Effects of feeding term infants low energy low protein formula supplemented with bovine milk fat globule membranes. Niklas Timby

Department of Clinical Sciences, Pediatrics

Umeå University 2014

Responsible publisher under swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7601-044-0

ISSN: 0346-6612

New series no: 1644

Electronic version available at http://umu.diva-portal.org/

Printed by: Print & Media

Umeå, Sweden 2014

i

Table of Contents

Table of Contents i Abstract iii Original papers iv Abbreviations v Sammanfattning på svenska vi Background 1

How to improve infant formulas? 2 Observed differences between formula-fed and breast-fed infants 2 Early growth and protein intake 2 Regulation of energy and volume intake 3 Neurodevelopment 4 Infections and immunology 6 Cardiovascular disease 6 The next step to improve infant formula, the rationale for the present study 10 The milk fat globule membrane 10 Hypotheses of the present trial 13

Materials and methods 14 Subjects 14 Formula composition 14 Measurements and analyses 15 Ethics 16 Statistics 16

Summary of results 18 Growth, macronutrient intakes and parental control of feeding (Papers I-II) 18 Cognitive, motor and verbal scores (Paper I) 19 Infections and pneumococcal antibodies (Paper III) 19 Blood lipids, adipokines, homocysteine, inflammatory markers and blood

pressure (Paper IV) 21 Gastrointestinal symptoms (Paper III) 21 Oral lactobacilli (Paper V) 22

Discussion 23 Energy regulation and growth 23 Cognitive development 24 Infections 24 Early metabolic programming 25 Oral lactobacilli 26 Limitations of the study 26 Strengths of the study 27 Conclusions from the present study 28 Future work on improvement of infant formulas 29

ii

Possible future areas for MFGM 31 Preterm infants 31 Acquired brain lesions 31

Acknowledgements 33 References 35

iii

Abstract

Background Observational studies have shown that early nutrition influences short- and long-term health of infants. Formula-fed infants have higher protein and energy intakes and lower intakes of several biologically active components present in human milk. Some of these are present in the milk fat globule membrane (MFGM). The aim of the present study was to examine the effects of feeding term infants an experimental low energy low protein formula supplemented with bovine milk fat globule membranes. Our hypothesis was that infants fed experimental formula (EF), compared to infants fed standard formula (SF), would have outcomes more similar to a breast-fed reference (BFR) group.

Methods In a double-blinded randomized controlled trial, 160 exclusively formula-fed, healthy, term infants were randomized to receive EF or SF from <2 to 6 months of age. A BFR group consisted of 80 breast-fed infants. Measurements were made at baseline, 4, 6 and 12 months of age. The EF had lower energy (60 vs. 66 kcal/100 mL) and protein (1.20 vs. 1.27 g/100 mL) concentrations, and was supplemented with a bovine MFGM concentrate.

Results At 12 months of age, the EF group performed better than the SF group in the cognitive domain of Bayley Scales of Infant Development, 3rd Ed. During the intervention, the EF group had a lower incidence of acute otitis media than the SF group, less use of antipyretics and the EF and SF groups differed in concentrations of s-IgG against pneumococci. The formula-fed infants regulated their intakes by increasing meal volumes. Thus, there were no differences between the EF and SF groups in energy or protein intakes, blood urea nitrogen, insulin or growth including body fat percent until 12 months of age. Pressure-to-eat score at 12 months of age was reported lower by parents of formula-fed infants than by parents of breast-fed infants, indicating a low level of parental control of feeding in the formula-fed groups. Neither high pressure-to-eat score nor high restrictive score was associated with formula feeding. During the intervention, the EF group gradually reached higher serum cholesterol concentrations than the SF group, and closer to the BFR group. At 4 months of age, there was no significant difference in the prevalence of lactobacilli in saliva between the EF and SF groups.

Conclusions Supplementation of infant formula with a bovine MFGM fraction enhanced both cognitive and immunological development in formula-fed infants. Further, the intervention narrowed the gap in serum cholesterol concentrations between formula-fed and breast-fed infants. The lower energy and protein concentrations of the EF were totally compensated for by a high level of self-regulation of intake which might, at least partly, be explained by a low level of parental control of feeding in the study population. The findings are of importance for further development of infant formulas and may contribute to improved short- and long-term health outcomes for formula-fed infants.

iv

Original papers

I. Timby, Domellöf E, Hernell O, Lönnerdal B, Domellöf M.

Neurodevelopment, nutrition, and growth until 12 mo of

age in infants fed a low-energy, low-protein formula

supplemented with bovine milk fat globule membranes: a

randomized controlled trial. Am J Clin Nutr. 2014;99(4):860-8.

II. Timby N, Hernell O, Lönnerdal B, Domellöf M. Parental feeding

control in relation to feeding mode and growth pattern in

early infancy. Submitted.

III. Timby N, Hernell O, Vaarala O, Melin M, Lönnerdal B, Domellöf M.

Infections in infants fed formula supplemented with

bovine milk fat globule membranes. A randomized

controlled trial. Submitted.

IV. Timby N, Lönnerdal B, Hernell O, Domellöf M. Cardiovascular

risk markers until 12 months of age in infants fed a

formula supplemented with bovine milk fat globule

membranes. Submitted.

V. Vestman NR, Timby N, Holgerson PL, Kressirer CA, Claesson R,

Domellöf M, Öhman C, Tanner AC, Hernell O, Johansson I.

Characterization and in vitro properties of oral lactobacilli

in breastfed infants. BMC Microbiol. 2013;13:193.

Paper I is reprinted with permission from American Journal of Clinical

Nutrition. Copyright © 2014 by the American Society for Nutrition.

Paper V is an open access article distributed under the terms of the Creative

Commons Attribution License. Copyright © 2013 Romani Vestman et al.;

licensee BioMed Central Ltd.

v

Abbreviations

AOM acute otitis media

ARA arachidonic acid

Bayley-III Bayley Scales of Infant and Toddler Development, Third

Edition

BFR breast-fed reference

BMI body mass index

BUN blood urea nitrogen

DHA docosahexaenoic acid

EEG electroencephalogram

EF experimental formula

ESPGHAN The European Society for Paediatric Gastroenterology,

Hepatology and Nutrition

hsCRP high sensitive C-reactive protein

IGF-1 insulin-like growth factor 1

LCPUFA long chain polyunsaturated fatty acids

MFGM milk fat globule membranes

SF standard formula

vi

Sammanfattning på svenska

Bakgrund

Från födseln fram till att man börjar med smakportioner, vanligen mellan 4-

6 månaders ålder, får barn som helammas all sin näring via bröstmjölken

vars sammansättning regleras i moderns bröstkörtel. Barn som inte ammas

bör istället få modersmjölksersättning som då blir den enda näringskällan.

Modersmjölksersättning baseras oftast på komjölk och eftersom varje art har

sin specifika sammansättning av mjölk, behöver komjölken ändras i sin

sammansättning för att ge en tillfredsställande näringstillförsel.

Ersättningen ska svara mot näringsbehovet för varje spädbarn som får den

och design av modersmjölksersättning, utan hjälp av regleringen i

bröstkörteln, är en av de största utmaningarna inom livsmedelsproduktion.

Faktorer som saknas eller finns i fel koncentration kan innebära negativa

hälsoeffekter.

Förbättringar av modersmjölksersättningar ska baseras på uppmätta

skillnader mellan ammade och ersättningsuppfödda barn. De skillnader man

ser är små och på individnivå oftast betydelselösa men på gruppnivå finns

klart mätbara skillnader som kan ha hälsoeffekter i befolkningen, såsom

skillnader i tillväxt, kognitiv utveckling och sjuklighet i infektioner. Amning

tycks också ha en skyddande effekt mot diabetes typ 1 och celiaki. Under de

senaste decennierna har konceptet tidig metabol programmering fått

uppmärksamhet där man kan koppla tidiga miljöfaktorers påverkan på

metabola system i kroppen till långsiktiga hälsoeffekter. Tidig nutrition och

tillväxt är några faktorer som har föreslagits kunna ha

programmeringseffekter som kan påverka risk för fetma, det metabola

syndromet och hjärt-kärlsjukdomar i vuxen ålder.

