Lymphocyte ecto-5'-nucleotidase deficiency in agammaglobulinemia.

Fresh peripheral blood lymphocytes from eight patients with congenital agammaglobulinemia demonstrate reduced ecto-5'-nucleotidase activity when compared to the mean activity of normal subjects and patients with other forms of immunoglobulin deficiency. A specific defect of ecto-5'-nucleotidase is further suggested by normal values for lymphocyte ecto-adenosinetriphosphatase and ecto-nonspecific phosphatase. The data provide evidence for an enzyme deficiency in this X-linked, B lymphocyte deficiency syndrome.

Purine nucleoside phosphorylase deficiency: altered kinetic properties of a mutant enzyme.

Erythrocyte purine nucleoside phosphorylase from two brothers had 0.5% of normal activity. It differed from the normal enzyme by a tenfold increase in the Michaelis constant for inosine, an inability of inosine to protect against thermal lability, and a more positive net charge. The altered kinetic properties may account for the milder disease in the patients compared to the previously described cases. The data provide evidence for a structural gene mutation and genetic heterogeneity in the new disease of purine nucleoside phosphorylase deficiency and T cell dysfunction.

Ethanol-induced activation of adenine nucleotide turnover. Evidence for a role of acetate.

Consumption of alcohol causes hyperuricemia by decreasing urate excretion and increasing its production. Our previous studies indicate that ethanol administration increases uric acid production by increasing ATP degradation to uric acid precursors. To test the hypothesis that ethanol-induced increased urate production results from acetate metabolism and enhanced adenosine triphosphate turnover, we gave intravenous sodium acetate, sodium chloride and ethanol (0.1 mmol/kg per min for 1 h) to five normal subjects. Acetate plasma levels increased from 0.04 +/- 0.01 mM (mean +/- SE) to peak values of 0.35 +/- 0.07 mM and to 0.08 +/- 0.01 mM during acetate and ethanol infusions, respectively. Urinary oxypurines increased to 223 +/- 13% and 316 +/- 44% of the base-line values during acetate and ethanol infusions, respectively. Urinary radioactivity from the adenine nucleotide pool labeled with [8-14C] adenine increased to 171 +/- 27% and to 128 +/- 8% of the base-line values after acetate and ethanol infusions. These data indicate that both ethanol and acetate increase purine nucleotide degradation by enhancing the turnover of the adenine nucleotide pool. They support the hypothesis that acetate metabolism contributes to the increased production of urate associated with ethanol intake.

Overproduction of uric acid in hypoxanthine-guanine phosphoribosyltransferase deficiency. Contribution by impaired purine salvage.

The contribution of reduced purine salvage to the hyperuricemia associated with hypoxanthine-guanine phosphoribosyltransferase deficiency was measured by the intravenous administration of tracer doses of [8-(14)C]adenine to nine patients with normal enzyme activity, three patients with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, and six patients with the Lesch-Nyhan syndrome. The mean cumulative excretion of radioactivity 7 d after the adenine administration is 5.6+/-2.4, 12.9+/-0.9, and 22.3+/-4.7% of infused radioactivity for control subjects, partial hypoxanthine-guanine phosphoribosyltransferase-deficient subjects, and Lesch-Nyhan patients, respectively. To assess relative rates of nucleotide degradation in control and hypoxanthine-guanine phosphoribosyltransferase-deficient patients two separate studies were employed. With [8-(14)C]inosine administration, three control subjects excreted 3.7-8.5% and two enzyme-deficient patients excreted 26.5-48.0% of the injected radioactivity in 18 h. The capacity of the nucleotide catabolic pathway to accelerate in response to d-fructose was evaluated in control and enzyme-deficient patients. The normal metabolic response to intravenous fructose is a 7.5+/-4.2-mmol/g creatinine increase in total urinary purines during the 3-h after the infusion. The partial hypoxanthine-guanine phosphoribosyltransferase-deficient subjects and Lesch-Nyhan patients show increases of 18.6+/-10.8 and 17.3+/-11.8 mmol/g creatinine, respectively. Of the observed rise in purine exretion in control subjects, 40% occurs from inosine excretion and 32% occurs from oxypurine excretion. The rise in total purine excretion with Lesch-Nyhan syndrome is almost entirely accounted for by an elevated uric acid excretion. Increases in urine radioactivity after fructose infusion are distributed in those purines that are excreted in elevated quantities.The observations suggest that purine salvage is a major contributor to increased purine excretion and that the purine catabolic pathway responds differently to an increased substrate load in hypoxanthine-guanine phosphoribosyltransferase deficiency. The purine salvage pathway is normally an important mechanism for the reutilization of hypoxanthine in man.

