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  1. 1. Antenatal supplementation with micronutrients and biochemicalindicators of status and subclinical infection in rural Nepal1–3Parul Christian, Tianan Jiang, Subarna K Khatry, Steven C LeClerq, Sharada R Shrestha, and Keith P West JrABSTRACT There is growing interest in elucidating the effect of dailyBackground: Previously we showed that women in rural Nepal antenatal supplementation with multiple micronutrients on preg-experience multiple micronutrient deficiencies in early pregnancy. nancy and newborn outcomes in areas of the world where mi-Objective: This study examined the effects of daily antenatal mi- cronutrient deficiencies are common. We conducted a cluster-cronutrient supplementation on changes in the biochemical status of randomized, double-masked, controlled trial in rural Nepal inseveral micronutrients during pregnancy. which we observed that an antenatal multiple micronutrient sup-Design: In Nepal, we conducted a randomized controlled trial in plement failed to provide any additional benefit above that seenwhich 4 combinations of micronutrients (folic acid, folic acid ѿ iron, Downloaded from by guest on October 6, 2012 with folic acid and iron for an outcome such as birth weight (4).folic acid ѿ iron ѿ zinc, and a multiple micronutrient supplement Furthermore, it failed to show an apparent reduction in infant mor-containing folic acid, iron, zinc, and 11 other nutrients) plus vitamin tality of Ȃ20%, which was observed with folic acid and iron (5),A, or vitamin A alone as a control, were given daily during preg- providing little evidence for the use of such an intervention fornancy. In a subsample of subjects (n ҃ 740), blood was collected enhancing pregnancy and infant outcomes in Nepal. Also, iron andboth before supplementation and at Ȃ32 wk of gestation. hematologic status in our trial (6) and another one in Mexico (7) didResults: In the control group, serum concentrations of zinc, ribo- not improve with multiple micronutrient supplementation beyondflavin, and vitamins B-12 and B-6 decreased, whereas those of cop- the improvement observed with iron and folic acid alone.per and ␣-tocopherol increased, from the first to the third trimester. Previously, we also published data from our trial in Nepal,Concentrations of serum folate, 25-hydroxyvitamin D, and under- which showed that micronutrient deficiencies in early pregnancycarboxylated prothrombin remained unchanged. Supplementationwith folic acid alone or folic acid ѿ iron decreased folate deficiency. are common and coexist in rural Nepal (8). The deficiencies areHowever, the addition of zinc failed to increase serum folate, which exacerbated due to increased metabolic demands as the preg-suggests a negative inhibition; multiple micronutrient supplemen- nancy advances. Plasma concentrations of micronutrients cantation increased serum folate. Folic acid ѿ iron ѿ zinc failed to also be modified by the extent of plasma volume expansion,improve zinc status but reduced subclinical infection. Multiple mi- which complicates the interpretation of serum biochemical indi-cronutrient supplementation decreased the prevalence of serum ri- cators in pregnancy. Some studies do show that supplementationboflavin, vitamin B-6, vitamin B-12, folate, and vitamin D deficien- with micronutrients enhance their circulating concentrations incies but had no effect on infection. pregnancy (9, 10).Conclusions: In rural Nepal, antenatal supplementation with mul- In this article we compare changes in maternal micronutrienttiple micronutrients can ameliorate, to some extent, the burden of de- status from the first to the third trimester of pregnancy in theficiency. The implications of such biochemical improvements in the control group, relative to that observed with daily supplementa-absence of functional and health benefits remain unclear. Am J tion with 4 combinations of micronutrients that included a mul-Clin Nutr 2006;83:788 –94. tiple micronutrient supplement. Serum indicators of B complex vitamins, copper, zinc, and vitamins D, E, and K are examined.KEY WORDS Micronutrients, pregnancy, Nepal, infection, 1supplementation From the Center for Human Nutrition, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD (PC, TJ, SCL, and KPW), and the Society for Prevention of Blindness, Tripureswor, Kathmandu, Nepal (SKK and SRS).INTRODUCTION 2 Supported by the Micronutrients for Health Cooperative Agreement no. Although it is generally accepted that multiple micronutrient HRN-A-00-97-00015-00 and the Global Research Activity Cooperativedeficiencies during pregnancy are common in developing coun- Agreement no. GHS-A-00-03-00019-00 between the Johns Hopkins Uni-tries, few studies have examined the changes in serum concen- versity and the Office of Health, Infectious Diseases and Nutrition, UStrations of micronutrients over the course of a pregnancy or the Agency for International Development, Washington, DC; grants from the Bill and Melinda Gates Foundation, Seattle, WA; and the Sight and Lifeeffect of antenatal micronutrient supplementation on these con- Research Institute, Baltimore, MD.centrations. Pregnancy is a period of numerous physiologic and 3 Address reprint requests to P Christian, 615 North Wolfe Street, Roommetabolic changes. Because micronutrients are involved in reg- W2041, Baltimore, MD 21205. E-mail: these processes, it is plausible that micronutrient defi- Received June 27, 2005.ciencies could alter pregnancy outcomes (1–3). Accepted for publication December 15, 2005.788 Am J Clin Nutr 2006;83:788 –94. Printed in USA. © 2006 American Society for Nutrition
  2. 2. ANTENATAL MICRONUTRIENTS AND BIOCHEMICAL STATUS 789Furthermore, we also present data on the effect of micronutrient on the effect of supplementation on iron-status indicators, in-supplementation on acute phase markers of subclinical infection cluding hemoglobin, serum ferritin, iron, and transferrin recep-during pregnancy. These data provide information regarding the tors, were published previously (6) and are not presented here.success or failure of such a supplementation strategy for enhanc- Details of the analytic methods are provided elsewhere (8).ing nutritional status and correcting micronutrient deficiencies Briefly, serum zinc and copper concentrations were analyzed byduring a critical life stage— one of the objectives for considering atomic absorption spectrometry (AAnalyst 600; Perkin-Elmer,its implementation in the developing world. It also allows the Wellesley, MA). Serum folate was measured with a microbio-exploration of nutrient-nutrient interactions that provide plausi- logical assay with the use of a chloramphenicol-resistant strain ofble pathways for understanding the mechanisms responsible for Lactobacillus rhamnosus (NCIMB 10463) (11). Homocysteinethe lack of beneficial effects and even perhaps adverse effects was analyzed with a microtiter plate assay (Calbiotech Inc,previously noted (4, 5) Spring Valley, CA), which is similar to an enzyme immunoassay and uses a genetically engineered homocysteine binding proteinSUBJECTS AND METHODS as the capturing agent. Serum vitamin B-12 was determined with a microbiological assay that uses a colistin sulfate–resistantStudy design and population strain of Lactobacillus lactis (NCIMB 12519) (12, 13). Serum This study was carried out in the Southeastern plains District 25-hydroxyvitamin D [25(OH)D] was determined by immuno-of Sarlahi in Nepal during 1999 –2001. Details of the trial are assay (Nichols Institute, San Juan Capistrano, CA). Serum reti-described elsewhere (4, 5). Briefly, the study area consisted of 30 nol and ␣-tocopherol were determined simultaneously byvillage development communities in the district that were di- reversed-phase HPLC (Beckman, System Gold, Columbia, MD)vided into 426 small units, called sectors. Married women of attached with an autosampler (717 Plus AS; Waters Corp, Mil- Downloaded from by guest on October 6, 2012reproductive age who were not already pregnant, menopausal, ford, MA) by using a procedure described by Yamini et al (14)sterilized, or widowed were enumerated. They were visited every with modifications. Serum riboflavin concentrations were mea-5 wk, and the amenstrual women were administered a urine- sured as a surrogate for vitamin B-2 with the use of reversed-based human chorionic gonadotropin test to identify new preg- phase HPLC (model 1100; Agilent Technologies, Foster City,nancies. Consenting pregnant women were