The Journal of Nutrition

Community and International Nutrition

Zinc, Gravida, Infection, and Iron, but Not Vitamin

B-12 or Folate Status, Predict Hemoglobin during
Pregnancy in Southern Ethiopia1,2
Rosalind S. Gibson,3* Yewelsew Abebe,4 Sally Stabler,5 Robert H. Allen,5 Jamie E. Westcott,6
Barbara J. Stoecker,7 Nancy F. Krebs,6 and K. Michael Hambidge6
3
Department of Human Nutrition, University of Otago, Dunedin, New Zealand 9015; 4College of Agriculture, Hawassa University,
Awassa, Ethiopia; 5Division of Hematology, Department of Medicine, and 6Section of Nutrition, Department of Pediatrics, University of
Colorado, Denver, CO 80262; and 7Department of Nutrition, Oklahoma State University, Stillwater, OK 74078

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Abstract
The etiology of anemia during pregnancy in rural Southern Ethiopia is uncertain. Intakes of animal-source foods are low and
infections and bacterial overgrowth probably coexist. We therefore measured the dietary intakes of a convenience sample
of Sidama women in late pregnancy who consumed either maize (n ¼ 68) or fermented enset (Enset ventricosum) (n ¼ 31)
as their major energy source. Blood samples were analyzed for a complete blood count, vitamin B-12 and folate status,
plasma ferritin, retinol, zinc, albumin, and C-reactive protein (CRP). The role of infection and gravida was also examined.
Dietary intakes were calculated from 1-d weighed records. No cellular animal products were consumed. Of the women,
29% had anemia, 13% had iron deficiency anemia, 33% had depleted iron stores, and 74 and 27% had low plasma zinc
and retinol, respectively. Only 2% had low plasma folate (,6.8 nmol/L) and 23% had low plasma vitamin B-12 (,150 pmol/L),
even though 62% had elevated plasma methylmalonic acid (MMA) (. 271 nmol/L). None had elevated plasma
cystathionine or total homocysteine (tHcys). Women with enset-based diets had higher (P ¼ 0.052) plasma vitamin B-12
concentration and lower (P , 0.05) cell volume, plasma cystathionine, and retinol than women consuming maize-based
diets, but mean hemoglobin, plasma ferritin, MMA, tHcys, and folate did not differ. Plasma zinc, followed by CRP (#5 mg/L),
gravida (#4), and plasma ferritin ($12 mg/L) status were major positive predictors of hemoglobin. Despite some early
functional vitamin B-12 deficiency, there was no macrocytic anemia. Consumption of fermented enset may have
increased vitamin B-12 levels in diet and plasma. J. Nutr. 138: 581–586, 2008.

