Effects of ascorbic acid on peroxidation of human erythrocyte membranes by lipoxygenase

The effects of ascorbic acid (AsA) on membrane phospholipids (PLs) and tocopherols (Tocs) of human erythrocyte during peroxidation by soybean lipoxygenase (LOX) were investigated. After extraction of the membrane lipids, alpha-tocopherol (alpha-Toc), gamma-tocopherol (gamma-Toc) and cholesterol were simultaneously measured by high-performance liquid chromatography (HPLC), and the changes of Tocs were expressed on the basis of cholesterol. The phospholipid classes and corresponding hydroperoxides (PL-OOHs) were detected simultaneously by HPLC, and the changes were calculated on the basis of sphingomyelin. These methods are sensitive to the changes of membrane Tocs and PLs by peroxidation. Control incubation without LOX and AsA was done for 45 min at 30 degrees C. After the incubation with LOX, alpha- and gamma-Tocs were exhausted, PLs decreased, and PL-OOHs and malondialdehyde (MDA) increased. Incubation with both AsA and LOX further increased MDA significantly, but it preserved about 30% of alpha-Toc and 45% of gamma-Toc of the control levels and did not decrease PLs or PL-OOHs from the levels after the incubation with LOX. Subsequent incubation with AsA for 45 min after the incubation with LOX (after Tocs were exhausted) showed a 240% increase in MDA, but it decreased PLs by only about 15% of the preincubation values and recovered gamma-Toc to about 13% of the control. The subsequent incubation with AsA after the control incubation increased PLs to higher than that of the control reaction. These results show that AsA protects and regenerates the membrane Tocs against enzymatic peroxidation. The results also indicate that repair of the membrane PLs is promoted in the presence of AsA.