695
Hypocalcemia and Pregnancy-Induced
Hypertension Produced by Low-Calcium Diet
Jorge A. Prada, Reginald C. Tsang, Kenneth E. Clark
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Abstract Recent studies from our laboratory in fasting pregnant ewes with twin gestation have implicated low serum
calcium concentration in the etiology of hypertension in pregnancy. We hypothesized that the reduction in serum calcium
concentration produced by feeding of a calcium-deficient diet in
twin gestation would lead to a significant increase in maternal
arterial blood pressure, vascular resistance, and protein in the
urine and decreased uterine blood flow. Twenty-five instrumented ewes were used in the present study. After surgery a
calcium-deficient diet and deionized water (calcium ion free)
were provided ad libitum to 19 animals. Blood pressure, cardiac
output, heart rate, and uterine blood flow were monitored every
other day. Six control animals were provided with standard
Rumilab diet and tap water (group 1). Animals on a lowcalcium diet (group 2) were subdivided according to the blood
ionized calcium response to low dietary calcium intake. Nonhypocalcemic animals were assigned to group 2a (n=10), and
hypocalcemic animals (calcium concentration below two stan-
dard deviations from the control group) were assigned to group
2b (n=9). In group 2b calcium concentration decreased from
1.03±0.04 mmol/L on day 110 of gestation to 0.77±0.03 mmol/L
by day 125 of gestation. Arterial blood pressure increased
significantly from 76±2 to 91 ±2 mm Hg, and uterine blood flow
decreased from 950±53 to 579±48 mL/min. Urinary protein
increased from 1.7±0.3 to 10.5±1.2g/L. Despite the fact that all
animals in group 2 had the same low dietary calcium intake,
group 2a did not develop hypocalcemia (by definition) or an
increase in arterial blood pressure. The control group (n=6)
showed no significant changes in the parameters studied. From
these data we suggest that calcium plays a significant role in
regulating systemic arterial blood pressure and uteroplacental
S
uteroplacental blood flow (UBF).2 The fetus requires a
considerable amount of calcium for its skeletal development, and fetal calcium requirements increase sharply
at approximately the 30th week in the human fetus. By
term, the fetus has accumulated as much as 28 g
calcium, 80% of this during the third trimester. 1517
Theoretically, calcium imbalance may occur in twin
pregnancy where increased calcium demands and limited dietary calcium intake situations coexist. We designed the present study to investigate the cardiovascular effects of a calcium-deficient diet in twin pregnant
ewes and to evaluate the effects of these changes on
parameters of fetal well-being such as BP, heart rate,
blood iCa concentration, and oxygenation.
everal studies in animals and humans link low
serum ionized calcium (iCa) concentration with
the development of pregnancy-induced hypertension (PIH).1-2 Furthermore, during pregnancy calcium intake may be inversely correlated with blood
pressure (BP) in humans and animals, 36 and dietary
calcium supplementation may reduce diastolic blood
pressure in humans.7 In human epidemiologic studies,
dietary calcium intake appears to be an important
variable in determining the incidence of PIH. 810
In humans PIH is a hypertensive disorder peculiar to
pregnancy that usually develops between the 30th week
of pregnancy and the end of the first week postpartum.
If associated with albuminuria, edema, or both, the
disorder is termed preeclampsia. The etiology of PIH is
still unknown; it develops in 10% to 15% of pregnant
women and characteristically occurs in young primigravidas,11'12 women with twin gestation,13 and women
with preexisting hypertension or vascular disease.14
In recent studies from our laboratory in pregnant
ewes with twin gestation, we have demonstrated that
fasting results in maternal hypocalcemia in half the
animals studied. These animals developed significant
increases in arterial BP and a significant decrease in
Received December 23, 1993; accepted in revised form March
23, 1994.
From The A.E. Seeds Perinatal Research Center and The
Perinatal Research Institute, Departments of Obstetrics and Gynecology, Pediatrics, University of Cincinnati (Ohio), College of
Medicine.
Correspondence to Jorge A. Prada, MD, Department of Pediatrics, University of Cincinnati, College of Medicine, 231 Bethesda
Ave, Cincinnati, OH 45267-0541.
blood flow in twin pregnant ewes. (Hypertension. 1994^23 [part 1]:
695-702.)
Key Words • calcium • diet • hypocalcemia • twins •
hypertension, pregnancy-induced
Methods
Animal Preparation
Twenty-five normal twin pregnant ewes (determined by
ultrasound examination) of mixed breed were obtained from
Morris & Co. Thirty minutes before surgery animals (88 to 92
days of gestation and weighing between 45 and 55 kg) were
sedated with diazepam (10 mg IV; Hoffmann-La Roche Inc)
and then anesthetized with sodium pentobarbital (15 mg/kg
IV; Steris Laboratories). When necessary, additional sodium
pentobarbital was administered to maintain anesthesia. Animals were instrumented with a Doppler flow probe (Transonic
Systems) on the pulmonary artery for monitoring of cardiac
output. On day 105±2 of gestation with animals under spinal
anesthesia (15 mg of 1% tetracaine hydrochloride, Winthrop
Pharmaceutical), all 25 ewes were instrumented with polyvinyl
catheters in the maternal and fetal femoral arteries and veins.
