Chia Supplementation and Non Alcoholic Fatty Liver Disease

Participants were chosen from the control group of the Genetics of the Atherosclerotic Disease (GEA, for its initials in Spanish) study, performed at the Institute National of Cardiology "Ignacio Chávez" in Mexico City, Mexico. The protocol was approved by the Research and Ethics Committee of the Instituto Nacional de Cardiología "Ignacio Chavez" under the number 16-980. Candidates that accepted to participate in the study signed voluntarily the informed consent.

In order to know eating habits and standardize macronutrient dietary composition, 24-hour dietary recalls will be applied in the first visit, considering two weekdays and one day of the weekend. Patients will be instructed to maintain constant their physical activity throughout the study. To standardize macronutrient dietary composition, an isocaloric diet (55% carbohydrate, 30% fat, and 15% protein diet) will be indicated two weeks before starting chia supplementation. After this, participants will be instructed to keep up this macronutrient composition during all the intervention period. 30 packages of 25 g of chia seeds will be provided to each patient monthly, with the instruction to mill one package per day, pointing out on the relevance of consume the milled chia accompanied by with water, salads, cereal, or other dishes, from breakfast through lunch but always before 6:00 PM. To favor treatment adherence and record adverse events (loss appetite loss, constipation, diarrhea, flatulence and nausea, allergy or chía chia intolerance), patients will be contacted once a week during the intervention. Adherence will be determined by counting empty chia packages, and evaluation of alpha linolenic acid (ALA) concentration in plasma, which is the chia's seeds main fatty acid compound.

Anthropometric evaluation, laboratory test, and computed tomography studies will be made at baseline and after the 8-wk intervention.

Nutritional intervention and food intake evaluation In order to know eating habits and standardize macronutrient dietary composition, 24-hour dietary recalls will be applied in the first visit, considering two weekdays and one day of the weekend. Patients will be instructed to maintain constant their physical activity throughout the study. To standardize macronutrient dietary composition, an isocaloric diet (55% carbohydrate, 30% fat, and 15% protein diet) will be indicated two weeks before starting chia supplementation. After this, participants will be instructed to keep up this macronutrient composition during all the intervention period. 30 packages of 25 g of chia seeds will be provided to each patient monthly, with the instruction to mill one package per day, pointing out on the relevance of consume the milled chia accompained by with water, salads, cereal, or other dishes, from breakfast through lunch but always before 6:00 PM. To favor treatment adherence and record adverse events (loss appetite loss, constipation, diarrhea, flatulence and nausea, allergy or chía chia intolerance), patients will be contacted once a week during the intervention.

Adherence will be determined by counting empty chia packages, and evaluation of alpha linolenic acid (ALA) concentration in plasma, which is the chia's seeds main fatty acid compound. Participants wre excluded when the adherence was lower tan 80% according to the package counting, or when plasma ALA concentration increased less than 30%.

Anthropometric evaluation, laboratory test, and computed tomography studies will be made at baseline and after the 8-wk of diet intervention.

Anthropometric evaluation: Weight and height will be recorded using a calibrated scale and wall stadiometer, with an accuracy of 0.1 Kg kg and 0.1 cm, after removing excess clothing and shoes. The body mass index (BMI) was calculated as weight (kg)/height (m2). Waist circumference will be measured with a non-stretch tape, at the midway between the lowest rib and the iliac crest without clothes around the waist.

Laboratory tests: After 10-h fasting and 20 min in sitting position, venous blood will be collected in assay tubes without anticoagulant and in tubes with K2-EDTA (1.8 mg/mL). Glucose, total cholesterol, triglyceride, and high density lipoprotein cholesterol (HDL-C) concentrations will be determined using direct standard enzymatic colorimetric methods on a COBAS c311 (Roche Diagnostics, Mannheim, Germany). Low density lipoprotein cholesterol (LDL-C) concentration was estimated using the De Long formula. The reproducibility and precision of these determinations in our laboratory is assessed by the Center for Disease Control and Prevention Lipids Standardization Program (LSP-CDC, Atlanta, GA, USA). Plasma free fatty acids (FFA) will be measured by an enzymatic-colorimetric assay (Wako Diagnostics, Chuo-Ku Osaka, Japan). Total fatty acids, including ALA will be extracted according to Folch method, and analyzed in a Shimadzu GC-8A gas chromatograph equipped with an SP2330 capilar column (25m x 0.25 mm x 0.25). Fatty-acid concentrations will be calculated in relation to heptadecanoic acid methyl ester as internal standard, fatty acids peaks will be identified by using the Supelco 37 component FAME Mix (CRM47885). A plasma control sample will be run in each extraction assay, to obtain an ALA inter-assay coefficient variation.

Computed tomography study: Computed tomography (CT) is a validated method for measuring visceral adipose tissue (VAT) and evaluate non alcoholic fatty liver disease. In the present study, these measurements will be obtained using a 64-slice scanner (Somatom Cardiac Sensation 64, Forcheim, Bavaria, Germany). To determine the liver and spleen attenuation index, a single slice CT scan is obtained at the level of T11-T12 or T12-L1. Fatty liver is defined as a liver/spleen attenuation ratio lower than 1.0. To calculate the amount of total abdominal tissue (TAT) and VAT, a single slice scan is obtained at the level of L4-L5, the area is expressed in square centimeters (cm2). Subcutaneous abdominal tissue (SAT) was calculated by subtracting the VAT from the TAT area.