1. Antenatal supplementation with micronutrients and biochemical
indicators of status and subclinical infection in rural Nepal1–3
Parul Christian, Tianan Jiang, Subarna K Khatry, Steven C LeClerq, Sharada R Shrestha, and Keith P West Jr
ABSTRACT There is growing interest in elucidating the effect of daily
Background: 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 in
several 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 seen
which 4 combinations of micronutrients (folic acid, folic acid ѿ iron,
Downloaded from ajcn.nutrition.org 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 for
nancy. In a subsample of subjects (n ҃ 740), blood was collected enhancing pregnancy and infant outcomes in Nepal. Also, iron and
both before supplementation and at Ȃ32 wk of gestation.
hematologic status in our trial (6) and another one in Mexico (7) did
Results: In the control group, serum concentrations of zinc, ribo-
not improve with multiple micronutrient supplementation beyond
flavin, 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 pregnancy
carboxylated prothrombin remained unchanged. Supplementation
with folic acid alone or folic acid ѿ iron decreased folate deficiency.
are common and coexist in rural Nepal (8). The deficiencies are
However, 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 can
tation 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 supplementation
boflavin, vitamin B-6, vitamin B-12, folate, and vitamin D deficien- with micronutrients enhance their circulating concentrations in
cies 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 micronutrient
tiple micronutrients can ameliorate, to some extent, the burden of de- status from the first to the third trimester of pregnancy in the
ficiency. 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,
1
supplementation 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 Cooperative
deficiencies 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, US
trations 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 Life
effect 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, Room
metabolic changes. Because micronutrients are involved in reg- W2041, Baltimore, MD 21205. E-mail: pchristi@jhsph.edu.
ulating 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. ANTENATAL MICRONUTRIENTS AND BIOCHEMICAL STATUS 789
Furthermore, 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 by
during 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 of
ble pathways for understanding the mechanisms responsible for Lactobacillus rhamnosus (NCIMB 10463) (11). Homocysteine
the 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 protein
SUBJECTS AND METHODS as the capturing agent. Serum vitamin B-12 was determined with
a microbiological assay that uses a colistin sulfate–resistant
Study 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 by
village 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 ajcn.nutrition.org by guest on October 6, 2012
reproductive 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 by
and infant survival and health. The pregnant women were ran- using HPLC. Serum (100 ␮L) was deproteinized by the addition
domly assigned to receive daily 400 ␮g folic acid, 60 mg folic of perchloric acid. Precolumn derivatization was performed with
acid ѿ iron, 30 mg folic acid ѿ iron ѿ zinc, a multiple micro- potassium cyanide. The fluorescent cyanide derivatives were
nutrient 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 assessed
thiamine, 1.8 mg riboflavin, 20 mg niacin, 2.2 mg vitamin B-6, with a commercial enzyme-linked immunoassay (ELISA) kit
2.6 ␮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-acid
alone as the control group. At baseline, an assessment of variables glycoprotein (AGP) and C-reactive protein (CRP). CRP was
reflecting 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 analysis
assessment of the newborn and mother was carried out by trained
teams 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 of
by 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 generalized
Blood 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 randomization
kept on ice, and brought to the project laboratory for centrifugation of sectors rather than individuals to treatment groups (15). Each
at 750 ҂ g for 20 min to separate the serum. Aliquots of serum were model was adjusted for the baseline concentration of the analyte
stored 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 (and
to 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 estimated
Laboratory 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 the
biochemical indicators of micronutrient and infection status. Data control (vitamin A alone) group as the reference category (15).

3. 790 CHRISTIAN ET AL
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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 exhibit
enrollment 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 Ȃ25
Nepal 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 as
RESULTS 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 multiple
agreed to have their blood drawn at baseline and 779 (59.2%) micronutrient group. Change in serum homocysteine was not
agreed 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 with
a 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 of
they 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 not
The high nonresponse at the third trimester was due to 1) women change in the other 3 supplementation groups. The exceptions were
having 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 concentration
significantly 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 who
control 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. ANTENATAL MICRONUTRIENTS AND BIOCHEMICAL STATUS 791
TABLE 1
Serum 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 micronturients
Maximum 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 10.4
Vitamin 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 136.7
Vitamin 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 11.0
Riboflavin (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 13.0
Homocysteine (␮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 3.8
Retinol (␮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 1.5
25(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 23.5
PIVKA-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 0.6
Zinc (␮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 2.1
Copper (␮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 6.9
AGP (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 0.22
CRP (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.
