Rapid quantification of CYP3A4 expression in human leukocytes by real-time reverse transcription-PCR.

The SD of residuals (S y͉x) was 73 ng/L in the concentration range of 0 –5000 ng/L and 22 ng/L in the range of 0 –500 ng/L. The agreement between the GC/MS and MEIA assays indicated low interference from other estrogenic compounds and their metabolites in the latter assay. In conclusion, we have developed a GC/MS method for the quantification of estradiol in patient serum samples and serum-based immunoassay calibrators; the method was validated by use of CRMs. The utility of the method was further demonstrated by the presented correlation between estradiol concentrations in patient samples measured by MEIA and GC/MS. We thank Dr. Greg Williams and his team at Abbott Laboratories for providing the immunoassay data for the patient samples. Determination of oestradiol-17b in human serum by isotope dilution-mass spectrometry. Analysis of unconjugated steroids in plasma by liquid-gel chromatography and glass capillary gas chromatography-mass spectrometry. binding in target tissues: a GC/MS method for assessing uptake, retention, and processing of estrogens in target cell nuclei under in vivo conditions. Gas chromatographic-mass spectrometric analysis of endogenoud levels of estradiol in plasma and in cytosol from rat uterus. Quantitative secondary ion monitoring gas chromatography/mass spectrometry of diethylstilbestrol in bovine liver. Cytochrome P450 3A4 (CYP3A4) contributes to the metabolism of a wide variety of drugs and endogenous substrates, such as steroid hormones (1, 2). Variations in the catalytic activity of CYP3A4 are predominantly caused by enzyme induction mediated by transcriptional activation or by competitive substrate inhibition. Such variation may strongly influence the bioavailability of drugs and may modulate drug interactions. CYP3A4 is one of the predominant CYPs in the human liver, accounting for ϳ30% of the total hepatic cytochrome P450 protein (2, 3). Relatively high CYP3A4 concentrations have been found in the small intestinal epithelium (70% of total CYP protein) and in the kidney (2). There are conflicting results concerning the amount of CYP3A4 in human peripheral blood lymphocytes. Several authors could not detect any CYP3A4 mRNA or protein, whereas some studies reported poor CYP3A4 expression in the white cell fraction (4 – 6). Thus, we assumed that CYP3A4 is expressed in lymphocytes in very small amounts and that only a very sensitive method could detect them. We developed a sensitive quantitative real-time reverse transcription -PCR (RT-PCR) method that allows rapid and correct determination of CYP3A4 mRNA expression in leukocytes. We investigated CYP3A4 mRNA expression in 31 human blood samples from healthy volunteers …