Imperial College London

DrPanagiotisVorkas

Faculty of MedicineDepartment of Metabolism, Digestion and Reproduction

Honorary Senior Lecturer
 
 
 
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p.vorkas

 
 
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Location

 

Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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36 results found

Giallourou N, Urbaniak C, Puebla-Barragan S, Vorkas P, Swann J, Reid Get al., 2021, Characterizing the breast cancer lipidome and its interaction with the tissue microbiota, Communications Biology, ISSN: 2399-3642

Journal article

Jukes Z, Freier A, Glymenaki M, Brown R, Parry L, Want E, Vorkas PA, Li JVet al., 2021, Lipid profiling of mouse intestinal organoids for studying APC mutations, Bioscience Reports: molecular and cellular biology of the cell surface, Vol: 41, Pages: 1-11, ISSN: 0144-8463

Inactivating mutations including both germline and somatic mutations in the adenomatous polyposis coli (APC) gene drives most familial and sporadic colorectal cancers. Understanding the metabolic implications of this mutation will aid to establish its wider impact on cellular behaviour and potentially inform clinical decisions. However, to date, alterations in lipid metabolism induced by APC mutations remain unclear. Intestinal organoids have gained widespread popularity in studying colorectal cancer and chemotherapies, because their 3D structure more accurately mimics an in vivo environment. Here, we aimed to investigate intra-cellular lipid disturbances induced by APC gene mutations in intestinal organoids using a reversed-phase ultra-high-performance liquid chromatography mass spectrometry (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from either wild-type (WT) or mice with APC mutations (Lgr5–EGFP-IRES-CreERT2Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Levels of phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and hexosylceramides (e.g. HexCer(d18:1/16:0), HexCer(d18:1/22:0)) were higher in Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), SM(d18:1/16:0)) were lower compared with WT. These observations indicate that cellular metabolism of sphingomyelin was up-regulated, resulting in the cellular accumulation of ceramides and production of HexCer due to the absence of Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of organoids and provided an enhanced insight into the effects of the APC mutations on lipid metabolism, making for a valuable addition to screening options of the organoid lipidome.

Journal article

Lau CH, Taylor-Bateman V, Vorkas PA, Gomes Da Graca G, Vu T-H, Hou L, Chekmeneva E, Ebbels T, Chan Q, Van Horn L, Holmes Eet al., 2020, Metabolic signatures of gestational weight gain and postpartum weight loss in a lifestyle intervention study of overweight and obese women, Metabolites, Vol: 10, ISSN: 2218-1989

BACKGROUND: Overweight and obesity amongst women of reproductive age are increasingly common in developed economies and are shown to adversely affect birth outcomes and both childhood and adulthood health risks in the offspring. Metabolic profiling in conditions of overweight and obesity in pregnancy could potentially be applied to elucidate the molecular basis of the adverse effects of gestational weight gain (GWG) and postpartum weight loss (WL) on future risks for cardiovascular disease (CVD) and other chronic diseases. METHODS: Biofluid samples were collected from 114 ethnically diverse pregnant women with body mass index (BMI) between 25 and 40 kg/m2 from Chicago (US), as part of a randomized lifestyle intervention trial (Maternal Offspring Metabolics: Family Intervention Trial; NCT01631747). At 15 weeks, 35 weeks of gestation, and at 1 year postpartum, the blood plasma lipidome and metabolic profile of urine samples were analyzed by liquid chromatography mass spectrometry (LC-MS) and 1H nuclear magnetic resonance spectroscopy (1H NMR) respectively. RESULTS: Urinary 4-deoxyerythronic acid and 4-deoxythreonic acid were found to be positively correlated to BMI. Seventeen plasma lipids were found to be associated with GWG and 16 lipids were found to be associated with WL, which included phosphatidylinositols (PI), phosphatidylcholines (PC), lysophospholipids (lyso-), sphingomyelins (SM) and ether phosphatidylcholine (PC-O). Three phospholipids found to be positively associated with GWG all contained palmitate side-chains, and amongst the 14 lipids that were negatively associated with GWG, seven were PC-O. Six of eight lipids found to be negatively associated with WL contained an 18:2 fatty acid side-chain. CONCLUSIONS: Maternal obesity was associated with characteristic urine and plasma metabolic phenotypes, and phospholipid profile was found to be associated with both GWG and postpartum WL in metabolically healthy pregnant women with overweight/obesity. Postpartu

