Study setting {9}
This is a single-centre, University of Glasgow, two-arm randomised controlled trial.
Eligibility criteria {10}
Potential participants will receive a mailed prestudy invitation which will provide details on study information and details around eligibility. If still interested, potential participants will be invited to the research site where eligibility will be determined based on the inclusion and exclusion criteria (Table 2).
Table 2
Inclusion and exclusion criteria
In order to be considered eligible for participation in the study they must:
|
Criterion
|
Characteristic of eligible participants
|
1
|
Be male or female aged 18-40 years (young group) or ≥65 years (older group) at time of consent
|
2
|
Participant is able and willing to sign the Informed Consent From
|
3
|
No plans to change lifestyle (activity and nutrition) during the study period
|
4
|
Not currently, or in the last year, participating in more than 1h per week of vigorous aerobic physical activity or any resistance exercise
|
5
|
BMI ≤ 30 kg/m2;
|
6
|
Not have diabetes mellitus; severe cardiovascular disease; dementia; seizure disorders; liver disease; uncontrolled hypertension (>150/90mmHg at baseline measurement); cancer or cancer that has been in remission <5 years
|
7
|
Not have ambulatory impairments which would limit ability to perform assessments of muscle function
|
8
|
Not be currently taking Vitamin K2 supplements
|
9
|
Not be currently taking Vitamin K antagonists/anticoagulants (e.g. warfarin);
|
10
|
Not be a current smoker
|
11
|
Not have a history of drug abuse
|
12
|
Not be currently taking medication known to affect muscle (e.g. steroids).
|
Who will take informed consent? {26a}
Written consent will be obtained by a member of the study team during a visit to the research site for the baseline assessment, with no study procedures taking place until consent has been obtained. At the baseline visit, a member of the study team will confirm eligibility.
Additional consent provisions for collection and use of participant data and biological specimens {26b}
Not applicable
Interventions
Explanation for the choice of comparators {6b}
The comparator chosen is a placebo capsule without the active ingredient.
Intervention description {11a}
Control
Participants in the control group will consume one 380mg micro-crystalline cellulose tablet per day.
Vitamin K2
Participants in the vitamin K2 380mg micro-crystalline cellulose tablet containing 240 µg vitamin K2 (K2VITAL® 0.2% DELTA powder) per day.
Criteria for discontinuing or modifying allocated interventions {11b}
In accordance with the Declaration of Helsinki, participants can withdraw from the trial at any time for any reason. Further, the investigator can withdraw a participant from the trial at any time if a withdrawal is considered in their best interest. Participants who have stopped the intervention prior to completion will be asked to follow study visits according to the protocol. Participants who choose to withdraw from the trial will be asked the reason why. However, it is emphasised that they are not obliged to state the reason.
Strategies to improve adherence to interventions {11c}
Participants will be sent a text message every 2 weeks to check how they are getting on taking their supplements and to serve as a reminder to do so.
Relevant concomitant care permitted or prohibited during the trial {11d}
Participants will be asked to maintain their usual physical activity and nutritional habits throughout the trial. Usual healthcare will also be maintained.
Provisions for post-trial care {30}
There are no specific provisions for post-trial care within this study, other than access to usual healthcare.
Outcomes {12}
Baseline characteristics will be collected – age, sex, blood pressure, weight, height, BMI, habitual diet, habitual physical activity levels, current medications and comorbidities.
All outcome measures will be assessed at baseline, and 12 weeks in all participants. Where the post-exercise recovery of outcomes is detailed, these will be measured fasted at baseline (pre-exercise) after which an exercise bout will take place. The exercise will involve bilateral leg extension exercises at 85% of one-repetition maximum (1RM), with 5 sets of 15 repetitions performed. Each repetition will be performed for 4s – 2s in the concentric and 2s in the eccentric phase. If necessary, the concentric phase can be assisted. Following this the same protocol will be performed for bench press. A standardised snack will then be consumed. Further measurements will be made at 3h, 24h, 48h and 72h post-exercise
Primary outcome measure
The primary outcome measure is change in post-exercise recovery of knee extensor muscle maximal isometric torque from baseline to 12 weeks.
Secondary outcome measures
Secondary outcome measures are the change post-exercise recovery from baseline to 12 weeks of:
Pain free range of motion
Time to complete 5 chair rises
Knee extensor surface electromyography (sEMG)
Systemic markers of inflammation (e.g. interleukin-6 and interleukin-1β)
Systemic markers of oxidative stress (e.g. peroxiredoxin 3 redox state and F2-isoprostanes)
And resting levels of:
Vitamin K levels
Carboxylated Osteocalcin
Uncarboxylated Osteocalcin
Carboxylate/Uncarboxylated Osteocalcin
Carboxylated matrix gla-protein
Dephosphorylated-uncarboxylated matrix gla-protein
Exploratory outcome measures
Exploratory outcome measures are the change from baseline to 12 weeks of:
Lean and fat mass (bioelectrical impedance)
Blood glucose
Blood lipids
Plasma insulin
Vastus lateralis muscle thickness
Substrate utilisation (carbohydrate and fat oxidation) during submaximal exercise
Participant timeline {13}
Sample size {14}
We have based our sample size on being able to (within age groups) detect a physiologically relevant difference in muscle strength of 10% with an SD of 10%, based on data from within our laboratory, with a power of 80% and alpha level of 0.05. This would require a sample size of 17 in each group and so we will aim to recruit 20 participants to each group (within age groups) to account for drop out.
