Alcohol Abuse, Oxidative Stress, and Zinc Deficiency in Lung Disease
Phase: NA
Conditions: Alcoholism
Sponsor: VA Office of Research and Development

 
 
 
Examination of Zinc, S-adenosylmethionine, and Combination Therapy vs. 
Placebo in Otherwise Healthy Alcoholic Patients and its Effect on the Alcoholic 
Lung Phenotype 
(ExZACTO Study) 
 
 
 
Protocol Version 2.3 
 August 21, 2017 
Principal Investigator 
Ashish Mehta, MD 
 
Co-Investigators 
David Guidot, MD 
Sushma Cribbs, MD 
Samantha Yeligar, PhD 
 
 
 

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1. Introduction: Clinical Background 
 
Alcohol abuse places a tremendous burden on society. According to the 2008 National 
Survey on Drug Use and Health, more than 50 percent of the adult population in the United 
States consumes alcohol, which would roughly translate into more than 125 million people.  In 
this same survey, almost 7 percent reported heavy drinking.1  Data from the 2001 National 
Epidemiologic Survey on Alcohol and Related Conditions reported that the lifetime prevalence 
of alcohol abuse was about 18 percent, making alcohol the most widely used and abused among 
all drugs.2  While average alcohol intake has decreased over time, more recent data suggest that 
the incidence of alcohol use disorders has not changed.3  Importantly, studies attribute more 
than 100,000 fatalities annually to alcohol consumption.4  Even among otherwise “healthy” 
individuals, alcohol abuse causes significant morbidity and dysfunction of many organ systems 
including the heart, liver, and skeletal muscle.5-8  In the past decade experimental evidence has 
emerged that elucidates the profound effects of chronic alcohol ingestion on the lung and how 
it creates a previously unrecognized ‘alcoholic lung’ phenotype that renders the individual 
susceptible to pulmonary infection and injury. 
 
Alcohol exerts a significant suppressive effect on the immune system, which leads to a 
vulnerability to a wide array of infections, especially pneumonia.  Studies in various organ 
systems have consistently demonstrated that alcohol abuse impairs the clearance of infectious 
organisms9 resulting in 1) an increased risk for community-acquired and nosocomial pneumonia, 
2) higher hospitalization-associated charges, 3) higher intensive care unit use and longer 
inpatient stays10, and 4) higher mortality rates.11  Alcohol abuse has a longstanding link with 
pulmonary infections, and in a recent study of patients admitted to a general hospital with 
pneumonia, 25-50% of such individuals were found to have a concomitant diagnosis of 
alcoholism.12, 13  Moreover, these alcoholic patients were more likely to be infected with serious 
Gram-negative bacteria such as Klebsiella pneumoniae.14  Despite this longstanding awareness, 
a detailed understanding of the mechanisms responsible for predisposing alcoholics to lung 
infections is lacking and may be the key to uncovering new intervention strategies that could 
reduce morbidity and mortality in these vulnerable subjects. 
 
The alveolar macrophage plays a key role in protecting the individual from developing 
pneumonia. The increased risk of pneumonia in the alcoholic host involves at least three 
important factors: 1) colonization of the oropharynx with pathogenic bacteria, which occurs in 
approximately one-third of alcoholics; 2) increased frequency of aspiration as a result of 
depressed level of consciousness and diminished gag and cough reflexes; and 3) impaired 
integrity of the host immune system.15  A crucial element of host defense in the lung is the 
alveolar macrophage. The lung is constantly exposed to the outside world, and the first cellular 
line of defense in protecting the alveolar space from invading pathogens is the resident 
macrophage. It is the primary phagocyte on the gas exchange surface of the lung and normally 
has the capacity to efficiently ingest and destroy inhaled pathogens. However, clinical and 
laboratory studies have identified that alveolar macrophages are impaired by chronic alcohol 

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ingestion. For example, we determined that alveolar macrophages isolated from rats on a 
chronic alcohol diet have significantly impaired immune function and are poorly equipped to 
phagocytose bacteria.16  Therefore, alveolar macrophage immune dysfunction is clearly 
implicated in the link between chronic alcohol consumption and the increased risk of pulmonary 
infections. 
 
Oxidative stress is a fundamental feature of the alcoholic lung phenotype and interferes with 
the function of numerous cell types including the alveolar macrophage. Our research group has 
identified in clinical and animal studies that chronic alcohol ingestion depletes the alveolar lining 
fluid levels of glutathione (GSH), a critical antioxidant, by ~80% and oxidizes the redox potential 
by 35-50 mV.17-19  Further investigations determined that alcohol-induced oxidant stress 
impaired alveolar macrophage terminal differentiation and plasma membrane expression of the 
receptors necessary to drive phagocytosis and the respiratory burst.16, 19-21  More importantly, 
oral GSH precursors attenuated the alcohol-induced oxidant stress and improved AM immune 
functions in animal models.19, 22  These findings suggest that dietary supplements that restore 
GSH availability within the alveolar space could improve alveolar macrophage immune function 
in alcoholics, although the potential benefit of this therapeutic modality has yet to be tested in 
clinical studies. 
 