Introduction
Pregnant women from subsistence farming households in rural
Africa usually consume predominantly plant-based diets; intakes
of animal-source foods are low. Consequently, micronutrient
intakes are often inadequate (1,2), a problem often exacerbated
in these settings by malabsorption induced by infections and
bacterial overgrowth (3,4). Hence, coexisting deficiencies of
iron, zinc, vitamin A, vitamin B-12, and folate have often been
reported (2,5,6) and associated with increased risk of maternal
complications and poor fetal outcomes (7).
1
Supported by NIH grants R21 TW06729 (Fogarty International Center and the
Office of Dietary Supplements), M01 RR00069 (General Clinical Research
Centers Program), and MRDDR Center grant P30 HD004024, and by a University
of Otago Research Grant.
2
Author disclosures: R. S. Gibson, Y. Abebe, J. E. Westcott, K. M. Hambidge,
B. J. Stoecker, and N. F. Krebs, no conflicts of interest; S. Stabler, R. H. Allen, and
the University of Colorado Denver hold patents on various aspects of the assays
of homocysteine and methylmalonic acid in the use of diagnosing vitamin B12
and folate deficiency. A company has been formed at the University of Colorado
to assay the metabolites.
* To whom correspondence should be addressed. E-mail: rosalind.gibson@
stonebow.otago.ac.nz.
0022-3166/08 $8.00 ª 2008 American Society for Nutrition.
Manuscript received 4 September 2007. Initial review completed 17 October 2007. Revision accepted 10 December 2007.
In the Sidama Zone of Southern Ethiopia, maize (Zea mays
L.) and fermented enset (Enset ventricosum) products are the
major staple foods, contributing up to 90% of energy. Animal
products often provide ,1% of the total energy (8). Neverthe-
less, dietary iron deficiency is not a major cause of anemia during
pregnancy in this region (8). Such low intakes of animal
products concomitant with infections and bacterial overgrowth
may place pregnant women in Sidama at high risk for vitamin
B-12 deficiency and possibly folate deficiency (3,6,9), especially
in this malarial endemic area, because some antimalarial drugs
interfere with folate metabolism (9). However, some vitamin
B-12 and folate-producing microorganisms have been isolated
from fermented enset (10). Hence, the consumption of fer-
mented enset may reduce the risk for vitamin B-12 and folate
deficiency. To our knowledge, however, there are no data on
vitamin B-12 and folate status of pregnant women in Sidama.
Our earlier cross-sectional study of subsistence pregnant
farmers from Sidama, Southern Ethiopia reported a high pre-
valence of protein and zinc deficiency but not iron deficiency (8).
Here, we extend the research by investigating the vitamin B-12
and folate status of these same subjects using a combination of
581
static (i.e. plasma vitamin B-12 and folate concentrations) and
functional [i.e. plasma methylmalonic acid (MMA),8 total
homocysteine (tHcys), and cystathionine] biochemical indices.
We also examined their vitamin B-12 and folate status according
to whether the women received more of their dietary energy
from unrefined maize (i.e. maize diet group) or from fermented
enset products (enset diet group). We also investigated the role of
vitamin B-12 and folate status, as well as protein, other
micronutrients (iron, vitamin A, zinc), infection, and gravida
on hemoglobin concentrations.
Subjects and Methods
Study site and subjects. Demographic and clinical details were given
earlier (8). Briefly, the women (n ¼ 99) were subsistence farmers from 3
rural communities in the Sidama Zone of southern Ethiopia who
volunteered to participate in the study. The women were all in the 3rd
Anemia was defined as a hemoglobin concentration ,115 g/L (cutoff
adjusted upward by 5 g/L for an altitude of 1800 m) (22). Macrocytosis
was defined as a mean cell volume (MCV) .100 fL (23) and microcytosis
as MCV ,81 fL (24). Anemia accompanied by a low plasma ferritin
concentration (i.e. ,12 mg/L) in the absence of infection or inflammation
was considered to reflect iron deficiency anemia (IDA); a low plasma
ferritin in the absence of anemia or infection was taken to indicate
depleted iron stores (8).
Statistical analysis. Values in the text are means 6 SD or median
[interquartile range (IQR)]. Plasma vitamin B-12, cystathionine, and
tHcys showed significant departures from normality and, hence, we
calculated medians and the Mann-Whitney U statistic to assess between-
group differences. Spearman correlation coefficients were used to
explore the relation between the variables. We assessed differences
between the 2 diet groups using the Student’s t test for independent
samples or Fisher’s exact test. Multiple linear regression analysis was
used to examine the predictors of hemoglobin concentration in all the
subjects. We used indicator variables in the regression analysis for

