Pyridoxine and pyridoxamine inhibits superoxide radicals and prevents lipid peroxidation, protein glycosylation, and (Na+ + K+)-ATPase activity reduction in high glucose-treated human erythrocytes.

Vitamin B(6) (pyridoxine) supplementation has been found beneficial in preventing diabetic neuropathy and retinopathy, and the glycosylation of proteins. Oxygen radicals and oxidative damage have been implicated in the cellular dysfunction and complications of diabetes. This study was undertaken to test the hypothesis that pyridoxine (P) and pyridoxamine (PM) inhibit superoxide radical production, reduce lipid peroxidation and glycosylation, and increase the (Na+ + K+)-ATPase activity in high glucose-exposed red blood cells (RBC). Superoxide radical production was assessed by the reduction of cytochrome C by glucose in the presence and absence of P or PM in a cell-free buffered solution. To examine cellular effects, washed normal human RBC were treated with control and high glucose concentrations with and without P or PM. Both P and PM significantly lowered lipid peroxidation and glycated hemoglobin (HbA(1)) formation in high glucose-exposed RBC. P and PM significantly prevented the reduction in (Na+ + K+)-ATPase activity in high glucose-treated RBC. Thus, P or PM can inhibit oxygen radical production, which in turn prevents the lipid peroxidation, protein glycosylation, and (Na+ + K+)-ATPase activity reduction induced by the hyperglycemia. This study describes a new biochemical mechanism by which P or PM supplementation may delay or inhibit the development of complications in diabetes.

Determination of superoxide and ascorbyl radicals in the circulation of animals under oxidative stress.

Quantitation of the superoxide radical and its related metabolites in vivo is practically difficult predominantly because of their short biological half-lives. Though oxidized cytochrome c (cyt c) has been used for determining superoxide radicals in vitro, it cannot be used for in vivo analysis because of its low specificity as an electron acceptor and rapid disappearance from the circulation. To measure superoxide radicals and related metabolites in normal and pathologic subjects, we have synthesized a cyt c derivative (SMAC) with prolonged half-life in the circulation (T1/2 = 130 min) by conjugating acetylated cyt c with poly(styreneco-maleic acid) butyl ester (SM). An SM-conjugated superoxide dismutase (SM-SOD) with prolonged in vivo half-life was also synthesized. When injected intravenously to the rat, SMAC was rapidly reduced in the circulation of normal rats. The rate of SMAC reduction was markedly increased by intravenous administration of menadione, a compound capable of redox cycling and generating superoxide. The rate of SMAC reduction was not inhibited by a large dose of SM-SOD (27,000 unit/kg) in both normal and menadione-treated animals. The rate of SMAC reduction also increased in animals which were administered alloxan, a diabetogenic agents. In contrast to the experiments with menadione, the alloxan-enhanced reduction of SMAC was significantly inhibited by SM-SOD. Kinetic analysis using ascorbate oxidase suggested that ascorbyl radical was principally responsible for the SM-SOD-insensitive reduction of SMAC. Streptozotocin, another diabetogenic agent, failed to increase the rate of SMAC reduction. Thus, the effect of streptozotocin on the redox state of animals and the mechanism of its diabetogenic action might differ from those of alloxan. Combined use of SMAC and SM-SOD might permit quantitative studies on the occurrence of ascorbyl and superoxide radicals in the circulation of animals challenged with oxidative stress.

Antioxidant properties of human colostrum.

Because it has recently been hypothesized that human milk is antiinflammatory, the effects of aqueous human colostrum on human polymorphonuclear leukocyte (PMN) respiratory burst activity and selected enzymatic activities was examined. Aqueous colostrum was found to spontaneously reduce ferricytochrome C in a concentration-dependent manner, prohibiting use of the standard assay to measure superoxide production. It also caused a significant concentration-dependent prolongation of the lagtime from stimulation of PMN with phorbol myristate acetate to the appearance of hydrogen peroxide. Substitution of an enzymatic peroxide-generating system for PMN did not alter the effect of colostrum. Colostrum also suppressed myeloperoxidase activity and lysozyme activity, but not beta-glucuronidase activity in PMN lysates. Inclusion of colostrum in an in vitro assay of PMN-mediated cell detachment significantly suppressed this PMN-mediated effect. These data demonstrate that aqueous human colostrum significantly interferes with PMN oxygen metabolic and enzymatic activities that are important in the mediation of acute inflammation.