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Rapid Quantification of CYP3A4 Expression in Human Leukocytes by Real-Time Reverse Transcription-PCR

¹ Institute of Clinical Pharmacology, Charité University Medical Center, Humboldt University of Berlin, 10098 Berlin, Germany

² Department of Clinical Pharmacology, University Medical Center, Georg-August University of Göttingen, 37075 Göttingen, Germany

^aaddress correspondence to this author at: Department of Clinical Pharmacology, University Medical Center, Georg-August-University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany; fax 49-551-3912767, e-mail jurgen.brockmoller@med.uni-goettingen.de

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 30% 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)(5)(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 (20 males and 11 females; mean age, 29 years; range, 20–64 years) and in three human liver samples obtained from the International Institute for the Advancement of Medicine (Exton, PA). Before blood collection, all volunteers signed informed consents that were accepted by the Ethical Committee of the Charité. Leukocytes were separated from 8 mL of whole blood in a Vacutainer^® cpt cell preparation tube system (Becton Dickinson). Small liver fragments were disrupted with a homogenizer (Potter; Braun). Samples were stored at -80 °C. Total cellular RNA was . . . [Full Text of this Article]

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