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trimester of pregnancy (based on fundal height), afebrile, and aged
27.8 6 4.6 y (mean 6 SD). Ethical approval of the study protocol was
obtained from the Human Ethics Committee of Hawassa University,
Awassa, Ethiopia, and the Colorado Multiple Institutional Review Board
of the University of Colorado, Denver, CO. Verbal informed consent was
obtained from all the participants. After recruitment, trained research
assistants interviewed the women using a pretested questionnaire and
observations to obtain sociodemographic and health data. The sample
size (n ¼ 99) allowed estimation of the prevalence of anemia to within
10 percentage points of the true value with a confidence level of 95%.
Dietary and anthropometric assessment. Dietary intakes were
calculated from 1-d weighed food records using an Ethiopian food
composition table based on literature and analyzed values for calcium,
iron, zinc, and phytate (Phy) (8,11). Observed intakes of energy and
protein were adjusted for digestibility and protein intakes for amino acid
score. The women were then classified into 2 groups according to
whether maize (n ¼ 68) or enset (n ¼ 31) was the predominant energy
source. Samples of each of the 2 fermented enset products, kocho and
bulla, were purchased from local markets and analyzed for folate and
vitamin B-12 content. We performed analysis for folate in duplicate on
each sample by microbiological assay using Lactobacillus rhamnosus
(formerly known as Lactobacillus casei, ATCC 7469) and the trienzyme
extraction method (12). Vitamin B-12 was assayed using a newly
developed liquid chromatography-MS method in the laboratory of
S. Stabler (R. H. Allen and S. P. Stabler, unpublished data). Briefly, D2-
labeled CN-cobalamin was added to samples of the kocho and bulla,
which were partially purified prior to liquid chromatography. The peak
areas for the internal standard were compared with that of the endogenous
cobalamin to calculate the concentration in the sample. We measured
weight and height and calculated BMI and height-for-age Z-scores (8).
infection, gravida, and ferritin because of the nonnormal distribution of
these independent variables. Differences were considered significant at
P , 0.05. Data were analyzed using SPSS version 12.0.
Results
Selected demographic and anthropometric characteristics of
the pregnant women did not differ between the 2 diet groups
(Table 1).
Dietary assessment. Both groups had a very monotonous
diet, low in energy, with .85% provided by foods from only 2
food sources: unrefined maize and enset products. Less than 1%
of energy in both diet groups was provided by dairy products
and no cellular animal foods were eaten. The enset diet group
had lower intakes (P , 0.001) of utilizable protein, fat, zinc,
dietary fiber, Phy, and lower Phy:Zn and Phy:Fe molar ratios
but higher intakes (P , 0.001) of calcium than the maize diet
group (Table 2).
Median intakes of kocho were 174 (129, 254) g/d for the
enset diet group compared with 54 (0, 132) g/d for the maize diet
group; neither group consumed bulla. The corresponding
median intakes of maize for the enset and maize diet groups
were 65 (25, 96) g/d and 107 (82, 158) g/d, respectively. The
mean analyzed content of folate for the samples of the 2 fer-
mented enset products, kocho and bulla, were 33.4 6 9.3 mg/100 g

Laboratory assessment. Anticoagulated whole blood and plasma

were collected from nonfasting morning venipuncture samples using TABLE 1 Selected demographic and anthropometric
rigorous collection and separation procedures. Methodological details characteristics of pregnant women who are
for the complete blood count, plasma albumin, ferritin, zinc, and subsistence farmers from Sidamo1
C-reactive protein (CRP) were reported previously (8). Plasma vitamin
B-12 was analyzed using the Solid Phase No Boil Dual count Maize-based Enset-based Mean difference2
Radioassay B-12/Folic acid kit (Diagnostic Products). MMA, tHcys, diet, n ¼ 68 diet, n ¼ 31 (95% CI)
and cystathionine were assayed in plasma by isotope dilution GC-MS
(13,14). Plasma retinol was measured by HPLC (15). Plasma CRP Age, y 27.6 6 4.3 28.4 6 5.4 20.86 (–3.10, 1.38)
concentrations .5 mg/L were used to indicate the presence of acute No schooling, % 77.9 74.2 0.793
infection or inflammation. Fundal height, cm 7.1 6 1.0 7.0 6 1.1 0.03 (–0.45, 0.50)
The following interpretive criteria were used to define risk of Gravida . 4, % 63.2 56.7 0.653
biochemical micronutrient deficiencies in late pregnancy: plasma vitamin Weight, kg 52.0 6 6.3 52.4 6 5.7 20.45 (–3.00, 2.11)
B-12 ,150 pmol/L (16); MMA .271 nmol/L (17); plasma tHcys .13.9 Height, cm 154.8 6 6.4 154.9 6 6.7 20.15 (–3.03, 2.73)
mmol/L (18); plasma cystathionine .342 nmol/L (17); plasma folate Height-for-age Z-score 21.48 6 1.07 21.46 6 1.12 20.03 (–0.51, 0.45)
,6.8 nmol/L (19); plasma zinc ,7.6 mmol/L (20); and plasma retinol
BMI, kg/m2 21.7 6 2.0 21.8 6 2.0 20.18 (–1.04, 0.69)
,0.70 mmol/L (21).
Rarely had enough food, % 39.7 32.3 0.513
8 1
Abbreviations used: ACD, anemia of chronic disease; CRP, C-reactive protein; Values are means 6 SD or %.
2
IDA, iron deficiency anemia; IQR, interquartile range; MCV, mean cell volume; From 2-tailed t test. Equality of variances not assumed.
3
MMA, methylmalonic acid; Phy, phytate; tHcys, total homocysteine. Significance of difference in proportion from Fisher’s exact test (2-sided).

