PYRIMIDINE METABOLISMIN MAN. IV. THE ENZYMATICDEFECTOF OROTIC ACIDURIA*

By LLOYD H. SMITH, JR., MARGARETSULLIVAN ANI) CHARLESM.HUGULEY, JR.

(From the Department of Medicine, Massachusetts General Hospital, and Harvard MedicalSchool, Boston, Mass., and the Department of Medicine, Emory University,

School of Medicine, Atlanta, Ga.)

(Submitted for publication November 8, 1960; accepted December 12, 1960)

Orotic aciduria is a rare disorder which has thusfar been described only in the propositus (1).This patient, a 9 month old child, exhibited re-tarded growth and development associated withmegaloblastic anemia unresponsive to vitamin B12,folic acid, ascorbic acid and iron. There washeavy crystalluria, demonstrated on subsequentstudy to be due to the excretion of large amountsof orotic acid, a pyrimidine nucleotide precursorabsent from normal urine. Improvement of theanemia without reversal of the megaloblasticmarrow followed treatment with glucocorticoidhormones. The administration of a mixture of py-rimidine nucleotides led to a complete hematologi-cal remission, a remarkable improvement in gen-eral well-being, and a prompt reduction in oroticacid excretion. The patient died of an overwhelm-ing varicella infection in 1958. His survivingparents and three siblings were normal hemato-logically and exhibited no orotic acid in the urineon chromatographic examination.

These clinical studies indicated the presence ofa metabolic block in the pathway of pyrimidinebiosynthesis, with impaired conversion of oroticacid to uridine-5'-phosphate (Figure 1). Thisreport describes the development of assays fororotidylic pyrophosphorylase and orotidylic de-carboxylase, the sequential enzymes which cata-lyze this conversion. Reduced activities of bothenzymes were found in circulating erythrocytesfrom the parents and two of the three siblings ofthe deceased propositus of orotic aciduria.

EXPERIMENTALPROCEDURE

Preparation of reactants

Carboxyl-labeled orotic acid-C14 (OA-C"4) was pur-chased from New England Nuclear Corporation. Car-

* This study was supported by Research Grant A-1606(C2) from the Institute of Arthritis and Metabolic Dis-eases, Bethesda, Md. A preliminary report of this workhas been published in abstract form (The enzymatic de-fect of orotic aciduria. J. clin. Invest. 1960, 39, 1029).

boxyl-labeled orotidine-5'-phosphate-C14 (05P-C"4) wasbiosynthetically prepared essentially as described byLieberman, Kornberg and Simms (2). Ammoniumsulfate fractionation was used in the purification oforotidylic pyrophosphorylase from yeast (3). The yieldwas increased (to about 10 per cent) by the inclusion ofazauridylic acid 1 (6 X 105M) in the incubation mediumto inhibit any residual orotidylic decarboxylase (4).Uniformly labeled L-aspartate-C4 was purchased fromNuclear Chicago Corporation. dl-Dihydroorotate-C1'was synthesized as previously described (5). 5-Phos-phoribosylpyrophosphate (PRPP) was purchased as themagnesium salt from Sigma Chemical Corporation,converted to the sodium salt on a Dowex-50 ion ex-change column, and assayed as described by Kornberg,Lieberman and Simms (6). Orotidylic decarboxylasewas purified from baker's yeast by the method of Hep-pel and Hilmoe (7).

Erythrocytes were separated from approximately 25ml of venous blood as previously described (5). Theerythrocytes were frozen and maintained at - 100 C un-til assays for enzymatic activities were carried out onaliquots of the whole hemolysates. Pilot studies revealedno loss of enzymatic activity over a period of 1 week.Saliva obtained using paraffin stimulation was cen-trifuged at 25,000 X G for 30 minutes, and the su-pernatant was assayed for enzymatic activity.

Assay procedures

The assay procedures, established after determiningthe characteristics of the enzyme systems in hemolysatesof circulating erythrocytes, are modifications of thosedescribed by Lieberman and associates (2).

