N. Lawrence Edwards

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.

Lymphocyte 5'-Nucleotidase Deficiency in Hypogammaglobulinemia: Clinical Characteristics'

Abstract Twelve patients with congenital agammaglobulinemia, 2 of 10 patients with selective IgA deficiency, and 4 of 15 patients with common variable immunodeficiency have reduced 5′-nucleotidase activity on lymphocyte plasma membranes. In congenital agammaglobulinemia and common variable immunodeficiency, enzyme-deficient patients have an early onset of immune dysfunction, low levels or an absence of circulating B-lymphocytes, and a positive family history of similar immunodeficiency. The enzyme deficiency involves peripheral E-rosette-forming lymphocytes, suggesting an abnormality of T-lymphocytes in these diseases. No systemic disorder of purine nucleotide degradation to uric acid is detectable in the enzyme-deficient patients. No direct relationship between 5′-nucleotidase activity and in vitro production of IgG is evident, since inhibition of the enzyme did not decrease IgG synthesis. At present the data do not allow a distinction between two hypotheses to explain the role of 5′-nucleotidase in hypogamma-globulinemia, that is, whether 5′-nucleotidase deficiency causes the immune dysfunction or is simply a marker for an intrinsic lymphocyte abnormality. However, this enzyme deficiency may provide a useful means of classifying disorders of immunoglobulin synthesis.

Differential metabolism of deoxyribonucleosides by leukaemic T cells of immature and mature phenotype

Summary. Experimental evidence has indicated that T lymphoblasts are more sensitive to deoxynucleoside toxicity than are B lymphoblasts. These data have led to the use of purine enzyme inhibitors as selective chemotherapeutic drugs in the treatment of T cell malignancies ranging from T cell acute lymphoblastic leukaemia to cutaneous T cell lymphomas. We have compared the toxicities of 2′‐deoxyadenosine, 2′‐deoxyguanosine, and thymidine for T cell lines derived from patients with T cell acute lymphoblastic leukaemia with those for mature T cell lines derived from patients with cutaneous T cell leukaemia/lymphoma. We have found that both deoxynucleosides are far less toxic to the mature T cell lies than to T lymphoblasts and that the mature cells accumulate much lower amounts of dATP and dGTP when exposed to deoxyadenosine and deoxyguanosine, respectively. Similar studies performed on peripheral blood cells from patients with T cell leukaemias of mature phenotype and on peripheral blood T cells demonstrate similar low amounts of deoxynucleotide accumulation. Measurements of the activities of several purine metabolizing enzymes that participate in deoxynucleoside phosphorylation or degradation do not reveal differences which would explain the toxicity of deoxynucleosides for immature, as compared to mature, T cells. We conclude that deoxynucleoside metabolism in leukaemic T cells varies with their degree of differentiation. These observations may be relevant to the design of chemotherapeutic regimes for T cell malignancies.

Increased Adenine Nucleotide Turnover in Duchenne Muscular Dystrophy

Summary: To investigate a possible disorder of adenine nucleotide turnover in Duchenne muscular dystrophy, we evaluated 15 patients with mild Duchenne muscular dystrophy, eight patients with severe muscular dystrophy, seven patients with other neuromuscular disorders, and eight patients with hypogammaglobulinemia but no muscle disease. The serum urate concentration was similar in all four groups. Base line urinary purine excretion was elevated in all patients with neuromuscular disease with values of 1.72 ± 0.15, 2.37 ± 0.22, 2.49 ± 0.35, and 2.60 ± 0.48 µmoles/100 ml glomerular filtration for control subjects, mild Duchenne muscular dystrophy, severe disease, and other neuromuscular diseases, respectively.Adenine nucleotide pool turnover was measured by labeling with [8-14C]adenine and then 5 days later administering intravenous fructose. Five-day cumulative mean radioactivity excretion was elevated in mild and severe Duchenne muscular dystrophy with excretion values of 11.4 ± 0.7 and 11.5 ± 1.1% of administered radioactivity, respectively, as compared to 9.0 ± 0.9% of administered radioactivity for control subjects. After intravenous fructose infusion, patients with Duchenne muscular dystrophy had a less than normal rise in serum urate concentration, a normal increase of urinary purine excretion, and a greater than normal elevation of urinary radioactivity excretion and urinary purine specific activity. Patients with other neuromuscular diseases had virtually no rise in plasma urate concentration, less than normal increase in urinary total purine excretion, and a greater than normal increase of urinary radioactivity excretion and urinary specific activity.These observations suggest that there is an increased rate of adenine nucleotide turnover in Duchenne muscular dystrophy. In patients with other neuromuscular disease an increased rate of adenine nucleotide turnover resembled the abnormality expected from a diminshed adenine nucleotide pool.Speculation: The accelerated turnover of adenine nucleotides in Duchenne muscular dystrophy may result from the proposed membrane defect. Treatment directed toward increasing the synthesis of ATP may be beneficial in Duchenne muscular dystrophy.

Lymphocyte 5'-nucleotidase deficiency: clinical and metabolic characteristics of the associated hypogammaglobulinemia.

Decreased activity of 5′-nucleotidase on the surface of peripheral blood mononuclear cells (PBM’s) has recently been observed in young males with congenital agammaglobulinemia1, primary non-familial hypogammaglobulinemia1–3, and selective IgA deficiency3,4. A partial deficiency of the ecto-enzyme occurred in 12 of 12 patients with congenital agammaglobulinemia, in 4 of 15 with acquired (common variable) immunodeficiency, and in 2 of 10 with selective IgA deficiency. The enzyme values and clinical classification of the 18 enzyme deficient subjects with primary hypogammaglobulinemia are shown in Table 1.

