Florida State University Libraries
Honors Theses
The Division of Undergraduate Studies
2013
The Effects of Ovarian Hormone Deficiency
on Lipid and Cholesterol Concentration
Aazim Arif
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Florida State University
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College of Human Sciences
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The Effects of Ovarian Hormone Deficiency On Lipid and
Cholesterol Concentration
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By
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Aazim S. Arif
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A Prospectus submitted to the Department of Nutrition,
Food and Exercise Sciences in partial fulfillment for the
requirements of Honors in the Major
Fall 2012
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The members of the Defense Committee approve the thesis
of Aazim Arif
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Dr. Bahram Arjmandi
Thesis Director
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Dr. Jacob VanLandingham
Outside Committee Member
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Dr. Jeong-Su Kim
Committee Member
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Dr. Michael Ormsbee
Committee Member
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CHAPTER 1
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INTRODUCTION
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Heart disease is the leading cause of death for both men and
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women in the United States (1). One of the many implications that
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persist in patients who suffer from coronary heart disease is the
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buildup of plaque within the blood vessels that supply blood to the
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heart. Atherosclerosis, which is the buildup of plaque within the
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blood vessels, causes the arteries to harden. This triggers the blood
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vessels to narrow. Consequently, the blood flow to the heart can
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become restricted partially or even completely, thus leading to a
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myocardial infarction. In postmenopausal women, the loss of
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estrogens has been associated with hypercholesterolemia. One of
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the
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hypercholesterolemia (FH). FH is an autosomal disorder associated
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elevated total cholesterol and low-density lipoproteins (LDL) (2).
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In addition, an FH phenotype has been associated with an
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increased risk of coronary heart disease and premature death (2).
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Low levels of high-density lipoprotein (HDL) and high levels of
critical
risk
factors
of
heart
disease
is
familial
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low-density lipoproteins have consistently been shown to be
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associated with coronary heart disease risk (3). The effects of
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estrogen vary depending on the route of administration and dose.
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Nonetheless, estrogen is frequently classified with an increase in
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HDL, as well as a decrease in low-density lipoproteins and
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triglycerides. Estrogens have also been shown to have beneficial
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effects
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Furthermore, estrogens have also well correlated with a decreased
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risk of developing atherosclerosis (4,9,10).
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on
plasma
lipid
and
lipoprotein
concentrations.
CHAPTER 2
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BACKGROUND
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Though the umbrella term for the female sex hormone is estrogen,
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there are various types of hormones that act in this manner. The
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most potent estrogen and in highest concentration is 17β-estradiol
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in premenopausal women (12). In postmenopausal women, the loss
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of 17β-estradiol causes the two other forms of estrogen, estriol and
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estrone, to be primarily found. These sterol derivatives make up a
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class of sex hormones synthesized from cholesterol that have an
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array of functions throughout the body. The association between
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protective health benefits and estrogen are defined by 17β-estradiol
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(13). Estrogens are known to have protective effects among
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women during their reproductive years (5). The effects of estrogen
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on lipid concentration, and more specifically, cholesterol
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concentration, will be discussed.
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As cholesterols are within the umbrella of lipids, a thorough
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understanding of what a lipid is needs to be fully explained. Lipids
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are any substance that has a low solubility in water and a high
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solubility in nonpolar organic solvents. As this definition is broad,
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the substances that are categorized as lipids are also broad. The
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many different types of lipids include phospholipids, steroids,
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which include cholesterol, glycolipids, terpenes (Vitamin A),
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triacylglycerol, and fatty acids. Each lipid provides their unique
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benefit within the human body. For example, phospholipids
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provide
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Triacylglycerol store metabolic energy, while steroids, including
structural
support
for
the
cell’s
membranes.
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cholesterol, regulate metabolic activities. A further explanation of
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one of the more important lipids for this research experiment,
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cholesterol, will be more thoroughly assessed.
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Cholesterol in the body is derived from two sources,
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endogenously or exogenously. The liver, which is a vital organ that
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ranges in numerous functions, produces eighty percent of the total
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cholesterol within the body (6). The diet provides the other twenty
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percent of cholesterol (6). It is extremely important to understand
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that self-produced cholesterol is sufficient for homeostasis. It is
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well known that the average American and other populations in the
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Western Hemisphere consume more than the recommended 300
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mg of cholesterol in the diet via the consumption of meats. This
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excess consumption has been associated with the increased risk of
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developing coronary heart disease (11).
