Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes
Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes
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DOI：
10.1016/j.steroids.2009.09.011
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发表时间：
2010-01-01
期刊：
影响因子：
2.7
通讯作者：
Taylor, Norman F.
中科院分区：
文献类型：
--
作者：
Christakoudi, Sofia;Cowan, David A.;Taylor, Norman F.
21-羟化酶缺乏的新生儿尿液中的类固醇代谢物主要是多羟基化17-羟孕酮和雄激素代谢物。并且大多数具有不完全限定的结构。这项研究形成了一个全面的项目，以表征和识别这些，以提高诊断和进一步阐明新生儿类型的类固醇metabolis.Steroids进行了分析，提取和酶共轭水解后，作为methyloximetrimethylsilicone醚衍生物的气相色谱仪耦合到四极杆和离子阱质谱仪。采用GC-MS和GC-MS/MS联用技术，在恒定激发条件下，对20-氧代和不含氧代基团的20-羟基十六烷（烯）的D环和侧链结构以及A环、B环和C环上的基团进行了分析，考虑了D环和侧链构型的所有可能组合。大多数碎片可以解释为部分或完全D-环裂解与侧链的损失，通过与氘代衍生物和硼氢化物还原代谢物的光谱比较的帮助。还讨论了可能的重排离子。GC-MS/MS特别有利于表征具有16，17-二羟基-20-氧代结构的化合物，解释为子宫内酶诱导的标志物。它还有助于16-羟基-20-氧代代谢产物的区分。存在于未受影响的新生儿的尿液中，来自诊断性17-羟基-20-氧代类固醇，并且能够检测低浓度的15，17-二羟基-20-氧代化合物。尽管CYP 21 A2的缺陷，17，21-二羟基化的藜芦烷（烯）的存在进行了讨论。我们得出结论，CC-MS结合GC-MS/MS允许可靠的鉴定的D-环和侧链的结构，而无需事先分离，即使在尿液中的低浓度。(C)2009 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 methyloximetrimethylsilyl 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 CC-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. (C) 2009 Elsevier Inc. All rights reserved.