Enzyme Replacement in the Lesch-Nyhan Syndrome with Long-Term Erythrocyte Transfusions

The pathogenesis of the neuro-behavioral abnormalities associated with the Lesch-Nyhan syndrome remains obscure despite recent reports of neurotransmitter abnormalities in these patients1. Many attempts to correct the characteristic manifestations of spasticity, mental retardation, choreoathetosis, and compulsive self-mutilation have been reported but none have reported sustained clinical efficacy. Many pathogenic mechanisms have been proposed over the past two decades to explain the relationship between the known aberration in purine metabolism and the observed neurologic dysfunction. One of these proposed mechanisms is that the absence of the purine salvage pathway in the central nervous system (CNS) results in (1) the accumulation of oxypurines in the spinal fluid which then may act as toxic endogenous mediators2 and (2) the depletion of guanine and adenine nucleotides that are important to normal CNS function3–5. Supplementation of purine intermediates with dietary adenine, guanosine, inosine, and GMP have not altered the clinical course of the disease.

Altered purine and pyrimidine metabolism in erythrocytes with purine nucleoside phosphorylase deficiency

Purine and pyrimidine metabolism was compared in erythrocytes from three patients from two families with purine nucleoside phosphorylase deficiency and T-cell immunodeficiency, one heterozygote subject for this enzyme deficiency, one patient with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, and two normal subjects. The erythrocytes from the heterozygote subject were indistinguishable from the normal erythrocytes. The purine nucleoside phosphorylase deficient erythrocytes had a block in the conversion of inosine to hypoxanthine. The erythrocytes with 0.07% of normal purine nucleoside phosphorylase activity resembled erythrocytes with hypoxanthine-guanine phosphoribosyltransferase deficiency by having an elevated intracellular concentration of PP-ribose-P, increased synthesis of PP-ribose-P, and an elevated rate of carbon dioxide release from orotic acid during its conversion to UMP. Two hypotheses to account for the associated immunodeficiency—that the enzyme deficiency leads to a block of PP-ribose-P synthesis or inhibition of pyrimidine synthesis—could not be supported by observations in erythrocytes from both enzyme-deficient families.

Hypoxanthine Salvage in Man: Its Importance in Urate Overproduction in the Lesch-Nyhan Syndrome

1. A daily urinary excretion of 0.8 percent of the administered radioactivity results from the turnover of the labeled adenine nucleotide pool. 2. A 4-fold increase of urinary radioactivity excretion occurs in patients with the Lesch-Nyhan syndrome and support the role of impaired hypoxanthine salvage in the purine overexcretion associated with HGPRT deficiency. 3. Our data do not support the possibility that the increased radioactivity excretion in the HGPRT deficient subjects results from an elevated rate of adenine nucleotide degradation.

Possible Role for 5′-Nucleotidase in Deoxyadenosine Selective Toxicity to Cultured Human Lymphoblasts

Adenosine deaminase deficiency and purine nucleoside Phosphorylase deficiency are relatively specific for causing a disorder of immune function. Several in vitro observations provide clues concerning the basis for this specificity. The addition of deoxyadenosine reduces the response of peripheral blood lymphocytes to mitogen stimulation when adenosine deaminase is inhibi ted1,2. The combination of deoxyadenosine and adenosine deaminase inhibition is also cytotoxic to T-lymphoblasts but not B-lymphoblasts3–5. Deoxyadenosine mediated cytoxicity in T-lymphoblasts is accompanied by increased concentrations of dATP.

29 METABOLISM OF CUANOSINE AND ITS C-8 SUBSTITUTED ANALOGUES IN HUMAN LYMPHOCYTE CYTOPLASM

The C-8 substituted guanine nucleosides are a new class of immunologically active purine analogues. Naturally occurring guanosine is immunosuppressive to B-cells. 8-Mercaptoguanosine (8-SGuo) and 8-bromoguanosine (8-BrGuo) have polyclonal B-cell activating properties in murine lymphocytes. 8-Aminoguanosine (8-NH Guo) has no immunologic effects on cells in culture but is a potent inhibitor of the purine catabolic enzyme, purine nucleoside phosphoylase. This study examined the metabolic fate of guanosine and the C-8 substituted analogues in dialyzed human splenic cytosol. Required cofactors for the involved enzyme systems were added to the cytosol. The metabolism of the ribonucleoside substrate to other purine forms was monitored by reverse-phase and anion exchange HPLC at 15 minute intervals for 2 hours. Guanosine declines linearly over 90 minutes with linear accumulations in guanine, xanthine, GTP and GDP when incubated at 37°C in the presence of PRPP. 8-SGuo and 8-BrGuo remain essentially non-metabolized to other forms after 120 minutes. 8-NH Guo is rapidly metabolized to 8-NH guanine and a minor peak of 8-NH GDP appearing after 60 minutes. These preliminary studies in human spleen demonstrate that 2 C-8 substituted purine analogues (8-SGuo and 8-BrGuo) that stimulate B-cell proliferation and differentiation are not substantially metabolized by cytoplasmic purine enzymes. Conversely guanosine is extensively metabolized to both higher (GTP and GDP) and lower (guanine and xanthine) forms. These findings may help define the molecular basis for both immune-suppressive and immune-potentiating effects of guanosine and its C-8 substituted analogues.