Clinical Chemistry
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Clinical Chemistry 43: 2397-2402, 1997;
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(Clinical Chemistry. 1997;43:2397-2402.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Age-specific distribution of plasma amino acid concentrations in a healthy pediatric population

Nathalie Lepage, Nancy McDonald, Louis Dallaire and Marie Lamberta

Service de Génétique Médicale, Département de Pédiatrie, Hôpital Sainte-Justine and Université de Montréal, Montréal, QC, Canada.
a Address correspondence to this author at: Service de génétique médicale, Hôpital Ste-Justine, 3175 Côte Ste-Catherine, Montréal, QC, Canada H3T 1C5. Fax 514-345-4766; e-mail lamberma{at}ere.umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Reference values were determined for 23 plasma free amino acids from measurements done in 148 healthy children ranging from 0 to 18 years of age. Amino acid analysis was performed by ion-exchange chromatography. We propose a graphic form of presenting the age-specific distribution of plasma amino acid concentrations where the 10th, 50th, and 90th quantiles are illustrated. Although each amino acid possesses its own pattern of distribution, we can identify five different profiles. Nine amino acids (alanine, arginine, asparagine, methionine, ornithine, phenylalanine, proline, threonine, and tyrosine) demonstrate a decrease in their concentrations during the first year of life; their concentrations then tend to increase throughout childhood and adolescence. Nine others (cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, tryptophan, and valine) show a steady increase throughout infancy, childhood, and adolescence. Five amino acids (aspartic acid, citrulline, glutamic acid, serine, and taurine) do not follow these two common profiles. For the first time, quantile curves are produced to illustrate the age-dependent variation of amino acid concentrations from infancy to adulthood. This alternative way of presenting amino acid concentrations may facilitate the follow-up of patients with inborn errors of amino acid metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Amino acid analysis by automated ion-exchange chromatography is a procedure extensively used for diagnosis and follow-up of inborn errors of metabolism. It is a technique that provides both highly reproducible and sensitive results. Normative values for plasma amino acid concentrations have been reported in adults (1)(2)(3)(4), in infants ages 0–2 months (5) and 1–5 months (6), and in older children ages 6–18 years (1)(7)(8). These data are usually presented in the form of mean concentrations with individual SDs for the selected population (1)(2)(4)(6)(7)(8). However, no data describe the age-specific distribution of amino acid concentrations from infancy to adulthood.

In the present paper, we propose an alternative method of analyzing results that takes advantage of all amino acid values. The distribution of amino acid concentrations is presented graphically from birth to 18 years of age, showing the 10th, 50th, and 90th quantile curves. Thus, the amino acid values of a patient with a particular metabolic disease can be easily compared from infancy to adulthood with those of the control population of the appropriate age.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
clinical specimens
Blood samples were collected in heparinized tubes from children of both sexes ranging from ages 0 to 18 years after a 12-h fast (Table 1 ). They were recruited frompatients undergoing minor elective surgery at Hôpital Sainte-Justine. Those known to have metabolic, renal, hepatic, cardiac, or muscular diseases were excluded from the study. Those who had presented a serious acute disease 3 months or less before surgery or who showed failure to thrive were also excluded. The study was approved by the Hospital Ethics Committee, and informed consent was obtained from the parents and (or) patients. Because only six children under age 1 year were recruited, we included in our study the results from 28 children ages 0 to 12 months for whom we received blood samples and who, after careful revision of their medical charts, showed no evidence of metabolic, renal, hepatic, cardiac, or muscular disorders.


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  		Table 1. Distribution of subjects according to their age and sex.

All plasma samples were analyzed fresh or stored at -80 °C until assayed. Before analysis, 2-aminoethyl cysteine hydrochloride (Calbiochem) was added to plasma and used as an internal calibrator. Then, plasma samples were deproteinized according to Mondino et al. (9) with sulfosalicylic acid (ICN Biomedicals) and brought to pH 2.2 with LiOH (Baker Chemical). After filtration, samples were ready for analysis.

amino acid analysis
Analysis was performed with the Beckman System 7300 High Performance Amino Acid Analyzer, according to manufacturer's specifications. Briefly, this analyzer utilizes cation-exchange chromatography with a step buffer elution and with ninhydrin detection. Absorbance of amino acid–ninhydrin complexes is detected at 570 nm for primary amino groups and at 440 nm for proline and hydroxyproline. Amino acid concentration is automatically calculated on the basis of both the calibrators and the internal calibrator.

