Biomedical subjects
H A Simmonds
Publications and source records attributed to H A Simmonds.
Demonstration of a combined deficiency of xanthine oxidase and aldehyde oxidase in xanthinuric patients not forming oxipurinol.
Genetic heterogeneity has been suggested in xanthinuria from the hitherto unexplained ability of some patients with this hereditary disorder to convert allopurinol to its active metabolite oxipurinol--an activity generally attributed to xanthine oxidase. This study provides evidence that the enzyme aldehyde oxidase is also deficient in xanthinuric patients not converting allopurinol to oxipurinol, whereas a xanthinuric patient with normal formation of oxipurinol had normal aldehyde oxidase activity. It is concluded that the enzyme aldehyde oxidase is the principal enzyme responsible for the formation of oxipurinol in man.
Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency.
This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.
The high performance liquid chromatography of enzyme systems relating to purine and pyrimidine metabolism: an overview.
Purines and pyrimidines are now routinely separated by HPLC. By careful selection of chromatographic conditions which match the expected changes in hydrophobicity and/or ionic nature of the substrate and products most enzymes of the purine and pyrimidine salvage pathways can be routinely and accurately determined. Ion-paired reversed-phase systems are often the most advantageous. The relevance of such assays to biomedical analysis including their role in the diagnosis of inborn errors of metabolism is stressed.
Inosine triphosphate pyrophosphohydrolase deficiency in a kindred with adenosine deaminase deficiency.
A complete deficiency of inosine triphosphate pyrophosphohydrolase (ITPase) has been identified, together with high concentrations (mean 157 mumol/l) of the unusual nucleotide ITP, in the erythrocytes of 3 members of a consanguineous United Kingdom kindred. The defect has been noted previously in North America and Sweden, but even in presumed homozygotes some residual ITPase activity was reported. Homozygosity for the defect has not been associated previously with any clinical abnormality. In this kindred it was co-existent with adenosine deaminase (ADA) deficient severe combined immunodeficiency. Since the genes for both ITPase and ADA are localised on the same chromosome, segregation analysis of ITPase and ADA activity was undertaken in available kindred members. The results confirmed an autosomal recessive mode of inheritance for ITPase deficiency, but suggested that the co-existence with ADA deficiency was coincidental.
Regulation of nicotinamide-adenine dinucleotide synthesis in erythrocytes of patients with hypoxanthine-guanine phosphoribosyltransferase deficiency and a patient with phosphoribosylpyrophosphate synthetase superactivity.
1. The synthesis of nicotinamide-adenine dinucleotide from nicotinamide and nicotinic acid was compared over different time scales at both physiological (0.7 mumol/l) and high (0.2-3 mmol/l) substrate concentrations in erythrocytes from three patients with hypoxanthine-guanine phosphoribosyltransferase (hypoxanthine phosphoribosyltransferase, EC 2.4.2.8) deficiency (including one Lesch-Nyhan patient) and from one patient with phosphoribosylpyrophosphate synthetase superactivity. The above disorders are associated with grossly altered erythrocyte nicotinamide-adenine dinucleotide levels. 2. At the physiological substrate concentration and incubation times up to 2 h, nicotinamide proved the most efficient nicotinamide-adenine dinucleotide precursor for erythrocytes from both patients and control subjects. The conversion of nicotinamide to its mononucleotide, but not further metabolism, was impaired in phosphoribosylpyrophosphate synthetase-mutant cells. The Lesch-Nyhan and phosphoribosylpyrophosphate synthetase-mutant cells were unusual in that both showed no further stimulation of nucleotide synthesis at 18 mmol/l Pi compared with 1 mmol/l. 3. At the high substrate concentrations, using 18 mmol/l Pi, nicotinamide was a poor precursor in all instances. Using nicotinic acid, nucleotide formation was 30-fold that from nicotinamide, reaching its maximum at 0.2 mmol/l. Conversion of nicotinic acid to nicotinamide-adenine dinucleotide in the phosphoribosylpyrophosphate synthetase-mutant cells was again grossly impaired. 4. There was no evidence for increased nicotinamide-adenine dinucleotide breakdown in the phosphoribosylpyrophosphate synthetase-mutant cells under any of the above conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Importance of the human erythrocyte in the diagnosis of inherited purine and pyrimidine disorders.
