Biomedical subjects
Publications and source records attributed to R W Watts.
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The subcellular distribution of 2-oxoglutarate:glyoxylate carboligase was investigated in a normal human liver, a liver from a patient with pyridoxine-resistant primary hyperoxaluria type I and rat livers subjected to various degrees and types of trauma. On continuous sucrose gradients most of the carboligase fractionated with a peak equilibrium density of 1.19-1.20 g/cm3 and paralleled the distribution of the major peaks of monoamine oxidase, glutamate dehydrogenase and cytochrome oxidase and can be considered to be mitochondrial. Various proportions of the carboligase and mitochondrial marker enzymes were found to be 'extramitochondrial' (at or near the top of the sucrose gradients), depending on the liver source and the severity of trauma to which they were subjected. Carboligase, monoamine oxidase (outer membrane marker) and glutamate dehydrogenase (matrix marker) were released from mitochondria by the homogenization and centrifugation procedures, to the extent of 19.9%, 32.4% and 11.5% respectively in hyperoxaluric liver, 12.5%, 17.9% and 8.2% in normal human liver and 3.0%, 4.9% and 3.8% in control rat liver. The proportion of extramitochondrial cytochrome oxidase (inner membrane marker) was virtually undetectable in both human and rat livers. However, sonication of rat liver homogenates or the addition of the detergent Triton X-100 caused a massive release of all four enzymes. The extramitochondrial carboligase was probably in the form of a free protein of very high molecular weight or aggregate, rather than associated with a mitochondrion-derived organelle. Subfractionation of a rat liver mitochondrial preparation indicated that most of the carboligase activity paralleled activities of 2-oxoglutarate decarboxylase, citrate synthase and glutamate dehydrogenase and was probably located in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
The overall activity of the purine de novo synthesis pathway and the activities of purine phosphoribosyltransferase in the rat testis were measured at different ages and were correlated with histological observations. Similar studies of the concentration of circulating gonadotrophins and testosterone were performed. The purine phosphoribosyltransferase activities were between two and three orders of magnitude greater than purine de novo synthesis. The peak activity of the purine de novo synthesis pathway coincided with the first appearance of meiosis in the spermatocytes immediately before the luteinising hormone (LH) level rose to its peak. The highest activity of the hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) - catalysed purine salvage pathway coincided with the first appearance of mature spermatozoa in the tubules just after the occurrence of peak levels of follicle-stimulating hormone (FSH). These findings are linked to the development of testicular atrophy in cases of severe HPRT deficiency in man.
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The metabolic pathways of pterin de novo synthesis, interconversion and salvage which lead to the tetrahydrobiopterin cofactor of phenylalanine 4-monooxygenase, tyrosine 2-monooxygenase and tryptophan 5-monooxygenase are reviewed and data on the enzymes which catalyze the individual steps are presented. Analogies drawn between the inborn errors of tetrahydrobiopterin production and the Lesch-Nyhan syndrome, in which purine salvage is deficient, are used as a basis for the hypothesis that the neurological manifestations of the Lesch-Nyhan syndrome are due to neurotransmitter imbalance which stems from an imbalance of the aromatic amino acid monooxygenase activities which are themselves due to impaired pterin biosynthesis. The latter arises because, in the absence of the hypoxanthine phosphoribosyltransferase catalyzed purine salvage pathway, the supply of GTP for the GTP cyclohydrolase reaction, which is the first reaction on the pterin de novo synthesis pathway, is reduced. It is proposed that the different aromatic amino acid monooxygenases are differentially affected by this constrained pterin production. The activities of those most directly related to the quantal production of the cerebral neurotransmitters dopamine, norepinephrine and 5-hydroxytryptamine are affected whereas liver phenylalanine 4-monooxygenase activity is not overtly impaired. The results of different lines of research which support this concept are cited, as is direct evidence for a selective reduction of dopamine production in the basal ganglia of patients with the Lesch-Nyhan syndrome. It is proposed that lack of GMP for functions, other than its role in pterin de novo synthesis, accounts for the features of the Lesch-Nyhan syndrome which do not occur when only tetrahydrobiopterin production is deficient as in the inborn errors of tetrahydrobiopterin synthesis.
