Lipid extraction and LC-MS measure of oxylipins

All sera samples at baseline were stored at − 80 °C, thawed once, and immediately used for free fatty acid and oxylipin isolation as previously described [8]. Briefly, 50 μL sera was spiked with a cocktail of 26 deuterated internal standards that also included some selected PUFAs (individually purchased from Cayman Chemicals, Ann Arbor, MI) and brought to a volume of 1 mL with 10% methanol. The samples were then purified by solid-phase extraction on Strata-X columns (Phenomenex, Torrance, CA), using an activation procedure consisting of consecutive washes with 3 mL of 100% methanol followed by 3 mL of water. The oxylipins were then eluted with 1 mL of 100% methanol, which was dried under a vacuum, dissolved in 50 μL of buffer A (consisting of water–acetonitrile–acetic acid, 60:40:0.02 [v/v/v]), and immediately used for analysis. Oxylipins in sera were analyzed and quantified by LC/MS/MS as previously described [8, 9]. Briefly, oxylipins were separated by reverse-phase chromatography using a 1.7 μm 2.1 × 100 mm BEH Shield Column (Waters, Milford, MA) and an Acquity UPLC system (Waters). The column was equilibrated with buffer A, and 10 μL of sample was injected via the autosampler. Samples were eluted with a step gradient starting with 100% buffer A for 1 min, then to 50% buffer B (consisting of 50% acetonitrile, 50% isopropanol, and 0.02% acetic acid) over a period of 3 min, and then to 100% buffer B over a period of 1 min. The LC was interfaced with an IonDrive Turbo V ion source, and mass spectral analysis was performed on a triple quadrupole AB SCIEX 6500 QTrap mass spectrometer (AB SCIEX, Framingham, MA). Oxylipins were measured using electrospray ionization in negative ion mode and multiple reaction monitoring (MRM) using the most abundant and specific precursor ion/product ion transitions to build an acquisition method capable of detecting 158 analytes and 26 internal standards. The ionspray voltage was set at − 4500 V at a temperature of 550 °C. Collisional activation of the oxylipin precursor ions was achieved with nitrogen as the collision gas with the declustering potential, entrance potential, and collision energy optimized for each metabolite. Oxylipins were identified by matching their MRM signal and chromatographic retention time with those of pure identical standards.

Oxylipins were quantitated by the stable isotope dilution method. Briefly, identical amounts of deuterated internal standards were added to each sample and to all the primary standards used to generate standard curves. To calculate the amount of oxylipins and free fatty acids in a sample, ratios of peak areas between endogenous metabolites and matching deuterated internal standards were calculated. Ratios were converted to absolute amounts by linear regression analysis of standard curves generated under identical conditions. Oxylipin levels are expressed in picomol/milliliter (pmol/mL). To account for batch effects, quality control samples were run in each batch; the average coefficient of variance for the quantified oxylipins was 4% (standard deviation 0.01).

The oxylipins identified and quantified in our study are derived from the fatty acid precursors: omega-6 polyunsaturated fatty acids (PUFA) - arachidonic acid (AA), linoleic acid (LA), and dihomo-gamma linolenic acid (DGLA), as well as omega 3 PUFAs: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (see Supplementary Figure 1)