a deficient maternal calcium intake during pregnancy increases blood pressure of the offspring in...
TRANSCRIPT
A deficient maternal calcium intake during pregnancy increasesblood pressure of the offspring in adult rats
Eduardo Bergel, Jose M. Belizan*
Objective To assess in an animal model the effect of maternal dietary calcium intake during pregnancy on theblood pressure of offspring.
Design Randomised controlled trial.
Sample Ninety-eight 20-week-old female Wistar–Kyoto rats, giving birth to a total of 119 pups that wereincluded in the follow up study.
Methods Rats were randomised to a calcium deficient diet, a diet with the recommended calcium levels, or adiet with calcium content much higher than the recommended levels. After one month on the experimentaldiet they were bred. After birth, systolic blood pressure in the offspring was measured monthly till 52 weeksof age.
Main outcome measures Blood pressure of the offspring.
Results The difference in blood pressure of the offspring between the normal and low maternal calcium groupincreased 0.49 mmHg per month (95% CI 0.18 to 0.84), 0.38 (0.07 to 0.68) between the low and highcalcium group, and 0.12 (�0.20 to 0.43) between the normal and high calcium group. At the end of thefollow up (52 weeks of age) blood pressure of the offspring in the deficient calcium group was 12.1 mmHg(95% CI 8.8 to 15.4, P < 0.0001) higher than in the normal calcium group and 7.5 mmHg (95% CI 4.4 to10.5, P < 0.001) higher than the high calcium group. Blood pressure of the offspring in the high calciumgroup was 4.3 mmHg (95% CI 1.0 to 7.5, P ¼ 0.01) higher than in the normal calcium group. In a multipleregression model maternal calcium intake during pregnancy was the strongest predictor of blood pressure ofthe offspring during adulthood.
Conclusions This experiment supports previous studies in humans suggesting a link between calcium intakeduring pregnancy and blood pressure in the offspring, and provides an animal model to explore themechanisms involved in such association.
INTRODUCTION
Our interest in the relationship between calcium intake
and blood pressure originated in observation of indigenous
women in Guatemala. Those women have a high calcium
intake due to the Mayan tradition of treating corn with lime
and have a very low incidence of hypertensive disorders
of pregnancy1. After this epidemiological observation
we performed a series of studies in which we showed an
association between dietary calcium intake and blood
pressure in rats2, young individuals3 and pregnant women4.
These observations lead us to state the hypothesis that the
incidence of one of the most severe forms of hypertension
during pregnancy, pre-eclampsia, can be reduced in popu-
lations of low calcium intake by calcium supplementation5.
In agreement with this hypothesis, a recent meta-analyses
including 10 randomised controlled trials has shown that in
communities with low dietary calcium intake calcium
supplementation during pregnancy is associated with a
68% (from 51% to 79%) reduction in the incidence of
pre-eclampsia6.
In 1987 our group performed a large, randomised,
placebo controlled trial of calcium supplementation during
pregnancy7 in a population with low calcium intake, and
seven years later we conducted the follow up of children
born from these mothers8. This study showed that the
offspring of calcium supplemented mothers had a lower
incidence of high blood pressure than offspring of mothers
receiving placebo, suggesting a role of fetal calcium
restriction on the genesis of hypertension in later life. This
observation is supported by a large number of epidemio-
logical studies linking an adverse fetal environment with an
increased risk of adult hypertension9 – 11, and by recent
animal studies showing that offspring life-long hyperten-
sion can be also determined during fetal life by maternal
protein restriction12.
In the present study an animal model is used to assess the
effect of maternal dietary calcium intake during pregnancy
on blood pressure of the offspring.
BJOG: an International Journal of Obstetrics and GynaecologyMay 2002, Vol. 109, pp. 540–545
D RCOG 2002 BJOG: an International Journal of Obstetrics and Gynaecology
PII: S1 4 7 0 - 0 3 2 8 ( 02 ) 0 1 1 5 5 - 2 www.bjog-elsevier.com
Latin American Centre for Perinatology, Pan American
Health Organization, World Health Organization,
Montevideo, Uruguay
* Correspondence: Dr J. M. Belizan, Latin American Centre for
Perinatology, Pan American Health Organization, World Health
Organization, Hospital de Clınicas s/n, 11000 Montevideo, Uruguay.
