Metabolismo Mitocondrial e Stress Oxidativo na Função do Exercício Físico e Aconselhamento Nutricional na DM2 (MITOX)

Sem intervenção: ND-INACT

Participantes que não receberam qualquer intervenção nutricional ou programa de exercício. Foram instruídos a manter os seus hábitos de vida normais no que diz respeito à atividade física e dieta.

Comparador Ativo: Treino contínuo de intensidade moderada (ND-MICT)

Participantes que não receberam intervenção nutricional, mas foram inscritos num programa de exercício de treino contínuo de intensidade moderada.

Outro: O Treino Contínuo de Intensidade Moderada (MICT)

Os participantes atribuídos ao MICT completaram 3 sessões semanais, com 1-2 dias de descanso entre sessões, num cicloergómetro durante 12 semanas.

O MICT consistiu em 50 minutos de pedalada contínua a uma intensidade aproximadamente 10% acima do limiar de lactato, com uma cadência entre 60-80 rpm. A carga de treino (CT) foi aumentada em 10% se estas duas condições fossem cumpridas: 1) nenhum aumento de pelo menos 2 batimentos da frequência cardíaca do minuto 20 ao minuto 40 da sessão de treino, e 2) níveis de lactato abaixo de 2 mmol/L no minuto 48 da sessão. A CT para a primeira sessão foi determinada por um teste de ajuste que consistiu num teste incremental com cinco fases consecutivas de 10 minutos a uma cadência constante (60-80 RPM) de 90% a 130% da potência do limiar ventilatório, calculada a partir de dados do teste FatOx previamente realizado, com incrementos de 10% em cada etapa. A CT foi determinada pela fase em que a FC aumentou ≥ 3 batimentos entre fases e os níveis de lactato no sangue aumentaram ≥ 1 mmol/L em relação à fase anterior.

Comparador Ativo: Treino intervalado de alta intensidade (ND-HIIT)

Participantes que não receberam intervenção nutricional, mas foram inscritos num programa de exercício de treino intervalado de alta intensidade.

Outro: 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.

Comparador Ativo: Intervenção nutricional (D-INACT)

Participantes que receberam intervenção nutricional, mas não um programa de exercício.

Comportamental: 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.

Experimental: Intervenção Nutricional Treino Contínuo de Intensidade Moderada (D-MICT)

Participantes que receberam intervenção nutricional e foram inscritos num programa de exercício de treino contínuo de intensidade moderada.

Outro: O Treino Contínuo de Intensidade Moderada (MICT)

Os participantes atribuídos ao MICT completaram 3 sessões semanais, com 1-2 dias de descanso entre sessões, num cicloergómetro durante 12 semanas.

O MICT consistiu em 50 minutos de pedalada contínua a uma intensidade aproximadamente 10% acima do limiar de lactato, com uma cadência entre 60-80 rpm. A carga de treino (CT) foi aumentada em 10% se estas duas condições fossem cumpridas: 1) nenhum aumento de pelo menos 2 batimentos da frequência cardíaca do minuto 20 ao minuto 40 da sessão de treino, e 2) níveis de lactato abaixo de 2 mmol/L no minuto 48 da sessão. A CT para a primeira sessão foi determinada por um teste de ajuste que consistiu num teste incremental com cinco fases consecutivas de 10 minutos a uma cadência constante (60-80 RPM) de 90% a 130% da potência do limiar ventilatório, calculada a partir de dados do teste FatOx previamente realizado, com incrementos de 10% em cada etapa. A CT foi determinada pela fase em que a FC aumentou ≥ 3 batimentos entre fases e os níveis de lactato no sangue aumentaram ≥ 1 mmol/L em relação à fase anterior.

Comportamental: 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.

Experimental: Intervenção Nutricional Treino intervalado de alta intensidade (D-HIIT)

Participantes que receberam intervenção nutricional e foram inscritos num programa de exercício de treino intervalado de alta intensidade.

Comportamental: 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.

Outro: 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.