Buffers designed for the Beckman System 7300 Amino Acid Analyzer, the amino acid calibrators, and ninhydrin were all purchased from Beckman Instruments. Tryptophan, glutamine, asparagine (Calbiochem), and 5-aminolevulinic acid (Sigma) were added to complete the amino acid calibrator mix.

cvs
To establish the mean CV for the individual amino acid concentrations obtained with the Beckman System 7300 High Performance Amino Acid Analyzer, six blood samples were used, and each sample was analyzed on five different occasions. Conservation of the deproteinized sample was at -80 °C. The mean CVs were calculated as follows: For every amino acid, the CVs were calculated for each blood sample; the mean CV was then computed for each amino acid.

statistical methods
Parametric statistical analyses were performed with SAS statistical software [release 6.09 (1989), SAS Institute]. Nonparametric analyses were done with the Mathematica program [release 2.2 (1988), Wolfram Research]

First, a descriptive univariate analysis was undertaken to assess the quality of the data, and to identify missing data, lack of continuity, or nonnormal distribution characteristics. Hence t-tests were performed on all amino acid concentrations, verifying for sex difference. Significant statistical differences were obtained only for isoleucine (P = 0.01) and for lysine (P = 0.03) when data from the 148 control children were analyzed. However, no significant statistical difference for these two amino acids was observed (isoleucine, P = 0.4; lysine, P = 0.08) when data from the 114 children over age 1 year were analyzed. Therefore, sex was not taken into consideration for subsequent analyses.

Next, a regression analysis was performed on each amino acid concentration to determine its polynomial relation to age (10). Where the assumptions needed to apply regression were met, the 10th and 90th quantiles were calculated. Then, the two-sided 90% confidence bands for the 10th and 90th quantile curves were computed (10). Because they would have been almost superimposed on the 50th quantile curve, the two-sided confidence bands were not calculated for the median curve.

Finally, when the assumptions needed to apply regression failed (alanine, arginine, asparagine, aspartic acid, citrulline, cystine, glutamic acid, isoleucine, leucine, methionine, ornithine, phenylalanine, proline, serine, taurine, threonine, tyrosine, and valine), parametric methods became inappropriate and we turned to kernel estimators to calculate the 10th, 50th, and 90th quantiles. Again, the two-sided 90% confidence bands were obtained (11).

Both methods, as expected by definition, led to 10% of amino acid concentrations being higher than the 90th quantile value, and to 10% of concentrations being lower than the 10th quantile value.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
This study presents the age-specific distribution, as well as the 10th, 50th, and 90th quantile curves, for concentrations of 23 amino acids. To verify the precision of our technique, we determined mean CVs for each amino acid tested. As shown in Table 2 , these mean CVs range from 4.0% to 12.1%. These results indicate that our technique is satisfactory considering that methods with manual pipetting, such as the one used for amino acid analysis, usually yield CVs between 10% and 15% (12).


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  		Table 2. CVs for plasma amino acid concentrations.

Values of amino acids tend to vary up to 18 years of age (Figs. 1 to 5). The concentrations rangefrom undetectable to 1000 µmol/L and each amino acid possesses its own maximum values. For convenience of illustration, we have used four different scales with respect to the maximum concentration shown by each amino acid (scale 1: maximum <100 µmol/L; 2: <250 µmol/L; 3: <500 µmol/L; and 4: <1000 µmol/L). A unique pattern of distribution is demonstrated for each amino acid. However, we can identify common tendencies. As shown in Fig. 1a –i, concentrations of this group of amino acids tend to decrease during the first years of life, then increase steadily up to 18 years of age. Alanine (a), arginine (b), asparagine (c), methionine (d), ornithine (e), phenylalanine (f), proline (g), threonine (h), and tyrosine (i) belong to this first group. Two amino acids, serine and taurine, show an initial reduction in their concentrations followed by stable concentrations (Figs. 2 a and 2b). Aspartic acid (Fig. 3 a) and glutamic acid (Fig. 3b ) demonstrate decreasing values throughout the studied period. Figs. 4 and 5 display amino acids with no initial decrease in their concentrations. Citrulline (Fig. 4 ) shows a modest two-step increment: The first increase occurs from 0 to 3 years of age, and the second from 13 to 15 years. The last group includes nine amino acids showing steadily increasing concentrations throughout infancy, childhood, and adolescence: cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, tryptophan, and valine (Figs. 5a to 5i). Table 3 gives the numerical values for the 10th, 50th, and 90th quantiles of plasma amino acid concentrations at four different ages: 6 months, 2 years, 6 years, and 16 years.



