Blood Collection and Eicosanoid Analysis

Six milliliter of lithium heparin anticoagulated blood was collected by jugular venipuncture with a butterfly catheter for AA metabolite assays. Blood tubes were placed on ice immediately after collection, and plasma separated within 1 hour of collection. All plasma samples were stored at −80°C until time of analysis.

Plasma eicosanoid concentrations were measured by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS)⁵ as previously described.26, 27, 28 Dog plasma (200 μL) was added to 700 μL of buffer (0.1 m NaH₂PO₄, 0.9% NaCl, 2.5 mm deferoxamine, pH 5) plus 100 μL of 0.1% butylated hydroxytoluene, then spiked with a deuterated internal standard (0.5 ng/μL × 10 μL). The samples were extracted by the Bligh‐Dyer technique, dried under a stream of nitrogen, and reconstituted in 100‐μL ethanol. A binary system set at a flow rate of 0.3 mL/min executed a gradient elution with 8.3 mm acetic acid, pH 5.7 with ammonium hydroxide (mobile phase A), and acetonitrile:2‐propanol (50 : 50) (mobile phase B) as follows: 3 minutes hold at 15% B, 10 minutes linear to 55% B, 15 minutes linear to 80% B, 5 minutes wash at 100% B, 7 minutes re‐equilibration at 15% B on a Zorbax SB‐C18 Narrow Bore column (2.1 × 100 mm, 5 micron) with a corresponding guard column at 40°C. The injection volume was 20 μL. Analysis was performed using multiple reaction monitoring (MRM). Individual calibration curves were generated for each analyte, and sample concentrations quantified by calculations from an external standard curve ranging from 0 to 2 ng/μL.

Metabolites measured included EET and DHET isomers, leukotrienes (B4, C4, D4, E4), lipoxin A4, prostaglandins (F2‐α, E2, and D2), thromboxane B2, 8‐iso‐prostaglandin F2‐α, hydroxyoctadecadienoic acids (9S‐ and 13(S)Hode), and hydroxyeicosatetraenoic acids (5(S)‐, 8(S)‐, 9(S)‐, 11(S)‐, 12(S)‐, 15(S)‐, and 20(S) HETE). A positive control standard solution was prepared containing all analytes at a concentration of 1 ng/μL in ethanol. Calibration standards were prepared at a range of 0.0–2.0 ng/μL. Similarly, a mixed deuterated internal standard was prepared at 0.5 ng/μL in ethanol, and was spiked into both samples and standards for a final concentration of 0.05 ng/μL. The limit of quantitation was 0.05 pmol/mL. Recovery based on spiking with deuterated arachidonic acid in plasma was 88%. Recovery for other metabolites in the internal standard mixture were as follows: TXB2‐d4 60.55%, PGF2α‐d4 97.97%, PGD2‐d4 84.42%, LTB4‐d4 66.7%, 5(S)HETE‐d8 66.36%, 13(S)Hode‐d4 70.36%, 5,6DHET‐d11 64.84%, and 5,6EET‐d11 88.33%. Intra‐assay reproducibility coefficient of variations ranged from 7.1 to 11.7% and interassay reproducibility ranged from 11.4 to 15.7%.