In humans, the cytochrome P450 3A (CYP3A) subfamily is involved in midazolam (MDZ) biotransformation into 1′‐ and 4‐hydroxy metabolites, and the former serves as a probe for CYP3A catalytic activity. In veterinary species is still crucial to identify enzyme‐ and species‐specific CYP substrates; thus, the aim of this study was to characterize MDZ oxidation in cattle liver. A HPLC‐UV method was used to measure 1′‐ and 4‐hydroxy MDZ (1′‐ and 4‐OHMDZ, respectively) formation in cattle liver microsomes and assess the role of CYP3A by an immunoinhibition study. Moreover, MDZ hydroxylation was evaluated in 300 cattle liver samples and results were correlated with testosterone hydroxylation. Formation of both metabolites conformed to a single‐enzyme Michaelis–Menten kinetics. Values of V_max and K_m were 0.67 nmol/min/mg protein and 6.16 μM for 4‐OHMDZ, and 0.06 nmol/min/mg protein and 10.08 μM for 1′‐OHMDZ. An anti‐rat CYP3A1 polyclonal antibody inhibited up to 50% and 94% 1′‐ and 4‐OHMDZ formation, respectively. MDZ oxidation in liver microsomes was poorly correlated with testosterone hydroxylation. In conclusion, cattle metabolized MDZ to 1′‐OHMDZ and 4‐OHMDZ. The immunoinhibition results indicated a major contribution of CYP3As to 4‐OHMDZ formation and the involvement of other CYPs in 1′‐OHMDZ production, paving the way for further investigations.

中文翻译：

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咪唑安定在牛肝微粒体中的氧化：细胞色素P450 3A的作用。

在人类中，细胞色素P450 3A（CYP3A）亚家族参与咪达唑仑（MDZ）生物转化为1'和4-羟基代谢物，而前者可作为CYP3A催化活性的探针。兽医物种对于鉴定酶和物种特异性CYP底物仍然至关重要。因此，本研究的目的是表征牛肝中的MDZ氧化。HPLC-UV方法用于测量牛肝微粒体中1'-和4-羟基MDZ（分别为1'-和4-OHMDZ）的形成并通过免疫抑制研究评估CYP3A的作用。此外，在300头牛肝样品中评估了MDZ羟基化，其结果与睾丸激素羟基化相关。两种代谢物的形成均符合单一酶Michaelis-Menten动力学。V _max和的值对于4-OHMDZ，K _m为0.67 nmol / min / mg蛋白和6.16μM;对于1'-OHMDZ，K _m为0.06 nmol / min / mg蛋白和10.08μM。抗大鼠CYP3A1多克隆抗体分别抑制高达50％和94％的1'-和4-OHMDZ形成。肝微粒体中的MDZ氧化与睾丸激素羟化关系较弱。总之，牛将MDZ代谢为1'-OHMDZ和4-OHMDZ。免疫抑制结果表明CYP3A对4-OHMDZ形成有重要贡献，而其他CYP参与1'-OHMDZ的产生，为进一步研究铺平了道路。