QUANTIFICATION OF CARNITINE, ACETYLCARNITINE, AND TOTAL CARNITINE IN TISSUES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - THE EFFECT OF EXERCISE ON CARNITINE HOMEOSTASIS IN MAN

A method for the quantitative determination of carnitine, acetylcarnit ine, and total carnitine in tissue was developed for application to cl inical research and diagnosis. Human skeletal muscle and heart specime ns (10-20 mg) were homogenized in 1 ml of water. Aliquots of the resul ting homogenates (50 mu l) were extracted with 1.0 ml of acetonitrile: methanol (3:1) and the carnitine-related compounds were isolated using columns containing 300 mg of silica gel. Samples were then derivatize d with 4'-bromophenacyl trifluoromethanesulfonate for spectrophotometr ic detection or 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate f or fluorescence detection and quantified by high-performance liquid ch romatography. Fluorometric detection of 2-(2,3-naphthalimino)ethyl est er derivatives afforded a 500-fold increase in sensitivity when compar ed to derivatization with 4'-bromophenacyl trifluoromethanesulfonate. This methodology permitted detection of acetylcarnitine in dilute huma n muscle homogenates at quantities of 790 fmol of acetylcarnitine inje cted. The method was applied to a series of human skeletal muscle biop sy samples obtained from subjects performing exercise at high work loa ds. The method permitted quantification of carnitine, acetyl- carnitin e, and total carnitine (sum of carnitine and all acylcarnitines) and d emonstrated the specific redistribution of the carnitine pool from car nitine to acetylcarnitine with exercise above the lactate threshold. T his HPLC method is facile, and provides a sensitive and specific appro ach for use in human biopsy specimens. (C) 1995 Academic Press, Inc.