Study setting
Healthy men and women of the age between 50 and 75 from the Klang Valley which is cantered in the federal territories of Kuala Lumpur and Putrajaya in Malaysia are recruited and divided into two cohorts which are supplemented with either placebo or tocotrienols for 6 months.
Eligibility criteria
Participants in this research must meet specific health criteria, including being generally healthy as determined by physical examination and blood lab tests, with satisfactory liver and renal function tests. Eligible participants can be of either gender, aged between 50 and 75 years. They should not have allergies to palm oil and vitamin E and should not have consumed vitamin E supplements in the past 3 months. Furthermore, they must be willing and able to adhere to the study's visit schedule and procedures, reside within a geographically feasible proximity for adequate follow-up (as determined by the investigator), and have a clear understanding of the study protocol, as indicated by their signed informed consent forms.
Participation in this research is restricted based on several exclusion criteria. Individuals with fat malabsorption, as well as those managing chronic conditions such as cardiac diseases, neurological diseases, diabetes, HIV infection, or psychiatric illness/social situations, are not eligible. Moreover, individuals adhering to a vegan diet, those who have smoked within the past 3 months, individuals awaiting surgery or who have undergone surgery within the same timeframe, or those with a current or past history of drug, alcohol abuse, or cancer, do not meet the study's eligibility requirements. Pregnant and lactating women, individuals with a history of bleeding tendencies or conditions that predispose to bleeding (e.g., thrombocytopenia, abnormal liver function, liver diseases such as chronic hepatitis, or gastrointestinal ulcers), those taking antibiotics or other medications, or dietary supplements that may interfere with tocotrienol action, individuals with a predisposition to inherited blood/circulation disorders, and those using anticoagulants and antithrombotic drugs are also ineligible for participation in this research.
Interventions
This research encompasses the evaluation of two investigation products that are supplied by KL-Kepong Oleomas Sdn. Bhd. The first product, known as DavosLifeE3 Complete (50mg), contains 50mg of tocotrienols that are sourced from palm oil and meet the specifications outlined in the US FDA GRAS exemption claim 21 CFR 170.36 (c) [80]. The second product, Placebo Softgel, serves as a vital control within the study.
Outcomes
The study encompasses a multifaceted array of assessments and procedures. Firstly, blood samples (15 ml) are collected as baseline samples for blood biochemistry analyses. Tubes containing samples are labelled with the subject's code, trial ID, and date for accurate identification. Of the collected blood samples, 4 ml is routed to the chemical pathology laboratory for further analysis. The remaining samples are stored appropriately on ice or in a 4⁰C refrigerator until they undergo centrifugation, facilitating the isolation of plasma samples. These collected plasma samples are meticulously apportioned into 1ml storage tubes and immediately stored in a -80 ⁰C freezer. This storage method ensures that the samples are preserved for the analysis of oxidative stress markers (MDA, AGE, F2-IsoPs, PC), inflammatory markers (TNF-α, IL6), vitamin E, leptin, and ghrelin levels, all to be conducted within 12 months of storage (Table 1).
Furthermore, the assessment of arterial stiffness is conducted by skilled laboratory assistants through arteriography during the subjects' visits at both the 0 and 6-month time points. Total body fat and lean mass measurements are accomplished using an Inbody (USA) device during the subjects' visits at 0, 3, and 6 months.
Subsequently, subjects undergo a cognitive function assessment, which includes the Montreal Cognitive Assessment (MoCA), Rey Auditory Verbal Learning Test (RAVLT), and digit span. This assessment is performed with the assistance of a trained research assistant during the subjects' visits at 0 and 6 months.
In addition, the analysis of skin health is performed using Cutometer (Germany) and Visioscan (Germany) during subjects' visits at 0, 3, and 6 months. Finally, the measurement of bone mineral density is conducted using a dual-energy X-ray absorptiometry device (DEXA) at 0 and 6 months. To monitor changes in dietary habits, subjects complete Food Frequency Questionnaires (FFQ) during their visits at 0, 3, and 6 months (Fig. 1).
