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In 21-hydroxylase deficiency, urinary metabolites of 21-deoxycortisol, mainly derived from its 11oxo form 21-deoxycortisone, are indicators of intra-adrenal overproduction of 17-hydroxyprogesterone. In affected neonates these metabolites are numerous and most have not been previously described. This work forms the concluding part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Cortisol metabolites found in untreated patients, similarly almost exclusively present in their 11oxo form in neonates, have been included for completeness. 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 11oxo-pregnane(enes). Few GC-MS features were associated with the presence of the non-derivatizeable 11oxo group in pregnane(ene)s. GC-MS/MS contributed only to the characterization of structures outside the C-ring, as described in the preceding parts of this study. Parallels were found between the metabolism of 21-deoxycortisone and cortisone. The major metabolic pathway was that of classical 3α,5β-reduction with a prominent further hydroxylation, predominantly at C6. Oxidation of the 6-hydroxyl was also common. We conclude that further oxygenated metabolites of 21-deoxycortisone have potential as more reliable markers of 21-hydroxylase deficiency in the early neonatal period, because their levels are higher during that period of life than for the classical marker 11oxo-pregnanetriol., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. 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 the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1β-, 2α-, 4β-, and 6β-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1β, 15β, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4β and 6β. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5β-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3β-hydroxy-5α-anes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. 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, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.

Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3beta-hydroxy-5-ene structure undergo transformation and yield of 5alpha-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime-trimethylsilylether derivatives by gas chromatography-mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3beta-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5alpha-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3beta-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5alpha-reduced steroids is low due to oxidative damage of beta-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested.