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• Title: Assay for Free and Total Carnitine in Human Plasma Using Tandem Mass Spectrometry (Technical Briefs)
• Author : Clinical Chemistry
• Release Date : January 01, 2000
• Genre: Chemistry,Books,Science & Nature,
• Pages : * pages
• Size : 175 KB

Description

Measurements of free and total carnitine in plasma are important in the diagnosis and clinical management of patients with carnitine deficiency syndromes and certain inborn errors of metabolism (1). The most popular analytical methods are based on the enzymatic reaction in which an acetyl group is transferred from acetyl-CoA to carnitine with release of free CoA (2). A spectrophotometric assay based on this method, using a Cobas centrifugal analyzer (Roche Diagnostics), has been in use in this laboratory for the past several years (3). Limitations of the Cobas enzymatic method in our view include the initial filtration step, which requires a minimum sample volume of 300 [micro]L, and the number and cost of the reagents required. Assay results are critically dependent on two labile reagents, the enzyme carnitine acetyltransferase and the color reagent 5,5'-dithiobis(2-nitrobenzoic acid). We have also observed that patients on valproic acid therapy sometimes give total carnitine values that are spuriously low for reasons that are not clear. Another limitation of the Cobas assay is that acylcarnitines with chain lengths 10 are lost in the filtration step (3), and the method will underestimate the total carnitine content of patients with defects in the oxidative degradation of long-chain or multiple acyl-CoAs, especially when metabolically decompensated. We have emphasized previously that the most valuable applications of the routine Cobas assay are the identification of patients with carnitine deficiency and the monitoring of patients on carnitine therapy (3). We have developed a new carnitine assay method based on isotope-dilution tandem mass spectrometry (TMS), which is inherently more straightforward and accurate than previously reported methods. This is true primarily because it has absolute molecular specificity, uses an isotope-labeled internal standard to compensate for any losses or variance resulting from the sample preparation, and has no known chemical interference. The new assay procedure has been developed using a total specimen volume of 150 [micro]L, but it can easily be scaled down to much smaller volumes. A fixed amount of stable-isotope-labeled free carnitine is added, and one aliquot of the mixture is used directly for the free carnitine assay. Another aliquot is subjected to base-catalyzed hydrolysis before measurement of total carnitine. The analyte is not subjected to acid-catalyzed esterification, thus avoiding any inaccuracies in free carnitine measurement from the hydrolysis of acylcarnitines or incomplete derivatization. The specimen can be either serum or plasma, and any anticoagulant can be used. In this report, the specimens from patients were predominantly heparinized plasma (green-topped tubes).

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