신체 운동 및 영양 상담이 제2형 당뇨병에서 미토콘드리아 대사와 산화 스트레스에 미치는 영향 (MITOX)

간섭 없음: 비활성 ND

영양 중재나 운동 프로그램을 받지 않은 참가자. 그들은 신체 활동과 식이에 관한 평소 생활 습관을 유지하도록 지시받았습니다.

활성 비교기: 중간 강도 지속 운동 (ND-MICT)

영양 중재를 받지 않았으나 중간 강도의 지속적 운동 프로그램에 참여한 참가자들.

다른: 중간 강도 지속 운동(MICT)

MICT에 배정된 참가자들은 12주 동안 사이클 에르고미터에서 주당 3회 세션을 수행했으며, 세션 사이에는 1-2일의 휴식을 취했습니다.

MICT는 젖산 역치보다 약 10% 높은 강도로 60-80 rpm의 케이던스로 50분간 지속적인 페달링으로 구성되었습니다. 훈련 부하(TL)는 다음 두 조건이 충족되면 10% 증가시켰습니다: 1) 훈련 세션 20분에서 40분 사이에 심박수가 최소 2회 이상 증가하지 않음, 2) 세션 48분 시점의 젖산 농도가 2 mmol/L 미만. 첫 번째 세션의 TL은 이전에 수행된 FatOx 테스트 데이터에서 계산된 환기 역치 파워의 90%에서 130%까지 일정한 케이던스(60-80 RPM)로 10분씩 5단계 연속 증분 테스트로 구성된 조정 테스트를 통해 결정되었으며, 각 단계마다 10%씩 증가시켰습니다. TL은 단계 간 심박수가 ≥ 3회 증가하고 혈중 젖산 농도가 이전 단계보다 ≥ 1 mmol/L 증가한 단계에서 결정되었습니다.

활성 비교기: 고강도 간헐적 운동 (ND-HIIT)

영양 중재를 받지 않았지만 고강도 인터벌 트레이닝 운동 프로그램에 참여한 참가자.

다른: 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.

활성 비교기: 영양 중재 (D-INACT)

영양 중재는 받았지만 운동 프로그램은 받지 않은 참가자.

행동: 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.

실험적: 영양 중재 중등도 지속 운동 (D-MICT)

영양 중재를 받고 중간 강도 지속 운동 프로그램에 등록된 참가자.

다른: 중간 강도 지속 운동(MICT)

MICT에 배정된 참가자들은 12주 동안 사이클 에르고미터에서 주당 3회 세션을 수행했으며, 세션 사이에는 1-2일의 휴식을 취했습니다.

MICT는 젖산 역치보다 약 10% 높은 강도로 60-80 rpm의 케이던스로 50분간 지속적인 페달링으로 구성되었습니다. 훈련 부하(TL)는 다음 두 조건이 충족되면 10% 증가시켰습니다: 1) 훈련 세션 20분에서 40분 사이에 심박수가 최소 2회 이상 증가하지 않음, 2) 세션 48분 시점의 젖산 농도가 2 mmol/L 미만. 첫 번째 세션의 TL은 이전에 수행된 FatOx 테스트 데이터에서 계산된 환기 역치 파워의 90%에서 130%까지 일정한 케이던스(60-80 RPM)로 10분씩 5단계 연속 증분 테스트로 구성된 조정 테스트를 통해 결정되었으며, 각 단계마다 10%씩 증가시켰습니다. TL은 단계 간 심박수가 ≥ 3회 증가하고 혈중 젖산 농도가 이전 단계보다 ≥ 1 mmol/L 증가한 단계에서 결정되었습니다.

행동: 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.

실험적: 영양 중재 고강도 간헐적 운동 (D-HIIT)

영양 중재를 받고 고강도 간격 운동 프로그램에 등록한 참가자들.

행동: 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.

다른: 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.

산화 스트레스 변화 평가

DNA 산화의 바이오마커로서 8-OHdG의 농도로 산화 스트레스 수준을 평가하였다. 혈청 샘플에서 경쟁적 ELISA 검출 방법을 사용하였다. 간단히 말하면, 표준물질과 샘플을 생티닐화된 검출 항체와 함께 미리 코팅된 마이크로플레이트에 첨가하였다. 세척 후, HRP-스트렙타비딘을 첨가하였다. 그런 다음, TMB 기질을 첨가하고 산성 용액으로 반응을 종료하였다. 색상 발생은 450nm 파장에서 분광광도계로 측정하였다. 샘플의 광학 밀도 값을 표준 곡선의 값과 비교하여 8-OHdG 농도(ng/ml)를 결정하였다.

