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. References Balasubramanian S, Shivbalan S and Kumar PS (2006) Hypocalcemia due to vitamin D deficiency in exclusively breastfed infants. Indian Pediatrics 43: 247-251. Barker AM (1996) Nutrition and Dietetics for Health care. 9th ed Churchill Livingstone PP. 203213. Cunningham FG, Leveno JK, Bloom LS, Hanth CJ, GilstrapIII CL and Wenstrom DK (2005) nd Williams Obstetrics 22 ed. Mc Graw Hill Companies USA. PP 700-701, 119811200. Das G, Crocombe S, McGrath, Berry JL and Mughal MZ (2006) Hypovitaminosis D among healthy adolescent girls attending an inner city school. Arch Dis Child,0000: 1-5 (www.archdischild.com) DeSantiago S, Alonso L, Halhali A, Larrea F, Isoard F and Bourges H (2002) Negative calcium balance during lactation in rural Mexican women. Am J Clin Nutr 76:845-851. Food and Nutrition Board, Institute of Medicine 1997, Dietary reference intakes for calcium phosphorus, Magnesium, vitamin D and Fluoride. Washington DC: National Academy Press. Frolisch A, Rudricki M, Fischer- Rasmassen W and Ottosson K. 1991, serum concentration of intact parathyroid hormone during late human pregnancy: a longitudinal study. Eur.J Obstet Gynecol Reprod Biol. 42: 8587. Institute of Medicine (1999) Dietary reference intakes. Calcium, phosphorus, Ikekpeazu et al. magnesium, vitamin, and fluoride. Washington, DC: National Academy Press. Kassi GM, Gross CL, Bork MD and Moses D 1998. Vitamins and minerals . In: Gleicher N, Buttin L (eds.) Principles of Medical rd therapy in pregnancy 3 edition Old tappan, NJ: Appleton and Lange, 311-319. Kametas N, McAuliffe F and Krampl E (2003) , Maternal electrolyte and liver function changes during pregnancy at high altitude. Clin Chem Acta 328: 21. Kerstetter J and Allen LH (1990), Journal of Nutrition 120:134-136. Kiebzak GM and Sacktor B (1985), Age related phosphaturia and adaptation to phosphorus deprivation in rat. New York Raven Press P 123. Laskey MA, Prentice A, Hanratty LA, Jarjou LM, Dibba B, Beavan SR and Cole TJ (1998) Bone changes after 3 mo of lactation; influence of calcium intake, breast-milk output, and vitamin D-receptor genotype. Am. J Clin. Nutr. 67: 685-692. National Research Council (NRC) 1989. th Recommended dietary allowances 10 ed Washington DC: National Academy Press. Nordin BEC 1976, Nutritional considerations; In calcium phosphate and magnesium metabolism. Nordin BEC (ed.) P 1- 35. Edinburgh: Churchill Livingstone. Pitkin RM, (1985) Calcium metabolism in pregnancy and the perinatal period: a review. Am. J Obstet. Gynecol. 151: 99- 109. Power ML, Heaney RP, Kalkwarf HJ, Pitkin RM, Repke JT, Tsang RC and Schulkin J (1999) Am J Obstet Gynecol 181: 15601569. 625 Robertson WG and Marshall RW (1981). Inonised calcium in body fluids. Crit. Revs. Clin Lab. Sci, 15: 85- 125. Roy M, Reynold WA, Gerard AW and Gary KH, (1979). Calcium metabolism in normal pregnancy. Ame. J Obstet. and Gynecol. 133: 783-789 Sower M, Crutchfield M, Jannausch M, Updike S and Carton G (1996). A prospective evaluation of bone mineral change in pregnancy. Obstet Gynaecol 77: 741-745. Stabile I, Chard T, Grudzinskas G eds. 1995 Clinical Obstetrics and Gynaecology. London Springer, 96-97. Sukonpan K and Phupong V (2005) Serum calcium and serum magnesium in normal and preeclamptic pregnancy. Arch Gynecol Obstet 273: 12-16. Villar J, Abdel-Aleem H, Merialdi M, Mathai M, Ali M, Zavalta N Buscemi N et al. 2006. World Health Organisation randomised trial of calcium supplementation among low calcium intake pregnant women. American Journal of Obstetrics and Gynaecology; 194, 639-49. Weisberg H and Rhodin J (1970) Relation of calcium to mucosal structure and B12 absorption in canine intestine. Am J Pathol. 61(2): 141-160. Weiss M, Eisenstein Z, Ramot Y, Piptz S, Shulman A and Frenkel Y 1998. Renal reabsorption of inorganic phosphorus in pregnancy in relation to the calciotropic hormones. Br. J Obstet Gynecol. 105: 195- 199. Wu DD, Boyd RD, Fix TJ and Burr DB (1990) Regional patterns of bone loss and altered bone remodelling in response to calcium deprivation in laboratory rabbits. Calcif Tissue Int. 47: 18-23