Effects of Acetate and Alcohol on Brain Function
Phase: NA
Conditions: Cerebral Blood Flow, Brain Function
Sponsor: University of Colorado, Denver

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Protocol #: 15-0933 
Project Title: Effects of acetate and alcohol on brain function 
 
 
 
Principal Investigator: Jody Tanabe, MD 
 
Version Date: 12/22/2017 
 
Overall goal: This is a pilot study to determine the feasibility of measuring effects of oral alcohol and intravenous 
acetate on cerebral blood flow and brain function in moderate drinkers and individuals with alcohol use disorder (AUD). 
Data from this pilot will be used to calculate the power needed for a larger, R21 application.  
 
I. Hypotheses and Specific Aims:   
Acute alcohol alters behavior and cognition through effects on brain metabolism and function. Although the 
risks of abuse are known, there is a gap in our understanding of the effects of acute alcohol and its metabolite, acetate, 
on brain function. Alcohol is first metabolized to acetaldehyde and then to acetate, primarily in liver but also in the gut. 
Alcohol consumption increases plasma acetate, which then accumulates in the brain (Volkow et al., 2013). Normally the 
brain utilizes glucose as its energy substrate. After acute alcohol there is a switch in brain energy substrate utilization 
from glucose to acetate. This switch may explain why moderate alcohol can decrease brain glucose metabolism by 25%-
30% with relatively little effect on cognitive processing  (Volkow et al., 2006). It has been hypothesized that brain 
acetate is a form of caloric “reward” that perpetuates drinking and that falling acetate levels associated with sobriety 
precipitate withdrawal (Jiang et al., 2013).  
 Alcohol increases endotoxin produced by the gut, stimulating inflammatory cytokine release from the liver 
which induces neuro-inflammation. The neurotoxicity of excessive alcohol is partially mediated through prolonged 
activation of microglia, the resident immune cells of the brain. The liver is critical in regulating the immune system’s 
response to alcohol since liver stimulates both pro-inflammatory and anti-inflammatory cytokines. Given the role of 
inflammation in alcohol neurotoxicity, it is intriguing that acetate has been shown to decrease microglial activation 
(Brissette et al., 2012) and inflammatory cytokine production (Soliman et al., 2012a), suggesting a neuroprotective role.  
Acute alcohol is associated with increased cerebral blood flow (CBF). It is unknown if changes in CBF are due 
to alcohol, acetate, or both. Determining the differential effects of alcohol and acetate on brain function is significant as 
it could lead to new treatments targeting alcohol metabolites. The goal of the proposed study is to determine feasibility 
of measuring effects of oral alcohol and intravenous acetate on cerebral blood flow and function in moderate drinkers 
with the objective to use the data collected for an R01or R21application. Although currently there is no data in the 
literature on this, finding such changes would be important and could direct future prevention and treatment plans for 
alcohol use disorders. For this feasibility pilot study we propose to investigate the effects of acetate and alcohol on CBF 
and brain function with two specific aims. 
 
Aim 1.  Investigate the effects of acetate on cerebral blood flow and function in controls and AUD individuals 
Preliminary data will be collected for the hypothesis that compared to placebo given IV acetate given IV will: a) 
increase CBF as measured by arterial spin labeling (ASL); and b) decrease blood oxygen level dependent (BOLD) 
activity during functional magnetic resonance imaging (fMRI); and c) these changes will be greater in AUD individuals 
than controls 
Aim 2. Investigate the effects of alcohol on cerebral blood flow and function in controls and AUD individuals 
Preliminary data will be collected for the hypothesis that compared to non-alcoholic jello shots, orally ingested alcohol 
jello shots will: a) increase CBF, and b) decrease BOLD activity; and c) these changes will be greater in AUD 
individuals than controls. 
 
II. Background and Significance:  
What is the problem and the critical barrier to progress in the field? Many studies have looked at the effects of acute 
alcohol on CBF (Sano et al., 1993; Strang et al., 2014; Volkow et al., 1988), activity (Bjork and Gilman, 2014; Van 
Horn et al., 2006) and metabolites (Biller et al., 2009). Although work is beginning to emerge on the brain effects from 
acetate  (Jiang et al., 2013), there have been no studies that fully evaluate the effects of acetate and compare them to that 
of alcohol. We review the literature to describe the gaps that exist and then describe how the proposed project will fill 
the gaps. Answering these questions will increase scientific knowledge and guide clinicians toward better strategies for 
the treatment and prevention of alcohol disorders. 
 
Alcohol and brain metabolism: In the United States, alcohol is the most widely abused addictive drug and the third 
leading cause of preventable deaths (CDC, 2014). Alcohol impairs cognition, likely through direct and indirect effects 
on brain metabolism and altered neurotransmitter function. The mechanisms for cognitive and behavior effects of an 

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acute alcohol challenge are partially understood. Moderate alcohol significantly reduces brain glucose metabolism up to 
25%, surprisingly high, given that these doses produce only mild intoxication (Volkow et al., 2008, 2006). The liver 
rapidly metabolizes alcohol to acetaldehyde, and then to acetate. Acetate then enters the circulation and can be used as 
an energy source. Volkow et al., demonstrated that the decrease in glucose metabolism resulting from alcohol ingestion 
is associated with an increase in brain acetate, suggesting a switch in brain substrate utilization (Volkow et al., 2013). 
Wang et al. (2013) found that rats exposed to alcohol had higher levels of brain acetate than control rats. 
 
