Postprandial modulation of dietary and whole-body nitrogen utilization by carbohydrates in humans

¹ From the UMR INRA, Institut National Agronomique Paris-Grignon, Unité de Physiologie de la Nutrition et du Comportement Alimentaire, Paris, and the Service d'Hépato-Gastroentérologie, Hôpital Avicenne, Bobigny, France.

² Address reprint requests to S Mahé, INRA, Unité de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon, 16 rue Claude Bernard, 75231 Paris Cedex 05, France. E-mail: sylvain.mahe{at}inapg.inra.fr.

ABSTRACT  
Background: Sucrose exerts a sparing effect on whole-body protein metabolism, mainly during the absorptive phase.

Objective: We aimed to characterize the acute postprandial effect of addition of sucrose on deamination of dietary and endogenous nitrogen, with particular consideration being given to the effects of bioavailability.

Design: Twenty-one subjects equipped with ileal tubes ingested ¹⁵N-labeled soy protein combined with [¹³C]glycine, with (n = 10) or without (n = 11) sucrose. Dietary and endogenous ileal flow of nitrogen were determined from the ileal effluents. The kinetics of dietary amino acid transfer to the blood were characterized by ¹³CO₂ enrichment in breath and ¹⁵N enrichment in plasma amino acids. Deamination of dietary and endogenous amino acid was determined from body urea, urinary nitrogen, and ¹⁵N enrichment.

Results: ¹³CO₂ recovery in breath and ¹⁵N plasma amino acid enrichments were highly correlated (R² 0.95, P < 0.001, for both meals) and markedly delayed by sucrose (half-¹³CO₂ recovery: 274 min compared with 167 min), whereas exogenous and endogenous ileal nitrogen kinetics and balances remained unchanged. Addition of sucrose halved the early (0–2 h) deamination peak of dietary nitrogen and reduced endogenous nitrogen oxidation over the first 4 h. Both were reduced by 18–24% over the 8-h period after the meal.

Conclusions: Without changing the nitrogen absorptive balance, sucrose markedly affected the bioavailability profile, which is governed by gastric emptying. Endogenous and dietary nitrogen were not spared in the same way and over the same periods, showing that the metabolism of endogenous and dietary nitrogen may be affected differently by nutritional modulation, even if the effects are of a similar magnitude over the entire postprandial period.

Key Words: Adults • nitrogen isotopes • carbon isotopes • amino acids • pharmacokinetics • oxidation • intestinal absorption • dietary protein • dietary carbohydrate • protein metabolism • nitrogen metabolism • sucrose • gastric emptying

INTRODUCTION  
The sensitivity of whole-body amino acid metabolism and nitrogen balance to energy intake has been recognized for many years (1–8). Carbohydrates are known to reduce amino acid catabolism during the postprandial phase, both directly and through their insulin-releasing effect. In a previous study with ¹⁵N-labeled milk protein, we showed that glucose diverts some dietary nitrogen from postprandial deamination (9). There are many reasons dietary and endogenous amino acids, which follow different routes and metabolic kinetics, may not be similarly affected by the addition of carbohydrates. Indeed, through their action on gastric emptying, carbohydrates in a meal modify the gastrointestinal kinetics, which are thought to play a key role in the subsequent metabolic utilization of dietary nitrogen (10–13). In addition, the presence of carbohydrates in a meal is thought to modulate the splanchnic metabolism and may thus also differently modify dietary and endogenous nitrogen utilization by the gut and liver and subsequent nitrogen utilization by peripheral tissues (5, 14). The present study aimed to investigate the effects of sucrose on postprandial deamination of dietary and endogenous amino acids in humans, with particular emphasis on bioavailability factors. Furthermore, this study provided a valuable opportunity to examine postprandial metabolism more closely, making a distinction between dietary and endogenous nitrogen utilization and their relative degrees of sensitivity to nutritional modulation.

SUBJECTS AND METHODS  
Diets
A purified soy protein isolate prepared from ¹⁵N-labeled soy seeds (variant Chandor) was provided by Nestec Research Centre (Lausanne, Switzerland) as described previously (15). Two experimental meals were prepared. The first meal, mixed with 500 mL water, contained 30 g (316 mmol N) soy isolate (protein meal, P). The second experimental meal, mixed with 500 mL water, contained 30 g soy isolate combined with 100 g sucrose (protein plus sucrose meal, PS). Both meals contained 15 g polyethylene glycol 4000 (PEG 4000) to mark their liquid phases and 75 mg [¹³C]glycine (L-[1-¹³C]glycine 99% enrichment; Euriso-Top, Gif-sur-Yvette, France) to evaluate the gastric-emptying kinetics of the liquid phase of the meals (16). The P meal provided 502 kJ and the PS meal provided 2174 kJ.

