HPLC profiles of products formed by platelet subcellular fractions. Platelet sonicates were centrifuged at 1,500g for 10 minutes and the supernatant centrifuged successively at 10,000gfor 10 minutes and 200,000g for 60 minutes as described in Materials and Methods. The 1,500g supernatant (A) and the 200,000g pellet (B) fractions (equivalent to 2 × 10⁷ platelets/mL) were incubated for 40 minutes at 37°C with 5-HETE (2 μmol/L) in the presence of NADP⁺ (1 mmol/L). The products were analyzed by precolumn extraction/RP-HPLC on a Novapak C₁₈ column (3.9 × 300 mm) using a gradient between water/acetonitrile/acetic acid (50:50:0.02) and water/acetonitrile/acetic acid (40:60:0.02) over 30 minutes, followed by isocratic elution with 60% acetonitrile for a further 10 minutes. PGB₂ (90 ng) was added as an internal standard. The inset to (A) shows the UV spectrum of biologically synthesized 5-oxo-12-HETE, which was identical to that of the chemically synthesized compound.