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Journal of Biochemical and Biophysical Methods, 21 (1990) 87-102
87
Elsevier BBM 00814
On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance Gheorghe Benga 1,2, Victor Ioan Pop, Octavian Popescu 1 and Victoria Borza i 1 Department of Cell Biology, Faculty of Medicine, Medical and Pharmaceutical Institute Cluj-Napoca, and 2 Institute of Hygiene and Public Health Cluj-Napoca, Cluj-Napoca, Romania (Received 3 July 1989) (Accepted 31 January 1990)
Summary The characteristics of water diffusional permeability ( P ) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2×10 -3 cm.s -1 at 5 ° C to 8.1×10 -3 cm-s-1 at 42 o C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3×10 -3 cm.s -1 at 20°C, 1.6x10 -3 cm.s -1 at 25°C, 1.9×10 -3 cm.s -1 at 3 0 ° C and 3.2x10 -3 cm-s -1 at 37°C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions. Key words: Water diffusion; Red blood cells; Human; Nuclear magnetic resonance
Introduction Because of its availability and simple structure, lacking internal membranes, the red blood cell (RBC) is ideally suited for investigating water permeability. In fact, Correspondence address: Gh. Benga, M.D., Ph.D., Department of Cell Biology, Faculty of Medicine Cluj-Nalxx~ 6 Pasteur St., 3400 Cluj-Napoca, Romania. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
88 for many years it has been one of the most favored cells for such investigations (for recent reviews see Refs. 1-5). There are two categories of methods for measuring water diffusion in RBCs: the radio-tracer methods [6,7] and the nuclear magnetic resonance (NMR) methods [8,11]. Studies on the water diffusional permeability of human RBCs have been performed by both methods. However, there are discrepancies in the permeability coefficients reported by various authors using different methods or the same method. One aim of this paper was to find the reasons for some of the discrepancies. However, all previous investigations involved measurements performed on a small number of subjects a n d / o r at one single temperature. Therefore, the second aim of our work was to perform NMR measurements of the water diffusion in erythrocytes obtained from a large group of subjects. This would reveal the characteristic permeability of human RBC membranes to water. Studies on the effects of mercury-containing sulfhydryl-reacting reagents (SH reagents) can contribute to a better understanding of the molecular mechanisms of the water permeability of red blood cells. For example, it has been shown that such reagents substantially inhibited the water exchange through erythrocyte membranes [12-17]. This has led to the suggestion that aqueous channels accommodated in the membrane proteins play a major role in the water exchange and that the mercurycontaining SH reagents act by closing the channels [18]. Moreover, recent studies using 2°3Hg-labeled p-chloromercuribenzene sulfonate (PCMBS) have allowed us to identify the membrane proteins that are involved in the water exchange through the human RBCs [19,20]. As pointed out by Stein [21], the permeability values obtained in conditions of maximal inhibition of transport of certain molecules through a membrane characterize the basal permeability of that membrane for the molecules involved, i.e., the permeability in the absence of any specific transport pathway. Consequently, in the case of red blood cells and water transport, measurements performed in the presence of mercurials would estimate the basal permeability of their membrane. The third aim of our paper was to provide an accurate value of this parameter for the human RBC membrane. In order to properly characterize all the above-mentioned aspects of the RBC water diffusional permeability (P), the measurements were performed in both erythrocytes and resealed ghosts, at a variety of temperatures. The data, representing the largest series available in the literature, can thus be taken as reference values for P of human red blood cells at various temperatures. Such values are important for comparing the characteristics of water permeability in various cells and also for further studies with potential clinical importance. Materials and Methods
Blood sample preparations Human blood from healthy males and females 2-50 years old was obtained by venapuncture and stored in heparinized tubes. The erythrocytes were isolated by
89 centrifugation, and washed three times in medium S (150 mM NaCI, 5.5 mM glucose, 5 mM Hepes (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), pH 7.4. For the preparation of resealed (pink) ghosts, the procedure of Bodemann and Passow as described by Schwoch and Passow [22] was used. The incubations of erythrocytes with PCMBS were performed as indicated in the tables. After incubation, the erythrocytes were washed three times in medium S, each followed by a centrifugation, in order to remove the reagent.
