Brain Small Chain Fatty Acid Metabolism in Parkinson Disease: Tributyrin Supplementation
Phase: PHASE1
Conditions: Parkinson Disease
Sponsor: Nicolaas Bohnen, MD, PhD

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
1 
 
Brain Small Chain Fatty Acid Metabolism in Parkinson Disease: Tributyrin Supplementation 
NCT05446168 
7/23/2024 
 
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
2 
CLINICAL PROTOCOL 
 
STUDY TITLE 
Brain Small Chain Fatty Acid Metabolism in Parkinson Disease: Tributyrin Supplementation 
Study Agents:  Tributyrin; [11C]Butyrate; [18F]FDG 
 
University of Michigan IRB HUM #00211320 
 
Principal Investigator:  
Nicolaas I Bohnen, MD, PhD  
 
Professor, Department of Radiology 
Professor, Department of Neurology 
University of Michigan 
Ann Arbor, MI  
 
Institution: University of Michigan 
 
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
3 
Brain Small Chain Fatty Acid Metabolism in Parkinson's Disease: Tributyrin supplementation 
 
Cognitive decline, progressing usually to dementia, is a common and disabling feature of Parkinson's 
disease (PD).  Progression to dementia generally reflects progressive cortical pathologies.  We and 
others showed that cognitive decline is accompanied by and heralded by deficits in regional 
neocortical glucose metabolism, demonstrated with [18F]fluorodeoxyglucose (FDG) PET imaging 1, 2.  
These deficits are widely believed to reflect synaptic loss.  Convergent imaging data, however, 
suggests relative preservation of neocortical synapses with abnormal neuronal glucose metabolism.  
Recent studies of PD and Alzheimer disease (AD) subjects with synaptic terminal SV2A PET ligands 
indicate preservation of cortical synaptic terminals at early to moderate disease stages.  The major 
alternative brain energy substrate is ketone bodies that are small chain fatty acids (SCFAs).  
Comparative studies of brain SCFA and glucose uptake in Mild Cognitive Impairment and early AD 
subjects indicates relative preservation of regional brain SFCA metabolism in subjects with abnormal 
regional glucose uptake, suggesting relative preservation of synapses.   
This accumulated PET data is broadly consistent with the concept that mitochondrial dysfunction is a 
major feature of, and likely pathogenetic mechanism in, PD.  Other data suggests relevant 
dysfunction of glucose uptake and metabolism mechanisms in PD.  One implication is that enhancing 
neuronal SCFA metabolism will circumvent abnormalities of glucose uptake and initial metabolism, 
enhancing-restoring neuronal mitochondrial function.  This would potentially provide symptomatic 
benefits and might be a disease-modifying therapy (Figure 1).  There are other good reasons to 
pursue SCFA therapy in PD.  SFCAs may be significant modulators of inflammatory, oxidative, and 
transcriptional regulatory processes linked to neurodegeneration in PD (Figure 1).  Recent 
experimental work suggests important links between mitochondrial dysfunction and pathogenic 
inflammatory mechanisms in PD.  SCFA therapy may address the brain-gut axis dysbiosis that seems 
to characterize PD.    
Capitalizing on our experience with PD clinical research and development 
of novel PET methods, we propose an integrated series of experiments to 
measure brain SCFA metabolism and to assess target engagement of 
SCFA therapy in older adults and in persons with Parkinson disease (PD).   
 
Aim 1: To explore [11C]butyrate PET and [18F]FDG PET glucose 
metabolism and clinical measures before and after open-label treatment 
with the SCFA prodrug tributyrin in a small pilot study in patients with PD 
(n=8) and normal control older adults (n=2). A separate subset of 10 participants may complete the 
study without the PET or brain imaging biomarkers but using the exploratory clinical or laboratory test 
battery only for explorative reasons. 
   
 
Hypothesis 2: [11C]butyrate PET and glucose metabolism may change in the brain, liver, heart and gut 
after approximately 30-day intervention with tributyrin 500 mg t.i.d. p.o. with meals in patients with PD 
and normal older adults.    
 
Exploratory hypothesis: Tributyrin supplementation may affect measures of cognition, autonomic, sleep 
metabolic, genetic, and other measures. 
 
Impact:  Positive findings in this small exploratory pilot trial may support future target engagement 
studies of SCFA supplementation in normal adults and PD.   
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
4 
 
