The determination of tissue malonyl-CoA (malCoA) has been performed by enzymatic assay using fatty acid synthase or more recently HPLC with UV detection. There are discrepancies in the values obtained; the HPLC procedure usually results in much higher tissue malCoA content. In developing a new malCoA determination method by HPLC/MS, we detected two forms of malCoA in commercial malCoA. The forms were purified by preparative HPLC and characterized by HPLC/MS. Both compounds had a molecular weight of 852 ([M-H]^-, malCoA) with production of a 808 MW fragment ([M-H]^-, acetyl-CoA) by collision induced dissociation. Since both peaks appeared to be malCoA, but were chromatographically resolved, we used NMR to structurally identify the two malCoA isoforms. One compound was identified as the naturally occurring 3'-phospho-malCoA (3'-malCoA) and the other was 2'-phospho-malCoA (2'-malCoA). With our purified 3'-malCoA and [¹³C₃]malCoA internal standard, we developed a method for the determination of malCoA. The 5% sulfosalicylic acid extract of powdered tissue was centrifuged, the supernatant processed using a reversed phase solid phase extraction column and the isolated malCoA was detected and quantified by HPLC/MS/MS. This HPLC/MS/MS method is specific, selective, and sensitive for the natural occurring malCoA in biological samples.

Supported in part by NIH P01 AG15885, DK35543 and VA Medical Research Service.