Tissue lipidomics is a rapidly advancing field in clinical and biomedical research that provides crucial information on the lipid-driven molecular mechanisms underlying physiological and pathological conditions. However, accurate MS-based analysis requires careful preanalytical handling due to the metabolic activity of tissue and analyte heterogeneity. Here, we introduce a robust tissue processing workflow with the pancreas as a model of a highly metabolically active organ. First, we evaluate lipid stability in porcine pancreatic tissue stored on ice, observing significant lysophospholipid formation after 60-120 min. Then, we compare sample handling using ice versus liquid nitrogen for both porcine and mouse pancreatic tissues, illustrating that processing temperature affects low-abundant lipid class levels, with liquid nitrogen providing better preservation. To enhance polar lipidome analysis, we optimize a hexane-methanol liquid-liquid extraction protocol and find that the addition of 2% (v/v) water to methanol yields the most effective recovery and reproducibility. Finally, the workflow is applied to mouse pancreatic tissue samples, enabling the identification of 209 polar lipid species across 10 classes, with 124 species quantified. Among these, hexosylceramides show clear sex-specific variation.

Department of Analytical Chemistry Faculty of Chemical Technology University of Pardubice Pardubice Czech Republic

Institute of Physiology Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Fahy E., Cotter D., Sud M., Subramaniam S. Lipid classification, structures and tools. Biochim. Biophys. Acta. 2011;1811:637–647. PubMed PMC

Fitzner D., Bader J.M., Penkert H., Bergner C.G., Su M., Weil M.T., et al. Cell-type- and brain-region-resolved mouse brain lipidome. Cell Rep. 2020;32 PubMed

Sarmento M.J., Llorente A., Petan T., Khnykin D., Popa I., Nikolac Perkovic M., et al. The expanding organelle lipidomes: current knowledge and challenges. Cell. Mol. Life Sci. 2023;80:237. PubMed PMC

Surma M.A., Gerl M.J., Herzog R., Helppi J., Simons K., Klose C. Mouse lipidomics reveals inherent flexibility of a mammalian lipidome. Sci. Rep. 2021;11 PubMed PMC

Naoe S., Tsugawa H., Takahashi M., Ikeda K., Arita M. Characterization of lipid profiles after dietary intake of polyunsaturated fatty acids using integrated untargeted and targeted lipidomics. Metabolites. 2019;9:241. PubMed PMC

Drouin G., Catheline D., Guillocheau E., Gueret P., Baudry C., Le Ruyet P., et al. Rioux V., Legrand P. Comparative effects of dietary n-3 docosapentaenoic acid (DPA), DHA and EPA on plasma lipid parameters, oxidative status and fatty acid tissue composition. J. Nutr. Biochem. 2019;63:186–196. PubMed

Stanley E.G., Jenkins B.J., Walker C.G., Koulman A., Browning L., West A.L., et al. Lipidomics profiling of human adipose tissue identifies a pattern of lipids associated with fish oil supplementation. J. Proteome Res. 2017;16:3168–3179. PubMed

May F.J., Baer L.A., Lehnig A.C., So K., Chen E.Y., Gao F., et al. Lipidomic adaptations in white and brown adipose tissue in response to exercise demonstrate molecular species-specific remodeling. Cell Rep. 2017;18:1558–1572. PubMed PMC

Eum J.Y., Lee J.C., Yi S.S., Kim I.Y., Seong J.K., Moon M.H. Aging-related lipidomic changes in mouse serum, kidney, and heart by nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A. 2020;1618 PubMed

Ooi G.J., Meikle P.J., Huynh K., Earnest A., Roberts S.K., Kemp W., et al. Hepatic lipidomic remodeling in severe obesity manifests with steatosis and does not evolve with non-alcoholic steatohepatitis. J. Hepatol. 2021;75:524–535. PubMed

