The goal of this observational study is to evaluate the presence of mitochondrial dysfunction related to oxidative stress and its possible role in frailty, with and without multimorbidity, and to identify possible frailty biomarkers correlated with mitochondrial dysfunction. The main questions it aims to answer are: * What is the role of oxidative stress-related mitochondrial dysfunction in frailty, taking into account the interaction with multimorbidity. * What could be the specific biomarkers associated with mitochondrial dysfunction in the assessment of frailty. In order to reach the study goals, we will enroll three categories of older adults: * Non-Frail without Multimorbidity (NFWoM); * Frail with Multimorbidity (FWM); * Frail without Multimorbidity (FWoM). Each individual will undergo an assessment of frailty phenotype and multimorbidity, and the collection of blood samples to isolate Peripheral Blood Mononuclear Cells (PBMCs). The identification of frailty biomarkers in each group of participants will be performed by combining untargeted metabolomics-based approaches and functional studies on specific mitochondrial dysfunctions performed on PBMCs and their subpopulations. Multivariate statistical and machine learning techniques will characterize the three clinical phenotype groups based on molecular data.
Study type: observational prospective study. Primary aim: is to evaluate the presence of mitochondrial dysfunction related to oxidative stress and its possible role in frailty, with and without multimorbidity. Primary endpoint: mitochondrial dysfunction in frailty. Secondary aims: to combine untargeted metabolomics-based approaches and functional studies on specific mitochondrial dysfunctions performed on PBMCs and PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes). The research activities are organized in the following tasks: * Task 1, Patient Enrollment: We will enroll individuals aged 65 years or older from geriatric outpatient clinics or geriatric wards. For each individual, we will perform an assessment of frailty and multimorbidity, and collect blood samples to isolate Peripheral Blood Mononuclear Cells (PBMCs). Three categories of individuals will be enrolled: 25 non-frail individuals without multimorbidity (NFWoM), 25 frail individuals with multimorbidity (FWM), and 25 frail individuals without multimorbidity (FWoM). * Task 2, Separation of PBMC Subpopulations: T lymphocytes, B lymphocytes, and monocytes will be separated from frozen PBMCs using the Cell Sorting Facility for Fluorescence-Activated Cell Sorting (FACS) separation and the MoFlo Astrios cell sorter. The analyses on CD45+/CD3+/CD19-/CD14- T lymphocytes, CD45+/CD3-/CD19+/CD14- B lymphocytes, CD45+/CD3-/CD19-/CD14+ monocytes. * Task 3, Mitochondrial Dysfunction Analysis on PBMCs and PBMC Subpopulations: For each individual, mitochondrial dysfunction will be evaluated by analyzing mtDNA damage (by Real-Time PCR), mitochondrial mass alteration (by Mitotracker staining), and intracellular and mitochondrial Reactive Oxygen Species (by DCF and MitoSOX staining). Moreover, we will evaluate alteration of glycolytic and mitochondrial metabolism using Agilent Seahorse Extracellular Flux Analyzer XFe96. * Task 4, Untargeted Metabolomics on PBMCs and PBMC Subpopulations: To assess metabolic signature of PBMCs and subpopulations and highlight metabolic dysregulations linked to frailty, we will perform untargeted LC-MS-based metabolomics on PBMCs, T lymphocytes, B lymphocytes, and monocytes. The analysis on the polar metabolome will allow us to understand better the metabolic alterations associated with mitochondrial dysregulation. * Task 5, Characterization of Biomarkers and Molecular Mechanism of Frailty: The potential biomarkers of frailty and the molecular mechanisms involved in mitochondrial dysfunction will be studied using statistical and machine learning techniques on molecular, metabolic and clinical data. This step will help characterize clinical phenotypes based on molecular measurements.
Study Type
OBSERVATIONAL
Enrollment
75
Difference in the mtDNA copy number between frail individuals with vs without multimorbidity
mtDNA copies isolated from total PBMCs and from T and B lymphocytes and monocytes, obtained from the participants, with JetQuick™ Blood and Cell Culture DNA Midiprep Kit (Invitrogen), and 10 ng of DNA will be used for analysis on QuantumStudio 7 Real Time PCR (Applied Biosystems). mtDNA copy number will be calculated by normalising the mitochondrial ND1 gene (mtND1) levels to nuclear Beta-2 microglobulin (B2M) levels. The number mtDNA copies will be compared between individuals with frailty and multimorbidity vs individuals with frailty without multimorbidity. Frailty will be derived based on the presence of at least three criteria among: involuntary weight loss ≥ 4.5 kg, muscle weakness measured by handgrip, self-reported fatigue on ≥ 3 days per week, low physical activity (assessed with the IPAQ questionnaire), and reduced gait speed (measured by the 4-m walking test). Multimorbidity will be defined as the presence of at least two chronic diseases.
Time frame: Baseline
Difference in the mtDNA copy number between non-frail vs frail individuals without multimorbidity
mtDNA copies isolated from total PBMCs and from T and B lymphocytes and monocytes, obtained from the participants, with JetQuick™ Blood and Cell Culture DNA Midiprep Kit (Invitrogen), and 10 ng of DNA will be used for analysis on QuantumStudio 7 Real Time PCR (Applied Biosystems). mtDNA copy number will be calculated by normalising the mitochondrial ND1 gene (mtND1) levels to nuclear Beta-2 microglobulin (B2M) levels. The number mtDNA copies will be compared between individuals with frailty and multimorbidity vs individuals with frailty without multimorbidity. Frailty will be derived based on the presence of at least three criteria among: involuntary weight loss ≥ 4.5 kg, muscle weakness measured by handgrip, self-reported fatigue on ≥ 3 days per week, low physical activity (assessed with the IPAQ questionnaire), and reduced gait speed (measured by the 4-m walking test). Multimorbidity will be defined as the presence of at least two chronic diseases.
