Effects of soy isoflavone consumption following a high fat meal on the oxidative resistance of healthy
young men
by Danielle Ann Dufner
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Health and Human Development
Montana State University
© Copyright by Danielle Ann Dufner (2003)
Abstract:
Coronary heart disease (CHD), also referred to as coronary artery disease (CAD) or atherosclerosis, is
the leading cause of death and disability in today’s society. The statistics of this progressive disease are
astounding; of the 13.9 million Americans who currently have CHD approximately 450,000 die
annually, consequently supporting the need for research leading to the prevention of atherogenesis. It
has been proposed that the oxidation of low-density lipoproteins (DDL) promotes this process of
atherosclerotic development. Soy foods and their components have become of particular interest in
impeding the atherogenic process through their ability to reduce oxidative stress. In 1979, Zilversfnit
hypothesized that atherosclerosis is a postprandial occurrence. Therefore, studies conducted on soy and
its ability to reduce oxidative stress should not only be performed in chronic settings but also
performed in the postprandial state. The purpose of this study was to determine if a difference exists
between 39 g soy (85 mg isoflavones) and 39.9 g milk protein (0 mg isoflavones) in combination with
a high-fat meal in relation to their protection against postprandial oxidation. Fifteen healthy
nonvegetarian men 20-47 years of age reported to the Nutrition Research Lab on the campus of
Montana State University on two nonconsecutive days to participate in a double blind crossover study.
Upon reporting to the lab the subjects had height and weight measurements completed, blood drawn
via venipunture, then were presented with their challenge meal in which they had 20 minutes to
consume. The challenge meal consisted of 2 apple muffins and a soy or milk shake (956 calories, 41%
fat, 41% carbohydrate, and 18% protein). Plasma samples were obtained at baseline (fasted) and hours
2, 4, and 6 postprandially. Isolated LDL were subjected to ex vivo copper-induced LDL oxidation. Lag
time, propagation rate, and initial absorbance were calculated for each time point analyzed. Results
showed no significant difference (p>0.05) between the main effects (protein type, time points) or their
interaction on LDL oxidation (lag time, propagation rate, or initial absorbance). Future studies are
needed to clearly define the role of soy in its ability to reduce postprandial LDL oxidation.. EFFECTS OF SOY ISOFLAVONE CONSUMPTION FOLLOWING A HGH FAT
MEAL ON THE OXIDATIVE RESISTANCE OF HEALTHY YOUNG MEN
by
Danielle Ann Dufner
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Health and Human Development
MONTANA STATE UNIVERSITY
Bozeman, Montana State University
May 2003
hr?5>
08745
ii
APPROVAL
of a thesis submitted by
Danielle Ann Dufner
This thesis has been read by each member of the thesis committee and has been found
to be satisfactory regarding content, English usage, format, citations, bibliographic style,
and consistency, and is ready for submission to the College of Graduate Studies.
Christina Gayer Campbell, Ph D., R D
Date
Approved for the Department of Health and Human Development
Ellen Kreighbaum, Ph.D.
? A
(Signature)
IJ
Date
Approved for the College of Graduate Studies
Bruce R. McLeod,
(Signature)
Date
/ o ?
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degree at Montana. State University, I agree that the Library shall make it available to
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TABLE OF CONTENTS
1. INTRODUCTION..........................................................................................................
2. REVIEW OF LITERATURE........................................................................................4
The Postprandial State.................................................................................................. 6
Soy Isoflavones............................................................................................................. 7
Soy and Oxidation........................................................................................................11
Lipid Turnover.............................................................................................................16
Summary.................................................................................................................... 17
3. METHODS...................................................................................................................18
Recruitment................................................................... ;............................................ 18
Screening......................................................................................................................19
Study Protocol.....................................................................................................
19
Diet Records.............................................................
21
Physical Activity Records........................................................................................... 21
Challenge Meal........................................................................................................... 21
Blood Samples............................................................................................................ 23
LDL-Oxidation........................................................................................................... 24
Lipid Analysis............................................................................................................. 27
Data Interpretation...................................................................................................... 28
Analysis of Data.......................................................................................................... 29
4. RESULTS.................................................................................................................... 30
5. DISCUSSION.............................................................................................................. 53
Implementation of a High SFA Meal vs. a High PUFA Meal.................................... 54
Time Frame of the Study.... ....................
58
Individual Variability................
59
Subject Antioxidant Status and Calorie Intake .......................................................... 60
Pre-Screening All Subjects to Determine Which Were Equol Producers.................. 61
Inappropriate Method of Assessment.........................................
63
Those in a High Risk Population................................................................................ 65
6. SUMMARY................................................................................................................. 69
REFERENCES CITED
72
V
TABLE OF CONTENTS - CONTINUED
APPENDICES................................................................ ..........................■...................... 79
APPENDIX A: Medical History Questionnaire.....................................................80
APPENDIX B: Human Subjects Consent Form.....................................................85
APPENDIX C: Diet Record Directions and Form ................................................ 90
APPENDIX D: Physical Activity Log......... .......................................................... 93
APPENDIX E: Challenge Meal Diet Analyses..................................................... 97
APPENDIX F: The Solae Company.................................................................... 100
APPENDIX G: LDL Oxidation Procedure......... .............. ...............].................. 105
APPENDIX H: Statistical D ata........................................................................... 112
LIST OF TABLES
Table
Page
1. Challenge Meal Nutrient Composition......... i..................................................22
2. Subject Characteristics...................................................................................... 30
3. Descriptive Statistics.....................................
31
4. Comparison of Soy vs. Milk Protein Study Days..............................
32
5. ANOVA Results for LDL Oxidation (Lag Time)............................................ .32
6. ANOVA Results for LDL Oxidation (Propagation Rate)................................. 33
7. Initial Absorbance ANOVA............................................................................. 33
8. Average Lag Time, Propagation Rate, and Initial Absorbance
(All Subjects).................................................................................................... 34
' LIST OF FIGURES
Figure
Page
1. Lipid Peroxidation...............................................................
...5
2. Comparison of Isoflavone Structural Similarity to Estrogen
...8
3. Timeline................................................................................
.20
4. Challenge Meal Nutrient Composition.................................
.23
5. Lag Time (Protection) Against Oxidation..... ......................
.28
6. Soy Protein Box Plot.............................................................
.35
7. Milk Protein Box Plot...........................................................
.35
8. Average Lag Times (Normalized) for All Subjects..............
.37
9. Average Lag Time (Normalized) for Subject I ....................
.38
10. Average Lag Time (Normalized) for Subject 2 ..................
.39
11. Average Lag Time (Normalized) for Subject 3 ..................
.40
12. Average Lag Time (Normalized) for Subject 4 ..................
.41
13. Average Lag Time (Normalized) for Subject 5 ..................
.42
14. Average Lag Time (Normalized) for Subject 6 ..................
.43
15. Average Lag Time (Normalized) for Subject 7 ..................
.44
16. Average Lag Time (Normalized) for Subject 8 ..................
,45
17. Average Lag Time (Normalized) for Subject 9 ..................
,46
18. Average Lag Time (Normalized) for Subject 10..............
.47
19. Average Lag Time (Normalized) for Subject 11................
,48
V lll
LIST OF FIGURES - CONTINUED
20. Average Lag Time (Normalized) for Subject 12........................................... .49
21. Average Lag Time (Normalized) for Subject 13.....;......................................50
22. Average Lag Time (Normalized) for Subject 14............................................ 51
23. Average Lag Time (Normalized) for Subject 15................
52
24. Structure of Daidzein and Equol.................................................................... 62
ABSTRACT
Coronary heart disease (CHD), also referred to as coronary artery disease (CAD) or
atherosclerosis, is the leading cause of death and disability in today’s society. The
statistics of this progressive disease are astounding; of the 13.9 million Americans who
currently have CHD approximately 450,000 die annually, consequently supporting the
need for research leading to the prevention of atherogenesis. It has been proposed that
the oxidation of low-density lipoproteins (DDL) promotes this process of atherosclerotic
development. Soy foods and their components have become of particular interest in
impeding the atherogenic process through their ability to reduce oxidative stress. In
1979, Zilversfnit hypothesized that atherosclerosis is a postprandial occurrence.
