Kremers P, De Graeve J, Azhir A, Gielen J E
Eur J Biochem. 1978 Jan 16;82(2):529-33. doi: 10.1111/j.1432-1033.1978.tb12047.x.
A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16alpha-hydroxylase activity. Specifically 16-tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16-3H]progesterone and [16-3H]pregnenolone. The incubation medium consists of a phosphate buffer (pH7; 150mM), NADPH (0.1 mM), nicotinamide (10mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]testosterone (50 micron) or [16-3H]dehydroepiandrosterone (100 micron). The enzymatically released tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform, very sensitive (50 pmol of 16alpha-hydroxylated metabolites) and is independent of any further metabolism of the 16alpha-hydroxylated products.
一种基于用羟基立体定向取代质子（3H）的新同位素方法已被开发出来，用于测量大鼠肝脏睾酮和脱氢表雄酮16α-羟化酶的活性。具体而言，通过微生物（圆柱状根霉菌）对[16-3H]孕酮和[16-3H]孕烯醇酮的转化制备了16-氚标记的底物。孵育培养基由磷酸盐缓冲液（pH7；150mM）、NADPH（0.1mM）、烟酰胺（10mM）和氯化镁（4mM）组成。吐温80（1mg/ml）用于溶解饱和浓度的[16-3H]睾酮（50微摩尔）或[16-3H]脱氢表雄酮（100微摩尔）。酶促释放的氚在孵育培养基中以氚化水的形式回收，该氚化水在减压下蒸馏并通过液体闪烁计数。该方法易于操作，非常灵敏（50皮摩尔的16α-羟基化代谢产物），并且与16α-羟基化产物的任何进一步代谢无关。
