JOURNAL OF

FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 20 (2007) 161168
Original Article
Phytate, zinc, iron and calcium content of selected raw and prepared
foods consumed in rural Sidama, Southern Ethiopia, and implications
for bioavailability
Yewelsew Abebe
a
, Alemtsehay Bogale
a
, K. Michael Hambidge
b
, Barbara J. Stoecker
c
,
Karl Bailey
d
, Rosalind S. Gibson
d,
a
Debub University, P.O. Box 5, Awassa, Ethiopia
b
University of Colorado Health Sciences Center, Denver, CO 80262, USA
c
Oklahoma State University, Stillwater, OK 74078, USA
d
Department of Human Nutrition, University of Otago, P.O. Box 56, Dunedin 9015, New Zealand
Received 10 April 2006; received in revised form 11 September 2006; accepted 29 September 2006
Abstract
Representative staple foods from Sidama, Southern Ethiopia, were analyzed for phytate using HPLC, and for Zn, Fe and Ca by ame
atomic absorption spectrophotometry (AAS). Enset starchy foods had the lowest phytic acid content, followed by fermented injera
prepared from tef. Oleaginous seeds (niger and sesame) had the highest phytate content (1600 mg/100 g). The iron content of raw tef
and tef injera, unlike barley our or corn bread, varied markedly, attributed to contaminant iron from soil. The foods prepared from
enset and tef were also rich sources of calcium. Most of the fermented foods prepared from enset and tef had low Phy:Zn and Phy:Fe
molar ratios, whereas corn bread (unleavened), kidney beans, sesame, and niger seeds had higher molar ratios. Absorption of intrinsic
Zn, Fe, and Ca as well as any exchangeable contaminant iron is unlikely to be compromised by phytate in the fermented foods prepared
from enset and tef, unless consumed together with high phytate foods such as corn bread, legumes, and oil seeds.
r 2006 Elsevier Inc. All rights reserved.
Keywords: Ethiopia; Foods; Enset; Maize; Tef; Barley; Kidney beans; Sesame; Niger; Calcium; Iron; Zinc; Phytate
1. Introduction
Cases of human zinc deciency were rst described as
early as the 1960s in male adolescent dwarfs from the
Middle East (Prasad et al., 1963). Since that time, zinc
deciency has been identied in many other regions of the
world, including Ethiopia (Umeta et al., 2000). Three
etiological factors are associated with the development of
zinc deciency: dietary inadequacies; disease states which
induce excessive losses or impair utilization of zinc; and
physiological states that increase zinc requirements (Hotz
and Brown, 2004). Of these, inadequate dietary intakes are
a major factor that can be attributed to low intakes of zinc
per se and/or to poor bioavailability.
Poor bioavailability is particularly a problem in diets
based on unrened cereals and legumes with a high content
of phytate. The latter refers to phytic acid (myo-inositol
hexaphosphate) as well as the associated magnesium,
calcium and potassium salts. It is the major storage form
of phosphorus in cereals, legumes and oleaginous seeds.
Phytate is negatively charged under physiological condi-
tions and thus strongly chelates metal ions, especially zinc,
but also iron and calcium, forming insoluble complexes in
the gastrointestinal tract that cannot be digested or
absorbed in humans because of the absence of intestinal
phytase enzymes (Iqbal et al., 1994). Phytate also
complexes endogenously secreted minerals such as zinc
(and calcium), making them unavailable for reabsorption
into the body (Sandstro m, 1997). High amounts of calcium
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Corresponding author. Tel.: +64 3 479 7955; fax: +64 3 479 7958.
E-mail address: Rosalind.Gibson@Stonebow.Otago.AC.NZ
(R.S. Gibson).
may exacerbate the inhibitory effect of phytate on zinc
absorption by forming a calciumzincphytate complex in
the intestine that is even less soluble than phytate
complexes formed by either ion alone (Lo nnerdal, 2000).
Calculating zinc intakes in developing countries is often
limited by the paucity of data on the zinc content of local
staple foods. Substitution of zinc values for staple foods
grown in western countries is not advisable because the
zinc content of plant-based foods tends to reect local soil
zinc levels (Sunanda et al., 1995; Alloway, 2004). In
Ethiopia, the zinc content of prepared foods is available in
the latest revision of the Ethiopian Food Composition
Table (Ethiopian Health and Nutrition Research Unit
(EHNRI), 1998), so that total zinc intakes can be
calculated from food consumption data. However, because
phytate data are not included in this publication, the
algorithms developed to estimate available zinc intakes
could not be used. They all require data on the proportion
of phytic acid to zinc in the habitual diets (Murphy et al.,
1992; World Health Organization (WHO), 1996; Food and
Agricultural Organization/World Health Organization
(FAO/WHO), 2002; Hotz and Brown, 2004). Phytate
values are also needed to calculate available iron intakes
(Tseng et al., 1997; Hallberg and Hulthe n, 2000; Reddy
et al., 2000), and to assess calcium bioavailability from
habitual diets (Murphy et al., 1992; WHO, 1996; FAO/
WHO, 2002; Hotz and Brown, 2004).
In many developing countries where zinc deciency has
been reported, nutritional iron deciency is also common.
This is not surprising because iron and zinc have a similar
distribution in the food supply, and several of the dietary
components that modify the bioavailability of zinc also
affect iron in a similar way (Gibson and Ferguson, 1999).
