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UME UNIVERSITY MEDICAL DISSERTATIONS

New Series No. 887 ISSN 0346-6612 ISBN 91-7305-631-6

From Epidemiology and Public Health Sciences,


Department of Public Health and Clinical Medicine
&
Pediatrics
Department of Clinical Sciences

Ume University, 901 87 Ume, Sweden

IRON AND ZINC IN INFANCY:


RESULTS FROM EXPERIMENTAL TRIALS IN
SWEDEN AND INDONESIA

Torbjrn Lind

Ume 2004

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Copyright Torbjrn Lind

Cover illustration: Mother breastfeeding


Oil on canvas. Yogyakarta 1999. Painter anonymous

Printed in Sweden by Print & Media, Ume 2004

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In spite of the spectacular advances in
scientific medicine which we have witnessed in
the last 20 years, there is still a need for
information about a number of fundamental, if
quite elementary, matters.
E M Widdowson and C M Spray, 1951

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ABSTRACT

Background: Iron and zinc are difficult to provide in sufficient


amounts in complementary foods to infants world-wide, resulting
in high prevalence of both iron and zinc deficiency. These
deficiency states cause anemia, delayed neurodevelopment,
impaired growth, and increased susceptibility to infections such as
diarrhea and respiratory infections.

Design: Two different intervention strategies; reduction of a


possible inhibitor of iron and zinc absorption, i.e. phytate, or
supplementation with iron and zinc, were applied to two different
populations in order to improve iron and zinc nutrition:
In a high-income population (Ume, Sweden), the amount of
phytate in commonly consumed infant cereals was reduced.
Healthy, term infants (n=300) were at 6 mo of age randomized to
phytate-reduced infant cereals, conventional infant cereals, or infant
formula and porridge.
In a low income population (Purworejo, Indonesia), daily iron and
zinc supplementation was given. Healthy, term infants (n=680)
were at 6 mo randomized to supplementation with iron, zinc, a
combination of iron and zinc, or placebo.

Blood samples, anthropometrical measurements, and data on infant


neurodevelopment and morbidity were collected. Also, in the
Swedish study, detailed information on the dietary intake was
recorded.

Results: In the Swedish study, the reduction of phytate had little


effect on iron and zinc status, growth, development or incidence of
diarrhea or respiratory infections, possibly due to the presence of
high contents of ascorbic acid, which may counteract the negative
effects of phytate. In the Indonesian study, significant negative
interaction between iron and zinc was evident for several of the
outcomes; Hb and serum ferritin improved more in the iron only
group compared to placebo or the combined iron and zinc group.
Further, supplementation with iron alone improved infant
psychomotor development and knee-heel length, whereas
supplementation with zinc alone improved weight and knee-heel
length compared to placebo. Combined iron and zinc
supplementation did decrease the prevalence of iron deficiency
anemia and low serum zinc, but had no other positive effects.
Vomiting was more common in the combined group.

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Analyses of dietary intake from the Swedish study showed that
dietary iron intake in the 6-11 mo period was significantly
associated with Hb, but not serum ferritin at 9 and 12 mo, whereas
the opposite was true in the 12-17 mo period, i.e. dietary iron
intake was significantly associated with serum ferritin, but not Hb
at 18 mo.

Conclusions: The phytate content of commercial infant cereals does


not seem to contribute to poor iron and zinc status of Swedish
infants as feared. However, the current definitions of iron and zinc
deficiency in infancy may overestimate the problem, and a change
in the recommended cutoffs is suggested. These studies also indicate
that dietary iron is preferably channeled towards erythropoiesis
during infancy, but to an increasing amount channeled towards
storage in early childhood. This suggests that in evaluating dietary
programs, Hb may be superior in monitoring response to dietary
iron in infancy, whereas S-Ft may respond better later in childhood.
However, as shown in this study, increasing Hb may not necessarily
be an indicator of iron deficiency, as more dietary iron increased
Hb regardless of iron status.

In the low-income setting combined supplementation with iron


and zinc resulted in significant negative interaction. Thus, it is not
possible to recommend routine iron-zinc supplementation at the
molar concentration and mode used in this study. It is imperative
that further research efforts are focused at finding cost-effective
strategies to prevent iron and zinc deficiency in low-income
populations.

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LIST OF ABBREVIATIONS
ANCOVA Analysis of co-variance
ANOVA Analysis of variance
ARI Acute respiratory infection
BRS The behavioral rating scale of the Bayley Scales of Infant
Development
BSID The Bayley Scales of Infant Development
CC In SINUS: commercially available MCD and porridge group
CHN-RL The Community Health and Nutrition Research Laboratories,
Gadjah Mada University, Yogyakarta, Indonesia
CI Confidence interval
DMT1 Divalent metal transporter 1
EZP Exchangeable zinc pool
HAZ Height-for-age z-score compared to the WHO/NHCS
reference population
Hb Hemoglobin
ID Iron deficiency
IDA Iron deficiency anemia
IF In SINUS: cows milk based infant formula and porridge
MCD Milk-based cereal drinks
MCV Erythrocyte mean cell volume
MDI The mental development index of the Bayley Scales of Infant
Development
NHANES The second National Health and Nutrition Examination
II Survey in the USA 1976-1980
PDI The psychomotor development index of the Bayley Scales of
Infant Development
PR In SINUS: phytate-reduced MCD and porridge group
RBC Red blood cells
RR Incidence rate ratio
S-Cu Serum copper
S-Fe Serum iron
S-Ft Serum ferritin
SINUS Study on Infant Nutrition in Ume, Sweden
S-TfR Serum transferrin receptor
S-Zn Serum zinc
TIBC Total iron binding capacity
WAZ Weight-for-age z-score compared to the WHO/NHCS
reference population
WHO The World Health Organization
WHZ Weight-for-height z-score compared to the WHO/NHCS
reference population
ZD Zinc deficiency
ZINAK Zinc and Iron Supplementation to Infants in Purworejo,
Indonesia

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ORIGINAL PAPERS
The thesis is based on the following papers:

I. Lind T, Lnnerdal B, Persson L, Stenlund H, Tennefors C,


Hernell O. Effects of weaning cereals with different phytate
contents on hemoglobin, iron stores, and serum zinc: a
randomized intervention in infants from 6 to 12 mo of age.
Am J Clin Nutr 2003;78:168-75.

II. Lind T, Persson L, Lnnerdal B, Stendlund H, Hernell O.


Effects of weaning cereals with different phytate content on
growth, development and morbidity: a randomized
intervention in infants from 6 to 12 mo of age.
Acta Paediatrica accepted with revisions.

III. Lind T, Hernell O, Lnnerdal B, Stenlund H, Domellf M,


Persson L. Dietary iron intake affects hemoglobin during
infancy but not during the second year of life. J Nutrition in
press..

IV. Lind T, Lnnerdal B, Stenlund H, Ismail D, Seswandhana


R, Ekstrm EC, Persson L. A community-based
randomized controlled trial of iron and zinc
supplementation in Indonesian infants: interactions between
iron and zinc. Am J Clin Nutr 2003;77:883-90.

V. Lind T, Lnnerdal B, Stenlund H, Gamayanti I, Ismail D,


Seswandhana R, Persson L. A community-based
randomized controlled trial of iron and zinc
supplementation in Indonesian infants: effects on growth
and development. Am J Clin Nutr in press.

The original articles have been reprinted with permission from the
publishers.

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CONTENTS
THE CHALLENGE............................................................................................................ 1
Infant nutrition ............................................................................................................... 1
Iron................................................................................................................................. 2
Zinc .............................................................................................................................. 11
Iron and zinc requirements............................................................................................ 15
Iron and zinc interaction ............................................................................................... 19

THE SCENE ................................................................................................................... 22


The situation in Sweden and Ume............................................................................... 22
The situation in Indonesia and Purworejo ..................................................................... 23

THE OBJECTIVE ............................................................................................................ 27

THE TRIALS .................................................................................................................. 28

METHODS .................................................................................................................... 30
Design and interventions............................................................................................... 30
Primary outcomes ......................................................................................................... 38
Baseline and follow-up data collection........................................................................... 39
Morbidity registration ................................................................................................... 40
Dietary intake ............................................................................................................... 40
Statistical methods ........................................................................................................ 41

FINDINGS ..................................................................................................................... 44
Baseline characteristics................................................................................................... 44
Iron and zinc status ....................................................................................................... 48
Growth ......................................................................................................................... 52
Incidence of diarrhea and respiratory infections............................................................. 56
Dietary intake ............................................................................................................... 56
Iron intake and iron status............................................................................................. 58

DISCUSSION ................................................................................................................. 63
Effects of phytate reduction........................................................................................... 66
Effects of iron and zinc supplementation....................................................................... 67
Iron and zinc interactions.............................................................................................. 72
Iron intake and iron status............................................................................................. 73
Monitoring of iron interventions................................................................................... 75

CONCLUSIONS AND RECOMMENDATIONS .................................................................. 76

POPULRVETENSKAPLIG SAMMANFATTNING ............................................................... 78

ACKNOWLEDGEMENTS ................................................................................................ 80

REFERENCES ................................................................................................................. 84

PAPERS I-V

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The Challenge

THE CHALLENGE
Exclusive breast-feeding, i.e. giving no additional food or drink but
breast milk, is the recommended type of feeding to term infants
during the first 6 mo of life (1). However, after 6 mo of age the
demand of the growing infants for some nutrients, particularly
micronutrients, i.e. vitamins and trace minerals present in low
concentrations in the diet, increases beyond the amounts supplied
from breast milk only, prompting addition of other foods or
supplements to the infants diet (2). The composition and provision
of these foods, ensuring adequate amounts of energy as well as
essential nutrients to sustain growth, development and a general
state of health of the growing infant or young child continue to be a
challenge to parents, health care providers and the scientific
community (3).

INFANT NUTRITION

After 6 mo of age, when breastmilk alone becomes increasingly


short in providing the infant with sufficient amounts of
micronutrients to sustain growth and development, the need for
complementary foods becomes evident. The term complementary
foods has in this thesis a broad definition, including both foods that
are given in addition to breast milk (2) and all other foods that are
used during the transition from the liquid diet typical of small
infants to family foods. Complementary foods may consist of
transitional foods, i.e. foods specifically designed to meet the
nutritional needs of infants and young children, or family foods,
meaning the that the infant consumes the same food as the rest of
the family.

The composition of complementary foods and how they are used


vary with country and culture, but there are some universal aspects.
One aspect, common to both low-income and high-income
settings, is that they are often plant-based (4-6). Although plants are
excellent sources of energy and nutrients, plant constituents can also
greatly affect nutrient bioavailability, i.e. the fraction of a particular
nutrient or other substance that is actually available for absorption
by the body. Polyphenols, for example, found in many vegetables
inhibit iron absorption (7). Another aspect of complementary foods
is that the content of meat and other sources of animal protein
tends to be low, also in the most affluent settings (5). Meats and

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The Challenge

other animal protein sources are not only excellent sources of


protein and energy, but also contain many essential nutrients, and
increase the absorption of others, for example, animal protein
enhances zinc absorption. The intestinal absorption of
micronutrients thus is affected by the composition of the diet, with
factors both enhancing and inhibiting absorption.

There are also many important differences among complementary


foods, particularly between high-income and low-income settings.
Whereas in the former setting it is common to give industrially
manufactured, fortified and nutritionally balanced foods, in the
latter it is more often home-produced from locally available staples.
Although studies of complementary foods in developing countries
indicate that the energy and protein content may be adequate, they
may be short in micronutrients; in most low-income settings,
intakes of iron, zinc and vitamin B6 are inadequate, and in some
populations the same is true for riboflavin, niacin, calcium,
thiamin, folate, vitamin C and vitamin A (6, 8, 9).

Although providing adequate amounts of energy and some


nutrients, diets may contain low absolute amounts of other
nutrients, or nutrients may have low bioavailability, leaving the
infant or child at risk of specific nutrient deficiencies. The
symptoms of these micronutrient deficiencies may appear subtle,
although the impact on health both in the short and long run is
substantial. Such vitamin and mineral malnutrition, sometimes
called hidden hunger, is amongst the most common nutritional
disorders in infancy and childhood world-wide (10). This thesis
concerns two of these micronutrients, iron and zinc, how they affect
health and well-being of term infants, and how two different
nutrient interventions can influence iron and zinc nutriture during
the second half of infancy and early childhood.

IRON
Iron is the fourth most abundant element in the Earths crust and
one of the cheapest (11), still iron deficiency is the most prevalent
nutritional disorder in the world (12). UNICEF estimates that 40-
50% of children under five years of age in low income countries
suffer from iron deficiency (ID), and that overall some 2 billion
people world-wide are anemic, with an even greater number
suffering from ID (Figure 1) (13).

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The Challenge

Iron deficinency

Anemia

Iron deficiency anemia

Figure 1. The distribution of anemia, iron deficiency and anemia in a fictitious


childhood population with an anemia prevalence of 30%, where iron
deficiency anemia would contribute to the majority of anemia cases. The
estimated prevalence of iron deficiency in such a population would be 40-60%.
Data from (260).

Iron is essential for a number of important proteins and


metalloenzymes in the body, which often use its ability to alternate
2+ 3+
between two oxidation states ferrous (Fe ) and ferric (Fe ) iron
in their function. Hemoglobin (Hb) and myoglobin are among the
more well-known iron-dependent compounds, but others include
cytochromes, peroxidases, and non-heme proteins such as
ribonucleotide reductase and lactoferrin. Thus, iron is essential in
the function of the living organism from oxygen transport to DNA
synthesis.

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The Challenge

Iron metabolism

The human iron pool is mainly regulated by intestinal uptake from


the diet (14), since epithelial losses and losses through urine, stool
and sweat under normal circumstances are small (15). The human
body has no active way to discharge excess iron.

Iron homeostasis has recently been reviewed (16-18) (Figure 2).


Absorption of non-heme iron is concentrated to the gastroduodenal
junction. The iron is transported across the apical membrane of
mature enterocytes by divalent metal transporter 1 (DMT1,
formerly Nramp2 or DCT1).

Fe Zn
Brain

Enterocyte

RBC
production

Fe2+
Zn2+ Ferritin? Bone
Phytate
Vitamin C
marrow
Calcium

Animal Zn Fe
Fe3+
protein
Tissue
Gut growth
Fe

Zn

Blood vessle

Resistance to
infections

Figure 2. Absorption of iron and zinc from the small intestine is affected by
enhancers and inhibitors in the intestinal fluid. Dietary iron is transported
across the apical membrane of the enterocyte by DMT1. The mechanism by
which dietary zinc crosses into the enterocyte is less clear, although the
transporter ZIP4 has been suggested. At the basolateral side of the enterocyte,
iron is exported to the portal circulation by ferroportin1, whereas zinc is
transported by Zn-T1. In the circulation iron is bound to transferrin and zinc
mainly to albumin. Because of their omnipresence in the human body,
deficiency of iron and zinc affect many different organs and tissues.

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This protein channel may also transport other metals, e.g. zinc,
cadmium, lead, manganese and cobalt (19). At the basolateral side
of the enterocyte, iron is transported to the portal circulation,
supposedly by ferroportin1 (formerly Ireg1 or MTP1). The export
2+
of Fe is facilitated by hephaestin, a transmembrane protein similar
2+ 3+
to ceruloplasmin, which also oxidizes Fe to Fe .

Ferric iron is then bound to transferrin and transported to tissues in


need of iron, most notably erythropoietic cells in the bone marrow
for the production of hemoglobin. Transferrin receptors on the cell
3+
surface bind the transferrin-Fe complex, and the complex is taken
into the cell using endocytosis. The ferric iron is again oxidized to
2+
Fe , which DMT1 transports from the endosomes to the cytosol.
The intracellular iron is then transported across the mitochondrial
membrane, where it is used for incorporation into heme, used for
other purposes in the cell or stored in cytosolic ferritin. Circulating
erythrocytes are destroyed by macrophages in the
reticuloendothelial system. Bound to ceruloplasmin, iron is moved
out of the reticuloendothelial macrophages and delivered to
transferrin for recycling.

Surplus iron may be stored in ferritin, a large multifunctional and


multiunit protein, capable of reversibly binding as many as 4500
iron atoms as iron oxide (20). Ferritin is found in the cytosol of
most cell types, most notably in the liver and various macrophages
found in the liver, spleen and bone marrow, but it is also the storage
form of iron in normal erythroblasts undergoing maturation. It
plays a key role in iron homeostasis because of its capability to
regulate the intracellular labile iron pool.
2+
Intracellular Fe can participate in the generation of oxygen
radicals, which can severely damage cell organelles including the
2+
DNA. Through its enzymatic properties, ferritin converts Fe to
3+
Fe , which is then stored in the ferritin mineral core as iron oxide
2+
(21). Ferritin may thus detoxify not only highly reactive Fe , but
also oxygen radicals harmful to the organism. Ferritin is found in
small quantities in human serum, although the origin of this ferritin
is unknown.

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The Challenge

Measures of iron status

Hematological indices: Hemoglobin and MCV

In infants as well as adults, about two-thirds of the body iron is


bound to Hb in the red blood cells. In the absence of available iron,
Hb begins to decline, thus Hb distinguishes between anemic and
non-anemic iron deficiency (ID) states. However, anemia, i.e. low
Hb, may be caused by deficiencies of other nutrients, e.g. vitamin
B12 and folic acid, as well as a number of other conditions including
hemolytic states, inflammation, and defective hemoglobin such as
thalassemia.

Thus, anemia alone does not necessarily imply iron deficiency.


Mean cell volume (MCV), a measure of the size of red blood cells,
can give guidance in discriminating between microcytic anemia,
caused by for example, ID; normoblastic anemia such as in
hemolytic states; and megaloblastic anemia, caused by for example
vitamin B12 deficiency. In ID, zinc is incorporated into
protoporphyrin instead of iron, thus zinc protoporphyrin (ZPP) to
heme ratio can be measured as an index of iron-deficient
erythropoiesis (22, 23).

Biochemical indicators: Serum ferritin, serum iron and serum


transferrin receptors

Although the exact origin and role of ferritin in serum is still


shrouded in uncertainty, serial phlebotomy in adults has indicated a
strong correlation between serum ferritin (S-Ft) and the magnitude
of iron stores, decreasing in iron deficiency and increasing in iron
overload states, such as hemochromatosis. Not surprisingly, it is
widely used as an indicator of iron stores, not only in adults, but
also in infants and children (24, 25). Serum ferritin is iron-poor
(<5% saturation) and carries carbohydrates on its surface indicating
active secretion of the protein to the serum. However, S-Ft acts as
an acute phase reactant and in the presence of inflammation it may
increase considerably, which may mask depleted iron stores (26-28).
This limits the value of serum ferritin as a marker of iron stores,
especially in the face of inflammation and infection, the latter being
common in childhood.

