Protein degradation and peptide release from milk proteins in human jejunum. Comparison with in vitro gastrointestinal simulation.

Human jejunal digests after oral ingestion of casein and whey protein were collected by a nasogastric tube and protein degradation and peptide release was compared with that found in the digests of the same substrates using a standardised protocol. No intact casein was detected in the jejunal nor in the in vitro samples taken during the intestinal phase, while ß-lactoglobulin was found in one hour-jejunal samples in agreement with the in vitro digestion. In vivo and in vitro digests showed comparable peptide profiles and high number of common sequences. A selective precipitation step was used to strengthen the identification of phosphorylated peptides. Most of the sequences found in jejunum, some of them not previously described, were also identified in the simulated digests. Common resistant regions to digestion were identified, revealing that the in vitro protocol constitutes a good approximation to the physiological gastrointestinal digestion of milk proteins.

Energy restriction only slightly influences protein metabolism in obese rats, whatever the level of protein and its source in the diet.

BACKGROUND: High protein (HP) diets during energy restriction have been studied extensively regarding their ability to reduce body fat and preserve lean body mass, but little is known about their effects on protein metabolism in lean tissues. OBJECTIVE: To determine the effects of energy restriction and protein intake on protein anabolism and catabolism in rats. METHODS: For 5 weeks, 56 male Wistar rats were fed an obesity induction (OI) diet . They were then subjected to a 40% energy restriction using the OI diet or a balanced HP diet for 3 weeks, whereas a control group was fed the OI diet ad libitum (n=8 per group). HP-restricted rats were divided into five groups differing only in terms of their protein source: total milk proteins, casein (C), whey (W), a mix of 50% C and W, and soy (n=8). The animals were then killed in the postprandial state and their body composition was determined. Protein synthesis rates were determined in the liver, gastrocnemius and kidney using a subcutaneous (13)C valine flooding dose. mRNA levels were measured for key enzymes involved in the three proteolysis pathways. RESULTS: Energy restriction, but not diet composition, impacted weight loss and adiposity, whereas lean tissue mass (except in the kidney) was not influenced by diet composition. Levels of neoglucogenic amino acids tended to fall under energy restriction (P<0.06) but this was reversed by a high level of protein. The postprandial protein synthesis rates in different organs were similar in all groups. By contrast, mRNA levels encoding proteolytic enzymes rose under energy restriction in the muscle and kidney, but this was counteracted by a HP level. CONCLUSIONS: In adult obese rats, energy restriction but not diet composition affected fat pads and had little impact on protein metabolism, despite marked effects on proteolysis in the kidney and muscle.

Daily delivery of dietary nitrogen to the periphery is stable in rats adapted to increased protein intake.

Dietary nitrogen was traced in rats adapted to a 50% protein diet and given a meal containing 1.50 g (15)N-labeled protein (HP-50 group). This group was compared with rats usually consuming a 14% protein diet and fed a meal containing either 0.42 g (AP-14 group) or 1.50 g (AP-50 group) of (15)N-labeled protein. In the HP group, the muscle nonprotein nitrogen pool was doubled when compared with the AP group. The main adaptation was the enhancement of dietary nitrogen transferred to urea (2.2 +/- 0.5 vs. 1.3 +/- 0.1 mmol N/100 g body wt in the HP-50 and AP-50 groups, respectively). All amino acids reaching the periphery except arginine and the branched-chain amino acids were depressed. Consequently, dietary nitrogen incorporation into muscle protein was paradoxically reduced in the HP-50 group, whereas more dietary nitrogen was accumulated in the free nitrogen pool. These results underline the important role played by splanchnic catabolism in adaptation to a high-protein diet, in contrast to muscle tissue. Digestive kinetics and splanchnic anabolism participate to a lesser extent in the regulation processes.

Energy nutrients modulate the splanchnic sequestration of dietary nitrogen in humans: a compartmental analysis.

We used a previously developed compartmental model to assess the postprandial distribution and metabolism of dietary nitrogen (N) in the splanchnic and peripheral areas after the ingestion of a single meal containing milk protein either alone (MP) or with additional sucrose (SMP) or fat (FMP). The addition of fat was predicted to enhance splanchnic dietary N anabolism only transiently, without significantly affecting the global kinetics of splanchnic retention and peripheral uptake. In contrast, the addition of sucrose, which induced hyperinsulinemia, was predicted to enhance dietary N retention and anabolism in the splanchnic bed, thus leading to reduced peripheral dietary amino acid availability and anabolism. The incorporation of dietary N into splanchnic proteins was thus predicted to reach 18, 24, and 35% of ingested N 8 h after MP, FMP, and SMP, respectively. Such a model provides insight into the dynamics of the system in the nonsteady postprandial state and constitutes a useful, explanatory tool to determine the region-specific utilization of dietary N under different nutritional conditions.

