Supplemental Material can be found at:

http://jn.nutrition.org/content/suppl/2015/07/15/jn.115.21241
5.DCSupplemental.html
The Journal of Nutrition
Nutrient Requirements and Optimal Nutrition

Early Protein Intake Is Associated with Body

Composition and Resting Energy Expenditure in
Young Adults Born with Very Low Birth Weight1–3
Hanna-Maria Matinolli,4,5* Petteri Hovi,4,6 Satu Männistö,4 Marika Sipola-Leppänen,4,5,7
Johan G Eriksson,4,8,9,10 Outi Ma¨ kitie,6,10 Anna-Liisa Ja¨ rvenpa¨ a¨ ,6 Sture Andersson,6 and Eero Kajantie4,6,11
4
Department of Health, Chronic Disease Prevention Unit, National Institute for Health and Welfare, Helsinki, Finland; 5Institute
of Health Sciences, University of Oulu, Oulu, Finland; 6Children’s Hospital, University of Helsinki and Helsinki University
Hospital,
Helsinki, Finland; 7Department of Pediatrics and Adolescence, Oulu University Hospital, Oulu, Finland; 8Department of General
Practice and Primary Health Care, Institute of Clinical Medicine, University of Helsinki, Helsinki, Finland; 9Unit of General
Practice, Helsinki University Hospital, Helsinki, Finland; 10Folkhä lsan Research Centre, Helsinki, Finland; and 11Department of
Obstetrics and
Gynaecology, MRC Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland

Downloaded from jn.nutrition.org by guest on November 8, 2016

Abstract
Background: Suboptimal nutrition during fetal life and early childhood may be important in early programming of health
and disease. Preterm infants born with very low birth weight (VLBW; <1500 g) frequently receive inadequate neonatal
nutrition; the long-term consequences are poorly known.
Objective: We evaluated the association between early macronutrient intake and body composition in young adults born with VLBW.
Methods: We collected comprehensive information on daily nutritional intake during the initial hospital stay for 127 participants
of the Helsinki Study of Very Low Birth Weight Adults. We calculated mean daily intakes of energy, protein, fat, and carbohydrate
during the first 9 wk of life. At the mean age of 22.5 y, the subjects underwent measurements of weight, height, body composition
by dual-energy X-ray absorptiometry, and resting energy expenditure. The associations were examined by linear regression.
Results: We found that energy, protein, and fat intakes during the first 3 wk of life, all below current recommendations, predicted
adult body composition. When adjusted for sex, age, birth weight SD score, and gestational age, a 1 g · kg21 · d21 higher protein
intake predicted 11.1% higher lean body mass (LBM) (95% CI: 3.7%, 18.9%) and 8.5% higher resting energy expenditure (REE)
(95% CI: 0.2%, 17.0%). Among those born before 28 wk of gestation, the numbers were 22.5% (95% CI: 1.9%, 47.4%) for LBM
and 22.1% (95% CI: 3.6%, 44.0%) for REE. Similar associations were seen with energy (P = 0.01, P = 0.05) and fat (P < 0.01, P =
0.03) but not with carbohydrate. Energy intake was also associated with BMI (P = 0.01) and fat intake with BMI (P < 0.01) and
percentage body fat (P = 0.05). The results were little changed when adjusted for prenatal and postnatal characteristics.
Conclusions: At relatively low neonatal protein intake levels, additional protein intake is reflected in a healthier body composition,
accompanied by a higher metabolic rate, in young adults born with VLBW 20 y earlier. J Nutr 2015;145:2084–91.

Keywords: preterm birth, very low birth weight, nutrition, early protein intake, body composition,
resting energy expenditure

Introduction
The developmental origins of the health and disease theory,
originally proposed by David Barker in the early 1980s, suggest
that certain early life events during critical time windows of
prenatal and postnatal
1
Supported by grants from the Academy of Finland, Jenny and Antti Wihuri
Foundation, Emil Aaltonen Foundation, the Finnish Government Special
Subsidiary for Health Sciences (EVO), Finnish Medical Societies (Duodecim and
Finska La¨ karesa¨ llskapet), Jalmari and Rauha Ahokas Foundation, Juho Vainio
Foundation, Novo Nordisk Foundation, Signe and Ane Gyllenberg Foundation,
Sigrid Jusélius Foundation, and Yrjö Jahnsson Foundation.
2
Author disclosures: H-M Matinolli, P Hovi, S Männistö, M Sipola-Leppänen, JG
Eriksson, O Mäkitie, A-L Järvenpää, S Andersson, and E Kajantie, no conflicts of interest.
3 12
Supplemental Tables 1–3 and Supplemental Figure 1 are available from the
‘‘Online Supporting Material’’ link in the online posting of the article and from the
same link in the online table of contents at http://jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: hanna-maria.matinolli@thl.fi.
life have a profound impact on a person’s health and well-being
in adulthood (1–5). The important role of early nutrition as an
exposure is supported by a large body of animal studies, in which
relatively modest alterations in early nutrient intake produce
substantial life-long effects on risk factors for adult cardiometabolic
disease (6–8). Despite the lack of direct human evidence,
modification of early nutrition is widely seen as a potential way to
intervene in early programming of adult disease. Particular
attention has recently been devoted to early protein intake.
Among infants born preterm (<37 gestational weeks) or with
low (LBW12; <2500 g) or, in particular, very low birth weight
Abbreviations used: BPD, bronchopulmonary dysplasia; LBM, lean body
mass; LBW, low birth weight; PBF, percentage body fat; REE, resting energy
expenditure; REE:LBM, ratio of REE to LBM; SGA, small for gestational age;
VLBW, very low birth weight (birth weight , 1500 g).

