31P核磁気共鳴法による生きたラットの脳における高エネルギーリン酸化合物（ATPクレアチンリン酸）の測定—ハロセン麻酔と低酸素状態の影響

31P-NMR STUDIES ON THE METABOLISM OF HIGH ENERGY PHOSPHORUS COMPOUNDS (PHOSPHOCREATINE, ATP) IN THE LIVING RAT BRAIN:THE EFFECT OF HALOTHANE ANESTHESIA AND HYFOXIC HYPDXIA

　抄録　著者らは³¹P核磁気共鳴法により生きた状態のラットの脳からアデノシン3リン酸（ATP）やクレアチンリン酸（Pcr．）などの高エネルギーリン酸化合物のシグナルを比較的短時間のうちに定量的かつ経時的に検知する方法を開発した。ここではその方法と応用として麻酔薬ハロセンや低酸素状態がラットの脳のエネルギー代謝に与える影響について述べる。装置の主な改良点は，計測点に実験動物が収納固定されるようにしたこと，検知コイルとして表面型コイルを採用したことである。動物の呼吸と麻酔の管理のために吸入麻酔器を用い，得られた脳のリンシグナルを定量的に分析するためにhexamethyl phosphoramideをexternal referenceとして用いるなどの工夫をした。ウイスター系雄ラット10匹で空気，純酸素ハロセン麻酔の影響を検討し，5匹について低酸素状態を負荷し，それぞれ脳のATPやPcr．を測定した。その結果1％ハロセン麻酔では，空気吸入時より脳のATPやPcr．は有意の増加を示した。低酸素状態では酸素濃度の低下に伴って脳のATPやPcr．が低下することが示された。³¹P-NMRは同一生体での脳エネルギー状態を経時的に追跡する手段として著しく有利な方法論と考えられた。

³¹Phosphorus nuclear magnetic resonance (³¹P-NMR) measurements have provided new and valu-able insights for studying the metabolism of living systems¹¹⁾. The aim of this paper is to introduce a technique of application of ³¹P-NMR measure-ments using a surface coil method, and to discuss the effects of halothane anesthesia and hypoxic hypoxia on the energetic metabolism of intact rat brains.

All measurements were made using a JEOI. FX 270 spectrometer with a super conducting magnet of 54-mm bore diameter. The magnetic field intensity of this machine is 6.3 tesla, and the resonance frequency used for ³¹P was 109.14 MHz. We remodelled an ordinary probe to take a live rat, and the animals were made to inhale anesthetic halothane or mixture of oxygen and nitrogen at various concentrations controlled by a flow regulator.

The best conditions for measurements with our surface coil method were determined in this study as follows : (1) 90° pulse width and selectivity, Fig. 1 shows signal selectivity in depthwise direction changed with 90° pulse width, which was set to 20 μsec. (2) Sensitivity and resolution ; To obtain a spectrum of ³¹P-NMR from a rat brain 500 accumulations of free induction decays were con-sidered suitable for both time and space resolution. Fig. 2 shows variations of signal intensity with pulse repetition time, which was set to 2 sec. It took about 17 min for averaging to get a spectro-gram. (3) Quantitative accuracy and qualification ; As shown in Fig. 3, a linear relationship was found between the signal intensity of β-phosphate of ATP and the concentration of ATP solutions, thus proving the quantitative accuracy of oursystems.

Wistar's male rats weighing 30 g to 150g were prepared for the halothane anesthesia and hypoxia experiments. A typical ³¹P-NMR spectrum of an intact rat cerebrum is shown in Fig. 4, in which seven peaks are identified. Peak 1, 2 and 3 are assigned to β-phosphate, α-phosphate and γ-pho-sphate of ATP, peak 4 to phosphocreatine (Pcr.), peak 5 to phosphodiesters, peak 6 to inorganic phosphate (Pi), and peak 7 to sugar phosphates^1,11). In order to calculate the changing rate of signal intensity of phosphates of Pcr. or β-ATP. we adopted a capillary tube filling with hexamethyl phosphoramide (HMPA) as an external reference to corrected the signal heights of each phosphate from the brain by the intensity of HMPA.

Effects of halothane anesthesia on the rat brains : As shown in Fig. 5 signal intensity of Pcr. and β-ATP increased as the rat was made to inhale air, pure oxygen and 1% halothane with oxygen, and the value of Pcr. and β-ATP decreased again when the concentration of halothane increased to 2 or 3%. The increasing effects of Pcr. and β-ATP with 1% halothane anesthesia was confirmed in 10 rat brains (Fig. 6).

Effects of hypoxia on the rat brains : We ex-amined the effects of hypoxia on the cerebellum in 4 rats, two were normal rats and the others were methyl mercury treated rats. The value of Pcr. and β-ATP decreased according to the diminu-tion of the concentration of oxygen in the inhaled gases (Fig. 7). The results of observations over longer time course of changes in phosphate com-pounds in a rat cerebrum with hypoxia are shown in Fig. 8.

The above data suggested that ³¹P-NMR meas-urements with a somewhat improved technique offer new insights for research on brain metabolism of intact animal, which would otherwise be hard to examine by conventional biochemical analytical methods.