A Novel Approach to Measuring Intestinal Production of Short Chain Fatty Acids: A Proof-of-concept Healthy Volunteer Study
Phase: NA
Conditions: Healthy
Sponsor: Indiana University

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STUDY PROTOCOL 
 
A novel approach to measuring intestinal 
production of short chain fatty acids: A proof-of-
concept healthy volunteer study 
 
Principal Investigator:  
Huiping Xu, PhD 
Indiana University School of Medicine 
410 W. Tenth Street, Suite 3000 
Indianapolis, IN 46202 
 
 
 
Support Provided by: 
Clinical and Translational Sciences Institute (CTSI) and National Institutes of Health (NIH) 
 
 
NCT05254340 
 
 
 
 
 

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Table of Contents: 
 
Study Schema 
1.0 
Background & Rationale 
2.0 
Objective(s) 
2.1 
Primary Objective 
2.2 
Secondary Objective 
3.0 
Outcome Measures 
3.1 
Primary Outcome Measures 
3.2 
Secondary Outcome Measures 
3.3 
Tertiary/ Exploratory/ Correlative Outcome Measures 
4.0 
Eligibility Criteria 
4.1 
Inclusion Criteria 
4.2 
Exclusion Criteria 
5.0 
Study Design 
6.0 
Enrollment/Randomization 
7.0 
Study Procedures 
8.0 
Study Calendar 
9.0 
Reportable Events 
10.0 Data Safety Monitoring 
11.0 Study Withdrawal/Discontinuation 
12.0 Statistical Considerations 
13.0 Data Management 
14.0 Privacy/Confidentiality Issues 
15.0 Follow-up and Record Retention 
16.0 References 
17.0 Appendix 
 

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 Background & Rationale 
 
Intestinal short-chain fatty acids (SCFA) are produced by microbial fermentation of 
undigested carbohydrates.1 SCFAs are an important in regulating numerous physiological 
processes of the human intestine through their effects fluid and electrolyte absorptio2,3 
motility4,5 and symptoms in IBS.6 Altered SCFA profiles have been identified in common 
gastrointestinal conditions such as irritable bowel syndrome (IBS); however, the pattern of 
these alternations is incompletely understood. Part of the challenge in accumulating consistent 
data regarding the significance fecal SCFAs in conditions such as IBS, lies in the technical 
challenges that accompany attempts to measure intestinal SCFAs. SCFAs are volatile, and 
luminal SCFAs serve as a primary metabolite for human colonocytes. Thus, direct measurement 
of fecal SCFAs may not accurately represent the quality or quantity of SCFAs that are produced 
in the colon. There remains a need to develop novel approaches for accurate assessment of 
colonic SCFA production that will be applicable for clinical and research purposes. Inulin is a 
non-digestible carbohydrate that is approved by the United States Food and Drug 
Administration as a dietary fiber and is fermented by colonic bacteria to produce SCFAs. There 
are no studies that have investigated inulin fermentation as a possible strategy for measuring 
fecal SCFA production. Furthermore, it is unclear if undigested fecal inulin can be recovered and 
measured in humans following oral ingestion. The aim of this proposal is to test a novel 
approach to the assessment of fecal SCFA production in humans through the measurement of 
fecal inulin after a one-time inulin ingestion among healthy adult volunteers.  
 
1.0 
Objective(s) 
1.1 
Primary Objective: To conduct a proof-of-concept study demonstrating the 
ability to measure fecal inulin and short chain fatty acid content in stool after a 
one-time ingestion of a standardized 10-gram dose of dietary inulin. 
1.2 
Secondary Objective(s):  
1.2.1 To assess associations of bowel symptoms with fecal inulin and SCFA 
content after a one-time ingestion of dietary inulin 
1.2.2 To assess associations of individual factors (baseline dietary intake, 
demographics, body-mass index) with fecal inulin and SCFA content after 
a one-time ingestion of dietary inulin. 
1.2.3 To examine characterize fecal microbiota composition following a one-
time ingestion of dietary inulin and associations of microbiota composition 
with fecal inulin and SCFA content 
 
