Mitochondriale stofwisseling en oxidatieve stress in functie van de fysieke inspanning en voedingsadvies bij T2DM (MITOX)

Geen tussenkomst: ND-INACT

Deelnemers die geen van beide voedingsinterventies of bewegingsprogramma's ontvingen. Zij kregen de instructie om hun normale levensgewoonten met betrekking tot fysieke activiteit en voeding te behouden.

Actieve vergelijker: Matige intensiteit continue training (ND-MICT)

Deelnemers die geen voedingsinterventie ontvingen, maar waren ingeschreven in een matig-intensief continu trainingsbewegingsprogramma.

Ander: De Matig-Intensieve Continue Training (MICT)

Deelnemers die aan de MICT werden toegewezen, voltooiden 3 wekelijkse sessies, met 1-2 dagen rust tussen de sessies, op een fietsergometer gedurende 12 weken.

MICT bestond uit 50 minuten continu trappen met een intensiteit van ongeveer 10% boven de lactaatdrempel, met een cadans tussen 60-80 rpm. De trainingsbelasting (TL) werd met 10% verhoogd als aan deze twee voorwaarden werd voldaan: 1) geen toename van ten minste 2 hartslagen van minuut 20 tot minuut 40 van de trainingssessie, en 2) lactaatniveaus onder 2 mmol/L in minuut 48 van de sessie. De TL voor de eerste sessie werd bepaald door een aanpassingstest bestaande uit een incrementele test met vijf opeenvolgende 10-minuten fasen bij een constante cadans (60-80 RPM) van 90% tot 130% van het vermogen bij de ventilatoire drempel, berekend uit eerder uitgevoerde FatOx-testgegevens, met 10% verhogingen in elke stap. TL werd bepaald door de fase waarin de HR toenam met ≥ 3 slagen tussen fasen en bloedlactaatniveaus toenamen met ≥ 1 mmol/L ten opzichte van de vorige fase.

Actieve vergelijker: Hoge intensiteit intervaltraining (ND-HIIT)

Deelnemers die geen voedingsinterventie kregen, maar wel ingeschreven waren in een hoog-intensieve intervaltraining oefenprogramma.

Ander: 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.

Actieve vergelijker: Voedingsinterventie (D-INACT)

Deelnemers die een voedingsinterventie ontvingen maar geen bewegingsprogramma.

Gedragsmatig: 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.

Experimenteel: Voedingsinterventie Matig-intensieve continue training (D-MICT)

Deelnemers die een voedingsinterventie ontvingen en werden ingeschreven in een matig-intensief continu trainingsprogramma.

Ander: De Matig-Intensieve Continue Training (MICT)

Deelnemers die aan de MICT werden toegewezen, voltooiden 3 wekelijkse sessies, met 1-2 dagen rust tussen de sessies, op een fietsergometer gedurende 12 weken.

MICT bestond uit 50 minuten continu trappen met een intensiteit van ongeveer 10% boven de lactaatdrempel, met een cadans tussen 60-80 rpm. De trainingsbelasting (TL) werd met 10% verhoogd als aan deze twee voorwaarden werd voldaan: 1) geen toename van ten minste 2 hartslagen van minuut 20 tot minuut 40 van de trainingssessie, en 2) lactaatniveaus onder 2 mmol/L in minuut 48 van de sessie. De TL voor de eerste sessie werd bepaald door een aanpassingstest bestaande uit een incrementele test met vijf opeenvolgende 10-minuten fasen bij een constante cadans (60-80 RPM) van 90% tot 130% van het vermogen bij de ventilatoire drempel, berekend uit eerder uitgevoerde FatOx-testgegevens, met 10% verhogingen in elke stap. TL werd bepaald door de fase waarin de HR toenam met ≥ 3 slagen tussen fasen en bloedlactaatniveaus toenamen met ≥ 1 mmol/L ten opzichte van de vorige fase.

Gedragsmatig: 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.

Experimenteel: Nutritionele interventie High-intensity interval training (D-HIIT)

Deelnemers die een voedingsinterventie ontvingen en waren ingeschreven in een hoog-intensief intervaltraining oefenprogramma.

Gedragsmatig: 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.

Ander: 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.

