Mitokondriell metabolisme og oksidativ stress i funksjon av fysisk trening og ernæringsveiledning ved T2DM (MITOX)

Ingen inngripen: ND-INAKT

Deltakere som ikke mottok verken ernæringsintervensjon eller treningsprogram. De ble instruert til å opprettholde sine vanlige livsvaner når det gjelder fysisk aktivitet og kosthold.

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

Deltakere som ikke mottok ernæringsintervensjon, men som ble inkludert i et treningsprogram med moderat intensitet kontinuerlig trening.

Annen: Moderat Intensitets Kontinuerlig Trening (MICT)

Deltakere som ble tildelt MICT fullførte 3 ukentlige økter, med 1-2 dagers hvile mellom øktene, på en sykkelergometer i 12 uker.

MICT besto av 50 minutter med kontinuerlig tråkking med en intensitet på omtrent 10 % over laktatterskelen, med en kadens mellom 60-80 rpm. Treningsbelastning (TL) ble økt med 10 % hvis disse to betingelsene var oppfylt: 1) ingen økning på minst 2 slag i hjertefrekvens fra minutt 20 til minutt 40 i treningsøkten, og 2) laktatnivåer under 2 mmol/L i minutt 48 av økten. TL for den første økten ble bestemt av en justeringstest som besto av en trinnvis test med fem påfølgende 10-minutters faser med en konstant kadens (60-80 RPM) fra 90 % til 130 % av ventilatorisk terskelkraft, beregnet fra tidligere utførte FatOx-testdata, med 10 % økninger i hvert trinn. TL ble bestemt av fasen der HR økte med ≥ 3 slag mellom fasene og blodlaktatnivåene økte med ≥ 1 mmol/L fra forrige fase.

Aktiv komparator: Høyintens intervalltrening (ND-HIIT)

Deltakere som ikke mottok ernæringstiltak, men som ble innlemmet i et høyt intensitets intervalltrening treningsprogram.

Annen: 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: Ernæringsmessig intervensjon (D-INACT)

Deltakere som mottok ernæringsintervensjon, men ikke et treningsprogram.

Atferdsmessig: 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.

Eksperimentell: Ernæringsmessig intervensjon Moderat intensitet kontinuerlig trening (D-MICT)

Deltakere som mottok ernæringsintervensjon og ble inkludert i et treningsprogram med moderat intensitet kontinuerlig trening.

Annen: Moderat Intensitets Kontinuerlig Trening (MICT)

Deltakere som ble tildelt MICT fullførte 3 ukentlige økter, med 1-2 dagers hvile mellom øktene, på en sykkelergometer i 12 uker.

MICT besto av 50 minutter med kontinuerlig tråkking med en intensitet på omtrent 10 % over laktatterskelen, med en kadens mellom 60-80 rpm. Treningsbelastning (TL) ble økt med 10 % hvis disse to betingelsene var oppfylt: 1) ingen økning på minst 2 slag i hjertefrekvens fra minutt 20 til minutt 40 i treningsøkten, og 2) laktatnivåer under 2 mmol/L i minutt 48 av økten. TL for den første økten ble bestemt av en justeringstest som besto av en trinnvis test med fem påfølgende 10-minutters faser med en konstant kadens (60-80 RPM) fra 90 % til 130 % av ventilatorisk terskelkraft, beregnet fra tidligere utførte FatOx-testdata, med 10 % økninger i hvert trinn. TL ble bestemt av fasen der HR økte med ≥ 3 slag mellom fasene og blodlaktatnivåene økte med ≥ 1 mmol/L fra forrige fase.

Atferdsmessig: 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.

Eksperimentell: Ernæringsmessig intervensjon Høyintensitet intervalltrening (D-HIIT)

Deltakere som mottok ernæringsintervensjon og ble inkludert i et treningsprogram med høyt intensitets intervalltrening.

Atferdsmessig: 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.

Annen: 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.