Surprisingly, nutritional iron deciency is not a serious
problem in Ethiopia (Hofvander, 1968; Gebre-Medhin
et al., 1976; Wolde-Gebriel et al., 1993), although
secondary anemia due to hookworm and malaria occurs
in some areas (Zein and Assefa, 1987). The absence of
nutritional iron deciency anemia has been attributed to
the consumption of the indigenous high-iron cereal tef
(Eragrostis tef), and some oleaginous seeds and kale, all
relatively high in iron.
To our knowledge, there is only one report on the
phytate content of Ethiopian plant-based foods (Umeta
et al., 2005). In this study, phytate was measured colori-
metrically (Haug and Lantzsch, 1983), a method that does
not distinguish between inositol phosphates with differing
numbers of phosphate groups. This is unfortunate because
only the higher inositol phosphates inhibit absorption of
zinc (Lo nnerdal et al., 1989) and iron (Sandberg et al.,
1999), and these will be hydrolyzed to the lower inositols in
the fermented staple foods (e.g., tef injera and enset)
consumed in several regions of Ethiopia, including Sidama.
Therefore, in this study we have analyzed the phytate,
and zinc, iron and calcium content of selected raw and
prepared foods consumed in rural Sidama in Southern
Ethiopia, a region where starchy fermented foods prepared
from enset (Enset ventroicosum), also known as false
banana, are major staples in the diet. We used high-
performance liquid chromatography (HPLC) (Lehrfeld,
1989) to separate and quantify the higher inositol
phosphates hexa-(IP-6) and penta-(IP-5) from the lower
inositol phosphates, which do not inhibit mineral bioavail-
ability. The relative bioavailability of zinc and iron in these
raw and prepared foods was also determined by calculating
their corresponding phytate:zinc and phytate:iron molar
ratios. The food composition data generated are part of a
larger study on the zinc status of pregnant women living in
this region of Southern Ethiopia.
2. Materials and methods
2.1. Sample collection
Five samples of 17 raw, non-perishable foods, each
weighing approximately 250 g, were purchased in August
2004 from local markets and households located in three
rural Sidama communities located in the Kolla or Rift
Valley lowlands of Southern Ethiopia. Each sample was
prepared as eaten by removing inedible husks, seeds,
leaves, rind, or pits, where necessary, using local utensils
and traditional methods. The ve as eaten samples of
each raw food type were then combined to form one
composite food sample per food type. After thorough
mixing of the composite sample, two representative
portions of each were removed, weighed and stored
individually in labeled, sealed trace-element-free polyethy-
lene bags. A complete description of the non-perishable
raw foods, including the common names with local
synonyms and scientic taxonomic names together with
details of the traditional methods used to prepare them as
eaten, where appropriate, are shown in Table 1.
Some perishable food items prepared using traditional
processing, preparation and cooking methods were also
purchased from households and local markets (details are
given in Table 3). Of these, a more detailed explanation of
the traditional processing methods used for the starchy
food staples prepared from enset (E. ventricosum), com-
monly known as false banana, are also shown in Table 1.
For some of the foods that were frequently consumed in
the households surveyed, several samples of the same food
type, each weighing about 150 g, were collected and
analyzed separately to provide information on variability
within one food type. For the three types of injera, a
pancake-like bread prepared from fermented tef dough, a
single composite of each type made up of ve samples as
noted above, was analyzed to reduce the analytical costs.
The perishable food samples were oven-dried (95105 1C)
in a laboratory in Debub University in order to conform to
the importation regulations of the Ministry of Food and
Agriculture, New Zealand. Drying at this temperature will
not affect the content of minerals of interest (Horwitz,
2002) or phytate (Hurell et al., 2004).
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Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 162
After recording the dry weight, individual foods or the
dried samples of injera prepared from tef, each weighing
about 125 g, were transferred to self-closing trace-element
free polyethylene bags, and the contents crushed on the
outside with a rolling pin to yield a ne powder. The
ground dried samples of each type of injera were combined
to form one composite sample per injera type, from which
two weighed portions were withdrawn, as noted above.
All food samples were transported to New Zealand where
they were stored at 481C in the trace element laboratory
at the University of Otago, Dunedin, New Zealand until
analysis.
2.2. Analysis
One portion (approximately 200 g) of each of the food
samples to be analyzed was ground again to a ne
homogenous powder using an acid-washed agate ball mill
(Brinkman Model MM2, Brinkman Instruments Division,
Sybron, Canada Ltd.); two aliquots of each were used for
the chemical analysis.
