Purine Nucleoside Metabolism in the Erythrocytes

of Patients with Adenosine Deaminase Deficiency

and Severe Combined Immunodeficiency

R. P. AGARWAL,G. W. CRABTREE,R. E. PARKS, JR., J. A. NELSON,R. KEIGHTLEy, R. PARKMAN,F. S. ROSEN, R. C. STERN, and S. H. POLMAR

From the Section of Biochemical Pharmacology, Division of Biological andMedical Sciences, Brown University, Providence, Rhode Island 02912;Kettering-Meyer Laboratory, Southern Research Institute, Birmingham,Alabama 35205; Department of Pediatric Immunology, University of Alabama,Birmingham, Alabama 35233; Immunology Division, Children's HospitalMedical Center, Department of Pediatrics, Harvard Medical School, Boston,Massachusetts 02115; Department of Pediatrics, Case Western ReserveUniversity School of Medicine, Cleveland, Ohio 44106

A B S T R A C T Deficiency of erythrocytic and lympho-cytic adenosine deaminase (ADA) occurs in some pa-tients with severe combined immunodeficiency disease(SCID). SCID with ADA deficiency is inherited asan autosomal recessive trait. ADA is markedly reducedor undetectable in affected patients (homozygotes), andapproximately one-half normal levels are found in in-dividuals heterozygous for ADA deficiency.

The metabolism of purine nucleosides was studied inerythrocytes from normal individuals, four ADA-deficientpatients, and two heterozygous individuals. ADA defi-ciency in intact erythrocytes was confirmed by a very sen-sitive ammonia-liberation technique. Erythrocytic ADAactivity in three heterozygous individuals (0.07, 0.08,and 0.14 i'molar units/ml of packed cells) was be-tween that of the four normal controls (0.20-0.37 umol/ml) and the ADA-deficient patients (no activity). Invitro, adenosine was incorporated principally into IMPin the heterozygous and normal individuals but into theadenosine nucleotides in the ADA-deficient patients.Coformycin (3-9-n-ribofuranosyl-6,7,8-trihydroimidazo-[4,5-d] [1,3]diazepin-8(R)-ol), a potent inhibitor ofADA, made possible incorporation of adenosine intothe adenosine nucleotides in normal and heterozygouserythrocytes. Coformycin did not alter the pattern of

Dr. Keightley's present address is: Department of Pedi-atrics, University of Texas, Health Sciences Center at SanAntonio, San Antonio, Tex. 78284.

Received for publication 29 August 1975 and in revisedform 1 December 1975.

nucleotide incorporation in the ADA-deficient cells.These results indicate that coformycin causes normaland heterozygous cells to behave like ADA-deficientcells. Similarly, p-nitrobenzylthioguanosine (2-amino-6-([4-nitrobenzyl]thio)-9-,-D-ribofuranosylpurine) (NB-TGR), an inhibitor of nucleoside transport, drasticallyreduced the incorporation of adenosine into the inosinenucleotides and enhanced the incorporation into adeno-sine nucleotides in the normal erythrocytes. NBGTRhad no effect upon ADA-deficient cells. NBTGR in-hibited the liberation of ammonia from adenosine in in-tact normal cells. In two patients previously treated withbone marrow or fetal liver transplantation, incubationof erythrocytes with inosine resulted in accumulation ofITP, whereas in the erythrocytes from the heterozy-gotes, the normal individuals and the two other ADA-deficient patients, IMP accumulated. Reexamination ofone of the transplanted patients 10 and 15 mo laterrevealed his erythrocytic inosine incorporation intoITP to be decreased from that observed at the initialexamination. Guanosine was incorporated into guano-sine nucleotides in all erythrocytes studied, suggestingthat the nucleoside transport system and the enzymespurine nucleoside phosphorylase, hypoxanthine-guaninephosphoribosyltransferase, and 5-phosphoribosyl-1-pyro-phosphate synthetase are functional in the erythrocytesof ADA-deficient patients. Formycin A (7-amino-3-9-D-ribofuranosylpyrazolo- [4,3-d]pyrimidine), an adenosineanalogue, was converted to formycin A-mono-, di-, andtriphosphate nucleotides in ADA-deficient patients'

The Journal of Clinical Investigation Volume 57 April 1976*1025-1035 1025

erythrocytes to a much greater extent than occurred innormal cells. This confirms the ability of formycin Ato act as an excellent substrate for both human erythro-cytic ADAand adenosine kinase.

These results are consistent with the hypothesis thatthe disposition of adenosine in human erythrocytes isthe result of the relative activities and Michaelis con-stants of the enzymes ADAand adenosine kinase. Vari-ous possible biochemical mechanisms by which ADAdeficiency causes cytotoxicity are discussed.

INTRODUCTION

Recently an inherited deficiency of the enzyme adenosinedeaminase (ADA) 1 has been described (1-6). De-ficiency of ADA is inherited as an autosomal recessivetrait, and patients with this disorder also have a severecombined immunodeficiency disease (SCID) character-ized by deficits both in thymus-derived and bone mar-row-derived cell-mediated immunity. In addition, mostof these children have striking skeletal abnormalities aswell as malfunction of the thymus gland (4). Approxi-mately one-third of the children with SCID studiedhave erythrocytes and lymphocytes that are deficient inADA (4, 5). This finding is of particular interest be-cause it is the first enzymic defect to be associated withan immune deficiency disease. Children with this condi-tion usually succumb to infection early in life. However,several children have survived for prolonged periodswith aseptic environmental conditions, and at least twochildren have become immunocompetent after bone mar-row transplantation (6).

It has been previously suggested that the dispositionof adenosine by human erythrocytes is, in part, deter-mined by the relative activities and Michaelis constantsof the enzymes ADA and adenosine kinase (7-10).Adenosine as well as adenosine analogues that are goodsubstrates for ADA were not incorporated into theerythrocytic nucleotide pools. On the other hand, adeno-sine analogues that are inhibitors or that lack substrateactivity with ADA but are good substrates for adeno-sine kinase in many cases are incorporated into thenucleotide pools (7-9). Several of the latter compoundsformed notably high concentrations of the respectiveanalogue triphosphate nucleotides (7, 9). When adeno-sine was incubated with normal erythrocytes, ammoniaand large concentrations of IMP accumulated, prob-ably as the result of the sequential reactions of the en-zymes ADA, purine nucleoside phosphorylase, and hy-poxanthine-guanine phosphoribosyl transferase (7-9,11, 12). On the other hand, when human erythrocytes

'Abbreviations used in this paper: ADA, adenosine de-aminase (adenosine aminohydrolase, EC 3.5.4.4); HPLC,high-pressure liquid chromatography; NBTGR, p-nitro-benzylthioguanosine; SCID, severe combined immunode-ficiency disease.

were treated with the potent ADA inhibitor, coformycin(8, 9, 12-14), adenosine and several adenosine ana-logues that are good substrates for ADA readily enteredthe nucleotide pools (9, 12). Thus, it is clear that ADAcan play a major role in the control of intracellularadenine nucleotide metabolism.