enrolled into a trial of CA) with a fluorescence detector (model FP-1520; Jasco Corp,antenatal and postnatal supplementation with alternative combi- Easton, MD). The serum concentration of pyridoxal 5-nations of micronutrients to examine the effect on birth weight phosphate, the active form of vitamin B-6, was measured byand infant survival and health. The pregnant women were ran- using HPLC. Serum (100 ␮L) was deproteinized by the additiondomly assigned to receive daily 400 ␮g folic acid, 60 mg folic of perchloric acid. Precolumn derivatization was performed withacid ѿ iron, 30 mg folic acid ѿ iron ѿ zinc, a multiple micro- potassium cyanide. The fluorescent cyanide derivatives werenutrient supplement containing the foregoing nutrients plus 11 detected by fluorometry. Undercarboxylated prothrombin (pro-other micronutrients (10 ␮g vitamin D, 10 mg vitamin E, 1.6 mg teins induced by vitamin K absence; PIVKA-II) were assessedthiamine, 1.8 mg riboflavin, 20 mg niacin, 2.2 mg vitamin B-6, with a commercial enzyme-linked immunoassay (ELISA) kit2.6 ␮g vitamin B-12, 100 mg vitamin C, 65 ␮g vitamin K, 2.0 mg (Asserachrom PIVKA II; Diagnostica Stago, Parsippany, NJ).Cu, and 100 mg Mg), or 1000 ␮g RE vitamin A and vitamin A Markers of inflammation that were examined included ␣1-acidalone as the control group. At baseline, an assessment of variables glycoprotein (AGP) and C-reactive protein (CRP). CRP wasreflecting household socioeconomic level, literacy, occupation, pre- measured by ELISA with a commercial kit from ADI (San An-vious pregnancy history, morbidity, diet, substance use, and stren- tonio, TX), and serum AGP was measured with a radial immu-uous work activities in the previous 7 d was made. Sector workers nodiffusion assay with commercially available kits (Kent Lab-delivered supplements twice a week to enrolled pregnant women in oratories, Bellingham, WA).their homes. Pregnancy outcome was monitored, and a day-of-birth Statistical analysisassessment of the newborn and mother was carried out by trainedteams of anthropometrists and interviewers. Statistical analyses were based on an intention-to-treat basis. In 25% of the sectors, a substudy involving blood collection The baseline biochemical status of pregnant women was com-was carried out to assess the effect of supplementation on the pared across treatment groups. The mean relative difference (de-women’s micronutrient status. Venous blood was drawn at home fined as the difference in the change in serum concentrations ofby trained phlebotomists at baseline (before supplementation) various analytes from baseline to follow-up for each supplemen-and again in the third trimester (scheduled at 32 wk of gestation). tation group compared with that in the control group) and 95%Detailed methods of this substudy were described previously (6, 8). confidence limits (CLs) were estimated by using generalizedBlood was collected into 7-mL trace metal–free vacuum test tubes estimating equations linear regression models with an identity(Vacutainer; Becton Dickinson Company, Franklin Lakes, NJ), link and exchangeable correlation to account for randomizationkept on ice, and brought to the project laboratory for centrifugation of sectors rather than individuals to treatment groups (15). Eachat 750 ҂ g for 20 min to separate the serum. Aliquots of serum were model was adjusted for the baseline concentration of the analytestored in liquid nitrogen tanks in trace element–free cryotubes (Nal- of interest. We used published cutoff values for defining defi-gene Company, Sybron International, New York, NY) and shipped cient concentrations of micronutrients. Prevalence ratios (andto the Johns Hopkins Bloomberg School of Public Health in Balti- 95% CLs) for micronutrient deficiencies and subclinical infec-more, MD, where they were stored at Ҁ80 °C until analyzed. tion, on the basis of a serum AGP concentration Œ1 g/L and a CRP concentration Œ5g/L in the third trimester, were estimatedLaboratory analyses by using a generalized estimating equations binomial regression Serum was analyzed over the course of 2–2.5 y for 11 different model with a log link and exchangeable correlation, with thebiochemical indicators of micronutrient and infection status. Data control (vitamin A alone) group as the reference category (15).
