Analytical Biochemistry 1995-11-01

Quantification of carnitine, acetylcarnitine, and total carnitine in tissues by high-performance liquid chromatography: the effect of exercise on carnitine homeostasis in man.

P E Minkler, E P Brass, W R Hiatt, S T Ingalls, C L Hoppel

Index: Anal. Biochem. 231 , 315, (1995)

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Abstract

A method for the quantitative determination of carnitine, acetylcarnitine, and total carnitine in tissue was developed for application to clinical research and diagnosis. Human skeletal muscle and heart specimens (10-20 mg) were homogenized in 1 ml of water. Aliquots of the resulting homogenates (50 microliters) 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 derivatized with 4'-bromophenacyl trifluoromethanesulfonate for spectrophotometric detection or 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate for fluorescence detection and quantified by high-performance liquid chromatography. Fluorometric detection of 2-(2,3-naphthalimino)ethyl ester derivatives afforded a 500-fold increase in sensitivity when compared to derivatization with 4'-bromophenacyl trifluoromethanesulfonate. This methodology permitted detection of acetylcarnitine in dilute human muscle homogenates at quantities of 790 fmol of acetylcarnitine injected. The method was applied to a series of human skeletal muscle biopsy samples obtained from subjects performing exercise at high work loads. The method permitted quantification of carnitine, acetylcarnitine, and total carnitine (sum of carnitine and all acylcarnitines) and demonstrated the specific redistribution of the carnitine pool from carnitine to acetylcarnitine with exercise above the lactate threshold. This HPLC method is facile, and provides a sensitive and specific approach for use in human biopsy specimens.