In vitro determination of the CYP 3A4 activity in rat hepatic microsomes by liquid-phase extraction and HPLC-photodiode array detection.
BACKGROUND
CYP3A4 is one of the most important of all drug-metabolizing enzymes. Although several direct or indirect quantification methods have been proposed for the determination of the CYP3A4 activity, the sample preparation is mostly tedious and usually requires time consuming separate extraction steps and solid-phase extraction.


METHODS
Here, we developed a simple and selective HPLC method, coupled to photodiode array detection for direct determination of CYP3A4-mediated testosterone hydroxylase activity in hepatic microsomes of rats. After microsome incubation, a single-step liquid-phase extraction of specific substrates, testosterone and its metabolite 6-hydroxytestosterone, together with the salicylamid acid used as an internal standard, was applied. The analytical method was fully validated by the determination of different parameters (intra- and inter-day variability of 6-β-OH-testosterone concentration, accuracy and limit of detection). Finally, this method was applied to quantify the CYP3A4 activity of rats intravenously administered with either the mesoporous iron(III) trimesate MIL-100 nanocarrier (MIL stands for Materials from Institut Lavoisier) or with its corresponding organic linker.


RESULTS
All analytes were simultaneously separated from a single run shorter than 10 min, reaching relative standard deviations of intra- and inter-day precision <18.5% and an accuracy of estimated 6-β-OH-testosterone concentrations ranging from 95 to 111%. The mean±standard deviation absolute recoveries of 6-β-OH-testosterone at 0.01, 1.00 and 20.00 μg/mL were 97±4%, 101±3% and 99±2%, respectively while it reached 98±3% for the internal standard.


CONCLUSIONS
The developed HPLC-PDA method enables the accurate and sensitive determination of the CYP3A4 activity through the quantification of the 6-β-OH-testosterone produced by the CYP3A4-mediated testosterone hydroxylase in rat hepatic microsomal suspensions. Additionally, the rapid procedure offers an economical advantage with respect to resources and operator time. Finally, the determination of CYP3A4 activity in hepatic microsomes of rats administered with nanoparticles of the porous iron(III) trimesate nanocarrier shows no significant differences between control, trimesic and nanoparticle groups, evidencing that the linker is not metabolized by CYP3A4.
UMR CNRS 8180, Institut Lavoisier, Université de Versailles Saint Quentin en Yvelines, France.
Tarek
T
Baati
Patricia
P
Horcajada
Ruxandra
R
Gref
Patrick
P
Couvreur
Christian
C
Serre
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D057166:High-Throughput Screening Assays; D006915:Hydroxytestosterones / Q000032:analysis / Q000378:metabolism; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D006915:Hydroxytestosterones; C025591:6 beta-hydroxytestosterone; D003577:Cytochrome P-450 Enzyme System; C465260:Cyp3a2 protein, rat; D051544:Cytochrome P-450 CYP3A