Quantitation of cytochrome P450 mRNAs in patients with suspected liver diseases as assessed by reverse transcriptase-polymerase chain reaction.
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 beta-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.
Finnström
N
N
Department of Medical Sciences, University Hospital, Uppsala, Sweden.
Bjelfman
C
C
Thörn
M
M
Lööf
L
L
Rane
A
A
D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D005260:Female; D005838:Genotype; D006801:Humans; D008107:Liver Diseases / Q000201:enzymology* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000502:physiology
D017931:DNA Primers; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System