The essential micronutrient zinc is crucial for normal immune function, and it is deficient in 
alcoholics. A key participant in normal host immune response is zinc, a trace element that is 
critical for normal protein metabolism, the function of more than 300 zinc metalloenzymes, and 
for membrane integrity.23-25  Alcoholics are prone to numerous nutritional deficiencies as the 
result of poor dietary intake, and zinc deficiency has long been recognized in this context. Zinc 
deficiency in the healthy, non-alcoholic population is uncommon in the United States, but 
contributes to a significant burden of disease in other parts of the world such as sub-Saharan 
Africa and Southeast Asia where dietary intake is severely limited26 and where children in 
particular are at high risk of pneumonia.27  Consistent with this association, there is evolving 
evidence that zinc is critical to airway health.23, 25, 28, 29  Importantly, even though there has been 
considerable attention to examining how alcohol-related zinc deficiency affects the liver, the 
important role of zinc deficiency in mediating the alcoholic lung phenotype has only been 
recently recognized in our animal studies.30, 31  Furthermore, we have preliminary data showing 
that human alcoholics show evidence of zinc deficiency in the alveolar space, and the next 
logical step would be to explore the potential role of zinc therapy in these patients. 
 
Despite the overwhelming evidence for zinc deficiency and oxidative stress in the alcoholic 
population, there are no trials evaluating the lung health effects of either dietary zinc or 
antioxidant supplementation in humans who abuse alcohol. If either of these strategies is able 
to reverse the immune dysfunction seen in the setting of chronic alcohol consumption, it offers 
a potentially simple therapeutic option for this high risk population. 
 
 

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2. Objectives 
 
2.1 Primary Aim 
 
ExZACTO is a four-arm (1:1:1:1), factorial design, placebo-controlled, double-blind 
randomized clinical trial.  The study is a clinical intervention trial that will determine the 
impact of both dietary zinc supplementation and dietary S-adenosylmethionine (SAMe) 
supplementation, individually and in combination, on alveolar macrophage immune 
function, intracellular zinc levels in the alveolar macrophage, and overall redox potential of 
the epithelial lining fluid in the lung. 
 
The primary null hypothesis is that there will be no difference in alveolar macrophage 
immune function with zinc treatment, SAMe treatment, or combination treatment when 
compared to placebo in patients with an alcohol use disorder.  This will be tested against the 
alternative hypothesis of a non-zero difference between zinc treatment, SAMe treatment, 
and combination treatment versus placebo.  The testing will be done at α < 0.05, two-sided, 
and 80% power to detect a 30% difference between treatment and placebo.  Based on our 
calculations, a total of 100 patients will need to be enrolled (25 in each of the four arms) to 
ensure adequate statistical power to detect this difference. 
 
2.2 Secondary Aims 
 
The primary aim is to determine if there improvement in phagocytic index with zinc 
treatment, SAMe treatment, and combination treatment.  An improvement in immune 
function would signify a decrease in vulnerability to infection.  There are other markers of 
the alcoholic lung phenotype, and these will be used as secondary aims.   Specifically, we 
will examine the effect of zinc, SAMe, and combination treatment on alveolar macrophage 
intracellular zinc levels, redox potential, and GM-CSF receptor expression.  The null 
hypothesis in each of these aims is that zinc, SAMe, and combination treatment will have no 
effect on the above outcomes, with the alternative hypotheses being that treatment will 
improve intracellular zinc levels, redox potential in the lung, and GM-CSF receptor 
expression in the alveolar macrophage. 
 
3. Trial Design 
 
3.1 Basic Design 
 
ExZACTO is a single center, double-blind, placebo-controlled, randomized clinical trial to 
determine the effect of zinc supplementation and SAMe supplementation (both alone and 
in combination) on alveolar macrophage immune function, redox stress, alveolar 
macrophage GM-CSF reception expression, and intracellular zinc status in outpatient 

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alcoholic subjects.  We will recruit a cohort of otherwise healthy patients with an alcohol 
use disorder from the Substance Abuse Treatment Program at the Atlanta VA Medical 
Center and randomize them to one of four treatment groups: 1) usual treatment plus 
placebo for two weeks; 2) usual treatment plus dietary zinc supplementation for two weeks; 
3) usual treatment plus dietary SAMe supplementation for two weeks; and 4) usual 
treatment plus dietary zinc and SAMe supplementation for two weeks.  The treatment 
allocation of these studies will be blinded to the patients and investigators.  In all, each 
participant will receive a total of two different pills: 1) zinc placebo and SAMe placebo; 2) 
active zinc and SAMe placebo; 3) active SAMe and zinc placebo; or 4) active zinc and active 
SAMe.  The placebo pills will appear identical to their active drug counterpart.  A simple 
schematic of the study design is depicted below.  While the goal of this study to evaluate the 
effect of two weeks of treatment, in certain instances subjects may receive treatment for 
longer than two weeks depending scheduling availability for the repeat bronchoscopy.  All 
subjects will remain on treatment from the time of enrollment to the time of repeat 
bronchoscopy.   
 
 

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3.1.1 
Power and sample size calculations 
 
The primary outcome for this clinical trial will be the phagocytic index of the alveolar 
macrophage before and after at least 14-days of treatment. Phagocytic index is the 
most relevant primary outcome as it is a well-established marker of immune 
function and its improvement with either zinc treatment or SAMe treatment would 
indicate reversal of the alcoholic lung phenotype. Secondary outcomes will include 
change in GM-CSF receptor expression in the alveolar macrophage, change in 
alveolar macrophage zinc levels (signifying improvement of alveolar zinc deficiency), 
and change in redox potential [as measured by ratios of the redox couples 
glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cysteine (Cys/Cyss) in 
the BAL and EBC fluid]. 
 
