Effect of Creatine+HMB and Creatine+GAA Supplementation in Presarcopenic and Sarcopenic Individuals

Study Overview
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Brief Summary
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Sarcopenia (SAR) is the loss of strength and muscle mass caused by aging. It is accompanied by a progressive loss of physical and cognitive abilities, increasing the risk of falls. This loss of muscle mass leads to pathophysiological changes at the neuromuscular and tendon level as a consequence of, among others, alterations in the protein synthesis/degradation balance, inflammation (INF), or alterations in the anabolic/catabolic state (EAC). These alterations are caused by oxidative stress (OS), when reactive O2 species, toxic metabolites produced by cells using O2, exceed the defense capacity of the antioxidant mechanism. Therapeutic strategies to modulate SAR are based on exercise and nutrition programs. Multicomponent physical exercise program has shown improvements in sarcopenia-related parameters. Likewise, the use of nutritional supplements such as creatine (CRE) has shown improvements in muscle function in the elderly. CRE could reduce INF and EO in the general population. Guanidinoacetate (GAA, also known as guanidinoacetate acid or glucosamine) is a naturally occurring creatine precursor with advanced transportability and an innovative dietary supplement that may increase the rate of creatine turnover. The CRE-GAA mixture outperforms creatine in increasing brain and muscle performance in adult men and women, but whether this mixture improves muscle function and quality in people with sarcopenia has not been addressed so far. Similarly, whether this mixture may promote oxidative stress and inflammation in adults with sarcopenia has not been studied. Similarly, beta-hydroxy-beta-methylbutyrate (HMB) also appears to improve muscle function in older people by enhancing myogenesis. However, the effects of these supplements on the elderly have only been shown to be seen when used in isolation. In this regard, our research team observed that a 10-week combination of 3 g/day of CRE+3 g/day of HMB (CRE-HMB) improved muscle recovery (better EAC) and physical performance in athletes exposed to heavy muscle wasting. However, not aware that it has been addressed whether this mixture improves muscle function, EO, INF, and EAC in women with SAR. Therefore, the hypothesize would be that the CRE-HMB combination could improve muscle function and physical performance, as well as OE, INF, and EAC in people with high muscle wasting such as those with SAR. Therefore, a randomized double-blind study is proposed to analyze the effect of 12 weeks of co-supplementation of 3 g/day of CRE + 3 g/day of HMB (CRE-HMB) and 3 g/day of CRE and 3 g/day of GAA (CRE-GAA) with 3 sessions/week of multicomponent physical exercise on muscle function, EO, INF, and EAC in 81 women with SAR over 70 years of age. These 81 women will be divided into 3 groups of 27 (27 placebo group, 27 CRE-HMB group, and 27 CRE-GAA group). At the control points (at baseline and after 12 weeks) participants will have their body composition, nutritional intake, strength, and performance tests analyzed. Blood will also be drawn to determine biochemical values of EO, INF, and EAC. The expected results are that co-supplementation with CRE-HMB and CRE-GAA for 12 weeks together with multicomponent physical exercise in individuals diagnosed with SAR will improve muscle strength, muscle quantity, and performance. In addition, improved EO, INF, and EAC levels are expected.

