Involvement of oxidative and nitrosative stress in promoting retinal vasculitis in patients with Eales' disease.

OBJECTIVES: Eales' disease (ED) is an idiopathic retinal vasculitis condition, which affects retina of young adult males. The histopathological hallmark in ED is the adhesion of leukocytes to the endothelium and the infiltration of these cells into the retinal parenchyma. Phagocyte generated free radicals have been implicated in mediating tissue damage associated with various inflammatory vasculopathies. In the present study, we have investigated the possible role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in causing retinal tissue damage in ED. DESIGN AND METHODS: 35 patients with ED and 20 healthy control subjects were included in the study. Monocytes (MC) were separated from peripheral blood of the respective study participants. Inducible nitric oxide synthase (iNOS) protein expression was assessed using Western blot and 3 nitrotyrosine (3NTYR) by reversed phase high performance liquid chromatography (RP HPLC). Thiobarbituric acid reactive substances (TBARS) were determined by measuring malondialdehyde (MDA) formed. Superoxide dismutase (SOD) activity was assayed based on the ability of SOD to inhibit auto-oxidation of epinephrine. Iron, copper and zinc content were determined using Atomic Absorption Spectrophotometer. Immunolocalization of iNOS and 3NTYR was performed on the surgically excised epiretinal membranes (ERM) from patients with ED. RESULTS: There was a significant increase in the expression of iNOS, as well as 3NTYR accumulation, diminished SOD activity, elevated lipid peroxides, iron, copper and decreased zinc content in the MC of patients with ED when compared with healthy control subjects. The elevated levels of ROS and RNS products correlated with diminished antioxidant status in patients with ED. Strong immunoreactivity for iNOS and 3NTYR was observed in inflammatory cells and endothelial cells in ERM obtained from patients with ED. CONCLUSIONS: Our findings from this study clearly reveal the involvement of RNS and ROS in the development of retinal vasculitis in ED. Based on our present study and earlier studies we confirm the role of free radicals in mediating retinal tissue damage in ED. Hence we believe selective inhibition of iNOS or supplementation with antioxidants vitamin E and C might be beneficial in controlling retinal vasculitis in patients with ED.

Glycation and glycoxidation studies in vitro on isolated human vitreous collagen.

BACKGROUND: Structural and functional impairment in vitreous collagen plays an important role in the pathogenesis of diabetic retinopathy. Collagen being a long-lived protein is prone to both glycation and glycoxidation, resulting in accumulation of advanced glycation end products (AGE). The objective of our study was to explore the extent of glycation by glucose, and iron- and copper-mediated glycoxidation of human vitreous collagen, and also to study the beneficial effects of lysine, inositol and aminoguanidine as antiglycating and anti-cross linking agents. MATERIAL/METHODS: Vitreous from human donor eyeballs was pooled and collagen was extracted using 0.9 M NaCl. Collagen was estimated by measuring the hydroxyproline content. The extracted collagen was used for glycation and glycoxidation studies. Glycation studies were conducted using U14C glucose, along with anti-glycating agents, such as lysine and aminoguanidine. Metal-mediated glycoxidation studies were done by measuring collagen content in cyanogen bromide insoluble fraction, in the presence and absence of an anti-cross linking agent, inositol. RESULTS: Human vitreous collagen extractable with 0.9 M NaCl was glycated by glucose at 5 and 10 mM concentrations under physiological conditions of temperature and pH. An anti-glycating effect was exhibited by lysine, inositol and aminoguanidine, of which lysine was the best (76% antiglycating activity) followed by inositol. Inositol was also found to be useful in inhibiting glycoxidation. CONCLUSIONS: Vitreous collagen undergoes glycation, as well as iron- and copper-mediated glycoxidation, leading to possible structural and functional impairment. Glycation and glycoxidation are inhibited, significantly by lysine and inositol respectively.

Effect of oral supplementation of free amino acids in type 2 diabetic patients-- a pilot clinical trial.

BACKGROUND: Oral amino acid intake reduces plasma glucose in Streptozotocin-induced diabetic rats. This study examined the effect of oral amino acid supplementation in patients with type 2 diabetes mellitus (DM). MATERIAL/METHODS: A double blind pilot clinical trial was conducted for a period of 2 months on 77 subjects with type 2 DM. Subjects of both sexes, ages 30-60, were included in the trial. All were receiving oral antidiabetic tablets. They were divided into groups on the basis of oral supplementation: (A) lysine, (B) essential amino acids, (C) amino acids and vitamins (fat and water-soluble), and (D) calcium phosphate (control). The subjects were periodically examined for fasting and post-prandial plasma glucose, fasting and post-prandial immunoreactive insulin, plasma amino acids, glycosylated haemoglobin (HbA1c), proteins and albumin in serum, urea and creatinine in plasma and sugar, and proteins and ketones in urine. RESULTS: The results revealed a significant decrease in PP plasma glucose (P<0.05) in group B when compared to groups C and D after 45 days. Plasma Arginine was increased in group C from 3.84 to 9.24 mg/dl. There were no statistically significant changes seen in other parameters between groups and visits. CONCLUSIONS: Oral supplementation with amino acids for patients with type 2 DM appears to decrease PP plasma glucose without any change in plasma insulin levels, perhaps due to improved insulin sensitivity. However, the long term effects of amino acids need further study.