The structural basis of the combined conductive and sensorineural deafness has been described in two patients with Hurler's disease. All parts of the ear contained numerous large vacuolated Hurler cells, the vacuoles being distended lysosomes from which accumulated glycosaminoglycans had been dissolved during fixation of the tissue. The external and middle ears also showed chronic inflammation. There was resorption of the bone in the mastoid process by masses of Hurler cells and abnormal new bone with prominent cement lines. The blood vessels were surrounded by a 'blue mantle' of osteoid tissue similar to that which is usually associated with otosclerosis. The stapes appeared deformed and was covered by thickened mucosa and granulation tissue. The bone structure of the ossicles resembled that of the mastoid process. The organ of Corti was degenerate and the Reissner's and tectorial membranes were adherent to one another and covered by haemorrhagic material near the vascular striae. The blood vessels in the striae were congested and the scalae media and tympani contained some blood. The neurons in the basal coil of the spiral ganglion were replaced by Hurler cells. The vestibulo-cochlear nerves were disrupted by numerous Hurler cells. These pathological findings adequately explain the combined conductive and sensorineural deafness in these cases. They are also discussed in relation to some other clinical and pathological aspects of these two specific patients.
We have measured glomerular filtration rate (GFR), extracellular fluid volume (ECF), oxalate distribution volume (OxDV), plasma oxalate concentration (POx.), plasma total clearance of oxalate (PCOx.), oxalate metabolic pool size [(OxDV) X (POx.)], renal clearance of oxalate (RCOx.), oxalate excretion, tissue clearance of oxalate (TCOx.) and tissue oxalate accumulation rate [(TOx.A) = (TCOx.) X (POx.)] in three patients with type I primary hyperoxaluria (hyperoxaluria with hyperglycollic aciduria) when they were taking pyridoxine and after discontinuation of the vitamin. Seven days after stopping pyridoxine the plasma oxalate concentration, oxalate metabolic pool size and the urinary excretion of oxalate had all increased between seven- and eight-fold in two of the patients. The third patient showed no changes on stopping pyridoxine. These results support the view that pyridoxine acts by reducing oxalate biosynthesis in some patients with type I primary hyperoxaluria. The possible biochemical basis for this effect is discussed.
A case is reported of a patient with renal failure and developing systemic and renal oxalosis due to pyridoxine-resistant type I primary hyperoxaluria. In spite of vigorous haemodialysis and hydration before and after operation, an allografted cadaveric kidney failed because of oxalate deposits in the transplant. The patient was treated by combined hepatic and renal transplantation. The liver allograft functioned well but the kidney had poor function due to primary acute tubular necrosis aggravated by steroid-associated acute pancreatitis, systemic cytomegalovirus infection and high cyclosporin A levels. The patient died from generalised cytomegalovirus infection. The early course after operation was associated with a reduced rate of oxalate production, which would slow the rate of oxalate deposition in the tissues. The size of the oxalate metabolic pool was also diminished. These observations are compatible with the grafted liver having corrected the metabolic lesion.
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We have measured the plasma oxalate concentration (POx), urinary oxalate excretion (UOx), oxalate equilibrium distribution volume (ODV), oxalate metabolic pool size [(ODV) X (POx)], total plasma oxalate clearance (PCOx), renal (or dialyser) oxalate clearance (RCOx), non-renal oxalate clearance (NRCOx) and the tissue oxalate accretion rate (TOA) = [(NRCOx) X (POx)] in three patients with severe renal failure due to primary hyperoxaluria who were being treated by peritoneal dialysis or haemodialysis, or by renal transplantation. The clearance (either GFR or dialyser) of [99mTc]diethylenetriaminepenta-acetate (DTPA) and the extracellular fluid volume (ECF) measured as [99mTc]DTPA distribution volume were also determined. Negligible amounts of 14C were found in faeces or as 14CO2 in expired air and hence (NRCOx) = (PCOx-RCOx). Haemodialysis removed oxalate more efficiently than peritoneal dialysis in the patient where a direct comparison was possible. Neither treatment could keep up with the TOA when performed for clinically acceptable times. The plasma oxalate concentrations calculated from 14C clearance through the dialyser and the chemically determined concentration of the oxalate in the dialysate were in the range 111-146 mumol/l. This is higher than in normals and in hyperoxaluric patients who are not in renal failure. Hence, although the ODV and ECF are similar to those of hyperoxaluric patients without renal failure and normal control subjects, the oxalate metabolic pool (ODV X POx) is grossly enlarged. In the patient treated by renal transplantation, the oxalate pool size diminished concurrently with the resumption of oxalate excretion but expanded again as renal function decreased due to oxalosis.(ABSTRACT TRUNCATED AT 250 WORDS)
