Clinical Science and Molecular Medicine (1973) 45, 173-181.
PLASMA VOLUME DECREASE A N D ELEVATED EVANS
BLUE DISAPPEARANCE RATE I N ESSENTIAL
HYPERTENSION
MILOS U L R Y C H
Institute for Cardiovascular Research, Prague, Czechoslovakia, and
University of Pittsburgh School of Medicine, Department of Medicine,
Pittsburgh, Pa., U.S.A.
(Received 14 November 1972)
SUMMARY
1. The disappearance rate of intravenously injected Evans Blue, plasma volume,
cardiac output, and blood pressure were measured in seven normotensive and eighteen
hypertensive subjects.
2. Plasma volume was found to be negatively correlated with the mean arterial
pressure, Evans Blue disappearance rate and packed cell volume.
3. Faster disappearance rate of Evans Blue in hypertensive subjects may be due to
an abnormality of mixing of the label or of the capillaries.
Key words : plasma volume, arterial hypertension, cardiac output, arterial pressure,
peripheral vascular resistance, capillaries, indicator-dilution technique.
Decrease in plasma volume in arterial hypertension has been described by several groups of
investigators (Tibblin, Bergentz, Bjure & Wilhelmsen, 1966; Frohlich, Ulrych, Tarazi, Dustan
& Page, 1967b; Tarazi, Frohlich & Dustan, 1968; Tarazi, Dustan & Frohlich, 1969; Tarazi,
Dustan, Frohlich, Gifford & Hoffman, 1970; Julius, Pascual, Reilly & London, 1971). There
seems to be little question that this exists in uncomplicated hypertension of mild and moderate
degree. A direct relationship exists between the amount of decrease of plasma volume and
increase in blood pressure. Unfortunately, there is almost no explanation available for the
phenomenon. Because of the possibility that the rate of protein exchange at the capillary level
could influence the plasma volume, the rate of disappearance of intravenously injected Evans
Blue was studied. This rate was found to be directly proportional to the blood pressure and
indirectly proportional to the plasma volume.
METHODS
Twenty-five subjects, seven with normal blood pressure (three males, four females) and eighteen
with essential hypertension (nine males, nine females), were studied. Their ages ranged from
Correspondence: Dr M. Ulrych, 231 Marion Drive, McMurray, Pa. 15317, U S A .
173
174
Milos Ulrych
21 to 59 years and their weights from 55 to 94 kg. All subjects underwent careful clinical
examination, urinalysis, plasma electrolytes, 24 h creatinine clearance and concentrating
ability measurements, rapid sequence excretion urography, chest radiography and ECG. None
of the subjects had grade 3 or 4 (Keith Wagener) eyeground changes, nor was there evidence
of heart failure in the present or past, myocardial infarction, stroke, or nephrosclerosis;
creatinine clearance exceeded 70 ml/min in all cases. Subjects were considered normal if this
examination failed to reveal any abnormalities and their blood pressures on repeated measurements were below 140/85; these subjects had been referred with diagnoses of neurocirculatory
asthenia or nonsignificant heart murmurs or because of suspicion of kidney disease which
could not later be substantiated. Hypertensives were those with blood pressures repeatedly
greater than 145/90 and because no cause of hypertension could be found they were considered
to have essential hypertension. All treatment was stopped at least 3 weeks before the examination was performed.
The nature and purpose of the procedure was explained to all subjects and only those who
were willing to participate on a voluntary basis were investigated. None of the subjects were
hospitalized and all performed their regular duties except on the day of examination. The
subjects fasted overnight and measurements were made in a quiet laboratory in the morning.
After at least 4 h of resting in a supine position the left brachial or radial artery and the right
antecubital vein were catheterized using the technique of Seldinger (1953). After the introduction of catheters a blank sample of blood for plasma volume determination was drawn and
10-20 mg of Evans Blue was injected into the vein. Blood samples were taken from the artery
exactly 5, 10,20 and 30 min after the injection. Cardiac output and blood pressure were then
measured in triplicate. Cardiac output was measured by indicator-dilution technique using
Indocyanine Green ; blood pressure was measured directly.
Plasma volume was measured by dilution of Evans Blue (Gibson & Evans, 1937). The
concentration of Evans Blue in samples was measured using an Optica Milano double-beam
spectrophotometer at 620 nm. Standards for these measurements were prepared using the
patient’s own plasma. The Evans Blue was prepared from powdered substance: four batches of
different origin were tried and the one which seemed most satisfactory as judged by paper
chromatography and linearity of calibration curve of the dye at a wide range of concentrations
was then used in all the subjects. The purity of the dye was tested by the State Institute for
Control of Drugs before being used. Patients with turbid plasma were excluded from the study.
