Britton Chance, PhD - US grants

The study of the metabolism of oxygen by cytochrome oxidase (aa3), a binuclear iron copper redox center as compared with the mononuclear cytochrome Omicron types of oxidases sheds light on the essential sequence reactions in oxygen reduction. In cytochrome (aa3) oxidase the formation of bound peroxides, the rupture of the peroxide bond and the generation of radical intermediates form steps where sequestered and nonsequestered radical intermediates can render the system biologically safe or dangerous, respectively. The optical study of the steps in these radical reactions are complemented by studies of the charge densities of the metal atoms, the nature of the ligand shells and the distance to the nearest and next to nearest ligands in the radical intermediates by the use of X-ray synchrotron radiation to determine the edge and EXAFS (Extended X-Ray Absorption Fine Structure) properties. These studies form the basis for the understanding of normoxic and hyperoxic metabolism of oxygen and are essential to molecular explanations of processes leading to the pathway of fibrotic and interstitial lung disease.

[unreadable]31[unreadable]P NMR presents a unique approach to non-invasive muscle biochemistry in normal and diseased patients. A 10 1/4" clear bore NMR spectrometer affords an opportunity to evaluate muscle biochemistry in the limbs of normal and diseased humans. The principal changes of muscle bioenergetics over the convenient range of exercise are phosphocreatine (PCr) decrease from approximately 6 times the ATP level to one times the APT level with corresponding rises of inorganic phosphate remaining practically constant. PCr/P[unreadable]i[unreadable] (or PCr/(PCr + P[unreadable]i[unreadable])) assesses the biochemical function of the resting and exercised human forearm and calf muscle and evaluates the competency of oxygen delivery to tissue and the capability of mitochondria to mobilize substrates and utilize oxygen for maintaining PCr levels high and the P[unreadable]i[unreadable] low. A study of the nature of errors in the [unreadable]31[unreadable]P NMR study of human tissues is here applied to the human leg under resting and exercise conditions in normal controls and in patients having unilateral peripheral vascular disease. The repeatability and reliability of the data from these groups under these conditions will afford a baseline for the use of NMR as a clinical analytical biochemical tool; the leg being the organ most available in a variety of diseased states, and the data will afford a baseline for future applications to other body organs. Thus the protocols involve appropriate resting states, maximum limb exercise feasible for the particular subject, normal or diseased, followed by insertion of the limb into the NMR spectrometer immediately following a quantitative exercise regimen. Categories of recovery times from this procedure are established and their reliability determined. On this data base, NMR reliability will be established by appropriate statistical methods, with correlations with disease categories obtained from other methods. Prediction of the course of peripheral vascular disease and the effectiveness of therapeutic procedures by the NMR technique may ultimately be possible.

The possibilities of optimizing the parameters for phosphorus, sodium and proton spectroscopy of biochemicals and of imaging tissue volumes perturbed by hypoxic/ischemic stress is a principle goal of this research. Using these techniques we propose to follow the course of hypoxic/ischemic injury which, as established by biochemical and morphological studies, has lacked an identification of the primary events on the pathway to cell damage. The technological developments necessary for this research have already been evaluated and approved under Research Resource activity and we propose to utilize the technology for this work as it develops. This progression is to be followed by spectroscopy and imaging of phosphorus metabolism as a primary event, of glycolytic metabolism involving lactate production as a secondary event and by sodium and water movements as a tertiary event. In the animal model system employed, we will minimize the multiple parameters that precipitate hypoxic/ischemic cell damage by employing a protocol of manual stabilization of the energy state of the tissue through NMR observation of the phosphocreatine/phosphate (PCr/Pi) at values of approximately 1, employing not only manual control of Fi02, but also complete monitoring of correlated biochemical and physiological parameters. The insult is quantitated by its duration (T) to intensity (PCr/Pi) quotient and is scored by its impact on the rate of recovery (PCr/Pi/min) in response to restoration of tissue oxygen levels, or in terms of the time required for complete loss of control of tissue oxidative metabolism (PCr/Pi falls to less than 0.05). The guiding hypothesis is that hypoxic stress sets a metabolic operating point (PCr/Pi value) that is inherently unstable because the metabolic load line (ATPase level) becomes nearly parallel to the asymptote of the Michaelis-Menten transfer function for oxidative metabolism in the steady-state condition (tissue respiration rate = tissue ATPase). With this approach we expect to identify unstable operating points (PCr/Pi), quantified insults T/PCr/Pi) that lead to impaired oxidative metabolism and increased ATPase. By a comparison of the multiple nucleic in spectroscopic and imaging modes. We propose to have a data base appropriate to an objective evaluation of the relative merits in the two modes. Furthermore, the biochemical data obtained in this study, we believe, will be predictive of metabolic "disasters" leading to acidosis, cell water and ion movements, and eventual cell disintegration and are applicable generally to animal models and neonate and adult human brains.

