Systematic Review and Meta Analysis
Neonatology 2018;113:187–205 Received: March 22, 2017
DOI: 10.1159/000481192
Low- versus High-Dose and Early versus Late
Parenteral Amino-Acid Administration in
Very-Low-Birth-Weight Infants: A Systematic
Review and Meta-Analysis
Erika K.S.M. Leenders a Marita de Waard a Johannes B. van Goudoever a, b 
a Department
of Pediatrics, VU University Medical Center, and b Department of Pediatrics, Emma Children’s Hospital,
Academic Medical Center, Amsterdam, The Netherlands
Keywords
Amino acids · Nutritional deficits · Parenteral administration ·
Preterm infants
Abstract
Objectives: Providing parenteral amino acids to very-low-
birth-weight infants during the first weeks of life is critical for
adequate growth and neurodevelopment. However, there is
no consensus about what dose is appropriate or when to
initiate supplementation. As a result, daily practice varies
among neonatal intensive care units. The objective of our
study was to determine the effects of early parenteral amino-
acid supplementation (within 24 h of birth) versus later ini-
tiation and high dose (>3.0 g/kg/day) versus a lower dose on
growth and morbidities. Methods: A systematic review and
meta-analysis of publications identified by searching
PubMed, EMBASE, and Cochrane databases was conducted.
Randomized controlled studies were eligible if information
on growth was available. Results: The search identified 14
studies. No differences were observed in growth or morbid-
ity after early or high-dose amino-acid supplementation, but
for several outcomes, meta-analysis was not possible due to
study heterogeneity. Initiation of amino acids within the first
24 h of life appeared to be safe and well tolerated, and leads
© 2017 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/neo
Accepted after revision: September 4, 2017
Published online: December 22, 2017
more rapidly to a positive nitrogen balance. Conclusions:
Administering a high dose (>3.0 g/kg/day) or an early dose
(≤24 h) of parenteral amino acids is safe and well tolerated
but does not offer significant benefits on growth. Further
large-scale randomized controlled trials in preterm infants
are needed to study the effects of early and high-dose amino
acids on growth and morbidity more consistently and exten-
sively. © 2017 S. Karger AG, Basel
Introduction
During the first weeks of life, premature neonates are
at highest risk of developing nutritional deficits. These
nutritional deficits may reach a daily shortage in energy
and proteins in comparison to the recommended dietary
intake [1]. Providing proteins or amino acids to preterm
infants during the early postnatal period is critical for ad-
equate growth and neurodevelopment [2, 3]. Reduction
in malnutrition results in higher postnatal growth rates
and is associated with favorable long-term neurodevelop-
mental outcome [4–8]. Supplementation of amino acids
E.K.S.M.L. and M.W. share first authorship.
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Univ. of California Santa Barbara
Johannes B. van Goudoever
VUmc, c/o Room ZH 9D-11
PO Box 7057
NL–1007 MB Amsterdam (The Netherlands)
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E-Mail h.vangoudoever @ vumc.nl
has been shown to improve protein balance by increasing
protein synthesis, improving the antioxidant defense sys-
tem [9], and potentially preventing a catabolic state and
neonatal growth retardation [10, 11]. However, no con-
sensus has yet been reached about what dose is appropri-
ate or when to initiate supplementation.
Recent studies showed that a protein dose of 3.5 or
even 4.0 g/kg/day is well tolerated [4, 12, 13]. This state-
ment is in contrast with suggestions from earlier studies
[14, 15] that raised concerns about the incidence of meta-
bolic acidosis and hyperammonemia in preterm infants
receiving high doses of amino acids through parenteral
nutrition. In addition, there is recent evidence suggesting
that high doses (3.5 g/kg/day) of amino-acid supplemen-
tation do not improve neonatal growth compared to low
doses (2.5 g/kg/day) [13, 16]. As a result, there is no con-
sensus about the optimal dose of parenteral amino-acid
administration, there is a concern regarding side effects
when higher doses are submitted, and, finally, it is uncer-
tain whether neonatal growth improves at all. There is
little assessment of long-term efficacy and safety, which
makes reliable recommendations difficult.
Consequently, the first objective of this systematic re-
view and meta-analysis is to identify the most suitable
dose of parenteral amino-acid administration to very-
low-birth-weight (VLBW; ≤1,500 g) infants within the
first days of life based on short-term safety and efficacy
and, when possible, to include long-term data, too. An-
thropometric data and side effects associated with low-
and high-dose amino acid supplementation were investi-
gated to determine the efficacy and safety of this interven-
tion.
In addition, there is as yet no determination of the op-
timal timing for initiating amino-acid administration to
premature infants. Delaying amino acid intake in a pre-
term infant could potentially result in a catabolic state of
nutrition and can lead to early postnatal growth failure.
The time frame for regaining birth weight (BW) could be
longer and the catch-up requirements greater [17]. In
clinical practice, the start of amino-acid administration
varies among institutions, and it is, therefore, important
to critically review the evidence and assess the benefits
and risks of early versus late amino-acid administration
[18–20]. Subsequently, the second objective of this sys-
tematic review is to determine if early (starting within
24 h after birth) compared to late (starting later than
24 h after birth) supplementation of amino acids has an
effect on VLBW infants’ growth and to define the benefits
and risks of this intervention.
188 Neonatology 2018;113:187–205
DOI: 10.1159/000481192
Methods
For this systematic review and meta-analysis, the requirements
of the PRISMA (Preferred Reporting Items for Systematic Reviews
and Meta-Analyses) statement were followed [21].
Search Strategy for Identification of Studies
Searches in PubMed (http://ncbi.nlm.nih.gov/pubmed), EM-
BASE (http://www.embase.com), and Cochrane Central Register
of Controlled Trials (CENTRAL, The Cochrane Library, http://
www.thecochranelibrary.com, latest issue) through September 21,
2016, were done by using the key terms (words in the title or ab-
stract of the study) “protein,” “peptide,” “amino acid,” “parenter-
al,” “intravenous,” and “infusion,” and the population-related
terms “very low birth weight,” “preterm,” “premature”, plus “in-
fant,” and “neonate.”
A manual search of reference lists of all relevant studies was
performed by E.K.S.M.L. and checked by M.W. The searches were
limited to human studies, and no language restriction was applied.
However, studies written in languages other than English were ex-
cluded later in the process. The citations with abstracts were up-
loaded into a reference database (Reference Manager, version
11.0) and checked for duplicates.
Data Collection
E.K.S.M.L. and M.W. independently selected the studies. Stud-
ies were included if they met all of the inclusion criteria: a random-
ized controlled trial (RCT) design; a study group of preterm infants
weighing <1,500 g who were admitted to a neonatal intensive care
unit, needed parenteral nutrition, and received any type of paren-
teral amino-acid solution within the first days of life; and weight
gain included as an outcome measure. No restriction on the dose
of amino acids was applied. Cohort studies, case series or reports,
and trials including only infants with congenital abnormalities
were excluded.
Data Extraction and Management
Both reviewers read the selected articles. E.K.S.M.L. extracted,
assessed, and coded all data for each study by using a form de-
signed specifically for this review. For each study, E.K.S.M.L. en-
tered final data into RevMan (version 5.2, 2012; The Nordic Co-
chrane Centre, The Cochrane Collaboration, Copenhagen, Den-
mark). M.W. checked each stage of the process.
The following study data were extracted from the studies in-
cluded:
1. Title, first author, journal, and year of publication
2. Study design and study participants’ characteristics (number of
participants, gestational age [GA], BW), and inclusion and ex-
clusion criteria per study
3. Type of intervention and control treatment (duration, start of
amino-acid administration, composition and initial and final
dose of amino acids administered, and co-interventions)
4. Short-term outcome measures:
a. Primary efficacy outcomes: anthropometric data (weight
gain, head circumference [HC] gain, linear growth, and lower-
limb length) and time to regain BW
b. Secondary safety outcomes: death, duration of respiratory
support and supplemental oxygen, necrotizing enterocolitis
(NEC), sepsis, intraventricular hemorrhage (IVH), metabolic
acidosis, hypo-/hyperglycemia, postnatal steroid use, blood
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 3,211 records identified until Sep 21, 2016
6 additional records identified
(PubMed, EMBASE, and Cochrane Library);
through searching of reference lists
2,270 records identified without duplicates

2,276 records screened

2,119 records excluded based

on title/abstract

157 full-text articles assessed 141 full-text articles

for eligibility
Reasons for exclusion:
• Language (n = 26)
• No RCT (n = 35)
16 studies included in quantitative
• Abstract only (n = 19)
and qualitative analysis
• Comment (n = 8)
• No anthropometric data (n = 18)
• Study protocol (n = 35)
Early vs. Low vs. Both
late high dose objectives
Fig. 1. Overview of the selection process 5 studies 8 studies 3 studies
throughout the study. RCT, randomized
controlled trial.

