Padieu P, Maume B F
Ann Biol Clin (Paris). 1976;34(1):63-77.
The most generalized methods in enzymology are based on the quantitative assay of compounds, substrate or coenzyme, by spectrophotometry without any separation. Such a method is ruled out if the colorimetric reaction is not specific of the compound. In liver enzymology, aside the classical metabolic pathways, such assays are difficult to apply, especially when several metabolic steps are investigated. It is therefore necessary to use separative methods to isolate the metabolized substrate(s). For instance, the reductive catabolism of corticosterone leads to fourteen isomers (two dihydrocompounds, four tetrahydrocompounds and eight hexahydrocompounds) in which their respective productions are sex and age-linked. A position isomer of corticosterone, the 18-hydroxy-11-deoxy-corticosterone, follows the same reductive route. In adrenals some reduced metabolites arise from these two steroid hormones and are age dependent. When such metabolites are amenable to volatilization for gas chromatography, the interfacing of the gas chromatograph to the mass spectrometer allows to identify each compound introduced in the spectrometer. Among the ions produced by fragmentation of a compound or of a family of compounds, several specific fragments can be selected to be monitored along the chromatographic run leading to mass peaks which are quantitatively proportional to the amount of compounds, as far as other foreign molecules do not contribute fo fragment productions. These methods called mass fragmentography or multiple ion detection, or selected ion monitoring, allow with the help of all the resources of gas chromatography such the derivatization of studied molecules with heavy isotope labeled reagents to use the same unlabeled derivatized molecules as carriers and internal standards at once. This method allows to quantitate at the level of the picomolecule or less. Examples will be given with the study of the metabolism hormone steroids and xenobiotic compounds by the liver and adrenals in the animal and by isolated liver and adrenal cell cultures.
酶学中最通用的方法是基于对化合物、底物或辅酶进行定量分析，采用分光光度法且无需任何分离。如果比色反应对该化合物不具有特异性，那么这种方法就不适用。在肝脏酶学中，除了经典的代谢途径外，这类分析方法很难应用，尤其是在研究多个代谢步骤时。因此，有必要使用分离方法来分离代谢底物。例如，皮质酮的还原分解代谢会产生十四种异构体（两种二氢化合物、四种四氢化合物和八种六氢化合物），它们各自的生成与性别和年龄有关。皮质酮的一种位置异构体，即18-羟基-11-脱氧皮质酮，遵循相同的还原途径。在肾上腺中，这两种甾体激素会产生一些还原代谢产物，且与年龄有关。当这些代谢产物适合挥发用于气相色谱分析时，将气相色谱仪与质谱仪联用，就可以识别引入质谱仪的每种化合物。在一种化合物或一类化合物裂解产生的离子中，可以选择几个特定的碎片在色谱运行过程中进行监测，从而得到与化合物量成正比的质谱峰，前提是其他外来分子不会对碎片产生有贡献。这些方法称为质量碎片分析法、多离子检测法或选择离子监测法，借助气相色谱的所有资源，例如用重同位素标记试剂对研究分子进行衍生化，能够同时将相同的未标记衍生化分子用作载体和内标。这种方法能够在皮摩尔或更低水平进行定量分析。将通过动物肝脏和肾上腺以及分离的肝脏和肾上腺细胞培养物对激素甾体和外源化合物代谢的研究给出实例。
