Mitokondriel Metabolisme og Oxidativ Stress i Funktion af Fysisk Træning og Ernæringsvejledning i T2DM (MITOX)

Ingen indgriben: ND-INAKTIV

Deltagere, som ikke modtog hverken ernæringsmæssig intervention eller motionsprogram.
De blev instrueret i at opretholde deres normale livsstilsvaner vedrørende fysisk aktivitet og kost.

Aktiv komparator: Moderat intensitet kontinuerlig træning (ND-MICT)

Deltagere, der ikke modtog ernæringsmæssig intervention, men som var tilmeldt et moderat intensivt kontinuerligt træningsprogram.

Andet: Moderat-intensitets kontinuerlig træning (MICT)

Deltagere tildelt MICT gennemførte 3 ugentlige sessioner, med 1-2 dages hvile mellem sessioner, på en cykelergometer i 12 uger.

MICT bestod af 50 minutters kontinuerlig pedaling med en intensitet på cirka 10 % over lactattærsklen, med en kadence mellem 60-80 rpm. Træningsbelastning (TL) blev øget med 10%, hvis disse to betingelser var opfyldt: 1) ingen stigning på mindst 2 slag i hjertefrekvens fra minut 20 til minut 40 af træningssessionen, og 2) lactatniveauer under 2 mmol/L i minut 48 af sessionen. TL for den første session blev bestemt af en justeringstest bestående af en inkrementel test med fem på hinanden følgende 10-minutters faser ved en konstant kadence (60-80 RPM) fra 90% til 130% af den ventilatoriske tærskelkraft, beregnet fra tidligere udførte FatOx-testdata, med 10% stigninger i hvert trin. TL blev bestemt af den fase, hvor HR steg med ≥ 3 slag mellem faser og blodlactatniveauer steg med ≥ 1 mmol/L fra den foregående fase.

Aktiv komparator: Højintensiv intervaltræning (ND-HIIT)

Deltagere, der ikke modtog ernæringsmæssig intervention, men var tilmeldt et højintensitetsintervaltræningsprogram.

Andet: 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: Ernæringsmæssig intervention (D-INACT)

Deltagere, der modtog ernæringsmæssig intervention men ikke et motionsprogram.

Adfærdsmæssigt: 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.

Eksperimentel: Ernæringsmæssig Intervention Moderat-Intensiv kontinuerlig træning (D-MICT)

Deltagere, der modtog ernæringsmæssig intervention og var tilmeldt et moderat intensivt kontinuerligt træningsprogram.

Andet: Moderat-intensitets kontinuerlig træning (MICT)

Deltagere tildelt MICT gennemførte 3 ugentlige sessioner, med 1-2 dages hvile mellem sessioner, på en cykelergometer i 12 uger.

MICT bestod af 50 minutters kontinuerlig pedaling med en intensitet på cirka 10 % over lactattærsklen, med en kadence mellem 60-80 rpm. Træningsbelastning (TL) blev øget med 10%, hvis disse to betingelser var opfyldt: 1) ingen stigning på mindst 2 slag i hjertefrekvens fra minut 20 til minut 40 af træningssessionen, og 2) lactatniveauer under 2 mmol/L i minut 48 af sessionen. TL for den første session blev bestemt af en justeringstest bestående af en inkrementel test med fem på hinanden følgende 10-minutters faser ved en konstant kadence (60-80 RPM) fra 90% til 130% af den ventilatoriske tærskelkraft, beregnet fra tidligere udførte FatOx-testdata, med 10% stigninger i hvert trin. TL blev bestemt af den fase, hvor HR steg med ≥ 3 slag mellem faser og blodlactatniveauer steg med ≥ 1 mmol/L fra den foregående fase.

Adfærdsmæssigt: 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.

Eksperimentel: Ernæringsmæssig Intervention Højintensiv intervaltræning (D-HIIT)

Deltagere, der modtog ernæringsmæssig intervention og blev tilmeldt et højintensivt intervaltræningsprogram.

Adfærdsmæssigt: 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.

Andet: 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.

