Proton Nuclear Magnetic Resonance Relaxation Measurements in Frog Muscle

Proton nuclear magnetic resonance (NMR) relaxation measurements are reported for frog muscle as a function of temperature and Larmor frequency. Each T_1ρ, T₂, and T₁ measurement covered a time domain sufficient to identify the average relaxation time for most intracellular water. Using regression analysis the data were fit with a model where intracellular water molecules are exchanging between a large compartment in which mobility is similar to ordinary water and a small compartment in which motion is restricted. The regression results suggest that: the restricted compartment exhibits a distribution of motions skewed toward that of free water; the residence time of water molecules in the restricted compartment is approximately 1 ms; and, the activation entropy for some water molecules in the restricted compartment is negative.     

Estimation of the Freezing Injury in Flower Buds of Evergreen Azaleas by Water Proton Nuclear Magnetic Resonance Relaxation Times

Temperature dependence of longitudinal relaxation times (T₁)of water protons in flower buds of six azalea species differingin cold hardiness and ecological distribution was investigatedby pulse nuclear magnetic resonance spectroscopy. Thermal hysteresiswas observed for T₁ following a slow freeze-thaw cycle. TheT₁ ratio (the ratio obtained from the difference between theoriginal T₁ value in an unfrozen sample and the final T₁ aftera freeze-thaw treatment, both at 20C, divided by the originalT₁) was closely correlated with the viability of florets innon-acclimated buds of R. kiusianum. If the buds were frozento a lethal temperature and then thawed to 20C, the T₁ ratioincreased. The T₁ ratios of acclimated winter buds for the sixspecies used were correlated with the level of cold hardiness(supercooling ability of florets determined by differentialthermal analysis). The T₁ ratio of deacclimated spring buds,especially those from hardier species, markedly increased uponcooling to a lethal temperature. Species differences observedin acclimated winter buds disappeared upon deacclimation. TheT₁ ratio appears to be related to the viability of florets andthe degree of freezing damage (membrane disruption) in florets. (Received December 28, 1984; Accepted May 24, 1985)     

Direct Evidence from Nuclear Magnetic Resonance Studies for Bound Sodium in Frog Skeletal Muscle

The recent work of Cope on ²³Na magnetic resonance studies of frog muscle has been repeated with the view of investigating certain objections which can be raised concerning the original studies. The present work leads to the conclusion that Cope's results concerning bound sodium are essentially correct in that a large fraction of the ²³Na present does not contribute normally to a detectable nuclear magnetic resonance (NMR) signal. This “missing” signal can be detected at high radio-frequency intensity however, and a signal-saturation study distinctly reveals its presence.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Transverse Nuclear Relaxation in Lecithin Bilayers

IN recent months a great deal of interest has been aroused by the study of the motion of the paraffinic chains in the lyotropic mesophases of the lipid-water and soap-water systems^1–6. Apart from being interesting in themselves, such systems can eventually lead to a better understanding of the structure and function of biological membranes. For this reason, the lecithin-water system has received special attention⁷.     

Nuclear Magnetic Resonance Evidence using D2O for Structured Water in Muscle and Brain

The electric quadrupole moment of the deuterium nucleus provides a nuclear magnetic resonance (NMR) probe of electric field gradients, and thereby of organization of tissue water. 8-17% of H₂O in rat muscle and brain was replaced by D₂O from 50% deuterated drinking water. The peak height of the steady-state NMR spectrum of D in muscle water was 74% lower than that of an equal concentration of D₂O in liquid water. Longitudinal NMR relaxation times (T₁) of D in water of muscle and brain averaged 0.092 and 0.131 sec, respectively, compared with 0.47 sec in D₂O in liquid water. Transverse NMR relaxation times (T₂) averaged 0.009 and 0.022 sec in D₂O of muscle and brain, respectively, compared with 0.45 sec in D₂O in liquid water. These differences cannot be explained by paramagnetic ions or by magnetic inhomogeneities, which leaves increased organization of tissue water as the only tenable hypothesis. Evidence was also obtained that 27% of muscle water and 13% of brain water exist as a separate fraction with T₂ of D₂O less than 2 × 10^-3 sec, which implies an even higher degree of structure. Each of the two fractions may consist of multiple subfractions of differing structure.     

