Nutrition 30 (2014) 291–296
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Applied nutritional investigation
Iron status of pregnant Indian women from an area of active iron supplementation Kavitha C. Menon Ph.D. a, b, Elaine L. Ferguson Ph.D. c, Christine D. Thomson Ph.D. a, Andrew R. Gray B.Com(Hons). d, Sanjay Zodpey Ph.D. e, Abhay Saraf M.D. e, Prabir Kumar Das M.D. f, Chandrakant S. Pandav M.D. g, Sheila A. Skeaff Ph.D. a, * a
Department of Human Nutrition, University of Otago, Dunedin, New Zealand Healthy Lifestyle Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Penang, Malaysia London School of Hygiene and Tropical Medicine, London, England d Department of Preventive and Social Medicine, University of Otago, Dunedin, New Zealand e Public Health Foundation of India, New Delhi, India f Health and Family Welfare Training Institute, Nagpur, India g All India Institute of Medical Sciences, New Delhi, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2013 Accepted 19 August 2013
Objective: The aim of this study was to investigate the iron status of pregnant tribal women from Ramtek, Nagpur, Maharashtra, India using a combination of indices. Methods: A community-based observational study was conducted to assess iron status using a convenience sample of pregnant Indian tribal women from Ramtek. Pregnant women were recruited at 13 to 22 wk gestation (first visit; n ¼ 211) and followed to 29 to 42 wk gestation (second visit; n ¼ 177) of pregnancy. Sociodemographic and anthropometric data; iron supplement intake; and blood samples for estimating hemoglobin (Hb), serum ferritin (SF), soluble transferrin receptor (sTfR), and C-reactive protein (CRP) were obtained. Results: The mean (SD) Hb concentration at recruitment was 106 (15) g/L and 106 (14) g/L at the second visit; 41% of the women at recruitment and 55% at second visit were anemic (14% higher, P < 0.001). No women at recruitment and 3.7% at second visit had SF concentration < 15 ng/mL; and 3.3% at recruitment and 3.9% at the second visit had sTfR > 4.4 ng/mL (0.6% higher, P ¼ 0.179). Almost 62% and 71% of pregnant women used iron supplements at both visits, respectively. Iron supplement intake > 7 d in the preceding month improved the Hb concentration by 3.23 g/L and reduced sTfR concentration by 13%; women who were breastfeeding at the time of recruitment had 11% higher SF concentration. Conclusions: The iron indices suggest that pregnant tribal women of central India, although anemic, had good iron status. Use of iron supplements > 7 d in the preceding month improved iron status; however, non–iron-deficiency anemia persisted in this group. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Anemia Iron deficiency anemia Iron status Pregnant women Dietary supplements
Introduction KCM conducted the research, analyzed the data and, along with SAS prepared the manuscript. SAS, ELF, and CDT designed and supervised the study, secured funding, and assisted in the interpretation of the data. ARG together with KCM conducted the statistical analysis. SZ, AS, and PKD helped with obtaining ethical approval and liaised with district health professionals in Nagpur. CSP supported this research in his capacity as regional director of ICCIDD (Southeast Asia). All authors commented on the final draft of the manuscript. The authors declare no conflicts of interest. * Corresponding author. Tel.: þ643 479 7944; fax:þ643 479 7958. E-mail address: [email protected] (S. A. Skeaff). 0899-9007/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nut.2013.08.015
According to the World Health Organization (WHO) 41.8% of pregnant women worldwide have anemia and about half of these women have iron-deficiency anemia (IDA) [1]. IDA continues to be a major public health challenge globally and is one of the most common nutritional disorders in pregnant women as iron requirements increase to meet the higher maternal–fetal demands [2]. Populations at highest risk for IDA during pregnancy are those with inadequate pre-pregnancy iron stores coupled with
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low dietary intakes of bioavailable iron. IDA during pregnancy also may have adverse effects on obstetric outcomes, making it a critical public health concern [3,4]. Research has shown that anemia during pregnancy is common in India. The 2006 national prevalence rate of anemia was 59% [5], with regional prevalence estimates between 30% and 71% [6,7]. Anemia is predominantly due to iron deficiency [8]; however, it may be attributed to concurrent micronutrient deficiencies of vitamin B12, folic acid, vitamin B6, or vitamin A, or high rates of infection, malaria, or hemoglobinopathies, all common in low-income countries [9]. To address IDA during pregnancy, the Indian government has implemented a free iron–folic acid supplementation program for all pregnant women. Despite this initiative, IDA in pregnancy remains a major public health challenge in India [7,10]. Many studies that have assessed the prevalence of anemia during pregnancy in India use hemoglobin (Hb) as the only measure [10–12], whereas a combination of indices is recommended to more accurately evaluate IDA. A combination of indices such as Hb, serum ferritin (SF), and soluble transferrin receptors (sTfR) along with a biomarker for infection, provides higher sensitivity and specificity to identify IDA than Hb alone [2]. Furthermore, to assess the iron status of pregnant women, the use of sTfR with other laboratory indices for iron depletion (ID) is a reliable method [2,13] because sTfR concentrations are unaltered during pregnancy, unaffected by infections, and their up-regulation is a sensitive index of stage II ID [14,15]. Despite having an active infant–maternal health care program in place, a recent study showed that 72% of young, nonpregnant tribal women living in central India were anemic based on their Hb concentration alone [16]. These results suggest that pregnant women in this region are likely to be at high risk for IDA and its associated negative effects on obstetric outcomes such as low birth weight, premature birth, and infant and maternal mortality [17,18]. Although there is abundant information on the prevalence of anemia in pregnant Indian women, there is a dearth of information on the prevalence of IDA in this group, especially from areas where an active iron–folic acid supplementation program is in place. To address this critical public health issue the present study aimed to (1) assess the prevalence of IDA and iron deficiency without anemia among pregnant tribal Indian women living in central India during the second and third trimesters of pregnancy; and (2) assess factors associated with indicators of maternal iron status during pregnancy. This study is part of a larger study, which also investigated the iodine status of tribal women during the second and third trimesters of pregnancy [19].
lost to follow-up, we needed to initially screen 225 pregnant women and enroll 191 into the study. A fasting blood sample, anthropometric measurements, and a general sociodemographic health questionnaire were collected at 13 to 22 wk gestation (first visit) and 29 to 42 wk gestation (second visit) of pregnancy. Ethical approval was obtained from the Ethics Committee of the Health and Family Welfare Training Centre, Nagpur, India. Participants gave informed written and verbal consent. Pregnant women who participated in this study belonged to the Gond tribe, the second largest tribe in India, which has its own language and culture. In the past 2 decades, members of the tribe have been resettled and subsequently integrated into the rural community of Ramtek where they earn their livelihood primarily from agriculture. Between February and May 2008, all women in the catchment areas of the three PHCs who were between 13 and 22 wk gestation and met the inclusion criteria were invited to take part in the study. The inclusion criteria for participation were healthy pregnant women ages between 18 and 30 y, who were between 13 and 22 wk gestation based on the self-reported date of their last menstrual period, were willing to participate in the study, and stated they would live in the research area throughout pregnancy and up to 1-mo postpartum. The exclusion criteria were non-pregnant women and women with self-reported HIV/AIDS, tuberculosis, fever lasting longer than 2 wk, or thyroid disease. A qualified medical practitioner examined the pregnant women for overt health issues before their recruitment into the study. Socioeconomic, health status, obstetric questionnaire, iron supplement intake, and anthropometry Trained Indian research assistants administered questionnaires to elicit information on socioeconomic and health status, including maternal age, number of years of completed schooling, annual household income, number of family members, dietary practices (i.e., vegetarian), parity, breastfeeding at the time of the first visit, use of dietary and medicinal supplements and health drinks, and lifestyle habits such as chewing pan (i.e., beetle leaf with lime and areca nut) with or without tobacco. Information on iron-containing supplement use for the previous month was collected at both visits via the questionnaire and for the previous day via a 24-h dietary recall (dietary data are not included in this article). The elemental iron content of supplements was obtained from the information provided on the labels or information by the manufacturer of the supplement. The daily elemental iron intake from supplements of the participant over the past month at both visits (i.e., medicinal and dietary supplements) was determined by summing the estimated elemental iron intake/d for each supplement over the previous month. Using the questionnaire data, women were categorized into one of two groups based on their supplement use; women who took supplements for >7 d in the previous month (>7 d) were classified as frequent users, whereas those who took supplements for < 7 d during the previous month (7 d) were classified as lessfrequent users. The classification was undertaken based on evidence that intermittent intake (one, two, or three times/wk) of iron-containing supplements improves iron status of pregnant women [20]. Supplement use was confirmed by examining the women’s 24-h recall. When the supplement intake in the questionnaire data disagreed with the 24-h recall data, the categorization was based on the questionnaire data (i.e., supplement intake over the past month for > 7 d/mo). Serial measurements of maternal weight using a digital flat scale (Seca Robusta 813, Seca corporation, Hamburg, Germany), and height using a portable stadiometer built by the University of Otago were taken at both visits by one trained research assistant [21]. Weight gain during pregnancy was calculated as the difference in maternal weight between the weights at the first visit and the second visit. Biochemical assessment
Materials and methods Study design and participant recruitment A longitudinal observational study was conducted from February to December 2008 with 225 pregnant women living in the catchment areas of three tribal primary health centers (PHCs) in Ramtek Block, Maharashtra State, India. These PHCs were purposefully selected because they were the most disadvantaged tribal PHCs in Ramtek Block. The tribal PHC had an active iron–folic acid supplementation program in place for pregnant women. The characteristics of this program included free distribution of iron (100 mg)–folic acid (0.5 mg) supplements to all the pregnant women for at least 100 d. Additionally, women were provided with deworming tablets twice during their pregnancy and a variety of other dietary supplements (e.g., calcium, liquid protein supplements, B-complex vitamins). Based on a reported prevalence of anemia in non-pregnant women of 72% [16], 170 women would be needed to estimate the prevalence of anemia in pregnant women to within 10% of the true value with 95% confidence. Assuming 85% would meet the inclusion criteria and 10% would be
A morning fasting peripheral venous blood sample was collected into 4.5 mL vacutainer containing EDTA (Becton Dickinson, Franklin Lakes, NJ, USA) and 7 mL trace element-free vacutainer (Becton Dickinson, Franklin Lakes, NJ, USA). A complete blood cell count (CBC) and assays for C-reactive protein (CRP) and sickle cell disease were done on the same day of blood sample collection at the National Reference Pathological Laboratory in Nagpur using a Sysmex automated hematology analyzer KX-21 for CBC; the Metabisulphide Slide test for sickle cell anemia [22]; and turbidimetry using CRP Turbilatex reagents (Agappe Diagnostics, India) for CRP. Blood samples collected in the trace element-free vacutainers were cooled to approximately 4 C in an ice box immediately after collection and then transported to the Nagpur Reference Pathological Laboratory where they were centrifuged at 3500g for 10 min, aliquoted into cryogenic vials (Sarstedt AG & Co., Nümbrecht, Germany), stored at 20 C and then transported on dry ice to Molecular Diagnostics, Lucknow, India for analysis. Maternal blood samples were analyzed for SF and sTfR. SF was measured by enzyme-linked immunosorbent assay (ELISA) (Biotron Diagnostic Inc. Hemet, CA, USA), which had a detection limit of 5.0 mg/L. The mean (SD) SF concentrations of
K. C. Menon et al. / Nutrition 30 (2014) 291–296 the low, medium, and high external standards (Bio-Rad quality control standards, Bio-Rad, Hercules, CA, USA) were: 48 (1.6) mg/L (expected range: 28–65.5 mg/L); 115 (7.1) mg/L (expected range: 54–125 mg/L); and 281 (20.1) mg/L (expected range: 148–336 mg/L), respectively. The interassay coefficients of variability (CVs) were 3.4%, 6.2%, and 7.2% at the low, medium, and high SF concentrations, respectively (n ¼ 6). sTfR was estimated by ELISA (BioVendor Laboratory Medicine Inc., Czech Republic) with functional assay sensitivity of 0.1 mg/L. The mean (SD) sTfR concentrations of the low- and high-quality control standards from Biovendor were: 2.0 (0.2) mg/L (expected range: 1.7–2.6 mg/L); and 6.4 (0.4) mg/ L (expected range: 4.9–7.3 mg/L), respectively. The interassay CVs were 10.8% and 6.4% at low and high sTfR concentrations (n ¼ 5). Anemia was defined as Hb < 110 g/L, Hb < 105 g/L, and Hb < 110 g/L in the first, second, and third trimesters of gestation, respectively [23]. The severity of anemia was categorized based on Hb concentration as mild (>100 to 4.4 mg/L, in the presence of anemia (Hb concentration 85 fL was used to identify macrocytic anemia [21]. Microcytic anemia was identified when the red cell distribution width was > 14% [21]. A CRP value of >10 mg/L defined the presence of infection [26]. Statistical analysis Descriptive statistics were used to summarize baseline characteristics of the participants. Differences in means for concentrations of Hb, SF, sTfR, and MCV between the two visits were examined using paired t tests. SF and sTfR were log transformed before analysis to improve the normality of model residuals. Differences in the prevalence of anemia, iron depletion, and tissue iron deficiency between the visits were tested using McNemar’s test for paired binary data. Random-effects models were used for unadjusted models and randomcoefficient models were used for adjusted models of maternal Hb, SF, and sTfR to account for differences in the timing of visits (i.e., first visit: 13–22 wk gestation and second visit: 33–37 wk) with gestation week included as a continuous variable and with a random intercept for participants. Exponential spatial covariance patterns were investigated in adjusted models to see whether they improved model fit as measured by the Akaike Information criterion. Denominator degrees of freedom were estimated using the Kenward-Rogers approach in all such models. Fractional polynomial transformations were applied to continuous predictor variables to test for and when appropriate model nonlinear associations. Predictors with relevance to maternal Hb, SF, and sTfR such as maternal age, parity (i.e., no child, one child versus two or more children), use of iron supplements (i.e., 7 d versus >7 d in the past month), maternal education (i.e., 8 y versus >8 y), annual household income, location (i.e., Hiwra Baazar, Karwahi versus Bhandarbodi), weight gain during pregnancy, food habits (i.e., vegetarian versus non-vegetarian), breastfeeding at first visit (i.e., breastfeeding versus non-breastfeeding), family size (i.e., five or fewer members versus six or more members), use of pan (i.e., users versus non-users), chewers of tobacco (i.e., chewers versus non-chewers), and duration of gestation [21,27] were tested initially using unadjusted univariate analyses (with a random participant effect to account for clustering) and associations with P < 0.20 were included in the adjusted models. The interactions between categorical predictors and duration of gestation were investigated in the final adjusted model for each outcome and included where P < 0.05. For six participants it was necessary to adjust for errors in self-reported last menstrual period to match a biologically possible time frame for length of pregnancy. Gestation week was adjusted by 1 mo backward for participants with unadjusted pregnancy duration 44 but 10 mg/L).
Results In all, 228 pregnant women were recruited into the study, 5 dropped out and 12 were excluded: 1 was HIV positive, 2 were expecting twins, and 9 had the sickle cell trait, thus baseline characteristics are shown for 211 women. Of these, 34 women were excluded after the second visit (i.e., 1 refused to give a blood sample; 4 suffered spontaneous abortions, 2 underwent medical
293
Table 1 Baseline characteristics of pregnant tribal women of Ramtek, Nagpur Variables
n
%
Gestation weeks at recruitment* Age (y) Anthropometry* Height (cm) Weight (kg) BMI (kg/m2) Annual household income (1000 INR)y Participant’s education 8 y Number of family members 5 members Vegetarian Parity No children One child Breastfeeding Pan At first visit At second visit Tobacco At first visit At second Iron supplements (>7 d in the past month) At first visit At second visit Elemental iron intake from supplementsz At first visit At second visit Weight gain (kg)y
211 211
17.5 (1.9) 23.0 (2.7)
211 211 211 210
152.3 (5.1) 44.9 (5.3) 19.3 (1.9) 30.5 (27.434.2)
104
49
142 56
67 27
101 80 38
48 38 18
36 4
17 2
29 33
14 19
130 126
62 71
130 126 175
70 (20157) 11 (37200) 4.2 (3.94.6)
BMI, body mass index; CI, confidence interval; INR, Indian rupees * Mean (SD). y Geometric mean (CI). z Median (25th, 75th percentiles).
termination of pregnancy, 2 had stillbirths, 9 women were lost to follow-up, and 16 delivered before the second visit), resulting in 177 eligible women (78%) who completed the study. Additionally, 16 women with a CRP concentration >10 mg/L were excluded for the determination of Hb and SF concentrations and for prevalence estimates for anemia and IDA (but were included in regression models). The mean (SD) unadjusted self-reported gestational age of women at the first visit was 17.5 (1.9) wk and at the second visit was 34.5 (0.5) wk (Table 1). The baseline characteristics showed that the majority of study participants were short and thin, were not breastfeeding, nulliparous, or primiparous and had low-level education. Weight gain at the time of the second visit (i.e., at approximately gestation week 35) ranged between 0.6 and 13.1 kg, with a geometrical mean (95% confidence interval) of 4.2 (3.9–4.6) kg. The PHCs freely distributed albandazole tablets twice to deworm all the pregnant women in this area. The majority of women reported taking an iron supplement > 7 d/mo (i.e., frequently) (Table 1), increasing from 62% at the first visit to 71% at the second visit (P ¼ 0.099). The estimated median (25th and 75th percentiles) elemental iron intake from supplements at the first and second visits was 70 mg/d (20 and 157 mg/ d) and 118 (37 and 200 mg/d), respectively. Self-reported adverse events (e.g., nausea, vomiting, constipation, and diarrhea) were reported in 15% of participants at the first visit and 32% of participants at the second visit. The government-sponsored supplement program was their primary source of iron supplements. Of the women who completed the study, 4.5% (n ¼ 8) had an unsuccessful pregnancy. The mean Hb concentration of the participants was 106 g/L (Table 2). The percentage of women with anemia increased significantly from 41% at the first visit to 55% at the second visit
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Table 2 Iron status of pregnant of Ramtek, Nagpur Variable
n
First visit
n
Second visit
P*,y
195
106 (15) 41
161
106 (14) 55
0.518 85 fl
CRP, C-reactive protein; Hb, hemoglobin; IQR, interquartile range; MCV, mean corpuscular volume; SF, serum ferritin; sTfR, serum transferrin receptor * P-value for difference in means from paired t test. y P-value for difference in proportion from McNemar’s test. z Women with CRP > 10 mg/L (n ¼ 16) both at first visit and at second visit excluded from analysis. x Hb < 105 g/L in second trimester; Hb < 110 g/L in third trimester [2]. k WHO cut-off for depleted iron stores [13]. { sTfR kit specific cut-off from Biovendor. # MCV cut-off to diagnose macrocytic anemia in non-pregnant women [21].
(Table 2). The percentage of women with moderate and severe anemia was 25% and 3% at the first visit and 28% and 1% at the second visit, respectively. Less than 5% of women had abnormal SF or sTfR concentrations during either visit (Table 2). The percentage of women with MCV > 85 fL significantly increased from 9% at first visit to 13.6% at second visit (P ¼ 0.020). Suboptimal iron status, when defined using a multiparametric index, was less than 10% at both visits (Table 3). The majority of anemic women had normal SF and sTfR concentrations (Table 3). In unadjusted analysis of maternal Hb concentration, only maternal age (P ¼ 0.159), breastfeeding at the first visit (P ¼ 0.137), parity (P ¼ 0.167), and use of iron supplements (P ¼ 0.017) had a P < 0.20 and so were included in the adjusted model. In unadjusted analysis of maternal SF concentration, duration of gestation (P ¼ 0.069), breastfeeding at the first visit (P ¼ 0.079), use of iron supplements (P ¼ 0.156), family size (P ¼ 0.118), and use of pan (P ¼ 0.140) had a P < 0.20. Likewise, unadjusted analysis of factors associated with maternal sTfR concentration found that only duration of gestation (P ¼ 0.001), breastfeeding at the first visit (P ¼ 0.032), and chewing tobacco during gestation (P ¼ 0.143) had a P < 0.20. These factors were included in adjusted analysis of Hb, SF, and sTfR concentration. Adjusted models showed that use of iron supplements >7 d in the past
month was associated with maternal Hb, SF, and sTfR concentration, and duration of gestation was associated with maternal SF and sTfR concentrations (Table 4). Maternal Hb concentration was 3.23 g/L higher (95% confidence interval [CI], 0.55–5.92; P ¼ 0.018), maternal sTfR was 13% lower (95% CI, 4%–22%; P ¼ 0.008), and SF was 6% higher (95% CI, 1%–14%; P ¼ 0.077) for women who used iron supplements for >7 d in the past month compared with those who used them 7 d (Table 4). Other factors associated with biochemical indices of iron status were duration of gestation (SF and sTfR) and lactational status (SF) (Table 4). SF concentration increased marginally (i.e., 0.2%; 95% CI, 0.03%–0.7%) per week as gestation advanced (Table 4). The SF concentration of women who were breastfeeding at the first visit was 11% higher (95% CI, 2%–20%; P ¼ 0.013) than women who were not breastfeeding (Table 4). Discussion This is the first study to evaluate the iron status of pregnant Indian tribal women using a combination of indicators (i.e., Hb, SF, sTfR) as recommended by WHO/Centers for Disease Control and Prevention (CDC) [2] in an area with an active iron supplementation program for pregnant women. In contrast to other studies of pregnant Indian women, the women in this study did not appear to be iron deficient. We suggest that the use of ironcontaining supplements had a positive effect on their iron status because their dietary intake of bioavailable iron was likely to be low and 72% of non-pregnant tribal women from this area were anemic [16]. The prevalence of anemia among these pregnant women (i.e., 41% and 55% at first and second visit, respectively) was lower than the national prevalence estimate of 59% [5] and other studies from India at any time during pregnancy [6–8,26]. Similarly, the prevalence of moderate (i.e., 4.4 mg/L)z Iron sufficient (Non-anemic; IS) % Normal Hb þ SF > 15 mg/L þ sTRf < 4.4 mg/Ly Noniron-deficiency anemia (NIDA) % Subnormal Hb þ SF > 15 mg/L þ sTRf < 4.4 mg/Lz
First visit (n ¼ 195)
Second visit (n ¼ 161)
P
0
3.7 (0.76.7)
–
0
2.5 (0.04.9)
–
3.6 (0.96.2)
5.6 (1.99.1)
0.059
59.0 (52.065.9)
42.2 (34.549.9)
10 mg/L at first and second visits excluded from Hb and SF analysis (n ¼ 195 at first visit; n ¼ 161 at second visit). y WHO [13]; sTfR kit specific cut-off from Biovendor. z Subnormal Hb concentration, Hb concentration < 110 gL in the first and third trimesters and < 105 g/L in the second trimester (CDC, [2]).
K. C. Menon et al. / Nutrition 30 (2014) 291–296 Table 4 Factors associated with maternal hemoglobin, serum ferritin, and serum transferrin receptor concentrations of pregnant tribal women of Ramtek, Nagpur, India* Factor Hemoglobin (g/L)y Iron supplements (> 7 d in the last month) Serum ferritin (mg/L)z,x,k Iron supplements (> 7 d in the last month) Duration of gestation (wk){ Not breastfeeding at first visit Serum transferrin receptor (mg/L)z,# Iron supplements (> 7 d in the last month) Duration of gestation (wk){
Ratio (CI)
P
3.23 (0.551.14)
0.018
1.06 (0.991.14) 1.00 (1.001.01) 0.89 (0.800.98)
0.077 0.033 0.013
0.87 (0.780.96) 1.01 (1.001.01)
0.008 0.001
CI, confidence interval * N ¼ 356 (n ¼ 195 at first visit; n ¼ 161 at second visit). y b-coefficient (95% CI) from mixed model analysis; adjusted for maternal age, duration of gestation, breastfeeding at first visit, parity, and weight gain. z Ratio of geometric means (95% CI) from mixed model analysis. x One unusual serum ferritin value excluded from analysis (n ¼ 355). k Adjusted for use of pan during gestation and family size. { Gestation week adjusted by 1 mo backwards for participants with >44 wk to 46 wk by 2 mo for >46 wk gestation estimated based on their self-reported last menstrual period (n ¼ 6). # Adjusted for chewing tobacco during gestation.
pregnancy, the use of iron supplements for >7 d in the past month predicted a 3.23 g/L higher Hb and 13% lower sTfR concentrations in these pregnant women. The absence of a significant relationship between SF and iron supplement intake may have been due to the confounding effect of infection although these women had a serum CRP concentration < 10 mg/L. CRP is a less-sensitive index of infection than a-glycoprotein, however, we could not measure a-glycoprotein as laboratory facilities for this analysis were unavailable in the study area. Our results contrast a previous study [29] that showed no change in mean Hb, SF, and sTfR concentrations with 60 mg of daily iron–folic acid supplement for 100 d in pregnant Indian women from Hyderabad; an increase in Hb was observed only in anemic pregnant women from Hyderabad. The differences between the two studies could be attributed to the following factors: Our participants had a lower Hb status at recruitment than the pregnant women from Hyderabad and might have responded better to iron supplementation; women with severe anemia were excluded from the Hyderabad study; more women in our study were consuming a variety of iron-containing supplements that had other micronutrients in them; a regular deworming of our participants was practiced but data on deworming was not reported in the Hyderabad study; and our participants were already consuming iron-containing supplements at the time of recruitment, whereas the women from Hyderabad started their supplements only after their recruitment at the 19th wk of gestation. We suggest that the iron from supplements given to women as part of the iron–folic acid supplementation program in India was used primarily to maintain the functional iron (i.e., Hb) concentration throughout gestation rather than to improve the storage iron (i.e., SF). In a recent Cochrane review of 18 trials involving 4072 pregnant women, it was concluded that intermittent consumption of iron and folic acid supplements during pregnancy produced similar effects as daily iron–folic acid supplementation [20]. The high elemental iron intake from supplements (i.e., median iron intake of 70 and 118 mg/d at first and second visits) taken by women in this study from the early stages of gestation also may have contributed to the improved iron status in these pregnant women compared with other Indian studies.
295
Tribal pregnant women from Ramtek have a high prevalence of anemia, despite their good iron status. Factors known to cause anemia include infection, hemoglobinopathies, and other micronutrient deficiencies [21]. The pregnant women in Ramtek were provided with two Albandazol doses during their pregnancy to prevent parasitic and protozoic infections. Only nine women were positive for the sickle cell trait and were excluded from the analysis; the presence of other hemoglobinopathies is uncommon in this area (personal communication, Dr. PK Das). In a case–control study, the iron, zinc, vitamin B12, copper, vitamin A, riboflavin, and folic acid status in 183 adults from Western India was evaluated [30]. The evaluation found that the odds of having a low Hb concentration was 7.2 and 1.1 times greater in subjects with riboflavin and copper deficiency, respectively, and there was a significant difference in vitamin B12 concentration between anemic and non-anemic individuals. In our earlier study conducted in the same area of India, non-pregnant women had a high prevalence of vitamin B12 deficiency (i.e., 34%). Thus, it is possible that vitamin B12 deficiency might have contributed to the high prevalence of anemia in pregnant women from this region of India. Additionally, the micronutrient status of vitamin A, folic acid, riboflavin, and vitamin B6 in pregnant women in the current study was unknown. Although breastfeeding did not affect Hb and sTfR concentrations, breastfeeding at first visit was significantly associated with SF concentrations of these women. Mothers who nursed their babies had 11% higher SF concentration than nonbreastfeeding mothers. A number of reasons might explain this apparent beneficial effect of breastfeeding. Lactational amenorrhea conserves iron loss, which could improve iron stores. Of more importance, however, is that as part of the Indian nutritional anemia prophylaxis program, both pregnant and lactating women receive iron–folic acid supplements, so lactating pregnant women in our study might have been taking iron supplements for a longer period of time (i.e., during the previous pregnancy followed by the period of lactation) than pregnant women who were not breastfeeding. Other studies have shown that iron–folic acid supplementation during pregnancy increased SF at term in pregnant Indian and Pakistani women [31]. The results of this study suggest that the use of iron supplements by women in this region is both widespread and effective in meeting the high iron requirements of pregnancy. More than 60% of women took a variety of iron supplements for >7 d/mo as a part of their antenatal care program. Often these women also consumed the supplements that contained other hemopoeitic micronutrients (such as vitamin A, some B vitamins (but not vitamin B12), and vitamin C) that may have increased the bioavailability of supplemental iron. Although the use of iron supplements was intermittent, primarily because women experienced side effects such as constipation, nausea, and vomiting, the maternal indicators of iron status suggested that the quantity of iron obtained from supplements was able to meet gestational iron requirements. Furthermore, the rate of iron absorption in pregnancy increases, especially when women are iron deficient and anemic before pregnancy. Our findings support a previous study [20] that showed that intermittent iron supplementation (i.e., any dose taken less frequently than daily) of pregnant women improves iron status. Without supplementation, good iron status in Indian pregnant women would be difficult to achieve as the typical Indian diet meets less than 50% of recommended dietary intake for iron and this dietary iron, predominantly from non-heme sources, has a low bioavailability [9]. In conclusion, although these pregnant women of central India were anemic, they were not iron deficient. Ideally, the iron
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status of pregnant women should be evaluated using a combination of Hb and SF rather than using anemia alone, because this combination of indices is specific to iron status. To our knowledge, this is the first study to demonstrate that pregnant Indian women have relatively good iron status as indicated by a combination of indices suggested by WHO/CDC [2]. Although the use of iron supplements was intermittent, the maternal indicators of iron status suggested that the quantity of iron obtained from supplements might have helped these women meet their gestational iron requirements. There is little doubt that many Indian women have IDA and will benefit from routine iron supplementation as outlined by the WHO [32]. The present study suggests that there is a need for further longitudinal studies to evaluate the current policy of daily iron–folic acid supplementation of all pregnant Indian women to assess the effect of such supplementation on pregnancy and infant outcomes. Acknowledgments The Department of Human Nutrition’s Performance Based Research Fund and a University of Otago Research Grant supported the study. The authors acknowledge the women and their families who participated and supported this study; our dedicated team of research assistants; Ramtek block Primary Health Centre Medical Officers and staff; and ICDS supervisors and staff. References [1] deBenoist B, McLean E, Egli I, Cogswell M. Worldwide prevalence of anaemia 1993–2005. Geneva, Switzerland: WHO (World Health Organization); 2008. [2] WHO/CDC (World Health Organization/Centers for Disease Control and Prevention). Assessment of iron status at the population level: report of a joint WHO/CDC technical consultation. Geneva, Switzerland: WHO; 2005. [3] Brabin BJ, Hakimi M, Pelletier D. An analysis of anemia and pregnancyrelated maternal mortality. J Nutr 2001;131:604S–14S. [4] Stoltzfus R, Barbour L, Black R. Iron deficiency anaemia. Comparative quantification of health risks: global and regional burden of disease attributable to selected major risk factors. Geneva, Switzerland: WHO; 2004. [5] IIPS (International Institute for Population Sciences). National Health and Family Survey India-3 (NFHS-3). Mumbai, India: IIPS; 2006. [6] Samuel TM, Thomas T, Finkelstein J, Bosch R, Rajendran R, Virtanen SM, et al. Correlates of anaemia in pregnant urban South Indian women: a possible role of dietary intake of nutrients that inhibit iron absorption. Pub Health Nutr; 2012:1–9. [7] Singh MB, Fotedar R, Lakshminarayana J. Micronutrient deficiency status among women of desert areas of western Rajasthan, India. Public Health Nutr 2009;12:624–9. [8] Pathak P, Kapil U, Yajnik CS, Kapoor SK, Dwivedi SN, Singh R. Iron, folate, and vitamin B12 stores among pregnant women in a rural area of Haryana State, India. Food Nutr Bull 2007;28:435–8. [9] Nair KM, Iyengar V. Iron content, bioavailability & factors affecting iron status of Indians. Indian J Med Res 2009;130:634–45.
