Protein Supplementation in Elderly With Sarcopenic Obesity Undergoing Caloric Restriction and Exercise

Fat-free mass evaluated through dual-energy x-ray absorptiometry (DEXA) and reported as percentage

Appendicular fat-free mass was evaluated through dual-energy x-ray absorptiometry (DEXA) and calculated as the sum of the fat-free mass of the upper and lower limbs.

The appendicular fat-free mass (AFFM) was assessed using dual-energy X-ray absorptiometry (DXA) and expressed in kilograms (kg). The body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m²). The AFFM/BMI ratio was computed by dividing AFFM (kg) by BMI (kg/m²), resulting in a unitless ratio. Higher values indicate greater muscle mass relative to body size

Appendicular fat-free mass was evaluated through dual-energy x-ray absorptiometry (DEXA) and calculated as the sum of the fat-free mass of the lower limbs.

Fat mass was evaluated trough dual-energy x-ray absorptiometry (DEXA) and reported as percentage.

Quadriceps cross-sectional area (CSA) was assessed by computed tomography imaging

Rectus femoris cross-sectional area (CSA) was assessed by B-mode ultrasound.

Vastus lateralis cross-sectional area (CSA) was assessed by B-mode ultrasound.

Muscle fiber cross-sectional area (type I) was assessed using an immunostaining assay of muscle tissue samples obtained through percutaneous muscle biopsy

Muscle fiber cross-sectional area (type II) was assessed using an immunostaining assay of muscle tissue samples obtained through percutaneous muscle biopsy

Bone turnover was assessed by an automated electrochemiluminescence method.

Bone turnover was assessed by an automated electrochemiluminescence method.

bone mineral density (whole-body) evaluated trough dual-energy x-ray absorptiometry (DEXA)

bone mineral density (femur neck) evaluated trough dual-energy x-ray absorptiometry (DEXA)

bone mineral density (total hip) evaluated trough dual-energy x-ray absorptiometry (DEXA)

bone mineral density (lumbar spine) evaluated trough dual-energy x-ray absorptiometry (DEXA)

Bone microarchitecture (total volumetric density) was assessed at the distal region of the radius of the non-dominant limb using a high-resolution peripheral quantitative computed tomography (HR-pQCT)

Bone microarchitecture (trabecular volumetric density) was assessed at the distal region of the radius of the non-dominant limb using a high-resolution peripheral quantitative computed tomography (HR-pQCT)

Bone microarchitecture (cortical volumetric density) was assessed at the distal region of the radius of the non-dominant limb using a high-resolution peripheral quantitative computed tomography (HR-pQCT)

Bone microarchitecture (BV/TV) was assessed at the distal region of the radius of the non-dominant limb using high-resolution peripheral quantitative computed tomography (HR-pQCT). Specifically, trabecular bone volume fraction (BV/TV) is computed as the ratio of the trabecular bone mineral density (Tb.vBMD in mg HA/cm3) and 1200 mg HA/cm3, which is assumed to be the density of fully mineralized bone. Afterwards, the values were multiplied by 100 to reflect the percentage of trabecular bone volume fraction.

The trabecular number (Tb.N) was analyzed using the ridge extraction technique in high-resolution peripheral quantitative computed tomography (HR-pQCT). In this approach, the trabeculae were treated as elongated structures resembling ridges. The technique involves detecting the central axis (ridge) of each trabecular element in a 3D image.

Trabecular separation (Tb.Sp), represents the average distance between trabeculae in the trabecular bone region. It was assessed using the distance transformation method, applied to the background (void space) of the trabecular structure. The transformation method measures the distance from each voxel (3D pixel) in the void space to the nearest trabecular element, and the average of these distances is then calculated. This method enables precise quantification of trabecular spacing in high-resolution 3D images.

The separation is inversely related to trabecular density, as closer trabeculae indicate a higher bone volume fraction (BV/TV) and a denser bone network. The calculation of trabecular separation can be expressed as:

Tb.Sp = 1- BV/TV : Tb.N

Trabecular thickness (Tb.Th) represents the average thickness of trabecular bone elements. It was assessed by calculating the mean thickness of the segmented trabecular structure, using the distance transformation method applied to the trabecular bone tissue.

The trabecular thickness is calculated as a ratio of the bone volume fraction (BV/TV) to trabecular number (Tb.N): Tb.Th =BV/TV : Tb.N

Cortical porosity (Ct.Po) is quantified using a density-based approach that segments bone into three compartments: compact cortex, transitional zone, and trabecular compartment. Voxels with a density below 1000 mg HA/cm³ indicate the presence of void space (pores), and porosity is estimated as the ratio of void space in each voxel. The mean of this ratio is calculated across all voxels in the compartment of interest, and the values were multiplied by 100. This method captures pores with diameters below the scanner's spatial resolution but relies on the assumption of fixed bone tissue mineral density and may be susceptible to image noise and beam hardening.

