Altered antioxidant-oxidant status in the aqueous humor and peripheral blood of patients with retinitis pigmentosa

Abstract

Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa.

Figure 1. Protein content of SOD3 in serum from RP patients and healthy controls.

(A) Fifty µg of total serum protein were subjected to electrophoresis and extracellular SOD3 content was analysed by inmunoblotting, as described in Materials and Methods. The immunological signal of SOD3 was normalized to transferrin, a loading control for serum samples; Correlation analysis between SOD3 activity and SOD3 content in serum from patients and healthy controls (B), healthy controls (C) or patients (D). Spearman’s correlation test was used (rs = correlation coefficient).

Figure 2. Blood free nitrotyrosine concentration in RP patients and healthy controls.

Free nitrotyrosine was measured by LC-MS/MS system as described in Materials and Methods. Values are expressed as mean ± SEM.

Figure 3. Gene expression of heme oxygenase I in peripheral blood mononuclear cells of RP patients and healthy controls.

mRNA expression was quantified by qPCR analysis as described in Materials and Methods. qPCR data were normalized to housekeeping gene, GAPDH. C; healthy controls, RP: patients with retinitis pigmentosa; PBMC: peripheral blood mononuclear cells. Mann-Whitney test was used (*p<0.05).

Figure 4. Heatmap representation of the fuzzy clustering results for antioxidant-oxidant markers in aqueous humor of RP patients.

The dark red to dark blue colors correspond to high to low values. Individuals classified as with higher oxidant status show low TAC, protein and SOD3 levels and medium to high NOX levels. Individuals classified as with lower oxidant status show low NOX levels and high SOD3, protein and TAC levels.