Brain Levels of NADH and NAD+ Under Hypoxic and Hypoglycaemic Conditions In Vitro

The effects of hypoxia and hypoglycaemia on the redox state in vitro have been studied. NADH and NAD+ were extracted simultaneously from superfused cerebral cortex slices and assayed by bioluminescence. The results show a nonsignificant increase in NADH and the redox ratio in "mild hypoxia," whereas "severe hypoxia" produced an increase of over 200% in NADH and in the NADH/NAD+ ratio. When the glucose in the incubation medium was reduced from its control value of 10 mM to 0.5 mM, significant decreases in NADH and the redox ratio to 60% of control value were observed. Further decreasing the glucose to 0.2 mM gave lower levels of NADH and the redox ratio (40% of control). The effects on the redox state of alternative substrates to glucose were also tested. Replacement of glucose by 10 mM pyruvate decreased the NADH by 77% and the NADH/NAD+ ratio by 79%. Replacement of glucose with 10 mM lactate gave decreases of 70% and 71%, respectively, whereas in the presence of 15 mM 2-deoxyglucose and 5 mM glucose, the NADH was decreased by 56% and the ratio by 50%. The results are discussed in relation to levels of creatine phosphate and ATP, as well as evoked action potentials, observed from parallel studies.     

A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD⁺ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD⁺/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD⁺/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD⁺/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD⁺/NADH, and pH. These distributions were calculated using physiological NAD⁺/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.     

Alleviating Redox Imbalance Enhances 7-Dehydrocholesterol Production in Engineered Saccharomyces cerevisiae

Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms. In this study, 7-dehydrocholesterol (7-DHC, a crucial precursor of vitamin D₃) biosynthesis pathway was constructed in Saccharomyces cerevisiae BY4742 with endogenous ergosterol synthesis pathway blocked by knocking out the erg5 gene (encoding C-22 desaturase). The deletion of erg5 led to redox imbalance with higher ratio of cytosolic free NADH/NAD⁺ and more glycerol and ethanol accumulation. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) and an alternative oxidase (AOX1) were employed in our system based on cofactor regeneration strategy. Consequently, the production of 7-dehydrocholesterol was increased by 74.4% in shake flask culture. In the meanwhile, the ratio of free NADH/NAD⁺ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L. This study provides a reference to increase the production of some desired compounds that are restricted by redox imbalance.     

Alteration of the NAD+/NADH ratio in CHO cells by stable transfection with human cytosolic glycerol-3-phosphate dehydrogenase: resistance to oxidative stress.

The intracellular level of the NAD+/NADH ratio plays a vital role in sustaining and coordinating the catabolic reaction of the cell, and reflects the redox state of cytosol. Antioxidants play a role to protect cytosol and membrane from free radicals. This role of antioxidants involves sustaining cell viability and the procedure is thought to be regulated by the equilibrium of the redox state of the cell. However, there is very little known about how the NAD+/NADH level is set and changed. To alter the ratio, human NAD-dependent glycerol-3-phosphate dehydrogenase (cGPDH) cDNA was transfected stably in CHO dhfr- cells. When compared to parental CHO cells, cGPDH activities of the transfected cells were increased 8-12 fold, but the NAD+/NADH ratio was decreased. Specific growth rate of the transfected cells was similar to or slight lower than that of wild type CHO cells. Cell viability of the stable transformants against H2O2 was increased without change of either catalase or glutathione peroxidase activity. However, the increase of cell viability was correlated with the decrease of NAD+/NADH ratio in transfectants. From these results, it is suggested that the overexpression of cGPDH changes the NAD+/NADH ratio toward a decrease, and by this change in the redox state the cell confers more resistance against H2O2.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state

Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.     

