ANALYTICALBIOCHEMISTRY

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The Determination of Intracellular Sodium Concentration in Human Red Blood Cells: Nuclear Magnetic Resonance Measurements Amira Rottman, Haggai Gilboa, Yael Schechter,* and Brian L. Silver Department of Chemistry, Technion-Israel Rambam Hospital, Haifa, Israel

Institute

of Technology,

and *Department

of

Hematology,

Received June 27, 1991

The intracellular sodium concentration and intracellular volume of human red blood cells were determined from “Na and ‘H NMR spectra. It is shown that sodium dissolved in the intracellular water has a concentration higher than that previously published. The intracellular sodium concentration measured was 11.4 f 3.1 mru. A comparison of different NMR methods used to determine sodium concentration is given. o 1992 Academic Press,

Inc.

The determination of intracellular concentrations of ions is an essential prerequisite for the quantitative understanding of many of the basic functions of cells. In particular, estimation of intracellular sodium concentrations in red blood cells has been the subject of much experimental effort (l-6). The determination of sodium concentrations requires the measurement of both the amount of intracellular sodium and the intracellular volume. Until recently, sodium was usually determined by rapidly separating cells from the medium and, after washing, using flame photometry or isotopic techniques to estimate sodium. Both methods are time consuming and neither is suitable for in uiuo measurements (5,6). Recently, the use of 23Na NMR and paramagnetic shift reagents has considerably simplified the determination of intracellular sodium (7-9). However, the determination of the intracellular volume available to sodium has often been approximate and not very accurate. For erythrocytes, for example, the method of determining volume is to measure the hematocrit, the proportion of the total volume of a cell suspension occupied by the cells when they are packed by centrifugation. However, the packed cells include extracellular solution, and the hematocrit therefore overestimates cellular volume. A

more direct method is to use a Coulter counter, but apart from the inaccuracy involved in using this apparatus to measure the volume of a nonspherical body, the volume obtained, as in the hematocrit method, is that of the whole cell, not of the internal space available to ions. The same arguments hold for the light-scattering-intensity method which is used for red blood cell volume determination (10). A similar criticism can be leveled at a recently described procedure involving the determination of cellular volume using 59Co NMR (2). As is shown below, the volume of the whole cell can be larger than the relevant intracellular volume. In the first attempt to measure the intracellular water space by NMR, Cowan et al. (3) induced a paramagnetic shift in the extracellular water by the use of dysprosium tripolyphosphate. The observed shift of the water NMR signal is a weighted average of this shift and the unshifted intracellular water. The method gives the ratio of internal to external volume but requires an accurate knowledge of the extracellular concentration of shift reagent, which must be determined indirectly from the 23Na shifts. Furthermore, three proton shifts must be measured, one of which must be corrected by taking into account the dysprosium concentration estimated from the sodium shifts. A further drawback of the method is the need for thorough deoxygenation in order to avoid paramagnetic shifts due to deoxyhemoglobin. Recently, a more accurate method for determining intracellular water space based on the use of deuterium NMR has been described (9,ll). However, in order to determine Na concentration by this method, three nuclei must be observed, 59Co, 2H, and 23Na (ll), or 2H and 23Na as was suggested by R. K. Gupta (9), where D,O is added to the red blood cells sample. We report here a straightforward method of determining intracellular concentrations of molecules or

48 All

ooo3-2697192 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.

NMR MEASUREMENT

OF Na IN RED BLOOD

CELLS

49

ions. The method is applied to the determination of so- ton signalsof the supernatant and the packedcells. This dium in human erythrocytes, for which purpose measurements of the NMR of two nuclei, 23Na and ‘H, are required. We compare different methods for red blood

quantity is proportional to the combined volume of the cell membranes and the nonaqueous contents of the cell, mainly hemoglobin in the case of erythrocytes. The

cell intracellular volume determination and show that the effective sodium concentration is higher than that quoted in the literature.

proton signals of the cell membrane and the cell’s macromolecules are too broad to contribute significantly to the observed proton resonances (12). The difference in intensity of the 23Na signals of the supernatant and the external solution of the sample is proportional to the total volume of the cells. The derivation of the intracellular sodium concentration from the observed spectra is performed as follows. We define: vint~vtotal = internal volume of cells/volume of the sample. vout~vtcltal = the intercellular volume/volume of the sample. vi.Jvtiti = p=t/INasup= = integrated intensity of external solution Na signal/integrated intensity of supernatant sodium signal. ( Vout+ Vint)l V,, = IW~mp’ellwSuper = integrated intensity of proton (water) signal of sample/integrated intensity of proton (water) signal of supernatant. /I NsStand = integrated intensity of internal Na sigI Neint nal/integrated intensity of solution of known Na concentration.

