Studies - Via Farma

Transcription

Studies - Via Farma

Clinical
Studies
Dairylytic® , ProHydrolase®,Glutalytic® são marcas registradas da Deerland Enzymes
ProHydrolase® Clinical
Phase II Study
Deaton, J.; Dawson, H.; Davidson, J.
ProHydrolase® Clinical—Phase II Study
University of Wisconsin - La Crosse
Office of Research and Sponsored Programs,
220 Morris Hall
1725 State St
La Crosse, WI 54601
Bindley Bioscience Center
1203 W. State Street
Purdue University
West Lafayette, IN 47907-2057
Correspondence to:
John Deaton, PhD
VP Technology
Deerland Enzymes
3800 Cobb International Blvd
Kennesaw, GA. 30152
[email protected]
770-919-8907 X 407 Phone
770-919-1194 Fax
2
Introduction
Whey protein supplementation is a common practice to promote muscle building and muscle
recovery for people with an active lifestyle (body-builders, fitness enthusiasts), as well as
others who experience muscle wasting due to disease or treatment of disease. Whey protein
supplementation has been shown to increase muscle protein synthesis and reduce muscle
protein degradation.
When whey protein is digested, it’s broken down in the digestive tract into small peptides,
and eventually into amino acids that will be absorbed in the intestines. To be effective, whey
protein must be broken down into a smaller particle size within approximately 90 minutes
of consumption; otherwise, the protein goes undigested and excreted from the body. During
digestion, the larger peptides composed of more than seven amino acids may trigger an immune
response, causing discomfort and even inflammation in the gut.
A proprietary digestive enzyme formulation has been developed to be consumed in conjunction
with a whey protein supplement in order to provide some pre-digestion of the protein, in order
to take full advantage of the availability of the essential amino acids for building muscle and
improving muscle recovery. The pre-digestion will also ensure that smaller, non-immunogenic
peptides will be formed, therefore reducing the potential for discomfort that is often associated
with protein consumption.
Purpose of the Study
The purpose of this study is to determine if the multi-enzyme formulation (ProHydrolase®)
combined with whey protein results in improved digestion and absorption of amino acids along
with reduction of the immunogenic responses associated with whey protein consumption,
compared to whey protein alone.
Procedure
Whey protein supplements were formulated with and without ProHydrolase, tested and
distributed by a third party to 20 test subjects with the following common characteristics:
• Ages 19-35 years
• Normal body weight (BMI = 18.5-25)
• No known food allergies or intolerance
• Non-weight training subjects who agree not to begin a new exercise program during the
course of the study
Two trials were completed by each test subject, one using whey protein alone and one with the
addition of ProHydrolase. Each trial occurred at the end of a nine-day period in which subjects
consumed a specific diet of approximately 2200 kcal; 40% carbohydrate, 25% protein, 35% fat.
Prior to each of the nine pretrial days, subjects were given a specific meal. On the day of each
trial, subjects were tested following a 12 hour fast. A catheter was placed in the arm or hand vein
and seven 5 ml blood samples were drawn at time 0 (before consumption), 1, 2, 3, and 4 hours
following consumption of the supplement. Blood samples were used to measure levels of amino
acids, C-reactive protein, and insulin.
3
Amino Acids Analysis – Results
Individual Time Points
Area Under the Curve
Leucine
n ProHydrolase n Control
30
Plasma Amino Acids μg hr/ml
Plasma Amino Acids μg/ ml
14
12
10
8
6
4
2
0
0
1
2
3
25
20
15
10
5
0
4
Control
ProHydrolase
Leucine is the strongest of the
branched chain amino acids
(BCAAs), and is responsible for
the regulation of blood-sugar
levels, the growth and repair
of tissues in skin, bones and
skeletal muscle. It’s a strong
potentiator to Human Growth
Hormone. It helps in healing
wounds, regulating energy
and assists in preventing the
breakdown of muscle tissue.
Time (Hours)
P value = .037
Isoleucine
14
12
10
8
6
4
2
0
0
1
2
3
Isoleucine is a BCAA that
promotes muscle recovery
after physical exercise. Alone,
it’s needed for the formation of
hemoglobin as well as assisting
with regulation of blood sugar
levels and energy levels. It’s
also involved in blood-clot
formation.
30
25
20
15
10
5
0
Control
4
Time (Hours)
ProHydrolase
P value = .036
Valine
140
60
50
40
30
20
10
0
1
2
Time (Hours)
P value = .028
4
100
80
60
40
20
0
0
Valine assists in the repair
and growth of muscle tissue,
as commonly attributed to
BCAAs. It’s not processed by
the liver; rather actively taken
up by muscle. It maintains the
nitrogen balance and preserves
the use of glucose.
120
3
4
Control
ProHydrolase
Aspartate
3.5
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
3
2.5
2
1.5
1
.5
0
0
0
1
2
3
4
Control
ProHydrolase
Aspartate is used to increase
absorption of the minerals with
which they combine, and to
enhance athletic performance.
It’s needed for stamina, brain
and neural health and assists
the liver by removing excess
ammonia and other toxins from
the bloodstream. It is also very
important in the functioning
of RNA, DNA, as well as the
production of immunoglobulin
and antibody synthesis.
Time (Hours)
P value = .036
Glutamate
45
18
20
16
14
12
10
8
6
4
2
40
35
30
25
20
15
10
5
0
0
0
1
2
3
Control
4
ProHydrolase
Time (Hours)
Glutamate is the most common
excitatory (stimulating)
neurotransmitter in the
central nervous system. More
than 50% of the amino acid
composition of the brain is
derived from glutamic acid and
its derivatives, providing fuel
for the brain. Glutamic acid
acts as carrier for potassium
across the brain/blood barrier.
Glutamic acid is instrumental in
the metabolism of other amino
acids as well as sugars and fats.
P value = .045
Cysteine
5
14
4.5
4
3.5
3
2.5
2
1.5
1
.5
0
1
2
Time (Hours)
P value = .04
5
10
8
6
4
2
0
0
12
3
4
Control
ProHydrolase
Cysteine is required in the
manufacture of the amino acid
taurine and is a component of
the antioxidant glutahione. It
helps remove harmful toxins
from the body and protect the
brain and liver from damage
from alcohol, drugs etc.
Research has shown that it
may help in strengthening the
protective lining of the stomach
as well as intestines, which may
help prevent damage caused
by aspirin and similar drugs.
Cysteine is also critical to the
metabolism of a number of
essential biochemicals including
coenzyme A, heparin, biotin,
lipoic acid, and glutathione.
Lysine
100
80
60
40
20
0
350
120
0
1
2
3
300
250
200
150
100
50
0
4
Control
Time (Hours)
ProHydrolase
P value = .05
Serine
100
30
25
20
15
10
5
Lysine is an essential amino
acid, important for proper
growth. It plays an essential
role in the production of
carnitine, a nutrient responsible
for converting fatty acids into
energy and helping to lower
cholesterol. Lysine helps the
body absorb calcium, and
it plays an important role in
the formation of collagen,
a substance important for
bones and connective tissues
including skin, tendon, and
cartilage.
Serine helps produce
immunoglobulins and
antibodies for a strong immune
system, and also aids in the
absorption of creatine, a
substance made from amino
acids that helps build and
maintain all the muscles in the
body, including the heart.
90
80
70
60
50
40
30
20
0
10
0
0
1
2
3
4
Control
Time (Hours)
ProHydrolase
P value = .029
Histidine
80
35
30
25
20
15
10
5
0
1
2
3
Time (Hours)
P value = .03
6
60
50
40
30
20
10
0
0
Histidine is needed for growth
and for the repair of tissue, as
well as the maintenance of the
myelin sheaths that act as a
protector for nerve cells. It’s
required for the manufacture
of both red and white blood
cells, and helps to protect the
body from damage caused
by radiation and in removing
heavy metals from the body.
70
4
Control
ProHydrolase
Asparagine
Asparagine is vital in nerve and
brain health and an important
building block for proteins and
muscle mass. It’s an important
aspect of metabolism, because
it aids in the removal of toxic
ammonia from the body.
