FORMULA INTAKE in EARLY INFANCY By

FORMULA INTAKE IN EARLY INFANCY

Susana Finkbeiner

A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Human Nutrition

Spring 2016

FORMULA INTAKE IN EARLY INFANCY

Susana Finkbeiner

Approved: ______Jillian C. Trabulsi, Ph.D., R.D. Professor in charge of the Advisory Committee

Approved: ______P. Michael Peterson, Ed.D. Chair of the Department of Behavioral Health and Nutrition

Approved: ______Kathleen S. Matt, Ph.D. Dean of the College of Health Sciences

Approved: ______Ann L. Ardis, Ph.D. Senior Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS

I would like to acknowledge my advisors, Dr. Jillian Trabulsi and Dr. Julie Mennella, for their continuous support, encouragement, and guidance. Their incredible mentorship and expertise were invaluable to my graduate school experience. I would also like to acknowledge the third committee member, Dr. Mia Papas, for dedicating her time and expertise.

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TABLE OF CONTENTS

LIST OF TABLES ...... vii LIST OF FIGURES ...... x ABSTRACT ...... xi

Chapter

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 7

2.1 History of Energy Requirements and Energy Intake in Early Infancy...... 7 2.2 Current Energy Requirements and Energy Intake in Early Infancy ...... 11 2.3 History of Protein Requirements and Protein Intake in Early Infancy.... 13 2.4 Current Protein Requirements and Protein Intake in Early Infancy ...... 15 2.5 Weight Gain in Infancy and Later Life Weight Status ...... 17 2.6 Association Between Diet Composition and Weight Gain in Infancy .... 21 2.7 Infant Formula Composition and its Effect on Feeding Behaviors and Growth ...... 23 2.8 Summary...... 27

3 STATEMENT OF THE PROBLEM...... 29

4 AIMS ...... 30

4.1 Specific Aims ...... 30

5 METHODS ...... 32

5.1 Subjects...... 32 5.2 Research Design ...... 33 5.3 Study Formulas...... 35 5.4 Testing Procedures Overview...... 35

5.4.1 Anthropometry ...... 36 5.4.2 Demographics and health history assessments ...... 36 5.4.3 Energy intake measurement ...... 37 5.4.4 Validation of energy intake measurement ...... 39

5.5 Statistical Analysis ...... 41

5.5.1 Preliminary analyses...... 41 5.5.2 Primary Analyses...... 41

6 RESULTS ...... 43

6.1 Subject Characteristics ...... 43

6.1.1 Characteristics of subjects who completed a 1-day diet record at 0.4 months of age...... 44 6.1.2 Characteristics of subjects who completed the 3-day TWbottle method at 0.6 months of age...... 44 6.1.3 Characteristics of mother-infant dyads by formula group...... 46

6.2 Dietary Intake at 0.4 Months (all Infants Feeding CMF), Assessed via a 1-Day Diet Record ...... 46

6.2.1 Energy intake of CMF fed infants at 0.4 months of age compared to historical data on energy intake ...... 47 6.2.2 Protein intake of CMF fed infants at 0.4 months of age compared to historical data on protein intake...... 48 6.2.3 Energy and protein intake of CMF fed infants at 0.4 months of age compared to the 2005 DRIs ...... 48

6.3 Dietary Intake at 0.6 Months (Infants Feeding Either CMF or EHF), Assessed via the 3-Day TWbottle Method ...... 49

6.3.1 Energy intake of CMF and EHF fed infants at 0.6 months of age compared to historical data on energy intake ...... 49 6.3.2 Protein intake of CMF and EHF fed infants at 0.6 months of age compared to historical data on protein intake ...... 49 6.3.3 Dietary intake of CMF and EHF fed infants at 0.6 months of age compared to 2005 DRIs ...... 50 6.3.4 Comparison of dietary intake by formula type (CMF vs. EHF fed infants) at 0.6 months of age ...... 50

7 DISCUSSION...... 52

7.1 Reported Energy and Protein Intake at 0.4 Months of Age in CMF Fed Infants Compared to Historical Data ...... 52 7.2 Reported Energy and Protein Intake at 0.4 Months of Age in CMF Fed Infants Compared to the Energy EER and Protein AI (2005 DRIs)...... 53

7.3 Measured Energy and Protein Intake at 0.6 Months of Age in CMF Fed Infants Versus Historical Data...... 53 7.4 Measured Energy and Protein Intake at 0.6 Months of Age in EHF Fed Infants Versus Historical Data...... 54 7.5 Measured Energy and Protein Intake at 0.6 Months of Age in CMF Fed Infants Compared to the Energy EER and Protein AI (2005 DRIs). 54 7.6 Measured Energy and Protein Intake of EHF Fed Infants at 6 Months of Age Compared to the Energy EER and Protein AI (2005 DRIs)...... 55 7.7 Comparison of Dietary Intake Variables at 0.6 Months of Age by Formula Type (CMF vs. EHF) ...... 56

8 CONCLUSIONS ...... 58

TABLES ...... 60 FIGURES ...... 88 REFERENCES ...... 92

Appendix

A IRB HUMAN SUBJECTS APPROVAL ...... 102 B INFORMED CONSENT ...... 105 C PARTICIPANT HANDOUTS ...... 113 D DATA COLLECTION FORM...... 120

LIST OF TABLES

Table 1: Summary of scientific literature on energy intake (kcal/kg/d) determined by the test weighing method in young infants...... 61

Table 2: Summary of scientific literature on protein intake (g protein/kg/d) determined by the test weighing method in young infants ...... 63

Table 3: Macronutrient composition of study formulas compared to breast milk...... 65

Table 4: Demographic characteristics of mother-infant dyads at study entry (Visit 1)…………………………………………….…………….….66

Table 5: Subject count for 1-day diet record when infants were 0.4 months of age (Visit 1)…………………………..………………...... …...... 67

Table 6: Characteristics of infants who returned a 1-day diet record (DR) versus those who did not returned a 1-day diet record (No DR)…....68

Table 7: Comparison of infant characteristics for infants randomized to a study formula versus those who withdrew from study after Visit 2. …...... 69

Table 8: Subject count for 3-day TWbottle method, when infants were 0.6 months of age (Visit 2)………………………………………..…….70

Table 9A: Demographic characteristics according to formula group in subjects who completed Visit 2….…………………………...... 71

Table 9B: Characteristics of infants who completed the TWbottle method per protocol at Visit 2 (N=99)…………………………………….…….72

Table 10: Infant dietary intake at 0.4 months of age (Visit 1, all infants fed CMF) determined by a 1-day reported diet record… …………...... 73

Table 11A: Dietary intake variables of CMF feedings at 0.4 months of age

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(Visit 1, all infants fed CMF) for subjects who completed the 1-day record per protocol…..……………………………….……..…..…..74

Table 11B: Dietary intake variables of CMF feedings at 0.4 months of age for subjects who completed the 1-day record per protocol.….………....75

Table 12: Comparison of historical data on energy intake to reported energy intake of CMF at 0.4 months of age (Visit 1)………………………76

Table 13: Comparison of historical data on protein intake to reported protein intake of CMF at 0.4 months of age (Visit 1)………………………77

Table 14A: Comparison of EER versus energy intake when all infants were fed CMF at 0.4 months of age (Visit 1)……………………………..….78 . Table 14B: Comparison of AI for protein versus protein intake when all infants were fed CMF at 0.4 months of age (Visit 1)…………………...... 78

Table 15A: Dietary intake variables for infants randomized to feed CMF at 0.6 months of age (Visit 2)……………………………………………..79

Table 15B: Dietary intake variables for infants randomized to feed EHF at 0.6 months of age (Visit 2)...... 80

Table 16A: Comparison of historical data on energy intake to energy intake of CMF fed infants at 0.6 months of age (Visit 2)……………...……..81

Table16B: Comparison of historical data on energy intake to energy intake of EHF fed infants at 0.6 months of age (Visit 2)…...... 82

Table 17A: Comparison of historical data on protein intake to protein intake of CMF fed infants at 0.6 months of age (Visit 2)...... 83

Table 17B: Comparison of historical data on protein intake to protein intake of EHF fed infants at 0.6 months of age (Visit 2)……………………..84

Table 18A: Comparison of EER to energy intake of CMF fed infants at 0.6 months of age (Visit 2)……………..…….…………………………………85

Table 18B: Comparison of EER to energy intake of EHF fed infants at 0.6 months of age (Visit 2)……………………….....….…………………….....85

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Table 19A: Comparison of AI for protein versus protein intake of CMF fed infants at 0.6 months of age (Visit 2)…………………………………….…86

Table 19B: Comparison of AI for protein versus protein intake of EHF fed infants at 0.6 months of age (Visit 2)…………………………….……....…86

Table 20: Dietary intake variables by formula group for infants who completed the TWbottle method at 0.6 months of age (Visit 2)……………….....87

LIST OF FIGURES

Figure 1: Schedule of events for Visit 1 and Visit 2……………………...…...89

Figure 2: Subject count for 1-day reported diet record……………….……….90

Figure 3: Subject count for the 3-day TWbottle method……………….….….…91

ABSTRACT

Background: Formula fed (FF) infants, the majority of whom are fed a standard cow milk infant formula (CMF), have greater weight gain than breast fed (BF) infants; this is of concern as accelerated weight gain in infancy is associated with greater obesity risk later in life. However, FF infants are not a homogenous group; infants fed an extensive protein hydrolysate formula (EHF) appear to have normative weight gain

(similar to that of BF infants), while infants fed CMF experience accelerated weight gain. The energy balance mechanisms contributing to differential weight gain are unknown; it is hypothesized to be due in part to differences in energy intake.

Aims: Determine energy (kcal/kg/day) and protein intake (g protein/kg/day), by formula type, in a contemporary cohort of healthy term FF infants in the first month of life. To compare nutrient intake to the estimated energy requirement (EER) and protein adequate intake (AI) of the 2005 Dietary Reference Intakes (DRIs).

Design: Infants in this analysis are part of an ongoing randomized controlled trial

(RCT) on the effect of CMF versus EHF formula on growth, energy balance, and satiety in healthy term infants.

Methods: At study entry, (Visit 1) all infants were fed CMF for approximately for one week; formula intake was determined by a 1-day diet record. At Visit 2, a 3-day supply of pre-weighed bottles with measured amounts of the randomized formula

(either CMF or EHF) were provided; infants were fed only from these bottles and formula intake was determined by 3-day test weighing of the bottle (TWbottle).

Results: When all infants were feeding CMF (Visit 1, mean infant age 12.2±2.8 days), energy intake was higher than the EER and protein AI by 20.7% and 83.0%, respectively. At Visit 2 (mean infant age19.3±3.0 days) infants randomized to feed

EHF had significantly (p<0.01) lower energy intake than infants fed CMF, 101.5±25.6 versus 125.2±26.1 kcal/kg/d, respectively. EHF fed infants had significantly (p<0.05) higher protein intake than CMF fed infants (2.9±0.7 versus 2.6±0.5 g/kg/d, respectively). Energy intake of CMF fed infants significantly (p<0.01) exceeded the

EER by 13.6%; energy intake of EHF fed infants was lower (6.6%) than but not significantly different (p=0.06) from the EER.

Conclusion: When all infants were feeding CMF, intake exceeded the energy EER and protein AI. Upon consumption of their randomized formula, CMF fed infants exceeded the energy EER and the protein AI, while EHF fed infants had an intake similar to the EER and greater than the protein AI. Energy intake of CMF fed infants was greater than energy intake of EHF fed infants, suggesting the protein composition of the formula influences intake.

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Chapter 1

INTRODUCTION

There is accumulating evidence that nutrition in early life has an impact on both child and adult health outcomes 1-3. Age appropriate weight gain, as defined by age and gender specific growth reference data from the World Health Organization

(WHO) and the Centers for Disease Control (CDC) is an indicator of appropriate nutritional status in infancy; however “rapid weight gain”, defined as a change in weight for age z-score or weight for length z-score of greater than 0.67 standard deviations, in the first six months of life, has been shown to be a risk factor for overweight/obesity in both child and adulthood4-9. Diet, and in particular energy and protein intake, are factors that contribute to infant weight gain10,11. Energy intake in excess of requirement may be a contributing factor to the accelerated weight gain observed in FF versus breastfed (BF) infants10-16.

The majority of standard infant formulas are made from cow’s milk; however cow’s milk has an amino acid pattern tailored to meet the nutrient needs of the calf. In order to provide the human infant with sufficient quantities of all essential amino acids, the total protein concentration of infant formula needs to be greater than the total protein content of breast milk; as such most CMFs contain 13 – 14 g protein/L as opposed to breast milk which averages 11-12 g protein/L17. The increased protein

1 content of infant formula translates into higher protein intake in FF infants compared to BF infant for the same total volume, and this is thought to play a role in the increased weight gain commonly observed in FF infants versus BF infants. This concept is referred to as the “Early Protein Hypothesis”, which proposes that greater weight gain in FF infants is partly caused by higher protein intake 18,19; it is thought that protein intake in excess of physiologic needs leads to excess circulating concentrations of amino acids which promotes higher plasma concentrations of insulin and IGF-1, which in turn causes increased weight gain and adipogenic activity.

Koletzko and colleagues19 tested the “Early Protein Hypothesis” in 1600 healthy term infants in the European Community. Infants were randomized to feed either a high protein infant formula (HP=20.5 g/L) and follow-on formula (HP=32 g/L) or a low protein infant formula (LP=12.5g/L) and follow-on formula (LP=16 g/L) for the first year of life. Significant differences in weight-for-length (WFL) Z-score between the

HP and LP formula groups emerged at 6 months of age, with infants in the HP group having a significantly higher WFL Z-score compared to infants in the LP group; and this difference was maintained to the end of the study period (two years of age). This study provides evidence that total protein intake in infancy, in addition to total energy intake, is also a driver of infant weight gain and growth.

In the United States (US), there are many types of infant formulas available in the market; one way that infant formulas can be classified is by protein composition and form, such as cow milk formula (CMF) and extensive protein hydrolysate formulas (EHF). For CMF, the protein source is intact casein and whey proteins, and

2 most CMF formulas have a total protein content of 13-14 g/L20. For EHF, the protein is extensively digested (hydrolyzed) by enzymes that cleave the peptide bonds, yielding small peptides and mostly free amino acids (FAAs). EHF has a total protein content of 19 g/L and it is often recommended for infants with a cow milk allergy20.

Ventura and colleagues20 reported total FAA concentration of common CMF to be

523-864 umol/L and EHF to be 80,000 to 85,000 umol/L. As a reference, breast milk has total FAA concentration of 3019 umol/L21.

Research by Mennella and colleagues22 suggests that differences in the protein composition (FAA content) between CMF and EHF plays a role in the growth and feeding behavior of FF infants. In their randomized controlled trial of healthy infants,

0.5 month old infants were randomized to feed CMF or EHF for seven months22. At seven months of age, infants fed EHF had significant lower WFL Z-score compared to infants fed CMF, this difference was first observed at two months of age and continued through 7.5 months of age. Moreover the weight gain trajectory of infants fed EHF was similar to that of BF infants, while infants fed CMF had a more rapid weight gain trajectory. With respect to energy intake, infants randomized to feed EHF consumed less volume of formula to satiation during laboratory test feedings, compared to CMF fed infants, starting at one month of age, suggesting that the higher rate of weight gain observed in CMF fed infants may have been due to increased energy intake. These findings are contrary to that of Koletzko and colleagues19 since

EHF formula has a higher total protein concentration than CMF, and the EHF formula has higher total protein than the LP formula used by Koletzko. Therefore it appears

3 that the form of protein in the infant formula, intact protein (found in standard CMF) versus extensively hydrolyzed protein (found in EHF), also plays a role in infant growth. Further, the study by Mennella and colleagues22 suggests that a greater understanding of energy and protein intake by formula type (CMF versus EHF) is needed.

