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

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 ( approximately 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-D(4)]tetracosanoic acid was solubilized in alpha-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.