Background 
 
Ketogenic diet and SCFA interventions in neurodegeneration: 
The ketogenic diet based on preferential intake of predominant medium and long-chain fatty acids 
and minimization of carbohydrates has become a subject of recent research in metabolic and 
neurodegenerative disorders. Preliminary positive findings have been reported mainly from short-
duration studies. Despite the benefits of this diet, health care professionals still question its safety due 
to the elevated serum ketones it induces and the limited dietary fiber intake 6. To compound the 
controversy, patient compliance with the program is poor due to the restrictive nature of the diet and 
symptoms related to energy deficit and gastrointestinal adversity during the introductory and energy 
substrate transition phase of the diet. Another class of fatty acids, so-called short chain fatty acid 
(SFCA), has been extensively studied for their role in the gut microbiome. For example SCFAs, in 
particular n-butyric acid (BA), acetic acid and propionic acid, are naturally present in the colon as a 
bacterial product of dietary fiber fermentation. Renewed interest in SCFAs has also emerged because 
of their ability to alter gene expression through histone deacetylase (HDAC) inhibition. HDAC has 
become a drug target in cancer as well as non-oncological diseases. These carboxylic acids are also 
of interest for their effects in the brain including memory enhancement and neuroprotection in rodent 
experiments 8. Animal and human ingestion studies of prebiotic fibers associated with brain health, 
such as inulin and resistant potato starch, found selective increases in the production of colonic BA 
and acetic acid 10, 11.  Similarly, intermittent fasting in MPTP-induced Parkinson's disease mice 
resulted in higher butyric acid as well as increased levels of 
BDNF 13. Although blood (and brain) levels of BA and acetic 
acid are naturally low 14, a 11C-acetate PET study in rats found 
significantly increased uptake in the brain (as well as heart and 
liver) after 8 weeks of inulin fiber supplemented feeding, see 
Figure 1 15. Effects of BA on the brain may be direct or indirect. 
Indirect actions of butyrate are believed to involve the hormono–
neuro–immuno system. For example, increased SCFA 
production in the colon may result in vagal nerve-mediated 
changes in the hypothalamus and induce blood-brain barrier 
changes. The 'gut brain axis' represents bidirectional 
communication between the gastrointestinal system and the 
brain and is based on a complex system, including the vagus 
nerve, sympathetic nerves, humoral, endocrine, and immune 
links. These observations suggest that SCFA may play a role in 
the so-called 'gut-brain axis' 19 and that interventions promoting 
SCFA may benefit patients with neurodegenerative or neuro-
inflammatory disorders.  
SCFAs can also be administered orally where gastric and  
duodenal absorption will result in increased portal vein and 
systemic plasma levels that may affect brain functions. For 
example, a 18F-FDG PET study in pigs found that 3 weeks of 
oral ingestion of sodium butyrate triggered basal brain glucose metabolism changes in the nucleus 
accumbens and hippocampus with histological evidence of increased hippocampal granular cell layer 
volume and neurogenesis 21. Intra-peritoneal administration of sodium butyrate in 6-OHDA-lesioned 
rats resulted in significant attenuation of motor deficits and increased striatal dopamine levels 22. 
Figure 2: Brain, heart and liver uptake 
of 11C-acetate before and after 8 weeks 
of inulin fiber supplemented feeding in 
rats (Frost et al. 2014 Nature 
Communications) 
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
5 
Moreover, sodium butyrate treatment attenuated oxidative stress and neuro-inflammatory markers. 
These effects occur concurrently with increased global H3 histone acetylation and BDNF levels 22.  
BA and its prodrug tributyrin are naturally present in butter. In spite of its promise, systemic butyrate 
supplementation suffers from several limitations as butyrate is metabolized rapidly as soon as it 
enters the colonocyte via its active transport system 24 and resulting plasma (or brain) concentrations 
are very low. Tributyrin, a stable and rapidly absorbed prodrug of butyric acid offers several 
advantages compared to butyrate. Tributyrin is a neutral short-chain fatty acid triglyceride that is likely 
to overcome the pharmacokinetic drawbacks of natural butyrate as a drug 25. Because it is rapidly 
absorbed and chemically stable in plasma, tributyrin diffuses through biological membranes and is 
metabolized by intracellular lipases, releasing therapeutically effective butyrate over time directly into 
the cell. Compared with butyrate, tributyrin has more favorable pharmacokinetics 25, 26 and is well 
tolerated 27. Liquid tributyrin filled into gelatin capsules and administered orally resulted in millimolar 
concentrations of butyrate both in plasma and inside the cell 27. Unlike BA, tributyrin is lipophilic with a 
plasma half-life of 40 minutes making it a preferred choice for systemic oral administration. 
Overall strategy 
 
Overview  
The overarching goal of this small exploratory open-label pilot study is to explore [11C]butyrate PET   
and [18F]FDG PET glucose metabolism and clinical measures before and after open-label treatment 
with the SCFA prodrug tributyrin in a small pilot study in PD (n=8) and normal control older adults 
(n=2). Positive findings in this small exploratory pilot trial may support target engagement study of 
SCFA supplementation in normal adults and PD. PET and/or MRI Imaging protocols will be optional 
for a separate subset of 10 participants may also complete the study based on clinical or laboratory 
test battery alone for the exploratory hypothesis. 
 
Timetable: This study, to be conducted over a 2-year period, will include a net total of n=20 subjects 
(NC, n=4; PD, n=16; total net; n=20; gross n=32 to allow for attrition). Details of the enrollment and 
major test procedures are listed in table 1 below. 
 
Milestone of recruitment success are net completion of 9 persons for year 1. 
 
Table 1: Study enrollment & main 
assessments 
NC 
PD 
TOTAL 
Baseline  [11C]butyrate PET and [18F]FDG 
PET-CT & brain MRI  with baseline clinical 
assessment 
NC=2 
PD=8 
 
n=10 
Post-intervention  [11C]butyrate PET and 
[18F]FDG PET-CT & MRI  
NC=2 
PD=8 
n=10 
30-day (± 7) tributyrin intervention with 
pre/post clinical assessments) 
NC=4 
PD=16 
n=20 
Exploratory clinical and laboratory test battery 
pre and post the tributyrin supplement 
intervention 
NC=4 
PD=16 
N=20 
Total subject net  recruitment aims 1 & 2 
NC=4 
 
PD=16 
n=20* 
*Net recruitment n=20; gross recruitment to account for attrition n=32 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
6 
 
Design: Exploratory open-label tributyrin supplementation pilot study.  
 
Methods: After obtaining informed consent and screening for study eligibility, study participants will 
undergo the baseline clinical assessment and imaging protocol (separate days). For patients with PD 
the motor component of the test battery will be performed in the dopaminergic medication 'off' state in 
the morning after overnight withholding of their dopaminergic medications in the PD, i.e., temporarily 
postponing the morning dose until the motor exam has completed.  The remainder of the test battery 
will be performed in the medication 'on' state. Participants will also complete some testing in a fasted 
state. For participants who were not consented prior to baseline, study staff will obtain a waiver of 
consent during screening call for fasting and withholding dopaminergic medications prior to their visit. 
Qualified personnel who have been trained in the use of the instruments and have undergone inter-
rater reliability evaluations will perform the assessments.  
 
