(2024) 24:529
Al‑Baiaty et al. BMC Pediatrics
https://doi.org/10.1186/s12887-024-04993-8

BMC Pediatrics

RESEARCH

Open Access

Assessing the efficacy of tocotrienol-richfraction vitamin E in obese children with nonalcoholic fatty liver disease: a single-blind,randomized clinical trial
Farah D. R. Al‑Baiaty1, Shareena Ishak2, Faizah Mohd Zaki3, Farin Masra2, Dayang Anita Abdul Aziz4,
Wan Nurulhuda Wan Md Zin2, Erica Yee Hing3, Azrina Syarizad Kuthubul Zaman4, Norhazlina Abdul Wahab1,
Khairul Najmi Muhammad Nawawi5,6, Zalina Hamid7, Raja Affendi Raja Ali6,8,9 and Norfilza Mohd Mokhtar1,6*

Abstract
Background Childhood obesity is a growing concern, and non-alcoholic fatty liver disease (NAFLD) is a significant
consequence. Currently, there are no approved drugs to treat NAFLD in children. However, a recent study exploredthe potential of vitamin E enriched with tocotrienol (TRF) as a powerful antioxidant for NAFLD. The aims of the present study were to investigate the effectiveness and safety of TRF in managing children with obesity and NAFLD.
Methods A total of 29 patients aged 10 to 18 received a daily oral dose of 50 mg TRF for six months (January 2020
to February 2022), and all had fatty liver disease were detected by ultrasonography and abnormally high alaninetransaminase levels (at least two-fold higher than the upper limits for their respective genders). Various parameters,
including biochemical markers, FibroScan, LiverFASt, DNA damage, and cytokine expression, were monitored.
Results APO-A1 and AST levels decreased significantly from 1.39 ± 0.3 to 1.22 ± 0.2 g/L (P = 0.002) and from 30 ± 12
to 22 ± 10 g/L (P = 0.038), respectively, in the TRF group post-intervention. Hepatic steatosis was significantly reducedin the placebo group from 309.38 ± 53.60 db/m to 277.62 ± 39.55 db/m (p = 0.048), but not in the TRF group. Comet
assay analysis showed a significant reduction in the DNA damage parameters in the TRF group in the post-intervention period compared to the baseline, with tail length decreasing from 28.34 ± 10.9 to 21.69 ± 9.84; (p = 0.049)
and with tail DNA (%) decreasing from 54.13 ± 22.1to 46.23 ± 17.9; (p = 0.043). Pro-inflammatory cytokine expression levels were significantly lower in the TRF group compared to baseline levels for IL-6 (2.10 6.3 to 0.7 1.0 pg/mL;
p = 0.047 pg/mL) and TNF-1 (1.73 5.5 pg/mL to 0.7 0.5 pg/mL; p = 0.045).
Conclusion The study provides evidence that TRF supplementation may offer a risk-free treatment option for children with obesity and NAFLD. The antioxidant and anti-inflammatory properties of TRF offer a promising adjuvant
therapy for NAFLD treatment. In combination with lifestyle modifications such as exercise and calorie restriction, TRF
could play an essential role in the prevention of NAFLD in the future. However, further studies are needed to explore
the long-term effects of TRF supplementation on NAFLD in children.

*Correspondence:
Norfilza Mohd Mokhtar
norfilza@ppukm.ukm.edu.my
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
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Al‑Baiaty et al. BMC Pediatrics(2024) 24:529

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Trial registration The study has been registered with the International Clinical Trial Registry under reference number(NCT05905185) retrospective registration on (15/06/2023).
Keywords Non-alcoholic fatty liver disease, Oxidative stress, Tocotrienols, Antioxidants, Anti-inflammatory agent

