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The recent Food and Agricultural Organization/World Health Organization/United Nations University expert consultations on protein requirements and quality have emphasized the need for the new Digestible Indispensable Amino Acid Score (DIAAS), as a measure of protein quality. This requires human measurements of the true ileal digestibility of individual indispensable amino acids (IAA) until the end of the small intestine. Digestibility is measured using standard oro-ileal balance methods, which can only be achieved by an invasive naso-ileal intubation in healthy participants or fistulation at the terminal ileum. Significant efforts have been made in last two decades to develop non-invasive or minimally invasive methods to measure IAA digestibility in humans. The application of intrinsically labeled (with stable isotopes like 13C, 15N and 2H) dietary proteins have helped in circumventing the invasive oro-ileal balance techniques and allowed for the differentiation between endo and exogenous protein digestibility. The non-invasive indicator amino acid oxidation (IAAO) technique, which is routinely employed to measure IAA requirements, has been modified to estimate metabolic availability (a sum of digestibility and utilization) of IAA in foods, but provide estimate for a single IAA at a time and burdensome for participants. The recently developed minimally invasive dual isotope tracer method measures small intestinal digestibility of multiple amino acids at once and is suitable for use in vulnerable groups and disease conditions. However, it remains to be validated against standard oro-ileal balance techniques. This review critically evaluates and compares the currently available stable isotope-based protein quality evaluation methods with a focus on the digestibility and metabolic availability measurements in humans. In the view of building reliable DIAAS database of various protein sources and subsequently supporting protein content claims in food labeling, a re-evaluation and harmonization of the available methods are necessary.

Sunflower is a promising protein source but data on amino acid (AA) digestibility are lacking in humans. Classically, the determination of AA digestibility requires ileal digesta sampling. The dual isotope method is minimally invasive but has not been compared to the conventional approach.This study aimed to determine the true ileal digestibility of sunflower AAs in healthy volunteers who ate biscuits containing 15nitrogen (N) protein isolate, in comparison with the dual isotope method.Twelve healthy volunteers (men and women; 40.4 ± 10.5 years old; BMI, 23.7 ± 2.9 kg/m2) were equipped with a naso-ileal tube. For 4 hours, they consumed 9 repeated meals comprising 15N-sunflower protein biscuits together with 13carbon (C)-AAs, carried either in chocolate (SUN + Ch; n = 7) or apple puree (SUN + P; n = 5). Ileal digesta and blood were sampled throughout 8 hours after ingestion of the first meal. The 15N and 13C AA enrichments were measured in digesta to determine ileal digestibility directly and in plasma to determine lysine and threonine digestibility using the dual isotope method. Differences between methods and between vector groups were analyzed using paired and unpaired t-tests, respectively.The ileal digestibility of sunflower indispensable AAs (IAA) was 89% ± 5.3%, with threonine and lysine having the lowest digestibility. In the SUN + Ch meal, IAA digestibility was 3% below that of SUN + P (P 0.05). The mean free 13C-AA ileal digestibility was 98.1% ± 0.9%. No matter which matrix was used to carry 13C-AAs, plasma 15N and 13C-AA kinetics displayed a 1-hour offset. Digestibility obtained with the dual isotope method (70.4% ± 6.0% for threonine and 75.9% ± 22.3% for lysine) was below the target values.The ileal digestibility of IAAs from a sunflower isolate incorporated in a biscuit was close to 90% in healthy adults. Under our experimental conditions, the dual isotope method provided lower values than the usual method. Further protocol developments are needed to validate the equivalence between both methods. This trial was registered at clinicaltrials.gov as NCT04024605.

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with

Background It is necessary to propose plant alternatives to animal proteins that are of good nutritional quality. Pea is a good candidate owing to its high protein content and its well-balanced amino acid (AA) profile. Objective This study aimed at assessing the real ileal AA and nitrogen digestibility (RIDAA and RIDN) of pea protein isolate as compared to milk casein in humans. It also aimed to evaluate their nutritional quality through calculation of the digestible indispensable amino acid score (DIAAS) and to determine the net postprandial protein utilization (NPPU). Methods Fifteen healthy volunteers were included in a randomized, single-blinded, 2-arms parallel design, trial. They were equipped with a naso-ileal tube. They ingested the test meals, which consisted of nine successive portions of mashed potatoes containing either pea protein or casein, intrinsically labelled with 15N. Ileal content, plasma and urine samples were collected regularly over an 8 h postprandial period. Results Mean RIDAA was 93.6 ± 2.9% for pea protein and 96.8 ± 1.0% for casein, with no difference between the sources (P = 0.22). Leucine, valine, lysine and phenylalanine were significantly less digestible in pea than in casein. The RIDN was 92.0 ± 2.7% and 94.0 ± 1.7% for pea protein and casein, respectively, and was not different (P = 0.11). The DIAAS was 1.00 for pea protein and 1.45 for casein. The NPPU was 71.6 ± 6.2% and 71.2 ± 4.9% for pea protein and casein, respectively (P = 0.88). Conclusion Although some AAs are less digestible in pea protein than in casein, the RID and the NPPU were not different. The DIAAS of 1.00 obtained for pea protein demonstrated its ability to meet all AA requirements. This study shows the potential of pea isolate as a high quality protein. This study was registered at clinicaltrials.gov as NCT04072770.

Resume Le PD-CAAS et le DIAAS rendent compte simplement de la qualite d’une proteine sur la base de sa digestibilite et de sa composition en acides amines indispensables comparee a une composition de reference. Le calcul de ces scores comporte des points critiques, parmi lesquels le profil de reference utilise selon la tranche d’âge. Par ailleurs, le facteur de conversion de l’azote en proteine utilise pour quantifier les proteines dans l’ingredient a un impact, le facteur 6,25 penalisant generalement les scores de qualite. De meme, l’utilisation du poids moleculaire libre ou « en chaine » des acides amines pour determiner le profil en acides amines de la proteine influence les scores. Il est donc primordial que ces parametres soient renseignes. Enfin, selon le modele (Homme, porc ou rat) et la methodologie (mesure ileale ou fecale, evaluation des pertes endogenes ou utilisation de traceurs isotopiques) utilises pour evaluer la digestibilite, les valeurs peuvent varier et il est important de considerer ces aspects pour interpreter ces scores et comparer les sources proteiques entre elles.

BACKGROUND In the context of developing plant protein sources for humans, sunflower is a good candidate in its form as an oilseed coproduct. OBJECTIVES We aimed to compare the real digestibility in rats of a sunflower isolate to that of goat whey protein. We also studied the efficiency of 15N and 2H intrinsic labeling in this assessment. METHODS Sunflower seeds and goat milk were labeled with 15N and 2H. Male Wistar rats (10 wk old) were fed a meal containing 12% of either sunflower isolate (n = 8) or whey (n = 8). Six hours after meal ingestion, protein and amino acid digestibility were assessed by measuring nitrogen, hydrogen, and amino acids in the digesta, as well as isotope enrichments in the bulk and individual amino acids. The differences between groups and isotopes were respectively tested with an unpaired and a paired t test. RESULTS Protein isolate purity was 87% for whey and 94% for sunflower. 2H and 15N enrichments were, respectively, 0.12 atom % (AP) and 1.06 AP in sunflower isolate and 0.18 AP and 0.95 AP in whey. Fecal 15N protein digestibility was 97.2 ± 0.2% for whey and 95.1 ± 0.5% for sunflower isolate. The use of 2H resulted in a lower digestibility estimate than 15N for whey (96.9 ± 0.2%, P 1.0 for whey and 0.84 for sunflower (lysine). CONCLUSIONS The protein and amino acid digestibility of sunflower isolate was high but its DIAAS reflected a moderate lysine imbalance. Despite slight differences with 15N, deuterium produced comparable results, making it suitable for in vivo digestion studies.

