Mitokondriell metabolism och oxidativ stress i funktion av fysisk träning och nutritionsrådgivning vid T2DM (MITOX)

Inget ingripande: ND-INAKTIV

Deltagare som inte fick någon nutritionell intervention eller träningsprogram. De fick instruktioner att behålla sina vanliga levnadsvanor när det gäller fysisk aktivitet och kost.

Aktiv komparator: Måttlig intensitet kontinuerlig träning (ND-MICT)

Deltagare som inte fick någon näringsintervention men som deltog i ett moderatintensivt kontinuerligt träningsprogram.

Övrig: Moderatintensiv kontinuerlig träning (MICT)

Deltagare som tilldelades MICT genomförde 3 veckovisa sessioner, med 1-2 dagars vila mellan sessionerna, på en cykelergometer i 12 veckor.

MICT bestod av 50 minuter av kontinuerlig trampning med en intensitet på cirka 10 % över laktattröskeln, med en kadens mellan 60-80 rpm. Träningsbelastningen (TL) ökades med 10 % om dessa två villkor uppfylldes: 1) ingen ökning av minst 2 hjärtslag från minut 20 till minut 40 av träningssessionen, och 2) laktatnivåer under 2 mmol/L i minut 48 av sessionen. TL för den första sessionen bestämdes av ett justeringstest som bestod av ett stegvis ökande test med fem på varandra följande 10-minutersfaser vid en konstant kadens (60-80 RPM) från 90 % till 130 % av den ventilatoriska tröskeleffekten, beräknad från tidigare utförda FatOx-testdata, med 10 % ökningar i varje steg. TL bestämdes av fasen där HR ökade med ≥ 3 slag mellan faser och blodlaktatnivåer ökade med ≥ 1 mmol/L från föregående fas.

Aktiv komparator: Högintensiv intervallträning (ND-HIIT)

Deltagare som inte fick någon nutritionell intervention men som deltog i ett träningsprogram med högintensiv intervallträning.

Övrig: 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.

Aktiv komparator: Nutritionell intervention (D-INACT)

Deltagare som fick näringsintervention men inte ett träningsprogram.

Beteende: 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.

Experimentell: Nutritionell intervention Måttlig intensitet kontinuerlig träning (D-MICT)

Deltagare som fick näringsintervention och deltog i ett träningsprogram med kontinuerlig träning av måttlig intensitet.

Övrig: Moderatintensiv kontinuerlig träning (MICT)

Deltagare som tilldelades MICT genomförde 3 veckovisa sessioner, med 1-2 dagars vila mellan sessionerna, på en cykelergometer i 12 veckor.

MICT bestod av 50 minuter av kontinuerlig trampning med en intensitet på cirka 10 % över laktattröskeln, med en kadens mellan 60-80 rpm. Träningsbelastningen (TL) ökades med 10 % om dessa två villkor uppfylldes: 1) ingen ökning av minst 2 hjärtslag från minut 20 till minut 40 av träningssessionen, och 2) laktatnivåer under 2 mmol/L i minut 48 av sessionen. TL för den första sessionen bestämdes av ett justeringstest som bestod av ett stegvis ökande test med fem på varandra följande 10-minutersfaser vid en konstant kadens (60-80 RPM) från 90 % till 130 % av den ventilatoriska tröskeleffekten, beräknad från tidigare utförda FatOx-testdata, med 10 % ökningar i varje steg. TL bestämdes av fasen där HR ökade med ≥ 3 slag mellan faser och blodlaktatnivåer ökade med ≥ 1 mmol/L från föregående fas.

Beteende: 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.

Experimentell: Nutritionell intervention Högintensiv intervallträning (D-HIIT)

Deltagare som fick näringsintervention och deltog i ett högintensivt intervallträningsprogram.

Beteende: 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.

Övrig: 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.

