A novel and less invasive technique to assess cytokine profile of vitreous in patients of diabetic macular oedema.

PurposeA pilot study to validate the collection of vitreous reflux (VR) after intravitreal injection using Schirmers tear strips was carried out. We assessed its efficiency for proteomics studies by estimating the differential expression of 27 cytokines using multiplexed bead array in diabetic macular oedema and proliferative diabetic retinopathy. To set, validate and assess the efficacy of Schirmer tear strips for collecting VR in patients undergoing intravitreal injections for diabetic macular oedema (DME).Patients and methodsVR samples were collected from 11 eyes of DME patients after intravitreal injections using Schirmer tear strips. Undiluted vitrectomy samples were obtained from six eyes of non-diabetic patients with idiopathic macular hole and seven eyes of diabetic patients with high-risk proliferative diabetic retinopathy (Hr-PDR), which were also subsampled on the Schirmer tear strips. Tear sampling was done in a subset of the DME patients. Total protein concentration between VR and vitrectomy samples was compared. Levels of the set of 27 cytokines in Schirmer tear strips samples were measured. Inter-group comparison for cytokines was done using Mann-Whitney U-test.ResultsSimilar protein concentration in VR samples and vitrectomy samples (P<0.05) was obtained. Tear protein contamination was not detected in VR samples. In comparison with no-DR patients, 25 and 20 of the measured 27 cytokines were significantly elevated (P<0.05) in the Hr-PDR and DME patients, respectively. As compared with no-DR patients, vascular endothelial growth factor was only moderately elevated in DME patients (P>0.05), but significantly elevated in Hr-PDR patients (P<0.05). Interleukin 1 receptor antagonist/interleukin 1b (IL1RA/IL1b) ratio was 13 times higher in DME patients as compared with Hr-PDR group.ConclusionWe demonstrated a simple, safe method of VR sampling. This technique provides a pure, albeit small, vitreous sample for proteomics. IL1RA/IL1b ratio was found to be 13-fold higher in the DME group as compared to the Hr-PDR.

Estimation of hydrogen sulphide in the human lymphocytes.

Hydrogen sulphide (H2S), a signaling gasotransmitter and a potent vasorelaxant is endogenously produced by the enzymes cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). CBS is a predominant source of H2S in the central nervous system, while CSE is the major H2S producing enzyme in the brain and other nervous tissues. Though the expression of these enzymes in the blood lymphocytes is known, H2S formation in the lymphocytes has not been reported so far. In the present study, H2S levels in the lymphocytes of healthy control subjects were estimated, after suitable modifications in a routine method [Stipanuk M H & Beck P W (1982) Biochem J 206, 267-277] used for detecting tissue levels of H2S. In this method, homocysteine (Hcys) due to its higher solubility was used as the substrate in place of L-cysteine and NaOH was used in place of zinc acetate to increase the entrapment of H2S in the central well. A mean H2S level of 11.64 +/- 6.36 microM/min/mg protein was detected in the lymphocytes of 8 subjects (mean age, 24 +/- 2; 2 male, 6 female). The modified method was found to be more sensitive for H2S estimation in human lymphocytes. As endogenous H2S is reported to be involved in the pathogenesis of various cardiovascular and pulmonary diseases, the levels of H2S in lymphocytes can be a marker of the endogenous tissue levels.

Biochemistry of homocysteine in health and diseases.

The amino acid homocysteine (Hcy), formed from methionine has profound importance in health and diseases. In normal circumstances, it is converted to cysteine and partly remethylated to methionine with the help of vit B12 and folate. However, when normal metabolism is disturbed, due to deficiency of cystathionine-beta-synthase, which requires vit B6 for activation, Hcy is accumulated in the blood with an increase of methionine, resulting into mental retardation (homocystinuria type I). A decrease of cysteine may cause eye diseases, due to decrease in the synthesis of glutathione (antioxidant). In homocystinurias type II, III and IV, there is accumulation of Hcy, but a decrease of methionine, thus, there is no mental retardation. Homocysteinemia is found in Marfan syndrome, some cases of type I diabetes and is also linked to smoking and has genetic basis too. In hyperhomocysteinemias (HHcys), clinical manifestations are mental retardation and seizures (type I only), ectopia lentis, secondary glaucoma, optic atrophy, retinal detachment, skeletal abnormalities, osteoporosis, vascular changes, neurological dysfunction and psychiatric symptoms. Thrombotic and cardiovascular diseases may also be encountered. The harmful effects of homocysteinemias are due to (i) production of oxidants (reactive oxygen species) generated during oxidation of Hcy to homocystine and disulphides in the blood. These could oxidize membrane lipids and proteins. (ii) Hcy can react with proteins with their thiols and form disulphides (thiolation), (iii) it can also be converted to highly reactive thiolactone which could react with the proteins forming -NH-CO- adducts, thus affecting the body proteins and enzymes. Homocystinuria type I is very rare (1 in 12 lakhs only) and is treated with supplementation of vit B6 and cystine. Others are more common and are treated with folate, vit B12 and in selected cases as in methionine synthase deficiency, methionine, avoiding excess. In this review, the role of elevated Hcy levels in cardiovascular, ocular, neurologial and other diseases and the possible therapeutic measures, in addition to the molecular mechanisms involved in deleterious manifestations of homocysteinemia, have been discussed.

The anti-oxidant activity of turmeric (Curcuma longa).

