ARTICLE
`
`Effects of Two Different Doses of Amino Acid
`Supplementation on Growth and Blood Amino Acid
`Levels in Premature Neonates Admitted to the
`Neonatal Intensive Care Unit: A Randomized,
`Controlled Trial
`
`Reese H. Clark, MDa, Donald H. Chace, PhD, MSFSb, Alan R. Spitzer, MDa, for the Pediatrix Amino Acid Study Group
`
`aPediatrix Medical Group, Sunrise, Florida; bPediatrix Analytical, Bridgeville, Pennsylvania
`
`Financial Disclosure: Drs Clark, Chace, and Spitzer are employees of Pediatrix Medical Group, which owns Pediatrix Screening, a company that offers newborn screening for inborn errors of metabolism and
`hearing loss.
`
`ABSTRACT
`
`OBJECTIVES. The goal was to measure the effects of 2 distinct strategies for parenteral
`nutrition on neonatal growth and blood amino acid profiles.
`METHODS. In a multicenter trial (n ⫽ 11 sites), we randomly allocated premature
`(23–29 weeks and 6 days of gestation) neonates to 1 of 2 approaches to intrave-
`nous amino acid administration. In one group, amino acid supplementation was
`started at 1.0 g/kg per day and advanced by 0.5 g/kg per day to a maximum of 2.5
`g/kg per day (2.5 g/kg per day group). The other group received amino acids
`starting at 1.5 g/kg per day and advancing by 1.0 g/kg per day to a maximum of
`3.5 g/kg per day (3.5 g/kg per day group). Filter paper blood spots were obtained
`from each infant on the day of random assignment and on days 7 and 28 of age,
`to monitor blood amino acid levels.
`
`RESULTS. We enrolled 122 neonates (64 in the 3.5 g/kg per day group and 58 in the
`2.5 g/kg per day group). There were no differences in demographic or baseline
`characteristics between the 2 treatment groups. There was no significant difference
`in growth by day 28 after birth (median weight gain: 12.9 and 11.4 g/kg per day
`for the 3.5 and 2.5 g/kg per day groups, respectively), and the incidences of
`secondary morbidities were similar in the 2 groups. On day 7, blood levels of
`several amino acids and the serum urea nitrogen level were higher in the 3.5 g/kg
`per day group, compared with the 2.5 g/kg per day group; none of the amino acid
`levels were lower.
`
`CONCLUSIONS. Higher doses of amino acid supplementation did not improve neonatal
`growth and were associated with increased blood amino acid and urea nitrogen
`levels.
`
`www.pediatrics.org/cgi/doi/10.1542/
`peds.2007-0545
`doi:10.1542/peds.2007-0545
`
`This trial has been registered at www.
`clinicaltrials.gov (identifier NCT00120926).
`
`Key Words
`neonates, parenteral nutrition, amino
`acids, acylcarnitines, nutrition
`
`Accepted for publication Jun 12, 2007
`
`Address correspondence to Reese H. Clark,
`MD, Pediatrix Medical Group, 1301 Concord
`Terrace, Sunrise, FL 33323-2825. E-mail:
`reese㛭clark@pediatrix.com
`PEDIATRICS (ISSN Numbers: Print, 0031-4005;
`Online, 1098-4275). Copyright © 2007 by the
`American Academy of Pediatrics
`
`1286
`
`CLARK et al
`
`Downloaded from
`
`
`
` at Reprints Desk on May 8, 2020www.aappublications.org/news
`
`Eton Ex. 1046
`1 of 13
`
`

`

`STUDIES HAVE INDICATED that premature neonates of-
`
`ten develop severe nutritional deficits during the
`first weeks after birth,1–5 and inhibited growth during the
`early postnatal period has been associated with poor
`long-term outcomes.2,5,6 Variations in dietary intake may
`account for 45% of the variations in growth.7,8 As a
`result, efforts have focused on determining whether nu-
`tritional deficiency and growth restriction in premature
`infants can be prevented through better nutritional in-
`take.6,9,10
`On the basis of animal studies that showed high levels of
`in utero amino acid flux during the later phases of gesta-
`tion, several authors have suggested that higher doses of
`amino acid supplementation may minimize extrauterine
`growth restriction and enhance the outcomes of very low
`birth weight infants.9–11 Although standard doses of amino
`acids may be inadequate to promote normal growth,
`higher doses may lead to elevated blood amino acid levels
`and increased toxicity. Given the limited metabolic capac-
`ity of very low birth weight infants, excess administration
`of amino acids (protein) may result in saturation of both
`catabolic and anabolic pathways of protein metabolism,
`leading to larger amino acid pools for longer periods of
`time. Through the implementation of a multicenter ran-
`domized trial and tandem mass spectrometric analysis of
`key amino acids from dried filter paper blood spots, the
`effects of 2 distinct strategies for amino acid supplementa-
`tion on amino acid profiles and growth in premature in-
`fants were evaluated.
`
`METHODS
`
`Study Subjects
`Eligible infants had estimated gestational ages between
`23 weeks 0 days and 29 weeks and 6 days, were inborn,
`and had parental consent for participation in the study.
`Patients were approached for random assignment during
`the first 48 hours after birth and were excluded if they
`were ⬎48 hours of age or had a major congenital anom-
`aly. Investigational review boards of each hospital (n ⫽
`11) approved the protocol.
`
`Random Assignment and Blinding
`We used an electronic system to assign a randomized code,
`which was delivered to the health care provider responsi-
`ble for preparing parenteral nutrition. That individual used
`the random assignment code to determine the treatment
`assignment and mixed the parenteral solution according to
`study and pharmacy protocols. The parenteral nutrition
`solution was labeled “study AA,” and the concentrations of
`amino acids were not indicated on the bag. A pharmacy log
`tracked the amounts of amino acids in the solution for each
`patient. This approach blinded the clinical care staff mem-
`bers to the amount of amino acid supplementation that
`each neonate received. Random assignment was stratified
`according to site.