particle accelerators; particle beam; metalloproteins; ultraviolet spectrometry; biomedical equipment resource; X ray crystallography; metalloenzyme; sulfur compounds; bacteriorhodopsins; photolysis; cytochrome oxidase; light scattering; DNA directed DNA polymerase; zinc; cytochrome c; iron; ligands; ferredoxin; hemocyanin; cobalt; copper; manganese; molybdenum; monitoring device; molecular site; chemical stability; online computer; pyruvate decarboxylase; carbonate dehydratase; chemical models; optics; alcohol dehydrogenase; superoxide dismutase; photosynthesis; flash photolysis; stop flow technique; Mossbauer spectrometry; photosynthetic bacteria; Escherichia coli; electron spin resonance spectroscopy;

The study of peroxide reactions becomes a major effort of this project in view of the ubiquity of peroxide in living cells and the new knowledge of the reaction of the unbridged form of cytochrome oxidase with peroxide. These are modelled by the reaction of the intermediates with peroxide, with hydrogen, alkyal and other peroxides, as oxidants and with a variety of heavy-atom-labelled substrates as reductants. The intermediates were studied in trapped states at low temperatures and at physiological temperatures in real time using rapid flow methods (regenerative flow). Horseradish peroxidase is of most interest because it forms two types of intermediates; as does lacto-peroxidase. In the case of those forming a single intermediate, yeast peroxidase and catalase are seriously to be considered, together with chloroperoxidase. Thus in this specific proposal, the structures of intermediates will be studied in the trapped state, and the relationship of these structures to their function will be demonstrated in room temperature rapid flow studies, with concomitant optical and epr monitoring of sample properties. Where possible, advantage will be taken of ongoing X-ray crystallography, magnetic and spin resonance methods, as well as Raman spectroscopic methods in order to ensure that conclusions based upon X-ray absorption spectroscopy are consistent with these other approaches. The results are expected to be significant in two respects: 1) the specific determination of the extent to which these compounds are appropriate models for the peroxidatic reaction of cytochrome oxidase and 2) the generalities of common mechanisms for the utilization of peroxide in biological systems.

The nature and function of metal atoms in enzymes and proteins has been a keystone of biomedical research since Warburg's initial discoveries in the 1930's on "heavy-metal enzyme catalysis" and Keilin's identification of hemoproteins. Many biophysical approaches to the study of these heavy metal enzymes have been in process. Optical and electron paramagnetic resonance studies are good examples of those that give definitive information on many properties but have "blind spots" that fail to give incisive information on the metal atoms in all valences or states, i.e., certain properties are "invisible" to most techniques. K edge absorption of X-rays by heavy metal atoms occurs in all valence states and gives definitive fingerprints of the nature of such atoms and their electronic configuration and chemical environment. Extended edge absorption studies (exafs) give many other properties, some of which can be interpreted as precise distance measurements to neighbors of the heavy metal atoms. While related data may ultmately be inferred from x-ray or nuclear magnetic resonance data, the great potential advantages of the exafs method are the possibilities of structural studies of the metalloproteins that are time resolved at enzymatically functional concentraions. Optical and epr sample monitoring detects any change of valence or liganding under synchrotron irradiation. This proposal seeks to set up instrumentation development and testing of components that increase the efficiency of photon collection from x-irradiated samples, the ultimate goal being to achieve edge absorption EXAFS and anomalous scattering studies in the range appropriate enzyme activity studies, i.e., less than 100 microns M and eventually extending down toward micromolar concentrations. Concomitant with this development are essential innovations necessary for kinetic studies of the state of mental atoms in enzymatic function. Last but not least are improved methods for on-line sample monitoring to ensure the integrity of the biological material throughout the course of the x-irradiation.