ã 2015 American Society for Nutrition.

2084 Manuscript received February 13, 2015. Initial review completed March 10, 2015. Revision accepted June 24, 2015.
First published online July 15, 2015; doi:10.3945/jn.115.212415.
(VLBW; <1500 g), protein requirements are relatively high, and
the key goal was to attain these requirements with formula
enrichments or, during early intensive care, with intravenous
nutrition. Although there is some evidence of benefits with more
healthy body composition #1 y of age (9–11), the long-term
effects on body composition and cardiometabolic health are
limited and contra- dictory (12–15). In infants born at term, who
have lower protein requirements, the discussion has centered on
the early protein hypothesis that suggests that excess protein
intake during infancy, by increasing weight gain in infancy,
results in increased risk of obesity later in life (16). The
hypothesis arose from observations that breast milk, which
contains lower amounts of protein than infant formulas, protects
from later obesity (17). This hypothesis has gained support
from randomized trials that compared high- and low-protein
formulas among healthy term-born children at 6 y of age (18)
and among term-born children small for gestational age (SGA;
birth weight < 22 SDs) at 5–8 y age (19). However, only the
SGA trials have thus far demonstrated a specific effect on fat
mass rather than lean mass. Again, evidence on longer-term
outcomes is lacking.
To assess the long-term effects of early protein intake, we
used the unique natural experiment of the Helsinki Study of
Very Low Birth Weight Adults (<1500 g). During the
postnatal hospital treatment of these preterm infants,
typically lasting for 2–4 mo, there is considerable variation in
nutrient intake, which is recorded in detail daily by the
medical staff. We have extracted these data for the first 9 wk
of life from the hospital records and assessed body
composition and resting energy expenditure (REE) in these
subjects when they became young adults. Our hypothesis was
that higher fat, protein, and total energy intakes in early life
predict overweight and obesity in early adulthood. Our
specific aim was to assess the role of protein intake in this
context.
Methods
The original study cohort, The Helsinki Study of Very Low Birth
Weight Adults, consisted of 335 consecutive infants born with
VLBW (<1500 g) between January 1978 and December 1985 and
cared for at the neonatal intensive care unit of Children’s Hospital at
Helsinki University Central Hospital (20) (Supplemental Figure 1).
When the study cohort reached young adulthood, a group for
comparison was selected from hospital records of infants born at
term and not SGA. Participants were group- matched for age, birth
hospital, and sex and were invited to take part in a clinical
examination to which 166 of the VLBW subjects and 172 of the
subjects born at term agreed to participate. All participants gave
their written informed consent, and the Ethics Committee of
Children and Adolescents’ Diseases and Psychiatry at the Helsinki
University Central Hospital approved the study protocol. In the
present study we did not use data collected for the comparison
group.
We collected the daily nutritional intake during the initial
hospital stay from hospital records for 158 subjects born preterm
and who underwent the clinical examination (data were
unavailable for 8 subjects). For 17 subjects we were not able to
find complete hospital records, and they were therefore excluded
from the analyses. Compar- ison between subjects included and
subjects excluded is shown in Supplemental Table 1. After 9 wk
there was a reduction in the number of participants with sufficient
data because of discharges from the hospital. Hence, we decided to
limit the data to the first 9 wk of life. We then decided to further
divide the 9-wk data into three 3-wk periods. We excluded the
subjects with neurosensory impairments (n = 14; cerebral palsy, n
= 13; mental retardation, n = 4; and blindness, n = 2, some of
these participants had $2 impairments) from the main analyses
because these conditions could have a separate association with
adult body composition and metabolism. The definitions of
cerebral palsy and mental retardation were based on self-report.
We collected detailed information on enteral and parenteral
nutri- tion, medications, and blood transfusions the infant received
and daily/ weekly growth measurements from the records of the
neonatal intensive care unit or follow-up units. We excluded
medications and blood transfusions from the analysis because of
minimal nutritional values for macronutrients. In our analysis, the
macronutrient content of the mothers’ own breast milk was based
on figures published by Anderson et al. (21) with measures drawn
from preterm milk. The corresponding content for banked human
breast milk was based on values published by Rö nnholm et al. (22,
23) with macronutrient content analyzed from the banked
pasteurized milk from the milk bank of the same hospital where the
infants of the present study were treated. The contents of fortifiers
were based on concentrations received from manufacturers
(Nutricia Baby Oy). For the contents of special preterm formulas
or protein fortifiers we used data published during the matching
time period in European countries (24, 25).