International audience; Physiological parameters such as adiposity and age are likely to infuence protein digestion and utilization. The aim of this study was to evaluate the combined efects of age and adiposity on casein protein and amino acid true digestibility and its postprandial utilization in ratsFour groups were included (n=7/8): 2 months/normal adiposity, 2 months/high adiposity, 11 months/normal adiposity and 11 months/high adiposity. Rats were given a calibrated meal containing 15N-labeled casein (Ingredia, Arras, France) and were euthanized 6 h later. Digestive contents were collected to assess protein and amino acid digestibilities. 15N enrichments were measured in plasma and urine to determine total body deamination. Fractional protein synthesis rate (FSR) was determined in diferent organs using a fooding dose of 13C valine.Nitrogen and amino acid true digestibility of casein was around 95–96% depending on the group and was increased by 1% in high adiposity rats (P=0.04). Higher adiposity levels counteracted the increase in total body deamination (P=0.03) that was associated with older age. Signifcant efects of age (P=0.006) and adiposity (P=0.002) were observed in the muscle FSR, with age decreasing it and adiposity increasing it.This study revealed that a higher level of adiposity resulted in a slight increase in protein and individual amino acid true digestibility values and seemed to compensate for the metabolic postprandial protein alterations observed at older age.

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International audience; Objectives Age and adiposity can impact the digestibility of dietary proteins and the metabolic response to their ingestion. The objective was to evaluate the effects of age and adiposity on casein digestibility and protein synthesis in tissues and organs. Methods Wistar rats of 1 month (n = 15) and 10 months (n = 15) at their arrival were fed ad libitum with a standard diet or High Fat diet to obtain rats of normal and high adiposity levels. Four groups were constituted (n = 7/8): 2 months/normal adiposity, 2 months/high adiposity, 11 months/normal adiposity and 11 months/high adiposity. At the end of the dietary intervention, they were fed the standard diet for 1 week before the test meal. The rats consumed a 4g meal containing 15N-labeled casein (Prodiet® 85B). Six hours after meal ingestion, the rats were euthanized. Intravenous injection of a massive dose of 13C-valine prior to euthanasia was used to determine protein synthesis rate in liver, kidneys, skin and muscle. Body composition was evaluated and digestive contents were collected to measure casein digestibility. Results No weight difference between rats of the same age was observed. However, a significant difference in adiposity was noted, with a surge in body fat of 3% in young rats and 7% in older rats. Digestibility increased with a higher adiposity level (P = 0.04). In young rats, it was 94.1 ± 1.1% in lean rats and 95.2 ± 1.7% in fat rats. In older rats, it was 94.5 ± 2.2% and 95.8 ± 0.7%, in lean and fat rats respectively. Significant effects of age (P

OBJECTIVES: The measurement of amino acid (AA) digestibility of protein through direct ileal sampling is highly invasive and inappropriate for vulnerable populations, such as children or elderly. The new dual tracer method relies on comparing meal and plasma isotopic ratios of 1/a test protein 2/a reference protein (or AA mix) of known digestibility, each one being labelled with a different isotope. The aim of this study was to compare this new indirect dual tracer method to standard ileal method, for the determination of AA digestibility of pea protein and milk casein. METHODS: Fifteen healthy adult volunteers completed the study and were equipped with a naso-ileal tube. They were given 9 portions of mashed potatoes containing either pea protein or casein isolates that were intrinsically labelled with (15)N and (2)H. A (13)C algal free AA mix was added in the meals as the reference for dual tracer method. Plasma samples were collected regularly from before the first ingestion to 8-h later, while ileal digesta were collected continuously. For ileal sampling method, the AA digestibility (RID(AA)) was determined using the recovery a non-absorbable marker (PEG-4000) perfused in the ileum, and the measurement of (15)N enrichment of the digesta. For the dual tracer method, the amount of AA absorbed (Ab(AA)) was calculated by the ratio of (2)H/(13)C enrichments in plasma and in meals. The isotopic enrichments were evaluated in digesta, plasma samples and meals by GC-C-IRMS. The AA content was measured in digesta and meals by U-HPLC. RESULTS: Mean Ab(AA) and RID(AA) of pea protein were 102.2 ± 3.1% and 94.3 ± 1.5%, respectively. Mean Ab(AA) and RID(AA) of casein were 91.9 ± 2.0% and 97.1 ± 0.8%, respectively. The dual tracer method overestimated by 10% and 5% the AA digestibility of pea protein and casein, respectively, and the variability was high. The mean ileal AA digestibility of the (13)C free AA mix was high (98.1 ± 1.1%), which validated our choice to use it as the reference ‘protein’ in the dual tracer method. CONCLUSIONS: Several AA digestibilities obtained with dual tracer method were in the same range as the digestibilities from ileal sampling method. The variability was high and the effect of the protein source was inconsistent. After further research and validation, the dual tracer method could lead to notable advances in the determination of protein quality in humans. FUNDING SOURCES: Roquette.

International audience; Objectives In the current context of finding plant alternatives to animal proteins, pea would be a good option regarding its high protein content and its well-balanced amino acid (AA) profile. However, we must examine its digestibility, a main criteria of protein nutritional quality. The aim of this study was to determine the real ileal AA and nitrogen (N) digestibility (RIDAA and RIDN) of pea protein as compared to milk casein in humans. We also evaluated their respective nutritional qualities through the calculation of the digestible indispensable amino acid score (DIAAS) and the net postprandial protein utilization (NPPU). Methods Fifteen healthy adult volunteers completed the study and were equipped with a triple-lumen naso-ileal tube. They were given 9 portions of mashed potatoes containing either pea protein or casein isolates that were intrinsically labelled with 15N. PEG-4000 was perfused in the ileum as a non-absorbable marker to measure the intestinal flow rate. Ileal digesta were collected continuously by aspiration with a syringe over an 8-h postprandial period, while plasma and urine were sampled regularly. N and AA contents of digesta and protein isolates were measured using EA and U-HPLC. PEG-4000 content of the digesta was measured by turbidimetric method. Urea was extracted from plasma and urine samples. 15N enrichment was assessed in digesta, urea and protein isolates by EA-IRMS and in individual AAs by GC-C-IRMS. Results Mean RIDAA was 93.6 ± 2.9% and 96.8 ± 1.0% for pea protein and casein, respectively, with no significant difference between groups (P = 0.22). RIDN was 92.0 ± 2.7% and 94.0 ± 1.7% for pea protein and casein, respectively, and were not different (P = 0.11). The DIAAS was 1.00 for pea protein and 1.45 for casein. The NPPU was 67.0 ± 6.2% for pea protein and 70.7 ± 1.9% for casein and the difference was not significant (P = 0.15). Conclusions Overall, the bioavailability was not different between pea protein and casein in healthy adults. The DIAAS of pea protein reached 1, revealing the absence of limiting AA in regard to the requirements. Considering its AA composition and the digestibility results we obtained, pea protein can be qualified as a high-quality protein. Funding Sources Roquette

OBJECTIVES: The direct assessment of ileal samples of amino acid (AA) digestibility is invasive in humans. A less invasive but indirect method, namely « dual tracer » was recently developed. It relies on the plasma isotopic enrichment ratio of two labeled protein, a reference protein or alternately AAs labeled with (13)C and the test protein labeled with (15)N. This recent method has not yet been challenged against direct measurement of ileal digestibility. METHODS: Seven healthy volunteers were intubated with naso-ileal tube. Every 30min for 4h, they ingested sunflower biscuits containing a total of 25g of (15)N intrinsically labeled sunflower protein isolate. They also ingested 60g chocolate containing a total of 400mg of a mix of (13)C algal individual AAs. Ileal contents were collected continuously for 8h following the first meal and plasma was sampled every 30min for 4h and hourly between 4 and 8h. (15)N and (13)C indispensable amino acid (IAA) ileal digestibility were determined by measuring (15)N and (13)C enrichment in AAs by GC-C-IRMS and AA content by UHPLC in ileal effluent. Plasma and meal (15)N and (13)C IAA enrichment were measured by GC-C-IRMS. Isotopic (15)N/(13)C ratio were determined using area under the curve value for each isotope. RESULTS: Using direct ileal sampling, average IAA ileal digestibility was: (i) 88.5 ± 5.0% for sunflower isolate ((15)N) with values ranged from 85.8 ± 5.1% for threonine to 91.1 ± 5.8% for methionine, and (ii) 97.6 ± 1.7% for free AAs (13C) with values ranged from 95.9 ± 2.3% for lysine to 98.8 ± 0.8% for phenylalanine. With the “dual tracer” method, digestibility of isoleucine, leucine, threonine and valine was significantly lower than with ileal determination (from 7.9% for threonine to 24.3% for leucine), Methionine and phenylalanine values were aberrant (over 100%) For lysine, the difference between the two methods was not statistically different (4.7%, p = 0.49). CONCLUSIONS: With our methodological conditions, the “dual tracer” method provides physiological values for most IAA except methionine and phenylalanine. However, values were low compared to ileal digestibility (about 10%) and interindividual variability was high. This less invasive method is promising but requires methodological improvements. FUNDING SOURCES: French Research National Agency (ANR), financial support of SOFIPROTEOL under the FASO Project PRODIAL.