Time frame: Baseline
Variation in the mean intensity of mitochondrial fluorescence between non-frail vs frail individuals without multimorbidity
500,000 PBMCs obtained from the study participants will be stained with CD45, CD3, CD19 and CD14 antibodies and incubated with 100 nM Mitotracker Deep Red (Thermo Fisher Scientific) for 30 minutes at 30°C. Cells will be also labelled with Live/Dead dye and analyzed by flow cytometry with MoFLO Astrios cell sorter. Mitochondrial mass, evaluated as the median fluorescence intensity (MFI) of Mitotracker Deep Red, will be assessed in total PBMCs, T and B lymphocytes and monocytes. 40,000 events in each population gate will be acquired and offline analysis will be performed with Kaluza software.
Time frame: Baseline
Variation in the mean intensity of mitochondrial fluorescence between frail individuals with vs without multimorbidity
500,000 PBMCs obtained from the study participants will be stained with CD45, CD3, CD19 and CD14 antibodies and incubated with 100 nM Mitotracker Deep Red (Thermo Fisher Scientific) for 30 minutes at 30°C. Cells will be also labelled with Live/Dead dye and analyzed by flow cytometry with MoFLO Astrios cell sorter. Mitochondrial mass, evaluated as the median fluorescence intensity (MFI) of Mitotracker Deep Red, will be assessed in total PBMCs, T and B lymphocytes and monocytes. 40,000 events in each population gate will be acquired and offline analysis will be performed with Kaluza software.
Time frame: Baseline
Difference of intracellular Reactive Oxygen Species (ROS) between non-frail vs frail individuals without multimorbidity
The fluorescent cell-permeable indicator 2',7'-dichlorofluorescin diacetate (DCFH-DA) will be used for detecting intracellular ROS. DCFH-DA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidised by ROS into fluorescent 2',7'-dichlorofluorescein (DCF). The intensity of the generated fluorescent signal correlates with the intracellular level of ROS. 500,000 PBMCs obtained from 25 NFWoM, 25 FWM and 25 FWoM subjects will be stained with CD45, CD3, CD19 and CD14 antibodies and incubated with 10 μM 2',7'-dichlorofluorescin diacetate (DCFH-DA) at 37°C for 30 minutes. Cells will be also labelled with Live/Dead dye and analyzed by flow cytometry. Intracellular ROS, evaluated as the median fluorescence intensity (MFI) of DCF, will be assessed in total PBMCs, T and B lymphocytes and monocytes. 40,000 events in each population gate will be acquired and offline analysis will be performed with Kaluza software.
Time frame: Baseline
Difference of intracellular Reactive Oxygen Species (ROS) between frail individuals with vs without multimorbidity
The fluorescent cell-permeable indicator 2',7'-dichlorofluorescin diacetate (DCFH-DA) will be used for detecting intracellular ROS. DCFH-DA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidised by ROS into fluorescent 2',7'-dichlorofluorescein (DCF). The intensity of the generated fluorescent signal correlates with the intracellular level of ROS. 500,000 PBMCs obtained from 25 NFWoM, 25 FWM and 25 FWoM subjects will be stained with CD45, CD3, CD19 and CD14 antibodies and incubated with 10 μM 2',7'-dichlorofluorescin diacetate (DCFH-DA) at 37°C for 30 minutes. Cells will be also labelled with Live/Dead dye and analyzed by flow cytometry. Intracellular ROS, evaluated as the median fluorescence intensity (MFI) of DCF, will be assessed in total PBMCs, T and B lymphocytes and monocytes. 40,000 events in each population gate will be acquired and offline analysis will be performed with Kaluza software.
Time frame: Baseline
Qualitative difference in metabolomics profiles of PBMCs and PBMC subpopulations between non-frail vs frail individuals without multimorbidity
Untargeted LC-MS-based metabolomics will be performed on PBMCs and on T and B lymphocytes and monocytes, obtained as described in Task 2. Cell samples will be quenched using cold methanol. After protein precipitation, metabolites will be extracted and analysed by liquid chromatography mass spectrometry (LC-MS). Hydrophilic interaction chromatography (HILIC) will be used to resolve the polar metabolome before MS detection using an Agilent 6546 lc/q-tof instrument (Agilent).The whole protein content will be quantified with NanoDrop™ (Thermofisher) and used to normalize the metabolic profile of each sample.
Time frame: Baseline
Qualitative difference in metabolomics profiles of PBMCs and PBMC subpopulations between frail individuals with vs without multimorbidity
Untargeted LC-MS-based metabolomics will be performed on PBMCs and on T and B lymphocytes and monocytes, obtained as described in Task 2. Cell samples will be quenched using cold methanol. After protein precipitation, metabolites will be extracted and analysed by liquid chromatography mass spectrometry (LC-MS). Hydrophilic interaction chromatography (HILIC) will be used to resolve the polar metabolome before MS detection using an Agilent 6546 lc/q-tof instrument (Agilent).The whole protein content will be quantified with NanoDrop™ (Thermofisher) and used to normalize the metabolic profile of each sample.
Time frame: Baseline
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