Changes In Glutathione Levels Research Articles

Changes in levels of glutathione and related compounds and activities of glutathione-related enzymes during rat liver regeneration.

The levels of glutathione and glutathione disulfide increased during the regeneration process of rat liver, reaching a maximum (about twice the control value) on day 2 and reverting to the normal level within 5 days. During this regeneration process, changes in the hepatic level of cysteine, glycine and glutamate, the substrates for glutathione synthesis, were determined. The cysteine level in liver increased, reaching a maximum on day 2 and returned to the normal level after 5 days. The levels of glycine and glutamate did not change. The enzyme activities of cystathionine-beta synthase and gamma-cystathionase for cysteine synthesis, and of gamma-glutamylcysteine synthetase, which is a limiting enzyme for glutathione synthesis, were clearly increased in regenerating liver. The increase of glutathione level could be clearly accounted for by the elevation of these enzyme activities.

The role of lipid peroxidation in menadione-mediated toxicity in cardiomyocytes

The role of lipid peroxidation in menadione-mediated toxicity was studied in neonatal rat Cardiomyocytes. Incubation of Cardiomyocytes with menadione resulted in depleted cellular glutathione levels, increased intracellular Ca 2+ and increased lipid peroxidation which all occurred prior to cell degeneration. Pre-treatment of cells with cysteine suppressed the menadione-induced cell degeneration and prevented changes in glutathione levels, intracellular Ca 2+. and lipid peroxidation. Pre-treatment of cells with fura-2 acetoxymethyl ester, a Ca 2+ chelator, reduced menadione-induced c cel degeneration and lipid peroxidation but it did not block cellular glutathione depletion. Pre-treatment of cells with deferoxamine mesylate. an iron chelator, also reduced both menadione-induced cell degeneration and lipid peroxidation: however, it did not prevent the menadione-induced increase in intracellular Ca 2+, nor the depletion of glutathione. Thus, the inhibition of menadione-induced lipid peroxidation by deferoxamine mesylate prevented cell degeneration even though intracellular Ca 2+ remained elevated and glutathione remained depleted. The protective effects of deferoxamine mesylate and fura-2 AM on menadione's toxicity were inhibited by addition of FeCl 3 to cells. Ferric ions did not inhibit the protective effect of cysteine. These data suggest that menadione-induced cardiomyocyte degeneration is directly linked to iron-dependent lipid peroxidation and less tightly coupled to elevation in intracellular Ca 2+ or depletion of glutathione.

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.

Effects of Aminotriazole on Lung Toxicity of Paraquat Intoxicated Mice

Background: Paraquat, a widely used herbicide, is extremely toxic, causing multiple organ failure in humans. Paraquat especially leads to irreversible progressive pulmonary fibrosis, which is related to oxygen free radicals. However, its biochemical mechanism is not clear. Natural mechanisms that prevent damage from oxygen free radicals include changes in glutathione level, G6PDH, superoxide dismutase(SOD), catalase, and glutathione peroxidase. The authors think catalase is closely related to paraquat toxicity in the lungs Method: The effects of 3-amino-1,2,4-triazole(aminotriazole), a catalase inhibitor, on mice administered with paraquat were investigated. We studied the effects of aminotriazole on the survival of mice administered with paraquat, by comparing life spans between the group to which paraquat had been administered and the group to which a combination of paraquat and aminotriazole had been administered. We measured glutathion level, glucose 6-phosphate dehydrogenase(G6PDH), superoxide dismutase(SOD), catalase, and glutathione peroxidase(GPx) in the lung tissue of 4 groups of mice: the control group, group A(aminotriazole injected), group B(paraquat administered), group C(paraquat and aminotriazole administered). Results: The mortality of mice administered with paraquat which were treated with aminotriazole was significantly increased compared with those of mice not treated with aminotriazole. Glutathione level in group B was decreased by 20%, a significant decrease compared with the control group. However, this level was not changed by the administration of aminotriazole(group C). The activity of G6PDH in all groups was not significantly changed compared with the control group. The activities of SOD, catalase, and glutathione peroxidase(GPx) in the lung tissue were significantly decreased by paraquat administration(group B); catalase showed the largest decrease. Catalase and GPX were significantly decreased by aminotriazole treatment in mice administered with paraquat but change in SOD activity was not significant(group C). Conclusion: Decrease in catalase activity by paraquat suggests that paraquat toxicity in the lungs is closely related to catalase activity. Paraquat toxicity in mice is enhanced by aminotriazole administration, and its result is related to the decrease of catalase activity rather than glutathione level in the lungs. Production of hydroxyl radicals, the most reactive oxygen metabolite, is accelerated due to increased hydrogen peroxide by catalase inhibition and the lung damage probably results from nonspecific tissue injury of hydroxyl radicals.

