Heavy Isotope-labeled-butyrate and Glucose in Breath Testing to Detect Mesenteric Ischemia, a Proof-of-concept Study in Healthy Volunteers.
Phase: NA
Conditions: Chronic Mesenteric Ischemia
Sponsor: Erasmus Medical Center

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13C-butyrate and 13C-glucose breath testing to detect 
mesenteric ischemia, a proof of principal study in 
healthy volunteers (CMI breath test) 
 (June 2020) 
 
 
 
 

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PROTOCOL TITLE ‘13C-butyrate and 13C-glucose breath testing to detect mesenteric 
ischemia, a proof of principal study in healthy volunteers’ 
 
Protocol ID 
Not applicable 
Short title 
CMI breath test 
EudraCT number  
Not applicable 
Version 
2.0 
Date 
08-06-2020 
Coordinating investigator/project 
leader 
 
L.G. Terlouw, MD  
Department of Gastroenterology & Hepatology, 
Erasmus MC University Medical Center, Rotterdam, 
the Netherlands  
l.terlouw@erasmusmc.nl  
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
<Multicenter research: per site> 
 
Erasmus MC University Medical Center 
M.J. Bruno, MD, PhD 
Department of Gastroenterology & Hepatology, 
 Erasmus MC University Medical Center, Rotterdam, 
the Netherlands 
m.bruno@erasmusmc.nl  
 
A. Moelker, MD, PhD 
Department of Radiology, 
Erasmus MC University Medical Center, Rotterdam, 
the Netherlands 
a.moelker@erasmusmc.nl  
 
M.P. Peppelenbosch, PhD 
Department of Gastroenterology & Hepatology, 
Erasmus MC University Medical Center, Rotterdam, 

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the Netherlands 
m.peppelenbosch@erasmusmc.nl 
 
H.J.M. Verhagen, MD, PhD 
Department of vascular surgery,  
Erasmus MC University Medical Center, Rotterdam, 
the Netherlands 
h.verhagen@erasmusmc.nl 
 
T.M. Luider, PhD 
Department of Neurology, 
Erasmus MC University Medical Center, Rotterdam, 
the Netherlands 
t.luider@erasmusmc.nl  
 
 
Franciscus Gasthuis & Vlietland 
D. van Noord, MD, PhD 
Department of Gastroenterology & Hepatology,  
Franciscus Gasthuis & Vlietland, Rotterdam, the 
Netherlands  
d.leemreis@franciscus.nl 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
Erasmus University Medical Center Rotterdam 
 
 
Subsidising party 
 
Independent expert (s) 
A.C. de Vries, MD, PhD 
Department of Gastroenterology & Hepatology,  
Erasmus MC University Medical Center, Rotterdam, 

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the Netherlands 
 
B.M. van Dalen, MD, PhD 
Department of Cardiology,  
Franciscus Gasthuis & Vlietland, Rotterdam,  
the Netherlands 
b.vandalen@franciscus.nl  
Laboratory sites <if applicable> 
Laboratorium MDL 
Erasmus MC University Medical Center,  
Wytemaweg 80, 
3015 CN Rotterdam, the Netherlands 
 
Da Vinci Laboratory Solutions B.V. 
Postbus 12103, 
3004 GC Rotterdam, the Netherlands 
 
 
Pharmacy <if applicable> 
Not applicable 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE .............................................................................11 
2. 
OBJECTIVES ................................................................................................................12 
3. 
STUDY DESIGN ...........................................................................................................12 
4. 
STUDY POPULATION ..................................................................................................14 
a. 
Population (base) .......................................................................................................14 
b. 
Inclusion criteria .........................................................................................................14 
c. 
Exclusion criteria ........................................................................................................14 
d. 
Sample size calculation .............................................................................................14 
5. 
TREATMENT OF SUBJECTS ......................................................................................16 
5.1 
Investigational product/treatment .......................................................................16 
5.2 
Use of co-intervention (if applicable) ..................................................................16 
5.3 
Escape medication (if applicable) .......................................................................17 
6 
INVESTIGATIONAL PRODUCT ...................................................................................18 
6.1 
Name and description of investigational  product(s).........................................18 
Not applicable ...................................................................................................................18 
6.2 
Summary of findings from non-clinical studies .................................................18 
6.3 
Summary of findings from clinical studies .........................................................18 
6.4 
Summary of known and potential risks and benefits ........................................18 
6.5 
Description and justification of route of administration and dosage ...............18 
Not applicable ...................................................................................................................18 
6.6 
Dosages, dosage modifications and method of administration .......................18 
Not applicable ...................................................................................................................18 
6.7 
Preparation and labelling of Investigational Medicinal Product .......................18 
Not applicable ...................................................................................................................18 
6.8 
Drug accountability ..............................................................................................18 
Not applicable ...................................................................................................................18 
7 
NON-INVESTIGATIONAL PRODUCT ..........................................................................19 
7.1 
Name and description of non-investigational product(s) ..................................19 
7.2 
Summary of findings from non-clinical studies .................................................19 
7.3 
Summary of findings from clinical studies .........................................................19 
7.4 
Summary of known and potential risks and benefits ........................................19 
7.5 
Description and justification of route of administration and dosage ...............20 
7.6 
Dosages, dosage modifications and method of administration .......................20 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ...............20 
7.8 
Drug accountability ..............................................................................................20 
8 
METHODS ....................................................................................................................21 
8.1 
Study parameters/endpoints ...............................................................................21 
8.1.1 
Main study parameter/endpoint ...................................................................21 
8.1.2 
Secondary study parameters/endpoints (if applicable) ..............................21 
Not applicable ...............................................................................................................21 
8.1.3 
Other study parameters (if applicable) ........................................................21 