Catheters were advanced to the level of the distal aorta and
inferior vena cava, respectively. Electromagnetic flow probes
(Dienco) of appropriate size were placed on the left and right
middle uterine artery through a 15-cm sterile lower abdominal
696
Hypertension
Vol 23, No 6, Part 1 June 1994
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incision for subsequent monitoring of blood flow to the uterus.
Flow probe cables and catheters were exteriorized through the
midline incision, passed subcutaneously to the ewe's left flank,
placed in a cloth pouch, and secured to the ewe's side.
Antibiotics (6 tnL IM) (Combiotics, GC Hartford Manufacturing Co) were administered on the day of and three days
after surgery. All animals were housed in individual portable
stainless steel cages. At the time of arrival, 6 animals chosen at
random were provided with standard laboratory diet (Rumilab, Ralston Purina) consisting of 0.71% calcium and tap water
ad libitum and were used as controls. Nineteen others were
provided with a calcium-deficient diet consisting of 0.21%
calcium (Rumilab chow 5508-2) and deionized water (calciumfree) ad libitum. All animals were allowed an adequate
recovery period before physiological recordings were made
and were studied 110±2 days of gestation to term. Animals
were not exposed to any experiments preceding the study.
Maternal catheters were flushed daily with 1000 USP units/mL
heparin sodium (Elkins-Sinn Inc) and fetal catheters with 500
USP units/mL heparin sodium for maintenance of patency. All
surgical and experimental procedures were performed in
accordance with the Institutional Animal Care and Use Committee guidelines of the University of Cincinnati.
Maternal and Fetal Measurements
Maternal cardiac output was measured by use of a Doppler
flow probe (Transonic Systems Inc), and maternal and fetal
systemic arterial BPs were measured via the appropriate
catheters with a model MP-15 BP transducer (Micron Instruments) anchored at the level of the sternum of the mother and
the fetal heart, respectively. Fetal BPs were corrected for
amniotic fluid pressure. Heart rates were determined with a
cardiotachometer (SensorMedics) triggered by an arterial
pressure pulse. Arterial UBF was measured by use of a
square-wave electromagnetic flowmeter (model RF 1000, Dienco). Electromagnetic flow probes (Dienco) were calibrated
with saline and were linear over the flow measured. BP, heart
rate, and blood flows were recorded continuously for 60
minutes on a pen writing dynograph physiological recorder
(Dynograph R612, SensorMedics).
Maternal and fetal arterial blood samples for blood iCa
were collected anaerobically into nonheparinized syringes and
immediately determined by use of an ionued calcium analyzer
(model ICA1, Radiometer). Blood iCa was adjusted to pH 7.4.
Samples for maternal and fetal arterial blood gas values (pH,
PaO2, and Paco 2 ) were collected anaerobically in heparinized
syringes and immediately determined with a blood gas analyzer (model BMS3 MK2 Micro System, Radiometer) operating at 39°C (ewes' body temperature). Oxygen content was
determined with a model LEX-O2 CON TL oxygen content
analyzer (Cavitron). Blood glucose concentrations were determined with a glucose analyzer (model 27, Yellow Springs
Instrument Co). Urine samples were collected every other day
(by clean catch) for determination of maternal urinary protein
concentrations with the use of a protein assay reagent (BCA
protein assay reagent, Pierce).
Experimental Protocol
We used 25 normotensive ewes with twin gestation (90+2
days of gestation) to study the effects of low dietary calcium
intake during pregnancy. BP, heart rate, cardiac output, and
UBF of the mother and BP and heart rate of the fetus were
measured once every other day between 9 and 11 AM for 60
minutes between days 110 and 135 of gestation. After thoracotomy, tap water and regular Rumilab chow were provided ad
libitum between 9 AM and 5 PM to 6 animals that were used as
controls. Deionized water (calcium free) and a calcium-deficient diet were provided ad libitum between 9 AM and 5 PM to
19 animals for the duration of the pregnancy. Blood samples
were taken once every other day in the morning (9 to 11 AM)
for determination of maternal and fetal glucose and lactate,
Pao 2 , PacOj, O2 content, pH, hematocrit, and iCa. Maternal
urine samples (100 to 200 mL) were collected every other day
in the morning (by clean catch) for determination of urinary
protein.
Histopathology of the Kidney
Kidney tissue specimens obtained from animals at the time
of death were collected and fixed in 10% neutral buffered
formalin solution (Sigma Chemical Co), Mossman's fixative
solution (Surgipath Medical Industries), and Zeus tissue fixative (Zeus Scientific, Inc) and were examined by light and
transmission electron microscopy at the Department of Surgical Pathology, University of (Cincinnati. The examiner was
blinded to the treatment groups.
Calculations and Statistical Analysis
All results are expressed as mean±SEM. For statistical
analyses, animals receiving a low-calcium diet (group 2) were
subdivided according to the blood iCa concentration response
to the low dietary calcium intake (normocalcemics to Group
2a and hypocalcemics to Group 2b; an animal was considered
to be hypocalcemic and assigned to group 2b if the ionized
calcium concentration decreased below two standard deviations from control, group 1). All animals on a low-calcium diet
that did not meet the criterion were considered to be normocalcemic and were assigned to group 2a. Cardiovascular and
biochemical responses of the two groups (2a and 2b) were
compared with each other and with the control group by
multiple ANOVA. The statistical significance of differences
between means was tested by Newman-Keuls test, and a value
of P=.Q5 was considered significant.
