WO1999047042A1 - A non-invasive method for the detection of intracellular sodium using multiple quantum nmr - Google Patents

Abstract

A non-invasive method for the measurement of intracellular sodium concentration using multiple quantum nuclear magnetic resonance (NMR) is disclosed. Unlike current methods, the present method does not use intervention or any chemical reagents. One example of multiple quantum NMR is TQF NMR. As an indicator of cell functioning, its viability the intracellular sodium can be used for diagnosis, and evaluating the efficiency of corresponding pharmaceutical interventions. Further, the method can be implemented for any tissue in both animals, and humans. Being completely non-invasive, the method has especially promising applications for brain study.

Description

A NON-INVASIVE METHOD FOR THE DETECTION OF INTRACELLULAR SODIUM USING MULTIPLE QUANTUM NMR

BACKGROUND OF THE INVENTION The present invention relates to a method of detecting and monitoring intracellular sodium using magnetic resonance spectroscopy. More particularly, the present invention relates to a non-invasive method of detecting and monitoring intracellular sodium using multiple quantum nuclear magnetic resonance without chemical shift reagents.

Intracellular sodium (Na;) homeostasis is an important indicator of the viability of cells in both animals and humans. However, existing methods for detecting and distinguishing between intracellular and extracellular sodium have been problematic. For example, chemical shift reagents (SR) are commonly used to detect changes in the levels of Na;. The greater the amount of shift reagents present, the greater the detectable differences between intra- and extracellular sodium. The presence of shift reagents, however. negatively influences cell physiology. In effect, the ions that are released from the shift reagents are very toxic to the cells being analyzed, often resulting in undesirable changes to the cells i.e., changes in heartbeat, metabolism etc.

Additionally, both single quantum nuclear magnetic resonance (NMR) with shift reagents or triple quantum filtered (TQF) and double quantum Na NMR (with or without chemical shift reagents) have been used to monitor Na;. Using Na TQF NMR in the detection of the Na; changes has been considered, however, the degree of correspondence between the Na TQF NMR signal and Naj has not been determined to the extent that a valid non-invasive method can be used for physiological studies in humans. There, thus, remains a need for a non-invasive method for detecting Na^ using multiple quantum filtered NMR without chemical shift reagents.

SUMMARY OF THE INVENTION

The present invention is directed to a non-invasive method for measuring intracellular sodium (Na^ concentration using multiple quantum NMR. The method is based on sodiumis ability to exhibit multiple quantum transitions when sodium ions experience the effect of the electric field of surrounding macromolecules. The present method does not use any chemical shift reagents, a fact which distinguishes this method from previously known NMR methods. The method can be implemented for any tissue, for example, in heart during normal contraction or in brain tissue. Further, the voxel localization for the analysis can be achieved using magnetic resonance localization technique.

5 Multiple quantum NMR can be implemented in several ways. 2D NMR pulse sequence allows one to detect single and multiple quantum Na signals at the same time. Double quantum or triple quantum filtering pulse sequence can filtrate NMR signals from those Na nuclei exhibiting multiple quantum transitions.

The feasibility of measuring Naj changes by multiple quantum NMR was o demonstrated by using Na TQF NMR without chemical shift reagents in an isolated rat heart during a variety of interventions for Naj loading. Perfusion with 1 mM ouabain or without K⁺ present in the perfusate for 30 minutes produced a rise of the Na TQF signal with a plateau of -190% and -228% relative to the pre-intervention level, respectively. Stop-flow ischemia for 30 minutes resulted in a TQF signal growth of -147%. The maximum Na TQF 5 signal increase of 460% was achieved by perfusion without K⁺/Ca²⁺, corresponding to an elimination of the Na trans-membrane gradient. The observed values of Na NMR TQF growth in the physiological and pathological range have a linear correlation with intracellular sodium concentration as determined in this study by Co-EDTA method and by sucrose-histidine washout of the extracellular space. Data further indicated that the increase 0 in the Na TQF NMR signal was determined by the growth of Naj, and the extracellular Na contribution to the total TQF signal was unchanged at -64%. Na multiple quantum NMR without chemical shift reagents provides a unique non-invasive method to measure alterations of intracellular sodium. It is contemplated that the method of the present invention may be used clinically in humans to assess a variety of pharmaceutical 5 interventions in normal and pathological tissue. The method also has promising applications for investigating the mechanism of synaptic transmission in brain.

In one embodiment of the present invention, a method for detecting and measuring Naj in humans using sodium multiple quantum NMR is disclosed, comprising the steps of: 1) proton-reference scanning a pre-selected region of the body of a person situated inside a 0 nuclear magnetic resonance scanner; 2) administering a pre-selected pulse sequence to the pre-selected region, resulting in a signal being generated from the bound and from the total sodium; 3) generating a spectrum from the scanner of the bound and total sodium; 4) comparing the spectrum generated from the person with a standard calibration curve as shown in FIG. 6, which illustrates a linear relationship between the intracellular Na concentration and the bound Na NMR TQF signal; and 5) thereafter determining the Na; concentration of the scanned region.

In one embodiment of the invention, the preferred multiple quantum NMR is TQF NMR.

In another embodiment of the invention, any pulse sequence can be used so long as the sequence separates the signal from bound Na. In yet another embodiment of the invention, the pulse sequence is

90°_φ -τ/2-180°_φ^ -τ/2- 90°_φ+ot -δ- 90°_ε where the delay δ = 40 μs, phase α = 90°, ε = 0°, phase ψ was incremented through 30°, 90°, 150°, 210°, 270°, 330° with receiver phase alternation by 180° each step.

Another application of the method of the present invention involved the assessment of the effect of multi-dose St. Thomas' cardioplegia on intracellular sodium homeostasis in a rat heart model. Eighteen isolated rat hearts were subjected to 50 minutes of ischemia at 37°C: (a) a multi-dose 2 minute infusion of St. Thomas' cardioplegia every 15 minutes (b) a single-dose cardioplegia and (c) a clamp ischemia. The non-invasive magnetic resonance spectroscopy method disclosed hereinabove was used for continuous detection of the intracellular sodium changes.

Multiple infusions of cardioplegic solution resulted in preservation of the heart's intracellular sodium concentration: the pre-ischemic level was 13.9 ± 1.2 mM and at the end of ischemia it was 14.3 ± ImM. This concentration only slightly increased during the following 50 minutes of reperfusion (16.7 ± ImM), yielding an outstanding recovery of the heart detected by rate-pressure product relative to pre-ischemic level (RPP = 85%). In comparison, a single-dose cardioplegia resulted in the intracellular sodium content of 30.2 ± 1 mM after 50 minutes of ischemia. This level continued to grow after reperfusion and reached 48.5 ± 1.7 mM at the end of 50 minutes perfusion (RPP = 29%). The corresponding values for clamp ischemia were 34.9 ± 1.3 mM at the end ischemia and 73.9 ± 1.9 mM at the end of reperfusion (RPP = 2%). The time course of intracellular sodium changes in a whole heart before, during and after multi-dose St. Thomas cardioplegia demonstrated the remarkable potential of cardioplegia to preserve intracellular sodium homeostasis. The intracellular sodium level during ischemia, as an indicator of the viability of the myocytes, can be prognostic for the heart's performance during reperfusion.

In another embodiment of the invention, a method is disclosed for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in normal and pathological tissues, comprising the steps of: a) administering to a mammal a pre-selected pharmaceutical; b) proton-reference scanning a pre-selected region of the body of the mammal following administration of the pharmaceutical, said mammal being situated inside a nuclear magnetic resonance scanner; c) administering a pre-selected pulse sequence to the pre-selected region, resulting in a signal being generated from the bound and from the total sodium: d) generating a spectrum from the scanner of the bound and total sodium; e) comparing the spectrum generated from the mammal with a standard calibration curve, which defines the relationship between the intracellular Na concentration and the bound Na NMR TQF signal; and thereafter f) determining the Naj concentration of the scanned region. It is contemplated that the present method can be used for evaluating any pharmaceutical for its effect on intracellular sodium.

The preceding and further objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood after a reading of the following description of the preferred embodiments when considered with the drawings.

