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The effect of pregnancy and pregnancy induced hypertension on active sodium transport in the erythrocyte
91
Clinica Chimica ACM, 132 (1983) 91-99 Elsevier
CCA 2583
The effect of pregnancy and pregnancy induced hypertension on active sodium transport in the erythrocyte Jill Rubython
and D. Brian Morgan
*
Department of Chemical Pathology, University of Leeds, Leeds (UK) (Received
October
25th, 1982; revision April 4th. 1983)
Summary (1) Active sodium transport was assessed in the erythrocytes of pregnant women with and without pregnancy-induced hypertension and in non-pregnant women. (2) The measurements made were the sodium content of washed erythrocytes (Nu”), the ouabain-sensitive efflux rate constant (K”“), the ouabain-sensitive sodium flux (MoS) and the number of sodium pumps (as the ouabain-binding capacity; OB). (3) The sodium content was reduced in the pregnant women due to an increase in the efflux rate constant and this increase was due to an increased number of sodium pumps (by 50%) which swamped a 20% reduction in the rate constant per pump. (4) There was a general reduction in the permeability of the erythrocyte to sodium but at the same time there was an increase in permeability in association with the increased number of sodium pumps. The greater variability of permeability and therefore of sodium flux in the pregnant women was explained by the variable increase in the number of sodium pumps. (5) There were no significant differences in these measurements between the hypertensive and the normotensive pregnant women and in particular there was no difference in the degree of pump inhibition. (6) These results suggest that the major change in active sodium transport in pregnancy is an increase in the number of sodium pumps. We suggest that this is a part of the wide ranging effect of pregnancy on protein synthesis and not a specific effect on sodium transport. There is an inhibition of the sodium pump in pregnancy but it does not cause an increase in the sodium content of the erythrocytes because there is also an increase in the number of sodium pumps. The inhibition is no greater in hypertensive than in normotensive pregnancies.
* Correspondence to: Prof. D.B. Morgan, Department University of Leeds, Leeds LS2 9JT, UK.
of Chemical
0009-8981/83/$03.00
B.V.
0 1983 Elsevier Science Publishers
Pathology,
Old Medical
School,
Introduction
Hypertension associated with pregnancy, particularly when accompanied by proteinuria, puts the mother and baby at risk [ lL3). It probably has an immunological basis [4,5] and might therefore be fundamentally different from essential hypertension. Nevertheless, sodium and water retention probably plays a role both in pregnancy-associated hypertension [6] and in essential hypertension [7.8]. A recent hypothesis for essential hypertension proposed by De Wardener and MacGregor [9] is as follows. In essential hypertension there is a genetically determined defect in the ability of the kidney to excrete sodium. The resulting sodium overload brings about an increased secretion of an inhibitor of sodium transport (probably natri-uretic hormone). The inhibition of active sodium transport in the kidney leads to diminished sodium reabsorption, sodium excretion returns to normal and sodium balance is achieved. However, the inhibition of active sodium transport in other tissues leads to a sustained increase in their sodium content and this has been observed in erythrocytes [ 10,111; lymphocytes [ 121 and leukocytes [I I]. It is not clear whether the sodium content of these tissues is increased in pregnancy-induced hypertension. Forrester and Alleyne [ 131 reported that the leucocyte sodium content was higher in pre-eclamptic than in normotensive primigravidae whereas MacGillivray and Campbell [ 141 found no difference between the two groups. Kaplay and Prema [ 151 and Weissberg et al [ 161 found a small but insignificant increase in erythrocyte sodium content in pre-eclamptic compared with normotensive pregnant women and Kuhnert et al [ 171 found a small but insignificant decrease in erythrocyte Na-K, ATPase activity in pre-eclamptic women. In the present study we have compared the sodium content, active sodium transport and the number of sodium pumps in the erythrocytes of pregnant women with and without hypertension and non-pregnant healthy women. Subjects
The 28 women with pregnancy-induced hypertension (PIH) were all primigravidae who had been admitted to hospital at 28 to 40 weeks gestation. PIH was defined as a diastolic blood pressure of more than 90 mm Hg on two separate occasions or an increase in diastolic pressure of at least 15 mm Hg from the value in early pregnancy [6]. The 16 normotensive pregnant women were primigravidae between 28 and 40 weeks gestation who were either in hospital for reasons unrelated to hypertension or were attending a routine ante-natal clinic. The non-pregnant women were 19 healthy hospital staff aged 16-41 years (mean 26 years). Methods The sodium content of washed erythrocytes (Na”; mmol . kg- ’ wet weight) was calculated from the concentration of sodium in haemolysates prepared from known weights of erythrocytes which had been centrifuged at 15 000 x g for 30 min [ 181.