Therefore, studies conducted on soy and its ability to reduce oxidative stress should not
only be performed in chronic settings but also performed in the postprandial state. The
purpose of this study was to determine if a difference exists between 39 g soy (85 mg
isoflavones) and 39.9 g milk protein (0 mg isoflavones) in combination with a high-fat '
meal in relation to their protection against postprandial oxidation. Fifteen healthy
nonvegetarian men 20-47 years of age reported to the Nutrition Research Lab on the
campus of Montana State University on two nonconsecutive days to participate in a
double blind crossover study. Upon reporting to the lab the subjects had height and
weight measurements completed, blood drawn via venipunture, then were presented with
their challenge meal in which they had 20 minutes to consume. The challenge meal
consisted of 2 apple muffins and a soy or milk shake (956 calories, 41% fat, 41%
carbohydrate, and 18% protein). Plasma samples were obtained at baseline (fasted) and
hours 2, 4, and 6 postprandially. Isolated LDL were subjected to ex vivo copper-induced
LDL oxidation. Lag time, propagation rate, and initial absorbance were calculated for
each time point analyzed. Results showed no significant difference (p>0.05) between the
main effects (protein type, time points) or their interaction on LDL oxidation (lag time,
propagation rate, or initial absorbance). Future studies are needed to clearly define the
role of soy in its ability to reduce postprandial LDL oxidation..
I
CHAPTER I
INTRODUCTION
Coronary heart disease (CHD)3also referred to as coronary artery disease (CAD)
or atherosclerosis, is the leading cause of death and disability in today’s society. The
statistics of this progressive disease are astounding; of the 13.9 million Americans who
currently have CHD approximately 450,000 die annually, supporting the need for
research leading to the prevention of atherogenesis (I). The process of athlerosclerotic
development can be explained in part by, but not limited to, heredity, aging, obesity,
hypertension, hypercholesterolemia, or diabetes (I). Lifestyle factors also play a major
role in the incidence of CHD: smoking, minimal exercise, and the consumption of a high
fat diet have also been identified as key contributors to this disease (1-5).
Recently, research in the area of CHD has focused on performing feeding studies
in the postprandial state, since the majority of humans exist primarily in this fed
condition throughout an average day (I). Americans are an extremely convenienceoriented population, typically choosing foods very high in fat (predominantly saturated
fat). The correlation between a high fat intake and atherosclerotic development has thus
been well documented (2-5, 6 ,1). A diet rich in polyunsaturated fatty acids (PUFA) has
been shown to reduce postprandial triglyceride (TG) response to fatty meals, while diets
rich in saturated fatty acids (SFA) increase the response (2, 3, 4). This is increasingly
important due to the fact that a high postprandial plasma TG concentration is considered
a risk marker of the athlerosclerotic. event (4).
2
Postprandial low-density lipoprotein (LDL) particles are more easily oxidized
than fasting LDL (3). This may be partially due to the fact that as LDL stays in the blood
for a longer period of time (as occurs with consumption of a high fat meal) the LDL is at
an increased risk of becoming oxidized. This oxidative event potentially leads to the
attachment of the now oxidized LDL to the arterial wall, and ultimately to foam cell
formation and blockage of the artery. This deposition of oxidatively-modified LDL in the
arterial intima has been identified as an important initial event in atherogenesis (3).
Soy foods and their components have become of particular interest in impeding
the atherogenic process; this has lead to the 1999 FDA health claim for the incorporation
of soy protein in relation to CHD reduction through its hypocholesterolemic effects (8).
Soy foods and their components have been recognized as containing potential
antioxidants known as isoflavones. A number of studies have demonstrated the
antioxidant properties of soy protein isoflavones both in vivo and in vitro (9, 10, 11).
This antioxidant property of soy in reducing oxidative stress provides a possible
mechanism by which consumption of soy foods may decrease the risk of CHD (9). It is
possible that the antioxidant property of soy isoflavones could play a role in prohibiting
the progression of CHD.
Epidemiological studies have consistently demonstrated a strong positive
correlation between the intake of SFA with the prevalence of CHD in various populations
(I). The combination of the LDL-Iowering effects and antioxidant effects of soy protein
and their isoflavones has been shown to contribute to lower rates of CHD among Asian
3
individuals who consume generous amounts of soy foods, compared to the rates among
Westernized societies who consume minor amounts (12, 13).
The purpose of this study was to determine if a difference exists between 39 g soy
protein (85 mg isoflavones) and 39.9 g milk protein (0 mg isoflavones). The soy or milk
protein was consumed in combination with a high saturated fat diet to determine their
relative protection against postprandial LDL oxidation. A high-fat meal (2 apple crumb
muffins, soy or milk shake, and a banana) was implemented containing 956 calories, 44 g
fat (41%), 26 g saturated fat (21% of total fat), 100 g carbohydrate (41%), 45 g protein •
(18%), 39 mg cholesterol, and 895 mg sodium.
Following a high saturated fat meal, soy protein (85 mg aglycone isoflavones)
consumption compared to milk protein (0 mg aglycone isoflavones) will significantly
enhance the oxidative, resistance of postprandial LDL as measured by copper-induced
LDL oxidation.
4
CHAPTER 2
REVIEW OF LITERATURE
According to the most widely accepted theory of atherogenesis, oxidatively
modified LDL activates a series of cellular events in the arterial wall ultimately leading to
plaque formation (13, 14). Generally, it is believed that the formation of a lesion in the
arterial wall is initiated by a response to some endothelial injury brought on by
hyperlipidemia, or by toxic or infectious agents (15). This oxidative event is thought to
occur once the LDL particle has become isolated from circulating water-soluble and fatsoluble (as in the otocopherol component of LDL) antioxidants (16). From this process
the formation of fatty streaks within the arterial wall is initiated due to the oxidation of
LDL occurring predominantly within the subendothelial space of the intima (16). The
LDL fraction is the principal carrier of cholesterol in the plasma. It is hypothesized that
LDL may play a role at several steps in atherogenesis; however, the precise mechanisms
by which LDL promotes the development of lipid laden foam cell in the fatty streak
lesion remains to be fully established (17).
Oxidative stress is a term used to describe any challenge in which pro-oxidants
(e.g. copper, hydrogen peroxide) predominate over antioxidants (e.g. soy isoflavones,
vitamin E). Pro-oxidants are compounds that interfere with normal metabolism by
oxidizing (removing electrons) normal cellular macromolecules (18). Oxidative stress is a
key factor in two processes leading to atherosclerosis: (I) LDL modification and (2)
endothelial dysfunction both caused by hypercholesterolemia and hypertriglyceridemia.
5
Because oxidized LDL appears to be such an important component of the atherogenic
process, it is valuable to examine the lipoprotein itself, and how it undergoes this
oxidative process (17).
One of the initial events in LDL oxidation is the free radical peroxidation of
PUFA and to lesser extents monounsaturated fatty acids (MUFA) in LDL. Initially, there
is conjugated diene formation due to hydrogen abstraction and molecular rearrangement.
After oxygen uptake, peroxyl radicals form, which in turn extracts hydrogen atoms from
fatty acids, initiating a reaction that leads to the formation of hydroperoxides (Figure I).
This event prevents the recognition of LDL by the LDL receptor; thus oxidized LDL is
processed by the scavenger receptor pathway, leading to cholesterol accumulation and
foam cell formation (17). Modified LDL (predominately oxidized) is taken up via the
scavenger receptors on macrophages creating the metabolically active foam cells found in
the early stages of atherosclerosis (5, 15, 17). This results in substantial accumulation of
cholesterol in the macrophages and smooth muscle cells in the arterial wall (4, 17, 19).
PLiFA wiih
three double bonds
HtO
Upid hy droperoxide
formation and propagation
<r.f Dipid peroxidation
H
I
Oz
A. Hydrogen
abstraction
D. Hydrogen abstraction
from adjacent PUFA
V z'=rX===yX __X \
9
O
B. Molecular
rearrangement to
conjugated diene
■-viih uv absorbance
at
nm
Figure I. Lipid Peroxidation
A.
C. Oxygen uptake
and peroxyl radical
formation
6
The Postprandial State
Over twenty years ago, Zilversmit stated that atherosclerosis is a postprandial
phenomenon with lipoprotein particles in the fed state contributing to atherosclerosis (I);
this concept has since been confirmed by others (20, 21). Because of our western
society’s consumption of regular meals throughout a day, we exist primarily in a
postprandial state. Zilversmit also supported the view that arterial lipid accumulated as
the result not only of abnormally high concentrations of LDL in the blood plasma, but
also as a consequence of the normal process of lipid absorption and transport. This
process has the potential to be pathogenic in persons who consume a diet rich in fat and
cholesterol (I).
