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We report on two Japanese brothers with hereditary deficiency in gamma-glutamyl transpeptidase. The propositus was 48 years old when he first visited our medical center and had a 51-year-old brother. The brothers were both tall and slender and had long limbs; the younger was diagnosed as having Marfan syndrome. Both patients both showed a tendency to retarded mental development. gamma-Glutamyltranspeptidase activity was below the detection limit of 1 IU/L in both patients. Glutathionaemia and glutathionuria were evident in both brothers. The analyses of sulphydryl compounds in the plasma (and serum for certain test items) and urine indicated high concentrations of glutathione, gamma-glutamylcysteine, cysteine and cysteinylglycine. Urine amino acid analysis on an automatic analyser showed a slightly increased excretion of cystine and a large peak in the citrulline position due, at least in part, to thio-compounds.

A mouse mutant with glutathionuria was discovered by screening for amino acidurias in the progeny of ethylnitrosourea-mutagenized mice. Total glutathione concentration was increased in both blood and urine but decreased in liver homogenates from affected mice. Glutathionuric mice exhibited lethargy, severe growth failure, shortened life spans and infertility. gamma-Glutamyl transpeptidase activity was deficient in kidney homogenates of glutathionuric mice. The glutathionuric phenotype in these mice is inherited as an autosomal recessive trait. This mouse mutant will be a useful animal model for the study of gamma-glutamyl transpeptidase physiology and glutathione metabolism.

1. Dosing rats with the gamma-glutamyl-transpeptidase inhibitor AT-125 results in the excretion of free glutathione in the urine of rat: this treatment did not lead to the excretion of glutathione conjugates of orally dosed xenobiotics, neither did AT-125 increase the biliary excretion of glutathione conjugates. 2. Dosing rat with AT-125 prior to dosing with 2-chloro-N-isopropylacetanilide decreased the excretion of 2-methylsulphonylacetanilide metabolites from 23% of the dose to < 0.5%. 3. We conclude that glutathione and glutathione-xenobiotic conjugates are probably not processed in vivo by the same pathway, and that AT-125 can alter the in vivo transport of mercapturic acid pathway metabolites.

A mentally retarded young woman with severe behaviour problems was found to excrete large amounts of glutathione due to a generalized gamma-glutamyl transpeptidase deficiency. As in the only other case described in detail, plasma levels and renal reabsorption of the amino acids were normal. In the parents' urine, plasma and leukocytes, enzyme activity was normal but in their cultured fibroblasts it was below the minimum for the control range. An autosomal recessive mode of inheritance is suggested. The implications of these findings for possible role of the gamma-glutamyl cycle in amino acid transport are briefly discussed.

Animals treated with potent gamma-glutamyl transpeptidase inhibitors and a patient with severe gamma-glutamyl transpeptidase deficiency excrete much larger than normal amounts of glutathione, gamma-glutamylcysteine, and cysteine in their urine; these compounds were found in disulfide forms. The findings indicate that the metabolic function of gamma-glutamyl transpeptidase is associated with the metabolism or transport (or both) of cysteine, gamma-glutamylcysteine, and glutathione, and that gamma-glutamylcysteine is a physiological substrate of the enzyme. The occurrence of gamma-glutamylcysteine in urine and other considerations suggest that this dipeptide is formed as an extracellular metabolite of glutathione in addition to its recognized role as an intrcellular precursor of glutathione. The dipeptide may be formed by a pathway involving transpeptidation or by cleavage of the Cys-Gly bond of glutathione. In the course of this work it was found that the mixed disulfide between glutathione and gamma-glutamylcysteine is a good substrate of glutathione reductase.

Acivicin (AT-125; 6.25-200 mumol/kg i.v.) inhibited hepatic, biliary and renal gamma-glutamyltranspeptidase (GGT) activity up to 88, 99 and 97%, respectively, in 4-week-old rats. This inhibition of GGT by acivicin resulted in a 10- to 12-fold increase in the biliary excretion of reduced (GSH) and oxidized glutathione. Because the biliary excretion of cysteinylglycine (Cys-Gly), Cys-Gly disulfide, cysteine (Cys) and cystine concomitantly decreased (63-99%), the biliary excretion rate of total glutathione-derived thiols and disulfides did not change. In contrast, acivicin treatment dramatically elevated the urinary excretion rate of glutathione-derived thiols in a dose-dependent fashion, resulting in a 390-fold increase at the highest dosage. This mainly originated from enhancement of urinary excretion of GSH (up to 7200-fold), although the excretion of Cys and Cys-Gly into urine was also increased. Acivicin treatment did not affect hepatic and renal levels of GSH but, at high dosages, reduced the concentration of Cys in these organs. GSH and oxidized glutathione concentrations in serum were increased, whereas cystine was diminished in acivicin-treated rats. Inhibition of GGT by acivicin (100 mumol/kg i.v.) failed to influence the biliary excretion of methylmercury but increased urinary excretion 34-fold. Even though the urinary thiol excretion was much higher than the biliary thiol excretion in the acivicin-treated rats, methylmercury was preferentially excreted into bile rather than urine, indicating the importance of the liver as an excretory organ for methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)

