Laboratory, Guy's Tower, London Bridge St., London,. SEI 9RT, U.K.; and %%Westminster Children's Hospital,. Westminster, London, SWI, U.K.. (Received 4 ...
Bioscience Reports I, 933-944 (1981) Printed in Great Britain
933
D e o x y a d e n o s i n e m e t a b o l i s m in t h e e r y t h r o c y t e s of children with severe, combined i m m u n o d e f i c i e n c y D. PERRETT, ~ A. SAHOTA, ~ H. A. SIMMONDS,% and K. HUGH-3ONES%% *Medical Unit, St. Bartholomew's Hospital, London, ECIA 7BE, U.K.; **University of Aston, Birmingham, U.K.; %Purine Laboratory, Guy's Tower, London Bridge St., London, SEI 9RT, U.K.; and %%Westminster Children's Hospital, Westminster, London, SWI, U.K. (Received 4 December 1981)
1. D e o x y a d e n o s i n e metabolism was compared in i n t a c t e r y t h r o c y t e s f r o m two children with severe combined immunodeficiency: one had normal adenosine deaminase (ADA; EC 3.5.Lt.4) levels and the other, a h o m o z y g o t e for ADA d e f i c i e n c y , had (following a bone-marrow graft) 15% of normal activity in lysed erythrocytes. 2. In contrast to previous studies with adenosine, deamination was the principal route of deoxyadenosine metabolism, under all incubation conditions, in intact e r y t h r o c y t e s from both patients and control subjects. 3. When the same studies were performed together with t he ADA inhibitor EHNA ( e r y t h r o - 9 [ 2 - h y d r o x y 3 - n o n y l ] a d e n i n e ) , f u r t h e r d i f f e r e n c e s b e t w e e n the m e t a b o l i s m of deoxyadenosine and adenosine became apparent: (i) deamination of deoxyadenosine was not c o m p l e t e l y inhibited by 5 pM EHNA even at the lowest s u b s t r a t e c o n c e n t r a t i o n s ; and (ii) significant deoxyn u c l e o t i d e formation occurred only at unphysiological phosphate levels (18 mM Pi ) and most of the substrate remained unmetabolized. 4. In t h e s e in v i t r o studies at high phosphate concentrations deoxyadenosine was incorporated into the d e o x y n u c l e o t i d e s dAMP:dADP:dATP in the same ratio (10:l.0:0.1) as had been found in the ADA-deficient e r y t h r o c y t e s prior to bone-marrow graft. These studies de m ons t r at e that under physiological conditions deoxyadenosineunlike a d e n o s i n e is deaminated~ not phosphorylated, and is thus dependent on intact ADA activity for its normal metabolism. The f a c t t h a t e v e n low levels of ADA (as assessed by lysate a c t i vi t y) are adequate in intact ceils to perform the important detoxifying function of this enzyme may e x p l a i n t h e e x i s t e n c e of immunocompetent children deficient in e r y t h r o c y t e ADA. Intact cells may thus give a b e t t e r indication of prognosis in suspected cases of ADA deficiency. 9
The Biochemical Society
934
PERRETT
ET AL.
Adenosine deaminase (ADA; EC 3.5.4.4.) deficiency is found in less than 20% of cases of inherited immunodeficiency and is invariably f a t a l unless bone-marrow or exchange transfusion can be e f f e c t e d (1,2). However, three subjects have been described with e r y t h r o c y t e ADA d e f i c i e n c y who are not immunodeficient (2,3). Erythrocyte nucleotide levels in such cases are normal (3), whereas grossly raised dATP ( 3 - 5 ) and also dADP ( 6- 8) and dAMP (9) levels have been f o u n d in t h e e r y t h r o c y t e s of ADA-deficient children with severe, c o m b i n e d i m m u n o d e f i c i e n c y (SCID). The disappearance of these e r y t h r o c y t e deoxyadenosine nucleotides a f t e r successful marrow graft, and the improvement in immune function, is in accord with the in v i t r o s t u d i e s d e m o n s t r a t i n g t h e e x q u i s i t e sensitivity of mitogenstimulated l y m p h o c y t e s to i n h i b i t i o n by d e o x y a d e n o s i n e ( 1 0 ) . However, while adenosine metabolism has been studied extensively in both normal (11-15) and ADA-deficient (13,15) e r y t h r o c y t e s , relatively little information exists concerning the metabolism of deoxyadenosine, even in normal e r y t h r o c y t e s (12). This paper reports studies of deoxyadenosine metabolism in normal e r y t h r o c y t e s compared with e r y t h r o c y t e s from two unrelated SCID children: one with normal e r y t h r o c y t e ADA levels, and one who was homozygous for ADA deficiency and had had a bone-marrow graft (9).