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illustrates the correlation between serum cholesterol levels and
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coronary heart disease (11).
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The graph below
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S
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r
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m
C
h
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l
e
s
t
e
r
o
l
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5
4
3
2
1
0
0
20
40
60
Age Related CAD Death
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Figure 1: As serum chol
olesterol increases, the mortality resulting
g from CHD
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also increases.
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With the incide
idence of menopause, the presence
ce of 17β-
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iminished (12). As stated before, 17β
7β-estradiol
estradiol is greatly dim
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has numerous protect
ctive effects within the female body
dy. One of
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these protective effe
ffects includes its regulation of HMG-CoA
H
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reductase, the rate-lim
limiting enzyme of cholesterol synthe
thesis. The
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iol causes cholesterol biosynthesi
esis to be
loss of 17β-estradio
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exacerbated in postme
menopausal women (14,15), and in addition
a
to
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the Western hemisphe
here diet, this can be extremely detri
trimental to
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result of menopause leading to an increase
in
in
one’s health. As a re
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cholesterol synthesis (15), it is important to understand cholesterol
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biosynthesis and how estrogen regulates this process. In order to
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understand cholesterol biosynthesis in the human body, and the
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importance of cholesterol, it is critical that the functions of
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cholesterol are known. Cholesterol is the precursor to our steroid
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hormones and bile salts. It also plays a significant role in our cell
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membranes by modulating its fluidity. Cholesterol can either be
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obtained in our diet, in mostly meats and dairy products, or it can
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be synthesized in the liver (6). There are numerous steps involved
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in cholesterol synthesis, which are illustrated in the diagram below.
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Figure 2: A summary of the synthesis of cholesterol via Acetyl-CoA
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The reaction that reduces 3-Hydroxy-3-Methyl-Glutaryl-Coa
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(HMG-CoA) to Mevalonate requires the rate-limiting enzyme,
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HMG-CoA reductase. This enzyme is tightly regulated in
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cholesterol synthesis. The rate by which cholesterol is synthesized
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depends on the levels of cholesterol in the cell. Increased levels of
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cholesterol in the cell control HMG-CoA reductase by three ways:
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inhibits the feedback of HMG-CoA reductase, decreases the
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translation of HMG-CoA reductase’s mRNA, which will reduce
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the amount of this rate-limiting enzyme and decreasing the amount
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of HMG-CoA reductase present by increasing its degradation.
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In addition to assessing HMG-CoA reductase activity, this
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research will also focus on the production of bile acids. 7-α
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hydroxylase is needed to synthesize 7-alphahydroxycholesterol
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from cholesterol itself. This reaction is critical because it is the
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rate-limiting step in the synthesis of bile acids. Bile acids are the
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major pathway for the elimination of cholesterol and its
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composition is composed of polar derivatives of cholesterol (6).
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The biosynthesis of bile acids occur in the liver, however, bile
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acids are stored in the gallbladder. In the liver, cholesterol is
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converted to 7-hydroxycholesterol, with the help of the enzyme 7-
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alpha-hydroxylase.
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converted to an intermediate cholyl-CoA, which may then react
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with an amino acid glycine to form glycocholate or it may react
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with taurine to form taurocholate (6). Bile acids are extremely
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important in the emulsification of dietary lipids within the small
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intestines. The main aspect that this research will focus on is if
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cholesterol and bile acids are increased ovarian hormone deficient
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Sprague-Dawley rats.
7-hydroxycholesterol
is
We propose the following hypothesis and specific aims to
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Furthermore,
address such questions:
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Hypothesis and Aims:
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Hypothesis: Ovarian hormone deficiency alters both lipid content
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and cholesterol concentration by up regulating the synthesis of
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each.
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Aim 1: To analyze if lipid content is greater in ovarian hormone
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deficient Sprague-Dawley liver.
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Aim 2: To analyze if cholesterol concentration is greater in ovarian
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hormone deficient Sprague-Dawley liver.
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Aim 3: To determine the amount of total lipid content and total
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cholesterol concentration in ovarian hormone deficient, E2-treated,
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and sham-control Sprague-Dawley livers.
