Mitochondriální metabolismus a oxidační stres ve funkci fyzického cvičení a nutričního poradenství u T2DM (MITOX)

Žádný zásah: ND-NEAKTIVNÍ

Účastníci, kteří neobdrželi ani nutriční intervenci, ani cvičební program. Bylo jim nařízeno zachovat si své běžné životní návyky týkající se fyzické aktivity a stravy.

Aktivní komparátor: Trénink střední intenzity s nepřetržitým zatížením (ND-MICT)

Účastníci, kteří neobdrželi nutriční intervenci, ale byli zařazeni do programu cvičení s mírnou intenzitou a kontinuálním tréninkem.

Jiný: Trénink střední intenzity s nepřetržitým zatížením (MICT)

Účastníci zařazení do skupiny MICT absolvovali 3 týdenní tréninkové jednotky s 1–2 dny odpočinku mezi jednotkami na cyklistickém ergometru po dobu 12 týdnů.

MICT sestával z 50 minut kontinuálního šlapání při intenzitě přibližně 10 % nad laktátovým prahem s kadencí mezi 60–80 ot./min. Tréninková zátěž (TL) byla zvýšena o 10 %, pokud byly splněny tyto dvě podmínky: 1) žádné zvýšení srdeční frekvence o alespoň 2 údery od 20. do 40. minuty tréninkové jednotky a 2) hladiny laktátu pod 2 mmol/l ve 48. minutě jednotky. TL pro první tréninkovou jednotku byla stanovena pomocí adjustačního testu sestávajícího z inkrementálního testu s pěti po sobě jdoucími 10minutovými fázemi při konstantní kadencji (60–80 ot./min) od 90 % do 130 % výkonu ventilačního prahu, vypočteného z dříve provedených údajů testu FatOx, s 10% přírůstky v každém kroku. TL byla stanovena fází, ve které se SF zvýšila o ≥ 3 údery mezi fázemi a hladiny laktátu v krvi vzrostly o ≥ 1 mmol/l oproti předchozí fázi.

Aktivní komparátor: Vysoko intenzivní intervalový trénink (ND-HIIT)

Účastníci, kteří neobdrželi nutriční intervenci, ale byli zařazeni do vysoce intenzivního intervalového tréninkového cvičebního programu.

Jiný: 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.

Aktivní komparátor: Nutriční intervence (D-INACT)

Účastníci, kteří obdrželi nutriční intervenci, ale nikoli cvičební program.

Behaviorální: 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.

Experimentální: Nutriční intervence Středně intenzivní kontinuální trénink (D-MICT)

Účastníci, kteří obdrželi nutriční intervenci a byli zařazeni do programu cvičení s mírnou intenzitou kontinuálního tréninku.

Jiný: Trénink střední intenzity s nepřetržitým zatížením (MICT)

Účastníci zařazení do skupiny MICT absolvovali 3 týdenní tréninkové jednotky s 1–2 dny odpočinku mezi jednotkami na cyklistickém ergometru po dobu 12 týdnů.

MICT sestával z 50 minut kontinuálního šlapání při intenzitě přibližně 10 % nad laktátovým prahem s kadencí mezi 60–80 ot./min. Tréninková zátěž (TL) byla zvýšena o 10 %, pokud byly splněny tyto dvě podmínky: 1) žádné zvýšení srdeční frekvence o alespoň 2 údery od 20. do 40. minuty tréninkové jednotky a 2) hladiny laktátu pod 2 mmol/l ve 48. minutě jednotky. TL pro první tréninkovou jednotku byla stanovena pomocí adjustačního testu sestávajícího z inkrementálního testu s pěti po sobě jdoucími 10minutovými fázemi při konstantní kadencji (60–80 ot./min) od 90 % do 130 % výkonu ventilačního prahu, vypočteného z dříve provedených údajů testu FatOx, s 10% přírůstky v každém kroku. TL byla stanovena fází, ve které se SF zvýšila o ≥ 3 údery mezi fázemi a hladiny laktátu v krvi vzrostly o ≥ 1 mmol/l oproti předchozí fázi.

Behaviorální: 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.

Experimentální: Nutriční intervence Vysokointenzivní intervalový trénink (D-HIIT)

Účastníci, kteří obdrželi nutriční intervenci a byli zařazeni do cvičebního programu s intervalovým tréninkem s vysokou intenzitou.

Behaviorální: 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.

Jiný: 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.

