The in-vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide was investigated in bovine liver homogenate. A metabolic reaction mixture was prepared that included a crude preparation of uridine diphosphate (UDP) glucuronyl transferase, 7-hydroxycoumarin and UDP-glucuronic acid. A HPLC method was developed to separate coumarin, 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide and an internal standard, 4-hydroxycoumarin. Samples were separated by reverse-phase HPLC, on a C18 column, with a 1 ml min-1 gradient elution with UV detection at 320 nm. The limit of quantification of the method, for 7-hydroxycoumarin-glucuronide, was 1.47 microM, and the linear range was from 0-295.7 microM. Concentrations of 7-hydroxycoumarin-glucuronide produced were calculated from a plot of 7-hydroxycoumarin-glucuronide concentration versus the mean absorbance ratio (n = 4) (7-hydroxycoumarin-glucuronide absorbance/4-hydroxycoumarin absorbance). It was possible to monitor the decrease in the 7-hydroxycoumarin content as it was metabolised as well as the increase in 7-hydroxycoumarin-glucuronide as it was produced enzymatically. The identity of the compound produced was confirmed by photodiode array spectral analysis. A plot of time versus 7-hydroxycoumarin-glucuronide produced indicates that the metabolism is linear for the first 90 min and reached a plateau at 150 min. The rate of reaction in the first 90 min was 2.96 +/- 0.06 (RSD 1.7%, n = 3) nmol of 7-hydroxycoumarin-glucuronide produced per minute per milligram of protein. After 150 min 0.34 +/- 0.005 mM (RSD 1.4%) 7-hydroxycoumarin-glucuronide was produced, from 0.77 mM 7-hydroxycoumarin introduced into the reaction mixture and 58.0% +/- 5.3% (or 0.44 +/- 0.02 mM) of the 7-hydroxycoumarin remained. These results show that it is possible to monitor the production of the phase II metabolite of coumarin with minimal sample clean-up and without the need for deconjugation of the glucuronide moiety. The method was very reliable and applicable for the direct determination of 7-hydroxycoumarin-glucuronide in an in-vitro metabolic assay.

中文翻译：

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通过HPLC分析7-羟基香豆素的葡糖醛酸糖苷化。

在牛肝匀浆中研究了7-羟基香豆素体外代谢为7-羟基香豆素-葡萄糖醛酸。制备代谢反应混合物，其包括尿苷二磷酸（UDP）葡糖醛酸转移酶，7-羟基香豆素和UDP-葡糖醛酸的粗制品。开发了一种HPLC方法以分离香豆素，7-羟基香豆素，7-羟基香豆素-葡糖苷酸和内标物4-羟基香豆素。通过反相HPLC在C18柱上分离样品，用1 ml min-1梯度洗脱，并在320 nm处进行UV检测。对于7-羟基香豆素-葡糖醛酸苷，该方法的定量限为1.47 microM，线性范围为0-295.7 microM。根据7-羟基香豆素-葡萄糖醛酸化物浓度对平均吸光度比（n = 4）（7-羟基香豆素-葡糖醛酸化物吸光度/ 4-羟基香豆素吸收度）的曲线计算出产生的7-羟基香豆素-葡糖醛酸浓度。可以监测被代谢的7-羟基香豆素含量的减少，以及通过酶促产生的7-羟基香豆素-葡萄糖醛酸含量的增加。通过光电二极管阵列光谱分析确认了产生的化合物的身份。时间与产生的7-羟基香豆素-葡糖醛酸的关系图表明，在最初的90分钟内代谢是线性的，并在150分钟达到平稳。在前90分钟内，每毫克蛋白质每分钟产生的7-羟基香豆素-葡萄糖醛酸的反应速率为2.96 +/- 0.06（RSD 1.7％，n = 3）nmol。150分钟后0。由引入到反应混合物中的0.77 mM 7-羟基香豆素和7种羟基化合物的58.0％+/- 5.3％（或0.44 +/- 0.02 mM）制得34 +/- 0.005 mM（RSD 1.4％）7-羟基香豆素-葡萄糖醛酸-羟基香豆素残留。这些结果表明，可以在不需对葡糖醛酸苷部分进行解偶联的情况下，以最少的样品净化来监测香豆素的II期代谢产物的产生。该方法非常可靠，可用于体外代谢测定中直接测定7-羟基香豆素-葡萄糖醛酸。这些结果表明，可以在不需对葡糖醛酸苷部分进行解偶联的情况下，以最少的样品净化来监测香豆素的II期代谢产物的产生。该方法非常可靠，可用于体外代谢测定中直接测定7-羟基香豆素-葡萄糖醛酸。这些结果表明，可以在不需对葡糖醛酸苷部分进行解偶联的情况下，以最少的样品净化来监测香豆素的II期代谢产物的产生。该方法非常可靠，可用于体外代谢测定中直接测定7-羟基香豆素-葡萄糖醛酸。