Proteome Dynamics to Inform Healthy Muscle Ageing
Phase: NA
Conditions: Ageing Well
Sponsor: Liverpool John Moores University

 
 
 
 
 
 
STUDY PROTOCOL 
 
Study title: Proteome Dynamics to Inform Healthy Muscle Ageing 
Document date: 12th December 2022 
Research Ethics Committee Reference Number: 22/SPS/075 

 

 
 
 
 
PARTICIPANT INFORMATION SHEET [Healthy, non-exercise trained adults.]  
 
Research Ethics Committee Reference Number: 22/SPS/075 Version 2 (23rd Jan 2023) 
 
Title of Study: Proteome Dynamics to Inform Healthy Muscle Ageing 
 
You are being invited to take part in a research study. You do not have to take part if you do not 
want to. Please read this information, which will help you decide. 
 
1. What is the purpose of the study? 
Losses in muscle function are a feature of ageing that negatively impact disease risk profile, 
resilience to injury, societal/economic contribution and quality-of-life. This project exploits a 
new technique (muscle proteome dynamics) that is unique to our group and capable of studying 
the quality of thousands of proteins in human muscle. This study hopes to identify any 
potential differences in skeletal muscle protein turnover due to aging, we hope this work could 
eventually lead to better care and lifestyle advice for older adults. 
 
2. Why have I been invited to participate?  
You have been invited because you are an otherwise healthy adult male that does not take part 
in very regular physical exercise training. 
Exclusion criteria – you must not participate if you: 
- female 
- aged <18 years, 35-65 years, or >75 years 
- take part in >3 bouts of endurance-/resistance-based training per week  
- are pregnant  
- have any previous diagnosis of ischaemic heart disease, diabetes mellitus myopathy or any    
neuromuscular disorder.  
- take any anticoagulant medications such as warfarin 
- have a current musculoskeletal injury.  
- currently unwell with cold or flu  
- have a chronic connective tissue disorder  
- have a cardiovascular disease  
- are currently enrolled in any other research study 
 
3. Do I have to take part?  
No. You can ask questions about the research before deciding whether to take part. If you do 
not want to take part that is OK. We will ask you to sign a consent form and will give you a 
copy for you to keep.   
You can stop being part of the study at any time, without giving a reason. You may withdraw 
from the study by contacting the research team. 
 
You must not take part if you have COVID-19 or symptoms of COVID-19 - 
https://www.nhs.uk/conditions/coronavirus-covid-19/symptoms/  
 
4. What will happen to me if I take part?  
 
We will talk you through all study protocols and procedures prior to your participating and 
give you the chance to ask any questions that you may have. 
 

 
Initial assessment 
You will first undergo an initial screening questionaire to check you meet the inclusion criteria 
prior to commencing the study. If you are eligible for the study, the first laboratory visit will take 
place 7 ± 2 days prior to the start of the experimental intervention. You will complete a biopsy 
screening form in the presence of a trained member of LJMU staff. In addition, basic assessments 
will be undertaken, including height, weight and blood pressure measurements.  
If you fit the inclusion criteria, we will assess your physical activity levels (Paffenbarger physical 
activity questionnaire), ability to exercise (Readiness to Exercise questionnaire), and typical 
dietary intake (Food Frequency Questionnaire). You will then undertake 6 assessments (body 
composition assessment, walking test, 5x sit-to-stand, grip strength, isokinetic dynamometry and 
10-repetition maximum leg press). 
 
Body composition assessment - we will assess your body composition using a bioelectrical 
impedance analysis (BIA) machine. This procedure is non-invasive, pain free and takes 
approximately five minutes, from a practical point of view it is like standing on a set of bathroom 
scales. From the test, we attain information on your height, body mass, fat mass and skeletal 
muscle mass. 
 
 The walk test - you will be required to complete seven 6-second walking trials over a 12-
metre distance, while the position of your whole body is monitored, and video recorded by 
multiple cameras.  
 
You will need to wear a t-shirt and cycling shorts. Cycling shorts 
can be provided if you don’t have any. You will then have some 
reflective markers attached to the skin on your legs and upper 
body/torso using medical grade double sided tape and elastic 
wrapping.  
 
 
The sit-to-stand test - Next, the 5 times sit-to-stand test will 
be completed while the position of your whole body is 
monitored, and video recorded by multiple cameras.  
 
