The development of an ultra performance liquid chromatography-coupled atmospheric pressure chemical ionization mass spectrometry assay for seven adrenal steroids.

An ultra performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (UPLC-APCI-MS) method was developed for the separation and quantification of adrenal steroid metabolites from heterologous expression media. Steroids were extracted by liquid-liquid extraction, separated on a Waters UPLC BEH C18 column, ionized by APCI, and detected using a triple quadrupole mass spectrometer in APCI positive mode with single ion monitoring. The incorporation of UPLC enabled the detection of seven structurally closely related steroids at between 5 and 40 ng/ml using run times of 11 min.

Functional expression and characterisation of human cytochrome P45017alpha in Pichia pastoris.

Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity.

Baboon cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17).

Human cytochrome P450 17alpha-hydroxylase (CYP17) catalyses not only the 17alpha-hydroxlation of pregnenolone and progesterone and the C17,20-side chain cleavage (lyase) of 17alpha-hydroxypregnenolone, necessary for the biosynthesis of C21-glucocorticoids and C19-androgens, but also catalyses the 16alpha-hydroxylation of progesterone. In efforts to understand the complex enzymology of CYP17, structure/function relationships have been reported previously after expressing recombinant DNAs, encoding CYP17 from various species, in nonsteroidogenic mammalian or yeast cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C19-steroids take place, in some species, principally in the gonads.