Journal article

Rahman MS, Haskard DO, Frost G, Vorkas P, Woollard KJet al., 2020, Acute dietary saturated fat intake suppresses human monocyte subset inflammatory and chemokine responses, Publisher: OXFORD UNIV PRESS, Pages: 3622-3622, ISSN: 0195-668X

Conference paper

Rahman MS, Vorkas P, Frost G, Morrison D, Haskard D, Woollard KJet al., 2019, OUTLINING THE HUMAN MONOCYTE INFLAMMATORY CYTOKINE RESPONSE TO DIETARY FAT INTAKE, Annual Conference of the British-Cardiovascular-Society (BCS) - Digital Health Revolution, Publisher: BMJ PUBLISHING GROUP, Pages: A159-A160, ISSN: 1355-6037

Conference paper

Qureshi M, Kaluarachchi M, Vorkas P, Holmes E, Davies Aet al., 2018, Metabolic Profiling of High-Risk Carotid Atherosclerosis, Vascular Annual Meeting of the Society-for-Vascular-Surgery (SVS), Publisher: MOSBY-ELSEVIER, Pages: E236-E236, ISSN: 0741-5214

Conference paper

Vorkas PA, 2018, Expanding lipidome coverage using MS/MS-aided untargeted data-independent RP-UPLC-TOF-MSE acquisition, Bioanalysis, Vol: 10, Pages: 307-319, ISSN: 1757-6180

Lipid function and importance in disease are being rediscovered due to modern advancements in chemicalanalysis. RP–UPLC–TOF–MSE is now the lipidomics tool of choice and can provide the demanded specificityfor detecting the great diversity of the lipidome. It can offer simplicity, rapidity, robustness and highthroughputness, without the need for further optimization in current sample preparation protocols. Thismethod can cover the major lipid categories with the ability to detect several corresponding subclasses.It can deliver adequate information for deciphering fatty chain length, unsaturation and regioisomerism.It has enabled the detection of a vast number of lipids, of which more than 250 are reported here. Theselipids were detected from applications in a variety of biological matrices and species.

Journal article

Vorkas PA, Abellona U MR, Li JV, 2018, Tissue Multiplatform-Based Metabolomics/Metabonomics for Enhanced Metabolome Coverage., Pages: 239-260

The use of tissue as a matrix to elucidate disease pathology or explore intervention comes with several advantages. It allows investigation of the target alteration directly at the focal location and facilitates the detection of molecules that could become elusive after secretion into biofluids. However, tissue metabolomics/metabonomics comes with challenges not encountered in biofluid analyses. Furthermore, tissue heterogeneity does not allow for tissue aliquoting. Here we describe a multiplatform, multi-method workflow which enables metabolic profiling analysis of tissue samples, while it can deliver enhanced metabolome coverage. After applying a dual consecutive extraction (organic followed by aqueous), tissue extracts are analyzed by reversed-phase (RP-) and hydrophilic interaction liquid chromatography (HILIC-) ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. This pipeline incorporates the required quality control features, enhances versatility, allows provisional aliquoting of tissue extracts for future guided analyses, expands the range of metabolites robustly detected, and supports data integration. It has been successfully employed for the analysis of a wide range of tissue types.

