Abstract
Purpose: :
Homocysteine thiolactone (HCTL) is proposed as the molecular basis for the vascular dysregulation caused by mild to moderate homocysteinemia. The role of HCTL as a risk factor for the microvascular complication in diabetic retinopathy has to be looked into. Paraoxonase (PON) enzyme activity (EC 3.1.8.1) associated with high density lipoprotein, exhibits esterase activity (PON-ARE) that can prevent LDL oxidation and a lactonase activity (PON-HCTLase) that can detoxify HCTL. Vitreous levels of HCTL and PON activity were analyzed in Proliferative Diabetic Retinopathy (PDR) to see if they are characteristic disease markers.
Methods: :
Undiluted vitreous obtained during vitrectomy in PDR and Macular Hole (n=14, n= 8) were used to determine HCTL levels by LC/MS (Q-Trap API 4000-App.Biosys, USA). PON-HCTLase activity and PON-ARE activity were measured spectrophotometrically. In vitro studies were done in primary cultures of bovine retinal capillary endothelial cells (BREC) to study the dose and time dependent effect of HCTL and Hcys (25 to 200 µM; 3 to 48 h) on the PON-HCTLase activity and mRNA expression at 200 µM at 24 h. MTT assay was done to assess the toxicity.
Results: :
A significant increase in the vitreous level of HCTL and PON-HCTLase activity was observed in PDR compared to MH (p=0.036, p=0.001), with a significant positive correlation between PON-HCTLase and the HCTL levels(r=0.96) only in PDR with corresponding lowering of the PON-ARE activity (p=0.000). The in vitro studies on BREC also showed a dose and time dependent increase in the PON-HCTLase activity on exposure to HCTL. The mRNA expression of PON2 in EC was found to be markedly increased by Hcys compared to HCTL correlating with the enzyme activity. The viability of the BREC cells was not altered even upto 200 µM Hcys and HCTL exposure till 48 h.
Conclusions: :
This is the first report of the elevated levels of vitreous HCTL and increased PON-HCTLase activity in the PDR, probably a protective effect to eliminate HCTL that can mediate EC dysfunction. Correspondingly low PON-ARE activity detected is indicative of low antioxidant activity that may contribute to the etiopathology of Diabetic Retinopathy. Vitreous levels of HCTL and PON activities can be a markers of diabetic retinopathy. However the structure - function that determines the dual enzyme activity of PON under pathological conditions warrants attention.
Keywords: vitreous • enzymes/enzyme inhibitors • diabetic retinopathy