BackgroundNon-alcoholic fatty liver disease (NAFLD) has emerged
as a growing health concern in children worldwide, with
its prevalence escalating alongside the rise in childhood
obesity and associated metabolic risk factors [1]. The
pathogenesis of pediatric NAFLD is unknown; however,
it is likely to be a multifactorial, multistep, and progressive condition. Accumulation of free fatty acids and triglycerides in the liver cells causes damage, leading to
inflammation, and cellular injury, biochemical and histological derangements seen in children with NAFLD [2].
This can trigger a cascade of events that can lead to fibrosis, cirrhosis, and liver failure [3, 4]. Alterations in gut
microbiota, genetic predisposition, and environmental
toxins are other variables that may be incorporated into
the development of pediatric NAFLD.
Globally, the frequency of NAFLD in children is rising,and in the overall pediatric population, it is believed tobe between 7 and 10% [5]. However, the incidence varies
according to age groups, racial and cultural backgrounds,
and geographic areas. In children who are overweightor obese, the prevalence of NAFLD is higher and is predicted to range from 34 to 76% [6]. Although there isa paucity of information on the incidence of NAFLD
among Malaysian children, it is likely that the condition
is becoming more common as a result of the country’s
rising rates of childhood obesity and bad lifestyle choices.
The percentage of overweight children in Malaysia ranging from 5 to 19 years old was 12.7% in a survey conducted by the United Nations International Children’s
Emergency Fund (UNICEF) [7]. A study by Mohamed
et al. (2020) investigated the prevalence and predictorsof NAFLD among children with obesity in Malaysia. The
study included 33 children ranging from 5.5 to 18.5 years
who were diagnosed with obesity and underwent screening for NAFLD. They found that of the 33 patients, 21(63.6%) had NAFLD. Several predictors of NAFLD wereidentified in this population, including age, waist circumference, body mass index (BMI), and high blood pressure[8]. It is crucial to highlight that this study focused on aobese children, and the prevalence of NAFLD in Malaysia’s general pediatric population may differ.
Currently, the FDA has authorized a new drug, Rezdiffra (resmetirom) for the treatment of NAFLD. The drug’s
safety and efficacy in children NAFLD are still up for
debate, and further research is needed to determine
whether or not this medication can be used in paediatricpopulations as it is primarily intended for adult patients[9]. As with previous clinical guidelines, the main strategy for paediatric NAFLD is still lifestyle changes [10,11]. A study by Ratziu et al. 2020 discussed the current
understanding of the pathophysiology of NAFLD and
NASH, including the role of oxidative stress, mitochondrial dysfunction, and gut microbiota in the development
and progression of these conditions [12]. Emerging therapies, such as GLP-1 receptor agonists and FXR agonists,are currently being evaluated in clinical trials [13]. Recent
studies have suggested that vitamin E, an antioxidantrich fat-soluble vitamin, is a potential therapy in pediatric NAFLD [14, 15]. The management of NAFLD was
discussed by a panel of experts from the American Association for the Study of Liver Disease (AASLD) who alsooffer pharmacological treatments including vitamin E,
pioglitazone, and GLP-1 receptor agonists as well as lifestyle strategies like diet and exercise [16]. Another randomized, placebo-controlled trial investigated the effectsof supplementing δ-tocotrienol on biochemical markers
of liver injury and steatosis in patients with NAFLD. In
comparison to the placebo group, δ-tocotrienol supplementation improved the level of ALT, AST, hs-CRP and
HOMA-IR levels [17]. The American Association of
Endocrinology Clinical Practice Guideline offered suggestions and methods for controlling NAFLD in pediatricpatients that were supported by research. These strategies include pharmacological methods like vitamin E andmetformin, as well as lifestyle changes, including healthier eating along with increased exercise. The recommendation prioritizes recognizing and managing comorbid
conditions, such as obesity and insulin resistance, which
frequently accompany NAFLD [18]. This study aimed
to investigate the supplementation of tocotrienol-richfraction vitamin E (TRF) as part of the management of
NAFLD among children with obesity in Malaysia.

Methods
Study design and patient recruitment

This is a randomized, single-blinded clinical trial involving children in the pediatric clinic at Universiti Kebangsaan Malaysia Medical Centre (UKMMC). The study
has been registered with the International Clinical Trial
Registry under reference number NCT05905185, retrospective registration on (15/06/2023). This study
was approved by the institutional review board ([UKMAl‑Baiaty et al. BMC Pediatrics(2024) 24:529

PPI/111/8/JEP-2018621]. Informed consent to participate in this study was obtained from all participants.
For participants who were younger than the age of 16,
consent was obtained from their parents or legal guardians. We ensured that all ethical guidelines for conducting research with minors were strictly followed. A total
number of 32 children between the ages of 10 and 18
who were overweight or obese (BMI between the 85thand 95th percentile for their age) were enrolled. Thisage range was selected because it includes the critical developmental period of late childhood and adolescence, during which it is most possible to study the onset
and progression of NAFLD [19, 20]. Previous research
has shown that the incidence of obesity and metabolic
syndrome, which are critical risk factors for the development of NAFLD, is greater among children and adolescents aged 10 to 18 [21, 22]. The inclusion criteria
included being able to swallow a small, soft gel capsuleand an abdominal ultrasound confirmed the diagnosis of NAFLD. Also, a controlled attenuation parameter(CAP) score on the FibroScan must be > 263 dB/m, with
an elevated alanine transaminase level of ≥ two-fold theupper limit (26 U/L for boys and 22 U/L for girls, respectively). We excluded subjects who had hepatitis C, hepatitis B, autoimmune hepatic disorders or primary chronic
liver disease. Prior to the start of the trial, the children
were instructed to avoid using any dietary supplements
for at least four weeks. According to the Malaysian Clinical Practice Guidelines, the Children’s clinical parameters, such as their height, weight, waist circumference,
and BMI was evaluated prior to the intervention [23]. All
study parameters were measured before the intervention and again six months after. Patient interviews weredone every two months to check on compliance. Using acomputer model built on Microsoft Excel, the recruitedpatients were enrolled in the study and randomly
assigned (1:1) to either the TRF treatment or the placebogroup. The randomized procedure was carried out by thelead investigator because she knew the allocation order.
The patients were given bottles containing either the TRF
or placebo supplements following completion of all baseline assessments for both groups.
Treatment product and procedure