NMR measurements Samples for NMR measurements were prepared by carefully mixing 0.2 ml erythrocyte suspension and 0.1 ml doping solution (40 mM MnC12, 100 mM NaC1). The water proton relaxation time of the cells (T2'a) was evaluated by the spin-echo method [8] as previously described [2,10,23]. T2'a is dominated by the exchange process through the erythrocyte membrane and is related to the water diffusion exchange time (Te) by the equation [10]: 1
1
1 T=i
= T2~
(1)
where T2i is the transverse relaxation time of the cell interior. T2i was measured by the 90-180 ° method using the Carr-Purcell-Meiboom-Gill sequence [24] on packed cells, from which the supernatant, with no added Mn, had been removed by centrifugation at 50 000 x g for 60 min. The membrane permeability for water diffusion, P, is related to 1/T3, the cell water volume, V (= 0.72 times the cell volume) and the call surface area, A, by: V V=~.g
1
(2)
Since different authors have used different values of V and A, we have used in the calculation of P three sets of values. On one hand, we have used the V/A ratio of 4.58 x 10 -5 cm, after Brahm [15] and the V/A ratio of 5.33 × 10 -5 cm, after Dix and Solomon [26]. On the other hand, we have performed our own estimations of the cell volume based on cell counts and measurement of the hematocrit, with correction for extracellular medium trapped between the packed cells. The values of the cell volume obtained in this way were within the ranges reported in the literature [26,27]. The value of A should be chosen from the values in the literature, and the most accepted present value is 1.35 × 10 -6 cm2 (see for discussion Ref. 3). When calculated with this value of A and with values of V measured as described above, the values of P were not significantly different from the values calculated by using the V/A ratios after Dix and Solomon [26]. The inhibition of water diffusion across human red blood ceil membranes was calculated in two ways. On the one hand, assuming that the permeability coefficient
90 is inversely r e l a t e d to I'2', we used the f o r m u l a [2,14,28]:
1
1
T2' ( c o n t r o l ) %inhibition =
T~ ( s a m p l e ) 1
× 100
(3)
T2~ ( c o n t r o l ) O n the o t h e r h a n d , in o r d e r to b e t t e r c o m p a r e o u r results w i t h those o f o t h e r a u t h o r s [12,13,15,24], a n o t h e r f o r m u l a was also used: %inhibition -- P ( c o n t r o l ) - P ( s a m p l e ) P(control)
(4)
T h e N M R m e a s u r e m e n t s were p e r f o r m e d with a n A R E M I - 7 8 s p e c t r o m e t e r ( m a n u f a c t u r e d b y the I n s t i t u t e of Physics a n d N u c l e a r E n g i n e e r i n g B u c h a r e s t Mfigurele, R o m a n i a ) at a f r e q u e n c y of 25 M H z . T h e t e m p e r a t u r e was c o n t r o l l e d to + 0 . 2 ° C b y air flow over a n electrical resistance, using the v a r i a b l e t e m p e r a t u r e unit a t t a c h e d to the spectrometer. T h e a c t u a l t e m p e r a t u r e in the s a m p l e was m e a s u r e d with a t h e r m o c o u p l e c o n n e c t e d to a m i c r o p r o c e s s o r t h e r m o m e t e r ( C o m a r k E l e c t r o n ics Limited, R u s t i n g t o n , L i t t l e h a m p t o n , U.K.). T h e values of T2~ were c a l c u l a t e d with a c o m p u t e r unit c o u p l e d o n - l i n e with the N M R s p e c t r o m e t e r .
Other procedures T h e calculations o f the c o r r e l a t i o n coefficients of the lines o b t a i n e d with the sets o f d a t a p o i n t s in the A r r h e n i u s plots were p e r f o r m e d with a n HP-41 C V c o m p u t e r ( H e w l e t t - P a c k a r d , U.S.A.).
