The Incorporation of Dietary Protein-Derived Amino Acids in Duodenal Epithelium

Study Overview
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Brief Summary
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Rationale: Aging is accompanied by a blunted muscle protein synthetic response to protein ingestion.This anabolic resistance may be related to decreased postprandial amino acid release in the circulation, due to greater amino acid extraction by splanchnic tissues in older individuals. It has been suggested that extracted amino acids are utilized by intestinal epithelial cells for cell proliferation, generating new cells to maintain healthy mucosa. So far, there is no evidence that dietary protein-derived amino acids are taken up and incorporated in intestinal mucosal protein in vivo in humans. Furthermore, there is no evidence that this process is impacted by age. Objective: To assess the postprandial incorporation of dietary protein-derived amino acids in intestinal mucosal protein in healthy young and older males. Study design: Cross-sectional, non-therapeutic intervention study design Study population: 5 healthy, non-obese (BMI 18.5-30kg/m2) young adult males (age: 18-35y inclusive) and 5 community dwelling older males (age: 67+y). Intervention: Continuous intravenous stable isotope amino acid tracer infusion will be applied, in combination with oral ingestion of 20g intrinsically labelled milk protein, with plasma, muscle and duodenal mucosa biopsy samples collected at different time points throughout the experimental test day. Main study parameters/endpoints: The primary study outcome is the postprandial (0-5h) incorporation of dietary protein-derived amino acids in duodenal mucosal protein following the ingestion of 20g intrinsically labelled milk protein. Secondary study parameters include postprandial plasma availability of dietary protein-derived amino acids and fractional duodenal mucosal protein synthetic rate.

Detailed Description
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Skeletal muscle tissue is in a constant state of remodelling, regulated by the balance between tissue protein synthesis and breakdown rates, with a turnover rate of 1-2% per day. It has been well established that protein ingestion is a major anabolic stimulus for muscle protein synthesis. Dietary protein ingestion provides amino acids that stimulate protein synthesis by both functioning as substrate and as signalling molecules that upregulate anabolic pathways. The anabolic properties of dietary protein largely depend on the protein digestion and amino acid absorption kinetics and subsequent increase in plasma amino acid availability. Following protein ingestion, protein is cleaved into small peptides and amino acids by digestive enzymes. Subsequently, these amino acids are absorbed across the intestinal mucosa by various membrane-bound transporters. The majority of dietary protein derived amino acids is released into the systemic circulation and transported and taken up by various peripheral tissues in the postprandial phase. However, a substantial part of the ingested protein does not become available in the circulation. This part is either not (yet) digested and absorbed, or the absorbed amino acids are taken up by splanchnic tissues, such as the gut and liver, providing precursors for de novo tissue protein synthesis, termed first-pass splanchnic extraction. Previous work in our laboratory has estimated that up to 40% of the protein derived amino acids are taken up and incorporated in the intestinal tract and liver tissues. The intestine is the most highly regenerative organ in the human body, regenerating its epithelium every 3 to 5 days. It has been suggested that the absorbed amino acids are utilized by epithelial cells for rapid cell proliferation, generating new cells to maintain healthy mucosa. Human studies on the incorporation of dietary protein-derived amino acids in intestinal mucosal protein have not been performed. Therefore, the aim of the present explorative in vivo study is to assess, for the first time, the incorporation of dietary protein-derived amino acids in duodenal mucosal protein in humans, and the systemic availability of dietary protein-derived amino acids. Aging is accompanied by a blunted muscle protein synthetic response to protein ingestion. This proposed anabolic resistance may be related to a decreased postprandial amino acid release in the circulation, due to impairments in protein digestion and amino acid absorption, and greater first-pass splanchnic amino acid extraction in older individuals. Whether dietary protein-derived amino acid incorporation in intestinal mucosal protein is increased with aging remains to be established. Therefore, the current study will use the data on incorporation of dietary protein-derived amino acids in duodenal mucosal protein and postprandial amino acid plasma availability obtained in young and older individuals to evaluate potential age-related differences. Two primary hypotheses will be tested: It is hypothesized that part of the absorbed amino acids will be directly incorporated in duodenal epithelium within 5 hours after protein ingestion. It is hypothesized that the extent of postprandial incorporation of dietary protein derived amino acids in duodenal epithelium will be greater in older compared to young males.

