Lipid peroxidation in porcine irises: Dependence on pigmentation

Abstract

Purpose. Pigmentation of the iris is caused by varying amounts of melanin pigment granula in a constant number of melanocytes in the superficial stroma. Melanin has been shown to act as antioxidant. We have now investigated lipid peroxidation in dependence on stromal pigmentation in isolated porcine irises. Methods. The same number of lightly and heavily pigmented porcine irises (visual selection) were homogenized (1:20w/v) in buffer (50mmol/l phosphate buffer and 4mmol/l sodium azide). 500µl homogenate were incubated at 37°C in duplicate for 5, 10, 20 and 40 min in absence and presence of Fe 2+ as inducer of lipid peroxidation. The amount of lipid peroxidation was assayed by the thiobarbituric acid (TBA) test. The results are expressed as nmol of TBA reactive material (TBAR) produced/mg protein. Fe 2+ concentration of the supernatant was determined spectrophotometrically with 1,10 orthophenanthroline. Concentrations of D -glucose and D -fructose in iris tissue homogenates were determined spectrophotometrically by enzymatic bioanalysis. Results. 70, 180 and 360 µmol/l Fe 2+ induced lipid peroxidation. A plateau region was reached after 20 min. The amount of lipid peroxidation differed in dependence on stromal pigmentation in porcine irises. The effect was most significant at 180 µmol/l Fe 2+, which induced 1.373 ± 0.138 nmol TBAR/mg protein in lightly compared to 0.491 ± 0.125 nmol TBAR/mg protein in heavily pigmented irises after 10 min incubation (p < 0.0001, n = 4). Similar effects (factor 2–3) were also measured after 20 and 40 min incubation. On the other hand, the content of Fe 2+ in the supernatant was the same within error. Sugar concentrations (D -glucose and D -fructose) did not differ significantly for the two differently pigmented iris tissues. Conclusions. There is a stronger induction of lipid peroxidation in lightly compared to heavily pigmented porcine irises. This effect may be related to the difference in stromal melanin content and its antioxidant activity.