Download A Combination High in Antioxidant Foods` Effects

A Combination High in Antioxidant Foods’ Effects on Blood Antioxidant and Oxidative
Stress Levels in Post-Menopausal Women.
by
Shelby Kloiber, B.S.
A Thesis
in
Health, Exercise, and Sport Sciences
Submitted in partial fulfillment of the requirements for the Degree of
Master of Sciences
Approved by
Melanie A. Hart, Ph.D.
Co-Chair of Committee
Jacalyn McComb, Ph.D.
Co-Chair
Yoonjung Park, Ph.D.
Peggy Gordon Miller
Dean of the Graduate School
August, 2011
Copyright
2011
Shelby Kloiber
Texas Tech University, Shelby Kloiber, August 2011
Acknowledgements
I would like to show my gratitude towards the individuals who supported and
assisted me during research. Dr. Robert Sawyer for guiding me through the research, Will
Martin, graduate student, for assisting me during my blood work and assay completion,
Dr. Jamie Cooper for allowing me to use her lab facilities during the research, and to my
parents Rick and Lydia Kloiber for their guidance and support during my entire
education. I would also like to send appreciation to Dr. Joaquin Gonzales for his help
and his direction with this project.
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Texas Tech University, Shelby Kloiber, August 2011
Table of Contents
Acknowledgements
ii
Abstract
vi
List of Tables
vii
I. Introduction
1
Statement of the Purpose
4
Significance of Study
4
Hypotheses
5
Delimitations
5
Limitations
6
Assumptions
6
Definition of Terms
6
II. Review of Literature
8
Oxidative Stress
9
Free Radicals (ROS)
10
Antioxidants
11
Menopause and Oxidative Stress
12
Antioxidants and Oxidative Stress
13
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Women and Antioxidants
15
Summary
20
III. Methodology
21
Participants
21
Procedure
22
Hematocrit and Anthropometric data collection
23
Antioxidant capacity and oxidative stress data collection
24
Intervention protocol
25
Antioxidant capacity assay protocol
25
Oxidative stress assay protocol
26
Data Analysis
26
IV. Results
28
Oxidative Stress
28
Antioxidant Capacity
29
V. Discussion
31
Oxidative Stress
31
Antioxidant capacity
33
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Summary
34
References
36
A. Recruitment Materials
41
B. Informed Consent
44
C. Health History Survey
47
D. Physical Activity Survey
50
E. Dietary Recall
52
F. Banned Food List
55
G. Food Check-Sheet
56
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Abstract
Oxidative stress brought on by free radicals can lead to an increased risk of
certain diseases such as heart disease and some cancers. Oxidative stress mediated
damage can be reduced by scavengers, or antioxidants that can eliminate the high
reactivity of free radicals by turning them into non-radical and nontoxic metabolites.
Many scientists have investigated the effects of different kinds of foods (whole, liquid, or
supplement) to measure the change in oxidative damage and antioxidant capacity. The
purpose of this study is to examine the effects of two types of foods high in antioxidants
on markers of oxidative stress and antioxidant capacity in postmenopausal women.
Healthy post-menopausal women, (N=16) were divided into four groups (i.e., fruits,
soymilk, fruits and soymilk and control). Oxidative stress and antioxidant capacity were
measured before and after the intervention. Oxidative stress results indicated no
significant differences. Antioxidant capacity results indicated a significant main effect
for Test with the mean for the pre-test (M = 0.28 units/ml, SD = 0.15) being significantly
lower than the mean for the post-test (M = 0.39 units/ml, SD = 0.23). The results from
this study did not support the effectiveness of fruits and soymilk on the oxidative stress
levels and antioxidant capacity in postmenopausal women.
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List of Tables
1. Weight and BMI
28
2. MDA values pre and post
29
3. SOD values pre and post
30
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Chapter I
Introduction
Within the human body there are a number of chemical reactions that occur
(Brooks, Fahey, & Baldwin, 2005). Most of the reactions are needed for the body to
function appropriately. However, some of these reactions produce by-products that have
the potential of being harmful to some of the body’s delicate tissues. One such byproduct is known as an oxidant. Oxidants are formed as a normal product of aerobic
metabolism, but can be produced at elevated rates under pathophysiological conditions.
These elevated rates may result in damage to cells. However, various compounds known
as antioxidants protect biological systems against the potential harmful effects of
processes or reactions associated with the presence of oxidants (Sies, 1997).
An imbalance between oxidants and antioxidants in favor of the oxidants is
known as “oxidative stress” (Sies, 1997). Oxidative stress biomarkers have been
recognized as a factor in many acute and chronic diseases. Antioxidants can prevent or
inhibit the effects of oxidation (i.e., oxidative stress) by decreasing localized oxygen
concentrations so that increased oxidation is less likely to occur. This prevents some of
the initial reactions of oxidation by scavenging free radicals that are capable of
abstracting hydrogen from molecules. Numerous studies have been conducted examining
the effects of antioxidants on various populations (Heitzer, Schlinizig, Krohn, Meinertz,
& Munzel 2001; Seifried, Anderson, Fisher, & Milner, 2007; Svilaas et al., 2004).
One benefit of antioxidants is that they have been found to reduce the damaging
effects of oxidative stress on deoxyribonucleic acid (DNA) (Ryan-Borchers et al, 2006).
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This finding is of particular importance as increased levels of oxidative stress result in an
imbalance of a body’s redox system. The redox system is important for oxygen
homeostasis, the balance between antioxidants and oxidants. Redox reactions involve the
transfer of electrons between two chemical species: compounds that lose electrons
(oxidized) and those that gain electrons (reduced). If the homeostasis is not maintained,
the cell becomes oxidatively stressed (Seifried et al., 2007). A disturbance in the
homeostasis can result in toxic levels of oxidative stress that produce free radicals, which
have been shown to damage all parts of a cell. Thus, oxidative stress may be involved in
many different types of diseases such as coronary artery disease, stroke, arthritis, and
cancer (Prior, 2003). Along with the increased risk of diseases, the cells of aging
organisms can accumulate increased levels of oxidant-damaged nuclear DNA (Finkel &
Holbrook, 2000), which may lead to mitochondrial DNA damage. This damage will
potentially compromise the mitochondrial function; thus, releasing more reactive
oxidative species (ROS) and increasing the cycle of more DNA damage. Termination of
ROS can only be halted by the removal of intermediates (i.e., antioxidants) within a
chain.
After menopause, there is a decline in estrogen concentration (Ryan-Borchers et
al, 2006). The sharp decline in endogenous estrogen production during menopause has,
historically, resulted in hormone replacement therapy to alleviate certain risks (e.g.,
cardiovascular diseases and osteoporosis; Frankenfeld et al. 2003). Although many
women engage in such therapy, others find this unnatural and/or are concerned with other
health risks (Mingo, Herman, & Jasperse, 2000). Increasing antioxidant levels in food
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such as soy isoflavones can help alleviate the signs and symptoms in menopausal and
postmenopausal women such as hot flashes (Han, Soares, Haidar, Rodrigues de Lima, &
Baracat, 2002). Additionally, oxidative stress often increases after menopause; thus,
further compromising a woman’s immune system. Many different antioxidants within a
diet can help break this chain. Additional research is needed to examine the effect of
antioxidants on oxidative stress in postmenopausal women.
There are various sources of antioxidants such as fruits and vegetables (Prior,
2003), fruit and vegetable juices (Cilla et al., 2009), red meat, poultry (Djuric et al.,
1998), soy products (Ryan-Borchers et al, 2006), and algae (Scoglio et al., 2009).
Antioxidants found within these foods have the potential to inhibit oxidation within the
cell. However without these antioxidants, the production of free radicals can set a chain
reaction of events that will damage cells. The damage or destruction of cells because of
the lack of antioxidants in a diet can result in many different diseases if antioxidants are
lacking in the diet. A variety of approaches have been examined to determine the effects
of antioxidant supplementation on oxidative stress (Seifried et al., 2007). Contradicting
results have been found among these studies, because different amounts of antioxidant
supplementation have been given during interventions. Antioxidants have been found to
play a vital role in a person’s health (Seifried et al., 2007); however, the specific type,
amount, and those that have an interdependent effect on each other are still being
researched.
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Statement of the Purpose
The purpose of the current study is to examine the effects of two types of foods
high in antioxidant on markers of oxidative stress and antioxidant capacity in
postmenopausal women.
Significance of the Study
An increase in oxidative stress levels as a result of menopause can lead to an
increased risk for diseases such as coronary heart disease and cancer (Ryan-Borchers et
al., 2006). Researchers have found positive effects within the body from foods high in
antioxidants (Seifried et al., 2007). Whole food supplementation including meat
products, fruits, vegetables, and walnuts were all shown to have a decrease in oxidative
stress, which can reduce the risk of breast cancer and coronary heart disease (Djuric et al.,
1998; McKay et al., 2010). Although there was a significant increase in antioxidant
levels after a soymilk intervention, there were no significant changes in antioxidant levels
with a fruit juice diet (Cilla et al., 2009; Ryan-Borchers et al., 2006). Grape powder,
algae extract, and tablets containing large amounts of vitamin E are just a few of the
antioxidant interventions that have been found to decrease oxidative stress, and increase
well-being in postmenopausal women (Ryan-Borchers et al., 2006).