582 Gibson et al.

TABLE 2 Intakes per day of energy, selected nutrients, concentration #5 mg/L, although plasma zinc, ferritin, albumin,
dietary fiber, Phy, and Phy:zinc and Phy:iron molar and none of the biochemical indices of folate or vitamin B-12
ratios for 2 groups of women consuming unrefined status did not differ. However, because plasma zinc, ferritin,
maize- or enset-based diets1 retinol, and albumin (but not folate and vitamin B-12 indices)
are all known to be influenced by inflammation or infection (15),
Maize-based Enset-based Mean difference2
women with elevated plasma CRP were excluded from the data
diet, n ¼ 68 diet, n ¼ 31 (95% CI)
in Table 3 but not from the multiple linear regression analysis in
Adjusted energy, kJ 4382 6 1481 3884 6 1273 497 (–83, 1078) Table 4, where an indicator variable for infection was included.
Utilizable protein, g 16.3 6 5.9 11.3 6 4.7 5.0 (2.8, 7.2)* Mean plasma vitamin B-12 level tended to be higher (P ¼
Fat, g 10.5 6 4.8 4.4 6 3.4 6.1 (4.5, 7.8)* 0.052), whereas MCV and plasma cystathionine were lower in
Carbohydrate, g 250.2 6 85.6 228.1 6 75.3 22.1 (–11.9, 56.2) the enset diet group compared with the maize diet group (P ,
Calcium, mg 384 6 9.3 773 6 324.1 –388 (–520, –256)* 0.05). There were no other significant differences between the 2
Iron, mg 28.2 6 11.3 27.7 6 9.9 0.5 (–4.0, 5.0) diet groups for hematological or biochemical indices or for the
Zinc, mg 6.3 6 2.5 3.7 6 1.6 2.59 (1.8, 3.4)* prevalence of abnormal values (Table 3).
Ascorbic acid,3 mg 2.2 (0.7, 5.0) 1.8 (1.0, 4.6) 0.87 Of the women, 29% (n ¼ 27) had anemia from all causes;
Total dietary fiber, g 29.0 6 11.8 17.0 6 8.7 12.0 (7.7, 16.2)* none had severe anemia (hemoglobin ,70 g/L). A total of 13%
Phy, mg 1356 6 603 446 6 335 910 (722, 1098)* (n ¼ 12) of the women had IDA with a mean cell hemoglobin