Chorell E., Otten J., Stomby A., Ryberg M., Waling M., Hauksson J., et al. Improved peripheral and hepatic insulin sensitivity after lifestyle interventions in type 2 diabetes is associated with specific metabolomic and lipidomic signatures in skeletal muscle and plasma. Metabolites. 2021;11:834. PubMed PMC

Ecker J., Benedetti E., Kindt A.S.D., Höring M., Perl M., Machmüller A.C., et al. The colorectal cancer lipidome: identification of a robust tumor-specific lipid species signature. Gastroenterology. 2021;161:910–923. PubMed

Jirásko R., Idkowiak J., Wolrab D., Kvasnička A., Friedecký D., Polański K., et al. Altered plasma, urine, and tissue profiles of sulfatides and sphingomyelins in patients with renal cell carcinoma. Cancers (Basel) 2022;14:4622. PubMed PMC

Eggers L.F., Müller J., Marella C., Scholz V., Watz H., Kugler C., et al. Lipidomes of lung cancer and tumour-free lung tissues reveal distinct molecular signatures for cancer differentiation, age, inflammation, and pulmonary emphysema. Sci. Rep. 2017;7 PubMed PMC

Cífková E., Brumarová R., Ovčačíková M., Dobešová D., Mičová K., Kvasnička A., et al. Lipidomic and metabolomic analysis reveals changes in biochemical pathways for non-small cell lung cancer tissues. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2022;1867 PubMed

Wolrab D., Chocholoušková M., Jirásko R., Peterka O., Holčapek M. Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography–mass spectrometry and hydrophilic interaction liquid chromatography–mass spectrometry. Anal. Bioanal. Chem. 2020;412:2375–2388. PubMed

Vaňková Z., Peterka O., Chocholoušková M., Wolrab D., Jirásko R., Holčapek M. Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS. Anal. Bioanal. Chem. 2022;414:319–331. PubMed

Contrepois K., Mahmoudi S., Ubhi B.K., Papsdorf K., Hornburg D., Brunet A., et al. Cross-platform comparison of untargeted and targeted lipidomics approaches on aging mouse plasma. Sci. Rep. 2018;8:1–9. PubMed PMC

Mainka M., Dalle C., Pétéra M., Dalloux-Chioccioli J., Kampschulte N., Ostermann A.I., et al. Harmonized procedures lead to comparable quantification of total oxylipins across laboratories. J. Lipid Res. 2020;61:1424–1436. PubMed PMC

Burla B., Arita M., Arita M., Bendt A.K., Cazenave-Gassiot A., Dennis E.A., et al. MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines. J. Lipid Res. 2018;59:2001–2017. PubMed PMC

Criscuolo A., Zeller M., Cook K., Angelidou G., Fedorova M. Rational selection of reverse phase columns for high throughput LC–MS lipidomics. Chem. Phys. Lipids. 2019;221:120–127. PubMed

Lippa K.A., Aristizabal-Henao J.J., Beger R.D., Bowden J.A., Broeckling C., Beecher C., et al. Reference materials for MS-based untargeted metabolomics and lipidomics: a review by the metabolomics quality assurance and quality control consortium (mQACC) Metabolomics. 2022;18:24. PubMed PMC

Alseekh S., Aharoni A., Brotman Y., Contrepois K., D’Auria J., Ewald J., et al. Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices. Nat. Methods. 2021;18:747–756. PubMed PMC

dos Santos A.C.A., Vuckovic D. Current status and advances in untargeted LC-MS tissue lipidomics studies in cardiovascular health. Trac Trends Anal. Chem. 2024;170

Lange M., Angelidou G., Ni Z., Criscuolo A., Schiller J., Blüher M., et al. AdipoAtlas: a reference lipidome for human white adipose tissue. Cell Rep. Med. 2021;2 PubMed PMC

Höring M., Krautbauer S., Hiltl L., Babl V., Sigruener A., Burkhardt R., et al. Accurate lipid quantification of tissue homogenates requires suitable sample concentration, solvent composition, and homogenization procedure-a case study in murine liver. Metabolites. 2021;11:365. PubMed PMC

