The Dose-response of Vitamin C-enriched Collagen on Markers of Collagen Synthesis in Healthy Young and Older Men and Women Following Resistance Exercise
Phase: NA
Conditions: Diet, Food, and Nutrition
Sponsor: Rob Erskine

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The Dose-response of Vitamin C-enriched Collagen on Markers of Collagen Synthesis in 
Healthy Young and Older Men and Women Following Resistance Exercise   
Study protocol, statistical analysis plan and informed consent form 
Document date: 06/06/2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Participant Flow 
The number of participants who started and completed this study is shown in Figures 1, 2, 3, 
and 4. 
 
Figure 1. A CONSORT flow diagram for young males.  

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Figure 2. A CONSORT flow diagram for young females.  
 
 
Figure 3. A CONSORT flow diagram for older males. 

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Figure 4. A CONSORT flow diagram for older females. 
 
Arm/Group Information 
Arm 1:  
Trial for young male participants (aged 18 – 39 years): Consuming hydrolyzed collagen (HC) 
with resistance exercise 
Arm 1 Description: 
Young male participants were asked to visit the laboratory on three different occasions to 
consume one of different doses of hydrolyzed collagen (HC) and performing resistance 
exercise (RE). 
Different doses of HC (0 grams, 15 grams and 15 grams) and 50 mg vitamin C were dissolved 
with 250 mL water in an opaque bottle. To match calories of 30 grams HC, 34.1 grams and 
15.4 grams maltodextrin was used in 0 grams HC and 15 grams HC, respectively. Also, to mask 
any potential difference in HC doses, 4 grams non-caloric sweetener was included in all HC 

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doses. 
Each trial (with a seven-day washout period) lasted for seven hours and immediately after 
participants consumed HC, they performed four sets of ten repetitions of barbell back squat 
exercise at 10-repetition maximum load and rested. Ten x 5 mL blood samples were collected 
as follows: at rest immediately prior to HC ingestion, 0.5 hour post HC ingestion, 1 hour post 
HC ingestion, 0.5 hour post exercise, 1 hour post exercise, 2 hours post exercise, 4 hours post 
exercise and 6 hours post exercise. All trials were performed at the same time of day (08:00 - 
15:00). 
Blood samples were analysed to measure the concentration of a biomarker of collagen synthesis 
(serum procollagen type Ⅰ N-terminal propeptide (PⅠNP)), a biomarker of collagen breakdown 
(plasma β-isomerized C-terminal telopeptide of type I collagen (β-CTX)), serum collagen 
amino acid concentration, and concentration of 17β-estradiol (for female participants only).  
The details of nutritional supplements used are as follows: 
Hydrolyzed collagen (Myprotein, Cheshire, UK), vitamin C powder (Holland and Barrett 
Retail Limited, Warwickshire, UK), maltodextrin (Myprotein, Cheshire, UK), and non-caloric 
sweetener (Truvia®, SilverSpoon, London, UK) 
Arm 2:  
Trial for young female participants (aged 18 – 39 years): Consuming HC with RE. 
Arm 2 Description: 
The intervention procedure is exactly same as Arm 1 except for the number of visits. Young 
female participants visited the laboratory on four occasions during two consecutive months. 
Therefore, there were two trials in each month, where female participants’ estrogen level was 
lower (i.e., onset of menses) or higher (i.e., ovulation). Dates for the trials were determined 

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based on self-report of onset of menses and previous menstrual cycle length. 
Arm 3:  
Trial for older male participants (aged 40 – 65 years): Consuming HC with RE. 
Arm 3 Description: 
The intervention procedure is the same as Arm 1.  
Arm 4:  
Trial for older female participants (aged 40 – 65 years): Consuming HC with RE.  
Arm 4 Description: 
The intervention procedure is the same as Arm 2.  
 
