Mitokondriális metabolizmus és oxidatív stressz a fizikai gyakorlat és táplálkozási tanácsadás funkciójában T2DM-ben (MITOX)

Nincs beavatkozás: ND-INAKT

Azok a résztvevők, akik nem kaptak sem táplálkozási beavatkozást, sem testmozgási programot. Utasítást kaptak, hogy tartsák meg szokásos életmódjukat a fizikai aktivitás és az étrend tekintetében.

Aktív összehasonlító: Közepes intenzitású folyamatos edzés (ND-MICT)

Azok a résztvevők, akik nem kaptak táplálkozási intervenciót, de beiratkoztak egy közepes intenzitású folyamatos edzésprogramba.

Egyéb: A közepes intenzitású folyamatos edzés (MICT)

A MICT-hez rendelt résztvevők heti 3 edzést végeztek, 1-2 nap pihenővel az edzések között, egy kerékpár ergométeren 12 hétig.

A MICT 50 perces folyamatos pedálozásból állt, körülbelül 10%-kal a laktátküszöb feletti intenzitással, 60-80 rpm-es fordulatszámmal. A terhelést (TL) 10%-kal növelték, ha ez a két feltétel teljesült: 1) nem történt legalább 2 ütés/min szívfrekvencia-növekedés az edzés 20. és 40. perce között, és 2) a laktátszint 2 mmol/L alatt maradt az edzés 48. percében. Az első edzés TL-jét egy beállítási teszt határozta meg, amely egy inkrementális tesztből állt öt egymást követő 10 perces fázissal állandó fordulatszámon (60-80 RPM) a ventilációs küszöb teljesítményének 90%-ától 130%-áig, amelyet korábban elvégzett FatOx teszt adataiból számítottak ki, 10%-os lépésekben. A TL-t az a fázis határozta meg, amelyben a szívfrekvencia ≥ 3 ütéssel nőtt a fázisok között, és a vér laktátszintje ≥ 1 mmol/L-rel emelkedett az előző fázishoz képest.

Aktív összehasonlító: Nagy intenzitású intervallum edzés (ND-HIIT)

A résztvevők, akik nem kaptak táplálkozási intervenciót, de beiratkoztak egy nagy intenzitású intervallum edzésprogramba.

Egyéb: 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.

Aktív összehasonlító: Táplálkozási intervenció (D-INACT)

A résztvevők, akik táplálkozási beavatkozásban részesültek, de nem vettek részt edzésprogramban.

Viselkedési: 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.

Kísérleti: Táplálkozási beavatkozás közepes intenzitású folyamatos edzés (D-MICT)

Azok a résztvevők, akik táplálkozási intervencióban részesültek és egy mérsékelt intenzitású folyamatos edzésprogramba jelentkeztek be.

Egyéb: A közepes intenzitású folyamatos edzés (MICT)

A MICT-hez rendelt résztvevők heti 3 edzést végeztek, 1-2 nap pihenővel az edzések között, egy kerékpár ergométeren 12 hétig.

A MICT 50 perces folyamatos pedálozásból állt, körülbelül 10%-kal a laktátküszöb feletti intenzitással, 60-80 rpm-es fordulatszámmal. A terhelést (TL) 10%-kal növelték, ha ez a két feltétel teljesült: 1) nem történt legalább 2 ütés/min szívfrekvencia-növekedés az edzés 20. és 40. perce között, és 2) a laktátszint 2 mmol/L alatt maradt az edzés 48. percében. Az első edzés TL-jét egy beállítási teszt határozta meg, amely egy inkrementális tesztből állt öt egymást követő 10 perces fázissal állandó fordulatszámon (60-80 RPM) a ventilációs küszöb teljesítményének 90%-ától 130%-áig, amelyet korábban elvégzett FatOx teszt adataiból számítottak ki, 10%-os lépésekben. A TL-t az a fázis határozta meg, amelyben a szívfrekvencia ≥ 3 ütéssel nőtt a fázisok között, és a vér laktátszintje ≥ 1 mmol/L-rel emelkedett az előző fázishoz képest.

Viselkedési: 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.

Kísérleti: Táplálkozási intervenció Nagy intenzitású intervallum edzés (D-HIIT)

A résztvevők, akik táplálkozási beavatkozásban részesültek és beiratkoztak egy nagy intenzitású intervallum edzésprogramba.

Viselkedési: 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.

Egyéb: 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.

