Analysis of lipoxygenase kinetics by high-performance liquid chromatography with a polymer column

Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio- and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX-catalyzed oxidation of mixtures of lipids containing linoleate, using high-performance liquid chromatography (HPLC) and a light-scattering detector (LSD). Previous literature suggested that reversed-phase HPLC with silica-based columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed-phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one-tenth the rate of linoleic acid. The diacylglycerol, 1,3-dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono- and dihydroperoxides as well as other unidentified products.