diet and nutrient status in infants and children with cow ......iv abstract background: cow’s milk...
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Diet and nutrient status in infants and children with cow’s milk protein
allergy
Results from a cross sectional study with emphasis on vitamin B12 and iron status
Mari Borge Eskerud
Master Thesis Department of Nutrition
Institute of Basic Medical Sciences Faculty of Medicine
II
© Mari Borge Eskerud
2015
Diet in nutrient status in infants and children with cow’s milk protein allergy.
Results from a cross sectional study with emphasis on vitamin B12 and iron status
Mari Borge Eskerud
http://www.duo.uio.no/
Trykk: CopyCat Forskningsparken
III
Acknowledgements
This work was conducted at the Department of Nutrition, Faculty of Medicine, at the
University of Oslo and at the Department for Paediatrics, Ullevål, Oslo University Hospital
between June 2014 and May 2015.
I would like to thank my supervisors Christine Henriksen and Janne Anita Kvammen for
giving me the opportunity to be a part of this project. Since this study and field of nutrition
research is so important to both of you it has been a great pleasure to be trusted with this
project.
I would also like to thank Rut Anne Thomassen for working so closely with me this year. I’ve
felt included in the work place, you’ve answered all my questions no matter how silly they
were and I’ve learned so much from you this year. I’ve had a really great time this year.
Without the participating families this work would not have been possible. I would therefore
like to thank the parents who genuinely care about their children and their nutrition status
enough to put them through the unpleasant experience of the blood sampling and who are
generous enough with their own time to spend it doing the dietary registration and visiting the
hospital. Your efforts will hopefully benefit other families and infants with cow’s milk protein
allergy in the future.
And last but not least, thanks to my family for always believing that I can do anything I set
my mind to. Because of you, I can.
Mari Borge Eskerud
Oslo, May 2015
IV
Abstract
Background: Cow’s milk protein allergy is the most common food allergy in early life. The
diet is restrictive and is known to pose a risk of malnutrition. Recent studies have proposed a
higher prevalence of B12 deficiency in Norwegian infants than previously thought. Dairy
products are the most important sources of B12 in this population so it is possible that
excluding cow’s milk poses a risk of deficiency. Vitamin B12 and iron have many similar
food sources and a lack of both nutrients affects hematologic parameters.
Objectives: The main objective of this thesis was to investigate diet, B12 and iron status in a
group of cow’s milk allergic infants and children aged less than two years old. The link
between dietary habits and nutrient status were also investigated.
Subjects and methods: Forty nine infants with cow’s milk allergy and primarily
gastrointestinal symptoms were included in this cross sectional study at Oslo University
Hospital, the Children’s Department, Ullevål. The participants had been attending a milk free
diet course and inclusion was set after at least three weeks on the milk free diet. Participants
were weighed and measured, blood samples were drawn for nutrient status, questionnaires
about diet and background information were filled out and a three-day dietary registration was
performed by the parents.
Results: B12 deficiency was present in 17% of the infants and iron deficiency was present in
24%. The non-breastfed infants met their nutritional requirements from their diet and had a
adequate B12 and iron status. Partially breastfed infants past the age of six months were
identified as a high risk group of B12 and iron deficiency. The median intake of iron was
below the recommended level for the partially breastfed infants. Extensively hydrolyzed
infant formula (eHF) was found to be an important source of nutrients in this population and
intake correlated with higher blood levels of B12 and iron.
Conclusion: Infants on a cow’s milk protein free diet who have received guidance from a
pediatric dietitian generally have a sufficient intake of energy and nutrients. B12 and iron
deficiency were prevalent in partially breastfed infants and children. Infants and children that
are not given an eHF and have a breast milk based diet after the age of six months have an
increased risk of deficiency.
V
Contents
List of figures and tables ................................................................................................... VIII
Abbreviations ........................................................................................................................ X
List of appendices ................................................................................................................. XI
1 Introduction ........................................................................................................................ 1
1.1 Cow’s milk protein allergy in infants and toddlers ..................................................... 1
1.1.1 Etiology, prevalence and symptoms ..................................................................... 1
1.1.2 Diagnosis .............................................................................................................. 2
1.1.3 Treatment ............................................................................................................. 2
1.1.4 Cow milks importance in the diet ........................................................................ 3
1.1.5 Nutrition status in cow’s milk protein allergy ...................................................... 3
1.2 Vitamin B12 ................................................................................................................ 3
1.2.1 Structure and function .......................................................................................... 3
1.2.2 Absorption and metabolism ................................................................................. 5
1.2.3 Recommended intake ........................................................................................... 5
1.2.4 Food sources ......................................................................................................... 6
1.2.5 B12 deficiency ...................................................................................................... 7
1.2.6 Groups at risk of deficiency ................................................................................. 8
1.2.7 B12 status in Norway ........................................................................................... 8
1.3 Iron............................................................................................................................... 9
1.3.1 Function ................................................................................................................ 9
1.3.2 Absorption and metabolism ................................................................................. 9
1.3.3 Recommended intake ......................................................................................... 10
1.3.4 Food sources ....................................................................................................... 10
1.3.5 Iron deficiency .................................................................................................... 11
1.3.6 Groups at risk of deficiency ............................................................................... 12
2 Objectives ......................................................................................................................... 14
3 Subjects and methods ....................................................................................................... 15
3.1 Study design .............................................................................................................. 15
3.2 Subjects ...................................................................................................................... 16
3.2.1 The milk free diet course .................................................................................... 17
3.3 Data collected ............................................................................................................ 17
VI
3.3.1 Dietary record ..................................................................................................... 18
3.3.2 Growth and development ................................................................................... 19
3.3.3 Blood samples .................................................................................................... 20
3.3.4 B12-status ........................................................................................................... 21
3.3.5 Iron status ........................................................................................................... 22
3.3.6 Urine samples ..................................................................................................... 24
3.3.7 Additional information ....................................................................................... 24
3.4 Follow-up................................................................................................................... 25
3.5 Statistical analysis...................................................................................................... 25
3.6 My contribution to the research project ..................................................................... 25
4 Results .............................................................................................................................. 27
4.1 Subject characteristics ............................................................................................... 27
4.1.1 Background information on the participants and parents ................................... 27
4.2 Biomarkers of nutrient status ..................................................................................... 30
4.2.1 B12 status ........................................................................................................... 30
4.2.2 Iron status ........................................................................................................... 33
4.3 Nutrient and food intake ............................................................................................ 37
4.3.1 Intake of macronutrients ..................................................................................... 38
4.3.2 Intake of micronutrients ..................................................................................... 39
4.4 Associations between nutrient status, feeding patterns and nutrient intake .............. 42
4.5 Dietary sources of iron and B12 ................................................................................ 43
4.5.1 B12 sources ........................................................................................................ 43
4.5.2 Iron sources ........................................................................................................ 44
4.6 Characteristics of the nutrient deficient infants and children .................................... 45
4.6.1 B12-deficient infants .......................................................................................... 45
4.6.2 Iron deficient infants .......................................................................................... 47
4.6.3 Association between the B12 and iron deficient participants ............................ 48
5 Discussion ........................................................................................................................ 49
5.1 Subjects and methods ................................................................................................ 49
5.1.1 Subjects and study design .................................................................................. 49
5.1.2 Strengths and limitations of the method ............................................................. 50
5.1.3 Statistics ............................................................................................................. 52
5.2 Discussion of results .................................................................................................. 52
VII
5.2.1 B12-status ........................................................................................................... 52
5.2.2 Iron status ........................................................................................................... 57
5.2.3 Nutrient intake .................................................................................................... 59
5.2.4 Dietary habits affecting B12 and iron status ...................................................... 61
5.2.5 Who are the infants at risk of deficiency? .......................................................... 63
5.2.6 Clinical implications .......................................................................................... 65
6 Conclusions ...................................................................................................................... 67
7 Future perspectives ........................................................................................................... 68
8 References ........................................................................................................................ 69
Appendices ............................................................................................................................... 75
VIII
List of figures and tables
Figures
Figure 1 B12’s functions in human metabolism. ….………………….………………………4
Figure 2 Flowchart of the study population.…………………………………………....…....15
Figure 3 Number of the infants with increased homocysteine, >6,5 µmol/L, and low B12,
<300 pmol/L………………………………………………………………………………..…31
Figure 4 Number of infants with decreased hemoglobin (below 9 g/100 mL for infants aged
2-5 months, below 10 g/100 mL for infants aged 6-11 months and below 11 g/100 mL for
infants aged 12-23 months) and ferritin (<25 µg/L for infants aged 0-11 months and <10 for
children aged 12-23 months)……………………………………………………….…………36
Figure 5 Distribution of infants with increased soluble transferrin(s-TfR, >4,4 mg/L for girls
and >5,0 mg/L for boys) receptor and low ferritin(<25 µg/L for infants aged 0-11 months and
<10 for children aged 12-23 months)………………………………………………………....36
Figure 6 Supplement use among the participants…………………………………….……...37
Figure 7 Food sources of B12, except for breast milk, in partially breastfed infants………..43
Figure 8 Food sources of B12 in non-breastfed infants……………………………………...43
Figure 9 Food sources of iron, except for breast milk, in partially breastfed infants………..44
Figure 10 Food sources of iron in non-breastfed infants…………………………………….44
Tables
Table 1 Recommended daily intake of B12 in different age groups…………………….……6
Table 2 Recommended iron intake in different age groups……………………………...…..10
Table 3 Reference intervals B12 and Hcy in infants and children………………………...…21
Table 4 Reference intervals for iron parameters in infants and children…………………….23
Table 5 Characteristics of the population…………………………………………………….28
Table 6 Background characteristics on the infants’ parents………………………………….29
Table 7 Vitamin B12, folate and vitamin D status presented based on breastfeeding..……...31
Table 8 B12 and Hcy in infants aged 6-8 months by feeding status………………………....32
Table 9 Biomarkers of iron status according to breastfeeding status…………………...……34
IX
Table 10 Intake of macronutrient from complimentary food in partially breastfed infants….38
Table 11 Total macronutrient intake in non- breastfed infants………………………………39
Table 12 Intake of micronutrients from complimentary foods in partially breastfed infants..40
Table 13 Total micronutrient intake in non-breastfed infants………………………………..41
Table 14 Factors correlated with s-B12……………………………………………………...42
Table 15 Factors correlated with s-ferritin…………………………………………………...42
Table 16 B12 deficient participants compared with the non-deficient participants………….46
Table 17 Characterization of the iron deficient participants and comparison with the non-
deficient………………………………………………………………………………………48
X
Abbreviations
Abbreviations
AAF Amino acid based formula
B12 Vitamin B12
CALIPER Canadian Laboratory Initiative on Pediatric Reference Intervals
CMP Cow’s milk protein
CMPA Cow’s milk protein allergy
CMPFD Cow’s milk protein free diet
CNS Central nervous system
CRP C Reactive Protein
E % Energy percent
eHF Extensively hydrolyzed formula
ESPGHAN European Society for Paediatric Gastroenterology, Hepatology and Nutrition
G grams
GIT Gastrointestinal tract
Hb Hemoglobin
Hcy Homocysteine
IDA Iron deficiency anemia
IF Intrinsic factor
Ig Immunoglobulin
Kcal Calories
MFDC Milk free diet course
MMA Metylmalonic acid
NHANESIII The third National Health and Nutrition Examination Survey
NFCT Norwegian Food Composition Table
OUS Oslo University Hospital
PA Pernicious anemia
RCT Randomized Controlled Trial
sTfR Soluble Transferrin Receptor
USA United States of America
WHO World Health Organization
XI
List of appendices
Appendix 1 Study invitation
Appendix 2 Consent form
Appendix 3 Questionnaire on background information
Appendix 4 Semi quantitative food frequency questionnaire
Appendix 5 Food diary
Appendix 6 Growth charts
1
1 Introduction
1.1 Cow’s milk protein allergy in infants and
toddlers
1.1.1 Etiology, prevalence and symptoms
Cow’s milk protein allergy (CMPA) is the most common food allergy in infancy with an
incidence of 2-3% in industrialized countries (1). It is difficult to assess changes in prevalence
of food allergy over time as the studies being compared need to be similar in both
methodology and population and is further complicated by the fact that tolerance often
develops in infants and children (2). A recent survey by the World Allergy Organization (3)
showed that solid data on the prevalence of food allergy is missing in many countries. More
than half of the 89 countries surveyed did not have updated information and just 10% had data
based on oral food challenges which is the diagnostic gold standard. The majority of the
countries reported an increase in food allergy prevalence in the past ten years, but this was
mainly due to an increased health care burden. The EuroPrevall birth cohort with data from
nine European countries found a challenge proven incidence of CMPA below 1% in infants
and toddlers less than two years old (4). The study only considered acute symptoms and not
gastrointestinal and therefore the true prevalence of adverse reactions to milk proteins is
probably higher.
In most cases the allergy is temporary as it is uncommon in adults. CMPA can either be
immunoglobulin (Ig)E-mediated, non-IgE-mediated or mixed. A recent study from Finland
(5) found that by five years of age all children with non-IgE-mediated allergy had developed
tolerance, and 74% of the children with IgE-mediated allergy. Symptoms may originate from
several organ systems and may be unspecific in infants. Gastrointestinal symptoms are
common in non-IgE-mediated allergy and include: colic, vomiting, anorexia, diarrhea, bloody
stools, constipation, failure to thrive, and iron deficiency anemia (IDA). Anaphylaxis is
uncommon in this population (6).
2
1.1.2 Diagnosis
Diagnosis can be challenging because of the diverse symptoms. Determination of specific Ig-
E by a blood sample or skin prick test can be useful, but does not speak to the presence of
non-IgE-mediated CMPA. Children with primarily gastrointestinal symptoms, which is
estimated to include 50% of all cases, are likely to not be identified by this test (6). The
recommended diagnostic test for these patients is elimination of cow’s milk protein (CMP)
from the patients’ or the breastfeeding mothers’ diet for two-four weeks. If there is an
improvement in symptoms after this period a home challenge can be performed. If the patient
is breastfed the mother can reintroduce dairy products into her own diet and if not the child
can be given a regular infant formula or a dairy product to confirm diagnosis (7).
1.1.3 Treatment
The only effective treatment in CMPA is a CMP-free diet. If the infant is breastfed the mother
must eliminate CMP from her diet. Breastfeeding is the recommended nutrition for infants
aged less than six months, but in CMPA the early introduction of an extensively hydrolyzed
hypo-allergenic formula (eHF) based on whey or casein is recommended (6). Depending on
the age of the infant the eHF can either supplement breastfeeding, replace breastfeeding or be
used as a cow’s milk substitute. It has been shown that introducing an eHF before three
months of age leads to better acceptance later in life, making it important to introduce this
even if the primary nutrition still comes from breast milk (8). Approximately 10% of infants
with CMPA, typically those with several food allergies, require an amino acid based formula
(AAF) for complete remission of symptoms (6). Soy-based formulas have recently been
found to be safe (9), but are no longer available in Norway.
Excluding CMP from the diet is more comprehensive than eliminating milk, yoghurt and
cheese. All other foods that have CMP as an added ingredient in any form must also be
avoided, like mixed meat products, baked goods with milk, chocolate, most baby porridges
and ready-made meals. This means that the parents must read the ingredient list on all foods
given to the child or eaten by the breastfeeding mother to ensure the child does not
accidentally ingest CMP. Milk from other animals is not a suitable substitute either, as cross
reactions in allergic children have been shown for sheep, goat and buffalo milk (10).
3
1.1.4 Cow milks importance in the diet
In Norwegian adults dairy products have been shown to contribute to 24% of the total B12
intake and 65% of the calcium intake (11). Updated data for infants and children are not
available but it is reasonable to believe that the contribution is similar. In infants, the use of
cow’s milk as a main drink is not recommended before 12 months age because it can replace
more iron rich foods (12). Milk is still included in the diet from an early age. Cow’s milk is
added to most common fortified baby porridges and therefore a part of the diet of Norwegian
infants and children. Fortified baby porridge is the most important source of iron for 12 month
old Norwegian infants and eliminating this food can make consuming enough iron difficult
(13).
1.1.5 Nutrition status in cow’s milk protein allergy
Several studies have found that children with current or previous CMPA have an impaired
nutritional status compared to children with milk in their diet (14-17). The diet is quite
restrictive and can be inadequate if suitable replacements are not used. In a recent prospective
multicenter intervention study (18) 91 otherwise healthy children with food allergy were
provided with counseling from a dietitian. At baseline, compared with healthy controls a
significantly larger percentage of the allergic children were underweight, defined as a weight
for height ratio below two standard deviations. Six months after the intervention this
difference was no longer significant. At baseline the allergic infants had a significantly lower
intake of energy, protein, calcium and zinc and after six months a significant increase in the
intakes of all these nutrients. This study shows that qualified nutritional guidance can reduce
the risk of malnutrition in allergic children and this is also recommended by the European
Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) (6).
1.2 Vitamin B12
1.2.1 Structure and function
Vitamin B12 (henceforth referred to as B12) includes a group of cobalamin compounds that
include methyl-cobalamin, adenosyl-cobalamin and hydroxycobalamin among others. All
4
these molecules contain a cobalt atom at the center of a corrin ring. Cyanocobalamin and
hydroxycobalamin are the most active forms (19).
B12 only has two known functions in humans. Methylcobalamin is a coenzyme for the
enzyme methionine synthase which converts homocysteine to methionine using methyl-
tetrahydrofolate (a form of the vitamin folate) as methyl donor. This is a key step in nearly all
biological methylations, and it occurs everywhere in the body, all the time.
Adenosylcobalamin is a coenzyme for methylamalonyl coenzyme A mutase which converts
methylmalonyl coenzyme A derived from branched-chain amino acids, odd-chain fatty acids
and cholesterol to succinyl coenzyme A. Succinyl coenzyme A then enters the tri-carboxylic
acid cycle and is therefore essential in human energy metabolism (20). A lack of B12 can
therefore lead to accumulation of homocysteine (Hcy) and methylmalonic acid (MMA). The
latter metabolite is a precursor of methylmalonic coenzyme A. The functions of of B12 are
shown in figure 4 below.
Figure 1 B12’s functions in human metabolism. Adapted from Nielsen et al, 2012 (20).
5
1.2.2 Absorption and metabolism
The absorption of B12 is a multistep process. Before entering the acidic stomach, B12 is
bound to haptocorrin which is believed to shield the vitamin structure from hydrolysis in the
acidic environment of the stomach. When this complex reaches the duodenum haptocorrin is
degraded by pancreatic enzymes and B12 is instead bound to intrinsic factor (IF). The B12-IF
complex is absorbed in terminal ileum by endocytosis mediated by the cubam receptor
complex (20).
Inside the lysosome B12 and IF are separated by proteases and B12 is released into the
cytosol. A number of proteins are involved in converting B12 to its coenzyme form inside the
cell and these processes are not yet fully understood. Transport mechanisms in the central
nervous system (CNS) are also largely unknown (20).
If the vitamin needs to be transported out of the intestinal cell to a different location for use or
storage this is performed via the multidrug resistant protein 1-transporter, and probably aided
by additional mechanisms that are currently unknown. After exiting the cell B12 is bound to
and transported by transcobalamin in the blood. This complex is readily taken up by cells via
the transmembrane receptor CD320 via endocytosis. Surplus B12 can either be excreted by
the kidneys or stored in the liver (20).