`
`Treatment
`For the group with a maximal dose of 2.5 g/kg per day
`(2.5 g/kg per day group), amino acid supplementation
`was started at 1.0 g/kg per day and advanced 0.5 g/kg
`per day to a maximum of 2.5 g/kg per day on day 4 of
`treatment. For the group with a maximal dose of 3.5
`g/kg per day (3.5 g/kg per day group), amino acid sup-
`plementation started at 1.5 g/kg per day of amino acids
`and advanced 1 g/kg per day to a maximum of 3.5 g/kg
`per day on day 3 of treatment.
`When enteral feedings were started, parenteral sup-
`plementation of amino acids continued, with the goal of
`delivering the maximal dose of amino acids allowed by
`the protocol. As feedings were advanced, intravenous
`fluids were decreased accordingly, to keep total fluids at
`ⱕ150 mL/kg per day. Parenteral nutrition was mixed to
`provide the maximal amount of protein allowed by the
`protocol. As feedings were advanced, the total amount
`of protein per kilogram per day increased as a result of
`the protein in the enteral feedings. When the parenteral
`nutrition was being administered at a rate of ⬍70 mL/kg
`per day, the amount of amino acids that could be added
`to the parenteral nutrition decreased (limited by the
`solubility of the elements in the parenteral nutrition).
`Therefore, when feedings reached 80 to 100 mL/kg per
`day, we decreased amino acid supplementation to 1 g/kg
`per day for the 2.5 g/kg per day group and to 2.0 g/kg per
`day for the 3.5 g/kg per day group. For both groups,
`amino acid supplementation was stopped when feedings
`reached 100 to 130 mL/kg per day. When enteral nutri-
`tion was at the level of 100 to 130 mL/kg per day, the
`patient was considered to have completed treatment.
`Subsequent parenteral nutrition was administered at the
`discretion of the health care team.
`
`Nutritional Support Guidelines
`We recommended starting parenteral lipid administra-
`tion at the same time that the study amino acid supple-
`mentation was started, beginning at a rate of 0.5 g/kg per
`day and advancing 0.5 g/kg per day to a maximum of 3.5
`g/kg per day.12–14 We discouraged the use of insulin and
`recommended limiting the glucose infusion rate to 8 to
`12 mg/kg per minute as tolerated. Although the quantity
`and quality of enteral nutritional support were not struc-
`tured in this trial, we offered the following feeding
`guidelines, in an attempt to maintain consistency: small-
`volume (⬍10 mL/kg per day) feedings should begin
`within the first week of life; low-dose dopamine treat-
`ment (defined as ⬍5 ␮g/kg per minute) was not consid-
`ered a contraindication for trophic feedings; feedings
`could be initiated even if umbilical catheters were in
`place; feedings could be advanced at a rate of as much as
`30 mL/kg per day and were not to be advanced at a rate
`of ⬍10 mL/kg per day; and 150 mL/kg per day, with
`consistent weight gain of 20 to 30 g/kg per day, was
`considered adequate nutrition.
`
`Downloaded from
`
`PEDIATRICS Volume 120, Number 6, December 2007
`
`
` at Reprints Desk on May 8, 2020www.aappublications.org/news
`
`1287
`
`Eton Ex. 1046
`2 of 13
`
`

`

`Study End Points
`Our primary outcome measure was growth, assessed as
`changes in weight, length, and head circumference over
`the first 28 days after birth. Weight gain was calculated as
`weight gain (in grams per kilograms per day) ⫽ (weight at
`28 days ⫺ birth weight) divided by birth weight divided by
`28 days. We also assessed length and head growth changes
`in centimeters per week, as (measurement at 28 days ⫺
`measurement at birth) divided by 4 weeks.
`Secondary outcome measures included blood amino
`acid profiles and incidence of major morbidities during
`the first 28 days after birth. Blood amino acid profiles
`were obtained on the day of random assignment (usu-
`ally day 1) and on day 7 (parenteral phase of nutrition)
`and day 28 (enteral phase of nutrition) of life.
`
`Data Collection and Monitoring
`All information was collected by using an Internet-based
`case report form that could not be finalized until all re-
`quired fields were completed. Site visits were conducted to
`monitor the accuracy of data collection and adherence to
`the International Conference on Harmonization of Techni-
`cal Requirements for Registration of Pharmaceuticals for
`Human Use good clinical practice guidelines.
`Study logs of amino acid contents in the parenteral
`nutrition were maintained for each subject for the first
`28 days of life, by pharmacy personnel. Amino acid
`contents were recorded each day that the subject re-
`ceived either study or nonstudy parenteral nutrition.
`Pharmacy logs and source documents were monitored to
`validate each of the growth measurements and the
`amount of amino acid supplementation that was re-
`corded on the case report form of each subject.
`
`Measurement of Amino Acid and Acylcarnitine Levels
`Amino acid and acylcarnitine profiles were analyzed
`from dried blood spots by using tandem mass spectrom-
`etry, as described by Chace and Kalas.15 All quantitative
`results were provided to study investigators and were
`reported as micromoles per liter.
`To provide “normal” reference values (reference values
`in Fig 1), we calculated the median and the 10th and 90th
`percentiles for each metabolite by using 1000 normal term
`newborn values (from samples obtained in the first 7 days
`after birth). These values were derived from samples sent
`to our laboratory for screening for inborn errors of metab-
`olism. The values represent our internal normal newborn
`control values (reference values). To try to identify the
`amino acid and acylcarnitine values that were commonly
`(⬎50% of the patients) abnormal, relative to our internal
`reference newborn levels, we calculated the proportion of
`patients with values greater than the 90th percentile (high
`levels) and the proportion of patients with values less than
`the 10th percentile (low values) for the overall study pop-
`ulation and each treatment group.