TABLE 1
Effect of preincubation of erythrocytes on the inhibition induced by subsequent incubation with PCMBS Washed erythrocytes were prepared as described in Materials and Methods, suspended in medium S at a hematocrit of 10%, and incubated with 1 mM PCMBS for 30 min at 37°C; these are 'control erythrocytes'. In the case of 'preincubated erythrocytes' the incubation with 1 mM PCMBS (for 30 rain at 37 o C) followed to a preincubation of red cells (suspended in medium S at a hematocrit of 10~) for 60 min at 37°C. A and B: %inhibitions were calculated according to Eqns. 3 and 4, respectively. The P values were obtained using the paired Student's t-test. Temperature
Sample
A
B
Statistical significance of the difference
~ Inhibition
20 ° C
control erythrocytes preincubated erythrocytes
54.2 ± 1.3 60.0 ± 1.4
59.1±1.5 65.0±1.6
P < 0.001
37 o C
control erythrocytes preincubated erythrocytes
43.1 ± 1.2 49.8 ± 0.4
45.2±1.2 52.1±0.5
P < 0.025
20 37 20 37 15 20 25 30 37 42 20 37 7 10 20 37 10 15 20
2 2 5 4 2 4 2 2 5 1 2 2 1 1 2 1 6 29 22
0.5 mM PCMBS
1.0 mM PCMBS
5 7 10 15 20 25 30 33 37 42
3 3 27 37 25 42 38 38 43 37
37
37
15
30
37
60
37
37
30
5
37
15
Temp. ( * (2)
Control
Time (rain)
Temp. of measurement
Conditions of incubation
No. of determinations
Compound
20.3 14.1 43.0 41.6 25.4 12.3 30.3 30.3 24.7
23.0 12.1 26.2 13.8 30.4 25.4 22.5 19.6 11.9 9.6
18.0+1.5 17.2:t:1.6 15.2+1.6 13.8+1.8 12.1 + 1.8 10.6+1.1 9.1 + 0.7 8.4 + 0.7 7.1+0.6 6.3 + 0.6
T2~ (ms)
25.0 15.6 97.8 81.7 33.2 13.4 47.4 + 4.2 41.2+6.3 32.0 4- 5.0
29.2 13.2 34.5 + 1.7 15.3 4-1.4 44.5 33.2+5.1 22.7 23.0 12.9+0.1 10.2
24.0+1.5 22.2::t:1.7 18.6-t-1.7 16.1-1-1.8 13.6 + 1.8 11.6+1.1 9.9 -t-0.7 8.9 + 0.7 7.5+0.6 6.6 + 0.6
Te (ms)
1.83 2.94 0.47 0.56 1.38 3.42 0.97 + 0.09 1.04+0.15 1.43 + 0.22
1.57 3.47 1.33 + 0.07 2.99 + 0.27 1.03 1.38+0.21 1.65 1.99 3.55+0.03 4.49
1.91+0.30 2.06-1-0.27 2.46+0.22 2.84+0.32 3.37 + 0.44 3.95+0.37 4.63 + 0.33 5.15 + 0.41 6.11+0.49 6.94 + 0.76
I
P cm. s - 1 x 10 3
2.13 3.42 0.54 0.65 1.61 3.98 1.12 + 0.10 1.21-1-0.17 1.67 4- 0.26
1.82 4.04 1.55 + 0.08 3.48 4- 0.32 1.20 1.61+0.25 1.92 2.32 4.13+0.03 5.23
2.22+0.35 2.40+0.31 2.87+0.26 3.31+0.37 3.92 + 0.52 4.59+0.43 5.38 + 0.38 5.99 + 0.47 7.11+0.57 8.08 + 0.88
II
53.4 43.2 60.1 51.0 63.8 59.0 58.1 56.9 41.9 40.2 45.6 51.9 76.8 77.2 59.0 44.0 60.8 63.6 57.5
47.5 41.1 53.9 48.6 54.7 52.5 53.0 53.0 39.9 38.7 30.5 49.5 62.6 63.4 52.4 42.0 49.7 54.4 51.1
%Inhibition A B
Washed red blood cells were prepared as described in Materials and Methods. The incubations with PCMBS were performed at a hematecrit of 10% in medium S. The cells were sedimented by centrifugation and aliquots were used for N M R measurements. Results are m e a n + SD. I and II: the permeabilities calculated from T~ using V/A ratios of 4.58 x 10-5 cm [15] and 5.33 × 10-5 cm [25], respectively. A and B: %inhibitions calculated according to Eqns. 3 and 4, respectively.
