Dietary supplementation with cysteine prodrugs selectively restores tissue glutathione levels and redox status in protein-malnourished mice1 1Supported in part by the OARDC Hatch Fund to T.M.B., National Institutes of Health Grant NS38315 to T.M.B (original) (raw)
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Prodrug Approach for Increasing Cellular Glutathione Levels
Molecules, 2010
Reduced glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and the preferred substrate for several enzymes in xenobiotic metabolism and antioxidant defense. It plays an important role in many cellular processes, such as cell differentiation, proliferation and apoptosis. GSH deficiency has been observed in aging and in a wide range of pathologies, including neurodegenerative disorders and cystic fibrosis (CF), as well as in several viral infections. Use of GSH as a therapeutic agent is limited because of its unfavorable biochemical and pharmacokinetic properties. Several reports have provided evidence for the use of GSH prodrugs able to replenish intracellular GSH levels. This review discusses different strategies for increasing GSH levels by supplying reversible bioconjugates able to cross the cellular membrane more easily than GSH and to provide a source of thiols for GSH synthesis.
Oxidative stress induces a reversible flux of cysteine from tissues to blood in vivo in the rat
FEBS Journal, 2009
Cysteine (Cys) and glutathione (GSH) are the most abundant low-molecular-mass thiols (LMM-SH), with GSH predominating intracellularly and cysteine predominating in extracellular fluids . These two compounds are metabolically inter-related, and GSH, in particular, determines the redox state and represents a defense against damage mediated by reactive oxygen species (ROS) or reactive nitrogen species.
Redox Biology
To determine if orally dosed γ-glutamylcysteine (γ-GC) can increase cellular glutathione (GSH) levels above homeostasis. Many chronic and age-related disorders are associated with down-regulation, or impairment, of glutamate cysteine ligase (GCL). This suggests that γ-GC supply may become limiting for the maintenance of cellular GSH at the normal levels required to effectively protect against oxidative stress and any resulting physiological damage. Methods: GSH levels were measured in lymphocytes of healthy, non-fasting participants before and after single oral doses (2 and 4 g) of γ-GC. Blood samples were immediately processed using high speed fluorescenceactivated cell sorting to isolate 10 6 lymphocytes that were then assayed for GSH content. Results: A single 2 g dose of γ-GC increased lymphocyte GSH content above basal levels (53 ± 47%, p < 0.01, n=14) within 90 min of administration. A randomized dosage (2 and 4 g γ-GC) crossover design was used to explore the pharmacokinetics of this GSH increase. In general, for both dose levels (n=9), GSH increased from initial basal levels over 3 h (t max) before reaching maximum GSH concentrations (C max) that were near two (2 g γ-GC) to three (4 g γ-GC) fold basal levels (0.4 nmol/10 6 lymphocytes). Beyond t max , GSH levels progressively declined reaching near basal levels by 5 h. The GSH half-life was between 2 and 3 h with exposure (AUC) to increased GSH levels of 0.7 (2 g γ-GC) and 1.8 (4 g γ-GC) nmol.h/10 6 lymphocytes. Conclusions: Oral γ-GC is a non-toxic form of cysteine that can be directly taken up by cells and transiently increase lymphocyte GSH above homeostatic levels. Our findings that γ-GC can increase GSH levels in healthy subjects suggests that it may have potential as an adjunct for treating diseases associated with chronic GSH depletion. This trial was registered at anzctr.org.au as ACTRN12612000952842.
Increasing evidences attribute a central role to oxidation/reduction (redox) homeostasis in controlling cell physiology, with redox transitions of glutathione and sensitive cysteines in protein constituting the major players. Thiol redox control of protein activity and gene expression ultimately extends to the proliferation/differentiation switch. Several diseases, including the highest causes of mortality, have been attributed to a shift toward a more oxidized environment. Interfering with thiol redox transitions in diseases therefore appears a major clinical objective, N-acetyl-cysteine (NAC) being the most obvious candidate drug. Targets comprise diseases related to altered control of proliferation/differentiation, adhesion, inflammation. To quote a few benign disorders, gynecological diseases can include endometriosis, polycystic ovary syndrome, sterility originating from defective embryo implantation; dermatological diseases can include acne, alopecia, psoriasis, and vitiligo. We review here our journey with NAC, from mechanisms identified in cells, though an animal model and finally to the clinic where cases of successful patient's treatment are reported, some unpublished before. NAC effect was eventually enhanced by a combination with melatonin and selenium, both involved in the pathway of redox regulation. Further studies can well extend NAC use to several other diseases, while providing better treatment modalities and helping in identifying further specifically targeted compounds.
Background: Children with severe edematous malnutrition have higher than normal oxidant damage and lower concentrations of the antioxidant reduced glutathione (GSH), which are associated with slower synthesis of GSH and with low extra-and intracellular concentrations of the precursor amino acid cysteine. Objective: We tested whether early dietary supplementation with cysteine could restore a normal GSH concentration and synthesis rate in these children. Design: Erythrocyte cysteine and GSH concentrations and the fractional and absolute synthesis rates of GSH were measured in 2 groups of 16 edematous malnourished children, 10 boys and 6 girls aged 6-18 mo, at 3 times after hospital admission: at Ϸ2 d (period 1), when they were malnourished and infected; at Ϸ11 d (period 2), when they were malnourished but cleared of infection; and at Ϸ50 d (period 3), when they had recovered. Supplementation with either 0.5 mmol · kg Ϫ1 · d Ϫ1 N-acetylcysteine (NAC group) or alanine (control group) started immediately after period 1 and continued until recovery. Results: From period 1 to period 2 the concentration and the absolute synthesis rate of GSH increased significantly (P < 0.05) in the NAC group but not in the control group. The increases in the GSH concentration and synthesis rate were Ϸ150% and 510% greater, respectively, in the NAC group than in the control group. The increases in the NAC group were associated with a significant effect of supplement (P < 0.03) on erythrocyte cysteine concentration. Conclusion: These results suggest that the GSH synthesis rate and concentration can be restored during the early phase of treatment if patients are supplemented with cysteine.