The slope of the Evans Blue disappearance curve was calculated after logarithmic transformation of E (optical density) units and expressed as a regression coefficient. A patient was
considered to have a satisfactory dye disappearance curve if the correlation coefficient between
time and the logarithm of E units was higher than 0.90. This was true whether all four samples
were used or only those at 10,20 and 30 min. Evans Blue disappearance slopes were calculated
from the latter data.
Plasma volume (PV) was then calculated in two ways; either from the 10 min dye concentration point (PV,,) or from the dye concentration value determined by the extrapolation
from 10,20 and 30 min samples to zero time (PV,,), i.e. as the intercept value in the regression
equation. Total blood volume was calculated from the plasma volume; haematocrit was
determined in duplicate. Clotting was prevented by placing approx. 500 units of dry heparin in
each tube. Haematocrit was not corrected for either trapped plasma or total body/peripheral
haematocrit ratio. Blood pressure was measured directly using a Statham P23DB transducer
Plasma volume in hypertension
175
attached to the Hellige Electromanometer and polygraph. Cardiac output was determined by
Indocyanine dilution using a Cambridge Mark I1 Dye Dilution Curve Recording Outfit. A
description of these procedures has been published elsewhere (Ulrych, Kasalicky & Widimsky,
1966; Ulrych, Frohlich, Tarazi, Dustan & Page, 1969).
For the injections of indicators, calibrated syringes were used. Plasma volumes in this study
were expressed as absolute and percentage deviations from the normal value as described
by Moore, Olesen, McMurrey, Parker, Ball & Boyden (1963). For the prediction of the normal
values, the regression equations PV =31.47 kg body weight +927 for males and PV=25.86 kg
body weight+849 for females were used. These regression equations are derived from measurements of plasma volume by Evans Blue distribution employing essentially the same technique as used here, and represent measurements on 222 normal males and 135 females of
ages between 16 and 90 with weights between 40 and 100 kg (Cohn & Shock, 1949; Gibson,
Peacock, Seligman & Sack, 1946; Gibson & Evans, 1937; Moore et al., 1963; von Porat,
1951; Schmidt, Iob, Flotte, Hodgson & McMath, 1956).
Correlation coefficients and the regression equation were calculated as described by Snedecor & Cochran (1971).
RESULTS
There was a statistically significant negative linear correlation between mean blood pressure
and Evans Blue disappearance rate (EBDR) (Fig. l), as well as a negative relationship
between mean blood pressure and plasma volume (Fig. 2), regardless of the technique used
for plasma volume calculations. Because cardiac output is inversely proportional to mean
blood pressure, blood pressure increase was a very close function of total peripheral vascular
resistance (Table 1).
Deviation of plasma volume from the normal predicted value is directly related to the EBDR
(Fig. 3), again regardless of the method of calculation of plasma volume.
A negative linear correlation between percentage deviation of plasma volume from predicted
values and haematocrit was found (r =0-581, y = -0*130x+ 56.1, P<O.Ol). This correlation
coefficient improves somewhat, if the values for the haematocrits of the females are raised by
3% each (to compensate for the sex difference), to r=0*621, P<O.OOl. No significant correlation was found, however, between haematocrit and blood pressure.
The ratio of cardiac output/total bloodvolume bears no relationship to EBDR, incontrast to
what would be expected if EBDR were mainly a function of indicator mixing due to differences
of cardiac outputs and plasma volumes.
As might be expected from the close relationship of mean blood pressure and total peripheral
vascular resistance there is also a negative linear correlation between EBDR and total peripheral resistance.
We also found a statistically significant correlation between plasma volume and cardiac
output using a partial correlation technique to exclude the influence of body weight on both
variables (r =0.482, P<O.O5).
DISCUSSION
Unfortunately, there is very little explanation why plasma volume is decreased in essential
hypertension and why this decrease is proportionate to the blood pressure increase. Julius
176
Milos Ulrych
et al. (1971) found a significant direct correlation between plasma volume/kg of body weight
and cardiac index in their labile hypertensive patients with increased cardiac outputs. This
correlation was reproduced in the present study when using the more correct statistical
techniques of partial correlation coefficients; calculating the relationship between plasma
volume and cardiac output while excluding the influence of body size on both variables.
Because our subjects were mild to moderate hypertensives with normal or subnormal cardiac
-0.001 L
0
A
-0~009-0.010 I
I
I
I
I
I
0
I
I
I
outputs (as follows from the negative correlation between blood pressure and cardiac output),
it seems that this finding may be a general feature of uncomplicated hypertension. This contrasts with the findings of Frohlich et al. (1967b) in renovascular hypertension where cardiac
output is increased and plasma volume decreased.
