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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 3 1146-1152
Copyright © 2004 by The Endocrine Society

Effects of Glutamine and Recombinant Human Growth Hormone on Protein Metabolism in Prepubertal Children with Cystic Fibrosis

Dominique Darmaun, Valerie Hayes, David Schaeffer, Susan Welch and Nelly Mauras

Divisions of Endocrinology (D.D., V.H., S.W., N.M.) and Pulmonology (D.S.), Nemours Children’s Clinic, Jacksonville, Florida 32207; and Institut National de la Santé et de la Recherche Médicale, Unité 539, Centre de Recherche en Nutrition Humaine (D.D.), 44093 Nantes, France

Address all correspondence and requests for reprints to: Dr. Dominique Darmaun, Nemours Children’s Clinic, Endocrine Research, 807 Children’s Way, Jacksonville, Florida 32207. E-mail: ddarmaun{at}nemours.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
To determine whether recombinant human GH (rhGH) and glutamine (GLN), alone or in combination, have a protein anabolic effect and whether rhGH alters GLN kinetics in cystic fibrosis (CF), nine 9.6 ± 0.5-yr-old children with CF who were either undernourished (weight/height, <50th percentile) or short (height, <5th percentile) received 2-h infusions of [13C]bicarbonate (to assess CO2 production), followed by 4-h infusions of [13C]leucine and [15N]GLN, on 4 separate days in the postabsorptive state: 1) at baseline, and after a 4-wk treatment with 2) oral GLN (0.7 g/kg·d), 3) rhGH (0.3 mg/kg·wk), and 4) GLN and rhGH combined (GLN and rhGH regimens were in randomized order). No significant effect of GLN on leucine kinetics was detectable. In contrast, rhGH induced a 32% reduction in leucine oxidation and a 13% stimulation of nonoxidative leucine disposal, an index of protein synthesis (P < 0.05), with no change in proteolysis or GLN kinetics. The combined GLN plus rhGH regimen had similar effects as rhGH alone. We conclude that in children with CF, 1) oral GLN may not promote protein gain in the fasting state; and 2) a short course of rhGH has a potent anabolic effect that is mediated by stimulation of protein synthesis and does not affect GLN kinetics.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
UNDERNUTRITION AND GROWTH failure are commonly found in children with cystic fibrosis (CF) (1). Contributing factors include anorexia associated with frequent infections, malabsorption due to exocrine pancreatic insufficiency, increased rates of energy expenditure or protein turnover, and the catabolic effects of corticosteroid treatment. Undernutrition, in turn, adversely affects prognosis by impairing immune function and altering pulmonary function as a consequence of a decrease in respiratory muscle strength. Although various nutritional regimens have been shown to improve weight gain and growth rates of CF patients, the difficulty in achieving a normal growth and nutritional status raises the question of whether anabolic agents would be useful in children with CF.

In studies by our group and others, recombinant human GH (rhGH) increased estimates of whole body protein synthesis in fed and fasted healthy adults and attenuated protein wasting in volunteers treated with prednisone, a model of stress-induced catabolism (2, 3). Similarly, rhGH improved lean body mass in glucocorticosteroid-dependent children with inflammatory bowel disease (4). More importantly, rhGH was found to improve both growth velocity and weight gain in children with CF (5, 6, 7). For instance, after 1 yr of rhGH treatment, height z-score improved from -1.86 ± 0.07 to -1.31 ± 0.9 in one study (6) and from -1.3 ± 0.2 to -0.76 ± 0.23 in another (7). Furthermore, the weight for height z-score improved from -0.04 ± 1.20 to +0.27 ± 0.70 in one study (5), and weight gain increased from 1.7 ± 0.9 to 3.8 kg/yr in another (6), whereas the trend toward a faster weight gain failed to reach statistical significance in a third study (7). Finally, recent studies using isotopic tracers carried out in CF patients receiving 1 yr of rhGH treatment demonstrate a sustained reduction in proteolysis, and protein oxidation is associated with rhGH treatment, as measured by labeled leucine infusion performed after 6 and 12 months of treatment (8).