80
35
30
25
20
15
10
5
70
60
50
40
30
20
10
0
0
0
1
2
3
Control
4
ProHydrolase
Time (Hours)
P value = .041
Glutamine
500
250
200
150
100
50
450
400
350
300
250
200
150
100
50
0
0
0
1
2
3
4
Control
Time (Hours)
ProHydrolase
P value = .021
Glutamine is produced in the
muscles and distributed by the
blood to the organs that need
it. Glutamine may help gut
function, the immune system,
and other essential processes
in the body, especially in times
of stress. It is also important
for providing “fuel” (nitrogen
and carbon) to many different
cells in the body. Glutamine
is needed to make other
chemicals in the body such as
other amino acids and glucose
(sugar).
Glycine
70
25
20
15
10
5
0
1
2
3
Time (Hours)
P value = .048
7
60
50
40
30
20
10
0
0
4
Glycine functions as a
neurotransmitter and a
precursor to metabolic
intermediates such as purines,
which are components of
DNA. Glycine increases
GH production, removing
unwanted substances from
the body (i.e. lactic acid),
enhancing energy levels and
calming the brain.
Control
ProHydrolase
Threonine
120
40
45
35
30
25
20
15
10
5
0
100
80
60
40
20
0
1
0
2
3
4
Control
Time (Hours)
ProHydrolase
P value = .041
Alanine
160
155
50
60
40
30
20
10
0
0
1
2
3
150
145
140
135
130
125
120
115
4
Control
Time (Hours)
ProHydrolase
P value = .045
Tyrosine
45
14
12
10
8
6
4
2
0
1
2
Time (Hours)
P value = .042
8
3
4
Alanine is an amino acid that
helps the body convert the
simple sugar glucose into
energy and eliminate excess
toxins from the liver. Alanine
has been shown to help protect
cells from being damaged
during intense aerobic activity,
when the body cannibalizes
muscle protein to help produce
energy.
Tyrosine initiates and
propels the development
of neurotransmitters and
hormones that work to
keep the mind sharp.
It’s also the precursor of
several neurotransmitters,
including L-dopa, dopamine,
norepinephrine, and
epinephrine.
40
35
30
25
20
15
10
5
0
0
Threonine is an essential
amino acid that is never
manufactured within the
body. Its main sources are
animal (dairy and meat), which
presents a challenge to those
on a vegan diet. It’s found
in heart, skeletal muscle and
nerve tissue in the central
nervous system. Threonine
is used to form the body’s
two most important binding
substances, collagen and
elastin. It’s involved in liver
function, lipotropic functions
(when combined with aspartic
acid and methionine) and
in the maintenance of the
immune system by helping in
the production of antibodies
and promoting growth
and activity of the thymus.
Arguably its most useful
property, it allows better
absorption of other nutrients,
making protein sources
containing threonine more bioavailable than others.
Control
ProHydrolase
Proline
Proline is needed for the
production of collagen and
cartilage. It keeps muscles and
joints flexible and helps reduce
sagging and wrinkling that
accompany UV exposure and
normal aging of the skin.
80
25
20
15
10
5
70
60
50
40
30
20
10
0
0
0
1
2
3
4
Control
ProHydrolase
Time (Hours)
P value = .038
Methionine
16
8
7
6
5
4
3
2
1
0
0
1
2
3
14
12
10
8
6
4
2
0
4
Control
Time (Hours)
ProHydrolase
P value = .049
Arginine
120
40
45
35
30
25
20
15
10
5
0
1
2
Time (Hours)
P value = .045
9
100
80
60
40
20
0
0
3
4
Methionine is an essential
sulfur amino acid, which
must be obtained from food
or supplement sources.
L-methionine contributes to
the synthesis of S-adenosyl-Lmethionine (SAMe), which may
elevate mood and support joint
health, and L-cysteine, which is
a component of glutathione, an
important antioxidant molecule
in the body. Methionine is also
a transporter of the antioxidant
mineral, selenium.
Control
ProHydrolase
Arginine has remarkable
nitrogen retention ability.
Nitrogen is one of the key
elements in muscle protein
synthesis. It enhances the
immune system and stimulates
the size and activity of the
thymus gland, making it a
prime choice for anyone in less
than optimal health, such as
those recovering from injury or
HIV patients. Arginine is also
a precursor of very important
molecules such as creatine
and gamma amino butric acid
(GABA, a neurotransmitter
in the brain). The hormonal
release properties include
releasing insulin from the
pancreas and a massive
stimulator in the manufacture
of GH (Growth Hormone)
from the anterior pituitary. It
increases blood flow. It also
improves the health of the liver,
skin and connective tissues,
and may lower cholesterol.
Primarily, it facilitates muscle
mass gain while limiting fat
storage, because it keeps fat
alive in the system and uses it.
It’s key in weight control.
Phenylalanine
7
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
Phenylalanine is the essential
building block of new cells that
help to restore damaged cells.
It helps to build up tissues in
muscles.
6
5
4
3
2
1
0
4
Control
ProHydrolase
Time (Hours)
P value = .033
Tryptophan
8
3.5
3
2.5
2
1.5
1
0.5
0
0
1
2
3
4
7
6
5
4
3
2
1
0
Control
ProHydrolase
Tryptophan is an essential
amino acid that helps support
mood, relaxation, and restful
sleep. It plays a part in the
synthesis of both melatonin
and serotonin, hormones
involved with mood and
stress response. It also
supports immune functions,
as the body’s precursor to
the kynurenines that regulate
immunity.
Time (Hours)
P value = .041
Total Serum Amino Acids
500000
450000
400000
TSAA μg
350000
300000
250000
200000
150000
100000
50000
0
P value = .05
10
Control
ProHydrolase
Total amino acid
concentrations increased
55mg in the blood over the
270 minutes after ingestion
of whey protein isolate with
ProHydrolase when compared
to taking whey protein isolate
alone. This is a 20% increase in
amino acids when whey isolate
is combined with ProHydrolase
vs. consuming whey isolate
alone. This correlates with
published results showing the
increase of amino acids when
taking whey hydrolysate vs.
whey protein unprocessed.
Amino Acids Analysis
Nine of the 20 amino acids the body uses are essential and must be
obtained through diet and/or supplementation. However, all amino
acids may become essential if the body is unable to produce them or
produce them fast enough to keep up with protein synthesis. People
with low-protein diets or eating disorders, liver disease, diabetes,
or genetic conditions that cause Urea Cycle Disorders (UCDs),
may need to take amino acid supplements to avoid a deficiency.
Physically active people may need additional amino acids due to
the high rate of protein synthesis that occurs when building and
repairing muscle and tissue during and after workouts. The most
common amino acid supplementation is protein in the form of whey,
egg, soy, etc. These proteins contain a high concentration of amino
acids, however they’re bound together in a peptide complex that
must be broken down in order to be utilized. ProHydrolase provides
the enzymes needed to break down these bound amino acids so they
can be fully utilized by the body for muscle building and recovery.
Bioactive Peptides
ProHydrolase is a unique blend of proteases that quickly and
effectively degrades proteins such as whey to small peptides. These
peptides can be used for both their amino acid content, as well as
bioactive peptides. Bioactive peptides have been defined as “specific
protein fragments that have a positive impact on body functions
and conditions”. Specifically, the peptides present in milk have been
shown to have intestinal anti-inflammatory activities.
Levels of C-reactive protein indicate general levels of inflammation in
the body, and can be determined by a blood test.
950
Figure 1: CRP levels start
at 750μ/l, and increase to
approximately 900μ/l after
consuming whey.
900
850
With the addition of
ProHydrolase, levels decrease
to 500μ/l, and then gradually
increase over the 4 hours to
.700μ/l.
μg/l
800
750
700
650
600
550
500
Whey +
ProHydrolase
Whey only
(control)
0
1
2
3
Time (Hours)
11
4
CRP levels were only affected in the test group that consumed
whey+ProHydrolase; these levels decreased significantly (p<0.039).
The control group that consumed only whey experienced no
significant change in CRP levels.
Insulin
In addition to insulin’s effect on entry of glucose into cells, it also
stimulates the active uptake of amino acids, again contributing to its
overall anabolic effect. When insulin levels are low, as in the fasting
state, the balance is pushed toward intracellular protein degradation.
ProHydrolase produces di, tri and tetra peptides but no single amino
acids. Di and tri peptides use proton driven active transporters
to pass from the intestine to the blood, which may or may not be
insulin driven.
To test whether insulin levels change with the consumption of whey
protein plus Prohydrolase, blood samples were tested for insulin
after consuming whey with and without ProHydrolase. Insulin levels
spike in both cases but no additional insulin increase/decrease is
detected versus the control, indicating the new peptides created by
ProHydrolase do not influence insulin levels.
