Design
The Nonalcoholic Fatty Liver Disease-Intermittent Calorie Restriction trial was designed as a prospective, two-arm, open-label RCT to investigate the efficacy of 12-week ICR in reducing liver fat content in NAFLD patients. The study will recruit 72 patients who will be followed up actively according to the protocol for 24 weeks. Patients will be classified according to their BMI (obese group: BMI ≥25 kg/m2 [36 patients] and non-obese group: BMI <25 kg/m2 [36 patients]). An enrolment period of 12 months is expected.
Study setting
The FLICR trial will recruit patients from Ewha Womans University Mokdong Hospital. This study will only recruit Korean patients. The Department of Nutritional Science and Food Management, Ewha Womans University will collaborate with this study.
Informed consent
The study nurse or investigators will be obtaining the informed consent. Each participant must have voluntarily given informed consent and signed by the patient and an investigator before any study-specific procedures are initiated. The patients will also be required to sign informed consent for the use of clinical and biological data in ancillary studies.
Recruitment process and allocation
The randomized allocation will be conducted at Ewha Womans University Mokdong Hospital.
Eligible patients will be screened by the principal and sub-investigator (hepatologists), and will will undergo screening to investigate whether they fulfill the eligibility criteria and none of the exclusion criteria for the study.
Eligibility criteria
Inclusion criteria:
- NAFLD diagnosed by (1) histologic assessment with a fat accumulation of >5% of the liver's weight on biopsy or (2) radiologic assessment with MRI-PDFF ≥8%
- Age between 19 and 75 years
- Capability to understand the study and the individual consequences of participation
- Signed and dated declaration of agreement in the forefront of the study
Exclusion Criteria:
- Daily alcohol consumption >30 g in men and >20 g in women
- Other causes of chronic liver disease (hepatitis B, C, D, and E virus infection, human immunodeficiency virus, autoimmune diseases, chronic cholestatic liver disease, drug induced liver injury, hereditary hemochromatosis, Wilson disease, and α-1-antitrypsin deficiency)
- Liver cirrhosis
- Hepatocellular carcinoma
- Medications that cause liver disease or secondary NAFLD (e.g., tamoxifen, systemic corticosteroids, methotrexate, tetracycline, estrogens, and valproic acid)
- Changes in body weight > 5% in the last three months
- Intake of medical treatment for NAFLD/NASH in the last six months (except vitamin E)
- Diabetes
- Pregnancy
- Patients after organ transplantation
- Missing or lacking consent capability
Randomization
For eligible patients, the principal- or sub-investigator will complete the patient enrollment form and enroll the patients. Patients will be randomly assigned to receive ICR or SOC via a computer program using a stratified block randomization method, adjusted for BMI (≥25 and <25 kg/m2). Therefore, the patient assignment will be concealed from the investigator, until the allocation of intervention. After the participants have been assigned to the ICR or SOC group through the randomization procedure, they will be informed by the principal- or sub-investigator. The FLICR trial is an open trial and no blinding will be used in any part of the study procedure.
Study procedures
The patients will be followed up for 24 weeks. Detailed dietary consultation will be conducted by a nutritionist at the baseline visit, and each participant will be provided with a written summary of the dietary advice. Dietary intervention will be performed during the first 12 weeks, and study visits will be performed at baseline and every week during the intervention period (telephone consultation with 24-hour recall or visit to clinic with a 3-day diet diary). Twelve weeks hereafter is the maintenance period, and patients will be followed up at the end of the study period. The schedule of the assessments of the study procedures is summarized in Table 1.
Follow-up at 12 weeks (end of treatment) will include the following mandatory tests: assessment of liver fat by MRI-PDFF, liver fibrosis by magnetic resonance elastography (MRE), anthropometrics, body composition analysis (BCA), visceral adipose tissue (VAT), food intake, quality of life (QOL), liver metabolites, microbiome, and hormones (leptin, adiponectin, ghrelin, GLP-1, apelin, and osteopontin). Follow-up will be terminated at the end of the maintenance period (24 weeks), and anthropometrics, BCA, VAT, food intake, and QOL will be assessed. All study participants, except those who withdraw consent for the study, will be followed up for the study period.
Interventions
Active comparator (ICR arm): On two non-consecutive days per week, participants in the ICR (5:2 diet) group will be instructed to consume 500 kcal/day for women and 600 kcal/day for men. Recipes will be provided with suggestions for meals that do not exceed calorie restriction. For the remaining five days of the week, they will receive instructions and recipes that follow the Korean Dietary Reference Intakes, with an intake limit of 2,000 kcal/day for women and 2,500 kcal/day for men. The percentage of energy (E%) from the different macronutrients in the recipes will be 45–60 E% carbohydrates, 25 E% fat, and 10–20 E% protein.
