Selective expression of CYP3A5 and not CYP3A4 in human blood.
Selective expression of CYP3A5 and not CYP3A4 in human blood.
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在人血液中选择性表达 CYP3A5 而不是 CYP3A4。
DOI：
10.1097/00008571-199610000-00001
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Watkins,PB
中科院分区：
文献类型：
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作者：
Janardan,SK;Lown,KS;Schmiedlin-Ren,P;Thummel,KE;Watkins,PB
肝脏和肠道中 CYP3A4 的活性存在明显的人与人差异。我们推断，如果 CYP3A4 在外周血细胞中表达，则基于血液的 CYP3A4 表型测试可能是可行的。我们制备了健康志愿者的外周血涂片，并用兔多克隆抗体进行了免疫染色，该抗体选择性地与 CYP3A 亚家族内的酶发生反应。仅在中性粒细胞 (PMN) 的细胞质内观察到染色。使用合成寡核苷酸作为引物，对从分离的 PMN 和单核细胞制备的 cDNA 进行聚合酶链式反应，选择性扩增每种已知 CYP3A cDNA（CYP3A3、CYP3A4、CYP3A5 和 CYP3A7）的片段。仅使用 CYP3A5 特异性寡核苷酸（主要在 PMN 中）获得扩增，并通过测序确认扩增片段的身份。接下来，将从人血中制备的全白细胞匀浆与识别所有 CYP3A 蛋白的单克隆抗体或仅识别 CYP3A5 的吸收多克隆抗体进行免疫印迹反应。两种抗体都能识别白细胞中与纯化的 CYP3A5 共迁移的蛋白质。然而，在分离的粒细胞匀浆、整个 WBC 级分匀浆或与未裂解的 WBC 制剂的孵育中，无法检测到咪达唑仑（CYP3A 底物）的代谢。我们的结论是 CYP3A4 在外周血中不表达，因此对该酶的血液表型测试不可行。我们发现 CYP3A5 的存在可能很重要，因为这种酶也存在于许多人的肝脏、肠道和肾脏中。
There is a marked variation between people in the activity of CYP3A4 in liver and intestine. We reasoned that if CYP3A4 was expressed in peripheral blood cells, a simple blood based test of CYP3A4 phenotype might be feasible. We prepared peripheral blood smears from healthy volunteers and performed immunostaining with a rabbit polyclonal antibody that selectively reacts with enzymes within the CYP3A subfamily. Staining was observed only within the cytoplasm of neutrophils (PMNs). cDNA prepared from isolated PMNs and mononuclear cells was subjected the polymerase chain reaction using as primers synthetic oligonucleotides that selectively amplify fragments of each known CYP3A cDNAs (CYP3A3, CYP3A4, CYP3A5, and CYP3A7). Amplification was only obtained with the CYP3A5 specific oligonucleotides, predominantly in PMNs, and the identity of the amplified fragment was confirmed by sequencing. Next, whole white cell homogenate prepared from human blood was reacted on immunoblots with a monoclonal antibody that recognizes all CYP3A proteins or an absorbed polyclonal antibody that recognizes only CYP3A5. Both antibodies recognized a protein in the white cells that comigrated with purified CYP3A5. However, metabolism of midazolam, a substrate of CYP3A, could not be detected in homogenates of isolated granulocytes, in homogenates of the whole WBC fractions, or in incubations with unlysed WBC preparations. We conclude that CYP3A4 is not expressed in peripheral blood and hence 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.