Sime Darby Oils Nutrition Sdn. Bhd. Malaysia supplied
the TRF pills utilized in the study. Each TRF pill, containing 50 mg of vitamin E isomers, comprised 7.4 mgof δ-tocotrienol, 2.02 mg of β-tocotrienol, 22.3 mg of
γ-tocotrienol, 18.28 mg of α-tocotrienol, and 17.1 mgof α-tocopherol. Over a six-month duration, participants were directed to ingest either the oral TRF (50 mg)
or a placebo after meals. The children were advised
to maintain their regular dietary habits, refrain fromPage 3 of 12

incorporating vitamin E-rich supplements, and also not
to alter their exercise routines. After randomization, follow-up visits were made every eight weeks for the duration of the trial. Every two weeks, a telephone follow-up
was conducted to check on the patient’s health. At theirinitial visit, patients were given 10 bottles containing 10
capsules (either TRF or placebo) for two distinct sessions,along with instructions on how frequently to take thesupplements, the patients were blinded to the contents ofthe bottles. There was no way to distinguish the placebofrom the TRF pills based on how they tasted, smelled, or
appearance because they were stored in similar bottles.
The two bottles each had an A or B label. The participants
were instructed to keep the pill sachets in dry conditions,out of the sun, at or below 25 °C. The pills’ shelf life was
two years. Before and after the study’s two-month session, pill counting was used to gauge the patient’s compliance. The acceptable compliance rate was 85 to 100%[24].
Outcome measures

The primary result was the mean difference (dB/m) in
hepatic steatosis as evaluated by the controlled attenuated parameter (CAP) after TRF supplementation versus placebo. Secondary outcomes were mean differencesin biochemical blood tests, FibroScan fibrosis score, andLIVERFASt calculated scores for fibrosis, activity, steatosis, DNA damage, and pro-inflammatory cytokines (IL-6,
TNF-α, and IFN-γ). Assessments were conducted on 29
participants (15 in the TRF group and 14 in the placebogroup) at baseline and end of trial.
Evaluation of biochemical blood and liverFASt

LiverFASt, a non-biopsy method, produces an algorithm
that correlates with the simple grading/staging of the
NAFLD based on the three basic features of the disease
which are: inflammation, fibrosis, and steatosis. To perform this test, the patients were instructed to fast overnight for at least eight hours before conducting the test.
A series of blood tests were conducted that included total
bilirubin, haptoglobin, apolipoprotein-A1 (ApoA1), fasting serum total cholesterol, fasting blood glucose, [25]and (ALT), alpha-2-macroglobulin (a2M), and fasting
triglyceride levels, gamma-glutamyl transferase (GGT)
were measured. These measurements were utilized in theLiverFASt algorithm along with BMI, gender, and age tocalculate the extent of the disease. These data were compared with liver stiffness measurements obtained by the
FibroScan device in order to provide an objective diagnosis of hepatic steatosis, fibrosis, and inflammatory activity
scores in NAFLD patients [26, 27].

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FibroScan examination

Sample size

Using FibroScan 502, data for liver stiffness measurement (LSM), a marker for liver fibrosis, and controlledattenuated parameter (CAP), an indicator of hepatic fat
deposition, were obtained (Echosens, Paris, France).
Liver stiffness of > 8 kPa indicates advanced fibrosis while CAP of > 263 indicates fatty liver [28]. The
measurement was considered reliable if the interquartile range/median (IQR/M) was < 20% and the successreading rate of ≥ 70%. The procedure was carried out
by a single, experienced operator to eliminate bias andinter-operator variability. The sizes of the patients wereconsidered when calculating the probe and CAP values.

According to Mofdi et al. (2017), the sample size wasestimated based on the mean of hepatic steatosis, witha controlled attenuation parameter (CAP) score evaluated by FibroScan as the primary outcome. To calculate the proper sample size for detecting a difference
between two independent samples, and based on theprevious study’s mean difference and standard deviation, the appropriate sample size for each arm shouldbe 14 patients for each group, TRF and placebo. The
sample size for each group has been determined to 16
patients per arm, taking into consideration 20% dropout and non-compliance [32].

Comet assay

Statistical analysis

The comet assay was employed to quantify DNA damage in an alkaline environment. The slides were prepared according to Singh et al. [29]. The slides wereobserved with the epifluorescence microscope (Olympus BH-2 System), equipped with a DPlanApo 20 UV
objective and an Olympus SPlan 10. The images were
captured with the aid of the OpenComet v1.3.1 program that was installed on the computer. For eachsample, 100 randomly chosen cells were examined (50cells from each of the two replicate slides) [30, 31]. The
degree of DNA damage was then measured based on
the length and percentage of the tail DNA.

The SPSS software ver. 20.0 from SPSS Inc. in Chicago,
Illinois, USA, was used to analyze the data. Independentt-tests or Mann–Whitney U tests and Wilcoxon Paired
Rank Test or Paired t-tests were used to compare differences between pre-and post-intervention values in eachgroup. Statistical significance was defined as a p-value ofless than 0.05.

Quantitative real‑time PCR (qPCR) of inflammatory
cytokines

Total RNA was isolated from fresh blood samples using
the RNA Blood Mini Kit (Qiagen, Germany). Using aDenovix spectrophotometer, the quantity and quality ofthe extracted RNA were determined by calculating its
absorbance at 260 and 280 nm. Complementary DNA(cDNA) was synthesized using the RT2 First Strand Kit(Qiagen, Germany). Quantitative PCR was carried out
using the RT2 SYBR Green Mastermix (Qiagen, Germany) via the Bio-Rad CFX96 instrument [Bio-Rad
Laboratories, CA, USA]. The PCR conditions were set
with the initial step at 95 °C for 15 s, followed by 60 °C
for 1 min. Finally, the melting curve of the product gradient analysis was calculated at a rate of 0.5 °C/10 s,starting from 65 °C to 95 °C. The RT-qPCR assess themRNA expression of inflammatory cytokines, including IL-6, TNF-α and IFN-γ (Qiagen, Germany). Allanalyses were done in triplicates after normalized withthe housekeeping genes (GAPDH). The relative gene
expression analysis was done by the ­2–ΔΔCT method.