Although L-glutamine (GLN) can be synthesized de novo by most tissues in the body and is thus classified as a nonessential amino acid, studies performed in the last decade suggest that GLN becomes conditionally essential (9) in numerous clinical situations. Although GLN is the most abundant free amino acid in muscle, the free GLN concentration plummets in situations of acute stress, such as major surgery and bone marrow transplant, and GLN supplementation improved nitrogen balance and/or clinical outcome in these clinical settings (10, 11, 12). Accordingly, GLN infusion was associated with a dramatic (~38%) inhibition of leucine oxidation along with a stimulation of nonoxidative leucine disposal (NOLD), an index of whole body protein synthesis, in healthy adults (13). Similarly, GLN acutely suppressed protein breakdown and oxidation in children suffering from disease associated with protein wasting, such as Duchenne muscular dystrophy (14), or extremely premature birth (15).

CF children may have depleted body GLN pools as a result of chronic undernutrition as well as of repeated bouts of infection that are associated with acute stress. As GLN is a free amino acid and does not require the action of any of the pancreatic proteolytic enzymes, GLN should be readily absorbed even in CF children presenting with exocrine pancreatic insufficiency. To our knowledge, neither GLN kinetics nor the potential benefit of GLN supplementation have been examined in CF children.

The putative protein-anabolic effect of GLN does not seem to be mediated by increased secretion of GH or IGF-I, because the circulating levels of these hormones failed to rise in healthy volunteers receiving large enteral doses of GLN (13). However, in acutely ill adults, rhGH treatment attenuates or prevents muscle GLN depletion (16) and decreases muscle GLN release (17) via a reduction in GLN de novo synthesis in skeletal muscle (18). Taken together, these studies collectively suggest that the anabolic effect of GLN is not mediated through increased secretion of rhGH. They also suggest that rhGH may reduce GLN availability to tissues other than muscle. Because GLN is an essential fuel for other tissues in the body, i.e. cells of the immune system, this may be considered an unwanted side-effect of rhGH.

The aim of this study therefore was to determine 1) whether a 4-wk supplement with oral GLN or sc rhGH enhances whole body protein synthesis in children with CF; 2) if so, whether a combination of GLN and rhGH has synergistic effects; and 3) whether rhGH affects GLN metabolism in this population.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patient population

Before inclusion into the study, each subject’s parents received detailed information about the purpose, objectives, and potential risks of the study and signed a written consent form, according to protocols approved by the Nemours Children’s Clinic research committee and the institutional review board of Baptist Medical Center, and each child gave his/her assent to participate in the study. To be enrolled, patients had to meet the following inclusion criteria: 1) diagnosis of CF by commonly accepted criteria, including a sweat chloride concentration greater than 60 mEq/liter on two occasions; 2) age between 7–13 yr, Tanner stage I; 4) significant growth delay (as defined either by height less than the 5th percentile or below -2 SD for age) and/or undernutrition (weight for height less than the 50th percentile); 5) stable lung disease over the last 3 months, defined as unchanged pulmonary function tests; 6) documented growth rate over the previous 2 yr; and 7) absence of clinically significant liver disease (bilirubin within normal limits and/or serum glutamate-pyruvate transaminase or serum glutamate-oxaloacetate transaminase less than twice the upper limit of normal), diabetes, or other organic disease.

Materials

L-[1-13C]Leucine (99% 13C) and L-[2-15N]-[2-15N]GLN (98% 15N) were obtained from Cambridge Isotope Laboratories (Woburn, MA) and were verified to be sterile and pyrogen free using the Limulus lysate assay. The day before each infusion study, tracer solutions were prepared using aseptic technique by dissolving accurately weighed amounts of labeled amino acids in known volumes of sterile 0.9% saline, passed through a 0.22-µm Millipore filter (Bedford, MA), and stored at 4 C in sterile sealed vials until infusion.