[10] Sharma A, Patnaik R, Garg S. Detection & management of anaemia in pregnancy in an urban primary health care institution. Indian J Med Res 2008;128:45–51. [11] Agarwal KN, Agarwal DK, Sharma A, Sharma K, Prasad K, Kalita MC, et al. Prevalence of anaemia in pregnant & lactating women in India. Indian J Med Res 2006;124:173–84. [12] Toteja GS, Singh P, Dhillon BS, Saxena BN, Ahmed FU, Singh RP, et al. Prevalence of anemia among pregnant women and adolescent girls in 16 districts of India. Food Nutr Bull 2006;27:311–5. [13] WHO/UNICEF/UNU (World Health Organization/ United Nationas Childrens Fund/ United Nations University). Iron deficiency anaemia, assessment, prevention and control: a guide for programme managers. Geneva, Switzerland: WHO/UNICEF/UNU; 2001. [14] Cook JD. Diagnosis and management of iron-deficiency anaemia. Best Pract Res Clin Haematol 2005;18:319–32. [15] Skikne BS. Serum transferrin receptor. Am J Hematol 2008;83:872–5. [16] Menon KC, Skeaff SA, Thomson CD, Gray AR, Ferguson EL, Zodpey S, et al. Concurrent micronutrient deficiencies are prevalent in nonpregnant rural and tribal women from central India. Nutrition 2011;27:496–502. [17] Allen LH. Anemia and iron deficiency: effects on pregnancy outcome. Am J Clin Nutr 2000;71:1280S–4S. [18] WHO (World Health Organization). Nutritional anaemias. Report of a WHO scientific group. Geneva, Switzerland: WHO; 1968. [19] Menon KC, Skeaff SA, Thomson CD, Gray AR, Ferguson EL, Zodpey S, et al. The effect of maternal iodine status on infant outcomes in an iodinedeficient Indian population. Thyroid 2011;21:1373–80. ~ a-Rosas JP, De-Regil LM, Dowswell T, Viteri FE. Intermittent oral iron [20] Pen supplementation during pregnancy. Cochrane Database Syst Rev 2012;7:CD009997. [21] Gibson R. Principles of nutritional assessment. New York: Oxford University Press; 2005. [22] McPherson R, Pincus M, Henry JB. Henry’s clinical diagnosis and management by laboratory methods. Saunders Elsevier; 2007. [23] CDC (Centers for Disease Control and Prevention). Recommendations to prevent and control iron deficiency in the United States. Centers for Disease Control and Prevention. MMWR Recomm Rep 1998;47:1–29. [24] WHO (World Health Organization). Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. Vitamin and mineral nutrition information system. Geneva, Switzerland: WHO; 2011. [25] WHO (World Health Organization). Report of the African Regional Consultation on control of anaemia in pregnancy. Geneva, Switzerland: WHO; 1989. [26] Picklesimer AH, Jared HL, Moss K, Offenbacher S, Beck JD, Boggess KA. Racial differences in C-reactive protein levels during normal pregnancy. Am J Obstet Gynecol 2008;199:1–6. [27] Subramoney S, Gupta PC. Anemia in pregnant women who use smokeless tobacco. Nicotine Tob Res 2008;10:917–20. [28] Rusia U, Flowers C, Madan N, Agarwal N, Sood SK, Sikka M. Serum transferrin receptors in detection of iron deficiency in pregnancy. Ann Hematol 1999;78:358–63. [29] Madhavan Nair K, Bhaskaram P, Balakrishna N, Ravinder P, Sesikeran B. Response of hemoglobin, serum ferritin, and serum transferrin receptor during iron supplementation in pregnancy: a prospective study. Nutrition 2004;20:896–9. [30] Chiplonkar A. Relative importance of micronutrient deficiencies in iron deficiency anemia. Nutr Res 2003;23:1355–67. [31] Bhutta ZA, Rizvi A, Raza F, Hotwani S, Zaidi S, Moazzam Hossain S, et al. A comparative evaluation of multiple micronutrient and iron-folic acid supplementation during pregnancy in Pakistan: impact on pregnancy outcomes. Food Nutr Bull 2009;30:S496–505. [32] WHO (World Health Organization). Guideline: daily iron and folic acid supplementation in pregnant women. Geneva, Switzerland: WHO; 2012.
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Applied nutritional investigation
Iron status of pregnant Indian women from an area of active iron supplementation Kavitha C. Menon Ph.D. a, b, Elaine L. Ferguson Ph.D. c, Christine D. Thomson Ph.D. a, Andrew R. Gray B.Com(Hons). d, Sanjay Zodpey Ph.D. e, Abhay Saraf M.D. e, Prabir Kumar Das M.D. f, Chandrakant S. Pandav M.D. g, Sheila A. Skeaff Ph.D. a, * a
Department of Human Nutrition, University of Otago, Dunedin, New Zealand Healthy Lifestyle Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Penang, Malaysia London School of Hygiene and Tropical Medicine, London, England d Department of Preventive and Social Medicine, University of Otago, Dunedin, New Zealand e Public Health Foundation of India, New Delhi, India f Health and Family Welfare Training Institute, Nagpur, India g All India Institute of Medical Sciences, New Delhi, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2013 Accepted 19 August 2013
Objective: The aim of this study was to investigate the iron status of pregnant tribal women from Ramtek, Nagpur, Maharashtra, India using a combination of indices. Methods: A community-based observational study was conducted to assess iron status using a convenience sample of pregnant Indian tribal women from Ramtek. Pregnant women were recruited at 13 to 22 wk gestation (first visit; n ¼ 211) and followed to 29 to 42 wk gestation (second visit; n ¼ 177) of pregnancy. Sociodemographic and anthropometric data; iron supplement intake; and blood samples for estimating hemoglobin (Hb), serum ferritin (SF), soluble transferrin receptor (sTfR), and C-reactive protein (CRP) were obtained. Results: The mean (SD) Hb concentration at recruitment was 106 (15) g/L and 106 (14) g/L at the second visit; 41% of the women at recruitment and 55% at second visit were anemic (14% higher, P < 0.001). No women at recruitment and 3.7% at second visit had SF concentration < 15 ng/mL; and 3.3% at recruitment and 3.9% at the second visit had sTfR > 4.4 ng/mL (0.6% higher, P ¼ 0.179). Almost 62% and 71% of pregnant women used iron supplements at both visits, respectively. Iron supplement intake > 7 d in the preceding month improved the Hb concentration by 3.23 g/L and reduced sTfR concentration by 13%; women who were breastfeeding at the time of recruitment had 11% higher SF concentration. Conclusions: The iron indices suggest that pregnant tribal women of central India, although anemic, had good iron status. Use of iron supplements > 7 d in the preceding month improved iron status; however, non–iron-deficiency anemia persisted in this group. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Anemia Iron deficiency anemia Iron status Pregnant women Dietary supplements
Introduction KCM conducted the research, analyzed the data and, along with SAS prepared the manuscript. SAS, ELF, and CDT designed and supervised the study, secured funding, and assisted in the interpretation of the data. ARG together with KCM conducted the statistical analysis. SZ, AS, and PKD helped with obtaining ethical approval and liaised with district health professionals in Nagpur. CSP supported this research in his capacity as regional director of ICCIDD (Southeast Asia). All authors commented on the final draft of the manuscript. The authors declare no conflicts of interest. * Corresponding author. Tel.: þ643 479 7944; fax:þ643 479 7958. E-mail address: [email protected] (S. A. Skeaff). 0899-9007/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nut.2013.08.015
According to the World Health Organization (WHO) 41.8% of pregnant women worldwide have anemia and about half of these women have iron-deficiency anemia (IDA) [1]. IDA continues to be a major public health challenge globally and is one of the most common nutritional disorders in pregnant women as iron requirements increase to meet the higher maternal–fetal demands [2]. Populations at highest risk for IDA during pregnancy are those with inadequate pre-pregnancy iron stores coupled with
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low dietary intakes of bioavailable iron. IDA during pregnancy also may have adverse effects on obstetric outcomes, making it a critical public health concern [3,4]. Research has shown that anemia during pregnancy is common in India. The 2006 national prevalence rate of anemia was 59% [5], with regional prevalence estimates between 30% and 71% [6,7]. Anemia is predominantly due to iron deficiency [8]; however, it may be attributed to concurrent micronutrient deficiencies of vitamin B12, folic acid, vitamin B6, or vitamin A, or high rates of infection, malaria, or hemoglobinopathies, all common in low-income countries [9]. To address IDA during pregnancy, the Indian government has implemented a free iron–folic acid supplementation program for all pregnant women. Despite this initiative, IDA in pregnancy remains a major public health challenge in India [7,10]. Many studies that have assessed the prevalence of anemia during pregnancy in India use hemoglobin (Hb) as the only measure [10–12], whereas a combination of indices is recommended to more accurately evaluate IDA. A combination of indices such as Hb, serum ferritin (SF), and soluble transferrin receptors (sTfR) along with a biomarker for infection, provides higher sensitivity and specificity to identify IDA than Hb alone [2]. Furthermore, to assess the iron status of pregnant women, the use of sTfR with other laboratory indices for iron depletion (ID) is a reliable method [2,13] because sTfR concentrations are unaltered during pregnancy, unaffected by infections, and their up-regulation is a sensitive index of stage II ID [14,15]. Despite having an active infant–maternal health care program in place, a recent study showed that 72% of young, nonpregnant tribal women living in central India were anemic based on their Hb concentration alone [16]. These results suggest that pregnant women in this region are likely to be at high risk for IDA and its associated negative effects on obstetric outcomes such as low birth weight, premature birth, and infant and maternal mortality [17,18]. Although there is abundant information on the prevalence of anemia in pregnant Indian women, there is a dearth of information on the prevalence of IDA in this group, especially from areas where an active iron–folic acid supplementation program is in place. To address this critical public health issue the present study aimed to (1) assess the prevalence of IDA and iron deficiency without anemia among pregnant tribal Indian women living in central India during the second and third trimesters of pregnancy; and (2) assess factors associated with indicators of maternal iron status during pregnancy. This study is part of a larger study, which also investigated the iodine status of tribal women during the second and third trimesters of pregnancy [19].