Recruitment {15}
Older and young people will be recruited:.
-
Using advertisements via multiple media outlets that include newspapers, magazines, flyers, social media, and posters.
-
Via local clubs, organization and centres.
Assignment Of Interventions: Allocation
Sequence generation {16a}
Following the baseline assessment, patients will be randomised to the control or vitamin K2 groups (1:1 ratio) using a computer-generated (sealedenvelope.com) randomisation sequence with randomly permuted blocks. Randomisation will be stratified by age group (18-40; ≥65 years) and by sex. The randomisation sequence will be generated by an independent statistician.
Concealment mechanism {16b}
Participants, research staff and trial statistician will be blind to allocation. The randomisation sequence will be generated by an independent statistician and the sequence passed to independent staff within the manufacturing facility who will label the supplement packets with a participant number. Staff and participants will be blinded to this sequence and all data will be analysed blind to allocation.
Implementation {16c}
Patients will be enrolled by a member of the research team and following randomisation the supplement packet for that participant ID will be given to them.
Assignment Of Interventions: Blinding
Who will be blinded {17a}
Participants, research staff and statistician will be blind to allocation.
Procedure for unblinding if needed {17b}
Unblinding will take place upon request of the participant or the clinical research staff. The independent statistician holding the list will be contact by the research team and will disclose the allocation to that participant. This will be logged in the study database.
Data Collection And Management
Plans for assessment and collection of outcomes {18a}
All outcome data will be collected according to developed standard operating procedures.
Plans to promote participant retention and complete follow-up {18b}
No further strategies, than those described to maintain engagement with intervention, are planned.
Data management {19}
All data will be kept in a secure storage area with access only to the study staff.
Confidentiality {27}
Access to the collated participants’ data will be restricted to the principal investigator and appropriate research study staff as required. All laboratory samples, completed forms, reports and other records will be identified using a unique participant ID number to maintain participant confidentiality.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
Blood samples will be collected via venepuncture and analysed in our clinical biochemistry laboratories.
Statistical Methods
Statistical methods for primary and secondary outcomes {20a}
The data analysis will be performed by the trial statistician (FH) who will be blinded to the group assignment. Participants’ characteristics will be described using mean and standard deviation (or median and interquartile range for skewed non-normal variables) for continuous variables and frequency tables for categorical variables. For each of the outcomes, two-sample t-tests will be used to compare between group differences in baseline and in follow-up assessments. Cohen’s d will be calculated to quantify effect sizes. Ad will also be used with group assignment, time, and group x time interaction as predictors with and without adjustment for prognostic covariates (e.g. age, sex).
Interim analyses {21b}
No interim analysis is planned in the current study.
Methods for additional analyses (e.g. subgroup analyses) {20b}
Subgroup analysis will be conducted for sex and age group (younger vs. older) and interactions between these subgroups and treatment groups will be tested.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
Adherence to supplements will be determined by capsule count following completion of the intervention period. Non-adherence will be handled through use of intention to treat analysis in the primary analysis and we will also add an additional instrumental variable analysis to examine effect sizes if the participant is fully compliant with supplementation [25]. Missing data will be handled by baseline observation carried forward in the primary analysis. Due to the randomised design, multivariable models will not be used to adjust for confounding, and therefore missing covariates will not impact the primary analysis.
Plans to give access to the full protocol, participant level-data and statistical code {31c}
The data sets analysed and the codes used for these analyses will be available from the corresponding author on reasonable request.
Oversight And Monitoring
Composition of the coordinating centre and trial steering committee {5d}
Principal investigator
Design and conduct of trial
Preparation of protocol and revision
Preparation of study materials and case report forms
Organise steering committee meetings
Publication of study reports
Trial Steering committee (Principal investigator, study coordinator, independent academic clinician)
Agreement of final protocol
All lead investigators will be members
Recruitment of patients and liaising with principal investigators
Reviewing progress of study and if needed agreeing changes to protocol
Trial Management Committee (Principal investigator, study coordinator, researchers)
Study planning
Adverse event reporting
Responsible for trial master file
Budget control
Data verification
Composition of the data monitoring committee, its role and reporting structure {21a}
Not applicable.
Adverse event reporting and harms {22}
There are unlikely to be major safety issues with our nutritional supplement interventions. However, if any safety concerns or incidents arise, we will follow our standard operating procedures for reporting adverse events in a non-Clinical Trial of an Investigational Medicinal Product (non-CTIMP) studies.
Frequency and plans for auditing trial conduct {23}
Formal audits will occur every 6 months.
Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}
Any amendments to the protocol will we approved by the local ethics committee prior to implementation. All investigators and patients enrolled in the trial will be informed.
Dissemination Plans {31a}
The study findings will be disseminated primarily via conference presentations and scientific papers.