Subjects
The study was performed in 21 healthy volunteers (14 men and 7 women) aged from 20 to 37 y ( ± SD: 26 ± 5 y), weighing from 55 to 89 kg (72 ± 11 kg), with body mass indexes (in kg/m²) ranging from 19.6 to 27.5 (23.1 ± 2.3), and total body water [TBW; as estimated by using the equation of Watson et al ( Clinical protocol
The volunteers were admitted to the hospital on the morning before the day of the study. An intestinal tube was passed through the nose and allowed to descend to the digestive tract, as described previously (18). The intestinal tube was used to 1) perfuse phenol red, a nonabsorbable intestinal marker, into the ileum and 2) collect intestinal samples by continuous suction 20 cm distally from the perfusion site. Volunteers ate dinner at 2000 and then fasted overnight. On the morning of the study, after the position of the intestinal tube at the terminal ileum had been checked by radioscopy, a catheter was inserted into a superficial forearm vein for blood sampling. The subjects then ingested the P or PS meal, and the postprandial sampling period lasted for 8 h. The study was performed while the subjects were resting; they were not allowed to ingest food or fluids until the end. Intestinal aspirates were collected over ice and pooled at 30-min intervals over an 8-h period; the first collection (before the meal) represented the initial period. Ileal effluents were freeze-dried and stored for future analysis. Blood samples were collected hourly during the 8-h period after ingestion of the meal, except for the first 2 postprandial hours when additional samples were taken. A final blood sample was collected the next morning, 22 h after the meal was ingested. Plasma was immediately separated from whole blood by centrifugation (2500 x g for 20 min at 4°C) and frozen at -20°C until analyzed. Breath samples were collected every 30 min and stored until later determination of ¹³CO₂ isotopic enrichment. Urine was collected over a 29-h period (0–2, 2–4, 4–6, 6–8, 8–12, 12–20, and 20–29 h after meal ingestion), treated with thymol crystals and liquid paraffin as preservatives, and stored at 4°C until analyzed.

Extraction of amino acids, urea, and ammonia from plasma and urine
Urea and ammonia were isolated by using a batch method, as described previously (19). Briefly, for the extraction of amino acids and urea, plasma proteins were pelleted by adding solid 5-sulfosalicylic acid. After centrifugation (2400 x g for 25 min at 4°C), the supernate was collected. Urinary ammonia was first extracted from the urine by using the Na-K form of cation exchange resin (Dowex AG-50X8, mesh 100–200; BioRad, Montluçon, France). The supernatant fraction was collected for the further extraction of urea. Urea was extracted from both the plasma supernatant fraction and the ammonia-free urine fraction by conversion into ammonium through hydrolysis with urease (Sigma Chemical Co, Saint-Quentin-Fallavier, France) for 2 h at 30°C on a cation-exchange resin. The part of the plasma fraction not retained in the resin was considered to be the plasma amino acid fraction. Ammonia and urea-derived ammonia were eluted from the resins by adding 2.5 mmol KH₂SO₄/L.

Analytic methods
The total nitrogen content of samples was determined by using an elemental nitrogen analyzer (NA 1500 series 2; Micromass, Manchester, United Kingdom) with atropine as the standard. Urea was assayed in both plasma and urine by an enzymatic method (urease and glutamate dehydrogenase) on a clinical analyzer (Dimension automate; Dupont de Nemours, Les Ulis, France). Ammonia was measured in urine by using an enzymatic method (glutamate dehydrogenase) on a clinical analyzer (Kone automate; Kone, Evry, France). Creatinine was measured by using a direct colorimetric method on a clinical analyzer (Dimension automate). Glucose was measured in plasma by using a glucose oxidase method (glucose GOD-DP kit; Kone). Insulin was measured in plasma by using a radioimmunoassay method (INSI-PR kit; Cis Bio International, Gif-sur-Yvette, France). The concentration of PEG 4000 in the digesta was measured by a turbidimetric method and that of phenol red by colorimetry, as described previously (12).

Isotopic nitrogen enrichments (¹⁵N:¹⁴N) were determined by isotope ratio mass spectrometry. An aliquot was burned in an elementary analyzer (NA 1500 series 2; Micromass) at 1020°C, interfaced with an isotope ratio mass spectrometer (Optima; Micromass). The ¹⁵N-¹⁴N ratios [mass-to-charge ratio (m/z) 28:29:30] were measured with reference to a calibrated ¹⁵N-¹⁴N tank.

Isotopic CO₂ enrichments (¹³CO₂:¹²CO₂) were measured by using a gas chromatograph coupled with an isotope ratio mass spectrometer. Samples were separated by gas chromatography (HP 5890 series II; Hewlett Packard, Les Ulis, France) on a 2.5 m x 3 mm Haysep Q column (Chrompak, Les Ulis, France) at 80°C and isotopic ratios (44:45:46) were determined by isotope ratio mass spectrometry with reference to a calibrated ¹³CO₂-¹²CO₂ tank.

