Митохондриальный метаболизм и оксидативный стресс в функции физических упражнений и нутрициологического консультирования при СД2 (MITOX)

Без вмешательства: НД-ИНАКТ

Участники, которые не получали ни пищевого вмешательства, ни программы упражнений. Им было рекомендовано сохранять свои обычные жизненные привычки в отношении физической активности и питания.

Активный компаратор: Тренировка умеренной интенсивности с непрерывной нагрузкой (ND-MICT)

Участники, которые не получали нутритивного вмешательства, но были включены в программу упражнений с умеренной непрерывной нагрузкой.

Другой: Умеренно-интенсивная непрерывная тренировка (МИТ)

Участники, назначенные на MICT, выполняли 3 еженедельных сессии с 1-2 днями отдыха между сессиями на велоэргометре в течение 12 недель.

MICT состоял из 50 минут непрерывного педалирования с интенсивностью примерно на 10% выше лактатного порога, с каденсом между 60-80 об/мин. Тренировочная нагрузка (TL) увеличивалась на 10%, если выполнялись два условия: 1) отсутствие увеличения ЧСС как минимум на 2 удара с 20-й по 40-ю минуту тренировочной сессии, и 2) уровень лактата ниже 2 ммоль/л на 48-й минуте сессии. TL для первой сессии определялась с помощью корректировочного теста, состоящего из инкрементального теста с пятью последовательными 10-минутными фазами при постоянном каденсе (60-80 об/мин) от 90% до 130% мощности вентиляторного порога, рассчитанной на основе ранее проведенных данных теста FatOx, с увеличением на 10% на каждом этапе. TL определялась по фазе, в которой ЧСС увеличивалась на ≥ 3 удара между фазами и уровень лактата в крови увеличивался на ≥ 1 ммоль/л по сравнению с предыдущей фазой.

Активный компаратор: Высокоинтенсивные интервальные тренировки (ВИИТ-НД)

Участники, которые не получали нутритивную интервенцию, но были включены в программу высокоинтенсивных интервальных тренировок.

Другой: 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.

Активный компаратор: Пищевая интервенция (D-INACT)

Участники, получившие нутритивное вмешательство, но не программу физических упражнений.

Поведенческий: 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.

Экспериментальный: Нутритивная интервенция Умеренно-интенсивная непрерывная тренировка (D-MICT)

Участники, которые получали нутритивную интервенцию и были включены в программу упражнений умеренной интенсивности с непрерывной тренировкой.

Другой: Умеренно-интенсивная непрерывная тренировка (МИТ)

Участники, назначенные на MICT, выполняли 3 еженедельных сессии с 1-2 днями отдыха между сессиями на велоэргометре в течение 12 недель.

MICT состоял из 50 минут непрерывного педалирования с интенсивностью примерно на 10% выше лактатного порога, с каденсом между 60-80 об/мин. Тренировочная нагрузка (TL) увеличивалась на 10%, если выполнялись два условия: 1) отсутствие увеличения ЧСС как минимум на 2 удара с 20-й по 40-ю минуту тренировочной сессии, и 2) уровень лактата ниже 2 ммоль/л на 48-й минуте сессии. TL для первой сессии определялась с помощью корректировочного теста, состоящего из инкрементального теста с пятью последовательными 10-минутными фазами при постоянном каденсе (60-80 об/мин) от 90% до 130% мощности вентиляторного порога, рассчитанной на основе ранее проведенных данных теста FatOx, с увеличением на 10% на каждом этапе. TL определялась по фазе, в которой ЧСС увеличивалась на ≥ 3 удара между фазами и уровень лактата в крови увеличивался на ≥ 1 ммоль/л по сравнению с предыдущей фазой.

Поведенческий: 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.

Экспериментальный: Пищевая интервенция Высокоинтенсивные интервальные тренировки (ВИИТ)

Участники, которые получили нутритивную интервенцию и были включены в программу высокоинтенсивных интервальных тренировок.

Поведенческий: 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.

Другой: 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.

