Development of a Method for Measuring Oxyngen Concentration in Biological Systems by Means of ESR

活性炭、丝素体和稳定的氮氧自由基TEMPONE作为ESR测定细胞和组织中溶解氧浓度的探针的适宜性进行了研究。另一方面，TEMPONE的ESR谱快速达到平衡，尽管其对氧浓度的依赖性较小。TEMPONE的全氘代类似物（TEMPONE-d_<16>）是最有用的，因为它的ESR信号较窄，因此在测定线宽或峰高时具有较高的灵敏度和准确度<16>。因此，它迅速分布在细胞内外。用顺磁性离子如铬酸盐（III）扩大细胞外信号使细胞内信号保持完整，因此能够测量细胞内氧浓度。氮氧自由基通常在细胞内发生氧化还原反应。这是非常有用的监测氧化还原酶的活动，而它需要一个测量线宽，而不是峰高，血氧测定法。将TEMPONE-d_的水溶液<16>密封在透气的小TPX管中。通过手术将探针并入小鼠腹膜腔中。对环隙谐振腔进行微调后，获得了满意的信噪比。根据呼吸气体中氧浓度的变化，ESR信号的峰高发生变化。氧浓度越高，峰高越小，反之亦然。

Active charcoal, fusinite and a stable nitroxide radical, TEMPONE, were examined for suitability as a probe for the measurement of dissolved oxygen concentrations in cells and in tissues using ESR.Fusinite and activated charcoal were very slow in reaching an equilibrium. On the other hand, the equilibrium was rapidly obtained with ESR spectrum of TEMPONE although its dependence on oxygen concentrations was smaller. Perdeuterted analog of TEMPONE (TEMPONE-d_<16>) is most useful since its ESR signals are narrower so that it gives higher sensitivity and higher accuracy in the determination of linewidth or peak height.Biological Membranes are permeable to TEMPONE-d_<16>. Therefore, it quickly distributes both inside and outside cells. Broadening extracellular signals with paramagnetic ions such as oxalatochromate (III) leaves intracellular signals intact and hence enables to measure intracellular oxygen concentrations. Nitroxide radicals are usually subject to redox reactions inside cells. This is very useful to monitor activities of redox enzymes while it necessitates one to measure linewidths, not peak heights, for oxymetry.Experiment with a mouse whole-body was performed using as L-band spectrometer. An aqueous solution of TEMPONE-d_<16> is sealed in a small TPX tube which is permeable to gas. The probe was incorporated in a mouse peritoneal cavity by operation. A satisfactory S/N ratio was obtained after a fine tuning of the loop-gap resonator. Depending on the change in oxygen concentrations of respiratory gas, peak heights of the ESR signal changed. The peak height was smaller with higher oxygen concentrations and vice versa.