Development of novel techniques for analysis of oxygen metabolism and oxygen-sensing mechanisms by use of positron-emitter oxygen-15

氧是生命所必需的分子，不仅用于细胞呼吸，而且用于各种重要生物分子的生物合成和代谢，如类固醇、类二十烷和神经活性物质。我们开发了一种新的氧代谢放射性示踪技术，利用正电子（β +）发射器^<15> O2，其物理半衰期为2.07 min。实验设置的targent回旋加速器的反应皿RI实验室一直在aseseed metapyrocatechase模型反应,著名的加双氧酶之一。用0.5%氧/99.5%氮的混合气体以250 ml/min的流速连续向反应容器中供应放射性标记氧，底物邻苯二酚转化为放射性产物，通过非常灵敏的LC-radio-UV-MS联用方法鉴定为α -羟苯二酚-半醛。接下来，我们尝试利用该技术跟踪神经内分泌肿瘤细胞系BON的细胞氧代谢。然而，很难从^<15>O2中识别出痕量放射性标记分子。我们必须获得10倍或更大的增益才能被发现。肾上腺嗜铬细胞在特定的K^+通道关闭、Ca^<2+>内流和短期缺氧释放儿茶酚胺方面具有氧感细胞的特征。在不久的将来，这种新颖的放射性示踪技术将应用于这种高氧敏感性细胞的研究中。

Oxygen, an essential molecule for life, is utilized not only for cellular respiration but also for biosynthesis and metabolism of various important biomolecules such as steroids, eicosanoids, and neuroactive substanes. We hare developed a novel radiotracer tecnique for oxygen metabolism that employs the positron (beta^+) emitter ^<15> O2, whose physical half-life is 2.07 min. The experimental set up from the targent in the cyclotron to the reaction vessel in the RI laboratory has been aseseed in a model reaction with metapyrocatechase, one of the well-known dioxygenases. The radiolabelled oxygen was continuously supplied to the reaction vessel with 0.5% oxygen/99.5% nitrogen mixed gas at a flow rate of 250 ml/min, and the substrate pyrocatechol was converted to the radioactive product which was identified as alpha-hydroxymuconic epsilon-semialdehyde by a very sensitive LC-radio-UV-MS combined method. Next, we tried to follow cellular oxygen metabolism in neuroendocrine tumor cell line BON by use of this technique. However, it was hard to idenify the trace radiolabelled molecule from ^<15>O2.We have to gain factor 10 or more for detection. Adrenal chromaffine cells were characterized as an oxygen-sensing cell in terms of specific K^+ channel closure, Ca^<2+> influx, and catecholamine release by a short term hypoxia. In the near future, this novel radiotracer technique will be applied to the research using such cells with high oxygen sensitivity.