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

开发利用正电子发射体Oxy-15分析氧代谢和氧传感机制的新技术

• 批准号: 07557198
• 负责人: WATANABE Yasuyoshi
• 金额: $ 2.56万
• 依托单位: Osaka Bioscience Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557198/
• 关键词: Positron emitter; Oxygen-15; Oxygen metabolism; Oxygenase; Oxygen sensor; Metapyrocatechase; Radio-LC-MS; Adrenal chromaffine cells

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.

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如类固醇、类花生酸和神经活性物质的生物合成和代谢。我们开发了一种新的氧代谢放射性示踪技术，该技术采用正电子（β ^+）发射体^<15>O2，其物理半衰期为2.07分钟。从回旋加速器中的靶到RI实验室的反应容器的实验装置已经在与著名的双加氧酶之一的间焦儿茶酶的模型反应中被证实。将放射性标记的氧以250 ml/min的流速用0.5%氧/99.5%氮混合气体连续供应到反应容器中，底物邻苯二酚转化为放射性产物，其通过非常灵敏的LC-radio-UV-MS组合方法鉴定为α-羟基粘康酸ε-半醛。接下来，我们试图通过使用该技术来跟踪神经内分泌肿瘤细胞系BON中的细胞氧代谢。但是，从~ 2O_2中很难分离出痕量的放射性标记分子<15>，必须放大10倍以上才能检测。肾上腺嗜铬细胞是一种氧敏感细胞，在短期缺氧条件下，其特异性K^+通道关闭、Ca^2+内流和儿茶酚胺释放。在不久的将来，这种新的放射性示踪技术将被应用于高氧敏感性细胞的研究。