Coffee Intake Biomarker Validation and Association With Respiratory Diseases
Phase: NA
Conditions: Coffee
Sponsor: National University of Singapore

Official title: Coffee Intake Biomarker Validation and Associations with Respiratory 
Diseases 
Approved Date: August 22, 2022 
 
 

 
Study Protocol and Statistical Analysis Plan 
Recruitment of study participants 
Study participants (n=100) will be recruited via flyers (e.g., shopping malls, MRT 
stations, neighbourhood shops, community centres, food centres, tertiary institutions 
such as NUS (Saw Swee Hock School of Public Health, National University of 
Singapore), and door-to-door), advertisements at on-campus notice boards, bus-stops 
and newspapers. Recruitment email within NUS  and NTU (Lee Kong Chian School of 
Medicine, Food Science & Technology and Office of Faculty Housing, Nanyang 
Technological University) will be sent by the respective administrative officers on behalf 
of the project team members. Recruitment will also be conducted through online 
platforms such as Telegram, Facebook and Instagram, as well as snowballing and 
convenience sampling through referrals. Participants will be screened for their eligibility 
before they are enrolled into the study. Exclusion criteria would include pregnancy, 
medication use, presence or history of diseases (e.g., cancer, cardiometabolic, 
endocrine), major surgical operations, depression, anxiety, caffeine/lactose intolerance, 
fear of needles and/or blood disorders. A short questionnaire will be administered to 
each participant to obtain information on their basic demographics, lifestyle and 
medical history. Inclusion criteria would include regular coffee drinkers aged 21-64 
years old and of Chinese ethnicity. All participants will be compensated for their time 
(S$300). Written informed consent will be obtained from each participant prior to the 
commencement of the study. Approvals from the Institutional Review Boards of NUS 
and NTU will also be obtained. 
 
Briefing session and informed consent (two weeks prior to intervention study)  
A briefing session, along with informed consent, will take place two weeks before 
commencing the intervention study at NTU Experimental Medicine Building level 1 
(interview room) or through an arranged Zoom call. Participants who opted for an 
arranged zoom call will be sent a scheduled meeting link, where he/she will be briefed 
by the recruited research assistant and/or PI/ Co-I. Researchers will explain the project 
aims and intervention study, and answer questions that the participants may have. If 

the participants agree, we will record their informed consent. Over zoom, informed 
consent will be conducted via a video zoom call, in presence of a third-party witness 
(either an individual added in the call or physically present with the participant at the 
time of the call) and the participant will be sign the consent with an e-signature. This 
will also be performed by the research assistant and should be completed within half 
an hour. Participants will be briefed on how to collect their 24-hour urine (during Day 0- 
the day before the intervention or otherwise known as Day 1) and provided with urine 
collection containers and esky bags. They will also be given or emailed handouts on 
foods/drinks not allowed to be consumed one week before the intervention study and 
during the study. 
 
Multi-dose intervention study design  
The accurate measurement of all coffee-derived compounds and metabolites in urine 
and blood will be carried out in a pilot intervention study, where coffee (Coffee A) will be 
fed to participants (n=100) in an increasing dose over three weeks (one dose per week, 
two dose per week and three dose per week). 
Their next coffee intervention will be Coffee B. Coffee B will be the highest dosage 
(three dose per week) of a different blend of coffee.  
 
Pre-study Commencement: 
Before commencing the study (Day 0), participants will be requested to collect their 
total volume urine (24h urine) in a ‘urine care package’ consisting of urine collection 
containers, zip lock bags, ice packs, an esky and an instruction sheet that would have 
been delivered to them prior. The participants will also be requested to fast overnight 
(8-12h) without caloric intake. 
 