Book chapter

Rahman S, Vorkas P, Morrison D, Frost G, Haskard D, Woollard Ket al., 2017, ACUTE DIETARY SATURATED FAT INTAKE CAN SUPPRESS THE INFLAMMATORY RESPONSE IN HUMAN CIRCULATING FOAMY MONOCYTES, 85th Congress of the European-Atherosclerosis-Society (EAS), Publisher: Elsevier, Pages: E116-E116, ISSN: 0021-9150

Conference paper

Anwar MA, Adesina-Georgiadis KN, Spagou K, Vorkas PA, Li JV, Shalhoub J, Holmes E, Davies AHet al., 2017, A comprehensive characterisation of the metabolic profile of varicose veins; implications in elaborating plausible cellular pathways for disease pathogenesis, Scientific Reports, Vol: 7, ISSN: 2045-2322

Metabolic phenotypes reflect both the genetic and environmental factors which contribute to the development of varicose veins (VV). This study utilises analytical techniques to provide a comprehensive metabolic picture of VV disease, with the aim of identifying putative cellular pathways of disease pathogenesis. VV (n = 80) and non-VV (n = 35) aqueous and lipid metabolite extracts were analysed using 600 MHz 1H Nuclear Magnetic Resonance spectroscopy and Ultra-Performance Liquid Chromatography Mass Spectrometry. A subset of tissue samples (8 subjects and 8 controls) were analysed for microRNA expression and the data analysed with mirBase (www.mirbase.org). Using Multivariate statistical analysis, Ingenuity pathway analysis software, DIANALAB database and published literature, the association of significant metabolites with relevant cellular pathways were understood. Higher concentrations of glutamate, taurine, myo-inositol, creatine and inosine were present in aqueous extracts and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in lipid extracts in the VV group compared with non-VV group. Out of 7 differentially expressed miRNAs, spearman correlation testing highlighted correlation of hsa-miR-642a-3p, hsa-miR-4459 and hsa-miR-135a-3p expression with inosine in the vein tissue, while miR-216a-5p, conversely, was correlated with phosphatidylcholine and phosphatidylethanolamine. Pathway analysis revealed an association of phosphatidylcholine and sphingomyelin with inflammation and myo-inositol with cellular proliferation.

Journal article

Qureshi MI, Vorkas P, Kaluarachchi M, Holmes E, Davies AHet al., 2017, Biomarker research in thromboembolic stroke, Annual Meeting of the Society-of-Academic-and-Research-Surgery (SARS), Publisher: WILEY, Pages: 12-13, ISSN: 0007-1323

Conference paper

Qureshi MI, Greco M, Vorkas PA, Holmest E, Davies AHet al., 2017, Application of metabolic profiling to abdominal aortic aneurysm research, Journal of Proteome Research, Vol: 16, Pages: 2325-2332, ISSN: 1535-3893

Abdominal aortic aneurysm (AAA) is a complex disease posing diagnostic and therapeutic challenges. Metabonomics may aid in the diagnosis of AAA, determination of individualized risk, discovery of therapeutic targets, and improve understanding of pathogenesis. A systematic review of the diversity and outcomes of existing AAA metabonomic research has been performed. Original research studies applying metabonomics to human aneurysmal disease are included. Seven relevant articles were identified: four studies were based on plasma/serum metabolite profiling, and three studies examined aneurysmal tissue. Aminomalonic acid, guanidinosuccinic acid, and glycerol emerge as potential plasma biomarkers of large aneurysm. Lipid profiling improves predictive models of aneurysm presence. Patterns of metabolite variation associated with AAA relate to carbohydrate and lipid metabolism. Perioperative perturbations in metabolites suggest differential systemic inflammatory responses to surgery, generating hypotheses for adjunctive perioperative therapy. Significant limitations include small study sizes, lack of correction for multiple testing false discovery rates, and single time-point sampling. Metabolic profiling carries the potential to identify biomarkers of AAA and elucidate pathways underlying aneurysmal disease. Statistically and methodologically robust studies are required for validation, addressing the hiatus in understanding mechanisms of aneurysm growth and developing effective treatment strategies.