In order to understand the role energy and protein intake play in influencing growth, it is imperative to have valid and reliable measures of intake. The gold standard for the measurement of energy and protein intake in infancy is the test- weighing (TW) method23. For the FF infant, the TW method measures the weight of the bottle prior to and after feeding the infant and the difference (after adjustment for density of infant formula) represents the volume of intake; herein referred to as

TWbottle. Using the TWbottle method, experimental research studies conducted from

1986 to 1998 reported that energy intake of FF infants, the vast majority being fed a standard CMF, at one month of age ranges from 108 – 125 kcal/kg/d (see Table 1).

Energy and protein intake of FF infants can also be compared to authoritative recommendations for intake such as those provided by the Recommended Dietary

Allowances (RDA), which are now known as the Dietary Reference Intakes (DRI).

The 1989 RDA based the estimated energy requirements (EER) of infancy on reported energy intake of ad libitum fed healthy infants (not the TWbottle method). The 1989

RDA was criticized for overestimating energy needs of infants, since the data were subjective rather than objective in nature17. The 2005 DRI based EER for infants on measurements of total energy expenditure (measured using the doubly labeled water

4 technique) and energy cost of growth24; this empirically derived equation is widely supported by the scientific community because it based the EER on measured versus self-reported data. Interestingly, the EER calculated by the 2005 DRI for infants is

15% lower than RDA for energy proposed in the 1989 RDA17,24.

The recommendation for protein requirements also decreased from the 1989

RDA to the 2005 DRI. The 1989 RDA determined protein requirements based on human milk protein intake of BF infants in the United States; the recommendation for infants from birth to three months of age was set to 1.68 g of protein/kg/d, using published data provided by Butte and colleagues in 198425. The 2005 DRIs for protein intake was based again on observed mean protein intake of infants fed with human milk, but the recommendation was set for a wider age range, birth to six months of age, and it was 1.53 g of protein/kg/d. This value is lower than the one proposed in

1989 RDA, because the 2005 DRIs used published data from several studies to estimate protein content in human milk25-28 (average protein content was set to 11.7

25,29 g/L) and the average volume of milk intake determined by the TWinfant ( 780 ml).

The overall aim of this thesis is to examine early measures of energy and protein intake of an ongoing randomized controlled trial (RCT) designed to examine the effect of formula composition on growth, energy balance, and satiety in healthy term infants.

This analysis will include the measurement of energy intake per kilogram of body weight (kcal/kg/day), protein intake per kilogram of body weight (g of protein/kg/day), feeding frequency (number of feedings/day) and average volume of intake per feeding (ml/feed). These measures will be obtained from a contemporary

5 cohort of healthy term FF infants within the first month of life when infants were feeding the same formula (CMF) and again after infants have been randomized to one of two formulas, which are isocaloric but differ in the protein concentration and form

(CMF, EHF).

Chapter 2

LITERATURE REVIEW

Diet in early infancy plays a role in infant weight gain and growth, yet there is a paucity of recent data on energy and protein intake during infancy. The present literature review discusses historic authoritative recommendations for energy and protein intake of infants and presents data on energy and protein intake of one month old infants during the same time period (1986 to 1998). Next, we review current recommendations for energy and protein intake of infants as outlined by the Dietary

Reference Intakes (DRIs) of 2005, and review data on actual energy and protein intake during the same time period. We conclude with a discussion of the literature to date on weight gain in early infancy and the effect of diet composition on energy intake and infant weight gain.

2.1 History of Energy Requirements and Energy Intake in Early Infancy

Equations used to calculate the energy requirements of infants (and adults) have been empirically derived and published by scientific bodies such as the National

Research Council (NRC) and the World Health Organization (WHO). In the United

States, the equations derived by the NRC are used; the NRC published nutrient

7 requirements as the Recommendation Dietary Allowances (RDA) from 1940 to 1989 and as the Dietary Reference Intakes (DRI) from 1999 to present.

The 1989 RDA for energy requirements endorsed recommendations made by the WHO of 198530, which derived energy requirement from observed energy intake associated with normal growth. The 1985 WHO compiled data on observed energy intake published in studies from 1940 to 1980; this dataset consisted of more than

9000 measurements in both breast fed (BF) and formula fed (FF) infants from studies carried out in the United States, Canada, the United Kingdom, and Sweden. The data on reported intake was analyzed by Whitehead and colleagues31, who added a 5% correction factor to reported energy intake values to account for underreporting, a flaw that at the time was commonly thought to occur in reported intake records in infants and children. In 1989, the RDA for energy intake in infants birth to 6 months of age was 108 kcal/kg/d and for infants >6 months of age to one year of age was 98 kcal/kg/d.

Determining energy requirements based on reported dietary intake has methodological limitations because it is subject to under and over reporting by the caregiver. The calculation of energy requirements based on measured energy expenditure and energy cost of growth is more accurate than calculating it based on reported energy intake, but at the time of the1989 RDA, it was not possible to set requirements based on measured energy expenditure due to the lack of studies17 .

Between 1986 and 1998, there were four scientific studies that provided data on energy intake in FF infants at one month of age. Energy intake in these studies was measured for at least 3 days by test weighing (TW) the bottle before and after feeding in the case of a FF infant, TWbottle. This method has been shown to be accurate for the measurement of formula intake in the FF infant13. Energy intake from each day

(typically 3 days) is averaged and divided by the infant’s weight in kilograms to provide kcal/kg/d.

One of the earliest studies that served as a basis for the 1989 RDA for energy was conducted by Montandon and colleagues 13. This study was designed to determine energy intake of FF infants (N=12), the majority of whom were fed standard cow milk formula (CMF), at one month of age (30±4 days). The energy content of the formula was measured using an adiabatic bomb calorimeter and ranged from 62.8/100ml to

70.6 kcal/100ml. Formula intake was measured by 5-day TWbottle of ready to feed infant formula bottles provided by research staff (the research staff performed pre- and post-weighs). This study found that measured energy intake of FF infants at one month of age was 125 ± 17 kcal/kg/d.

Butte et al 199015 also studied energy intake of FF (N=10) at 1 month of age, with a cross-sectional design. Infant formulas used in the study were CMF-1 (Enfamil,

Mead Johnson Nutrition, Evansville, IN) and CMF-2 (Similac, Ross Laboratories,

Columbus, OH)]. The energy content of the isocaloric CMF-1 and CMF-2 was determined by an adiabatic bomb calorimeter and was 67 kcal/100 g. Energy intake

9 was measured by 5-day TWbottle by the mother. This study found that measured energy intake of FF infants at one month of age was 118±17 kcal/kg/d.

In another study by Butte and colleagues14, authors measured energy intake in

FF(N=17) infants at one month of age in a cross-sectional study. Infant formulas used in the study were CMF with or without iron (Enfamil, Mead Johnson Nutrition,

Evansville, IN).The energy content of the CMF determined by bomb calorimetry was

69.0±0.3 kcal/100 g. Intake was measured by 3-day TWbottle and the study found that measured energy intake at one month of age of FF was 108±18 kcal/kg/d.

Finally, de Bruin et al 199816 examined nutrient intake for the first year of life in exclusively FF (N=23) infants. Energy intake at one month of age was assessed by

5-day TWbottle method. All infants were fed CMF Nutrilon Premium (Nutricia Inc,

Zoetermeer, Netherlands). Energy content of infant formula was determined by calculation of the fat (Rose-Gotlieb procedure), protein (Kejdahl method) and carbohydrate content (lactose by enzymatic procedures and total carbohydrates by difference). The average energy intake for FF infants by gender was 111 ±18 kcal/kg/d for males and 117 ±27 kcal/kg/d for females.

Taken together, these studies found that energy intake of FF infants at approximately at one month of age ranges from 108±18 kcal/kg/d to 125±17 kcal/kg/d, see Table 1. In 1989 the RDA for energy requirements from birth to six months of age was suggested to be 108 kcal/kg/d, and based on these studies, it appears that energy intake of FF infants was either close to or above the recommendation.

2.2 Current Energy Requirements and Energy Intake in Early Infancy

The ability to calculate energy requirements based on actual (measured) total energy expenditure (TEE), as opposed to reported energy intake, became possible due to the validation of the doubly labeled water (DLW) technique in the late 1980s, followed by numerous studies that used this technique to measure (TEE) in infants and adults. The DLW technique, validated for use in humans by Schoeller in 198632, is considered the most accurate method for the measurement of energy requirements in free living individuals; the technique was validated for use in infants by Jones and colleagues in 198733 and Roberts and colleagues in 198634. TEE measured by the

DLW technique includes energy required for basal metabolism, thermic effect of food, thermoregulatory needs, physical activity, and in infants and children, the energy required for growth.

Based upon analysis of the DLW data for infants and very young children, the

2005 DRIs derived a single equation for the calculation of total energy expenditure in infants regardless of gender and based solely on weight. The empirical equation proposed for TEE is the following:

TEE= 89 x weight (kg) -100 (Equation-1)

Since infants and very young children are growing, an allowance for energy deposition was added to the TEE to derive EER. This energy deposition allowance is the average of energy deposition for boys and girls of similar ages. The EER is equal

11 to the sum of TEE from “Equation-1” plus energy deposition. Specific EERs for age proposed by the 2005 DRI are:

Infants 0 to 3 months of age: [89 x weight (kg) – 100] + 175 kcal

Infants 4-6 months of age: [89 x weight (kg) – 100] + 60 kcal

Infants 7 to 12 months of age: [89 x weight (kg) – 100] + 22 kcal

EER calculated by these equations are15% lower than energy requirements calculated by the 1989 RDA recommendation.

A study carried out by Butte and colleagues in 201035, suggest that energy intake in early infancy may be higher than estimated energy needs (EER). In brief, the authors examined data from the 2008 Feeding Infants and Toddlers Study (FITS), a cross sectional survey from national random sample of children in the United States

(US) under the age of four years, which included 382 BF and FF infants, birth to five months of age. Diet intake was assessed by a 24-hour dietary recall in which caregivers recall all feedings, feeding times, and amounts of each feeding for the past

24-hours. Dietary intake data was analyzed using Nutrition Data System for Research

(NDSR). Using median weights and heights of US children from the Center of Disease

Control (CDC) growth charts36, estimated energy requirement (EER) was calculated and compared to reported energy intake. Mean reported energy intake exceeded the

EER by 8% in infants from birth to five months of age and by 22% for infants aged six to 11 month. It is important to note that energy intake was reported for all infants as a group; energy intake was not stratified by feeding type (breast fed, formula fed, or mixed fed infants). Findings presented by Butte and colleagues35 suggest that energy

12 consumption in early infancy may be higher than energy needs. However, findings should be interpreted with caution since energy intake was assessed through a recall method (a 24-hr diet recall), rather than the gold standard TWbottle method, and reported intake may be biased due to over reporting or overestimation of portion size by caregivers17,37. Another limitation of Butte et al 201035, is that EER was determined using the median weights of US infants from the CDC growth charts, as actual weights of infants in the 2008 FITS study were not collected.

2.3 History of Protein Requirements and Protein Intake in Early Infancy

The 1989 RDA determined protein requirements for the first months of life was based on reported intake data of BF infants, because at that time it was not possible to estimate protein allowances for growth and maturation of body composition in infancy. The scientific community at that time, concluded that healthy infants of well-nourished mothers who were either breastfed or fed human milk by the bottle, grew at a satisfactory rate for the first four months of life25,38 and that the range of protein intake by breastfed infants varied from 2.43 g/kg/d in the first month of life to 1.51 g/kg/d in the fourth month of life, averaging 2.04 g/kg/d in the first three months of life30. The RDA of 1989 took into consideration previous reports25,30,38 but based its recommendation on one study conducted in the United States by Butte and colleagues in 198425, which concluded that BF infants grew satisfactorily from birth to three months of life at a mean protein intake of 1.68 g/kg/d.

Between 1986 and 1998, there were three scientific studies that included measurements of protein intake of infants at one month of age. Protein intake in these studies was measured for at least 3 days by TWbottle method. Protein content in infant formula or breast milk was determined by the Kjedahl method in all the three studies.

The Kjeldahl method involves the determination of total nitrogen in infant formula; total nitrogen is then converted to total protein, using the factor of 6.25 (which corresponds to 0.16 g of nitrogen per gram of protein).

Montadon et al 198613 studied protein intake at one month of age in infants

(N=12) who were being fed cow milk formula (75%) or soy formula (25%). Total protein in formulas used in the study varied from 10.6 to 15.5 g of protein/L and 15.2 to 19.6 g of protein/L for soy formula (SF). Authors measured formula intake using a

5-day TWbottle method and the average estimated protein intake for these FF infants was 2.6±0.7 g/kg/d.

Butte et al 199014 studied protein intake in FF (N=10) infants at one month of age in a cross-sectional study. Protein content of infant formulas was determined by the Kjeldahl method and total protein content for CMF 1 (Enfamil™, Mead Johnson

Nutrition, Evansville, IN) was 14.9±0.3 g/kg and CMF 2 (Similac™, Ross

Laboratories, Columbus, OH) was 14.6±0.2 g/kg. Intake was measured by 5-day

TWbottle method and the estimated total protein intake for these FF infants was 2.2±0.3 g/kg/d.

Finally, de Bruin et al 199816 examined protein intake in FF infants (N=23) fed a standard CMF (Nutrilon Premium (Nutricia Inc, Zoetermeer, Netherlands). Protein

14 content of the formula was determined by the Kjeldhal method and was 14 g/L. Intake at one month of age was assessed by 5-day TWbottle and the average protein intake for

FF infants, reported by gender, was 2.06±0.39 g/kg/d for males and 2.10 ±0.46 g/kg/d for females,

In summary, protein intake of FF infants measure by TWbottle method ranged from 2.06±0.39 g/kg/d to 2.6±0.7 g/kg/d (see Table 2), a value that was higher than the protein allowance of 1.68 g/kg/d recommended from birth to three months of age by the 1989 RDAs. It is important to note the vast majority of infants in these studies were fed a standard CMF.

2.4 Current Protein Requirements and Protein Intake in Early Infancy

Protein requirements in infancy were re-evaluated in the 2005 Dietary

Reference Intakes. The 2005 DRI for protein intake recognizes human milk as the gold standard for infant nutrition and recommends it as the sole nutritional source for infants from birth to six months of life 39. Therefore, the recommended intake of protein is based on an Adequate Intake (AI) that reflects the mean protein intake of infants fed with human milk. The AI for protein was calculated from data provided by studies in which the intake volume of human milk was measured by test weighing of the infants, before and after each feed. Then the average intake volume was multiplied by the average protein concentration of the human milk reported by several studies25-

27,28.

The protein AI for infants from birth to six months of age is based on an estimated average volume of milk intake of 0.78 L/d10,29 for this age group, and an average protein content of human milk of 11.7 g/L. This is the average protein content of human milk during the first six months of lactation from studies 25-28 in which the sample size was at least 10 subjects and actual data were provided. These values are in the range of protein content reported in other studies. Multiplying this amount by the estimated average volume of intake of human milk for infants form birth to six months, the AI would be 11.7 g/L * 0.78 L/d= 9.1 g/d and considering a reference weight of six kilograms24, the recommendation for protein intake for infants from birth to six months of age is 1.53 g/kg/d

Butte and colleagues35 assessed protein intake in a contemporary sample of US infants (N=382) from birth to 5 months of age, using data from the 2008 Feeding

Infants and Toddlers Study (FITS). Diet was assessed by 24-hour dietary recall. Diet data was analyzed using Nutrition System Data for Research (NDSR) 2008, and protein intake was expressed in grams per day. Infants in this study had a mean protein intake of 11.5±0.20 g/day (mean±SEM) with the lowest and highest quartile being 8.4 g/d and 16.9 g/d, respectively. Protein intake of these infants was above the recommendation of 9.1 g/d set by the DRIs of 2005. The infants in the study were reported to be BF, FF and mixed fed, however protein intake was reported for the total sample and not by feeding group (BF, FF or mixed fed).

