Enzymatic cycling assay for D-carnitine determination.
An enzymatic method for d-carnitine determination using the enzyme d-carnitine dehydrogenase is described. The assay is based on the amplified signal produced during NAD(+) cycling in the presence of a tetrazolium salt and using phenazine methosulfate as electron carrier. Optimum assay conditions were studied with two tetrazolium salt pairs: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)/MTT-formazan and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT)/INT-formazan. The first pair (MTT) showed higher sensitivity. The calibration curve was linear from 0.1 to 5 mM d-carnitine, with a quantification limit of 0.1 mM and a relative standard deviation of 1.51%. The procedure is simple, rapid, accurate, and easily automated. It was satisfactorily applied to following d-carnitine levels during the microbial transformation of d-carnitine into l-carnitine and to determining the d-carnitine content of pharmaceutical preparations.
Obón
J M
JM
Department of Biochemistry and Molecular Biology B, Faculty of Chemistry, University of Murcia, Murcia, E-30100, Spain.
Buendía
B
B
Cánovas
M
M
Iborra
J L
JL
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D001331:Automation; D001711:Biotransformation; D002331:Carnitine / Q000032:analysis*; D002332:Carnitine O-Acetyltransferase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005562:Formazans; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D015203:Reproducibility of Results; D012231:Rhizobium / Q000201:enzymology; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D013237:Stereoisomerism
D005562:Formazans; D007202:Indicators and Reagents; D009243:NAD; D000429:Alcohol Oxidoreductases; C019842:carnitine dehydrogenase; D002332:Carnitine O-Acetyltransferase; D002331:Carnitine