The comprehensive evaluation of the feasibility, reliability and diagnostic capability of phosphorus NMR spectroscopy in the study of independently diagnosed peripheral vascular disease over an adequate population to identify categories of severity, etiology, therapy, and prognosis, is a general goal of an intensive study using phosphorus NMR spectroscopy and a standardized protocol for evaluation of oxygen delivery to claudicated limbs. An 11 1/2" clear bore NMR spectrometer, complemented by access to a 30" clear bore spectrometer affords an opportunity to evaluate muscle biochemistry in the limbs of over 85% of the population of peripheral vascular diseased patients and normal controls. The standard protocol of "top-up" exercises outside the magnet and insertion of the leg in the magnet, used in critical evaluation of over 209 limbs provides significant correlations with diagnosis by ankle pressure index. We plan to extend the study population by 400 patients so that populations can be divided based on severity, etiology and therapeutic procedures, and, in particular post-operative recovery. Two variations of the basic "toe-up" exercise protocol are specialized for particular patient populations; 1) resting phosphocreatine/phosphate determinations in severly diseased patients and 2) in-magnet graded exercise protocols will be developed for patients with subclinical symptoms, and for those whose degree of claudication is moderate. A new method of data analysis, based upon the Michaelis-Menten hypothesis and standard enzyme-substrate kinetics affords insight into the results of these 3 portocols; recovery kinetics, graded exercise and resting state values, which allows an estimation of the maximal oxygen delivery capability to the tissue. The goals of this study are to establish the feasibility and reliability of performing clinical NMR studies, and to quantify categories of severity, etiology, and the effectiveness of therapeutic measures. The ultimate goal is to collect a reliable data bank for NMR evaluation of peripheral vascular disease, which can be used as a basis for further evaluation of the use of NMR in noninvasive "in vivo" detection of disease and monitoring of therapeutic intervention.

The Society of Magnetic Resonance in Medicine was formed five years ago by scientists working in the fields of nuclear magnetic resonance (NMR), electron spin resonance (ESR) and in vivo spectroscopy with magnetic resonance. The membership is comprised of prioneers in in vivo NMR spectroscopy and imaging and draws both clinical and basic scientists. A major purpose of the Society is to maintain a strong basic science foundation for clinical in vivo diagnostic and experimental studies. The Society has held four annual conferences thus far, and attendance has grown tremendously at each one. A "teach-in" session is held the day before the conference officially begins. It has become apparent that a major function the annual meetings play is in the education of young scientists, both M.D.'s and Ph.D.'s. To this end we seek support from NIH for student travel stipends as matching support to funds the Society will also provide for this purpose. We believe student attendance at this meeting will double in 1986, and that without the matching support, a multitude of promising young scientists will miss the opportunity to attend this important meeting, and participate in the exciting interchange of the ideas and latest developments in the field. NMR has emerged as a powerful new technique with great clinical potential, and it is drawing researchers from many disciplines. We want to be able to open our doors to all of them, and to fulfill our goal of bringing together the finest scientists in the field to disseminate their knowledge.

This proposal seeks support for a MicroVAX II and Evans and Sutherland PS300 graphics system to be located at the University City Sciene Center adjacent to the University of Pennsylvania. These instruments will provide major data processing and graphics capabilities for research projects directed by four investigators: N.R. Kallenbach, J. Leigh, S.W. Englander and B. Chance, focussing on molecular structure determination and dynamics of proteins and nucleic acids. Three areas will particularly benefit from the computer graphics facility: 1) structure determinations using high resolution 2D NMR techniques relying on distance-geometry algorithms; 2) EXAFS/XANES spectroscopy of metalloprotiens, and 3) simulation and interpretation of metal ion EPR and ESEEM spectra in enzyme substrate complexes. Together these projects place heavy emphasis on numerical data reduction, modeling and analysis. In addition the system will support software development and an EXAFS data bank made generally available through the National Biostructures Research Resource.