1. Orotidylic pyrophosphorylase. In this assay therelease of carbon dioxide-C14 from carboxyl-labeled oro-tic acid-C14 was measured in the presence of optimal con-centrations of Mg++ and PRPP and of excess purifiedyeast orotidylic decarboxylase: OA-C"4, 2.5 X 10AM (3.6X 106 cpm per jumole); PRPP, 2.5 X 10'M; Mg++, 2.5X 10-M; yeast orotidylic decarboxylase, approximately0.1 U (2); Tris buffer (pH 7.8), 0.25 M, in a final vol-ume of 1 ml.

2. Orotidylic decarboxylase. Enzymatic activity wasmeasured as the release of carbon dioxide-C14 from bio-synthetically prepared carboxyl-labeled oritidine-5'-phos-phate: 05P-C14, 5 X 101M (3.6 X 10' cpm per ,umole);

1 Weare grateful to Dr. R. E. Handschumacher for agift of azauridylic acid.

656

ENZYMATIC DEFECTOF OROTIC ACIDURIA

phosphate buffer (pH 6.6), 0.25 M, in a final volume of1 ml.

Incubations were carried out under air in the outerchamber of stoppered center-well 10-ml Erlenmeyer flaskswith constant shaking at 37° C for 60 minutes. The re-actions were stopped by the introduction of excess sul-furic acid and shaking was continued for 45 minutesfor completion of carbon dioxide trapping in center-wellhyamine base. (8). Radioactivity was measured usinga Tricarb liquid scintillation spectrometer.2 Assays foraspartate carbamyltransferase and dihydroorotase andfor isolation of orotic acid were carried out as previouslydescribed (5).

SubjectsControl studies were carried out on erythrocytes from

laboratory personnel. Erythrocytes were obtained fromthe parents and the 3 surviving siblings (ages 16, 12 and8) of the deceased patient with orotic aciduria. All of

2 Packard Instrument Company, La Grange, Ill.

01°

HN'CH + PRPP, "I i -

0e N SCOOHH

OA

OROrIDYLICPrROfPWPHORrLASE

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HCOH HCOHH

lH G- o

I IHC-O-P-OH HC-O-P-OH

H 105P OHUMP

OROTIDYLIC DECARBOXYLASE

FIG. 1. THE ENZYMATICCONVERSIONOF OROTIC ACID TO

URIDINE-5'-PHOSPHATE. OA, orotic acid; PRPP, 5-phos-phoribosylpyrophosphate; PP, pyrophosphate; 05P, oroti-dine-5'-phosphate; UMP, uridine-5'-phosphate.

these subjects were normal hematologically and had ex-hibited normal growth and development. The parentsand 2 brothers of another subject, presumably heterozy-gous for orotic aciduria, were also investigated. The

3

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OROTIC ACID 1 /0-5 M

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ERiYTHROCYTESx /0 7

D

FIG. 2A) SUBSTRATECONCENTRATIONCURVEOF ERYTHROCYTEOROTIDYLIC PYROPHOSPHORYLASE.B) PH CURVE OF

ERYTHROCYTEOROTIDYLIC PYROPHOSPHORYLASE.C) TIME CURVE OF ERYTHROCYTEOROTIDYLIC PYROPHOSPHORYLASE,D) ENZYMECONCENTRATIONCURVEOF ERYTHROCYTEOROTIDYLIC PYROPHOSPHORYLASE,

657

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TIME IN MINUTESC

LLOYD H. SMITH, JR., MARGARETSULLIVAN AND CHARLESM. HUGULEY, JR.

5.4 5. 6.2 6.6

pHB

7.0 7.4 7.8

3 26

ERYTHRoCYrTES J /07D

39 52

FIG. 3A) SUBSTRATECONCENTRATIONCURVEOF ERYTHROCYTEOROTIDYLIC DECARBOXYLASE. B) PH CURVEOF ERYTH-

ROCYTEOROTIDYLIC DECARBOXYLASE. C) TIME CURVEOF ERYTHROCYTEOROTIDYLIC DECARBOXYLASE. D) ENZYMECON-CENTRATION CURVE OF ERYTHROCYTEOROTIDYLIC DECARBOXYLASE.

father and 1 of the 2 brothers of the latter subject hadhyperuricemia and clinical gout.