Detailed Description
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Participants: Based on the analysis performed with the G*Power package (version 3.1.9.2), there will be a total of 66 volunteer persons (50% women) diagnosed with sarcopenia between 65 and 80 years of age from the 4 nursing homes supervised by the Diputación de Burgos, Spain, which have a total of 745 places. The total sample is obtained when the effect size among the 3 groups is medium (D = 0.25), assuming the power analysis as a result of the study, at least 66 persons (at least 22 persons per group) with 95% confidence level (with margin of error of 0.05) and a power of 80% is obtained. However, due to the possible loss of participants during the study, it is proposed to recruit 81 sarcopenic individuals between 65 and 80 years of age (42 women). INTERVENTION T0-T1 period: during the T0-T1 period, meetings will be held with potential participants to introduce the procedure. In the first meeting, the project, its objectives and the intervention exercises will be explained, as well as its voluntary nature. They will be invited to participate in the study and, if they accept, to sign the informed consent form. In subsequent sessions, the various tests will be conducted to determine if they meet the inclusion criteria for sarcopenia. In addition, the next meetings will include instructions on the content and development of the training sessions. During this period, data on the sociodemographic characteristics of the participants will be collected using a form that will include: first name, last name, age, diseases, medications taken, dominant side, smoking, alcohol consumption, previous occupation, marital status, education level, income level, number of children, social security, number of falls in the last year, and whether there is a need for security or special care if requested. Once all 81 people have been recruited for the study, randomization will be performed to form the groups. Period T1-T2-T3: all participants will undergo 2 assessments: an initial assessment at enrollment (T1), another 12 weeks later (T2) and a final assessment (T3) at 24 weeks after the start of the study. During the T1-T2 period, all 3 groups will conduct a 12-week multicomponent training intervention using the facilities and means available at the residence. G1 participants will receive supplementation with a CRE-HMB mixture (3 g/day of CRE and 3 g/day of HMB), G2 participants will receive a CRE-GGA mixture (3 g/day of CRE and 3 g/day of GAA), while G3 participants will receive a placebo (6 g/day of inulin). During T2 and T3 (12 weeks after the supplementation intervention), members of all three groups (G1, G2 and G3) will continue to perform the multicomponent training, but will not take any supplements. Multicomponent training: the intervention will be carried out in the senior center itself and will be periodized in 1 Macrocycle with 3 Mesocycles and 12 Microcycles (weeks) of 36 sessions of strength, endurance, balance and flexibility exercises, composed of 3 parts and with different exercises according to the means available in the center's facilities. Regarding the distribution of the sessions, all participants will receive three sessions per week during the whole study (T1 to T3). These sessions will be held on alternate days, preferring the frequency of Monday, Wednesday and Friday. The approximate duration of each session will be 40 to 60 min. All sessions will be directed and supervised at all times by the center's instructor or therapist. During the sessions, participants will work on different cognitive processes, such as coordination, memory and attention. In addition, implicitly, participants are expected to improve strength, aerobic capacities, flexibility and balance during the different activities and exercises of the training. All sessions will have three main parts: (I) Warm-up: it will start with static joint mobility exercises and will continue with dynamic muscle activation exercises with different forms of gait movements (coordination, balance and dynamic flexibility), which are intended to increase in complexity and effort as the training progresses. If possible, the warm-up could be gamified in order to increase motivation, socialization, affectivity and adherence to the training protocol. (II) Main part: it will be a concurrent training of strength and aerobic endurance, giving priority to strength in the order in which the exercises are performed. Strength: Frequency: 3d/s. Monday, Wednesday and Friday. Duration: 20-30 minutes. Method: intervalic. Distribution: upper and lower hemispheres. Intensity: start at 20% 1RM and progressively increase up to 80% 1RM. Volume: start with 1-2 exercises per muscle group, 1-2 sets of 12-15 reps each up to 3-4 g/m exercises, 3-4 sets of 6-8 reps. Density: starts low and increases according to the volume, intensity and training level of each subject. Inter-set and Inter-exercise recovery starts at 2-3 min to decrease to 30 s as far as possible. Execution speed: 2:2, 2:1 and 1:1. Material: body weight. TRX. Elastic bands. Medicine balls. Dumbbells. Dumbbells. Pulleys and machines. Upper body exercises: Pectorals: thrusts and adductions. Examples: pulldowns, bench presses, openings, contractor, pulley crossovers, etc. Dorsal: pull-ups, rowing and pull-ups. Examples: pull-ups, seated rowing, rowing on point, rowing with barbell or