International audience; Objectives The availability of indispensable amino acids (IAA) modulates protein turnover. More particularly AAI deficiency reduces protein synthesis while the consequence on proteolysis remains unclear. The present study aims to evaluate the specific response of both protein synthesis and proteolysis to a diet restricted on one strictly indispensable IAA, either lysine or threonine Methods Sixty-four growing rats were divided into 8 groups (n = 8/group). They were fed for 3 weeks isocaloric diets composed with different levels of lysine or threonine (L or T), 15, 25, 40, 60, 75, 100 or 170% of the theoretical lysine/threonine requirements. At the end of the experiment, rats were injected with valine13C and tissues and biological fluids were collected for gene expression measurement and blood amino acids (AA). Protein synthesis rate (Fractional and Absolute rate synthesis, ie FSR, ASR) were determined in liver and muscle. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Results Severe (L/T15, L/T25) and moderate (T40) lysine or threonine deficiency resulted in a decrease in body weight gain due to a decrease in lean body mass. Severe restriction (L15, T15, T25) decreased the muscle FSR whereas no effect was observed in the liver. When the rate of protein synthesis was expressed per tissue, the ASR was decreased by severe restriction of lysine and threonine in liver and muscle and by moderate threonine deficiency (T40, T60, T75) in muscle. In liver, no effect of lysine and threonine on proteolysis was observed. In muscle, only severe lysine (L15) deficiency increased proteolysis. Dietary lysine deficiency induced a decrease in lysine concentration in the portal vein and in the vena cava whereas for threonine deficiency, all AAIs except threonine were decreased in the portal vein and vena cava. Conclusions These results indicate that the decreased protein synthesis is the primary mechanism involved in decreased lean body mass in response to the severe deficiency in a single AAI. Deficiency of a single AAI reproduce the effect of the low protein diet on protein synthesis. Lysine and threonine deficiency differently affect for a part protein turnover probably in relation with the tissue where they are metabolized. Funding Sources This study was funded by the doctoral school ABIES and AlimH-INRAE department.

Background Insects have recently been identified as a more sustainable protein-dense food source and may represent a viable alternative to conventional animal-derived proteins. Objectives We aimed to compare the impacts of ingesting lesser mealworm– and milk-derived protein on protein digestion and amino acid absorption kinetics, postprandial skeletal muscle protein synthesis rates, and the incorporation of dietary protein–derived amino acids into de novo muscle protein at rest and during recovery from exercise in vivo in humans. Methods In this double-blind randomized controlled trial, 24 healthy, young men ingested 30 g specifically produced, intrinsically l-[1-13C]-phenylalanine and l-[1-13C]-leucine labeled lesser mealworm– or milk-derived protein after a unilateral bout of resistance-type exercise. Primed continuous l-[ring-2H5]-phenylalanine, l-[ring-3,5-2H2]-tyrosine, and l-[1-13C]-leucine infusions were applied, with frequent collection of blood and muscle tissue samples. Results A total of 73% ± 7% and 77% ± 7% of the lesser mealworm and milk protein–derived phenylalanine was released into the circulation during the 5 h postprandial period, respectively, with no significant differences between groups (P 0.05). Incorporation of mealworm and milk protein-derived l-[1-13C]-phenylalanine into de novo muscle protein was greater after exercise than at rest (P 0.05). Conclusions Ingestion of a meal-like amount of lesser mealworm–derived protein is followed by rapid protein digestion and amino acid absorption and increases muscle protein synthesis rates both at rest and during recovery from exercise. The postprandial protein handling of lesser mealworm does not differ from ingesting an equivalent amount of milk protein concentrate in vivo in humans. This trial was registered at www.trialregister.nl as NL6897.

International audience; Background: Whey protein and zein are of nutritional interest due to their high leucine content, but little data are available on their amino acid (AA) ileal digestibility.Objective: This study aimed to determine ileal digestibility of whey protein isolate (WPI) and zein in healthy volunteers by use of the naso-ileal intubation method, which allows continuous collection of postprandial ileal digesta.Methods: Twenty-two healthy volunteers were intubated with a naso-ileal sampling device positioned at the terminal ileum level. They received a single meal of protein-free biscuits and a drink containing zein (n = 8), WPI (n = 7), or no protein (protein free, n = 7). Ileal effluents and plasma samples were collected over a 9-h postprandial period. Total nitrogen and AA contents were quantified in effluents. True ileal digestibility was calculated after correction for endogenous losses evaluated in the protein-free group.Results: True ileal nitrogen digestibility of zein was markedly lower than WPI (60.2 ± 4.5% and 91.2 ± 2.6%, respectively, P = 0.0003). True ileal digestibility of AAs ranged from 87.4 ± 2.7% for threonine to 98.4 ± 1.0% for methionine in the WPI group, and from 59.3 ± 5.6% for methionine to 69.0 ± 5.8% for arginine in the zein group. The digestible indispensable AA (IAA) score was 1.03 (histidine) for WPI and close to 0 for zein, owing to its negligible lysine content. Plasma IAA concentration significantly increased after WPI intake (P = 0.0319), whereas no effect of zein on aminoacidemia was observed, including plasma leucine, despite its high leucine content.Conclusions: Our findings provide data on ileal digestibility of WPI and zein AAs in healthy humans and, in contrast to WPI, zein is poorly digestible. This study was registered at clinicaltrials.gov as NCT03279211.

International audience; Purpose: Spirulina is often used as dietary supplement for its protein content and quality. However, in vivo data on protein digestibility are lacking. This study aims to determine nitrogen and amino acid digestibility in rats. A secondary objective was to test the effect of sonication prior to ingestion to break cell walls.Methods: Wistar rats were fed a single test meal containing 15N Spirulina that was either sonicated (n = 11) or not (control, n = 13). Rats were euthanized 6 h after the meal ingestion. Spirulina nitrogen digestibility was measured by assessment of 15N recovery in digestive contents. Amino acid digestibility was measured by quantification of the caecal amino acid content and their 15N enrichment.Results: Real fecal nitrogen digestibility was 86.0 ± 0.7%, without any differences between groups. Mean 15N amino acid caecal digestibility was 82.8 ± 1.3%, and values ranged between 77.9 ± 1.9% for serine and 89.4 ± 1.0% for methionine. No effect of sonication was observed. The most limiting AA was histidine, with a chemical score of 0.98 and a PD-CAAS of 0.84. Lysine was also limiting in a lesser extent.Conclusion: The nitrogen and amino acid digestibility of Spirulina is relatively low, and showed no effect of prior sonication. Its amino acid composition is relatively well balanced but not enough to compensate for the poor digestibility.

