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

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.