dumbbell, pulley halon, pulley halon with straight arms, pullover with dumbbells or barbell, dead weight, etc. Deltoids: vertical thrusts, abductions and flights. Examples: military press, lateral raises, front raises, birds, flights, etc. Biceps, triceps and forearms: flexions, extensions and rotations. Examples: biceps curl, French press, supinate and pronate, etc. Lumbosacral girdle: push-ups, extensions, tilts and twists. Examples: crunch, hollow, sit-up, good morning, Russian twist, prone trunk extensions, lateral bends, etc. Lower body exercises: Quadriceps: push-ups, extensions and thrusts. Examples: squats, lunges, knee extensions, press, etc. Hamstrings: push-ups and thrusts. Examples: squats, lunges, femoral curls, Romanian deadlifts, Nordic curls, etc. Glutes: extensions, lifts and thrusts. Examples: squats, lunges, deadlifts, kicks, hip extensions, lateral raises, etc. Adductors: adductors. Examples: squats, lateral lunges, leg locks on a machine, with elastic bands or pulleys, etc. Calf muscles: extensions. Examples: squats on point, extensions with machine, with dumbbells, barbell or elastic bands, etc. Aerobic endurance: Duration: 10 to 20 min. Method: continuous. Periodization: wavy. Intensity: 50% to 80 % FCmax. Equipment: exercise bike, treadmill, audio equipment. Exercises: walking, dancing, exercise bike, slow jogging if possible. (III) Return to calm: return to a state of rest using active flexibility exercises, static or dynamic, and relaxation exercises. If possible, it is also recommended to use games or activities that stimulate cognitive and affective capacities. During the intervention period, all participants will continue with their conventional treatments and therapies provided in the nursing homes, such as physiotherapy, occupational therapy and gym sessions. Nutrition: All study participants will receive a Mediterranean-style diet prepared by a team of research dietitians and/or researchers. This diet will be sent weekly to the nursing homes to be prepared in the central kitchens. MEASUREMENT AND DATA COLLECTION Data will be collected at three control points (T1, T2 and T3), and will include body composition, nutritional assessment, physical performance assessment and blood analysis. Data collection will be performed by specialized personnel from the research team (an occupational therapist, a physiotherapist, a physician, a strength and conditioning trainer, a psychologist and a dietician-nutritionist), accompanied by the reference personnel of the residences, at points T1, T2 and T3. Body composition. Nutritional evaluation. Assessment of muscle strength and performance: The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. Assessment of fragility. Blood tests: Blood analysis will be performed by the head physician of the nursing homes and who belongs to the research group. Blood samples will be taken from the antecubital vein of all participants at each time point of the study between 8:30 and 10:00 am under basal conditions, followed by an overnight fast and 36 hours without exercise. The blood sample will be kept at room temperature for 10 minutes before being centrifuged at 4 °C and 3,000 rpm for 15 minutes. The serum will then be separated and stored in aliquots at -80 °C until further analysis in the physiology laboratory of the University of Burgos. All analyses should be carried out according to the manufacturer's instructions. Biochemical analysis of blood samples: Inflammation markers, Oxidative stress markers and hormones. Statistical analysis: Data will be reported as mean ± standard deviation of the mean (SD). The percentage change of each variable between baseline and end of treatment will be calculated as Δ (%): ((Post - pre)/pre × 100). Normality of the data will be tested using the Kolmogorov-Smirnov test. Levene's test will also be employed to ensure homogeneity of variance. Once confirmed, two-way ANNOVA will be performed to explore time × treatment group (t × g) interactions between both treatments (placebo and experimental) throughout the study for all parameters studied. As age and amount of fat are factors that influence strength and physical performance variables, these will be included as potential confounders in the different analyses). Also, the 95% confidence interval (95% CI) and statistical power will be calculated. Differences between study periods in each experimental situation individually (placebo and supplemented), will be determined by the reduced Student's t-test or the Wilcoxon Sign Test. Differences in Δ (%) and other tests in each study period will be contrasted between treatment groups using the independent sample Student's test or the Mann-Whitney U test with Treatment Group as a fixed factor. Finally, a linear regression test will be performed to determine the relationship between changes in inflammation and oxidative stress parameters and changes in strength and physical performance. For this purpose, strength and physical performance parameters will be the dependent variables and changes in inflammation and oxidative stress parameters will be the independent variables. Statistical analysis will be completed using SPSS version 25 software (IBM-Inc., Chicago, IL, USA). In addition, if graphs are necessary, they will be performed using Graphpad Prism 6 software (Graphpad Software, Inc., San Diego, CA, USA.). Statistical significance will be determined with a p value <0.05.

