Targeted Metabolomics Oxylipins/Eicosanoids/Bioactive Lipids

Oxylipins are fatty acid metabolites which include a metabolically important class of compounds encompassing oxysterols and eicosanoids.  Oxylipins have been implicated in many biological processes such as inflammation1, immunity2, and pain3, along with cardiovascular disease4 and Alzheimer’s disease5.  Their role in these pathways along with the ability to modulate oxylipin bioavailability in the diet, result in the increasing recognition of oxylipins as targets for metabolic studies.   

The Duke Proteomics and Metabolomics Shared Resource performs oxylipin analysis using a UPLC-MS/MS method to analyze oxylipins.  The method utilizes an Acquity UPLC coupled to a Xevo TQ-S triple quadrupole mass spectrometry by Waters Corporation to perform quantitative multiplexed analysis of up to 109 oxylipins inbiofluid or tissue samples.  Typically, healthy plasma or serum samples will measure approximately 30 of these above the lower limit of quantification (10 pg/mL), while more may be detected under conditions of disease or inflammation.  Tissue or cell line samples may allow detection of 70 or more species6

Pricing is performed on a per sample basis, plus the cost of running calibration curves.  Solid samples will require an additional sample preparation step, typically homogenization.  For more details, see our pricing page.

TECHNICAL INFORMATION

A 96 well plate is used for analysis, which includes calibration standards and isotopically labeled internal standards, along with quality control samples and blanks.  Approximately 20 mg of tissue or 500 uL of biofluids are reuired per sample.  Negative electrospray ionization allows for the oxylipins to be readily and specifically detected by MS/MS instrumentation.

The typical concentration range detected with the method is 10 pg/mL to 5000 pg/mL, although this range can vary depending on the specific compound.  We typically observe and quantify approximately 40-50 of these compounds in any given sample, potentially because many of them are natively at very low abundance (<10 pg/mL) or nonexistent.

Quantitation of oxylipins is performed using the calibration curve generated as part of the standard analysis.  The table below provides abbreviations used in the oxylipin assay, for both analytical standards and internal standards.  Note that one internal standard can be used to quantify several analytical standards within the same chemical class of compounds.  

    Figure 1 shows a representative chromatogram illustrating the separation of the quality control standards at 40 ng/mL concentration.

    Oxylipin chromatogram

    Figure 2 below shows an example of data generated from the oxylipin assay.  The figure shows boxplots with ANOVA testing performed using the data divided into six groups, which was unique to this experimental design.  This visualization makes clear the statistical differences between the sample groups with respect to the metabolite 12(13)-EpOME, derived from linoleic acid and produced by neutrophils.

    oxylipin data

    References

    1. Walker, R., Richter, C., Skulas-Ray, A., et al.  Effects of Long Chain Omega-3 Fatty Acid Supplementation on the Lipoprotein Oxylipin Response to an Inflammatory Challenge in Humans.  Current Developments in Nutrition 3, 1 (2019). https://doi.org/10.1093/cdn/nzz044.P08-127-19

    2.  Fang, J., Cui, L., Sun, Y. et al. Targeted metabolomics reveals altered oxylipin profiles in plasma of mild cognitive impairment patients. Metabolomics 13, 112 (2017). https://doi.org/10.1007/s11306-017-1249-0

    3. Hellström, F., Gouveia-Figueira, S., Nording, M.L. et al. Association between plasma concentrations of linoleic acid-derived oxylipins and the perceived pain scores in an exploratory study in women with chronic neck pain. BMC Musculoskelet Disord 17, 103 (2016). https://doi.org/10.1186/s12891-016-0951-9

    4. Caligiuri, S. P. B., Aukema, H. M., Ravandi, A., et al.  Specific plasma oxylipins increase the odds of cardiovascular and cerebrovascular events in patients with peripheral artery disease.  Canadian J. Phys. And Pharm., 95, 961 (2017).  https://doi.org/10.1139/cjpp-2016-0615

    5. Morris, J. K., Piccolo, B. D., John, C. S. et al.  Oxylipin Profiling of Alzheimer’s Disease in Nondiabetic and Type 2 Diabetic Elderly.  Metabolites 9, 9, (2019).  https://doi.org/10.3390/metabo9090177

    6. Harrison, A., Dubois, L. G., St. John-Williams, L., et al.  Comprehinsive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment.  Mol. Cell. Proteomics 15, 3 (2016).  https://doi.org/10.1074/mcp.M115.052498

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