Eftersom det är oetiskt att randomisera barn till amning eller

modersmjölksersättning är alla studier som jämför skillnaderna

observationsstudier. Det innebär att det alltid finns en möjlighet att de

skillnader man ser beror på skillnader mellan grupperna som inte har med

uppfödningssätt att göra. Mycket talar dock för att åtminstone en del av

skillnaderna orsakas av skillnader i sammansättning mellan

modersmjölksersättning och bröstmjölk, där bröstmjölk har lägre energi-

och proteininnehåll och innehåller bioaktiva ämnen som saknas i

modersmjölksersättning. En bioaktiv fraktion i bröstmjölk är

mjölkfettsmembranet (MFGM) som innehåller bl a glykoproteiner och

fosfolipider som både har immunologiska effekter och är byggstenar i

nervsystemet. MFGM saknas i modersmjölksersättning eftersom

vegetabiliskt fett, och inte mjölkfett, används som fettkälla. Nyligen har

renade MFGM-fraktioner från komjölk börjat produceras kommersiellt.

vii

Hypotes

Vår huvudhypotes var tvådelad: 1) att en experimentell

modersmjölksersättning (EF) med sänkt energi- och proteininnehåll skulle

ge ett lägre energi- och proteinintag och därmed att barn som fått EF skulle

ha ett tillväxtmönster närmare en ammad referensgrupp (BFR) jämfört med

barn som fått standard-modersmjölksersättning (SF) och 2) att tillsats av en

MFGM- fraktion från komjölk till EF skulle påverka neurologisk utveckling

positivt för barn som fått EF. För de sekundära utfallsvariablerna var vår

hypotes att barn som fått EF, jämfört med barn som fått SF, skulle få utfall

närmare BFR-gruppen i riskmarkörer för hjärt-kärlsjukdom,

infektionssjuklighet och kolonisation av munfloran med laktobaciller.

Metoder

I en dubbelblindad studie randomiserades 160 barn som slutat ammas innan

2 månaders ålder till att få EF eller SF fram till 6 månaders ålder. En ammad

referensgrupp (BFR) med 80 barn rekryterades parallellt. Besök gjordes vid

inklusionen (<2 mån), 4, 6 och 12 månaders ålder. EF innehöll mindre

energi (60 jämfört med 66 kcal/100 mL) och protein (1.20 jämfört med 1.27

g/100 mL) än SF samt hade tillsats av en MFGM-fraktion från komjölk.

Resultat

Vid 12 månaders ålder hade EF-gruppen i medeltal högre resultat på den

kognitiva domänen av testinstrumentet Bayley III.

Under interventionen hade EF-gruppen signifikant lägre incidens av akut

öroninflammation än SF-gruppen, mindre användande av febernedsättande

medicin och skilde sig i nivåer av antikroppar mot pneumokocker.

Barnen som fick modersmjölksersättning reglerade sitt intag genom att öka

måltidsvolymerna. Detta ledde till att energi- och proteinintag för EF- och

SF-grupperna inte skiljde sig åt, och inte heller tillväxt inklusive

kroppssammansättning, urea- eller insulinnivåer. BFR-gruppen hade lägre

vikt- och längdökning mellan inklusionen och 6 månaders ålder och lägre

urea- och insulinnivåer än de ersättningsuppfödda grupperna (EF+SF).

viii

Föräldrarna till de ersättningsuppfödda barnen rapporterade lägre

“pressure-to-eat score”, ett mått på föräldrarnas oro för undervikt och vilja

att påverka barnets intag, vid 12 månader jämfört med föräldrarna till BFR-

gruppen tydande på en låg grad av föräldrakontroll för de

ersättningsuppfödda grupperna.

Under interventionen nådde EF-gruppen gradvis högre serum-

kolesterolnivåer än SF-gruppen och det var ingen signifikant skillnad mellan

EF- och BFR-grupperna vid 6 månaders ålder.

Vid 4 månaders ålder hade BFR-gruppen oftare laktobaciller i sin munflora

jämfört med EF- och SF-grupperna.

Slutsatser

Tillsats med MFGM till modersmjölksersättning gav en positiv effekt både på

den kognitiva och immunologiska utvecklingen hos ersättningsuppfödda

barn. Interventionen minskade också skillnader i kolesterolkoncentrationer

mellan ersättningsuppfödda och ammade barn.

Det sänkta energi- och proteininnehållet i EF kompenserades fullständigt av

de ersättningsuppfödda barnens egenreglering. Den oväntat goda

egenregleringen kan åtminstone delvis förklaras av låg grad av

föräldrakontroll i vår studiepopulation.

Resultaten är en viktig del i det fortsatta arbetet med utveckling av

modersmjölksersättningar och skulle kunna bidra till att förbättra

ersättningsuppfödda barns hälsa på kort och lång sikt.

1

Background

The nutritional support needed for growth and development of the fetus and

infant is based on the mother´s diet and supplied via the placenta and breast

milk. When introduced to complementary food, typically between 4 and 6

months of age, the infant’s diet becomes more and more diversified and is

expected to meet the needs for growth and development independently of

the mother´s diet. When breast milk is not available for the infant, the

nutritional support during the nursing period is solely dependent on the

composition and amount of the infant formula given and, therefore, the

functional composition of an infant formula is one of the greatest challenges

in nutrition. The composition of the formula has to meet the need for every

infant fed the formula, without help from the regulation in the mother´s

mammary gland. Any compound with too low or too high concentration may

result in unfavourable functional outcomes for the recipient infant.

Figure 1. Breast-feeding vs. formula-feeding.

2

How to improve infant formulas?

The European Society for Paediatric Gastroenterology, Hepatology and

Nutrition (ESPGHAN) Committee on Nutrition has commented on how

future work on improvement of infant formulas should be carried out. The

Committee stresses that the composition of human milk should not be the

reference in studies on infant formulas, but the outcome of healthy breast-

fed infants. A systematic review of existing information should be the base

for the introduction of any modifications to infant formulas, and appropriate

studies of efficacy and safety should be performed. Blind randomization

between study and control formula is important (1).

Observed differences between formula-fed and breast-fed infants

As randomization between formula-feeding and breast-feeding is unethical,

the knowledge on differences between formula-fed and breast-fed infants is

based on observational studies. In all populations, there are

sociodemographic differences between the parents of formula-fed and

breast-fed infants and different studies adjust for theses differences in

different ways. Due to the observational design of the studies, it is uncertain

if any found difference is caused by differences in the composition of infant

formula and breast milk or due to differences in background factors not

adequately adjusted for.

Early growth and protein intake

The protein content of infant formula has always been higher than that of

breast milk, mainly to ensure sufficient levels of essential amino acids. With

improved protein quality, the protein concentration has gradually been

lowered. In the DARLING study from the late 1980s, formula-fed infants

showed a more rapid gain in weight and lean body mass between 3 and 9

months of age, a significantly higher total energy intake and 70% higher

protein intake compared to breast-fed infants (2). In a large European multi-

center study recruiting infants between 2002 and 2004, protein: energy

ratios in iso-caloric infant and follow-on formulas were reduced from 2.9 to

1.8 and from 4.4 to 2.2 g/100 kcal, respectively. This resulted in a reduction

of weight-for-length z-score at 2 years by 0.20 in the low protein compared

to the high protein group. However, the low protein group still had

significantly higher weight and BMI than a breast-fed reference group at 2

3

years of age (3). The protein intake has been suggested to be the most

important macronutrient factor for the accelerated growth in formula-fed

infants since high protein intake leads to high levels of growth-promoting

hormones like insulin and IGF-1 (4), and higher protein intake has been

linked to future overweight (5, 6).

Regulation of energy and volume intake

The average energy density of mature breast milk has long been considered

to be about 67 kcal/100 ml and virtually all infant formulas have the same

energy density. Current regulations of the composition of infant formulas (7-

10) are based on this assumption, albeit with some allowance for variation,

resulting in a minimum of 60-67 kcal/100 ml (8, 10, 11). However, several

studies strongly suggest that the metabolizable energy of breast milk is

considerably lower, and possibly over-estimated by 10-30% (12-14).

It has been reported that healthy infants have the capacity to self-regulate

meal size depending on energy density of the food during the first year (15),

and this was noted already during the first week in premature newborns (16).

However, early studies on infants fed formulas with large differences in

energy density have shown that 1.5 – 3 months old infants were able to fully

regulate the intake of a low and high energy formula (54 vs 100 kcal/100 ml),

but the regulation was incomplete before 1.5 months of age (17). Infants 4-6

months who received a very low energy formula (36 vs 67 kcal/100 ml) plus

unlimited complementary food were not able to fully up-regulate the volume

intake and had a lower energy intake and weight gain than infants fed

standard formula (18).

