Development of Oral Amino Acid Tracers to Study Protein Turnover in Humans

Study Overview
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Brief Summary
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Previous studies have used a combination of oral L-[1-13C]leucine and intravenous labeled L-[5,5,5-2H3]leucine to assess the acute postprandial changes in whole-body protein turnover (12, 19). Intravenous and dietary-labeled amino acid tracers have also been used in tandem to assess rates of myofibrillar protein synthesis in response to bolus protein ingestion and resistance exercise (46). By validating whole-body net balance to myofibrillar protein synthesis, our proposed multi-tracer approach will develop minimally invasive models to study protein turnover in a variety of populations in which traditional infusions and/or repeated blood samples are not possible (i.e. pediatric, free-living populations).

Detailed Description
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The primary objective of the proposed study is to validate the use of a novel oral tracer model to accurately and reliably measure myofibrillar protein synthesis. It is hypothesized that oral L-[1-13C]leucine and L-[ring-2H5]phenylalanine, ingested as a bolus to mimic an intravenous 'pulse dose' administration (51, 65), will reveal similar rates of myofibrillar protein synthesis when compared to traditional intravenous L-[5,5,5-2H3]leucine infusion. Moreover, it is hypothesized that both methods will reveal the expected graded changes in myofibrillar protein synthesis in response to feeding and resistance exercise (i.e. fasted<feeding<exercise & feeding). The secondary objective of the proposed study is to develop and validate non-invasive models to measure whole-body amino acid oxidation and net balance in response to feeding and resistance exercise. It is hypothesized that whole-body net balance, as determined by a novel oral tracer model (i.e. L-[1-13C]leucine) , will align with traditional intravenous tracer methodology (i.e. L-[5,5,5-2H3]leucine) and reveal the expected physiological changes in whole-body protein turnover in response to feeding and resistance exercise (i.e. fasted<feeding<exercise & feeding).

Official Title
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Development of Oral Amino Acid Tracers to Study Protein Turnover in Humans

Conditions
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Healthy Adult Males

Intervention / Treatment
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* Dietary Supplement: Crystalline amino acids to assess myofibrillar protein synthesis rates
* Dietary Supplement: Crystalline amino acids to assess whole-body protein turnover, amino acid oxidation, and net protein balance


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male Currently performing structured physical activity 2-5 days per week Exclusion Criteria: Unable to safely perform exercise as per PARQ+ guidelines Currently using tobacco products Currently using or have history of anabolic steroid use Diagnosed with medical condition including type 2 diabetes, cancer, heart disease Unable to abstain from supplement use (HMB, branched chain amino acids, phosphatidic acid) for at least three weeks prior to trial currently using medications known to affect protein metabolism e.g. corticosteroids, NSAID, prescription-strength acne medication allergic to local anesthetics female: Hormonal fluctuations associated with the menstrual cycle have been reported to alter protein metabolism during exercise and may influence indices of the post-exercise myofibrillar protein synthetic response. Accordingly, the study will include males to ensure a stable hormonal environment and to increase the homogeneity of the physiological response.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 35 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Three groups: Fast, Fed, Ex-Fed Two trials per group
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Healthy, recreationally-active adult males - Muscle Trial<br>Subjects receive 0.25g/kg crystalline amino acids modelled after egg protein, enriched with L-[1-13C]leucine and L-ring-[2H5]phenylalanine with a primed-constant infusion of L-[555-2H3]leucine to assess myofibrillar protein synthesis rates | Dietary Supplement: Crystalline amino acids to assess myofibrillar protein synthesis rates<br>* Amino acid dose = 0.25g/kg bodyweight<br>|
| Experimental: Healthy, recreationally-active adult males - Whole Body Trial<br>Subjects receive 0.25g/kg crystalline amino acids modelled after egg protein, enriched with L-[1-13C]leucine with a primed-constant infusion of L-[555-2H3]leucine to whole-body protein turnover, amino acid oxidation, and net protein balance | Dietary Supplement: Crystalline amino acids to assess whole-body protein turnover, amino acid oxidation, and net protein balance<br>* Amino acid dose = 0.25g/kg bodyweight<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Myofibrillar protein synthesis rates | Myofibrillar protein synthesis rates assessed by oral and intravenous tracers during Fast, Fed, and Ex-Fed | 5 hours | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Whole-body protein turnover | Whole-body protein turnover assessed by oral and intravenous tracers during Fast, Fed, and Ex-Fed | 5 hours | 
| Amino acid oxidation and net protein balance | Amino acid oxidation and net protein balance assessed by oral tracers during Fast, Fed, and Ex-Fed. Net protein balance is derived from the difference between amino acid intake (known) and total amino acid oxidation over the 5h measurement period. | 5 hours | 
| Muscle anabolic signalling | Immunoblotting for mTORC1/key downstream targets of mTORC1 | 2 and 5 hours | 
| Amino acid transporter expression | Immunoblotting for amino acid transporters LAT1/SNAT2 | 2 and 5 hours |