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American Journal of Clinical Nutrition, Vol. 87, No. 3, 666-678, March 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Increasing habitual protein intake results in reduced postprandial efficiency of peripheral, anabolic wheat protein nitrogen use in humans1,2,3

Barbara Juillet, Hélène Fouillet, Cécile Bos, François Mariotti, Nicolas Gausserès, Robert Benamouzig, Daniel Tomé and Claire Gaudichon

1 From INRA, AgroParisTech, UMR914 Nutrition Physiology and Ingestive Behavior, CRNH-IdF, Paris, France (BJ, HF, CB, FM, DT, and CG); Danone Vitapole, Massy-Palaiseau, France (NG); and the Department of Gastroenterology, Avicenne Hospital, Bobigny, France (RB)

2 Supported by grants from Danone (Paris, France) and ITCF (Institut Technique des Céréales et des Fourrages, Paris, France).

3 Address reprint requests to H Fouillet, AgroParisTech, UMR914 INRA-AgroParisTech Nutrition Physiology and Ingestive Behavior, 16 rue Claude Bernard, F-75005 Paris, France. E-mail: helene.fouillet{at}agroparistech.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 REFERENCES
 
Background: The postprandial retention of dietary protein decreases when the prevailing protein intake increases.

Objective: We investigated the influence of the prevailing protein intake on the regional utilization and anabolic use of wheat protein during the postprandial non–steady state in humans.

Design: Healthy adults (n = 8) were adapted for 7 d, first to a normal-protein diet (NP: 1 g · kg–1 · d–1) and then to a high-protein diet (HP: 2 g · kg–1 · d–1). After each adaptation period, the subjects received the same single, solid mixed meal containing [15N]-labeled wheat protein. The postprandial kinetics of dietary nitrogen were then measured for 8 h in blood and urine. These data were further analyzed by using a multicompartmental model to predict the postprandial kinetics of dietary nitrogen in unsampled pools.

Results: The postprandial whole-body retention of wheat protein nitrogen, measured 8 h after meal ingestion, decreased by 10% when the subjects switched from the NP diet to the HP diet. According to modeling results, this resulted from an increased splanchnic utilization of dietary nitrogen for urea production, whereas its incorporation into splanchnic proteins was unchanged, leading to a 20–30% decrease in peripheral availability and anabolic use in HP-adapted compared with NP-adapted subjects having ingested the same protein load.

Conclusions: By combining clinical experimentation with compartmental modeling, we provide a global overview of postprandial dietary protein metabolism. Increasing prior protein intake was shown to reduce the postprandial retention of wheat protein nitrogen, mainly by diminishing the efficiency of its peripheral availability and anabolic use.

Key Words: High-protein diets • protein metabolism • urea production • protein quality • compartmental model


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 REFERENCES
 
Considerable attention has already been paid to the metabolic consequences on whole-body nitrogen metabolism of changes to the prevailing protein intake, leading to the conclusion that habituation to a high-protein diet results mainly in higher deamination losses (1-5). However, it remains unclear whether this increase in deamination losses results from stimulated urea production (3, 5, 6) or from control over urea hydrolysis (7, 8). Adaptation to high protein intakes has also been shown to induce an increase in the daily cycling of protein gains and losses. In particular, this involves a higher protein accretion through the inhibition of proteolysis and slight activation of protein synthesis (3, 9-12). However, these studies were carried out with the use of steady state tracer methodologies, which did not allow investigation of the acute metabolic responses to the ingestion of a normal single meal in the postprandial non–steady state (13, 14). In addition, because of the limited access in humans to some regional nitrogen pools of interest, such as muscular and hepatic amino acids and proteins, most of these investigations were conducted at the whole-body level, thus making it impossible to determine the specific contribution of the splanchnic or peripheral areas to total protein accretion. Few data are thus available regarding the influence of prior protein intake on postprandial inter-organ metabolism and the specific utilization of dietary proteins in humans.

After the ingestion of a meal, the inter-organ distribution and assimilation of dietary amino acids throughout the body constitutes a crucial step for protein homeostasis, ensuring protein repletion by counterbalancing postabsorptive losses (15, 16). This involves a cascade of dynamic metabolic processes, which are modulated by numerous nutritional and physiologic factors, including the amino acid composition of the ingested load (17-19), its kinetic behavior within the gastrointestinal tract (20-22), and habitual protein and energy intakes (9, 23, 24). In particular, it was recently shown that increasing the dietary protein content from a moderate (1 g · kg–1 · d–1) to a high (2 g · kg–1 · d–1) level diminished the whole-body postprandial retention of both milk and soy proteins in humans, when assessed as the fraction of ingested nitrogen that was not eliminated through ileal or deamination losses at the end of the 8-h postprandial phase (23). This phenomenon was much more pronounced when the protein source in the meal was soy rather than milk, which indicates that an elevation of the habitual protein intake accentuates the gap in nutritional quality between dietary protein sources. According to this assumption, the nutritional value of wheat protein—which is known to be lower than those of milk and soy proteins (25-27)—may be drastically decreased in the context of a Western diet, which is generally rich in protein (28, 29).

The aim of the present work was to investigate in humans the degree of influence of habitual protein intake on the postprandial inter-organ metabolism of wheat protein nitrogen under realistic, physiologic conditions of ingesting a single, solid mixed meal. For this purpose, healthy adults were adapted for 7 d, first to a normal protein intake (NP: 1 g · kg–1 · d–1) and then to a high protein intake (HP: 2 g · kg–1 · d–1). At the end of each adaptation period, all subjects received the same test meal containing [15N]-labeled wheat protein and then underwent an 8-h investigation to measure the postprandial kinetics of dietary nitrogen in blood and urine. The experimental data were used to calculate the postprandial retention of wheat protein nitrogen at the whole-body level in NP- and HP-adapted subjects. The data were then analyzed by compartmental modeling to further explore the postprandial distribution of dietary nitrogen between unsampled pools of the splanchnic and peripheral areas, and thus to clarify the influence of habitual protein intake on the regional metabolic utilization of wheat protein nitrogen in humans.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 APPENDIX A
 REFERENCES
 
Subjects
Eight adults (4 women, 4 men) in good health, as determined by a medical examination and routine blood tests, were enrolled in this study. The subject characteristics are detailed in Table 1Go. The design and aims of the study were fully explained to each subject, and written informed consent was obtained. All procedures during the study were approved by the Institutional Review Board for St-Germain-en-Laye Hospital, France.


