271
Clinica
Chimica
Acta,
74 (1977)
0 ElsevierlNorth-Holland
271-279
Biomedical
Press
CCA 8339
A PATIENT WITH PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY: ENZYMOLOGICAL AND METABOLIC ASPECTS
L.H. SIEGENBEEK VAN HEUKELOM &*, J.W.N. AKKERMAN a, G.E.J. STAAL C.H.M.M. DE BRUYN b, J.W. STOOP c, B.J.M. ZEGERS c, P.K. DE BREE c and S.K. WADMAN c
a,
a Unif of Medical Enzymology, University Hospital, Utrecht (The Netherlands), b Department of Human Geneiics, Faculty of Medicine, University of Nijmegen (The Netherlands) and c University Children’s Hospital “Het Wilhelmina Kinderziekenhuis”, Utrecht (The Netherlands) (Received
September
lOth,
1976)
Summary 1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. Incubation of intact NP-deficient red cells with [14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. Incubation with [ 14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. Incubation with [ 14C]hypoxanthine and [ 14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples. -. Introduction In 1972 Giblett deaminase (ADA) * To
whom
et al. [l] described a deficiency of the enzyme adenosine in a patient with a severe combined immunodeficiency
correspondence
should
be addressed.
(SCID). In 1975 Giblett. et al. 121 reported the deficiency ol’ anothrlr t’nzymr. nucleoside phosphorylase (NP), of purine metabolism in the erythrocytes of a child with a severe selective cellular irn~~lu~ledeficiency while the ADA activity was normal. Recently [3] we also were able to detect a patient with a NP deficiency in the erythrocytes and lymphocytes in association with a severely defective T-cell immunity. The parents and a healthy brother of the propositus have NP activity in the heterozygote range [3]. Two girls of this family died at early age: one of lymphosar~oma, the other one of a graft-versus-host reaction after bloodtransfusion [ 41. In this paper we describe enzymological abnormalities in the patient’s red cells. In addition the effect of NP deficiency on the overall purine metabolism has been studied by urinary and serum purine analysis. Clinical and immunological details will be reported elsewhere (Stoop et al., in prep~ation). Materials and methods Enzyme assays in ery throcyte lysates All enzyme activity measurements of purine metabolism were carried out with radioactive substrates (Radiochemi~al Centre, Amersham, England). Lysates from 3 times washed erythrocytes were used as enzyme source. HGPRT, APRT, ADA and NP were determined according to De Bruyn [5] (for abbreviations see Table I). Adenosine kinase. Endvolume 100 ~1; concentrations of the compounds 0.005 mM 8-[‘4C]adenosinc; 1.25 mM ATP, 0.25 M sodium acetate, pH 5.7; 0.5 mM MgC12, 100 yl lysate protein. High voltage ~lectrophoresis of 5 1-11on Whatman 3 MM paper (60 V/cm; 40 min) in 50 mM sodium citrate buffer, pH 3.5. Glucose-6-phosphate dehydrogenase, glutathione reduclase and ATP were determined according to Beutler [ 61.
Erythrocytes were washed three times in saline and finally resuspended in Ca-free tyrode buffer (pH 7.3) containing 137 mM NaCl; 11.9 mM NaHCO,; 1 g/l glucose and 2 g/l bovine albumin in a final volume of about 5000 000 cell/ ~1. These cells were incubated with 50 PM [U-14Cladenosine (spec. act. 557 mCi~m~no1) at 37” C for 120 min. At different incubation periods 100 ,ul samples were withdrawn and immcdiately mixed with 200 ~~11 EDTA-ethanol (9 volumes of 96% ethanol to 1 volume 0.1 M EDTA-Na2, pH 7.6, freshly prepared, 0°C). Samples were mixed for 10 min at 0°C and centrifuged (2 min, 10 000 X g, room temperature). Adenosine and related compounds were separated by electrophoresis at 60 V/cm according to Holmsen and Weiss [ 7 1. Incubation experiments with [ ‘“C]hypoxanthine, j’JC]hypoxanthine, [“Cladenine and [ “C]inosine were performed as described elsewhere [ 51. PRPP concentration TI?e determination
and generation of PRPP levels in erythrocytes
was based upon the release
273
of 14C02 from carboxylic[‘4C]orotic acid, catalysed by purified orotate phospho~bosyl transferase and orotidylic acid decarboxylase. The PRPP synthetase assay was based on the same principle; PRPP formed from ribose-5-P (1 mM) and ATP (1 mM) under optimal conditions (10 mM MgClz; 35 mM Tris/HCl, pH 7.4) was measured by trapping the 14C02 released from labelled erotic acid. The details of this method are to be reported elsewhere (Tax et al., in preparation).
