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

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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.