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8.2 
Randomisation, blinding and treatment allocation ............................................21 
8.3 
Study procedures .................................................................................................21 
8.4 
Withdrawal of individual subjects .......................................................................23 
8.4.1 
Specific criteria for withdrawal (if applicable) .............................................24 
Not applicable ...............................................................................................................24 
8.5 
Replacement of individual subjects after withdrawal ........................................24 
Inclusion will end after inclusion of 5 volunteers in each of the six groups. ........................24 
8.6 
Follow-up of subjects withdrawn from treatment ..............................................24 
Follow-up is not performed. ...............................................................................................24 
8.7 
Premature termination of the study ....................................................................24 
9 
SAFETY REPORTING ..................................................................................................25 
9.1 
Temporary halt for reasons of subject safety ....................................................25 
9.2 
AEs, SAEs and SUSARs ......................................................................................25 
9.2.1 
Adverse events (AEs) ...................................................................................25 
9.2.2 
Serious adverse events (SAEs) ....................................................................25 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) ...................26 
9.3 
Annual safety report ............................................................................................26 
9.4 
Follow-up of adverse events ...............................................................................26 
9.5 
[Data Safety Monitoring Board (DSMB) / Safety Committee] ............................26 
10 
STATISTICAL ANALYSIS.........................................................................................27 
10.1 
Primary study parameter(s) .................................................................................27 
10.2 
Secondary study parameter(s) ............................................................................27 
10.3 
Other study parameters .......................................................................................27 
10.4 
Interim analysis (if applicable) ............................................................................27 
11 
ETHICAL CONSIDERATIONS ..................................................................................28 
11.1 
Regulation statement ...........................................................................................28 
11.2 
Recruitment and consent ....................................................................................28 
11.3 
Objection by minors or incapacitated subjects (if applicable) ..........................28 
11.4 
Benefits and risks assessment, group relatedness ...........................................28 
11.5 
Compensation for injury ......................................................................................28 
11.6 
Incentives (if applicable) ......................................................................................29 
12 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ........................30 
12.1 
Handling and storage of data and documents ...................................................30 
12.2 
Monitoring and Quality Assurance .....................................................................30 
12.3 
Amendments ........................................................................................................30 
12.4 
Annual progress report........................................................................................30 
12.5 
Temporary halt and (prematurely) end of study report .....................................30 
12.6 
Public disclosure and publication policy ...........................................................31 
13 
STRUCTURED RISK ANALYSIS ..............................................................................32 
13.1 
Potential issues of concern .................................................................................32 
13.2 
Synthesis ..............................................................................................................33 
14 
REFERENCES ..........................................................................................................33 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; 
in Dutch: Algemeen Beoordelings- en Registratieformulier (ABR-
formulier) 
AE 
Adverse Event 
AR 
Adverse Reaction 
ATP 
Adenosine triphosphate 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CMI 
Chronic mesenteric ischemia 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics; in Dutch: officiële 
productinformatie IB1-tekst 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; 
in Dutch: Uitvoeringswet AVG 

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VLS 
Visible light spectroscopy 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
 
Rationale: Chronic mesenteric ischemia (CMI) is an incapacitating disease that can 
progress to potentially fatal acute mesenteric ischemia. The yearly incidence of CMI is 9.2 
per 100.000 and will increase due to the aging population and the rising prevalence of 
cardiovascular risk factors. A gold standard diagnostic test to diagnose CMI is currently 
lacking, causing both undertreatment and overtreatment of patients and thereby superfluous 
healthcare expenses. Since oxygen is needed to absorb and metabolise butyrate and 
glucose in the enterocyte, a 13C-butyrate and 13C -glucose breath test could theoretically 
quantify mucosal oxygen content and thereby identify patients with CMI.  
Objective: To explore the possibility of detecting mesenteric ischemia by 13C-butyrate and 
13C -glucose breath testing by comparing timing and concentration of the peak of expired 
13CO2. 
Study design: randomised intervention study 
Study population: healthy human volunteers, >18 old 
Intervention (if applicable): The intervention group will perform a standardized bicycle 
ergometry exercise test with a duration of 30 minutes. The aim of the exercise test is to 
induce temporary mesenteric ischemia. 
Main study parameters/endpoints: To explore the possibility of detecting mesenteric 
ischemia by 13C-butyrate and 13C -glucose breath testing by comparing timing and 
concentration of the peak of expired 13CO2. 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: All participating volunteers will perform 2 breath tests at baseline, 0.5, 1, 
1.25 1.5, 1.75, 2, 2.25, 2.5, 3, 3.5 and 4 hours after ingestion of 13C-butyrate or 13C-glucose. 
A peripheral intravenous cannula will be inserted in patients in the intervention group, in 
order to obtain a serum blood sample at baseline and after the exercise test. The cannula will 
also be used to obtain blood for the lactate measurements during the exercise test. 
Participating volunteers will not experience any benefits of participation. No adverse events 
are expected and risks associated with participation are deemed very low to non-existent. 
Insertion of the peripheral intravenous cannula can result in a hematoma. 
 