1.2.3 Recommended intake
The recommended intake of B12 in the Nordic countries is shown in table 1. There is no
recommendation for infants aged less than six months as exclusive breastfeeding is the
preferable source of nutrition. If complimentary feeding starts at four-five months of age the
recommended intakes for 6-11 month old infants should be used (21).
6
Table 1 Recommended daily intake of B12 in different age groups1
Recommended
daily intake,
µg
Lower
intake
level, µg
Upper
intake
level2, µg
6-11 mo 0,5 - -
12-23 mo 0,6 - -
2-5 y 0,8 - -
6-9 y 1,3 - -
<18 y 2,0 1,0 -
Pregnancy 2,0 - -
Lactation 2,6 - -
µg: micrograms, mo: months, y: years
1Nordic Nutrition Requirements 2012 (21).
2Not established
1.2.4 Food sources
B12 is only synthesized by bacteria, but can be stored in animal tissue. Important dietary
sources in Norway include liver, meat, milk and dairy products, fish, shellfish and eggs. B12
from dairy and fish seem to be more bioavailable than B12 from sources such as meat and
eggs. Milk has a low B12 concentration, 0,2-0,4µg/100g, compared to for example beef which
contains about 1,5 µg/100g, but milk is consumed in large quantities in Norway and therefore
constitutes and important source (22). Dairy products, vitamin supplements and liver pate
have the strongest correlation with B12 status in children (23).
Certain types of algae contain B12 but these forms are probably inactive. A few types of
seaweed on the other hand do contain B12 and has recently been found to constitute a
possible plant based source of B12 for people who do not consume enough animal foods to
meet their requirements, such as people with food allergies (24).
7
1.2.5 B12 deficiency
Pernicious anemia (PA) used to be the most common reason for B12 deficiency because it
involves autoimmune gastritis that impairs IF-secretion. In recent years several other
underlying conditions have emerged such as celiac disease, tropical sprue, helicobacter pylori
infection, gastric surgery and human immunodeficiency virus infection. Diseases of the liver
or kidneys can lead to inadequate synthesis of B12 binding proteins and therefore increased
excretion. Inadequate diet can also result in deficiency, most commonly in vegans and strict
vegetarians, in alcoholism and in poor regions were animal foods are unavailable in sufficient
quantities (19). If these conditions occur in a pregnant or breastfeeding mother, the child
could be at risk of deficiency.
B12 deficiency affects the blood, gastrointestinal tract (GIT) and the CNS. The most
common biomarker in a screening setting is s-B12, but this measure has low sensitivity and
specificity, especially for lower concentrations. Therefore deficiency cannot be ruled out by
this measure alone. The most commonly used biomarkers are Hcy and MMA which
accumulate when B12 is in short supply as a coenzyme. In infants Hcy has been shown to be
a more accurate biomarker than MMA (25). In severe deficiency megaloblastic anemia is
present. This occurs when Hcy is no longer converted to methionine and DNA-synthesis is
slowed down. This primarily affects cells with high turnover such as cells in the GIT or
erythropoietic cells in bone marrow (26).
In infants the most common symptoms of deficiency are fatigue, anorexia, failure to thrive,
developmental delay, hypotonia, seizures, vomiting and diarrhea (27). These are unspecific
symptoms which can lead to delayed diagnosis of deficiency. Infants with B12 deficiency are
also at increased risk of damage to the CNS due to reduced myelination. Myelin contains fatty
acids that are synthesized with B12 as a coenzyme. Lack of B12 leads to synthesis of
abnormal fatty acids that are incorporated into myelin but alters the structure of myelin.
Methylcobalamin is a cofactor in the synthesis of phosphatidylcholine which is also an
important part of myelin. A shortage of phosphatidylcholine may lead to impaired
myelination and even demyelination. Myelin protects the nerve cells and facilitates nerve
communication and a shortage of myelin can lead to degenerations of axons. Myelination is
most active in the first six months of life making adequate B12-status important at this age
(28).
8
Consensus on diagnostic criteria for B12 deficiency in infants and children are currently
missing, which makes diagnosis challenging (29). A retrospective search of medical journals
at the Department of Paediatric Medicine at Oslo University Hospital found that the diagnosis
B12 deficiency had been documented in 20 patients in the last 12 years, indicating that this
condition is underdiagnosed in Norway (30).
1.2.6 Groups at risk of deficiency
Population groups at increased risk of deficiency are elderly people, infants and several
patient groups mentioned above. Infants are at risk due to insufficient intake, malabsorption
or inborn errors of B12-metabolism. Insufficient intake usually stems from the mother having
B12 deficiency or PA and can lead to both low stores in the infant at birth and low supply
thereafter if the mother is breastfeeding. Malabsorption can occur if the infant is given drugs
that affect gastric acid secretion, if it’s undergone gastric resection or lacks IF. Inborn errors
of metabolism can affect many different proteins involved in B12 metabolism and therefore
both the presentation of and the treatment for these conditions are quite diverse (27).
1.2.7 B12 status in Norway
Estimated B12 intake in the general population in North America and Europe exceed the
recommended daily allowance of around 2,0 µg per day for the adult population and B12
deficiency is not considered a large problem in these areas (31). The mean total intake in
middle-aged and elderly Norwegians was found to be 6 µg per day in the Hordaland
Homocysteine Study and there was a low prevalence of deficiency in this population (22).
The Norwegian Mother and child cohort found that the mean B12 intake in pregnant
Norwegian women was 8,8 µg per day. Less than one percent of the participants had an intake
below the recommended 2 µg/day (32).
In a study by Hay et al (23) in healthy Norwegian two-year olds the median B12 intake was
found to be 3,1 µg per day and none of the children had an intake below the recommended
intake level for their age (23). In this study 1,4% of infants were found to be deficient in B12,
defined as s-B12 below 150 pmol/L. A new study on B12-status in Norwegian infants
suggests that more than two thirds of healthy, breastfed infants have a low B12-status, defined
as Hcy > 6,5 µmol/L (33). To our knowledge B12-status in cow’s milk allergic infants or
children has not previously been studied.
9
1.3 Iron
1.3.1 Function
Iron is an essential trace element in the human body. Iron has the ability to participate in
oxidation and reduction reactions, which are essential in many chemical reactions in the body.
This high degree of reactivity also makes iron a pro-oxidant that can damage cells and DNA if
it is not bound to proteins. Iron is involved in many functions in the body such as red blood
cell function, oxygen transport, cognitive function, immune function and several heme and
non-heme enzymes. Cytochromes are enzymes present in all cells of the body and they are
part of the mitochondrial respiratory chain’s electron transfer and therefore necessary for
energy metabolism (34).
1.3.2 Absorption and metabolism
Dietary iron exists in two forms, heme and non-heme iron. These two forms are absorbed
differently. Heme iron is the form of iron present in hemoglobin (Hb), myoglobin and some
enzymes and is found in animal foods in the diet. Heme iron is absorbed easily and quickly
bound to transferrin. Transferrin is absorbed by cells via the transferrin receptor 1. Non-heme
iron is found primarily in plant foods and requires some digestion before it can be absorbed.
The iron is reduced by the acidic conditions in the stomach or brush border enzymes to its
ferrous state, which is then absorbed by the brush border iron transporter divalent metal
transporter 1. The absorption of non-heme iron can also be increased or decreased according
to the body’s needs. This is controlled by factors such as the hormone hepcidin and by total
iron-binding capacity (TIBC) (34). The iron stores in the body have been estimated to be 250
mg at birth, and doubles as the infant grows over the first year of life. Infants absorb about
10% of the iron ingested from a mixed diet but the absorption from breast milk is generally
assumed to be 50% (35). In the blood iron is transported bound to transferrin to the tissues
that require iron. There major iron storage molecule is ferritin, and iron is stored in the liver,
bone marrow, spleen and the muscles. Since iron is highly conserved there are no routes of
regulatory excretion. Therefore iron is only lost from the body through bleeding, non-
absorbed iron in the GIT and normal loss of hair and skin (34).
10
1.3.3 Recommended intake
The recommended intakes for different age groups in the Nordic countries are presented in
table 2. There is no recommendation for infants aged less than six months as exclusive
breastfeeding is the preferable source of nutrition. If complimentary feeding starts at four-five
months of age the recommended intakes for 6-11 month old infants should be used (21).
Table 2 Recommended iron intake in different age groups1
Recommended
daily intake,
mg
Lower
intake
level, mg
Upper
intake
level, mg
6-11 mo 8 - -
12-23 mo 8 - -
2-5 y 8 - -
6-9 y 9 - -
10-13 y 11 - -
Women 15/93
51
60
Men 9 - 60
Pregnancy -2
- -
Lactation 15 - -
Y: years
1Nordic Nutrition Requirements 2012 (21)
2Requirements should be evaluated individually
3Post menopause
1.3.4 Food sources
The best sources of iron in the diet are animal tissues that store and contain iron, such as liver,
muscle tissue, fish and eggs. Good plant sources of non-heme iron are beans and legumes,
whole grain products and to a certain degree vegetables and fruit. Proteins from animal foods
have also been found to enhance non-heme iron absorption by an unknown mechanism called
the meat-fish-poultry factor. Vitamin C also enhances non-heme iron absorption by forming a
11
chelate with the iron that aids absorption. Other food components impair iron absorption, such
as phenols in coffee and tea and phytates in grain products (19).
The most important source or iron for infants, like all other nutrients, in the first six months of
life is breast milk. Breast milk of healthy Swedish mothers have been found to contain about
0,3 mg iron/L (36). This is a low concentration, but iron is breast milk is highly bioavailable
which has an important impact on the contribution from this source. To compensate for the
biological compounds in breast milk that increase absorption commercial infant formulas
contain 2-8,5 mg iron/L (37). Infants born at term are thought to have enough iron stored to
double their birth weight, which takes about four months at a normal growth rate. Since the
iron content in breast milk is quite low breastfed infants are in a state of negative iron balance
for the first months of their life. After about four to six months they require iron rich foods to
avoid deficiency when their stores are low (35).
Iron intake correlates with total energy intake in a normal western, varied diet. In Norway
iron fortification is limited to infant porridge and formula because infants need iron rich food
due to large requirements compared to their body size. This is illustrated by the recommended
intake of eight mg/day in childhood compared to the nine mg/day recommended to adult men
(21).
With the exception of cow’s milk B12 and iron have similar food sources. In infants and
young children the sources are even more overlapping as quantitatively important foods such
as fortified baby porridge and eHF contain both. It is therefore interesting to investigate these
nutrients together.
1.3.5 Iron deficiency
Iron status is a continuum. The status at present in an individual is therefore difficult to
describe without several measurements and evaluation of different parameters. Status ranges
from IDA to iron overload, and a number of biological markers can be used to describe the
iron conditions in the body at present. For the measurement of iron stores and ID s-ferritin is
widely recognized as the best marker and is recommended by European, British and
American diagnostic guidelines even though it has not been fully validated in infants (38-40).
12
The stages of ID can be divided into three. The first stage is low iron stores, reliably assessed
by ferritin. The second stage occurs when there is a shortage of iron available to the cells, and
this can be assessed by measuring the soluble transferrin receptor (sTfR) or TIBC. When the
cells lack iron they produce more of their iron receptor in an effort to obtain more iron. TIBC
is a measure of the total iron binding capacity in the blood and is based on the transferrin
concentration. A high TIBC value means that the cells in the body need iron. A reduced
concentration of Hb is also evident in the second stage of ID but may still be within the
normal range. The last stage is IDA, when the Hb production is severely reduced and
microcytosis is present (41).
1.3.6 Groups at risk of deficiency
IDA, commonly defined as low hemoglobin with or without low ferritin, is the most common
of all nutritional deficiencies. Population groups at risk are infants, adolescent girls, women of
childbearing age and the elderly. During pregnancy and lactation iron needs are increased
because the blood volume increases, the fetus grows and there is blood loss associated with
giving birth (21). This means that sufficient iron intake in the pregnant and breastfeeding
mother helps prevent deficiency in the infant (42). All premature infants need iron
supplements as the iron stores in the infant accumulate at a late stage of pregnancy (43).
Infants born with low birth weight, commonly defined as less than 2500g, are also at
increased risk of deficiency in the first months of life (44).
ID is not a common problem in the first six months of life in healthy breastfed infants because
they are born with iron stores and iron in breastmilk is highly bioavailable (21). Young
children continue to have high iron requirements compared to their energy intake for the first
few years of their life. This means that the iron concentration of their total food intake needs
to be higher than it does for adults. In a Norwegian study ID was present in 12% of two year
olds, indicating that this condition is a problem today (45).
The World Health Organization (WHO) estimates that anemia affects a quarter of the total
population. In children aged 0-5 years the worldwide prevalence is 76%. In Europe the
prevalence in pre-school aged children is 19%. As other factors also can lead to anemia iron
deficiency is not the only reason, but it is the most prevalent one and data on iron status are
scarce (46). Data from the third National Health and Nutrition Examination Survey
(NHANES III) in the USA performed between 1988-1994 found a prevalence of ID of 9% in
13
infants aged 1-2 years, in a population with iron fortification of flour (47). They concluded
that ID is prevalent in toddlers, adolescent girls and pregnant women. In a European study on
12 month old infants performed in the early 1990’s the prevalence of ID was found to be 7%
(48). To our knowledge iron status in infants and children with CMPA has not been studied
previously.
14
2 Objectives
The main objective of this thesis is to provide more knowledge about the diet and nutrition
status of infants following a cow’s milk protein free diet (CMPFD). As B12 and iron come
from similar food sources and both nutrients affect hematological parameters, emphasis was
put on these two nutrients. The specific aims of this thesis are:
To describe the B12- and iron-status by measuring relevant biomarkers.
To describe nutrient intake in a population of infants and children with CMPA.
To identify dietary patterns and food choices that influences B12- and iron-
status in this population
Identify groups at increased risk of deficiency.
15
3 Subjects and methods
3.1 Study design
This thesis is part of a cross sectional study investigating dietary habits and nutritional status
in infants and children aged 0-24 months old on a cow’s milk protein free diet. Nutritional
status was determined using anthropometric measures, blood samples and urinary samples.
Diet was recorded by a three day dietary record. Recruiting and collection of data was
conducted in the period of March 2014 to February 2015. The study is a collaborative project
between the Department of Nutrition at the University of Oslo and the Department of
Paediatric Medicine, Women and Children’s Division at Oslo University Hospital. A flow
chart of the recruitment process is shown in figure 2.
Figure 2 Flowchart of the study population.
16
Participants were primarily recruited among patients referred to a hospital group education
session on dietary management of cow’s milk protein allergy (CMPA), here forth abbreviated
Milk Free Diet Course (MFDC). Participants were also recruited from hospitalized patients or
by doctors at the children’s outpatient clinic. In total, 79 infants and children were invited to
participate and 49 (62%) completed the study.
All the materials for the study were either given to the parents at the Milkschool or mailed if
they were included at a later time, for example if they had not been on the diet long enough to
be included yet.
The collection of data was mainly done at one visit to the outpatient clinic where growth was
recorded by research workers and blood samples were taken by hospital staff. Urine samples
were collected at home by the parents and either brought to the hospital or mailed afterwards.
The dietary registration and the questionnaires were filled out at home by the parents.
Ethics
The study was approved by the Regional Committee for Medical and Health Research Ethics
in Norway (REC nr. 2013/1579) and the Research Committee management at the Women and
Children’s Division, Department of Paediatric Medicine, OUS. Eligible candidates for
participation were contacted once by phone about participation. Written informed consent to
participate was obtained from both parents of each child or the primary caregiver. Each
participant was randomly assigned an ID-number used on all data collected. The key
connecting names and ID-numbers was kept locked in at the Department of Paediatrics, and
not removed from the hospital at any time. All digital study material was stored on a
designated research server in a folder only accessible by study personnel. Participation was
voluntary and in accordance with good clinical practice participants were allowed to withdraw
from the study at any time without supplying any further information as to why they decided
to drop out.
3.2 Subjects
Gastroenterology patients aged 0-24 months old being treated for CMPA at Oslo University
Hospital, Department of Paediatric Medicine, were invited to participate in the study if they
were eligible for inclusion. The infants and children had been diagnosed with CMPA by
17
pediatricians, based on symptoms, and were on an elimination diet. Provocations with milk
proteins are not commonly performed if the elimination diet has a definitive effect.
Inclusion criteria:
Had been following a CMPFD for at least three weeks.
Aged less than two years old.
Born at term or no earlier than 37 weeks of gestation after an uncomplicated
pregnancy.
Exclusion criteria:
Known thyroid disease.
Currently were or previously had been receiving tube feeding or parenteral nutrition
for a substantial amount of time.
Use of contrast fluid in the past six months.
Need of interpreter, due to financial limitations.
3.2.1 The milk free diet course
The MFDC is a two hour class held by registered dietitians. The aim is to ensure that children
diagnosed with CMPA are given a diet that completely excludes cow’s milk protein and also
is nutritionally adequate. Emphasis is put on avoidance of milk proteins in the diet, the
importance of a eHF as a cow’s milk or breast milk substitute, a varied diet to ensure
nutritional adequacy, use of foods rich in iron, calcium and protein and recommendations for
dietary supplements. Mothers of children with CMPA who breastfeed must also follow a diet
free from cow’s milk protein. Therefore the class also covers the mother’s diet and how to
ensure she gets enough energy and nutrients to meet her requirements while breastfeeding.
3.3 Data collected
Diet and nutritional status was recorded after the children had been following a cow’s milk
protein free diet for a minimum of three weeks. The data collected included a three day
dietary registration, a questionnaire on diet, a questionnaire on background information, two
18
urine samples collected at home by the parents and weight, length, head circumference and
blood samples that were recorded at the hospital outpatient clinic.
3.3.1 Dietary record
The participants parents were asked to complete a three day dietary record for their child. A
three day dietary record has been found to accurately estimate food intake in one year old
infants (49). The parents were instructed to use household measurements but asked to
describe the type and amount of food eaten as accurately as possible. The front page of the
food diary given to the participants contained information about details and foods easily
forgotten when doing the registration, such as type of bread used, use of butter or margarine,
snacks between meals, type of salt if used, amount of porridge and type as well as mixing
ratio of eHF if used. If the child was breastfed this was also registered as time of and duration
of feeding, but volumes were not recorded. See appendix 5.
The food diaries were entered into ”Kostholdsplanleggeren” (50), a web based food diary
launched by the Norwegian Food Safety Authority and the Norwegian Directorate of Health
in 2014, based on the Norwegian Food Composition Table (NFCT) (51). This website is
aimed at the general public and is easy to use. The food database currently contains 1469 food
items and information on 36 different nutrients. The nutrient content of the chosen portion is
calculated automatically. Once a year new foods are added and nutritional information on
existing foods are updated.
The parents were asked to provide accurate recipes for homemade dishes. These recipes were
then entered into ”Kostholdsplanleggeren” and the amount the child ate was recorded.
