Fluorescence Detection of Free Radicals by Nitroxide Scavenging

The fluorescence quantum yield of 4-(1-napthoyloxy)-2,2,6,6-tetramethylpiperidine-l-oxyl (I) in acetonitrile and hexane is 55 and 30-fold lower, respectively, than those of diamagnetic analogs. Experiments described herein demonstrate that this property makes possible the fluorescence detection of radical scavenging reactions in which the paramagnetic nitroxide-substituted naphthalene is converted to a diamagnetic N-alkoxy derivative. 2-Cyanopropyl free radicals were generated by the thermal decomposition of azobisisobutyronitrile (AIBN) in cyclohexane or in acetonitrile containing 1. The fluorescence intensity of the sample increased proportionally to the decrease in its ESR signal intensity, indicating the conversion of the paramagnetic nitroxide to the diamagnetic product. The linear relationship between the increase in fluorescence intensity and decrease in ESR signal intensity shows that the changes in the fluorescence intensity can serve as a sensitive means for optically detecting radicals.     

Effects of vitamin C,vitamin E,zinc gluconate,and selenomethionine supplementation on muscle function and oxidative stress biomarkers in patients with facioscapulohumeral dystrophy: A double-blind randomized controlled clinical trial

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease characterized by progressive weakness and atrophy of specific skeletal muscles. As growing evidence suggests that oxidative stress may contribute to FSHD pathology, antioxidants that might modulate or delay oxidative insults could help in maintaining FSHD muscle function. Our primary objective was to test whether oral administration of vitamin C, vitamin E, zinc gluconate, and selenomethionine could improve the physical performance of patients with FSHD. Adult patients with FSHD (n=53) were enrolled at Montpellier University Hospital (France) in a randomized, double-blind, placebo-controlled pilot clinical trial. Patients were randomly assigned to receive 500 mg vitamin C, 400 mg vitamin E, 25 mg zinc gluconate and 200 μg selenomethionine (n=26), or matching placebo (n=27) once a day for 17 weeks. Primary outcomes were changes in the two-minute walking test (2-MWT), maximal voluntary contraction, and endurance limit time of the dominant and nondominant quadriceps (MVC_QD, MVC_QND, T_limQD, and T_limQND, respectively) after 17 weeks of treatment. Secondary outcomes were changes in the antioxidant status and oxidative stress markers. Although 2-MWT, MVC_Q, and T_limQ were all significantly improved in the supplemented group at the end of the treatment compared to baseline, only MVC_Q and T_limQ variations were significantly different between groups (MVC_QD: P=0.011; MVC_QND: P=0.004; T_limQD: P=0.028; T_limQND: P=0.011). Similarly, the vitamin C (P<0.001), vitamin E as α-tocopherol (P<0.001), vitamin C/vitamin E ratio (P=0.017), vitamin E γ/α ratio (P=0.022) and lipid peroxides (P<0.001) variations were significantly different between groups. In conclusion, vitamin E, vitamin C, zinc, and selenium supplementation has no significant effect on the 2-MWT, but improves MVC_Q and T_limQ of both quadriceps by enhancing the antioxidant defenses and reducing oxidative stress. This trial was registered at clinicaltrials.gov (number: NCT01596803).     

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H₂O₂), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS² mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.