Study Visits and Blood Collection: 
On Day 1 of the intervention study, participants will be split into small groups, 
consisting of at most 50 participants each visit over at least two days. This will take 

place at the NTU Experimental Medicine Building. Upon arrival, participants will be 
required to go to the seminar room to provide us with their filled urine containers and 
complete questionnaires. They will then proceed to level 1 (team-based learning  
room) for blood drawing. This is a designated, safe, clean, quiet/comfortable room. 
Qualified personnel/registered nurses from Caring Hearts Nursing Pte Ltd have been 
engaged to provide mobile phlebotomy service through the duration of the intervention 
study and will perform the blood-taking procedure. 
A total of 11 blood collections will be performed. Subjects will be divided into two 
groups (Group A and Group B) for logistics reason. For Group A, blood will be collected 
into 6 mL vacutainers on Days 1, 8, 15,22, 29, 36, 43, 50, 57, 60, 63 of the intervention 
study (over 10 consecutive weeks). For Group B, blood will be collected into 6 mL 
vacutainers on Days 2, 9, 16, 23, 30, 37, 44, 51, 58, 61, 64 of the intervention study 
(over 10 consecutive weeks). 
 
Coffee Intervention: 
Subjects will then proceed to a designated area outside the seminar room for coffee 
drinking and to receive coffee grounds for the remaining days of the week. They will 
receive pre-packaged coffee grounds to prepare the low coffee dose at home for the 
remainder of the week. Ground coffee will be purchased from a local coffee provider 
and packed into individual coffee sachets. The coffee grounds will be packed by an 
external food packing service, in a food-grade certified premise. The letters/ 
documentation indicating Hazard Analysis Critical Control Point (HACCP) certification 
of the vendor’s premise has been provided.  
The starting dose of coffee A per dose is ≤10 g coffee powder for low dose, ≤20 g 
coffee powder for medium dose and ≤40 g coffee powder for high dose. On the other 
hand, coffee B will be of high dosage (≤40g). Participants will be asked to brew the 
coffee for a maximum of 10 minutes in a cup of hot water, minimum 150 mL. The 
concept of this intervention study design is to stimulate habitual drinking of coffee. 
Hence, participants are required to consume the coffee doses daily but are not 
restricted in the time of coffee consumption, they may take the coffee in the morning or 
afternoon. 

At the end of the low coffee dose intervention (Day 7), participants will be requested to 
collect their 24h urine and return on Day 8, where fasting serum samples will be 
collected from them. This is followed by a week without any coffee consumption to 
investigate the post consumption kinetics, i.e., how long the candidate biomarkers 
remain present in the biofluids after stopping coffee consumption. At the end of the 
‘coffee abstinence’ week (Day 14), again, participants will be requested to collect their 
24h urine and fast overnight for the serum sampling on Day 15. On the same day (Day 
15), participants will be provided with the medium dose coffee and the next set of pre-
packaged coffee grounds to prepare the medium coffee dose at home for the 
remainder of the week. This is repeated for the high dose coffee A and high dose coffee 
B interventions. Each study visit is estimated to be completed within a maximum of two 
hours. 
 
Tea Intervention: 
A single dose will be provided to participants, in the form of one tea-bag from a generic 
brand. Pre-packaged commercial tea bags will be purchased from a supermarket. 
Participants will be asked to brew the tea-bag for a maximum of 5 minutes in a cup of 
hot water (200 mL). From two days before commencing the study until the end of the 
intervention study, participants will be requested to avoid certain foods that may 
confound the study design including other coffee and tea that are not provided by us, 
coffee or tea-flavoured foods, caffeine-containing beverages (i.e., Coca-Cola, energy 
drinks), supplements containing caffeine or of herbal/botanical origin, chocolate and 
chocolate-containing foods. We will organise a briefing with the participants before 
commencing the study, and provide a pamphlet containing pictures and a detailed list of 
“allowed” and “non-allowed” foods/beverages. We will also ask the participants to keep 
an electronic daily dietary record and take photos of their meals to ensure that they 
maintain compliance with the study design. Participants who consume coffee products 
outside of our study design will be flagged and excluded in the sensitivity analysis. With 
a sample size of 100 participants, we will stagger their visit for the serum 
collection/handing out of coffee grounds into morning sessions for 3 days, i.e., 33-34 
participants per day. The session (08.00-11.00) will consist of serum sampling, briefing, 
consent taking, etc from 08.00-11.00 and processing of serum/urine samples in the 

laboratory from 09.00-13.00. 
 
