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

Abstract

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.

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System

“…Recent advances in the development of stabilized/protected nitrone spin traps bound to cyclodextran or calixpyrrole groups offer promise in addressing this limitation. 32,33 Alternative approaches for the detection of spin trap adducts or their metabolites include high-performance liquid chromatography (HPLC) and mass spectroscopy, 34,35 high-field nuclear magnetic resonance, 36 and antibody-immune based detection. 37,38 …”

Engineering a Therapy‐Induced “Immunogenic Cancer Cell Death” Amplifier to Boost Systemic Tumor Elimination

“…NIR photothermal heating of Cu‐LDH nanocarrier showed very limited effect on the catalytic generation of hydroxyl radical (•OH, the most toxic ROS) from H 2 O 2 , as demonstrated by classic 1:2:2:1 •OH signal in DMPO assay (Figure S4, Supporting Information). [ 18 ] However, while combining the NIR photothermal heating to FeOOH Fenton agents by loading on the Cu‐LDH nanocarrier, •OH stimulation of FeOOH (Figure S4, Supporting Information) was induced remarkably (Figure 1d), indicating the significant role of photothermal heating in Fenton reaction catalyzed by FeOOH nanodots in pH 7.4 buffer and confirming the pivotal role of moderately increased temperature in Fenton reaction. [ 19 ] In pH 5.0 buffer, Fenton reaction was enhanced even more remarkably than that in pH 7.4.…”

Hydrogen Peroxide Generation from Hydrated Protein Drink Mixes

“…As can be seen in Figure and A, the highest levels of ROS occur in protein drink mixes that contain a substantial amount of ascorbic acid. Michail and Siraki () also demonstrated the ability of several reducing agents (including ascorbate) to rapidly convert DMPO radical adducts into EPR‐silent species. Thus the absence of a DEPMPO‐adduct EPR signal is likely due to the high concentrations of ascorbate included in the commercial protein beverages.…”