The intact erythrocyte has proved invaluable in the diagnosis of inherited purine and pyrimidine disorders. Not only can it be used to establish the integrity of a particular pathway where enzyme activity is undetectable in disrupted cells, the characteristic alterations in nucleotide patterns in some disorders have provided valuable insight into the mechanism by which the abnormal gene product produces disease. The severe GTP depletion associated with neurological deficits may mirror the situation in the brain, which like the erythrocyte is largely dependent on salvage to sustain its GTP levels. The ability of analogues to accumulate in erythrocytes may likewise give some clue as to the metabolic basis for the associated clinical manifestations, or the efficacy of a particular drug. In addition to their use in determining prognosis as well as providing a diagnosis, such studies have enabled demonstration of a novel route of ATP formation in human cells. The above changes may be masked if immediate processing is not possible.
Precocious familial gout with reduced fractional urate clearance and normal purine enzymes.
Gout is primarily a disease of middle-aged males and is unusual in females and adolescent males. We describe 21 young men and women (mean age 28 years) referred because of precocious onset of gout or hyperuricaemia. Fifteen patients had a family history, but known hereditary causes of primary or secondary hyperuricaemia were excluded. A high proportion (14/21) also had reduced renal function, but there was no clear relationship between this and the hyperuricaemia. Only two patients were hypertensive. A striking abnormality was that the uric acid clearance relative to creatinine [FEur] was subnormal (mean 4.4 +/- 1.4 per cent) in 15/21 patients, including all seven females. A dominantly inherited defect in renal tubular urate handling, leading to a reduced endogenous urate clearance, is the most likely explanation for the hyperuricaemia in this group. Only six patients, all male had a normal [FEur] (mean 8.6 +/- 1.0 per cent). The results confirm that young men and women with gout-or hyperuricaemia disproportionate to the renal dysfunction-should always be investigated, not only for purine defects, but also for abnormalities of renal urate handling. Since the abnormality described here was found also in symptomless subjects, the families of such patients must also be studied. Early identification is important since treatment may ameliorate the renal damage. Although rare, the number of such patients seen in a single referral unit suggests that this type of renal tubular defect in urate handling is more common than suspected and may only come to attention through an isolated episode of precocious gout, when renal function may already be impaired.
NAD synthesis by erythrocytes in phosphoribosylpyrophosphate synthetase (PRPPs) superactivity.
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Further evidence for a 'new' purine defect, inosine triphosphate (ITP) pyrophosphohydrolase deficiency.
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Neurodevelopmental impairment and deranged PRPP and purine nucleotide synthesis in inherited superactivity of PRPP synthetase.
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Deoxy GTP accumulates in thymocytes, but not in T or B lymphocytes in simulated PNP deficiency.
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Optimised conditions for the routine HPLC separation of nucleotides in cell extracts.
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Purine enzyme defects as a cause of acute renal failure in childhood.
Acute renal failure (ARF) is not listed as a usual form of presentation in hypoxanthine-guanine phosphoribosyltransferase deficiency, despite the gross uric acid overproduction in the defect. We found that a third of such patients may present in ARF when the urinary uric acid/creatinine ratio may be normal, not raised, and the defect may be suspected from the disproportionate increase in plasma uric acid. This is important in view of the potential confusion of uric acid with 2,8-dihydroxyadenine, the even more insoluble purine excreted in the other salvage enzyme disorder, adenine phosphoribosyltransferase deficiency. In that disorder, presentation in ARF is well recognized, the uric acid/creatinine ratio is also normal, but plasma urate is not raised. Our combined experience in these two disorders underlines the importance of early recognition and treatment with carefully adjusted doses of allopurinol, which may reverse or postpone renal failure.
ATP formation from deoxyadenosine in human erythrocytes: evidence for a hitherto unidentified route involving adenine and S-adenosylhomocysteine hydrolase.
A novel route of ATP formation has been identified using erythrocytes from patients deficient in four different enzymes associated with ATP formation. It entails prior adenine production from deoxyadenosine (or adenosine) in a reaction involving S-adenosylhomocysteine hydrolase. The postulated route has been demonstrated in human erythrocytes which, unlike other human cells, cannot form ATP from IMP. It is based on studies by others using purified S-adenosylhomocysteine hydrolase preparations in vitro. The results provide the first confirmation that this reaction occurs in intact human cells in vitro and thus most probably in vivo. This adenine production is normally masked in intact cells by further metabolism to ATP. Clinical significance for such a route is suggested by the fact that some adenosine analogues with potent oncostatic and antiviral properties also release adenine (or analogues) in vitro.
Altered erythrocyte nucleotide patterns are characteristic of inherited disorders of purine or pyrimidine metabolism.
