Spin-trapping in vivo

For in vivo work, the nitrone spin traps have been favoured. Thus, in general, only the l4N and H coupling constants of the parent trap are measured and these are matched against reference compounds. 

There are of course many problems. One is that metabolism of the trap may occur before it has captured the radicals of interest. Another is that the trap itself may upset the delicate balance of reactions which serve to produce radicals in its absence. It seems that for most traps, toxicity is not a serious problem. 

One of the first in vivo studies was that of Lai et al. (1979) using the PBN trap together with tetrachloromethane fed to a rat via a stomach tube. After 2 h the liver was extracted with a methanol/chlo-roform mixture, and the chloroform layer was studied. The spectrum was identified as that of the CC13 radical adduct. This identification was fully proven using 13CC14 (Albano et al., 1982 Tomasi et al., 1985). This remains one of the most successful studies of this type, but in situ detection not involving sacrifice and extraction would be most desirable. 

Other similar halogenated radicals have been detected in this way, and it seems that the high toxicity of such compounds is due to radical formation probably via electron capture. The great advantage of extraction in non-aqueous solvents such as toluene is that much larger volumes can be used than with aqueous extracts. However, this discriminates against water-soluble nitroxides, which may be missed entirely. 

Ron Mason and his co-workers have greatly improved this technique by using an improved (TMn0) ESR cavity which enables them to use 17-mm flat cells containing ca. 100 fi of fluid. Because of the low solubility of oxygen in water, deoxygenation is not necessary, and radical metabolites can be detected in urine, blood and bile fluids with no difficulty (LaCagnin et al., 1988). 

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The primary anion radical of Scheme 3-69 produces the l-chloro-2,2,2-trifluoroethyl radical. Having been spin-trapped in vivo, this radical was detected by the ESC method (Poyer et al. 1981). Ahr et al. (1982) has presented additional evidence for the formation of the radical as an intermediate in halothane metabolism and identified 2-chloro-l,l-difluo-... 

The ability to use these spin traps in vivo without the occurrence of other complicating metabolic effects. 

Furthermore, application of the spin trapping technique in intact animals require an understanding of the stability of the spin traps and the spin adducts in vivo. A new class of a-phosphorus-containing DMPO analogues, 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline N-oxide (DEPMPO) has been... 

Albano, E., Lott, K.A.K., Slater, T.F., Stier, A., Symons, M.C.R.and Tomasi, A. (1982). Spin trapping studies on the free radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions, isolated hepato-cytes and in vivo in the rat. Biochem. J. 204, 593-603. 

Poyer, J.L., McCay, P.B., Lai, E.K., Janzen, E G. and Davis, E.R. (1980). Confirmation of assignment of the trich-loromethyl radical spin adduct detected by spin trapping during carbon tetrachloride metabolism in vim and in vivo. Biochem. Biophys. Res. Commun. 94, 1154-1160. 

On the other hand, microsomes may also directly oxidize or reduce various substrates. As already mentioned, microsomal oxidation of carbon tetrachloride results in the formation of trichloromethyl free radical and the initiation of lipid peroxidation. The effect of carbon tetrachloride on microsomes has been widely studied in connection with its cytotoxic activity in humans and animals. It has been shown that CCI4 is reduced by cytochrome P-450. For example, by the use of spin-trapping technique, Albani et al. [38] demonstrated the formation of the CCI3 radical in rat liver microsomal fractions and in vivo in rats. McCay et al. [39] found that carbon tetrachloride metabolism to CC13 by rat liver accompanied by the formation of lipid dienyl and lipid peroxydienyl radicals. The incubation of carbon tetrachloride with liver cells resulted in the formation of the C02 free radical (identified as the PBN-CO2 radical spin adduct) in addition to trichoromethyl radical [40]. It was found that glutathione rather than dioxygen is needed for the formation of this additional free radical. The formation of trichloromethyl radical caused the inactivation of hepatic microsomal calcium pump [41]. 

A series of 2//-imidazole-l-oxides, isoquinoline-A-oxides and pyrrolidine-A-oxides were investigated as to their specificity and efficiency at spin trapping HO and 02 as well as the stability of the corresponding spin-trapped adducts. 2,2-Dimethyl-4-methoxycarbonyl-2A-imidazole (42) has been found to be the most selective of the spin traps investigated for the in vivo, in situ detection of HO at the expense of 02. ... 

Iba MM, Ghosal A, Poyer JL, et al. 1991. In vivo spin-trapping of the radieal metabolites of 3,3, diehlorobenzidine and related compoimds in the rat. Progress in Pharmaeology and Clinical Pharmacology 8(3) 255-266. 

Tripathi GNR (1998) Electron-transfer component in hydroxyl radical reactions observed by time resolved resonance Raman spectroscopy. J Am Chem Soc 120 4161-4166 TsaiT, Strauss R, Rosen GM (1999) Evaluation of various spin traps for the in vivo in situ detection of hydroxyl radical. J Chem Soc Perkin Trans 2 1759-1763 Tsay L-Y, Lee K-T, Liu T-Z (1998) Evidence for accelerated generation of OH radicals in experimental obstructive jaundice of rats. Free Rad Biol Med 24 732-737 Ulanski P, von Sonntag C (2000) Stability constants and decay of aqua-copper(lll) - a study by pulse radiolysis with conductometric detection. Eur J Inorg Chem 1211-1217 Veltwisch D, Janata E, Asmus K-D (1980) Primary processes in the reactions of OH radicals with sul-phoxides. J Chem Soc Perkin Trans 2 146-153... 

MPTP decreases glutathione levels and increases the levels of reactive oxygen species and the degree of lipid peroxidation in mouse brain slices in vitro and increases the levels of reactive oxygen species in mouse brain in vivo. MPTP neurotoxicity in vitro is reduced by glutathione. In vitro studies have shown that MPP neurotoxicity can be reduced by vitamin E, vitamin C, coenzyme Q, and mannitol (but not by superoxide dismutase, catalase, allopurinol, or dimethyl sulfoxide). P-Carotene, vitamin C, and /V-acctylcystcine partially protect against the neurotoxic effects of MPTP in mice, as do nicotinamide, coenzyme Q, and the free-radical spin trap A-tert-butyl-a-(sulfophenyl) nitrone. 

Kadiiska MB, Xiang Q-H, Mason RP. 1994. In vivo free radical generation by chromium(VI) an electron spin resonance spin-trapping investigation. Chem Res Toxicol 7 800-805. 

Spin traps come in basically two types nitroso compounds and nitrone compounds. Reactive free radicals react with the carbon of the nitrone functional group to form a radical adduct that always has a nitroxide group, which is an unusually stable type of free radical. Nitrones are the most useful spin traps for the in vivo detection of free radical metabolites because of the stability of the resulting radical adduct. However, identification of the parent radicals can be difficult because adducts derived from different radicals often have very similar EPR spectra. A comprehensive review of this area through 1992 has recently been published [48]. 

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