Abstract
Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).
Highlights
Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify
Lipid-derived radicals generated in the polyunsaturated fatty acid/lipoxygenase system were trapped with nitroxyl radicals as follows
Twenty microliters of 2 mM polyunsaturated fatty acid emulsion in 0.1 M phosphate buffer containing 2% ethanol was mixed with 10 l of 4 mM nitroxyl radical in 0.1 M phosphate buffer and 10 l of 4 M soybean lipoxygenase-1 in 0.1 M phosphate buffer in a glass vial tube with screw cap, and the solution was left to stand at room temperature (25–28ЊC) for 10 min
Summary
Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. We established a method for the determination of nitroxyl radical-lipid-derived carbon-centered radical adducts by the combination of HPLC and thermal decomposition. Lipid-derived radicals generated in the polyunsaturated fatty acid/lipoxygenase system were trapped with nitroxyl radicals as follows.
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