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Original Research Communications |
1 From the Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany; the Institute of Nutrition, University of Bonn, Germany; and the Centre de Recherche en Nutrition Humaine, Hotel-Dieu, Nantes, France.
2 Supported by Fresenius-Kabi, Bad Homburg, Germany.
3 Address reprint requests to KS Kuhn, Institute for Biological Chemistry and Nutrition, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Germany. E-mail: kkuhn{at}uni-hohenheim.de.
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ABSTRACT |
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70% of overall glutamine production in healthy adults; the contribution of de novo synthesis being estimated at 60%. However, measurement of the de novo synthesis rate in muscle tissue requires knowledge of the appearance rate of glutamine in plasma and the quantity of glutamine derived from intracellular proteolysis. Thus, the content of glutamine in muscle protein is a prerequisite for an accurate calculation. Objective: The objective of the study was to measure glutamine in muscle protein.
Design: Muscle specimens (open biopsies) were obtained from humans (10 men and 4 women), rats (n = 4), cows (n = 4), and pigs (n = 4). Glutamine was assessed via prehydrolysis derivatization, rapid microwave-enhanced acid hydrolysis, and 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) reversed-phase HPLC, and expressed per mg alkali-soluble protein (ASP) and DNA.
Results: Glutamine concentrations in muscle cell protein of various species ranged from 41 to 49 µg/mg ASP; the differences were not species related. The combined means (±SDs) for the 4 species were 43.6 ± 4.9 µg/mg ASP and 11.9 ± 2.0 mg/mg DNA, respectively. In humans, there was no apparent influence of age, sex, or BMI.
Conclusions: Direct and specific measurements of glutamine in intact muscle protein were 50% lower than assumed previously. We used data compiled from earlier studies to recalculate the contributions of proteolysis and de novo synthesis to the endogenous production of glutamine in selected age groups of healthy humans; these contributions remained remarkably constant at 13% and 87%, respectively.
Key Words: Glutamine • glutamic acid • muscles • biopsy • proteins • kinetics • de novo synthesis • humans • animals • alkali-soluble protein
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INTRODUCTION |
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Because glutamate–ammonia ligase (or, glutamine synthetase) is a nearly ubiquitous enzyme in mammalian cells, glutamine can be synthesized de novo and is considered to be a nonessential amino acid. Under physiologic conditions, sufficient amounts of glutamine are produced endogenously to allow maintenance of the large intracellular free glutamine stores and the demands of the glutamine-consuming tissues to be met. During episodes of catabolic stress (eg, surgery, burns, infection, and malnutrition), however, there is much evidence that the rate of endogenous glutamine production becomes insufficient to meet the increased glutamine demand. This is reflected by decreased concentrations of extra- and intracellular free glutamine (11, 12).
To gain more insight into rates of glutamine synthesis and utilization in humans, numerous approaches in which radioactive or stable-isotope tracer techniques, or both, are used are available (13), showing an average glutamine turnover rate of 350 µmol• kg-1•h-1, which exceeds that of all other amino acids studied to date (14, 15). In the postabsorptive state, endogenous production (appearance rate) of glutamine (Gln Ra) can be either derived by de novo synthesis via the cytosolic enzyme glutamine synthetase or released via the breakdown of proteins. The proportion of glutamine synthesized de novo has been indirectly estimated by using essential amino acid and glutamine tracers (16), assuming that protein breakdown in the postabsorptive state is the only endogenous source of an essential amino acid and that the relation of the essential amino acid tracee and glutamine in lean tissue is known. The fraction of glutamine turnover that cannot be accounted for by glutamine release from protein breakdown is then attributed to de novo synthesis.
However, correct estimation of glutamine release is not possible because of the lack of accurate analytic data concerning the glutamine concentration in muscle protein. We developed a novel analytic method enabling the determination of protein-bound glutamine (17, 18). In the present study, we used this method to assess the glutamine content of human muscle specimens as well as of various mammalian species. The data obtained will facilitate accurate assessment of glutamine turnover.