The 5 x sit-to-stand test will be conducted using a standard height (43-45 cm) chair with straight 
back. You will start in the seated position and, after confirming your ability to perform 1 sit-to-
stand action, you will be instructed to stand and sit 5 times as quickly as possible, ensuring your 
feet remain flat on the floor. The time taken to complete 5 sit-to-stand movements will be 
measured using a stopwatch.  
 
While seated, your grip strength of your dominant arm will be measured using a hand 
dynamometer. During this test you will be instructed to grip the dynamometer as tightly as 
possible without moving from the seat or raising your legs from the floor. Next, isokinetic torque 
of the knee extensor muscles will be assessed via isokinetic dynamometry (IKD). You will first 
perform a warm-up (5 min at 50 W on a cycle ergometer) and then be seated in an upright position 
on the isokinetic dynamometer (similar to a gym leg-extension exercise machine). Non-extendable 
straps crossing the chest, abdomen and thighs will be used to minimise upper body movements 
during the leg exercise. The test protocol will consist of four consecutive maximal knee extension 
movements of each leg at two different fixed movement velocities, 1.05 rad.sec-1 (60 deg.sec-1) 
and 3.14 rad·sec-1 (180 deg.sec-1) from which peak torque (Nm) data will be recorded. Each 
movement will be separated by a five-minute rest.  
We will then assess your single-leg leg press ten-repetition maximum (10RM) on a leg press 
machine, which will be the same equipment that will be used in the subsequent exercise sessions - 
this will help to provide familiarization to the apparatus. You will perform warm-up exercises 
including cycling (5 min at 50 W) and leg press (10 repetitions at <50 % of your predicted 10RM 

estimated from normative data). Repetitions of leg press will be performed to a metronome-based 
cadence of 3 seconds for the concentric (push) phase of the lift and 3 seconds for the eccentric 
(return) phase of the lift. If you are able to complete 10 repetitions at a given weight, a greater 
weight will be selected and the test repeated after a 5 min period of rest. The greatest weight with 
which you are able to complete ten repetitions is recorded as the 10RM.  
 
Figure 1 – schematic illustration of the experimental protocol. 
 
Experimental Intervention 
Baseline muscle biopsy 
On day -1 of the experimental intervention (around 7 days after the initial assessment), you will 
need to arrive at the laboratory at ~8 am after an overnight fast. A blood sample (7 ml) will be 
collected via venipuncture and muscle samples will be collected by percutaneous biopsy of the 
vastus lateralis (quadriceps) of each leg (see below for more detail).  
 
Deuterium Oxide consumption 
On the next day (Experiment Day 0) you will consume the stable isotope, deuterium oxide (D2O – 
see below for more details), which is a tracer that can be used to measure the turnover rate of 
proteins inside you muscle. You will consume a dose of 13.3 ml D2O per kg body weight on Day 
0. This equates to ~1 L of liquid for a person of 75 kg body weight. The liquid must be consumed 
in 4 doses of ~250 ml each at 2 h intervals, during which time you will be monitored for potential 
side effect (see below) by the research team, this means you will need to be available for 6 h on 
this day. (N.B. during this period you will have access to office and catering facilities on the 
LJMU Byrom Street site). On subsequent days of the experiment you will need to consume one 
maintenance dose of 0.67 ml D2O per Kg body weight (e.g. 50 ml for a person of 75 kg body 
weight) – self-administered each day, at a standardised time of day until day 14. 
 
Unilateral leg-press exercise 
On days 1, 4, 7, 10 and 13 of the experimental intervention, you will perform supervised single-
legged resistance exercise (unilateral leg-press) at a standardised time of day. Each session will 
begin with a warm-up consisting of 5 repetitions at 70 % of 10RM. You will then perform three 
sets of 10 repetitions at a lifting intensity of 90 % of 10 RM with 2- to 3-min rest between sets. On 
days 1 and 7 we will collect a venous blood sample (7 ml) and a finger-prick blood sample (~0.2 
ml).   
 
Post-intervention muscle biopsy 
On day 15 of the experimental intervention, you will arrive at the laboratory at 8 am after an 
overnight fast. A blood sample (7 ml) will be collected via venipuncture and muscle samples will 
be collected by percutaneous biopsy of the vastus lateralis (quadriceps) of each leg. 
 