Journal of the American Society of Nephrology
Glutathione is a major cellular antioxidant that protects protein thiols and inhibits cellular damage due to oxygen free radicals. It has been reported previously that patients undergoing dialysis have low levels of blood glutathione, which may lead to increased susceptibility to oxidant stress. L-2-oxothiazolidine-4-carboxylic acid (OTZ) is a cysteine prodrug that raises cellular glutathione levels by increasing delivery of cysteine, the rate-limiting substrate for glutathione synthesis. This study investigates the effect of OTZ on blood glutathione in a blinded, placebo-controlled study of patients with chronic renal failure treated by peritoneal dialysis. Twenty patients were randomly selected to receive OTZ (0.5 g three times a day orally with meals) or placebo for 14 d. Patients visited the clinic for predose blood collection and safety evaluation at baseline (days 3, 7, and 14 and again at 14 d from the last dose [follow-up]). Glutathione concentrations were determined in whol...
Diabetes Care, 2010
OBJECTIVE Sustained hyperglycemia is associated with low cellular levels of the antioxidant glutathione (GSH), which leads to tissue damage attributed to oxidative stress. We tested the hypothesis that diminished GSH in adult patients with uncontrolled type 2 diabetes is attributed to decreased synthesis and measured the effect of dietary supplementation with its precursors cysteine and glycine on GSH synthesis rate and oxidative stress. RESEARCH DESIGN AND METHODS We infused 12 diabetic patients and 12 nondiabetic control subjects with [2H2]-glycine to measure GSH synthesis. We also measured intracellular GSH concentrations, reactive oxygen metabolites, and lipid peroxides. Diabetic patients were restudied after 2 weeks of dietary supplementation with the GSH precursors cysteine and glycine. RESULTS Compared with control subjects, diabetic subjects had significantly higher fasting glucose (5.0 ± 0.1 vs. 10.7 ± 0.5 mmol/l; P < 0.001), lower erythrocyte concentrations of glycine (...
Increasing evidences attribute a central role to oxidation/reduction (redox) homeostasis in controlling cell physiology, with redox transitions of glutathione and sensitive cysteines in protein constituting the major players. Thiol redox control of protein activity and gene expression ultimately extends to the proliferation/differentiation switch. Several diseases, including the highest causes of mortality, have been attributed to a shift toward a more oxidized environment. Interfering with thiol redox transitions in diseases therefore appears a major clinical objective, N-acetyl-cysteine (NAC) being the most obvious candidate drug. Targets comprise diseases related to altered control of proliferation/differentiation, adhesion, inflammation. To quote a few benign disorders, gynecological diseases can include endometriosis, polycystic ovary syndrome, sterility originating from defective embryo implantation; dermatological diseases can include acne, alopecia, psoriasis, and vitiligo. We review here our journey with NAC, from mechanisms identified in cells, though an animal model and finally to the clinic where cases of successful patient's treatment are reported, some unpublished before. NAC effect was eventually enhanced by a combination with melatonin and selenium, both involved in the pathway of redox regulation. Further studies can well extend NAC use to several other diseases, while providing better treatment modalities and helping in identifying further specifically targeted compounds.
Journal of Nutrition, 2008
Most methods of determining amino acid (AA) requirements are based on endpoints that determine adequacy for protein synthesis. However, the sulfur AA (SAA) cysteine is believed to be the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant, glutathione (GSH). Our objectives were to determine whether supplementation of cysteine in a diet containing adequate SAA for protein synthesis, as methionine, increased GSH synthesis by measuring the fractional and absolute synthesis rates, and if concentration of GSH changed in response to feeding 5 graded intakes of cysteine (0, 10, 20, 30, and 40 mgÁkg 21 Ád 21 ) in a random order with a fixed methionine intake of 14 mgÁkg 21 Ád 21 and a protein intake of 1 gÁkg 21 Ád 21 . Each subject received a multivitamin and choline supplement during the study. Four healthy adult men each underwent 5 isotope infusion studies of 7-h duration after a 2-d adaptation to the level of cysteine intake being studied on the isotope infusion day. The isotope used was [U-13 C 2 -15 N]glycine. Analyses included erythrocyte GSH synthesis rates and concentration and urinary sulfate excretion. The GSH synthesis rates and concentration, measured at a methionine intake of 14 mgÁkg 21 Ád 21 , did not change with increasing intakes of cysteine. Urinary sulfate excretion showed a significant positive relationship with cysteine intake (r ¼ 0.92; P , 0.01). In conclusion, this study provides preliminary evidence that consumption of SAA adequate to meet the requirement for protein synthesis does not limit GSH synthesis in healthy adult men receiving an otherwise adequate diet.