Table 1
Summary of tests performed at 0, 3 and 6 months
No | Time points | Parameters | Sample | Methodology |
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1 | Screening | Blood biochemistry test • Liver function (ALT, ALP, albumin, protein, bilirubin) • Lipid profile (TG, HDL, LDL, cholesterol) • Renal function (Na, K, Cl, Urea, Creatinine) • Full blood count (WBC, RBC, haematocrit, MCV, platelet) • Fasting glucose • C-reactive protein Physical examination (blood pressure, heart rate, BMI) Past medical history (medication, supplements, history of illness) Social history (smoking, alcoholic consumption, exercise, diet pattern) | Blood | Clinical chemistry analyser Blood pressure monitor, weighing and height scale |
2 | 0, 3 and 6 months | Physical examination (blood pressure, heart rate, BMI) | | Blood pressure monitor, weighing and height scale |
| | Body composition measurement • Body mass index • Segmental and visceral fat • Basal metabolic rate | Whole body | Body composition analyser |
| | Blood biochemistry test Oxidative stress markers • Advanced glycation end-products (AGEs) • Protein carbonyl (PC) • F2-isoprostanes (F2-IsoPs) • Malondialdehyde (MDA) | Blood Blood | Clinical chemistry analyser ELISA kit ELISA kit ELISA kit High performance Liquid Chromatography (HPLC) |
| | Inflammation markers • Tumor necrosis factor-α (TNFα) • Interleukin-6 (IL-6) | Blood | ELISA kit ELISA kit |
| | Weight Management • Ghrelin • Leptin | Blood | ELISA kit ELISA kit |
| | Vitamin E level | Blood | HPLC |
| | Skin assessment • Elasticity • Hydration • Transepidermal water loss • Pigmentation • Sebum secretion • Wrinkles and Roughness | Face and Arm Face and Arm Face and Arm Face and Arm Face and Arm Face and Arm | Cutometer Corneometer Tewameter Mexameter Sebumeter Visioscan |
5 | 0 and 6 months | Arterial stiffness assessment | Whole body | Arteriograph device /Photoplethysmography |
| | Bone density assessment | Whole Body | Dual-energy x-ray absorptiometry |
| | Cognitive function assessment • MoCA • RAVLT • Digit Span | | Questionnaires |
Participant timeline
Over the study period, three assessments at 0, 3, and 6 months, are carried out across various domains of inquiry, encompassing oxidative stress, inflammation, skin attributes, arterial stiffness, cognitive function, weight management, bone mineral density, and vitamin E levels. To ensure comprehensive oversight of the assessments, a delegation of authority log is established and formally endorsed by the respective investigators, each of whom possesses expertise in their specific area of assessment.
Sample Size
Sample size is calculated using Power and Sample Size Program. The total number of subjects calculated per group of experimental design is 93 when dichotomous outcomes is presumed with Type I error probability, α of 0.05 and power of 0.8. To maintain the power of study, 20% dropout rate of subjects is factored in. Consequently, a total of 110 subjects will be enrolled to receive the placebo, while an additional 110 subjects will be recruited to receive TRF supplements.
Recruitment
The selection of potential trial subjects is conducted by the Principal Investigator (PI) based on the inclusion and exclusion criteria. Informed consents are obtained from subjects by contract research assistance. During enrolment, general information (name, gender, age, address and contact information) is obtained. To ensure security and confidentiality, each subject is assigned a subject-specific code. The code is used to label the subject’s laboratory results. Each subject is reimbursed with standard transportation fees.
Subjects who fail to meet the inclusion and exclusion criteria are defined as screening failures. The principal investigator (PI) maintains a Screening Log which includes screen failures to ensure a systematic selection of subjects.
Subjects are free to withdraw from the study at any time for any reason. Subjects may also be withdrawn at any time at the discretion of the investigator. Possible reasons for withdrawal include sample events, abnormal laboratory values, protocol violation, subject requiring the use of unacceptable concomitant medication, subject not complying with protocol procedures, subject developing a condition during the study that violates the inclusion/exclusion criteria, lost to follow up, death or any other reason in the investigator’s opinion that would impede the subject’s participation in the study.
Assignment of interventions
Tocotrienols and placebo supplements are prepared and labelled with codes by the sponsor. Assignment of supplements to subjects is made by chance through the drawing of lots by contract research assistants. The treatment is double-blinded throughout the study period until all data has been collected and analysed after which the supplementation codes are revealed to the PI by the sponsor. However, in case of any serious adverse event reported during the study period, the unmasking of code is done immediately.
A set of numbers from 1 to 60 is randomised using the randomization application for every block of 60 subjects. The subjects are assigned to the randomised number according to numerical sequence 1 to 60 in the list of randomised number. All even numbers are treated with the same IP while odd numbers are treated with another IP. Thus, every block randomization has an equal number of subjects for each IP. The randomization is repeated until 220 subjects are recruited.
The unblinding in emergency situations is only permitted in case of a suspected, unexpected serious adverse reaction (SUSAR) or other important adverse event, when the knowledge of the supplemented product in question is required for therapeutic decisions for the management of the subject.
In the event of an emergency, an emergency decoding option is accessible to both the Investigator and designated personnel at the Sponsor. The responsibility for breaking the blind for individual subjects in emergency situations lies with the Investigator. If the need arises to break the blind, the Investigator will communicate this to the Sponsor. Before the treatment code is broken, the Investigator responsible for unblinding must document the reason and date for unblinding in the CRF to ensure proper documentation of the unblinding event, including a clear explanation of the reason for unblinding.
In the event that the Sponsor requires unblinding of the treatment, the reason and date of unblinding are recorded in the CRF. In situations where it is necessary to unblind an individual subject's treatment for the purpose of expedited reporting to the authorities, only designated individuals within the Sponsor, who are responsible for reporting this information, will have access to the identity of the product. Every effort is made to ensure that all other members of the clinical trial remain blinded throughout the entire duration of the trial.