Official Title
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Effect of 12 Weeks of Creatine+HMB and Creatine+GGA Supplementation on Physical Performance, Oxidative Stress and Inflammation in Presarcopenic and Sarcopenic Individuals Performing a Multicomponent Physical Exercise Program

Conditions
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Aging, Sarcopenia, Nutrition, Healthy

Intervention / Treatment
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* Dietary Supplement: CRE + HMB
* Dietary Supplement: CRE + GAA
* Dietary Supplement: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Participants will be age between 65 and 80 years. Presarcopenics. With sarcopenia, i.e., those with grip strength <16 kg and appendicular skeletal muscle mass (ASM)/height2 <6 kg/m2. Voluntarily agreeing to participate in the study and having signed the informed consent. Not suffering any physical impediment to perform multicomponent physical exercise. Exclusion Criteria: Patients diagnosed with: Dementia. Infection. Vascular or hematologic disease. Heart disease and cerebrovascular disease. Endocrine disorders. Electrolyte imbalance and infection. Tumors. Chemotherapy and radiation therapy. Delirium or depression, those with loss of protective sensation (those with Semmes- weinstein monofilament of 4.56 or greater). Lower or upper extremity amputations at any level.

Ages Eligible for Study
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Minimum Age: 65 Years
Maximum Age: 80 Years

Sexes Eligible for Study
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All

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: Prevention
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Randomized double-blind study
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Intervention Group 1<br>CRE-HMB mixture | Dietary Supplement: CRE + HMB<br>* 3 g/day of CRE and 3 g/day of HMB<br>|
| Experimental: Intervention Group 2<br>CRE-GGA mixture | Dietary Supplement: CRE + GAA<br>* 3 g/day of CRE and 3 g/day of GAA<br>|
| Placebo Comparator: Intervention Group 3<br>placebo | Dietary Supplement: Placebo<br>* 6 g/day of inulin<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Body composition (Day 0 and at 12 weeks of intervention). | The evaluation of body composition will be performed by a dietician-nutritionist of the research group at each control point. Prior to the other tests, participants will be weighed and measured by seaside telescopic staghater MOD 220. A bioelectrical impedance analysis (BIA) of body composition will also be performed to minimize the effect of hydration status on all measurements. Participants will be instructed to refrain from caffeine and alcohol 24 h before, as well as, if performed 12 h before the test according to published guidelines for BIA. To predict the MMEA, a tanita TA-MC980MA- 1 PLUS impedance meter (Tanita Europe B.V., UK) will be used according to the manufacturer's guidelines. The MMEA will be predicted through the Equation of Sergi et al, (80): MMEA (kg) = -3.964 + (0.227 RI) + (0.095 * body mass) + (1.384 * sex) + (0.064 * XC), where RI is the standardized resistance for height and XC is the reactance. | 12 weeks | 
| Body composition (12 weeks after the intervention). | The evaluation of body composition will be performed by a dietician-nutritionist of the research group at each control point. Prior to the other tests, participants will be weighed and measured by seaside telescopic staghater MOD 220. A bioelectrical impedance analysis (BIA) of body composition will also be performed to minimize the effect of hydration status on all measurements. Participants will be instructed to refrain from caffeine and alcohol 24 h before, as well as, if performed 12 h before the test according to published guidelines for BIA. To predict the MMEA, a tanita TA-MC980MA- 1 PLUS impedance meter (Tanita Europe B.V., UK) will be used according to the manufacturer's guidelines. The MMEA will be predicted through the Equation of Sergi et al, (80): MMEA (kg) = -3.964 + (0.227 RI) + (0.095 * body mass) + (1.384 * sex) + (0.064 * XC), where RI is the standardized resistance for height and XC is the reactance. | 24 weeks | 