It has been shown in studies with both experimental animals and humans that
LDL becomes elevated when atherogenic (e.g. high fat) diets are incorporated (I). A
proposed mechanism by which a high-fat (especially saturated fat) diet leads to
atherosclerosis is by the meal’s influence in elevating serum triglycerides (TG). A high
postprandial plasma TG concentration is considered a risk marker of coronary heart
disease (CHD). It is known that a diet rich in PUFA (predominantly n-3 PUPA) can
reduce the postprandial TG response to fatty meals while diets rich in SFA has the
potential to increase the response (4).
In a recent study completed by Ursini et al. (5), a test meal was adopted mirroring
a typical English/American breakfast including bacon, eggs, bread with butter and coffee.
The nutrient composition was 11% protein, 34% carbohydrate, and 55% fat, comprising
approximately 1200 kcal. Nine male volunteers participated in this high fat feeding study
7
in which the primary endpoint was the accumulation of plasma peroxides, reflective of
lipid peroxidation (oxidation). The results demonstrated an increase in the plasma
peroxide level from baseline (207 ± 96.7 pmol/ml) vs. those obtained two hours after
breakfast (412 ± 195.8 pmol/ml) in all subjects (153% average change). The results of
this study highlights that a high saturated fat diet is a direct source of lipid
hydroperoxides in lipoproteins, and thus this diet could become atherogenic (5).
Despite past evidence for the significance of postprandial studies, most research
has been completed on subjects in the fasted state, because it is thought to be more
reproducible for research studies. The level of TG in the LDL fraction is increased in the
postprandial state, which increases the susceptibility of LDL to oxidative modification
(2). Since the absorption and transport of dietary fat is mediated by plasma lipoproteins
and that intestinally derived lipoproteins have been implicated in the development of
atherogenesis, investigations into lipoprotein metabolism need to be conducted in the fed
state (3).
• ■Sov Isoflavones
Isoflavones are naturally occurring plant chemicals belonging to the
phytoestrogen class. Recent epidemiological evidence and experimental data from both
human and animal studies are highly suggestive of beneficial effects of isoflavones on
human health. Specific claims have been made for the physiological properties of
isoflavones to aide in reducing the incidence of atherosclerosis that ultimately leads to
CHD (26, 27).
8
Isoflavones are found almost exclusively in legumes; the soybean in particular
provides an abundant source. The major isoflavones, daidzein and genistein are found in
four chemical forms: aglycone (genistein and daidzein), glycoside (daidzin and genistin),
acetylglucoside and malonylglucoside (27). Isoflavones occur predominantly as
glycosides in plants and consequently are highly polar (water-soluble) compounds.
The
chemical form in which isoflavones occur is an important consideration because it may
influence the biological activity, such that the aglycone form has increased activity and
absorption over other forms (26).
The two primary soy phytoestrogens, genistein and daidzein, bind to estrogen
receptors (Ers) with low affinity (28). Genistein and daidzein both bind to Ers,
explaining their structural similarity with estrogens (Figure 2). Setchell et al. (29)
suggested that soy protein foods could lower cholesterol since they exhibit weak
estrogenic activity due to the phytoestrogens genistein and daidzein binding to Ers.
OH
Figure 2. Comparison of Isoflavone Structural Similarity to Estrogen
Isoflavone structure*:
*Genistein = OH at positions 4’, 5, 7
Estrogen structure
*Daidzein = OH at positions 4’, 7
9
Anthony et al. (25) fed a diet containing casein, intact soy protein isolates (SP+)
and soy protein isolates from which the phytoestrogens were removed (SP-) to 160
cynomolgus monkeys. All monkeys were fed the diets for 14 months, during which time
cardiovascular disease risk factors, including plasma lipids were measured. The diets
were identical in the percentages of energy from protein (18.5%), fat (40.6%), and
carbohydrate (40.9%), and had the same amount of cholesterol (0.31 mg/kcal). There
were no isoflavones in the casein diet, low amounts (equivalent to 16 mg per individual
per day) in the SP- diet, and approximately a 10-fold higher amount of isoflavones
(equivalent to 143 mg per individual per day) in the SP+ diet. Serum lipoproteins
(VLDL and LDL) in the SP+ animals were significantly reduced (426 ± 23.94 mg/dL
casein, 394 ± 22.00 mg/dL SP-, and 276 ± 22.39 mg/dL SP+; p<0.05) and high density
lipoprotein (HDL) cholesterol levels were significantly increased as compared to SP- and
control animals (40.2 ± 3.47 mg/dL casein, 47.9 ± 3.09 mg/dL SP-, and 59.8 ± 3.08
mg/dL SP+; p<0.05). The mean atherosclerotic plaque size was reduced in SP+ animals,
suggesting the possibility of antiatherogenic effects caused by alteration in the serum
lipoprotein profile (25). This study shows that the incorporation of soy isoflavones in
conjunction with soy protein as compared to soy protein alone has the ability to reduce
serum lipoprotein (very low-density lipoproteins or VLDL and LDL) levels while
simultaneously increasing HDL cholesterol levels.
Analyses of the isoflavone content of numerous soy foods generally indicate that
most contain 0.1 - 3.0 mg/g of total isoflavone (26). Although a high proportion of foods
contain soy products, these are mostly soy oils and soy lecithin; these soy products are
10
devoid of isoflavones, so the average daily dietary intake of isoflavones in Western
populations is typically negligible (<1 mg/d). Isoflavones migrate with the protein
fraction of the soybean during its processing, and because soy protein is rarely a normal
component of the average Western diet, this accounts for the low daily intake reported in
the American population (26). The typical Asian diet contains approximately 50-100 mg
of isoflavones per day (30, 31). This is typical of the Asian diet excluding metropolitan
areas that have adopted westernized eating patterns, in which isoflavone consumption
drops dramatically to 25 mg per day. In westernized societies, such as our own, the
typical consumption is noted to be less than 5 mg per day. Tofu and tempeh have 38.3
and 60.5 mg isoflavones per serving respectively vs. second generation products such as
soy hot dogs or soy-based ice creams which have substantially lower amounts of
isoflavones (approximately 6 mg per serving) because they frequently contain
considerable amounts of non-soy ingredients (30, 31).
The serum cholesterol and LDL cholesterol lowering effects of soy proteincontaining foods have been investigated in a large number of studies since the 1940s. In
1995, Anderson et al. (32) conducted a meta-analysis; including 38 clinical studies
reported in 29 articles. The mean daily intake of soy protein in the meta-analysis was
47g (17 - 124 g/d), however 14 of the studies (37%) reported an intake of <31 g per day.
The meta-analysis showed an overall net reduction in serum LDL cholesterol of 12.9%.
The decrease was said to be related to the initial serum cholesterol level (pO.001), such
that insignificant changes were found in individuals with low initial serum cholesterol
and the greatest reductions occurred in those with the highest levels of initial cholesterol
11
(32). Additionally, soy protein consumption was associated with significant reductions in
total cholesterol (23.2 mg/dL, 9.3%); and triglycerides (13.3 mg/dL, 10.5%), whereas a
nonsignificant increase of 2.4% (p>0.05) in serum concentrations of HDL-cholesterol
was apparent. The results of the meta-analysis supported the theory that the consumption
of soy protein (average of 47 g/d) when compared to animal protein significantly
decreases serum concentrations of total cholesterol, LDL cholesterol, and TG (22, 32).
Sov and Oxidation
Clinical and epidemiological studies indicate that lipoprotein oxidation may
contribute to the development of inflammatory lesions that are typical of the first stages
of atherosclerosis (24). Along with these studies it has also been shown that antioxidant
supplementation with antioxidants such as vitamin E and soy may reduce the risk for
atherosclerotic events (12).
Tikkanen et al. (16) hypothesized that isofiavone antioxidants derived from soy
could be incorporated into lipoproteins and possibly protect them against oxidation. One
way isoflavones can do this is by the binding of copper to apo B of LDL in vivo, thereby
initiating lipid peroxidation. This type of mechanism has also been suggested for
dehydroascorbic acid (also a water-soluble antioxidant) which, when coincubated in vitro
with LDL, renders it resistant to oxidation (16). The possibility that isofiavone intake has
an antiatherogenic effect has received support from the existence of plausible underlying
mechanisms of protection, such as plasma lipid risk laqtor modification, and antioxidant
protection of LDLs (11, 12, 22-25).
12
The oxidation of LDL results in a lipoprotein particle that is generally thought to
be more atherogenic than the native LDL (33). Isoflavones, primarily genistein, have
been reported to inhibit oxidative modification of LDL by macrophages, enhancing the
resistance of LDL to oxidation and exhibiting antioxidant activities in the human by
being incorporated in small quantities (~1%) in the LDL molecule itself (16, 23).