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  		TABLE 1. Subject characteristics1

 
Study design
The subjects first underwent dietary standardization for 7 d with an NP intake, as already detailed (23). At the end of this adaptation period, they attended the hospital for a postprandial metabolic test: on the morning of day 8, after an overnight fast, the NP-adapted subjects ingested a test meal containing [15N]-labeled wheat protein and then underwent a postmeal investigation for 8 h to measure dietary nitrogen postprandial kinetics in blood and urine. They were then adapted for the next 7 d to an HP diet. On the morning of day 16, ie, at the end of the second adaptation period, the same metabolic test was repeated (same test meal, same experimental protocol).

Adaptation diets
Both NP and HP adaptation diets were adjusted for body weight and were designed to be isoenergetic (138 kJ · kg–1 · d–1). They supplied the same amount of carbohydrate (4.5 g · kg–1 · d–1), whereas an isoenergetic exchange occurred between protein (NP: 1 g · kg–1 · d–1 and HP: 2 g · kg–1 · d–1) and fat (NP: 1.2 g · kg–1 · d–1 and HP: 0.8 g · kg–1 · d–1). Total energy was distributed into 12% protein, 55% carbohydrate, and 33% fat for the NP diet versus 24% protein, 54% carbohydrate, and 22% fat for the HP diet. All subjects were asked to adhere to 3 main meals with few intermittent snacks and were provided with a detailed diet program for each day, weighing scales, and daily record sheets.

Postprandial metabolic test
The subjects were admitted to the hospital on the morning of days 8 and 16 after they had fasted overnight. A catheter was inserted into their superficial forearm vein for blood sampling, and they ingested a test meal containing intrinsically and uniformly [15N]-labeled wheat protein, obtained and prepared as previously described (27). This 15N methodology allows us to specifically follow the metabolic fate of the amino groups moiety of the dietary amino acids, whatever their intermediary fate and independently of the fate of the carboxyl groups and carbon skeletons of the amino acids. This method has been widely used by our group as an excellent tracer of dietary nitrogen (17, 27, 30-32). The test meal was a solid mixed meal containing wheat protein in the form of biscottes (a typical French breakfast ingredient resembling toast) and it was adjusted for body weight, providing 46 kJ/kg (ie, one-third of the previous daily energy intake). The meal composition corresponded to dietary allowances as follows: 15% of total energy as protein, 54% as carbohydrate (one-quarter in the form of sucrose and three-quarters in the form of maltodextrins), and 31% as fat (sunflower oil). The test meal provided 0.41 g protein/kg, 1.51 g carbohydrate/kg, and 0.38 g fat/kg. The mean amount of ingested nitrogen was 4.85 mmol/kg. At time zero, after the collection of baseline blood and urine samples, the test meal was ingested. Blood was sampled every 30 min for 3 h and then hourly for 5 h. Plasma and serum were separated by centrifugation, divided into aliquots, and frozen at –20 °C. Total urine was collected every 2 h throughout the 8-h postprandial period. Urine specimens were weighed, divided into aliquots, and stored at –4 °C until analysis.

Analytic methods
Urea concentrations were measured in both serum and urine samples with the use of an enzymatic method on a Dimension automat (du Pont de Nemours, Les Ulis, France). Ammonia was measured in urine on a Kone instrument (Kone automat, Evry, France). 15N enrichments in serum urea, protein, and free amino acids and in urinary samples were measured by isotopic ratio mass spectrometry (Optima; Fisons Instruments, Manchester, UK), coupled to an elemental nitrogen analyzer (NA 1500 series 2; Fisons Instruments), as detailed elsewhere (31).

Calculations
Dietary nitrogen incorporation (Ndiet) into the sampled pools (serum protein and free amino acids, body urea, urinary urea, and ammonia) was evaluated at each sampling time as follows:

Formula 1(1)
where Ntot (t) was the total nitrogen content (in mmol N) of the sampled pool considered at sampling time t, APE(t) is the [15N] enrichment above baseline at time t, APEmeal is the [15N] enrichment of the meal, and Ningested is the nitrogen content of the meal (in mmol N). The total nitrogen content in serum protein was calculated from the nitrogen concentration measured in this fraction and from plasma volume, estimated at 5% of body weight (33). The total nitrogen content in body urea was calculated knowing the plasma urea nitrogen concentration and its volume of distribution (total body water), corrected by the multiplying factor 0.92, which represents the water content in blood. The total nitrogen content in urinary urea (ammonia) was determined from the volume of urine and from the urea (ammonia) nitrogen concentration. For compartmental analysis, urinary data were interpolated in such a way as to obtain the same 1-h step size.

At the end of the 8-h experimental period, total urea production (ie, the production of urea nitrogen from both dietary and endogenous sources) was assessed from urinary urea excretion over the 8-h period, corrected for changes to the body urea pool size (17, 23, 34). Moreover, the net postprandial protein utilization (NPPU), defined as the fraction of dietary nitrogen that was not recovered in ileal effluents, urine, and body urea 8 h after the meal, was calculated by using the following formula (23, 27, 30):

Formula 2(2)
where Ndiet-urea (Ndiet-ammonia) was the amount of dietary nitrogen present at 8 h in blood or urine in the form of urea (ammonia), and D is the ileal digestibility of wheat protein, evaluated at 90.3% of ingested nitrogen during a previous study (27). NPPU constitutes an index of the postprandial retention of dietary protein nitrogen and represents the fraction of wheat protein nitrogen truly retained in the body, ie, absorbed but not lost through deamination after 8 h.

Analysis of kinetic data
Data concerning the postprandial kinetics of dietary nitrogen into the sampled blood and urinary pools were analyzed by multicompartmental modeling to predict the postprandial distribution of dietary nitrogen from its ingestion to its disposal or incorporation into the main regional nitrogen pools of the body, as previously detailed and documented (35-37). Thus, a linear multicompartmental model with 13 compartments and 19 pathways was previously developed by use of similar experimental data obtained after the ingestion of a liquid, mixed soy protein meal in humans (36). In the present work, we tested this basic model with the current experimental data obtained after ingestion of a solid, mixed wheat protein meal in humans so as to obtain a new—either reduced or expanded—model that could adequately fit these data.