lost to follow-up, we needed to initially screen 225 pregnant women and enroll 191 into the study. A fasting blood sample, anthropometric measurements, and a general sociodemographic health questionnaire were collected at 13 to 22 wk gestation (first visit) and 29 to 42 wk gestation (second visit) of pregnancy. Ethical approval was obtained from the Ethics Committee of the Health and Family Welfare Training Centre, Nagpur, India. Participants gave informed written and verbal consent. Pregnant women who participated in this study belonged to the Gond tribe, the second largest tribe in India, which has its own language and culture. In the past 2 decades, members of the tribe have been resettled and subsequently integrated into the rural community of Ramtek where they earn their livelihood primarily from agriculture. Between February and May 2008, all women in the catchment areas of the three PHCs who were between 13 and 22 wk gestation and met the inclusion criteria were invited to take part in the study. The inclusion criteria for participation were healthy pregnant women ages between 18 and 30 y, who were between 13 and 22 wk gestation based on the self-reported date of their last menstrual period, were willing to participate in the study, and stated they would live in the research area throughout pregnancy and up to 1-mo postpartum. The exclusion criteria were non-pregnant women and women with self-reported HIV/AIDS, tuberculosis, fever lasting longer than 2 wk, or thyroid disease. A qualified medical practitioner examined the pregnant women for overt health issues before their recruitment into the study. Socioeconomic, health status, obstetric questionnaire, iron supplement intake, and anthropometry Trained Indian research assistants administered questionnaires to elicit information on socioeconomic and health status, including maternal age, number of years of completed schooling, annual household income, number of family members, dietary practices (i.e., vegetarian), parity, breastfeeding at the time of the first visit, use of dietary and medicinal supplements and health drinks, and lifestyle habits such as chewing pan (i.e., beetle leaf with lime and areca nut) with or without tobacco. Information on iron-containing supplement use for the previous month was collected at both visits via the questionnaire and for the previous day via a 24-h dietary recall (dietary data are not included in this article). The elemental iron content of supplements was obtained from the information provided on the labels or information by the manufacturer of the supplement. The daily elemental iron intake from supplements of the participant over the past month at both visits (i.e., medicinal and dietary supplements) was determined by summing the estimated elemental iron intake/d for each supplement over the previous month. Using the questionnaire data, women were categorized into one of two groups based on their supplement use; women who took supplements for >7 d in the previous month (>7 d) were classified as frequent users, whereas those who took supplements for < 7 d during the previous month (7 d) were classified as lessfrequent users. The classification was undertaken based on evidence that intermittent intake (one, two, or three times/wk) of iron-containing supplements improves iron status of pregnant women [20]. Supplement use was confirmed by examining the women’s 24-h recall. When the supplement intake in the questionnaire data disagreed with the 24-h recall data, the categorization was based on the questionnaire data (i.e., supplement intake over the past month for > 7 d/mo). Serial measurements of maternal weight using a digital flat scale (Seca Robusta 813, Seca corporation, Hamburg, Germany), and height using a portable stadiometer built by the University of Otago were taken at both visits by one trained research assistant [21]. Weight gain during pregnancy was calculated as the difference in maternal weight between the weights at the first visit and the second visit. Biochemical assessment
Materials and methods Study design and participant recruitment A longitudinal observational study was conducted from February to December 2008 with 225 pregnant women living in the catchment areas of three tribal primary health centers (PHCs) in Ramtek Block, Maharashtra State, India. These PHCs were purposefully selected because they were the most disadvantaged tribal PHCs in Ramtek Block. The tribal PHC had an active iron–folic acid supplementation program in place for pregnant women. The characteristics of this program included free distribution of iron (100 mg)–folic acid (0.5 mg) supplements to all the pregnant women for at least 100 d. Additionally, women were provided with deworming tablets twice during their pregnancy and a variety of other dietary supplements (e.g., calcium, liquid protein supplements, B-complex vitamins). Based on a reported prevalence of anemia in non-pregnant women of 72% [16], 170 women would be needed to estimate the prevalence of anemia in pregnant women to within 10% of the true value with 95% confidence. Assuming 85% would meet the inclusion criteria and 10% would be
A morning fasting peripheral venous blood sample was collected into 4.5 mL vacutainer containing EDTA (Becton Dickinson, Franklin Lakes, NJ, USA) and 7 mL trace element-free vacutainer (Becton Dickinson, Franklin Lakes, NJ, USA). A complete blood cell count (CBC) and assays for C-reactive protein (CRP) and sickle cell disease were done on the same day of blood sample collection at the National Reference Pathological Laboratory in Nagpur using a Sysmex automated hematology analyzer KX-21 for CBC; the Metabisulphide Slide test for sickle cell anemia [22]; and turbidimetry using CRP Turbilatex reagents (Agappe Diagnostics, India) for CRP. Blood samples collected in the trace element-free vacutainers were cooled to approximately 4 C in an ice box immediately after collection and then transported to the Nagpur Reference Pathological Laboratory where they were centrifuged at 3500g for 10 min, aliquoted into cryogenic vials (Sarstedt AG & Co., Nümbrecht, Germany), stored at 20 C and then transported on dry ice to Molecular Diagnostics, Lucknow, India for analysis. Maternal blood samples were analyzed for SF and sTfR. SF was measured by enzyme-linked immunosorbent assay (ELISA) (Biotron Diagnostic Inc. Hemet, CA, USA), which had a detection limit of 5.0 mg/L. The mean (SD) SF concentrations of
K. C. Menon et al. / Nutrition 30 (2014) 291–296 the low, medium, and high external standards (Bio-Rad quality control standards, Bio-Rad, Hercules, CA, USA) were: 48 (1.6) mg/L (expected range: 28–65.5 mg/L); 115 (7.1) mg/L (expected range: 54–125 mg/L); and 281 (20.1) mg/L (expected range: 148–336 mg/L), respectively. The interassay coefficients of variability (CVs) were 3.4%, 6.2%, and 7.2% at the low, medium, and high SF concentrations, respectively (n ¼ 6). sTfR was estimated by ELISA (BioVendor Laboratory Medicine Inc., Czech Republic) with functional assay sensitivity of 0.1 mg/L. The mean (SD) sTfR concentrations of the low- and high-quality control standards from Biovendor were: 2.0 (0.2) mg/L (expected range: 1.7–2.6 mg/L); and 6.4 (0.4) mg/ L (expected range: 4.9–7.3 mg/L), respectively. The interassay CVs were 10.8% and 6.4% at low and high sTfR concentrations (n ¼ 5). Anemia was defined as Hb < 110 g/L, Hb < 105 g/L, and Hb < 110 g/L in the first, second, and third trimesters of gestation, respectively [23]. The severity of anemia was categorized based on Hb concentration as mild (>100 to 4.4 mg/L, in the presence of anemia (Hb concentration 85 fL was used to identify macrocytic anemia [21]. Microcytic anemia was identified when the red cell distribution width was > 14% [21]. A CRP value of >10 mg/L defined the presence of infection [26]. Statistical analysis Descriptive statistics were used to summarize baseline characteristics of the participants. Differences in means for concentrations of Hb, SF, sTfR, and MCV between the two visits were examined using paired t tests. SF and sTfR were log transformed before analysis to improve the normality of model residuals. Differences in the prevalence of anemia, iron depletion, and tissue iron deficiency between the visits were tested using McNemar’s test for paired binary data. Random-effects models were used for unadjusted models and randomcoefficient models were used for adjusted models of maternal Hb, SF, and sTfR to account for differences in the timing of visits (i.e., first visit: 13–22 wk gestation and second visit: 33–37 wk) with gestation week included as a continuous variable and with a random intercept for participants. Exponential spatial covariance patterns were investigated in adjusted models to see whether they improved model fit as measured by the Akaike Information criterion. Denominator degrees of freedom were estimated using the Kenward-Rogers approach in all such models. Fractional polynomial transformations were applied to continuous predictor variables to test for and when appropriate model nonlinear associations. Predictors with relevance to maternal Hb, SF, and sTfR such as maternal age, parity (i.e., no child, one child versus two or more children), use of iron supplements (i.e., 7 d versus >7 d in the past month), maternal education (i.e., 8 y versus >8 y), annual household income, location (i.e., Hiwra Baazar, Karwahi versus Bhandarbodi), weight gain during pregnancy, food habits (i.e., vegetarian versus non-vegetarian), breastfeeding at first visit (i.e., breastfeeding versus non-breastfeeding), family size (i.e., five or fewer members versus six or more members), use of pan (i.e., users versus non-users), chewers of tobacco (i.e., chewers versus non-chewers), and duration of gestation [21,27] were tested initially using unadjusted univariate analyses (with a random participant effect to account for clustering) and associations with P < 0.20 were included in the adjusted models. The interactions between categorical predictors and duration of gestation were investigated in the final adjusted model for each outcome and included where P < 0.05. For six participants it was necessary to adjust for errors in self-reported last menstrual period to match a biologically possible time frame for length of pregnancy. Gestation week was adjusted by 1 mo backward for participants with unadjusted pregnancy duration 44 but 10 mg/L).
