Pathophysiology/Complications ORIGINAL ARTICLE Glutathione Synthesis Is Diminished in Patients With Uncontrolled Diabetes and Restored by Dietary Supplementation With Cysteine and Glycine 1,2 1,2 RAJAGOPAL V. SEKHAR, MD VASUMATHI T. REDDY, PHD polyol pathway, advanced glycation end 3,4 1,2 SIRIPOOM V. MCKAY, MD ASHOK BALASUBRAMANYAM, MD product formation, protein kinase C acti- 1,2 3,4 SANJEET G. PATEL, MD FAROOK JAHOOR, PHD 1,2 vation, and the hexosamine pathway, a ANURADHA P. GUTHIKONDA, MD common feature is increased oxidative stress marked by elevated levels of reac- tive oxygen species (ROS) (1). The ability OBJECTIVE — Sustained hyperglycemia is associated with low cellular levels of the antiox- of a cell to resist damage caused by oxida- idant glutathione (GSH), which leads to tissue damage attributed to oxidative stress. We tested tive stress is determined by the capacity of the hypothesis that diminished GSH in adult patients with uncontrolled type 2 diabetes is an array of antioxidant defense systems, attributed to decreased synthesis and measured the effect of dietary supplementation with its precursors cysteine and glycine on GSH synthesis rate and oxidative stress. among which reduced glutathione (GSH) is the most ubiquitous and abundantly RESEARCH DESIGN AND METHODS — We infused 12 diabetic patients and 12 available within human cells. GSH is a 2 nondiabetic control subjects with [ H2]-glycine to measure GSH synthesis. We also measured tripeptide synthesized from glutamate, intracellular GSH concentrations, reactive oxygen metabolites, and lipid peroxides. Diabetic cysteine, and glycine in two steps cata- patients were restudied after 2 weeks of dietary supplementation with the GSH precursors lyzed by ␥-L-glutamyl-L-cysteine:glycine cysteine and glycine. ligase and glutathione synthetase. Diabe- tes is associated with decreased cellular RESULTS — Compared with control subjects, diabetic subjects had significantly higher fast- ing glucose (5.0 Ϯ 0.1 vs. 10.7 Ϯ 0.5 mmol/l; P Ͻ 0.001), lower erythrocyte concentrations of glutathione concentrations (2–5), but the glycine (514.7 Ϯ 33.1 vs. 403.2 Ϯ 18.2 ␮mol/l; P Ͻ 0.01), and cysteine (25.2 Ϯ 1.5 vs. 17.8 Ϯ cause of GSH deficiency currently is 1.5 ␮mol/l; P Ͻ 0.01); lower concentrations of GSH (6.75 Ϯ 0.47 vs. 1.65 Ϯ 0.16 ␮mol/g Hb; unknown. P Ͻ 0.001); diminished fractional (79.21 Ϯ 5.75 vs. 44.86 Ϯ 2.87%/day; P Ͻ 0.001) and Oxidative stress and ROS formation absolute (5.26 Ϯ 0.61 vs. 0.74 Ϯ 0.10 ␮mol/g Hb/day; P Ͻ 0.001) GSH synthesis rates; and are markedly increased by uncontrolled higher reactive oxygen metabolites (286 Ϯ 10 vs. 403 Ϯ 11 Carratelli units [UCarr]; P Ͻ 0.001) hyperglycemia (2,6); conversely, lower- Ϯ Ϯ Ͻ and lipid peroxides (2.6 0.4 vs. 10.8 1.2 pg/ml; P 0.001). Following dietary supplemen- ing blood glucose concentrations lowers tation in diabetic subjects, GSH synthesis and concentrations increased significantly and plasma oxidative stress (7,8). Decreased oxida- oxidative stress and lipid peroxides decreased significantly. tive stress could be an important mecha- CONCLUSIONS — Patients with uncontrolled type 2 diabetes have severely deficient syn- nism whereby glycemic control thesis of glutathione attributed to limited precursor availability. Dietary supplementation with diminishes the incidence of diabetic mi- GSH precursor amino acids can restore GSH synthesis and lower oxidative stress and oxidant crovascular complications (9,10). How- damage in the face of persistent hyperglycemia. ever, there are practical limitations to blunting oxidative stress through glyce- Diabetes Care 34:162–167, 2011 mic control alone, despite strenuous at- tempts to implement evidence-based iabetes is the leading worldwide macrovascular complications including guidelines, a majority of patients are un- cause of blindness, end-stage renal myocardial ischemia and strokes. Al- able to achieve the glycemic goals (e.g., D disease, and amputations. Diabetes though multiple pathways are involved in A1C Ͻ7%) advocated by the American also is associated with an elevated risk of mediating tissue damage, including the Diabetes Association (11). Consequently, despite the clear message of landmark tri- ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● als such as the Diabetes Control and Com- From the 1Translational Metabolism Unit, Baylor College of Medicine, Houston, Texas; the 2Division of plications Trial and the UK Prospective Diabetes, Endocrinology, and Metabolism, Department of Medicine, Baylor College of Medicine, Hous- Diabetes Study regarding the need for ex- ton, Texas; the 3Department of Pediatrics, Baylor College of Medicine, Houston, Texas; and the 4Chil- dren’s Nutrition Research Center, Agriculture Research Service, U.S. Department of Agriculture, Baylor cellent glycemic control, diabetes remains College of Medicine, Houston, Texas. the leading