Alcohol, acetate and knowledge gaps about acetate’s functional significance: While acetaldehyde toxicity is firmly 
established as a mechanism for tissue injury due to alcohol, less is known about the effects of acute and chronic acetate. 
The current proposal will systematically study the effects of acetate and alcohol on CBF and brain activity. Acetate is a 
short chain fatty acid normally produced by gut bacteria. Basal acetate 
levels are low and modulated by diet. In the brain, acetate is nearly 
exclusively metabolized by glia, being preferentially taken up by glia 18 
times faster than by synaptosomes (Waniewski and Martin, 1998). In fact, a 
major interest in acetate arises from its potential as a specific marker of 
astrocyte activity (Wyss et al., 2011). After it is absorbed from the gut, 
alcohol is metabolized to acetate in the liver resulting in a 3-5 fold increase 
in plasma acetate (Sarkola et al., 2002). It enters the TCA cycle and can 
eventually be used in the glutamate-glutamine cycle (Håberg et al., 1998) 
(Figure 1). Acetate is not an inert substance. Its biological actions on the 
CNS can potentiate and antagonize the effects of alcohol in animals 
(Carmichael et al., 1991; Correa et al., 2003; McLaughlin et al., 2008). 
Large amounts of acetate have adverse effects on neural function (Israel et 
al., 1994). In alcoholics, one hypothesis is that chronically high brain 
acetate may be a form of caloric “reward” that may perpetuate drinking and 
that falling acetate levels from acute sobriety may precipitate withdrawal 
(Jiang et al., 2013). Acetate also exhibits anti-inflammatory properties. Specifically, acetate reduces microglial 
activation, pro-inflammatory cytokine release (Brissette et al., 2012) and endotoxin production (Soliman et al., 2012b). 
In addition, acetate decreases alcohol and thiamine deficiency-related thalamic neurodegeneration (Qin and Crews, 
2014). Outside the CNS, acetate and other short chain fatty acids have been shown to suppress inflammation (Ishiguro et 
al., 2014; Vinolo et al., 2011). These findings suggest that acetate may have protective effects against alcohol-induced 
neuro-inflammation. 
Acetate is also implicated in neurovascular “decoupling” observed after acute alcohol. Normally, lowering 
glucose metabolism results in a decrease in CBF due to tight neurovascular coupling. Instead, most studies have shown 
that alcohol increases CBF (Mathew and Wilson, 1986; Newlin et al., 1982; Strang et al., 2014). One explanation 
involves differences in the time scale of PET studies of glucose metabolism compared to blood flow assays. Another 
explanation involves the vasodilatory actions of acetate-derived adenosine (Schwartz et al., 1993). Studying the alcohol-
induced changes in CBF will help disentangle the effects of flow and metabolism and clarify alcohol- induced changes 
in neural circuitry (Gilman et al., 2012; Marxen et al., 2014). 
 
 
Acute alcohol and brain MRI studies: Structural and functional MRI and MRS brain studies of alcohol users has given 
us valuable insight into cumulative toxic effects of alcohol on the CNS (Meyerhoff, 2014; Sullivan et al., 2013). There is 
less information on brain effects of acute alcohol. Acute alcohol decreases fMRI BOLD activity in cortical regions 
implicated in cognitive control (Calhoun et al., 2004; Schuckit et al., 2012; Van Horn et al., 2006). One fMRI study 
showed that acute alcohol increased activity in the striatum during decision-making (Gilman et al., 2012).  
 
How will the project improve scientific knowledge or practice? This is a pilot study to determine the feasibility of 
measuring effects of oral alcohol and intravenous acetate on CBF and brain function in moderate drinkers and 
individuals with alcohol use disorder (AUD). Data from this study will be used to apply for funds to conduct a larger 
study. The proposed experiments will allow us, in the larger study, to parse out the effects of alcohol and acetate on 
brain function. We will test if acetate increases CBF, as was suggested in a prior alcohol administration study (Schwartz 
et al., 1993).  
 
Theoretical Model 
Figure 1. Glucose is taken up by neurons 
and astrocytes. Astrocytes have a unique 
ability to take up acetate as substrate for 
the TCA cycle (from Haberg et al. (1998)). 

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The working model is shown in Figure 2. Possible pathways through 
which brain function is altered following alcohol intake include 
through hepatic metabolism of alcohol to acetate (Aim 1) and through 
the direct effect of alcohol on the brain, including the effect from 
acetate (Aim 2). 
  
 
 
 
 
 
 
III. Preliminary Studies  
 
Practice effects: It is possible that differences in brain activity could reflect learning effects. To test for learning effects 
we administered two versions of the decision-making task to a healthy control, 2 hours apart. The two versions differed 
in the color and position of the decks and the scale of the magnitude of outcomes, while keeping the value of the decks 
balanced. The frequency of wins and losses was identical. There was no difference in Play response on good cards (93 
vs. 95), bad cards (54 vs. 55), or on net score (good minus bad cards played) (39 vs. 40). This lack of significant practice 
effect is consistent with a prior study showing stable performance on the Iowa Gambling Task, on which our task is 
based, given multiple times to controls (Xiao et al., 2013) and patients (Waters-Wood et al., 2012) and, together, 
suggest that there will not be a significant learning effect.   
 
 
IV. Research Methods 
 
A.  Outcome Measure(s):   
 
Our goal in this pilot study is feasibility. 
 
