20 hete d6

An aliquot of platelet concentrate (1 mL) was transferred into a 15 mL Falcon tube for two different treatment types. Ice cold ethanol (EtOH) (4 mL) was added, containing 10–100 nM of each internal standard (12S-hydroxyeicosatetraenoic acid (HETE)-d8, 15S-HETE-d8, 5-oxo-eicosatetraenoic acid (ETE)-d7, 11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (DiHETrE)-d11, prostaglandin E2 (PGE2)-d4, 20-HETE-d6 (Cayman Chemical, Tallinn, Estonia), for protein precipitation over night at −20 °C. The samples were then centrifuged for 30 min at 4536× g and 4 °C, protein pellets were discarded, and EtOH was evaporated via vacuum centrifugation at 37 °C until the original sample volume was restored. Solid-phase extraction (SPE) was performed with 30 mg/mL StrataX SPE columns (Phenomenex, Torrance, CA, USA). The columns were washed with 2 mL absolute methanol (abs. MeOH) equilibrated with 2 mL MS grade H₂O before sample loading with Pasteur pipettes. The samples were washed with 5 mL MS grade H₂O before eluting the eicosanoids into 1.5 mL glass vials with 500 µL abs. MeOH containing 2% formic acid (FA). The eluates were stored at −80 °C until further analysis. Before measurement, the organic phase was evaporated at room temperature using nitrogen steam and reconstituted in 150 µL reconstitution buffer (H₂O/acetonitrile (ACN)/MeOH + 0.2% FA—65:31.5:3.5).

Plasma and homogenized tissue (kidney and liver) samples were subjected to alkaline hydrolysis and solid-phase extraction was performed as described previously [41 (link)]. 10 ng of each 20-HETE-d6, 14,15-EET-d8, 14,15-DHET-d11, and 15-HETE-d8 (Cayman Chemicals, Ann Arbor, MI, USA) served as internal standards. Subsequent analysis of the endogenous eicosanoid profiles was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS; Lipidomix GmbH, Berlin, Germany) as established previously [42 (link)]. Results are given in ng metabolites per ml plasma or per g of organ wet weight.