Journal article

Qureshi M, Vorkas P, Kaluarachchi M, Holmes E, Davies Aet al., 2017, Biomarker research in thromboembolic stroke (BRUITS), Publisher: KARGER, ISSN: 1015-9770

Conference paper

Qureshi M, Vorkas P, Jenkins I, Holmes E, Davies Aet al., 2016, Biomarker research in thromboembolic stroke, Publisher: SAGE PUBLICATIONS LTD, Pages: S32-S32, ISSN: 1747-4930

Conference paper

Qureshi M, Coupland A, Vorkas P, Jenkins I, Holmes E, Davies Aet al., 2016, Metabolic profiling of ischaemic stroke, Publisher: SAGE PUBLICATIONS LTD, Pages: S32-S32, ISSN: 1747-4930

Conference paper

Qureshi MI, Vorkas PA, Coupland AP, Jenkins IH, Holmes E, Davies AHet al., 2016, Lessons from metabonomics on the neurobiology of stroke, Neuroscientist, Vol: 23, Pages: 374-382, ISSN: 1073-8584

The application of metabonomic science to interrogate stroke permits the study of metabolite entities, small enough to cross the blood-brain barrier, that provide insight into neuronal dysfunction, and may serve as reservoirs of biomarker discovery. This systematic review examines the applicability of metabolic profiling in ischemic stroke research. Six human studies utilizing metabolic profiling to analyze biofluids from ischemic stroke patients have been included, employing 1H-NMR and/or mass spectrometry to analyze plasma, serum, and/or urine in a targeted or untargeted fashion. Three are diagnostic studies, and one investigates prognostic biomarkers of stroke recurrence following transient ischemic attack. Two studies focus on metabolic distinguishers of depression or cognitive impairment following stroke. Identified biomarkers from blood and urine predominantly relate to homocysteine and folate, branched chain amino acid, and lipid metabolism. Statistical models are well fitted and reproducible, with excellent validation outcomes, demonstrating the feasibility of metabolic profiling to study a complex disorder with multicausal pathology, such as stroke.

Journal article

Anwar MA, Vorkas PA, Li J, Adesina-Georgiadis KN, Reslan OM, Raffetto JD, Want EJ, Khalil RA, Holmes E, Davies AHet al., 2016, Prolonged Mechanical Circumferential Stretch Induces Metabolic Changes in Rat Inferior Vena Cava, EUROPEAN JOURNAL OF VASCULAR AND ENDOVASCULAR SURGERY, Vol: 52, Pages: 544-552, ISSN: 1078-5884

Journal article

Djekic D, Pinto R, Vorkas PA, Henein MYet al., 2016, Replication of LC-MS untargeted lipidomics results in patients with calcific coronary disease: an interlaboratory reproducibility study, International Journal of Cardiology, Vol: 222, Pages: 1042-1048, ISSN: 1874-1754

BackgroundRecently a lipidomics approach was able to identify perturbed fatty acyl chain (FAC) and sphingolipid moieties that could stratify patients according to the severity of coronary calcification, a form of subclinical atherosclerosis. Nevertheless, these findings have not yet been reproduced before generalising their application. The aim of this study was to evaluate the reproducibility of lipidomics approaches by replicating previous lipidomic findings in groups of patients with calcific coronary artery disease (CCAD).MethodsPatients were separated into the following groups based on their calcium score (CS); no calcification (CS: 0; n = 26), mild calcification (CS: 1–250; n = 27) and severe calcification (CS: > 250; n = 17). Two serum samples were collected from each patient and used for comparative analyses by 2 different laboratories, in different countries and time points using liquid chromatography coupled to mass spectrometry untargeted lipidomics methods.ResultsSix identical metabolites differentiated patients with severe coronary artery calcification from those with no calcification were found by both laboratories independently. Additionally, relative intensities from the two analyses demonstrated high correlation coefficients. Phosphatidylcholine moieties with 18-carbon FAC were identified in lower intensities and 20:4 FAC in higher intensities in the serum of diseased group. Moreover, 3 common sphingomyelins were detected.ConclusionThis is the first interlaboratory reproducibility study utilising lipidomics applications in general and specifically in patients with CCAD. Lipid profiling applications in patients with CCAD are very reproducible in highly specialised and experienced laboratories and could be applied in clinical practice in order to spare patients diagnostic radiation.