Protocol design (Fig. 1Go)

Each patient underwent four separate admissions: 1) an initial study under baseline conditions and three separate studies after a 4-wk regimen consisting of 2) oral GLN (0.7 g/kg·d), given as a flavored drink, three times a day, 3) rhGH (0.3 mg/kg·wk, sc, daily), or 4) GLN/rhGH combined. The order of the GLN and rhGH regimens was randomized, and study periods were separated by a 2-wk washout period. When there was an exacerbation of the disease or an acute intercurrent illness during a washout or a treatment phase, studies were interrupted and restarted 2 wk after the child had recovered from the intercurrent episode. All of the patients ’ medications remained unchanged during the studies.



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  		FIG. 1. Protocol design.

 
On the night before each isotope infusion study, the child was admitted to the Clinical Investigational Unit at Wolfson Children’s Hospital. A dietary record was obtained to estimate protein and calorie intake over the 3 d before each study, and body fat mass was assessed by determination of skinfold thickness (Harpenden calipers), dual energy x-ray absorptiometry (DEXA), and bioelectrical impedance analysis (BIA). Pulmonary function tests were carried out under baseline conditions and after the fourth isotope infusion study. Compliance with the regimens was monitored by frequent phone contact and by having the parents return any unused GLN packets or rhGH vials at interim visits.

At 0630 h on the morning of each isotope infusion day, two iv lines were placed: one in a forearm vein for isotope infusion, and the second in a superficial vein of the contralateral hand for blood sampling. During the blood-sampling period, the hand was kept in a heated pad (air temperature, ~55 C) to obtain arterialized venous blood. At 0654 and 0700 h, duplicate blood and expired air samples were obtained to determine the background isotopic enrichments in plasma amino acids and breath CO2, and each child then received two consecutive primed infusions of stable isotopes. First, as indirect calorimetry may be laborious and inaccurate for these subjects, a 2-h, primed, continuous infusion (~6 µmol/kg prime; 6.5 ± 0.2 µmol/kg·h infusion) of H13CO3Na was administered from 0700–0900 h to assess the total CO2 production rate (18, 19, 20). This was immediately followed by a 4-h concomitant infusion of [13C]leucine (prime, ~5 µmol/kg; infusion rate, 4.9 ± 0.1 µmol/kg·h) and 15N-GLN (prime, ~6 µmol/kg; infusion rate, 5.7 ± 0.1 µmol/kg·h) between 0900–1300 h. Blood sampling was performed at 20-min intervals between 1100–1300 h to measure plasma isotopic enrichments. Breath aliquots were obtained to determine 13CO2 enrichment at baseline and at 20-min intervals between 0800–0900 and 1200–1300 h, i.e. during the last hour of labeled bicarbonate and labeled amino acid infusions, respectively.

Analytical methods

Standard RIAs were used to measure serum testosterone, estradiol, insulin, IGF-I, and IGF-binding protein-3 (Mayo Medical Laboratories, Rochester, MN). Measurement of 13CO2 enrichment from collections of expired air was performed by gas chromatography-isotope ratio mass spectrometry, and stable isotopic enrichments of plasma leucine, {alpha}- ketoisocaproate (KIC), and GLN were measured by gas chromatography-mass spectrometry as previously described (14). Plasma amino acid concentrations were measured using a Beckman-6300 amino acid analyzer (Beckman Instruments, Fullerton, CA).

Calculations

The leucine appearance rate [Raleu; expressed as micromoles per kilogram of lean body mass (LBM) per minute] was calculated as: RaLeu = ileu [(Eileu/ Epkic) - 1], where ileu is the [13C]leucine infusion rate, and Eileu and Epkic are the isotopic enrichments (molar percent excess) in the infused leucine tracer solution and plasma KIC at steady state, respectively.