  3. 3. 790 CHRISTIAN ET AL Downloaded from by guest on October 6, 2012 FIGURE 1. Study participation and follow-up by supplementation group. R, randomization.Data were analyzed by using SAS version 8.1 (SAS Institute Inc, Serum concentrations of fat-soluble vitamin E (␣-tocopherol) in-Cary, NC). creased by 69%, but vitamins D and K (indicated by PIVKA-II) Informed consent was obtained from all participants before remained unchanged. Trace mineral concentrations did not exhibitenrollment in the study. The study received ethical approval by a consistent pattern of change, serum zinc concentrations decreased,the Committee on Human Research of the Johns Hopkins and serum copper concentrations increased.Bloomberg School of Public Health, Baltimore, MD, and the Serum folate concentrations increased significantly, by Ȃ25Nepal Health Research Council, Kathmandu, Nepal. nmol/L, in the groups receiving folic acid or folic acid ѿ iron or the multiple micronutrient supplement (Table 2). The combina- tion of folic acid ѿ iron ѿ zinc failed to increase serum folate asRESULTS indicated by an increment of Ȃ10 ␮mol/L with CLs that did not Of 1361 pregnant women in the substudy area, 1165 (85.6%) overlap those in the other folic acid groups or in the multipleagreed to have their blood drawn at baseline and 779 (59.2%) micronutrient group. Change in serum homocysteine was notagreed to having their blood drawn during the third trimester significantly different across supplementation groups. Zinc con-(Figure 1). A total of 740 women contributed both a baseline and centrations did not increase in response to supplementation witha follow-up blood sample. A smaller number of women had both zinc in combination with folic acid and iron relative to the con-baseline and follow-up blood samples collected because women trol, but did so with multiple micronutrient supplementation (0.5;were eligible to contribute blood at the follow-up visit even if 95% CL: 0.1, 0.99 ␮mol/L) (Table 2). Serum concentrations ofthey had not at baseline. The mean (ȀSD) gestational age at baseline most micronutrients included in the multiple micronutrient sup-and at follow-up was 10.2 Ȁ 4.1 and 32.6 Ȁ 3.9 wk, respectively. plements increased in that group relative to the control but did notThe high nonresponse at the third trimester was due to 1) women change in the other 3 supplementation groups. The exceptions werehaving gone to their parental home for delivery, 2) early pregnancy ␣-tocopherol and PIVKA-II, which remained unchanged with sup-loss, 3) migration, and 4) refusal; the nonresponse rates did not differ plementation. There was a significant increase in the concentrationsignificantly by treatment group (Figure 1). of 25(OH)D in the women who received folic acid alone, which did The baseline mean (ȀSD) concentrations of micronutrients not occur in response to folic acid given with zinc or iron (Table 2).and CRP and AGP did not differ by treatment group (Table 1). In Folate deficiency decreased by 75– 86% in the 4 groups whocontrol subjects, serum concentrations of water-soluble vitamins received folic acid (Table 3). This effect was not accompanied(including riboflavin and vitamins B-12 and B-6) decreased signif- by a reduction in the prevalence of high homocysteine concen-icantly by 32– 48% from early to late gestation (data not shown). trations. Zinc deficiency, defined as a serum zinc concentration
  4. 4. ANTENATAL MICRONUTRIENTS AND BIOCHEMICAL STATUS 791TABLE 1Serum concentrations of micronutrients and markers of subclinical infection at baseline by supplementation group1 Folic acid ѿ Folic acid ѿ Multiple Control Folic acid iron iron ѿ zinc micronturientsMaximum observations (n ҃ 270) (n ҃ 199) (n ҃ 200) (n ҃ 244) (n ҃ 252)Folate (nmol/L) 16.6 Ȁ 11.8 17.0 Ȁ 12.6 17.2 Ȁ 12.6 17.7 Ȁ 10.8 15.7 Ȁ 10.4Vitamin B-12 (pmol/L) 246.0 Ȁ 141.5 238.9 Ȁ 136.2 232.4 Ȁ 123.5 228.8 Ȁ 138.3 232.8 Ȁ 136.7Vitamin B-6 (nmol/L) 23.4 Ȁ 12.5 24.3 Ȁ 12.2 24.1 Ȁ 13.9 24.5 Ȁ 13.3 22.8 Ȁ 11.0Riboflavin (nmol/L) 18.7 Ȁ 14.3 19.1 Ȁ 14.9 18.6 Ȁ 17.0 19.1 Ȁ 15.3 17.9 Ȁ 13.0Homocysteine (␮mol/L) 10.6 Ȁ 3.8 11.0 Ȁ 3.7 11.1 Ȁ 4.2 10.6 Ȁ 4.0 11.0 Ȁ 3.8Retinol (␮mol/L) 1.20 Ȁ 0.4 1.24 Ȁ 0.4 1.18 Ȁ 0.4 1.19 Ȁ 0.4 1.17 Ȁ 