Hydrolysis of polyhydroxy polyunsaturated fatty acid-glycerophosphocholines by Group IIA, V, and X secretory phospholipases A2

It is widely accepted that unesterified polyunsaturated omega-6 and omega-3 fatty acids (PUFA) are converted through various lipoxygenases, cyclooxygenases, and cytochrome P450 enzymes to a range of oxygenated derivatives (oxylipins), among which the polyhydroxides of unesterified PUFA have recently been recognized as cell signaling molecules with anti-inflammatory and pro-resolving properties, known as specialized pro-resolving mediators (SPMs). This study investigates the mono-, di-, and trihydroxy 16:0/PUFA-GPCs, and the corresponding 16:0/SPM-GPC, in plasma lipoproteins. We describe the isolation and identification of mono-, di-, and trihydroxy AA, EPA, and DHA-GPC in plasma LDL, HDL, HDL3, and acute phase HDL using normal phase LC/ESI-MS, as previously reported. The lipoproteins contained variable amounts of the polyhydroxy-PUFA-GPC (0-10 nmol/mg protein), likely the product of lipid peroxidation and the action of various lipoxygenases and cytochrome P450 enzymes on both free fatty acids and the parent GPCs. Polyhydroxy-PUFA-GPC was hydrolyzed to variable extent (20%-80%) by the different secretory phospholipases A(2) (sPLA(2)s), with Group IIA sPLA(2) showing the lowest and Group X sPLA(2) the highest activity. Surprisingly, the trihydroxy-16:0/PUFA-GPC of APHDL was largely absent, while large amounts of unidentified material had migrated in the free fatty acid elution area. The free fatty acid mass spectra were consistent with that anticipated for branched chain polyhydroxy fatty acids. There was general agreement between the masses determined by LC/ESI-MS for the polyhydroxy PUFA-GPC and the masses calculated for the GPC equivalents of resolvins, protectins, and maresins using the fatty acid structures reported in the literature.

Automated summary

This study investigates the presence of mono-, di-, and trihydroxy PUFA-GPCs in plasma lipoproteins and their hydrolysis by different sPLA(2)s enzymes. It was found that lipoproteins contain varying amounts of polyhydroxy-PUFA-GPC, likely the product of lipid peroxidation and the action of enzymes. Interestingly, the trihydroxy-16:0/PUFA-GPC was largely absent in one type of HDL, while unidentified material was present in the same area. LC/ESI-MS analysis showed agreement between the masses of polyhydroxy PUFA-GPC and the calculated masses for related GPCs.