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1 From the Research Institute, The Hospital for Sick Children, Toronto, Canada (JMT, MAH, RE, MR, VL, and PBP); the Department of Nutritional Sciences, University of Toronto, Toronto, Canada (PBP); and the Department of Agricultural, Food and Nutritional Science, University of Alberta, Alberta, Canada (ROB)
2 Presented in part at the annual meeting of the Federation of American Societies for Experimental Biology, San Diego, April 2005. 3 Supported by grant MT 10321 from the Canadian Institutes for Health Research. Mead Johnson Nutritionals (Canada) donated the protein-free powder for the experimental diets. 4 Address reprint requests to PB Pencharz, Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail: paul.pencharz{at}sickkids.on.ca.
ABSTRACT
Background: Current total sulfur amino acid (TSAA) requirements of children are based on a factorial estimate that involves several assumptions.
Objective: The objective was to determine the TSAA requirement (methionine alone) of healthy school-age children by measuring the appearance of 13CO2 (F13CO2) in breath after the oxidation of L-[1-13C]phenylalanine in response to graded methionine intakes.
Design: Six healthy school-age children randomly received each of 6 methionine intakes (0, 5, 10, 15, 25, and 35 mg · kg–1 · d–1) along with an amino acid mixture to give a final protein intake of 1.5 g · kg–1 · d–1 and an energy intake of 1.7 x resting energy expenditure. The diet was devoid of cysteine. The mean TSAA requirement was determined by applying a biphase linear regression crossover analysis on F13CO2 data, which identified a breakpoint at minimal F13CO2 in response to graded methionine intakes.
Results: The mean and population-safe (upper 95% CI) intakes of TSAA (as methionine) were determined to be 12.9 and 17.2 mg · kg–1 · d–1, respectively.
Conclusions: The current study suggests that children of this age group have a mean TSAA requirement similar to that of adults (12.6 mg · kg–1 · d–1). Therefore, it is valid to use a factorial approach, which assumes that maintenance requirements in childhood are similar to adult requirements, to estimate TSAA requirements in school-age children.
Key Words: Total sulfur amino acid • methionine • indicator amino acid oxidation • amino acid requirement • stable isotope • phenylalanine • children
INTRODUCTION
The sulfur amino acids methionine and cysteine are important in human nutrition; both are required for protein synthesis. Methionine also supplies the methyl groups to choline, creatine, both DNA and RNA intermediates (1–4), and the sulfur atom for cysteine synthesis (2–5). Similarly, cysteine is a precursor for the synthesis of several metabolites, such as glutathione, coenzyme A, taurine, and inorganic sulfate.
Currently there is only one published estimate of the total sulfur amino acid (TSAA) requirement in children, 27 mg · kg–1 · d–1 (6), and it was determined by using nitrogen balance. There are concerns about this estimate because it was derived over short adaptation periods that might not have allowed adaptation to take place in the body urea pool and, thus, may have biased the nitrogen balance–based estimate of TSAA (7). For this reason the recent Institute of Medicine/Food and Nutrition Board Dietary Reference Intake for Macronutrients chose to use a factorial approach to estimate dietary indispensable amino acid requirements in children (7). The factorial approach assumes that the maintenance requirement for children is the same as in adults (7), to which is added a growth component. In school-age children the growth component is small, 10% of the maintenance value (7).
It is widely recognized that the classic nitrogen balance technique has important limitations when it is used to determine amino acid requirements (7). Therefore, alternative carbon oxidation techniques have been developed (7, 8). The 2 most acceptable of these carbon oxidation techniques are indicator amino acid oxidation (IAAO) and 24-h IAAO and indicator amino acid balance (24-h IAAO/IAAB) (7, 9). Both techniques have been applied to determine TSAA in adults, and similar mean estimates were obtained: 12.6 mg · kg–1 · d–1 with IAAO (10) and 14 mg · kg–1 · d–1 with 24-h IAAO/IAAB (11). The application of the IAAO method to determine dietary indispensable amino acid requirements in children was made possible by the development of the minimally invasive IAAO model (12). We applied this model to determine total branched-chained amino acid (BCAA) requirements in school-age children (13) and compared the estimates obtained with estimates in adults studied with the use of the same technique (14). Similar estimates were obtained for total BCAA in children and adults, which constitute the first experimental evidence to support the assumption that maintenance amino acid requirements are the same in children as they are in adults. The present study was designed to determine the TSAA requirement in school-age children and to compare the estimates obtained with those previously reported by our group in adults with the use of the same experimental model (10).