Definitions and calculations
The model compartments represent kinetically homogeneous amounts of dietary nitrogen present at a particular site in the body (eg, in the intestinal lumen), in a particular metabolic form (eg, in body urea), or a combination of both (eg, in a free amino acid form in the splanchnic bed). The compartments are linked by dietary nitrogen exchange pathways, each of which is characterized by a constant diffusion coefficient (or transfer rate) ki,j, representing the fraction of dietary nitrogen transferred from compartment j to compartment i per unit of time (in min–1). The size of compartment i at time t is denoted by qi(t), in % of dose, the flux of dietary nitrogen from compartment j to compartment i at time t is calculated as

Formula 3(3)
in % of dose per min, and the total amount of dietary nitrogen that has been transferred from compartment j to compartment i at time t is calculated as

Formula 4(4)
in % of dose. A compartmental model is a mathematical construct to be tested against experimental data, its structure and parameters being modified until satisfactory fits to the data and reliable modeling results are obtained.

Structure identification and a priori global identifiability
The methods used to identify the adequate structure for the model were based on the principle of parsimony and consisted in testing the goodness-of-fit of different models of increasing order, starting with the structure of the basic model (36) and retaining the simplest reduced or expanded structure that adequately fitted the data versus higher-order models that did not significantly improve the fit (35, 38). To achieve this, different candidate models with a firm physiologic basis were all confronted with the mean of individual experimental data of each group (NP and HP) and then discriminated by using the Akaike and the Schwarz criteria (38, 39) and the likelihood ratio test (40). Furthermore, the a priori global identifiability of the selected structure was tested by using the GLOBI2 software package (41) to ensure that all model parameters had a unique solution in the ideal context of an error-free structure and noise-free, continuous data (42). This identifiability test was necessary to avoid the choice of a structure that would allow several or an infinite number of parameter sets to reproduce the data.

Data fitting and model validation
Model parameters were estimated by using both mean and individual experimental data (NP and HP) so that they would produce model predictions closest to the experimental observations for all sampled compartments simultaneously. The objective function used to quantify the goodness-of-fit of model predictions was the logarithm of the likelihood function. During the fitting process, the parameters were allowed to vary simultaneously until a maximum of the objective function was reached, using an optimization strategy that we recently developed to solve such a complex problem (37, 43). The goodness-of-fit of model predictions were tested as previously detailed and documented (35, 37): first by visual inspection of the model-simulated curves versus experimental data and analysis of the percentage variations explained for each sampled compartment (R2 > 95% considered as satisfactory), and second through a further analysis of standardized residuals (ie, the differences between observed and predicted data divided by the corresponding standard deviations) to detect any systematic deviations between data and predictions (35, 40). The reliability of parameter estimates was also evaluated from the inverse of the Fisher information matrix (44): their precision was expressed in terms of a percent fractional SD or CV, parameter values with a CV < 50% being usually considered as estimated with sufficient precision (45). Quantitative assessment of model validity was completed with an external validation of the model. Model simulations regarding the sizes and evolution of unsampled compartments and the values of dietary nitrogen fluxes between them were strictly validated to ensure that they captured most known features of the postprandial metabolism of dietary nitrogen. Model predictions concerning the postprandial fate of dietary nitrogen after each adaptation period were deduced from the parameter estimation process carried out on the mean of individual data, whereas individual fits were used to assess statistical differences between the 2 adaptation conditions.

Statistical analysis
Results are presented as means ± SDs. Differences between adaptation periods (HP versus NP) concerning the postprandial kinetics of dietary nitrogen in the sampled and unsampled pools, and the instantaneous and cumulated fluxes of dietary nitrogen between compartments, were analyzed by using mixed models for repeated-measure analysis (version 9.1; SAS Institute Inc, Cary, NC) with time and diet (NP or HP) as independent factors. Differences with P values < 0.05 were deemed to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 APPENDIX A
 REFERENCES
 
Dietary nitrogen postprandial kinetics in sampled pools
Few differences were seen between the 2 adaptation periods (HP versus NP) in terms of postprandial kinetics and dietary nitrogen incorporation into the serum protein and free amino acid pools (Figure 1Go, A and B). In particular, the incorporation of dietary nitrogen into plasma proteins reached {approx}8.0 ± 1.1% of ingested nitrogen at the end of the 8-h investigation period after both adaptation levels (Figure 1AGo). The kinetics of dietary nitrogen appearance in plasma free amino acid was also similar under both adaptation conditions, with a maximum value reaching {approx}0.6 ± 0.1% of the dose between the second and third hours after the meal (Figure 1BGo). By contrast, dietary nitrogen orientation toward the deamination pools was markedly influenced by the habitual protein intake (Figure 1Go, C and D). The amount of dietary nitrogen recovered in body urea was significantly higher (P < 0.01) in HP- than in NP-adapted subjects as early as the second hour after the meal, with maximum values reaching 24.9 ± 4.0% and 21.4 ± 3.5% of the dose at {approx}5 h, respectively (Figure 1CGo). Dietary nitrogen excretion in the form of urinary urea was subsequently increased (P < 0.01) by the HP diet, reaching values at 8 h of 11.9 ± 3.1% and 15.7 ± 4.2% of ingested nitrogen after NP versus HP adaptation, respectively (Figure 1CGo). There was also a trend (0.05 < P < 0.1) toward an increased excretion of dietary nitrogen in the form of urinary ammonia during the second half of the postprandial period in HP- versus NP-adapted subjects, with maximum values reaching 0.9 ± 0.3% and 0.7 ± 0.2% of the dose at 8 h, respectively (Figure 1DGo). As a result, the amount of wheat protein nitrogen retained in the body 8 h after meal ingestion was reduced by 10% (P < 0.001) when the subjects switched from the NP to the HP diet, as reflected by the NPPU (fraction of dietary N retained in the body, ie, used or available for anabolic purposes), which decreased from 61.3 ± 3.7% to 55.2 ± 4.8% of ingested nitrogen, respectively.


