Stephen P. Spielberg

Hyperphenylalaninemia Due to a Deficiency of Biopterin

We studied the components of the hepatic phenylalanine hydroxylating system in a child with phenylketonuria who showed substantial neurologic impairment despite early dietary control of elevated blood phenylalanine levels. Phenylalanine hydroxylase, dihydropteridine reductase and dihydrofolate reductase activities were normal. In contrast the level of hydroxylation cofactor, tetrahydrobiopterin, in liver was only 10 per cent of normal. In addition to this hepatic deficiency, serum and urinary levels of biopterin-like compounds were low, and the serum biopterin did not increase in response to a phenylalanine load as it does in normal and phenylketonuric subjects. The phenylalanine hydroxylase activity in this child, as determined by an in vivo tritium-release assay, was 2.3 per cent of the normal value. These results indicate that the child suffers from a variant form of phenylketonuria--a deficiency of a functional phenylalanine hydroxylating system secondary to a defect in biosynthesis of biopterin.

5-Oxoprolinuria: Biochemical observations and case report

We have studied a patient with 5-oxoprolinuria who presented with hemolysis and metabolic acidosis as a neonate; he has had normal growth and development to one year of age. Compensated hemolytic anemia persists, and he requires alkalinizing agents for correction of acidosis. Biochemical studies have confirmed that a deficiency of glutathione synthetase is responsible for the 5-oxoprolinuria. Genetic heterogeneity was apparent on comparative study of glutathione synthetase kinetics in cells from two patients with this disorder. The consequences of the deficiency of glutathione synthetase, decreased intracellular glutathione, and overproduction of 5-oxoproline are discussed with reference to the possible cellular roles of these compounds.

Endocrine studies in cystinosis: Compensated primary hypothyroidism

Children with nephropathic cystinosis exhibit marked growth retardation. Improved medical management and renal transplantation have increased their life expectancy beyond the second decade. We have studied endocrine function in seven patients with cystinosis and reviewed autopsy findings of four patients and medical records of 24 others. One 10-year-old boy was overtly hypothyroid. The six other patients had normal studies of peripheral thyroid function but two had borderline and two had frankly elevated serum TSH levels. Stimulation tests of cortisol and growth hormone secretion and basal levels of serum NSILA-s were normal. Postmortem histology of the thyroid glands revealed extensive destruction and infiltration of the epithelium with cystine crystals. Despite the presence of cystine crystals in other endocrine tissues, there was no destruction of epithelium in glands other than in the thyroid. We conclude that in nephropathic cystinosis compensated primary hypothyroidism occurs frequently and early and may be diagnosed by measurement of serum TSH concentrations.

Normal amino acid uptake by cultured human fibroblasts does not require gamma-glutamyl transpeptidase

The uptake kinetics for four amino acids (cystine, glutamine, methionine, and alanine) which are among the best gamma-glutamyl acceptors have been determined for normal human fibroblasts and for a cell line containing undetectable quantities (< 0.5% normal mean) of gamma-glutamyl transpeptidase activity. Apparent Km and V(max) for uptake for each of the four amino acids were normal in the mutant fibroblasts. Insulin increased the uptake of alpha-aminoisobutyrate as in control cells. levels of 16 amino acids were also normal in this cell strain; the intracellular concentrations of phenylalanine, cystine, and cysteine were increased. In human fibroblasts, amino acid transport appears to proceed normally in the absence of active gamma-glutamyl transpeptidase.

HYPERPHENYLALANINEMIA DUE TO PHENYLALANINE HYDROXYLASE COFACTOR DEFICIENCY

A child with phenylketonuria diagnosed early developed severe hypotonia with minimal signs of spasticlty and retardation in all developmental patterns despite adequate dietary management. At 4 years of age, a liver biopsy was performed and the components of the phenylalanine hydroxylase system were measured in vitro. Normal levels of phenylalanine hydroxylase, dihydropteridine reductase and dihydrofolate reductase were found. However, there was only 5% of the normal level of active phenylalanine hydroxylase cofactor. This cofactor deficiency was also demonstrable in plasma and urine. Since the phenylalanine hydroxylase cofactor (tetrahydrobiopterin) is probably also necessary for the hydroxylation of tyrosine and tryptophan, the rate limiting steps in the biosynthesis of dopamine and norepinephrine and serotonin, this child may be suffering from a lack of these neurotransmitters.The childs phenylalanine hydroxylase activity has also been measured with a new in vivo method. In this procedure, hydroxylase activity is assayed by following the rate of formation of tritiated water from ring tritium labelled phenylalanine. Phenylalanine hydroxylase activity 1-5% of normal was detected in vivo confirming the incomplete block in the conversion of phenylalanine to tyrosine predicted by the in vitro assays on the liver biopsy.

Glutathione metabolism in normal and cystinotic fibroblasts.

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.

