Bioavailability of Protein and Amino Acids From Oilseeds in Healthy Volunteers

Study Overview
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Brief Summary
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The study aims to determine in healthy subjects the bioavailability of protein and amino acids of 4 protein sources: sunflower, rapeseed, lupin, flax. For this purpose, the investigators will compare two methods: the standard method consisting in measuring the ileal digestibility using ileal tubes an alternative method that has been proposed by an Food and Agriculture Organization (FAO) expert group: the dual isotope method

Detailed Description
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4 groups of healthy volunteers are recruited (males and females from 18 to 65 y old), BMI from 18 to 30. Each group tests a protein source (20g) incorporated in a biscuit that is divided in small portions to perform a repeated meal protocol. Protein sources (sunflower, rapeseed, flaxseed or lupin) are intrinsically labeled with 15N and deuterium (2H). The day before the meal test, the volunteers are equipped with an intestinal tube that migrates until the ileum. On the day of the experiment, the position of the intestinal tube is checked by radiography in order to verify its location at the terminal ileum. A catheter is inserted in the forearm vein for blood sampling. A saline solution containing polyethylene glycol 4000 (PEG-4000, 20 g/L), used as a non-absorbable marker of the intestinal flow, is infused into the ileum in order to calculate the flow rate of the intestinal effluents. After a basal blood and urine sampling, as well as a basal collection of ileal effluents performed for 30 min, subject ingests at t=0 the first biscuit dose. The small biscuit portions are ingested every 30 min for 4h, to achieve an isotopic plateau. Tracer doses of carbon 13 (13C) amino acids are ingested concomitantly with the biscuit doses. Non absorbable markers are added to the test meal to correct for incomplete recovery at the ileal level. The postprandial sampling period lasts for 8 h after the meal ingestion. The intestinal content is continuously collected over ice and pooled every 30 min. Blood is sampled every 30 min for 4 h, and hourly thereafter.Total urine is collected every 2 h. Measurements: In the effluents, the investigators measure PEG 4000, nitrogen (N) and 15N, carbon and 13C, amino acids (AA), 15N and 13C in amino acids and the non-absorbable markers of the meal. The investigators calculate the ileal flow rate, the overall real ileal protein digestibility and the real ileal digestibility of individual amino acids. In the plasma, the investigators measure uremia and 15N in urea, AA and protein to measure the transfer of dietary N in plasma protein pools; 15N, 2H and 13C in individual amino acids too measure the relative bioavailability of 15N/2H amino acids compared to free 13C amino acids. In the urine, the investigators measure urea and 15N in the urea ammonia to calculate the postprandial deamination losses. These measurements will allow to determine the protein and amino acid bioavailability from 4 different plant protein sources, using the classical method implying ileal sampling and intrinsic tracer of dietary protein (15N). Additionally, it will allow to test a less invasive procedure using multiple tracers to assess the amino acid bioavailability.

Official Title
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Bioavailability of Protein and Amino Acids From Oilseeds in Healthy Volunteers

Conditions
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Protein

Intervention / Treatment
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* Dietary Supplement: Sunflower
* Dietary Supplement: Rapeseed
* Dietary Supplement: Flaxseed
* Dietary Supplement: Lupin


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: 18<BMI<30 Healthy Insured under the French social security system for women: use of birth control signed informed consent Exclusion Criteria: Any dietary allergy Latex allergy Positive serology to HIV, hepatite C virus antibody, hepatite B virus surface antigen and core antibodies Gluten intolerance Anemia Abusive use of drugs or alcohol Hypertension, diabetes, digestive disease, hepatic or renal disease, severe cardiac disease Pregnancy High sport practicing (>7h/wk) Blood donation in the 3 months prior to the study Participation in a clinical study in the 3 months prior to the study No signed informed consent

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Sunflower<br>Biscuits containing sunflower isolate intrinsically labelled with 15N and 2H | Dietary Supplement: Sunflower<br>* Plant proteins are intrinsically enriched with 15N and 2H. There are incorporated into biscuits. The biscuits are formulated to supply 20 g of plant protein per volunteer.<br>|
| Experimental: Rapeseed<br>Biscuits containing rapeseed isolate intrinsically labelled with 15N and 2H | Dietary Supplement: Rapeseed<br>* Plant proteins are intrinsically enriched with 15N and 2H. There are incorporated into biscuits. The biscuits are formulated to supply 20 g of plant protein per volunteer.<br>|
| Experimental: Flaxseed<br>Biscuits containing flaxseed isolate intrinsically labelled with 15N and 2H | Dietary Supplement: Flaxseed<br>* Plant proteins are intrinsically enriched with 15N and 2H. There are incorporated into biscuits. The biscuits are formulated to supply 20 g of plant protein per volunteer.<br>|
| Experimental: Lupin<br>Biscuits containing lupin flour intrinsically labelled with 15N and 2H | Dietary Supplement: Lupin<br>* Plant proteins are intrinsically enriched with 15N and 2H. There are incorporated into biscuits. The biscuits are formulated to supply 20 g of plant protein per volunteer.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Percentage of ileal digestibility of plant proteins | Total N and 15N will be measured by Elemental Analyzer coupled to an Isotopic Ratio Mass Spectrometer (EA-IRMS) and the percentage of dietary N reaching the terminal ileum will be then determined. This percentage will be corrected by the ileal flow rate which is estimated with measurement of PEG concentration in effluents by a turbidimetric methods. Ileal digestibility of plant proteins (i.e. N digestibility) will be calculated as 100 - corrected dietary N percentage. | time -30 minutes to 8 hours after the meal | 
| Percentage of ileal digestibility of amino acids from plant proteins | Amino acid concentrations will be measured by Ultra high performance liquid chromatography (UHPLC) and 15N, 2H and 13C enrichment in individual amino acids will be measured by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) in order to determine the percentage of dietary individual amino acids reaching the terminal ileum. This percentage will be corrected by the ileal flow rate which is estimated with measurement of PEG concentration in effluents by a turbidimetric methods. Ileal digestibility of plant amino acid will be calculated as 100 - corrected dietary amino acid percentage. | time -30 minutes to 8 hours after the meal | 
| Percentage of net postprandial protein utilization of plant proteins | Urea in plasma and urine (by enzymatic methods) will be determined and associated with 15N enrichment values (by EA IRMS) to determine the 15N transfer in plasma and urinary N pools and calculate the net postprandial protein utilization. | time -30 minutes to 8 hours after the meal | 
| Postprandial plasma kinetics of dietary amino acids | Amino acids in plasma will be determined by UHPLC and associated with 15N enrichment values in individual amino acids (by GC C IRMS) to determined the kinetics of amino acids from plant proteins. | time -30 minutes to 8 hours after the meal | 
| Postprandial metabolism of glucose 1 | Glycemia (by enzymatic methods) will be determined in plasma. | time -30 minutes to 8 hours after the meal | 
| Postprandial metabolism of glucose 2 | Insulinemia (by ELISA) will be determined in plasma. | time -30 minutes to 8 hours after the meal | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Validation of the dual isotope method to assess the bioavailability of individual amino acids | Measurements of 15N, 2H and 13C amino acids in the plasma by GC-C-IRMS | time -30 minutes to 8 hours after the meal |