SUBJECTS AND METHODS
Subjects
Six (5 boys, 1 girl) healthy school-age children were studied in the Clinical Investigation Unit at the Hospital for Sick Children (HSC), Toronto, Canada. Subject characteristics, body composition, and energy intakes are described in Table 1. None of the subjects had a history of recent weight loss or illness, and none were using any medication at the time of entry into the study. The Ethics Review Board of the HSC approved all procedures. Informed written consent was obtained from a parent or guardian and the assent of the participating children was also obtained. The parent or guardian of each participating subject received financial compensation for their inconvenience.
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Table 1. Characteristics and energy intakes of 6 preadolescent children1
Experimental design and tracer protocol
The study design was based on the minimally invasive IAAO model (12) that was recently used in healthy adults (10, 14, 15) and children (13). Two days before the study day, the subjects consumed a maintenance diet that supplied 1.5 g · kg–1 · d–1 protein and 1.7 x REE. On the study day, after a 12-h fast, the subjects randomly received 1 of 6 dietary intakes of methionine (0, 5, 10, 15, 25, and 35 mg · kg–1 · d–1) along with an L-amino acid mixture to give a final protein intake of 1.5 g · kg–1 · d–1 and an energy intake of 1.7 x REE. The protein intake was devoid of cysteine. The study day diet was consumed as 8 isonitrogenous and isocaloric hourly meals, each meal representing one-twelfth of the subject’s total daily protein and energy requirements. The subjects were not allowed to eat or drink anything else except water.
The tracer protocol was started with the fifth meal to measure phenylalanine kinetics with the use of L-[1-13C]phenylalanine (99 atom% excess; Cambridge Isotope Laboratories, Woburn, MA). Oral priming doses of 0.176 mg/kg NaH13CO3 (99 atom% excess; Cambridge Isotope laboratories) and 1.09 mg/kg L-[1-13C]phenylalanine were given with the fifth hourly meal. In addition, an hourly oral dosing protocol of L-[1-13C]phenylalanine (1.958 mg · kg–1 · d–1) was commenced simultaneously (with the fifth meal) and continued for the remaining 3 h of the study. The amount of L-[1-13C]phenylalanine given during the study day was subtracted from the dietary provision of phenylalanine such that the total intake of phenylalanine was 25.13 mg · kg–1 · d–1 and that of tyrosine was 61.10 mg · kg–1 · d–1 (to ensure an excess of tyrosine). Study day periods were separated by 1 wk, and all subjects completed all 6 studies within a 2-mo period.
Study diets
The maintenance diet (energy: REE x 1.7; protein: 1.5 g · kg–1 · d–1) for the 2 d before the study day was prescribed by the study dietitian on the basis of the participant’s 3-d food records. REE was measured by open-circuit indirect calorimetry (2900 Computerized Energy measurement System; Sensormedics, Yorba Linda, CA). For the entire duration of the 6 studies, the subjects also consumed a daily supplement of B vitamins (Replavite; Landmark Medical Systems Inc, Unionville, Canada) that contained 1.0 mg folic acid, 10 mg vitamin B-6, and 6 µg vitamin B-12 to ensure adequate folate and B-vitamin intakes as cofactors for the metabolism of methionine and homocysteine (10).
As described previously (10), the study day diet consisted of a protein-free liquid formula (flavored with soft drink crystals), corn oil, the crystalline amino acid mixture (based on the amino acid composition of egg protein), and protein-free cookies. The energy and protein intakes on the study day were the same as in the maintenance diet. The macronutrient composition of the diet (expressed as a percentage of dietary energy) was 53% for carbohydrate, 37% for fat, and 10% for protein.
Sample collection and analysis
Breath and urine samples were collected as described previously (10). Breath samples were stored at room temperature until analyzed. Urine samples were stored at –20°C. During each study day, open-circuit indirect calorimetry (2900 Computerized Energy Measurement System; Sensormedics, Yorba Linda, CA) was performed for 20 min to measure the rate of carbon dioxide production (·VCO2).
