Quantitation of cytochrome P450 mRNAs in patients with suspected liver diseases as assessed by reverse transcriptase-polymerase chain reaction

• Published in: The Journal of laboratory and clinical medicine Vol. 134; no. 2; pp. 133 - 140
• Main Authors: Finnström, Niklas, Bjelfman, Catarina, Thörn, Mari, Lööf, Lars, Rane, Anders
• Format: Journal Article
• Language: English
• Published: Saint Louis, MO Mosby, Inc 01-08-1999; Elsevier
• Subjects: Biological and medical sciences; Cytochrome P-450 Enzyme System - genetics; DNA Primers; Female; Genotype; Humans; Investigative techniques, diagnostic techniques (general aspects); Liver Diseases - enzymology; Liver Diseases - genetics; Male; Medical sciences; Medicin och hälsovetenskap; Middle Aged; Miscellaneous. Technology; Nucleic Acid Hybridization; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Phenotype; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Messenger - analysis; Sensitivity and Specificity; Transcription, Genetic - physiology; rcRNA; cDNA; CYP; RT-PCR; Human; Polymerase chain reaction; Messenger RNA; Cytochrome P450; Reverse transcription; Digestive diseases; Hepatic disease; Diagnosis; Technique; Molecular biology; Quantitative analysis
• ISSN: 0022-2143; 1532-6543
• DOI: 10.1016/S0022-2143(99)90117-8

The cytochrome P450 system (CYP) is vital for the oxidation and detoxification of numerous drugs and other xenobiotics in the liver. Many of the CYP enzymes are polymorphically expressed and may be induced or inhibited by xenobiotics including drugs and alcohol. The measurement of gene expression is thus important in studies of the mechanisms of interaction with and function of the CYP system. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for the study of the mRNA expression of three CYP enzymes—2E1, 1A2, and 3A4—in snap-frozen percutaneous liver biopsy samples. The method was made quantitative by the introduction of a recombinant RNA internal standard that contains a transcript of the β-globin gene and sequences specific for the studied CYP enzymes. The method allows the analysis of mRNA expression of several enzymes in as little as 5 mg of liver tissue. Liver tissue specimens from 19 patients with suspected liver disease were analyzed for CYP-specific mRNA expression. The mean mRNA concentrations for CYP1A2, 2E1, and 3A4 were 0.16, 0.74, and 0.32 amol of specific mRNA per nanogram of poly (A+) mRNA, respectively, but a large interindividual variation was observed. CYP3A7 primers were included in the internal standard. However, because of low expression it was not possible to quantitate the enzyme. This quantitative RT-PCR method is of value for studies of the mechanisms of variation and interactions with the members of the CYP enzyme family in healthy and diseased liver and other organs.