The test meal will consist of 60 g of lyophilized whole egg powder which contains 30 g of
2H-egg protein and 800 mg [U-13C]-spirulina or 400 mg of 13C free AA mixture, mixed with
water and cooked as an omelet. The 2H-egg powder has been produced by our collaborators of
the St John's Research Institute in Bangalore (India). Celite (acid insoluble ash, Advanced
Minerals Corporation) will be added to the meal as an indigestible marker to check the
recovery of the meal in effluents. The test-meal will be given as a bolus or in a plateau
feeding (repeated mini-meals) depending of the groups of subjects. 600 g of 2H-labeled
lyophilized eggs will be obtained from layer hens administered a uniformly 2H-labeled AA mix
orally for 60 d with their daily feed. Microbiological analyses will be performed to ensure
their safety for human consumption.
18 volunteers will be included at the end of the study and tested at the Human Nutrition
Research Centre of Avicenne Hospital (France). The main inclusion criteria are men and women,
18 to 45 year-old, body mass index (BMI) 18 to 30 kg/m2. The main exclusion criteria are any
digestive or hepatic pathology, allergy against eggs, allergy against latex, pregnant women.
Due to the invasive procedure of intubation, the investigators plan to recruit 30 volunteers
to accommodate for the usual 40% dropouts.
The subjects will be divided in 3 groups of n = 6:
Bolus-spi, n = 6, the volunteers will consume the test-meal as a bolus in one time at t
= 0, with 13C-spirulina as the reference protein
Plateau-spi, n = 6, the volunteers will consume the test-meal in a plateau feeding
protocol, with 13C-spirulina as the reference protein
Plateau-AA, n = 6, the volunteers will consume the test-meal in a plateau feeding
protocol, with 13C-free AA mixture as the reference "protein"
One week before the experiment, the volunteers will follow a standard diet adapted to their
body weight to control their protein intake (1.3 g protein/kg body weight).
The volunteers will arrive at the Human Nutrition Research Centre of Avicenne Hospital on the
morning before the day of the experiment. They will be equipped with a triple lumen
intestinal tube that will be allowed to progress through the intestinal tract for 24 h. One
of the lumen is radio opaque and serves to perfuse a non-absorbable maker in the intestine
(slow marker method). The second lumen is dedicated to inflate a weighted balloon that
facilitate the migration of the tube. The third lumen is dedicated to the continuous
aspiration of the effluents, 15 cm below the perfusion site. The measurement of the
non-absorbable marker in the effluents allows the determination of the effluent flow rate.
On the day of the experiment, the position of the tube will be checked by radiography to
verify its location at the terminal ileum. A catheter will be inserted in the forearm vein
for blood sampling. The perfusion of the non-absorbable marker, polyethylene glycol (PEG)
4000 (20 g/l), will start at a flow rate of 1 ml/min. The intestinal flow and the basal ileal
sample will be collected during 30 min. Basal plasma and urine sample will be collected.
At t = 0, the volunteers of the group "Bolus-spi" will consume all the test-meal in less the
20 min. For the volunteers of the groups "Plateau-spi" and "Plateau-AA", the test-meal will
be divided into 22 equal portions and they will consume the first meal as a priming dose of 6
mini-portions. Then, they will consume single mini-portions every 30 min for 7.5 h. One
portion will be dedicated to meal analyses. Until t = 8, intestinal content will be
continuously collected by aspiration and pooled every 30 min. Blood will be sampled hourly
during 4 h and every 30 min from t = 5 to t = 8. Urine will be collected every 2 h until t=8
(total volume of urine output will be noted at each time point for proportional pooling).
Breath samples will be collected every 30 min. Carbon dioxide output (VCO2) will be evaluated
hourly during 10 min by indirect calorimetry (Canopy). Urine sample will be collected at the
start of the experiment and during the study protocol. Fecal samples will be collected before
the start of the experiment and within the next 24 h. The naso-ileal tube will be removed
after the last collection of effluent at t = 8.The naso-ileal tube will be removed after the
last collection of effluent at t=8.
The following analyses will be performed:
Concentration of PEG-4000 by turbidimetric method in digesta samples
Total hydrogen content and 2H enrichment in meals and digesta samples by isotope-ratio
mass spectrometry (IRMS) coupled to pyrolysis analyzer
Total carbon content and 13C enrichment in meals and digesta samples by Elemental
analyzer (EA) coupled to IRMS
AA concentration in meals and digesta samples by Ultra High Performance Liquid
Chromatography (UHPLC)
13C-AA and 2H-AA enrichment plasma and meal samples by Liquid Chromatography coupled to
tandem Mass Spectrometry (LC-MS/MS)
13C-AA and 2H-AA enrichment in digesta and meal samples by gas
chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS)
Celite content in pooled (0-8 h) digesta samples
13CO2 in breath sample by Multiflow-IRMS
Amino-acidome and peptidome of digesta, plasma, urine, and fecal samples
Part of the analyses (13C-AA and 2H-AA enrichment plasma and meal by LC-MS/MS, -omic
analyses) will be done by our collaborators of the St John's Research Institute in Bangalore
(India).