Period 
Period title:  
Overall study 
Participants were recruited from a university student population (young participants) and from 
the local area (older participants). Recruitment began in January 2019 and data collection was 
completed in September 2021. 
2. Baseline Characteristics  
Arm 1: 10 young men (mean ± SD; age: 26 ± 3 years, height: 1.77 ± 0.04 m, body mass: 79.7 
± 7.0 kg).  
Arm 2: One young woman (age, 36 years; height, 1.61 m; body mass, 82.6 kg).  

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Arm 3: Eight older men (mean ± SD: age, 49 ± 8 years; height, 178 ± 2 cm; body mass, 90 ± 4 
kg).  
Arm 4: Two older women. One participant (age, 43 years; height, 160.5 cm; body mass 63.5 
kg) was a European masters level karate athlete, while the other was a recreational distance 
runner (age, 42 years; height, 172.6 cm; body mass 69.4 kg). 
 Age 
o young participants > 18 and < 40 years;  
o older participants > 40 and < 65 years 
 Sex 
o male and female 
 Ethnicity 
o Race and Ethnicity Information Not Collected 
 
 Region of Enrollment 
 
o Liverpool, United Kingdom 
Variables 
Outcome 
measure type 
Number of participants analyzed 
Arms 1 and 3 
Serum PⅠNP 
Primary 
18 
Plasma β-CTX 
Primary 
18 
Serum amino acid concentration 
Primary 
18 

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3. Outcome Measure Information 
Table 1 shows data for each primary and secondary outcome measure by arm. 
Table 1. Data for outcome measures. 
 
Outcome Measure Title and description 
Concentrations of serum procollagen type Ⅰ N-terminal propeptide (PⅠNP) (ng/mL)  
Concentration of plasma β-isomerized C-terminal telopeptide of type I collagen (β-CTX) (µg/L)   
Concentration of 18 amino acids that constitute collagen (µmol/L): 
Glycine, proline, hydroxyproline, glutamic acid, alanine, arginine, aspartic acid, lysine, serine, 
leucine, valine, phenylalanine, threonine, isoleucine, histidine, tyrosine, methionine, and 
glutamine.  
Concentration of 17β-estradiol (pg/mL) 
Outcome Measure Time Frame 
Outcome measures were analyzed from September 2019 to August 2022 
Arm 1 Information 
Arms 2 and 4 
Serum PⅠNP 
Primary 
3 
Plasma β-CTX 
Primary 
3 
Serum amino acid concentration 
Primary 
3 
Serum 17β-estradiol 
Secondary 
3 

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Arm 1 title: 
Young male trials 
Arm 1 description: 
Thirteen male participants volunteered for this study but three were excluded prior to 
participation due to not meeting the inclusion criteria (n = 1) and declining to proceed with 
participation (n = 2). Therefore a total of 10 male participants completed the study, which 
included three separate trials.  
Arm 2 Information 
Arm 2 title: 
Young female trials 
Arm 2 description:  
One female participant completed this study, which included four separate trials. 
Arm 3 Information 
Arm 3 title: 
Older male trials 
Arm 3 description: 
Ten male participants volunteered for this study but two were excluded prior to participation 
due declining to proceed with participation. Therefore a total of eight male participants 
completed the study, which included three separate trials.  
Arm 4 Information 
Arm 4 title: 

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Older female trials 
Arm 4 description:  
Two female participants completed this study, which included four separate trials. 
 
Analysis Population Information 
In Arm 1, data from 10 male participants were analyzed and in Arm 2, data from one female 
participant were analyzed. In Arm 3, data from 8 male participants were analyzed and in Arm 
4, data from two female participants were analyzed. 
Outcome Measure Data Table 
Outcome measure data are shown in Table 2. 
 