RESULTS

Orotidylic pyrophosphorylase. Orotidylic py-

rophosphorylase, previously studied as a partiallypurified enzyme from yeast (2, 3), catalyzes theformation of orotidine-5'-phosphate from oroticacid and 5-phosphoribosylpyrophosphate in thepresence of Mg++ (Figure 1). This reaction, a

reversible pyrophosphorolysis, has also been stud-ied less rigorously in experimental tumors (4)and in rat liver homogenates (9). Studies on the

characteristics of this enzyme in human erythro-cyte hemolysates are summarized by the series ofgraphs presented in Figure 2. The substrate con-

centration curve showed enzyme saturation to beattained at approximately 5 x 10-5M orotic acid.The calculated Km for orotic acid in this systemwas 0.4 X 10-5. Similarly the Kmfor PRPPwas

calculated to be 1.6 x 10-5, although the instabilityof this compound and the wide variety of com-

peting reactions in such a crude enzyme prepara-

tion detract from the significance of this value.In dialyzed preparations, Mg++ was required withan optimal concentration of 2 X 10-3M. The pH

658

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T/ME IN MINUTESC

ENZYMATICDEFECTOF OROTIC ACIDURIA

activity curve demonstrated maximal synthesis of05P at 7.8. The reaction was linear with timeover a period of 1 hour. It is to be noted that 05Psynthesis was not proportional to enzyme con-centration (number of hemolyzed erythrocytes) atrates of synthesis less than 1 mumole per hour.This lack of a linear response at these extremelylow rates of synthesis was also noted in studies ofthe orotidylic decarboxylase reaction on erythro-cyte hemolysates (see below) and of dilute prepa-rations of rat liver homogenates and partiallypurified yeast enzymes. The following manipu-lations failed to lead to a linear response in thissection of the curve: use of an internal bicarbonatecarrier; addition of glutathione, cysteine or hu-man albumin; addition of a boiled extract of ahemolysate; incubation at 240 C or collectingCO2at 560 C. In all assay procedures, determina-tions were made on the linear part of the enzymeconcentration curve. The reversibility of this re-action in erythrocyte preparations was demon-strated as the release of orotic acid-C14 from 05P-C14 in the presence of inorganic pyrophosphate,but the characteristics of the backward reaction(pH curve, time curve, substrate concentrationcurve) were not determined.

Orotidylic decarboxylase. Orotidylic decar-boxylase catalyzes the irreversible decarboxylationof orotidine-5'-phosphate to uridine-5'-phosphate(2). This reaction has no known cofactors. Re-cent evidence suggests that the activity of the en-zyme is influenced by the concentration of uridine-5'-phosphate (9), and is competitively inhibitedby 6-azauridylic acid (4). Some of the biochem-ical characteristics of orotidylic decarboxylase inhuman erythrocyte hemolysates are summarizedin Figure 3. The substrate concentration curveindicated enzyme saturation at approximately5 X 10-5M 05P. The calculated Kmfor 05P is1.1 X 10-6. There was a broad, flat pH curvewith the optimal pH in the range 6.2 to 6.8. Thedecarboxylation was linear with time over a pe-riod of 2 hours. The enzyme concentration curvedeviated from a linear response at rates of car-bon dioxide liberation of less than 1 miumole perhour, as discussed concerning orotidylic pyro-phosphorylase above. The reaction was not af-fected by ethylenediamine tetraacetic acid (10-2M), the addition of Mg++, or preincubation of theenzyme preparation with pyridoxalphosphate (2 X

10-3 M), biotin (2 x 10-3 M), or avidin (1 U perml).