urea nitrogen (BUN), hypoalbuminemia, hypocalcemia, hypo- Sensitivity Analysis

phosphatemia, nitrogen balance, and creatinine and ammonia In cases of poor study quality, a sensitivity analysis was per-
levels formed by removing the particular study to examine whether this
5. Long-term outcomes: anthropometrics; neurodevelopmental significantly changed the results.
outcome at ≥12 months of corrected age

Assessment of Study Quality

The level of evidence of each article was established following the
Oxford Center for Evidence-Based Medicine Level of Evidence scale.
RCT quality was assessed by E.K.S.M.L. and M.W. using the Jadad
criteria (0- to 5-point rating scale, with 5 as maximum score) [22].
Data Analysis
Review Manager software was used to perform analyses (Rev-
Man, version 5.2, 2012; The Nordic Cochrane Centre). A 2-sided
value of p < 0.05 was considered significant.
Measures of Treatment Effect
The first comparison of this study was a higher-dose amino-
acid administration (>3.0 g/kg/day) versus a lower dose (≤3.0 g/
kg/day). A second comparison was performed to compare early
(≤24 h) to late (>24 h) initiation of amino acids in VLBW infants.
For both comparisons, all outcomes were assessed. To analyze
treatment effect and calculate a pooled mean of outcomes reported
in ≥2 studies, the Mantel-Haenszel method was used for categori-
cal outcomes, and the inverse variance method was used for con-
tinuous outcomes.
Assessment of Heterogeneity
For all outcome measures, we assessed the statistical heteroge-
neity of data from the included studies by calculating the I2 statis-
tic (I2 >50% was considered heterogeneous).
Results
A total of 3,211 titles and abstracts were screened in the
databases, and 2,270 individual articles were identified.
Six additional articles were found through a search of ref-
erence lists. After screening titles and abstracts, 2,119 ar-
ticles were excluded. The remaining 157 full-text articles
were assessed for eligibility (Fig. 1).
Data Collection
Sixteen of the 157 studies met the predefined inclusion
criteria (Table  1). Eight studies compared low- versus
high-dose amino-acid supplementation [13, 16, 23–28], 5
studies compared early versus late supplementation [12,
29–32], and 3 studied both [16, 33–35]. Three studies,
from Blanco et al. [33–35], were based on the same study
population and were, therefore, seen as one. Six studies
included co-interventions in their feeding protocol: 2
compared early versus late amino-acid supplementation
[12, 32] and 4 studied low- versus high-dose supplemen-
tation [13, 25, 27, 28]. All authors were contacted for ad-
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Parenteral Amino-Acid Administration to Neonatology 2018;113:187–205 189

VLBW Infants DOI: 10.1159/000481192
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Table 1. Characteristics of studies assessing the effect of low- versus high-dose amino-acid (AA) supplementation and early versus late
AA introduction

First author, Study Study Design Population n Intervention and duration

year location period

Studies assessing the effect of low- versus high-dose AA supplementation

Morgan [25], England 2009 – 2012 RCT Preterm infants with 150 AA and lipids 3.8 vs. 2.8 g/kg/day,
2013 BW <1,200 g, glucose 15.6 vs. 13.5 g/kg/day
and <29 weeks GA
Scattolin [26], Italy 2009 – 2010 RCT Preterm infants with 115 AA 2.0 g/kg/day on DOL 1 advancing to 4.0 g/kg/day
2012 BW <1,250 g on DOL 4 vs. AA 1.5 g/kg/day on DOL 1 advancing to
3.0 g/kg/day on DOL 4
Vlaardingerbroek The 2008 – 2012 RCT Preterm infants with 96 AA 3.6 g/kg/day on DOL 1 vs. AA 2.4 g/kg/day on
[13], 2013 Netherlands BW <1,500 g DOL 1
Tan [27], UK 2004 – 2007 RCT Preterm infants 142 AA and lipids 1.0 g/kg/day on DOL 1 advancing to 4.0
2008 <29 weeks GA g/kg/day in 7 days, and dextrose 16.3 g/kg/day vs. AA
and lipids 1.0 g/kg/day on DOL 1 advancing to 3.0 g/
kg/day in 5 days, and dextrose 13.5 g/kg/day
Burrattini [24], Italy 2006 – 2009 RCT Preterm infants with 131 AA 2.5 g/kg/day on DOL 1 advancing to 4.0 g/kg/day
2013 BW 500 – 1,249 g on DOL 4 vs. 1.5 g/kg/day on DOL 1 advancing to 2.5
g/kg/day on DOL 3
Clark [16], USA 2005 – 2006 RCT Preterm infants 122 AA 1.5 g/kg/day on DOL 1 advancing with
2007 born from 1.0 g/kg/day to 3.5 g/kg/day on DOL 4 vs. AA
23 weeks 0 days to 1.0 g/kg/day on DOL 1 advancing with 0.5 g/kg/day to
29 weeks 6 days GA 2.5 g/kg/day on DOL 4
Blanco[33 – 35], USA 2002 – 2005 RCT Preterm infants 61 AA 2.0 g/kg/day on DOL 1 advancing with
2012 ≥24 weeks GA (after inclu- 1 g/kg/day to 4.0 g/kg/day vs.
ding 20 infants start AA 0.5 g/kg/day on DOL 2 advancing with 0.5 g/
<24 weeks GA, kg/day to 3.0 g/kg/day
<1,000 g BW,
<12 h of age)
Bellagamba [23], Italy 2006 – 2008 RCT Preterm infants born bet- 164 AA at 1.5 g/kg/day on DOL 1 advancing
2016 ween 28 and 29 weeks GA with 0.5 g/kg/day to 3.5 g/kg/day vs.
and AA 1.5 g/kg/day on DOL 1 advancing with
BW 500 – 1,249 g 0.5 g/kg/day to 2.5 g/kg/day
Uthaya [28], UK 2010 – 2013 RCT Preterm infants 168 Start with AA at max. 3.6 g/kg/day on
2016 <31 weeks GA DOL 1 vs. start AA at 1.7 g/kg/day on DOL 1
advancing to 2.7 g/kg/day on DOL 3
Studies assessing the effect of early versus late AA introduction
Wilson [32], Ireland 1990 – 1992 RCT Preterm infants with 125 AA 0.5 g/kg/day on DOL 1 advancing to
1997 BW <1,500 g 2.5 g/kg/day on DOL 5 or 3.5 g/kg/day on DOL 7
requiring mechanical venti- depending on caloric intake vs.
lation AA 1.0 g/kg/day on DOL 3 advancing to
2.5 g/kg/day on DOL 6
Te Braake [31], 2005 The 2003 – 2004 RCT Preterm infants with 135 AA 2.4 g/kg/day at DOL 1 vs.
Netherlands BW <1,500 g AA 1.2 g/kg/day on DOL 2 and
2.4 g/kg/day on DOL 3
Heimler [29], USA Unknown RCT Preterm infants 20 AA 1.5 g/kg/day on DOL 1 advancing to
2010 <34 weeks GA, 2.5 g/kg/day on DOL 3 vs.
requiring respiratory sup- AA 1.0 g/kg/day on DOL 4 advancing to 2.5 g/kg/day
port on DOL 7
Ibrahim [12], USA 2001 – 2002 RCT Preterm infants, 32 AA 3.5 g/kg/day and lipids 3.0 g/kg/day on DOL 1 vs.
2004 24 – 32 weeks GA, AA 2.0 g/kg/day and intralipid 0.5 g/kg/day on DOL
requiring mechanical venti- 3
lation for RDS advancing to 3.5 g/kg/day AA and
3.0 g/kg/day intralipid on DOL 6 and 9
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Table 1 (continued)
First author, Study Study Design Population
year location period
Blanco [33 – 35], USA 2002 – 2005 RCT
2012
Saini [30], England unknown RCT
1989
GA, gestational age; BW, birth weight; RDS, respiratory distress syndrome; DOL, days of life; RCT, randomized controlled trial.
ditional nonpublished data on growth and secondary
outcome measures to enhance the amount of consistent-
ly defined data for meta-analysis.
Assessment of Reporting Biases
To detect publication bias, a funnel plot was construct-
ed. However, there was an insufficient number of studies
to permit proper evaluation of publication bias and to
evaluate potential asymmetry of the funnel plot using
Begg and Egger tests.
Low- versus High-Dose Amino-Acid Supplementation
Characteristics of the included studies assessing the ef-
fect of low- (<3.0 g/kg/day) versus high-dose (>3.0 g/kg/
day) parenteral amino-acid supplementation are shown
in Table 1. The inclusion of preterm infants was based on
GA in 3 studies, BW in 4 studies, and both BW and GA
in 2 other studies.
The maximum doses in the intervention groups ranged
from 3.5 to 4.0 g/kg/day. In 6 of 9 studies, the dose was
slowly increased by 0.5–1.0 g/kg/day over several days,
starting with doses between 1.0 and 3.0 g/kg/day on the
first day of life (DOL). Morgan et al. [25], Vlaardinger-
broek et al. [13], and Uthaya et al. [28] started with the
maximum doses of 3.8, 3.6, and 3.6 g/kg/day on DOL 1,
respectively.
Three studies scored 5 points on the Jadad assessment
[22]. For the other studies, points were subtracted be-
cause the randomization method was not discussed, the
study was not double blind, or no appropriate blinding
method was described (Table  2). Dropouts and with-
drawals were described in all but 1 study. Baseline char-
acteristics and outcome measures of the studies are shown
Parenteral Amino-Acid Administration to
VLBW Infants
n Intervention and duration
Preterm infants 61 AA 2.0 g/kg/day on DOL 1 advancing to
≥24 weeks GA 4.0 g/kg/day on DOL 3 vs.
(after including 20 infants AA 0.5 g/kg/day on DOL 2 advancing to
<24 weeks GA, <1,000 g 3.0 g/kg/day on DOL 7
BW,
<12 h of age)
Ventilator-dependent 21 AA 1.0 g/kg/day on DOL 1 advancing to
preterm infants, 3.0 g/kg/day on DOL 3 vs.
<30 weeks GA AA 1.0 g/kg/day >72 h advancing to
3.0 g/kg/day
in Table 3. Blood levels of ammonia and creatinine, nitro-
gen balance, duration of oxygen support, and incidences
of metabolic acidosis and hypoglycemia are not shown
because none or only 1 of the included studies discussed
these outcomes. Overall, 1,149 infants were included in
the studies: 573 in the intervention groups and 576 in the
control groups.
Outcome Measures
Primary Outcome Measures
In all studies, weight gain was described as an outcome
measure. Tan and Cooke [27] did not find a difference in
weekly weight gain rates between the 2 groups in the first
7 weeks of life but did not specify exact growth rates. Bu-
rattini et al. [24], Clark et al. [16], Vlaardingerbroek et al.
[13], Blanco et al. [33–35]. and Bellagamba et al. [23] re-
ported actual rates in gram per kilogram per day. A meta-
analysis including these 5 studies showed no differences
between the intervention and control groups (Fig.  2a).
However, a lack of consistency in the timeline of this out-
come could have influenced the results. Vlaardinger-
broek et al. [13], Clark et al. [16], and Blanco et al [33–35]
reported mean weight gain for the first 28 days, Burattini
et al. [24] compared mean weight gain at 36 weeks post-
menstrual age (PMA), and Bellagamba et al. [23] reported
mean weight gain rates from birth and regain of BW to
1,800 g (54 days in both groups). Scattolin et al. [26] also
reported growth rates, but only for the 2nd and 3rd week
of life separately and was, therefore, not included in the
meta-analysis. They described a higher growth rate in the
intervention group that became significant in the 3rd
week of life (18.76 [SD 6.83] vs. 14.70 [SD 8.99] g/kg/day,
p < 0.01).
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 HC growth rates were measured in 3 studies [13, 16,

