Ohta M, Fujii S, Wada A, Ohnishi T, Yamano T, Okamoto M
J Steroid Biochem. 1987 Jan;26(1):73-81. doi: 10.1016/0022-4731(87)90033-1.
Incubation of 11-deoxycorticosterone with a cytochrome P-450(11)beta-reconstituted system yielded, in addition to corticosterone and 18-hydroxy-11-deoxycorticosterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-11-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-11-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450(11)beta was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-11-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-11-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450(11)beta. Therefore, we next incubated 19-hydroxy-11-deoxycorticosterone with cytochrome P-450(11)beta and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-oxo-11-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and 1H NMR analyses. Its field desorption mass spectrum (FD-MS) showed a M+ peak at m/e 344. The 1H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450(11)beta can catalyze the 19-hydroxylation of 11-deoxycorticosterone, and the 19-hydroxy-11-deoxycorticosterone produced is further oxidized at the C-19 position to 19-oxo-11-deoxycorticosterone.
将11-脱氧皮质酮与细胞色素P-450(11)β重组系统一起温育，除了生成皮质酮和18-羟基-11-脱氧皮质酮外，还产生了一种新的类固醇产物。该新产品在高效液相色谱(HPLC)上的保留时间与 authentic 19-羟基-11-脱氧皮质酮相同。19-羟基-11-脱氧皮质酮形成的周转数为7.0 mol/分钟/摩尔P-450。当使用大量细胞色素P-450(11)β进行反应并通过HPLC分析产物时，色谱图中19-羟基-11-脱氧皮质酮峰消失，同时出现新的未鉴定峰。这些结果表明，19-羟基-11-脱氧皮质酮被细胞色素P-450(11)β进一步代谢为其他类固醇。因此，我们接下来将19-羟基-11-脱氧皮质酮与细胞色素P-450(11)β一起温育，并通过HPLC分析反应产物。上述未鉴定峰再次出现在色谱图中。其中一个峰的保留时间与 authentic 19-氧代-11-脱氧皮质酮的保留时间一致。该峰物质通过反复HPLC纯化，并进行质谱和1H NMR分析。其场解吸质谱(FD-MS)在m/e 344处显示M+峰。1H NMR谱显示在C-19位置有醛基质子的信号，而不是羟甲基质子的信号。这些结果表明，细胞色素P-450(11)β可以催化11-脱氧皮质酮的19-羟化反应，并且产生的19-羟基-11-脱氧皮质酮在C-19位置进一步氧化为19-氧代-11-脱氧皮质酮。
