Oral glutamine and amino acid supplementation inhibit whole-body protein degradation in children with Duchenne muscular dystrophy

¹ From the Centre d'Investigation Clinique 9202 INSERM, Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Paris, France (EM, CE-DV, CD, and RH); the LAPHAP EA3813, Université de Poitiers, Poitiers, France (EM, RH); the Clinique de Pédiatrie, CHR&U de Lille, Hôpital Jeanne de Flandre, Lille, France (FG); the Biochimie, Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Paris, France (OR); the Pharmacie, Assistance Publique-Hôpitaux de Paris, Hôpital Jean-Verdier, Paris, France(J-EF); the Service de Neuropédiatrie, CHR&U de Lille, Hôpital Roger-Salengro, Lille, France (J-MC); and the Clinical Research Center, CHU de Poitiers, Poitiers, France (JG)

² Supported by the Assistance Publique-Hôpitaux de Paris and a grant from the Institut National de la Santé et de la Recherche Médicale #33104700 (RAF 01005) (to RH) and by the Association Française Contre les Myopathies (to RH). EM is supported by Le Prix de Nutrition de la Fédération Association Nationale pour les Traitements à Domicile, les Innovations et la Recherche, awarded by La Société Francophone de Nutrition Entérale et Parentérale and Les Fonds de la Recherche en Santé Québec PhD Fellowship.

³ Reprints not available. Address correspondence to R Hankard, Pédiatrie Multidisciplinaire-Nutrition de l'Enfant, Centre Hospitalier Universitaire de Poitiers, 2 rue de la Milétrie, 86021 Poitiers Cedex, France. E-mail: r.hankard{at}chu-poitiers.fr.

ABSTRACT  
Background: Glutamine has been shown to acutely decrease whole-body protein degradation in Duchenne muscular dystrophy (DMD).

Objective: To improve nutritional support in DMD, we tested whether oral supplementation with glutamine for 10 d decreased whole-body protein degradation significantly more than did an isonitrogenous amino acid control mixture.

Design: Twenty-six boys with DMD were included in this randomized, double-blind parallel study; they received an oral supplement of either glutamine (0.5 g · kg^–1 · d^–1) or an isonitrogenous, nonspecific amino acid mixture (0.8 g · kg^–1 · d^–1) for 10 d. The subjects in each group were not clinically different at entry. Leucine and glutamine metabolisms were estimated in the postabsorptive state by using a primed continuous intravenous infusion of [1-¹³C]leucine and [2-¹⁵N]glutamine before and 10 d after supplementation.

Results: A significant effect of time was observed on estimates of whole-body protein degradation. A significant (P < 0.05) decrease in the rate of leucine appearance (an index of whole-body protein degradation) was observed after both glutamine and isonitrogenous amino acid supplementation [ Conclusion: Oral glutamine or amino acid supplementation over 10 d equally inhibits whole-body protein degradation in DMD.

Key Words: Duchenne muscular dystrophy • children • randomized controlled clinical trial • supplement • oral administration • stable isotopes • protein metabolism • glutamine • leucine • amino acids

INTRODUCTION  
Nutritional support was shown to improve outcome in several clinical conditions (1). Certain nutrients, such as glutamine, have distinct properties that are separate from their role of providing calories or nitrogen (2, 3), and they are called "pharmaconutrients." Glutamine has a protein-sparing effect in cell cultures, animal models, and humans (4). This effect may be useful in catabolic situations, when cellular glutamine concentrations decline (5, 6).

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle wasting and a reduced functional capacity. Protein metabolism in DMD, which has been studied by using stable isotope tracers and nitrogen balance, suggests that the cause of muscle wasting is a reduction in muscle protein synthesis or an increase in protein degradation (7-9). Furthermore, muscle wasting in DMD is associated with a decrease in glutamine turnover and a more negative whole-body leucine balance (10). Because glutamine is mainly produced by the muscle, and muscle mass is severely reduced in DMD, the need for glutamine may be increased in persons who have this disease. Moreover, in DMD, as in other protein-wasting situations, the intramuscular glutamine concentration is low (11). Thus, in DMD, as in situations of catabolic stress, glutamine may be considered a conditionally essential amino acid (10).

In a previous study using stable isotope methodology, we showed that acute (5 h) oral glutamine decreased whole-body protein degradation in DMD compared with placebo (12). The objectives of the present study were to test, in the same population, whether this effect persists when glutamine is administered over longer periods (10 d) and whether the effect is specific to glutamine.