Erythrocyte enzymatic activities in control sub-jects and in heterozygotes of orotic aciduria. Thenormal range of activity of orotidylic pyrophos-phorylase per 109 erythrocytes is summarized inFigure 4. Erythrocytes from the parents andtwo of the three siblings of the propositus of oro-tic aciduria (R. family) exhibited reductions ofenzymatic activity while that of the third siblingwas in the normal range. These determinationswere carried out repeatedly and at varying en-zyme concentrations. In the course of these stud-ies, one subject (J.S.), chosen as an apparentlynormal control, was found to have consistentlylow levels of erythrocyte orotidylic pyrophos-phorylase activity on five determinations over aperiod of several months. This subject was nor-mal clinically and hematologically and was foundto have a normal serum uric acid concentration.Studies of the parents and two brothers of J.S.(the S. family) revealed a tendency toward re-duced activity of orotidylic pyrophosphorylase,but this was not statistically significant.

Orotidylic decarboxylase was also studied inthe same subjects, and the enzyme activities per109 erythrocytes are summarized in Figure 5. A

CONTROLS

myM/109 RBC

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C-

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0

0

0

0.0000

0.0*

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R. FAMILY S. FAMILY

0

0

0

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OROTIDYLIC PYROPHOSPHORYLASE

FIG. 4. OROTIDYLIC PYROPHOSPHORYLASEACTIVITIES INERYTHROCYTESFROM CONTROL SUBJECTS AND PRESUMEDHETEROZYGOTESOF OROTIC ACIDURIA. R. family: parentsand 3 siblings of propositus of orotic aciduria. S. family:parents, siblings, and 5 determinations on a new heterozy-gote of orotic aciduria.

659

I !

LLOYD H. SMITH, JR., MARGARETSULLIVAN AND CHARLESM. HUGULEY, JR.

mpUM/109 RBC

15

I'.

10

5

CONTROLS R. FAMILY S. FAMILY

OROTIDYLIC DECARBOXYLASE

FIG. 5. OROTIDYLIC DECARBOXYLASE ACTIVITIES IN

ERYTHROCYTESFROM CONTROL SUBJECTS AND PRESUMED

HETEROZYGOTESOF OROTIC ACIDURIA.

pattern similar to that for orotidylic pyrophos-phorylase was observed with a reduction of en-

zyme activity in the erythrocytes of four of the fivesurviving members of the R. family and in Sub-ject J.S.

Since two consecutive enzymes in pyrimidinebiosynthesis were found to be reduced in activity,it was imperative to demonstrate the specificityof these abnormalities. In Figure 6 are summa-

rized studies of the enzymatic activities (per 109erythrocytes) of aspartate carbamyltransferaseand dihydroorotase, enzymes prior to orotic acidbiosynthesis. With the exception of one valuethat was unusually high, erythrocyte levels ofthese enzymes were normal in all subjects.

Mixtures of hemolysates from normal and"defective" erythrocytes were additive in enzy-

matic activities, evidence against the presence ofan inhibitory agent. Within the limits imposedby nonpurified enzyme preparations, the Michaelisconstants determined for orotic acid and orotidylicacid were the same for enzyme preparations fromnormal and "defective" erythrocytes.

Levels of enzymatic activities in saliva. Oroti-dylic pyrophosphorylase and orotidylic decarboxy-lase activities were also studied in saliva from a

series of control subjects and from Subject J.S.As might be anticipated, there was a wide scatterof enzyme activity per milligram of N in the25,000 x G supernatant from saliva. Althoughthe lowest activities of both enzymes were found

in studies of J.S. (Figure 7), these values were

not statistically significant due to the wide normalrange.