We have measured the rate of purine synthesis de novo in blood mononuclear cells in vitro and the activities of the purine salvage enzymes [hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), adenine phosphoribosyltransferase (APRT; EC 2.4.2.7)] and ribosephosphate pyrophosphokinase (PP-ribose-P synthetase; EC 2.7.6.1)] and the concentration of phosphoribosylpyrophosphate (PP-ribose-P) in the erythrocytes of affected family members. These subjects belong to families where hyperuricaemia and renal failure occur together early in life, and the genetic transmission follows an autosomal dominant mode of inheritance. We term this syndrome, familial hyperuricaemic nephropathy. No significant differences were detected in either the rates of purine synthesis de novo in vitro between the index patients and the control subjects with respect to the enzyme activities or the PP-ribose-P concentrations. Two groups of controls were used, healthy individuals and patients with a comparable degree of renal failure due to non-immune complex renal disease. Mononuclear cells from patients with Lesch-Nyhan syndrome (congenital HPRT deficiency) showed the expected acceleration of purine synthesis de novo in vitro. The accelerated purine synthesis de novo in vitro associated with phytohaemagglutinin-induced lymphocyte transformation was detectable by the method used. We conclude that familial hyperuricaemic nephropathy is not due to a metabolic lesion which causes accelerated purine synthesis de novo. This suggests that the primary abnormality may be a failure of the renal tubular net excretion of urate.
Using an in vitro method, the uptake of radio-labelled Ca2+ by jejunal biopsy specimens from control subjects, patients with idiopathic hypercalciuria and patients with renal stones without hypercalciuria, were compared. Radio-labelled Ca2+ uptake was investigated over the concentration range 0.1-5.0 mmol/l. For all subjects there was a linear relationship between Ca2+ uptake and medium concentration suggesting that Ca2+ uptake was a passive process. There was no significant difference in Ca2+ uptake between control subjects and patients with renal stones without hypercalciuria. Patients with idiopathic hypercalciuria, both absorptive and renal subtypes, showed increased Ca2+ uptake at all incubation medium concentrations. Assays of various biochemical parameters including alkaline phosphatase, Ca2+-activated ATPase, cyclic AMP and Ca2+-binding protein, in jejunal biopsy specimens showed no significant differences between control subjects and patients with idiopathic hypercalciuria. The results suggest that the intestinal abnormality in idiopathic hypercalciuria is due to enhanced permeability of the brush border membrane to Ca2+, possibly mediated by alterations in membrane lipids.
This communication reports studies on the composition of the urinary glycosaminoglycans and oligosaccharides in mucopolysaccharidosis patients who were being treated by fibroblast transplantation. The urinary glycosaminoglycans were precipitated with 9-aminoacridine, the oligosaccharides remaining in solution. Both fractions were further subfractionated by gel filtration. The glycosaminoglycan subfractions were examined for their content of iduronic acid, glucuronic acid, galactosamine and glucosamine. We found no changes in these parameters in a patient who had been treated by repeated fibroblast transplantations over the course of 4 1/2 years. The amino sugar composition of the oligosaccharide fraction was examined and shown to be unchanged. We also found no changes in the degree of sulphation of the urinary glycosaminoglycans specifically related to the transplant in four patients with Hurler disease and two with Hunter disease. We conclude that fibroblast transplantation does not produce detectable changes in the carbohydrate content or degree of sulphation of the urinary glycosaminoglycans and oligosaccharides.