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Phy:zinc molar ratio 21.1 6 4.4 10.8 6 4.8 10.3 (8.2, 12.3)* concentration of 343 6 11.7 g/L and a MCV of 88.3 6 5.6 fL.
Phy:iron molar ratio 4.1 6 1.2 1.3 6 0.7 2.8 (2.4, 3.2)* Interestingly, the mean values for MCV, plasma vitamin B-12,
Energy from maize products, % 74.0 6 19.1 20.8 6 15.4 plasma folate, MMA, and tHcys concentrations for the anemic
Energy from enset products, % 15.4 6 16.2 66.1 6 15.6 and nonanemic women were comparable (data not shown),
1
whereas the mean plasma cystathionine was higher in the
Values are means 6 SD and mean differences (95% CI) unless noted otherwise.
2
From 2-tailed t test. Equality of variances not assumed. *Means differ, P , 0.001.
anemic group than the nonanemic group (173 vs. 128 nmol/L;
3
Median (IQR). In the right-hand column, the test statistic is the 2-tailed asymptotic mean difference: 43.8, 95% CI: 6.9, 80.7).
significance from the Mann-Whitney U test.
Interrelations among laboratory variables. Spearman rank
correlation coefficients were significant between plasma folate
and 15.0 6 4.3 mg/100 g (fresh weight), respectively, whereas the and plasma cobalamin (r ¼ 0.265; P ¼ 0.015), plasma folate and
corresponding analyzed values for vitamin B-12 were 3.9 mg/100 g MMA (r ¼ 0.298; P ¼ 0.006), and plasma zinc and hemoglobin
and 3.7 mg/100 g, respectively. Hence, kocho contributed 58.1 (r ¼ 0.328; P ¼ 0.001). Negative correlations were noted
mg/d folate and 6.8 mg/d vitamin B-12 for the enset diet group between hemoglobin and plasma cystathionine (r ¼ –0.281; P ¼
compared with 18.0 mg/d folate and 2.1 mg/d vitamin B-12 for 0.007) and plasma folate and plasma cystathionine (r ¼ –0.357;
the maize diet group. P ¼ 0.001). No significant correlations existed between plasma
zinc and ferritin or between plasma tHcys and any of the other
Biochemical indices. Women with an elevated plasma CRP folate or vitamin B-12 indices.
indicative of acute infection or inflammation (.5 mg/L; n ¼ 8)
had a significantly lower mean hemoglobin, RBC count, and Predictors of hemoglobin status. In the multiple regression
plasma retinol (P , 0.05) than did those with a plasma CRP model with the largest explanatory effect on hemoglobin (Table 4),

TABLE 3 Selected hematologic and biochemical indices in the maize- and enset-based diet groups1,2

Mean difference for

maize vs. enset
All subjects Maize-based diet Enset-based diet (95% CI)

n % n % n %
Hemoglobin, g/L 84 17 121 6 11.1 56 14 122 6 10.6 28 21 118 6 11.9 3.7 (–1.6, 9.1)
RBC count, 3 1012/L 84 37 3.9 6 0.41 56 36 3.9 6 0.40 28 39 3.9 6 0.44 20.01 (–0.21, 0.19)
MCV, fL 84 5 88.9 6 4.2 56 2 89.6 6 3.7 28 11 87.3 6 4.8 2.30 (0.21, 4.39)*
Mean cell hemoglobin 83 0 349 6 10 55 0 350 6 10 28 0 346 6 10 4.36 (–0.32, 9.04)
concentration, g/L
Plasma zinc, mmol/L 84 74 6.99 6 1.09 57 77 7.05 6 1.03 27 67 6.86 6 1.22 0.19 (-0.36, 0.73)
Plasma ferritin, mg/L 86 33 13.9 (10.7, 23.7) 58 38 13.6 (9.8, 24.4) 28 21 14.5 (12.5, 21.8) 0.396
Plasma retinol, mmol/L 85 27 25 (19, 31) 62 21 0.96 6 0.35 23 39 0.76 6 0.35 0.20 (0.04, 0.36)*
Plasma vitamin B-12, pmol/L 83 23 268 (152, 372) 56 25 238 (147, 345) 27 18 291 (220, 449) 0.888
Plasma MMA, nmol/L 94 62 407 6 206 65 60 398 6 211 29 66 425 6 198 227 (–117, 64)
Plasma cystathionine, nmol/L 83 0 126 (94, 165) 65 3 135 (109, 172) 29 0 111 (88, 132) 0.020*
Plasma folate, nmol/L 94 2 26.1 6 12.6 56 0 25.4 6 12.1 27 0 27.6 6 13.7 22.2 (–8.4, 4.0)
Plasma homocysteine, mmol/L 94 0 3.5 (3.1, 4.1) 65 0 3.5 (3.0, 4.2) 29 0 3.3 (3.1, 4.1) 0.703
1
Values are means 6 SD or median (IQR). The right-hand column is the mean difference (95% CI) or the 2-tailed asymptotic significance for the differences in the medians from
the Mann-Whitney U test. *Means differ, P , 0.05.
2
For the hematologic variables, plasma zinc, plasma ferritin, and plasma retinol, only subjects with plasma CRP #5 mg/L are included. Cutoffs used: hemoglobin ,115 g/L; RBC
count ,3.8 3 1012/L; MCV ,81 fL; mean cell hemoglobin concentration ,320 g/L; plasma zinc ,7.6 mmol/L; plasma ferritin $12 mg/L and plasma retinol ,0.70 mmol/L; plasma
vitamin B-12 ,150 pmol/L; plasma MMA .271 nmol/L; plasma cystathionine .342 nmol/L; plasma folate ,6.8 nmol/L; and plasma homocysteine .13.9 mmol/L.