Results
Animals in group 2 started a low-calcium diet at 90 ±2
days of gestation. Although all animals on the lowcalcium diet demonstrated lower blood iCa concentration after 20 days of low dietary calcium intake (110
days of gestation), only 9 animals (47%, group 2b)
developed hypocalcemia and hypertension, and 10 others (53%, group 2a) remained nonnocalcemic and
normotensive.
Effect of Low-Calcium Diet on Maternal
Cardiovascular and Blood Gas Values
The six control animals (group 1) demonstrated no
significant changes in mean arterial BP (Fig 1, top) or
blood iCa concentration (Fig 1, middle). UBF increased
with the progression of gestation (916±74 to 1152±58
mL/min) (Fig 1, bottom). Cardiac output also increased
with the progression of gestation (5.94±0.42 L/min at
day 110 of gestation to 7.16±0.34 L/min by day 135) (Fig
2, top), and systemic vascular resistance decreased
(14±1 to 11±1 mm Hg/L per minute) (Fig 2, middle).
Urinary protein concentration increased slightly but not
significantly (1.5 ±0.2 g/L on day 110 of gestation to
2.5±0.5 g/L on day 135). (Fig 2, bottom). Maternal
heart rate, blood glucose concentration, and hematocrit
did not change significantly (data not shown).
As also shown in Fig 1 (top), group 2a (n = 10 twins)
demonstrated no significant changes in arterial BP
(from 80±2 mm Hg at day 110 of gestation to 75±1
mm Hg on day 135). In this group, blood iCa concentration remained stable throughout the experimental
period (1.04±0.02 mmol/L at day 110 of gestation and
1.07+0.02 mmol/L by day 135) (Fig 1, middle). UBF
showed initial increases with the progression of gestation (800+40 mL/min at day 110 of gestation to 889 ±38
Prada et al Calcium and Hypertension in Pregnancy
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Fra 1. Line graphs show maternal mean arterial blood pressure
(top), maternal blood ionized calcium (middle), and uterine
blood flow (bottom) of groups I (control, n=6), lla (normocalcemic, n=10), and lib (hypocalcemic, n=9). Group II animals
received low-calcium diet from day 90±2 of gestation. Values
are mean±SEM. *Signiflcant difference from control (P=.O1,
Newman-Keuls test).
mL/min by day 120). However, UBF did not continue to
increase as in control animals. By 125 and 130 days of
gestation significant differences (/>=.O1) were established between control and group 2a (Fig 1, bottom).
Cardiac output and systemic vascular resistance changes
were similar to those observed in the control group (Fig
2, top and middle). Urinary protein was found in all 10
animals at the beginning of the experiment (2.3±0.2
g/L) and remained constant throughout gestation, ending at 2.4 ±0.4 g/L by day 135 of gestation (Fig 2,
bottom). As shown in Table 1, maternal heart rate,
blood glucose, and hematocrit values did not change
significantly during the study. These animals remained
on a low-calcium diet until death (average, 138±2 days)
and showed stable blood concentrations of iCa and no
significant changes in cardiovascular or blood gas
parameters.
110
115
120
125
130
135
DAYS OF GESTATION
FIG 2. Line graphs show percent change in cardiac output
(top), maternal vascular resistance (middle), and urinary protein
(bottom) of groups I (control, n=6), lla (normocalcemic, n=10),
and lib (hypocalcemic, n=9). Group II animals received lowcalcium diet from day 9 0 ± 2 of gestation. Values are
mean±SEM. *Signiflcant difference from control (P=.O1, Newman-Keuls test).
In contrast, in group 2b (n=9, 47%) mean arterial BP
increased significantly by 20%. The peak values were
seen between 120 and 125 days of gestation in eight
animals and 115 days of gestation in another one (Fig 1,
top). Blood iCa concentration decreased with the progression of gestation, starting at 1.03±0.04 and falling to
0.77±0.03 mmol/L by day 125 of gestation (Fig 1,
middle). UBF demonstrated gestational increases from
110 to 120 days of gestation, being 758±38 and 950±53
mL/min, respectively. However, after day 120 we observed a significant reduction of UBF from 950±53 to
579±48 mL/min (Fig 1, bottom). We observed a reduction in cardiac output from 6.33±0.29 to 5.78±0.45
L/min by day 120 of gestation and a significant (^=.05)
increase in systemic vascular resistance from 12.01 ±0.75
at 120 days of gestation to 14.35±0.73 mm Hg/L per
698
Hypertension
Vol 23, No 6, Part 1 June 1994
TABLE 1. Cardiovascular and Blood Measurements In Group 2a (Normocalcemic)
Day 110
Day 115
Day 120
Day 125
Maternal
98±2
Heart rate, bpm
101 ±2
97±2
Glucose, mmol/L
3.77±0.33
3.71+0.27
3.71 ±0.38
3.55±0.27
Hematocrit
0.28±0.01
0.28±0.01
0.27±0.01
0.27±0.01
96±1
Fetal
MBP, mm Hg
Heart rate, bpm
44±2
44±2
45 ±3
45±2
166±5
160±5
165±8
168±4
7.371 ±0.010
PH
7.384±0.012
7.379±0.006
7.354±0.007
37.0±2.2
38.9±1.8
36.5±1.2
0.062±0.004
0.052±0.006
37.7±1.2
PacO2, mm Hg
0.061 ±0.005
O2 content
0.061 ±0.004
bpm Indicates beats per minute; MBP, mean blood pressure. Values are mean±SEM, n=10.