       Preliminary outcome measures: 
1.   CBF before and after acute alcohol or acetate 
2.   Brain activity during decision-making before and after acute alcohol or acetate  
 
B. Description of Population to be Enrolled:   
 
This is a single blinded placebo controlled test of the effects of acetate and alcohol on brain function. 
 
Inclusions-all: (1) age 21-55, (2) English proficient, (3) Participants must understand the nature of the study and must 
sign an informed consent; (4) be currently non-smoking. 
 
Inclusions-moderate drinkers: (1) participants must be moderate drinkers (male: 1-14 drinks per week: female: 1-7 
drinks per week); and (2) have drunk at least 2 drinks in one hour in the past; and have no history of dependence on 
drugs or alcohol. 
 
Inclusions-AUD: (1) must meet DSM-V criteria for lifetime history of Alcohol Use Disorder (AUD) 
 
Exclusions-all: Participants will be excluded if they: (1) have a “facial flushing” response to alcohol; (2) are pregnant or 
nursing; (3) have major medical problems including diabetes, uncontrolled high blood pressure (> 150/90), prior 
neurological or psychiatric history (schizophrenia, bipolar, or current major depressive disorder, epilepsy (alcohol 
withdrawal seizures is not an exclusion)); (4) have ever been told they have or been diagnosed with liver or kidney 
disease; (5) have a diagnosis of chronic gastrointestinal disease; (6) are obese (BMI>30 kg/m2); (7) have prior head 
trauma resulting in loss of consciousness>15 min.; (8) have MR exclusions which include claustrophobia, intracranial, 
orbital, or spinal metal, pacemakers, cochlear implants, cardiac stents or other non-MR-compatible implants or devices. 
 
Figure 2. Working Model 

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C. Study Design and Research Methods   
 
 
Study Design 
This is a pilot study to determine the feasibility of measuring effects of acetate alone and acetate obtained as a byproduct 
of alcohol metabolism on CBF and brain activity. Our recruitment goal will be 50 subjects: 40 healthy controls who are 
social drinkers (1-14 drinks per week) to achieve a target of n=20 scanned for Aim 1 and n=20 scanned for Aim 2, with 
a final usable healthy control target of n=15 for Aim1 and n=15 for Aim 2 (a few subjects may not qualify for data 
analysis due to excessive head motion in scanner); and 10 AUD individuals, to achieve a target of n=7 scanned for Aim 
1. AUD participants seeking treatment will be excluded from Aim 2 (alcohol arm). Moderate drinkers will be recruited 
through advertisements and flyers. AUD subjects will be recruited from the Center for Dependency, Addiction and 
Rehabilitation (CeDAR) at the University of Colorado Anschutz Medical Campus.  
Order effects: Before the study commences, test-retest reliability will be conducted on two healthy controls on MR 
measures by scanning them twice in a single day, with no intervention. The null hypothesis of no difference in brain 
activity and CBF will be tested, between the two sessions. Notably, the literature shows high reliability for test-retest of 
CBF using ASL at various time intervals (Floyd et al, 2001, Chen et al., 2011). 
Session 1: Consent and diagnostic assessment (total time ~1 hour) 
Potential participants will take part in a short interview at the Anschutz Medical Campus consisting of explanation of the 
study, screening for MRI compatibility, signing informed consent, and answering questions on drug and alcohol use.. 
Control participants will be randomized to the acetate or alcohol branch of the study but will not know which branch 
they are assigned to until Session 2. AUD participants will be assigned only to the acetate branch of the study. 
Participants will be asked to not drink alcohol for 48 hours prior to the study and to eat a light low-fat breakfast before 
coming to the University for Session 2. Session 2 (scan day) will be scheduled as soon as possible, about 1-2 weeks after 
Session 1.  
Questionnaires: 
(1) Composite International Diagnostic Interview Substance Abuse Module (CIDI-SAM). Dependence on alcohol, 
cannabis and nicotine will be measured using the computerized CIDI-SAM, a structured interview designed for trained, 
lay interviewers (Cottler et al. 1995). (30 min)   
(2) Alcohol Use Disorders Identification Test (AUDIT). This test screens for alcohol abuse and average alcohol use (5 
minutes) 
(3) Timeline Followback assessment. This assessment measures frequency and quantity of alcohol consumption for the 
most recent 1-3 months of alcohol use. 
 
 
Session 2: Acetate or Alcohol and MRI scanning (~7 hours)  
Research participants will arrive at the Clinical and Translational Research Clinic (CTRC) in the Leprino Building on 
the University of Colorado Anschutz Medical Campus (AMC) at 7:30 am, turn in their stool samples and 2 day food log, 
and provide urine samples to screen for alcohol, drugs and pregnancy.  
 