Characteristic Changes in Relaxation Times of Water Protons in Vigna radiata Seedlings Exposed to Temperature Stress

Water protons in hypocotyl tissues from etiolated seedlingsof Vigna radiata that were exposed to temperature stress showedcharacteristic relaxation behaviors for ¹H-NMR. Cold stresstreatment (0C) caused gradual prolongation of NMR relaxationtimes (T₁). After exposure of tissues to cold stress for 24h, T₁ returned to the initial value as a result of subsequentincubation at normal temperature (20C). By contrast, heat stresstreatment (40C) induced a time-dependent decrease in T₁, whichdid not return completely to the initial value upon subsequentincubation at 20C after exposure to heat stress for 4 h. Weexamined changes in various physical factors that influencethe response of T₁ to temperature stress, namely, water contentand the concentrations of protein, diamagnetic (K⁺, Na⁺, Ca²⁺and Mg²⁺) and paramagnetic (Mn²⁺ and Fe²⁺) ions in the tissues.From the relationships between T₁ and these factors in vitro,we could not interpret the responses of T₁ to the temperaturestress only in terms of a change in water content. A synergisticeffect of an Mn²⁺ -protein complex and pH might be essentialfor the mechanism of changes in T₁ that are due to cold stress.The influence of heat stress on structural water in tissuesis discussed in terms of water-protein interactions. (Received December 28, 1992; Accepted May 6, 1993)     

A Nuclear Magnetic Resonance Study of Water in Cold-acclimating Cereals

Continuous wave nuclear magnetic resonance (NMR) studies indicated that the line width of the water absorption peak (Δv½) from crowns of winter and spring wheat (Triticum aestivum L.) increased during cold acclimation. There was a negative correlation between Δv½ and crown water content, and both of these parameters were correlated with the lowest survival temperature at which 50% or more of the crowns were not killed by freezing (LT₅₀). Regression analyses indicated that Δv½ and water content account for similar variability in LT₅₀. Slow dehydration of unacclimated winter wheat crowns by artificial means resulted in similarly correlated changes in water content and Δv½. Rapid dehydration of unacclimated crowns reduced water content but did not influence Δv½. The incubation of unacclimated winter wheat crowns in a sucrose medium reduced water content and increased Δv½. The increase in Δv½ appears to be dependent in part on a reduction in water content and an increase in solutes.     

Relationship of Nuclear Magnetic Resonance Relaxation Time to Water Content and Cold Hardiness in Flower Buds of Evergreen Azalea

The longitudinal relaxation time (T₁) of water protons in floretsof R. ? akebono flower buds was measured with a pulse NMR spectrometerto determine the relationship of T₁ to water content and coldhardiness (supercooling ability). Seasonal changes of T₁ inflorets were closely correlated with water content and supercoolingability of florets. T₁ of florets was short for acclimated budshaving a low water content and long for non-acclimated budshaving a high water content. Flower buds collected in Novemberand stored at 0 and 5?C for 4 weeks had shorter T₁ values thanbuds stored at 10?C even though the floret water content andsupercooling ability were similar. Thus, the short T₁ of coldacclimated buds hardened naturally or by storage at low temperaturesis due to a combination of both reduced water content and temperature. (Received August 27, 1983; Accepted May 26, 1984)     

Hyperpolarized Functional Magnetic Resonance of Murine Skeletal Muscle Enabled by Multiple Tracer-Paradigm Synchronizations

Measuring metabolism''s time- and space-dependent responses upon stimulation lies at the core of functional magnetic resonance imaging. While focusing on water''s sole resonance, further insight could arise from monitoring the temporal responses arising from the metabolites themselves, in what is known as functional magnetic resonance spectroscopy. Performing these measurements in real time, however, is severely challenged by the short functional timescales and low concentrations of natural metabolites. Dissolution dynamic nuclear polarization is an emerging technique that can potentially alleviate this, as it provides a massive sensitivity enhancement allowing one to probe low-concentration tracers and products in a single-scan. Still, conventional implementations of this hyperpolarization approach are not immediately amenable to the repeated acquisitions needed in real-time functional settings. This work proposes a strategy for functional magnetic resonance of hyperpolarized metabolites that bypasses this limitation, and enables the observation of real-time metabolic changes through the synchronization of stimuli-triggered, multiple-bolus injections of the metabolic tracer ¹³C₁-pyruvate. This new approach is demonstrated with paradigms tailored to reveal in vivo thresholds of murine hind-limb skeletal muscle activation, involving the conversion of ¹³C₁-pyruvate to ¹³C₁-lactate and ¹³C₁-alanine. These functional hind-limb studies revealed that graded skeletal muscle stimulation causes commensurate increases in glycolytic metabolism in a frequency- and amplitude-dependent fashion, that can be monitored on the seconds/minutes timescale using dissolution dynamic nuclear polarization. Spectroscopic imaging further allowed the in vivo visualization of uptake, transformation and distribution of the tracer and products, in fast-twitch glycolytic and in slow-twitch oxidative muscle fiber groups. While these studies open vistas in time and sensitivity for metabolic functional magnetic resonance studies in muscle, the simplicity of our approach makes this technique amenable to a wide range of functional metabolic tracer studies.     