Journal article

Vorkas PA, Shalhoub J, Lewis MR, Spagou K, Want EJ, Nicholson JK, Davies AH, Holmes Eet al., 2016, Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk – An Exploratory Study, European Journal of Vascular and Endovascular Surgery, Vol: 52, Pages: 5-10, ISSN: 1532-2165

Objectives: Stroke is a major cause of death and disability. The fact that three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, we assess the utility of comprehensive metabolic phenotyping to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples.Design: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n=5), and asymptomatic patients (n=5). Two adjacent biological replicates were obtained from each tissue.Materials and Methods: Organic and aqueous metabolite extracts were separately obtained and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were utilised for statistical analysis.Results: The two studied groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p=10-4-10-5). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine and palmitoylcarnitine), intermediates of the β-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected.Conclusions: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and β-oxidation) were implicated and will be further investigated. These results indicate that metabolic

Journal article

Lamour SD, Gomez-Romero M, Vorkas PA, Alibu VP, Saric J, Holmes E, Sternberg JMet al., 2015, Discovery of Infection Associated Metabolic Markers in Human African Trypanosomiasis., PLOS Neglected Tropical Diseases, Vol: 9, ISSN: 1935-2735

Human African trypanosomiasis (HAT) remains a major neglected tropical disease in Sub-Saharan Africa. As clinical symptoms are usually non-specific, new diagnostic and prognostic markers are urgently needed to enhance the number of identified cases and optimise treatment. This is particularly important for disease caused by Trypanosoma brucei rhodesiense, where indirect immunodiagnostic approaches have to date been unsuccessful. We have conducted global metabolic profiling of plasma from T.b.rhodesiense HAT patients and endemic controls, using 1H nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography, coupled with mass spectrometry (UPLC-MS) and identified differences in the lipid, amino acid and metabolite profiles. Altogether 16 significantly disease discriminatory metabolite markers were found using NMR, and a further 37 lipid markers via UPLC-MS. These included significantly higher levels of phenylalanine, formate, creatinine, N-acetylated glycoprotein and triglycerides in patients relative to controls. HAT patients also displayed lower concentrations of histidine, sphingomyelins, lysophosphatidylcholines, and several polyunsaturated phosphatidylcholines. While the disease metabolite profile was partially consistent with previous data published in experimental rodent infection, we also found unique lipid and amino acid profile markers highlighting subtle but important differences between the host response to trypanosome infections between animal models and natural human infections. Our results demonstrate the potential of metabolic profiling in the identification of novel diagnostic biomarkers and the elucidation of pathogenetic mechanisms in this disease.

Journal article

Anwar MA, Vorkas PA, Li JV, Shalhoub J, Want EJ, Davies AH, Holmes Eet al., 2015, Optimization of metabolite extraction of human vein tissue for ultra performance liquid chromatography-mass spectrometry and nuclear magnetic resonance-based untargeted metabolic profiling, Analyst, Vol: 140, Pages: 7586-7597, ISSN: 1364-5528