0.4␣-Tocopherol (␮mol/L) 11.9 Ȁ 4.0 12.3 Ȁ 4.2 11.8 Ȁ 4.2 12.3 Ȁ 3.6 12.3 Ȁ 4.3␥-Tocopherol (␮mol/L) 1.6 Ȁ 1.2 1.6 Ȁ 1.1 1.6 Ȁ 1.2 1.7 Ȁ 1.3 1.8 Ȁ 1.525(OH)D (nmol/L) 46.5 Ȁ 23.3 54.6 Ȁ 24.0 53.7 Ȁ 26.8 55.0 Ȁ 24.6 47.4 Ȁ 23.5PIVKA-II (ng/mL) 1.93 Ȁ 0.6 1.88 Ȁ 0.6 1.92 Ȁ 0.7 1.97 Ȁ 0.6 1.94 Ȁ 0.6Zinc (␮mol/L) 8.1 Ȁ 1.8 8.1 Ȁ 2.1 8.5 Ȁ 2.1 8.2 Ȁ 2.0 8.3 Ȁ 2.1Copper (␮mol/L) 23.8 Ȁ 7.4 23.4 Ȁ 6.3 23.4 Ȁ 6.9 23.6 Ȁ 6.8 23.2 Ȁ 6.9AGP (g/L) 0.69 Ȁ 0.22 0.67 Ȁ 0.21 0.66 Ȁ 0.21 0.65 Ȁ 0.22 0.68 Ȁ 0.22CRP (g/L) 1.53 Ȁ 2.15 1.32 Ȁ 2.05 1.50 Ȁ 2.31 1.74 Ȁ 2.65 1.32 Ȁ 1.72 1 All values are x Ȁ SD. 25(OH)D, 25-hydroxyvitamin D; PIVKA-II, proteins induced by vitamin K absence (undercarboxylated prothrombin); AGP, ៮␣1-acid glycoprotein; CRP, C-reactive protein. There were no significant differences at baseline between supplementation groups. Downloaded from by guest on October 6, 20127.6␮mol/L, did not decrease with the folic acid ѿ iron ѿ zinc Serum AGP concentrations decreased in the control groupor the multiple micronutrient supplement, which contained zinc. (Table 2). Relative to this decrease, serum AGP decreased sig-Deficiencies of vitamins B-12, B-6, riboflavin, and 25(OH)D nificantly more in response to folic acid alone or folic acid withwere 35–77% lower in the multiple micronutrient group than in zinc with or without iron (P  0.05). Supplementation withthe control group. Relative to the control group, the prevalence of a multiple micronutrients failed to influence serum AGP concen-PIVKA-II concentration Œ2.7 ng/mL was lower in the folic acid ѿ trations relative to the control supplement. Unlike AGP, CRPiron ѿ zinc group, but not in the multiple micronutrient group, increased during pregnancy in all groups, and the decrementwhich received a supplement that provided an RDA of vitamin K. relative to the control group was statistically significant only inTABLE 2Change in serum concentrations of micronutrients and markers of subclinical infection from the first (baseline) to the third trimester and differencesbetween the supplementation groups and the control subjects1 Folic acid ѿ Multiple Control3 Folic acid4 Folic acid ѿ iron4 iron ѿ zinc4 micronutrients4Maximum observations2 (n ҃ 164) (n ҃ 126) (n ҃ 127) (n ҃ 167) (n ҃ 156)Folate (nmol/L) Ҁ1.8 Ȁ 15.4 25.9 (19.4, 32.5)5 24.9 (19.2, 30.8)5 10.3 (6.9, 13.7)5 27.6 (22.7, 32.5)5Vitamin B-12 (pmol/L) Ҁ113.6 Ȁ 1296 12.2 (Ҁ3.2, 27.6) 7.3 (Ҁ10.5, 25.1) 11.0 (Ҁ5.3, 27.2) 49.6 (27.8, 71.4)5Vitamin B-6 (nmol/L) Ҁ11.4 Ȁ 12.46 0.9 (Ҁ0.5, 2.4) Ҁ0.02 (Ҁ1.5, 1.5) Ҁ0.3 (Ҁ1.7, 1.1) 9.9 (7.0, 11.2)5Riboflavin (nmol/L) Ҁ6.2 Ȁ 12.06 0.9 (Ҁ1.0, 2.8) Ҁ1.1 (Ҁ3.0, 0.9) 0.05 (Ҁ2.1, 2.2) 10.8 (8.3, 13.4)5Homocysteine (␮mol/L) Ҁ1.6 Ȁ 4.5 0.17 (Ҁ0.51, 0.84) 0.26 (Ҁ0.41, 0.93) Ҁ0.06 (Ҁ0.73, 0.61) 0.05 (Ҁ0.68, 0.77)Retinol (␮mol/L) 0.18 Ȁ 0.5 Ҁ0.06 (Ҁ0.17, 0.05) 0.05 (Ҁ0.04, 0.14) Ҁ0.05 (Ҁ0.14, 0.03) 0.06 (Ҁ0.03, 0.14)␣-Tocopherol (␮mol/L) 7.9 Ȁ 4.66 Ҁ0.3 (Ҁ1.4, 0.7) 0.2 (Ҁ0.7, 1.2) Ҁ0.6 (Ҁ1.5, 0.3) 0.3 (Ҁ0.7, 1.3)␥-Tocopherol (␮mol/L) 0.00 Ȁ 1.3 Ҁ0.00 (Ҁ0.18, 0.17) Ҁ0.07 (Ҁ0.26, 0.12) Ҁ0.12 (Ҁ0.28, 0.04) Ҁ0.38 (Ҁ0.56, Ҁ0.21)525(OH)D (nmol/L) 3.2 Ȁ 28.7 15.2 (5.7, 24.7)5 5.1 (Ҁ1.9, 12.1) 5.9 (Ҁ0.2, 12.0) 17.8 (11.7, 23.8)5PIVKA-II (ng/mL) Ҁ0.12 Ȁ 0.9 0.07 (Ҁ0.18, 0.34) Ҁ0.03 (Ҁ0.21, 0.15) Ҁ0.10 (Ҁ0.25, 0.04) 0.00 (Ҁ0.19, 0.21)Zinc (␮mol/L) Ҁ1.6 Ȁ 2.56 0.1 (Ҁ0.5, 0.6) 0.1 (Ҁ0.4, 0.5) 0.2 (Ҁ0.3, 0.6) 0.5 (0.1, 0.99)5Copper (␮mol/L) 11.1 Ȁ 9.36 0.9 (Ҁ0.9, 2.8) 0.3 (Ҁ1.3, 1.9) Ҁ0.3 (Ҁ1.8, 1.2) Ҁ0.5 (Ҁ2.0, 1.00)AGP (g/L) Ҁ0.22 Ȁ 0.296 Ҁ0.04 (Ҁ0.08, Ҁ0.002)5 Ҁ0.03 (Ҁ0.06, Ҁ0.001)5 Ҁ0.05 (Ҁ0.08, Ҁ0.01)5 Ҁ0.01 (Ҁ0.05, 0.03)CRP (g/L) 0.74 Ȁ 3.466 Ҁ0.41 (Ҁ1.04, 0.23) Ҁ0.25 (Ҁ0.81, 0.32) Ҁ0.57 (Ҁ1.07, 0.56)5 0.00 (Ҁ0.74, 0.74) 1 25(OH)D, 25-hydroxyvitamin D; PIVKA-II, proteins induced by vitamin K absence (undercarboxylated prothrombin); AGP, ␣1-acid glycoprotein; CRP,C-reactive protein. 2 Serum values for some analytes missing in 2–5 samples per group. 3 All values are the mean (Ȁ SD) change from baseline to follow-up. 4 All values are mean differences (and 95% confidence limits) in the change from baseline to follow-up relative to the control group, calculated by usinga generalized estimating equations linear regression model adjusted for baseline concentration. 5 P  0.05 (generalized estimating equations linear regression model adjusted for baseline concentrations). 6 P  0.05 (paired t test).