Transverse Impedance of Single Frog Skeletal Muscle Fibers

The transverse electrical impedance of single frog skeletal muscle fibers was measured at 31 frequencies that ranged from 1 to 100,000 Hz. Each fiber was bathed entirely in Ringer's solution, but it was positioned so that a central length of 5 mm was in a hollow plastic disk and was electrically isolated from the ends of the fiber. The diameter of the segment of the fiber in the disk was measured and then the segment was pressed from opposite sides by two insulating wedges. Electrical current was passed transversely through the segment between two platinum-platinum black electrodes that were located in the pools of Ringer's solution within the disk. The results were corrected for stray parallel capacitance, series resistance of the Ringer's solution between the fiber and the electrodes, parallel shunt resistance around the fiber, and the phase shift of the measuring apparatus. A nonlinear least-squares routine was used to fit a lumped equivalent circuit to the data from six fibers. The equivalent circuit that was chosen for the fibers contained three parallel branches; each branch was composed of a resistor and a capacitor in series. The model also included a seventh adjustable parameter that was designed to account for the degree of compression of the fibers by the insulating wedges. The branches of the equivalent circuit were assumed to represent the electrical properties of: (a) the myoplasm in series with the membrane capacitance that was exposed directly to the pools of Ringer's solution; (b) the capacitance and series resistance of the transverse tubules that were exposed directly to the pools of Ringer's solution; (c) the membrane capacitance in series with the shunt resistance between the fibers and the insulating wedges. The results gave no indication that current entered the sarcoplasmic reticulum.     

Nuclear Magnetic Resonance Studies of Sodium Ions in Isolated Frog Muscle and Liver

Nuclear magnetic resonance (NMR) was used to determine Na⁺ complexing in muscle and liver (at 23°C) from bullfrogs (Rana catesbeiana) and to study the influence of temperature on Na⁺ complexing in muscle from leopard frogs (Rana pipiens). The Na⁺ complexed in muscle and liver was found to be 36.6 ± 4.6% and 66.1 ± 3.5% respectively. A temperature decrease from +34°C to -2°C results in a 20% decrease in the mobility of the free Na⁺ in the fresh muscle. This 20% decrease in mobility results in about 50% of the free Na⁺ at 34°C being complexed at the lower temperature.     

Nuclear Magnetic Resonance Studies on Intracellular Sodium in Human Erythrocytes and Frog Muscle

The nuclear magnetic resonance (NMR) spectrum of sodium was determined in muscle and erythrocytes using conventional continuous wave techniques. NMR spectra of fresh intact muscle revealed a single line with a width of about 38 Hz equivalent in intensity to about 53% of the total muscle sodium, in general agreement with previous work. Prolonged washing with sodium-free solutions led to a marked loss of both total and NMR-detectable sodium. The NMR-visible sodium remaining in the muscle was somewhat larger than the fraction calculated to remain extracellular and, presumably, was intracellular. The original sodium signal is thus interpreted as arising from both extracellular sodium and the narrow line portion of the signal from intracellular sodium. NMR spectra of sodium were also obtained for human erythrocytes under conditions preserving the sodium transport system. The intensity of the sodium signal in fresh cells was 98% of that present in the same samples after complete hemolysis of the cells. The NMR sodium present in intact cells was 92% of the sodium recovered by flame photometric determination of sodium from ashed samples. It is concluded that no NMR-“invisible” sodium occurs in human erythrocytes and that the presence of such sodium is not necessary for the normal functioning of the sodium transport system in erythrocytes.     

17O Nuclear Magnetic Resonance Spectrum of H217O in Frog Striated Muscle

Whole striated muscles from the frog Rana esculenta were bathed in Ringer's solution enriched with H₂¹⁷O; the muscle water was subsequently collected by vacuum distillation. The integrated intensity of the nuclear magnetic resonance (NMR) signal of ¹⁷O in the muscle was measured to be approximately ¾ of the signal observed in the distilled water. The phenomenon may arise either from immobilization of a population of the water molecules which may be a very small fraction or as much as ¼ of the total, or may reflect tumbling of ⅓ of the water molecules in a compartment containing an anisotropic medium. Such an effect was demonstrated for H₂¹⁷O using the model system of sodium linoleate in water.     