Sample processing and extraction 
Urine will be collected (weekly, 20mL) in urine specimen containers, transferred to 
50mL tubes, centrifuged (1500g, 10 min, 4°C) and aliquoted into 1.5mL tubes. Blood 
will be collected (weekly, 6mL) in Vacutainer® serum tubes and allowed to clot/stand at 
room temperature. After 30-40 min, serum will be separated from entire blood by 
centrifugation (2000g, 10 min, 24°C) and aliquoted into 1.5mL tubes. All samples will 
be processed on the same day that they are collected. Coffee and tea samples 
provided to the participants will also be collected (weekly, 50mL each) with a portion 
aliquoted into 1.5mL tubes for a detailed chemical analysis as the composition of local 
coffee brews has not been studied. Samples will be stored at -80°C until all samples 
have been collected at the end of the intervention study and processed in a single 
batch. Briefly, thawed samples will be added with deuterated internal standards (for 
measure of control for extraction). After, urine samples will be centrifuged (4000g, 10 
min, 4°C) and transferred into glass vials. Serum samples will be de-proteinised with 
acidified methanol, centrifuged (4000g, 10 min, 4°C) and transferred into glass vials. 
Coffee and tea aliquots will be centrifuged (4000g, 10 min, 4°C) to separate the 
particulate pellet from aqueous phase and fat layer on the surface, if present. The fat 
layer will be removed with a micro-spatula, diluted 50-fold and transferred into glass 
vials. All samples will be stored at -80°C until ready for analysis. 
 
Targeted and untargeted metabolomic profiling 
Urine and serum samples will undergo a combination of targeted and untargeted 
metabolomic profiling at the Singapore Phenome Centre located at NTU, which has 
state-of-the-art mass spectrometers and strong technical support. Firstly, targeted 
detection and quantification of a list of previously proposed coffee biomarkers  will be 
conducted on the intervention urine and serum samples using a Waters Acquity Ultra-
High Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry 
(UHPLC-TQ-MS), and commercially available standards and deuterated standards. For 
atractyligenin, its lack of standard previously limited its detection but we have 

successfully synthesised this compound from its atractyloside precursor, which can be 
used for level l identification and accurate quantification in this project. Briefly, thawed 
urine, serum, coffee and tea samples will be analysed directly on the UHPLC-TQ-MS. 
Secondly, to explore potentially novel biomarkers and/or metabolites related to the 
consumption of the two types of coffee relevant to Singapore (black coffee and coffee 
with additives), untargeted metabolomics will be conducted on the urine and serum 
samples using a Waters Acquity UHPLC-QTOF-MS, operating in positive and negative 
electrospray ionisation modes with a mass scan range from 50 to 1000 m/z. Samples 
will be processed as described above. For each matrix, a quality control extract of 
pooled urine/serum from all study participants will be injected at the beginning and end 
of each run, and in-between every 10 samples. 
 
Data Processing and Compound Identification 
Quantification of the biomarker compounds from the targeted method will be processed 
using MassLynx (Waters-specific software). Processing of the untargeted 
metabolomics data will be carried out using an open access platform, Galaxy 
WorkFlow4Metabolomics (http://workflow4metabolomics.org/). Only de-identified 
datasets without subject identifiers will be uploaded to the open access platform. In 
untargeted metabolomics, the identity of detected ions is a priori unknown and the 
multi-step identification process will follow as previously described using publicly 
available databases, e.g., Metlin, PhytoHub, HMDB, m/zCloud, etc. As identification of 
unknown ions is still very much a bottleneck in metabolomics studies, an in-house 
database of all known coffee precursors and metabolites will also be created to 
facilitate this identification process, and will be built with monoisotopic mass, chemical 
formula, m/z, and fragmentation pattern and spectra obtained from commercially 
available and deuterated standards. 
 
Statistical analysis 
The concentrations of compounds identified and quantified from the targeted method 
will be used to build calibration curves relative to their dose-response in Microsoft Excel 
2016 and R version 3.4. Assuming a drop-out rate of 33% (one-third), we will have a 

total of 67 participants in for this pilot study. Cross-validated principal component 
analysis and multivariate linear regression models will be used to assess the 
compounds that best discriminate the multi-dose coffee consumption in the untargeted 
approach (e.g., having a variable importance in projection (VIP) >2) using SIMCA 
version 15, and prioritised for identification.