Leucine oxidation (OxLeu) was calculated based on the rate of 13CO2 excretion (Excr13CO2) during labeled leucine infusion. The latter is commonly calculated as: Excr13CO2 = (VCO2 x ECO2)/FRCO2, where VCO2 is the total rate of CO2 production determined using indirect calorimetry, ECO2 is the steady state 13CO2 enrichment in breath over the last 2 h of labeled leucine infusion, and FRCO2 is the fractional recovery of 13CO2 in breath. In our patients, VCO2 was not directly measured by indirect calorimetry, but the RaCO2 was determined by isotope dilution, based on the appearance of 13CO2 in expired air over the course of a primed 2-h infusion of H13CO3Na, as previously described (19, 20, 21): RaCO2 = iBicarb [(EBicarb/ECO2) - 1], where iBicarb is the H13CO3Na infusion rate (micromoles per kilogram per minute), and ECO2 and EBicarb are the 13C enrichments (molar percent excess) in expired air at steady state during the last 30 min of the labeled bicarbonate infusion and in the infused bicarbonate solution, respectively. As shown by us and others (19, 20), RaCO2 = VCO2/FRCO2; therefore, FRCO2 = VCO2/RaCO2. In our patients, 13CO2 excretion was thus calculated as: Excr13CO2 = (VCO2 x ECO2)/(VCO2/RaCO2) = RaCO2 x ECO2. It follows that Excr13CO2 = RaCO2 x ECO2, assuming the FRCO2 does not vary between the labeled bicarbonate infusion and labeled leucine infusion performed immediately thereafter on the same day. OxLeu was therefore calculated as: OxLeu = Excr13CO2 x [1/EKIC - 1/EiLeu]. NOLD was calculated as NOLD = RaLeu - OxLeu.

The Ragln was calculated as: Ragln = igln [(Eigln/Epgln) - 1], where igln is the [15N]GLN infusion rate, and Eigln and Epgln are the isotopic enrichments (molar percent excess) in the infused GLN tracer solution and plasma GLN at steady state, respectively.

Because GLN is a nonessential amino acid, two sources contribute to its endogenous production: GLN release from protein breakdown (Bgln) and GLN from de novo synthesis (Dgln). As proteolysis releases amino acids in proportion to their relative abundance as bound residues in body protein, Bgln was calculated as: Bgln = RaLeu x 0.423, where 0.423 is the ratio of GLN/leucine abundance in human muscle protein (22). Bgln was then calculated as Dgln= Ragln - Bgln. The GLN metabolic clearance rate (MCRgln; milliliters per kilogram per hour) was calculated as: (MCRgln) = 1000 x Ragln/[GLN], where [GLN] is the plasma GLN concentration (micromoles per liter).

Body composition was assessed using anthropometry, BIA, and DEXA on each of the four admissions. First, the percentage of fat mass (%FM) was determined from the sum of four skinfolds (SF) as: %FM = [4.95/(a - b x log10 SF)] - 4.5, where the values for a and b are 1.1533 and 0.0643 in boys and 1.1369 and 0.0598 in girls, respectively (23). FM was then calculated as: FM = Wt x %FM/100, where Wt is body weight. LBM was then calculated as: LBM = Wt - FM, where WT is body weight. LBM was also assessed from bioelectrical impedance as: LBM = (0.61x Ht2/R) + (0.25 x Wt) + 1.31, where Ht is height (centimeters), and R is resistance (ohms), as proposed by Houtkooper et al. (24).

Statistics

Results are expressed as the mean ± SE. Parameters were compared between treatments using repeated measures ANOVA and paired t tests. Significance was established at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Characteristics of patients

Twelve patients were recruited, and nine completed the full set of studies. Three patients withdrew their consent for the study after only two isotope infusion studies, and the data from these subjects were therefore not analyzed. The clinical characteristics of the nine patients who completed the trial are shown in Table 1Go. As attested by clinical exam and confirmed by sex steroid concentrations (Table 2Go), all patients were prepubertal (Tanner I) upon inclusion in the study, and remained so over the 4-month duration of their enrollment.