For the power calculations, we used data from previous animal studies, in which the 
estimated standard deviation for change in phagocytic capacity is between 0.2 
(20%) and 0.40 (40%) depending on baseline values versus follow-up values, 
respectively. We assume a correlation coefficient of 0.85 between baseline and 14-
day follow-up values within a treatment group.  Given inherent variability in human 
data compared to animal data, our assumption will be that the separation of groups 
will not be as robust.  Therefore, we took these predictions, as well as our some of 
our preliminary data in human subjects, into consideration when calculating sample 
size requirements.  Assuming that we will detect on average a 30% increase in 
treated (zinc and/or SAMe supplemented) subjects and on average no change in the 
untreated subjects, a sample size of 19 alcoholics in each group will ensure 
statistical power exceeding 80% to detect a treatment difference of 30% at the 2-
sided 5% significance level if the true difference between groups is 30%. However, 
in order to account for limitations including dropout and inadequacy of sampling, 
we will use a sample size of 25 subjects in each group (dietary zinc, dietary SAMe, 
and placebo) for a total of 100 subjects randomized for the entire study. This 
additional recruitment will ensure adequate statistical power is maintained at the 
completion of the study. 
 
3.1.2 
Interim analyses  
 
There are no interim analyses planned as there would be no grounds for stopping 
the study early for therapeutic benefit.  The DSMB will continually monitor the study 
for any adverse events and will make any determinations for early termination in 
the unlikely occurrence of harm. 
 
3.1.3 
Test statistic for the primary null hypothesis 
 

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The primary null hypothesis will be tested in an intent-to-treat analysis using ANOVA 
to compare in phagocytic function across the four treatment groups. 
 
3.2 Study Population 
 
3.2.1 
Inclusion criteria 
 
1. Age 18-60 years 
2. Active alcohol use disorder (with last alcoholic drink within 8 days of 
randomization) 
 
3.2.2 
Exclusion criteria 
 
1. Any active and uncontrolled medical problem(s) 
2. Known zinc deficiency 
3. Primary substance of abuse something other than alcohol 
4. Current abnormal chest x-ray 
5. HIV-positive 
6. Any disorder of blood coagulation 
7. Currently on medical treatment with anti-coagulants (including warfarin, 
heparin, direct thrombin inhibitors, or anti-platelet agents) 
8. Daily use of vitamins or other nutritional supplements (Clarification: patients 
who are prescribed vitamins as part of their treatment for alcohol abuse will not 
be excluded and will be allowed to continue their prescribed vitamins 
throughout the study. Only patients on daily vitamin supplements prior to 
screening will be excluded.) 
9. Renal impairment (GFR < 60) 
10. Active bipolar disorder 
11. Active Parkinson’s disease 
12. Current pregnancy 
13. Contraindication to treatment with zinc or S-adenosylmethionine 
14. Inability to give informed consent (i.e., limited cognitive capacity) 
15. Non-English speaking 
 
3.3 Randomization 
 
A statistician who is otherwise uninvolved with this study will provide a randomization list, 
which will be prepared using a computer-based pseudo-random number generator.  Our 
scheme will be permuted-block randomization with a block size of 8 and an allocation ratio 
of2:2:2:2.  Based on pilot information, we expect a minority (10-15%) of participants to be 
non-smokers.   To ensure equivalent representation of smoking and non-smoking subjects in 
each group, we will stratify randomization on current smoking status.  After the patient is 

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enrolled in the study, but prior to randomization, he/she will undergo an initial 
bronchoscopy procedure.  The randomization will occur only after this initial bronchoscopy 
procedure.  The patient will be assigned to one of four treatment groups based on the 
randomization list. 
  
3.4 Blinding 
 
Treatment assignment will be masked to investigator, analyzing statistician, study staff, and 
subject.  Only the dispensing pharmacist and the statistician who provides the 
randomization list will be aware of the treatment assignment. 
 
4. Drugs and Dosages 
 
4.1 Study Medications 
 
There are a total of four treatment groups, and each randomized patient receives two types 
of pills. The first medication is either a tablet containing 50 mg of elemental zinc (zinc 
sulfate), or an identical appearing placebo tablet.  The second medication is either 400 mg S-
adenosylmethionine (SAMe), or an identical appearing placebo tablet.  Patients assigned to 
the combination treatment arm will receive both active medications (i.e., zinc and SAM), 
and those assigned to the placebo arm will receive placebo tablets only.  The remaining two 
groups will receive one placebo and one active medication (i.e., either zinc or SAMe). 
 
4.2 Medication Administration 
 
4.2.1 
Zinc sulfate 
 
A zinc sulfate tablet (containing 50 mg elemental zinc) or identically appearing 
placebo will be taken once daily for a total of two weeks.  This medication can be 
taken at the same time as the other study medication below (i.e., SAMe).  After 
randomization, subjects will receive 14+ tablets of active zinc or placebo, which is 
enough to complete the minimum 14-day course of therapy until the repeat 
bronchoscopy procedure can be performed. 
 
4.2.2 
S-Adenosylmethionine 
 
A 400 mg tablet of s-adenosylmethionine (SAMe) or identically appearing placebo 
will be taken twice daily for a total of two weeks.  This medication can be taken at 
the same time as the other study medication.  After randomization, subjects will 
receive 28+ tablets of active SAMe or placebo, which is enough to complete the 
minimum 14-day course of therapy until the repeat bronchoscopy procedure can be 
performed. 

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4.3 Storage and Handling of Medications 
 
The trial medications will be stored in a locked area with limited access.  The investigational 
drug services at the VA will be responsible for maintaining an inventory of study medication. 
The study coordinator and investigators will distribute the medications after the pharmacist 
dispenses it.  In the event of any emergency, the investigational drug services at the VA 
(including the dispensing pharmacist) will be able to break the blind.  The study medication 
will not be used for any purpose other than this study. 
 