FIG. 1 illustrates the time courses of the Na TQF NMR signal from a rat heart during

20 minutes (n = 4) and 30 minutes (n = 4) of K⁺- free perfusion;

FIG. 2 illustrates Na TQF NMR signal in a rat heart, during 30 minutes of 1 mM ouabain supplementation to KH perfusate (•, n = 8). For comparison, a 30 minute K⁺-free perfusion of the heart is also present (o, n = 4);

1 FIG. 3 illustrates a time course of the Na TQF NMR signal during Ca²⁺-free perfusion in a rat heart at 37_C (•, n = 6) and 20_C (o, n = 3);

FIG. 4 illustrates time dependence of the Na TQF NMR signal from a rat heart during K⁺-free and the subsequent (K⁺/Ca²⁺)-free perfusion at 37_C (n = 4). The increase in the TQF signal of 460 ± 30% corresponds to elimination of the Na trans-membrane gradient;

FIG. 5 illustrates Na SQ NMR signal during 2 cycles of sucrose- histidine washout of extracellular Na in a rat heart before and after 30 minutes K⁺ -free perfusion. Growth of the residual signal, presented in percent to the pre- ischemic level, demonstrate the growth of Na, ;

FIG. 6 illustrates the correlation of intracellular Na concentration in crystalloid perfused isolated rat heart and the Na NMR TQF signal, presented as a percent of pre-intervention level: a) normal, b) ouabain plus reperfusion, c) stop-flow ischemia, d) ouabain, e) K⁺-free, f) Ca²⁺-free plus reperfusion, g) K /Ca -free (Na, value assigned as perfusate value);

FIG. 7 illustrates a time course of intracellular Na concentration in rat heart before intervention, during multi-dose cardioplegia and after reperfusion in comparison to single- dose cardioplegia and stop-flow ischemia;

FIG. 8 illustrates a time course of Na TQF MR signal from a rat heart during multi- dose cardioplegic arrest. The drop in the signal represents changes of the extracellular sodium contribution to the total TQF MR signal;

FIG. 9 illustrates the validation of intracellular sodium stability during the first 15 minutes after application of cardioplegia: washouts accomplished before CP and after CP; and FIG. 10 illustrates the validation of intracellular sodium stability during the first 15 minutes after application of cardioplegia: washouts performed after CP and during reperfusion. The residual Na MR signal in a rat heart after washout of extracellular space, representing intracellular sodium content, remains unchanged before and after cardioplegic arrest. DETAILED DESCRIPTION OF THE INVENTION

In the studies of the present invention, one objective was to determine the extent of the correlation, if any, between Na multiple quantum filtered NMR without chemical shift reagents, in particular TQF NMR, and intracellular Na under various physiological and 5 pathological conditions in the perfused rat heart model. A limitation of the TQF NMR is the relatively low intensity of the signals. However, even at magnetic fields of 2 T, the detection of changes in intracellular sodium is feasible. The peak intensity of the Na NMR TQF signal in a rat heart corresponds to -3% of the heart Na single quantum (SQ) signal. Both intracellular and extracellular compartments of the heart contribute to the Na NMR

10 TQF signal. If extracellular Na contributes as much as -64% to the total TQF signal, then the intracellular Na TQF NMR signal (peak amplitude) from the heart is -1% of the total SQ signal.

Only sodium nuclei experiencing the electric field of membranes and macromolecules contribute to the TQF signal. Under physiological conditions, the

15 extracellular binding places are exposed to a sodium concentration of -145 mM. while the intracellular space has a Na concentration of only about 10 to about 15 mM. Thus, sodium entering the intracellular space increases the amount of Na nuclei experiencing non- averaged quadrupolar interactions, and can be detected by growth of the total Na TQF NMR signal.

20 For validation of the method of the present invention, reversible and irreversible Na, loading of the myocardium was achieved by depleting the concentration of extracellular K\

7+ 7+

Ca and Mg ions by applying ouabain during continuous perfusion and by inducing stop flow ischemia. Independent methods of validating intracellular sodium changes were used: an extracellular sodium washout procedure combined with SQ NMR, application of a

? <; chemical shift reagent, and the use of Co-EDTA (ethylene-diamine-tetraacetate) extracellular space marker.

MATERIALS AND METHOD USED FOR THE DETECTION OF INTRACELLULAR SODIUM

J 0

Male Sprague-Dawley rats weighing 250-300 g were anesthetized with 75 mg/kg pentobarbital and anticoagulated with 1000 U/kg sodium heparin bv intraperitoneal injection. The heart was connected to the Langendorff system and the aorta was retrograde perfused with Krebs-Henseleit (KH) modified solution (niM/L): 118.5 NaCl, 25.0 NaHCO₃, 4.9 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.2 CaCl₂, 11.1 D-Glucose (pH 7.4, 37_C, aerated with a gas mixture of 5% CO₂ and 95% O₂). The effluent around the heart was evacuated through tubing connected to the bottom of the NMR probe-head coil. The hearts beat spontaneously. To continuously monitor hemodynamic function, a balloon catheter was placed in the left ventricle and connected to a pressure transducer. The temperature around the hanging heart was kept at 37°C (or 20°C) using a water jacketed NMR probe-head.

+ 7+

The extracellular ion interventions were performed with K -free, Ca -free, Ca²⁺/Mg²⁺-free, K⁺/Ca²⁺-free and ouabain perfusions introduced through a second thermostatic water-jacketed reservoir. In the K⁺-free experiments, NaH₂PO₄ was used to substitute for KH₂PO₄. The corresponding osmotic changes were less than 5%. and were not corrected. The modified solutions were continuously perfused during 20, 30 or 60 minute interventions. For independent Naj assay, the extracellular marker KCo(III)EDTA was added to the perfusate at a final concentration of 1 mM and the Na TQF NMR measurements were repeated again. KCoEDTA was synthesized according to Dwyer et al. (32). The perfusion with this modified solution was performed -25 minutes before the heart was taken for analysis of intracellular Na⁺ (31). The mass of Co and the total content of Na in the heart were determined within the same sample by atomic absorption methods (AAS) after nitric acid digestion of the dried heart at the University of California Department of Chemistry Micro-analytical Laboratory. The resulting intracellular Na concentration in mM was obtained from the formula: [Naj](mM) = 10³*{Na(mg) - 23mg/mmol*V_ex*[Na]_ex}/{23mg/mmol*(N_t- V_ex)}; where Νa(mg) is the amount of sodium in the heart determined by AAS, V_t is the water volume in a rat heart in ml, obtained by drying a rat heart in an oven at 85_C until a constant weight is achieved (-24 hours); the extracellular volume V_ex is Co(mg)*(59mg/mmol*(lmmol/l))^~1; extracellular sodium concentration: [Na]_ex = 143.5 mM. The washout of the extracellular Na from the rat heart was performed using a procedure of others (30, 33). Ten ml of the ice-cold washout solution (350 mM sucrose, 5 mM histidine, pH 7.4) was perfused through the heart for 1-2 minutes.

NMR experiments were performed with the NMR imaging system with a 2.35 T Bruker magnet and a 25 cm clear bore. Resonance frequency for sodium was 26.47 MHz. An observation of TQF signal was achieved by the pulse sequence: 90°_φ -τ/2-180°φ_+α -τ/2- 90°_φ+α -δ- 90°_ε , where the delay δ = 40 μs, phase α = 90°, ε = 0°, phase φ was incremented through 30°, 90°, 150°, 210°, 270°, 330° with receiver phase alternation by 180° each step. Duration of 90° pulse was 40 μs, and the dead time of the receiver was 180 μs. The NMR signal was acquired by 2K points in a spectral width of 4 KHz, and a line broadening of 15 Hz was applied before processing. Repetition time of the pulse sequence was 310 ms. Each observation point required - 1.5 minutes, and had 192 acquisitions of Na TQF signal and subsequent 48 acquisitions of SQ signal. The peak intensity of the NMR TQF signals and an initial FID amplitude for the SQ NMR signals were used to monitor the sodium time course changes. NMR acquisition extended for 20 minutes before interventions and for durations up to 30 minutes after reperfusion. Each experiment required 2 hours of data acquisition and consisted of about 80 observation points. Data are presented as a mean ± SE relative to the pre-ischemic level.