Introduction et but de l’etude La proteine de pois est une source proteique interessante en alimentation humaine par son profil en acides amines (AA) bien equilibre. Pour evaluer sa qualite, il est necessaire d’evaluer la digestibilite des AA. Chez l’Homme, la mesure de digestibilite au niveau ileal est possible par l’intermediaire d’une sonde naso-ileale, mais cette procedure est tres invasive. Une methode indirecte dite « double traceur », se basant uniquement sur des prelevements plasmatiques, a ete developpee recemment (Devi et al. Am J Clin Nutr. 2018). Le premier objectif de cette etude est de determiner la digestibilite ileale reelle de la proteine de pois chez l’Homme en comparaison d’une proteine animale de bonne qualite, la caseine. Un second objectif est de confronter les resultats obtenus a la methode double traceur. Materiel et methodes Du lait et du pois ont ete doublement marques au 15N et 2H, a partir desquels ont ete produits de la caseine et un isolat de proteines de pois. Quinze volontaires intubes avec une sonde naso-ileale ont recu un repas fractionne en 9 portions distribuees sur 4 h contenant une proteine test, aleatoirement l’isolat de pois (n = 8) ou la caseine (n = 7). Le contenu ileal et le plasma ont ete recueillis pendant 8 h. La digestibilite de l’azote et des AA a ete determinee par mesure des quantites d’azote et d’AA retrouves dans les effluents ileaux et de leurs enrichissements en 15N, par EA-IRMS pour l’azote et U-HPLC et GC-C-IRMS pour les AA. Le DIAAS a ete calcule a partir du profil de besoin adulte (FAO 2011). Pour mettre au point la methode double traceur, des AA libres marques au 13C et supposes totalement absorbes, ont ete integres aux repas et les enrichissements des AA en 2H et 13C dans le repas et le plasma ont ete evalues par GC-C-IRMS. Resultats et analyse statistique Les enrichissements en 15N et 2H des isolats proteiques etaient respectivement de 0,7 et 0,1AT % pour le pois et 1,8 et 0,1AT % pour la caseine. La digestibilite ileale de la proteine de pois a ete evaluee a 92,4 ± 3,1 %, legerement inferieure a celle de la caseine (95,7 ± 1,2 %, t test p = 0,02). La digestibilite ileale des AA variait de 95,2 % a 98,0 % (glycine et acide glutamique) pour la proteine de pois et de 96,2 % a 99,4 % (acide glutamique et phenylalanine) pour la caseine. En revanche, la digestibilite ileale moyenne des AA etait comparable entre les 2 proteines (97,1 ± 1,5 % pour le pois et 97,5 ± 1,0 % pour la caseine). Le DIAAS etait de 1,03 pour le pois et 1,45 pour la caseine. Les analyses des enrichissements isotopiques dans les AA plasmatiques sont en cours pour calculer la digestibilite des AA des 2 proteines par la methode double traceur. Conclusion L’isolat de pois presente une tres bonne biodisponibilite, avec une digestibilite ileale de l’azote legerement inferieure a celle de la caseine et equivalente pour les acides amines. En consequence, son DIAAS est superieur a 1, ce qui en fait une des rares sources vegetales sans acide amine limitant, confirmant ainsi son interet pour l’alimentation humaine.

The objective of this study was to assess the nutritional quality of pea protein isolate in rats and to evaluate the impact of methionine (Met) supplementation. Several protein diets were studied: pea protein, casein, gluten, pea protein–gluten combination and pea protein supplemented with Met. Study 1: Young male Wistar rats (n 8/group) were fed the test diets ad libitum for 28 d. The protein efficiency ratio (PER) was measured. Study 2: Adult male Wistar rats (n 9/group) were fed the test diets for 10 d. A protein-free diet group was used to determine endogenous losses of N. The rats were placed in metabolism cages for 3 d to assess N balance, true faecal N digestibility and to calculate the Protein Digestible-Corrected Amino Acid Score (PDCAAS). They were then given a calibrated meal and euthanised 6 h later for collection of digestive contents. The true caecal amino acid (AA) digestibility was determined, and the Digestible Indispensable Amino Acid Score (DIAAS) was calculated. Met supplementation increased the PER of pea protein (2·52 v. 1·14, P < 0·001) up to the PER of casein (2·55). Mean true caecal AA digestibility was 94 % for pea protein. The DIAAS was 0·88 for pea protein and 1·10 with Met supplementation, 1·29 for casein and 0·25 for gluten. Pea protein was highly digestible in rats under our experimental conditions, and Met supplementation enabled generation of a mixture that had a protein quality that was not different from that of casein.

International audience; Objectives The global demand for protein is growing and it seems mandatory to find alternatives to animal proteins that are of good nutritional quality, implying a good digestibility. Legumes are potential candidates, as their indispensable amino acid (IAA) profile is relatively balanced. The aim of this study is to compare the protein and AA ileal digestibility of pea protein and casein in humans using ileal sampling and intrinsically labelled proteins. Methods Sixteen healthy volunteers were intubated and were given twelve doses of mashed potatoes containing either pea protein or milk casein isolates. The proteins were intrinsically labelled with 15N. Polyethylene glycol (PEG) was perfused as a non-absorbable marker to measure the intestinal flow, and ileal digesta were collected during 8 hours after the ingestion of the first meal. PEG content was assessed using a turbidimetric assay. Real ileal nitrogen (N) digestibility was measured by assessing N content and 15N enrichment by EA-IRMS and calculated as follows: $$\begin{eqnarray} \begin{array} \text{Ileal fiow (mL/30 min)} &=& \rm{[PEG]_{solution}/[PEG]_{digesta}}\\ &&\times \text{ infusion rate} \times \rm{time} \end{array} \end{eqnarray}$$$$\begin{eqnarray} \begin{array} \text{Exogenous N fiow (mmol/30 min) } &=& \rm {N_{exo} = (\% DM \times \%N)}\\ &&/(14 \times 10) \times \text{ileal fiow} \times \rm{APE_{digesta}/APE_{meal}} \end{array} \end{eqnarray}$$$$\begin{eqnarray} \rm{Digestibility\ (\%)} = (\rm{N_{ingested} - N_{exo} / N_{ingested} \times 100}) \end{eqnarray}$$$$\begin{eqnarray} \text{WithDM = DryMatterofdigesta, APE = AtomPercentExcess} \end{eqnarray}$$ Results The first results were obtained on 7 subjects that were given pea protein (N = 3) or milk casein (N = 4). The mean ileal flow for all the volunteers was 1.83 ± 0.70 mL/30 min (mean ± SD), and was quite stable over time. The total N exogenous flow was 15.50 ± 5.83 mmol for pea protein and 13.56 ± 3.48 mmol for casein. The true ileal N digestibility was 95.40 ± 1.73% for pea protein and 95.42 ± 1.17% for casein. Ileal IAA digestibility is in progress. Conclusions The intermediate results show no difference between pea protein and casein in terms of ileal digestibility, suggesting that pea protein could be a promising plant-based alternative. AA digestibilities and data from the remaining 9 volunteers to be tested will complete the study. Funding Sources Roquette.

OBJECTIVES: The aim of this study was to evaluate the digestibility of casein and its amino acids (AA) in rats of different ages (2 months vs 11 months) and adiposity levels (normal vs high). METHODS: Wistar rats of 1 month (n = 15) and 10 months (n = 15) at their arrival were fed ad libitum for 28 days either with a standard diet (14% P/E, 75% C/E, 11% L/E) or a Western diet (14% P/E, 51% C/E, 35% L/E) in order to obtain respectively rats of normal and high adiposity levels. Four groups were constituted (n = 7/8): 2 months/normal adiposity (2 M/NA), 2 months/high adiposity (2 M/HA), 11 months/normal adiposity (11 M/NA) and 11 months/high adiposity (11 M/HA). After a week on the standard diet, the rats consumed a 4 g meal containing (15)N labeled casein (Prodiet® 85B). Blood samples were taken at t = 0, t = 1 h, t = 3 h after meal consumption, and the rats were euthanized at t = 6 h. Digestive contents were collected and body composition was determined. Nitrogen quantity and (15)N enrichment were analyzed in the meal and digestive contents by EA-IRMS, allowing for the estimation of casein digestibility. Concentrations in AA and their (15)N enrichments were determined respectively by UHPLC and GC-C-IRMS to obtain the digestibility of individual AA. RESULTS: Weight did not differ between groups of the same age, but body composition analysis showed a significant difference in adiposity (P = 0.0008 between the 2 M groups; P = 0.0004 between the 11 M groups). Nitrogen fecal digestibility of casein increased significantly in rats with higher adiposity levels (2 M/NA = 94.1 ± 1.1%; 2 M/HA = 95.2 ± 1.7%; 11 M/NA = 94.5 ± 2.2% for 11 M/HA = 95.9 ± 0.7%; P = 0.0339). No difference was found in the digestibility of individual AA. 6 hours after meal ingestion, there was significantly more exogenous nitrogen in the stomach of rats aged 11 months compared to those of 2 months (3,9 ± 6,9% for the 2 M groups and 11,3 ± 9,2% for the 11 M groups P = 0.0188;). Except for methionine, there was no difference in plasma concentration levels of essential AA over time between the groups. CONCLUSIONS: Our results suggest that age slows down digestion, in accordance with the literature. We showed that adiposity increased casein digestibility but without any effect at the level of individual AA digestibility. FUNDING SOURCES: Ingredia.