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
minute by day 125 compared with control (Fig 2, top
and middle). A negative correlation (r= — .477, P=.OO1)
was found between blood iCa concentration and arterial
BP (Fig 3). Maternal heart rate, blood glucose concentration, and hematocrit remained constant during the
experimental period (Table 2).
Urinary protein concentration began to rise before
the appearance of increased systemic arterial BP, from
1.7±0.3 to 10.5 + 1.2 g/L, and this continued until the
animals were killed (Fig 2, bottom). After the BP
increase and the UBF decrease, premature labor occurred within 96 hours, and the fetuses were aborted.
This occurred in 1 animal on day 115, 3 animals on day
127, and 4 animals on day 129, and a mother and its
fetus were found dead on day 131 of gestation, for an
average gestational length of 127 ±2 days. After abortion, there was an abrupt (48 hours) return of maternal
BP and iCa toward normal.
Histopathology of the Kidney
Kidney tissue specimens were obtained from seven
animals at the time of death and were examined by light
and transmission electron microscopy. All control animals studied (n=3) demonstrated no significant histological changes (Fig 4). However, all animals receiving
the low-calcium diet (n=4, groups 2a and 2b) demonstrated significant histopathologic tissue alterations
characterized by glomerular swelling, segments of foot
process effacement along the basal lamina of the glo-
r = - 0 477
p - 0001
110
90
70
Group lib
50
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0.6
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0.8
0.9
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MATERNAL BLOOD IONIZED CALCIUM (mM)
FIG 3. Scatterpkrt shows mean arterial blood pressure in relation to maternal blood ionized calcium concentration during
experimental period in group lib (hypocalcemic).
merular capillaries (group 2a, Fig 5), and electrondense paramesangial deposits that probably represent
protein deposits and mesangial hypercellularity (group
2b, Fig 6).
Fetal Changes
In group 1 fetuses demonstrated no significant
changes in fetal blood iCa when compared with the
baseline period (1.50±0.03 to 1.43+0.03 mmol/L, Fig 7,
top). No significant changes in fetal BP, pH, Paco 2 ,
PaO2, or O2 occurred during the experimental period
when compared with baseline (data not shown). Heart
rate decreased significantly with the progression of
gestation (183±3 to 157±6 beats per minute by day 135
of gestation, P=.Q5).
In contrast, in group 2 fetuses demonstrated reductions in blood iCa concentrations (1.29±0.05 mmol/L at
day 135 of gestation for group 2a and 1.31 ±0.04 mmol/L
at day 125 for group 2b; />=.O1, groups 2a and 2b versus
group 1; Fig 7, top). In group 2b fetal BP remained
constant (Table 2) despite maternal hypertension, but
Pao2, pH, and O2 content decreased with the decrease
in maternal UBF and reached significance by day 120 of
gestation (F=.O1, Fig 7, bottom, and Table 2).
Discussion
In the present study we have demonstrated that when
pregnant ewes with twin gestation are placed on a
calcium-deficient diet, hypocalcemia occurs in approximately one half of the animals between days 120 and
125 of gestation. This is the equivalent of 32 weeks of
human gestation, a time when significant fetal bone
calcification is occurring. The inability of these ewes to
maintain blood calcium concentrations indicates that
they were unable to mobilize adequate calcium to meet
the needs of both the mother and the twin fetuses. In
this study hypocalcemia was associated with significant
increases in arterial BP, systemic and uterine vascular
resistance, proteinuria, and significant reductions in
cardiac output and UBF. These hemodynamic and renal
changes are similar to those reported in human PIH.18
Once animals became hypocalcemic, systemic arterial
BP increased and UBF decreased, resulting in fetal
hypoxia. Mothers of hypoxic fetuses went into premature labor and aborted within 48 to 96 hours, for an
average gestation of 127±2 days. In fetal sheep, hypoxia
Prada et al Calcium and Hypertension in Pregnancy
TABLE 2.
699
Cardiovascular and Blood Measurements In Group 2b (Hypocalcemic)
Day 115
Day 110
Day 120
Day 125
Maternal
Heart rate, bpm
96±2
93±2
92+2
93±3
Glucose, mmol/L
3.96±0.27
3.60+0.11
3.01+0.16
3.38±0.11
Hematocrit
0.29±0.01
0.29±0.01
0.28+0.01
0.28±0.01
44±2
45±2
47+1
48±3
Fetal
MBP, mm Hg
Heart rate, bpm
pH
PacOj, mm Hg
O2 content
176±4
7.392±0.017
179±2
7.376±0.010
178+6
7.379±0.012
34.2±3.1
37.8±1.5
40.6+3.2
0.055+0.007
0.060±0.004
0.054+0.006
161±7
7.338±0.025*
38.0±1.3
0.048±0.005*
bpm indicates beats per minute; MBP, mean blood pressure. Values are mean±SEM, n=9.
*P=.01 by ANOVA (group 1 vs group 2b).
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is known to lead to significant increases in corticotropin
and cortisol,19 which are intimately involved in the
initiation of labor.20 Once sheep aborted, maternal BP
and blood iCa concentration returned to normal. In
contrast, animals that managed to maintain stable blood
iCa concentration and BP had an average gestational
length of 138 ±2 days, when they were killed (term,
144±2 days).