If the subject is randomized to acetate: Control participants will need to arrange for a driver to and from the second 
session or we will provide a voucher for up to $50 for them to take a taxi to Session 2 and up to $50 for them to take a 
taxi home from Session 2. AUD participants will be accompanied to and from CeDAR for the second session. Two IVs 
will be placed by a CTRC nurse or a physician on the study. One IV will be for infusion and one will allow blood draws 
without repeat sticks. The infusion IV and blood draw IV will be placed in separate arms to prevent infusate from 
confounding blood sample metabolite measures. The Session 2 timeline is illustrated in Figure 2. The first blood draw 
will be immediately after IVs are placed. Participant will then be escorted over to the Brain Imaging Center at the 
University of Colorado AMC for MRI scans. Participant will be blinded as to when they are receiving placebo and when 
they are receiving acetate. A placebo infusion will be started and continued through the first scan. The participant will be 
given the Subjective High Assessment Scale 7 (SHAS7), an analogue scale evaluating 7 subjective feelings of 
intoxication, before the first scan and after each scan (Schuckit et al., 2000). After each scan, participants will perform 
several motor tasks (psychomotor vigilance task, simple and choice reaction time task, and grooved pegboard task) to 
measure attention and arousal after placebo and drug.  The participant will have the first MR scan of the brain. 
Immediately after the scan, the second blood sample will be obtained. One hour later, at 11:00 am, the placebo infusion 
will be switched to an acetate infusion and the participant will be placed in the MR scanner. The acetate will be prepared 
by the University of Colorado Hospital Research Pharmacy and will be based on methods of Jiang et al.(Jiang et al., 
2013) (See below). The participant will have the second MR scan using the same protocol. Immediately following the 
second MRI scan, the third blood sample will be obtained. Then both IVs will be removed, participants will be given 

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lunch and observed in the Brain Imaging Center for 1 hour. Participants will be given a stool sample kit and a prepaid 
return envelope for mailing the sample back to us. Participants will be reimbursed and allowed to go home with a driver 
or by taxi (details at top of paragraph). AUD participants will be accompanied back to CeDAR. All participants will 
receive a follow up call that evening. They will collect a stool sample the day after the scans and mail it back to us that 
same day. 
 
If the subject is randomized to alcohol: The procedure is identical to that described for acetate with the following 
differences: (a) only one IV will be placed, for blood draws, (b) participant will receive a jello shot rather than an IV 
infusion. The participant is blinded as to when they will receive a placebo jello shot and when they receive an alcohol 
jello shot. The participant will be given a placebo jello shot 15 min before the first scan, and an alcohol jello shot 15 min 
before the second scan (Van Horn et al., 2006). Following the MRI, breath alcohol (BrAC) will be taken every 30 min 
until it reaches a level ≤0.02%, at which time participant will be allowed to go home with a driver or will be given a 
voucher for up to $50 for a taxi (as described in first sentence of acetate section, above). We expect BrAC levels to be 
≤0.02% by 3 pm. All participants will receive a follow up call that evening. 
 
 
Figure 2. Procedure for acetate and alcohol administration 
 
Research Methods 
 
IV acetate and placebo: Infusions will be prepared by the University of Colorado Hospital Research Pharmacy at the 
University Hospital the morning of the study. To maintain the single blind, the placebo bag will be labeled “Morning” 
and the acetate bag labeled “Afternoon.” A study team member will bring the infusion bags to the CTRC immediately 
before the study. Based on Jiang et al., acetate infusion will be 5 mg/kg/min for the first 7 minutes, followed by 
continuous infusion of 2.5 mg/kg/min for 68 minutes (Jiang et al., 2013). This rate results in safe plasma acetate levels 
of ~1.2 mM, comparable to acetate levels after moderate drinking (Jiang et al., 2013; Mason et al., 2006; Smith et al., 
2007).   
 
Alcohol and control jello shots: Methods will be as described by Van Horn et al (Van Horn et al., 2006). For the 
alcohol condition, participants will consume more or less of the jello shot based on the Widmark formula which takes 
into account their weight and sex, so as to bring the breath alcohol concentration (BAC) for each subject to the same 
level of 0.07%. Jello shots will be prepared with 100 proof Vodka, according to calculations based on Widmark's 
equation: Number of fl.oz. EtOH = [[BAC g%/ml + (hours since last drink) × Widmark b] × Weight in lbs. × Widmark 
r] / [0.0514 lb/fl.oz. EtOH × 100% × 1.055 g%/h]. The Widmark b factor will be set to 0.017. The Widmark r factor will 
be set to 0.68 for male and 0.55 for female participants, respectively. Two mL (< ½ tsp) of alcohol will be spread on top 
of the nonalcoholic jello shot to give the smell and taste of alcohol, but keep BAC at 0%. 
 
 
Blood samples: Figure 2 shows the timing of blood draws. Five cc of blood will be drawn three times during the MR 
study. Samples will be kept on ice until the end of Session 2. They will then be processed and assayed for acetate and 
alcohol levels.   
 
Behavioral response to alcohol (SHAS7): After each blood draw, participants will be given the Subjective High 
Assessment Scale 7 (SHAS7) (Eng et al., 2005; Schuckit et al., 2000) which is an analogue scale evaluating 7 subjective 
feelings of intoxication. The SHAS7 includes three general items (Effects of Alcohol, Drunk, and High) and four 
specific aspects of the effects of alcohol (Clumsy, Confused, Dizzy, and Difficulty Concentrating). Items are rated on a 

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36-point Likert scale. The SHAS has been shown to have high reliability and validity and may be predictive of possible 
alcohol use disorders (Schuckit et al., 2000; Schuckit and Smith, 2000).  
 
Motor tasks to measure arousal and attention: After each scan, participants will perform three motor tasks, 
(psychomotor vigilance task, simple and choice reaction time task, and grooved pegboard task) to measure arousal and 
attention after placebo and drug.  These tasks will measure how alcohol and acetate affect arousal and attention.  
 