 

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1. INTRODUCTION AND RATIONALE 
 
Chronic mesenteric ischemia (CMI) is an invalidating disease, causing severe complaints of 
post-prandial pain, food fear and weight loss(1, 2). When left untreated CMI has a substantial 
risk  of bowel infarction in 26-67% of patients, resulting in high mortality and morbidity (3, 4). 
Diagnosis of CMI remains difficult, since no gold standard diagnostic test exists. The gold 
standard diagnosis described in current literature is relief of symptoms after endovascular or 
surgical revascularisation. In order to take treatment decisions a consensus diagnosis is 
used. Consensus is reached during a multidisciplinary meeting attended by 
gastroenterologists, vascular surgeons and interventional radiologists. Consensus is based 
on history, presence of mesenteric artery stenosis on abdominal imaging and in dedicated 
centres a functional test, such as visible light spectroscopy (VLS) and tonometry. In patients 
with a consensus diagnosis of CMI based on a stenosis of a single mesenteric artery, 
treatment is successful in 73% of patients(5). In the remaining 27% not benefitting from 
treatment, CMI was never the cause of the patients symptoms.  
 
A more accurate functional test detecting intestinal ischemia is of great value to improve 
the used consensus diagnosis. Current functional tests have several downsides and are 
therefore not widely used. VLS, a technique measuring mucosal saturations, is limited by a 
specificity of 60%, causing a substantial amount of false positive test results(6). Tonometry, a 
technique measuring luminal carbon dioxide concentrations in stomach and jejunum, has a 
better specificity of 92%(6). Yet this test will cease to exist in a few years, since tonometry 
equipment is no longer produced. Stressing the urgency of development of a new and 
improved functional test. 
 
Breath tests are accurate, simple and widely available tests for diseases, such as 
helicobacter pylori infection and small intestinal bacterial overgrowth(7, 8). Breath tests using 
glucose and butyrate could possibly be used to diagnose CMI. Butyrate is metabolized by the 
intestinal epithelium(9). Metabolism of butyrate is oxygen dependent, since oxidation is one 
of the steps during the production of adenosine triphosphate (ATP). 14C-butyrate can be used 
to measure the oxidation rate of butyrate(10). During the oxidation process the 14C atom is 
transferred to an oxygen molecule, producing 14C-carbon dioxide. The labelled carbon 
dioxide molecule is exhaled and can be measured in the air exhaled by the patient, thereby 
quantifying the presence of mucosal oxygen.  
 
Administration of deuterated glucose (13C-glucose) is a second potential method to 
measure mucosal oxygen content. Transport of glucose from the lumen into the epithelial cell 
is driven by an active process consuming ATP and thus oxygen(11). The transport of glucose 
is driven by a sodium gradient. At the basolateral side of the cell, Na+/K+-ATPase extrudes 
sodium, thereby creating a low sodium concentration within the cell. At the luminal side a 
Na+/glucose cotransporter, driven by the low intracellular sodium concentration, transports 
sodium and glucose into the cell. Further transport of glucose into the mesenteric circulation 
occurs by diffusion facilitated by passive glucose transporters. When 13C-glucose is 
metabolized the 13C atom is transferred to an oxygen molecule, producing 13C-carbon 
dioxide. The labelled carbon dioxide molecule is exhaled and can be measured in the air 
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In subjects with low mucosal oxygen concentrations, the peak in exhaled 13CO2  of 
butyrate and glucose is expected to be delayed and lower compared to control subjects with 
normal mucosal oxygen concentrations. Thereby creating a functional test measuring 
mucosal oxygen content, which could be of great value in diagnosing CMI. 
 
 
2. OBJECTIVES 
 
Primary Objective:  
To explore the possibility of detecting mesenteric ischemia by 13C-butyrate and 13C-glucose 
breath testing by comparing timing and concentration of the peak of expired 13CO2. 
 
3. STUDY DESIGN 
 
This study is a multi-center randomized interventional proof of principal study, exploring the 
possibility of quantifying mucosal oxygen content by 13C-butyrate and 13C-glucose breath 
testing. 
 
Inclusion 
The study is conducted in healthy volunteers without gastrointestinal complaints and unre-
markable medical history. Volunteers are recruited by distribution of patient information fold-
ers, containing study information and the investigators contact information, at the gastroen-
terology and Hepatology outpatient clinics of Erasmus MC University Medical Center and 
Franciscus Gasthuis & Vlietland. When volunteers contact the investigator the study outline 
is discussed and inclusion and exclusion criteria are checked. Volunteers are asked whether 
they have a medical history or are currently experiencing any complaints, they do not under-
go additional tests or check-ups to ensure they are healthy. When a healthy volunteer de-
cides to participate in the study an Informed Consent Form will be signed. 
 
