SELECTIVE EXPRESSION OF CYP3A5 AND NOT CYP3A4 IN HUMAN BLOOD

There is a marked variation between people in the activity of CYP3A4 i n liver and intestine. We reasoned that if CYP3A4 was expressed in per ipheral blood cells, a simple blood based test of CYP3A4 phenotype mig ht be feasible. We prepared peripheral blood smears from healthy volun teers and performed immunostaining with a rabbit polyclonal antibody t hat selectively reacts with enzymes within the CYP3A subfamily. Staini ng was observed only within the cytoplasm of neutrophils (PMNs). cDNA prepared from isolated PMNs and mononuclear cells was subjected the po lymerase chain reaction using as primers synthetic oligonucleotides th at selectively amplify fragments of each known CYP3A cDNAs (CYP3A4, CY P3A5, and CYP3A7). Amplification was only obtained with the CYP3A5 spe cific oligonucleotides, predominantly in PMNs, and the identity of the amplified fragment was confirmed by sequencing. Next, whole white cel l homogenate prepared from human blood was reacted on immunoblots with a monoclonal antibody that recognizes all CYP3A proteins or an absorb ed polyclonal antibody that recognizes only CYP3A5. Both antibodies re cognized a protein in the white cells that comigrated with purified CY P3A5. However, metabolism of midazolam, a substrate of CYP3A, could no t be detected in homogenates of isolated granulocytes, in homogenates of the whole WBC fractions, or in incubations with unlysed WBC prepara tions. We conclude CYP3A4 is not expressed in peripheral blood and hen ce a blood phenotyping test for this enzyme will not be feasible. Our discovery that CYP3A5 is present may be important since this enzyme is also present in the liver, intestine and kidney of many people.