Effects of ascorbate on membrane phospholipids and tocopherols of intact erythrocytes during peroxidation by t‐butylhydroperoxide: Comparison with effects of dithiothreitol

Abstract

Peroxidation of intact human erythrocytes by t-butylhydroperoxide (tBHP)) was studied. By incubation of the erythrocytes with 1 mM tBHP, reduced glutathione (GSH) was exhausted within 1 min, and tocopherols (Toc) and phospholipids (PL) decreased to nearly their lowest levels (in this study) within 5 min. The rate of decrease of alpha-Toc was faster than that of gamma-Toc, but alpha-Toc was never exhausted. The rates of decrease of Toc were faster than that of PL. Malondialdehyde increased slowly to reach a maximal value at 30 min. Methemoglobin (metHB) reached a maximum at 15 min. The maximal levels of these substances were maintained until 90 min incubation, which indicated that the peroxidation by tBHP had stopped spontaneously until at least 90 min. By the incubation with tBHP for 30 min, phosphatidylethanolamine (PE) and alpha-Toc decreased to about 70 and 30% of control levels, respectively, and gamma-Toc and GSH were almost exhausted. Ascorbate (0.1 mM) afforded protection of 92% to PE, 50% to alpha-Toc, and 65% to gamma-Toc against peroxidation, but ascorbate had no preventive effect at all on the formation of metHB and the decrease of GSH. These results may indicate that ascorbate-mediated protection of the membrane PL against the peroxidation depends primarily on Toc. On the other hand, dithiothreitol (DTT) (5 mM) almost completely prevented the formation of metHB, and DTT completely protected the PL and Toc against peroxidation, indicating the importance of sulfhydryl groups in erythrocytes.

Verapamil Prevents Impairment in Filterability of Human Erythrocytes Exposed to Oxidative Stress

“…Measurements were performed at room temperature (22.0Ϯ1.5°C). The formation of methemoglobin (met-Hb, in %) was calculated as previously reported [25].…”

“…Extraction of total phospholipid from erythrocytes was performed as previously reported [25,26]. Briefly, packed erythrocytes were hemolyzed by addition of 1 ml of 5 mM hypotonic phosphate buffer (pH 7.4), and by further addition of 1 ml of 80 mM sodium dodecyl sulfate (SDS).…”

High-cholesterol Diets Induce Changes in Lipid Composition of Rat Erythrocyte Membrane Including Decrease in Cholesterol, Increase in α-Tocopherol and Changes in Fatty Acids of Phospholipids

“…The extraction of the total lipids including Toc from the packed erythrocytes was done immediately after the preparation of the washed erythrocytes, and as described in our previous report. 13) Brie‰y, the packed erythrocytes were hemolyzed by addition of 1 ml of 5 mM phosphate buŠer (pH 7.4) containing 5 mM ascorbic acid, which was followed by addition of 1 ml of 80 mM sodium dodecyl sulfate. Then, 2 ml of ethanol containing 1.2 mM BHT was added, and the mixture was left at room temperature for 60 min.…”

“…Cholesterol and Toc were measured simultaneously by the HPLC method as described in our previous reports. 13,14) The HPLC system (HP-1100, Yokogawa Electric Co., Tokyo, Japan) was connected in series to a ‰uorescence detector (FS-8020, Tosoh, Tokyo, Japan) and an evapolative light scattering detector (ELSD) (SEDEX-55, Vitry sur Seine, France).…”

“…The separation and measurement of each PL class was done by the HPLC method reported previously. 13,15) The column was a Wakosil 5 NH2, 150× 4.6 mm (Wako Pure Chemical Co.) and the guard column was a m-Bondapak NH2, 20×4 mm (Waters Co., Tokyo, Japan). A portion (5 ml) of the total lipid extract, which was used for the detection of Toc and cholesterol, was injected into the HPLC system.…”