Researchers (Zern et al., 2005) have used individual antioxidant supplementation
to increase total antioxidant capacity (TAC). However, the influences of the combination
of foods high in antioxidants have not been examined. Therefore, it is important to
determine if there can be increased antioxidant levels because of a combination of
antioxidants, such as increased fruit intake and soymilk. It is important to assess the
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effects of a combination of antioxidants in postmenopausal women, because an increased
risk for certain diseases may occur due to high oxidative stress levels (Scoglio et al.,
2009).
Hypotheses
The experimental design of this study will allow for the comparison of three
antioxidant supplementations on antioxidant levels and levels of oxidative stress. There
will be a soymilk intervention, a fruit intervention, an intervention that includes a
combination of the two antioxidants, and a control group.
Hypothesis I: All experimental groups will see an increase in antioxidant levels.
Hypothesis II: All experimental groups will see a decrease in oxidative stress
Hypothesis III: A combination of antioxidants will elicit a greater increase in
antioxidant levels than a single supplementation.
Hypothesis IV: A combination of antioxidants will elicit a greater decrease in
oxidative stress markers than single supplementation.
Delimitations
This study was delimited to the following:
1. Females in amenorrhea for at least 12 months with climacteric symptoms of at least 4
months.
2. Women participating in hormonal treatments or therapies were excluded from the
study.
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3. Participants had to have an absence of uterus dysfunctions, cardiovascular diseases,
hypertension, or diabetes.
4. The antioxidant interventions were fruits, soymilk and a combination of the two.
Limitations
The following are potential limitations of the study:
1. The accuracy of the results was dependent on the participants adhering to the
intervention protocols (i.e., consuming the proper antioxidants and the correct amount
each day).
2. The participant’s truthfulness on the compliance check sheet.
3. A relatively low sample size may result in insufficient power to identify a significant
effect.
Assumptions
The following assumptions were made during this study:
1. The daily food record was truthfully answered.
2. The participants adhered to the protocol intake of their assigned antioxidant group.
3. The participants purchased the appropriate food and/or beverages to complete the
antioxidant supplementation.
4. Superoxide Dismutase (SOD) is an accurate detection for antioxidant levels.
5. Thiobarbituric Acid Reactive Species (TBARS) is a well-established method for
screening and monitoring lipid peroxidation, an indication of oxidative stress.
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Definition of Terms
1. ROS – reactive oxygen species – a free radical with an unpaired electron that is
potentially dangerous as they are likely to react indiscriminately with any molecule
nearby including DNA (Brooks, Fahey, & Baldwin, 2005).
2. Postmenopausal is the point after which a woman has reached menopause or cessation
of the menstrual cycle for one year (Robert-McComb, Norman, & Zumwalt, 2008).
3. SODs – superoxide dismutase- are metalloenzymes that catalyze the dismutation of the
superoxide anion to molecular oxygen and hydrogen peroxide and this form a crucial part
of the cellular antioxidant defense mechanism (McCord & Fridovich, 1969).
4. TBARS – Thiobarbituric Acid Reactive Substances- is a protocol that monitors lipid
peroxidation, which is a well-established mechanism of cellular injury in both plants and
animals and is used as an indicator of oxidative stress in the cells and tissue (Kosugi,
Kojima, & Kikugawa, 1989).
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Chapter II
Literature Review
According to the World Health Organization, 605 million persons are currently 60
years of age or older (Kennedy, 2006), which is partially due to an increase in life
expectancy. This increase in life expectancy is predominately as a result of better health
care and preventative strategies, or overall wellness. Wellness is defined by Conrad
(1994) as a conscious and deliberate approach to an advanced state of physical and
psychological/spiritual health. One component of wellness is health, which is not
suffering from disease, pain, or other defects (Hansen-Kyle, 2005). Living a healthy life
can contribute to prolonged wellness and life expectancy. Some strategies include
lifestyle changes such as engaging in regular physical activity and having a well-balanced
diet. Health and health-promotion behaviors are frequently depicted as the good while
disease and putatively disease-producing behaviors are seen as bad (Conrad, 1994).
Infectious diseases of the early 1900s, such as pneumonia and tuberculosis, have
been replaced by cardiovascular disease and cancers as the major causes of mortality
(Conrad, 1994). Globally, chronic diseases such as obesity, type 2 diabetes,
hypertension, coronary artery disease, and cancers are becoming more prominent
(Kennedy, 2006). Many of these chronic diseases are preventable when individuals
engage in healthy lifestyle behaviors. Engaging in physical activity has shown to
increase the physiological component of healthy aging. Individuals who maintain
physical conditioning and exercise regularly tend to have fewer medical problems than
individuals who live a sedentary lifestyle. Along with physical activity, a nutritionally
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balanced diet high in protein and fiber has been found to be a factor that contributes to
overall improved health (Hansen-Kyle, 2005). This improved health can lead to a higher
quality of life, especially as one gets older. However as one ages, a number of diseases
associated with an altered immune system, such as cardiovascular diseases, osteoporosis,
and some cancers, may be prevented by a well-balanced diet. Micronutrients have been
found to be associated with the reduction of physiological responses such as oxidative
stress (Hansen-Kyle, 2005).
Oxidative Stress
Oxidative stress is defined as an imbalance between production of free radicals
and reactive metabolites, so-called oxidants, and their elimination by protective
mechanisms, referred to as antioxidative systems (Durackova, 2010). Three conditions
that deal with the role of oxidative stress in aging include: 1) levels of molecular
oxidative damage increase during aging; 2) a relatively longer life expectancy within and
among species is associated with a correspondingly lower accrual of oxidative damage;
and, 3) a prolongation of life-span by regimens such as caloric restriction in mammals is
associated with the amelioration of oxidative damage (Sohal & Weindruch, 1996).
Oxidative stress biomarkers have been recognized in many acute and chronic diseases,
and increase throughout various phases in the human lifespan (Finkel & Holbrook, 2000).
This finding is of particular importance as increased levels of oxidative stress result from
an imbalance of a body’s redox system.
The redox system is important for oxygen homeostasis; the balance between
antioxidants and oxidants (Seifried et al., 2007). Redox reactions involve the transfer of
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electrons between two chemical species: compounds that lose electrons (oxidized) and
those that gain electrons (reduced). If the homeostasis is not maintained, the cell
becomes oxidatively stressed. This oxidative stress has been associated with several
diseases, such as neurodegenerative diseases, diabetes mellitus, metabolic syndromes,
skin and tumor diseases, and psychic impairments. Additionally, oxidative stress is
associated with the aging process (Durackova, 2010). Currently, there is no possible way
to measure ROS directly. Oxidative stress can be assessed by measurement of reaction
products of oxidative damage. As noted previously, lipid peroxidation is the most
studied reaction product for oxidative stress markers with a variety of techniques
including thiobarbituric acid-reactive material assays, commonly known as TBARS
(Betteridge, 2000).
Free Radicals (ROS)
Free radicals can be defined as any chemical species that contains unpaired
electrons. Unpaired electrons increase the chemical reactivity of an atom or molecule
(Betteridge, 2000). Free radicals, also known as reactive oxygen species (ROS), have
long been assumed to have only negative functions in the organism. An increase in free
radicals may lead to irreversible damage of mitochondrial DNA, membrane lipids and
proteins; thus, resulting in mitochondrial dysfunction and ultimately cell death
(Kowaltowski & Vercesi, 1999). Lipid peroxidation is perhaps the most extensively
studied consequence of free radical attack. Reactive free radicals have the capacity to
abstract a hydrogen atom from fatty acids, leaving behind an unpaired electron on the
carbon. The remaining carbon undergoes molecular rearrangement resulting in
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conjugated dienes, which combine with oxygen and become radical. This production
starts a chain reaction that continues until the substrate is consumed or the reaction is
terminated by a chain-breaking antioxidant. This lipid peroxidation can have profound
effects on cellular function (Betteridge, 2000). In summary, oxidative stress occurs when
the balance between free radicals and antioxidative protection is disturbed leading to
damage within the organism (Durackova, 2010). However, the effects of oxidative stress
can be reduced when specific compounds are present.
Antioxidants
Various compounds known as antioxidants protect biological systems against the
potential harmful effects of processes or reactions that can cause excessive oxidations
(Durackova, 2010). Scavengers of free radicals (i.e., antioxidants) can eliminate the high
reactivity of free radicals by turning them into non-radical and nontoxic metabolites.
These antioxidants prevent oxidation by free radicals of biologically important molecules
(Durackova, 2010). Antioxidant function can be described in terms of prevention,
interception, and repair. The preventative function of antioxidation channels involves an
attacking species by turning over or elimination of the whole cell, hence lowering the risk
for further damage. The antioxidant function of interception involves transferring the
radical function away from a more sensitive target sites (e.g., cell membrane ) to
compartments of the cell in which an oxidative challenge would be less deleterious (e.g.,
cytosol). Lastly, repairing the damage of oxidation includes assisting multiple enzyme
systems involved in DNA repair, lipolytic and proteolytic enzymes, that are capable of
serving the functions of restitution or replenishment (Sies, 1997). The three major
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classes of antioxidant enzymes are the superoxide dismutases, catalases and glutathione
peroxidases, and those involved in the transportion and elimination of reactive
compounds. Antioxidant enzymes can be found in varying amounts in different
subcellular sites and types of cell (Sies, 1997). Antioxidants such as vitamin E, βcarotene, and coenzyme Q are highly effective in interrupting the chain reaction
associated with lipid peroxidation (Betteridge, 2000). Without the antioxidants, oxidative
stress may impair the body’s immune function.