METHODS
Virgin Wistar–Kyoto rats (Taconic, Germantown, New
York, USA) were housed individually in wire mesh cages,
and maintained at 24�C on natural light cycles. All rats
had free access to water. Animals were housed in the
centre’s animal facility in strict compliance with institu-
tional regulations.
At 20 weeks of age, the rats were randomly assigned to
one of three experimental synthetic diets: a calcium defi-
cient diet, a diet with the recommended calcium levels for
rat chow, or a diet with a calcium content much higher than
the recommended levels. Synthetic diets (Dyets Inc, Beth-
lehem, Pennsylvania, USA) were prepared by supplement-
ing a calcium deficient rat chow with 0 ppm added calcium
(low calcium diet), 5000 ppm added calcium (normal
calcium diet) and 10,000 ppm added calcium (high calcium
diet).
A sample of each synthetic diet was sent to an inde-
pendent laboratory (Biochemistry Faculty, National Uni-
versity of Rosario, Rosario, Argentina) to determine
calcium and protein content. The measurement in the
samples showed a value of 10.5mg% calcium and
16.8g% protein in the low calcium diet, 473mg% calcium
and 16.1g% protein in the normal calcium diet, and
785mg% calcium and 17.7g% protein in the high calcium
diet. Synthetic diets were identical in colour, odour and
taste.
To conceal the randomisation, a unique number was
assigned to each dam and imprinted in the tail. An external
statistician used this number to allocate the dams to three
groups using a random-numbers table from a statistical
textbook. Rats were then housed in groups of four dams
per cage, according to the allocated group, and cages were
labelled as group I, II, and III. An external statistician
labelled rat chow containers as group I, II and III, and kept
treatment codes concealed till the end of the study. All
the investigators involved in implementing the study re-
mained blinded with respect to the calcium content of the
experimental diets, including the statistician that analysed
the results. A period of 30 days was allowed to habituate
the female rats to the experimental diets before mating. The
experimental feeding regimen was maintained throughout
the mating period and pregnancy.
Systolic blood pressure and weight was measured before
randomisation and at weekly intervals till birth. After birth,
we recorded the birthweight for all surviving pups and their
mothers were transferred to a standard laboratory rat chow.
The pups were removed from their mothers when they were
four weeks old. In litters with two or more pups, two pups
were selected at random from each litter, and when pos-
sible, one male and one female pup were chosen. The
excess was discarded. Selected pups were identified by
unique numbers and housed in groups of four regardless of
maternal diet. After weaning, the pups were fed on a diet
with the recommended calcium levels for standard rat
chow, and maintained under standard laboratory condi-
tions. The same person recorded the systolic blood pressure
and weight of the offspring monthly until they were 52
weeks old.
Blood pressure was measured in conscious rats using
an automatic rat tail blood pressure monitor (Kent Sci-
entific, Litchfield, USA). The rats were placed in a
darkened restraint tube, maintained at 28�C. To reduce
the effects of stress on blood pressure, the animals were
conditioned to handling and measurements were taken
routinely 10 minutes after initial handling. An occlusion
cuff was placed over the tail and inflated to 300 mmHg.
As the rats were different sizes, care was taken to use
cuffs of appropriate size for each animal. A separate
piezo-electric pulse sensor was also attached to the tail.
The blood pressure monitor was linked to a personal
computer that recorded deflation rate and pulses and
computed systolic blood pressure. The computer produced
a graph of cuff pressure and pulses that was used to assess
whether the recordings were valid. At least five valid
measurements were recorded for each rat at each time and
the average was used for the analysis.
Statistical methods
The original hypothesis was that the minimal difference
worth detecting between a low or high calcium diets,
compared with a normal calcium diet, was 5 mmHg. The
sample size required to detect such difference, with a type I
error of 5% and type II error of 10%, was 30 mothers per
group. To evaluate the statistical significance of different
blood pressure patterns of the offspring over time, a multi-
level modelling approach was used13,14. Multilevel mod-
elling is an extension of ordinary multiple regression where
data have a hierarchical or clustered structure. A hierarchy
consists of units grouped at different levels. Repeated
measures are one example of hierarchical structured data.