Results In all, 228 pregnant women were recruited into the study, 5 dropped out and 12 were excluded: 1 was HIV positive, 2 were expecting twins, and 9 had the sickle cell trait, thus baseline characteristics are shown for 211 women. Of these, 34 women were excluded after the second visit (i.e., 1 refused to give a blood sample; 4 suffered spontaneous abortions, 2 underwent medical
293
Table 1 Baseline characteristics of pregnant tribal women of Ramtek, Nagpur Variables
n
%
Gestation weeks at recruitment* Age (y) Anthropometry* Height (cm) Weight (kg) BMI (kg/m2) Annual household income (1000 INR)y Participant’s education 8 y Number of family members 5 members Vegetarian Parity No children One child Breastfeeding Pan At first visit At second visit Tobacco At first visit At second Iron supplements (>7 d in the past month) At first visit At second visit Elemental iron intake from supplementsz At first visit At second visit Weight gain (kg)y
211 211
17.5 (1.9) 23.0 (2.7)
211 211 211 210
152.3 (5.1) 44.9 (5.3) 19.3 (1.9) 30.5 (27.434.2)
104
49
142 56
67 27
101 80 38
48 38 18
36 4
17 2
29 33
14 19
130 126
62 71
130 126 175
70 (20157) 11 (37200) 4.2 (3.94.6)
BMI, body mass index; CI, confidence interval; INR, Indian rupees * Mean (SD). y Geometric mean (CI). z Median (25th, 75th percentiles).
termination of pregnancy, 2 had stillbirths, 9 women were lost to follow-up, and 16 delivered before the second visit), resulting in 177 eligible women (78%) who completed the study. Additionally, 16 women with a CRP concentration >10 mg/L were excluded for the determination of Hb and SF concentrations and for prevalence estimates for anemia and IDA (but were included in regression models). The mean (SD) unadjusted self-reported gestational age of women at the first visit was 17.5 (1.9) wk and at the second visit was 34.5 (0.5) wk (Table 1). The baseline characteristics showed that the majority of study participants were short and thin, were not breastfeeding, nulliparous, or primiparous and had low-level education. Weight gain at the time of the second visit (i.e., at approximately gestation week 35) ranged between 0.6 and 13.1 kg, with a geometrical mean (95% confidence interval) of 4.2 (3.9–4.6) kg. The PHCs freely distributed albandazole tablets twice to deworm all the pregnant women in this area. The majority of women reported taking an iron supplement > 7 d/mo (i.e., frequently) (Table 1), increasing from 62% at the first visit to 71% at the second visit (P ¼ 0.099). The estimated median (25th and 75th percentiles) elemental iron intake from supplements at the first and second visits was 70 mg/d (20 and 157 mg/ d) and 118 (37 and 200 mg/d), respectively. Self-reported adverse events (e.g., nausea, vomiting, constipation, and diarrhea) were reported in 15% of participants at the first visit and 32% of participants at the second visit. The government-sponsored supplement program was their primary source of iron supplements. Of the women who completed the study, 4.5% (n ¼ 8) had an unsuccessful pregnancy. The mean Hb concentration of the participants was 106 g/L (Table 2). The percentage of women with anemia increased significantly from 41% at the first visit to 55% at the second visit
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Table 2 Iron status of pregnant of Ramtek, Nagpur Variable
n
First visit
n
Second visit
P*,y
195
106 (15) 41
161
106 (14) 55
0.518 85 fl
CRP, C-reactive protein; Hb, hemoglobin; IQR, interquartile range; MCV, mean corpuscular volume; SF, serum ferritin; sTfR, serum transferrin receptor * P-value for difference in means from paired t test. y P-value for difference in proportion from McNemar’s test. z Women with CRP > 10 mg/L (n ¼ 16) both at first visit and at second visit excluded from analysis. x Hb < 105 g/L in second trimester; Hb < 110 g/L in third trimester [2]. k WHO cut-off for depleted iron stores [13]. { sTfR kit specific cut-off from Biovendor. # MCV cut-off to diagnose macrocytic anemia in non-pregnant women [21].
(Table 2). The percentage of women with moderate and severe anemia was 25% and 3% at the first visit and 28% and 1% at the second visit, respectively. Less than 5% of women had abnormal SF or sTfR concentrations during either visit (Table 2). The percentage of women with MCV > 85 fL significantly increased from 9% at first visit to 13.6% at second visit (P ¼ 0.020). Suboptimal iron status, when defined using a multiparametric index, was less than 10% at both visits (Table 3). The majority of anemic women had normal SF and sTfR concentrations (Table 3). In unadjusted analysis of maternal Hb concentration, only maternal age (P ¼ 0.159), breastfeeding at the first visit (P ¼ 0.137), parity (P ¼ 0.167), and use of iron supplements (P ¼ 0.017) had a P < 0.20 and so were included in the adjusted model. In unadjusted analysis of maternal SF concentration, duration of gestation (P ¼ 0.069), breastfeeding at the first visit (P ¼ 0.079), use of iron supplements (P ¼ 0.156), family size (P ¼ 0.118), and use of pan (P ¼ 0.140) had a P < 0.20. Likewise, unadjusted analysis of factors associated with maternal sTfR concentration found that only duration of gestation (P ¼ 0.001), breastfeeding at the first visit (P ¼ 0.032), and chewing tobacco during gestation (P ¼ 0.143) had a P < 0.20. These factors were included in adjusted analysis of Hb, SF, and sTfR concentration. Adjusted models showed that use of iron supplements >7 d in the past
month was associated with maternal Hb, SF, and sTfR concentration, and duration of gestation was associated with maternal SF and sTfR concentrations (Table 4). Maternal Hb concentration was 3.23 g/L higher (95% confidence interval [CI], 0.55–5.92; P ¼ 0.018), maternal sTfR was 13% lower (95% CI, 4%–22%; P ¼ 0.008), and SF was 6% higher (95% CI, 1%–14%; P ¼ 0.077) for women who used iron supplements for >7 d in the past month compared with those who used them 7 d (Table 4). Other factors associated with biochemical indices of iron status were duration of gestation (SF and sTfR) and lactational status (SF) (Table 4). SF concentration increased marginally (i.e., 0.2%; 95% CI, 0.03%–0.7%) per week as gestation advanced (Table 4). The SF concentration of women who were breastfeeding at the first visit was 11% higher (95% CI, 2%–20%; P ¼ 0.013) than women who were not breastfeeding (Table 4). Discussion This is the first study to evaluate the iron status of pregnant Indian tribal women using a combination of indicators (i.e., Hb, SF, sTfR) as recommended by WHO/Centers for Disease Control and Prevention (CDC) [2] in an area with an active iron supplementation program for pregnant women. In contrast to other studies of pregnant Indian women, the women in this study did not appear to be iron deficient. We suggest that the use of ironcontaining supplements had a positive effect on their iron status because their dietary intake of bioavailable iron was likely to be low and 72% of non-pregnant tribal women from this area were anemic [16]. The prevalence of anemia among these pregnant women (i.e., 41% and 55% at first and second visit, respectively) was lower than the national prevalence estimate of 59% [5] and other studies from India at any time during pregnancy [6–8,26]. Similarly, the prevalence of moderate (i.e., 4.4 mg/L)z Iron sufficient (Non-anemic; IS) % Normal Hb þ SF > 15 mg/L þ sTRf < 4.4 mg/Ly Noniron-deficiency anemia (NIDA) % Subnormal Hb þ SF > 15 mg/L þ sTRf < 4.4 mg/Lz
First visit (n ¼ 195)
Second visit (n ¼ 161)
P
0
3.7 (0.76.7)
–
0
2.5 (0.04.9)
–
3.6 (0.96.2)
5.6 (1.99.1)
0.059
59.0 (52.065.9)
42.2 (34.549.9)
10 mg/L at first and second visits excluded from Hb and SF analysis (n ¼ 195 at first visit; n ¼ 161 at second visit). y WHO [13]; sTfR kit specific cut-off from Biovendor. z Subnormal Hb concentration, Hb concentration < 110 gL in the first and third trimesters and < 105 g/L in the second trimester (CDC, [2]).
