Metabolismo Mitocondrial y Estrés Oxidativo en Función del Ejercicio Físico y la Asesoría Nutricional en la DM2 (MITOX)

Sin intervención: ND-INACT

Participantes que no recibieron ni la intervención nutricional ni el programa de ejercicio. Se les indicó que mantuvieran sus hábitos de vida normales en cuanto a actividad física y dieta.

Comparador activo: Entrenamiento continuo de intensidad moderada (ND-MICT)

Participantes que no recibieron intervención nutricional pero fueron inscritos en un programa de ejercicio de entrenamiento continuo de intensidad moderada.

Otro: El Entrenamiento Continuo de Intensidad Moderada (MICT)

Los participantes asignados al MICT completaron 3 sesiones semanales, con 1-2 días de descanso entre sesiones, en un cicloergómetro durante 12 semanas.

El MICT consistió en 50 minutos de pedaleo continuo a una intensidad aproximadamente un 10 % por encima del umbral de lactato, con una cadencia entre 60-80 rpm. La carga de entrenamiento (TL) se aumentó en un 10% si se cumplían estas dos condiciones: 1) no hubo un aumento de al menos 2 latidos de la frecuencia cardíaca desde el minuto 20 al minuto 40 de la sesión de entrenamiento, y 2) niveles de lactato por debajo de 2 mmol/L en el minuto 48 de la sesión. La TL para la primera sesión se determinó mediante una prueba de ajuste que consistió en una prueba incremental con cinco fases consecutivas de 10 minutos a una cadencia constante (60-80 RPM) desde el 90% hasta el 130% de la potencia del umbral ventilatorio, calculada a partir de datos de la prueba FatOx previamente realizada, con incrementos del 10% en cada paso. La TL se determinó por la fase en la que la FC aumentó en ≥ 3 latidos entre fases y los niveles de lactato en sangre aumentaron en ≥ 1 mmol/L respecto a la fase anterior.

Comparador activo: Entrenamiento por intervalos de alta intensidad (ND-HIIT)

Participantes que no recibieron intervención nutricional pero fueron inscritos en un programa de ejercicio de entrenamiento en intervalos de alta intensidad.

Otro: 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 activo: Intervención nutricional (D-INACT)

Participantes que recibieron intervención nutricional pero no un programa de ejercicio.

Conductual: 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: Intervención Nutricional Entrenamiento Continuo de Intensidad Moderada (D-MICT)

Participantes que recibieron intervención nutricional y se inscribieron en un programa de ejercicio de entrenamiento continuo de intensidad moderada.

Otro: El Entrenamiento Continuo de Intensidad Moderada (MICT)

Los participantes asignados al MICT completaron 3 sesiones semanales, con 1-2 días de descanso entre sesiones, en un cicloergómetro durante 12 semanas.

El MICT consistió en 50 minutos de pedaleo continuo a una intensidad aproximadamente un 10 % por encima del umbral de lactato, con una cadencia entre 60-80 rpm. La carga de entrenamiento (TL) se aumentó en un 10% si se cumplían estas dos condiciones: 1) no hubo un aumento de al menos 2 latidos de la frecuencia cardíaca desde el minuto 20 al minuto 40 de la sesión de entrenamiento, y 2) niveles de lactato por debajo de 2 mmol/L en el minuto 48 de la sesión. La TL para la primera sesión se determinó mediante una prueba de ajuste que consistió en una prueba incremental con cinco fases consecutivas de 10 minutos a una cadencia constante (60-80 RPM) desde el 90% hasta el 130% de la potencia del umbral ventilatorio, calculada a partir de datos de la prueba FatOx previamente realizada, con incrementos del 10% en cada paso. La TL se determinó por la fase en la que la FC aumentó en ≥ 3 latidos entre fases y los niveles de lactato en sangre aumentaron en ≥ 1 mmol/L respecto a la fase anterior.

Conductual: 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: Intervención nutricional Entrenamiento de intervalos de alta intensidad (D-HIIT)

Participantes que recibieron intervención nutricional y se inscribieron en un programa de ejercicio de entrenamiento interválico de alta intensidad.

Conductual: 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.

Otro: 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.