Calculations
Exogenous and endogenous nitrogen flux at the terminal ileum
Assessment of phenol red dilutions between the perfusion solution and samples collected by using the ileal perfusion technique enabled the calculation of flow rates in the ileum (calculation of the average flux for 30 min), as described previously (20). The fraction of exogenous and endogenous nitrogen in ileal samples was calculated from both total nitrogen and the isotopic ¹⁵N-¹⁴N ratio. Exogenous nitrogen (N_exo-ileal, in mmol) and endogenous nitrogen (N_endo-ileal, in mmol) transiting through the terminal ileum were thus calculated by using the following equations:

RESULTS  
Liquid phase and dietary nitrogen passing the terminal ileum and ileal digestibility
The first level of comparison between the P and PS meals was the effect of sucrose on soy protein digestibility in the ileum and endogenous ileal flow (Figure 1). At the terminal ileum, transit of the liquid phase of the meal, represented by PEG 4000 flux (Figure 1A), did not differ significantly between the 2 meals. Half-recovery times were 2 h 32 ± 40 min and 2 h 47 ± 57 min for the P and PS meals, respectively. The cumulative quantities of dietary nitrogen passing at the terminal ileum after ingestion of the P and PS meals are shown in Figure 1B. A significant amount of exogenous nitrogen was detected 1 h after ingestion of the P meal and 2 h after ingestion of the PS meal. At 8 h, the total exogenous nitrogen that had transited through the terminal ileum was 25.9 ± 6.1 and 28.6 ± 6.9 mmol for the P and PS meals, respectively. Consequently, true oroileal digestibility was 91.8 ± 1.9% and 90.9 ± 2.2% for the P and PS meals, respectively, and did not differ significantly between meals. Parameters of curves fitted onto data showed that plateaus were reached at 467 and 417 min, and that asymptotic values were 25.8 and 28.2 mmol, for the P and PS meals, respectively. When data were pooled independently with regards to meal, the digestibility value was 91.5 ± 2.0%.

View larger version (23K):
FIGURE 1. . Mean (±SD) cumulative polyethylene glycol (PEG, a marker of the liquid phase of the meal) transiting at the terminal ileum (A), cumulative dietary nitrogen transiting at the terminal ileum (B), and endogenous nitrogen transiting at the terminal ileum (C) after the ingestion of soy protein alone (; n = 9) or soy protein plus sucrose (•; n = 8). There were no significant differences between meals.

 
The endogenous nitrogen transiting at the terminal ileum is represented in Figure 1C. The type of meal had no effect on endogenous nitrogen fluxes, and no significant difference was found between the 2 meals at any time during the 8-h experiment. The mean endogenous nitrogen flow was 5.8 ± 3.8 mmol/h for both the P and PS meals.

Dietary glucose and amino acids entering the blood
A second important feature was the kinetics of nutrient transfer to the blood, ie, glucose and amino acids. Plasma glucose concentrations of both groups are given in Figure 2. The plasma glucose concentration of the P meal group was stable for 4 h, fell significantly between 4 and 5 h, and remained significantly lower than the initial concentration for the remaining 4 h. The plasma glucose concentration of the PS meal group showed a rapid increase and remained significantly higher than the basal value during the first 3 h. After meal ingestion, plasma glucose concentrations were always significantly higher in the PS meal group than in the P meal group.

View larger version (16K):
FIGURE 2. . Mean (±SD) plasma glucose concentration and plasma insulin concentration in subjects receiving soy protein alone (; n = 11) or soy protein plus sucrose (•; n = 10). ^*Significantly different from initial value, P < 0.05. ^#Significant difference between meals, P < 0.05.

 
Plasma insulin concentrations in the P meal group never differed significantly from baseline, whereas those of the PS meal group were significantly higher than the initial concentration for the first 4 h and significantly higher than the insulin concentration of the P meal group for the first 5 h.

The transfer of amino acids to the blood was measured from the ¹⁵N enrichment of amino acids in blood and ¹³CO₂ enrichment in breath (Figure 3). With the P meal, [¹⁵N]amino acid enrichment rose rapidly, peaked at 2 h, and then decreased significantly every subsequent hour measured. With the PS meal, the peak was smoother and appeared later (around 3 h). Compared with that after the P meal, enrichment after the PS meal was significantly lower at 1 h and significantly higher from 5 to 8 h. The shape of the curve of ¹³CO₂ enrichment in breath was different after the P and PS meals. Except when the curves crossed each other (time 150 and 180 min), the differences were always significant. For both meals, plasma amino acid ¹⁵N enrichment and ¹³CO₂ excretion in breath showed very similar profiles. These 2 variables were highly correlated (R² = 0.949 and 0.958 for P and PS meals, respectively; P < 0.001 for both meals).

View larger version (19K):
FIGURE 3. . Mean (±SD) ¹³C recovery of [¹³C]glycine dose as ¹³CO₂ in the breath of subjects receiving soy protein alone (; n = 11) or soy protein plus sucrose (•; n = 10), and mean (±SD) ¹⁵N enrichment of plasma amino acids of subjects receiving soy protein alone (; n = 11) or soy protein plus sucrose (; n = 10). ^*Significant difference in ¹³C recovery between meals, P < 0.05. ^#Significant difference in ¹⁵N enrichment between meals, P < 0.05.