DISCUSSION

In this study the metabolism of orotic acid inhemolysates of human erythrocytes was shownto follow the same pathway previously demon-strated in bacteria (2) and rat liver homogenates(9). Orotidylic pyrophosphorylase in man re-

sembles the enzyme from E. coli in its optimalpH range, requirement for magnesium ions and5-phosphoribosylpyrophosphate, and in its rever-

sibility in the presence of inorganic pyrophosphate.The estimated Kmfor PRPP (1.6 X 10-5) agrees

closely with that previously reported (2 X 10-5),but the Km for orotic acid (0.4 X 10-5) was

lower than that found for the partially purifiedbacterial enzyme (2 x 10-5). Orotidylic decar-boxylase exhibited a broad optimal pH range

(pH 6.2 to 6.8). No cofactors could be demon-strated. Specifically the addition of pyridoxal-phosphate or biotin and preincubation of the en-

zyme preparation with avidin did not alter enzyme

activity. The Km for orotidine-5'-phosphate inhuman erythrocytes (1.1 x 105) approximatesthat found with the bacterial enzyme (1.4 x 10-5)(3). Low but reproducible activities of both en-

zymes were found in normal human erythrocytes.Assay procedures have now been developed

for five sequential enzymes in hemic cells leadingto the formation of uridylic acid from simple pre-

cursors (Figure 8). Four of these enzymes-

300

m,uM/109 RBC

200

100(3

CONTROLSR.FAMILY CONTROLSR. FAMILY+ J. S. + J. s.

m,uM/109 RBC

100

75 Z

501~tQ

ASPARTATE DIHYDROOROTASE

CARBAMYLTRANSFERASE

FIG. 6. ASPARTATE CARBAMYLTRANSFERASEAND DI-

HYDROOROTASEACTIVITIES IN ERYTHROCYTESFROMCONTROL

SUBJECTS AND PRESUMEDiTETEROZYGOTES OF OROTIC ACI

DURIA,

@00

000 0

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00 ~~~~~0

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660

ENZYMATIC DEFECTOF OROTIC ACIDURIA

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2

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CONTROLS J.S. CONTROLS J.S.

mpM/mj N6

5

4 $

3 4

21

I.I

OROTIDYLIC OROTIDYLCPYROPHOSPHORYLASEDECARBOKYLASE

SALIVA

FIG. 7. LEVELS OF OROTIDYLIC PYROPHOSPHORYLASEAND

OROTIDYLIC DECARBOXYLASEACTIVITIES IN SALIVA FROM

CONTROL SUBJECTS AND A SUBJECT (J.S.) PRESUMABLY

HETEROZYGOUSFOR OROTIC ACIDURIA.

aspartate carbamyltransferase, dihydroorotase,orotidylic pyrophosphorylase and orotidylic de-carboxylase-are present in reproducible activitiesin the mature human erythrocyte. Dihydrooroticdehydrogenase, which catalyzes the oxidation ofdihydroorotic acid to orotic acid, is absent in themature erythrocyte leading to a complete blockin the de novo biosynthesis of pyrimidine nucleo-tides (Figure 8) (5). This erythrocyte enzyme

pattern parallels that found in an E. coli mutant(ATCC no. 12632) which has a genetic block indihydroorotic dehydrogenase activity (10).Erythrocytic dihydroorotic dehydrogenase seems

to be lost during the process of maturation.Orotidylic pyrophosphorylase and orotidylic de-carboxylase seem to be vestigial in the matureerythrocyte, which is unable to form precursor

orotic acid, and is unique in containing no nucleicacids.

The propositus of orotic aciduria died beforethis study was undertaken (1). No subsequentcases of this unusual syndrome have been de-scribed. Clinical investigation of the originalpatient seemed to establish quite clearly that therewas a block in the conversion of orotic acid touridine-5'-phosphate. 1) Large amounts of oro-

tic acid (0.5 to 1.5 g) were excreted in the pa-

tient's urine. This pyrimidine cannot be detected

in normal uirine. 2) 'There was a striking clinicaland henmatological remission during treatmentwith a mixture of pyrimidine nucleotides, sub-stances distal to the presumed metabolic block.3) Pyrimidine nucleotide treatment resulted in areduction of orotic acid excretion, indicating aspecificity of the therapeutic action. This find-ing is consistent with negative feedback controlof pyrimidine biosynthesis, as demonstrated inE. coli in the classical studies of Yates and Pardee(11). The accumulation and excretion of oroticacid by the patient is analogous to the pattern of"pyrimidine starvation" seen in E. coli mutantsgrown in minimal media. A less marked patternof pyrimidine starvation in man has been sug-gested from studies on pernicious anemia (12).

