Preparation and purification of soybean lipoxygenase-derived unsaturated hydroperoxy and hydroxy fatty acids and determination of molar absorptivities of hydroxy fatty acids

Abstract

A method was developed for the preparation and purification of polyunsaturated hydroperoxy and hydroxy fatty acids from 18:2n-6, 18:3n-6, 18:3n-3, 20:3n-6, 20:3n-3, and 20:4n-6 with soybean lipoxygenase. This method involved incubation of unsaturated fatty acids in Tris-HCl buffer with soybean lipoxygenase and extraction of reaction products on C18 solid-phase columns. The yields of conversion of fatty acid substrate to oxygenated products were in all cases greater than or equal to 95%. C18 solid-phase extracted reaction products were purified by C18 HPLC, yielding 3-4 mg of products with purities of greater than or equal to 98%. Purified polyunsaturated hydroxy fatty acids were characterized by HPLC and mass spectral analysis and showed absorption spectra with maximum absorption occurring between 234.5 and 237.5 nm due to the presence of a conjugated diene function. Polyunsaturated hydroxy fatty acid potassium salts were converted to (4-bromobenzoyl)methyl esters by reaction with 2,4'-dibromoacetophenone in the presence of 18-crown-6. Molar absorptivities for the conjugated diene functions were determined by relating their absorbance to the absorbance contributed by the 4-bromobenzoyl chromophore and its molar absorption intensity of 17.6 X 10(3) (M.cm)-1. The molar absorptivities determined for 13-OH-9,11-18:2, 13-OH-6,9,11-18:3, 13-OH-9,11,15-18:3, 15-OH-11,13-20:2, 15-OH-8,11,13-20:3, 15-OH-11,13,17-20:3, and 15-OH-5,8,11,13-20:4 ranged from 23.2 x 10(3) to 24.6 x 10(3) (M.cm)-1. The molar absorbtivity values of 18.8 x 10(3) and 20.3 x 10(3) (M.cm)-1 determined for commercial, chemically synthesized 12-OH-5,8,10,14-20:4 [12-(S)-hydroxyeicosa-5,8,10,14-tetraenoic acid (12(S)-HETE)] and 5-OH-6,8,11,14-20:4 ((+/-)-5-HETE) were lower than that of soybean lipoxygenase-derived 15-OH-5,8,11,13-20:4 (15-HETE), which exhibited a molar absorptivity of 23.3 x 10(3) (M.cm)-1. The molar absorptivity values determined for 5-, 12-, and 15-HETE are considerably lower than 30.5 x 10(3) (M.cm)-1, which was reported previously.