As a reference, single quantum Na NMR was performed on 12 rat hearts in the presence of chemical shift reagent Tm(DOTP)^5" to assess intracellular sodium. Multiple component analysis of SQ NMR signals in the presence of chemical shift reagents was accomplished using time domain data. A linear prediction singular value decomposition method (LPSVD) was applied to NMR free induction signals (36). The SQ NMR signals were modeled as a sum of the exponentially decaying complex sinusoids. The data and noise subspaces were identified, and the noise components were filtered from the signal (37, 38). The initial starting estimates of parameters (amplitudes, relaxation rates and phases) obtained on LPSVD stage were further improved using a nonlinear least squares fit to the signal by Levenberg-Marquart's algorithm (39). The errors of the subsequent parameters were estimated from the statistics of residuals as the difference between the measured signal and the model. i The time course of the TQF NMR signal during a variety of Na; loading is presented in the first four sections below. The validation of Naj changes is provided in sections 5 and 6, using a washout of the extracellular space together with SQ NMR or Co-EDTA with atomic absorption spectroscopy.

(1) Potassium-free perfusion

The sodium TQF signals under conditions of potassium-free perfusion are shown in FIG. 1. Asystole occurred within - 7-10 min after the onset of intervention. During 20 minutes of K⁺-free perfusion, the TQF signal increased to 220 ± 5% of the initial pre- intervention level (n = 4). Reperfusion resulted in almost full recovery of TQF signal (115 ± 7%). After 30 min of K⁺-free perfusion, the TQF signal was 228 ± 5% (n = 4). and there was only a partial return of the signal to baseline level on reperfusion (135 ± 7%). The rat heart recovery estimated by rate-pressure product (RPP) at the end of reperfusion was 81 ± 10%) of pre- intervention level. The growth rate of the sodium TQF signal determined during the first -10 minutes of perfusion was ~9%/min. Two experiments with KJfree perfusion up to 60 minutes resulted in a growth of TQF signal to -265%. On reperfusion. there was only a partial recovery of the TQF signal to -203% and no functional recovery of the heart. At 20_C (n = 2), the rate of TQF signal growth was 2.3 times less than at 37_C and the maximum TQF signal was only -190%). The improved RPP recovery of heart (-92%) was observed at the end of reperfusion.

(2) Effect of ouabain

The results of 30 minute perfusion with 1 mM ouabain in KH solution and subsequent reperfusion is shown in FIG. 2 (n = 8). The rat heart stopped beating in 3-4 minutes, and the growth of the TQF signal was at a rate of ~9%/min during this period. After 30 minutes of ouabain perfusion, the maximum TQF signal was 190 ± 9%. Reperfusion resulted in recovery of the TQF signal to 120 ± 9% and the corresponding rat heart RPP recovery was 45 ± 9%.

(3) Ca²⁺-free perfusion

A much higher level of the Na TQF signal in the rat heart was obtained during calcium-free perfusion and subsequent reperfusion at 37_C. The Na TQF signal εrowth showed a delay of -8 minutes and later an intensive initial growth rate of ~8J%/min. These results are shown in FIG. 3. The signal reached a plateau in - 30 minutes at ~220±25% (n = 6). The TQF signal deviations, from heart to heart, had a much larger amplitude than the error of the TQF signal measurements. A further growth of the TQF signal to -270% was usually observed on reperfusion with no recovery of the heart. The large value of Na; at reperfusion was confirmed by Co-EDTA analysis (n = 3) performed after 10 minutes of Ca²⁺ repletion ( [Na;] = 95 ± 16 mM).

Decrease of the temperature to 20_C did not yield additional growth in TQF signal, and gave a remarkably lower rate of growth in the signal (0.6%/min) for the whole period of

7+ 60 minutes of Ca -free perfusion. These results are shown in FIG. 3. There was no growth of the TQF signal upon reperfusion, and the RPP rat heart recovery was 60 ± 20% (n = 3).

In preliminary experiments, when both divalent ions (Ca²⁺, Mg²⁺) were removed from the KH perfusate solution at 37_C, a further increase of the Na TQF signal was achieved (n=2). The plateau of -360% for Na TQF signal was reached in this case with the initial rate of the TQF signal growth of ~17.5%/min. The delay in the Na TQF signal growth was - 4 minutes, which is two times less than for the Ca²⁺ depleted medium.

(4) K⁺/Ca²⁺-free perfusion

The combined use of K⁺-free and Ca²⁺-free perfusion has been used (24, 25), wherein by the coupled effect of Ca²⁺/K⁺-free perfusion, the intracellular space of ferret ventricular muscle was loaded to the level of [Na;] = [Na^]. The largest amplitude of the TQF signal was achieved in the present invention when the K⁺-free perfusion was applied together with Ca ⁺-free perfusion. These results are shown in FIG. 4. The Na TQF signal reached a plateau of 460 ± 30% with an initial rate of TQF signal growth of ~16J%/min (n=4).

(5) Sucrose-histidine washout of extracellular sodium

The intracellular sodium growth during K⁺-free perfusion was validated using sucrose-histidine washout procedure and SQ NMR signal. The results are illustrated in FIG. 5. The time course of the continuous washout procedure observed by SQ Na NMR (15 sec/point) in a normal rat heart is approximated by the sum of two decaying exponential functions (fast component: A_f = 88.3 ± 1%, T_f = 22.3 ± 0.5 sec and slow component: A_s = 11.7 ± 0.7%, T_s = 602 ± 84 sec). This experiment demonstrated adequate extracellular sodium washout by 10 ml of sucrose-histidine solution going through the heart during -1-2 minutes. A single cycle of washout and reperfusion performed in a normal rat heart (FIG. 5) showed a full recovery of Na SQ signal after -10 minutes. There was also nearly complete restoration of heart performance, so a second washout intervention was applied to the same heart. The 30 minutes of K⁺- free perfusion resulted in growth of the residual Na SQ NMR signals (n = 4) determined from the washout values by a factor of 3.6 of the pre- intervention level (i.e., 11.7 ± 0.3% to 41.9 ± 0.3%).

For a normal heart, the residual washout Na SQ NMR signal was compared with the signal from Na, using SR. Introduction of the chemical shift reagent Tm(DOTP)^5" to a normally perfused rat heart produces a stable splitting of Na NMR line in -3 minutes. The area of non-shifted NMR line, corresponding to Na,, was 11.6 ± 0.4% (n = 12) of the pre- intervention total Na SQ NMR signal. The result closely corresponds to the residual washout Na SQ NMR signal in a normal rat heart 11.1 ± 0.3% (see FIG. 5).

(6) Correlation of Na TQF NMR signal and Na_j determined by Co-EDTA method

Co-EDTA method for a normally perfused heart (n = 4) yielded [Na = 16.2 ± 4 mM and the intracellular volume fraction of 0.5 ± 0.015 ml/heart. At the end of 30 minutes of K⁺-free perfusion, [Na,] increased to 67.9 ± 3 mM (TQF signal = 217 ± 5%, n = 4), while the intracellular volume fraction was relatively unchanged at 0.46 ± 0.025 ml/heart. In experiments with ouabain, two time points during the experiment were taken for Co-EDTA analysis. The first point at the end of 30 minutes of ouabain perfusion (TQF signal = 159 ± 15%), n = 3) yielded [Na,] = 40.0 ± 0J mM and the second after 20 minutes of a subsequent reperfusion (TQF signal = 120 ± 9%, n = 3) gave [Na,] - 20.5 ± 5 mM. 30 minutes of global ischemia at 37_C produced a 147 ± 10% increase of the TQF signal. The corresponding [Na,] concentration determined at the end of global ischemia by Co-EDTA method was 45 ± 6 mM (n = 3). Co-EDTA analysis after 40 minutes of Ca-free perfusion at 37_C and subsequent 10 min Ca²⁺ re-introduction resulted in [Na,] = 95 ± 16 mM (TQF signal = 332 ± 26%. n=3).

