PMID- 32738524
OWN - NLM
STAT- MEDLINE
DCOM- 20210322
LR  - 20210322
IS  - 1873-376X (Electronic)
IS  - 1570-0232 (Linking)
VI  - 1153
DP  - 2020 Sep 15
TI  - UPLC-MS/MS analysis of CYP1A-mediated ethoxyresorufin-O-deethylation activity in 
      the rat kidney microsomes.
PG  - 122272
LID - S1570-0232(20)30748-0 [pii]
LID - 10.1016/j.jchromb.2020.122272 [doi]
AB  - 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 microL reaction mixture, 
      rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1-5 microM) 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 C(18) 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 microM (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.
CI  - Copyright (c) 2020 Elsevier B.V. All rights reserved.
FAU - Venkatapura Chandrashekar, Devaraj
AU  - Venkatapura Chandrashekar D
AD  - Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, Chapman 
      University, Irvine, USA.
FAU - Mehvar, Reza
AU  - Mehvar R
AD  - Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, Chapman 
      University, Irvine, USA. Electronic address: mehvar@chapman.edu.
LA  - eng
PT  - Journal Article
DEP - 20200716
PL  - Netherlands
TA  - J Chromatogr B Analyt Technol Biomed Life Sci
JT  - Journal of chromatography. B, Analytical technologies in the biomedical and life 
      sciences
JID - 101139554
RN  - 0 (Oxazines)
RN  - 5725-91-7 (ethoxyresorufin)
RN  - 635-78-9 (resorufin)
RN  - EC 1.14.14.1 (Cytochrome P-450 CYP1A1)
SB  - IM
MH  - Animals
MH  - Chromatography, High Pressure Liquid/*methods
MH  - Cytochrome P-450 CYP1A1/*analysis/metabolism
MH  - Kidney/cytology/*metabolism
MH  - Kinetics
MH  - Limit of Detection
MH  - Linear Models
MH  - Male
MH  - Microsomes/enzymology/*metabolism
MH  - Oxazines/analysis/metabolism
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Reproducibility of Results
MH  - Tandem Mass Spectrometry/*methods
OTO - NOTNLM
OT  - CYP1A
OT  - Ethoxyresorufin-O-deethylase
OT  - Kidney
OT  - Michaelis-Menten kinetics
OT  - Microsomes
OT  - UPLC-MS/MS
COIS- Declaration of Competing Interest The authors declare that they have no known 
      competing financial interests or personal relationships that could have appeared 
      to influence the work reported in this paper.
EDAT- 2020/08/02 06:00
MHDA- 2021/03/23 06:00
CRDT- 2020/08/02 06:00
PHST- 2020/05/15 00:00 [received]
PHST- 2020/07/03 00:00 [revised]
PHST- 2020/07/12 00:00 [accepted]
PHST- 2020/08/02 06:00 [pubmed]
PHST- 2021/03/23 06:00 [medline]
PHST- 2020/08/02 06:00 [entrez]
AID - S1570-0232(20)30748-0 [pii]
AID - 10.1016/j.jchromb.2020.122272 [doi]
PST - ppublish
SO  - J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 15;1153:122272. doi: 
      10.1016/j.jchromb.2020.122272. Epub 2020 Jul 16.