All infants received pooled, banked, and pasteurized human
milk. The practice was to start enteral feedings through a
nasogastric tube during the first or second day of life with pooled
banked human milk. The daily milk intake was thereafter
increased according to individual tolerance until the amount of
200 mL · kg21 · d21, which was then maintained until discharge.
Calcium, phosphate, and multivitamin supplementation was used
throughout the study period (23). Milk fortifiers or preterm
formulas were being introduced; 28 of the participants (22%)
received these during the 9-wk period and only 1 participant
during the first 3 wk of life. If targeted enteral feeding was not
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possible, intravenous fluids were started with glucose up to 15% [44
participants (35%)]. Parenteral nutrition with amino acids to 22
participants (17%) and lipids to 19 participants (15%) were
gradually introduced from the second or third day.
Clinical measurements. At the mean age of 22.5 y the subjects
attended a clinical examination, after a fasting period of $8 h. During
the examination height, weight, and waist and hip circumferences
were measured, and BMI was calculated. The participants completed
a detailed questionnaire on socioeconomic status, smoking, medical
history, and use of medication. Detailed information on maternal
pregnancy was collected separately from the records of birth
hospitals and maternity clinics.
A whole-body DXA (Hologic Discovery A, software version
12.3:3) was used to measure body composition (n = 118) (20, 26).
Calibration of the measurements was performed with a spine
phantom; inter-CV for the phantom bone mineral content, area,
and bone mineral density were 0.35%, 0.21%, and 0.41%,
respectively. The reproducibility of DXA measurement for bone,
fat, and lean mass is 1.2%, 1.9%, and 0.7%, respectively (27).
REE was measured at rest by indirect calorimetry (Deltatrac II;
Datex) when the device was available (n = 96) (28).
Statistical analyses. All statistical analyses were run on IBM SPSS
for Windows 21, and P < 0.05 was used to indicate significance. The
chi- square test was used to compare proportions, and Student’s t test
was used to compare means. We used multiple linear regression
analysis to assess associations between energy and nutrient intakes at
early age (1–3 wk, 4–6 wk, and 7–9 wk) as predictors and body
composition measurements in young adulthood as outcomes. Results
of the linear regression are presented as B (95% CIs). For the
outcome variables with skewed distributions [BMI, lean body mass
(LBM), and REE] we used log-transformations for the distributions
to attain normality. After analyses the results were back-transformed.
The results are therefore expressed as percentages. Potential
confounders included in the adjusted analyses were sex, age at
clinical examination (model 1), gestational age, and birth weight SD
score (model 2). In addition to the potential confounders used in the
full model, we included highest parental education as an indicator of
socioeconomic status in childhood, maternal smoking during
pregnancy, and maternal preeclampsia and neonatal exposure to
treatment with ventilator (days), bronchopulmonary dysplasia
(BPD; prospectively confirmed by a neonatologist), septicemia
(diagnosed if the infant had symptoms and if the blood culture was
positive), exchange transfusion, or persistent ductus arteriosus. We
ran the analyses also with current smoking and leisure time physical
exercise
Early protein intake and adult body composition 2085
(assessed by questionnaire) (29) as covariates. The potential TABLE 1 Characteristics of the young adults born with very low
mediating role of height was also tested in the analyses of LBM. birth weight1
We compared the body size and composition of the subjects included
in the study (n = 127) with the VLBW members of the original Values Missing values, n
cohort who attended the clinical examination but who were
excluded from our analyses because of missing data of early Women 73 (58) 0
nutrition (n = 25), and no statistically significant differences were At delivery
found between the groups. Gestational age, wk 29.0 6 2.1 0
Birth weight, g 1105 6 218 0
Birth weight SD score 21.20 6 1.51 0
Results Small for gestational age 37 (29) 0
Sample description. The characteristics of the study Twins 17 (13) 0
partici- pants are shown in Table 1. At the mean 6 SD age of Triplets 5 (4) 0
22.5 6 2.1 y, as expected, men and women differed in the Apgar score 1 min 5.70 6 2.69 2
body composition measurements and REE. Because the Maternal smoking during pregnancy 25 (20) 0
interaction between sex and early nutrient intake in the
Maternal preeclampsia 23 (18) 0
further analysis was not sig- nificant, we did not perform
sex-specific analyses. Maternal chorioamnionitis 8 (6) 3
Premature rupture of membranes 20 (16) 3
Energy. Mean energy intake during weeks 1–3 was lower Neonatal
than currently recommended, but it reached the Supplemental oxygen, d 15 [4, 35] 5
recommended levels after week 4 (Table 2). All except 6 of Mechanical ventilation, d 6 [0, 17] 2
the study participants met the recommended level of energy Bronchopulmonary dysplasia 24 (19) 0
intake by the end of the 9-wk period. The associations Sepsis 8 (6) 3
between energy intake and adult body composition are shown
Weight SD score at term age 22.51 6 1.16 1