Several recent technical advances have greatly facili-tated the study of nucleoside and nucleotide metabolism.Especially useful is the technique of high-pressure liquidchromatography (HPLC) for the study of nucleotidepools in cells (7, 15, 16). Also of value is the avail-ability of a variety of purine nucleoside analogs andspecific inhibitors, such as coformycin, a tight-bindinginhibitor of ADA (13, 14), and p-nitrobenzylthioguano-sine (NBTGR), a potent inhibitor of the facilitated nu-cleoside transport system of human erythrocytes (17).The present report describes studies in which severalof these new biochemical tools have been applied to anexamination of purine nucleotide metabolism in eryth-rocytes of four SCID-ADA-deficient patients as well astwo heterozygous individuals.

METHODSAdenosine, inosine, and guanosine were purchased from P-LBiochemicals, Inc., Milwaukee, Wis. [8-'C]Adenosine (51mCi/mmol) and [8-1C]inosine (35 mCi/mmol) were ob-tained from Schwarz/Mann Div., Becton, Dickinson & Co.,Orangeburg, N. Y., and [8-G'C]guanosine was a product ofNew England Nuclear, Boston, Mass. A mixture of peni-cillin (10,000 U/ml) and streptomycin (10,000 ,ug/ml) wasobtained from Grand Island Biological Co., Grand Island,N. Y. Formycin A (7-amino-3-,8--ribofuranosylpyrazolo-[4,3-d]pyrimidine) and coformycin(3-p-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-d] [1,3]diazepin-8(R) -ol) were gifts ofDr. H. Umezawa, Institute of Microbial Chemistry, Tokyo,Japan, and NBTGR (2-amino-6-[ (4-nitrobenzyl)thio]-9-,B-D-ribofuranosylpurine) was provided by Dr. Harry B. Wood,Jr., of the Drug Development Branch, Drug Research andDevelopment, Division of Cancer Treatment, National Can-cer Institute.

Heparinized or citrated blood used in the present studywas obtained from the donors listed below. In each case,the blood specimens were placed on ice immediately afterphlebotomy and were hand-carried to the laboratory forstudy. All experiments were initiated as soon as practicalafter drawing the blood, i.e., within a period of 2-20 h. Ineach experiment, control blood specimens from normal sub-jects were included and were handled in a similar manner.It should be noted that in unpublished experiments in thislaboratory, nucleotide profiles obtained by HPLC were per-formed daily on the same refrigerated normal blood sampleover a 5-day period. No significant differences in thesenucleotide profiles were seen. Also in other experiments theADA activity was measured by the ammonia liberationprocedure described here, with aliquots of the same specimenof refrigerated normal erythrocytes on several occasionsover a 2-wk period. No significant difference in the activityof the enzyme was detected.

Patient 1 (A. W.) is a 5-yr-old male. The diagnosis ofSCID was reinforced by the occurrence of this disorder ina previous sibling. He was treated successfully by trans-plantation of bone marrow from an older sister. Although

1026 Agarwal et al.

this child had a successful bone marrow transplant about 4yr ago and now has normal thymus-derived and bone mar-row-derived cell function, his erythrocytes still display theADA deficiency. Details of the history, diagnosis, andtreatment were reported previously (6).

Patient 2 (M. R.), who was a male infant diagnosed atbirth as having SCID by the absence of lymphocytes incord blood, had a family history of SCID. Successful en-graftment of fetal liver, transplanted at 3 mo of age, resultedin the gradual development of functional thymus-derivedand bone marrow-derived lymphocytes with ADA activity.Red cell chimerism was transient, and his erythrocyteslacked ADA activity at the time of study. 1 yr post-trans-plantation he developed an ultimately fatal nephrotic syn-drome associated with immune complex nephritis. A de-tailed report of this case is in press (17a).

Patient 3 (C. W.) is a male who was 5-mo-old at thetime of study (Feb. 1975). ADA deficiency was diagnosedprenatally by amniocentesis (18), and SCID was subse-quently confirmed by immunologic studies.

Patient 4 (I. B.) is a female whose diagnosis of SCIDwas suggested by profound lymphopenia and absent thymusshadow on X-ray study (6). Familial history and immuno-logical studies confirming the diagnosis have been reportedby Parkman et al. (6).

Heterozygotic indiniduals. Characteristics of all threeheterozygotic individuals used in the present study havebeen described by Parkman et al. (6). G. W. and M. W. arethe brother and mother, respectively, of A. W. (patient 1).The third heterozygotic individual (M. B.) is the motherof I. B. (patient 4). With respect to M. W., only ADAactivity studies were performed. No nucleoside incorpora-tion studies were done with this heterozygotic individual'serythrocytes.

Preparation of cells. Plasma-free erythrocytes werewashed 2-3 times with equal volumes of 0.9% NaCl solu-tion and then suspended in the incubation medium (potas-sium phosphate buffer, 50 mM, pH 7.5; MgSO4, 2 mM;NaCl, 75 mM; and glucose, 10 mM). These cell suspensionswere used for studies of ADA activity and nucleoside in-corporation.

Assay of ADA actizvity in intact erythrocytes. The ac-tivity of ADA in intact cells was calculated from the rateof liberation of ammonia during the deamination of adeno-sine. The ammonia was determined by slight modificationsof the microdiffusion method of Seligson and Seligson (19)and the colorimetric procedure of Chaney and Marbach (20)as described below. Erythrocytic suspensions (2% in theabove medium) were incubated with 1 mMadenosine. 1-mlsamples were withdrawn at various time intervals after in-cubation in a shaking water bath at 30°C and were added toflasks containing 1 ml of saturated K2CO3 solution andequipped with glass rods having flared ground tips andcoated with 1 M citric acid. The flasks were placed on arotator for 30 min and the ammonia was allowed to diffuseand be trapped on the citric acid-coated rods (19). Theammonia collected on the glass rods was washed into testtubes with 5-ml aliquots of the phenol-nitroprusside solu-tion (20), and the color was developed by the addition of5 ml of the sodium hydroxide-sodium hypochlorite solutionas described by Chaney and Marbach (20).

Incorporation of nucleosides into nucleotide pools. Eryth-rocytic suspensions (15-25% in the above medium contain-ing penicillin, 10 U/ml, and streptomycin, 10 Ag/ml) wereincubated with or without coformycin (1 jug/ml) or NB-TGR (10 AM) in a shaking water bath at 30°C for 20 min.Then one of the following nucleosides was added: adeno-

sine, 0.5 mM (0.98 mCi/mmol); inosine, 0.27 mM (1.8mCi/mmol); guanosine, 0.12 mM (8.3 mCi/mmol), orformycin A, 1.0 mM. Aliquots (200 bd1) were withdrawnafter incubation at 30'C for 0, 1, 2, and 4 h after additionof the nucleoside and were added to 100 Al of cold 12%perchloric acid. The samples were stirred on a Vortexmixer (Scientific Industries, Inc., Springfield, Mass.), cen-trifuged, and the resulting supernatant solutions were neu-tralized with KOH with phenol red as the pH indicator.Precipitates of KC104 were removed by centrifugation andthe neutralized supernatant fluids were stored frozen at- 20'C for further study.