Sepurut~~~ u~udenosi~e, inosine and h~poxu~thine An aliquot (20 ~1) of the extract * was applied together with 2 ~1 of a solution containing adenosine, inosine and hypoxanthine, each 2 mg/ml, on a 10 X 10 cm thin-layer chromatogram (DC alufolien Merck AC, Darmstadt, No. 5552). The two-dimensional chromatogram was developed in isopropanol/ammonia (8 : 2, v/v) respectively butanol/acetic acid/water (8 : 2 : 2, v/v), twice in each direction. After drying the chromatograms were sprayed with mercuric acetate (0.2 g in 100 ml ethanol containing 5 drops of glacial acetic acid) and dried again. Finally a spray of diphenyl carbazone (0.1 g in 200 ml of ethanol 96%) was applied and the ~hromato~am was heated at 120°C until complete decoloration of the background. Purines and pyrimidines present as blue to pink spots. The spots of adenosine, inosine and hypoxanthine were scratched off. The cellulose powder was suspended in 10 ml of counting fluid. Counting of the samples was performed with a Packard Tri-carb 2425 liquid scintillation counter.
Urinary purines were analyzed by conventional automated cation exchange column chromatography with a Technicon TSM 1 Auto Analyzer, combined with a Schoeffel model SF 770 UV spectroflow monitor, an Infotronics CRS 309 integrator, and a printer [8]. The column (69 cm, diameter 0.5 cm) was packed with Technicon spherical resin Chromosorb C3 (12 pm). Elution was performed with three 0.067 M trisodium-citrate buffers adjusted to pH 3.25, 4.00 and 6.50 with 6 M HCl, for 60,45 and 104 min, respectively. The urine (3-10 ~1) was applied directly on the cartridge, which was equilibrated with buffer pH 3.25. The pump rate was 27 ml/h. Serum was deproteinized with sulfosalicylic acid, 40 mg per ml; 0.1 ml of the supernatant was applied on the cartridge. With the here described elution program uric acid travels in the same position as xanthosine and therefore it was determined enzymatically with uricase. Results
Incubation of intact red cells with adenosine Fig. 1 shows the results of the incubation
of normal erythrocytes with 50 PM [ 14C]adenosine. After about 15 min 70% of the radioactivity was found in the electrophoretic fraction of inosine + hypoxanthine (Ino + Hx) whereas about 30% was present in the adenosine nucleotides (mainly in ATP). There* The extract
was prepared
as described
in the section
Metabolism
of adenosine.
total
normdl radsoact,vty
0 Hyx .
loo-
%
/:no
AMP,
ADP,
ATP
d Ado
-A-A-A-L 1-1-------!-,__(-I-
10
20
30
LO
Fig. 1. The uptake of [14Cjadenosine the adenosine nudeotides (*A
50
(50 FM) f&y--a) by normal intact ) and the inosine-hypoxanthine fraction
red cells and its conversion ((j.I.
to
after the percentage of radioactivity in the (Ino + Hx) fraction decreased with time. In contrast, after incubation of the patient’s red cells with 10 PM [14C]adenosine almost all radioactivity was found in the (Ino f Hx) fraction already after 10 min. This remained so during the whole experiment. Almost no labeling of the adenosine nucleotides occurred (Fig. 2). Analysis of the patient’s (Ino + Hx) fractions by thin-layer ~hromato~aphy (see Methods} revealed the total absence of radioactivity in the hypoxanthine spot; all radioactivity was present in the inosine fraction, which was in contrast with the results in the normal red cells. Determination of enzymes of purine interconversion To give a possible explanation for the results found with the erythrocytes patient