Pregnancy is associated with substantial changes in
calcium metabolism that include increased urinary calcium excretion and significant placental transfer and
fetal uptake of calcium.21'22 It has been reported that in
humans the transport of calcium from mother to fetus
increases from approximately 50 mg/24 h at 20 weeks of
gestation to a maximum of approximately 300 mg/24 h
at 35 weeks of gestation.1517 This increased maternal
calcium requirement theoretically may be met by enhanced intestinal absorption. However, in a situation in
which calcium intake is seriously decreased (ie, lowcalcium diet and deionized water, as in the present
study), parathyroid hormone (PTH) would be released
in response to a falling serum iCa concentration. PTH
would act on bone cells to increase the mobilization rate
of calcium ions out of bone to a point at which calcium
from bone is no longer available, thus causing an undue
disturbance to maternal calcium homeostasis. It is unclear why some animals on a low-calcium diet developed
low blood iCa concentrations whereas others did not.
The initial bone mineral reserves and the differences
between animals in the PTH response to a falling blood
FIG 4. Electron micrograph shows small part of a glomerulus
from group 1 (control). Part of a glomerular capillary is seen, with
the basal lamina (common to the capillary and podocyte) that
possesses numerous foot processes of podocytes on its outer
surface. No significant histological alterations were found (magnification x3000).
FIG 5. Electron micrograph shows small part of a glomerulus
from group 2a (normocalcemic). Arrows show segments of foot
process effacements on its outer surface. M indicates mesangial
cell; E, endothelial cell (magnification x2500).
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Vol 23, No 6, Part 1 June 1994
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125
130
135
DAYS OF GESTATION
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24
23
22
21
20
19
18
17
16
15
-1
I
i
A
T
A Control
O.
6 Group lla
Group lib
13
FIG 6. Electron micrograph shows very small part of a glomerulus from group 2b (hypocalcemic). The picture is characterized
by dense paramesangial deposits (arrows) that probably represent protein deposits (magnification X8000).
iCa concentration (not studied in the present report)
may in part explain some of the differences.
Vascular smooth muscle cells contract when intracellular free calcium concentration ([Ca2+],) is increased,
either by calcium release from intracellular stores or by
an influx of [Ca2+], from the extracellular compartment.
Conversely, vascular smooth muscle relaxation results
from a decrease in [Ca2+]i, which may occur by efflux
across the cell membrane and/or sequestration of
[Ca2+], into intracellular organelles.23'24 It has been
proposed that increasing the concentration of blood iCa
decreases [Ca2+], by calcium-regulated mechanisms.25
Theoretically, decreasing the concentration of blood
iCa increases [Ca2+]|.
The physiological concentration of active free calcium
ion in the blood of healthy pregnant women compared
with women with PIH is currently unclear. Fogh-Andersen and Schultz-Larsen26 reported that the blood concentration of free calcium ion in healthy pregnant
women is significantly increased, whereas Richards et
al27 reported no differences in the levels of blood iCa in
either healthy gravid women or women with PIH. In
contrast, Moodley et al28 have reported that calculated
blood iCa concentration in women with eclampsia was
significantly lower than in healthy pregnant and nonpregnant women with or without hypertension. Recently, Sowers et al29 reported that erythrocyte [Ca2+], is
higher in patients with PIH compared with healthy
pregnant women. These results were interpreted to
indicate that in response to the low extracellular calcium, cells such as erythrocytes increase their [Ca2+]|.
This effect appeared specific for calcium because no
changes were observed in intracellular sodium, potassium, or magnesium concentrations.
12
115
120
125
130
135
DAYS OF GESTATION
FIG 7. Line graphs show fetal blood ionized calcium (top) and
fetal PaO2 (bottom) of groups I (control, n=6), lla (normocalcemic, n=10), and lib (hypocalcemic, n=9). Group II animals
received low-calcium diet from day 90±2 of gestation. Values
are mean±SEM. *Signitlcant difference from control (P=.O1,
Newman-Keuls test).
The hypothesis that an increased need for calcium
during pregnancy is accompanied by physiological hyperparathyroidism was first expressed by Bodansky and
Duff in 1939.30 However, more recently reported
changes in PTH concentration during pregnancy are not
unanimous. Part of the differences seen in these measurements is due to the use of different PTH assay
methodologies.3'-33 Moreover, dietary intake of calcium
and vitamin D as well as age differences could be
confounding variables among studies. Similar deficits
exist in our knowledge concerning PTH and calcitriol
production in PIH. August et al34 reported increased
PTH and decreased 1,25 -dihydroxyvitamin D concentrations in 11 women with preeclampsia, but Lalau et
al33 reported no changes in mild PIH, and increased
PTH and calcitriol in severe PIH. Nevertheless, the
reported increase in serum PTH concentration during
pregnancy and PIH may be the result of a low dietary
calcium intake, increased calcium needs, or increased
calcium loss. It is not clear how PTH could directly
contribute to the development of hypertension during
pregnancy. PTH actions would include stimulation of
renal reabsorption of calcium, bone resorption, and
production of calcitriol (1,25-dihydroxyvitamin D), the
physiologically active form of vitamin D. Calcitriol
would enhance intestinal absorption of calcium. Moreover, PTH infusions to isolated vascular preparations
result in vasodilatation.35-36 However, Kawashima37 has
reported that PTH can induce transient increases in
[Ca2+], in vascular smooth muscle cells. Calcitriol is also
a positive modulator of contractile activity of vascular
Prada et al
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smooth muscle cells.38 Nevertheless, the specific role of
PTH and calcitriol, if any, in the development of
hypertension during pregnancy is unresolved.
