Quantification of F2-isoprostanes as a reliable index of oxidative stress in vivo using gas chromatography–mass spectrometry (GC-MS) method

Abstract

Free radical-induced lipid peroxidation has been implicated in a number of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. F2-Isoprostanes (IsoPs) are isomers of prostaglandin PGF2α that are generated in vivo from the free radical-initiated peroxidation of arachidonic acid independent of cyclooxygenase enzymes. Since the discovery of the IsoPs in the early 1990s, a large body of evidence has been accumulated to indicate that quantification of these F2-IsoPs represents the most reliable biomarker to assess oxidative stress in vivo. A variety of analytical approaches have been developed for the quantification of these novel compounds; these methods include mass spectrometry (MS) detection coupled to gas chromatography (GC) or liquid chromatography (LC) separation, and detection using immunological approaches. This article summarizes our current methodology to quantify F2-IsoPs in biological fluids and tissues using GC-MS. This method includes solid phase extraction (SPE), thin layer chromatography (TLC) purification, chemical derivatization, and MS detection using negative ion chemical ionization (NICI) coupled with GC. The protocol described herein has been optimized and validated to provide the best sensitivity and selectivity for quantification of F2-IsoPs from a variety of biological sources.

Про-антиокcидантна рівновага в печінці та м’язах cтерляді за дії штучного гіпобіозу й анестезії

“…Гальмування ж активноcті ПОЛ може бути причиною порушення cинтезу деяких біологічно активних речовин в організмі та cвідченням зниження фагоцитозу, як важливої ланки імунної cиcтеми (Sunderman, Zaharia, 1988;Tushnytska et al, 2006). Пероксидне окиснення ліпідів на cьогодні вважаєтьcя однією з оcновних причин пошкодження і загибелі клітини внаcлідок дії активованих форм окcигену (АФО) (Kirichek, Zubova, 2004;Liu et al, 2009;Nekrasov, 2012). До антиокcидантів, які перешкоджають утворенню радикальних форм окcигену (АФО) і тим cамим запобігають відповідним патологічним впливам на cтруктури тканин організму, відносяться різні водорозчинні і гідрофобні cполуки, дія яких у кінцевому результаті гальмує утворення АФО і зменшує концентрацію продуктів перокcидного окиcнення (Baraboy, 2006;Kostiuk, 2014).…”

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids

“…The reactions were quenched with 2.0 ml of CH 3 OH containing butylated hydroxytoluene (0.005%, w/v) and 1.0 ml of aqueous KOH (15%, w/v). The mixtures were then mixed with a vortex device, purged with Ar, and incubated at 37°C for 30 min to hydrolyze the oxidized LPCs and release the oxidized fatty acids ( 35 ). The mixtures were acidifi ed to pH 2 with HCl, and the oxidized fatty acids were extracted into CH 2 Cl 2 .…”

“…Oxidation products of FFAs and LPCs were extracted as described above. The products were derivatized with 20 l of 10% (v/v) N , N -diisopropylethylamine in CH 3 CN and 40 l of 10% (v/v) pFBB in CH 3 CN at 37°C for 20 min ( 35 ). pFBB-derivatized samples were dried under a N 2 stream and then derivatized with 20 l of BSTFA and 7 l of dry dimethylformamide at 37°C for 20 min ( 35 ) and analyzed by GC/MS in the chemical ionization mode.…”

“…The products were derivatized with 20 l of 10% (v/v) N , N -diisopropylethylamine in CH 3 CN and 40 l of 10% (v/v) pFBB in CH 3 CN at 37°C for 20 min ( 35 ). pFBB-derivatized samples were dried under a N 2 stream and then derivatized with 20 l of BSTFA and 7 l of dry dimethylformamide at 37°C for 20 min ( 35 ) and analyzed by GC/MS in the chemical ionization mode. FFA C17:0 and C19:0 standards were used to LC/MS is one of the most widely used analytical methods for metabolomic analysis and has proved to be a powerful approach in substrate searches (15)(16)(17)(18).…”

A Simple and Highly Sensitive Quantitation of Eicosanoids in Biological Samples Using Nano-flow Liquid Chromatography/ Mass Spectrometry

“…In the past, eicosanoids were mainly analyzed by gas chromatography-mass spectrometry (GC/MS), 8,9 enzyme-linked immunosorbent assays (ELISA) 10,11 and LC/MS/MS. The drawback of ELISA is being able to detect only a single analyte in a single set of analysis.…”