Hemoglobin in Ethiopian pregnant women 583

 TABLE 4 Multiple linear regression analysis with hemoglobin as the dependent variable1,2
Unstandardized coefficients
Model B SE
(Constant) 6.93 0.74
Plasma zinc, mmol/L 0.42 0.10
CRP # 5 mg/L 1.35 0.37
Gravida # 4 0.73 0.22
Plasma ferritin $ 12 mg/L 0.60 0.23
1
n ¼ 88, R2 ¼ 0.37, adjusted R2 ¼ 0.34.
2
The pairwise correlation coefficients for all of the independent variables in the linear regression model are all nonsignificant (data not
shown), suggesting that colinearity does not compromise the regression model.
3
Standardized coefficients are estimates from the analysis performed on the standardized variables so that each variable has a variance of 1.0.
there was no evidence of multiple colinearity or interactions
among the independent variables. Plasma zinc had the largest
standardized b coefficient, followed by CRP, gravida, and
plasma ferritin (Table 4). None of the biochemical indices of
folate or vitamin B-12 status, plasma retinol, or plasma albumin
were significant predictors of hemoglobin concentrations. Re-
moval of plasma zinc from the multiple regression model
produced the largest reduction in the percentage of variance
explained, from 37.3 to 24.0%.
Discussion
The most notable finding related to this group of women from
subsistence farming households in Sidama, Ethiopia was the low
prevalence of IDA and the absence of any hematopoietic defects
associated with folate or vitamin B-12 deficiency during preg-
nancy. This is not withstanding the negligible consumption of
animal products. Consumption of fermented enset products may
have increased vitamin B-12 levels in the diet and plasma and
thus reduced the risk of vitamin B-12 deficiency anemia,
although there was some evidence of an early stage of functional
vitamin B-12 deficiency, based on elevated MMA values.
Interrelationship between diet and vitamin B-12 and folate
status. Several vitamin B-12-producing microorganisms have
been isolated during the fermentation of enset, including
Lactobacillus planatarum (10). Hence, the tendency for women
in the enset diet group to have higher plasma vitamin B-12
concentrations than those in the maize diet group might be
associated with the microbial contribution of vitamin B-12 from
fermented kocho. Certainly, based on our intake data and
chemical analysis of kocho, vitamin B-12 intakes in the enset-
diet group were more than twice the level set by WHO/FAO (25)
for the estimated average requirement during pregnancy (i.e. 2.2
mg/d). Even in the maize diet group, the contribution of vitamin
B-12 from fermented enset was considerable (i.e. 2.1 mg vitamin
B-12/d).
Notwithstanding the apparently large contribution to the diet
of vitamin B-12 by fermented enset, mean MMA levels did not
differ between the 2 diet groups. Indeed, the prevalence of
elevated MMA concentrations, considered to be the most
sensitive indicator of vitamin B-12 deficiency (26), was very
high in both groups (Table 4). This apparent discrepancy
suggests that the fermented enset might have contained some
nonfunctional analogs of vitamin B-12 as well as the cobalamins
that were measured in the radioassay. This may have led to the
apparently normal plasma vitamin B-12 concentrations in some
584 Gibson et al.
Standardized coefficients3 95% CI for B
b Lower bound Upper bound
5.36 8.50
0.37 0.23 0.62
0.31 0.60 2.09
0.28 0.28 1.17
0.23 0.14 1.05
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of the women whose plasma MMA values were unexpectedly
high.
L. planatarum is also a folate-producing microorganism (27)
so it is not surprising that fermented enset also contributed some
folate, albeit a much smaller amount in relation to the WHO/
