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

考察了活性碳、丝光沸石和稳定的氮氧自由基Tempone作为探针，用ESR.法测量细胞和组织中的溶解氧浓度。丝光沸石和活性碳达到平衡的速度非常慢。另一方面，Tempone的ESR谱对氧浓度的依赖性较小，但能快速达到平衡。全氚类似物Tempone(Tempone-d&&lt；16&gt；)最有用，因为它的ESR信号更窄，因此在测定线宽或峰高时具有更高的灵敏度和更高的准确性。生物膜对Tempone-d_&lt；16&gt；具有渗透性。因此，它迅速地在细胞内外分布。用顺磁性离子加宽细胞外信号，如草酸铬(III)，使细胞内信号保持不变，从而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。这对于监测氧化还原酶的活性是非常有用的，但它需要测量氧气的线宽，而不是峰高。用小鼠全身进行的实验使用了L波段光谱仪。Tempone-d&lt；16&gt；的水溶液被密封在一个对气体渗透的小TPX管中。通过手术将该探针植入小鼠腹膜腔内。对环隙谐振器进行微调后，获得了满意的S/N比。根据呼吸气体中氧浓度的变化，ESR信号的峰高也会发生变化。氧气浓度越高，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.