Diffusional permeability of the human red blood cells
TABLE 2
37
37
37
37
37 37
37
37 37
60
5
15
30
1 5
0
1 5
4.0 mM PCMBS
10 mM PCMBS
2.0 mM PCMBS
Temp. ( ° C)
Conditions of incubation
Time (rain)
Compound
T A B L E 2 (continued)
1 1 1 1
1 1 1 1 1 10 20 20 20
20 7 10 20 20
6 10 20 37 15 20 25 30 37 20 37
25 30 33 37 42 10 15 20 25 30 33 37 42
21 20 5 30 12 3 5 7 4 5 4 8 5 1 1 1 1 1 5 1 1 5 1 1
Temp. of measurement
No. of determinations
26.8 17.9 23.2 20.7
27.3 38.2 38.5 27.7 31.6
39.0 36.7 23.9 12.8 32.1 26.3 23.7 20.7 13.3 28.3 12.8
20.8 17.4 14.4 12.4 10.6 31.0 25.4 26.1 20.5 16.8 15.6 12.8 9.7
(ms)
1.824-0.20 2.29 4- 0.27 2.84+0.55 3.39 5:0.25 4.024-0.28 0.93 1.33 4- 0.48 1.33 4- 0.28 1.854-0.46 2.39 4- 0.17 2.62 4- 0.42 3.27 5:0.21 4.45 + 0.60
1.49 3.27 0.95 1.32 5:0.21 1.55 1.87 3.144-0.11 1.21 3.27 1.25 0.60 0.64 1.23 1.03 1.16 2.14 1.55 1.79
81.2 64.6 30.7 14.0 48.2 34.8 4- 5.5 29.5 24.5 14.64-0.5 37.8 14.0 36.5 76.3 71.1 37.2 44.6 39.4 21.4 29.5 25.6
I
c m . s -1 x 10 3
25.25:2.7 20.0 4- 2.4 16.15:3.1 13.5 4-1.0 11.44-0.8 49.2 34.5 4-12.5 34.4 4- 7.3 24.75:6.1 19.2 5:2.0 17.5 4- 2.8 14.0 4- 0.9 10.3 4-1.4
re (ms)
1.35 2.49 1.81 2.09
1.46 0.70 0.75 1.43 1.20
1.74 3.81 1.11 1.53 4- 0.24 1.81 2.18 3.654-0.12 1.41 3.81
2.124-0.23 2.67 4- 0.32 3.315:0.64 3.95 4- 0.29 4.684-0.33 1.08 1.54 +__0.56 1.55 4- 0.33 2.164-0.53 2.78 4- 0.20 3.05 4- 0.49 3.81 4- 0.24 5.17 4- 0.70
II
43.2 32.5 47.9 41.6
55.8 57.9 60.4 56.4 61.8
55.9 58.5 49.5 44.2 57.0 54.2 55.3 55.5 46.3 56.9 44.2
49.2 47.0 42.0 42.3 44.8 50.9 45.6 53.7 51.6 45.1 46.2 44.2 39.3
A
52.7 36.6 53.9 46.8
62.7 73.3 73.8 63.4 69.5
72.1 71.2 55.7 46.4 66.6 60.9 60.8 59.6 48.6 64.0 46.4
54.0 50.5 44.7 44.4 46.5 62.2 53.3 60.6 53.0 48.4 49.1 46.4 40.8
B
%Inhibition
to
,~
25 30 33 37
17.9±2.8 15.7±2.4 14.5±2.2 12.5±1.6 10.8±1.3 9.9±1.1 8.7±1.0 7.6±0.7
22.8±2.9 18.8±2.5 16.8±2.3 14.0±1.6 11.8±1.3 10.7±1.1 9.3±1.1 8.0±0.7
T2a
(ms)
(°C)
10 15
Te
(ms)
Control
Temp.