Experimental Procedures Patients
Two children with SClD were studied: 1. The f i r s t p a t i e n t (LS) was a 12-month-old female child identified as ADA-deficient at 5 months immediately prior to successful g r af t with bone marrow from an unrelated donor (9). The child subsequently received periodic transfusions with normal red cells, over the 10 weeks prior to our investigations. At the time of the study, e r y t h r o c y t e lysate ADA a c t i v i t y was approximately 15% of the normal mean (Table 1). Table i. Erythrocyte lysate adenosine deaminase activity and erythrocyte nucleotide levels in SCID patients (LS, SM) compared with the ranges for healthy controls (numbers given in parentheses) from the local population by the methods described LS I = pre-treatment; LS 2 = 7 months after marrow graft and I0 weeks after last red blood cell transfusion.
Subject
Lysate ADA activity nmol/mg Hb/h
Nucleotide ATP
concentrations
ADP
AMP
(nmol/l packed RBC) dATP
dADP
dAMP
750
116
15
LS 1
0.6
760
130
30
LS 2
11.4
1260
131
i0
not detectable
SM
104.1
1526
137
13
not detectable
114•
10• (n=9)
not detectable
Controls
69.7• (n = 20)
1278•
DEOXYADENOSINE
METABOLISM
935
2. The second was a 6-month-old male (SM) with SCID but normal ADA in erythrocyte lysates activity. The controls (VR and AS) were healthy adult laboratory personnel. Materials
P u r i n e bases~ nucleosides, and nucleotides were obtained from Sigma Limited. P o t a s s i u m dihydrogen phosphate ( A r i s t a r ) and potassium c h l o r i d e ( A n a l a r ) were obtained from BDH, Poole. [8-1~C]deoxya d e n o s i n e ( 3 5 - 4 5 IJCi/IJmol) was from New England Nuclear, and [8-1~C]adenosine and [8-1~C]inosine (both 55-60 lJCi/lJmol) were from the R a d i o c h e m i c a l C e n t r e , A m e r s h a m . Erythro-9-(2-hydroxy-3-nonyl) a d e n i n e ( E H N A ) was a gift Irom Dr. Wood (NIH, Bethesda, MD, U.S.A.). Earle's Medium containing 1 or 18 m m o l / l phosphate (pH 7.4) but without b i c a r b o n a t e was p r e p a r e d in the l a b o r a t o r y . Separation of red cells
F r e s h blood was rapidly c e n t r i f u g e d and the plasma and bully coat r e m o v e d and all studies c o m m e n c e d within 1 h of v e n i p u n c t u r e in order to minimize ATP breakdown (16). For the incubation studies, the cells were washed once with 1 mM Pi medium and the packed-cell v o l u m e was d e t e r m i n e d . Red-cell nucleotides were e x t r a c t e d with t r i c h l o r o a c e t i c acid (TCA) according to Dean et al. (16). Whole-cell studies
Red cells were incubated with [8-1~C]nucleosides (5-8 tJmol/l final c o n c e n t r a t i o n ) with and without EHNA (5 IJmol/l) according to the m e t h o d of D e a n and P e r r e t t ( 1 7 ) u n d e r t w o d i f f e r e n t sets of conditions: (i) physiological, i.e., 1 m m o l / l Pi b u f f e r for 5 rain; and (ii) at 18 m m o l / l Pi b u f f e r for 40 rain to s t i m u l a t e phosphoribosylp y r o p h o s p h a t e ( P P - r i b o s e - P ) production. Incubations were t e r m i n a t e d by t h e a d d i t i o n of 20% TCA, and the TCA was e x t r a c t e d with water-saturated ether. The n e u t r a l i z e d samples were stored at - 2 0 ~ until analysed. Enzyme activities
E n z y m e a c t i v i t i e s in e r y t h r o c y t e lysates were d e t e r m i n e d using r a d i o c h e m i c a l methods. The s u b s t r a t e (0.1 m m o l / l adenosine for ADA or 1.5 m m o l / l inosine for PNP) was i n c u b a t e d with fresh l y s a t e for 15 rain in 50 m m o l / l phosphate b u f f e r (pH 7.4) at 37~ The incubation was t e r m i n a t e d by heating for 2 rain. The lysate c o n c e n t r a t i o n was such t h a t 5 - 1 0 % of the inosine and 10-20% of the adenosine were utilized. All m e a s u r e m e n t s were p e r f o r m e d on fresh cell lysate, since on storage at -20~ ADA a c t i v i t y d e c r e a s e s rapidly (15). Analytical methods
P u r i n e bases and nucleosides were separated by high-voltage electrophoresis on thin-layer plates using the method of Simmonds (9). The radioactive bands were scraped into scintillation vials and counted in a toluene-based scintillant.
