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 spectrophotometically 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 μmol/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 (r2 = 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.