The suggested role for [Ca ]i in the synthetic regulation of several vasodilator and vasoconstrictor humoral factors (eicosanoids, nitric oxide, and endothelins) may be significant in the control of systemic and
uterine hemodynamics during abnormal pregnancy.
Preeclampsia is characterized by alterations in the
equilibrium and production of prostacyclin and thromboxane A2.39 Although prostacyclin synthesis decreases,
concentrations of the potent vasoconstrictor thromboxane A2 increase in conjunction with platelet aggregation,40 resulting in a shift in the ratio of prostaglandin I2
to thromboxane A2, which could increase arterial BP
and reduce blood flow. Plasma concentration of thromboxane A2 seems to play an important role in ovine
pregnancy-induced hypertension, as its hemodynamic
effects are reversed with thromboxane synthetase inhibitors.41 Hassid and Oudinet,42 and more recently Magness and Rosenfeld,43 have shown a direct relation
between prostacyclin synthesis and blood calcium concentration. The vascular reactivity to angiotensin II is
known to be increased in PIH, whereas prostacyclin
synthesis is reduced. The reduction of prostacyclin
production theoretically might be the result of low
blood iCa concentration, because calcium supplementation blunts the vascular reactivity to angiotensin II.44
The synthetic alteration in a second potent vasodilator
agent, nitric oxide, may also play a major role in
controlling systemic arterial BP and UBF. Yallampalli
and Garfield45 have reported a syndrome consistent
with the hemodynamics and renal alterations seen in
preeclampsia: increased BP, proteinuria, and intrauterine growth retardation after the infusion of an inhibitor
of nitric oxide synthesis during rat pregnancy.
Vascular endothelial cells also produce endothelin-1,
a potent vasoconstrictor agent in a number of vascular
beds whose synthesis is significantly increased by the
elevation of [Ca2*],.46-48 Endothelin-1 has been shown
to be elevated in both essential hypertension49 and
PIH.50-51 Endothelin-1 might be involved in the renal
vascular alterations leading to proteinuria.52 Thus, increased calcium concentrations in the underlying vascular smooth muscle cell and changes in humoral factors
in the endothelial cells may explain some of the hemodynamic alterations seen in PIH.
Examination of the kidneys and urine of animals
receiving a low-calcium diet revealed glomerular hypercellularity, paramesangial deposits, and fusion of mesangial cell podocytes as well as increased urinary protein
concentration and red blood cells in the urine of hypertensive animals. Such changes are characteristic of reductions in glomerular blood flow and cellular damage.
Thatcher and Keith53 have also reported that after the
induction of hypertension by fasting pregnant ewes for 72
hours, proteinuria was present in all animals and averaged 3.5+ (>7.75 mmol/L). Ferris et al54 have reported
similar histological abnormalities of the glomeruli in
stress-induced hypertensive pregnant ewes, with deposits
of amorphous materials in the cytoplasm of the endothelial cells, which are characteristic of human preeclampsia. They have reported tubular necrosis and glomerular
lesions secondary to ischemia, which in turn may explain
the presence of increased protein in the urine. We have
Calcium and Hypertension in Pregnancy
701
demonstrated paramesangial deposits, mesangial hypercellularity, and segments of foot process effacement in all
animals studied that received a low-calcium diet. The
histopathologic alterations were not present in animals
on a normal calcium diet, suggesting that low dietary
calcium intake per se is capable of producing significant
glomerular alterations during twin pregnancy.
Hypertension in pregnancy and preeclampsia are
associated with high perinatal morbidity and mortality,55 with an increased incidence (as high as 40%) of
intrauterine growth retardation.56 Studies in an ovine
model of intrauterine growth retardation have shown a
clear relation between reduced UBF and fetal growth.57
In the present study we have observed a 39% decrease
in UBF in hypocalcemic and hypertensive animals,
which, if maintained throughout the latter part of
gestation, theoretically would lead to fetal growth
retardation.
In summary, from the evidence presented here as
well as from previous studies from our laboratory2 we
conclude that reductions in dietary calcium intake during ovine pregnancy with twin gestation can produce
symptoms similar to human preeclampsia, including
increases in maternal arterial BP, protein in the urine,
and decreases in cardiac output and UBF. The present
animal model will allow us to investigate the underlying
mechanisms responsible for the hemodynamic changes
observed in PIH.
Acknowledgments
This research was supported by a grant from the National
Institute of Child Health and Human Development (HD17742) and the National Heart, Lung, and Blood Institute
(HL-40083). We thank the staff of the Perinatal Research
Center for their expert technical assistance and Dr Mark
Weiss at the Department of Surgical Pathology for the histological examination of the kidneys.
References
1. McCarrom DA. Low serum concentrations of ionized calcium in
patients with hypertension. N Engl J Med. 1982307:226-228.
2. Prada JA, Ross R, Clark KE. Hypocalcemia and pregnancyinduced hypertension produced by maternal fasting. Hypertension.
1992;20:620-626.
3. McCarron DA. Calcium metabolism and hypertension. Kidney Int.
1989^5:717-736.
4. Villar J, Belizan JM, Repke J. The effect of calcium intake on the
blood pressure of young healthy individuals. Ann N Y Acad Sci.
1983;435:509-511.
5. Ackley S, Barret-Connor E, Suarez L. Dairy products, calcium and
blood pressure. Am J Clin Nuir. 1983^8:457-461.