 
Gastric-emptying parameters were calculated from breath ¹³CO₂ recovery kinetics (Table 1). Gastric-emptying coefficients, times of maximum recovery, and times of half recovery all differed significantly between P and PS meals and indicated that there was a marked delay of gastric emptying with the PS meal compared with the P meal.

View this table:
TABLE 1.. Parameters of ¹³CO₂ excretion in breath after [¹³C]glycine ingestion in humans¹  
Postprandial endogenous and exogenous amino acid deamination
Amino acid deamination was measured from urea and ammonia production after meal ingestion. Total and exogenous urea nitrogen were measured in TBW and urine, and total and exogenous ammonia nitrogen were measured in urine. After meal ingestion, total body urea nitrogen values did not differ significantly between meals, but sucrose modulated (P = 0.07) the time effect on total-body urea kinetics, and cumulative total urea nitrogen recovered in urine was significantly lower 4 h after the meal than after the P meal (Figure 4). Cumulative total ammonia recovered in urine showed only a trend toward reduction by sucrose (P < 0.1) from 4 h after the meal (data not shown). Whatever time point was considered, exogenous urinary urea nitrogen excretion was always lower when protein was ingested with sucrose (Figure 5). Exogenous urea in TBW was lower during the first 3 h after ingesting a PS meal than after a P meal. The peak (assessed from curve fitting) was reached 3 h 50 min after the P meal but delayed to 5 h with the PS meal.

View larger version (16K):
FIGURE 4. . Mean (±SD) body urea in subjects receiving soy protein alone (; n = 11) or soy protein plus sucrose (•; n = 10), and cumulative urinary urea in subjects receiving soy protein alone (; n = 11) or soy protein plus sucrose (; n = 10). ^*Significant difference between meals, P < 0.05.

 

View larger version (22K):
FIGURE 5. . Mean (±SD) exogenous urea nitrogen in body water, cumulative exogenous urea nitrogen in urine, and cumulative exogenous ammonia nitrogen in urine of subjects receiving soy protein alone (; n = 11) or soy protein plus sucrose (•; n = 10). ^* Significant difference between meals, P < 0.05.

 
Exogenous deamination flux (Figure 6) increased quickly after meals and peaked at 25 min in the P meal group and at 55 min in the PS meal group. The 2 fluxes were similar from 2 h after ingestion. At 2 h, cumulative exogenous nitrogen deamination was halved when sucrose was included in the meal (24.8 ± 16.4 mmol for the PS meal compared with 48.8 ± 13.3 mmol for the P meal) and finally reached 60.3 ± 15.4 mmol with the PS meal and 73.9 ± 12.5 mmol with the P meal at 8 h.

View larger version (12K):
FIGURE 6. . Theoretical estimates of mean dietary nitrogen deamination flux in subjects receiving soy protein alone (solid line) or soy protein plus sucrose (dotted line).

 
Endogenous deamination was then calculated for the 8 h after meal ingestion and compared with exogenous deamination (Figure 7). During the first 2 h after ingestion of a meal, total, endogenous, and exogenous deamination were significantly lower with PS than with P meals, by 41%, 39%, and 50%, respectively. Between 2 and 4 h after the meal, endogenous deamination was again lower with PS than with P meals (by 27%), although the effect was not significant (P = 0.094). During this same period, dietary nitrogen deamination showed an opposite tend (53% higher with PS than with P meals), but this was not significant. Total dietary deamination was not significantly different during this period. After that period (from 4 to 8 h), sucrose had no significant effect on total, endogenous, or exogenous deamination. Over the whole 8-h period, total, endogenous, and exogenous deamination were lower with the PS meal than with the P meal by 23%, 24%, and 18%, respectively, with a lower level of significance (0.5 < P < 0.1).

View larger version (30K):
FIGURE 7. . Mean (±SD) deamination of dietary and endogenous nitrogen in subjects receiving soy protein alone (P; n = 11) or soy protein plus sucrose (PS; n = 10) over the periods 0–2, 2–4, 4–6, and 6–8 h after the meal. ^*Significantly different from P meal, P < 0.05.

 

DISCUSSION  
The aim of this study was to closely examine the effect of adding sucrose to a pure protein meal on dietary and endogenous nitrogen utilization to assess whether exogenous and endogenous nitrogen utilization would be different in terms of sensitivity. The supposition was that the mechanism of sparing might differ between the 2 types of meals. Particular attention was paid to the balance and kinetics of nitrogen bioavailability.