Official Title
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The Incorporation of Dietary Protein-Derived Amino Acids in Duodenal Mucosal Protein in Young and Older Males

Conditions
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Protein Malabsorption

Intervention / Treatment
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* Dietary Supplement: Intrinsically labelled milk protein


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male sex Aged 18-35 years or 67+ years Body mass index (BMI) between 18.5 and 30 kg/m2 Exclusion Criteria: History of cardiovascular, respiratory, gastrointestinal, urogenital, neurological, psychiatric, dermatologic, musculoskeletal, metabolic, endocrine, haematological, immunologic disorders, allergy, major surgery and/or laboratory assessments which might limit participation in or completion of the study protocol, interfere with the execution of the experiment, or potential influence the study outcomes (to be decided by the principal investigator and responsible physician) Major abdominal surgery interfering with gastrointestinal function (upon judgement of the principal investigator and responsible physician) Use of medication which limit participation in or completion of the study protocol, interferes with the execution of the experiment, or potential influences the study outcomes (to be decided by the principal investigator and responsible physician) Use of supplementation (i.e. vitamin, pre- and probiotic supplementation) within 14 days prior to testing Administration of investigational drugs or participation in any scientific intervention study in the 14 days prior to the study, which may interfere with this study (upon judgement of the principal investigator and responsible physician) Specific diet (e.g. vegetarian, vegan, gluten free, no diary) within the study period Planning to lose weight during the study period Lactose intolerance Excessive alcohol consumption (defined as > 14 alcoholic consumptions per week) Smoking Drug use Donated blood two months prior to the test day Recent (<1 year) participation in amino acid tracer (L-[ring-2H5]-phenylalanine, L-[ring-2H3]-leucine, L-[ring-2H4]-lysine, L-[ring-2H2]-tyrosine) or intrinsically labelled protein ([1-13C]-phenylalanine, [1-13C]-leucine, [1-13C]-lysine) studies No given permission to register participation in electronic patient file at MUMC+ and to add records of gastroduodenoscopy

Ages Eligible for Study
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Minimum Age: 18 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: Basic Science
Intervention Model: Single Group Assignment
Interventional Model Description: Young vs. old
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Intrinsically labelled milk protein<br>20 grams of protein dissolved in 500 mL of water | Dietary Supplement: Intrinsically labelled milk protein<br>* 20 grams of protein dissolved in 500 mL of water<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Postprandial incorporation of dietary protein-derived amino acids in duodenal mucosal protein | Assessed by duodenal mucosal protein-bound [1-13C]-phenylalanine enrichment (expressed as MPE) | 0-5 hours | 
| Impact of age on postprandial incorporation of dietary protein-derived amino acids in duodenal mucosal protein | Assessed by duodenal mucosal protein-bound [1-13C]-phenylalanine enrichment (expressed as MPE) in young and older adults | 0-5 hours | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Postprandial plasma amino acid concentrations | Appearance of milk protein-derived amino acids in plasma over the full assessment period (5 h), as determined using stable isotope tracer methodology | 0-5 hours | 
| Fractional duodenal mucosal protein synthetic rate | Mucosal protein synthesis rates are calculated using L-ring-2H5-phenylalanine tracer and provided as 1 integrated value over the specified timeframe using plasma as precursor | 0-5 hours | 
| Fractional muscle protein synthetic rate | Muscle protein synthesis rates are calculated using L-ring-2H5-phenylalanine tracer and provided as 1 integrated value over the specified timeframe using plasma as precursor | 0-5 hours | 
| Fecal [1-13C]-phenylalanine enrichments (expressed as MPE) | Fecal protein excretion, assessed by fecal [1-13C]-phenylalanine enrichments (expressed as MPE) | Fecal sample of first feces after endoscopy | 
| Fecal nitrogen content | Fecal protein excretion, assessed by fecal nitrogen content | Fecal sample of first feces after endoscopy | 
| Fecal microbial metabolites | Microbial metabolites, including short-chain fatty acids, assessed by analyzing fecal samples | Fecal sample of first feces after endoscopy | 
| Plasma glucose concentrations | Plasma glucose concentrations | 0-5 hours | 
| Plasma insulin concentrations | Plasma insulin concentrations | 0-5 hours | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Protein, Gastrointestinal tract, Intestinal mucosa, Digestion, Absorption, Aging, Muscle protein synthesis