An in vitro microdialysis methodology to study 11 beta-hydroxysteroid dehydrogenase type I enzyme activity in liver microsomes
by Sun, Li; Stenken, Julie Ann; Yang, Amy Y.; Zhao, Jamie J.; Musson, Donald G.
Microdialysis sampling coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS) was used to observe in vitro I I beta-hydroxysteroid dehydrogenase type 1 (HSDI) enzyme-catalyzed conversion of stable-isotope-labeled cortisone to cortisol in liver microsomes from dog, monkey, and human. Experimental conditions that would affect the microdialysis sampling approach including probe length, perfusion fluid flow rate, extraction efficiency (E-d), substrate concentration, and enzyme reaction conditions were evaluated. Dialysates containing high salt concentrations (> 1 50 mM) were directly assayed using LC/MS/MS without additional sample cleanup. The sensitivity (with lower level of quantitation at 0. 1 ng/mL) and selectivity of this assay allowed detection of the enzyme reactants at physiologically relevant levels. The interconversion from M+4 cortisone to M+4 cortisol was detected in dog, human, and monkey liver microsomes. Results show species-specific reaction profiles, with a five times higher conversion rate in dog liver microsomes than in human and monkey liver microsomes. Based on M+4 cortisol production rate obtained using a microdialysis infusion of M+4 cortisone to the microsomes coincubated with a proprietary I I beta-HSDI inhibitor of different concentrations, the degrees of enzyme inhibition were found to be 40 and 85%, consistent with values obtained by a traditional in vitro incubation method. The microdialysis sampling methodology with LC/MS/MS provided extensive information about I I beta-HSDI activities in microsomes from different mammalian species. (c) 2007 Elsevier Inc. All rights reserved.
- Analytical Biochemistry
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