2.2.1. Atomic absorption analysis
Approximately 5 g of each powdered composite food
sample was dry-ashed at 450 1C in a mufe furnace
following procedures described earlier (Ferguson et al.,
1989). Ashed samples were dissolved in 6 mL Hyper-
pure HCl (Arista, BDH Laboratory Supplies), and
the solutions quantitatively transferred into acid-
washed 100 mL volumetric asks containing 2 mL 6 M
Hyperpure HCl, and then adjusted to volume with
distilled, deionized water. The zinc, calcium and iron
content of the solutions were analyzed by ame atomic
absorption spectrophotometry (AAS) (AA-800, Perkin-
Elmer Corp., Norwalk, CT, USA). Standards were
prepared from stock standard solutions of zinc, iron and
calcium (BDH Laboratory Supplies). For calcium analysis,
lanthanum chloride (1%) was added to both samples and
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Table 1
Local and scientic names of raw staple foods and processed enset starchy foods analyzed from Sidama, Ethiopia
Food type Local name Scientic name Description
Cereals
Barley, white, our: roasted and
then milled
Gebs, nech Hordeum vulgare L. Dehusked barley, roasted on wood re and milled
locally
Tef, red: whole grain Tyef, qeyy Eragrostis tef (Zucc.) Trott Threshed under the hooves of cattle. After
removing visible soil contamination, tef grains
washed with tap water
Tef, mixed: whole grain Tyef, dibilliq, duqyet Eragrotis tef (Zucc.) Trott As above
Tef, white: whole grain Tyef, nech, duqyet Eragrotis tef (Zucc.) Trott As above
Maize, white: whole dried kernels Bequolo, nech Zea mays L. Maize kernels removed from cob and sun dried
Maize, white, 95% extraction our Bequolo, nech, duqyet Zea mays L. Sun dried maize kernels milled locally
Legumes
Broad beans: whole, dried Baqyela, din, dereq Vicia faba L. Includes seed coat; sun dried
Chickpeas: whole, dried Shimbra, diffn, dereq Cicer arietinum Includes seed coat; sun dried
Fenugreek, white: roasted and
powdered
Abish, duqyet Trigonella foenum-graecum L. Roasted on wood re; powdered in pestle and
mortar
Haricot beans, white: whole, dried Adengwarrye Phaseolus adenguarre Includes seed coat; sun dried
Kidney beans, red: whole, dried Din, dereq Phaseolus vulgaris L. Includes seed coat; sun dried
Lentils :whole, dried Missir, din, dereq Lens culinaris Med Sun dried
Peas, eld: whole, dried Ater, diffn, dereq, nifro Pisum sativum L. Includes seed coat; sun dried
Oleaginous seeds
Sesame seed: whole Selyit, dereq Sesamum indicum L.
Niger seed: whole Zeyt, nug Guizotia abyssinica
Enset starchy foods
False banana: pulp, fresh Kocho, fresh Enset ventricosum Mixture of decorticated enset pseudostem and
pulverized corm
False banana: pulp, fermented Kocho, fermented Enset ventricosum Pre-fermented pseudostem base added as starter
to fresh kocho which is left to ferment in an earth
pit from 3 weeks to 1 year. After fermentation,
mixture squeezed, shredded and sifted to remove
unwanted ber
False banana: desiccated juice from
enset pulp
Bulla Enset ventricosum Desiccated juice squeezed from decorticated
pseudostem and corm mixture fermented while
exposed to air and light to retain white colour
False banana: decorticated Amicho Enset ventricosum Boiled decorticated corm (root part) located at
base of pseudostem of enset
Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 163
standard solutions to overcome interference from phos-
phorus.
2.2.2. Phytate analysis
For the analysis of inositol hexa-(IP6) and penta-(IP5)
phosphates, the HPLC procedure of Lehrfeld (1989), as
modied by Hotz and Gibson (2001), was followed.
Aliquots of the powdered food samples (0.5 g) were
analyzed in duplicate using a Waters 2690 separations
Module (Waters, Milford, MA, USA) and a differential
refractometer (410 Differential Refractometer, Waters,
MA, USA), after extraction and separation of the inositol
phosphates by a Hypersil column (H30DS-250A, Hi-
CHROM, Berkshire, England). IP5 and IP6 concentrations
were calculated from regression equations derived from
the different concentrations of the standard solutions and
peak areas of the samples. Samples of sesame seed and
niger seeds were defatted prior to extraction and separation
of the inositol phosphates using standard procedures
(Horwitz, 2002).
2.2.3. Quality control
To minimize the risk of adventitious contamination, all
glassware used for the analytical methods was acid-washed,
and sterile disposable powder-free plastic gloves were worn
when handling the foodstuffs during the sampling and
analyses stages.
Aliquots of two standard reference materials (SRMs)
citrus leaves (SRM-1572) and rice our (SRM-1568a)
purchased from the National Institute of Standards and
Technology (Gaithersburg, MD, USA) were analyzed
with each batch of analysis to check on the accuracy
and precision of the AAS procedures. The mean7SD and
coefcient of variation (CV as %) for the zinc, iron and
calcium content of citrus leaves (SRM-1572) (n 5) were:
Zn, 29.071.3 mg/g, (CV 4.5%); Fe, 82.373.9 mg/g (CV
4.7%); and Ca, 3198872666 mg/g (CV 8.3%) compared to
certied values of Zn, 29.272 mg/g; Fe, 90710 mg/g; and
Ca, 31,50071000 mg/g. Corresponding values for rice our
(SRM 1568a) (n 5) were: Zn, 19.170.2 mg/g (CV 1.0 %);
Fe, 6.970.6 mg/g (CV 4.7%); and Ca, 11176 mg/g (CV
5.1%) compared to certied values of Zn, 19.470.5 mg/g;
Fe, 7.470.9 mg/g; and Ca, 11876 mg/g.
The HPLC method for phytate has an inter-run
coefcient of variation of 7.2%. Our analyzed value
(mean7SD) for maize our (65% extraction) (n 3) was
292721 mg/100 g IP57IP6 compared to our previously
reported value of 284722 mg/100 g.