MR scan: Studies will be performed on the research-dedicated 3T MR system (Skyra, Siemens Healthcare, Erlangen, 
Germany) and 32-channel head coil at the Brain Imaging Center (Bldg. 400). An anatomic 3D T1-weighted image will 
be acquired for image registration and normalization. Pulsed arterial spin labeling (ASL) will be used to measure brain 
CBF. Blood oxygen level dependent (BOLD) functional MRI will be acquired to measure brain activity during the 
decision-making task.  
 
MR acquisition (Total MR time = 60 minutes): Linear and second order shimming will be performed to reduce static 
field inhomogeneities at the skull base prior to scans. 
1.  Anatomic: Structural high-resolution isotropic 3D T1-weighted image will be acquired for image registration and 
normalization with TR/TE/FA 45 ms/20ms/45°, 256x256 matrix, 240x240 mm2 field-of-view (0.9 x 0.9mm2 in-plane 
resolution). Scan time = 5 minutes 
2.  Pulsed arterial spin labeling (ASL) CBF images: 2D Pulsed arterial spin labeling (ASL) using: TR/TE/TI 
4500/10/1800 ms, 240 mm FOV, 128 x 128 matrix (1.8 x1.8 mm2 in-plane), 4 mm thick slices, sensitivity encoding 
(SENSE) factor= 2, and 60 pairs of interleaved control and tag images, axial plane. Scan time=~6 minutes 
3.  Blood oxygen level dependent (BOLD) fMRI: T2* weighted echo-planar imaging BOLD fMR images will be 
acquired with 3.4 x 3.4 x 3 mm3 voxel size, TR/TE/FA 2100/30/70. Images will be acquired to measure brain activity 
during the decision-making with which we have extensive experience (see below for details). Scan time=24 minutes 
 
Decision-making task (Fig 2): Participants will play a modified version of 
the Iowa Gambling Task (Bechara et al., 1994) during fMRI scanning. The 
modified IGT assesses risk avoidance and sensitivity to negative outcomes 
(Tanabe et al., 2013; Thompson et al., 2012). We have many years of 
experience giving the task to substance users and controls. Briefly, 
participants are presented four decks of cards and instructed to earn as much 
hypothetical money as possible by choosing to either Play or Pass on a given 
deck. “Play” results in a single positive or negative monetary value, along 
with the running total. “Pass” results in no change. To perform well, 
participants must learn to Pass on the two bad decks that result in a net loss 
and Play on the two good decks that result in net gain over time. The payout 
of the decks is controlled on the frequency and magnitude of wins and 
losses. The decision period is 2 seconds, and the outcome period is 4 seconds, separated by a variable jitter of 0 to 4 
seconds. There are 50 trials of each deck, 200 trials total, plus 40 baseline trials to control for motor effects, and 10 six-
second fixation crosses presented in pseudorandom order. Total task scan time is 24 minutes consisting of two 12 minute 
runs. The task will be given twice, once in the morning and once in the afternoon. To reduce practice effects for the 
afternoon study, the color, win/loss amounts (but not the ratio of wins:losses), and the order of the decks will be 
changed.  
 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
      Session 1 
1. Consent Procedures: Participants will have time to ask questions and have the study explained by PI. They will 
fill out an MRI screening form to ensure safety prior to entrance to the scanner.  
2. The session will be conducted in a private room to facilitate confidentiality. Data collected during the initial 
screening and between and after the MR sessions will be associated with the participant number only and will 
assess alcohol use (frequency, amount, and abuse/dependence symptoms). 
3. Participants will be paid $35 for completing Session 1. 
 
Session 2 
Figure 3. Decision-making task and 
timing parameters. 

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1. Participants will be asked to not drink alcohol for 48 hr prior to Session 2 and to eat a light low-fat breakfast on 
the day of Session 2 to avoid interference with alcohol absorption. 
2. Participants will need to arrange for a driver to and from the second session or we will provide a voucher for up 
to $50 for them to take a taxi to Session 2 and up to $50 for them to take a taxi home from Session 2. 
3. CTRC nurse or a physician co-investigator will start IV, monitor infusions, and perform blood draws. 
4. Participants will change into scrubs and provide a urine sample. Participants will be excluded from the MRI if 
positive for drugs, alcohol, or pregnancy.   
5. Participant will enter MR scanner. There will be two scans, each ~1 hour. The first scan will be from 9-10am, 
the second scan will be from 11:30am-12:30pm.     
6. Participants will either be in the acetate arm or in the alcohol arm of the study. 
7. Participants receiving alcohol will have BrAC samples measured at 30 min intervals until BAC is ≤0.02%, at 
which time they will be allowed to go home with a driver or will be given a voucher for up to $50 for a taxi 
(details in 2., above).  
8. Participants receiving acetate will be observed for 1 hr to give acetate time to metabolize and then will be 
allowed to go home with a driver or will be given a voucher for up to $50 for a taxi (details in 2., above). 
9. The participant will change back into clothes and will be paid $95 per scan. Thus they will be paid $190 for 
completing all scans 
      
  Follow-up 
1. Participant will receive a follow-up email or call from the PI on the evening of Session 2. 
 