Control 
Two different control groups both consisting of 5 volunteers will be used during the study. 
The first group will receive 13C-butyrate and the second group 13C-glucose. The control 
groups will perform the breath tests without performing any physical exercise. 
 
Intervention  
Two different intervention groups both consisting of 5 volunteers will be used during the 
study. The first group will receive 13C-butyrate, the second group 13C-glucose. All intervention 
groups will be performing a standardized bicycle ergometer exercise test with a total duration 

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of 30 minutes(12). The exercise test has been proven to elicit mesenteric ischemia in 
athletes. Previous studies suggest an additional 10 minute period of splanchnic hypo 
perfusion after discontinuation of exercise(13, 14).   
The exercise test consist of 3 phases.  
- 
Phase 1: The first 10 minutes of exercise are used to gradually increase the workload 
until submaximal exercise intensity is reached. Submaximal exercise is defined as 
lactate between 3 and 5.5 mmol/L. Lactate measurements are performed every 2 
minutes, using a rapid lactate measurement kit. 
- 
Phase 2: From minute 10 until 20 submaximal exercise intensity is maintained by 
adjusting the workload based on lactate measurements. The lactate measurements 
are performed every 3 minutes. 
- 
Phase 3: Minute 20 until 30 are used to reach maximal exercise intensity. Every 3 
minutes the workload is increased by 10% of the submaximal workload until 
exhaustion. Lactate measurements will be continued with 3 minute intervals. 
 
Randomisation 
Allocation to the 13C-butyrate or 13C-glucose group and allocation to the control or 
intervention group will be performed by randomisation. Blinding of the healthy volunteers will 
not be possible, since the intervention group has to perform an exercise test. 
 
Breath test 
Both intervention and control group will perform breath tests at the following time points 
baseline, 0.5, 1, 1.25 1.5, 1.75, 2, 2.25, 2.5, 3, 3.5 and 4 hours after ingestion of 13C-butyrate 
or 13C-glucose (figure 1). A breath sample is obtained by blowing a single breath of air 
through a straw into a test tube. Two breath samples are collected at all mentioned time 
points. The decision to perform breath testing for a total of 4 hours was based on  a study 
that determined 14CO2 elimination patterns after instillation of butyrate in human caecum and 
a study describing 13CO2 elimination patterns in healthy volunteers(10, 15). The tracer could 
be measured in exhaled air at the first time point, which was 30 minutes after instillation. A 
peak in expired 14CO2 was measured after 2 hours. Four hours after instillation 
approximately 55% of the total 14CO2 dose had been expired, 24 hours after instillation 70% 
of the instilled dose had been expired. Since we are interested in the peak of expired 13CO2 a 
measurement period of 4 hours seems sufficient, even in the intervention group, in whom a 
delayed or lower peak is expected. The frequency of breath testing is intensified between 1 
and 2.5 hours after ingestion, in order to detect the timing of the 13CO2  peak more precisely.  
 
 

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Figure 1. timeline collection of breath samples. 
 
 
Dosages 
All volunteers will be instructed to take their last meal 6 hours before start of the breath test. 
Volunteers in the 13C-butyrate group receive an oral dose of 0.03 µmol 13C-butyrate(10).  
Volunteers in the 13C-glucose group receive an oral dose of 20 mmol 13C-glucose(16).  
 
Follow-up 
There is no follow-up of healthy volunteers. 
 
4. STUDY POPULATION 
a. Population (base)  
Healthy volunteers with no gastrointestinal complains and unremarkable medical history 
will be asked to participate in our study through information folders. 
 
b. Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
• 
≥18 years of age 
• 
Experience with cycling 
• 
Signed informed consent  
• 
Unremarkable medical history (no gastrointestinal diseases, no cardiac or 
pulmonary diseases) 
• 
No gastrointestinal complaints 
 
c. Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
• 
Unable to sign informed consent 
• 
Pregnancy 
 
d. Sample size calculation 
This study is a proof of principal study, no previous studies exist on this topic, therefore a 
sample size calculation could not be performed. We have chosen to include a total of 20 
healthy volunteers in order to be able to compare 5 control subjects and 5 intervention 

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subjects for each breath test. In case the number of 5 subjects per group is insufficient to 
detect a statistical difference, the generated data can be used to perform a sample size 
calculation for a second study.   
 
