzymol., 36: 203-268, 1972.

34. Tatibana, M., and Shigesada, K. Two Carbamyl Phosphate Synthetases of Mammals: Specific Roles in Control of Pyrimidine and UreaBiosynthesis. Advan. Enzyme Regulation. 10. 249-271, 1972.

35. Ternberg. J. L., and Hershey, F. B. Colorimetric Determination ofBlood Ammonia.J. Lab. Clin. Med., 56: 766-776, 1960.

36. Thomson, R. Y. Chemical Aspects of Compensatory Hypertrophy inLiver and Kidney. In: W. W. Nowinski and R. J. Goss, (eds.),Compensatory Renal Hypertrophy, pp. 87-100. New York: AcademicPress, Inc., 1969.

37. Thomson, J. F.. and Moss. E. M. Effect of Adrenalectomy onTryptophan Peroxidase, Adenosine Deaminase and Arginase Contentof Regenerating Rat Liver. Proc. Soc. Exptl. Biol. Med.. 89: 230 -233,1955.

38. Weber, G., Queener, S. F., and Morris. H. P. Imbalance in OrnithineMetabolism in Hepatomas of Different Growth Rates as Expressed inBehavior of L-Ornithine Carbamyl Transferase Activity. Cancer Res.,32:1933-1940.1972.

39. Williams-Ashman,H. G., Coppoc.G. L., and Weber.G. ImbalanceinOrnithine Metabolism in Hepatomas of Different Growth Rates asExpressed in Formation of Putrescine, Spermidine. and Spermine.Cancer Res., 32: 1924- 1932, 1972.

40. Williams-Ashman,H. G., Jänne,J., Coppoc,G. L.. Geroch,M.and Schenone,A. New Aspectsof PolyamineBiosynthesisin Eukaryotic Organisms. Advan. Enzyme Regulation, 10: 225-245. 1972.

404 CANCER RESEARCH VOL. 35

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1975;35:397-404. Cancer Res   Nelson Fausto, John T. Brandt and Leo Kesner  Pyrimidines and Polyamines in Regenerating LiverPossible Interactions between the Urea Cycle and Synthesis of

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