Interventional Strategy in Tackling Emerging Non-alcoholic Fatty Liver Disease in Childhood Obesity
Condition: Obesity
MeSH terms: Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease; Obesity; Pediatric Obesity
Study type: Interventional
Phase: Phase 4

Summary: 
      The goal of this clinical trial is to investigate the effects of tocotrienol-rich fraction
      vitamin E supplementation on liver enzymes in overweight and obese children with
      non-alcoholic fatty liver disease as compared to placebo.

      The main question[s] it aims to answer are:

        1. Does supplementation of tocotrienol-rich fraction vitamin E reduce the level of liver
           enzymes and improve liver steatosis in non-alcoholic fatty liver disease among
           overweight and obese children?

        2. Does tocotrienol-rich fraction vitamin E supplementation improve the level of liver
           steatosis by reducing the level of DNA damage?

      Participants will :

        1. consume daily either a dose of 50 mg of tocotrienol-rich fraction (TRF) vitamin E or a
           placebo for 6 months.

        2. Routine clinical assessments include weight, height, waist circumference, and BMI.
           Fasting glucose, and fasting serum lipid.

        3. The following investigations were performed upon recruitment and following 6 months of
           intervention: (i) liver biomarker and enzymes; (ii) DNA damage; (iii) TNFα, IL-6 and
           IFN-gamma genes; (iv) Fibroscan.
    

Detailed description: 
      Methodology This study enrolled a diverse group of participants aged 10 to 18, regardless of
      gender, who were diagnosed with fatty liver disease detected via ultrasonography and
      abnormally high alanine transaminase levels (at least two-fold higher than the upper limits
      for their respective genders). The trial consisted of 29 patients, with 15 receiving a daily
      oral dose of 50 mg TRF and 14 receiving a placebo for a duration of six months. Various
      clinical parameters, LIVERFASt in vitro diagnostic test, fibroscan, biochemical parameters,
      DNA damage, and gene expression, both at the outset and at the end of the study were
      monitored.

      The amount of blood sample taken was 13mls each at entry and on completion at 6 months.
      Details of these tests are as shown below:

      Transient Elastography (FibroScan):

      This is a non-invasive test to measure liver stiffness, kPa (indicator of liver fibrosis) and
      to detect degree of fat accumulation (CAP range value of 100-400 dB/m), liver stiffness of >
      8kPa indicates advanced fibrosis while CAP of > 263 indicates fatty liver. During the
      screening, the participant lied down on the examination bed with his/her right hand behind
      his/her head. A probe was placed by the investigator between the right ribs of the patient.
      Measurements were recorded into the machine (FibroScan® 502 Touch). The screening is quick
      and easy, usually taking less than 15 minutes. The scanned steatosis was scored and graded.

      Determination of DNA Damage The DNA Damage pre and post intervention were conducted by using
      CometAssay Kit (Trevigen, Gaithersburg, USA) following the manufacturer's protocol. Briefly,
      blood cells in 5 μL medium was suspended in 70 μL warm 0.6% low-melting point (LMA) agarose
      (Boehringer Mannheim, Germany) (DNAse-free, RNAsefree). Slides were allowed to sit in the
      alkaline buffer for 20 min to allow unwinding of DNA strands and expression of alkali-labile
      damage. Electrophoresis was performed for 20 min at 300 mA and 25 V. Following dropwise
      neutralization (TrisHCl, pH 7.5) for 5 min, cells were stained by applying 30 μL 1X ethidium
      bromide. The slides were examined and the tail length was measured with a fluorescence
      microscope (Carl Zeiss, Germany). The DNA migration of 100 randomly selected cells were
      examined for each sample. A total damage score was determined by multiplying the number of
      cells assigned to each grade of damage by the numeric value of the grade according to methods
      described by Heaton et al. [19]. Total DNA damage score was calculated as follows. Total DNA
      damage = [(0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)] where n0 =cells with Score 0,
      n1 = cells with Score 1, n2 = cells with Score 2, n3 = cells with Score 3, and n4 = cells
      with Score 4.

      Determination of liver biomarker and enzyme The levels of liver enzymes including alanine
      aminotransferase, ALT, aspartate aminotransferase, AST, gamma glutamyl transferase, GGT, and
      alkaline phosphatase, ALP, pre and post intervention were determined from the blood samples
      of the patients. Other biomarkers such as lipid profile, α-2 macroglobulin and haptoglobin
      were also be analysed. The blood samples were collected in a BD Vacutainer and centrifuge at
      1500 xg for 15 mins for the serum separation. The serum was stored in -80°C if it is not
      processed immediately. The levels of the liver enzymes were determined by using ADVIA 1800
      (Siemens, USA).

      Quantitative Polymerase Chain Reaction (qPCR):

      Total RNA was extracted from blood samples of NAFLD patients with or without tocotrienol.
      Primers and probes for TNFα, IL-6 and IFN-gamma genes were designed. 1 ug/ul of total RNA was
      converted to cDNA which then be used for PCR reaction. All samples were analysed in
      duplicate. Expression of genes was determined after normalised with the housekeeping genes.
    

Intervention name(s): Tocotrienol-rich fraction vitamin E (TRF); Placebo
Intervention type(s): Drug; Drug
Intervention description(s): The 50 mg of vitamin E isomers in the TRF included 17.1 mg of α-tocopherol ,18.28 mg of α- tocotrienol, 2.02 mg of β-tocotrienol, 22.3 mg of γ-tocotrienol and 7.4 mg of δ-tocotrienol.; 50mg of Placebo
Intervention MeSH terms: Vitamin E; Tocopherols; Tocotrienols
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Treatment
Keywords: obesity; Non-alcoholic Fatty Liver Disease; Children; Tocotrienol-Rich Fraction Vitamin E