 
2.0 
Outcome Measures/Endpoints  
2.1 
Primary Outcome Measures: Fecal inulin, total and individual fecal SCFAs 
2.2 
Secondary Outcome Measures: Bowel symptoms (stool frequency, stool 
form, ease of passage) based on a validated bowel diary, baseline dietary intake 
(Automated Self-Administered Dietary Assessment) 
2.3 
Covariates for inclusion: Demographics and body mass index 
 
3.0 
Eligibility Criteria 
3.1 
Inclusion Criteria 
• 
Healthy adults ages 18-75 years with no prior history of GI disease or 
symptoms.  

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• 
Participants should be on a stable and consistent diet regimen and should not 
be following an extreme diet intervention such as gluten-free or a low 
fermentable oligo-, di-, monosaccharides, and polyols diet (FODMAP) diet at 
the time of study participation 
 
3.2 
Exclusion Criteria 
• 
History of microscopic colitis, inflammatory bowel disease, celiac disease, 
visceral cancer, chronic infectious disease, immunodeficiency, uncontrolled 
thyroid disease, history of liver disease or history of elevated AST/ALT > 2.0x 
the upper limit of normal 
• 
Prior radiation therapy of the abdomen or major abdominal surgeries except 
for C-section, tubal ligation, vaginal hysterectomy and appendectomy or 
cholecystectomy, or other minimally invasive procedures (as deemed by the 
PI) > 6 months prior to study initiation. 
• 
Ingestion of any prescription, over the counter, or herbal medications which 
can affect study interpretation within 6 months of study initiation for 
asymptomatic volunteers. Rescue therapy to facilitate stool collection will be 
permitted where needed. 
• 
Any females who are pregnant or breast-feeding 
• 
Antibiotic usage within 3 months prior to study participation 
• 
Prebiotic or probiotic usage within the 2 weeks prior to study initiation 
• 
Inulin usage within the 2 weeks prior to study initiation 
• 
Regular use of tobacco products within the past 6 months 
 
 
4.0 
Study Design 
 
We will conduct a prospective study of fecal inulin, fecal SCFAs, bowel symptoms, fecal 
microbiota in healthy adults. Baseline data on dietary intake, demographics, body mass index, 
medical history and medication-use will be collected in adults who meet the eligibility criteria 
defined above. The accessible population includes individuals from the local community (Marion 
County, IN within a 150 mile radius). Participants may be identified by public advertisement 
(e.g. newspaper advertisement, flyers, social media platforms) or during clinical visits to 
any Indiana University site. Participants may also be recruited from the local community, the 
Indiana CTSI Research Network, Regenstrief Data Core, and the national ResearchMatch 
research registry. All volunteers will sign written informed consent prior to enrollment and 
initiation of study procedures after study explanation.  
 

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Flow diagram for cross-sectional survey-based study 
 
Visit 1 
 
 
 
 
 
 
Active Study  
 
 
 
 
 
 
 
Visit 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.0 
Enrollment 
Up to 50 healthy volunteers will be recruited by public advertisement (e.g. newspaper 
advertisement, flyers, social media advertisements) and through notification of CTSI and 
ResearchMatch research participants. Subjects may also be identified and invited during clinical 
visits to any Indiana University site. Flyers will be made available at our clinical Indiana 
University sites. Flyers may also be posted in public common areas throughout the Indiana 
University campus and local community (within a 150 mile radius) or posted on other public or 
forums where such free advertisements are allowed including social media websites (e.g. 
Facebook, Craigslist). 
 