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CHAPTER 3
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METHODS
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There are several aspects of this research that require the use and
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knowledge of several scientific procedures. The Folch method and
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Western blot are two procedures that will explicitly be used in this
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research. The Folch method is used to analyze lipid extractions
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from a given sample. To understand how the Folch method of
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extraction, a basic understanding of lipid composition must be
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understood. Lipid’s associations with carbohydrates, proteins, or
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other lipids contain van der Waal interactions, hydrogen bonding,
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or covalent bonds (7). As a result, the separation of lipids from
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these interactions requires different chemical treatments. Lipids are
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composed of a neutral and polar portion, respectively. The neutral
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portions of the lipid are hydrophobically bound and are separated
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from tissues by nonpolar solvents (7). On the other hand, the polar
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portion of the lipid involves polar solvents capable of breaking its
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bonds (7). Therefore, to guarantee a thorough and quantitative
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retrieval of all tissue lipids, the Folch method will be used.
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After using the Folch method, determination of liver total
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cholesterol will be assessed. Part of the Folch method procedure
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involves a 25 ml lipid sample that will be used in order to
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determine cholesterol concentration. The reagents involved in this
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procedure included a saponification solution, which consisted of
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15% alcoholic KOH solution and 3% pyrogallic acid, hexane,
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acetone:ethanol (1:1) mixture, saturated FeSO4 7H2O, and
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concentrated sulfuric acid. The standard solutions that were
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necessary in order to compare the samples were 7 mg/dl, 5 mg/dl,
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4 mg/dl, 3 mg/dl, 2 mg/dl, 1 mg/dl and 0 mg/dl. The absorbance of
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these standards, at 490 nm, provided a means to calculate the
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concentration of the cholesterol samples.
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CHAPTER 4
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RESULTS
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Part A: Lipid Concentration
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To answer the basic question if estrogen has an effect on total lipid
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concentration, an experimental analysis of lipid concentration
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within each of the rat’s liver was analyzed. In order to fully
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understand the effects of estrogen within each of the livers, three
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differentially treated rat livers were examined. These three
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different types of rat livers consisted of an estrogen-treated liver
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(E2-treated), a sham-control liver, and lastly, an ovarian hormone
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deficient (OVX) rat liver. The analysis of these three livers will
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fully explain, through the findings in each type of rat livers, the
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effects of estrogen on not only lipid concentration, but as well as
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cholesterol concentration. The Folch method for lipid extraction
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provided a statistical examination of the results.
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The comparison between E2-treated, Sham-control, and OVX will
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be analyzed within the table and graph seen below
Lipids (mg/g
liver)
Weight of Lipid
OVX
O21
O11
O18
O3
O20
O10
Mean
Std. Dev
SE
E2
O19-2
O4
OE2-#1
O9
O7
O6
Mean
Std. Dev
SE
Sham
O23
O13
Old Sham
#15
O5
Mean
Std. Dev
SE
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Total Lipids (mg/whole
liver)
0.5
0.67
0.52
0.8
0.5
0.53
190
254.6
197.6
304
190
201.4
222.93333
46.58166
18.40541386
1181.8
2650.39
1545.23
5535.2
1233.1
1355.42
2250.19
1698.52654
633.32947
0.52
0.56
0.48
0.57
0.49
0.49
197.6
212.8
182.4
216.6
186.2
186.2
196.96667
14.70098
7.871361593
1383.2
1649.2
1253.09
1695.98
1629.25
1146.99
1459.61833
231.00352
87.50108465
0.69
0.48
262.2
182.4
2766.21
1417.25
0.48
0.52
182.4
197.6
206.15
38.04747
18.01344984
2083.01
1337.75
1901.055
666.57848
289.5366719
Table 1 Lipid concentration within each individual type of rat liver
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Table 1 numerically presents the weight of the lipids extracted, the
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lipid concentration within each gram of liver, and in addition the
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total lipids within each liver. The weight of the lipid was
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experimentally derived, however the lipids within each gram of
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liver, as well as the total lipids within each whole liver were
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mathematically derived. The calculation use to determine the lipids
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within each gram of liver and the total lipids within each liver is as
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follows:
.
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"
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/
.
0.76 1000
/
Lip
Lipids Per Gram of Liver
Sham
E2
OVX
300
(mg/g liver)
250
200
150
100
50
0
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Fig. 3 Analysis of lipids
ds within each gram of liver
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Figure 3 illustrates a comparison of the lipid content within
wi
each
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gram of liver betwee
een OVX, E2-treated, and Sham-co
control. As
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seen in the graph, thee estrogen deficient (OVX) liver con
ontained the
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pids per gram of liver. In addition,, E2-treated
E
most amounts of lipid
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liver contained the lea
east amount of lipids per gram of liver
ver.