Genistein is a naturally occurring polyphenolie compound known to have antioxidant
properties. Genistein exhibits antioxidant effects in vitro including the inhibition of ADP
and NADPH-dependent lipid peroxidation in rat liver microsomes and inhibition of the
coupled oxidation of (3-carotene and linoleic acid (34). This antioxidative activity has
been related to the ability of isoflavones to scavenge free radicals. Recent work by
Kapiotis (35) has documented the ability of genistein to inhibit LDL oxidation in vitro
when challenged with copper ions or superoxide radicals as measured by thiobarbituric
acid reactive substance (TEARS) formation, altered electrophoretic mobility and lipid
hydroperoxides. The general endogenous antioxidants presumably present in LDL
particles have not been fully characterized. It is hypothesized that dietary antioxidants
such as soy derived isoflavones could play a role in protecting LDL against oxidation
(23).
Anderson et al. (12) compared the effects of various diets containing antioxidant
rich foods on serum lipids and lipoprotein oxidation in 60 male rats for 3 weeks. The
purpose of this study was to determine if a diet containing isoflavone-rich soy protein
could influence lipoprotein oxidation in rats. Comparison die(s included a diet void of
vitamin E (the control group), a diet high vitamin E (1000 units/kg), a diet low in soy
13
isoflavones (0.08 mg genistein/g of protein), a diet high in the soy isoflavone genistein
(1.45 mg genistein/g protein), a diet containing 2% green tea, and a diet containing 50
mg/kg /3 carotene. Oxidative damage to lipoproteins was determined by measuring the
lag phase and formation of conjugated dienes, lipid peroxides, and TEARS. A significant
reduction in triglyceride values occurred (pO.OOl) for the high-genistein group vs. the
control group. A significant prolongation in the lag phase for the high and low genistein
groups were observed with an increase of 83 min (49%, p=0.002) and 80 min (p=0.002),
respectively. The high genistein group also had significantly lower (p=0.01) conjugated
diene values (0.49 OD units) than the control group (0.68 OD units). High genistein soy
protein intake also significantly decreased (p=0.006) lipid peroxides in lipoprotein
fractions from control values from 982 nmoles/mg protein to 677 nmoles/mg protein,
respectively. This study shows that the incorporation of soy isoflavones (particularly the
isoflavone genistein) has a strong antioxidant characteristic to decrease the susceptibility
of VLDL-LDL to oxidation (12).
Hwang et al. (37) conducted a study to determine if soy isoflavones could inhibit
in vitro LDL oxidation. The LDL was incubated with genistein, daidzein, or equol at
concentrations ranging from 0.5 to 10 pM. The LDL samples were isolated from one
adult male volunteer; copper was then added to the cuvettes and formation of conjugated
dienes was monitored at 234 nm for up to 16 hours. The results demonstrated a
significant prolongation of lag time (p<0.05) at concentrations greater than 0.5 pM for
equol, I pM for genistein, and 2.5 pM for daidzein. The researchers also noted that it
was equol, a daidzein metabolite formed in vivo by gut microflora that proved to be the
14
most potent antioxidant tested. There has been speculation that interindividual variability
in gut microflora may influence the conversion of daidzein to equol, leading to varying
results among subjects. The use of samples from one individual is a limitation to this
study (36, 37).
Six volunteers (three men, three women) participated in a study by Tikkanen et al.
(16) to determine whether isoflavone antioxidants derived from soy could be
incorporated into lipoproteins possibly protecting them against oxidation (16). Subjects
received three soy bars (7.1 g soy protein, 12 mg genistein and 7 mg daidzein per bar)
daily for two weeks. Fasting blood samples were collected following a 2 week run in,
during the 2 weeks of the soy-feeding period as well as after a twelve day washout
period. Following two weeks of soy feeding, a significant prolongation for lag time was
observed for the soy feeding (147 ± 9 min) as compared to baseline to the soy feeding
(173 ± 19 min) (p<0.02). Although large increases in plasma isoflavone levels occurred
following two weeks of consumption, the incorporation of isoflavones into the LDL was
determined to be less than 1% of total plasma isoflavones (16). These results indicate
that following chronic consumption of soy, the LDL was resistant to oxidation for a
longer period, however the mechanism for which soy isoflavones impacted the resistance
of LDL to oxidation was not apparent.
Jenkins et al. (9) determined whether consumption of a moderate amount of soy
protein (36 g/d) and associated isoflavones (168 mg/d) in breakfast cereal can favorably
alter markers of increased cardiovascular disease risk in 25 hyperlipidemic subjects.
Total conjugated dienes in the LDL fraction, a marker of oxidized LDL cholesterol was
15
significantly reduced on the soy diet compared with the control (9.2% ± 4.3%, p=0.42),
and the ratio of conjugated dienes to cholesterol in the LDL fraction was also reduced
(8.7% ± 4.2%, p=.050) at the end of the three-week treatment. This study indicates a
beneficial effect of soy in reducing indices of LDL oxidation; supporting previous reports
that soy isoflavone consumption may protect LDL cholesterol from oxidative damage (9).
In a similar study, Jenkins et al. (38) recruited 31 hyperlipidemic subjects (19
men; 12 postmenopausal women) in a randomized crossover study investigating the
effects of soy protein foods on LDL oxidation. The subjects were asked to consume a
test meal of 33g/d of soy protein (86 mg isoflavones/2000 kcal/d) for a month, followed
by a control diet with no soy protein or isoflavones for a second month. The test diet
decreased both oxidized LDL measured as conjugated dienes in the LDL fraction (56 ± 3
test vs. 63 ± 3 pmol/L control, p<0.001) and the ratio of conjugated dienes to LDL
cholesterol (15.0 ±1.0 test vs. 15.7 ± 0.9 control, p=.032), even in subjects who had
already been using vitamin E supplements (400-800 mg/d). The consumption of high
isoflavone foods appears to be associated with reduced levels of circulating oxidized
LDL (38).
Wiseman et al. (39) also studied the use of isoflavones to increase the resistance
of LDL to oxidation in humans. In this randomized crossover study, the effects of a
textured soy protein high in isoflavones (HI, 21.2 mg daidzein, 34.8 mg genistein) were
compared with a similar product yet low in soy isoflavones (LI, 0.9 mg daidzein, LO mg
genistein), both of which were consumed for 17 days. Plasma concentrations of Fzisoprostanes, a biomarker of in vivo lipid peroxidation, as well as the resistance of LDL
16
to copper induced oxidation were evaluated. A 9% longer lag time for the copperinduced LDL oxidation (p=0.017), as well as a 19.5% lower concentration of 8-epiprostaglandin F2a (p=0.028) was apparent with the high isoflavone diet. In agreement
with previous long-term feeding studies, these results indicate that soy isoflavones act to
protect lipoproteins against oxidative damage (39).
Linid Turnover
The atherogenic effect of various fatty acids has been reported in numerous
studies (17). Several lines of evidence indicate that diets rich in SFA raise serum lipid
concentrations whereas MUFA and PUFA have cholesterol lowering antiatherogenic
actions (lowering total and LDL-cholesterol, and finally lowering the risk of
cardiovascular disease) (17, 40-44). Avoiding excess intake of SFA has thus been
recommended because hypercholesterolemia appears to be critical to the atherogenic
process and subsequently leads to serious cardiovascular disease.
A project by Gallon et al. (6) was done to test the hypothesis that dietary
cholesterol ingested during a given meal is resecreted into chylomicrons and plasma after
several subsequent meals. Seven healthy subjects ingested 3 comparable mixed test
meals (at three separate test times) each containing a given amount of fat (49 g) and
cholesterol (157 mg); blood samples were then taken 3 and 6 hours after each test meal.
Plasma triacylglycerols in this study and chylomicron friacylglycerols increased
markedly 3 h after the test meals (up to 50.9 ± 23.1 mg/dL after hour I, 42.5 ± 15.4
mg/dL after hour 2, and 43.2 ± 16.6 mg/dL after hour 3; p<0.05) and then returned to
17
baseline values after 6 h. This study indicates that if several subsequent meals result in
an increase in plasma triacylglycerols that possibly that the inclusion of a single high fat
challenge meal may have the same effect (6).
Summary
Each of the events discussed in the studies presented is subject to a substantial
amount of individual variability. Nevertheless, a high fat meal (predominantly saturated
fat) leads to a general increase in plasma VLDL and LDL. The level of postprandial lipid
hydroperoxides likely depends on the incorporation of the specific fatty acid incorporated
into the diet, as various fatty acid compositions give rise to considerable differences in
peroxide content. It has been shown that SFA causes the greatest increase in postprandial
peroxides.