SUBJECTS AND METHODS  
The protocol was approved by the Paris-Bichat Ethics Committee (Comité Consultatif pour la Protection de la Personne dans la Recherche Biomédicale) and conducted according to the principles of the Declaration of Helsinki. All families gave their written informed consent after a thorough explanation of the study protocol by the investigator to both the parents and the children. The study was performed at the Robert Debré Hospital Clinical Investigation Center (CIC 9202 INSERM-Assistance Publique-Hôpitaux de Paris). Subjects were selected on the basis of the following eligibility criteria: male sex, aged 7–15 y, and diagnosis of DMD by clinical history, muscle biopsy sample, or molecular biology. Children with acute pathology who had either cardiac or respiratory insufficiency or those who were taking corticosteroids at the time were excluded from the study. Children had stable weights and clinical status before study entry.

The children were hospitalized for 24 h on 2 separate days, before and after a 10-d oral supplement of either L-glutamine or an isonitrogenous control made of a mixture of amino acids. The children were admitted the night before the tracer infusion. They fasted after dinner (with the exception of ad libitum water) until the end of the study (1300 the following day). Whole-body leucine and glutamine metabolisms were studied by using a primed continuous infusion of L-[1-¹³C]leucine (99%: 3 µmol/kg, 3 µmol · kg^–1 · h^–1; Euriso-top,CEA group, St-Aubin, France) and L-[2-¹⁵N]glutamine (98%: 8 µmol/kg, 8 µmol · kg^–1 · h^–1; Euriso-top, CEA group) for 5 h (0800 to 1300). Arterialized blood samples (5 mL) were withdrawn at baseline and every 10 min over the last hour of the infusion (during the isotopic enrichment plateau). Breath samples were obtained simultaneously with blood samples, and the carbon dioxide production rate ( L-Glutamine (0.5 g · kg^–1 · d^–1; ADP Pharm Lab, Reventin Vaugris, France) and the isonitrogenous amino acid mixture (0.8 g · kg^–1 · d^–1, ie, 96 mg N · kg^–1 · d^–1; Cooper, Melun, France) were prepared according to good pharmaceutical practices by a pharmacist independent of the investigator and were randomly allocated to patients in a double-blind fashion. The children were instructed to take the supplemental glutamine or control amino acid mixture once per day in the morning mixed with yogurt at breakfast. The composition of the control amino acid mixture was 0.78 g L-arginine, 0.21 g L-cysteine [chloride (Cl)], 0.66 g L-histidine (Cl), 1.30 g L-isoleucine, 2.08 g L-leucine, 1.85 g L-lysine (Cl), 0.92 g L-phenylalanine, 0.83 g L-threonine, 1.00 g L-tyrosine, 1.47 g L-valine, 0.78 g L-alanine, 2.66 g L-glutamate, 0.40 g L-glycine, which provided a total of 15.64 g, ie, 1.92 g N. The 2 supplements were prepared as powders of identical taste, odor, and appearance and were flavored with banana, caramel, and artificial sweetener (Aspartame; Cooper). Comments from the children concerning taste and appearance of the supplements varied and were independent of whichever treatment the child received.

Plasma -ketoisocaproic acid (KIC), the intracellular metabolite of leucine (MTBSTFA derivative; Sigma-Aldrich, St Quentin Fallavier, France), and glutamine enrichments were measured by gas chromatography–mass spectrometry (Automass system 2-Benchtop Quadrupole Mass spectrometer and DB1 25–30 M Finnigan Mat column; ThermoFinnigan, Les Ulys, France) (13-15). The ¹³CO₂ enrichment in expired air (E¹³CO₂) was measured with an infrared spectrometer (IRIS; Wagner Analysen Technik Vertriebs GmbH, Bremen, Germany).

The rate of leucine appearance (Ra_Leu, in µmol/kg/h) and leucine oxidation (Ox_Leu) were calculated with plasma KIC enrichments (E_pKIC). Ra_Leu was calculated as:

RESULTS  
The baseline characteristics of the 26 children with DMD are presented in Table 1. No significant differences in age, weight, height, BMI, and BMI z scores were observed between the 2 supplemental groups at baseline (Table 1). No statistically significant differences were observed between the groups at baseline for all outcome measures except for plasma isoleucine concentrations, which were significantly lower in the glutamine group than in the amino acids group at baseline (56 ± 3 µmol/L for the glutamine group compared with 69 ± 3 µmol/L for the amino acids group, P < 0.05) (Table 2).