936
PERRETT
ET AL.
R e d - c e l l n u c l e o t i d e s were quantified by high-performance liquid chromatography (HPLC) using a 5- x 100-mm column of APS Hypersil (18), A linear phosphate gradient at a flow rate of 1 ml/min was used to elute the nucleotides which were d e t e c t e d using a Cecil CE 212 variable-wavelength monitor, Nucleotides were identified by their e l u t i o n t i m e s under d i f f e r i n g c h r o m a t o g r a p h i c conditions and by co-chromatography. For r a d i o a c t i v e sample% 0.5-ml fractions of column eluant were collected and the radioactivity was measured using a gelling scintillant.
Results Enzyme activities and erythrocyte nucleotide levels
Table 1 indicates that the lysed e r y t h r o c y t e s from LS at the time of the study contained approximately 15% of the control mean ADA activ ity while the level for SM was within the normal range. It also compares nucleotide levels at the time of the study (LS) with those prior to marrow graft (LSI). It is evident that despite the original p r e s e n c e of c o m p a r a b l e amounts of deoxyadenosine and adenosine nucleotides (resulting in low adenosine nucleotide levels)~ e r y t h r o c y t e nucleotide levels at the time of the study were well within the control range~ as were those for the SCID child (SM) with normal ADA activity, Fig. 1 shows the separation of standards by HPLC. Figs, 2a and 2b compare the red-cell nucleotide profile of patient LS prior to transplantation with that of SM~ the SCID child with normal ADA levels. Abnormal peaks eluting a f t e r 3.0~ 7.0~ and 11.0 minutes in LS (Fig, 2b) were identified as dAMP~ dADP~ and dATP respectively. The profile in SM was normal, Simultaneous nucleotide profiles and radioactivity measurement in column fractions from e r y t h r o c y t e s aft er incubation with labelled substrate confirmed that significant deoxyadenosine nucleotide formation occurred only in the presence of EHNA and high phosphate ( l g mM Pi)~ with the counts principally in dATP. Metabolic studies
Studies of the metabolism of [8-t#C]deoxyadenosine in intact cells at physiological phosphate levels ( l mM Pi ) and short incubation times in the ADA-deficient child (LS) and control e r y t h r o c y t e s are compared in Table 2. At three different deoxyadenosine concentrations there w a s : n e g l i g i b l e synthesis of deoxyadenosine or adenosine nucleotides, D e o x y a d e n o s i n e was m e t a b o l i z e d p r i n c i p a l l y by d e a m i n a t i o n to h y p o x a n t h i n e e v e n in the ADA-deficient cells~ which at the time possessed low e r y t h r o c y t e Iysate activity (
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942
PERRETT
ET AL.
