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Cyst(e)ine Requirements in Enterally Fed Very Low Birth Weight Preterm Infants

^a Division of Neonatology, Department of Pediatrics, University Medical Center
^b Mass Spectrometry Laboratory, Division of Neonatology, Department of Pediatrics, Erasmus Medical Center–Sophia Children's Hospital, Rotterdam, Netherlands
^c Department of Pediatrics, Amphia Hospital, Breda, Netherlands
^d Department of Pediatrics, Medical Center Rijnmond-Zuid, Rotterdam, Netherlands
^e Department of Pediatrics, Albert Schweitzer Hospital, Dordrecht, Netherlands

	ABSTRACT

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OBJECTIVE. Optimal nutrition is of utmost importance for the preterm infant's later health and developmental outcome. Amino acid requirements for preterm infants differ from those for term and older infants, because growth rates differ. Some nonessential amino acids, however, cannot be sufficiently synthesized endogenously. Cyst(e)ine is supposed to be such a conditionally essential amino acid in preterm infants. The objective of this study was to determine, at 32 and 35 weeks’ postmenstrual age, cyst(e)ine requirements in fully enterally fed very low birth weight preterm infants with gestational ages of <29 weeks.

METHODS. Infants were randomly assigned to 1 of the 5 graded cystine test diets that contained generous amounts of methionine. Cyst(e)ine requirement was determined with the indicator amino acid oxidation technique ([1-¹³C]phenylalanine) after 24-hour adaptation.

RESULTS. Fractional [1-¹³C]phenylalanine oxidation was established in 47 very low birth weight preterm infants (mean gestational age: 28 weeks ± 1 week SD; birth weight: 1.07 kg ± 0.21 kg SD). Increase in dietary cyst(e)ine intake did not result in a decrease in fractional [1-¹³C]phenylalanine oxidation.

CONCLUSIONS. These data do not support the hypothesis that endogenous cyst(e)ine synthesis is limited in very low birth weight preterm infants with gestational ages of <29 weeks, both at 32 and 35 weeks postmenstrual age. It is safe to conclude that cyst(e)ine requirement is <18 mg/kg per day in enterally fed very low birth weight preterm infants who are older than 32 weeks’ postmenstrual age and whose methionine intake is adequate. Therefore, cyst(e)ine is probably not a conditionally essential amino acid in these infants.

Key Words: requirements • amino acids • indicator amino acid oxidation • nutrition

Abbreviations: VLBW—very low birth weight • IAAO—indicator amino acid oxidation • PMA—postmenstrual age • GA—gestational age • APE—atom percentage excess

Early nutrition is pivotal for preterm infants’ survival but also has profound influence on their later developmental and intelligence outcomes.^1,2 The quantity of administered essential amino acids is also of relevance; however, some nonessential amino acids are considered conditionally essential during specific circumstances (eg, rapid growth, critical illness).³ Endogenous synthesis then will temporarily not be sufficient to meet the requirement. Cyst(e)ine^* is believed to be such a conditionally essential amino acid in preterm infants, because preterm infants show biochemical immaturity of cystathionase (EC 4.4.1.1), the enzyme catalyzing the final step in cyst(e)ine synthesis pathway (ie, transsulfuration pathway).^4–6 Cyst(e)ine is a sulfur-containing amino acid that is nonessential in humans. It is synthesized de novo from methionine, which is the only essential sulfur-containing amino acid, and from 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 1 of the amino acid components of the tripeptide glutathione, an important intracellular antioxidant. It is also a precursor for taurine and sulfate. It is, therefore, important to know the exact cyst(e)ine requirements of preterm infants at various postnatal ages, considering that cystathionase maturation occurs postnatally. Previous estimates of amino acid requirements were based on less accurate methods than are currently available. We performed a study aiming at estimating cyst(e)ine requirements in very low birth weight (VLBW) preterm infants at 4 and 8 weeks postnatally, using the indicator amino acid oxidation (IAAO) method. This method was used to reestimate individual essential amino acid requirements in adults^7–9 and was introduced by Zello et al.¹⁰ We hypothesized that cyst(e)ine is an essential amino acid for VLBW preterm infants early in life; however, it depends on postmenstrual age (PMA), and it becomes a nonessential amino acid after maturation of the transsulfuration pathway.

