Abstract
This chapter describes the production and the radioimmunoassay (RIA) for 12-hydroxyeicosatetraenoic acid (12-HETE) and presents two high-performance liquid chromatography (HPLC) procedures to separate mono- and di-hydroxy acids from other arachidonic acid metabolites. In RIA a double-antibody technique is used to separate free antigen from antibody-bound antigens in solution. In HPLC the resolution of cyclooxygenase products and hydroxyeicosatetraenoic acids, two Waters Model 6000A pumps, Model 660 solvent programmer, Model U6K injector, and a Waters μ-Porasil column is used. The solvent system consisted of hexane-acetic acid eluted isocratically for 11 min followed by linear gradient from solvent A to chloroform-methanol-acetic acid for the total time of 76 min. Final elution with 100% solvent B is run isocratically for total time of 100 min. The flow rate for the entire program was set at 1 ml/min per tube. Resolution of lipoxygenase products 12-HETE, 5-HETE, and LTB 4 , as well as several cyclooxygenase products are carried out by this normal-phase HPLC. It is suggested that reversed-phase system does not separate the mono-HETEs; their resolution is achieved by normal-phase HPLC.
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