Post-intervention muscle function test 
On day 18 of the experimental intervention, isokinetic torque of the knee extensor muscles will be 
assessed via isokinetic dynamometry (IKD). Consistent with the initial assessment (Experiment 
day -7), you will first perform a warm-up (5 min at 50 W on a cycle ergometer) and then complete 
a test protocol consisting of four consecutive maximal knee extension movements of each leg at 
two different fixed movement velocities, 1.05 rad.sec-1 (60 deg.sec-1) and 3.14 rad·sec-1 (180 
deg.sec-1) from which peak torque (Nm) data will be recorded. This assessment will be scheduled 
at a time of day roughly equivalent to that of your initial visit. 
 
5. Are there any potential risks in taking part? 
The main risks to you as a participant are associated with the collection of muscle and blood 
samples. All these measures are, however, routinely used in research and carry a low risk (outlined 
below). If at any point during your participation you feel uncomfortable, unwell, or unable to 
continue participating for any reason, you should contact the research team immediately and 
testing can be ceased if necessary. 
 
Muscle biopsy procedure: 
In total you will have 4 muscle biopsies taken during the study (one sample from each leg at two 
different time points). Briefly, you will undergo a short period of discomfort whereby you receive 
a local anaesthetic to numb the area on the vastus lateralis (thigh) muscle. The researcher will then 
make a ~4-5 mm incision using a scalpel and extract an extremely small (approximately the size of 
a grain of rice) piece of muscle tissue for analysis. 
 
These procedures are carried out by fully qualified personnel, who have been previously trained 
and signed off by a Physician. The biopsy sites will be fully dressed upon completion of the 
procedure, and you will be able to walk without any pain immediately after the dressing is applied. 
The day after the biopsies you may feel a ‘dead leg’ like sensation and may experience minimal 
amounts of bruising which is normal. If you do experience any pain, this normally disappears 
within 2 days of the biopsy and if you have any concerns, you can contact a member of the 
research team and come back in at any time to get the biopsy site checked. 
 
There is an extremely low risk (1 in 1,000,000) of allergic reaction to the local injection. The 
chance of a local skin infection in less than 1 in 1000. Carefully cleaning the skin and keeping the 
area clean until the skin heals will minimize this. Infection can be serious and if you therefore 
experience a lot of bleeding from the biopsy site, swelling or infection around the biopsy site, 
faintness, light headedness, heart pain, chest pain or increasing pain in your leg which is not 
relieved by Paracetamol, you must contact the researcher who did the biopsy right away. However, 
if for some reason, you are not able to contact this individual then you should contact your family 
doctor or go to the Accident and Emergency Department. More detailed information on the muscle 
biopsy procedure can be found on the accompanying muscle biopsy information sheet. This 
information sheet will provide advice on what to do following your muscle biopsy and it is 
essential these guidelines are followed. 
 
All risks can be discussed with the research team, and we advise all participants to visit their GP 
before consenting to take part in the study. This will give you the opportunity to discuss any 
medical history that may prevent you from taking part. 
 
Because you will be given an anaesthetic, we will request your consent to notify your GP about 
your involvement in this study 
 
Blood sampling 
You will feel a sharp pain when the needle is inserted, though this will be short-lived. The 
researchers are accredited and experienced in this technique, so the pain you experience should be 
minimal. You may develop a small bruise on your arm, which can be prevented by applying 
pressure on the site following removal of the needle (the researcher will either do this themselves 

or ask you to do so if you are comfortable to). The risk of haematoma or bruising has been shown 
to occur in ~12% of blood collections, with minor bruising most common at the site. There is also 
a small risk of fainting, which typically occurs in less than 1% of blood collections. If you develop 
any issues at a blood collection site, you should contact the research team, who will decide if there 
is an issue for medical referral. 
 
Deuterium (D2O) Consumption: 
You will be asked to orally consume the stable isotope, deuterium (D2O), also called ‘heavy 
water’, which is similar to regular water (H2O) but contains a stable (non-radioactive) isotope of 
hydrogen with one more neutron (2H instead of 1H) and it is a bit heavier than regular water (~1.1 
kg per litre instead of 1 kg per litre). Heavy water behaves like water in your body, but because of 
its slightly heavier isotope, we can detect how much of it is retained in your body. Also, because 
there is decay in the amount of circulating D2O you consume, we will ask you to consume small 
top-up doses on a daily basis. 
 
D2O consumption is safe but there are risks associated with deuterium consumption when 
enrichment exceeds ~20%, or when large amounts (e.g. > 500 ml) are taken as a single dose. These 
side-effects typically include dizziness and nausea. Human research, including this study, typically 
aims to achieve an enrichment of 1 - 3%, which is significantly below this threshold. 
 