There will be a total of 5 visits for this pilot study. The first two visits will consist of the initial general 
clinical assessment (e.g., vital signs, vital capacity lung/breath holding tests) and optional imaging split 
over 2 days (no specific sequence or preset time interval).  At visit 1 participants will be instructed to 
wear physical activity and sleep trackers (called ActivPal a sensor will be put on the upper leg and a 
ring called Oura ring, which will be worn at a finger) for 1 week prior to visit 2. Participants will also wear 
a continuous glucose monitor during the same time. After the participants complete the 1 week of 
physical activity, sleep and glucose monitoring they will return for visit 3, where a limited repeat clinical 
test battery will be administered. At visit 3 the participant will also be instructed to start the 
approximately 30 day supplementation of tributyrin. The physical activity and sleep trackers and the 
continuous glucose monitor will also be worn during the last week of the tributyrin supplementation 
period. At the end of the approximately 30-day tributyrin supplementation the participants will return for 
visits 4 (repeat clinical assessment) and 5 (repeat optional imaging) again split over 2 days. There is 
no pre-set sequence which assessment (clinical or imaging) comes first. Blood and/or saliva tests will 
be collected for assessments of genetic variables (e.g. ApoE). Blood assessment will also be used for 
metabolomics. Blood continuous glucose data will be collected blinded to the participant. 
 
Details for the schedule of activities for the tributyrin supplement intervention study are shown below 
(Table 2). 
 
 
Table 2 Schedule 
of activities 
Visit 1* / 
Visit 2*: 
Baseline 
assessm
ent 
(clinical) 
 
Visit 1* / Visit 
2* 
Baseline 
assessment 
(imaging – 
OPTIONAL) 
At-home 
wearable 
sensor 
use (1 
week ± 3 
days) 
Visit 3 
Pre- 
intervention  
assessment 
At-home 
4 (± 1) week 
Tributyrin 
(500 mg tid 
po) 
intervention 
Visit 4* / Visit 
5* 
Post- 
intervention 
clinical 
assessment 
(while on 
tributyrin) 
Visit 4* / Visit 
5* 
Post- 
intervention 
imaging 
assessment 
(while on 
tributyrin, 
OPTIONAL) 
Approximate time 
commitment 
4-6 
hours 
3-6 hours 
- 
4-5 hours 
- 
4-6 hours 
3- 6 hours  
Test location 
Domino
s 
Farms 
UM hospital 
Home 
Dominos 
Farms 
Home 
Dominos 
Farms 
UM hospital 
Informed Consent 
(or prior 
eConsent) 
X 
 
 
 
 
 
 
Eligibility 
X 
 
 
 
 
 
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
7 
OPTIONAL  
[11C]butyrate, 
[18F]FDG 
PET-CT 1.2 
(brain/body)* 
 
X 
 
 
 
 
X (after 
taking 
tributyrin) 
In case of  
[11C]butyrate PET, 
obtain vital signs 
pre- & post 
injection of tracer 
 
X 
 
 
 
 
X 
OPTIONAL MRI 
brain* 
 
X 
 
 
 
 
X 
DEXA Bone 
Density Scan 
X 
 
 
 
 
X 
 
Blood/saliva/urine 
sample collection1 
X 
 
 
 
 
X 
 
Continuous 
glucose monitor  
for 7±3 days 
 
 
X 
 
X (last 7 
days) 
X 
 
Sleep tracking ring 
 
 
 
X 
X 
X 
X 
 
Activity tracker for 
7±3 days 
 
 
X 
 
X (last 7 
days) 
 
 
Motor MDS 
UPDRS2 
X 
 
 
X 
 
X 
 
Balance test 
MiniBESTest 
(sensored)2 
X 
 
 
X 
 
X 
 
Pegboard testing2 
X 
 
 
X 
 
X 
 
Foot Tapping2 
X 
 
 
X 
 
X 
 
Finger Tapping2 
X 
 
 
X 
 
X 
 
Electronic Gait 
Mat/sensored 
walk2 
X 
 
 
X 
 
X 
 
Beck Depression 
Inventory II scale 
X 
 
 
X 
 
X 
 
Spielberger Trait 
Anxiety Scale 
X 
 
 
X 
 
X 
 
Sleepiness scales 
X 
 
 
 
 
X 
 
 MoCa, 
PDCRS (limited 
cognition) 
X 
 
 
X (optional) 
 
X 
 
Cognitive test 
battery 
X 
 
 
X (optional) 
 
X 
 
PCFRS functional 
scale 
X 
 
 
 
 
X 
 
Quality of life 
scale PDQ-39 
X 
 
 
 
 
X 
 
Adverse event 
assessment 
X 
 
 
X 
 
X 
 
Phone Call/Email 
Drug monitoring 
 
 
 
 
X 
 
 
Drug 
accountability 
 
 
 
X 
 
X 
X 
Pregnancy test in 
women of 
 
X 
 
X 
 
 
X 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
8 
childbearing 
potential 
(within 48 
hours of 
PET) 
(within 48 
hours of 
PET) 
Note 
1: Fasting in morning 
2: Dopaminergic off state in PD  
*Visits 1-2 and 4-5. There is no pre-set sequence which assessment (clinical or imaging) comes first. 
 
 
Details of the neuropsychological test battery is shown in Table 3. 
 