Results
Baseline characteristics

A total of 32 patients were randomly assigned to either
the placebo or TRF groups. Two patients withdrew fromthe trial; their excuse was that they were unable to attend
follow-up appointments because of scheduling conflicts
with their parents’ jobs and academic exams. As a result,they did not get the intervention. One patient dropped
out of the trial because their parents’ work obligations
prevented them from attending the follow-up appointments. According to the consort diagram for patient
recruitment, no patient reported issues with complianceduring the 6-month intervention period due to TRF orplacebo supplements adverse effects (Fig. 1). The baseline demographic information gathered from the patientsis shown in Table 1. There was no statistically significant
difference in baseline values between the two groups.
Blood biochemical measurements

The biochemical tests demonstrated that patients who
took TRF supplements daily for six months had significantly lower serum apolipoprotein- APO-A1 andaspartate aminotransferase AST levels than the baseline(P = 0.002) and (P = 0.038) respectively. Total cholesterollevels did not differ significantly post-TRF supplementation compared to the baseline. The level was, however,significantly higher in the placebo group (p < 0.05) whencompared to the baseline after six months. However,no significant difference was observed in other serum

Al‑Baiaty et al. BMC Pediatrics(2024) 24:529

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Fig. 1 CONSORT flow diagram depicts how participants progressed through each stage of the randomized controlled trial, which usedeither tocotrienol-rich fraction vitamin E (TRF) or placebo

Table 1 Patient characteristics at the baseline in tocotrienoltreated (TRF) and placebo groupTRF (n = 15)

Placebo (n = 14)

p value
0.591

Age (years)

11.8 (2.1)

12.5 (2.6)

BMI (Percentile)a99.5 (2.0)

99.8 (0.7)

0.400Fasting glucose, mmol/L

4.6 (0.3)

4.7(0.4)

0.354

29.0 (15)30.4(10.7)0425

biomarkers such as ALT, fasting blood glucose, α-2-M,
GGT, haptoglobin, total bilirubin and triglycerides wasseen at the end of the intervention for either group, asshown in (Table 2).
LiverFASt scores

Haptoglobin g/L

1.29 (0.5)

1.40(0.5)

0.235

Total bilirubin µmol/La

7.9 (2.1)

7.7(3.1)

0.476

α-2-M g/L a

2.38(0.3)

2.46(0.4)

0.780

Triglycerides mmol/L a

1.53(0.6)

1.29(0.4)

0.477

The LiverFASt test was used to determine three hepaticparameters, i.e., hepatic fibrosis, activity scores and liver
steatosis. The test results showed that patients in thetreatment group did not significantly improve in any
parameters (i.e., fibrosis score: p = 0.094; activity score:p = 0.955; steatosis score: p = 0.078). Also, there was no
improvement was noted in the placebo group (fibrosis
score: p = 0.31; activity score: p = 0.424; steatosis score:p = 0.060) (Table 2).

    Steatosis ­Scorea

0.33 (0.2)

0.39 (0.5)

0.425    Activity ­Scorea

0.11 (0.1)

0.12 (0.06)

0.377

Transient elastography    Fibrosis Score

0.13 (0.07)

0.13 (0.04)

0.902    CAP dB/m

307.14(50.4)

309.38(53.6)

0.518    Liver stiffness ­kPa

4.85(1.17)

4.58(1.07)

0.201

ALT IU/La

AST IU/LaTotal Cholesterol mmol/L

30.0 (12)27.4(9.7)

0.505

4.54(0.8)

4.41(1.0)

0.523

APO-A1g/L

1.39 (0.3)

1.32(0.3)

0.405

GGT IU/La

29 (20)27(20.7)

0.652

Serum LiverFASt

Transient elastography

Data are presented as the mean (SD)ALT Alanine Aminotransferase, AST Aspartate Aminotransferase, APOA1 Apolipoprotein A1, GGT​ Gamma-glutamyl transferase, α-2-M alpha-2Macroglobulin, CAP Controlled Attenuation Parameter, BMI Body mass indexaData are presented as the medianAfter six months of intervention, the hepatic steatosis
score decreased from 307.14 ± 50.4 to 286.07 ± 67.5 db/m.
Yet, it was not statistically significant. In comparison,
after six months of intervention, the score in the placebogroup decreased significantly from 309.38 ± 53.6 db/m to277.62 ± 39.5 db/m (p = 0.048). There was no improvement in fibrosis score in the NAFLD group supplemented
with TRF compared to the baseline (4.85 ± 1.17 kPa vs4.65 ± 1.53 kPa; p = 0.167). After six months of intervention, however, there was a trend toward an increase inAl‑Baiaty et al. BMC Pediatrics(2024) 24:529

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Table 2 Comparisons of the changes from baseline to endpoint of study outcomes of NAFLD patientsTRF (n = 15)
Baseline

Placebo (n = 14)
After 6 months

p-value

Baseline

After 6 months

pvalueBMI (Percentile)a99.5(2.0)