3. Results
The zinc, iron, calcium and phytate contents and the
phytate:zinc and phytate:iron molar ratios for the raw,
non-perishable and perishable prepared food samples,
expressed on a fresh weight basis, are shown in Tables 2
and 3. Moisture values are given for the prepared foods
only.
Of special note is the very high and variable iron content
of the indigenous Ethiopian cereal tef (E. tef) compared to
that of the other cerealsmaize or barley (Table 2). The
iron content of the oleaginous seedssesame (Sesamum
indicum L.) and niger (Guizotia abyssinica)was also high
in comparison to levels in the maize, barley and legumes,
except lentils. Of all the raw foods analyzed, both sesame
and niger seeds had the highest calcium content, followed
by chickpeas, fenugreek, and then tef, kidney beans and
eld peas. Whole dried kernels of white maize and
unrened maize our had the lowest calcium content.
The zinc content of the raw food samples varied the least,
values ranging from 1.83 mg/100 g for whole dried broad
beans to 4.75 mg/100 g for dry kidney beans. Zinc values
for the three varieties of tef were much lower
(2.864.02 mg/100 g) and much less varied than their
corresponding values for iron. The zinc content of barley
our was comparable to that of mixed tef, and slightly
higher than that of unrened maize our. Not surprisingly,
the oleaginous seedssesame and nigerhad the highest
phytate (i.e., IP5+IP6) content (1600 mg/100 g), whereas
dried eld peas had the lowest. The phytate content of raw
tef ranged from 842 mg/100 g for white tef to 528 mg/100 g
for mixed tef.
Most of the raw foods had phytate:zinc molar ratios
above 15, with the exception of roasted barley our, certain
legumes (broad beans, chick peas, fenugreek, kidney beans,
lentils and eld peas) and mixed tef. Field peas had the
lowest phytate:zinc molar ratio and sesame and niger seeds
had the highest. Similarly, only two of the raw foods (red
tef and mixed tef) had phytate:iron ratios below the critical
ratio of 1:1 suggested by Hurrell (2004).
Table 3 presents the zinc, iron, calcium and phytate
content (mg per 100 g fresh weight) and the phytate:zinc
and phytate:iron molar ratios of the perishable foods
prepared from the major plant-based staples of the region:
enset (E. ventroicosum), commonly known as false banana,
tef (E. tef) and maize (Zea mays L.). There was a wide
range in the mineral content of the three starchy fermented
foods prepared from enset; details are given in Table 1.
Again, the content of iron of these staple foods was more
varied than that of zinc, with amicho, the decorticated
corm (Table 1) often boiled and served as a snack, having
the lowest iron content (0.7 mg/100 g), and fermented
kocho baked into bread, having the highest (6.2 mg/100 g).
In contrast, their zinc content ranged from 0.07mg/100 g for
bulla (the white low-bre desiccated juice from enset pulp) to
1.33 mg/100g fresh weight for amicho. Amicho, followed by
bulla, had a much lower content of calcium than fermented
baked kocho. Indeed, fermented, baked kocho had the
highest level of calcium of all the perishable, prepared foods
analyzed in Table 3.
Of the three types of fermented tef injera samples analyzed,
only one had a very high iron content (71mg/100g)
compared to 7.0mg/100g for the other two injera samples
and 3.4mg/100g for unleavened corn bread. The zinc content
of all the prepared cereal products was very similar, ranging
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Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 164
from 0.93mg/100g for injera prepared from a mixture of red
and white tef to 1.75mg/100g for injera prepared from red
tef. The calcium content of the unleavened corn bread was
lower than that of fermented tef injera. Not surprisingly, all
the starchy fermented foods prepared from enset and
fermented tef injera had levels of IP5+IP6 that were much
lower than those of the raw staples (Table 2), and the
unleavened corn bread. Indeed the IP5+IP6 content of all
prepared foods analyzed, with the exception of unleavened
corn bread and boiled kidney beans, was relatively low.
Hence, only unleavened corn bread had a calculated
phytate:zinc molar ratio above 15, whereas corn bread,
boiled kidney beans and injera prepared from white tef had
phytate:iron ratios above 1.0.