Figure 1
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  		FIGURE 1.. Experimental data and model-predicted kinetics of dietary nitrogen (N) in the sampled compartments in response to a single, solid mixed meal containing [15N]-labeled wheat protein after adaptation for 7 d to a normal protein (NP: 1 g · kg–1 · d–1) or high-protein (HP: 2 g · kg–1 · d–1) diet. A: Plasma proteins. B: Plasma free amino acids (AA). C: Body (BU) and urinary (UU) urea. D: urinary ammonia. Observed data (circles: NP; triangles: HP) and model predictions (lines: NP; dashes: HP) are expressed as a percentage of ingested N over time. Each experimental data point is plotted by value ± SD. Model predictions were obtained after optimization by use of the mean of individual experimental data. For a description of the model and abbreviations, see Figure 2Go. *,#Significant difference between the NP and HP conditions: *P < 0.05 (mixed models for repeated-measures analysis), #P < 0.1.

 
Model identification and validation
According to the principle of parsimony, the model presented in Figure 2Go was the simplest structure that adequately fitted both sets of data (NP and HP). The selected model integrated the 5 sampled compartments, as well as 7 other compartments that were not monitored experimentally, in particular, splanchnic and peripheral free amino acids and proteins. Because of the solid nature of the test meal, the gastric emptying of dietary nitrogen was represented by a linear second-order ordinary differential equation (see Appendix A), which was inspired by current knowledge of the literature (46) and was validated by using experimental data obtained previously with the use of duodenal tubes for the gastric emptying of dietary nitrogen after the ingestion of a similar solid, wheat protein meal in humans (27). The model was selected from numerous candidate structures with a firm physiologic basis, as being the minimum structure that adequately fitted the data versus higher-order structures that did not significantly improve the fit. Compared with the structure selected, one of the lower-order models was characterized by an absence of the pathway representing urea hydrolysis (k4,10), whereas one of the higher order models integrated an additional intestinal compartment, in line with the classic method used to model the gastrointestinal tract with a catenary structure (36, 47). The results of the comparisons between these nested models are shown in Table 2Go. The Akaike and Schwarz criteria and ratio tests all led to selection of the chosen model, which significantly improved the fit by comparison with lower-order models, there being no significant improvement in the fit between the chosen model and higher-order models.


Figure 2
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  		FIGURE 2.. Compartmental model of postprandial dietary nitrogen (N) distribution in humans. Circles indicate compartments representing kinetically homogeneous amounts of dietary N, arrows between compartments represent transfer pathways, and coefficients ki,j next to the arrows indicate constant transfer rates from compartment j to compartment i. Bullets S1 to S5 indicate sampled compartments. The model is divided into 3 subsystems: the gastrointestinal tract, deamination, and retention. The gastrointestinal tract subsystem consists of 3 compartments representing dietary N in the stomach (G), in the intestinal lumen (IL), and in ileal effluents (E). It includes 2 unidirectional pathways representing the gastric emptying of dietary N (G to IL) and its subsequent intestinal transit to ileal effluents (IL to E) and one bidirectional pathway representing the metabolic phenomena of dietary N intestinal absorption to splanchnic free amino acids (SA) and its reciprocal secretion into the intestinal lumen (IL to SA and SA to IL, respectively). Dietary N is considered to be evacuated from the stomach according to a second-order ordinary differential equation involving parameters k2,1 and B (see Appendix A). The deamination subsystem is made up of 3 compartments representing dietary N in body urea (BU), urinary urea (UU), and urinary ammonia (UA). It includes 5 metabolic or exchange pathways representing urea production (SA to BU), urea hydrolysis and enterohepatic recycling (BU to SA), urea excretion into urine (BU to UU), and ammonia production from the splanchnic and peripheral free amino acids (SA to UA and PA to UA, respectively). The retention subsystem is made up of 6 compartments representing dietary N in the splanchnic free amino acids (SA), splanchnic constitutive proteins (SCP), splanchnic exported plasma proteins (SEP), plasma free amino acids (PL), peripheral free amino acids (PA), and peripheral proteins (PP). It includes 9 pathways representing bidirectional transfers of dietary N between splanchnic and plasma free amino acids (SA to PL and PL to SA) and between plasma and peripheral free amino acids (PL to PA and PA to PL); dietary N incorporation into splanchnic constitutive proteins (SA to SCP), splanchnic exported proteins (SA to SEP), and peripheral proteins (PA to PP); or dietary N release from the degradation of splanchnic constitutive proteins (SCP to SA) and peripheral proteins (PP to PA). The synthesis pathway from SA to SEP integrates a variable delay representing the secretion time of plasma proteins, ie, the time elapsing between the synthesis of plasma proteins and their appearance in the blood. AA, amino acids.

 

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  		TABLE 2. Objective criteria governing the choice of the model structure from a series of candidate models1

 
In addition, the parameters of the selected model (transfer rates) were all globally uniquely identifiable, as checked by a priori identifiability analysis. The a posteriori, numerical identifiability of the model was also checked to ensure confidence in its results and ensuing predictions. As shown in Table 3Go, for both sets of experimental data (NP and HP), all model parameters were estimated with satisfactory precision. Their average and maximum CV values (relative errors between optimum and estimated parameters) were about 10% and 40%, respectively. Besides, the goodness-of-fit of the model predictions was deemed satisfactory for each sampled compartment (serum protein and free amino acids, body urea, urinary urea, and urinary ammonia) of each data set (NP and HP) by visual inspection of the simulated curves versus observed data (Figure 1Go) and further analysis of the standardized residuals (not shown). For both data sets, the percentage variations explained reached {approx}96% for compartment PL and were superior to 98.5% for the other sampled compartments, which indicates that model predictions were highly satisfactory. Finally, the model predictions were strikingly in line with our current knowledge of the system and the scarce data available in the literature regarding 1) the gastrointestinal kinetics of dietary nitrogen, characterized by its moderate gastric emptying and rapid intestinal absorption (46, 48, 49) and also 2) the regional distribution and metabolism of dietary nitrogen, with its predominant splanchnic uptake during the early postprandial phase (50-52), modulating the subsequent delivery of dietary amino acid to peripheral tissues (53-57). The range of variation covered by the model predictions for both diets in terms of splanchnic and peripheral metabolisms of dietary nitrogen are presented in Table 4Go, together with the corresponding previous findings in the literature (2-4, 50-55, 58-70). The paucity of such findings in the literature highlights the need for additional data to further validate the model predictions. However, the plausibility and physiologic relevance of the model are supported by the consistency of its predictions with respect to our current knowledge of the system (71).