FAO (25) estimated average requirement for folate during
pregnancy (i.e. 520 mg/d). There were several additional sources
of folate in the diets of both groups, most notably kale (Brassica
carinata), kidney beans (Phaseolus vulgaris L.), and haricot
beans (Phaseolus adenguarre) (8), which probably accounted for
the absence of folate deficiency, based on low plasma folate and
elevated tHcys concentrations, in both diet groups (Table 3).
Predictors of hemoglobin. Plasma zinc concentrations were
the strongest predictor of hemoglobin, followed by the indicator
variables for infection (CRP #5 mg/L), gravida (#4), and
plasma ferritin ($12 mg/L) (Table 4). Moreover, there were no
significant pairwise correlations linking the independent vari-
ables, including plasma ferritin and zinc (data not shown). The
predictors of hemoglobin in Table 4 are discussed in turn below,
along with protein and vitamin A, also required for normal
hematopoiesis.
The strong positive association between plasma zinc and
hemoglobin was unexpected, although it has been reported in
some earlier studies (28–31). There have also been a few
intervention studies in which the addition of zinc alone (32), or
zinc and iron relative to iron alone (33,34), has improved the
hematological response of young children who were thought to
be zinc deficient. Several mechanisms may be involved whereby
zinc affects hemoglobin concentrations. Zinc is implicated in
hemoglobin synthesis through the activity of several zinc-
dependent enzyme systems, including aminolevulinic acid dehy-
drase that mediates a step in the synthesis of heme (35) and
thymidine kinase and DNA polymerase, which are involved in
DNA synthesis. More recently, the zinc-finger transcription
factor, GATA-1, has also been confirmed as essential for normal
erythropoiesis (36). Other potential mechanisms may involve
the stimulation of hematopoiesis by zinc-induced increases in
plasma insulin-like growth factor-1 levels (37) and the role of
zinc in stabilizing cell membranes (38).
An elevated CRP (.5 mg/L) was negatively associated with
hemoglobin, notwithstanding the relatively small number of
subjects (n ¼ 8) with elevated values indicative of underlying
acute infection or inflammation. The latter is often accompanied
by anemia, commonly termed anemia of chronic disease (ACD),
which results from the effects of cytokines. Apparently normal
or increased iron stores are present in ACD (4).
 Gravida also had an independent and significant inverse
association with hemoglobin (Table 4), a relationship that has
also been reported in pregnant women in rural Malawi (2). This
negative relationship has been attributed to the cumulative
demands on iron stores of successive pregnancies and short
interpregnancy intervals (39).
Plasma ferritin was also a significant predictor of hemoglo-
bin, although its importance, based on the absolute value of its
standardized coefficient, was less than that for plasma zinc
(Table 4). Dietary factors probably account in part for the
positive association between plasma ferritin and hemoglobin
noted here. Certainly, dietary iron intake in Ethiopia is charac-
teristically high (8,40,41), some of which is absorbed. In
Sidama, absorption of nonheme iron may be facilitated by
colonization of L. planatarum from fermented enset (42).
Several other reports have confirmed that IDA is not a serious
problem in Ethiopia, even during pregnancy (40,41), compared
with other countries in Sub-Saharan Africa (e.g. Malawi) (2). Of
the nondietary factors, helminth parasitic infections simulta-
neously reduce hemoglobin and ferritin concentrations and thus
could account for the positive b coefficient observed here. In
