LipidomicsReveals Dramatic Physiological KineticIsotope Effects during the Enzymatic Oxygenation of PolyunsaturatedFatty Acids Ex Vivo

摘要

Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-cis,cis-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AAisotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneouslyquantify native and deuterated lipid products, we were able to demonstratethat the magnitude of the PKIE measured in macrophages for COX andLOX oxygenation of AA is similar to KIEs determined in previous reportsusing the AA isotopologue deuterated at carbon 13 (C13). However,for the first time we show that increasing the number of deuteratedbis-allylic carbons to include both C10 and C13 leads to a massiveincrease in the PKIE for COX oxygenation of AA. We provide evidencethat hydrogen(s) present at C10 of AA play a critical role in thecatalysis of prostaglandin and thromboxane synthesis. Furthermore,we discovered that deuteration of C10 promotes the formation of theresolving lipid mediator lipoxin B4, likely by interfering with AAcyclization and shunting AA to the LOX pathway under physiologicalconditions.