Research in
Res Exp Med (1986) 186 : 71-77
Experimental Medicine
© Springer-Verlag 1986
Erythrocyte Na+-Li ÷ Countertransport
and Blood Viscosity in Arterial Hypertension
F. Fallo 1, M. P r o c i d a n o 1, V. de A n g e l i s 2, F. M a n o n i 3, F. M a n t e r o 1,
and A . Girolami I
1 Institute of Medical Semeiotics, University of Padova, Via Ospedale 105, 1-35100 Padova,
Italy
2 Blood Bank, Civic Hospital, Pordenone, Italy
3 Central Laboratory, Military Hospital, Padova, Italy
Summary. The aim of this study was to evaluate together the main hemorheologic parameters and one of the transmembrane ion transport systems
in erythrocytes of subjects with normal and elevated blood pressure. Three
sex-, age-, and weight-matched groups consisting of 15 normotensive subjects (NT) with no parental hypertension, 15 patients with essential hypertension (EH) at stage 1-II W H O , and eight patients with secondary hypertension (Sec.H), respectively, were studied. Red blood cell Na+-Li + countertransport (CTT), blood viscosity (~B) at shear rates of 230. s-1, 115. s-1,
and 46. s-1 and plasma viscosity (TIp) at shear rate of 46- s-1 were measured.
Plasma proteins and fibrinogen were also evaluated. CTT was higher in E H
than in N T (P < 0.01), while no significant difference was found between N T
and Sec.H patients. ~B at 115.s -1 and 46. s-1 was higher in E H , but not in
Sec. H, than in N T patients ( P < 0.05). No difference in TIp, plasma proteins
and fibrinogen levels was observed between E H and NT. Elevated VlBand/or
CTT may indicate a structural alteration in the erythrocyte membrane of
some essential hypertensive patients. This is consistent with the hypothesis
that a widespread membrane disorder is involved in the pathogenesis of primary hypertension.
Key words: Na+-Li + countertransport - Blood viscosity - Hypertension
Introduction
Several ion transport abnormalities have been described in erythrocyte membrane of patients with essential hypertension [1-3]. It has been hypothesized
Offprint requests to: Francesco Fallo, MD (address see above).
72
F. Fallo et al.
that similar a l t e r a t i o n s affecting o t h e r types of cells (vascular, renal) m a y play a
p r o m i n e n t role in the p a t h o g e n e s i s of the h y p e r t e n s i v e disease [4--6]. Modifications in physical p r o p e r t i e s of red cells have b e e n o b s e r v e d in association with
such ion t r a n s p o r t disorders [7-9]. O t h e r studies r e p o r t h e m o r h e o l o g i c changes
in h y p e r t e n s i o n which can be ascribed to alterations of the cellular c o m p o n e n t
of the b l o o d [10, 11]. T h e aim of o u r study was to e v a l u a t e the m a i n h e m o r h e o logic factors a n d o n e of the e r y t h r o c y t e t r a n s m e m b r a n e i o n t r a n s p o r t systems,
n a m e l y N a + - L i ÷ c o u n t e r t r a n s p o r t , in subjects with n o r m a l a n d e l e v a t e d b l o o d
pressure.
Patients and Methods
Three groups of sex-, age- (range 18--43 years), and weight-matched subjects were studied.
The first group of 15 (ten males and five females) healthy normotensive subjects (NT) with
both parents normotensive; the second group, 15 patients (nine males and six females) with
essential hypertension (EH) at stage I-II according to WHO classification; the third group,
eight patients (five males and three females) suffering from different forms of secondary hypertension (Sec. H), i.e., three primary aldosteronism, two Cushing's disease, two renoparenchymal disease, two renovascular hypertension. All three patients with primary aldosteronism
had a serum potassium level at a limit lower than normal. None of the patients had clinical
symptoms of regional vascular damages or positive history of stroke or myocardial infarction;
patients with other causes of hemorheologic abnormalities (i.e., diabetes, hemolytic disease,
etc.) were excluded from the study. All subjects were white and all were off any medication
for at least 2 weeks before the study.
Whole-blood and plasma viscosity, hematocrit, red blood cell (RBC) Na+-Li ÷ countertransport (CCT), plasma protein and fibrinogen concentrations were measured in all subjects.