93
The erythrocytes had been washed three times with iso-osmolar magnesium chloride before they were centrifuged and haemolysed. The rate constant of ouabain-sensitive sodium efflux (KO”, hh ‘) was measured with the technique of Cumberbatch and Morgan [ 181. The principle of this technique is that lop4 mol. 1-l ouabain completely inhibit the active sodium transport so that the rate of subsequent increase in erythrocyte sodium content is determined by, and is therefore a measure of, the sodium flux through the pumps (M”“, mmol kg- ’ . h- ‘). This flux is close to the total sodium flux through the cell. The ouabain-sensitive efflux rate constant (K”“, hh’) was calculated as the ratio of the flux (A4”‘) to the erythrocyte sodium content (Nu”). The number of available sodium pumps in the erythrocyte was estimated as the specific ouabain-binding capacity which was measured with a technique modified from Gardner and Conlon [ 191. The erythrocytes were incubated with [ 3Hlouabain at a final concentration of 2 x lo-’ mol. 1-l which was sufficient to saturate the ouabain-binding sites. The ouabain was in a potassium-free buffer so that the steady-state binding of ouabain was achieved in 1 h. The percentage non-specific binding, which was measured in the presence of a large excess of non-radioactive ouabain, was subtracted from the observed binding at lower concentrations to give the specific ouabain-binding capacity (dpm per 10’ cells). [ 3H]Ouabain was purchased from New England Nuclear (lot Nos. 1191-064 and 1227-076) and had a stated specific activity of 11.6 and 14.0 Ci . mmol- ‘. Plasma potassium was measured by flame photometry. Statistics The results in each group are given as the mean and standard deviation. The differences between two groups were assessed by Wilcoxon’s test. The correlation between two variables was assessed by the product-moment correlation coefficient (r ). The linear relationship between two variables was calculated as the major axis of symmetry by the method of Brace [20]. Results Table I and Fig. 1 summarise the results in the two groups of pregnant women compared with the non-pregnant controls. In both groups of pregnant women there was a significant decrease in the erythrocyte sodium content, with an increase in the ouabain-sensitive rate constant which was significant in the hypertensive group. There was a significant increase in the number of free sodium pumps as measured by the ouabain-binding capacity in both groups of pregnant women, and a decrease in the ratio of the ouabain-sensitive efflux rate constant to the ouabain-binding capacity ( K”“/OB). In neither group of pregnant women was the ouabain-sensitive sodium flux significantly different from that in the non-pregnant controls. The plasma potassium concentration was significantly lower in the pregnant than in the non-pregnant women. The results were similar in the hypertensive and normotensive women except that
I
deviation
of the measured
0.321
K”$
KP
OB
K”’
controls
0.188
0.33
4.21
31.4
0.044
0.115
0.85
SD
1.688
191.9
1.797
M””
OB
5.70
No”
x
Non-pregnant
16
19
19
19
19
19
n
(C)
0.388
1.690
4.53
3.74
1.297
298.5
X
Pregnant
in non-pregnant
controls
(C). and normotensive
(N) and hypertensive
1
0.21
0.243
47.9
0.107
0.189
0.8
SD
13
IO
10
10
10
10
n
(N)
1
0.429
1.864
4.5
3.88
1.314
325.0
x
Pregnant
0.24
0.123
93.1
0.094
0.122
0.86
SD
hypertensive
21
11
14
1I
11
1I
”
(H)
3.425
3.579
4.261
1.377
1.583
2.891
Z
C vs N
0.0 1
0.01
0.001
ns
ns
0.01
P
3.054
4.153
4.189
3.335
1.141
3.378
Z
C vs H
I
‘. h
N vs H
1.921
I 0.0
0.247
1.464
I .408
2.113
0.070
Z
0.001
0.00 1
women
(H)
ns
ns
ns
ns
0.05
ns
P
‘: K”‘. ouabain-sen-
pregnant
0.01
ns
0.0
P
sodium content. mmol’ kg- ‘; M”‘. ouabain-sensitive sodium efflux. mmol kg capacity. dpm IO’ cells; K,, plasma potassium. mmol I - ‘.
variables
normotensive
The measured variables are Na”, washed erythrocyte sitive efflux rate constant. h ‘; OB, ouabain-binding
The mean and standard
TABLE
P
95
the ouabain-sensitive sodium efflux was significantly greater in the hypertensive group. The variability in several of the measurements was greater in the pregnant women
t&J=
2.0
; : 1 : :.
r, 1.5L
*
:
I
OB
500
:
i
Kos
) 0.2vi .
I
:
i
:
: : !I . 2.0
p 100
! 0.6 i .. 0.4
!
: ""y& .I. . _;. :;i_
1.0
@z'x'Fi
7?7Y'x
Fig. 1. The values of erythrocyte sodium content (Nu”, mmol kg- ’ wet weight), ouabain-sensitive efflux ), ouabain-sensitive efflux rate constant (K"',h-l), ouabain-binding capacity (MoS, mmol.kg-‘.h-’ (OB, dpm IO’ cells) and the ratio of the ouabain sensitive efflux rate constant to the ouabain-binding capacity (KoS/OB) in the non-pregnant controls (C), and the normotensive (N) and hypertensive (H) pregnant women.
than in the non-pregnant wpmen, which indicates that the changes were much greater in some pregnant women than others. However, there was no relation between any of the variables and the period of gestation (not shown). The relationship between the various measurements were separately examined in the non-pregnant and in the pregnant women. Fig. 2 shows that in each group of women there was an inverse and close relation between the ouabain-sensitive efflux rate constant and the erythrocyte sodium content. Fig. 3 shows that there was a relationship between the ouabain-sensitive rate constant of sodium efflux and the number of sodium pumps (measured as ouabainbinding capacity) in both the non-pregnant women (y = 0.0014x + 0.05; r = 0.713; p < 0.001) and the pregnant women (y = 0.0015x - 0.06; r = 0.807; p -C 0.001). However, there was a parallel shift between the two regression lines so that the average KoS/OB was lower in the pregnant women than it was in the controls. This decrease in K”“/OB could be due to a decrease in the activity of all pumps or to a greatly decreased activity only of those pumps which are in excess of the normal number. Fig. 4 shows that there was no relationship between the KoS/OB and OB which suggests that there was a decreased activity of all pumps rather than two populations of pumps of very different activity. One cause of a decreased activity of the sodium pump is hypokalaemia [21]. The
96
6 Na
rbc
I\
-1
“\
6-1
a\
(mm01 kg-‘)
r
I
1
0.6
0.4
0.2 Kos
0
(h-l)
250 06
750
1250
(dpm/i07
cells)
The relation between the erythrocyte sodium content (Now) and the ouabain-sensitive efflux rate constant (K”“) in the non-pregnant controls (A) and normotensive (0) and hypertensive (0) pregnant women. The line is the relationship when the ouabain-sensitive efflux is the mean value observed in the Fig.