2.66±0.34 3.22±0.43 3.61±0.49 4.33±0.49 5.14±0.57 5.66±0.58 6.52±0.47 7.58±0.66
I 3.09±0.39 3.75±0.50 4.20±0.57 5.~±0.58 5.97±0.66 6.59±0.58 7.58±0.54 8.81±0.77
II
P ( c m - s - I ×103 )
24.4±1.5 22.9±0.6 21.2±1.0 17.2±2.3 14.0±1.7 12.6±1.7 11.0±1.0 9.7±0.8
(ms)
T2'a
PCMBS T~
34.4±1.5 30.1±0.6 26.3±1.0 20.1±2.3 15.6±1.7 13.8±1.7 11.9±1.0 10.3±0.8
(ms)
1.75±0.08 2.01±0.~ 2.32±0.09 3.01±0.34 3.88±0.42 4.39±0.54 5.09±0.43 5.88±0.~
I
2.05±0.09 2.34±0.05 2.68±0.10 3.51±0.~ 4.52±0.49 5.11±0.63 5.92±0.50 6.84±0.53
II
P (cm-s-1 ×103 )
33.7±4.8 37.5±5.6 36.1±4.3 30.3±1.5 21.3±4.1 22.5±5.3 21.8±6.1 22.3±7.7
%Inhibition
The measurements have been performed on duplicate blood samples from 3-5 donors. The incubation with 0.1 mM PCMBS was performed for 20 rain at 37 o C at a cytocrit of 10% in medium S. After incubations three washes of resealed ghosts in medium S were performed, each followed by a centrifugation. Aliquots from the sediment were used for NMR measurements as described in Materials and Methods. The inhibition was calculated according to Eqn. 4. Results are expressed as mean± SD. The permeability was calculated from Te using a V/A ratio of 6.06 X 10 -5 cm [15] in column T and a slightly higher V/A ratio, 7.05 x 10 -5 cm, in colunm II, corresponding to the values for volume and membrane area of human red cells given by Dix and Solomon [26].
Diffusional permeability of the human resealed ghosts
TABLE 3
94 Results
In order to obtain accurate values for the basal permeability to water of the RBC membrane, it is important for the measurements to be performed under conditions of maximal inhibition of water transport. We have previously studied some factors influencing the inhibition by mercurials of water diffusion through erythrocyte membranes and found that the conditions for the exposure of erythrocytes to mercurials (concentration, temperature, duration) are critical for the degree of inhibition [14,16,20]. We report here new features of the development of PCMBS-induced inhibition of water diffusion. The degree of inhibition appeared to increase by decreasing the temperature at which water permeability is measured (Table 1). In addition, as documented by data in the same table, a higher degree of inhibition could be obtained if the exposure to PCMBS was performed after a preincubation of erythrocytes for 60 min at 37 ° C without inhibitor. Systematic studies were performed to determine in a large number of subjects the values of the water permeability of erythrocytes and ghosts, in the absence and in the presence of SH reagents, at various temperatures. In order to enable comparison with values reported by various authors, based on measurements performed by NMR or other techniques, the values of T2'a, T~ and the corresponding permeability at various temperatures are listed in Table 2. It should be noticed that the same value of permeability could be obtained under various conditions of exposure of erythrocytes to PCMBS. The lowest values of permeability were around 1.3 × 10 -3 cm- s -1 at 20°C, 1.6 × 10 -3 cm. s -1 at 25°C, 1.9 × 10 -3 cm- s -1 at 30° C and 3.2 × 10 -3 c m - s -1 at 37°C. These values seem to be the best estimates for the basal permeability of human red blood cells to water. Resealed ghosts have a permeability similar to erythrocytes, both in the absence and in the presence of PCMBS (Table 3), longer values of T~ in ghosts being due to a higher intracellular solvent volume caused by the removal of hemoglobin [15,23].
