Acute hypercapnia/ischemia alters the esterification of arachidonic acid and docosahexaenoic acid epoxide metabolites in rat brain neutral lipids

Authors: OTOKI, YURIKA - University Of California, Davis; METHEREL, ADAM - University Of Toronto; PEDERSEN, THERESA - University Of California, Davis; YANG, JUN - University Of California, Davis; HAMMOCK, BRUCE - University Of California, Davis; BAZINET, RICHARD - University Of Toronto; Newman, John; TAHA, AMEER - University Of California, Davis

Submitted to: Lipids
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2019
Publication Date: 11/6/2019
Citation: Otoki, Y., Metherel, A.H., Pedersen, T., Yang, J., Hammock, B.D., Bazinet, R.P., Newman, J.W., Taha, A.Y. 2019. Acute hypercapnia/ischemia alters the esterification of arachidonic acid and docosahexaenoic acid epoxide metabolites in rat brain neutral lipids. Lipids. 55(1):7-22. https://doi.org/10.1002/lipd.12197.
DOI: https://doi.org/10.1002/lipd.12197

Interpretive Summary: Oxylipins are oxygenated metabolites of polyunsaturated fatty acids, many of which have important biological properties which include blood flow regulation and inflammation mediation in the brain. In the brain, over 90% of these oxylipin fatty acids are chemically incorporated in to complex lipids through ester bonds. However, the type of lipids they are esterified to, and the significance of this esterification process is not known. In the present study, we validated a method to separate esterified lipids and oxylipins using columns packed with an aminopropyl resin, a compound with a weak positive charge (i.e. a cation) bound to particles by a small hydrophobic linker. We then used the method to measure the distribution of esterified oxylipins within phospholipids and neutral lipids (i.e. triacylglycerol and cholesteryl ester) in rats euthanized my two distinct, but approved methods: 1) head-focused microwave fixation (instant termination controls); 2) CO2-induced asphyxiation (delayed termination treatment). We hypothesized that oxylipin esterification into these lipid pools is an active process, and that the distribution of these lipids in the brain would be effected by the mode of sacrifice. Total lipids were extracted from control (n=8) and CO2-asphyxiated (n=8) rat brain, and separated on aminopropyl solid phase extraction cartridges to yield phospholipids and neutral lipids. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced solid phase extraction resins, and analyzed with ultra-high pressure liquid chromatography coupled to tandem mass spectrometry. Phospholipid-bound oxylipins were 9 times more abundant than oxylipins within neutral lipids. Three epoxides of arachidonic acid were increased and one epoxide of docosahexaenoic acid was decreased in neutral lipids of CO2-asphyxiated rats compared to control rats. No differences in phospholipid-bound oxylipins were detected. This study demonstrates that the majority of brain oxylipins are esterified to phospholipid pools, and that CO2-induced asphyxiation selectively alters the concentration of fatty acid epoxides in neutral lipids, suggesting the presence of an active turnover process observable in viable postmortem tissues, which regulates oxylipin esterification in brain. Targeting the esterification of oxylipins that reduce inflammation and increase cerebral blood flow might protect the brain against ischemic injury.

Technical Abstract: In the brain, over 90% of oxylipins are esterified to complex lipids. However, the type of lipids they are esterified to, and the significance of this esterification process is not known. In the present study, we 1) validated an aminopropyl column method for separating esterified lipids, and 2) applied the method to quantify the distribution of esterified oxylipins within phospholipids and neutral lipids (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) and CO2-induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools is an active process. Total lipids were extracted from control (n=8) and CO2-asphyxiated (n=8) rat brain, and separated on aminopropyl solid phase extraction cartridges to yield phospholipids and neutral lipids. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced solid phase extraction, and analyzed with ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Phospholipid-bound oxylipins were 9 times more abundant than oxylipins within neutral lipids. Three epoxides of arachidonic acid were significantly increased and one epoxide of docosahexaenoic acid was significantly decreased in neutral lipids of CO2-asphyxiated rats compared to control rats. There were no significant differences in phospholipid-bound oxylipins. This study demonstrates that the majority of brain oxylipins are esterified to phospholipid pools and that hypercapnia / ischemia selectively alters the concentration of fatty acid epoxides within neutral lipids, reflecting an active turnover process regulating oxylipin esterification in brain. Targeting the esterification of pro-resolving arachidonic acid epoxides might protect the brain against ischemic injury.