4
Figure 2. Insulin levels
increased both when
consuming whey isolate alone
and with ProHydrolase. There
was no difference in insulin
levels between the two groups
during the four hours.
3.5
IU/ml
3
2.5
2
1.5
1
Whey +
ProHydrolase
0.5
Whey only
(control)
0
0
1
2
3
Time (Hours)
12
4
Conclusion
In summary, this clinical study has demonstrated the benefits of adding the protease
blend ProHydrolase to whey protein isolate. When whey protein isolate is consumed
along with ProHydrolase, amino acid concentrations in the blood increase by 20%,
compared to consuming whey protein alone. In addition, C-reactive protein levels
decrease as a result of the bioactive peptides created by the hydrolyzed protein,
indicating a reduction of inflammation in the body. ProHydrolase has been determined
to have no effect on insulin levels.
13
3800 Cobb International Boulevard
Kennesaw, GA 30152
770.919.8901
800.697.8179
[email protected]
DeerlandEnzymes.com
Deerland Enzymes, Inc., based in Kennesaw,
GA, is a specialty formulator and contract
manufacturer of enzyme-based dietary
supplements. The company specializes in
customized formulations, collaborating with
customers to develop innovative and often
proprietary solutions. Deerland Enzymes also
performs specialty contract manufacturing
services, including bulk powders, liquids, hard
shell capsules, and tablets; as well as bottling
and labeling. The company is GMP certified by
NSF for dietary supplements and for sport.
Glutalytic Clinical Trial for Normal Consumption
of Gluten Containing Foods
Martin Hudson, Colin King
Department of Biology, Kennesaw State University,
1000 Chastain Road, Kennesaw Georgia 30144, United States
*Running title: Glutalytic Clinical Trial
Keywords: Gluten Sensitive, Celiac Disease
Background
Gluten sensitivity and the incidence of Celiac
disease are ever increasing. The aim was to
measure the effectiveness of an enzyme based
dietary supplement on the reduction of symptoms
encountered upon the consumption of gluten.
Results
The data showed a statistically significant
improvement when compared to the placebo group
in the following categories: pain, bloating, emptying
of bowels, hunger pains, rumbling of stomach, lower
energy levels, headaches, and food cravings. There
was no statistically signficant improvement in the
following symptoms: comfort, nausea, and loss of
appetite.
Conclusion
Overall, the dietary supplement, Glutalytic, reduced
many of the symptoms that are encountered upon
the consumption of gluten.
Introduction
Gluten is the most important protein component of
several grains, most notably wheat, rye, and barley.
These grains are the basis for a range of flour and
wheat derived products consumed throughout
the world. In some individuals, gluten components
cause significant gastrointestinal distress and
other symptoms. Non-Celiac gluten sensitivity
is a syndrome characterized by gastrointestinal
and extraintestinal symptoms occurring within
a few hours to days after the ingestion of gluten
and rapidly improving after gluten withdrawal.
Celiac disease, on the other hand, is classified as
a genetically linked autoimmune disorder that
damages the lining of the small intestine and
prevents it from absorbing parts of food that are
important for staying healthy. The damage is due to
a reaction to eating gluten, which is found in wheat,
barley, rye, and possibly oats.[1-3]
Non-Celiac gluten sensitivity and Celiac disease
appear to be different due to epidemiologic and
pathogenetic aspects. Although non-Celiac gluten
sensitivity prevalence is still poorly defined in
society, it is thought to be more frequent than Celiac
disease. The Center of Celiac Research estimates
that approximately six percent of the U.S. population
suffers from gluten sensitivity, while the prevalence
of Celiac disease is approximately one percent of the
population in developed and developing countries.
Due to the ever increasing prevalence of gluten
sensitivity and Celiac disease, there is ongoing
research on the subject. There is currently no cure or
treatment outside of a gluten free diet. [2,4]
The purpose of this double-blind, randomized,
placebo controlled study is to determine if a dietary
supplement, namely Glutalytic, included with a
normal diet results in reduced symptoms of gluten
intolerance in a standard population, as compared
to the placebo group. For those who are gluten
sensitive, the hope is to enable them to lead a normal
life without any food restrictions by incorporating
the dietary supplement into their daily regimen. The
dietary supplement should aid in the breakdown of
the gluten complex to help minimize the symptoms.
For those diagnosed with Celiac disease, the goal
is to minimize the risk of persistent or recurring
symptoms due to contamination through the
crossover effect, thus giving the individuals peace of
mind when eating away from home.
Methods
Questionnaire Design - The questionnaire used in
this clinical was a modified version of the Internal
Review Board (IRB) approved, Celiac diseasespecific symptom index which was developed and
validated by Daniel A. Leffler and his colleagues. The
questionnaire was modified to account for non-celiac
gluten sensitivity instead of narrowing only on Celiac
sensitivity, and was designed to cover a spectrum
of symptoms that could occur upon the ingestion of
gluten. [1]
Clinical Design - This survey was administered to
the entire group of individuals prior to beginning
the supplement regimen. In conjunction with the
questionnaire, questions regarding the participants’
demographics were also completed. The participants
were not asked to change their diets in any way.
The clinical was designed for twenty individuals
and the data presented represents the eleven
participants who achieved full completion of the
clinical. Individuals proceeded to ingest a dosage of
the placebo or Glutalytic with every meal for a week.
During this time, the participants maintained a meal
log of everything they ingested. After seven days,
the participants completed the questionnaire.
Results
Sample frequency was calculated using Microsoft
Excel 2010 (Table 1, 2, 3) and samples were analyzed
using IBM SPSS Statistics for the Wilcoxon Signedrank test and descriptive statistics (Table 2, 3). The
level of significance (α) was chosen to be 5%. Initially
frequencies of responses were calculated, followed
by the Wilcoxon Signed-rank test in order to see
if there was any improvement on a question by
question basis between subjects.
The results of the initial questionnaire were tallied
and converted into percentages to be used as a
control against the data gathered during the placebo
time as well as the dietary supplement regimen. This
data is exhibited in Table 1.
To examine the placebo effect, the raw data
gathered at the end of week two were converted into
percentages. This can be viewed in Table 2.
To determine the effectiveness of the dietary
supplement, Glutalytic, the raw data was transformed
into percentages and displayed in Table 3.
Before Regimen
Question
None of
the time
A little of
the time
Some of
the time
Most of
the time
All of
the time
Have you been bothered by pain or discomfort in the upper
abdomen or the pit of the stomach during the past 4 weeks?
18%
55%
27%
0%
0%
2. Have you been bothered by nausea during the past 4 weeks?
82%
9%
9%
0%
0%
3. Have you been bothered by rumbling in your stomach during the
past 4 weeks?
0%
45%
55%
0%
0%
1.
4. Has your stomach felt bloated during the past 4 weeks?
0%
55%
36%
9%
0%
5. When going on the toilet, have you had the sensation of not
completely emptying your bowels during the past 4 weeks?
27%
45%
18%
9%
0%
6. Have you been bothered by hunger pains during the past 4 weeks?
9%
64%
27%
0%
0%
7. Have you been bothered by low energy during the past 4 weeks?
9%
27%
64%
0%
0%
8. Have you been bothered by headaches during the past 4 weeks?
36%
36%
18%
9%
0%
9. Have you had food cravings in the last 4 weeks?
18%
36%
9%
36%
0%
10. Have you had a loss of appetite during the past 4 weeks?
36%
64%
0%
0%
0%
Excellent
Good
Fair
Poor
Terrible
18%
82%
0%
0%
0%
Terrible
Question
11. Overall, how is your health?
Question
Excellent
Good
Fair
Poor
12. How much more physical pain have you had during the past 4 weeks?
55%
36%
9%
0%
0%
Question
Strongly
agree
Somewhat
agree
Neither
agree nor
disagree
Somewhat
disagree
Strongly
disagree
13. I am comfortable
18%
73%
9%
0%
0%
14. I am as healthy as anybody I know
55%
27%
18%
0%
0%
Table 1
Frequency of answers to questionnaire given before both placebo and Glutalytic.
After Placebo
Question
None of
the time
A little of
the time
Some of
the time
Most of
the time
All of
the time
27%
18%
55%
0%
0%
55%
27%
18%
0%
0%
past 4 weeks?
18%
64%
18%
0%
0%
0%
73%
9%
18%
0%
55%
18%
18%
9%
0%
55%
36%
9%
0%
0%
9%
27%
64%
0%
0%
55%
36%
0%
9%
0%
18%
18%
55%
9%
0%
1.