4.4 Concomitant Medications  
 
In general, the supplements used in this study are safe, but there may be a potential for 
some drug interactions.  First, zinc salts may alter the absorption of some antibiotics, and 
this caution will be communicated with the participants.  A full review of patient medication 
will be done prior to the study.  The subjects in this study will be otherwise healthy, so we 
expect there to be minimal use of other medications.  We will also advise patients not to 
take any other nutritional supplements during the time that they are in this trial as not to 
complicate the study.   Second, SAMe is also a safe, readily available nutritional supplement, 
but can cause problems for patients with bipolar disorder and Parkinson’s disease.  Such 
subjects will be excluded from the study.   
 
5. Trial Procedures 
 
5.1 Recruitment and Pre-Screening 
Study recruitment will primarily occur at the Atlanta VA Substance Abuse Treatment 
Program (SATP) clinic. IRB-approved recruitment posters will be displayed, and study 
brochures will be utilized. Interested patients will be pre-screened either by telephone 
evaluation or face-to-face during clinic visit with SATP provider.  The purpose of the pre-
screening is to identify patient who meet inclusion criteria and do not have any exclusions 
(listed in section 3.2).  IRB-approved patient recruitment letters may also be utilized. 
 
Recruitment will also occur in other parts of the VA hospital. Study brochures will be placed 
in VA clinics and other patient care areas. Interested patients may self-refer for the study by 
calling the study staff directly. 
 
5.2 Baseline Visit 
 
At (or prior to) the baseline visit, the study investigators in conjunction with the study 
coordinator will: 
- 
Recheck to ensure that the patient meets all inclusion criteria and none of the exclusion 
criteria for the study (listed in section 3.2). 

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- 
Obtain informed consent: 
o Written informed consent will be obtained from each patient prior to 
performing any baseline evaluations.  In certain instances the coordinator may 
ask screening questions to determine eligibility for the study, but no actual 
evaluation will take place prior to obtaining informed consent.  A copy of the 
signed informed consent is given to the patient, and the study coordinator 
retains the original and has it sent to medical records for scanning. The 
informed consent document will be reviewed and approved by the Institutional 
Review Board as well as the Atlanta VA Medical Center Research and 
Development Committee prior to the initiation of the trial. 
 
- 
Collect baseline demographic information and medical history. 
- 
Obtain AUDIT and SMAST questionnaires on participants to grade alcohol consumption 
(see appendix 2 and 3). 
- 
Obtain Drinking History Questionnaire, Alcohol Dependence Scale (ADS), Food 
Frequency Questionnaire (FFQ), and Timeline Followback Form-90 to better understand 
the drinking pattern. (see appendix 4-8; appendix 7 is provided to participants to define 
“standard drinks”) 
- 
Perform a physical examination. 
- 
Order a chest x-ray (if one is not available from the past one year prior to enrollment). 
- 
Collect urine for pregnancy testing (in women of childbearing age). 
- 
Compensate the subject for the baseline visit with cash. 
In certain instances, the patient will also undergo the bronchoscopy procedure described 
below on the same day as the baseline visit, but this would need to be scheduled in advance 
as the patient is required to not eat or drink anything for a minimum of 6 hours prior to the 
procedure.  The actual randomization into treatment group will occur only after the initial 
bronchoscopy is done since the treatment follows the initial procedure.  A schematic of the 
schedule of assessments is shown in appendix 1. 
5.3 Bronchoscopy Procedure 
 
5.3.1 
Technical Procedure 
 
- 
Study participants must not eat or drink anything for a minimum of 6 hours prior to the 
procedure. Prior to each bronchoscopy, blood will be collected for serum zinc, blood 
alcohol, biomarker testing, and measurement of redox stress. Urine will be collected for 
the presence of recreational drugs and ethyl glucuronide. Exhaled breath condensate 
will also be collected (described in section 5.4). 
Patients will undergo a flexible fiberoptic bronchoscopy with standardized 
bronchoalveolar lavage (BAL) technique (180 ml of isotonic saline in a sub-segment 

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of the right middle lobe or lingula) using standard conscious sedation techniques.  
Lidocaine will be used for topical anesthesia.  To ensure safety, participants will be 
closely monitored during the procedure per the routine protocol in the hospital 
bronchoscopy suite.  Bronchoscopy is a common procedure performed at the 
Atlanta VA Medical Center for clinical and research purposes with a published 
complication rate of less than 0.5% and 0% mortality rate.33  The bronchoscopy will 
be supervised by one of the study investigators or an attending physician with 
credentials to perform the procedure.  Subjects will be monitored for up to 4 hours 
after completion of the procedure per standard conscious sedation recovery 
protocol until fully recovered.  An optional “no sedation” bronchoscopy will be 
considered when requested by the patient. The usual post-procedure recovery 
period would not be required for cases done without sedation. 
For the initial bronchoscopy, subjects will be randomized after the procedure and 
given the study medication prior to discharge.  Study volunteers will be 
compensated with $25 cash for each study visit and either a check or direct deposit 
after each bronchoscopy procedure.  In addition, the study team will offer a $5 VA 
canteen voucher to the volunteer’s driver (the friend or family member who must 
accompany the volunteer to the procedure). 
 
5.3.2 
Measurements 
 
BAL fluid will be processed and a standard cell count and leukocyte differential will 
be determined.  BAL fluid will be separated into cell component (i.e., alveolar 
macrophage isolation) for assays of zinc, GM-CSF receptor expression, and function 
(phagocytic index).   The supernatant will be aliquoted and frozen at -70C for assays 
of Glutathione (GSH/GSSG), Cysteine (Cys/CySS).   All experiments performed are 
common research protocols within our Alcohol research group.  The excess cells and 
BAL fluid, if any, will be frozen for future analysis.  After all enrollment and analyses 
are completed and all record-keeping requirements expire, all collected samples will 
be discarded. 
 