Hricko J., Rudl Kulhava L., Paucova M., Novakova M., Kuda O., Fiehn O., et al. Short-term stability of serum and liver extracts for untargeted metabolomics and lipidomics. Antioxidants. 2023;12:986. PubMed PMC

Dorochow E., Gurke R., Rischke S., Geisslinger G., Hahnefeld L. Effects of different storage conditions on lipid stability in mice tissue homogenates. Metabolites. 2023;13:504. PubMed PMC

Pradas I., Huynh K., Cabré R., Ayala V., Meikle P.J., Jové M., et al. Lipidomics reveals a tissue-specific fingerprint. Front. Physiol. 2018;9:1165. PubMed PMC

Bögl T., Mlynek F., Himmelsbach M., Buchberger W. Comparison of one-phase and two-phase extraction methods for porcine tissue lipidomics applying a fast and reliable tentative annotation workflow. Talanta. 2022;236 PubMed

Cífková E., Holčapek M., Lísa M. Nontargeted lipidomic characterization of porcine organs using hydrophilic interaction liquid chromatography and off-line two-dimensional liquid chromatography-electrospray ionization mass spectrometry. Lipids. 2013;48:915–928. PubMed

Falcomatà C., Saur D. Self-renewal equality in pancreas homeostasis, regeneration, and cancer. Cell Rep. 2021;37 PubMed

Caldart F., de Pretis N., Luchini C., Ciccocioppo R., Frulloni L. Pancreatic steatosis and metabolic pancreatic disease: a new entity? Intern. Emerg. Med. 2023;18:2199–2208. PubMed PMC

Petrenko V., Sinturel F., Loizides-Mangold U., Montoya J.P., Chera S., Riezman H., et al. Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets. PLoS Biol. 2022;20 PubMed PMC

Unger K., Mehta K.Y., Kaur P., Wang Y., Menon S.S., Jain S.K., et al. Metabolomics based predictive classifier for early detection of pancreatic ductal adenocarcinoma. Oncotarget. 2018;9:23078–23090. PubMed PMC

Salhi A., Amara S., Mansuelle P., Puppo R., Lebrun R., Gontero B., et al. Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices. Biochimie. 2020;169:106–120. PubMed

de Haas G.H., Postema N.M., Nieuwenhuizen W.V., van Deenen L.L.M. Purification and properties of phospholipase A from porcine pancreas. Biochim. Biophys. Acta Enzymol. 1968;159:103–117. PubMed

Gluchowski N.L., Becuwe M., Walther T.C., Farese R.V. Lipid droplets and liver disease: from basic biology to clinical implications. Nat. Rev. Gastroenterol. Hepatol. 2017;14:343–355. PubMed PMC

Tong X., Liu S., Stein R., Imai Y. Lipid droplets’ role in the regulation of β-cell function and β-cell demise in type 2 diabetes. Endocrinol. 2022;163 PubMed PMC

Gerst F., Wagner R., Oquendo M.B., Siegel-Axel D., Fritsche A., Heni M., et al. What role do fat cells play in pancreatic tissue? Mol. Metab. 2019;25:1–10. PubMed PMC

Want E.J., Masson P., Michopoulos F., Wilson I.D., Theodoridis G., Plumb R.S., et al. Global metabolic profiling of animal and human tissues via UPLC-MS. Nat. Protoc. 2013;8:17–32. PubMed

Liebisch G., Fahy E., Aoki J., Dennis E.A., Durand T., Ejsing C.S., et al. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures. J. Lipid Res. 2020;61:1539–1555. PubMed PMC

Folch J., Less M., Sloane Strnley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957;226:497–509. PubMed