Nucleotides were resolved either by thin-layer chroma-tography or by HPLC. The method of Crabtree and Hen-derson (21) was used to separate the nucleotides on PEI-cellulose thin-layer plates (J. T. Baker Chemical Co.,Phillipsburg, N. J.). This method separates cleanly theadenine-, hypoxanthine-, and guanine-containing nucleotides.In studies with isotopically labeled compounds, unlabeled,known nucleotides were added as carriers to facilitate theidentification of the labeled compounds. After developmentof the chromatograms, the individual nucleotide spots werevisualized by UV light at 2537 A, cut out, and the radio-activity was counted in an Omnifluor (98%o PPO and 2%op-bis- (o-methylstyryl)benzene, New England Nuclear) -tol-uene (4 g/liter) scintillation fluid in a liquid scintillationcounter (Packard Tri Carb, Packard Instrument Co., Inc.,Downers Grove, Ill.). HPLC was performed on a VarianLCS 1000 analyzer (Varian Aerograph, Palo Alto, Calif.)(15), and the concentrations of the nucleotides were calcu-

lated by planimetery using known nucleotides as standards.The recent availability of a multiwave-length detection sys-tem (model SF 770, Schoeffel Instrument Corp., Westwood,N. J.) adaptable to HPLC has made practical the readydetection and estimation of the concentrations of intracellu-lar analogue nucleotides that have absorbancy maxima dif-ferent from those of the natural nucleotides. For example,nucleotides of formycin A were detected and estimated at295 nm, a wave length at which the natural nucleotides donot absorb (See Fig. 8).

In some instances, incorporation of inosine into IMP,IDP, and ITP was followed by HPLC with a WatersAssociates liquid chromatograph equipped with BondapakAX columns (2.3 mm ID X 122 cm, Waters Associates,Inc., Milford, Mass.). The flow rate was 2 ml/min, and alinear gradient from 5 mMNH4H2PO4, pH 3.0, to 125mMNH4H2PO4plus 250 mMKC1 was formed in 15 min.This system permits the separation of inosine ribonucleo-tides from adenosine ribonucleotides, but does not separateadenosine nucleotides from guanosine nucleotides. However,in control erythrocytic extracts, the guanosine nucleotidepool is considerably lower than that of the adenosine com-pounds (16).

RESULTS

ADAactivity in intact erythrocytes of SCID-ADA-deficient patientsFig. 1 presents measurements of ADA activity in

intact erythrocytes from six normals, three ADA-de-ficient patients (A. W., C. W., and I. B.), and threeheterozygous individuals. The normal intact erythro-cytes liberated ammonia from adenosine at a mean rateof 0.29 imol/min/ml of packed cells (values rangedfrom 0.2 to 0.4 umol/min/ml), which is in good agree-

Purine Nucleoside Metabolism in ADA-Deficient Erythrocytes 1027

0 5 0 46 6

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------9------ ------ 9---AW. X B.-PATIENTS0 15 30 45 60

INCUBATION TIME ("in)

FIGURE 1 ADA activity in intact erythrocytes. Erythrocyticsuspensions (2% in a medium consisting of potassium phos-phate buff er, 50 mM, pH 7.5; MgSO4, 2 mM; NaCi, 75mM; and glucose, 10 mM) were incubated with 1 mMadenosine at 300C in a shaking water bath. At various timesafter addition of adenosine, 1-ml aliquots were removedand the ammonia liberated by the deamination of adenosinewas determined as described in Methods. "Normal" line isthe mean of six samples. Ranges from the mean are indi-cated by vertical bars.

ment with the values reported earlier for normal hemo-lysates (8, 9, 11). No detectable liberation of ammoniafrom adenosine was observed with the ADA-deficientcells. This confirms the reports of Parkman et al. (6)and Hirschhorn et al. (18), that the erythrocytes ofthese patients lack the enzyme ADA. The activity ofADA in the intact erythrocytes from the heterozygousindividuals ranged from 0.07 to 0.14 Amol/min/ml ofpacked erythrocytes. Interestingly, the heterozygous in-dividuals (G. W. and M. W.) of the same familyshowed similar rates of ammonia liberation. Thesevalues are substantially below the range of ADA ac-tivity usually observed by this method with normalerythrocytes (see above). These results are consistentwith earlier reports which have examined ADA activi-ties in heterozygous individuals (6, 18, 22).

Metabolism of purine nucleosides in theerythrocytes of ADA-deficient SCID patients

Adenosine metabolism. The nucleotide levels oferythrocytes from six normal, two heterozygous, andfour ADA-deficient individuals are shown in Table I.With the exception of one blood sample from M. R.,ATP concentrations from all samples fell within therange normally found in human erythrocytes. In mostcases the ratios of ATP/ADP were similar in theerythrocytes of all three types of donors. When firstexamined, the erythrocytes of A. W. had an ATP/ADPratio of 17:1, but on reexamination 10 and 15 mo later,the ATP/ADP ratio was about 4: 1 and 9:1, respec-tively, which is close to the normal range. Although inmost samples examined the adenine nucleotides fell

within the normal range, individual deviations from thisrange were seen. Therefore, it will be interesting toexamine additional patients as they become availableand to do serial adenine nucleotide determinations onindividual patients. As discussed below, it seems pos-sible that fluctuations in adenosine nucleotide concentra-tions may occur more readily with ADA-deficient thanwith normal cells.

The nucleotide profiles obtained by HPLCwere simi-lar in the normal and the ADA-deficient patients (Fig.2). Figs. 2 and 3 illustrate the response to incubationwith adenosine of erythrocytes from one patient (A.W.). Similar findings were obtained with erythrocytesfrom all four patients. After incubation of erythrocyteswith [8-14C]adenosine (0.5 mM) at 30'C for 4 h sub-stantial differences were observed both in the nucleotideprofiles and in the concentrations of nucleotides betweennormal and ADA-deficient cells. Fig. 2 illustrates thatwith normal erythrocytes, in agreement with earlierfindings (7, 9), a large new peak appears with a re-tention time consistent with that of the monophosphatenucleotide, IMP. The identity of this peak as IMP wasconfirmed by collecting effluent from the HPLC andresolving the contents of this peak by thin-layer chroma-tography according to the method of Crabtree andHenderson (21). On the other hand, in the ADA-de-ficient cells, this monophosphate nucleotide peak was notpresent. In these cells the ADP and ATP peaks in-creased in size. These observations are supported by

TABLE IAdenine Nucleotide Concentrations in Human Erythrocytes

from Normal Donors, SCID-ADA-Deficient Patients,and Heterozygous Individuals

ADP ATP

Mmol/ml packed erythrocytesNormal 0.18 ±0.07* 1.06±i0.30*SCID-ADA-deficient patients

No. 1 (A. W.)t 0.08 1.34(A. W.)§ 0.24 0.91(A. W.)II 0.10 0.89

No. 2 (M. R.)¶ 0.13 1.28(M. R.)** 0.52 1.99

No. 3 (C. W.) 0.23 1.30No. 4 (I. B.) 0.10 0.88

HeterozygotesG. W. 0.27 1.14M. B. 0.09 0.78

* Values from six normal donors (mean-±SD).Blood drawn on 7 July 1974.