 
5.4 Exhaled Breath Condensate Collection 
 
5.4.1 
Technical procedure 
 
After the participant has abstained from food and beverages for at least one hour, 
they will rinse their mouth with distilled or de-ionized water. Participants will be 
seated upright, and breathe normally in and out through the mouthpiece. The 
mouthpiece is connected to an exhaled breath condensate (EBC) collection tube, 
called the R-tube, which consists of a plastic tube placed inside a chilled cooling 
sleeve. The total collection period, with the patient breathing normally through the 

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mouthpiece, is approximately 10–15 minutes.  After the collection session, an 
evacuation plunger is utilized to collect the sample and a pipette is used to aliquot 
samples into 1 ml tubes. The samples are stored at –70° C until analyzed. 
 
5.4.2 
Measurements 
 
The EBC samples will be analyzed for oxidative stress through the measurement of 
redox pairs of Glutathione (GSH/GSSG) and Cysteine (Cys/CySS).  These particular 
measurements are feasible and have previously been done in similar studies of 
alcoholic patients.32 
 
 
5.5 Follow-up Visits 
 
Patients will have up to two follow-up visits during the two weeks that they are randomized 
to receive a study medication.  These follow-up visits will occur prior to the second 
bronchoscopy procedure.  In general, the first follow-up visit will occur sometime during the 
first week of therapy, and the second follow-up visit will occur sometime during the second 
week of therapy. At the discretion of the PI, the second follow-up visit may be performed on 
the day of the second bronchoscopy procedure.  During the follow-up visit(s), the study 
coordinator will: 
- 
Review the subject’s compliance with study medication and assess alcohol use.  
- 
Review the subject’s alcohol intake and use of any other over-the-counter dietary or 
nutritional supplements. 
- 
Review any adverse events or side effects from using study medications. 
- 
Collect blood for alcohol level testing and biomarker testing (Follow-up Visit #1 only). 
- 
Collect exhaled breath condensate using the procedure described above (Follow-up Visit 
#1 only). 
- 
Compensate the subject for the visit with cash. 
 
5.6 Second Bronchoscopy Procedure 
 
- 
After at least two weeks of therapy with study medication (or placebo), the participants 
will undergo a repeat bronchoscopy procedure using the same method as described in 
section 5.3.1. Prior to the bronchoscopy, blood will be collected for serum zinc, blood 
alcohol, biomarker testing, and measurement of redox stress. Urine will be collected for 
ethyl glucuronide. Exhaled breath condensate will also be collected (described in section 
5.4). 
   
Subjects will again be compensated for undergoing the procedure, and the volunteer’s 
driver will be offered a $5 VA canteen voucher.  No further clinical follow-up will be 

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performed after the second procedure, but the patient will receive a phone call within the 
next week to ensure no issues arise after completion of the study requirements. 
 
6. Adverse Events and Serious Adverse Events 
 
6.1 Definition of Adverse Event 
 
An adverse event (AE) is any noxious, pathologic, or unintended change in anatomical, 
physiologic, or metabolic function, as indicated by physical signs, symptoms, or laboratory 
changes occurring in any phase of the clinical trial, whether associated with study 
medications or placebo, and whether or not considered related to the study medications. 
 
Adverse events include, but are not limited to: 
1. Worsening (change in nature, severity, or frequency) of conditions present at the start 
of the trial. 
2. Acute illness occurring during the trial. 
3. Drug reactions. 
4. Experiences related or possibly related to concomitant medications. 
5. Clinically significant abnormalities in physical examination, vital signs, or laboratory 
values (examination of laboratory values will not be done on a routine basis during 
study visits, but may be performed if any concern for adverse event arises). 
 
6.2 Serious Adverse Events 
 
In general, serious adverse events are not expected with the use of these over the counter 
dietary supplements.  However, if a serious adverse event does occur, the Principal 
Investigator report all serious adverse events (SAEs) that are unexpected (not listed in the 
most up-to-date Physician’s Desk Reference) and potentially related to the study medication 
to the DSMB within two to three business days after becoming  aware of the event. The 
study investigators will also conform to the AE and SAE reporting requirements of the IRB 
and the Research and Development Committee of the Atlanta VA.   
 
6.3 Describing Adverse Events and Serious Adverse Events 
 
The Principal Investigator will make a determination on reporting the relationship of the 
adverse event to study medication as one of the following: 
- 
None:  Unrelated to study medication 
- 
Possible:  Possible association with study medication is known 
- 
Probable:  Study medication has a known association with this type of event, and the 
event in question is probably due to the medication 
- 
Definite:  Study medication has a known association with this type of event, and the 
event in question is definitely due to the medication 

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The Principal Investigator will also make a determination of the severity of the adverse 
event as one of the following: 
- 
Mild:  No limitation of usual activities 
- 
Moderate:  Some limitation of usual activities 
- 
Severe:  Effects ranging from inability to carry out usual activities to life-threatening. 
 
7. Trial Termination 
 
7.1 Trial Termination 
 
Termination of the trial may be recommended by Data and Safety Monitoring Board during 
one of their bi-annual meetings if there are concerns regarding safety or if the trial has 
failed to reach its declared goals.  
 
7.2 Withdrawal of Consent 
 
Participants who withdraw their consent during the course of the trial will discontinue their 
study medications and follow-up visits, but they will receive phone-call follow to monitor for 
adverse events and ensure appropriate safety surveillance. 
 
The primary investigator will retain the right to withdraw a subject for any clinical/safety 
concerns or non-adherence to study protocol. 
 
