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Title Nutritional assessment of Einkorn ancient wheat for gluten and anti-gliadin antibodies (AGA) to be used in gluten sensitive population Doctor of Philosophy in Clinical Research

Authors:

Abstract

Gluten sensitivity (gluten intolerance) is a spectrum of disorders, including celiac disease in which gluten has an adverse effect on the body. Symptoms include bloating, abdominal discomfort or pain, diarrhea, muscular disturbances and bone or joint pain. [12-13]. Methodology of this study include 120 subjects, medical examination done by investigators before enrolment. Blood samples collected for gluten antibodies (anti-gliadin antibodies) to rule out pre-existence of disease, those are elisa positive were excluded from study. After fitness all subjects were randomly assigned to two arms consisting of four groups 1. BMI < 18.5, 2. BMI>18.5 to 25 & 3. BMI < 18.5, & 4. BMI>18.5 to 25. Arm 1 Group 1 & 2 provided Einkorn Wheat Chapati while group Arm2 , Group 3 & 4 with normal available wheat chapatti, 300 gms each for21 days. After three months again 3 ml blood collected for gluten antibodies (anti-gliadin antibodies) to rule out impact of Einkorn and local market wheat flour. SPSS was used for Statistical analysis.
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TEXILA AMERICAN UNIVERSITY, GEORGETOWN
A MATHEWS, M.SC., K YADAV, PH. D., R BHAMRA, PH.D. , N. MURTHY, M. PHIL
KEY WORDS: ANTI-GLIADIN ANTIBODIES, GLUTEN ALLERGY, GLUTEN SENSITIVITY, WHEAT, NUTRITION
ABSTRACT:
Gluten sensitivity (gluten intolerance) is a spectrum of disorders, including celiac disease in which gluten
has an adverse effect on the body. Symptoms include bloating, abdominal discomfort or pain, diarrhea,
muscular disturbances and bone or joint pain. Methodology of this study included 120 subjects. Subject
were consented and screened to undergo screening procedure and assessment conducted before enrolment.
Blood samples collected for gluten antibodies (anti-gliadin antibodies) to rule out pre-existence of disease,
those are Elisa positive were excluded from study. After assessment at baseline visit subjects were randomly
assigned to two arms consisting of four groups 1. BMI < 18.5, 2. BMI>18.5 to 25 & 3. BMI < 18.5, & 4.
BMI>18.5 to 25. Arm 1 Group 1 & 2 provided Einkorn Wheat Chapati while group Arm2, Group 3 & 4
with normal available wheat chapatti, 300 gms each for21 days (3 Weeks) with 2 weeks safety follow up.
At the baseline 3 ml of blood collected and to perform and tests performed to check anti gliadin antibodies
and gluten reactivity in both arms. After three months again 3 ml blood collected for gluten antibodies (anti-
gliadin antibodies) to rule out impact of Einkorn and local market wheat flour. SPSS was used for Statistical
analysis.
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INTRODUCTION
Malnutrition is the condition that results from
eating a diet in which certain nutrients are
lacking, in excess (too high in intake), or in the
wrong proportions. Several different nutrition
disorders may arise, depending on which
nutrients are under- or over-abundant in the diet.
In most of the world, malnutrition is present in the
form of under-nutrition, which is caused by a diet
lacking adequate calories and protein, not enough
food, and of poor quality. Extreme
undernourishment is starvation, and its symptoms
and effects are inanition. While malnutrition is
more common in less-developed countries, it is
also present in industrialized countries. It is more
likely to be caused by unhealthy diets with excess
energy, fats, and refined carbohydrates.
In this study subjects having mild to moderate
Gluten sensitivity will be provided with einkorn
and normal wheat. Purpose of selection of this
population is to minimize deviation in data their
health assessment.
Wheat gluten, also called seitan, wheat meat,
mock duck, gluten meat, or simply gluten, is a
food made from gluten, the main protein of
wheat. It is made by washing wheat dough with
water until all the starch dissolves, leaving
insoluble gluten as an elastic mass which is then
cooked before being eaten.
A gluten-free diet (GF diet) is a diet that excludes
foods containing gluten. Gluten is a protein
complex
A Gluten-free diet (GF diet) is a diet that excludes
foods containing gluten. Gluten is a protein
complex found in wheat (Including einkorn,
kamut and spelt), barley, rye and triticale. A
gluten-free diet is the only medically accepted
treatment for celiac disease. [2] Being gluten
intolerant can often mean a person may also be
wheat intolerant as well as suffer from the related
inflammatory skin condition dermatitis
herpetiformis, [3] there are a smaller minority of
people who suffer from wheat intolerance alone
and are tolerant to gluten
METHOD
A randomized, double-blind comparative study
of Ancient Einkorn wheat and modern wheat.
Study groups: Total 120 Subjects are sub-divided
in four different groups as given, subjects were
randomized in to two arms. Approx. 340 subjects
screened to allow the 60 subjects randomized in
each arm. 10% drop out rate.