MATERIALS

AND

METHODS

Fresh blood from human donors was washed three times with Tris buffer containing the appropriate marker nuclei as described below. The washed erythro-

cytes were packed by centrifugation and transferred to a

lo-mm NMR tube in which there was a 5-mm tube containing the detected nuclei and shift reagent (see below) as an intensity reference. The supernatant was preserved. Unless otherwise mentioned, 23Na and ‘H NMR spectra of the sample were recorded at room temperature. The 23Na and ‘H spectra of the supernatant were also recorded. The measuring pulse gave =30” flip for sodium and z-3” flip for protons, allowing full relaxation when the delay between the measuring pulses was 400 ms for sodium and 1.5 s for protons. All compounds were analytically pure, except for Vi:,,,/ v,,, = pamPle/f%Per - pext/p.wer* Na5P3010. Technical-grade Na,P,O,, (Alfa Products) We thus use was recrystallized three times from a water ethanol solution. The washing solution was 20 mM Tris buffer, pH 7.4, [Na], = INaintlINastand* [Na],wd . Vm,/Vint [l] containing 1 mM Na,HPO,, 1 mM MgCl,, 140 mM NaCl, 5 mM KCl, 12 mM Na,P,O,,, and 5 mM Dy (NO,),. to calculate the intracellular sodium ion concentration. When 5gCo NMR measurements were carried out, the The 5gCoNMR spectra contained two lines, one of the washing solution contained 2 mM K,Co(CN),. When ‘H inner reference tube and the other of the sample. spectra were measured, the buffer was prepared in a Co(CN)i3 does not penetrate the red blood cell mem1:50 D,O:H,O ratio. brane and the intensity of cell suspension signal related The reference solution was 35 mM NaCl, 10 mM to that of the supernatant gave the ratio of intracellular 24 mM Na,P,O,, dissolved at a 1:1.5 volume to the total volume (V,,,IV,,),, as obtained DYNU,, D,O:H,O ratio. For 5gCoNMR measurements the referfrom the 5gCo NMR measurements. ence solution was 56 mM K, EDTA, 1 mM K,Co(CN),, 2H NMR spectra contained the reference and the sam20 mM Dy(NO,), dissolved at a 1:1.5 D,O:H,O ratio. The ple line originated from the D,O in the sample and the reference tubes were calibrated against a solution of 35 reference tube. As mentioned in Ref. (9), the D,O unmM sodium chloride. dergoes fast exchange between the inter- and intracelluNMR measurements were made within 4 to 6 h of lar media the same as H,O (3). donation and within an hour of the addition of shift The spectra of the packed cells were not time depenreagent. ‘H, 23Na, 5gCo, and 2H were recorded on a dent over a period of at least an hour, indicating that no Bruker AM 400 WB NMR spectrometer at 400.13, settling of cells occurred during the measurements and 105.8, 94.91, and 61.43 MHz, respectively. that there was no flux of sodium through the membrane. The 23Na spectra of the cells showed three peaks due to the reference solution, the intracellular sodium, and AND DISCUSSION the sodium in the external solution. The reference and RESULTS external sodium peaks are shifted by the paramagnetic Typical spectra of sodium and protons in red blood shift reagent dysprosium tripolyphosphate. The intencells are presented in Fig. 1. The sodium spectrum presity of the peaks was related to the reference signal. sented in Fig. la shows three signals; Ns, the intracelThe method of determining intracellular volume is lular sodium; Naout, the extracellular sodium; and the partially based on the difference in intensity of the pro- inner tube signal serving as an intensity reference. Fig-

50

ROTTMAN

ET AL.

a

FIG. 1. NMR spectra of red blood cells. (a) Sodium spectrum the extracellular space; and Ref, from the inner reference tube. red blood cells and the water in the reference tube.

shows three signals: (b) Proton spectrum

ure lb shows the proton spectrum of the red blood cells sample. The spectrum represents the water inside and outside the cells and the inner tube reference signal. As can be seen, the lines are well separated in both spectra. The origin of the shift, i.e., hyperfine shift or bulk susceptibility shift (13), is not relevant for this study since the interesting feature is the separation between the signals. Some experimentally determined intracellular 23Na concentrations in samples of human erythrocytes were calculated according to Eq. [l] and are shown below: Sample: Na,, (mM):

1 5.5

2 19.3

3 14.0

Sample: Na,, (mM):

8 8.9

9 10.3

10 12.6

4 13.4

5 8.3

6 11.3

7 11.4

11 12 9.4 12.7

13 12.1

14 10.7

The mean value of intracellular sodium concentration calculated is 11.4 + 3.1 mM. This value is about 20-30% higher than those cited in the literature [7-8.5 InM; Refs. (1,2,5,6)]. On the other hand, our results and the results obtained by Cowan et al. (3), i.e., 10.7 f 1.9 mM, agree within experimental error. The volume determined in this study is that occupied by mobile water and it is exactly this volume that is relevant to the estimation of intracellular concentrations of ions and other molecular species. The method gives the fraction of the total volume of a sample that is occupied by the aqueous portions of the cells. The absolute “aqueous volume” of one cell can be obtained if a cell count is performed. In the case of red blood cells this is readily carried out on a Coulter counter or by counting under a microscope. The intracellular sodium concentrations usually measured take into account the

Na,,, resulting from the intracellular shows two signals, the water signal

volume; Na,,, from inside and outside the

whole cell volume (1,2,5,6) in contrast to the measurements by Cowan et al. (3) and those by us where only the volume of the mobile water is considered. To compare our method to other NMR methods (2,11), we performed the following experiments using 5gCoand ‘H NMR studies. From the 5gComeasurements we obtain the extracellular volume of the sample and compare it to the result obtained from sodium NMR of the same sample. Table 1 presents measurements on two different samples (different rows). It can be seen from Table 1 that with regard to the extracellular volume, we obtain almost the same results whether we use 59Coor 23Na; therefore, there is no need to add CO(CN),~ to the washing media for the determination of sodium concentration. Moreover, if Eq. [l] of Ref. (2) is used, sodium concentrations close to those published in the literature are obtained.