Despite extensive investigation, atherosclerosis is a complex multifactorial
disease that is not well understood. Progress in this area has been limited by the lack of
animal models that fully reproduce all of the important features of CHD in the human
(24). Nonetheless, the incorporation of soy protein isoflavones into the diet of an
individual is hypothesized to be one dietary intervention that could possibly lead to an
increased protective effect against the progression of atherogenesis due to a possible
reduction in oxidative stress.
18
CHAPTER 3
METHODS
Fifteen healthy, nonvegetarian men 18-50 years of age were recruited from the
surrounding Bozeman area. It had been indicated in a similar study (36), from a power
calculation based on each subject’s best response to soy protein, that 1 5 -2 5 subjects
would be needed to detect a significant difference in postprandial LDL oxidation.
Males were chosen as the population to be used in the present study as a result of a study
by Cohn et al. (3) who showed that males tend to have greater postprandial
triglyceridemia than females. There is increasing evidence that TG-rich lipoproteins are
of importance in the pathogenesis of atherosclerosis. Due to the fact that postprandial
hypertriglyceridemia has been directly linked to atherosclerotic development, it is
especially important that the male population be studied (45, 46).
Recruitment
Recruitment of subjects was accomplished through local advertising. Flyers were
distributed throughout the campus of Montana State University and at various
establishments throughout the Bozeman area detailing the components of the study and
requirements for prospective participants. Newspaper advertisements were also used in
the recruitment of subjects outlining similar information as the fryer. Interested
participants contacted the Nutrition Research Lab (NRL) to receive further information
about the study.
19
Screening
Exclusion from the study occurred if the potential participants met any of the
following criteria: I) regular use (>3 times per week) of a vitamin or mineral supplement
in the previous 3 months; 2) regular consumption of soy protein ( >2 servings of 6.25
g/day); 3) known existence of metabolic disorders (thyroid disease, diabetes, liver or
renal disease); 4) use of oral prescription medications (including any antibiotic use within
the previous 3 months) (47); 5) obesity (BMI >30 kg/m2); 6) cigarette smoking within the
last 3 years (48); 7) known food allergies (soy protein, milk protein, peanuts); 8) a strict
vegetarian status (49); 9) regular alcohol consumption (>2 drinks/d; I drink = 12 oz beer,
6 oz wine, or 1.5 oz distilled alcohol); 10) or regular use of vitamin or herbal supplement
(>3 times/week) with antioxidant capacity.
Study Protocol
Participants not fulfilling any of the exclusion criteria were asked to complete a
medical history questionnaire and to sign a consent form (approved by the Montana State
University Human Subjects Committee on September 5, 2002). After these steps were
accomplished, participants underwent a fasted blood lipid screening that was assessed for
total cholesterol (TC), HDL, LDL, and TC. Upon analysis of the blood lipid screening
further inclusion criteria was implemented, by means of a TC <200 mg/dL, HDL >40
mg/dL, LDL <100 mg/dL, and TG <200 mg/d (51).
Approved subjects reported to the NRL on two separate, nonconsecutive days in a
fasted state (no food or beverage, with the exception of water, for 10 hours) for
20
participation in the double blind crossover feeding study. The participants arrived at
0700 in a fasted state, and had refrained from any strenuous physical activity or alcohol
consumption for at least 24 hours prior to the study date, the participants also were asked
to bring with them their completed 3-day weighed diet records and activity logs for the
three days just prior to the study date. At this time the investigator recorded weight and
height; a fasted venipuncture was taken from the antecubital vein of the subjects by a
trained phlebotomist.
After baseline blood was drawn (at approximately 0720), the participants were
given 20 minutes to consume the challenge meal. Blood was taken via the same method
used at baseline at hours 2, 4, and 6 (see Figure 3). Research has shown that
triacylglycerol concentrations (4, 6) and isoflavones (26, 27, 52, 53) typically peak at
approximately 2-4 h after the test meal and returning to baseline values after 6-8 h,
O
supporting the need to collect blood samples for a minimum of six hours at the 0, 2, 4,
and 6 time periods. During the study, a television and videos were available for the
subjects to watch. The subjects were allowed to leave the NRL at approximately 1330,
after their final (hour 6) blood draw.
Meal
And
___Shake___________________________________________ ____________ ____
0
I
2
3
4
5
6
BD
BD
Figure 3. Timeline (BD = blood draws)
BD
BD
21
Diet Records
Participants were given instructions on how to weigh and record all food and
beverages (excluding water) consumed on the three days prior to the study date.
Participants were provided with a Cuisinart SA-IIOA dietary scale (Cuisinart, East
Windsor, NI) for the process. The weighed diet records were analyzed using the
Nutritionist Pro (First Data Bank, Wadsworth, Belmont, CA) software package.
Physical Activity Records
Participants were also asked to complete a three-day activity log for the three days
just prior to the study date. The Bouchard Three Day Physical Activity Record (50) was
used to approximate mean energy expenditure and to assess compliance to the study
protocol. The procedure for recording the data was thoroughly explained to the subjects
and a list of activities along with their corresponding ratings (1-9) was provided.
Challenge Meal
The test meal consumed at 0720 included 2 high-fat apple crumb muffins made in
the Food Science Laboratory on the campus of Montana State University, and either a
soy or milk protein shake with an added banana for payability. The powdered
beverages were provided by The Solae Company. The soy beverage was made with
SUPRO®SOY (part of the Sdlae™ brand) isolated soy protein that has been water
washed to retain the naturally occurring isoflavones. Two 44 gram servings were
consumed, providing 39.0 grams of soy protein, containing 85 mg of isoflavones
22
(aglycone weight). The placebo beverage was made with milk protein isolate. Two 44
gram servings were consumed, providing 39.9 grams of milk protein and no isoflavones.
A banana (100 g) was added to both shakes. The participants were then given 20 minutes
to consume the meal, in order to keep all time to completion constant. The nutrient
composition of the apple crumb muffins was as follows: 544 calories, 42.0 g total fat
(68.2% of total calories), 26.2 g saturated fat (66.3% of total fat), 6.7 g polyunsaturated
fat, 40.6 g carbohydrates (29.2% of total calories), 3.6 g protein (2.6% of total calories),
and 33.8 mg cholesterol (see Table I). Subjects were also allowed water ad lib
throughout the day. The challenge meal, consisting of 2 muffins, shake, and a banana,
had a total calorie amount of 956 kcal (41% fat, 41% carbohydrate, 18% protein) (see
Figure 4) with a P/S (polyunsaturated/saturated ratio) of 0.25. This specific calorie
amount is representative of approximately 1/3 of the RDA for this group of individuals.
Table I. Challenge Meal Nutrient Composition
Nutrient
Weight
Kilocalories
Carbohydrate
Protein
Total Fat
Saturated Fat
Polyunsaturated Fat
Monounsaturated Fat
Cholesterol
Sodium
Total Dietary Fiber
Value
312
956
100
45
44
26
7
7
39
895
4
Unit
R
kcal
g
g
g
g
g
g
mg
mg
g
23
Carbohydrate
41%
T otal Fat
41%
PU FA
17 %
M UFA
17%
18%
SFA
66 %
Figure 4. Challenge Meal Nutrient Composition Including Breakdown of Fat
Blood Samples
Baseline blood was collected in two vacutainer tubes (six mL each) containing
ethylenediaminetetraacetic acid (EDTA) (Greiner Bio-one, Monroe, NC). The same
procedure was also used in the following 2, 4, and 6 hours from baseline, for a total of 48
mL of blood.
Blood samples were centrifuged in a 21000 Marathon centrifuge (Fisher
Scientific, Pittsburgh, PA) for 15 minutes at 2500 rpm and 16°C. Plasma supernatant
was then aspirated off, and aliquots were placed into labeled plastic vials and stored in a
Revco Ultima II freezer (Legaci Refrigeration Systems, Ashville, NC) at -SO0C until the
time of analysis. Each microcentrifuge tube was labeled with the subject’s identification
number, the sample time point, and the date of collection.