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  		TABLE 3. Main parameter estimates and their precision for each adaptation condition1

 

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  		TABLE 4. External validation: comparison of findings in the literature and model predictions concerning the regional metabolism of dietary nitrogen1

 
Model predictions for the postprandial distribution of dietary nitrogen
The model made it possible to obtain additional information about the postprandial fate of dietary nitrogen in the body under both adaptation conditions. These model predictions—in terms of both parameter values (transfer rate constants between compartments) and compartment sizes (kinetics of dietary nitrogen in the unsampled compartments)—were not accessible from the experimental data themselves.

Parameter estimates and compartment sizes
During the compartmental analysis of the experimental data, significant differences between the NP and HP diets in the sampled compartments of the deamination subsystem (BU, UU, and UA, Figure 1Go, C and D) led to important differences in their closely related model parameters (k4,10, k10,4, k12,4, and k12,8, which varied from 20% to 90% between diets, Table 3Go), and the absence of significant differences in the sampled compartments of the retention subsystem (SEP and PL, Figure 1Go, A and B) led to lesser differences in their neighborhood parameters (k6,4, k7,4, and k8,7 that varied by <15% between diets, Table 3Go). Because nonsignificant differences in primary data generated no significant differences within the corresponding model parameters, the modeling process proved to comply with the principle of parsimony. Results concerning the predicted compartment sizes cannot be interpreted so easily inasmuch as each of them was the complex result of all model parameters of all subsystems varying simultaneously. Indeed, the diet effect on a given compartment was the consequence of integrated diet-induced effects on parameters of the gastrointestinal, deamination, and retention subsystems.

Dietary nitrogen losses and regional retention
By construction, model predictions first confirmed the experimental results regarding both ileal (E) and deamination (calculated as BU + UU + UA) losses (Figure 3AGo). The delivery of dietary nitrogen to the ileum was predicted to be relatively similar under both adaptation conditions: it was only transiently accelerated by the HP diet (between 2 and 4 h, P < 0.005), before reaching at 8 h for both adaptation levels the same value of {approx}10% of the dose that was previously observed for the test meal (27). In contrast, deamination losses were significantly increased (P < 0.02) after HP adaptation, as early as the second postmeal hour, with this increase reaching 20% at the end of the postprandial period (from 29% to 35% of the dose). Furthermore, the model allowed a distinction between the splanchnic and peripheral parts of the postprandial whole-body retention of dietary nitrogen (Figure 3BGo). The splanchnic retention of dietary nitrogen (calculated as SA + SCP + SEP) was not significantly affected by habitual protein intake, whereas its peripheral retention (calculated as PA + PP) was significantly lowered by the HP diet at the end of the 8-h investigation period. The final postprandial value was reduced (P < 0.0001) from 17 ± 2% to 13 ± 1% of the dose when the subjects switched from NP to HP intake.


Figure 3
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  		FIGURE 3.. Model-predicted kinetics of dietary nitrogen (N) losses and retention in response to a single, solid mixed meal containing [15N]-labeled wheat protein after adaptation for 7 d to a normal protein (NP: 1 g · kg–1 · d–1) or high-protein (HP: 2 g · kg–1 · d–1) diet. A: Ileal (E) and deamination (BU + UU + UA) losses of dietary N. B: Gastric emptying of dietary N (G) and its consecutive incorporation into the splanchnic (S, calculated as SA + SCP + SEP) and peripheral (P, calculated as PA + PP) areas. Model predictions (lines: NP; dashes: HP) were obtained after optimization by using the mean of individual experimental data and are expressed as a percentage of ingested N over time. For a description of the model and abbreviations, see Figure 2Go. *Significant difference between the NP and HP conditions, P < 0.05 (mixed models for repeated-measures analysis).

 
Gastrointestinal kinetics of dietary nitrogen
Dietary nitrogen disappearance from the stomach was predicted to be accelerated when habitual protein intake was increased, the half-time of dietary nitrogen gastric emptying falling from {approx}105 to {approx}85 min when the subjects switched from the NP to the HP diet (Figure 3BGo). This was mainly due to more rapid emptying of the gastric liquid phase, because parameter k2,1 was increased by {approx}20% after transition from the NP to the HP diet (Table 3Go). As a result, the flux of dietary nitrogen delivered to the splanchnic bed from intestinal absorption [fABS, calculated as flux4,2(t) = k4,2 x IL(t)] was sharper after HP adaptation: it was increased by {approx}15% (P < 0.01) during the first postmeal hours and then decreased (P < 0.02) during the second half of the postprandial period (Figure 4AGo). However, the total amount of dietary nitrogen absorbed from the intestinal lumen 8 h after meal ingestion (calculated as {int}08hfABS) finally reached a similar value of {approx}85% of ingested nitrogen with both adaptation levels.


Figure 4
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  		FIGURE 4.. Model-predicted fluxes of dietary nitrogen (N) absorption and metabolism in the splanchnic and peripheral areas in response to a single, solid mixed meal containing [15N]-labeled wheat protein after adaptation for 7 d to a normal protein (NP: 1 g · kg–1 · d–1) or high-protein (HP: 2 g · kg–1 · d–1) diet. A: Flux of intestinal absorption of dietary N [fABS = k4,2 x IL(t)] and its flux of utilization for urea production [fUP = k10,4 x SA(t)]. B: Fluxes of dietary N utilization for the synthesis of splanchnic constitutive [fSCP = k5,4 x SA(t)] and exported [fSEP = k6,4 x SA(t)] proteins. C: Flux of peripheral delivery of dietary N [fPD = k7,4 x SA(t)] and its flux of peripheral anabolic use [fPA = k9,8 x PA(t)]. Model predictions (lines: NP; dashes: HP) were obtained after optimization by using the mean of individual experimental data and are expressed as a percentage of ingested N per min. For a description of the model and abbreviations, see Figure 2Go. *Significant difference between the NP and HP conditions, P < 0.05 (mixed models for repeated-measures analysis).