 

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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
Intervention 
Two different intervention groups both consisting of 5 volunteers will be used during the 
study. The first group will receive 13C-butyrate and the second group 13C-glucose. Both 
intervention groups will be performing a standardized bicycle ergometer exercise test with a 
total duration of 30 minutes(12). The exercise test has been proven to elicit mesenteric 
ischemia in athletes. Previous studies suggest an additional 10 minute period of splanchnic 
hypo perfusion after discontinuation of exercise(13, 14).   
The exercise test consist of 3 phases.  
- 
Phase 1: The first 10 minutes of exercise are used to gradually increase the workload 
until submaximal exercise intensity is reached. Submaximal exercise is defined as 
lactate between 3 and 5.5 mmol/L. Lactate measurements are performed every 2 
minutes, using a rapid lactate measurement kit. 
- 
Phase 2: From minute 10 until 20 submaximal exercise intensity is maintained by 
adjusting the workload based on lactate measurements. The lactate measurements 
are performed every 3 minutes. 
- 
Phase 3: Minute 20 until 30 are used to reach maximal exercise intensity. Every 3 
minutes the workload is increased by 10% of the submaximal workload until 
exhaustion. Lactate measurements will be continued with 3 minute intervals. 
 
Control 
Two different control groups both consisting of 5 volunteers will be used during the study. 
The first group will receive 13C-butyrate and the second group 13C-glucose. The control 
groups will perform the breath tests without performing any physical exercise. 
 
Dosages 
All volunteers will be instructed to take their last meal 6 hours before start of the breath test. 
Volunteers in the 13C-butyrate group receive an oral dose of 0.03 µmol 13C-butyrate(10).  
Volunteers in the 13C-glucose group receive an oral dose of 20 mmol 13C-glucose(16).  
 
5.2 Use of co-intervention (if applicable) 
Not applicable 

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5.3 Escape medication (if applicable) 
Not applicable 

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6 INVESTIGATIONAL PRODUCT  
Not applicable 
  
6.1 Name and description of investigational  product(s) 
Not applicable 
6.2 Summary of findings from non-clinical studies 
Not applicable 
6.3 Summary of findings from clinical studies 
Not applicable 
6.4 Summary of known and potential risks and benefits 
Not applicable 
6.5 Description and justification of route of administration and dosage 
Not applicable 
6.6 Dosages, dosage modifications and method of administration 
Not applicable 
6.7 Preparation and labelling of Investigational Medicinal Product 
Not applicable  
6.8 Drug accountability 
Not applicable 
 

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7 NON-INVESTIGATIONAL PRODUCT 
7.1 Name and description of non-investigational product(s) 
13C-butyrate is a stable isotope used as a tracer molecule. 
https://www.sigmaaldrich.com/catalog/product/aldrich/488380?lang=en&region=NL  
 
D-Glucose-1-13C is a stable isotope used as a tracer molecule.  
https://www.sigmaaldrich.com/catalog/product/aldrich/297046?lang=en&region=NL 
 
7.2 Summary of findings from non-clinical studies 
13C-butyrate  
We refer to the safety information statement on the website of Sigma-Aldrich  
https://www.sigmaaldrich.com/catalog/product/aldrich/488380?lang=en&region=NL  
 
D-Glucose-1-13C  
We refer to the safety information statement on the website of Sigma-Aldrich  
https://www.sigmaaldrich.com/catalog/product/aldrich/297046?lang=en&region=NL 
 
7.3 Summary of findings from clinical studies 
13C-butyrate  
A study including 12 healthy volunteers and 12 Crohn’s disease patients showed that 13C-
butyrate absorption metabolism can be measured by 13CO2 breath testing(17). They detect-
ed 13CO2 at 30 minutes after oral administration of 13C-butyrate, with a peak 13CO2 concentra-
tion at 1 hour after administration. A study showed similar results and timing of expired 14CO2 
concentrations after instillation of 14C-butyrate in the cecum(10).  
 
D-Glucose-1-13C  
A study including 8 healthy highly trained volunteers examined the oxidation of 13C-glucose 
during exercise(16). This study showed that 13CO2 could be measured reliably, in expired air, 
after administration of a 13C-glucose dose of 0.05g/kg bodyweight. 
 
7.4 Summary of known and potential risks and benefits 
13C-butyrate  
We refer to the safety information statement on the website of Sigma-Aldrich  
https://www.sigmaaldrich.com/catalog/product/aldrich/488380?lang=en&region=NL  
 

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D-Glucose-1-13C  
We refer to the safety information statement on the website of Sigma-Aldrich  
https://www.sigmaaldrich.com/catalog/product/aldrich/297046?lang=en&region=NL 
 
7.5 Description and justification of route of administration and dosage 
13C-butyrate  
A study performing 14CO2 breath testing after instillation of 14C-butyrate in the cecum showed 
that a tracer dose of 0.03 µmol was sufficient and could be detected in expired air(10).  
 
D-Glucose-1-13C  
A study performing 13CO2 breath testing during exercise showed that 13CO2 could be meas-
ured reliably after administering an oral dose of 0.05g/kg bodyweight 13C-glucose(16). This 
dosage equals approximately 20mmol of glucose, when using a bodyweight of 70kg. 
 
7.6 Dosages, dosage modifications and method of administration 
13C-butyrate  
A study performing 14CO2 breath testing after instillation of 14C-butyrate in the cecum showed 
that a tracer dose of 0.03 µmol was sufficient and could be detected in expired air(10).  
 