Question
Question
73%
27%
0%
0%
0%
Excellent
Good
Fair
Poor
Terrible
18%
82%
0%
0%
0%
Excellent
Good
Fair
Poor
Terrible
9%
91%
0%
0%
0%
Question
Strongly
agree
Somewhat
agree
Neither
agree nor
disagree
Somewhat
disagree
Strongly
disagree
9%
82%
9%
0%
0%
18%
73%
9%
0%
0%
None of
the time
A little of
the time
Some of
the time
Most of
the time
All of
the time
36%
64%
0%
0%
0%
Table 2
Frequency of questionnaire response after placebo was given.
After Glutalytic Regimen
Question
1.
91%
9%
0%
0%
0%
past 4 weeks?
45%
45%
9%
0%
0%
18%
73%
9%
0%
0%
64%
36%
0%
0%
0%
73%
27%
0%
0%
0%
36%
45%
18%
0%
0%
64%
27%
9%
0%
0%
36%
55%
0%
9%
0%
Question
64%
18%
18%
0%
0%
Excellent
Good
Fair
Poor
Terrible
64%
27%
9%
0%
0%
Terrible
Question
Excellent
Good
Fair
Poor
64%
36%
0%
0%
0%
Question
Strongly
agree
Somewhat
agree
Neither
agree nor
disagree
Somewhat
disagree
Strongly
disagree
36%
55%
9%
0%
0%
45%
45%
9%
0%
0%
Table 3
Frequency of questionnaire response after regimen was given. Frequencies which differed from the
“before-regimen questionnaire” statistically (p<0.05) are indicated via *. Questions which additionally
did not differ between the pre-regimen and placebo group are indicated with**.
Discussion
As was expected, the highest frequency of
symptoms occurred prior to the clinical, then there
was a slight placebo effect vs. the initial results and
compared to the Glutalytic results. When Glutalytic
was administered, the participants exhibited the
least amount of symptoms (Figure 1). The frequency,
and severity of all of the symptoms were reduced
when Glutalytic was administered.
While there were numerous symptoms that were
reduced by Glutalytic, many were also reduced by
the placebo; for the purposes of this study only
those symptoms that were reduced by Glutalytic,
while not being reduced by the placebo will be
examined. These symptoms included pain in the
upper abdomen, feeling bloated, trouble emptying
bowls, and food cravings (Table 3).
stomach, hunger pains, lower energy levels, and
headaches. The comparison of the placebo effect
to Glutalytic can be seen in Figure 2. Overall the
dietary supplement reduced many of the symptoms
including: pain in the abdominal area, rumbling of
the stomach, feeling bloated, trouble emptying
bowels, hunger pains, lower energy levels, headaches
and food cravings.
Although it was not statistically significant, the
placebo group saw an increase in pain compared
to the pre-regimen. The pain anomaly cannot be
explained without further investigation.
It can be concluded that while Glutalytic cannot
completely diminish the symptoms associated with
gluten sensitivity, it can reduce the frequency and
severity.
Symptoms that were reduced by both the
placebo and Glutalytic included: rumbling of the
Changes from the Initial Questionnaire
n Initial Questionnaire n Placebo n Glutalytic
n Placebo n Glutalytic
Some of the time
A Little of the time
Bloa
ting
Diffi
culty
Emp
tying
Bow
els
Freq
uenc
y of
Hun
ger P
ains
Freq
uenc
y of
Low
Ener
gy
Freq
uenc
y of
Head
ache
s
Freq
uenc
y of
Foo
d Cr
avin
gs
Freq
uenc
y of
Loss
of A
ppet
ite
Freq
uenc
y of
Phys
ical P
ain
Rum
bling
Freq
uenc
y of
Freq
uenc
y of
Nau
sea
Freq
uenc
y of
Freq
uenc
y of
Freq
uenc
y of
Stom
ach
Pain
None of the time
Figure 1. Direct Comparison of the Three
Questionnaires
100
80
60
40
20
0
-20
-40
-60
Som
ach
Pain
Dec
reas
e in
Nau
sea
Dec
reas
e in
Rum
bling
Dec
Dec
reas
reas
e in
e in
B
loatin
Diffic
g
ulty
Emp
tying
B
o
wels
Dec
reas
e in
Hun
ger P
ains
Incre
ase
in En
ergy
Leve
ls
Dec
reas
e in
Head
a
Dec
ches
reas
e in
Foo
d Cr
avin
gs
Incre
ase
in A
ppet
Incre
ite
ase
in O
vera
ll He
alth
Dec
reas
e in
Phys
ical P
Incre
ain
ase
in Co
mfo
rt Le
vel
Most of the time
Dec
reas
e in
All of the time
Percent difference from the initial response
Direct Comparison of the Three Questionnaires
Figure 2. Changes from the Initial Questionnaire to
the Placebo and Glutalytic
References
1. Clin Gastroenterol Hepatol. 2009 Dec;7(12):1328-34, 1334.e1-3. doi: 10.1016/j.cgh.2009.07.031. Epub 2009 Aug 7
2. Caio G, Volta U, Tovoli F, De Giorgio R. Effect of gluten free diet on immune response to gliadin in patients with non-celiac gluten
sensitivity. BMC Gastroenterology. 2014;14(1):1-15.
3. Mooney PD, Aziz I, Sanders DS. Non-celiac gluten sensitivity: Clinical relevance and recommendations for future research.
Neurogastroenterology & Motility. 2013;25(11):864-871.
4. Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large
multicenter study. Arch Intern Med 2003;163:286–292.
Funding
This study was supported by Deerland Enzymes
Tolerance and Efficacy of Glutalytic®
A Randomized, Double-Blind, Placebo-Controlled Study
John Deaton, Ph.D., Gina Labellarte
Deerland Enzymes
3800 Cobb International Boulevard, Kennesaw, Georgia 30152, United States
Study Design
The tolerance and efficacy of Glutalytic® was
assessed in a double-blind, randomized, and placebocontrolled fashion with daily consumption of 3
capsules, with meals, for 30 days. Tolerance and
efficacy were evaluated through a comprehensive
metabolic panel, C-reactive protein levels (CRP-an
inflammatory marker), a gliadin antibody profile (IgG,
IgA, EIA), and a series of gastrointestinal symptom
index questionnaires.
Subject Dynamics
Thirty-seven subjects were recruited for this study.
Subject ages ranged from 19 to 64 years of age with
a mean age of 33.2 ± 12.2 years. Randomization of
capsule assignments, placebo and product groups,
were performed using the SPSS randomizer function.
Blood Sample
For the purposes of this study, blood samples were
obtained and analyzed using Walk-In-Lab LLC, a
certified CLIA lab. Certified phlebotomists drew
blood from the subjects and results were sent
electronically and in accordance with HIPAA Policy.
Study Inclusion Criteria
Criteria for inclusion in the study were adult subjects
(≥18 years at the time of participation), with or
without self-reported gluten intolerance and/or
sensitivity or undefined gastrointestinal problems.
Subjects with a known diagnosis of Celiac Disease
were excluded from the study. Subject participation
was voluntary and subjects were able to terminate
their participation in the study at any time without
penalty.
Prior to Capsule Consumption and Capsule
Distribution
Participants reviewed, signed, and completed the
initial documents packet, which consisted of the
following forms: informed consent, authorization to
use or disclose health information, consent to blood
draw, health questionnaire, and a W9 (Appendix
A). A diagnostic baseline was achieved through the
completion of a self-reported initial gastrointestinal
symptom index questionnaire (Appendix B1) and
an initial, 12-hour fasted, blood sample, taken and
tested, by a third party. After individual baselines
were established, randomly assigned capsules were
distributed in person or by mail. If capsules were sent
by mail, subjects verified the capsule type received
(i.e. Product A or B).
Capsule Consumption Participants were instructed to consume one of
the assigned capsules 3 times daily, with meals, for
30 days. If a dose was missed, participants were
instructed to report it to the researcher immediately.
Recurring incidences of missed doses were taken
into consideration for possible exclusion from the
study. Participants were also instructed to maintain
a dietary intake record throughout the duration of
the study. The intermediate gastrointestinal index
symptom questionnaire was completed 14 days
after the first capsule was taken (Appendix B2). If
an adverse reaction beyond any gluten intolerant
and/or sensitive symptoms a participant may
experience occurred, participants were instructed to
report all questions, concerns, and symptoms to the
researchers immediately and a medical professional
would be consulted if necessary.
