Experience of measuring glutathione peroxidase activity in surgically induced endometrial-like lesions in rats

Abstract

BACKGROUND: Endometriosis is known to be linked with altered activities of antioxidant enzymes and with their gene polymorphisms. Progestins are known to induce glutathione peroxidase activity in the endometrium and promote reduction of endometrial lesions. It could be useful to estimate the correlation between the activity of glutathione peroxidase within endometrial lesions and their degree of reduction.
 AIM: The present study was aimed at estimating glutathione peroxidase activity in surgically induced endometrial-like lesions of different degree of reduction in rat model of endometriosis.
 MATERIALS AND METHODS: The method for determining glutathione peroxidase activity using hydrogen peroxide as a substrate and 5,5-dithiobis(2-nitrobenzoic acid) for estimation of residual reduced glutathione was applied for quantitative analysis of the enzyme activity in endometriotic foci, surgically induced in female Wistar rats. An assay of glutathione peroxidase activity in tissue homogenates was performed at 37C in a reaction medium containing Tris-HCl buffer supplemented with tetrasodium ethylenediaminetetraacetate and sodium azide (pH 8.5) in the presence of 0.55 mM reduced glutathione and 0.192 mM hydrogen peroxide. Before adding trichloroacetic acid, 40-second incubation was used. The correlation between the specific activity of the enzyme and protein amount in endometriotic foci was estimated.
 RESULTS: In a rat model of endometriosis, there was a high, well-determined glutathione peroxidase activity in endometriotic foci. For the same endometriotic tissue sample, the enzymatic activity was proportional to the amount of protein in the reaction mixture. The range of specific glutathione peroxidase activity was 2.436.45 micromoles of consumed glutathione per minute per milligram of protein (n = 7). In most reduced endometriotic foci (with the minimum amount of endometriotic tissue), the highest specific activity of glutathione peroxidase was found (the Spearmans rho of 0.93 with p = 0.0067).
 CONCLUSIONS: The method for determining glutathione peroxidase activity using hydrogen peroxide and 5,5-dithiobis(2-nitrobenzoic acid) is convenient for working with the endometriotic tissue in a rat model of endometriosis. We can accept, with p 0.01, that weight of endometriotic foci is negatively linked with specific glutathione peroxidase activity within their tissue. The results are analogous to the previously obtained data on catalase activity and suggest the involvement of both antioxidant enzymes in reduction of endometrial lesions.

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“…Determination of GSH: About 15 μL of tissue homogenate was added to 260 μL assay buffer (0.1 M sodium phosphate and 1 mM EDTA, pH: 8) and 5 μL Ellman reagents. Samples were kept at room temperature for 15 min, and the TNB 2− formation was quantified in a spectrophotometer by taking the absorbance of visible light at 412 nm 12 .…”