If the infant´s self-regulation of energy intake is disturbed, the growth

pattern can be affected. Both parental handling of the feeding dynamic and

intrinsic characteristics of the child (“appetite”) influence weight gain in

early infancy (19). The temperament of the newborn infant affects weight

gain; “fear” (rejection of new objects or people) is associated with poor

weight gain and “distress to limitations” (negative emotionality and the

infant’s reactions to frustrating situations) with fast weight gain (20). Several

studies have shown a difference in self-regulation between formula-fed and

breast-fed infants suggesting that formula-feeding is a risk factor for

disturbed self-regulation. Data on milk intake for breast-fed infants are

available from stable isotope methodology and shows an increase in volumes

from 1 to 4 months reaching a plateau after 6 months with a great variability

within and between populations (21). Bottle-fed infants showed less

4

variability in volume between feedings, an earlier daytime-concentration of

feedings and larger total daily volume of feeding (22). Further, children who

were bottle-fed in early infancy had reduced self-regulation in late infancy

(23). Mothers who had breast-fed their infants showed less restrictive

behavior regarding child feeding at 1 year compared to mothers who had

exclusively formula-fed their infants (24). A high level of maternal control at

6 months of age worsens both underweight (“failure to thrive”) and

overweight problems between 6 and 12 months of age (25), and the level of

maternal control explains 13% of the association between breast-feeding

duration and BMI at 3 years of age (26). In rats, maternal behavior has been

shown to result in epigenetic changes in the pups resulting in an altered

response to stress (27). Intake of formula can also be affected by the

concentration of certain nutrients, e.g. supplementation of formula with free

glutamate decreases volume intake (28).

Neurodevelopment

Experimental data from animal models and short-term follow-up studies

indicate a relationship between early nutrition and neurodevelopment (29).

Many nutrients affect the development of the nervous system in early

childhood including protein, energy, fat, iron, zinc, copper, iodine, selenium,

vitamin A, choline and folate. Deficits in certain nutrients can have either

global or circuit-specific negative effects on brain development (30), and the

importance of choosing the right measurements of neural function in

nutrition-cognition studies has been stressed (31). Breast-fed and formula-

fed infants have been shown to differ in both global and specific areas. For

preterm infants, total brain volume and especially the white matter volume

in late childhood is positively correlated to the amount of breast milk

ingested during the first month (32). During the first year, healthy breast-fed

and formula-fed infants have different developmental profiles of EEG

spectral power (33). Several large studies have shown better psychomotor

development and cognitive function in breast-fed compared to formula-fed

infants. In a meta-analysis of observational studies adjusting for potential

confounders (socioeconomic, educational and hereditary factors), a

difference of 3.16 points in cognitive testing to the breast-fed children´s

advantage was found at 6-23 months of age. The difference was persistent at

least until 15 years of age. The advantage increased with duration of breast-

feeding and low birth weight infants benefitted most (34). Due to the

observational design of the included studies, the external validity of the

meta-analysis is controversial, though (35, 36). However, one single

randomized study from the 1980’s showed higher developmental scores in

5

preterm infants receiving banked breast-milk supporting the hypothesis that

there are factors in breast milk that enhance neural development (37). In the

PROBIT study in Belarus where the rate of breast-feeding was low, hospitals

were cluster randomized to breast-feeding promotion or no intervention.

The breast-feeding promotion significantly increased the rate of breast-

feeding and was associated with 7.5 points higher verbal IQ at 6.5 years (38).

Several factors have been suggested to explain the difference in

neurodevelopment between formula-fed and breast-fed infants. Any

candidate factor should be present at a different concentration in breast milk

than in formula, and have an effect on neurodevelopment shown in animal

or human studies. Supplementation with the long chain polyunsaturated

fatty acids (LCPUFA) docosahexaenoic acid (DHA) and arachidonic acid

(ARA) to infant formula has been subjected to several randomized controlled

trials. However, Cochrane reviews have not confirmed that LCPUFA

supplementation has a positive effect on neurodevelopment neither in term,

nor in preterm infants (39, 40), leaving the field open for other candidate

factors. Children aged 4-6 years who were breast-fed as infants have a

greater likelihood of foveal stereo acuity at 4-6 years compared to those who

were fed formula with or without DHA supplementation, further suggesting

that breast milk factors besides DHA might be of importance for visual

development (41).

Sphingomyelin, choline, sialic acid, gangliosides and cholesterol are all

present in higher concentration in human milk than in infant formula (42-

45). Sphingomyelin-fortified milk had a positive association with

neurobehavioral development in very low birth weight infants (46). Choline

supplementation improved memory and learning in rats (47). Sialic acid is

important for the development and growth of neural tissue (48), and

supplementation with sialic acid in a pig model improved memory and

learning (49). Gangliosides are known to play a role in neuronal growth,

migration and maturation, neuritogenesis, synaptogenesis, and myelination

(50). One double-blinded randomized study showed a positive effect of

supplementation with complex lipids containing gangliosides to infant

formula, from 2 to 8 weeks until 24 weeks of age, on cognitive function

measured with Griffith Mental Development Scale at 24 weeks of age (51).

Cholesterol increases myelination in the mouse brain (52) and is protective

against neural degradation in elderly humans (53). The concentration of iron

is considerably higher in infant formula than in human milk, and the results

from one study suggests that too much iron to iron replete infants may have

a negative effect on neurodevelopment (54).

6

Infections and immunology

The preventive effect of breast-feeding on infections, especially in developing

countries, has been pointed out as its most important health benefit (55).

Even in developed countries, formula-fed infants have higher risk of

infections during the first year of life, e.g. respiratory tract infections (56),

acute otitis media (AOM) (57, 58) and gastroenteritis (59), compared to

breast-fed infants and promoting breast-feeding has been shown to reduce

infectious diseases at the community level (60). Several antibacterial,

antiviral and immune-modulating factors in human milk have been

described; proteins including immunoglobulins, cytokines, lactoferrin,

lysozyme, κ-casein, lactoperoxidase, haptocorrin, α-lactalbumin,

osteoprotegerin and bile salt-stimulated lipase (61-65), but also

oligosaccharides, glycoconjugates (66) and lipids (67, 68). These factors can

act directly by intervening with the adhesion of microbes to the mucosa, by

killing the pathogenic microbes, by modulating the intestinal microbiota, or

by affecting other parts of the systemic immune system (69-73).

Breast-fed and formula-fed infants differ in their immune responses (74)

where breast-feeding is associated with an anti-inflammatory cytokine

milieu (75), Th1 type response (76) and better serological protection after

vaccination (77). Infant feeding and weight gain pattern during the first year

of life has been shown to influence the risk of type 1 diabetes where

breastfeeding seems to be protective (78). Breastfeeding also seems to

reduce the risk of developing celiac disease (79).

Cardiovascular disease

Observational studies on the relationships between perinatal factors and

health or disease in adult life have given rise to the concept of metabolic

programming resulting from environmental exposures during certain early

time windows causing irreversible effects on the individual that remain

through life. The suggested possible mechanisms are many, from injuries

causing suboptimal development of organs to epigenetic changes resulting in

modified expression of genes. Early feeding is one important factor affecting

metabolic programming. Many observed relationships are related to

metabolic factors or growth, highly dependent on early diet, even in previous

generations or during pregnancy or early infancy.

Barker et al. found that low birth weight is a risk factor for ischemic heart

disease later in life (80). The fetal origins hypothesis stated that fetal under-

7

nutrition leads to disproportionate fetal growth and programs later coronary

heart disease (81). The same phenomenon was found for type 2 diabetes

mellitus which led to the thrifty phenotype hypothesis (82). Besides fetal

environmental factors, a genetic predisposition of insulin resistance giving

both an intrauterine growth restriction and later insulin resistance has been

proposed to be a part of the explanation of the association between low birth

weight and later metabolic syndrome (83). Follow-up of children to mothers

who were pregnant during the Dutch famine 1944-1945 has shown that the

timing of the nutritional insult is important; starvation during early

gestation was associated with more coronary heart disease, raised blood

lipids, altered clotting and obesity while mid-gestation starvation was

associated with obstructive airway disease and micro-albuminuria and late-

gestation starvation with decreased glucose tolerance (84-86). Even a trans-

generational effect has been reported: children of fetuses exposed to in utero

starvation during the Dutch famine suffer from neonatal obesity and poor

health later in life (87).

The findings of the importance of early growth velocity, where a relationship

between fast growth in childhood and cardiovascular disease (88), impaired

glucose tolerance (89), obesity (90), increased blood pressure (91, 92) and

poor endothelial function (93), further contributed to the programming

concept. Singhal and Lucas formulated the growth acceleration hypothesis,

i.e. that accelerated growth in childhood is the common denominator for

programming cardiovascular disease (94). The growth acceleration can start

already in fetal life and high maternal weight gain during pregnancy

increases birth weight independently of genetic factors (95). Another way of

describing the idea of metabolic programming is an imbalance between on

one hand the metabolic capacity which is set in fetal life and infancy, and on

the other hand the metabolic load which is mainly dependent on the growth

pattern from infancy to adulthood (96).