Outcome measures:  
 
Primary outcome measures:  
• 
[11C]butyrate uptake 
• 
Change in average blood glucose 
metabolism measured by continuous 
glucose meter 
Exploratory outcome measures: 
• 
[18F]FDG PET glucose metabolism 
• 
Cognition dependent mobility 
functions: postural sway (iSWAY), 
Timed Up and Go test (iTUG) 
measured using Mobility Lab modules 
(APDM, Inc., Portland, OR) 28-30 
• 
Dynamic balance using the Mini-
BESTest 31 
• 
Spatiotemporal gait measurements 
using Protokinetics Zeno™ Walkway 
(Protokinetics LLC, Havertown, PA) 
• 
Remainder of the clinical and laboratory test battery 
 
 
 
Tributyrin intervention aim: BA and its prodrug tributyrin are naturally present in butter. In spite of its 
promise, systemic butyrate supplementation suffers from several limitations as butyrate is 
metabolized rapidly as soon as it enters the colonocyte via its active transport system 24 and resulting 
plasma (or brain) concentrations are very low. Tributyrin is a stable and rapidly absorbed prodrug of 
butyric acid offers several advantages compared to butyrate. Tributyrin is a neutral short-chain fatty 
acid triglyceride that is likely to overcome the pharmacokinetic drawbacks of natural butyrate as a 
drug 25. Because it is rapidly absorbed and chemically stable in plasma, tributyrin diffuses through 
biological membranes and is metabolized by intracellular lipases, releasing therapeutically effective 
butyrate over time directly into the cell. Compared with butyrate, tributyrin has more favorable 
pharmacokinetics 25, 26 and is well tolerated 27. Liquid tributyrin filled into gelatin capsules and 
administered orally resulted in millimolar concentrations of butyrate both in plasma and inside the cell 
27. Unlike BA, tributyrin is lipophilic with a plasma half-life of 40 minutes making it a preferred choice 
for systemic oral administration. Tributyrin is available over-the-counter as a supplement for patients 
with inflammatory bowel disease. Healus complete biotic butyrate supplement (500 mg tributyrin per 
Table 3: Neuropsychological characterization 
Overall mental status 
Montreal Cognitive Assessment Scale 3 
PD-Cognitive Rating Scale 4  
Intelligence 
WAIS-IV: Information5 
Attention & Cognitive 
Control 
n-back test (OPTIONAL) 7, Stroop Color Word 
Interference Test 9 
WAIS-IV: Digit Span (5)  
WAIS-III: Digit Symbol-Coding 5 
Language 
Boston Naming Test (30 item) 12 
Controlled Oral Word Association Test (Phonemic & 
Semantic) 
Learning & Memory 
California Verbal Learning Test – II 16 
 
Visuospatial/Construction 
Clock drawing 17 
Benton Judgment of Line Orientation 18 
Executive/Working 
Memory 
D-KEFS: Color Word Test, Verbal Fluency Test, Trail 
Making Test, Sorting Test 20 
WAIS-IV Matrix Reasoning, Digit Backwards 5 
Information processing 
speed 
D-KEFS: Trail Making Test 20 
WAIS-III: Digit Symbol-Coding 5, 23 
Reaction time  
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
9 
capsule) will be used as 1 capsule tid with meals (total of 1.5 g of tributyrin per day). If the patient 
cannot tolerate tributyrin we will lower the dose to one bid and if this is not tolerated than to once per 
day (e.g. at evening) and if this is not tolerated we will withdraw the study participant from the study. 
The participant will be asked to complete a daily drug diary while taking tributyrin. All supplement 
bottles will be returned at the end of the study for a drug accountability check. 
Magnetic resonance imaging (MRI) will be performed on a 3 Tesla Philips Achieva system (Philips, 
Best, The Netherlands). A 3D inversion recovery-prepared turbo-field-echo was performed in the 
sagittal 
plane 
using 
TR/TE/TI=9.8/4.6/1041ms; 
turbo 
factor=200; 
single 
average; 
FOV=240x200x160mm; acquired Matrix = 240x200x160 slices and reconstructed to 1mm isotropic 
resolution. Resting-state fMRI scans will serve as a secondary outcome measure and be acquired using 
a 32-channel head coil and multi-band sequence 32, with nominal parameters: (TR/TE/FA = 720/34/52, 
2mm isotropic resolution, 72 slices). Data are collected with eyes fixated on a cross for 10 minutes 33. 
Pre-processing follows our standard pipeline that includes physiologic noise reduction and thorough 
motion correction 34, 35.  Graph theory analysis will be used to identify the most highly connected nodes 
following published methods. Briefly, ROIs will be represented as nodes and pairwise correlations, 
based upon the mean time series for each ROI, will serve as weights over corresponding edges, 
thresholded by FDR correction, p<0.05 34, 35. Change from baseline will serve as the primary measure 
of interest. 
 
PET imaging will be performed in 3D imaging mode on a Biograph 6 TruePoint PET/CT scanner 
(Siemens Molecular Imaging, Inc., Knoxville, TN), which acquire 63 transaxial slices (slice thickness: 
2.4 mm) over a 15.2 cm axial field-of-view. The i.v. injection dose for [11C]butyrate PET is 18 mCi and 
for [18F]FDG is 5 mCi.  Vital signs will be obtained pre- and post-[11C]butyrate injection. Images will be 
corrected for scatter and motion. The PET imaging frames will be spatially coregistered within subjects 
with a rigid-body transformation to reduce the effects of subject motion during the imaging session 36. 
Statistical parametric mapping (SPM) software (SPM12; Wellcome Trust Centre for Neuroimaging, 
University College, London, England [https://www.fil.ion.ucl.ac.uk/spm/software/spm12/] will be used 
for PET-MRI registration using the cropped T1-weighted MR volumetric scan. Freesurfer software 
(http://surfer.nmr.mgh.harvard.edu) will be used to define cortical and subcortical volumes-of-interest 
(VOI).  
 