International audience; Background: Assessment of amino acid bioavailability is of key importance for the evaluation of protein quality; however, measuring ileal digestibility of dietary proteins in humans is challenging. Therefore, a less-invasive dual stable isotope tracer approach was developed.Objective: We aimed to test the assumption that the 15N:13C enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of 15N test protein.Methods: In a crossover design, 10 healthy adults were given a semi-liquid mixed meal containing 25 g (low protein) or 50 g (high protein) of 15N-labeled milk protein concentrate simultaneous with 0.4 g of highly 13C-enriched spirulina. The meal was distributed over multiple small portions, frequently provided every 20 min during a period of 160 min. For several amino acids, the blood 15N- related to 13C-isotopic enrichment ratio was determined at t = 0, 30, 60, 90, 120, 180, 240, 300, and 360 min and differences between the 2 meals were compared using paired analyses.Results: No differences in 13C AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas 15N AUC of amino acids was ∼2 times larger on the high-protein meal (P < 0.001). Doubling the intake of 15N-labeled amino acids increased the 15N:13C ratio by a factor of 2.04 ± 0.445 for lysine and a factor between 1.8 and 2.2 for other analyzed amino acids, with only phenylalanine (2.26), methionine (2.48), and tryptophan (3.02) outside this range.Conclusions: The amino acid 15N:13C enrichment ratio in the peripheral circulation increased proportionally to the quantity of 15N-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using 15N-labeled protein, correction for, e.g., α-carbon 15N atom transamination is advised for determination of bioavailability of individual amino acids. This trial was registered at www.clinicaltrials.gov as NCT02966704.

Introduction:Sunflower protein is not used in human nutrition despite a relatively good amino acid composition. However, the bioavailability of sunflower isolate has never been measured in Humans. The goal of this work was to determine ileal digestibility of protein and amino acids from a sunflower isolate in healthy volunteers and to challenge newly developed dual isotope method.Materials and methods:Eight healthy volunteers were equipped with a naso-ileal tube. They received during four hours twelve doses of biscuits containing, in total, 25 g of 15N sunflower protein isolate together with 400 mg of a mixture of free 13C amino acids incorporated in chocolates. Polyethylene glycol was perfused as non-absorbable marker and ileal contents were collected during 8 hours after ingestion of the first meal. Real ileal digestibility was measured by assessing nitrogen and carbon content as well as 15N and 13C enrichments by EA-IRMS. Amino acid digestibility was determined by measuring 15N and 13C enrichments by GC-C-IRMS and quantity of amino acids by UPLC. Blood was collected for 8 h to determine 15N and 13C enrichments by GC-C-IRMS.Results:The ileal nitrogen flow was 2.7 ± 0.5 mL/min (mean ± SD). In average, 53.1 ± 12.0 mmol of exogenous nitrogen was recovered during the eight hours of experiment, resulting in an ileal digestibility of sunflower isolate was 85.6 ± 2.6 % of nitrogen ingested. 13C amino acids were also recovered at the ileal level. The mixture of free 13C revealed an ileal digestibility of 94.9 ± 0.9 %. Ileal indispensable amino acids digestibility and DIAAS are in progress.Discussion:Ileal digestibility of sunflower isolate incorporated in toasted biscuits was lower than the value found or a raw isolate in a rat model (94.5%). The study revealed that 5 % of free amino acids were not absorbed in the ileum. Amino acid digestibility will complete the study to evaluate the DIAAS of sunflower isolate and to compare values obtained with the standard method and with the dual isotope method.

Spirulina is often used as dietary supplement for its protein content and quality. However, in vivo data on protein digestibility are lacking. This study aims to determine nitrogen and amino acid digestibility in rats. A secondary objective was to test the effect of sonication prior to ingestion to break cell walls.Wistar rats were fed a single test meal containingReal fecal nitrogen digestibility was 86.0 ± 0.7%, without any differences between groups. MeanThe nitrogen and amino acid digestibility of Spirulina is relatively low, and showed no effect of prior sonication. Its amino acid composition is relatively well balanced but not enough to compensate for the poor digestibility.

International audience; Objectives Severely low-protein (LP) diets induce a decrease in body weight and an increase in relative food including intake (FI) in rat. In the liver, changes in anabolic and catabolic protein pathways could transitorily participate to compensate for amino acid (AA) deficiency. The present study investigated these liver and muscle protein metabolic pathways on LP diet fed growing rats. Methods Growing rats were fed for three weeks different diets containing 3–5–8–12–15 or 20% energy from milk protein. Body weight and FI were measured daily. At the end of the experiment, rats were injected with 13C valine and tissues and biological fluids were collected for gene expression measurement, blood AA UPLC analysis and protein synthesis rate determination in liver and muscle. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Results P3, P5 and P8% diets resulted in significant growth retardation and significant decrease in lean mass. Severe protein deficiency induced a decrease in the rate of protein synthesis in the liver and muscle. In addition, the results showed activation of the GCN2 pathway, via ATF4-CHOP-TRB3 both in the liver and in the muscle, which suggests the inhibition of the initiation of translation at the level of the binding of the RNAt-Met. Liver proteolytic pathways were up-regulated including the ubiquitin-proteasome, the caspase system and the autophagy. In muscle, both the ubiquitin-proteasome pathway, and autophagy were increased as well as the calpain system. The GCN2 pathway, via ATF4-CHOP-TRB3 was activated in both liver and muscle, confirming the activation of protein degradation by the ubiquitin-proteasome pathways, and autophagy. In portal vein, indispensable AA were lower in severe protein deficient diet whereas in vena cava no difference was observed. Conclusions Severe protein restriction lowered protein synthesis and activated protein catabolism in both liver and muscle whereas no effect was observed for moderate protein restriction. These results confirm that the liver and muscle play a major role in supplying the body with indispensable AA in response to severe protein restriction. Funding Sources This study was funded by the doctoral school ABIES and AlimH-INRAE department.

Adequate protein intake during development is critical to ensure optimal bone gain and to attain a higher peak bone mass later. Using a mild protein restriction model in Balb/C mice consuming 6% of their total energy intake as soy protein (LP‐SOY)—for which we observed a significantly lower femoral cortical thickness, bone volume, trabecular number, and thickness reduction—we evaluated the effects of monosodium glutamate (MSG) supplementation at different concentrations (0.5, 1, 5, 10, and 20 g/kg of diet) on bone characteristics in LP‐SOY‐fed mice. After 6 and 12 weeks, LP‐SOY‐fed mice had lower BMD and reduced body weight related to lower lean mass, which was associated with a reduced IGF‐1 level. The negative effect of the LP‐SOY diet on BMD correlated with impaired bone formation. MSG supplementation, at 5, 10, and 20 g/kg of diet, and PTH injection, used as a positive control, were able to improve BMD and to increase osteoblast activity markers (P1NP and osteocalcin), as well as glutamine plasma concentration. An analysis of bone microarchitecture found that cortical bone was less sensitive to protein restriction than trabecular bone, and that MSG ingestion was able to preserve bone quality through an increase of collagen synthesis, although it did not allow normal bone growth. Our study reinforces the view that glutamate can act as a functional amino acid for bone physiology and support clinical investigation of glutamate supplementation in adults characterized by poor bone status, notably as a result of insufficient protein intake. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

In the context of developing plant protein sources for humans, sunflower is a good candidate in its form as an oilseed coproduct.We aimed to compare the real digestibility in rats of a sunflower isolate to that of goat whey protein. We also studied the efficiency of 15N and 2H intrinsic labeling in this assessment.Sunflower seeds and goat milk were labeled with 15N and 2H. Male Wistar rats (10 wk old) were fed a meal containing 12% of either sunflower isolate (n = 8) or whey (n = 8). Six hours after meal ingestion, protein and amino acid digestibility were assessed by measuring nitrogen, hydrogen, and amino acids in the digesta, as well as isotope enrichments in the bulk and individual amino acids. The differences between groups and isotopes were respectively tested with an unpaired and a paired t test.Protein isolate purity was 87% for whey and 94% for sunflower. 2H and 15N enrichments were, respectively, 0.12 atom % (AP) and 1.06 AP in sunflower isolate and 0.18 AP and 0.95 AP in whey. Fecal 15N protein digestibility was 97.2 ± 0.2% for whey and 95.1 ± 0.5% for sunflower isolate. The use of 2H resulted in a lower digestibility estimate than 15N for whey (96.9 ± 0.2%, P 0.05) and sunflower (94.2 ± 0.5%, P 0.01). For both isotopes, protein digestibility was about 2% higher for whey than for sunflower isolate. Mean 15N amino acid caecal digestibility was 97.5 ± 0.2% for whey and 96.3 ± 0.2% for sunflower isolate. The values obtained with 15N and 2H resulted in significant differences ranging from -0.1% to 3.5%. The DIAAS was1.0 for whey and 0.84 for sunflower (lysine).The protein and amino acid digestibility of sunflower isolate was high but its DIAAS reflected a moderate lysine imbalance. Despite slight differences with 15N, deuterium produced comparable results, making it suitable for in vivo digestion studies.