Vurderede ændringer i oxidativ stress

Niveauet af oxidativ stress blev vurderet ved koncentrationen af 8-OHdG som en biomarkør for DNA-oxidation. En kompetitiv ELISA-detektionsmetode blev anvendt i serumprøver. Kort fortalt blev standarden og prøven tilsat en forcoateret mikroplade sammen med den biotinylerede detektionsantistof. Efter vask blev HRP-streptavidin tilsat. Derefter blev TMB-substratet tilsat, og reaktionen blev afsluttet med en syreopløsning. Farveudviklingen blev målt spektrofotometrisk ved en bølgelængde på 450 nm. De optiske densitetsværdier af prøverne blev sammenlignet med dem fra standardkurven for at bestemme 8-OHdG-koncentrationen (ng/ml).

Muskelbiopsiprøve: Citratsyntaseaktivitet

2-5 mg frossen muskelvæv blev homogeniseret i en 1:20 fortynding af CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, UK) med 1 % protease inhibitor cocktail (P8340, Sigma-Aldrich, Dorset, UK) ved 4 °C. Lysatet blev centrifugeret ved 12.000 × g i 10 minutter ved 4 °C for at pelletere affald, og supernatantet blev opbevaret ved -80 °C. CS-aktivitet blev kvantificeret i duplikater ved hjælp af et analysekit efter producentens vejledning (CS0720: Sigma-Aldrich, Dorset, UK). Kort fortalt blev endogen aktivitet registreret gennem farveemission fra DTNB, og total aktivitet blev beregnet efter tilsætning af oxalacetat. Proteinkoncentration blev bestemt i duplikater ved hjælp af et bicinchoninsyre-analysekit (BCA1: Sigma-Aldrich, Dorset, UK), hvilket muliggjorde normalisering af CS-aktivitetsværdier til totalt proteinindhold (µmol/g/min). CS-aktivitet og proteinkoncentration blev målt i 96-brænderglasplader ved hjælp af en mikropladelæser ved henholdsvis 412 nm og 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.

Vurderede ændringer i insulinresistens: HOMA-IR

Insulinresistens blev estimeret ved hjælp af Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), beregnet som fastende insulin (µU/mL) ganget med fastende glukose (mg/dL) divideret med 405.

Vurderede ændringer i insulinsensitivitet: Matsuda-indeks

Hele krops insulinfølsomhed blev estimeret gennem Matsuda-indekset, beregnet som 10.000 / √[(fastende glukose (mg/dL) × fastende insulin (µU/mL)) × (gennemsnitlig glukose under Oral Glukose Tolerance Test (mg/dL) × gennemsnitlig insulin under Oral Glukose Tolerance Test (µU/mL))].

Vurderede ændringer i Insulinfølsomhed: Quicki

Insulinfølsomhed blev estimeret gennem den Kvantitative Insulinfølsomheds Kontrolindeks (QUICKI), beregnet som 1 / [log(fastende insulin (µU/mL)) + log(fastende glukose (mg/dL))].

Vurderede ændringer i Body Mass Index

Body Mass Index (BMI) i kilogram per kvadratmeter (kg/m²) blev beregnet som kropsvægt (kg) divideret med højden i anden potens (m²).

Vurderede ændringer i antropometri: Omkredse

Taljemål (cm) blev målt på midtpunktet mellem den nederste laterale margin af den 10. ribben og iliac-kammen, vinkelret på kroppens længdeakse. Maveomkreds (cm) blev målt på niveau med navlen, vinkelret på kroppens længdeakse. Hofteomkreds (cm) blev målt på niveau med den største posteriore fremspring på ballerne, vinkelret på kroppens længdeakse. Låromkreds (cm) blev målt på midtpunktet mellem trochanter major og den laterale tibia condyle. Alle målinger blev udført med deltageren stående oprejst og afslappet. Hver måling blev foretaget to gange, og en tredje måling blev udført, hvis forskellen oversteg 1%. Gennemsnittet af de to mest ens målinger blev registreret.

Vurderede ændringer i antropometri: Talje-hofte-forhold

Talje-hofte-forholdet (WHR) blev beregnet som taljeomkreds (cm) divideret med hofteomkreds (cm).

Vurderet højde

Højde (meter, m) blev målt ved hjælp af en vægmonteret stadiometer (TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Japan) med deltagerne stående i henhold til producentens 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).