Both historical and recent studies have consistently found that FF infants have greater protein intake when compared to the 1989 RDAs and 2005 DRIs protein

16 recommendation. This is not surprising since both the RDA and the DRI based recommendations for protein intake on the protein intake on the BF infant; and mature breast milk has a lower protein concentration than most infant formulas. The majority of infant formulas are made from cow’s milk, which contains different amounts of certain essential amino acids compared to human milk. In order to provide the human infant with sufficient quantities of all essential amino acids, the total protein content of infant formula has to be higher than breast milk; most CMFs contain 13 – 14 g protein/L as opposed to breast milk, which averages 11-12 g protein/L. The increased protein intake of FF infants is thought to play a role in the increased weight gain commonly observed in FF, versus BF infants.

2.5 Weight Gain in Infancy and Later Life Weight Status

Breastfeeding is considered the gold standard for infant feeding and early nutrition, however not every mother is able to breastfeed. When a mother is not able to breastfeed, infant formula is considered the next best feeding alternative. In the United

States (US), there is a high prevalence of formula feeding; about 60% of infants have been exposed to formula either exclusively or as a supplement of breastfeeding by 4 months of age40.

Several prospective cohort studies provide evidence that increased or “rapid weight gain” during early infancy, birth to six months of age, affect weight status later in life at three, four, seven, eight, 17 and 20 years of age. “Project Viva”, a cohort study of 559 mother-child pairs in the United States, followed infants from birth to

17 three years of age5. More rapid increases in weight for length Z-scores (WLZ) in the first 6 months of life were associated with higher odds for obesity, higher BMI Z-score and higher adiposity at 3 years of age5. The obesity prevalence at three years of age was 40% among infants in the highest quartile of WLZ at both birth and 6 months of age. The use of WLZ is the strength of this study since it is a better proxy of infant adiposity as opposed to weight alone.

A cohort of 248 Swedish children followed from birth to 17 years was studied by Ong and colleagues9. “Rapid weight gain” in infancy (birth to six months) and in early childhood (three to six years), defined as gain in weight-for-age Z-score (WAZ) of > 0.67, was associated with larger fat mass, relative fat mass, fat free mass, waist circumference, and BMI at 17 years of age9. This study also found a higher prevalence of “rapid weight gain” during infancy (25.4%) than during early childhood (8.8%), suggesting infancy as opposed to childhood, as a more beneficial period for obesity prevention programs.

“The Dortmund Nutritional and Anthropometric Longitudinal Design Study” known as DONALD study in Germany, followed subjects (n = 206) from birth to seven years of age6. In brief, this study collected repeated anthropometric measurements between six months and seven years of age. Children whose WAZ increased more than 0.67 standard deviations between birth and two years of age were defined as “rapid growers” and children with an increase of less than 0.67 in their

WAZ were defined as “normal growers”. When compared to “normal growers”, “rapid growers” had a significantly greater BMI and percent body fat by two years of age,

18 and such differences persisted until seven years of age, when “rapid growers” also had higher risk of overweight.

A cohort of 616 children participating in the Special Supplemental Nutrition

Program for Women, Infants and Children (WIC) in New York were examined at birth, six months and four years of age41. A higher rate of weight gain between birth and six months of age was significantly associated with being obese (BMI ≥95th percentile) at four years of age (odds ratio [OR]: 1.4; 95%CI, 1.3-1.6). The study sample was multi-ethnic (32% Hispanic, 19% Black and 49% White), which allowed authors to explore ethnic differences in weight gain during infancy. Hispanic children had twice the odds of being overweight at 4 years of age than children of other ethnicities (OR): 2.2; 95%CI, 1.5-3.3). This study is of particular interest because it had a sample similar to the projected US population (increased Hispanic population) and identified a higher risk for being overweight for the Hispanic ethnic group.

A cohort study of European American infants, N=653, who were exclusively

FF and were measured at eight, 14, 28, 42, 56, 84 and 112 days of life, with a follow up anthropometric assessment at 20-30 years of age, was conducted by Stettler and colleagues42. Weight gain in the first week of life was identified as an important factor in weight status at 20-30 years of age. After adjusting for cofounder variables (gender, birth weight, type of infant formula used, age at interview, gestational age, maternal pre-pregnancy BMI), greater weight gain during the first week of life (birth to eight days of age) was associated with adulthood overweight status (OR for each 100 g

19 increase was 1.28 (85%CI: 1.08-1.52) as was weight gain during the first 112 days of life (OR of 1.04 (95%CI: 1.01-1.08).

A subsample of 300 African Americans subjects, who were part of the

Collaborative Perinatal Project cohort and who were measured at birth, four months, seven years and 20 years of age were studied by Stettler and collegues43. A rapid rate of weight gain in infancy, defined as increase in WAZ >1.0 between birth and four months, was associated with greater odds for obesity at 20 years of age (OR: 5.22;

95% CI 1.55-17.6, p=0.008), weight status at seven years of age was also associated with odds for obesity at 20 years of age (OR: 11.4; 95% CI 2.23-57.6, p=0.003).

Stettler and colleagues44 also analyzed the entire population from the

Collaborative Perinatal Project cohort, which had N=19,397children that were followed from birth to seven years of age. After adjusting for several confounding factors (birth weight, maternal BMI and socio-economic status), rapid weight gain from birth to four months of age was associated with 38% increased risk for overweight at seven years of age (OR:1.38 (95% CI: 1.32-1.44). This association was independent from birth weight and weight at 1 year of age.

Taken together, these studies provide consistent evidence, across different populations, time periods, and study designs, that rapid weight gain in infancy is a risk factor of overweight/obesity in both childhood and adulthood. Early infancy, defined as younger than 6 months of age, is of interest since it corresponds to time when the diet of the infant is predominantly milk (breast milk or infant formula), and therefore a time period where the composition of the milk has a large impact on growth. While

20 there are many risk factors for rapid weight gain during early infancy such as pre- pregnancy maternal BMI, weight gain during pregnancy, paternal BMI, socio- economic status, education, gestational age, birth weight; we are most interested in how diet in early life influences nutrient intake, weight gain in infancy and subsequent growth.

2.6 Association Between Diet Composition and Weight Gain in Infancy

Prospective cohort studies have shown that breastfeeding has a protective effect against obesity. For example, a study of a cohort of 32,000 Scottish children in the Child Health Surveillance Program found that breastfeeding status at less or equal to two months of age was associated with reduced risk of obesity at ≤ 10.5 months of age45. In brief, children who had a anthropometric and health data at age six to eight weeks and at age 39 to 42 weeks and whose parents reported infant’s diet were categorized as exclusively BF or exclusively FF; mixed fed infants were excluded from the analysis. At 39 to 42 weeks, BMI was calculated and categorized using

United Kingdom reference data of 1990; obesity was defined as BMI>95th percentile and severe obesity was defined as BMI>98th percentile. Prevalence of obesity was significantly lower in BF infants compared to FF children, with an odds ratio of

0.72(95%CI: 0.65-0.79) for BMI>95th percentile and an odds ratio of 0.70 (95%CI:

0.61-0.80) for BMI>98th percentile.

Similar results were also found in a community-based prospective cohort study in China designed to evaluate long-term impact of early feeding mode on BMI status

21 at two years of age46. Weight and length were collected in 1,098 infants at birth, three months, and 24 months of age; parents also reported feeding pattern of their children.

When comparing exclusive breastfeeding versus exclusive formula feeding at one month of age, breastfeeding was associated with 47% decreased risk of becoming overweight or obese by two years of age.

A protective effect of breastfeeding on later life weight status has been evaluated in numerous studies leading to several systematic reviews and meta- analyses on the topic. A systematic review of 28 research studies, with a total sample of about 300 000 subjects, showed consistently across all reviewed studies, that breastfeeding was associated with reduced risk of obesity when compared to formula feeding (OR: 0.87; 95% confidence interval [CI]:0.85-0.89)47; where and OR <1.0 indicates reduced odds for obesity and a confidence interval that does not include 1.0 indicates significance.

One of the first studies that found growth differences between FF and BF was conducted by Dewey and colleagues and is referred to as the Davis Area Research on

Lactation Infant Nutrition and Growth (DARLING) study12. Authors analyzed anthropometric data from birth to 18 months of age in infants who were BF (N=46) or

FF (N=41) for the first year of life. Feeding groups were matched for socio-economic status, education, ethnic group, and for anthropometric characteristics of the mother and for the infant by sex, birth weight and by age of solid food introduction >4months.

All infants were born with appropriate weight (2.5-3.5 kg) and gestational age (38 to

42 weeks). This study found that the mean weight of BF infants was significant lower

22 than that of the FF group starting at 6 months of age and such differences were maintained until 18 months of age, while length and head circumference values were similar between groups at all time points. Weight for length Z-scores (WLZ) were significantly lower in the BF group from seven to 18 months; suggesting that BF infants were leaner. FF and BF infants had similar weight gain during the first 3 months of life, but BF infants gained weight less rapidly during the remainder of the year. Cumulative weight gain in the first 12 months was 0.65 kg less in the BF group and was significant different when compare to the FF group (p<0.05), also 15% of FF infants and 7% of BF infants had WLZ higher than 90th percentile.

This study was one of the first to characterize the growth differences between

BF and FF infants, in a cohort matched meticulously for most cofounders. Since this study, several other prospective studies have documented that FF infants tend to be larger than BF infants by the end of the first year of life22,45-47 providing substantial evidence that FF infants grow at a faster rate compared to BF infants. However, a recent study by Mennella and colleagues22 has found that FF infants are not a homogenous group in terms of growth; the type of infant formula fed to the infant can differentially effect weight gain.

2.7 Infant Formula Composition and its Effect on Feeding Behaviors and

Growth

There are many infant formulas available in the United States (US) and a common method to classify infant formulas is by the type and form of protein. Cow-

23 milk formula (CMF) is made from cow’s milk and hydrolysate formulas (HF) are made from hydrolyzed milk proteins. In CMF, the protein source is intact casein and whey proteins, this formula is commonly known as standard cow milk formula in the

US, and it accounts for 80% of sales in the formula market48. HF are formulas in which the protein has been either partially or extensively digested by enzymes that cleave the peptide bonds in the milk proteins and then ultrafiltered by molecular size, finally yielding: 1) partial hydrolysate formula (PHF), which are composed mainly of small peptides, and 2) extensively hydrolysate formulas (EHF) composed mostly of free amino acids (FAA). EHF accounts for about 6% of the US market48 and usually recommended by physicians to infants with a cow-milk allergy.

Ventura and colleagues20 determined the FAA profile of the most commonly consumed formulas in the United States. FAA concentration was measured with an automatic amino acid analyzer (model L-8900; Hitachi, Tokyo, Japan), which has a limit of detection (LOD) of 3 picomolar (pM). Total FAA concentration in the formulas varied greatly, please see Table 3. EHF had the highest concentrations of

FAAs (80,375 umol/L), CMF had a total FAA concentration of 864 umol/L, and breastmilk had a total FAA concentration of 3020 umol/L21.

Experimental research suggests that the type of formula the infant feeds affects growth. Menella and colleagues carried out a randomized controlled trial (RCT) with healthy infants, in which 0.5 month old infants were randomized to feed CMF (N=35) or EHF (N=29) for seven months22. The macronutrient composition of the CMF and the EHF formula used in the Mennella study and the present study, were identical and

24 are presented in Table 3. Data on growth and formula intake were collected monthly.

Growth data were converted to Z-scores using WHO growth reference standards and intake volume of a feeding (volume consumed to satiation) was determined through laboratory test feeds. The groups were matched by race, ethnicity and sex; also there were no differences in age at study entry, birth weight or birth length. Results showed that infants fed EHF had significant lower WAZ starting at 3.5 months of age and significant lower WLZ starting at 2.5 months of age compared to infants fed CMF, these differences in Z-scores were maintained until the conclusion of the study (7.5 months). This study found that infants fed EHF showed a normative growth trajectory, similar to the growth of BF infants, while infants fed CMF had a more rapid growth trajectory. The researchers found no differences in number of formula feeding per day across all time points, but infants fed EHF consumed less volume of formula to satiation, during laboratory test feeds, compared to CMF fed infants starting at one month of age; suggesting that the higher rate of weight gain observed in CMF fed infants may be due to increased energy intake.

In order to expand of these findings, Ventura et al 201249 conducted a within- subject experimental study to determine if differences in the free amino acid concentration of the formula contribute to satiation. Infants less than four months of age (n=30) who were currently being fed a standard CMF or standard soy formula completed 3 study visits. At each visit, infants were fed 1 of 3 formulas: CMF, CMF with added glutamate, or EHF. When infants signaled hunger again, they were fed a second meal of CMF. Infants consumed significantly less CMF with added glutamate,

25 and less EHF, than CMF during the first meal. Infants also showed greater levels of satiety after consuming CMF with glutamate or EHF. The results of this study suggest that the free amino acids found in EHF may play a role in satiation.

Finally, other studies have reported that healthy infants randomized to feed

EHF have less daily volume and energy intake when compared to infants fed CMF.

Vandenplas and colleagues50 found that the mean daily volume of intake of infants fed a whey hydrolysate formula (WHF) was significantly smaller than the mean volume of intake of infants fed a whey predominant formula (WF). Authors conducted a double blind randomized prospective study with the aim to examine the nutritional value of two infant formulas: 1) the WF had casein to whey ratio of 40/60 and had a protein content of 1.4 grams of protein per 100 ml and 2) the WHF contained hydrolyzed whey, although the degree of hydrolysis was intermediate between partly hydrolyzed

(i.e., “Good Start, Nestle) and extensively hydrolyzed (i.e., Nutramigen, Mead

Johnson), this WHF had a protein content of 1.5 grams of protein per100 ml. Healthy infants, with no family history of atopic disease were included in the study and were randomized to feed WF (N=20) or WHF (N=25) for the first 13 weeks of life. During this trial, the WF group had a mean daily intake of protein of 2.05 g/kg/d and a mean volume intake is 147.2 ml/kg/day. The WHF group had a mean protein intake was

1.80 g/kg/day and the mean volume of intake was 120.3 ml/kg/day. Mean daily volume intake was estimated through parental report, and was found to be significantly smaller in the WHF group when compared to the WF group (p<0.001).

Hyams and colleagues51 found that daily volume of intake of infants randomized to feed an EHF (Nutramigen) was significant lower than daily intake volume of infants randomized to feed standard CMF or standard soy formula. This study was a prospective double blind study with the aim to investigate stool characteristics by formula type. Two hundred and thirty-four healthy one month old infants were randomized to feed one of four infant formula for 12-14 days: Enfamil

(CMF), Enfamil with Iron (CMF), ProSobee (standard soy formula), or Nutramigen

(EHF). Parents were asked to keep a daily formula intake record for the last 7 days of feeding. Results showed a significant difference in total volume of intake per day between the four formula groups (p<0.03), with infants in the Nutramigen (EHF) group consuming the least amount of formula per day.

2.8 Summary

Research conducted between 1986 and 1998 shows that reported energy and protein intakes of FF infants at one month of age tended to be greater than the recommended energy requirements proposed by the 1989 RDA. At that time, energy and protein recommendations were based on reported intake of healthy infants. The

1989 RDA was criticized for overestimating energy and protein needs of infants and children. Authoritative recommendations for energy and protein were revised in the

2005 DRI. Energy requirements are now based on measured TEE and the energy cost of growth is added to the TEE to obtain the EER. Protein requirements in the 2005

DRI are based on the protein intake of the BF infant measured by the TW method.

“Rapid weight gain” in early infancy has been shown to be a risk factor for overweight/obesity later in life. Diet, and in particular energy and protein intake, are factors that contribute to infant weight gain. Research suggests that energy and protein intake of formula fed (FF) infants, the vast majority being fed a standard cow milk formula (CMF), are in excess of requirements, which may be a contributing factor to the accelerated weight gain observed in FF versus breastfed (BF) infants. In particular, research by Koletzko and colleagues19 suggests that the total protein intake in FF infants is a driver of infant weight gain and growth. On the other hand, research by

Mennella and colleagues22 suggest that total protein intake is not the full story, and that the form of the protein in the infant formula also plays a role; since healthy infants randomized to feed EHF, which contains hydrolyzed proteins (small peptides and free amino acids) experienced normative growth, that is growth similar to BF infants, compared to infants fed CMF who demonstrate greater weight gain.

