ENZYME REACTOR FOR URINARY ACYLCARNITINES ASSAY BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

An immobilized enzyme reactor, made up acylcarnitine hydrolase, carnit ine dehydrogenase and diaphorase in sequence, was developed for the se nsitive and selective determination of urinary free and individual acy lcarnitines by a reversed-phase high-performance liquid chromatography . A 100-mul urine sample was directly injected onto the TSKgel ODS 80T s column and eluted by a step-gradient procedure. The eluent was mixed with the substrate solution of beta-NAD+ (1.0 mmol/l), resazurin (25 mumol/1) and Tris acetate (0.2 mol/l, pH 9.0). The mixture was passed through the immobilized enzyme reactor at 40-degrees-C. Acylcarnitines were hydrolyzed and then converted to rezorufin which was measured by monitoring the fluorescence intensity at lambda(EX) = 560 nm and lamb da(EM) = 580 nm. Free, acetyl-, glutaryl-, propionyl-, butyryl-, isobu tyryl-, valeryl- and isovalerylcarnitine were determined within 55 min with detection limits (< 1 mumol/l) and within-run and day-to-day imp recision (C.V. < 6%). Free, acetyl- and isobutyrylcarnitine were found in normal urine. On the other hand, propionylcarnitine was detected i n the urine of children with propionic aciduria and methylmalonic acid uria and multiple acylcarnitines were found in the urine of children w ith glutaric aciduria (type II).