Bio-Research, 8(1): 622 – 625
622
Evaluation of Serum Calcium and Inorganic Phosphate Levels in Pregnant and
Lactating Women in Enugu Metropolis
1
2
3
Ikekpeazu, J. E., Ibegbu, M. D. and Ugwoke, H. A.
Department of Medical Biochemistry College of Medicine, University of Nigeria Enugu campus, Enugu, Nigeria;
3
Department of Chemical Pathology School of Medical Laboratory Science UNTH Enugu, Nigeria.
1,2
Corresponding author: Ikekpeazu, J. E. Department of Medical Biochemistry, College of Medicine, University of
Nigeria Enugu campus, Enugu, Nigeria. Email: iebelejoy@yahoo.com
Abstract
The importance of calcium and inorganic phosphate in pregnancy cannot be overemphasized. Their
adequacy or otherwise amongst pregnant and lactating women in Enugu metropolis receiving their
routine antenatal supplements was the focus of this study. Two hundred subjects (forty in each
trimester; forty lactating and forty controls) were used for this study with informed consent. All subjects
were within the age range of 19-40 years. Serum samples were analysed for calcium and inorganic
phosphate using titrimetric and colorimetric methods respectively. Our result revealed a steady decrease
in calcium from first trimester to lactating period with statistically significant values in second and third
trimesters, and lactation (P<0.05) when compared with control (non-pregnant non-lactating women).
Statistically significant level of inorganic phosphate (P<0.05) were observed only in the second and third
trimesters. The significantly reduced level of calcium and inorganic phosphate during pregnancy and
lactation (for calcium) observed in this study is indicative of inadequate calcium intake (dietary) during
pregnancy or poor adherence to antenatal prescriptions. Higher provision of these elements and
enlightenment on the need for supplementation within the studied metropolis is suggested to avoid the
documented consequences of their deficiency to both the mother and the foetus.
Keywords: Serum, Calcium, Inorganic phosphate, Pregnancy, Lactation, Enugu
Introduction
Calcium is a divalent cation and the most abundant
mineral in human body, with a total body weight of
1.9% (Nordin, 1976) of which about 99% are found
in the bones and the remaining 1% in the
extracellular fluid (Robertson and Marshall, 1981).
Calcium absorption is vitally dependent upon
vitamin D, which the most important function is to
maintain serum calcium concentrations within the
normal range by stimulating its absorption from the
diet (Das et al., 2006). During pregnancy and
lactation, adequate calcium and inorganic
phosphate are necessary to meet the requirement
of the foetus and for the secretion in the growing
infant (Barker, 1996). In addition, calcium plays vital
roles as a cofactor to ATP, acts as an essential
cofactor in various enzymatic conversions [which
occur during blood clotting] in nerve transmission
and in assisting intrinsic factor in absorption of
vitamin B12. (Weisberg and Rhodin, 1970).The
recommended daily intake of calcium for pregnant
and lactating mothers is placed at 1200mg/day with
a tolerable upper limit of 2500mg/day (Food and
Nutrition Board, 1997). Calcium has its abundant
sources for humans in foods like milk, soya beans,
nuts, cheese, green leafy vegetables, and bread
(Villar et al., 2006)
Physiological conditions associated with
life cycle changes such as growth, pregnancy and
lactation are associated with increase in phosphate
absorption (Kiebzam and Sacktor, 1985). Findings
have shown that no major changes in either bone
mass or blood inorganic phosphate have been
observed during pregnancy (Sower et al., 1996;
Frolich et al., 1991) suggesting that phosphorus
supplementation is not recommended as it remains
Bio-Research
constant because of maternal adaptations to the
mineral (Weiss et al., 1998; Institute of Medicine,
1999); and it is abundant in many foods (NRC,
1981).
Developing infant needs calcium and
phosphate for structural formation of bones and
teeth, as low serum calcium during pregnancy may
result
in
skeletal
demineralised
foetus
(Cunningham, 2005), and in decreased bone
density in the mother with consequent high risk of
osteoporosis (Wu et al, 1990). It has been observed
that total serum calcium declines during pregnancy
reflecting lowered plasma albumin concentrations;
thus, the consequent decreases in the amount
bound to protein (Power et al, 1999); hence the
need for calcium supplementation to maintain
normal maternal serum levels and for normal
mineralization of the foetus. Furthermore, low
calcium intake has been implicated in the
development of hypertension, colon cancer, and
premenstrual syndrome (Power et al., 1999), and
hypocalcaemia has been shown to cause seizures
in children (Balasubramanian et al., 2004).