Human vein tissue is an important matrix to examine when investigating vascular diseases with respect to understanding underlying disease mechanisms. Here, we report the development of an extraction protocol for multi-platform metabolic profiling of human vein tissue. For the first stage of the optimization, two different ratios of methanol/water and 5 organic solvents – namely dichloromethane, chloroform, isopropanol, hexane and methyl tert-butyl ether (MTBE) solutions with methanol – were tested for polar and organic compound extraction, respectively. The extraction output was assessed using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and a panel of Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) methodologies. On the basis of the reproducibility of extraction replicates and metabolic coverage, the optimal aqueous (methanol/water) and organic (MTBE/methanol) solvents identified from the first stage were used in a sequential approach for metabolite extraction, altering the order of solvent-mixture addition. The combination of organic metabolite extraction with MTBE/methanol (3 : 1) followed by extraction of polar compounds with methanol/water (1 : 1) was shown to be the best method for extracting metabolites from human vein tissue in terms of reproducibility and number of signals detected and could be used as a single extraction procedure to serve both NMR and UPLC-MS analyses. Molecular classes such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, sphingolipids, purines, and pyrimidines were reproducibly extracted. This study enabled an optimal extraction protocol for robust and more comprehensive metabolome coverage for human vein tissue. Many of the physiological and pathological processes affecting the composition of human vein tissue are common to other tissues and hence the extraction method developed in this study can be generically applied.

Journal article

Vorkas PA, Isaac G, Holmgren A, Want EJ, Shockcor JP, Holmes E, Henein MYet al., 2015, Perturbations in fatty acid metabolism and apoptosis are manifested in calcific coronary artery disease: An exploratory lipidomics study, International Journal of Cardiology, Vol: 197, Pages: 192-199, ISSN: 1874-1754

BackgroundControversy exists concerning the beneficial or harmful effects of the presence of ectopic calcification in the coronary arteries. Additionally, further elucidation of the exact pathophysiological mechanism is needed. In this study, we sought to identify metabolic markers of vascular calcification that could assist in understanding the disease, monitoring its progress and generating hypotheses describing its pathophysiology.MethodsUntargeted lipid profiling and complementary modeling strategies were employed to compare serum samples from patients with different levels of calcific coronary artery disease (CCAD) based on their calcium score (CS). Subsequently, patients were divided into three groups: no calcification (NC; CS = 0; n = 26), mild calcification (MC; CS:1–250; n = 27) and severe (SC; CS > 250; n = 17).ResultsPhosphatidylcholine levels were found to be significantly altered in the disease states (p = 0.001–0.04). Specifically, 18-carbon fatty acyl chain (FAC) phosphatidylcholines were detected in lower levels in the SC group, while 20:4 FAC lipid species were detected in higher concentrations. A statistical trend was observed with phosphatidylcholine lipids in the MC group, showing the same tendency as with the SC group. We also observed several sphingomyelin signals present at lower intensities in SC when compared with NC or MC groups (p = 0.000001–0.01).ConclusionsThis is the first lipid profiling study reported in CCAD. Our data demonstrate dysregulations of phosphatidylcholine lipid species, which suggest perturbations in fatty acid elongation/desaturation. The altered levels of the 18-carbon and 20:4 FAC lipids may be indicative of disturbed inflammation homeostasis. The marked sphingomyelin dysregulation in SC is consistent with profound apoptosis as a potential mechanism of CCAD.

Journal article

Qureshi MI, Vorkas P, Shalhoub J, Anwar M, Holmes E, Davies AHet al., 2015, Biomarker research in thromboembolic stroke (bruits), Annual Meeting of the Society-of-Academic-and-Research-Surgery (SARS(, Publisher: WILEY-BLACKWELL, Pages: 25-25, ISSN: 0007-1323

Conference paper

Vorkas PA, Shalhoub J, Isaac G, Want EJ, Nicholson JK, Holmes E, Davies AHet al., 2015, Metabolic phenotyping of atherosclerotic plaques reveals latent associations between free cholesterol and ceramide metabolism in atherogenesis., Journal of proteome research, Vol: 14, Pages: 1389-1399, ISSN: 1535-3893

