Metabolismo Mitocondriale e Stress Ossidativo nella Funzione dell'Esercizio Fisico e del Counseling Nutrizionale nel Diabete di Tipo 2 (MITOX)

Nessun intervento: ND-INATTIVO

Partecipanti che non hanno ricevuto né l'intervento nutrizionale né il programma di esercizio. Sono stati istruiti a mantenere le loro normali abitudini di vita riguardo all'attività fisica e alla dieta.

Comparatore attivo: Allenamento continuo a intensità moderata (ND-MICT)

Partecipanti che non hanno ricevuto l'intervento nutrizionale ma sono stati arruolati in un programma di esercizio di allenamento continuo di intensità moderata.

Altro: L'Allenamento Continuo a Intensità Moderata (MICT)

I partecipanti assegnati al MICT hanno completato 3 sessioni settimanali, con 1-2 giorni di riposo tra le sessioni, su un cicloergometro per 12 settimane.

Il MICT consisteva in 50 minuti di pedalata continua a un'intensità di circa il 10% superiore alla soglia del lattato, con una cadenza tra 60-80 rpm. Il carico di allenamento (TL) è stato aumentato del 10% se queste due condizioni erano soddisfatte: 1) nessun aumento di almeno 2 battiti della frequenza cardiaca dal minuto 20 al minuto 40 della sessione di allenamento, e 2) livelli di lattato inferiori a 2 mmol/L al minuto 48 della sessione. Il TL per la prima sessione è stato determinato da un test di adattamento consistente in un test incrementale con cinque fasi consecutive di 10 minuti a una cadenza costante (60-80 RPM) dal 90% al 130% della potenza della soglia ventilatoria, calcolata dai dati del test FatOx precedentemente eseguito, con incrementi del 10% in ogni fase. Il TL è stato determinato dalla fase in cui la FC è aumentata di ≥ 3 battiti tra le fasi e i livelli di lattato nel sangue sono aumentati di ≥ 1 mmol/L rispetto alla fase precedente.

Comparatore attivo: Allenamento a intervalli ad alta intensità (ND-HIIT)

Partecipanti che non hanno ricevuto un intervento nutrizionale ma erano iscritti a un programma di esercizio ad alta intensità di interval training.

Altro: The High-Intensity Interval Training (HIIT)

Participants assigned to the HIIT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks.

The training load (TL) was determined from an incremental test until volitional exhaustion with one-minute work intervals (from 85% to 165% of maximal power output from a previously performed VO2max test [Wmax] and with 10% increments) separated by 1.5-minute rest periods (at 20% Wmax).

The program consisted of a 3 minutes warm-up with the first 2 minutes at 20% and last minute at 40% of the TL, followed by 10 series of 1 min duration at 90% of TL, with 1 minute of rest between sets at 20% of TL and ending with 2 minutes of cool-down at 20% of TL (estimated total time of the session: 25 minutes). A 5% increase in workload was applied when, for two consecutive sessions, the first three intervals failed to exceed 85%, and at least five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

Comparatore attivo: Intervento nutrizionale (D-INACT)

Partecipanti che hanno ricevuto un intervento nutrizionale ma non un programma di esercizi.

Comportamentale: Nutritional intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

Sperimentale: Intervento Nutrizionale Allenamento Continuo di Intensità Moderata (D-MICT)

Partecipanti che hanno ricevuto l'intervento nutrizionale e sono stati arruolati in un programma di esercizio di allenamento continuo di intensità moderata.

Altro: L'Allenamento Continuo a Intensità Moderata (MICT)

I partecipanti assegnati al MICT hanno completato 3 sessioni settimanali, con 1-2 giorni di riposo tra le sessioni, su un cicloergometro per 12 settimane.

Il MICT consisteva in 50 minuti di pedalata continua a un'intensità di circa il 10% superiore alla soglia del lattato, con una cadenza tra 60-80 rpm. Il carico di allenamento (TL) è stato aumentato del 10% se queste due condizioni erano soddisfatte: 1) nessun aumento di almeno 2 battiti della frequenza cardiaca dal minuto 20 al minuto 40 della sessione di allenamento, e 2) livelli di lattato inferiori a 2 mmol/L al minuto 48 della sessione. Il TL per la prima sessione è stato determinato da un test di adattamento consistente in un test incrementale con cinque fasi consecutive di 10 minuti a una cadenza costante (60-80 RPM) dal 90% al 130% della potenza della soglia ventilatoria, calcolata dai dati del test FatOx precedentemente eseguito, con incrementi del 10% in ogni fase. Il TL è stato determinato dalla fase in cui la FC è aumentata di ≥ 3 battiti tra le fasi e i livelli di lattato nel sangue sono aumentati di ≥ 1 mmol/L rispetto alla fase precedente.