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in Table 3. Higher energy intake during weeks 1–3 predicted
higher BMI and higher LBM as determined by DXA (model Current
1, adjusted for sex and age, and model 2, additionally Age, y 22.5 6 2.1 0
adjusted for gestational age and birth weight SD score). The Height, cm
association between energy intake and LBM persisted after Men 174.9 6 7.0 0
adjustment for parental education and prenatal and neonatal Women 162.9 6 7.2 0
factors (full model). The results remained the same after BMI, kg/m2
further adjustments for current smoking and leisure time
Men 21.6 6 3.0 0
physical activity (data not shown). Energy intake was not
associated with height, percentage body fat (PBF), or waist Women 22.4 6 3.9 0
circumference. Waist circumference, cm
Among the subset of 96 subjects who underwent Men 81.3 6 8.1 0
indirect calorimetry, higher energy intake tended to be Women 78.3 6 10.1 0
associated with higher REE (model 2; P = 0.05) and lower Percentage body fat
ratio of REE to LBM (REE:LBM) (model 2; P = 0.05) (Table Men 18.9 6 5.0 4
4). From week 4 onward energy intake did not predict adult Women 31.7 6 6.0 5
body size, composition, or REE. Lean mass, kg
Protein. Mean protein intake increased throughout the Men 53.5 6 7.7 4
first 9 postnatal weeks, although it remained substantially Women 39.9 6 5.5 5
below current recommendations with only 1 study REE, kcal/d
participant reaching the currently recommended level Men 1810 6 218 17
during the assessed 9-wk period (Table 2). During weeks Women 1468 6 215 14
1–3 higher protein intake predicted higher LBM such that REE:LBM, kcal · kg21 · d21
1 g · kg21 · d21 higher intake predicted 11.2% higher LBM Men 33.7 6 2.3 18
(Table 3). The association persisted after adjustment for
Women 37.0 6 3.7 17
sex; age; parental education; and prenatal, neonatal, and
current lifestyle factors and also after adjustment for Parental education
height. The associations of protein intake with height (P = Elementary 12 (10) 3
0.08) and BMI (P = 0.06) were borderline statistically High school 32 (26)
significant (model 1) but attenuated after adjustments (full Intermediate 45 (36)
model). The associations between protein intake and PBF University 35 (28)
or waist circumference were not statistically significant. Smoking 34 (27) 3
Higher protein intake also predicted higher total REE Leisure time physical activity2
and lower REE:LBM (Table 4). Protein intake from week 4
Competing 5 (4) 3
onward did not predict adult body size, composition, or
Brisk, .3 h/wk 30 (24)
REE.
Light, .4 h/wk 43 (35)
Fat. Mean fat intake during weeks 1–3 was slightly below Not much 46 (37)
current recommendations, but it reached the recommended 1
Values are n (%), means 6 SDs, or mean [25th, 75th percentiles]; n = 127 unless
levels after week 4 (Table 2). Higher fat intake during weeks observations were missing. REE, resting energy expenditure; REE:LBM, ratio of
1–3 predicted higher BMI and higher LBM (Table 3); 1 g · resting energy expenditure to lean body mass.
kg21 · d21 higher intake predicted 3.8% higher BMI and 2
Assessed by the following question: 1) How much do you exercise and stress yourself
3.6% higher LBM. The associations persisted after physically in your leisure time? 2) I regularly train for competitive sports several times a week; 3)
adjustment for sex, age, I exercise to maintain my physical condition for at least 3 h/wk; I walk, cycle, or perform other
exercise not causing substantial perspiration at least 4 h/wk; or 4) I do not exercise much.

2086 Matinolli et al.

TABLE 2 Intakes of energy and macronutrients among the young adults born with very low birth weight 1

Energy, kcal · kg21 · d21 Protein, g · kg21 · d21 Fat, g · kg21 · d21 Carbohydrate, g · kg21 · d21
From human From human From human From human
n Total intake milk Total intake milk Total intake milk Total intake milk

Weeks 1–3 127 94.1 6 15.5 (64.6, 120) 78 6 24 1.4 6 0.4 (0.8, 2.1) 1.2 6 0.4 4.3 6 1.1 (2.2, 6.0) 3.9 6 1.2 11.1 6 1.29 (9.04, 12.9) 8.08 6 2.47
Weeks 4–6 117 119 6 14.6 (97.1, 142) 108 6 22 1.9 6 0.4 (1.4, 2.8) 1.7 6 0.4 5.9 6 1.0 (4.3, 7.6) 5.5 6 1.2 12.4 6 1.30 (10.1, 14.9) 11.2 6 2.27
Weeks 7–9 96 124 6 13.3 (106, 147) 109 6 25 2.1 6 0.5 (1.6, 3.1) 1.7 6 0.4 6.1 6 0.9 (5.0, 7.6) 5.4 6 1.3 12.9 6 1.28 (10.8, 15.5) 11.3 6 2.56
Current 110–135 1) 4.0–4.4 4.8–6.6 11.6–13.2
recommendations2 2) 3.5–4.0
1
Values are means 6 SDs (5th, 95th percentile).
2
Recommendations published (30); for protein, infant weight: 1) ,1 kg; 2) 1.0–1.8 kg.