8. Data Collection and Statistical Considerations 
 
Demographic data will be collected from patients undergoing initial screening for the study. 
During the study, we will collect data from enrolled subjects from blood samples, exhaled 
breath condensate and BAL fluid that are collected.  The information collected for this trial will 
be recorded on Case Report Forms (CRFs) and entered in a web-based database called REDCAP.  
REDCAP is a secure, HIPAA-compliant, data management software website that requires 
appropriate VA credentials for access. All data entered in the REDCAP database will reside on VA 
servers. 
 
For data analysis, the VINCI (VA Informatics and Computing Infrastructure) Workspace will be 
utilized. The VINCI Workspace will allow the team access to SAS (Statistical Analysis Software) 
while keeping all data on the VA’s secured servers, thereby ensuring Veterans' privacy and data 
security.  
 
 
 
 

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9. Trial Administration 
 
9.1 Data and Safety Monitoring Board (DSMB) 
 
An independent Data and Safety Monitoring Board (DSMB) will be appointed and charged 
with oversight responsibility for the performance and safety of the trial.  The board will be 
comprised of three members (including a statistician) with clinical trial experience.  An 
independent, experienced physician with no involvement in the study will serve as the 
chairman of the board.    They will meet formally prior to the study to approve the protocol 
and thereafter convene about every six months or more frequently as needed.  In general, 
the DSMB will receive reports comprised of: (1) the number of subjects that have signed 
consent, (2) number of screen fails, (3) number of drop-outs, (4) number of completers, (5) 
listing of reasons for drop-out, (6) a tabulated list of all adverse events, their frequency and 
severity, that occur during the study, (7) complete description on an IRB template of all 
serious adverse events that occur during the study, and (8) a written description of any 
known breaches of confidentiality of study subjects.   
 
After each meeting, the DSMB will make formal recommendations regarding trial 
continuation.  As there are no interim analyses, there will be no grounds for stopping the 
trial early for efficacy.  However, the trial may be stopped early for any safety or 
performance issues.  A special responsibility of the DSMB is to review serious adverse events 
(SAEs) as defined in this protocol.  SAEs considered reportable per the Reportable Events 
policy will be reported to the chairman of the DSMB or designee within 48–72 hours for 
review.  This initial review will determine whether there is a recommendation for a change 
in the protocol (or halt the study) until the matter can be considered by a quorum of the 
DSMB. All SAEs (reportable and non-reportable) SAEs will be available for review at the 
semi-annual DSMB meetings. 
 
9.2 Institutional Review Board Approval 
 
The Principal Investigator is responsible for obtaining and maintaining IRB approval 
throughout the entire course of this study. 
 
9.3 VA Research and Development Committee Approval 
 
In addition to IRB approval, the Principal Investigator will ensure that the Research and 
Development Committee at the Atlanta VA Medical Center approves the study and receives 
notification of IRB approval along with letters of approval for all amendments to the study 
per VA guidelines. 
 
9.4 ClinicalTrials.gov Registration 
 

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This application includes a trial which requires registration in ClinicalTrials.gov. This 
registration will occur prior to study initiation. 
 
9.5 Trial Documentation 
 
All worksheets, supporting source documents and administrative records will be retained by 
the Principal Investigator for a minimum of two years following the last notification of 
approval by an appropriate regulatory authority.  The consent forms and other research 
documents are scanned permanently into the medical record for veteran participants of the 
study per usual VA protocol. 
 
9.6 Confidentiality 
 
9.6.1 
Patient Anonymity/Protected Health Information (PHI) 
 
All PHI collected and processed for the purposes of this study will be managed by 
the investigators and study staff.  The anonymity of participating patients will be 
strictly maintained.  All subjects are identified by an assigned unique patient ID 
number on all worksheets, CRFs, and other documents.  Documents that identify 
the patient and contain PHI (e.g., the signed informed consent form) will be 
maintained in strict confidence by the study investigators and study coordinator, 
except to the extent necessary to allow auditing by the appropriate regulatory 
authority.  All electronic documents containing patient information will be stored 
electronically on VA-encrypted drives.  All data collected as a hard copy will be kept 
in a locked file cabinet in a locked office.  Only the investigators and study staff will 
have access to this information. 
 
Auditors and other authorized agents as well as that of any other applicable 
government agencies will be granted direct access to the study subjects’ original 
medical records for verification of clinical trial procedures and/or data, without 
violating the confidentiality of the subjects, to the extent permitted by law and 
regulations.  Any presentations of results from this study will only exhibit de-
identified data.  
 
9.6.2 
Website and Database Security  
 
Access to the web-based database is provided by password protected individual 
account only.  There are no group or generic accounts.  Permission for each account 
is granted on the basis of its user’s role in the trial, and the web site itself is 
accessed via a secure connection. No personal identifiers (names, addresses, 
medical record numbers, etc.) are ever transmitted to the web-based database, and 
all participant data is accompanied only by the unique study ID. 