There is a lack of data on energy and protein intake of a contemporary cohort of FF infants fed CMF or EHF, and how this intake relates to current recommendations for protein and energy intake as outlined in the 2005 DRIs.

Chapter 3

STATEMENT OF THE PROBLEM

There is accumulating evidence that nutrition in early life and early rapid weight gain can have substantial influences on adult health outcomes such as weight status and obesity. Rapid weight gain in infancy is a risk factor for overweight and obesity in later life, and formula fed (FF) infants as a group, have been shown to have greater weight gain in the first year of life compared to breast fed infants. However, there is a paucity of research on energy and protein intake of FF infants in early infancy (first month of life) and how such intake compares to current guidelines for energy/protein intakes recommended in the Dietary Reference Intakes. Further, there is a lack of research on feeding related behaviors; such as frequency of feedings

(number of feeding per day) and average volume per feeding (ml/feed), in a contemporary sample of FF infants fed formulas of differing protein composition.

Chapter 4

AIMS

The overall aim of this thesis is to determine energy intake (kcal/kg/day), protein intake (g protein/kg/day), feeding frequency (number of feedings/day) and average volume of intake per feeding (ml/feed); in a contemporary cohort of healthy term FF infants within the first month of life when infants were feeding the same formula

(CMF) and again after infants have been randomized to one of two formulas, which are isocaloric but differ in the protein concentration and form (CMF, EHF).

4.1 Specific Aims

Primary Aim 1: Compare measured energy intake to historical data on energy intake, at a time point when all infants are fed CMF (1-2 weeks of age), and again when infants are randomized to CMF or EHF (2-3 weeks of age).

Primary Aim 2: Determine if measured energy intake meets or exceeds the current

Dietary Reference Intake (DRI) for estimated energy requirements (EER), at a time point when all infants are fed CMF (1-2 weeks of age), and again when infants are randomized to CMF or EHF (2-3 weeks of age).

Primary Aim 3: Compare measured protein intake to historical data on protein intake,

30 at a time point when all infants were fed CMF (1-2 weeks of age), and again after infants were randomized to CMF or EHF (2-3 weeks of age).

Primary Aim 4: Determine if measured protein intake of this contemporary cohort of infants meets or exceeds the current DRI for protein requirement, at a time point when all infants were fed CMF (1-2 weeks of age), and again after infants were randomized to CMF or EHF (2-3 weeks of age).

Primary Aim 5: Determine energy intake, protein intake, feeding frequency and average volume of intake per feeding, by type of formula, CMF or EHF, at 2-3 weeks of age.

Chapter 5

METHODS

5.1 Subjects

Newly parturient women who intended to exclusively formula feed their infants were recruited from hospital nurseries in the Philadelphia area, WIC (Special supplemental nutrition program for women, children and infants) clinics, advertisement in newspapers, and mass mailing to pregnant and newly parturient women. Mothers interested in participating in the study called the Monell Center and were screened to determine if they met the inclusion/exculsion criteria

Inclusion critera included that infants must be: healthy, born at full term (≥37 weeks to ≤42 weeks), with appropriate birth weight for gestational age (2500-4500 grams or 5.5 to 9.9 lbs), and be feeding a standard cow milk formula (CMF) for at least two days prior to enrollment. Exclusion criteria included the presence of medical condition that interferes with feeding (i.e.: congenital malformations like cleft palate or hemangiomas) or metabolism [i.e.: intolerance to cow milk intact protein (casein) or cow milk sugar (lactose)] or infants feeding an extensive protein hydrolysate formula (EHF). Infants were also excluded if there was presence of infectious or systemic diseases, documented systemic congenital infections (i.e., cytomegalovirus), or evidence of significant cardiac, respiratory, endocrinologic, hematologic,

32 gastrointestinal disease; if they were receiving supplements, insulin, or growth hormone; if their mother had gestational diabetes; or if they were a relative (son, daughter, nice, nephew, cousin, aunt, uncle, sibling) of ancillary personnel connected to the study.

5.2 Research Design

This research is part of randomized, controlled, double blind (mothers and investigators are blinded to formula type) longitudinal study of energy balance and growth in healthy term infants randomized to feed one of two infant formulas for the first year of life. The study formulas were a standard cow milk formula (CMF) and an extensive hydrolyzed formula (EHF), referred as formula A and formula B, respectively. Infants were approximately one to two weeks of age at enrollment and followed until they were 18.5 months of age. Mothers, who were unaware of the hypothesis, went to the Monell Center each month and during each visit research personnel weighed and measured the infants, distributed the next month's supply of formula, and performed scheduled tests. Study visits to assess total energy expenditure

(TEE) and energy/protein intake by the TWbottle method occurred at approximately 2-3 weeks of age, three months of age, and 12 months of age. For the purpose of this thesis, we focus on the measurements collected during Visit 1 (study entry) and Visit

2, which occurred approximately one week apart, please see Figure 1, for schedule of events.

During Visit 1, mothers were consented and data on anthropometry, demography, health history, diet and feeding practices were collected. Mother-infant dyads were randomized into groups that differed in the type of formula that mothers were asked to feed their infants for the remainder of the first year of life (formula A or formula B). Randomization was stratified by gender and race of infant, and both mothers and investigators were blinded to which formula the infant was assigned. At the end of this visit mothers were given enough standard CMF, namely Enfamil™

(Mead Johnson Nutrition, Evansville, IN) to feed their infants until Visit 2, which occurred approximately one week later. Mothers were also given a 1-day diet record form to record the time and amount of formula consumed of each feeding for a 24- hour period. Mothers were expected to return this diet record during their second visit to the Monell Center (Visit 2).

Visit 2 occurred when infants were 2-3 weeks of age, and at the conclusion of this visit mothers were sent home with a 3-day supply of feeding bottles pre-filled with the assigned formula and instructed to add water to level designated on the bottle when the infant was ready to feed. Mothers were instructed to feed as they usually would, and place all bottles, empty or partially empty, into a bag for return to the

Monell Center (see Section 5.5 for more details on the TW bottle method). Mothers also kept a written log of each bottle offered and the time that is was offered. Data on anthropometry, health history and feeding practices were also collected during this visit.

5.3 Study Formulas

Formulas included a standard cow milk protein formula (CMF; Enfamil, Mead

Johnson Nutrition, Evansville, IN; Formula A) or an extensive protein hydrolysate formula (EHF; Nutramigen, Mead Johnson Nutrition, Evansville, IN, Formula B).

Study formulas were labeled by the manufacturer as infant formula A or formula B and were packaged in otherwise identical containers; these formulas had identical formulations to those used by Mennella and colleagues 22 , who made the initial discovery that healthy infants randomly assigned to feed EHF ingested less formula to satiation and had a normative growth pattern (similar to the growth of BF infant) when compared to infants randomized to feed a standard CMF.

Table 3 lists the macronutrient composition and amino acid profile of the study formulas. The formulas were isocaloric and contained a similar fat blend but differed in the structure of the protein. CMF contains two sources of protein, nonfat milk and whey protein concentrate, which provide protein in the form of intact casein and whey proteins. The sole source of protein in EHF is casein hydrolysate, which is a casein protein that has been enzymatically treated (cleaving the peptide bonds), resulting in higher levels of free amino acids (FAAs)20 and small peptides52.

5.4 Testing Procedures Overview

During Visit 1 and Visit 2 to the Monell Center, anthropometric demographic, health history, dietary intake, and feeding practices data were collected. Mothers were unaware of the study hypothesis and research personnel did not provide any

35 instruction to how or when to feed infants. Figure 1, details the testing procedures occurred and a more detailed description of each method then follows.

5.4.1 Anthropometry

Infant birth weight and birth length were determined by maternal report. Infant head circumference, recumbent length and weight at study Visit 1 (1-2 weeks of life) and Visit 2 (2-3 weeks of life) were measured in triplicate by trained personnel using standard anthropometry techniques53 as described below.

a) Weight: measured with a digital scale accurate to 0.001 kg.

b) Recumbent length: measured with a length board accurate to 0.1 cm.

c) Head circumference: measured with a tape measure accurate to 0.1 cm.

Raw anthropometric measures were compared to the World Health Organization growth standard54 and the following Z-scores were calculated: weight for age (WFA), length for age (LFA), head circumference for age (HFA), and weight for length

(WFL).

5.4.2 Demographics and health history assessments

At study entry (Visit 1) demographic and health history data were collected using the following forms.

a) Demographics form: collected data related to: the mother’s age, education,

family income and race.

b) General interview form: collected data related to maternal weight gain during

pregnancy, smoking status, number of children in the home, and the birth order

of the infant enrolled in the study.

c) Diet history form: collected data about the diet (i.e. cow milk infant formula,

soy formula, breastmilk, etc.) fed to the infant from birth until enrollment in

the study.

d) Health history form: collected data related to maternal and infant health at the

start of each visit using a 5-likert scale (Very healthy=1, Healthy=2, Slightly

sick=3, Sick=4 and Very sick=5). Data on any illnesses since last visit (i.e. ear

infection, cold, diaper rash, diarrhea, constipation etc.) and prescription or over

the counter medication use.

5.4.3 Energy intake measurement

a) Energy Intake (1-day or 24-hour record), CMF infant formula:

Formula intake immediately following Visit 1, (when all infants were feeding

CMF) was determined using a 1-day diet record in which mothers were asked

to record the time and amount of all feedings for a 24-hour period. Infants were

approximately 1-2 weeks of age at this diet assessment.

b) Energy Intake (TWbottle): CMF (Formula A) versus EHF (Formula B)

Formula intake immediately following Visit 2, (when infants were feeding

their assigned formula) was determined by a 3-day TWbottle method. For the

TWbottle method, mothers were given a 3-day supply of pre-weighed bottles

with a measured amount of the assigned formula powder. Each bottle was

labeled with a number from one to 30 and the pre-weight of each bottle was

measured and recorded by bottle number. Immediately before feeding, mothers

were instructed to add water to a level designated on the bottle (6 ounces of

water). Mothers were also instructed to feed their infant as usual and keep all

bottles (empty and/or partial consumed) for eventual return to the study site.

Upon return of bottles, study personnel weighed each bottle and determine the

post-weight, from which formula intake volume was then determined55,56,57.

Mothers were also given a diet record form to record the time of their infants’

feeding, the bottle number, the amount left in the bottle at each feeding, and if

anything was added to the bottles. Detailed verbal and written instructions for

measuring and recording intake were given. Upon return of bottles, study

personnel reviewed the completed diet records and queried the family for

missing or additional information when necessary. They also queried the

mother regarding infant acceptance and consumption of the assigned formula

and use of the TW bottles. Study personnel also gathered information about

health status of the infants during the testing period. If the infant was reported

sick, the mother was asked about the type sickness and if infant ate

significantly less than usual due to sickness.

Both diet records, 1-day diet record and 3-day TWbottle diet records were analyzed for nutrient intake using the Nutrient Data System for Research (NDSR,

Minneapolis, MN) at the University of Delaware. NDSR is a Windows-based dietary analysis program designed for the collection and analyses of dietary intake data.

5.4.4 Validation of energy intake measurement

All data from 1-day and 3-day intake records were reviewed by study personnel for validity. Decisions about inclusion or exclusion of the data were taken based on the following criteria:

For 1-day diet records:

a) Compliance in feeding assigned formula: if mother did not feed assigned

formula, data was excluded.

b) Completeness of record: if mother did not record feeding for the entire 24-

hour period, data was excluded.

c) Validity of data due to infant’s health: if mother reported that infant was sick

requiring hospitalization during testing period, data was excluded.

d) Validity of data due to record not well kept: if mother filled a bottle log that

was not logical, e.g., multiple bottles recorded as consumed at the same time.

e) Physiological plausibility: was determined using Montandon et al 198613

reported values for average volume of intake per day for infants mostly fed

standard CMF at one month of age (30±4 days) using the TWbottle method;

intake data for infants <30 days old is not available in the literature. The

mean reported intake plus four standard deviations was used to calculate a

maximum intake volume per day of 1115 ml/day. Reported intake over this

amount was excluded from the analysis as being physiologically not possible.

For 3-day TW measures:

a) Compliance in feeding assigned formula: if mother did not feed assigned

formula, data was excluded.

b) Completeness of record: if mother did not record feeding for the entire 24-hour

period, data for that day was excluded.

c) Validity of data due to infant’s health: if mother reported that infant was sick

requiring hospitalization during testing period, data was excluded.

d) Validity of data due to deviations of the TW protocol: if mother rinsed out

bottles, did not return used bottles, or bottles leaked significantly (therefore

affecting the measurement of intake volume), data was excluded

e) Validity of data due to record not well kept: if mother filled a bottle log that

was not logical, i.e.: same bottle fed to the infant on different days, data was

excluded.

f) Physiological plausibility: was determined using Montandon et al 198613

reported values for average volume of intake per day for infants mostly fed

standard CMF at one month of age (30±4 days) using the TWbottle method;

intake data for infants <30 days old is not available in the literature. The mean

reported intake plus four standard deviations was used to calculate a maximum

volume of intake per day of 1115 ml/day. Reported intake over this amount

was excluded from the analysis as being physiologically not possible.

5.5 Statistical Analysis

5.5.1 Preliminary analyses

Missing data and out-of-range values were assessed with univariate statistics

(means, standard deviations, ranges and frequencies). Summary tables of continuous variables and frequencies for categorical variables were provided using descriptive statistics. Data was checked for normality of the distribution and no mathematical transformation was required.

5.5.2 Primary analyses

NDSR software was used to compute dietary intake variables: energy intake

(kcal/day and kcal/kg/day), protein intake (grams of protein/day and grams of protein/kg/day), feeding frequency (number of feedings/day) and average volume of intake per feeding (ml), in a contemporary cohort of healthy term FF infants within the first 1 month of life; when infants were feeding the same formula (CMF), Visit 1, and then again after infants have been randomized to groups of formula that have identical caloric content but differ in protein concentration and form (CMF, EHF), Visit 2.

Energy intake data at Visit 1 and Visit 2 were compared to: 1) historical data on energy intake in one month old infants, and 2) estimated energy requirements

(EER) calculated from the 2005 Dietary Reference Intake (DRI). Protein intake data at

Visit 1 and Visit 2 were compared to: 1) historical data on protein intake in one month old infants, and 2) the 2005 DRI for protein intake. Energy and protein intake at Visit

2 were stratified by formula type, to determine if intake differed by formula group

(independent sample t-test). Energy and protein intake at each time point is expressed per kilogram (kg) of body weight since nutrient requirements are, in part, based on body size24.

For continuous variables, independent t-tests or a Mann-Whitney U test were used, depending on the distribution of the variable, to assess for differences between means; Chi-square test was used to test for differences in categorical variables. All data was analyzed using Dell Statistica (data analysis software system), version 13

(Dell Inc. 2015). To describe the anthropometric characteristics of the study population, infant weight, length, head circumference were normalized to z-scores using WHO Anthro software version 3.2.2 (www.who.int/childgrowth/en/) to calculate age- and sex-specific Z-scores (WHO growth standards). Z-scores for weight-for-age

(WAZ), length-for age (LAZ) and weight-for-length (WLZ), were calculated at two time points, Visit 1 (1-2 weeks of age) and Visit 2 (2-3 weeks of age).