The mechanisms responsible for the changes in central circuits and

peripheral tissues are not fully understood (97). From a molecular point of

view, epigenetic changes resulting in altered expression of certain genes are

likely to play a role (98). From the phenotypical point of view, changes in the

development and organization of hypothalamic circuits regulating body

weight and energy balance are probably important (99) besides the possible

changes in peripheral tissues listed below:

Children born small for gestational age with catch-up growth have

elevated insulin levels and increased proportion of body fat,

characteristics probably important for any programming effect of

8

accelerated early growth (100). Increased body fat is partly mediated

by suppressed thermogenesis (101).

Inflammation plays a key role in the development of atherosclerosis,

and increased levels of several inflammatory markers are associated

with cardiovascular disease, e.g. C-reactive protein (CRP), tumor

necrosis factor α (TNF-α) and interleukin-6 (IL-6) (102, 103). CRP

itself can also be a direct mediator of endothelial injury (104).

Children and young adults aged 5-25 years of parents with essential

hypertension had higher CRP-levels than offsprings to normotensive

parents (105).

Elevated homocysteine levels are hypothesized to cause toxic

endothelial damage resulting in both reactive oxygen species

inducing oxidative damage to endothelial cells and decreasing

endothelial production of nitric oxide leading to impaired vascular

reactivity and platelet activation and thrombosis (104). In children

with familial hypercholesterolemia, total homocysteine is related to

carotid intima-media thickness (106). Children born small for

gestational age have elevated homocysteine levels at 8-13 years

(107).

Overweight children aged 5-18 years have an unfavorable blood

lipid profile, as do adults with metabolic syndrome, i.e. high total

cholesterol, high low-density lipoprotein cholesterol, low high-

density lipoprotein cholesterol and high triglycerides (108).

In children born small for gestational age, low serum adiponectin is

associated with insulin resistance at 12 years (109), and with

elevated leptin at 8-13 years (107).

Chronic stress is linked to metabolic syndrome in a dose-dependent

manner (110). Both elevated cortisol levels and autonomic

dysfunction are involved (111). In infants born small for gestational

age, increased glucocorticoid activity is associated with insulin

resistance at 12 years (109).

Overweight children with metabolic syndrome have elevated levels

of 8-iso-prostane, a marker for systemic oxidative stress (112).

Blood pressure is significantly correlated to fasting insulin in

children 3-18 years, and fasting insulin levels predict the level of

blood pressure 6 years after (113). Systolic blood pressure in

childhood has been shown to predict hypertension and risk of

metabolic syndrome in adulthood (114).

Children with pulmonary hypertension have elevated plasma nitrate

levels (115). Nitric oxide (NO) is the key molecule in the relaxation of

smooth muscles in the vascular wall and the exogenous pathway

from dietary nitrate and nitrite and the reduction of nitrate to nitrite

9

by bacterial nitrate reductases in the oral cavity has been suggested

as a potential factor affecting cardiovascular disease (116).

Veillonella spp. is the most prevalent nitrate reductase positive taxa

in the oral cavity (117).

Vascular changes in blood vessels associated with the development

of atherosclerosis can be found in early childhood. Anatomic

(intima-media thickness) as well as physiologic (flow-mediated

dilation and endothelial performance) and mechanical (arterial

distensibility) changes can be measured with a non-invasive

ultrasound technique (104). Aortic intima-media thickness was

greater in newborn infants with low birth weight compared to

normal birth weight babies. Aortic intima-media thickness was also

greater in neonates of smoking mothers (118). Pre-pubertal children

with obesity had an increased carotid intima-media thickness, which

decreased after weight loss (119). Children aged 10-19 years with

familial hypercholesterolemia had greater carotid intima-media

thickness than a control group (106).

Differences in the infant´s intestinal flora predict later overweight

(120), and has been suggested as an important risk factor for obesity

(121, 122). On a group level, there are differences in the colonization

pattern of the gastrointestinal tract between breast-fed and formula-

fed infants. In the oral flora, lactobacilli are more common in breast-

fed infants (123). In the gut flora, L. rhamnosus, clostridia,

bacterioides, enterococci and enterobacteriaceae are more common

in formula-fed infants, while staphylococci are more common in

breast-fed infants (124).

A twin study has shown that both genetic and environmental factors

can explain the clustering of metabolic factors in metabolic

syndrome (125). Children aged 11-15 years with at least one parent

with metabolic syndrome had lower glucose disposal in a

euglycemic- hyperinsulinemic clamp and higher fasting insulin

levels compared with children with parents without metabolic

syndrome (126). Children of parents with early coronary artery

disease were overweight in childhood and had unfavourable

cardiovascular risk factors profiles (127). Mitochondrial DNA

abnormalities are related to both placental dysfunction and vascular

changes in atherosclerosis and have been considered as a possible

genetic/intrauterine environmental factor explaining metabolic

programming effects (128). Both maternal and fetal genotypes have

been associated to early infant growth (129). Children with genetic

markers of known adult obesity risk genes show faster weight and

10

length gain during the first 6 weeks of life, suggesting that the

genetic risk profile affects growth directly after birth (130).

According to the growth acceleration hypothesis (94), formula-fed infants

are at risk of future cardiovascular disease, because of a more rapid weight

gain compared to breast-fed infants (131, 132). A relation between formula-

feeding and higher risk for cardiovascular disease has been suggested from

several studies. There is a dose-dependent association between longer

duration of breast-feeding and decreased risk of overweight (133). According

to Owen et al., adults that were formula-fed had 39% higher risk of type 2

diabetes (134), 13% higher risk of obesity (135) and 1.1 mm Hg higher

systolic blood pressure (136). Breastfed infants had higher blood cholesterol

during infancy but lower levels in adulthood (137). Another study showed

that breastfed infants had higher cardiorespiratory fitness in childhood and

adolescence even after correction for background factors, weight and

physical activity (138). Other possible programming mechanisms have not

been shown to be related to infant feeding mode, for example it should be

noted that one large study could not find any association between

breastfeeding in infancy and inflammatory status in adolescence (139).

The next step to improve infant formula, the rationale for the present study

The most obvious differences in developed countries between formula-fed

and breast-fed infants found in observational studies are the differences in

growth pattern, cognitive development and infections, particulary AOM. This

indicates that lowering energy and protein intakes of formula-fed infants

aiming at mimicking the growth of breast-fed infants and to add functional

components similar to those in breast milk to infant formula to enhance

neurodevelopment and the defense against infections would be the way to

further narrow the gap in performance between formula-fed and breast-fed

infants.

The milk fat globule membrane

The lactating mammary gland cell forms and releases lipids by a unique

mechanism. In the cytoplasm of the epithelial cells, droplets of the

hydrophobe triacylglycerol core are surrounded by a coating

phospholipid/cholesterol layer with incorporated proteins. These lipid

droplets are secreted from the cells by fusion with the apical plasma

11

membrane or by exocytosis over the apical plasma membrane after being

surrounded by secretory vesicles. This gives the lipid droplet in the lumen a

triple phospholipid layer membrane consisting of the coating layer and the

double phospholipid/cholesterol layer with proteins and glycoproteins

derived from the cell membrane or secretory vesicles. The proteins are

located to different layers in the membranes with the carbohydrates of the

glycoproteins directed outwards on the hydrophilic surface of the lipid

droplet. The membrane surrounding the secreted fat droplets is called the

milk fat globule membrane (MFGM). The lipid: protein weight ratio of the

MFGM is approximately 1:1 (140, 141)

The lipid fraction of the MFGM is rich in various phospholipids and

cholesterol. Phospholipids make up 30% of the total lipid weight.

Sphingomyelin, phosphatidylcholine and phosphatidylethanolamine make

up 30% each of the phospholipid content in MFGM (140).

Choline is a highly methylated compound and is a precursor for the

biosynthesis of the membrane constituents phosphatidylcholine,

sphingomyelin and choline-containing plasmalogens as well as the

neurotransmitter acetylcholine. In foods, choline exists unesterified or

esterified as phosphocholine, glycerophosphocholine, phosphatidylcholine

and sphingomyelin. The choline and choline ester content is different in

breast milk than in infant formula; the sphingomyelin and phosphocholine

concentrations being higher in breast milk (142). Choline is closely related to

folate and is, like folate, essential for the development of the nervous system.

The fetus and the neonate have high concentrations of choline in blood and

tissues. In a mouse model, experimentally inhibited uptake and metabolism

of choline in embryos was associated with neural tube defects (143). In

humans, women in the lowest quartile for daily choline intake had twice the

risk of having a baby with a neural tube defect than women in the highest

quartile (144). Choline status of the mother in the first half of pregnancy was

associated with cognitive development measured with the Bayley Scales of

Infant and Toddler Development at 18 months of age (145). In a rat model,

supplementation with choline improved memory and learning (146). In rats,

there are two sensitive periods when oral choline supplementation gives

positive effects on brain function, during neurogenesis and synaptogenesis.