A 13-element Germanium detector and its associated electronics are requested for X-ray absorption spectroscopic studies of small sample volumes of dilute metalloprotein solutions. The system will be used in our studies on native and modified Cytochrome Oxidases, Ni and Fe enzymes isolated from methanogenic bacteria and Cytochrome c1 - Cytochrome c complexes. The detector will also be made available for use by 1 members of the National Biostructures PRT (NBPRT) at the National Synchrotron Light Source, Brookhaven National Labs. Use and maintenance of the instrument will be coordinated and supervised by the NBPRT. This detector will reduce the time required to obtain data by a factor between two and five compared to existing detectors by resolving the difference in energy between signal and background X-ray photons, which will improve efficiency and lessen radiation damage to the sample. The energy resolution of the detector is approximately 250 eV, and the maximum count rate is in excess of 106 per second.

No Abstract Provided

The possibility of quantitative measurements of deoxyhemoglobin concentrations in the neonate and adult brain depends upon picosecond pulse of phase modulation spectroscopy that determines the optical path traveled by photons exiting from the brain. Algorithms, based upon a program of milk and animal models, will be developed to permit localization of pathological states due to brain bleeding, i.e., hematomas or aneurysms. Thus, the possibility of on-radioactive, non-magnetic, non-confining studies based upon innocuous low-powered deposition near red illumination on the surface of the head is the general goal of these studies. These developments can lead to a new technology of quantitation and localization of normal and pathological levels of deoxyhemoglobin in the human brain. It is conceived that this instrument has clinical applications and safety aspects that would allow it to be used wherever needed in research and clinical studies.

The need for X-ray synchrotron beams of high intensity and positional stability is a logical sequel to the construction of high intensity, low emission synchrotrons throughout the world and particularly at NSLS (National Synchrotron Light Source at Brookhaven National Laboratory, NY). The concept that the "second generation" synchrotrons would deliver beams of high positional stability has been falsified at the Photon Factory and NSLS> Furthermore the requirements for measuring fluorescence changes of dilute or very small samples now is a paramount question which affects not only the efficiency of data collection but, in some cases, the ability to collect an effective data set. Our position operates with beam interception of a few percent. This device is required at many synchrotrons and particularly at the bending magnet beam lines such as X9A and B at NSLS where possibilities of wideband electronically stabilized photon and other positional effects that are implicit in long transport lines containing monochromators, mirrors, slits, small samples, etc. A unique feature of this study is the availability of white beam (Beam Line X9B) and a transported monochromated beam (Beam Line X9A) supplied by the same bending magnet section of NSLS. Evaluations and comparisons of positional errors attributable to the electron beam on the one hand and of the transported beam on the other will allow of table tracking, devices with slow readily available servos, fast servos and finally a piezo electrically controlled glass plate will be used to compensate for positional fluctuations, the intermediate goal being a few microns and a few Hz, the ultimate being 50-100 Hz. An impact of these studies upon the efficiency and precision of X9A and upon other beam lines is expected to occur through the dissemination procedures of RR-01633 (National Biostructures PRT, J.K. Blasie, P.I.).

