Cysteine: a conditionally essential amino acid in low-birth-weight
`preterm infants? 1- 4
`
`Maaike A Riedijk, Ron HT van Beek, Gardi Voortman, Henrica MA de Bie, Anne CM Dassel, and
`Johannes B van Goudoever
`
`ABSTRACT
`Background: Cyst( e )ine can be synthesized de novo from methio(cid:173)
`nine and serine and is, therefore, a nonessential amino acid in human
`adults. Several studies have suggested that cyst(e)ine might be a
`conditionally essential amino acid in preterm infants because of
`biochemical immaturity. No data are available on cyst( e )ine require(cid:173)
`ments in low-birth-weight (LBW) preterm infants.
`Objective: The aim was to determine cyst( e )ine requirements in LBW
`infants with gestational ages from 32 to 34 wk, measured 1 mo after birth
`with the use of the indicator amino acid oxidation technique.
`Design: LBW infants were randomly assigned to 1 or 2 of the 5
`formulas containing graded cystine concentrations (11, 22, 32, 43, or
`65 mg cyst(e)ine/100 mL) and generous amounts of methionine.
`After 24-h adaptation, cyst( e )ine requirement was determined by
`13CO2 release from [l- 13C]phenylalanine in expired breath. 13CO2
`enrichment was measured by isotopic ratio mass spectrometry.
`Results: Cyst( e )ine requirement was determined in 25 LBW infants
`withamean(±SD)gestationalageof33 ± 1 wkandbirthweightof
`1.78 ± 0.32 kg. Fractional oxidation of [1 - 13C]phenylalanine did not
`differ between the 5 groups.
`Conclusions: There is no evidence for limited endogenous
`cyst(e)ine synthesis in 4-wk-old LBW preterm infants born at ges(cid:173)
`tational ages from 32 to 34 wk. It is safe to conclude that the
`cyst( e )ine requirement is < 18 mg · kg- 1 • d- 1 providing generous
`amounts of methionine and that cyst( e )ine is probably not a condi(cid:173)
`tionally essential amino acid in fully enterally fed LBW preterm
`Am J Clin Nutr 2007;86:1120-5.
`infants born at 32-34 wk.
`
`KEY WORDS
`oxidation, nutrition
`
`Amino acid requirements, indicator amino acid
`
`INTRODUCTION
`Cyst(e)ine is a sulfur-containing amino acid that is not essen(cid:173)
`tial in humans. The human body synthesizes cyst( e )ine de novo
`from methionine, the only essential sulfur-containing amino
`acid, and serine. Cyst( e )ine has several important metabolic
`functions. First, like all other amino acids it is involved in growth
`and protein synthesis. Furthermore, it is a precursor for the tri(cid:173)
`peptide glutathione, an important intracellular antioxidant. Then,
`it is also a precursor for the production of taurine, another anti(cid:173)
`oxidant, and sulfate.
`Some nonessential amino acids are classified as conditionally
`essential. These amino acids may become temporarily essential
`when synthesis during rapid growth or critical illness is insufficient.
`
`Cyst( e )ine is believed to be conditionally essential in preterm infants
`because of biochemical immaturity of the enzyme cystathionase
`(EC 4.4.1.1) that is involved in cyst(e)ine synthesis (1-3). It is,
`therefore, important to know the exact cyst( e )ine requirement of
`preterm infants and also in view of the higher amino acid re(cid:173)
`quirement as a result of rapid growth and development.
`Different methods are used to estimate individual amino acid
`requirements, eg, the nitrogen balance method, growth rate,
`plasma amino acid patterns, and the factorial approach. The first
`reports on amino acid requirements in neonates were based on
`nitrogen balance and weight gain rate ( 4, 5). Nitrogen balance has
`several limitations (6), for instance, it requires 7-10 d to adaptto
`the test diet (7). Because present-day practice will not allow
`neonates to be maintained on either deficient or excess intakes of
`amino acids for a minimum of 7 d, no recent requirement studies
`have used this method in preterm infants.
`At present, the factorial approach is used to define amino acid
`requirements in infants (8). This model is based on fetal protein
`accretion during normal intrauterine development. Accretion
`data are derived from body carcass analysis of stillborn preterm
`infants, some born > 100 y ago (9, 10). Not all data are suitable
`as reference material; gestational age and cause of death were not
`accurately obtained (11), and, in view of this study, cysteine
`content of the body was not determined. The current recommen(cid:173)
`dation for cyst(e)ine requirement for preterm infants, ie, 66-95
`mg · kg- 1 • d- 1, is based on the minimum and maximum
`amounts of each amino acid present in the amounts of human
`
`1 From the Department of Pediatrics, Division ofNeonatology (MAR and
`JBvG), Mass Spectrometry Laboratory (GV), Erasmus MC-Sophia Chil(cid:173)
`dren' s Hospital, University Medical Center, Rotterdam, the Netherlands; the
`Department of Pediatrics, Amphia Hospital, Breda, the Netherlands
`(RHTvB); the Department of Pediatrics, Vlietland Hospital, Vlaardingen, the
`Netherlands (HMAdB); and the Department of Pediatrics, Deventer Hospi(cid:173)
`tal, Deventer, the Netherlands (ACMD).