Hyperuricemia in glycogen storage disease type I. Contributions by hypoglycemia and hyperglucagonemia to increased urate production.

Studies were performed to determine whether hypoglycemia or the glucagon response to hypoglycemia increases uric acid production in glycogen storage disease type I (glucose-6-phosphatase deficiency). Three adults with this disease had hyperuricemia (serum urate, 11.3-12.4 mg/dl) and reduced renal clearance of urate (renal urate clearance, 1.1-3.1 ml/min). These abnormalities were improved in one patient by intravenous glucose infusion for 1 mo, suggesting a role for hypoglycemia and its attendant effects on urate metabolism and excretion. A pharmacologic dose of glucagon caused a rise in serum urate from 11.4 to 13.0 mg/dl, a ninefold increase in urinary excretion of oxypurines, a 65% increase in urinary radioactivity derived from radioactively labeled adenine nucleotides, and a 90% increase in urinary uric acid excretion. These changes indicate that intravenous glucagon increases ATP breakdown to its degradation products and thereby stimulates uric acid production. To observe whether physiologic changes in serum glucagon modulate ATP degradation, uric acid production was compared during saline and somatostatin infusions. Serum urate, urinary oxypurine, radioactivity, and uric acid excretion increased during saline infusion as patients became hypoglycemic. Infusion of somatostatin suppressed these increases despite hypoglycemia and decreased the elevated plasma glucagon levels from a mean of 81.3 to 52.2 pg/ml. These data suggest that hypoglycemia can stimulate uric acid synthesis in glucose-6-phosphatase deficiency. Glucagon contributes to this response by activating ATP degradation to uric acid.

Partial purine nucleoside phosphorylase deficiency. Studies of lymphocyte function.

Immune function in two brothers with a deficiency of purine nucleoside phosphorylase was evaluated in vivo and in vitro. Both patients had a history of recurrent infections and profound lymphopenia. Studies of cell-mediated immunity revealed an absence of delayed cutaneous reactivity to a number of antigens, including dinitrochlorobenzene, and significantly reduced lymphocyte proliferative responses to nonspecific mitogens, specific antigen, and allogeneic cells. E-rosetting cells were present but reduced in number (20.0% and 31.5%). Serum immunoglobulin levels, percentages of circulating immunoglobulin-and C3-receptor-bearing B cells, as well as the ability to produce antibody in response to specific antigen in vivo were normal. Moreover, studies of the in vitro induction of specific IgM antibody delineated the presence of T-helper and T-regulator cells. The normal induction of bone marrow precursor T-cell maturation by human thymic epithelium-conditioned medium or thymosin suggested that the initial stages of T-cell generation were intact in these patients. Attempts to reconstitute the in vitro proliferative response with a variety of reagents, including purine nucleoside phosphorylase itself, were unsuccessful. Selective impairment of certain aspects of T-cell function in these patients and a less severe clinical picture than previously described may be explained by the presence of a partial deficiency of nucleoside phosphorylase activity and incomplete block of purine catabolism.

Inactivation of S-adenosylhomocysteine hydrolase during adenine arabinoside therapy.

To assess for possible inhibition of cellular transmethylation during adenine arabinoside (ara-A) therapy, S-adenosylhomocysteine hydrolase activity was analyzed in 10 patients with chronic hepatitis B virus infection. In six patients receiving ara-A, enzyme activity was suppressed to 0-2% of control erythrocyte enzyme activity. This decrease in enzyme activity was evident within 4 h of starting the drug infusion and continued for 7 d after cessation of therapy. S-adenosylhomocysteine hydrolase activity of peripheral mononuclear cells was also measured in two patients receiving ara-A. Suppression to as low as 3.5% of pretreatment levels was found; however, marked fluctuations with partial return of enzyme activity during therapy was also observed in mononuclear cells. Inhibition of an enzyme involved in transmethylation reactions was observed in patients during ara-A therapy. This could contribute to the side effects and antiviral properties of ara-A.

Hereditary xanthinuria. Evidence for enhanced hypoxanthine salvage.