parental education, and prenatal and neonatal factors and for
current lifestyle factors. Higher fat intake tended to be associ-
ated with PBF (P = 0.08 for model 1, P = 0.05 for model 2)
and waist circumference (P = 0.07 for model 1, P = 0.05 for
model 2); however, the adjustments for neonatal and current
characteris- tics attenuated the results. Fat intake was not
associated with height.
Higher fat intake was associated with higher REE and
lower REE:LBM (Table 4). Fat intake from week 4
predicted adult height, there were statistically significant
inter- actions between the effects of gestational age and
energy (P =
0.01 in the full model), protein (P = 0.01), and fat intake
(P = 0.01). We conducted the analyses separately among
subjects born before 28 wk of gestation (characteristics of
this group are presented in Supplemental Table 2), the
commonly used limit for extremely low gestational age,
and those born thereafter. The associations with height

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onward did not predict adult body size, composition, or
REE.
Carbohydrate. Mean carbohydrate intake during weeks 1–
3 was slightly below current recommendations, but it reached
the recommended levels after week 4 (Table 2).
Carbohydrate intake did not predict adult body size,
composition, or REE.
Length of gestation, fetal growth, and neonatal condi-
tions. We found no associations between gestational age at
birth or fetal growth as assessed by birth weight SD score and
nutrient intake during weeks 1–3, a period during which
nutrient intake predicted adult outcomes in our study.
We further assessed whether the association of nutrient
intakes during postnatal weeks 1–3 with the adult outcomes
differed according to the degree of prematurity. In analyses
that
were confined to subjects born before 28 wk of gestation.
For example, among these subjects 1 g · kg21 · d21 higher
protein intake predicted 10.8 cm (95% CI: 1.01, 20.5 cm)
taller adult height, 29.1% (95% CI: 7.85%, 54.6%) higher
LBM, and 22.3% (95% CI: 5.34%, 41.9%) higher REE
when adjusted for sex and age, whereas no associations were
present among subjects born after 28 wk of gestation. No
other statistically significant interactions were found
between gestational age and nutrient intake.
The numbers of subjects with specific neonatal conditions
are shown in Table 1. Infants with lung disease that later
met the criteria for BPD had 9.91 kcal · kg21 · d21 (95% CI:
3.23, 16.7 kcal · kg21 · d21; P = 0.004) lower energy intake,
0.19 g · kg21 · d21 (95% CI: 0.03, 0.35 g · kg21 · d21; P =
0.018) lower protein intake, and 0.76 g · kg21 · d21 (95% CI:
0.29, 1.23 g · kg21 · d21; P = 0.002) lower fat intake during
the weeks 1–3 than infants without BPD. No difference in
carbohydrate intake was found

TABLE 3 Linear regression models for the association between nutrient intake between weeks 1 and 3 and body composition
measurements in young adulthood in young adults born with very low birth weight 1

Height, cm (n = 127) P BMI, % (n = 127) P LBM, % (n = 118) P PBF (n = 118) P Waist, cm (n = 127) P