Arm 1
Group I:BMI15-18.5 (Very severely underweight
to Underweight) given three CHAPPATIS(twice
a day), total of 300 grams per day for 21 days,
Einkorn wheat flour 30 subjects (Mix population
of equal sex).
Group II:BMI 18.5-25 (NORMAL) GIVEN three
CHAPATTI’S(TWICE a day), total of 300 grams
per day for 21 days, Einkorn wheat flour , 30
subjects( Mix population of equal sex)
Arm 2
Group III: BMI 15-18.5 (very severely
underweight to underweight) given three
CHAPATIS (TWICE a day), total of 300 grams
per day for 21 days, wheat procured from local
market and grinded by conventional flour mill
available locally, 30 subjects (Mix population of
equal sex).
Group IV: BMI 18.5-25 (NORMAL) GIVEN
three CHAPPATIS (TWICE a day), total of 300
grams per day for 21 days, wheat procured from
local market and grinded by conventional flour
mill available locally, 30 subjects(Mix population
of equal sex).
Subject were consented and screened to undergo
screening procedure and medical examination
conducted by investigator for enrolment. Blood
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samples collected for gluten antibodies (anti-
gliadin antibodies) to rule out pre-existence of
disease, those are Elisa positive were excluded
from study. After assessment at baseline visit
subjects were randomly assigned to two arms
consisting of four groups 1. BMI < 18.5, 2.
BMI>18.5 to 25 & 3. BMI < 18.5, & 4. BMI>18.5
to 25.
Arm 1 Group 1 & 2 provided Einkorn Wheat
Chapati while group Arm2, Group 3 & 4 with
normal available wheat chapatti, 300 gms each
for21 days (3 Weeks) with 2 weeks safety follow
up. At the baseline 3 ml of blood collected and to
perform and tests PERFORMED TO check anti
gliadin antibodies and gluten reactivity in both
arms. After end of treatment again 3 ml blood
samples collected for gluten antibodies (anti-
gliadin antibodies) to rule out impact of Einkorn
and local market wheat flour.
SPSS was used for Statistical analysis.
RESEARCH DESIGN
SAMPLE SIZE and STASTICAL ANALYSIS
Primary analysis: Test gluten levels of einkorn
wheat compared with standard wheat using the
response rate at week3, assuming reduction in
gluten level Arm 1 when compared to Arm2,
power 80% USED for sample size calculation.
Data recorded was assessed by t test Inferential
statistics to draw inferences about the population
represented, accounting for randomness. These
inferences may take the form of: answering
yes/no questions about the data (hypothesis
testing), estimating numerical characteristics of
the data (estimation), describing associations
with THE data (CORRELATION) and
Modelling relationships within the data using
regression analysis.
This STUDY ENROLLED approximately 60
patients in each treatment Arm.
Sample size calculations were performed for a
two-group t-test comparing means for the dose
group (300 gms of EINKORN) versus normal
wheat (assuming equal variances). In order to
detect a difference of -0.5% in Gluten between
treatment groups, 60 randomized patients per
treatment group given a power of 80% with a
common standard deviation of 1% and an alpha
level of 0.05 (2-sided).
INCLUSION/EXCLUSION CRITERIA
Inclusion criteria:
1. ≥ 18 years old at the time of screening
2. Must be able to understand and
communicate with the investigator and
comply with the requirements of the
study and must give a written, signed,
and dated informed consent before any
study-related activity is performed.
Where relevant, a legal representative
will also sign the informed study consent
according to local laws and regulations
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3. BMI15-18.5 (Very severely underweight
to Underweight) and BMI15-18.5-25
(Normal)
4. Free from any metabolic disorders
Exclusion criteria:
1. Subjects not willing to provide written
informed consent or have problem
adhering to study procedures
2. Suffering with chronic infectious
disease.
3. Liver, kidney and cardiac diseases.
4. Obese and below 18 years age
5. Subject is a family member or employee
of the investigator
6. Non-cooperative during study
STUDY POPULATION AND DATA
COLLECTION
All subjects recruited in this study from
residential hostels were examined by physician.
Those subjects declared fit for study criteria
matching inclusion and exclusion criteria were
enrolled into study after written and audio-visual
consent as per the recent guidelines
recommended by DCGI. ICMR guidelines for
human research and ICH GCP were followed
strictly. Height, Weight, Age, and Sex are
recorded in patient’s data sheet.
Blood glucose, total proteins, albumin measured
by photometry, globulin and albumin- globulin
ratio calculated from standard measurements,
complete Blood Count (CBC) for evolution of
Hemoglobin and other blood indices measured on
first day of study and on the last DAY (21st day)
of the study.
Wheat levels for Gluten is measured in triplicates
for einkorn wheat flour and wheat procured from
local market and grinded by conventional flour
mill. Blood anti-gliadin antibodies (AGA)
measured on first day of study and on the last day
(21st day) of the study. ELISA test done to assess
antibodies at baseline and end of treatment.