cause of blindness, renal fail- Corresponding author: Rajagopal V. Sekhar, [email protected]. ure, and amputations. There is an urgent Received 26 May 2010 and accepted 30 September 2010. Published ahead of print at http://care. need for novel strategies to reduce the rate diabetesjournals.org on 7 October 2010. DOI: 10.2337/dc10-1006. The contents of this article do not necessarily reflect the views or the policies of the USDA. Mention of trade of diabetes complications in patients un- names, commercial products, and organizations does not imply endorsement by the U.S. Government. able to achieve stable glycemic control. © 2011 by the American Diabetes Association. Readers may use this article as long as the work is properly We therefore investigated whether oxida- cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons. tive stress associated with low levels of org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby GSH could be ameliorated through the marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. alternative strategy of increasing cellular 162 DIABETES CARE, VOLUME 34, NUMBER 1, JANUARY 2011 care.diabetesjournals.org Sekhar and Associates GSH levels in diabetic patients with un- The GCRC protocol consisted of intrave- and left in the dark at room temperature controlled hyperglycemia. nous infusions of stable isotopes to mea- for 20 min for development of the GSH- Because circulating concentrations of sure GSH synthesis in the fasted state. All MBB derivative. After adding 0.5 ml of a protein depend on the balance between subjects were studied under baseline con- 20% perchloric acid, the sample was cen- its rates of production and consumption, ditions; only the diabetic subjects were trifuged, and the supernatant containing we hypothesized that GSH deficiency in studied again after 14 days of dietary sup- the MBB derivative was sealed and frozen uncontrolled diabetes occurs because of plementation with 0.81 mmol/kg/day of at Ϫ80°C for later analysis of GSH. Con- diminished synthesis. We further hy- cysteine (given as n-acetylcysteine) and centrations of GSSG were measured by pothesized that short-term dietary sup- 1.33 mmol/kg/day of glycine. Subjects first converting oxidized glutathione to plementation of two key amino acid were asked to consume their usual habit- reduced glutathione with the addition of a precursors of GSH, glycine and cysteine, ual diets from 2 weeks before beginning reducing agent (5 mmol/l dithiothreitiol) would increase intracellular GSH synthe- the study to the end of the study period. and measuring this as total GSH; the cal- 2 sis and concentrations and thus lower ox- Sterile solutions of [ H2]glycine culated difference between total GSH and idative stress, despite continuing (Cambridge Isotope Laboratories, reduced GSH is the concentration of hyperglycemia. To test these hypotheses, Woburn, MA) were prepared. After a GSSG. we used stable isotope methods to com- 10-h fast, subjects were admitted to the Erythrocyte GSH was isolated as an pare GSH synthesis rates and concentra- GCRC for the study, where two intrave- red blood cell-free aliquot, and the con- tions within erythrocytes, as well as nous catheters were inserted into superfi- centration measured high-performace plasma markers of oxidant damage, in cial veins for continuous infusion of the liquid chromatography (Waters, Milford, adult patients with poorly controlled type tracer solutions and blood sampling. After MA) using a 717 Plus autosampler com- 2 diabetes matched to nondiabetic con- a basal blood sample was drawn, a primed plexed to a 2475 fluorescent detector and trol subjects. The diabetic patients were constant intravenous infusion of equipped with a reverse-phase ODS Hy- 2 ␮ ␮ ϫ studied before and after 14 days of dietary [ H2]glycine (prime dose 20 mol/kg; in- persil column (5 m, 4.6 200 mm; Wa- supplementation with cysteine and fusion dose 15 ␮mol/kg/h) was main- ters). Elution of GSH was accomplished glycine. tained for 8 h. Additional blood samples with a 3–13.5% acetonitrile linear gradi- were taken at 2, 3, 4, 5, 6, 7, and8hfor ent in 1% acetic acid (pH 4.25) at a flow RESEARCH DESIGN AND measurement of erythrocyte GSH derived rate of 1.1 ml/min. The GSH eluate was METHODS — The study was ap- glycine isotopic enrichments. collected using a fraction collector, dried, proved by the institutional review board The primary outcome variables were and hydrolyzed for4hin4mol/l HCl at for Human Studies at Baylor College of fractional and absolute synthesis rates of 110°C. Medicine. Twelve adults with uncon- GSH within erythrocytes, erythrocyte trolled type 2 diabetes (A1C 8–10%) and GSH, cysteine, glycine and glutamate Erythrocyte free amino acids 12 nondiabetic control subjects matched concentrations, plasma lipid peroxide A 1-ml aliquot of blood was centrifuged, for age, sex, and BMI were recruited.