Here, monthly blood pressure measurements are clustered
within rats that represent the level 2 unit, with the monthly
measurements being the level 1 units. Thus, because rats
were measured on more than one occasion, two levels of
variability accounted for a single rat’s departure from the
fitted curve (level 1) and the differences between blood
pressure curves of different rats (level 2). The model fitted
accounted for complex level 2 variation that allows each rat
to have their own intercept and slope14. Because two rats
per litter were selected, and blood pressure variability
within offspring belonging to the same litter might be
smaller than variability in offspring from different litters,
litter was included in the multilevel model as level 3 in
hierarchy. The fitted model included maternal calcium diet
as a fixed effect, the interaction between maternal calcium
diet and rat’s age, and a cuadratic and cubic term for the
rat’s age. Multilevel residuals were calculated for model
checking and diagnosis14.
DEFICIENT MATERNAL CALCIUM INTAKE DURING PREGNANCY 541
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 540–545
Multiple linear regression analysis was used to assess the
statistical significance of the differences in systolic blood
pressure between treatment groups at the end of follow up
(52 weeks), and to compare the magnitude of the effect of
treatment with other predictors of blood pressure. The
models included maternal calcium diet during pregnancy,
offspring’s weight at 52 weeks of age, sex, birthweight, and
maternal blood pressure during pregnancy and maternal
blood pressure at 28 weeks of age. Regression residuals
were used to assess the validity of the regression models.
Regression coefficients were standardised to allow the
comparison of independent variables with different scale
of measurement, and the coefficient represents the change
in rat’s systolic pressure (in mmHg) for one standard
deviation shift in the value of the independent variable.
Statistical analyses were performed using MLWIN (Multi-
level Models Project, Institute of Education, London, UK)
and SAS (Cary, North Carolina, United States) packages
for IBM-PC.
RESULTS
Ninety-eight female rats were included in the experi-
ment. Pregnancy was not achieved in 18 rats (six in each
group), and in another 18 rats (six in the high calcium
group, five in the low calcium group and seven in the
normal calcium group) all pups died soon after birth due to
cannibalism and neglect (Table 1). The number of pups per
litter and the average birthweight was similar among
experimental groups (Table 1). In five litters only one
pup was available for follow up. A total of 119 pups were
finally included in the study: 40 in the high calcium group,
42 in the low calcium group and 37 in the normal calcium
group. Offspring groups were similar with respect to
proportion of male pups and birthweight (Table 1).
Figure 1 shows offspring systolic blood pressure pat-
terns over time in the three groups of maternal dietary
calcium during pregnancy. At one month of age systolic
blood pressure was similar between offspring of rats with
different dietary calcium intake during pregnancy, but
differences between groups became evident with increas-
ing age.
A multilevel model was used to analyse the patterns of
blood pressure of the offspring over time, according to
maternal calcium during pregnancy (Fig. 1). These analyses
showed a significant interaction between maternal calcium
diet during pregnancy and offspring age ( P < 0.001). The
interaction coefficients were used to evaluate if the differ-
ence between groups changes with increasing age. The
difference in blood pressure of the offspring between the
normal and low maternal calcium group increased
0.49 mmHg per month (95% CI 0.18 to 0.84). The
difference between the high and low calcium group also
significantly increases over time (difference between
groups increased 0.38 mmHg per month, 95% CI 0.07 to
0.68). The difference between the normal and high mater-
nal calcium group did not significantly change over time
(difference between groups increased 0.12 mmHg per
month, 95% CI �0.20 to 0.43).
At the end of follow up (i.e. 52 weeks of age) blood
pressure of the offspring in the low calcium groups was
significantly higher than in the normal calcium group. On
average, the blood pressure of offspring in the low calcium
group was 12.1 mmHg (95% CI 8.8 to 15.4, P < 0.0001)
higher than in the normal calcium group (Fig. 1). Blood
pressure in the high calcium group was 4.3 mmHg (95% CI
1.0 to 7.5, P ¼ 0.01), higher than in the normal calcium
group (Fig. 1).