Enrichment of 13C in breath was analyzed by continuous-flow isotope ratio mass spectrometry (20/20 Isotope Analyzer; PDZ Europa Ltd, Cheshire, United Kingdom). Enrichments were expressed as atom percent excess (APE) compared with a reference standard of compressed carbon dioxide. L-[1-13C]Phenylalanine enrichment in urine samples was analyzed with a triple quadrupole mass analyzer API 4000 (Applied Biosystems/MDS SCIEX, Concord, Canada) coupled to an 1100 HPLC system (Agilent, Mississauga, Canada). A 62.5-µL aliquot of urine sample was deproteinized with 200 µL methanol and centrifuged at 7000 x g for 5 min. The supernatant fluid was freeze-dried and then reconstituted in 1 mL water containing 0.1% formic acid. The samples were then injected into the 1100 HPLC system (Agilent, Mississauga, Canada). The individual amino acids were separated by Waters (Milford, MA) Xterra MS C18 3.5 µm, 2.1 x 150 mm column. The amino acids were eluted with a binary LC gradient (40–60% aqueous-acetonitrile containing 0.1% formic acid, 3 min isocratic). L-[1-13C]Phenylalanine enrichment was then analyzed by triple quadrupole mass analyzer API 4000 (Applied Biosystems/MDS SCIEX) operated in positive ionization mode. All aspects of system operation and data acquisition were controlled by ANALYST NT version 1.2 software (Applied Biosystems/MDS SCIEX). Selected-ion chromatograms were obtained by monitoring mass-to-charge ratios of product ions of 165 and 166 for [1-13C]phenylalanine corresponding to the unenriched (M) and enriched (M+1) peaks, respectively. The areas under the peaks were integrated by using ANALYST NT software (version 1.2). Isotopic enrichment was expressed as molecule % excess and calculated from peak area ratios at isotopic steady state at plateau and baseline.
Tracer kinetics
Phenylalanine kinetics was calculated according to the stochastic model of Matthews et al (16), as previously used by Zello et al (17). Isotopic steady state in the tracer enrichment at baseline and plateau was represented by unchanging values of L-[1-13C]phenylalanine in urine and 13CO2 in breath. At plateau, the APE was calculated by subtracting the mean breath 13CO2 enrichments of the 3 baseline samples from the 4 plateau samples.
Phenylalanine flux (µmol · kg–1 · h–1) was calculated from the dilution of orally administered L-[1-13C]phenylalanine into the metabolic pool (at steady state) by using enrichments of L-[1-13C]phenylalanine in urine (16, 17). The rate of appearance of 13CO2 in breath (F13CO2, µmol · kg–1 · h–1) after the oxidation of ingested L-[1-13C]phenylalanine was calculated according to the model of Matthews et al (16) with the use of a factor of 0.82 to account for the retention of 13CO2 in the bicarbonate pool of the body in the fed state (18). The rate of L-phenylalanine oxidation (µmol · kg–1 · h–1) was calculated from F13CO2 and urinary L-[1-13C]phenylalanine enrichment (16, 17).
Statistical analysis
Data were analyzed by using PROC MIXED (SAS version 8.2; SAS Institute Inc, Cary, NC). Repeated-measures analysis of variance was performed on primary and derived variables to assess the effects of methionine intake, of subject, and of interactions. Tukey’s test was used for post hoc analysis of variance results. Results are expressed as means ± SDs. Statistical significance was assumed at the 5% level of significance (P < 0.05).
The TSAA requirement (breakpoint) was determined by applying a biphase linear regression crossover model on F13CO2 data (17). This model selects for the minimum residual SE in a stepwise partitioning of data points between 2 regression lines. The safe intake (upper 95% CI, equivalent to the Recommended Dietary Allowance) was calculated by using Fieller’s theorem (19).
Because the study design included repeated graded levels within a subject (6 levels per subject), we reasoned that the 6 subjects providing a total of 36 data points should be adequate to estimate the mean and population safe requirements of TSAA in children by applying a 2-phase linear regression crossover analysis on the data as determined previously in adults (10).
RESULTS
Subject characteristics
Six healthy school-age children (9.1 ± 2.2 y) completed the study. The anthropometric measurements of the subjects (Table 1) were within the normal range for age (20). Similarly, energy and protein intakes of the subjects were adequate. According to self- and parent-rated Tanner staging, all subjects were in early puberty except for 1 boy, who was in midpuberty (21).
