Characterisation of novel oxysterols by mass spectrometry
Lead Research Organisation:
Swansea University
Department Name: Institute of Life Science Medical School
Abstract
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Technical Summary
Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilities the flux of oxysterols between tissue and blood. Oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. It has been suggested that by interacting with the orphan nuclear receptors, LXR alpha and LXR beta, oxysterols may be involved in the control of cholesterol balance by affecting cholesterol synthesis, absorption and metabolism. Endogenous oxysterols are difficult to analyse by modern techniques, not only are they present at very low levels, but they are also present amongst a very large excess of cholesterol (approximately 0.1ng oxysterol per micro g cholesterol). In the proposed study we will develop new mass spectrometry (MS) based methods to allow the identification, characterisation and quantification of known and novel endogenous oxysterols. Initially it will be necessary to develop an extraction and purification scheme to obtain an oxysterol rich fraction from rat tissue (or human plasma). The oxysterol fraction will then be analysed by MS. Using modern MS methods oxysterols are very difficult to analyse in that they are poorly ionised by both atmospheric pressure ionisation eg. ESI, and by MALDI. However, we have recently developed a derivatisation protocol suitable for 3-oxo-4-ene steroids which allows their ESI- or MALDI-MS analysis on the sub pg level. Most oxysterols do not posses a 3-oxo-4-ene group, but can easily be converted to 3-oxo-4-ene analogues by the action of cholesterol oxidase. They can then be subsequently derivatised for ESI-(or MALDI-) MS analysis. It will be necessary to optimise both of the oxidation and derivatisation steps for the compatibility of oxysterol analysis with ESI-MS. For oxysterols unsuitable for oxidation with cholesterol oxidase alternative derivatisation methods based on derivatisation of the alcohol group will be employed. The preferred technique for the analysis of low levels of biomolecule mixtures extracted from biological matrixes is nano-scale-LC-ESI-MS MS (this is preferable to GC-MS on account of the higher sample capacity of the LC column, and the compatibility of LC-ESI to the analysis of previously unidentified conjugated oxysterols). It will be necessary to develop a nano-LC-ESI-MS MS method which is suitable for derivatised oxosterols and will differentiate between oxosterol stereoisomers. Once an LC-MS MS method for oxysterol analysis is optimised, quantitative studies will be performed using stable-isotope dilution methodology. Synthesis of stable-isotope labelled and reference oxysterols will be performed where necessary. Once identified, the interaction between oxysterols and the LXR will be investigated using mass spectrometry methods. We have recently developed mass spectrometry based assays to investigate ligand ¿ nuclear receptor interactions, and also to pull-out ligands from biological extracts. We will employ these methods in the study.
Publications
Abdel-Khalik J
(2014)
Studies on the analysis of 25-hydroxyvitamin D(3) by isotope-dilution liquid chromatography-tandem mass spectrometry using enzyme-assisted derivatisation.
in Biochemical and biophysical research communications
Chen Y
(2013)
Shotgun cholanomics of ileal fluid.
in Biochimie
Griffiths WJ
(2013)
Analytical strategies for characterization of oxysterol lipidomes: liver X receptor ligands in plasma.
in Free radical biology & medicine
Griffiths WJ
(2011)
Analysis of oxysterol metabolomes.
in Biochimica et biophysica acta
Griffiths WJ
(2011)
On the future of "omics": lipidomics.
in Journal of inherited metabolic disease
Griffiths WJ
(2013)
Methods for oxysterol analysis: past, present and future.
in Biochemical pharmacology
Griffiths WJ
(2010)
Analytical strategies for characterization of bile acid and oxysterol metabolomes.
in Biochemical and biophysical research communications
Griffiths WJ
(2010)
Bile acids: analysis in biological fluids and tissues.
in Journal of lipid research
Karu K
(2011)
Nano-liquid chromatography-tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation.
in Chemistry and physics of lipids
Description | In this work we developed a method for the high sensitivity measurement of cholesterol metabolites in biological systems. This has lead to three key discoveries. 1. We have identified 24S,25-epoxycholesterol, a cholesterol like molecule, in embryonic rodent brain that promotes the formation of dopaminergic neurons during brain development. Dopaminergic neurons are the cells that die in Parkinson's disease. Our results may have implications for stem cell replacement therapy in the treatment of this disease. This work was published in the journal Nature Chemical Biology in 2012. 2. We have identified the cholesterol metabolite, 25-hydroxycholesterol, to be secreted by macrophages upon virus infection, and to be inhibitor of viral infection as part of the body's defense mechanism against pathogens. This work was published in the journal Immunity in 2013 3. We have discovered that the acidic metabolite of cholesterol, 3ß,7a-dihydroxycholestenoic acid, is protective towards motor neurons. This may have potential in the treatment of motor neuron disease. This work was published in the Journal of Clinical Investigation in 2014. |
Exploitation Route | The analytical methods developed in this work can be used by others interested in steroid, sterol and oxysterol analysis. The cholesterol-like molecules we have identified to (1) promote neurogenesis and (2) to have pro-survival properties are potential lead candidates in drug discovery programs. |
Sectors | Agriculture, Food and Drink,Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |
URL | http://sterolanalysis.org.uk/?page=Home |
Description | The analytical method developed is being used by us and by others in the identification of and screening for in born errors of metabolism and in support of clinical trials. The method has been taken up by the pharmaceutical industry as part of their research programs. |
First Year Of Impact | 2012 |
Sector | Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | BBSRC Responsive mode |
Amount | £460,386 (GBP) |
Funding ID | BB/N015932/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2016 |
End | 09/2019 |
Description | KESS II |
Amount | £53,476 (GBP) |
Funding ID | EGR817 |
Organisation | Government of Wales |
Sector | Public |
Country | United Kingdom |
Start | 10/2016 |
End | 09/2019 |
Description | MS Society Research Grant Award |
Amount | £93,333 (GBP) |
Funding ID | 94 |
Organisation | Multiple Sclerosis Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2019 |
End | 03/2022 |
Title | EADSA |
Description | We have developed a charge-tagging approach to greatly enhance mass spectrometry sensitivity for biomolecule analysis. |
Type Of Material | Technology assay or reagent |
Year Produced | 2011 |
Provided To Others? | Yes |
Impact | Allowed the identification of novel cholesterol metabolites in biological systems. |
Title | Sterolanalysis |
Description | Collection of sterol mass spectrometry data (MS3). |
Type Of Material | Database/Collection of data |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | The database has been used by researchers using the EADSA technology developed in Swansea. |
URL | http://sterolanalysis.org.uk/ |
Description | CHORI: Inborn errors of metabolism |
Organisation | Children's Hospital Oakland Research Institute (CHORI) |
Country | United States |
Sector | Hospitals |
PI Contribution | Analysis of patient and mouse model samples. |
Collaborator Contribution | Provision of patient and mouse model samples. Expertise in inborn errors of metabolism. |
Impact | Three publications |
Start Year | 2007 |
Description | ICH: Inborn errors of metabolism |
Organisation | University College London |
Department | Institute of Child Health |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Investigation of inborn errors of cholesterol biosynthesis and metabolism. |
Collaborator Contribution | Clinical information concerning inborn errors of metabolism |
Impact | Four publications |
Start Year | 2009 |
Description | Karolinska |
Organisation | Karolinska Institute |
Country | Sweden |
Sector | Academic/University |
PI Contribution | We have brought essential data and insight to the collaboration. We are bringing new technology |
Collaborator Contribution | They have brought essential data and insight to the collaboration. Our partners are bringing transgenic animal material and expertise to the collaboration. |
Impact | This is a multidisciplinary collaboration between neuroscientists and clinical chemists at Karolinska Institute and analytical scientists in Swansea. This colaboration has resulted in papers in Nature Chemical Biology and the Journal of Clinical Investigation. |
Start Year | 2006 |
Title | KIT AND METHOD FOR QUANTITATIVE DETECTION OF STEROIDS |
Description | The invention relates to a kit and methods for quantitative detection of steroids in a sample. The kit comprises quantitative charge tags and an oxidising agent. |
IP Reference | WO2014037725 |
Protection | Patent application published |
Year Protection Granted | 2014 |
Licensed | Yes |
Impact | The kit will be commercialised by Avanti Polar Lipids |
Description | ASBMB Cholesterol and ALS |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Article in ASBMB monthly magazine |
Year(s) Of Engagement Activity | 2016 |
Description | Cholesterol and ALS |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Press release to communicate latest scientific discovery. Many email responses from motor neuron disease sufferers or carers. |
Year(s) Of Engagement Activity | 2016 |
Description | Cholesterol metabolites regulate motor neuron function |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | A research highlight was published in Nature Reviews Endocrinology. NA |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.nature.com/nrendo/journal/vaop/ncurrent/full/nrendo.2014.184.html |
Description | New steroid discovery could improve Parkinson's treatment; |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Scientists at Swansea University and Karolinska Institutet in Stockholm have identified two steroid-type molecules that play an important role in the survival and production of nerve cells in the brain. The discovery, published in the journal Nature Chemical Biology, could help in the development of new treatments for neurological diseases such as Parkinson's disease. no actual impacts realised to date |
Year(s) Of Engagement Activity | 2013 |
Description | Steroids: Scientists examine controversial substance's potential to treat Parkinson's, http://www.walesonline.co.uk/news/wales-news/steroids-scientists-examine-controversial-substances-2496776 |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Steroids could be used to treat a debilitating disease that affects thousands in Wales today. Researchers at a University in Wales have highlighted the controversial substance's positive side after discovering two steroid type molecules that could treat Parkinson's disease no actual impacts realised to date |
Year(s) Of Engagement Activity | 2013 |