Comparison of the rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide and tamazepam-glucuronide catalyzed by E. coli beta-D-glucuronidase using the on-line benzodiazepine screening immunoassay on the Roche/Hitachi 917 analyzer.
The catalytic rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide, and temazepam-glucuronide when catalyzed by E. Coli. beta-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 microM, followed by oxazepam-glucuronide, which has a turnover rate of 2.4 s(-1) with an associated Km of 60 microM. Temazepam-glucuronide has the lowest rate of 0.94 s(-1) with an associated Km of 34 microM. In buffered drug-free urine, a similar trend was observed. In addition, an optimal pH for beta-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 beta-glucuronidase. This is consistent with other observations made for monosubstituted phenyl-beta-glucuronides by Wang et al. (1).
Dou
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Roche Diagnostic Corporation, Indianapolis, IN 46250, USA.
Bournique
J S
JS
Zinda
M K
MK
Gnezda
M
M
McNally
A J
AJ
Salamone
S J
SJ
D014151:Anti-Anxiety Agents / Q000378:metabolism* / Q000493:pharmacokinetics; D004926:Escherichia coli / Q000201:enzymology*; D005554:Forensic Medicine / Q000379:methods; D005966:Glucuronidase / Q000378:metabolism; D020719:Glucuronides / Q000032:analysis / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007118:Immunoassay; D008140:Lorazepam / Q000378:metabolism* / Q000493:pharmacokinetics; D010076:Oxazepam / Q000378:metabolism* / Q000493:pharmacokinetics; D013693:Temazepam / Q000378:metabolism* / Q000493:pharmacokinetics; D013696:Temperature; D016482:Urinalysis
D014151:Anti-Anxiety Agents; D020719:Glucuronides; D010076:Oxazepam; D013693:Temazepam; D005966:Glucuronidase; D008140:Lorazepam