D-Glucose-1-13C  
A study performing 13CO2 breath testing during exercise showed that 13CO2 could be meas-
ured reliably after administering 0.05g/kg bodyweight 13C-glucose(16). This dosage equals 
approximately 20mmol of glucose, when using a bodyweight of 70kg. 
 
Both 13C-butyrate and D-Glucose-1-13C are administered orally, since metabolism and ab-
sorption by the enterocytes are the processes that are of interest. 
 
 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
Preparation and labelling of the non-investigational medicinal products will be done 
according to the relevant GMP guidelines by licensed staff.  
 
7.8 Drug accountability 
13C-butyrate and D-Glucose-1-13C solutions will be prepared at the Gastroenterology & Hepa-
tology department. 

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8 METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
To explore the possibility of detecting mesenteric ischemia by 13C-butyrate and 13C-glucose 
breath testing by comparing timing and concentration of the peak of expired 13CO2. 
 
8.1.2 Secondary study parameters/endpoints (if applicable) 
Not applicable 
8.1.3 Other study parameters (if applicable) 
Not applicable 
 
 
8.2 Randomisation, blinding and treatment allocation 
Participating volunteers will not be blinded. Randomization will be performed into four groups 
of five volunteers.  
 
Randomisation will be performed through a random list generated by an online randomisa-
tion tool and is executed by a person not actively involved in the study. The randomisation 
tool can be found at https://www.sealedenvelope.com/simple-randomiser/v1/lists. The used 
seed will be noted and saved, the treatment groups are: BC, BI, GC, GI. Block sizes will be 5 
blocks of 4, resulting in a randomized list of 20 subjects (5 per group). The created list will be 
saved and used to fill 20 envelopes numbered 1-20. Each envelope will contain the treatment 
group as specified in the randomly generated list. Envelope 1 will be for the first subject en-
rolled in the study, envelope 2 for the second subject enrolled in the study, etc.  
 
8.3 Study procedures  
Inclusion 
The study is conducted in healthy volunteers without gastrointestinal complaints and unre-
markable medical history. Volunteers are recruited by distribution of patient information fold-
ers, containing study information and the investigators contact information, at the gastroen-
terology and Hepatology outpatient clinics of Erasmus MC University Medical Center and 
Franciscus Gasthuis & Vlietland. When volunteers contact the investigator the study outline 
is discussed and inclusion and exclusion criteria are checked. Volunteers are asked whether 
they have a medical history or are currently experiencing any complaints, they do not under-
go additional tests or check-ups to ensure they are healthy. When a healthy volunteer de-
cides to participate in the study an Informed Consent Form will be signed. 
Volunteers will be randomized and assigned to one of four study groups. 

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Control 
Two different control groups both consisting of 5 volunteers will be used during the study. 
The first group will receive 13C-butyrate and the second group 13C-glucose. The control 
groups will perform the breath tests without performing any physical exercise. 
 
Intervention  
Two different intervention groups both consisting of 5 volunteers will be used during the 
study. The first group will receive 13C-butyrate and the second group 13C-glucose. All 
intervention groups will be performing a standardized bicycle ergometer exercise test with a 
total duration of 30 minutes(12). The exercise test has been proven to elicit mesenteric 
ischemia in athletes. Previous studies suggest an additional 10 minute period of splanchnic 
hypo perfusion after discontinuation of exercise(13, 14).   
The exercise test consist of 3 phases.  
- 
Phase 1: The first 10 minutes of exercise are used to gradually increase the workload 
until submaximal exercise intensity is reached. Submaximal exercise is defined as 
lactate between 3 and 5.5 mmol/L. Lactate measurements are performed every 2 
minutes, using a rapid lactate measurement kit. 
- 
Phase 2: From minute 10 until 20 submaximal exercise intensity is maintained by 
adjusting the workload based on lactate measurements. The lactate measurements 
are performed every 3 minutes. 
- 
Phase 3: Minute 20 until 30 are used to reach maximal exercise intensity. Every 3 
minutes the workload is increased by 10% of the submaximal workload until 
exhaustion. Lactate measurements will be continued with 3 minute intervals. 
 
All volunteers will be instructed to take their last meal 6 hours before start of the breath test. 
Volunteers in the 13C-butyrate group receive an oral dose of 0.03 µmol 13C-butyrate(10).  
Volunteers in the 13C-glucose group receive an oral dose 20 mmol 13C-glucose.  
 
Randomisation 
Allocation to the 13C-butyrate and 13C-glucose group and allocation to the control or 
intervention group will be performed by randomisation. Blinding of the healthy volunteers will 
not be possible, since the intervention group has to perform an exercise test. 
 