This study involves  answering questionnaires, performing several motor tasks, having oral alcohol or an acetate 
infusion, and having a functional MRI of the brain. Breaches of confidentiality are a risk. It is necessary for participants 
to answer questions about private things like drug use, to rule out or diagnose substance dependence and other 
psychiatric conditions. Participants' names will be associated with a participant number to insure confidentiality. 
Behavioral data and imaging data will be stored using the participant number. All documents indicating the identity of 
participants will be kept in a locked room inside a locked file cabinet. Computer files include no identifiers except a 
code number, and will be password-protected. If the results of the study are published, data that might reveal the identity 
of any particular subject will be disguised. An MRI is a non-invasive routinely used procedure that poses minimal risk to 
participants.  
 
Potential Risks 
Risks of alcohol. Ethyl alcohol in the amount used in the current study presents minimal risk to the participants. The 
amount of alcohol they receive will be less than the legal limit and is expected to produce mild intoxication. There is a 
chance of gastrointestinal irritation, nausea, vomiting, light-headedness, diarrhea with alcohol. These symptoms are self-
limited and are expected to dissipate with time. Any subject who has never had alcohol in the past will be excluded. The 
PI will be with the participants for the length of the study until they leave. 
Risks of acetate. Sodium acetate presents minimal risk to the participants. It can cause eye and skin irritation in the 
crystal form and ingesting large amounts may cause gastrointestinal irritation. The acetate will be in solution, and thus 
not expected to be an irritant. There is a rare possibility that a reaction may occur during or immediately after the 
infusion of acetate characterized by flushing or redness of the skin which may resemble a rash. No subject will receive 
both acetate and alcohol as they will be randomized to one arm. An established protocol will be used for the acetate 
infusions (Jiang et al., 2013; Mason et al., 2006).The infusion rate for this protocol results in plasma acetate levels of 
~1.2mM, lower than another previous study (Akanji and Hockaday, 1990) that resulted in plasma acetate levels of 1.38 
mM for healthy participants and 1.65 for diabetics. One study reported a mild metabolic alkalosis which was well 
tolerated by healthy participants (Smith et al., 2007). Participants will be observed until about 2:00 pm, 1.5 hours after 
infusion, for any adverse effects. The PI will be with the participants for the length of the study until they leave. 
Venous Cannulation. Venous cannulation is a routine clinical procedure that carries minimal risks when performed by 
trained personnel. The major risk is pain and discomfort. It is possible bruising could occur in some participants. The 
risk of phlebitis or infection is very remote. 
Blood draws. Blood will be drawn through the venous access catheter. Fifteen cc of blood per person will be taken, 
which is a minimal amount that does not pose significant risk to the participants. 
Brain Imaging. The University of Colorado Anschutz Medical Campus research dedicated Brain Imaging Center (BIC) 
is run by full time trained technicians with extensive experience and training to conduct the proposed studies safely. In 
addition, all research personnel involved in this study are/will be trained in MR safety by the imaging center staff. The 
primary physical risk is the risk of injury from metal objects being drawn into the magnet. Additionally, some 

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participants may experience transient peripheral nerve stimulation or nausea in the bore of the magnet. Nausea can be 
minimized by stabilizing the head. The participant may experience discomfort due to noise or confined environment of 
the scanner (claustrophobia), or discomfort and frustration carrying out the tasks. 
Exposure to a high magnetic field. The only known hazard associated with exposure to a static high magnetic field is 
that the magnet exerts a strong force on ferromagnetic objects. For this reason, ferromagnetic objects are excluded from 
the vicinity of the magnet so that they will not become projectiles. At our Center for Functional MRI, the research 
systems for human use have field strengths of 3T. Imaging at these field strengths is not considered a significant risk 
according to FDA guidelines. The scanning sequences applied are within the FDA guidelines for human MR scanning. 
In addition, every subject undergoes extensive safety screenings to determine whether he/she has any implanted 
materials or braces and dental retainers that may pose a risk. This screening will be done once the subject is consented, 
and another time on each imaging study day. If there is any doubt about the nature of any implanted material, or any 
other contraindication to MR scanning, the subject will not be scanned. 
Heating from radiofrequency (RF) pulses. The RF pulses that are used for creating the MR signal deposit some energy in 
the body in the form of heat, but no ionizing radiation is used with MRI. For the same pulse sequence, the RF power 
deposited is higher at higher magnetic field strengths. However, the pulse sequences that will be used at 3T have 
relatively low power depositions. In the future, pulse sequences with higher RF power depositions may be developed, 
but it will be insured that the power deposited is always below the FDA guidelines. 
Peripheral nerve stimulation from rapidly switched magnetic fields (dB/dt). Magnetic field gradients are switched on and 
off during imaging to encode the spatial distribution of the MR signal. Gradient switching rate depends on the gradient 
coil used, but does not depend on field strength. For this reason, the gradient switching rates will be similar to those for 
the 1.5 T scanners we have used in the past, and these rates will not exceed FDA recommendations. The FDA guideline 
states that a significant risk is involved only when “dB/dt sufficient to produce severe discomfort or painful stimulation” 
is used. All of the fMRI studies performed in the past on our 3T system are well below this threshold. 
Acoustic noise. Acoustic noise is an unwanted side effect of MR imaging. As currents are pulsed through the gradient 
coils within the magnetic field, the system acts like a loudspeaker, making a repetitive tapping sound. In all of our 
studies, participants will wear ear plugs to reduce the noise to a comfortable, safe level. Additionally, head phones will 
be placed over the ears. The FDA guideline for a significant risk due to acoustic noise is “peak acoustic noise over 140 
dB”, and we will ensure that the acoustic levels remain well below this value. 
Potential risk of loss of confidentiality. Since this study includes drug and alcohol screening, and pregnancy test 
assessment, there is the potential that this information may not be kept confidential (for instance by theft of study 
material). The investigator team will make every effort to keep all information confidential. All study material will be 
stored in locked cabinets in the Research-2 Building, University of Colorado Anschutz Medical Campus sponsored 
facilities. Furthermore, a unique study number will be used for each person in data sets and spreadsheets that do not 
readily identify a name. The identifying name information containing material will be locked. 
Psychological risk: These studies do not pose any significant psychological risk to the patients. If patients are anxious 
they can be trained in the mock scanner located adjacent to the MR scanner to mitigate anxiety and help the subject 
adjust to the scanning environment.  
Social Risk. There is no social risk associated with participation in this study; all results will be kept strictly confidential 
and will not be used to influence clinical decision making. There are no other known risks associated with participation 
in this study. 
 