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  		TABLE 1. Selected clinical characteristics of the population studied upon inclusion

 

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  		TABLE 2. Lean body mass (kilograms), as determined by skinfold thickness, BIA, and DEXA at baseline and after a 4-wk treatment with either GLN, rhGH, or the combination of both

 
In retrospect, the population was somewhat heterogeneous. For instance, subject 4 failed to meet the inclusion criteria, as she was neither wasted nor stunted (Table 1Go) based on Waterlow’s criteria (25). Nevertheless, as her results could not be distinguished from the rest of the group (data not shown), this subject was not excluded from the analysis.

Only one patient (no. 5) among the nine patients was receiving corticosteroids during the study period, albeit at a small dose (10 mg prednisone every other day). The dose received by this patient was identical throughout the study period. When data for this single corticosteroid-treated patient were excluded, and data from the eight remaining patients were analyzed, the direction and magnitude of the changes observed were unaltered (see below). Data from the whole group of nine patients as a whole are therefore reported in this paper.

There was no order effect in any of the parameters measured; hence, the data were grouped for analysis.

Body composition (Table 2Go)

As shown in Table 2Go, the data obtained from anthropometry, BIA, and DEXA were in close agreement. Using the average results from the three methods, both GLN and GH regimens were found to be associated with a slight, but significant, increase in LBM (+1.6% and +3.8%, with GLN and rhGH, respectively) despite the short duration (4 wk) of the treatment. Although the gain in LBM tended to be greater with the combined (rhGH and GLN) treatment than with rhGH alone (5.6% vs. 3.8%), the difference failed to reach statistical significance (P = 0.056, using one-tail paired t test).

Glucose metabolism in CF children (Table 3Go)

The GLN regimen did not alter the blood glucose level (90 ± 2 vs. 90 ± 2 mg/dl; P = NS). In contrast, blood glucose tended to rise after treatment with rhGH either alone (90 ± 2 vs. 97 ± 4 mg/dl; P = NS), or in combination with GLN (90 ± 2 vs. 95 ± 2 mg/dl; P = 0.05). Fasting insulin levels rose with GH treatment (4.4 ± 0.8 vs. 7.4 ± 1.1µU/ml; P = 0.04), but not with the GLN regimen (4.4 ± 1.6 vs. 5.0 ± 0.9 µU/ml), and tended to rise less with the combined regimen (5.1 ± 0.5 vs. 7.4 ± 1.1µU/ml). Accordingly, the insulin/glucose ratio rose with rhGH (0.05 ± 0.01 vs. 0.07 ± 0.01 µU/mg; P = 0.05), but not with the GLN regimen (0.05 ± 0.01 vs. 0.05 ± 0.01 µU/mg; P = 0.36) or with the combined regimen (0.06 ± 0.01 vs. 0.07 ± 0.01 µU/mg; P = 0.20).


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  		TABLE 3. Plasma concentrations of glucose and selected hormones and growth factors in CF children at baseline and after 1-month treatment with GLN, rhGH, or the combination of both regimens

 
Plasma amino acid concentrations

The plasma GLN concentration increased slightly (5.5%), but significantly (P < 0.05), with the GLN regimen. The 10% rise in the concentration of citrulline, a nonessential amino acid known to arise from GLN metabolism in the gut (26), was significant using Wilcoxon nonparametric test, but failed to reach statistical significance using the paired t test (P = 0.11). The concentrations of other amino acids derived from GLN metabolism (glutamate, proline, and arginine) remained unaltered. Although plasma taurine and ornithine declined during rhGH treatment, most amino acid concentrations remained unaffected regardless of the regimen (Table 4Go).


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  		TABLE 4. Plasma amino acid concentrations (micromoles per liter) in CF children at baseline and after 1-month treatment with GLN, rhGH, or the combination of both regimens

 
Leucine kinetics (Fig. 2Go) were calculated as a function of both body weight (data not shown) and LBM. As the changes were similar regardless of the expression of results, leucine kinetics are solely reported per kilogram of LBM in the following.



