Harmonized procedures lead to comparable quantification of total oxylipins across laboratories

Authors: MAINKA, MALWINA - University Of Wuppertal; DALLE, CELINE - Institut National De La Recherche Agronomique (INRA); PETERA, M. - Institut National De La Recherche Agronomique (INRA); DALLOUX-CHIOCCIOLI, JESSICA - Institut National De La Sante Et De La Recherche Medicale (INSERM); KAMPSCHULTE, N. - University Of Wuppertal; OSTERMANN, ANNIKA - University Of Wuppertal; ROTHE, MIKAEL - Lipidomix; BERTRAND-MICHEL, JUSTINE - Institut National De La Sante Et De La Recherche Medicale (INSERM); Newman, John; GLADINE, CECILE - Institut National De La Recherche Agronomique (INRA); SCHEBB, NILS - University Of Wuppertal

Submitted to: Journal of Lipid Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2020
Publication Date: 11/1/2020
Citation: Mainka, M., Dalle, C., Petera, M., Dalloux-Chioccioli, J., Kampschulte, N., Ostermann, A.I., Rothe, M., Bertrand-Michel, J., Newman, J.W., Gladine, C., Schebb, N.H. 2020. Harmonized procedures lead to comparable quantification of total oxylipins across laboratories. Journal of Lipid Research. 61(11):1424-1436. https://doi.org/10.1194/jlr.RA120000991.
DOI: https://doi.org/10.1194/jlr.RA120000991

Interpretive Summary: The quantitative analysis of broad swaths of low abundance metabolites involved in the regulation of inflammation and energy metabolism can provides unique insights into normal biological processes associated with health and disease. However, current approaches are labor intensive, and not amenable for the study of large sample sets needed to understand the variability in the general population. The goal of this research was to develop a high-throughput, cost-effective method for metabolic profiling of lipid mediators and hormones involved in the regulation of inflammation and energy metabolism, along with polyunsaturated fatty acids and common over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs). We describe a rapid 96-well plate based method for 50 µL of plasma or serum that can evaluate 145 compounds, including regulators of inflammation and energy metabolism along with a handful of common anti-inflammatory drugs. Measurement precision was excellent for the majority of measured compounds at concentrations above the influence of instrumental variability. Measurement accuracy as compared to a standard reference material of metabolites in human plasma provided by the National Institutes of Standards and Technology was also very good. Using this method, we provide new data for ~40 lipid mediators in important and commercially available reference material. Application to two independent cohorts of elderly men and women showed the routine detection of 86 metabolites, identified fasting state influences on essential fatty acid-derived oxylipins, N-acylethanolamides and conjugated bile acids, identified rare presence of high and low testosterone levels and the presence of NSAIDs in ~10% of these populations. The described method appears valuable for investigations in large cohort studies to provide insight into metabolic cross-talk between the array of mediators assessed here.

Technical Abstract: Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, this will require the use of standardized and harmonized protocols and to assess the technical and interlaboratory variability. We performed an interlaboratory comparison for the MS-based quantitative analysis of esterified oxylipins. Five independent labs were involved to assess the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e. 7 quality control plasmas), standard calibration series and analytical methods. The analysis was performed using a sensitive LC-ESI(-)-MS/MS method with optimized mass spectrometric and chromatographic parameters. The quantitative analysis is based on a standard calibration series with isotopically labelled internal standards. For 73% of oxylipins the technical variance was within ±15%, however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of results across laboratories was examined using PCA and PLS-DA. Inter-lab variability was very limited and did not interfere with our ability to distinguish the different plasma pools. Moreover, all laboratories were able to identify similar differences between plasma pools. In summary, we could show that using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all steps of the oxylipin analysis leads to reliable, reproducible and comparable oxylipin concentrations in different laboratories allowing the generation of biologically meaningful oxylipin patterns.