Discussion
As shown above, the inhibition by mercurials of water diffusion increased when measurements are performed at lower temperatures. This should be interpreted by taking into account the mechanisms of water diffusion across erythrocyte membranes and the action of mercurials. Two parallel pathways have been considered for water transport [1]. One pathway, nonspecific, is the diffusion across the lipid region, the water molecules riding along with free-volume pockets created in the membrane interior by thermally generated mobile structural defects in the hydrocarbon chains, the so-called 'kinks' [29]. The second pathway is represented by hydrophilic channels for water movement, the so-called 'pores' localized in membrane proteins. There is general agreement that PCMBS inhibition reflects the closure of water channels in proteins. We suggest that the proportion of water diffusing across the
95 lipid pathway relative to the protein pathway decreases by decreasing the temperature. This can be explained by taking into account the effect of temperature upon the lipid counterpart of membrane. It is possible that by decreasing the temperature, the fluidity of the lipid bilayer decreases. Then, since only the protein pathway is inhibited by mercurials, a higher degree of inhibition of water diffusional permeability is expected to occur at lower temperatures. The results presented above represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature. A comparison with previous reports of other authors using various methods for measuring the water permeability is of interest. There are several reasons for the rather large variation in permeability values reported by various authors (see the section 'Data as reported' in Table 4) and also the compilation of values given in some publications [3,5,25,33]. Sometimes a permeability value has been calculated from the parameter originally reported; however, little attention was paid to the way the parameter itself was actually estimated. This is the case with the data taken from the original reports of Paganelli and Solomon [6] and Morariu and Benga [10] (see the explanation in the footnotes to Table 4). In other cases the water permeability values have been calculated using rather different values for the RBC volume and membrane area, or in case of radiotracer measurements, without taking into account the difference in the bulk water coefficient between 3HHO and H20. This is the case of the permeability value given by Brahm [15], that was considered by Dix and Solomon [26] to be in disagreement with other authors' determinations. Although, when recalculated as described above, the values of permeability obtained by tracer 3HHO diffusion do not differ from those obtained by other methods (Table 4), the flow-tube approach is technically demanding, requires large quantities of blood and is inaccurate when used to measure exchange times much under 10 ms. In addition, the presence of extracellular unstirred layer could result in an underestimate in the value of diffusional permeability [47]. Another source of apparently divergent results resides in the differences of the Ea,d value taken as basis of calculation when measurements performed at different temperatures are converted to another temperature (usually 20°C). In order to ensure a reliable basis for comparison, we have recalculated the values available in the literature taking into account all the above-mentioned factors and using the same V/A value and the s a m e Ea, d = 25 kJ/mol, that seems to be the best estimate for human RBC membrane [17]. For example, there are several complications that occur in the water exchange measurement by NMR in conditions different from those using the Conlon and Outhred [8] method. The use of NMR to measure water-exchange rates in red cell suspensions involves the measurement of the rate that nuclear spins of water exchange from inside the cell to outside the cell. If the water exchange is measured by 170 T 1 or T2 methods, or by 1H T2 methods, then there are only two exchanging spin systems, intracellular and extracellular water. If, however, the water exchange is measured by 1 H T 1 methods, then there is a third spin system which contributes to the exchanging spin system. The third spin system is the hemoglobin protons, which are able to exchange with the intracellular water
96 TABLE 4
Diffusional permeability of the human red blood cell membrane In the section 'Data as reported' the number of experiments, given in parentheses, is placed in the column under the parameter that was actually reported. When there are no parentheses, the number of experiments was not recorded. Conversion to the standard temperature of 20 o C was made using the Ea, d of 25 k J / m o l [17]. The value of P in the section 'Converted to standard cell' was calculated from Te using a V/A ratio of 5.33X10 -5 cm [25]. The radiotracer coefficients were increased by 14% to take account of the difference in the bulk water diffusion coefficients between 3HHO and H 2 0 [6]. Cell sample method
Data as reported Temp. (°C)
Te (ms)
Human red cells radiotracer (rapid flow)
23
radiotracer (rapid flow)
23
radiotracer (rapid flow) radiotracer (rapid flow)
22 room temp. 25
11.5 a (n = 7) 10.9 (n =10) 13.1 15.3
radiotracer (rapid flow) radiotracer (linear diffusion) radiotracer (linear diffusion)
13.0
Converted to 20 o C H20 , standard cell P (cm.s -1 x103)
2.4
20
3.71
20
5.3
Ref.