K. C. Menon et al. / Nutrition 30 (2014) 291–296 Table 4 Factors associated with maternal hemoglobin, serum ferritin, and serum transferrin receptor concentrations of pregnant tribal women of Ramtek, Nagpur, India* Factor Hemoglobin (g/L)y Iron supplements (> 7 d in the last month) Serum ferritin (mg/L)z,x,k Iron supplements (> 7 d in the last month) Duration of gestation (wk){ Not breastfeeding at first visit Serum transferrin receptor (mg/L)z,# Iron supplements (> 7 d in the last month) Duration of gestation (wk){
Ratio (CI)
P
3.23 (0.551.14)
0.018
1.06 (0.991.14) 1.00 (1.001.01) 0.89 (0.800.98)
0.077 0.033 0.013
0.87 (0.780.96) 1.01 (1.001.01)
0.008 0.001
CI, confidence interval * N ¼ 356 (n ¼ 195 at first visit; n ¼ 161 at second visit). y b-coefficient (95% CI) from mixed model analysis; adjusted for maternal age, duration of gestation, breastfeeding at first visit, parity, and weight gain. z Ratio of geometric means (95% CI) from mixed model analysis. x One unusual serum ferritin value excluded from analysis (n ¼ 355). k Adjusted for use of pan during gestation and family size. { Gestation week adjusted by 1 mo backwards for participants with >44 wk to 46 wk by 2 mo for >46 wk gestation estimated based on their self-reported last menstrual period (n ¼ 6). # Adjusted for chewing tobacco during gestation.
pregnancy, the use of iron supplements for >7 d in the past month predicted a 3.23 g/L higher Hb and 13% lower sTfR concentrations in these pregnant women. The absence of a significant relationship between SF and iron supplement intake may have been due to the confounding effect of infection although these women had a serum CRP concentration < 10 mg/L. CRP is a less-sensitive index of infection than a-glycoprotein, however, we could not measure a-glycoprotein as laboratory facilities for this analysis were unavailable in the study area. Our results contrast a previous study [29] that showed no change in mean Hb, SF, and sTfR concentrations with 60 mg of daily iron–folic acid supplement for 100 d in pregnant Indian women from Hyderabad; an increase in Hb was observed only in anemic pregnant women from Hyderabad. The differences between the two studies could be attributed to the following factors: Our participants had a lower Hb status at recruitment than the pregnant women from Hyderabad and might have responded better to iron supplementation; women with severe anemia were excluded from the Hyderabad study; more women in our study were consuming a variety of iron-containing supplements that had other micronutrients in them; a regular deworming of our participants was practiced but data on deworming was not reported in the Hyderabad study; and our participants were already consuming iron-containing supplements at the time of recruitment, whereas the women from Hyderabad started their supplements only after their recruitment at the 19th wk of gestation. We suggest that the iron from supplements given to women as part of the iron–folic acid supplementation program in India was used primarily to maintain the functional iron (i.e., Hb) concentration throughout gestation rather than to improve the storage iron (i.e., SF). In a recent Cochrane review of 18 trials involving 4072 pregnant women, it was concluded that intermittent consumption of iron and folic acid supplements during pregnancy produced similar effects as daily iron–folic acid supplementation [20]. The high elemental iron intake from supplements (i.e., median iron intake of 70 and 118 mg/d at first and second visits) taken by women in this study from the early stages of gestation also may have contributed to the improved iron status in these pregnant women compared with other Indian studies.
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Tribal pregnant women from Ramtek have a high prevalence of anemia, despite their good iron status. Factors known to cause anemia include infection, hemoglobinopathies, and other micronutrient deficiencies [21]. The pregnant women in Ramtek were provided with two Albandazol doses during their pregnancy to prevent parasitic and protozoic infections. Only nine women were positive for the sickle cell trait and were excluded from the analysis; the presence of other hemoglobinopathies is uncommon in this area (personal communication, Dr. PK Das). In a case–control study, the iron, zinc, vitamin B12, copper, vitamin A, riboflavin, and folic acid status in 183 adults from Western India was evaluated [30]. The evaluation found that the odds of having a low Hb concentration was 7.2 and 1.1 times greater in subjects with riboflavin and copper deficiency, respectively, and there was a significant difference in vitamin B12 concentration between anemic and non-anemic individuals. In our earlier study conducted in the same area of India, non-pregnant women had a high prevalence of vitamin B12 deficiency (i.e., 34%). Thus, it is possible that vitamin B12 deficiency might have contributed to the high prevalence of anemia in pregnant women from this region of India. Additionally, the micronutrient status of vitamin A, folic acid, riboflavin, and vitamin B6 in pregnant women in the current study was unknown. Although breastfeeding did not affect Hb and sTfR concentrations, breastfeeding at first visit was significantly associated with SF concentrations of these women. Mothers who nursed their babies had 11% higher SF concentration than nonbreastfeeding mothers. A number of reasons might explain this apparent beneficial effect of breastfeeding. Lactational amenorrhea conserves iron loss, which could improve iron stores. Of more importance, however, is that as part of the Indian nutritional anemia prophylaxis program, both pregnant and lactating women receive iron–folic acid supplements, so lactating pregnant women in our study might have been taking iron supplements for a longer period of time (i.e., during the previous pregnancy followed by the period of lactation) than pregnant women who were not breastfeeding. Other studies have shown that iron–folic acid supplementation during pregnancy increased SF at term in pregnant Indian and Pakistani women [31]. The results of this study suggest that the use of iron supplements by women in this region is both widespread and effective in meeting the high iron requirements of pregnancy. More than 60% of women took a variety of iron supplements for >7 d/mo as a part of their antenatal care program. Often these women also consumed the supplements that contained other hemopoeitic micronutrients (such as vitamin A, some B vitamins (but not vitamin B12), and vitamin C) that may have increased the bioavailability of supplemental iron. Although the use of iron supplements was intermittent, primarily because women experienced side effects such as constipation, nausea, and vomiting, the maternal indicators of iron status suggested that the quantity of iron obtained from supplements was able to meet gestational iron requirements. Furthermore, the rate of iron absorption in pregnancy increases, especially when women are iron deficient and anemic before pregnancy. Our findings support a previous study [20] that showed that intermittent iron supplementation (i.e., any dose taken less frequently than daily) of pregnant women improves iron status. Without supplementation, good iron status in Indian pregnant women would be difficult to achieve as the typical Indian diet meets less than 50% of recommended dietary intake for iron and this dietary iron, predominantly from non-heme sources, has a low bioavailability [9]. In conclusion, although these pregnant women of central India were anemic, they were not iron deficient. Ideally, the iron
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status of pregnant women should be evaluated using a combination of Hb and SF rather than using anemia alone, because this combination of indices is specific to iron status. To our knowledge, this is the first study to demonstrate that pregnant Indian women have relatively good iron status as indicated by a combination of indices suggested by WHO/CDC [2]. Although the use of iron supplements was intermittent, the maternal indicators of iron status suggested that the quantity of iron obtained from supplements might have helped these women meet their gestational iron requirements. There is little doubt that many Indian women have IDA and will benefit from routine iron supplementation as outlined by the WHO [32]. The present study suggests that there is a need for further longitudinal studies to evaluate the current policy of daily iron–folic acid supplementation of all pregnant Indian women to assess the effect of such supplementation on pregnancy and infant outcomes. Acknowledgments The Department of Human Nutrition’s Performance Based Research Fund and a University of Otago Research Grant supported the study. The authors acknowledge the women and their families who participated and supported this study; our dedicated team of research assistants; Ramtek block Primary Health Centre Medical Officers and staff; and ICDS supervisors and staff. References [1] deBenoist B, McLean E, Egli I, Cogswell M. Worldwide prevalence of anaemia 1993–2005. Geneva, Switzerland: WHO (World Health Organization); 2008. [2] WHO/CDC (World Health Organization/Centers for Disease Control and Prevention). Assessment of iron status at the population level: report of a joint WHO/CDC technical consultation. Geneva, Switzerland: WHO; 2005. [3] Brabin BJ, Hakimi M, Pelletier D. An analysis of anemia and pregnancyrelated maternal mortality. J Nutr 2001;131:604S–14S. [4] Stoltzfus R, Barbour L, Black R. Iron deficiency anaemia. Comparative quantification of health risks: global and regional burden of disease attributable to selected major risk factors. Geneva, Switzerland: WHO; 2004. [5] IIPS (International Institute for Population Sciences). National Health and Family Survey India-3 (NFHS-3). Mumbai, India: IIPS; 2006. [6] Samuel TM, Thomas T, Finkelstein J, Bosch R, Rajendran R, Virtanen SM, et al. Correlates of anaemia in pregnant urban South Indian women: a possible role of dietary intake of nutrients that inhibit iron absorption. Pub Health Nutr; 2012:1–9. [7] Singh MB, Fotedar R, Lakshminarayana J. Micronutrient deficiency status among women of desert areas of western Rajasthan, India. Public Health Nutr 2009;12:624–9. [8] Pathak P, Kapil U, Yajnik CS, Kapoor SK, Dwivedi SN, Singh R. Iron, folate, and vitamin B12 stores among pregnant women in a rural area of Haryana State, India. Food Nutr Bull 2007;28:435–8. [9] Nair KM, Iyengar V. Iron content, bioavailability & factors affecting iron status of Indians. Indian J Med Res 2009;130:634–45.
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