Menopause and Oxidative Stress
Menopause can lead to a decrease in estrogen production (Pick, 2005). Estrogen,
an immune-modulating hormone is associated with proper functioning of the immune
system. Because estrogen production decreases following menopause, the immune
system of post-menopausal women may be compromised. The immune system
encompasses an array of defenses that help to guard against the development of a number
of diseases, some of them age-related (Ryan-Borchers et al. 2006). An increase in
oxidative stress and a decrease in estrogen place postmenopausal women at increased risk
for several diseases. Oxidative stress may be involved in many different types of diseases
such as coronary artery disease, stroke, arthritis, and cancer (Prior, 2003). The damage to
the cell as a result of decreased estrogen production combined with DNA damage from
reactive oxygen species alters the mitochondrial physiology that may contribute to a
greater cellular stress response, cell growth arrest, and subsequent apoptosis (Yakes &
Van Houten, 1997). The life expectancy for women has increased leading to
approximately 30 years of ceased production of estrogen from ovaries, and there is
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overwhelming evidence that indicates that estrogen has a protective effect against
coronary artery disease (Robert-McComb et al. 2008). Based on these changes,
postmenopausal women may benefit from a high antioxidant diet, which may reduce the
amount of DNA damage brought on by the production of oxidative stress after
menopause (Ryan-Borchers et al., 2006).
Antioxidants and Oxidative Stress
Some studies have examined antioxidant effects by assessing the diets of
individuals. Most of these studies used a food recall or diary as opposed to a diet
intervention or antioxidant supplementation to measure the effects of antioxidants (Djuric
et al., 1998; Svilaas et al., 2004). In a study examining the effects of the consumption of
various food groups in Norwegian adults on the total antioxidant capacity, antioxidant
intake was determined by collecting a 7- day weighed dietary record (Svilaas et al.,
2004). The results of the data analysis indicated that the total intake of antioxidants was
significantly correlated with plasma lutein, zeaxanthin, beta carotene, and alpha carotene,
all of which have strong antioxidant properties within the body. Although coffee
consumption has been shown to increase plasma homocysteine, and is likely associated
with a small increase in blood pressure after many years of consumption, the results of
the dietary analysis showed coffee along with fruits such as berries were the greatest
contributors to the total antioxidant intake. These antioxidants were found to be
associated with a reduced risk of major chronic degenerative diseases.
Another study by Prior (2003) examined the effects of fruits and vegetables on
antioxidant levels. Specifically, Prior examined the amount of absorption between two
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different flavonoids, anthocyanins and flavonols. The effects of vegetables containing
quercitin (e.g., onions) and fruits containing cyaniding glycosides (e.g., pears and dark
fruits such as blueberries and blackberries) on cyaniding 3-glucoside were examined in a
sample of rats. The major findings were a decrease in antioxidant levels and a measure
of lipid peroxidation (i.e., TBARs) with dark berries. Additionally, an increase in
antioxidant activity and a decrease in hydroperoxides, a measure of oxidative stress, were
found with the consumption of onions.
Tesoriere and colleagues (Tesoriere, Butera, Pintaudi, Allegra, & Livrea, 2004)
studied the effects of cactus pear fruit on oxidative stress levels in healthy middle-aged
men and women. Participants were given either 250g of cactus pear pulp or a vitamin C
supplementation to be taken for 2 weeks. After a 6-week wash-out period participants
received the other treatment. Blood samples were taken to measure hydroperoxides. The
results indicated the cactus pear fruit intervention showed significant decreases in
hydroperoxide, but no significant decrease was found with vitamin C supplementation.
These results showed the antioxidant effect was due to the fruit and not the vitamin C in
the fruit.
Another study examined specific supplement antioxidant activity in both men and
women (Cornell et al., 2001). The contents of the antioxidant supplementations included
either capsules prepared with different types of antioxidants or a fluid formula with the
same types of antioxidants. Formula 1 contained Zinc, Selenium, L-cysteine, Vitamin A
and E, and beta-carotene. Formula 2 contained citrus flavanoids, Vitamin C and B-6, and
Coenzyme Q10. The final formula contained all ingredients from formulas one and two.
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The type of supplementation and amount of antioxidants in the capsules and fluid were
similar to that of an average amount of antioxidants consumed in a normal diet. The
participants were asked to consume the supplementation every day during breakfast.
Each participant’s treatment included the capsules, followed by a washout period, and
ending with the fluid supplementation. Organic hydroperoxides within the blood serum
were examined. The results indicated that low dosages of antioxidant supplementation
given in fluid form showed the largest decrease in oxidative stress. Based on the studies
summarized thus far, there is evidence that antioxidants in the diet, whether naturally
consumed or supplemented, are associated with decreased levels of ROS.
Women and Antioxidants
The research examining the role of antioxidants in women is limited. Djuric and
colleagues (1998) investigated the relationship between specific food intakes (e.g., meat,
vegetables, and fruits) and the levels of oxidative DNA damage in female participants
between the ages of 18 and 65 years who had at least one first-degree relative who had
been diagnosed with breast cancer. Two groups were randomly assigned to a normal diet
or diet low in fat (15% fat). Each group was given a list of appropriate foods to consume
based on the group requirements and appropriate cooking methods for each of the foods.
Dietary intake per day and the preparation method (e.g., raw, cooked, or high temperature
cooked) were recorded. Analyses examined how individual food items and various
combinations of the items related to DNA damage. The results showed a significant
association of beef and pork with oxidative stress, and no association with cooked
vegetables. Additionally, fish and poultry showed no or a negative association to DNA
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damage. Although there were many combinations with dietary options, a low fat diet
(e.g., high in fruits, vegetables, fish and poultry) resulted in reduction in oxidative DNA
damage. Thus, reduced oxidative DNA damage could decrease the possible cancer risks
in women with a family history of breast cancer.
Lycopene found in tomato products, was studied to identify its degree of DNA
protection in women (Porrini & Riso, 2000). The researchers required the women to
consume 25g of tomato puree daily for 14 days. Carotenoid concentrations and DNA
strand breaks were analyzed before and after the intervention. The results indicated that
tomato consumption increased plasma and lymphocyte lycopene concentrations, but betacarotene concentration increased only in plasma. These changes suggest that the tomato
puree was taken up within the cell, which in turn increased the total antioxidant level
significantly. The role of tomato puree in this study is shown to have benefits that protect
against cancer and chronic diseases. These researchers concluded that small amounts of
tomato puree might increase the resistance of lymphocytes to oxidative stress.
Diets rich in antioxidants have been shown to reduce the risk of chronic
degenerative diseases. Cilla’s (2009) research evaluated how the consumption of fruit
beverages and the addition of milk and iron can affect the antioxidant status. Women in
their twenties were assigned to one of three studies based on the consumption of different
beverages that occurred twice a day for 3 weeks. In the first study the women consumed a
fruit juice, while in the second study they consumed juice and milk, and the third study
included the consumption of fruit juice, milk, and iron supplementation. Serum
antioxidant capacity was measured. In the first two studies the fruit juice did not increase
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antioxidant capacity; however, there was an increase in superoxide dismutase (SOD)
activity (measurement of antioxidant activity). SOD activity increased in the first two
studies, and decreased with the iron supplementation initially then increased after 6
weeks. The induction of SOD activity and the complementary source of iron added to the
drink in the third study lead to a conclusion that habitual consumption of fruit juice and
iron can have beneficial effects against oxidative stress in women. This research was
successful in finding new and creative ways to change a woman’s diet so she can reduce
the risk of certain diseases.
Another study used strawberries, spinach, and red wine as the antioxidant
supplementation to determine if the total antioxidant level would increase (Cao, Russell,
Lischner, & Prior, 1998). Diets of elderly women (N=8) were supplemented with 240 g
of strawberries, 1250 mg of ascorbic acid, 294 g of raw spinach, and 300 ml of red wine.
Blood samples were taken within 24 hours of consuming the supplement, and the total
antioxidant levels were determined. Results were that the consumption of these four
foods, which are rich in antioxidant phenolic compounds, can increase the serum
antioxidant capacity in humans; thus, providing the potential to reduce the risk of some
diseases associated with aging. Although this study was conducted on older women there
was no menopause criteria, nor did they analyze markers of hormone/ hormone
replacement therapy.
Alternative plant products, specifically walnuts, have been shown to have high
amounts of polyphenols (McKay et al., 2010). Healthy postmenopausal women, as well
as men 50 years of age and older, were given 21-24g of raw walnuts per day. Although
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linoleic acid increased, which would be assumed to be a result of the consumption of
walnuts, the lipid peroxidation only minimally affected antioxidant capacity. Overall the
results concluded no significant increase in antioxidant levels. In another study
researchers used grape polyphenol supplementation and its effects on the reduction of
coronary heart disease (Zern et al., 2005). Lyophilized grape powder (LGP) has been
reported to decrease triglyceride levels in animals. Zern and colleagues examined the
effects of LGP on plasma lipids, lipoprotein metabolism, LDL oxidation, inflammation,
and oxidative stress in twenty-four premenopausal and twenty postmenopausal women.
The women were involved in a single-blind crossover study in which they consumed
LGP or placebo for 4 weeks, a 3-week washout period, and finally the alternate treatment
for 4 weeks. Lyophilized grape powder had no effect on triglyceride or HDL levels,
glucose levels, IL-6, and C-reactive protein in both groups. However, LGP treatment
decreased the tumor necrosis factor, cholesterylester transfer protein, and isoprostane in
both pre- and postmenopausal groups. Isoprostane, formed from free radicals, was
decreased showing a reduction in oxidative stress with LGP treatment. These results
showed a reduction in major coronary heart disease risk factors that are typically elevated
when women reach postmenopausal status. Based on the results of these two studies,
further research is needed with controlled diets to determine the antioxidant capabilities
of these and other products.
