Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 May 15;879(17-18):1384-92. doi: 10.1016/j.jchromb.2010.11.019. Epub 2010 Nov 23.
In this study we profile free 3-oxo sterols present in plasma from patients affected with the neurodegenerative disorder of sterol and bile acid metabolism cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girard's P (GP) reagent 1-(carboxymethyl) pyridinium chloride hydrazide, an approach uniquely suited to studying the 3-oxo sterols that accumulate in CTX, as Girard's reagent reacts with the sterol oxo moiety to form charged hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MS(n) analysis of these sterols in the presence of a large excess (3 orders of magnitude) of cholesterol. Often cholesterol detected in biological samples makes it challenging to quantify minor sterols, with cholesterol frequently removed prior to analysis. We derivatized plasma (10 μl) without SPE removal of cholesterol to ensure detection of all sterols present in plasma. We were able to measure 4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of bile acid precursors were identified in plasma using this technique. GP-tagged sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) (M→) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS(3) (M→M-79→) spectra that provided additional structurally informative fragment ions.
在这项研究中，我们利用电荷标记和 LC-ESI-MS(n)技术，结合 LTQ-Orbitrap Discovery 仪器，对患有神经退行性胆固醇和胆汁酸代谢脑腱黄瘤病（CTX）的患者血浆中的游离 3-氧代固醇进行了分析。此外，我们还对缺乏 CTX 缺陷酶的 Cyp27A1 基因敲除小鼠 24 个月大的血浆中的固醇进行了分析。电荷标记是通过与阳离子吉拉德 P（GP）试剂 1-（羧甲基）吡啶鎓氯化氢酰肼反应来实现的，这种方法特别适合研究 CTX 中积累的 3-氧代固醇，因为吉拉德试剂与固醇的氧代部分反应形成带电荷的腙衍生物。选择性生成 GP 标记的 3-氧代-4-烯和 3-氧代-5（H）饱和血浆固醇的能力使我们能够在胆固醇大量过剩（3 个数量级）的情况下对这些固醇进行 ESI-MS(n)分析。通常，在生物样本中检测到的胆固醇使得定量分析少量固醇变得具有挑战性，因为通常在分析之前去除胆固醇。我们对未经 SPE 去除胆固醇的血浆（10 μl）进行了衍生化，以确保检测到所有存在于血浆中的固醇。我们能够测量未经治疗的 CTX 患者血浆中的 4-胆甾烯-3-酮（1207±302 ng/ml，平均值±标准差，n=4），以及提出的 5α-胆甾醇途径中的其他中间产物。此外，还使用该技术在血浆中鉴定出了许多胆汁酸前体。GP 标记的固醇是利用高分辨率精确质量谱（±5 ppm）以及具有特征性的 79Da（吡啶）碎片离子中性丢失的 MS(2)（M→）谱和提供额外结构信息的 MS(3)（M→M-79→）谱来鉴定的。
