N-Formyl-L-methionyl-L-leucyl-L-phenylalanine → (5Z,8Z,11Z,14Z)-16-Hydroxy-5,8,11,14-eicosatetraenoic acid

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Reaktionsschritte

16(R)-HETE (0.01-1.0 μM) was preincubated for 10 minutes with the neutrophil suspension (3×106/ml) prior to the addition of formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were then incubated for an additional 10 minutes with 5×10−7M fMLP. All incubations took place at 37° C. Plasma samples for the measurement of LTB4 were collected by centrifugation of the whole blood samples at 1200 rpm for 15 minutes. Samples were stored at 70° C. until analyzed. The plasma was then acidified to pH3 with 1M Hcl. The plasma was then placed on C2 reverse phase columns (Amprep for Amersham Corp., Arlington Heights, Ill.) to extract LTB4. LTB4 was then measured using the Biotrak LTB4 enzyme immunoassay system (Amersham Corp., Arlington Heights, Ill.). All standards and samples were performed in duplicate. Following incubation, all samples were read at 450 nm and the LTB4 concentration in each sample was determined following the generation of a standard curve. LTB4 concentration was expressed as picograms LTB4 per 1000 neutrophils.

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