MRI-based PET partial volume correction: We will use the Müller-Gärtner partial volume correction 
technique, which is based on the assumption that white matter uptake is homogeneous and uses 
gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF) to correct the partial volume effect 
in GM tissues 37. Using the segmented images and the assumed PET resolution, the method 
calculates the spill-out from WM to GM and subsequently subtracts it from the GM. Similarly, it also 
calculates the spill-out from the GM to the surrounding tissues and compensates for the difference to 
the GM. The result is a grey matter image with corrected activity values in all voxels.  
 
Statistical analysis 
We will use mixed linear modeling to assess for changes in butyrate and glucose metabolism before 
and after the tributyrin supplementation.  
 
As a post hoc and only exploratory analyses we will regress differences in with tributyrin-induced 
changes of the various clinical and laboratory outcome measures.  
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
10
Sample size estimation: This experiment is a first in human study of this drug for treatment of glucose 
changes in a neurodegenerative condition. There is a preliminary experiment of this safe drug 
conducted in high-fat fed mice that we base our same size justification off that showed mice receiving 
tributyrin showed a reduction in glucose levels compared to a comparator arm of approximately 13% 
with a standard deviation of 9% (PMID 22621868). If estimate a similar effect size in our cohort, 
estimating a mean plasma glucose of 110mg/dl at baseline, using a power of 0.8 and a two-sided 
alpha of 0.05 for our primary analysis that will compare pre and post tributyrin, we arrive at a total 
sample size of 20.  
 
 
Pitfalls, problems and alternative strategies 
 
Ineligibility and attrition rate: 
 
Protection of Human Subjects 
 
i) Human Subjects 
Description of subject populations:  
i) 
Healthy control persons, M/F, age 45 years and over without a history of neurological or 
psychiatric disease or significant GI disease or uncontrolled medical comorbidity will be 
recruited (net n=4).  
ii) 
PD (net n=16) Diagnosis of PD in PD participants (M/F, age 45 years and over) will based 
on the United Kingdom Parkinson's Disease Society Brain Bank Diagnostic Research Criteria 
(patients with clinically significant dementia will be excluded (i.e., fails informed assent 
screening criteria and has significant evidence of iADL functions, such as ability to take own 
medications while administering the pre-consent screening questionnaire).  
 
Exclusion criteria: 
1. Exclusion: (a) subjects with contra-indications to MR imaging, including pacemakers or severe 
claustrophobia (for participants undergoing imaging); (b) Evidence of large vessel stroke or mass 
lesion on MRI; (c) Regular use of anti-cholinergic, benzodiazepines or neuroleptic drugs, (d) history of 
significant GI disease, (e) significant metabolic or uncontrolled medical cormorbidity, (f) poorly 
controlled diabetes, (g) pregnancy or breast feeding, (h) dementia requiring informed assent, (i) 
suicidal ideation. 
  
Recruitment: Subjects will be recruited from the University of Michigan Movement Disorder clinics, UM 
research studies website, and/or advertisement/flyers. 
 
ii) Sources of Materials: 
 
Information gathered specifically for this research project includes test results from clinical test scales, 
psychometric cognitive measures, neurobehavioral rating scales, clinical motor data, questionnaires,  
and data derived from the butyrate and FDG PET and from brain  MRI. 
 
iii) Potential Risks: 
 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
11
Confidentiality of Research Information:  The research data to be collected from subjects will consist of 
confidential information relating to clinical, motor, cognitive, neurobehavioral and neuro-imaging 
functions.  These research data are not intended for entry into the subjects’ clinical medical records.  
However, the data remain potentially discoverable. This may lead to violation of privacy and 
embarrassment of the subject. 
 
Clinical Testing: Risks in regard to the clinical assessment are limited to fatigue, frustration, discomfort, 
and momentary embarrassment that may occur when one experiences difficulty performing a task or 
learning a new skill.  
 
Risks associated with clinical, neuropsychological and behavioral testing: This study involves cognitive 
tests and questionnaires. The cognitive tests are not harmful but some people find them frustrating and 
concerning. Risks in regard to the neuropsychological and behavioral assessment are limited to fatigue, 
frustration and momentary embarrassment that may occur when one experiences difficulty performing 
a task or learning a new skill. Some people may be uncomfortable/embarrassed disclosing personal 
information or become a little nervous about memory testing, and may experience discomfort or 
become tired. Study team members are very well experienced in the assessment of older individuals 
and persons with dementia, understanding the need for breaks, gentle reassurance, or reinforcement. 
All assessments will be stopped if requested by the participant.   
 
MRI scan: There is a substantial risk to persons who have metallic objects inside their bodies, as the 
magnet in the MR scanner can cause these to move. Consequently, participants with pacemakers or 
metallic objects located in the eye, ear, brain or blood vessel walls will be excluded. Participants who 
developed claustrophobic anxiety during scanning found that this fear dissipated within 15 min while 
remaining in the scanner, or as necessary, after exiting the scanner. There also is the potential that 
imaging could reveal a previously unrecognized but pre-existing abnormality. Many such abnormalities 
are not clinically significant, but they may cause anxiety or require further investigation by a personal 
physician. If one of the investigators identifies such an abnormality, they will contact the personal 
physician, who will arrange for appropriate care. 
 