`2 "Cyst( e )ine" is used throughout to designate any undefined combination
`of cysteine and cystine. The terms "cysteine" and "cystine" are used to
`designate the specific amino acids.
`3 Supported by the Sophia Foundation for Medical Research (grant 417),
`the Netherlands, and the Nutricia Research Foundation, Wageningen, the
`Netherlands.
`4 Reprints not available. Address correspondence to JB van Goudoever,
`Erasmus MC-Sophia Children's Hospital, Department of Pediatrics, Divi(cid:173)
`sion ofNeonatology, Dr Molewaterplein 60, 3015 GJ Rotterdam, the Neth(cid:173)
`erlands. E-mail: j.vangoudoever@erasmusmc.nl.
`Received January 22, 2007.
`Accepted for publication June 6, 2007.
`
`1120
`
`Am J Clin Nutr 2007;86: 1120-5. Printed in USA. © 2007 American Society for Nutrition
`
`Eton Ex. 1049
`1 of 6
`
`

`

`CYST(E)INE REQUIREMENTS IN LBW INFANTS
`
`1121
`
`TABLE 1
`Amino acid profiles of the study formulas
`
`Amino acid
`
`Formula 1
`
`Formula2
`
`Formula3
`
`Formula4
`
`Formula5
`
`L-Alanine (mg/170 mL)
`L-Arginine (mg/170 mL)
`L-Aspartic acid (mg/170 mL)
`L-Cystine (mg/170 mL)
`L-Glycine (mg/170 mL)
`L-Histidine (mg/170 mL)
`L-lsoleucine (mg/170 mL)
`L-Leucine (mg/170 mL)
`L-Lysine (mg/170 mL)
`L-Methionine (mg/170 mL)
`L-Proline (mg/170 mL)
`L-Phenylalanine (mg/170 mL)
`L-Serine (mg/170 mL)
`L-Threonine (mg/170 mL)
`L-Tryptophan (mg/170 mL)
`L-Tyrosine (mg/170 mL)
`L-Valine (mg/170 mL)
`L-Asparagine (mg/170 mL)
`L-Citrulline (mg/170 mL)
`L-Carnitine (mg/170 mL)
`L-Taurine (mg/170 mL)
`L-Glutarnine/glutamate (mg/170 mL)
`Total (g/170 mL)
`
`166
`292
`274
`18
`259
`168
`266
`435
`300
`70
`313
`197
`194
`215
`88
`197
`282
`0
`0
`3
`8
`371
`4.12
`
`166
`292
`274
`36
`259
`168
`266
`435
`300
`70
`313
`197
`194
`215
`88
`197
`282
`0
`0
`3
`8
`371
`4.13
`
`166
`292
`274
`54
`259
`168
`266
`435
`300
`70
`313
`197
`194
`215
`88
`197
`282
`0
`0
`3
`8
`371
`4.14
`
`166
`292
`274
`72
`259
`168
`266
`435
`300
`70
`313
`197
`194
`215
`88
`197
`282
`0
`0
`3
`8
`371
`4.17
`
`166
`292
`274
`109
`259
`168
`266
`435
`300
`70
`313
`197
`194
`215
`88
`197
`282
`0
`0
`3
`8
`371
`4.21
`
`milk protein corresponding to the recommended minimum and
`maximum protein contents of 3.0 g/120 kcal and 4.3 g/120 kcal,
`respectively (8).
`Because methionine is the precursor of cyst(e)ine, the methi(cid:173)
`onine intake needs to be taken into account as well. The current
`estimated methionine requirement for preterm infants is 48-69
`mg· kg- 1 • d- 1 (8). To ensure an adequate methionine intake, the
`study formula supplied an amount of70 mg methionine · kg- 1 • d- 1•
`Shoveller et al (12) determined the total sulfur amino acid require(cid:173)
`ment in enterally fed neonatal piglets when no dietary cysteine was
`provided. The extrapolation from this piglet study results in a me(cid:173)
`thionine intake of 84 mg • kg - i • d- 1 for the human neonate, which
`is slightly above the current recommendations (8).
`The objective of the present study was to use indicator amino
`acid oxidation (IAAO) with [1- 13C]phenylalanine to estimate
`cyst(e)ine requirements in fully enterally fed low-birth-weight
`(LBW) infants supplied with an adequate methionine intake. We
`hypothesized that cyst(e)ine is a conditionally essential amino
`acid for these infants.