4. Discussion
One of the most striking ndings of this study was the
high and variable iron content of both the indigenous
small-grained cereal tef (E. tef), and injera, the traditional
pancake-like thin leavened bread prepared from fermented
tef. These ndings are not unexpected: published food
ARTICLE IN PRESS
Table 2
Zinc, iron, calcium and phytate content (mg/100 g fresh weight) and Phy:Zn and Phy:Fe molar ratios of selected raw cereals, legumes and oleaginous seeds
from Sidama, Ethiopia
Food type (n) Zinc
(mg/100 g)
Iron
(mg/100 g)
Calcium
(mg/100 g)
Phytate
(mg/100 g)
Phy:Zn Phy:Fe
Cereals
Barley, white our: roasted, milled with cardomen 3.57 8.4 45 370 10.3 3.7
Barley, white our: roasted, milled 3.29 9.4 46 452 13.6 4.1
Barley, white our: roasted with fenugreek 3.54 10.4 40 553 15.5 4.5
Red tef: whole grain 4.02 4150.0 155 675 16.6 o0.4
Mixed red and white tef: whole grain 3.86 4150.0 147 528 13.6 o0.3
White tef: whole grain 2.86 37.7 124 842 29.2 1.9
Maize, white: whole dried kernels 4.04 4.4 16 1443 35.4 27.8
Maize, white: 95% extraction our 2.15 4.9 12 661 30.5 11.4
Legumes
Broad beans: whole, dried 1.83 5.0 101 478 11.2 8.1
Chickpeas: whole, dried 3.35 13.4 213 305 9.0 1.9
Fenugreek, white: roasted and powdered 3.76 18.5 179 480 12.6 2.2
Haricot beans, white: whole, dried 2.92 10.8 78 604 20.5 4.8
Kidney beans, red: whole, dried 4.75 12.2 126 716 14.9 5.0
Lentils: whole, dried 3.84 38.4 42 561 14.5 1.2
Peas, eld: whole, dried 4.13 5.0 90 235 5.6 4.0
Oleaginous seeds
Sesame seed: whole 2.48 64.2 359 1525 60.9 2.0
Niger seed: whole 2.73 58.4 291 1690 61.4 2.5
Table 3
Median (1st, 3rd quartile) zinc, iron, calcium and phytate contents (mg/100 g fresh weight) of perishable prepared foods from Sidama, Ethiopia
Food type (n) Moisture
(g/100 g)
Zinc
(mg/100 g)
Iron
(mg/100 g)
Calcium
(mg/100 g)
Phytate
(mg/100 g)
Phy:Zn Phy:Fe
Enset starchy foods
Kocho, fresh: pulp (3) 85 (82, 86) 0.09 (0.08, 0.15) 1.1 (0.8, 1.6) 52 (46, 60) 7 (6, 9) 8.4 0.9
Kocho, fermented: pulp, baked (16) 36 (26, 44) 0.52 (0.32, 0.72) 6.2 (3.6, 10.1) 162 (140, 226) n.d.
Bulla: desiccated juice from enset pulp (4) 57 (54, 58) 0.07 (0.05, 0.09) 4.8 (3.6, 6.5) 44 (40, 47) n.d.
Amicho: decorticated tuber before
fermentation (4)
76 (71, 77) 1.33 (1.03, 1.61) 0.7 (0.6, 1.1) 25 (24, 29) 0.9 (0.2, 13.0 0.1 0.6
Cereals
Tef dough unfermented (3) 57 (35, 66) 1.38 (1.19, 1.96) 6.8 (6.7, 26.4) 65 (51, 91) 139 (136, 144) 8.4 0.6
Injera: from fermented white tef dough (1) 71.6 1.11 6.8 36 102 9.1 1.3
Injera: from fermented white and red tef
dough (1)
67.8 0.93 7 35 36 3.8 0.4
Injera: from fermented red tef dough (1) 56.5 1.75 70.9 76 117 6.6 0.1
Corn bread, unleavened (16) 35 (29, 49) 1.66 (1.36, 1.84) 3.4 (3.0, 5.5) 14 (12, 20) 394 (366, 442) 21.6 5.4
Legumes
Kidney beans, red: whole, dried, boiled (7) 38 (34, 59) 1.52 (1.23, 1.94) 2.7 (2.4, 4.3) 69 (55, 96) 219 (176, 253) 14.6 6.0
Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 165
composition data also report consistently high iron levels
that uctuate markedly in these indigenous Ethiopian
foods (Interdepartmental Committee on Nutrition for
National Defense (ICNND), 1959; A

gren and Gibson,

1968; Ethiopian Nutrition Institute (ENI), 1981; Pijls et al.,
1995; EHNRI, 1998; Umeta et al., 2005).
Contamination from soil rather than iron intrinsic to the
food is believed to be responsible for these high iron levels
in tef. Certainly, the iron contents of both mixed raw red
and white tef (Table 2) and injera prepared from such a
mixture (Table 3) were not consistent with the analytical
differences noted between the iron content of red and white
tef in Table 2. Contaminant iron may arise from the
traditional method of threshing tef under the hooves of
cattle (Bothwell et al., 1979). Indeed, careful washing with
hydrochloric acid has been reported to reduce the iron
content of tef from 39.7 mg/100 g to 3.5 mg/100 g (Hallberg
and Bjorn-Rasmussen, 1981). The extent to which the
contaminant iron from soil in these Sidamo tef samples
joins the common non-heme iron pool (i.e., is exchange-
able), and is thus available for absorption is uncertain
(Hallberg and Bjorn-Rasmussen, 1981). No simple method
of predicting the exchangeability of contaminant iron has
been reported. Most of the studies examining the
bioavailabilty of contaminant iron from soil are based on
in vitro models (Aufreiter et al., 1997; Hooda et al., 2004);
very few have actually measured in vivo absorption of
contaminant iron separately from that of the iron intrinsic
to the food (Harvey et al., 2000). Results of an early radio-
iron absorption study in which soil was added to rice-based
Thai meals, however, led investigators to conclude that at
least some of the iron from clay is available for absorption
(Hallberg et al., 1977). Nevertheless, some investigators
believe that iron from soil is not bioavailable because the
soil minerals have an inherently low solubility in the
intestine (Bothwell et al., 1979). Indeed, when consumed
with food, certain soil minerals may even prevent the
absorption of non-heme iron derived both from the soil
and intrinsic to the food (Hooda et al., 2004).
