UPLC-MS/MS analysis of CYP1A-mediated ethoxyresorufin-O-deethylation activity in the rat kidney microsomes
| Autor: | Devaraj Venkatapura Chandrashekar, Reza Mehvar |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Male
Formic acid Clinical Biochemistry Kinetics Kidney 030226 pharmacology & pharmacy 01 natural sciences Biochemistry High-performance liquid chromatography Michaelis–Menten kinetics Analytical Chemistry Rats Sprague-Dawley 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Limit of Detection Tandem Mass Spectrometry Microsomes Oxazines medicine Cytochrome P-450 CYP1A1 Animals Trichloroacetic acid Chromatography High Pressure Liquid Chromatography biology Chemistry 010401 analytical chemistry Cytochrome P450 Reproducibility of Results Cell Biology General Medicine 0104 chemical sciences Rats medicine.anatomical_structure biology.protein Microsome Linear Models |
| Zdroj: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 1153 |
| ISSN: | 1873-376X |
| Popis: | Ethoxyresorufin (ER)-O-deethylation (EROD) activity has been widely used to assess cytochrome P450 1A (CYP1A) activity. The kinetics of CYP1A activity have been well characterized in the liver microsomes. However, studies in kidney microsomes are limited due to the much lower EROD activity in this organ. Here, we developed and validated a sensitive UPLC-MS/MS assay for the characterization of the EROD activity in the rat kidney microsomes. In a 50 µL reaction mixture, rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1–5 µM) and NADPH (1 mM) for 10 min. Acidic solvents, such as trichloroacetic acid or formic acid, used for quenching of the metabolic reactions and precipitation of the proteins, unexpectedly caused a spontaneous formation of resorufin (RES) from ER. Therefore, the metabolic reactions were terminated by adding acetonitrile, containing a deuterated internal standard (IS). Chromatographic separation was achieved on a C18 UPLC column, and the MS/MS ion transitions were 213.9/185.9 for RES and 220.0/192.0 for IS. The assay was validated in the linear range of 0.5 nM to 75 nM of RES and had a lower limit of quantitation of 0.5 nM. The overall recoveries of RES (90%–99%) and IS (85%–103%) were relatively high, with minimal matrix effect. The assay was successfully applied to the estimation of the Michaelis-Menten (MM) kinetics of EROD activity in the rat kidney microsomes (n = 3), which showed a maximum velocity of 2.68 ± 0.17 pmol/min/mg and a MM constant of 1.72 ± 0.24 µM (mean ± SD). It is concluded that our sensitive and specific analytical method, coupled with the optimized microsomal incubation conditions, provides a robust platform for further investigations of the effects of xenobiotics, environmental factors, or pathophysiologic conditions on the kinetics of EROD activity in the kidney microsomes. |
| Databáze: | OpenAIRE |
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