Phenylalanine flux and oxidation
Phenylalanine flux was not affected by TSAA intake (Table 2), which is evidence that the precursor pool for indicator oxidation did not change in size in response to the test amino acid (ie, TSAA). Phenylalanine oxidation declined in response to graded increases in TSAA intakes of up to 10 mg · kg–1 · d–1; however, the changes were not statistically significant by analysis of variance.
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Table 2. Phenylalanine flux and oxidation at 6 methionine intakes (n = 36 observations) in 6 school-age children1
L-[1-13C]Phenylalanine oxidation
Conversely, the effect of dietary methionine intake on the oxidation of L-[1-13C]phenylalanine measured as the rate of label appearance in breath (F13CO2) changed significantly in response to graded intakes of TSAA (Figure 1, representing individual responses). There were no sex effects on the response of F13CO2 to graded intakes of TSAA. At a methionine intake of 0 mg · kg–1 · d–1, the oxidation of L-[1-13C]phenylalanine was highest. As the methionine intake increased, the oxidation of L-[1-13C]phenylalanine decreased steadily (representing increased incorporation of label into protein synthesis) until a point was reached (at a methionine intake between 10 and 15 mg/kg), after which there was no further decrease in L-[1-13C]phenylalanine oxidation with the increase in methionine intake (representing no further increase in the incorporation of label into protein synthesis). Application of biphase linear regression crossover analysis on the F13CO2 data resulted in the identification of a breakpoint (mean TSAA requirement) at 12.9 mg · kg–1 · d–1 and a safe level of intake (the upper 95% CI, equivalent to the Recommended Dietary Allowance) at 17.2 mg · kg–1 · d–1 (Figure 2).
FIGURE 1.. Relation between the rate of breath 13CO2 excretion and methionine intake in 6 school-age children (n = 36 observations). The filled circle represents a female subject.
FIGURE 2.. Mean (±SD) breath 13CO2 excretion after the oxidation of orally administered L-[1-13C]phenylalanine over methionine intakes of 0, 5, 10, 15, 25, and 35 mg · kg–1 · d–1 in 6 school-age children (n = 36 observations). The dashed arrow indicates the mean total sulfur amino acid requirement of 12.9 mg · kg–1 · d–1.
DISCUSSION
This was the first report of the use of the IAAO technique to determine the TSAA requirement in healthy children. The major finding of the present study was that the mean TSAA requirement of healthy children was 12.9 mg · kg–1 · d–1. This value is similar to that determined in adults (12.6 mg · kg–1 · d–1) by our group using a similar protocol (10) and by others (14.0 mg · kg–1 · d–1) using the 24-h IAAO/IAAB method (11). However, our value in school-age children is 52% lower than that determined in healthy children (27 mg · kg–1 · d–1) with the nitrogen balance method (6). The similarity between the adult IAAO method, the 24 h-IAAO/IAAB method, and the current school-age IAAO estimate suggests that the previous nitrogen balance–based TSAA requirement was an overestimate. The limitations of nitrogen balance studies are well recognized (7, 22). Prolonged adaptation periods (at the level of the test amino acid) are particularly impractical in studies of children. The short adaptation periods, the few numbers of subjects studied, and the extremely high nitrogen retention reported at adequate methionine intakes all raise concerns regarding the validity of this earlier study undertaken by Nakagawa et al (6). The authors of the recent Dietary Reference Intake for macronutrients chose not to include this study when determining the estimated average requirement (EAR) for TSAA in children. Therefore, the current EAR for indispensable amino acids in children (18 mo through 18 y of age) is based on factorial prediction that includes a maintenance component derived from studies conducted in adults and a growth component derived from studies in children.
The approach used to calculate the factorial estimates of indispensable amino acid requirements included the addition of an average estimate of amino acid requirements in adults to a growth component, which was based on estimates of protein deposition from body-composition studies at different ages and then converted to a dietary requirement for growth by dividing by a factor of 0.58 for efficiency of dietary protein utilization (7, 23). By this approach in boys aged 9–13 y, the maintenance TSAA requirement for adults (15 mg · kg–1 · d–1) was added to the calculated product of average protein deposition (49 mg · kg–1 · d–1) by TSAA composition in whole-body protein (0.035%) divided by 0.58 to give an EAR for TSAA of 18 mg · kg–1 · d–1 (7). This value is also significantly greater than the mean requirement calculated in the current study.
