Structural Diversity and Analytical Characterization of Acylhomocarnitines

Homocarnitine is a five-carbon analog of carnitine produced in mammals through hydroxylation of the microbiome-derived metabolite δ-valerobetaine. Here, we describe liquid chromatography-mass spectrometry methods for the measurement of fatty acyl-homocarnitines, a previously uncharacterized family of mammalian metabolites. These acyl-homocarnitines are homologs of acyl-carnitines, in which the fatty acid is extended by one carbon. We show that short-chain fatty acyl-CoAs are converted to corresponding acyl-homocarnitines by carnitine acetyltransferase and that these enzyme-generated standards exhibit retention times and ion dissociation patterns identical to acyl-homocarnitines produced by mammalian cells. In vitro ¹³C₃-homocarnitine isotope tracer studies showed that mammalian cells produce short-, medium-, and long-chain acyl-homocarnitines. Ion dissociation analyses established diagnostic product ions to distinguish acyl-homocarnitines from isomeric acyl-carnitines. Sample preparation and chromatographic methods are provided to separate and analyze isomers in extracts of mouse tissues. These findings expand knowledge of carnitine analogs and establish analytical strategies to differentiate acyl-homocarnitines from isomeric acyl-carnitines.