An Integrated platform for Quantitative Sterolomics: From Oxysterols to Bile Acids and Steroids

胆固醇是每个动物细胞的基本成分。它是细胞膜上的一种结构脂质，是氧化甾醇、胆汁酸和类固醇激素的前体。细胞胆固醇稳态是通过胆固醇吸收、生物合成和代谢之间的平衡来维持的。所有胆固醇代谢的第一步是氧化为氧甾醇。氧甾醇是胆固醇的氧合衍生物，过去一直被认为是胆固醇的转运形式，将其返回肝脏转化为胆汁酸。然而，最近的数据表明，氧化甾醇具有生物活性，介导一些胆固醇诱导的代谢作用。此外，下游酸性胆固醇代谢物，由许多不同类型的细胞生物合成，也具有生物活性。新的研究结果表明，氧化甾醇参与了许多生物学领域，例如，在发育中的大脑中减少祖细胞的增殖，减少初始B细胞的增殖和阻断免疫系统中的类开关重组，对神经退行性疾病和记忆丧失提供保护，并在恶性细胞中表现出差异表达。此外，经肠肝系统循环利用的胆汁酸已被证明可作为激素，通过激活G蛋白偶联受体TGR5，引发能量消耗的增加和饮食引起的肥胖的衰减。重要的是要认识到，氧化甾醇是一类分子组成的广泛的不同的化学实体。它们的下游代谢物也是如此，这是胆固醇分子上许多潜在位点中的任何一个发生初始氧化反应的结果，以及随后酶生物转化的顺序是可变的。这导致了大量可能的代谢物。这种复杂性同样反映在胆汁酸中，胆汁酸可以被肠肝系统中的细菌进行结构转化。胆固醇代谢产物是生物系统中具有挑战性的分子分析。这是由于它们在高胆固醇背景下丰度较低（例如，脑内的ng氧甾醇/微量胆固醇，血浆中的ng胆汁酸/ mg胆固醇，脑脊液中的pg神经类固醇/微量胆固醇），胆固醇在空气中氧化为氧甾醇（以及C19和C21类固醇）的倾向，从而产生分析人工产物，以及它们缺乏强发色团，但热不稳定性。其结果是，在体液、组织和细胞类型中，全面的胆固醇代谢谱描述得很差。在本提案中，我们打算通过开发一个基于集成质谱的平台来应对这一挑战，该平台用于生物系统中胆固醇代谢物的超高灵敏度定量和结构测定。我们将引入基于化学标记的新技术来加强胆固醇代谢物的分析、结构测定和定量。通过利用电荷标签中的稳定同位素标记，我们将能够确定特定代谢物的绝对数量，也可以对不同样品之间的非靶向代谢物进行相对定量，例如在大脑的不同位置或不同细胞类型之间。通过国际合作，我们将在定义的生物分析中研究鉴定的代谢物的生物活性。该项目可能会对健康老龄化产生影响，因为紊乱的胆固醇合成和代谢与许多疾病状态（神经退行性疾病、动脉粥样硬化、糖尿病）和畸形综合征有关，这将对英国制药公司和那些参与生物标志物发现和临床筛选的人有益。

Cholesterol is an essential component of every animal cell. It is a structural lipid in cell membranes and the precursor of oxysterols, bile acids and steroid hormones. Cellular cholesterol homeostasis is maintained by the balance between cholesterol absorption, biosynthesis and metabolism. The first step of all cholesterol metabolism is oxidation to an oxysterol. Oxysterols are oxygenated derivatives of cholesterol which in the past have been regarded as transport forms of cholesterol returning it to the liver for conversion to bile acids. However, recent data indicates that oxysterols have biological activity, mediating a number of cholesterol-induced metabolic effects. Furthermore, down-stream acidic cholesterol metabolites, biosynthesised by many different cell types, are also biologically active. New results show that oxysterols are involved in many areas of biology e.g. acting to reduce proliferation of progenitor cells in developing brain, reducing proliferation of naive B cells and blocking class switch recombination in the immune system, offering protection against neurodegenerative disease and memory loss, and showing differential expression in malignant cells. Furthermore, bile acids, recycled by the enterohepatic system, have been shown to act as hormones by activating the G protein coupled receptor TGR5 and triggering an increase in energy expenditure and attenuation of diet-induced obesity. It is important to realise that oxysterols are a class of molecule consisting of a wide-range of distinct chemical entities. This is also true of their down-stream metabolites, and is a consequence of the initial oxidation reaction occurring at any one of many potential sites on the cholesterol molecule and the order of subsequent enzymatic biotransformations being variable. This leads to a multitude of possible metabolites. This complexity is similarly reflected in bile acids which can be structurally-transformed by bacteria in the enterohepatic system. Cholesterol metabolites are challenging molecules to analyse in biological systems. This is a consequence of their low abundance against a high background of cholesterol (e.g. ng oxysterol / microg cholesterol in brain, ng bile acid / mg cholesterol in plasma, pg neurosteroid / microg cholesterol in CSF), the propensity of cholesterol to be oxidised in air to oxysterols (and also to C19 & C21 steroids) there-by generating analytical artefacts, and their lack of a strong chromophore but thermal lability. The consequence of this is that comprehensive cholesterol metabolite profiles are poorly described in body fluids, tissues and cell types. In this proposal we intend to meet this challenge by developing an integrated mass spectrometry-based platform for the ultra-high sensitivity quantitative and structural determination of cholesterol metabolites in biological systems. We will introduce new technology based on chemical-tagging to enhance the analysis of cholesterol metabolites, their structural determination, and quantification. By exploiting stable-isotope labelling in the charge-tags we will be able to determine absolute quantities of specific metabolites and also perform relative quantification of untargeted metabolites between different samples e.g. between different locations in brain or between different cell types. Through international collaboration we will investigate the biological activity of the identified metabolites in defined biological assays. This project is likely to have impact with respect to healthy aging, and as deranged cholesterol synthesis and metabolism is implicated in numerous disease states (neurodegenerative disease; atherosclerosis; diabetes) and malformation syndromes will be of benefit to UK pharma and those involved in biomarker discovery and clinical screening.