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METHODS |
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All muscle specimens were stored on ice for transportation and were frozen in liquid nitrogen within 30 min. Samples were freeze-dried and then stored in cold-resistant ampoules under liquid nitrogen until analyzed further. To facilitate protein and DNA extraction, portions of the freeze-dried muscle samples were defatted in hexane for 2 h at room temperature and subsequently ground in a mortar while all visible blood and connective tissue were removed, yielding the dry, fat-free solids (DFFS) (19). For alkali-soluble protein (ASP) extraction, 12–18 mg DFFS was incubated for 18 h at room temperature with 2 mL of 0.05 mol NaOH/L, resulting in a nearly complete dissolution of the "muscle powder" (20). Duplicate extractions were carried out whenever possible. For 9 muscle specimens, the limited amount of DFFS allowed only a single protein extraction. ASP extracts were dialyzed overnight against 2 L ultrapure water by using a Spectrapor 3 dialysis membrane (MWCO 3500; Serva, Heidelberg, Germany). Measurements of glutamine concentrations expressed per mg ASP, assessed according to the method of Bradford (21), and per mg DNA in the muscle specimens were obtained with a modified diphenylamine assay (22, 23).
We determined protein-bound glutamine concentrations according to the method of Kuhn et al (17), as described previously. Dialyzed ASP extracts were diluted to a final protein concentration of 2.5 g/L and protein-bound glutamine was assessed after treatment with bis-1,1-(trifluoroacetoxy)iodobenzene (BTI; Sigma Deisenhofen, Germany), hydrolysis, and reversed-phase HPLC. Briefly, an aliquot of ASP extract was mixed with an equal amount of a freshly prepared solution of BTI in dimethylformamide (optimized BTI concentration: 11.63 mmol/L). After the reaction (4 h at 50°C), the sample was evaporated and the precipitate was redissolved in water containing an internal standard (norleucine). Subsequently, the specimens were hydrolyzed by using a novel microwave technology (1200 mega high-performance microwave digestion unit; MLS, Leutkirch, Germany) (24, 25). The amino acid composition was determined by reversed-phase HPLC by using precolumn derivatization with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) and fluorescence detection (26). In the present study, glutamine, glutamic acid, and leucine were quantitatively assayed. We measured free intracellular glutamine in DFFS extracts after deproteinization in perchloric acid (0.2 mol/L) by reversed-phase HPLC and o-phthaldialdehyde derivatization as described previously (26, 27). After deproteinization, the extracts were stored at -80°C until analyzed by HPLC.
Stable-isotope methods
The kinetic data for leucine and glutamine reported here were determined by using a primed, continuous, 4-h intravenous infusion of L-[1-13C]leucine and L-[2-15N]glutamine, respectively, in several studies performed by our group and published elsewhere (16, 28–30). All studies were performed after informed consent was obtained from the adult subjects or the children's parents, according to procedures approved by the local ethical committee at the relevant institution. Infusions were performed in the resting, postabsorptive state after an overnight fast, except in the neonates who underwent isotope infusions while receiving intravenous nutrition. Briefly, isotope infusions were carried out in 4 different populations: 1) 5 very-low-birth-weight (<1250 g) infants studied on the 10th day of life while they were receiving total parenteral nutrition providing a glutamine-free amino acid mixture [2.8 g•kg-1•d-1, 201 kJ (48 kcal)•kg-1•d- 1] supplied as 50% glucose and 50% long-chain triacylglycerols (28); 2) 4 infants aged 8–20 mo who had no evidence of endocrine or gastrointestinal disease (29); 3) 6 healthy 13-y-old boys who were prepubertal on the basis of a physical examination (Tanner stage 1) and on a baseline plasma testosterone concentration <10.4 pmol/L (30 ng/dL), and who had normal variant short stature without any detectable endocrine or other organic illness (30); and 4) 6 healthy 22–30-y-old normal-weight men (16).
Calculation of leucine kinetic indexes
The Ra of leucine in plasma (µmol•kg-1•h-1) was calculated by using standard isotope-dilution equations for steady state conditions (31):
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| (1) |
where iLeu is the infusion rate of [13C]leucine and Ei and Ep are the enrichments of 13C (mol% excess) determined in the infused isotope solution and in plasma leucine or in 
-ketoisocaproate (KIC) at isotopic steady state, respectively. The isotopic enrichment in plasma KIC is thought to reflect intracellular [13C]leucine enrichment better than does plasma [13C]leucine enrichment (31, 32). In studies in which [13C]KIC determinations were performed, the latter were therefore used to calculate leucine kinetics. In studies in which only plasma [13C]leucine enrichments were determined, the latter were corrected assuming a ratio of [13C]KIC to [13C]leucine of 0.77, on the basis of the results of Matthews et al (32). Because leucine is an essential amino acid, the only source of plasma leucine is leucine release from protein breakdown and exogenous leucine intake. Thus, for studies performed in the postabsorptive state, Leu Ra = leucine release from protein breakdown. For studies performed during parenteral nutrition (16, 29, 30), leucine release from protein breakdown was calculated by subtracting the intravenous leucine intake from Leu Ra (28).