Energy, 10 kcal
Model 12 0.58 (20.26, 1.41) 0.18 2.12 (0.30, 3.87) 0.02 2.33 (0.70, 3.98) 0.01 0.33 (20.35, 1.00) 0.34 0.80 (20.28, 1.88) 0.14
Model 23 0.52 (20.30, 1.34) 0.21 2.32 (0.50, 4.19) 0.01 2.43 (0.70, 4.08) 0.01 0.43 (20.27, 1.13) 0.22 0.97 (20.13, 2.06) 0.08
Full model4 0.42 (20.52, 1.36) 0.38 1.92 (20.10, 3.98) 0.07 2.22 (0.30, 4.19) 0.02 0.26 (20.53, 1.06) 0.51 0.67 (20.59, 1.94) 0.30
Protein, g
Model 12 3.20 (20.38, 6.78) 0.08 7.36 (20.40, 15.8) 0.06 11.2 (3.77, 19.1) ,0.01 0.15 (22.78, 3.09) 0.92 2.53 (22.11, 7.17) 0.28
Model 23 2.74 (20.74, 6.22) 0.12 6.72 (21.92, 16.0) 0.12 10.4 (2.43, 19.1) 0.01 0.62 (23.86, 2.63) 0.71 1.97 (23.21, 7.16) 0.45
Fat, g
Model 12 0.87 (20.32, 2.07) 0.15 3.77 (1.20, 6.29) ,0.01 3.56 (1.21, 5.97) ,0.01 0.85 (20.11, 1.81) 0.08 1.41 (20.12, 2.94) 0.07
Model 23 0.70 (20.47, 1.86) 0.24 3.87 (1.31, 6.61) ,0.01 3.56 (1.21, 5.97) ,0.01 0.97 (20.01, 1.95) 0.05 1.56 (0.01, 3.10) 0.05
Full model4 0.55 (20.77, 1.87) 0.41 3.56 (0.70, 6.61) 0.01 3.25 (0.60, 5.97) 0.02 0.80 (20.29, 1.90) 0.15 1.22 (20.55, 2.98) 0.18
Carbohydrate, g
Model 12 0.41 (20.59, 1.40) 0.42 0.40 (21.71, 2.53) 0.69 1.31 (20.70, 3.36) 0.19 20.26 (21.07, 0.55) 0.53 0.34 (20.94, 1.62) 0.60
Model 23 0.59 (20.36, 1.55) 0.22 0.50 (21.61, 2.63) 0.63 1.71 (20.30, 3.67) 0.09 20.19 (21.02, 0.63) 0.64 0.49 (20.80, 1.77) 0.46
Full model4 0.43 (20.57, 1.42) 0.40 0.00 (22.02, 2.12) 0.96 1.11 (20.60, 3.46) 0.28 20.41 (21.26, 0.45) 0.35 0.20 (21.14, 1.55) 0.76
1
Values are B (95% CIs). Data were analyzed with linear regression. LBM, lean body mass; PBF, percentage body fat.
2
Adjusted for sex and age at clinical examination.
3
Additionally adjusted for gestational age and birthweight SD score.
4
Additionally adjusted for highest parental education, maternal smoking during pregnancy, and maternal preeclampsia and for neonatal exposures of treatment with ventilator
(days), bronchopulmonary dysplasia, septicemia, exchange transfusion, or persistent ductus arteriosus.

Early protein intake and adult body composition 2087

TABLE 4 Linear regression models showing the association predicted higher BMI, LBM, and REE. The most robust
between nutrient intake between weeks 1 and 3 and REE and associations were seen between early protein and fat
REE:LBM in young adults born with very low birth weight 1 intakes, which in most infants were low by today’s
standards, and adult LBM (Figure 1). Nutrient intakes were
REE, % (n = 96) P REE:LBM, % (n = 92) P lower in infants who were born more immature or who
had complications of prematurity, and, for example, the
Energy, 10 kcal association between early protein intake and adult LBM
Model 12 1.34 (20.43, 3.14) 0.13 21.35 (22.46, 20.22) 0.02 was confined to infants born before 28 wk of gestation.
Model 23 1.82 (0.00, 3.87) 0.05 21.15 (22.28, 20.01) 0.05
Otherwise, the associations were independent of prenatal
and neonatal factors. This natural experiment elucidates
Full model4 1.91 (20.18, 4.06) 0.07 21.02 (22.33, 0.31) 0.13
the early protein hypothesis from a different angle by
Full model extended5 2.13 (0.03, 4.28) 0.05 21.11 (22.43, 0.22) 0.10 showing that at relatively low neonatal protein intake
Protein, g levels, additional protein intake is reflected by a more
Model 12 7.02 (20.86, 15.5) 0.08 26.46 (211.0, 21.74) 0.01 healthy body composition 20 y later.
Model 23 8.52 (0.16, 17.0) 0.05 25.52 (210.0, 20.81) 0.02 Our findings should be compared with findings in other
Full model4 8.53 (20.18, 18.0) 0.05 24.73 (29.72, 0.52) 0.08 studies in the light of physiologic protein intakes specific to
the developmental period. In the healthy fetus, daily fetal
Full model extended5 9.03 (0.30, 18.5) 0.04 24.88 (29.85, 0.38) 0.07
protein accretion increases from 1.25 g · kg21 · d21 in
Fat, g
gestational week 24 to 4.34 g · kg21 · d21 in gestational week 37
Model 12 2.41 (20.18, 5.07) 0.07 22.08 (23.69, 20.44) 0.01 and slowly decreases thereafter (31). This, together with increased
Model 23 2.89 (0.23, 5.62) 0.03 21.70 (23.32, 20.05) 0.04 metabolic requirements, has resulted in the current guideline of
Full model4 3.13 (0.14, 6.21) 0.04 21.50 (23.35, 0.39) 0.12 ;4 g · kg21 · d21 protein (Table 2) in small preterm infants.
Full model extended5 3.39 (0.41, 6.46) 0.03 21.64 (23.48, 0.24) 0.09 The mean intakes in our study were less than one-half of this:
Carbohydrate, g 1.4 g · kg21 · d21 during weeks 1–3, only gradually rising to 2.1 g
Model 12 1.21 (20.93, 3.23) 0.28 20.28 (21.63, 1.08) 0.68 · kg21 · d21 by week 9, accompanied by a much slower growth
Model 23 than would take place in utero. After term birth, physiologic