contrast in ACD and malaria there is a negative relationship
between hemoglobin and ferritin (2,4). Hence, our observations
imply that neither ACD nor malaria were significant in these
pregnant women, a suggestion supported by the relatively small
number of subjects (n ¼ 8) with elevated CRP values.
Unlike ferritin, neither plasma vitamin B-12 nor MMA was a
significant predictor of hemoglobin, despite the high prevalence
of elevated concentrations of MMA (i.e. 62%). This discrepancy
is attributed to nonfunctional vitamin B-12 analogs present in
the plasma of women consuming fermented enset, as discussed
earlier, as well as the absence of any hematopoietic defects
associated with vitamin B-12-deficiency anemia. Plasma folate
was also not a significant predictor of hemoglobin, although
levels were negatively correlated with plasma cystathionine (r ¼
–0.357; P ¼ 0.001) (but not tHcys). This finding may indicate
some folate (or pyridoxine) deprivation, although plasma folate
may not reflect true folate status in malarial endemic areas such
as Sidama, where levels may be elevated through erythrocyte
hemolysis induced by malaria.
Plasma retinol concentrations were not positively associated
with hemoglobin (Table 3) in contrast to some earlier findings in
Ethiopia (43) and elsewhere (44). Likewise, plasma albumin
levels were also unrelated to hemoglobin concentrations despite
low levels, attributed to low protein intakes (8), hemodilution of
pregnancy, infection, and zinc deficiency.
Our findings are based on an observational study conducted
on a self-selected sample of women. Hence, causal inferences
cannot be made from the associations reported in this study and
the results cannot be extrapolated to all women in the 3rd
trimester of pregnancy living in the Sidama Zone of Ethiopia
(45). Also, our sample size was small and this may have
contributed to the null findings of differences in micronutrient
status between the 2 groups. However, fermented enset products
are reported to be the staple food for more than more than 10
million Ethiopians (46) and further investigation into the
presence of vitamin B-12 analogs in the plasma of women
consuming fermented enset and in the food itself is warranted.
In summary, there was some evidence of early functional
deficiency of vitamin B-12 but not folate deficiency among these
pregnant women and no vitamin B-12 deficiency anemia, despite
very low intakes of animal products. The prevalence of IDA was
also low compared with elsewhere in Africa. Consumption of
enset fermented by vitamin B-12-producing microorganisms
may have increased vitamin B-12 levels in the diets and in
plasma while at the same time enhancing nonheme iron
absorption. Two micronutrients (zinc and iron status) and 2
nondietary factors, infection and gravida, were the major
predictors of hemoglobin. A well–designed, randomized, con-
trolled trial of multi-micronutrient supplements with and
without zinc, conducted in a population group at high risk for
zinc deficiency such as the Sidama pregnant women studied here,
is needed to establish the role of zinc deficiency in the etiology of
anemia.
Acknowledgments
The authors acknowledge the invaluable contributions of
Alemtsehay Bogale, and Elsa and Meberat, the two senior
community research workers, as well as Keneni Fufa (labora-
tory coordinator), Isabel Arbide, and Akilu Teshome from
Downloaded from https://academic.oup.com/jn/article/138/3/581/4670259 by guest on 11 June 2023
Busholo Health Center. The folate analysis of kocho and bulla
was performed in the laboratory of Professor T. Tamura,
University of Alabama, Birmingham. We are particularly
grateful for this assistance.
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