Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.10.3.3 (ascorbate oxidase)
 778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vivo spin trapping of radical metabolites has become a promising tool in understanding and predicting toxicities caused by different xenobiotics. However, in biological systems radical adducts can be reduced to electron paramagnetic resonance (EPR)-silent hydroxylamines. To overcome this difficulty, different procedures for reoxidation of the reduced radical adducts were systematically investigated and some metabolic inhibitors of nitroxide reduction were tested. As a test system, carbon tetrachloride (CCl4), a known hepatotoxic substance, was used. CCl4 is metabolized by liver to .CCl3 and, in the presence of the spin trap phenyl N-t-butylnitrone (PBN), forms the PBN/.CCl3 and PBN/.CO2- radical adducts. These radical adducts were measured in the bile using electron paramagnetic resonance after administration of CCl4 and PBN to the rat. We have shown that these radical adducts were reduced to the corresponding hydroxylamines in vivo, since immediately after the collection of bile only traces of the radical adducts could be detected, but after oxidation by different procedures such as bubbling with oxygen, addition of mild oxidant potassium ferricyanide or autoxidation the EPR spectra intensity increases, indicating that the hydroxylamines had been re-oxidized back to nitroxides. The collection of bile into plastic Eppendorf tubes containing the sulfhydryl reagent N-ethylmaleimide (NEM) or the enzyme ascorbate oxidase did not increase the intensity of the spectra significantly, demonstrating that neither reduction by reduced glutathione (GSH) nor ascorbic acid occurred ex vivo. However in the presence of NEM faster re-oxidation was observed. A new radical adduct that was not observed previously in any in vivo experiment and which exhibited 13C hyperfine coupling was detected when the rats were injected with 13CCl4. We have proven that this is the same adduct detected previously in vitro in microsomal incubations of CCl4, PBN, GSH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). As a general rule, we have shown that a variety of oxidation procedures should be tried to detect the different radical adducts which are otherwise not observable due to the in vivo reduction of radical adducts.
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PMID:Inhibition of radical adduct reduction and reoxidation of the corresponding hydroxylamines in in vivo spin trapping of carbon tetrachloride-derived radicals. 132 96

Rats were treated with CCl4 and the spin trapping agent alpha-phenyl-N-t-butyl nitrone (PBN), followed by ESR investigations on samples of heparinized blood. The major signal detected was the ascorbate semidione radical, but smaller concentrations of the carbon dioxide radical anion spin adduct of PBN could also be detected. The ESR signals were more intense when experiments were conducted with plasma, rather than blood. The spin adducts detected were not associated with the red blood cells, and their apparent concentrations increased when the cells were removed by centrifugation. The addition of ascorbate oxidase to the samples markedly diminished the intensity of the ascorbate semidione radical. When plasma samples from CCl4-treated rats were extracted into toluene, the ESR spectrum of the trichloromethyl adduct of PBN was observed in the extract. Because the spectrum of this adduct was not observed in direct ESR studies of plasma, it is possible that immobilization occurred in the presence of plasma proteins. Evidence to support this hypothesis was developed by adding bovine serum albumin (BSA) to an aqueous solution of the trichloromethyl radical adduct of PBN. As the BSA concentration was increased, the intensity of the ESR spectrum was markedly diminished, and displayed features of an immobilized adduct.
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PMID:Detection of spin adducts in blood after administration of carbon tetrachloride to rats. 164 21