Chapter 6

RESULTS

6.1 Subject Characteristics

One hundred and forty-one mother-infant dyads were enrolled in the study and completed Visit 1. Demographic characteristic of mother-infant dyads are presented in

Table 4. At study entry (visit 1), infants were between the ages of 0.1 and 0.6 months

(mean age was 12.3 ±3.0 days), and a similar number of male (N=67) and female

(N=74) infants were enrolled. Birth weight ranged from 2.2 to 4.3 kg, with a mean of

3.2±0.4 kg. The racial distribution of the infants was 62.2% African American, 21.2%

Caucasian and 16.3% other race (Asian and Mixed Race). The age range of the mothers at study entry was 18.1 to 41.3 years, with a mean of 27± 5.6 years. Pre- pregnancy body mass index (BMI) ranged from 17.2 to 58.8 kg/m2, with a mean of

28.5±8.0 kg/m2. Mean weight gain during pregnancy was 13.4±9.0 kg. Almost half of the families, 47% (N=64), had an income below $15,000; and about a third of the mothers, 37.8% (N=53), reported to have graduated High School.

One hundred and thirty-three mother-infant dyads returned to the Monell

Center for Visit 2, the number of days between Visit 1 and Visit 2 ranged from one to

19 days with a mean of 7.1±3.2. Age of infants at Visit 2 ranged from 0.2 to 0.9 months with a mean age of 19.3±2.9 days.

6.1.1 Characteristics of subjects who completed a 1-day diet record at 0.4

months of age

As seen in Figure 2 and Table 5, of the 141 mothers-infant dyads who consented to participate in the study, eight mother-infant dyads chose to withdraw from the study after the first visit (Visit 1). Of the 133 mother-infant dyads who returned for Visit 2, only 124 of them returned the Visit 1, 1-day diet record of infant feeding. The records were then evaluated for validity, upon which 16 diet records were excluded from the analysis because they were determined to be physiological not possible (N=8), incomplete (N=5), mothers were not feeding assigned formula (N=2), or unreliable (N=1). To this end 108 diet records were included in the analysis to determine intake of CMF at Visit 1.

Characteristics of the infants whose mothers’ returned a 1-day diet record

(N=124) versus those who did not (N=17, where nine subjects did not return the record and eight subjects withdrew), were compared in Table 6. There were no significant differences in (gender distribution, birth weight, age at study entry, and growth parameters in those who returned a 1-day diet record (DR) versus those who did not (no DR).

6.1.2 Characteristics of subjects who completed the 3-day TWbottle method at 0.6

months of age

At Visit 2, mothers were sent home with a 3-day supply of pre-weighed bottles of the assigned study formula (formula A or formula B). Mothers were instructed to

44 feed their infant as usual and keep all bottles (empty and/or partial consumed) for return to the Monell Center; this method of measuring formula intake is known as test weighing of the bottle (TWbottle).

One hundred and thirty-three mother-infant dyads returned to the Monell

Center for Visit 2, sixty-seven mother-infant dyads were sent home with formula A, also referred to as group A, and 66 mother-infants dyads were sent home with formula

B, also known as group B. As seen in Figure3, five mother–infants dyads withdrew from group A and ten mother-infant dyads withdrew from group B; leading to a sample size of 118 mother-infant dyads that completed the TWbottle method (62 infants randomized to group A and 56 infants randomized to group B). Characteristics of the infants that were randomized to study formulas (N=118) versus infants that withdrew

(N=15) were compared in Table 7 and there were no differences in gender distribution, birth weight, age at visit 1, age at Visit 2 and growth parameters between these two groups.

One hundred and eighteen mother-child dyads completed at least one day of the 3-day TWbottle method. Research personnel evaluated the returned materials

(empty/partially consumed bottles and 3-day diet records) for validity using a standardized protocol (see Section 5.4.3 and 5.4.4). As seen in Table 8 and Figure

3), 19 infants were excluded from the analysis (nine infants were excluded from group

A and ten infants were excluded from group B); the reasons for exclusion were: unreliable data (N=13), incompleteness of record (N=3), mothers were not feeding assigned formula (N=2) and diet intake exceeded the physiologically plausible range

(N=1). As such, a total of 99 infants were included in the analysis of dietary intake by the 3-day TWbottle method (53 randomized to group A and 46 randomized to group B), this sample is referred as the per protocol population.

6.1.3 Characteristics of mother-infant dyads by formula group

Demographic characteristics of the subjects who completed the 3-day TWbottle method (N=118) and the sample that completed it according to protocol (N=99) were compared by formula group in Table 9A and Table 9B, respectively. There were no statistically significant differences were found in infant demographic characteristics

(gender distribution, infant age at randomization and raced/ethnicity) and anthropometric Z-scores (weight-for-age (WAZ), length-for age (LAZ) and weight- for-length (WLZ)), maternal/familial demographic characteristics (maternal age, income and education) and maternal anthropometry characteristics (pre-pregnancy

BMI and weight gain during pregnancy).

6.2 Dietary Intake at 0.4 Months (all Infants Feeding CMF), Assessed via a 1-

Day Diet Record

Dietary intake at Visit 1 (study entry) was determined from a 1-day diet record in which mothers recorded time and amount of each feeding for a 24 hour period. At this assessment all infants were receiving CMF; see Table 10 for summary of dietary intake variables. In brief, infant age at Visit 1 ranged from 4 to 19 days, with a mean of 12.2±2.8 days; the mean frequency of feeding was 8.8±1.1 feedings/d, and mean

46 total volume of formula intake was 703.3±166.0 (ml/d). Mean total energy intake was

134.3±32.2 kcal/kg/d and mean protein intake was 2.8±0.6 (g of protein/kg/d). No significant differences in energy intake (kcal/kg/d) and protein intake (gram of protein/kg/day) were observed between the sexes (p=0.09 for energy and p=0.15 for protein).

CMF dietary intake variables assessed with the 1-day diet record we compared by assigned formula group, in Table 11A (intent to treat sample, N=108) and Table

11B (sample who completed the 1-day record per protocol, N=90). In both populations

(intent to treat and per protocol), there were no significant differences in number of formula feedings per day, total volume of intake per day (ml/d), total energy intake per day (kcal/day and kcal/kg/day) and total protein intake per day (g protein/day and g protein/kg/day), which confirms that prior to randomization both groups of infants had similar CMF intake.

6.2.1 Energy intake of CMF fed infants at 0.4 months of age compared to

historical data on energy intake

We compared reported energy intake collected with 1-day diet record to the historical data in Table 12. At 0.4 months of age, when all infants were fed a standard

CMF, their energy intake was 134kcal/kg/day; this value was higher than the range of measured energy intake reported by historical data of FF fed infants at 1 month of age, which was 108 to 125kcal/kg/day.

6.2.2 Protein intake of CMF fed infants at 0.4 months of age compared to

historical data on protein intake

We compared reported protein intake from the 1-day diet record to historical data (see Table 13). At 0.4 months of age, when all infants were fed a standard CMF, their protein intake was 2.8±0.6 g/kg/d; this value was higher than the range of measured protein intake reported by historical data of FF fed infants at 1 month of age, which was .0±0.3 g/kg/d to 2.6±0.7 g/kg/d.

6.2.3 Energy and protein intake of CMF fed infants at 0.4 months of age

compared to the 2005 DRIs

We compared reported energy and protein intake from the 1-day diet record to

Estimated Energy Requirements (EER). The EER for each infant was calculated using the formula proposed by the 2005 Dietary Reference Intakes (DRIs) for infants from birth to three months of age: [89 x weight (kg) – 100] + 175 kcal. At 0.4 months of age, when all infants were feeding CMF, the estimated total energy intake, measured by the 1-day diet record, was significantly higher than the calculated EER:

464.7±109.6 kcal/day versus 384.9±37.4 kcal/day, respectively (p<0.001), exceeding the EER by 20.7 (see Table 14A). The reported protein intake (2.8g/kg/d) was also higher than the Adequate Intake (AI) proposed by the 2005 DRI of 9.1 g/d or 1.52 g/kg/d by 84% (see Table 14B).

6.3 Dietary Intake at 0.6 Months (Infants Feeding Either CMF or EHF),

Assessed via the 3-Day TWbottle Method

6.3.1 Energy intake of CMF and EHF fed infants at 0.6 months of age

compared to historical data on energy intake

Dietary intake variables for infants randomized to feed CMF (N=53) are presented in Table 15A and for infants randomized to feed EHF (N=46) are in Table

15B. At Visit 2, as shown in Table 16A, infants randomized to feed CMF had an energy intake of 125.2±26 kcal/kg/d, this value is similar to one reference in the historical data on energy intake, Montandon et el 198613 but higher than the values reported in the other three references14-16, see Table 1. Infant randomized EHF had an energy intake of 101.5±25.6 kcal/kg/d, this value is lower than the range reported by historical data on energy intake, see Table 16B.

6.3.2 Protein intake of CMF and EHF fed infants at 0.6 months of age

compared to historical data on protein intake

At Visit 2, as shown in Table 17A, infants feeding CMF had a total protein intake of 2.6±0.5 g/kg/day, this value is similar to one of the references in the historical data, Montandon et al 198613 but higher than the values reported by the other two references in the historical data, see Table 215,16. Infant feedings EHF had a total protein intake of 2.9±0.7 g/kg/day see Table 17B, this values is higher than the range of protein intake reported by all the references in the historical data, see Table 2.

6.3.3 Dietary intake of CMF and EHF fed infants at 0.6 months of age

compared to 2005 DRIs

For infants randomized to feed CMF, the measured energy intake was higher than their calculated EER: 459.9±93.8 kcal/day versus 404.9±42.2 kcal/day, respectively (p<0.01), exceeding the calculated EER by 13.5% (see Table 18A). The measured energy intake for infants feeding EHF was lower but not different to their calculated EER: 378.6±59.4 kcal/d versus 405.7±34.3 kcal/d, respectively (p=0.06), falling below their calculated EER by 6.6% (see Table 18B). The protein intake in both groups, CMF and EHF, was significantly higher than the AI for protein in the

2005 DRI, 1.52 g/kg/d, by 69.9% and 89.5%, respectively (see Table 19A and Table

19B).

6.3.4 Comparison of dietary intake by formula type (CMF vs. EHF fed infants)

at 0.6 months of age

Average daily dietary intake at Visit 2 was measured by the 3-day TWbottle method and results by formula group are shown in Table 20. In brief, mean infant age at Visit 2 was similar between the formula groups; it was 19.6±3.2 days for infant randomized to CMF and 19.1±2.8 days for infants randomized to EHF. Frequency of feedings per day was also similar between the formula groups, 7.7±1.1 for CMF fed infants and 7.7±1.2 for EHF fed infants. However, infants randomized to feed CMF

50 had a higher total volume of intake per day (ml/day) and total energy intake (kcal/day and kcal/kg/day) than infants randomized to feed EHF (all p-values<0.01).

Interestingly, infants feeding CMF had a significantly higher average volume of intake per feeding when compared to infants feeding EHF; 91.8±21.7 ml/feed versus

75.1±20.5 ml/feed, respectively (p<0.01). While infants randomized to EHF consumed less total volume per day, they had a higher total protein intake (grams of protein/day and grams of protein/kg/day) than infants feeding CMF.

Chapter 7

DISCUSSION

7.1 Reported Energy and Protein Intake at 0.4 Months of Age in CMF Fed

Infants Compared to Historical Data

Energy and protein intake in a contemporary cohort of formula fed infants at

0.4 months of age, when all were fed a standard CMF, was higher than the range reported by historical data for one month old infants (see Table 12 and Table 13)13-16.

This finding however must be interpreted with caution for several reasons. Firstly, historical data on energy and protein intake is provided for one month old infants, versus our infants who were 0.4 months old, and it is well established that energy and protein requirements per kilogram body weight decline with age24. Secondly, the composition of infant formula has changed over time (current formulas versus historical data), and compositional differences may impact intake. Thirdly, difference in energy intake between infants in our study and historical data may also be due to differences in the method used to measure dietary intake. We assessed energy intake at

0.4 months of age using a 1-day reported diet record, and this method can be subject to over-report and over-estimate of true intake35, while the historical data used the 3-day test weighing of the bottle (3-day TWbottle) method to determine intake.

7.2 Reported Energy and Protein Intake at 0.4 Months of Age in CMF Fed

Infants Compared to the Energy EER and Protein AI (2005 DRIs)

Estimated energy requirements (EER) were calculated using the equation proposed by the 2005 DRI for infants 0 to 3 months of age. At 0.4 months of age, when all infants were feeding CMF, reported energy intake was significantly higher than the EER by 20.7% (see Table 14A); this result should be interpreted with caution however, as intake at this time point was determined by a 1-day diet record, which may be subject to over-reporting37. Protein intake was 84.0% higher than the protein

AI of the 2005 DRIs (see Table 14B), a finding similar to many studies of protein intake in formula fed infants10,12 since the protein concentration of infant formulas is greater than the protein concentration of breastmilk and the protein AI is based on the protein intake of breastfed infants24. Additionally, as mentioned above, intake at this time point was determined by a 1-day diet record.

7.3 Measured Energy and Protein Intake at 0.6 Months of Age in CMF Fed

Infants Versus Historical Data

Dietary intake when infants were approximately 0.6 months of age, was measured using the 3-day TWbottle method, a method comparable to historical literature data. Energy intake of infants randomized to feed CMF is shown in Table 15A. We found that infants randomized to feed CMF, had a mean energy intake of 125.2±26.1 kcal/kg/d which was similar to one historical reference, Montandon et al 198613 and was higher than three other historical references14-16(see Table 16A). Protein intake at

0.6 months of age, for infants randomized to feed CMF, (see Table 17A) was similar to one historical reference paper on protein intake (Montandon et al 1986) and was higher than three other historical references (see Table 2). Historical studies of energy and protein intake of formula fed infants have primarily used the 3-day TWbottle method to determine intake (similar to our study), and the majority of infants in the historical literature were fed a standard CMF.

7.4 Measured Energy and Protein Intake at 0.6 Months of Age in EHF Fed

Infants Versus Historical Data

Energy intake of infants randomized to feed EHF had an energy intake of

101.5±25.6 kcal/kg/d (see Table 15B), which is lower than three historical reference studies13-163-16 (see Table 17B), in which most infants were fed CMF. Protein intake of

EHF fed infants was higher than the protein intake reported by historical literature (see

Table 2). This is because EHF has higher in protein content (1.9 g of protein in 100 ml) than CMF (about 1.4 g of protein in 100 ml) and the vast majority of infants in the historical data were fed a standard CMF formula.

7.5 Measured Energy and Protein Intake at 0.6 Months of Age in CMF Fed

Infants Compared to the Energy EER and Protein AI (2005 DRIs)

Estimated energy requirements (EER) were calculated using the formula proposed by the 2005 DRIs and energy intake, measured by the 3-day TWbottle method, was higher than the respective EER by 13.6%. This finding suggest that as a group

54 infants fed CMF have an intake exceeding their energy requirements; and energy intake in excess of needs, over time, is likely a factor that contributes to accelerated weight gain observed in CMF fed infants10,58. CMF fed infants had a protein intake that was 69.9% higher than the protein AI (2005 DRIs), a finding consistent with other studies10,12.The higher protein intake of formula fed infants is partly due to the fact that protein requirements of the 2005 DRI are based on the total protein intake of the breastfed infant, and protein concentration of human milk steadily decreases during the course of lactation whereas the protein concentration of infant formula is constant during the first year of life. Additionally, most infant formulas contain a higher total protein concentration than mean total protein concentration of human milk, in order to provide the infant with sufficient quantities of all essential amino acids.

7.6 Measured Energy and Protein Intake of EHF Fed Infants at 6 Months of

Age Compared to the Energy EER and Protein AI (2005 DRIs)

At 0.6 months of age, EHF fed infants had a measured energy intake that was lower than, but not significantly different (p=0.06) from their EER. This finding suggests that as a group, EHF-fed infants are meeting and not exceeding their energy needs, and adequate energy intake over time is a factor in the normative weight gain observed in the EHF fed infants reported by Mennella et al 201122. Measured protein intake of EHF-fed infants was higher than protein AI (2005 DRIs) by 90.7%, which is not surprising given the total protein concentration of EHF formula (1.9g/100 ml), coupled with the fact that the protein AI is based on protein intake of the breastfed

55 infant.