To clarify the peritubular mechanism for renal handling of plasma glutathione (GSH), variation of GSH levels in plasma, urine, kidney and liver was examined after intravenous administration of GSH to three groups of animals; control, acivicin-treated and rats treated with buthionine sulfoximine (BSO). Treatment of animals with BSO, a potent inhibitor of de novo GSH synthesis, markedly reduced hepatorenal GSH levels. Acivicin did not affect these levels. Upon intravenous injection of GSH (0.1 mmol/kg), renal GSH levels did not appreciably change in any of three animal groups. The rate of GSH disappearance from the circulation was rapid in control and BSO-treated rats, while it was markedly retarded in animals whose renal gamma-glutamyltransferase was extensively inactivated by acivicin. At 30 min after administration a significant amount of injected GSH was localized extracellularly (urine and plasma) in acivicin-treated animals. By contrast, most of the GSH rapidly disappeared from the extracellular space in control and BSO-treated animals. Together with the immunocytochemical evidence for the peritubular gamma-glutamyltransferase [Spater, H.W., Poruchynsky, M.S., Quintana, N., Inoue, M. & Novikoff, A.B. (1982) Proc. Natl Acad. Sci. USA 79, 3547-3550] the present results are fully consistent with the contention that the catalytic function of this enzyme is principally responsible for the peritubular mechanism for the renal handling of plasma GSH.

Treatment of mice with various gamma-glutamyl amino acids leads to marked urinary excretion of glutathione and other gamma-glutamyl compounds. There is good correlation between the affinity of gamma-glutamyl transpeptidase for various gamma-glutamyl amino acids and the extent of glutathionuria. The findings indicate that the administered gamma-glutamyl compounds effectively compete with glutathione (exported from kidney cells and present in the glomerular filtrate) for the enzyme. The administration of certain gamma-glutamyl amino acids appears to be a specific and nontoxic procedure for in vivo inhibition of gamma-glutamyl transpeptidase that may be useful in experimental work on glutathione metabolism and function and also for treatment of certain toxicities and for modulation of the metabolism of endogenous glutathione conjugates.

S-(2-chloroethyl)glutathione (CEG; 270 mumol/kg) produced renal lesions that were confined to the proximal tubules of the outer stripe of the outer medulla and were similar to those lesions produced by the cysteine analog S-(2-chloroethyl)cysteine or by the nephrotoxic glutathione (GSH) adduct of 2-bromohydroquinone. These histopathologic changes in the kidney were correlated with alterations in renal function as reflected by dose- and time-dependent elevations in blood urea nitrogen levels as well as by the increased urinary excretion of protein, glucose and lactate dehydrogenase activity. The role of renal GSH metabolism as a mediating factor in the nephrotoxicity of these GSH conjugates was investigated by administering the gamma-glutamyltranspeptidase inhibitor AT-125 [L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid]. Treatment with AT-125 led to a dose-dependent decrease in renal gamma-glutamyltranspeptidase activity that correlated inversely with increased GSH concentrations in the urine and kidney. Pretreatment with AT-125 ameliorated 2-bromohydroguinone-induced renal toxicity but did not protect against the CEG-induced renal lesion. In fact, pretreatment with AT-125 produced a dose-dependent potentiation of CEG renal toxicity. The CEG-induced renal lesion was dependent on a probenecid-sensitive transport system that was not involved in the toxicity of 2-bromohydroguinone. These studies demonstrate that CEG need not be metabolized by gamma-glutamyltranspeptidase to the corresponding cysteine adduct [S-(2-chloroethyl)cysteine] in order to enter renal tubule cells and ultimately exert its nephrotoxic action.

Several inhibitors of gamma-glutamyl transpeptidase in vitro [L-serine plus borate, 6-diazo-5-oxo-L-norleucine, and L- and D-gamma-glutamyl-(o-carboxy)phenylhydrazide] are active in vivo, as indicated by their effect in decreasing the conversion of administered D-gamma-glutamyl-L-alpha-amino[(14)C]butyrate to respiratory (14)CO(2) in mice. The hydrazides (both L and D isomers) are the most potent inhibitors in vitro and in vivo. Inhibition of gamma-glutamyl transpeptidase in vivo by the hydrazides is accompanied by extensive glutahionuria. The evidence suggests that a substantial fraction of the urinary glutathione arises from the kidney. The findings support the view that renal intracellular glutathione is normally translocated to the membrane-bound gamma-glutamyl transpeptidase as a separate step in the gamma-glutamyl cycle. Studies on in vivo inhibition of glutathione synthesis and of gamma-glutamyl transpeptidase provide direct evidence that glutathione is normally translocated from tissues to the blood plasma and that the turnover of plasma glutathione is relatively high. The data suggest that the low but significant steady-state level of glutathione in the plasma reflects synthesis of glutathione (predominantly in the liver) and its utilization by gamma-glutamyl transpeptidase (predominantly in the kidney). Thus, glutathione synthesized in cells that have transpeptidase may be translocated to and used by the membrane-bound enzyme, whereas glutathione synthesized in cells that lack the transpeptidase may be transported via the plasma to transpeptidase located on the membranes of other cells.