This research program calls for an intensive development and quantitation of a new technology for noninvasive measurements of hemoglobin/myoglobin deoxygenation in skeletal and cardiac muscle. A continuous light method (CWS) is base upon the dual wavelength spectrophotometry principle and can be attached directly to the limb of the exercising animal or human subject. This instrument is a "trend indicator" and is calibrated in relation to cuff ischemia and hyperemia where possible. The optical path is unknown and only relative responses are obtained with human subjects with peripheral vascular disease or congestive heart failure. The results can be closely correlated with those obtained by phosphorus magnetic resonance spectroscopy. In order to improve quantitation by direct measurement of the optical path, time resolved spectroscopy (TRS) involving picosecond or phase modulation spectroscopy (PMS) technologies afford a quantitation of concentration changes for a single wavelength device and direct measurements of concentrations and their changes for a dual and multi- wavelength animal models in normoxic and ischemic limbs. Furthermore, the separate contributions of Hb/Mb will be evaluated by 1) chemical modification of myoglobin, 2) highly resolved laser spectroscopy, 3) synchronization of hemoglobin signals with the arteriolar pulse, and 4) freeze trapping and redox scanning at low temperatures. The imaging potential of TRS will be studied in models, in normal animals and in limbs with an induced compartment syndrome and in model infarcts of the myocardium using single channel CWS and PMS. Upgrading PMS and TRS to multichannel spectroscopy of these models will provide the data sets which can be analyzed by appropriate algorithms by which the imaging potential can be evaluated with the ultimate goal providing a 3-D motion picture of Hb/Mb changes over the contraction cycle. The hypoxic tissue volume of each model will be evaluated by freeze trapping and 3-D redox scanning under other support. Observations through the chest wall will be attempted where synchrony with systole-distole may afford identification of myocardial tissue volumes, with an end in view of providing methods applicable to human subjects. Thus safe, economical and efficient evaluation of muscle hypoxia/ischemia may become generally available to general and speciality medical evaluations without the use of large magnets or radioactive materials.

Photon migration in tissues has afforded a novel approach to the measurement of scattering (mus') absorption (mua) properties of human tissues. Localization depends upon the effects of localized absorbers upon the photon migration pattern. In this rapidly developing field, localization by the effect of localized absorbers/scatterers upon the mean pathlength observed between separated input-output points has afforded a primary approach. The pathlength is lengthened for a proximal object and shortened for an object distal with respect to the banana- shaped photon probability distribution. Such patterns are highly localized when the mean pathlength is sharply identified as by single frequency phase modulation systems. However, significant improvements are obtained when the optical pattern is more sharply defined. The possibility of localizing low concentrations of small volumes of blood and contrast agents is provided by the phased array optical system. The interference properties of the photon diffusion waves of wavelength at ~7 cm are described in experiments in collaboration with Gratton et al. (1). An array of laser diode transmitters with phases alternating between 0o and 180o in a four element array gives an antenna pattern containing a sharp null of 2 cm FWHM. When the array extends over one wavelength with transmitters at lambda/4, the sharpness of the null point is 5o of phase shift per min of displacement of the receiver with respect to the transmitter. the sensitivity in the detection of a 5-mm object containing a model contrast agent at the expected tissue concentrations is 1.5o/mm motion of the object midway between a 5-cm separation transmitter-receiver array in 0.5% intralipid, conditions closely mimicking those of breast or brain and at an agent concentration approximating the absorption of blood in tissue. No loss of sensitivity is obtained by measuring the time resolved fluorescence emission of a localized contrast agent. The estimated amount of cardiogreen required for 1.5o/mm sensitivity is 0.7 mug or 1.0 nmol. The localized perturbation of the phase array null by focused ultrasound will also be studied.