In the absence of blood cells or tissue from thedeceased patient, studies were carried out on hissurviving family members in an attempt to dem-onstrate a heterozygous defect. The data obtaineddemonstrated significant reductions of both oro-tidylic pyrophosphorylase and orotidylic decar-boxylase activities in erythrocytes from the pa-rents and two of the three siblings of the propo-situs. Both enzyme activities were normal in the

MATUREERYTHROCYTE

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0

OA'uria

ASPARTIC ACID + CAP

E I

CARBAMYLASPARTICACID

E 2

DIHYDROOROTICACID

E 3

OROTIC ACID

E] [4

OROTIDINE-5'-PHOSPHATE

E 5

URIDINE-5'- PHOSPHATE

0

+

LENETICBLOOK

FIG. 8. SUMMARYOF LEVELS OF ENZYMESINVOLVED IN

URIDINE-5'-PHOSPHATE BIOSYNTHESIS IN NORMALHUMAN

ERYTHROCYTESAND IN ERYTHROCYTESOF SUBJECTS HETER-

OZYGOUSFOR OROTIC ACIDURIA. OA'uria, orotic aciduria;El, aspartate carbamyltransferase; E2, dihydroorotase;E3, dihydroorotic dehydrogenase; E4, orotidylic pyro-phosphorylase; E5, orotidylic decarboxylase.