Serum iron (S-Fe) measures the transferrin-bound iron in serum


and together with the transferrin concentration, which determines
how much iron can be bound in plasma, i.e. the total iron binding

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The Challenge

capacity (TIBC); it is used as another functional assessment of iron


status. These measures are however subject to considerable
variation; for S-Fe there is a diurnal variation with lower
concentrations in the afternoon and also during acute phase
reactions, and similar to S-Ft, TIBC increases during acute phase
reactions, which limits their usefulness, and its discontinuation in
the diagnosis of ID has been suggested (23, 29).

In the search for more specific tools to measure iron stores serum
transferrin receptor (S-TfR) has been identified as an indicator of
intracellular iron demand and hence a measure of iron status (30).
These receptors are found most abundantly on cells involved in
erythropoiesis (31), and they are progressively found in serum as
iron stores are depleted. Unlike S-Ft, S-TfR is not affected by
inflammation (32). However, the measurement of S-TfR has not
yet been standardized and various commercial kits have different
cut-offs. This together with insufficient data on S-TfR as an
indicator of ID in infants and young children has so far limited its
use (23). A novel indicator of iron status, especially in the diagnosis
of IDA in children, is the reticulocyte hemoglobin content (33),
although its exact role remains to be elucidated.

Iron deficiency

Iron deficiency is considered to develop in discreet steps, which are


reflected by the biochemical markers of iron status in blood. First,
with iron needs surpassing demands, iron stores are depleted, which
is seen in the blood as diminishing S-Ft values. Several cut-off
values for S-Ft have been proposed to determine when depleted
iron stores are present; ranging from 10-16 Pg/l, but so far no
consensus has been reached, in part because of lacking information
on the functional consequences of different levels of iron stores. As
iron status deteriorates, there is also evidence of increasing
hematological and biochemical changes due to iron-deficient
erythropoiesis (changing blood indices, e.g. decreasing MCV,
decreasing transferrin saturation and increasing erythrocyte zinc
protoporphyrin) as well as signals of increasing cellular iron demand
(increasing S-TfR). The last stage is frank anemia, reflected in a low
Hb (34). This is the indicator most commonly used when
evaluating ID, although a low Hb is, as already stated, frequently
caused by other factors, i.e. infections, other non-nutritional causes
or non-iron nutritional causes (26, 35).

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The Challenge

Definition of iron deficiency

With the plethora of iron status measures available, multiple


definitions of ID have been proposed (36-41) (Table 1). In this
thesis the definitions suggested by WHO (40) are used, i.e. from 6
mo of age; Hb <110 g/L, defining anemia; and S-Ft <12 g/L,
defining depleted iron stores. In conjunction to these, when
available, the following are used: MCV <73 fL, defining iron-
deficient erythropoesis (41), transferrin saturation <16%, indicating
low levels transferrin-bound iron, or as recently has been suggested
S-TfR >11 mg/L, indicating increased cellular need for iron (36).
However, all these definitions and cutoffs have been challenged in
infants and young children as they are to a large extent extrapolated
from values in older children and adults (23, 36). Hence, there is a
need for age appropriate cut-offs for the various iron-status
indicators (23, 36, 37), particularly in infants.
Table 1. Suggested cut-offs for various iron status indicators in infancy
according to various authors.

Criteria

Hb (g/L) S-ferritin MCV (fL) Transferrin S-TfR


(g/L) saturation (%) (mg/L)

WHO (40) <110 <12 73 16 NA

Domellf et al 2002 6 mo: 105 6 mo:  9 71 NA >11


(36) 9 mo: 110 9 mo:  5

Dallman et al 1980 110  10-12 70 NA NA


(59)

Emond et al 1996 97 17 NA NA NA


(37)

Looker et al 1997 110 10 10


(261)

Virtanen et al 1999 106 9 72 5 >11


(259)
NA not available

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The Challenge

Consequences of iron deficiency

There are a number of adverse functional consequences of ID alone


or in conjunction with anemia, including decreased work capacity
(42-44), impaired regulation of body temperature and heat
production in cold environments (45), and increased uptake of lead
(46). ID reportedly also affects growth (47) and the immune system
(48), but it is the suggested, and possibly irreversible effect of ID on
cognitive and psychomotor development of infants and young
children that brought it onto the public health agenda (49-52).
Studies on auditory brainstem responses and visual evoked
potentials suggest that ID alters neuron myelination in infants (53,
54). Effects of ID on cognitive functioning may also be evident in
school-aged children (55).

Causes of iron deficiency

Iron deficiency is caused by inadequate intake or low bioavailability


of dietary iron to meet the organism's metabolic needs, or by non-
nutritional factors such as parasite infestation, particularly intestinal
parasites such as hook worm, or blood loss, including obstetric
practices (56-58). The metabolic needs for iron are affected by
physiological demands such as rapid growth, pregnancy, lactation,
and increased losses (e.g., menstruation). Thus, infants, children
and women during their fertile years are particularly prone to
develop ID. In children, the highest prevalence of ID is found
between 4 mo and 3 years of age (59). There are estimates that
more than half of the children worldwide have ID, mainly due to
poor iron stores to begin with due to low birth weight and other
fetal factors, low absolute iron intake in the diet, poor
bioavailability of the dietary iron, and, in endemic areas infestations
with hookworm, or a combination of these factors (40).

In infants and young children, dietary factors are strongly


contributing to the problem of ID. These factors include short
breastfeeding periods, early introduction of foods that interfere with
the uptake of iron from breast milk, and use of complementary
foods with low iron content or with low bioavailability of iron (60,
61). In some high-income settings, the prevalence of ID among
infants and children has decreased due to increased prevalence of
exclusive breastfeeding, increased use of iron-fortified foods and
decreased use of cow's milk in infancy and early childhood (62, 63),
although low absolute iron intake has also been reported from

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The Challenge

affluent societies (64). Early introduction of and high intake of


unmodified, i.e. non-iron fortified cows milk is associated with
increased risk of ID also in high-income settings (65). In other parts
of the world, complementary foods continue to be low in iron, or
contain large amounts of inhibitors or inadequate quantities of
enhancers, leading to an inadequate intake and/or absorption of
iron and eventually to high prevalence of ID (66, 67).

Iron in the diet

Two types of iron are available in the diet: heme and non-heme
iron. Heme iron is mainly found in animal products, while non-
heme iron is largely found in plant foods. The bioavailability of
heme iron is high and not influenced much by the composition of
the food (68), although calcium has been shown to decrease its
absorption (69). In contrast, the bioavailability of non-heme iron is
greatly influenced by various dietary enhancers (e.g. meat and
vitamin C) and inhibitors (e.g. polyphenols, phytate, calcium,
tannins) (70). Therefore, absorption of non-heme iron can vary
between <1% to >50% (7). Despite this, non-heme iron is the
quantitatively most important source of iron in diets world wide,
especially among infants and children in low-income countries (66).

Ascorbic acid (vitamin C) is an important enhancer of iron


absorption (71), acting in the stomach and duodenum primarily by
3+ 2+
reducing Fe to Fe , thus improving iron solubility, which is higher
2+
in the Fe -state. At an ascorbic acid:Fe molar ratio of 4:1, ascorbic
acid prevents the negative effects of inhibitors of iron absorption
inhibitors, such as calcium, polyphenols, phytate and soy (72-74).
However, the effects of ascorbic acid on non-heme iron from the
complete diet has been more modest than from single meal studies
(75). A high intake of ascorbic acid, either as fruit or vegetables is
associated with higher Hb in children up to 18 mo (76), and
increased uptake of ascorbic acid is one of the suggested strategies to
improve iron bioavailability, also in low-income settings (77, 78).

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The Challenge

ZINC
Zinc is like iron a divalent metal, but considerably less abundant
(79). Although zinc has been know to be essential for living
th
organisms since the latter part of the 1900 century (80), it is only
within the last 40 years that it has been established as an essential
nutrient for humans (81). Reasons for this, though zinc is
ubiquitously present in the cells of living organisms, including
humans, are that actual zinc status is difficult to measure and
symptoms of deficiency are subtle. Thus, an estimate of the global
prevalence of zinc deficiency (ZD) has been difficult to achieve.
Estimates using indirect measures, such as the zinc content in the
global food supply, shows that 49% of the worlds population is at
risk of inadequate zinc intake (79), of which the majority is found
in low-income countries. An estimate based on the disability
adjusted life years lost (DALY) places ZD behind vitamin A
deficiency, but before ID in importance (82).

Zinc is essential to the function of a number of enzymes, structural


components and regulatory proteins vital to the human organism. It
has unique properties, although in contrast to iron it does not
display redox chemistry. Thus, zinc functions in milieus where
generation of free radicals would be deleterious. Zinc is involved in
several of the basic functions of the cell and acts catalytically as well
as structurally and regulatory, affecting cell growth and
differentiation. Examples of zinc-dependent compounds are RNA
nucleotide transferases, carbonic anhydrases, and zinc fingers in
various proteins. Zinc may also act as a factor regulating gene
expression, e.g. for metallothionein, and may regulate apoptotic cell
death (83). About 60% of total body zinc is found in muscle tissue
and 30% in bone, whereas plasma zinc only accounts for 0.1%
(84).

Zinc metabolism

Zinc homeostasis is primarily regulated through zinc absorption


and excretion in the gastrointestinal tract, although some zinc is
excreted in the urine. Epithelial losses and losses in sweat are small,
but in adult men losses in semen can be substantial (85). The
jejunum has the highest rate of absorption (Figure 2), but zinc is
also absorbed from the duodenum and ileum (83). Both absorption
and excretion are actively, and maybe independently, regulated.
Decreasing zinc intake increases fractional absorption of zinc and

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increasing intake increase fecal excretion. Excretion responds faster


to changes in dietary zinc intake, but absorption of zinc has the
capacity to adjust for larger fluctuations in zinc intake (86). In
infants, fractional absorption varied from 29% to 54% as zinc
intakes decreased from 111 to 15 mol/d (87).

Several classes of zinc transporters have been identified (88). The


first transporter identified, ZnT-1 (89), is found in virtually all
organs. In the duodenum and jejunum it is located to the
basolateral membrane, exporting zinc from the enterocyte to the
circulation. Increased dietary zinc intake increases ZnT-1 levels in
the intestine indicating a function of the transporter in zinc
retention (90). A recently discovered transporter, ZIP4, mutated in
patients with acrodermatitis enteropathica, a sometimes lethal
disease of zinc metabolism, is located at the apical membrane of
enterocytes and may function as the main transporter of zinc into
the enterocyte, and has been suggested to have a key regulatory
function in dietary zinc absorption and cellular zinc homeostasis
(91, 92).

Experimental studies indicate that humans can regulate their whole


body zinc over a 10-fold change in intake. In adult men fed a zinc-
restricted diet homeostatic mechanisms to control body zinc
included reduced urinary and fecal excretion and reduced plasma
zinc concentration, with urinary and plasma zinc concentrations
responding faster than fecal zinc (93).

Through stable isotope studies in adults an exchangeable zinc pool


(EZP) has been identified (94). This pool mixes with plasma and is
thought to ascertain zinc supply to zinc-dependent biological
processes. In infants, the EZP also seems to relate to crucial
variables of zinc homeostasis (87). Hence, EZP is positively
correlated to the total amount of absorbed zinc and endogenous
fecal zinc excretion, but is not correlated to fractional absorption of
zinc. This would indicate that the regulation of total absorbed zinc
is limited, that endogenous zinc excretion is both dependent on and
responsive to the individuals zinc status, and that intraluminal zinc
and, possibly, presence of factors affecting absorption such as
phytate, affect fractional absorption more than the infants zinc
status (87).

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Measures of zinc status

There is still no single, sensitive and specific measure of zinc status.


Plasma or serum zinc (S-Zn) has been most commonly used. A
zinc-restricted diet significantly decreases S-Zn (85), but the assay is
affected by a number of other factors, e.g. infections, environmental
contamination, and time since last meal, which limits its value (95,
96). However, in a study from Peru, children with clinical signs of
infection (fever, respiratory infections, diarrhea or anorexia) did not
differ significantly in S-Zn from those without infection, whereas
those with biochemical evidence of infection, i.e. elevated C-
reactive protein or leukocytosis had significantly lower S-Zn
compared to those with normal laboratory tests (26). S-Zn
decreases after meals (97), although this effect may be blunted in
infants; Michaelsen et al found no correlation between duration of
fasting and S-Zn in Danish infants 2-9 mo of age (98). Technical
predicaments such as hemolysis of the blood sample or zinc
contamination from syringes, needles, test tubes, etc., may falsely
elevate S-Zn (99). Other measures used in the assessment of zinc
status have been hair zinc, urine zinc, zinc content of leukocytes,
and the activity of zinc-containing enzymes such as alkaline
phosphatase, or the protein metallothionein and serum thymulin.
In most cases sensitivity and specificity of these tests have been low,
the analyses laborious to perform, or the results difficult to interpret
(95). Assessment of dietary zinc intake has also been used (66).
Although this method provides a feasible way to quantify zinc
intake, which may be an indirect proxy for zinc status, it requires
information on the zinc content of local foods as well as presence of
dietary factors affecting zinc bioavailability and the likely absorption
of zinc from these diets. Thus, in spite of these short-comings, from
a pragmatic point of view, determination of serum or plasma zinc
concentration remains the best reasonable method to assess zinc
status, especially at the group level (100).

Zinc deficiency

Aspergillus niger, a fungus causing black mold on foodstuffs, was the


first biological system where zinc was shown to be essential [J
Raulin 1869, cited in (80)], but it was not until the early 1960s that
ZD was described in humans (81). Dietary factors, i.e. low absolute
intake of zinc, the presence of phytate or other inhibitors of zinc
absorption, absence of enhancers of zinc absorption such as animal

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protein, and increased losses as in diarrheal states, all contribute to


the development of significant ZD (101, 102).

Definition of zinc deficiency

With the problems of assessing zinc status, defining ZD has been


equally difficult. Based on data from the second National Health
and Nutrition Examination Survey (NHANES II), a much used
definition of low S-Zn has been <10.7 mol/L (<70 g/dL) in
persons sampled in the morning after an overnight fast and <9.2
mol/L (<60 g/dL) in non-fasting persons sampled in the
afternoon (103). A recent reanalysis of the NHANES II data (104)
has suggested a lower 2.5 percentile cutoff of 9.9 mol/L (65
g/dL) in children <10 years. However, the children examined in
NHANES II were all 3 years or older. Population-based data in
infants are restricted to two surveys; one from Canada (n=62, age 1-
6 years), which observed the 2.5 percentile at 10 mol/L (105), and
a more recent from Australia (n=467, age 9-62 mo.), which
suggested the lower 2.5 percentile at 9 mol/L (106).

Consequences of zinc deficiency

Due to the ubiquitous presence of zinc in the cell, ZD affects the


basic mechanisms of the organism including growth, appetite,
skeletal maturation, sexual development, and the immune system
(81, 83, 107). Acrodermatitis enteropathica, a potentially lethal
inborn error of metabolism causing malabsorption of zinc,
instigates desquamating dermatitis, diarrhea, mood changes,
anorexia, growth retardation, alopecia, and recurrent infections.
The condition is readily treatable with oral or parenteral zinc (108,
109). However, the vast majority of cases with ZD has much more
obscure symptoms including growth failure, increased
susceptibility, severity and duration of infectious disease episodes,
and possibly altered behavior (110-112).

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IRON AND ZINC REQUIREMENTS

Iron requirements

The healthy, full-term infant is born with iron stores which, in


breast-fed infants, last up to 6 mo of age (113, 114). The growing
infant will increase its total body iron from about 250 mg at 6 mo
of age to about 420 mg at 12 mo. The need for iron is estimated to
45 mg per kg weight gain (115). Infants with low birth weight have
smaller stores and require supplemental iron from an earlier date
(116). The total requirements of absorbed iron in infants (6-12 mo;
80 g/kg body weight/d) and young children (1-3 years; 35
g/kg/d) can be divided into those needed for growth (infants 6-12
mo; 60 g/kg/d, and children 1-3 years; 20 g/kg/d) and those to
cover iron losses (infants 6-12 mo; 20 g/kg/d, and children 1-3
years; 15 g/kg/d). Based on these assumptions and an assumed
bioavailability of 5-15% depending on the presence or absence of
inhibitors or enhancers of absorption in the food, the recommended
daily iron intake of infants 6-12 mo is 0.7-2.1 mg/kg/d and of
children 1-3 years 0.3-0.9 mg/kg/d (117) (Table 2).

Table 2. Iron and zinc requirements (mg/d) in infants and young children 6-36
mo of age according to bioavailability. From (117).

Bioavailability

Fe Age (mo) High 15% Intermediate 12% Low 10% Very low 5%

6-11 6.2 7.7 9.3 18.6

12-36 3.9 4.8 5.8 11.6

Zn Age (mo) High 50% Moderate 30% Low 15%

6-11 2.5 4.1 8.4

12-36 2.4 4.1 8.3

Zinc requirements

Although breast milk contains relatively low concentrations of zinc,


it is highly available (118). The combination of high fractional
absorption of available zinc in breast milk, low endogenous fecal
zinc excretion and, possibly, stores from birth, ensure adequate zinc
status to sustain growth through the first 5 mo of infancy (118,

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119). Even in mothers with marginal zinc intake, adequate breast


milk zinc output is sustained during these first months of lactation
(120). However, in mothers from both high-income and low
income settings, zinc concentrations in breast milk fall dramatically
during the infants first half year (121-125) and zinc supplied from
complementary foods is deemed necessary after 6 mo of age (2).

Zinc requirements of infants and children have been extrapolated


from adults, as data on zinc metabolism of younger age-groups are
lacking (84). In the age-group 6-12 mo some 35 g zinc/kg body
weight/d are needed to cover basal losses and another 35 g/kg/d
are needed for growth, adding up to a total basal need of around 70
g absorbed zinc/kg/d. In young children, 1-3 years old, the
corresponding figure for maintenance of basal losses is 35 g/kg/d
and for growth 15 g/kg/d, which gives a total of 50 g absorbed
zinc/kg/d. However, prolonged provision at that rate will leave the
individual with no reserves in case of further reductions in zinc
intake or increased losses as is seen in diarrheal states (102). Hence,
a normative physiologic requirement, which is 40% above the basal
requirement, has been calculated. In 6-12 mo old infants, this
corresponds to 95 g zinc/kg/d, and in children 1-3 years of age, 70
g/kg/d.

In the last trimester of pregnancy, the fetus accumulates zinc. Thus,


infants born prematurely (<38 weeks of gestation) presumably have
smaller zinc stores. Studies show that preterm infants need to retain
(i.e. absorption minus endogenous excretion and other losses) at
least 326 g zinc/kg/d to grow at the expected rate (126), and that a
higher zinc intake is beneficial to very-low-birth-weight infants
(127). Other infants requiring catch-up growth, e.g. infants
recovering from malnutrition or infection also have increased zinc
th
needs. A modest estimate has been that an underweight (5
percentile of the WHO reference population) infant at one year
th
requires 114 g absorbed zinc/kg/d to reach the 40 weight-
percentile within a reasonable amount of time (84). Zinc
supplementation has been efficient in the rehabilitation of
malnourished infants (128), though high doses may have
detrimental effects (129).

As for iron, zinc absorption is influenced by a number of dietary


factors. Animal and fish protein are both rich sources of zinc and
important stimulators of zinc uptake (130). On the other hand,
phytate is one of the most important inhibitors, and is likely to
contribute significantly to the world-wide problem of inadequate

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zinc status [reviewed in (101)]. With diets differing in zinc


bioavailability, different recommendations regarding the lower
limits of population mean zinc intake per day are given for diets
with high (i.e. 50%), moderate (30%) or low bioavailability (15%)
(117). Table 2 gives the recommended intakes of infants and young
children. A high zinc bioavailability diet is low in cereal fiber, has a
low phytate:zinc molar ratio (i.e. <5), and has adequate protein
from mainly animal sources. A diet with moderate bioavailability
has a phytate:zinc molar ratio of 5-15, though not exceeding 10 if a
considerable part of the energy intake comes from unrefined cereals,
and does contain animal protein sources. A low bioavailability diet
is high in unrefined cereals, low in animal protein and has a
phytate:zinc molar ratio >15. Especially phytate:zinc molar ratios
>15 are known to compromise zinc status (131, 132).

Phytic acid

Phytic acid (myo-inositol hexaphosphate; Figure 3) is a constituent


of plants such as seeds, roots, tubers and vegetables, where it
functions as the storage form of phosphorus. The focus on phytic
acid in human nutrition has largely been due to its ability to chelate
metals. In the intestinal lumen it is negatively charged over a wide
2+ 2+ 2+
pH-range and can thus chelate cations such as Ca , Zn , Fe and
2+
Mg . These chelates are either insoluble or difficult to hydrolyze
during digestion, and thus difficult to absorb by humans and other
monogastric animals. A chelate of phytic acid and a mineral is
called phytate. In grains such as barley, wheat, rye and rice, most of
the phytate (90%) is found in the outer layers of the seed. Thus,
whole meals of cereals have higher contents of phytic acid than do
flours with lower extraction rates, roots, tubers and vegetables.
Western diets typically include low extraction flour and roots,
tubers and vegetables with low phytate content, whereas the typical
diet in many low-income countries include whole meal cereals and
other plant-based foods with high phytate content. The difference
in daily phytate intake between a Western country, e.g. Sweden,
and a less affluent country may be >10-fold (133, 134). Table 3
shows the phytate content in common staple foods. Phytate, in the
absence of ascorbic acid, inhibits iron absorption in a dose-
dependent manner above a phytate:Fe molar ratio of 1:7 (135),
whereas phytate:zinc molar ratios >15:1 have been shown to inhibit
zinc absorption (131, 132). Large intakes of phytate have also been
associated with large fecal zinc losses (136).

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Table 3. The phytate content in common staple foods.

Staple foods Phytate (mol/100 g)

Rye 1280

Wheat 1450

Oat 1430

Barley 940

Wheat bran 4330

Rice, brown 1120

Rice, polished 1000

Maize 1300

Soy bean 2000

Lentils 1400

Roots and tubers 180-530

Calculated from (133, 262).

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OH
OH
O P O-
O- P O
O-
O O
O- P O

O O-

O- P O
O
O

O P O-
O
O-
O P O-

OH

Figure 3. Phytic acid is negatively charged in the intestinal lumen, giving it an


ability to chelate various metal cations. These complexes, called phytates, are
difficult or impossible to hydrolyse during digestion, rendering the metals
unavailable for absorption.

IRON AND ZINC INTERACTION


Sharing several chemical characteristics, it has been suspected that
iron and zinc may interfere with one another with regard to
absorption and metabolism. It has been shown that high
concentrations of inorganic iron given in water inhibit zinc
absorption (137-139) and zinc given in water inhibits iron
absorption (140, 141). Solomons and Jacob (137) found that 25
mg Fe added to a water solution with 25 mg Zn decreased plasma
zinc, whereas lower total amounts of minerals (10 mg Fe and 5 mg
Zn) and an iron:zinc ratio of 2:1 had no effect on plasma zinc levels
(142). However, Sandstrm et al (139) found no effect on zinc
absorption when the iron:zinc ratio was 1:1 or 2.5:1 in a water
solution, but decreased zinc absorption when the iron:zinc ratio was
25:1. Crofton et al showed that iron and zinc given in a 1:1 ratio
decreased iron absorption (141), but that a 2:1 iron:zinc ratio had
no effect on iron absorption. Rossander-Hultn et al found that an
iron:zinc ratio of 1:4 significantly reduced iron absorption when
given as a water solution but with no effect on iron absorption

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when given as a meal (140). No effect was seen of a high iron:zinc


molar ratio on zinc absorption when given as infant foods (143-
145).

Exactly by which mechanism iron and zinc interact is not known.


One may be through a common transport mechanism. Previous
studies indicated that iron and zinc shared a common transporter,
i.e. DMT1 (19). However, later research has shown that though
DMT1 does not transport zinc per se, zinc does affect the DMT1
level but there may be other, more important, common iron-zinc
pathways (146). It is also not known why iron and zinc given
together in foods seem not to interact. The two minerals are
affected by some of the same inhibitors, e.g. phytate, though iron to
a greater extent than zinc. In addition, the effects of iron and zinc
may counteract each other in some situations, i.e. zinc has been
shown to improve growth in nutritionally deprived infants (110),
whereas iron may negatively affect growth in iron replete children
(147-149). However, with both iron and zinc deficiency being
highly prevalent and possibly present in the same groups of infants,
the interaction between the two minerals is an important research
issue.

Strategies to prevent iron and zinc deficiencies

Interventions to improve iron and zinc nutriture in infancy and


childhood include optimizing complementary food nutrient density
and enhancing the bioavailability of the minerals present, as well as
avoiding foods that inhibit or displace iron and zinc in the diet.
Complementary foods are cereal- or vegetablebased in most
settings. If these foods are centrally produced, fortification is an
attractive alternative, especially in the combination with ascorbic
acid (78, 150). Adding other enhancers such as meat or other
animal protein sources may also improve iron and zinc
bioavailability (130, 151). Unmodified cows milk has a low
content of iron, and consumption of large amounts of such milk
has been associated with poor iron status in infancy and childhood
(65), whereas iron-fortified cows milk formula is associated with
improved iron status (152, 153). Food processing techniques such
as fermentation, germination and milling may affect iron and zinc
bioavailability through phytate degradation (154, 155).

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If the complementary foods are home produced, supplementation


with iron and zinc, or mixing it with foods as sprinkles or fat-based
spreads may be feasible alternatives (9, 156, 157). However, which
strategy to ensure adequate iron and zinc nutriture is most effective
depends on the setting; fortification, addition of meat to
complementary foods, and avoidance of large amounts of
unmodified cows milk seem to work well in more affluent settings.
Although iron and zinc supplementation has been suggested in low
income settings (9), no consensus exists on how to effectively
accomplish this, with remaining questions concerning dosage,
interactions, avoidance of accidental poisonings, costs, compliance
and distribution. Further, knowledge about which indicators are
most accurate when evaluating such interventions is also missing. In
the present thesis, two strategies, phytate-reduction of infant cereals
in a high-income setting, and giving supplemental iron and zinc in
a water solution to infants in a low income setting, are evaluated.

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The Scene

THE SCENE

THE SITUATION IN SWEDEN AND UME


The kingdom of Sweden is a high income country with a
population of about 9 million. Infant and child health has
improved greatly during the past century with an infant and under-
five mortality rate at 3/1000 live born being among the lowest in
the world (158). The nutritional situation among infants and
children is considered good (159-163).

Ume is a university town in the northern part of Sweden with a


population of 105 000 (Figure 4). The town hosts a university
hospital and 7 primary health care centers. In 1997, the infant
mortality rate in the municipality of Ume was 2.5/1000 live born
and the rate of low birth weight (<2 500 g) was 31.6/1000 live
born. At one week of age 94% of infants were exclusively breast-fed,
and at 6 mo 46% were exclusively and 32% partially breast-fed
(164). The average annual income for people employed in the
public sector was USD 29 457 (165).

Ume, Sweden

Purworejo, Indonesia

Figure 4. Location of Ume and Purworejo.

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Surprisingly, a cross-sectional study, performed in the 1990s (166)


under the umbrella of the Euro-Growth multi-center study (65),
showed that 26% of 12 months-old term infants had low S-Ft (<12
Pg/l) and 36% low S-Zn (<10.7 Pmol/l). Interpreting low S-Ft as
depleted iron stores and low S-Zn as indicative of sub-optimal zinc
status, these were unexpectedly high prevalence figures in seemingly
well-nourished infants fed according to current recommendations.
It was concluded that the most likely explanation to the findings
was that the high intake of infant cereals, rich in phytate, impaired
the bioavailability of iron and zinc leading to iron and zinc
deficiency.

In Sweden, the intake of commercially manufactured, cereal-based


products is high from 6 mo of age, as both milk-based cereal drinks
(MCDs, essentially a liquid cereal eaten from a bottle) and
porridges are consumed, but the intakes of infant formula,
conventional follow-on formula and unmodified cows milk are low
(161). The MCDs and porridges are composed of cooked cereals,
skim milk powder, and vegetable fat. They are fortified with
minerals (iron and calcium) and vitamins (vitamin A, C, D, E, B6,
B12, thiamine, niacin, folic acid and pantothenic acid). With this
composition they comply with European Union legislation (167).
These products contribute substantially to the intakes of
micronutrients during the second half of infancy (161, 168, 169).

THE SITUATION IN INDONESIA AND PURWOREJO


The republic of Indonesia is the forth most populous country in the
world with a population of over 210 million. About 6 000 of the
nations 13 500 islands are inhabited, Java being the most
populated, harboring almost 2/3 of the population. Being a low
income country, malnutrition among infants, children and women
are important public health problems in Indonesia.

In 1997, the infant mortality rate was 52.2 and 45.2 per 1 000 live
births and the under-five mortality rate was 70.6 and 59.9 per 1
000 live births at the national level and in Central Java, respectively
(170). Malnutrition reportedly contributed to 40% of the infant
mortality and 60% of the child mortality. The differences between
regions and between social groups are substantial in Indonesia;
children in eastern Nusa Tenggara and Kalimantan, in the rural
areas and among the poorest socio-economic groups have the lowest

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weight-for-age, while the situation is somewhat better on Java and


Bali, in the urban areas and among the more affluent.

Data on anthropometrical status from 1995 show that at the


national level, 11% and 13% were wasted [weight-for-height z-
score (WHZ) < -2 SD compared to the WHO/NHCS reference
population], 25% and 42% were stunted [height-for-age z-score
(HAZ) < -2 SD], and 18% and 34% were underweight [weight-for-
age z-score (WAZ) < -2 SD] in infants 6-12 mo and children <5
years, respectively (171). In Central Java Province, 29% of children
<5 years were reported underweight (172) and that 38% of the
children were stunted (173).

Purworejo is a predominantly agricultural district in the southern


2
part of Central Java (Figure 4). The district covers 1 034 km and
the population was some 730 000 in 1994. The district is the
location of a health surveillance project run by the Community
Health and Nutrition Research Laboratories (CHN-RL), Gadjah
Mada University, Yogyakarta, Indonesia. For this surveillance
14 868 households have been selected. The infant mortality rate in
the surveillance area is reported at 49.2 (174) and the prevalence of
low birth weight (< 2 500 g) was 60/1000 live born in 1995-96
(175). The prevalence of malnutrition in children <24 mo (Figure
5), based on two longitudinal cohort studies within the surveillance
system (176, 177), indicates a pattern of increasing malnutrition in
the population during the first 2 years of life typical of many low-
income Asian countries (178). The median breastfeeding duration
was 22 mo, but only 7% received colostrum, and at 1 mo of age a
quarter of the infants were receiving complementary foods, a figure
that increased to 70% at 4 mo (179). These figures are similar to
those reported at the national level (170).

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40

35 Stunted
Underweight
30
Wasted

25

20

15

10

0
6 9 12 18 24

Figure 5. Prevalence (%) of stunting, underweight and wasting among infants


and young children in the CHN-RL surveillance area at 6, 9, 12, 18 and 24
mo of age.

The frequency of bottlefeeding at 12 mo was reported at 16% in


the surveillance area (179). The complementary foods used in the
area are plant-based and contain little animal protein and low
amounts of iron and zinc (180). This, together with large amounts
of phytate renders the diet inadequate in terms of iron and zinc,
and together with early introduction of complementary foods,
reducing the bioavailability of breastmilk iron (60), places the
infants at high risk of developing micronutrient deficiencies.

A report from Semarang on the north coast of Central Java (181),


shows that even though breastfeeding initiation was close to 100%,
61% received pre-lacteal feeds. More than one third of breastfed
infants were given supplementary food within the first two weeks,
and this number increased steadily during the first year of life.
Exclusive breastfeeding was uncommon, as was the use of
colostrum. The principal foods given were grains, fruits and sugar,
with little or no meat, fish or other animal source of nutrients.
Close to half of the mothers gave tea with or without sugar to their
infants.

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Nationwide household surveys in 1992 and 1995 report anemia


prevalence in under-fives at 56% and 40%, respectively [referred in
(182)]. Dijkhuizen et al reported from a cross-sectional study in
West Java (183) in infants 2.4-10.5 mo of age (n=155), who were
assessed together with their mothers, that 57% of the infants had
Hb <110 g/L, 54% had plasma retinol <0.70 mol/L, but only
20% had S-Ft <15 g/L and 17% S-zinc <10.7 mol/L. In Central
Java, Dibley and co-workers found 38% with Hb <110 g/L among
children 6-48 mo of age (n=666) (Dibley 1995, unpublished data).
From the same cross-sectional study in Central Java, Kjolhede et al
reported that 58% had S-retinol <0.70 mol/L, with the lowest S-
retinol levels found in infants 6-11 mo of age (184). Since the late
1970s, Indonesia has a national program for vitamin A
supplementation of children after the first year of life (185). Goiter
due to iodine deficiency is endemic on all larger Indonesian islands,
and some 20% of the population lives in areas with the goiter rate
being greater than 10% (186). Among elementary school children
in Purworejo, 11.5% of boys and 7.6% of girls were found to have
mild to moderate goiter (D. Ismail 1996, personal comm.).

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The Objective

THE OBJECTIVE
The overall objective of the two randomized trials reported in this
thesis was to prospectively investigate the effect of dietary or
supplemental iron and zinc on iron and zinc status, infant linear
growth, cognitive development, and incidence of childhood
infectious diseases during the first 6-12 mo of age in a high-income
(Ume, Sweden; the SINUS study) and a low-income (Purworejo,
Indonesia; the ZINAK study) setting.

The more specific aims were to:


x Assess whether phytate-reduced MCD and phytate-reduced
porridge or infant formula and conventional porridge fed to
Swedish infants from 6 to 12 mo of age would improve iron
(Hb, MCV, S-Ft, S-TfR) and zinc (S-Zn) status, affect growth
(weight, length, knee-heel length), mental and psychomotor
development or the incidence of diarrheal and acute respiratory
infections, measured during the second half of infancy compared
to infants fed conventional MCD and porridge.
x Record the dietary intake of Swedish infants to explore the effects
of dietary iron intake on iron status until 18 mo of age.
x Examine if daily supplementation with iron and zinc given alone
or in combination from given to Indonesian infants 6 to 12 mo
of age would improve iron (Hb, S-Ft, S-TfR) and zinc (S-Zn)
status, growth (weight, length, knee-heel length), mental and
psychomotor development or the incidence of diarrhea and
acute respiratory infections compared with placebo, with
emphasis on possible interactions between the two minerals for
the biochemical and functional outcomes.

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The Trials

THE TRIALS
Two trials compose the basis of this thesis. The SINUS (Study on
Infant Nutrition in Ume, Sweden) study is described in detail in

papers I III and the ZINAK (Zinc and Iron Supplementation to
Infants in Purworejo, Indonesia) study is described in papers IV
V. The papers are summarized in Table 4. The two trials originate
from a common problem of presumed or actual iron and zinc
deficiencies due to inadequacies in the complementary diet during
the latter half of infancy and their functional consequences and
possible prevention.

The studies focus on iron and zinc nutritional status and deficiency
with two different intervention strategies; reduction of a possible
inhibitor of iron and zinc absorption, i.e. phytate, or
supplementation with iron and zinc, appropriate for the different
circumstances in which the trials took place. The studies share
several common features; both use a randomized, controlled trial
design; both follow infants from 6 mo of age, the age at which
complementary feeding is recommended to commence; and both
assess several of the same biochemical as well as functional
outcomes.

There are also several differences in the design and execution of the
trials due to the conditions of the populations studied, the actual
problems prevalent in these settings, and the presumed ways to
alleviate these problems. Thus, this thesis is not a presentation of an
intervention trial in two different settings, but an effort to address a
common problem in two different populations by different
intervention strategies. Results and consequences are discussed
separately as well as across the studies and inferences are drawn to
the local populations as well as to the global scene, when
appropriate.


The acronym ZINAK stands for Zinc, Iron and aNAK, Indonesian for child.

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Table 4. Summery of papers.

Paper Title Objective Sample Outcomes assessed

I Effects of weaning cereals with different Examine if extensively phytate-reduced 267 infants completing a Hb, MCV, S-Ft, S-Zn, S-Cu, and
phytate contents on hemoglobin, iron infant cereals, or infant formula and regular dietary intervention trial prevalence of anemia, low S-Ft
stores, and serum zinc: a randomized porridge reduce the prevalence of depleted from 6 to 12 mo of age and low S-Zn
intervention in infants from 6 to 12 mo of iron stores and low serum zinc compared to
age regular infant cereal

II Effects of weaning cereals with different Examine if extensively phytate-reduced 263 infants completing a 6 WAZ, HAZ, knee-heel length,
phytate content on growth, development infant cereals, or infant formula and regular mo dietary intervention BSID MDI and PDI, incidence of
and morbidity: a randomized intervention porridge improve growth, development and trial and follow-up until 18 diarrhea and respiratory
trial in infants from 6 to 12 months of age health compared to regular infant cereal mo of age infections

III Dietary iron intake affects hemoglobin A secondary data analysis, assessing 245 children completing a Hb, MCV, S-Fe, S-Ft, S-TfR in
during infancy but not during the second possible associations between dietary iron 6 mo dietary intervention relation to dietary iron intake

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year of life intake during infancy and early childhood and follow-up until 18 mo
The Trials

and subsequent hematological and iron


status.

IV A community-based randomized controlled Examine if daily supplementation with iron 549 infants completing a Hb, S-Ft, S-TfR, S-Zn, S-Cu,
trial of iron and zinc supplementation in and zinc improve iron and zinc status supplementation trial from prevalence of low Hb, low S-Ft
Indonesian infants: interactions between compared to iron or zinc treatment alone or 6 to 12 mo of age and low S-Zn, interaction
iron and zinc placebo between iron and zinc

V A community-based randomized controlled Examine if daily supplementation with iron 650 infants completing a WAZ, HAZ, WHZ, knee-heel
trial of iron and zinc supplementation in and zinc improve growth, development or supplementation trial from length, BSID MDI and PDI,
Indonesian infants: effects on growth and reduces the incidence of diarrhea or 6 to 12 mo of age incidence of diarrhea and
development respiratory infections compared to iron or respiratory infections
zinc treatment alone or placebo
Methods

METHODS
Design and interventions

Table 5 summarizes the main characteristics of SINUS and


ZINAK. Table 6 summarizes the data collection and Table 7 shows
methods used for analysis of blood samples and collection of
anthropometrical and developmental data in the studies.

SINUS

In the Swedish study, infants were assigned to one of 3 study


groups; regular infant cereals (commercially available MCD and
porridge; CC group), low-phytate infant cereals (phytate-reduced
MCD and porridge; PR group) or cows milk based infant formula
and porridge (IF group). The rationale for this design was to allow
for comparisons between both the regular Swedish complementary
diet, i.e. phytatecontaining infant cereals and a diet with phytate-
reduced infant cereals, but also with the internationally more
common practice of using a combination of cows milk formula and
infant cereal, evaluating benefits as well as detriments of these
different feeding regimes.

Both breast-fed and non-breast-fed infants were recruited. If the


participating infants were breast-fed when entering the study, the
mothers were recommended to breast-feed as long as they wished.
At the mothers own discretion, the study products were introduced
into the infants diets from 6 mo of age and continued until 12 mo
of age, with no other interventions being done. Research nurses
supplied the families with the predetermined MCD/formula and
porridge ad libitum and free of charge. Semper AB, Stockholm,
Sweden, manufactured all study products.

Table 8 summarizes the study products used in SINUS. Figure 6


shows the processes used to reduce the phytate content in the study
products. The phytate content of the MCD and porridge was
analyzed as individual inositol phosphates by HPLC according to
Sandberg and Ahderinne (187).

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Methods

Oat Wheat Rye

Low extraction
wheat
Drum drying

Figure 6. The process of phytate reduction used in the SINUS study. Oat,
whole meal wheat and rye flours were scalded and soaked at pH 4.5, allowing
endogenous phytases to act. The slurry was then drum-dried and mixed with
low-extraction wheat flour.

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Table 5. Main characteristics of SINUS and ZINAK.

Study
Study of infant nutrition in Ume, Sweden Zinc and iron supplementation to infants in Purworejo,
Indonesia

Acronym SINUS ZINAK

Study area Ume, Sweden Purworejo, Indonesia

Problem Swedish infants show a 26% prevalence of low S-Ft and a 35% The infants of Purworejo, consuming a diet a high vegetable
prevalence of low S-Zn, indicating suboptimal micronutrient diet with little animal protein, are at a high risk of developing
status at one year of age. A high intake of phytate-containing micronutrient deficiencies during their first year of life.
cereals may contribute to this situation. Supplementation with iron and zinc may be a feasible way to
provide these micronutrients.

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Methods

Design Randomized dietary intervention trial with 3 study groups; Randomized supplementation study with 4 supplementation
groups in a factorial design;
x regular infant cereals (commercially available MCD and
porridge; CC group) x 10 mg of iron as ferrous sulfate (Fe group)
x low-phytate infant cereals (phytate-reduced MCD and x 10 mg of zinc as zinc sulfate (Zn group)
porridge; PR group)
x 10 mg of both iron and zinc (Fe+Zn group)
x cows milk based infant formula and porridge (IF group).
x placebo
All in sweet-tasting syrup.

Study population Healthy, term infants <6 mo of age from 6 well-baby clinics in Healthy, singleton infants from the CHN-RL surveillance
Ume (gestational age at birth 38 42 weeks) with birth system in Purworejo district who had not passed 6 mo of age
weights >2 500 g. Infants who at 6 mo had IDA were not and whose mothers had been monitored during pregnancy and
recruited. Exclusion criteria were severe and protracted illness birth were considered eligible. Exclusion criteria were Hb <90
and allergy or intolerance to the study products. g/L and severe and protracted illness.
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Randomization Block randomization; 12 infants per block Block randomization; 20 infants per block

Sample size calculation Prevalence of low S-Ft of 25% and of low S-Zn of 35% in the Primary outcomes of physical growth (knee-heel difference, 2
high phytate group, and an estimated prevalence of low S-Ft of mm in 6 months), psychomotor development (Bayley scales of
5% and low S-Zn of 15% in the low phytate group. With 100 development, 5 points difference) and diarrheal disease
infants per group we could allow for a 20% dropout rate morbidity (relative risk 0.65 in comparison with placebo). With a
(=0.05, power 80%) to show a significant difference in the dropout rate of 20%, 170 per group (=0.05, power 80%) would
prevalence of low S-Ft and low S-Zn between groups. detect significant differences between groups.

Outcomes Major outcomes: Main biochemical outcomes:


Iron status (Hb, MCV, S-Ft, S-TfR and S-Fe) Iron status (Hb, S-Ft, S-TfR)
Zinc status (S-Zn) Zinc status (S-Zn)

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Methods

Prevalence of Hb <110 g/l, S-Ft <12 Pg/l and S-Zn <10.7 Prevalence of Hb <110 g/l, S-Ft <12 Pg/l and S-Zn <10.7
Pmol/l Pmol/l
Secondary outcomes: Major functional outcomes
Somatic and linear growth Somatic and linear growth
Attained infant development Attained infant development
Incidence of diarrheal disease and lower respiratory infections Incidence of diarrheal disease and lower respiratory infections
Methods

Infants in ZINAK.

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Methods

Table 6. Data collection in SINUS and ZINAK.

Age (mo)

6 7 8 9 10 11 12 13 14 15 16 17 18

SINUS

Intervention

5-d dietary x x x x x x x x x x x x x
record

Blood sample x x x x

Anthropometry x x x x x x x x x x

Morbidity x x x x x x x x x x x x x

Development x x x

SES x x

ZINAK

Intervention

24-h recall x x x x x x x

Blood sample x x

Anthropometry x x x x x x x

Morbidity

Development x x

SES1 x

1
Socioeconomic status.

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Methods

Table 7. Analytical methods used in SINUS and ZINAK.

Study site

SINUS ZINAK

Hemoglobin Sysmex Sulfolyser automated Hemocue photometer


concentration hemoglobin reagent

Serum iron Ferrozine method NA

Serum ferritin Immunoturbidometric technique Radioimmunoassay

Serum transferrin Enzyme-linked immunosorbent Enzyme-linked immunosorbent


receptor assay assay

Serum zinc Atomic absorption Atomic absorption


spectrophotometry spectrophotometry

Serum copper Atomic absorption Atomic absorption


spectrophotometry spectrophotometry

Weight Seca 835 digital baby scale Seca 835 digital baby scale

Length Harpenden infantometer Locally produced wooden


board

Knee-heel length Infant knemometer BK5 Infant knemometer BK5

Head Non-stretchable plastic measuring Non-stretchable plastic


circumference tape measuring tape

Mid upper arm Non-stretchable plastic measuring Non-stretchable plastic


circumference tape measuring tape

Infant Bayley Scales of Infant Bayley Scales of Infant


development Development Development

Mental development (MDI) Mental development (MDI)

Psychomotor development (PDI) Psychomotor development


(PDI)

Behavior (BRS)

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Table 8. The energy and nutrient content in the infant cereals and infant formula per 100 g ready-to-feed product.

Milk cereal drink/Formula Porridge

6 7 mo. 8 11 mo. 6 7 mo. 8 11 mo.

CC1 PR2 IF3 CC PR IF CC PR IF CC PR IF

Energy (kcal) 66 66 65 65 65 65 101 101 101 96 98 96


Protein (g) 2.3 2.3 1.3 2.4 2.4 1.3 2.5 2.5 2.5 2.8 3.2 2.8
Fat (g) 2.4 2.4 3.4 2.5 2.5 3.4 2.8 2.8 2.8 2.7 2.9 2.7
Carbohydrate (g) 8.6 8.6 7.3 8.0 8.0 7.3 16.5 16.5 16.5 15.0 14.7 15.0
Iron (mg) 1.24 1.24 0.45 1.24 1.44 0.45 1.66 1.66 1.66 1.76 1.36 1.76
Zinc (mg) 0.3 0.3 0.5 0.4 0.3 0.5 0.4 0.4 0.4 0.5 0.5 0.5

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Calcium (mg) 93 93 44 106 106 44 149 149 149 149 149 149
Methods

Vitamin C (mg) 17 17 13 17 17 13 28 28 28 28 28 28
Phytate (Pmol) 30.0 9.9 0 30.0 3.4 0 23.1 23.1 23.1 78.5 9.7 78.5
% IP 67 65 87  75 83  88 88 88 86 86 86

Phytate:iron molar 1.4:1 1:2.2 0 1.4:1 1:7.4 0 1:1.2 1:1.2 1:1.2 2.6:1 1:2.4 2.6:1
ratio

Phytate:zinc molar 6.5:1 2.2:1 0 4.9:1 1:1.3 0 3.8:1 3.8:1 3.8:1 10.3:1 1.3:1 10.3:1
ratio

1
Commercial MCD and porridge. 2Phytate-reduced MCD and phytate-reduced porridge .3Infant formula and porridge with regular phytate level.
4 5 6 7
Iron pyrophosphate. Iron sulfate Iron orthophosphate (EKA-Fer). Percentage myo-inositol hexaphosphate [IP6] of total phytate.
Methods

ZINAK

In the Indonesian study, infants were randomly assigned to one of 4


supplementation groups: iron (Fe group), zinc (Zn group), iron +
zinc (Fe + Zn group), or placebo. The four supplements provided
the infants with a daily dose of either 10 mg of iron as ferrous
sulfate (Fe group), 10 mg of zinc as zinc sulfate (Zn group), 10 mg
each of iron and zinc (Fe + Zn group), or placebo, in a sweet-tasting
syrup. All supplements included, per dose (1.6 ml, i.e. 2 measuring
pipettes), 30 mg of ascorbic acid, sugar and water. The supplements
would then contain approximately the recommended daily intake of
iron and vitamin C and between 1-2 times the recommended daily
intake of zinc for infants 6-11 mo of age (9, 117).

PT Konimex, Solo, Indonesia, manufactured the supplements in


association with the Department of Pharmacology, Gadjah Mada
University. The supplements were administered by the
parents/caretakers once daily from 6 to 12 mo of age (180 days of
supplementation). Field workers oversaw and administered the daily
dose every third day and monitored the intake the previous two
days by means of parent recall. Empty bottles were replaced every 2
weeks and the remaining syrup, if any, was measured and registered.

Primary outcomes

Iron and zinc status

In both studies, venous blood was collected in zinc-free vacuum


systems. In the Swedish study iron status was analyzed at the
Department of Clinical Chemistry, Ume University Hospital by
use of a Sysmex SE 9000 Autoanalyzer (Tillqvist, Kista, Sweden).
Hb was analyzed using Sysmex Sulfolyser automated hemoglobin
reagent (Toa Medical Electronics Co, Los Alamitos, CA, USA) and
MCV was automatically calculated from the erythrocyte particle
concentration and hematocrit. S-Fe was analyzed by the ferrozine
method (Iron kit 1553712 and UIBC kit 1030600, Boehringer
Mannheim, Scandinavia AB, Bromma, Sweden). S-Ft was analyzed
by an immunoturbidometric technique (BM/Hitachi 704/717/911,
Boehringer Mannheim) calibrated against WHO standard 80-602.

In ZINAK, Hb was analyzed using a portable Hemocue


photometer (Hemocue AB, ngelholm, Sweden) and S-Ft was
analyzed by radioimmunoassay (Diagnostic Products, San Diego,

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Methods

CA). In both studies S-TfR was analyzed by enzyme-linked


immunosorbent assay (ELISA) (Ramco, Houston, TX), and S-Zn
and S-Cu were assessed by atomic spectrometry (188).

Anthropometry

In both studies, anthropometrical measurements were performed on


a monthly basis from 6 to 12 mo of age, and in SINUS, bi-monthly
from 14-18 mo of age. Naked weight was measured to the nearest
0.02 kg using a Seca 835 digital baby scale (Seca, Hamburg,
Germany). Recumbent length was in the Swedish study measured
using a Harpenden infantometer (CMS Weighing Equipment,
London, UK), and in the Indonesian study using a locally produced
wooden board. Knee-heel length was measured in both studies
using an infant knemometer (Infant Knemometer BK5, FORCE
Instituttet, Brndby, Denmark). Head circumference and mid-
upper arm circumference were measured using a non-stretchable
plastic measuring tape.

Infant development

Both studies used the Bayley Scales of Infant Development (BSID)


(189) to assess development, recording the mental development
index (MDI) and the psychomotor development index (PDI). In
ZINAK the behavioral rating scale (BRS) was also recorded. In
SINUS developmental testing was done at ages 7, 13 and 18 mo,
whereas in ZINAK it was assessed at 6 and 12 mo of age.

Baseline and follow-up data collection

In SINUS, background data including family size, parental


education and occupation were collected at 12 mo and then again
at 18 mo, with birth weight and birth length collected
retrospectively from birth records. In ZINAK, socio-economic
information was retrieved from the CHN-RL surveillance system at
baseline. CHN-RL field workers or community midwives measured
birth weight at the time of delivery. After the end of the
supplementation period, the Indonesian families were interviewed
on perceived side-effects of the supplements (abdominal pain,
decreased appetite, vomiting, diarrhea, constipation, increased
crying or fussiness) as well as the general health condition of the
child before and after supplementation.

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Methods

Morbidity registration

Parents recorded daily symptoms of fever, coryza, cough, fast or


difficult breathing, ear discharge and change in stool consistency in
the Swedish study. Records were collected at monthly intervals and
checked for consistency.

In ZINAK, field workers visited the families every third day,


recording compliance to supplementation as well as symptoms of
illness for the day of visit, and by parental recall, for the two days
preceding the visit. Symptoms of fever (mothers own definition);
coryza, cough, difficult or fast breathing, ear-discharge, diarrhea and
vomiting were recorded.

Dietary intake

Much emphasis was put on collecting dietary data in the Swedish


study. Each month, starting from 6 mo of age, parents or caregivers
recorded the type and amount of each food item consumed by the
infant during five consecutive days. Household measures were used
for quantities, and the participating family was encouraged to serve
the study child all meals from a standardized plate. The families
were also given a booklet with photos of different portion sizes of
common infant foods, using the standardized plate, to facilitate
registration (Figure 7).

Breast-feeding was recorded as meal, equivalent to a full meal, or


snack, i.e. a short feed mainly for comfort or other non-nutritive
purposes, according to the mothers own perception. Daily energy
and nutrient intake was calculated using the MATs software
(Rudans Lttdata, Vsters, Sweden), which uses the food
composition tables of the Swedish National Food Administration
(190). The database was complemented with baby foods, formulas
and other recipes not originally included according to information
from the participating families and the manufacturers.

Intakes of breast milk were set to 134 g per meal up to 8 mo of age,


to 102 g per meal beyond 8 mo, and to 25 g per snack at all ages
(191). Nutrient intake from breast milk was calculated from Jensen
(192) and Tsang et al (193). Nutrient and energy intake from breast
milk was added to the total daily nutrient and energy intake of the
study subjects for the 6-11 mo period, but not in the subsequent
period.

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Methods

Figure 7. Pictures from the booklet of different portion sizes of infant foods
used in the recording of dietary intake in the SINUS study.

In the Indonesian study, 24-h recalls of dietary intake were


collected monthly, but analyses of that data were not included in
this thesis.

Statistical methods

SPSS for Windows 11 (SPSS Inc, Chicago, IL) was used for all
statistical computations. Non-normally distributed variables, e.g. S-
Ft and S-TfR values, were log-transformed. Main outcomes are
shown as means and standard deviations, geometric means, when
applicable, and proportions. Anthropometrical data are shown as z-
scores compared to the WHO/NCHS reference population (194).
Prior to analysis, the anthropometrical data were interpolated to
correspond to each completed month of age. Conversion to
anthropometrical z-scores was done using Epi Info 2000 version
1.1.1 (Centers for Disease Control and Prevention, Atlanta, GA)
using the 2000 CDC reference growth data (194). Development
was assessed using the BSID.

This scale measures three facets of infant development, mental


development using the MDI, psychomotor development using the
PDI and behavior using the BRS. Developmental data are shown as
mean MDI and PDI, and for the Indonesian study, also median
BRS. BRS data were highly skewed so in order to achieve normal
distribution, rank was used as outcome in the analyses. Morbidity
was analyzed with Poisson regression using Stata 6.0 (Stata Corp,
College Station, TX) and is shown as incidence and incidence rate
ratios (95% confidence interval; CI) for diarrhea and acute

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Methods

respiratory infections (ARI) with the control groups, i.e. in SINUS


the CC group and in ZINAK the placebo group, as reference. In
both studies, an episode of illness was defined as the symptom for at
least one day, followed by at least three disease-free days. Diarrhea
was defined as three or more loose or watery stools in 24 hours,
while ARI was defined as fever in combination with cough and/or
fast or difficult breathing.

Factor analysis was used on the monthly diet registrations. The


Swedish dietary data are shown as mean or geometric mean daily
intakes of energy and selected nutrients during the 6-8, 9-11 and
12-17 months periods, respectively. When comparing proportions,
2
Chi -test and/or Fishers exact test were used in both studies.
Statistical significance was set at p <0.05.
In SINUS, one-factor ANOVA was used in the intention-to-treat
analysis for the continuous variables comparing the study groups.
To adjust for effects over time, analysis of co-variance (ANCOVA),
using initial values at 6 mo as covariates for results at 12 mo was
used. Significant differences between study groups were Bonferroni
corrected to allow for multiple comparisons.

In paper III the combined data from all three SINUS groups was
used. The rationale behind this was that although the three groups,
i.e. CC, PR and IF differed in which study products they
consumed, the group allocation had little effect on the main
outcomes. For Hb, there was a significant difference between PR
and IF, but this became non-significant when adjusting for mean
daily iron intake, indicating that it was iron intake and not group
allocation per se that had an impact.

The associations between dietary iron intake, iron status variables


and background variables were analyzed with linear regressions.
Multivariate regression models were constructed, using variables
with biologically known or plausible relevance to the main
outcomes and dietary iron intake and those with a univariate p-
value <0.20, i.e. gender, birth weight, growth (i.e. relative weight
change since birth), intake of cows milk and breast milk, baseline
values of main outcomes, study group and intakes of energy,
protein, calcium, ascorbic acid and phytate, testing for interaction
and confounding.

In ZINAK, two-factor ANOVA was applied to examine main


effects and interactions between iron and zinc supplementation for
the continuous outcome variables. When a significant interaction (p

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Methods

<0.10) was found, follow-up test using Bonferroni adjustment was


performed. To adjust for possible confounding, the covariates
gender, birth weight, initial values for the main outcomes,
compliance measured as volume of supplement consumed, presence
of vomiting, both as reported in the follow-up interview on side-
effects and as recorded in the morbidity registrations, mothers
education and as a proxy for socioeconomic status, the location of
the households water source were included in the analysis.

Lowess smoothed plots, which uses an iterative locally weighted


least-squares method to fit a curve to a set of points (195), were
used to visualize the dose-effect relationship between daily iron
intake and the main outcomes in paper III, and the dose-effect
relationship between volume taken and outcome for the different
treatment groups in paper IV. In the former paper, this was
followed by construction of multivariate regression models, as
discussed above. In the latter paper, the Lowess smoothed plots
were followed by multivariate linear regression analyses to
statistically assess the associations between iron and/or zinc
supplementation, and S-Ft and S-Zn. Adjustments were made for
potential confounding of the dose-effect. In the regression analysis,
presence of vomiting reported as a sideeffect at the follow-up
interview with the mother, educational level of the mother, water
source of the household, and birth weight were identified as
possible confounders and thus included in the model.

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Findings

FINDINGS

BASELINE CHARACTERISTICS
This thesis reports the main findings of the two studies until the
end of the interventions at 12 mo of age. However, in the Swedish
study some functional findings of the follow-up until 18 mo of age
are also reported. Figure 8 summarizes the flow of infants in
SINUS. There were no significant differences between infants who
completed the trial and those who did not with regard to birth
weight, birth length, breast-feeding duration, parental level of
education, family size, baseline biochemistry, anthropometry or
development, nor were there any statistically significant differences
at baseline between the three study groups for any of the socio-
economic, anthropometrical, hematological or biochemical
variables, except for low S-Zn being more common in the CC
group than in the other groups (31% vs. 16% and 19% in the PR
and IF groups, respectively, p=0.04).

Figure 9 summarizes the flow of participating infants in ZINAK.


Baseline biochemistry (Hb, S-Ft, S-TfR, S-Zn), anthropometry,
psychomotor development and socio-demographic characteristics
did not differ between those who completed and did not complete
the trial, nor were there differences between the four treatment
groups in baseline characteristics.

Two study sites differed significantly in several of the background


characteristics (family size, mothers educational level, birth weight
and number of infants breast fed at study start) and baseline
measurements, including S-Ft, S-TfR, S-Zn, and prevalence of
anemia, low S-Ft, IDA, low S-Zn, and the anthropometrical
measurements (Table 9). However, baseline Hb did not differ
significantly between sites and there were no statistical differences
in mental or psychomotor development index.

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Findings

Randomly assigned
n=300

Commercial Phytate-reduced Infant formula


cereal group (CC) group (PR) group (IF)
n=100 n=100 n=100

Discontinued Discontinued Discontinued


intervention intervention intervention
Moved (n=2) Moved (n=1) Moved (n=2)
Refused (n=2) Refused (n=4) Refused (n=10)
CMA (n=1) CMA (n=2)

Excluded from Excluded from Excluded from


analysis at 12 mo analysis at 12 mo analysis at 12 mo
Incomplete diet Incomplete diet Incomplete diet
registration (n=1) registration (n=3) registration (n=3)
Missing blood
Missing anthrop Missing anthrop
sample n=2
data (n=5) data (n=2)

Discontinued Discontinued Discontinued


follow-up follow-up follow-up
Refused (n=5) Refused (n=2) Refused (n=6)

Excluded from Excluded from Excluded from


analysis at 18 mo analysis at 18 mo analysis at 18 mo
Missing blood Missing blood Missing blood
sample (n=3) sample (n=3) sample (n=3)

Analysed at 18 mo Analysed at 18 mo Analysed at 18 mo


Anthrop. n=90 Anthrop. n=91 Anthrop. n=82
Biochemistry n=86 Biochemistry n=85 Biochemistry n=74

Figure 8. Flow of infants in the SINUS study. CMA cows milk allergy

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Assessed for eligibility (n=1067)

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Chronic illness (n=16)
Hb <90 g/L (n=18)
Twins (n=8)
Refused blood sampling (n=214)
Eligible but beyond the monthly
recruitment of 50 infants (n=131)

Randomly assigned (n=680)

Allocated to iron group Allocated to zinc group Allocated to iron + zinc group Allocated to placebo
(n=170) (n=170) (n=170) (n=170)

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Discontinued intervention (n=3) Discontinued intervention (n=6)
Findings

Discontinued intervention (n=4) Discontinued intervention (n=1)


Refused supplement n=1, Refused supplement n=3,
Refused supplement n=4 Refused supplement n=1
died n=2 moved n=3

Anthropometry (n=163) Anthropometry (n=162) Antropomerty (n=161) Anthropometry (n=164)


Excluded from analysis (n=3) Excluded from analysis (n=5) Excluded from analysis (n=3) Excluded from analysis (n=5)
Incomplete anthopometrical Incomplete anthopometrical data Incomplete anthopometrical data Incomplete anthopometrical
data n=3 n=5 n=3 data n=5

Biocheistry (n=136) Biochemistry (n=134) Biochemistry (n=136) Biochemistry (n=143)


Excluded from analysis (n=30) Excluded from analysis (n=33) Excluded from analysis n=28 Excluded from analysis n=26
Refused blood sample n=10 Refused blood sample n=13 Refused blood sample n=9 Refused blood sample n=14
Insufficient serum volume n=20 Insufficient serum volume n=20 Insufficient serum volume n=19 Insufficient serum volume n=12

Figure 9. Flow of infants in the ZINAK study.


Findings

Table 9. Baseline characteristics of infants in SINUS and ZINAK.

SINUS (n=267) ZINAK (n=549) P-value

Persons per household (n)1 3.7 (0.84) 4.2 (1.35) <0.001

Mothers education 94 44 <0.001


>9 years (%)

Girls (%) 52 48 0.33

Birth weight (kg)1 3.62 (0.483) 3.21 (0.462) <0.001


ZINAK n=509

Breast fed at 6 mo (%) 75 97 <0.001

Hb (g/L)1 115 (8.2) 114 (13.3) 0.084

S-ferritin (g/L)2 44.6 (2.49) 33.6 (2.58) <0.001

S-TfR (mg/L)2 6.17 (1.43) 7.08 (1.39) <0.001

S-zinc (mol/L)1 13.2 (3.69) 9.3 (2.52) <0.001

Hb <110 g/L (%) 28 41 <0.001

S-ferritin <12 g/L (%) 9 15 0.018

IDA (%) 3 8 0.008

S-zinc <10.7 mol/L (%) 22 78 <0.001

MDI1 99.1 (5.66) 99.7 (8.08) 0.35

PDI1 95.6 (10.2) 95.7 (11.67) 0.94

WLZ1 0.12 (1.01) -0.09 (1.13) 0.009

WAZ1 0.48 (0.93) -0.41 (0.99) <0.001

HAZ1 0.69 (0.77) -0.31 (0.84) <0.001

Data are 1 mean (SD),2 geometric mean (antilog SD).

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Findings

Deviation from protocol


In SINUS, there were no differences in consumption of the study
products between study groups (Table 10), nor were there any
significant difference between study groups in the number of
infants who left the study before 12 mo (5%, 7% and 12%, p=0.17
for the CC, PR and IF groups, respectively). No adverse effects of
the different diets were reported.

In ZINAK, vomiting was more common in the Fe + Zn group


(Table 11). Compared to the placebo group, the Fe + Zn group had
a 4.1 (95% CI 3.4-4.9) higher risk for vomiting, and the Zn group
a 1.9 (95% CI 1.5-2.5) higher risk, respectively. Other perceived
sideeffects (abdominal pain, diarrhea, constipation, poor appetite,
increased crying, and fuzziness) did not differ between treatment
groups (data not shown). Vomiting was negatively associated with
total volume of given supplement (r = -0.387, p<0.001), which
implies the possibility of loss of supplement after the daily dose. For
consumption of supplement, main effects for both iron treatment
and zinc treatment were significant, but interaction was not.

IRON AND ZINC STATUS


At baseline, overall prevalence of Hb <110 g/L, S-Ft <12 g/L and
S-Zn <10.7 mol/L was 28%, 9% and 22%, respectively among the
Swedish infants. Six infants could be classified as having IDA (Hb
<110 g/L, S-Ft <12 g/L and MCV <73 fL). After 6 mo of
consuming the diets with different phytate content, neither iron
status (Hb, S-Ft, S-Fe, and S-TfR) nor S-Zn differed significantly
between the groups consuming lower amounts of phytate (PR and
IF) and the CC group (Table 11).

Mean Hb was slightly but significantly lower in the IF group than


in the PR group (117 vs. 120 g/L, p=0.015) (Table 12) at 12 mo of
age. The diet in the IF group had a lower iron content than the
other two groups, and total iron intake was significantly lower in
the IF group than in the other groups. When adjusting the
intention-to-treat analysis for total iron intake, the group
differences in effect on Hb between the PR and IF groups became
non-significant (p=0.48) and the effect estimates were reduced from
a difference of 3.3 g/L to 0.8 g/L for Hb.

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Findings

Table 10. Mean daily consumption of study formula/milk cereal drink and porridge,
frequency of breast feeding, and intake of breast milk, energy and selected nutrients at 6-
8 and 9-11 months of age (n=267).

6 8 months 9 11 months

CC1 PR2 IF3 CC PR IF


(n=94) (n=90) (n=83) (n=94) (n=90) (n=83)

Formula/MCD (g/d)4 329 (275.5) 321 (268.2) 310 (262.1) 441 (226.1) 455 (231.6) 388 (229.5)

Porridge (g/d)4 94 (50.9) 85 (51.4) 85 (55.1) 85 (59.4) 80 (52.7) 87 (62.0)

Breast feeding 3.9 (2.5) 4.1 (2.4) 4.2 (2.7) 2.5 (2.3) 2.5 (1.8) 3.0 (2.8)
frequency (times/d)4,5,10

Breast milk intake 413 (276) 424 (254) 440 (277) 201 (194) 215 (175) 240 (235)
(g/d)4,5,10

Energy (kcal/kg/d)4 89.9 (15.27) 89.8 (18.50) 87.9 (16.53) 85.2 (15.27) 87.4 (15.74) 84.7 (17.83)

Protein (g/kg/d)4 2.4 (0.67) 2.4 (0.82) 2.0 (0.56)6 3.0 (0.71) 3.0 (0.66) 2.3 (0.57)7

Fat (g/d)4 30.9 (6.03) 31.5 (6.49) 33.8 (6.28)6 29.4 (5.35) 31.3 (6.22) 33.0 (7.33)9

Carbohydrates (g/d)4 98.0 (17.34) 95.8 (22.15) 91.3 (18.90) 107.3 (21.71) 106.5 (20.91) 97.9 (18.45)9

Iron (mg/d)4 7.5 (3.69) 7.6 (4.27) 4.7 (2.13)6 9.9 (3.40) 10.3 (3.66) 6.2 (2.00)7

Zinc (mg/d)4 2.8 (0.78) 2.8 (0.98) 3.3 (1.27)6 4.0 (1.02) 4.1 (1.02) 4.3 (1.21)

Vitamin C (mg/d)4 124 (48.9) 121 (54.0) 104 (39.8)6 140 (48.7) 139 (46.7) 109 (34.5)7

Calcium (mg/d)4 661 (228.5) 649 (261.1) 473 (138.7)6 832 (248.2) 830 (225.0) 556 (146.1)7

Phytate (Pmol/d)4,8 124 (82.4) 48 (31.0)9 26 (18.2)6 189 (86.8) 36 (18.9)9 62 (46.8)7

1 2 3
Commercial MCD and porridge. Phytate-reduced MCD and phytate-reduced porridge. Infant formula
4 5 6
and porridge with regular phytate level. Mean (SD). Geometric mean for 9-12 mo. period. Significantly
7
different from other groups 6-8 mo. (ANOVA p<0.05, Bonferroni corrected). Significantly different from
8
other groups 9-12 mo. (ANOVA p<0.05, Bonferroni corrected). Phytate from study products only.
9 10
Significantly different from Commercial cereal (ANOVA p<0.05, Bonferroni corrected). At 6-8 mo. CC
n=70, PR n=67, IF n=62. At 9-12 mo. CC n=34, PR n=35, IF n=33.

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Findings

Table 11. Details of follow-up at the end of supplementation.

Study group Main effect Interaction


P-value P-value

Placebo Fe Zn Fe+Zn Fe Zn Fe Zn
(n=164) (n=163) (n=162) (n=161)
Treatment

Total 242 (51) 218 (54) 232 (57) 202 (70) <0.001 0.004 0.60
supplement
volume (ml)1
Health

Breast fed at 134 (94) 154 (94) 155 (95) 148 (92) 0.12 0.04 0.99
12 months
[n (%)]

Illness within 2 40 (26) 43 (28) 43 (29) 40 (26) 0.82 0.90 0.53


weeks prior to
endpoint
[n (%)]
Side-effects

Any perceived 94 (57) 96 (59) 102 (63) 112 (70) 0.001 0.34 0.88
side effect
[n (%)]

Vomiting [n 44 (27)a 49 (30)a 56 (35)a 84 (53)b 0.004 <0.001 0.043


(%)]2

1 2
Data are mean (SD), defined as reported vomiting as side effect during
supplementation at follow-up interview. Means not sharing a common
superscript letter are significantly different at P<0.05 based on 2-factor
ANOVA (Bonferroni adjusted).

S-Cu was significantly higher in the CC group compared to the PR


group (17.0 vs. 15.6 Pmol/L, p=0.004). Overall, from 6 to 12
months of age, the prevalence of Hb <110 g/L decreased
significantly (28% vs. 15%, p=0.001), the prevalence of S-Ft <12
g/L increased (9% vs. 16%, p=0.012) and the prevalence of S-Zn
<10.7 mol/L remained unchanged (23% vs. 29%, p=0.09). The
proportion of infants with Hb <110 g/L at 12 months tended to be
higher in the IF group compared to the other two groups (23% vs.
11% and 13%, in the CC and PR groups, respectively, p=0.06).

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There were no differences between study groups in prevalence of


low S-Ft or low S-Zn at 12 months.

Among the Indonesian infants, 41% had Hb <110 g/L, 15% had S-
Ft <12 g/L and 78% had S-Zn <10.7 mol/L at baseline. After 6
mo of supplementation, there was a significant negative interaction
between iron and zinc treatment for Hb (main effects: iron
treatment p=0.020, zinc treatment p=0.56; iron u zinc interaction
p=0.021) (Table 13). The Fe group had a significantly higher Hb
concentration at the endpoint compared to placebo and Fe + Zn.
The prevalence of Hb <110 g/L in the Fe group was significantly
lower than in the placebo and Fe + Zn groups (25%, 36%, 38%,
and 44% in the Fe, Zn, Fe + Zn and placebo groups, respectively,
p<0.05).

For ln S-Ft, the interaction was also negative and significant (main
effects: iron treatment p<0.001, zinc treatment p=0.13; ironuzinc
interaction p=0.023). S-Ft was significantly higher in the Fe group
compared to the Fe + Zn group. For S-TfR, the main effects (iron
treatment p<0.001, zinc treatment p=0.003) were significant, but
there was no significant interaction (iron zinc interaction p=0.76).

At the endpoint, the prevalence of S-Ft <12 Pg/L was 5%, 35%,
10%, and 37% in the Fe, Zn, Fe + Zn and placebo groups,
respectively. The Fe and Fe + Zn groups had significantly lower
prevalence of IDA (Hb <110 g/L and S-Ft <12Pg/L) as compared
to placebo (2%, 16%, 3% and 21% in the Fe, Zn, Fe + Zn and
placebo groups, respectively, p<0.001). The prevalence of IDA
increased significantly from 6 to 12 mo of age in the placebo group
(p=0.002).

Two-factor ANOVA for S-Zn showed significant main effects (iron


treatment p=0.01, zinc treatment p<0.001), but no significant
interaction (iron zinc interaction p=0.15). In the Zn and Fe + Zn
groups, significantly fewer infants had low S-zinc levels as compared
to placebo and the Fe group (87%, 47%, 54%, and 78% in the Fe,
Zn, Fe + Zn, and placebo groups, respectively, p<0.001).

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GROWTH
The Swedish infants grew at the level of or above Swedish national
(196) and international reference populations (194) for height and
weight throughout the study period (Figure 10). The overall mean
WAZ decreased significantly from 6-18 mo, both compared to
Swedish national data (WAZ 0.34 vs. -0.17, p<0.001) and an
international reference population (WAZ 0.49 vs. 0.18, p=0.001).
Neither during the intervention, i.e. from 6 to 12 mo of age, nor
during the subsequent follow-up until 18 mo of age, were there any
significant differences among study groups in any of the
anthropometrical measures (Table 12).

Overall, anthropometrical status deteriorated significantly in the


Indonesian infants from 6 to 12 mo of age (Figure 10). The
prevalence of wasting increased from 4% to 16% (p<0.001), of
underweight from 4% to 38% (p<0.001) and of stunting from 4%
to 10% (p<0.001). Between study groups, the proportion of
wasting at 12 mo was significantly higher in the Fe group compared
to the Zn group (21.5 vs. 11.1%, p=0.044).

At the end of the 6 mo supplementation period there was a


significant negative interaction between iron and zinc treatment for
WAZ (main effects: iron treatment p=0.046, zinc treatment
p=0.11; ironuzinc interaction p=0.020). In the Zn group, WAZ
was significantly higher compared to the placebo and Fe + Zn
groups (Table 13).

Further, two-factor ANOVA showed significant negative


interaction between iron and zinc treatment for knee-heel length
(main effects: iron treatment p=0.65; zinc treatment p=0.59;
ironuzinc interaction p=0.001). Knee-heel length was higher in the
Zn and Fe groups compared to placebo. Controlling for potential
confounders, such as consumption of supplement and occurrence of
vomiting, did not significantly change the results. For WHZ, there
was a significant main effect of zinc treatment (p=0.004), but no
effect of iron treatment and no interaction (data not shown). HAZ,
head circumference, or mid-upper arm circumference were not
significantly different among the groups.

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0.5

0
6 7 8 9 10 11 12 13 14 15 16 17 18
Z-score

-0.5
SINUS WAZ
SINUS HAZ
-1 ZINAK WAZ
ZINAK HAZ

-1.5

-2
Age (mo)

Figure 10. Weight-for-age and height-for-age z-scores in the ZINAK and


SINUS studies from 6 to 12 mo, and 6 to 18 mo of age, respectively.

Mental and psychomotor development

Of the 300 infants in the Swedish trial, 276 were ever tested and
194 (65%) had measurements at 7, 13 and 18 mo of age. There
were no significant differences in mean BSID MDI or PDI between
study groups at any time point (Table 12), nor were there any
significant associations between iron or zinc status and
developmental scores at any age.

In ZINAK, there was significant negative interaction between iron


and zinc treatment for BSID PDI at 12 mo (main effects: iron
treatment p=0.39; zinc treatment p=0.82; ironuzinc interaction
p=0.009). In the Fe group, attained PDI at 12 mo was significantly
higher than in the placebo group (difference in mean 2.86,
p=0.042) (Table 13).

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Table 12. Outcomes at 12 mo of age in the SINUS study.

Study group

CC PR IF P-value1

Biochemistry n = 94 n = 90 n = 83

Hemoglobin (g/L)2 119 (8.0)a,b 120 (8.4)b 117 (9.5)a 0.039

MCV (fL)2 76 (3.4) 76 (2.8) 75 (3.8) 0.065

Serum iron 9.1 (4.62)a 11.2 (4.34)b 9.7 (4.16)a 0.005


(mol/L)2

Serum ferritin 25.3 (1.99) 21.3 (1.92) 25.2 (1.97) 0.145


(g/L)3

Serum TfR (mg/L)2 6.88 (1.44) 7.11 (1.41) 6.78 (1.41) 0.66

Serum zinc 12.2 (2.56) 12.5 (2.80) 12.4 (2.68) 0.79


(mol/L)2

Serum copper 17.1 (3.53)a 15.6 (3.40)b 16.4(3.25)a,b 0.014


(mol/L)2

Anthropometry n = 90 N = 91 n = 82

WAZ2 0.16 (0.96) 0.13 (0.97) 0.19 (0.96) 1.00

HAZ2 0.69 (0.66) 0.61 (0.86) 0.57 (0.66) 0.59

WHZ2 0.17 (1.10) 0.23 (0.90) 0.26 (1.03) 0.85

Mid-upper arm 15.7 (1.20) 15.6 (1.01) 15.7 (1.06) 0.81


circumference (cm)2

Knee-heel length 20.74 (0.860) 20.67(0.919) 20.73 (0.790) 0.96


(cm)2

Development n = 69 n = 68 n = 57

MDI2 108.2 (7.49) 108.7 (8.59) 108.4 (9.54) 0.81

PDI2 100.3(12.44) 99.5(12.45) 99.9 (12.65) 1.00

Data are 1ANOVA P-value for difference between groups, 2mean (SD),
3
geometric mean. Means not sharing a common superscript letter are
significantly different at P0.05 (Bonferroni adjusted).

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Findings

Table 13. Outcome of supplementation on biochemical indicators of iron and


zinc status, anthropometry and development at 12 months of age.

Study group Main effect P-value Interaction


P-value
Placebo Fe Zn Fe+Zn Fe Zn Fe Zn

Biochemistry n = 143 n = 136 n = 134 n = 136

Hemoglobin (g/L)1 113 119 116 115 0.020 0.56 0.021


(16.0)a (15.3)b (15.2)a (13.9)a
Serum ferritin (g/L)2 12.9 46.5 13.3 32.3 <0.001 0.13 0.023
(3.7)a (2.0)b (3.6)a (2.9)c
Serum TfR (mg/L)2 9.02 6.71 9.78 7.56 <0.001 0.005 0.87
(1.73) (1.33) (1.42) (1.36)
Serum zinc (mol/L)1 9.06 8.76 11.58 10.80 0.011 <0.001 0.23
(1.27) (1.24) (1.41) (1.34)
Serum copper (mol/L)1 15.2 15.2 15.0 14.7 0.65 0.34 0.55
(5.1) (4.8) (5.1) (4.5)
Anthropometry n = 164 n = 163 n = 162 n = 161
(n = 650)
WAZ 1,3 -1.72 -1.65 -1.46 -1.68 0.092 0.079 0.004
(1.00)a (1.08)a (1.08)b (1.02)a
HAZ 1,4 -0.81 -0.66 -0.77 -0.90 0.84 0.16 0.16
(0.86) (0.91) (0.92) (0.90)
WHZ 1,5 -1.01 -1.07 -0.70 -0.86 0.26 0.004 0.14
(1.16) (1.23) (1.06) (1.06)
Mid arm circumference 14.7 14.7 14.8 14.6 0.36 0.77 0.039
(cm)1 (1.18) (1.12) (1.14) (1.04)
Knee-heel length (cm)1 19.30 19.45 19.50 19.40 0.46 0.62 0.003
(0.95)a (0.96)b (0.99)b (0.94)a,b
Development n = 165 n = 163 n = 167 n = 160
(n = 655)
Mental development 99 101 101 100 0.76 0.63 0.069
index1 (10.0) (9.7) (9.3) (9.8)
Psychomotor 103 106 105 103 0.82 0.39 0.009
development index1 (10.8)a (11.0)b (10.6)a,b (10.3)a,b
Behavioural rating 42 42 39 35 0.55 0.062 0.091
scale6,7 (20-62) (22-69) (20-66) (19-53)

Data are 1mean (SD), 2geometric mean (antilog SD), 3weight-for-age z-score,
4
height-for-age z-score, 5weight-for-height z-score, 6median (25-75 percentile),
7
and main effect and interaction calculated from group ranks. Means not
sharing a common superscript letter are significantly different at P<0.05 based
on 2-factor ANOVA (Bonferroni adjusted).

Controlling for potential confounders, such as volume consumed of


supplement, initial iron status, vomiting and mothers education,
did not significantly change the result. There were no significant
differences for MDI or BRS between groups.

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INCIDENCE OF DIARRHEA AND RESPIRATORY INFECTIONS


Among the Swedish infants, we recorded an overall incidence of
0.97 episodes of diarrhea per person year and 1.55 episodes of ARI
per person year from 6 to 11 mo, and from 12-17 mo, the
incidence of diarrhea and ARI was 0.70 and 0.89 per person year,
respectively. The decreases in both diarrhea and ARI incidences
from 6-11 to 12-17 mo were significant. There were no differences
in incidence of diarrhea or ARI among the study groups during the
6-11 mo. period. However, the IF group had significantly higher
incidence rate ratio (RR) for diarrhea during the 12-17 mo period
compared to the PR group (RR=1. 77, 95% CI 1.05 2.97), but
not to the CC group. There was a trend, albeit non-significant, in
the relative risk for diarrhea between the IF and PR groups; during
the 10-11, 12-13, 14-15 and 16-17 mo periods RR was 1.12 (95%
CI 0.66-1.90), 2.56 (0.90-7.27), 1.66 (0.75-3.69) and 1.41 (0.56-
3.58), respectively.

Among the Indonesian infants, the incidence of diarrhea (2.9, 3.0,


2.7 and 2.8 episodes/person year for the Fe, Zn, Fe + Zn and
placebo groups, respectively) and ARI (3.5, 3.6, 3.4 and 3.7
episodes/person year for Fe, Zn, Fe + Zn and placebo, respectively)
did not differ significantly between treatment groups, nor did
duration of diarrhea or ARI (data not shown). Compared to the
Swedish infants during the 6-11 mo period, the incidence was three
times higher for diarrhea and more than two times higher for ARI
(Figure 11). Anthropometrical status was not associated with
incidence of infectious disease among the Indonesian infants, nor
was there any significant interaction between treatment group and
level of nutritional status in respect to morbidity.

DIETARY INTAKE
Among the Swedish infants, 75% of the participating infants were
breast-fed at baseline. Median duration of breast-feeding was 8.9
mo. Total duration and daily frequency of breast-feeding, as well as
daily intake of breast milk was similar between the three groups.
Energy intake from the total diet was similar in all groups
throughout the study, but intake of protein, iron, vitamin C,
calcium and phytate differed, basically mirroring the differences in
nutrient content between the infant cereals and infant formula
(Table 10).

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Zinc intake also differed, but only during the 6-8 mo period. Of
the total energy intake, the study products contributed with on
average 41% during the 6-8 mo period and 44% during the 9-11
mo period. A significant proportion of the daily intake of iron and
zinc came from MCD or infant formula and porridge. On average,
62% (4.5 mg) and 59% (5.6 mg) of the daily iron intake came
from the study products during the 6-8 mo and 9-11 mo periods,
respectively. For zinc, 45% (1.5 and 2.0 mg) of the daily intake
came from the study products during the 6-8 mo and 9-11 mo
periods, respectively.

4
SINUS
3.5 ZINAK

3
Episodes/person year

2.5

1.5

0.5

0
Diarrhea Acute respiratory infection

Figure 11. Incidence of diarrhea and acute respiratory infections


(episodes/person year) during 6-12 mo of age in the SINUS and ZINAK
studies.

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Findings

IRON INTAKE AND IRON STATUS


In the Swedish infants, mean daily iron intake during 6-8 mo was
significantly correlated to Hb at 9 mo (r=0.27, p<0.001), and iron
intake during 9-11 mo was significantly associated with Hb at 12
mo (r=0.21, p=0.001). However, 12-17 mo iron intake was not
correlated to Hb at 18 mo (r=0.082, p=0.22) (Figure 12). Daily
iron intake during 6-8 mo, or 9-11 mo was not associated with S-Ft
at 9 or 12 mo, respectively. However, the 12-17 mo iron intake was
significantly correlated to S-Ft at 18 mo (r=0.14, p=0.032) (Figure
13). Iron intake during 6-8 mo was significantly positively
correlated to S-Fe at 9 mo (r=0.16, p=0.011) and the 9-11 mo
intake was correlated to S-Fe at 12 mo (r=0.20, p=0.001).
However, the 12-17 mo iron intake was not associated with S-Fe at
18 mo (r=-0.03, p=0.65). Dietary iron intake was also not
associated with MCV or S-TfR at any time point.

In a multivariate linear regression model, the relationship between


dietary iron intake and Hb was explored further. At 9 mo, Hb was
significantly associated with daily iron intake during 6-8 mo
(E=4.0, p<0.001), adjusting for baseline Hb and relative weight
change from birth to 9 mo, explaining 36% of the variation in Hb
at 9 mo. Hemoglobin at 12 mo was significantly associated with
daily iron intake from 9-11 mo (E=5.3, p<0.001), adjusting for
initial Hb and relative weight change from birth to 12 mo.
2
Adjusted r for this model was 0.32. Hemoglobin concentration at
18 mo was not associated with iron intake during 12-17 mo period.

The association between dietary intake and S-Ft was also tested in a
multivariate model. No association was found between ln S-Ft at 9
or 12 mo and dietary iron intake during 6-8 mo or 9-11 mo,
respectively. However, ln S-Ft at 18 mo was significantly associated
with dietary iron intake from 12-17 mo (E=0.44, p=0.010),
adjusting for ln S-Ft at 12 mo and relative weight increase from 12
to 18 mo. This model explained 16% of the variation of ln S-Ft at
18 mo of age.

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Findings

130
18 mo
Hemoglobin concentration (g/L)

120

12 mo

9 mo
110

100
0 1.0 2.0
Dietary iron intake [mg/(kgxd)]

Figure 12. Lowess curves of the effects of iron intake [mg/(kg body weight
d)] during 6-8 mo, 9-11 mo and 12-18 mo on Hb (g/L) at 9 mo, 12 mo, and
18 mo of age. At 9 mo, the dose-response relationship was linear between iron
intake 6-8 mo and Hb (regression coefficient E=3.3, p=0.005), without evident
difference in slope below or above a daily intake of 0.8 mg/(kg body weight
d). At 12 mo, the Hb-slope was significant up to an iron intake 9-11 mo of 1.2
mg/(kg body weight d) (E=8.8, p<0.001). At 18 mo, Hb was not significantly
associated with iron intake 12-18 mo (E=1.8, p=0.41).

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60
Serum ferritin concentration (ug/L)

18 mo

40
9 mo

20

12 mo

0
0.0
0 1,0
1.0 2,0
2.0
Dietary iron intake [mg/(kgxd)]

Figure 13. Lowess curves of the effects of iron intake [mg/(kg body weight
d]) during 6-8 mo, 9-11 mo and 12-18 mo on S-Ft (g/L) at 9 mo, 12 mo,
and 18 mo of age. At 9 and 12 mo, iron intake and ln S- Ft was not
significantly associated (iron intake 6-8 mo: E=-0.1, p=0.36 and iron intake 9-
11 mo: E=-0.06, p=0.59, respectively). At 18 mo, ln S-Ft was significant
associated with iron intake 12-18 mo (E=0.38, p=0.034), but without evident
difference for intakes < or > 1.0 mg/(kg body weight d).

We assessed the relationship between total amounts of iron


consumed from 6-8 mo, from 6-11 mo, and from 6-17 mo. and
Hb and ln S-Ft at 9, 12 and 18 mo of age, respectively. These
analyses confirmed the significant associations between total iron
intake at 6-8 mo and Hb at 9 mo (p<0.001) and the 6-11 mo total
iron intake and Hb at 12 mo (p<0.001). However, there was no
association between total iron intake during 6-8 , 6-11, or 6-17 mo,
and subsequent ln S-Ft, whereas the 12-18 mo total iron intake was
significantly associated with ln S-Ft at 18 mo (p=0.015).

We also stratified the multiple linear regressions of Hb and ln S-Ft


for initial iron status, i.e. those infants with S-Ft in the lowest
quartile (S-Ft <23 g/L) and highest quartile (S-Ft >80 g/L). This
did not significantly change the results. Neither did inclusion or
exclusion of the 6 infants who at 6 mo fulfilled the WHO criteria
for IDA (Hb <110 g/L, S-Ft <12 g/L and MCV <73 fL).

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In the Indonesian study, we explored the association between


supplemental iron and S-Ft. To evaluate the efficacy of the Fe and
Fe + Zn regimens on S-Ft at the endpoint, the dose-effect over the
range of total supplement intake during the treatment period was
studied (Figure 14). Lowess curves for the Fe and Fe + Zn groups
indicated an initial dose-effect, followed by a plateau with no
further increased effect. In the ordinary least square regression
model, the dose effect up to 200 ml was compared with that above
200 ml of total supplementation.
In the Fe group there was a significant dose-effect up to the level of
200 ml (corresponding to a total dose of 1250 mg of iron over a
period of 6 months, or 7 mg per day). At higher total doses no
further increase in effect was seen. Above 200 ml of total
supplement, the effect on the Fe group was significantly higher than
on the Fe + Zn group (E = 0.171, P = 0.003), but there was no
significant interaction between treatment group and volume. In
spite of the appearance of the Lowess curves, no difference in effect
for a given dose and no interaction in dose-effect between the
groups for supplementation amounts below 200 ml could be shown
between Fe and Fe + Zn (data not shown).

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Findings

300
200

100

Fe group
50
40
Serum ferritin (g/L)

30
Fe + Zn group
20

Zn group
10
Placebo
5
4
3

1
0 50 100 150 200 250 300 350

Total amount of consumed supplement (mL)

Figure 14. Lowess curves of the effect of the volume of supplementation on


serum ferritin (log scale) at 12 mo of age. In the Fe group there was a
significant dose effect up to 200 ml, after which no further increase in serum
ferritin was seen. Above 200 ml of consumed supplement the effect on serum
ferritin was significantly higher in the Fe group compared to the Fe + Zn
group, without significant treatment group volume interaction. Below 200
ml there were no significant difference in serum ferritin between the Fe and Fe
+ Zn groups.

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DISCUSSION

In the present thesis the effects of two different approaches to


improve iron and zinc nutriture in infants, i.e. phytate-reduction of
commonly consumed infant cereals, and iron and zinc
supplementation are reported. The results were obtained from two
intervention trials. Both studies used a randomized, double-masked
design and included infants from 6 mo of age. In evaluating these
interventions, multiple outcomes were measured, and several of the
same variables, i.e. Hb, S-Ft, S-TfR, S-Zn, growth, development
and incidence of diarrhea and respiratory infections were assessed at
both sites using similar or identical methods.

The common challenge to improve iron and zinc nutriture in


infants and the many similarities in design and execution of the
trials allow for some comparisons of the two studied cohorts.
However, the several differences between study sites also call for
separate discussions of the two studies, the practical implications of
the findings at the different settings and their consequences, both at
the local level and in a broader perspective.

In the Indonesian study a factorial design was used. This is a cost-


effective study design when evaluating the effects of several
treatments and several outcomes at once. It also allows for testing of
interactions between different treatments. The latter characteristic is
especially important because of the plausible interactions between
iron and zinc.

In the Swedish study, where the main problem was if the phytate
content of complementary foods caused the unexpected high
prevalence of low S-Ft and low S-Zn (166), a more traditional trial
design was used with three study groups, although a factorial design
with different levels of e.g. both phytate and iron could have been
reasonable. However, the primary objectives were to compare the
present Swedish infant feeding practice to one with reduced
phytate-levels, but also compare current Swedish infant feeding
practices, i.e. phytate-containing infant cereals with the
internationally common usage of iron-fortified formula and
porridge. Thus, the design using three groups was deemed the most
appropriate.

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The Swedish cohort was recruited from well-baby clinics and could
be viewed as a healthy volunteer sample, whereas the recruitment of
the Indonesian sample was community-based, although from a
cohort of women followed during pregnancy. The Swedish cohort
may therefore differ in some characteristics as compared to the
infant population in the study area. However, using double-masked
randomization to the various study groups should have handled
much of this bias in the interpretation of the results of the actual
interventions. However, generalization of the results to populations
with nutritional and health status very different from the
populations studied should be made with caution.

Baseline randomization was successful in both studies, although


there was a significant difference between randomization groups in
the prevalence of low S-Zn in the Swedish study. However,
adjusting for initial zinc status in SINUS did not affect the main
outcomes of S-Zn or prevalence of low S-Zn. Compliance was
measured in both studies. In SINUS, the dietary intake was
recorded, including the intakes of the study products.

These records show that the intake of MCD and formula was
approximately two servings per day, and for porridge d1 serving per
day. These intakes are at the level of previous Swedish studies (169)
(J Svahn, personal communication 2002) and indicate that the
study products were consumed as part of a mixed diet. However, a
higher than average intake of these iron-fortified study products can
not be ruled out completely, as the prevalence of low S-Ft and low
S-Zn at 12 mo was lower in the CC group compared to what was
reported in a previous study (166).

In ZINAK, the intake of the study supplements was measured both


through records of self-reported compliance and measurement of
actual consumed supplement. Further, careful follow-up of possible
sideeffects of the supplementation with both day-to-day recordings
of symptoms, and a comprehensive interview at the end of the
supplementation, were done. Analysis of these data revealed that
there were substantial differences in the sideeffects of the different
supplements.

The group consuming the combined iron and zinc supplement had
a substantially higher prevalence of vomiting, which could
potentially reduce the effect estimates of the intervention. However,
adjusting for these factors in the analysis modified the results only
slightly. Adverse effects in the combined group have been reported

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from other studies of similar design and indicate one level of


possible interaction between iron and zinc in the combined
supplement. Dijkhuizen et al reported a higher drop-out rate in the
combined Fe+ Zn group (197), and Penny et al reported that
vomiting within 30 min of receiving supplement was significantly
more common in the combined zinc, iron and vitamins group
(198). Also, Baqui et al, when supplementing Bangladeshi infants
from 6 mo of age with either iron, zinc, iron + zinc, a mix of iron,
zinc and vitamins, or placebo found higher drop-out rate in the
group consuming the mineral and vitamin mix, and higher drop-
out rates due to vomiting in the groups receiving either the mineral
and vitamin mix or iron + zinc (199).

At baseline, there were significant differences between the study


sites for several background and nutritional parameters, e.g.
household size, maternal education, S-Ft, S-TfR, S-Zn, prevalence
of anemia and IDA, and anthropometrical measurements such as
WAZ and HAZ. These differences were not surprising given the
large socio-economical differences between the study sites. It is
noteworthy, though, that there were no differences in Hb or
development. In addition, mean WAZ and HAZ at 6 mo of age in
the Indonesian cohort were higher than those reported from other
low-income Asian countries (178). Both studies dispute the
common notion that the prevalence of ID is several times higher
than the prevalence of IDA (200).

A striking although not unexpected difference in the development


of health indicators was seen between the Swedish and Indonesian
infants during the 6-12-mo period. Whereas the Swedish infants
remained stable or improved in the measures of nutritional status,
the Indonesian infants deteriorated from a fairly low degree of
malnutrition to high prevalence of anemia, IDA, low S-Zn, low
weight, and wasting. Further, morbidity from both diarrhea and
ARI were several times higher in the Indonesian cohort compared
to their Swedish peers.

Whereas several of these differences can be attributed to


socioeconomic differences, e.g., the maternal level of education,
hygienic conditions, accessibility to health care, etc., many can be
attributed directly to dietary factors, which could be addressed in
appropriate food interventions programs. The deterioration in
anthropometrical status was considerable among the Indonesian
infants with the prevalence of stunting doubling from 6-12 mo, the
prevalence of wasting becoming 4 times greater in the same time

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period, the prevalence of underweight increasing more than 9-fold.


Compared to other studies from the same area (176, 177), we
report higher prevalence of both underweight and wasting, with
particularly wasting being very high, i.e. >8%. The reasons for this
are unclear. Indonesia was struck by a severe economic crisis
beginning in late 1997.

Studies on food consumption among pregnant women in the


CHN-RL surveillance area before and during the crisis show that
the women, especially poor women, decreased their intake of most
nutrients (201). For infants or children no similar analyses are
available. Although it seems plausible that the economic crisis also
affected the nutrient intakes of infants and children, directly or
indirectly, we could not show any differences in the prevalence of
malnutrition in infants born before and during the economic crisis.

EFFECTS OF PHYTATE REDUCTION


Though phytate has been shown to reduce iron and zinc
bioavailability in several single-meal studies (202, 203), phytate-
reduction of infant cereals in this long term follow-up had no effect
on iron and zinc status, growth, development or incidence of
diarrhea or respiratory infections in Swedish infants until 12 mo of
age.
Phytate, in the absence of ascorbic acid, inhibits iron absorption in
a dose-dependent manner above a phytate:Fe molar ratio of 1:7
(135). However, ascorbic acid counteracts the effect of phytate
when the ascorbic acid:Fe molar ratio exceeds 4:1 (73, 74). In
SINUS, all infant cereals except PR, had phytate:Fe molar ratios
>1:7, implying that, theoretically the phytate could bind all
available iron, except in the phytate-reduced MCD. On the other
hand, ascorbic acid:Fe molar ratios were high, in all cereals >4:1.

Thus, the ascorbic acid content may have limited the phytate effect,
ensuring similar long-term iron absorption from the regular and
phytate-reduced cereals. Davidsson et al (74) showed that increasing
the ascorbic acid content from an ascorbic acid:Fe molar ratio of
2:1 to 4:1 increased Fe incorporation into red blood cells in a
similar fashion as completely removing the phytate from regular,
phytate-containing soy formula.

Studies have shown that a phytate: Zn molar ratio above 15:1 may
be associated with increased risk of zinc deficiency (131). In

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SINUS, the phytate: Zn molar ratios were generally below 10:1 and
a further reduction in phytate content had no effect on S-Zn.
However, the PR group had significantly lower S-Cu than the CC
group. A possible explanation is that zinc absorption may have been
higher in the phytate-reduced group although not evident from
measurement of S-Zn, causing lower copper absorption. In a study
on infant rhesus monkeys, zinc absorption was higher, plasma zinc
was higher, and plasma copper concentration was lower in the
group fed phytate-reduced soy formula than in the group fed
regular soy formula (204).

Comparing the internationally more common feeding pattern of


using iron-fortified infant formula and cereals (the IF group) to the
Swedish regime with phytate-containing infant cereals (the CC
group) revealed no significant differences. This indicates that the
present Swedish practice of feeding does not compromise iron or
zinc status compared to an infant formula with 4 mg iron/L when
the phytate-containing infant cereal has a sufficiently high ascorbic
acid: Fe molar ratio.

EFFECTS OF IRON AND ZINC SUPPLEMENTATION


In low-income settings, where complementary foods are mostly
home-prepared from primary products with low iron and zinc
bioavailability, supplementing these minerals may be a feasible and
effective way of improving iron and zinc nutriture during infancy
(9). However, in ZINAK we found significant negative interactions
when combining iron and zinc in, both for the biochemical results,
i.e. Hb and S-Ft as well as the functional outcomes, i.e. WAZ,
knee-heel length and psychomotor development.

In a recently published evaluation of the efficacy and effectiveness


of iron supplementation as prevention for anemia in infancy, it is
stated that iron supplementation to infants usually improves Hb
and S-Ft (205). In the present study, iron given alone had a
significant positive effect on Hb and S-Ft compared to both placebo
and the Fe + Zn group. For Hb, the group mean differences to the
placebo and Fe + Zn groups were 5.9 g/L (p=0.001) and 3.7 g/L
(p=0.042), respectively. A difference of >5 g/L usually indicates
public health significance. For S-Ft the group, mean differences to
the placebo and Fe + Zn groups were 34 g/L (p<0.001) and 10
g/L (p=0.007), respectively. There was also a significant main
effect of iron treatment on S-TfR, with TfR decreasing significantly

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Discussion

compared to placebo. This indicates that the iron supplement was


consumed by the participating infants, and that iron
supplementation is effective in preventing anemia and IDA during
the 6-12 mo age period in Indonesian infants. However, the Fe
group had a significantly higher prevalence of S-Zn <10.7 mol/L
(87% vs. 78%, p=0.033), indicating that iron supplementation may
have adversely affected zinc absorption.

Zinc treatment had a significant main effect on S-Zn with no


evidence of interaction between iron and zinc, suggesting that zinc
supplementation is effective in improving zinc status measured as S-
Zn in Indonesian infants. The finding is in agreement with a recent
meta-analysis showing that supplementation of zinc to infants and
young children has been associated with significant increases in S-
Zn (110).

In the present study, iron supplementation had a positive, small,


but significant effect on knee-heel length compared to placebo
(difference in means 1.9 mm), but it had no effect on body weight
or overall length. From a public health point of view, the small
difference in knee-heel length between the Fe and placebo groups is
negligible, but indicates that iron may be one of several growth-
limiting factors in this population. Iron deficiency has been implied
to contribute to growth retardation, but different studies have given
conflicting results, where some have showed an effect of iron
supplementation on growth, while others have failed to
demonstrate any difference.

Aukett et al (206) showed an increased weight gain in anemic,


British toddlers receiving iron, and supplementation to Indonesian
preschoolers resulted in increased linear growth (207). In the
former study, the weight gain was greatest in the group with the
greatest increase in Hb, and in the latter the positive results on
linear growth were associated with decreased morbidity in the iron
group. Studies in school children have also shown effects of iron
supplementation on growth (208-210). A recent randomized trial
in Ethiopian children assessing the effects of consuming foods
cooked in iron pots showed improved iron status and growth (211).

On the other hand, Moffatt et al (212) in a carefully designed


prospective study did not find any differences in growth between
infants given iron fortified and unfortified formula until 15 mo of
age, despite differences in both hematological parameters and
psychomotor development. Similarly, neither Rosado et al (213),

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Discussion

nor Rahman et al (214) or Sungthong et al (215) found any effect


of iron supplementation on growth compared to placebo.

A recent meta-analysis on the effects of zinc supplementation on


growth has shown positive effects on both height and weight (110).
The meta-analysis shows that the positive effect on the HAZ was
almost two times greater if the child was stunted at enrollment, and
that the effect on WAZ was more than two times greater if the child
was underweight at inclusion. Likewise, the effect on growth was
greater in those with S-Zn <12.2 mol/L. However, neither the
differences between stunted and non-stunted nor underweight and
non-underweight or those with S-Zn below or above 12.2 mol/L
were statistically significant.

Zinc supplementation to the infants in the ZINAK study


significantly improved growth (WAZ and knee-heel length)
compared to placebo. The differences in means between the zinc
and placebo groups were 0.26 for WAZ and 2.0 mm for knee-heel
length, which agree with earlier results [referred in (110)]. Again,
and as with the effects of iron supplementation, the public health
significance of this difference may be limited, but point toward that
zinc is one of several growth-limiting factors in this population.

Iron supplementation in combination with zinc had no effect on


growth. These results are thus in line with several other studies
which have failed to show growth-benefits of simultaneous iron and
zinc supplementation (197, 213, 216), but contrary to Perrone et al
(217), who showed improved growth in the combined iron and
zinc group. However, in that study the iron and zinc
supplementation was separated by 12 h and the subjects were older
(4-11 years) and all were stunted as compared to the present study.
The separation in time between the administration of iron and zinc
is likely to have eliminated interactions at the intestinal absorption
level, allowing zinc to act independently as growth-promoting
agent.

In the last three decades, a large body of evidence has accumulated


on the association between iron deficiency and impaired
neurodevelopment in infants and children (49, 50, 52-55, 206,
218-239). The effects of iron on brain development have been
reviewed recently (240). Animal studies indicate that monoamine,
especially dopamine function and the myelination process seem
especially vulnerable to iron depletion. Early iron depletion in rats,
i.e. from birth to 21 d of age, induced irreversible changes in brain

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iron (241). In human infants such window of vulnerability to


iron deficiency may lie between ages 3 and 23 mo, ages where iron
deficiency is most common world-wide (59, 240).

Early studies by Oski et al (221) and Walter et al (222) indicated


that even mild iron deficiency, i.e. without anemia, was correlated
with poor cognitive development, but later studies (224, 225) have
indicated that anemia in combination with iron deficiency is
needed to cause developmental effects. Some studies implicate that
at least part of the effect on mental and psychomotor development
is irreversible, even after treatment with iron and an excellent
hematological response (49, 50), although others have disputed this
(228).

However, iron deficiency has in many of the studies been associated


with other socioeconomic risk factors of poor development, and
though attempts to adjust for these have been made, confounding
of the causal relationship between ID and development by these
other factors can not be ruled out (232). Martins et al conclude that
though few of the studies fulfill stringent scientific criteria to allow
us to draw strong inferences on the association between ID and
infant development, the data presented could imply significant
positive neurodevelopmental effects of iron supplementation in
infants with IDA (238).

Several animal studies have shown that zinc deficiency induces


mental and behavioral changes. Similar to iron deficiency, the
effects are clearly manifested in overt zinc deficiency. However,
studies in young rhesus monkeys show that even marginal zinc
deficiency can induce behavioral changes (242). Studies on zinc
deficient adult humans have implicated impaired short-term
memory and changes in food preferences (243). Studies of zinc on
infant development and behavior in infants and children have been
inconclusive (244-250).

In ZINAK, there was a small but significant positive effect of iron


supplementation compared to placebo on psychomotor
development measured with the Bayley Scales of Infant
Development. The difference was 3 points, which may be
insignificant from a public health point of view. No effect was seen
on cognitive development or behavior. However, the results point
in the same direction as those published by Idjradinata and Pollitt
(228) and Moffatt et al (212), which showed significant effects of
iron supplementation on psychomotor development in iron-

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deficient children or infants at high risk of developing IDA. In


ZINAK, combining iron and zinc supplements had no effect on
psychomotor development compared to placebo, nor was there any
effect of zinc supplementation on either development or behavior.

There was no decline in psychomotor development with age in the


infants not treated with iron, although anthropometrical status
declined and the prevalence of IDA increased significantly, which is
contrary to other studies (212, 234). This, together with a low
baseline prevalence of IDA, possibly diminishing the differences in
psychomotor development between iron treatment and placebo,
may imply that other environmental or nutritional factors, e.g. long
breast-feeding duration and a high degree of formal education
among mothers may have moderated the effects of iron deficiency
in the non-iron treated groups, as has been described in the case of
protein-energy malnutrition and psychomotor development (251).

Several in vitrostudies have shown that iron deficiency impairs


immune function, and that iron administration restores function
but clinical studies in children, with few exceptions, have not been
able to demonstrate significant positive effects of iron
supplementation. In a review on the effects of iron on immunity,
Oppenheimer (252) concludes that; a) parental iron treatment to
newborns can cause sepsis and meningitis, and should not be used;
b) iron-fortified infant foods do not reduce infectious disease
morbidity compared to breastfeeding, but has other beneficial
effects; that there is some, although not very compelling evidence
that oral iron supplementation in non-malarious, disadvantaged
populations may be of benefit; and c) that high doses of oral iron
(>2 mg/kg/d) to children in malaria-endemic populations carries
increased risk of both clinical malaria and other infections, such as
pneumonia.

In a recent meta-analysis, Gera and Sachdev examined the effects of


iron supplementation on infectious disease morbidity in children
(48). They found no benefits of iron supplementation, and though
it did not significantly increase the overall risk of infection, there
was a small, but significant increase in diarrheal disease (incidence
rate ratio 1.11, 95% CI 1.01-1.23). Iron supplementation increased
the odds ratio for malaria parasitemia (1.43, 95% CI 1.08-1.91).
However, in the meta-regression analysis, adjusting for baseline
parasitemia, iron supplementation was not significantly associated
with malaria-positive blood smears (p=0.076).

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Adults fed a zinc-restricted diet display signs of increased


susceptibility to infections (253). In urban slum children in India,
low plasma zinc (8.4 mol/L) significantly increased the risk of
diarrhea and acute lower respiratory infections, with boys being
more vulnerable than girls (254). A recent meta-analysis has shown
that zinc supplementation significantly reduces diarrheal morbidity
and pneumonia in children in low-income settings (111). Diarrheal
disease has also been found to induce zinc deficiency through
massive fecal losses of zinc (102).

In ZINAK, neither iron, nor zinc supplementation, alone or in


combination had any effect on the incidence of diarrhea or
respiratory infections. One possible reason may be that the
preventive effect of zinc on diarrheal disease has been more
pronounced among children t12 mo than in younger children
(111, 255). However, Baqui et al found that combined iron and
zinc supplementation reduced the incidence of severe diarrhea and
acute lower respiratory infections, particularly in less well-nourished
infants, i.e. WAZ <-1, when given once weekly to infants from 6
mo of age, although a combination of iron, zinc and minerals
increased the risk of diarrhea and significantly contributed to
vomiting (199).

Further, the diarrheal incidence in ZINAK was lower than in some


other studies (199, 255, 256). Also, the high proportion of infants
in this study who were still being breast-fed together with high
accessibility to safe water as well as family factors, such as the
relatively high educational level of the mothers, might have added a
protective effect. Taken together, these factors may have made it
difficult to achieve further reductions in the morbidity incidences
through zinc supplementation in this population.

IRON AND ZINC INTERACTIONS


The results in ZINAK indicate that interaction between iron and
zinc is possible for several outcomes, biochemical as well as
functional, when combining the two minerals in the same
supplement. Several possible levels of iron-zinc interactions can be
identified. First, compliance to the various supplements was
different, with vomiting in relation to the supplements being
reported more often from the combined Fe + Zn group. We
adjusted for this as well as consumption of supplements in the
analysis and the interactions remained significant. However, it is

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Discussion

not know what proportion of the different supplements was lost


through vomiting and thus the possibility can not be ruled that
vomiting, i.e. losing an unknown proportion of the ingested
supplement before absorption, may have affected the results. As
discussed earlier, decreased compliance or increased adverse effects
of combined iron-zinc supplementation has been reported by others
(197-199).

A second level of interaction is intestinal absorption. Several studies


have shown that giving iron and zinc together in a water solution
affects absorption of both minerals (137, 139-141). However, when
the minerals were given as part of infant foods, no effect of high
iron:zinc molar ratios on iron or zinc absorption was seen (140,
143-145). Common iron-zinc pathways have been identified (146),
but the substrate specificity and how these transporters act in vivo,
particularly in human infants is not known.

A third possible level of interaction is the effects and counter-effects


of the two minerals on the functional outcomes. Zinc
supplementation to zinc deficient infants has been shown to
improve growth (110), whereas iron supplementation to iron
replete infants has been shown to negatively affect growth (147-
149). Given the high prevalence of low S-Zn (78%), but the low
prevalence of ID and IDA (15% and 8%, respectively) at baseline,
this level of interaction may also have been present in the ZINAK
study.

IRON INTAKE AND IRON STATUS


In the analysis of the association between dietary iron intake and
iron status, we expected to find little effect on variables like Hb,
since all infants were well-nourished and IDA was uncommon, and
infants with presumably significant IDA (Hb <100 g/L, S-Ft <12
g/L and MCV <70 fL) were excluded, but to find an increase in S-
Ft, the storage form of iron. Instead the opposite was found in
infants below 12 mo of age there was a significant increase in Hb
with increasing iron intake, but no effect on S-Ft.

Only at 18 mo was an association between iron intake and S-Ft


found, whereas the correlation between iron intake and Hb had
disappeared at this time. From 6-8 mo the relationship between
dietary iron intake and Hb was linear and with no significant
interaction between other nutrients and iron intake. Later, during

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the 9-11 mo period, there was still a significant, but now a non-
linear relationship between daily iron intake and Hb. These data
suggest that the regulation of iron metabolism during the first years
of life is highly dynamic, and it is tempting to speculate that dietary
iron is to a higher degree channeled to erythropoiesis and
incorporated into Hb during infancy, as indicated by the almost
linear relationship between iron intake and Hb concentration, than
later in childhood. Interestingly, S-TfR was positively associated
with Hb at both 9 and 12 mo.

The concentration of transferrin receptors in serum are thought to


mirror the expression of transferrin receptors on iron-requiring
cells, most abundantly on cells involved in erythropoiesis, and is
believed to reflect intracellular iron needs (31, 257, 258). A positive
association between Hb and S-TfR may thus indicate increased
erythropoiesis. Virtanen et al have described higher S-TfR in infants
than in pre-pubertal boys and men, and increased erythropoiesis is
one of several possible explanations (259).

Little of the dietary derived iron seems to be directed to storage, i.e.


to ferritin, in infancy, as there was no correlation between iron
intake and S-Ft until after 12 mo of age. Gradually less of iron
obtained from the diet is incorporated into Hb during 9-11 mo,
seen as a plateau in the iron intake-Hb curve at 12 mo of age.
Thereafter, dietary iron seems to be channeled towards storage to an
increasing extent, seen as an increase in S-Ft at 18 mo with
increasing iron intake during 12-17 mo. A shift in the channeling at
around 1 yr of age is also indicated by the finding that not even the
total iron dose given until 11 or 17 mo of age did affect S-Ft, but
only the amount of iron given after 12 mo of age.

The way iron is provided, i.e. as a food constituent or through


supplementation, may modify the effects on hematological
parameters. In the Indonesian study, iron intake from the
supplements was more than 5.5 times lower than in the Swedish
study (iron intake 0.15 mg/kg/d vs. 0.85 mg/kg/d in the ZINAK
and SINUS studies, respectively). However, there were significant
dose-response relationships for both iron supplementation and Hb,
and iron supplementation and S-Ft in the Fe group, whereas in the
Swedish study dietary iron only associated significantly to Hb but
not S-Ft in infancy.

One explanation may be that iron sulfate provided as a supplement


had much higher bioavailability than does the fortificant iron of

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Swedish infant cereals, providing sufficient iron to increase both Hb


and S-Ft. Another explanation may be that whereas the increase in
Hb indicates that iron is absorbed and utilized in the erythropoiesis,
the increase in S-Ft may indicate the organisms attempt to detoxify
highly reactive iron in the intestinal lumen.

MONITORING OF IRON INTERVENTIONS


Several authors have questioned the current definitions of ID and
IDA, suggesting lower and more age-appropriate cut-offs (23, 36-
38). Hence, the prevalence of ID and IDA reported in this thesis
may have overestimated the initial problem. Such overestimation
may also have as a consequence that true differences in iron status
and effects of intervention become less clear. However, using the
alternative definitions suggested did not result in significant
differences between the groups. The data presented indicate that
when evaluating the effects of dietary iron interventions in infancy,
Hb correlates closer to dietary iron intake than does S-Ft and S-
TfR, whereas the opposite may be true in the second year of life.
Further, the data indicate that Hb increases regardless of iron status,
at least before 9 mo of age, suggesting that the reference value of a
population at that age is dependent on its iron intake, whether or
not actual ID is present or not. Using reference populations with
high intakes of dietary iron may thus increase the Hb distribution
beyond what is physiological at that age, which should be
considered when defining anemia in infants.

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Conclusions and recommendations

CONCLUSIONS AND RECOMMENDATIONS

Providing sufficient iron and zinc to infants after the period of


exclusive breastfeeding remains a challenge. In high-income settings
these problems can be partly overcome by use of fortified
complementary foods. However, the composition of the
complementary foods, often cereal-based and thus containing
phytate may infringe on the bioavailability of iron and zinc.

The data presented show that in the high-income setting, reduction


of the phytate content of commonly consumed infant cereals has
little effect on iron and zinc status, growth, development or
incidence of diarrhea or respiratory infections. A possible
explanation to this is the high content of ascorbic acid in the
commercially available infant cereals, increasing the bioavailability
of iron. Given a sufficient ascorbic acid:Fe molar ratio there is no
urgent need to reduce the phytate-content of infant cereals
currently used in the Swedish infant population. Thus, the phytate
content of complementary foods of Swedish infants does not
contribute to poor iron and zinc status as feared. Instead the
definitions of iron and zinc deficiency in infancy, at least before 12
mo of age, may overestimate the problem, and a change in the
recommended cutoffs is suggested.

In low-income settings, where the complementary foods are more


often than not home-prepared from staples such as vegetables and
cereals, with low absolute amounts and low bioavailability of iron
and zinc, the problem of sufficient nutrient density is considerable.
Supplementation may thus be a feasible and effective alternative.
However, interaction between iron and zinc is possible when they
are given together.

In the low-income setting, combined supplementation with iron


and zinc resulted in significant negative interaction, not only for
biochemical outcomes such as Hb and S-Ft, but also for functional
outcomes such as growth, i.e. weight and knee-heel length, and
psychomotor development. Several levels of interaction are possible;
from competition for absorptive pathways in the intestine to
differences in compliance due to side effects of the combined
supplement. The study also indicates that the chosen level of iron
supplementation, i.e. 10 mg/d may be unnecessarily high as no
further effect on iron status, i.e. S-Ft, was seen beyond 7 mg/d.
From these results it is not possible to recommend routine iron-zinc

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Conclusions and recommendations

supplementation at the molar concentration and mode used in this


study. Instead, alternative approaches should be investigated,
including intermittent supplementation with iron and zinc, e.g.
weekly dosage of the two minerals, separating the supplements in
time, e.g. zinc supplementation only during diarrheal episodes, the
use of iron and zinc compounds with different absorptive properties
than iron sulfate and zinc sulfate, administration of iron and zinc at
other molar ratios than those applied here or food-based strategies
where the additional iron and zinc is mixed with home-prepared
complementary foods, maybe together with other vitamins and
minerals as well. With the high prevalence of ID and the
presumably high prevalence of ZD among infants and young
children in low income countries and their various and severe
consequences for child health and development, it is imperative that
research efforts are focused to finding solutions to prevent these
deficiency states.

Finally, studies have indicated that iron homeostasis undergoes


developmental changes during infancy. In the present thesis, this
theory is supported by the finding that dietary iron intake is
significantly positively associated with Hb, but not serum ferritin
until 12 mo, although the opposite is true after 12 mo of age. This
may indicate that dietary iron is preferably channeled towards
erythropoiesis during infancy, but to an increasing amount
channeled towards storage in early childhood. This suggests that in
evaluating dietary programs, Hb may be superior in monitoring
response to dietary iron in infancy, whereas S-Ft may respond better
later in childhood. However, as shown in this study, increasing Hb
is not necessarily an indicator of iron deficiency, as dietary iron
increased Hb regardless of iron status.

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Populrvetenskaplig sammanfattning

POPULRVETENSKAPLIG SAMMANFATTNING

Jrn- och zinkbrist bland spdbarn r mycket vanligt i ett globalt


perspektiv. Dessa mineralbrister orsakar bl a anemi (blodbrist),
frsenad utveckling, frsmrad tillvxt och kad sjuklighet i diarr-
och luftvgsinfektioner. En starkt bidragande orsak till att jrn- och
zinkbrist uppstr r att tillggskosten r otillrcklig.

Vi har underskt tv stt att frbttra intaget av jrn och zink hos
spdbarn i tv olika befolkningar; i en svensk grupp barn minskade
vi mngden fytinsyra, en hmmare av jrn- och zinkupptaget i
vlling och grt, medan vi i en indonesisk grupp barn underskte
effekten av dagligt jrn- och zinktillskott.

I den svenska studien randomiserades 300 6-mnadersbarn till att


ta antingen vanlig vlling och grt, fytinsyrareducerad vlling och
grt, eller brstmjlksersttning och grt. I den indonesiska studien
randomiserades 680 6-mnadersbarn till att antingen f
jrndroppar, zinkdroppar, jrn- och zinkdroppar eller placebo. Fr
att utvrdera effekten av dessa interventioner tog vi blodprover,
mtte och vgde barnen, underskte hur de utvecklades samt fljde
symptom p vanliga infektionssjukdomar. I den svenska studien
registrerade vi ocks terkommande barnens kost- och nringsintag.

Minskning av mngden fytinsyra i vlling och grt till de svenska


barnen hade en frsumbar effekt p jrn- och zinkstatus, tillvxt,
utveckling, eller sjuklighet i diarr eller luftvgsinfektioner. Detta
kan bero p nrvaron av askorbinsyra i studiekosten, som motverkar
fytinsyrans negativa effekt p jrn- och zinkupptaget. I den
indonesiska studien sg vi en negativ vxelverkan nr jrn och zink
gavs tillsammans; blodvrdet (Hb) och ett mtt p jrnfrrdens
storlek (serum ferritin) var betydligt hgre i den grupp som bara ftt
jrn n i den grupp som fick placebo eller de kombinerade jrn- och
zinkdropparna. Enbart jrntillskott frbttrade ocks ett av
tillvxtmtten samt barnens psykomotoriska utveckling, och enbart
zinktillskott kade vikten och benens tillvxt jmfrt med placebo.
De kombinerade jrn- och zinkdropparna minskade andelen barn
med jrnbristanemi och lgt zinkvrde, men hade inga andra
positiva effekter.

Bland de svenska barnen var jrnintaget under perioden 6-11 mn


kopplat till Hb, men inte till serum ferritin vid 9 och 12 mn,
medan det omvnda gllde senare, d v s att jrnintaget under

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Populrvetenskaplig sammanfattning

perioden 12-17 mn var kopplat till ferritin men inte Hb vid 18


mn. Detta tyder p att jrn i kosten verkar g till produktion av
rda blodkroppar under spdbarnsret, medan det senare i strre
utstrckning lggs i jrnfrrden. Nr man utvrderar effekten av
kostinterventioner kan det drfr vara bttre att flja Hb under
spdbarnsret, medan serum ferritin kan svara bttre efter 12 mn
lder. Dock behver stigande Hb inte automatiskt betyda jrnbrist,
d mer jrn i kosten gav kat Hb oberoende av tidigare jrnstatus.

Vi drar vidare slutsatsen att mngden fytinsyra i vlling och grt till
svenska barn inte verkar bidra till frsmrat jrn- och zinkstatus
givet tillrckliga mngder askorbinsyra. De nuvarande
definitionerna av jrn- och zinkbrist kan behva justeras, d de
tenderar att verdriva andelen barn med otillrckligt jrn- och
zinkstatus. Frn den indonesiska studien drar vi slutsatsen att vi inte
kan rekommendera att ge jrn och zink tillsammans i de
koncentrationer och p det stt vi gjort, d detta ger upphov till
negativ vxelverkan, vilket motverkar syftet med tillskotten. D
bde jrn- och zinkbrist r vanligt r det dock av strsta vikt att
studera hur man bst frebygger dessa tillstnd i lginkomstlnder.

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Acknowledgements

ACKNOWLEDGEMENTS
It is evident that this dissertation is the effort of many people. For
me, having the honor to compile and present it, it is a proud
moment, but also a time to recognize the people who with their
knowledge, support, patience, diligence, generosity, skill, and
struggle made it at all possible. To these people I extend my most
cordial gratitude and my deepest respect as well as a hope for
continued collaboration in the years to come:
First and foremost this must go to the parents and children of
Ume and Purworejo, who with such generosity and unselfishness
contributed with literally their blood and tears. Without their help
and compliance this project would truly have been impossible.
Olle Hernell and Lars-ke Persson, my main supervisors, for their
gentle but unswerving supervision of this whole project. Their
profound knowledge together with an ability to see the greater
picture, and not lose track of the goal when their apprentice was
already adrift, has been a great lesson. Also, for their great scientific
skills in combination with great humanity and a genuine interest in
improving child health, which have made an everlasting impression
and a legacy to carry on.
Bo Lnnerdal, Department of Nutrition, University of California,
Davis for his unique combination of a sharp mind, an enormous
knowledge, and an even larger, empathetic heart. A true role model
for any aspiring researcher. Also for his assistance in the analyses of
the blood samples, as well as tips and tricks how to carry thousands
of them across customs, and the meticulous feed-back on the papers
in this thesis, and the friendly and hospitable atmosphere I have
encountered each time I have visited him and his laboratory.
Hans Stenlund for his enormous patience and pedagogic skills in
explaining the deepest and sometimes not so deep secrets of
biostatistics. Also for his significant contributions from the earliest
planning stages of this work to its completion.
Anna Winkvist, coordinator of the Swedish-Indonesian
collaboration and the person responsible for giving me the
opportunity to work within this great cooperation. Her
organizational skills and research capabilities are renowned as well
as her gardeners skills in making her fellow co-workers grow and
flourish. Another true role model. Also for her indispensable input
throughout the ZINAK project, for her diplomatic dexterity, and
for her initiated and valuable criticism at the seminar.

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Acknowledgements

Stig Wall, head of Epidemiology and Public Health Science, and


Gisela Dahlquist, head of the Department of Clinical Sciences for
their wise criticism and continued support also at the most difficult
of times.
Leif Gothefors, Stellan Hkansson and Erik Bergstrm, in turn
heads of the clinical department of pediatrics for always supporting
me and showing great flexibility, allowing me to mix research with
clinical work.
Djauhar Ismail, Rosadi Seswandhana and Indria Gamayanti, my
co-authors, friends and significant Indonesian collaborators for
believing in this project and for the hard work they have put in
seeing it to the finish. Their great effort should also be seen in the
light that ZINAK took place during one of the most turbulent
times in Indonesian history, which further increased their toil.
Detty Nurdiati, Agung Nugroho, Tunjung Wibono, Abdul
Wahab, Z Eko Firdos and Ninuk Sri Hartini, at CHN-RL, and
Adi Utarini, at the Department of Public Health, Gadja Mada
University, my friends and colleagues, for their generosity and
welcoming attitude towards a sometimes confused and ill-fitting
foreigner, for house-keeping me, and for being so truly helpful and
supportive during this whole process. Also for their great company
and cooking during their stays here in Ume.
Mohammad Hakimi, Achmad Surjono, Soenarto Sastrowijoto,
Siswanto Agus Wilopo and Djaswadi Dasuki, all professors at
CHN-RL, for allowing me to collaborate and work within the
CHN-RL infrastructure, for inviting me to the largest wedding I
believe I will ever witness, for always finding time for discussion,
and for looking out for me at times of unrest.
Pithok, Utari Marlinawati, Sudharto, R Kristiono, Susetyo,
Budiantoro, Apurwo Pamungkas, Budiarto, Swasti Lastini, Haryo
Bambang, Pratiwi, Edi Siswanto, Ermy Puspitaningrum, Ratna
Asiyati, Tri Wardani, Dani Herliana, Arifah Budi Utami,
Wartono, Muh Arifin, Tari, Siwi Budi Prihantari, Emy Zulaika
Kartikasari, Istiti, Wati Wusana, Yusuf, Fajar Nur Hidayat, Fatah,
Arifin, Sigit, Daniek, Solikhin, Eka Surya, and all the rest of the
CHN staff for their friendship, and diligent and skilful work in the
field as well as administratively bringing ZINAK safely to shore.
To all my Indonesian friends I want to send my deepest
appreciation for showing me their wonderful country and culture,
including dawns at the peak of volcanoes and how to enjoy ripe
durian, causing me to consider Indonesia my second home, and to

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Acknowledgements

where I always long and where I always will keep a significant part
of my heart.
Margareta Henriksson and Margareta Bckman, research nurses in
SINUS for being such wonderful friends and co-workers, and true
magicians in dealing with parents and their children.
Inger hlund, dietician, for valuable contributions in the planning
and performance of SINUS and for sharing her great knowledge in
pediatric nutrition with me.
Maria Sehlstedt, Anna Karlsson, Maj-Britt Nyberg and Agneta
Frngsmyr, dieticians in SINUS, for their hard and almost endless
work in preparing the dietary records.
Carina Andersson, for the enormous task of taking care of all the
SINUS blood samples and analyzing a main part of them, and Erik
Domellf for assisting me doing the developmental tests of the
SINUS infants.
The staff at the Department of Clinical Chemistry, Ume
University Hospital, and Shannon Kelleher, Department of
Nutrition, University of California, Davis for invaluable assistance
in analyzing the SINUS blood samples, and Sunarti, Weng-In
Leong and Michael Crane, Department of Nutrition, University of
California, Davis for an excellent job with the biochemical analyses
in ZINAK.
Birgitta strm, administrative coordinator of the Swedish-
Indonesian collaboration for her enthusiasm and help in completing
the project including (as usual) some last minute hard work, Jerzy
Pilch for entering the main body of data in SINUS as well as
providing emergency assistance at various times of severe computer
malfunctioning, Lennart Nystrm for generating and safe-keeping
of the ZINAK randomization key as well as valuable statistical
advice, and Barbro Skog and Susanne Walther for supplying
invaluable administrative support, helping me compile this thesis.
All the staff of Epidemiology and Public Health, and Pediatrics, for
always encouraging and supporting me despite severe absence on
my part, and for helping to create the creative atmospheres which
are so typical of these to fine departments.
Catharina Tennefors, Lars-Brje Sjberg, and Hkan Ahlmn at
Semper AB for invaluable input during the development of SINUS,
including the development of the study products and for their great
support of this whole project.

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Acknowledgements

My colleague PhD-students and resident pediatricians over the


years for being such great, reliable friends, always ready to listen and
provide support at times of need.
Kerstin, Gunnar, Maria, Lars and Johan, my parents and siblings,
for always allowing me to chose my own path in life and supporting
me in that choice.
Frida and Hanna, my beloved daughters, for being the finest
daughters in the World and my eternal inspiration.
Katarina, their mother, who although our paths have separated,
have shown great patience and who has given me invaluable support
throughout these long years.

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