A high-protein meal exceeds anabolic and catabolic capacities in rats adapted to a normal protein diet.

The postprandial fixation of dietary nitrogen in splanchnic and peripheral tissues as well as its dynamic transfer to the nitrogen pools of the body were quantified in rats subjected to an acute augmentation of dietary protein. For this purpose, we traced the dietary protein and studied the immediate fate of exogenous nitrogen in many tissues and biological fluids. Rats were adapted to a diet providing an adequate protein level (14 g/100 g), and then fed a meal containing either 0.42 g (Group A) or 1.50 g (Group H) of [(15)N]-labeled milk protein. The amounts of exogenous nitrogen transferred to urea (0.32 +/- 0.04 vs. 2.46 +/- 0.25 mmol, respectively), incorporated in splanchnic (0.41 +/- 0.02 vs. 0.87 +/- 0.10 mmol) and peripheral (1.65 +/- 0.84 vs. 2.36 +/- 0.49 mmol) tissue protein were higher in group H than in group A. Individual plasma amino acids (AA) [(15)N]-enrichments showed that AA respond differentially to an acute augmentation of dietary intake. This work provides new descriptive and quantitative information on the metabolic fate of dietary nitrogen in the postprandial state. It highlights the higher integration of a surplus of dietary nitrogen in the tissues even if it is rapidly limited by saturation of the protein synthesis capacities. The main metabolic response remains the stimulation of AA degradation, leading to a large rise in urea production. However, both anabolic and catabolic systems are exceeded, resulting in an elevation of peripheral AA and negative feedback on the gastric emptying rate.

Compartmental modeling of postprandial dietary nitrogen distribution in humans.

A linear 11-compartment model was developed to describe and simulate the postprandial distribution of dietary nitrogen. The values of its 15 constant diffusion coefficients were estimated from the experimental measurement of (15)N nitrogen kinetics in the intestine, blood, and urine after the oral administration of (15)N-labeled milk protein in humans. Model structure development, parameter estimation, and sensibility analysis were achieved using SAAM II and SIMUSOLV softwares. The model was validated at each stage of its development by testing successively its a priori and a posteriori identifiability. The model predicted that, 8 h after a meal, the dietary nitrogen retained in the body comprised 28% free amino acids and 72% protein, approximately 30% being recovered in the splanchnic bed vs. 70% in the peripheral area. Twelve hours after the meal, these values had decreased to 18 and 23% for the free amino acid fraction and splanchnic nitrogen, respectively. Such a model constitutes a useful, explanatory tool to describe the processes involved in the metabolic utilization of dietary proteins.

Short-term protein and energy supplementation activates nitrogen kinetics and accretion in poorly nourished elderly subjects.

BACKGROUND: An increase in protein intake exerts a stimulating effect on protein kinetics in children, young adults, and healthy elderly persons. However, there are few data on the response to such dietary changes in malnourished elderly subjects, despite important medical implications in this population. OBJECTIVE: The objective of this study was to determine the metabolic response to short-term nutritional supplementation in moderately malnourished elderly subjects. DESIGN: The influence of 10 d of supplementation (1.67 MJ/d and 30 g protein/d) on body composition, resting energy expenditure, and whole-body protein kinetics was studied in 17 malnourished elderly patients and 12 healthy young adults. A control group of 6 malnourished elderly patients received no supplementation. RESULTS: Supplemented elderly subjects had a significantly greater fat-free mass gain than did unsupplemented elderly subjects (1.3 and 0.1 kg, respectively; age effect, P < 0.05; diet effect, P < 0.02) and a significantly greater increase in fasting rate of protein synthesis than did young supplemented subjects (0.6 and 0.2 g*kg FFM(-1)*11 h(-1); age effect, P < 0.05). The net protein balance in the supplemented elderly subjects in the fed state was positively correlated with protein intake (r(2) = 0.46) and in the fasted state was negatively correlated with protein intake (r(2) = 0.27). The sum of these regressions is a line with increasingly positive net diurnal protein balance produced by increasing protein intake. CONCLUSION: These data provide evidence of a short-term anabolic response of protein metabolism to dietary supplementation in malnourished elderly patients that is likely to improve muscle strength and functional status.

Nutritional and physiological criteria in the assessment of milk protein quality for humans.