International audience; Objectives The global demand for protein is growing and it seems necessary to find new alternatives for animal proteins. Legumes are good candidates, as their indispensable amino acid (IAA) profile is relatively balanced. The aim of the study is to evaluate the nutritional quality of a pea protein isolate (NUTRALYS® pea protein), with or without methionine (Met) supplementation, using various indexes. We measured the Protein Efficiency Ratio (PER), the Digestible Indispensable Amino Acid Score (DIAAS) and the nitrogen balance in rats. Methods Study 1: 40 Wistar male rats weighing ∼50 g were fed ad libitum for 28 days with a diet containing 10% protein with variation in protein sources only. 5 groups (n = 8) were included: pea, casein, wheat gluten, pea-gluten combination, pea supplemented with Met. PER is obtained by the ratio between weight gain and protein intake throughout the experimental period. Study 2: 45 Wistar male rats weighing ∼250 g were housed in metabolic cages for 2 days and fed with a diet containing 14% protein of different sources. 5 groups (n = 9) were included: pea, casein, gluten, pea supplemented with Met, and protein-free diet for endogenous losses. Nitrogen content of diets, feces and urines was measured with an elementary analyzer for nitrogen balance (N ingested – N excreted). Then, rats were given a calibrated meal containing an indigestible marker and were euthanized 6 h later. Stomach, intestine, ileum, cecum, and colon contents were collected. DIAAS was calculated as follows: mg digestible IAA in 1 g of test protein*100/mg of IAA in 1 g of reference protein. IAA in ileum contents and diets were assayed by UPLC. Results Values are means ± SD. Met supplementation allows pea protein to reach the PER of casein, but association with gluten is not sufficient. Nitrogen balance values are higher for casein and pea + Met. Fecal digestibility of pea protein is higher than casein in our conditions. Analysis for DIAAS calculation are ongoing. Conclusions Pea protein is highly digestible in our conditions and Met supplementation can improve its capacity to insure growth. Funding Sources Roquette.

International audience; ObjectivesIn order to establish DIAAS in humans, the FAO recommended to develop a new method to measure indispensable amino acid (IAA) digestibility. This method uses two isotopic labeling, one for the protein to test and one for a reference protein. Spirulina was chosen as the 13C reference protein due to its commercial availability and affordability. However, the real digestibility of spirulina protein has not been measured in vivo. This work aims to assess the digestibility of spirulina and its repeatability in different meal tests in rats. Methods 23 Wistar male rats were fed a test meal containing 0.5 g of 15 N protein from either spirulina (n = 7), sunflower n = 8) or goat milk isolate (n = 8) and 10 mg of 13C labeled spirulina. Rats were euthanized 6 h after the meal and their digestive luminal contents (stomach, small intestine, ileum, caecum, colon) were collected. Protein digestibility was determined for the test and the reference proteins by measuring 15 N and 13C enrichments in the digesta by EA-IRMS. Caecal IAA digestibility of 13C spirulina was determined by measuring the quantity of AA in the caecum by UPLC and the 13C enrichment in AA by GC-C-IRMS. Group effects were tested using one way ANOVA and differences between groups using Bonferroni test. Results Six hours after ingestion, most of the dietary 15 N and 13C were found in the caecum and colon. But there at least twice more 15 N nitrogen in the caecum and colon in the spirulina group than in the two other groups. Therefore, spirulina protein digestibility (86.0 ± 0.7%) was lower (P

Bariatric surgery may induce protein malabsorption, although data are scarce. This study aims at evaluating dietary protein bioavailability after different bariatric surgeries in rats. Diet-induced obese Wistar rats were operated for vertical sleeve gastrectomy (VSG) or Roux-en-Y gastric bypass (RYGB). The control group was composed of pair-fed, sham-operated rats (Sham). Two weeks after surgery, rats were fed a 15N protein meal. Protein bioavailability was assessed by determination of 15N recovery in the gastrointestinal tract and organs 6 h after the meal. Fractional protein synthesis rate (FSR) was assessed using a flooding dose of 13C valine. Weight loss was the highest in RYGB rats and the lowest in Sham rats. Surprisingly, RYGB (95.6 ± 0.7%) improved protein digestibility ( P = 0.045) compared with Sham (93.5 ± 0.5%) and VSG (93.8 ± 0.6%). In contrast, 15N retained in the liver ( P = 0.001) and plasma protein ( P = 0.037) was lower than in Sham, with a similar trend in muscle ( P = 0.052). FSR was little altered by bariatric surgery, except for a decrease in the kidney of RYGB ( P = 0.02). The 15N distribution along the small intestinal tissue suggests that dietary nitrogen was considerably retained in the remodeled mucosa of RYGB compared with Sham. This study revealed that in contrast to VSG, RYGB slightly improved protein digestibility but altered peripheral protein bioavailability. This effect may be ascribed to a higher uptake of dietary amino acids by the remodeled intestine. NEW & NOTEWORTHY Using a sensitive 15N meal test, we found that gastric bypass slightly improved protein digestibility compared with sleeve gastrectomy or control but, in contrast, lowered protein retention in the liver and muscles. This paradox can be due to a higher uptake of dietary nitrogen by the intestinal mucosa that was hypertrophied. This study provides new insight on the digestive and metabolic fate of dietary protein in different models of bariatric surgery in rats.

Foals born to primiparous mares are lighter and less mature than those born to multiparous dams. Factors driving this difference are not totally understood. Using 7 multiparous and 6 primiparous standardbred mares, we demonstrated that, in late gestation, primiparous mares were less insulin resistant compared to multiparous mares, and that their foals had reduced plasma amino-acid concentrations at birth compared to foals born to multiparous mares. Vascular development, as observed through structure and gene expression, and global DNA methylation were also reduced in primiparous placentas. Another group of 8 primiparous mares was orally supplemented with L-arginine (100 g/day, 210d to term). L-arginine improved pregnancy-induced insulin resistance and increased maternal L-arginine and L-ornithine plasma concentrations but foal plasma amino acid concentrations were not affected at birth. At birth, foal weight and placental biometry, structure, ultra-structure and DNA methylation were not modified. Placental expression of genes involved in glucose and fatty acid transfers was increased. In conclusion, maternal insulin resistance in response to pregnancy and placental function are reduced in primiparous pregnancies. Late-gestation L-arginine supplementation may help primiparous mares to metabolically adapt to pregnancy and improve placental function. More work is needed to confirm these effects and ascertain optimal treatment conditions.

International audience; Objectives Sunflower protein has a relatively well-balanced amino acid profile but it is little used in human food. Nutritional quality of sunflower protein isolate has never been directly measured in humans. This study aims to determine the ileal digestibility of protein and amino acid of a sunflower isolate incorporated in biscuits, in healthy humans. Methods Healthy volunteers (n = 7) were intubated with a naso-ileal tube. They ingested every 30 min for 4 h, sunflower biscuits for a total of 156 g of biscuits and 25 g of 15N intrinsically labeled sunflower protein isolate. A non-absorbable marker was perfused into the ileum. Ileal samples were continuously collected during 8 h after the first meal. Blood was sampled every 30 min for 4 h and every hour after and total urine was collected every 2 h. To measure nitrogen ileal digestibility, the percentage of nitrogen and 15N enrichment in ileal contents were assessed by an elemental analyzer coupled with and isotope ratio mass spectrometer (EA-IRMS). Indispensable amino acid (IAA) ileal digestibility was determined by measuring amino acid 15N enrichment by GC-C-IRMS and their quantification by UHPLC. Metabolic losses of dietary nitrogen were assessed by measuring 15N in urine and plasma. The digestible indispensable amino acid score (DIAAS) and the net post-prandial utilization (NPPU) were calculated. Results Exogenous nitrogen appeared in the ileum 1 h after the first meal, its flow was stable between 4 h and 7 h. Cumulated dietary nitrogen in the ileum was 14.0 ± 4.0% over 8 h. Sunflower nitrogen real ileal digestibility was 86.0 ± 4.0% of ingested nitrogen. Mean IAA ileal digestibility was 87.0 ± 6.1% with values ranging from 84.4% for lysine and threonine to 89.3 ± 7.8% for methionine. DIAAS was 0.93 for lysine. After 8 h, 4.0 ± 1.0% of dietary nitrogen was incorporated in plasma protein. Cumulated exogenous nitrogen in urinary urea was 4.0 ± 1.4% and exogenous nitrogen in body urea after 8 h was 9.8 ± 2.5%. The NPPU was 72.2 ± 3.7% at 8 h. Conclusions Sunflower isolate ileal digestibility is relatively low compared to other protein isolate assessed in similar conditions (ileal tube and 15N labeling of protein). Despite a moderate deficiency in lysine, sunflower protein isolate has a good post-prandial metabolic utilization. Funding Sources French Research National Agency (ANR), financial support of SOFIPROTEOL under the FASO Project PRODIA.