`
`SUBJECTS AND METHODS
`
`Subjects
`Subjects eligible for the study were LBW infants with gesta(cid:173)
`tional age (GA) between 32 and 34 wk, admitted to the neonatal
`department of the Amphia Hospital, Breda; Vlietland Hospital,
`Vlaardingen; or Deventer Hospital, Deventer (all: the Nether(cid:173)
`lands). The infants needed to be clinically stable during the study,
`and those with any congenital or gastrointestinal disease were
`excluded. All tolerated full enteral feeding and were partly fed
`through a nasogastric feeding tube and partly bottle-fed. The
`study protocol was approved by The Central Committee on Re(cid:173)
`search Involving Human Subjects (CCMO) and the Erasmus MC
`
`Institutional Review Board. Written and informed consent was
`obtained from both parents of each subject.
`
`Study formula
`We used 5 study formulas that contained graded cyst( e )ine
`concentrations: 11, 22, 32, 43, and 65 mg cystine/100 mL (Xcys/
`Neocate; Nutricia Nederland BV, Zoetermeer, the Netherlands/
`SHS International, Liverpool, United Kingdom). Infants were
`enrolled in the study when they tolerated full enteral feeding
`(> 150mL · kg- 1 • d- 1). Exceptforthecyst(e)ineconcentration,
`the 5 formulas did not differ in amino acid composition
`(Table 1), and consequently the nitrogen content increased
`slightly from 0.37 mg nitrogen/100 mL (formula 1) to 0.38 mg
`nitrogen/100 mL (formula 5). The graded cyst(e)ine concentra(cid:173)
`tions of the study formulas were based on the current estimated
`cyst(e)ine requirement for preterm infants (8), which is 66-95
`mg · kg- 1 • d- 1, and is based on the minimum and maximum
`amounts of each amino acid present in the amounts of human
`milk protein corresponding to the recommended minimum and
`maximum protein contents of 3.0 g/120 kcal and 4.3 g/120 kcal,
`respectively. We decided to test 3 cyst(e)ine intakes above and 2
`cyst( e )ine intakes below the estimated cyst( e )ine requirements
`for formula-fed preterm infants. Methionine content was similar
`in all formulas and was supplied generously.
`
`Study design and tracer protocol
`Cyst( e )ine requirement was measured ""1 mo after birth
`(range: 35-37 wk postmenstrual age). Infants were randomly
`assigned to at least one of the study formulas. The study diet was
`initiated 24 h before start of the oxidation study, and the dietary
`intake was not changed until the tracer protocol was finished. All
`subjects received ""170 mL formula· kg- 1 • d- 1 to ensure that
`all essential amino acids other than cyst( e )ine were in excess
`
`Eton Ex. 1049
`2 of 6
`
`

`

`1122
`
`RIEDUK ET AL
`
`(Table 1) and, therefore, were not limiting for protein synthesis.
`Before the introduction of the study formula, almost all infants
`received their mothers' (expressed) breast milk, and only a few
`infants received standard preterm formula (Nenatal; Nutricia,
`Zoetermeer, the Netherlands). The cyst(e)ine concentration of
`human milk varies widely, but the preterm formula provided 35
`cyst(e)ine/100 mL.
`The IAAO technique uses as indicator a labeled essential
`amino acid that is different than the test amino acid. The indicator
`is independent of the different intake amounts of the test amino
`acid. If the test amino acid is deficient in the diet, this will limit
`overall protein synthesis and all other essential amino acids will
`be oxidized. Increasing the dietary intake of the test amino acid
`will linearly decrease oxidation of the indicator until requirement
`of the test amino acid is met. We chose [l- 13C]phenylalanine as
`the indicator (13).
`After 24-h adaptation, subjects received a primed (10 µ,moll
`kg) continuous (10 µ,mo!· kg- 1 • h- 1) enteral infusion of
`[ 13C]bicarbonate [sterile pyrogen free, 99 atom percent excess
`(APE); Cambridge Isotopes, Woburn, MA] for 2.5 h to quantify
`individual carbon dioxide production. We infused the tracer en(cid:173)
`terally to minimize invasiveness, which was validated by our
`group (14). The labeled sodium bicarbonate infusion was di(cid:173)
`rectly followed by a primed (30 µ,mol/kg) continuous (30
`µ,mo!· kg- 1 • h- 1) enteral infusion of [1- 13C]phenylalanine (93
`APE; Cambridge Isotopes) for 5 h. One hour before the start of
`the oxidation study, the feeding regimen was changed into con(cid:173)
`tinuous drip-feeding. Enterally infused tracer was mixed with the
`study formula and infused continuously by an infusion pump
`through the nasogastric tube. All infants were breathing sponta(cid:173)
`neously, and 14 infants needed supplemental oxygen by a nasal
`prong.