Extrapolating to humans, these periods would correspond to a period from

in utero to 4 years of age (147). It has been suggested that the effects of

choline on neurodevelopment could be due to epigenetic mechanisms (47).

The sphingomyelin concentration of human milk is higher than in formula

based on cow´s milk and much higher than in soy-based formula (42).

Sphingomyelin and its metabolites ceramide, sphingosine, ceramide-1-P and

12

sphingosine-1-P act as second messengers in cell signalling and have

regulating effects on apoptosis, cell-cycle arrest, cell survival, cell

proliferation and inflammation (148). In a rat model with experimentally

inhibited myelination of the nervous system, oral sphingomyelin increased

myelination (149). Oral sphingomyelin increased maturation of the intestine

in rats (150). Sphingomyelin has also been shown to have cholesterol

lowering effects in rats by inhibiting intestinal absorption of cholesterol (151,

152).

Gangliosides are sialic acid-containing glycosphingolipids found in breast

milk and known to play a role in neuronal growth, migration and

maturation, neuritogenesis, synaptogenesis and myelination (50). MFGM

contains gangliosides and ganglioside concentrations in intestinal mucosa

and plasma were elevated after MFGM supplementation in a mouse model

(153). Supplementation of an infant formula with complex lipids containing

gangliosides has in one study, in which the formula was fed from 2-8 weeks

until 24 weeks of age, shown increased plasma levels of the gangliosides

GM3 and GD3 and a higher Griffith Scales score on hand-eye coordination,

performance IQ and general IQ at 6 months compared to a control formula

(51). Ceramide, a component of gangliosides, constitutes 10% of the lipid

mass of the brain (48).

MFGM has high capacity to carry lipophilic compounds, and therefore

MFGM-enrichment has the potential to increase lipid-soluble nutrients in

food (154).

The proteome of the human MFGM revealed 191 different identified proteins

(155). In a study with bovine MFGM-rich fractions, 244 proteins were

identified in a whey protein concentrate and 133 in a buttermilk protein

concentrate. Combined, the proteins of the MFGM only represent 1-4% of

the total milk protein content, but they have gained much interest because of

studies showing that many of them have potential beneficial health effects.

Almost 50% of the MFGM-proteins have membrane/protein trafficking or

cell signaling functions (156). Fatty acid binding protein (FABP), BRCA1,

BRCA2 and beta-glucuronidase inhibitor have proposed anticancer effects,

helicobacter pylori-inhibitor prevents gastric diseases and butyrophilin

suppresses multiple sclerosis (157). In addition, MFGM has been suggested

to have anti-infective properties. Butyrophilin, MUC1, PAS6/7 (lactadherin),

CD14, toll-like receptor 1 and 4 and xanthine oxidase are proteins in MFGM

with antimicrobial effect (156-159).

Sialic acid is a nine-carbon sugar present in free form or as a structural and

functional component of gangliosides, glycoproteins and oligosaccharides.

13

The oligosaccharide concentration is considerably lower in bovine than

human milk (160), and sialic acid in infant formula is mainly bound to

glycoproteins (48). The total sialic acid concentration in infant formula is

typically <25% of that in breast milk, and the protein-

bound/oligosaccharide-bound ratio is 3:1 in infant formula compared to 1:3

in breast milk (44). MUC1 and MUC15 are highly glycosylated proteins with

high content of sialic acid that are present in the MFGM (161, 162). Sialic

acid concentrations in body fluids reflect metabolic status and body tissue

levels. Formula-fed infants have lower concentration of both free and total

sialic acid in saliva compared to breast-fed infants (163). The human brain

has the highest known concentration of sialic acid in nature, and sialic acid

has been suggested as a potential breast milk factor supporting optimal

neural function (48). In a pig model, sialic acid supplementation improved

memory and learning (49).

Traditionally, during the manufacturing of infant formula, the MFGM

fraction has been discarded as vegetable oil instead of milk fat has been used

as fat source. With new dairy industry techniques, bovine MFGM-rich

fractions are now commercially available and can be supplemented to food

(164). MFGM supplementation to complementary food fed from 6 to 11

months of age gave a reduction of diarrhea in a study in Peru (165). MFGM-

enriched formula fed to preschool children for 4 months gave a reduction in

febrile episodes and behavioural disturbancies (166). MFGM

supplementation during 4 weeks to adults reduced the trend towards

increasing blood lipids concentrations (167).

Hypotheses of the present trial

Our main hypotheses were that 1) an experimental infant formula (EF) with

reduced energy and protein concentrations would yield a lower energy and

protein intake of infants fed EF compared to infants fed standard formula

(SF) and a growth closer to that of a breast-fed reference (BFR) group and 2)

that supplementation with a bovine MFGM fraction to the EF would enhance

neurodevelopment for EF-fed infants. Primary outcomes were weight at 6

months, fat percentage at 4 months and Bayley III-scores at 12 months.

We further hypothesized that the EF group, compared to the SF group,

would perform more similar to the BFR group in the secondary outcomes

cardiovascular risk markers, infectious morbidity and oral colonization of

lactobacilli.

14

Materials and methods

Subjects

From March 2008 to February 2012, 160 formula-fed infants (80 girls and

80 boys) and a breast-fed reference (BFR) group with 80 infants (40 girls

and 40 boys), all born at Umeå University Hospital, Umeå, Sweden, were

recruited after inviting parents by telephone. Inclusion criteria were <2

months of age, gestational age at birth 37-42 weeks, birth weight 2500-4500

g, absence of chronic illness, and exclusive formula-feeding or, for the BFR

group, exclusive breastfeeding at inclusion and mother´s intention to breast-

feed until 6 months of age, with only small amounts (taste portions)

complementary foods, but no formula, allowed between 4 and 6 months. The

same recommendations with respect to complementary feeding were given

to parents of formula-fed infants. Formula-fed infants were stratified for sex

and randomized to receive experimental formula (EF) or standard formula

(SF) from inclusion until 6 months of age. Twins were co-randomized to the

same intervention group. The intervention was blinded to both parents and

staff until all infants had finished the intervention. Powdered formula was

distributed to families together with preparation instructions in identical

boxes marked with a code number.

Formula composition

BabySemp 1 (Semper AB, Sweden) was used as the SF, and the EF was

modified from this formula. Compared to the SF, the EF had reduced energy

(60 vs. 66 kcal/100ml) and protein (1.20 vs. 1.27 g/100 ml) densities and

was supplemented with a bovine MFGM-enriched whey concentrate

(Lacprodan MFGM-10, Arla Foods Ingredients, Viby, Denmark). MFGM

proteins constituted 4% (wt/wt) of the total protein content in the EF. The

macronutrient contents, fatty acid compositions and amino acid contents of

the formulas are shown in detail in Paper I (Tables 1 and 2).

15

Measurements and analyses

Visits were made at baseline (<2 months), 4 months, 6 months and 12

months of age. A summary of measurements at each visit is shown in Table 1.

Details of the methods used are presented in respective paper (I-V).

Analysis Baseline 4 mo 6 mo 12 mo In

paper

Anthropometrics,

blood pressure

● ● ● ● I, IV

Body fat% ● ● I

Insulin, BUN ● ● ● I

Plasma amino

acids

● ● ● I

Blood lipids,

homocysteine,

hsCRP

● ● ● ● IV

Adipokines ● IV

Faecal

calprotectin

● ● ● ● IV

Bayley-III ● I

S-IgG against

pneumococci

● III

Oral microbial

sampling

● V

Table 1. Analyses in the study presented by age at measurement.

16

Ethics

This study was approved by the Regional Ethical Review Board, Umeå,

Sweden. During inclusion, which was made by telephone with parents of

newly born infants, we were careful not to influence any breast-feeding

negatively. This was ascertained by first asking the parent an open question

about whether the infant was breast-fed or formula-fed. Parents that had

already totally stopped breast-feeding were asked to participate in the

randomized part of the study and those exclusively breast-feeding were

asked to participate in the BFR group.

Statistics

A pre-study power analysis showed that a sample size of 63 in each group

was needed to detect a difference of 0.5 SD in the primary outcomes with

80% power at the significance level of 0.05. Expected drop-out rate was 25%,

hence 80 infants was included in each group. Most analyses are presented on

an intention-to-treat basis, i.e. infants that stopped the intended

intervention or breast-feeding were kept in the study and included in

analyses. Statistical calculations were made using IBM SPSS Statistics

Version 19 (©IBM 1989, 2010) and methods are described in the respective

paper (I-V).

The sample size according to the power analysis above was chosen to have a

low risk (5%) of a type 1 error, i.e. find a difference when no difference exists.

The risk of a type 2 error, i.e. not to find a true difference, is 20%, which is

important when interpreting the results. A lack of difference is more

uncertain than any found difference.

