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This study tested the ability of MR relaxography (MRR) to discriminate intra- (Nai+) and extracellular (Nae+)23Na+ signals using their longitudinal relaxation time constant (T1) values. Na+-loaded yeast cell (Saccharomyces cerevisiae) suspensions were investigated. Two types of compartmental 23Na+T1 differences were examined: a selective Nae+T1 decrease induced by an extracellular relaxation reagent (RRe), GdDOTP5-; and, an intrinsic T1 difference. Parallel studies using the established method of 23Na MRS with an extracellular shift reagent (SRe), TmDOTP5-, were used to validate the MRR measurements. With 12.8 mM RRe, the 23Nae+T1 was 2.4 ms and the 23Nai+T1 was 9.5 ms (9.4 T, 24 degrees C). The Na+ amounts and spontaneous efflux rate constants were found to be identical within experimental error whether measured by MRR/RRe or by MRS/SRe. Without RRe, the Na+-loaded yeast cell suspension 23Na MR signal exhibited two T1 values, 9.1 (+/-0.3) ms and 32.7 (+/-2.3) ms, assigned to 23Nai+ and 23Nae+, respectively. The Nai+ content measured was lower, 0.88 (+/-0.06); while Nae+ was higher, 1.43 (+/-0.12) compared with MRS/SRe measures on the same samples. However, the measured efflux rate constant was identical. T1 MRR potentially may be used for Nai+ determination in vivo and Na+ flux measurements; with RRe for animal studies and without RRe for humans., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Frequency shift/concentration calibration curves applicable to the use of shift reagents (SRs) for in vivo 23Na MRS studies can be obtained from experiments with whole blood. Here, they are reported for titrations of rat blood with the SRs DyTTHA3- and TmDOTP5-. There are a number of considerations that must be made in order to derive accurate calibration curves from the experimental data. These include the effects of bulk magnetic susceptibility (BMS, since the SRs are paramagnetic), the effects of water flux (since addition of the SR stock solution to blood renders the plasma hyperosmotic), and the consequences of restricted distribution of the SR anion in the erythrocyte suspension. We give in some detail the BMS shift theory that obtains in this case and show also how it applies to excised perfused organ as well as in vivo studies. Also, we report significant effects of adjuvant Ca2+ additions in the TmDOTP5- titrations. These are very important to the successful use of this SR in vivo. Finally, our considerations of BMS lead naturally to an understanding of its manifestations in the shifts of the 1H2O resonance frequencies of cell suspensions and tissues induced by SRs. Since these are being increasingly reported, and often misinterpreted, we devote an experiment and some discussion to this subject. We show that this phenomenon cannot be used to quantitatively discriminate intra- and extracellular 1H2O signals.

The comprehensive approach described in the accompanying paper is illustrated here with the 23Na signal of a concentrated solution of bovine serum albumin (BSA) in saline and the intracellular (Nai) 23Na resonance of a dense suspension of Na(+)-loaded yeast cells. We use frequency shift reagents to discriminate the latter from the extracellular resonance. We find that the Nai signal corresponds to that of an effective single population of Na+ ions exhibiting a single type c spectrum. This is true despite the fact that the yeast protoplasm is too large and too compartmentalized for a given Na+ ion to sample its entirety on the relevant NMR timescale. Our results show clearly that, in addition to the decay of transverse magnetization, the recovery of longitudinal magnetization is biexponential. This is required for a type c spectrum but has not often been detected. The temperature dependence of the relaxation rate constants of the Nai resonance is not consistent with either a simple Debye process or a discrete exchange mechanism connecting two sites in the fast limit. We have fitted the data using an asymmetric continuous distribution of correlation times for the fluctuations of electric field gradients sensed by the Nai nuclei. The analogous distribution function for the Na+ in a 44% (w/w) BSA solution is quite similar to that of the Nai at the same temperature. This suggests that while the macromolecular environment of the Nai ions is quite congested, it is also isotropic on quite a small spatial scale. Also, one can use the correlation time distribution function, obtained from fitting the relaxation data, to calculate a relaxometry curve. This is useful because experimental 23Na relaxometry is difficult. The calculated curve may be a reasonable model for the mostly extracellular 23Na resonance encountered in vivo.

An extensive protocol for the study of tissue resonances of spin 3/2 nuclei is described. The roles of the most relevant multiple pulse experiments are indicated. Their theory is organized in terms of irreducible tensor operators and the pulse and quadrupolar relaxation transfer functions which relate them for a type c spectrum. A systematic approach to the interpretation of the temperature and/or magnetic field dependences of all six of the relaxation rate constants of the resonance of a single population of isolated spins in fast exchange, and giving rise to a type c spectrum, is presented. An experimental calibration and an application of this protocol are presented in an accompanying paper. The comprehensive method we describe has a number of practical benefits in the interpretation of the physiological spectra obtained from conventional one pulse experiments. A consideration of the appropriate transverse relaxation transfer function leads to an analytical expression for the heretofore empirical NMR visibility factor. This includes factors which account for relaxation during the receiver 'dead' time and relaxation during the pulse itself. Also, consideration of realistic transverse relaxation times likely to be observed in tissue leads to a reasonable strategy for the quantitative resolution and integration of in vivo spectra obtained in the presence of hyperfine shift reagents.

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)

A new nuclear magnetic resonance (NMR) method for monitoring transmembrane metal cation transport is reported. It is illustrated with a study of Na(+) efflux from Na(+)-rich yeast cells. The technique involves the use of an anionic paramagnetic shift reagent, present only outside the cells, to induce a splitting of the sodium-23 NMR peak, in this case, into components representing intra- and extracellular Na(+). The time course of the efflux is in good agreement with the literature and can be well fitted with a double exponential decay expression. Splitting of the lithium-7 NMR signal from a suspension of Li(+)-rich respiratory-deficient, petite yeasts is also reported.

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%.

23Na NMR studies of large unilamellar vesicles of egg lecithin in salt solutions are reported. A shift reagent, the dysprosium nitrilotriacetate ion Dy[N(CH2CO2)3]3-2 has been used to distinguish between 23Na+ inside and outside the vesicles. When both are present and the shift reagent is present on only one side, two clearly distinct resonances are observed. Creation of a Na+ concentration gradient and subsequent catalysis of passive transport induced by the introduction of gramicidin can be monitored easily by using the relative intensities of the two resonances. We report the observation of transport both out of and into vesicles.