total radicactlvity t”cl
0-o-o-o-Q
Fig. 2. The uptake of [14Cladenosine sion to the adenosine nucleotides (*A
-0-o
of
0 Ino .
AMP,
A
Ado
ADP,
ATP
0
1) by NP deficient intact red cells and its conver(50 PM)
Clinica
Chimica
Acta,
74 (1977)
0 ElsevierlNorth-Holland
271-279
Biomedical
Press
CCA 8339
A PATIENT WITH PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY: ENZYMOLOGICAL AND METABOLIC ASPECTS
L.H. SIEGENBEEK VAN HEUKELOM &*, J.W.N. AKKERMAN a, G.E.J. STAAL C.H.M.M. DE BRUYN b, J.W. STOOP c, B.J.M. ZEGERS c, P.K. DE BREE c and S.K. WADMAN c
a,
a Unif of Medical Enzymology, University Hospital, Utrecht (The Netherlands), b Department of Human Geneiics, Faculty of Medicine, University of Nijmegen (The Netherlands) and c University Children’s Hospital “Het Wilhelmina Kinderziekenhuis”, Utrecht (The Netherlands) (Received
September
lOth,
1976)
Summary 1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. Incubation of intact NP-deficient red cells with [14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. Incubation with [ 14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. Incubation with [ 14C]hypoxanthine and [ 14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples. -. Introduction In 1972 Giblett deaminase (ADA) * To
whom
et al. [l] described a deficiency of the enzyme adenosine in a patient with a severe combined immunodeficiency
correspondence
should
be addressed.
(SCID). In 1975 Giblett. et al. 121 reported the deficiency ol’ anothrlr t’nzymr. nucleoside phosphorylase (NP), of purine metabolism in the erythrocytes of a child with a severe selective cellular irn~~lu~ledeficiency while the ADA activity was normal. Recently [3] we also were able to detect a patient with a NP deficiency in the erythrocytes and lymphocytes in association with a severely defective T-cell immunity. The parents and a healthy brother of the propositus have NP activity in the heterozygote range [3]. Two girls of this family died at early age: one of lymphosar~oma, the other one of a graft-versus-host reaction after bloodtransfusion [ 41. In this paper we describe enzymological abnormalities in the patient’s red cells. In addition the effect of NP deficiency on the overall purine metabolism has been studied by urinary and serum purine analysis. Clinical and immunological details will be reported elsewhere (Stoop et al., in prep~ation). Materials and methods Enzyme assays in ery throcyte lysates All enzyme activity measurements of purine metabolism were carried out with radioactive substrates (Radiochemi~al Centre, Amersham, England). Lysates from 3 times washed erythrocytes were used as enzyme source. HGPRT, APRT, ADA and NP were determined according to De Bruyn [5] (for abbreviations see Table I). Adenosine kinase. Endvolume 100 ~1; concentrations of the compounds 0.005 mM 8-[‘4C]adenosinc; 1.25 mM ATP, 0.25 M sodium acetate, pH 5.7; 0.5 mM MgC12, 100 yl lysate protein. High voltage ~lectrophoresis of 5 1-11on Whatman 3 MM paper (60 V/cm; 40 min) in 50 mM sodium citrate buffer, pH 3.5. Glucose-6-phosphate dehydrogenase, glutathione reduclase and ATP were determined according to Beutler [ 61.
Erythrocytes were washed three times in saline and finally resuspended in Ca-free tyrode buffer (pH 7.3) containing 137 mM NaCl; 11.9 mM NaHCO,; 1 g/l glucose and 2 g/l bovine albumin in a final volume of about 5000 000 cell/ ~1. These cells were incubated with 50 PM [U-14Cladenosine (spec. act. 557 mCi~m~no1) at 37” C for 120 min. At different incubation periods 100 ,ul samples were withdrawn and immcdiately mixed with 200 ~~11 EDTA-ethanol (9 volumes of 96% ethanol to 1 volume 0.1 M EDTA-Na2, pH 7.6, freshly prepared, 0°C). Samples were mixed for 10 min at 0°C and centrifuged (2 min, 10 000 X g, room temperature). Adenosine and related compounds were separated by electrophoresis at 60 V/cm according to Holmsen and Weiss [ 7 1. Incubation experiments with [ ‘“C]hypoxanthine, j’JC]hypoxanthine, [“Cladenine and [ “C]inosine were performed as described elsewhere [ 51. PRPP concentration TI?e determination
and generation of PRPP levels in erythrocytes
was based upon the release
273
of 14C02 from carboxylic[‘4C]orotic acid, catalysed by purified orotate phospho~bosyl transferase and orotidylic acid decarboxylase. The PRPP synthetase assay was based on the same principle; PRPP formed from ribose-5-P (1 mM) and ATP (1 mM) under optimal conditions (10 mM MgClz; 35 mM Tris/HCl, pH 7.4) was measured by trapping the 14C02 released from labelled erotic acid. The details of this method are to be reported elsewhere (Tax et al., in preparation).
