Thomas Jue - US grants

Oxygen is a fundamental regulator of cellular bioenergetics and intermediary metabolism. Observing tissue oxygen level, however, has faced many hurdles. Extant techniques confront major obstacles in sampling intracellular oxygenation in a localized tissue region. Consequently a key question cannot be posed: Is the accepted view of the interaction between oxygen level and the regulation of bioenergetics, derived from model studies, also accurate in vivo? NMR offers a unique opportunity to answer such questions in vivo. Our preliminary data have demonstrated the 1H NMR visibility of the myoglobin proximal histidyl F8 NH and Val Ell methyl groups in heart muscle. These signals directly reflect intracellular oxygenation, in contrast to the commonly used 31P peaks, which monitor only the cellular interaction. We propose then to establish firmly the 1H NMR techniques to observe oxygen tension in specific tissue regions in vivo. Clearly the capacity to measure tissue oxygen opens many applications with both physiological and pathophysiological perspectives.

This grant proposal requests funding for a vertical bore high field NMR spectrometer for cell, perfused organ, and intact animal studies. The instrument will consist of a highest proven field magnet, 9.4T, that is in standard use for in vivo NMR application, homogeneous magnet bore diameter to accommodate at least 20 mm sample tubes, and a console with all features necessary for implementing multinuclear observation of metabolic process in vivo. The spectrometer will be utilized by a core group of seven investigators (Primary Users) as well as several secondary users. The individual projects of the investigators cover a wide range of fundamental research in physiology and biochemistry. Every investigator has an established research program. The proposed instrument will come under the auspices of the UCD NMR Facility. The Facility has an established record over ten years in providing investigators state of the art NMR instruments that are well maintained and reliable. UCD has also demonstrated the continuing support of NMR research on campus.

DESCRIPTION: A fundamental issue in muscle physiology is the regulation of oxygen transport to the mitochondria, since oxygen consumption can vary over a wide range, depending upon stimulation or exercise condition. Yet measuring vascular and intracellular oxygen has been extremely elusive. For vascular p02, investigators have recently noted that the water signal in leg muscle shifts with ischemia. The observation implies that the source of the shift may originate from Hb deoxygenation. If so, that paramagnetic transition during Hb deoxygenation can enhance the T2*, as functional magnetic resonance imaging studies have demonstrated, but also can shift the water line. The effect arises from the bulk magnetic susceptibility and the given limb geometry relative to main magnetic field. Consequently a calibrated curve of the water shift could then reflect the vascular p02. The project will specifically aim to determine the relationship between the chemical shift of water as a function of Mb and Hb oxidation states in analytical solutions and to establish the theoretical basis for utilizing the magnetic susceptibility induced H20 chemical shift as a p02 indicator. The results would open a new approach to measuring vascular p02 in muscle in other tissues.

This project will combine animal physiology with biophysics and biochemistry in order to examine oxygen transport and consumption from whole animal to subcellular levels during the prolonged, spontaneous breath holds of elephant seals during sleep. Unique adaptations in these animals include enhanced oxygen storage and exquisite regulation of heart rate, blood flow and tissue metabolic rates. The physiological processes and biochemical mechanisms underlying these adaptations are relevant to the basic principles of oxygen transfer within tissues as well as to the remarkable diving abilities of seals and whales.
A variety of minimally- and non-invasive techniques, including blood/tissue sampling, blood flow measurements, and nuclear magnetic resonance imaging/spectroscopy, will allow study of these seals while they sleep undisturbed. This will allow: 1) examination of the regulation of heart rate, cardiac output and muscle blood flow throughout the ten-minute breath holds of these seals, 2) measurement of the rate of blood oxygen depletion throughout the breath-hold, 3) examination of blood-to-muscle oxygen transfer as the blood oxygen level decreases, 4) determination of the oxygen desaturation rate of myoglobin - the oxygen storage protein in muscle, 5) examination of metabolic regulation in muscle, as reflected in the depletion of the high-energy phosphate, phosphocreatine, and the formation of lactate, and 6) examination of the mobility of the myoglobin molecule within the muscle cell.

Description: (Verbatim from the applicant's abstract) 1-H NMR techniques have detected the myoglobin signals in perfused myocardium and have opened a unique opportunity to investigate the fundamental question of oxygen regulation in tissue. How the cell responds to varying oxygen levels and meets the complex energy demand is central to a broad range of issues in cardiac/skeletal muscle physiology and metabolism. Perfused heart experiments have established the spectroscopic techniques to measure the intracellular oxygen in myocardium with the proximal histidyl NH and Val E11 signals of Mb and have set the stage for investigating the regulation of oxidative metabolism in the intact animal. They have also established a critical basis to measure the intracellular pO2 in blood perfused organs and to determine the regulation of respiration in the myocardium in situ. The proposal addresses a key question about the mechanism regulating respiration in myocardium. It will specifically assess whether the oxygen supply actually determines the respiration rate. Developing and utilizing the Mb technique to measure intracellular pO2 will then shape our understanding of oxidative phosphorylation in vivo.

nuclear magnetic resonance spectroscopy; biomedical equipment purchase;