2-4-dinitrophenylhydrazine and Alcoholism

Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat.. Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins.. Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively.. This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.

Topics: Alcoholism; Animals; Central Nervous System Depressants; Cytosol; Ethanol; Glutathione; Glutathione Peroxidase; Humans; Male; Mitochondria, Liver; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Acetaldehyde, the first metabolite of ethanol, reacts with haemoglobin in vitro to produce acetaldehyde-haemoglobin adducts. Some clinical studies on the minor haemoglobins have suggested that these adducts may be formed in people abusing alcohol. Under hydrolysis of haemoglobin, with oxalic acid at 100 degrees C in sealed vials, some acetaldehyde was released and then specifically determined by HPLC. The kinetics of hydrolysis were studied using haemoglobin previously labelled with 14[C] acetaldehyde. The maximum liberation of 14 [C] acetaldehyde was obtained after 3 hr 30 min hydrolysis and this time factor was then utilized in the analysis of alcoholic and control haemoglobin. Thus, we have confirmed the formation of acetaldehyde haemoglobin adducts in vivo. It must be noted that the released acetaldehyde corresponds only to an index of the stable adducts. The levels were higher in alcoholics than in controls (1.417 +/- 0.171 and 1.295 +/- 0.139 nmol/mg Hb, respectively, P less than 0.001). In conclusion, this marker is not a convenient tool for the monitoring of alcohol exposure levels because of the low differences between alcoholic and control haemoglobins.

Topics: Acetaldehyde; Adult; Aged; Alcoholism; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Hemoglobins; Humans; Hydrolysis; Male; Middle Aged; Oxalates; Oxalic Acid; Phenylhydrazines