7.7 Comparison of Dietary Intake Variables at 0.6 Months of Age by Formula

Type (CMF vs. EHF)

EHF fed infants had lower measured energy intake than CMF fed infants

(kcal/kg/d) (see Table 20). When feeding related behaviors were evaluated by formula group, we found that EHF fed and CMF fed infants had the same average number of feedings per days, but EHF fed infants had a lower mean volume of formula intake per feeding (ml/feed), which suggests that these infants reached satiation at a lower volume per feeding than CMF fed infants. This finding is consisted with the study by

Mennella and colleagues22 which found infants fed EHF from 0.5 months to 7.5 months of age consumed less formula (ml) to satiation at test feedings, compared to

CMF fed infants, starting at one month of age. Additionally, Ventura and colleagues49 also found infants satiate on lower volumes of EHF and CMF with added glutamate, compare compared to CMF formula.

CMF and EHF are isocaloric but they differ greatly in their protein composition, see Table 3. While CMF has a lower protein concentration than EHF (14 g/L versus 19g/L, respectively), the protein in CMF is intact casein protein with very few free amino acids (FAAs), about 864 umol/L total FAAs. On the other hand, EHF has higher protein content but the protein is extensively hydrolyzed, there is no intact casein, and the protein is mainly in the form of small peptides and free amino acids;

EHF has high content of total FAAs, about 80,375 umol/L. The difference between

CMF and EHF in total FAA content is 90 fold. The FAA acid content of EHF formula leads to differences in the taste of the formula59 and absorption rates of protein60.

Therefore there are several hypotheses to explain why infants consume less of EHF versus CMF at a feeding.

Some may speculate that infants fed EHF consume less due to the taste of the formula, since EHF formulas are characterized for having offensive a strong odor and unpleasant taste59. However previous research has shown that when infants are introduced to EHF at a young age (<4 months of age) they readily accept EHF61 through infancy and consume it to satiation22,49. Thus, we believe that is very unlikely that infants satiate earlier for reasons having to do with the taste of the formula.

The lower energy intake when fed EHF may be due to post-ingestive effects of

FAAs versus intact cow milk protein (casein) on infant feeding behavior, namely satiation. FAAs may regulate energy intake by the following two mechanisms:

1) FAAs can be sensed by receptors in the oral cavity, gastric walls, and intestinal

walls and some amino acids promote metabolic and digestive processes

through the gut-nutrient sensing system62-64 thereby leading to earlier satiation

signals.

2) Ingestion of EHF (versus intact protein), has been shown to have faster

absorption and metabolism65 leading to faster and greater rise in plasma amino

acid concentrations60 during feeding, which may lead to earlier satiation49.

Chapter 8

CONCLUSIONS

We found that energy intake of less than one month old CMF fed infants was consistent with historical data13-16 when energy intake was measured using the gold standard, 3-day TWbottle method, and less consistent with the historical data when measured using a 1-day reported diet record24,37. This finding indicates that the method used to measure intake in early infancy plays a very important role in validity and interpretation of dietary intake data. Additionally, the infants in our study were younger than infants in the literature, limiting our ability to make a direct comparison to historical data. We also found that infants randomized to feed CMF had an energy intake significantly greater than the EER (2005 DRI)24, while EMF fed infants had an energy intake similar to the EER (2005 DRI)24, which suggests that EHF fed infants, as a group, met their energy requirements whereas CMF infants, as group, exceeded their energy requirements. Intake of energy in excess of requirements over time is a likely factor in the accelerated weight gain observed in many CMF fed infants10-12. Protein intake of this contemporary sample of formula fed infants (based on 3-day TWbottle method) was above the recommendation proposed by the 2005 DRI24 for both CMF and EHF fed infants, this is not surprising since the 2005 DRI24 recommendation was based solely on protein intake of the breastfed infant.

To compare intake of CMF fed versus EHF fed infants, we measured energy intake during the first three days of consuming their randomized formula, and found

58 that infants fed EHF have a significant lower volume of intake (ml/day), volume per feed (ml/feed), and lower energy intake (kcal/day and kcal/kg/day) when compared to infants randomized to feed a standard CMF. These findings adds to the results of

Mennella et al 201122 and Ventura et al 201249, in that when exposed to EHF, infants decrease their feeding volume and thus their daily energy intake. The mechanism that drives the lower intake volume of EHF-fed versus CMF fed infants is thought to be due to the high FAA concentration of EHF versus CMF, since FFA may influence nutrient absorption and metabolism62-64,66 and may lead to earlier satiation signals.

Whether the differential energy intake of CMF versus EHF fed infants observed at 2-3 weeks of age will be maintained throughout the first year of life and result in differential weight-gain trajectories between CMF and EHF fed infants throughout the first year of life, is the main goal of the randomized controlled trial from which these data were obtained. Additional research into the effect of formula composition on physiological mechanisms that drive feeding behaviors such as satiation and satiety are needed.

TABLES

Table 1: Summary of scientific literature on energy intake (kcal/kg/d) determined by the test weighing method in young infants. Type of Subject Age in Study Method to Energy Intake References Feeding Characteristics Days Location Assess kcal/kg/d (Number, sex, Energy race/ethnicity) Mean±SD Intake Mean±SD Formula N=12 30±4 5-day TW of 125±17 kcal/kg/d Montandon et al. ready to feed Feeds(n)=6.5±1.0 J of Pediatr 75% of N=6 Males bottles by Gastroenterol and infants N=6 Females research staff Nutr.1986;5(3):43 United consuming 4-438. States CMF Race was not 25% of reported 61 infants

consuming SF Formula N=10 33±4 5-day TW of 118±17 kcal/kg/d ready to feed Butte et al. CMF N=8 Males bottle Feeds(n)=7.8±1.0 Pediatr N=2 Females United by mothers Res1990;28(6):63 States and verified 1-640. N=7 Black by research N=1 Caucasian staff N=2 Hispanic Cow milk formula (CMF), soy formula (SF), test weigh (TW).

Table 1: Summary of scientific literature on energy intake (kcal/kg/d) determined by the test weighing method in young infants. Type of Subject Age in Study Method to Energy Intake References Feeding Characteristics Days Location Assess kcal/kg/d (Number, sex, Energy race/ethnicity) Mean±SD Intake Mean±SD Formula N=17 35±4 days 3 day TW of 108±18 kcal/kg/d Butte el al. ready to feed AJCN. CMF N=9 Males bottles by 1990;51(3):350- N=8 Females mother 358. United N=1 Black States N=16 Caucasian 62 N=0 Hispanic

Formula N=23 Not reported 5-day TW of 111 ± 18 in days but as bottles kcal/kg/d CMF N=15 Males 1 month containing Males N=8 Females assessment reconstituted powdered 117 ± 27 Bruin et al. AJCN N=23 Caucasian The formula by kcal/kg/d 1998;67:885-896. N=0 Hispanic Netherlands mothers. Females.

Mean of Males and Females combined was not provided. Cow milk formula (CMF), test weigh (TW).

Table 2: Summary of scientific literature on protein intake (g protein/kg/d) determined by the test weighing method in young infants Type of Feeding Subject Age in Study Method to Protein Intake References Characteristics Days Location Assess g/kg/d (Number, sex, Protein race/ethnicity) Mean±SD Intake Mean±SD Formula N=12 30±4 5-day TW of 2.6±0.7 g/kg/d Montandon et al. ready to feed J of Pediatr 75% of infants N=6 Males bottles by Gastroenterol United consuming CMF N=6 Females research staff and States 25% of infants Nutr.1986;5(3):4 consuming SF Race was not 34-438. reported 63 Formula N=10 33±4 5-day TW of

ready to feed 2.2±0.3 g/kg/d Butte et al. CMF N=8 Males bottle Pediatr Res. N=2 Females United by mothers 1990;28(6):631- States and verified 640. N=7 Black by research N=1 Caucasian staff N=2 Hispanic Cow milk formula (CMF), soy formula (SF), test weigh (TW).

Table 2: Summary of scientific literature on protein intake (g protein/kg/d) determined by the test weighing method in young infants Type of Feeding Subject Age in Study Method to Protein Intake References Characteristics Days Location Assess g/kg/d (Number, sex, Protein race/ethnicity) Mean±SD Intake Mean±SD Formula N=23 Not 5-day TW of 2.0± 0.3 g/kg/d reported in bottles Males CMF N=15 Males days but as containing Bruin et al. N=8 Females 1 month reconstituted 2.1 ± 0.4 g/kg/d The AJCN assessment powdered Females. Netherlands 1998;67:885- N=23 formula by 896. Caucasian mothers. Mean of Males 64 N=0 Hispanic and Females

combined was not provided. Cow milk formula (CMF), test weigh (TW).

Table 3. Macronutrient composition of study formulas compared to breast milk

CMF1 EHF1 Breast milk2 Total calories (kcal/100 ml) 67.7 67.7 62.0-70.0 Carbohydrates (g/100 kcal) 7.4 7.0 6.5-7.8 Fat (g/100 kcal) 3.6 3.6 3.0-3.6 Protein or protein equivalent (g/100 1.4 1.9 1.0-1.3 ml) Essential FAA: (umol/L) Histidine 9 1880 8 Isoleucine tr 5327 33 Leucine nd 11886 56 Lysine 22 8254 39 Methionine nd 2854 9 Phenylalanine 11 4283 24 Threonine 5 4653 98 Tryptophan nd 1348 nr Valine 10 7038 73 Semi-essential FAA: Arginine 10 3489 35 Cystine nd 542 nr Taurine 529 496 301 Tyrosine 9 1170 3 Nonessential FAA: Alanine 31 4905 228 Asparagine tr 3705 nr Aspartic acid 9 1535 183 Glutamic acid 109 7472 1184 Glutamine nd nd 285 Glycine 38 1658 125 Proline 63 2667 64 Serine 9 5213 274 Total FAAs 864 80375 3020 Tr: trace amount; nd: not detected; nr; not reported. 1CMF, cow milk formula; EHF, extensively hydrolyzed formula, values from Ventura et al. 201220. Breast milk estimates for mothers of 3-month-old infants. Macronutrient estimates from Nommsen et al. 199128, Riordan 200567. FAA values from Agostoni et al. 200021.

Table 4: Demographic characteristics of mother-infant dyads at study entry (Visit 1) All infants Minimum- (N=141) Maximum Infant characteristics Gender, male, %(n) 47.5(67) Age at Visit 1, study entry 0.4±0.1 0.1-0.6 mean ± SD, months Birth weight, mean± SD, Kg 3.2±0.4 2.2-4.3 Racial/ethnic category, %(n) Black 62.2(88) White 21.2(30) Other/more than one race 16.3(23) Ethnicity, %(n) Hispanic 12.7(18) Not Hispanic 87.2(123) Infant Z-scores at Visit 1a Weight-for-age (WAZ),mean ± SD -0.2±0.7 -2.4-1.6 Length-for age (LAZ),mean ± SD -0.4±0.9 -3.0-2.1 Weight-for-length (WLZ),mean ±SD 0.1±0.8 -3.1-1.9 Maternal/familial characteristics Age at study entry, years 27.3±5.6 18.1-41.3 Pre-pregnancy BMIa, 28.5±8.0 17.2-58.8 mean ± SD, kg/m2 Weight gain during pregnancyb, 13.4±9.0 -18.6-45.4 mean±SD, kg Family income level, % (n)c Less than $ 15, 000 47.0(64) $15,000 – $35,000 26.4(36) $35,000 – $75,000 9.5(13) More than $75,000 16.9(23) Maternal education, % (n)d Grade School 15.7(22) High School 37.8(53) Trade School 20.7(29) College/Graduate School 25.7(36) BMI=Body Mass Index, a Infant Z-score data at Visit 1 is not available for N=1 infant. bPre-pregnancy is not available for N=5 mothers. cWeight gained during pregnancy is not available for N=4 mothers. dIncome is not available for N=5 families. eMaternal education is not available for N=1 mother

Table 5. Subject count for 1-day diet record when infants were 0.4 months of age (Visit 1) N Mothers that signed the informed consent 141 Mother-infant dyads that completed Visit 1 141 Mother-infant dyads that withdrew after Visit 1 8 (did not returned to Visit 2) Mother-infant dyads that returned to Visit 2 133 Number of diet records that were returned for Visit 1 124 Number of diet records that were not returned for Visit 1 9 Number of diet records included in the analysis 108 Number of diet records excluded in the analysis 16 Number of diet records excluded for physiological criteria 8 Number of diet records excluded for incomplete 5 Number of diet records excluded for not compliance in feeding 2 assigned formula Number of diet records excluded for being unreliable 1 N=108 1-day diet records from infants aged 1-2 weeks of age that are included for analysis; for Visit 1 for this diet record all infants are feeding CMF.

Table 6: Characteristics of infants who returned a 1-day diet record (DR) versus those who did not return a 1-day diet record (No DR). Chi-value(df) DR No DR P N=124 N=17 T-value(df) value

Infant characteristics at study entry Gender, male, %(n) 46.7(58) 52.9(9) 0.22(1) 0.63

Birth weight, mean ± SD, kg 3.2±0.4 3.1±0.3 1.06(139) 0.29

Age, mean ± SD, months 0.4±0.09 0.4±0.1 -1.79(139) 0.07

68 a

Weight, mean ± SD, kg 3.4±0.4 3.4±0.3 0.72(138) 0.46

Length, mean ± SD, cm 50.7±1.9 50.3±1.9a 0.85(138) 0.39

Weight-for-age (WAZ), mean ± SD -0.2±0.7 -0.5±0.7a 1.41(138) 0.16

Length-for age (LAZ), mean ± SD -0.4±0.9 -0.8±0.9a 1.50(138) 0.13

Weight-for-length (WLZ), mean ± SD -0.1±0.8 -0.1±0.8a -0.31(138) 0.75

DR= returned 1-day diet record, No DR=withdrew or not retuned 1-day diet record. a weight and length at study entry data in this group is not available for N=1. An independent t-test (for continuous variables) or Chi-square test (categorical variables) was used to determine differences between mother-infant dyad who returned a 24-hour diet record (R-DR) versus those that did not (No-DR).

Table 7: Comparison of infant characteristics for infants randomized to a study formula versus those who withdrew from study after Visit 2. All Randomized Withdrew Chi value (df) P N=133 N=118 N=15 or T-value(df) value Infant characteristics Gender, male, %(n) 48.1(64) 50(59) 30(5) 1.48(1) 0.22 Birth weight, mean ± SD, Kg 3.2±0.4 3.2±0.4 3.1±0.3 0.89(131) 0.37 At Visit 1 Age, mean ± SD, months 0.4±0.1 0.4±0.0 0.3±0.1 0.42(131) 0.66 Time elapse between Visit 1 and Visit 7.1±3.2 7.0±3.2 8.0±3.2 -1.09(131) 0.27 2, mean ± SD

69 At Visit 2

Age, mean ± SD, months 0.6±0.0 0.6±0.1 0.6±0.0 -0.71(131) 0.47 Weight, mean ± SD, cm 3.7±0.4 3.7±0.4 3.6±0.2 0.28(131) 0.77 Length, mean ± SD, cm 51.5±2.0 51.6±2.0 51.1±1.7 0.81(131) 0.41 Weight-for-age (WAZ), mean ± SD -0.3±0.7 -0.3±0.8 -0.3±0.5 0.10(131) 0.91 Length-for age (LAZ), mean ± SD -0.5±1.0 -0.5±1.0 -0.7±0.9 0.79(131) 0.31 Weight-for-length (WLZ), mean ± SD -0.0±0.9 0.0±0.9 0.2±0.7 -1.01(131) 0.42 N=118 who were randomized to receive CMF or EHF at Visit 2; N=15 subject who withdrew from the study after Visit 1. An independent t-test (for continuous variables) or Chi-square test (categorical variables) was used to determine differences between randomized sample (N=118) and those that withdrew (N=15).