This paper compares erythrocyte nucleotide levels in patients with eight different inherited purine or pyrimidine enzyme defects identified amongst a variety of patients referred predominantly for investigation of severe neurological abnormalities, or immunodeficiency syndromes. Characteristic nucleotide patterns were identified only in the six disorders (four involving purine and two pyrimidine metabolism) where there was clinical evidence of cellular toxicity. They were frequently related to the accumulation of abnormal metabolites in body fluids. These erythrocyte studies have demonstrated the following. 1. ATP depletion is not an invariable feature of adenosine deaminase (ADA) deficiency, but the accumulation of the deoxyribonucleotides dATP, or dGTP, is diagnostic of ADA, or purine nucleoside phosphorylase (PNP) deficiency, respectively. The early accumulation of dATP in foetal blood is a valuable aid to prenatal diagnosis of ADA deficiency. 2. GTP depletion appears to reflect the degree of CNS involvement in hypoxanthine-guanine phosphoribosyltransferase and PNP deficiency, as well as PP-ribose-P synthetase superactivity. Other diagnostic changes involving increased pyrimidine sugars and increased or decreased NAD levels, or ZTP in Lesch Nyhan erythrocytes, show no consistent correlation with the clinical manifestations. 3. These altered nucleotide levels afford a novel means for carrier detection of the X-linked defect associated with aberrant PP-ribose-P synthetase activity, where no other test is yet available. Measurement of erythrocyte nucleotide levels thus provides a simple and rapid aid to diagnosis and may sometimes be essential for determining prognosis, carrier detection, or monitoring therapy. These characteristic 'fingerprints' may give some insight into the mechanism by which the abnormal gene product produces disease. Such grossly altered nucleotide levels could also result in loss of erythrocyte flexibility, increased destruction and hence the anaemia, or other clinical manifestations, observed in some disorders.
Inherited superactivity of phosphoribosylpyrophosphate synthetase: association of uric acid overproduction and sensorineural deafness.
PURPOSE: Superactivity of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) synthetase, inherited as an X chromosome-linked trait, has been reported in nearly 20 families in which overproduction of uric acid is invariably present in hemizygous affected males. Clinical manifestations of PP-Rib-P synthetase superactivity are mainly limited to gout in early adulthood. Neurologic deficits, including sensorineural deafness, have rarely been described. We herein document the association of PP-Rib-P synthetase superactivity, gout with excessive uric acid synthesis, and sensorineural deafness in an additional family. PATIENTS AND METHODS: Two members of a Spanish family were studied: an eight-year-old boy (Patient 1) with tophaceous gout, purine nucleotide and uric acid overproduction, and sensorineural deafness, and his 27-year-old mother (Patient 2), who had gout. Fibroblast cultures were initiated from skin biopsy specimens, and measurements of PP-Rib-P and purine nucleotide metabolism in the fibroblasts were performed. RESULTS: A labile but superactive PP-Rib-P synthetase was demonstrated in the fibroblasts cultured from both Patients 1 and 2. The kinetic basis of PP-Rib-P synthetase superactivity in this family was resistance to purine nucleotide inhibition of enzyme activity. More severe derangements in the enzyme and in PP-Rib-P and purine synthesis in Patient 1's cells than in Patient 2's cells suggest that Patient 1 is hemizygous and Patient 2 is heterozygous for an X chromosome-linked genetic defect. Limited pedigree data support this view. Compared with affected members of seven other families with PP-Rib-P synthetase superactivity, these patients are intermediate in the range of clinical expression and in the severity of the enzyme defect as measured by the degree of aberration of PP-Rib-P and purine nucleotide synthesis in fibroblasts. Metabolic abnormalities were more severe in Patient 1's cells than in the cells of most male patients (in whom clinical expression is limited to early adult-onset gout) but were less severe than in the cells of two patients in whom more complex enzyme defects were associated with uric acid overproduction and neurodevelopmental abnormalities (including deafness) in male children and adult women. CONCLUSION: Certain defects resulting in PP-Rib-P synthetase superactivity may be causally related to neurologic impairment, most commonly sensorineural deafness.
Allopurinol kinetics after massive overdosage.
A 15-year-old girl with normal renal function took 75 tablets of allopurinol 300 mg. She did not suffer any ill-effects. Samples obtained over 84 h showed prolonged elimination of allopurinol, with a half-life of 3.6 h, and oxipurinol, with a half-life of 26 h.