We tested the hypothesis that there is an enhanced rate of hypoxanthine salvage in two siblings with hereditary xanthinuria. We radiolabeled the adenine nucleotide pool with [8-14C]adenine and examined purine nucleotide degradation after intravenous fructose. The cumulative excretion of radioactivity during a 5-d period was 9.7% and 9.1% of infused radioactivity in the enzyme-deficient patients and 6.0 +/- 0.7% (mean +/- SE) in four normal subjects. Fructose infusion increased urinary radioactivity to 7.96 and 9.16 X 10(6) cpm/g creatinine in both patients and to 4.73 +/- 0.69 X 10(6) cpm/g creatinine in controls. The infusion of fructose increased total urinary purine excretion to a mean of 487% from low-normal baseline values in the patients and to 398 +/- 86% in control subjects. In the enzyme-deficient patients, the infusion of fructose elicited an increase of plasma guanosine from undetectable values to 0.7 and 0.9 microM. With adjustments made for intestinal purine loss, these data support the hypothesis that there is enhanced hypoxanthine salvage in hereditary xanthinuria. Degradation of guanine nucleotides to xanthine bypasses the hypoxanthine salvage pathway and may explain the predominance of this urinary purine compound in xanthinuria.

Adenosine triphosphate turnover in humans. Decreased degradation during relative hyperphosphatemia.

The regulation of ATP metabolism by inorganic phosphate (Pi) was examined in five normal volunteers through measurements of ATP degradation during relative Pi depletion and repletion states. Relative Pi depletion was achieved through dietary restriction and phosphate binders, whereas a Pi-repleted state was produced by oral Pi supplementation. ATP was radioactively labeled by the infusion of [8(14)C]adenine. Fructose infusion was used to produce rapid ATP degradation during Pi depletion and repletion states. Baseline measurements indicated a significant decrease of Pi levels during phosphate depletion and no change in serum or urinary purines. Serum values of Pi declined 20 to 26% within 15 min after fructose infusion in all states. Urine measurements of ATP degradation products showed an eightfold increase within 15 min after fructose infusion in both Pi-depleted and -supplemented states. Urinary radioactive ATP degradation products were fourfold higher and urinary purine specific activity was more than threefold higher during Pi depletion as compared with Pi repletion. Our data indicate that there is decreased ATP degradation to purine end products during a relative phosphate repletion state as compared to a relative phosphate depletion state. These data show that ATP metabolism can be altered through manipulation of the relative Pi state in humans.

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

The mutation in a young gouty male with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase has been evaluated. The serum uric acid was 11.8 mg/100 ml, and the urinary uric acid excretion was 1,279 mg/24 h. Erythrocyte hypoxanthine-guanine phosphoribosyltransferase was 34.2 nmol/h/mg, adenine phosphoribosyltransferase was 36.5 nmol/h/mg and phosphoribosylpyrophosphate was 2.6 muM. Hypoxanthine-guanine phosphoribosyltransferase from peripheral leukocytes and cultured diploid skin fibroblasts was within the normal range, but enzyme activity in rectal mucosa was below the normal range. Initial velocity studies of the normal enzyme and the mutant enzyme from erythrocytes with the substrates hypoxanthine, guanine, or phosphoribosylpyrophosphate showed that the Michaelis constants were similar. Product inhibition studies distinguished the mutant enzyme from the normal enzyme. Hyperbolic kinetics with increasing phosphoribosylpyrophosphate were converted to sigmoid kinetics by 0.2 mM GMP with the mutant enzyme but not with the normal enzyme. The mutant erythrocyte hypoxanthine-guanine phosphoribosyltransferase was inactivated normally at 80 degrees C and had a normal half-life in the peripheral circulation. The mol wt of 48,000 was similar to the normal enzyme mol wt of 47,000. With isoelectric focusing, the mutant erythrocyte enzyme had two major peaks with isoelectric pH's of 5.50 and 5.70, in contrast to the isoelectric pH's of 5.76, 5.82, and 6.02 of the normal isozymes. Isoelectric focusing of leukocyte extracts from the patient revealed the presence of the mutant enzyme. Cultured diploid fibroblasts from the propositus appeared to function normally, as shown by the inability to grow in 50-100 muM azaguanine and by the normal incorporation of [14C]hypoxanthine into nucleic acid. In contrast, erythrocytes from the patient displayed abnormal properties, including the increased synthesis of phosphoribosylphyrophosphate and elevated functional activity of orotate phosphoribosyltransferase and orotidylic decarboxylase. These unique kinetic, physical, and functional properties provide support for heterogeneous structural gene mutations in partial deficiencies of hypoxanthine-guanine phosphoribosyltransferase.

Familial hyperuricemia and renal disease.