Posouzené změny oxidačního stresu

Hladina oxidačního stresu byla hodnocena koncentrací 8-OHdG jako biomarkeru oxidace DNA. K detekci byla použita kompetitivní ELISA metoda v sérových vzorcích. Stručně řečeno, standard a vzorek byly přidány do předem potažené mikrotitrační destičky spolu s biotinylovanou detekční protilátkou. Po promytí byl přidán HRP-Streptavidin. Poté byl přidán TMB substrát a reakce byla zastavena kyselým roztokem. Barevná produkce byla měřena spektrofotometricky při vlnové délce 450 nm. Hodnoty optické hustoty vzorků byly porovnány s hodnotami standardní křivky pro stanovení koncentrace 8-OHdG (ng/ml).

Vzorek svalové biopsie: Aktivita citrát syntázy

2-5 mg zmraženého svalu bylo homogenizováno v 1:20 ředění CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, UK) s 1 % koktejlem inhibitorů proteáz (P8340, Sigma-Aldrich, Dorset, UK) při 4 °C. Lyzát byl centrifugován při 12 000 × g po dobu 10 minut při 4 °C k sedimentaci úlomků a supernatant byl skladován při -80 °C. Aktivita CS byla kvantifikována ve dvou opakováních pomocí testovací sady podle pokynů výrobce (CS0720: Sigma-Aldrich, Dorset, UK). Stručně řečeno, endogenní aktivita byla zaznamenána prostřednictvím barevné emise DTNB a celková aktivita byla vypočtena po přidání oxalacetátu. Koncentrace proteinu byla stanovena ve dvou opakováních pomocí testovací sady s kyselinou bicinchoninovou (BCA1: Sigma-Aldrich, Dorset, UK), což umožnilo normalizaci hodnot aktivity CS na celkový obsah proteinu (µmol/g/min). Aktivita CS a koncentrace proteinu byly měřeny v 96-jamkových destičkách pomocí mikrodestičkového čtečky při 412 nm a 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.

Posouzené změny inzulinové rezistence: HOMA-IR

Inzulinová rezistence byla odhadnuta pomocí Homeostatického modelu pro hodnocení inzulinové rezistence (HOMA-IR), vypočteného jako inzulin nalačno (µU/ml) vynásobený glukózou nalačno (mg/dl) děleno 405.

Posouzené změny inzulinové senzitivity: Matsudův index

Celková tělesná citlivost na inzulin byla odhadnuta pomocí Matsuda indexu, vypočítaného jako 10 000 / √[(hladina glukózy nalačno (mg/dL) × hladina inzulinu nalačno (µU/mL)) × (průměrná hladina glukózy při orálním glukózovém tolerančním testu (mg/dL) × průměrná hladina inzulinu při orálním glukózovém tolerančním testu (µU/mL))].

Posouzené změny inzulinové senzitivity: Quicki

Citlivost na inzulin byla odhadnuta pomocí Kvantitativního indexu kontroly citlivosti na inzulin (QUICKI), vypočteného jako 1 / [log(hladina inzulinu nalačno (µU/mL)) + log(hladina glukózy nalačno (mg/dL))].

Hodnocené změny v indexu tělesné hmotnosti

Index tělesné hmotnosti (BMI) v kilogramech na metr čtvereční (kg/m²) byl vypočítán jako tělesná hmotnost (kg) dělená výškou na druhou (m²).

Posouzené změny v antropometrii: Obvody

Obvod pasu (cm) byl měřen v polovině vzdálenosti mezi dolním postranním okrajem 10. žebra a hřebenem kosti kyčelní, kolmo k podélné ose trupu. Obvod břicha (cm) byl měřen v úrovni pupku, kolmo k podélné ose trupu. Obvod boků (cm) byl měřen v úrovni největšího zadního vyklenutí hýždí, kolmo k podélné ose trupu. Obvod stehna (cm) byl měřen v polovině vzdálenosti mezi velkým chocholíkem a postranním kondylem kosti holenní. Všechna měření byla provedena s účastníkem ve vzpřímeném stoji a v uvolněném stavu. Každé měření bylo provedeno dvakrát a třetí měření bylo provedeno, pokud rozdíl přesáhl 1 %. Byl zaznamenán průměr dvou nejpodobnějších záznamů.

Posouzené změny v antropometrii: Poměr pasu k bokům

Poměr pasu k bokům (WHR) byl vypočítán jako obvod pasu (cm) děleno obvodem boků (cm).

Posouzená výška

Výška (metry, m) byla měřena pomocí nástěnného stadiometru (TANITA-LEICESTER HR-001, Tanita Corp., Tokio, Japonsko) s účastníky stojícími v souladu s pokyny výrobce.

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).