Ewelina Borkowska

NUTR 732: Research Protocol

Inhibitory Effects of Sulforaphane Present in Broccoli Sprouts on Helicobacter

pylori in Adult Human Subjects.
Purpose
Helicobacter pylori (H. pylori) is a microorganism responsible for most frequent and highly
persistent bacterial infections worldwide, associated with a number of diseases and
complications including: iron deficiency anemia, diabetes, gastritis, peptic ulcer disease and
gastric adenocarcinoma (1, 2, 3, 4). H. pylori infections are common in both developing and
developed countries. It is estimated that as many as 90% of the population carries H. pylori in
developing countries, whereas the prevalence of infections in developed countries ranges from
25-50%(5).Typically H. pylori infection is acquired during childhood and persist lifelong, if not
treated and eradicated properly. The most common eradication methods are based principally
on the use of antibacterial drug therapies consisting of 2 or more antibiotics, and often proton
pomp inhibiting medication. However, their effectiveness is often limited due to patients
compliance, antibiotic intolerance and occurrence of multiple side effects such as: nausea,
diarrhea, dyspepsia, headache and disturbance in intestinal microflora and/or antibiotic
resistance (6).Moreover, the widespread overuse of antibiotic therapies to eradicate H. pylori
infection in developing countries can contribute to a further escalation of an economic burden.
Considering the consequences of H. pylori infection, as well as the success rate of the existing
treatment therapies, the use of novel eradicating agents and treatment strategies is essential in
clinical advancement. Therefore, antibacterial plant derivatives, such as Sulforafphane (SF), has
been recently examined. SF is obtained from cruciferous vegetables such as uncooked broccoli
and broccoli sprouts, Brussel sprouts or cabbages. SF, the biologically active isothiocyanate, is
produced when the enzyme myrosinase transforms glucoraphanin, the SF precursor, into its
active form upon mastication or other mechanical damage to the plant, which allows the
conversion (7).
Previous research findings reported potential bactericidal properties of SF against H. pylori
infections and related complications. Yanaka, et al suggested that SF contributed to reduced
gastric bacterial colonization, decreased mucosal expression of tumor necrosis, decreased
corpus inflammation, and prevented expression of high salt-induced gastric corpus atrophy, in
mice treated with broccoli sprouts (8). Haristoy et al, showed that H. pylori was completely
eradicated in 8 of the 11 sulforaphane-treated gastric human grafts, implanted in mice (9).
Fahey et al investigation of inhibitory effects of SF on urease, the enzyme produced by H. pylori,
failed to revel decreased urease activity, however it did reveal bactericidal effects of SF on H.
pylori strains (10).
Additional studies suggested protective properties of SF against H.pylori. Chang et al, examined
inhibitory effects of broccoli sprout extract containing SF on H. pylori infection and anti- oxidative
effect on gastric mucosal damage in human subject. The results reveled significant reduction in
mucosal malondialdehyde (MDA) concentration, a byproduct of oxidative stress, therefore
suggesting reduced oxidative damage caused by H. pylori (11). Bahadoran et al investigated
effectiveness of high-SF broccoli sprouts powder, as both complementary and alternative

treatment to the standard triple therapy, on H. pylori eradication in type 2 diabetic patients. His
results showed significantly increased eradication rates in the SF powder treatment group (12).
Despite of the promising results of several previous investigations supporting the hypothesis of
bactericidal properties against H. pylori, findings from human trails are still very limited,
inconclusive and show only temporary eradication and reoccurrence of infection after the
treatment. Moreover, sufficient data on larger doses of SF administration for prolong periods of
time, to provide long term inhibition of H. pylori, is lacking. Therefore effects of SF on H. pylori
eradication require further investigation. Consequently, the purpose of this study is to investigate
whether a SF, derived from glucoraphanin rich broccoli sprouts, will inhibit the H. pylori infection
in H. pylori positive adult human subjects, when administered increased doses of SF during
extended intervention duration, compared to preliminary studies.
Null Hypothesis: Sulphoraphane will not exhibit inhibitory effects on H. pylori infection in H.
pylori positive adult human subjects.
Working Hypothesis: Sulphoraphane will exhibit inhibitory effects on H. pylori infection in H.
pylori positive adult human subjects.