View this table:
TABLE 1. Baseline characteristics of children with Duchenne muscular dystrophy¹

 

View this table:
TABLE 2. Plasma amino acid concentrations before and after 10 d oral glutamine or isonitrogenous amino acid supplementation in children with Duchenne muscular dystrophy¹

 
A significant effect of time on estimates of whole-body protein degradation was observed (Figure 1). After 10-d supplementation with either glutamine or isonitrogenous amino acids, a significant (P < 0.05) decrease in Ra_Leu, an index of whole-body protein degradation ( ± SEM:136 ± 9 to 124 ± 6 µmol · kg FFM^–1 · h^–1 for the glutamine group and 136 ± 6 to 131 ± 8 µmol · kg FFM^–1 · h^–1 for the amino acids group), and in B_Gln (91 ± 6 to 83 ± 4 µmol · kg FFM^–1 · h^–1 for the glutamine group and 91 ± 4 to 88 ± 5 µmol · kg FFM^–1 · h^–1 for the amino acids group, P < 0.05) was observed (Figure 1). The decrease in whole-body leucine and glutamine turnover did not differ significantly between the 2 supplemental groups (Figure 1). Other estimates of leucine and glutamine turnover were not affected, namely Ox_Leu (30 ± 2 compared with 31 ± 4 µmol · kg FFM^–1 · h^–1 for the glutamine group and 29 ± 3 compared with 27 ± 4 µmol · kg FFM^–1 · h^–1 for the amino acids group), NOLD (105 ± 9 compared with 91 ± 6 µmol · kg FFM^–1 · h^–1 for the glutamine group and 105 ± 4 compared with 102 ± 6 µmol · kg FFM^–1 · h^–1 for the amino acids group), Ra_Gln (402 ± 28 compared with 413 ± 22 µmol · kg FFM^–1 · h^–1 for the glutamine group and 445 ± 40 compared with 461 ± 23 µmol · kg FFM^–1 · h^–1 for the amino acids group), and D_Gln (308 ± 30 compared with 327 ± 23 µmol · kg FFM^–1 · h^–1 for the glutamine group and 352 ± 38 compared with 369 ± 25 µmol · kg FFM^–1 · h^–1 for the amino acids group). Furthermore, measures of body composition (muscle mass, weight, FFM, and percentage fat mass) were not significantly affected after glutamine or isonitrogenous amino acid administration for 10 d (Table 3
View larger version (21K):
FIGURE 1.. Mean (±SEM) whole-body leucine and glutamine metabolism before and after 10-d oral glutamine or isonitrogenous amino acid supplementation in children with Duchenne muscular dystrophy. There were no significant differences between the groups at baseline (Mann-Whitney U test). There was a significant effect of time on estimates of whole-body protein degradation and a significant decrease in the rate of appearance of leucine (Ra_Leu) and in the rate of endogenous (body) glutamine arising from protein degradation (B_Gln) after 10-d glutamine and isonitrogenous amino acid supplementation, P < 0.05 (2-factor repeated-measures ANOVA). There was no significant effect of treatment and no significant time-by-treatment interaction. FFM, fat free mass.

 

View this table:
TABLE 3. Body composition before and after 10 d oral glutamine or isonitrogenous amino acid supplementation in children with Duchenne muscular dystrophy¹

 
Apart from plasma taurine concentrations, which showed a significant (P < 0.01) decrease over time in both supplemental groups (63 ± 4 to 47 ± 4 µmol/L for the glutamine group and 58 ± 4 to 50 ± 6 µmol/L for the amino acids group), plasma amino acid concentrations were not significantly affected after 10-d supplementation with either glutamine or amino acids (Table 2).

A significant effect of time was observed on blood urea nitrogen. Blood urea nitrogen significantly (P < 0.001) increased (but remained within the normal range) after 10-d supplementation with either glutamine or the amino acid mixture (3.7 ± 0.2 to 4.9 ± 0.3 mmol/L for the glutamine group and 3.9 ± 0.3 to 4.6 ± 0.3 mmol/L for the amino acids group). The increase in blood urea nitrogen did not differ significantly between the supplemental groups.

Glutamine or amino acid supplementation for 10 d did not affect plasma insulin (5.1 ± 0.4 compared with 6.5 ± 0.6 mUI/L for the glutamine group and 7.5 ± 1.3 compared with 7.4 ± 1.1mUI/L for the amino acids group), insulin growth factor I (284 ± 47 compared with 289 ± 60 ng/mL for the glutamine group and 209 ± 30 compared with 224 ± 27 ng/mL for the amino acids group), energy intake (1660 ± 93 compared with 1670 ± 80 kcal/d for the glutamine group and 1715 ± 116 compared with 1728 ± 83 kcal/d for the amino acids group), or protein intake (1.8 ± 0.1 compared with 2.0 ± 0.1 g · kg^–1 · d^–1 for the glutamine group and 2.0 ± 0.2 compared with 1.9 ± 0.1 g · kg^–1 · d^–1 for the amino acids group). Glutamine and amino acid supplementations were safe and well tolerated at the doses prescribed.