Vurderte endringer i oksidativt stress

Nivået av oksidativt stress ble vurdert ved konsentrasjonen av 8-OHdG som en biomarkør for DNA-oksidasjon.
En konkurransedyktig ELISA-deteksjonsmetode ble brukt i serumprøver.
Kort fortalt ble standarden og prøven tilsatt en forhåndsbelagt mikroplate sammen med den biotinylerte deteksjonsantistoffet.
Etter vasking ble HRP-Streptavidin tilsatt.
Deretter ble TMB-substratet tilsatt, og reaksjonen ble avsluttet med en syreløsning.
Fargeproduksjonen ble målt spektrofotometrisk ved en bølgelengde på 450 nm.
De optiske tetthetsverdiene til prøvene ble sammenlignet med standardkurven for å bestemme 8-OHdG-konsentrasjonen (ng/ml).

Muskelbioprøve: Sitratsyntase-aktivitet

2-5 mg frossen muskel ble homogenisert i en 1:20 fortynning av CelLytic™ MT vevslyse (C3228, Sigma-Aldrich, Dorset, Storbritannia) med 1 % proteasehemmermiks (P8340, Sigma-Aldrich, Dorset, Storbritannia) ved 4 °C. Lysatet ble sentrifugert ved 12 000 × g i 10 minutter ved 4 °C for å pelletere avfall, og supernatanten ble lagret ved -80 °C. CS-aktivitet ble kvantifisert i duplikater ved bruk av et analysesett i henhold til produsentens instruksjoner (CS0720: Sigma-Aldrich, Dorset, Storbritannia). Kort fortalt ble endogen aktivitet registrert gjennom fargeemisjon av DTNB, og total aktivitet ble beregnet etter tilsats av oksaloacetat. Proteinkonsentrasjon ble bestemt i duplikater ved bruk av et bikinkoninsyreanalysesett (BCA1: Sigma-Aldrich, Dorset, Storbritannia), noe som muliggjorde normalisering av CS-aktivitetsverdier til totalt proteininnhold (μmol/g/min). CS-aktivitet og proteinkonsentrasjon ble målt i 96-brønnsplater ved bruk av en mikroplateleser 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.

Vurderte endringer i insulinresistens: HOMA-IR

Insulinresistens ble estimert ved hjelp av Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), beregnet som fastende insulin (µU/mL) multiplisert med fastende glukose (mg/dL) dividert med 405.

Vurderte endringer i Insulinfølsomhet: Matsuda-indeksen

Helkroppens insulinfølsomhet ble estimert gjennom Matsuda-indeksen, beregnet som 10 000 / √[(fasting glukose (mg/dL) × fasting insulin (µU/mL)) × (gjennomsnittlig glukose under Oral Glukosetoleransetest (mg/dL) × gjennomsnittlig insulin under Oral Glukosetoleransetest (µU/mL))].

Vurderte endringer i insulinfølsomhet: Quicki

Insulinsensitivitet ble estimert gjennom Quantitative Insulin Sensitivity Check Index (QUICKI), beregnet som 1 / [log(fastende insulin (µU/mL)) + log(fastende glukose (mg/dL))].

Vurderte endringer i kroppsmasseindeks

Kroppsmasseindeks (KMI) i kilogram per kvadratmeter (kg/m²) ble beregnet som kroppsvekt (kg) delt på høyde i annen (m²).

Vurderte endringer i antropometri: Omkretser

Midjeomkrets (cm) ble målt på midtpunktet mellom den nedre laterale kanten av den 10. ribben og iliac-kammen, vinkelrett på kroppens lengdeakse. Bukomkrets (cm) ble målt på nivået med navlen, vinkelrett på kroppens lengdeakse. Hofteomkrets (cm) ble målt på nivået med den største bakre fremspringet på skinkene, vinkelrett på kroppens lengdeakse. Låromkrets (cm) ble målt på midtpunktet mellom trochanter major og den laterale tibialcondylen. Alle målingene ble utført med deltageren stående oppreist og avslappet. Hver måling ble tatt to ganger, og en tredje måling ble utført hvis forskjellen oversteg 1 %. Gjennomsnittet av de to mest like registreringene ble registrert.

Vurderte endringer i antropometri: Midje-til-hofte-forhold

Midje-til-hoftetall (WHR) ble beregnet som midjeomkrets (cm) delt på hofteomkrets (cm).

Vurdert høyde

Høyde (meter, m) ble målt ved hjelp av en veggmontert stadiometer (TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Japan) med deltakere stående i henhold til produsentens instruksjoner.

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).