The relationship between measured Na, concentration using the Co-EDTA method and the total TQF signal for the above interventions are summarized in FIG. 6. The straight line fit to these data gives a slope of 0.35 ± 0.015 raM/% and an intercept of 50 ± 5% (R = 0.995). The latter represents an average extracellular Na TQF signal contribution to the total Na TQF signal. As global ischemia was the only experiment, where there was no perfusion of extracellular space during intervention, these data were omitted from the calculation of the correlation line.

Results of FIG. 6 demonstrate that the TQF NMR signal in a rat heart has a linear dependence on a wide range of Na, concentrations. The results also indicate that all increases in the Na TQF signal are due to the growth of intracellular Na content, and extracellular Na can be regarded as unchanged. These results provide the basis for applying the TQF NMR to monitor Na, changes without using a chemical shift reagent.

The KH perfusion either with a modified K⁺ content (0 mM) or with ouabain (1 mM) produces inhibition of the Na⁺/K⁺ ATPase, which resulted in the growth of the Na, and corresponding growth of the sodium TQF signal. This is illustrated in FIGS. 1 and 2. The detected increase of the TQF signal in both cases was substantially reversed upon reperfusion, reflecting a restoration of Na⁺/K⁺ pump activity. The Na TQF signal for both interventions had similar initial growth rates of ~9%>/min, while the heart was beating. Thereafter, however, a lower rate of growth and a lesser value for the maximum signal were detected for ouabain perfusion (see FIG. 2). The decrease of intracellular Na on reperfusion in this case was verified by the Co-EDTA method. The fact that the TQF signal did not decrease to normal level and that 81%) of normal rate pressure product was obtained after reperfusion are indicative of some irreversible damage in a rat heart associated with K⁺-free perfusion. The heart injury in this case is supported by the rapid decline of ATP and PCr metabolites and the K⁺ leakage detected by ³¹P and ³⁹K NMR during 30 minutes of K⁺- free perfusion. The estimated rate of [Na,] growth during K⁺-free perfusion as shown in FIG. 1 was

-33%o/min. The [Na⁺], growth rate of 46%>/min was measured relative to the normal state during the first 5 minutes of K⁺-free perfusion in sheep cardiac muscle (43) and 43%./min during the first 6 minutes of K⁺- free perfusion in rabbit myocytes (44), indicating comparable results with other methods of Na, detection. The observed 2.3 times decrease in the rate of Na, growth at 20_C corresponds to the -2 times decline in the electro genie

13- sodium pump current obtained by others in sheep cardiac Purkinje fibers for the temperature range from 36.7 to 26.4_C (27).

Ca -free perfusion has been shown to produce a very intensive rise of Na, as was demonstrated by the ion selective micro-electrode method (ISME) (24, 25, 45, 46) and the washout of extracellular sodium method (30, 47). The present invention also revealed a dramatic increase in Na,, which was demonstrated by the growth of the Na TQF signal during Ca-free perfusion. This is shown in FIG. 3. A further growth of Na TQF signal detected at reperfusion is indicative of Na influx into the cell possibly due to the

94- 74- reintroduction of Ca . Na, growth was confirmed during Ca -free perfusion using the CoEDTA method. All the above alterations in Na,, however, contradict the previous results of others (18) where no changes of Na, were observed by SQ NMR during 30 minutes of Ca²⁺- free perfusion in the presence of a chemical shift reagent.

Our initial SQ NMR experiments using Tm(DOTP)^5" also revealed no changes in intracellular Na during 20 minutes of Ca ⁺-free perfusion at 37°C. A possible explanation is that the presence of the shift reagent might attenuate Na influx into the cell.

Hypothermia protects against the calcium paradox (24, 30, 45, 48). A decrease in the growth rate of the TQF signal from 8.2%>/min to 0.6%/min was observed when the temperature was changed from 37°C to 20°C. The observed rates of the TQF signal growth correspond to the [Na,] growth rates of -30%/min (37°C) and ~2J%/min 20°C). This 14- fold decline in the rate of sodium build-up corresponds to the 4-fold decrease in the rate of

Na, build-up in ferret ventricular muscle detected by ISME (24). These experiments show

94- that hypothermia helps maintain Na, homeostasis during Ca -free perfusion.

A plateau for K⁺-free perfusion is a common feature for Na, growth which has been observed for sheep heart Purkinje fibres (22, 26) and in the rat heart (10). The decline in the rate of TQF signal growth after -15 minutes of K⁺-free perfusion may be due to activation of Na7Ca²⁺ exchange and lowering of intracellular Na concentration by exchange with calcium. The Na⁺/Ca ⁺ exchange, however, is less effective for sodium efflux than the Na⁺/K⁺ pump (22). An additional increase in the TQF signal detected when Ca²⁺-free perfusion was added to the ongoing K⁺-free perfusion (shown in FIG. 4) is in accord with ISME experiments in sheep heart Purkinje fibres (22, 23). Removal of Ca²⁺ from the

13 extracellular fluid would inactivate the reverse mode of Na⁺/Ca²⁺ exchanger, thus preventing another mechanism of Na efflux.

So it is reasonable that the highest level of the Na TQF signal of 460% (shown in

FIG. 4) represents the highest level in Na; corresponding to the Na concentration in KH solution (143.5 mM). Using this data point, the ratio of 3.58 for Na; growth in the washout experiments (FIG. 5) and the Na NMR TQF signal growth of 228% for 30 minutes of K⁺- free perfusion resulted in Na; = 17.5 mM for the normal heart myocytes and an extracellular

Na contribution to the total Na TQF signal of 50%.

The weighted average of all the determinations of the extracellular contribution to Na TQF signal is 64% (including the previous value of 73% (7)), and the corresponding average amount for intracellular Na in a rat heart is 13.0 mM. This value is in accordance with reported ranges of between about 5 and 17 mM (7, 11, 13, 18. 20, 28, 41, 44).

All the interventions, except for global ischemia, were done during continuous perfusion of extracellular space with the perfusate having a constant content of Na. The results of global ischemia (wherein there is no replacement of Na entering the cell and consequently a decrease of extracellular Na content is expected) are close to the observed linear dependence. So, in the case when there is no perfusion of the extracellular space, Na; growth by TQF NMR was still detected, because Naj has about 10 times larger contribution per atom to the TQF signal than extracellular Na. Thus, for global ischemia an influx of extracellular Na -10% may give a corresponding decrease of extracellular TQF signal and, at the same time, a -100% increase in TQF signal from Na_j. As the sum of these TQF signals is detected, the underestimation for Na; changes of -10%) is possible and needs to be considered for accurate measurements in such cases.

Though the Na NMR TQF signal change can also report an alteration in the state of tissue other than Na; (e.g., electric field gradient changes associated with macromolecule assemblage modification), the linear correspondence of the TQF signal and the independently measured Naj confirm the applicability of the TQF Na signal to studies of Na; physiology in the heart.

Regarding the visibility of Na NMR, practically all NMR signals from Na in the heart can be observed during experiments. In the present invention, rat hearts after sucrose histidine washout of extracellular space have been used to study the visibility of intracellular sodium. There is a minor decrease in Na SQ NMR signal (-2.8%) as the amplitude of the radio-frequency field decreases in the range from 33 KHz to 6 KHz, indicating practically full visibility for sodium in this region (dead time of the receiver was 30 μs). However, at 0.8 KHz, the decrease of the Na SQ signal was 18%, suggesting that the problem of Na visibility in rat heart can be explained solely by the experimental setting. In conclusion with respect to this aspect of the present invention, Na TQF NMR signal in a rat heart has a linear correlation with intracellular Na concentration as determined by washout and Co-EDTA methods. The results are in accord with the previously reported data of ion selective electrode and fluorescent methods. TQF NMR without chemical shift reagents is a reliable tool in developing cardioprotective strategies, and may yield additional insights into cardiac physiology.

The method of detecting intracellular sodium using TQF NMR was then used to assess the effect of multi-dose St. Thomas cardioplegia on intracellular sodium homeostasis in a rat heart model. Crystalloid cardioplegic solution is an effective tool of myocardial preservation during heart surgery (50-52). Multi-dose cardioplegia (CP), first introduced in 1976 (53), enhances preservation of the heart and improves the recovery of heart function after ischemia. An objective of the present invention was to investigate whether improvement in heart recovery provided by multi-dose CP is correlated with sodium homeostasis of myocardium in comparison to single dose CP and clamp ischemia, using a rat heart model.

