J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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A more recent version of this article appeared on July 1, 2005

Papers In Press, published online ahead of print May 1, 2005
J. Lipid Res., doi:10.1194/jlr.D500013-JLR200
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Submitted on March 21, 2005
Revised on April 20, 2005
Accepted on April 20, 2005

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

Nathan L. Alderson, Michael D. Walla, and Hiroko Hama

Biochemistry and Molecular Biology, Medical Univeristy of South Carolina, Charleston, SC 29425

Corresponding Author: hama{at}musc.edu

Fatty acid 2-hydroxylase, encoded by the FA2H gene, is an enzyme responsible for 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 uniquely high proportion (~50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. Physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study roles of fatty acid 2-hydoxylase and 2-hydroxy sphingolipids in various tissues, we have developed a highly sensitive in vitro fatty acid 2-hydroxylase 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 following 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, fatty acid 2-hydroxylase activities were reproducibly measured in murine brains and tissue culture cell lines.


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