Beoordeelde veranderingen in oxidatieve stress

Het niveau van oxidatieve stress werd beoordeeld door de concentratie van 8-OHdG als biomarker van DNA-oxidatie. Een competitieve ELISA-detectiemethode werd toegepast in serummonsters. Kort samengevat werden de standaard en het monster toegevoegd aan een vooraf gecoate microplaat samen met het gebiotinyleerde detectieantilichaam. Na het wassen werd HRP-Streptavidine toegevoegd. Vervolgens werd het TMB-substraat toegevoegd en werd de reactie gestopt met een zuuroplossing. De kleurontwikkeling werd spectrofotometrisch gemeten bij een golflengte van 450nm. De optische dichtheidswaarden van de monsters werden vergeleken met die van de standaardcurve om de 8-OHdG-concentratie (ng/ml) te bepalen.

Spierbiopt monster: Citraat Synthase activiteit

2-5 mg bevroren spier werd gehomogeniseerd in een 1:20 verdunning van CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, VK), met 1% protease-inhibitorencocktail (P8340, Sigma-Aldrich, Dorset, VK) bij 4 °C. Het lysaat werd gecentrifugeerd bij 12.000 × g gedurende 10 minuten bij 4 °C om debris te pelletten, en het supernatant werd opgeslagen bij -80 °C. CS-activiteit werd in duplo gekwantificeerd met behulp van een assaykit volgens de instructies van de fabrikant (CS0720: Sigma-Aldrich, Dorset, VK). Kort samengevat werd endogene activiteit geregistreerd via kleuremissie door DTNB, en totale activiteit werd berekend na toevoeging van oxaloacetaat. Eiwitconcentratie werd in duplo bepaald met behulp van een bicinchoninezuur assaykit (BCA1: Sigma-Aldrich, Dorset, VK), waardoor normalisatie van CS-activiteitswaarden naar totale eiwitinhoud mogelijk werd (µmol/g/min). CS-activiteit en eiwitconcentratie werden gemeten in 96-wells platen met behulp van een microplatenlezer bij respectievelijk 412 nm en 562 nm (MQX200R2, Biotek instruments, Burlington, VS).

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.

Beoordeelde veranderingen in insulineresistentie: HOMA-IR

Insulineresistentie werd geschat met behulp van de Homeostatische Modelbeoordeling voor Insulineresistentie (HOMA-IR), berekend als nuchtere insuline (µU/mL) vermenigvuldigd met nuchtere glucose (mg/dL) gedeeld door 405.

Beoordeelde veranderingen in Insulinegevoeligheid: Matsuda-index

De algehele insulinegevoeligheid van het lichaam werd geschat via de Matsuda-index, berekend als 10.000 / √[(nuchtere glucose (mg/dL) × nuchtere insuline (µU/mL)) × (gemiddelde glucose tijdens orale glucosetolerantietest (mg/dL) × gemiddelde insuline tijdens orale glucosetolerantietest (µU/mL))].

Beoordeelde veranderingen in insulinegevoeligheid: Quicki

Insulinegevoeligheid werd geschat via de Kwantitatieve Insulinegevoeligheidscontrole-index (QUICKI), berekend als 1 / [log(nuchtere insuline (µU/mL)) + log(nuchtere glucose (mg/dL))].

Beoordeelde veranderingen in Body Mass Index

Body Mass Index (BMI) in kilogram per vierkante meter (kg/m²) werd berekend als lichaamsgewicht (kg) gedeeld door lengte in het kwadraat (m²).

Beoordeelde veranderingen in Antropometrie: Omtrekken

De middelomtrek (cm) werd gemeten op het midden tussen de onderste laterale rand van de 10e rib en de iliacale kam, loodrecht op de longitudinale as van de romp. De buikomtrek (cm) werd gemeten ter hoogte van de navel, loodrecht op de longitudinale as van de romp. De heupomtrek (cm) werd gemeten ter hoogte van de grootste posterieure uitstulping van de billen, loodrecht op de longitudinale as van de romp. De dijomtrek (cm) werd gemeten op het midden tussen de grote trochanter en de laterale tibiale condylus. Alle metingen werden uitgevoerd met de deelnemer rechtopstaand en ontspannen. Elke meting werd tweemaal uitgevoerd, en een derde meting werd uitgevoerd als het verschil meer dan 1% bedroeg. Het gemiddelde van de twee meest vergelijkbare metingen werd geregistreerd.

Beoordeelde veranderingen in Antropometrie: Taille-heupverhouding

Taille-heupverhouding (THV) werd berekend als tailleomtrek (cm) gedeeld door heupomtrek (cm).

Gemeten Lengte

Lengte (meters, m) werd gemeten met een aan de wand gemonteerde stadiometer (TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Japan) met deelnemers staand in overeenstemming met de instructies van de fabrikant.

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).