The randomized design is powerful when comparing the EF and SF groups

since background variables are likely to be equally distributed between the

groups. Some comparisons between the formula groups are adjusted for

relevant co-variates to further ensure a causal relationship between the

intervention and outcome.

All comparisons with the BFR group are observational and as always in

observational studies, there is a risk of differences in background factors not

adequately corrected for. We chose to collect background information to

ensure to have enough information to allow comparisons in the randomized

design. Many of the comparisons with the BFR group are unadjusted

17

because we did not have enough background information for an adequate

adjustment. Other comparisons are adjusted for relevant co-variates.

18

Summary of results

Growth, macronutrient intakes and parental control of feeding (Papers I-II)

In a longitudinal analysis, the EF and SF groups did not differ significantly in

any of the growth parameter z-scores (weight, length, BMI or head

circumference). However, the BFR group differed from the combined

formula-fed (EF+SF) groups. The BFR group had higher ΔSDS for weight

and length between baseline and 6 months of age. In a cross-sectional

analysis, there were no differences between the EF and SF group in any of

the growth parameters (weight, length, BMI and head circumference) at

baseline, 4, 6 or 12 months of age. The BFR group had a higher length at

baseline compared to the formula-fed (EF+SF) groups. There were no

differences between the EF, SF or BFR groups in body fat percentage at

baseline or 4 months of age as assessed by plethysmography (PeaPod).

Mean daily intake of study formula was larger in the EF group compared to

the SF group, resulting in virtually identical energy and protein intakes from

formula in the EF and SF groups. When data on intakes of complementary

foods were added, there were still no significant differences in energy or

protein intakes between 4-6 months of age between the formula groups. The

difference in mean daily intake was attributed to larger mean formula meal

sizes between 2 and 6 months of age for the EF group. Meal frequency did

not differ significantly between the EF and SF groups.

There were no differences in fasting plasma insulin, fasting plasma glucose

or blood urea nitrogen (BUN) between the EF and SF groups, but compared

to the BFR group, the combined formula-fed (EF+SF) groups had higher

plasma insulin and BUN concentrations indicative of higher protein intake.

Parental pressure-to-eat score 12 months postpartum was lower in the

combined formula-fed (EF+SF) groups than in the BFR group. After

dichotomizing parental control scores to high and low, both high restrictive

score and high pressure-to-eat score at 12 months postpartum was

associated with infant growth parameters but not with feeding mode.

19

Cognitive, motor and verbal scores (Paper I)

Cognitive score was 4.0 (95% confidence interval: 1.1-7.0) points higher in

the EF than in the SF group. No other significant differences between the EF

and SF groups were found in the Bayley-III scales. The BFR group

performed significantly better in the cognitive domain compared to the SF

group, but not compared to the EF group. Further, the BFR group performed

better in the receptive verbal subscale compared to both formula-fed

(EF+SF) groups, even after adjustment for psychosocial background

variables.

Figure 2. Cognitive, motor and verbal scores at 12 months of age,

measured with the Bayley Scales of Infant and Toddler Development, Third

Edition, for the experimental formula (EF), standard formula (SF) and

breast-fed reference (BFR) groups.

Infections and pneumococcal antibodies (Paper III)

During the intervention, from inclusion until 6 months of age, parents of

infants fed the EF reported fewer episodes of acute otitis media (AOM)

treated with antibiotics compared to parents of infants fed the SF. The total

number of episodes of bacterial infections leading to antibiotics and viral

20

infections leading to hospitalization during the intervention was also lower

in the EF than the SF group. There were no significant differences between

the formula groups in other specific bacterial infections treated with

antibiotics or viral infections leading to hospitalization before 6 months of

age, nor between 6 and 12 months of age.

Figure 3. Proportion of infants in the experimental formula (EF),

standard formula (SF) and breast-fed reference (BFR) groups whose

parents reported bacterial infections leading to antibiotic treatment or

viral infections leading to hospitalization.

Further, the EF group reported significantly less antipyretics use during

intervention compared to the SF group. Serum concentrations of

immunoglobulins against pneumococci differed between the EF and SF

groups. The concentrations of IgG against serotypes ST1, ST5 and ST14 were

significantly lower in the EF than the SF group.

21

Figure 4. Longitudinal prevalence of antipyretics use for the infants in the

experimental formula (EF), standard formula (SF) and breast-fed reference

(BFR) groups.

Blood lipids, adipokines, homocysteine, inflammatory markers and blood pressure (Paper IV)

The mean total s-cholesterol concentration gradually reached higher levels

during the intervention in the EF group compared to the SF group, and did

not differ significantly in a cross-sectional analysis between the EF and BFR

groups at 6 or 12 months. The LDL cholesterol to HDL cholesterol (LDL:

HDL) ratio did not differ significantly between the EF and SF groups during

the intervention, but the BFR group had higher LDL: HDL ratio than both

the EF and SF groups until 6 months of age. Homocysteine levels were lower

in the EF group compared to the SF group during intervention. Adipokines

and hsCRP-levels were similar in all three groups. Fecal calprotectin was

significantly higher in the BFR group compared to the formula-fed groups at

baseline. Blood pressure did not differ between the three groups.

Gastrointestinal symptoms (Paper III)

There were no significant differences in the frequency of hard stools,

abdominal pain, vomiting or consumption of laxatives or probiotic drops

between the EF and SF groups. The BFR group had lower frequency of hard

22

stools, higher stool frequency and consumed less laxatives compared to the

formula-fed (EF+SF) groups.

Oral lactobacilli (Paper V)

Lactobacilli were cultured less often from saliva of infants in the EF group

(9%) and the SF group (5%) compared to the BFR group (34%), p<0.001.

The most prevalent lactobacillus was L. gasseri found in 88% of the

lactobacilli-positive infants. In vitro, L. gasseri bound to parotid and

submandibular saliva, the proteins salivary gp340 and MUC7, purified

MFGM and adhered to epithelial cells. Further, L. gasseri inhibited

Streptococcus mutans, Streptococcus sobrinus, Actinomyces naeslundii,

Actinomyces oris, Candida albicans and Fusobacterium nucleatum.

23

Discussion

In the present study we tested our hypotheses that feeding healthy term

infants with a novel formula with lower energy and protein concentrations

and supplemented with bovine milk fat globule membranes (MFGM) would

yield functional outcomes in different areas closer to breast-fed infants when

compared to infants fed standard formula, by use of a double-blinded

randomized controlled trial.

Energy regulation and growth

Infants fed EF fully compensated for the reduced energy and protein

concentrations and showed a growth pattern not different from infants fed

SF. This did not confirm our original hypothesis that infants fed EF would

gain less weight due to a lower energy and protein intake. Previous studies

have shown that bottle-feeding is associated with poor self-regulation of

intake (22, 24-26, 168-170). Our findings indicate, on the contrary, that

bottle-fed infants may have very precise energy regulation in the range from

60 to 66 kcal/100 ml (the range of breast milk) by adopting mean meal sizes

throughout the intervention. Further, parents of the formula-fed infants had

a low level of parental control of feeding during the first year, and even a

significantly lower pressure-to-eat score at 12 months postpartum than

parents of breast-fed infants. High parental pressure-to-eat and restrictive

scores 12 months postpartum were associated with infant growth but not

with formula-feeding. This low level of parental control differs from studies

in other populations where parents of formula-fed infants reported higher

level of parental control than parents of breast-fed infants (24, 171). The low

level of parental control in our study population might explain the

unexpected high level of self-regulation for the formula-fed infants, and

suggests that, depending on study population, changes in formula energy

concentration can be totally compensated for. In such a population, a

reduced protein: energy ratio is needed to reduce the protein intake.

The EF and SF groups did not differ in any growth parameters until 12

months of age. However, the BFR group had a slower weight and length gain

between baseline and 6 months of age compared to the combined formula-

fed groups (EF+SF). Body fat percentage did not differ significantly between

any of the groups at baseline or 4 months of age. This is well in line with

other studies on formulas with a low protein: energy ratio where no or small

differences in growth between formula-fed and breast-fed infants were found

24

(3, 172). Plasma insulin and BUN concentrations were higher in the formula-

fed than in the breast-fed infants, indicative of a higher protein intake of

formula-fed infants, in support of previous findings (173-175).

Cognitive development

We found that infants fed the EF performed better on cognitive testing at 12

months compared to infants fed the SF, and at a level similar to the BFR

group. The size of the difference in cognitive score between the EF and SF

group of 4.0 (95% CI: 1.1-7.0) points, is in the same range as the calculated

difference in cognitive function between normal-birth-weight formula-fed

and breastfed infants of 2.66 (95% CI: 2.15-3.17) points after adjusting for

socioeconomic factors in the meta-analysis of observational studies by

Anderson et al (34). On an individual basis, an improvement of 4.0 points in

cognitive score does probably not give any practical benefits, but on a group

level this effect size of the intervention is substantial and to our knowledge,

this is the first randomized controlled trial that has shown such a large

positive effect of any supplementation to infant formula on cognitive

function in term infants measured by the Bayley Scales of Infant and Toddler

Development test.

