Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC-MS and GC-MS/MS, of androstanes and androstenes

TY - JOUR

T1 - Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC-MS and GC-MS/MS, of androstanes and androstenes

AU - Christakoudi, Sofia

AU - Cowan, David A.

AU - Taylor, Norman F.

PY - 2012/11

Y1 - 2012/11

N2 - 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-triinethylsilyl 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 5 alpha(beta) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes.The major endogenous 3 beta-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5 alpha(beta)-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. (C) 2012 Elsevier Inc. All rights reserved.

AB - 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-triinethylsilyl 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 5 alpha(beta) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes.The major endogenous 3 beta-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5 alpha(beta)-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. (C) 2012 Elsevier Inc. All rights reserved.

KW - CYP21A2

KW - GC-MS/MS

KW - Neonate

KW - Urine

KW - Steroidomics

KW - 'Backdoor pathway'

KW - CONGENITAL ADRENAL-HYPERPLASIA

KW - CYTOCHROME-P450 ISOFORMS

KW - MASS-SPECTROMETRY

KW - BACKDOOR PATHWAY

KW - LIVER MICROSOMES

KW - HUMAN PLACENTA

KW - IDENTIFICATION

KW - METABOLISM

KW - FETAL

KW - BIOSYNTHESIS

U2 - 10.1016/j.steroids.2012.08.012

DO - 10.1016/j.steroids.2012.08.012

M3 - Article

SN - 0039-128X

VL - 77

SP - 1487

EP - 1501

JO - Steroids

JF - Steroids

IS - 13

ER -