2.
non-pregnant
controls.
Fig. 3. The relation between the ouabain-sensitive efflux rate constant (KC”) and the ouabain-binding capacity (OB) in the three groups of women (symbols are as in Fig. 2). The dotted line indicates the relationship in the non-pregnant women ( _Y= 0.0014~ +0.05, r = 0.713) and the continuous line indicates the relationship in both groups of pregnant women combined (); = 0.0015~ - 0.06. r = 0.807).
plasma potassium was lower in the pregnant women than in the controls but the average decrease in plasma potassium (0.4 mmol . I-‘) was too small to explain the observed differences in K”“/OB [22]. The increase in the number of sodium pumps in the pregnant women was not associated with a significant increase in the sodium flux in either group but the flux was significantly greater in the hypertensive than in the normotensive pregnant women. Fig. 5 shows that within the pregnant women there was a relationship
2
2.0
Kos / 06
MO%
1
1.5
(mm01 kg- 1 h-1) 0
1 .o 0
250 06
Fig. 4. The relation capacity ( KoS/OB)
750 (dpml107
1250 cells)
0
250 OB
750 (dpmli
1250
07cells)
between the ratio of the ouabain-sensitive efflux rate constant to the ouabain-binding and the ouabain capacity (OB) in the three groups of women (symbols are as in Fig.
Fig. 5. The relation between the ouabain-sensitive efflux (M”‘) and the ouabain-binding capacity (OB) in the three groups of women (symbols are as in Fig. 2). The continuous line is the best fit linear relationship between the two variables in the two groups of pregnant women combined.
97
between MO” and OB (y = 0.0027x + 0.94; r = 0.542; p < 0.02). The values in the controls all lay above this regression line so that in the pregnant women compared with the controls the flux was lower at a given ouabain-binding capacity but the flux in the individual pregnant women was determined by the extent of the increase in the number of sodium pumps. Discussion This study demonstrates large and complex effects of pregnancy on erythrocyte sodium transport, which have not been previously studied in detail. Kaplay and Prema [15] and Weissberg et al [16] found a small but insignificant decrease in erythrocyte sodium content in normotensive pregnant women compared with non-pregnant controls, whereas in the present study this decrease was clear cut and significant. This decrease in erythrocyte sodium content in the pregnant women was due to an increase in the ouabain-sensitive rate constant of sodium efflux which in turn was due to an increase in the number of sodium pumps. An increase in the number of sodium pumps in pregnancy has been recently reported by two other groups [23,24]. There was a decrease in the ratio ( KoS/OB) which we suggest was common to all sodium pumps and suggests a decreased activity of each pump. An alternative explanation would be an inhibition of some pumps by a substance which was washed off during the in vitro manipulations so that the measured ouabain-binding capacity overestimates the number of free binding sites in vivo. Studies of the time curves of ouabain binding gave no indication of displacement or release of a competitor from the ouabain-binding sites. At best however, we can only measure the number of pumps with unoccupied ouabain-binding sites; the possibility that there was a large number of pumps with occupied binding sites cannot be excluded. The reduction of K’“/OB of these pumps with unoccupied binding sites is however probably real and indicates a decrease in the activity of each pump by about 20%. Hypokalaemia causes a reduction in K”“/OB. No other mechanism for a reduction in K”“/OB is commonly discussed. However, recently we have shown that digoxin which has usually been thought to act entirely by blocking the ouabain-binding site (reducing OB) also greatly reduces the KoS/OB [25]. It is therefore possible that in pregnancy there is a circulating substance like digoxin which occupies some of the ouabain-binding sites and completely inhibits the associated pumps but also reduces the K”“/OB of other pumps. There would be no reduction in the number of unoccupied ouabain-binding sites because of the large increase in the total number of sodium pumps. In healthy subjects there is a relation between the sodium efflux and the rate constant of sodium efflux [26] and, therefore, between sodium efflux and ouabainbinding capacity. We attributed this relationship to an increase in local permeability around each sodium pump. There was a similar relationship in the pregnant women (Fig. 5), but the flux was lower at any ouabain-binding capacity in the pregnant women compared with the controls. We suggest that there is a a decrease in the
9x
general membrane permeability in pregnancy on which is superimposed an increase in permeability directly related to the increase in the number of sodium pumps. When the number of pumps was greatly increased then the permeability was increased above that in the non-pregnant women which is what was reported by Weissberg et al [ 161. The increased number of active sodium pumps might be the result of the emergence of previously latent pumps in the erythrocyte membrane. The alternative is that the erythrocytes which develop during pregnancy have an increased number of sodium pumps. The sodium pump is a protein complex, and an increased synthesis of these proteins would presumably lead to an increased number of pumps in the membrane. The increased number of pumps could therefore be considered alongside the increased cell content and plasma concentrations of a range of proteins observed in pregnancy. The change in the number of pumps would by this argument be part of the change in protein synthesis in pregnancy and not a primary disorder of sodium balance. This suggestion receives some support from the observation that in pregnancy there is an increase in the sodium-dependent lithium efflux from the erythrocytes which is also presumably due to changes in a membrane protein [24]. One of the aims of this study was to examine the effect of pregnancy hypertension on sodium transport in the erythrocyte in the light of the changes which had been reported in essential hypertension. The two studies on the sodium content of leucocytes in pregnancy hypertension are in conflict [ 13,141. Both Kaplay and Prema [15] and Weissberg et al [16] found that the erythrocyte sodium content was higher in hypertensive than in non-hypertensive pregnant women but the change was not significant. We found no difference between the two groups. The present study makes it clear, however, that the sodium content in pregnancy is determined by the increase in the number of sodium pumps, and is not an indication of the activity of each pump. Indeed when the activity of each pump was assessed it was reduced in pregnancy, although, like all the other changes, it was the same in the hypertensive as in the normotensive pregnant women. Acknowledgements We thank Professor J. Scott and Mr. Karl Hancock for their permission their patients, and the women themselves for their generous cooperation.