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  		FIG. 2. Effect of GLN, GH, and GLN plus GH on leucine metabolism in nine prepubertal children with CF. Data (mean ± SE) are expressed as micromoles per kilogram of LBM per minute. *, P < 0.01 vs. baseline values.

 
GLN supplementation did not affect leucine release from proteolysis (baseline vs. GLN, 2.89 ± 0.22 vs. 2.82 ± 0.18 µmol/kg LBM·min; P = 0.48). Even though leucine oxidation declined in six of nine patients after the GLN regimen (data not shown), the overall, approximately 9% decline failed to reach statistical significance (0.71 ± 0.05 vs. 0.64 ± 0.10 µmol/kg LBM·min; P = 0.36). As a consequence, NOLD remained unaltered with the GLN regimen (2.18 ± 0.22 vs. 2.18 ± 0.18 µmol/kg LBM·min).

GH treatment

Whereas rhGH did not alter rates of proteolysis (2.89 ± 0.22 vs. 2.96 ± 0.27 µmol/kg LBM·min), rhGH treatment was associated with a sharp, 32% drop in leucine oxidation (0.72 ± 0.05 vs. 0.49 ± 0.09 µmol/kg LBM·min; P = 0.004). As a consequence, NOLD increased 13% with the rhGH regimen (2.13 ± 0.22 vs. 2.41 ± 0.22 µmol/kg LBM·min; P = 0.01).

Effects of combined GLN and rhGH on leucine kinetics

In the same way as with rhGH alone, with the combined GLN plus GH regimen, proteolysis remained unaltered (2.89 ± 0.22 vs. 2.98 ± 0.30 µmol/kg LBM·min; P = 0.69), leucine oxidation declined approximately 34% (0.70 ± 0.05 vs. 0.46 ± 0.08 µmol/kg LBM·min; P = 0.01), and NOLD increased (2.13 ± 0.22 vs. 2.52 ± 0.21 µmol/kg LBM·min; P = 0.05). Neither leucine oxidation (P = 0.60) nor NOLD (P = 0.71) differed with the combined treatment compared with rhGH alone.

Regardless of the regimens tested, excluding the single patient (subject 5) who was receiving low dose prednisone treatment did not alter the overall directional changes observed. In fact, in the remaining group of eight subjects, Ra was 2.82 ± 0.19 µmol/kg LBM·min at baseline vs. 2.80 ± 0.19, 2.89 ± 0.26, and 3.01 ± 0.28 µmol/kg LBM·min after GLN, rhGH, and the combined regimen, respectively (P = NS); OxLeu was 0.71 ± 0.05µmol/kg LBM·min at baseline vs. 0.64 ± 0.10 (P = NS), 0.50 ± 0.08 (P = 0.005), and 0.47 ± 0.07 (P = 0.01) µmol/kg LBM·min after GLN, rhGH, and the combined regimen, respectively; and NOLD was 2.11 ± 0.18 µmol/kg LBM·min at baseline vs. 2.16 ± 0.19 (P = NS), 2.38 ± 0.20 (P = 0.01), and 2.54 ± 0.25 (P = 0.07) µmol/kg LBM·min after GLN, rhGH, and the combined regimen, respectively.

GLN metabolism in CF children

The plasma GLN concentration rose 5.5 ± 2.3% (P = 0.045) after the GLN regimen, but remained unaltered with all other regimens (Table 4Go). None of the regimens affected the RaGLN, GLN release from proteolysis, or GLN de novo synthesis (Table 4Go).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The findings of the current study suggest that a 4-wk course of GH treatment improves protein balance through a stimulation of protein synthesis in prepubertal children with CF. They further suggest that oral GLN supplementation may not affect body protein turnover measured in the fasting state in this population. Although these findings do not provide any evidence for a synergistic effect of GLN and GH, they suggest that rhGH does not impair whole body GLN metabolism.

