An HPLC method forin vivo quantitation of oxygen free radicals using spin and chemical traps in biological systems

Rao, P. Syamasundar; Weinstein, Gregory S.; Rujikarn, Nisa; LuberJr., J. M.; Tyras, Denis H.

doi:10.1007/bf02270443

Cited by 9 publications

(1 citation statement)

References 5 publications

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“…Previous attempts to detect free radical intermediates were focused intensively on the use of combinations of detectors and/or techniques in which EPR played a central role, such as the determination of chemically generated phenyl radicals, hydroxyl radical, and metal-catalyzed hydrazine-derived pentyl and phenyl radicals by liquid chromatography/electron paramagnetic resonance/mass spectrometry (LC/EPR/MS), tandem LC/EPR–LC/MS with dual spin trapping, and HPLC/ECD/UV/ESR/MS, respectively. − In biological settings, 5,5-dimethyl-1-pyrroline N -oxide (DMPO) and N - tert -butyl-α-phenylnitrone (PBN) are the two most commonly used spin traps for detecting free radicals. Only few studies not involving EPR spectroscopy employed DMPO, to detect oxygen- or sulfur-centered radicals, − whereas the majority of studies utilized open chain nitrones, viz. PBN and α-(4-pyridyl-1-oxide)- N - tert -butylnitrone (4-POBN), to determine carbon-centered radicals. − Nevertheless, adequate detection and quantification of spin-trapped radicals was not attainable.…”

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confidence: 99%

Post-Trapping Derivatization of Radical-Derived EPR-Silent Adducts: Application to Free Radical Detection by HPLC/UV in Chemical, Biochemical, and Biological Systems and Comparison with EPR Spectroscopy

Michail

Siraki

2012

Anal. Chem.

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Free radicals are conventionally detected by electron paramagnetic resonance (EPR) spectroscopy after being trapped as spin adducts. Albeit this technique has demonstrated utmost efficacy in studying free radicals, its application to biological settings is intrinsically hampered by the inevitable bioreduction of radical-derived paramagnetic adducts. Herein, we describe a reliable technique to detect and quantify free radical metabolites, wherein reduced alkyl- and phenyl-5,5-dimethyl-1-pyrroline N-oxide (DMPO) adducts are converted into ultrastable N-naphthoate esters. To mimic the ubiquitous in vivo microenvironment, bioreductants, exogenous thiols, and sodium borohydride were studied. Nitroxyl reduction was confirmed using EPR and triphenyltetrazolium chloride. The formation of the N-naphthoyloxy derivatives was established by liquid chromatography/mass spectrometry (LC/MS). The derivatives were chromatographed using a binary eluent. HPLC and internal standards were synthesized using Grignard addition. The labeled DMPO adduct is (1) fluorescent, (2) stable as opposed to nitroxyl radical adducts, (3) biologically relevant, and (4) excellently chromatographed. Applications encompassed chemical, biochemical, and biological model systems generating C-centered radicals. Different levels of phenyl radicals produced in situ from whole blood were successfully determined. The method is readily applicable to the detection of hydroxyl radical. Analogously, DMPO, the spin trap, could be detected with extreme sensitivity suitable for in vivo applications. The developed method proved to be a viable alternative to EPR, where for the first time the reductive loss of paramagnetic signals of DMPO-trapped free radicals is transformed into fluorescence emission. We believe the proposed methodology could represent a valuable tool to probe free radical metabolites in vivo using DMPO, the least toxic spin trap.

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confidence: 99%

Post-Trapping Derivatization of Radical-Derived EPR-Silent Adducts: Application to Free Radical Detection by HPLC/UV in Chemical, Biochemical, and Biological Systems and Comparison with EPR Spectroscopy

Michail

Siraki

2012

Anal. Chem.

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Simple and rapid catalytic spectrophotometric determination of superoxide anion radical and superoxide dismutase activity in natural medical vegetables using phenol as the substrate for horseradish peroxidase

Tang

Wang

2004

Analytical and Bioanalytical Chemistry

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The coupling reaction of 4-aminoantipyrine (4-AAP) with phenol using the superoxide anion radical (O2-*) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, O2-* produced by irradiating vitamin B2 (VB2) was spectrophotometrically determined at 510 nm. Under the optimum experimental conditions, the relationship between A510 and O2-* concentration was linear in the range 9.14x10(-6)-1.2x10(-4) mol L(-1). The detection limit was determined to be 1.37x10(-6) mol L(-1). A possible reaction mechanism was discussed. The effect of interferences and surfactants on the determination of O2-* was also investigated. The proposed method was applied to determine superoxide dismutase activity in garlic, scallion, and onion with satisfactory results.

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Noninvasive measures of oxidative stress status in humans

Pryor

Godber

1991

Free Radical Biology and Medicine

207

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An HPLC method forin vivo quantitation of oxygen free radicals using spin and chemical traps in biological systems

Cited by 9 publications

References 5 publications

Post-Trapping Derivatization of Radical-Derived EPR-Silent Adducts: Application to Free Radical Detection by HPLC/UV in Chemical, Biochemical, and Biological Systems and Comparison with EPR Spectroscopy

Post-Trapping Derivatization of Radical-Derived EPR-Silent Adducts: Application to Free Radical Detection by HPLC/UV in Chemical, Biochemical, and Biological Systems and Comparison with EPR Spectroscopy

Simple and rapid catalytic spectrophotometric determination of superoxide anion radical and superoxide dismutase activity in natural medical vegetables using phenol as the substrate for horseradish peroxidase

Noninvasive measures of oxidative stress status in humans

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