Final Day of Capsule Consumption
Upon completion of capsule consumption
participants submitted dietary intake records and
completed the final gastrointestinal index symptom
questionnaire (Appendix B3). Participants provided a
12-hour fasted blood sample, taken and tested, by the
same third party that performed the initial sampling
and testing.
Blood Analysis
A comprehensive metabolic (or safety) panel was
performed in order to ensure Glutalytic was tolerated
by the experimental population. Glucose is measured
to determine blood sugar levels and is the most
direct way of determining a patient’s likelihood
of developing diabetes as well as maintaining it.
Biomarkers such as BUN, creatinine, and BUN/
creatinine ratio, were used to determine kidney
function. Liver biomarkers (protein, albumin, globulin,
albumin/globulin ratio, bilirubin, alkaline phosphatase,
AST, and ALT) were used to determine the state of
nutrition in the body. Fluids and electrolytes were
indicators of nerve and muscle activity as well
as electrolyte balance. C-reactive protein (CRP)
was examined as CRP is an inflammatory marker.
Common causes of elevated CRP include: burns,
trauma, infections, inflammation, arthritis, and certain
cancers. Finally, a gliadin antibody panel was used to
detect anti-gliadin antibodies. The gliadin antibody
panel aids in the diagnosis of celiac disease, wheat
allergy, and non-celiac disorders.
Gastrointestinal Symptom Index Questionnaire
Gastrointestinal Symptom Index Questionnaires
were modified from the Celiac Symptom Index
Questionnaire (Leffler et al. 2009). Questionnaires
were completed by each participant prior to, on day
15, and on the final day of capsule consumption.
Statistical Analysis
Several assumptions were tested out such as Box’s
M, which indicates the homogeneity of covariance
matrices, and the within-subjects contrast, which
indicates that significance of the error between
the time points. In the case where both of these
assumptions were supported a statistical analysis
was performed using the general linear model, more
specifically a MANOVA, to determine the product’s
effect on time by test group. Significance was
determined where α < 0.05 for Wilk’s Lambda. The
analysis was performed on SPSSS Version 22.0 (IBM
Corp., Armonk, NY)
Data Transfer
For the purpose of this study, the first round of
participants’ data was manually inputted and double
checked by senior staff. Subsequent volunteers
were electronically added to the study via direct
import from SurveyMonkey.com in a .csv file. Upon
importation, data was pooled into a master data file
for analysis.
Results
Blood Analysis
The inflammatory marker, CRP, is highly variable
from not only day-to-day but from the time of day
the sample is provided. With that said, initial CRP
levels were statistically indistinguishable between
groups while there was a significant difference
between the final time points of the two CRP levels
using an independent T-Test (α ≤0.05) (Figure 1).
The deamidated gliadin, IgA, is rated high in both
sensitivity and specificity, thus resulting in a high
positive predictive value. Significant differences
between the test groups over time was demonstrated
with an independent T-test p value of 0.024
(Figure 2). As anticipated, all other biomarkers had
insignificant changes as a result of both time and test
group.
Gastrointestinal Symptom Index Questionnaire
The most significant change relates to the amount
of gastrointestinal reflux experienced by subjects.
Specifically, between the beginning and the
intermediate questionnaire, subjects on Glutalytic
experience a significant steep mean drop of 1.64
to 1.14 (p = 0.038) while subjects on the placebo
only experienced a slight change of 1.36 to 1.27.
Additionally, there was a significant effect of
Glutalytic on cravings with a p value of 0.04. There
was a nearly significant effect on bloating with
subjects consuming Glutalytic (p = 0.065). All
averaged answers can be found in Figure 3.
References
Leffler, D.A., Dennis, M., Edwards, G.J., Jamma, S., Cook, E.F., Schuppan, D., Kelly, C.P. (2009). A validated disease-specific symptom index
for adults with celiac disease. Clinical Gastroenterology and Hepatology, 7(12), 1328-1334. doi: 10.1016/j.cgh.2009.09.031
Nabili, S.N. & Shiel, W.C. (2014). C-Reactive Protein. Medicinenet. Html link: http://www.medicinenet.com/script/main/art.
asp?articlekey=47579
www.walkinlab.com
www.webmd.com
Table 1
Comprehensive Metabolic Panel, Gliadin IgG/IgA Antibody Profile, and C-Reactive Protein. Values are expressed
as mean ± standard deviation of the mean. Individual results with an absolute z-score of above 3.29 are not
reflected.
Reference
Interval
Baseline
Average
Enzyme Group
Baseline
Average
Placebo Group
Final
Average
Enzyme Group
89.80 ± 6.78
89.60 ± 5.94
18%
9%
Final
Average
Placebo Group
Comprehensive Metabolic Panel
Glucose, Serum (mg/dL)
65 - 99
BUN (mg/dL)
6 - 20
Creatinine, Serum (mg/dL)
0.57 - 1.00
eGFR (NonAfrican American)
55%
(mL/min/1.73)
> 59
eGFR (African American) (mL/min/1.73)
> 59
BUN/Creatinine Ratio
8 -20
Sodium, Serum (mmol/L)
134 - 144
Potassium, Serum (mmol/L)
3.5 - 5.2
Chloride, Serum (mmol/L)
97 - 108
Carbon Dioxide, Total (mmol/L)
18 - 29
Calcium, Serum (mg/dL)
8.7 - 10.2
Protein, Total, Serum (g/dL)
6.0 - 8.5
Albumin, Serum (g/dL)
3.5 - 5.5
Globulin, Total (g/dL)
1.5 - 4.5
A/G Ratio
1.1 - 2.5
Bilirubin, Total (mg/dL)
0.0 - 1.2
Alkaline Phosphatase, S (IU/L)
39 - 117
AST (SGOT) (IU/L)
0 - 40
ALT (SGPT) (IU/L)
0 - 32
Gliadin IgG/IgA Antibody Profile, EIA
Deamidated Gliadin Abs, IgA
Negative
0 - 19
Weak Positive
90.60 ± 9.91
13.60 ± 4.04
14.00 ± 5.08
97.8 ± 19.23
84.50 ± 7.15
13.40 ± 6.31
14.50 ± 5.60
113.00 ± 22.33
87.70 ± 21.46
101.00 ± 24.65
100.80 ± 19.43
116.20 ± 22.30
86.10 ± 22.76
89.40 ± 37.67
139.80 ± 1.30
141.00 ± 2.11
139.80 ± 0.84
140.00 ± 2.67
101.20 ± 3.19
101.70 ± 2.11
101.20 ± 0.84
100.30 ± 2.31
9.14 ± 0.26
9.52 ± 0.40
9.18 ± 0.28
9.44 ± 0.28
4.36 ± 0.18
4.48 ± 0.30
1.78 ± 0.29
2.00 ± 0.32
53.60 ± 18.19
68.80 ± 20.29
55.40 ± 17.54
59.10 ± 24.76
15.20 ± 5.54
14.20 ± 3.04
19.20 ± 8.44
15.60 ± 4.81
3.20 ± 1.30
5.00 ± 1.63
3.00 ± 1.22
5.30 ± 2.26
2.60 ± 1.34
3.00 ± 1.33
3.00 ± 1.00
2.90 ± 1.10
1.12 ± 1.29
5.27 ± 12.06
0.74 ± 0.59
1.84 ± 1.19
0.92 ± 0.14
18%
14.60 ± 2.61
4.04 ± 0.34
23.80 ± 2.17
6.84 ± 0.36
2.48 ± 0.40
0.60 ± 0.35
18.80 ± 2.78
0.96 ± 0.26
0.90 ± 0.19
13.44 ± 3.29
4.35 ± 0.47
14.60 ± 4.16
4.06 ± 0.21
24.10 ± 1.79
6.78 ± 0.36
2.27 ± 0.24
2.44 ± 0.36
18.10 ± 4.90
0%
23.00 ± 1.41
6.75 ± 0.31
0.48 ± 0.25
0.97 ± 0.30
4.34 ± 0.15
1.78 ± 0.25
0.58 ± 0.30
20.40 ± 5.68
13.67 ± 4.42
4.25 ± 0.30
24.00 ± 1.63
6.81 ± 0.41
4.48 ± 0.15
2.33 ± 0.29
1.94 ± 0.30
0.50 ± 0.25
18.20 ± 3.58
20 - 30
Moderate to Strong Positive
> 30
Deamidated Gliadin Abs, IgG
Negative
0 - 19
Weak Positive
20 - 30
Moderate to Strong Positive
> 30
C-Reactive Protein, Quantitative (mg/L)
0.0 - 4.9
Figure 2. Deamidated Gliadin, IgA Averages.