Correction of plasma volume for body weight and sex is better performed by predictions
from regression equations calculated by Moore et al. (1963) than by expressing plasma volume
per unit of body weight, since the regression lines do not pass through the origin. These equations are based on a large series of 357 subjects, with age and body-weight fluctuations similar
to those of our subjects.
177
Plasma volume in hypertension
Becauseblood pressure in the hypertensivesubjectswas raised mainly due to a total peripheral
vascular resistance increase, it is virtually impossible to decide whether the plasma volume
decrease is mainly related to blood pressure or to a total peripheral resistance increase. If the
assumption that plasma volume decrease is related to a total peripheral vascular resistance
increase is accepted, one is tempted to interpret this as a consequence of diminished vascular
capacity connected with an increase in total peripheral resistance. Because the capillary blood
pressure in hypertension is unknown, there is no way of telling whether the resistance increase
is on the pre-capillary level alone or on the post-capillary segment as well. If the former is
true, then it is very difficult to assume that this small change could lead to a clearly measurable
‘7
I30
0
-
A
0
0
60
I
I
I
I
I
I
I
I
I
volume change, and only in the case where post-capillary resistance is increased as well would
this result in an appreciable decrease of blood volume. Tarazi, Melsher, Dustan & Frohlich
(1970) and Eisenberg & Wolf (1965) made two completely different findings concerning
decrease of plasma volume during an upright tilt, which apparently was caused by filtration
of water out of the vessels. Tarazi et al. (1970) did not find any difference between hypertensive
and normotensive subjects, whereas Eisenberg & Wolf (1965) saw twice as great a drop in
plasma volume in hypertensive as in normotensive subjects. In studies of adjustment to upright
tilt in hypertension (Frohlich et al., 1967b; Frohlich, Tarazi, Ulrych, Dustan & Page, 1967a),
a complex haemodynamic situation was revealed; for these reasons the use of Evans Blue as a
label for plasma albumin and the measurement of the disappearancerate as an index of exchange
of protein between the intravascular and extravascular pools were preferred. This simplifies
TABLE1. Correlation coefficients ( r ) and regression equations between some of the measured variables. 6 =
regression coefficient; a = intercept in the regression equation where y = bx a and mean blood pressure is x
in the upper half of the table and Evans Blue disappearance rate is x in the lower half. PVlo=plasma volume
calculated from the 10 min concentration point; PV., = plasma volume calculated by extrapolation (see the text).
ns., not significant
+
Deviation of plasma volume from
predicted value
Evans Blue disappearance
PVlO
rate
[In(,? units)]
(ml)
(%I
Mean blood
pressure
(mmHd
Females
n
b
a
r
P
Total
Cardiac
peripheral
output
resistance
(I/min) (arbitrary units)
PVe x
(%)
(ml)
0.0024
-0.0385
< 0.1
13
-0.958
2732
111.7
-0.665 -0.700
<0.001
< 0.05
13
13
-25.1
-1.013
113.2
2785
-0.664 - 0.697
<0.05 < 0.01
12
-0.oooO851
0.0049
-0.683
< 0.05
12
- 7.9
807
- 0.470
> 0.01
12
-0.248
25.8
-0.453
> 0.1
12
-9.8
882
-0.547
<0.1
25
25
-0.oooO767 -15.3
0,004
1755
-0557
-0.575
<0.01
<0.01
25
-00572
66.7
-0.580
<0.01
25
25
-17.1
-0.626
1827
68.3
-0.602 -0.602
<0.01 <0.01
13
13
-O~oooO601
- 23.5
12
12
-0.0312
0.310
9.1
-14.2
-0.587
0.866
<0.05
<0.001
-
Males
n
b
a
r
P
Females + Males n
b
a
r
P
12
-0.306
28.1
-0.541
< 0.1
12
12
0.235
8.6
0.916
- 0.0205
8.7
-0.579
<0.05
<0.001
24
-0'0205
8.3
-0.439
<0.05
24
0.249
8.8
0.832
<0.001
Deviation of plasma volume from
predicted value
Evans Blue
Cardiac output
PVlO
disappearancerate Total blood ________
[In (E units)]
volume
(ml)
PA)
PV.,
~
Females
n
6
r
1.23
-0.083
P
n.s.
a
Males
12
- 7.2
r
12
12.0
1.15
0.156
P
n.s.
n
b
a
+
Females Males n
b
a
r
24
8.9
1.2
0.102
P
ns.
