Comparison of the Rates of Hydrolysis of Lorazepam-Glucuronide, Oxazepam-Glucuronide and Temazepam-Glucuronide Catalyzed by E. Coli. β-D-Glucuronidase Using the On-line Benzodiazepine Screening Immunoassay on the Roche/Hitachi 917 Analyzer

    Volume 46, Issue 2 (March 2001)

    ISSN: 0022-1198

    CODEN: JFSOAD

    Page Count: 6


    McNally, AJ
    director of research and development, Roche Diagnostic Corporation, Indianapolis, IN

    Dou, C
    Research scientists, Roche Diagnostic Corporation, Indianapolis, IN

    Bournique, JS
    Research scientists, Roche Diagnostic Corporation, Indianapolis, IN

    Zinda, MK
    Research scientists, Roche Diagnostic Corporation, Indianapolis, IN

    Gnezda, M
    Research scientists, Roche Diagnostic Corporation, Indianapolis, IN

    Salamone, SJ
    vice president of research and development, Roche Diagnostic Corporation, Indianapolis, IN

    (Received 15 February 2000; accepted 28 April 2000)

    Abstract

    The catalytic rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide, and temazepam-glucuronide when catalyzed by E. Coli. β-glucuronidase both in phosphate buffer and buffered drug-free urine were compared as well as the pH dependence of enzyme activity. In 50 mM phosphate buffer pH 6.4, lorazepam-glucuronide has the highest turnover rate of 3.7 s−1 with an associated Km of about 100 µM. followed by oxazepam-glucuronide, which has a turnover rate of 2.4 s−1 with an associated Km of 60 µM. Temazepam-glucuronide has the lowest rate of 0.94 s−1 with an associated Km of 34 µM. In buffered drug-free urine, a similar trend was observed. In addition, an optimal pH for β-glucuronidase was determined to be between 6 and 7 when the enzyme hydrolyzes the benzodiazepine conjugates in buffered drug-free urine. Effects of temperature and incubation time were also examined. It can be concluded that the electron donating or withdrawing of the individual benzodiazepine structure may play an important role in the reactivity of the lorazepam-glucuronide, oxazepam-glucuronide and temazepam-glucuronide catalyzed by β-glucuronidase. This is consistent with other observations made for monosubstituted phenyl-β-glucuronides by Wang et al. (1).


    Paper ID: JFS14967J

    DOI: 10.1520/JFS14967J

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    Title Comparison of the Rates of Hydrolysis of Lorazepam-Glucuronide, Oxazepam-Glucuronide and Temazepam-Glucuronide Catalyzed by E. Coli. β-D-Glucuronidase Using the On-line Benzodiazepine Screening Immunoassay on the Roche/Hitachi 917 Analyzer
    Symposium , 0000-00-00
    Committee E30