Saini

1989
[30],
27]. Meta-analysis was not possible due to a lack of con-

+
–

2
sistency in the timeline, but none of the individual studies

Blanco

2012a
found a significant difference in head growth rate be-

[35],

–
3
tween groups. Mean HC per group at 36 weeks PMA was

Table 2. Quality assessment of the randomized controlled trials included using the Jadad criteria (0- to 5-point rating scale, with 5 as the maximum score)

Ibrahim
described in 5 studies [23–27]. All 5 studies indicated no

2004
[12],
Early versus late introduction
difference in HC at birth. One study (Morgan et al. [25])

+
–

2
described a significant difference in mean HC in favor of

Heimler
the intervention group (p = 0.007). However, a meta-

2010
[29],
analysis of the 5 studies showed no significant difference

–
2
in mean HC at 36 weeks PMA between groups (p = 0.99,

Wilson Te Braake
with I2 = 39%; Fig. 2b). One study described HC at term

2005
[32], 1997 [31],

–
age and reported on a smaller mean HC in the interven-

1
tion group (mean difference: 0.8 cm, p = 0.02) [28]. Fi-
nally, Blanco et al. [33–35] described no significant dif-

+
–

3
ference in HC between groups at birth and discharge.
Mean linear growth rates (mm/day) using lower-limb-

Uthaya

2016
[28],
length gain, were measured in 2 studies [13, 27]. In Tan

+
5
and Cooke [27], lower-limb-length gain was higher in the

[23], 2016
intervention group in the first 2 weeks of life (0.28 vs. 0.20

gamba
Bella-
mm/day, p = 0.05). Vlaardingerbroek et al. [13] did not

+
–

3
find a difference between groups (mean 0.26 [0.16 SD] vs.

Blanco
0.27 [0.13 SD] mm/day) in the first 28 days of life. No

2012a
[35],

–
3
meta-analysis was possible due to a lack of consistency in
the definition of the outcome measure. Mean length at 36 Clark

2007
[16],

+
weeks PMA was described in 4 studies [23, 24, 26, 27]. In

5
3 studies, no significant differences were found. Tan and
tini [24],
Burrat-

Cooke [27] found a significant difference in mean length

2013

+
–

3
Low- versus high-dose supplementation

at 36 weeks PMA in favor of the intervention group. Me-

ta-analysis showed no significant difference between the
2008
[27],
Tan

+
groups (p = 0.09, Fig. 2c).
–

3
The same 4 studies [23, 24, 26, 27] reported the mean
Vlaardingerbroek

number of days to regain BW. Meta-analysis showed no

[13], 2013

significant difference between both groups (p = 0.20;

Fig. 2d). The heterogeneity of these studies was shown by

1 Jadad score calculated for 3 articles combined.

+

+
–

3
an I2 of 66%.
Scattolin

2012
[26],

Secondary Outcome Measures

+

+
–

All studies reported on death during hospital admis-

Morgan

2013
[25],

sion. In the individual studies, no significant differences

+

+
5

in overall mortality were found. In total, 50 of 491 (10%)

Was the randomization scheme

Was there description of drop-

described and appropriate?

participants in the high-dose group died vs. 60 of 494

Was the study described as

Was the study described as

Was the method of double

(12%) participants in the low-dose group. Meta-analysis

outs and withdrawals?
blinding appropriate?

did not show a significant difference in mortality before

discharge or DOL 28 (p = 0.86; Fig. 2e).
double blind?

Serum BUN levels were reported in 7 studies. All stud-

ies found elevated levels in the intervention group in
random?

comparison to the control group. Blanco et al. [33–35]

measured BUN levels at DOL 1 and 7 and found a sig-
Score
a

nificant difference on both days (mean differences of 1.4

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 a Mean weight gain, g/kg/day*
Study or subgroup Experimental Control Weight, Mean difference Mean difference
% IV, fixed, 95% CI IV, fixed, 95% CI
mean SD total mean SD total

Bellagamba [23], 2016 12.6 1.7 82 12.3 1.6 82 68.9 0.30 (–0.21, 0.81)
Blanco [35], 2012 11.76 4.2 24 11.66 5.65 28 2.4 0.10 (–2.58, 2.78)
Burattini [24], 2013 16.6 2.4 56 16 2.7 58 20.0 0.60 (–0.34, 1.54)
Clark [16], 2007 12.42 4.9 64 11.83 5.94 58 4.7 0.59 (–1.35. 2.53)
Vlaardingerbroek [13], 2013 12.3 5.8 47 13.4 4.7 49 3.9 –1.10 (–3.22, 1.02)

Total (95% CI) 273 275 100.0 0.31 (–0.11, 0.73)

Heterogeneity: χ2 = 2.18, df = 4 (p = 0.70), I2 = 0%
Test for overall effect: z = 1.47 (p = 0.14) –4 –2 0 2 4
Favors experimental Favors control

b Mean head circumference at 36 weeks PMA

Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Bellagamba [23], 2016 306 12 82 308 16 82 23.9 –2.00 (–6.33, 2.33)

Burattini [24], 2013 305 14 56 306 13 58 20.4 –1.00 (–5.96, 3.96)
Morgan [25], 2014 316 13 63 311 15 64 20.8 5.00 (0.12, 9.88)
Scattolin [26], 2013 308.5 13.4 60 307.1 19.4 55 15.4 1.40 (–4.75, 7.55)
Tan [27], 2008 311 15 55 314 13 60 19.5 –3.00 (–8.15, 2.15)

Total (95% CI) 316 319 100.0 –0.01 (–2.88, 2.86)

Heterogeneity: τ2 = 4.13, χ2 = 6.51, df = 4 (p = 0.16), I2 = 39%
Test for overall effect: z = 0.01 (p = 0.99) –10 –5 0 5 10
Favors experimental Favors control

c Mean length at 36 weeks PMA

Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, fixed, 95% CI IV, fixed, 95% CI
mean SD total mean SD total