Comportamentale: Nutritional intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

Sperimentale: Intervento nutrizionale Allenamento a intervalli ad alta intensità (D-HIIT)

Partecipanti che hanno ricevuto l'intervento nutrizionale e sono stati arruolati in un programma di esercizio ad intervalli ad alta intensità.

Comportamentale: Nutritional intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

Altro: The High-Intensity Interval Training (HIIT)

Participants assigned to the HIIT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks.

The training load (TL) was determined from an incremental test until volitional exhaustion with one-minute work intervals (from 85% to 165% of maximal power output from a previously performed VO2max test [Wmax] and with 10% increments) separated by 1.5-minute rest periods (at 20% Wmax).

The program consisted of a 3 minutes warm-up with the first 2 minutes at 20% and last minute at 40% of the TL, followed by 10 series of 1 min duration at 90% of TL, with 1 minute of rest between sets at 20% of TL and ending with 2 minutes of cool-down at 20% of TL (estimated total time of the session: 25 minutes). A 5% increase in workload was applied when, for two consecutive sessions, the first three intervals failed to exceed 85%, and at least five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

Valutazione delle variazioni dello Stress Ossidativo

Il livello di stress ossidativo è stato valutato mediante la concentrazione di 8-OHdG come biomarcatore dell'ossidazione del DNA. È stato utilizzato un metodo di rilevamento ELISA competitivo nei campioni di siero. In breve, lo standard e il campione sono stati aggiunti a una micropiastra pre-rivestita insieme all'anticorpo di rilevamento biotinilato. Dopo il lavaggio, è stato aggiunto HRP-Streptavidin. Quindi, è stato aggiunto il substrato TMB e la reazione è stata interrotta con una soluzione acida. La produzione di colore è stata misurata spettrofotometricamente a una lunghezza d'onda di 450nm. I valori di densità ottica dei campioni sono stati confrontati con quelli della curva standard per determinare la concentrazione di 8-OHdG (ng/ml).

Campione di biopsia muscolare: attività della citrato sintasi

2-5 mg di muscolo congelato sono stati omogeneizzati in una diluizione 1:20 di CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, UK), con 1% di cocktail di inibitori delle proteasi (P8340, Sigma-Aldrich, Dorset, UK) a 4 °C. Il lisato è stato centrifugato a 12.000 × g per 10 minuti a 4 °C per sedimentare i detriti, e il surnatante è stato conservato a -80 °C. L'attività CS è stata quantificata in duplicati utilizzando un kit di saggio seguendo le istruzioni del produttore (CS0720: Sigma-Aldrich, Dorset, UK). In breve, l'attività endogena è stata registrata attraverso l'emissione di colore da DTNB, e l'attività totale è stata calcolata dopo l'aggiunta di ossalacetato. La concentrazione proteica è stata determinata in duplicati utilizzando un kit di saggio con acido bicinconinico (BCA1: Sigma-Aldrich, Dorset, UK), consentendo la normalizzazione dei valori di attività CS al contenuto proteico totale (μmol/g/min). L'attività CS e la concentrazione proteica sono state misurate in piastre a 96 pozzetti utilizzando un lettore per micropiastre a 412 nm e 562 nm, rispettivamente (MQX200R2, Biotek instruments, Burlington, USA).

Muscle biopsy samples: mitochondrial respiratory capacity

Skin was locally anesthetized, followed by an incision of approximately 6-7mm on the dermal surface. Using sterilized Bergstrom needles, approximately 3-6 mg of skeletal muscle from the lateral vastus were extracted and placed in a BIOPS buffer for immediate analysis of mitochondrial respiration (MR) through high-resolution respirometry. Before, the fibers were mechanically separated in BIOPS buffer using sterilized needles and chemically permeabilized with a 50 μg/ml saponin solution.