Dietary protein quality is influenced by several factors and especially amino acid composition as well as the bioavailability of the protein. The method to assess the dietary protein quality recommended by the FAO/WHO (1985, 1990) is based on the ability of the protein to satisfy the indispensable amino acid requirements. The Protein Digestibility Corrected Amino Acid Score (PD-CAAS) has been proposed as a quality index and takes into account both the indispensable amino acid composition and the protein digestibility. This index can easily be used routinely, but some conceptual and methodological limits must be considered, such as the determination of both nitrogen and indispensable amino acid requirements, the bioavailability of dietary protein and the validation of the quality indexes. Another level in the evaluation of protein quality considers more specific activities related to specific protein-derived components. The compounds responsible for these activities include enzymes, immunoglobulins, mediator and hormone-like substances. These actions are linked to native proteins or to peptides cleaved from protein during digestion.

Nutritional value of [15N]-soy protein isolate assessed from ileal digestibility and postprandial protein utilization in humans.

The purpose of this work was to assess the true oro-ileal digestibility, and to concurrently quantify the deamination of absorbed dietary nitrogen to examine the postprandial nutritional value of a soy protein isolate (SPI) in humans. To assess bioavailability and bioutilization of SPI, 10 healthy volunteers ingested 30 g of SPI, intrinsically and uniformly [15N]-labeled, added with 100 g of sucrose and water up to a final volume of 500 mL. True ileal digestibility was assessed by the [15N]-dilution method for 8 h by means of a naso-intestinal intubation technique. To describe and quantify exogenous nitrogen deamination for the same time period, urine and plasma samples were collected. True oro-ileal digestibility of SPI nitrogen was 91%. The amount of absorbed SPI amino acids used for nonoxidative disposal, i.e., postprandial biological value, was 86% 8 h after meal ingestion. Hence, net postprandial protein utilization of SPI was 78%. Compared to previous data that were assessed under the same condition in humans, the nutritional value of SPI is 92% of that in milk protein concentrate.

Assessment of net postprandial protein utilization of 15N-labelled milk nitrogen in human subjects.

The nutritional quality of milk proteins, evaluated both in terms of digestibility and postprandial oxidation and retention in human subjects, was investigated in this study. Five healthy adult volunteers were given 480 ml 15N-labelled milk (i.e. 190 mmol N). 15N was subsequently determined at the ileal level, using a naso-intestinal intubation technique, as well as at the faecal level. Plasma and urine were sampled for 8 h after meal ingestion. Dietary exogenous N recovered at the terminal ileum after 8 h reached 8.6 (SE 0.8) mmol while the amount collected in the faeces was 6.5 (SE 0.7) mmol after 5 d. The true ileal and faecal digestibilities were 95.5 (SE 0.4)% and 96.6 (SE 0.4)% respectively. The appearance of [15N]amino acids in the plasma was rapid and prolonged. The measurement of 15N in the body urea pool and in the N excreted in the urine allowed us to calculate the deamination occurring after [15N]milk protein absorption. The net postprandial protein utilization (i.e. NPPU = (Nabsorbed-Ndeaminated)/Ningested), calculated as an index of protein quality 8 h after milk ingestion, was 81.0 (SE 1.9)%. Our data confirm that milk protein has a high oro-ileal digestibility in man and demonstrate that milk protein has a high NPPU, an index corresponding to a period in which the dietary protein retention is maximal.

Net postprandial utilization of [15N]-labeled milk protein nitrogen is influenced by diet composition in humans.

The aim of this study was to follow the fate of dietary nitrogen to assess the postprandial utilization of purified milk protein and to determine the acute influence of energy nutrients. For this purpose, a [15N]-labeling dietary protein approach was used. Twenty-five subjects swallowed an ileal tube and ingested [15 N]-milk protein alone or supplemented with either milk fat or sucrose. The absorption and postprandial deamination of dietary protein was monitored for 8 h. Sucrose delayed the absorption of protein longer than fat, but the ileal digestibility did not differ among groups (94.5-94.8%). Sucrose, but not fat, significantly reduced the postprandial transfer of [15N]-milk nitrogen to urea. Consequently, the net postprandial protein utilization (NPPU) of milk protein calculated 8 h after meal ingestion was 80% when ingested either alone or supplemented with fat and was significantly greater with sucrose (NPPU = 85%). This study shows that energy nutrients do not affect the nitrogen absorption but modify the metabolic utilization of dietary protein in the phase of nitrogen gain. Our method provides information concerning the deamination kinetics of dietary amino acids and further allows the detection of differences of dietary protein utilization in acute conditions. The diet composition should be carefully considered, and protein quality must be determined under optimal conditions of utilization.

Pattern of metabolism and composition of the fecal microflora in infants 10 to 18 months old from day care centers.