Áttekintett oxidatív stressz változások

Az oxidatív stressz szintjét a DNS-oxidáció biomarkerének, a 8-OHdG koncentrációjával értékeltük. Versenyképes ELISA érzékelési módszert alkalmaztunk a szérummintákban. Röviden: a standardot és a mintát hozzáadtuk egy előre bevont mikrocitoplathoz a biotinilezett érzékelő antitesttel együtt. Mosás után HRP-Streptavidint adtunk hozzá. Ezután hozzáadtuk a TMB szubsztitútot, és a reakciót savas oldattal leállítottuk. A színképződést spektrofotometriailag mértük 450 nm hullámhosszon. A minták optikai sűrűségi értékeit a standard görbe értékeivel hasonlítottuk össze a 8-OHdG koncentráció (ng/ml) meghatározásához.

Izombiopszia minta: Citrát Szintáz aktivitás

2-5 mg fagyasztott izmot homogenizáltunk 1:20 hígítású CelLytic™ MT szöveti lízissel (C3228, Sigma-Aldrich, Dorset, UK), 1 %-os proteáz gátló koktéllal (P8340, Sigma-Aldrich, Dorset, UK) 4 °C-on. A lizátumot 12 000 × g centrifugálással 10 percig 4 °C-on tisztítottuk, a lebegő részecskéket kicsapva, majd a szupernatánst -80 °C-on tároltuk. A CS aktivitást duplikátumban kvantifikáltuk egy tesztkészlet segítségével a gyártó utasításait követve (CS0720: Sigma-Aldrich, Dorset, UK). Röviden, az endogén aktivitást a DTNB által kibocsátott színen keresztül rögzítettük, és a teljes aktivitást az oxalacetát hozzáadása után számítottuk ki. A fehérjekoncentrációt duplikátumban határoztuk meg egy bicinkoninsav alapú tesztkészlettel (BCA1: Sigma-Aldrich, Dorset, UK), lehetővé téve a CS aktivitási értékek normalizálását a teljes fehérjetartalomra (µmol/g/perc). A CS aktivitást és a fehérjekoncentrációt 96-os mikrotányérban mértük mikrotányér olvasóval 412 nm-en, illetve 562 nm-en (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.

Értékelt inzulinrezisztencia változások: HOMA-IR

Az inzulinrezisztenciát a Homeosztatikus Modell Az Inzulinrezisztencia Felmérésére (HOMA-IR) használatával becsülték, amelyet úgy számítottak ki, hogy a születés utáni inzulin (µU/mL) szorozva a születés utáni glükózzal (mg/dL) osztva 405-tel.

Értékelt inzulin érzékenység változások: Matsuda Index

A teljes test inzulinérzékenységét a Matsuda-index alapján becsülték meg, amelyet a következő képlettel számítottak ki: 10 000 / √[(ehőmérsékleti glükóz (mg/dL) × ehőmérsékleti inzulin (µU/mL)) × (átlagos glükóz a szájon át történő glükóztolerancia-teszt során (mg/dL) × átlagos inzulin a szájon át történő glükóztolerancia-teszt során (µU/mL))].

Felmért változások az inzulinérzékenységben: Quicki

Az inzulinérzékenységet a Kvantitatív Inzulin Érzékenységi Ellenőrző Index (QUICKI) segítségével becsülték meg, amelyet 1 / [log(éhomi inzulin (µU/mL)) + log(éhomi glükóz (mg/dL))] képlettel számítottak ki.

A testtömegindex változásainak értékelése

A testtömegindex (BMI) kilogramm per négyzetméterben (kg/m²) a testtömeg (kg) osztva a magasság négyzetével (m²) alapján számítva.

Értékelt antropometriai változások: Kerületek

A derékbőséget (cm) a 10. borda alsó oldalsó szélét és a csípőcsont gerincét összekötő vonal középpontjában mérték, a törzs hossztengelyére merőlegesen. A haskörfogatot (cm) a köldök szintjén mérték, a törzs hossztengelyére merőlegesen. A csípőbőséget (cm) a fenék legnagyobb hátsó kiemelkedésének szintjén mérték, a törzs hossztengelyére merőlegesen. A combkörfogatot (cm) a nagy csípőcsont dudor és a külső sípcsont kondilus közötti vonal középpontjában mérték. Minden mérést a résztvevő egyenesen álló, ellazult helyzetében végezték. Minden mérést kétszer végeztek el, és harmadszori mérés történt, ha az eltérés meghaladta az 1%-ot. A két leginkább hasonló rekord átlagát rögzítették.

Felmért változások az antropometriában: derék-csípő arány

A derék-csípő arány (WHR) a derék kerület (cm) és a csípő kerület (cm) hányadosaként lett kiszámítva.

Megmért magasság

A magasságot (méter, m) egy falra szerelt sztadiométerrel (TANITA-LEICESTER HR-001, Tanita Corp., Tokió, Japán) mérték, a résztvevők a gyártó utasításainak megfelelően állva.

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).