The first result of this study was that gastric emptying markedly modified nitrogen absorption kinetics without affecting the overall ileal digestibility of dietary nitrogen. Indeed, as expected (21), the addition of sucrose to the meal induced a marked delay in the gastric-emptying profile. The excretion of ¹³CO₂ arising from [¹³C]glycine oxidation was shown to closely reflect gastric emptying of the liquid phase of a meal (22). In this experiment, we showed that gastric emptying was markedly reduced by sucrose because the half-recovery time for ¹³CO₂ was delayed by 108 min after the PS meal compared with the P meal. As mentioned above, ¹³CO₂ excretion is considered to be governed solely by gastric emptying, other events (glycine absorption, oxidation, and excretion of carbon dioxide from the blood) being rapid and not limiting. Conversely, ¹⁵N plasma amino acid enrichments were expected to depend on gastric emptying, absorption kinetics, and first-pass metabolism in the splanchnic area. However, ¹⁵N plasma amino acid enrichments were highly correlated with ¹³CO₂ excretion, thus showing that gastric emptying was the major determinant of the shape of the plasma amino acid enrichment. These results confirmed, at least for highly digestible protein, that gastric emptying is the limiting step in the absorption process and that nitrogen is principally absorbed in the proximal part of the intestine (23).

Despite this initial delay in gastric emptying with sucrose ingestion, no delay persisted at the ileal level. Both liquid phase and exogenous nitrogen showed identical passage profiles at the terminal ileum and were passed completely 8 h after the 2 meals. This implies that with the addition of sucrose, the initial delay was gradually reduced as the meal moved through the intestine; thus, the reduction in intestinal residence time entirely made up for the delay in gastric emptying. These findings agree with other reports showing that changes in small bowel transit time and gastric emptying may occur independently and even in opposite directions (24, 25). This effect may depend on the type of protein because ileal passage was shown to be delayed when the same amount of sucrose was added to a milk protein concentrate meal (9).

Although gastroileal nitrogen transit was changed considerably by the presence of sucrose, we showed that dietary nitrogen absorption was the same from both meals. This agrees with various reports indicating that digestibility is not very sensitive to variations in meal composition, gastric release, or intestinal transit (9, 26–28). Assessing digestibility at the terminal ileum presents many advantages. It avoids the need to take into account colonic transformations that are of minimal nutritional value (29). It also avoids the long-term collection of feces necessary to assess dietary nitrogen recovery, which may be biased by recycling (30). However, the recycling of ¹⁵N into gastrointestinal secretions might have introduced inaccuracies into the estimation of endogenous and dietary ileal nitrogen flow. This potential methodologic problem was discussed previously (19), and it is likely that our estimates of endogenous and dietary ileal nitrogen flux may be slightly low and high, respectively. In our study, the presence of sucrose in the meal did not affect the true ileal digestibility of purified soy protein, which reached 91–92%. The addition of sucrose could have modified the efficiency of absorption according to 2 different and opposing mechanisms. First, nitrogen delivery in the upper intestine is markedly slowed by sucrose, whereas it is accelerated further along the intestinal tract. This modification is likely to favor absorption in the upper tract, which is considered to be the principal site for absorption. Second, it has long been thought that the presence of a high load of sucrose may alter amino acid absorption (31, 32).

We showed that the endogenous nitrogen flux at the terminal ileum did not vary with time or between meals. This indicates that during the absorptive period, the endogenous contribution to total nitrogen inflow was not modified by sucrose. Nitrogen entering the colon is considered an important route for nitrogen and amino acid losses, with meaningful consequences for amino acid homeostasis (33). The mean endogenous nitrogen flux was 5.8 mmol N/h, and it could be seen that over a 24-h period 12 g (N x 6.25) endogenous protein entered the colon. Thus, the endogenous nitrogen escaping absorption in the small intestine may be an important factor influencing the net nitrogen balance. In this experiment, we showed that the absorptive balances of both endogenous and dietary nitrogen were not modified by sucrose.

In addition to quantifying the modulation of bioavailability and its consequences on systemic dietary amino acid concentrations, our study was designed to precisely quantify endogenous and dietary nitrogen utilization. Estimations of exogenous (or endogenous) deamination were calculated from serial measures of both exogenous (and endogenous) body urea and exogenous (and endogenous) urinary nitrogen. In fact, this assessment of deamination differs from net amino acid catabolism because of the disposal and salvage of urea nitrogen. Because we assumed that both of these fluxes were constant over the 8 postprandial hours and between groups, our calculation of deamination should closely reflect net amino acid catabolism. Furthermore, if a variation occurs, we see no reason under the conditions of the present experiment why this should apply differently to endogenous or exogenous nitrogen or to different meals, and it should not affect the validity of any of the comparisons that we present.

