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Localization of Protein‐Acetaldehyde Adducts on Cell Surface of Hepatocytes by Flow Cytometry
Author(s) -
Lin Renee C.,
Sidner Richard A.,
Fillenwarth Michael J.,
Lumeng Lawrence
Publication year - 1992
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.1992.tb00708.x
Subject(s) - cyanamide , acetaldehyde , aldehyde dehydrogenase , paraformaldehyde , chemistry , ethanol , flow cytometry , biochemistry , cytosol , alcohol dehydrogenase , fluorescein isothiocyanate , microbiology and biotechnology , hepatocyte , isothiocyanate , microsome , fluorescence , biology , enzyme , in vitro , physics , organic chemistry , quantum mechanics
Acetaldehyde, a highly reactive intermediate of ethanol metabolism, has been shown to form adducts with liver proteins (e.g., a cytosolic 37 kDa protein and the microsomal cytP450IIE1) in rats fed alcohol chronically. In this study, flow cytometry was utilized to test for the presence of protein‐acetaldehyde adducts (‐AAs) on the surface of hepatocytes and immunotransblot was used to detect for the 37 kDa protein‐AA in cytosol as was previously described. For flow cytometric analysis, rabbit anti‐hemocyanin‐AA IgG and fluorescein isothiocyanate‐conjugated goat anti‐rabbit serum IgG were used as the primary and secondary antibodies to label surface protein‐AAs on hepatocytes at 0° to 4°C. After labeling and washing, hepatocytes were fixed with paraformaldehyde‐cacodylate and analyzed with a flow cytometer. In an experiment wherein hepatocytes isolated from rats pair‐fed liquid diets with and without ethanol were treated by adding both the primary and secondary IgGs, some hepatocytes from both alcohol‐fed and control rats exhibited positive fluorescence but no significant difference in fluorescence intensity was noted. In another experiment, hepatocytes were isolated from rats pair‐fed cyanamide (a selective aldehyde dehydrogenase inhibitor) with and without ethanol. The number of hepatocytes showing positive fluorescence in the presence of both primary and secondary IgGs was significantly higher in rats fed cyanamide plus ethanol than in rats fed cyanamide only. Of note, the 37 kDa protein‐AA could be detected by immunotransblot in liver cytosol of alcohol‐fed rats but not in the controls of both experiments with and without cyanamide supplementation. When hepatocytes from rats fed cyanamide plus ethanol were incubated with 0.5% trypsin for 5 min at 37°C after being labeled with fluorochrome, the population of cells with positive fluorescence disappeared. Our data thus indicate that hepatocytes contain surface protein‐AAs when in vivo acetaldehyde concentration is elevated by feeding alcohol and cyanamide simultaneously but not by feeding alcohol alone. The implication is that at least in the rat, only high concentrations of acetaldehyde in vivo can lead to presentation of protein‐AAs to liver cell surface to serve as neoantigens.