Bellagamba [23], 2016 42.7 2.3 82 42.8 2 82 31.4 –0.10 (–0.76, 0.56)
Burattini [24], 2013 42.7 2.4 56 42.7 1.9 58 21.5 0.00 (–0.80, 0.80)
Scattolin [26], 2013 43.06 2.19 60 42.03 2.19 55 21.3 1.03 (0.23, 1.83)
Tan [27], 2008 42.9 2.3 68 42.4 2.1 74 25.9 0.50 (–0.23, 1.23)

Total (95% CI) 266 269 100.0 0.32 (–0.05, 0.69)

Heterogeneity: χ2 = 5.43, df = 3 (p = 0.14), I2 = 45%
–2 –1 0 1 2
Test for overall effect: z = 1.68 (p = 0.09)
Favors experimental Favors control

Fig. 2. a–n Meta-analysis of the effects of supplementation of low-
dose amino acids (≤3.0 g/kg/day) compared to high-dose amino
acids (>3.0 g/kg/day). IV, inverse variance; M-H, Mantel-Haenszel;
PMA, postmenstrual age; NEC, necrotizing enterocolitis; IVH, in-
traventricular hemorrhage; DOL, days of life. * Bellagamba et al.
[23]: mean weight gain from birth to 1,800 g (mean 54 days both
groups). Blanco et al. [35], Clark et al. [16], and Vlaardingerbroek
et al. [13]: mean weight gain on DOL 28. Burratini et al. [24]: mean
weight gain at 36 weeks PMA. ** Bellagamba et al. [23] excluded
the 25 infants who deceased before 36 weeks PMA and 8 with NEC
from all further analysis.
(Figure continued on next pages.)
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 d Mean number of days to regain birth weight
Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Bellagamba [23], 2016 12.7 5 82 12.2 5.1 82 28.7 0.50 (–1.05, 2.05)
Burattini [24], 2013 11.2 4.5 56 11.7 4.1 58 28.3 –0.50 (–2.08, 1.08)
Scattolin [26], 2013 14.82 5.77 60 16.15 7.25 55 21.0 –1.33 (–3.74, 1.08)
Tan [27], 2008 10.3 6.1 55 13.9 6.3 59 22.0 –3.60 (–5.88, –1.32)

Total (95% CI) 253 254 100.0 –1.07 (–2.71, 0.56)

Heterogeneity: τ2 = 1.80, χ2 = 8.85, df = 3 (p = 0.03), I2 = 66%
Test for overall effect: z = 1.28 (p = 0.20) –10 –5 0 5 10
Favors experimental Favors control

e Death before discharge, DOL 28 or 36 weeks PMA**

Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, fixed, 95% CI M-H, fixed, 95% CI
events total events total

Bellagamba [23], 2016 13 99 12 98 17.0 1.08 (0.47, 2.51)

Blanco [35], 2012 6 30 4 31 5.1 1.69 (0.42, 6.70)
Burattini [24], 2013 4 64 5 67 7.5 0.83 (0.21, 3.23)
Clark [16], 2007 2 64 1 58 1.7 1.84 (0.16, 20.83)
Morgan [25], 2014 11 74 12 76 16.4 0.93 (0.38, 2.27)
Scattolin [26], 2013 0 60 1 55 2.5 0.30 (0.01, 7.53)
Tan [27], 2008 15 68 16 74 19.4 1.03 (0.46, 2.28)
Uthaya [28], 2016 5 84 11 84 16.8 0.42 (0.14, 1.27)
Vlaardingerbroek [13], 2013 7 47 10 49 13.6 0.68 (0.24, 1.97)

Total (95% CI) 590 592 100.0 0.89 (0.62, 1.28)

Total events 63 72
0.01 0.1 1 10 100
Heterogeneity: χ2 = 3.98, df = 8 (p = 0.86), I2 = 0%
Favors experimental Favors control
Test for overall effect: z = 0.65 (p = 0.51)

f Blood urea nitrogen days 1 – 2

Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Blanco [35], 2012 7.14 3.2 30 5.7 2.1 31 48.2 1.44 (0.08, 2.80)
Vlaardingerbroek [13], 2013 11.7 3.2 47 8.3 2.5 49 51.8 3.40 (2.25, 4.55)

Total (95% CI) 77 80 100.0 2.46 (0.54, 4.37)

Heterogeneity: τ2 = 1.51, χ2 = 4.63, df = 1 (p = 0.03), I2 = 78%
Test for overall effect: z = 2.51 (p = 0.01) –20 –10 0 10 20
Favors experimental Favors control
2
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 g Blood urea nitrogen days 6 – 7
Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Blanco [35], 2012 12.5 4.28 30 6.07 3.21 31 31.9 6.43 (4.53, 8.33)
Scattolin [26], 2013 12.35 4.8 60 10.5 5.24 55 32.2 1.85 (0.01, 3.69)
Vlaardingerbroek [13], 2013 7.7 2.7 47 6.1 1.9 49 35.8 1.60 (0.66, 2.54)

Total (95% CI) 137 135 100.0 3.22 (0.39, 6.05)

Heterogeneity: τ2 = 5.59, χ2 = 20.36, df = 2 (p < 0.0001), I 2 = 90%
Test for overall effect: z = 2.23 (p = 0.03) –20 –10 0 10 20
Favors experimental Favors control

h Hyperglycemia
Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, fixed, 95% CI M-H, fixed, 95% CI
events total events total

Burattini [35], 2013 6 56 20 58 65.5 0.23 (0.08, 0.62)

Vlaardingerbroek [13], 2013 10 47 12 49 34.5 0.83 (0.32, 2.17)

Total (95% CI) 103 107 100.0 0.44 (0.22, 0.85)

Total events 16 32
Heterogeneity: χ2 = 3.36, df = 1 (p = 0.07), I 2 = 70% 0.01 0.1 1 10 100
Test for overall effect: z = 2.42 (p = 0.02) Favors experimental Favors control

j Number of infants on additional oxygen at 36 weeks PMA

Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, random, 95% CI M-H, random, 95% CI
events total events total

Blanco [35], 2012 15 24 8 27 30.0 3.96 (1.23, 12.73)

Burattini [24], 2013 Nov 7 56 13 58 33.0 0.49 (0.18, 1.35)
Tan [27], 2008 40 55 36 59 37.0 1.70 (0.77, 3.76)

Total (95% CI) 135 144 100.0 1.46 (0.49, 4.33)

Total events 62 57
Heterogeneity: τ2 = 0.67, χ2 = 7.40, df = 2 (p = 0.02), I 2 = 73% 0.01 0.1 1 10 100
Test for overall effect: z = 0.68 (p = 0.50) Favors experimental Favors control

and 6.4 mmol/L, respectively, p < 0.001). Clark et al. [16]
reported a significant difference between groups mea-
sured on DOL 7 (p = 0.01). Bellagamba et al. [23] and Bu-
rattini et al. [24] found a significantly higher BUN level in
the intervention groups throughout the first 5 and 10 days
of life, respectively. Uthaya et al. [28] found that signifi-
cantly more infants had BUN levels of >7 and >10 mmol/L
in the intervention groups (p < 0.01). Vlaardingerbroek
et al. [13] and Scattolin et al. [26] reported serum BUN
levels on days 2, 4, and 6, and 7 and 21, respectively. A
meta-analysis was performed for days 1–2 and 6–7
(Fig. 2f, g), which showed a significant effect on days 1–2
(p = 0.01) and days 6–7 (p = 0.03). Heterogeneity of the
studies was shown by an I2 of 78 and 90%, respectively
(Fig.  2f, g). Nitrogen balance was addressed in 1 study
[13], which reported a significantly higher positive bal-
ance on DOL 2 in the intervention group in comparison
to the control group. This difference disappeared on days
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 k NEC ≥ stage 2**
Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, fixed, 95% CI M-H, fixed, 95% CI
events total events total

Bellagamba [23], 2016 4 98 4 99 10.3 1.01 (0.25, 4.16)

Blanco [35], 2012 3 30 2 31 4.8 1.61 (0.25, 10.39)
Burattini [24], 2013 2 64 2 76 4.8 1.19 (0.16, 8.72)
Clark [16], 2007 7 64 3 58 7.6 2.25 (0.55, 9.15)
Morgan [25], 2014 5 74 9 76 22.4 0.54 (0.17, 1.69)
Scattolin [26], 2013 4 60 3 55 7.9 1.24 (0.26, 5.80)
Tan [27], 2008 6 55 6 59 14.0 1.08 (0.33, 3.58)
Uthaya [28], 2016 5 84 7 84 17.8 0.70 (0.21, 2.29)
Vlaardingerbroek [13], 2013 1 47 4 49 10.4 0.24 (0.03, 2.27)

Total (95% CI) 576 587 100.0 0.93 (0.58, 1.48)

Total events 37 40
Heterogeneity: χ2 = 4.61, df = 8 (p = 0.80), I 2 = 0% 0.01 0.1 1 10 100
Test for overall effect: z = 0.31 (p = 0.76) Favors experimental Favors control

l Sepsis
Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, random, 95% CI M-H, random, 95% CI
events total events total

