Application of the reverse dept polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy.
Using the reverse 13C----1H DEPT polarization-transfer pulse sequence the metabolism of 13C ethanol in vitro and in vivo has been monitored by 1H-NMR spectroscopy. Using yeast alcohol dehydrogenase, acetaldehyde, the hydrated form of acetaldehyde and acetate were identified as metabolites of [2-13C]-ethanol. The ratio of hydrated to free acetaldehyde was dependent upon the protein concentration of the reaction mixture. Binding of acetaldehyde in an irreversible Schiffs base resulted in optimal enzyme activity. Hepatocytes from rats fasted for 20 h, metabolised [1-13C] and [2-13C]ethanol in a linear fashion, but no [13C]acetaldehyde was detected. Metabolic integrity of the hepatocytes was confirmed with [2-13C]acetate. The addition of disulfiram (50 micron) to hepatocyte suspensions which had been incubated with [1-13C]ethanol, resulted in the resynthesis of [13C]ethanol. The amount of [13C]ethanol resynthesized under these conditions represents intracellular acetaldehyde whose concentration was in the range of 400-800 mumol/g wet weight of hepatocytes when 50 mM ethanol had been originally incubated with the hepatocyte suspension. These studies show how NMR-polarization transfer pulse sequences can be used to monitor the metabolism of 13C-ethanol in vivo, and provide a unique tool to measure in vivo concentrations of acetaldehyde. The studies also suggest that cytoplasmic aldehyde dehydrogenase may play a major role in hepatic ethanol metabolism.
Irving
M G
MG
Simpson
S J
SJ
Brooks
W M
WM
Holmes
R S
RS
Doddrell
D M
DM
D000079:Acetaldehyde / Q000378:metabolism; D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors; D000818:Animals; D004221:Disulfiram / Q000494:pharmacology; D000431:Ethanol / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D000085:Acetates; D000431:Ethanol; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D000444:Aldehyde Dehydrogenase; D000079:Acetaldehyde; D019342:Acetic Acid; D004221:Disulfiram