During the past ten years there has been an increasing awareness of the biochemical functions of L-carnitine, and of the clinical consequences of carnitine deficiency. See, e.g., Bieber et al., Fed. Proc. 41, 2858 (1982); Stanley, Adv. Pediatr. 34, 59 (1987). Carnitine is produced in the body in the form of L-carnitine. Both free L-carnitine and acylcarnitine are required for proper fatty acid oxidation within cells. A convenient, automated assay for free L-carnitine and total short chain acylcarnitine is needed for both research and clinical purposes. Certain inborn errors of metabolism result in the accumulation of toxic acetyl-coA; carnitine may be given to detoxify the acetyl-CoA. Patients receiving total parenteral nutrition and those on renal dialysis may have L-carnitine deficiencies. In each situation an assay for free L-carnitine and total short-chain acylcarnitine concentrations would be useful.
Several methods for assaying free L-carnitine and total short chain acylcarnitine are known in the art. See. e.g., Marzo et al., J. Chromatogr. 527, 247 (1990); Hoppel, In: Hommes (ed.), Techniques in Diagnostic Human Biochemical Genetics, New York, Wiley-Liss, 309-326 (1991). The most widely used methods are dependent on the enzyme carnitine acetyltransferase (CAT; EC 2.3.1.7), which catalyzes the reaction: EQU acetyl-coA+L-carnitine.fwdarw.acetylcarnitine+coASH
where coASH represents free coenzyme A liberated.
One widely used spectrophotometric assay for carnitine is based on the reaction of the liberated coASH with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). This reaction produces the thiophenylate ion, which absorbs light at 412 nm. Spectrophotometric evaluation of a sample after reacting with DTNB provides an indirect measurement of free L-carnitine. See. e.g., Marquis and Fritz, J. Lipid Res. 5, 184 (1964). To assay acylcarnitine, alkaline hydrolysis of a sample can be used to convert acylcarnitine to free carnitine, and the steps above can then be used to measure total short-chain acylcarnitine.
Most methods currently in use are based on a partially automated version of these procedures, but require that several essential steps be performed manually by a technician. See, e.g., Seccombe et al., Clin. Chem. 22, 1589 (1976); Rodriguez-Segade et al., Clin. Chem. 31, 754 (1985); Cederblad et al., Clin. Chem. 32, 342 (1986). The manual steps required increase the potential for error and limit the convenience of these methods. Additionally the samples must be diluted, treated with thiol oxidizing reagent, or otherwise treated prior to analysis to remove biological interferences that affect the chemical reactions utilized in the assay.
The most widely used alternative to the spectrophotometric assay is a radioisotopic exchange assay (REA) that uses 1-.sup.14 C-acetyl-coA as substrate for the enzyme and measures the 1-.sup.14 C-acetylcarnitine produced. See Cederblad and Lindstedt, Clin. Chim. Acta. 37, 235 (1972). This method is generally accepted in the art as one of the most accurate, and several modifications have been described. See, e.g., Hoppel, In: Hommes (ed.), Techniques in Diagnostic Human Biochemical Genetics, New York: Wiley-Liss, 309 (1991); McGarry and Foster, J. Lipid Res. 22, 1589 (1976). However, this assay method is labor intensive and not suited to automation, and is therefore not routinely employed in clinical laboratories.
Mass spectrometry can also be used to measure total carnitine. While this method is currently used clinically, it is expensive and the number of hospitals equipped to perform it is limited.
In view of the foregoing, there is a continued need for new automated methods of assaying carnitine.