Ryan-Borchers and colleagues (2006) examined the effect of isoflavones found in
soy products such as soymilk or soy supplementation. The problem under investigation
was to determine the effects of soy isoflavones on immune and oxidative markers in
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postmenopausal women. The participants included 52 healthy postmenopausal women.
This double blind study randomly generated 3 groups; first group consumed cow’s milk
and placebo (control), second group consumed soymilk and placebo, and third group
consumed cow’s milk and isoflavones supplements. Participants were instructed to
consume 706 ml of the specified milk per day as well as oral ingestion of
supplement/placebo every day for 16 weeks. At the end of the 16 weeks, B cell
populations, which heighten humoral immune response, were higher among women in
both the soymilk and supplement groups than in the control group. Because of the
stimulation of B cells from soy isoflavones, DNA oxidative damage in postmenopausal
women was inhibited. Isoflavones intervention did not influence cytokine production,
interleukin 2, interferon γ, TNF-α, lipid peroxidation, or CRP concentrations. Also,
subjects receiving soymilk/supplementation did see a lower plasma 8-OHdg
concentration than the control group suggesting a protective effect of soy isoflavones
against oxidative stress in postmenopausal women.
The studies that have been more successful with reduced oxidative stress with
antioxidant consumption involve the intake of whole fruits and vegetables (Porrini &
Riso, 2000; Prior, 2003; Svilaas et al., 2004). People who have an allergy to a fruit or
vegetable, or those who are selective with their own diet need a different means to
increase their antioxidant consumption. Scoglio and others (2009) researched the effects
of a certain algae extract, Klamath, which is known to have antioxidant qualities. The
aim of this investigation was to see the effects of a 2-month treatment with the Klamath
algae extract on the general and psychological well-being of menopausal women, as well
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Texas Tech University, Shelby Kloiber, August 2011
as on their oxidative stress status and level of antioxidants. Subjects participating in the
study included twenty-one females between the ages of 47-54 years, amenorrhea for a
year, no hormonal treatments, absence of uterus dysfunction, and no cardiovascular
disease hypertension or diabetes. Subjects were given a Klamin tablet to be taken twice a
day for two months. Results included a decrease in Malondialdehyde (MDA) as an index
of lipid peroxidation and antioxidant levels were shown to increase. Menopausal
symptoms evaluated before and after the study by the Green Scale were shown to
decrease after treatment. Lastly, hormone plasma levels showed no significant change
during the supplementation period. These researchers concluded that Klamin
supplementation, a Klamath algae extract, is capable of reducing oxidative processes, and
improving the well-being of menopausal women. The researchers proposed this
alternative treatment for hormonal therapy as a way to overcome climacteric symptoms
(Scoglio et al., 2009).
Summary
The previously reviewed studies generally indicated positive results with different
types of supplementation and amounts increasing a person’s antioxidant levels. In most
of the studies, whole foods, liquids, and supplementations were found to decrease the
levels of oxidative stress (Cao, Russell, Lischner, & Prior, 1998; Cilla et al., 2009;
Stampfer et al., 1993). The studies examining the role of antioxidants in postmenopausal
women have typically found changes to oxidative stress markers, but most of the studies
have examined the effect of only one type of antioxidant on measures of oxidative stress
(Cornelli et al., 2001). The purpose of this current study was to see if there is a greater
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Texas Tech University, Shelby Kloiber, August 2011
decrease in oxidative stress measures with a combination of antioxidants (i.e., increased
levels of fruit/vegetables with soy supplementation).
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Texas Tech University, Shelby Kloiber, August 2011
Chapter III
Methodology
It has been confirmed that an increased amount of antioxidant within the diet can
reduce ROS levels and reduce the risk of diseases such as coronary heart disease and
cancers (Ryan-Borchers et al., 2006). The purpose of the current study is to investigate
the effects of a combination of antioxidants on levels of antioxidant levels and oxidative
stress measures in post-menopausal women.
Participants
Recruitment of the participants by flyers and oral presentations (see Appendix A)
took place at health and fitness clubs such as Body Works, Zach’s Gym, Freedom
Fitness, Lifestyle Center, Zum Fitness, Fusion Fitness, and Studio 57. Local health care
businesses, such as Grace Clinic and other gynecology practices were other locations for
recruitment. Flyers were also placed within churches in the Lubbock area, and
throughout the Texas Tech University campus and the Texas Tech University Health
Sciences Center. There were also advertisement in the Daily Toreador, Tech Announce
and the Lubbock Avalanche Journal. Participants signed informed consent (see Appendix
B) in accordance with the committee of the use of human subject’s requirement.
The participants (N=37) consisted of women who were given a health history
survey (see Appendix C) to determine if they met the inclusion criteria. The inclusion
criteria included: (1) currently in the postmenopausal phase whether naturally or
surgically induced with cessation of menses greater than one year; (2) had a hematocrit
level greater than 38% (i.e., not anemic); (3) weighed at least 110 pounds; (4) were not
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Texas Tech University, Shelby Kloiber, August 2011
participating in any type of hormonal treatment, nor had been participating in hormone
replacement therapy within the last 5 years; (5) had a BMI between 18 and 40; (6)
nonsmoker for at least one year; (7) consumed two or more alcoholic drinks per day; and,
(8) had no known uterus dysfunctions, cardiovascular diseases, hypertension, diabetes,
gastrointestinal, renal disease, or HIV. There were 18 participants excluded based on the
previously mentioned criteria. One participant was excluded because we were unable to
collect a blood sample from the vein. The participants were asked to complete a physical
activity survey (Appendix D) to insure that none of them were engaged in heavy physical
activity. One additional participant was excluded because she regularly took a type of
NSAIDS. Participants were then asked to complete a food recall record to help the
researchers determine if the participant already had a high antioxidant diet in fruits and
soymilk (Appendix E). Those who were found to consume a diet similar to the
intervention were excluded from the research (n=1). The participants that were excluded
from the study were thanked for their willingness to participate. One additional
participant chose not to continue with the study after the initial meeting. Therefore, there
were 16 participants who completed the study.
Procedure
All participants set up a meeting with the investigator at the participant’s
availability. The first meeting consisted of completion of the written consent (Appendix
B), the health history questionnaire (Appendix C), the physical activity survey (Appendix
D), and the food recall (Appendix E). The participants that qualified for continuation
with the study were then familiarized with the lab. These participants were given
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Texas Tech University, Shelby Kloiber, August 2011
instructions on the procedures for the duration of the study. The study took place over a
period of approximately 5 weeks.
After the initial meeting, a one-week wash-out period where the participants
refrained from foods on the Banned Food List (see Appendix F) took place. During the
second and third meeting measurements (i.e., height, weight, heart rate and blood
pressure) were taken, and blood draws were completed to obtain levels of antioxidant
capacity and oxidative stress. The laboratories were in compliance with OSHA standards
for blood collection procedures, and the investigators involved in blood collection
procedures were appropriately trained. Pre- (i.e., second meeting) and post-dietary (i.e.,
third meeting) intervention testing was performed at the same time of day following an 812 hours fast. Heights and weights were obtained by a standard doctor’s scale. Body
mass index (BMI) was calculated by weight (kg) divided by height (m2). Participant’s
heart rates were measured using Polar Heart Rate Monitors, and blood pressure for each
participant was obtained by a stethoscope and sphygmomanometer. Anthropometric
measurements, blood draws, and a finger prick were repeated post-intervention (i.e., third
meeting) following the same procedures and guidelines.
Hematocrit and anthropometric data collection. Hematocrit levels were
determined via blood collected by finger prick protocol. Alcohol pads were used to
sterilize the finger site followed by a sterilized finger stick to draw the blood sample (2-3
drops) to determine hematocrit concentration. First, a finger is lanced and a small drop of
blood is allowed to accumulate. Blood is drawn into a capillary glass tube, and one end
of the blood filled tube is sealed with clay. The blood-filled tube is placed in a centrifuge
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Texas Tech University, Shelby Kloiber, August 2011
and spun until the plasma and formed elements separate. The glass tube is moved across
a reader board until the bottom of the red blood count column is at zero and the top of the
plasma column is at 100. The percentage of red blood cells in the blood is determined by
tracing the grid lines near the top of the red column in the tube to the scale (i.e.,
percentage; Hematocrit Test, 2011). If the participant weighed less than 110 pounds or
if her hematocrit was less than 38% she was excluded from the study. The hematocrit
levels, or the volume of red blood cells as a percentage of total blood volume, were
assessed to exclude anemic participants. This was in accordance to the research
guidelines of the Human Research Protection Office of Texas Tech University.
Antioxidant capacity and oxidative stress data collection. Antioxidant capacity
and oxidative stress levels were determined before and after the antioxidant intervention
via blood samples that were taken during the second and third meetings. In preparation
for the venipuncture procedure, the participant was asked to come having fasted for at
least 8-12 hours. She was asked to refrain from any anti-inflammatory (NSAIDS) such
as Ibuprofen because of blood thinning prior to the data collection. A phlebotomy chair
and arm rests designed for blood draws was used during the blood draw procedure.