International audience; Objectives Severely low-protein diets (LP) induce behavioral and metabolic changes including a decrease in body weight, an increase in relative food intake (FI) and alterations in hepatic metabolism. During such protein restriction, changes in hepatic anabolic and catabolic protein pathways could transitory participate to compensate for amino acid (AA) deficiency. In the present study, liver expression of gene involved in proteosynthesis and proteolysis pathways, were related to FI, blood AA levels and body composition in rats fed LP diet. Methods Growing rats were fed for three weeks different diets containing 3-5-8-12-15 or 20% energy of milk protein. Body weight and FI were measured daily. At the end of the experiment, tissues and biological fluids were removed for gene expression measurement and blood AA UPLC analysis. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Results Despite an increase in relative food intake under P3 and P5% diets, P3, P5 and P8% diets resulted in significant growth retardation compared to other groups. Lean mass was significantly decreased in rats under P3, P5 and P8% compared to P12, P15 and P20% diets, while there was no difference in fat mass between all groups. P3, P5 and P8% diets induced a decrease in essential amino acid concentrations in portal vein, whereas there was no significant difference between groups in veina cava. Severely protein restricted P3% and P5% diets induced an increase in hepatic gene expression involved in proteolysis as calpain 2 and ubiquitin, and an activation of ATF4-CHOP-TRB3 pathway. Conclusions These results suggested that under severe protein restriction, hepatic protein catabolism became a source of plasma amino acid that could partially compensate for the AA not provided by the diet. These observations confirm that liver plays a major role in the adaptation of the body to dietary protein restriction and highlight that severe dietary protein restriction induced liver protein catabolism by inducing an activation of ATF4-CHOP-TRB3 pathway in order to provide amino acids to body tissues. Funding Sources ABIES, AlimH-INRAE.

Relaxin-3 (RLN3) is an orexigenic neuropeptide that produces sex-specific effects on food intake by stronger stimulation of feeding in female compared with male rats. This study determined which hypothalamic nuclei and associated neuropeptides may be involved in the sex-specific orexigenic effects of RLN3. Relaxin-3 (800 pmol) or vehicle was injected into the lateral ventricle of female and male rats. Food and water intake were measured after the first injection, and rats were euthanized after the second injection to determine the mRNA expression of the hypothalamic neuropeptides. Food but not water intake showed sex-specific effects of RLN3. Stimulation of food intake by RLN3 was significantly higher in female than in male rats. No effect of RLN3 injection was found on c-fos mRNA expression in the arcuate, dorsomedial and ventromedial hypothalamic nuclei. Increased c-fos mRNA expression was observed in the paraventricular hypothalamic nucleus (PVN) in both sexes and in the lateral hypothalamic area (LHA) in female rats. Relaxin-3 injections led to a sex-nonspecific increase in the expression of oxytocin mRNA in the magnocellular PVN. Conversely, RLN3-induced expression of anorexigenic neuropeptide arginine vasopressin (AVP) was significantly higher in the parvocellular PVN in male compared with female rats. Finally, RLN3 administration significantly increased the expression of orexin (ORX) mRNA in the LHA in female but not in male rats. Stronger expression of anorexigenic AVP in the PVN in male rats and increased expression of ORX in the LHA in female rats may contribute to stronger orexigenic effects of RLN3 in female rats compared with male rats.

Sous presse; The neuropeptide relaxin-3 (RLN3) binds with high affinity to its cognate receptor, relaxin-family peptide receptor 3 (RXFP3), and with lower affinity to RXFP1, the cognate receptor for relaxin. Intracerebroventricular (icv) administration of RLN3 in rats strongly increases food and water intake and alters the activity of the hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes, but the relative involvement of RXFP3 and RXFP1 in these effects is not known. Therefore, the effects of icv administration of equimolar (1.1 nmol) amounts of RLN3 and the RXFP3-selective agonist RXFP3-A2 on food and water intake, plasma levels of corticosterone, testosterone, and oxytocin, and c-fos mRNA expression in key hypothalamic regions in male rats were compared. Food intake was increased by both RLN3 and RXFP3-A2, but the orexigenic effects of RXFP3-A2 were significantly stronger than RLN3 30 and 60min after injection. Water intake and plasma corticosterone and testosterone levels were significantly increased by RLN3, but not by RXFP3-A2. Conversely, RXFP3-A2 but not RLN3 decreased oxytocin plasma levels. RLN3, but not RXFP3-A2, increased c-fos mRNA levels in the parvocellular (PVNp) and magnocellular (PVNm) paraventricular and supraoptic (SON) hypothalamic nuclei, in the ventral medial preoptic area (MPAv), and in the organum vasculosum of the lamina terminalis (OVLT). A significant increase in c-fos mRNA expression was induced in the perifornical lateral hypothalamic area (LHApf) by RLN3 and RXFP3-A2. These results suggest that RXFP1 is involved in the RLN3 stimulation of water intake and activation of the HPA and HPG axes. The reduced food intake stimulation by RLN3 compared to RXFP3-A2 may relate to activation of both orexigenic and anorexigenic circuits by RLN3.

OBJECTIVES: There are limited data available on true ileal nitrogen and amino acid digestibility of protein sources. The goal of this study was thus to determine for the first time in healthy volunteers the ileal digestibility of zein and whey proteins. METHODS: Twenty-two volunteers (10 women and 12 men; aged 37 ± 12y) were equipped with a double lumen intestinal tube positioned at the ileal level. They received a single meal of protein-free biscuits and a drink containing zein (n = 8), whey protein isolate (WPI, n = 7) or no protein (protein-free, n = 7). 13C-inuline was added to the drink as a non-absorbable marker. Ileal effluents were collected over a 9-h period after meal ingestion. Total nitrogen content was measured in effluents and correction for endogenous losses evaluated with the protein-free group was used to determine true digestibility of zein and WPI proteins. RESULTS: The mean ileal endogenous nitrogen flow was 3.89 ± 1.41mmol/h (mean ± SD). For zein, mean dietary nitrogen flow rate was 12.1 ± 6.9mmol/h, reaching 23.6 ± 13.5mmol/h 4h after the meal. In comparison, mean dietary nitrogen flow rate for WPI was significantly lower (1.6 ± 1.2mmol/h, P