The urgent need for a fast, safe, economical, noninvasive approach to global and localized brain studies of the desaturation of hemoglobin together with the possibility of early detection of hemorrhage, aneurysm and stroke volumes can be met by research on spectroscopy and imaging with three novel technologies. The scattering of light by the skin, skull and brain tissue requires these recently developed technologies: time domain (TD), frequency domain (FD) spectroscopy and phased array (PA) imaging. Multiwave TD/FD affords a global or hemispheric quantitative determination of hemoglobin saturation and blood volume already demonstrated in human brain to be highly precise measure of clinical utility. This is also the case in early detection of brain bleeds, hematoma, hemorrhages, etc. in emergency health care. The theoretical basis for these methods is the diffusion equation. The point-by-point fitting of experimental data deconvolutes the absorption (mu a) and the scattering (mus') coefficients for TD. A novel "substitution' method allows the precise determination of mu a and mu s" for small excised portions of brain tissue identifying the eventual imaging contrast obtainable for gray/white matter in the brain. Using these values of brain mu a and mu s' a "brain slab" model provides a definitive evaluation of the ability of TD and FD to determine the oxygen saturation of hemoglobin in localized volume (o.5 gram) and to determine its position in two dimensions. A hundred-fold increase in sensitivity is obtained using Phased Array (PA), the recently discovered low frequency, long wave (10 cm) diffusive waves of light in highly scattering media (such as human brain). Interference between such diffusive waves arising from two light sources that are coded in phase gives highly sensitive amplitude nulls or phase transitions. Positional detection to less than 1 mm obtained in volumes of localized absorbers of less than 0.1 gram. Subnanomole amounts of a model contrast agent or hemoglobin at hematocrits of a few percent can be detected in a brain slab model. TD and FD systems optimally quantitate global and localized hemoglobin saturations and blood concentrations. The phased array system (PA) localizes absorption and extremely small scattering and absorption anomalies in brain tissue due to bleeding, aneurysm, stroke volume, on the one hand, and diseases which alter the scattering or the organelle content of brain tissue, for example, Alzheimers disease and white matter disease, on the other.

The novel technology of photon diffusion or migration in tissue (diffusometry) permits the detection and localization of a variety of parameters that permit a characterization of the structural and functional properties of normal host and embedded tumor tissue. The two parameters, apparent scattering coefficient (mus', cm-1) and absorption coefficient (mua, cm-1) are determined from photon kinetics in tissues in response to a light pulse (time resolved spectroscopy, TRS). Their ratio is determined by high frequency sinusoidal oscillation of the light intensity and phase sensitive detection of the signal (phase modulation spectroscopy, PMS). The location of the absorber is triangulated by in- phase and anti-phase laser diode arrays (phased arrays) based upon photon density interference (PDI) that afford angular accuracies of < 0.5o in 100 microliter volumes of 1 nmole absorber. Saturation of hemoglobin with oxygen (Y) is measured by the ratio of mua's at two appropriate wavelengths by TRS or the phase shifts at two wavelengths by PMS. The research program proposes to quantify mua and mus' of small samples of organelles, cells and biopsy/autopsy tissue samples that cover the expected range of parameters of host tissue and tumors. The research has three stages: Aim 1) Homogeneous scattering/absorber systems at multiple input/output positions, mua, mus', mus'/mua. Lipid vesicles, intracellular organelles (mitochondria etc. endoplasmic reticulum) and cells (ascites tumors and cytochrome alphaalpha3 free and cytochrome alphaalpha3 sufficient yeast (petit and wild) are used with varying amounts of hemoglobin or carboycyanine dyes as absorbers; Aim 2) The sensitivity in the detection of the differences of saturation of a localized blood volume with respect to a hemoglobin containing background will be evaluated by TRS, PMS, and PDI; and Aim 3) Heterogeneous systems with single and multiple localized absorbers (scatterers, fluorochromes) of various volumes and locations. In-phased and anti-phased arrays causing photon density interference (PDI) will be tested for precision of angular position as a function of sample volume, mua, mus', etc. Thus, the detectable fraction of the optical field containing altered mus' and mua and fluorescence will be measured in model systems and will afford a solid basis for prediction of resolution and precision of human tumor spectroscopy is to be obtained.

