Acylcarnitine in a given sample is converted to carnitine by Acylcarnitine Esterase (ACE). The generated carnitine is specifically oxidized by Carnitine Dehydrogenase (CDH) under the presence of oxidized β-Thionicotinamide Adenine Dinucleotide (Thio-NAD+) to dehydrocarnitine and reduced β-Thionicotinamide Adenine Dinucleotide (Thio-NADH). Dehydrocarnitine in turn is specifically reduced by CDH under the presence of reduced β-Nicotinamide Adenine Dinucleotide (NADH) to carnitine and oxidized β-Nicotinamide Adenine Dinucleotide (NAD+). The following enzymatic cycling reaction leads to the accumulation of Thio-NADH. Because the generated amount of Thio-NADH is proportional to the carnitine concentration in the sample, total carnitine concentration (acylcarnitine and free carnitine) can be determined by the specific absorption of Thio-NADH.