`
`Statistical Analyses and Sample Size Calculation
`Our hypothesis was that the higher-dose group would
`have a growth velocity (13.0 g/kg per day) 3 g/kg per
`day more than that of the lower-dose group (10 g/kg per
`day). On the basis of previous work with premature
`neonates,6 we calculated that a sample size of 108 pa-
`tients (54 patients in each group) would allow us to
`detect a difference of 3.0 g/kg per day in weight gain
`between the 2 treatment groups (␣ ⫽ .025, power ⫽
`80%, assuming SD ⫽ 5.0 g/kg per day). The ␣ level was
`set at .025 on the basis of a 2-sided, 2-sample t test. We
`anticipated a 10% dropout rate resulting from morbidity
`and death; therefore, we enrolled 122 subjects.
`We performed an intent-to-treat analysis. Categorical
`variables were evaluated by using a 2-tailed ␹2 test and
`Fisher’s exact test. Continuous variables were compared by
`using a 2-tailed t test for parametrically distributed data and
`Kruskal-Wallis analysis of variance for nonparametrically
`distributed data. Rank data were assessed by using a
`2-tailed, Mann-Whitney, nonparametric test. Analysis of
`variance for repeated measures was used to evaluate the
`changes in all laboratory values over time, from the day of
`random assignment to days 7 and 28 of age. Both time and
`treatment groups were evaluated. To evaluate the influ-
`ence of covariates (gestational age, birth weight, gender,
`and postnatal exposure to steroids) on our study results, we
`performed a linear regression analysis. Our goal was to
`determine the treatment-influenced weight gain when
`these covariates were included in the regression model.
`
`RESULTS
`
`Enrollment
`Between September 1, 2005, and June 1, 2006, site logs
`showed that 230 neonates were screened and 122 neo-
`nates (53%) were enrolled from 11 sites, 64 in the 3.5
`g/kg per day group and 58 in the 2.5 g/kg per day group.
`The 2 reasons for nonenrollment were parent refusal
`(n ⫽ 57) and missed opportunities for obtaining consent
`(n ⫽ 51). The median enrollment per site was 11 pa-
`tients (range: 3–16 patients).
`
`Report on Patients Who Did Not Complete the Study
`Of the 122 patients enrolled, 11 patients did not complete
`the study. Three patients died before 28 days. Five patients
`were withdrawn from the study (3 in the 3.5 g/kg per day
`group and 2 in the 2.5 g/kg per day group). One patient
`was considered by the attending neonatologist to be too
`sick to continue in the study, and 2 patients were trans-
`ferred out of the study facility before 28 days. Follow-up
`laboratory assessments were not available for those pa-
`tients. The primary reason for a patient being withdrawn
`from the study was a serum urea nitrogen level of ⬎50
`mg/dL (n ⫽ 4). Two of the 11 patients who did not com-
`plete the study did not receive study parenteral nutrition.
`Removal of the 11 patients who failed to complete the
`
`1288
`
`CLARK et al
`
`Downloaded from
`
`
`
` at Reprints Desk on May 8, 2020www.aappublications.org/news
`
`Eton Ex. 1046
`3 of 13
`
`

`

`75th
`Median
`25th
`
`2.5day28
`
`3.5day28
`
`2.5day7
`
`3.5day7
`
`2.5day1
`
`3.5day1
`
`Reference
`
`75th
`Median
`25th
`
`2.5day28
`
`3.5day28
`
`2.5day7
`
`3.5day7
`
`2.5day1
`
`3.5day1
`
`75th
`Median
`25th
`
`2.5day28
`
`3.5day28
`
`2.5day7
`
`3.5day7
`
`2.5day1
`
`3.5day1
`
`90th
`
`10th
`
`Reference
`
`90th
`
`10th
`
`Reference
`
`40
`35
`30
`25
`20
`15
`10
`
`05
`
`0.45
`0.40
`0.35
`0.30
`0.25
`0.20
`0.15
`0.10
`0.05
`0.00
`
`160
`140
`120
`100
`80
`60
`40
`20
`0
`
`B
`
`BloodUreaNitrogen(mg/dL)
`
`D
`
`Isovalerylcarnitine
`
`F
`
`Tyrosine
`
`3.5 g/kg group
`2.5 g/kg group
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10 12 14 16 18 20 22 24 26 28
`Days since birth
`
`75th
`Median
`25th
`
`2.5day28
`
`3.5day28
`
`2.5day7
`
`3.5day7
`
`2.5day1
`
`3.5day1
`
`75th
`Median
`25th
`
`2.5day28
`
`3.5day28
`
`2.5day7
`
`3.5day7
`
`2.5day1
`
`3.5day1
`
`90th
`
`10th
`
`Reference
`
`90th
`
`10th
`
`Reference
`
`4.0
`3.5
`3.0
`2.5
`2.0
`1.5
`1.0
`0.5
`0.0
`
`A
`
`Averageparenteralproteindose,
`
`g/kg per d
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`C
`
`Leucine-isoleucine
`
`350
`300
`250
`200
`150
`100
`50
`0
`
`E
`
`Alanine
`
`FIGURE 1
`A, Mean ⫾ SE of the dose of amino acids over time for neonates receiving a maximal dose of 3.5 g/kg per day and neonates receiving a maximal dose of 2.5 g/kg per day. B, Median and 25th and
`75th percentile values for changes in serum urea nitrogen levels over time. C–F, representative changes in levels of key amino acids and acylcarnitines over time (C, leucine/isoleucine; D, isovaleryl
`carnitine; E, alanine; F, tyrosine). All values are reported in micromoles per liter. Statistical details are presented in Table 3. Graphs represent median and 25th and 75th percentile values for
`measured values and median and 10th and 90th percentile values for reference values. 3.5 indicates 3.5 g/kg per day group; 2.5, 2.5 g/kg per day group.