Placebo comparator (SOC arm): The SOC group will receive 80% of the standard calories (1200–1500 kcal/day or reducing 500–1000 kcal/day from standard calories). They will receive individualized guidance from a hepatologist on how to choose a healthy diet, reduce the intake of sweets and saturated fatty acids, increase sources of unsaturated fat, avoid large portions, and regularly eat three meals per day.
Criteria for discontinuing allocated interventions
Patients may be discontinued from the study at any time if, in the opinion of the investigator, it is medically necessary or if it is an expressed wish of the patient. The patients are free to discontinue their participation in the trial at any time.
Outcomes
The primary objective is to evaluate the impact of ICR on liver steatosis measured using MRI-PDFF (time frame: baseline and 12 weeks), and the outcome of interest is a change in the MRI-PDFF value of at least 30% with ICR. Secondary objectives include changes in liver fibrosis by MRE (time frame: baseline and 12 weeks); anthropometrics including BMI (kg/m2) (time frame: baseline, 12, and 24 weeks); BCA measured using Inbody (time frame: baseline, 12, and 24 weeks); VAT measured using fat computed tomography (CT) (time frame: baseline, 12, and 24 weeks); QOL score measured using Chronic Liver Disease-NAFLD questionnaire (CLDQ) (time frame: baseline, 12, and 24 weeks); hormones including leptin, adiponectin, ghrelin, GLP-1, apelin, and osteopontin (time frame: baseline and 12 weeks); liver metabolites including diglycerides, fatty acid, phosphatidylethanolamines, phosphatidylcholines, methionine, S-adenosyl-l-methionine, and methylthioadenosine (time frame: baseline and 12 weeks); and gut microbiota analyzed with 16S rRNA gene sequencing (time frame: baseline and 12 weeks). Polymorphisms of PNPLA3, TM6SF2, TM4SF5, SREBF2, MBOAT7-TMC4, HSD17B13, and adenine insertion (A-INS) will also be analyzed using next-generation sequencing (NGS). Objectives of this study are summarized in Table 2.
Observation of adverse events (AEs)
All AEs during the study will be recorded with the following data: date of onset and date of completion (if applicable), severity of AEs, investigator’s view on the relationship to dietary intervention, taken actions on AEs, treatment of the AEs, cause of the event (if known), and outcome. The AE intensities will be graded according to the National Cancer Institute’s Adverse Event Common Terminology version 4.03.
Sample size determination
In a previous study, the percentage of participants who reached the endpoint of a relative reduction in liver fat content >30% measured using MRI-PDFF after 12 weeks was 80% in the ICR group and 29% in the control group [17]. Based on this assumption, 72 participants (36 in the obese group and 36 in the non-obese group) are planned to be randomized into the two treatment groups in a 1:1 ratio (ICR:SOC) to achieve 80% power for superiority comparison (Fisher’s exact test) of the primary objective between the two treatment groups, with a two-sided type-I error of 5% and allowing for a 10% dropout rate. G*Power 3.1.9.4 was used to calculate the sample size.
Data management
Investigators will enter the data required by the protocol into the case report forms (CRF). The principal investigator is responsible for assuring that data entered into the CRF is complete and accurate, and that entry is performed in a timely manner. The signature of the investigator will attest the accuracy of the data on each CRF. All data management activities will be completed prior to the final closure of the database.
Data monitoring committee
An independent data monitoring committee will be established with two individuals from the Department of Biostatistics, Ewha Womans University Mokdong Hospital. The management team will observe the progress and data monthly by mail and web conferencing. If the monitoring committee decides that on-site monitoring is necessary, members will visit the site for face-to-face monitoring.
Confidentiality
All patient data collected and processed for the purposes of this study will be managed by the investigators with adequate precautions to ensure the confidentiality of the data and in accordance with applicable national laws and regulations on personal data protection. No patient-identifiable data will be obtained. In any presentation of the results of this study, at meetings, or in publications, the patients’ identities will remain confidential. In all activities, the General Data Protection Regulation will be followed to ensure protection of sensitive personal information, and the study will be performed in accordance with the World Health Organization guidelines for good clinical practice.
Statistical analyses
Descriptive statistics will be computed for all variables: mean and standard deviation for continuous variables, unless stated otherwise, and frequency and percentage for categorical variables. Intention-to-treat will be performed mostly. Comparisons will be made within groups (post- vs. pre-intervention for each group) and between groups (ICR vs. SOC). Independent and paired t-tests will be used to compare continuous variables between and within groups, respectively. SPSS version 23 (SPSS Inc., Chicago, United States) will be used for statistical analysis. All two-sided P value <0.05 will be considered as statistically significant.