Calcium deficiency is rare in pregnancy but
appears in cases of hypoparathyroidism, severe
dietary inadequacy and in individuals who are
unable to eat a diet rich in dairy products (Kazzi et
al., 1998; Stabile et al, 1995). Some factors that can
impair absorption of calcium can lead to its
deficiency; these include substances that can form
complexes with it. Phytates which can form
insoluble
calcium
phytate
reduce
calcium
availability. Smoking also reduces its absorption as
cadmium in cigarette smoke is a calcium
antagonist. High protein diet stimulates bone
calcium resorption and encourages long term bone
loss (Kerstette and Allen, 1990) and high fibre diet
Published June 2010
ISSN 1596-7409
Ikekpeazu et al.
has also been implicated in reducing serum calcium
levels (De Santiago et al, 2001)
In this study, we evaluated the serum levels of
calcium and inorganic phosphate in different
trimesters of pregnancy and during lactation in
South Eastern Nigerian women to relate the
findings to some of the established knowledge
about
calcium
and
inorganic
phosphate
concentration during pregnancy and lactation.
Materials and Methods
Two hundred subjects were involved in the study, of
which one hundred and twenty were pregnant (forty
in each trimester); forty lactating (at first six months
of postpartum) and all within the age range of 19-40
years and attended Agbani District Hospital, Balm
of Gilead Maryland, University of Nigeria Teaching
Hospital Ituku-Ozalla and St. Mary’s Maternity
Abakpa; all in Enugu metropolis.
For comparative purposes, blood samples
were collected from forty non-pregnant; nonlactating women within the same age range. None
of the subjects had any disorder that affected
metabolism of calcium or bone, no history of
endocrine, renal or liver illness, hypertension of
pregnancy or gestational diabetes. None was
regularly taken medications or using hormonal
contraceptives. Those with any of these disorders
were excluded from the study and participation was
with informed consent. All the subjects were on their
routine antenatal supplements.
Collection and processing of samples: 5ml of
venous blood samples were collected from
antecubital fossa vein (with minimum toniquet
occulsion) by vein puncture with sterile needles.
Collected blood samples in the syringes were gently
discarded into clean plain glass tubes after removal
of needle to avoid heamolysis. All the collected
blood samples were allowed to clot at room
temperature for two hours and then centrifuged at
3000rpm for 5 (five) minutes and the serum
separated immediately to minimize the effect of red
cells phosphate on phosphate level. Blood samples
with any degree of haemolysis were deemed
unsuitable for the study and were discarded. The
o
separated sera were stored at -20 C in a deep
freezer until when needed. The samples were
thawed and brought to room temperature before
analysis, which was normally done within two days.
Calcium estimation: Complexometric titration
method with ethylene diamine tetra-acetic acid
(EDTA) by Appleton et al, (1959) was used. Serum,
de-ionized water and standard (each 0.5ml) were
placed in three universal containers labelled test,
blank and standard respectively and were diluted to
5.0 ml with 1.25M KOH to ensure alkaline
environment needed for the reaction. 0.25ml of
calcein indicator was added to each container and
mixed together; then titrated using .02N EDTA from
1.0ml graduated pipette. Colour change from
yellow-green fluorescence to non-fluorescence
salmon pink colour was noted and the volume of
EDTA added to change the colour was also noted.
623
Calculation: Test-blank/standard-blank
mg/100ml, Mg/100ml= Mmol/L,
x
10
=
Inorganic phosphate estimation: The method
used was based on modified ammonium molybdate
of Goldenberg et al, (1966). 0.2ml each of serum,
standard and water were pipette into three
centrifuge tubes labelled test, standard and blank
respectively. 5.0ml of TCA was added to them and
then centrifuged for ten (10) minutes. The
supernatant was decanted carefully and completely
into another clean test tube, 0.5ml of ammonium
molybdate was added and then allowed to stand for
15 minutes for colour development and was read
colorimetrically at 710 nm against blank.
Calculation: Absorbance of test/absorbance of
standard x concentration of standard x 0.58. After
the estimation data were collated and statistical
analysis was done using ANOVA and where
significant Turkey was used for mean comparisons
(P< 0.05).
Results
Table 1 shows the mean and standard deviation of
the serum calcium and inorganic phosphate levels
obtained from apparently healthy non-pregnant nonlactating women (control), in the trimesters and in
lactation. The mean serum calcium and inorganic
phosphate decreased during pregnancy when
compared with control; during lactation calcium
level also decreased while inorganic phosphate
increased.
Table 1: Mean levels of calcium and inorganic
phosphate in pregnant, lactating mothers and nonpregnant non-lactating women
Subject
N
Serum
Serum
calcium
inorganic
(Mmol/L)
Phosphate
(Mmol/L)
Control
40
2.512±0.155
1.221±0.109
First trimester
40
2.460±0.154
1.207±0.142
Second trimester
40
*2.324±0.143
*1.111±0.142
Third trimester
40
*2.213±0.128
*1.085±0.135
Lactation
40
*2.180±0.112
1.197±0.128
*Statistically significant when compared with control
(P<0.05)
Table 2 showed that mean serum calcium levels
have some changes throughout pregnancy and
nd
lactation. The changes were significant in 2 and
rd
3 trimesters and during lactation. The mean
inorganic phosphate was statistically significant at
nd
rd
the 2 and 3 trimesters.
