After alcohol ingestion, ethanol is oxidized to acetal-Acetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to dehyde in the liver mainly by the action of alcohol dehybe covalently modified by acetaldehyde in vivo. We have drogenase 1 and, on prolonged alcohol consumption, by previously reported the detection of a cytosolic 37-kd microsomal cytochrome P450IIE1. 2 Little acetaldehyde protein-acetaldehyde adduct (-AA) in the liver of alcoholaccumulates during alcohol oxidation because this infed rats. The liver extract from an alcohol-fed rat was termediate is rapidly converted to acetate by aldehyde subjected to 2-dimensional (2D) sodium dodecylsulfate dehydrogenase. Nevertheless, acetaldehyde is highly (SDS)-polyacrylamide gel electrophoresis (PAGE), transreactive. For many years, investigators have shown ferred to polyvinylidene difluoride (PVDF) membrane, that acetaldehyde can covalently modify proteins in and the 37-kd protein-AA spot was digested with trypsin vitro to form protein-acetaldehyde adducts (-AAs). The and sequenced for amino acids. Degenerate oligonucleoformation of protein-AAs in vivo has been demontides corresponding to a peptide sequence of the proteinstrated more recently. [5] Our laboratory was the first AA were used as the probe to screen a lgt11 rat liver to report the detection of a liver protein-AA (apparent complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. MW, 37,000)
by Western immunoblot in rats fed alcohol
The open reading frame was 978 nucleotides long, encodchronically. The 37-kd liver protein-AA can be deing 326 amino acid residues. The sequence matched that tected in rats within 1 week of alcohol feeding (either of rat liver D 4 -3-ketosteroid 5b-reductase. The cloned the high-fat Lieber-DeCarli or the low-fat AIN'76 liquid cDNA was expressed in Escherichia coli using pGEX-KG diet containing alcohol [Bio-Serv, Inc., Frenchtown, as the vector. The expressed protein was found to be of NJ]). 10 The bond between the 37-kd protein and acetalcorrect molecular weight. It reacted with an antibody dehyde was very stable, and its stability was not enthat recognized the unmodified liver 37-kd protein by hanced by incubating with cyanoborohydride as a re-Western blotting. Peptide profiles of tryptic-digested reducing agent. Adding pyrazole (an inhibitor of alcohol combinant protein and the purified rat liver 37-kd prodehydrogenase) 11,12 to the alcohol-containing diet abrotein were similar and yielded the same peptide segated the formation of the liver adduct, indicating that quence. D 4 -3-ketosteroid 5b-reductase catalyzes the ethanol provided acetaldehyde as the precursor via the reduction of key intermediates during bile acid biosynaction of alcohol dehydrogenase. 13 Feeding rats an alcothesis. Whether modification of the 5b-reductase by acetaldehyde affects the enzyme activity and bile acid syn-hol-containing liquid diet supplemented with cyanathesis remains to be studied. (HEPATOLOGY 1996;23: mide (a specific inhibitor of the low K M form of aldehyde 115-122.) dehydrogenase) 14 raised plasma acetaldehyde concentrations more than sixfold and greatly increased the intensity of the 37-kd protein-AA band on immunoblots. 13 These experiments therefore provide strong