Metabolizm mitochondrialny i stres oksydacyjny w funkcji ćwiczeń fizycznych i poradnictwa żywieniowego w T2DM (MITOX)

Brak interwencji: ND-INAKT

Uczestnicy, którzy nie otrzymali ani interwencji żywieniowej, ani programu ćwiczeń. Zostali poinstruowani, aby utrzymać swoje normalne nawyki życiowe dotyczące aktywności fizycznej i diety.

Aktywny komparator: Trening o umiarkowanej intensywności w trybie ciągłym (ND-MICT)

Uczestnicy, którzy nie otrzymali interwencji żywieniowej, ale zostali zapisani do programu ćwiczeń o umiarkowanej intensywności w formie ciągłego treningu.

Inny: Trening ciągły o umiarkowanej intensywności (MICT)

Uczestnicy przydzieleni do grupy MICT wykonywali 3 sesje treningowe tygodniowo, z 1-2 dniami odpoczynku między sesjami, na ergometrze rowerowym przez 12 tygodni.

MICT składał się z 50 minut ciągłego pedałowania o intensywności około 10% powyżej progu mleczanowego, z kadencją między 60-80 obr/min. Obciążenie treningowe (TL) zwiększano o 10%, jeśli spełnione były dwa warunki: 1) brak wzrostu tętna o co najmniej 2 uderzenia od 20. do 40. minuty sesji treningowej oraz 2) poziom mleczanów poniżej 2 mmol/L w 48. minucie sesji. TL dla pierwszej sesji określono za pomocą testu dostosowawczego, składającego się z testu przyrostowego z pięcioma kolejnymi 10-minutowymi fazami przy stałej kadencji (60-80 obr/min) od 90% do 130% mocy progowej wentylacyjnej, obliczonej na podstawie wcześniej wykonanych danych testu FatOx, z 10% przyrostami na każdym etapie. TL określano na podstawie fazy, w której tętno wzrosło o ≥ 3 uderzeń między fazami, a poziom mleczanu we krwi wzrósł o ≥ 1 mmol/L w porównaniu z poprzednią fazą.

Aktywny komparator: Trening interwałowy o wysokiej intensywności (ND-HIIT)

Uczestnicy, którzy nie otrzymali interwencji żywieniowej, ale zostali zapisani do programu ćwiczeń interwałowych o wysokiej intensywności.

Inny: 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.

Aktywny komparator: Interwencja żywieniowa (D-INACT)

Uczestnicy, którzy otrzymali interwencję żywieniową, ale nie program ćwiczeń.

Behawioralne: 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.

Eksperymentalny: Interwencja żywieniowa Trening ciągły o umiarkowanej intensywności (D-MICT)

Uczestnicy, którzy otrzymali interwencję żywieniową i zostali zapisani do programu ćwiczeń z umiarkowaną intensywnością treningu ciągłego.

Inny: Trening ciągły o umiarkowanej intensywności (MICT)

Uczestnicy przydzieleni do grupy MICT wykonywali 3 sesje treningowe tygodniowo, z 1-2 dniami odpoczynku między sesjami, na ergometrze rowerowym przez 12 tygodni.

MICT składał się z 50 minut ciągłego pedałowania o intensywności około 10% powyżej progu mleczanowego, z kadencją między 60-80 obr/min. Obciążenie treningowe (TL) zwiększano o 10%, jeśli spełnione były dwa warunki: 1) brak wzrostu tętna o co najmniej 2 uderzenia od 20. do 40. minuty sesji treningowej oraz 2) poziom mleczanów poniżej 2 mmol/L w 48. minucie sesji. TL dla pierwszej sesji określono za pomocą testu dostosowawczego, składającego się z testu przyrostowego z pięcioma kolejnymi 10-minutowymi fazami przy stałej kadencji (60-80 obr/min) od 90% do 130% mocy progowej wentylacyjnej, obliczonej na podstawie wcześniej wykonanych danych testu FatOx, z 10% przyrostami na każdym etapie. TL określano na podstawie fazy, w której tętno wzrosło o ≥ 3 uderzeń między fazami, a poziom mleczanu we krwi wzrósł o ≥ 1 mmol/L w porównaniu z poprzednią fazą.

Behawioralne: 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.

Eksperymentalny: Interwencja żywieniowa Trening interwałowy o wysokiej intensywności (D-HIIT)

Uczestnicy, którzy otrzymali interwencję żywieniową i zostali zapisani na program ćwiczeń interwałowych o wysokiej intensywności.

Behawioralne: 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.

Inny: 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.