The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli

Escherichia coli MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT 相似文献   

Increased estradiol-estrone quotient in rat liver by hydroxysteroids: an effect of the specific hydrogen transfer between steroids]

After addition of estrone to rat liver slices, a quotient of estradiol/estrone of ca. 0.1 is reached within 1 - 2 min. By additional application of 17 beta-hydroxysteroids this quotient is changed in the direction of estradiol, although the applied concentrations of both steroids are far below the concentration of the cytoplasmic redox couple NADH/NAD. Of all the steroids tested, testosterone had the strongest influence on the quotient, especially in the liver of female rats. This influence is smaller in the livers of male rats and infantile animals. The changing of the E2/E1 quotient by testosterone can be inhibited by the antiandrogen cyproteron acetate. Steroids with hydroxy groups at C-3 or C-20 or high concentrations of non-steroids, which can be oxidized by NAD, change the E2/E1 quotient only minimally. The experiments demonstrate that in liver, the redox couple estradiol/estrone is not in equilibrium with the main redox couple of the cytoplasmic NADH/NAD. Only on account of this fact it is possible that relatively low concentrations of testosterone change the E2/E1 quotient via the C-17 leads to C-17 hydrogen transfer between steroids. Biological consequences are discussed.     

L-NAME co-treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation

Based on the fact that vitamin A in clinical doses is a potent pro-oxidant agent to the lungs, we investigated here the role of nitric oxide (NO?) in the disturbances affecting the lung redox environment in vitamin A-treated rats (retinol palmitate, doses of 1000-9000 IU?kg(-1)?day(-1)) for 28 days. Lung mitochondrial function and redox parameters, such as lipid peroxidation, protein carbonylation and the level of 3-nytrotyrosine, were quantified. We observed, for the first time, that vitamin A supplementation increases the levels of 3-nytrotyrosine in rat lung mitochondria. To determine whether nitric oxide (NO ?) or its derivatives such as peroxynitrite (ONOO-) was involved in this damage, animals were co-treated with the nitric oxide synthase inhibitor L-NAME (30 mg?kg(-1), four times a week), and we analysed if this treatment prevented (or minimized) the biochemical disturbances resulting from vitamin A supplementation. We observed that L-NAME inhibited some effects caused by vitamin A supplementation. Nonetheless, L-NAME was not able to reverse completely the negative effects triggered by vitamin A supplementation, indicating that other factors rather than only NO? or ONOO- exert a prominent role in mediating the redox effects in the lung of rats that received vitamin A supplementation.     

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP⁺/reduced NADP and NAD⁺/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD⁺/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples. cell proliferation; contact inhibition; glutathione     

Vitamin E deficiency in the rat. IV. Alteration in mitochondrial membrane and its relation to respiratory decline

The respiratory control and rate of oxidation of exogenous NADH in vitro by liver mitochondria from vitamin E deficient rats were studied as a means of providing information concerning possible mitochondrial membrane alterations due to the deficiency.When mitochondria were aged at different temperatures for various periods of time, half-maximal inhibition of respiratory control occurred at lower temperatures and shorter aging periods in deficient mitochondria than in normal ones. Also, respiratory control was lost more rapidly in deficient mitochondria than in normal ones in the presence of either digitonin or low (hypotonic) concentrations of mannitol.Microsomes, both freshly prepared and boiled, dramatically lowered respiratory control and the effect was greater in the deficient mitochondria. Bovine serum albumin overcame the suppressed respiratory control, and exogenously added fatty acids mimiced the action of the microsomes.NADH oxidation by normal mitochondria proceeded slowly in isotonic media, while mitochondria of vitamin E deficient rats oxidized NADH much more rapidly. When mitochondria were subjected to ultrasonic disruption or incubated in hypotonic media, the rates of NADH oxidation by both types of mitochondria were similar.Respiratory decline associated with oxidation of β-hydroxybutyrate by the deficient mitochondria was decreased by including in the medium either a high concentration of NAD⁺, 0.5 mm oxalacetate, or 2 mm aspartate plus 1 mm α-ketoglutarate. This observation, plus the finding of similar activities of malate dehydrogenase and glutamic-oxalacetic transminase in normal and deficient livers, suggests that the action of each was due to an elevation of the mitochondrial NAD⁺/NADH ratio via a malate shuttle and cytoplasmic and mitochondrial glutamic-oxalacetate transaminase. It is postulated that the marked mitochondrial respiratory decline in the deficient rats is attributed to a limiting availability of NAD⁺ and a low ratio of NAD⁺ to NADH.     