Information on a familial syndrome of hyperuricemia and renal disease with or without gout was obtained on 33 of 41 blood relatives: Nine had renal disease; abnormalities of the urinary sediments were minimal; serum uric acid levels were elevated in seven and were not measured in two. Hyperuricemia was noted in three additional family members without evidence of renal disease. Goulty arthritis (three patients) did not precede renal disease. One individual had hyperuricosuria. The following erythrocyte purine enzyme levels were normal: adenine phosphoribosyltransferase, hypoxanthine-guanine phosphoribosyltransferase, phosphoribosylpyrophosphate, synthetase, adenosine deaminiase, and purine nucleoside phosphorylase. Renal biopsy specimens showed focal global and segmental sclerosis of glomeruli, occasional hypercellularity, foci of atrophic tubules, chronic interstitial inflammation, and folding and wrinkling of glomerular basement membrane without electron-dense deposits. There were no immunofluorescent abnormalities.

Duchenne muscular dystrophy: normal ATP turnover in cultured cells.

We examined ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients, and there was no difference in ATP synthesis or degradation. Although there was a significant decrease in radioactively labeled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration or ADP metabolism.

ATP degradation products after ischemic exercise: hereditary lack of phosphorylase or carnitine palmityltransferase.

We measured purine degradation products of ATP in plasma after ischemic exercise in eight normal subjects, one patient with myophosphorylase deficiency (McArdle's disease), and one with carnitine palmityltransferase deficiency. Normal subjects increase hypoxanthine and inosine, but not xanthine. Plasma purine levels were elevated above the normal range after ischemic exercise in McArdle's disease and in carnitine palmityltransferase deficiency after fasting. Those changes implied abnormally accelerated ATP degradation in these two myopathies.

Chronic allopurinol and adenine therapy in Duchenne muscular dystrophy: effects on muscle function, nucleotide degradation, and muscle ATP and ADP content.

Prompted by the controversy on the efficacy of allopurinol in Duchenne muscular dystrophy and by our observations of an abnormal adenine nucleotide turnover in this disease, we conducted an 18 month, double-blind clinical trial with allopurinol and adenine in 14 Duchenne boys paired according to age and functional activity. Detailed clinical evaluation was performed trimonthly. Muscle ATP and ADP content was measured before and after 1 year of treatment. The effect of therapy on adenine nucleotide turnover was determined. No significant difference was observed between the treated and placebo groups, but both showed a significant deterioration (p less than 0.05) in most clinical parameters. Muscle ATP was reduced in Duchenne dystrophy (p less than 0.02) but did not change with therapy, and no correction of the abnormal adenine nucleotide degradation was observed.

Increased body fluid purine levels during hypotensive events. Evidence for ATP degradation.

Tissue ischemia leads to adenosine triphosphate (ATP) breakdown with elevation of body fluid ATP metabolites. This study tests the hypothesis that there is a direct relationship between periods of hypotension and body fluid uric acid and oxypurine levels in 19 prospectively studied patients. Significant elevations in urine oxypurine/creatinine clearance were found during periods of hypotension as compared with nonhypotensive periods (p less than 0.05). During severe episodes of hypotension, the serum urate level was significantly elevated as well (p less than 0.05). The increase in these body fluid products of ATP degradation may reflect cellular ischemia during hypotensive periods. There was a weak correlation (r = -0.31, p less than 0.001) between the systolic blood pressure and urine oxypurine/creatinine clearance. However, variability in the appearance of body fluid ATP breakdown products during episodes of hypotension suggests the interplay of multiple factors in the degradation of ATP. The use of ATP degradation products to quantitate the physiologic significance of clinical events remains tantalizing but not proved.

Hyperuricemia in acute illness: a poor prognostic sign.

To clarify the role of the serum urate level and its change as a potential marker for severe tissue hypoxia, we have measured serum urate levels and urine uric acid excretion in 16 patients with acute cardiovascular disease. The six patients who died had a baseline mean serum urate level of 11.1 mg/lg (range, 6.6 to 15.5 mg/dl) and reached a peak mean value of 20.7 mg/dl (range, 13.6 to 33.0 mg/dl). Five of these patients had findings to suggest increased production of uric acid, in addition to decreased excretion of uric acid from impaired renal function. The 10 survivors had a baseline mean serum urate level of 6.8 mg/dl (range, 1.3 to 14.0 mg/dl) and a maximal mean peak value of 7.1 mg/dl (range, 2.9 to 14.0 mg/dl). There was no consistent evidence for increased production or decreased excretion of uric acid. Patients who died had a lower systolic blood pressure, arterial pH and plasma bicarbonate level and a higher heart rate and serum creatinine level compared with the patients ho survived. The observations suggest that marked hyperuricemia at the height of an illness may predict a fatal outcome. Tissue hypoxia may contribute to this sequence of events by leading to the depletion of adenosine triphosphate (ATP) and activation of purine nucleotide degradation to uric acid.

