Hidden β-γ Dehydrogenation Products in Long-Chain Fatty Acid Oxidation Unveiled by NMR: Implications on Lipid Metabolism

We present a comprehensive analysis of the initial α,β-dehydrogenation step in long-chain fatty acid β-oxidation (FAO). We focused on palmitoyl-CoA oxidized by two mitochondrial acyl-CoA dehydrogenases, very-long-chain acyl-CoA dehydrogenase (VLCAD) and acyl-CoA dehydrogenase family member 9 (ACAD9), both implicated in mitochondrial diseases. By combining MS and NMR, we identified the (2E)-hexadecenoyl-CoA as the expected α-β-dehydrogenation product and also the E and Z stereoisomers of 3-hexadecenoyl-CoA: a “γ-oxidation” product. This finding reveals an alternative catalytic pathway in mitochondrial FAO, suggesting a potential regulatory role for ACAD9 and VLCAD during fatty acid metabolism.