Two pathways for chromium(VI)-induced DNA damage in 14 day chick embryos: Cr-DNA binding in liver and 8-oxo-2'-deoxyguanosine in red blood cells.

Previous studies have shown that chromium(VI) induced cell-specific types of DNA damage, i.e. DNA cross-links in liver and DNA strand breaks in red blood cells, in 14 day chick embryos. Direct and indirect pathways for chromium(VI)-mediated DNA damage, in the form of Cr-DNA binding and 8-oxo-2'-deoxyguanosine (8-oxo-dG) respectively, were examined in liver and red blood cells of 14 day chick embryos. Levels of hepatic Cr-DNA binding increased in a Cr(VI) dose-dependent manner. Cr-DNA binding in red blood cells was 10-fold lower than in liver, although the Cr-uptake in red blood cells was only 2-fold lower than in liver. The level of 8-oxo-dG formation in red blood cells increased at all Cr(VI) doses tested but peaked at 0.10 mmol Cr(VI)/kg, whereas no increase in 8-oxo-dG levels over background levels was observed in liver of Cr(VI)-treated embryos. The possible role of glutathione in modulating Cr(VI)-induced DNA damage was examined by using L-buthionine-R,S-sulfoximine (BSO) to deplete glutathione. No changes in glutathione levels were observed in either liver or red blood cells of embryos treated with Cr(VI) in the presence or absence of BSO pretreatment. Ascorbate levels in liver and red blood cells were not affected by treatment of embryos with chromium(VI), BSO or Cr(VI) and BSO. Depletion of glutathione by BSO resulted in a small increase of chromium uptake in liver of embryos treated with 0.050 and 0.10 mmol Cr(VI)/kg, but had no effect on hepatic chromium uptake at 0.20 mmol Cr(VI)/kg. BSO had no effect on chromium uptake in red blood cells. Depletion of glutathione had no effect on hepatic or red blood cells Cr-DNA binding in embryos treated with Cr(VI). However, depletion of glutathione significantly decreased the 8-oxo-dG levels in red blood cells at all Cr(VI) doses tested. Levels of 8-oxo-dG in liver of Cr(VI)-treated embryos remained at background in the presence or absence of BSO pretreatment. These results indicate that Cr(VI)-induced DNA damage in 14 day chick embryos is through a direct interaction of chromium with DNA in liver, but is through an indirect oxidative pathway in red blood cells. It appears that glutathione plays an important role in chromium(VI)-induced formation of 8-oxo-dG in red blood cells.

L-BOAA induces selective inhibition of brain mitochondrial enzyme, NADH-dehydrogenase