Under baseline conditions, the RaLeu, an index of whole body proteolysis, did not differ in our CF patients from that measured in a group of six healthy, 11.3 ± 0.4-yr-old children in previous studies (2.89 ± 0.21 vs. 2.86 ± 0.26 µmol/kg LBM·min1 in CF and controls, respectively) (27). Earlier studies reported either normal (28, 31) or elevated rates of proteolysis in patients with CF (8, 31, 32). Enhanced rates of proteolysis seem to correlate with the degree of malnutrition (32) or insulin resistance (31); the latter is an index of decreased clinical status (33). However, we observed approximately 80% higher OxLeu in the current study compared with healthy children in earlier studies (0.71 ± 0.03 vs. 0.32 ± 0.03 µmol/kg LBM·min; P < 0.05) (27), suggesting a higher rate of protein oxidation in CF children. This difference must be interpreted with caution, however, because 1) the determination of OxLeu involved measurement of CO2 production using 13C-labeled bicarbonate infusion in the current study, instead of indirect calorimetry in our earlier studies (27); 2) in direct comparison with a group of healthy age-matched controls, Hardin et al. (8) found identical rates of OxLeu in children with CF and healthy, age-matched controls using indirect calorimetry for determination of CO2 production (VCO2); and 3) although Kien et al. (29) used the labeled bicarbonate method, they report identical OxLeu in children with CF and controls, yet their results were not expressed per unit of LBM, so that differences in body composition may obscure the comparison between patients and controls.

To our knowledge, the current study is first to report on GLN metabolism in patients with CF. The average RaGLN of 7.83 ± 0.38 µmol/kg LBM·min was approximately 11% higher than the 7.05 ± 36 µmol/kg LBM·min observed in healthy 11-yr-old controls (27). As elevated rates of OxLeu and GLN turnover are hallmarks of stress-associated protein wasting, the slightly elevated values observed in our patients suggest that CF may be associated with a mild degree of hypercatabolism, even though our patients were studied under baseline conditions in the absence of intercurrent illness or any exacerbation of the disease.

The lack of a protein anabolic effect of GLN in CF children in the current study contrasts with the positive effects observed in children suffering from other conditions associated with protein wasting or stress, such as Duchenne muscular dystrophy (14) or premature birth (15). Several explanations can account for the lack of any protein anabolic effect of GLN in the current study. 1) The anabolic or anticatabolic effects of GLN reported in earlier studies were all documented over the course of continuous GLN administration at a time when plasma GLN concentration was elevated about 25 (15) to about 67% (14) above baseline values. In contrast, in the current study, protein kinetics were assessed in the postabsorptive state, more than 12 h after the last dose of oral GLN, and the plasma GLN concentration was only approximately 5% above baseline values after 4 wk of GLN supplementation; accordingly, the concentrations of glutamate, citrulline, and arginine, all of which are known to rise as a result of GLN metabolism, remained essentially unaltered. The slight (1.1%), but significant, increase in LBM associated with GLN treatment suggests that GLN may have had a protein anabolic effect in the fed state, an effect that was missed by measurements performed by isotope dilution in the postabsorptive state. 2) Wide intersubject variability may preclude the detection of GLN's effect, because although the overall effect failed to reach statistical significance, OxLeu declined in six of nine patients after the GLN regimen. 3) Finally, the oral GLN supplement was not given during periods of acute exacerbations of the disease. The current results therefore do not rule out a potential benefit of GLN during periods of acute stress in CF children.