The maintenance component used in calculating the EAR for indispensable amino acids was based on direct measurements in adults of mean requirements for each amino acid. The adult values were considered to represent maintenance values for children given that the maintenance nitrogen requirement in childhood (110 mg · kg–1 · d–1 based on nitrogen balance data) does not change significantly with age and is not significantly different from that of adults (105 mg · kg–1 · d–1) (7). In calculating the adult EAR for TSAA, the average of 20 mg · kg–1 · d–1 (based on a reanalysis of nitrogen balance data; 24), 13 mg · kg–1 · d–1 (based on methionine balance at the reported level; 25), and 12.6 mg · kg–1 · d–1 (based on the IAAO method; 10) was used to derive the value of 15 mg · kg–1 · d–1. This value is biased upward by using a reanalysis of the nitrogen balance data of Reynolds et al (24). We previously reported that a reanalysis of the nitrogen balance data of Rose et al (26) gives a mean requirement of 13.2 mg · kg–1 · d–1 (10). This value is more consistent with the results of methionine balance (25), the IAAO study in adults (10) and reported here, and the IAAO/IAAB study of Kurpad et al (11), which was reported in 2003—a year after the Institute of Medicine Report. Considering the limitations of the nitrogen balance technique and the various assumptions that may be applied when reanalyzing nitrogen balance data, it might be argued that the adult EAR should be based on the average of those studies, other than the nitrogen balance studies, which are in fact more consistent and provided an average value of 13.2 mg · kg–1 · d–1.
Maintenance requirements of adults and children are similar (on the basis of weight) and represent the predominant part of TSAA requirements in children. In the view of the Institute of Medicine, TSAA requirements in children should only differ by growth requirement compared with adults, and the growth requirement for TSAA, in fact, represents only a modest percentage (10%) of the TSAA requirement (7). If the growth requirement for children is added to the maintenance requirement for adults of 12.6 mg · kg–1 · d–1, a TSAA requirement of 13.9 mg · kg–1 · d–1 is expected in children. This is slightly higher than the value determined in the current study (12.9 mg · kg–1 · d–1) and suggests that the requirement determined by the current study primarily represents the maintenance requirement. Because children of this age group are growing slowly and the duration of the current study protocol was only a few hours, it is probable that this approach was unlikely to be sensitive enough to detect growth requirements. We previously reported that the BCAA requirement of children in a similar age group was 147 mg · kg–1 · d–1 compared with the requirement of 144 mg · kg–1 · d–1 we had determined earlier for adults (13, 14). These results are not significantly different from those for adults and are also likely to represent only the maintenance requirements for both age groups. Only long-term studies should be able to account for the growth requirement; in this regard it may be expected that values derived from nitrogen balance studies, or perhaps from 24-h IAAO/IAAB studies in children, would be higher (in the range of that predicted by factorial estimation or 10% above maintenance) than those derived with the use of the IAAO method.
In the current study, the mean and upper safe level of TSAA (as methionine) was estimated to be 12.9 and 17.2 mg · kg–1 · d–1. The mean requirement is similar to that determined in adults (10, 11). Therefore, this finding supports the use of the factorial approach, which assumes that the maintenance requirements in childhood are similar to those in adulthood and thus can be used to estimate the TSAA requirement in school-age children (7). However, the results of the current study and other studies in adults (10, 11, 25) suggest that the maintenance component of the proposed EAR for TSAA may also be biased because it is based on nitrogen balance data. It is suggested that the EAR for TSAA in school-age children be revised to take into account the results of direct measurements undertaken in children in the current study.
ACKNOWLEDGMENTS
We thank the subjects who participated in the study and Linda Chow in the Department of Nutrition and Food Services (HSC) for preparing the protein-free cookies. JMT and MAH were involved in the study design, data collection, sample and data analysis, and manuscript writing. RE was involved in the data collection and sample and data analysis. MR was involved in the sample and data analysis. VR was involved in subject recruitment and looked after the subjects and data analysis. ROB and PBP were involved in the study design, data analysis, and manuscript writing. The authors had no conflict of interest.
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