Significance
The acuteness of the prevalence of worldwide bacterial infections, including one of the most
persistent infections with H. pylori, still remains a major epidemiological concern. Severe health
complications related to such infections can contribute no only to economical, societal and
environmental burden but also to decline in both life quality and expectancy of the communities.
Lack of successful medical treatment strategies and insufficient evidence confirming prosperous
prospective use of alternative treatments, such as SF, only confirms the urgency of continuing to
seek new regimen strategies and expansion of already existing research. Consequently, our
goal is to better address the existing gaps related to use of SF and extend the duration of
treatment dose in order to promote long term eradication of H. pylori and to pave the way to new
successful treatment of H. pylori with dietary component-SF.

Innovation
Widely used antibiotic therapies, in H. pylori treatment, is some cases decrease symptoms of
the infection and provides short term eradication. However in many cases the first line treatment
is not efficient and a second or third line treatments with multiple antibiotic combinations are
offered. Yet, even with currently most effective treatment strategies 10-20% of patients fail to
obtain a cure from such regimens (6, 16). Additionally, the side effects related to prolonged
antibiotic use and the corresponding consequences are highly concerning and therefore using
innovative and less detrimental eradicating agents, such as SF, is essential in improving health
and quality of life of our communities, and in advancement of the medical field.
Although the use SF in treatment of H. pylori infection has been previously examined and
described by many researchers, the exact dose and duration of interventions, which would
promote long-term eradicating effects, has not yet been established. Additionally many
eradicating antibiotics treatment as well SF interventions, described in the preliminary studies,
often focus on the results of the primary therapy and not overall, final eradication rate.
Therefore, additional follow up data to determine long-term eradication, is insufficient in current
research. Preliminary studies, on humans and animals, used variable doses and intervention
periods and focused on either higher dose/ shorter intervention time or lower dose/ slightly

longer intervention time (8, 9, 11,12). While some results of these studies shown decreased
colonization of H. pylori during broccoli sprout treatment, in some cases the reoccurrence of H.
pylori infection was detected shortly after broccoli sprouts administration was terminated (8, 9,
11, 12).
Considering the demand for a better specified dosage and intervention duration, as well as a
follow up data indicating reoccurrence, or lack of infection post intervention, we intend to
implement a new approach that will address these demands.

Research Strategy
Participants. 50 H. pylori- positive adult volunteers will be recruited from a GI Health clinic, our
collaborating site, specializing in digestive health and digestive disorders, localized in New York
City. The participants will include both males and females, (age 21+) of a diverse ethnicity,
allowing us to observe responses to treatment in diverse group rather than one specific gender
or ethnicity, which could affect the results and prevent potential findings from being
generalizable to more than one population, gender or age group. The study will include
participants who underwent unsuccessful antibiotic therapy, 30 days or longer, prior to
enrolment in the study, and whose post treatment endoscopic biopsy reveled reoccurrence of H.
pylori infection. The rational related to inclusion of adult subjects was because of the previous
statistical findings which suggest the increased prevalence of H. pylori among adult population
compared to children and young adults (17).Therefore we wanted to include mainly the
population that is most prone for infection and related health consequences.
The exclusion criteria will include participants who are undergoing any antibiotic therapy upon
enrollment, are current users of proton pump inhibitors, non-steroidal anti-inflammatory drugs
(NSAIDs) and/or anticoagulant or antihypertensive drugs. Patients with past history of gastric
resection surgery, peptic ulcer disease, severe gastritis, DB, HTN, renal disease, heart disease,
gastric malignancy or a history of malabsorptive disease, such as celiac disease, will also be
excluded from the study. The exclusion criteria related to drug use were determined based on
potential side effects of drugs involving damage to the stomach mucosa, formation of peptic
ulcers and drug nutrient interactions that could possibly arise during supplementation of sprouts
and interfere with absorption and metabolism of glucoraphanin (18, 19). We want to assure
safety of the participant as well as avoid covariates that could possibly affect the outcome of the
intervention. The exclusion of participants with stated health conditions was related to
avoidance of potential disturbance in metabolism, absorptions and excretion of SF, or
interference with pathophysiology of the diseases with our intervention strategies.
We will assure protection of the rights and welfare of the human subjects included in this study.
Written informed consent will be obtained from each participant prior to enrolment. Approval
from the Institutional Review Board (IRB) is pending.