 
Splanchnic handling of dietary nitrogen
Both splanchnic fluxes for the catabolic and anabolic utilization of dietary nitrogen followed the same pattern as fABS and peaked higher and sooner after meal ingestion in HP- than in NP-adapted subjects (Figure 4Go, A and B). After transition from the NP to the HP diet, dietary nitrogen use fluxes for the synthesis of constitutive and exported proteins [fSCP and fSEP, calculated as flux5,4(t) = k5,4 x SA(t) and flux6,4(t) = k6,4 x SA(t), respectively] were transiently increased by {approx}10% (P < 0.002) during the first postmeal hours before declining (P < 0.05) during the second half of the feeding period (Figure 4BGo). The HP-induced stimulation of urea production [fUP, calculated as flux10,4(t) = k10,4 x SA(t)] was more pronounced and sustained than that of protein synthesis: it increased by {approx}25% during the first 3 postmeal hours (P < 0.005) but no significant decline was observed during the second half of the postprandial period (Figure 4AGo). In the splanchnic area, the catabolic use of dietary nitrogen was thus more markedly stimulated after HP adaptation than its anabolic use, as also indicated by the 20% increase in the rate of dietary urea nitrogen production (parameter k10,4) compared with the 5% increase in the rates of dietary nitrogen incorporation into splanchnic proteins (parameters k5,4 and k6,4) after HP versus NP adaptation (Table 3Go). As a consequence, the splanchnic utilization of dietary nitrogen for anabolic purposes, ie, the total amount of dietary nitrogen used to synthesize proteins in the splanchnic bed (calculated as {int}08hfSCP + {int}08hfSEP), finally reached a similar value at 8 h of 37 ± 6% of ingested nitrogen after both adaptation levels (Figure 5Go). By contrast, the splanchnic utilization of dietary nitrogen for catabolic purposes, ie, the total amount of dietary nitrogen used for urea production (calculated as {int}08hfUP), was significantly increased by the HP diet (P < 0.05) and finally reached values of 41 ± 3% and 47 ± 3% of ingested nitrogen 8 h after meal ingestion in NP versus HP-adapted subjects, respectively (Figure 5Go). Besides, the augmentation of the postprandial transfer into urea of both dietary and endogenous nitrogen was within the same range after transition from the NP diet to the HP diet, because dietary and total urea nitrogen production were increased by 15% (from 2.0 ± 0.1 to 2.3 ± 0.2 mmol N · kg–1 · 8 h–1) and 20% (from 3.9 ± 0.4 to 4.7 ± 0.6 mmol N · kg–1 · 8 h–1) after HP versus NP adaptation, respectively. Furthermore, the rate of dietary urea nitrogen reincorporation into the splanchnic precursor free amino acid compartment through urea hydrolysis and enterohepatic recycling (parameter k4,10) was decreased by {approx}15% after transition from the NP to the HP diet (Table 3Go). Because of the higher production of dietary urea nitrogen after HP adaptation, the total amount of dietary urea nitrogen recycled (calculated as {int}08hfREC = k4,10 x {int}08hBU) did not, however, differ between the 2 adaptation conditions and reached {approx}10% of ingested nitrogen at 8 h. As a consequence, the recycling efficiency, calculated as the proportion of dietary urea nitrogen produced that was salvaged at the end of the postprandial period (ie, {int}08hfREC/{int}08hfUP) was decreased from {approx}30% to 25% (P < 0.0001) when switching from the NP to the HP diet.


Figure 5
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  		FIGURE 5.. Model-predicted 8-h balance for the postprandial utilization of dietary nitrogen (N) in the splanchnic and peripheral areas in response to a single, solid mixed meal containing [15N]-labeled wheat protein after adaptation for 7 d to a normal protein (NP: 1 g · kg–1 · d–1) or high-protein (HP: 2 g · kg–1 · d–1) diet. Open bars: postprandial utilization of dietary N for splanchnic catabolic, splanchnic anabolic, and peripheral anabolic purposes at 8 h. Hatched bars: dietary N recycling into the metabolic N pool through urea hydrolysis at 8 h. Model predictions were obtained after optimization by using the mean of individual data and are expressed as a percentage of ingested N. Values were calculated as the integral over 8 h of the fluxes of dietary N utilization for urea production [k10,4 x {int}08hSA(t)], dietary urea N reincorporation into splanchnic free AA through urea N enterohepatic recycling [k4,10 x {int}08hBU(t)], dietary N utilization for the synthesis of splanchnic both constitutive and exported proteins [(k5,4+k6,4) x {int}08hSA(t)], and dietary N utilization for the synthesis of peripheral proteins [k9,8 x {int}08hPA(t)]. For a description of the model and abbreviations, see Figure 2Go. *Significant difference between NP and HP conditions, P < 0.05 (mixed models for repeated-measures analysis).

 
Peripheral availability and anabolic use of dietary nitrogen
The flux of dietary nitrogen delivery to the periphery, ie, the flux of dietary nitrogen transferred from splanchnic to systemic free amino acids [fPD, calculated as flux7,4(t) = k7,4 x SA(t)], was decreased as early as the second postmeal hour (P < 0.005) after HP adaptation (Figure 4CGo). This resulted from both the higher splanchnic extraction of dietary nitrogen after HP versus NP adaptation (mainly for catabolic purposes) and the concomitant {approx}15% decrease in the rate of dietary nitrogen delivery to the periphery (parameter k7,4, Table 3Go). The HP diet thus induced a 20% decrease (P < 0.0001) in the total amount of dietary nitrogen delivered to the periphery after 8 postprandial hours (calculated as {int}08hfPD), which at that time reached 18 ± 2% and 14 ± 1% of ingested nitrogen in NP- versus HP-adapted subjects, respectively. Moreover, the flux for the peripheral anabolic use of dietary nitrogen [fPA, calculated as flux9,8(t) = k9,8 x PA(t)] was decreased (P < 0.05) as early as the third postmeal hour after HP adaptation (Figure 4CGo), mainly because of a 40% reduction in the rate of dietary nitrogen incorporation into peripheral proteins (parameter k9,8, Table 3Go). Finally, the peripheral anabolic use of dietary nitrogen, ie, the total amount of dietary nitrogen used to synthesize peripheral proteins after 8 postprandial hours (calculated as {int}08hfPA), was decreased by {approx}30% (P < 0.005) from 15 ± 2% to 10 ± 2% of ingested nitrogen when switching from the NP to the HP diet (Figure 5Go). Furthermore, the efficiency of peripheral protein synthesis, defined as the fraction of the peripheral intracellular amino acid flux of appearance incorporated into protein (19, 63, 64), calculated here as the peripheral anabolic use of dietary nitrogen divided by its cumulated availability in the precursor free amino acid compartment for anabolic purposes {ie, {int}08hflux9,8(t)/[{int}08hflux8,7(t)+ {int}08hflux8,9(t)]}, was reduced (P < 0.05) from 50% to 37% after transition from the NP to the HP diet.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
APPENDIX A
 REFERENCES
 