`
`study protocol did not change our results, and we report
`only the intent-to-treat analysis.
`
`support or the total number of days of any parenteral
`nutrition (Table 1).
`
`Protocol Experience
`Fifty percent of the patients were enrolled during the
`first 24 hours after birth, and there was good separation
`of the 2 treatment groups regarding the amounts of
`amino acids delivered after random assignment (Fig 1A).
`By design, from day 1 to day 28, neonates in the 3.5 g/kg
`per day group received a larger dose of amino acids than
`did those in the 2.5 g/kg per day group (Fig 1). There was
`no difference in the number of days of study amino acid
`
`Demographic Data and Primary and Secondary Morbidity
`Outcomes
`We used an intent-to-treat approach, and all tables re-
`flect all enrolled infants, categorized according to treat-
`ment group. There were no significant differences in the
`demographic or baseline characteristics of the 2 treat-
`ment groups (Table 1). On day 7, there were no differ-
`ences in weight, length, head circumference, or degree
`of nutritional support (Table 1). Similarly, there was no
`
`Downloaded from
`
`PEDIATRICS Volume 120, Number 6, December 2007
`
`
` at Reprints Desk on May 8, 2020www.aappublications.org/news
`
`1289
`
`Eton Ex. 1046
`4 of 13
`
`

`

`TABLE 1 Demographic Characteristics
`
`N
`EGA, median (IQR), wk
`Birth weight, median (IQR), g
`Birth length, median (IQR), cm
`Birth head circumference, median (IQR), cm
`1-min Apgar score, median (IQR)
`5-min Apgar score, median (IQR)
`Prenatal steroid use reported, n (%)
`Cesarean section, n (%)
`Male gender, n (%)
`Product of multiple gestation, n (%)
`Race, n (%)
`Asian
`Black
`Hispanic
`Other
`White
`Surfactant use reported, n (%)
`Age at random assignment, median (IQR), d
`Type of amino acid, n (%)
`Aminosyn
`TrophAmine
`Carnitine added to parenteral nutrition, n (%)
`Received protein before study enrollment, n (%)
`Total parenteral amino acid dose during first 7 d, median
`(IQR), g/kg
`Total parenteral amino acid dose during first 28 d,
`median (IQR), g/kg
`Time on treatment protocol, median (IQR), d
`Total time on parenteral amino acid support, median
`(IQR), d
`Day 7 growth parameters
`Weight, median (IQR), g
`Length, median (IQR), cm
`Head circumference, median (IQR), cm
`Day 7 nutritional support
`Glucose infusion, median (IQR), g/kg per d
`Lipids, median (IQR), g/kg per d
`Receiving some feedings, n (%)
`Enteral feedings, median (IQR), mL/kg per d
`Intravenous fluids, median (IQR), mL/kg per d
`Total fluids, median (IQR), mL/kg per d
`Day 7 laboratory data
`Serum bicarbonate level, median (IQR), mEq/L
`Serum blood urea nitrogen level, median (IQR), mg/dL
`Serum creatinine level, median (IQR), mg/dL
`Serum total bilirubin level, median (IQR), mg/dL
`EGA indicates estimated gestational age; IQR, interquartile range.
`
`Maximal Dose of
`3.5 g/kg per d
`64
`27 (26–28)
`961 (780–1187)
`35.5 (33.5–37.5)
`25 (23.1–26.5)
`5 (4–7)
`8 (7–8)
`53 (82.8)
`39 (60.9)
`38 (59.4)
`17 (26.6)
`
`1 (1.6)
`20 (31.3)
`8 (12.5)
`4 (6.3)
`31 (48.4)
`50 (78.1)
`1 (0.5–1.4)
`
`5 (7.8)
`59 (92.2)
`24 (37.5)
`42 (65.6)
`21.5 (19.5–21.5)
`
`Maximal Dose of
`2.5 g/kg per d
`58
`27 (25–28)
`918 (788–1231)
`35 (32–38)
`24.5 (23–27)
`5 (3–7)
`7 (6–8)
`49 (84.5)
`39 (67.2)
`32 (55.2)
`17 (29.3)
`
`4 (6.9)
`17 (29.3)
`5 (8.6)
`4 (6.9)
`28 (48.3)
`51 (87.9)
`1 (0.5–1.4)
`
`3 (5.2)
`55 (94.8)
`26 (44.8)
`38 (65.5)
`14.5 (13.5–15.4)
`
`51 (28–74)
`
`34.5 (22.5–58)
`
`15 (10–22)
`18 (11.8–25)
`
`14.5 (10–25)
`17.5 (11.8–26)
`
`910 (760–1092)
`36 (33.5–37.8)
`24.5 (22.9–26)
`
`11.3 (8.6–13.7)
`2.3 (1.6–2.9)
`44 (68.8)
`12.8 (0–57)
`127 (98.6–153.6)
`151 (134–171)
`
`22 (19–24)
`27 (20–36)
`0.9 (0.7–1)
`5.6 (4.5–7.3)
`
`880 (768–1105)
`35 (32.1–37.7)
`24.5 (23.1–26.5)
`
`11.2 (8.5–13.1)
`2.4 (1.7–2.9)
`40 (69)
`12.9 (0–34)
`140 (123–160.3)
`158 (147–174)
`
`22 (20–25)
`21.5 (12.8–30)
`1 (0.73–1.18)
`5.5 (4.1–6.6)
`
`P
`
`.8
`.99
`.6
`.9
`.4
`.1
`.8
`.6
`.7
`.7
`.7
`
`.2
`.8
`
`.7
`.7
`.37
`.9
`.01
`
`.01
`
`.5
`.6
`
`.99
`.7
`.5
`
`.8
`.7
`.99
`.2
`.4
`.99
`
`.24
`.01
`.04
`.26
`
`significant difference in growth (or any growth param-
`eter) on day 28 (Table 2). The incidences of morbidities
`were similar for the 2 treatment groups (Table 2). Mul-
`tivariate regression analysis showed that gestational age,
`birth weight, and postnatal exposure to steroids each
`had an independent influence on weight gain. The ne-
`onates with the poorest weight gain were small neonates
`who were gestationally immature and who were ex-
`posed postnatally to dexamethasone. In multivariate
`analyses, treatment group did not influence growth or
`
`the occurrence of the patient’s weight being below the
`10th percentile for weight at 28 days of age.