6. Belizan JM, Pineda O, Saenz E, Menendez LA, Villar J. Rise of
blood pressure in calcium-deprived pregnant rats. Am J Obstet
Gynecol. 1981;141:163-169.
7. Belizan JM, Villar J, Pineda O, Gonzales AE, Saenz E, Garrera G,
Sibrian R. Reduction of blood pressure with calcium supplementation in young adults. JAMA. 1983;249:1161-1165.
8. Belizan JM, Villar J. The relationship between calcium intake and
edema-, proteinuria-, and hypertension-gestosis: an hypothesis.
Am J Clin Nutr. 1980^3:2202-2210.
9. Belizan JM, Villar J, Repke J. The relationship between calcium
intake and pregnancy-induced hypertension: up-to-date evidence.
Am J Obstet Gynecol. 1988;158:898-902.
10. Villar J, Belizan JM, Fisher PJ. Epidemiology observations on the
relationship between calcium intake and eclampsia. Int J Gynaecol
Obstet. 1983;21:271-278.
11. Guzick DS, Klein VR, Tyson JE, Lasky RE, Gant N, Rosenfeld
CR. Risk factors for the occurrence of PIH in pregnancy. Clin Exp
Hypertens B. 1987;6:281-297.
12. Sarkar CS, Gin AK, Sarkan B. Outcome of teenage pregnancy and
labor, a retrospective study. J Indian Med Assoc. 1991;89:197-199.
702
Hypertension
Vol 23, No 6, Part 1 June 1994
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
13. Long PA, Oats JN. Pre-eclampsia in twin pregnancy: severity and
pathogenesis. Aust NZ J Obstet Gynaecol. 1987;27:l-5.
14. Sibai BM, Moretti MM. PIH: still common and still dangerous.
Contemporary OBIGYN. February 1988:57-70.
15. Twardock AR, Symonds HW, Samsom BF, Rowlands GJ. The
effect on litter size upon foetal growth rate and the placental
transfer of calcium and phosphorus in superovulated Scottish
half-bred ewes. BrJNutr. 1973;29:437-446.
16. Forbes GB. Calcium accumulation by the human fetus. Pediatrics.
1975;57:976-977. Utter.
17. Widdowson WM, Dickerson T. Mineral Metabolism. New York,
NY: Academic Press Inc; 1961;2:l-247.
18. Fairlie FM, Sibai BM. Hypertensive diseases of pregnancy. In:
Reece EA, ed. Medicine of the Fetus and Mother. Philadelphia, Pa:
JB Lippincott; 1992:925-942.
19. Challis JR, Richardson BS, Rurak D, Wlodek ME, Patrick JE.
Plasma adrenocorticotropic hormone and cortisol and adrenal
blood flow during sustained hypoxia in fetal sheep. Am J Obstet
Gynecol. 1986;155:1332-1336.
20. Thorburn GD, Challis JR. Endocrine control of parturition.
Physiol Rev. 1979;59:863-918.
21. Chesley LC. Renal Functional Changes in Normal Pregnancy. New
York, NY: Appleton-Century Crofts; 1978:349-363.
22. Pitkin RM, Reynolds WA, Williams GA, Hargis GK. Calcium
metabolism in normal pregnancy: a longitudinal study. Am J Obstet
Gynecol. 1979;133:781-790.
23. Somlyo AP, Somlyo AV. Vascular smooth muscle: normal
structure, pathology, biochemistry, and biophysics. Pharmacol Rev.
1968;20:197-272.
24. Breemen CV, Cauvin C, Johns A, Leijten P, Yamamoto H. Ca ++
regulation of vascular smooth muscle. Fed Proc. 1986;45:2746-2751.
25. Katz AM. Mechanisms of action and differences in calcium
channel blockers. Am J Cardiol. 1986;58:20D-22D.
26. Fogh-Andersen N, Schultz-Larsen P. Free calcium ion concentration in pregnancy. Acta Obsta Gynecol Scand. 1981;60:309-312.
27. Richards SR, Nelson M, Zuspan FP. Calcium levels in normal and
hypertensive pregnant patients. Am J Obstet Gynecol. 1984;149:
168-171.
28. Moodley J, Rampersadh S, Becker P, Norman RJ, O'Donell D.
Serum calcium ion concentrations. S Afr Med J. 1987;72:382-385.
29. Sowers JR, Zemel MB, Bronsteen RA, Zemel PC, Walsh MF,
Standley PR, Sokol RJ. Erythrocyte cation metabolism in preeclampsia. Am J Obstet Gynecol. 1989;161:441-445.
30. Bodansky M, Duff VB. Regulation of the level of calcium in the
serum during pregnancy. JAMA. January 1939:223-229.
31. Pitkin RM. Calcium metabolism in pregnancy and the perinatal
period: a review. Am J Obstet Gynecol. 1985;151:99-109.
32. Davis O, Hawkins D, Rubin LP, Posillico JT, Brown EM, Schiff I.
Serum parathyroid hormone in adult pregnant women determined
by an immuno-radiomimetic assay for intact PTH. J Clin Endocrinol Metab. 1988;67:850-852.
33. Lalau JD, Jans I, Esper NE, Bouillon R, Foumier A. Calcium
metabolism, parathyroid hormone, and calcitriol in transient
hypertension of pregnancy. Am J Hypertens. 1993;6:522-527.