99.7(0.9)

0.859

99.8 (0.7)

99.9(0.5)

0.527

Fasting glucose, mmol/L

4.6 (0.3)

4.8(0.8)

0.156

4.7(0.4)

4.8(0.6)

0.591

ALT IU/L a

29.0(15)34.0(16)0.726

30.4(10.7)

33.2(10.3)

0.450

AST IU/L a

30.0(12)22.0(10)0.038*

27.4(9.7)

23.9(7.7)

0.138

Total Cholesterol,mmol/L

4.54(0.8)

4.59(0.9)

0.670

4.41(1.0)

4.70(0.9)

0.021*APO-A1g/L a

1.39(0.3)

1.22(0.2)

0.002*

1.32(0.3)

1.22(0.2)

0.077

GGT U/L a29(20)

32(19)

0.975

27(20.7)21(30.5)

0.345

Haptoglobin g/L

1.29(0.5)

1.28(0.4)

0.941

1.43(0.5)

1.51(0.6)

0.366

Total bilirubin µmol/La

7.9(2.1)

7.2(5.7)

0.156

7.7(3.1)

8.6(2.6)

0.370

α-2-M g/L

2.38(0.3)

2.40(0.2)

0.126

2.46(0.4)

2.35(0.4)

0.283

Triglycerides mmol/L

1.53(0.6)

1.70(0.8)

0.131

1.29(0.4)

1.35(0.4)

0.609

Serum LiverFASt
Steatosis Score

0.33 (0.2) S0

0.35(0.3)

0.078

0.39(0.5)

0.39(0.5)

0.060

Activity Score

0.11 (0.1) A0

0.15(0.1)

0.955

0.12(0.06)

0.14(0.09)

0.424

Fibrosis Score

0.13 (0.07) S0

0.18(0.1)

0.094

0.13(0.04)

0.14(0.04)

0.31

307.14(50.4)

286.07(67.5)

0.189

309.38(53.6)

277.62(39.5)

0.048*

4.85(1.17)

4.65(1.53)

0.167

4.58(1.07)

5.42(2.01)

0.075

Transient elastography
CAP dB/m
Liver stiffness kPaaaData are presented as the medianFig. 2 Alkaline single-cell gel electrophoresis was used to assess DNA damage. A Baseline TRF-treated group cells with DNA damage visible
as comets. B Baseline placebo-treated group cells with DNA damage shown as comets. After six months, cells with TRF supplementation had
less DNA damage. Cells with increased DNA damage following six months of placebo supplementation

Al‑Baiaty et al. BMC Pediatrics(2024) 24:529

the fibrosis score in NAFLD supplemented with placebo(4.58 ± 1.07 kPa to 5.42 ± 2.01 kPa; p = 0.075) (Table 2).
Comet assay analysis

Fluorescence microscopy was used to assess the DNA
fragmentation following six months of intervention
with either TRF or a placebo (Fig. 2A, B). Based onthese results, the TRF group had less DNA damage thanthe control group, as shown by the reduced amount of
stained comet tails (Fig. 2C). A high amount of stained
comet tails, as seen in (Fig. 2 D), which suggests the placebo group experienced more DNA damage.
The ImageJ software was used to analyze the cometand measure the DNA amount in the tail length (TL)
and tail (% DNA) of the digital comet image. The results

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showed that after six months of the intervention, themean tail length in the TRF group decreased significantly from 28.34 ± 10.9 to 21.6 ± 9.84 (p = 0.04), compared to the baseline. In the placebo group, however,
the mean tail length increased from 23.67 ± 11.15 to
27.72 ± 8.7 (p = 0.19), yet this increase was not statistically significant. The percentage of tail DNA (% DNA) in
the TRF group decreased significantly from 54.13 22.1
to 46.23 ± 17.9 (p 0.045), but not in the placebo group48.56 ± 21.1 vs 51.78 ± 21.1 (p 0.48) (Fig. 3).
Quantitative real‑time PCR analysis

Figure 4 shows the fold change in mRNA levels of
inflammatory cytokines before and after TRF and placebo intervention. The TRF group showed significant

Fig. 3 DNA damage was determined using computerized image analysis for TRF and placebo at baseline and post-intervention. The comet assay
method detected DNA damage in a whole blood sample by calculating the tail length (TL) and percentage of DNA in the tail (DNA%) of 100
randomly selected cells. A fluorescence microscope was used to examine the slide. ImageJ software was used to capture the photos and measurethem. Data are presented as mean ± SEM, and a statistically significant value is deemed to be P < (0.05)

Fig. 4 Relative fold change of gene expression measured by quantitative real-time PCR for the vitamin E group & Placebo group at baseline and six
months’ post-intervention. A Relative expression of IL-6 gene. B Relative expression of TNF-α. Relative expression of IFN-γ gene. Relative expression
levels were normalised to GAPDH, and fold changes were calculated against the baseline. Values represent the means and SEM. Significant
differences are highlighted as ∗ (P < 0.05)

Al‑Baiaty et al. BMC Pediatrics(2024) 24:529

downregulation in the IL-6 expression (p < 0.05). A
comparable trend was noted for the TNF-α value in the
TRF group, which showed significant downregulation of
TNF-α gene expression after the intervention comparedto the baseline values (p < 0.05). In both the pre and postintervention TRF and placebo groups, the results indicated no significant difference in the IFN-γ expression.