Table 8. Subject count for 3-day TWbottle method when infants were 0.6 months of age (Visit 2) N Mother-infant dyads that returned to Visit 2 133 Mother-infant dyads sent home with bottle kits 133 Mothers-infants dyads that used bottle kits (randomized subjects), intent 118 to treat sample Mother-infant dyads that withdrew after Visit 2: 15 Summary of reason why subject withdrew: Mother did not give a reason, n=10 Mother reported infant did not tolerate assigned formula, n=3 PI withdrew for no compliance, n=2 Number of infants included in the TWbottle method: 99 Number of infant with 3 days included in the analysis 84 Number of infant with 2 days included in the analysis 13 Number of infant with 1 day included in the analysis 2 # of infants excluded from the analysis 19 Number of mother-infant dyads excluded for incomplete record 3 Number of mother-infant dyads excluded for not physiological 1 Number of mother-infant dyads excluded for no compliance-formula 2 Number of mother-infant dyads excluded for not reliable: 13 Bottles leaked significantly, N=4 Mother rinsed all bottles, N=3 Bottles were used for more than 3 days and there is no bottle log, N=2 Mother filled bottles to 9oz N=1 Record was not well kept N=1 No variability in intake, N=1 Not reliable reported by Monell RAs, N=1 N= 118 subjects returned a completed bottle kit, 15 subjects withdrew from study after Visit 2 and 19 subjects were not included in the analysis due to incomplete records, non-physiological data, non-compliance with study formula, or not reliable, leaving N=99 infants with TWbottle data.

Table 9A: Demographic characteristics according to formula group in subjects who completed Visit 2. CMF EHF Chi (df) P group group or t(df) value N=62 N=56 Infant characteristics Gender, male, %(n) 51.6(32) 48.2(27) 0.13 (1) 0.71 Age at Visit 2, mean ± SD, months 0.6±0.1 0.6±0.0 0.88(116) 0.37 Birth weight, mean± SD, Kg 3.2±0.4 3.2±0.4 -0.25(116) 0.80 Racial/ethnic category, %(n) 3.80(2) 0.14 Black 56.4(35) 66.0(37) White 29.0(18) 14.2(8) Other/mixed 14.5(9) 19.6(11) Ethnicity, %(n) 0.59(1) 0.43 Hispanic 90.3(56) 85.7(48) Not Hispanic 9.6(6) 14.2(8) Infant Z-scores at Visit 2 Weight-for-age (WAZ),mean ± SD -0.4±0.8 -0.2±0.7 -0.92(116) 0.35 Length-for age (LAZ),mean ± SD -0.5±1.0 -0.5±1.0 -0.16(116) 0.86 Weight-for-length (WLZ),mean -0.1±0.8 0.0±1.0 -1.21(116) 0.22 ±SD Maternal/familial characteristics Age at study entry, y 27.3±5.9 26.8±5.3 0.43(116) 0.66 Pre-pregnancy BMIa, 28.5±7.8 29.6±9.0 -0.71(115) 0.47 mean ± SD, kg/m2 Weight gain during pregnancyb, 13.4±10.0 14.9±14.9 -0.64(114) 0.51 mean±SD, Kg Family income level, % (n)c 1.92(3) 0.58 Less than $ 15, 000 41.6 (25) 50.9 (28) $15,000 – $35,000 30.0 (18) 20.0 (11) $35,000 – $75,000 10.0 (6) 12.7 (13) More than $75,000 18.3 (11) 16.36(9) Maternal education, % (n) 3.47 (3) 0.32 Grade School 19.3 (12) 8.6 (5) High School 33.8 (21) 44.6 (25) Trade School 17.7 (11) 21.4 (12) College/Graduate School 29.0 (18) 25.0 (14) BMI=Body Mass Index, CMF=Cow Milk Formula, EHF=Extensively Hydrolyzed Formula. a Pre-pregnancy BMI is missing for N=1 mother. b weight gain during pregnancy is missing for n=2 mothers. c Family income is missing for N=3 family. An independent t-test (continuous variables) or Chi-square test (categorical variables) was used to determine differences between infants randomized to CMF versus EHF group.

Table 9B: Characteristics of infants who completed the TWbottle method per protocol at Visit 2 (N=99). CMF EHF Chi (df) or P group group t(df) N=53 N=46 Infant characteristics Gender, male, %(n) 52.8(28) 43.4(20) 0.86 (1) 0.35 Age at Visit 2, mean ± SD, months 0.6±0.1 0.6±0.0 0.75(97) 0.45 Birth weight, mean ± SD, Kg 3.2±0.4 3.2±0.4 -0.13(97) 0.89 Racial/ethnic category, %(n) 3.58(2) 0.16 Black 49.0(26) 58.7(27) White 33.9(18) 17.3(8) Other/More than one race 16.9(9) 23.9(11) Ethnicity 0.74(1) 0.38 Hispanic 88.6(47) 82.6(38) Not Hispanic 11.3(6) 17.3(8) Infant Z-scores at Visit 2 Weight-for-age (WAZ),mean ± SD -0.4±0.8 -0.2±0.7 -0.69(97) 0.48 Length-for age (LAZ),mean ± SD -0.6±1.0 -0.5±1.0 0.13(97) 0.83 Weight-for-length (WLZ),mean ±SD 0.0±0.8 0.1±0.9 -0.92(97) 0.35 Maternal/familial characteristics Age at study entry, years 27.6±5.9 27.4±.6 -0.17(97) 0.85 Pre-pregnancy BMIa, 28.4±8.0 29.5±9.1 -0.63(96) 0.52 mean ± SD, kg/m2 Weight gain during pregnancyb, 13.7±10.5 15.3±15.4 -0.61(95) 0.54 mean±SD, Kg Family income level, % (n)c 1.43(3) 0.69 Less than $ 15, 000 38.4 (20) 47.8 (22) $15,000 – $35,000 28.8 (15) 19.5 (9) $35,000 – $75,000 11.5 (6) 13.0 (6) More than $75,000 21.1 (11) 19.5 (9) Maternal education, % (n) 2.28(3) 0.51 Grade School 18.8 (10) 10.8 (5) High School 32.0 (17) 43.4 (20) Trade School 16.9 (9) 19.5 (9) College and Graduate School 32.0 (17) 26.0 (12) BMI=Body Mass Index, CMF=Cow Milk Formula, EHF=Extensively Hydrolyzed Formula. a Pre-pregnancy BMI is missing for N=1 mother. b Weight gain during pregnancy is missing for N=2 mothers. cFamily income is missing for N=1 family. An independent t-test (continuous variables) or Chi-square test (categorical variables) was used to determine differences between infants randomized to CMF versus EHF.

Table 10: Infant dietary intake at 0.4 months of age (Visit 1, all infants fed CMF) determined by a 1-day reported diet record. CMF, N=108 Minimum and Maximum

Age at study entry (Visit 1), months 0.4±0.0 (0.0) 0.13-0.62

Age at study entry(Visit 1), days 12.2±2.8 (0.2) 4-19

Average number of formula feedings per day 8.8±1.6 (0.1) 6-13

Total volume of intake in a day, ml/day 703.3±166.0 (15.9) 330.0 -1095.0

Average volume per feeding, ml/fee 80.9±21.1 (2.03) 34.0-135.0

Total Energy intake, kcal/day 464.7±109.6 (10.5) 217.8-722.7

Energy intake, kcal/kg/day 134.3±32.2 (3.0) 71.64-228.4

Protein intake, g/day 9.8±2.3 (0.2) 4.6-15.1

Protein intake, g/kg/day 2.8±0.6 (0.0) 1.5-4.8

CMF=Cow Milk Formula; all infants at Visit 1 were feeding CMF. Dietary intake variables were measured using a 1-day diet record. Values presented as mean±SD(SEM).

Table 11A: Dietary intake variables of CMF feedings at 0.4 months of age (Visit 1, all infants fed CMF) for subjects who completed the 1-day record CMF group EHF group T-value(df) or P-value N=55 N=53 U-value(df) Age of infant, days 12.2 2.8 12.2±2.9 -0.022 0.97 Number of formula feedings per day 8.9±1.6 (0.2) 8.8±1.5 (0.2) 0.43(106) 0.66 Total volume intake in a day, ml 691.7±161.3 (21.7) 715.0±171.4 (23.5) -0.73(106) 0.46 Total Energy intake, kcal/day 457.7±106.7 (14.3) 472.1±113.1 (15.5) -0.68(106) 0.49 Total energy intake, kcal/kg/day 133.1±33.8 (4.5) 135.6±30.7 (4.2) -0.40(106) 0.68 Protein intake, g/day 9.6±2.2 (0.2) 9.9±2.3 (0.3) -0.83(106) 0.40

Protein intake, g/kg/day 2.8±0.7 (0.0) 2.8±0.6 (0.0) -0.53(106) 0.59 CMF=Cow Milk Formula, EHF=Extensively Hydrolyzed Formula. Dietary intake variables were measured using a 1- day diet record. All values presented as mean±SD (SEM). An independent t-test (for continuous variables) or Mann- Whitney U-test (ordinal variables) was used to determine differences between infants randomized to CMF versus EHF.

Table 11B: Dietary intake variables of CMF feedings at 0.4 months of age for subjects who completed the 1-day record per protocol CMF group EHF group T-value(df) or P-value

N=47 N=43 U-value(df)

Number of formula feedings per day 8.9±1.6 (0.2) 8.9±1.5 (0.2) 0.08 (88) 0.94

Total volume intake in a day, ml 681.9±160.7 (23.4) 704.1±171.5 (26.1) -0.63 (88) 0.53

Total energy intake, Kcal/day 451.4±106.4 (15.5) 464.7±113.2 (17.2) -0.57 (88) 0.57

Total energy intake, kcal/kg/day 134.1±33.6 (4.9) 131.5±31.5 (4.8) 0.39 (88) 0.70

Protein intake, g/day 9.5±2.2 (0.3) 9.8±2.3 (0.3) -0.57 (88) 0.57

Protein intake, g/kg/day 2.7±0.7 (0.1) 2.8±0.6 (0.0) -0.38 (88) 0.70

Dietary intake variables were measured using a 1-day diet record. N=90 infants in the per protocol population. Values presented as mean±SD (SEM). An independent t-test (for continuous variables) or Mann-Whitney U-test (ordinal variable) was used to determine differences between infants randomized to CMF versus EHF.

Table 12. Comparison of historical data on energy intake to reported energy intake of CMF at 0.4 months of age (Visit 1)

Historical data on energy intake, TWbottle method Present study, 1-day reported record (N=108)

Median, Mean±SD(SEM), Quartiles Reference Energy Intake Age 25th Median Energy Intake 75th Age (days) kcal/kg/d kcal/kg/d Mean±SD Mean±SD(SEM) Mean ±SD Butte el al.199014 108±18 35±4 days 112.4 131.3 134.3±32.2(3.1) 153.1 12.2±2.8

Bruin et al. 199816 Males: 111±18 1 month 112.4 131.3 134.3±32.2(3.1) 153.1 12.2±2.8

Bruin et al. 199816 Females: 117±27 1 month 112.4 131.3 134.3±32.2(3.1) 153.1 12.2±2.8 Butte et al. 199015 118±17 33±4 days 112.4 131.3 134.3±32.2(3.1) 153.1 12.2±2.8

Montandon et al. 125±17 30±4 days 112.4 131.3 134.3±32.2(3.1) 153.1 12.2±2.8 198613

TWbottle=test weighing of the bottle, kcal=kilocalories, kg=kilogram, d=day

Table 13: Comparison of historical data on protein intake to reported protein intake of CMF at 0.4 months of age (Visit 1)

Historical data, TWbottle method Present study, 1-day reported record (N=108) Median, Mean±SD(SEM), Quartiles

Reference Protein intake Age 25th Median Protein intake 75th Age (days) (g/kg/d) Mean ±SD (g/kg/d) Mean ±SD Mean± SD(SEM) Mean ±SD Bruin et al. Males: 1 month 2.3 2.7 2.8±0.6 (0.0) 3.2 12.2±2.8 199816 2.0± 0.3

77 Bruin et al. Females: 2.1±0.4 1 months 2.3 2.7 2.8±0.6 (0.0) 3.2 12.2±2.8 199816 Butte et al. 2.2±0.3 33±4 days 2.3 2.7 2.8±0.6 (0.0) 3.2 12.2±2.8 199015 Montandon et 2.6±0.7 30±4 days 2.3 2.7 2.8±0.6 (0.0) 3.2 12.2±2.8 al. 198613

TWbottle=test weighing of the bottle, kg=kilogram, d=day

Table 14A: Comparison of EER versus energy intake when all infants were fed CMF at 0.4 months of age (Visit 1) 2005 DRI24 Present study, 1-day reported record (N=108) Paired t-test EER (kcal/d) Energy Intake (kcal/d) Age (days) T-value(df) P-value Mean ±SD Mean ±SD

384.9±37.4 464.7±109.6 12.2±2.8 7.82 (107) <0.001 EER=Estimated energy requirements, CMF=cow milk formula, DRI=dietary reference intake, kcal=kilocalories. A paired comparison t-test was used to determine differences between infants’EER and reported energy intake.

Table 14B. Comparison of AI for protein versus protein intake when all infants were fed CMF at 0.4 months of age (Visit 1) 2005 DRI24 Present study, 1-day reported record (N=108) Single sample t-test

AI Protein intake Age (days) T-value(df) P-value (gram of (grams of protein/d) Mean ±SD protein/d) Mean ±SD 1.52 2.8±0.6 12.2±2.8 19.87 (107) <0.001 AI=adequate intake, CMF=cow milk formula, DRI=dietary reference intake. A single sample t-test was used to determine differences between infants AI and reported protein intake.

Table 15A: Dietary intake variables for infants randomized to feed CMF at 0.6 months of age (Visit 2) CMF group Minimum and Maximum N=53 Age, months 0.6±0.1 (0.0) 0.4-0.9 Age, days 19.6±3.2 (0.4) 13-29 Number of formula feedings per day (feeds/day) 7.7±1.1 (0.1) 5.0-11.5 Total volume of intake in ml/day 696.8±141.2 (19.3) 461.8-1051.3 Average volume per feeding, ml/feed 91.8±21.7 (2.9) 56.4-151.5

79 Total energy intake, kcal/day 459.9±93.3 (12.8) 304.8-693.8

Energy intake, kcal/kg/day 125.2±26.1 (3.5) 78.7-207.4 Protein intake, g/day 9.7±1.9 (0.2) 6.4-14.7 Protein intake, g/kg/day 2.6±0.5 (0.0) 1.6-4.4 All values presented as mean±SD (SEM).

Table 15B: Dietary intake variables for infants randomized to feed EHF at 0.6 months of age (Visit 2)

EHF group Minimum and Maximum N=46 Age, months 0.5±0.0 (0.0) 0.2-0.8 Age, days 19.1±2.8 (0.4) 9-27 Number of formula feedings per day 7.7±1.2 (0.1) 6-11 Total volume of intake in ml/day 573.7±161.6 (23.8) 228.6-935.9 Average volume per feeding, ml/feed 75.1±20.5 (3.02) 34.2-120.5

80 Total energy intake, kcal/day 378.6±106.6 (15.7) 150.9-617.7 Energy intake, kcal/kg/day 101.5±25.6 (3.7) 43.1-161.7 Protein intake, g/day 10.9±3.0 (0.4) 4.3-17.7 Protein intake, g/kg/day 2.9±0.7 (0.1) 1.2-4.6 All values presented as mean±SD (SEM).