The risks associated with deuterium consumption in the doses provided in this study are therefore 
minimal. You will be monitored during the initial enrichment (4x 250 ml drinks over 6 h on 
Experiment Day 0) in case you experience side-effects. During the remaining days of the 
experiment you will be provided with 50 ml doses to consume daily at home. In the unlikely event 
that you experience any side-effects whilst not in the lab, you should contact the research team 
immediately and if necessary, we will advise you to seek further medical advice from your GP or 
by dialling 999. 
 
 
6. Are there any benefits in taking part? 
The potential or hoped for benefits of the study for the wider society are to show there detectable 
age-related differences in the turnover of proteins in human muscle during basal conditions or 
when performing regular exercise. If this is the case, our new technique for assessing protein 
turnover in humans could help other researchers to better develop and test interventions aimed at 
improving healthy ageing. 
 
7. Payments, reimbursements of expenses or any other benefit or incentive for taking part 
You will receive up to £15 per laboratory visit for reasonable travel costs 
 
8. What will happen to information/data provided? 
The information you provide as part of the study is the study data.  Any study data from which 
you can be identified (e.g. from identifiers such as your name, date of birth, audio recording etc.), 
is known as personal data. Your participation in this study will involve the collection/use of 
personal data.  
 
We will keep personal data safe and secure. People who do not need to know who you are will not 
be able to see your name or contact details. The personal data collected will include: 
• Contact details. With your agreement, we would like to store your contact details so that 
you may be contacted about future opportunities to participate in research studies. 
• A record of consent (which will include your name) 
 
Study data. We will use a code/pseudonym so that you cannot be directly identified from the data. 
Study data / records of consent / contact details will be kept for up to three years after the study has 
finished.  
 

Once we have finished the study, we will keep some of the data so we can check the results.  
 
9. What will happen to the tissue samples? 
It will be necessary to retain the consent form (personal data) until the sample has been depleted or 
destroyed, in order to meet the traceability requirements of the Human Tissue Act. 
Your DNA and tissue samples will be assigned a code and your data will also be identified only by 
this number. You will not be identifiable from reading the code; however, your DNA is unique to 
you so it can never be completely anonymous. 
Your  muscle biopsy sample(s) (or derivatives of) will be  stored securely at LJMU indefinitely 
and might be shared anonymously with other investigators, in ethically approved studies that take 
place in hospitals, universities, non-profit institutions or commercial laboratories worldwide. If 
you agree to your samples being used in future studies, your consent form will be held until the 
samples have been used up. 
 
10. What if we find something unexpected? 
The investigator will not suggest the presence or absence of diagnosable conditions. The study is 
not a clinical or personal assessment and the investigators are not appropriately qualified to 
provide meaningful individual assessments. If the investigator suspects they have found something 
unexpected, they will consult with senior researchers and may advise you to seek appropriate 
advice if they think that could help you gain access to services that might be of help. 
 
 
11. Who is organising and who is funding/commissioning the study? 
This study is organised by Liverpool John Moores University and funded by the ART of Healthy 
Aging Network who focus on addressing novel questions on underlying biological processes 
during ageing. 
 
12. Whom do I contact if I have a concern about the study or I wish to complain? 
If you have a concern about any aspect of this study, please contact Dr Jamie Pugh, and we will do 
our best to answer your query.  You should expect a reply within 10 working days.  If you remain 
unhappy or wish to make a formal complaint, please contact the Chair of the Research Ethics 
Committee at Liverpool John Moores University who will seek to resolve the matter as soon as 
possible: 
Chair, Liverpool John Moores University Research Ethics Committee; Email: 
FullReviewUREC@ljmu.ac.uk; Tel: 0151 231 2121; Research Innovation Services, Liverpool 
John Moores University, Exchange Station, Liverpool L2 2QP 
 
13. Data Protection 
 
Liverpool John Moores University is the data controller with respect to your personal data. 
Information about your rights with respect to your personal data is available from:  
• https://www.ljmu.ac.uk/legal/privacy-and-cookies/external-stakeholders-privacy-
policy/research-participants-privacy-notice  
• by asking one of the study team or contacting us using the information below 
 
14. Contact details  
Principal Investigator: Dr Jamie Pugh (Member of LJMU staff) 
LJMU Email address: j.pugh@ljmu.ac.uk 
LJMU School/Faculty: Sport and Exercise Science 
LJMU Central telephone number: 0151 231 2121