The overall aim of our study is to identify the impact of VitC on liver fibrosis and/or steatosis. This is achieved by confirming liver fibrosis and/or steatosis by fibroscan and providing these patients with VitC supplements for 6 months after which liver fibrosis and/or steatosis will be repeated by fibroscan. Also inflammatory markers, liver enzymes and blood metabolic parameters will be measured pre and post intervention. This will help identify drivers of NAFLD in the Middle Eastern population and apply that knowledge in the development of more effective approaches to diagnosis, treatment, and prediction of disease prognosis.
Therefore, specifically we will:
Ø Identify the impact of VitC on improvement of liver fibrosis and steatosis in NAFLD patients as reported by fibroscan
Ø Determine the level of liver enzymes in patients receiving daily doses of VitC in comparison to control
Ø Identify the impact of VitC on inflammation by measuring serum TNFα, IL-1 and IL-6 in NAFLD patients and controls
Ø Identify impact of VitC on metabolic parameters including HbA1c, fasting blood sugar, serum insulin, triglycerides, LDL, cholesterol and iron
This is a prospective double blind randomized control trial comparing the VitC use to placebo in patients with NAFLD. Post screening and consent process, the patients will be randomized in 1:1 ratio to receive placebo or VitC supplements.
o Adults patients with confirmed NAFLD as reported by fibroscan at the AUBMC hepatobiliary unit
o Patients with advanced steatosis
o History of liver surgery
o History of kidney stones
o Presence of kidney conditions
o Presence of gout
In order to detect a 1 degree decrease in liver fibrosis or steatosis by fibroscan in the study group in comparison to control group and considering a 10% dropout rate with a margin of error of 5%, a power of 0.80, the desired sample size is 70 with 35 patients per group. Dr. Hani Tamim from CRI unit was consulted for sample size calculation.
- Assignment of interventions
Participants will be randomly assigned to their treatment in a 1:1 ratio, according to a computer generated sequence, stratified by type of supplement, using permuted blocks of variable sizes. Random sequence generation will be selected by biostatistician independent of the research team using computer software and will hold details of the blocking and block sizes in a separate document not shared with the study team.
The allocation arm will be blinded to the PI, co-PIs, the research team, and the participants will be blinded about the allocation arm. If any complications or side effects occur, the treatment arm will be revealed.
- Blood withdrawal and laboratory tests
Blood will be collected from patients at baseline and at 6 months follow up or at withdrawal time. A total of 4 tubes of blood will be collected from each patient at two time points each with 5 ml of blood; one hematology tube, one chemistry tube, one EDTA tube will be sent directly to AUBMC laboratory to run the samples on the spot as per laboratory medicine protocol. One EDTA tube will be used to collect blood for ELISA samples and will be centrifuged for 15 minutes at 1000 x g within 30 minutes of collection. Serum will be collected, put in aliquots and preserved at -20⁰C. All the stored samples will be coded. Only the principal investigator and his research team will have a list linking the participants’ names to their codes.
- TNF-α, IL-1 and IL-6 ELIZA tests
TNF-α, IL-1 and IL-6 will be measured with the enzyme-linked immunosorbent assay technique (Quantikine ELISA; R&DSystems, Minneapolis, MN, USA) and will be done according to manufacture’s protocol. Using a microplate reader set to 450 nm absorbance will be measured. The duplicate readings for each standard, control, and sample will be averaged and subtracted from the average zero standard optical density. A standard curve will be created by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit and concentrations will be calculated. We will request approval to use the AUB Faculty of Medicine Molecular Biology Core Facility Lab to perform the ELISA experiments.