Bone microarchitecture (cortical thickness) wwas assessed at the distal region of the radius of the non-dominant limb using a high-resolution peripheral quantitative computed tomography (HR-pQCT)

Bone microarchitecture (cortical pore diameter) was assessed at the distal region of the radius of the non-dominant limb using a high-resolution peripheral quantitative computed tomography (HR-pQCT)

Stiffness (kN/mm) is defined as the total reaction force of the model divided by the applied displacement. It represents the resistance of a material or structure to deformation under an applied load. A higher stiffness value indicates greater resistance to deformation, while a lower value suggests more flexibility.

Estimated failure load is indirectly calculated from linear finite element (FE) models using a yield criterion. The failure load is estimated when a specified volume of bone tissue (critical volume) exceeds a critical strain threshold, at which point the model is assumed to have yielded. This approach is often based on the Pistoia criterion, which is used to predict the point of failure in the material based on its mechanical properties and deformation behavior.

Isometric muscle strength was evaluated using a handgrip dynamometer (Jamar®, Sammons Preston Rolyan, USA).

Muscle strength was evaluated using maximal dynamic strength test [1RM])

Muscle strength was evaluated using maximal dynamic strength test [1RM])

Muscle function evaluated through battery of tests - Short Physical Performance Battery (SPPB). The SPPB is a standardized assessment of lower extremity function that includes three components: balance tests, gait speed over 4 meters, and the five-times sit-to-stand test. Each component is scored from 0 to 4, with a total score ranging from 0 to 12. Higher scores indicate better physical performance. The SPPB is widely used to evaluate physical function, predict disability, and monitor health status in older adults.

The 30-second sit-to-stand test is a simple measure of lower body strength and functional capacity. Participants are asked to rise from a seated position and sit back down as many times as possible within 30 seconds. The total number of complete sit-to-stand repetitions performed in the given time is recorded. This test is commonly used to assess physical fitness and mobility, particularly in older adults or individuals with health conditions.

The Timed Up and Go (TUG) test is a simple and widely used assessment of mobility and balance. Participants are asked to stand up from a seated position, walk 3 meters, turn around, walk back to the chair, and sit down again, all as quickly as possible. The total time taken to complete the task is recorded. The TUG test is commonly used to evaluate functional mobility, fall risk, and the ability to perform daily activities, particularly in older adults or individuals with mobility impairments.

Muscle function evaluated through 4-m gait speed test. The 4-meter usual gait speed test measures the time it takes for a participant to walk a distance of 4 meters at their usual pace. The test is commonly used to assess walking speed, which is an important indicator of mobility, physical function, and overall health. The time taken to complete the 4-meter walk is recorded and used to evaluate the individual's functional capacity, with slower times potentially indicating mobility impairments or a higher risk of adverse health outcomes.

Cardiorespiratory fitness was evaluated by maximal oxygen uptake (VO²max) during a maximal exercise test on a treadmill

Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) is a method used to estimate insulin resistance based on fasting plasma glucose and fasting insulin levels. It is calculated using the following formula:

HOMA-IR = (fasting insulin [µU/mL] × fasting glucose [mg/dL]) / 405. Higher values of HOMA-IR indicate greater insulin resistance and are considered worse. There is no fixed theoretical maximum value, but typical reference ranges in healthy individuals are usually <2. Values above this threshold may suggest impaired insulin sensitivity or metabolic dysfunction. The HOMA-IR is widely used in clinical and research settings as a surrogate marker for insulin resistance.

Brachial flow-mediated dilation (FMD) is a non-invasive measure of endothelial function, assessed using high-resolution B-mode ultrasound. The test evaluates the percent change in brachial artery diameter in response to increased blood flow (reactive hyperemia) following 3 minutes of cuff occlusion, on the forearm. An increase in arterial diameter after cuff release indicates vasodilation mediated by nitric oxide. Higher FMD values reflect better endothelial function, whereas lower values are associated with cardiovascular risk and impaired vascular health.

Lipid profile (i.e; HDL, LDL, VLDL, and triglycerides) were evaluated by colorimetric enzymatic methods

Lipid profile (i.e; HDL, LDL, VLDL, and triglycerides) were evaluated by colorimetric enzymatic methods

Lipid profile (i.e; HDL, LDL, VLDL, and triglycerides) were evaluated by colorimetric enzymatic methods

Lipid profile (i.e; HDL, LDL, VLDL, and triglycerides) were evaluated by colorimetric enzymatic methods

Relative telomere length was measured using quantitative polymerase chain reaction (qPCR), which determines the ratio of telomeric repeat copy number (T) to a single-copy gene number (S) in a given sample. This T/S ratio is a unitless index that reflects the average telomere length relative to the reference gene. Higher T/S ratios indicate longer telomeres, while lower values indicate shorter telomeres. Although the T/S ratio does not provide absolute telomere length in base pairs, it is a widely used, validated method to assess relative telomere length in epidemiological and clinical research.