In order to compare the contribution of maternal dietary
calcium intake to the development of high blood pressure in
offspring with other predictors of high blood pressure, a
multiple regression model was fitted with blood pressure of
offspring at 52 weeks of age as the dependent variable and
maternal calcium diet during pregnancy, maternal blood
pressure during pregnancy, maternal blood pressure at
28 weeks of age, offspring weight at 52 weeks of age,
offspring sex and offspring birthweight as explanatory
variables. In this model maternal dietary calcium during
pregnancy was the strongest predictor of blood pressure of
offspring at 52 weeks of age, followed by rat’s weight
(Fig. 2). Birthweight showed an inverse association with
Table 1. Number and characteristics of rats included in the study.
High calcium Low calcium Normal calcium All
No. of randomised mothers 33 33 32 98
Pregnancy not achieved 6 6 6 18
Pregnancy achieved but all pups dead 6 5 7 18
No. of mothers with at least one live pup 21 22 19 62
Total no. of pups 119 131 108 358
Average no. of pups per litter 5.7 5.9 5.7 5.8
Mean [SD] birthweight (g) 50.9 [11.6] 49.3 [11.0] 49.2 [8.4] 49.8 [10.2]
Pups included in the follow up study
No. of pups 40 42 37 119
No. of male pups 20 22 18 60
Mean [SD] birthweight (g) 52.2 [12.1] 50.6 [11.6] 52.3 [8.8] 51.7 [10.9]
542 E. BERGEL & J.M. BELIZAN
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 540–545
blood pressure that was borderline significant (Fig. 2). No
association was found between blood pressure of the
offspring and maternal blood pressure (Fig. 2). An analysis
fitting a multilevel model, with mother as level 2 and
offspring as level 1, produced similar results.
DISCUSSION
The present study shows in an animal model that
maternal dietary calcium intake during pregnancy has a
modelling effect on the offspring’s blood pressure. Calcium
deficit during pregnancy involves offspring with higher
blood pressure values, an effect that amplifies in adult life.
Furthermore, maternal dietary calcium during pregnancy
was the main predictor of blood pressure in the adult rat.
These findings are in agreement with findings of our
previous study on children and provide support to our
statement that the effect of maternal calcium intake during
pregnancy on blood pressure of the offspring could be
amplified throughout later life and could contribute to
hypertension on adulthood8. On the other hand a dietary
calcium intake much higher than the recommended levels
showed no further benefits.
Fig. 1. Offspring systolic blood pressure from one month of age till 52 weeks of age, according to maternal calcium intake during pregnancy. Data represents
mean (SEM). At 52 weeks of age blood pressure of the offspring in the low maternal calcium group was 12.1 mmHg (95% CI 8.8 to 15.4, P < 0.0001) higher
than in the normal maternal calcium group and 7.5 mmHg (95% CI 4.4 to 10.5, P < 0.001) higher than the high calcium group. At 52 weeks the blood
pressure of offspring in the high maternal calcium group was 4.3 mmHg (95% CI 1.0 to 7.5, P ¼ 0.0l), higher than in the normal calcium group.
Fig. 2. Independent predictors of blood pressure of offspring at the end of
follow up (i.e. 52 weeks of age). Figures are adjusted standardised
regression coefficients and 95% confidence intervals representing the
change in the rat’s systolic blood pressure for one standard deviation
increase in the independent variable, adjusted for the other independent
variables. The rat’s weight and sex were included in two different models
to avoid colinearity.
DEFICIENT MATERNAL CALCIUM INTAKE DURING PREGNANCY 543
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 540–545
There are differences between this experimental model
and human populations. In human populations with a
calcium deficit, there is usually a chronic restriction in
calcium consumption, from birth to adult life, and for both
mother and offspring. In contrast, in this animal model the
restriction was only for a short period of time before and
during pregnancy, and offspring received an adequate
amount of calcium in the diet. On the other hand, calcium
restriction during pregnancy was more severe in our model
than in human populations, because the low calcium diet
was almost calcium-free. Further research is needed to
address the impact of chronic calcium restriction on moth-
er’s and their offspring, in a model with conditions more
similar to human populations. This model should also
explore the effect of increasing dietary calcium beyond
the recommended levels.
To our knowledge, there is only one randomised study in
rats that assessed the effect of maternal calcium diet on
blood pressure of the offspring15. This study is in agree-
ment with our results, showing no effect of maternal
calcium restriction during pregnancy on blood pressure of
the offspring at one month of age15.
