Nuclear Magnetic Resonance Visibility Research Articles

In vitro and in vivo studies of 1H NMR visibility to detect deoxyhemoglobin and deoxymyoglobin signals in myocardium

1H nuclear magnetic resonance (NMR) spectroscopy can be used noninvasively to detect the proximal histidyl Nδ proton signals of deoxymyoglobin in the myocardium. However, the quantification of deoxymyoglobin is based on the assumption that the deoxymyoglobin signal detected is not contaminated by the deoxyhemoglobin signals contributed from the blood. The purpose of this study was to conduct in vitro and in vivo 1H NMR studies to examine the in vivo NMR visibility of deoxyhemoglobin in the myocardium. The results demonstrate that the NMR visibility of α and β subunits of deoxyhemoglobin is sensitive to the pulse width for spin excitation because of short T2 relaxation times, and they are not NMR visible in the canine myocardium in vivo at 4.7 T when a 0.5–1.0 msec long Gaussian excitation pulse is used. Therefore, the resonance peak detected at ∼72 ppm (relative to the water resonance) in the ischemic canine myocardium in vivo is dominated by deoxymyoglobin. Magn Reson Med 42:1–5, 1999. © 1999 Wiley-Liss, Inc.

In vitro and in vivo studies of 1H NMR visibility to detect deoxyhemoglobin and deoxymyoglobin signals in myocardium.

1H nuclear magnetic resonance (NMR) spectroscopy can be used noninvasively to detect the proximal histidyl N delta proton signals of deoxymyoglobin in the myocardium. However, the quantification of deoxymyoglobin is based on the assumption that the deoxymyoglobin signal detected is not contaminated by the deoxyhemoglobin signals contributed from the blood. The purpose of this study was to conduct in vitro and in vivo 1H NMR studies to examine the in vivo NMR visibility of deoxyhemoglobin in the myocardium. The results demonstrate that the NMR visibility of alpha and beta subunits of deoxyhemoglobin is sensitive to the pulse width for spin excitation because of short T2 relaxation times, and they are not NMR visible in the canine myocardium in vivo at 4.7 T when a 0.5-1.0 msec long Gaussian excitation pulse is used. Therefore, the resonance peak detected at approximately 72 ppm (relative to the water resonance) in the ischemic canine myocardium in vivo is dominated by deoxymyoglobin.

Effects of aminooxyacetate on glutamate compartmentation and TCA cycle kinetics in rat hearts.

The nonspecific transaminase inhibitor aminooxyacetate (AOA) has multiple influences on the dynamics of 13C appearance in glutamate in rat hearts as measured by 13C nuclear magnetic resonance (NMR) without altering O2 consumption or tricarboxylic acid (TCA) cycle flux. These include the following: 1) a reduced rate of 13C enrichment at glutamate C3 and C4; 2) a near coalescence of the C3 and C4 fractional enrichment curves; 3) a dramatic alteration in the time-dependent evolution of the glutamate C4 multiplets, C4S and C4D34; and 4) a decrease in the NMR visibility of glutamate. A fit of the 13C fractional enrichment curves of glutamate C4 and C3 in the absence of inhibitor to a kinetic model of the TCA cycle gave values for transaminase flux of 7.5 mumol.min-1.g dry wt-1 and TCA cycle flux of 7.5 mumol.min-1.g dry wt-1, thereby confirming reports by others that the kinetics of 13C enrichment of glutamate C3 and C4 in heart tissue is significantly affected by flux through reactions other than TCA cycle. The 13C fractional enrichment data collected in the presence of 0.5 mM AOA could not be fitted using this same kinetic model. However, kinetic simulations demonstrated that the time-dependent changes in C4S and C4D34 are only consistent with a 10-fold reduction in the size of intermediate pools undergoing rapid turnover in the TCA cycle. We conclude that inhibition of glutamic-oxalacetic transaminase by AOA effectively reduces the size of the alpha-ketoglutarate pool in rapid exchange with the TCA cycle. Our data indicate that changes in glutamate multiplet areas in the 13C NMR spectra of heart (as demonstrated by glutamate C4S and C4D34) are more sensitive to alterations in metabolic pool sizes in exchange with the TCA cycle than are measurements of 13C fractional enrichment at glutamate C3 and C4.

NMR visibility of Pi in perfused rat hearts is affected by changes in substrate and contractility.