Bellagamba [23], 2016 23 82 20 82 20.9 1.21 (0.60, 2.43)

Blanco [35], 2012 4 30 4 31 4.6 1.04 (0.23, 4.59)
Burattini [24], 2013 9 56 9 58 10.0 1.04 (0.38, 2.85)
Clark [16], 2007 15 64 11 58 13.3 1.31 (0.55, 3.14)
Morgan [25], 2014 26 74 28 76 22.8 0.93 (0.48, 1.81)
Scattolin [26], 2013 9 60 9 55 10.0 0.90 (0.33, 2.47)
Uthaya [28], 2016 2 84 8 84 4.1 0.23 (0.05, 1.13)
Vlaardingerbroek [13], 2013 16 47 17 49 14.3 0.97 (0.42, 2.26)

Total (95% CI) 497 493 100.0 0.99 (0.72, 1.36)

Total events 104 106
Heterogeneity: τ2 = 0.00, χ2 = 4.04, df = 7 (p = 0.77), I 2 = 0% 0.01 0.1 1 10 100
Test for overall effect: z = 0.06 (p = 0.95) Favors experimental Favors control

m Postnatal steroid use

Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, random, 95% CI M-H, random, 95% CI
events total events total

Bellagamba [23], 2016 3 82 9 82 20.0 0.31 (0.08, 1.18)

Burattini [24], 2013 4 56 2 58 14.5 2.15 (0.38, 12.26)
Clark [16], 2007 12 64 18 58 30.8 0.51 (0.22, 1.19)
Scattolin [26], 2013 1 60 4 55 10.1 0.22 (0.02, 2.00)
Tan [27], 2008 9 55 6 59 24.6 1.73 (0.57, 5.22)

Total (95% CI) 317 312 100.0 0.70 (0.32, 1.56)

Total events 29 39
Heterogeneity: τ2 = 0.35, χ2 = 7.21, df = 4 (p = 0.13), I 2 = 45% 0.01 0.1 1 10 100
Test for overall effect: z = 0.86 (p = 0.39) Favors experimental Favors control

2
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 n IVH grade III or IV
Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, random, 95% CI M-H, random, 95% CI
events total events total

Bellagamba [23], 2016 8 82 3 82 15.9 2.85 (0.73, 11.14)

Blanco [35], 2012 3 30 2 31 8.5 1.61 (0.25, 10.39)
Burattini [24], 2013 1 56 2 58 5.0 0.51 (0.04, 5.78)
Clark [16], 2007 8 64 7 58 25.2 1.04 (0.35, 3.07)
Morgan [25], 2014 5 74 2 76 10.6 2.68 (0.50, 14.28)
Scattolin [26], 2013 0 60 1 55 2.8 0.30 (0.01, 7.53)
Tan [27], 2008 6 55 8 59 23.2 0.78 (0.25, 2.41)
Vlaardingerbroek [13], 2013 2 47 3 49 8.8 0.68 (0.11, 4.27)

Total (95% CI) 468 468 100.0 1.18 (0.68, 2.03)

Total events 33 28
0.01 0.1 1 10 100
Heterogeneity: τ2 = 0.00, χ2 = 4.70, df = 7 (p = 0.70), I2 = 0%
Favors experimental Favors control
Test for overall effect: z = 0.58 (p = 0.56)
2

4 and 6. Two studies reported on creatinine levels [16, 26];
neither study found a significant difference between in-
tervention and control groups. Because of a lack of con-
sistency in outcome measures, it was not possible to per-
form a meta-analysis.
The definition of hyperglycemia differed between
studies. Tan and Cooke [27] defined hyperglycemia by a
blood glucose level >12 mmol/L that was treated with in-
sulin. They found a significant difference between the
number of infants that needed insulin treatment in the
groups – 33 in the intervention group vs. 12 in the control
group (p < 0.01) – while dextrose intake was higher in the
intervention group. Burattini et al. [24] defined hypergly-
cemia as blood glucose levels >175 mg/dL (∼9.7 mmol/L)
at 2 consecutive measurements. They also found a sig-
nificant difference, but in favor of the intervention group,
which had significantly fewer episodes of hyperglycemia
(6 vs. 20) during hospital admission (p < 0.001). Glucose
intake was marginally but significantly higher in the in-
tervention group. Scattolin et al. [26], Vlaardingerbroek
et al. [13], Uthaya et al. [28], and Blanco et al. [33–35]
found no differences between groups. Blanco et al. [33–
35] reported no actual data with this statement. Meta-
analysis was possible for 2 studies with a similar glucose
intake between groups [13, 24] and showed an association
between high-dose amino-acid supplementation and a
lower incidence of hyperglycemia (p = 0.02, I2 = 70%;
Fig. 2h).
The number of infants needing oxygen supplementa-
tion at 36 weeks PMA was reported by 3 studies. Blanco
et al. [33–35] found a significant difference in favor of the
control group (p = 0.01). Burattini et al. [24] and Tan and
Cooke [27] did not find a difference. Meta-analysis
showed no overall effect (p = 0.5, with an I2 of 73%;
Fig. 2j). Four studies reported on duration of mechanical
ventilation. Lack of consistency in outcome measures
made meta-analysis impossible. Individual studies did
not show a significant effect.
All studies reported on NEC incidence. Studies used
different definitions, including need for surgical inter-
vention, strong clinical suspicion leading to medical
treatment, and Bell stage >II. In 4 studies, no definition
was given. None of the individual studies reported on a
significant difference, which was confirmed by meta-
analysis including 966 infants (p = 0.80; Fig. 2k).
Additionally, all studies reported on sepsis incidence.
Vlaardingerbroek et al. [13] defined sepsis according to
criteria described by Stoll et al. [36]. Additionally, a blood
culture positive for coagulase-negative staphylococci to-
gether with elevated C-reactive protein (>10 mg/L) was
considered true sepsis. Scattolin et al. [26] defined sepsis
as deterioration of clinical condition with a positive blood
culture. The remaining studies defined sepsis as a positive
blood, urine, or cerebrospinal fluid culture or did not re-
port a definition. None of the studies found a significant
difference between groups. Meta-analysis underlined this
finding (p = 0.95; Fig. 2l).
Eight studies reported on IVH grade ≥III. No signifi-
cant differences in overall incidence were found. This was
supported by meta-analysis (p = 0.99; Fig. 2m). Neuro-
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 a Death before discharge
Study or subgroup Experimental Control Weight Odds ratio Odds ratio
% M-H, random, 95% CI M-H, random, 95% CI
events total events total

Blanco [35], 2012 6 30 4 31 22.7 1.69 (0.42, 6.70)

Ibrahim [12], 2004 1 16 2 16 6.9 0.47 (0.04, 5.73)
Saini [30], 1989 1 11 2 10 6.5 0.40 (0.03, 5.25)
Wilson [32], 1997 15 64 15 61 64.0 0.94 (0.41, 2.13)

Total (95% CI) 121 118 100.0 0.97 (0.50, 1.86)

Total events 23 23
Heterogeneity: τ2 = 0.00, χ2 = 1.41, df = 3 (p = 0.70), I2 = 0% 0.01 0.1 1 10 100
Test for overall effect: z = 0.10 (p = 0.92) Favors experimental Favors control

b Blood urea nitrogen days 1 – 2

Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Blanco [35], 2012 7.14 3.2 30 5.7 2.1 31 46.6 1.44 (0.08, 2.80)
Te Braake [31], 2005 9.6 2.8 66 6 1.8 69 53.4 3.60 (2.80, 4.40)

Total (95% CI) 96 100 100.0 2.59 (0.48, 4.71)

Heterogeneity: τ2 = 2.01, χ2 = 7.19, df = 1 (p = 0.007), I2 = 86%
Test for overall effect: z = 2.41 (p = 0.02) –20 –10 0 10 20
Favors experimental Favors control

c Blood urea nitrogen days 3 – 4

Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Heimler [29], 2010 7.2 3.4 8 3.2 1.2 9 17.6 4.00 (1.52, 6.48)
Te Braake [31], 2005 9.4 3.5 66 6 3.3 69 82.4 3.40 (2.25, 4.55)

Total (95% CI) 74 78 100.0 3.51 (2.46, 4.55)

Heterogeneity: τ2 = 0.00, χ2 = 0.18, df = 1 (p = 0.67), I2 = 0%
Test for overall effect: z = 6.59 (p < 0.00001) –20 –10 0 10 20
Favors experimental Favors control

d Blood urea nitrogen days 6 – 7

Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Blanco [35], 2012 12.5 4.3 30 6.1 3.2 31 48.7 6.40 (4.49, 8.31)
Te Braake [31], 2005 8.4 3.8 66 6.7 3.1 69 51.3 1.70 (0.53, 2.87)