High-resolution visualization of oxygen distribution in the liver in vivo

Microcirculatory failure after stress events results in mismatch in oxygen supply and demand. Determination of tissue oxygen distribution in vivo may help elucidate mechanisms of injury, but present methods have limited resolution. Male Sprague-Dawley rats were anesthetized, prepared for intravital microscopy, and received intravenously the oxygen-sensitive fluorescent dye Tris(1,10-phenanthroline)ruthenium(II) chloride hydrate [Ru(phen)3(2+)]. An impaired hepatic oxygen distribution was induced by either phenylephrine or hemorrhage. Intensity of Ru(phen)3(2+) fluorescence was compared with NADH autofluorescence indicating changes in the mitochondrial redox potential. Ethanol was injected to affect the NADH-to-NAD+ ratio without altering the P(O2). Infusion of Ru(phen)3(2+) resulted in a heterogeneous fluorescence under baseline conditions reflecting the physiological acinar P(O2) distribution. A decrease in oxygen supply due to phenylephrine or hemorrhage was paralleled by an increase in Ru(phen)3(2+) and NADH fluorescence reflecting an impaired mitochondrial redox state. Ethanol did not alter Ru(phen)3(2+) fluorescence but increased NADH fluorescence indicating independence of P(O2) and redox state imaging. Intravenous administration of Ru(phen)3(2+) for intravital videomicroscopy represents a new method to visualize the hepatic tissue P(O2). Combined with NADH autofluorescence, it provides additional information regarding the tissue redox state.     

Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

Energy-conserving reactions in phosphorylating electron-transport particles from Nitrobacter winogradskyi. Activation of nitrite oxidation by the electrical component of the protonmotive force.

1. In electron-transport particles (ET particles) prepared from Nitrobacter winogradskyi, the uncoupling agent carbonyl cyanide phenylhydrazone increased the rate of NADH oxidation but decreased the rate of oxidation of NO2-. Its effectiveness in stimulating NADH oxidation closely paralleled its effectiveness in inhibiting NO2- oxidation. 2. In the presence of ADP and phosphate the oxidation of NADH was stimulated, whereas the oxidation of NO2- was inhibited. In the presence of excess of Pi the concentration dependence with respect to ADP was the same for acceleration of NADH oxidation and inhibition of NO2- oxidation. 3. Oligomycin inhibited NADH oxidation and stimulated the oxidation of NO2-. The concentration of oligomycin required to produce half-maximal effect in both systems was the same. 4. The apparent Km for NO2- was not affected by ADP together with Pi, by uncoupling agent or by oligomycin. 5. With NADH as substrate, classical respiratory control was observed. With NO2- as substrate the respiratory-control ratio was less than unity. 6. A reversible uptake of H+ accompanied the oxidation of NO2- by ET particles. 7. In the presence of NH4Cl or cyclohexylamine hydrochloride, H+ uptake was abolished and increased rates of NO2- oxidation were observed. When valinomycin was present in the reaction medium, low concentrations of NH4Cl inhibited NO2- oxidation. 8. Pretreatment of ET particles with oligomycin enhanced the stimulation of NO2- oxidation induced by NH4Cl or by cyclohexylamine hydrochloride. Pretreatment with the uncoupler carbonyl cyanide phenylhydrazone prevented these stimulations. 9. In the presence of dianemycin together with K+, the uptake of H+ was abolished and the rate of NO2- oxidation was increased. In contrast, in the presence of valinomycin together with K+, the uptake of H+ was increased, and the rate of NO2- oxidation decreased. 10. Sodium tetraphenylboron was found to be an inhibitor of NO2- oxidation, but caused a stimulation of NADH oxidation which was dependent on the presence of NH4Cl or cyclohexylamine hydrochloride. 11. It is concluded that the enhanced rate of NO2- oxidation observed in the absence of energy-dissipating processes clearly relates to some state before the involvement of adenine nucleotides, and it is suggested that the oxidation of NO2- generates a protonmotive force, the electrical component of which controls the rate of NO2- oxidation.     