Superoxide dismutase (SOD) activity was measured using an enzyme-linked immunosorbent assay (ELISA), following the manufacturer's instructions. The assay is based on the competitive binding of SOD present in the sample and a SOD standard to a monoclonal antibody coated on a microplate.

Catalase activity was assessed using an enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's protocol. The assay quantifies catalase based on the competition between the sample catalase and a catalase standard for binding to specific antibodies coated on the microplate. The detection is achieved through a colorimetric reaction measured at a specific wavelength.

Glutathione peroxidase activity was measured using a colorimetric assay according to the manufacturer's instructions. The method is based on the enzyme-catalyzed reduction of hydrogen peroxide by reduced glutathione (GSH), forming oxidized glutathione (GSSG). In the presence of glutathione reductase and NADPH, GSSG is converted back to GSH with concomitant oxidation of NADPH to NADP⁺.

Oxidative stress markers (SOD, CAT, glutathione, GPx, GST and TBARS) were assessed through ELISA assay.

Glutathione S-transferase (GST) activity was assessed using a colorimetric assay based on the conjugation of the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione (GSH). The reaction results in a yellow product that is quantified by measuring the absorbance at 340 nm.

Thiobarbituric Acid Reactive Substances (TBARS): Lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances (TBARS), following the manufacturer's instructions. This colorimetric assay detects malondialdehyde (MDA), a byproduct of lipid peroxidation, which reacts with thiobarbituric acid to form a colored complex measurable at 532-535 nm. Results are expressed as micromoles of MDA equivalents per liter (µmol/L), with higher values indicating greater oxidative stress.

Inflammatory profile (i.e.; IL1β, IL-10, IL-6, and TNF-α) were quantified using the Luminex xMAP technology.

Inflammatory profile (i.e.; IL1β, IL-10, IL-6, and TNF-α) were quantified using the Luminex xMAP technology.

Inflammatory profile (i.e.; IL1β, IL-10, IL-6, and TNF-α) were quantified using the Luminex xMAP technology.

Inflammatory profile (i.e.; IL1β, IL-10, IL-6, and TNF-α) were quantified using the Luminex xMAP technology.

Inflammatory profile (i.e.; C-Reactive Protein ) was quantified via an immunoturbidimetric assay.

The area under the curve (AUC) of blood glucose was measured during a 2-hour oral glucose tolerance test (OGTT). Blood samples were collected at baseline (0 minutes, following a 12-hour overnight fast), and at 30, 60, 90, and 120 minutes after ingestion of a 75 g glucose bolus. The AUC was calculated using these time points (0, 30, 60, 90, and 120 minutes) to assess the blood glucose response over the 2-hour period following glucose ingestion.

The area under the curve (AUC) of insulin was measured during a 2-hour oral glucose tolerance test (OGTT). Blood samples were collected at baseline (0 minutes, following a 12-hour overnight fast), and at 30, 60, 90, and 120 minutes after ingestion of a 75 g glucose bolus. The AUC was calculated using these time points (0, 30, 60, 90, and 120 minutes) to assess the insulin response over the 2-hour period following glucose ingestion.

Anxiety was assessed using the Geriatric Anxiety Inventory (GAI), a 20-item self-report questionnaire designed to measure anxiety symptoms in older adults. The total score ranges from 0 to 20, with higher scores reflecting more severe anxiety symptoms.

Depression was assessed using the Geriatric Depression Scale (GDS-15), a 15-item self-report questionnaire designed to measure depressive symptoms in older adults. The total score ranges from 0 to 15, with higher scores indicating more severe depression.

Health-related quality of life was evaluated using the 36-Item Short Form Health Survey (SF-36), a widely used questionnaire designed to assess various dimensions of health in adults. The total score ranges from 0 to 100, with higher scores indicating better health-related quality of life.

Health-related quality of life was evaluated using the 36-Item Short Form Health Survey (SF-36), a widely used questionnaire designed to assess various dimensions of health in adults. The total score ranges from 0 to 100, with higher scores indicating better health-related quality of life.

Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI), a self-report questionnaire that evaluates various aspects of sleep quality. The PSQI is scored by summing the scores of seven components, each ranging from 0 to 3. The total score ranges from 0 to 21, with higher scores indicating poorer sleep quality.