§ Blood drawn on 12 May 1975.II Blood drawn on 13 October 1975.¶ Blood drawn on 22 December 1973.** Blood drawn on 19 January 1974.

1028 Agarwal et al.

2

IEc

a

w0

nm

RETENTION TIME (min)

FIGURE 2 Erythrocytic nucleotide profiles obtained by HPLC. Erythrocytic suspensions (15-25% in the medium described in the legend to Fig. 1 supplemented with penicillin, 10 U/mland streptomycin, 10 Ag/ml) were incubated with 0.5 mMadenosine. Aliquots (200 ul) wereremoved at 0 and 4 h after addition of adenosine and added to 100 al of cold 12%70 perchloricacid. After thorough mixing the samples were centrifuged and the supernatant solutions wereneutralized with KOH. After removal of precipitated KCIO4 by centrifugation, 20 /l of theneutralized extracts were chromatographed on a Varian LCS-1000 liquid chromatographequipped with a Reeve-Angel AS-Pellionex SAX (3-m X 1-mm) column. A linear elutiongradient was used which consisted of 0.002 M potassium phosphate, pH 4.5, as the low con-centrate buffer and 0.5 M potassium phosphate, pH 4.5, in 1.0 M KCl as the high concentratebuffer. The starting volume was 40 ml and the flow rates were 14 and 7 ml/h for the columnand gradient, respectively. Although the profile of only one SCID-ADA-deficient patient(A. W.) is shown here, the profiles of erythrocytes from all other SCID-ADA-deficientpatients examined were similar. The nucleotide profile of erythrocytes from two heterozygousindividuals (G. W. and M. B.) were similar to those of erythrocytes from normal individuals.

data shown in Fig. 3, which illustrates the incorporationof [8-14C]adenosine into the various nucleotides. Innormal and heterozygous cells, large amounts of adeno-sine were converted to IMP (and ITP) rather thanto adenine nucleotides. In ADA-deficient cells, however,the isotope was incorporated principally into the adeninenucleotides (ADP and ATP) with negligible incorpora-tion into the inosine nucleotides. In this experiment,after 4 h of incubation, the ATP levels in the patients'cells increased from about 1.1 to about 2.6 IAmol/ml,i.e., an increase of approximately 135%. In contrast, theincreases in ATP levels in the heterozygous and normalcells were 29 and 15%, respectively. In ADA-deficient

erythrocytes from all four patients studied, the ATPlevels increased about 130-150% after 4 h of incubationwith 0.5 mMadenosine.

Coformycin, a tight-binding inhibitor of ADA, hasbeen shown capable of inhibiting this enzyme in intacterythrocytes and of significantly modifying the patternsof adenosine and adenosine analogue metabolism inthese cells (9, 12). The effect of coformycin on theadenosine metabolism of ADA-deficient erythrocyteswas also examined (Fig. 4). As expected, pretreatmentof these cells with coformycin (1 ,tg/ml reaction mix-ture) did not affect either the nucleotide profiles or theamount of adenosine incorporated into the nucleotide

Purine Nucleoside Metabolism in ADA-Deficient Erythrocytes 1029

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FIGURE 3 Incorporation of adenosine into nucleotides byintact erythrocytes. Erythrocytes were incubated and ex-tracts were prepared as described in Fig. 2 except thatlabeled [8-"C]adenosine (0.5 mM, 0.98 mCi/mmol) wasused. Aliquots (25 pl) of the 4-h samples were chromato-graphed on PEI-cellulose thin-layer sheets and developedwith a stepwise gradient of sodium formate solutions ac-cording to the method of Crabtree and Henderson (21).Concentrations of nucleotides refer only to the incorporationof the precursor and do not represent total erythrocyticconcentrations. Results are presented for one of the SCID-ADA-deficient patients (A. W.), his heterozygous sibling(G. W.), and a normal individual.

pools. However, in normal and heterozygous cells, co-formycin abolished the formation of inosine nucleotidesand markedly enhanced the incorporation of adenosineinto the adenine nucleotides. In fact, treatment of nor-mal cells with coformycin caused these cells to metabo-lize adenosine in a manner very similar to that of eryth-rocytes from the ADA-deficient patients.

It has been shown recently that NBTGR, a potentinhibitor of the facilitated nucleoside transport systemof erythrocytes (17), greatly inhibits the deaminationof adenosine and enhances adenosine incorporation intothe adenine nucleotides in intact normal erythrocytes(11). To examine whether NBTGRaffects the adeno-

15

jewon.+CoF

0AMP AlP WPN IDP IT

FIGURE 4 Effects of coformycin on incorporation of adeno-sine into nucleotides by intact erythrocytes. Cells were in-cubated with and without coformycin (1 jtg/ml of reactionmixture) for 20 min before addition of [8-14C]adenosine.Conditions for incubation, extraction, and analysis are de-scribed in Figs. 2 and 3. Concentrations of nucleotides referonly to incorporation of precursor. Results from only one ofthe SCID-ADA-deficient patients (A. W.) are representedhere. However, similar findings were obtained with the othertwo patients studied. "Hetero" refers to heterozygote G. W.and "CoF" refers to coformycin.

r l NormalNorrna+NBTGR

OMPatientmPatient+NBTGR

AMP ADP ATP IMP IDP ITPFIGURE 5 Effects of NBTGR on the incorporation ofadenosine into nucleotides by intact erythrocytes. Cell wereincubated with and without NBTGR(10 AM) for 20 minbefore addition of [8-14C]adenosine. Conditions for incuba-tion, extraction, and analysis are presented in Figs. 2 and 3.Concentrations of nucleotides refer only to incorporation ofprecursor. The patient studied was A. W.

sine metabolism of ADA-deficient erythrocytes, cellsfrom A. W. and normal cells were pretreated with 10AM NBTGR, after which they were incubated with[8-'4C]adenosine. As shown in Fig. 5, the NBTGRdidnot alter the incorporation of adenosine into the nucleo-tides of patients' cells whereas in normal cells the in-corporation of adenosine into adenine nucleotide poolswas enhanced and the formation of IMP was decreased.The latter observation is consistent with earlier reportswhich proposed that ADA is physically associated withthe cell membrane (23) at a site close to the nucleosidetransporter of human erythrocytes (11).