Participants assumed a seated position in the phlebotomy chair. The participant’s nondominant arm was examined for the most prominent vein at the antecubital area of the
elbow by a certified technician. After proper disinfectant by an alcohol pad, a tourniquet
was positioned approximately 3-4 inches above the puncture site. The needle was angled
15-30 degrees with the surface of the arm. The needle was inserted through the skin and
into the lumen of the vein. As the last tube is filling, the tourniquet was removed. The
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Texas Tech University, Shelby Kloiber, August 2011
needle was removed from the arm swiftly, and gauze was pressed firmly to avoid
formation of a hematoma. A total of 5 cc or about 5 ml per vial of blood was collected
in three separate vials. This amount did not exceed the 550 ml limit specified be the
research guidelines of the Human Research Protection Office at Texas Tech University
Health Sciences Center (2007). Serum samples were separated by centrifugation and
samples were frozen at -80° for later analysis.
Intervention protocol
At the second meeting participants were randomly assigned to one of four groups:
a control group (C) who were asked to maintain their normal diet; daily soymilk
consumption group (S); an increase in daily fruit consumption group (F); and, a
combination of daily soymilk and increased fruits group (S+F). The S+F and S groups
were instructed to consume a total of 706 ml of approved soymilk throughout the day.
This amount was based on three dairy servings per day from the United States
Department of Agriculture’s (USDA) MyPyramid (2011). The participants were asked to
complete a daily check-off calendar to insure that they followed instructions (see
Appendix G). Those participants who were in the F and S+F groups were instructed to
increase their daily fruit intake to 5 or more servings per day based on serving sizes from
the USDA’s MyPyramid. The participants were asked to choose a variety of fruits and
check off the fruits from a daily check-off calendar along with given serving sizes (see
Appendix G). All participants were informed of the importance of compliance with
dietary requirements of the study for 4 weeks.
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Texas Tech University, Shelby Kloiber, August 2011
Antioxidant capacity assay protocol
Blood samples were assayed for superoxide dismutase (SOD) using the
Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI). A
volume of 200 µl of diluted radical detector and 10 µl of standard were added to each
designated standard well. Sample wells had 200µl of the diluted radical detector and 10
µl of sample in each well. Reactions were initiated by adding 20 µl of diluted xanthine
oxidase to each well. The 96-well plate was covered and incubated on a shaker (LabLine Instruments Titer Plate Shaker; Melrose Park, Illinois) for 20 min at room
temperature. The mixture was read by a spectrophotometer (Spectramax 384 Plus,
Molecular Devices; Sunnyvale, CA) at 450nm. Standards of known SOD concentration
were used to establish a standard curve.
Oxidative stress assay protocol
Blood samples were also assayed by the TBARs Assay Kit (Cayman Chemical
Company; Ann Arbor, MI). A volume of 100 µl of each standard was added to each
properly labeled 5ml vial followed by an additional 100 µl of sodium dodecyl sulfate
(SDS) solution. The same procedure was followed for each sample. The vials were then
swirled to mix the contents followed by the addition of 4 ml of color reagent. The vials
were then capped and boiled in a water bath for 60 min. The vials were removed and
immediately placed in an ice bath for 10 min. After removal from the ice bath, the
mixtures were centrifuged for 10 min at 1600 g at 4°C. In duplicate, 150 µl of the
mixture from each vial was added to a 96 well plate and read by a spectrophotometer
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Texas Tech University, Shelby Kloiber, August 2011
(Spectramax 384 Plus, Molecular Devices; Sunnyvale, CA) at 535 nm. Standards of
known TBARS concentration were used to establish a standard curve.
Data Analysis
The pretest data for oxidative stress and antioxidant capacity were analyzed using
separate one-way ANOVAs to insure that the groups were equal at the beginning of the
study. The data were then analyzed using two separate 4 X 2 (Group X Test) analyses of
variance with repeated measures on the last factor. Alpha for this study was set at .05.
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Texas Tech University, Shelby Kloiber, August 2011
Chapter IV
Results
The purpose of the current study was to examine the effects of two types of foods
high in antioxidants on markers of oxidative stress and antioxidant capacity in
postmenopausal women. The women were divided into three intervention groups (i.e.,
fruits, soymilk, and a combination of fruits and soymilk) and one control group. The
oxidative stress was measured by the TBARS assay, and the antioxidant capacity was
measured by the SOD assay. The mean heights, weights and BMI can be found in Table
1.
Table 1. Means and standard deviations for weights and BMI.
Weight (pounds)
Pre
Post
168.7±29.3 168.3±28.8
BMI
Pre
Post
28.3±3.7 28.2±3.7
Oxidative Stress
The concentrations of MDA found using the TBARS assay were analyzed to
determine the effects of the food high in antioxidants on oxidative stress. To determine if
there was a difference among the groups, the pre-test data were analyzed using a one-way
ANOVA. The results of the analysis indicated no significant differences among the
groups, F (3, 12) < 1.0, p = .655. To determine the effects of the interventions, the data
were analyzed using a 4 X 2 (Group X Test) ANOVA with repeated measures on the last
factor. The levels of Group were the three intervention groups and the control group.
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Texas Tech University, Shelby Kloiber, August 2011
The levels of Test were pre-test and post-test. The results of the analysis indicated no
significant main effect for Group, F (3,12) < 1.0, p = .604 and Test F (1,12) < 1.0, p =
.629. Additionally, the interaction was not significant, F (3,12) < 1.0, p = .539. The
means and standard deviations for each group and test can be found in Table 2.
Group
Fruit
Soymilk
Fruit and Soymilk
Control
N
5
3
4
4
Pre-Test
Post-Test
139.6 ± 136.5
47.4 ± 48.5
93.4 ± 95.0
207.4 ± 291.8
71.0 ± 76.0
22.2 ± 27.0
210.5 ± 337.5
52.4 ± 40.5
Table 2. Means and standard deviations for MDA (µM).
Antioxidant Capacity
The detection of superoxide radicals generated by xanthine oxidase and
hypoxanthine found using the SOD assay were analyzed to determine the effects of the
food high in antioxidants on antioxidant capacity. To determine if there was a difference
among the groups, the pre-test data were analyzed using a one-way ANOVA. The results
of the analysis indicated no significant differences among the groups, F (3,12) < 1.0, p =
.680. To determine the effects of the interventions, the data were analyzed using a 4 X 2
(Group X Test) ANOVA with repeated measures on the last factor. The levels of Group
were the three intervention groups and the control group. The levels of Test were pre-test
and post-test. The results of the analysis indicated a significant main effect for Test F
(3,12) = 5.180, p = .042. The mean for the pre-test (M = 0.28 units/ml, SD = 0.15) was
significantly lower than the mean for the post-test (M = 0.39 units/ml, SD = 0.23). The
results indicated no significant main effect for Group, F (3,12) = 2.377 p = .121.
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Texas Tech University, Shelby Kloiber, August 2011
Additionally, the interaction was not significant, F (3,12) = 1.225, p = .343. The means
and standard deviations for each group and test can be found in Table 3.
Group
N
Pre-Test
Post-Test
Fruit
Soymilk
Fruit and Soymilk
Control
5
3
4
4
0.24 ± 0.07
0.33 ± 0.12
0.35 ± 0.10
0.24 ± 0.27
0.29 ± 0.16
0.53 ± 0.19
0.53 ± 0.09
0.24 ± 0.31
Table 3. Means and standard deviations of units/ml SOD.
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Texas Tech University, Shelby Kloiber, August 2011
Chapter V
Discussion
Free radicals can be defined as any chemical species that contains unpaired
electrons. Free radicals, also known as reactive oxygen species (ROS), have long been
assumed to have only negative functions in the organism. An increase in free radicals
may lead to irreversible damage of mitochondrial DNA, and membrane lipids and
proteins; thus, resulting in mitochondrial dysfunction and ultimately cell death
(Kowaltowski & Vercesi, 1999). Oxidative stress biomarkers have been recognized in
many acute and chronic diseases, and increase throughout various phases in the human
lifespan (Finkel & Holbrook, 2000). Scavengers of free radicals (i.e., antioxidants) can
eliminate the high reactivity of free radicals by turning them into non-radical and
nontoxic metabolites. These antioxidants prevent oxidation by free radicals of
biologically important molecules (Durackova, 2010). The purpose of this study was to
examine the effects of two types of foods high in antioxidant on markers of oxidative
stress and antioxidant capacity in postmenopausal women. The diet manipulations
included increasing the servings of fruit to five per day, adding soymilk or both.
Oxidative Stress
It was hypothesized that adding more fruits that are high in antioxidants, soymilk
and a combination of these two would decrease the levels of oxidative stress in
postmenopausal women. The data did not support this hypothesis, as no change in any of
the groups was indicated in the levels of oxidative stress. These results are similar to
those in Engleman (Engleman, Alekel, Hanson, Kanthasamy, & Reddy; 2005) who had 4
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Texas Tech University, Shelby Kloiber, August 2011
groups of different amounts of isoflavone and phytate. These soy protein isolates were
taken at 40g/day and showed no significant decreases in oxidative stress marers.