Thirty milliliters of venous blood was drawn into a plastic syringe at 9a.m. after the subjects
had fasted from midnight. Ten milliliters of blood was anticoagulated with dry edetic acid
(EDTA 0.004tool/I) for measurement of whole-blood viscosity (riB) and plasma viscosity 01P),
in a Wells-Brookfield LTV Cone-plate Viscometer (Brookfield Lab., Stoughton, MA, USA)
at 37°C. The determination was carried out in duplicates, and the average value was kept as
the final reading. The shear rates were 230. s-1, 115. s-1, 46-s -1, for ~B, and 46. s-1 for rip, respectively, being qp shear-rate independent [12]. riB was corrected at a standard hematocrit of
45% using the mathematical formula by Nicolaides et al. [13]. Values are expressed in centipoise (cps). The intra-individual coefficient of variation was estimated to be 8.9, 9.4, and
9.1% for riB at 230' s-1, 115- s-1, 46" s-1, respectively, and 6.7% for qp. Hematocrit was measured by the Haswkley microhematocrit technique in duplicate sample.
Ten milliliters of blood was centrifuged, plasma and bully coat were removed, and 2-3 ml
of RBC were used for CTT measurements according to the method of Adragna et al. [14]. In
this procedure RBC were first washed in a washing solution containing: MgC1 75mM, TrisMOPS 10raM, pH 7.4 at 4°C, sucrose 85mM. The cells were incubated in the presence of a
lithium containing solution (LiC1 150mM, Tris-MOPS 10mM, pH 7.4 at 37°C, glucose
10mM) for 3h at 37°C in order to load the cells with lithium at the expense of intracellular
sodium: Then they were re-washed and resuspended to - 5 0 % hematocrit in the washing solution. An aliquot of the 50% suspension was added to two media at a final hematocrit of - 5%.
One medium was sodium-free (MgC1 75mM, Tris-MOPS 10mM, pH 7.4 at 37°C, sucrose
85mM, ouabain 10mM), the other contained sodium (NaC1 150mM, Tris-MOPS 10mM, pH
7.4 at 37°C, glucose 10 mM, ouabain 10 mM). Duplicates of the suspension were used as initial
incubation time; the remaining was incubated for lh. The suspensions were centrifuged, and
lithium was measured by atomic absorption spectrophotometry. The measurement of CTT
was derived from the efflux in the presence of sodium minus the efflux into the sodium-free
medium. The rates of efflux were expressed as mmol lithium/1 RBC x l h of incubation
Na+-Li + Countertransport and Blood Viscosity in Hypertension
73
(mmol/1 RBC. h-l). All the solutions used in effiux measurements were adjusted to 300 ±
5mOsM. The coefficient of variation for this assay is 9.7% as calculated from the formula
~ ( d
= difference for each pair, N = number of pairs) and expressed as a percentage
of the mean for 28 pairs of samples assayed in a blind fashion.
Plasma protein concentrations were determined at 37°C using a Bausch and Lombe refractometer [15]. Plasma fibrinogen was estimated in aliquots of heparinized plasma using the
method of Ratnoff and Menzie [16]. All the values were analyzed as mean + standard error
(M _+SEM). Intergroup comparison was analyzed by Student's unpaired t-test and correlation
by linear regression analysis. A P value greater than 0.05 was not significant.
Results
T a b l e 1 shows clinical d a t a a n d p l a s m a m e a s u r e m e n t s in the v a r i o u s groups of
subjects. M e a n values for qp a n d its m a i n c o m p o n e n t s , i.e., f i b r i n o g e n , total alb u m i n , a n d g l o b u l i n c o n c e n t r a t i o n s , were n o t different in N T in respect to E H
patients. P l a s m a f i b r i n o g e n was significantly higher in p a t i e n t s with s e c o n d a r y
h y p e r t e n s i o n t h a n in N T a n d in E H patients.
A s s h o w n in T a b l e 2, m e a n values for C T T a n d rib at shear rates of 115-S -1
a n d 46. s-1 were significantly higher in E H t h a n in N T a n d Sec. H patients. I n d i vidual data for C T T a n d for qB, are s h o w n in Figs. 1 a n d 2, respectively. N o cor-
Table 1. Clinical data and determinants of plasma viscosity (TIp) in normotensive subjects
(NT) and in patients with essential hypertension (EH) and with secondary hypertension
(Sec.H)
Subjects
Age
(yr)
Weight Systolic/diastolic Albumin
(kg)
blood pressure
(g/dl)
(mm Hg)
Globulin Fibrinogen
(g/dl)
(g/dl)
NT
EH
Sec.H
25+1
26+2
31_+8
79+7
70+4
71+3
2.4±0.3
2.2+-0.3
2.5+0.4
132+3/ 8 7 + 2 6.7+0.5
168+6/102+4" 6 . 4 + 0 . 4
176+8/104+3" 6 . 5 + 0 . 4
Plasma
viscosity
(cps)
0.344+0.1 1.4+0.04
0.453+0.1 1.5+0.03
0.569+0.3"'1.5+0.09
* P<0.01, EH and Sec.H vs. NT.
