Patients
In the present study, patients were consecutively recruited by local advertisement from overweight/obese (25 ≤ body mass index [BMI] < 40 kg/m2) adult (18 ≤ age < 60 years) volunteers visiting the gastrointestinal outpatient clinic of Golestan Hospital of Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran. Eligible NAFLD patients required to have a grade 2–3 (i.e., moderate-to-severe) hepatic steatosis on ultrasonography (US) in the absence of any secondary causes of hepatic steatosis, including excessive alcohol intake (≥ 30 g/day for males and ≥ 20 g/day for females) and other etiologies of liver disease and steatosis.
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008), as reflected in a priori approval by the AJUMS’s human research committee (reference number: IR.AJUMS.REC.1396.138). Written informed consent was obtained from each patient included in the study. Patient recruitment took place from September 23, 2017 through January 01, 2018, and the last recruited patient completed the final follow-up visit on March 26, 2018. This trial was registered at https://www.irct.ir as IRCT2017060210181N10, in which the full trial protocol can be accessed.
Study design
In the present single-center, multi-arm, parallel-group RCT, a total of 100 eligible NAFLD patients were initially studied for a 1-week run-in phase to establish their current energy requirements. Throughout this phase, patients were instructed to provide a 3-days dietary record (2 weekdays and 1 weekend day). Of these, 8 patients declined to participate in the study (Fig. 1), leaving a total of 92 eligible patients (52.2% female; mean age 37.2 years) available for randomization at the end of the run-in phase. Using computer-generated random permuted blocks of size 4 or 8 stratified by gender (male or female) and hepatic steatosis (grade 2 or 3), patients were assigned in a 1:1:1:1 fashion to 4 groups (n = 23) labeled as control, β-cryptoxanthin, HP-diet, and HP-diet and β-cryptoxanthin to receive their corresponding outpatient interventions for 12 weeks. Patients in the HP-diet and β-cryptoxanthin group received a hypocaloric HP-diet supplemented with 6 mg/day of β-cryptoxanthin, while those in the HP-diet group were given the same diet along with a β-cryptoxanthin placebo. On the other hand, those in the β-cryptoxanthin group received a hypocaloric normal protein (NP)-diet with 6 mg/day of β-cryptoxanthin, whereas patients in the control group were given the same diet and a β-cryptoxanthin placebo. The 12-week intervention period of this study was chosen according to a recent work in which an intervention period of > 10 weeks was required for a hypocaloric HP-diet to improve liver enzymes in patients with NAFLD [22].
The random allocation sequence was generated by an independent statistician at the AJUMS School of Health and was given in sequentially numbered, opaque, and sealed envelopes to the study clinician responsible for assessing and enrolling the patients at the Golestan Hospital. After enrollment and completion of all baseline assessments, each patient received a sealed envelope with his/her full name written on it and was referred to an independent dietician at the AJUMS School of Paramedical Sciences who opened the envelopes and assigned patients to their allocated interventions. The random allocation sequence was concealed, and patients, healthcare providers, data collectors, and outcome adjudicators remained blinded to the allocated interventions until the last recruited patient completed the final follow-up visit.
This study adheres to CONSORT guidelines and included a completed CONSORT checklist as an additional file.
Interventions
The patients’ weight maintenance level of energy intake at baseline was calculated using the standard equations for prediction of total energy expenditure (TEE) in overweight and obese individuals 19 years and older according to their gender, age, weight, height, and physical activity level [29]. The calculated TEE value was then used by an independent dietician to prescribe an individually tailored, food exchange-based, 500 kcal/day (i.e., 2092 kJ/day) deficit hypocaloric diet for each patient. The percent of energy intake from carbohydrate, fat, and protein in diets prescribed to the patients in the control and β-cryptoxanthin groups were approximately 55%, 30%, and 15%, respectively. The corresponding values in diets prescribed to those in the HP-diet and HP-diet and β-cryptoxanthin groups were approximately 45%, 30%, and 25%, respectively. These values were chosen since they are within the acceptable macronutrient distribution ranges presented in the dietary reference intake tables [29], and also because they are easily achievable. In all study groups, animal and plant sources of protein each contributed to about 50% of overall dietary protein intake.
β-Cryptoxanthin powder, containing 1% β-cryptoxanthin and approximately 99% starch, was purchased from the Shanghai Tianfu Chemical Co., Shanghai, China. β-cryptoxanthin powder and pure starch, as β-cryptoxanthin placebo, were then encapsulated in 600 mg, tasteless, odorless, and identical-in-appearance oral capsules at the AJUMS School of Pharmacy. Before randomization, the capsules were packed in identical, opaque, and sealed containers by a person other than the researchers, coded with letters A or B, and handed over to the person responsible for assigning patients to their allocated interventions. Patients were asked to take 1 capsule/day of β-cryptoxanthin or β-cryptoxanthin placebo with a glass of water after dinner. The decision to choose a 6 mg/day dosage of β-cryptoxanthin supplementation in the present work was based on a previous dose-finding study in which this particular dosage was associated with the most significant rise in serum β-cryptoxanthin level without causing any serious adverse events [30].