| Nutritional evaluation (Day 0 and at 12 weeks of intervention). | The nutritional evaluation will be performed by a dietitian-nutritionist of the research group at each control point. Food consumption will be assessed by a 3-day dietary record, applied on three non-consecutive days of the week prior to data collection. A photographic manual of food portion sizes will be used to improve the accuracy of the dietary intake report. Home measurements of nutritional values of food and supplementation will be converted into grams and milliliters using Virtual Dietopro online software (Dietopro® , Valencia, Spain) for diet analysis. | 12 weeks | 
| Nutritional evaluation (12 weeks after the intervention). | The nutritional evaluation will be performed by a dietitian-nutritionist of the research group at each control point. Food consumption will be assessed by a 3-day dietary record, applied on three non-consecutive days of the week prior to data collection. A photographic manual of food portion sizes will be used to improve the accuracy of the dietary intake report. Home measurements of nutritional values of food and supplementation will be converted into grams and milliliters using Virtual Dietopro online software (Dietopro® , Valencia, Spain) for diet analysis. | 24 weeks | 
| Assessment of muscle strength and performance. Short Physical Performance Battery (Day 0 and at 12 weeks of intervention). | Short Physical Performance Battery (SPPB): consists of 3 tests to be used to assess lower extremity function following standard protocols: Balance by balance test. Muscle strength test in the legs, sit to up test. Mobility test using gait speed, gait speed test. It is important to follow the sequence of the tests, because if the patient starts to stand and sit, he/she may become fatigued and perform falsely poorly in the other two tests. The average test duration is between 6 and 10 min. Each test will be scored from 0 to 4. A score of 0 will represent an inability to complete the test, and a score of 4 will represent the highest level of performance. Therefore, total scores (SPPB scores) range from 0 to 12, and a higher SPPB score will indicate a higher level of physical fitness. | 12 weeks | 
| Assessment of muscle strength and performance. Short Physical Performance Battery (12 weeks after the intervention). | Short Physical Performance Battery (SPPB): consists of 3 tests to be used to assess lower extremity function following standard protocols: Balance by balance test. Muscle strength test in the legs, sit to up test. Mobility test using gait speed, gait speed test. It is important to follow the sequence of the tests, because if the patient starts to stand and sit, he/she may become fatigued and perform falsely poorly in the other two tests. The average test duration is between 6 and 10 min. Each test will be scored from 0 to 4. A score of 0 will represent an inability to complete the test, and a score of 4 will represent the highest level of performance. Therefore, total scores (SPPB scores) range from 0 to 12, and a higher SPPB score will indicate a higher level of physical fitness. | 24 weeks | 
| Assessment of muscle strength and performance. Timed-up-and-go (Day 0 and at 12 weeks of intervention). | The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. The tests to be performed to the participants will be: Timed-up-and-go (TUG): Timed-up-and-go is used to assess mobility, balance and agility. Participants are instructed to get up from a chair without assistance (starting point), walk forward three meters (m), walk back to the chair and sit down again (end of test). They are asked to perform the test at a normal, everyday pace, using marks on the floor to indicate where to turn. The total time used from the beginning to the end of the test is recorded. | 12 weeks | 
| Assessment of muscle strength and performance. Timed-up-and-go (12 weeks after the intervention). | The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. The tests to be performed to the participants will be: Timed-up-and-go (TUG): Timed-up-and-go is used to assess mobility, balance and agility. Participants are instructed to get up from a chair without assistance (starting point), walk forward three meters (m), walk back to the chair and sit down again (end of test). They are asked to perform the test at a normal, everyday pace, using marks on the floor to indicate where to turn. The total time used from the beginning to the end of the test is recorded. | 24 weeks | 
