Impact of Canola Protein Processing on Plasma Amino Acid Responses

To assess whether volunteers are eligible to participate in this study, the investigator will invite them to the University for a screening. At the start of the screening session, the entire experimental trial will be explained, and any potential questions will be answered. Thereafter, the volunteers are asked to read, fill out, and sign the informed consent form. After signing the informed consent form, the participant's eligibility will be assessed based on the in- and exclusion criteria. To further asses their eligibility, participants will be asked to fill out a medical questionnaire to assess their general health, use of medication, habitual food intake, and physical activity. Next, blood pressure will be assessed. Body mass (with accuracy of 0.1 kg) and height (with accuracy of 0.01 m) will be determined. The screening sessions will take place in the morning and participants are instructed to not have any breakfast in the morning in order to avoid perturbations in body weight and possibly in the DEXA scan. In the event of an unexpected medical finding during the screening, subjects will always be notified. If a subject does not want to receive this notification, she cannot participate in the study. When the screening is successfully performed, the test days will be scheduled. Protein and amino acid metabolism in women can be influenced by the menstrual cycle. Therefore, the experimental test days will be standardized in a specific period of the menstrual cycle. The test days will be planned in the first 10 days after the start of the menses (day 1 of the follicular phase), when hormones levels (i.e., estrogen) are low. In practice, this means that the test days will be planned based on the subjects' menstrual cycle. Therefore, 2 test days can be performed within 1 menstrual cycle (allowing 2 days between test days for standardization) and further test days will be planned approximately 28 days thereafter. This allows the investigators to finish testing for a single participant within a relatively short time frame while still taking the menstrual cycle and accompanying hormonal influences into consideration. A similar approach was used in our lab in previous studies that included young healthy women (METC17-3-047 \& METC19-087). Throughout the study period, participants will also undergo a fasted Dual Energy X-ray Absorptiometry (DEXA) scan to assess body composition. A DEXA scan is a simple, non-invasive procedure. At the beginning of the procedure, subjects will be asked to lie down on a scanning table wearing underwear and they need to remain motionless during the measurements. As the scanner moves, a dual energy beam passes through the targeted skeletal muscle section and is measured by a detector. This procedure takes approximately 3 minutes. The DEXA-scan provides valuable insight in participants' body composition, with minimal radiation burden.

When the screening is successfully performed, the test day will be scheduled. In order to prevent major impact from the menstrual cycle on the measurements of protein metabolism, the test day will be scheduled to take place in the first 10 days of the follicular phase.

Each subject will participate in 4 experimental test days lasting ~5h. Subjects will be instructed to arrive at the university at 8:45 AM in an overnight fasted and rested state, meaning that participants are not allowed to eat and drink (except for water) from 22:00 the night prior to the experimental trial. They will be instructed to come to the university by car or public transportation. After the subjects arrive at the University, subjects will rest in a seated position and a Teflon catheter will be inserted in a heated dorsal hand vein and placed in a hot-box (60°C) for arterialized blood sampling. A baseline blood sample (Serum + plasma = 20 mL) will be collected at t=0 min to determine the amino acid concentrations prior to the intervention.

Then, subjects will ingest the assigned drink (20g native canola protein, 20g processed canola protein #1, 20g processed canola protein #2, or 20g whey protein). Subjects will be instructed to consume the drink within ~5 min. Upon drink ingestion, the stopwatch will be started (t= 0 min), and arterialized blood samples (10mL) will be collected at t = 15, 30, 45, 60, 90, 120, 180, 240, and 300 min. During the entire experimental test day, the hand with the dorsal hand catheter will be pre-heated for 10 minutes prior to every blood draw in a hot box at 60°C to increase blood flow. This makes it easier to collect blood and minimizes nutrient exchange from blood into other tissues. Therefore, it allows the investigators to collect nutrient-rich (arterialized) blood from a vein.

In total 10 times blood samples will be taken during the test day, the first sample that will be collected will be 20mL and subsequent samples will be 10mL, making a total of 110mL.

Additionally, gastro-intestinal (GI) symptoms will be assessed at t = 0, 30, and 180 minutes and palatability at t = 15 min by questionnaires.