to study
References 1 Page EW, Christianson R. Influence of blood pressure changes with and without proteinuria upon outcome of pregnancy. Am J Obstet Gynecol 1976; 126: 821-833. 2 Studd J. Pre-eclampsia. Br J Hosp Med 1977; July: 52-62. 3 Chamberlain G. Raised blood pressure in pregnancy. The fetus in hypertension. Br J Hosp Med 198 1: Aug: 127-133. 4 Scott JS, Jenkins DM, Need JA. Immunology of pre-eclampsia. Lancet 1978; 704-706. 5 Feeney JG, Scott JS. Pre-eclampsia and changed paternity. Eur J Obstet Gynecol Reprod Biol 1980; II: 35-38. 6 Chesley LC. Hypertensive disorders in pregnancy. New York: Appleton-Century-Crofts, 1978.
99
7 Editorial. New evidence linking salt and hypertension. Br Med J 1981; 282: 1993-1994. 8 Lever AF, Beretta-Piccoli C, Brown JJ, Davies DL, Fraser R, Robertson JIS. Sodium and potassium in essential hypertension. Br Med J 1981; 283: 463-468. 9 De Wardener HE, MacGregor GA. Dahl’s hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. Kidney Int 1980; 18: 1-9. 10 Fadeke Aderounmu F, Salako LA. Abnormal cation composition and transport in erythrocytes from hypertensive patients. Eur J Clin Invest 1979; 9: 369-375. 11 Poston L, Sewell RB, Wilkinson SP et al. Evidence for a circulating sodium transport inhibitor in essential hypertension. Br Med J 1981; 282: 847-849. 12 Ambrosioni E, Cost FV, Montebugnoli L, Tatagni F, Magnani B. Increased intralymphatic sodium content in essential hypertension: an index of impaired Na+ cellular metabolism. Clin Sci 1981; 61: 181-186. 13 Forrester TE, Alleyne GAO. Leucocyte electrolytes and sodium efflux rate constant in the hypertension of pre-eclampsia. Clin Sci 1980; 59: 199s-201s. 14 MacGillivray I, Campbell DM. The relevance of hypertension and oedema in pregnancy. Clin Exper Hypertens 1980; 897-914. 15 Kaplay SS, Prema K. A study of erythrocyte membrane cation transport adenosine triphosphatase in pregnancy-induced hypertension and of in vivo effects of diuretic treatment. Clin Chim Acta 1981; 110: 27-33. 16 Weissberg PL, Weaver J, West MJ, Woods KL. Intracellular electrolytes and sodium flux changes caused by normal and hypertensive pregnancy and the contraceptive pill. Clin Sci 1982; 63: 68P. 17 Kuhnert BR, Kuhnert PM, Murray BA, Sokal RJ. Na/K and Mg-ATPase activity in the placenta and in maternal and cord erythrocytes of pre-eclamptic patients. Am J Obstet Gynecol 1977; 127: 56-60. 18 Cumberbatch M, Morgan DB. A simple technique for the measurement of ouabain-sensitive sodium transport in red cells. Clin Chim Acta 1978; 89: 221-230. 19 Gardner JD, Conlon TO. The effects of sodium and potassium on ouabain binding by human erythrocytes. J Gen Physiol 1972; 60: 609-629. 20 Brace RA. Fitting straight lines to experimental data. Am J Physiol 1977; 233: R94-R99. 21 Cumberbatch M, Morgan DB. Erythrocyte sodium and potassium in patients with hypokalaemia. Clin Sci 1983; 64: 167-176. 22 Rubython EJ, Morgan DB. The effect of hypokalaemia on the ouabin sensitive sodium transport and the ouabain binding capacity in human erythrocytes. Clin Sci 1983; 64: 177-182. 23 Aronson JK, Moore MP, Redman CWG, Harper C. Sodium-lithium counter transport in erythrocytes of pregnant women (letter). N Engl J Med 1982; 307: 1645. 24 Smith JB, Ash KO, Hentschel WM, Worley RJ, Astle CD, Williams RR. Sodium lithium counter transport in erythrocytes of pregnant women (letter). N Engl J Med 1982; 307: 1645-1646. 25 Rubython JE, Burrows G, Swaminathan R, Morgan DB. The acute effect of digoxin on the sodium pump in erythrocytes. 1.R.C.S Med Sci 1982; 10: 830. 26 Cumberbatch M, Morgan DB. Relations between sodium transport and sodium concentration in human erythrocytes in health and disease. Clin Sci 1981; 60: 555-564.