`
`Changes in Amino Acid Levels
`
`Differences in Levels According to Treatment Group
`There were no differences in amino acid levels on the day
`of random assignment (Table 3). On day 7 (parenteral
`phase of nutrition), blood levels of several amino acids
`(alanine, arginine, glutamate, leucine/isoleucine, methio-
`
`1290
`
`CLARK et al
`
`Downloaded from
`
`www.aappublications.org/news
`
` at Reprints Desk on May 8, 2020
`
`Eton Ex. 1046
`5 of 13
`
`

`

`TABLE 2 Outcomes to 28 Days
`
`Maximal Dose of
`3.5 g/kg per d
`
`Maximal Dose of
`2.5 g/kg per d
`
`P
`
`.99
`.9
`.99
`.9
`.6
`.6
`.6
`.6
`.6
`
`.8
`.99
`
`.2
`.9
`.8
`
`.3
`
`.6
`
`.7
`.99
`.051
`.5
`
`.2
`
`.6
`
`.6
`
`1170 (973–1559)
`27 (24.6–29.4)
`37.2 (35–41)
`310 (162–448)
`2 (1.1–2.9)
`3 (1.5–4.2)
`11.4 (7.2–14.9)
`0.5 (0.3–0.7)
`0.8 (0.4–1.1)
`
`49 (84.5)
`145 (102–155)
`
`12 (20.7)
`6 (10.3)
`11 (19)
`
`20 (34.5)
`7 (12.1)
`25 (43.1)
`6 (10.3)
`
`8 (13.8)
`4 (6.9)
`1 (1.7)
`6 (10.3)
`19 (32.8)
`7 (12.1)
`4 (6.9)
`
`0 (0)
`2 (3.4)
`1 (1.7)
`3 (5.2)
`55 (94.8)
`44 (75.9)
`
`1 (1.7)
`17 (29.3)
`39 (67.2)
`1 (1.7)
`5 (8.6)
`
`1276 (1079–1629)
`27.5 (25–28.5)
`38 (35.9–41)
`317 (241–443)
`2 (1.2–3)
`3 (1.9–3.5)
`12.9 (9.4–14.9)
`0.5 (0.3–0.8)
`0.8 (0.5–0.9)
`
`55 (85.9)
`145 (104–155.2)
`
`Primary outcome measure, growth
`Weight, median (IQR), g
`Head circumference, median (IQR), cm
`Length, median (IQR), cm
`Weight change, median (IQR), g
`Length change, median (IQR), cm
`Head circumference change, median (IQR), cm
`Weight gain, median (IQR), g/kg per d
`Head growth, median (IQR), cm/wk
`Length growth, median (IQR), cm/wk
`Secondary outcomes at 28 d of age
`Receiving some feedings, n (%)
`Amount of enteral feedings, median (IQR), mL/kg per day
`Steroids used in first 28 d, n (%)
`Dexamethasone
`Hydrocortisone
`Positive blood or CSF culture, n (%)
`PDA, n (%)
`No PDA reported
`PDA reported but no report of treatment
`PDA treated with indomethacin
`PDA ligated
`Intracranial hemorrhage, n (%)
`Subependymal
`Intraventricular
`Intraventricular with dilation
`Intraparenchymal
`Any hemorrhage
`Severe (grade 3 or 4)
`Cystic periventricular leukomalacia
`Bowel disease, n (%)
`Isolated intestinal perforation
`Necrotizing enterocolitis, medical
`Necrotizing enterocolitis, surgical
`Any bowel disease
`None reported
`Received blood transfusion, n (%)
`Status at 28 d, n (%)
`Missing data
`Alive without oxygen therapy
`Alive with oxygen therapy
`Died
`Cholestasis (direct serum bilirubin level of ⬎5 mg/dL at
`any time in first 28 d), n (%)
`PDA indicates patent ductus arteriosus; IQR, interquartile range; CSF, cerebrospinal fluid.
`
`7 (10.9)
`5 (7.8)
`15 (23.4)
`
`30 (46.9)
`9 (14.1)
`18 (28.1)
`7 (10.9)
`
`8 (12.5)
`6 (9.4)
`5 (7.8)
`3 (4.7)
`22 (34.4)
`8 (12.5)
`0 (0)
`
`1 (1.6)
`5 (7.8)
`2 (3.1)
`8 (12.5)
`56 (87.5)
`47 (73.4)
`
`0 (0)
`24 (37.5)
`38 (59.4)
`2 (3.1)
`8 (12.5)
`
`nine, ornithine, phenylalanine, serine, tyrosine, and va-
`line) were higher in the 3.5 g/kg per day group than in the
`2.5 g/kg per day group. On day 28 (enteral phase of nutri-
`tion), tyrosine levels were higher for the neonates in the
`3.5 g/kg per day group, compared with those in the 2.5
`g/kg per day group. None of the amino acids levels was
`lower in the 3.5 g/kg per day group, compared with the 2.5
`g/kg per day group, on either day 7 or day 28.