MR was measured in a specific buffer (Mir05) in duplicate in hyperoxygenated chambers (250-450 nmol/mL of oxygen) in an Oxygraph-2k at 37ºC (Oroboros, Innsbruck, Austria). A substrate-uncoupler titration protocol was employed by adding (final concentration): malate (2mM), pyruvate (5mM), glutamate (2mM), MgCl2 (3mM), and ADP (5mM), cytochrome C (10μM), succinate (10mM), and p-trifluoromethoxyphenylhydrazone. Oxygen flux was presented as mass-specific (pmol·s/mg wet weight) and normalized to CS activity.

Valutazione delle variazioni della resistenza all'insulina: HOMA-IR

La resistenza all'insulina è stata stimata utilizzando il Modello di Valutazione Omeostatica della Resistenza all'Insulina (HOMA-IR), calcolato come insulina a digiuno (µU/mL) moltiplicata per glucosio a digiuno (mg/dL) diviso per 405.

Valutazione delle variazioni della sensibilità all'insulina: Indice di Matsuda

La sensibilità all'insulina dell'intero corpo è stata stimata attraverso l'Indice di Matsuda, calcolato come 10.000 / √[(glucosio a digiuno (mg/dL) × insulina a digiuno (µU/mL)) × (glucosio medio durante il Test di Tolleranza al Glucosio Orale (mg/dL) × insulina media durante il Test di Tolleranza al Glucosio Orale (µU/mL))].

Valutate le variazioni della sensibilità all'insulina: Quicki

La sensibilità all'insulina è stata stimata tramite l'Indice di Controllo Quantitativo della Sensibilità all'Insulina (QUICKI), calcolato come 1 / [log(insulina a digiuno (µU/mL)) + log(glucosio a digiuno (mg/dL))].

Valutate le variazioni dell'Indice di Massa Corporea

L'indice di massa corporea (BMI) in chilogrammi per metro quadrato (kg/m²) è stato calcolato come peso corporeo (kg) diviso per l'altezza al quadrato (m²).

Valutate le variazioni in Antropometria: Perimetri

La circonferenza della vita (cm) è stata misurata nel punto medio tra il margine laterale inferiore della decima costa e la cresta iliaca, perpendicolare all'asse longitudinale del tronco. La circonferenza addominale (cm) è stata misurata a livello dell'ombelico, perpendicolare all'asse longitudinale del tronco. La circonferenza dei fianchi (cm) è stata misurata a livello della maggiore sporgenza posteriore dei glutei, perpendicolare all'asse longitudinale del tronco. La circonferenza della coscia (cm) è stata misurata nel punto medio tra il grande trocantere e il condilo tibiale laterale. Tutte le misurazioni sono state eseguite con il partecipante in piedi, in posizione eretta e rilassato. Ogni misurazione è stata effettuata due volte, e una terza misurazione è stata eseguita se la differenza superava l'1%. È stata registrata la media dei due record più simili.

Modifiche valutate nell'antropometria: rapporto vita-fianchi

Il rapporto vita-fianchi (WHR) è stato calcolato come circonferenza vita (cm) divisa per circonferenza fianchi (cm).

Altezza valutata

L'altezza (metri, m) è stata misurata utilizzando uno stadiometro a parete (TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Giappone) con i partecipanti in piedi in conformità alle istruzioni del produttore.

Assessed changes in Glucose concentration

Glucose concentration (mg/dL) was measured from blood samples collected from the antecubital vein after at least 8 hours of fasting and at 30, 60, 90, 120, 150, and 180 minutes after the ingestion of 75 grams of glucose (Nuter-tec, 75 g/200 mL, orange flavor). Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine consumption for 24 hours, and avoid vigorous physical activity for at least 48 hours. Blood samples were centrifuged and plasma was analyzed using the colorimetric method performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).

Assessed changes in Insulin concentration

Insulin concentration (µU/mL) was measured from blood samples collected from the antecubital vein into serum separator tubes after at least 8 hours of fasting and at 30, 60, 90, 120, 150, and 180 minutes after the ingestion of 75 grams of glucose (Nuter-tec, 75 g/200 mL, orange flavor). Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine consumption for 24 hours, and avoid vigorous physical activity for at least 48 hours. Blood samples were centrifuged at 4000 rpm for 7 minutes, and serum was analyzed. Insulin concentrations were determined by chemiluminescent immunoassays performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).

