This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.

A growing body of evidence suggests that oxidative stress plays a key role in the pathogenesis of micro- and macrovascular diabetic complications. The increased oxidative stress in subjects with type 2 diabetes is a consequence of several abnormalities, including hyperglycemia, insulin resistance, hyperinsulinemia, and dyslipidemia, each of which contributes to mitochondrial superoxide overproduction in endothelial cells of large and small vessels as well as the myocardium. The unifying pathophysiological mechanism that underlies diabetic complications could be explained by increased production of reactive oxygen species (ROS) via: (1) the polyol pathway flux, (2) increased formation of advanced glycation end products (AGEs), (3) increased expression of the receptor for AGEs, (4) activation of protein kinase C isoforms, and (5) overactivity of the hexosamine pathway. Furthermore, the effects of oxidative stress in individuals with type 2 diabetes are compounded by the inactivation of two critical anti-atherosclerotic enzymes: endothelial nitric oxide synthase and prostacyclin synthase. Of interest, the results of clinical trials in patients with type 2 diabetes in whom intensive management of all the components of the metabolic syndrome (hyperglycemia, hypercholesterolemia, and essential hypertension) was attempted (with agents that exert a beneficial effect on serum glucose, serum lipid concentrations, and blood pressure, respectively) showed a decrease in adverse cardiovascular end points. The purpose of this review is (1) to examine the mechanisms that link oxidative stress to micro- and macrovascular complications in subjects with type 2 diabetes and (2) to consider the therapeutic opportunities that are presented by currently used therapeutic agents which possess antioxidant properties as well as new potential antioxidant substances.

The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 micrograms GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 +/- 2 mg/cm2 in OVX and 192 +/- 2 in OVX with 5 micrograms GEN/g b.w. per day; p < 0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 micrograms and 25 micrograms GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 micrograms GEN dose, but not with the 1 microgram and 5 micrograms doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 +/- 0.7% of total bone volume in SHAM-Veh vs 3.3 +/- 0.2% in OVX-Veh; p < 0.01) and less bone loss in OVX rats injected with 5 micrograms GEN per gram per day (3.3 +/- 0.2% of total bone volume in OVX-Veh vs 5.2 +/- 0.4% in OVX-GEN; p < 0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNF alpha) was tested in monocytic cells from the same four rat groups. Production of TNF alpha was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone.

Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.