Te (ms)
P ( c m - s - ! x103)
12.8
4.16
[6[
12.2
4.37
[301
14.0 16.5
3.81 3.73
[71 [31]
16.0
3.80
[151
3.71
[32]
15.0
4.13
[33]
NMR, T 2, doping (15.4 or 30.8 mM MnCI2)
25
11.0+0.4 (n = 9)
13.0
4.10
[8]
NMR, doping (40 or 80 mM MnCI z)
20
12.2 + 1.0 (n = 12) 6.0 + 0.5 b (n = 35) 11.0+0.6 (n = 9) 7.5 (n = 5) 7.1 + 0.4
12.2
4.37
[34]
13.3
4.00
[101
13.0
4.10
[351
13.6
3.92
[36]
13.2
4.03
[16]
7.3 (n = 8) 10.9 + 1.8 (n = 7) 6.6 + 0.4 (n =11) 11.0
13.4
3.98
[28]
13.6
3.92
[25]
12.7
4.19
[37]
13.0
4.10
[38]
13.3 + 1.3 (n = 12) 7.75 + 0.48
13.3
4.01
[231
13.8
3.86
[17]
NMR, Tz, doping (20 mM MnC12) NMR, Tz, doping (35 or 53 m M MnC12) NMR, '1'2,doping (20 mM MnClz) NMR, T2, doping (20 mM MnC12) NMR, doping (20 mM MnC12) NMR, I'2, doping (20 mM MnC12) NMR, Tz, doping (20 mM MnClz) NMR, T2, doping (30 mM MnC12) NMR, doping (20 mM MnC12) NMR, doping
37 25 37 37 37 27 37 25 20 37
97 TABLE 4 (continued) Cell sample method
Data as reported Temp. (°C)
(20 mM MnCl2) NMR, doping (20 mM MnC12) NMR, doping (20 mM MnC12) radiotracer (rapid flow) NMR, 170, T 1 NMR, field gradient NMR, T1, doping (2 mM MnC12) Ibid, cells suspended in 50% diluted plasma 2 rain after disaggregation Ibid, cells suspended in isotonic NaCI/KC1, with 4% serum albumin, 2 rain after disaggregation Ibid, cells suspended in isotonic NaC1/KCI with 4% serum albumin and 2% Dextran T40, immediately following disaggregation Resealed ghosts radiotracer (rapid flow) NMR, '1'1,'I'2,doping (2 mM MnCI2, ceils suspended in plasma) Ibid, cells suspended in isotonic NaC1/KC1 with 3% serum albumin NMR, T 1, doping (20 mM MnC12) NMR, T2, doping (1.7 mM MnC12) Ibid NMR, T2, doping (1.7 mM MnC12) NMR, T2, doping (4.4 mM MnCl 2, cells suspended in plasma 2 min after disaggregation)
37 20 25 25 25 25
T~ (ms) (n =12) 6.21 + 0.71 (n = 15) 13.6 + 1.8 (n = 43) 19.0 16.7 17 21.7 + 2.5
Converted to 20 o C H20 , standard cell P ( c m . s - l x l 0 3)
2.40
Ref.
T~ (ms)
P ( c m . s - l × 1 0 3)
12.3
4.33
[39]
13.6
3.92
this paper
21.0 18.7 19 22.7
2.54 2.85 2.80 2.35
[151 [401 [41] [9]
room temp.
15
16.2
3.24
[42]
ibid
12.8
14.1
3.78
[42]
ibid
12.2
13.5
3.95
[42]
25
16.3
2.9
12.9
4.43
[151
25
18.4
21 +0.2
20.4
2.61
[43]
25
12.9
3.0 + 0.2
14.9
3.57
[43]
25
17.7
19.7
2.70
[351
23
21.0 + 0.6
22.3
2.39
[11]
25 25
20.0 20.8 + 0.3 (n = 20) 20.0
22.0 22.8
2.42 2.34
[441 [45]
23.2
2.30
[42]
room temp.
98 T A B L E 4 (continued) Cell sample method
Data as reported Temp. (°C)
T¢ (ms)
37
8.83 + 0.39 (n =19) 19.3+3.3 (n =12) 16.8 + 2.3
N M R , T2, doping (20 m M MnC12) Ibid
20
Ibid
20
Converted to 20 o C H 2 0 , standard cell P ( c m . s -1 x 10 3)
Ref.