We found that the deamination of dietary nitrogen peaked during the first 2 h and then began a slow decline. Early deamination was halved when sucrose was added. This suggests that the first-pass effect is a major determinant of both total dietary amino acid retention within the body and any subsequent improvement in retention when carbohydrates are present. Other studies have provided indications of the important role of the splanchnic area in dietary nitrogen sparing by carbohydrates. Using an intraduodenal diet infusion, Krempf et al (14) reported that carbohydrates decreased the splanchnic uptake of dietary leucine and lowered whole-body leucine oxidation. This effect, which is not present for phenylalanine, may be dependent on the amino acid studied, particularly for amino acids that are taken up differently by the splanchnic area (34). Furthermore, because the diet was infused intraduodenally, the carbohydrate-modified bioavailability kinetics were not considered and the effect under investigation corresponded to the specific action of carbohydrates. Data on pigs support the role of the gut in the sparing of dietary amino acids by the splanchnic area (35, 36). It was shown in catheterized pigs that the addition of carbohydrates to a meal increased intestinal amino acid retention (37), an effect attributed to the stimulation by insulin of protein retention in the gut. Furthermore, according to recent studies in piglets conducted by Stoll et al (38, 39), the gut mucosa oxidizes rather than incorporates dietary amino acids, as reflected by a portal ammonia balance accounting for 18% of total nitrogen intake; portal amino acids are mainly utilized by the liver for synthetic purposes. These studies suggest that the diversion of dietary amino acids from intestinal deamination, whether this is achieved through increased intestinal synthesis or through increased gut output of amino acids taken up by the liver or extrasplanchnic tissues, may be crucial to dietary nitrogen sparing. Although the present study did not directly address intestinal or liver protein metabolism, our findings are in line with these hypotheses because they show that deamination occurs very early and that sucrose acts by lowering this early deamination peak.

Our findings show that the sparing of dietary nitrogen occurred alongside a sparing effect on endogenous nitrogen that was of similar importance over the entire 8-h period (a reduction of 24% compared with a reduction of 18% in deamination). However, endogenous and exogenous sparing effects did not display the same pattern, and thus, the mechanisms may not be totally shared. The reduction in endogenous nitrogen deamination took place during the first 4 h of hyperinsulinemia and hyperglycemia, and is likely to be linked to the following mechanisms. First, insulin stimulation, by inhibiting protein catabolism, reduces the intracellular concentration of endogenous amino acids and thus their oxidation (6, 40, 41). Furthermore, gluconeogenesis may be reduced by sucrose and by the inhibition of glucagon release (42, 43). We observed that the amount of sparing of endogenous nitrogen matched the blood insulin and glucose profiles. These factors may also account for the sparing of dietary nitrogen during the first hours after a meal. Nevertheless, compared with endogenous sparing, sparing of exogenous nitrogen appeared to be more effective during the first 2 postprandial hours but less effective later. We suggest that this difference originates with the kinetics of amino acid inflow and the hyperinsulinemic profile. During the first 2 postprandial hours, the inflow of amino acids from the PS meal was lower than that from the P meal, whereas blood insulin was at its highest. As a consequence, the endogenous intracellular amino acid pool was reduced and dietary amino acids, resulting from slower gastric emptying, may more readily have been diverted from oxidation (44). In contrast, during the later hours, because the insulin effect is dose dependent (45), the insulin-mediated inhibition of proteolysis was reduced, whereas during this period, the PS meal (compared with the P meal) provided a larger quantity of dietary amino acids that were relatively less spared than were endogenous amino acids. These combined effects may explain why the sparing effect on dietary nitrogen appeared to be concentrated during the first 2 h, whereas the effect on endogenous nitrogen showed a more widely spread pattern. This is in line with studies proposing that gut amino acid output is a major factor accounting for variations in amino acid utilization (46, 47), and others reporting that the kinetics of amino acid or carbohydrate supply may modulate postprandial nitrogen retention (13, 48). The diversion of dietary amino acids from oxidation may arise from increased retention in the splanchnic area and also from improved transfer to the systemic circulation. The preferential diversion of dietary amino acids from oxidation, during the first 2 h after a meal, may also result from the muscle uptake of dietary amino acids not extracted by the splanchnic area. In particular, dietary branched-chain amino acids, poorly extracted in the splanchnic area, are extensively taken up by muscle, particularly if insulin concentrations are high (49, 50).

In conclusion, these different effects of the addition of carbohydrates to a protein test meal halved the oxidative peak of dietary nitrogen during the early absorptive phase (0–2 h), whereas there was little absolute difference later. The effect of sucrose on endogenous and exogenous amino acid deamination was of similar amplitude over the 8-h postprandial period. However, the sparing of dietary and endogenous amino acids did not occur at the same time or in the same way, thus showing that the same mechanisms do not govern utilization of endogenous and dietary nitrogen.

ACKNOWLEDGMENTS  
We acknowledge the Gastroenterology Unit and the Biochemistry Laboratory at Avicenne Hospital (Bobigny, France) for their skillful assistance and S Daré for her technical assistance.

