CARNITINE PALMITOYLTRANSFERASE-I ACTIVITY MONITORING IN FIBROBLASTS AND LEUKOCYTES USING ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Carnitine palmitoyltransferase I (CPT I) is one of the enzymes associa ted with normal mitochondrial membrane transport of certain metabolite s. The importance of the enzyme in normal energy production is well il lustrated during fasting conditions when a large flux of long-chain fa tty acids must be transported over the mitochondrial membrane to under go beta-oxidation. Up to now CPT I activity has been assayed in variou s tissues, including liver, leukocytes, platelets, and fibroblasts by the use of an isotope exchange forward assay which measures the rate o f palmitoyl-L-[methyl-H-3]carnitine formation from palmitoyl-CoA and L -[methyl-H-3]carnitine. We have developed an electrospray ionization m ass spectrometric method for detecting palmitoylcarnitine formation fr om palmitoyl-CoA and carnitine, thus avoiding the use of radiolabeled isotopes. In this assay, time-dependent conversion of free carnitine b y CPT I to palmitoylcarnitine is measured quantitatively, relative to isotopically labelled palmitoylcarnitine, by parent ion monitoring of fragment ion m/z 85. The specific activity of CPT I in fibroblasts and leukocytes compared well with the activity determined with the isotop e exchange method, however, the combination of high sensitivity and se lectivity of tandem mass spectrometry along with the environment-frien dly nature of the electrospray method makes it an ideal technique to m easure CPT I activity. (C) 1998 Academic Press.