24
LDL-Oxidation
Isolation of LDL occurred through a sequential density-gradient
ultracentrifugation in an Optima TLX Ultracentrifuge (Beckman Instruments, Palo Alto,
CA). First, 0.5 ml of plasma was placed into a I ml centrifuge tube and 0.5 ml of 0.9%
sodium chloride solution was then added, then up to ten centrifuge tubes were placed into
the 120K KPM rotor and secured in the centrifuge. The centrifuge ran for 2.5 hours at
100,000 rpm and 16°C. The rotor was removed upon completion of the run. The
centrifuge tubes were removed from the rotor and sliced at the 0.5 ml mark using a
Beckman CentriTube Sheer (Beckman Instruments, Palo Alto, CA). The top fraction,
containing VLDL, was discarded and the bottom layer containing LDL and HDL, was
transferred to a new centrifuge tube. A volume of 0.5 ml of 16.7% sodium chloride was
added to the HDL and LDL in each new centrifuge tube, the tubes were again placed in
the rotor and spun in the centrifuge under the same conditions used previously. Upon
completion of the run, the centrifuge tubes containing the samples were removed and
sliced in the same manner as before, retaining the top layer, the LDL, for analysis (54).
The isolated LDL was then desalted using prepackaged columns (Econo-Pac 10
DG, Bio-Rad, Richmond, CA) filled with bio-Gel Pb desalting gel. The column was
preconditioned with 20 ml phosphate-buffered saline (PBS). After preconditioning, 350
gl of LDL was added to the column, followed by 2650 pi PBS with the first 3 ml of
effluent will be discarded. A 525 pi volume of PBS was then added and used to elute the
desalted LDL.
25
A bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) for the
detection and quantification of total protein was used to determine the protein
concentration of the LDL elute (55, 56). A bovine serum albumin (BSA) standard of
!mown concentration was diluted to encompass a working range of 25 to 2000 jag/ml.
Twenty-five pi of each standard, of known concentration, and each sample, of unknown
concentration was added to individual wells on a microwell plate (Nalge Nunc
International, Rochester, NY). Working reagent (comprised of sodium carbonate, sodium
bicarbonate, bicinchoninic acid, sodium tartrate, sodium hydroxide and cupric sulfate), in
the amount of 200 pi, was then added to each well. The microwell plate was then placed
on a 6115 Rotator-Incubator (Eberbach, Ann Arbor, MI), shaken for 30 seconds, covered
and incubated at 37°C for 30 minutes. Following incubation the microwell plate was
placed on a pQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments,
Winooski, VT) and absorbance was measured at 562 nm. The BCA is a chelating agent,
which combined with the copper product in the reduction caused by protein in alkaline
medium. The colored product absorbs strongly at 562 nm, the absorbance is proportional
to the number of cations bound and therefore to the amount of protein percent. A
standard curve, corrected for a PBS blank, was then be plotted and the sample protein
concentrations, based on sample absorbance corrected for PBS blank absorbance,
determined from the standard curve using a KCJunior (Bio-Tek Instruments, Winooski,
VT) software package. Using the results of the protein assay, the isolated and desalted
LDL samples were then diluted with PBS to achieve a final concentration of 0.10 mg
protein/ml for each sample (57).
26
The susceptibility of the LDL to oxidation was determined by measuring the
formation of conjugated dienes following copper induced oxidation of the LDL (16, 17).
In vivo LDL oxidation is mimicked in vitro by adding a prooxidant, in this case copper, to
the LDL. The isolated double bonds in the PUFA of the LDL are converted to fatty acid
hydroperoxides with conjugated double bonds (conjugated dienes). The conjugated
dienes absorb light at 234 nm, therefore monitoring the increase in absorbance at this
wavelength reflecting the formation of conjugated dienes and susceptibility of the LDL to
oxidation (17, 33, 58, 59, 60). This was accomplished by pipetting the samples and blank
into separate wells of a microwell plate (Costar, Coming Incorporated, Coming, NY). A
10 pi volume of 500 mM cupric chloride solution was then added to each well. The
plate was then placed on the pQuant Universal Microplate Spectrophotometer and the
absorbance was read every ten minutes for eight hours at 234 nm at 37°C. The lag time,
propagation rate, and initial absorbance for each sample was calculated using Excel
(Microsoft Corporation, Redmond, WA) for each time point analyzed in which the
absorbance readings were plotted against time for each individual well. The LDLoxidation procedure was mn one subject per analysis day (both treatment days analyzed
in same day) using an identical plasma control sample throughout all analysis days.
Analyses were mn so that any percent coefficient of variance (CV) over 10% was remn.
The average CV for interaassay was 4%, while the intrassay was 2% for all 15 subjects
analyzed.
27
Lipid Analysis
Using the Kodak Ektachem DT HDL-C kit (Johnson & Johnson, Rochester, NY),
HDL-C, TC, and TG were analyzed. The HDL kit uses 50,000 MW dextran sulfate and
magnesium chloride to precipitate VLDL and LDL. The remaining HDL-C is then
measured by the slide. The procedure for the HDL-C follows: I) pipette EDTA plasma
to the 0.5 mL mark on the Ektachem HDL tube; 2) cap and vortex for 30 seconds; 3) let
stand for a minimum of five minutes; 4) centrifuge the tube for ten minutes at 1500 x g;
5) if the supernatant is clear use the Ektachem DT Pipette to transfer 10 pi of clear
supernatant directly from the HDL Tube. The TC and TG of the sample were read by
simply pipetting IOpl of plasma on its respective slide. In the analysis, cholesterol esters
undergo a series of reactions to produce a colored compound. The intensity of the color
is proportional to the amount of HDL-C, TC, or TG in the sample. The printer then
produced a printout of the analyzed serum and the calculated value for the subject. The
Friedwald equation (LDL = TC - HDL - (TG/5) was used to determine LDL-C for each
subject (61).
The accuracy of the Ektachem machine used in this preliminary analysis,
however, must be addressed. Following the lipid analysis of all subjects we had observed
some participant’s HDL levels as being uncharacteristically below normal for their age
group (<40 mg/dL). To confirm the accuracy of our results we submitted some plasma
samples to Bozeman Deaconess Hospital for a secondary analysis. Their follow-up
analysis demonstrated HDL levels consistently 10 mg/dL higher than the results that were
28
obtained from the Ektachem machine in the NRE. However, due to lack of funds we
were unable to run all samples in this manner.
Data Interpretation
The oxidizability of the plasma was characterized using typical parameters: lag
phase duration, rate of propagation, and initial absorbance. The lag phase represents the
time during which antioxidants protect the unsaturated fatty acids against oxidation
(Figure 5), a longer duration of lag time thus represents longer protection and is therefore
favorable. The propagation phase is representative of the time during which oxidation
occurs and conjugated dienes are forming (58). Initial absorbance represents the level of
oxidation that the subject began (time 0) with, so that from this initial point we are able to
observe how oxidized the sample becomes over the time of the read from the baseline
absorbance measurement.
Raw Data and StraightLine (2 pts)
Lag time
Time (min)
Figure 5. Lag Time (Protection) Against Oxidation
29
Analysis of Data
A two-way analysis of variance (ANOVA) with repeated measures was used to
assess treatment differences for LDL oxidation parameters (lag time, propagation rate,
and initial absorbance) for the 4 time points analyzed using the SPSS statistical program
(LEAD Technologies Inc., Chicago, IL). A paired t-test was also used to determine
significance between the means of weight, BMI, energy expenditure, total energy
expenditure, energy intake, and total energy intake measurements between the two
treatment types (soy vs. milk) (Synergy Software, Reading, PA). The a level was set at
0.05.
30
CHAPTER 4
RESULTS
The characteristics for each individual subject are displayed below in Table 2,
while the descriptive statistics of each individual are illustrated in Table 3.
Table 2. Subject Characteristics
JE
i
M ilk
2
Milk
Soy
3
M ilk
m
l a
23
71.5
47
73.75
21
75.5
37
70.5
20
64.75
23
67.5
34
72.25
Sov
4
5
Milk
Soy
M ilk
^ S o ^ ^
6
Milk
Soy
7
M ilk
Soy
8
9
Milk
Soy
M ilk
24
74
20
73
28
74
25
70.5
46
67.5
24
68.5
22
71.5
23
68.75
27.8
70.9
^ S o ^ ^
10
11
Milk
Soy
M ilk
^ S o ^ ^
12
Milk
Soy
13
M ilk
Sov
14
Milk
Soy
15
M ilk
Soy
Avg.
