Supplementation of a Leucine-Enriched Dairy Protein Blend

Experimental approach:

8 participants will be included in this study. Each participant will consume the supplement only once in a randomized fashion during three separate visits and there will be approximately 1 week between each visit.

Standardized meal:

Participants will be provided a standardized diet one day prior to experimentation. When performing research involving human metabolism it is important that the participants be tested in a weight-stable state. Therefore, the investigators will estimate the participants resting energy expenditure using a widely reference prediction formula, known as the Harris-Benedict equation. This formula takes into account gender, body mass, height, age, and self-reported activity level. Once resting energy expenditure is calculated the investigators will provide each participant with a pre-packaged standardized diet for the day prior to the experiment that is designed to ensure a sufficient energy balance.

Dual Energy X-Ray Absorptiometry (DXA) Scans:

DXA scans will be used to determine the participants body composition. The DXA procedures use a small amount of radiation to determine how much fat, bone, and lean mass the participants have in their body. The procedure takes approximately seven minutes and involves the participants lying still on an open bed while the sensor passes over the participants body.

Arterialized blood sampling:

All blood sampling will be achieved through an intravenous indwelling cannula. This method provides the least trauma to the participant when repeated blood sampling is required and is convenient for the investigators. In theory, repeated needle sticks may stress the participant thereby increasing sympathetic nervous system activity, thus causing changes in metabolism. Additionally, another potential drawback from venous blood sampling is the influence by regional tissue specific metabolism, thereby making it difficult to interpret whole-body metabolism on the basis of metabolites measured in venous blood. Therefore, arterial blood sampling is deemed the ideal method for metabolic studies. Arterial cannulation, however, may be unethical for research purposes due to increased risk of complications, such as damage to the arterial wall, thrombosis, and clot formation, all of which may cause death. Arterializing the blood being sampled can be achieved through heating either the superficial antecubital vein or dorsal hand vein with a heating blanket or specialized heat box. By doing so, blood flow increases as a result of vasodilatation of vessels in the arms skeletal musculature, as well as arteriovenous anastomoses in skin. Due to the increased blood flow and negligible muscle mass at the hand and elbow, results in venous blood being similar in composition to an arterial sample. This method is a widely used surrogate for safe direct arterial blood sampling. Once arterialized blood samples are obtained they will be preserved and used in analyses. The investigators will utilize heating blankets (Theratherm Large Digital Moist Heat Pad [14" x 27"]) to arterialize the blood samples and an infrared thermometer (Nubee, NUB8380) to determine the skin temperature (45-68∘C) prior to drawing the participant's blood.

Per-protocol conditions will include:

Consumption of a standardized meal the evening prior to each trial and consistent exercise/activity 2d before each trial There will be ~1wk between trials (washout periods) Blood samples will be analyzed for plasma amino acids, glucose, and insulin.

Experimental Interventions (randomized order):

• Participants will consume a Low Protein Containing Breakfast (10% of the Acceptable Macronutrient Distribution Range according to the Food and Nutrition Board of the Institute of Medicine) and 2 hours later the participants will consume 17g of a leucine enriched whey protein- hydrolyzed whey protein-micellar casein blend (50:43:7 whey:hydrolyzed-whey:casein) containing 600 IU Vitamin D and 3.5g leucine
• Participants will consume a High Protein Containing Breakfast (25% of the Acceptable Macronutrient Distribution Range according to the Food and Nutrition Board of the Institute of Medicine)
• Participants will consume a Low Protein Containing Breakfast (10% of the Acceptable Macronutrient Distribution Range according to the Food and Nutrition Board of the Institute of Medicine)

Proteins were provided by Covance Laboratories, Inc. owned by Eurofins.

The whey protein and micellar casein are milk proteins derived from cow's milk. The participants will consume the 17g of protein contained in a semi-solid bar equal to ∼40g, which is the size of a typical sports recovery bar.

Sample size and data analyses:

The sample size was determined by a statistical power analysis (G*Power 3.1 software, version 3.1.9, 2014) that revealed a large effect size. This study was powered on the basis of previous studies. Thus, a total of 8 (n = 8/group) participants will be used in analyses. See Below:

Analysis: A priori: Compute required sample size ANOVA: Repeated measures, within-between interaction Input Effect size f = 0.5 α err prob = 0.05 Power (1-β err prob) = 0.95 Number of groups = 3 Number of measurements = 14 Corr. Among rep. measures = 0.5 Nonsphericity correction ε = 1 Output Noncentrality parameter ƛ = 63 Critical F = 1.6380186 Numerator df = 26 Denominator df = 78 Total sample = 9 Actual power = 0.997

Inclusion criteria

• 8 participants will be included in this study. Each participant will consume the supplement only once in a randomized fashion during one of three separate visits and there will be approximately 1 week between each visit. In order to participate in this study, each participant must be male or female, 65 years or older (inclusive) and cannot be a smoker or user of tobacco products.

Exclusion criteria

• The exclusion requirements for this study include the following conditions:

• Take any analgesic or anti-inflammatory drugs(s), prescription or non-prescription, chronically will be excluded. However, a washout period of 4 weeks will be suitable for participation.
• A history of neuromuscular problems or muscle and/or bone wasting diseases
• Any acute or chronic illness, cardiac, pulmonary, liver, or kidney abnormalities, uncontrolled hypertension, insulin- or non-insulin dependent diabetes or other metabolic disorders-all ascertained through medical history screening questionnaires
• Use medications known to affect protein metabolism (i.e. corticosteroids, non-steroidal anti-inflammatories, or prescription strength acne medications)