0-81

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TOTAL , '

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FIGURE 6 Time-course of incorporation of inosine into nu-cleotides by intact erythrocytes. Incubation, extraction, andanalysis conditions are as described in Figs. 2 and 3 exceptthat [8-14C]inosine (0.27 mM, 1.8 mCi/mmol) was used inplace of [8-14C] adenosine. "Total" refers to the sum ofIMP+IDP+ITP. "Patient" was A. W.

1030 Agarwal et al.

IMP IDP ITP

i

I-nosine netabolism. Usually, the concentrations ofinosine nucleotides in both normal and ADA-deficienterythrocytes are too low for ready detection by HPLC,i.e., below 10 nmol/ml packed cells. However, a largeamount of IMP accumulates in normal but not in ADA-deficient erythrocytes when these cells are incubatedwith adenosine (See Fig. 3). To examine whetherADA-de. cient cells have the capacity to metabolize ino-sine and guanosine, the incorporation of these com-pounds into nucleotide pools was studied.

Timie-courses of the incorporation of inosine intothe nucleotides of normal and ADA-deficient erythro-cytes (A. W.) are presented in Fig. 6. Cells were incu-bated at 300C with [8-'4C]inosine (0.27 mM) for vari-ous times up to 4 h. Aliquots were extracted, the nu-cleotides were separated by thin-layer chromatography,and the radioactivity in the various purine nucleotideswas determined. As shown in Fig. 6, after 4 h of incu-bation with inosine, IMP accumulated in the normalerythrocytes to a concentration of about 0.8 /mol/ml ofpacked cells, and ITP to a concentration of about 0.05Amol/ml of packed cells, i.e., an ITP/IMP ratio ofabout 0.06. The accumulation of IDP was negligible atall time periods examined. On four occasions we haveperformed similar studies with normal erythrocytes andhave found ITP/IMP ratios between 0.06 and 0.4 withan average of about 0.2. The question of the normalvariation in this parameter has not yet been subjectedto detailed study by this laboratory, but results to datesuggest that significant variability exists. We have hadthe opportunity to perform similar inosine incorporationexperiments with the erythrocytes of patient A. W. onthree separate occasions over a 15-mo period, and Fig.6 illustrates the first of these experiments. On thesethree occasions, ITP/IMP ratios of about 1.2. 0.4, and0.5 were obtained. With the erythrocytes of a secondpatient, M. R., after 17 h of incubation with inosinea ratio of ITP/IMP of about 4.0 was seen. In contrast,with the erythrocytes of the two other ADA-deficientpatients (C. W. and I. B.), after 4 h of incubation withinosine, ITP/IMP ratios of about 0.02 and 0.07 wereobserved. In addition, in similar experiments, the ITP/IMP ratios of the erythrocytes of two of the heterozy-gous subjects studied similarly were about 0.08 and 0.2.It is not vet possible to evaluate the significance of theap)parentlv rapid synthesis of ITP seen with the erythro-cvtes of two of the patients (A. W. and M. R.). Pos-sible reasons for these differences in inosine metabolismare discussed below.

The ATP concentrations remained unchanged inboth normal and patients' cells during the course ofincubation with inosine, suggesting that the formationof inosine nucleotides was not at the expense of cellularATP under conditions used here. Although the rates ofglycolysis were not determined in the various erythro-

15r0

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mNORMALM PATIENT

Fl r- FImIMP GMP GDP

XMP

FIGURE 7 Incorporation of guanosine into nucleotides byintact erythrocytes. Conditions for incubation, extraction,and analysis are as described in Figs. 2 and 3 except that[8-'4C]guanosine (0.12 mM, 8.3 mCi/mmol) was used inplace of [8-'4C]adenosine. "Patient" refers to A. W.

cytes in this study, it has been shown that during theincorporation of large amounts of 2-fluoroadenosine into

erythrocytic nucleotide pools, the rate of glycolysis in-creased about threefold, whereas the ATP and 2,3-di-phosphoglycerate concentrations were unaffected (7).

Guanosine metabolismi. Incubation of both normaland ADA-deficient erythrocytes with [8-'4C] guanosine(0.16 mM) resulted in the rapid incorporation of labelinto the nucleotide pools. The total amounts of guano-

sine incorporated were similar and represented virtuallytotal conversion of the added guanosine to nucleotidesafter 4 h of incubation. At the end of this incubationtime most of the [8-'4C]guanosine had accumulated as

GTP in both types of cells (Fig. 7).The results of this experiment and that of inosine

incorporation noted above indicate that the purine nu-

cleoside transport process, purine nucleoside phosphory-lase, hypoxanthine-guanine phosphoribosyl transferase,and 5-phosphoribosyl-1-pyrophosphate synthetase, allfunction normally in the erythrocytes of the ADA-deficient patients studied. Fig. 7 also shows that a smallamount of label appears as IMP + XMP. Although thesignificance of this observation is not known at thepresent tille, a trace contamination of the [8-'4C]guano-sine by hypoxanthine, xanthine, or their nucleotidescannot be ruled out. It should be noted that ATP levelsremained unchanged during these guanosine incorpora-

tion experiments (data not shown).Metabolism of form ycin A. Fig. 8 shows the incor-

poration of the C-nucleoside analogue of adenosine, for-nycin A, into the nucleotide pools of ADA-deficienterythrocytes (from A. W.). Cells were incubated with1 nllM fornwvcin A at 30'C and nucleotide profiles were

obtained at various time intervals. As shown in Fig. 8,the profile at 0 time does not have a peak in the nu-

cleotide region that absorbs at 295 nm (the Xniax forfornlycin A). Incubation of normal cells for 4 h pro-

duced small peaks in the di- and triphosphate regions.By contrast, in ADA-deficient erythrocytes, throughoutthe 4-h incubation period, substantial quantities of di-

Purine Nucleoside Metabolism in ADA-Deficient Erythrocytes

GTP

1031

010

ot04 l t =0\F 005

ADP

I~k-------------- ----- ----- ---- ----- ----- --- -- -- - --

04 0 04 60

008- ~~~~~Normol-4 h Patient (A.W)-4 h 010

FTP-4:.

D>04-0-05

FD

RETENTON TIME (min)

FIGURE 8 HPLC profiles of erythrocytes incubated with formycin A. Conditions of incubation,extraction, and chromatography are described in Fig. 2 except that formycin A (1 mM) wasused in place of adenosine. Column effluents were monitored at both 254 nm and 295 nm (theXmax for formycin A). "FDP" and "FTP" refer to the di- and triphosphate nucleotides offormycin A.

and triphosphate nucleotides of formycin A were formed.The amount of analogue monophosphate nucleotide thataccumulated was insignificant during this period. Thetotal concentrations of formycin nucleotides reachedabout 4.5 smol/ml of erythrocytes (about three timesthe normal ATP concentration) after 4 h of incubationwhich represents almost complete conversion of the ad-ded formycin A to analogue nucleotides. As with guano-

sine and 2-fluoroadenosine, the ATP levels remainedunchanged during the incubation period. Similar resultswere obtained with the erythrocytes of two other SCID-ADA-deficient patients (C. W. and I. B.) studied.

