Abstract
Fatty acid synthase (FASN or FAS, EC 2.3.1.85) is the sole mammalian enzyme to synthesize fatty acids de novo from acetyl- and malonyl-coenzyme A (CoA) esters. This article describes a new method that directly quantifies uniformly labeled (¹³C&sub1;₆-labeled palmitate ([¹³C&sub1;₆]palmitate) by tracing [¹³C&sub2;]acetyl-CoA and [¹³C&sub3;]malonyl-CoA using an in vitro FASN assay. This method used gas chromatography-mass spectrometry (GC-MS) to detect [¹³C&sub1;₆]palmitate carboxylate anions (m/z 271) of pentafluorobenzyl (PFB) derivatives and was highly sensitive at femtomole quantities. Uniformly incorporated [¹³C&sub1;₆]palmitate was the primary product of both recombinant and crude tissue lysate FASN. Quantification of FASN protein within crude tissue lysates ensured equal FASN amounts, preserved steady-state kinetics, and enabled calculation of FASN-specific activity. FASN activity determined by [¹³C&sub1;₆]palmitate synthesis was consistent with values obtained from β-nicotinamide adenine dinucleotide 2'-phosphate (NADPH) oxidation assays. Analysis of FASN activity from tissue extracts was not hampered by contaminating enzymes or preexisting fatty acids. Crude mammary gland and liver lysates had significantly different activities at 82 and 65 nmol min⁻¹ mg⁻¹, respectively, suggesting that tissue-specific activity levels differ in a manner unrelated to FASN amount. GC-MS quantification of [¹³C&sub1;₆]palmitate synthesis permits sensitive evaluation of FASN activity from tissues of varied physiological states and of purified FASN activity in the presence of modifying proteins, enzymes, or drugs.