PET-CT scans & venipuncture: Insertion of a catheter for intravenous injection of the PET 
radiopharmaceutical may be commonly associated with slight pain or bruising at the puncture site and 
rare chance of infection. Participation in this research study will involve low level exposure to radiation 
associated with the PET, CT and DEXA scans.  
 
Certified staff will be in attendance at all times during the study.  A physician will be available and an 
emergency cart is located in the PET Facility for treatment of any adverse reactions that may occur. 
 
During the course of this study, the participant will be exposed to radiation from the two [11C]butyrate 
PET, the two [18F]FDG PET-CT and two DEXA scans. The risks associated with the amount of 
radiation exposure participants receive in this study are considered very rare and comparable to 
every day radiation exposure risks.  
The biological effect of radiation in humans is measured in terms of Sieverts (Sv) or mSv (1/1000 Sv), 
which is a unit of uniform whole body exposure. The maximum radiation exposure (assuming a 
person will complete all 4 PET scans) a participant will receive from the CT, [11C]butyrate PET and 
[18F]FDG PET scans is equivalent to a uniform whole body dose of 21.2 mSv, which is approximately 
42.4% of the annual radiation exposure (50 mSv) permitted to radiation workers by federal 
regulations. The participant will be instructed to use the bathroom and urinate as soon as possible 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
12
after the PET scans to minimize bladder exposure. The radiation the participant will be exposed to 
from each DXA scan is 0.0310 mSv, which is significantly less than 1% of the permitted annual 
radiation exposure.  
 
There is no known minimum level of radiation exposure that is recognized as being totally free of the 
risk of causing genetic defects (cellular abnormalities) or cancer.  However, the risk associated with 
the amount of radiation exposure that you will receive from this study is considered to be low. The risk 
of a side effect from this level of radiation exposure is very rare. The risk from radiation exposure of 
this amount is considered to be similar to other everyday risks, such as driving a car. 
 
No PET studies will be performed on pregnant, nursing, or potentially pregnant women. 
A urine pregnancy test will be performed on all women of childbearing potential within 48 hours prior 
to the PET scanning session, and urine sampling may be performed on subjects at baseline and post-
interventions as an additional screening measure. 
 
Risks associated with tributryin: The risks to participants are very minimal. Although tributyrin is taken 
for symptoms of irritable or inflammatory bowel disease it is possible that nausea or indigestion may 
occur.  However, these gastrointestinal side effects are both rare and mild. Tributyrin is received 
GRAS (Generally Regarded As Safe) certification from the FDA, is commercially available in the 
United States and prior work has shown that the dosing regimen proposed in this study has been 
found to be safe and generally well-tolerated.  
 
Data Study Monitoring Plan (DSMP): Drs. Bohnen and Albin will act as the Data Safety Monitoring 
Plan members for this study. All adverse events and other study related events will be discussed at 
monthly investigator meetings. Study team will document any adverse events specifically related to 
study participation (imaging, clinical visit, at-home supplement and/or device usage, etc.). Adverse 
events will be classified according to standard criteria and reported to the IRBMED (relevant 
University of Michigan IRB) according to standard guidelines. Serious adverse events will be reported 
within 24 hours. 
For risk of suicidality (if participants select answer responses 2 or 3 for question 9 on the Beck 
Depression Inventor, i.e. 'I would like to kill myself' (answer 2) or 'I would like to kill myself if I had a 
chance' (answer 3) then the staff will hand out the UM Depression brochure, refer to the contact 
information and also inform Dr. Bohnen or Dr. Albin who will discuss this with the participant. The face 
to face conversation with the physicians must occur during that visit and must not be deferred. There 
must be a safety plan in place before the patient can leave the building. 
 
