In Vitro Conjugation of Ethanolamine with Fatty Acids by Rat Liver Subcellular Fractions

Abstract

Previous studies from our laboratory have shown the enzymic formation of fatty acid (FA) conjugates of xenobiotic alcohols and amines. In the present study, the formation of FA conjugates of a bifunctional compound, ethanolamine was investigated by incubating [1-¹⁴C]oleic acid (1 mM) with ethanolamine (25 mM) at 37°C in the presence of various rat liver subcellular fractions. The resultant product (or products) was separated by thin-layer chromatography (TLC) and the radioactivity corresponding to the relative flow of fatty acid amide was determined. Under similar conditions, formation of ethanolamides of palmitic, stearic, linoleic, linolenic, and arachidonic acids were also examined. The formation of ethanolamine conjugate with oleic acid was found to be 16.3 nmol/h/mg protein as compared to 6.7, 6.2, 8.1, 8.3, and 7.6 nmol/h/mg protein for palmitic, stearic, linoleic, linolenic, and arachidonic acids, respectively. The formation of oleoyl ethanolamide was found to be 18.9, 40.1, 65.9, and 0.3 nmol/h/mg protein in postnuclear, mitochondrial, microsomal, and cytosolic fractions, respectively. Mass spectrometric and nuclear magnetic resonance spectroscopic data of the TLC-purified product confirm the formation of oleoyl ethanolamide, and amidation appeared to be a preferred reaction over esterification. The results of this study suggest that the enzyme responsible for the amidation of fatty acids resides mainly in the microsomal fraction of the liver, and that oleic acid is a better substrate than other fatty acids used in the present study.

This work was supported by grants ES 04815 from National Institute of Health and Environmental Health Sciences (G.A.S.A.) and AA13171 from the National Institute of Alcohol Abuse and Alcoholism (B.S.K.), and its contents are solely the responsibility of authors and do not necessarily represent the official views of NIEHS or NIAAA. The authors thank Drs. Richard Hodge and Alex Kurosky of NIEHS Center, supported by the grant P30ES06676, for the NMR and mass spectral analysis.

Notes

This work was supported by grants ES 04815 from National Institute of Health and Environmental Health Sciences (G.A.S.A.) and AA13171 from the National Institute of Alcohol Abuse and Alcoholism (B.S.K.), and its contents are solely the responsibility of authors and do not necessarily represent the official views of NIEHS or NIAAA. The authors thank Drs. Richard Hodge and Alex Kurosky of NIEHS Center, supported by the grant P30ES06676, for the NMR and mass spectral analysis.