Total (95% CI) 96 100 100.0 3.99 (–0.62, 8.59)

Heterogeneity: τ2 = 10.39, χ2 = 16.93, df = 1 (p < 0.0001), I2 = 94%
–20 –10 0 10 20
Test for overall effect: z = 1.70 (p = 0.09)
Favors experimental Favors control

Fig. 3. a–e Meta-analysis of the effects of early supplementation (≤24 h) of amino acids compared to late supplementation (24 h) of
amino acids (random effects). IV, inverse variance; M-H, Mantel-Haenszel.
(Figure continued on next page.)
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 e Mean nitrogen balance in the first 7 days
Study or subgroup Experimental Control Weight Mean difference Mean difference
% IV, random, 95% CI IV, random, 95% CI
mean SD total mean SD total

Heimler [29], 2010 217 109 8 –132 51 9 24.2 349.00 (266.45, 431.55)
Ibrahim [12], 2004 385 20 15 –203 21 14 25.3 588.00 (573.05, 602.95)
Saini [30], 1989 120 6 11 –133 23 10 25.3 253.00 (238.31, 267.69)
Te Braake [31], 2005 150 50 66 –98 50 69 25.3 248.00 (231.13, 264.87)

Total (95% CI) 100 102 100.0 359.62 (166.59, 552.66)

Heterogeneity: τ2 = 38,324.25, χ2 = 1,261.81, df = 3 (p < 0.0001), I2 = 100%
Test for overall effect: z = 3.65 (p = 0.0003) –1,000 –500 0 500 1,000
Favors experimental Favors control
3

logical outcome at 2 years of age was studied by Blanco et
al. [33–35], Burattini et al. [24], and Bellagamba et al. [23].
No significant differences were found between groups.
Postnatal steroid use was reported by 5 studies. Indi-
vidual studies did not find a significant difference be-
tween groups. Meta-analysis supported this finding (p =
0.39, I2 = 45%; Fig. 2n).
Data on metabolic acidosis was reported by Bellagam-
ba et al. [23]. Metabolic acidosis was defined as a standard
base excess <7.5 mmol/L in 2 consecutive gas analyses.
Bicarbonate treatment was started when these conditions
were met. Bicarbonate treatment during parenteral nutri-
tion was required in more infants in the intervention
group than in the control group (73 vs. 63; p = 0.038).
Data on hypoglycemia, hypoalbuminemia, hypocalce-
mia, hypophosphatemia, or ammonia levels were report-
ed by only 1 study or none.
Sensitivity Analysis
After removing the study with a Jadad score <3 [26],
serum BUN levels on days 6–7 were no longer signifi-
cantly different between groups (p = 0.10). All other re-
sults did not change.
Early Introduction versus Late Introduction of
Parenteral Amino Acids
Characteristics of the included studies assessing the ef-
fect of early (<24 h after birth) versus late (>24 h) introduc-
tion of parenteral amino acids are shown in Table 1. In 2
studies, the included infants had a BW of <1,500 g, in 1
study the infants had a BW of <1,000 g, and in the remain-
ing studies inclusion was based on GA rather than BW.
Amino-acid supplementation was initiated on the
first DOL in all intervention groups and in the control
groups on DOL 2–4. Ibrahim et al. [12], Heimler et al.
[29], and Wilson et al. [32] described unblinded RCT. Te
Braake et al. [31] used a single-blinded approach, while
the approach of Blanco et al. [33–35] was double blinded
but the blinding method was not mentioned. In Saini et
al. [30], blinding was not mentioned. Four of the 6 stud-
ies were randomized in an appropriate way according to
the Jadad criteria [22]. Te Braake et al. [31] did not de-
scribe their randomization method, and Saini et al. [30]
randomized sequentially and, therefore, inappropriately
(Table 2).
Baseline characteristics and outcome measures of the
studies are shown in Table 4. Creatinine, postnatal steroid
use, hypoglycemia, and metabolic acidosis are not includ-
ed in this table because the included studies did not report
data on these outcomes. Overall, 405 infants were includ-
ed in the studies: 203 in the intervention groups and 202
in the control groups.
Outcome Measures
Primary Outcome Measures
In 3 of 6 studies (Blanco et al. [33–35], Ibrahim et al.
[12], and Saini et al. [30]), mean weight gain was given as
outcome measure for growth. No statistical difference be-
tween groups was found by the individual studies. Due to
a lack of consistency in the published data, no meta-anal-
ysis could be performed. Additional nonpublished data
on growth became available for 1 study [33–35], but this
was insufficient for further statistical analysis.
HC was measured by Heimler et al. [29] and Saini et
al. [30]; neither found a significant difference between
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 Table 3. Baseline characteristics and outcome measures of patients in studies comparing low- versus high-dose administration of amino acids

200
First author In- Male, GA1, BW1, Weight gain1, HC1, LLL1, Linear Time to Death, Respiratory NEC, Sepsis, IVH, Hyper- Post- BUN,
fants, n (%) weeks g g/kg/day mm/ mm/ growth1, regain BW1, n support1, n n n glycemia, natal mmol/L
n day day mm/day days total days n steroids,
n

Intervention

Morgan [25] 74 44 (59) 26.8 (1.3) 909 (154) 1,269 (222)12 – – – – 89 – 3 26 5 – – –

Tan [27] 68 39 (57) 26 (1.5) 911 (224) ns ns 0.285 – 10.3 (6.1) 1514 10.0 (25.0)4 6 – 6 33 9 –

Scattolin [26] 60 – 27.8 (2.0) 945 (194) 13.3 (7.4)2 – – – 14.8 (5.8) 08 – 4 9 0 – 1 12.4 (4.8)6
18.8 (6.8)3

Burattini [24] 56 33 (59) 28.7 (2.0) 974 (182) 16.6 (2.4) – – – 11.2 (4.5) 48 40.55 2 9 1 6 4 –

Clark [16] 64 38 (59) 27 (26 – 28) 961 12.42 (4.9)15 0.5 (0.3 – – – 29 – 7 15 8 – 12 –
(780 – 1,187) – 0.8)13

Vlaardinger-broek 47 21 (45) 27.2 (2.1) 867 (223) 12.3 (5.8) 8.4 (1.3) 0.26 (0.16) 0.27 (0.13) – 78 10.4 (11.4) 1 16 2 10 – 11.7 (3.2);

DOI: 10.1159/000481192
[13] 7.7 (2.7)7

Blanco [33 – 35] 30 20 (67) 25.7 (2.0) 768 (124) 11.76 (4.2)15 – – – – 69 22.0 (19.0) 3 4 3 – – 7.1 (3.2);

Neonatology 2018;113:187–205
12.5 (4.3)7

Bellagamba [23] 82 44 (54) 27 (26 – 28) 885 (150) 12.6 (1.7) – – – 12.7 (5) 1314 – 4 23 8 – 3 64 (35)10
8
Uthaya [28] 84 43 (51) 28.1 (2.1)/ 1,060 (290) ns – – – – 2 – 5 2 – 3 – –
27.8 (2.1)11 1,040 (280)

Control

Morgan [25] 76 39 (51) 26.6 (1.4) 898 (172) 1,212 (242)9 – – – – 79 – 2 28 2 – – –

Tan [27] 74 40 (54) 26.2 (1.5) 914 (219) ns ns 0.205 – 13.9 (6.3) 1614 4.0 (5.0)4 6 – 8 21 6 –

Scattolin [26] 55 – 27.6 (2.0) 926 (216) 12.3 (7.8)2 – – – 16.2 (7.3) 18 – 3 9 1 – 4 10.5 (5.2)6
14.7 (9.0)3

Burattini [24] 58 32 (55) 28.7 (2.14) 994 (194) 16.0 (2.7) – – – 11.7 (4.1) 58 40.05 2 9 2 20 2 –

Clark [16] 58 32 (55) 27 (25 – 28) 918 11.83 (5.94)15 0.5 (0.3 – – – 19 – 3 11 7 – 18 –
(788 – 1,231) – 0.7)13

Vlaardinger-broek 49 19 (39) 27.2 (2.2) 876 (209) 13.4 (4.7) 8.1 (1.5) 0.27 (0.13) 0.26 (0.16) – 108 10.4 (12.3) 4 17 3 12 – 8.3 (2.5); 6.1
[13] (1.9)7

Blanco [33 – 35] 31 17 (55) 26.3 (2.0) 783 (140) 11.66 (5.65)15 – – – – 49 15.0 (15.0) 2 4 2 – – 5.7 (2.1); 6.1
(3.2)7

Bellagamba [23] 82 49 (60) 27 (26 – 29) 906 (157) 12.3 (1.6) – – – 12.2 (5.1) 1214 – 4 20 3 – 9 56 (33)10

Uthaya [28] 84 54 (64) 27.5 (2.4) 1,030 (290) ns – – – – 48 – 7 8 – 8 – –

27.8 (1.9)11 1,050 (340)

GA, gestational age; BW, birth weight; HC, head circumference; LLL, lower-limb length; NEC, necrotizing enterocolitis; IVH, intraventricular hemorrhage; BUN, blood urea nitrogen; DOL, day of life; –, no data; ns, no data,
only described as “not significant.”
1 Mean (SD or range). 2 Data for DOL 1 – 7. 3 Data for DOL 8 – 14. 4 Median (IQR). 5 SD, range or IQR was not presented and could not be calculated from available data. 6 Data for DOL 6 – 7. 7 Data for DOL 1 – 2 and DOL

6 – 7. 8 Death before discharge. 9 Death <28 days. 10 mg/dL. 11 Study consists of 2 high- and 2 low-dose amino-acid groups. 12 Mean weight at DOL 28 (no data on weight gain). 13 cm/week. 14 Death before 36 weeks postmenstrual
age. 15 Additional nonpublished data received from author.