DISCUSSION  
Oral glutamine or isonitrogenous amino acid supplementation for 10 d equally inhibit whole-body protein degradation in children with DMD. The present study showed an effect on protein metabolism that was not measured during glutamine or amino acid administration but was measured 24 h after supplementation ceased while plasma amino acid concentrations were normal. Similarly, a protein feeding pattern induced chronic regulation of protein turnover that persisted 1 d after the end of dietary treatment (22). The response of protein metabolism to supplemental protein intake can also be compared with the studies by Motil et al (23, 24). These previous studies used a similar methodology to the present work and showed an effect of increased protein intake on whole-body amino acid metabolism after a 12 h fast. To date, mechanisms of the "chrono-biological" regulation of whole-body protein metabolism are not fully understood.

In the present study, plasma glutamine and amino acid concentrations did not increase after supplementation. Hence, the effect of glutamine on whole-body protein metabolism cannot be attributed to increased plasma glutamine concentrations, as shown in previous studies (25). We used the same glutamine dose as that used in previous studies (4, 12, 26), which was selected to double the plasma glutamine concentration and concurrently explore whole-body protein metabolism. The present study suggests that the effects of glutamine are not solely driven by substrate availability. Alternatively, glutamine uptake by the intestine might be sparing other amino acids and substrates. Previous studies of enterally administered glutamine in very-low-birth-weight infants showed similar findings; that is, there was no change in plasma glutamine concentration after glutamine administration, thereby suggesting uptake of glutamine by the gut (27, 28).

The significant decrease in plasma taurine concentrations after supplementation with either glutamine or amino acids may reflect an improvement in muscle cellular status, because the plasma taurine concentration was shown to be elevated in the mdx mouse model of DMD (29). Moreover, mdx mice fed a high-protein diet showed a decrease in the elevated plasma taurine concentrations, which was associated with decreased muscle protein catabolism due to decreased protein degradation (29).

Although the decrease in whole-body protein degradation did not significantly differ between the supplemental groups, the glutamine group showed a larger decrease in Ra_Leu than did the amino acids group (9% compared with 4%). Moreover, the decrease in Ra_Leu (9%) observed in the present study after 10-d glutamine supplementation was the same as that observed in a previous study, in which the same dose of oral glutamine was given to patients with DMD over a 5-h period (12). Hormonal changes (ie, changes in insulin and insulin growth factor I) cannot account for the decrease in leucine turnover (30) that was observed in the present study.

The lack of significant difference in leucine turnover between the glutamine and amino acid control groups highlights the need for dose and time course data on glutamine administration in patients with DMD. The route of administration (enteral in the present study) could partly explain the lack of a significant difference. Previous studies in patients without DMD who were given enteral glutamine showed less dramatic anabolic effects on protein metabolism (27, 31, 32) than when patients without DMD were given intravenous glutamine (2, 33). Also, glutamine treatment in DMD may have different effects depending on the stage of the disease (34). However, we could not stratify by age in the present study because of the sample size. Larger age-stratified studies are needed to test this hypothesis.

The present study showed no significant effect of glutamine or amino acids on the Ra_Gln measured 24 h after the last dose (ie, while the plasma glutamine concentration was normal). In our previous study (12), oral glutamine administration was associated with a decrease in Ra_Gln due to a decrease in both glutamine release from proteolysis (B_Gln) and glutamine de novo synthesis (D_Gln). In contrast with the previous study, we observed a decrease only in B_Gln, without any effect on D_Gln. Experimental conditions could account for the difference in findings. In the previous study, glutamine kinetic variables were measured during glutamine administration, and plasma glutamine concentrations doubled. In the present study, the glutamine concentration remained within the normal range.

The present study compares the effects of oral glutamine and isonitrogenous supplementation over 10 d on whole-body protein metabolism in patients with DMD. The results suggest that nitrogen supplementation might have a protein-sparing effect in DMD that results from a decrease in protein degradation. This short-term approach provides a mechanism to explain the recent results from a randomized, placebo-controlled trial that showed less deterioration in measures of function with long-term (6 mo) nitrogen supplementation (glutamine and creatine) in younger children with DMD (34). Decreased body protein breakdown after short-term nitrogen supplementation has implications for present disease therapies and could be a possible route for therapeutic modulation in DMD as well as in other protein-wasting situations.

ACKNOWLEDGMENTS  
EM contributed to the analysis of data and the writing of the manuscript. CE-DV contributed to the collection and analysis of data. CD, FG, OR, J-EF, and J-MC contributed to the collection of data. JG contributed to the analysis of data. RH contributed to the design of the experiment, collection and analysis of data, and the writing of the manuscript. The authors had no conflicts of interest to report.

REFERENCES