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Literature Cited 
1. 
Results from the 2008 National Survey on Drug Use and Health: National Findings. 2009, 
Substance Abuse and Mental Health Services Administration: Rockville, MD. 
2. 
Hasin, D.S., et al., Prevalence, correlates, disability, and comorbidity of DSM-IV alcohol abuse 
and dependence in the United States: results from the National Epidemiologic Survey on Alcohol 
and Related Conditions. Arch Gen Psychiatry, 2007. 64(7): p. 830-42. 
3. 
Zhang, Y., et al., Secular trends in alcohol consumption over 50 years: the Framingham Study. Am 
J Med, 2008. 121(8): p. 695-701. 
4. 
Horgan, C.M., Brandeis University. Schneider Institute for Health Policy., and Robert Wood 
Johnson Foundation., Substance abuse : the nation's number one health problem : key indicators 
for policy update. 2001, Princeton, NJ: The Foundation. 128 p. 
5. 
Lieber, C.S., Medical disorders of alcoholism. N Engl J Med, 1995. 333(16): p. 1058-65. 
6. 
Lieber, C.S., Biochemical factors in alcoholic liver disease. Semin Liver Dis, 1993. 13(2): p. 136-53. 
7. 
Lieber, C.S., Biochemical and molecular basis of alcohol-induced injury to liver and other tissues. 
N Engl J Med, 1988. 319(25): p. 1639-50. 
8. 
Otis, J.S., L.A. Brown, and D.M. Guidot, Oxidant-induced atrogin-1 and transforming growth 
factor-beta1 precede alcohol-related myopathy in rats. Muscle Nerve, 2007. 36(6): p. 842-8. 
9. 
Brown, L.A., et al., Acute and chronic alcohol abuse modulate immunity. Alcohol Clin Exp Res, 
2006. 30(9): p. 1624-31. 
10. 
Saitz, R., W.A. Ghali, and M.A. Moskowitz, The impact of alcohol-related diagnoses on 
pneumonia outcomes. Arch Intern Med, 1997. 157(13): p. 1446-52. 
11. 
Fine, M.J., et al., Prognosis and outcomes of patients with community-acquired pneumonia. A 
meta-analysis. JAMA, 1996. 275(2): p. 134-41. 
12. 
Fernandez-Checa, J.C., Alcohol-induced liver disease: when fat and oxidative stress meet. Ann 
Hepatol, 2003. 2(2): p. 69-75. 

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13. 
Goss, C.H., et al., Cost and incidence of social comorbidities in low-risk patients with community-
acquired pneumonia admitted to a public hospital. Chest, 2003. 124(6): p. 2148-55. 
14. 
Chen, C.W., et al., Adult bacteremic pneumonia: bacteriology and prognostic factors. J Formos 
Med Assoc, 1992. 91(8): p. 754-9. 
15. 
Joshi, P.C. and D.M. Guidot, The alcoholic lung: epidemiology, pathophysiology, and potential 
therapies. Am J Physiol Lung Cell Mol Physiol, 2007. 292(4): p. L813-23. 
16. 
Joshi, P.C., et al., Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-
stimulating factor receptor expression and downstream signaling in the alveolar macrophage. J 
Immunol, 2005. 175(10): p. 6837-45. 
17. 
Moss, M., et al., The effects of chronic alcohol abuse on pulmonary glutathione homeostasis. Am 
J Respir Crit Care Med, 2000. 161(2 Pt 1): p. 414-9. 
18. 
Yeh, M.Y., et al., Chronic alcoholism alters systemic and pulmonary glutathione redox status. Am 
J Respir Crit Care Med, 2007. 176(3): p. 270-6. 
19. 
Brown, L.A., et al., Glutathione availability modulates alveolar macrophage function in the 
chronic ethanol-fed rat. Am J Physiol Lung Cell Mol Physiol, 2007. 292(4): p. L824-32. 
20. 
Brown, L.A., et al., Chronic ethanol ingestion and the risk of acute lung injury: a role for 
glutathione availability? Alcohol, 2004. 33(3): p. 191-7. 
21. 
Kamat, P.P., et al., Mechanical ventilation exacerbates alveolar macrophage dysfunction in the 
lungs of ethanol-fed rats. Alcohol Clin Exp Res, 2005. 29(8): p. 1457-65. 
22. 
Ping, X.D., et al., In vivo dysfunction of the term alveolar macrophage after in utero ethanol 
exposure. Alcohol Clin Exp Res, 2007. 31(2): p. 308-16. 
23. 
Zalewski, P.D., Zinc metabolism in the airway: basic mechanisms and drug targets. Curr Opin 
Pharmacol, 2006. 6(3): p. 237-43. 
24. 
Tudor, R., P.D. Zalewski, and R.N. Ratnaike, Zinc in health and chronic disease. J Nutr Health 
Aging, 2005. 9(1): p. 45-51. 

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25. 
Zalewski, P.D., et al., Zinc metabolism in airway epithelium and airway inflammation: basic 
mechanisms and clinical targets. A review. Pharmacol Ther, 2005. 105(2): p. 127-49. 
26. 
Maverakis, E., et al., Acrodermatitis enteropathica and an overview of zinc metabolism. J Am 
Acad Dermatol, 2007. 56(1): p. 116-24. 
27. 
Ezzati, M. and World Health Organization., Comparative quantification of health risks : global 
and regional burden of disease attributable to selected major risk factors. 2004, Geneva: World 
Health Organization. 
28. 
Truong-Tran, A.Q., et al., New insights into the role of zinc in the respiratory epithelium. Immunol 
Cell Biol, 2001. 79(2): p. 170-7. 
29. 
Jayaram, L., et al., Sputum zinc concentration and clinical outcome in older asthmatics. 
Respirology, 2011. 
30. 
Mehta, A.J., et al., Zinc Supplementation Restores PU.1 and Nrf2 Nuclear Binding in Alveolar 
Macrophages and Improves Redox Balance and Bacterial Clearance in the Lungs of Alcohol-Fed 
Rats. Alcohol Clin Exp Res, 2011. 
31. 
Joshi, P.C., et al., Zinc deficiency mediates alcohol-induced alveolar epithelial and macrophage 
dysfunction in rats. Am J Respir Cell Mol Biol, 2009. 41(2): p. 207-16. 
32. 
Yeh, M.Y., et al., Non-invasive evaluation of pulmonary glutathione in the exhaled breath 
condensate of otherwise healthy alcoholics. Respir Med, 2008. 102(2): p. 248-55. 
33. 
Pue, C.A. and E.R. Pacht, Complications of fiberoptic bronchoscopy at a university hospital. Chest, 
1995. 107(2): p. 430-2. 
 