Lathyrism, a human neurological disorder has been linked to the excessive consumption of a plant toxin, β-oxalylamino- l-alanine ( l-BOAA) present in Lathyrus sativus. The present study was carried out to elucidate the biochemical mechanisms underlying l-BOAA-induced toxic insult. Incubation of sagittal slices of mouse brain with l-BOAA resulted in dose and time-dependent inhibition of mitochondrial NADH-dehydrogenase (NADH-DH). Significant inhibition of NADH-DH was seen following incubation of brain slices with very low concentration of l-BOAA (0.1 pM). l-BOAA also induced lactate dehydrogenase (LDH) leakage from the slice into the medium in dose-dependent manner. The inhibition of NADH-DH preceded LDH leakage from the slices into the medium. l-BOAA had no effect on other mitochondrial enzymes, namely, isocitrate dehydrogenase or cytochrome c oxidase. Incubation of isolated mouse brain mitochondria with l-BOAA also resulted in inhibition of NADH-DH. l-BOAA-induced inhibition of NADH-DH was prevented by non- N-methyl- d-aspartate (non-NMDA) glutamate receptor antagonists in general and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist (NBQX) in particular. Other glutamate agonists examined namely, N-methyl- d-aspartate, β- N-methylamino- l-alanine ( l-BMAA), l-glutamic acid, N-acetylaspartylglutamate (NAAG), quisqualic acid, kainic acid or AMPA did not have any effect on NADH-DH activity in slices although they induced LDH leakage from the slice into the medium. Incubation of brain slices with l-BOAA did not induce lipid peroxidation or changes in glutathione levels. Prior incubation of slices with glutathione (GSH) or GSH-isopropyl ester did not prevent l-BOAA-induced inhibition of NADH-DH. However, incubation of isolated mitochondria with l-BOAA in the presence of GSH-isopropyl ester prevented l-BOAA-induced inhibition of NADH-DH, indicating the protective effect of mitochondrial glutathione in the prevention of l-BOAA-induced toxicity.

Mechanism for the changes in levels of glutathione upon exposure of cultured mammalian cells to tertiary-butylhydroperoxide and diamide.

Qualitative and quantitative changes associated with cellular glutatione (GSH) in response to oxidants were investigated in cultured Chinese hamster V79 cells. Incubation of cells with benzoylperoxide (BZP), tert-butylhydroperoxide (t-BuOOH), hydrogen peroxide or diamide for 1 h reduced the level of total GSH (GSH + GSSG). Among the oxidants, t-BuOOH and diamide caused an increase in levels of glutathione disulfide (GSSG) and a resultant increase in the ratio of the level of GSSG to the level of total GSH, suggestive of the induction within the cells of a pro-oxidant state by the oxidants. o-Phenanthroline, a chelator of divalent ion, almost completely suppressed the decrease in levels of total GSH caused by t-BuOOH while it did not suppressed either increases in levels of GSSG or increases in the ratio of the levels of GSSG to that of total GSH caused by the hydroperoxide. These results suggest that reactive oxygen radicals are involved in the decrease in levels of GSH by treatment with t-BuOOH but not in the increase in the level of GSSG. After treatment with either t-BuOOH or diamide for 1 h, the level of GSH rapidly increased to more than twice the control level during 15-45 min of post-treatment incubation. o-Phenanthroline almost completely suppressed the increase in levels of GSH caused by t-BuOOH, while it did not affect the changes caused by diamide, suggesting a difference between the mechanisms by which t-BuOOH and diamide cause increases in levels of GSH. It seems likely that reactive oxygen radicals participate not only in the decrease in levels of GSH caused by t-BuOOH but also in the rapid increase that occurs after such treatment. Hence, the first decrease in levels of GSH by the hydroperoxide may be causally related to the latter increase. The amount of [35S]-cysteine taken up by cells after treatment with t-BuOOH was about one half of that taken up by control cells. By contrast, the rate of incorporation of radioactive cysteine into acid-soluble material increased to more than twice that of the controls after treatment with t-BuOOH. The increase in the rate of incorporation of [35S]cysteine into acid-soluble material caused by t-BuOOH was not a consequence of inhibition by the hydroperoxide of utilization of cysteine for protein synthesis. Inhibition of protein synthesis by cycloheximide caused neither an increase in the incorporation of cysteine into acid-soluble material nor an increase in rate of biosynthesis of GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Antioxidant response and winter hardiness in red spruce (Picea rubens Sarg.)