Experimental Approach.
We propose our study to be a randomized control trial. Participants will be randomized into two
groups: intervention (n=25), broccoli sprouts group, and control (n=25), alfalfa sprouts group.
The study will be conducted over 10 weeks span with a follow up session at week 20 and 30. All
participants will be required to visit the clinic for collection of samples (described in detail in the
specific aims section) at day 0, 35 (week 5), 70 (week 10), 140 (week 20), 210 (week 30). The
estimation of the duration of the study was determined based on preliminary interventions (8, 9,

10, 13). Additionally physical examination will be performed at each visit, at which participant will
be physically assessed for signs of fatigue, confusion or lethargy. Measurements of blood
pressure will also be obtained at each visit and an interview with a physician, regarding
occurrence of any gastrointestinal distress or any unusual symptoms, during and after the
intervention period, will be conducted. This will allow us to control for any signs of intolerance of
the sprouts and digestive issues and terminate the intervention for subjects who develop
symptoms that can potentially be detrimental to their health.
Our purposed experimental approach will be non-invasive, and therefore the tests that we are
proposing to use are of that nature. Preliminary studies often use endoscopic biopsies and other
invasive methods which are costly, often risky and distressing (3,5,8,10,11). Our experimental
approach will focus on use of tests that are non-invasive yet deliver identical or similar resistivity
and specificity, in detecting H. pylori infection, to those using biopsy specimens (21,22,23).
To test the stated hypothesis consecutive aims will be followed:
Specific Aims
Aim 1. Administer raw broccoli sprouts to subjects in the treatment group, and alfa-alfa
sprouts to subjects in the control group, and evaluate the compliance to the intervention
by urine test detecting SF metabolites-dithiocarbamates (DTC).
Participants in the intervention group will be administered broccoli sprouts in the amount of 100
g/day (x 10 weeks) in form of raw, at least 3-day germinated sprouts, and the control group will
be administered the equivalent amount of alfalfa sprouts. The sprouts will be delivered to the
participants home twice a week from Windfall Farms, located in Montgomery, NY via New York
Transport Company. The participants will be educated on storage and consumption of sprouts
and will be asked to keep a daily record of the consumption of sprouts, including time of
consumption, amount (all of it or less) as well as incompliance and reason behind it. Additionally
each participant will receive a list or other cruciferous vegetables/ foods which consumption also
will be required to be reported. No other dietary manipulation to patients existing diet will be
executed.
The rationale behind the use of raw broccoli sprouts rather than cooked sprouts was determined
based on statistics showing bioavailability of SF in urine, suggesting 37% bioavailability from
raw sprouts versus 3.45% from cooked sprouts (14). Lack of reports of adverse symptoms
during intake of broccoli or alpha-alpha sprouts and no evidence of toxicity related to intake of
such sprouts was also a determining factor (14). Glucoraphanin is also present in many other
cruciferous vegetables such as Brussel sprouts, cabbages etc. and therefore keeping record of
consumption of such vegetables will allow us to better control and evaluate the results of our
intervention.
Cruciferous vegetables such as cabbages, broccoli, broccoli sprouts undergo enzymatic
conversion of glucosinolates to isothiocyanates (ITC), which further get metabolized to
dithiocarbamates (DTC) (20).Urinary DTC biomarkers have been found to be sensitive,
biomarkers detecting cruciferous vegetable consumption, which can help to determine
adherence to intervention in a non-invasive manner (21,22).The dose of the broccoli sprouts
(100g/day) was determined based on data from preliminary studies (2,8,9,10,11,12,13). A
human feeding study by Yanaka and colleagues was specifically used as a model for our study.
It was estimated in the previous study that intake of 70 g of broccoli sprouts will deliver