Occasionally foods eaten were not included in the food composition table. If accurate
nutritional information about the product was available this was entered into
Kostholdsplanleggeren and that product was added to the database, for example the different
infant formulas commonly used in this population. If only macronutrient content was
available for a specific food item information on micronutrients was extrapolated from similar
foods in the database or a product with similar ingredients was used. When information on the
amounts of food eaten, typically for dinner, was incomplete a standard serving of food, based
on the size of a glass of baby food for the appropriate age was used. If information about the
amounts of other foods was not accurately provided, amounts were either entered based on
other entries in that child’s food diary or on an approximate normal serving for a child that
19
age. When information on special serving sizes was available, these were used. For example
for the baby porridges from “Holle”, the weight per deciliter was less than for regular flour
and the information from the manufacturer was used.
The NFCT does not contain information about all foods in both cooked and raw form. This
difference is relevant because cooking decreases the water content of the food and therefore
changes the nutrient density. If information was available for just one form of the food, i.e.
cooked or raw the available form was chosen. When the recipe for a glass of baby food was
added, the raw form was used because this seemed to be the best match with the nutrient
information supplied by the manufacturer. If for example the infant was reported to have
eaten “about 30 grams of chicken mince” this was assumed to be in the cooked form.
The output from “Kostholdsplanleggeren” does not provide information on consumption of
different food groups. Therefore a manual calculation of the contribution from different foods
to the total intake of B12 and iron was performed.
To obtain more information about the infants and children’s habitual intake a semi
quantitative food frequency questionnaire about diet was administered, see appendix 4. This
questionnaire included 27 questions about dietary habits. Duration of breastfeeding was
recorded as well as the age when solids were introduced. If homemade porridge was used the
frequency of consumption was recorded along with what types of liquid that was commonly
used in the porridge. Consumption of dinner, fish, caviar, shellfish, roe paste, eggs, chicken
and salt were recorded. Frequency of consumption was recorded as either daily, three-six
times per week, one-two times per week, less than once per week or never. Additional
questions covered foods intentionally avoided, eating development, frequency and use of
dietary supplements, natural supplements and sources of information about infant and child
nutrition.
Recommendations for intake are from the Nordic Nutrition Recommendations 2012 and the
age ranges are chosen based on the median age of the group (21).
3.3.2 Growth and development
Weight, length and head circumference was measured at time of inclusion by a pediatric
dietitian, research worker (master’s student) or by trained pediatric nurses. Weight was
recorded in the nude on a children’s scale to the nearest 0,005 kg. Weight was measured on a
20
Data Baby Scale 930 (Oriola, Espoo, Finland). Weight was recorded lying down for most
children, but older children that were too long to lay on the scale were weighted sitting
upright.
Recumbent length was measured without clothing on a measuring board to the nearest
completed millimeter. Usually the child’s parents held the baby’s head by the head piece on
the measuring board as the research worker held the child’s legs with the ankles at 90o and
recorded the length. The length board stood on a flat and stable surface.
Head circumference was measured with a polyfibre measuring tape (Hoechtsmass, Germany)
to the nearest millimeter three times and the average of these measurements was recorded.
The tape was placed just above the glabella, the area between the eyebrows, and around the
largest protuberance of the head. The tape was firmly tightened to compress hair.
When growth could not be recorded at the time of inclusion or growth had been recorded
recently at the hospital or outpatient clinic these data were used. Growth data was plotted on
charts from the Bergen Growth Study (52), see appendix 6, as these are based on data from
Norwegian children. These charts are believed to be more appropriate for Norwegian children
who are generally heavier, longer and in particular have greater head circumference than the
standard that the WHO charts are based on (53). Weigh, length and head circumference for
age and weight for length was plotted on the charts. Birth weight was either found in medical
records, found in the child’s health visitor card (Helsestasjonskort), a growth record provided
to all children born in Norway, or obtained directly from the parents who tend to accurately
remember this number.
3.3.3 Blood samples
Serum was obtained from venous non-fasting samples by personnel at the outpatient clinic
according to routine procedures. This was done to gain information about the child’s
nutritional status, particularly B12 and iron status. The following blood parameters were
included: b-Hb, s-iron, s- TIBC, s-ferritin, b-mean corpuscular volume (MCV), b-mean
corpuscular haemoglobin (MCH), s-transferrin, s-sTfR, s-Vitamin B12, p-homocysteine, s-
folate, s-zinc, s-albumin, s-C Reactive Protein (CRP) and s-25-OH vitamin D. The blood
samples were analyzed by the Department of Medical Biochemistry at Ullevål, OUS except s-
21
25-OH vitamin D and urinary iodine that were analyzed by the Hormone Laboratory at Aker,
OUS. The same laboratories were used for all samples.
3.3.4 B12-status
Vitamin B12 status was assessed by measuring s-B12 and p-homocysteine. B12 status was
determined based on the reference intervals and recommended limits for deficiency
recommended by the Norwegian Paediatric Association (54), as shown in table 3. These
ranges are similar to the ones provided by the Norwegian Association for Medical
Biochemistry (55), except that the latter states 300 pmol/L as the upper s-B12 level where a
deficiency is possible. Therefore 300 pmol/L was used as the upper limit where deficiency
was possible in this study. The reference intervals provided by the laboratory at Oslo
University hospital (56) are also presented in table 3. In adults MMA is commonly used to
diagnose vitamin B12 deficiency, but MMA has previously been shown to not have
significant diagnostic value in children under 1,5 years old (33). Conversely Hcy is associated
with both folate and vitamin B12 status in adults, but correlates better with vitamin B12 in
young children (25). For a few infants Hcy was not measured and s-B12 alone was used to
determine status. There is unfortunately not a consensus on which values constitute a
deficiency in this age group.
Table 3 Reference intervals B12 and Hcy in infants and children
s-B12, pmol/L p-Hcy, µmol/L
Norwegian Paediatric Association
0-1 y 99-7451
3,4-12,02
1-9 y 278-11151
2,3-7,22
Department of medical biochemistry, OUS
0-120 y 150-650
0-15 y 3,0-11,6
y: years, Hcy: homocysteine
1Values <250 pmol/L in children aged <1,5 years old requires measurement of the metabolic
markers Hcy and MMA as deficiency is possible.
2Values >6,5µmol/L in younger children requires measurement of B12 to and/or folate to rule
out deficiency.
22
The B12 concentration in serum was determined by an electrochemiluminescence
immunoassay. In short the analysis involves B12 from serum being bound to ruthenium
marked IF. The luminescence that is emitted from this reaction is measured and used to
quantify the concentration of B12 in the sample. The analysis is done on a Cobas 8000 e602
modular analyzer (serial number AL1123-04, MTU number 30898), Roche Diagnostics (57).
The Hcy concentration in plasma is determined enzymatically on a Cobas 8000 c502 modular
analyzer (serial number AL1020-02, MTU-number 30897), Roche Diagnostics. Hcy is
quantified in a reaction where serine acts as a catalyst and the enzymes cystathionin beta-
synthase and cystathionine beta-lyase convert Hcy to pyruvate and nicotinamide adenine
dinucleotide, the latter of which is then quantified to determine the Hcy concentration in the
sample (kit number FHRWR100 from Axis-Shield Diagnostic Systems) (58).
MCV and MCH were measured in this population because high values can indicate B12
deficiency and low values IDA. Increased MCV and MCH are diagnostic markers for
macrocytosis, resulting from impaired DNA synthesis which can be caused by deficiency of
cobalamin or folate. MCV and MCH will not be increased above normal levels in subclinical
deficiency, and neurological changes due to vitamin B12 deficiency can occur without
megaloblastic anemia (59). Low values are seen in IDA but ferritin is a more reliable measure
(60).
3.3.5 Iron status
Ferritin is the most accurate measurement of iron stores and a low ferritin is diagnostic of ID
and low iron stores is the only known reason for low ferritin (61). The cutoff values for
different biomarkers of iron status used in this study are shown in table 4.
The ferritin concentration was determined by an electrochemiluminescence immunoassay
utilizing the sandwich principle. The ferritin in the sample is bound to biotinylated
monoclonal ferritin specific antibodies marked with a rhutenium complex, which creates the
sandwich complex. The addition of streptavidin coated micro particles creates a stable
complex that can attach to a magnetic electrode where the chemiluminescence emitted can be
measured by a photomultiplier. The procedure is performed on a Cobas 8000 e602 modular
analyzer (serial number AL1123-04, MTU-number 30898), Roche Diagnostics (62).
23
The sTfR concentration in serum was determined by immunoturbidimetry. The reagent kit is
from Roche Diagnostics, catalog number 12148315122. Two reagents are added to the sample
and this creates an antigen-antibody complex. After agglutination the concentration is
measured by turbidimetry (63).
Table 4 Reference intervals for iron parameters in infants and children.
s-ferritin1,
µg/L
s-sTfR,
mg/L
b-Hb,
g/100ml
s-TIBC MCH, pg MCV, pg
2-5 mo 25-790 9,0 – 14,0
25-35 74-115
6-11 mo 25-790 10,0 – 13,5 23-31 70-87
0-11 mo 25-790 23-31 70-87
12-24 mo 10-501 11,0-15,5 23-31 70-87
0-24mo M: 2,2-5,0
F: 1,9-4,4
49-83
Mo: months age, sTfR: soluble transferrin receptor, Hb: hemoglobin, TIBC: Total Iron
Binding Capacity, MCH: Mean Corpuscular Hemoglobin, MCV: Mean Corpuscular Volume
M: male, F: female
1 Reference ranges are from the Department of Clinical Biochemistry, OUS except for and s-
ferritin which is from the Norwegian Guidelines in General Paediatrics by the Norwegian
Paediatric Association (54).
Hb was measured to investigate anemia in this population. TIBC is another parameter that is
used in clinical practice to evaluate iron status, but was not used as the main determinant of
iron status in this study.
The sTfR is a measure of the cells iron needs and will be increased in iron deficiency, before
anemia appears. STfR is not affected by inflammation or infection but can be elevated in
conditions such as leukemia, hemolysis and myeloplastic syndrome. When and infection was
present, as evidenced by an increased CRP, sTfR was used to diagnose ID.
24
3.3.6 Urine samples
Two spot urine samples collected by the parents on two separate days at the same time as the
food diary was used to investigate iodine status in this population, but these results are not
discussed in this thesis.
3.3.7 Additional information
A second questionnaire was filled out by the parents and covered background information, see
appendix 3. Parents were asked questions on symptoms and diagnosis of CMPA, other
diseases, mother’s age and smoking habits, marital status, ethnicity, educational level and
family history of atopy. If the child was breastfed there were questions regarding the
breastfeeding mother’s intake of dairy products and dietary supplements.
Breastfed infants and children
Fifty-seven percent of the participants were currently breastfed. No attempt was made to
estimate the amount of breastmilk ingested by these infants and children. This was due to
individual variations in nutrient content of breast milk as well as the practical difficulties with
assessing the amount of milk taken at every meal. Therefore the dietary registrations for these
infants and children are not complete. The breastfed children were therefore analyzed
separately and the results only reflects nutrient intake from solid foods, eHF and supplements
if used.
Dietary supplements
Eighty-six percent of the participants were reported to use dietary supplements such as cod-
liver oil and multivitamins/-minerals, as this is encouraged at the MFDC. For participants
using supplements two separate files on nutrient intake was generated, one including nutrients
from supplements and one without. Supplement use was determined from the questionnaire
on diet and the food diaries. The questionnaire recorded frequency of use in times per week
but not duration of use.
25
3.4 Follow-up
All participants were given feedback on their results by mail or. If the participants were found
to be deficient of any nutrient such as vitamin B12 or iron they were contacted by study staff
and recommended proper treatment. B12 injections were prescribed by doctors and
administered by nursing staff at the outpatient clinic. If the participant was iron deficient a
liquid supplement containing hem-iron was recommended. After treatment nutrient status was
assessed again after two-three months and parents were informed of the results. Breastfeeding
mothers of B12 deficient infants were also recommended to check their own B12 status.
3.5 Statistical analysis
Statistical analyses in this thesis are mostly describing. All data was analyzed using IMB
SPSS Statistics version 22 (IBM Corp, Released 2013, Armonk, NY). For most analyses non-
parametric testing was used, as the data material is quite small. When normal distribution was
present as evidenced by a histogram and parametric tests were used this is shown in the text.
Results are presented as median (25 th – 75 th percentile), percentages or n. When the
distribution in smaller subgroups is described data is presented as median (min-max). Fishers
exact test was used for the difference between two categorical variables, Wilcoxen signed
rank test was used for the difference between two related samples, Mann Whitney U-test was
used for the difference between two unrelated sample and Spearman’s correlation coefficient
was used to describe the correlation between two continuous variables. A two-sided p-value
<0,05 was considered statistically significant.
Results are shown for the whole population or are separated based on breast feeding or
nutrient status.
3.6 My contribution to the research project
I started working on the project in June 2014 and was responsible for recruiting, data
collection and follow up of study participants from mid-June to December. To include
participants I held the MFDC about three times per month in this period and contacted
eligible participants by phone. After completing the dietary records and filling out the
questionnaires I set up appointments for subjects to return study material and take blood
samples as well as anthropometric measurements. During this visit to the hospital the
26
participants were welcome to ask any questions about their child’s diet or nutritional needs.
When the results from the blood samples were ready I discussed them with more experienced
pediatric dietitians and the participants’ doctor and gave feedback to the participants’ parents
on the findings and whether or not there was need for treatment such as supplements or
dietary adjustments.
27
4 Results
4.1 Subject characteristics
4.1.1 Background information on the participants and parents
Participant characteristics
Background information on the 49 infants included in the study is shown in table 5. The
study population is diverse in terms of age and this creates heterogeneity in the study material.
The majority of the participants, 61%, are between 6-12 months of age. As all participants in
this study are less than two years old the data is not separated based on sex. The majority of
the infants, 65%, were reported to have received the diagnosis of CMPA from a doctor, while
the rest were still excluding milk for diagnostic purposes. The infants had spent a median time
of 4,8 months on the milk free diet before inclusion in the study. Six percent of the infants
were exclusively breastfed at the time of the study, 51% were receiving breast milk and
complementary feeding and 43% were weaned.
Sixty-nine percent of the infants used an eHF. Of these, 65% used extensively hydrolyzed
formulas such as Pepticate, Althera or Nutramigen 2 DHA and 35% used amino acid based
formulas such as Neocate LCP or Neocate Active. In addition to these, 4% used Nutramigen
Spoon, which is an amino acid based fortified porridge rich in iron and calcium.
The infants were recruited at the pediatric gastroenterology department at Ullevål, OUS. The
most common symptoms were colic or abdominal pain, reflux and loose stools, and the
median number of symptoms was four. More than 90% of the infants were reported by
parents to have had complete or some improvement in symptoms of allergy after starting a
milk free diet.
There was no significant difference in age or breastfeeding status, partial or no breastfeeding,
between boys and girls.
28
Table 5 Characteristics of the population.
n with
available
data
% (n) or
median
(25 th – 75 th percentile)
Study subjects 49
Girls 46,9 % (23)
Median age, mo
Age group
- 0-5 mo
- 6-11mo
- 12-23 mo
8,0 (6,0-12,0)
10,2 % (5)
61,2 % (30)
28,5 % (14)
CMPA diagnosed by a doctor
Milk free diet for diagnostic purposes 45 65,3 % (32)
26,5 % (13)
Time on milk free diet, mo 47 4,8 (2,7-5,7)
Breast feeding status at inclusion
- Breast milk only
- eHF only
- Breast milk and eHF
- Breast milk and solids
- Breast milk, eHF and solids
- eHF and solids
- Solid food only
49
6,1 % (3)
-
-
20,4 (10)
30,6 (15)
38,8 (19)
4,1 (2)
Birth weight, g
- Boys
- Girls
Birth length, cm
- Boys
- Girls
47
26
21
40
21
19
3446 (3087-3782)
3260 (3005-3534)
51,0 (50,0-52,0)
50,0 (48,0-52,0)
Weight at inclusion, g
- Boys
- Girls
Length at inclusion, cm
- Boys
- Girls
49
26
23
49
26
23
8780 (7492-10199)
8250 (7080-9335)
72,1 (69,4-75,3)
71,0 (66,5-77,0)
Most frequently parent-reported symptoms of
CMPA
- Colic/abdominal pain
- Reflux
- Loose stools
- Problems sleeping
- Skin affection
- Faltering growth
- Feeding problems
48
75,5 % (37)
67,3 % (33)
51,0 % (25)
49,0 % (24)
49,0 % (24)
38,8 % (19)
34,7 % (17)
Number of symptoms
- >1 symptom
- Median number of symptoms
48
96 % (47)
4 (3,0 - 5,7)
Effect of milk free diet
- Complete remission of symptoms
48
40,8 % (20)
29
- Some improvement
- No effect
- Don’t know
49,0 % (24)
2,9 % (1)
6,1 % (3)
Mo: months, CMPA: cow’s milk protein allergy, g: grams, cm: centimeters
Parent characteristics
Background information about the infants’ parents is presented in table 6. In this population
the majority of the parents were of Norwegian or other Scandinavian decent. The parents in
this study had a high education level, with 65% of the fathers and 87% of the mothers having
finished higher education. Eighty-one percent of the breastfeeding mothers did not have any
dairy products in their own diet, and 18% used small amounts. Eighty-nine percent of the
breastfeeding mothers reported using dietary supplements weekly, primarily calcium and
different multivitamin and -mineral products. Thirty-seven percent of the mothers using
supplements used products containing B12. None of the mothers reported smoking.
Table 6 Background characteristics on the infants’ parents
n with
available
data
% (n) or median
(25 th – 75 th
percentile)
Mothers age, years 48 33 (30,0 – 35,0)
Mother smoking 48 0
Parents civil status
- Married or cohabitating 48
95,9 % (47)
Ethnicity
- Scandinavian mother
- Scandinavian father
- Neither mother nor father are
Scandinavian
96
83,7 % (41)
77,6 % (38)
8,1 % (4)
University or college level education
- Mother
- Father
48
48
87,7 % (43)
65,3 % (32)
Milk in the diet of breast feeding mothers
- No milk
- Consumes small quantities - Consumes milk as normal
27
81,4 % (22)
18,5 % (5)
0
Dietary supplement use in breastfeeding
mothers
- Yes
- Of these, supplements contained B12
27
88,8 % (24)
37,5 % (9)
30
4.2 Biomarkers of nutrient status
Blood parameters analyzed in the study are presented in tables 7-9. The results are presented
for all infants and also separated based on breast feeding status for comparison.
4.2.1 B12 status
Vitamin B12 status was investigated by measuring the p-Hcy and s-B12 concentrations.
Median s-B12 was 441 pmol/L and median p-Hcy was 6,1 µmol//L in this population, see
table 7. Twenty-one percent of the infants and children were found to have s-B12 below 300
pmol/L which warrants further inspection of metabolic markers of deficiency, in this case
Hcy. Seventeen percent had both a s-B12 below 300 pmol/L and p-Hcy above 6,5 µmol/L
which is diagnostic of B12 deficiency. These results are shown in figure 3. Half of these
infants had a s-B12 below 200 pmol/L and the other half had values between 200-300 pmol/L
with the latter being indicative of a subclinical deficiency. As shown in table 9, none of the
infants had macrocytic anemia as no cases of increased MCV was found.
There was borderline significantly more participants with increased homocysteine in the
partially breastfed group compared with the non-breastfed group.
All infants had adequate folate and vitamin D status.
There were no significant differences between boys and girls for these measures.
31
Table 7 Vitamin B12, folate and vitamin D status presented based on breastfeeding status.
25-OH vitamin D: 25-hydroxy vitamin D
1Blood samples are missing for one participant
2Fisher’s exact test comparing the number of infants with sub-optimal levels of vitamins or
Hcy in the non-breastfed and the partly breastfed group.
3Reference ranges are from the Department of Clinical Biochemistry, OUS (56) except for s-
Vitamin B12 and p-homocysteine that are from the Norwegian Guidelines in General
Paediatrics by the Norwegian Paediatric Association (54).
Figure 3 Number of the infants with increased homocysteine, >6,5 µmol/L, and low B12, <300 pmol/L.
Hcy
20
B12
10
Total
n:481
Non-
breastfed
n:20
Partially
Breastfed
n:25
p-
value2
Exclusively
Breastfed
n:3
Reference
Range3
0-24 mo
n
Median (25-75)
s-B12, pmol/L
n below reference
n:47
441,0
(350,0-
555,0)
10
n:19
526,0 (383,0-
623,0)
1
n:19
398,0(314,0-
506,5)
6
0,119 n:3
250,0
3
300-650
pmol/1
p-homocystein,
µmol/L
n above reference
n:42
6,1 (4,7-
7,7)3
20
n:17
5,3 (4,6-6,6)
4
n:22
6,6 (5,1-8,5)
13
0,050 n:3
7,5
3
<6,5 umol/l
s-folate, nmol/L
n below reference
n:46
39,0 (33,9-
45,0)
0
n:19
41,4 (36,7-
45,0)
0
n:24
36,4 (32,0-
44,6)
0
- n:3
39,6
0
7-27 nmol/L
25-OH vitamin D,
nmol/L
n below reference
n:46
80,0 (61,5-
89,5
0
n:19
78,0 (60,0-
87,0)
0
n:24
85,0 (63,5-
103,0)
0
- n:3
68,0
0
37-131
nmol/L
8
32
Data on maternal serum levels was not available, and no significant difference in serum B12-
levels was found between the breastfed infants who had mothers that took oral B12
supplements and those who did not (data not shown).
B12 and Hcy in infants aged six to eight months
Forty percent of the infants in this study were between six and eight months old. To study the
effect of diet on B12 status in a less heterogeneous group a subgroup analysis was performed.
The difference in B12 and Hcy with regards to diet is shown in table 8.
Table 8 B12 and Hcy in infants aged 6-8 months by feeding status.
Group 1
Breast milk and
solids
n: 71
Group 2
Breast milk, eHF and
solids
n: 7
Group 3
eHF and
solids
Group
1 vs 2
Group
1 vs 3
Group
2 vs 3
Median (min-max) p: difference between the
groups2
s-B12,
pmol/L
392,0
(133,0-565,0)
397,0
(172,0-459,0)
526,0
(482,0-784,0)
0,565 0,015 0,003
p-Hcy,
µmol/L
8,4
(6,6-11,5)
6,6
(4,7-10,3)
5,0
(3,1-7,4)
0,073 0,018 0,099
eHF: extensively hydrolyzed formula, Hcy: homocysteine
1Missing two values for Hcy
2Difference assessed by the Mann-Whitney U-Test.
n: 6
33
4.2.2 Iron status
In this population 24% of the infants were found to have ID. ID was defined as s-ferritin
below 25 µg/L for infants aged 0-12 months and below 10 for infants aged 12-24 months. The
results are presented in table 9. If measurements of either ferritin or CRP was not available,
or CRP was increased, sTfR, >4,4 mg/L for girls and >5,0 mg/L for boys, was used to
determine status. Seventy-five percent of the iron deficient infants were currently being
breastfed, none exclusively.
There were significantly more infants with low s-ferritin in the partially breastfed group than
in the non-breastfed group.
34
Table 9 Biomarkers of iron status according to breastfeeding status.
Total:48
Non-breastfed
n:20
Partially
Breastfed
n:25
p-
value1
Exclusively
Breastfed
n:3
Reference
Range2
Parameter n
Median (25-75)
s-hemoglobin,
g/100ml
2-5 mo
6-11 mo
12-24 mo
n below reference
n:48
n:5 11,1 (10,1
– 11,6)
n:30 11,6
(10,9-14,2)
n:13 12,0
(11,7-12,4)
2
n:20
n:2 11,6
n:8 12,3 (11,8-
12,8)
n:10 11,9
(11,5-12,5)
0
n:25
n:0
n:22 11,25
(10,8-12,3)
n:3 12,1
2
0,119 n:3
n3: 10,2
n:0
n:0
0
9,0 – 14,0
g/100ml
10,0 – 13,5
g/100ml
11,0-15,5
g/100ml
MCH, pg
2-5 mo
6-24 mo
n below reference
n:44
n:5 26,5
(25,3-28,0)
n:39 26,4
(24,8-27,0)
1
n:17
N:2 25,9
n:15 26,7
(24,7-27,2)
0
n:24
N:0
n:24 26,0
(24,8-26,9)
1
1,00
n:3 27,2
n:0
0
25-35 pg
21-31pg
MCV, fl
2-5 mo
6-24 mo
n below reference
n:44
n:5 80,0
(78,0-81,0)
n:39 77,0
(74,0-79,0)
1
n:17
n:2 79,5
n:15 78,0
(75,0-79,0)
0
n:24
n:0
n:24 77,0
(74,0-78,9)
1
1,00
n:3 79,5
n:0
0
74-115 fl
70-87 fl
S-iron, µmol/L
4-52 wk
12-24 mo
n below reference
n above reference
n:47
n:35 9,6 (6,3-
11,7)
n:12 8,2 (5,7-
13,1)
16
2
n:19
n:10 11,3 (8,7-
17,7)
n:9 9,8 (5,1-
14,1)
6
2
n:25
n:22 9,2 (6,2-
10,8)
n:3 8,1
9
0
1,00 n:3
n:3 7,5
n:0
1
0
7-18 µmol/L
9-34 µmol/L
s-TIBC
0-24 mo
n below reference
n above reference
n:47
71,0 (66,0-
79,0)
2
8
n:19
71,0 (66,0-
79,0)
0
3
n:25
72,0 (67,2-
83,0)
1
5
1,00 n:3
66,0
1
0
49-83
35
Mo: months, MCH: Mean Corpuscular Hemoglobin, MCV: Mean Corpuscular Volume,
TIBC: Total Iron Binding Capacity, CRP: C-Reactive Protein, soluble TfR: soluble transferrin
receptor
1Fisher’s exact test comparing the number of infants with sub-optimal levels for iron status in
the non-breastfed and the partly breastfed groups.
2Reference ranges are from the Department of Clinical Biochemistry, OUS (56) except for
and s-ferritin which is from the Norwegian Guidelines in General Paediatrics by the
Norwegian Paediatric Association (54).
Boys had significantly higher TIBC-values than girls, p=0,021 otherwise there were no
observable differences related to sex.
s-ferritin µg/L
0-11 mo
12-23 mo
n below reference
n:47
n:34 31,5
(20,2-59,2)
n:13 30 (25,0-
38,0)
10
n:19
n:9 43 (28,0-
71,5)
n:10 31,5
(20,7-38,5)2
1
n:25
n:22 27,0
(17,2-43,2)
n:3 30,0
9
0,027 n:3
n:3 123,0
n:0
0
25-790 µg/L
10-501 µg/L
s-CRP, mg/L
0-24 mo
n above reference
n:47
<0,6 (<0,6-
<0,6)
3
n:19
<0,6 (<0,6-
<0,6)
2
n:25
<0,6(<0,6-
<0,6)
1
0,570 n:3
<0,6
0
0-4 mg/L
s-soluble TfR,
mg/L
Girls
Boys
n above reference
n:42
n:20 4,1 (3,3-
5,0)
n:17 4,8 (4,4-
5,5)
16
n:16
n:9 4,0 (3,5-
5,1)
n:7 4,8 (3,6-
5,0)
4
n:19
n:10 4,6 (3,8-
5,3)
n:9 5,5 (4,5-
5,5)
10
0,166
n:3
n:1 2,9
n:2 5,7
2
1,9-4,4 mg/L
2,2-5,0 mg/L
36
Two infants, 4%, had non-normal values for hemoglobin and s-ferritin, suggesting iron
deficiency anemia, see figure 4. One infant had subnormal levels of MCV and MCH
indicating microcytosis not related to iron status as all other measures of iron status were
normal.
Figure 4 Number of infants with decreased hemoglobin (below 9 g/100 mL for infants aged 2-5 months, below
10 g/100 mL for infants aged 6-11 months and below 11 g/100 mL for infants aged 12-23 months) and ferritin
(<25 µg/L for infants aged 0-11 months and <10 for children aged 12-23 months).
sTfR is increased above the reference range in 38% of the infants. STfR was used to
determine iron status when the CRP was increased indicating falsely high ferritin values due
to inflammation. Low ferritin and increased TfR occurs simultaneously in 12% of infants, see
figure 5.
Figure 5 Distribution of infants with increased soluble transferrin(s-TfR, >4,4 mg/L for girls and >5,0 mg/L for
boys) receptor and low ferritin(<25 µg/L for infants aged 0-11 months and <10 for children aged 12-23 months) .
Hb
2
Ferritin
10
STfR
14
Ferritin
10
2
5
37
4.3 Nutrient and food intake
The intake of macro- and micronutrients in this population is presented separately based on
breastfeeding status. The intake for the children being partially breastfed is not complete as
the breast milk intake was not recorded; therefore these data represent the intake from foods
other than breastmilk. There is no data on nutrient intake for the exclusively breastfed infants.
To highlight the contribution from dietary supplements the intake of micronutrients is
presented with and without supplements. Thirty-nine out of the 45 (86%) participants that had
dietary information available used supplements. The most common combinations of
supplements used in this population are shown in figure 6. A vitamin D supplement used
alone was most common in this population, followed by the combination of a fish oil
supplement and a multivitamin. None of the infants used supplements containing B12. Two
infants in the partially breastfed group and four in the non-breastfed group did not use any
dietary supplements which results in a different number of infants included in each
supplement-category.
Figure 6 Supplement use among the participants.
0
5
10
15
20
25
30
35
Fish oil Vitamin D Fish oil andmultivitamin
Vitamin Dand
multivitamin
Multivitamin Fish oil andvarious
supplements
Nosupplements
Percentage of the participants using the different supplement combinations
38
4.3.1 Intake of macronutrients
The macronutrient intake of the infants in this study is shown in tables 10-11.
The intake of macronutrients for the partially breastfed infants is shown in table 10.
Macronutrients from supplements such as cod liver oil are included in the calculations. There
are no recommendations for the macronutrient composition of complimentary foods in
Norway.
Table 10 Intake of macronutrient from complimentary food in partially breastfed infants.
Nutrient intake1
n: 24
Age, mo 8,0 (7,0-11,0)
Kilojoule, KJ 1719 (1208-2539)
Calories, kcal
- Kcal/kg
408 (285-632)
53,1 (37,1-87,8)
Carbohydrates, g
- %
60,3 (40,7-83,4)
53,0 (50,2-57,7)
Added sugar, g
- %
0,1 (0,0-2,0)
0,1 (0,0-1,9)
Dietary fiber, g
- %
5,2 (4,0-8,0)
2,5 (2,0-3,0)
Protein, g
- %
- g/kg
14,4 (9,4-20,7)
13,0 (10,8-14,0)
1,8 (1,2-2,8)
Fat, g
- %
12,8 (10,1-24,8)
30,5 (26,0-35,7)
Saturated fat, g
- %
3,2 (2,3-6,2)
8,2 (6,2-10,0)
MUFA, g 5,0 (3,2-9,8)
PUFA, g 2,6 (1,9-5,0)
Mo: months, KJ: kilo joule, Kcal: kilocalories, MUFA: Mono Unsaturated Fatty Acid, PUFA:
Poly Unsaturated Fatty Acid
1Values presented as median (25 th – 75 th percentile)
The intake of macronutrients for the infants that are fully weaned is shown in table 11. Both
with and without dietary supplements they meet the recommendations for macronutrient
composition (21). The energy percentage from fat is significantly higher when supplements
are included.
39
Table 11 Total macronutrient intake in non- breastfed infants
Nutrient intake1
n:21 Recommended
intake2
Age, mo 12,0 (6,5-16,5)
Kilojoule, KJ 3282 (2695-4069) -
Calories, kcal
- Kcal/kg
779,0 (638,0-971,0)
96,7(85,7-120,8)
80
Carbohydrates, g
- %
101,3 (85,7-117,9)
52,0(47,0-55,0)
45-60
Added sugar, g
- %
1,4 (0,0-9,5)
0,2 (0,0-4,2)
<10
Dietary fiber, g
- %
6,2 (3,3-10,2)
1,5 (1,0-2,1) -
Protein, g
- %
- g/kg
24,4 (18,8-30,4)
12,0 (11,0-14,0)
3,1 (2,6-4,0)
10-15
1,0-1,1
Fat, g
- %
27,9 (24,2-38,4)
34,0 (31,0-37,0
30-40
Saturated fat, g
- %
8,8 (4,7-12,3)
9,9 (6,3-12,2)
<10
MUFA, g 12,0 (5,5-14,0) -
PUFA, g 6,0 (3,3-8,4) -
Mo: months, KJ: kilo joule, Kcal: kilocalories, MUFA: Mono Unsaturated Fatty Acid, PUFA:
Poly Unsaturated Fatty Acid
1Values presented as median (25 th – 75 th percentile)
2Recommended intakes for infants aged 12-23 months from the Nordic Nutrition
Recommendations 2012 (21).
4.3.2 Intake of micronutrients
For the infants being partially breastfed the median intake of most micronutrients from solid
food exceeds the recommended intake level for infants aged 6-12 months (64), as shown in
table 12. When supplements are included there was a significant difference in the intake of
salt per MJ, vitamin A, D, E, C, B6, thiamin, riboflavin, niacin, and folate. The median iron
and zinc intake was below the recommended intake level, and there was not a significant
contribution of supplements of these nutrients.
40
Table 12 Intake of micronutrients from complimentary foods in partially breastfed infants.
With
supplements
n:22
Without
Supplements
n:24
P1
Recommended
Intake2
Median (25 th-75 th percentile)
Age, mo 8,0 (7,0-11,0)
Sodium, mg 234,3 (106,7-
388,1)
225,3 (93,8-
362,8)
1,000 -
Salt, g/MJ 0,3 (0,2-0,5) 0,3 (0,2-0,5) 0,023 <0,5
Vitamin A,
RAE
399,2 (257,9-
686,9)
310,0 (233,2-
455,7)
0,001 300
Vitamin D, µg 13,4 (9,4-16,8) 3,1 (1,6-5,7) <0,001 10
Vitamin E,
αTE
8,8 (3,1-15,2) 3,0 (2,2-5,1) <0,001 3
Thiamin, mg 0,7 (0,4-1,0) 0,6 (0,3-0,7) 0,003 0,4
Riboflavin, mg 0,6 (0,3-1,4) 0,4 (0,3-0,8) 0,004 0,5
Niacin, NE 7,0 (4,1-11,8) 4,6 (2,9-7,1) 0,011 5
Vitamin B6,
mg
0,8 (0,4-1,3) 0,5 (0,3-0,7) 0,004 0,4
Folat, µg 85,2 (60,7-130,2) 64,0 (34,1-96,6) 0,005 50
Vitamin B12,
µg
0,9 (0,5-1,6) 0,8 (0,4-1,5) 0,414 0,5
Vitamin C, mg 76,6 (60,8-90,5) 48,2 (24,7-69,3) 0,002 20
Calcium, mg 300,3 (149,7-
431,6)
263,2 (167,8-
373,3)
1,000 540
Iron, mg 7,2 (3,8-9,7) 6,1 (3,6-8,4) 0,273 8
Zinc, mg 4,3 (2,6-5,5) 3,5 (2,4-5,2) 0,593 5
Iodine, µg 26,6 (11,9-47,6) 24,1 (13,3-42,7) 0,593 50
Selenium, µg 10,0 (3,6-15,2) 8,8 (4,0-12,7) 1,000 15
Copper, mg 0,5 (0,2-0,6) 0,4 (0,2-0,6) 0,593 0,3
Phosphorus,
mg
372,5 (196,0-
471,2)
300,5 (212,7-
440,2)
0,655 420
Magnesium,
mg
87,7 (52,8-118,8) 83,8 (44,7-112,5) 0,593 80
Potassium, mg 865,8 (477,9-
1156,1)
829,0 (474,4-
1140,3)
0,593 1100
Mo: months, MJ: Mega Joule, RAE: Retinol Activity Equivalents, αTE: α-Tocopherol
Equivalents
1Wilcoxen signed-rank test for two related samples.
2Recommended intakes for infants aged 6-11 months from the Nordic Nutrition
Recommendations 2012 (21).
41
The children that were not breastfed had median intakes for all micronutrients that matched
the recommended intakes for children aged 12-24 months (21), except for iodine, selenium
and potassium, as shown in table 13. There was a significant contribution to total intake from
supplements for vitamin D, E, C, B6 and thiamin.
Table 13 Total micronutrient intake in non-breastfed infants.
With supplements
N: 17
Without
supplements
N:21
P for
difference1
Recommended
intake2
Median (25 th-75 th percentile)
Age, mo 12,0 (6,5-16,5)
Vitamin A,
RAE
540,6 (447,1-
941,0)
521,7 (318,5-808,5) 0,237 300
Vitamin D, µg 15,4 (13,3-18,4) 7,5 (5,3-9,2) <0,001 10
Vitamin E, αTE 8,0 (6,0-13,9) 6,3 (5,8-8,0) 0,017 4
Thiamin, mg 1,0 (0,8-1,1) 0,8 (0,7-1,0) 0,046 0,5
Riboflavin, mg 0,9 (0,7-1,4) 0,9 (0,7-1,1) 0,116 0,6
Niacin, NE 10,6 (8,7-12,2) 9,8 (6,7-11,1) 0,144 7
Vitamin B6,
mg
0,9 (0,7-1,2) 0,8 (0,5-0,9) 0,046 0,5
Folate, µg 99,7 (85,2-158,0) 94 (82,7-110,7) 0,173 60
Vitamin B12,
µg
1,7 (0,9-2,8) 1,7 (1,0-2,7) 0,317 0,6
Vitamin C, mg 76,7 (65,8-90,5) 72,7 (52,8-89,5) 0,043 25
Calcium, mg 631,7 (535,3-
693,2)
601,0 (425,2-660,6) 0,180 600
Iron, mg 11,0 (9,0-12,3) 10,1 (7,9-12,3) 0,285 8
Zinc, mg 6,7 (5,6-7,4) 6,5 (5,3-7,4) 0,317 5
Iodine, µg 52,9 (38,5-92,4) 55,4 (38,5-89,1) 1,000 70
Selenium, µg 19,0 (13,5-22,5) 18,0 (14,0-21,5) 0,655 20
Copper, mg 0,6 (0,5-0,7) 0,6 (0,5-0,8) 0,180 0,3
Phosphorus, mg 520,7 (432,0-
686,3)
480,5 (430,3-664,7) 0,317 470
Magnesium,
mg
115,0 (77,6-139,0) 102,0 (77,7-135,5) 0,317 85
Potassium, mg 992,3 (683,7-
1259,8)
987,0 (692,0-
1176,5)
0,180 1400
Mo: months, MJ: Mega Joule, RAE: Retinol Activity Equivalents, αTE: α-Tocopherol
Equivalents
1Wilcoxen signed-rank test for two related samples.
2Recommended intakes for infants aged 12-23 months from the Nordic Nutrition
Recommendations 2012 (21).
42
4.4 Associations between nutrient status, feeding
patterns and nutrient intake
Correlation coefficients for s-B12 and background or dietary factors are shown in table 14 for
all the participants. Kcal per kilo body weight, gram protein per kilo body weight and eHF
intake have a significant positive correlation with B12 status in this population.
Table 14 Factors correlated with s-B12.
r Sig
Age 0,181 0,228
Time on diet -0,019 0,902
Number of symptoms 0,112 0,463
Kcal/kg 0,410 0,007
Protein, g/kg 0,430 0,004
eHF, g 0,341 0,027
Liver patè, g 0,199 0,206
Eggs, g 0,134 0,398
Fish, g 0,164 0,298
eHF: extensively hydrolyzed formula, r: Spearman’s correlation coefficient.
Correlation coefficients for s-ferritin and relevant background or dietary factors are shown in
table 15 for all infants and children. Intake of eHF and fortified porridge are significantly
correlated with iron status. The correlation with porridge is negative. Duration of diet, age,
calories per kilo bodyweight and grams of protein per kilo body weigh are borderline
significantly correlated with iron status.
Table 15 Factors correlated with s-ferritin.
r Sig
Age -0,270 0,067
Time on diet 0,088 0,567
Number of symptoms -0,151 0,316
Kcal/kg 0,272 0,077
Protein, g/kg 0,273 0,073
eHF, g 0,362 0,017
Liver patè, g 0,248 0,108
Fortified porridge, g -0,325 0,034
eHF: extensively hydrolyzed formula, r: Spearman’s correlation coefficient.
43
4.5 Dietary sources of iron and B12
4.5.1 B12 sources
The food sources of B12 in the partially breastfed infants are shown in figures 7-8. The
partially breastfed infants obtain 23% of their total B12 intake from eHF, 22% from fortified
baby porridge and 16% from both liver pate and fish, shellfish and caviar combined. The
contribution from breastmilk is unknown.
Figure 7 Food sources of B12, except for breast milk, in partially breastfed infants.
The non-breastfed infants get a larger portion of their B12 from eHF, 35%. The other large
sources for these infants are fortified baby porridge which contributes to 18% of total intake;
liver pate contributes with 20% and fish, shellfish or caviar with 11%.
Figure 8 Food sources of B12 in non-breastfed infants.
Fortified Porridge
eHF
Fish, shellfish,
caviar
Red meat
White meat
Egg
Soy/oat products
Misc Liver patè
Fortified porridge
eHF Fish, shellfish,
caviar
Red meat
White meat
Egg
Soy/oat products Misc. Liver patè
44
4.5.2 Iron sources
The food sources of iron in the partially breastfed infants are shown in figures 9-10. The
charts are based on the mean contribution from the different foods in the infants that eat those
foods. The partially breastfed infants obtain 39% or their iron from fortified baby porridge,
19% from eHF, 13% from grain products including rice and 8% from fruit or berries and
vegetables, respectively. The contribution from breast milk is unknown.
Figure 9 Food sources of iron, except for breast milk, in partially breastfed infants.
The non-breastfed infants get the majority of their iron, 41%, from eHF. Fortified baby
porridge contributes with 24% of the total intake and grain products including rice contribute
14%. As two infants use iron supplements this source contributes to 6 % of the total intake for
the non-breastfed infants.
Figure 10 Food sources of iron in non-breastfed infants.
F.porridge
eHF Fish, shellfish,
caviar Red meat White meat
Egg Supplement
s
Vegetables
Fruit, berries
Misc.
Grain products
Liver patè
F.porridge
eHF Fish, shellfish,
caviar
Red meat
White meat 15
Supplements Vegetables
Fruit, berries
Misc.
Grain products
Liver patè
45
4.6 Characteristics of the nutrient deficient infants
and children
4.6.1 B12-deficient infants
In table 16 factors related to B12 status is shown for the infants that are B12 deficient. All the
B12 deficient infants were breastfed and the B12 intake from breast milk is not measured in
this study. Three of the B12 deficient infants were only receiving breast milk so no data on
their dietary intake is available. The median intake of calories per kilo from complementary
foods was 31, indicating that these infants receive over half of their daily energy and nutrient
requirement from breastmilk. Twenty five percent of the deficient infants were given an eHF
in addition to breast milk, and they consumed significantly less eHF than the non-deficient
infants and children.
46
Table 16 B12 deficient participants compared with the non-deficient participants.
B12-deficient
infants1 (n:8)
Non deficient
infants (n:395)
P2
Age, mo 6,5 (2,0-11,0) 9,0 (5-23) 0,032
Breastfeeding, %
- Breastmilk
- Breastmilk, solids
- Breastmilk, eHF,
solids
- eHF, solids
- Solids
37,5
37,5
25,0
0
0
0
17,9
33,3
46,2
2,6
s-B12, pmol/L 217,0 (133-278) 482,0 (204-1475) <0,001
p-Hcy, µmol/L
8,2 (6,7-10,9) 5,6 (3,1-11,5) 0,002
s-folate nmol/L 35,5 (24,2-45,0) 40,1 (13,0-45,0) 0,279
B12 supplements,
breastfeeding mothers, %
50 20 0,020
Feeding patterns
Use of eHF 3
, % 25,0 68,4 0,3244
eHF, g 0,0 (0,0-27,0) 33,6 (0,0-126,0) 0,034
Kcal/kg 30,9 (22,2-54,8) 81,9 (28,1-163,2) 0,007
B12 intake, µg 0,5 (0,4-1,0) 1,4 (0,0-5,2) 0,017
Fortified porridge, g 35,0 (0,0-70,0) 18,0 (0,0-138,0) 0,405
Liver pate, g 0,0 (0,0-2,0) 0,8 (0,0-33,0) 0,309
Fish and shellfish, g 0,0 (0,0-33,0) 2,3 (0,0-56,0) 0,313
Most common
symptoms:
- Reflux
- Colic
62,5 %
62,5 %
69,2 %
79,5 %
1,000
0,636
Mo: months, eHF: extensively hydrolyzed formula, Hcy: homocysteine, sTfR: soluble
transferrin receptor, TIBC: Total Iron Binding Capacity, Kcal: kilocalories.
Values presented as median (min – max) or % due to the small sample size in the B12
deficient group.
1Defined as a s-B12 < 300 pmol/L and a p-Hcy > 6,5
2Difference between groups tested with Mann Whitney U-Test for continuous variables and
Fisher’s exact test for categorical data.
3 Defined as using more than 15 grams of eHF powder per day.
4Defied condition, 2 cells have expected value less than 5.
5B12-status is missing for two infants.
47
4.6.2 Iron deficient infants
Table 17 shows characteristics of the iron deficient infants and children compared to the non-
deficient infants and children. In this population 24 % of the infants were categorized as iron
deficient. The iron deficient participants had significantly lower ferritin and higher TIBC than
the non-deficient participants and a borderline significantly lower intake of iron. Seventy-five
percent of the iron deficient infants and children were breastfed compared with 54% of the
non-deficient. The intake of eHF was significantly lower in the iron deficient infants and
children. The iron deficient infants and children had a lower intake of calories per kilo than
the non-deficient participants, indicating that they get more of their energy from breastmilk.
None of the infants with ID used iron supplements.
48
Table 17 Characterization of the iron deficient participants and comparison with the non-deficient.
Iron deficient
Infants1 (n:12)
Non-iron
deficient
infants (n:35)2
P for difference3
Age, mo 8,5 (7,0-10,7) 8,0 (6,0-12,0) 0,980
Breastfeeding, %
- Breastmilk
- Breastmilk and
solids
- Breastmilk, eHF
and solids
- eHF and solids
- Solids
0
33,3
41,7
25,0
0
8,6
17,1
28,6
42,9
2,9
Blood values
s-ferritin, µg/L 17,0 (10,0-23,2) 33,0 (28,0-58,0) <0,001
High sTfR, % 58,3 25,7 0,062
s-TIBC
79,0 (76,0-88,0) 68,0 (62,0-72,0) <0,001
Dietary habits and iron rich food sources
Iron intake, mg 6,8 (4,8-10,5) 8,9 (4,7-10,8) 0,465
Protein, g/kg 1,8 (1,4-2,9) 2,5 (1,4-3,0) 0,386
eHF, g 10,0 (0,0-27,0) 36,0 (1,5-73,1) 0,049
Fortified porridge, g 33,5 (0,0-60,8) 15,3 (0,0-38,0) 0,249
eHF and porridge
combined, g
38,0 (21,7-88,2)
65,8 (32,0-107,4)
0,285
Kcal/kg 51,8 (41,3-88,8) 81,7 (36,3-100,7) 0,330
Mo: months, eHF: extensively hydrolyzed formula, sTfR: soluble transferrin receptor, TIBC:
Total Iron Binding Capacity, Kcal: kilocalories.
Values are presented as median (25 th – 75 th percentile)
1Defined as a s-ferritin <25 µg/L for infants aged 0-11 months and <10 µg/L for children aged
12-24 months.
2Measurement of iron status is missing for two infants.
3Difference between groups tested with Mann Whitney U-Test for continuous variables and
Fisher’s exact test for categorical data.
4.6.3 Association between the B12 and iron deficient participants
There was no significant association between the infants with ID and the B12 deficient
infants, investigated by Fisher’s Exact Test, p=0,374.
49
5 Discussion
In this study 17% of the infants and children were found to be B12 deficient and 24% had ID.
Both deficiencies were most common in the infants less than one year old. Breastfed infants
aged six to eight months were found to have lower serum levels of B12 and higher levels of
Hcy than non-breastfed infants of the same age. Use of an eHF were found to secure nutrient
intake and was associated with better B12- and iron-status in this population with CMPA.
In this section the method and results will be discussed, in that order.
5.1 Subjects and methods
5.1.1 Subjects and study design
This study is a cross sectional study. These studies are not designed to determine causality but
are suitable for describing a population and generating hypotheses for further research (65).
Selection bias occurs when there is an imbalance in the recruitment of participants to a study
(65). In this study all infants attending the MFDC in the inclusion period that fit the inclusion
criteria were invited to participate. Infants and children were also recruited by doctors and
other health care personnel at the hospital that were informed of the study, both at the
outpatient clinic and among inpatients. Since the hospital where the study was performed is
large, all eligible infants and children may not have been referred to a dietitian. Families that
did not speak Norwegian were excluded due to financial limitations and this is a source of
bias.
The parents of the infants in this study are very highly educated. They had an education level
that was higher than in Oslo and more than twice that of the general public in Norway, as
75% had finished higher education (66). This might make the results less generalizable.
A large portion of the participating infants were reported to have colic and trouble sleeping as
symptoms of their CMPA. These conditions were alleviated by the milk free diet for most of
the participants, but there is a risk of selection bias if parents with the most demanding
children said no to participate due to the extra effort required for the study. If the infant had
been admitted to hospital previously or already had undergone blood sampling at the
50
outpatient clinic this was also a reason provided by some parents for not wanting to
participate and put the child through more blood samples. On the other hand, one of the
benefits of participating in the study was blood samples to investigate nutritional status and
extra attention from a dietitian, which could mean that parents who were worried about the
nutritional status of their child or struggled with feeding were more inclined to participate.
The median time the participants had been on the CMPFD at inclusion was almost five
months, but as participants were recruited at the MFDC a few had only been following the
diet for three weeks at inclusion. The median age of the participants was eight months, so
most of them had been on the CMPFD for a substantial part of their life. To study the effect of
following this diet for a long time it would have been ideal if a larger proportion of the
participants had been between 12-24 months old. But as parents in Norway get the first year
off work to take care of their child it is likely that some parents of older infants said no to
participate due to being back at work and having less time during the day to do the dietary
registration and meet at the hospital for blood samples. The infants that had been on the
CMPFD for some time were also harder to reach for the study staff, as they are normally not
followed further by doctors at the outpatient clinic.
Most of the participants were recruited after the parents had received guidance from a
dietitian about how to secure the infants’ nutritional intake when excluding cow’s milk. This
is probably the strongest factor that might make the results from this study less generalizable
to infants who are on a CMPFD without proper guidance. Infants and children with CMPA
are commonly found to have poorer nutritional status than healthy infants but dietary
counseling by a dietitian can secure adequate nutrition even with these dietary restrictions (18,
67, 68). This is probably the case in this study and it would have been interesting to also have
dietary records from the period before the MFDC if a CMP-free diet was initiated beforehand.
5.1.2 Strengths and limitations of the method
A limitation in this study is the lack of a healthy control group for comparison of the findings,
as there is little data especially on B12 status in infants in Norway. This was not possible due
to practical and financial reasons.
Dietary intake was assessed by a three day dietary record completed by the parents. It is
challenging to assess intake in infants since they are messy eaters. To simplify the procedure
51
participants were allowed to use household measurements and did not need to weight all
foods eaten. Some parents chose to weigh the food before serving it and weigh the remaining
food after the meal and some only gave vague approximations of serving sizes, e.g. one
serving of breakfast cereal. This complicated the calculation of nutrient intake and is a
possible source of error in the data material.
A systematic review of dietary assessment methods in children found that assessment of
dietary intake by weighed food record provides a better measure of energy intake than doubly
labeled water in children between 0,5-4 years old (69). This indicates that the method used in
this study provides a good estimation of intake, although it was not a weighed dietary record.
The number of days needed to obtain an accurate measure of intake in infants has been
evaluated by Erkkola and colleagues (49). They found that the variation in intake increases
with age and that three days record provide an accurate measure of intake in 1-year old
children. As the mean age of the infants in our study is less than one year it is reasonable to
assume that the dietary record provides a good estimation of intake in this population. A
picture pamphlet showing different serving sizes of common infant foods might have made
the estimation easier for the parents.
Dietary intake data is only complete for the infants that were not breastfed during the study.
There are ways to approximate dietary intake from breast milk, for example by recording the
number of feedings per day, the length of the feedings or by subtracting the energy intake
from food from the estimated daily requirements of the child and assume the remaining
energy intake equals that from breast milk. In this study we chose not to approximate the
intake from breast milk as all these methods have great room for error. For example; the
nutrient content of the breast milk varies throughout the breastfeeding period, as is the case
with zinc (19). The nutritional quality of the breast milk is also dependent on the adequacy of
the mothers’ diet and being on a milk free diet might affect this. Breast milk from women on
a vegetarian diet have been shown to have a lower B12 content than that of omnivorous
women (70).
The questionnaires on diet and background information used in this study had not been
validated before use. Therefore there is a risk of the questionnaire not measuring exactly what
it was designed to, i.e. lack of validity. For this paper data was primarily obtained from the
questionnaire on background information and validation is probably of less importance for
52
that kind of data, for example age, current breastfeeding, symptoms and parental education,
than for the questionnaire on diet.
The dietary records were entered into “Kostholdsplanleggeren” by the master’s student and an
experienced pediatric dietitian working in close proximity to one another. The fact that only
two people were involved and that one of them is an experienced pediatric dietitian increases
the quality of the data on dietary intake.
Symptoms of CMPA were reported by parents and were not necessarily confirmed by a
doctor. It can be difficult for parents to assess for example whether or not the child has reflux,
unless a doctor has diagnosed the condition. Another general symptom that was asked about
was trouble sleeping. This is also a very subjective symptom the parents’ answers to this must
be interpreted with care.
5.1.3 Statistics
Due to the small sample size in this study, n=49, non-parametric statistics were used. When
there were outliers in the data material, statistical analysis were performed with and without
these, and if no considerable difference was found they were included in the analysis. The
study was powered to describe the population, but not for subgroup assessments like the
difference between boys and girls or between different diet groups. Therefore there is a risk of
type II errors where true differences are not found.
5.2 Discussion of results
5.2.1 B12-status
Cutoff points and reference intervals
One of the main objectives in this thesis was to investigate B12-status in this population with
CMPA, as milk products previously has been shown to be the most important source of B12
for young infants in Norway (23). This is complicated by the fact there are no widely
accepted guidelines for diagnosis of B12-deficiency in infants, and even less firm evidence
for determining cutoffs for subclinical deficiency (29). The biochemical measurements used
to determine B12-status are s-B12, p-Hcy and s-MMA. MMA is more strongly correlated
53
with B12-status in adults than in infants where Hcy has been shown to be the strongest
predictor of B12-status (25).
The laboratory at OUS states that the reference interval for s-B12 is 150-650 pmol/L for all
ages (56). They state that s-B12 is at its lowest the first year of life. Values <100 pmol/L can
be considered as definitive B12-deficiency. Values between 100-300 pmol/L represent a grey
area with the possibility of a functional B12-deficiency and Hcy and MMA should be
measured in such cases. The reference intervals recommended by the Norwegian Paediatric
Association states that for infants 0-12 months s-B12 should be between 99-745 pmol/L and
for children 1-9 years the reference interval is 278-1115 pmol/L (54). They also state that
values <250 pmol/L should lead to further investigation by measuring Hcy and MMA in
infants < 1,5 years old. They provide Hcy >6,5 µmol/L as the limit in younger children that
warrants analysis of s-B12 and s-folate to investigate deficiency. Another Norwegian
guideline published by the Norwegian Association for Medical Biochemistry suggest a
reference interval of 233-883 pmol/L for children 1,5-15 years old (55).
A recent study preformed in Canada is providing updated reference intervals for infants and
children (71). This study found the reference interval for B12 in infants aged less than one
year old to be 140-858 pmol/L and for children 1-9 years old 154-878 pmol/L (72).
Unpublished data from Sørlandet Hospital in Kristiansand, where measurements from 7888
healthy children are included, show that for s-B12 in infants <12 months old the 2,5 percentile
and the 97,5 percentile is between 106-847 pmol/L. For children aged 1-9 years old the
reference interval is 237-1053 pmol/L. Using a statistical approach to compute reference
intervals described by Bolann (73) they suggest a reference interval of 256-1053 pmol/L for
children 1-9 years old.
For this thesis cutoffs for deficiency were chosen based on the most recent research in this
field, conducted at Haukeland University Hospital and the University of Bergen. These
cutoffs are recommended by the Norwegian Paediatric Association and the Norwegian
Association for Medical Biochemistry. Their most noteworthy finding is that a Hcy >6,5
constitutes impaired B12-status in infants. This value represents the 97,5th
percentile post
intervention, a 400µg hydroxycobalamin injection, that was administered to six week old
infants to examine the response in blood markers of B12-status (33). These infants were then
assumed to have an optimal B12 status.
54
The same group also published a randomized placebo-controlled intervention study (RCT) in
2013 showing that in infants aged less than eight months with feeding difficulties, subtle
neurological symptoms and Hcy > 6,5 µmol/L, supplementation with B12 improved motor
development and regurgitations (74). One month after an intramuscular injection with 400 µg
hydroxycobalamin mothers of infants in the intervention group reported a significantly greater
reduction in regurgitations. The score for motor development was also significantly higher in
the intervention group than the placebo group after one month. These findings are interesting
because this was a well-designed study performed on Norwegian infants that have previously
been thought to be B12 replete.
These different cutoffs and age intervals illustrate the challenge facing pediatric dietitians and
pediatricians in determining the right treatment for their patients. The main dispute regarding
B12 status in infancy stems from the fact that several have found low status at various times
during the first years of life, but it is not clear whether this represents a deficiency or is a
normal physiologic finding (23, 25, 75, 76). For instance, Hay et al found that cobalamin
status is strongly correlated with breastfeeding status during infancy (77). In breastfed infants
cobalamin status declined the first year and in non-breastfed infants it increased, but the
researchers found no indication that this low s-B12 had any harmful effect. They speculate
that B12 in breastmilk is more bioavailable or that breastmilk contains factors that lead to
more efficient use of the vitamin. Others have also found that breastfed infants have
biomarkers of B12 status that indicate a lower B12 status than eHF-fed infants have (78).
A Danish research group recently published a study on B12-status in mother, child and the
concentration in breast milk during the first nine months postpartum (76). Measurements were
performed at birth, four months and nine months postpartum. At these times they found no
change in s-B12 in the mothers, but they found a significantly lower content of B12 in the
breast milk and lower s-B12 in the infants at four months than at the other time points. The
infants also had higher MMA levels at four months indicating a functional deficiency. The
mothers in this study were healthy and probably had an adequate B12 status themselves, as
most of them used dietary supplements containing B12 at all time points in the study. They
also found that four month old infants consuming other foods in addition to breast milk had a
better B12 status than the infants only receiving breastmilk. These data indicate that exclusive
breastfeeding at four months may not provide enough B12 to the infant.
55
Results in this study
In this population the median s-B12 was 441 pmol/L. The non-breastfed infants had higher
serum levels than the partially breastfed infants and the three exclusively breastfed infants all
had s-B12 <300 pmol/L suggesting possible deficiency. In the infants between six-eight
months of age, s-B12 was significantly higher in infants receiving an eHF and solid foods
with and without breast milk, and Hcy was significantly lower in the infants not receiving
breastmilk.
S-B12 alone is not a good measure of B12-status Hcy was also measured in this study.
Seventeen percent of the infants had both low s-B12 and increased Hcy, and are classified as
B12 deficient. All of the exclusively breastfed infants, 21% of the partially breastfed infants
and none of the non-breastfed infants were B12 deficient. Only two of the deficient infants
and children were given any eHF. The median age of the B12 deficient infants was 6,5
months. These findings correlate well with those of others, particularly Greibe et al (76) who
found a median s-B12 of 240 pmol/L in infants four months postpartum (76). This was
significantly lower than the serum levels at birth and nine months postpartum. The
concentration of cobalamin in breast milk was also lower at four months postpartum than at
the other time points. The median s-B12 of the B12 deficient participants in this thesis was
217 pmol/L.
The median Hcy value in this population was 6,1 µmol/L. Forty percent of the infants had
Hcy > 6,5 µmol/L. According to the research group from Bergen these infants and children all
have an impaired B12 status. The RCT from Bergen was performed on six week old infants
and status was controlled at four months. Since the first years of life are a period of rapid
development it is not sure that this value applies all the way through the first two years of life.
For this study the decision was made to measure s-B12 and Hcy, in accordance with the
guidelines available, and to evaluate the results in our population based on this . Better
designed studies reflecting more age groups are necessary to determine the optimal cutoffs at
different ages.
According to the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER)
database infants aged 0-12 months have Hcy values between 2,8-9,9 µmol/L, and for the ages
one-seven years the reference range is 2,7-7,6 µmol/L. They do not suggest cutoffs for B12
deficiency. A nationwide study on Canadian children’s diets found that less than five percent
56
of children aged 1-3 years had an inadequate B12 intake, indication that deficiency is rare in
this population (79).
Since there was no control group in this study, an effort was made to find data on B12 status
in a comparable population. B12 and Hcy measurements from pediatric patients at
Rikshospitalet, OUS was obtained from Dr. Lars Mørkrid in April 2015. One hundred and
fifty infants under two years of age had both B12 and Hcy measurements available. In this
population the median B12 concentration was 357 (242-522) pmol/L, and the Hcy
concentration was 7,0 (6,0-9,0) µmol/L. These findings are not significantly different from the
findings in this study. These data suggest that the infants with CMPA in this study do not
have a different B12-status than other infants referred to hospital in Oslo, although this is not
a perfect reference population.
In this study no information on the breastfeeding mothers’ diet except for use of dairy
products and supplements which was recorded. A third of the mothers used supplements
containing B12 and all had excluded or significantly limited milk from their diet. Greibe et al
(76) found that even in mothers who took B12 supplements the breast milk concentration of
B12 decreased at four months postpartum (76). They did not find a correlation between the
dietary intake of B12 and breastmilk B12 at the time points they investigated. As the B12
content of breastmilk was not investigated in this study one can only speculate that these
infants were not receiving enough B12 from the breastmilk. The partially breastfed infants
and children in this study had a median dietary intake of B12 from solid foods of 0,47 µg
which is just below the recommended intake of 0,5 µg, and they would not need much from
breastmilk to meet the recommendation. But it is possible that the mothers in this study have a
lower B12 intake than mothers who consume milk products in greater quantities and thus has
a lower B12 content in their breastmilk.
The study protocol did not include collection of data after the measurements were done. The
infants who were found to be B12 deficient were offered treatment in the form of an
intramuscular injection of 400 µg hydroxycobalamin (Vitamin B12-depot, Takeda Nycomed),
prescribed by a pediatrician. When the research worker called the parents to inform of the
findings, a few of the mothers (data not recorded) were unsurprised and said they had either
had low B12 levels themselves their whole life or been told they did during pregnancy.
Previous research has found that maternal B12 status predicts infant status at six months
postpartum in B12 replete mothers (80). In a study on mothers in Guatemala where B12
57
deficiency is prevalent, Deegan et al (81) found significantly lower levels of B12 in milk from
deficient, 22 pmol/L, or marginally deficient, 25 pmol/L, mothers compared to mothers with
normal B12 status, 63 pmol/L (81). To my knowledge these are the only clinical studies on
the B12 content in human milk, as the high content of unsaturated haptocorrin has hindered
the results of previous assays. The new method for analysis has recently been developed in
Denmark and has been described by Lildballe (82).
All (n=3) of the exclusively breastfed infants, 21% (n=5) of the partially breastfed infants and
none of the non-breastfed infants were B12 deficient. A low B12 concentration in breast milk
can become a problem especially in infants who receive substantial amounts of their nutrition
from breast milk between six and twelve months of age. In this study 50% of the mothers of
B12 deficient infants reported to use supplements containing B12 themselves. Only 20% of
the other breastfeeding mothers took B12-supplements, and this indicates that the mothers of
B12 deficient infants had a poorer B12-status than the other mothers. The use of supplements
was reported before blood samples were drawn and the parents were informed of the results.
Due to the limitations of the method used and the amount of data collected in this study it is
difficult to draw firm conclusions from these findings. But, it seems that breastfed infants on a
CMPFD not receiving an eHF are at risk of low B12 status around 6 months of age.
Folate
A B12 deficiency can cause a secondary folate deficiency because folate is trapped. This is
due to the intersecting roles of these two nutrients in their metabolism. Conversely, treatment
with folic acid can improve symptoms that were actually caused by a B12 deficiency, and
delay the right diagnosis of B12 deficiency (83). Infants are born with large stores of folate,
and folate deficiency is seldom observed in newborns (84). In this study folate intake and
status was adequate. The use of a multivitamin mixture containing folate was also common.
5.2.2 Iron status
In total were 24% of the infants in this study found to have ID based on their ferritin level or
sTfR when ferritin was missing or affected by inflammation. The cutoffs for ID chosen for
this study was <25 ug/L for infants 0-11 months and <10 ug/L for infants 12-23 months old
(54). IDA was present in 4% of the participants. Two more infants, 4%, reported using an iron
58
supplement. This indicates that these two had been diagnosed with ID previously as iron
supplements are not commonly used in healthy infants in Norway.
A cutoff of 30 µg/L for ferritin has been suggested as an action threshold in otherwise healthy
individuals with no active inflammation (85). They also recommend sTfR as an additional
measure in inflammation. Using those criteria in this population of infants 42% can be
classified as iron deficient. Concerns have been voiced against this definition because it might
lead to over diagnosis of ID and under diagnosis of causes of low iron stores other than low
intake (86). As other causes such as myeloplastic syndrome, leukemia or hemolytic anemia
are rare in infants it is unlikely that other illnesses pose a problem in interpreting findings in
this population.
In this study several markers of iron status were analyzed as these are used in clinical practice,
but few are validated in infants and the cutoffs used are generally based on adult data. STfR is
a measure of iron status that is unaffected by inflammation. Unfortunately reference ranges
for infants and children are not provided at OUS, and the adult cutoffs are used in clinical
practice as well as in this study. STfR has been found to improve diagnosis of ID, especially
in the presence of chronic disease or gastrointestinal malignancies (87). Using the adult
cutoffs for s-sTfR set by the laboratory at OUS 38% of the infants have a functional iron
deficit, but only 12% of these also have low ferritin levels. The Paediatric guidelines sites an
article from 2001 (88) investigating sTfR in Finish infants, using the same technique for the
analyses as the laboratory at OUS uses. Based on data from 52 children aged six months to
four years old they suggest a reference interval of 1,5-3,3 mg/L. This is considerably lower
than the adult cutoffs and no difference between boys and girls was found in this age group.
Using this cutoff and excluding the infants younger than six months 94% of the participants in
the present study had increased levels of sTfR. These findings indicate that the iron status in
this population is poorer than what is indicated by the ferritin values.
The most recent study describing iron status in Norwegian infants was performed in Oslo by
Hay et al (45) on infants born in the spring of 1997 and included approximately 250
participants (45). This longitudinal study followed the participants through their first two
years of life. The study showed an increasing incidence of ID with age. Using a cutoff of 15
ug/L for s-ferritin they found an incidence of ID of 6, 21 and 29% at 6, 12 and 24 months of
age respectively. In the present study, a cutoff of 15 ug/L for the infants aged 12-24 months
gives a prevalence of ID of 14%, less than half of prevalence in the healthy population at 24
59
months age. If the same cutoff is applied to the infants between 0-12 months in the present
study, 12% can be classified as ID, which is comparable to the average of the findings at six
and 12 months of age by Hay et al. These data imply that among infants aged 12-24 months
cow’s milk allergic children seem to have a better iron status than healthy infants, but these
data are based on just 14 cow’s milk allergic children. One possible explanation for the lower
prevalence of ID in infants <12 months old could be that many use an iron fortified eHF. In
infants and young children one of the most established risk factors of ID and IDA are high
intakes of cow’s milk (48). Since the infants in this study for the most part use iron fortified
eHF as a cow’s milk substitute this could lead to better iron status after the age of 12 months
when healthy infants usually start drinking cow’s milk in greater quantities.
5.2.3 Nutrient intake
Macronutrients
It’s difficult to discuss the macronutrient intake of the partially breastfed infants and children
as the total intake of energy and nutrients is unknown. Assuming that the infants meet their
total energy requirements they obtain about 60% of their nutrition from complimentary foods.
They meet their protein requirements from complementary foods as a common rule of thumb
is that infants and children older than six months of age need 1,5 g protein/kg, and the infants
in this study get about 1,7 g/kg. The infants also have a low intake of added sugar, as sugar
contributes to less than 1% of energy intake from solid food and breastmilk contains none.
The infants and children in the non-breastfed group have a median age of 12 months. The
intakes of macro- and micronutrients in this group is compared to the recommendations from
NNR (21) for children from 12-23 months of age. This choice was made so that any results
would not be missed and because the recommendations are quite similar.
The total energy intake of the non-breastfed infants and children was 94 kcal/kg which
exceeds the recommended intake of 80 kcal/kg for this age group (21). This could be because
the parents know from the MFDC that being on a CMPFD poses a risk of malnutrition, and
they put in the extra effort to make sure their child eats enough. They might do this all the
time, or they might have been more aware during the dietary registration, in which case the
high energy intake does not reflect the habitual intake. The percentage of energy from
carbohydrates, protein and fat are within the recommended ranges.
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The median age of the non-breastfed infants was 12 months, therefore the intake data from
this group can be compared to data from “Spedkost 12 måneder”, a nationwide study with
1635 12 month old participants (13). Dietary intake in “Spedkost 12 måneder” was registered
via a food frequency questionnaire (FFQ) which has been shown to overestimate intake (89).
Because of this and because of the small sample size in the present study the comparison
should be performed with some care. In “Spedkost 12 måneder” the average contribution to
total energy intake was 13% from protein, 32% from fat, 12% from SFA, 52% from
carbohydrates and 4% from added sugar. In this study the median contribution to total energy
was 12% from protein, 34% from fat, 9% from SFA, 52% from carbohydrates and 0,5% from
added sugar. From these data it looks like the non-breastfed children in this study have a
healthier diet with regards to macronutrient intake than the non-allergic children in “Spedkost
12 måneder” as the contribution to energy from SFA is below 10% and the intake of added
sugar is lower.
Micronutrients
The partially breastfed infants have a median intake of micronutrients, including supplements,
that meets their daily requirements for most nutrients. The exceptions are; calcium, iron, zinc,
iodine, selenium, phosphorus and potassium. Without supplements they also come out short
of the requirement for Vitamin D, riboflavin and niacin. Seeing as these infants also obtain
nutrients from breast milk these findings are probably not a concern, except for iron and zinc.
Four to six months postpartum the iron stores of the infant becomes depleted and at this time
breast milk is a poor source of both iron and zinc (64). As 36% of the infants in the partially
breastfed group have ID it is reasonable to assume that many do not obtain their
recommended intake of iron. Fortified porridge is the greatest source of iron for these infants.
There is only one milk-free iron fortified baby porridge available in Norway today (Sinlac,
Nestlé). It is therefore possible that parents limit the use of this to create variety or reduce the
sweetness in their child’s diet, but at the same time reducing the iron intake of their child.
Another possible reason is that parents hold off on introducing new foods that can be
potentially allergenic, typically fish and eggs, which could be important iron sources in this
age group. The use of liver pate as a source of iron is common in Norway but only contributes
to 3% of iron intake from complimentary foods in this group.
61
The non-breastfed infants’ average intake of micronutrients exceeds all recommended intakes
when supplements are included except for iodine, potassium and selenium. The NFCT does
not have complete information on the iodine content of all food items; therefore the estimated
iodine intake is probably too low. Norwegian children have been shown to obtain 70% of
their iodine from milk and milk products so the low intake could potentially be a problem in
this population (90). The selenium intake is just below the recommended intake of 20 µg/day.
The potassium intake is 70% of the recommended intake level. Dairy products are an
important source of potassium and a possible reason for the low intake (21).
The intake of B12 in this group was almost three times the recommended daily intake and
none of the non-breastfed infants were B12 deficient. The iron intake also exceeds the
recommendation and only 5% of the non-breastfed participants had low ferritin levels
compared to 36% of the partially breastfed participants.
Most of the participants in this study did not have a very low intake of B12 or iron, yet
deficiencies were still prevalent. Recent research suggests that this could be due to increased
intestinal permeability in non-IgE-mediated CMPA (91). The children in that study were
asymptomatic and had been avoiding the allergen for some time yet one third had increased
gastrointestinal permeability. More research is needed concerning this mechanism but it is a
possible explanation for the poor status that was found in many of the infants in this study
even though their nutrient intake was mostly adequate. The high vitamin D status in this study
suggests that the participants do not have a fat malabsorption.
5.2.4 Dietary habits affecting B12 and iron status
As dairy products are an important food group in Norway and milk is added to many common
baby foods an objective of this thesis was to see what these infants actually eat. Emphasis was
put on sources of B12 and iron.
B12
eHF was the greatest source of B12 in this population. It was the only food item to have a
significant correlation with s-B12. This is probably because the eHF is a quantitatively
important food in this age group and that the majority of the participants in this study
consume it. None of the dietary supplements used by the infants provide B12.
62
The second most important source of B12 in this population was fortified baby porridge for
the partially breastfed infants and liver pate for the non-breastfed infants. Seafood was the
third most important iron source for the partially breastfed infants and fortified porridge was
third for the non-breastfed infants.
Hay et al (23) performed a study on healthy two year old Norwegian children and found that
they obtain 43% of their B12 from dairy products, 25% from meat products including liver
pate, 11% from fish and 7% from supplements. The non-breastfed infants in this group,
though on average a year younger, obtain 35% from eHF, 20% from liver pate, 18% from
baby porridge and 11% from fish, shellfish and caviar. It appears that the infants in this study
successfully replace cow’s milk with eHF and soy or oat products which added together
provide 41% of the total B12 intake. None of the infants used supplements containing B12.
The lower age of the infants in this study probably explains the high contribution from baby
porridge.
A significant correlation was found between both kcal/kg and gram protein/kg and s-B12.
This could mean that eating enough food helps secure B12-status but it could also be that
breastfeeding is a confounding factor. The correlation disappears when the analysis is
performed with just the non-breastfed infants (data not shown).
It’s important to keep in mind that the majority of the parents in this study had been educated
by a pediatric dietitian about nutrition in CMPA at the MFDC. Emphasis is put on the
importance of introducing an eHF to supplement breastfeeding and to replace cow’s milk if
the allergy persists after cessation of breastfeeding. The use of a suitable formula is
recommended by ESPGHAN (6).
It’s possible that parents who are not educated about the importance of an eHF on a CMPFD
do not include this in their child’s diet. In Norway these products are available in pharmacies
but a prescription from a pediatrician is required to get them free of charge. Replacing cow’s
milk with a B12 fortified soy, oat, or rice based product would provide a substantial amount
of B12 but not all the other nutrients found in an eHF and therefore pose a risk of
malnutrition.
Iron
63
The partially breastfed infants get the majority of their iron from fortified porridge, eHF and
grain products in that order. The most important sources for the non-breastfed infants were
eHF, fortified porridge and grain products.
The partially breastfed infants also obtain 8% of their iron from fruit and berries. This should
also increase the absorption of non-heme iron from grains and therefore contribute positively
to iron status. Liver pate is generally mentioned as an important iron source for Norwegian
infants and children but only contributes with 3% of the iron from complementary foods in
this population. This is probably because less than half of the partially breastfed infants use it.
For the non-breastfed infants and children the contribution to total iron intake from foods can
be compared with data from “Spedkost 12 måneder” (13). In “Spedkost 12 måneder” fortified
porridge contributed to 39%, eHF 15%, bread 14% and meat products including liver pate 9%
of total iron intake. The infants and children in our study obtain 41% of their iron from eHF
making it the most important source. Formula is iron fortified with 2,0-8,5 mg/L (37). It has
also been shown that iron from fortified powdered cow’s milk is more bioavailable than iron
from red meat in infants (92). This underscores the importance of this food to maintain a good
iron status in the early years of life. The negative significant correlation found between intake
of fortified porridge and ferritin could be due to porridge being more frequently used in the
younger infants that were also found to have a poorer iron status in this study. It could also be
that parents feed their child with more porridge. which is known as a good iron source, if they
are worried about their child’s iron intake.
For the non-breastfed infants and children supplements contributed to 6% of total intake as
three infants use iron supplements in doses higher than what they could obtain from food
alone.
5.2.5 Who are the infants at risk of deficiency?
It has recently been shown that infants and children with non-IgE mediated food allergy
struggle to meet their nutritional requirements without an eHF in the diet (93). This is the
main finding in this population as well.
B12
64
The infants characterized as B12 deficient are significantly younger than the rest of the
population. This coincides with the feeding patterns, where the deficient infants all receive
breast milk, and to a lesser degree an eHF. The amount of breastmilk used can be
approximated from the dietary registrations via the calories consumed per kilo body weight.
Infants between 0-24 months need approximately 80 kcal/kg/day (21). The B12 deficient
infants consume almost 40% of their energy from complimentary food, and the non-deficient
infants and children receive 100%. The deficient infants have a B12 intake that is
significantly lower than the non-deficient infants but seeing as they rely so heavily on breast
milk for their nutrients, and the B12 contribution from this source is unknown, it is not
possible to read too much into this value.
The deficient infants have a significantly lower intake of eHF than the non-deficient infants
do. This is not surprising as they are younger and therefore likely to receive more breast milk.
This finding coincides with the findings by Hay et al (77), who found that breastfed infants
have a lower B12 status than non-breastfed infants (77). The intake of fortified porridge in the
deficient infant is higher, but not significantly, than it is in the non-deficient infants and
children. This could be because the infants are younger, as fortified porridge is one of the
most important sources of B12 in this population.
To eliminate the effect of age and eating development a subgroup analysis was performed on
the infants aged six to eight months as this constitutes 40% of the study participants. This
analysis underscores the differences in B12 status with regards to breastfeeding, because the
breastfed infants had significantly lower B12 than the infants not receiving breast milk. The
Hcy is significantly higher in the infants not receiving any eHF. Whether this means that the
breastfed infants have a lower B12 status or that this is a normal finding and breast milk
contains other bioactive compounds that secure status is currently unknown. It is however
clear, and has been shown previously (77), that the inclusion of an eHF and/or cessation of
breastfeeding leads to a biochemical profile that indicates a better B12 status. This has also
been shown for B12 supplements (74).
As this is a cross sectional study firm conclusions about causality from these findings should
not be drawn. But, it seems that infants with CMPA between six and twelve months of age
who receive a large part of their nutrition from breastmilk are at risk of low B12 status.
65
Iron
The infants and children with ID are similar in age to the rest of the population in this study,
making the comparison of the two groups easier. The majority of the iron deficient
participants were in the feeding category “breast milk, eHF and solids”, while the majority of
the non-deficient participants received eHF and solids. The amount of eHF used was the only
dietary variable that was significantly different between the iron deficient and the non-
deficient participants. The iron deficient participants had significantly lower serum levels of
ferritin, sTfR and TIBC.
The iron intake from foods other than breastmilk was not different between the groups. The
only food component to significantly differ between the deficient and the non-deficient
participants was eHF. The intake was significantly higher in the non-deficient infants, which
coincides with eHF being one of the two most important sources of iron in this population.
Known risk factors of low iron status in early life include; mother smoking (42), early cord
clamping (94), low birth weight (44), male sex (95), insufficient intake of iron rich foods after
six months age (48) and high intake of cow’s milk (96). None of the mothers in this study
reported smoking at present, making it unlikely that they did during the pregnancy. Only one
participant had a birth weight <2500 g, which is the common cutoff for low birth weight, and
this participant also had ID. Delayed cord clamping is recommended by the Norwegian
Association of Gynecologists (97) but whether or not this was performed in the participants in
this study was not registered. None of the infants in this study consume cow’s milk so this
risk factor does not apply to them. As the eHF provided 19% of the iron in partially-breastfed
infants and children and 40% in the non-breastfed this was a quantitatively important source
of iron in this population, and the only food where there was a significant difference in intake
between the iron deficient participants and the iron replete participants.
5.2.6 Clinical implications
The results from this study show that infants’ and children whose parents have been to a
MFDC led by a pediatric dietitian meet their requirements for energy and most nutrients. The
use of vitamin D supplements was common and this dietary guideline seems to be
implemented well in this population. The only exception was infants and children that still
receive the majority of their nutrition from breast milk around six months age, who are at risk
66
of B12 deficiency and ID. An eHF seems to protect against nutrient deficiencies and the
infants or young children that refuse this should be followed closely by a dietitian to
determine if nutritional needs are being met. If they are not, nutrition therapy should be
tailored to meet the child’s needs.
The results of this study and others suggest that B12 deficiency is a condition that needs to be
considered in infants and young children that receive a substantial amount of their nutrition
from breast milk after six months age. Infants and children that are breastfed by mothers with
insufficient B12 status should also be investigated. Infants of deficient mothers should
probably be supplemented with B12 to make sure they meet their requirements. Based on the
results of others, it seems that investigating biochemical markers of B12 status in infants who
have symptoms such as delayed neurological development and feeding difficulties is
indicated. Treating these infants with B12 is a low risk intervention that could be an
inexpensive and easy solution to these problems for the infant, parents and medical
professionals involved, but more research is needed to confirm these findings.
ID is common in Norwegian infants and children. In infants and children with CMPA ID
seems to be more prevalent in the first year of life than in the second, probably because the
use an iron fortified eHF is more common in these children than in children consuming cow’s
milk. Dietary guidance with an emphasis on iron rich foods such as fortified baby porridge
should therefore be a part of the nutrition therapy. Infants and children who are picky eaters
and do not consume an eHF should have their iron status investigated. Liquid iron
supplements are indicated if ID has been diagnosed.
Based on the results from this study it seems that dietary advice regarding CMPA should
focus on introducing an eHF because it is better tolerated when introduced early (8). The
mother does not have to stop breastfeeding, but including formula as a supplement may lead
to better nutrition over time. Fortified milk free baby porridge is another important source of
nutrients in this age group, and the use of this should be promoted over unfortified porridges
that are more commonly milk free. To secure nutrition status in infants and children excluding
staple foods from their diet, close follow-up from dietitians and pediatricians is important and
this has also been found by others (68).
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6 Conclusions
The main objectives of this thesis were to investigate B12 and iron status in infants on a
CMPFD, and the dietary habits that influence these nutrients. The conclusions that can be
drawn from this study are summarized below.
Twenty-one percent of the participants were found to have s-B12 < 300 pmol/L.
Seventeen percent also had p-Hcy > 6,5 µmol/L which is diagnostic of B12
deficiency. Half of these participants, 8%, had a s-B12 below 200 pmol/L which
constitutes a true deficiency and the other half had values between 200-300 pmol/L
which is indicative of a subclinical deficiency.
Twenty four percent of the infants had ID. The prevalence of low iron status is
probably higher, based on the sTfR values.
The participants met their requirements for most nutrients. The partially breastfed
infants had a low intake of iron from complimentary foods and the majority probably
do not meet their requirements. In the non-breastfed infants intake of both B12 and
iron reached the recommended daily intake.
The foods contributing substantially to B12 intake were eHF, fortified porridge, liver
pate and fish. The foods contributing to the iron intake were fortified infant porridge,
eHF and grain products.
Use of an eHF was positively associated with both B12 and iron status and should be
recommended to infants and young children with CMPA. Infants and children with
CMPA that do not consume any eHF should be referred to a pediatric dietitian.
Infants receiving a substantial amount of their nutrition from breastmilk between six
and twelve months age are at increased risk of deficiency of B12 and iron.
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7 Future perspectives
As this study had a cross sectional design without a control group several observations need
confirmation from better-designed studies before conclusions can be drawn. Inclusion of
participants is ongoing and may provide more robust data at a later time. More participants
would allow for a multiple regression analysis that could provide more information about
exactly which dietary factors impact nutrient status. Another master thesis is looking at iodine
status and growth which are other relevant areas to investigate in this population.
The need for studies to determine reference intervals in infants and children is urgent, as is
evident from this study. The fact that using reference intervals from the most recent studies on
sTfR in children classifies > 90% of the participants as having too little iron available to the
cells compared to 40% when using the adult cutoffs illustrates this well. This means that the
findings regarding B12 and iron status are difficult to interpret without a reference population
and the inclusion of a control group would strengthen the findings in this study.
If we consider this present study a pilot study, future research should include nutrient status in
the breastfeeding mothers, dietary records of their intake as well as the nutrient concentration
in breast milk. To my knowledge this has not previously been investigated in breastfeeding
mothers on a milk free diet. Symptoms before and after B12 treatment of deficient individuals
should also be recorded. Inclusion before birth is difficult as CMPA is only present in
between two and three percent of infants, but the outcome could be included in a larger study
on maternal and infant nutrition. A multicenter design would also be preferable, as the
population from central Oslo probably does not represent the population in the rest of the
country.
The most important factor limiting the generalizability of the findings in this study is the fact
that the parents had been to the MFDC and this probably improved their children’s diet.
Comparing the findings in this population to those in allergic infants who have not been
evaluated by a pediatric dietitian would be very interesting as this probably is the case for
most infants on a CMPFD. Showing a discrepancy between these groups in a Norwegian
population of infants could potentially lead to better care for allergic infants and children and
increased use of pediatric dietitians.
69
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75
Appendices
76
Appendix 1 Study invitation
1
Forespørsel om deltakelse i forskningsprosjektet
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
Bakgrunn og hensikt
Dette er en forespørsel om å delta i en forskningsstudie for å kartlegge kosthold, vekst og
ernæringsstatus hos barn under 2 års alder som følger melkeproteinfri diett. Melk og andre
melkeprodukter er viktige kilder til mange næringsstoffer. Vi ønsker å øke kunnskapen om
hvordan diett påvirker barnets inntak av næringsstoffer, deres vekst og ernæringsstatus.
Studien vil kunne gi viktig informasjon som kan bidra til å forbedre behandlingen av barn
som må følge diett.
Studien er forankret i Kvinne- og barneklinikken, og Oslo universitetssykehus HF er ansvarlig
for studien sammen med Avdeling for Ernæringsvitenskap ved Universitetet i Oslo. Studien
vil ledes av kliniske ernæringsfysiologer og leger ved sykehuset.
Hva innebærer studien?
Inklusjon i prosjektet vil skje i forbindelse med at barnet er til undersøkelse ved sykehuset.
Kravet for å få være med er at barnet følger melkeproteinfri diett. Når barnet har fulgt dietten i
minst 3 uker kan kostholdet og urinen undersøkes. Studien er knyttet til behandling ved
Kvinne Barn klinikken. Opplysninger vil i tillegg til det dere oppgir på Skjema om
bakgrunnsinformasjon, også hentes fra barnets journal (f.eks om symptomer,
blodprøvesvar) av prosjektmedarbeidere.
Kostholdsundersøkelse: Barnets kosthold må registreres nøye (med husholdningsmål, dvs
med f.eks 200 ml morsmelkerstatning (Althera, blandet etter oppskrift på pakken)) i tre dager.
Alt som spises og drikkes må skrives ned. Det deles også ut et spørreskjema om barnets
vanlige kosthold, inkludert noen spørsmål om mors kosthold dersom barnet ammes. Dette
skjemaet returneres i posten sammen med urinprøvene.
Urinprøver: Vi trenger to urinprøver fra barnet. Disse tas helst to av de samme dagene som
kostholdet registreres. For å samle urin legges et innlegg (bind) i bleien. Når barnet har tisset
tas dette ut og urinen suges ut vha en sprøyte og samles i et urinprøveglass. Dette kan gjøres
hjemme. Utstyr og frankert konvolutt deles ut ved sykehuset og prøven sendes tilbake til
studiekoordinator i den frankerte konvolutten.
Blodprøve: En blodprøve tas for å få svar på hvordan barnets nivå av jern, vitamin D og
noen få andre næringsstoffer og stoffskifteprøver er. Prøven tas som hovedregel samtidig med
andre prøver legen ønsker i forbindelse med utredning på sykehuset, eller dere kommer til
Barneklinikken og tar prøvene når det passer best for dere.
Vekst: Vekst kartlegges ved at vekt, lengde og hodeomkrets måles på nytt dersom disse ikke
allerede er målt. Dette kan skje enten på helsestasjonen eller på sykehuset.
2
Mulige fordeler og ulemper
Prøvene som tas er relevante i forhold til barnets alder og aktuell diett. Derfor vil mange barn
som undersøkes på sykehuset ta disse prøvene rutinemessig og vi ber da om å bruke
prøvesvarene til denne undersøkelsen. En mulig ulempe kan være at barnet må avgi en ekstra
blodprøve. Fordelen er at dersom blodprøvene skulle vise avvik på verdier vil dere få beskjed
om dette, samt råd om behandling. Ved problemer med dietten vil dere få direkte tilgang til
klinisk ernæringsfysiolog som kan besvare spørsmål.
Hva skjer med informasjonen om deg? Informasjonen som registreres om barnet skal kun brukes slik som beskrevet i hensikten med
studien. Alle opplysningene vil bli behandlet uten navn og fødselsnummer eller andre direkte
gjenkjennende opplysninger. En kode knytter barnet til opplysningene gjennom en navneliste.
Det er kun autorisert personell knyttet til prosjektet som har adgang til navnelisten og som kan
finne tilbake til barnet. Navnelisten vil slettes innen 31.12.28. Det vil ikke være mulig å
identifisere barnet når resultatene presenteres/publiseres.
Frivillig deltakelse
Dersom du/dere ønsker at barnet deltar, undertegner du og den du eventuelt deler
foreldreansvaret med samtykkeerklæringen på siste side. Vi ber også om at ammende mødre
signerer samtykke da vi har noen få spørsmål omkring kosthold og diett hos ammende mødre.
Det er frivillig å delta i studien. Foreldrene/foresatte kan når som helst, og uten å oppgi noen
grunn, trekke deres samtykke til at barnet deltar i studien eller ditt eget samtykke (ammende
mor). Dette vil ikke få konsekvenser for videre behandling av barnet.
Rett til innsyn og sletting av opplysninger om deres barn og sletting av prøver
Hvis du/dere sier ja til å delta i studien, har du/dere rett til å få innsyn i hvilke opplysninger som er
registrert om deres barn/deg (mor). Du/dere har videre rett til å få korrigert eventuelle feil i de
opplysningene vi har registrer. Dersom du/dere trekker barnet/mor fra studien, kan du/dere kreve å få
slettet innsamlede prøver og opplysninger, med mindre opplysningen allerede er inngått i analyser
eller brukt i vitenskaplige publikasjoner.
Kontakt informasjon:
Dersom du/dere har noen spørsmål, kan du/dere kontakte klinisk ernæringsfysiolog:
Janne Kvammen, Kvinne- og barneklinikken, Oslo universitetssykehus HF, 0027
Oslo. Tlf: 23015744. ([email protected])
Rut Anne Thomassen, Kvinne- og barneklinikken, Oslo universitetssykehus HF, 0027
Oslo. Tlf: 23015743. ([email protected]).
Med vennlig hilsen
Janne Kvammen Rut Anne Thomassen Mari Eskerud
Klinisk ernæringsfysiolog Klinisk ernæringsfysiolog Masterstudent klinisk
Barneklinikken Barneklinikken ernæring
OUS OUS UiO
Christine Henriksen Jarle Rugtveit Hans Jacob Bangstad
Klinisk ernæringsfysiolog/ Dr Med Professor og Dr Med.
Førsteamanuensis Barneklinikken Barneklinikken
Universitetet i Oslo OUS OUS
Samtykke til deltakelse i studien
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
Jeg/vi gir min/vår tilslutning til at barnet
kan delta i studien
Mor: Far:
(Signert av de foreldre/foresatte som har foreldreansvar, dato)
Jeg er villig til å delta i studien da jeg ammer barnet som deltar i studien
__________________________________________________________________________
(Signert av mor, dato)
Jeg bekrefter å ha gitt informasjon om og innhentet samtykke til studien
___________________________________________________________________________
(Signert av prosjektmedarbeider, dato)
Barnets navnelapp
(navn, adresse, personnummer)
Appendix 2 Consent form
Appendix 3 Questionnaire on background information
Kosthold, vekst og ernæringsstatus hos barn Idnr som følger melkeproteinfri diett
1
Spørreskjema om bakgrunnsinformasjon
Vi ber deg/dere svare på følgende spørsmål som del av studien ”Kosthold, vekst og
ernæringsstatus hos barn som følger melkefri diett”.
Dato for utfylling av skjemaet: 201
Skjemaet er utfylt av:
Barnets mor Barnets far Både mor og far Annen person
Spørsmål 1: Om diagnosen (sett et kryss)
Barnet har fått diagnosen melkeproteinallergi av lege.
eller
Barnet forsøker diett for å finne ut om det er melkeproteiner som gir plager.
Spørsmål 2: Hvilke symptomer/reaksjon har barnet (eller har barnet hatt) på sin (mulige)
melkeproteinallergi (du kan angi flere symptomer/reaksjoner)?
Reflux/gulp/oppkast Kolikk/magesmerter
Løs mage/diarre Hard mage/forstoppelse
Blod i avføring Spisevegring
Vokser dårlig/øke lite i vekt Eksem/kløe
Puste problemer/anafylaxi Søvnproblem
Ørebetennelse/rennende nese
________________________________________________________________
________________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr som følger melkeproteinfri diett
2
________________________________________________________________
Spørsmål 3: Er barnets plager/symptomer bedre etter oppstart av melkeproteinfri diett?
Ja, helt bra
Ja, noe bedring
Nei, ingen forskjell
Vet ikke
Spørsmål 4: Har barnet noen andre sykdommer?
Ja Nei
Hvis ja, angi hvilke(n):
________________________________________________________________
________________________________________________________________
________________________________________________________________
Spørsmål 5: Foreldrenes sivilstatus:
Gift/samboere med hverandre
Skilt/bor ikke sammen
Spørsmål 6: Foreldrenes etnisitet:
Mor:____________________________________________________________________
Far: _____________________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr som følger melkeproteinfri diett
3
Spørsmål 7: Mors alder:
år
Spørsmål 8: Røyker mor?
Ja Nei
Spørsmål 9: Mors høyeste fullførte utdanning
Mindre enn 9-årig grunnskole 9-årig grunnskole
Videregående skole
Høyskole/universitet inntil 4 år (cand.mag. bachelor, lærer, sykepleier etc)
Høyskole/universitet mer enn 4 år (hovedfag, master, embetseksamen)
Spørsmål 10: Fars høyeste fullførte utdanning
Mindre enn 9-årig grunnskole 9-årig grunnskole
Videregående skole
Høyskole/universitet inntil 4 år (cand.mag. bachelor, lærer, sykepleier etc)
Høyskole/universitet mer enn 4 år (hovedfag, master, embetseksamen)
Kosthold, vekst og ernæringsstatus hos barn Idnr som følger melkeproteinfri diett
4
Spørsmål 11: Er det kjent atopisk sykdom (astma, allergier, eksem) hos foreldre eller
søsken?
Nei
Mor har hatt/har astma/allergi/eksem
Far har hatt/har astma/allergi/eksem
Barnets søsken har/har hatt astma/allergi/eksem
Kosthold, vekst og ernæringsstatus hos barn Idnr som følger melkeproteinfri diett
5
Spørsmålene på denne siden (side 5) gjelder kun hvis barnet ammes nå:
Spørsmål 12: Melk og melkprodukter i mors kosthold
Mor unngår melk og melkeprodukter, selv i små mengder.
Mor bruker bare litt melk og/eller melkeprodukter.
Mor inntar melk og/eller melkeprodukter som vanlig, mengdene begrenses ikke.
Spørsmål 13: Bruker mor kosttilskudd (vitaminer, mineraler, omega-3 etc)?
Ja Nei
Hvis ja, angi hvilke kosttilskudd (produktnavn) og hvor ofte de vanligvis tas i løpet av en uke:
________________________________________________________________
________________________________________________________________
________________________________________________________________
Spørsmål 14: Bruker mor helsekostpreparater (angi produktnavn, f.eks Spirulina)?
Ja Nei
Hvis ja, angi hvilke (produktnavn) og hvor ofte de vanligvis tas i løpet av en uke:
________________________________________________________________
________________________________________________________________
________________________________________________________________
Takk for at du/dere besvarte spørreskjemaet!
Appendix 4 Semi quantitative food frequency questionnaire
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
1
Spørreskjema om kosthold
Vi ber deg/dere svare på følgende spørsmål som del av studien ” Kosthold, vekst og ernæringsstatus hos barn som følger melkeproteinfri diett”.
Dato for utfylling av skjemaet: 201
Skjemaet er utfylt av:
Barnets mor Barnets far Både mor og far Annen person
Spørsmål 1: Får barnet morsmelk?
Ja, bare morsmelk (og eventuelt tran eller annet kosttilskudd) Gå til spørsmål 2
Ja, morsmelk og vann/juice/saft o.l. Gå til spørsmål 2
Ja, morsmelk og fast føde samt eventuelt vann/juice/saft Gå til spørsmål 2
Ja, morsmelk og morsmelkerstatning/annen melk Gå til spørsmål 2
Ja, morsmelk og morsmelkerstatning/annen melk og fast føde samt eventuelt
vann/juice/saft o.l Gå til spørsmål 2
Nei, men barnet har tidligere fått morsmelk Gå til spørsmål 2
Nei, barnet har aldri fått morsmelk Gå til spørsmål 5
Spørsmål 2: Ble barnet fullammet fra fødsel?
Ja Nei
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
2
Spørsmål 3: Hvor mange ganger per døgn drikker barnet morsmelk?
12 ganger eller mer pr døgn
9-11 ganger pr døgn
6-8 ganger pr døgn
3-5 ganger pr døgn
1-2 ganger pr døgn
sjeldnere enn 1 gang pr døgn
Dersom barnet fullammes, gå til spørsmål 23, s 12.
Spørsmål 4: Dersom barnet ikke får morsmelk lenger, hvor gammelt var barnet når det sluttet å få morsmelk?
Måneder
Spørsmål 5: Dersom barnet får annet å drikke enn morsmelk (vann, juice, saft, morsmelkerstatning, melk), hvor gammelt var barnet når annen drikke ble gitt for første gang?
måneder
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
3
Spørsmål 6: Drikker barnet en annen melketype enn morsmelk?
Ja Nei
Hvis ja, hvilke(n) type(r) melk drikker barnet?
Morsmelkerstatning
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Hvor mange ganger per døgn drikker barnet morsmelkerstatning?
Eller
Hvor mange ganger pr uke drikker barnet morsmelkerstatning?
Plantemelk (av f.eks havre, soya, ris)
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Hestemelk, geitemelk, annen melk fra dyr
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
De neste spørsmålene handler om fast føde (mat). Hvis barnet ditt ikke får fast føde, gå til spørsmål 23 s 12.
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
4
Spørsmål 7: Dersom barnet startet med fast føde/mat, hvor gammelt var barnet når fast føde (annen mat enn morsmelk/ kosttilskudd) ble gitt for første gang?
måneder
Spørsmål 8: Hvor ofte spiser barnet industrifremstilt grøt/velling?
3 ganger eller mer pr dag
1-2 ganger pr dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn ukentlig
Aldri
Hvilke(n) type(r) industrifremstilt grøt/velling benyttes (angi produktnavn, f.eks Hipp, Nestle, Semper, Holle)?
_____________________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
5
Spørsmål 9: Dersom barnet spiser industrifremstilt grøt/velling nå, hva slags væske tilsettes vanligvis ved tilberedning, (sett flere kryss hvis flere produkter benyttes)?
Vann
Morsmelk
Morsmelkerstatning
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Plantemelk (f.eks havre, soya, ris)
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Hestemelk, geitemelk, annen melk fra dyr
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Spørsmål 10: Hvor ofte spiser barnet hjemmelaget grøt/velling?
3 ganger eller mer pr dag
1-2 ganger pr dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn ukentlig
Aldri
Hvilke(n) type(r) hjemmelaget grøt spiser barnet vanligvis (angi type grøt, f.eks havregrøt, hirsegrøt)?
_____________________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
6
Spørsmål 11: Dersom barnet spiser hjemmelaget grøt/velling nå, hva slags væske tilsettes vanligvis ved tilberedning. (sett flere kryss hvis flere produkter benyttes)?
Vann
Morsmelk
Morsmelkerstatning
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Plantemelk (f.eks havre, soya, ris)
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Hestemelk, geitemelk, annen melk fra dyr
Hvilke(n) type(r) benyttes (angi produktnavn)?
_____________________________________________________________________
Spørsmål 12: Hvor ofte spiser barnet fisk til middagsmat?
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
7
Spørsmål 13: Hvor ofte spiser barnet fisk som pålegg? (f.eks makrell i tomat, sild, laks)
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Spørsmål 14: Hvor ofte spiser barnet kaviar som pålegg?
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Spørsmål 15: Hvor ofte spiser barnet skalldyr? (f.eks blåskjell, reker, Scampi, kamskjell)
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
8
Spørsmål 16: Hvor ofte spiser barnet rognleverpostei som pålegg? (Svolværpostei-/Lofotpostei)
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Spørsmål 17: Hvor ofte spiser barnet egg som pålegg eller retter med mye egg i (f.eks omelett eller pannekaker med egg)?
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Spørsmål 18: Hvor ofte spiser barnet kylling til middag?
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
9
Spørsmål 19: Tilsettes det salt i middagsmaten barnet spiser?
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Hvilke(n) type(r) salt benyttes vanligvis (angi produktnavn)? (f.eks jozo, jozo med jod, Seltin, havsalt)
_____________________________________________________________________
Spørsmål 20: Hvor ofte tilsettes salt i annen mat enn i middagsmaten til barnet? (f.eks til kokt egg, pålegg, i hjemmelaget grøt)
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Hvilke(n) type(r) salt benyttes vanligvis (angi produktnavn)?
_____________________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
10
Spørsmål 21: Hvilke matvarer gir dere IKKE til barnet?
Glutenholdig korn/mel (hvete, spelt, rug, bygg)
Vanlig kumelk /yoghurt/ost
Appelsin/appelsinjuice/annen sitrusfrukt
Fisk/skalldyr
Nøtter/nøtteprodukter (peanøttsmør o.l.)
Belgfrukter (erter, bønner o.l.)
Egg
Soya
Salt
Matvarer med tilsetningsstoffer
Mat som ikke er økologisk dyrket
Annet ______________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
11
Spørsmål 22: Barna som er med i denne studien er fra 0-2 år. Hva de spiser og drikker vil avhenge av alder. Har barnet problemer i forhold til mat/spising som ikke er naturlige i forhold til barnets alder?
Nei, har ikke noen problemer
Ja, dårlig matlyst/småspist
Ja, liker få matvarer
Ja, vanskelig med tilvenning til familiens kosthold
Ja, allergi/intoleranse mot flere matvarer i tillegg til melk
Oppgi hvilke:
___________________________________________________________________________
Ja, andre problemer
Oppgi hvilke:
___________________________________________________________________________
___________________________________________________________________________
(f.eks problemer med å suge fra brystet, barnet vil ikke ta flaske, svelgeproblemer, brekker seg lett)
Dersom barnet ved noe tidspunkt har vært sondeernært, i hvilken tidsperiode (fra alder- til alder) var dette?
___________________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
12
Spørsmål 23: Hvor ofte tar barnet kosttilskudd?
Hver dag
3-6 ganger pr. uke
1-2 ganger pr. uke
Sjeldnere enn 1 gang pr uke
Aldri
Hvilke(n) type(r) benyttes (angi produktnavn og mengde)?
_____________________________________________________________________
_____________________________________________________________________
(Eksempel Nycoplus multi flytende, Sanasol, Nycoplus multi vitamin og mineraltbl for barn, kalsium 250 mg, kalsium 500 mg, NeoFer (jerntilskudd), omega-3 tilskudd, vitamin C, Møllers tran med sitronsmak)
Spørsmål 24: Tar barnet helsekostprodukter (f.eks probiotika, alger el.) nå (beskriv hvilke produkter og dosering)?
Ja Nei
Hvis ja, hvilke(n) type(r) benyttes (angi produktnavn og mengde)?
______________________________________________________________
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
13
Spørsmål 25: Hvor har du/dere fått informasjon om amming/spedbarnsernæring, og hvordan vurderer du/dere denne informasjonen? Sett et kryss pr rad
Ikke fått informasjon
Svært nyttig Nyttig Lite nyttig Unyttig
Føde-barselavdeling
Helsestasjon
Helsepersonell utenforhelsestasjon/ sykehus f.eks kiropraktor
Homøopat
Familie/kjente
Ammehjelpen
Bøker/oppslagsverk
Aviser/TV/ukeblad
Reklamemateriell
Butikken
Mattilsynets hjemmeside
Helsedirektoratets hjemmeside
Norges astma- og allergiforbund www.naaf.no
www.matportalen.no
Andre internettsider
Klinisk ernæringsfysiolog
Lege
Sykepleier
Kosthold, vekst og ernæringsstatus hos barn Idnr:
som følger melkeproteinfri diett
14
Spørsmål 26: Hvor lang tid måtte du/dere vente på kostholdsveiledning fra det ble bestemt at barnet skulle følge melkeproteinfri diett?
Tid (angi dager, uker, måneder): _________________________________________________
Ta kontakt på telefon 23015744/ 23015743 dersom du har spørsmål.
Tusen takk for at du/dere besvarte spørsmålene!
ID-nummer:
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
1
Kostregistreringsskjema
Alt barnet spiser og drikker i 3 dager registres på dette skjemaet.
Instruksjon: Beskriv all mat og drikke, samt mengdene som inntas så nøyaktig som mulig. Tidspunktet for
inntaket skal også noteres.
Type grøt (f.eks 2 strøkne spiseskjeer Hipp hirsegrøt 6 måneder laget med 40 ml
Althera, ½ dl lettkokte havregryn med 2 dl vann). Angi antall spiseskjeer/ml grøtpulver
og vannmengde/morsmelk/morsmelkerstatningsmengde pr porsjon som ble tilberedt, og
evt hvor stor del av porsjonen barnet spiste opp (f.eks halv porsjon, 2 små teskjeer)
Type brød (f.eks loff, mellomgrovt brød (f.eks hjemmebakt brød), kneippbrød). Angi
mengde som f.eks ¼ skive Speltbrød (Goman) uten skorpe, ca 0,5 cm tykk skive.
Margarin og hvilken type (f.eks Soft spesial, melkefri melange ), tynt lag på brødet.
Type pålegg (f.eks tynt lag kaviar (Mills blå), ½ hardkokt egg, 1 ss jordbærsyltetøy).
Hvordan maten er tilberedt (kokt, stekt, grillet, panert) (kokt fullkornpasta, ½ dl, med
havsalt i kokevannet).
Tilbehør (Toro sjysaus 1 ss, olje/balsamico dressing ½ ts, Mills majones 1 ts, ¼ ts
sukker og 1 ts Soft spesial som smørøye til havregrøten).
Diverse (f.eks 1 stk Marie kjeks, 1 appelsinbåt, 1 plomme, 10 rosiner)
Middagsmat beskrives så godt som mulig (f.eks 1 eggstor kokt potet uten skall, 1
kjøttkake (angi gjerne hele oppskriften på hjemmelagde retter eller f.eks Gilde
ferdigkjøpt), 2 ss hjemmelaget brun saus, 1 cm kokt gulrot. 1 glass Nestle Min første
gulrot potet 4 mnd.
Dersom maten saltes, angi type salt (Jozo med jod, Jozo uten jod, havsalt, Seltin etc)
10 ml Sanasol
Ved amming angis tidspunktet for amming, og om det var før/etter annen mat hvis
barnet spiser begge deler. Angi om du opplever at barnet spiste et fullt måltid, litt etc.
Drikke angis så nøyaktig som mulig (f.eks 50 ml vann, 1 dl Neocate LCP)
Beskriv mengdene på de vanlige glassene/flaskene:
1 flaske = __________ ml
1 glass = ____________ml
Dersom barnet drikker melkeerstatning der man blander pulver og vann, angi hvor mye
pulver du blander i hvor mye vann
Type morsmelkerstatningspulver: _______________________________
Antall måleskjeer pulver ____________ blandets i ___________ml vann
Glem ikke: Det barnet spiser og drikker mellom måltidene.
Appendix 5 Food diary
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
2
Barnehagen må også føre registrering dersom barnet spiser/drikker der.
Bruk ett nytt ark for hver ny dag, og begynn på ny linje for hver matvare.
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
8:00 1/2 skiver Kneippbrød Uten skorpe
1/2 SS Lettmargarin Soft spesial (blå pakke)
1 ss Leverpostei (Go og mager)
4 stk druer
8:20 Morsmelk Ammet ca 20 minutter
10:15 20 ml Vann
Ved spørsmål ta kontakt med
Klinisk ernæringsfysiolog ved Barneklinikken Ullevål:
Rut Anne Thomassen på tlf 23015743
eller
Janne Kvammen på tlf 23015744
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
3
DATO:……………………………………
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
4
DATO:……………………………………
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
5
DATO:……………………………………
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
6
DATO:……………………………………
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
7
DATO:……………………………………
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
8
DATO:……………………………………
TIDSPUNKT
MENGDE
MAT OG DRIKKE
BESKRIVELSE, FOR EKSEMPEL
TILBEREDNING, PRODUKTNAVN
ID-nummer
Kosthold, vekst og ernæringsstatus hos barn
som følger melkeproteinfri diett
9
Egne oppskrifter:
Appendix 6 Growth charts
Kilde: Júlíusson PB, Roelants M, Eide GE, Moster D, Juul A, Hauspie R, Waaler PE, Bjerknes R. Tidsskr Nor Legeforen 2009;129:281-6.
0 1 2 3 4 5 6 7 8 9 10 11 122
3
4
5
6
7
2
3
4
5
6
7
8
9
10
11
12
13
44
46
48
50
52
54
56
58
60
62
64
66
68
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72
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78
80
82
84
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
42
43
44
45
46
47
48
49
50
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
Vekstk
urv
e je
nte
r (0 −
1 å
r), Veksts
tudie
n i B
erg
en, 2
007 P
B J
úlíu
sson, M
Roela
nts
, R B
jerk
nes ©
2007
BG
S1−
20071206N
/0−
1/F
Vekt
(kg
)L
en
gd
e (
cm
)H
od
eo
mkre
ts (
cm
)
kg
cm
Navn
Født
jenterVekstkurve 0 − 1 år
9 10 11 12
alder vekt lengde hodeomkrets
Alder (mnd)
Kilde: Júlíusson PB, Roelants M, Eide GE, Moster D, Juul A, Hauspie R, Waaler PE, Bjerknes R. Tidsskr Nor Legeforen 2009;129:281-6.
0 1 2 3 4 5 6 7 8 9 10 11 122
3
4
5
6
7
2
3
4
5
6
7
8
9
10
11
12
13
44
46
48
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
42
43
44
45
46
47
48
49
50
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
Vekstk
urv
e g
utte
r (0 −
1 å
r), Veksts
tudie
n i B
erg
en, 2
007 P
B J
úlíu
sson, M
Roela
nts
, R B
jerk
nes ©
2007
BG
S1−
20071206N
/0−
1/M
Vekt
(kg
)L
en
gd
e (
cm
)H
od
eo
mkre
ts (
cm
)
kg
cm
Navn
Født
gutterVekstkurve 0 − 1 år
9 10 11 12
alder vekt lengde hodeomkrets
Alder (mnd)
Kilde: Júlíusson PB, Roelants M, Eide GE, Moster D, Juul A, Hauspie R, Waaler PE, Bjerknes R. Tidsskr Nor Legeforen 2009;129:281-6.
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 2 4 6 8 101 2 3 4 5
7
8
9
10
11
12
13
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98
100
102
104
106
108
110
112
100
102
104
106
108
110
112
114
116
118
120
122
42
43
44
45
46
47
48
49
50
51
52
53
54
55
47
48
49
50
51
52
53
54
55
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
Vekt
(kg
)H
øyd
e (
cm
)H
od
eo
mkre
ts (
cm
)
kg
cm
Vekstk
urv
e je
nte
r (1 −
5 å
r), Veksts
tudie
n i B
erg
en, 2
007 P
B J
úlíu
sson, M
Roela
nts
, R B
jerk
nes ©
2007
BG
S1−
20071206N
/1−
5/F
Navn
Født
Høyde far Høyde mor MFH *
* MFH = (høyde far + mor − 13)/2 & Målhøyde = MFH ± 9cm
jenterVekstkurve 1 − 5 år
10 2 4 6 8 104 5alder vekt høyde hodeomkrets
Alder (år, mnd)
Kilde: Júlíusson PB, Roelants M, Eide GE, Moster D, Juul A, Hauspie R, Waaler PE, Bjerknes R. Tidsskr Nor Legeforen 2009;129:281-6.
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 2 4 6 8 101 2 3 4 57
8
9
10
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14
13
14
15
16
17
18
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21
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23
24
25
26
27
28
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98
100
102
104
106
108
110
112
100
102
104
106
108
110
112
114
116
118
120
122
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
48
49
50
51
52
53
54
55
56
57
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
P3
P10
P25
P50
P75
P90
P97
−2.5s
−2s
+2s
+2.5s
Vekt
(kg
)H
øyd
e (
cm
)H
od
eo
mkre
ts (
cm
)
kg
cm
Vekstk
urv
e g
utte
r (1 −
5 å
r), Veksts
tudie
n i B
erg
en, 2
007 P
B J
úlíu
sson, M
Roela
nts
, R B
jerk
nes ©
2007
BG
S1−
20071206N
/1−
5/M
Navn
Født
Høyde far Høyde mor MFH *
* MFH = (høyde far + mor + 13)/2 & Målhøyde = MFH ± 10cm
gutterVekstkurve 1 − 5 år
10 2 4 6 8 104 5alder vekt høyde hodeomkrets
Alder (år, mnd)
50 55 60 65 70 75 80 85 90 95 100 105 110 115
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
P3
P10
P25
P50
P75
P90
P97
Veksts
tudie
n i B
erg
en. P
B J
úlíu
sson, M
Roela
nts
, R B
jerk
nes ©
2010
BG
S−
WF
L−
20100919N
/0−
5/F
Vekt
(kg
)
Lengde/høyde (cm)
jenterVekt for lengde 0 − 5 årNavn
Født
50 55 60 65 70 75 80 85 90 95 100 105 110 115
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
P3
P10
P25
P50
P75
P90
P97
Veksts
tudie
n i B
erg
en. P
B J
úlíu
sson, M
Roela
nts
, R B
jerk
nes ©
2010
BG
S−
WF
L−
20100919N
/0−
5/M
Vekt
(kg
)
Lengde/høyde (cm)
gutterVekt for lengde 0 − 5 årNavn
Født