It has been shown (7,54) that single dose cardioplegia may delay the Na MR triple quantum filtered (TQF) signal growth pertinent to ischemia. In the present invention, the experiment was enlarged by using multi-dose cardioplegia, and extended evidence is presented on the preservation of intracellular sodium by cardioplegia. A novel non-invasive method of intracellular sodium detection is disclosed herein, and the success of the method has been validated as shown by the corresponding results. The selection of multiple quantum NMR was essential for observation of the effects of the cardioplegia on intracellular sodium. This was due, in part, to the fact that high concentrations of Mg⁺ ions, as in cardioplegia (16 mM), reduces chemical shift for extracellular sodium (10), and makes it impossible to detect the [Na⁺]_j in the heart using chemical shift reagents. MATERIALS AND METHOD TO ASSESS THE EFFECT OF MULTI-DOSE CARDIOPLEGIA ON INTRACELLULAR SODDJM HOMEOSTASIS

Eighteen isolated rat hearts were subjected to 50 minutes of ischemia at 37°C: (a) a multi-dose 2 minute infusion of St. Thomas' cardioplegia every 15 minute (b) a single-dose cardioplegia and (c) a clamp ischemia. The non-invasive magnetic resonance spectroscopy (MRS) method was used for the continuous detection of the intracellular sodium changes. This method is based on the property of sodium, bound to macromolecules, to exhibit MR spectroscopic triple quantum transitions and on the corresponding growth in bound sodium when a sodium ion passes from extracellular to intracellular space.

Male Sprague-Dawley rat (250-300 g) hearts were rapidly excised and loaded onto the Langendorff system. Perfusion and reperfusion were performed by modified Krebs- Henseleit (K-H) solution (mM/L): 1 18.5 NaCl, 25.0 NaHCO3, 4.9 KCl, 1.2 KH2PO4, 1.2 MgSO4. 1.2 CaC12, 1 1.1 D-Glucose (pH 7.4, with oxygenation 95% 02:5% CO2). Cardiac arrest was achieved by single (2 min) infusion of warm oxygenated St. Thomas cardioplegic solution (mM/L): 110 NaCl, 10 NaHCO3, 16 KCl, 1.2 CaC12, 16 MgC12 at pH 7.8 with stop- flow later. In multi-dose experiments the CP was repeatedly applied for 2 min at 17 min intervals. Performance of the heart was monitored by the heart rate and LVDP pressure product (RPP) relative to the pre-ischemic level.

A non-circulating perfusion system was used. The rat heart was not submerged in a buffer but was suspended in the air in the temperature controlled chamber at 37=^ C. An additional water jacketed reservoir has been used in the experiments to introduce warm, oxygenated cardioplegic solution. Perfusate was evacuated through the tubing connected to the bottom of the MR coil.

The intracellular sodium changes were detected on the MR imaging system (2.35 T) using TQF pulse sequence 90°_φ -τ/2-180°_φ+α -τ/2- 90°_φ+α -δ-90°_ε , where τ = 4.4 ms, δ - 40 μs. Repetition time of the whole pulse sequence was 310 ms. Each observation point used 192 acquisitions and required 1.5 minutes. Na TQF NMR peak intensities were used to assess the effects of interventions. The following linear relationship was used between [Na_j] and Na TQF NMR signal: [Na_i](mM) = [Na_i]₀ + A₁(S/S₀ - l), where A_[ is a calibration constant which is equal to 45mM for continuous perfusion. The

S/S₀ is the ratio of the Na TOF MR signal to its pre-ischemic level, [Na_j]₀ is another calibration constant which equals to the initial intracellular concentration of 13.9 mM. For stop-flow conditions in the heart, the transport of sodium to intracellular space decreases for the same amount the extracellular sodium content. Thus, for the heart in a range of the cell volume ratio N_j/N_ex = 0.5 - 1, the MR TQF signal growth is only 0.84 of the value for normal perfusion, representing the situation that each intracellular sodium ion is more efficient in contributing to the TQF signal than extracellular sodium. These coefficients were obtained from a linear relationship between TQF signal and intracellular sodium content (55). Consequently, it gives the following relationship between the TQF signal and intracellular sodium concentration for stop-flow conditions:

[Νa_jKmM) = [Νa^ + A₂(S/S₀ - C), where A = 54 ± 5 mM, C=l for clamp ischemia or 0.73 for cardioplegic arrest. The washout of the extracellular Νa from the rat heart was performed using a procedure of others (30,33). Ten ml of the ice-cold washout solution (350 mM sucrose, 5 mM histidine, pH 7.4) was perfused through the heart for 1-2 minutes.

The time courses of [Νa changes in a rat heart before, during 3 different ischemic interventions and the following reperfusion at 37°C are presented in FIG. 7. Clamp ischemia (FIG. 7) produced a remarkably large increase in intracellular Νa concentration which started growing almost immediately and gave the largest Νa_; concentration at the end of reperfusion (34.9 ± 1.3 mM). The concentration continued to grow after reperfusion and reached the level of 73 ± 1.9 mM at the end of reperfusion with final RPP recovery of a rat heart of2 ± 2% (n=3). Several effects of single dose CP can be seen in comparison to simple stop-flow ischemia as shown in FIG. 7. First, the delay of -15 min in intracellular sodium growth was reproduced as was reported earlier (7). After 50 minutes of cardioplegic arrest the intracellular sodium concentration was 30.2 ± 1 mM. During reperfusion the growth rate of intracellular sodium was substantially reduced, and in 50 minutes, the resulting intracellular

\ 1 sodium concentration was 48.5 ± 1.1 mM. The heart rate pressure product recovery was correspondingly better than for clamp ischemia (29 ±7%. n= 8).

Application of the second and third infusion of CP extended dramatically the period of no growth in Naj. This is shown in FIG. 7. Fifty minutes of ischemia resulted in non- significant changes in intracellular sodium (14J ± 1 mM), and at the end of reperfusion, the intracellular sodium concentration was not far from the pre-ischemic level (16J ± 1 mM). The corresponding RPP recovery of the heart at the end of reperfusion was also remarkable 85 ± 7% (n=7).

Turning attention to the Na TQF MR signal itself during multi-dose cardioplegic arrest (see FIG. 8), one can see that the infusion of CP produced an immediate decrease in the TQF signal of -30% and a period -50 minutes of no growth in the signal. Reperfusion almost restored the pre-ischemic TQF MR signal. The above changes in the signal represent the changes in extracellular sodium contribution to the TQF signal. This observation was validated using an independent method of Na_j detection performed during the first 15 minutes after CP arrest (see FIGS. 9A and 9B). The sucrose-histidine wash-out of extracellular space gave the same intensity of residual Na MR signal observed before and after cardioplegic arrest. Two wash-outs were performed on the same heart in two sets of experiments. In the one set, the first wash-out was performed before CP arrest and the second was done after CP infusion (FIG. 9, n=2). In another set of experiments, the first wash-out was accomplished during CP and the second wash-out at the following reperfusion (FIG. 10. n=2). All experiments gave the residual Na signal of -1 1.6%, relative to the Na MR signal from the normally perfused heart corresponding to unchanged intracellular sodium content.

The results of this study represent, to our knowledge, the first observation of the effects of multi-dose cardioplegic solution on intracellular sodium in the whole rat heart, and demonstrate the possibility of performing these studies without additional chemical interventions. The preservation of intracellular sodium yields insights into the mechanism of action of St. Thomas CP, which may also be common to other types of cardioplegia.

A short delay of -5 minutes in irreversible damage during clamp ischemia is a well known phenomena. The observed delay of -15 minutes in intracellular sodium growth during single-dose CP arrest is an indication of the additional delay produced bv cardioplegia in comparison to clamp ischemia. One may expect this delay because of the known ability of CP to induce the diastolic heart arrest which decreases metabolic demands and slows down the oxygen consumption by more than ten times relative to stop flow ischemia (52, 57). At the same time, the stimulation of Na efflux through the sodium/potassium pump by the high potassium content of cardioplegia can be another important mechanism of intracellular sodium preservation process.