~420mol of glucoraphanin (8). Based on these calculations we estimated that 1gm of broccoli
sprouts will contain about ~6mol of glucoraphanin which will yield~ 600 mol of glucoraphanin
per 100g of broccoli sprouts. For the purpose of this study a urine collected over a 24 hour
period will be evaluated. The patients will be instructed on the collection of urine and storage.
The test will be performed at each visit to the clinic during and post intervention.
The DTC, which is based on the cyclocondensation reaction, will allow us to determine not only
the compliance of the consumption of the sprouts (and perhaps other glucoraphanin rich
vegetables) but also the successfulness of the conversion of glucoraphanin to SF and the
excreted amount. These findings will contribute to better evaluation of the results of further tests
(described in Aim 2&3).
Aim 2. Evaluate the effects of SF on H. pylori infection by determination of CO2
concentration in the expired air through Urea Breath Test (UBT).
H. pylori is a microorganisms that adapts itself to living in a very acidic environment with a low
pH 2.0 or lower. Urease is central to H. pylori's metabolism and virulence and helps the
microorganism colonize the gastric mucosa, by the mechanism in which H. pylori uses it to
convert urea into ammonia, and bicarbonate, to counteract the low acidity of the stomach. With
high urease activity this microorganism can protect the bacterium from acid damage by
buffering the cell and the environment. The hydrolysis of urea molecules in the gastric juices
creates ammonia which increases pH in the stomach. Therefore enzyme must be consistently
available for the organism to survive in the acidic environment (23).
The urea breath test (UBT) is a none-invasive test that uses H.pyloris survival mechanism, to
detect its presence in the stomach. In this test a solution of a non-radioactive, carbon-13
isotope-labeled urea is given to the participant. Fasting, and delaying the gastric emptying,
through administration of citric acid solution, is very important in this procedure. The solution is
rapidly hydrolyzed by Pylori, if this organisms is present in the stomach the labeled CO2 is
absorbed from the stomach, to the blood and further lungs, and exhaled in expired air. In case
of lack of infection the isotope is not detected in the expired air, and instead it is eliminated in
the urine (23).
The rationale behind implementation of this methods is its non-invasive, easy and safe
approach, as well as high sensitivity (95%), comparable with more complexed invasive methods
(23).
Aim 2. Evaluate the effects of SF on H. pylori infection by determination of H. pylori
antigens in stool through Helicobacter pylori stool antigen test (HpSA).
Stool tests have the advantage of being a more direct, yet still non-invasive form of testing. H.
pylori, alike many other pathogens and digestives enter/ occupy the stomach, are excreted via
stool. For the purpose of this study the stool samples will be collected from the evening, or
morning void prior to the visit. The test will be based on a micro wells-based immunoassay that
detects H. pylori antigens in stool. This test utilizes a mixture of monoclonal anti-Pylori
antibodies and an enzyme conjugate (peroxidase) which allows the change of the color to
yellow, indicating the presence of H.pylori in the sample (23).Rationale behind the use of this
method is that this stool assay is a reliable (sensitivity 96.1% and specificity 95.7%) and easyto-use tool for diagnosis of H. pylori infection, which supports our non-invasive approach for this

study. HpSa will allow us to confirm the presence of absents of H. pylori infection post
intervention and further at follow up visits.
All statistical analysis will be carried out using SPSS system. Data will be expressed in means
and standard deviations and multiple analytical procedures such as: one way NOVA,
independednt and paired-t test will be used to evaluate the results.

Predicted outcomes and Speculations

Based on the results from the previous human feeding studies, focusing on determining the
effects of Sf on H. pylori eradication, we expect our results to show and increase levels of DTC
in the urine post intervention, in the broccoli sprout group, and no change of DTC levels in the
alfalfa group. We also expect to see very limited or no adverse symptoms from the treatment in
both groups, and high adherence to the intervention. Additionally we predict that the UBT as
well as HpSA test will show change in the intervention group, from baseline, and will suggest
lack of H.pylori infection post intervention and consequently at follow up sessions. We predict no
changes in the control group in the UBT results as well as the HpSA. Any results that will that
our experimental dose and duration of the intervention was inappropriate to contribute to
complete eradication of the infection. An evidence of eradication during intervention and
reoccurrence of the infection at follow up visits will confirm the finding of preliminary studies, the
inaccurate selection of dosage and intervention length. Moreover the sampling can potentially
affect the results as well. The sampling will be performed by the participants and delivered to the
testing center, which can possibly affect the quality and vitality of the samples, but also can
question the accurateness of the sampling and possible manipulation to the procedures. It is
also important to consider the impact of bowel movements on the results of the HpSA
measurements. It is suggested that short transit time will favor the excretion of unaltered
antigens, while constipation can possible contribute to destruction of the antigens (23). Similarly
UBT test can be affected by inadequate following of the protocol of the test which indicated
importance of delayed gastric emptying prior to the test, which maximizes to contact between
labeled urea solution and the bacteria. Non fasting state ad elimination of the delayed gastric
emptying could possible result in false negative results, which could skew the overall results of
the intervention (23).
The results of the purposed study can be a good indicator for the future research. The dosage
and length of intervention suggested in this study can be a precursor of future research
evaluating the actual mechanisms behind the effects of higher doses on the H.pylori strains.
The purpose of this study was solely focus on investigation of the eradication of H. pylori on
participants with history of prior antibiotic use. In case of desired results of this intervention our
research can largely contribute to growth of clinical field. However, in case of undesired results
proving unsuccessful intervention this research will still add additional information to the already
existing evidence, and perhaps created a question if antibiotic resistant H. pylori strains treated
with SF require a different type of intervention and more complex approach.

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