2-4-dinitrophenylhydrazine and ferric-chloride

Exposure of bovine aortic endothelial cells in vitro to oxidative stress causes a cascade of changes in cell function, culminating in cell death if the stress is sufficiently severe. Oxidative modification of proteins, as measured by the reaction of 2,4-dinitrophenylhydrazine with carbonyl groups of oxidized proteins, increased three- to fourfold in endothelial cells exposed to hydrogen peroxide or to a xanthine/xanthine oxidase system. The increase in oxidative modification of protein occurred rapidly, preceding loss of cellular ATP and eventual cell death. Oxidative modification of protein was paralleled by loss of activity of the key metabolic enzymes, glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The finding that oxidative modification of protein is an early event following oxidative stress suggests that oxidative modification of protein is not only a marker for oxidative damage but also a causal factor in oxidative injury.

Topics: Adenosine Triphosphate; Animals; Aorta; Cattle; Cell Death; Cells, Cultured; Chlorides; Chromium; Endothelium, Vascular; Ferric Compounds; Glucosephosphate Dehydrogenase; Glyceraldehyde-3-Phosphate Dehydrogenases; Hydrogen Peroxide; Oxidative Stress; Oxyquinoline; Phenylhydrazines; Proteins

Application ot the alpha,alpha'- dipyridyl method for determination of ascorbic acid in urine is described. The urine sample was acidified with trichloracetic acid and shaken with activated carbon to remove interfering substances. The acid filtrate was first neutralized (pH 7.0) by adding Na2HPO4. The dehydroascorbic acid was then reduced back to ascorbic acid by incubation with dithiothreitol. After removal of the excess dithiothreitol with N-ethylmaleimide, ascorbic acid was determined by measuring the reduction of ferric ion. The ferrous ion produced was coupled to alpha,alpha'-dipyridyl in the presence of H3PO4. Ferrous ion in urine samples, which theoretically interferes with the method, was removed by a combination of Na2HPO4 and H3PO4.

Topics: 2,2'-Dipyridyl; Ascorbic Acid; Chlorides; Dehydroascorbic Acid; Ferric Compounds; Oxidation-Reduction; Phenylhydrazines; Pyridines; Specific Gravity; Spectrophotometry; Urine