The unique delay in intracellular sodium growth produced by single-dose cardioplegia is effectively extended by multi-dose cardioplegia, which correlates with the known cardioprotective effect of multi-dose cardioplegia Consequently, the remarkable effect in stabilization of sodium homeostasis during ischemia correlates with the outstanding recovery of the heart in the present invention.

Some experimental factors can also affect the outcome of multi-dose cardioplegic arrest. Auxiliary oxygen supply and washout of extracellular space by each infusion of CP are taking place. The above conditions may augment the effects of cardioplegia. Overall, regarding preservation of the heart, from FIG. 7, the general tendency can be seen the that the recovery of heart is better the closer one is to the pre-ischemic intracellular sodium level. The observed value of the intracellular sodium content during ischemia can be predictive of future heart recovery upon reperfusion.

The wash-out technique is a known method of intracellular sodium detection (30,33). Here, the technique was applied in combination with MR observation of sodium, which allowed one to see immediately the results of wash-out and, moreover, to perform double intervention. The observed unchanged residual signal before and after CP arrest indicates that there is no change of intracellular sodium because of the infusion of cardioplegia. The detected drop in the Na TQF signal during CP arrest (FIG. 8), as follows from validation experiment (FIGS. 9 and 10), is due to the change of the extracellular sodium contribution to the TQF signal. Half of this drop is determined by the lower sodium content in cardioplegia than in the KH solution and the other half is mainly due to the presence of 16 mM of magnesium, which may compete with sodium for its binding places.

The time course of Na TQF signal during multi-dose CP and rat heart recovery are comparable to single dose CP arrest at 21 °C, thus suggesting that multiple CP at 37°C is as efficient in a rat heart preservation as single dose cardioplegia at 21°C. In conclusion, multi-dose St. Thomas cardioplegia is an effective measure of protecting the sodium homeostasis. Single-dose cardioplegia preserves sodium content for the first - 15 minutes of ischemia at 37°C and multi-dose cardioplegia dramatically extends this period. The detected intracellular sodium accumulation during ischemia can be an 5 effective predictive factor for heart recovery on reperfusion. The observed intracellular sodium during ischemia and reperfusion inversely correlates with rat heart functional recovery. Na multiple quantum MR has great potential as a non-invasive tool to monitor intracellular sodium changes and early cell responses to pharmaceutical treatment.

o IMPLEMENTATION OF THE INVENTION FOR EVALUATING A SPINAL CORD INJURY

Spinal cord injury involves both primary (mechanical) and secondary (delayed) injury. The secondary injury results from a number of factors including metabolic energy 5 failure, neuronal depolarization and an increased release of endogenous excitatory amino acids. Altogether, these conditions trigger abnormal ion Na fluxes which potentiate neuron damages.

Sodium channel blockers such as tetrodotoxin and saxitoxin or the application of anesthetics such as lidocaine and procaine (which can reversibly block 0 sodium channels) are protective against hypoxic ischemic injury. Conversely, increasing Na channel conductivity during anoxia resulted in greater injury (59). Consequently, the control of Naj can be of specific value for the non- invasive evaluation of the viability of the cell, especially in the spinal cord (60, 61).

An embodiment of the present invention comprises the following steps: 5 1. Placement of the patient in the MR scanner with the surface coil tuned for Na frequency.

2. Completion of a multi-slice proton localizer which images the region of interest.

3. Set localizer to image the region for intracellular sodium analysis.

4. Perform a Na multi-slice scan using both a single quantum (SQ) and multiple 0 quantum (MQ) pulse sequences for detecting total and bound sodium, respectively, in region of interest.

5. The growth of the MQ/SQ ratio will represent intracellular Na growth which can be evaluated according to its linear relationship with Na; concentration.

The SQ signal serves as a reference to exclude many parameters relevant to the specific hardware and software used during scanning and processing. The MQ signal, as a measure of bound Na. is proportional to the concentration of the intracellular Na. Both SQ and MQ are scanner dependent. Consequently, the MQ/SQ ratio can be a facile parameter to avoid the effects of the instrument settings on Naj concentration measurements.

The ability of the cells to maintain intracellular homeostasis is an indicator of their viability. Knowledge of the Na; changes, i.e., the Na, level and the Na, response to the pharmaceutical intervention can be an independent symptom for evaluating the extent of spinal cord injury and the efficiency of drug administration.

All references referred to herein are hereby incorporated by reference in their entirety.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. By way of example, it is contemplated that the method of the present invention may be used to observe changes following an acute stroke or spinal cord injury by monitoring intracellular sodium levels.

Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

References

1. L. A. Jelicks, R. K. Gupta, Nuclear magnetic resonance measurement of intracellular sodium in the perfused normotensive and spontaneously hypertensive rat heart. Am. J. Hypertens. 7, 429-435 (1994). 2. G. S. Payne, A. M. L. Seymour, P. Styles, G. K. Radda, Multiple quantum filtered

²³Na NMR spectroscopy in perfused heart. NMR in Biomed. 3, 139-146 (1990).

3. R. Reddy, L. Bolinger, M. Shinnar, E. Noyszewski, J. S. Leigh, Detection of residual quadrupolar interaction in human skeletal muscle and brain in vivo via multiple quantum filtered sodium NMR spectra. Magn. Reson. Med. 33, 134-139 (1995).

4. K. J. Brooks, T. R. M. Pirttila, R. A. Kauppinen, Delayed increase in intracellular Na⁺ in cerebral cortical slices during severe hypoxia as measured by double quantum filtered ²³Na NMR. NeuroReport 4, 139-142 (1993).

5. V. Seshan, M. J. Germann, A. D. Sherry, N. Banal, Effect of ischemia on triple quantum filtered intra- and extracellular Na⁺ in rat liver, in "Porch.. SM. and

ESMRMB, Nice, France, 1995," p. 217.

6. R. L. Tyson, G. R. Sutherland, J. Peeling, Na nuclear magnetic resonance spectral changes during and after forebrain ischemia in hypoglycemic, normoglycemic, and hyperglycemic rats. Stroke 27, 957-964 (1996). 7. V. D. Schepkin, I. O. Choy, T. F. Budinger, Sodium alterations in isolated rat heart during cardioplegic arrest. J. Appl. Physiol. 81, 2696-2702 (1996). 8. T. L. Dowd, R. K. Gupta, NMR studies of the effect of Mg²⁺ on post-ischemic recovery of ATP and intracellular sodium in perfused kidney. Biochim. et Biophys. Acta 1272, 133-139 (1995). 9. J. Pekar, P. F. Renshow, J. S. Leigh Jr., Selective detection of intracellular sodium by coherence-transfer NMR. J. Magn. Reson. 72, 159-161 (1987). 10. M. M. Pike, J. C. Frazer, D. F. Dedrick, J. S. Ingwall, P. D. Allen, C. S. J.

Springer, T. W. Smith, Na and K nuclear magnetic resonance studies of perfused rat hearts (Discrimination of intra- and extracellular ions using a shift reagent). Biophys. J. 48, 159-173 (1985). 1 1. L. A. Jelicks, R. K. Gupta, Multinuclear NMR studies of Langendorff perfused rat heart. J. Biol. Chem. 264, 15230-15235 (1989).

12. L. A. Jelicks, R. K. Gupta, On the extracellular contribution to multiple quantum filtered ²³Na NMR of perfused rat heart. Magn. Reson. Med. 29, 130-133 (1993). 13. C. R. Malloy, D. C. Buster, M. M. Castro, C. F. Geraldes, F. M. Jeffrey, A. D.

Sherry, Influence of global ischemia on intracellular sodium in the perfused rat heart. Magn. Reson. Med. 15, 33-44 (1990).

14. G. Navon, J. G. Werrmann, R. Maron, S. M. Cohen, ³¹P NMR and triple quantum filtered Na NMR studies of the effects of inhibition of Na⁺/H⁺ exchange on intracellular sodium and pH in working and ischemic hearts. Magn. Reson. Med.

