Disulfide nitroxide biradicals, DNB, have been used for glutathione, GSH, measurements by X-band electron paramagnetic resonance, EPR, in various cells and tissues. In the present paper, the postulated potential use of DNB for EPR detection of GSH in vivo was explored. Isotopic substitution in the structure of the DNB was performed for the enhancement of its EPR spectral properties. (15)N substitution in the NO fragment of the DNB decreased the number of EPR spectral lines and resulted in an approximately two-fold increase in the signal-to-noise ratio, SNR. An additional two-fold increase in the SNR was achieved by substitution of the hydrogen atoms with deuterium resulting in narrowing the EPR lines from 1.35 G to 0.95 G. The spectral changes of DNB upon reaction with GSH and cysteine were studied in vitro in a wide range of pHs at room temperature and "body" temperature, 37 degrees C, and the corresponding bimolecular rate constants were calculated. In in vivo experiments the kinetics of the L-band EPR spectral changes after injection of DNB into ovarian xenograft tumors grown in nude mice were measured by L-band EPR spectroscopy, and analyzed in terms of the two main contributing reactions, splitting of the disulfide bond and reduction of the NO fragment. The initial exponential increase of the "monoradical" peak intensity has been used for the calculation of the GSH concentration using the value of the observed rate constant for the reaction of DNB with GSH, k(obs) (pH 7.1, 37 degrees C)=2.6 M(-1)s(-1). The concentrations of GSH in cisplatin-resistant and cisplatin-sensitive tumors were found to be 3.3 mM and 1.8 mM, respectively, in quantitative agreement with the in vitro data.

The reactions of the reversible addition of thiols and thiyl radicals to the nitrone spin traps DMPO (5,5dimethyl-1-pyrroline N-oxide) and DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide) are described. Addition of the thiols to the double CdN bond of the nitrones results in the formation of the corresponding hydroxylamines, measured using 31 P NMR and the phosphorus-containing trap DEPMPO. Subsequent mild oxidation of these hydroxylamines into the paramagnetic adducts may interfere with genuine spin trapping of thiyl radicals representing the Forrester-Hepburn mechanism. The reverse decomposition of hydroxylamines to the parent nitrone and thiol and of paramagnetic adducts to the nitrone and thiyl radical were observed for the first time. The recycling of reduced thiols from thiyl radicals by nitrones may comprise the mechanism of their effective antioxidant activity, in vivo. The release of thiyl radicals upon the breakdown of the paramagnetic adduct may significantly affect not only the quantitative analysis of the spin trapping data but even the conclusions regarding the origin of short-lived radical intermediates. The equilibrium constant for the reactions of the formation of the product of DEPMPO with S-centered nucleophiles decreases in the series: sulfite > thioglycolic acid > cysteine > glutathione. The rate constants for the reaction of the monomolecular decomposition of the radical adducts back to the nitrone and glutathiyl radical were found to be equal to 0.3 ( 0.1 s -1 and 0.02 s -1 for DMPO/GS • and DEPMPO/GS • radical adducts, correspondingly.