Nuclear magnetic resonance (NMR) spectroscopy does not always detect the total metabolite content in isolated perfused rat hearts. Alterations in NMR peak areas therefore could be caused by a change either in the metabolite content per se or in its NMR visibility. We have therefore compared the ATP, phosphocreatine (PCr), and Pi content of hearts, as determined by 31P-NMR spectroscopy, with the total, chemically determined ATP, PCr, and Pi contents of the same hearts to determine the fractions, if any, that are NMR invisible under different perfusion conditions. The three perfusion buffers used contained 1) glucose, 2) glucose plus high K+, and 3) pyruvate. Fully relaxed 31P-NMR spectra were collected during the final 10 min of perfusion in each group, and the ATP, PCr, and Pi contents were quantified using methylene diphosphonate as an external standard. The hearts were then freeze-clamped and chemically assayed for ATP, PCr, and Pi. Under all three conditions, the NMR-determined ATP and PCr contents were almost identical to the chemically determined values. However, only a portion of the chemically determined Pi was NMR visible. During perfusion with glucose-containing buffer, 39 +/- 8% of the total Pi was NMR visible, and this decreased to 12 +/- 2% (P less than 0.01) during K+ arrest and to 9 +/- 5% (P less than 0.01) during perfusion with pyruvate-containing buffer. In conclusion, whereas the total cellular content of ATP and PCr is always NMR visible during normoxic perfusion, alterations in substrate and contractile status can change the fraction of NMR-visible Pi.

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment

Single and multiple quantum nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the motional dynamics of sodium and potassium ions in concentrated protein solution, represented in this study by cortical and nuclear bovine lens tissue homogenates. Both ions displayed homogeneous biexponential magnetic relaxation behavior. Furthermore, the NMR relaxation behavior of these ions in lens homogenates was consistent either with a model that assumed the occurrence of two predominant ionic populations, "free" and "bound," in fast exchange with each other or with a model that assumed an asymmetric Gaussian distribution of correlation times. Regardless of the model employed, both ions were found to occur in a predominantly "free" or "unbound" rapidly reorienting state. The fraction of "bound" 23Na+, assuming a discrete two-site model, was approximately 0.006 and 0.017 for cortical and nuclear homogenates, respectively. Corresponding values for 39K+ were 0.003 and 0.007, respectively. Estimated values for the fraction of "bound" 23Na+ or 39K+ obtained from the distribution model (tau C greater than omega L-1) were less than or equal to 0.05 for all cases examined. The correlation times of the "bound" ions, derived using either a two-site or distribution model, yielded values that were at least one order of magnitude smaller than the reorientational motion of the constituent lens proteins. This observation implies that the apparent correlation time for ion binding is dominated by processes other than protein reorientational motion, most likely fast exchange between "free" and "bound" environments. The results of NMR visibility studies were consistent with the above findings, in agreement with other studies performed by non-NMR methods. These studies, in combination with those presented in the literature, suggest that the most likely role for sodium and potassium ions in the lens appears to be the regulation of cell volume by affecting the intralenticular water chemical potential.

Determination of fixed charge density in cartilage using nuclear magnetic resonance.

Many biomechanical and chemical properties of cartilage are dependent on the fixed charge density (FCD) of the extracellular matrix. In this study, nuclear magnetic resonance (NMR) spectroscopy was investigated as a nondestructive technique for determining FCD in cartilage. Sodium content was measured by NMR in cartilage explants and was compared with sodium content measured by inductively coupled plasma emission spectroscopy (ICP) in order to verify the total NMR visibility of sodium in cartilage. The ratio of NMR to ICP results was 1.02 +/- 0.04 (calf, mean +/- SD, n = 7) and 1.04 +/- 0.11 (adult bovine, n = 8). Sodium concentration as measured by NMR was then used with ideal Donnan theory to compute estimates of FCD. For calf articular cartilage (AC) near physiological conditions, calculated FCD was -0.28 +/- 0.03 M (n = 10). NMR measurements were then made for individual cartilage specimens sequentially equilibrated in baths of differing salt composition, pH, or ionic strength. For calf and adult AC, calculated FCD decreased dramatically between pH 3 and 2, with adult specimens becoming positively charged but calf tissue retaining a net negative charge. For calf AC equilibrated in 0.3-0.015 M NaCl, calculated FCD was observed to decrease slightly with decreasing bath ionic strength. For epiphyseal cartilage, FCD varied with the position of origin of the explant within the joint, ranging from -0.19 to -0.35 M in a manner that correlated with tissue glycosaminoglycan content. Preliminary NMR imaging experiments demonstrated similar variations of sodium concentration in intact ulnar epiphyseal cartilage. Collectively, these results demonstrate the ability of NMR to nondestructively follow FCD in cartilage. The technique is applicable to dynamic studies as well as to both in vitro and in vivo studies on living tissue.