Clinica Chimica ACM, 132 (1983) 91-99 Elsevier
CCA 2583
The effect of pregnancy and pregnancy induced hypertension on active sodium transport in the erythrocyte Jill Rubython
and D. Brian Morgan
*
Department of Chemical Pathology, University of Leeds, Leeds (UK) (Received
October
25th, 1982; revision April 4th. 1983)
Summary (1) Active sodium transport was assessed in the erythrocytes of pregnant women with and without pregnancy-induced hypertension and in non-pregnant women. (2) The measurements made were the sodium content of washed erythrocytes (Nu”), the ouabain-sensitive efflux rate constant (K”“), the ouabain-sensitive sodium flux (MoS) and the number of sodium pumps (as the ouabain-binding capacity; OB). (3) The sodium content was reduced in the pregnant women due to an increase in the efflux rate constant and this increase was due to an increased number of sodium pumps (by 50%) which swamped a 20% reduction in the rate constant per pump. (4) There was a general reduction in the permeability of the erythrocyte to sodium but at the same time there was an increase in permeability in association with the increased number of sodium pumps. The greater variability of permeability and therefore of sodium flux in the pregnant women was explained by the variable increase in the number of sodium pumps. (5) There were no significant differences in these measurements between the hypertensive and the normotensive pregnant women and in particular there was no difference in the degree of pump inhibition. (6) These results suggest that the major change in active sodium transport in pregnancy is an increase in the number of sodium pumps. We suggest that this is a part of the wide ranging effect of pregnancy on protein synthesis and not a specific effect on sodium transport. There is an inhibition of the sodium pump in pregnancy but it does not cause an increase in the sodium content of the erythrocytes because there is also an increase in the number of sodium pumps. The inhibition is no greater in hypertensive than in normotensive pregnancies.
* Correspondence to: Prof. D.B. Morgan, Department University of Leeds, Leeds LS2 9JT, UK.
of Chemical
0009-8981/83/$03.00
B.V.
0 1983 Elsevier Science Publishers
Pathology,
Old Medical
School,
Introduction
Hypertension associated with pregnancy, particularly when accompanied by proteinuria, puts the mother and baby at risk [ lL3). It probably has an immunological basis [4,5] and might therefore be fundamentally different from essential hypertension. Nevertheless, sodium and water retention probably plays a role both in pregnancy-associated hypertension [6] and in essential hypertension [7.8]. A recent hypothesis for essential hypertension proposed by De Wardener and MacGregor [9] is as follows. In essential hypertension there is a genetically determined defect in the ability of the kidney to excrete sodium. The resulting sodium overload brings about an increased secretion of an inhibitor of sodium transport (probably natri-uretic hormone). The inhibition of active sodium transport in the kidney leads to diminished sodium reabsorption, sodium excretion returns to normal and sodium balance is achieved. However, the inhibition of active sodium transport in other tissues leads to a sustained increase in their sodium content and this has been observed in erythrocytes [ 10,111; lymphocytes [ 121 and leukocytes [I I]. It is not clear whether the sodium content of these tissues is increased in pregnancy-induced hypertension. Forrester and Alleyne [ 131 reported that the leucocyte sodium content was higher in pre-eclamptic than in normotensive primigravidae whereas MacGillivray and Campbell [ 141 found no difference between the two groups. Kaplay and Prema [ 151 and Weissberg et al [ 161 found a small but insignificant increase in erythrocyte sodium content in pre-eclamptic compared with normotensive pregnant women and Kuhnert et al [ 171 found a small but insignificant decrease in erythrocyte Na-K, ATPase activity in pre-eclamptic women. In the present study we have compared the sodium content, active sodium transport and the number of sodium pumps in the erythrocytes of pregnant women with and without hypertension and non-pregnant healthy women. Subjects
The 28 women with pregnancy-induced hypertension (PIH) were all primigravidae who had been admitted to hospital at 28 to 40 weeks gestation. PIH was defined as a diastolic blood pressure of more than 90 mm Hg on two separate occasions or an increase in diastolic pressure of at least 15 mm Hg from the value in early pregnancy [6]. The 16 normotensive pregnant women were primigravidae between 28 and 40 weeks gestation who were either in hospital for reasons unrelated to hypertension or were attending a routine ante-natal clinic. The non-pregnant women were 19 healthy hospital staff aged 16-41 years (mean 26 years). Methods The sodium content of washed erythrocytes (Na”; mmol . kg- ’ wet weight) was calculated from the concentration of sodium in haemolysates prepared from known weights of erythrocytes which had been centrifuged at 15 000 x g for 30 min [ 181.