Table 2. Outcome Measure Data. 
Variable 
Measure type 
Measure of Dispersion 
Arm 1 
Serum PⅠNP 
 (ng/mL) 
Mean 
Standard deviation 
Plasma β-CTX 
(µg/L)   
Mean 
Standard deviation 
18 Serum amino acids 
(µmol/L) 
Mean 
Standard deviation 
Arm 2 
Serum PⅠNP 
n/a 
n/a 

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 (ng/mL) 
Plasma β-CTX 
(µg/L)   
n/a 
n/a 
18 Serum amino acids 
(µmol/L) 
n/a 
n/a 
Serum 17β-estradiol  
(pg/mL) 
n/a 
n/a 
Arm 3 
Serum PⅠNP 
 (ng/mL) 
Mean 
Standard deviation 
Plasma β-CTX 
(µg/L)   
Mean 
Standard deviation 
18 Serum amino acids 
(µmol/L) 
Mean 
Standard deviation 
Arm 4 
Serum PⅠNP 
 (ng/mL) 
n/a 
n/a 
Plasma β-CTX 
(µg/L)   
n/a 
n/a 
Serum 18 amino acids 
(µmol/L) 
n/a 
n/a 
Serum 17β-estradiol  
(pg/mL) 
n/a 
n/a 
 
Statistical Analyses 

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Due to the sample size (n=1) in Arm 2, only data from Arm 1 were analyzed.  
Two-way within-subject ANOVA models (dose × time) were performed to detect changes in 
serum PⅠNP, amino acid and plasma β-CTX concentration. One-way repeated measures 
ANOVA models were performed to detect differences in area-under-the-curve between trials 
for PⅠNP and each of the 18 amino acids. If an interaction effect or a main effect of HC dose 
existed, Bonferroni post-hoc pairwise comparisons were performed. The level of statistical 
significance was set at P < 0.05. 
4. Adverse Event Information 
During data collection period, any adverse event such as all-cause mortality or serious adverse 
event did not occur.  
5 Certain Agreements 
Are all PIs Employees of Sponsor? 
No: The principal investigator is a PhD student, not an employee of the sponsor 
Results Disclosure Restriction on PI(s)? 
No 
6. Results Point of Contact 
Dr Rob Erskine 
Research Institute for Sport and Exercise Sciences, Liverpool John Moores University  
Tel: +44 (0)151 904 62; E-mail: R.M.Erskine@ljmu.ac.uk 
 
 

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Informed Consent Form (Human Tissue Act 2004) 
Study Title: 
The dose-response of vitamin C-enriched collagen on markers of collagen 
synthesis in healthy young and older men and women following resistance 
exercise 
LJMU Ethics code:  18/SPS/059 
Name of Principal Investigator: 
Joonsung Lee 
Faculty & School: 
School of Sport and Exercise Sciences 
Contact details: 
J.Lee1@2017.ljmu.ac.uk 
 
1. I confirm that I have read and understand the information provided for the above 
study. I have had the opportunity to consider the information, ask questions and 
have had these answered satisfactorily 
 
2. I understand that my participation is voluntary and that I am free to withdraw at any 
time, without giving a reason and that this will not affect my legal rights. 
 
3. I understand that any personal information collected during the study will be 
anonymised and remain confidential.  
 
4. I understand that providing a muscle biopsy is optional and thus, I do not have to 
provide a muscle to take part in the study. 
 
5. I consent to my skeletal muscle and blood samples being stored securely at LJMU 
for the duration of this ethically approved research and used for the research 
purposes outlined in the Participant Information Sheet. 
 
6. I give the consent for my skeletal muscle and blood samples to be used for DNA 
analysis or other genetic testing as described in the Participant Information Sheet. 
 
7. I agree for my skeletal muscle and blood samples to be stored for the research 
purposes this ethically approved research and future projects under the regulation 
of the Human Tissue Act. 
 
8. I agree to my GP being informed of my participation in the study (if appropriate). 
 
9. I agree to take part in the above study. 
 
Name of Participant 
 
 
 
 
Date  
 
 
Signature 
 
Name of Researcher 
 
 
 
 
Date 
 
 
Signature 
 
Name of Person taking consent 
 
 
 
Date 
 
 
Signature 
(If different from researcher 
FORM: Informed Consent - Human Tissue 
 (HT-CONSENT-003) 
Approved by 
 
Date effective 
 
Version 
 
Date of next review 
 
 
Please Initial