In the present study the ability of normal erythrocytes to form deoxynucleotides in vitro, using conditions simulating ADA deficiency (5 tJM EHNA), confirms that a kinase does exist in the erythrocyte for the phosphorylation of deoxyadenosine (12). Unlike adenosine~ however ( l l - 1 5 ) , deoxyadenosine is phosphorylated only when the a l t e r n a t i v e c a t a b o l i c p a t h w a y is blockeG and then only at high~ unphysiological phosphate levels (lg mM Pi), which must call into question the in vivo significance of these observations. At physiol o g i c a l levels of s u b s t r a t e and phosphate, deoxyadenosine~ unlike a d e n o s i n e , which is primarily phosphorylated under these conditions ( / 4 ) , is almost completely deaminated (>90%). In this in vitro study, EHNA at 5 IJmol, although a potent inhibitor ol adenosine deamination, did not completely inhibit the deamination of deoxyadenosine by the intact erythrocyte. Furthermor% 20-60% of the deoxyadenosine remained unmetabolized. This was again unlike adenosine, which was almost completely metabolized under the same conditions (14~15). These results support the suggestion that there may be two independent mechanisms 5or deamination of deoxyadenosine in the human red cell (19) and require 5urther investigation. The ability of the intact erythrocytes (aSter marrow transplant) from the child with inherited ADA deficiency to completely deaminate deoxyadenosine9 even at extremely high substrate levels~ a n d despite the low ADA activity as measured in haemolysed erythrocyte extracts~ is noteworthy. It was also found in similar studies using adenosine and consequently may provide an explanation for the existence of subjects w i t h low to u n d e t e c t a b l e e r y t h r o c y t e ADA levels who are not immunodeficient but who do have low ADA lysate activity in other cell types (1-3). Studies using intact rather than lysed cells may thus provide a better guide to ADA competence~ and hence prognosis, in children with suspected ADA deficiency. Several reports originally suggested that ADA-deficient erythrocytes c o n t a i n e d raised ATP levels (1,20). Other workers reported the presence of dATP (3-5) or dATP and also dADP (6-8) in ADAd e f i c i e n t e r y t h r o c y t e s ~ t o g e t h e r with a n o r m a l c o m p l e m e n t of a d e n o s i n e nucleotides. In this study an improved HPLC separation system has confirmed that dATP, dADP~ and dAMP are present~ not only as previously noted in ADA-deficient erythrocytes (9)~ but also in these in vitro studies in normal ceils at 18 mM Pi using EHNA to simulate ADA deficiency. Their ratio 05 approximately 10"l.0"0.1 is likewise identical with that of the adenosine nucleotides, and indicates that these deoxyadenoxine nucleotides are also in rapid equilibrium and maintained at a high ratio of the triphosphate to the other t w o presumably by the same non-specific mono- and diphosphate kinases
(9). No difference from normal was noted in the metabolism of either adenosine or deoxyadenosine by the erythrocytes from the SCID child with normal ADA activity~ which indicates that the activities o5 the r e s p e c t i v e a d e n o s i n e and a d e n y l a t e kinases were not impaired. E r y t h r o c y t e n u c l e o t i d e levels were also within the normal ranger c o n I i r m i n g the a b s e n c e of any d e t e c t a b l e abnormality of purine metabolism from these studies in the intact erythrocytes of a SCID child who was not ADA-deIicient. These in vitro studies have thus demonstrated the formation of all three deoxyadenosine nucleotides by
DEOXYADENOSINE METABOLISM
943
the intact erythrocyte - but only when ADA was inhibited or absent, and then only at high, unphysiological, phosphate levels. By contrast adenosine was generally phosphorylated, even at physiological phosphate concentrations. It would therefore appear that the main physiological f u n c t i o n of adenosine deaminase (14) lies in the deamination of deoxyadenosine and that the enzyme should more appropriately be called deoxyadenosine deaminase, at least in the human erythrocyte.
