Abstract

Urine from neonates with 21-hydroxylase deficiency contains a large range of androstane(ene)s, many of which have not been previously described. We present their characterization as the third part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed androstane(ene)s. GC-MS/MS was pivotal for the structural characterization of 2-hydroxylated androstenediones but GC-MS was generally more informative for androstane(ene)s, in contrast to 17-hydroxylated pregnane(ene)s. Parallels were found between the GC-MS and GC-MS/MS characteristics of structurally similar androstenediones and progesterones without a substituent on the D-ring, but not with those of 17-hydroxylated progesterones. Assignment of 5α(β) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes. The major endogenous 3β-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5α(β)-androstanes and 3-oxo-androst-4-enes. Major positions of hydroxylation in these were C(2), C(6), C(11), C(16) and C(18). Additional oxo-groups were common at C(6), C(11) and C(16). We conclude that the presence of multiple further oxygenated metabolites of androstenedione in urine from neonates with 21-hydroxylase deficiency and their pattern indicate a predominance of the classical pathway of androgen synthesis and reflect an increased demand for clearance. The positions of oxygenation in androstane(ene)s are dependent on the configuration at C(3)-C(5).

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Published: 2012 Nov;77(13):1487-501. doi: 10.1016/j.steroids.2012.08.012. Epub 2012 Sep 3.