Alterações avaliadas no Stress Oxidativo

O nível de stress oxidativo foi avaliado pela concentração de 8-OHdG como biomarcador da oxidação do ADN. Foi utilizado um método de deteção ELISA competitivo em amostras de soro. Resumidamente, o padrão e a amostra foram adicionados a uma microplaca pré-revestida juntamente com o anticorpo de deteção biotinilado. Após lavagem, foi adicionado HRP-Streptavidina. Em seguida, foi adicionado o substrato TMB, e a reação foi terminada com uma solução ácida. A produção de cor foi medida espectrofotometricamente a um comprimento de onda de 450nm. Os valores de densidade ótica das amostras foram comparados com os da curva padrão para determinar a concentração de 8-OHdG (ng/ml).

Amostra de biópsia muscular: Atividade da Citrato Sintase

2-5 mg de músculo congelado foi homogeneizado numa diluição de 1:20 de CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, Reino Unido), com 1% de cocktail de inibidores de protease (P8340, Sigma-Aldrich, Dorset, Reino Unido) a 4 °C. O lisado foi centrifugado a 12.000 × g durante 10 minutos a 4 °C para sedimentar os detritos, e o sobrenadante foi armazenado a -80 °C. A atividade da CS foi quantificada em duplicado usando um kit de ensaio seguindo as instruções do fabricante (CS0720: Sigma-Aldrich, Dorset, Reino Unido). Brevemente, a atividade endógena foi registada através da emissão de cor por DTNB, e a atividade total foi calculada após a adição de oxaloacetato. A concentração de proteína foi determinada em duplicado usando um kit de ensaio de ácido bicinconínico (BCA1: Sigma-Aldrich, Dorset, Reino Unido), permitindo a normalização dos valores de atividade da CS para o conteúdo total de proteína (µmol/g/min). A atividade da CS e a concentração de proteína foram medidas em placas de 96 poços usando um leitor de microplacas a 412 nm e 562 nm, respetivamente (MQX200R2, Biotek instruments, Burlington, EUA).

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.

Alterações avaliadas na Resistência à Insulina: HOMA-IR

A resistência à insulina foi estimada utilizando o Modelo de Avaliação Homeostática da Resistência à Insulina (HOMA-IR), calculado como insulina em jejum (µU/mL) multiplicada pela glicose em jejum (mg/dL) dividida por 405.

Alterações avaliadas na Sensibilidade à Insulina: Índice de Matsuda

A sensibilidade à insulina de corpo inteiro foi estimada através do Índice de Matsuda, calculado como 10 000 / √[(glicemia em jejum (mg/dL) × insulina em jejum (µU/mL)) × (glicemia média durante o Teste de Tolerância à Glucose Oral (mg/dL) × insulina média durante o Teste de Tolerância à Glucose Oral (µU/mL))].

Avaliação das alterações na Sensibilidade à Insulina: Quicki

A sensibilidade à insulina foi estimada através do Índice Quantitativo de Verificação da Sensibilidade à Insulina (QUICKI), calculado como 1 / [log(insulina em jejum (µU/mL)) + log(glicose em jejum (mg/dL))].

Avaliou as alterações no Índice de Massa Corporal

O Índice de Massa Corporal (IMC) em quilogramas por metro quadrado (kg/m²) foi calculado como o peso corporal (kg) dividido pela altura ao quadrado (m²).

Alterações avaliadas na Antropometria: Perímetros

O perímetro da cintura (cm) foi medido no ponto médio entre a margem lateral inferior da 10.ª costela e a crista ilíaca, perpendicular ao eixo longitudinal do tronco. O perímetro abdominal (cm) foi medido ao nível do umbigo, perpendicular ao eixo longitudinal do tronco. O perímetro da anca (cm) foi medido ao nível da maior proeminência posterior das nádegas, perpendicular ao eixo longitudinal do tronco. O perímetro da coxa (cm) foi medido no ponto médio entre o grande trocânter e o côndilo tibial lateral. Todas as medições foram realizadas com o participante de pé, na posição vertical e relaxado. Cada medição foi realizada duas vezes, e uma terceira medição foi efetuada se a diferença excedesse 1%. A média dos dois registos mais semelhantes foi registada.

Alterações avaliadas na Antropometria: Relação Cintura-Anca

O rácio cintura-ança (WHR) foi calculado como a circunferência da cintura (cm) dividida pela circunferência da anca (cm).

Altura Avaliada

A altura (metros, m) foi medida usando um estadiómetro de parede (TANITA-LEICESTER HR-001, Tanita Corp., Tóquio, Japão) com os participantes em pé de acordo com as instruções do fabricante.

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).