This paper reports the clinical and biochemical results in six patients with Hurler disease (Mucopolysaccharidosis IH; McKusick 25280), two patients with Hunter disease (Mucopolysaccharidosis II; McKusick 25285) and one patient with Sanfilippo B disease (Mucopolysaccharidosis IIIB; McKusick 25292) who were treated by fibroblast transplantation. Except for one patient who died for a coincidental reason, the patients have been studied for between 2.5 and 4.5 years. The clinical course of the disease was not materially altered. There was no evidence that the patients had developed immune responses against the transplanted fibroblasts. Transplantation did not produce measurable levels of either alpha-L-iduronidase (EC 3.2.1.76) in the leukocytes from patients with Hurler disease or of N-acetyl-alpha-D-glucosaminidase (EC 3.2.1.50) in the plasma of the patients with Sanfilippo B disease. Under the conditions used for the assay, leukocytes from the patients with Hunter disease had detectable levels of residual alpha-L-idurono-2-sulphate sulphatase activity which were increased after the transplants, although these changes were of inconstant size and their time course was not consistently related to the transplantations. Cytogenetic studies in cases where the donor was of the opposite sex detected only cells of the recipient's sex among the fibroblasts grown from biopsies of the transplantation sites. The technique used would have detected a donor to recipient cell ratio of 1:100. We found no consistent long-term trends in the excretion patterns of glycosaminoglycans and oligosaccharides from either a quantitative or qualitative point of view which could be specifically related to the transplantation. The combined administration of immunosuppressive doses of prednisolone and azathioprine was associated with an increased excretion of the lower molecular weight glycosaminoglycans. We conclude that fibroblast transplantation is not therapeutically useful in the diseases studied.
The regulation and integration of purine nucleotide biosynthesis is considered from the viewpoint of the main groups of reaction sequences involved and with respect to some specific organs and tissues. Inhibiting either IMP dehydrogenase or adenylosuccinate synthetase in rat liver in vitro reduced the rate of purine do novo synthesis with respect to the purine remaining in the tissue and did not materially affect the rate with respect to the purines extruded into the incubation medium. These results are considered in contrast to the results of previous studies in cultured lymphoblasts. The relative activities of purine de novo synthesis and of purine salvage have been assessed in different tissues by the activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase (HPRT), respectively. Changes in purine de novo synthesis as measured by [14C]formate incorporation into cellular purines were reflected in the amidophosphoribosyltransferase activities. The capacity of different tissues to synthesize purines de novo is widespread and the role of the liver as the main site of purine de novo synthesis in vivo and exporting purines to other tissues appears questionable. Regulatory mechanisms may well be tissue specific. The age-related changes in the activity of the purine de novo synthesis and purine salvage pathways, respectively, in the brain suggest that it is physiological or neuropharmacological functions of the developed brain rather than cell division and organogenesis which require a high level of purine salvage relative to purine de novo synthesis. This is compatible with the observation that purine de novo synthesis alone can meet the needs for additional purine nucleotides which lectin induced lymphocyte transformation involves. The mechanism whereby purine de novo synthesis is initiated during lectin induced lymphoblast transformation remains obscure.
The term immune function is taken to mean the process by which lymphocytes respond to an antigenic challenge. This response involves cell division and differentiation, which depend on purine nucleotides. The present state of knowledge relating events at the cell surface to the necessarily increased rate of purine de novo synthesis, and the modulation of purine nucleotide interconversions through the mediation of putative intracellular messengers, is reviewed. These physiological processes are related, where possible, to some genetic and pharmacologically induced perturbations of the systems involved.
We have measured the total plasma clearance, renal clearance and equilibrium distribution volume of [14C]oxalate, and the urinary oxalate excretion rate and plasma oxalate levels at approximately 6 months intervals for up to 2.5 years in five patients with primary hyperoxaluria. The renal clearance and distribution volumes of [99mTc]DTPA (diethylenetriaminepenta-acetate) were measured simultaneously to provide estimates of glomerular filtration rate (GFR) and extracellular fluid volume (ECF). The same measurements were made on each of five normal volunteers. Clearances and distribution volumes were measured with a modified single injection technique. The oxalate clearance was two to three times the simultaneously measured GFR in the patients and control subjects. The renal clearance of oxalate was less than the total plasma clearance in the patients. The oxalate distribution volume was approximately 1.5 times the ECF in both the patients and controls. Only small changes were observed over a 2.5 years period in these particular patients. The plasma oxalate concentration was derived from the urinary oxalate excretion rate and the plasma [14C]oxalate clearance. It was raised in the patients. The oxalate removal rate was derived from the total plasma clearance and the plasma oxalate concentration.
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