Enzymatic cycling assay for D-carnitine determination

Anal Biochem. 1999 Oct 1;274(1):34-9. doi: 10.1006/abio.1999.4233.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases* / isolation & purification
  • Alcohol Oxidoreductases* / metabolism
  • Automation
  • Biotransformation
  • Carnitine / analysis*
  • Carnitine O-Acetyltransferase / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Formazans
  • Indicators and Reagents
  • Kinetics
  • NAD / metabolism
  • Reproducibility of Results
  • Rhizobium / enzymology
  • Sensitivity and Specificity
  • Spectrophotometry / methods
  • Stereoisomerism

Substances

  • Formazans
  • Indicators and Reagents
  • NAD
  • Alcohol Oxidoreductases
  • carnitine dehydrogenase
  • Carnitine O-Acetyltransferase
  • Carnitine