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7.6␮mol/L, did not decrease with the folic acid ѿ iron ѿ zinc Serum AGP concentrations decreased in the control group
or 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 with
were 35–77% lower in the multiple micronutrient group than in zinc with or without iron (P  0.05). Supplementation with
the 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, CRP
iron ѿ zinc group, but not in the multiple micronutrient group, increased during pregnancy in all groups, and the decrement
which received a supplement that provided an RDA of vitamin K. relative to the control group was statistically significant only in
TABLE 2
Change in serum concentrations of micronutrients and markers of subclinical infection from the first (baseline) to the third trimester and differences
between the supplementation groups and the control subjects1
Folic acid ѿ Multiple
Control3 Folic acid4 Folic acid ѿ iron4 iron ѿ zinc4 micronutrients4
Maximum 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)5
Vitamin 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)5
Vitamin 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)5
Riboflavin (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)5
Homocysteine (␮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)5
25(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)5
PIVKA-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)5
Copper (␮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 using
a 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. 792 CHRISTIAN ET AL
TABLE 3
Prevalence 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)3
Vitamin 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)3
Vitamin 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)3
Riboflavin  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)3
Homocysteine 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)3
PIVKA-II Π2.7 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 Π1 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 Π5 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 3
subjects (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.
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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 in
the trend was evident with the other 2 groups who received folic copper, as shown before (30).
acid. The frequencies of third-trimester concentrations of AGP
and CRP above the thresholds considered to reflect infection (Œ1 Effects of supplementation on status
and 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. Another
nutrients 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 a
DISCUSSION
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 by
ring 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 without
combinations of micronutrients among rural pregnant Nepali altering maternal status. Other reasons for a nonresponse could be
women living with chronic dietary deficits. The trial that gener- related to an inadequate dose or an inhibitory effect of one or more
ated 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 an
and perhaps even an adverse effect on infant survival (5). Thus, increase in serum folate concentrations. A significant attenuation
the biochemical data need to be interpreted in light of these of this effect was apparent in combination with zinc, which points
previous findings. to a negative interaction between zinc and folate. Old in vitro and
in vivo studies have shown that a mutual inhibition exists at the
Change 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 deficiency
vitamins decreased by 20 –50% from early to late gestation, a (33), although one study of short term folic acid supplementation
finding that has also been recorded in healthy populations (24). found no adverse effects on zinc status (34). The same study also
Concentration of homocysteine did not decrease, unlike the de- showed that folic acid utilization was not influenced by zinc intake
creases that have been described due to pregnancy-related endo- (34), which is contrary to our study findings. We found no published
crinologic changes (25). Fat-soluble vitamins E and K trans- evidence that zinc supplementation per se (alone or in combination
ported by plasma lipoproteins are known to increase during with folic acid or iron) affects folate metabolism. In the present
pregnancy, 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 could
creased 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 of
increase during pregnancy. With regard to the 2 minerals, their B vitamins as indicated by their circulating concentrations. With
change was expectedly in the opposite direction; serum zinc the exception of folate, this has not been demonstrated to our

6. ANTENATAL MICRONUTRIENTS AND BIOCHEMICAL STATUS 793
knowledge for other vitamins in pregnancy. Daily supplementa- that folic acid and zinc may ameliorate the inflammatory process
tion with the Recommended Dietary Allowance (RDA) of these in pregnancy, which has implications for reproductive health
vitamins, 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 nutrients
mocysteine 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 Conclusion
acid supplementation and fortification in the United States and In addition to dietary interventions, supplementation may be a
other countries (35–37), although deficiencies of both vitamin reasonable approach for addressing the problem of micronutrient
B-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 goal
effect 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 our
ceived 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 (vitamins
described 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 ajcn.nutrition.org by guest on October 6, 2012
the 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 of
which has been shown before in pregnancy (40). It is likely that biochemical indicators for assessing benefits of supplementation
the bioavailability of zinc was compromised by the presence of is limited. Instead, functional outcomes as true indicators of the
iron 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 Π2.7 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 their
and 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 responsible
tions of copper, which increased significantly during pregnancy. for computer programming and data management, and Lee Wu provided
Previously, 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 was
altering 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 the
sure to a high copper index in men showed no changes in plasma
study. SCL participated in the procedure development, study design, and
concentration of copper, although other indicators, such as uri-
edits to the article. SRS supervised the field work and data collection. KPW
nary copper, ceruloplasmin activity, benzylamine oxidase, and assisted in the development of the research idea, study design, protocol, and
superoxide dismutase, were significantly elevated (49). Our manuscript preparation. None of the authors had a personal or financial
study showed no evidence of copper status being affected by zinc interest to declare.
supplementation at 30 mg/d. Neither was there evidence that
copper supplementation affects zinc status because serum zinc
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