OVX,
X, E
E2, Sham Total Liver Lipids
Sham
E2
OVX
(mg/whole liver)
3500
3000
2500
2000
1500
1000
500
0
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Fig 4. Analysis of totall llipids within each whole liver
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Figure 4 further dep
epicts the effects of estrogen on total
to
lipids
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liver. As seen in figure 3, OVX cont
ntained the
within each whole liv
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most amounts of lipid
pids. Furthermore, E2-treated liverss contained
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the least amounts of to
total lipids within each liver.
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Both figure 3, as well as figure 4, offer a graphically
gr
289
representation of the
he effects of estrogen within each liver.
l
The
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estrogen absent (OVX
X) livers contained the most amount
nt of lipids.
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In addition the estro
trogen treated (E2) liver contained
d the least
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amount of lipids. This
his depicts the importance of estrogen
gen on lipid
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concentration.
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Part B: Cholesterol Concentration
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To further breakdown the type of lipids within each liver, an
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analysis of cholesterol concentration was examined. As stated
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before, cholesterol is an extremely important lipid because it helps
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support the cell membrane’s structure. The effects of estrogen on
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lipid concentration are evident, but will estrogen have the same
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effects on a more particular form of lipid such as cholesterol. In
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order to determine the cholesterol concentration in the livers, Folch
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method, as well, as a liver total cholesterol protocol were
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experimentally assessed. The table and figure below illustrates the
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research findings.
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Concentration
OVX
O21
O11
O18
O3
O20
O10
Mean
2.294
1.699
3.754
3.829
2.337
3.505
2.903
O19-2
O4
OE2-#1
O9
O7
O6
Mean
2.723
3.05
3.346
4.439
3.016
3.073
3.2745
O23
O13
Old Sham #15
O5
Mean
4.829
1.428
3.05
3.636
3.236
E2
Sham
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315
Table 2 Cholesterol concentrations within each individual type of rat
liver
A graphically representation of table 2 is shown below.
Cholest
lesterol Concentration
Sham
E2
OVX
Concentration (mg/dl)
4.5
316
317
4
3.5
3
2.5
2
1.5
1
0.5
0
Fig. 5 Cholesterol conce
centrations within each individual typee of
o rat liver
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319
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Figure 5 depicts OVX
X, E2-treated, as well as Sham-contr
ntrol having
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similar cholesterol co
concentration. Unlike the lipid conce
centrations,
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which are significantly
tly different between each category of rat liver,
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total cholesterol conce
centration are not statistically signific
ficant.
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CHAPTER 5
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DISCUSSION/FUTURE
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The findings of this research clearly depict the intriguing, yet
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complicated, role of estrogen within the body. The effects of
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estrogen are numerous, as this research as shown its specific
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effects on lipid concentration as well as cholesterol concentration.
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Estrogen’s role in lipid formation is clearly illustrated in figure 3
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and figure 4. The ovarian hormone deficient Sprague-Dawley rat
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liver’s contained the most amount of lipid concentration. The loss
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of estrogen increased the lipid concentration in those livers. In
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addition, the liver’s treated with estrogen significantly had lower
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lipid concentration when compared to sham-control, as well as
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OVX. The second portion of this research analyzed the effects of
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estrogen on cholesterol concentration. As cholesterol is a form of
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lipid, the effects are extremely surprising. It is understood that
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estrogen protects the body against the increase of cholesterol. As
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seen in postmenopausal women, the rise of cholesterol is extremely
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detrimental in our older female population. However, the research
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findings showed the cholesterol concentration to be quite similar
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between each of the three groups. It is expected that the OVX
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livers would have a substantially higher amount of cholesterol
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when compared to E2-treated or sham-control. In addition, E2-
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treated livers would contain the least amount of cholesterol.
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However, it should be noted that the different subcategories of
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cholesterol could explain the discrepancy. In the future, a further
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analysis of the different types of cholesterol should be evaluated.
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OVX livers could possibly contain a higher amount of LDLs with
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a lower amount of high-density lipoproteins HDL. In addition, E2-
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treated livers could possibly contain a higher amount of HDLs with
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a lower amount of LDLs. This lipoprotein variation would appear
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to have the same cholesterol concentration, but a higher amount of
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HDLs would be more beneficial as it provides a more positive role
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in the body. Lastly, a more thorough understanding of whether
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HMG-CoA reductase’s activity is increased in ovarian hormone
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deficient Sprague-Dawley rats will be crucial. This understanding
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will help combat the harmful effects that the loss of estrogen has
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on the female body.
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CHAPTER 6
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