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1.53 (20.57, 3.68) 0.15 20.41 (21.73, 0.93) 0.55
Full model4 1.29 (20.93, 3.56) 0.25 20.10 (21.53, 1.36) 0.89
Full model extended5 1.52 (20.72, 3.81) 0.18 20.12 (21.58, 1.36) 0.87
1
Values are B (95% CIs). Data were analyzed with linear regression. REE, resting
energy expenditure; REE:LBM, ratio of resting energy expenditure to lean body mass.
2
Adjusted for sex and age at clinical examination.
3
Additionally adjusted for gestational age and birthweight SD score.
4
Additionally adjusted for highest parental education, maternal smoking during
pregnancy, and maternal preeclampsia and for neonatal exposures of treatment
with ventilator (days), bronchopulmonary dysplasia, septicemia, exchange transfusion,
or persistent ductus arteriosus.
5
Additionally adjusted for leisure time exercise and current daily smoking.
between infants diagnosed with BPD and infants without the
diagnosis. Infants who were treated longer with ventilators or
who were supplied oxygen for a longer time had lower early
intakes of energy, protein, and fat (P < 0.01) and had (P =
0.04) or tended to have (P = 0.07) a lower intake of
carbohydrate. Infants who received indomethacin or who
underwent surgery because of persistent ductus arteriosus had
lower intakes of energy, protein, and fat (P < 0.001 for all
associations). Neonatal sepsis or exposure to exchange
transfusion was not associated with nutrient intake during
weeks 1–3 (P > 0.10 for all associations).
We further assessed the effects of key prenatal
conditions on our main outcomes. No associations were
found between nutrient intake and being born from
preeclamptic or multiple pregnancy or between nutrient
intake and maternal smoking during pregnancy.
Adjustments for these neonatal and prenatal conditions are
shown in Table 3 (full model). In general, the results
showed little change.
To take the important role of growth into account, we
adjusted for growth during the first 3 wk [difference between
birth weight SD score and weight SD score at age 21 d,
available for 122 participants (96%)]. The adjustments did
not change our results.
Discussion
We found that macronutrient intake during the first 3 wk
after preterm birth predicted body composition and energy
metabo- lism 20 y later. Higher intakes of total energy,
protein, and fat all
2088 Matinolli et al.
protein intake is reduced. For example, a study showed that
protein intake in exclusively breastfed infants at 3 mo is 1.1 g
· kg21 · d21, corresponding to 6.8 g/d (32). In formula-fed
infants, protein intake can be roughly 2- fold (18).
The European Childhood Obesity Project included 1138
formula-fed infants randomly assigned to receive isocaloric
low- or high-protein formula and were compared with
breastfed infants. At 6 y, those who received high-protein
formula had higher BMI than the low-protein formula group,
whose BMI was similar to that of the breastfed infants (18).
However, among a subset of 66 infants who underwent body
composition measurements by isotope dilution at 6 mo, no
difference was found in either fat or LBM between the low-
and high-protein groups, although both groups had higher fat
mass than breastfed infants (33). Although these results are
consistent with the early protein hypothesis, firm evidence of
the effects on body composition awaits further follow-up.
Clearer evidence was provided by trials that randomly
assigned infants born at term SGA to receive protein- and
energy-enriched formula. At 5–8 y, those receiving the
enriched formula had higher fat mass but similar LBM, as
assessed by bioimpedance or isotope dilution (19).
Only a few previous studies have used a similar design
and reported long-term follow-up of early nutrient intake
in small preterm infants. One study assessed adult body
composition by DXA among 61 adolescents born before 34
wk of gestation. No associations were found between
neonatal energy or protein intake and fat mass, although
those who had received more energy were taller and
heavier (13). However, the study used a dichotomous
comparison of high and low intakes which could mask
existing associations that could be revealed by other
methods. Another study assessed insulin sensitivity among
37 4–10-y-olds born before 32 wk of gestation and found
no associations with early macronutrient intakes and
insulin sensitivity (14). Short-term effects of early
nutrition on body composition in preterm LBW infants
were reported. The results suggest an association between
higher protein provision in both hospital (9, 10) and post-
discharge (11) formulas and lower fat mass and higher lean
mass after relatively short follow-up up to