  5. 5. 792 CHRISTIAN ET ALTABLE 3Prevalence ratios of deficiency and subclinical infection in the third trimester in the supplementation groups relative to the control group1 Folic acid ѿ Folic acid ѿ Multiple Control Folic acid2 iron2 iron ѿ zinc2 micronutrients2 (n ҃ 173) (n ҃ 133) (n ҃ 135) (n ҃ 173) (n ҃ 165) %Folate  6.8 nmol/L (16) 22.5 0.22 (0.08, 0.58)3 0.14 (0.03, 0.72)3 0.25 (0.11, 0.60)3 0.24 (0.10, 0.55)3Vitamin B-12  150 pmol/L (17) 69.9 0.95 (0.79, 1.14) 0.96 (0.80, 1.16) 0.87 (0.71, 1.04) 0.65 (0.52, 0.81)3Vitamin B-6  19 nmol/L (18) 88.4 0.98 (0.91, 1.06) 0.99 (0.90, 1.09) 1.03 (0.94, 1.11) 0.66 (0.55, 0.78)3Riboflavin  11.3 nmol/L (19) 63.6 0.90 (0.69, 1.16) 1.07 (0.86, 1.32) 0.92 (0.75, 1.13) 0.37 (0.26, 0.52)3Homocysteine Œ 12 ␮mol/L (16) 16.2 0.96 (0.56, 1.64) 0.90 (0.54, 1.50) 0.79 (0.44, 1.42) 0.85 (0.48, 1.52)Retinol  0.7 ␮mol/L (20) 3.7 1.54 (0.63, 3.74) 0.44 (0.11, 1.76) 1.47 (0.53, 4.10) 0.88 (0.28, 2.75)25(OH)D  25 nmol/L (21) 24.3 0.48 (0.25, 0.92)3 0.71 (0.41, 1.20) 0.48 (0.30, 0.77)3 0.23 (0.12, 0.45)3PIVKA-II Œ 2.7 ng/mL4 28.8 0.99 (0.54, 1.81) 0.98 (0.54, 1.81) 0.44 (0.19, 0.99)3 0.68 (0.32, 1.47)Zinc  7.6 ␮mol/L (22) 75.1 0.98 (0.84, 1.14) 0.90 (0.78, 1.05) 0.94 (0.81, 1.07) 0.87 (0.75, 1.01)AGP Œ 1 g/L 3.1 0.25 (0.03, 2.11) 0.54 (0.11, 2.58) 0.20 (0.02, 1.67) 1.12 (0.32, 3.95)CRP Œ 5 g/L 13.4 0.57 (0.28, 1.19) 0.84 (0.49, 1.45) 0.48 (0.25, 0.90)3 1.18 (0.71, 1.97) 1 25(OH)D, 25-hydroxyvitamin D; PIVKA-II, proteins induced with vitamin K absence (undercarboxylated prothrombin). AGP, ␣1-acid glycoprotein;CRP, C-reactive protein. None of the subjects had a serum copper concentration 15 ␮mol/L. ␣-Tocopherol concentrations are not presented because only 3subjects (0.4%) had a concentration below the cutoff of 9.3 ␮mol/L (23). ␥-Tocopherol concentrations are not presented because of no known suitable cutoff. 2 All values are prevalence ratios (and 95% confidence limits) calculated by using a generalized estimating equations logistic regression model. Downloaded from by guest on October 6, 2012 3 P  0.05. 4 Cutoff selected on the basis of a normal value of 2 ng/mL.the folic acid ѿ iron ѿ zinc group (0.57 g/L; P  0.05), although concentrations decreased in contrast with significant increases inthe trend was evident with the other 2 groups who received folic copper, as shown before (30).acid. The frequencies of third-trimester concentrations of AGPand CRP above the thresholds considered to reflect infection (Œ1 Effects of supplementation on statusand Œ5 g/L, respectively) were lower in the folic acid ѿ iron ѿ Improvements in maternal biochemical status during preg-zinc group than in the control group, but the 95% CL for AGP did nancy associated with micronutrient supplementation may pri-not exclude 1.0 (Table 3). Supplementation with multiple micro- marily be due to correction of underlying deficiency. Anothernutrients failed to affect either of these indexes of infection. mechanism may be related to the effect on subclinical infection known to lower circulating concentrations of micronutrients caused by an acute phase response. A lack of response in aDISCUSSION measured indicator may, perhaps, be masked by the plasma volume Our community-based study provides data on changes occur- expansion of pregnancy that may in turn have been influenced byring in circulating concentrations of vitamins and minerals and micronutrient status (3), or changes in endocrine regulations of preg-the biological response to daily supplementation with 4 different nancy may facilitate channeling of nutrients to the fetus withoutcombinations of micronutrients among rural pregnant Nepali altering maternal status. Other reasons for a nonresponse could bewomen living with chronic dietary deficits. The trial that gener- related to an inadequate dose or an inhibitory effect of one or moreated these data showed no benefit of a multiple micronutrient nutrients when provided simultaneously.supplement over folic acid ѿ iron in improving birth weight (4) Folic acid singly or in combination with iron resulted in anand perhaps even an adverse effect on infant survival (5). Thus, increase in serum folate