Cellular immune deficiency with autoimmune hemolytic anemia in purine nucleoside phosphorylase deficiency.

Immunologic and metabolic abnormalities were studied in a five year old boy with 0.07 per cent of normal erythrocyte purine nucleoside phosphorylase activity. The clinical course is characterized by severe autoimmune hemolytic anemia, a transient neurologic disorder with tremor and ataxia, and minor infectious illnesses. There is severe lymphopenia with decreased absolute numbers of T and B lymphocytes. Mitogen-stimulated blastogenesis is reduced, but response to allogeneic lymphocytes is normal. A monoclonal IgG protein is present. There is hypouricemia, elevated plasma inosine level, hypouricosuria and an increase in the urinary concentration of inosine and guanosine. The pattern of heterozygote distribution in the patient's family is compatible with an autosomal recessive trait in which heterozygotes are identifiable. In addition, the unusual laboratory and clinical manifestations of this patient illustrate the heterogeneity of the clinical syndrome associated with purine nucleoside phosphorylase deficiency.

Altered properties of human T-lymphoblast soluble low Km 5'-nucleotidase: comparison with B-lymphoblast enzyme.

Soluble low Km 5'-nucleotidases have been purified from human cultured T- and B-lymphoblasts to compare their properties and to examine the mechanism of different rates of nucleotide dephosphorylation. The enzyme from B-lymphoblasts (MGL-8) was 4385-fold purified with a specific activity of 114 mumol/min/mg, while the enzyme from T-lymphoblasts (CEM, MOLT-4) was 4355-fold purified with a specific activity of 35 mumol/min/mg. The activity of both enzymes have an absolute requirement for Mg++. The B-cell enzyme has maximum activity with Mg2+ > Mn2+ > Co2+, while the T-cell enzyme had maximum activity with Co2+ > Mn2+ > Mg2+. The optimum activity was at pH 7.4-9.0 for the B-cell enzyme and pH 9.0 for the T-cell enzyme. Substrate specificity was the same for both enzymes with the following relative Vmax values: CMP > UMP > dUMP > dCMP > dAMP > IMP > GMP > dIMP > dGMP. The Km values for AMP and IMP were 12 and 25 microM for the B-cell enzyme, and 7.0 and 12 microM for the T-cell enzyme. ATP and ADP are competitive inhibitors of these enzymes with apparent Ki values of 100 and 20 microM for the B-cell enzyme, and 44 microM and 8 microM for the T-cell enzyme, respectively. The apparent molecular mass by gel filtration column chromatography is 145 kD for the B-cell enzyme and 72 kDa for the T-cell enzyme. The subunit molecular masses by Western blots are 69.2 kD for both enzymes. These properties suggest that the B-lymphoblast enzyme is identical or similar to the enzyme from human placenta. However, the T-cell enzyme has some different properties. We conclude that these differences plus a lower content of low Km 5'-nucleotidase in T-cells may account for the decreased ability of T-lymphoblasts to dephosphorylate nucleotides and may contribute to the selective cytotoxicity of deoxyribonucleosides for T-lymphoblasts as compared to B-lymphoblasts.

Evidence for adenosine triphosphate degradation in critically-ill patients.

Alterations of cellular energy metabolism may provide important markers during the clinical course of critically ill patients. To determine whether adenosine triphosphate (ATP) degradation occurs in critically ill patients, we measured levels of adenosine, inosine, hypoxanthine, and xanthine in 18 patients and seven control subjects. The mean concentration of hypoxanthine (3.8 microM) in the critically ill patients was elevated (p less than 0.01) compared to that of our control population (0.1 microM). A subgroup of seven critically ill patients had levels of hypoxanthine, xanthine, or inosine higher than those of any member of the control group. This subgroup was characterized by a lower systolic blood pressure, an increased requirement for vasopressors, and a markedly decreased survival rate when compared to the other critically ill patients. Arterial and mixed venous blood gas values were not helpful in predicting survival and did not correlate with levels of ATP degradation products. In two patients who showed subsequent clinical improvement, the initially elevated levels of hypoxanthine and xanthine returned to normal. This study indicates that critically ill patients have elevated levels of ATP degradation products. These increased levels may indicate cellular hypoxia.

Human placental nucleoside kinase activities.

Our studies have indicated that normal human placental cytosol contains a complex mixture of nucleoside kinase enzymes, some of which conform to previously characterized activities. Deoxyadenosine is phosphorylated by deoxycytidine kinase, adenosine kinase, and two as yet uncharacterized activities. Deoxyguanosine phosphorylation is associated with deoxycytidine kinase. More complete and detailed studies will be necessary to characterize these enzymes fully.