DISCUSSION

The above results with erythrocytes from SCID-ADA-deficient patients as well as normal cells treated withcoformycin, a tight-binding inhibitor of erythrocyticADA, are consistent with the hypothesis presented pre-

viously that the disposition of adenosine in human eryth-rocytes is the result of the relative activities and Mi-chaelis constants of the enzymes, ADA and adenosinekinase (7-9, 12). When normal erythrocytes were incu-bated with adenosine, principally the monophosphatenucleotide, IMP, accumulated. Furthermore, only small

quantities of analogue nucleotides were formed fromformycin A, a C-nucleoside analogue of adenosine thatis an excellent substrate for ADA (8, 12). In contrast,with SCID-ADA-deficient erythrocytes or normal cellstreated with coformycin, marked increases in the ade-nine nucleotides were produced on incubation withadenosine (Fig. 4), and when formycin A was used as

precursor, quantities of analogue nucleotides were pro-

duced that exceeded the normal adenine nucleotides byabout two- to threefold (Fig. 8).

Mature human erythrocytes cannot synthesize purinenucleotides de novo but actively salvage purine basesand purine nucleosides to form nucleotides. Naturalpurine bases, as well as several base analogues, can re-

act with 5-phosphoribosyl-1-pyrophosphate and the ap-propriate phosphoribosyl transferase to form directlythe 5'-monophosphate nucleotides. Nucleosides of hy-poxanthine and guanine as well as many related ana-

logues can be phosphorolyzed by purine nucleoside phos-phorylase to form the purine base and ribose-l-phos-phate. The existence of inosine or guanosine kinaseshas not been demonstrated in human erythrocytes-rather the formation of nucleotides from inosine and

guanosine or their analogues is readily explained by

1032 Agarwal et al.

04

the sequential actions of the enzymes, purine nucleosidephosphorylase and hypoxanthine-guanine phosphoribo-syl transferase. On the other hand, adenosine, which isa very poor substrate for purine nucleoside phosphory-lase (24), can be converted directly to AMPby reactionwith adenosine kinase. This enzyme is also capable ofreacting with many adenosine-like analogues. Since theactivity of ADA is about eightfold higher than that ofadenosine kinase, normal human erythrocytes, in thepresence of high concentrations of adenosine, principallydeaminate adenosine to inosine with the subsequentformation and accumulation of IMP by the reactionsnoted above (7, 9). Thus, the results presented abovewith SCID-ADA-deficient erythrocytes and with co-formycin-treated normal erythrocytes effectively dem-onstrate the key role played by ADA in the dispositionof adenosine in the human erythrocyte.

Despite considerable investigation and speculationsince the discovery of the first ADA-deficient patient, asatisfactory explanation of the relationship betweenADA deficiency and the deficit in thymus-derived andbone marrow-derived cell function is not yet available.It seems likely, however, that the inability of ADA-deficient lymphocytes, or perhaps lymphatic stem cells,to deaminate and detoxify adenosine is somehow in-volved. Adenosine has been reported to cause inter-ference with pyrimidine biosynthesis in lymphoid celllines (25, 26) and with RNA, DNA, and protein syn-thesis in phytohemagglutinin-stimulated peripheral lym-phocytes (27). Incubation with adenosine produced anincrease in cyclic AMPlevels in lymphocytes and an in-hibition of lymphocyte-mediated cytolysis (28). As re-ported elsewhere (29), the lymphocytes of an SCID-ADA-deficient individual (patient C. W., this paper)lacked responsiveness to the mitogens phytohemagglu-tinin and concanavalin A. Significantly, addition of calfintestinal or human erythrocytic ADA to cultures ofthis patient's lymphocytes restored their responsivenessto mitogen stimulation, suggesting a causal relationshipbetween the ADA deficiency and the cellular immunedefects.

These studies deal with purine metabolism in theerythrocytes of SCID-ADA-deficient patients, althoughthe major abnormality in such patients appears to in-volve primarily lymphatic cells. ADA-deficiency in theerythrocyte does not appear to affect either the functionor survival of such cells. Therefore, one must exercisecaution in extrapolating directly to the lymphocyteconclusions derived from the study of erythrocytes.However, it should be noted that, although the purinemetabolism of human erythrocytes is very limited, e.g.,they lack the de novo purine biosynthetic pathway anddo not synthesize nucleic acids, they represent an idealcell for the study of many facets of purine salvagemetabolism. Already several important differences be-tween the purine salvage mechanisms of normal human

erythrocytes and those of peripheral lymphocytes havebeen detected in this laboratory. For example, erythro-cytes can synthesize very large quantities of GTP or ofanalogue nucleotides such as 2-fluoroATP, without caus-ing significant alterations in the normal adenine nu-cleotide pools (7, 9). However, when peripheral lym-phocytes are incubated with 2-tiuoroadenosine, 2-fluoro-adenine nucleotides are synthesized rapidly and largelyreplace the normal adenine nucleotides (9). Also, asnoted elsewhere, the activity of ADA in peripherallymphocytes is about 10-fold greater than that of humanerythrocytes, which is further indication of an importantrole of this enzyme in the lymphocyte.

Perhaps the most significant observation made inthese experiments with ADA-deficient erythrocytes(either from SCID-ADA-deficient patients or withnormal erythrocytes treated with coformycin) is thatthese cells, on exposure to adenosine, undergo rapidsurges in the adenine nucleotide concentrations withincreases in the ATP levels of the order of 130 to 150%(see Fig. 4). By contrast, normal ADA-containingerythrocytes convert most of the adenosine to IMPwith only minor increases (about 15%) in the ATPlevels (see Fig. 3). Thus, one might speculate that thecells of SCID-ADA-deficient patients might also besubject to major surges in the adenine nucleotide levelsafter exposure to adenosine, as might occur after apurine-rich meal or after tissue breakdown. Thus, inaddition to the proposed explanations of the cytotoxicaction of adenosine noted above, other possibilities mustbe considered. For example, if effects such as those seenwith erythrocytes also occur in lymphatic cells, theymight have drastic effects on intracellular metabolism.It is well established that many key metabolic functionsare sensitive to the adenylate energy charge (30) andto fluctuations in the adenine nucleotide levels of thecell, e.g., in many tissues the enzyme 5-phosphoribosyl-1-pyrophosphate synthetase is subject to strong allo-steric regulation by these nucleotides (30, 31). Thus, ifmarked elevations in the ATP levels occur, there mightbe an inhibition of this enzyme with consequent dele-terious effects on such vital reactions as pyrimidine nu-cleotide biosynthesis, as well as on the salvage mecha-nisms for purine and pyrimidine bases. Also, it has beenknown for about 20 yr that the key glycolytic enzymephosphofructokinase in various tissues is strongly in-hibited by ATP (32). It is now generally accepted thatallosteric regulation of this enzyme by ATP, as well asby a number of other metabolites, plays a major rolein the control of glycolysis in many tissues (for a recentthorough review see 33). It is intriguing to speculatethat elevated intracellular adenine nucleotides might nothave damaging effects in the resting peripheral lympho-cyte. However, when such cells are subjected to greatmetabolic demands imposed by cellular division ortransformation during the immune response or in re-

Purine Nucleoside Metabolism in ADA-Deficient Erythrocytes 1033

sponse to mitogens, blockade of glycolysis by high levelsof ATP might have protounc errects. For example,during these events there is a marked increase in intra-cellular membranous structures that require the avail-ability of large amounts of fatty acids and glycerophos-phate. Thus, blockade of glycolysis at the phospho-fructokinase reaction might drastically impair membranesynthesis. It also seems likely that similar damagingeffects might be produced on the process of maturationof lymphoid stem cells into mature lymphocytes. Thus,as mentioned previously ADA might offer a logicaltarget enzyme for the design of immunosuppressiveagents (8, 9).