| Assessment of muscle strength and performance. 400-meter Walk Test Perfomance (Day 0 and at 12 weeks of intervention). | The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. The tests to be performed to the participants will be: 400-meter Walk Test Perfomance: measures mobility and cardiovascular fitness through a 400-meter timed walk (min). Cones are placed 20 m apart, around which participants must move 10 times, back and forth, as fast as possible. Only one attempt is allowed. | 12 weeks | 
| Assessment of muscle strength and performance. 400-meter Walk Test Perfomance (12 weeks after the intervention). | The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. The tests to be performed to the participants will be: 400-meter Walk Test Perfomance: measures mobility and cardiovascular fitness through a 400-meter timed walk (min). Cones are placed 20 m apart, around which participants must move 10 times, back and forth, as fast as possible. Only one attempt is allowed. | 24 weeks | 
| Assessment of muscle strength and performance. Grip strength (Day 0 and at 12 weeks of intervention). | The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. The tests to be performed to the participants will be: Grip strength: isometric hand strength is a commonly used measure to establish upper body skeletal muscle function and has been widely used as a general indicator of physical fitness. Grip strength will be measured using a validated hydraulic hand-held dynamometer (Jamar plus +, if instruments, Adelaide, Australia). Values will be recorded in kilograms. Grip strength will be measured while seated, elbow in 90° flexion and wrist in neutral position. Participants will be asked to apply maximum grip strength in 3 different attempts with the left and right hands. Between each attempt, a 30 s rest will be allowed. The measurement in which the maximum grip strength is obtained from the 3 attempts will be used. | 12 weeks | 
| Assessment of muscle strength and performance. Grip strength (12 weeks after the intervention). | The evaluation of muscular strength and physical performance will be performed by a strength and conditioning coach graduated in physical activity and sport sciences, which will be carried out at each control point. The tests to be performed to the participants will be: Grip strength: isometric hand strength is a commonly used measure to establish upper body skeletal muscle function and has been widely used as a general indicator of physical fitness. Grip strength will be measured using a validated hydraulic hand-held dynamometer (Jamar plus +, if instruments, Adelaide, Australia). Values will be recorded in kilograms. Grip strength will be measured while seated, elbow in 90° flexion and wrist in neutral position. Participants will be asked to apply maximum grip strength in 3 different attempts with the left and right hands. Between each attempt, a 30 s rest will be allowed. The measurement in which the maximum grip strength is obtained from the 3 attempts will be used. | 24 weeks | 
| Assessment of fragility. Frailty phenotype (Day 0 and at 12 weeks of intervention). | Using Fried Fragility Scale examines several factors: Unintentional weight loss > 4.5 kg or > 5% in the last year. Self-perceived exhaustion. Weakness quantified using maximal grip strength. Weakness is assessed while seated, preferably with the hand and elbow dominant at 90°. The highest value of 3 measurements (separated by one minute) is used. Slow walking speed, based on the time required to walk 4.57 m at a normal pace. Walking time ≥7 s for men ≤173 cm tall or women ≤159 cm tall or walking time ≥6 s for men whose height is >173 cm or women whose height is >159 cm is considered slow. Low activity level, physical activity energy expenditure per week <383 kcal for men (at least 2.30 h/week of physical activity), or <270 kcal for women (at least 2 h/week of physical activity). The presence of one or two of the above properties is considered pre-frailty state. The presence of three or more of these characteristics is considered frailty. | 12 weeks | 