All study tests and visits will be paid for by the study without cost to the participant. 
Participants will receive a total reimbursement of $50 for completing study participation and will 
Visit 1 (Day 1)  
Provide explanation of study procedures and obtain informed consent. Collect baseline data 
on demographics, medical history, medications, baseline dietary data. Dispense bowel diary, 
food diary, inulin supplement, and study materials including calendar of events 
Day 2-Day 5 
All participants record dietary intake and bowel symptoms for 4 days (Day 2-5). Participants 
will consume a one-time 10 gram inulin supplement on the morning of Day 2. Perform first 
24H stool collection from (Day 2-3). Perform second 24H stool collection (Day 3-4). Perform 
third stool collection (Day 4-5). Perform fourth stool collection (Day 5-6) 
Final Assessments 
Review data for completion and 
accuracy. Measure fecal inulin, SCFAs, 
and microbiota composition.  
Conduct data analyses 
Visit 2 (Day 6) 
Return stool collection (on ice), bowel diaries, dietary intake diaries 

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be reimbursed $10 for Visit 1 and $40 for Visit 2. Volunteers who present for a screening visit, 
but are ultimately determine ineligible will be provided a parking voucher to cover parking costs 
for that visit.  
 
6.0 
Study Procedures 
 
6.1 
VISIT 1 (Screening Visit and Informed Consent) - Eligible volunteers will 
undergo a screening visit by a physician, be asked to sign informed consent after 
explanation of the study. This visit may be completed in-person or as a virtual 
video visit. All questions will be answered. Inclusion/exclusion criteria will be 
reviewed. Medical history and medication intake will be reviewed. Upon signing 
the consent (paper version or via REDCap), participants will be scheduled for 
study-days and participants will fill out a validated bowel disease questionnaire 
(BDQ). All participants will complete the web-based Automated Self-Administered 
Dietary Assessment Tool developed by the National Cancer Institute 
(https://epi.grants.cancer.gov/asa24/) and a food frequency questionnaire 
(https://www.epic-norfolk.org.uk/for-researchers/ffq/). Study materials will be 
dispensed or mailed after completion of the visit: (1) stool kits and instructions 
for stool collection, (2) inulin supplement, (3) a bowel pattern diary including the 
Bristol stool form scale for participants to record stool symptoms over four days 
overlapping with stool collections, (4) a dietary intake diary. The bowel pattern 
and dietary intake diaries will be made available in several forms for the 
participant in order to allow flexibility for diary completion (i.e. link to secure 
web-based diary, paper diary or instructions for phone surveys). The participant 
may opt to complete the diaries using the method of their choice.  
 
6.2 
Day 2 – Day 2 reflects the first day of active study. Day 2 may occur anytime up 
to 30 days after Day 1. This will depend whether the patient may need washout 
for medications or on the day that the patient can return for the final visit. It will 
also depend upon the discretion of the PI or Sub I. The participant will consume 
the inulin supplement before 9 am on Day 2. The first 24 hour stool collection 
will begin starting at 7 am on Day 2 and will continue until 7 am on Day 3. All 
stool collections will be refrigerated. The participant will begin recording their 
bowel symptoms and dietary intake data using 4-day diaries (recorded using 
either paper diaries or the web-based Automated Self-Administered Dietary 
Assessment Tool (https://epi.grants.cancer.gov/asa24/). 
 
6.3 
Day 3 – The participant will start their second 24 hour stool at 7 am on Day 3 
and continue until 7 am Day 4. 
 
6.4 
Day 4 - The participant will start their third 24 hour stool at 7 am on Day 4 and 
continue until 7 am Day 5. 
 
6.5 
Day 5 – The participant will start their fourth 24 hour stool collection at 7 am on 
Day 5 and continue until 7 am Day 6. 
 
6.6 
VISIT 2 (Day 6): Participants will return will return bowel diaries, dietary intake 
diaries, and stool samples. Diaries may be returned by mail, fax, or by electronic 

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submission. will complete the Automated Self-Administered Dietary Assessment. 
Study exit will be confirmed at this time.  
 
Sample collection: Stool samples will be collected from all participants after study initiation. 
Stool will be refrigerated by participants and brought to the laboratory on ice within 4 days of 
collection. Once received, stool specimens each daily specimen into 4 samples for fecal 
microbial (brown screw-top tube), fecal SCFA (microcentrifuge tube), a fecal inulin 
(microcentrifuge tube with ethanol), and one back-up residual sample (microcentrifuge tube) 
and immediately frozen at -80ο Celsius.  
 
