This program is aimed at the development and application of in vivo NMR spectroscopic methods for studying metabolism and its perturbation by chemical toxins. A principal focus of these studies has been the development of NMR active, intracellular indicator molecules to allow determination of metabolic parameters of interest in intact cells. Research has focused on the use of fluorinated indicators as a consequence of the inherent sensitivity of fluorine for NMR detection and the essential absence of background fluorine resonances from untreated cells. During the past year, progress was made in the development of fluorinated indicators for cytosolic calcium, magnesium, and pH. One of the fluorinated pH indicators synthesized during the past year appears to have more ideal kinetic, shift sensitivity, and loading properties than previously described pH indicators, and should provide valuable insight into this important metabolic parameter. We have also continued work on the use of perfluorocarbons, several of which are used clinically in surgical eye procedures, as indicators of cellular oxygen concentration, and evaluated the sensitivity of the fluorine chemical shift values to temperature as well. It appears that both oxygen concentration and temperature can be determined from NMR measurements on tissues containing these perfluorocarbons. A new effort initiated over the past year is aimed at determining the molecular basis for the toxicity of borate and several organic boron derivatives. Boric acid, which has a number of important commercial applications, has been shown to cause testicular atrophy in rats and dogs after ingestion of diets containing > 1000 ppm. During the past year we have carried out NMR studies of boric acid and phenyl boric acid in the presence of intact erythrocytes, enzymes, and other cellular materials thought to be potential targets, in order to quantify and evaluate the biochemical significance of these interactions. In addition, studies of the carbohydrate metabolism of sertoli cells using [1-13C] and [2-13C] labeled glucose have been initiated to investigated possible alterations related to borate toxicity.