TABLE Intracellular

Sodium Concentrations: 5gCo and 23Na NMR

0.28 0.23

Comparison

p~x,/p.,, 0.27 0.17

between

Measurements

Intracellular Na concentration (mM)a

voutJvti p=qpb”P.

1

Intracellular Na concentration (mM)*

c

d

c

d

7.36 8.83

7.26 8.19

12.6 14.4

11.5 12.6

’ As calculated by Eq. [l] from Ref. (2). * Using Eq. [l] in this study. c Values were taken from column 1. d Values were taken from column 2.

NMR MEASUREMENT

We attempted to apply this method to the determination of intracellular sodium concentrations in young and old erythrocytes separated on the density gradient

TABLE 2 Intracellular

Sodium Concentration:

51

OF Na IN RED BLOOD CELLS

Results Obtained

from ‘H and ‘H NMR Studies

of structan. The results did not show any differences VJVm Sample

1 2 3 4

Na,, (mM)

‘H

2H

‘H

2H

0.40 0.47 0.33 0.42

0.40 0.41 0.30 0.44

10.30 9.40 12.70 12.10

10.30 9.40 14.00 11.52

between young and old cells of the same donor (in collaboration with J. Hochman and D. Gershon, unpublished results). The method presented here was applied to red blood cells. It is questionable whether the method could be applied to other types of smaller cells or to multicellular organisms. REFERENCES

Another suggestion was to use D,O for the determination of the intracellular volume accessible to water (9,ll). Since H,O was used for the same purpose in this study, a comparison of the two methods was carried out and the results are presented in Table 2. The calculations in Table 2 are based on Eq. 111of this study. As can be seen from Table 2, there is no need to use D,O for the determination of the intracellular volume. As can be deduced from the results presented in this research, measurement of 23Na and ‘H NMR signals is sufficient to study the intracellular sodium concentration. Since most NMR probes contain ‘H decoupling coils, it is very simple to use this method, It has been suggested that the same detection coil should be used for volume determinations to obtain reliable results (9,ll). It is shown here that if one uses a reference tube and the sample is homogeneous, the results obtained are reliable and the difference in coil size has no effect. Our results are similar to those of Cowan et al. (3), probably because in both methods the volume accessible to water is measured. We think that our method is easier to use.

1. Trevisan, M., Ostrow, D., Cooper, R., Liu, K., Sparks, S., and Stamler, J. (1981) Clin. Chim. Actu 116,319-351. 2. Shinar, H., and Navon, G. (1984) Biophys. Chem. 20,275-283. 3. Cowan,

B. E., Sze, D. Y., Mai,

M.

T., and Jardetzky,

0. (1985)

FEBS Lett. 184,130-133. 4. Rottman, A., Gilboa, H., Schechter, Y., and Silver, B. L. (1986) Proceedings of the XXIII Congress Ampere on Magnetic Resonance, Roma (Maraviglia, B., De Luca, F., and Campanella, R., Eds.), p. 532-533. 5. Khalit-Manesh, U. G. (1987) 6. Tuck,

F., Venkataraman,

K., Samat,

D. R., and Gadgil,

Hypertension 9, 18-23.

M. L., Corry,

D. L., Maxwell,

M.,

and Stern,

L. (1987)

Hy-

pertension 10, 204-211. 7. Gupta, 8. Pettegrew, T. (1984) 9. Gupta, (Gupta, 10. Bartels,

R. K., and Gupta, J. W., Woessner, J. 1Liagn. Reson.

J. Magn.

Reson. 47,344-350.

D. E., Minshow, 57, 185-196.

N. J., and Glonek,

P. (1982)

R. K. (1987) in Nmr Spectroscopy of Cells and Organisms R. K. Ed.), Vol. II, pp. l-32, CRC Press, Boca Raton, FL. P. C., Helleman, P. W., and Soons, J. B. J. (1989) Scan. J.

Clin. Lab. Invest. 49, 225-231. H., and Navon, G. (1985) FEBS Lett. 193, 75-78.

11. Shinar,

12. Sullivan, Winston,

S. G., Stern, A., Rosenthal, A. (1988) FEBS Lett. 234,

13. Chu, S. C., Pike, M. M., Fossel, and Springer, C. S., Jr. (1984)

J. S., Minkoff,

L. A., and

349-352.

E. T., Smith,

T. W., Balschi,

J. Mugn. Reson. 56, 33-47.

J. A.,