Cambios evaluados en el Estrés Oxidativo

El nivel de estrés oxidativo se evaluó mediante la concentración de 8-OHdG como biomarcador de la oxidación del ADN. Se empleó un método de detección ELISA competitivo en muestras de suero. Brevemente, el estándar y la muestra se añadieron a una microplaca previamente recubierta junto con el anticuerpo de detección biotinilado. Después del lavado, se añadió HRP-Estreptavidina. Luego, se añadió el sustrato TMB y la reacción se detuvo con una solución ácida. La producción de color se midió espectrofotométricamente a una longitud de onda de 450 nm. Los valores de densidad óptica de las muestras se compararon con los de la curva estándar para determinar la concentración de 8-OHdG (ng/ml).

Muestra de biopsia muscular: actividad de la citrato sintasa

Se homogeneizaron 2-5 mg de músculo congelado en una dilución 1:20 de CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, Reino Unido), con 1 % de cóctel inhibidor de proteasas (P8340, Sigma-Aldrich, Dorset, Reino Unido) a 4 °C. El lisado se centrifugó a 12.000 × g durante 10 min a 4 °C para sedimentar los restos celulares, y el sobrenadante se almacenó a -80 °C. La actividad de CS se cuantificó por duplicado utilizando un kit de ensayo siguiendo las instrucciones del fabricante (CS0720: Sigma-Aldrich, Dorset, Reino Unido). Brevemente, la actividad endógena se registró mediante la emisión de color por DTNB, y la actividad total se calculó tras la adición de oxalacetato. La concentración de proteína se determinó por duplicado utilizando un kit de ensayo de ácido bicinconínico (BCA1: Sigma-Aldrich, Dorset, Reino Unido), lo que permitió normalizar los valores de actividad de CS al contenido total de proteína (μmol/g/min). La actividad de CS y la concentración de proteína se midieron en placas de 96 pocillos utilizando un lector de microplacas a 412 nm y 562 nm, respectivamente (MQX200R2, Biotek instruments, Burlington, EE. UU.).

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.

Cambios evaluados en la Resistencia a la Insulina: HOMA-IR

La resistencia a la insulina se estimó utilizando el Modelo de Evaluación Homeostática de la Resistencia a la Insulina (HOMA-IR), calculado como insulina en ayunas (µU/mL) multiplicado por glucosa en ayunas (mg/dL) dividido por 405.

Cambios evaluados en la Sensibilidad a la Insulina: Índice de Matsuda

La sensibilidad a la insulina de todo el cuerpo se estimó mediante el Índice de Matsuda, calculado como 10,000 / √[(glucosa en ayunas (mg/dL) × insulina en ayunas (µU/mL)) × (glucosa media durante la Prueba de Tolerancia Oral a la Glucosa (mg/dL) × insulina media durante la Prueba de Tolerancia Oral a la Glucosa (µU/mL))].

Cambios evaluados en la Sensibilidad a la Insulina: Quicki

La sensibilidad a la insulina se estimó mediante el Índice Cuantitativo de Sensibilidad a la Insulina (QUICKI), calculado como 1 / [log(insulina en ayunas (µU/mL)) + log(glucosa en ayunas (mg/dL))].

Evaluados los cambios en el Índice de Masa Corporal

El Índice de Masa Corporal (IMC) en kilogramos por metro cuadrado (kg/m²) se calculó como el peso corporal (kg) dividido por la altura al cuadrado (m²).

Evaluación de cambios en Antropometría: Perímetros

El perímetro de la cintura (cm) se midió en el punto medio entre el margen lateral inferior de la décima costilla y la cresta ilíaca, perpendicular al eje longitudinal del tronco. El perímetro abdominal (cm) se midió a la altura del ombligo, perpendicular al eje longitudinal del tronco. El perímetro de la cadera (cm) se midió a la altura de la prominencia posterior más grande de los glúteos, perpendicular al eje longitudinal del tronco. El perímetro del muslo (cm) se midió en el punto medio entre el trocánter mayor y el cóndilo tibial lateral. Todas las mediciones se realizaron con el participante de pie y relajado. Cada medición se tomó dos veces, y se realizó una tercera medición si la diferencia superaba el 1%. Se registró la media de los dos registros más similares.

Cambios evaluados en Antropometría: Relación Cintura-Cadera

La relación cintura-cadera (RCC) se calculó como la circunferencia de la cintura (cm) dividida por la circunferencia de la cadera (cm).

Altura evaluada

La altura (metros, m) se midió utilizando un tallímetro de pared (TANITA-LEICESTER HR-001, Tanita Corp., Tokio, Japón) con los participantes de pie de acuerdo con las instrucciones del 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).