Assessment of fecal microbiota: 
 
a) Nucleic acid extraction: When received at the lab, frozen aliquots of stools samples will 
undergo processing for isolation of nucleic acids using the appropriate DNA isolation kit. 
Controls will be included at all steps to monitor for potential reagent contamination. DNA quality 
and quantity will be monitored by gel electrophoresis and fluorescent dsDNA assay. Genomic 
DNA will be stored at -80ºC for further use in construction of sequencing libraries and qPCR. 
Back up stool specimens will also be stored at -80οC. 
 
b) Multiplex 16S allele PCR and sequencing: The V4 region of 16S alleles will be PCR amplified 
using the appropriate library preparation kit from stool genomic DNA to be sequenced in pools 
on an Ilumina MiSeq as previously described.7  
 
c) qPCR validation experiments: We will design primers for specific microbial taxa of interest, for 
example specific taxa that contribute to the biosynthesis of SCFAs, so that their absolute 
abundance can be determined by quantitative PCR (q-PCR). Q-PCR targets will be chosen from 
the study 16S sequences using Primrose and PrimerQuest (IDT Inc.). Specificity of the primers 
will be assessed by Blast against the study 16S sequences and the NCBI database. Target 
sequences will be then be synthesized by a commercial vendor (e.g. Genescript) and cloned 
into a carrier vector. Q-PCR assays will be benchmarked against characterized fecal specimens 
that do or do not contain the target microorganism to assess assay specificity, and against fecal 
specimens that were determined not to contain the target taxa by 16S sequencing and which 
were spiked with different concentrations of the target vector to assess assay sensitivity. These 
assays will be used to assess presence and loads of specific organisms in the study fecal 
specimens. 
 
d) Shotgun Metagenomic Sequencing (SGS): In order to overcome limitations of 16S allele PCR 
and sequencing which may include: (1) biases in the range of taxa amplified by universal 
primers, (2) limited resolution of the 16S rRNA gene between closely related species, and (3) 
dependence on the presence of a single marker gene, we propose further analysis of the 
microbial communities using coupled shotgun metagenomics sequencing. Specimens will be 
paired-end (2 x 150 bp) sequenced using the Illumina HiSeq and one lane of the sequencer 
allocated to the pooled sample. Sequencing data will be transmitted to the Bioinformatics Team 
for further analysis. 
 
Data analysis of molecular methods:  
 

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a) 16S sequencing data will be processed by the DADA2 package8 to generate separate lists of 
microbial taxa and their relative abundances in stool.9 For downstream biostatistical analysis, to 
minimize unequal sampling effects, 16S sequences from each individual will be sub-sampled to 
equal sequencing depth. Using the statistical R packages, phyloseq75 and vegan, bacterial taxa 
richness and α/β diversity indices will be computed to quantify community-level variability 
among samples. Principal coordinate analysis, non-metric dimensional scaling and heatmap 
methods will be applied to cluster samples based on β diversity. 
 
b) For SGS data, taxonomy will be assigned using MG-RAST to generate lists of bacterial, viral, 
fungal, protozoal and phage tax. Calculations for relative abundance will be similar to those 
used for 16S sequences. Total abundance will be estimated from numbers of taxa-specific reads 
and reads from internal standards.   
 
SCFA measurement: Stool samples will be processed for extraction of fecal SCFAs to be 
quantitatively analyzed by high performance liquid chromatography-mass spectrometry or NMR 
and mass spectrometry as previously described.10  
 
Inulin measurement: Fecal inulin content will be measured using short acid hydrolysis and 
high-performance liquid chromatography to measure inulin as hydroxymethylfuraldehyde by the 
Complex Carbohydrate Research Center at the University of Georgia. This approach is adapted 
from a method that has previously been published to measure inulin in plasma and urine.11  
 
Inulin (Orafti®) is officially recognized by The United States Food and Drug Administration as 
an approved dietary fiber. It is naturally sourced from chicory root fiber; 92% inulin content, 
granulated powder. This fiber occurs naturally in a great number of plants and vegetables. 
Orafti® inulin is extracted through hot water processing and is of 100 % vegetable origin. In 
order to minimize any potential symptom exacerbation that may occur with consumption of 
such a dietary fiber, inulin powder supplementation will be limited to a one-time ingestion of a 
10-gram dose with water. 
 