Breath test 
Both intervention and control group will perform breath tests at the following time points 
baseline, 0.5, 1, 1.25 1.5, 1.75, 2, 2.25, 2.5, 3, 3.5 and 4 hours after ingestion of 13C-butyrate 

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or 13C-glucose. Two breath samples are collected at all mentioned time points. The decision 
to perform breath testing for a total of 4 hours was based on  a study that determined 14CO2 
elimination patterns after instillation of butyrate in human caecum and a study describing 
13CO2 elimination patterns in healthy volunteers(10, 15). The tracer could be measured in 
exhaled air at the first time point, which was 30 minutes after instillation. A peak in expired 
14CO2 was measured after 2 hours. Four hours after instillation approximately 55% of the 
total 14CO2 dose had been expired, 24 hours after instillation 70% of the instilled dose had 
been expired. Since we are interested in the peak of expired 13CO2 a measurement period of 
4 hours seems sufficient, even in the intervention group, in whom a delayed or lower peak is 
expected. The frequency of breath testing is intensified between 1 and 2.5 hours after 
ingestion, in order to detect the timing of the 13CO2  peak more precisely.  
 
Follow-up 
There is no follow-up of healthy volunteers. 
 
Study procedures 
Control 
group 
Intervention 
group 
Oral dose of either 0.03 µmol 13C-butyrate or 20 mmol 
13C-glucose  
Yes 
Yes 
Insertion of venous canule  
(measurement of lactate, 2 serum samples: 1 at baseline 
and 1 after the exercise test) 
No 
Yes 
Bicycle ergometer exercise test 
No 
Yes 
Collection of 2 breath samples at baseline, 0.5, 1, 1.25 
1.5, 1.75, 2, 2.25, 2.5, 3, 3.5 and 4 hours after ingestion 
of 13C-butyrate or 13C-glucose 
Yes 
Yes 
 
Analysis of breath samples 
Measurement of 13CO2 will be performed using mass spectrometry (IDMicro Breath, Com-
pact Science Systems, Newcastle-Under-Lyme, United Kingdom). The mass spectrometry 
procedure is equal to the procedures used to measure 13CO2 in testing for Helicobacter Pylo-
ri, which is current clinical practice.  
 
8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without 
any consequences. The investigator can decide to withdraw a subject from the study 
for urgent medical reasons. 

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8.4.1 Specific criteria for withdrawal (if applicable) 
Not applicable 
8.5 Replacement of individual subjects after withdrawal 
Inclusion will end after inclusion of 5 volunteers in each of the four groups. 
8.6 Follow-up of subjects withdrawn from treatment 
Follow-up is not performed. 
8.7 Premature termination of the study 
The study will be terminated prematurely when participation is associated with serious health 
risks, which are not expected. 
 

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9 SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety.  The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed.  
 
9.2 AEs, SAEs and SUSARs 
9.2.1 Adverse events (AEs) 
Adverse events are defined as an undesirable experience occurring to a subject dur-
ing the study, related to the study. All adverse events reported spontaneously by the 
subject or observed by the investigator or his staff will be recorded. 
 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention but could have been based upon 
appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
The sponsor will report the SAEs through the web portal ToetsingOnline to the ac-
credited METC that approved the protocol, within 7 days of first knowledge for SAEs 
that result in death or are life threatening followed by a period of maximum of 8 days 
to complete the initial preliminary report. All other SAEs will be reported within a peri-
od of maximum 15 days after the sponsor has first knowledge of the serious adverse 
events. 
 

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9.2.3 Suspected unexpected serious adverse reactions (SU-
SARs) 
Not applicable. 
 
9.3 Annual safety report 
 
Not applicable 
 
9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical pro-
cedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till end of study within the Netherlands, as defined in the proto-
col  
 
9.5 [Data Safety Monitoring Board (DSMB) / Safety Committee] 
Not applicable 

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10 STATISTICAL ANALYSIS 
Baseline characteristics of the intervention and control group will be shown and com-
pared. Categorical data is compared by chi-square or Fisher exact testing. Numerical da-
ta is shown as mean and standard deviation or median and interquartile range. Compari-
son of the numerical data of intervention and control group will be performed by the inde-
pendent sample t-test or the Mann-Whitney-U test. 
 
10.1 Primary study parameter(s) 
The height of the 13CO2 peak of the intervention and control groups will be compared by 
the independent sample t-test or Mann-Whitney-U test. Repeated measurement correc-
tion will be performed with the Bonferroni method. The area under the 13CO2 curves will 
be compared by the independent sample t-test or Mann-Whitney-U test.  
 
10.2 Secondary study parameter(s)  
Not applicable 
10.3 Other study parameters 
Not applicable 
 
10.4 Interim analysis (if applicable) 
Not applicable 

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11 ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
The study will be performed according to the principles of the Declaration of Helsinki, of 
Good Clinical Practice, and the applicable laws and regulations of the Netherlands, 
including but not limited to the Medical Research Involving Human Subjects Act (WMO) 
and the Personal Data Protection Act. 
 
11.2 Recruitment and consent 
Volunteers are recruited by distribution of patient information folders, containing study infor-
mation and the investigators contact information, at the gastroenterology and Hepatology 
outpatient clinics of Erasmus MC University Medical Center and Franciscus Gasthuis & 
Vlietland. When volunteers contact the investigator the study outline is discussed and inclu-
sion and exclusion criteria are checked. Volunteers are asked whether they have a medical 
history or are currently experiencing any complaints, they do not undergo additional tests or 
check-ups to ensure they are healthy. When a healthy volunteer decides to participate in the 
study an Informed Consent Form will be signed. 
 