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  		Figure 1. Age-specific distributions of plasma concentrations of alanine (a), arginine (b), asparagine (c), methionine (d), ornithine (e), phenylalanine (f), proline (g), threonine (h), and tyrosine (i) from 148 control children.

The upper curve represents the 90th quantile (solid line) with its 90% confidence bands (dotted lines). The lower curve represents the 10th quantile with its 90% confidence bands, and the line enclosed by the 10th and the 90th quantiles represents the 50th quantile of amino acid concentrations.



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  		Figure 2. Age-specific distributions of plasma concentrations of serine (a) and taurine (b) from control children ages 0 to 18 years.

Curves are as defined in Fig. 1Up .



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  		Figure 3. Age-specific distributions of plasma concentrations of aspartic acid (a) and glutamic acid (b) from control children ages 0 to 18 years.

Curves are as defined in Fig. 1Up .



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  		Figure 4. Age-specific distributions of plasma concentrations of citrulline from control children ages 0 to 18 years.

Curves are as defined in Fig. 1Up .



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  		Figure 5. Age-specific distributions of plasma concentrations of cystine (a), glutamine (b), glycine (c), histidine (d), isoleucine (e), leucine (f), lysine (g), tryptophan (h), and valine (i) from control children ages 0 to 18 years.

Curves are as defined in Fig. 1Up .


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  		Table 3. Plasma amino acid values (µmol/L) from control children of selected age.

The potential for comparison of these results with others is limited because previous reports did not take into account the 10th, 50th, and 90th quantile values, and amino acid reference values were usually presented as mean concentrations with their individual SDs for the selected population. Scott et al. (13) studied amino acid concentrations in infants at five different ages: 11 days, 21 days, 7 weeks, 11 weeks, and 15 weeks. The age distribution from 0 to 4 months that can be estimated from the study is consistent with our results except for alanine, asparagine, glycine, and lysine. In the Scott study, alanine and asparagine showed a continuous increase in concentrations, whereas we observed a decrease up to age 2 years for asparagine (Fig. 1cUp ) and up to age 3 years for alanine (Fig. 1aUp ). Scott et al. found a decrease in concentration for glycine and lysine (13), whereas we observed an increase in concentration (Fig. 5cUp and g). However, Janas et al. (5) measured amino acid concentrations in infants of three different ages: 2 weeks, 4 weeks, and 8 weeks. In comparison with our results, discordant profiles are only seen for arginine and cystine: arginine showed a slight increase, whereas we observed a decrease (Fig. 1dUp ), and cystine showed a slight decrease, whereas we observed an increase in concentration (Fig. 5aUp ). Finally, a third report compared amino acid concentrations at ages 8 and 16 years (7). The conclusions reached in that study were similar to ours except for phenylalanine, which showed a decrease in concentration where we found an increase (Fig. 1fUp ). Thus, our results compare adequately with previous reports.

Many analytes, including plasma amino acids, show an age-related distribution of their concentrations. This underlines the importance of using appropriate reference values when working with a pediatric population. Quantile curves that illustrate the age-dependent variation of the median (50th quantile) and the extremes (10th and 90th quantiles) of plasma amino acid concentrations should be a useful tool for the follow-up of patients with disorders of amino acid metabolism. Variations in amino acid concentrations according to age should be considered by clinical chemists studying amino acids in children.


   Acknowledgments
 
Statistical analysis was performed by Marie-Claude Guertin from the Départment de Mathématiques et Statistiques de l'Université de Montréal. These statistical services were made possible through special funding from the Fonds de Recherche en Santé du Québec (FRSQ) and the Interservice Club Council (Telethon of Stars) granted to the Group on Evaluative, Clinical, and Epidemiologic Research at Hôpital Sainte-Justine. We thank Louise Lortie and the anesthesiologists of Hôpital Sainte-Justine for their help in recruiting subjects.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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  8. Walker V, Mills GA. Quantitative methods for amino acid analysis in biological fluids. Ann Clin Biochem 1995;32:28-57.
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