[Enzymatic activity assays in the hepatic cell by mass fragmentography associated with gas-liquid chromatography].
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.
D000305:Adrenal Cortex Hormones / Q000378:metabolism; D000367:Age Factors; D000818:Animals; D002478:Cells, Cultured; D002849:Chromatography, Gas / Q000379:methods*; D003345:Corticosterone / Q000378:metabolism; D005260:Female; D006801:Humans; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D051381:Rats; D012733:Sex Differentiation; D012737:Sex Factors; D013739:Testosterone / Q000378:metabolism
D000305:Adrenal Cortex Hormones; D013739:Testosterone; D003345:Corticosterone
Mesure d'activités enzymatiques dans la cellule hépatique par la fragmentographie de masse couplée à la chromatographie gaz-liquide