`
`(day of random assignment, day 7, and day 28), gluta-
`mate, glycine, phenylalanine, and tyrosine levels de-
`creased. Arginine, aspartate, ornithine, and serine levels
`increased. Alanine and citrulline levels were relatively
`constant. Histidine, leucine/isoleucine, methionine, and
`valine levels increased and then decreased back to base-
`line (day of random assignment) levels. There was no
`treatment group/sample time interaction.
`
`Changes Over Time
`There were significant changes over time in most of the
`blood amino acid levels, and these changes were similar
`for the 2 treatment groups (Table 3). In the 3 samples
`
`Differences From Our Reference Sample of Normal Term
`Newborns
`In the study population, blood alanine and glutamate
`levels were commonly (⬎50% of the patients) low
`
`Downloaded from
`
`PEDIATRICS Volume 120, Number 6, December 2007
`
`
` at Reprints Desk on May 8, 2020www.aappublications.org/news
`
`1291
`
`Eton Ex. 1046
`6 of 13
`
`

`

`TABLE 3 Serum Amino Acid and Acylcarnitine Levels
`Daya
`Level, Median (IQR), mol/L
`
`Pb
`
`Maximal Dose of
`3.5 g/kg per d
`
`Maximal Dose of
`2.5 g/kg per d
`
`Kruskal-Wallis
`Test Between
`Groups
`
`Repeated-Measures
`Analysis of Changes
`Over Time
`
`Days Compared in
`Repeated-
`Measures Analysis
`
`Direction of
`Change
`
`Normal Newborn Level, mol/L
`
`10th
`Percentile
`
`50th
`Percentile
`
`90th
`Percentile
`
`Amino acids
`Alanine
`Alanine
`Alanine
`Arginine
`Arginine
`Arginine
`Aspartate
`Aspartate
`Aspartate
`Citrulline
`Citrulline
`Citrulline
`Glutamate
`Glutamate
`Glutamate
`Glycine
`Glycine
`Glycine
`Histidine
`Histidine
`Histidine
`Leucine/isoleucine
`Leucine/isoleucine
`Leucine/isoleucine
`Methionine
`Methionine
`Methionine
`Ornithine
`Ornithine
`Ornithine
`Phenylalanine
`Phenylalanine
`Phenylalanine
`Serine
`Serine
`Serine
`Tyrosine
`Tyrosine
`Tyrosine
`Valine
`Valine
`Valine
`Acylcarnitines
`Acetyl (C2)
`Acetyl (C2)
`Acetyl (C2)
`Butyryl (C4)
`Butyryl (C4)
`Butyryl (C4)
`Free carnitine
`Free carnitine
`Free carnitine
`Glutaryl (C5DC)
`Glutaryl (C5DC)
`Glutaryl (C5DC)
`Isovaleryl (C5)
`Isovaleryl (C5)
`Isovaleryl (C5)
`3-Hydroxyisovaleryl
`(C5OH)
`3-Hydroxyisovaleryl
`(C5OH)
`3-Hydroxyisovaleryl
`(C5OH)
`Linoleyl (C18:2)
`Linoleyl (C18:2)
`Linoleyl (C18:2)
`Oleyl (C18:1)
`Oleyl (C18:1)
`Oleyl (C18:1)
`Palmitoyl (C16)
`Palmitoyl (C16)
`Palmitoyl (C16)
`Palmitoleoyl (C16:1关N-7兴)
`Palmitoleoyl (C16:1关N-7兴)
`Palmitoleoyl (C16:1关N-7兴)
`Propionyl (C3)
`Propionyl (C3)
`Propionyl (C3)
`
`1292
`
`CLARK et al
`
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`1
`7
`28
`
`133.7 (98.2–191.1)
`160.8 (117.9–199.1)
`159.5 (129.9–196.8)
`9.9 (7.6–14.3)
`17.6 (13.6–28.4)
`12.5 (8.8–16.3)
`13.5 (10.2–18.7)
`15.6 (13.3–19.2)
`20.9 (15.3–26.2)
`9.1 (6.8–10.7)
`8.36 (6.97–12.09)
`8.5 (6.8–10.7)
`145.1 (120.4–176)
`131.7 (109.7–156.1)
`141.1 (115.3–164)
`143.7 (113.9–176.3)
`120.3 (101.4–145.6)
`116.2 (87.3–145)
`32.5 (28.6–42.4)
`41.8 (34.4–53.6)
`29.7 (22.5–43)
`128.6 (104–169.5)
`179.1 (152.5–203.9)
`135.3 (108.5–166.9)
`21.3 (13.9–33.1)
`29.9 (23.4–37.8)
`25 (18.2–33.2)
`32.1 (24.9–40.5)
`51.5 (41.4–69.4)
`44.6 (30.9–63)
`59.1 (50.1–72.8)
`59.6 (48.6–69.5)
`38.8 (32.7–49.1)
`104.4 (76.2–134.2)
`120.3 (94.5–155.7)
`122.6 (89.1–162.6)
`78.3 (54.3–110.5)