The MFGM fraction contains several components that individually have been

associated to brain development, and our interpretation is that MFGM-

supplementation has enhanced the cognitive development in the formula-fed

infants. From the present study, we cannot deduce the exact mechanism. It

could be one single responsible factor (e.g. sialic acid (44, 49), gangliosides

(43, 50, 51), sphingomyelin (42, 46, 149), choline (42, 145, 147) or

cholesterol (45, 52, 53), a combination of several factors, or it could be

different limiting factors for different infants explaining the effect size. The

EF and the SF were both supplemented with LCPUFAs to the same level, and

it is possible that there could be a synergistic effect of MFGM and LCPUFAs

on cognitive development.

Infections

Our hypothesis, that MFGM supplementation of infant formula would give a

reduction in infectious morbidity, was verified by our findings that infants

fed EF had a lower incidence of AOM and a reduced number of days with

antipyretic medication during the intervention period than infants fed SF.

25

Several components of MFGM have been described to have an antimicrobial

effect. In a proteomic characterization of human MFGM, 191 proteins were

identified of which 20% are involved in immune function (155). In bovine

MFGM, 120 different proteins were identified of which 4% have

immunological effects and 21% unknown effect (156). Further, the lipid

fraction of bovine MFGM had antiviral effect in vitro (67), and gangliosides

of the MFGM have been suggested to play an important role in the

establishment of intestinal microbiota, gut immunity, and consequently, in

the defense of infections (176). The mechanism behind the effect on AOM

seen in the present study is unclear. The differences between the EF and SF

groups in s-IgG concentrations against pneumococcal serotypes 1, 5 and 14

indicates that a possible mechanism for the protective effect against

infections is mediated by or reflected in the humoral immune system, a

difference previously shown between breast-fed and formula-fed infants

(77). Breast-feeding reduces immune responses, limits hyper-responsiveness

and promotes tolerance (74, 75). In the present study, factors in MFGM

could have an inhibiting effect on the humoral immune system yielding

lower pneumococcal s-IgG concentrations after one or two vaccine doses.

Another possible explanation could be that anti-microbial factors of the

MFGM exert a local effect on the microbiota in the upper airways in line with

numerous identified factors of breast milk (69), which might change the

humoral immune response. Growing evidence suggests a link between the

gut microbiota and the peripheral immune system (177), and anti-microbial

and immunostimulating factors from the MFGM could, by affecting the gut

microbiota, have a general effect on the immune system that affects s-IgG

concentrations against pneumococci.

Early metabolic programming

The present study shows that infants fed the EF differ in serum total

cholesterol (s-Ch) concentrations compared to infants fed the SF. By

increasing the cholesterol content of the EF, we achieved higher s-Ch,

gradually reaching the level of breast-fed infants during the intervention.

Theoretically, this could lead to a programming effect and change the

cholesterol trajectory for the EF group closer to the BFR group. Breast-fed

infants have, compared to formula-fed infants, higher cholesterol levels in

infancy, a difference that disappears during childhood to result in lower

levels in adolescence and adulthood (137).

However, it is noteworthy that the LDL: HDL ratio did not follow that of the

BFR group. In fact, the LDL: HDL ratio did not differ significantly between

26

the EF and SF group, and the BFR group had higher LDL: HDL ratio from

baseline until 6 months of age. This contrasts to the findings in a previous

study, with a smaller sample size, where infants fed a formula with high

cholesterol content showed elevated total cholesterol at 4 months of age, but

also a trend towards elevated LDL: HDL ratio compared to infants fed a low

cholesterol formula (178). The impact of early LDL: HDL ratio on any

programming effect affecting later cholesterol levels has not been studied in

infants, and a long term follow up until adolescence would be desirable.

Oral lactobacilli

Oral lactobacilli were more often found in the BFR group than the EF and SF

groups, and there was no significant difference between the EF and SF

groups. It should be noted that cultures were taken from a subsample in the

study (EF: n=47, SF: n=43 and BFR: n=43), so the study could be under-

powered for detecting a difference between the EF and SF groups. Our

results contribute to the field of microbiota and health. The colonization of

the gastrointestinal tract from the oral cavity to colon is known to be

influenced by different environmental factors including nutrition (124). The

interpersonal variability of bacterial community composition is high while

the intrapersonal variability is low (179), and the intestinal microbiota has

been suggested to contribute to health parameters in different areas, e.g.

obesity, atopic diseases, inflammatory bowel disease and intestinal cancers

(121). The health consequences of differences in the oral microbiota are less

studied. Lactobacilli are often found in caries lesions (180) but also seem to

have protective effect against caries (181). Systemic health effects could be

mediated by nitrate reducing oral bacteria that influence the concentration

of circulating nitric oxide involved in vasodilation, nerve transmission, host

defence and cellular energetics (182).

Limitations of the study

Some differences between the formula-fed groups and the BFR group, e.g.

insulin and BUN levels, were seen already at baseline. This is probably

caused by the fact that all infants in the EF and SF groups had a period of

formula feeding before inclusion. As exclusive formula-feeding was an

inclusion criterion, which was formulated to avoid affecting duration of

partial breast-feeding in a negative way, only parents of infants that already

were exclusively formula-fed were asked to participate in the study. Most

parents in all three study groups chose to introduce complementary foods in

27

the interval 4-6 months. This dilution of the intervention is a weakness of the

study leading to an underestimation of the biological effect of the nutritional

intervention, but, together with calculations on an intention-to-treat basis,

gives a picture of the effect in a population.

Another limitation of the study is the double intervention with both lowered

protein/energy content and supplementation with MFGM, with two primary

outcomes. The study was designed this way for economical and resource

reasons. We predicted that any effect on growth would be caused by the

changed energy and protein contents, and that any effect on

neurodevelopment or infections would be caused by the MFGM

supplementation. As the EF group up-regulated their ingested volumes

resulting in similar energy and protein intakes for both formula groups, the

probability that the energy or protein intervention had an effect on

neurodevelopmental outcomes or infections is low. We cannot, however,

exclude an effect of the MFGM on growth or intake.

Due to the study design with randomization between the formula groups, a

limited number of background variables were collected, and comparisons

with the BFR group should be done with caution. We have chosen to adjust

some comparisons for relevant background variables in different

multivariate models. Other comparisons were only performed unadjusted

when we judged that the collected background variables were insufficient for

an adequate adjustment.

Strengths of the study

The double-blinded randomized design of the study is a major strength

giving high validity to causal relationships of the intervention. All analyses

were done on an intention-to-treat basis, i.e. parents of infants that

interrupted the intervention before 6 months of age were asked to stay in the

study for measurements, giving the true effect of the intervention in a

population. The homogenous, high socioeconomic study population is also a

strength when assessing the true biological effect of the intervention with

little disturbance of background differences.

28

Conclusions from the present study

We have found a positive effect of MFGM supplementation of infant formula

on cognitive function, and this nutritional intervention eradicated the gap in

cognitive performance between breast-fed and formula-fed infants at 12

months of age.

Supplementation of infant formula with a bovine MFGM fraction decreased

the incidence of AOM in formula-fed infants between 0-6 months of age to a

level similar to breast-fed infants, and modulated the concentration of s-IgG

antibodies against pneumococci.

Formula-fed infants had the capacity to compensate for a reduction of

energy density from 66 to 60 kcal/100 ml by increasing ingested volumes, at

least in a population with low parental control of feeding.

Parental control of feeding was influenced by infant growth pattern. An

improved formula composition leading to an early growth similar to breast-

fed infants might influence not only metabolic programming effects, but also

patterns of parental control, and yield beneficial long term effects on growth

and metabolic status.

MFGM supplementation of infant formula might influence the higher long-

term risk of cardiovascular disease in formula-fed infants by intervening

with mechanisms of early programming. We have shown that increasing

cholesterol intake between 2 and 6 months of age leads to s-Ch similar to

breast-fed infants, but does not change the LDL: HDL ratio towards the level

of breast-fed infants.

MFGM supplementation of infant formula did not significantly change the

presence of lactobacilli in saliva of formula-fed infants, but lactobacilli were

more often present in breast-fed infants. Possible health effects of this are

unknown.