Ascorbic acid-rich foods such as kale and tomatoes are
often consumed with injera, and the enset starchy foods
bulla and kotcho. It is possible that this ascorbic acid
together with organic acids (e.g., lactic acid) produced
during fermentation of injera act as chelating agents,
increasing the solubility of both the contaminant iron and
intrinsic iron, thus resulting in increased amounts of the
iron being absorbed (Harvey et al., 2000; Teucher et al.,
2004). Support for this suggestion comes from an in vivo
radio-iron absorption study conducted by Derman et al.
(1982) in which the absorption of iron from two common
compounds of contaminant ironferric oxide and ferric
hydroxideadded to maize-meal porridge to simulate
contaminant iron, was substantially increased by the
addition of ascorbic acid to the meal. Further studies are
needed to conrm this suggestion, but the absence of
nutritional iron deciency anemia in Ethiopia in general
(Hofvander, 1968; Gebre-Medhin et al., 1976; Wolde-
Gebriel et al., 1993), and indeed in the pregnant women of
this study (K.M. Hambidge, pers. comm.) provides strong
support for this hypothesis.
Most of the prepared foods analyzed had varied
but often high iron concentrations and consequently
phytate:iron molar ratios below 1.0, with the exception of
the unleavened corn bread, boiled kidney beans and white
tef injera (Phy:Fe 1.27). Phytate begins to lose its inhibitory
effect on iron absorption when phytate:iron molar ratios
are less than 1.0, although even ratios as low as 0.2 exert
some negative effect (Hurrell, 2003). Our results suggest,
therefore, that in general, absorption of intrinsic non-heme
iron from fermented kocho and tef injera as well as any
exchangeable contaminant iron from tef injera, will not be
signicantly inhibited by phytate, unless these foods are
consumed together with others such as unleavened corn
bread, sesame or niger seeds and/or certain legumes.
Unlike for iron, there is no evidence that inadvertent
ingestion of soil affects the zinc or calcium content of
fermented tef injera. Indeed, the zinc and calcium content
of the three varieties of tef injera were analyzed (Table 3),
as well as in the raw cereals, legumes and oleaginous seeds
analyzed in Table 2, are consistent with earlier published
values (ENI, 1981; EHNRI, 1998; Umeta et al., 2005). It is
of interest that the zinc content of all the enset- and tef-
based indigenous foods analyzed was very low (i.e.,
0.071.75 mg/100 g fresh weight). Not surprisingly, the
hexa-inositol (IP6) and penta-inositol (IP5) content of
the fermented tef injeras was lower than that for the
unfermented tef dough, and the three varieties of whole
grain tef (Table 2), and negligible for the starchy enset
products. As a result, the phytate:zinc molar ratios of these
traditional tef and enset food staples were all below 15
(Table 3). Indeed, of the prepared foods analyzed, only
unleavened corn bread had a phytate:zinc molar ratio at a
level likely to markedly compromise zinc absorption.
Similarly, unleavened corn bread, together with boiled
kidney beans, and white tef injera had phytate:iron ratios
above 1.0, a level said to compromise iron absorption
(Hurrell, 2004).
The foods prepared from tef and enset analyzed here are
notably rich in calcium, consistent with earlier reports
(EHNRI, 1998; Umeta et al., 2005). Further, the relative
consistency of the calcium content within each food type,
strongly suggests that soil contamination did not con-
tribute to their calcium content. This is not surprising
because the soils in this Sidama region are clay-rich and not
calcareous (WHO, 1996; Hooda et al., 2004).
It is likely that the bioavailability of calcium from these
indigenous fermented enset- and tef-based foods is high
because of their low phytate content (Table 3). Oxalate
found in high concentrations in certain green leafy
vegetables such as spinach (Bohn et al., 2004), and in
rhubarb is a particularly potent inhibitor of calcium
absorption, even stronger than phytate. Ethiopian kale
(Brassica carinata braun), however, the major vegetable
staple of Sidama households is a low-oxalate vegetable
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Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 166
containing no phytate, and hence will also be a relatively
good source of highly bioavailable calcium (Weaver et al.,
1999; Bohn et al., 2004). In contrast, although the varieties
of dried beans analyzed all contain signicant concentra-
tions of calcium, they are also rich in phytate (Table 2), and
hence will have substantially lower calcium bioavailability
than the foods prepared from tef, enset and kale. The
critical molar ratio above which calcium absorption is
compromised by phytate is uncertain.
Both the calcium and phytate content of the prepared
plant-based foods consumed in Sidama (e.g., kocho, bulla,
injera and corn bread) are unlikely to be high enough
to play a role in adversely affecting zinc bioavailability
(Lo nnerdal, 2000).
In general the phytate values for the indigenous Sidama
fermented foods analyzed here were lower than those
reported earlier by Umeta et al. (2005) for similar foods.
This discrepancy is not unexpected: we used HPLC to
analyze the IP-5 and IP-6 content of our food samples,
whereas Umeta and co-workers measured the total phytate
content, including the lower inositol phosphates arising
from phytase-induced hydrolysis of IP-6 and IP-5 during
fermentation. Further, our results highlight the marked
variability in IP-5 and IP-6 content of injera prepared from
white and/or red fermented tef dough, probably arising
from differences in the fermentation processes. Whether
the adverse effect of inositol phosphates on calcium
absorption (Reinhold et al., 1976) is restricted to IP-5
and IP-6 is uncertain.