0

0

* ~~~~000

.00

0 0~~~0

661

LLOYD H. SMITH, JR., MARGARETSULLIVAN AND CHARLESM. HUGULEY, JR.

R. FAMILY

d

o orTCACIDURIA

FIG. 9. PRESUMEDPATTERN OF INHERITANCE OF OROTICACIDURIA IN THE R. FAMILY.

third sibling. This pattern is consistent with thetransmission of orotic aciduria as an autosomalrecessive trait, with the deceased patient pre-

sumed to have been homozygous (Figure 9).There was no close consanguinity in the family,although the parents had a common ancestor whowas the grandfather of the father and the great-grandfather of the mother.

It is unlikely that the defect in the conversionof orotic acid to uridine-5'-phosphate was limitedto the patient's erythropoietic system in view ofhis diversity of symptoms and failure of growth(1). Gastrointestinal symptoms were prominent,suggesting involvement of intestinal epithelium,a tissue which is known to undergo rapid regenera-

tion. Preliminary observations suggest that re-

duced activities of orotidylic pyrophosphorylaseand orotidylic decarboxylase are found in poly-morphonuclear leukocytes from heterozygotes oforotic aciduria. In the syndrome of primaquine-sensitive hemolytic anemia, deficiency of glucose-6-phosphate dehydrogenase activity has beenfound in erythrocytes, platelets and leukocytes(13). Recent studies have shown the enzyme

defect to be present also in saliva (14). Simi-larly, salivary levels of orotidylic pyrophosphory-lase and orotidylic decarboxylase were found tobe low in a heterozygote of orotic aciduria, al-though the wide range of normal values reducesthe significance of this observation.

In previous studies of enzymatic abnormalitiesassociated with human "inborn errors of metabo-lism," defects of single enzymes have been found,in keeping with the one gene-one enzyme hypothe-sis (15). Possible exceptions to this generaliza-

tion have been reported as combined deficienciesof 1)lood coagulation factors. Similarly, muta-tions in bacteria and Neurospora almost uniformlyare specific for a single enzyme (16). In thecurrent studies two sequential enzymes werefound to be equally deficient. Specificity of theseenzyme defects was indicated by finding normalerythrocyte levels of aspartate carbamyltransferaseand dihydroorotase, earlier enzymes in the pyrimi-dine biosynthetic pathway. Studies of mixtures ofhemolysates from normal and defective cells gaveno evidence for the presence of an inhibitoryagent. Further studies carried out in an attemptto determine whether the deficiency of one of theseenzymes is more likely to be primary are reportedelsewhere ( 17).

In the course of these studies another subject(J.S.) was found whose erythrocytes have re-peatedly exhibited over a period of months re-duced activities of orotidylic pyrophosphorylaseand orotidylic decarboxylase with levels compara-ble to those found in the family of the patient withorotic aciduria. Preliminary observations sug-gest that the defect is also demonstrable in hisleukocytes. The enzyme defects were "specific"in that normal erythrocyte levels of aspartatecarbamyltransferase and dihydroorotase werefound. It seems probable that this subject isalso heterozygous for orotic aciduria. It is to benoted, however, that no clear-cut pattern of in-heritance could be demonstrated in his immediatefamily. No large survey has as yet been under-taken to document the true incidence of the oroticaciduric defect. The finding of another presumedheterozygote in the current small series suggeststhat the abnormal gene may have wide distribu-tion. If this is so, the rarity of the syndrome oforotic aciduria suggests that the genetic defect isusually lethal in the homozygous state. It maywell be asked how the propositus of orotic aci-duria was able to survive with a homozygous de-fect in the main pathway for the de novo synthesisof pyrimidine nucleotides. An analogous mu-tant in E. coli (ATCC no. 11548) exhibits the"orotic aciduria syndrome" in that it accumulatesorotic acid in the medium and demonstrates ablock in the conversion of orotic acid to uridine-5'-phosphate ( 11, 17). This organism fails togrow in the absence of an exogenous source ofuracil or pyrimidine nucleotides. The patient

662

ENZYMATICDEFECTOF OROTIC ACIDURIA

failed to resl)on(l to the administration of largeamounts of uracil, indicating an ineffective "sal-vage synthesis" of pyrimidine nucleotides fromfree pyrimidine bases, which are liberated in nu-cleic acid degradation (18). In the absence of anopportunity for direct measurements, it must beassumed that although homozygous, the patientwas able to form orotidylic pyrophosphorylaseand orotidylic decarboxylase in amounts sufficientfor marginal survival. The phenomenon of inter-allele complementation in enzyme formation hasrecently been demonstrated in Neurospora crassa(19) and in some bacterial mutants. Here, thecrossing of different mutants, each without de-tectable enzymatic activity, resulted in progenywith the ability to synthesize the enzyme in ques-tion. Enzyme production by interallele comple-mentation has always resulted in levels of ac-tivity markedly reduced below those found inwild-type strains. It is possible that some suchform of "genetic leakage" permitted the survivalof the propositus of orotic aciduria.

