Award details

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

Reference	BB/I001735/1
Principal Investigator / Supervisor	Professor William Griffiths
Co-Investigators / Co-Supervisors	Professor Yuqin Wang
Institution	Swansea University
Department	Institute of Life Science Medical School
Funding type	Research
Value (£)	326,463
Status	Completed
Type	Research Grant
Start date	01/09/2011
End date	31/08/2014
Duration	36 months

Abstract

The primary objective of the current proposal is to develop an integrated mass spectrometry (MS)-based platform for the quantitative and structural determination of cholesterol metabolites in biological systems. This will be supplemented by assays of their bioactivity. Over the last 20 years the focus of the PI's research has been the identification and quantification of oxysterols, bile acids and steroids in body fluids, tissues and cell types. While many molecules in these classes can be analysed at high sensitivity in targeted analysis by MS, usually utilising multiple reaction monitoring (MRM) or selected-ion recording (SIR), such analysis require pre-defined targets and are not suitable for profile analysis or the identification of unknown or unexpected components. Furthermore accurate quantification requires an isotope-labelled internal standard. In recent years we have developed an alternative strategy based on charge-tagging and LC-MSn for the ultra-high sensitivity identification and quantification of cholesterol metabolites. By utilising simple chemistry we tag a charged group to a ketone group on cholesterol metabolites, this improves the LC-MS response by two - three orders of magnitude. Further, the nature of the derivative enhances the information content of MSn spectra, allowing structure determination and identification of unknowns. In this proposal we seek to move this methodology to the next level by incorporating stable-isotope labelling in the charge-tag thereby allowing absolute quantification of individual samples and relative quantification between samples. Further, we will extend the number of metabolites amenable to charge-tagging by incorporating enzymatic conversion of alcohol functions to ketone groups (thus available for derivatisation).

Summary

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.

Impact Summary

The primary objective of the current proposal is to develop an integrated mass spectrometry (MS)-based platform for the quantitative and structural determination of cholesterol metabolites in biological systems. As part of this proposal we will create a series of isotope-coded charge tags for use in ultra-high sensitivity quantitative studies. The isotope-coded charge tags will offer improved analyte sensitivity and allow absolute quantification of defined analytes and relative quantification (between samples) of undefined targets. We will further develop the tagging chemistry to allow charge-tagging to alcohol (3beta and 3alfa hydroxyl groups on the steroid skeleton) functionalities in addition to oxo (ketone/aldehyde) groups. Although in this study we will concentrate our attention on the analysis of cholesterol metabolites, the developed isotope-coded charge tags will be equally useful in other areas of lipidomics and metabolomics. The likely beneficiaries of our analytical platform are analysts working in the areas of metabolomics, lipidomics, biomarker discovery, clinical screening and doping control. The methods generated by the present study will be valuable to those working in the sterol bile acid and steroid fields, including contract research, pharmaceutical companies, and doping and clinical laboratories. The analytical methods are likely to be important in neuroscience, particularly for the prediction and monitoring of neurodegenerative disease, and in stem cell biology, cancer and immunology where specific cholesterol metabolites have been shown to be important in influencing cell proliferation. We will publish our data in peer review journals, make presentations at international meetings and also establish a website where data and methods will be freely available. We will also present our data to the general public at public seminars, open days and visits to schools. With respect to commercialisation we are collaborating with Proteome Sciences plc in the area of isotope-coded tags for tandem mass spectrometry in metabolomics, and we would negotiate with them to commercialisation our isotope-coded charge tags. The research has considerable potential impact on the nation's health and wellbeing. We plan (in future studies) to utilise our analytical platform to validate potential cholesterol derived metabolites as markers for the early diagnosis of Alzheimer's disease. Early diagnosis will be important as new treatments; both pharmacological and psychological are being developed. The research may also have an impact in the development of regenerative therapies for Parkinson's disease as cholesterol metabolites have been found to be important in the development of dopaminergic neurons from stem cells. The PDRA employed in this project will gain an expert education in biological mass spectrometry; which can in the future be exported to all areas of analytical science. Further, the project is at the interface of chemistry, biology and medicine and is thus multidisciplinary. Cholesterol metabolites identified to have biological activity will be patented in association with Swansea University. We will seek to exploit commercially our isotope-coded charge tags in collaboration with Proteome Sciences plc.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Structural Biology, Technology and Methods Development
Research Priority	Technology Development for the Biosciences
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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