In conclusion, our results suggest that contaminant iron,
probably from soil, contributes signicantly to the iron
content of the indigenous cereal tef, and hence injera
prepared from this staple. There is some evidence that
some of this contaminant iron may be exchangeable, and
thus absorbed, perhaps facilitated by the chelating action
of organic acids produced by fermentation of tef and enset,
as well as by the consumption of ascorbic acid-rich foods at
the same meal. Because of the low content of IP-5 and IP-6
in these fermented staple foods, absorption of intrinsic
non-heme iron, zinc and calcium, as well as any exchange-
able contaminant iron in the foods will not be compro-
mised by phytate. Only in the unleavened corn bread,
certain legumes and oil seeds had phytate:zinc and
phytate:iron molar ratios at levels likely to markedly
compromise zinc and iron absorption.
References
A

gren, G., Gibson, R., 1968. Food composition table for use in Ethiopia.
I, Childrens Nutrition Unit, Addis Ababa, Ethiopia and Institute of
Medical Chemistry, University of Uppsala, Sweden.
Alloway, B.J., 2004. Zinc in Soils and Crop Nutrition. International Zinc
Association, Brussels, Belgium.
Aufreiter, S., Hancock, R.G.V., Mahaney, W.C., Stambolic-Robb, A.,
Sanmugadas, K., 1997. Geochemistry and mineralogy of soils eaten by
humans. International Journal of Food Sciences and Nutrition 48,
293305.
Bohn, T., Davidsson, L., Walczyk, T., Hurell, R.F., 2004. Fractional
magnesium absorption is signicantly lower in human subjects from a
meal with an oxalate-rich vegetable, spinach, as compared with a meal
served with Kale, a vegetable with a low oxalate content. British
Journal of Nutrition 91, 601606.
Bothwell, T.H., Charlton, R.W., Cook, J.D., 1979. Iron Metabolism in
Man. Blackwell Scientic Publications, Oxford, UK.
Derman, D.P., Bothwell, T.H., Torrance, J.D., Macphail, A.P., Bezwoda,
W.R., Charlton, R.W., Mayet, F.G., 1982. Iron absorption from
ferritin and ferric hydroxide. Scandinavian Journal of Haematology
29, 1824.
Ethiopian Health and Nutrition Research Unit (EHNRI), 1998. Food
Composition Table for use in Ethiopia. Part IV. 19951997, Ethiopian
Health and Nutrition Research Unit/Food Agriculture Organization,
Addis Ababa, Ethiopia.
Ethiopian Nutrition Institute (ENI), 1981. Expanded Food Composition
Table for Use in Ethiopia, Ethiopian Nutrition Institute, Addis
Ababa, Ethiopia.
Ferguson, E.L., Gibson, R.S., Weaver, S.D., Heywood, P., Heywood, A.,
Yaman, C., 1989. The mineral content of commonly consumed
Malawian and Papua New Guinean foods. Journal of Food
Composition and Analysis 2, 260272.
Food and Agricultural Organization/World Health Organization (FAO/
WHO), 2002. Human Vitamin and Mineral Requirements, Food and
Nutrition Division, FAO, Rome, Italy.
Gebre-Medhin, M., Killander, A., Vahlquist, B., Wuhib, E., 1976. Rarity
of anemia in pregnancy in Ethiopia. Scandinavian Journal of
Haematology 16, 168175.
Gibson, R.S., Ferguson, E.L., 1999. An Interactive 24-h Recall Method
for Assessing Intakes of Iron and Zinc in Developing Countries. ILSI
Press, Washington, DC.
Hallberg, L., Bjorn-Rasmussen, E., 1981. Measurement of iron absorption
from meals contaminated with iron. American Journal of Clinical
Nutrition 34, 28082815.
Hallberg, L., Hulthe n, L., 2000. Prediction of dietary iron absorption: an
algorithm for calculating absorption and bioavailability of dietary
iron. American Journal of Clinical Nutrition 71, 11471160.
Hallberg, L., Bjorn-Rasmussen, E., Rossander, L., Suwanik, R., 1977.
Iron absorption from Southeast Asian diets. II. Role of various factors
that might explain low absorption. American Journal of Clinical
Nutrition 30, 539548.
Harvey, P.W.J., Dexter, P.B., Darnton-Hill, I., 2000. The impact of
consuming iron from non-food sources on status in developing
countries. Public Health Nutrition 3, 375383.
Haug, W., Lantzsch, H.J., 1983. A sensitive method for the rapid
determination of phytate in cereals and cereal products. Journal of the
Science of Food and Agriculture 34, 14231426.
Hofvander, Y., 1968. Hematological investigations in Ethiopia, with
special reference to a high iron intake. Acta Medica Scandinavica 494
(Suppl.), 174.
Hooda, P.S., Henry, C.J.K., Seyoum, T.A., Armstrong, L.D.M.,
Fowler, M.B., 2004. The potential impact of soil ingestion on
human mineral metabolism. The Science of the Total Environment
333, 7587.
Horwitz, W. (Ed.), 2002. Ofcial Methods of Analysis of the AOAC, 17th
ed. Arlington, VI.
Hotz, C., Brown, K.M. (Eds.), 2004. Assessment of the risk of zinc
deciency in populations and options for its control. Food and
Nutrition Bulletin 25, S95S203.
Hotz, C., Gibson, R.S., 2001. Assessment of home-based processing
methods to reduce phytate content and phytate/zinc molar ratios of
white maize (Zea mays). Journal of Agricultural and Food Chemistry
49, 692698.
Hurell, R.F., 2003. Inuence of vegetable protein sources on trace element
and mineral bioavailability. Journal of Nutrition 133, 2973S2977S.
Hurell, R.F., 2004. Phytic acid degradation as a means of improving iron
absorption. International Journal for Vitamin and Nutrition Research
74, 445452.
ARTICLE IN PRESS
Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 167
Interdepartmental Committee on Nutrition for National Defense
(ICNND), 1959. Ethiopia. Nutrition Survey Report, Interdepartmen-
tal Committee on Nutrition for National Defense (ICNND),
Washington, DC.
Iqbal, T.H., Lewis, K.O., Cooper, B.T., 1994. Phytase activity in the
human and rat small intestine. Gut 35, 12331236.
Lehrfeld, J., 1989. High-performance liquid chromatography analysis of
phytic acid on a pH-stable, macroporous polymer column. Cereal
Chemistry 66, 510515.
Lo nnerdal, B., 2000. Dietary factors inuencing zinc absorption. Journal
of Nutrition 130, 1378S1383S.
Lo nnerdal, B., Sandberg, A.-S., Sandstro m, B., Kunz, C., 1989. Inhibitory
effects of phytic acid and other inositol phosphates on zinc and
calcium absorption in suckling rats. Journal of Nutrition 119, 211214.
Murphy, S.P., Beaton, G.H., Calloway, D.H., 1992. Estimated mineral
intakes of toddlers: predicted prevalence of inadequacy in village
populations in Egypt, Kenya and Mexico. American Journal of
Clinical Nutrition 56, 565572.
Pijls, L.T., Timmer, A.A.M., Wolde-Gebriel, Z., West, C.E., 1995.
Cultivation, preparation and consumption of Enset (Enset ventroico-
sum) in Ethiopia. Journal of the Science of Food and Agriculture 67,
111.
Prasad, A.S., Miale, A., Farid, Z., Sandstead, H.H., Schulert, A.R., 1963.
Zinc metabolism in patients with syndrome of iron deciency anemia,
hepatosplenomegaly, dwarsm and hypogonadism. The Journal of
Laboratory and Clinical Medicine 61, 537549.
Reddy, M.B., Hurell, R.F., Cook, J.D., 2000. Estimation of non-heme-
iron bioavailability from meal composition. American Journal of
Clinical Nutrition 71, 937943.
Reinhold, J.G., Faradji, B., Abadi, P., Ismail-Beigi, F., 1976. Decreased
absorption of calcium, magnesium, zinc and phosphorus by humans
due to increased ber and phosphorus consumption as wheat bran.
Journal of Nutrition 106, 493503.
Sandberg, A.-S., Brune, M., Carlsson, N.-G., Hallberg, L., Skoglund, E.,
Rossander-Hulthe n, L., 1999. Inositol phosphates with different
numbers of phosphate groups inuence iron absorption in humans.
American Journal of Clinical Nutrition 70, 240246.
Sandstro m, B., 1997. Bioavailability of zinc. European Journal of Clinical
Nutrition 51, S17S19.
Sunanda, L., Sumathi, S., Venkatasubbaiah, V., 1995. Relationship
between soil zinc, dietary zinc and zinc nutritional status of humans.
Plant Foods for Human Nutrition 48, 201207.
Teucher, B., Olivares, M., Cori, H., 2004. Enhancers of iron absorption:
ascorbic acid and other organic acids. International Journal for
Vitamin and Nutrition Research 74, 403419.
Tseng, M., Chakraborty, H., Robinson, D.T., Mendez, M., Kohlmeier,
L., 1997. Adjustment of iron intake for dietary enhancers and
inhibitors in population studies: bioavailable iron in rural and urban
residing Russian women and children. Journal of Nutrition 127,
14561468.
Umeta, M., West, C.E., Haidar, J., Deurenberg, P., Hautvast, J.G., 2000.
Zinc supplementation and stunted infants in Ethiopia: a randomised
controlled trial. Lancet 355, 20212026.
Umeta, M., West, C.E., Fufa, H., 2005. Content of zinc, iron, calcium and
their absorption inhibitors in foods commonly consumed in Ethiopia.
Journal of Food Composition and Analysis 18, 803817.
Weaver, C.M., Proulx, W.R., Heaney, R., 1999. Choices for achieving
adequate dietary calcium with a vegetarian diet. American Journal of
Clinical Nutrition 70, 543S548S.
World Health Organization (WHO), 1996. Trace Elements in Human
Nutrition and Health, World Health Organization, Geneva, Switzer-
land.
Wolde-Gebriel, L.Z., West, C.E., Gebru, H., Tadesse, A.-S., Fisseha, T.,
Gabre, P., Aboye, C., Ayana, G., Hautvast, J.G.A.J., 1993.
Interlationship between vitamin A, iodine and iron status in school
children in Shoa Region, Central Ethiopia. British Journal of
Nutrition 70, 593607.
Zein, A.Z., Assefa, M., 1987. The prevalence of anaemia among
populations living in different altitudes in north-western Ethiopia.
Ethiopian Medical Journal 25, 105111.
ARTICLE IN PRESS
Y. Abebe et al. / Journal of Food Composition and Analysis 20 (2007) 161168 168