** P<0.01, Sec.H vs. NT.
Table 2. Erythrocyte Na+-Li + countertransport (CTT) and blood viscosity (rib) at three different shear rates (. s-1) in normotensive subjects (NT) and in patients with essential hypertension
(EH) and with secondary hypertension (Sec.H)
Subjects
NT
EH
Sec.H
CTT
(mmol/1 RBC. h-1)
Blood viscosity (cps)
230. S-1
115" S-1
46" S-1
0.258 + 0.01
0.399 + 0.03**
0.249 + 0.04
4.4 + 0.1
4.7 + 0.1
4.3 + 0.1
5.7 + 0.1
6.5 + 0.2*
5.7 + 0.2
* P<0.05 EH vs. NT and Sec.H.
** P<0.01.
4.9 + 0.1
5.3 +__0.1"
4.8 + 0.5
74
F. Fallo et al.
0.7
0.6
.1=
d
0.5
m
n,
..I
-o
0.4
I'm
G3
"X-
E
E
I.O
ee
+]_
--I 0.2
eee
÷Z
0.1
* p , , o.o, E H vs
NT,~
NT
EH
S,cH
Sec.H
Fig. 1. Individualdata for erythrocyteNa+-Li+ countertransport (CTI') in normotensivesubjects (NT) and in patients with essentialhypertension(EH) and with secondaryhypertension
(Sec.H)
relation between CTT and ~B, at whatever shear rate value, was found in the
three separate groups of subjects. Systo-diastolic blood pressure levels did not
correlate with CTT or 11Bin the same group.
Discussion
Our results, confirming previous reports [14, 17-19], show elevated erythrocyte
CTT in patients with essential hypertension as compared with normotensive
subjects or patients affected by secondary hypertension. However, CTT alteration appears not to be shared by all subjects. Analysis of individual data show
in fact that in nine of 15 patients (60%) with essential hypertension, CTT values
are higher than maximum CTT level measured in the group of normotensive
control. It has been hypothesized that this erythrocyte membrane abnormality
represents a marker of a widespread disorder, genetically transmitted and in
various ways influenced by humoral and environmental factors, which has a direct role in the pathogenesis of primary hypertension [5, 6]. The increase in lIB
in our group of patients with essential hypertension suggests also a specific
structural alteration of the red cell. Firstly, normal levels of TIp and its main
plasma determinants indicate that the erythrocyte component may primarily ac-
Na+-Li + Countertransport and Blood Viscosity in Hypertension
230
9
s e c -1
118
75
sec -I
46
sec -1
8
2
v
>.
Fm
(n
o
o
>
5
.i-
|
•
(/)
•
I.
tl-
#
!
v v -
ee
qPq)
!
0
.J
rn
•
IfpcO.O5
EH ~
•
NTand
S.~H
3.
I
NT
I
EH
I
S~H
NT
I
i
EH
S,c.H
NT
EH S,c.H
Fig.2. Individual data for blood viscosity (rIB) at three different shear rates in normotensive
subjects (NT) and in patients with essential hypertension (EH) and with secondary hypertension (Sec. H)
count for this rheologic abnormality. Furthermore, qB is increased at intermediate and low shear rates, and this has been maintained to be a real index of
spontaneous red cell aggregation [20]. This finding confirms also the increased
relative blood viscosity previously observed in a large population of patients
with essential hypertension [21]. Finally, the influence of hematocrit can be
reasonably excluded by its correction to a standard level for calculation of riB.
In terms of individual data, riB values of essential hypertensive patients also
overlap those of normotensive controls, being in five out of 15 patients (33.3%)
higher than the maximum value observed in the group of normotensives. However, similar values of riB and CTT found in the majority of patients with secondary hypertension and in normotensive could confirm this hypothesis that the
alteration of these parameters is not attributable to the presence of high blood
pressure "per se".
Since biophysical properties of erythrocyte membrane skeleton are known
to be dependent on normally active ion transport systems [22, 23], we assessed
a possible relationship between CTT and riB. In spite of the fact that no statistical correlation was found in the three groups considered separately, one could
speculate that the abnormality of both parameters in some of our patients with
essential hypertension reflects a common erythrocyte membrane defect. It is
also conceivable that the methods for measuring C T T and riB have different
technical limitations so that a comparative evaluation is not feasible.
76
F. Fallo et al.
I n contrast to o t h e r reports [24], n o statistical c o r r e l a t i o n of C T T a n d ~B
with the systo-diastolic b l o o d p r e s s u r e was f o u n d in the t h r e e groups of subjects. M o r e o v e r , p r e v i o u s o b s e r v a t i o n s showed that either C T T [25] or rib [26]
are r a t h e r directly r e l a t e d to the total p e r i p h e r a l resistance. F u r t h e r studies are
n e e d e d to b e t t e r elucidate the significance of these findings.
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Received May 31, 1985 / Accepted October 23, 1985