93
The erythrocytes had been washed three times with iso-osmolar magnesium chloride before they were centrifuged and haemolysed. The rate constant of ouabain-sensitive sodium efflux (KO”, hh ‘) was measured with the technique of Cumberbatch and Morgan [ 181. The principle of this technique is that lop4 mol. 1-l ouabain completely inhibit the active sodium transport so that the rate of subsequent increase in erythrocyte sodium content is determined by, and is therefore a measure of, the sodium flux through the pumps (M”“, mmol kg- ’ . h- ‘). This flux is close to the total sodium flux through the cell. The ouabain-sensitive efflux rate constant (K”“, hh’) was calculated as the ratio of the flux (A4”‘) to the erythrocyte sodium content (Nu”). The number of available sodium pumps in the erythrocyte was estimated as the specific ouabain-binding capacity which was measured with a technique modified from Gardner and Conlon [ 191. The erythrocytes were incubated with [ 3Hlouabain at a final concentration of 2 x lo-’ mol. 1-l which was sufficient to saturate the ouabain-binding sites. The ouabain was in a potassium-free buffer so that the steady-state binding of ouabain was achieved in 1 h. The percentage non-specific binding, which was measured in the presence of a large excess of non-radioactive ouabain, was subtracted from the observed binding at lower concentrations to give the specific ouabain-binding capacity (dpm per 10’ cells). [ 3H]Ouabain was purchased from New England Nuclear (lot Nos. 1191-064 and 1227-076) and had a stated specific activity of 11.6 and 14.0 Ci . mmol- ‘. Plasma potassium was measured by flame photometry. Statistics The results in each group are given as the mean and standard deviation. The differences between two groups were assessed by Wilcoxon’s test. The correlation between two variables was assessed by the product-moment correlation coefficient (r ). The linear relationship between two variables was calculated as the major axis of symmetry by the method of Brace [20]. Results Table I and Fig. 1 summarise the results in the two groups of pregnant women compared with the non-pregnant controls. In both groups of pregnant women there was a significant decrease in the erythrocyte sodium content, with an increase in the ouabain-sensitive rate constant which was significant in the hypertensive group. There was a significant increase in the number of free sodium pumps as measured by the ouabain-binding capacity in both groups of pregnant women, and a decrease in the ratio of the ouabain-sensitive efflux rate constant to the ouabain-binding capacity ( K”“/OB). In neither group of pregnant women was the ouabain-sensitive sodium flux significantly different from that in the non-pregnant controls. The plasma potassium concentration was significantly lower in the pregnant than in the non-pregnant women. The results were similar in the hypertensive and normotensive women except that
I
deviation
of the measured
0.321
K”$
KP
OB
K”’
controls
0.188
0.33
4.21
31.4
0.044
0.115
0.85
SD
1.688
191.9
1.797
M””
OB
5.70
No”
x
Non-pregnant
16
19
19
19
19
19
n
(C)
0.388
1.690
4.53
3.74
1.297
298.5
X
Pregnant
in non-pregnant
controls
(C). and normotensive
(N) and hypertensive
1
0.21
0.243
47.9
0.107
0.189
0.8
SD
13
IO
10
10
10
10
n
(N)
1
0.429
1.864
4.5
3.88
1.314
325.0
x
Pregnant
0.24
0.123
93.1
0.094
0.122
0.86
SD
hypertensive
21
11
14
1I
11
1I
”
(H)
3.425
3.579
4.261
1.377
1.583
2.891
Z
C vs N
0.0 1
0.01
0.001
ns
ns
0.01
P
3.054
4.153
4.189
3.335
1.141
3.378
Z
C vs H
I
‘. h
N vs H
1.921
I 0.0
0.247
1.464
I .408
2.113
0.070
Z
0.001
0.00 1
women
(H)
ns
ns
ns
ns
0.05
ns
P
‘: K”‘. ouabain-sen-
pregnant
0.01
ns
0.0
P
sodium content. mmol’ kg- ‘; M”‘. ouabain-sensitive sodium efflux. mmol kg capacity. dpm IO’ cells; K,, plasma potassium. mmol I - ‘.
variables
normotensive
The measured variables are Na”, washed erythrocyte sitive efflux rate constant. h ‘; OB, ouabain-binding
The mean and standard
TABLE
P
95
the ouabain-sensitive sodium efflux was significantly greater in the hypertensive group. The variability in several of the measurements was greater in the pregnant women
t&J=
2.0
; : 1 : :.
r, 1.5L
*
:
I
OB
500
:
i
Kos
) 0.2vi .
I
:
i
:
: : !I . 2.0
p 100
! 0.6 i .. 0.4
!
: ""y& .I. . _;. :;i_
1.0
@z'x'Fi
7?7Y'x
Fig. 1. The values of erythrocyte sodium content (Nu”, mmol kg- ’ wet weight), ouabain-sensitive efflux ), ouabain-sensitive efflux rate constant (K"',h-l), ouabain-binding capacity (MoS, mmol.kg-‘.h-’ (OB, dpm IO’ cells) and the ratio of the ouabain sensitive efflux rate constant to the ouabain-binding capacity (KoS/OB) in the non-pregnant controls (C), and the normotensive (N) and hypertensive (H) pregnant women.
than in the non-pregnant wpmen, which indicates that the changes were much greater in some pregnant women than others. However, there was no relation between any of the variables and the period of gestation (not shown). The relationship between the various measurements were separately examined in the non-pregnant and in the pregnant women. Fig. 2 shows that in each group of women there was an inverse and close relation between the ouabain-sensitive efflux rate constant and the erythrocyte sodium content. Fig. 3 shows that there was a relationship between the ouabain-sensitive rate constant of sodium efflux and the number of sodium pumps (measured as ouabainbinding capacity) in both the non-pregnant women (y = 0.0014x + 0.05; r = 0.713; p < 0.001) and the pregnant women (y = 0.0015x - 0.06; r = 0.807; p -C 0.001). However, there was a parallel shift between the two regression lines so that the average KoS/OB was lower in the pregnant women than it was in the controls. This decrease in K”“/OB could be due to a decrease in the activity of all pumps or to a greatly decreased activity only of those pumps which are in excess of the normal number. Fig. 4 shows that there was no relationship between the KoS/OB and OB which suggests that there was a decreased activity of all pumps rather than two populations of pumps of very different activity. One cause of a decreased activity of the sodium pump is hypokalaemia [21]. The
96
6 Na
rbc
I\
-1
“\
6-1
a\
(mm01 kg-‘)
r
I
1
0.6
0.4
0.2 Kos
0
(h-l)
250 06
750
1250
(dpm/i07
cells)
The relation between the erythrocyte sodium content (Now) and the ouabain-sensitive efflux rate constant (K”“) in the non-pregnant controls (A) and normotensive (0) and hypertensive (0) pregnant women. The line is the relationship when the ouabain-sensitive efflux is the mean value observed in the Fig.