Assessed changes in Tissue Oxygenation Index

A NIRO-200NX device was used to measure skeletal muscle tissue oxygenation index (%) through near-infrared spectroscopy (NIRS), the modified Beer-Lambert law (MBLL), and spatially resolved spectroscopy. The NIRS sensor (emitter-sensor distance of 3cm) was placed at two-thirds of the distance from the anterior superior iliac spine to the superolateral patellar border, over the vastus lateralis muscle belly. The NIRS data were continuously acquired throughout a resting energy expenditure test for 30 minutes, a maximal fat oxidation (MFO) test on a cycloergometer, and a maximal oxygen consumption (VO2max) test on a cycloergometer. Briefly, the MFO test consisted of an incremental task with 3-minute phases starting from 15W and increasing by 15W each phase until a respiratory exchange ratio of 1. After a 5-minute rest, the VO2max test was initiated at last MFO load with 15W increases each minute until VO2max determination.

Assessed changes in haemoglobin saturation delta

A NIRO-200NX device was used to measure skeletal muscle changes on oxygenated, desoxygenated, and total haemoglobin (μmol/L) through near-infrared spectroscopy (NIRS) and the modified Beer-Lambert law. The NIRS sensor (emitter-sensor distance of 3cm) was placed at two-thirds of the distance from the anterior superior iliac spine to the superolateral patellar border, over the vastus lateralis muscle belly. The NIRS data were continuously acquired throughout a resting energy expenditure test for 30 minutes, a maximal fat oxidation (MFO) test on a cycloergometer, and a maximal oxygen consumption (VO2max) test on a cycloergometer. Briefly, the MFO test consisted of an incremental task with 3-minute phases starting from 15W and increasing by 15W each phase until a respiratory exchange ratio of 1. After a 5-minute rest, the VO2max test was initiated at last MFO load with 15W increases each minute until VO2max determination.

Assessed changes in Body Weight

Body weight (kilograms, kg) was measured using a multifrequency bioelectrical impedance device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat Mass (kilograms of Fat Mass)

Fat mass (kilograms, kg) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat Mass (percentage of Fat Mass)

Fat mass (percentage of total body weight, %) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat-free Mass (kilograms of Fat-free Mass)

Fat-free mass (kilograms, kg) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat-free Mass (percentage of Fat-free Mass)

Fat-free mass (percentage of total body weight, %) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Anthropometry: Skinfold thickness

Thigh skinfold thickness was measured at two-thirds of the distance from the iliospinale point (the lowest part of the anterior superior iliac spine) to the superolateral border of the patella, over the belly of the vastus lateralis muscle. Measurements were taken on the dominant leg. Two measurements were obtained, and a third was taken if the difference between the first two exceeded 1mm. The mean of the two most similar records was recorded.

Assessed changes in interleukin-6

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum interleukin-6 (pg/mL) was measured by electrochemiluminescence immunoassay using a Cobas e 411 (Roche Diagnostics, Mannheim, Germany).

Assessed changes in C-reactive protein

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum C-reactive protein (mg/L) was determined by immunoturbidimetric assay on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in alanine aminotransferase

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum alanine aminotransferase (U/L) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in aspartate aminotransferase

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum aspartate aminotransferase (U/L) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in gamma-glutamyl transferase

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum gamma-glutamyl transferase (U/L) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in tumor necrosis factor-alpha

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® EDTA K2 separator tubes (Greiner Bio-One, Kremsmünster, Austria) to obtain plasma. Plasma was aliquoted into 500 µL tubes with 5 µL protease inhibitors, then stored at -80 °C. Plasma tumor necrosis factor-alpha (pg/mL) was measured using multiplex magnetic bead-based immunoassays (MILLIPLEX® Metabolic Hormone Panel V3, HMH3-34K-06, Merck Millipore, Darmstadt, Germany) and analyzed with a Luminex® xMap IntelliFlex System (Luminex Corp., Austin, TX, USA).

Assessed changes in triglycerides

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum triglycerides (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in total cholesterol

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum total cholesterol (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in high-density lipoprotein

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum high-density lipoprotein (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in low-density lipoprotein

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum low-density lipoprotein (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).