`Breath samples were obtained with the use of the direct sam(cid:173)
`pling method described by van der Schoor et al (15). In brief, a
`6-French gastric tube (6 Ch Argyle; Cherwood Medical, Tul(cid:173)
`lamore, Ireland) was inserted 1-1.5 cm into the nasopharynx, and
`end-tidal breath was taken slowly with a syringe connected at the
`end. Collected air was transferred into 10-mL sterile, non(cid:173)
`silicon-coated evacuated glass tubes (Van Loenen Instruments,
`Zaandam, the Netherlands) and stored at room temperature until
`analysis. Baseline samples were obtained 15 and 5 min before
`starting tracer infusion. Duplicate 13C-enriched breath samples
`were collected every 30 min and every 15 min during the last 45
`min of the tracer infusion.
`
`Analytic methods and calculations
`13CO2 isotopic enrichment in expired air was measured by
`isotope ratio mass spectrometry (ABCA; Europe Scientific, Van
`Loenen Instruments, Leiden, the Netherlands) and expressed as
`APE above baseline (15). APE was plotted relative to time.
`It is necessary to determine the individual carbon dioxide
`production to determine the rate of substrate oxidation. In gen(cid:173)
`eral, the rate of oxidation is calculated by multiplying the isotopic
`enrichment of carbon dioxide in breath by the total rate of carbon
`dioxide excreted; the latter needs to be determined by indirect
`calorimetry to estimate the rate of carbon dioxide production.
`Alternatively, body carbon dioxide production can be deter(cid:173)
`mined by quantifying the excretion of 13CO2 in expired air during
`[ 13C]sodium bicarbonate infusion, which avoids the quantifica(cid:173)
`tion of total expired air (15).
`
`(in
`production
`dioxide
`carbon
`body
`Estimated
`mmol · kg- 1 • h- 1) was calculated as described previously (14).
`The rate of fractional [l- 13C]phenylalanine oxidation was cal(cid:173)
`culated as follows:
`
`Fractional pheny !alanine oxidation ( % )
`
`= [IErHE X iB]/[iPHE X IEB] X 100
`
`(1)
`
`where IErHE is the 13C isotopic enrichment in expired air during
`[1-13C]phenylalanine infusion (in APE), is is the infusion rate of
`[ 13C]bicarbonate (in µ,mo!· kg- 1 • h- 1), irHE is the infusion rate
`of [l- 13C]phenylalanine (in µ,mo! · kg- 1 • h- 1), and IE8 is the
`13C isotopic enrichment in expired air during [13C]bicarbonate
`infusion (16).
`
`Statistical analysis
`Descriptive data are expressed as mean ± SD. Steady state of
`13CO2 in expired breath during the [ l -13C]phenylalanine was
`achieved when the linear factor of the slope was found not to be
`significantly different from zero (P 2:: 0.05). The cyst(e)ine re(cid:173)
`quirement was determined with the IAAO method. The indicator
`oxidation rate is plotted against the varying dietary intakes of
`cyst( e )ine. The inflection or breakpoint in the indicator oxidation
`rate represents the physiologic requirement of cyst( e )ine (17).
`Data were analyzed with the use of mixed model analysis of
`variance in SPSS SOFTWARE (version 14.0; SPSS Inc, Chi(cid:173)
`cago, IL), while encoding the patients who participated twice
`with the same number. Repeated measures analysis of variance
`was performed on primary and derived variables to assess the
`effects of dietary intake and of subjects. Regression analysis was
`performed to analyze oxidation rates. Power calculation showed
`that, assuming 5 formula groups with a group variance of 16, an
`intergroup variance of 5.5, and a power of 80%, a breakpoint
`should be detected with 6 subjects per group. Statistical signifi(cid:173)
`cance was assumed at the 5% level of significance (P :5 0.05).
`
`RESULTS
`We included 25 LBW infants (12 boys, 13 girls) born at mean
`(±SD) GA of33 ± 1 wk (range: 32-34 wk). They were studied
`at mean postmenstrual age of 36 ± 1 wk (range: 35-38 wk), ie,
`approximately at postnatal age 1 mo. Subject characteristics are
`depicted in Table 2. Five infants participated twice and received
`2 different study formulas. Aiming at 6 measurements per for(cid:173)
`mula, we performed a total of 30 labeled phenylalanine oxidation
`rate measurements in these 25 infants.