Table 3 showed a negatively low degree of
correlation between first, third trimesters and
lactation phosphate; while others showed positively
low degree of correlation.
Discussion
Pregnancy and lactation have been observed to
induce dynamic changes in calcium metabolism.
Adequate calcium and inorganic phosphate are
necessary for protection of maternal bone density
and teeth; and reduce the risk of pregnancy induced
Serum calcium and inorganic phosphate levels in pregnant and lactating women
Table 2: Comparisons of mean values of calcium and
inorganic phosphate between the controls, the
trimesters of pregnancy and during lactation
Serum
Subject
Serum
inorganic
calcium
phosphate
(Mmol/L)
(Mmol/L)
P-value
(Sig.)
P-value (Sig.)
st
Control vs.1 trimester
0.438
0.990
Controlvs.2nd trimester
0.001
0.002
rd
Controlvs.3 trimester
0.001
0.001
Control vs. Lactation
0.001
0.926
(P<0.05) shows significant value of mean difference.
Table 3: Comparisons of serum calcium and inorganic
phosphate mean values between first, second and
third trimesters of pregnancy and lactation
Subject
Serum
Serum
calcium
inorganic
(Mmol/L)
phosphate
P-value
(Mmol/L)
P-value (Sig.)
st
nd
1 vs. 2 trimester
0.001
0.011
1st vs. 3rd trimester
0.001
0.001
st
1 vs. Lactation
0.001
0.997
2nd vs. 3rd trimester
0.004
0.909
2nd tri. vs. Lactation
0.001
0.031
3rd tri. vs. Lactation
0.818
0.002
(P<0.05) indicates significant values of mean difference
hypertension and pre-eclampsia (Sukonpan and
Phupong, 2005). This is also important in
mineralization during the foetal bone formation
(Pitkin, 1985). Findings have shown a downward
trend of serum calcium levels in pregnancy from
first trimester to third trimester. This trend has been
attributed to several factors including the decrease
in serum albumin that accompanies hemodilution in
pregnancy (Power et al 1999). Research has also
shown that intestinal absorption of calcium is
doubled during pregnancy from, as early as 12
weeks of gestation, reflecting the mineral’s
importance and high need for its appropriate dietary
intake and supplementation.
Our study did not show any statistical
significant difference in serum calcium between the
non pregnant women and first trimester of
pregnancy; this may probably be due to less need
of calcium during the period. Statistical significant
differences were, however, recorded when non
pregnant, non lactating women were compared with
second and third trimesters as well as during
lactation (P<0.05). This observation is in line with
the work of Laskey et al. (1998) that reported
increased need of calcium in late pregnancy and
during lactation. It is evident that the rate of transfer
of calcium into the developing foetus or infant
exceeds the maternal absorption (bioavailable
calcium), thereby altering the maternal calcium
homeostasis.
Serum inorganic phosphate levels also
showed statistical significant decreases in second
and third trimesters as compared to non pregnant,
non lactating (P<0.05). This is in agreement with
Roy et al. (1979) who reported that phosphate
concentrations generally fall during the 29 to 32
weeks of pregnancy. This view and our data
contradict some findings, which indicated that
phosphate levels during pregnancy remain constant
(Sower et al, 1996; Frolich et al, 1991). However,
624
there was no significant difference in inorganic
phosphate level during lactation when compared to
non-lactating non-pregnant rather a rapid increase
in inorganic phosphate level was observed during
lactation. This is in consonant with the work of
Kametas et al. (2003) which showed that serum
phosphate levels are within the non-pregnant range;
possibly because of increased renal reabsorption
and skeletal resorption during lactation.
Our data support the view that there is a
decreasing trend in serum calcium level during
pregnancy from first trimester reaching nadir at the
third trimester. This was made manifest in the
statistical significant differences between the nonpregnant, non-lactating, and second trimester, third
trimester and during lactation.
Though
supplementation of calcium (as calcium lactate )
has been a practice during pregnancy, as part of
the routine medication, the significantly reduced
calcium level observed in the second and third
trimesters and during lactation in this study could
either be due to inadequate dietary intake or poor
adherence to anti-natal supplementation.
In conclusion therefore, this study further
emphasized the much needed attention towards
adequate dieting and supplementation of calcium
and inorganic phosphate in pregnancy and during
lactation to avoid the documented deleterious
effects of their deficiency or reduced levels to both
mother and foetus.
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