Discussion
The aim of this study was to get more insight into thepotential use of tocotrienol-rich fraction vitamin E (TRF)
as a supplement in children with obesity who are suffering from non-alcoholic fatty liver disease (NAFLD). Our
study demonstrated that the TRF supplementation was
safe and more effective as compared to the placebo after
24 weeks by significantly lowering apolipoprotein A1 andaspartate aminotransferase levels in children with obesity
and NAFLD. The strength of this study lies in its comprehensive examination of inflammatory and oxidative stressmarkers, which provides robust evidence for the antiinflammatory and antioxidant benefits of tocotrienols
observed in our research. Our study showed that patients
who received TRF supplementation had much less DNA
damage in their blood samples based on the comet assay
results. This test provides a relative measure of the number of DNA strand breaks. The substantial therapeuticadvantages of TRF in NAFLD in children with obesity,
particularly in cases of severe liver damage [11, 33]. Thiseffect is likely due to TRF’s ability to act as an antioxidantby reducing oxidative damage. Additionally, Vitamin E
may modulate mitochondrial biogenesis and regulate theproduction of superoxide and other reactive oxygen species [34] by influencing various mitochondrial enzymes
and pathways. Vitamin E can enhance the activity of
mitochondrial superoxide dismutase (SOD), an enzymethat converts superoxide radicals into less harmful molecules. It also helps to stabilize the inner mitochondrial
membrane, thereby reducing electron leakage and subsequent ROS formation. These mechanisms collectively
contribute to the reduction of oxidative stress within cells[35, 36].
Furthermore, it was also noted that the TRF supplementation decreased DNA damage in healthy subjects
after 12 and 24 weeks of supplementation. This effect was
greater in females and older individuals above the age of50 [37]. When patients with hypercholesterolemia were
given four progressively increasing dosages of tocotrienol-rich fraction derived from palm oil (TRF): 125 mg/
day, 250 mg/day, 500 mg/day, and 750 mg/day overfour weeks, this provided more evidence that tocotrienol could also function as an anti-inflammatory agent.
There were significantly lower levels of serum nitricoxide [38], C-reactive protein (CRP), interleukin-6 (IL-6),

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malondialdehyde (MDA) and gamma glutamyl-transferase compared to the placebo group for all concentrations of TRF [39].
Oxidative stress plays a crucial role in the pathophysiology of NAFLD. Imbalances between the production ofreactive oxygen species [34] and the body’s antioxidant
defense capacity can disrupt normal lipid metabolism,
contributing to the onset and progression of NAFLD[40]. Excess fat accumulation in adipose tissue leads to
the release of free fatty acids into the bloodstream. The
liver receives these fatty acids via transport, where they
are normally processed and stored as triglycerides. However, individuals with insulin resistance, often associated with obesity, face challenges in the liver’s ability toefficiently metabolize and export these lipids. Consequently, fat builds up within the liver [41]. Imbalances in
the gut microbiota composition can escalate the severityof NAFLD. This disruption has the capacity to enhance
fatty acid synthesis in the gut, elevate small bowel permeability, and facilitate increased absorption of fatty
acids into the bloodstream. These combined effects contribute to the overall accumulation of fat in the liver, further deteriorating the condition. Presently, there is no
FDA-approved medication for the treatment of this disease in pediatric population [42]. The primary approachto manage NAFLD involves lifestyle modifications suchas weight loss, regular exercise, and the adoption of ahealthy diet [43]. In addition, vitamin E, a well-known
fat-soluble antioxidant, is utilized as an alternative in the
management of pediatric NAFLD with obesity [44].
The present study revealed no differences in blood biochemical parameters after TRF supplementation, including ALT, fasting blood glucose, α-2-M, GGT, haptoglobin,
total bilirubin, and triglycerides. A meta-analysis in 2020of nine randomized controlled studies similarly indicatedthat vitamin E failed to show any reduction in biochemical tests such as ALT, GGT, and TG[45]. Furthermore,
several other factors should be considered, including
therapy dosage, average patient age, sample size, gender
distribution, duration of the intervention, research location, and dietary patterns. Individual variability, including
genetic factors, diet, lifestyle, and NAFLD severity, also
influences how liver enzymes respond to TRF and vitamin E supplementation [46, 47]. A 96-week randomized
controlled trial comparing the effects of 800 IU vitamin
E and 1000 mg metformin on pediatric NAFLD foundthat neither vitamin E nor metformin outperformed placebo in terms of ALT levels. Lack of significant changes
could be explained by the particular patient cohort orthe characteristics of the therapies. This emphasizes thenecessity for alternate therapeutic approaches, such as
TRF, that may possess different mechanisms of action or
efficacy profiles [48]. Magosso et al. (2013) conducted aAl‑Baiaty et al. BMC Pediatrics(2024) 24:529