2.
non-pregnant
controls.
Fig. 3. The relation between the ouabain-sensitive efflux rate constant (KC”) and the ouabain-binding capacity (OB) in the three groups of women (symbols are as in Fig. 2). The dotted line indicates the relationship in the non-pregnant women ( _Y= 0.0014~ +0.05, r = 0.713) and the continuous line indicates the relationship in both groups of pregnant women combined (); = 0.0015~ - 0.06. r = 0.807).
plasma potassium was lower in the pregnant women than in the controls but the average decrease in plasma potassium (0.4 mmol . I-‘) was too small to explain the observed differences in K”“/OB [22]. The increase in the number of sodium pumps in the pregnant women was not associated with a significant increase in the sodium flux in either group but the flux was significantly greater in the hypertensive than in the normotensive pregnant women. Fig. 5 shows that within the pregnant women there was a relationship
2
2.0
Kos / 06
MO%
1
1.5
(mm01 kg- 1 h-1) 0
1 .o 0
250 06
Fig. 4. The relation capacity ( KoS/OB)
750 (dpml107
1250 cells)
0
250 OB
750 (dpmli
1250
07cells)
between the ratio of the ouabain-sensitive efflux rate constant to the ouabain-binding and the ouabain capacity (OB) in the three groups of women (symbols are as in Fig.
Fig. 5. The relation between the ouabain-sensitive efflux (M”‘) and the ouabain-binding capacity (OB) in the three groups of women (symbols are as in Fig. 2). The continuous line is the best fit linear relationship between the two variables in the two groups of pregnant women combined.
97
between MO” and OB (y = 0.0027x + 0.94; r = 0.542; p < 0.02). The values in the controls all lay above this regression line so that in the pregnant women compared with the controls the flux was lower at a given ouabain-binding capacity but the flux in the individual pregnant women was determined by the extent of the increase in the number of sodium pumps. Discussion This study demonstrates large and complex effects of pregnancy on erythrocyte sodium transport, which have not been previously studied in detail. Kaplay and Prema [15] and Weissberg et al [16] found a small but insignificant decrease in erythrocyte sodium content in normotensive pregnant women compared with non-pregnant controls, whereas in the present study this decrease was clear cut and significant. This decrease in erythrocyte sodium content in the pregnant women was due to an increase in the ouabain-sensitive rate constant of sodium efflux which in turn was due to an increase in the number of sodium pumps. An increase in the number of sodium pumps in pregnancy has been recently reported by two other groups [23,24]. There was a decrease in the ratio ( KoS/OB) which we suggest was common to all sodium pumps and suggests a decreased activity of each pump. An alternative explanation would be an inhibition of some pumps by a substance which was washed off during the in vitro manipulations so that the measured ouabain-binding capacity overestimates the number of free binding sites in vivo. Studies of the time curves of ouabain binding gave no indication of displacement or release of a competitor from the ouabain-binding sites. At best however, we can only measure the number of pumps with unoccupied ouabain-binding sites; the possibility that there was a large number of pumps with occupied binding sites cannot be excluded. The reduction of K’“/OB of these pumps with unoccupied binding sites is however probably real and indicates a decrease in the activity of each pump by about 20%. Hypokalaemia causes a reduction in K”“/OB. No other mechanism for a reduction in K”“/OB is commonly discussed. However, recently we have shown that digoxin which has usually been thought to act entirely by blocking the ouabain-binding site (reducing OB) also greatly reduces the KoS/OB [25]. It is therefore possible that in pregnancy there is a circulating substance like digoxin which occupies some of the ouabain-binding sites and completely inhibits the associated pumps but also reduces the K”“/OB of other pumps. There would be no reduction in the number of unoccupied ouabain-binding sites because of the large increase in the total number of sodium pumps. In healthy subjects there is a relation between the sodium efflux and the rate constant of sodium efflux [26] and, therefore, between sodium efflux and ouabainbinding capacity. We attributed this relationship to an increase in local permeability around each sodium pump. There was a similar relationship in the pregnant women (Fig. 5), but the flux was lower at any ouabain-binding capacity in the pregnant women compared with the controls. We suggest that there is a a decrease in the
9x
general membrane permeability in pregnancy on which is superimposed an increase in permeability directly related to the increase in the number of sodium pumps. When the number of pumps was greatly increased then the permeability was increased above that in the non-pregnant women which is what was reported by Weissberg et al [ 161. The increased number of active sodium pumps might be the result of the emergence of previously latent pumps in the erythrocyte membrane. The alternative is that the erythrocytes which develop during pregnancy have an increased number of sodium pumps. The sodium pump is a protein complex, and an increased synthesis of these proteins would presumably lead to an increased number of pumps in the membrane. The increased number of pumps could therefore be considered alongside the increased cell content and plasma concentrations of a range of proteins observed in pregnancy. The change in the number of pumps would by this argument be part of the change in protein synthesis in pregnancy and not a primary disorder of sodium balance. This suggestion receives some support from the observation that in pregnancy there is an increase in the sodium-dependent lithium efflux from the erythrocytes which is also presumably due to changes in a membrane protein [24]. One of the aims of this study was to examine the effect of pregnancy hypertension on sodium transport in the erythrocyte in the light of the changes which had been reported in essential hypertension. The two studies on the sodium content of leucocytes in pregnancy hypertension are in conflict [ 13,141. Both Kaplay and Prema [15] and Weissberg et al [16] found that the erythrocyte sodium content was higher in hypertensive than in non-hypertensive pregnant women but the change was not significant. We found no difference between the two groups. The present study makes it clear, however, that the sodium content in pregnancy is determined by the increase in the number of sodium pumps, and is not an indication of the activity of each pump. Indeed when the activity of each pump was assessed it was reduced in pregnancy, although, like all the other changes, it was the same in the hypertensive as in the normotensive pregnant women. Acknowledgements We thank Professor J. Scott and Mr. Karl Hancock for their permission their patients, and the women themselves for their generous cooperation.