There have been studies that have found reduced levels of oxidative stress by
increasing foods or supplements with antioxidants. Increasing the consumption of whole
fruits and vegetables (Porrini & Riso, 2000; Prior, 2003; Svilaas et al., 2004) has resulted
in the most decrease in oxidative stress. More specifically, in relation with studies on
postmenopausal women who typically have an increased rate of oxidative stress
production, can be reduced with diets high in antioxidants. Many of the studies (RyanBorchers et al., 2006; Scoglio et al., 2009) concluded that the reduction of oxidation
because of the increase in antioxidant levels reduce the risk of diseases such as cancer
and CHD as well as increasing the well-being of women. Ryan-Borchers (2006) used a
soymilk intervention similar to the current study in regards to 706mL soymilk, however
their study laster 16 weeks. It is possible that the current study was not long enough to
see effects on oxidative stress levels. Scoglio (2009) found significant decreases in
oxidative stress with a 2 month intervention of a 0.8g tablet of algae extract. These
studies show evidence that a diet including foods high in antioxidants such as fruit and/or
soymilk can significantly increase blood antioxidant levels and decrease oxidative stress
levels. It is possible that in the current study, the fruit consumption of five servings a day
was not met, or a variety of the given fruit list was not consumed. Also, it is possible that
those in the soymilk and combination group consumed less than the required three cups
per day. The soymilk brand and flavor was not monitored, and could have increased or
decreased the antioxidant availability within the body. The non-significant changes in
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Texas Tech University, Shelby Kloiber, August 2011
oxidative stress and antioxidant levels between groups could be because of a cap of
antioxidant absorption in the body.
Oxidative stress in the blood was measured by the TBARs assay. Although this
assay has been used in studies that have found significant changes in oxidative stress
(Kasapoglu & Ozben, 2001) it should be noted that this assay has been found to be nonspecific as the level of TBARs seen in the blood can be increased by other chemical
reactions occurring in the body not specific to reactive oxygen species (Vollaard,
Shearman, & Cooper, 2005). It is currently unclear the certain amount of food products
and specific types of antioxidants are needed to decrease the risk for certain diseases,
however, the guidelines for this study were taken from the Food and Drug Administration
recommended daily intake for fruit and dairy products (MyPyramid, 2011). Guidelines
for the participants in this study were given a check off list to remind them of at least five
servings of fruit a day and/or three servings of soymilk per day. The participants’ check
list of consumed foods indicated a compliance rate of 96.8% in the fruit group, 81% in
the soy milk group, and 92.3% for the combination group. No significant results for the
current study could be a result of the decreased compliance rate along with the 4-week
length of the study.
Antioxidant Capacity
It was hypothesized that adding more fruits high in antioxidants, soymilk and a
combination of these two would increase the antioxidant capacity in postmenopausal
women. The data did not support this hypothesis, as no change in any of the groups was
indicated in the antioxidant capacity. These results are similar to McKay (2010) who
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Texas Tech University, Shelby Kloiber, August 2011
found no significant increase in antioxidant capacity in the cross-over study of either
21g/day or 42g/day of raw walnuts. Insignificance in our results could be a result of the
compliance rate, and the possibility of the wash out week directions not properly being
followed. However examining the data, indicated the means were moving in the
expected directions. The means of the control group from pretest to posttest were
essentially unchanged, while the means for the three control groups increased from
pretest to posttest. It would be expected that if a larger sample size was examined these
results of the SOD analyzes would produce a significant interaction. Additionally, a
longer intervention period could possibly allow for a greater time for the antioxidant
capacity to increase.
Diets rich in antioxidants have been shown to reduce the risk of chronic
degenerative diseases. Cilla’s (2009) research indicated the consumption of fruit
beverages and the addition of milk and iron can positively affect the antioxidant status.
The study did use the same assay, SOD, to measure antioxidant capacity; however the
study lasted longer than the current study at a total of 12 weeks. Another study used
strawberries, spinach, and red wine as the antioxidant supplementation to determine if the
total antioxidant level would increase (Cao, Russell, Lischner, & Prior, 1998). Results
found that the consumption of these four, which are rich in antioxidant phenolic
compounds, can increase the serum antioxidant capacity in humans and help reduce the
risk of some diseases associated with aging. Compared to the current study, the assay
used to determine antioxidant capacity was not similar and the study lasted 10 weeks.
During specimen collection, Cao (1998) blood samples as well as urine samples were
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Texas Tech University, Shelby Kloiber, August 2011
taken to measure possible changes hourly during test consumption day. In another study
researchers used grape polyphenol supplementation and its effects on the reduction of
coronary heart disease (Zern,et al., 2005). The study was similar to the current study with
an intervention of 4 weeks. These results showed a reduction in major coronary heart
disease risk factors that are shown to be elevated when women reach postmenopausal
status.
Summary
The results of this study indicate that consumption of foods high in antioxidants
such as fruits and/or soymilk increased, though not statistically significant, blood
antioxidant levels measured by superoxide dismutase, but the levels of oxidative stress
did not change. Combining the fruit and soymilk, combination group, showed no
significant difference compared to the single food of fruit or soymilk. It is apparent in
the results that diets high in antioxidant rich food, specifically fruits and soymilk, did not
reduce oxidative stress after a 4 week time period as measured by TBARs. It is apparent
from other studies (Cao et al. 1998; Prior 2003; Tesoriere et al 2004; Zern et all 2005)
that a diet high in antioxidant foods plays a large role in oxidative stress compared to a
non-intervention group. More research is necessary to further investigate the specific
types of fruit and soymilk, and amount of each needed to show a greater reduction on
oxidative stress. Also, studies combining two different types of foods rich in antioxidants
other than fruits and soymilk should be researched further.
In conclusion, the results of the current study showed no significant differences
among groups and between groups on TBARs measuring oxidative stress and SOD
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Texas Tech University, Shelby Kloiber, August 2011
measuring total antioxidant capacity. Antioxidant levels for each group showed a
significant main effect for mean pre antioxidant levels compared to post measures.
Lastly, using a blood assay for measuring blood antioxidant levels and oxidative stress
levels may not be sensitive or reliable enough to look at the change of antioxidant and
oxidative stress properties between our studies’ groups. According to the results, a diet of
five servings of fruits high in antioxidant and/or a daily intake of 3 cups of soymilk do
not significantly increase antioxidant levels. Additional research closely monitoring the
participants for compliance of the appropriate consumption of the fruits and soymilk is
needed to thoroughly understand the protective properties of foods high in antioxidants
from the risks associated with oxidative stress in general, but specifically in
postmenopausal women.
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Mingo, C., Herman, C. J., & Jasperse, M. (2000). Women's stories: Ethnic variations in
women's attitudes and experiences of menopause, hysterectomy, and hormone
replacement therapy. Journal of Women's Health & Gender-Based Medicine, 9,
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MyPyramid. (2011, February 3). Retrieved May 4, 2011, from United States Department
of Agriculture: http://www.mypyramid.gov/pyramid/milk_amount_table.html
Pick, M. (2005, January 11). Menopause and perimenopause. Retrieved June 29, 2011,
from Women to Women:
http://www.womentowomen.com/menopause/estrogendominance.aspx
Porrini, M., & Riso, P. (2000). Lymphocyte lycopene concentration and DNA protection
from oxidative damage is increased in women after a short period of tomato
consumption. The Journal of Nutrition, 130, 189-192.
Prior, R. L. (2003). Fruits and vegetables in the prevention of cellular oxidative damage.
The American Journal of Clinical Nutrition, 78, 570-578.
Robert-McComb, J. J., Norman, R., & Zumwalt, M. (2008). The Active Female: Health
Issues throughout the Lifespan. Totowa: Humana Press.
Ryan-Borchers, T. A., Park, J. S., Chew, B. P., McGuire, M. K., Fournier, L. R., &
Beerman, K. A. (2006). Soy isoflavones modulate immune function in healthy
postmenopausal women. The American Journal of Clinical Nutrition, 1118-1125.
Scoglio, S., Benedetti, S., Canino, C., Santagni, S., Rattighieri, E., Chierchia, E.,
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Seifried, H. E., Anderson, D. E., Fisher, E. I., & Milner, J. A. (2007). A review of the
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Sies, H. (1997). Oxidatve Stress: Oxidants and Antioxidants. Experimental Physiology,
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Sohal, R. S., & Weindruch, R. (1996). Oxidatve Stress, Caloric Restriction, and Aging.
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Stampfer, M. J., Hennekens, C. H., Manson, J. E., Colditz, G. A., Rosner, B., & Willett,
W. C. (1993). Vitamin E consumption and the risk of coronary disease in women.
The New England Journal of Medicine, 328(20), 1444-1449.
Svilaas, A., Sakhi, A. K., Frost Anderson, L., Svilaas, T., Strom, E. C., Jacobs Jr., D. R.,
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Tesoriere, L., Butera, D., Pintaudi, A. M., Allegra, M., & Livrea, M. A. (2004).
Supplementation with cactus pear (Opuntia ficus-indica) fruit decreases oxidative
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pre- and postmenopausal women by lowering plasma lipids and reducing
oxidative stress. The Journal of Nutrition, 135, 1911-1917.
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Appendix A
Recruitment Materials
SCRIPT USED TO RECRUIT VOLUNTEERS FOR THE
The effects of a combination of foods high in antioxidants on blood antioxidant and
oxidative stress levels in post-menopausal women
PROTOCOL
Drs. Melanie Hart, Jacalyn McComb, and Yoonjung Park, and Shelby Kloiber, graduate
student in the Department of Health, Exercise and Sport Sciences are conducting a study
for which they are interested in seeking volunteers. The purpose of this study is to
determine if there is a greater benefit in consuming a combination of sources of
antioxidants compared to just one type of food source in postmenopausal women.
Participation in this study will require three visits to the Exercise Physiology Laboratory
housed within the Exercise Sciences Center on the Texas Tech campus. The first visit
will include a complete explanation of the study protocol, completion of the medical
history questionnaire and consent form, and familiarization with the testing equipment
and procedures. At the end of this meeting you will take home a no food list. We ask
that you do not consume anything that is on this last for the week before your second
visit. You will have a food diary to be completed during this week as well. This visit
will last about 20 min. Visit 2 will consist of blood collection at a vein in the elbow. We
will also gather measurements such as height, weight, BMI, blood pressure, heart rate,
and hematocrit levels by finger prick. At the end of this visit you will take home the list
of foods/liquids along with the daily servings determined by the USDA. This will allow
you to keep track of the required diet you shall complete depending on the group you are
randomly placed. Half way through the intervention the research group will contact you
for updates and any questions you may have. The third and final visit will include the
last blood draw and measurements. The second and third visit will last about an hour.