SUMMARYAntioxidant metabolism has been examined in red spruce growing at two sites with similar levels of ozone pollution but different winter temperatures. The northern site was Whiteface Mountain, New York with a latitude of 44° 31′(NY). The southern site was Whitetop Mountain, Virginia with a latitude of 36° 54′(VA). The northern site has lower winter temperatures. At the NY site two populations of trees were denned with respect to cold tolerance. An effort was made to establish a temporal relationship between the antioxidant capacity of conifer needles on one hand and the different stages of winter hardening/dehardening, and the induction of dormancy, on the other. The hypothesis tested was that winter injury of red spruce was due to an increased demand for glutathione at levels exceeding the inherent capacity of the needles and/or the altered activities of glutathione reductase.In the autumn, as temperatures began to decrease, photosynthetic potential decreased as spruce entered dormancy. At the same time, cold tolerance (measured as needle electrolyte leakage) gradually increased. Glutathione metabolism appeared to be coupled to these processes. At both sites, seasonal changes in glutathione levels were observed. These changes were found to coincide temporally with the onset and cessation of dormancy (as measured by decline in photosynthetic activity). The same pattern was observed at the northern and the southern site with the winter increases being greater at the former. Glutathione began to increase as soon as needles entered dormancy and began hardening. Glutathione reductase activities closely followed the dehardening process during spring, while glutathione concentration continued to stay relatively high with prehardening levels reached only by summer. At the northern site during the winter, cold‐tolerant trees had significantly higher glutathione reductase activities compared with cold‐sensitive ones. Based on these data, it is proposed that individuals that cannot respond to the increased demand for glutathione reductase activity during the hardening, winter and dehardening periods, become susceptible to winter injury.

Biochemical responses of skin to allergenic and non-allergenic nitrohalobenzenes. Evidence that an NADPH-dependent reductase in skin may act as a prohapten-activating enzyme.

Using a selection of 'classic' haptens (dinitrohalobenzenes and picryl chloride) and related non-sensitizing analogous, we examined changes in levels of glutathione (GSH) and glutathione disulphide (GSSG) in mouse skin 12 h after their epicutaneous application. We observed that elevation of GSSG levels and/or depletion of GSH levels correlated well with contact allergenic potential. Non-sensitizing analogous failed to perturb GSH/GSSG status. In vitro assays using mouse skin and rat liver microsomal preparations indicated that only the allergenic nitrohalobenzenes initiated NADPH-dependent oxygen utilization, with the activity falling off in the order picryl chloride >> DNIB > DNBB > DNCB > DNFB. In addition, an examination of the colour of mouse skin homogenates ex vivo after application of the dinitrohalobenzenes showed significant yellowing (consistent with aromatic nucleophilic substitution) only with DNFB. Our results indicate that, while an aromatic nucleophilic substitution reaction with skin protein can possibly account for the allergenicity of DNFB, it does not seem to occur with DNCB, DNBB or DNIB. These may instead behave mainly as prohaptens which are activated enzymically by NADPH-dependent reductase(s) within the skin, with the concomitant generation of superoxide and hydrogen peroxide, to form potentially protein-reactive free radical and other metabolites. Picryl chloride appears capable of both conjugating directly with proteins by aromatic nucleophilic substitution and undergoing NADPH-dependent metabolism to other potentially protein-reactive metabolites.

Glutathione in Parkinson's disease: a link between oxidative stress and mitochondrial damage?

Several links exist between the two mechanisms of neuronal degeneration (i.e., oxygen radical production and mitochondrial damage) proposed to have a role in Parkinson's disease. Indeed, mitochondria are critical targets for the toxic injury induced by oxygen radicals, and experimental evidence suggests that mitochondrial damage may cause an increased generation of oxygen radicals. A potentially important link between these two mechanisms of neurodegeneration is glutathione. Because of the scavenging activity of glutathione against accumulation of oxygen radicals, its decrease in the brains of parkinsonian patients has been interpreted as a sign of oxidative stress; however, this change may also result from or lead to mitochondrial damage. It is conceivable therefore that regardless of whether oxidative stress or mitochondrial damage represents the initial insult, these toxic mechanisms may both contribute to neuronal degeneration via changes in glutathione levels.