Appearance rate of glutamine
The Gln Ra in plasma (µmol•kg-1•h-1) was calculated as follows:
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| (2) |
where iGln is the infusion rate of [15N]Gln and Ei and Ep are the enrichments of 15N determined in the infused isotope solution and in plasma glutamine at isotopic steady state, respectively. Because glutamine is a nonessential amino acid, both the release of glutamine from protein breakdown and the de novo synthesis of glutamine contribute to the Gln Ra. If one assumes that the release of amino acids from proteolysis is directly proportional to their abundance in whole-body protein, the release of glutamine from protein breakdown (B) can be estimated as follows:
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| (3) |
where K is the ratio of the abundance of glutamine to leucine in proteins (both expressed in mmol amino acids/g protein). In the present study, on the basis of determinations made in human muscle, the abundance of leucine was assumed to be 91.8 mg/g protein, ie, 700 µmol/g body protein, and the glutamine content of body protein was assumed to be the mean value measured for human muscle protein, ie, 43.2 mg (296 µmol)/g body protein (see Results). Thus, K = 296/700 = 0.423. The fraction of the Gln Ra that cannot be accounted for by release of glutamine protein breakdown was attributed to the de novo synthesis of glutamine. Thus,
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Statistical analysis
For each muscle specimen, we carried out protein extraction and all analytic methods in duplicate. The relative error of an analytic method (100 x method error/overall mean) was calculated according to known formulas (33). Results are presented as means ± SDs (n = 4–10) for each species. The relative method error of the protein extraction procedure [including the protein determination method of Bradford (21)] was 9.3%. The relative method error of the diphenylamine assay was 0.18%. The relative error of the BTI method, including derivatization, hydrolysis, and HPLC analysis was 11.2%. Group comparisons were performed by using Student's t test for unpaired samples. The level of significance was set at P < 0.05. Coefficients of correlation between glutamine content and age or BMI were calculated by linear regression analysis; the significance of the linear regression was evaluated by analysis of variance.
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RESULTS |
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. We determined protein-bound glutamine concentrations in the ASP extracts and found similar values in human and animal specimens, ranging from 40.5 to 48.5 µg Gln/mg ASP, as shown in Table 2. Glutamic acid concentrations ranged from 111.1 to 171.5 µg/mg ASP. Glutamine and glutamic acid concentrations were also expressed per mg DNA, as summarized in Table 2.
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and are expressed per mg DFFS.
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, the concentration of protein-bound glutamine + glutamic acid (Glx) in the various species and, more importantly, the proportion of glutamine (black section of the bars) are depicted. For comparison, the concentration of Glx in human muscle protein as determined by conventional analytic methods is also given (34). During acid hydrolysis of muscle protein without prior BTI treatment, glutamine was quantitatively converted to glutamic acid. Consequently, differentiation between glutamine and glutamic acid was not possible.
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(16, 28–30). To perform these calculations, the mean values of 43.2 and 91.8 µg/mg ASP obtained from human (men and women) muscle were used as estimates of body protein glutamine and leucine contents, respectively, in all age groups.
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DISCUSSION |
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Glutamine release from protein breakdown and glutamine de novo synthesis were estimated previously by using a Glx concentration of 139 µg/mg body protein (16). However, because it is known that glutamate is a component of whole-body protein, use of this value results in underestimation of de novo synthesis. Therefore, more recent studies have assumed that glutamine residues contribute to one-half of total Glx, ie, 69.5 µg/mg protein (2). In the present study, however, the measured true concentrations of protein-bound glutamine in the ASP fraction of human and selected mammalian muscle protein ranged from 40.5 to 48.5 mg Gln/mg ASP, with an overall average of 4.36%. These concentrations are considerably lower than previously assumed. In fact, glutamine only amounts to 25% of total Glx (Figure 1
). Concentrations of Glx, as determined in various species, compared well with results obtained by conventional analytic methods in mixed human muscle protein (34).
Mixed muscle protein is made up of hundreds of protein fractions, each presumably having different glutamine contents. We measured glutamine in the ASP fraction, which is the cell protein fraction (20). We did not consider the non-ASP fraction of muscle protein. This insoluble fraction contains 50% collagen and yields 16% of total muscle protein. Collagen thus accounts for 0.4% of total protein-bound glutamine in muscle (35); however, this fraction is metabolically inactive and may not be relevant when calculating turnover.
