Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

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 spintrapping. 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 (Cm ⌬ P) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting Cm ⌬ P-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 ؇ 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). Peroxidation of polyunsaturated fatty acids, which are components of cellular membrane and lipoproteins, leads to vital damage to several types of cells. Reactive oxygen/nitrogen species (1), such as hydroxyl radical, hypochlorous acid (2-4), peroxynitrite (5, 6), and singlet oxygen molecule (7-9) are involved in the peroxidation of polyunsaturated fatty acids in the inflammatory lesions. On the other hand, lipoxygenases are speculated to be involved in the peroxidation of polyunsaturated fatty acids in the atheromatous plaque (10). Commonly, lipid alkyl radicals seem to be necessarily produced as an intermediate in these reactions.To detect labile free radicals via ESR, spin-trapping techniques have been established, in which unstable radicals react with spin-trapping agents to form relatively stable radical adducts (11). To date, nitrone spin-trapping agents, including ␣ -[4-pyridyl-1-oxide] N -tert-butyl nitrone and 5,5 Ј -dimethyl-1-pyroline-N -oxide, have been used to detect these lipid-derived radicals (12-17). Furthermore, Mason and coworkers (13-15) established an HPLC/ESR system to identify the resulting radical adducts. However, the trapping efficiency of these nitrone compounds toward lipid alkyl radicals is comparatively low. Consequently, these spin-trapping agents are not applicable to the quantification of lipid alkyl radicals. Five-or six-membered cyclic nitroxyl radicals are relatively stable. Nitroxyl radicals appeared to possess an ability to scavenge carbon-centered radicals (18). This special character of nitroxyl radicals makes it possible to quantitatively trap carbon-centered radicals. Johnson, Caron, and Blough (19) used hydrophilic 3-aminomethyl-2,2,5,5-tetramethylpyrrolidine-N -oxyl to evaluate carbon-centered radical generation: in this method, the adducts were fluorometrically detected after derivatization of amino groups by fluorescamine.The nitroxyl radical-carbon-centered radi...

Feedback activation of ferrous 5-lipoxygenase during leukotriene synthesis by coexisting linoleic acid

“…FA-derived carbon-centered radical-CmDP adducts are commonly cleaved into a hydroxylamine form of CmDP (molecular mass, 184) and residual cationic FA derivatives (12). Therefore, it is possible to selectively detect adducts by LC-MS/MS with PIS for m/z 185.…”

“…Subsequently, the ferrous lipoxygenase should be reoxidized to ferric one by cycling hydroperoxides through a pseudoperoxidase reaction, generating a FA alkoxyl radical, which may be intramolecularly rearranged by the addition of a double bond to form the FA epoxyallyl radical (carbon-centered radical) (16,17). Nitroxyl radical can trap FA epoxyallyl radical to the FA epoxyallyl radicalnitroxyl radical adduct through radical-radical conjunction (12). Namely, each revolution of the redox cycle reaction between ferrous lipoxygenase and ferric one produces one FA allyl radical-nitroxyl radical adduct and one FA epoxyallyl radical-nitroxyl radical adduct.…”

“…In our previous reports (11,12), we established a novel method for converting ferric lipoxygenase into its ferrous form. When soybean ferric lipoxygenase was incubated with PUFAs in the presence of nitroxyl radical, which selectively traps carbon-centered radicals, FA allyl radical on the ferrous lipoxygenase (13-15) was trapped by nitroxyl radical, generating ferrous lipoxygenase.…”

Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

“…In our recent study (12), we demonstrated that the nitroxyl radical, CmDP: M.W., 183, stoichiometrically trapped lipid-derived carbon-centered radicals, including lipid allyl radicals and lipid epoxyallyl radicals, in the linoleate/lipoxygenase system through radical-radical conjunction. We confirmed that the presence of the nitroxyl radical did not prevent the lipoxygenase reaction as follows: when 1.0 mM linoleate was incubated with 0.1 mM lipoxygenase in the presence or absence of CmDP (1.0 mM), there is no significant difference in the initial rate of hydroperoxide generation between the two systems (approximately 2.4 mM/sec).…”

“…For this purpose, we established a novel method for the detection of fatty acid-derived carbon-centered radicals via the combination of the nitroxyl radical spin-trapping technique with the precursor ion-scanning (PIS) technique in liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The nitroxyl radical, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDP), was employed to selectively trap carbon-centered radicals, although the resulting adducts are comparatively thermolabile (12).…”