The turmeric anti-oxidant protein (TAP) had been isolated from the aqueous extract of turmeric. The anti-oxidant principle was found to be a heat stable protein. Trypsin treatment abolished the anti-oxidant activity. The anti-oxidant principle had an absorbance maximum at 280 nm. After gel filtration, the protein showed a 2-fold increase in anti-oxidant activity and showed 2 bands in the SDS-PAGE with approximate molecular weight range of 24,000 Da. The protein showed a concentration-dependent inhibitory effect on the promoter induced lipid peroxidation. A 50% inhibitory activity of lipid peroxidation was observed at a protein concentration of 50 micrograms/ml. Ca(2+)-ATPase of rat brain homogenate was protected to nearly 50% of the initial activity from the lipid peroxidant induced inactivation by this protein. This protection of Ca(2+)-ATPase activity was found to be associated with the prevention of loss of -SH groups.

Serum PON1 arylesterase activity in relation to hyperhomocysteinaemia and oxidative stress in young adult central retinal venous occlusion patients.

AIM: To estimate the arylesterase activity of serum paraoxonase-1 (PON1-ARE), which is reported to have an antioxidant and antiatherogenic potential and to correlate with plasma homocysteine (Hcys) and plasma TBARS in young adult central retinal venous occlusion (CRVO) patients. METHODS: A case-control prospective study carried out in 10 CRVO patients (mean age 27+/-5 years; 7 males, 3 females) and 20 healthy controls (mean age 29+/-5 years; 15 males, 5 females). RESULTS: The CRVO patients showed a significantly lowered serum PON1-ARE activity (P=0.009) along with a significant increase in the levels of plasma Hcys (P=0.018) when compared to the control subjects. There was a negative correlation between serum PON1-ARE and plasma Hcys levels (P=0.058) as well as between PON1-ARE and plasma TBARS levels (P=0.001) in the CRVO patients. CONCLUSION: This is the first report of lowered serum PON1-ARE level as a risk factor for CRVO (OR= 1.108, CI=0.914, 1.314; P=0.296), which is found to correlate with oxidative stress.

Plasma homocysteine and total thiol content in patients with exudative age-related macular degeneration.

PURPOSE: Exudative age-related macular degeneration (ARMD) is one of the debilitating ocular complications, which results in permanent blindness. Elevated homocysteine (Hcys) levels have been associated in the development of several vascular diseases. Vascular and oxidative stress theories have been implicated for the development of choroidal neovascularization in exudative ARMD. The aim of the present study was to investigate the possible role of plasma Hcys and thiol content (tSH) as a risk factor for the development of exudative ARMD. METHOD: A total of 16 patients with exudative ARMD and 20 age-matched controls were recruited for the study. Plasma Hcys levels were analysed using Reverse Phase High Performance Liquid Chromatography. Plasma glutathione (GSH) content was determined using o-phthalaldehyde (OPA) derivatization and subsequent detection by fluorimeter. Plasma tSH levels were determined by using thiol-specific reagent dithionitrobenzoic acid (DTNB) spectrophotometrically. RESULTS: Plasma Hcys levels in exudative ARMD were elevated three-fold (18+/-5.0 microM) when compared to healthy controls (6.7+/-1.8 microM). There was a two-fold decrease in the GSH and tSH in exudative ARMD when compared with controls. Negative correlation was observed between diminished tSH and Hcys levels (r=-0.4837, P=0.05). Similarly plasma Hcys levels negatively correlated with GSH content (r=-0.6620, P<0.05). CONCLUSION: Results from our present study revealed that there is an elevated Hcys level and diminished thiol pool content in exudative ARMD that are significant.

Effect of gamma-glutamyl carboxylation of renal microsomes on calcium oxalate monohydrate crystal binding in hyperoxaluria.

AIM: The gamma-carboxy glutamic acid (gla) containing proteins have been shown recently to have a role in calcium oxalate urolithiasis. Therefore, the effect of carboxylation on calcium oxalate monohydrate (COM) binding was studied in experimental hyperoxaluria. METHODS: Urolithiasis was induced in experimental rats by oral feeding 1% ethylene glycol (EG). The microsomes were carboxylated in presence of reduced vitamin K and COM binding activity was determined. RESULTS: Microsomal proteins adsorbed maximally with COM crystals. On carboxylation of the renal microsomal proteins, a significant increase in the COM crystal adsorption by 2.5-fold (p < 0.001) was observed in the hyperoxaluric condition. Further cyboxylated microsomes of EG-treated rats showed significant binding with calcium oxalate. CONCLUSION: This study presents an increased COM adsorption after the carboxylation of renal microsomes of hyperoxaluric rats.

Enhanced renal vitamin-K-dependent gamma-glutamyl carboxylase activity in experimental rat urolithiasis.

OBJECTIVE: To detect the role of the enzyme gamma-glutamyl carboxylase in an experimental stone-forming condition. METHODS: Urolithiasis was induced in experimental rats by (i) oral feeding of 1% ethylene glycol (EG) and (ii) feeding a calculus-producing diet containing 3% sodium glycolate. RESULTS: A significant enhancement in the activity of renal vitamin-K-dependent gamma-glutamyl carboxylase was observed in both groups of experimental urolithic rats. Dicoumarol as well as EG treatment enhanced the accumulation of the endogenous substrate for the enzyme. The carboxylase activity was stimulated by sodium oxalate as well as calcium oxalate in vitro. A positive correlation was observed between lipid peroxidation and the renal gamma-glutamyl carboxylase activity. CONCLUSION: The enhanced carboxylase activity observed in the hyperoxaluric condition is suggested to be due to stimulation of the enzyme by oxalate/calcium oxalate, increased concentration of endogenous carboxylase substrate and lipid peroxidation.