Diffusion of Exchangeable Water in Cortical Bone Studied by Nuclear Magnetic Resonance

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D_a = (7.8 ± 1.5) × 10⁻⁷ cm²/s) measured at 40°C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D_a measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D_a perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (∼30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.     

Estimation of Chilling Sensitivity and Injury in Gloxinia Leaves by the Thermal Hysteresis of NMR Relaxation Times of Water Protons

Thermal hysteresis of longitudinal relaxation times (T₁) ofwater protons in leaf tissues of gloxinia was investigated bypulse nuclear magnetic resonance (NMR) spectroscopy. The profilesof T₁ hysteresis during a slow cool-warm cycle (20 to 0?C) variedwith the degree of chilling injury. General trends of T₁ changesupon warming were as follows: (1) a small increase of T₁ wasobserved when no chilling injury occurred or the degree of injurywas minor, (2) a large increase of T₁ occurred when injury increased,(3) a large decrease of T₁ occurred from the earlier stage ofwarming when serious injury occured. The sum of T₁ ratio (theratio obtained from the difference between the T₁ value in thecooling process and that of the wanning process for eight stepsof cool-warm cycle, divided by the former) reflecting T₁ increaseor decrease during the warming process was related to varietaldifference and seasonal changes of chilling sensitivity. Therefore,T₁ hysteresis can be used as a diagnostic tool in detectingthe chilling sensitivity and the degree of chilling injury. (Received August 27, 1986; Accepted February 24, 1987)     

Evanescent Waves Nuclear Magnetic Resonance

Nuclear Magnetic Resonance spectroscopy and imaging can be classified as inductive techniques working in the near- to far-field regimes. We investigate an alternative capacitive detection with the use of micrometer sized probes positioned at sub wavelength distances of the sample in order to characterize and model evanescent electromagnetic fields originating from NMR phenomenon. We report that in this experimental configuration the available NMR signal is one order of magnitude larger and follows an exponential decay inversely proportional to the size of the emitters. Those investigations open a new road to a better understanding of the evanescent waves component in NMR with the opportunity to perform localized spectroscopy and imaging.     

Transverse Relaxation Time of Leaf Water Protons and Membrane Injury in Wheat (Triticum aestivum L.) in Response to High Temperature

The relationship between high temperature stress injury andtemperature dependence of the transverse relaxation time (T₂)of leaf water was examined using NMR in four cultivars of wheatdiffering in their sensitivity to high temperature stress. TheT₂declined with increasing temperature between 25 and 35 °C.A comparison of relative injury based on electrolyte leakageand T₂, between 40 and 50 °C, indicated that while membranepermeability increased with increasing temperature there wasan increase in T₂until 44 and 48 °C in susceptible and tolerantcultivars respectively, followed by a sharp decline. This patternof change in T₂with increasing temperature was consistent whetherthe same or different samples were used for each treatment temperature.Loss of temperature dependence of T₂after heat killing indicatedirreversible changes in T₂, probably due to the loss of membraneintegrity. Heat tolerant varieties, which suffered less membraneinjury, had a higher T₂compared to susceptible varieties. Tolerantvarieties also maintained the T₂of leaf water protons to highertemperatures than did sensitive varieties. This NMR-based, non-invasive,rapid technique could be used to efficiently detect heat injuryin leaf tissues. Copyright 1999 Annals of Botany Company Membrane integrity, transverse relaxation time, high temperature stress, Triticum aestivum L.     

A Nuclear Magnetic Resonance Study of Hydrated Systems Using the Frequency Dependence of the Relaxation Processes

A practical method is described for determining some characteristics of the spectrum of proton mobilities in a hydrated system from the frequency dependence of the nuclear magnetic resonance (NMR) relaxation processes. The technique is applied to water in association with agarose and gelatin. The results for agarose are consistent with the hypothesis that a fraction of the protons is distributed over states of reduced mobility and exchanges rapidly with the remaining fraction which is attributed to water in the normal state. No variation in the characteristics of the modified fraction could be detected for water concentrations in the range 1.2-50 g H₂O/g agarose. Within the modified fraction, higher mobilities are more common than low mobilities; at 1.2 g H₂O/g agarose, not more than 10% of the proton population has mobilities more than 100 times smaller than normal. The modified proton fraction is tentatively identified with agarose hydroxyl protons and possibly water molecules bound to the polymer. Proton states with mobilities intermediate between water and ice have also been detected in hydrated gelatin. As in agarose, higher mobilities are the most common. In contrast to agarose, the characteristics of the modified proton states are markedly dependent on water concentration. They are tentatively attributed to gelatin protons coupled for spinlattice relaxation with those of the bulk phase by exchange and spin diffusion.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement