Diet and nutrient status in infants and children with cow’s milk protein allergy

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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

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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

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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

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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.

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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

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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

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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

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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

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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).

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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).

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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

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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).

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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).

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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).

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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).

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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.

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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).

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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

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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).

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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

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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.

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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.

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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.

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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

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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

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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

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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

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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 90^o 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-

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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.

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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).

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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.

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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.

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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

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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.

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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.

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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)

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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)

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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.

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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

Diet and nutrient status in infants and children with cow’s milk protein allergy