`GA, study age, birth weight, and study weight did not differ
`between the 5 formula groups as shown in Table 2. In addition,
`the total enteral intake did not differ between the 5 formulas (P =
`0.07). Although the nitrogen content of study formula 5 was
`somewhat higher, the nitrogen intake did not differ significantly
`between the formula groups (P = 0.25).
`To detect any differences between the 5 groups receiving the
`different formulas we corrected for sex, study age, and study
`weight. The baseline 13C enrichment in expired breath did not
`differ between the 5 formula groups ( -17 .24 ± 0.56, -16. 77 ±
`0.62, -17.95 ± 1.38, -17.87 ± 1.01, and -17.58 ± 1.55 Pee
`Dee Belemnite, respectively; P = 0.67) after correction for birth
`weight and study weight (Table 3). Each subject reached a pla(cid:173)
`teau during both [13C]bicarbonate and [l- 13C]phenylalanine
`tracer infusions. As an illustration, the 13C enrichments in
`
`Eton Ex. 1049
`3 of 6
`
`

`

`CYST(E)INE REQUIREMENTS IN LBW INFANTS
`
`1123
`
`TABLE 2
`Patient characteristics1
`
`Formula
`
`Subjects
`
`GA
`
`1
`2
`3
`4
`5
`All
`
`n
`
`6
`6
`6
`6
`6
`30
`
`wk
`33 ± I2
`32 ± 1
`33 ± 1
`33 ± 1
`32 ± 1
`33 ± 1
`
`SA
`
`wk
`
`36 ± 1
`35 ± 0
`36 ± 1
`36 ± 0
`36 ± 1
`36 ± 1
`
`BW
`
`SW
`
`kg
`1.88 ± 0.28
`1.81 ± 0.36
`1.81 ± 0.38
`1.81 ± 0.24
`1.58 ± 0.38
`1.78 ± 0.32
`
`kg
`2.26 ± 0.24
`2.10 ± 0.38
`2.17 ± 0.38
`2.17 ± 0.27
`2.04 ± 0.31
`2.15±0.31
`
`Enteral intake
`mL·kg- 1 •r 1
`170 ± 6
`167 ± 1
`167 ± 2
`163 ± 5
`166 ± 3
`167 ± 5
`
`Cyst(e)ine
`intake
`mg·kg- 1 •r 1
`18 ± 1
`36 ± 0
`54 ± 1
`70 ± 2
`108 ± 2
`
`SAA intake
`mg·kg- 1 -r 1
`89 ± 3
`105 ± 1
`123 ± 1
`138 ± 4
`177 ±4
`
`1 GA, gestational age; SA, study age; BW, birth weight; SW, study weight; SAA, sulfur amino acid. No significant differences were detected in GA
`(P = 0.20), SA (P = 0.38), BW (P = 0.55), SW (P = 0.86), or enteral intake (P = 0.07) between the 5 formula groups.
`2 i ± SD (all such values).
`
`expired breath during the infusion of [l- 13C]phenylalanine of
`6 subjects receiving 54 mg cyst(e)ine · kg- 1 • d- 1 are shown in
`Figure 1.
`The mean fractional [ 1-13C]pheny lalanine oxidation did not
`differ between the groups (P = 0.73). Regression of the data did
`not show a linear decrease in indicator oxidation rate, and con(cid:173)
`sequently a breakpoint is missing (Figure 2). A trend in the
`formula could not be detected (P = 0.90). This implies that the
`cyst( e )ine requirement under these circumstances is already met
`at an intake of 18 mg cystine · kg - I • d- 1• At the intakes of
`18-109 mg · kg- 1 • d- 1, cyst( e )ine is not the limiting amino acid
`for protein synthesis and is therefore not deficient in the diet.
`
`DISCUSSION
`To our knowledge this is the first study to determine the exact
`cyst( e )ine requirementin LBW infants. With the use of the IAAO
`method, we found it to be < 18 mg · kg- 1 • d- 1 1 mo after birth,
`which suggests that cyst(e)ine is not a conditionally essential
`amino acid in these infants at this age who are receiving generous
`amounts of methionine. The IAAO method is based on the as(cid:173)
`sumption that the partition of any essential amino acid between
`oxidation and protein synthesis is sensitive to the amount of the
`most limiting amino acid in the diet (17, 18). Thus, a limitation
`of this method is the necessity of providing all amino acids in
`excess, except for the one under study. Accordingly, insufficient
`
`amounts of essential amino acids could have resulted in another
`essential amino acid than cystine being limiting for protein syn(cid:173)
`thesis. However, we do not believe this is the case in this study,
`seeing that the fractional [1- 13C]phenylalanine oxidation did not
`differ between the 5 test diets and that essential amino acids were
`supplied in excess of the estimate dietary requirements for pre(cid:173)
`term infant formula ( 19). Although the dietary intake of formula
`1 (170 mL·kg- 1 ·d- 1) was higher than formula 4 (163
`mL · kg- 1 • d- 1),itdidnotsignificantlydiffer. Wedo not believe
`that this had a major influence on the obtained results. The higher
`intake of formula 1 could have resulted in a lower phenylalanine
`oxidation rate because of a slightly higher cyst( e )ine intake com(cid:173)
`pared with formula 2. However, we did not find a difference in
`the phenylalanine oxidation.
`The current guidelines on requirements of individual amino
`acids for infants are based on the factorial method. To calculate
`the deposit of each amino acid, growth rate is assumed to be
`constant at 15 g · kg- 1 • d- 1 for a fetus from 900 to 2400 g and
`protein retention at 70% of total protein intake. Furthermore, the
`protein maintenance requirement in preterm infants ranges from
`0.55 to0.75 g • kg- 1 • d- 1 (20). Fetal amino acid accretion during
`normal intrauterine growth was determined by Widdowson (21).
`Cyst( e )ine accretion by the human fetus was not determined,
`however; thus, the requirement could not be estimated by the
`factorial approach.
`
`TABLE 3
`Whole-body carbon dioxide production and fractional [13C]phenylalanine (phe) oxidation rates of 5 different cyst(e)ine intakes1
`
`Cyst( e )ine intake
`
`Baseline 13CO2
`
`PDB
`
`13CO2
`[ 13C]bicarb
`
`APE
`
`-17.24 ± 0.56
`
`0.0331 ± 0.0046
`
`Carbon dioxide
`production
`
`mmol • kg- 1 • d- 1
`30.78 ± 5.31
`
`13CO2
`[l-13C]phe
`
`APE
`
`0.0209 ± 0.0080
`
`Fractional oxidation
`of [ I- 13C]phe
`
`%
`20.32 ± 5.26
`
`18mg·kg- 1 ·d- 1
`(n = 6)
`36 mg· kg- 1 • d- 1
`(n = 6)
`54 mg· kg- 1 • d- 1
`(n = 6)
`72mg·kg- 1 ·d- 1
`(n = 6)
`109 mg· kg- 1 • d- 1
`(n = 6)
`1 All values are i ± SD. 13CO2, enrichment of 13C in expired air; PDB, Pee Dee Belemnite; bicarb, sodium bicarbonate; APE, atom percent excess. No
`significant differences were detected in baseline 13CO2 (P = 0.67), carbon dioxide production (P = 0.61), or fractional oxidation of labeled phenylalanine (P
`= 0.73) between the 5 formula groups.
`
`-16.77 ± 0.62
`
`0.0353 ± 0.0030
`
`28.19 ± 2.61
`
`0.0200 ± 0.0058
`
`18.73 ± 5.03
`
`-17.95 ± 1.38
`
`0.0339 ± 0.0035
`
`29.83 ± 3.08
`
`0.0206 ± 0.0043
`
`20.33 ± 4.15
`
`-17.87 ± 1.01
`
`0.0329 ± 0.0023
`
`30.20 ± 1.97
`
`0.0210 ± 0.0049
`
`21.48 ± 5.70
`
`-17.58 ± 1.55
`
`0.0344 ± 0.0028
`
`29.24 ± 2.22
`
`0.0197 ± 0.0054
`
`19.07 ± 4.93
`
`Eton Ex. 1049
`4 of 6
`
`

`

`1124
`
`0.04
`
`RIEDIJK ET AL
`
`iii' 0.03
`
`I I I I I I II
`~ I
`
`8 0.02
`..,
`-c
`Ill
`~ 0.01
`
`o.01o-1----.---------"'I""---,
`200
`250
`400
`450
`300
`350
`Time (min)
`FIGURE 1. Mean 13C enrichments in expired breath during enteral
`receiving 54 mg
`[l- 13C]-phenylalanine
`infusion
`in 6
`infants
`cyst(e)ine·kg- 1 ·d- 1 . Data are expressed as means± SDs. APE, atom
`percent excess.
`
`Little is known, too, about the biosynthetic capacity of non(cid:173)
`essential amino acids in preterm infants. In the early 1970s sev(cid:173)
`eral investigators reported that the transsulfuration pathway
`might be immature in preterm infants because of limited enzyme
`activity of cystathionase (1-3). Some found cystathionase activ(cid:173)
`ity to be absent in fetal liver tissues (2, 3). Zlotkin and Anderson
`(1) showed that cystathionine activity was limited but not iso(cid:173)
`lated to the liver and was also present in extrahepatic tissues
`(kidneys and adrenals). They also found in term and preterm
`infants that this activity increased after birth. Furthermore,
`Stegink and Den Besten (22) showed that plasma cysteine con(cid:173)
`centrations in adult humans fed a cystine-deficient diet intragas(cid:173)
`tricall y were significantly higher than in those fed the same diet
`intravenously. This finding suggests an important role in
`cyst( e )ine production for the gut and was confirmed in animal
`experiments. Neonatal piglets fed a cysteine-free diet enterally
`showed significantly higher plasma cysteine concentrations than
`did parenterally fed piglets (23). In addition, dietary methionine
`and plasma cysteine concentrations were positively correlated in
`enterally fed piglets but not in parenterally fed piglets.
`Earlier studies that investigated whether cyst(e)ine is an es(cid:173)
`sential amino acid in preterm infants were all performed during
`parenteral nutrition. Several of those studies reported low plasma
`
`. •
`-,----·-"···---:---------------------·-
`.
`.
`
`•
`
`I
`
`..-.
`,fl.
`C
`._..
`l!! 3
`_g
`a, ·-
`'C C
`·- a,
`>< -
`~ ';.2
`a, C
`§ 1!
`;,9; e .P 1
`IL-, =-
`
`O
`
`100
`75
`50
`25
`Cyst(e)ine intake (mg. kg-1 • d·')
`FIGURE 2. Fractional [l- 13C]phenylalanine oxidation plotted against
`increasing cyst(e)ine intake. Data are expressed as means± SDs. No differ(cid:173)
`ences could be detected between the fractional oxidation of [ l- 13C](cid:173)
`phenylalanine of the 5 different cyst(e)ine intakes (n = 6 per intake; P =
`0.73).
`
`125
`
`0
`
`a.
`
`~ ::, g
`(D a. a 3
`
`cysteine concentrations, with or without cysteine supplementa(cid:173)
`tion (24-26). Pohlandt (26) observed no differences in plasma
`cystine concentrations between preterm infants receiving only
`glucose intravenously and preterm infants fed a mixture of syn(cid:173)
`thetic amino acids free from cystine. In both groups cystine
`plasma concentrations were low, <5 µmol/L, indicating limited
`endogenous synthesis from methionine. Furthermore, Vina et al
`(25) and Miller et al (27) reported impaired cysteine metabolism
`in premature infants on total parenteral nutrition. All those stud(cid:173)
`ies indicate that limited cystathionase activity makes cyst( e )ine a
`conditionally essential amino acid in preterm infants. Zlotkin et
`al (28), nevertheless, reported that cysteine supplementation did
`not affect growth rate and nitrogen balance in parenterally fed
`term and preterm infants. It did slightly increase urinary
`3-methylhistidine excretion, however, suggesting that either
`muscle protein catabolism or muscle mass had increased. Also
`Malloy et al (29) did not find an improved nitrogen balance with
`cysteine supplementation, although plasma-free cyst(e)ine con(cid:173)
`centration increased. Contradictorily, findings from a recent
`study suggest that the transsulfuration pathway produces suffi(cid:173)
`cient amounts of cysteine in the parenterally fed preterm infant
`(30).
`Our study was performed when neonates received full enteral
`feeding supplied with a generous amount of methionine and
`shows that the cyst(e)ine synthesis pathway is active and suffi(cid:173)
`cient at this time. We studied the indicator oxidation during 5 h
`when infants were continuously fed. The standard feeding regi(cid:173)
`men of these infants in our neonatal intensive care unit dictates
`feeding every hour or every 2 h, depending on clinical consid(cid:173)
`erations. Thus, we studied neonates under physiologic circum(cid:173)
`stances. In our view, therefore, the absence of a postabsorptive
`state in these preterm infants does not necessitate the study of
`oxidation during 24 h.
`In conclusion, our findings reject our hypothesis that cys(cid:173)
`t( e )ine is a conditionally essential amino acid in 1-mo-old fully
`enterally fed LBW preterm infants with a mean GA of 33 wk.
`These findings show indirectly that activity of the enzyme cys(cid:173)
`tathionase is sufficient to synthesize adequate amounts of
`cyst(e)ine in these infants supplied with a generous amount of
`dietary methionine.
`
`We thank Ineke van Vliet for her support in performing the studies, Ko
`Hagoort for critical review of the manuscript, and Paul Mulder for statistical
`help. We also thank all the parents who consented for their infants to partic(cid:173)
`ipate in this study.
`The author's responsibilities were as follows-MAR: collected and ana(cid:173)
`lyzed the data and wrote the manuscript; RHTvB, HMAdB, and ACMD:
`recruited all patients; GV: analyzed the data; JBvG: designed the study and
`provided helpful comments on the manuscript. None of the authors had any
`financial or personal interest in any company or organization sponsoring the
`research.
`
`REFERENCES
`1. Zlotkin SH, Anderson GH. The development of cystathionase activity
`during the first year of life. Pediatr Res 1982; 16:65-8.