`59.8 (42.8–75)
`77.1 (53.8–103.9)
`90.1 (70.8–111.5)
`118.7 (96.7–142.7)
`80.6 (61.7–101)
`
`136.7 (109.5–180.7)
`132.5 (96.5–160.7)
`149.8 (107.8–193.5)
`8.8 (6.2–13)
`11.9 (8.8–16.3)
`11.1 (8.1–17.6)
`12.4 (9.7–17.1)
`14.2 (11.1–18.1)
`18.7 (14.9–26)
`8.9 (6.9–11.4)
`8.22 (6.51–9.75)
`10.2 (6.9–13.2)
`148.7 (124.1–195.7)
`115 (98.3–137.1)
`128 (96.9–154.6)
`146.7 (113.8–173.6)
`111.6 (88.1–129.7)
`108.7 (89.8–132.5)
`35.7 (25.8–44.7)
`39.7 (32.1–45.5)
`29.1 (21.5–36.4)
`126.7 (106.6–160.8)
`154.7 (133.3–167.2)
`130.5 (103–154.9)
`20.7 (15.7–29.4)
`27.2 (20.8–31.2)
`25.4 (16.3–29.7)
`30.5 (24.1–43.4)
`41 (36.5–51.1)
`44 (32.9–57.8)
`57.4 (48.1–67.3)
`51.5 (44.5–60.1)
`39.4 (33.4–46.6)
`104.7 (87.3–133.6)
`104.3 (77.9–137.6)
`126.6 (94.5–155.2)
`85.6 (56.9–136.4)
`42.8 (28.9–64.7)
`66.2 (41.3–78.6)
`85.1 (68.1–108.1)
`94.3 (83.2–110.2)
`81.5 (66–95.9)
`
`20.7 (15.6–26.5)
`21.8 (16.2–28.6)
`1
`11.3 (8.6–16.5)
`13 (10.3–17.4)
`7
`12.3 (9.1–19.2)
`15.2 (10.7–21.5)
`28
`0.375 (0.299–0.523)
`0.391 (0.328–0.487)
`1
`0.265 (0.192–0.395)
`0.296 (0.235–0.423)
`7
`0.201 (0.149–0.262)
`0.195 (0.153–0.253)
`28
`23.3 (17.2–29.9)
`24.6 (19.1–32.5)
`1
`14.4 (8.7–30.9)
`18.9 (12.2–29.8)
`7
`21.8 (13.2–30.4)
`21.1 (16.3–35.8)
`28
`0.021 (0.017–0.0285) 0.0195 (0.015–0.034)
`1
`7 0.0245 (0.019–0.044) 0.0235 (0.015–0.037)
`28 0.0268 (0.019–0.036) 0.0243 (0.016–0.034)
`1
`0.262 (0.193–0.34)
`0.252 (0.185–0.321)
`7
`0.307 (0.235–0.427)
`0.244 (0.183–0.37)
`28
`0.156 (0.113–0.198)
`0.157 (0.117–0.194)
`1
`0.136 (0.108–0.159)
`0.117 (0.101–0.153)
`
`7
`
`0.169 (0.12–0.208)
`
`0.136 (0.108–0.177)
`
`28
`
`0.13 (0.113–0.173)
`
`0.127 (0.1–0.174)
`
`0.109 (0.079–0.167)
`0.121 (0.079–0.184)
`1
`0.526 (0.366–0.716)
`0.61 (0.423–0.835)
`7
`0.231 (0.136–0.365)
`0.251 (0.176–0.383)
`28
`0.475 (0.368–0.611)
`0.501 (0.385–0.742)
`1
`0.584 (0.405–0.731)
`0.661 (0.476–0.847)
`7
`0.351 (0.24–0.575)
`0.439 (0.291–0.643)
`28
`0.96 (0.77–1.35)
`1.14 (0.74–1.5)
`1
`0.595 (0.44–0.76)
`0.651 (0.46–1.0)
`7
`0.419 (0.31–0.62)
`0.519 (0.36–0.68)
`28
`0.091 (0.067–0.14)
`0.105 (0.08–0.14)
`1
`0.0365 (0.029–0.049)
`7 0.0465 (0.03–0.07)
`28 0.0448 (0.029–0.064) 0.0328 (0.025–0.056)
`1
`2.41 (1.84–2.99)
`2.49 (1.6–3.03)
`7
`0.94 (0.65–1.39)
`0.72 (0.51–1.08)
`28
`0.73 (0.49–1.29)
`0.56 (0.37–1.16)
`
`.6
`.004
`.2
`.2
`.0001
`.7
`.7
`.051
`.5
`.9
`.08
`.2
`.5
`.016
`.1
`.9
`.07
`.3
`.8
`.1
`.4
`.6
`.0001
`.4
`.99
`.029
`.9
`.7
`.0001
`.9
`.3
`.002
`.99
`.5
`.018
`.8
`.3
`.011
`.039
`.7
`.0001
`.9
`
`.4
`.09
`.07
`.8
`.1
`.99
`.3
`.09
`.4
`.99
`.2
`.4
`.6
`.044
`.7
`.3
`
`.023
`
`.3
`
`.5
`.2
`.4
`.2
`.09
`.1
`.4
`.2
`.1
`.3
`.034
`.09
`.8
`.010
`.08
`
`.4
`.4
`.4
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`.21
`.21
`.21
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`.05
`.05
`.05
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`.005b
`.005b
`.005b
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`.94
`.94
`.94
`.05
`.05
`.05
`⬍.001
`⬍.001
`⬍.001
`.10
`
`.10
`
`.10
`
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`
`No change
`No change
`
`Increasing
`Increasing
`
`Increasing
`Increasing
`
`No change
`No change
`
`Decreasing
`Decreasing
`
`Decreasing
`Decreasing
`
`Increasing
`
`Increasing
`
`Increasing
`
`Increasing
`Increasing
`
`Decreasing