It is conceivable that measured glutamine concentrations are closely related to the composition of the alkaline protein extract. Proportions of the various fractions in the alkaline extract may vary according to their occurrence in muscle tissue and, of course, according to the completeness of the extraction procedure. Consequently, slight differences in the glutamine concentrations, as observed between the various species, were not considered to be species related. When expressed per mg DNA as a base of reference, no influence of sex, age, or BMI on human muscle protein-bound glutamine was seen. Although glutamine concentrations expressed per mg ASP were significantly higher in women than in men, the ratio of the abundance of glutamine to leucine in the muscle ASP was not, which is pertinent for the calculation of glutamine kinetics.
Our data strongly suggest that calculations of the proportion of de novo synthesized glutamine in muscle must be revised by using the assayed concentrations of glutamine. Darmaun et al (16) calculated a 61.6% contribution of de novo synthesis in total muscle glutamine turnover in healthy humans, assuming a 13.9% glutamine concentration. Considering the considerably lower glutamine concentrations found in the present study, a similar calculation yields an increased proportion of de novo synthesis of 86% of total glutamine turnover.
Considering the amount of glutamine (4.32%) and leucine (9.18%) in human muscle protein assayed in the present study, we used data compiled from selected earlier studies to recalculate the contribution of proteolysis and de novo synthesis to the overall Gln Ra in selected age groups of healthy humans (Table 4). Even though both the Gln Ra and estimated rates of glutamine de novo synthesis decline continuously through development—from infancy through adulthood—the relative contributions of proteolysis and de novo synthesis remain remarkably constant at 13% and 87%, respectively (Table 4). This suggests that the ratio of leucine to glutamine in muscle protein remains constant throughout growth and development. Although skeletal muscle contributes only 25% to whole-body protein turnover in humans (36), 70% of overall glutamine production in healthy adults arises from skeletal muscle (37). The relative contribution of muscle and other tissues to Gln Ra, however, has not yet been determined in other age groups, and the relative size of body organs changes dramatically throughout growth (38). The fact that the relative contribution of de novo synthesis and proteolysis to the Gln Ra remains constant throughout growth suggests that the ratio of leucine to glutamine in all tissues must be very similar to that measured in muscle protein in adults.
The present findings emphasize the prominent role of de novo synthesis of glutamine in the maintenance of glutamine homeostasis. There is ample evidence in the literature to suggest that under conditions of stress, the demand for glutamine in glutamine-consuming tissues is dramatically enhanced. Assuming that, in response to a severe illness, a fasting healthy adult must abruptly double his or her glutamine production (eg, from 325 to 650 µmol•kg-1•h-1), the extra amount (325 µmol•kg-1•h-1) of glutamine could, in theory, be produced either by 1) an 7-fold increase in his or her rate of glutamine release from proteolysis (from 46 to 325 µmol•kg-1•h-1) with no change in glutamine de novo synthesis, or, alternatively, 2) through a mere 2-fold increase in the rate of glutamine de novo synthesis (from 279 to 604 µmol•kg-1•h-1), with no alteration in proteolysis. From a teleologic standpoint, it thus seems much more economical to enhance glutamine de novo synthesis. In an earlier study (16), we observed that when healthy volunteers received an intravenous infusion of cortisol sufficient to raise plasma cortisol to concentrations seen under conditions of moderate stress, glutamine release from proteolysis increased by 13%, whereas glutamine de novo synthesis increased by only 44% (from 279 to 401 µmol•kg-1•h-1) and accounted for the bulk of the extra glutamine produced.
In the present study, we provided new data on whole-body protein glutamine concentrations as determined in mixed muscle protein from various species. We pointed out the heterogeneity of mixed muscle protein, consisting of >100 fractions, each having not only different glutamine concentrations but also different turnover rates. Myofibrillar proteins have the lowest turnover rate, close to 1%/d, whereas mitochondrial proteins and cytosolic enzymes generally have higher turnover rates (36). During the various stages of muscle protein catabolism, these fractions are affected differently. Although in the beginning stages, mainly short-lived proteins such as enzymes are degraded, a loss of myofibrillar protein is characteristic in the severe muscle wasting occurring under conditions of prolonged catabolic stress. Under these conditions, the amount of sarcoplasmic soluble protein might even increase slightly. In future studies, concomitant measurement of endogenous glutamine production and analyses of arteriovenous balances of 3-methylhistidine might enable the development of new kinetic models.
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ACKNOWLEDGMENTS |
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