NMR STUDIES OF CELLULAR METABOLISM

本项目旨在开发和应用活体核磁共振技术 研究代谢及其扰动的光谱方法 化学毒素 这些研究的一个主要重点是 开发NMR活性的细胞内指示剂分子， 在完整细胞中确定感兴趣的代谢参数。 因此，研究的重点是氟化指标的使用 的固有灵敏度的氟核磁共振检测和必要的 不存在来自未处理细胞的背景氟共振。 期间 去年，氟化指标的制定取得了进展 细胞溶质钙，镁和pH值。氟化pH值之一 过去一年综合的指标似乎更理想， 动力学、换档灵敏度和装载性能优于以前 描述的pH指示剂，并应提供有价值的见解， 重要代谢参数。 我们还继续努力使用 全氟化碳，其中几种在临床上用于眼科手术 程序，作为细胞氧浓度的指标，并评估 氟化学位移值对温度的敏感性， 好. 看来，氧气浓度和温度都可以 通过对含有这些化合物的组织的NMR测量确定 全氟化碳。 过去一年发起的一项新的努力旨在 确定硼酸盐毒性的分子基础， 有机硼衍生物 硼酸，它具有许多重要的 商业应用，已显示可导致大鼠睾丸萎缩 和狗摄入含> 1000 ppm的饲料后。 在过去 2004年，我们对硼酸和苯基硼酸进行了核磁共振研究， 存在完整的红细胞、酶和其他细胞物质 被认为是潜在的目标，以量化和评估 这些相互作用的生物化学意义。 此外，研究 用[1- 13 C]和[2- 13 C]研究支持细胞的糖代谢 已开始使用标记葡萄糖来研究可能的改变 与硼酸盐毒性有关。

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.