Additional evidence of autoxidation as a possible mechanism of neutrophil locomotory dysfunction in blunt trauma

Previous studies in victims of blunt injury suggest that the observed neutrophil (PMN) locomotory dysfunction is, in part, due to autoxidation. To further clarify the occurrence and significance of autoxidation, we studied changes in levels of glutathione in PMN and of ascorbic acid and alpha-tocopherol in serum and blood cells of postsurgical and blunt trauma patients. Levels of total, reduced, and oxidized glutathione in PMN from trauma patients were similar to normal controls. Serum and cellular ascorbic acid and alpha-tocopherol levels dropped significantly after injury and remained below normal control levels during the 7 to 8-day study period. Low serum alpha-tocopherol was partially explainable on the basis of changes in serum lipids. When serum samples of trauma patients were thawed unprotected without pyrogallol, there was significant loss of recoverable alpha-tocopherol, whereas no significant losses occurred with unprotected thawed normal sera. Less total reducing capacity was observed in PMN of trauma patients compared with normal controls. These findings indicate that synthesis and regeneration capacity of glutathione are intact but that the levels of the consumable antioxidants, ascorbic acid, and alpha-tocopherol are compromised after injury. These results add further support to the hypothesis that autoxidation occurs in trauma.

Glutathione metabolism under the influence of hydroperoxides in the lactating mammary gland of the rat. Effect of glucose and extracellular ATP.

Tert-butyl hydroperoxide decreases GSH and total free glutathione (GSH + 2GSSG) contents of acini from lactating mammary glands. The decrease in total free glutathione can be explained by an increase in mixed disulfide formation and by excretion of GSSG to the extracellular medium, and subsequent degradation catalyzed by gamma-glutamyl transpeptidase. Low concentrations of glucose prevented the changes in glutathione levels induced by the peroxide. In the presence of extracellular ATP, glucose did not prevent these changes. However, incubations with the peroxide, did not alter the rate of other metabolic pathways by acini.

The importance of membrane sulfhydryl groups to calcium homeostasis in the lens.

This study focused on whether changes in lens levels of glutathione and calcium, early events associated with cataract formation, were related or that one might cause the other. The first part of the investigation was concerned with the extent to which an increase in levels of intracellular calcium might alter GSH levels in lens fiber and epithelial cells. The results demonstrate that calcium accumulation, either at 19 degrees C or 37 degrees C, did not diminish the concentration of GSH. More importantly, GSH levels did not decline in opaque regions of a calcium-loaded lens. The reciprocal part of the problem focused on whether a decline in lens thiol might lead to an increase in levels of calcium and subsequent opacification. In particular, it was shown that treatment of lenses with parachloromercuribenzene sulphonic acid (PCMBS), a nonpenetrating sulphydryl probe, resulted in a 10-30% loss of membrane SH groups in the epithelium. Diminished numbers of SH groups was accompanied by chloride fluxes and an increase in membrane permeability to sodium and calcium with an influx of sodium and calcium leading to opacities. It is important to note that these changes occurred in the absence of any change in cellular levels of soluble protein-SH or GSH. Additional experiments suggest that calcium transport was not impaired, as evidenced by lack of inhibition of Ca-ATPase activity in lenses treated with PCMBS. The results suggest that one explanation for opacification is that oxidative insults, which diminish GSH levels, leads to a loss of important membrane SH groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between liver and kidney levels of glutathione and metallothionein in rats

The purpose of this study was to test if the tissue levels of glutathione and metallothioneins were inter-related. In rats, intraperitoneal administration of diethyl maleate or bromobenzene decreased glutathione levels in both the liver and the kidney before doubling the metallothionein concentration in the liver and increasing that in kidneys by 40% starting from 6 h after intraperitoneal administration. Both Zn and Cd produced an increase in hepatic and renal metallothionein levels. However, unlike the responses to diethyl maleate and bromobenzene, the increase in metathionein caused by the metals was not preceded by any significant changes in glutathione levels. Cd decreased the concentration of glutathione in the liver (at 36 h) and kidneys (at 24 h). In contrast, Zn produced an increase and no change in hepatic and renal glutathione concentrations, respectively. The conclusion is that tissue levels of metallothionein and glutathione are not always interrelated.