concentrations. A significant attenuationthe biochemical data need to be interpreted in light of these of this effect was apparent in combination with zinc, which pointsprevious findings. to a negative interaction between zinc and folate. Old in vitro and in vivo studies have shown that a mutual inhibition exists at theChange in micronutrient status during pregnancy site of intestinal transport (31, 32). Folic acid supplements have In the present study, concentrations of most water-soluble shown to increase zinc excretion in men with mild zinc deficiencyvitamins decreased by 20 –50% from early to late gestation, a (33), although one study of short term folic acid supplementationfinding that has also been recorded in healthy populations (24). found no adverse effects on zinc status (34). The same study alsoConcentration of homocysteine did not decrease, unlike the de- showed that folic acid utilization was not influenced by zinc intakecreases that have been described due to pregnancy-related endo- (34), which is contrary to our study findings. We found no publishedcrinologic changes (25). Fat-soluble vitamins E and K trans- evidence that zinc supplementation per se (alone or in combinationported by plasma lipoproteins are known to increase during with folic acid or iron) affects folate metabolism. In the presentpregnancy, parallel to the increases in serum concentrations of study, however, multiple micronutrients, which also contained zinc,lipids and triacylglycerols (24, 26). An increase in vitamin D, completely reversed the negative effect of zinc on serum folate,acting as a calciotropic hormone, is crucial for meeting the in- although which one or more micronutrients in this mixture couldcreased need for calcium during pregnancy (27–29). In our study, have alleviated this inhibition remains unclear.unlike vitamin E, concentrations of vitamins D and K did not Multiple micronutrients succeeded in enhancing the status ofincrease during pregnancy. With regard to the 2 minerals, their B vitamins as indicated by their circulating concentrations. Withchange was expectedly in the opposite direction; serum zinc the exception of folate, this has not been demonstrated to our
  6. 6. ANTENATAL MICRONUTRIENTS AND BIOCHEMICAL STATUS 793knowledge for other vitamins in pregnancy. Daily supplementa- that folic acid and zinc may ameliorate the inflammatory processtion with the Recommended Dietary Allowance (RDA) of these in pregnancy, which has implications for reproductive healthvitamins, however, was insufficient to lower deficiency for some outcomes. However, the multiple micronutrient supplements,by much. For example, the prevalence of vitamin B-6 and B-12 which included the above nutrients, failed to show this reduction,deficiencies was reduced by only 30 –35% in late gestation. Ho- which suggests an inhibitory interaction with the other nutrientsmocysteine did not decrease with folic acid or in combination present in the supplement.with vitamins B-6 and B-12 supplementation. Unlike these find-ings, changes in homocysteine were previously noted with folic Conclusionacid supplementation and fortification in the United States and In addition to dietary interventions, supplementation may be aother countries (35–37), although deficiencies of both vitamin reasonable approach for addressing the problem of micronutrientB-12 and vitamin B-6 may also affect homocysteine concentra- deficiencies in pregnancy. The data presented in our article sup-tions (38, 39). Persisting deficiencies of these vitamins, despite port this conclusion, although several considerations are neces-supplementation, may provide an explanation for the lack of sary before such an approach is adopted broadly. First, if the goaleffect on homocysteine in our study. of such a strategy were just to alleviate deficiency (on the basis Concentrations of 25(OH)D increased in the group that re- of known indicators of status), then the formulation tested in ourceived an RDA of vitamin D. This increase was also observed study achieved this goal for some nutrients (folate, riboflavin,with folic acid supplementation alone. We found no previously and vitamin D), but had only a modest effect on others (vitaminsdescribed evidence linking folate status with