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 Table 4. Baseline characteristics and outcome measures of patients in studies comparing early with late introduction of amino acids
First In- Male, GA1, BW1, Mean weight HC1, LLL, Linear Days to Death, Respir. Oxygen NEC, Sepsis, IVH, Hyper- Mean Nitrogen Ammonia1,
author fants, n (%) weeks g gain, cm/day cm/day growth, regain birth n support, support, n n n glyce- BUN, balance1, µmol/L
n g/kg/day cm/ weight1 days days mia, mg/dL g/kg/day
day n

VLBW Infants
Intervention

Wilson [32] 64 34 (53) 27 (2.4) 925 (221) 2,111 (904)8 – – – 9 (6 – 11)5 156 7 (1 – 18) 26 (3 – 48)⁵ 4 – – 18 – – –

Blanco 30 20 (66) 25.7 (2.0) 768 (124) 11.76 (4.2)10 – – – – 66 22 – 3 4 3 – 7.14 (3.2); – –
[33 – 35] 12.5 (4.28)3

Ibrahim [12] 16 10 (63) 27 (1.6) 846 (261) ns – – – – 16 – – – 7 5 – – 385 (20) –

Te Braake 66 34 (52) 28.4 (2.0) 1,039 (235) – – – – 8 (2 – 25)⁵ – – – – – – – 9.6 (2.8); 150 (50) –
[31] 9.4 (3.5);
8.4 (3.8)2

Heimler [29] 8 – 29.6 (2.3) 1,258 (339) – 0.5 (0.5)9 – – 12 (3.2) 156 – – – – – – 7.2 (3.4) 217 (109) 30 (9.1)

Parenteral Amino-Acid Administration to

Saini [30] 11 9 (82) 28 (1) 1,087 (95) – 0.4 (0.1) – 0.7 (0.1) – 16, 7 – – – – – – – 120 (6) –

Control

Wilson [32] 61 32 (52) 27.4 (2.3) 933 (242) 2,080 (809)8 – – – 12 (9 – 17) 156 7 (1 – 18) 19 (3 – 51) 4 – – 24 – – –

Blanco 31 17 (55) 26.3 (2.0) 783 (140) 11.66 (5.65)10 – – – – 47 15 – 2 4 2 – 5.7 (2.1); – –
[33 – 35] 6.1 (3.2)3

Ibrahim [12] 16 9 (56) 26.8 (1.5) 968 (244) ns – – – – 26 – – – 6 4 – – –203 (21) –

Te Braake 69 31 (45) 28.4 (1.9) 989 (252) – – – – 10 (2 – 26)⁵ – – – – – – – 6.0 (1.8); –98 (50) –
[31] 6.0 (3.3);
6.7 (3.1)2

Heimler [29] 9 – 30.2 (1.9) 1,182 (214) – 0.25 (0.27)9 – – 13.7 (2.7) 156 – – – – – – 3.2 (1.2) –132 (51) 32.2 (10.7)

Saini [30] 10 6 (60) 28 (1) 990 (78) – 0.4 (0.1) – 0.7 (0.2) – 26,7 – – – – – – – –133 (23) –

GA, gestational age; BW, birth weight; HC, head circumference; LLL, lower-limb length; NEC, necrotizing enterocolitis; IVH, intraventricular hemorrhage; BUN, blood urea nitrogen; –, no data; ns, no data, only described
as “not significant.”
1 Mean (SD or range). 2 Data for day of life (DOL) 1 – 2, 3 – 4, and 6 – 7. 3 Data for DOL 1 – 2 and 6 – 7. 4 SD, range, or IQR was not presented and could not be calculated from available data. 5 Median; (IQR or range). 6 Death

before discharge. 7 Death <28 days. 8 Mean weight at DOL 28 (no data on weight gain). 9 HC gain in 2 weeks time. 10 Additional nonpublished data received from author.