Ethanol is remarkable for the wide range of structural and functional changes induced in the liver. The metabolic changes are manifest as fatty infiltration, alcoholic hepatitis, cirrhosis, and fibrosis. These are attributable in part to the direct effects of ethanol upon the redox state of the cytosolic and mitochondrial components which in turn can impact upon major pathways of metabolism. Of great interest is the effect of ethanol upon the phosphorylation potential, a topic which can be investigated in great detail in this project because of the availability of transfected creatine kinase in the liver of the mice under study. Thus, this transfection has made it possible to compare metabolic control by inorganic phosphate (Pi) with control by adenosine diphosphate (ADP). A comparison of the response of these different controls to imposed stresses offer new opportunities to evaluate the metabolic effects of ethanol, free radicals and metabolic overload. At the same time, we will explore metabolic control in a system in which the phosphate potential and hence the thermodynamic properties have been altered ten fold by the transfection. A series of stresses are described, particularly that of the redox state by ethanol. A phosphorus nuclear magnetic resonance spectroscopy (31P NMR) and analytical biochemistry study of the PCr/Pi ratio and the pH values of perfused mouse liver over a range of metabolic and redox stresses will be conducted to evaluate the range of ADP control. Steady state and equilibrium values of substrate-couples, especially ethanol/aldehyde that control PP will be evaluated by 31P NMR, analytical biochemistry and freeze-trap redox studies of the flavoprotein/NADH (FP/NADH) ratio, especially ethanol. Natural or enhanced Chemiluminescence (CL) of radical oxidizing species (ROS) will be correlated with the redox state of the liver, and perturbed by ornithine stress. T-butyl-peroxide stress and alcohol stresses will be evaluated by 3-D freeze-trapped redox scanning. Resistance to stress will be evaluated by ROS and histopathological measurements.

DESCRIPTION (Adapted from Applicant's Abstract): The quantitative assessment of functional activity of skeletal tissues has been the subject of developments over many years of exercise oriented research: Many global whole body responses to exercise, particularly the body oxygen utilization, heart rate and power output. Invasive procedures of localized oxygen/pH plus needle biopsies and quick freezing provide insight on localized muscle function. Phosphorous Nuclear Magnetic Resonance (31P NMR) has provided both global and localized evidence of biochemical responses to muscle activity, particularly the rise of adenosine diphosphate and inorganic phosphate as a function of exercise level during exercise, and of recovery times thereafter, particularly in vascular disease. Optical measurements of muscle function originated in 1935 with studies of hemoglobin/myoglobin changes in the cat soleus muscle on a relative basis. Progress on quantifying Hb/Mb measurements enabled as the phenomenon of photon migration was recognized: separation, absorption and scattering properties of exercising muscle were made possible by application of the diffusion theory. This research emphasizes the application of quantitative tissue oximetry to muscle exercise in terms of Hb desaturation and blood concentration characteristic of the gamut of performance from dystrophies to elite. Furthermore, initial transients of muscle oxygenation (warm-up), together with the dynamics of recovery, will be quantified as they appear to a remarkable extent in mitopathies. Finally and most important, imaging of functional muscle is possible with simple multi-source multi-detector combinations using continuous light and as, the physiology may require, using frequency and time domain optical techniques. Thus, images of Hb/Mb deoxygenation and blood concentration in muscle function will be available. The population to be studied ranges from elite Olympic performers (skaters, sprinters, endurance performers) through the normal sedentary population to the geriatric population with special emphasis on exercise protocols for the mitochondrial myopathy and CHF populations. In sum, the two major obstacles of optical studies of the limb may be overcome in these studies, namely quantitation and localization. Furthermore, the application to medically needful populations where outcomes can be altered by quantitative localized studies is consonant with the goals of "Healthy People 2000".

technology /technique development; infrared radiation; model design /development; biomedical resource; biomedical equipment development;