Table 16A: Comparison of historical data on energy intake to energy intake of CMF fed infants at 0.6 months of age (Visit 2)

Historical data, TWbottle method Present study, 3-day TWbottle method (N=53) Median, Mean±SD(SEM), Quartiles

Reference Energy Age 25th Median Energy Intake 75th Age (days) Intake (kcal/kg/d) Mean± (kcal/kg/d) SD(SEM) Mean ±SD

Butte el 108±18 35±4 days 110.1 128.1 125.2±26.1(3.6) 136.5 19.6±3.2 al.199014

Bruin et al. Males: 1 month 110.1 128.1 125.2±26.1(3.6) 136.5 19.6±3.2 81 199816 111±18

Bruin et al. Females: 1 months 110.1 128.1 125.2±26.1(3.6) 136.5 19.6±3.2 199816 117±27

Butte et al. 118±17 33±4 days 110.1 128.1 125.2±26.1(3.6) 136.5 19.6±3.2 199015 Montandon et 125±17 30±4 days 110.1 128.1 125.2±26.1(3.6) 136.5 19.6±3.2 al. 198613

TWbottle=test weighing of the bottle, kcal=kilocalories, kg=kilogram, d=day

Table16B: Comparison of historical data on energy intake to energy intake of EHF fed infants at 0.6 months of age (Visit 2)

Historical data, TWbottle method Present study, 3-day TWbottle method (N=46) Median, Mean±SD(SEM), Quartiles

Reference Energy Age 25th Median Energy Intake 75th Age (days) Intake (kcal/kg/d) Mean ±SD (kcal/kg/d) Mean ±SD

Butte el 108±18 35±4 days 88.0 97.8 101.5±25.6(3.7) 123.2 19.1±2.8 al.199014

Bruin et al. Males: 1 month 88.0 97.8 101.5±25.6(3.7) 123.2 19.1±2.8 16 82 1998 111±18

Bruin et al. Females: 1 months 88.0 97.8 101.5±25.6(3.7) 123.2 19.1±2.8 199816 117±27 Butte et al. 118±17 33±4 days 88.0 97.8 101.5±25.6(3.7) 123.2 19.1±2.8 199015 Montandon et 125±17 30±4 days 88.0 97.8 101.5±25.6(3.7) 123.2 19.1±2.8 al. 198613

TWbottle=test weighing of the bottle, kcal=kilocalories, kg=kilogram, d=day

Table 17A: Comparison of historical data on protein intake to protein intake of CMF fed infants at 0.6 months of age (Visit 2)

Historical data on protein intake, TWbottle method Present study, 3-day TWbottle method (N=53) Median, Mean±SD(SEM), Quartiles

Reference Protein Intake Age 25th Median Protein Intake 75th Age (g/kg/d) (g/kg/d) (days) Mean ±SD Mean ±SD Mean ±SD

Bruin et al. Males: 1 month 2.3 2.7 2.6±0.5(0.0) 2.8 19.6±3.2 199816 2.0± 0.3

83 Bruin et al. Females: 1 months 2.3 2.7 2.6±0.5(0.0) 2.8 19.6±3.2 16 1998 2.1±0.4

Butte et al. 2.2±0.3 33±4 days 2.3 2.7 2.6±0.5(0.0) 2.8 19.6±3.2 199015

Montandon et 2.6±0.7 30±4 days 2.3 2.7 2.6±0.5(0.0) 2.8 19.6±3.2 al. 198613

TWbottle=test weighing of the bottle, kcal=kilocalories, kg=kilogram, d=day

Table 17B: Comparison of historical data on protein intake to protein intake of EHF fed infants at 0.6 months of age (Visit 2)

Historical data on protein intake, TWbottle method Present study, Energy Intake 3-day (g/kg/d)TWbottle method (N=46) Median, Mean±SD(SEM), Quartiles Reference Protein intake Age 25th Median Protein intake 75th Age (days) (g/kg/d) (g/kg/d) Mean ±SD Mean±SD(SEM) Mean ±SD Bruin et al. Males: 1 month 2.5 2.8 2.9±0.7(3.7) 3.5 19.1±2.8 199816 2.0± 0.3

84 Bruin et al. Females: 1 months 2.5 2.8 2.9±0.7(3.7) 3.5 19.1±2.8 16 1998 2.1±0.4 Butte et al. 2.2±0.3 33±4 days 2.5 2.8 2.9±0.7(3.7) 3.5 19.1±2.8 199015

Montandon et 2.6±0.7 30±4 days 2.5 2.8 2.9±0.7(3.7) 3.5 19.1±2.8 al. 198613

TWbottle=test weighing of the bottle, AI=adequate intake, kg=kilogram, d=day

Table 18A: Comparison of EER to energy intake of CMF fed infants at 0.6 months of age (Visit 2)

24 2005 DRI 3-day TWbottle method (N=53) Paired t-test EER (kcal/d) Energy Intake Age (days) T-value(df) P-value (kcal/d) Mean ±SD Mean ±SD 404.9±42.3 459.9±93.3 19.6±3.2 4.47(52) <0.001

EER=Estimated energy requirements, CMF=cow milk formula, DRI=dietary reference intake, kcal=kilocalories. An paired comparison t-test was used to determine differences between infants’EER and reported energy intake.

Table 18B: Comparison of EER to energy intake of EHF fed infants at 0.6 months of age (Visit 2)

24 2005 DRI 3-day TWbottle method (N=46) Paired t-test EER (kcal/d) Energy Intake Age (days) T-value(df) P-value (kcal/d) Mean ±SD Mean ±SD 405.7±34.3 378.6±106.6 19.1±2.8 -1.92(45) 0.06

EER=Estimated energy requirements, EHF=extensively hydrolyzed formula, DRI=dietary reference intake, kcal=kilocalories. A paired comparison t-test was used to determine differences between infants’EER and reported energy intake.

Table 19A: Comparison of AI for protein versus protein intake of CMF fed infants at 0.6 months of age (Visit 2)

24 2005 DRI 3-day TWbottle method (N=53) Single sample t-test

AI Protein intake Age (days) T-value(df) P-value (gram of (grams of protein/d) Mean ±SD protein/d) Mean ±SD 1.53 2.6±0.5 19.6±3.2 14.77 (52) <0.001

AI=adequate intake, CMF=cow milk formula, DRI=dietary reference intake. A single sample t-test was used to determine differences between infants AI and reported protein intake.

Table 19B: Comparison of AI for protein versus protein intake of EHF fed infants at 0.6 months of age (Visit 2)

24 2005 DRI 3-day TWbottle method (N=46) Single sample t-test

AI Protein intake Age (days) T-value(df) P-value (gram of (grams of protein/d) Mean ±SD protein/d) Mean ±SD 1.53 2.9±0.7 19.1±2.8 12.82 (45) <0.001

AI=adequate intake, EHF=extensively hydrolyzed formula, DRI=dietary reference intake. A single sample t-test was used to determine differences between infants AI and reported protein intake.

Table 20: Dietary intake variables by formula group (CMF vs. EHF) for infants who completed the TWbottle method at 0.6 months of age (Visit 2) CMF group EHF group T-value P N=53 N=46 (df) or U- value value(df) Age, months 0.6±0.1 (0.0) 0.5±0.0 (0.0) 0.75(97) 0.45 Age, days 19.6±3.2 (0.4) 19.1±2.8 (0.4) 0.75(97) 0.45 Number of formula 7.7±1.1 (0.1) 7.7±1.2 (0.1) -0.10(97) 0.92 feedings per day Total volume of intake 696.8±141.2 573.7±161.6 4.04(97) <0.01 in ml/day (19.3) (23.8) Average volume per 91.8±21.7 (2.9) 75.1±20.5 (3.02) 3.89 (97) <0.01 feeding, ml/feed Total energy intake, 459.9±93.3 378.6±106.6 4.04(97) <0.01 kcal/day (12.8) (15.7) Total energy intake, 125.2±26.1 (3.5) 101.5±25.6 (3.7) 4.53(97) <0.01 kcal/kg/day Protein intake, g/day 9.7±1.9 (0.2) 10.9±3.0 (0.4) -2.23(97) 0.03 Protein intake, g/kg/day 2.6±0.5 (0.0) 2.9±0.7 (0.1) -2.06(97) 0.04

Dietary intake variables were measured by a 3-day TWbottle method, N=99 infant in the per protocol population. All values presented as mean±SD (SEM). An independent t-test (for continuous variables) or Mann-Whitney U-test (ordinal variable) was used to determine differences between infants randomized to CMF versus EHF.

FIGURES

Figure 1. Schedule of Events

Visit 1: Infants’ Age 0.4±0.1 months Informed consent obtained. Measures: anthropometry, demographic and health history. Infants were randomized to formula A or B. At end of Visit 1, all mothers were given CMF (Enfamil, Mead Johnson Nutrition, Evansville, IN) to feed their infants until Visit 2 (approximately 1 week apart) and a diet record for for mothers to report CMF intake for 24-hours.

Visit 2: Infants’ age 0.6±0.0 months Return of 1-day diet record (CMF). Measures: Anthropometry and health history. At end of Visit 2, mothers were sent home with TWbottle kits (30 pre-filled/pre-weighed bottles of assigned formula A or B to feed their infants over the next three 3 days). They were given forms to record time and frequency of feedings

Sent home with Sent home with Formula A Formula B

Completed TWbottle Completed TWbottle method for Formula A method for Formula B

TWbottle=Test weighting of the bottle.

Figure 2: Subject count for 24-hour reported diet record.

Visit 1: Infants’ Age 0.4±0.1 N=141 mother-infant dyads completed visit 1. At the end of Visit 1, all mothers were given CMF (Enfamil, Mead Johnson Nutrition, Evansville, IN) to feed their infants until visit 2 (approximately 1 week apart) and a diet record form for mothers to report CMF intake for 24 hours.

Visit 2: Infants’ Age 0.6±0.02 N=8 mother-infant dyads withdrew N=133 mother-infant dyads returned after visit 1. to visit 2

N=124 diet records for N=9 mothers did not returned infants' feeding CMF were infants' 24-hour -diet record for returned. CMF feedings.

N=108 diet N=16 diet records were excluded records were from the analysis included in the Reasons: analysis. N=8, physicological criteria N=5, incomplete N=2, no compliance-formula N=1, unreliable

1 Mean age was (mean±SD) 12.3±3.0 days. 2 Mean age was (mean±SD) 19.3±2.9 days.

Figure 3: Subject count for 3-day TWbottle method. Visit 2: Infants’ age Age 0.6±0.01 Subjects that returned to visit 2, N=133

Subjects randomized Subjects randomized to to Formula A, N=67 Formula B, N=66

91 Subjects that Subjects that returned Subjects that Subjects that returned

withdrew, used bottle kits, N=62 withdrew, N=5 used bottle kits, N=56 N=10

Subjects excluded, Subjects excluded, Subjects Subjects included in N total =9 included in N total =10 analysis, N=7, not reliable analysis, N=6, not reliable N=53 N=1, no compliance- N=46 N=1, no compliance- formula formula N=1, incomplete N=1, physiological criteria N=2, incomplete

1 Mean age was (mean±SD) 19.3±2.9 days.

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Appendix A

IRB HUMAN SUBJECTS APPROVAL

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Appendix B

INFORMED CONSENT

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109

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Appendix C

PARTICIPANT HANDOUTS

113

Demographics Questionnaire

Are you a student? YES NO If no, when was the last time you attended school? ______How many years of schooling have you had? (Circle the last grade completed.) Grade School: 1 2 3 4 5 6 7 8 High School: 9 10 11 12 Trade School: 1 2 3 4 If a trade school, how long was the program in years or months? ______College/University: 1 2 3 4 (Name of college: ______) Graduate education (Master’s or Doctoral degree): ______What is your occupation? ______

What is your child’s father’s age? ______How many years of schooling has your child’s father had? (Circle the last grade completed.) Grade School: 5 6 7 8 High School: 9 10 11 12 Trade School: 1 2 3 4 If a trade school, how long was the program in years or months?______College: 1 2 3 4 (Name of college: ______) Graduate education (Master’s or Doctoral degree): ______What is your child’s father’s occupation? ______

What is your family’s total yearly income? _____ Under $10,000 _____$35,000 - $49,999 _____$10,000 - $14,999 _____$50,000 - $74,999 _____$15,000 - $24,999 _____$75,000 - $99,999 _____$25,000 - $34,999 _____$100,000 or more

Do you currently participate in federal nutrition education programs such as WIC? Yes No

114

If so, but it is not WIC, please specify the name: ______

If not participating presently, have you participated in the past? Yes No

If yes, when did you participate (dates)?______

If your child is adopted, please inform the study investigator of your child’s ethnicity and race.

______

1. A. What is YOUR ethnic category?  Hispanic or Latino  Not Hispanic or Latino

B. If you checked “Hispanic or Latin,” do you consider yourself to be any of the following? Check all that apply  Mexican American or Mexican  Central American  South American  Puerto Rican  Cuban  Dominican  Spaniard or Portuguese  Other (please specify country):- ______ Don’t Know

2. A. What is YOUR racial background? (Check all that apply)  White or Caucasian  Black or African American  American Indian or Alaskan Native  Asian or Asian American  Native Hawaiian or Pacific Islander  Other (please specify)

B. If you checked “Black or African American,” do you consider yourself to be any of the following? (Check all that apply)

115

 American  African (please specify country)______ Haitian  Jamaican  Cuban  Puerto Rican  Dominican  Other Caribbean Island  Central/South American  Other (please specify country):______ Don’t Know

C. If you checked “Asian or Asian American,” do you consider yourself to be any of the following? Check all that apply  Chinese  East Indian/South Asian  Japanese  Filipino  Korean  Southeast Asian  Other (please specify country):______ Don’t Know 3. A. What is YOUR CHILD’S FATHER’S ethnic category?  Hispanic or Latino  Not Hispanic or Latino

B. If you checked “Hispanic or Latin,” do you consider the father to be any of the following? Check all that apply  Mexican American or Mexican  Central American  South American  Puerto Rican  Cuban  Dominican  Spaniard or Portuguese  Other (please specify country):______ Don’t Know

4. A. What is YOUR CHILD’S FATHER’S racial background? (Check all that apply)

116

 White or Caucasian  Black or African American  American Indian or Alaskan Native  Asian or Asian American  Native Hawaiian or Pacific Islander  Other (please specify) B. If you checked “Black or African American,” do you consider the father to be any of the following? (Check all that apply)  American  African (please specify country)______ Haitian  Jamaican  Cuban  Puerto Rican  Dominican  Other Caribbean Island  Central/South American  Other (please specify country):______ Don’t Know

C. If you checked “Asian or Asian American,” do you consider the father to be any of the following? Check all that apply  Chinese  East Indian/South Asian  Japanese  Filipino  Korean  Southeast Asian  Other (please specify country):______ Don’t Know

117

GENERAL QUESTIONNAIRE

1. First, what is your date of birth? ______Age:______2. Do you have a religious preference? ______3. Are you single, divorced, widowed, or married, co-habitating?

______

4. Does your child’s biological father live in your household? YES NO Is father part of child’s life? YES NO How often does child see biological father?

If no, find out if male adult (Stepfather), other than father lives in household? ______

5. How many times have you been pregnant?______

6. How many children did you give birth to?______

7. What is the age and gender of each child? age______gender ♀ ♂ age______gender ♀ ♂ age______gender ♀ ♂ age______gender ♀ ♂ age______gender ♀ ♂ 8. What is the birth order of the child currently enrolled in the study?______9. Your child's date of birth: ______

10. Her/his birth weight: ______in pounds (______kg)

11. Her/his birth length: ______in inches (______cm)

12. Your length of pregnancy (in weeks): ______

13. Your height: ft. in.

14. Pre-pregnancy weight:

15. How many pounds did you gain during this pregnancy?

16. Post-pregnancy weight:

118

17. Did you have a pre-pregnancy history of diabetes? Yes No

18. Did you develop diabetes during pregnancy? Yes No

19. Did you smoke during pregnancy? Yes No

20. Type of delivery: Natural C-Section

Was mom put on antibiotics during delivery? Yes No

If yes, please list:

Was baby put on antibiotics during delivery? Yes No

If yes, please list:

Any complications during pregnancy/birth? Yes No

If yes, please list:

21. Has your child ever been breastfed? Yes No a. If Yes, for how long? ______days/weeks

22. Has your child ever been formula fed? Yes No a. If Yes, for how long? ______days/weeks b. What kinds of formula did your child receive (ask for the formula the child had the most of and also any others s/he tried)? ______

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Appendix D

DATA COLLECTION FORM

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