`Decreasing
`
`Increasing
`Increasing
`
`Decreasing
`Decreasing
`
`Increasing
`Decreasing
`
`Decreasing
`Decreasing
`
`Decreasing
`Decreasing
`
`No change
`No change
`
`Increasing
`
`Increasing
`Decreasing
`
`No change
`
`No change
`
`Increasing
`Decreasing
`
`Increasing
`Decreasing
`
`Decreasing
`Decreasing
`
`Decreasing
`Decreasing
`
`Decreasing
`Decreasing
`
`154
`154
`154
`4.2
`4.2
`4.2
`10.5
`10.5
`10.5
`5.9
`5.9
`5.9
`147
`147
`147
`92
`92
`92
`28.1
`28.1
`28.1
`89.7
`89.7
`89.7
`16.4
`16.4
`16.4
`18.8
`18.8
`18.8
`40.6
`40.6
`40.6
`71.6
`71.6
`71.6
`48.6
`48.6
`48.6
`62.9
`62.9
`62.9
`
`17.2
`17.2
`17.2
`0.129
`0.129
`0.129
`14.5
`14.5
`14.5
`0.016
`0.016
`0.016
`0.054
`0.054
`0.054
`0.077
`
`0.077
`
`0.077
`
`0.101
`0.101
`0.101
`0.919
`0.919
`0.919
`1.671
`1.671
`1.671
`0.10
`0.10
`0.10
`1.1
`1.1
`1.1
`
`216
`216
`216
`7.8
`7.8
`7.8
`15.1
`15.1
`15.1
`8.3
`8.3
`8.3
`195
`195
`195
`143
`143
`143
`39.8
`39.8
`39.8
`120.2
`120.2
`120.2
`21.9
`21.9
`21.9
`29.0
`29.0
`29.0
`51.5
`51.5
`51.5
`116.7
`116.7
`116.7
`74.7
`74.7
`74.7
`82.2
`82.2
`82.2
`
`25.9
`25.9
`25.9
`0.218
`0.218
`0.218
`21.8
`21.8
`21.8
`0.039
`0.039
`0.039
`0.093
`0.093
`0.093
`0.124
`
`0.124
`
`0.124
`
`0.201
`0.201
`0.201
`1.396
`1.396
`1.396
`2.716
`2.716
`2.716
`0.2
`0.2
`0.2
`1.8
`1.8
`1.8
`
`312
`312
`312
`17.2
`17.2
`17.2
`23.5
`23.5
`23.5
`12.1
`12.1
`12.1
`259
`259
`259
`209
`209
`209
`61.2
`61.2
`61.2
`166.6
`166.6
`166.6
`29.4
`29.4
`29.4
`44.9
`44.9
`44.9
`66.5
`66.5
`66.5
`220.5
`220.5
`220.5
`119.8
`119.8
`119.8
`111.3
`111.3
`111.3
`
`38.9
`38.9
`38.9
`0.387
`0.387
`0.387
`33.4
`33.4
`33.4
`0.074
`0.074
`0.074
`0.16
`0.16
`0.16
`0.201
`
`0.201
`
`0.201
`
`0.407
`0.407
`0.407
`2.093
`2.093
`2.093
`4.129
`4.129
`4.129
`0.33
`0.33
`0.33
`2.9
`2.9
`2.9
`
`7, 28
`1, 28
`1, 7
`28
`28
`1, 7
`
`7, 28
`1
`1
`7, 28
`1
`1
`7
`1, 28
`7
`7
`1, 28
`7
`7
`1, 28
`7
`7, 28
`1
`1
`7, 28
`1, 28
`1, 7
`28
`28
`1, 7
`7, 28
`1, 28
`1, 7
`7, 28
`1, 28
`1, 7
`
`7, 28
`1
`1
`28
`28
`1, 7
`
`7
`1
`
`7, 28
`1, 28
`1, 7
`
`7, 28
`1, 28
`1, 7
`7
`1, 28
`7
`7, 28
`1, 28
`1, 7
`7, 28
`1
`1
`7, 28
`1
`1
`
`Downloaded from
`
`www.aappublications.org/news
`
` at Reprints Desk on May 8, 2020
`
`Eton Ex. 1046
`7 of 13
`
`

`

`TABLE 3 Continued
`
`Daya
`
`Level, Median (IQR), mol/L
`
`Pb
`
`Maximal Dose of
`3.5 g/kg per d
`
`Maximal Dose of
`2.5 g/kg per d
`
`Stearoyl (C18)
`Stearoyl (C18)
`Stearoyl (C18)
`Succinyl (C4DC)
`Succinyl (C4DC)
`Succinyl (C4DC)
`
`1
`7
`28
`1
`7
`28
`
`0.553 (0.437–0.724)
`0.611 (0.387–0.709)
`0.549 (0.426–0.714)
`0.553 (0.416–0.744)
`0.31 (0.205–0.375)
`0.302 (0.203–0.384)
`0.0683 (0.057–0.098)
`0.066 (0.058–0.092)
`0.08 (0.053–0.1318) 0.0925 (0.059–0.149)
`0.116 (0.076–0.141)
`0.114 (0.086–0.165)
`
`Kruskal-Wallis
`Test Between
`Groups
`.9
`.8
`.99
`.9
`.3
`.3
`
`Repeated-Measures
`Analysis of Changes
`Over Time
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`⬍.001
`
`Days Compared in
`Repeated-
`Measures Analysis
`28
`28
`1, 7
`7, 28
`1, 28
`1, 7
`
`Direction of
`Change
`
`Normal Newborn Level, mol/L
`
`10th
`Percentile
`
`50th
`Percentile
`
`90th
`Percentile
`
`0.62
`0.62
`0.62
`0.067
`0.067
`0.067
`
`0.96
`0.96
`0.96
`0.128
`0.128
`0.128
`
`1.47
`1.47
`1.47
`0.219
`0.219
`0.219
`
`Decreasing
`
`Increasing
`Increasing
`
`DC indicates dica