Homocysteinethiolactone and paraoxonase: novel markers of diabetic retinopathy

Abstract

Objective: Paraoxonase (PON) exhibits esterase activity (PON-AREase) and lactonase activity (PON-HCTLase), which prevent LDL oxidation and detoxify homocysteine thiolactone (HCTL). The role of HCTL and PON-HCTLase as a risk factor for the microvascular complication in diabetic retinopathy at the level of vitreous has not been investigated.

Research design and methods: Undiluted vitreous from patients with proliferative diabetic retinopathy (PDR) (n = 13) and macular hole (MH) (n = 8) was used to determine PON-HCTLase and PON-AREase activity spectrophotometrically. HCTL levels were detected by liquid chromatography-tandem mass spectrometry. In vitro studies were done in primary cultures of bovine retinal capillary endothelial cells (BRECs) to determine the dose- and time-dependent effect of HCTL and homocysteine (Hcys) on PON-HCTLase activity, as well as to determine mRNA expression of PON by RT-PCR.

Results: A significant increase in HCTL and PON-HCTLase activity was observed in PDR compared with MH (P = 0.036, P = 0.001), with a significant positive correlation between them (r = 0.77, P = 0.03). The in vitro studies on BRECs showed a dose- and time-dependent increase in the PON-HCTLase activity and mRNA expression of PON2 when exposed to HCTL and Hcys.

Conclusions: This is the first study showing elevated levels of vitreous HCTL and PON-HCTLase activity in PDR. These elevations are probably a protective effect to eliminate HCTL, which mediates endothelial cell dysfunction. Thus, vitreous levels of HCTL and PON activity can be markers of diabetic retinopathy. The bioinformatics analysis reveals that the structure and function of PON that can be modulated by hyperhomocysteinemia in PDR can affect the dual-enzyme activity of PON.

Figure 1

PON activity and HCTL levels in the vitreous of PDR and MH case subjects. Distribution graphs show the reciprocal relationship of HCTLase and AREase in PDR (n = 13) and MH (n = 8). A: PON-AREase activity. B: PON-HCTLase activity. C: Activity staining for PON protein in the vitreous using phenylacetate as substrate and parasoaniline as chromogen. The band was observed at 66 kDa (lane 1: MH; lanes 2–4: PDR; lane 5: high–molecular weight marker). Representative liquid chromatography–tandem mass spectrometry chromatogram showing the HCTL (left) and the corresponding internal standard, namely homatropine (right). D: Standard vitreous. E: MH vitreous. F: PDR vitreous. The m/z of HCTL is 118.2 and homatroprine is 276.1 (seen as the peak). G: Distribution of HCTL levels in PDR (n = 9) and MH (n = 3) case subjects. Correlation between HCTL and PON-HCTLase is shown. H: PDR (n = 9), ♦; MH (n = 3), ●. (A high-quality color representation of this figure is available in the online issue.)

Figure 1

PON activity and HCTL levels in the vitreous of PDR and MH case subjects. Distribution graphs show the reciprocal relationship of HCTLase and AREase in PDR (n = 13) and MH (n = 8). A: PON-AREase activity. B: PON-HCTLase activity. C: Activity staining for PON protein in the vitreous using phenylacetate as substrate and parasoaniline as chromogen. The band was observed at 66 kDa (lane 1: MH; lanes 2–4: PDR; lane 5: high–molecular weight marker). Representative liquid chromatography–tandem mass spectrometry chromatogram showing the HCTL (left) and the corresponding internal standard, namely homatropine (right). D: Standard vitreous. E: MH vitreous. F: PDR vitreous. The m/z of HCTL is 118.2 and homatroprine is 276.1 (seen as the peak). G: Distribution of HCTL levels in PDR (n = 9) and MH (n = 3) case subjects. Correlation between HCTL and PON-HCTLase is shown. H: PDR (n = 9), ♦; MH (n = 3), ●. (A high-quality color representation of this figure is available in the online issue.)

Figure 2

In vitro experiments. PON-HCTLase activity in BRECs exposed to Hcys and HCTL. Graphs showing the dose- and time-dependent increase in PON-HCTLase activity after treatment with HCTL (A) or Hcys (B). C: PON-AREase and PON-HCTLase activity in BRECs exposed to Hcys and HCTL at 200 μmol/l compared with the baseline control activity. D: mRNA expression of PON2 in BRECs exposed to Hcys and HCTL (200 μmol/l at 24 h). The PCR was carried out using the following primers for bovine glyceraldehyde 3-phosphate dehydrogenase (GAPDH): forward primer 5′-TGTTCCAGTATGATTCCACCC-3′ and reverse primer 5′-GTCTTCTGGGTGGCAGTGAT-3′ corresponding to 424 bp, and for PON2: forward primer 5′-CCT TCC TAA TTG CCA CCT GA-3′ and reverse primer 5′-TGG AGG CCT GGA CAT TTT AG-3′, corresponding to ∼150 bp. The bands obtained were quantified using National Institutes of Health ImageJ software after normalization to GAPDH. (A high-quality color representation of this figure is available in the online issue.)

Figure 3

Bioinformatic analysis of PON2 interaction with Hcys. A: Residues of the PON2 protein that have hydrogen bonding with the ligand Hcys. B: Residues of the PON2 protein that have hydrophobic interaction with the ligand Hcys.