Оцененные изменения оксидативного стресса

Уровень окислительного стресса оценивался по концентрации 8-OHdG как биомаркера окисления ДНК. В сывороточных образцах использовался конкурентный метод ELISA-детекции. Кратко: стандарт и образец добавляли в предварительно покрытую микропланшет вместе с биотинилированным детектирующим антителом. После промывки добавляли HRP-стрептавидин. Затем добавляли субстрат TMB, и реакцию останавливали кислотным раствором. Образование цвета измеряли спектрофотометрически при длине волны 450 нм. Оптические плотности образцов сравнивали со значениями стандартной кривой для определения концентрации 8-OHdG (нг/мл).

Образец биопсии мышцы: активность цитратсинтазы

2-5 мг замороженной мышечной ткани гомогенизировали в 1:20 разведении CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Дорсет, Великобритания) с 1% коктейлем ингибиторов протеаз (P8340, Sigma-Aldrich, Дорсет, Великобритания) при 4 °C. Лизат центрифугировали при 12 000 × g в течение 10 минут при 4 °C для осаждения дебриса, и супернатант хранили при -80 °C. Активность CS определяли в дубликатах с использованием набора для анализа в соответствии с инструкциями производителя (CS0720: Sigma-Aldrich, Дорсет, Великобритания). Кратко, эндогенную активность регистрировали по цветовой эмиссии DTNB, а общую активность рассчитывали после добавления оксалоацетата. Концентрацию белка определяли в дубликатах с использованием набора для бицинхониновой кислоты (BCA1: Sigma-Aldrich, Дорсет, Великобритания), что позволило нормализовать значения активности CS к общему содержанию белка (мкмоль/г/мин). Активность CS и концентрацию белка измеряли в 96-луночных планшетах с использованием микропланшетного ридера при 412 нм и 562 нм соответственно (MQX200R2, Biotek instruments, Берлингтон, США).

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.

Оцененные изменения инсулинорезистентности: HOMA-IR

Инсулинорезистентность оценивалась с использованием Гомеостатической модели оценки инсулинорезистентности (HOMA-IR), которая рассчитывается как произведение уровня инсулина натощак (мкЕд/мл) на уровень глюкозы натощак (мг/дл), делённое на 405.

Оцененные изменения инсулиновой чувствительности: Индекс Мацуда

Чувствительность к инсулину всего организма оценивалась с помощью индекса Мацуда, рассчитываемого как 10 000 / √[(глюкоза натощак (мг/дл) × инсулин натощак (мкЕд/мл)) × (средняя глюкоза во время перорального теста на толерантность к глюкозе (мг/дл) × средний инсулин во время перорального теста на толерантность к глюкозе (мкЕд/мл))].

Оцененные изменения чувствительности к инсулину: Quicki

Чувствительность к инсулину оценивалась с помощью Количественного индекса чувствительности к инсулину (QUICKI), рассчитываемого как 1 / [log(инсулин натощак (мкЕд/мл)) + log(глюкоза натощак (мг/дл))].

Оцененные изменения индекса массы тела

Индекс массы тела (ИМТ) в килограммах на квадратный метр (кг/м²) рассчитывался как масса тела (кг), деленная на квадрат роста (м²).

Оцененные изменения в антропометрии: Обхваты

Окружность талии (см) измерялась в средней точке между нижним латеральным краем 10-го ребра и гребнем подвздошной кости, перпендикулярно продольной оси туловища. Окружность живота (см) измерялась на уровне пупка, перпендикулярно продольной оси туловища. Окружность бедер (см) измерялась на уровне наибольшего заднего выступа ягодиц, перпендикулярно продольной оси туловища. Окружность бедра (см) измерялась в средней точке между большим вертелом и латеральным мыщелком большеберцовой кости. Все измерения проводились при вертикальном положении участника в расслабленном состоянии. Каждое измерение проводилось дважды, и третье измерение выполнялось, если разница превышала 1%. Регистрировалось среднее значение двух наиболее близких записей.

Оцененные изменения в антропометрии: соотношение талии и бедер

Соотношение талии и бедер (WHR) рассчитывалось как окружность талии (см), деленная на окружность бедер (см).

Измеренный рост

Рост (в метрах, м) измеряли с помощью настенного ростометра (TANITA-LEICESTER HR-001, Tanita Corp., Токио, Япония) при положении участников стоя в соответствии с инструкциями производителя.

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).