 
References 
 
1. 
Albin RL, Minoshima S, D'Amato CJ, Frey KA, Kuhl DE, Sima AAF. Fluoro-deoxyglucose positron emission 
tomography in diffuse Lewy body disease. Neurology. 1996;47:462-6. 
2. 
Bohnen NI, Koeppe RA, Minoshima S, Giordani B, Albin RL, Frey KA, Kuhl DE. Cerebral glucose metabolic features 
of Parkinson disease and incident dementia: longitudinal study. J Nucl Med. 2011;52(6):848-55. Epub 2011/05/17. doi: 
jnumed.111.089946 [pii] 
10.2967/jnumed.111.089946. PubMed PMID: 21571793. 
3. 
Nasreddine ZS, Phillips NA, Bédirian V, Charbonneau S, Whitehead V, Collin I, Cummings JL, Chertkow H. The 
Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment. J Am Geriatr Soc. 
2005;53(4):695-9. doi: 10.1111/j.1532-5415.2005.53221.x. PubMed PMID: 15817019. 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
13
4. 
Pagonabarraga J, Kulisevsky J, Llebaria G, Garcia-Sanchez C, Pascual-Sedano B, Gironell A. Parkinson's disease-
cognitive rating scale: a new cognitive scale specific for Parkinson's disease. Mov Disord. 2008;23(7):998-1005. Epub 
2008/04/03. doi: 10.1002/mds.22007. PubMed PMID: 18381647. 
5. 
Wechsler D. Wechsler Adult Intelligence Scale-Third Edition. San Antonio, TX: Psychological Corporation; 1997. 
6. 
Cavaleri F, Bashar E. Potential Synergies of beta-Hydroxybutyrate and Butyrate on the Modulation of 
Metabolism, Inflammation, Cognition, and General Health. J Nutr Metab. 2018;2018:7195760. Epub 2018/05/29. doi: 
10.1155/2018/7195760. PubMed PMID: 29805804; PMCID: PMC5902005. 
7. 
Kirchner WK. Age differences in short-term retention of rapidly changing information. J Exp Psychol. 
1958;55(4):352-8. Epub 1958/04/01. doi: 10.1037/h0043688. PubMed PMID: 13539317. 
8. 
Reolon GK, Maurmann N, Werenicz A, Garcia VA, Schroder N, Wood MA, Roesler R. Posttraining systemic 
administration of the histone deacetylase inhibitor sodium butyrate ameliorates aging-related memory decline in rats. 
Behav Brain Res. 2011;221(1):329-32. Epub 2011/03/23. doi: 10.1016/j.bbr.2011.03.033. PubMed PMID: 21421011; 
PMCID: PMC3093300. 
9. 
Bohnen N, Jolles J, Twijnstra A. Modification of the Stroop Color Word Test improves differentiation between 
patients with mild head injury and matched controls. The Clin Neuropsychologist. 1992;6:178-84. 
10. 
Matt SM, Allen JM, Lawson MA, Mailing LJ, Woods JA, Johnson RW. Butyrate and Dietary Soluble Fiber Improve 
Neuroinflammation Associated With Aging in Mice. Front Immunol. 2018;9:1832. Epub 2018/08/30. doi: 
10.3389/fimmu.2018.01832. PubMed PMID: 30154787; PMCID: PMC6102557. 
11. 
Baxter NT, Schmidt AW, Venkataraman A, Kim KS, Waldron C, Schmidt TM. Dynamics of Human Gut Microbiota 
and Short-Chain Fatty Acids in Response to Dietary Interventions with Three Fermentable Fibers. MBio. 2019;10(1). Epub 
2019/01/31. doi: 10.1128/mBio.02566-18. PubMed PMID: 30696735; PMCID: PMC6355990. 
12. 
Saxton J, Ratcliff G, Munro CA, Coffey EC, Becker JT, Fried L, Kuller L. Normative data on the Boston Naming Test 
and two equivalent 30-item short forms. Clin Neuropsychol. 2000;14:526-34. 
13. 
Zhou ZL, Jia XB, Sun MF, Zhu YL, Qiao CM, Zhang BP, Zhao LP, Yang Q, Cui C, Chen X, Shen YQ. Neuroprotection 
of Fasting Mimicking Diet on MPTP-Induced Parkinson's Disease Mice via Gut Microbiota and Metabolites. 
Neurotherapeutics. 2019;16(3):741-60. Epub 2019/03/01. doi: 10.1007/s13311-019-00719-2. PubMed PMID: 30815845. 
14. 
Kim SW, Hooker JM, Otto N, Win K, Muench L, Shea C, Carter P, King P, Reid AE, Volkow ND, Fowler JS. Whole-
body pharmacokinetics of HDAC inhibitor drugs, butyric acid, valproic acid and 4-phenylbutyric acid measured with 
carbon-11 labeled analogs by PET. Nucl Med Biol. 2013;40(7):912-8. Epub 2013/08/03. doi: 
10.1016/j.nucmedbio.2013.06.007. PubMed PMID: 23906667; PMCID: PMC3769509. 
15. 
Frost G, Sleeth ML, Sahuri-Arisoylu M, Lizarbe B, Cerdan S, Brody L, Anastasovska J, Ghourab S, Hankir M, Zhang 
S, Carling D, Swann JR, Gibson G, Viardot A, Morrison D, Louise Thomas E, Bell JD. The short-chain fatty acid acetate 
reduces appetite via a central homeostatic mechanism. Nat Commun. 2014;5:3611. Epub 2014/05/02. doi: 
10.1038/ncomms4611. PubMed PMID: 24781306; PMCID: PMC4015327. 
16. 
Delis DC, Kramer JH, Kaplan E, Ober BA. California Verbal Learning Test Manual, Adult Version. Second ed. San 
Antonio: The Psychological Corporation; 2000. 
17. 
Freedman M, Leach L, Kaplan E, Winocur G, Shulman KI, Delis DC. Clock drawing: A neuropsychological analysis. 
New York: Oxford University Press; 1994. 
18. 
Benton AL, Hamsher K, Varney NR, Spreen O. Contributions to Neuropsychological Assessment. New York: 
Oxford University Press; 1983. 
19. 
Agusti A, Garcia-Pardo MP, Lopez-Almela I, Campillo I, Maes M, Romani-Perez M, Sanz Y. Interplay Between the 
Gut-Brain Axis, Obesity and Cognitive Function. Front Neurosci. 2018;12:155. Epub 2018/04/05. doi: 
10.3389/fnins.2018.00155. PubMed PMID: 29615850; PMCID: PMC5864897. 
20. 
Delis DC, Kaplan E, Kramer JH. Delis-Kaplan Executive Function System (D-KEFS): Examiner's manual. San 
Antonio, TX: The Psychological Corporation; 2001. 
