Determination of free and total carnitine in plasma by an enzymatic reaction and spectrophotometric quantitation spectrophotometric determination of carnitine.

OBJECTIVES: Carnitine initiates the beta-oxidation of fatty acids and its deficiency is a problem in several metabolic diseases. This work describes a validated quick and simple enzymatic assay for the determination of free and total carnitine in plasma.
METHODS: Carnitine reacts with acetyl-CoA catalized by carnitine acetyltransferase. The coenzyme A liberated combines with 5,5'-dithiobis-(2-nitrobenzoic acid) and forms thiophenolate ion, measured spectrophotometrically at 412 nm. The method requires precipitation of proteins and the alkaline hydrolysis of acylcarnitines for total carnitine.
RESULTS: The detection limit is 1.7 micromol/L in plasma and inter- and intra-day imprecision is less than 5%. The recovery of spiked plasma samples is 97%. The method was tested with an inter-laboratory assay, yielding excellent correlation (r(2)=0.994), and it was applied to the determination of normal values of carnitine in plasma.
CONCLUSIONS: A reliable spectrophotometric method has been validated with very good precision, with simple instrumental and easy sample preparation.