Measurement of rat-intestinal cefuroxime axetil esterase activity: comparison of an h.p.l.c. and coupled-enzyme assay.
An alternative method to h.p.l.c. for determining cefuroxime axetil esterase (CAE) activity has been developed which involves coupling acetaldehyde, produced in the esterase reaction, with alcohol dehydrogenase (ADH) to provide a direct reading spectrophotometric assay. The optimum temperature and concn. of NADH, cefuroxime axetil and ADH for the assay are 37 degrees C, 160 microM, 2.9 mM and 160 U/ml, respectively. The coupled assay was more reproducible but less sensitive than the h.p.l.c. assay, and the two methods gave results that were not significantly different (P greater than 0.05). Both assays responded linearly when CAE activity was measured as a function of protein concn., however, the coupled assay was impaired at ionic strengths greater than 0.2 M NaCl, whereas no adverse effects were seen with the h.p.l.c. assay up to 0.5 M NaCl.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002444:Cefuroxime / Q000031:analogs & derivatives / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004950:Esterases / Q000378:metabolism*; D008433:Mathematics; D009243:NAD / Q000378:metabolism; D051381:Rats; D013696:Temperature
D009243:NAD; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D004950:Esterases; D002444:Cefuroxime; C040738:cefuroxime axetil