Several studies have demonstrated that GH treatment enhances the growth rate in children with CF (6, 7, 8). Because protein accretion is a prerequisite for growth, this implies that rhGH must increase the net protein gain. The findings of the current study show that a short course of rhGH acutely enhances protein gain by decreasing OxLeu and stimulating NOLD, an estimate of whole body protein synthesis. This is consistent with the effect observed after rhGH treatment in healthy adult volunteers (2, 3). In contrast, Hardin et al. (8) observed that after 6 and 12 months of treatment, the main effect of rhGH was a reduction in the rates of whole body proteolysis and protein oxidation, with no change in protein synthesis. Differences in protocol design can account for the discrepancy. First, the determination of OxLeu relied on rates of CO2 production measured using indirect calorimetry in earlier studies, whereas the infusion of labeled bicarbonate in the current study takes into account any potential change in the rate of 13C recovery in breath. Secondly, and more to the point, the current study only assessed the acute effect of rhGH over the first 4 wk of the regimen, and the mechanisms by which rhGH affects protein gain may change with the more extended periods of treatment studied by Hardin et al. (8). In fact, long-term rhGH treatment was found to promote protein gain without enhancing whole body protein synthesis in the postabsorptive state in children receiving long-term prednisone treatment for inflammatory bowel disease (4), another situation characterized by poor growth and undernutrition.

Impaired glucose tolerance is one of the main potential side-effects associated with the long-term use of GH (33, 34). In past studies, impaired carbohydrate tolerance reportedly occurred in two of 24 CF patients receiving rhGH (5). Plasma insulin, glucose, and the insulin/glucose ratio rose significantly, albeit well within normal limits, after 4 wk of GH treatment in our patients. In that context, it is of interest to note that the increases in glucose and the insulin/glucose ratio were blunted when rhGH was combined with oral GLN. Previous studies have indeed reported that although GLN plays a major role as a source of carbon for gluconeogenesis in humans (35), the infusion of large doses of GLN failed to affect either plasma glucose or insulin (13) or the glucose production rate (26). Moreover, GLN was shown to improve insulin sensitivity in euglycemic, hyperinsulinemic clamp studies in vivo in dogs (36). The potential benefit of GLN to preserve carbohydrate tolerance and/or prevent the occurrence of insulin resistance during long-term GH treatment therefore warrants further investigation.

Finally, because GLN is an essential fuel for rapidly replicating cells, such as cells of the immune system (37), and as rhGH was found to alter the synthesis rate of GLN in critically ill adults (18), it is of clinical relevance to observe that under the conditions tested we found no evidence for a blunting effect of rhGH treatment on GLN synthesis or utilization in pediatric patients with CF. This implies that rhGH may not deprive the immune system of its preferred source of energy.

In conclusion, the chronic administration of oral GLN had no measurable protein anabolic effects in the fasting state in children with CF who were either malnourished or growing poorly. However, rhGH administration potently stimulated whole body protein synthesis, without any effect on proteolysis. These data suggest that rhGH may play a beneficial role in the treatment of chronic debilitating conditions, improving anabolism in childhood. Further studies will be needed to better determine the benefit of that approach.


   Acknowledgments
 
We are grateful to the patients and parents who agreed to participate in these very demanding studies. We gratefully acknowledge the help of Bernice Rutledge and her nursing staff in the performance of these labor-intensive studies. We are thankful for the superb technical help of Brenda Sager, Shawn Sweeten, and Lynda Everlyne with laboratory work, and for Dr. Jaya Punati for assistance with patient recruitment.


   Footnotes
 
This work was supported in part by grants from the Genentech Foundation for Growth and Development (Charlottesville, VA) and the Nemours Research Programs (Jacksonville, FL).

Abbreviations: BGLN, Glutamine release from protein breakdown; BIA, bioelectrical impedance analysis; CF, cystic fibrosis; DEXA, dual energy x-ray absorptiometry; Excr13CO2, 13CO2 excretion; %FM, percentage of fat mass; FRCO2, fractional recovery of 13CO2 in breath; GLN, glutamine; KIC, {alpha}-ketoisocaproate; LBM, lean body mass; NOLD, nonoxidative leucine disposal; OxLeu, leucine oxidation; Ra, appearance rate; rhGH, recombinant human GH.

Received August 12, 2003.

Accepted December 16, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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