 
Stool Frequency and Consistency:  Participants will complete a daily diary to record bowel 
habits (frequency, consistency and form).12 Participants will be given the option of completing 
the diary on paper, which will be dispensed at Visit 1 and collected at study conclusion.  
 
Dietary Intake: Participants will be instructed to complete a food record during active study 
participation (using either a paper record or the electronic ASA24 website). Participants will 
further be instructed to maintain a stable diet throughout the duration of the study; and to 
avoid other extreme diet interventions such as a gluten-free diet or low FODMAP diet.  
 
7.0 
Study Calendar 
 
We will conduct a prospective study that will require participants to complete a 
screening/baseline visit followed by 4-day active study period and a final follow-up visit. The 
active study may start anywhere up to 30 days after the initial study visit. Thus, participants 
may be active in the study for up to 35 days. Up to 50 healthy volunteers may be enrolled. We 
plan to conduct the study over a 24 month time period. 
 

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8.0 
Reportable Events 
 
All potential research concerns will be monitored by the study research team from the time of 
initiation until completion. Potential risks associated with this study include loss of 
confidentiality, discomfort associated with stool collections, or discomfort associated with 
dietary intervention. The study team will take care to recognized and minimize any discomfort 
that collecting stool or completing questionnaires may cause for participants. Inulin ingestion 
will be kept to a one-time ingestion of 10 grams and participants will be counseled appropriately 
prior to its use. Any potential adverse events will be followed by the study physician until they 
normalize. All serious events will be reported immediately to regulatory authorities and the 
Indiana University IRB. Any incidental findings that may require medical or profession 
intervention will be discussed with the participant by the study physician and the participant will 
be referred to the appropriate care team.  
 
 
9.0 
Data Safety Monitoring 
 
The study PI (Huiping Xu, PhD) and all members of the study team will perform safety 
monitoring of all research subjects. Data quality, subject recruitment, subject accrual, subject 
retention, outcomes, procedures designed to protect privacy of subjects, outcomes, and 
adverse events data will be monitored by the PI and designated members of the study team on 
a regular basis.  
 
10.0 Study Withdrawal/Discontinuation 
 
Drop-outs may occur due to withdrawal of consent or because of discomfort related to study 
procedures. In order to compensate for withdrawals and loss to follow-up, up to 50 patients 
could be enrolled to meet the target of 30 completed volunteers. 
 
11.0 Statistical Considerations 
 
We plan to enroll 30 healthy volunteers. In order to compensate for withdrawals and loss to 
follow up, up to 50 patients could be enrolled. As the primary objective of this study is to 
describe the technical feasibility of measuring, sample size calculations are not based on the 
primary objective. Instead, the sample size estimation is based on secondary endpoints of the 
association between fecal SCFAs after inulin supplementation and fecal microbiota composition. 
Our sample size of 30 subjects provides an 80% power to detect a Pearson correlation of 0.5 
based on the Fisher’s z test at the 5% significance level. We consider the correlation of 0.5 an 
important correlation. Such a correlation is large but achievable based on data from a 
preliminary study of 14 healthy volunteers conducted by our lab, where the Pearson correlation 
between microbiota composition (e.g. individual taxa) and individual fecal SCFA levels were 
found to be above 0.5 for 5% of the taxa found in the stool sample.   
 
12.0 Statistical Data Management 
 
All study data will be recorded onto an electronic database (REDCap) which will be accessible 
only by certified research personnel. Only de-identified anonymized data will be analyzed. 
Quality assurance steps will include: built in range checks, testing of the database prior to 

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moving to production mode. The following quality control methods will be used: extraction and 
cleaning of data every 6 months.  
 