You will be asked not to consume any non-steroidal anti-inflammatory medications (such
as ibuprofen or Aleve (Naproxen)) 2 days prior to the blood draws. You will be asked to
fast 8-12 hours before each blood draw.
Recruitment includes the completion of a food recall record that will allow us to exclude
those who already consume a diet high in antioxidants. Recruitment for this study is
limited to postmenopausal women, healthy subjects who are not participating in
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Texas Tech University, Shelby Kloiber, August 2011
hormonal therapy. (Cessation of menses > 1 year) Participants need to be willing to
participate in a diet high in antioxidant rich food/liquid. Anyone willing to participate
must be free of any known disease, and a non-smoker. Those interested cannot consume
more than 2 alcohol drinks per day and do not engage in heavy exercise. There is
absolutely no penalty if you should decline to participate. Participation in this study is
completely voluntary and participants are free to quit the study at any time.
Thank you for your attention and time.
Newspaper Announcement
Are you a postmenopausal woman not taking part in hormone replacement therapy? The
Health, Exercise, & Sport Sciences department is asking adult females who are in their
postmenopausal stage to join in a research study examining the effects of a combination
of foods high in antioxidants. The study will be three (3) meetings, and each visit will
last less than an hour. Women will be asked to add to their normal diet five or more
servings of fruit every day and/or three servings of soymilk every day.
Measurements taken at the last two meetings include height, weight, body mass, blood
pressure, heart rate, and hydration levels. Blood will be taken to measure antioxidant and
oxidative stress levels.
Your taking part in this study will be voluntary and greatly appreciated. You may say no
or stop your involvement at any time during the study. If you are interested and would
like to see if you can take part in the study or want more information contact Shelby
Kloiber in the Department of Health, Exercise, and Sport Sciences at (806) 787-5070 or
by email at [email protected]
Thank you for your interest,
Shelby Kloiber
Graduate Teaching Assistant at Texas Tech University
[email protected]
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Texas Tech University, Shelby Kloiber, August 2011
The Department of Health, Exercise,
and Sport Sciences at Texas Tech
University is studying the effect of a
high antioxidant diet on total
antioxidant and oxidative stress
levels
Study includes:
-Total of three (3) visits
Study Involves:
-Each visit takes
-Blood draws during visits
Less than 1 hour
two and three
-Consuming ≥ 5 fruit
-Measurements taken:
servings
Height, weight, BMI, hydration,
soymilk a
and/or ~ 3cups of
Heart rate, blood pressure
Shelby Kloiber
day for 4 weeks
[email protected]
44
806-787-5070
Texas Tech University, Shelby Kloiber, August 2011
Appendix B
Informed Consent
You are being invited to participate in the research project entitled: The effects of a
combination of foods high in antioxidants on blood antioxidant and oxidative stress levels
in post-menopausal women. The people responsible for this research project are: Drs.
Melanie Hart, Jacalyn McComb, and Yoonjung Park, and Shelby Kloiber, graduate
student in the Department of Health, Exercise and Sport Sciences at Texas Tech
University, (806) 742-3371.
I. Purpose and explanation of the study
The purpose of this research is to determine if there is a difference in markers of
oxidative stress and antioxidant capacity in individuals with single or a combination of
food source interventions. Oxidative stress occurs when substances called free radicals
occur in large amounts and cause damage to cell DNA. Postmenopausal women have a
decrease in estrogen, lowering their immune function, increasing their risk for disease
caused by these free radicals. Antioxidant capacity is the body’s ability to take free
radicals out of the system.
You will be asked to complete the following procedures:
1. Insertion of a needle in a vein at the elbow for blood collection at the beginning
and end of the research project.
2. Completion of one week of following no consumption from a No food list, and
4 weeks of a specific diet and intake record.
All blood draws and measurements will be conducted in the Exercise Physiology Lab at
Texas Tech University Exercise Sciences Center. The first visit will include a complete
explanation of the study protocol, completion of the medical history questionnaire and
consent form, and familiarization with the testing equipment and procedures. You will
also be asked to start recording a food diary for one week, and follow a list of foods not
to consume (wash out period). This visit will last about 20 min. Visit 2 will consist of
blood collection at a vein in the elbow. We will also gather measurements such as height,
weight, BMI, blood pressure, heart rate, and hematocrit by finger prick. At the end of
this visit you will take home the list of foods/liquids along with the daily servings
determined by the USDA. Fruit consumption includes 5 or more fruits and/or 706mL of
soymilk. Food and liquid will be purchased by the participant. The food check off list
will allow you to keep track of the required diet. Half way through the intervention the
research group will contact you for updates and any questions you may have. The third
and final visit will include the last blood draw and measurements. At this point you are
no longer required to following the intervention.
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Texas Tech University, Shelby Kloiber, August 2011
Before you undergo the diet intervention, you must certify to the researchers that you are
in good health to the best of your knowledge. You will fill out a medical history
questionnaire that will be reviewed by trained professionals prior to undergoing the
intervention and blood work. Based on the medical history you may be disqualified from
the study because of increased risk to you during the diet intervention. Consequently, it
is important that you provide complete and accurate responses to the interviewer and
recognize that your failure to do so could lead to possible unnecessary health problems
during the procedures.
You will have a blood draw of about 3 tubes at 5 ml each (about 1-2 teaspoons each)
taken from a needle that will be inserted in a vein at your elbow to determine markers of
free radical activity and antioxidant activity. There will be a blood draw at the second
and third meeting.
You will be asked not to take anti-inflammatory drugs (such as ibuprofen and Aleve
(Naproxen)) 3 days prior to, and during the blood draws. Lastly, you will be asked to
come in fasting 8-12 hours before each blood draw.
II. Risks
There exists the possibility of adverse changes during the intervention. These changes
could include diarrhea, constipation, minor GI discomfort such as bloating and cramping,
hyperglycemia, hypoglycemia, and gastro-esophageal reflux. Some minor discomfort
may be experienced with a needle prick for the collection of blood samples and
hematocrit testing. There is the possibility of infection at the needle prick site; however
this is greatly minimized by following strict aseptic technique.
In the event of any problems such as physical, psychological, financial, etc, Texas Tech
University or the Student Health Center may not be able to treat your injury. You will
have to pay for treatment from your own insurance. The university does not have
insurance to cover such injuries. More information about these matters may be obtained
from Dr. Kathleen Harris, Associate Vice President for Research, (806) 742-3884, Room
203 Holden Hall, Texas Tech University, Lubbock, Texas 79409.
III. Benefits to be expected
The results of this research may or may not benefit you. Potential benefits relate mainly
to a possible decrease in your risk for developing chronic diseases. Those who are
increasing their fruit intake will meet their requirement for fruit servings per day. Those
who are increasing their soymilk consumption will meet their requirements for dairy
servings per day.
IV. Confidentiality and use of information
All information obtained from these procedures will be treated as privileged and
confidential and will consequently not be released or revealed to any person without your
written consent. By signing this form, you are agreeing to the use of any data recorded
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Texas Tech University, Shelby Kloiber, August 2011
for research or statistical purposes so long as it does not provide facts that could lead to
your identification. A numeric code will be used as an identifier for statistical purposes
and these will be deleted upon completion of the analysis. The information contained in
the medical history form will be maintained with complete confidentiality during the
course of the study and destroyed upon completion of the study. Any other information
obtained, however, will be used only by the program staff to evaluate your diet
intervention. Any information that can lead to your identification will be kept in the
exercise physiology laboratory in the Exercise Science Center in a locked file cabinet,
with access controlled by Drs. McComb and Park, and Shelby Kloiber.
V. Inquiries and freedom of consent
Dr. Hart, Dr. McComb, Dr. Park, or Shelby Kloiber will answer any question that you
may have about this study. For questions about your rights as a participant or about
illness caused by this research, you should contact TTU Institutional Review Board for
the Protection of Human Subjects, Office of Research Services, Texas Tech University,
Lubbock, Texas 79409. Or you can call (806) 743-3884.
Participation in this research project is voluntary and refusal to participate involves no
penalty or loss to which you may be entitled and you may discontinue participation at any
time without penalty or loss of benefits.
By signing this form, you are acknowledging that you have read this document in its
entirety and that the investigator reviewing this document has made certain that you
understand it.
____________________________________ Date____________
Participant’s signature
This consent form is not valid after December 31, 2011.
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Texas Tech University, Shelby Kloiber, August 2011
Appendix C
Health History Survey
MEDICAL HISTORY QUESTIONNAIRE
Demographic Information
Last Name __________________________
First Name __________________________
Middle Initial ________________________
Date of Birth ________________________
Sex _______________________________
Home Phone ________________________
Address ____________________________
City ______________________________
State ______________________________
Zip Code ____________________________
Work Phone ________________________
Family Physician ____________________
The following questions are to ensure that you are healthy enough to participate in the
intervention in this study, and are for your protection.
Section A
1. When was the last time you had a physical examination?
*2. If you are allergic to any medications, foods, or other substances, please name them.