FIGURE 1 Results from linear regression analyses with measure-
ments of body composition of the young adults born with very low
birth weight as dependent variables and nutritional intakes of energy
(A), protein (B), fat (C), and carbohydrate (D) during the first 3 wk of life
as independent variables. Data represent the unstandardized regres-
sion coefficient with 95% CI. *P , 0.05. BMI and LBM were log-
transformed in the analyses so the values presented are expressed as
back-transformed percentages. All models were adjusted for sex, age,
birth weight SD score, gestational age, highest parental education,
1 y. The consistent evidence of these associations is still
lacking (12, 15).
The results of our study could suggest an extension to
the early protein hypothesis by proposing that in the light of
present results and past literature the relations of early
protein intake with body composition are nonlinear. At
subphysiologic intake levels, such as those in our study, a
higher protein intake predicts an increase in lean rather than
fat mass. At rather high levels, relative to the
developmental period, such as in formula-fed infants,
increasing protein intake however will result in higher fat
rather than lean mass in later life (19, 33). An analogous
phenomenon is seen in studies that assessed the long-term
effects of growth during infancy. In studies in subjects
born between 1934 and 1944 in Helsinki, Finland (34), and
between 1970 and 1973 in New Delhi, India (35), a more
rapid increase in BMI between birth and 2 y predicts
higher LBM but not fat mass or percentage in adult life.
These cohorts are likely to include a substantial proportion
of infants with subphysiologic growth. By contrast, among
contemporary Western populations, supra- physiologic
weight gain is more common, and in these popula- tions a
more rapid growth during infancy will result in an increase
in both lean and fat mass later in life (36). Accordingly,
when interventions to reduce infant growth by reducing
protein intake are planned, which may be appropriate in
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today’s infants born at term as a group, care should be
taken that infants who are at the lower end of the protein
intake distribution do not attain too low levels that could
endanger the growth of lean tissue.
Our finding of the association between higher energy,
protein, and fat intakes and higher REE but lower
REE:LBM is also of particular interest when discussing
potential pathways in the development of overweight and
obesity. To our knowledge no other study has yet assessed
this association. We have previously shown that, in
addition to lower LBM (26), VLBW adults in this cohort
have lower REE and higher REE:LBM than adults born at
term (28); we now show that this phenomenon is most
pronounced among VLBW adults with lowest early protein
and fat intakes. The main constituents of LBM include
muscle and internal organs, most of which are at rest
metabolically more active than muscle (37). Our finding of
the higher LBM but lower REE:LBM of subjects who
received more protein supports the view included in the
developmental origins of adult health and disease
hypothesis that materno-fetal undernutrition influ- ences
the metabolic phenotype later in life so that undernutrition
during early life increases the metabolic activity, resulting
in more active energy metabolism in adulthood.
The design of our study holds a set of strengths and
limitations. Our cohort is unique in that the data on neonatal
nutrient intake are based on objective recordings by medical
staff. However, data for weeks 1–3 were unavailable for 25
subjects. In addition, a small proportion of infants received
nutritional products for which we have not been able to trace
the exact compositions, and we have used compositions of a
closely corresponding product (e.g., the same label with
composition available for a different study year). All these
inaccuracies would, however, only be expected to increase
random error and
maternal smoking during pregnancy, and maternal preeclampsia and
for neonatal exposure to treatment with ventilator (days), broncho-
pulmonary dysplasia, septicemia, exchange transfusion, or persistent
ductus arteriosus. n = 127 for height, BMI, and waist circumference;
n = 118 for LBM and PBF. LBM, lean body mass; PBF percentage
body fat.
Early protein intake and adult body composition 2089
result in a more conservative conclusion. Although selection
bias cannot be excluded, a comparison of participants and
nonpar- ticipants (20) showed no difference in perinatal,
neonatal, and socioeconomic variables. Participants who
were diagnosed with cerebral palsy by 15 mo were less
likely to participate, which is not an issue in the present
study that focused on unimpaired subjects. As to
confounding, the most important concern is whether the
associations could be due to a medical condition that
affected both neonatal nutrition and the adult outcomes.
Although residual confounding remains a possibility, the
asso- ciations remained remarkably similar after careful
adjustment for such conditions. Confounding by genetic and
lifestyle factors is unlikely because these would not be
expected to affect neonatal nutrition. Because the severity
of illness is an important factor that affects the nutrient
requirements of VLBW infants, it is possible that there
remains some factors about the early weeks of life we have
not been able to assess even though we have made careful
adjustments for covariates that describe the severity of
illness. A further limitation of our study is that we have no
data on body composition in childhood; hence, we cannot
assess body composition trajectories.
Despite the limitations of the historical perspective, our
data represent the nutrition the VLBW infants received in
the 1970s and early 1980s and how it has affected their
body composition in adult age. Because of the
comprehensive data, we were able to take into account
potential confounders at different stages of our subjects’
lives, prenatal, neonatal, and current, which seem to have
little effect on our results. Because of substantial changes
in the nutritional regimen of the VLBW infants, our
findings may not be representative of VLBW neonates
born today.
In conclusion, our results suggest that early nutritional
management of the preterm infants predicts body
composition 20 y later by showing that higher intakes of
energy, protein, and fat are associated with especially LBM
and energy metabolism They also extend the early protein
hypothesis by showing that at protein intake levels that are
low for the developmental period a higher protein intake is
associated with a more healthy body composition. When
reducing protein and energy intakes in infancy, care should
be taken that some infants do not attain levels low enough
to endanger the development of healthy body composition.
Acknowledgments
PH, SM, MS-L, JGE, A-LJ, SA, and EK designed the
research;
PH, SM, MS-L, JGE, OM, and EK conducted the research;
H-MM analyzed the data; H-MM and EK wrote the paper;
H-MM had primary responsibility for final content. All
authors read and approved the final manuscript.
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