13.0 Privacy/Confidentiality Issues 
 
Potential risks include breach of confidentiality or invasion of privacy. To minimize risk of loss of 
confidentiality, participant records will be assigned a unique identifier in a coded manner 
accessible only by study personnel. All data collected on paper will be kept in a locked drawer 
within the GI Research Office until data collection is complete and transferred to an electronic 
storage system, after which papers will be discarded and destroyed. Data will also be captured 
and stored electronically in REDCap and in a protected Microsoft Excel spreadsheet on a 
Department Server.  The storage location will be backed up manually every month. Electronic 
data will be automatically exported to common statistical packages such as SPSS, SAS, Stata, 
R/S-Plus at the time of conducting analyses. Only de-identified or anonymized clinical data will 
be transferred processing laboratories.  
 
14.0 Follow-up and Record Retention 
 
The study is expected to last 24 months in duration. All electronic records will be retained 
indefinitely. All data collected on paper will be kept in a locked drawer within the GI Research 
Office until data collection is complete and transferred to an electronic storage system, after 
which papers will be discarded and destroyed. 
 
15.0 References 
 
1. Roy C, Kien L, Bouthillier L, Levy E. Short-chain Fatty Acids: Ready for Prime Time? Nutr Clin 
Pract 2006;21:351-66 
 
2. Musch MW, Bookstein C, Xie Y, Sellin JH, Chang EB. SCFA increase intestinal Na absorption 
by induction of NHE3 in rat colon and human intestinal C2/bbe cells. Am J Physiol Gastrointest 
Liver Physiol 2001;280:G687-93 
 
3. Murray RD, McClung HJ, Li Buk, Ailabuoni A. Stimulatory effects of short-chain fatty acids on 
colonic absorption in newborn piglets in vivo. J Pediatr Gastroenterol Nutr 1989;98:95-101 
 
4. Fukumoto S, Tatewaki M, Yamada T, Fujimiya M, Mantyh C, Voss M, et al. Short-chain fatty 
acids stimulate colonic transit via intraluminal 5-HT release in rats. Am J Physiol Regul Integr 
Comp Physiol 2003;284:R1269-76 
 
5. Mitsui R, Ono S, Karaki S, Kuwahara A. Neural and non-neural mediation of propionate-
induced contractile responses in the rat distal colon. Neurogastroenterol Motil. 2005;17:585-94 
 
6. Banasiewicz T, Krokowicz Ł, Stojcev Z, Kaczmarek BF, Kaczmarek E, Maik J, et al. 
Microencapsulated sodium butyrate reduces the frequency of abdominal pain in patients with 
irritable bowel syndrome. Colorectal Dis 2013;15:204-9 
 

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7. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD, Development of a dual-index 
sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq 
Illumina sequencing platform. Appl Environ Microbiol 2013. 79:5112-20 
 
8. Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: High-resolution sample inference from 
Illumina amplicon data. Nat Methods 2016;13:581-3 
 
9. Gao X, Lin H, Revanna K, et al. A Bayesian taxonomic classification method for 16S rRNA 
gene sequences with improved species-level accuracy. BMC Bioinformatics 2017;18:247 
 
10. Park J, Kim M, Kang SG, Jannasch AH, Cooper B, Patterson J, et al. Short-chain fatty acids 
induce both effector and regulatory T cells by suppression of histone deacetylases and 
regulation of the mTOR-S6K pathway. Mucosal Immunol 2015;8:80-93 
 
11. Dall'Amico R, Montini G, Pisanello L, et al. Determination of inulin in plasma and urine by 
reversed-phase high-performance liquid chromatography. J Chromatogr B Biomed Appl 
1995;672:155-9 
 
12. Heaton KW, Radvan J, Cripps H, Mountford RA, Braddon FE, Hughes AO. Defecation 
frequency and timing, and stool form in the general population: a prospective study. Gut 
1992;33:818-24