Figure 1. C-Reactive Protein Averages
Figure 2. Deamidated Gliadin, IgA Averages.
Figure 1. C-Reactive Protein Averages.
C-Reactive Protein
Deamidated Gliadin, IgA
6
6
5
5
4
4
3
3
2
2
1
1
0
0
Initial
Final
Enzyme
Placebo
Initial
Final
Enzyme
Placebo
Figure 3. Gastrointestinal Symptom Questionnaire Averages.
1 = None of the time; 2 = Less than 7 days; 3 = 7-14 days; 4 = More than 14 days; 5 = all of the time
Figure 3. Gastrointestinal Symptom Questionnaire Averages.
1 = None of the time; 2 = Less than 7 days; 3 = 7-14 days; 4 = More than 14 days; 5 = all of the time
2.00
abdomen or the pit of the stomach during the past 4
(2) weeks?
Have you been bothered by nausea during the past 4 (2)
weeks?
1.40
1.80
1.20
1.60
1.00
1.40
1.20
0.80
1.00
0.60
0.50
0.60
0.40
0.40
0.20
0.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Enzyme
Placebo
Initial
Final
When going on the toilet, have you had the sensation of not
completely emptying your bowels during the past 4 (2) weeks?
Intermediate
Final
Has your stomach felt bloated during the past 4 (2) weeks?
2.5
2.00
1.80
2
1.60
1.40
1.5
1.20
1.00
1
0.50
0.60
0.5
0.40
0.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Enzyme
Placebo
Initial
Final
Intermediate
Final
Have you been bothered by low energy levels during the past 4
(2) weeks?
Have you been bothered by hunger pains during the past 4 (2)
weeks?
2.50
2.00
1.80
1.60
2.00
1.40
1.20
1.50
1.00
0.50
1.00
0.60
0.50
0.40
0.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Enzyme
Placebo
Initial
Final
Have you been bothered by headaches during the past 4 (2)
weeks?
Intermediate
Final
Have you had food cravings in the past 4 (2) weeks?
2.50
1.95
2.00
1.90
1.85
1.50
1.80
1.00
1.75
0.50
1.70
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Final
Enzyme
Placebo
Initial
Intermediate
Final
Agree;
= Somewhat
Agree;
3 = Neither
Agree
Disagree;
4 =4Somewhat
Disagree;
= Strongly
11 = Strongly
None of
the2time;
2 = Less
than
7 days;
3 =or7-14
days;
= More than
14 5days;
5 = Disagree
all of the time
Have you experienced a loss of appetite during the past 4 (2)
weeks?
1.60
During the past 4 (2) weeks, how often have you been feeling
down, depressed or hopeless?
1.80
1.60
1.40
1.40
1.20
1.20
1.00
1.00
0.80
0.80
0.60
0.60
0.40
0.40
0.20
0.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Enzyme
Placebo
Initial
Final
Final
During the past 4 (2) weeks, how often have you felt a sense of
irritability?
During the past 4 (2) weeks, how often have you been
bothered by a feeling of nervous anxiety or on edge?
2.00
Intermediate
2.50
1.95
1.90
2.00
1.85
1.80
1.50
1.75
1.70
1.00
1.65
1.60
0.50
1.55
1.50
0.00
0.00
Enzyme
Enzyme
Placebo
Initial
Intermediate
Placebo
Initial
Final
During the past 4 (2) weeks, how frequently have you had difficulty
concentrating on things, such as reading the newspaper
or watching TV?
2.00
Intermediate
Final
During the past 4 (2) weeks, how frequently have you been
bothered with skin problems, such as acne, rashes, or itchiness?
1.60
1.40
1.80
1.60
1.20
1.40
1.00
1.20
0.80
1.00
0.50
0.60
0.60
0.40
0.40
0.20
0.20
0.00
0.00
Enzyme
Enzyme
Placebo
Initial
Intermediate
Final
Placebo
Initial
bothered by joint or muscle aches?
2.00
Intermediate
Final
During the past 4 (2) weeks, how often have you experienced acid
reflux (heartburn)?
1.80
1.80
1.60
1.60
1.40
1.40
1.20
1.20
1.00
1.00
0.80
0.50
0.60
0.60
0.40
0.40
0.20
0.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Final
Enzyme
Placebo
Initial
Intermediate
Final
Stronglyof
Agree;
= Somewhat
Agree;
3 = Neither
Agree
Disagree;
= Somewhat
Disagree;
5 = Strongly
Disagree
1 1== None
the 2time;
2 = Less
than
7 days;
3 = or
7-14
days;4 4
= More than
14 days;
5 = all
of the time
bothered with diarrhea?
During the past 4 (2) weeks, how often have you been bothered
with constipation?
1.60
1.55
1.40
1.50
1.20
1.45
1.00
1.40
0.80
1.35
0.60
1.30
0.40
1.25
0.20
1.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
Enzyme
Placebo
Initial
Final
During the past 4 (2) weeks, how frequently have you been
bothered with gas?
Final
During the past 4 (2) weeks, how frequently have you
experienced difficulty sleeping?
2.50
2.50
2.00
2.00
1.50
1.50
1.00
1.00
0.50
0.50
0.00
Intermediate
0.00
Enzyme
Placebo
Initial
Intermediate
Enzyme
Placebo
Initial
Final
How much physical pain have you had during the past 4 (2)
weeks?
2.00
Intermediate
Final
During the past 4 (2) weeks, have you experienced any
unexplained weight loss or weight gain?
1.40
1.80
1.20
1.60
1.00
1.40
1.20
0.80
1.00
0.60
0.80
0.60
0.40
0.40
0.20
0.20
0.00
0.00
Enzyme
Placebo
Initial
Intermediate
I am as healthy as anybody I know
2.50
2.00
1.50
1.00
0.50
0.00
Enzyme
Placebo
Initial
Intermediate
Final
Enzyme
Placebo
Initial
Final
Intermediate
Final
APPENDIX A
All Initial Documents
Informed Consent for Participating in a Glutalytic®
Clinical Research Study
Effect of Consumption of a Snack
with a Dietary Supplement or Placebo
I understand that I am being asked to be a volunteer in a research study. I have been
informed that the purpose of this research is to study the effects of a specific dietary
supplement on my body when included with a meal. It is expected that up to 60
adults will be recruited to participate in this study. The supplement may (if I’m in the
treatment group) or may not (if I’m in the placebo group) contain digestive enzymes.
An enzyme is a chemical that helps modify other chemicals. Digestive enzymes
that are normally in our gut modify nutrients in our foods. My participation in this
study will involve two trips to the designated clinic. On the first day, I will review this
informed consent and if I give informed consent to participate in the study, I will fill
out a brief health history, after which, I will be informed that I will or will not be eligible
to participate in the study. Criteria for inclusion in the study are adult age (18-75
years of age at time of participation), no illnesses at the time of recruitment, and no
use of antibiotics for at least seven days prior to recruitment. Subjects must report
any illnesses or use of antibiotics prior to recruitment and at any point throughout
the study and will be excluded if an illness or antibiotic use is reported at any point
throughout the study. Subjects with known health conditions must consult with their
physician prior to participating in this study. If eligible, I will sign up for a second day,
on which I will be given capsules containing a dietary supplement, or placebo (I will not
be told or know which), to take prior to my meal for 4 weeks. I will consume these two
items within a defined time frame prescribed by the researchers (no more than one
hour) and use these items as indicated. I will not open the capsules, as contact with
the capsule ingredients may cause skin irritation. I will abstain from any other dietary
supplements throughout the timeframe of the trial. I will then be asked to complete
two surveys asking about how my body feels at specified times throughout the same
day. I will have my blood collected at the beginning and end of the trial. I will then
return my completed surveys to the investigators and will be offered $100 as a thank
you for my participation.
I realize that my participation in this study is voluntary, though if I complete the study,
I may accept $100 as a thank you for my participation. I may also learn something
about the effectiveness of a dietary supplement after the conclusion of the study. The
results of this study may be published in scientific journals or presented at professional
meetings. However, none of my data will be linked to my name or any personally
identifying information. The reporting of experimental results will only contain group
mean results and will contain no personal information about individual participants
including performance on the study. My records will be kept in locked files and only
study staff will be allowed to look at them.