32, 556-564 (1994).

91

15. M. M. Pike, M. Kitakaze, E. Marban, Na-NMR measurements of intracelllular sodium in intact perfused ferret hearts during ischemia and reperfusion. Am. J. Physiol. 259, HI 767- 1773 (1990). 16. M. M. Pike. C. S. Luo, S. Yanagida, G. R. Hageman, P. G. Anderson, ²³Na and

P nuclear magnetic resonace studies of ischemia-induced ventricular fibrilation (Alteration of intracellular Na⁺ and cellular energy). Circ. Res. 11, 394-406 (1995).

17. R. Ramasamy, H. Liu, S. Anderson, J. Lundmark, S. Schaefer, Ischemic preconditioning stimulates sodium and proton transport in isolated rat hearts. J.

Clin. Invest. 96, 1464-1472 (1995).

18. C. J. A. van Echteld, J. H. Kirkels, M. H. J. Eijgelshoven, P. van der Meer, T. J. C.

Ruigrok, Intracellular sodium during ischemia and calcium-free perfusion: a Na

NMR study. J. Mol. Cell. Cardiol. 23, 297-307 (1991). 19. M. M. Pike, C. S. Luo, M. D. Clark, K. A. Kirk, M. Kitakaze, M. C. Madden, E. J.

J. Cragoe, G. M. Pohost, NMR measurements of Na⁺ and cellular energy in ischemic rat heart: role of Na⁺-H⁺ exchange. Am. J. Physiol. 265, H2017-2026

(1993).

20. J. S. Ingwall, K. Clarke, L. C. Stewart, Cation movements across cell walls of intact tissues using MRS. In: "Encyclopedia of Nuclear Magnetic Resonance "

(D. Grant and R. Harris Eds.), p. 1 189-1 197, Wiley, Chichester, England. 1996. 3 21. R. Kalyanapuram, V. Seshan, N. Bansal, Effect of ischemia on 3D SQ and TQF Na images of the dog head, in "Proc, ISMRM, Vancouver. Canada. 1997," p. 611.

22. J. W. Deitmer, D. Ellis, The intracellular sodium activity of cardiac Purkinje fibres during inhibition and re-activation of the Na⁺-K⁺ pump. J. Physiol. [Lond] 284.

241-259 (1978).

23. J. W. Deitmer, D. Ellis, Changes in the intracellular sodium activity of sheep heart Purkinje fibres produced by calcium and other divalent cations. J. Physiol. [Lond] 277, 437-453 (1978). 24. M. S. Suleiman, R. A. Chapman, Effect of temperature on the rise in intracellular sodium caused by calcium depletion in ferret ventricular muscle and the mechanism of the alleviation of the calcium paradox by hypothermia. Circ. Res. 67, 1238-1246 (1990).

25. I. H. Bhojani, R. A. Chapman, The effects of bathing sodium ions upon the intracellular sodium activity in calcium- free media and the calcium paradox of isolated ferret ventricular muscle. J. Mol. Cell. Cardiology 22, 507-522 (1990).

26. D. Ellis, The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres. J. Physiol. [Lond] 273, 211-240 (1977).

27. D. A. Eisner, W. J. Lederer, Characterization of the electrogenic sodium pump in cardiac Purkinje fibres. J. Physiol. [Lond] 303, 441 -474 ( 1980).

28. J. S. Ingwall. How high does intracellular sodium rise during acute myocardial ischemia? A view from NMR spectroscopy. Cardiovasc. Res. 27, 278 (1995).

29. C. Steenbergen, M. E. Perlman, R. E. London, E. Murphy. Mechanism of preconditioning. Ionic alterations. Circ. Res. 72, 112-125 (1993). 30. L. E. Alto, N. S. Dhalla, Myocardial cation contents during induction of calcium paradox. Am. J. Physiol. 237, H713-719 (1979). 31. E. Scheufler, T. Peters, Determination of the extracellular space with nonradiactive Co ⁺EDTA and simultaneous estimation of Na, K, Ca, and Mg contents in isolated guinea-pig heart preparations by atomic absorption spectrometry. Basic Res. in Cardiol. 82, 341-347 (1987). 32. F. P. Dwyer, E. C. Gyarfas, D. P. Mellor, The resolution and racemization of potassium ethylenediaminetetraacetatocobaltate (III). J. Phys. Chem. 59. 296-297 (1955).

33. M. Tani, J. R. Neely, Role of intracellular Na⁺ in Ca²⁺ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H⁺-Na⁺ and Na⁺-Ca²⁺ exchange. Circ. Res. 65, 1045-56 (1989).

34. J. W. C. van der Veen, S. Slegt, J. H. N. Creyghton, A. F. Mehlkopf, M. J. Bovee, Optimal pulse angles for the detection of sodium multiple-quantum coherence filtered by phase cycling and gradients. J. Magn. Reson. B101, 87-90 (1993). 35. J. L. Allis, A. M. L. Seymour, G. K. Radda, Absolute quantification of intracellular Na⁺ using triple-quantum-filtered sodium-23 NMR. J. Magn. Reson. 93, 71-76 (1991).

36. M. Joliot, B. M. Mazoyer, R. H. Huesman, In vivo NMR spectral parameter estimation: a comparison between time and frequency domain methods. Magn. Reson. Med. 18, 358-370 (1991).

37. R. de Beer, D. van Ormondt, W. W. F. Pijnappel, Quantification of 1-D and 2-D magnetic resonance time domain signals. Pure andAppl. Chem. 64, 815-823 (1992).

38. E. B. Cady, Determination of absolute concentrations of metabolites from NMR spectra. In: "NMR: Basic Principles and Progress " (P. Diehl, E. Fluck and R.

Kosfekd Eds.), p. 249-281, Springer- Verlag, Berlin, New York, 1993.

39. W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Veterling, "Numerical Recipes: The Art of Scientific Computing," Cambridge University Press, New York, 1986. 40. J. S. Tauskela, E. A. Shoubridge, Response of the ² Na-NMR double-quantum filtered signal to changes in Na⁺ ion concentration in model biological solutions and human erythrocytes. Biochim. et Biophys. Acta 1158, 155-165 (1993). 41. J. M. Dizon, J. S. Tauskela, D. Wise, D. Burkhoff, P. J. Cannon, J. Katz,

91

Evaluation of triple-quantum-filtered Na NMR in monitoring of intracellular Na content in the perfused rat heart: comparison of intra- and extracellular transverse relaxation and spectral amplitudes. Magn. Reson. Med. 35, 336-345 (1996). 42. E. W. Peterson, K. G. Chen, N. Elkins, R. B. Hutchison, J. I. Shapiro. NMR spectroscopy studies of severe hypokalemia in isolated rat heart. Am. J. Physiol. 269, H1981-1987 (1995).

43. C. T. January, H. A. Fozzard, The effects of membrane potential, extracellular potassium, and tetrodotoxin on the intracellular sodium ion activity of sheep cardiac muscle. Circ. Res. 54, 652-665 (1984).

44. A. J. Levi, C. O. Lee, P. Brooksby, Properties of the fluorescent sodium indicator "SBFI" in rat and rabbit cardiac myocytes. J. Cardiovasc. Electrophysiol. 5, 241- 257 (1994). 45. M. S. Suleiman, New concepts in the cardioprotective action of magnesium and taurine during the calcium paradox and ischaemia of the heart. Magnesium Res. 7, 295-312 (1994).

46. R. A. Chapman, H. A. Fozzard, I. R. Friedlander, C. T. January, Effects of

Ca /Mg removal on aj(Na), aj(K), and tension in cardiac Purkinje fibers. Am. J. Physiol. 251, C920-927 (1986).

47. W. G. Nayler, S. E. Perry, J. S. Elz, M. J. Daly, Calcium, sodium and calcium paradox. Circ. Res. 55, 227-237 (1984).

48. A. B. T. J. Boink, T. J. C. Ruigrok, D. de Moes, A. H. J. Maas, A. N. E. Zimmerman, The effect of hypothermia on the occurrence of the calcium paradox. Pflugers Arch. 385, 105-109 (1980).