| Assessment of fragility. Frailty phenotype (12 weeks after the intervention). | Using Fried Fragility Scale examines several factors: Unintentional weight loss > 4.5 kg or > 5% in the last year. Self-perceived exhaustion. Weakness quantified using maximal grip strength. Weakness is assessed while seated, preferably with the hand and elbow dominant at 90°. The highest value of 3 measurements (separated by one minute) is used. Slow walking speed, based on the time required to walk 4.57 m at a normal pace. Walking time ≥7 s for men ≤173 cm tall or women ≤159 cm tall or walking time ≥6 s for men whose height is >173 cm or women whose height is >159 cm is considered slow. Low activity level, physical activity energy expenditure per week <383 kcal for men (at least 2.30 h/week of physical activity), or <270 kcal for women (at least 2 h/week of physical activity). The presence of one or two of the above properties is considered pre-frailty state. The presence of three or more of these characteristics is considered frailty. | 24 weeks | 
| Inflammation markers. CRP (Day 0 and at 12 weeks of intervention). | CRP will be assessed in duplicate from frozen stored serum. PCR levels using an enzyme-linked immunostorbent assay based on purified proteins and purified anti-CRP antibodies (Calbiochem, San Diego, CA). | 12 weeks | 
| Inflammation markers. CRP (12 weeks after the intervention). | CRP will be assessed in duplicate from frozen stored serum. PCR levels using an enzyme-linked immunostorbent assay based on purified proteins and purified anti-CRP antibodies (Calbiochem, San Diego, CA). | 24 weeks | 
| Inflammation markers. Cytokine levels and cytokine soluble receptors (Day 0 and at 12 weeks of intervention). | IL-1 (soluble IL-1 receptor [IL-1SR]), IL-2 (soluble IL-2 receptor [il-2SR]), IL-6 (soluble IL-6 receptor [IL-6SR]), IL-7 (soluble IL-7 receptor [IL-7SR]) will be measured using enzyme-linked immunostorbent assay kits (Research and Development Systems, Minneapolis, MN). | 12 weeks | 
| Inflammation markers. Cytokine levels and cytokine soluble receptors (12 weeks after the intervention). | IL-1 (soluble IL-1 receptor [IL-1SR]), IL-2 (soluble IL-2 receptor [il-2SR]), IL-6 (soluble IL-6 receptor [IL-6SR]), IL-7 (soluble IL-7 receptor [IL-7SR]) will be measured using enzyme-linked immunostorbent assay kits (Research and Development Systems, Minneapolis, MN). | 24 weeks | 
| Inflammation markers. TNF-α (Day 0 and at 12 weeks of intervention). | TNF-α (TNFSR1 and TNFSR2) levels will be assessed in duplicate from frozen stored serum. | 12 weeks | 
| Inflammation markers. TNF-α (12 weeks after the intervention). | TNF-α (TNFSR1 and TNFSR2) levels will be assessed in duplicate from frozen stored serum. | 24 weeks | 
| Oxidative stress markers. Reduced (GSH) and oxidized glutathione (GSSG) (Day 0 and at 12 weeks of intervention). | Reduced (GSH) and oxidized glutathione (GSSG) will be determined using colorimetric kits. Reducing power will be calculated by the equation GSH/(GSH + GSSG). | 12 weeks | 
| Oxidative stress markers. Reduced (GSH) and oxidized glutathione (GSSG) (12 weeks after the intervention). | Reduced (GSH) and oxidized glutathione (GSSG) will be determined using colorimetric kits. Reducing power will be calculated by the equation GSH/(GSH + GSSG). | 24 weeks | 
| Oxidative stress markers. TBAR (Day 0 and at 12 weeks of intervention). | Lipid peroxidation will be analyzed in plasma as thiobarbituric acid reactive substances (TBAR). The TBAR will be determined after treatment of the sample with 4% butylated hydroxytoluene and an additional reaction with 0.375% thiobarbituric acid in 0.25 M HCl and 1% Triton X-100 (15 min, at 100°C). | 12 weeks | 
| Oxidative stress markers. TBAR (12 weeks after the intervention). | Lipid peroxidation will be analyzed in plasma as thiobarbituric acid reactive substances (TBAR). The TBAR will be determined after treatment of the sample with 4% butylated hydroxytoluene and an additional reaction with 0.375% thiobarbituric acid in 0.25 M HCl and 1% Triton X-100 (15 min, at 100°C). | 24 weeks | 