REFERENCES  

1. Munro HN. Energy and protein intakes as determinants of nitrogen balance. Kidney Int 1978;14:313–6.
2. Garlick PJ, McNurlan MA, Ballmer PE. Influence of dietary protein intake on whole-body protein turnover in humans. Diabetes Care 1991;14:1189–98.
3. Motil KJ, Bier DM, Matthews DE, Burke JF, Young VR. Whole body leucine and lysine metabolism studied with [1-¹³C]leucine and [-¹⁵N]lysine: response in healthy young men given excess energy intake. Metabolism 1981;30:783–91.
4. Vazquez JA, Paul HS, Adibi SA. Regulation of leucine catabolism by caloric sources. Role of glucose and lipid in nitrogen sparing during nitrogen deprivation. J Clin Invest 1988;82:1606–13.
5. Young VR. Nutrient interactions with reference to amino acid and protein metabolism in non-ruminants; particular emphasis on protein-energy relations in man. Z Ernahrungswiss 1991;30:239–67.
6. Nair KS, Halliday D, Ford GC, Heels S, Garrow JS. Failure of carbohydrate to spare leucine oxidation in obese subjects. Int J Obes 1987;11:537–44.
7. Welle S, Matthews DE, Campbell RG, Nair KS. Stimulation of protein turnover by carbohydrate overfeeding in men. Am J Physiol 1989;257:E413–7.
8. Zillikens MC, van den Berg JW, Wattimena JL, Rietveld T, Swart GR. Nocturnal oral glucose supplementation. The effects on protein metabolism in cirrhotic patients and in healthy controls. J Hepatol 1993;17:377–83.
9. Gaudichon C, Mahé S, Benamouzig R, et al. Net postprandial utilization of [¹⁵N]-labeled milk protein nitrogen is influenced by diet composition in humans. J Nutr 1999;129:890–5.
10. Kies C. Bioavailability: a factor in protein quality. J Agric Food Chem 1981;29:435–40.
11. Darcy B. Availability of amino acids in monogastric animals. Variations of digestive origin. Diabete Metab 1984;10:121–33.
12. Mahé S, Roos N, Benamouzig R, et al. Gastrojejunal kinetics and the digestion of [¹⁵N]ß-lactoglobulin and casein in humans: the influence of the nature and quantity of the protein. Am J Clin Nutr 1996;63:546–52.
13. Boirie Y, Dangin M, Gachon P, Vasson MP, Maubois JL, Beaufrere B. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci U S A 1997;94:14930–5.
14. Krempf M, Hoerr RA, Pelletier VA, Marks LM, Gleason R, Young VR. An isotopic study of the effect of dietary carbohydrate on the metabolic fate of dietary leucine and phenylalanine. Am J Clin Nutr 1993;57:161–9.
15. Mariotti F, Mahé S, Benamouzig R, et al. Nutritional value of [¹⁵N]-soy protein isolate assessed from ileal digestibility and postprandial protein utilization in humans. J Nutr 1999;129:1992–7.
16. Maes BD, Ghoos YF, Geypens BJ, Hiele MI, Rutgeerts PJ. Relation between gastric emptying rate and energy intake in children compared with adults. Gut 1995;36:183–8.
17. Watson PE, Watson ID, Batt RD. Total body water volumes for adult males and females estimated from simple anthropometric measurements. Am J Clin Nutr 1980;33:27–39.
18. Mahé S, Huneau JF, Marteau P, Thuillier F, Tome D. Gastroileal nitrogen and electrolyte movements after bovine milk ingestion in humans. Am J Clin Nutr 1992;56:410–6.
19. Gausseres N, Mahé S, Benamouzig R, et al. [¹⁵N]-labeled pea flour protein nitrogen exhibits good ileal digestibility and postprandial retention in humans. J Nutr 1997;127:1160–5.
20. Gausseres N, Mahé S, Benamouzig R, et al. The gastro-ileal digestion of ¹⁵N-labelled pea nitrogen in adult humans. Br J Nutr 1996;76:75–85.
21. Elias E, Gibson GJ, Greenwood LF, Hunt JN, Tripp JH. The slowing of gastric emptying by monosaccharides and disaccharides in test meals. J Physiol (Lond) 1968;194:317–26.
22. Maes BD, Ghoos YF, Geypens BJ, et al. Combined carbon-13-glycine/carbon-14-octanoic acid breath test to monitor gastric emptying rates of liquids and solids. J Nucl Med 1994;35:824–31.
23. Gaudichon C, Roos N, Mahé S, Sick H, Bouley C, Tome D. Gastric emptying regulates the kinetics of nitrogen absorption from ¹⁵N-labeled milk and ¹⁵N-labeled yogurt in miniature pigs. J Nutr 1994;124:1970–7.
24. Read NW, Cammack J, Edwards C, Holgate AM, Cann PA, Brown C. Is the transit time of a meal through the small intestine related to the rate at which it leaves the stomach? Gut 1982;23:824–8.