Ocenione zmiany w stresie oksydacyjnym

Poziom stresu oksydacyjnego oceniano na podstawie stężenia 8-OHdG jako biomarkera utleniania DNA. W próbkach surowicy zastosowano konkurencyjną metodę wykrywania ELISA. W skrócie, standard i próbkę dodano do wstępnie pokrytej mikropłytki wraz z biotynylowanym przeciwciałem detekcyjnym. Po przemyciu dodano HRP-Streptawidynę. Następnie dodano substrat TMB, a reakcję zatrzymano za pomocą roztworu kwasowego. Produkcję koloru mierzono spektrofotometrycznie przy długości fali 450 nm. Wartości gęstości optycznej próbek porównano z wartościami krzywej standardowej w celu określenia stężenia 8-OHdG (ng/ml).

Próbka biopsji mięśnia: aktywność syntazy cytrynianowej

2-5 mg zamrożonego mięśnia homogenizowano w rozcieńczeniu 1:20 CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, Wielka Brytania) z 1% koktajlem inhibitorów proteaz (P8340, Sigma-Aldrich, Dorset, Wielka Brytania) w temperaturze 4 °C. Lizat odwirowywano przy 12 000 × g przez 10 minut w temperaturze 4 °C w celu osadzenia pozostałości, a supernatant przechowywano w temperaturze -80 °C. Aktywność CS oznaczano w duplikatach przy użyciu zestawu testowego zgodnie z instrukcjami producenta (CS0720: Sigma-Aldrich, Dorset, Wielka Brytania). W skrócie, aktywność endogenną rejestrowano poprzez emisję barwy przez DTNB, a całkowitą aktywność obliczano po dodaniu szczawiooctanu. Stężenie białka oznaczano w duplikatach przy użyciu zestawu testowego kwasu biureinowego (BCA1: Sigma-Aldrich, Dorset, Wielka Brytania), umożliwiając normalizację wartości aktywności CS do całkowitej zawartości białka (µmol/g/min). Aktywność CS i stężenie białka mierzono w płytkach 96-dołkowych przy użyciu czytnika mikropłytek odpowiednio przy 412 nm i 562 nm (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.

Ocena zmian w insulinooporności: HOMA-IR

Oporność na insulinę szacowano za pomocą modelu oceny homeostazy oporności na insulinę (HOMA-IR), obliczanego jako insulina na czczo (µU/mL) pomnożona przez glukozę na czczo (mg/dL) podzielona przez 405.

Ocena zmian w wrażliwości na insulinę: wskaźnik Matsudy

Wrażliwość na insulinę całego ciała została oszacowana za pomocą wskaźnika Matsudy, obliczanego jako 10 000 / √[(glukoza na czczo (mg/dL) × insulina na czczo (µU/mL)) × (średnia glukoza podczas doustnego testu tolerancji glukozy (mg/dL) × średnia insulina podczas doustnego testu tolerancji glukozy (µU/mL))].

Ocenione zmiany w wrażliwości na insulinę: Quicki

Wrażliwość na insulinę została oszacowana za pomocą Wskaźnika Ilościowej Kontroli Wrażliwości na Insulinę (QUICKI), obliczanego jako 1 / [log(insulina na czczo (µU/mL)) + log(glukoza na czczo (mg/dL))].

Ocenione zmiany wskaźnika masy ciała

Wskaźnik masy ciała (BMI) w kilogramach na metr kwadratowy (kg/m²) został obliczony jako masa ciała (kg) podzielona przez wzrost do kwadratu (m²).

Ocena zmian w antropometrii: Obwody

Obwód talii (cm) mierzono w punkcie środkowym między dolnym bocznym brzegiem 10 żebra a grzebieniem kości biodrowej, prostopadle do podłużnej osi tułowia. Obwód brzucha (cm) mierzono na poziomie pępka, prostopadle do podłużnej osi tułowia. Obwód bioder (cm) mierzono na poziomie największego tylnego uwypuklenia pośladków, prostopadle do podłużnej osi tułowia. Obwód uda (cm) mierzono w punkcie środkowym między krętarzem większym a bocznym kłykciem kości piszczelowej. Wszystkie pomiary wykonano z uczestnikiem stojącym wyprostowanym i rozluźnionym. Każdy pomiar wykonano dwukrotnie, a trzeci pomiar przeprowadzono, jeśli różnica przekroczyła 1%. Zarejestrowano średnią z dwóch najbardziej zbliżonych wyników.

Ocena zmian w antropometrii: stosunek talii do bioder

Wskaźnik talia-biodro (WHR) był obliczany jako obwód talii (cm) podzielony przez obwód bioder (cm).

Zmierzony wzrost

Wysokość (metry, m) mierzono za pomocą stadiometru ściennego (TANITA-LEICESTER HR-001, Tanita Corp., Tokio, Japonia), przy czym uczestnicy stali zgodnie z instrukcjami producenta.

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).