Homocysteine & its metabolite homocysteine-thiolactone & deficiency of copper in patients with age related macular degeneration - A pilot study.

BACKGROUND & OBJECTIVES: Age related macular degeneration (ARMD) is a leading cause of blindness, particularly in persons above 60 yr of age. Homocysteine is implicated in many ocular diseases including ARMD. This study was undertaken to assess the status and relationship between plasma homocysteine, homocysteine - thiolactone, homocysteinylated protein and copper levels in patients with ARMD. METHODS: A total of 16 patients with ARMD and 16 age-matched controls were recruited for the study. Plasma glutathione, homocysteine, homocysteine - thiolactone and extent of homocysteine conjugation with proteins, copper and thiobarbituric acid reactive substances were measured. RESULTS: Homocysteine levels were elevated with increase in homocysteine-thiolactone, thiobarbituric acid reactive substances and a decrease of glutathione. The levels of homocysteinylated protein were elevated in ARMD. The elevated homocysteine, homocysteine-thiolactone correlated with the decrease in copper level. INTERPRETATION & CONCLUSIONS: Elevated homocysteine and its metabolite homocysteine-thiolactone and decreased levels of copper may play an important role in the pathogenesis of ARMD.

Increased homocysteine, homocysteine-thiolactone, protein homocysteinylation and oxidative stress in the circulation of patients with Eales' disease.

BACKGROUND: Eales' disease (ED) is an idiopathic retinal vascular disorder. It presents with inflammation and neovascularization in the retina. Adult men, aged between 15 and 40 years are more susceptible than women. Homocysteine has been implicated in other ocular diseases including age-related macular degeneration (ARMD), central retinal vein occlusion (CRVO) and optic neuropathy. The present study investigates the role of homocysteine in ED. METHODS: Forty male subjects, 20 with ED and 20 healthy controls, were recruited to the study. Their blood samples were used to measure thiobarbituric acid reactive substances (TBARS), glutathione (GSH), homocysteine, homocysteine-thiolactone, extent of homocysteine conjugation with proteins and plasma copper concentration. RESULTS: In the ED group, plasma homocysteine (18.6 ± 1.77 µmol/L, P < 0.001) and homocysteine-thiolactone (45.3 ± 6.8 nmol/L, P < 0.0001) concentrations were significantly higher compared to homocysteine (11.2 ± 0.64 µmol/L) and homocysteine-thiolactone (7.1 ± 0.94 nmol/L) concentrations in control subjects. TBARS (P < 0.011) and protein homocysteinylation (P < 0.030) were higher in the ED group while GSH (5.9 ± 0.44 µmol/L, P < 0.01) and copper (6.6 ± 0.42 µmol/L, P < 0.001) were lower compared to GSH (8.1 ± 0.41 µmol/L) and copper (15.4 ± 0.73 µmol/L) concentrations in control subjects. CONCLUSIONS: Increased homocysteine, and its metabolite thiolactone, is associated with the functional impairment of protein due to homocysteinylation in ED.

Total antioxidant capacity in Eales' disease, uveitis & cataract.

BACKGROUND & OBJECTIVES: The human system possesses antioxidants that act harmoniously to neutralize the harmful oxidants. This study was aimed to evaluate the serum total antioxidant capacity (TAC) as a single parameter in Eales' disease (ED) and in an acute inflammatory condition such as uveitis and in cataract which is chronic, compared to healthy controls. METHODS: The TAC assay was done spectrophotometrically in the serum of Eales' disease cases (n=20) as well as in other ocular pathologies involving oxidative stress namely, uveitis and cataract (n=20 each). The oxidative stress measured in terms of TBARS, was correlated with the TAC. Individual antioxidants namely vitamin C, E and glutathione were also estimated and correlated with TAC. RESULTS: TAC was found to be significantly lower in Eales' disease with active vasculitis (0.28 ± 0.09 mM, P<0.001), Eales' disease with healed vasculitis (0.67 ± 0.09 mM), uveitis (0.46 ± 0.09 mM, P<0.001) and cataract (0.53 ± 0.1 mM, P=0.001) compared to the healthy controls, with a TAC level of 0.77 ± 0.09 mM. The TAC was found to correlate positively with vitamin E levels (P=0.05), GSH (P=0.02) but not with vitamin C, as seen in ED cases. In ED cases supplemented with vitamin E and C, there was a significant increase in the TAC level (P=0.02). INTERPRETATION & CONCLUSIONS: The TAC measurement provided a comprehensive assay for establishing a link between the antioxidant capacity and the risk of disease as well as monitoring antioxidant therapy. This method is a good substitute for assay of individual antioxidants as it clearly gives the status of the oxidative stress in the disease process.

Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.

PURPOSE: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Iron chelation abrogates excessive formation of hydroxyl radicals and lipid peroxidation products in monocytes of patients with Eales' disease: direct evidence using electron spin resonance spectroscopy.