We investigated the effects of doubling the habitual protein intake on the acute postprandial mechanisms involved in the metabolic handling of wheat protein nitrogen after ingestion of a single, solid mixed meal in humans. Our results indicated that the postprandial whole-body retention of wheat protein nitrogen was decreased after adaptation to a high versus a normal protein intake. This effect was accompanied by 1) an acceleration in the kinetics of dietary nitrogen delivery from the gut, 2) an increase in its splanchnic extraction via its enhanced utilization for urea production, and thus 3) a reduction in its availability and consecutive anabolic use in peripheral tissues.

Repeated ingestion of the test wheat protein meal after adaptation to the 2 protein levels produced differences that mainly concerned postprandial deamination losses of both dietary and endogenous nitrogen, which were enhanced by {approx}20% when elevating the prevailing protein intake. This result confirms our previous finding of a 20–40% increase in the postprandial deamination losses of both dietary and endogenous nitrogen after the ingestion of milk or soy protein with the same NP-HP pattern (23). During both studies, the meal ingested at the end of each week of adaptation consisted of the same fixed dose (0.41 g/kg) of milk, soy, or wheat protein. Our results thus showed that the acute ingestion of a reduced, but adequate, protein intake after chronic adaptation to a higher protein level in the usual diet led to an increase in the postprandial losses of both endogenous and dietary nitrogen. This result is in line with findings in the literature concerning the 24-h excretion of total nitrogen (dietary + endogenous), which increases with prior protein intake (2-5, 66), inasmuch as being too high to maintain nitrogen balance when acute protein intake is only adequate (72-74). In addition, compartmental analysis of our experimental data made it possible to distinguish between the metabolic phenomena of dietary nitrogen utilization for urea production and its reincorporation into the splanchnic precursor pool through urea hydrolysis, resulting in its net elimination in the form of urinary urea when corrected for changes in the body urea content. Doubling the prevailing protein intake was thus predicted to induce (8 h after ingestion of the same protein load) a slightly higher catabolic use of dietary nitrogen for urea synthesis (+15%), with no change in its salvage through urea hydrolysis, but a 15% reduction in its recycling efficiency (ie, the proportion of dietary nitrogen used for urea production that was recycled), which fell from 30% to 25%, 8 h after the meal (Figure 5Go). These results do not enable furtherance of the debate on a quantitative relation between nitrogen intake and the 24-h production of urea nitrogen (5, 8), but are in line with previous findings in the literature that reported that the amount of urea nitrogen that is hydrolyzed constitutes a significant fraction ({approx}10–50%) of the 24-h production of urea nitrogen for various protein levels (normal to high) in the diet (2-4, 66-70), recycling efficiency being decreased when the habitual protein intake is increased from normal (3) or low levels (2, 4, 65).

As a consequence of increased postprandial deamination losses, the whole-body fractional retention (ie, NPPU) of wheat protein nitrogen was reduced by {approx}10% (from 61% to 55% of the dose) when shifting from the NP to the HP diet. Because of the specificity and limitations of our 15N labeling method, tracer exchange by transamination between dietary and endogenous amino acids with different composition profiles is possible and implies that measurement of NPPU may not strictly reflect the fate of the carbon skeletons of dietary amino acids. This remains theoretical, however, and cannot be practically verified. We have also no reason to believe that this phenomenon may vary in magnitude between the 2 NP and HP study periods and influence our finding that increasing the prior protein intake reduces the postprandial retention of wheat protein N. This reduction in the NPPU reflected the weaker drive of wheat protein N toward anabolic uses: a shift was observed in its relative postprandial orientation in favor of increased catabolic versus anabolic use after HP adaptation. This feature was a consequence of the increase in the habitual level of amino acid catabolism that is known to occur after a sustained increase in the habitual protein intake, so that it equilibrates nitrogen balance (73, 75). In line with this, we recently found a drastic change in the NPPU of soy protein nitrogen; this fell by {approx}15% (from 71% to 61% of the dose) after transition from the same NP to HP diets (23), whereas the NPPU of milk protein nitrogen fell only moderately, by 5% (from 74% to 71% of the dose). When compared with milk and soy proteins, wheat protein remained the protein with lower nutritional value in both adaptation conditions. In addition, the NPPU of wheat protein nitrogen fell from 82% to 77% of the value of milk protein nitrogen in NP- versus HP-adapted subjects, thus confirming the previously formulated hypothesis (23) that increasing the habitual protein intake accentuates differences in metabolic utilization between dietary proteins. However, the effects of an increase in the prevailing protein intake on the postprandial retention of dietary protein nitrogen appeared to be less pronounced for wheat than for soy protein. This may be because of the solid nature of the test meal used in the present study, compared with the liquid nature of the soy protein meal used during our previous work (23): because the fluxes of dietary nitrogen intestinal absorption and its subsequent appearance in splanchnic free amino acids are delayed and softened after ingesting a solid meal (compared with what occurs after a liquid meal), adaptation to the HP versus NP diet may have induced less drastic changes in the postprandial kinetics of dietary amino acid appearance in the splanchnic precursor pool, leading to a reduced activation of catabolism between the 2 adaptation conditions, as described earlier for variations in the digestion kinetics of proteins (19, 20, 22, 32, 59). In line with this, our modeling results indicated that the splanchnic handling of dietary nitrogen was markedly controlled by kinetic factors, because the differences between the 2 adaptation levels regarding the splanchnic kinetics of dietary nitrogen utilization for both anabolic and catabolic purposes reflected those of its availability from the gut (Figure 4Go, A and B). In addition to these kinetic effects, the splanchnic catabolic capacity for urea production exhibited specific stimulation after HP adaptation, which led to a higher extraction of dietary nitrogen in the splanchnic bed and a subsequent decrease in its availability for peripheral tissues (Figure 5Go). These results confirmed the role of the splanchnic zone in preventing sharp increases in blood amino acid levels and in regulating the amount of dietary amino acids made available to the periphery (36, 76), particularly in the event of a high protein intake (23). In the peripheral area, downstream of all the successive metabolic processes of disposal and release, the dietary nitrogen uptake was modulated by the diet, because it was dependent on both prior delivery kinetics and the deamination level.