| Oxidative stress markers. Malondialdehye equivalents (MDA) (Day 0 and at 12 weeks of intervention). | Malondialdehye equivalents (MDA) will be calculated according to the absorbance at 535 nm against a blank (lacking TBA) and using 1,1,2,2-tetroxyethylpropane as standard. | 12 weeks | 
| Oxidative stress markers. Malondialdehye equivalents (MDA) (12 weeks after the intervention). | Malondialdehye equivalents (MDA) will be calculated according to the absorbance at 535 nm against a blank (lacking TBA) and using 1,1,2,2-tetroxyethylpropane as standard. | 24 weeks | 
| Oxidative stress markers. Superoxide dismutase (SOD) (Day 0 and at 12 weeks of intervention). | Superoxide dismutase (SOD) will be estimated by inhibition of adrenaline autooxidation, read at 480 nm in a spectrophotometer. | 12 weeks | 
| Oxidative stress markers. Superoxide dismutase (SOD) (12 weeks after the intervention). | Superoxide dismutase (SOD) will be estimated by inhibition of adrenaline autooxidation, read at 480 nm in a spectrophotometer. | 24 weeks | 
| Oxidative stress markers. Nitric oxide (NO) (Day 0 and at 12 weeks of intervention). | Nitric oxide (NO) will be estimated spectrophotometrically as a function of nitrite generation. | 12 weeks | 
| Oxidative stress markers. Nitric oxide (NO) (12 weeks after the intervention). | Nitric oxide (NO) will be estimated spectrophotometrically as a function of nitrite generation. | 24 weeks | 
| Oxidative stress markers. Total antioxidant capacity (TAC) (Day 0 and at 12 weeks of intervention). | Total antioxidant capacity (TAC) will be analyzed in duplicate samples, and absorbance will be measured using a microplate reader. | 12 weeks | 
| Oxidative stress markers. Total antioxidant capacity (TAC) (12 weeks after the intervention). | Total antioxidant capacity (TAC) will be analyzed in duplicate samples, and absorbance will be measured using a microplate reader. | 24 weeks | 
| Hormones. Total serum testosterone (Day 0 and at 12 weeks of intervention). | Total serum testosterone will be measured using the enzyme from commercially available enzyme-linked immunosorbent assay (ELISA) kits (DRG Testosterone ELISA KIT, DRG Instruments GmbH, Marburg,® Germany). | 12 weeks | 
| Hormones. Total serum testosterone (12 weeks after the intervention). | Total serum testosterone will be measured using the enzyme from commercially available enzyme-linked immunosorbent assay (ELISA) kits (DRG Testosterone ELISA KIT, DRG Instruments GmbH, Marburg,® Germany). | 24 weeks | 
| Hormones. Sex hormone binding globulin (SHBG) (Day 0 and at 12 weeks of intervention). | Sex hormone binding globulin (SHBG) will be measured by chemiluminescence based on the immunometric method (immunite, 2000), which will be from the same supplier as testosterone. | 12 weeks | 
| Hormones. Sex hormone binding globulin (SHBG) (12 weeks after the intervention) | Sex hormone binding globulin (SHBG) will be measured by chemiluminescence based on the immunometric method (immunite, 2000), which will be from the same supplier as testosterone. | 24 weeks | 
| Hormones. Cortisol (Day 0 and at 12 weeks of intervention). | Cortisol will be measured by enzyme-linked fluorescent assay in a microplate reader (Cytation 5 Cell Imaging Multi-Mode Reader -Biotek Instruments, USA). | 12 weeks | 
| Hormones. Cortisol (12 weeks after the intervention). | Cortisol will be measured by enzyme-linked fluorescent assay in a microplate reader (Cytation 5 Cell Imaging Multi-Mode Reader -Biotek Instruments, USA). | 24 weeks | 
| Hormones. Total testosterone/cortisol and total testosterone/SHBG ratios (Day 0 and at 12 weeks of intervention). | Total testosterone/cortisol and total testosterone/SHBG ratios will be calculated from serum testosterone, cortisol and SHBG concentrations. | 12 weeks | 
| Hormones. Total testosterone/cortisol and total testosterone/SHBG ratios (12 weeks after the intervention). | Total testosterone/cortisol and total testosterone/SHBG ratios will be calculated from serum testosterone, cortisol and SHBG concentrations. | 24 weeks | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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GAA, HMB, aging, sarcopenia, nutrition, creatine, exercise