Kazuo Yoshizaki

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.

Significance of proton relaxation time measurement in brain edema, cerebral infarction and brain tumors

We examined the proton relaxation times in vitro in various neurological diseases using experimental and clinical materials, and consequently obtained significant results for making a fundamental analysis of magnetic resonance imaging (MRI) as followings. 1) In the brain edema and cerebral infarction, T1 prolonged and T2 separated into two components, one fast and one slow. Prolongation of T1 referred to the volume of increased water in tissue. The slow component of T2 reflects both the volume and the content of increased edema fluid in tissue. 2) In the edematous brain tissue with the damaged Blood-Brain-Barrier (BBB), the slow component of T2 became shorter after the injection of Mn-EDTA. Paramagnetic ion could be used as an indicator to demonstrate the destruction of BBB in the brain. 3) After the i.v. injection of glycerol, the slow component of T2 became shorter in the edematous brain with the concomitant decrease of water content. The effects of therapeutic drug could be evaluated by the measurement of proton relaxation times. 4) Almost all tumor tissue showed a longer T1 and T2 values than the normal rat brain, and many of them showed two components in T2. It was difficult to determine the histology of tumor tissue by the relaxation time alone because of an overlap of T1 and T2 values occurred among various types of brain tumors. 5) In vivo T1 values of various brain tumor were calculated from the data of MRIs by zero-crossing method, and they were compared with the in vitro T1 values which were measured immediately after the surgical operation. Though the absolute value did not coincide with each other due to differences in magnetic field strength, the tendency of the changes was the same among all kinds of tumors. It is concluded that the fundamental analysis of proton relaxation times is essentially important not only for the study of pathophysiology in many diseases but also for the interpretation of clinical MRI.

High-resolution proton magnetic resonance spectra of muscle.

High-resolution proton magnetic resonance spectra of intact muscles of frog and rat were obtained with selective saturation of the water signal. The spectra consisted of the superposition of a broad component and a high-resolution portion. The line width of the former was about 5 ppm and is assumed to originate from the protons of the macromolecules in muscle. The high-resolution portion showed well-resolved signals arising from creatine phosphate, creatine, carnosine, lactate and lipids. It is suggested that this technique could be used to monitor the intracellular pH by measuring the chemical shift of carnosine and the lipid consumption due to muscular contraction. When the spectrum of 31P-NMR is prepared simultaneously, the ratio of creatine phosphate to total creatine can also be determined.

Proton nuclear magnetic resonance spectra of brain tumors

Proton nuclear magnetic resonance (NMR) spectra were successfully measured in human brain tumor tissues and experimental rat brain tumors. The investigation was performed on clinical materials which consisted of tissue from one normal brain and 36 brain tumors. Normal rat brain tissue and rat glioma implanted in the brain were also analysed. NMR measurements were carried out at the resonance frequency of 99.54 MHz. The proton NMR spectrum of the normal brain consisted of one broad component and eight superimposed sharp peaks. The sharp peaks obtained from the brain tumors varied from those of the normal brain. A decrease in the signal intensity from N-acetyl aspartate was the most common finding in all tumors. Spectral patterns were similar within the same histological types, but varied among the different types. Therefore, 1H-NMR spectra might indicate the metabolism characteristic of each tumor type which would be invaluable for clinical differential dagnosis of brain tumors.

Fatigue and recovery of phosphorus metabolites and pH during stimulation of rat skeletal muscle: an evoked electromyography and in vivo31P-nuclear magnetic resonance spectroscopy study

Abstract31P-nuclear magnetic resonance spectroscopy and evoked electromyography were applied to rat skeletal muscle to examine the mechanism of muscle fatigue and the recovery of muscle phosphorus metabolites and pH during fatigue. When the sciatic nerve was electrically stimulated at 1 Hz, the contraction force of the gastrocnemius muscle decreased gradually to 46% of the maximal force, accompanied by a decrease in phosphocreatine (PCr) and a corresponding increase in inorganic phosphate (Pi) and diprotonated inorganic phosphate (H2PO4−). Neither the amplitudes of compound muscle action potentials (CMAP) nor muscle pH changed significantly. At 10-Hz stimulation, contraction force rapidly decreased to 26% of maximal force, accompanied by a decrease in PCr and increases in Pi and H2PO4−. Muscle pH decreased for a few minutes, then gradually recovered during continued stimulation. The amplitude of the CMAP also decreased for a few minutes and then reached steady values. At 100-Hz stimulation, the contraction force decreased to 6% of the maximal force and there was a decrease in the amplitude of the CMAP. However, the changes in the phosphorus metabolites and pH were transient and recovered to the control value during the stimulation. These results indicated that fatigue at 1 and 100-Hz stimulation was mainly caused by the change in phosphorus metabolite concentrations and electrical failure, respectively, and that fatigue at 10-Hz stimulation might have been due to both of the these factors. These results also indicated that electrical failure might have been the cause of the recovery of the phosphorus metabolites and pH during 100-Hz stimulation and of pH during 10-Hz stimulation.

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 4880 using 1H NMR

1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis.