Gardi J. Voortman

Effects of Early Amino Acid Administration on Leucine and Glucose Kinetics in Premature Infants

We previously showed that, in prematurely born infants, an anabolic state without metabolic acidosis can be achieved upon intravenous amino acid (AA) administration in the immediate postnatal phase, despite a low energy intake. We hypothesized that the anabolic state resulted from an increased protein synthesis and not a decreased proteolysis. Furthermore, we hypothesized that the energy needed for the higher protein synthesis rate would be derived from an increased glucose oxidation. To test our hypotheses, 32 ventilated premature infants (<1500 g) received intravenously either solely glucose or glucose and 2.4 g AA/kg/d immediately postnatally. On postnatal d 2, each group received primed continuous infusions of either [1-13C]leucine or [U-13C6]glucose. 13CO2 enrichments in expiratory air and plasma [1-13C]α-KICA (as an intracellular leucine precursor) and [U-13C6]glucose enrichments were measured by mass spectrometry techniques. The AA administration resulted in an increased incorporation of leucine into body protein and a higher leucine oxidation rate, whereas leucine release from proteolysis was not affected. Glucose oxidation rate did not increase upon AA administration. In conclusion, the anabolic state resulting from AA administration in the immediate postnatal period resulted from increased protein synthesis and not decreased proteolysis. The energy needed for the additional protein synthesis was not derived from an increased glucose oxidation.

Lysine requirement of the enterally fed term infant in the first month of life

BACKGROUND Infant nutrition has a major impact on child growth and functional development. Low and high intakes of protein or amino acids could have a detrimental effect. OBJECTIVE The objective of the study was to determine the lysine requirement of enterally fed term neonates by using the indicator amino acid oxidation (IAAO) method. L-[1-(13)C]phenylalanine was used as an indicator amino acid. DESIGN Twenty-one neonates were randomly assigned to lysine intakes that ranged from 15 to 240 mg · kg(-1) · d(-1). Breath, urine, and blood samples were collected at baseline and during the plateau. The mean lysine requirement was determined by using biphasic linear regression crossover analysis on the fraction of (13)CO(2) recovery from L-[1-(13)C]phenylalanine oxidation (F(13)CO(2)) and phenylalanine oxidation rates calculated from the L-[1-(13)C]phenylalanine enrichment of urine and plasma. RESULTS The mean (±SD) phenylalanine flux calculated from urine and plasma L-[1-(13)C]phenylalanine enrichment data were 88.3 ± 6.9 and 84.5 ± 7.4 μmol · kg(-1) · h(-1), respectively. Graded intakes of lysine had no effect on phenylalanine fluxes. The mean lysine requirement determined by F(13)CO(2) was 130 mg · kg(-1) · d(-1) (upper and lower CIs: 183.7 and 76.3 mg · kg(-1) · d(-1), respectively). The mean requirement was identical to the requirement determined by using phenylalanine oxidation rates in urine and plasma. CONCLUSIONS The mean lysine requirement of enterally fed term neonates was determined by using F(13)CO(2) and phenylalanine oxidation rates calculated from the L-[1-(13)C]phenylalanine enrichment of urine and plasma. These methods yielded a similar result of 130 mg lysine · kg(-1) · d(-1). This study demonstrates that sampling of (13)CO(2) in expired air is sufficient to estimate the lysine requirement by using the IAAO method in infants. This trial was registered at www.trialregister.nl as NTR1610.

Cyst(e)ine requirements in enterally fed very low birth weight preterm infants

OBJECTIVE. Optimal nutrition is of utmost importance for the preterm infants 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.

Methionine requirement of the enterally fed term infant in the first month of life in the presence of cysteine

BACKGROUND The essential amino acid methionine can be used for protein synthesis but also serves as a precursor for homocysteine and cysteine. OBJECTIVE The objective of this study was to determine the minimal obligatory methionine requirement of infants in the presence of excess cysteine (91 mg ⋅ kg(-1) ⋅ d(-1)) by using the indicator amino acid oxidation (IAAO) method with l-[1-(13)C]phenylalanine as the indicator. DESIGN Fully enterally fed term infants <1 mo of age were randomly assigned to methionine intakes that ranged from 3 to 59 mg ⋅ kg(-1) ⋅ d(-1) as part of an elemental formula. After 1 d of adaptation to the test diet, [(13)C]bicarbonate and l-[1-(13)C]phenylalanine tracers were given enterally. Breath samples were collected at baseline and during isotopic plateaus. The mean methionine requirement was determined by using biphasic linear regression crossover analysis on the fraction of (13)CO(2) recovery from l-[1-(13)C]phenylalanine oxidation (F(13)CO(2)). Data are presented as means ± SDs. RESULTS Thirty-three neonates (gestational age: 39 ± 1 wk) were studied at 13 ± 6 d. With increasing methionine intakes, F(13)CO(2) decreased until a methionine intake of 38 mg ⋅ kg(-1) ⋅ d(-1); additional increases in methionine intake did not affect F(13)CO(2). The mean methionine requirement was determined at 38 mg ⋅ kg(-1) ⋅ d(-1), and the upper and lower CIs were 48 and 27 mg ⋅ kg(-1) ⋅ d(-11), respectively (P < 0.0001, r(2) = 0.59). CONCLUSIONS Although the current recommended methionine intake of 28 mg ⋅ kg(-1) ⋅ d(-1) is within the CIs of our study, the estimated mean requirement is substantially higher. However, most of the infant formulas provide a methionine intake of 49-80 mg ⋅ kg(-1) ⋅ d(-1), which is above the upper CI of our study. This trial was registered at www.trialregister.nl as NTR1610.

Use of [13C]bicarbonate for metabolic studies in preterm infants : Intragastric versus intravenous administration

The metabolic fate of substrates in humans can be examined by the use of stable isotopes, one of which, [13C]bicarbonate, may serve to estimate CO2 production rate. In view of minimizing the burden of metabolic studies for preterm infants, the authors determined whether intragastric and intravenous infusions of [13C]bicarbonate would achieve the same 13CO2 enrichment in expired air during steady state. A second aim of this study was to determine the minimum time required to reach steady state during intragastric infusion. Ten preterm infants received a primed continuous [13C]bicarbonate infusion intragastrically, followed by an intravenous infusion the next day. Breath samples were obtained every 30 min by the direct sampling method. 13CO2 isotopic enrichment, expressed as atom percent excess, was measured by isotopic ratio mass spectrometry. Two-tailed t tests were used to detect statistically significant differences between the infusion routes. The isotopic enrichment at plateau did not differ between intragastric and intravenous infusion. A steady state of 13CO2 enrichment was achieved after 60 min of intravenous infusion and after 120 min of intragastric infusion. In conclusion, intragastric infusion of [13C]bicarbonate may serve to estimate the whole-body CO2 production rate in preterm infants. To reach 13CO2 steady state, a minimum of 120 min of bicarbonate administration is required.

Simultaneous analysis of 13C-glutathione as its dimeric form GSSG and its precursor [1-13C]glycine using liquid chromatography/isotope ratio mass spectrometry

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine (13)C-glutathione as its dimeric form (GSSG) and its precursor [1-(13)C]glycine in a small volume of erythrocytes in one single analysis. After having transformed (13)C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the (13)C-isotopic enrichment of GSSG and glycine in 150 microL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of micromol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 micromol/mL. The (13)C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3 per thousand) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants.

Branched-chain amino acid requirements for enterally fed term neonates in the first month of life

BACKGROUND Knowledge of essential amino acid requirements in infants is important because excessive intake of protein can lead to increased long-term morbidity such as obesity. A deficient intake may lead to suboptimal growth and impaired neurodevelopment. The current recommended branched-chain amino acid requirements in infants aged 0-1 mo are based on the amino acid content of human milk. OBJECTIVE We quantified the requirements for isoleucine, leucine, and valine for term neonates by using the indicator amino acid oxidation method with [1-(13)C]phenylalanine as the indicator. DESIGN Fully enterally fed term infants received randomly graded amounts of isoleucine (5-216 mg · kg(-1) · d(-1)), leucine (5-370 mg · kg(-1) · d(-1)), or valine (5-236 mg · kg(-1) · d(-1)) as part of an elemental formula. Data are expressed as means ± SDs. RESULTS Eighty-three Asian, term neonates (mean ± SD birth weight: 3.3 ± 0.4 kg; gestational age: 39.4 ± 1.3 wk) were studied at a postnatal age of 13 ± 5 d. Mean requirements for isoleucine, leucine, and valine (measured in boys only) were 105 mg · kg(-1) · d(-1) (r(2) = 0.61, P < 0.001), 140 mg · kg(-1) · d(-1) (r(2) = 0.26, P < 0.01), and 110 mg · kg(-1) · d(-1) (r(2) = 0.35, P = 0.001), respectively. CONCLUSIONS Current human milk-based recommendations for isoleucine and valine in term infants aged 0-1 mo are correct. However, the current recommendation for leucine (166 mg · kg(-1) · d(-1)) is higher than the mean requirement of 140 mg · kg(-1) · d(-1) that we determined in this study. This trial was registered at www.trialregister.nl as NTR1610.

New insights into the methodological issues of the indicator amino acid oxidation method in preterm neonates

Background:We determined the effect of adaptation to the study diet on oxidation of the indicator amino acid and the required tracer washout time in preterms.Methods:Subjects received a study diet for 6 d that entailed a 50% reduction in leucine. Tracer studies using enterally infused [13C]bicarbonate and [1-13C]phenylalanine were performed on days 1, 2, 4, and 6. Breath samples containing 13CO2 were collected during steady state and measured by infrared spectrometric analysis, and the fraction of 13CO2 recovery from 13C oxidation (F13CO2) was calculated.Results:Preterm infants (n = 11, birth weight 1.9 ± 0.1 kg, gestational age 32.6 ± 1.5 wk) received 166 mg/kg/d of leucine. Baseline enrichment changed significantly at day 1 of the study diet. F13CO2 did not change significantly between days 2 and 4 but was significantly lower at day 6. The tracer washout time was determined to be 7.5 h using a biphasic regression analysis.Conclusion:One day of adaptation to a new diet is necessary to adapt to the 13C enrichment of the study formula before starting infant requirement studies. Adaptation for a period of 5 d results in a protein-sparing response. The minimal time between two studies within the same subject is 7.5 h.

Tryptophan Requirement of the Enterally Fed Term Infant in the First Month of Life

Objectives: Tryptophan not only is an amino acid essential to protein synthesis but also serves as a precursor in 2 important metabolic pathways: the serotonin and the kynurenine pathways. Tryptophan is related to sleeping patterns. The objective of the present study was to determine the tryptophan requirement of term infants using the indicator amino acid oxidation (IAAO) method with L-[1-13C]phenylalanine as the indicator. Methods: Enterally fed infants were randomly assigned to tryptophan intakes ranging from 0.5 to 73 mg · kg−1 · day−1 as part of an elemental diet. After 1-day adaptation to the test diet, [13C]bicarbonate and L-[1-13C]phenylalanine tracers were given enterally. Breath samples were collected at baseline and during isotopic plateaus. The mean tryptophan requirement was determined by using the biphasic linear regression crossover analysis on the fraction of 13CO2 recovery from L-[1-13C]phenylalanine oxidation (F13CO2). Data are presented as mean ± standard deviation. Results: A total of 30 term neonates (gestational age 39 ± 1 weeks) were studied at 9 ± 4 days. F13CO2 decreased until a tryptophan intake of 15 mg · kg−1 · day−1; additional increases in tryptophan intake did not affect F13CO2. Mean requirement was determined to be 15 mg · kg−1 · day−1. Conclusions: The mean tryptophan requirement for elemental formula-fed term infants is 15 mg · kg−1 · day−1. This requirement is lower than the present recommended intake of 29 mg · kg−1 · day−1, which is based on the average intake of a breastfed infant.

Phenylalanine requirements of enterally fed term and preterm neonates

BACKGROUND Phenylalanine, which is an essential aromatic amino acid, is either used for protein synthesis or irreversibly hydroxylated to tyrosine. The provision of optimal amounts of dietary phenylalanine is not only important for growth and development but might also influence catecholamine synthesis and release rates. The current recommended aromatic amino acid requirement for infants aged 0-6 mo is based on the amino acid content of human milk. OBJECTIVE We quantified the requirements for phenylalanine in the presence of excess tyrosine (166 or 177 mg/kg per day for term and preterm infants, respectively) for term and preterm neonates by using the indicator amino acid oxidation method with l-[1-(13)C]lysine 2HCl as an indicator. Hence, we determined the minimum obligatory phenylalanine requirement. DESIGN Fully enterally fed term and preterm infants received randomly graded amounts of phenylalanine (5-177 mg/kg per day) as part of an elemental formula. Data are expressed as means ± SDs. RESULTS Twenty term (birth weight: 3.19 ± 0.34 kg; gestational age: 38.9 ± 1 wk) and 16 preterm (birth weight: 1.75 ± 0.17 kg; gestational age: 32.5 ± 0.6 wk) Asian infants participated at a postnatal age of 17 ± 8 d. In total, 44 studies were performed. The minimum obligatory phenylalanine requirement was 58 mg/kg per day (95% CI: 38-78 mg/kg per day) and 80 mg/kg per day (95% CI: 40-119 mg/kg per day) for term and preterm infants, respectively. CONCLUSION The determined mean phenylalanine-requirement estimates are lower than the contents of term and preterm formulas currently on the market. This trial was registered at www.trialregister.nl as NTR1610.