At present we cannot explain the apparently increasedrates of synthesis of ITP found in the erythrocytes oftwo of the four SCID-ADA-deficient patients studiedabove. No distinct IMP kinase activity has been dem-onstrated in human erythrocytes. However, IMP servedas a weak substrate for several guanylate kinase iso-zymes purified from pooled-human erythrocytes but hadunfavorable kinetic characteristics compared with thoseof the natural substrate GMP (34, 35). The Vnax forIMP was 1% or less than that of GMP, and the Mi-chaelis constants (about 1 X 10' M) were about 50-fold greater than for GMP. Furthermore, normal humanerythrocytes have an ITPase capable of the pyrophos-phorolysis of ITP to IMP (36). The metabolic regula-tion and physiological role of this enzyme are stillpoorly understood. Additional investigations will benecessary to determine whether the apparently elevatedrates of ITP accumulation are a common occurrence inSCID-ADA-deficient patients and to determine themechanism, e.g., whether the elevated ITP levels aredue to increased rates of synthesis or to an impairmentof the activity of ITPase. The two patients who dis-played elevated rates of ITP synthesis from inosine hadbeen subjected to transplantation procedures with tem-porary and long-term restoration of immuncompetence.The erythrocytes from one patient (A. W.), were ex-amined on three occasions over a period of 15 mo. Onthe second and third examinations, the rates of ITPaccumulation had decreased markedly.

It will be both of interest and importance to examineother patients with SCID who display normal ADAactivity. Recently, such a patient has been reportedwhose erythrocytes are deficient in purine nucleosidephosphorylase (37). It is tempting to speculate thatdefects in the facilitated nucleoside transport mechanism(17) or in the postulated binding of ADA to the cellmembrane at a site near the nucleoside transporter (11)might be involved in other SCID patients who displaynormal ADA activity in hemolysates. It appears thatthe ammonia liberation procedure for the measurementof ADA activity in intact erythrocytes and hemolysates

might be of particular value in the identification of suchporstulated- defects.

It seems likely that the adenosine analogue, formycinA, will prove of special value in future studies of SCIDcells with defects in adenosine metabolism. This analogueis deaminated about eight times more rapidly thanadenosine by ADA (8, 9) and readily forms the 5'-monophosphate nucleotide by reaction with adenosinekinase. In ADA-competent cells, deamination predomi-nates over phosphorylation and little analogue nucleotideis formed (see reference 7 and Fig. 8 of this paper). Onthe other hand, in ADA-deficient cells, very largeamounts of formycin A nucleotides can accumulate(Fig. 8). Of great potential value is the fact that for-mycin A and its nucleotides fluoresce strongly at 340nm upon excitation with ultraviolet light at 295 nmat pH 7 (38). Therefore, after incubation with for-mycin A, it should be possible to distinguish betweennormal cells and ADA-deficient cells (that contain largeamounts of formycin A nucleotides). In addition to theuse of fluorescence, it may be possible to distinguishbetween normal and ADA-deficient cells by radioautog-raphy after incubation with tritiated formycin A. Sucha technique may prove of clinical value in followingpatients with mixed-cell populations as might occurafter procedures such as blood transfusions or bonemarrow transplantations.

ACKNOWLEDGMENTSThe authors wish to acknowledge Mr. Jeff rey Traubermanand Mr. Todd Savarese for their excellent technical assist-ance and Ms. Sandra Bobick and Mr. Oliver Curme ofBrown University and Ms. Lucy M. Rose of the SouthernResearch Institute for the nucleotide analyses by HPLC.

This work was supported by the following U. S. PublicHealth grants: CA 07340, CA 13943, CA 16673, AM08305,HL 06009, and AL 10343; by RR-32, Clinical ResearchCenters Program, Division of Research Resources, Na-tional Institutes of Health; and by grant L-354, NationalFoundation, March of Dimes.

REFERENCES

1. Giblett, E. R., J. E. Anderson, F. Cohen, B. Pollara,and H. J. Meuwissen. 1972. Adenosine-deaminase de-ficiency in two patients with severely impaired cellularimmunity. Lancet. 2: 1067-1069.

2. Dissing, J., and B. Knudsen. 1972. Adenosine-deaminasedeficiency and combined immunodeficiency syndrome.Lancet. 2: 1316.

3. Ochs, H. D., J. E. Yount, E. R. Giblett, S. H. Chen,C. R. Scott, and R. J. Wedgwood. 1973. Adenosine-deaminase deficiency and severe combined immunode-ficiency syndrome. Lancet. 1: 1393-1394.

4. Meuwissen, H. J., B. Pollara, and R. J. Pickering. 1975.Combined immunodeficiency disease associated withadenosine deaminase deficiency. J. Pediatr. 86: 169-181.

5. Combined Immunodeficiency Disease and Adenosine De-aminase Deficiency: A Molecular Defect. 1975. H. J.Meuwissen, R. J. Pickering, B. Pollara, and I. H.Porter, editors. Academic Press, Inc., New York.

1034 Agarwal et al.

6. Parkman, R., E. W. Gelfand, F. S. Rosen, A. Sanderson,and R. Hirschhorn. 1975. Severe combined immunode-ficiency and adenosine deaminase deficiency. N. Engl. J.Med. 292: 714-719.

7. Parks, R. E., Jr., and P. R. Brown. 1973. Incorporationof nucleosides into the nucleotide pools of human eryth-rocytes. Adenosine and its analogs. Biochemistry. 12:3294-3302.

8. Agarwal, R. P., S. M. Sagar, and R. E. Parks, Jr.1975. Adenosine deaminase from human erythrocytes:purification and effects of adenosine analogs. Biochem.Pharmacol. 24: 693-701.

9. Parks, R. E., Jr., G. W. Crabtree, C. M. Kong, R. P.Agarwal, K. C. Agarwal, and E. M. Scholar. 1975.Incorporation of analog purine nucleosides into theformed elements of human blood: erythrocytes, platelets,and lymphocytes. Ann. N. Y. Acad. Sci. 255: 412-434.

