Quantitative Analysis of Arachidonic Acid Release

Quantification of arachidonic acid release from cellular membranes was performed as previously described [31 (link), 32 (link)]. Briefly, freshly isolated PMNL (10⁷ cells/ml, in HBSS containing 1.6 mM CaCl₂ and 0.1% BSA) were incubated with adenosine deaminase (0.3 U/ml) and test compounds for 5 min at 37°C. Stimulation was initiated with the addition of 1 μM thapsigargin, followed by incubation at 37°C for 5 min. The reactions were stopped with the addition of two volumes of cold MeOH and 300 ng of arachidonic acid-d₈ (Cayman Chemicals) as internal standard. The samples were stored at −20°C overnight and then centrifuged the next day at 1000 g for 10 min. The supernatants were diluted with four volumes of acidified water (0.1%) then processed on an octadecyl (C18) column. Samples were eluted with the addition of 3 ml of MeOH and dried under nitrogen. Pentafluorobenzylesters were prepared with the addition 50 μl N,N-diisopropylethylamine (20% in acetonitrile) and 50 μl of 2,3,4,5,6-pentafluorobenzylesters (20% in acetonitrile). Samples were heated for 40 min at 40°C, then dried under nitrogen and resuspended in 100 μl of hexane. Samples were quantified by negative ion chemical ionization gas chromatography/mass spectrometry using TraceGC ultra column (Thermo) and a Polaris Q mass spectrometer (Thermo).