Nuclear magnetic resonance monitoring of sodium in biological tissues.

In recent year, the 23Na nuclear magnetic resonance (NMR) technique has been applied to the study of biological tissues. The advantages of this method are noninvasiveness and good sensitivity. The resonances of the intra- and extra-cellular sodium can be separated by the addition of shift reagents to the extracellular compartment. The method has been mostly applied to cell suspensions, kidney tubules, glands, and small organs. Owing to line-broadening effects, the NMR visibility of the intracellular sodium is reduced to 40% in most cases but can be lower or higher. Time-dependent measurements are possible with adequate life-supporting equipment, allowing the determination of transport parameters. 23Na relaxation times are short in tissues (below 50 ms) and highly dependent on the medium composition. The application of the 23Na NMR technique to intact organs can be hampered by the difficulty of getting a good distribution of the shift reagent in the extracellular milieu. A summary of the studies performed is presented with specific examples to illustrate typical applications.

Assessment of the NMR visibility of intraerythrocytic sodium by flame photometric and ion-competitive studies.

23Na nuclear magnetic resonance (NMR) studies of intraerythrocytic sodium concentrations were performed at 37 degrees C and the accuracy of the method was evaluated through concomitant emission flame photometric analysis. NMR visibility of intraerythrocytic sodium was estimated to be 75 +/- 3%. Verification of the presence of an NMR invisible intraerythrocytic sodium pool and assessment of the mechanisms explaining this invisibility was achieved by measurement of 23Na NMR signal intensities and T1 relaxation times of erythrocyte hemolysate during ion-competitive sodium release from association sites. Signal intensities after addition of small quantities of K+ and Mg2+ to the hemolysates were significantly larger than that expected from dilution of the sample, implying that the additions have resulted in the release of formerly invisible sodium. Furthermore, the intensities following the addition of K+ were significantly larger than those after the addition of Li+ and choline+. This signifies a specificity of the association sites at which sodium has decreased visibility. Increased T1 values following the additions confirmed the release of bound sodium.

Compartmentation of Nucleotides in Corn Root Tips Studied by 31P-NMR and HPLC

Corn (Zea mays L.) root tips were subjected to different conditions so that nucleotide levels varied over a wide range. Levels of nucleotides in corn root tips were measured using (31)P nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography. Results indicate: (a) Similar amounts of NTP and sugar nucleotides were observed by in vivo NMR and in extracts. In contrast, a significant amount of NDP observed in root tip extracts was not detected by in vivo NMR. Thus, for a given sample, [NTP]/[NDP] ratios determined in vivo by (31)P-NMR are always higher than ratios observed in extracts, deviating by approximately 4-fold at the highest ratios. The NMR-invisible pool of NDP appeared quite metabolically inert, barely changing in size as total cell NDP changed. We conclude that NDP in corn root tips is compartmented with respect to NMR visibility, and that it is the NMR-visible pool which responds dynamically to metabolic state. The NMR-invisible NDP could either be immobilized (and so have broad, undetectable NMR signals), or be complexed with species that cause the chemical shift of NDP to change (so it does not contribute to the NMR signal of free NDP), or both. (b) (31)P-NMR cannot distinguish between bases (A, U, C, and G) of nucleotides. HPLC analysis of root tip extracts showed that the relative amount of each base in the NTP and NDP pools was quite constant in the different samples. (c) In extracts, for each of the nonadenylate nucleotides, [NTP]/[NDP] was linearly proportional to [ATP]/[ADP], indicating near equilibrium in the nucleoside diphosphokinase (NDPK) reaction. However, the apparent equilibrium constants for the phosphorylation of GDP and UDP by ATP were significantly lower than 1, the true equilibrium constant for the NDPK reaction. Thus, for a given sample, [ATP]/[ADP] approximately [CTP]/[CDP] > [UTP]/[UDP] > [GTP]/[GDP]. This result suggests that the different NDPs in corn root tips do not have equal access to NDPK.