I realize that I may withdraw from this study at any time, for any reason. I realize that
the researchers may want me to contact them by email or phone with any questions
or concerns I have before, during, and after the study. Questions regarding study
procedures may be directed to the Investigators, John Deaton, PhD, RD (678-3917407).
I understand that there are minimal risks of participation in the study. I will have
blood drawn on two occasions. The blood will be taken from the antecubital fossa
(arm). The risks of taking blood include pain, a bruise at the point where the blood
is taken, redness and swelling of the vein and infection, and a rare risk of fainting.
There is also a risk of mild physical symptoms. Should an adverse reaction to the
supplement occur, it should be reported immediately to the researcher and a medical
professional at the Science Center will be consulted if necessary. If the symptoms are
severe, emergency (911) care may be requested. In the unlikely event that any injury or
illness occurs as a result of this research, Deerland Enzymes, their officers, agents and
employees, will not automatically provide reimbursement for medical care or other
compensation. Payment for treatment of any injury or illness must be provided by the
subject or subject’s third-party payer, such as health insurer or Medicare. If any injury
or illness occurs in the course of research, or for more information, I should notify the
investigator in charge.
I have been informed of and understand the following:
• I have the right to change my mind and leave the study at any time, without giving
any reason and without penalty.
• I will be given a copy of this consent form to keep.
• I am not waiving any rights that I may have for injury resulting from negligence of any
person.
Questions regarding study procedures may be directed to the Investigator, John
Deaton, PhD, RD (678-3917407) Deerland Enzymes, 3800 Cobb International Blvd,
Questions regarding the protection of human subjects may be addressed to jwarren@
deerlandenzymes.com.
By signing below, I am acknowledging that I have read (or had read to me) the
information in this consent form, that I do not have any of the conditions that this form
indicate would exclude me from participating in the study, and I would like to be a
participant in this study.
Participant’s Signature: Date:
Participant’s Printed Name: Researcher’s Signature: Date:
Authorization to Use or Disclose (Release) Health Information
that Identifies You for a Research Study
If you sign this document, you give permission to Deerland Enzymes, Inc. to use or disclose (release) health or
other information that identifies you for the research study described here:
Gluten is a protein complex found in commonly consumed grains such as barley, rye, and wheat. In some
individuals, gluten components cause significant gastrointestinal distress and other symptoms. The purpose
of this study is to determine if a dietary supplement (compared to a placebo) included with a normal diet
results in reduced symptoms of gluten sensitivity in a standard population. Briefly, up to 60 adult subjects
meeting the following criteria will be recruited: 1) Willing to abstain from other dietary supplements through
the four week clinical, 2) willing to come between 7:00 am and 6:00 pm at the beginning and end of the
trial. 3) Willing to consume dietary supplement or placebo in capsule forms 4) willing to complete three
questionnaires (modified versions of the attached Celiac Symptom Index questionnaire from Leffler et
al.2009, describing symptoms hours (versus weeks) following consumption of meal and supplement) at
specified times on the same day as meal consumption and return them to the study investigators. The dietary
supplement and placebo will be supplied by Deerland Enzymes, Kennesaw GA (who fill fund the study, if
approved). 5) Willing to have their blood sampled at the beginning and end of the trial. The study will be
conducted during the spring of 2015.
You have agreed to participate in the study described above and have signed or will sign a separate informed
consent that explains the procedures of the study and the risks and benefits of participation.
The health information that may be collected for this research includes personal identifiers such as name,
date of birth, and race, and general health history data. To protect your privacy, your records will be kept
in locked files and only study staff will be allowed to look at them. Although certain data will be used and
disclosed for research purposes, your name and other personally identifying information will not be used
other than in initial documents. No data released for purposes of evaluating and publishing the results of the
study will be linked to your personal identifying information. Such data will be presented in de-identified form
only that cannot identify you. This information may be used and/or disclosed to: Kennesaw State University,
LabCorp, and WalkinLab.
By signing this document, you authorize Deerland Enzymes to use and/or disclose (release) your health
information as described above for this research. Those persons who receive your health information may
not be required by Federal privacy laws (such as the HIPAA Privacy Rule) to protect it and may share your
information with others without your permission, if they are permitted to do so by other laws governing them.
Please note that:
• You are not required to sign this Authorization. If you refuse to sign the Authorization you will not be able to
participate in the research study, but will not suffer any loss of benefits to which you are otherwise entitled
• You may cancel this Authorization and withdraw your approval to allow the use and disclosure of your
health information as described here at any time. You must do so in writing to Allison Healey at 3800
Cobb International Blvd, Kennesaw, GA 30152. Even if you withdraw your permission, your personal health
information that was collected before we received your written request may still be used and disclosed, as
necessary for the research. If you withdraw your permission to use your health information, you may also be
withdrawn from the research study.
• You will be provided a copy of this signed Authorization.
This Authorization will expire at the end of the research study.
Signature of participant or participant’s personal representative Date
Printed name of participant or participant’s personal representative If applicable, a description of the personal representative’s authority to sign for the participant.
Consent to Blood Draw for Clinical Research
Study Title: Glutalytic® Clinical Trial
Study Investigator: John Deaton, Ph.D., RD
You have volunteered to participate in a dietary supplement clinical trial being conducted by Deerland
Enzymes. This study includes the collection of blood samples to learn about the effects of a dietary
supplement on the status of your liver, kidneys, and electrolyte and acid/base balance. The blood will also be
analyzed for the detection of gliadin antibodies to monitor certain gluten-sensitive enteropathies.
Your donation of blood for this clinical trial is voluntary, and you do not have to participate in the study or
donate blood samples. Any personal information provided by you in connection with the collection and
donation of blood will be held in confidence. For reasons of safety, you should not donate if:
• You know, or think that you might be infected with hepatitis B or hepatitis C.
• You know, or think that you might be infected with HIV – the AIDs virus
• You have a sexual partner who is infected with hepatitis or HIV
• You are unwell at the moment
• You are anemic or receiving treatment for anemia or iron deficiency
• You are, or may be, pregnant
• You have given blood in the last 1 month (if more than 100 ml is requested)
What is the Procedure?
Blood will be collected at the beginning and end of the trial. If you give a blood sample, you will be seated
and blood will be drawn by putting a needle into a vein in your arm. One small tube of blood will be taken.
This will take about five minutes.
Are there risks?
The needle stick may hurt. There is a small risk of bruising, a rare risk of infection, and you may feel
lightheaded.
Are there benefits?
There is no benefit to you. The blood will be used only for clinical research. CONSENT TO PARTICIPATE
You have been given copies of this consent form to keep.
You have signed a separate Informed Consent that explains the procedures of the study and a separate form
authorizing access, use, or disclosure of health information about you.
BY SIGNING BELOW, I ACKNOWLEDGE I HAVE READ THIS CONSENT FORM OR HAD IT READ OR
EXPLAINED TO ME AND I UNDERSTAND THIS FORM. I HAVE BEEN GIVEN AMPLE OPPORTUNITY TO
ASK QUESTIONS AND ANY QUESTIONS I HAVE ASKED HAVE BEEN ANSWERED SATISFACTORILY. I
VOLUNTARILY CONSENT TO THE DRAWING OF BLOOD FOR THE REASONS SET FORTH HEREIN. Participant’s Signature and Date
Participant’s Name Printed
Person Obtaining Consent Signature and Date
Person Obtaining Consent Printed
HEALTH AND FOOD QUESTIONNAIRE
Name: First MI Last
Address: House Number and Street Email: Phone: City State
PERSONAL BACKGROUND INFORMATION
1.
( ) Male ( ) Female
2.
Age (years): DOB (MM/DD/YYYY): 3.
Height (ft’ in’’): 4.
Current weight (lbs):
Lowest weight for height (lbs): Recent change in weight (lbs)? (
5.
6.
Which of the following categories best describes your racial/ethnic background?
( ) Caucasian
( ) Asian or Pacific Islander
( ) African American ( ) American Indian/Native American
( ) Hispanic/Latino ( ) Other. Please specify
Do you smoke?
( ) Yes ( ) No ( ) On occasion. Please specify: 7.