In addition to the scheduled follow-up visits at week 6 and 12 of the intervention period, weekly phone follow-ups were performed to minimize the drop-out rate. Adverse events and adherence, defined as taking ≥ 90% of prescribed capsules, were recorded at every scheduled follow-up visit.
Outcome measures
Objective assessment of the primary (i.e., serum levels of liver enzymes and grade of hepatic steatosis) and secondary (i.e., improvement in hepatic steatosis: achieving grade 0 hepatic steatosis) outcome measure was conducted at baseline and study endpoint. Hepatic steatosis was diagnosed via upper abdominal US performed by a trained radiologist at the Golestan Hospital using a Toshiba Nemio 30 ultrasound system (Toshiba Co., Tokyo, Japan) equipped with a 3.5 MHz linear transducer and was classified into 4 grades as follows: grade 0 (no steatosis); grade 1 (mild steatosis); grade 2 (moderate steatosis); and grade 3 (severe steatosis). Fasting serum levels of liver enzymes (IU/L) including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) were measured via enzymatic methods using commercially available kits (Pars Azmun Co., Tehran, Iran) on an Alcyon 300 automated analyzer (Abbott Laboratories, Abbott Park, IL, USA) by a trained lab technician at the Golestan Hospital on the same day as the blood sampling.
Other variables
Serum β-cryptoxanthin level (μg/mL) was measured at baseline and week 12 of the intervention period using isocratic reverse-phase high-performance liquid chromatography. The data on demographics including gender (male, female) and age (year) were obtained by a general questionnaire at baseline. Anthropometric variables including weight (kg), height (cm), and waist circumference (WC; cm) were recorded at baseline and every scheduled follow-up visit in accordance to the standard guidelines. BMI (kg/m2) was then computed using the following standard formula: BMI = weight (kg)/[height (m)]2. In addition to the run-in phase, each patient provided a 3-days dietary record at week 6 and week 12 of the intervention period. The dietary records were then reviewed and analyzed by the Nutritionist IV (First Databank, San Bruno, CA, USA) to determine patients’ daily energy intake (Kcal/day) and percent of energy intake from carbohydrate, fat, and protein ((% of energy). Physical activity (metabolic equivalent-min/week) was assessed at baseline and every scheduled follow-up visit using the valid and reliable Iranian version of the International Physical Activity Questionnaire-Short Form [31,32,33]. The study clinician at the Golestan Hospital administered the general and physical activity questionnaires via face-to-face interviews, reviewed and analyzed the dietary records, and recorded the anthropometric variables.
Statistical analysis
The sample size was estimated based on one of the primary outcome measures (i.e. serum ALT level). Using the standard deviations obtained from previous studies [24, 27], a minimum sample size of 18 patients per group was required to detect a minimum difference of 20 IU/L in mean serum ALT level among study groups with a statistical power of 80% and a type-1 error of 5%. However, to allow for a drop-out rate of about 20%, it was finally decided that 23 patients per group would be recruited.
Statistical analyses were performed using the intention-to-treat approach, including all randomized patients (N = 92). The chi-square test or the Fisher’s exact test was used for comparison of categorical variables among study groups as appropriate, while pairwise within group comparison of grade of hepatic steatosis was conducted using the Wilcoxon signed-rank test. For continuous variables, the normality assumption was initially evaluated by the Shapiro–Wilk test, and those with a non-normal distribution were normalized by log-transformation prior to any further analysis. Pairwise within group comparison of continuous variables was then performed using the paired sample T-test. The one-way analysis of variance (ANOVA) was used for comparison of continuous variables among study groups, while the one-way analysis of covariance (ANCOVA) was applied to compute and compare the multivariable adjusted means of continuous outcomes (e.g., serum levels of liver enzymes at study endpoint) among study groups. For each continuous outcome, the corresponding baseline value, gender, age, and 12-week changes in BMI and WC were included as covariates in the ANCOVA. In case of any significant differences, the ANOVA and the ANCOVA were followed by pairwise between group comparisons using the Bonferroni post-hoc test to adequately adjust for multiple comparisons. The IBM SPSS Statistics 21 (IBM Corp., Armonk, NY, USA) was used to conduct all analyses, considering a 2-sided p value of < 0.050 as statistically significant. Data were presented as n (%) or mean [95% confidence intervals].