The results of the present study are of importance for future improvements

of infant formulas that should be based on differences in performance

between formula-fed and breast-fed infants (1). We found that

supplementation with MFGM narrowed the gap between formula-fed and

breast-fed infants in cognitive development and infectious morbidity, but the

results need to be confirmed by other studies. Only one dose of MFGM was

tested. If the effects would be more pronounced with a higher concentration

of MFGM remains to be studied.

29

A long-term follow-up of the present study is important. A new assessment

of cognitive function close to school-age is needed to find out if the

difference between the formula groups persists with age. Further, a long time

follow-up of growth and metabolic status during childhood, adolescence and

adulthood would further elucidate possible programming effects of this

nutritional intervention.

Future work on improvement of infant formulas

The results of the present study suggest that the MFGM fraction might be

responsible for some or all of the observed differences in cognitive

development and infectious morbidity between formula-fed and breast-fed

infants. Suggestions for future improvements of infant formulas include:

To further decrease differences in growth between formula-fed and

breast-fed infants by lowering the protein: energy ratio. The high

protein intake of formula-fed infants is still the most striking

difference in macronutrient intakes between formula-fed and breast-

fed infants. The high level of self-regulation of energy intake in the

present study indicates that moderate changes in energy density

probably have little or no effect on energy intake, at least in some

populations. Staging of infant formulas with different

macronutrient content at different ages during the first 12 months of

life would be more physiological and could help to lower the protein:

energy ratio, even if this is not allowed according to present

regulations (8).

To test the effect of MFGM supplementation of infant formula in

new randomized controlled trials in different settings to support or

contradict the findings of the present study, and to test different

concentrations of MFGM in infant formulas.

To test the effects of supplementation with other bioactive milk

fractions, preferably in combination with MFGM supplementation to

examine additive effects:

o Lactoferrin in human milk has positive effects on iron

absorption and has anti-microbial and anti-inflammatory

effects. Rice-expressed recombinant human lactoferrin has

been expressed and characterized and has been suggested as

a beneficial supplementation to infant formula (183).

Lactoferrin supplementation of very low birth-weight

premature infants reduced the risk of sepsis (184) and one

30

double-blinded, placebo-controlled pilot study has shown

that bovine lactoferrin supplementation of infant formula

fed to term infants gave a reduction in lower respiratory

tract infections during the first year and higher hematocrit

at 9 months (185).

o Bile salt-stimulated lipase (BSSL) from human milk has

antimicrobial effect in vitro (65). Supplementation of

premature infants with recombinant human BSSL improved

growth and fat absorption (186).

o Other bioactive components of human milk that are lacking or

present in a lower concentration in infant formula do not

have convincing evidence of positive health effects for

supplementation of formula-fed infants. Cytokines may give

possible beneficial effects on immunology, inflammation

and host defense (187). Transforming growth factor-beta

(TGF-β) (188), soluble CD14 (189), IL-10 (190) are examples

of cytokines where supplementation in animal models have

yielded immunological effects, but there is no support from

human studies. Numerous hormones and growth factors

have been the subject of studies. Insulin (191), IGF-1(192)

and epidermal growth factor (EGF) (193) stimulate cell

growth and maturation. Leptin, adiponectin, IGF-1, ghrelin,

obestatin and resistin are also hormones present in breast

milk but probably not in formula and have been suggested

as likely or possible hormonal mediators of metabolic

programming (194). However, there is still no evidence that

supplementation with hormones or growth factors to infants

would yield any health benefit. Differences in the

development of the intestinal flora between breast-fed and

formula-fed infants give room for a potential beneficial

supplementation with pre- or probiotics to infant formula

(195). Human milk contains probiotic bacteria and

modulation of the intestinal flora with probiotic bacteria has

been shown to affect immune function and enhance defense

against pathogens (196). One study has shown that

enrichment with Lactobacillus rhamnosus GG to infant

formula increases weight and height between 0-6 months of

age (197). Oral supplementation with prebiotic

oligosaccharides to formula-fed infants until 6 months of

age gave a reduction of allergic manifestations and

infections during the first two years of life indicating an

effect on the immune system as well as the maturation of the

intestinal flora (198). Many studies have also been

31

performed with negative results and the total body of

evidence on supplementation of infant formula with

probiotics and prebiotics is weak (199). Supplementation

with nucleotides to infant formula has been shown to give a

gut flora more similar to that of breast-fed infants with a

high proportion of Bifidobacteria (200) and to increase

head circumference during the first half-year of life (201),

but evidence of health benefits are lacking.

Possible future areas for MFGM

Preterm infants

The health benefits of breast-feeding are larger for preterm than for term

infants (34, 55), and human milk is recommended as enteral feeding for

preterm infants (202, 203). However, it is not breast-feeding, but the trans-

placental transport from the mother´s blood that is the physiological route of

delivering nutrients to the fetus. Concentrations of nutrients in the cord

blood is less examined than concentrations in breast milk, but the placental

regulation of different nutrients is of course of greatest importance for fetal

growth and development (204, 205). Factors of MFGM might be needed in

higher concentrations in the fetus or premature infant than is delivered by

breast milk and MFGM supplementation of breast milk to premature infants

could theoretically improve outcomes in different areas beyond the benefit of

breast-feeding. Possible areas where breast milk has been shown or

suggested to have a positive clinical effect and MFGM theoretically could

have an additive effect include necrotizing enterocolitis (203), brain

development (34), retinopathy of prematurity (206) and infections (207).

Further, fat intake has been shown to be positively associated with head

circumference growth in extremely preterm infants (208), and components

from MFGM could be involved.

Acquired brain lesions

The positive effect of MFGM on cognitive development suggested by our

results suggests that MFGM may be used to enhance neurodevelopment in

other situations. A number of different dietary factors have been tested for

injury recovery after acquired lesions in the central nervous system (209),

and MFGM could be of interest as supportive in the healing process after

32

traumatic, vascular, inflammatory, malignant or iatrogenic injuries in the

central nervous system.

33

Acknowledgements

Even if my name is on the front page, research is, like most other areas in

life, a team work. I would like to express my greatest gratitude to …

All participating infants and their families for your contributions for

the possible benefit for future generations.

Magnus Domellöf, my main supervisor, for helping me to discover the joy

of science, for giving me responsibility, and for your never-ending

enthusiasm and believe that everything is possible.

Olle Hernell, my co-supervisor, for your dedicated work in all aspects of

the study, for sharing your great experience in the field, and for always

having time for discussions.

Bo Lönnerdal, my co-supervisor, for your invaluable contributions to the

study, for always being supportive, for helping me to improve my written

English, and for being present despite your geographical position on the

other side of the Atlantic Ocean.

Carina Forslund, research nurse and TUMME queen, for your enthusiasm

and devotion for the study, for giving the infants and parents personal and

professional support, and for your company and cooperation during the

study. Camilla Steinwall-Lindberg, research nurse, for your invaluable

contribution during part of the study.

Yvonne Andersson, Carina Lagerqvist, Catarina Lundell and the

rest of the staff at the Pediatriklab for analyses, preparations and

storing of the samples, for the best cooperation and for help with economical

and biobank issues.

Erik Domellöf for sharing your knowledge on psychological development,

for great cooperation and for excellent Bayley-testing together with Frida

Tunlind, Linda Leek and Kalle Hörnström.

Catharina Tennefors and Lars-Börje Sjöberg, Semper AB, for

professional and friendly cooperation during the entire study.

Outi Vaarala and Merit Melin for immunological input and analyses.

Nelly Romani Vestman, Pernilla Lif Holgerson, Carina Öhman,

Ingegerd Johansson and the rest of the co-workers in the oral lactobacilli-

part of the study.

Karin Moström, Elisabeth Torstensson, Carina Jonsson and Ulla

Norman for keeping everything, even PhD students, in order.

All other researchers and staff at the Unit for Pediatrics for great

company, a friendly atmosphere and the 9.30 fika.

Staffan Berglund, Jenny Alenius Dahlqvist, Elisabeth Stoltz-

Sjöström, Frida Karlsson Videhult, the PhD group at the department of

clinical sciences and all other fellow PhD students during the years for nice

34

moments during courses, journal clubs, seminars, PhD weeks and the 14.30

fika.

Erik Bergström and Per-Erik Sandström, heads of the Pediatric clinic

for understanding the dilemma of combinating clinical work and research.

All my colleagues at the Pediatric clinic and the Kolbäcken

habilitation center for a good working place.

Olof, Torbjörn, Mattias, Antti, Staffan, Frans, Lasse, Mats, Johan

Magnus, Magnus, Kjell and Anders, the curling team, for sharing wins

and losses, takes and draws, beer and gurka.

My parents Lena and Björn and my parents-in-law Anita and Lasse for

all support.

My beloved family Erika, Otto and Moa, you are the best.

The study was funded by grants from Sweden´s Innovation Agency (Vinnova), the Västerbotten County Council and Semper AB.

35

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