근육 생검 샘플: 시트르산 합성효소 활성

2-5 mg의 동결 근육을 CelLytic™ MT 조직 용해액(C3228, Sigma-Aldrich, Dorset, UK)의 1:20 희석액에 1 % 프로테아제 억제제 칵테일(P8340, Sigma-Aldrich, Dorset, UK)을 첨가하여 4 °C에서 균질화하였다. 용해물은 4 °C에서 12,000 × g로 10분간 원심분리하여 침전물을 제거하고, 상등액은 -80 °C에 보관하였다. CS 활성은 제조사의 지침에 따라 시험 키트(CS0720: Sigma-Aldrich, Dorset, UK)를 사용하여 이중으로 정량하였다. 간단히 말해, 내인성 활성은 DTNB에 의한 색소 발광으로 기록되었으며, 옥살아세테이트 첨가 후 총 활성을 계산하였다. 단백질 농도는 bicinchoninic acid 시험 키트(BCA1: Sigma-Aldrich, Dorset, UK)를 사용하여 이중으로 측정하여 CS 활성 값을 총 단백질 함량(μmol/g/min)으로 정규화하였다. CS 활성과 단백질 농도는 각각 412 nm 및 562 nm에서 마이크로플레이트 리더(MQX200R2, Biotek instruments, Burlington, USA)를 사용하여 96-웰 플레이트에서 측정하였다.

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.

인슐린 저항성 변화 평가: HOMA-IR

인슐린 저항성은 HOMA-IR(Homeostatic Model Assessment for Insulin Resistance)을 사용하여 추정되었으며, 공복 인슐린(μU/mL)에 공복 혈당(mg/dL)을 곱한 값을 405로 나누어 계산되었습니다.

인슐린 민감도 변화 평가: 마츠다 지수

전신 인슐린 감수성은 마츠다 지수를 통해 추정되었으며, 10,000 / √[(공복 혈당(mg/dL) × 공복 인슐린(µU/mL)) × (경구 당 부하 검사 중 평균 혈당(mg/dL) × 경구 당 부하 검사 중 평균 인슐린(µU/mL))]로 계산되었습니다.

인슐린 민감도 변화 평가: Quicki

인슐린 민감도는 Quantitative Insulin Sensitivity Check Index(QUICKI)를 통해 추정되었으며, 1/[log(공복 인슐린(μU/mL)) + log(공복 혈당(mg/dL))]로 계산되었습니다.

체질량 지수의 변화 평가

체질량 지수(BMI)는 체중(kg)을 신장의 제곱(m²)으로 나눈 값으로 킬로그램 매 제곱미터(kg/m²) 단위로 계산되었습니다.

인체 측정학적 변화 평가: 둘레

허리둘레(cm)는 10번째 갈비뼈의 아래쪽 측면 가장자리와 장골능 사이의 중간 지점에서, 몸통의 세로 축에 수직으로 측정되었습니다. 복부둘레(cm)는 배꼽 높이에서, 몸통의 세로 축에 수직으로 측정되었습니다. 엉덩이둘레(cm)는 엉덩이의 가장 돌출된 후면 부위에서, 몸통의 세로 축에 수직으로 측정되었습니다. 허벅지둘레(cm)는 대전자와 외측 경골과의 중간 지점에서 측정되었습니다. 모든 측정은 참가자가 똑바로 서서 편안한 상태에서 수행되었습니다. 각 측정은 두 번 수행되었으며, 차이가 1%를 초과할 경우 세 번째 측정이 수행되었습니다. 가장 유사한 두 기록의 평균이 등록되었습니다.

인체 측정학적 변화 평가: 허리-엉덩이 비율

허리-엉덩이 비율(WHR)은 허리 둘레(cm)를 엉덩이 둘레(cm)로 나눈 값으로 계산되었습니다.

평가된 신장

신장(미터, m)은 제조사의 지침에 따라 참가자가 서 있는 상태에서 벽걸이식 신장계(TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Japan)를 사용하여 측정되었습니다.

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).