either the synthesis B-6 and B-12) and even failed to affect it for some (zinc, copper,of vitamin D in the skin, the synthesis of the vitamin D– binding and vitamin K). Second, both negative and positive nutrient-protein, or the hydroxylation of vitamin D, which suggests that nutrient interactions may occur. Knowledge of these interactions Downloaded from by guest on October 6, 2012the mechanism remains to be elucidated. is critical in creating combinations that will work best together Zinc concentrations were not responsive to supplementation, and perhaps even synergize each other. Finally, the usefulness ofwhich has been shown before in pregnancy (40). It is likely that biochemical indicators for assessing benefits of supplementationthe bioavailability of zinc was compromised by the presence of is limited. Instead, functional outcomes as true indicators of theiron and folic acid (33, 41, 42). Zinc, in combination with other effect are needed and should be assessed as endpoints in studies.micronutrients, did increase serum zinc concentrations by 0.5 Methods for the safe delivery of micronutrients to correct the␮mol/L. high levels of deficiency that are clearly apparent among women The combination of folic acid ѿ iron ѿ zinc reduced the risk in South Asia are urgently needed. Testing different combina-of PIVKA-II Œ 2.7 ng/mL, which suggests that zinc promotes tions and doses of micronutrients and alternative delivery mech-vitamin K status. Previously, an in vitro study showed that zinc anisms (food fortification, sprinkles) on both short- and long-sulfate caused a dose-dependent prolongation of prothrombin term health and functional outcomes in the mothers and theirand partial thromboplastin times as well as shortened thrombin infants should receive priority.clotting time (43). A rat experiment of the effect of vitamin K2 Apart from the authors, several members of the Nepal study team helped(menaquinone-7) on bone metabolism showed an enhancement in the successful implementation of the study and laboratory analysis, in-with zinc (44). In patients with alcoholic cirrhosis, zinc supple- cluding the field managers, supervisors, and phlebotomy team. Tracey Wag-mentation increased plasma prothrombin and serum alkaline ner conducted the laboratory analyses, Joanne Katz was a co-investigator,phosphatase concentrations (45). Kerry Schulze performed the PIVKA-II analysis and provided comments on Copper supplementation did not change plasma concentra- the paper, Elizabeth K Pradhan and Gwendolyn Clemens were responsibletions of copper, which increased significantly during pregnancy. for computer programming and data management, and Lee Wu providedPreviously, copper supplementation in pregnant ewes, mares, or statistical support.cows resulted in increases in liver copper concentrations without PC was the principal investigator and analyzed and wrote the paper. TJ wasaltering plasma concentrations (46 – 48). Even long-term expo- the laboratory director, oversaw all the biochemical analyses, and provided edits for the manuscript. SKK was country director and implemented thesure to a high copper index in men showed no changes in plasma study. SCL participated in the procedure development, study design, andconcentration of copper, although other indicators, such as uri- edits to the article. SRS supervised the field work and data collection. KPWnary copper, ceruloplasmin activity, benzylamine oxidase, and assisted in the development of the research idea, study design, protocol, andsuperoxide dismutase, were significantly elevated (49). Our manuscript preparation. None of the authors had a personal or financialstudy showed no evidence of copper status being affected by zinc interest to declare.supplementation at 30 mg/d. Neither was there evidence thatcopper supplementation affects zinc status because serum zinc REFERENCESresponse was the highest in the multiple micronutrient group, 1. Allen LH. Multiple micronutrients in pregnancy and lactation: an over-who received 2 mg Cu. Recently, zinc supplementation was view. Am J Clin Nutr 2005;81(suppl):1206S–12S.found not to affect plasma copper concentrations in infants (50) 2. Keen CL, Clegg MS, Hanna LA, et al. 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