Interventional Strategy in Tackling Emerging Non-alcoholic Fatty Liver Disease in Childhood Obesity

• ClinicalTrials.gov Identifier: NCT05905185
• Recruitment Status: Completed
• First Posted: June 15, 2023
• Last Update Posted: June 15, 2023
• Sponsor: National University of Malaysia
• Information provided by (Responsible Party): National University of Malaysia

Study Description

Brief 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.

Condition or disease	Intervention/treatment	Phase
Obesity	Drug: Tocotrienol-rich fraction vitamin E (TRF); Drug: Placebo	Phase 4

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.

Study Design

• Study Type: Interventional (Clinical Trial)
• Actual Enrollment: 29 participants
• Allocation: Randomized
• Intervention Model: Parallel Assignment
• Masking: Triple (Participant, Care Provider, Investigator)
• Primary Purpose: Treatment
• Official Title: Interventional Strategy in Tackling Emerging Non-alcoholic Fatty Liver Disease in Childhood Obesity
• Actual Study Start Date: January 1, 2020
• Actual Primary Completion Date: January 1, 2022
• Actual Study Completion Date: January 21, 2022

Arms and Interventions

Arm	Intervention/treatment
Experimental: Treatment; Sime Darby Bioganic Sdn. Bhd, Malaysia provided the TRF pills that were used in the study. 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. During a period of six months, the patients were instructed to consume oral TRF (50 mg) daily	Drug: Tocotrienol-rich fraction vitamin E (TRF); 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.
Placebo Comparator: Placebo; During a period of six months, the patients were instructed to consume oral placebo (50 mg) pills daily.	Drug: Placebo; 50mg of Placebo

Outcome Measures

Primary Outcome Measures :

1. Mean difference of fibrosis score, inflammation (activity) score and steatosis score [ Time Frame: Six months ]
   Serum LiverFASt level is a blood test to detect the degree and stage of: fibrosis (0-1), inflammation (0-1), and steatosis (0-1) scores. The higher the score indicates the higher the degree of fibrosis, inflammation and steatosis. These measurements were taken at the baseline and at the end of trial.
2. Mean difference of fasting blood glucose [ Time Frame: 6 months ]
   Fasting blood glucose levels were measured at the baseline and at the end of trial
3. Mean difference in aspartate aminotransferase & alanine aminotransferase levels [ Time Frame: 6 months ]
   Aspartate aminotransferase & alanine aminotransferase levels were measured at the baseline and at the end of trial.
4. Mean difference in apolipoprotein-A1, triglycerides, total cholesterol levels [ Time Frame: 6 months ]
   Total cholesterol, triglycerides, apolipoprotein-A1 levels were measured at the baseline and at the end of trial
5. Mean difference of liver steatosis and liver stiffness, measured by controlled attenuation parameter (CAP) score in percentage and kPa respectively, by transient elastography [ Time Frame: 6 months ]
   Transient elastography was performed to measure controlled attenuation parameter (%) and liver stiffness (kPa) at the baseline and at the end of trial

Secondary Outcome Measures :

1. Mean difference in tail length to represent DNA damage [ Time Frame: 6 months ]
   Determination of DNA damage using comet assay was done at the baseline and at the end of trial
2. Mean difference in the interleukin-6, TNF-alpha and interferon-gamma [ Time Frame: 6 months ]
   IL-6, TNF-alpha and interferon-gamma levels were measured at the baseline and at the end of trial
3. Mean difference in the body mass index (percentile) [ Time Frame: 6 months ]
   Weight measurement based on age was measured at the baseline and at the end of trial

Eligibility Criteria

• Ages Eligible for Study: 13 Years to 18 Years (Child, Adult)
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: No

Criteria

Inclusion Criteria:

1. Children aged 13 - 18 years old
2. Overweight or obese (BMI percentile range ≥ 85%)
3. Able to swallow small oral soft gel capsule
4. Diagnosis of NAFLD confirmed by presence of fatty liver detected by abdominal ultrasound and controlled attenuation parameter (CAP) score of >263
5. Elevated alanine transaminase (ALT) ≥ 2 fold upper limits (26 U/L for boys and 22 U/L for girls)

Exclusion Criteria:

• Evidence of other primary chronic liver diseases (as determined by clinical and standard investigations) - e.g. Hepatitis B, C infections, autoimmune hepatic disorders.
• Not on any dietary supplements.

Contacts and Locations

Locations

Malaysia

National University Of Malaysia

Kuala Lumpur, Malaysia

Sponsors and Collaborators

National University of Malaysia

Investigators

• Principal Investigator: Norfilza Mohd Mokhtar; National University of Malaysia

More Information

• Responsible Party: National University of Malaysia
• ClinicalTrials.gov Identifier: NCT05905185
• Other Study ID Numbers: GUP 2018-052
• First Posted: June 15, 2023
• Last Update Posted: June 15, 2023
• Last Verified: June 2023

Individual Participant Data (IPD) Sharing Statement:

• Plan to Share IPD: No

• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No

Keywords provided by National University of Malaysia:

obesity; Non-alcoholic Fatty Liver Disease; Children; Tocotrienol-Rich Fraction Vitamin E

Additional relevant MeSH terms:

Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease; Obesity; Pediatric Obesity; Overweight; Overnutrition; Nutrition Disorders; Body Weight; Digestive System Diseases; Vitamin E; Tocopherols; Tocotrienols; Vitamins; Micronutrients; Physiological Effects of Drugs; Antioxidants; Molecular Mechanisms of Pharmacological Action; Protective Agents