BACKGROUND: From 5 months of age, infants are progressively introduced to a variety of foods which influence the equilibrium of the intestinal microflora. METHODS: Thirty-five children age 10-18 months from 8 day care centers in France were studied. Fecal specimens were examined for their biochemical and microbiological criteria. RESULTS: Bifidobacteria and Bacteroides belong to the predominant populations (9.7 and 8.6 log 10 cfu/g, respectively). The mean level of enterobacteria and enterococci were 8.0 and 7.8 log 10 cfu/g, respectively. Only 10% of the infants presented a lactobacilli amount above 6 log 10 cfu/g. Most feces had similar pH values (mean 6.4), percentage of water (mean 76.4%), and short chain fatty acid, ammonia and lactic acid concentrations (means 77, 6.7 and 2.3 mumol/g, respectively). beta-galactosidase had the highest activity (106 IU/g of protein) and nitroreductase, the lowest (0.1 IU/g of protein). alpha-glucosidase and nitrate reductase showed intermediate values of 17 and 4 IU/g of protein, respectively. With the exception of 4 infants, beta-glucosidase and beta-glucuronidase values were low (4 and 2 IU/g of protein). Age and day care center were not significant factors for most parameters studied, except that rotavirus was related to day care center, with detection in 5 infants from the same center. CONCLUSIONS: Many biochemical parameters were comparable to those found in adults, with the exception of ammonia concentration and beta-galactosidase activity. The fecal bacterial profile was different than in adults, with more Bifidobacteria than Bacteroides and higher levels of facultative anaerobes. One infant suffering from gastroenteritis had distinctive biochemical and bacterial parameters.

A 15N-leucine-dilution method to measure endogenous contribution to luminal nitrogen in the human upper jejunum.

OBJECTIVE: This study was done to investigate whether an intraveinous infusion of 15N-leucine was accurate to differentiate between endogenous and exogenous nitrogen in the human jejunum after meal ingestion. SUBJECTS: Four healthy human volunteers equipped with an upper jejunal tube. INTERVENTIONS: The jejunal effluents were collected both under fasting conditions and after ingestion of 300g of yoghurt. The nitrogen, amino acid composition and 15N-leucine enrichment were determined in the digesta. RESULTS: During fasting, the jejunal flow rates (mmol/h) of both total nitrogen and amino acids were stable (6.9 +/- 2.7 and 1.88 +/- 0.79, respectively). After yoghurt ingestion, the flow rate of total nitrogen increased to 28.6 +/- 5.8mmol/h at 2h. The 15N-leucine enrichment in plasma reached a plateau at 4.3 mole % excess after one hour and did not vary significantly after meal ingestion. The 15N-leucine enrichment of the endogenous secretion (Ee) in the jejunum was fitted by the equation: Ee = 2.18[1 - 2.05 x exp( - 0.42 x t)]. After yoghurt ingestion, the enrichment in jejunal secretions decreased during the first 80 min. The endogenous nitrogen, calculated from the 15N-enrichment, significantly increased from 20 to 40min after meal ingestion compared to the basal value (P < 0.05). The estimation of the exogenous nitrogen and amino acid yield 300min after yoghurt ingestion indicated that 62 +/- 30% of the exogenous nitrogen and 75 +/- 12% of the amino acids were absorbed in the upper jejunum. CONCLUSIONS: The 15N-leucine-dilution method appears to be a convenient method to differentiate between the exogenous and endogenous contributions to nitrogen fluxes in the intestinal digesta of humans. It can be used in association with dietary protein labelling or in substitution when no labelled dietary proteins are available to compare the digestion as well as the absorption of meals at different levels of the intestine.

Exogenous and endogenous nitrogen flow rates and level of protein hydrolysis in the human jejunum after [15N]milk and [15N]yoghurt ingestion.

Milk and yoghurt proteins were 15N-labelled in order to measure the flow rate of exogenous N during digestion in the human intestine. After fasting overnight, sixteen healthy volunteers, each with a naso-jejunal tube, ingested either [15N]milk (n 7) or [15N]yoghurt (n 9). Jejunal samples were collected every 20 min for 4 h. A significant stimulation of endogenous N secretion was observed during the 20-60 min period after yoghurt ingestion and the 20-40 min period after milk ingestion. The endogenous N flows over a 4 h period did not differ between the groups (44.3(SEM 6.5) mmol for milk and 63.5(SEM 5.9) mmol for yoghurt). The flow rates of exogenous N indicated a delayed gastric emptying of the yoghurt N compared with N from milk. The jejunal non-protein N (NPN) flow rate increased significantly after milk and yoghurt ingestion due to an increase in the exogenous NPN flow rate. The NPN fraction of exogenous N ranged between 40 and 80%. The net gastro-jejunal absorption of exogenous N did not differ significantly between milk (56.7(SEM 8.5)%) and yoghurt (50.9(SEM 7)%). The high level of exogenous N hydrolysis is in accordance with the good digestibility of milk products. Fermentation modifies only the gastric emptying rate of N and does not affect the level of diet hydrolysis, the endogenous N stimulation or the digestibility rate.