The father and one brother of Subject J.R.have idiopathic hyperuricemia with typical at-tacks of gouty arthritis. The occurrence of a"pyrimidine disease" and of the most frequent"purine disease" in the same family is a provoca-tive finding. The deceased patient with oroticaciduria exhibited no evident abnormality of uricacid metabolism. Subsequent studies of erythro-cytes from ten subjects with idiopathic gout havefailed to reveal the enzyme pattern of orotic aci-duria (20). At the present time no definite re-lationship of orotic aciduria to gout has been es-tablished.

SUMMARY

1. Two enzymes which catalyze the conversionof orotic acid to uridine-5'-phosphate, orotidylicpyrophosphorylase and orotidylic decarboxylase,were found to be present in reproducible activitiesin circulating human erythrocytes. Both enzymeswere also detected in saliva.

2. Reduced activities of orotidylic pyrophos-phorylase and orotidylic decarboxylase were foundin erythrocytes from the parent and two of thethree siblings of the deceased propositus of oro-tic aciduria. The erythrocytes exhibited normalactivities of aspartate carbamyltransferase and di-

hydroorotase, earlier enzymes in the l)yrillidinebiosynthetic pathway.

3. The pattern of inheritance of orotic aciduriais consistent with that of an autosomal recessivetrait.

4. In a small control series another heterozy-gote of orotic aciduria was discovered. It is sug-gested that the genetic disorder of orotic aciduriais usually lethal in its homozygous state.

ACKNOWLEDGMENT

The authors would like to express their appreciationto Dr. James A. Bain for his advice during the prepara-tion of this manuscript.

REFERENCES

1. Huguley, C. M., Jr., Bain, J. A., Rivers, S. L., andScoggins, R. B. Refractory megaloblastic anemiaassociated with excretion of orotic acid. Blood1959, 14, 615.

2. Lieberman, I., Kornberg, A., and Simms, E. S. En-zymatic synthesis of pyrimidine nucleotides. Oro-tidine-5'-phosphate and uridine-5'-phosphate. J.biol. Chem. 1955, 215, 403.

3. Holmes, W. L. Studies on the mode of action ofanalogues of orotic acid: 6-Uracilsulfonic acid,6-uracilsulfonamide, and 6-uracil methyl sulfone.J. biol. Chem. 1956, 223, 677.

4. Handschumacher, R. E. Orotidylic acid decarboxy-lase: Inhibition studies with azauridine 5'-phos-phate. J. biol. Chem. 1960, 235, 2917.

5. Smith, L. H., Jr., and Baker, F. A. Pyrimidinemetabolism in man. I. The biosynthesis of oroticacid. J. clin. Invest. 1959, 38, 798.

6. Kornberg, A., Lieberman, I., and Simms, E. S. En-zymatic synthesis and properties of 5-phosphori-bosylpyrophosphate. J. biol. Chem. 1955, 215, 389.

7. Heppel, L. A., and Hilmoe, R. J. Purification andproperties of 5-nucleotidase. J. biol. Chem. 1951,188, 665.

8. Eisenberg, F., Jr. Round-table on "The preparationof the alkaline absorbent for radioactive CO2 inliquid scintillation counting" in Liquid ScintillationCounting, C. G. Bell, Jr. and F. N. Hayes, Eds.New York, Pergamon Press, 1958, p. 123.

9. Blair, D. G. R., Stone, J. E., and Potter, V. R.Formation of orotidine-5'-phosphate by enzymesfrom rat liver. J. biol. Chem. 1960, 235, 2379.

10. Yates, R. A., and Pardee, A. B. Pyrimidine bio-synthesis in Escherichia coli. J. biol. Chem. 1956,221, 743.

11. Yates, R. A., and Pardee, A. B. Control by uracilof formation of enzymes required for orotate syn-thesis. J. biol. Chem. 1957, 227, 677.

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LLOYD 14. SMITH, JR., MARGARETSULLIVAN AND CHARLESM. HUGULEY, JR.

12. Smith, L. H., Jr., and Baker, F. A. Pyrimidine me-

tabolism in man. III. Studies on leukocytes anderythrocytes in pernicious anemia. J. clin. Invest.1960, 39, 15.

13. Ramot, B., Fisher, S., Szeinberg, A., Adam, A.,Sheba, C., and Gafni, D. A study of subj ectswith erythrocyte glucose-6-phosphate dehydroge-nase deficiency. II. Investigation of leukocyte en-

zymes. J. clin. Invest. 1959, 38, 2234.14. Ramot, B., Sheba, C., Adam, A., and Ashkenasi, I.

Erythrocyte glucose-6-phosphate dehydrogenase-de-ficient subjects: Enzyme-level in saliva. Nature(Lond.) 1960, 185, 931.

15. Stanbury, J., Wyngaarden, J. B., and Fredericksen,D., Eds. Metabolic Basis of Inherited Diseases.New York, McGraw-Hill, 1960.

16. Fincham, J. R. S. Genetically controlled differ-ences in enzyme activity. Advanc. Enzymol. 1960,22, 1.

17. Smith, L. H., Jr., and Sullivan, M. Studies on theenzyme defect of orotic aciduria. Submitted forpublication.

18. Reichard, P., and Skdld, 0. Enzymes of uracil me-

tabolism in the Ehrlich ascites tumour and mam-

malian liver. Biochim. biophys. Acta 1958, 28,376.

19. Pateman, J. A., and Fincham, J. R. S. Gene-enzymerelationships at the am locus in Nettrospora crassa.

Heredity 1958, 12, 317.20. Smith, L. H., Jr., and Sullivan, M. Unpublished

observations.

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