11.3 Objection by minors or incapacitated subjects (if applicable) 
Not applicable 
 
11.4 Benefits and risks assessment, group relatedness 
Participation in this study will not be beneficial for the healthy volunteers. However, this 
study will provide a valuable insight into the feasibility of 13C-butyrate and 13C-glucose 
breath testing. This proof-of-principal study is the first step in the development of a more 
accurate and less cumbersome and less invasive functional test for the detection of CMI. 
 
There are no known side-effects of the administration of 13C-glucose. Administration of 
high concentrations of 13C-butyrate could result in diarrhoea, however tracer dosages are 
used in this study. 
 
11.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of 
the WMO. 
  

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The sponsor (also) has an insurance which is in accordance with the legal requirements 
in the Netherlands (Article 7 WMO). This insurance provides cover for damage to re-
search subjects through injury or death caused by the study. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
11.6 Incentives (if applicable) 
Not applicable 

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12 ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
The handling of personal data will be compatible with the Code of Good Behaviour (Code 
goed gedrag) and the Dutch Personal Data Protection Act (Wet Bescherming 
Persoonsgegevens). The collected data will be recorded in an electronic database, which 
contains all relevant clinical data of the volunteers. This database will be provided with a 
password, which is only available for the investigators. The database will be anonymized 
by replacing all names of volunteers by a code and will be used for statistical analysis. Only 
these codes will be used for references in reports and publications about this 
investigation. Volunteers will be asked for their permission to store the material for 15 years 
and that it could be used for scientific research in the future with the same goal. 
 
12.2 Monitoring and Quality Assurance  
Not applicable 
 
12.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the accred-
ited METC has been given. All amendments will be notified to the METC that gave a fa-
vourable opinion.  
 
12.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accred-
ited METC once a year. Information will be provided on the date of inclusion of the first 
subject, numbers of subjects included and numbers of subjects that have completed the 
trial, serious adverse events/ serious adverse reactions, other problems, and amend-
ments.  
 
12.5 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last patient’s last visit.  
 
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action.  
    

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In case the study is ended prematurely, the sponsor will notify the accredited METC with-
in 15 days, including the reasons for the premature termination. 
 
 Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC.  
 
12.6 Public disclosure and publication policy 
Results of the study will be published in an international journal. There are no limitations or 
restrictions for publication mention the arrangements made between the sponsor and the in-
vestigator concerning the public disclosure and publication.

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STRUCTURED RISK ANALYSIS  
 
12.7 Potential issues of concern 
 
a. Level of knowledge about mechanism of action 
Both 13C-butyrate and 13C-glucose are stable isotopes  
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
13C-butyrate and 13C-glucose have been administered in human subjects(10). Adverse 
events were not reported. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo hu-
man cell material? 
Not applicable 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
Both 13C-butyrate and 13C-glucose can be metabolized in cells other than enterocytes, yet 
in this case the timing of the peak is expected to be delayed, since absorption and trans-
portation through the bloodstream are required. 
 
e. Analysis of potential effect 
A study performing 14CO2 breath testing after instillation of 14C-butyrate in the cecum 
showed that a tracer dose of 0.03 µmol was sufficient and could be detected in expired 
air(10). A study has shown that a 13C-glucose dose of 0.05 gr/kg bodyweight (approxi-
mately 3.5gr in an individual of 70 kg) could be detected in expired air. The effects of ex-
ercise induced mesenteric ischemia on the breath test patterns have not been studied. 
 
f. Pharmacokinetic considerations 
Not applicable 
 
g. Study population 
Heathy volunteers have been used in previous studies administering 13C-butyrate and 13C-
glucose. 
 
h. Interaction with other products 
No drug interactions are expected, since 13C-butyrate and 13C-glucose are metabolized by 
the enterocytes. 
 
 
 
i. Predictability of effect 

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A study that determined 14CO2 elimination patterns after instillation of butyrate in human 
caecum and a study described 13CO2 elimination patterns in healthy volunteers(10, 15). 
The tracer could be measured in exhaled air at the first time point, which was 30 minutes 
after instillation. A peak in expired 14CO2 was measured after 2 hours. Four hours after in-
stillation approximately 55% of the total 14CO2 dose had been expired, 24 hours after in-
stillation 70% of the instilled dose had been expired. Since we are interested in the peak 
of expired 13CO2 a measurement period of 4 hours seems sufficient, even in the interven-
tion group, in whom a delayed or lower peak is expected. 
 
j. Can effects be managed? 
No effects are expected, but when unexpected effects do occur the exercise test can be 
stopped. 
 
12.8 Synthesis 
Not applicable 
 
 
 
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2. Mensink PB, Moons LM, Kuipers EJ. Chronic gastrointestinal ischaemia: shifting 
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3. Thomas JH, Blake K, Pierce GE, Hermreck AS, Seigel E. The clinical course of 
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Chen L, Tuo B, Dong H. Regulation of Intestinal Glucose Absorption by Ion 
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