Introduction et but de l’etude La spiruline est couramment utilisee comme complement alimentaire chez l’Homme. Sa production et sa consommation sont en augmentation dans le monde. Il existe peu de donnees sur sa qualite nutritionnelle in vivo, mais sa biodisponibilite, mesuree chez l’Homme par une methode indirecte, apparait mediocre (Engelen et al., 2014). L’objectif de cette etude est de quantifier in vivo la digestibilite reelle des proteines et acides amines de spiruline, et de determiner si une sonication prealable pour rompre les parois cellulaires peut l’ameliorer. Materiel et methodes Douze rats Wistar mâles, habitues aux conditions d’animalerie, recoivent un repas test contenant 0,77 g de spiruline, soit 0,41 g de proteines, marquee intrinsequement au 15N. Les rats sont repartis en deux groupes de 6 : spiruline prealablement soniquee pendant 1,5 h ou spiruline non soniquee. Les animaux sont euthanasies 6 h apres l’ingestion du repas test et les contenus des differents segments du tractus gastro-intestinal (estomac, intestin grele, ileon, caecum, colon) sont collectes. Afin de determiner la digestibilite globale des proteines de spiruline, la quantite d’azote et l’enrichissement en 15N des contenus digestifs sont determines par un analyseur elementaire couple a un spectrometre de masse a ratio isotopique (EA-IRMS). Les acides amines sont doses par HPLC dans le caecum et leurs enrichissements en 15N mesures par chromatographie gazeuse couple a un four de combustion et a un IRMS (GC-C-IRMS), afin de determiner leur biodisponibilite. Les resultats sont exprimes en moyennes (± ESM), comparees par un test t (p Resultats et analyse statistique Trois rats sur 12 n’ont pas consomme la totalite de leur repas test, et ce malgre une habituation prealable, du fait de l’odeur de la spiruline. La quantite d’azote alimentaire dans les differents segments du tractus gastro-intestinal est presentee dans le Tableau 1 . La quantite d’azote 15N retrouvee dans le caecum et le colon est relativement importante, et la sonication ne permet pas de la reduire significativement. En consequence, la digestibilite reelle oro-fecale des proteines de spiruline est faible (82,1 ± 1,9 %) sans amelioration significative par la sonication (86,6 ± 2,1 %), malgre une tendance. L’acide amine le moins digestible est la glycine (79,3 ± 1,5 %) et le plus digestible la tyrosine (92,7 ± 0,6 %). La digestibilite des acides amines indispensables varie de 85,1 ± 1,2 % pour la lysine a 89,6 ± 1,0 % pour la phenylalanine. Conclusion Cette etude confirme que la digestibilite oro-fecale de la spiruline est faible et inferieure a celle de proteines testees dans les meme conditions (86 et 97 % pour le colza et le lait, respectivement). Nous n’avons pas pu mettre en evidence d’amelioration par la sonication, peut-etre du fait d’un manque de puissance. Nous avons aussi mis en evidence que la lysine avait la plus faible biodisponibilite parmi les acides amines indispensables.

Metabolizable energy in dry dog food is best predicted by NRC 2006 equation Calvez J, Weber M, Ecochard C, Biourge V, Royal Canin Research Center, Aimargues, France. Metabolizable energy (ME) of petfoods can be determined by animal trials or predicted using published equations. To date, the most commonly used equation is the modified Atwater factorial equation (3.5 kcal/g of protein and carbohydrates, 8.5 kcal/g of fat) proposed by the National Research Council (NRC) in 1985. This equation is still recommended by the American Association of Feed Control Officials (AAFCO). In 2006, however, NRC has recommended newer predictive equations of ME based on digestible energy estimated as a function of dietary fiber (total dietary fiber (TDF) or crude fiber (CF)). Our objective was to compare ME determined by those different equations versus ME determined by animal trials (gold standard) in dry dog food. A dataset containing 389 observations with dry expanded dog foods was included in the study. For each dietary trial, proximate analysis, gross energy of the diet and the feces and digestion trials on 6 dogs were available. ME was predicted by modified Atwater, NRC2006 with CF or with TDF equations and compared with the experimentally determined ME by simple linear regression analysis. The mean absolute difference between measured and predicted ME was also calculated as an index of error of prediction. The mean (±SD) measured ME was 3872±298 kcal/kg of diets as fed (range: 2871-4352 kcal/kg). A good correlation was found between ME measured and predicted by modified Atwater equation (R²=0.838). However, mean error of prediction was quite high (205±111 kcal/kg, 5.3%) and this equation did underestimate ME, especially for diets with an energy density above 3500 kcal/kg. ME predicted by NRC 2006 equations using CF or TDF showed a good correlation with measured ME (R²=0.817 and 0.875 respectively) and the precision of the prediction was improved as the mean error were 105±84 kcal/kg (2.7%) and 95±76 kcal/kg (2.5%) respectively. Our results suggest that the equations recommended by the NRC in 2006 (using CF or TDF) are better than the modified Atwater equation to predict ME in dry dog food, especially for diet above 3500 kcal/kg as fed.

This study examined the effects of chronic central administration of relaxin-3 (RLN3) on food intake, body weight and fat mass in intact and sterilized male and female rats as well as on the hypothalamo-pituitary-gonadal (HPG) axis activity in intact male and female rats that received intracerebroventricular (icv) infusions of RLN3 (400 pmol/day) or vehicle during a 14-day period. The intact RLN3-injected rats displayed higher body weight than the vehicle-treated groups, and this increase was statistically significantly stronger in the female rats compared to the male rats. In addition, feed efficiency and gonadal white adipose tissue weight were higher in the female RLN3-injected rats. Chronic icv administration of RLN3 activated the HPG axis in the intact male rats whereas inhibition of the HPG axis was observed in the intact female rats. RLN3 significantly increased the plasma levels of luteinizing hormone and follicular stimulating hormone in the male rats but not in female rats. Conversely, hypothalamic expression of gonadotropin-releasing hormone mRNA was decreased by RLN3 in the female rats but not in the male rats. In addition, the plasma levels of estradiol were significantly decreased by RLN3 administration in the female rats. Consequently, the intact RLN3-injected female rats failed to display phasic inhibition of eating during estrus. Sex-specific effects of RLN3 on food intake and body weight were also observed in ovariectomized female and orchidectomized male rats suggesting that the sex-specific effects of RLN3 on energy metabolism are independent on the differential effects of RLN3 on HPG axis activity in male and female rats. This article is protected by copyright. All rights reserved.

Relaxin-3 (RLN3) is a neuropeptide that is strongly expressed in the pontine nucleus incertus (NI) and binds with high affinity to its cognate receptor RXFP3. Central administration of RLN3 in rats increases food intake and adiposity. In humans, RLN3 polymorphism has been associated with obesity and hypercholesterolaemia. Emerging evidence suggests that the effects of RLN3 may have sex-specific aspects. Thus, the RLN3 knockout female but not male mice are hypoactive. RLN3 produced stronger orexigenic and obesogenic effects in female rats compared with male rats. In addition, female rats demonstrated higher sensitivity to lower doses of RLN3. Repeated cycles of food restriction and stress were accompanied by an increase in RLN3 expression and hyperphagia in female but not in male rats. Furthermore, stress-induced binge eating in female rats was blocked by an RXFP3 receptor antagonist. RLN3 increased the expression of corticotropin releasing factor in the paraventricular hypothalamic nucleus in male but not in female rats. Conversely, in female rats, RLN3 increased the expression of orexin in the lateral hypothalamus. There is evidence that orexin directly activates the RLN3 neurons in the NI. The positive reinforcement of the RLN3 effects by orexin may intensify behavioural activation and feeding in females. Sex-specific effects of RLN3 may also depend on differential expression of RXFP3 receptors in the brain. Given the higher sensitivity of females to the orexigenic effects of RLN3 and the stress-induced activation of RLN3, the overall data suggest a possible role for RLN3 in eating disorders that show a higher propensity in women. Linked articles This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Binge eating is frequently stimulated by stress. The neuropeptide relaxin-3 (RLN3) and its native receptor RXFP3 are implicated in stress and appetitive behaviors. We investigated the dynamics of the central RLN3/RXFP3 system in a newly established model of stress-induced binge eating. Female Sprague-Dawley rats were subjected to unpredictable intermittent 1-h access to 10% sucrose. When sucrose intake stabilized, rats were assessed for consistency of higher or lower sucrose intake in response to three unpredictable episodes of foot-shock stress; and assigned as binge-like eating prone (BEP) or binge-like eating resistant (BER). BEP rats displayed elevated consumption of sucrose under non-stressful conditions (30% > BER) and an additional marked increase in sucrose intake (60% > BER) in response to stress. Conversely, sucrose intake in BER rats was unaltered by stress. Chow intake was similar in both phenotypes on 'non-stress' days, but was significantly reduced by stress in BER, but not BEP, rats. After stress, BEP, but not BER, rats displayed a significant increase in RLN3 mRNA levels in the nucleus incertus. In addition, in response to stress, BEP, but not BER, rats had increased RXFP3 mRNA levels in the paraventricular and supraoptic nuclei of the hypothalamus. Intracerebroventricular administration of a selective RXFP3 antagonist, R3(B1-22)R, blocked the stress-induced increase in sucrose intake in BEP rats and had no effect on sucrose intake in BER rats. These results provide important evidence for a role of the central RLN3/RXFP3 system in the regulation of stress-induced binge eating in rats, and have therapeutic implications for eating disorders.