to study
References 1 Page EW, Christianson R. Influence of blood pressure changes with and without proteinuria upon outcome of pregnancy. Am J Obstet Gynecol 1976; 126: 821-833. 2 Studd J. Pre-eclampsia. Br J Hosp Med 1977; July: 52-62. 3 Chamberlain G. Raised blood pressure in pregnancy. The fetus in hypertension. Br J Hosp Med 198 1: Aug: 127-133. 4 Scott JS, Jenkins DM, Need JA. Immunology of pre-eclampsia. Lancet 1978; 704-706. 5 Feeney JG, Scott JS. Pre-eclampsia and changed paternity. Eur J Obstet Gynecol Reprod Biol 1980; II: 35-38. 6 Chesley LC. Hypertensive disorders in pregnancy. New York: Appleton-Century-Crofts, 1978.
99
7 Editorial. New evidence linking salt and hypertension. Br Med J 1981; 282: 1993-1994. 8 Lever AF, Beretta-Piccoli C, Brown JJ, Davies DL, Fraser R, Robertson JIS. Sodium and potassium in essential hypertension. Br Med J 1981; 283: 463-468. 9 De Wardener HE, MacGregor GA. Dahl’s hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. Kidney Int 1980; 18: 1-9. 10 Fadeke Aderounmu F, Salako LA. Abnormal cation composition and transport in erythrocytes from hypertensive patients. Eur J Clin Invest 1979; 9: 369-375. 11 Poston L, Sewell RB, Wilkinson SP et al. Evidence for a circulating sodium transport inhibitor in essential hypertension. Br Med J 1981; 282: 847-849. 12 Ambrosioni E, Cost FV, Montebugnoli L, Tatagni F, Magnani B. Increased intralymphatic sodium content in essential hypertension: an index of impaired Na+ cellular metabolism. Clin Sci 1981; 61: 181-186. 13 Forrester TE, Alleyne GAO. Leucocyte electrolytes and sodium efflux rate constant in the hypertension of pre-eclampsia. Clin Sci 1980; 59: 199s-201s. 14 MacGillivray I, Campbell DM. The relevance of hypertension and oedema in pregnancy. Clin Exper Hypertens 1980; 897-914. 15 Kaplay SS, Prema K. A study of erythrocyte membrane cation transport adenosine triphosphatase in pregnancy-induced hypertension and of in vivo effects of diuretic treatment. Clin Chim Acta 1981; 110: 27-33. 16 Weissberg PL, Weaver J, West MJ, Woods KL. Intracellular electrolytes and sodium flux changes caused by normal and hypertensive pregnancy and the contraceptive pill. Clin Sci 1982; 63: 68P. 17 Kuhnert BR, Kuhnert PM, Murray BA, Sokal RJ. Na/K and Mg-ATPase activity in the placenta and in maternal and cord erythrocytes of pre-eclamptic patients. Am J Obstet Gynecol 1977; 127: 56-60. 18 Cumberbatch M, Morgan DB. A simple technique for the measurement of ouabain-sensitive sodium transport in red cells. Clin Chim Acta 1978; 89: 221-230. 19 Gardner JD, Conlon TO. The effects of sodium and potassium on ouabain binding by human erythrocytes. J Gen Physiol 1972; 60: 609-629. 20 Brace RA. Fitting straight lines to experimental data. Am J Physiol 1977; 233: R94-R99. 21 Cumberbatch M, Morgan DB. Erythrocyte sodium and potassium in patients with hypokalaemia. Clin Sci 1983; 64: 167-176. 22 Rubython EJ, Morgan DB. The effect of hypokalaemia on the ouabin sensitive sodium transport and the ouabain binding capacity in human erythrocytes. Clin Sci 1983; 64: 177-182. 23 Aronson JK, Moore MP, Redman CWG, Harper C. Sodium-lithium counter transport in erythrocytes of pregnant women (letter). N Engl J Med 1982; 307: 1645. 24 Smith JB, Ash KO, Hentschel WM, Worley RJ, Astle CD, Williams RR. Sodium lithium counter transport in erythrocytes of pregnant women (letter). N Engl J Med 1982; 307: 1645-1646. 25 Rubython JE, Burrows G, Swaminathan R, Morgan DB. The acute effect of digoxin on the sodium pump in erythrocytes. 1.R.C.S Med Sci 1982; 10: 830. 26 Cumberbatch M, Morgan DB. Relations between sodium transport and sodium concentration in human erythrocytes in health and disease. Clin Sci 1981; 60: 555-564.