Changes in glutathione in acute and chronic alcohol intoxication

After 5 hours of acute ethanol intoxication, the levels of reduced glutathione of liver, heart, kidney and brain decreased to 60, 71, 70 and 83% of controls, respectively. The decrease was less when animals were pretreated with pyrazole. Pretreatment with disulfiram potentiated the effect of ethanol. Levels of GSSG were not altered by these treatments. Chronic ethanol administration, in a liquid diet, also produced a decrease in GSH levels. The lowest levels were observed at 4 weeks in liver (65%). However, there was a slow recovery to 75% and 80%, respectively, at 10 and 14 weeks of treatment. Withdrawal from alcohol administration after 10 and 14 weeks intoxication resulted in a complete recovery in GSH levels within 24 hours. Similar changes were seen in other tissues, although they were less marked than in liver, and no changes were observed in GSSG. To clarify the changes in glutathione levels, we measured the main enzymes connected with the synthesis (γ-glutamylcysteine synthetase + glutathione synthetase) and utilization (γ-glutamyl transpeptidase) of glutathione. In liver, the activity of the glutathione synthesizing system was unchanged after 4 weeks, but increased to 140% within 10 weeks; γ-glutamyl transpeptidase was slightly elevated (120%) at 4,10 and 14 weeks. Similar changes in these enzymes were observed in kidney and brain. γ-glutamyl transpeptidase was elevated in serum (120%) at 4 weeks, but reached 140% within 10 weeks. The levels of the glutathione synthesizing system returned to normal in all tissues within 2 days after alcohol withdrawal; however, γ-glutamyl transpeptidase levels did not revert to normal until one week after withdrawal.

Changes in glutathione in acute and chronic alcohol intoxication

After 5 hours of acute ethanol intoxication, the levels of reduced glutathione of liver, heart, kidney and brain decreased to 60, 71, 70 and 83% of controls, respectively. The decrease was less when animals were pretreated with pyrazole. Pretreatment with disulfiram potentiated the effect of ethanol. Levels of GSSG were not altered by these treatments. Chronic ethanol administration, in a liquid diet, also produced a decrease in GSH levels. The lowest levels were observed at 4 weeks in liver (65%). However, there was a slow recovery to 75% and 80%, respectively, at 10 and 14 weeks of treatment. Withdrawal from alcohol administration after 10 and 14 weeks intoxication resulted in a complete recovery in GSH levels within 24 hours. Similar changes were seen in other tissues, although they were less marked than in liver, and no changes were observed in GSSG. To clarify the changes in glutathione levels, we measured the main enzymes connected with the synthesis (γ-glutamylcysteine synthetase + glutathione synthetase) and utilization (γ-glutamyl transpeptidase) of glutathione. In liver, the activity of the glutathione synthesizing system was unchanged after 4 weeks, but increased to 140% within 10 weeks; γ-glutamyl transpeptidase was slightly elevated (120%) at 4,10 and 14 weeks. Similar changes in these enzymes were observed in kidney and brain. γ-glutamyl transpeptidase was elevated in serum (120%) at 4 weeks, but reached 140% within 10 weeks. The levels of the glutathione synthesizing system returned to normal in all tissues within 2 days after alcohol withdrawal; however, γ-glutamyl transpeptidase levels did not revert to normal until one week after withdrawal.

Blood glutathione in mild diabetes mellitus before treatment and during sulfonylurea-induced hypoglycemia.

Summary1. Blood glucose and reduced glutathione determinations were performed at intervals on 9 non-diabetic patients and on 15 patients with moderately uncontrolled diabetes following oral administration of 3 g of tolbutamide or carbutamide. 2. Mean maximal decrease from fasting blood glucose value was 14% in patients with normal carbohydrate metabolism, and 43% in the diabetic group. 3. In control patients there was no change in glutathione levels during the test period. In diabetic patients a minimal, insignificant rise had occurred 5 hours after ingestion of the hypoglycemic agent. 4. The mean blood reduced glutathione concentration was strikingly lower in patients with diabetes than in those whose carbohydrate metabolism was intact. In 4 patients subnormal values persisted despite restoration of normal fasting blood glucose levels by daily therapeutic doses of tolbutamide.