Measurement of tissue potassium in vivo using 39K nuclear magnetic resonance

39K nuclear magnetic resonance (NMR) spectra were readily obtained, in vivo, from rat muscle, kidney, and brain in 5-10 min with signal-to-noise ratios of approximately 20:1. Quantitation of the K+ signal was achieved by reference to an external standard of KCl/dysprosium nitrate as well as by reference to the proton signal from tissue water. In vitro NMR studies of isolated tissue showed a K+ visibility (NMR K+/total tissue K+) of 96%, 62 +/- 8%, 47 +/- 1.9%, 45 +/- 3.5%, and 43 +/- 2.5% for blood, brain, muscle, kidney, and liver, respectively. Absolute tissue K+ was determined by flame photometry of acid-digested tissue. Changes in tissue K+ status by chronic K+ depletion or acute K+ loading produced changes of 39K NMR signal intensity that were equal to changes of absolute tissue K+. Acidosis, alkalosis, mannitol, or RbCl infusion did not significantly change the NMR K+ signal. These results indicate that the changes in K+ detected by NMR were specifically and accurately detected. To investigate the factors that affect the 39K NMR signal, the effects of liver homogenate on 39K NMR signal intensity were studied. Addition of homogenate produced a 60% loss of signal intensity, suggesting that a large portion of cell K+ may be only 40% visible. Addition of RbCl to undiluted homogenate increased the NMR K+ signal by 11 +/- 2 mumol/g. Addition of H2O or NaCl had no effect, suggesting that Rb+ was replacing K+ in sites of low (less than 40%) NMR visibility. These results demonstrate that 39K NMR experiments can be performed using intact organs. To explain the lack of detectable K+ and changes in K+ NMR visibility, a three compartment model is proposed.

NMR measurements of intracellular sodium in the rabbit proximal tubule

The present study evaluated the use of nuclear magnetic resonance (NMR) spectroscopy to monitor directly and continuously intracellular sodium levels in rabbit renal cortical tubule suspensions. When the paramagnetic shift reagent dysprosium tripolyphosphate was added to the extracellular medium it was possible to resolve signals from intracellular and extracellular sodium without adversely affecting cellular viability. An efflux of intracellular sodium against a significant concentration gradient was observed when sodium-loaded cells were warmed from 4 to 37 degrees C. At 37 degrees C in steady state, inhibition of Na+-K+-ATPase activity by ouabain increased intracellular sodium content in a dose-dependent and time-dependent manner. A biphasic time course of increased intracellular sodium following ouabain (10(-3) M) suggested that the sodium permeability of the plasma membrane may decrease following pump inhibition, thus limiting sodium influx. Nystatin, an agent known to facilitate sodium entry across cell membranes, increased intracellular sodium fivefold. In another series of experiments several maneuvers were performed to ascertain the fraction of intracellular sodium that was NMR visible. Quantitative assessment of either an efflux or influx of sodium indicated that the NMR visibility of the transported sodium was 100%. Furthermore, disruption of the cell membranes with Triton X-100 showed that the entire pool of intracellular sodium was 100% NMR visible.(ABSTRACT TRUNCATED AT 250 WORDS)

23Na and 39K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intra- and extracellular ions using a shift reagent

High-resolution 23Na and 39K nuclear magnetic resonance (NMR) spectra of perfused, beating rat hearts have been obtained in the absence and presence of the downfield shift reagent Dy(TTHA)3- in the perfusing medium. Evidence indicates that Dy(TTHA)3- enters essentially all extracellular spaces but does not enter intracellular spaces. It can thus be used to discriminate the resonances of the ions in these spaces. Experiments supporting this conclusion include interventions that inhibit the Na+/K+ pump such as the inclusion of ouabain in and the exclusion of K+ from the perfusing medium. In each of these experiments, a peak corresponding to intracellular sodium increased in intensity. In the latter experiment, the increase was reversed when the concentration of K+ in the perfusing medium was returned to normal. When the concentration of Ca2+ in the perfusing medium was also returned to normal, the previously quiescent heart resumed beating. In the beating heart where the Na+/K+ pump was not inhibited, the intensity of the intracellular Na+ resonance was less than 20% of that expected. Although the data are more sparse, the NMR visibility of the intracellular K+ signal appears to be no more than 20%.

In vivo NMR imaging of sodium-23 in the human head.

We report the first clinical nuclear magnetic resonance (NMR) images of cerebral sodium distribution in normal volunteers and in patients with a variety of pathological lesions. We have used a 1.5 T NMR magnet system. When compared with proton distribution, sodium shows a greater variation in its concentration from tissue to tissue and from normal to pathological conditions. Image contrast calculated on the basis of sodium concentration is 7 to 18 times greater than that of proton spin density. Normal images emphasize the extracellular compartments. In the clinical studies, areas of recent or old cerebral infarction and tumors show a pronounced increase of sodium content (300-400%). Actual measurements of image density values indicate that there is probably a further accentuation of the contrast by the increased "NMR visibility" of sodium in infarcted tissue. Sodium imaging may prove to be a more sensitive means for early detection of some brain disorders than other imaging methods.