Current optimum medical treatments have had limited success in the primary prevention of cardiovascular events, underscoring the need for new pharmaceutical targets and enhanced understanding of mechanistic metabolic dysregulation. Here, we use a combination of novel metabolic profiling methodologies, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) followed by chemometric modeling, data integration, and pathway mapping, to create a systems-level metabolic atlas of atherogenesis. We apply this workflow to compare arterial tissue incorporating plaque lesions to intimal thickening tissue (immediate preplaque stage). We find changes in several metabolite species consistent with well-established pathways in atherosclerosis, such as the cholesterol, purine, pyrimidine, and ceramide pathways. We then illustrate differential levels of previously unassociated lipids to atherogenesis, namely, phosphatidylethanolamine-ceramides (t-test p-values: 3.8 × 10(-6) to 9.8 × 10(-12)). Most importantly, these molecules appear to be interfacing two pathways recognized for their involvement in atherosclerosis: ceramide and cholesterol. Furthermore, we show that β-oxidation intermediates (i.e., acylcarnitines) manifest a pattern indicating truncation of the process and overall dysregulation of fatty acid metabolism and mitochondrial dysfunction. We develop a metabolic framework that offers the ability to map significant statistical associations between detected biomarkers. These dysregulated molecules and consequent pathway modulations may provide novel targets for pharmacotherapeutic intervention.

Journal article

Vorkas PA, Isaac G, Anwar MA, Davies AH, Want EJ, Nicholson JK, Holmes Eet al., 2015, Untargeted UPLC-MS Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to Cardiovascular Disease., Analytical Chemistry, Vol: 87, Pages: 4184-4193, ISSN: 1086-4377

Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaround time of the two methods contributed toward obtaining highly reproducible features with exceptional chromatographic performance (CV % < 0.5%), making this pipeline suitable for metabolic profiling applications. We structurally assigned 226 metabolites from a range of chemical classes (e.g., carnitines, α-amino acids, purines, pyrimidines, phospholipids, sphingolipids, free fatty acids, and glycerolipids) which were mapped to their corresponding pathways, biological functions and known disease mechanisms. The combination of the two untargeted UPLC-MS methods showed high metabolite complementarity. We demonstrate the application of this pipeline to cardiovascular disease, where we show that the analyzed diseased groups (n = 120) of arterial tissue could be distinguished based on their metabolic profiles.

Journal article

Mirnezami R, Spagou K, Vorkas PA, Lewis MR, Kinross J, Want E, Shion H, Goldin RD, Darzi A, Takats Z, Holmes E, Cloarec O, Nicholson JKet al., 2014, Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects, Molecular Oncology, Vol: 8, Pages: 39-49, ISSN: 1574-7891

Matrix‐assisted laser desorption ionisation imaging mass spectrometry (MALDI‐MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI‐MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI‐MSI; (3) To determine whether MALDI‐MSI profiling of tumour‐adjacent tissue can identify novel metabolic ‘field effects’ associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour‐adjacent and tumour‐remote healthy mucosa. We have referred to this ‘field effect’, exhibited by the tumour locale, as cancer‐adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

Journal article

Shalhoub J, Sikkel MB, Davies KJ, Vorkas PA, Want EJ, Davies AHet al., 2014, Systems Biology of Human Atherosclerosis, VASCULAR AND ENDOVASCULAR SURGERY, Vol: 48, Pages: 5-17, ISSN: 1538-5744

Journal article

Anwar MA, Vorkas P, Li J, Shalhoub J, Lim CS, Want E, Holmes E, Davies AHet al., 2014, Differential Metabolic Phenotype of Human Varicose Veins Tissue and Their Utility in Understanding Disease Pathogenesis and Identifying Potential Prognostic Biomarkers., Pages: 113-113, ISSN: 2213-333X

Conference paper

Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou Eet al., 2013, Clinical evaluation of microRNA expression profiling in non small cell lung cancer, LUNG CANCER, Vol: 81, Pages: 388-396, ISSN: 0169-5002

Journal article

Anwar MA, Vorkas P, Li J, Resslan O, Want E, Raffetto JD, Khalil RA, Holmes E, Davies AHet al., 2013, Prolonged Mechanical Stretch Alters the Metabolic Profile in Rat Inferior Vena Cava., Pages: 105-106, ISSN: 2213-333X

Conference paper

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