 
E.   Potential Scientific Problems:   
 
1. Sex effects: Alcohol metabolism differs in men and women and several studies find an interaction between sex and 
alcohol’s effect on terms of brain morphometry (Hommer et al., 2001), function, and CBF (Marxen et al., 2014; 
Rickenbacher et al., 2011). It should be noted, however, that not all studies have confirmed sex differences in brain 
morphometry (Demirakca et al., 2011). There will not be power to detect sex effects. Our goal is to demonstrate 
feasibility. 
2. Vasomotor effect: The BOLD signal reflects complex interactions between CBF, cerebral blood volume, and 
oxygenation. We plan to study differences in global and regional vascular effects in acetate vs. alcohol treatment. The 
fMRI BOLD measurements are insensitive to time lags or shifts in the hemodynamic response of the brain during 
decision-making. Therefore in exploratory studies a finite impulse response (FIR) model will be used, according to prior 
work (Yamamoto et al., 2014).  

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3. Diurnal variations: We cannot exclude the possibility of diurnal fluctuations confounding our findings. The alternative 
to conduct blinded placebo and treatment studies on two separate days in the same participant would be a hardship on 
participants.  
4. Dose-response: A dose-response for acetate or alcohol will not be obtained. Fortunately, the pharmacokinetics of 
alcohol are well established and the timing of the MR scans is planned according to Figure 2. The pharmacokinetics of 
acetate are not as well established. The IV infusion protocol will give us better control of plasma levels, and serum 
acetate and alcohol will be assayed. 
 
F.   Data Analysis Plan:   
 
Blood alcohol and acetate: Levels of blood alcohol and acetate will be analyzed using colorimetric enzyme-linked 
immunosorbent assays (ELISA). 
   
MR measures: MR CBF and fMRI BOLD activity data will be processed in SPM12 (The FIL Methods Group, 2014). 
First, each participants’ T1-weighted anatomic image will be skull-stripped then coregistered to the Montreal 
Neurological Institute (MNI) MNI152 template. The transformation matrix generated from this step will be applied to 
CBF and BOLD contrast maps of interest. 
MR CBF: Motion-corrected ASL CBF images (mL/100g/min) will be generated by the scanner software. CBF maps will 
be skull-stripped and co-registered to each participant’s T1-weighted anatomic image in native space followed by 
application of the transformation matrix to MNI space. 
BOLD fMRI pre-processing: After discarding the first 4 volumes for saturation effects, images will be corrected for 
movement with a 3-dimensional rigid body transformation. Participants with scan-to-scan head movement >2 mm will 
be excluded. Motion parameters will be modeled as nuisance variables. Images will be normalized to MNI space using 
the two step transformation procedure described for MR CBF. Images will be spatially smoothed with a 6 mm full-
width-half-maximum isotropic Gaussian kernel, filtered to remove low-frequency fluctuations (128 s high pass filter), 
and corrected for temporal autocorrelation. 
BOLD fMRI: Model specification and contrast of interest: The time series will be predicted by nine regressors, 
consisting of decision and outcome for each of the four decks, plus motor control. Motion parameters will be modeled as 
nuisance regressors. The stimulus function will be convolved with the canonical hemodynamic response function (HRF). 
We are primarily interested in effects of acetate or alcohol on brain activity during the decision phase of the task. The 
primary outcomes will be the contrast of Decision>motor control baseline and Outcome>motor control baseline. For 
each participant these contrast maps will be brought to the second level for subsequent ROI and whole-brain analyses. 
 
Regions-of-interest (ROI): ROI and whole brain analyses will be conducted for outcome 
measures of CBF and fMRI BOLD activity. Six anatomically- defined ROIs will be based 
on the neural circuitry found to be involved during decision-making on the modified IGT 
and which overlap with brain regions showing altered signal after acute alcohol (Anderson 
et al., 2011; Bjork and Gilman, 2014). ROIs will be bilateral ventral striatum (VST), dorsal 
striatum, insula, and anterior cingulate cortex (ACC), orbitofrontal cortex (OFC), and 
dorsolateral prefrontal cortex (DLPFC). VST and DST will be manually drawn, based on 
published landmarks (Mawlawi et al., 2001). For the OFC, insula, ACC, and DLPFC, the automated anatomical labeling 
atlas (AAL) (Tzourio-Mazoyer et al., 2002) will be used. Figure 3 shows examples of VST=green, DST=orange, 
OFC=blue, insula=red. 
 