clinical trial in 87 patients suffering from mild untreatedhypercholesterolemic with NAFLD using a mixtureof vitamin E (61.5 mg of α-tocotrienol, 112.8 mg of
γ-tocotrienol, and 25.7 mg of δ-tocotrienol and 61.1 mgof α-tocopherol), taken twice a day for 12 months. The
tocotrienol-treated group significantly outperformed
the placebo groups in terms of normalization of thehepatic echogenic response. However, as the study did
not include a pediatric population, there may be lessdirect relevance between the results and our investigation ​[49]. According to a study conducted by Murer et al.
(2014), antioxidant supplementation decreased oxidative stress and stabilized liver function tests in children
with NAFLD. The study enrolled 90 participants aged 8
to 17 years’ old who were randomly assigned to receive
either an antioxidant supplement containing vitaminC (500 mg), alpha-tocopherol (400 IU), and selenium(50 mg) or a placebo for 12 weeks. One of its key advantages is that it focuses on a paediatric population similar
to ours, and the findings are relevant to our study [50].
It is important to keep in mind that the dosage andduration of vitamin E supplementation may also influence its effectiveness. We employed 50 mg/kg TRF forsix months as the intervention in the present study. In arandomized, double-blind, placebo-controlled trial study,
80 adolescents with biopsy-proven NAFLD received alower dosage of vitamin E (10 mg) combined with 7.5 mgof hydroxytyrosol for a shorter duration (16 weeks). They
examined the impacts of antioxidant supplementation onoxidative stress, liver function, and inflammation. The
trial included 80 participants ranging in age from 4 to
16 years. Participants were randomly assigned to receive
either two capsules containing vitamin E plus hydroxytyrosol or a placebo. The study demonstrated enhanced
insulin resistance, reduced steatosis, and improved oxidative stress markers in children with NAFLD [51].
The genetic variants associated with NAFLD in children were revealed in a review that was published in
2019, emphasizing the importance of genes involved inlipid metabolism, inflammation, and insulin resistance.
They hypothesized that investigating these genes could
aid in discovering new treatment targets for NAFLD.
Non-invasive diagnostic modalities, such as imaging techniques and biomarkers, emphasize the importance of accurate and non-invasive tools for detecting
and monitoring of NAFLD in children [52]. This study
employed the LiverFASt and FibroScan tests to detect
hepatic steatosis, fibrosis, and activity scores for assessment of liver steatosis in children, as previously reported[53]. There was no significant improvement in the degree
and grade of hepatic steatosis observed in the tocotrienol
group after six months of the intervention in this study.
This suggests that the reduction in intrahepatic fat may

Page 9 of 12

not have been significant enough to bring about a visible improvement in the radiological grade of hepatic
steatosis. This finding is supported by a clinical study
conducted by Pervez et al. in 2018, which revealed that
δ-tocotrienol supplementation significantly decreased
high-sensitivity C-reactive protein, malondialdehyde,
and aminotransferase levels in NAFLD patients but not
hepatic steatosis on ultrasound examination. The trial
also emphasized the safety of tocotrienol administration
because no negative effects were observed. This study
provides a solid foundation and additional validation for
the therapeutic potential of tocotrienol in managing conditions related to oxidative stress, and the consistency
between studies highlights the dependability and relevance of our results [54].
A meta-analysis was conducted to investigate theimpact of vitamin E supplementation on aminotransferase levels in adults and children. Interestingly, no significant decrease in aminotransferase levels was found,particularly in the pediatric age group [55]. Anothermeta-analysis of 15 RCTs involving NAFLD patients ofvarious ages found that the group receiving vitamin E had
a significant drop in ALT and AST levels among adults
following short-, intermediate-, and long-term followups. The findings of the study suggested that a significant
change in biochemical markers in children occurred after
12 months [56]. Furthermore, increased ALT serum levels correlate with NAFLD activity score, liver triglyceride,and fasting insulin, establishing it as a reliable predictor of NAFLD [57]. In a randomized placebo-controlled
trial involving 86 participants, Bril et al. (2019) revealed
that vitamin E (tocopherol) administration did not significantly improve hepatic steatosis compared to the placebo group [58]. This could be explained by a short study
period or an inadequate dosage of vitamin E administration, however keep in mind that high dosages of this vitamin especially tocopherol, may have unfavorable effects,
including bleeding and an increased risk of prostate cancer especially in adults [59].
Surprisingly, after the intervention in the present
study, the CAP scores in the placebo group significantly decreased. The suggested etiology of the placebo
effect included patients in the placebo arms changing
their behavior in response to their knowledge of being
observed [60]. This phenomenon, known as the Hawthorne effect, typically causes patients’ BMIs to fall withtime. In the current study, the BMI of the individuals in
the placebo group, however, remained the same.
In a rat model of NAFLD, the effect of tocotrienol on
inflammatory response and oxidative stress was examined [61]. They discovered that tocotrienol was able toregulate the inflammatory response and reduce oxidative stress in rats with NAFLD. These findings offer