67.3
67.3
88.6
88.2
88.2
87.7
78.6
78.9
65.5
65.0
77.5
76.4
79.5
79.1
95.9
97.3
88.2
89.5
87.0
86.8
79.1
77.7
74.5
75.0
836
82.3
103.6
105.0
81.6
81.4
20.4
20.4
25.3
25.1
24.0
23.9
24.5
24.6
24.2
24.0
26.4
26.0
23.6
23.5
27.1
27.5
25.6
26.0
24.6
24.6
24.7
24.2
25.4
25.5
27.6
27.2
31.4
31.8
26.8
26.7
82.5
25.4
* information did not change between study days
65.8
62.9
52.6
47.7
46.1
53.6
37.5
42.6
36.9
37.5
46.5
44.5
70.3
73.1
41.5
413
50.4
46.1
51.4
414
39.2
41.3
415
33.7
43.7
35.4
41.2
40.4
43.4
319
46.6
4429
4232
4660
4202
4066
4706
2952
3358
2417
2440
3607
3398
5589
5782
3985
4216
4442
4126
4478
4206
3102
3213
3021
2529
3659
2915
4269
4240
3543
3166
3463
2340
1802
3802
2023
1781
1781
2427
2354
2048
3654
2613
3048
2269
1854
2500
43.1
39.3
18.2
21.3
29.3
28 9
35.0
46.2
27.6
45.2
40.3
38.1
57.5
52.9
36.1
24.1
20.4
42.5
212
215
22.5
31.2
31.6
213
43.7
31.8
29.4
21.6
22.7
30.7
3832
2662
32.7
2897
2644
1616
1881
2583
2536
2756
3641
1807
2941
3125
2906
4572
41X2
31
Table 3. Descriptive Statistics
Age
27.8
20
47
9.0
Height (in)
70.9
64.75
75.5
3.0
Weight (kg) 1
82.6
65.5
103.6
10.0
Weight (kg) 2
82.5
65
105
10.5
BM I 1
25.4
20.4
31.4
2.4
BM I 2
25.4
20.4
31.8
2.5
Energy Expenditure
(kcal/kg) 1
47.2
36.9
70.3
9.8
Energy Expenditure
(kcal/kg) 2
46.0
33.7
73.1
10.5
Total Energy
Expenditure (kcal) 1
3881.3
2417
5589
814.3
Total Energy
Expenditure (kcal) 2
3781.9
2440
5782
889.7
Intake (kcal) 1
3155.7
1616
5897
1527.0
Intake (kcal) 2
2967.4
1781
5644
1110.9
Total Intake (kcal/kg) 1
38.2
18.2
87.7
219
Total Intake (kcal/kg) 2
36.0
21.3
819
17.3
= milk protein treatment;2 = soy protein treatment
32
There were no significant differences for weight, BMI, energy expenditure, total
energy expenditure, or energy intake between study treatment days as seen through the ttest analysis (see Table 4)
Table 4. Comparison of Soy vs. Milk Protein Study Days
Variable
t-value
Weight
0.31
BMI
0.53
Energy expenditure (keal/kg)
1.02
Total expenditure (Iccal)
1.03
Total energy intake (kcal)
0.84
Energy intake (keal/kg)
0.53
14
14
0.75
0.59
0.32
0.31
0.41
0.60
Results of the 2-way analysis of variance (ANOVA) with repeated measures on
both factors (treatment, time points) for lag time, propagation rate and initial absorbance
are presented in Tables 5, 6, and 7, respectively. No significant differences exist between
the main effects (treatment and time points) or in the interaction between the factors
(treatment and time) on LDL oxidation parameters (lag time, propagation rate, or initial
absorbance) (p>0.05).
The lag time occurs during the time in which antioxidants are able to protect the
LDL against oxidation; meaning that the longer the lag time, the more protection
available for the subject at the specific time point analyzed (as expressed in minutes).
Table 5. ANOVA Results for LDL Oxidation (Lag Time)
Factor
Time Points
52.1
2.2
23.2
Error
1126.9
31.3
35.9
Treatment
0.0
I
0.0
Error
1139.4
14
81.3
Interaction
13.1
2.6
5.0
Error
1029.7
36.8
27.9
0.64
0.54
0.00
0.98
0.17
0.88
33
The propagation phase follows the lag phase and occurs as the antioxidants are
consumed; resulting in a rapid conversion to lipid hydroperoxides (conjugated dienes).
Consequently a less steep propagation rate (expressed as slope) is more favorable due to a
limited amount of time to accumulate the hydroperoxides.
Table 6. ANOVA Results for LDL Oxidation (Propagation Rate)
Factor
Time Points
Error
Treatments
Error
Interaction
Error
2.6E-07
7.5E-06
2.8E-07
6.5E-06
2.9E-07
4.4E-06
3
42
I
14
3
42
8.9E-08
1.7E-07
2.8E-07
4.6E-07
9.7E-08
1.0E-07
0.50
0.68
0.59
0.45
0.92
0.43
Initial absorbance was also analyzed to determine the preliminary measure of
oxidation the subjects were experiencing at each of the 4 time points during the
respective study day. Initial absorbance was calculated by subtracting the initial read of
each time point from the initial read of the blanked well.
Table 7. Initial Absorbance ANOVA
Factor
Time Points
3.8E-04
Error
1.7E-02
Treatments
1.2E-04
Error
5.9E-03
Interaction
1.1E-03
Error
1.2E-02
3
42
I
14
3
42
1.2E-04
4.2E-04
1.2E-04
4.2E-04
3.6E-04
2.9E-04
0.30
0.82
0.28
0.60
1.23
0.30
34
Averages for lag time, propagation rate, and initial absorbance are expressed in
Table 8 below. This information illustrates how similar the averages of all LDL
oxidation parameters were for all 15 subjects analyzed.
Table 8. Average Lag Time, Propagation Rate, and Initial Absorbance (All Subjects)
Lag
Time
Prop.
Rate
Initial
Abs.
Protein
TO
T2
T4
T6
Soy
68.9 ±0.5
66.5 ± 0.5
66.8 ± 0.4
67.4 ±0.5
Milk
68.1 ±0.4
67.5 ± 0.4
66.7 ±0.4
67.1 ±0.4
Soy
0.0041 ±0.0001
0.0042 ± 0.0001
0.0041 ±0.0001
0.0040 ± 0.0001
Milk
0.0041 ± 0.0001
0.0040 ± 0.0001
0.0039 ± 0.0001
0.0040 ±0.0001
Soy
1.116 ±0.009
1.116 ±0.009
1.118 ±0.009
1.114±0.010
Milk
1.117 ±0.011
1.114 ±0.008
1.115 ±0.009
1.126 ±0.013
Values = mean ± SEM
Box plots were used to compare the distributions for soy (Figure 6) and milk
'
protein (Figure 7) at hours 0, 2, 4, and 6. All data was normalized by subtracting the lag
time for the zero hour time point of that study day from the lag time for each of the other
time points (2, 4, and 6). A positive number reflects a lag time greater than the baseline
value and a negative number reflects a shorter lag time. Each box encloses 50% of the
data with the median value of the variable displayed as a line. The top and bottom of the
box mark the limits of ± 25% of the variable population. The lines (whiskers) extending
from the top and bottom of each box mark the minimum and maximum values within the
data set. The outliers are the points that fall outside of the box by more than 1.5 times the
middle 50% of the sample values. From the box plots we can visually deduce that the
soy protein treatment did not have a significant positive effect on lag time. In fact, the
35
box plot shows a decrease in lag time at time 2, rising to near baseline levels by time 6.
The milk protein treatment also did not have a significant positive affect on lag time.
From the box plot we can see a minor drop at hour 4, rising to near baseline at hour 6.
40
30
E
I
20
H
$
I0
•S
N
0
15
-10
O
Z
-20
-30
0
2
4
6
2
4
6
Figure 6. Soy Protein Box Plot
40
30
.S
E
<
L
>
E
H
I
i
O
Z
20
I0
0
- 10
-20
-30
0
Figure 7. Milk Protein Box Plot
36
Although all statistical analysis was found to be nonsignificant (p>0.05), valuable
information may still be provided. Examining individual patterns of each of the subjects
independently, may serve to provide this information. This information is helpful in
aiding us to decipher possible reasons for the insignificance of the results presented, and
to provide direction for future acute research studies.
Figure 8 depicts the average normalized lag times for all subjects, and shows no
appreciable change with either treatment. Furthermore, Figures 9-23 describe each
individual’s response to the soy and milk treatment. From these figures two distinct
visual patterns can be deduced. Individuals exhibiting a pattern A response (7 subjects,
46.7%) had a greater lag time (whether positive or negative) when fed the soy protein vs.
the placebo (see Figures 9, 10-12, 15, 18, and 21). Individuals defined in the pattern B
category (8 subjects, 53.3%) had a greater lag time (whether positive or negative) when
fed the milk protein vs. the soy (see Figures 13-17, 19, 20, 22, and 23).