 
Statistical Analysis: We have three outcomes of interest: 1) CBF, 2) fMRI BOLD signal during decision-making, 3) 
behavioral measures. A bio-statistician employed part-time by the Department of Radiology will provide statistical 
support. The aim of this pilot study is simply to determine the feasibility of performing the experiment and to acquire the 
necessary data to calculate power needed for a larger study. 
 
CBF: ROI analyses: For this feasibility pilot study, a paired t-test will be used to investigate the effect of acetate and the 
effect of alcohol separately. In an exploratory manner, drug x time interactions will be examined through descriptive and 
graphical analyses, though we acknowledge insufficient power to reject the null hypothesis of no interaction. 
Nevertheless, a noticeable difference in outcomes based on factors of drug or time could provide data to compute power 
for a larger, more powered study.  
 
Fig 3. Regions of interest 

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To provide context for this pilot, the statistical plan for the later, larger study is included: We will fit a general linear 
multivariate model with repeated measurements of CBF as the outcomes. Each participant will have 12 repeated 
measurements (6 ROIs x 2 treatments (acetate and placebo or alcohol and placebo)). Age and number of drinks per week 
will be included as covariates. MCMC imputation (Li, 1988) will be used to address missing data. A Chi-Muller test will 
be conducted (Chi and Muller, 2013) for the main effect of treatment (acetate versus placebo or alcohol versus placebo) 
at any ROI. Residual analysis will be used, and Box-Cox transformations will be considered to account for deviations 
from multivariate normality. An alpha spending approach will be used to handle multiple comparisons problems, by 
testing the overall hypothesis of no effect at any region at the alpha = 0.04 level, and then, if we attain significance, 
testing the treatment effects at each ROI at the alpha =0.01/6 level. This yields a total Type I error for this aim of 0.05 
=0.04 + 6*(0.01/6).  
 
CBF: Whole brain analyses: The main effects of acetate on CBF will be analyzed in SPM12 using a repeated-measures 
ANCOVA with drug (alcohol, or acetate vs. placebo) as within-subject factor and adjusting for age and number of 
drinks per month. As this is a feasibility study, a less stringent threshold of p<0.05, uncorrected will be used to explore 
for possible main effects and interaction effects as well as nonparametric approaches. In the later, larger study, 
significance levels will be set at p<0.05, corrected for multiple comparisons with family-wise error (FWE) using 
AlphaSim Monte Carlo simulations, 10,000 iterations, to determine cluster size criterion. The voxel-level significance 
will be set at p<0.005.  
 
fMRI BOLD activity: ROI analyses: For each ROI and each subject, the percent signal change will be extracted using 
Marsbar toolbox. The feasibility analyses are the same as CBF: ROI approach. To provide context for this pilot, the 
statistical plan for the later, larger study is included: To examine the main effect of alcohol or acetate on BOLD 
response, a general linear multivariate model with 12 repeated BOLD response measurements as the outcomes will be 
employed. Covariates will include education level and number of drinks per week. From prior experience, BOLD 
activity during decision-making correlates with education level. Analyses are otherwise the same as CBF: ROI 
approach.  
 
fMRI BOLD activity whole brain analyses: The feasibility analyses are the same as CBF: whole brain analyses. In the 
later, larger study, the main effects of alcohol or acetate on fMRI BOLD signal during decision- making will be 
analyzed in SPM12 using a repeated-measures ANCOVA with drug (alcohol or acetate vs. placebo) as within-subject 
factor and adjusting for education and number of drinks per month. Analyses are otherwise the same as for the CBF: 
whole brain analyses.  
 
Behavioral: Decision-making performance will be analyzed with repeated measures ANOVA on net score, adjusted for 
education level. Net score is the number of plays on good decks minus bad decks. The behavioral effects of treatment 
will be analyzed using repeated measures ANOVA on SHAS7, on the psychomotor vigilance task, simple and choice 
reaction time task, and grooved pegboard task. 
 
 
Power and Confidence Interval Width:  This study is a pilot study, designed to estimate the size of the effect, and 
provide accurate parameter estimates for a later, more definitive study.  The power analysis here is based on the work of 
Schwartz et al. (Schwartz et al., 1993), who studied CBF before and after drinking and observed a mean difference in 
flow measured as a ratio of cortex/cerebellum (after – before ) of  0.054 with standard deviation of the difference of 
0.085. The smallest difference in CBF that a study with 10 people can detect using repeated measures ANCOVA with 
90% power and a Type 1 error of 0.05 is a difference of 0.09 (ratio of cortex/cerebellum).  Therefore we should have 
adequate power to detect a difference with alcohol. There is no prior CBF data on acetate in the literature to perform 
power analysis for acetate.  This study is designed to provide preliminary data for a later study and an estimation of the 
N necessary for that study to provide sufficient power for analysis.  
 
G.  Summarize Knowledge to be Gained:   
 
This proposal is relevant to public health because of the considerable impact alcohol has on the health of individuals and 
in terms of legal, medical, and social costs to the country. Again, this is a pilot study to determine the feasibility of 
measuring effects of oral alcohol and intravenous acetate on CBF and function in moderate drinkers and AUD 
individuals. Data from this study will be used to apply for funds to conduct a larger study. The results from this the later, 

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larger project will add to our knowledge about mechanisms of acute alcohol’s effects on brain function and the role of 
acetate in these effects.  
 
 
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