The association between birthweight and elevated blood
pressure in later life found in our study is in agreement with
a weight of evidence in humans and in animals16. Rat
models with low protein feeding during pregnancy have
been used extensively to study mechanisms that may be
involved in such an association17. Such studies have
provided a considerable body of evidence implicating
glucocorticoids in the programming of blood pressure by
the maternal diet18. Maternal under-nutrition reduces activ-
ity of 11 beta-hydroxysteriod dehydrogenase in the pla-
centa and may hence lead to overexposure of the fetus to
glucocorticoids19. Offspring of rats which receive low
amounts of protein during pregnancy remain hypersensitive
to glucocorticoids into adult life, and have increased
glucocorticoids receptor numbers at several sites, including
the vasculature20. Intrauterine steroid exposure thus may
establish an increased sensitivity to angiotensin II in early
postnatal life, which in turn establishes lifelong raised
blood pressure17. In a recent study it has been shown that
losartan, a specific angiostensin II receptor antagonist,
prevents raised blood pressure in offspring of rats that
received low amounts of protein during pregnancy, while
nifedipine, a calcium-channel blocker with negligible long
term effects upon the renin–angiostensin system, had no
effect on the blood pressure elevation in these animals21.
The data are consistent with the hypothesis that angiosten-
sin II plays a major role in the prenatal programming of
hypertension by low levels of protein, and that a different
mechanism might be involved in low calcium models. It
has been shown that treatment of hypertension simultane-
ously with calcium antagonists and a high calcium diet is
synergistic, with a mechanism of action that might be
mediated by calciotropic hormones regulating calcium-
channel activity in vascular smooth muscle cells22. In line
with these arguments, it has been suggested that all forms
of hypertension are associated with and dependent on
cytosolic-free calcium excess that is either extra cellular or
intracellular in origin23. In intracellular calcium-dependent
hypertension (identified clinically with low renin forms
of hypertension) the operative mechanism seems to involve
excess net cellular calcium accumulation from the extra-
cellular space, mediated by the action of calcium regulating
hormones such as 1,25(OH)2D, and parathyroid hyperten-
sive factor (PHF)23,24. The mechanism of action of PHF
involves an increase in calcium-channel activity in vascular
smooth muscle cells. PHF level explains why a high
calcium diet may be effective in lowering blood pressure
in patients who respond to calcium-channels blockers:
dietary calcium might inhibit the production of PHF (and
parathyroid hormone), whereas calcium-channels blockers
would inhibit PHF at its target site24,25. In support for this
hypothesis, studies in SHR rats have shown that the effects
of dietary calcium on blood pressure may be mediated by
PHF, such that a high calcium diet inhibits, and a low
calcium diet stimulates, the expression of this factor25. In
summary, an animal model of protein restriction during
pregnancy have been used to explore the mechanisms
linking a deficient fetal environment and hypertension in
adult life and evidence suggest the effect of maternal
calcium restriction on blood pressure of the offspring
operates through a different mechanism of action. In our
model calcium restriction during pregnancy might generate
alterations in cellular ion transport systems inducing a
metabolic set-point of calcium regulating hormones that
can result in a predisposition to high blood pressure. These
are intriguing hypotheses and the detailed mechanism of
the involvement of calcium-regulating hormones in the
genesis of hypertension and in the programming of blood
pressure of the offspring await further studies. The present
study provides an animal model for such studies.
The confirmation of these findings could have strong
public health implications. The deleterious effects of hyper-
tension on survival, disabilities, quality of life, and costs of
health care are widely known. On the other hand calcium
intake in the world is well below the requirements during
pregnancy.
It is estimated that the mean calcium intake in the world
is 472 mg per day, with an average intake of 860 mg per day
in the developed world and 346 mg per day in developing
countries26. These figures contrast with the recommended
calcium intake during pregnancy of 1200 mg per day27. In
view of the present findings, the achievement of such
requirements could imply relevant effects on the survival
and quality of life of future generations.
Acknowledgements
The authors would like to thank Dr R. Perez for her
excellent contributions to the experiment; Dr J. Jost for
544 E. BERGEL & J.M. BELIZAN
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 540–545
his valuable methodological suggestions; and N. Dorf and
A. Decker for their laboratory support. They would also
like to thank Dr H. Piriz for providing facilities to conduct
the experiment. The study was partially supported by a
grant from the Argentinean Research Council (CONICET).
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