13
119491
620
0.524
<0 1
13
4447
24.7
0.508
12
60447
181
0.446
> 0.1
12
1923
6.4
0.438
> 0.1
25
97737
450
0.507
<0.02
25
3488
17.7
0.485
<0.02
< 0.1
~
(ml)
(%)
Total
peripheral
resistance
(arbitrary units)
13
127980
556
0.529
<0.1
13
4813
21.7
0.517
<0.1
n.s.
12
82018
146
0.571
12
2568
5.2
0.566
<0*07
12
1660
10.5
-0.806
<0.01
<0.06
12
-450.7
18.4
-0.197
25
25
24
113397
- 940
3971
403
15.4
15.3
0.584
0.525 -0.435
<0.01 <0.01
<0.05
179
Plasma volume in hypertension
the analysis, since otherwise the difference between circulation in the supine and tilted positions
would have to be considered.
The following three possibilities to explain why the Evans Blue disappearance rate is
increased in arterial hypertension should be considered. (1) An abnormality in mixing. If there
were a mixing abnormality related to changes of cardiac output in hypertension, one would
expect some degree of association between the cardiac output/total blood volume ratio and
EDBR. Our finding of virtually no association between these variables makes this possibility
unlikely. There is, however, another mechanism to be considered. As shown previously
(Ulrych et al., 1969), in many hypertensives there is a tendency towards a shift of blood volume
A
i 30
A
0
A
Evans Blue disappearance rate [In(€ units)/min]
FIG.3. Relationship between Evans Blue disappearance rate and plasma volume as a percentage
of the predicted normal value (r = 0.584, P < 0.01). Symbols are as in Fig. 1.
from the peripheral vascular bed to the lungs and heart. In further analysis of these data a
highly significant indirect relationship (r = -0.8 14, P ~ 0 . 0 0 1 )was found between total blood
volume and its percentage in the lungs and heart. This relationship remains practically unchanged when corrected for the influences of body size on both variables using a partial
correlation coefficient (r= -0.807, P<0401).It is possible that redistribution of blood from
the peripheral to the central circulation may influence the mixing of the dye. It is likely that
mixing is fast in the increased cardiopulmonary blood volume and may be markedly slowed
down in the constricted peripheral vascular bed. Therefore a possibility that some parts of the
peripheral vascular bed may equilibrate dye at a much slower rate has to be considered as an
explanation for the differences in the Evans Blue disappearance.
(2) The possibility that water might be shifted from the interstitial space into the intravascular space while the measurements are done. This may occur on resuming a supine position
180
Milos Ulrych
after an upright position. There are two basic reasons which make this explanation unlikely.
First, our subjects were always in a supine position for more than 30 min before the measurement was started and in this time a steady state between plasma volume and interstitial fluid
volume should have been achieved. Secondly, the length of the interval between the time when
the subjects lay down and the beginning of the plasma-volume measurement was variable and
was not correlated with the EDBR.
(3) The possibility of an increased rate of protein exchange between intravascular and extravascular compartments. Floyer (1966) has shown very clearly in renal hypertensive rats that
they have increased capillary pressure and a decreased rate of water escape from the intravascular compartment and that their interstitial pressure goes up more after saline infusion
than in normal rats. Thus the evidence points to an abnormality in the interstitial fluid, possibly
in its protein content or composition. The previous findings of Tarazi et al. (1969) show that
there may be a relative increase of the interstitial fluid compartment in hypertension connected
with a normal or decreased plasma volume. Tibblin et al. (1966) showed an increase in
haematocrit, plasma protein and blood viscosity in their hypertensive subjects. The present
data, especially when the inverse relationship between plasma volume deviation from predicted
value and haematocrit is considered, are in good agreement with all these findings. A common
interpretation for these findings and the present ones may be that there is an increased amount
of protein in the interstitial fluid in hypertensive subjects and an increased rate of protein
exchange between intravascular and extravascular compartments. Further work is necessary
to advance our understanding of this aspect of circulation in hypertensives.
It seems, however, that the plasma volume decrease in hypertension does not have to be a
permanent one, because Parry 8c Dollery (1968) and Cranston & Brown (1963) have shown
that hypertensives have a greater diurnal fluctuation of plasma volume compared to normal
subjects. This adds another aspect to the problem of plasma volume decrease in essential
hypertension, as it seems to be present mainly in the morning and much less so in the afternoon
and in the evening. Unfortunately, we have almost no knowledge of the haemodynamic
variables in hypertensivesin the afternoon and evening, because practically all studies have been
performed in the morning.
These arguments suggest that the problem of plasma volume in essential hypertension should
be further studied and more attention paid to the microcirculation.
ACKNOWLEDGMENT
I am indebted to Mrs V. Chrpovh for excellent technical assistance.
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