Sepurut~~~ u~udenosi~e, inosine and h~poxu~thine An aliquot (20 ~1) of the extract * was applied together with 2 ~1 of a solution containing adenosine, inosine and hypoxanthine, each 2 mg/ml, on a 10 X 10 cm thin-layer chromatogram (DC alufolien Merck AC, Darmstadt, No. 5552). The two-dimensional chromatogram was developed in isopropanol/ammonia (8 : 2, v/v) respectively butanol/acetic acid/water (8 : 2 : 2, v/v), twice in each direction. After drying the chromatograms were sprayed with mercuric acetate (0.2 g in 100 ml ethanol containing 5 drops of glacial acetic acid) and dried again. Finally a spray of diphenyl carbazone (0.1 g in 200 ml of ethanol 96%) was applied and the ~hromato~am was heated at 120°C until complete decoloration of the background. Purines and pyrimidines present as blue to pink spots. The spots of adenosine, inosine and hypoxanthine were scratched off. The cellulose powder was suspended in 10 ml of counting fluid. Counting of the samples was performed with a Packard Tri-carb 2425 liquid scintillation counter.
Urinary purines were analyzed by conventional automated cation exchange column chromatography with a Technicon TSM 1 Auto Analyzer, combined with a Schoeffel model SF 770 UV spectroflow monitor, an Infotronics CRS 309 integrator, and a printer [8]. The column (69 cm, diameter 0.5 cm) was packed with Technicon spherical resin Chromosorb C3 (12 pm). Elution was performed with three 0.067 M trisodium-citrate buffers adjusted to pH 3.25, 4.00 and 6.50 with 6 M HCl, for 60,45 and 104 min, respectively. The urine (3-10 ~1) was applied directly on the cartridge, which was equilibrated with buffer pH 3.25. The pump rate was 27 ml/h. Serum was deproteinized with sulfosalicylic acid, 40 mg per ml; 0.1 ml of the supernatant was applied on the cartridge. With the here described elution program uric acid travels in the same position as xanthosine and therefore it was determined enzymatically with uricase. Results
Incubation of intact red cells with adenosine Fig. 1 shows the results of the incubation
of normal erythrocytes with 50 PM [ 14C]adenosine. After about 15 min 70% of the radioactivity was found in the electrophoretic fraction of inosine + hypoxanthine (Ino + Hx) whereas about 30% was present in the adenosine nucleotides (mainly in ATP). There* The extract
was prepared
as described
in the section
Metabolism
of adenosine.
total
normdl radsoact,vty
0 Hyx .
loo-
%
/:no
AMP,
ADP,
ATP
d Ado
-A-A-A-L 1-1-------!-,__(-I-
10
20
30
LO
Fig. 1. The uptake of [14Cjadenosine the adenosine nudeotides (*A
50
(50 FM) f&y--a) by normal intact ) and the inosine-hypoxanthine fraction
red cells and its conversion ((j.I.
to
after the percentage of radioactivity in the (Ino + Hx) fraction decreased with time. In contrast, after incubation of the patient’s red cells with 10 PM [14C]adenosine almost all radioactivity was found in the (Ino f Hx) fraction already after 10 min. This remained so during the whole experiment. Almost no labeling of the adenosine nucleotides occurred (Fig. 2). Analysis of the patient’s (Ino + Hx) fractions by thin-layer ~hromato~aphy (see Methods} revealed the total absence of radioactivity in the hypoxanthine spot; all radioactivity was present in the inosine fraction, which was in contrast with the results in the normal red cells. Determination of enzymes of purine interconversion To give a possible explanation for the results found with the erythrocytes patient
total radicactlvity t”cl
0-o-o-o-Q
Fig. 2. The uptake of [14Cladenosine sion to the adenosine nucleotides (*A
-0-o
of
0 Ino .
AMP,
A
Ado
ADP,
ATP
0
1) by NP deficient intact red cells and its conver(50 PM)