Check any health problems that you have (or have had):
( ) Celiac Disease
( ) Gastroesophageal Reflux Disease (GERD)
( ) Gluten Intolerance/Sensitivity
( ) Irritable Bowel Syndrome (IBS)
( ) Diverticular Disease
( ) Inflammatory Bowel Disorder
( ) Chronic Liver Disease
( ) Crohn’s Disease
( ) Heart Failure
( ) Ulcerative Colitis
8.
Check any autoimmune disorders that may apply:
( ) Type 1 Diabetes
( ) Hashimoto’s Thyroiditis
( ) Psoriac or Rheumatoid Arthritis
9.
Check any blood-borne diseases that may apply:
( ) HIV/AIDS ( ) Hepatitis
10.
Please list all prescription and over the counter medications or supplements you are currently taking:
Medicine/Supplement ) gain Highest weight for height (lbs): ( ) loss ( )no change
Reason for taking Dose Frequency of dose
APPENDIX B
Gastrointestinal Symptom Index Questionnaires
B1. Initial Questionnaire
B2. Intermediate Questionnaire
B3. Final Questionnaire
APPENDIX C
Protease: Literature Review
Anamaria Cuentas
Gastrointestinal System
The gastrointestinal (GI) system, or alimentary canal, is a hollow tube which consists of: the mouth, pharynx,
esophagus, stomach, small intestine, large intestine, and anus. The aligned system is responsible for chemical
and mechanical breakdown of foods and liquids into useable energy sources. Mechanical digestion consists
of mastication, propulsion or peristalsis, and mixing of foodstuff and chemical digestion exploits the harsh
conditions of the GI system (i.e. varying pH levels and secreted enzymes). Subsequent breakdown of ingested
food into a semiliquid, or chyme, nutrients are able to be absorbed in order to provide energy, build, and
repair tissue.
Extensive digestion and absorption occurs in the small intestine. Measuring 6 meters in length, the small
intestine utilizes digestive enzymes secreted from the pancreas, liver, and gall bladder. Millions of finger-like
projections, also known as villi, provide a remarkable 250 square-meter surface area for optimal absorption.
Various GI conditions are prevalent worldwide across the socio-economic spectrum, many of which affect the
small intestine (Angus 2015). Small intestinal inflammation, with or without, villous atrophy can be deleterious
to the body’s ability to absorb essential nutrients and may be a diagnostic marker for inflammatory disorders,
such as celiac disease (Allen 2004 and Ludvigsson et al. 2009).
Gluten-Related Disorders and Diets
Celiac disease (CD) is an immune-mediated disorder triggered by ingestion of the protein, gluten, which
results in hypersensitivity to certain antigens. Predisposed individuals are unable to break down gluten;
further leading to inflammatory injury or atrophy of villi and results in various clinical ramifications (Lahdeaho
et al. 2014 and Ludvigsson et al. 2009). Therefore, gluten has been identified as a leading cause of a variety
of GI and extraintestinal conditions (Holmes 2009). However, CD frequently goes undiagnosed due to the
varying symptoms.
Hypersensitivity to gluten has forced the afflicted remove gluten from their diets, hence gluten-free diets
(GFDs). Gluten is a protein compound, which stems from the endosperm of wheat, rye, and barley, and its
prominent in the food industry makes complete avoidance challenging (Jurgelewicz, 2015 and Lahdeaho
2014). Subsequently, GFDs can be expensive and therefore, may be unattainable for people in many
countries and compliance may be difficult in certain age-specific populations, such as adolescents (Holmes
et al. 2009). Hence, a strong need for alternatives that allow for the unavoidable consumption of gluten. Even
after the transition to a GFD, many patients continue to experience, requiring other measures to be taken
(Allen, 2015).
Proteolytic Enzymes
A potential cost-effective solution to the aforementioned problem is the implementation of a protease as an
oral supplement. Proteases are enzymes that catalyze hydrolysis of the peptide bonds that link amino acids
together. Proteases aid in the breakdown of substances, such as gluten, and offer affected individuals an
alternative approach to their dietary needs without extreme dietary restrictions. Lahdeaho et al. developed a
study to test the possibility of Glutenase ALV003 reducing gluten-induced small intestinal injury in patients
with celiac disease. The study revealed that the use of “orally administered gluten-specific proteases was able
to reduce gluten-induced mucosal injury in celiac disease patients.”
Other studies have demonstrated that the use of bacterial and fungal proteases have the capability to
improve gluten-related inflammation by reducing gluten concentration in foods (M’hir et al, 2009). The use
of proteases also shows a reduction in autoantibodies of patients with persistent seropositivity caused by
nonadherence to a GFD (Horowitz, 2011). A combination of two common (unspecified) enzyme supplements
may result in appropriate breakdown of gluten before absorption in the GI tract; and therefore, produce
a clinically significant decrease in autoantibodies of patients with celiac disease (Horowitz, 2011). Oral
formulations of enzymes have proposed an innovative and likely alternative to the rising issue (Holmes,
2009).
Conclusion
Alleviation of gluten-induced symptoms is an unmet need that could be supplemented with enzymatic
support. Although GFDs are positively enforced, in certain cases this is not enough. While new ingredients
for the GFD are currently under evaluation and new technologies are constantly improving the quality of
food that is suitable for individuals with celiac disease, results are still not fully satisfactory (Jurgelewicz,
2015). Oral proteases or protease blends that are composed exclusively of commercially available food-grade
enzymes could be a viable alternative (Horowicz, 2011). An enzyme blend that can nullify “immunogenic
gluten peptides” may be clinically valuable (Lahdeaho et al, 2014). As observed in various studies, patients
who have strictly followed a GFD but still experience symptoms may benefit from the implementation of
protease supplements and in turn greatly increase an individual’s quality of life.
References
Allen, P. J. (2015). Primary Care Approaches. Gluten-Related Disorders: Celiac Disease, Gluten Allergy, Non-Celiac Gluten Sensitivity.
Pediatric Nursing, 41(3), 146-150.
Angus, K., Asgharifar, S., Gleberzon, B. (2015). What effect does chiropractic treatment have on gastrointestinal (GI) disorders: a narrative
review of the literature? Journal of the Canadian Chiropractic Association, 59(2), 122-133.
Ehren, J., Moron, B., Martin, E., Bethune, M. T., Gray, G. M., & Khosla, C. (2009). A Food-Grade Enzyme Preparation with Modest Gluten
Detoxification Properties. Plos ONE, 4(7), 1-10. doi:10.1371/journal.pone.0006313
Holmes, G., Catassi, C., & Fasano, A. (2009). Chapter 10: Future trends. Fast Facts: Celiac Disease (pp. 116-123). Health Press Limited.
Horowitz, S. (2011). Celiac Disease New Directions in Diagnosis, Treatment, and Prevention. Alternative & Complementary Therapies, 17(2),
92-98. doi:10.1089/act.2011.17205
Johanson, L. (2015). The Gluten-Free Frenzy: Fad or Fitting? MEDSURG Nursing, 24(4), 213-217.
Jurgelewicz, M. (2015). Is a Gluten-Free Diet Enough for Patients with Celiac Disease? Nutritional Perspectives: Journal of the Council on
Nutrition, 38(1), 8-10. Retrieved from: http://eds.b.ebscohost.com/eds/
Lahdeaho, M., Kaukinen, K., Laurila, K., Vuotikka, P., Koivurova, O., Karja-Lahdensuu, T., Marcantonio, A., Adelman, D. C., & Maki, M. (2014).
Glutenase ALV003 Attenuates Gluten-Induced Mucosal Injury in Patients With Celiac Disease. Gastroenterology, 146, 1649-1658.
Ludvigsson, J.F., Montgomery, S.M., Ekbom, A., Brandt, L., Granath, F. (2009). Small-Intestinal histopathology and Mortality Risk in Celiac
Disease. The Journal of the American Medical Association, 302(11), 1171-1178. doi: 10.1001/jama.2009.1320
McAllister, C., & Kagnoff, M. (2012). The immunopathogenesis of celiac disease reveals possible therapies beyond the gluten-free diet.
Seminars in Immunopathology, 34(4), 581. doi:10.1007/s00281-012-0318-8
M’hir, S., Rizzello, C. G., Di Cagno, R., Cassone, A., and Hamdi, M. (2009). Use of selected enterococci and Rhizopus oryzae proteases
to hydrolyse wheat proteins responsible for celiac disease. Journal of Applied Microbiology, 106(2), 421-431. doi:10.1111/j.13652672.2008.04008.x

Studies - Via Farma

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