Pyridine nucleotide pool size changes in iron-deficient Plantago lanceolata roots during reduction of external oxidants

The involvement of pyridine nucleotides in the reduction of extracytoplasmatic electron acceptors by iron-deficient Plantago lanceolata L. roots has been examined by measuring the changes in NAD(P)H and NAD(P)⁻ induced by various external acceptors. Exposure of the plants to FeEDTA, ferricyanide, ferric citrate or hexachloroiri-date resulted in a transient decrease in NADPH and an increase in NAD⁻. No major differences in this pattern were observed between acceptors which were assumed to be reduced by different enzymes. The application of the membrane-permeable oxidant nitro blue tetrazolium led to similar changes in reduced and oxidized pyridine nucleotides and decreased the reduction of external acceptors. The amino acid analog p -fluorophenylalanine caused a transient decline in both NADPH level and NADPH/ NADP⁻ ratio and a decrease in the ratio of NADH to NAD⁻ without affecting the level of NADH. Exposure of the plants to the translation inhibitor cycloheximide increased both NADH and NADPH concentrations. A comparison of the redox activities and pyridine nucleotide fractions after inhibitor treatment revealed that the constitutive, but not iron stress-induced redox activity correlates with NADPH levels. These results are interpreted as confirming that the redox systems on the root plasma membrane are separately regulated. Possible metabolic reactions during the reduction processes are discussed.     

Vitamin E stimulates luteinizing hormone-releasing hormone and ascorbic acid release from medial basal hypothalami of adult male rats

Vitamin E, a dietary factor, is essential for reproduction in animals. It is an antioxidant present in all mammalian cells. Previously, we showed that ascorbic acid (AA) acted as an inhibitory neurotransmitter in the hypothalamus by scavenging nitric oxide (NO). Earlier studies have shown the antioxidant synergism between vitamin E and ascorbic acid (AA). Therefore, it was of interest to evaluate the effect of vitamin E on luteinizing hormone-releasing hormone (LHRH) and AA release. Medial basal hypothalami from adult male rats of the Sprague Dawley strain were incubated with Krebs-Ringer bicarbonate buffer or graded concentrations of a water soluble form of vitamin E, tocopheryl succinate polyethylene glycol 1000 (TPGS, 22-176 microM) for 1 hr. Subsequently, the tissues were incubated with vitamin E or combinations of vitamin. E + N-methyl-D-aspartic acid (NMDA), an excitatory amino acid for 30 min to study the effect of prior and continued exposure to vitamin E on NMDA-induced LHRH release. AA and LHRH released into the incubation media were determined by high-performance liquid chromatography and radioimmunoassay, respectively. Vitamin E stimulated both LHRH and AA release. The minimal effective concentrations were 22 and 88 microM, respectively. NMDA stimulated LHRH release as previously shown and this effect was not altered in the combined presence of vitamin E plus NMDA. However, AA release was significantly reduced in the combined presence of vitamin E plus NMDA. To evaluate the role of NO in vitamin E-induced LHRH and AA release, the tissues were incubated with vitamin E or combinations of vitamin E + NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NO synthase. NMMA significantly suppressed vitamin E-induced LHRH and AA release indicating a role of NO in the release of both LHRH and AA. The data suggest that vitamin E plays a role in the hypothalamic control of LHRH and AA release and that the release is mediated by NO.     

Effect of vitamin E on the degradation of hydrogen peroxide in cultured human umbilical vein endothelial cells

Vitamin E reacts with radicals such as lipid peroxyl radical (LOO*) and singlet oxygen ((1)O2), and plays a role in inhibiting lipid peroxidation in cell membranes and preventing the oxidation of low-density lipoproteins (LDL). However, only a few studies have investigated the effect of vitamin E on the degradation of hydrogen peroxide (H2O2). Therefore, we examined the effect of vitamin E on glutathione redox cycle-dependent H2O2 degradation activity in human umbilical vein endothelial cells (HUVEC). Confluent HUVEC were cultured for seven days in media containing various concentrations of vitamin E (alpha-tocopherol). The level of glutathione redox cycle-dependent H2O2 degradation activity and the intracellular glutathione level were determined. HUVEC that had been cultured in the presence of higher concentrations of vitamin E had a higher level of H2O2 degradation activity and a higher intracellular content of the reduced form of glutathione (GSH). Therefore, it is suggested that the vitamin E-induced increase in H2O2 degradation activity in HUVEC results from an increase in intracellular GSH level.