Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques.

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.

Quantitative determination of SH groups using 19F NMR spectroscopy and disulfide of 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid.

A new method of measurement of thiol concentration by 19F NMR spectroscopy is developed. The method is based on the detection of products of the exchange reaction of thiols with a newly synthesized fluorinated disulfide, 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (BSSB). A significant broadening of the 19F NMR signal of BSSB in the presence of thiols was observed and attributed to the exchange reaction between the parent disulfide and 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid. The rate constant for this reaction was found to be equal to (63 +/- 11) x 10(3) M(-1) s(-1) at pH 7.0. The method was applied for the measurement of concentration of glutathione and albumin in rat blood.

Nonradical mechanism of (bi)sulfite reaction with DEPMPO: cautionary note for SO3*- radical spin trapping.

Knowledge of the formation of radicals from sulfites, in vivo, is of interest in understanding the allergic and inflammatory responses to environmental sulfur dioxide exposure. Sulfite anion trioxide (SO(3)(-*)) radical formation was measured in mice, preloaded with the spin trap, 5-(diethoxy-phosphoryl)-5-methyl-pyrrolidine-N-oxide, (DEPMPO). Based on spin trapping NMR, a surprising quantity of reduced SO(3)(-*)-adduct was observed that did not depend on co-administration of oxidizing agents, suggesting a possible nonradical reaction between (bi)sulfite and DEPMPO. The products of the reversible nucleophylic addition of (bi)sulfite to the nitrone functional group were identified using (31)P-NMR, (1)H-NMR, and (13)C-NMR spectroscopy as cis- and trans- stereoisomers of hydroxylamine and confirmed by quantum chemical calculations. Oxidation of the hydroxylamines results in the formation of two corresponding cis- and trans-isomeric nitroxides, only one of which has been earlier described as the paramagnetic adduct of genuinely trapped SO(3)(-*) radical. The results demonstrate that SO(3)(-*) detection using nitrone spin traps such as DEPMPO and DMPO may involve nonradical addition reactions except in cases when the required controls unambiguously prove a radical mechanism.