DESCRIPTION (Adapted from Applicant's Abstract): Neurological activity underlying the function of the brain in a cognitive task can be reliably indicated by the closely associated local hemodynamic response. This response can characterize, not only functional activity, but also functional anatomy. PET is a technique that employs intravascular radio labeled positron emitting tracers and can measure regional cerebral blood flow. Functional (f)MRI uses blood oxygen level dependent imaging (BOLD) and affords information primarily dependent upon both blood oxygen, blood deoxyhemoglobin and on flow effects. For fMRI, particularly studies of the response of the primary visual cortex by photic stimulation where 5 sec after the stimulus, there is a large change of signal which is interpreted as a decrease of deoxyghemoglobin. Thus fMRI signal allows spatio and temporal coregistration and validation of the optical signal and will be used in 50 tests per year. Based upon optical studies of the exposed animal brain (Grinvald, A. et al. (1991)), both blood concentration and its oxygenation can be determined spectophotometrically. Transcranial studies of the adult human brain is made possible by photon migration techniques using continuous or rapidly modulated light, first used in the frequency domain to identify characteristics frequencies induced by functional activity of the prefrontal cortex (Change, B. et al.(1993)). Localization of the response in the pre-frontal cortex, a chronic problem with the optical method, is studied here by a 9-source, 4-detector continuous light imager (CWI) operating with a modified C.T. algorithm. This imager acquires data at 0.4 sec per point with an overall imaged acquisition and display time of ~10 sec per image. The imager gives shapes of blood oxygenation and blood concentration responses resolved to ~1 cm and showing magnitudes of response, approximately 0.1 deltaOD for sensory motor 0.03 deltaOD for occipital responses, and 0.03 deltaOD for cognitive responses based upon 182 images. The histogram displays validate the blood volume and oyxgenation increases in the pre-frontal region. The apparatus affords a measure of localized changes of these two parameters in response to a variety of cognitive tasks, particularly paired word analogies, and two types of pattern recognition. Since the apparatus is highly portable, economical and the data presented appear to be highly efficacious, basic research on pre-frontal responses of normal controls (200 tests/yr) and patients with mild traumatic injury (30 tests/yr) is proposed for year 1-4 with a more quantitative apparatus in year 3, and a hair penetrating apparatus in year 4 using fMRI co-registration as the "Gold Standard" (50/year).

neoplasm /cancer diagnosis

DESCRIPTION (Adapted from Applicant's Abstract): An intensive program of key improvement of the current TRS breast cancer imager will increase the number of detectors, the dynamic range, and the number of parameters measured leading to increased accuracy, resolution, area of coverage, dynamic range and feature detection ability based upon both absorption and fluorescence studies of intrinsic and extrinsic contrast agents. These improvements will be evaluated one by one by computer simulation and anecdotal human subject studies. Increased imaging performance will be studied using perturbative diffusive optical tomography (DOT) and will be tested with single and multiple optical heterogeneities as a function of source detector pairs, projection angles, frequencies and signal to noise ratios. These improvements will also be tested with anecdotal DOT images of human breast tumor exploiting these instrumental and theoretical improvements in which coregistration with gadolinium enhanced MRI fluorodeoxy glucose PET and histopathology afford gold standards. Anecdotal clinical data will then be processed for a) image reconstruction, and b) fitting procedures in MRI designated voxels. The primary images and fits will be based upon evaluation with gadolinium enhanced MRI or and with histopathology respectively. Other features, especially tumor oxygenation as estimated from hemoglobin saturation will be compared, for imaging reconstruction, with PET and, for tumor diagnostic capability, with histopathology. A similar two tiered analysis will be made of light scattering in normal and hormonally activated breast. In this way the capability of DOT for image reconstruction and feature extraction will be evaluated with adequate studies to ensure validation of these novel approaches to breast cancer detection. Large scale tests are available under other support where a direct comparison with not only MRI but also digital mammography, ultrasound and PET will secure the validation studies.

Algorithms; Body Tissues; Brain; Brain imaging; CRISP; Complement; Complement Proteins; Computer Retrieval of Information on Scientific Projects Database; Coupling; Data; Data Set; Dataset; Development; Elements; Encephalon; Encephalons; Flying body movement; Functional Imaging; Funding; Grant; Human; Human, General; Image; Imaging technology; Institution; Investigators; Man (Taxonomy); Man, Modern; Methods and Techniques; Methods, Other; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Optics; Physiologic Imaging; Research; Research Personnel; Research Resources; Researchers; Resolution; Resources; Scanning; Source; Spottings; System; System, LOINC Axis 4; Techniques; Technology; Tissues; Transmission; United States National Institutes of Health; brain visualization; detector; flying; imaging; novel; reconstruction; remote sensing; transmission process

brain imaging /visualization /scanning; prosencephalon; biomedical facility; bioimaging /biomedical imaging;

ABSTRACT NOT PROVIDED