10. Lomax, C. A., and J. F. Henderson. 1973. Adenosineformation and metabolism during adenosine triphosphatecatabolism in Ehrlich ascites tumor cells. Cancer Res.33: 2825-2829.

11. Agarwal, R. P., and R. E. Parks, Jr. 1975. A possibleassociation between the nucleoside transport system ofhuman erythrocytes and adenosine deaminase. Biochem.Pharmacol. 24: 547-550.

12. Agarwal, R. P., N. Burgess, M. Driesman, and R. E.Parks, Jr. 1974. Inhibition of human erythrocytic adeno-sine deaminase by coformycin: studies with the isolatedenzyme and the intact cells. Pharmacologist. 16: 266.(Abstr.)

13. Cha, S., R. P. Agarwal, and R. E. Parks, Jr. 1975.Tight-binding inhibitors. II. Non-steady state natureof inhibition of milk xanthine oxidase by allopurinol andalloxanthine and of human erythrocytic adenosine de-aminase by coformycin. Biochem. Pharmacol. 24: 2187-2197.

14. Sawa, T., Y. Fukagawa, I. Homma, T. Takeuchi, andH. Umezawa. 1967. Mode of inhibition of coformycinon adenosine deaminase. J. Antibiot. (Tokyo) Ser. A.20: 227-231.

15. Brown, P. R. 1970. The rapid separation of nucleo-tides in cell extracts using high-pressure liquid chro-matography. J. Chromatogr. 52: 257-272.

16. Scholar, E. M., P. R. Brown, R. E. Parks, Jr., andP. Calabresi. 1973. Nucleotide profiles of the formedelements of human blood determined by high-pressureliquid chromatography. Blood. 41: 927-936.

17. Paterson, A. R. P., and J. M. Oliver. 1971. Nucleotidetransport. II. Inhibition by p-nitrobenzylthioguanosineand related compounds. Can. J. Biochem. 49: 271-274.

17a. Keightley, R., A. R. Lawton, E. J. Yunis, and M. D.Cooper. 1976. Successful fetal liver transplantation in achild with severe combined immunodeficiency. Lancet.In press.

18. Hirschhorn, R., N. Beratis, F. S. Rosen, R. Parkman,R. Stern, and S. Polmar. 1975. Adenosine-deaminasedeficiency in a child diagnosed prenatally. Lancet. 1:73-75.

19. Seligson, D., and H. Seligson. 1951. A microdiffusionmethod for the determination of nitrogen liberated asammonia. J. Lab. Clin. Med. 38: 324-330.

20. Chaney, A. L., and E. P. Marbach. 1962. Modified re-agents for determination of urea and ammonia. Clin.Chem. 8: 130-132.

21. Crabtree, G. W., and J. F. Henderson. 1971. Rate-limiting steps in the interconversion of purine ribo-nucleotides in Ehrlich ascites tumor cells in vitro. CancerRes. 31: 985-991.

22. Chen, S-H., C. R. Scott, and E. R. Giblett. 1974. Adeno-sine deaminase: demonstration of a "silent" gene asso-ciated with combined immunodeficiency disease. Am. J.Hum. Genet. 26: 103-107.

23. Schrader, J., R. M. Berne, and R. Rubio. 1972. Theuptake and metabolism of adenosine by human erythro-cyte ghosts. Am. J. Physiol. 223: 159-166.

24. Zimmerman, T. P., N. B. Gersten, A. F. Ross, and R.P. Miech. 1971. Adenine as substrate for purine nucleo-side phosphorylase. Can. J. Biochem. 49: 1050-1054.

25. Aronow, L. 1961. Reversal of adenine toxicity by py-rimidine nucleosides. Biochim. Biophys. Acta. 47: 184-185.

26. Green, H., and T-S. Chan. 1973. Pyrimidine starvationinduced by adenosine in fibroblasts and lymphoid cells:role of adenosine deaminase. Science (Wash. D. C.).182: 836-837.

27. Hirschhorn, R., J. Grossman, and G. Weissman. 1970.Effect of cyclic 3',5'-adenosine monophosphate and the-ophylline on lymphocyte transformation. Proc. Soc. Exp.Biol. Med. 133: 1361-1365.

28. Wolberg, G., T. P. Zimmerman, K. Hiemstra, M. Win-ston, and L-C. Chu. 1975. Adenosine inhibition of lym-phocyte-mediated cytolysis: possible role of cyclic adeno-sine monophosphate. Science (Wash. D. C.). 187: 957-959.

29. Polmar, S. H., E. M. Wetzlar, R. C. Stern, and R.Hirschhorn. 1975. Restoration of in-vitro lymphocyte re-sponses with exogenous adenosine deaminase in a patientwith severe combined immunodeficiency. Lancet. 2: 743-746.

30. Atkinson, D. E. 1969. Regulation of enzyme function.Annu. Rev. Microbiol. 23: 47-68.

31. Switzer, R. L. 1974. Phosphoribosylpyrophosphate syn-thetase and related pyrophosphokinases. In The En-zymes. P. D. Boyer, editor. Academic Press, Inc., NewYork. 1o: 607-629.

32. Lardy, H. A., and R. E. Parks, Jr. 1956. Influence ofATP concentration on rates of some phosphorylationreactions. In Enzymes: Units of Biological Structureand Function. 0. H. Gaebler, editor. Academic Press,Inc., New York. 584-587.

33. Ramaiah, A. 1974. Pasteur effect and phosphofructo-kinase. Curr. Top. Cell. Regul. 8: 297-345.

34. Agarwal, R. P., E. M. Scholar, K. C. Agarwal, andR. E. Parks, Jr. 1971. Identification and isolation on alarge scale of guanylate kinase from human erythro-cytes: effects of monophosphate nucleotides of purineanalogs. Biochem. Pharmacol. 20: 1341-1354.

35. Agarwal, K. C., and R. E. Parks, Jr. 1972. Adenosinetriphosphate-guanosine 5'-phosphate phosphotransferase.IV. Isozymes in human erythrocytes and sarcoma 180ascites cells. Mol. Pharmacol. 8: 128-138.

36. Vanderheiden, B. S. 1965. Phosphate esters of humanerythrocytes. IV. Sedoheptulose-1,7-diphosphate, octu-lose-1,8-diphosphate, inosine triphosphate and uridinediphosphate. Biochem. Biophys. Res. Commun. 21: 265-270.

37. Giblett, E. R., A. J. Ammann, D. W. Wara, R. Sand-man, and L. K. Diamond. 1975. Nucleoside-phosphorylasedeficiency in a child with severely defective T-cell im-munity and normal B-cell immunity. Lancet. 1: 1010-1013.

38. Ward, D. C., and E. Reich. 1969. Fluorescence studiesof nucleosides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, andtheir derivatives. J. Biol. Chem. 244: 1228-1237.

Purine Nucleoside Metabolism in ADA-Deficient Erythrocytes 1035