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groups. Heimler et al. [29] described a mean HC incre-
ment in the first 2 weeks of 0.50 cm (0.50 SD) in the in-
tervention group vs. 0.25 cm (0.27 SD) in the control
group (p = 0.22). Saini et al. [30] found a mean HC incre-
ment of 0.40 cm (0.1 SD) in the first 10 days in both
groups.
Number of days to regain BW was assessed by Te
Braake et al. [31], Heimler et al. [29], and Wilson et al.
[32]. The latter reported, as a single study, a significant
difference between groups in favor of the intervention
group (median 9 vs. 12 days, p < 0.001). Because of a lack
of consistency in these measurements, meta-analysis was
not possible.
Secondary Outcome Measures
Death during hospital stay was reported in 4 studies.
Meta-analysis did not show a significant effect of early
introduction of amino-acid supplementation on overall
mortality (p = 0.63; Fig. 3a). Individual studies either did
not find significant differences in overall mortality or did
not report a p value.
Serum BUN levels were reported by Blanco et al. [33–
35], Te Braake et al. [31], and Heimler et al. [29]. All
found significantly elevated serum BUN levels in the in-
tervention groups in comparison to the control groups.
First, Te Braake et al. [31] measured serum BUN on DOL
2, 4, and 6 and found differences (p < 0.05) on all days.
Second, Heimler et al. [29] collected blood samples at a
mean age of 78 h and found a mean difference of 4.0
mmol/L in favor of the intervention group (p < 0.001).
Third, Blanco et al. [33–35], who measured levels on DOL
1 and 7, found differences on both days (mean: 1.43 and
6.4 mmol/L, respectively; p < 0.001). Meta-analyses per-
formed for days 1–2, 3–4, and 6–7 showed a significant
effect on days 1–2 (p = 0.02) and 3–4 (p < 0.00001), but
not on days 6–7 (p = 0.09; Fig. 3b–d).
Nitrogen balance was described by Heimler et al. [29],
Ibrahim et al. [12], Saini et al. [30], and Te Braake et al.
[31]. Premature infants who received early amino-acid
supplementation had a positive nitrogen balance, where-
as a negative nitrogen balance was recorded in infants
who received supplementation late (>24 h). In the study
by Heimler et al. [29], urine was collected on DOL 3, be-
fore the administration of amino acids to the control
group. The study showed a positive mean nitrogen bal-
ance in the intervention group and a negative mean bal-
ance in the control group. In addition, Ibrahim et al. [12]
reported a significant difference between groups on days
1, 3, and 5 (urine was collected in 29 infants [90.6%]) but
not on day 7. The intervention group started with a mean
202 Neonatology 2018;113:187–205
DOI: 10.1159/000481192
nitrogen balance of 384.5 mg/kg/day on DOL 1, which
remained stable until day 7. The control group started
with a negative nitrogen balance (mean –203.4 mg/kg/
day), but the level increased until day 7. On day 7 a 4th
measurement showed no statistically significant differ-
ence (no p value available). Te Braake et al. [31] reported
a significant difference in favor of the intervention group
on day 2. Contrarily, on day 4, a significant difference was
found in favor of the control group. Saini et al. [30] de-
scribed a significant effect in favor of the intervention
group on days 1–3 but not thereafter. Meta-analysis
showed a significant difference between groups: p =
0.0003 (Fig. 3e). χ2 statistics revealed substantial hetero-
geneity (I2 = 100%).
Serum glucose levels were reported by all studies. Wil-
son et al. [32] described hyperglycemia as blood glucose
levels >11 mmol/L with glycosuria >+++ or 55 mmol/L.
The number of infants requiring insulin treatment was
reported separately. Only the latter was significantly dif-
ferent between both groups in favor of the control group,
of which 2% were treated with insulin in comparison to
33% of infants in the intervention group. Blanco et al.
[33–35] defined hyperglycemia as blood glucose levels
>150 mg/dL and found no difference between groups
(p = 0.51). Ibrahim et al. [12], Te Braake et al. [31], and
Heimler et al. [29] did not report the incidence of hyper-
glycemia but rather the mean serum glucose values of
both groups. Te Braake et al. [31] found a significantly
lower value in the intervention group on DOL 2. Meta-
analysis was not possible due to inconsistency in defini-
tions.
Neurodevelopmental outcome after 2 years was re-
ported by Blanco et al. [35]. No significant difference was
found between groups. The same holds for the duration
of mechanical ventilation, duration of supplemental oxy-
gen, incidence of NEC, sepsis, IVH, and levels of ammo-
nia. Te Braake et al. stated that there was no difference in
metabolic acidosis between groups without presenting
actual data. None of the included studies reported on hy-
poglycemia, postnatal steroid use, creatinine levels, hypo-
albuminemia, hypophosphatemia, or hypocalcemia.
Sensitivity Analysis
When removing the studies with a Jadad score <3 [12,
29, 30], only 1 meta-analysis (on overall mortality) could
be performed. The outcome of this analysis did not
change.
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 Discussion
In this systematic review, the efficacy and safety of
both high- versus low-dose and early versus late paren-
teral amino-acid supplementation in VLBW infants was
studied. The benefits of these interventions, as well as the
number and diversity of adverse events, were reported.
The results suggested that administering a high dose
(>3.0 g/kg/day) or an early dose (≤24 h) of parenteral
amino acids is well tolerated, but does not offer signifi-
cant benefits on growth when compared to a lower dose
or later initiation. On the other hand, it does not cause a
higher incidence of adverse events either.
Unfortunately, for several outcome measures, a (mean-
ingful) meta-analysis could not be performed due to con-
siderable heterogeneity in reported outcome measures in
the included RCT despite our attempt to include addi-
tional, nonpublished data by contacting the authors. Fur-
thermore, Jadad quality ratings of the included studies
varied, and removal of low-quality studies for sensitivity
analysis resulted in even less obtainable meta-analyses.
One possible reason why higher doses of amino acids
did not translate into improved growth outcomes might
be that the solutions used were of suboptimal composi-
tion for the needs of preterm infants. Limited data were
available on the parenteral amino-acid compositions
used by the different RCT. In 2 studies [13, 31], the source
was Primene 10% (Baxter Healthcare Corporation), in 1
study Aminosyn PF (Abbott Laboratories, Chicago, IL,
USA) [33–35], in 2 studies TrophAmine (Baxter Health-
care Corporation or McGaw Inc.) [24, 29], and in anoth-
er study both Aminosyn and TrophAmine [16]. Due to
this variability, comparing the results across studies is dif-
ficult. Aminosyn, for example, only contains essential
amino acids, while Primene and TrophAmine both con-
tain essential and nonessential amino acids. None of the
studies compared different compositions for superiority;
therefore, no definitive statement could be made on
which amino-acid composition should be used. All stud-
ies compared plasma concentrations of amino acids to
previously reported levels in healthy term breastfed in-
fants. For example, in Clark et al. [16], supplementation
of amino acids resulted in excess levels of arginine, leu-
cine/isoleucine, methionine, and ornithine but inade-
quate concentrations of tyrosine. Remarkably, the com-
bined plasma concentrations of leucine and isoleucine
were less than normal human fetal plasma concentrations
[37]. This was documented for more (non)essential ami-
no acids in other studies [10, 38] and might suggest that
the current solutions are not optimal for VLBW infants,
Parenteral Amino-Acid Administration to
VLBW Infants
since even high doses do not result in plasma amino-acid
concentrations that are sufficient to promote growth.
Nevertheless, Poindexter et al. [11] did document on sig-
nificantly better growth outcomes at 36 weeks of PMA in
VLBW infants who received greater early amino-acid
supplementation (≥3 g/kg/day at ≤5 days of age) com-
pared to lower late supply. These results were based on a
secondary analysis (including 1,018 infants) of data from
a RCT with a study design similar to that of Clark et al.
[16]. Since the study maintained a higher total protein
and amino-acid supply for a longer period, it has been
proposed that both early and sustained higher rates of
amino acids are necessary to achieve growth in VLBW
infants [39].
On the other hand, the lack of growth improvement
after higher-dose amino-acid supplementation has also
been suggested to be due to the existence of a threshold
above which the delivery of protein results in amino-acid
oxidation without improvement in nitrogen retention or
growth [13, 28]. Based on fetal human data, this threshold
is suggested to be ∼4 g/kg/day of protein [40]. Indeed, 7
studies on low- versus high-dose amino-acid supplemen-
tation found statistically significantly elevated serum
BUN levels in the intervention groups in comparison to
the control groups. In 1 study, the amino-acid intake of
23 infants (28%) was reduced due to elevated BUN levels
in comparison to 14 infants (17%) in the control group
(p = 0.093) [23]. In the study by Blanco et al. [33] in ex-
tremely low-birth-weight infants, 6 infants in the early
and high-dose group (4 g/kg/day on day 3) were with-
drawn from the study protocol due to elevated serum
BUN levels and hyperammonemia. However, in this
study, plasma amino acid concentrations were remark-
ably higher than those reported by others at the same dose
of amino acids [10, 16, 34], which may reflect an ineffi-
cient metabolism in these infants. Also, the study by Blan-
co et al. [33] was the only one in which Aminosyn was
used as the source of amino acids, which might provide
different BUN and ammonia values from those using
TrophAmine or Primene. Furthermore, the study has
been criticized on its design [41], and the results have not
been replicated by others. Higher BUN concentrations
are to be expected when a higher dose of amino acids is
administered immediately after birth, reflecting amino-
acid oxidation and protein turnover and not toxicity. It
remains debatable as to what constitutes a clinically sig-
nificant state of uremia [42]. Very recently, van den Ak-
ker et al. [43] reported no association between early-life
urea levels and neurocognitive outcome at 2 years of cor-
rected age.
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 As a consequence of amino-acid oxidation after ad-
ministration of high doses of amino acids, metabolic aci-
dosis may occur [14, 15]. Indeed, infants in the supple-
mented groups of 2 included RCT needed more sodium
acetate or bicarbonate than infants in the control groups,
suggesting lower pH values in the supplemented groups
[23, 33–35]. However, no adverse effects were described.
Meta-analysis of the data on early versus late amino-
acid initiation also showed a significantly higher nitrogen
balance in the early supplementation group (p = 0.003).
Premature infants who received amino-acid supplemen-
tation early had positive nitrogen balances, and negative
balances were recorded in infants who received supple-
mentation later. The potential clinical significance of an
early positive nitrogen balance is not known. Saini et al.
[30] stated that it may be reasonable to assume an advan-
tage, particularly during the critical transitional phase of
extrauterine adaptation.
We found no significant differences in overall mortal-
ity, duration of mechanical ventilation or oxygen supple-
mentation, incidence of NEC, sepsis, IVH, hyperglyce-
mia, postnatal steroid use, or neurodevelopmental out-
come for both research questions.
In 2013, a Cochrane review was published on early
(<24 h after birth) versus late (>24 h) parenteral amino-
acid supplementation in premature infants [20]. In addi-
tion, in a subgroup analysis, the authors compared high-
(≥2.0 g/kg/day) versus low-dose (<2.0 g/kg/day) amino-
acid supplementation. Consistent with our results, the
authors found no short-term difference in length and HC
between groups for both research questions. No state-
ments were made regarding our other primary outcomes.
In our review, 10 additional studies were included, of
which 6 were recent studies. Therefore, we were able to
make additional and more extensive statements on the
outcome measures. Nevertheless, due to the great vari-
ability in study designs and outcome measurements as
well as the general low quality of the available studies, it
remained difficult to perform true meta-analyses. Fur-
thermore, although differences in enteral diet, lipid in-
take, and energy dosing between studies may be con-
founding factors, no subgroup analyses could be done
due to insufficient data or heterogeneity.
In summary, from available data, it can be concluded
that a combination of early and high-dose supplementa-
tion of amino acids in VLBW infants is safe and well tol-
erated but yields no benefits on anthropometric out-
comes when compared to later or lower-dose supplemen-
tation. It leads more rapidly to a higher nitrogen balance
and results in higher serum BUN levels without detri-
204 Neonatology 2018;113:187–205
DOI: 10.1159/000481192
mental effects. Although exact beneficial effects of these
findings are not known, it is suggested that a more posi-
tive nitrogen balance has advantages in the transitional
phase of extrauterine adaptation [30]. High serum BUN
levels are presumably a reflection of a higher amino-acid
oxidation rate and not a sign of intolerance [31].
This systematic review highlights the considerable
variability in study design, interventions, and outcomes
of available RCT, whereby meta-analysis of the results
was often not possible, and thus no firm recommenda-
tions could be made.
Further research including larger numbers of infants
is needed to study the effects of early and high-dose ami-
no acids on both short-term and long-term anthropo-
metric outcomes more consistently and extensively.
These studies should also take the influence of parenteral
lipids and dextrose, as well as enteral nutrition, into ac-
count. Finally, future research is warranted to determine
the optimal amino-acid composition of parenteral nutri-
tion.
Disclosure Statement
The authors declare no conflicts of interest.
Funding Sources
No funding was received for this project.
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