Another valuable outcome of the model was that it enabled further exploration of the effects of the prevailing protein intake on dietary nitrogen retention and utilization at the regional level (Figure 5Go). The model predicted a predominant splanchnic anabolic use of dietary nitrogen ({approx}40% of the dose at 8 h), leading to a net accretion of splanchnic proteins ({approx}35% of the dose at 8 h) that was within the range of values (15–50%) previously reported in both animals (51, 52, 61, 77) and humans (21, 36). Despite its temporary increase after HP adaptation as the result of kinetic factors, the retention of dietary nitrogen in splanchnic proteins reached similar values 8 h after meal ingestion under both adaptation conditions, in line with previous findings showing that the fractional splanchnic uptake of certain amino acids remained stable for markedly different protein intakes (62). Similarly, the incorporation of dietary nitrogen into serum proteins was not significantly affected by the prior protein intake, as previously reported for other protein sources (23). As a result, the lowering effect of the HP diet on whole-body wheat protein nitrogen retention occurred in peripheral tissues. Indeed, the peripheral utilization of dietary nitrogen for anabolic purposes was predicted to be reduced by {approx}30% at the end of the postprandial period, from {approx}15% to 10% of ingested nitrogen, when switching from the NP to the HP diet (Figure 5Go). Peripheral tissues were thus predicted to receive only a small fraction of the oral load of wheat protein nitrogen, which was lower than that previously predicted to be taken up by the peripheral area when the protein in the meal was milk or soy (19). Further experimental investigations are required to confirm these model predictions. However, our results are in line with other findings in the literature reporting that an increase in the prior protein intake does not enhance the growth of skeletal muscle in rats (78-80), but reduces muscle protein synthesis in dogs (81) and rats (54, 82). Furthermore, our modeling results indicated that the lower efficiency of the peripheral anabolic use of dietary nitrogen in HP- versus NP-adapted subjects who had ingested the same protein load resulted from both a reduced availability of dietary nitrogen for peripheral tissues—as previously reported for certain dietary amino acids in animals and humans (23, 54, 83)—and impaired ability to retain the dietary nitrogen available in peripheral proteins when switching from the NP to the HP diet. This latter finding could be compared with previous reports of a reduced fractional synthesis rate in the muscle of rats adapted to a normal versus a high-protein diet (82) or human subjects adapted to a moderate versus a high-protein diet (84).

In conclusion, we showed that increasing the habitual protein intake influenced the postprandial utilization of wheat protein nitrogen in humans, leading to a significant reduction in its availability and anabolic use for peripheral tissues. In addition, habituation to a high protein intake widened the gap between the nutritional values of wheat and milk proteins, even though the HP-induced reduction in the postprandial retention of wheat protein nitrogen was less pronounced than expected. Finally, our study showed that the combination of an experimental protocol enabling the follow-up of dietary nitrogen under the physiologic conditions of ingesting a single, solid mixed meal with compartmental modeling of the data obtained constituted an effective and noninvasive method to further investigate the notion of protein quality under various nutritional or physiopathologic conditions in humans.


   APPENDIX A
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
REFERENCES
 
The linear, second-order ordinary differential equation presented in this Appendix was used in our model to describe the gastric evacuation of dietary N after ingestion of the solid test meal (model in Figure 2Go, transfer of dietary N from G to IL).

Gastric emptying of a solid meal mainly involves 2 phenomena (46): (i) grinding of large particles of the meal by antral mechanics and (ii) incorporation of the newly formed, small particles into the gastric liquid phase, which is gradually evacuated from the stomach into the duodenum. During the present study, we modeled these metabolic phenomena as follows:

Let GNdiet(t) be the total amount of dietary N present in the stomach at time t after ingestion of the solid meal. A that time, dietary N is either trapped in large solid particles Formula 4, or has been released into the gastric liquid phase Formula 4.

The amount of solid particles ground by antral mechanics during a short period of time dt between times t and t + dt is assumed to be proportional to the amount of solid particles present in the stomach at time t (46):

Formula A1(EA1)
where B(t) is the time-dependent coefficient of antral grinding of solid particles in the stomach.

Ground particles are then evacuated from the stomach in the gastric liquid phase, which follows a simple law of exponential decrease, as described earlier for liquid meals (46, 85):

Formula A2(EA2)
where k2,1 is the constant fraction of dietary N transferred from the stomach to the intestinal lumen per unit of time.

As the total amount of dietary N present in the stomach at time t [GNdiet(t)] is the sum of its solid Formula A2 and liquid Formula A2 fractions, its temporal evolution is finally described by the following equation:

Formula A3(EA3)
In the present study, B was chosen to depend linearly on time, ie B(t) = a + b x t, with a the coefficient of antral grinding of solid particles at time zero, and b its acceleration over time (46).

This equation was validated using experimental data previously obtained through duodenal tubes in healthy subjects (n = 5) for the gastric emptying of dietary N in response to a wheat protein load similar to our experimental meal (27).


   ACKNOWLEDGMENTS
 
We thank the staff in the Gastroenterology Unit at Avicenne Hospital in Bobigny and the volunteers who participated in this study.

The contributions of the authors were as follows—BJ and HF: planned and conducted the modeling work, data analysis, and interpretation; CB and CG: planned and conducted the human experiments and data collection and contributed to data interpretation; FM: contributed to the modeling work and data interpretation; NG, RB, and DT: contributed to study design and the collection of funds; BJ and HF: wrote the initial draft; HF: supervised the manuscript writing and editing, with review and input from all authors. None of the authors had any financial or personal conflicts of interest.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 REFERENCES
 

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Received for publication March 30, 2007. Accepted for publication October 4, 2007.




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H. Fouillet, B. Juillet, C. Gaudichon, F. Mariotti, D. Tome, and C. Bos
Absorption kinetics are a key factor regulating postprandial protein metabolism in response to qualitative and quantitative variations in protein intake
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2009; 297(6): R1691 - R1705.
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