Wolrab D., Chocholoušková M., Jirásko R., Peterka O., Mužáková V., Študentová H., et al. Determination of one year stability of lipid plasma profile and comparison of blood collection tubes using UHPSFC/MS and HILIC-UHPLC/MS. Anal. Chim. Acta. 2020;1137:74–84. PubMed

Chocholoušková M., Vivó-Truyols G., Wolrab D., Jirásko R., Antonelli M., Peterka O., et al. Lipid Quant 2.1: Open-source software for identification and quantification of lipids measured by lipid class separation QTOF high-resolution mass spectrometry methods. Chemom. Intell. Lab. Syst. 2024;251

Chocholoušková M., Wolrab D., Jirásko R., Študentová H., Melichar B., Holčapek M. Intra-laboratory comparison of four analytical platforms for lipidomic quantitation using hydrophilic interaction liquid chromatography or supercritical fluid chromatography coupled to quadrupole - time-of-flight mass spectrometry. Talanta. 2021;231 PubMed

Kopczynski D., Ejsing C.S., McDonald J.G., Bamba T., Baker E.S., Bertrand-Michel J., et al. The lipidomics reporting checklist a framework for transparency of lipidomic experiments and repurposing resource data. J. Lipid Res. 2024;65:1–14. PubMed PMC

Liu J., Qin Z., Wang J. Liquid-liquid equilibria for methanol + water + hexane ternary mixtures. J. Chem. Eng. Data. 2002;47:1243–1245.

Murakami M., Sato H., Miki Y., Yamamoto K., Taketomi Y. A new era of secreted phospholipase A2. J. Lipid Res. 2015;56:1248–1261. PubMed PMC

Kinkaid A., Wilton D.C. Comparison of the catalytic properties of phospholipase A2 from pancreas and venom using a continuous fluorescence displacement assay. Biochem. J. 1991;278:843–848. PubMed PMC

Römisch-Margl W., Prehn C., Bogumil R., Röhring C., Suhre K., Adamski J. Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics. Metabolomics. 2012;8:133–142.

Turtoi E., Jeudy J., Henry S., Dadi I., Valette G., Enjalbal C., et al. Analysis of polar primary metabolites in biological samples using targeted metabolomics and LC-MS. STAR Protoc. 2023;4 PubMed PMC

De Caro J., Eydoux C., Chérif S., Lebrun R., Gargouri Y., Carrière F., et al. Occurrence of pancreatic lipase-related protein-2 in various species and its relationship with herbivore diet. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 2008;150:1–9. PubMed

Rugivarodom M., Geeratragool T., Pausawasdi N., Charatcharoenwitthaya P. Fatty pancreas: linking pancreas pathophysiology to nonalcoholic fatty liver disease. J. Clin. Transl. Hepatol. 2022;10:1229–1239. PubMed PMC

Schwartz P.B., Barrett-Wilt G.A., Ronnekleiv-Kelly S.M. Chronic jetlag alters the landscape of the pancreatic lipidome. Pancreas. 2022;51:80–89. PubMed PMC

Omar A.M., Zhang Q. Evaluation of lipid extraction protocols for untargeted analysis of mouse tissue lipidome. Metabolites. 2023;13:1002. PubMed PMC

Moon S.H., Dilthey B.G., Liu X., Guan S., Sims H.F., Gross R.W. High-fat diet activates liver iPLA2γ generating eicosanoids that mediate metabolic stress. J. Lipid Res. 2021;62 PubMed PMC

Choi J., Yin T., Shinozaki K., Lampe J.W., Stevens J.F., Becker L.B., et al. Comprehensive analysis of phospholipids in the brain, heart, kidney, and liver: brain phospholipids are least enriched with polyunsaturated fatty acids. Mol. Cell. Biochem. 2018;442:187–201. PubMed PMC

Muralidharan S., Shimobayashi M., Ji S., Burla B., Hall M.N., Wenk M.R., et al. A reference map of sphingolipids in murine tissues. Cell Rep. 2021;35 PubMed