`2. Sturman JA, Gaull G, Raiha NC. Absence of cystathionase in human
`fetal liver: is cystine essential? Science 1970;169:74-6.
`3. Gaull G, Sturman JA, Raiha NC. Development of mammalian sulfur
`metabolism: absence of cystathionase in human fetal tissues. Pediatr Res
`1972;6:538-47.
`4. Snyderman SE, Boyer A, Norton PM, Roitman E, Holt LE Jr. The
`essential amino acid requirements of infants. IX. Isoleucine. Am J Clin
`Nutt 1964;15:313-21.
`
`Eton Ex. 1049
`5 of 6
`
`

`

`CYST(E)INE REQUIREMENTS IN LBW INFANTS
`
`1125
`
`5. Snyderman SE, Boyer A, Norton PM, Roitman E, Holt LE Jr. The
`essential amino acid requirements of infants. X. Methionine. Am J Clin
`Nutr 1964;15:322-30.
`6. Fuller MF, Garlick PJ Human amino acid requirements: can the contro(cid:173)
`versy be resolved? Annu Rev Nutr 1994;14:217-41.
`7. Rand WM, Young YR, Scrimshaw NS. Change of urinary nitrogen
`excretion in response to low-protein diets in adults. Am J Clin Nutr
`1976;29:639-44.
`8. Garlick P, Pencharz PB, Reeds P. Protein and amino acid. In: Panel on
`macronutrients: dietary reference intakes for energy, carbohydrates, fi(cid:173)
`ber, fat, fatty acids, cholesterol, protein and amino acids. Washington,
`DC: Institute of Medicine, National Academy of Sciences, 2005:589-
`768.
`9. Sparks JW. Human intrauterine growth and nutrient accretion. Semin
`Perinatol 1984;8:74-93.
`10. Widdowson EM, Spray CM. Chemical development in utero. Arch Dis
`Child 1951 ;26:205-14.
`11. Ziegler EE, O' Donnell AM, Nelson SE, Fomon SJ. Body composition of
`the reference fetus. Growth 1976;40:329-41.
`12. Shoveller AK, Brunton JA, Pencharz PB, Ball RO. The methionine
`requirement is lower in neonatal piglets fed parenterally than in those fed
`enterally. J Nutr 2003;133:1390-7.
`13. Ball RO, Bayley HS. Tryptophan requirement of the 2.5-kg piglet de(cid:173)
`termined by the oxidation of an indicator amino acid. J Nutr 1984;114:
`1741-6.
`14. Riedijk MA, Voortman G, van Goudoever JB. Use of [13C]bicarbonate
`for metabolic studies in preterm infants: intragastric versus intravenous
`administration. Pediatr Res 2005;58:861-4.
`15. van der Schoor SR, de Koning BA, Wattimena DL, Tibboel D, van
`Goudoever JB. Validation of the direct nasopharyngeal sampling
`method for collection of expired air in preterm neonates. Pediatr Res
`2004;55:50-4.
`16. van der Schoor SR, Reeds PJ, Stellaard F, et al. Lysine kinetics in preterm
`infants: the importance of enteral feeding. Gut 2004;53:38-43.
`17. Brunton JA, Ball RO, Pencharz PB. Determination of amino acid re(cid:173)
`quirements by indicator amino acid oxidation: applications in health and
`disease. Curr Opin Clin Nutr Metab Care 1998;1:449-53.
`
`18. Pencharz PB, Ball RO. Different approaches to define individual amino
`acid requirements. Annu Rev Nutr 2003;23: 101-16.
`19. Klein CJ. Nutrient requirements for preterm infant formulas. J Nutr
`2002; l 32(suppl): 1395S-577S.
`20. Zello GA, Menendez CE, Rafii M, et al. Minimum protein intake for the
`preterm neonate determined by protein and amino acid kinetics. Pediatr
`Res 2003;53:338-44.
`21. Widdowson E. Body composition of the fetus and infant. In: Visser H,
`ed. Fifth Nutricia Symposium: nutrition and metabolism of the fetus and
`infant. The Hague, the Netherlands: Martinus Nijhoff, 1979:169-77.
`22. Stegink LD, Den Besten L. Synthesis of cysteine from methionine in
`normal adult subjects: effect of route of alirnentation. Science 1972; 178:
`514-6.
`23. Shoveller AK, Brunton JA, House JD, Pencharz PB, Ball RO. Dietary
`cysteine reduces the methionine requirement by an equal proportion in
`both parenterally and enterally fed piglets. J Nutr 2003;133:4215-24.
`24. Van Goudoever JB, Sulkers EJ, Tinunerman M, et al. Amino acid so(cid:173)
`lutions for premature neonates during the first week of life: the role of