34. August P, Marcaccio B, Gerthner JM. Abnormal 1,25-dihydroxyvitamin D metabolism in preedampsia. Am J Obstet Gynecol. 1992;
166:1295-1299.
35. Anderson S, Grady JR, Ellison DH, McCarron DA. Calcium
balance and parathyroid hormone mediated vasodilatation in the
spontaneously hypertensive rat. Hypertension. 1983;5:59-63.
36. Pang PKT, Narvey S, Doris PA. Plasma parathyroid hormone
during the development of spontaneous hypertension in rats. Can
J Physiol Pharmacol. 1987;65:2386-2389.
37. Kawashima H. Parathyroid hormone causes a transient rise in
intracellular ionized calcium in vascular smooth muscle cell.
Biochem Biophys Res Common. 1990;166:709-714.
38. Bukoski RD, Xue H, McCarron DA. Effect of 1,25 (OH^ vitamin
D and ionized calcium on <5Ca uptake by primary cultures of aortic
myocytes of spontaneously hypertensive and Wistar-Kyoto normotensive rats. Biochem Biophys Res Commun. 1987; 146:
1330-1335.
39. Yamaguchi M, Mori N. 6-Keto prostaglandin Fl, thromboxane B2,
and 13,14-dihydro-15-keto prostaglandin F concentration of normotensive and preeclamptic patients during pregnancy, delivery,
and the postpartum period. Am J Obstet Gynecol. 1985;151:121-127.
40. Kinlough-Rathbone RL, Packham MA, Mustard JF. Synergism
between platelet aggregating agents: the role of the arachidonate
pathway. Thromb Res. 1977;ll:567-580.
41. Keith JC, Miller K, Eggleston MK, Kutruff J, Howerton T,
Konczal C, McDaniels C. Effects of thromboxane synthetase inhibition on maternal-fetal homeostasis in gravid ewes with ovine
pregnancy-induced hypertension. Am J Obstet Gynecol. 1989;161:
1305-1313.
42. Hassid A, Oudinet JP. Relationship between cellular calcium and
prostaglandin synthesis in cultured vascular smooth muscle cells.
Pmstaglandins. 1986^2:457-478.
43. Magness RR, Rosenfeld CR. Calcium modulation of endotheliumderived prostacyclin production in ovine pregnancy. Endocrinology. 1993;132:2445-2452.
44. Kawasaki N, Matsui K, Ito M, Nakamura T, Yoshimura T,
Ushijima H, Maeyama M. Effect of calcium supplementation on
the vascular sensitivity to angiotensin II in pregnant women. Am J
Obsta Gynecol. 1985;153:576-582.
45. Yallampalli C, Garfield RE. Inhibition of nitric oxide synthesis in
rats during pregnancy produces signs similar to those of preeclampsia. Mm J Obstet Gynecol. 1993;169:1316-1320.
46. Rubanyi GM, Botelho LHP. Endothelins. FASEB J. 1991;5:
2713-2720.
47. Lerman AF, Hildebrand K, Margulies B, O'murchu M, Perrella D,
Heublein T, Burnett J. Endothelin: a new cardiovascular regulatory peptide. Mayo Clin Proc. 1990;65:1441-1455.
48. Gouville A, Lippton HI, Cavero W, Hyman SA. Endothelin: a new
family of endothelium-derived peptides with widespread biological
properties. Ufe Sci. 1989;45:1499-1513.
49. Schiffrin E, Thibault G. Plasma endothelin in human essential
hypertension. Am J Hypertens. 1991;4:303-308.
50. Kamoi K, Sudo N, Ishbashi M, Yamaji T. Plasma endothelin-1
levels in patients with pregnancy-induced hypertension. N Engl J
Med. 1990-323:1486-1487.
51. Taylor RN, Varma M, Teng NNH, Roberts JM. Women with
preeclampsia have higher plasma endothelin levels than women
with normal pregnancies. J Clin Endocrinol Metab. 199O;71:
1675-1677.
52. Cornet S, Braillon A, Guilmard C, Chabrier PE, Pirotzky E,
Braquet P. Involvement of endothelin in renal processes.
Hypertension. 1990;5:724-728.
53. Thatcher CD, Keith JC. Pregnancy-induced hypertension: development of a model in the pregnant sheep. Am J Obstet Gynecol.
1986;155:201-203.
54. Ferris TF, Herdson PB, Dunnill MS, Lee MR. Toxemia of
pregnancy in sheep: a clinical physiological and pathological study.
J Clin Invest. 1969;48:1643-1655.
55. Kaunitz AM, Hughes JM, Grimes DA, Smith JC, Rochat RW,
Kafrissen ME. Causes of maternal mortality in the United States.
Obstet Gynecol. 1985;65:605-612.
56. Cunningham FG, MacDonald PC, Gant NF. Hypertensive disorders in pregnancy. In: Williams Obstetrics. 18th ed. Norwalk,
Conn: Appleton & Lange; 1989:653-694.
57. dapp JF, McLaughlin MK, Larrow R, Farnham J, Mann LI. The
uterine hemodynamic response to repetitive unilateral vascular
embolization. Am J Obsta Gynecol. 1982;144:3O9-318.
Hypocalcemia and pregnancy-induced hypertension produced by low-calcium diet.
J A Prada, R C Tsang and K E Clark
Hypertension. 1994;23:695-702
doi: 10.1161/01.HYP.23.6.695
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