Nitrogen movements in the upper jejunum lumen in humans fed low amounts of casein or beta-lactoglobulin.

OBJECTIVES AND METHODS: To compare the progression of milk proteins in the upper part of the digestive tract, gastro-jejunal nitrogen movements were studied in 6 healthy human volunteers after beta-lactoglobulin and casein ingestion. 400 mL of water (control), purified beta-lactoglobulin (20 g/L) or casein (20 g/L), each adjusted to 25 microCi with 14C-polyethylene glycol, were given per os. Samples were collected in the stomach and 20 cm below the Treitz ligament every 20 min for 2 hours and measured for volume, osmolarity, ions and nitrogen content. RESULTS: The jejunal flow rate peaked in the 0-20 min period following water and beta-lactoglobulin ingestion, and in the 20-40 min period after casein ingestion. The gastric half-emptying time (T1/2 min) of the liquid phase was significantly different (P < 0.05) for water (12.1 +/- 0.8), beta-lactoglobulin (14.5 +/- 3.3) and casein (26.5 +/- 9.3). Before ingestion of the test meals, the basal rate of nitrogen was 9.14 +/- 4.09 mmol/h in the jejunum. The total nitrogen content in the jejunum peaked significantly in the 0-20 min period after beta-lactoglobulin ingestion and the 20-40 min period after casein ingestion. The apparent gastro-jejunal protein absorption values were 63% for casein and 66% for beta-lactoglobulin in the 120 min period. CONCLUSIONS: These results show that beta-lactoglobulin and casein behave differently in the upper part of the digestive tract due to different gastric emptying rates.

Gastric emptying regulates the kinetics of nitrogen absorption from 15N-labeled milk and 15N-labeled yogurt in miniature pigs.

Thirty-six miniature pigs divided into two groups of 18 animals were fed 15N-labeled milk or yogurt. Polyethylene glycol 4000 was added to the diets as a non-absorbable marker of the liquid phase. Animals were slaughtered 1, 2, 4, 8 and 12 h after meal ingestion, and the gastrointestinal tract was removed and divided into 10 parts. Polyethylene glycol, total nitrogen and 15N enrichment were measured in the digesta. Both the intestinal delivery of the liquid phase and the nitrogenous fraction of the chyme were delayed more in pigs fed yogurt than in pigs fed milk. No stimulatory effect of diet ingestion on endogenous nitrogen secretion was found. Both milk proteins and yogurt proteins were highly digestible: 93% of the exogenous nitrogen disappeared 12 h after feeding. The kinetics of exogenous nitrogen delivery into the intestine was correlated (r = 0.999 for milk and r = 0.974 for yogurt) with that of exogenous nitrogen absorption. These results suggest that milk proteins are rapidly absorbed after they reach the intestine. Gastric emptying is a major factor controlling the kinetics of milk nitrogen absorption.

Rate of [15N]leucine incorporation and determination of nitrogenous fractions from gastro-jejunal secretion in fasting humans.

The aim of this study was to quantify the nitrogen fraction flow rates in gastro-jejunal secretions in fasting humans and to determine the [15N]leucine incorporation into the secreted proteins. A double lumen intestinal perfusion method was used in 5 healthy volunteers. Plasma and gastro-jejunal juices were collected during a 15-h intravenous [15N]leucine infusion. Total, soluble and insoluble nitrogen, amino acids and [15N]leucine enrichment were measured. The total nitrogen flow rate was 7.2 +/- 1.9 mmol.h-1 and 58% was ethanol soluble. The amino-acid composition remained constant and glycine was the most abundant. The plasma [15N]leucine enrichment at the isotopic plateau was 4.8 +/- 0.9 mol% excess. The [15N]leucine enrichment in the intestinal chyme increased asymptotically to reach a plateau after 5 h. The [15N]leucine enrichment at the plateau and the fractional synthesis rate of secreted proteins were 1.6 +/- 0.5 mol% excess and 21.5 +/- 3.3%.h-1, respectively. These results show that the composition of the basal gastro-jejunal secretion is very stable. A part of this secretion is composed of proteins with rapid synthesis rates.