21. 
Val-Laillet D, Guerin S, Coquery N, Nogret I, Formal M, Rome V, Le Normand L, Meurice P, Randuineau G, 
Guilloteau P, Malbert CH, Parnet P, Lalles JP, Segain JP. Oral sodium butyrate impacts brain metabolism and 
hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs. Faseb j. 2018;32(4):2160-71. Epub 
2017/12/16. doi: 10.1096/fj.201700547RR. PubMed PMID: 29242276. 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
14
22. 
Sharma S, Taliyan R, Singh S. Beneficial effects of sodium butyrate in 6-OHDA induced neurotoxicity and 
behavioral abnormalities: Modulation of histone deacetylase activity. Behav Brain Res. 2015;291:306-14. Epub 
2015/06/07. doi: 10.1016/j.bbr.2015.05.052. PubMed PMID: 26048426. 
23. 
Chriett S, Dabek A, Wojtala M, Vidal H, Balcerczyk A, Pirola L. Prominent action of butyrate over beta-
hydroxybutyrate as histone deacetylase inhibitor, transcriptional modulator and anti-inflammatory molecule. Sci Rep. 
2019;9(1):742. Epub 2019/01/27. doi: 10.1038/s41598-018-36941-9. PubMed PMID: 30679586; PMCID: PMC6346118. 
24. 
Stein J, Zores M, Schroder O. Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and 
carrier mediated transport mechanism. Eur J Nutr. 2000;39(3):121-5. Epub 2000/08/05. PubMed PMID: 10918994. 
25. 
Schroder C, Eckert K, Maurer HR. Tributyrin induces growth inhibitory and differentiating effects on HT-29 colon 
cancer cells in vitro. Int J Oncol. 1998;13(6):1335-40. Epub 1998/11/24. doi: 10.3892/ijo.13.6.1335. PubMed PMID: 
9824654. 
26. 
Egorin MJ, Yuan ZM, Sentz DL, Plaisance K, Eiseman JL. Plasma pharmacokinetics of butyrate after intravenous 
administration of sodium butyrate or oral administration of tributyrin or sodium butyrate to mice and rats. Cancer 
Chemother Pharmacol. 1999;43(6):445-53. Epub 1999/05/13. doi: 10.1007/s002800050922. PubMed PMID: 10321503. 
27. 
Conley BA, Egorin MJ, Tait N, Rosen DM, Sausville EA, Dover G, Fram RJ, Van Echo DA. Phase I study of the orally 
administered butyrate prodrug, tributyrin, in patients with solid tumors. Clin Cancer Res. 1998;4(3):629-34. Epub 
1998/05/14. PubMed PMID: 9533530. 
28. 
Salarian A, Horak FB, Zampieri C, Carlson-Kuhta P, Nutt JG, Aminian K. iTUG, a sensitive and reliable measure of 
mobility. IEEE Trans Neural Syst Rehabil Eng. 2010;18(3):303-10. Epub 2010/04/15. doi: 10.1109/TNSRE.2010.2047606. 
PubMed PMID: 20388604; PMCID: 2922011. 
29. 
Mancini M, Salarian A, Carlson-Kuhta P, Zampieri C, King L, Chiari L, Horak FB. ISway: a sensitive, valid and 
reliable measure of postural control. J Neuroeng Rehabil. 2012;9:59. Epub 2012/08/24. doi: 10.1186/1743-0003-9-59 
1743-0003-9-59 [pii]. PubMed PMID: 22913719; PMCID: 3481400. 
30. 
Mancini M, Fling BW, Gendreau A, Lapidus J, Horak FB, Chung K, Nutt JG. Effect of augmenting cholinergic 
function on gait and balance. BMC Neurol. 2015;15(1):264. doi: 10.1186/s12883-015-0523-x. PubMed PMID: 26697847; 
PMCID: 4690312. 
31. 
Franchignoni F, Horak F, Godi M, Nardone A, Giordano A. Using psychometric techniques to improve the Balance 
Evaluation Systems Test: the mini-BESTest. J Rehabil Med. 2010;42(4):323-31. Epub 2010/05/13. doi: 
10.2340/16501977-0537. PubMed PMID: 20461334; PMCID: 3228839. 
32. 
Feinberg DA, Setsompop K. Ultra-fast MRI of the human brain with simultaneous multi-slice imaging. J Magn 
Reson. 2013;229:90-100. Epub 2013/03/12. doi: 10.1016/j.jmr.2013.02.002. PubMed PMID: 23473893; PMCID: 
PMC3793016. 
33. 
Patriat R, Molloy EK, Meier TB, Kirk GR, Nair VA, Meyerand ME, Prabhakaran V, Birn RM. The effect of resting 
condition on resting-state fMRI reliability and consistency: a comparison between resting with eyes open, closed, and 
fixated. Neuroimage. 2013;78:463-73. Epub 2013/04/20. doi: 10.1016/j.neuroimage.2013.04.013. PubMed PMID: 
23597935; PMCID: PMC4003890. 
34. 
Roy CW, Seed M, Kingdom JC, Macgowan CK. Motion compensated cine CMR of the fetal heart using radial 
undersampling and compressed sensing. J Cardiovasc Magn Reson. 2017;19(1):29. Epub 2017/03/21. doi: 
10.1186/s12968-017-0346-6. PubMed PMID: 28316282; PMCID: PMC5357808. 
35. 
Hillary FG, Medaglia JD, Gates KM, Molenaar PC, Good DC. Examining network dynamics after traumatic brain 
injury using the extended unified SEM approach. Brain Imaging Behav. 2014;8(3):435-45. Epub 2012/11/10. doi: 
10.1007/s11682-012-9205-0. PubMed PMID: 23138853. 
36. 
Minoshima S, Koeppe RA, Fessler JA, Mintun MA, Berger KL, Taylor SF, Kuhl DE. Integrated and automated data 
analysis method for neuronal activation studying using O15 water PET. In: Uemura K, Lassen NA, Jones T, Kanno I, editors. 
Quantification of brain function to tracer kinetics and image analysis in brain PET. Tokyo: Excerpta Medica; 1993. p. 409-
18. 
37. 
Muller-Gartner H, Links J, Prince J, Bryan R, McVeigh E, Leal J, Davatzikos C, Frost J. Measurement of radiotracer 
concentration in brain gray matter using positron emission tomography:  MRI based correction of partial volume effects. 
J Cereb Blood Flow Metab. 1992;12:571-83. 

Nicolaas Bohnen, MD, Ph.D. 
UM HUM #00211320 
PROTOCOL VERSION #: 1.6 
 
15