	METHODS

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Patients
Patients who were eligible for the study were VLBW infants who were born at gestational age (GA) of <29 weeks and admitted to the NICU of the Erasmus Medical Center–Sophia Children's Hospital (Rotterdam, Netherlands) in the first days of life. At the time of the study, a number of patients had already been transferred to affiliated hospitals in the region: Amphia Hospital (Breda), Albert Schweitzer Hospital (Dordrecht), or the Medical Center Rijmond-Zuid (Rotterdam), all in the Netherlands. In that case, the study was conducted in the hospital of relevance.

We determined cyst(e)ine requirements in patients who were aged 1 month (group 1) and patients who were aged 2 months (group 2) postnatally. They needed to be clinically stable and were excluded in case of congenital or gastrointestinal diseases. Infants were enrolled in the study when they tolerated full enteral feeding (>150 mL/kg per day). Feeding was either completely through a nasogastric feeding tube or partly by bottle, depending on postconceptual age. During the study, all infants were breathing spontaneously for at least 8 hours.

The study protocol was approved by the Central Committee on Research Involving Human Subjects, the Erasmus Medical Center institutional review board, and review boards from the affiliated hospitals. Written informed consent was obtained from both parents of each patient.

Study Formula
In this study, we used 5 elemental study formulas that contained graded cyst(e)ine concentrations: 11, 22, 32, 43, and 65 mg cyst(e)ine/100 mL (Xcys/Neocate; Nutricia Nederland BV, Zoetermeer, Netherlands/SHS International, Liverpool, United Kingdom). The cyst(e)ine content was monitored by SHS International. The study formulas were not totally isonitrogenous. Except for cyst(e)ine concentration, the 5 formulas did not differ as to amino acid composition. Methionine intake was similar for all formula groups (71 mg/kg per day) and was supplied generously according to the estimated methionine requirement for preterm infants (48–69 mg/kg per day).¹¹

Study Design and Tracer Protocol
Infants who were eligible for the study were included, and cyst(e)ine requirement was determined 1 month after birth (PMA range: 30–32 weeks) or 2 months after birth (PMA range: 35–37 weeks). Infants were randomly assigned to at least 1 of the study formulas. The study diet was initiated 24 hours before start of the study so that the patient could adapt to the diet. Cyst(e)ine intake and the dietary intake was not changed until the tracer protocol was finished. The adaptation period of 24 hours was selected according to studies by the group of Pencharz.^12,13

All patients received 170 mL/kg per day formula to ensure that all other essential amino acids, particularly methionine, were in excess and, therefore, not limiting for protein synthesis. Before the introduction of the study formula, almost all infants received their mother's (expressed) breast milk, and only a few infants received standard preterm formula (Neonatal; Nutricia, Zoetermeer, Netherlands). The cyst(e)ine concentration of breast milk varies widely, but the preterm formula provided 35 cyst(e)ine/100 mL.

The IAAO method is based on a labeled essential amino acid that is different from the test amino acid. This indicator is independent of the different intake levels of the test amino acid. If the test amino acid is deficient in the diet, then this will limit overall protein synthesis and all other essential amino acids will be oxidized. As dietary intake of the test amino acid increases, oxidation of the indicator will decrease linearly until requirement of the test amino acid is met. We chose [1-¹³C]phenylalanine as the indicator.¹⁴

After 24-hour adaptation, patients received a primed (10 µmol/kg) continuous (10 µmol/kg per hour) enteral infusion of [¹³C]bicarbonate (sterile pyrogen free, 99% ¹³C APE; Cambridge Isotopes, Woburn, MA) for 2.5 hours to quantify individual CO₂ production. We infused the tracer enterally to minimize invasiveness of the experiment. This method has been validated by our group.¹⁵ The labeled sodium bicarbonate infusion was directly followed by a primed (30 µmol/kg) continuous (30 µmol/kg per hour) enteral infusion of [1-¹³C]phenylalanine (93% ¹³C APE; Cambridge Isotopes) for 5 hours. One hour before start of the oxidation study, the feeding regimen was changed to continuous drip-feeding. Enterally infused tracer was mixed with the study formula and infused continuously by an infusion pump via the nasogastric tube.

Breath samples were obtained by using the direct sampling method described by Van der Schoor et al.¹⁶ In brief, a 6F gastric tube (6 Ch Argyle; Cherwood Medical, Tullamore, Ireland) was inserted 1.0 to 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–silicon-coated evacuated glass tubes (Van Loenen Instruments, Zaandam, Netherlands) and stored at room temperature until analysis. Baseline samples were obtained 15 and 5 minutes before start of tracer infusion. Duplicate ¹³C-enriched breath samples were first collected every 30 minutes but every 15 minutes during the last 45 minutes of tracer infusion.

Analytical Methods and Calculations
¹³CO₂ isotopic enrichment in expired air was measured by isotope ratio mass spectrometry (ABCA; Europe Scientific, Van Loenen Instruments, Leiden, Netherlands) and expressed as atom percentage excess (APE) above baseline.¹⁶ APE was plotted relative to [1-¹³C]phenylalanine infusion time.

Estimated body CO₂ production (mmol/kg per hour) was calculated as described previously.¹⁵ The rate of fractional [1-¹³C]phenylalanine oxidation was calculated as fractional phenylalanine oxidation (%) = (IE_PHE x i_B)/(i_PHE x IE_B) x 100%, where IE_PHE is the ¹³C isotopic enrichment in expired air during [1-¹³C]phenylalanine infusion (APE), i_B is the infusion rate of [¹³C]bicarbonate (µmol/kg per hour), i_PHE is the infusion rate of [1-¹³C]phenylalanine (µmol/kg per h), and IE_B is the ¹³C isotopic enrichment in expired air during [¹³C]bicarbonate infusion.

Statistical Analysis
Descriptive data are expressed as means ± SD. The steady state of ¹³CO₂ release in expired breath during the [¹³C]bicarbonate and [1-¹³C]phenylalanine infusions was achieved when the linear factor of the slope was found not to be significantly different from 0 (P > 0.05). The cyst(e)ine requirement was determined with the use of the IAAO method. The indicator oxidation rate was plotted against varying dietary cyst(e)ine intakes (mg/kg per day). The inflection or breakpoint in the indicator oxidation rate represents the physiologic cyst(e)ine requirement.¹⁷

Data were analyzed with the use of mixed-model analysis of variance in SPSS 14.0 (SPSS Inc, Chicago, 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 patients. Regression analysis was performed to analyze oxidation rates. Power calculation revealed 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 5 patients per group. Statistical significance was assumed at 5% level of significance (P .05).

	RESULTS

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We determined the cyst(e)ine requirements in VLBW infants 1 month after birth (group 1) and 2 months after birth (group 2). We included in total 47 VLBW infants: 20 infants in group 1 and 27 infants in group 2. Patient characteristics are depicted in Tables 1 and 2. Seven infants were studied at both time points, and in each group, 3 children participated twice and were assigned to 2 different formulas. Infants were randomly assigned to 1 or 2 study formulas providing 18, 36, 54, 72, or 109 mg cystine/kg per day at an intake of 168 mL/kg per d. The mean GA of all infants in both groups was 28 ± 1 week, and mean birth weight was 1.07 ± 0.21 (data not shown). Weight gain rate of the infants during the days before the study was >10 g/kg per day.

View this table: [in this window] [in a new window]   TABLE 1 Patient Characteristics at 1 Month's PMA for Group 1

View this table: [in this window] [in a new window]   TABLE 2 Patient Characteristics at 2 Months’ PMA for Group 2

In both groups, GA, birth weight, and study weight did not differ among the 5 formula groups (Tables 1 and 2); however, by chance, in group 1, the PMA of formula 2 was slightly higher compared with formula 5 (P = .04). We do not believe that this would have had any affect on the results because of the small difference in PMA and because the patients’ cyst(e)ine intake was randomly selected. The total enteral intake of both groups did not differ among the 5 formula groups (group 1: P = .14; group 2: P = .08).

To compare outcome parameters among the 5 formulas within each group, we corrected the model for gender, study age, and study weight. In both groups, the baseline ¹³C enrichment in expired breath did not differ among the formulas (Table 3). Each patient reached plateau during both [¹³C]bicarbonate and [1-¹³C]phenylalanine tracer infusions. For 6 patients (group 2) who received 72 mg/kg per day cystine, the ¹³C enrichments in expired breath during the infusion of [1-¹³C]phenylalanine are shown in Fig 1.

View larger version (10K): [in this window] [in a new window]   FIGURE 1 Mean 13C enrichments in expired breath during enteral [1-13C]phenylalanine infusion in 6 infants (mean PMA: 35 weeks) who received 72 mg/kg per day cyst(e)ine (formula 4).

View larger version (10K): [in this window] [in a new window]   FIGURE 2 Relationship between the cystine intake and the individual fractional [1-13C]phenylalanine oxidation in VLBW infants with a mean GA of 28 weeks at 32 (A) and 35 (B) weeks’ PMA. A, The fractional oxidation of [1-13C]phenylalanine in VLBW infants at 32 weeks’ PMA (group 1). Five, 5, 4, 5, and 4 measurements were taken for cyst(e)ine intakes of 18, 36, 54, 72, and 109 mg/kg per day, respectively. B, The fractional oxidation of [1-13C]phenylalanine in VLBW infants at the PMA of 35 weeks (group 2). Six measurements were taken for each cyst(e)ine intake.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
In this study, application of the IAAO method showed that a cyst(e)ine intake of 18 mg/kg per day did not limit endogenous cyst(e)ine synthesis and that the cyst(e)ine requirement was <18 mg/kg per day. Hence, it is presumably safe to say that cyst(e)ine is not a conditionally essential amino acid in fully enterally fed VLBW preterm infants who are older than 32 weeks’ PMA.

The IAAO method is based on the assumption that essential amino acids participate between protein synthesis and oxidation.¹⁸ If 1 essential amino acid is deficient in the diet, then this will limit overall protein synthesis and all remaining essential amino acids are in excess and thus will be oxidized. Generous amounts of each essential amino acid must be applied, therefore, except for the 1 under study. We could not determine a decrease in fractional [1-¹³C]phenylalanine oxidation with increasing cystine intake. Thus, an amino acid other than cystine or other co-factors for protein synthesis could have been limiting and hence no change in oxidation rate could be detected; however, weight gain rates in both groups were not compromised, indicating no major limitations for protein synthesis. In addition, the amount of methionine provided by a daily intake of 168 mL/kg per day was 70 mg/kg per day, 20% in excess of the methionine requirement estimated on fetal accretion rate and obligatory losses (59 mg/kg per day).¹⁹ We propose, therefore, that methionine intake is not the limiting factor for endogenous cyst(e)ine synthesis. Another explanation for our finding might lie in the duration of the adaptive period to a different cystine intake. At 24 hours, it might have been too short; however, Zello and colleagues^18,20 did not see an effect of previous adaptation to various levels of the test amino acid and suggested 4 hours to be enough to establish a new steady state.

Over the years, various methods have been used to estimate individual amino acid requirements (eg, the nitrogen balance method, growth rate, plasma amino acid patterns, the factorial approach). In 1971, Snyderman et al²¹ reported amino acid requirements for neonates on the basis of nitrogen balance and weight gain rate, yet nitrogen balance usually overestimates nitrogen retention, and growth rate also depends on factors other than amino acid intake. Moreover, 7 to 10 days’ adaptation is needed to establish nitrogen equilibrium.²² Because current clinical practice does not accept maintaining neonates on either deficient or excess amino acid intakes for a minimum of 7 days, no such requirement studies have been reported in preterm infants since then.

Current dietary requirement estimates for humans so far are based on the factorial approach.¹¹ For preterm infants, this approach uses data on body composition of the fetus in utero of approximately the same age. These data are derived from body carcass analysis of stillborn preterm infants, some born >100 years ago.^23–25 In many cases, GA of the analyzed fetuses was not accurately known; therefore, not all data could serve as standard reference.²⁶ Because fetal accretion rate of cyst(e)ine is not available, the current cyst(e)ine requirement for preterm infants (66–95 mg/kg per day) is based on the minimum and maximum amounts of each amino acid present in the amounts of breast milk protein corresponding to the recommended minimum and maximum protein contents (g/503 kJ) of 3.0 and 4.3 g, respectively.²⁷ This estimation, however, does not take into account the influence of postnatal maturation.

Until now, cyst(e)ine was believed to be a conditionally essential amino acid in preterm infants. Snyderman²¹ was the first to show that cyst(e)ine might be required for preterm infants. She found lower rates of nitrogen retention and weight gain in 2- to 4-month-old infants who were born preterm and enterally fed a synthetic diet without cyst(e)ine. The cyst(e)ine intakes of 44 or 66 mg/kg per day did not restore nitrogen retention and weight gain to control values. She recommended a minimal intake of 85 mg cyst(e)ine/kg per day; however, because she did not provide methionine intake, this recommendation might be overestimated in view of the risk of inadequate methionine intake. Several in vitro studies then reported that the enzyme cystathionase was absent in fetal liver in these infants, in contrast to term infants.^4–6 Cystathionase activity seems to be a postnatal phenomenon, reaching mature levels at 3 months of age.^5,6 Cyst(e)ine requirement in these infants thus would depend on GA and should decrease with postnatal age. Obviously, we did not confirm this hypothesis.

Several in vivo studies demonstrated low plasma cyst(e)ine concentrations in preterm infants with or without cyst(e)ine supplementation, suggesting that limited cystathionase activity had impaired cyst(e)ine synthesis.^28–31 Conversely, Zlotkin et al³² did not find differences in nitrogen balance for parenterally fed term and preterm infants with or without cysteine supplementation. They showed slightly higher urinary 3-methylhistidine excretion in cysteine supplemented infants but failed to detect an evident relation. Although Malloy et al³³ showed that cysteine supplementation increased free cysteine plasma concentration and sulfur balance in preterm infants, it did not improve nitrogen retention. A study in parenterally fed VLBW infants who received an isotopically labeled glucose infusion showed incorporation of isotopic label in plasma cysteine and in hepatically derived apo B-100 cysteine.³⁴ In agreement with our results, the authors concluded that these infants were certainly capable of sufficient endogenous cyst(e)ine synthesis, which was directly related to birth weight.

Cyst(e)ine is an important sulfur-containing amino acid. Indispensable for protein synthesis, it also serves as a significant precursor for glutathione synthesis. If cyst(e)ine is a conditionally essential amino acid in preterm infants, then it could be the limiting factor for adequate glutathione production. Preterm birth and critical illness including oxygen supplementation might lead to higher glutathione requirement in these infants. If cyst(e)ine concentration is inadequate, then glutathione quantity might be insufficient to prevent oxidative stress; however, Shew et al³⁴ considered the minimum capacity for cysteine synthesis enough to counteract oxidative stress. For confirmation of this statement, future studies should investigate incorporation of quantities of cysteine in glutathione in both parenterally and enterally fed preterm infants.

A limitation of this study is that we did not include a formula providing for cyst(e)ine intake of <18 mg/kg per day. The formulas used were based on current nutrient recommendations for preterm formula.²⁷ At the onset of the study, we decided not to incorporate a study formula without cyst(e)ine for ethical reasons. Whether cyst(e)ine requirement is >0 but <18 mg/kg per day has to be determined with the aid of a cyst(e)ine-free formula yet with sufficient methionine.

	CONCLUSIONS

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From the findings of this study, we may safely conclude that the cyst(e)ine requirement for enterally fed VLBW preterm infants who are older than 32 weeks’ PMA is <18 mg/kg per day, provided that methionine intake is adequate.

	ACKNOWLEDGMENTS

 
This study was supported by Sophia Foundation for Medical Research (Rotterdam, Netherlands) grant 417 and the Nutricia Foundation (Wageningen, Netherlands).

We thank Ineke van Vliet for assistance in collecting the data, Ko Hagoort for critical review of the manuscript, and Paul Mulder for statistical help. We also thank the parents for giving consent for participation of their infants in this study.

	FOOTNOTES

 
Accepted Aug 1, 2007.

Address correspondence to Johannes B. van Goudoever, MD, PhD, Erasmus MC–Sophia Children's Hospital, Department of Pediatrics, Division of Neonatology, Dr Molewaterplein 60, 3015 GJ, Rotterdam, Netherlands. E-mail: j.vangoudoever{at}erasmusmc.nl

The authors have indicated they have no financial relationships relevant to this article to disclose.

^* Cyst(e)ine is used throughout to designate any undefined combination of cysteine and cystine.

	REFERENCES

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