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Journal of Lipid Research, Vol. 46, 1569-1575, July 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry

Nathan L. Alderson^*, Michael D. Walla and Hiroko Hama^1,*

^* Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, 29208

Published, JLR Papers in Press, May 1, 2005. DOI 10.1194/jlr.D500013-JLR200

¹ To whom correspondence should be addressed. e-mail: hama{at}musc.edu

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion (50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D₄]tetracosanoic acid was solubilized in -cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner.

With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.

Abbreviations: FA2H, fatty acid 2-hydroxylase; TMS, trimethylsilyl

Supplementary key words FA2H • fatty acid alpha-hydroxylase • 2-hydroxy fatty acid

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