Height, weight and BMI assesses at baseline and
end of treatment
Data recorded in Paper case report forms
ETHICAL CONSIDERATION
Ethical approval was taken by the guide from the
Institutional review board, D.Y. Patil prior to
commence of the study. Those subjects declared
fit for study criteria matching inclusion and
exclusion criteria were enrolled into study post
consent. ICMR guidelines for human research
and ICH GCP were followed strictly. No conflict
of interest by any investigators
RESULTS AND INTERPRETATION
IGE and AGA reactivity was found to be
significant p<0.05 for Arm 1 when compared to
Arm 2
Gliadin antibodies were higher in Arm 2
compared to Arm 1
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FIG 1 BARGRAPH OF DIFFERENCE IN
GROUPS IGE/AGA REACTIVY IN TWO
ARMS
FIG 2 BARGRAPH OF COMPARASIOM IN
GROUPS ANTIGLIADIN ANTIBODIES
REACTIVY IN TWO ARMS AT BASELINE
AND END OF TREATMENT
BMI index mean between various groups at
baseline and end of treatment p>0.005
mean of BMI in
each group AT
BASELINE(n=30)
mean of BMI in
each group at end of
treatment (N=30)
Group
1
16
16.5
Group
2
23
22
Group
3
16.5
18.5
Group
4
23.5
25
Table 1 MEAN BMI INDEX AT BASELINE
AND END OF TREATMENT
FIG 3. PLOT OF CHANGE IN BMI INDEX
FROM BASELINE TO EOT
In term of toxicity profile, in difference with more
modern forms of wheat the gliadin protein of
einkorn did not show significance in toxicity.
However, we haven’t come across any
recommendation for celiac disease profile if this
would be safe to consume. It has not yet been
recommended in any gluten-free diet. Einkorn
wheat exhibits contents of gluten, but quantity
was different from modern wheat, as it consists of
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14 chromosomes as opposed to 42 in modern
wheat. This alters the gluten structure which may
be why it does not affect those with gluten
intolerance as much as other wheat.
DISCUSION AND CONCLUSION
Data available from research shows that there is
not much available on the contents and
compositions of gluten components in ancient
wheats. However, the data that are available
demonstrates that there is a difference in terms of
contents from modern wheat species mostly
bioactive components and may be lower in some
components (such as dietary fiber).
The remarkable difference from normal wheat
other than gluten is high contents of the
carotenoid lutein in einkorn. These analyses do
suggest that einkorn wheats which are ancient
wheat are generally “healthier” in terms of their
gluten contents than normal modern wheat.
However, we would recommend further detailed
studies needed with multiple genotypes of
einkorn and modern wheat species grown in
replicate multi-site field trials and analyzed with
standard methods. Also, a future research should
include gluten sensitive population to compare
Einkorn level sensitivity for its equivalence when
compared to gluten free diet. This will validate
further to prove if Einkorn wheat products are
safe and efficacious and could be used as a
substitute to gluten free diet in population have
gluten sensitivity or wheat allergy. So far, the
studies on einkorn’s toxicity have been mixed,
although it is considered less toxic than modern
wheat. But we do not recommend celiac patients
to consume einkorn.
CONCLUSION
Einkorn does not have the crease that’s present on
the side of modern wheat grains, which arose in
modern wheat due to being genetically altered by
choosing seeds that delivered more gluten and
higher yields that are ideal for large-scale
production and distribution in larger farms.
Einkorn has gluten, but it may be a healthier
version, making it easier to digest compared
to the gluten found in modern wheat. It doesn’t
contain the D genome but rather the A genome, a
significant difference because the most popular
test for detecting the presence of gluten is based
on the presence of the D genome. Although
Einkorn does contain gluten, it’s a different type
of gluten and passes the ELISA test which is a
commonly used laboratory test to detect
antibodies in the blood. Einkorn is a diploid like
most plants, meaning it has two sets of
chromosomes, while modern bread wheat has six
sets. Einkorn is clearly the most ancient and
purest type of wheat with only two sets of
chromosomes, meaning its natural gluten content
is low, making it a healthy food. Einkorn contains
more carotenoids, which can help in preventing
serious diseases, such as cancer, whereas
carotenoids are harder to find in modern whole
wheat.
LIMITATIONS
Sample size (wider population and centres
required)
Data collection (TIMING and support from
staff was challenging)
Funding
Limited review data or study on modern and
ancient wheat
Supply of product
Cost
FUTURE SCOPE
A future research should include gluten sensitive
population to compare Einkorn level sensitivity
for its equivalence when compared to gluten free
diet. This will validate further to prove if Einkorn
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wheat products are safe and efficacious and could
be used as a substitute to gluten free diet in
population have gluten sensitivity or wheat
allergy.
Celiac disease and population in Celiac disease
are yet to be assessed to further confirm if
Einkorn is suitable to use in such population
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