49. R. A. Chapman, Cardiovascular controversies. Cardiovasc. Res. 29, 284 (1995).

50. G. D. Buckberg, Update on current technique of myocardial protection. Ann. Thoracic Surg. 60, 805-814 (1995). 51. M. M. Gebhard, H. J. Bretschneider, P. A. Schnabel, Cardioplegia priciples and problems. In: "Physiology and Pathology of the Heart (N. Sperelakis Eds.), p. 731-743, Kluwer Academic, Boston, MA, 1995. 52. J. C. Cleveland Jr., D. R. Meldrum, R. T. Rowland, A. Banerjiee, A.H.

Harken, Optimal myocardial preservation: cooling, cardioplegia, and conditioning. Ann Thorac Surg 61 , 760-768 ( 1996). 53. R. L. Nelson, K. H. Fey, D. M. Follette, J. J. Livesay, E. C. DeLand, J. V. Maloney Jr., G. D. Buckberg, Intermittent infusion of cardioplegic solution during aortic cross-clamping. Surgical Forum 27, 241-243 (1976).

54. I. O. Choy, V. D. Schepkin, T. F. Budinger, D. Y. Obayashi, J. N. Young. W. M. DeCampli, Effects of specific sodium/hydrogen exchange inhibitor during cardioplegic arrest. Ann. Thoracic Surg. 64, 94-99 (1997).

55. V. D. Schepkin, I. O. Choy, T. F. Budinger, S. E. Taylor, Intracellular sodium in isolated perfused rodent heart and Na TQF NMR., in "Proc, Sixth Meeting of ISMRM, Sydney, Australia, 1998J p. 56. V. D. Schepkin. I. O. Choy, T. F. Budinger, D. Y. Obayashi, S. E. Taylor. W. M. DeCampli, S. C. Amartur, J. N. Young, Sodium TQF NMR and intracellular Na in isolated crystalloid perfused rat heart. Magn. Reson. Med. April, (1998).

57. G. D. Buckberg, J. R. Brazier, R. L. Nelson, S. M. Goldstein, D. H. McConnell, N. Cooper, Studies of the effects of hypothermia on regional myocardial blood flow and metabolism during cardiopulmonary bypass. I. The adequately perfused beating, fibrilating, and arrested heart. J Thorac Cardiovasc Surg 73, 87-94 (1977).

58. T. Ko, H. Otani, H. Imamura, K. Omori, C. Inagaki, Role of sodium pump activity in warm induction of cardioplegia combined with reperfusion of oxigenated cardioplegic solution. J. Thorac. Cardiovasc. Surg. 110, 103-110 (1995). 59. P.K. Stys, S.G. Waxman, and B.R. Ransom, Ionic mechanism of anoxic injury in mammalian CNS white matter: role of Na channels and Na-Ca exchanger. The

Journal of Neuroscience 12(2), 430-439 (1992). 60. M.C. Fehlings. S. Agrawal, Role of sodium in the pathology of secondary spinal cord injury. Spine 20, 20, 2187-2197 ( 1995). 61. S.K. Agarwal. M.G. Fehling, The effect of sodium channel blocker QX-314 on recovery after acute spinal cord injury. Journal of Neurotrauma 14, 2, 81-88 (1997).

»7

Claims

WHAT IS CLAIMED IS:

1. A method for detecting and measuring Na, in mammals using sodium multiple quantum NMR, comprising the steps of: a) proton-reference scanning a pre-selected region of the body of a mammal situated inside a nuclear magnetic resonance scanner; b) administering a pre-selected pulse sequence to the pre-selected region, resulting in a signal being generated from the bound and from the total sodium; c) generating a spectrum from the scanner of the bound and total sodium; and d) comparing the spectrum generated from the mammal with a standard calibration curve, which defines the relationship between the intracellular Na concentration and the bound Na NMR TQF signal; and thereafter e) determining the Na, concentration of the scanned region.

2. The method for detecting and measuring Na, in accordance with claim 1 , wherein the relationship between the intracellular Na concentration and the bound Na NMR TQF signal is linear.

3. The method for detecting and measuring Na, in accordance with claim 1 , wherein the standard calibration curve is illustrated in FIG. 6.

4. The method for detecting and measuring Na, in accordance with claim 1 , wherein the preferred multiple quantum NMR is TQF NMR.

5. The method for detecting and measuring Na, in accordance with claim 1 , wherein any pulse sequence can be used so long as the sequence separates the signal from bound Na.

6. The method for detecting and measuring Na, in accordance with claim 1 , wherein the pulse sequence is:

90°_φ -τ/2-180°_φ+α -ill- 90°_φ._HΪ -δ- 90°_ε where the delay δ = 40 μs, phase α = 90°, ε = 0°, and phase φ was incremented through 30°, 90°, 150°, 210°, 270°, 330° with receiver phase alternation by 180° each step.

7. A method for assessing the effect of multi-dose St. Thomas' cardioplegia on intracellular sodium homeostasis, comprising the steps of: a) proton-reference scanning a pre-selected region of the body of a mammal situated inside a nuclear magnetic resonance scanner; b) administering a pre-selected pulse sequence to the pre-selected region, resulting in a signal being generated from the bound and from the total sodium; c) generating a spectrum from the scanner of the bound and total sodium; and d) comparing the spectrum generated from the mammal with a standard calibration curve, which defines the relationship between the intracellular Na concentration and the bound Na NMR TQF signal; and thereafter e) determining the Naj concentration of the scanned region.

8. The method for assessing the effect of multi-dose St. Thomas' cardioplegia in accordance with claim 7, wherein the relationship between the intracellular Na concentration and the bound Na NMR TQF signal is linear.

9. The method for assessing the effect of multi-dose St. Thomas' cardioplegia in accordance with claim 7, wherein the standard calibration curve is illustrated in FIG. 6.

10. The method for assessing the effect of multi-dose St. Thomas' cardioplegia in accordance with claim 7, wherein the preferred multiple quantum NMR is TQF NMR.

11. The method for assessing the effect of multi-dose St. Thomas' cardioplegia in accordance with claim 7, wherein any pulse sequence can be used so long as the sequence separates the signal from bound Na.

12. The method for assessing the effect of multi-dose St. Thomas' cardioplegia in accordance with claim 7, wherein the pulse sequence is: 90°_φ -τ/2-180°_φ+α -τ/2- 90 ^ -δ- 90°_ε where the delay δ = 40 μs, phase α = 90°, ε = 0°, and phase φ was incremented through 30°, 90°, 150°, 210°, 270°, 330° with receiver phase alternation by 180° each step.

13. A method for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in normal and pathological tissues, comprising the steps of: a) administering to a mammal a pre-selected pharmaceutical; b) proton-reference scanning a pre-selected region of the body of the mammal following administration of the pharmaceutical, said mammal being situated inside a nuclear magnetic resonance scanner; c) administering a pre-selected pulse sequence to the pre-selected region, resulting in a signal being generated from the bound and from the total sodium: d) generating a spectrum from the scanner of the bound and total sodium; e) comparing the spectrum generated from the mammal with a standard calibration curve, which defines the relationship between the intracellular Na concentration and the bound Na NMR TQF signal; and thereafter f) determining the Na, concentration of the scanned region.

14. The method for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in accordance with claim 13, wherein the relationship between the intracellular Na concentration and the bound Na NMR TQF signal is linear.

15. The method for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in accordance with claim 13, wherein the standard calibration curve is illustrated in FIG. 6.

16. The method for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in accordance with claim 13, wherein the preferred multiple quantum NMR is TQF NMR.

17. The method for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in accordance with claim 13, wherein any pulse sequence can be used so long as the sequence separates the signal from bound Na.

18. The method for evaluating the effects of pharmaceutical intervention therapy on intracellular sodium in accordance with claim 13, wherein the pulse sequence is: 90°_φ -τ/2-180°_φ^ -τ/2- 90°_φ+α -δ- 90°_ε where the delay δ = 40 μs, phase α = 90°, ε = 0°, and phase φ was incremented through 30°, 90°, 150°, 210°, 270°, 330° with receiver phase alternation by 180° each step.

yv