PURPOSE: Eales' disease (ED) is an idiopathic retinal vasculitis condition, which affects the retina of young adult males. Retinal changes include perivasculitis, non-perfusion and neovascularization. Disruption of blood-retinal barrier (BRB) is the common feature in intra-ocular inflammatory diseases. Disruption of BRB results in vascular hyper permeability and infiltration of circulating leukocytes into the retinal parenchyma. Monocyte (MC) activation results in oxidant thrust and subsequent tissue damage. This has been reported in various intra-ocular inflammatory diseases such as uveitis and Behcet's disease. However, there are no such reports available in ED. Hence in the present study we have investigated the role of MC activation and hydroxyl radicals (OH) production and its possible involvement in promoting the development of retinal vasculitis in patients with ED. METHODS: Twelve patients with ED and twelve healthy volunteers were recruited for the study. MC was separated from their peripheral blood. MC from patients with ED and control subjects was stimulated with phorbol-12-myristate-acetate (PMA) and OH generated was analyzed using an electron spin resonance spectrometer (ESR). Superoxide dismutase (SOD), thiobarbituric acid reactive substances (TBARS), and iron content was determined in MC to assess the oxidant thrust and antioxidant defense. RESULTS: OH generation was elevated in MC from patients with ED, which coincided with diminished SOD activity and elevated levels of iron and TBARS, when compared with healthy control subjects. OH generation was abrogated when MC from ED were co-incubated with PMA and iron chelators such as diethylenetriaminepentacetic acid (DTPA) and desferrioxamine. Iron chelation also inhibited TBARS accumulation restored SOD activity in MC of patients with ED. CONCLUSIONS: For the first time we have demonstrated the production of OH generation in MC of patients with ED using ESR. Further we have shown the beneficial effect of iron chelation in mitigating free radical mediated changes in cellular metabolism. Based on our findings, we provide further evidence for the role of oxidant thrust in promoting retinal tissue damage in patients with ED.

Accumulation of 8-hydroxydeoxyguanosine and its relationship with antioxidant parameters in patients with Eales' disease: implications for antioxidant therapy.

PURPOSE: Eales' disease (ED) is an idiopathic retinal inflammatory disease, which affects the retina of young adult males. Oxidative stress has been associated with the development of various inflammatory diseases. Accumulation of DNA oxidation products has been considered as an early biomarker for assessing oxidative tissue damage. To our knowledge there are no human clinical reports available on the accumulation of DNA adducts in ocular inflammatory diseases. Hence the aim of the present study was to determine the levels of 8-hydroxy-2'deoxyguanosine (8-OHdG) and to correlate it with the antioxidant and oxidant parameters in patients with ED and healthy control subjects. METHODS: Twenty-two patients with ED [12 in active perivasculitis (AV) and 10 in healed vasculitis stages (HV)] were recruited for the study. 14 healthy volunteers were included as control. 8-OHdG was determined in their leukocytes by Gas chromatography-Mass Spectrometry (GC/MS) technique. Glutathione levels were determined in plasma, while Superoxide dismutase (SOD) activity and thiobarbituricacid reactive substances (TBARS) content were determined in their erythrocytes. RESULTS: Our results indicated that 8-OHdG levels were elevated by 3 and 1.9 folds respectively in AV and HV stages of patients with ED, when compared with healthy control subjects. Increased 8-OHdG levels correlated with diminished SOD activity and decreased GSH content in patients with ED. CONCLUSIONS: To our knowledge, this is the first report of DNA adduct accumulation in patients with ED. Increased DNA adduct accumulation correlated with the severity of the disease, and they lie in parallel with diminished antioxidant capacity observed in patients with ED. Based on the observations from our present work and our earlier studies, we reiterate that oxidative stress is involved in the disease process. Hence we believe antioxidant vitamin E and C supplementation might be beneficial to patients with ED.

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.

Presence of a 88 kDa Eales protein in uveitis, tuberculosis, leprosy and rheumatoid arthritis

BACKGROUND: Eales disease (ED) is an idiopathic retinal vasculitis affecting young adult males. We have earlier reported the identification, purification and partial characterization of a novel 88 kDa protein found in the serum of patients with ED. The aim of the present study was to look for the 88 kDa protein in serum samples obtained from cases of retinal vasculitis mimicking ED and in other systemic inflammatory diseases. MATERIAL/METHODS: Serum samples from healthy volunteers and from patients with ED, uveitis, parsplanitis ocular sarcoidosis, toxoplasmosis, leprosy, diabetic retinopathy, viral hepatitis, and rheumatoid arthritis were analyzed for the presence of the 88 kDa protein by polyacralymide gel electrophoresis (PAGE). The immunological identity of the 88 kDa protein found in ED and in other diseases was investigated by Western blot. Immunohistochemistry was performed on epiretinal membranes (ERM) obtained from ED patients to localize the 88 kDa protein. RESULTS: 88 kDa protein were detected in serum samples obtained from patients with posterior uveitis, tuberculosis, leprosy and rheumatoid arthritis. The 88 kDa protein found in serum from patients with ED is immunologically identical to that found in other systemic inflammatory conditions. 88 kDa protein was localized in inflammatory cells and in nonvascular endothelium in ERMs obtained from patients with ED. CONCLUSIONS: We have identified a novel acute phase reactant, which is elaborated in ocular and systemic inflammatory conditions other than Eales disease. Further work is necessary to decipher the precise role of the 88 kDa protein in the pathophysiology of these inflammatory diseases.