Pharmacogenetics and the Fetus

It is well accepted in clinical medicine that there is wide variation between patients in the handling of and response to drugs; these differences are based in part on genetic mechanisms. Pharmacogenetic differences among patients contribute to altered drug kinetics, resulting in failure of therapy or in side effects related to blood levels after standard doses. Predisposition to idiosyncratic reactions (hepatotoxicity, aplastic anemia, or even carcinogenesis) may also be genetically determined.1 Similarly, not all fetuses exposed to a potential teratogen in utero will have birth defects, and the pattern of any abnormalities may vary. Some of the differences may result .xa0.xa0.

Genetic Disorders of Glutathione and Sulfur Amino-Acid Metabolism: New Biochemical Insights and Therapeutic Approaches

Important insights have recently been derived from studies of inborn human defects of sulfur metabolism. Metabolic lesions responsible for homocystinuria have been elucidated, with possible implications for understanding atherogenesis in the general population. The cause of cystinosis remains enigmatic, but important information has been gained on the origin of some stored cystine from degraded protein. Cysteamine and ascorbic acid deplete the cystine content of cystinotic fibroblasts in vitro, and clinical trials with these agents have been undertaken. Studies of patients with glutathione synthetase deficiency have provided new understanding of the roles of glutathione as in antioxidant and as a modulator of microtubule-related processes. Studies of patients with this disorder and glucose-6-phosphate dehydrogenase deficiency, in which the capacity to maintain glutathione in the reduced state is compromised, indicate that pharmacologic doses of vitamin E can correct certain functional consequences of an inadequate supply of reduced glutathione both in erythrocytes and polymorphonuclear leukocytes. Much remains to be learned about the mechanisms of membrane damage in these states of enhanced oxidative susceptibility.

Accumulation of cystine from glutathione-cysteine mixed disulfide in cystinotic fibroblasts; blockade by an inhibitor of γ-glutamyl transpeptidase

Cystinotic and normal skin fibroblasts in tissue culture were treated with varying concentrations of reduced glutathione, oxidized glutathione and glutathione-cysteine mixed disulfide, substrates of gamma-glutamyl transpeptidase, the catabolic enzyme of the gamma-glutamyl cycle. Cystine accumulated more rapidly and to a greater extent from the glutathione-cysteine mixed disulfide in cystinotic than in normal cells. Inhibition of gamma-glutamyl transpeptidase activity by serine in a borate buffer partially blocked this accumulation of cystine. Reduced glutathione and oxidized glutathione have lesser effects on cystine accumulation. Stored cystine in cystinotic tissues may derive in part from glutathione-cysteine mixed disulfide via transpeptidation.

Depletion of Cystine in Cystinotic Fibroblasts by Drugs Enclosed in Liposomes

Summary: Lysosomally compartmentalized cystine can be removed from cystinotic cells by the use of reducing agents. In further investigation of means for converting cystine to a form or forms capable of penetrating the lysosomal membrane, a number of chemical agents either enclosed in phospholipid vesicles (liposomes) or free in solution were presented to cystinotic cells in tissue culture. After a 2-hr incubation, cystine content of cultured cystinotic cells was generally reduced more effectively by such agents in liposomes than in the medium. The most effective combination was cysteamine (MEA) in liposomes: a 0.5 mM dose of MEA reduced the cystine content of cystinotic cells 86% more when enclosed in liposomes than when dissolved at the equivalent dose in the medium. This observation could not be repeated when serum was omitted from the incubation medium, indicating that serum binds or otherwise inactivates cysteamine and that the liposome-enclosed cysteamine is protected from this action. Other liposome-entrapped compounds tested showed little if any depletion of intracellular cystine beyond that caused by non-liposome-enclosed drug action. Some agents increased the intracellular cystine content. Others of low molecular weight proved to be poorly retained by liposomes, a factor which may have been responsible for their relative ineffectiveness. Cysteamine, which is positively charged at neutral pH, was retained effectively when enclosed in “negative” liposomes made by inclusion of phosphatidic acid in the lipid mixture.[35S]Cysteamine (2.5 μCi, 23 μg/g), both free and enclosed in liposomes, was injected intravenously into mice. Ratios of MEA uptake by phagocytic as compared to non-phagocytic tissues (e.g., kidney/brain) were higher in liposome-injected than in control mice, indicating preferential uptake of liposome-enclosed MEA by selected tissues of mice corresponding to those exhibiting high levels of cystine accumulation in cystinotic patients. Cell fractionation of liver and kidney from MEA-liposome-treated mice showed a much higher proportion of radioactivity in the mitochondrial-lysosomal fraction than in control animals receiving the same drug dissolved in medium.Speculation: Drugs such as MEA and dithiothreitol (DTT) have been used in treatment of cystinotic patients, as they are effective in reducing intracellular accumulations of cystine in vitro. They are, however, toxic, particularly to brain tissue. Cyctine is known to accumulate in large amounts in certain actively endocytic tissues in cystinosis. The administration of liposomes containing drugs aimed at reducing or otherwise altering the accumulated cystine might prove advantageous as a therapeutic measure if selectivity of uptake into target tissues could be improved, particularly since these lipid carriers are nonallergenic and degradable by lysosomal enzymes after their uptake by phagocytic cells.