Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition.
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).
Koshiishi
Ichiro
I
Nihon Pharmaceutical University, Ina-machi, Kita-Adachi-gun, Saitama 362-0806, Japan. ikoshi@nichiyaku.ac.jp
Tsuchida
Kazunori
K
Takajo
Tokuko
T
Komatsu
Makiko
M
D016718:Arachidonic Acid / Q000737:chemistry; D001391:Azo Compounds / Q000737:chemistry; D001671:Biochemistry / Q000295:instrumentation* / Q000379:methods; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004563:Electrochemistry; D005609:Free Radicals / Q000737:chemistry*; D006358:Hot Temperature; D019787:Linoleic Acid / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008084:Lipoxygenase / Q000378:metabolism; D013058:Mass Spectrometry; D008956:Models, Chemical; D009570:Nitriles / Q000737:chemistry; D009589:Nitrogen Oxides; D000442:Octanols / Q000737:chemistry; D012680:Sensitivity and Specificity; D013113:Spin Labels; D019097:Spin Trapping; D013696:Temperature; D013997:Time Factors; D014867:Water / Q000737:chemistry
D001391:Azo Compounds; D003497:Cyclic N-Oxides; D005609:Free Radicals; D008055:Lipids; D009570:Nitriles; D009589:Nitrogen Oxides; D000442:Octanols; D013113:Spin Labels; C055792:2,2'-azobis(2,4-dimethylvaleronitrile); D014867:Water; D016718:Arachidonic Acid; C047253:3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxyl; D019787:Linoleic Acid; C101084:lipoxygenase L-1; D008084:Lipoxygenase; C039900:nitroxyl