 
 

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Appendix 1 – Schedule of Assessments 
 
 
 
 
 
 
 
 
 

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Appendix 2 – AUDIT Questionnaire 
 
 

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Appendix 3 – SMAST Questionnaire 
 

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Appendix 4 – Drinking History Questionnaire 
CURRENT DRINKING 
 
 
 
 
 
1. How many alcohol beverages do you consume per day on average?  
 
2. How long have you used at this rate (in years)?  
 
 
 
 
3. If Male: In the past 12 months, how many times have you had 5 or more drinks in a day?  
 
 
 
4. If Female: In the past 12 months, how many times have you had 4 or more drinks in a day? 
 
 
 
5. In the past 12 months, what is the largest amount of alcohol that you have had in one day? 
a. Amount (in standard drinks, refer to chart):  
 
 
b. Over what time period (in hours):   
 
c. Type of beverage:   
 
 
d. Brand: 
 
 
 
6. Beverage preferences 
 
✓ 
Beverage List 
Preferred Manner 
of Drinking 
Method of 
Preparation 
Brand(s) 
 
Beer or ale 
 
 
 
 
Brandy 
 
 
 
 
Gin 
 
 
 
 
Hard Cider 
 
 
 
 
Liqueurs (Cordials) 
 
 
 
 
Malt Liquor 
 
 
 
 
Rum 
 
 
 
 
Sake 
 
 
 
 
Sparkling Wine 
 
 
 
 
Special Fortified Wine 
 
 
 
 
Tequila 
 
 
 
 
Vodka 
 
 
 
 
Whiskey 
 
 
 
 
Red, Dry Wine 
 
 
 
 
Red, Sweet Wine 
 
 
 
 
Rose Wine 
 
 
 
 
White, Dry Wine 
 
 
 
 
White, Sweet Wine 
 
 
 
 
Wine Coolers 
 
 
 
 
Other (specify): 
 
 
 
 
Other (specify): 
 
 
 
 
 
 
 

V ersi o n 2. 3 8 / 2 1 / 2 0 1 7  
P a g e 2 5  
 
P A S T D R I N K I N G  
 
 
 
 
 
1.  At w h at a g e di d y o u first h a v e 1 or m or e dri n ks of al c o h ol ?  
 
   
 
 
2.  H o w ol d w er e y o u w h e n y o u first b e c a m e i nt o xi c at e d:   
    N e v er i nt o xi c at e d  
 
3.  If a p pli c a bl e:  At w h at a g e di d dri n ki n g b e gi n t o h a v e a n eff e ct o n y o ur lif e, w hi c h y o u di d 
n ot a p pr o v e of ? W h e n di d dri n ki n g first b e c o m e a pr o bl e m f or y o u ?  
 
A g e w h e n first pr o bl e m:  
 
 
 
  N e v er a pr o bl e m  
 
 
4.  H a v e y o u e v er willi n gl y q uit dri n ki n g f or a p eri o d l o n g er t h a n a f e w d a ys ?  
 N o   Y es  
 
 
 
 
5.  H o w m a n y p ast q uit att e m pts h a v e y o u h a d ?  
 
  N ot a p pli c a bl e, n e v er  att e m pt e d 
t o q uit 
 
 
 
 
 
 
6.  H a v e y o u e v er b e e n t hr o u g h al c o h ol tr e at m e nt ?   N o     Y es  
 
 
If Y es, pr o vi d e d et ails:  
 I n p ati e nt: _ _ _ _ _ y e ar(s) of tr e at m e nt 
 O ut p ati e nt: _ _ _ _ _ y e ar(s) of tr e at m e nt  
      
 Al c o h oli cs A n o n y m o us ( A A): _ _ _ _ _ y e ar(s) of tr e at m e nt  
 
 

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Appendix 5 – Timeline Followback Form-90 
Instructions: 
▪ Think back over the past 90 days.  
▪ Write down how many drinks you consumed each day on the calendar. 
▪ Record the drinks you consumed in terms of standard drinks. This is important because 
different types of beverages contain different concentrations of alcohol. Refer to the 
Standard Drink Chart as necessary. 
▪ Record a “0” for days when you did not drink any alcohol.  
▪ Before you begin, please look at the SAMPLE CALENDAR on the next page. 
 
Your Best Estimate: 
We know it isn’t easy to recall things with 100% accuracy. If you are not sure if you had 3 or 4 
drinks on a given day, give it your best guess! Our goal is to get a sense of how frequently you 
drink alcohol, how much you drink, and your patterns of drinking. 
 
Holidays such as Thanksgiving and Christmas are marked on the calendar to help you recall 
your drinking. Also, think about how much you consumed on personal holidays and events such 
as birthdays, vacations, or parties.  
 
 

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Sample calendar 
 
July 2012 
S 
M 
T 
W 
T 
F 
S 
1 
 
2 
2 
 
1 
3 
 
0 
4 
Independence Day  
2 
5 
 
2 
6 
 
4 
7 
 
2 
8 
 
2 
9 
 
2 
10 
 
2 
11 
 
2 
12 
 
2 
13 
 
2 
14 
 
2 
15 
 
2 
16 
 
1 
17 
 
1 
18 
 
0 
19 
 
0 
20 
 
3 
21 
 
3 
22 
 
2 
23 
 
0 
24 
 
0 
25 
 
1 
26 
 
3 
27 
 
2 
28 
 
0 
29 
 
2 
30 
 
0 
31 
 
2 
 
 
 
 
 
 

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Appendix 6 – Alcohol Dependence Scale (ADS) 
 

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Appendix 7 – Standard Drink Chart 
 
 
 

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Appendix 8 – Food Frequency Questionnaire 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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