Utvärderade förändringar i Oxidativ Stress

Nivån av oxidativ stress bedömdes genom koncentrationen av 8-OHdG som en biomarkör för DNA-oxidation. En kompetitiv ELISA-detektionsmetod användes i serumprover. Kortfattat tillsattes standarden och provet tillsammans med den biotinylerade detektionsantikroppen i en förbehandlad mikroplatta. Efter tvättning tillsattes HRP-Streptavidin. Därefter tillsattes TMB-substratet och reaktionen avslutades med en syralösning. Färgutvecklingen mättes spektrofotometriskt vid en våglängd på 450 nm. Optiska densitetsvärdena för proverna jämfördes med standardkurvan för att bestämma 8-OHdG-koncentrationen (ng/ml).

Muskelbiopiprov: Citratsyntasaktivitet

2–5 mg fryst muskelvävnad homogeniserades i en 1:20 utspädning av CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, Storbritannien), med 1 % proteasinhämmarkocktail (P8340, Sigma-Aldrich, Dorset, Storbritannien) vid 4 °C. Lysatet centrifugerades vid 12 000 × g i 10 minuter vid 4 °C för att sedimentera debris, och supernatantet förvarades vid –80 °C. CS-aktivitet kvantifierades i dubbletter med hjälp av ett analyskit enligt tillverkarens instruktioner (CS0720: Sigma-Aldrich, Dorset, Storbritannien). Kortfattat registrerades endogen aktivitet genom färgavgivning från DTNB, och total aktivitet beräknades efter tillsats av oxaloacetat. Proteinkoncentration bestämdes i dubbletter med hjälp av ett bicinchoninsyraanalyskit (BCA1: Sigma-Aldrich, Dorset, Storbritannien), vilket möjliggjorde normalisering av CS-aktivitetsvärden till totalt proteininnehåll (µmol/g/min). CS-aktivitet och proteinkoncentration mättes i 96-välsplattor med hjälp av en mikroplattläsare vid 412 nm respektive 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.

Utvärderade förändringar i insulinresistens: HOMA-IR

Insulinresistens uppskattades med hjälp av Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), beräknat som fastande insulin (µU/mL) multiplicerat med fastande glukos (mg/dL) dividerat med 405.

Bedömda förändringar i insulinkänslighet: Matsuda-index

Hela kroppens insulinkänslighet uppskattades genom Matsuda-indexet, beräknat som 10,000 / √[(fastande glukos (mg/dL) × fastande insulin (µU/mL)) × (medelglukos under oralt glukostoleranstest (mg/dL) × medelinsulin under oralt glukostoleranstest (µU/mL))].

Utvärderade förändringar i insulinkänslighet: Quicki

Insulinkänsligheten uppskattades med hjälp av det kvantitativa insulinkänslighetsindexet (QUICKI), beräknat som 1 / [log(fastande insulin (µU/mL)) + log(fastande glukos (mg/dL))].

Utvärderade förändringar i kroppsmassaindex

Body Mass Index (BMI) i kilogram per kvadratmeter (kg/m²) beräknades som kroppsvikt (kg) dividerat med längd i kvadrat (m²).

Utvärderade förändringar i antropometri: Omkretsar

Midjemått (cm) mättes vid mittpunkten mellan den nedre laterala kanten av den 10:e revbenet och iliacakammen, vinkelrätt mot kroppens längdaxel. Bukomfång (cm) mättes på nivån med naveln, vinkelrätt mot kroppens längdaxel. Höftomfång (cm) mättes på nivån med den största bakre utbuktningen av skinkorna, vinkelrätt mot kroppens längdaxel. Låromfång (cm) mättes vid mittpunkten mellan trochanter major och den laterala tibiakondylen. Alla mätningar utfördes med deltagaren stående upprätt och avslappnad. Varje mätning utfördes två gånger, och en tredje mätning gjordes om skillnaden översteg 1 %. Medelvärdet av de två mest lika mätningarna registrerades.

Utvärderade förändringar i antropometri: Midje-höftkvot

Midja-höftkvot (WHR) beräknades som midjeomkrets (cm) dividerat med höftomkrets (cm).

Bedömd Längd

Längd (meter, m) mättes med en väggmonterad stadiometer (TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Japan) med deltagare som stod i enlighet med tillverkarens instruktioner.

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).