Al‑Baiaty et al. BMC Pediatrics(2024) 24:529

more details on tocotrienol’s possible application in thetreatment of NAFLD [61]. Since NAFLD is linked to theaccumulation of DNA damage, changes in the genetic
material encoding proteins related to repair pathways
may contribute to the development of the disease. This isparticularly relevant when considering the intricate relationship between oxidative stress, DNA repair, and liver
steatosis. One important factor that may be important in
the development of NAFLD is the impairment of DNA
repair mechanisms [62].
Tocotrienols have been well-established, and promisingresearch has demonstrated that it has anti-inflammatory
properties [63]. In comparison to the placebo group, TRF
supplementation for 24 weeks reduced the expression of
IL-6 and TNF-α in children with obesity with NAFLD
in the current study. This finding was verified by anin vivo study that found γ-that tocotrienol supplementation reduced adipose tissue inflammation in mice fed ahigh-fat diet by lowering the production of cytokines andchemokines that promote inflammation. Additionally,
γ-tocotrienol supplementation reduced the recruitmentof pro-inflammatory M1 macrophages in adipose tissue[64]. It was proven that tocotrienol can reduce inflammation in NAFLD in a double-blinded, active-controlled
experiment utilizing 300 mg δ-tocotrienol daily for
48 weeks by significantly lowering serum IL-6 and TNF-α[65]. Inflammation induced by TNF-α in differentiated
3T3-L1 adipocytes exposed to various concentration ofγ-tocotrienol (0.024—2.4 µM) for 6 h showed a significant reduction in the expression of MCP-1 and IL-6 [66].
There were limitations in this study that could havean impact on the outcomes and should be mentioned
to create chances for improvement in future researchendeavors. This study exhibits a limited sample size, andconducting a larger-scale study could mitigate the risk of
random findings, offering more robust evidence regarding the efficacy of supplementation. Additionally, ourstudy has inherent limitations, including a single-dose
administration, which may impact the generalizability
and comprehensiveness of the results. The use of a single
dosing approach might not fully capture the broad spectrum of responses or account for variations that could
arise under different dosing regimens. The duration of
supplementation could impact the robustness of the findings. A longer duration might provide a more thoroughcomprehension of the impacts of the supplementation
over time. The absence of therapeutic drug monitoring(TDM) in this study is a notable limitation, as TDM is
crucial for ensuring optimal therapeutic levels, adjusting
dosages based on individual responses, and monitoring
potential fluctuations in drug concentrations over time. It
is important to highlight that the experiment was limited
to a single health center, and the majority of participants

Page 10 of 12

were Malays. Therefore, the conclusions regarding therole of vitamin E could not fully reflect the views of thelarger Malaysian population. Another study limitation
was that we did not measure other oxidative stress markers such as glutathione (GSH), glutathione peroxidase(GPX), catalase (CAT), superoxide dismutase (SOD), andmalondialdehyde (MDA).

Conclusion
This study demonstrates that children with obesityand having NAFLD receiving tocotrienol-rich vitamin
E supplements at a dose of 50 mg/day experienced noside effects, indicating that these supplements are typically safe and well tolerated. Evidence of its effectivenessthroughout the course of the six-month study included
improvements in serum apolipoprotein-A1, aspartate
aminotransferase levels, decreased DNA damage, andlow expression of pro-inflammatory cytokines. Thesefindings offer preliminary support for the superiority of
tocotrienol fraction of vitamin E as an adjuvant therapy
in the medical management of pediatric NAFLD.
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12887-​024-​04993-8.
Supplementary Material 1
Acknowledgements
We would like to acknowledge the Sime Darby Oil Nutrition Sdn. Bhd. (FF2020-180) and Fundamental Research Grant, Faculty of Medicine, Universiti
Kebangsaan Malaysia (FF-2020-180/1) and to all specialists and medical officers of the Department of Pediatrics, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Center.
Authors’ contributions
N.M.M designed, give technical advice, writing an original draft preparationand data analysis, F.D.R. conducted the experiments and analyzing the data,
N.M.M., and R.A.R.A supervised the study, K.N.M. involved in sample collection,and conducted FibroScan. All authors have read and agreed to the publishedversion of the manuscript.
Funding
This research was funded by the Sime Darby Oil Nutrition Sdn. Bhd. andResearch University Grant, Universiti Kebangsaan Malaysia (project code:
FF-2020–180 and FF-2021–180/1).
Availability of data and materials
All supporting data this study is included in the paper.

Declarations
Ethics approval and consent to participate
The study was conducted according to the guidelines of the Declaration of
Helsinki, and was approved by the Universiti Kebangsaan Malaysia ethics
committee [UKM PPI/111/8/JEP-2018621]. Informed consent to participate
in this study was obtained from all participants. For participants who wereyounger than the age of 16, consent was obtained from their parents or legal
guardians.

Al‑Baiaty et al. BMC Pediatrics(2024) 24:529

Consent for publication
Not applicable.
Competing interests
The authors hereby declare no conflict of interest.
Author details
1
Department of Physiology, Faculty of Medicine, Universiti Kebangsaan
Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, Cheras, Bandar Tun RazakKuala
Lumpur 56000, Malaysia. 2 Department of Pediatrics, Faculty of Medicine,
Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia. 3 Departmentof Radiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Kuala
Lumpur, Malaysia. 4 Department of Surgery, Faculty of Medicine, Universiti
Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia. 5 Gastroenterology Unit,
Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia,
56000 Kuala Lumpur, Malaysia. 6 GUT Research Group, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia. 7 Sime Darby Oils
Nutrition Sdn. Bhd, Jalan PJU 1A/7, 47301 Petaling Ara DamansaraSelangor,
Malaysia. 8 School of Medical & Life Sciences, Sunway University, Sunway, Selangor, Malaysia. 9 Division of Gastroentero‑Hepatology, Department of Internal
Medicine, Universitas Airlangga, Surubaya, Indonesia.
Received: 16 February 2024 Accepted: 5 August 2024

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