Data collection and management
During the screening, potential subjects are assessed for eligibility to enter the study according to the inclusion and exclusion criteria. A total of 10 ml blood samples is taken from each volunteer after an overnight fast. The screening tests consist of comprehensive evaluations, encompassing blood lipid profile, liver function test, renal profile, full blood count, and fasting blood sugar measurements. Additionally, the records of past supplementation and medical history are reviewed to obtain a holistic understanding of the individual's health status. The results of blood tests are revealed to volunteers upon request. Volunteers who meet the inclusion criteria are selected for baseline sampling typically within a timeframe of 1 to 2 months following the screening process.
Eligible subjects are contacted to schedule appointments for data collection at baseline, 3rd and 6th month. During each visit, a comprehensive medical evaluation is conducted by a physician, encompassing medical history assessment, documentation of any concurrent medications, measurements of subjects' weight, height, resting blood pressure, pulse rate, and temperature, along with the evaluation of vital signs. Subsequently, blood samples are drawn from each subject for immediate analysis of blood biochemistry, and additional samples are preserved for future analysis of oxidative stress markers, inflammatory markers, vitamin E, leptin, and ghrelin levels. Furthermore, on the same day as the visit, assessments are performed for arterial stiffness, body fat composition, skin health, bone mineral density, and cognitive function.
The records of subjects and data of analyses are kept in separate folders according to the protocol and transferred into the CRF of each subject by the contract research assistants. The result for each assessment is monitored by specific investigators with expertise in the field, to facilitate comprehensive monitoring of the data.
A disclosure
contractual agreement signed between the sponsors and investigators outline the sponsors' access rights to the trial dataset and source data. Both investigators and sponsors should collaborate and jointly author publications reporting on the trial's results to ensure an accurate representation of the results and the drawing of appropriate conclusions, thereby preserving the integrity of the findings. All data are reported in a collective manner with no reference to any subjects in the trial to ensure that the identity of subjects remain confidential.
Analyses
Statistical analysis will be conducted using SPSS software (Chicago, IL, USA). The Kolmogorov-Smirnov test will be performed to assess the normality distribution of the data. Type III sum of squares will be set to evaluate the hypotheses. A mixed-design ANOVA will be used to determine whether supplementation affects the blood biochemistry parameters (inflammation and oxidative stress) and physical health (body composition, skin, cognitive function, and vascular health) at baseline, 3 months follow-up, and 6 months follow-up among the supplementation groups. Mauchly’s test of sphericity will be conducted to assess homogeneity of variance and compare the means of the supplemented group to the placebo group. To control against an increase in the risk of Type I errors during significance evaluation, Tukey's post hoc test for multiple comparisons will be applied. Dunnett’s pairwise multiple comparison test will be utilized to assess the changes of the means within groups. Prior to statistical analysis, any missing data will be excluded. All data will be presented as mean ± standard error of the mean, with statistical significance defined as p < 0.05.
Monitoring
Subjects are instructed to maintain their regular lifestyle throughout the study duration. It is recommended that they adhere to their usual routine regarding the number of meals and dietary plan. If there is a necessity to use any medication, they are required to inform the PI to ensure proper documentation in the CRF.
To ensure compliance, subjects are requested to submit the provided supplement bottles during their visits at the 3rd and 6th month. Additionally, plasma vitamin E levels are measured at 0, 3, and 6 months to assess the subjects' response to supplementation. In the event of suspected side effects or adverse reactions, subjects should promptly report to the PI and seek immediate medical assistance. The PI will assess whether the observed effects are related to the trial and determine if the subject should be excluded from the study accordingly.
Ethics and dissemination
This research upholds the principles of Good Clinical Practice (GCP) and adheres to the pertinent regulatory guidelines established by the Malaysia Ministry of Health (MOH). The research is approved by the Research Ethics Committee of Universiti Kebangsaan Malaysia (UKM), registered with the Malaysia National Medical Research Register (NMRR) and National Pharmaceutical Regulatory Agency (NPRA). Funding for the study is provided by KL-Kepong Oleomas Sdn. Bhd., with the research itself being independently conducted by scholars and experts from a higher learning institution. Before the clinical trial commenced, the two parties sign a distinct legal agreement designed to safeguard the rights of the sponsor and researchers involved.
It is important to note that the investigators have no financial or other competing interests in relation to the overall trial, ensuring impartiality and scientific integrity. Any alterations to the protocol are communicated by the investigators to both the Research Ethics Committee and the sponsor prior to implementation.
Upon completion of the trial, the PI and sponsor collaborate in publishing the results and deriving pertinent conclusions, all while ensuring the strict confidentiality of subjects' personal information.
The PI is responsible for retaining the data for a minimum duration of seven years from the date of closure of the database. The sponsor possesses exclusive rights to utilize the data for commercial purposes, except for any personal information related to the subjects. Concurrently, researchers are granted the privilege to employ the data for non-commercial purposes, encompassing but not limited to patient care and treatment, academic pursuits, and publication.