Te (ms)
P ( c m - s -1 × 10 3)
17.8
3.96
[37]
19.3
3.6-1-0.2
[23]
16.8
4.2 + 0.5
this work
0.98
[38]
(n = 5) H 2 0 / 2 H 2 0 exchange
20
1.2
' As pointed out by Solomon and co-workers [46] there was an error in the original Paganelli and Solomon [6] paper that arose from including a calculated zero-time point. Subsequently, Barton and Brown [30] repeated the Paganelli and Solomon experiment using original apparatus; their corrected value is used in the table. b In the original Morariu and Benga [10] paper a m e a n value of T2'a = 7.0 m s at 3 7 ° C , was found, i.e., very close to values obtained in the present work and in other studies [8,16,17,20,24,25,28,37]. The value of T~ = 6.0 ms was calculated using a different formula compared to all other publications. W h e n calculated in the same way as in other studies a similar value of T~ ( = 7.35 ms) was obtained (see p. 304 in Ref. [10]).
protons by a process known as spin diffusion [43]. Neglect of spin diffusion leads to an overestimate of the exchange time, as in the measurements of Fabry and Eisenstadt [9] and Chien and Macey [35] by T 1 methods. When the exchange of hemoglobin protons with water protons is taken into account, the values of T~ obtained by the T1 methods [25] do not differ from those obtained by the T 2 methods when relatively high concentrations of Mn 2÷ are used. The second complication occurs when water exchange is measured in red cells suspended in plasma or albumin-containing media with a low concentration of Mn 2÷. As Conlon and Outhred [8] have pointed out, red cells form a rouleaux in albumin-containing media and this would provide a l~/rger effective cell volume, thus leading to an overestimate in the exchange time. The values of T~ reported by Goldstein and co-workers [11,44,45], based on N M R measurements using low concentrations of Mn 2÷, are reflective of such errors. In fact, when a procedure for dispersing the rouleaux (by stirring) and to inhibit reaggregation by Dextran T4o was found [42], the value of permeability calculated was identical to that obtained by the N M R methods that used high Mn 2÷ concentrations (Table IV). The measurements of water diffusion by 170-NMR, [40] are subject to large 17, . . . . errors, because the intrinsic decay of water O is similar to the exchange Umes. The method of diffusion in a magnetic field gradient [41] is particularly insensitive at relatively short exchange times, and this is the case in human erythrocytes [2]. The doping N M R method using relatively high Mn 2÷ concentrations [8] avoids all these complications. It is clear now that the suggestion of some authors [9,11]
99 that high concentrations of Mn 2+ could affect the water exchange rates has proved to be unfounded. Brahm [15] could not detect any effect of 19 m M M n 2÷ on the water exchange time measured by the isotopic method. On the other hand, no significant penetration of Mn 2+ occurs after a short exposure of red cells to MnC12 at 2 0 ° C or at 3 7 ° C [10,17,48]. Even in PCMBS-treated cells the penetration of M n z+ is taking place in a time that is longer than that required for the measurements of T2] to be completed [17]. Consequently, the values of water-diffusional permeability and of Ea,d obtained by the doping N M R method of Conlon and Outhred [8] are not subject to methodological errors. In fact, an excellent agreement of values reported by several laboratories using this method and also with radiotracer measurements (using either the flow tube or the linear diffusion method) is obvious from Table IV in this paper and Table IV in Ref. 17. From the above-mentioned considerations it is clear that the N M R method based on doping with Mn 2÷ (in concentrations higher than 10 m M ) is the method of choice for studying the water exchange in h u m a n red blood cells. It has the advantage of relative technical simplicity, reproducible results and speed of data collection, allowing an almost unrestrained number of experiments. Besides, it required a very small amount of blood and is the only method that can be performed with equipment that is commercially available (in fact any pulsed N M R spectrometer can be used). These are essential features in view of measurements with potential clinical significance. Abnormal erythrocyte permeability has already been reported in pathological conditions [2,10,39,45,49,50] and further characterization of this process in human subjects (including children) in disease processes could be of great importance.
Sim#ified description of the method The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (with or without exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for studies of water permeability in various cells or in pathological conditions.
Acknowledgements The authors thank Rodica G a b o r and Marieta D a n for technical assistance and to the Ministry of Education and Teaching, the Academy of Medical Science (Romania) and the Wellcome Trust for financial support.
lOO
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