References I. Enzyme Defects and Immune Dysfunction. (1979) Ciba Symposium 68 (new series), Excerpta Medica, Amsterdam. 2. Hirschhorn R (1979) in Inborn Errors of Specific Immunity, (Pollard B, Pickering RJ, Meuwissen HJ & Porter IH, eds), pp 5-15, Academic Press, New York. 3. Cohen A, Hirschhorn R, Horowitz SD, Rubenstein A, Polmar SH, Hong R & Martin DW Jr (1978) Deoxyadenosine triphosphate as a potentially toxic metabolite in adenosine deaminase deficiency. Proe. Natl. Acad. Sei. U.S.A. 75, 472-476. 4. Hirschhorn R & Roegner V (1980) Plasma deoxyadenosine, adenosine and erythrocyte deoxy-ATP are elevated at birth in an adenosine deaminase-deficient child. J. Clin. Invest. 65, 768-771. 5. Rich KC, Richman CM, Mejias E & Daddona P (1980) Immunoreconstitution by peripheral blood leukocytes in adenosine deaminase-deficient severe combined immunodeficiency. J. Clin. Invest. 66, 389-395. 6. Coleman MS, Donofrio J, Hutton JJ, Hahn L, Daoud A, Lampkin B & Dyminski J (1978) Identification and quantitation of adenine deoxynucleotides in erythrocytes of a patient with adenosine deaminase deficiency and severe combined immunodeficiency. J. Biol. Chem. 253, 1619-1626. 7. Chen S-H, Ochs HD & Scott CR (1978) Adenosine deaminase deficiency. Disappearance of adenine deoxynucleotides from a patient's erythrocytes after successful marrow transplantation. J. Clin. Invest. 62, 1386-1389. 8. Ziegler JB, Lee CH, Van der Weyden MB, Bagnara AS & Beveridge J (1980) Severe combined immunodeficiency and adenosine deaminase deficiency: failure of enzyme replacement therapy. Arch. Dis. Child 55, 452-457. 9. Simmonds HA~ Sahota A~ Potter CF, Perrett D, Hugh-Jones K & Watson JG (1979) Purine metabolism in adenosine deaminase deficiency. In Enzyme Defects and Immune Dysfunction, Ciba Symposium 68 (new series)~ Exeerpta Medica, pp 255-262, Amsterdam. i0. Simmonds HA, Panayi GS & Corrigall V (1978) A role for purine metabolism in the immune response: adenosine deaminase activity and deoxyadenosine Catabolism. Lancet i, 60-63. ii. Meyskens FL & Williams HE (1971) Adenosine metabolism in human erythrocytes. Biochim. Biophys. Acta 240, 170-179.
9#4
PERRETT
ET AL.
12. Snyder FF & Henderson JF (1973) Alternative routes of deoxyadenosine and adenosine metabolism. J. Biol. Chem. 248, 5899-5904. 13. Agarwal RP, Crabtree GW, Parks RE Jr, Nelson JA, Keightley R, Parkman R~ Rosen FS, Stern RC & Polman SH (1976) Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency. J. Clin. Invest. 57, 1025-1035. 14. Perrett D & Dean BM (1977) The function of adenosine deaminase in the human erythrocyte. Biochem. Biophys. Res. Commun. 77, 374-378. 15. Sahota A, Simmonds HA, Potter CF, Watson JG, Hugh-Jones K & Perrett D (1979) Abnormal purine metabolism in adenosine deaminase deficiency. Correction following marrow transplantation. In Purine Metabolism in Man, IliA, (Rapado A, De Bruyn C & Watts RWE, eds), pp 397-403, Plenum Press, New York. 16. Dean BM~ Perrett D & Sensi M (1978) Changes in nucleotide concentrations in the erythrocytes of man, rabbit and rat during short-term storage. Biochem. Biophys. Res. Commun. 80~ 147-154. 17. Dean BM & Perrett D (1976) Studies on adenine and adenosine metabolism by intact human erythrocytes using high performance liquid chromatography. Biochim. Biophys. Acta 4379 1-15. 18. Perrett D (1979) Application of high performace liquid chromatography (HPLC) to biochemical anslysis. In Techniques in Metabolic Research, (Kornberg HL, Metcalfe JC, Northcote DH, Pogson CI & Tipton KF, eds), pp 1-22, B 215~ Elsevier, North Holland. 19' Schrader WP, Pollara B & Meuwissen HJ (1978) Characterisation of the residual adenosine deaminating activity in the spleen of a patient with combined immunodeficiency disease and adenosine deaminase deficiency (enzyme deficiency). Proc. Natl. Acad. Sci. U.S.A. 75, 446-450. 20. Schmalstieg FC~ Mills GC~ Nelson JA~ May LT~ Goldman AS & Goldblum RM (1978) Limited effect of erythrocyte and plasma infusions in adenosine deaminase deficiency. J. Pediatrics 93~ 597-603.