25. McIntyre A, Vincent RM, Perkins AC, Spiller RC. Effect of bran, ispaghula, and inert plastic particles on gastric emptying and small bowel transit in humans: the role of physical factors. Gut 1997; 40:223–7.
26. Lien KA, McBurney MI, Beyde BI, Thomson AB, Sauer WC. Ileal recovery of nutrients and mucin in humans fed total enteral formulas supplemented with soy fiber. Am J Clin Nutr 1996;63:584–95.
27. Zhao XT, McCamish MA, Miller RH, Wang L, Lin HC. Intestinal transit and absorption of soy protein in dogs depend on load and degree of protein hydrolysis. J Nutr 1997;127:2350–6.
28. Weber E, Ehrlein HJ. Relationships between gastric emptying and intestinal absorption of nutrients and energy in mini pigs. Dig Dis Sci 1998;43:1141–53.
29. Rowan AM, Moughan PJ, Wilson MN, Maher K, Tasman-Jones C. Comparison of the ileal and faecal digestibility of dietary amino acids in adult humans and evaluation of the pig as a model animal for digestion studies in man. Br J Nutr 1994;71:29–42.
30. Bos C, Mahé S, Gaudichon C, et al. Assessment of net postprandial protein utilization of ¹⁵N-labelled milk nitrogen in human subjects. Br J Nutr 1999;81:221–6.
31. Vinardell MP. Mutual inhibition of sugars and amino acid intestinal absorption. Comp Biochem Physiol A 1990;95:17–21.
32. Rerat A, Vaissade P, Vaugelade P. Comparative digestion of maltitol and maltose in unanesthetized pigs. J Nutr 1991;121:737–44.
33. Fuller MF, Reeds PJ. Nitrogen cycling in the gut. Annu Rev Nutr 1998;18:385–411.
34. Biolo G, Tessari P, Inchiostro S, et al. Leucine and phenylalanine kinetics during mixed meal ingestion: a multiple tracer approach. Am J Physiol 1992;262:E455–63.
35. Sève B, Leterme P. Amino acid fluxes in the pig. In: Laplace JP, Février C, Barbeau A, eds. Digestive physiology in pigs. Proceedings of the VIIth international symposium on digestive physiology in pigs. INRA Editions ed. Saint Malo, France: EAAP, 1997:304–15.
36. Soeters PB, de Blaauw I, van Acker BA, von Meyenfeldt MF, Deutz NE. In vivo inter-organ protein metabolism of the splanchnic region and muscle during trauma, cancer and enteral nutrition. Baillieres Clin Endocrinol Metab 1997;11:659–77.
37. Deutz NEP, Ten Have GAM, Soeters PB, Moughan PJ. Increased intestinal amino-acid retention from the addition of carbohydrates to a meal. Clin Nutr 1995;14:354–64.
38. Stoll B, Burrin DG, Henry J, Yu H, Jahoor F, Reeds PJ. Dietary amino acids are the preferential source of hepatic protein synthesis in piglets. J Nutr 1998;128:1517–24.
39. Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J Nutr 1998;128:606–14.
40. Garlick PJ, McNurlan MA, Bark T, Lang CH, Gelato MC. Hormonal regulation of protein metabolism in relation to nutrition and disease. J Nutr 1998;128:356S–9S.
41. De Feo P. Hormonal regulation of human protein metabolism. Eur J Endocrinol 1996;135:7–18.
42. Charlton MR, Adey DB, Nair KS. Evidence for a catabolic role of glucagon during an amino acid load. J Clin Invest 1996;98:90–9.
43. Hamberg O, Vilstrup H. Effects of insulin and glucose on urea synthesis in normal man, independent of pancreatic hormone secretion. J Hepatol 1994;21:381–7.
44. Millward DJ, Fereday A, Gibson NR, Pacy PJ. Post-prandial protein metabolism. Baillieres Clin Endocrinol Metab 1996;10:533–49.
45. Tessari P, Trevisan R, Inchiostro S, et al. Dose-response curves of effects of insulin on leucine kinetics in humans. Am J Physiol 1986;251:E334–42.
46. Bloomgarden ZT, Liljenquist J, Lacy W, Rabin D. Amino acid disposition by liver and gastrointestinal tract after protein and glucose ingestion. Am J Physiol 1981;241:E90–9.
47. Rerat A, Simoes-Nunes C, Mendy F, Vaissade P, Vaugelade P. Splanchnic fluxes of amino acids after duodenal infusion of carbohydrate solutions containing free amino acids or oligopeptides in the non-anaesthetized pig. Br J Nutr 1992;68:111–38.
48. van der Meulen J, Bakker JG, Smits B, de Visser H. Effects of source of starch on net portal flux of glucose, lactate, volatile fatty acids and amino acids in the pig. Br J Nutr 1997;78:533–44.
49. Capaldo B, Gastaldelli A, Antoniello S, et al. Splanchnic and leg substrate exchange after ingestion of a natural mixed meal in humans. Diabetes 1999;48:958–66.
50. Meek SE, Persson M, Ford GC, Nair KS. Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects. Diabetes 1998;47:1824–35.