3. If you have been told that you have any chronic or serious illnesses, please list them.
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Texas Tech University, Shelby Kloiber, August 2011
4. Give the following information pertaining to the last time you were hospitalized.
Reason for hospitalization:
Month and year of hospitalization:
Hospital:
City and State:
5. Are you affected with hemophilia?
*6. Have you participated in any type of hormone replacement therapy in the last five
years?
If yes, explain_____________________________________
*7. Please list the date of your last menstrual cycle. _____________________________
Section B
For questions 1-13 have any of the following situations happened during the past 12
months?
1. Has a physician prescribed any form of medication for you? Yes___ No ___
If yes, were the medications for a cardiovascular condition? Yes ___ No ___
2. Has your weight fluctuated more than a few pounds? Yes ___ No ___
If yes, did you attempt to bring about this weight change through diet or exercise?
Yes___ No___
3. Have you experienced any faintness, light-headedness, or blackouts? Yes___ No ___
If yes, what were the circumstances?
4. Have you occasionally had trouble sleeping? Yes ___ No ___
5. Have you experienced any blurred vision? Yes ___ No ___
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Texas Tech University, Shelby Kloiber, August 2011
6. Have you any severe headaches? Yes ___ No ___
7. Have you experienced any temporary change in your speech pattern, such as slurring
or loss of
speech? Yes ___ No ___
8. Have you felt unusually nervous or anxious for no apparent reason? Yes___ No___
*9. Have you smoked cigarettes? Yes___ No ___ If yes, how often?
For questions 10-14 are you currently experiencing any of the following situations?
*10. Have you ever been told that your blood pressure was abnormal? Yes ___ No ___
11. Have you ever been told that your serum cholesterol or triglyceride level was high?
Yes ___ No ___
*12. Do you have diabetes? Yes ___ No ___
___ Dietary means
If yes, how is it controlled? (Check One)
___Insulin injection ___Oral medication
___Uncontrolled
13. How often would you characterize your stress level as being high? (Check One)
___ Occasionally
___Frequently
___Constantly
*14. Have you ever been told that you have any of the following illnesses? Yes ___
No___
__ Cardiovascular Disease __Uterus dysfunctions __ Hypertension __ Gastrointestinal
disease
__ Renal disease __ HIV
Section C
BMI= body weight (kg)/height (m2) = ___________
Is your BMI within 18-40? Yes ___ No ___
Exclusion Criteria
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Texas Tech University, Shelby Kloiber, August 2011
Questions with an asterisk * can exclude the person from the study unless clarified or
cleared by a physician. Other questions will help the researchers determine if the subject
does not fit the apparently healthy criterion.
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Texas Tech University, Shelby Kloiber, August 2011
Appendix D
Physical Activity Survey
General Practice Physical Activity Questionnaire
Date_____________
Name _________________
1. Please tell us the type and amount of physical activity involved in your work.
Please mark one only
a. _______I am not in employment (e.g. retired, retired for health reasons, unemployed, fulltime carer etc.)
b. _______I spend most of my time at work sitting (such as in an office)
c. _______ I spend most of my time at work standing or walking. However, my work does
not require much intense physical effort (e.g. show assistant, hairdresser, security
guard, childminder, etc.)
d. _______My work involves definite physical effort including handling of heavy objects and
use of tools (e.g. plumber, electrician, carpenter, cleaner, hospital nurse, gardener,
postal delivery workers, etc.)
e. _______My work involves vigorous physical activity including handling of very heavy
objects (e.g. scaffolder, construction worker, refuse collector, etc.)
2. During the last week, how many hours did you spend on each of the following activities?
Please answer whether you are in employment or not.
None
Physical exercise such as swimming,
jogging, aerobics, football, tennis,
gym workout, etc.
Cycling, including cycling to work
and during leisure time
Walking, including walking to work,
shopping, for pleasure, etc.
Housework/childcare
52
Some but
less than 1
hour
1 hour but less 3 hours
than 3 hours
or more
Texas Tech University, Shelby Kloiber, August 2011
Gardening? DIY
3. How would you describe your usual walking pace? Please mark one only
_______ Slow pace (i.e. less than 3 mph)
_______ Steady average pace
_______ Brisk pace
_______ Fast pace (i.e. over 4 mph)
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Texas Tech University, Shelby Kloiber, August 2011
Appendix E
Dietary Recall
This questionnaire will give us information about your eating habits. There are no “right”
or
“wrong” answers. Accurate and thoughtful responses will allow us to pinpoint your eating
habits.
• Use the past month as your standard for how you eat.
• Recall the times during the day when you ate, and what you had.
• Include snacks and “nibbles” as well as meals and beverages.
• If you ate out regularly or traveled, remember to include those foods too.
• Be sure to answer every item on this form. If you did not eat a food listed
below— or ate it less than once a week — write a “0” in the space provided. Please do
not leave blanks.
Part I. We want to know how often you ate certain foods. For each of the foods listed,
please
indicate how many servings per week you usually ate in the past month. (If you ate a
food less
than once a week, write a “0” in the space provided.)
Food
Amount YOU eat/month
Typical Serving Size
Fresh fruit
1 whole piece or ½ cup cut-up
Dried fruit
½ cup
Fruit juice
½ cup or 4 ounces
Food
Amount YOU eat/month
Typical Serving Size
Cooked vegetables
½ cup
Raw vegetables
1 cup
Food
Amount YOU eat/ month
Red meat (beef, pork, ham,
veal, lamb)
Typical Serving Size
4 ounces (size of a deck of
cards)
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Texas Tech University, Shelby Kloiber, August 2011
Dried beans, split peas, or
lentils
Food
¾ cup cooked
Amount YOU eat/month
Typical Serving Size
Chocolate or candy bar
1 regular candy bar
Alcoholic drinks
1 drink, 1 can of beer, 1 glass
of wine
Soymilk
½ cup or 4 ounces
Soy products (please give food type, amount consumed and how many times you consumed the
soy product per week) _________________________________________________________
Part II. We want to know the different types of fruits and vegetables consumed in the last
month. For each of the foods listed, please indicate how many servings per week you usually
ate in the past month. (If you ate a food less than once a week, write a “0” in the space
provided.) Please place a number in the blank cell that indicates how many times you have
consumed this food in the past month. If the food is not mentioned please list the food and
amount of times consumed per month in others section.
Apple
Apricot
Blackberry
blood orange
Blueberry
boysenberry
Cactus Pear
Cantaloupe
Cherry
cranberry
grapefruit
Green grape
guava
Honeydew melon
kiwi
lemon
Lime
mango
artichokes
arugula
asparagus
avocadoes
broccoli rabe
broccoli
brussel sprouts
celery
cucumbers
endive
green beans
cabbage
green peppers
leafy green (kale)
leeks
lettuse
okra
peas
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Texas Tech University, Shelby Kloiber, August 2011
nectarine
Orange
papaya
Peach
Pear
Persimmon
pineapple
Plum
Pomegranate
prunes
purple grape
Raspberry
starfruit
Strawberry
tangerine
Watermelon
kumquat
Litchis
rambutan
spinach
zucchini
cauliflower
mushroom
onions
parsnips
potatoes
white corn
beets
red peppers
red potatoes
tomatoes
squash
sweet corn
yellow pepper
eggplant
carrots
red onion
sweet potato
Others:
_____________________________________________________________________________
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Texas Tech University, Shelby Kloiber, August 2011
Appendix F
Banned Food List
During the first week of this study, we ask you to refrain from the foods below.
Multivitamins
Alcohol
Tea
Coffee
Orange
Pomegranate
Blueberry
Blackberry
Cherry
Soy Products
Beans
Chocolate
Vegetables orange in color
Dark green leafy vegetables
Tomatoes
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Texas Tech University, Shelby Kloiber, August 2011
Appendix G
Daily Food Check-Sheet
Soymilk daily check-off
DATE
15-Jan
16-Jan
17-Jan
18-Jan
19-Jan
20-Jan
21-Jan
22-Jan
23-Jan
24-Jan
25-Jan
26-Jan
27-Jan
28-Jan
29-Jan
30-Jan
1-Feb
2-Feb
3-Feb
4-Feb
5-Feb
6-Feb
7-Feb
8-Feb
9-Feb
10-Feb
11-Feb
12-Feb
*dates subject to change
Soymilk Brand:
Amount
Total amount per day: 706 mL
Can by split up into 2 or 3
servings
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Texas Tech University, Shelby Kloiber, August 2011
Fruit List ( dates subject to change)
Fruit
Apple
Apricot
Blackberry
blood orange
Blueberry
boysenberry
Cactus Pear
Cantaloupe
Cherry
cranberry
grapefruit
Green grape
guava
Honeydew
melon
kiwi
lemon
Lime
mango
nectarine
Orange
papaya
Peach
Pear
Persimmon
pineapple
Plum
Pomegranate
Serving
1 small
1/2 cup
1/2 cup
1 small
1/2 cup
1/2 cup
1 cup
1 cup
1/2 cup
1/2 cup
0.5
large
15
3/4 cup
15-Jan
16Jan
1 cup
1 whole
3/4 cup
2 whole
1/2
small
1 small
1 large
0.50
1 large
1
mediu
m
2
1 cup
2 large
1/3
59
17Jan
18Jan
19Jan
20-Jan
21-Jan
Texas Tech University, Shelby Kloiber, August 2011
prunes
purple grape
Raspberry
starfruit
Strawberry
tangerine
Watermelon
kumquat
Litchis
rambutan
whole
3
dozen
1/2 cup
2 large
6
berries
2 small
1 1/4
cup
5
10
1/3 cup
60