Soy
M ilk
Change in Lag Time from Baseline (min
35
15 ■
5■
V
-15 -
-25
Time Point (hours)
Figure 8. Average Lag Times (Normalized) for All Subjects
35
Change in Lag Time from Baseline (min
25 -
15 -
52
4»
-5
4
2
-15 -
-25
T im e P o in t (h o u r s )
Figure 9. Average Lag Time (Normalized) for Subject I
Change in Lag Time from Baseline (min)
M ilk
T im e P o in t ( h o u r s )
Figure 10. Average Lag Time (Normalized) for Subject 2
M ilk
= -5-
Time Point (hours)
Figure 11. Average Lag Time (Normalized) for Subject 3
.5
-5 -
Time Point (hours)
Figure 12. Average Lag Time (Normalized) for Subject 4
Soy
.2
M ilk
-5 -
Time Point (hours)
Figure 13. Average Lag Time (Normalized) for Subject 5
M ilk
Change in Lag Time from Baseline (min
Soy
T im e P o in t (h o u r s)
Figure 14. Average Lag Time (Normalized) for Subject 6
M ilk
Change in Lag Time from Baseline (min)
Soy
T im e P o in t (h o u r s )
Figure 15. Average Lag Time (Normalized) for Subject 7
Soy
'j Z
15
M ilk
■
.E -5 *
Time Point (hours)
Figure 16. Average Lag Time (Normalized) for Subject 8
Soy
M ilk
TZ 15 ■
.5
-5-
Time Point (hours)
Figure 17. Average Lag Time (Normalized) for Subject 9
35
25 -
-25
T im e P o in t (h o u r s)
Figure 18. Average Lag time (Normalized) for Subject 10
Change in Lag Time from Baseline (min)
T im e P o in t (h o u r s )
Figure 19. Average Lag Time (Normalized) for Subject 11
Change in Lag Time from Baseline (mi
35
T im e P o in t (h o u r s)
Figure 20. Average Lag Time (Normalized) for Subject 12
.5
-5
T im e P o in t (h o u r s )
Figure 21. Average Lag Time (Normalized) for Subject 13
.5
-5
Time Point (hours)
Figure 22. Average Lag Time (Normalized) for Subject 14
Soy
'1Z
Milk
15 -
6
Time Point (hours)
Figure 23. Average Lag Time (Normalized) for Subject 15
53
CHAPTER 5
DISCUSSION
The purpose of this experiment was to determine the acute effects of a high
saturated fat meal in combination with either a soy protein (85 mg aglycone isoflavones)
or milk protein (0 mg aglycone isoflavones) shake (956 kcal, 41% fat, 41% carbohydrate,
18% protein) on the oxidative resistance of fifteen healthy men. We examined these
effects with the copper induced LDL oxidation method and calculated lag time,
propagation rate, and initial absorbance. Our results found no significant difference
between the two treatments (soy vs. milk) in the protective resistance of the LDL particle
to oxidative stress.
There are several possible reasons for why, as a group, no statistical significance
was found in the comparison of the fat meal and the implementation of soy or milk
protein. This could possibly be due to the I) the implementation of a high SFA meal vs.
a high PUFA meal; 2) time frame of the study (short term vs. long term); 3) the
individual variability between the subjects; 4) the antioxidant status and calorie intake of
the subjects before the study days; 5) not pre-screening subjects to determine which were
equol producers; 6) inappropriate method of assessment; and 7) the use of
normocholesterolemic men vs. using hypercholesterolemic men or those representing a
high-risk oxidative stress population.
54
Implementation of a High SFA Meal vs. a High PUFA Meal
The atherogenic effect of various fatty acids has been reported in numerous
studies (17). Several lines of evidence indicate that diets rich in SFA raise serum lipid
concentrations whereas MUFA and PUFA have cholesterol lowering antiatherogenic
actions, through their ability to lower total and LDL cholesterol reducing the risk of CVD
(17, 40-44). Avoiding excess intake of SFA has thus been recommended because
hypercholesterolemia appears to be critical to the atherogenic process. It was our thought
that since SFA raises serum lipids that a meal rich in SFA (typical in the American diet)
would cause more oxidative stress.
On the contrary, it is well known that PUFA’s are more susceptible to lipid
peroxidation than are SFA’s. The rearrangement of double bonds in PUFA, i.e., the
formation of conjugated dienes, is an early event of lipid peroxidation (60, 62), therefore
one could logically assume that the implementation of a high PUFA diet (predominately
n-6 PUFA) could lead to increased oxidation of the LDL molecule. Saturated fat, on the
other hand cannot become directly oxidized due to its lack of double bonds.
To illustrate this point, Lu et al. (62) set out to investigate the influence of
different P/S (polyunsaturated/saturated) ratios on lipid peroxidation of LDL in rats using
dietary fats with different saturation levels. Thirty male rats were divided randomly into
five groups: COlOO (100% com oil), C075 (75% com oil, 25% lard), CO50 (50% com
oil, 50% lard), C025 (25% com oil, 75% lard), and LA (100% lard), with P/S ratios of
3.81, 2.13, 1.21, 0.64, and 0.28, respectively. The results showed that the rats fed P/S
ratio <1.21 (i.e., CO50, C025, and LA groups) had lower TEARS than rats fed P/S ratio
55
>1.21 (i.e., COlOO and C075 groups) and experienced significantly increased lag time of
conjugated diene formation in LDL than those fed P/S >1.21 (62). This is suggestive
that the major products at the earliest stages of LDL oxidation in vitro might result from
the PUFA hydroperoxides (an increased P/S ratio), and conjugated diene formation.
From this study we observe that a P/S ratio <1.21 could effectively retard LDL
peroxidation in the initial step of lipid peroxidation in rats suggesting that a more
saturated fat diet would lengthen the LDL oxidation in the early stage. The P/S ratio in
our experiment was 0.25 (approximately a 1/4 ratio) and judging by the results of Lu et
al. it may be assumed that at this ratio a shorter lag time period of conjugated diene
formation in the LDL would have been apparent if we were to use a meal with a higher
P/S ratio.
To further support the need for a high PUFA meal to see an acute increase in
oxidative LDL modification, Nielsen et al. (4) examined the use of low fat test meals
enriched with various oils (sunflower oil [SO], rapeseed oil [RO], olive oil [00], palm
oil [PO], and butter [B], each individually rich in PUFA, MUFA, and SFA). On six
separate days, 18 healthy male subjects were given low-fat meals or low-fat meals
enriched with one of the various oils mentioned above. The low-fat meal alone provided
7% of energy from fat, 12% from protein, and 81% from carbohydrate which was
enriched with one of the test fats to a total of 41% energy from fat, 10% from protein and
49% from carbohydrate. Following ingestion of the test fats the plasma level of TG was
in the highest concentration after B (high SFA), and the lowest after the SO-rich meal
(high PUFA). The fat rich meals all resulted in similar postprandial TG responses with
56
peak value about 4.5 hours postprandially followed by a return to fasting levels 8.5 h
postprandially. It was found that the resistance of VLDL particles to oxidation in the
postprandial state (as assessed from lag time) was decreased after the SO meal (PUfArich) (123.5 ± 11.8 min) as compared to the PO meal (SFA-rich) (157.9 ± 12.0 min)
(pO.OOl). However, the resistance of LDL particles to oxidation, as determined by lag
time, were not significantly different in the fasting state and were not affected by the
ingestion of different test meals (SO, 79.3 ± 3 min lasting vs. 76.7 ±5.1 min
postprandial; RO, 78.8 ± 4.8 min fasting vs. 75.2 ±3.6 min postprandial; 0 0 , 76.3 ±3.6
min fasting vs. 77 ± 4.1 min postprandial; PO, 78.8 ± 4.9 min fasting vs. 75.1 ± 7.8 min
postprandial; B, 86.7 ± 10.6 min fasting vs. 82.8 ± 4.6 min postprandial; p>0.05). This
study concludes that VLDL particles become more susceptible to oxidation after the
ingestion of a PUFA-rich meal and less after a SFA-rich meal (4).
From the Nielsen study described above, it is apparent that another important
parameter in acute studies may be to measure the susceptibility of VLDL to oxidation in
conjunction with LDL. The study tested one fat treatment for one day (a protocol similar
to our study) and found no significant differences in lag time of the LDL from the
ingestions of the test meals (also similar to our study). However it was found that only
the resistance of VLDL particles to oxidation was decreased after the SFA-rich meal
compared to the PUFA-rich meal. Some findings indicate that postprandial LDL
particles are more readily oxidized than fasting LDL (4). Similarly, postprandial VLDL
particles may be more prone to oxidation than fasting VLDL. This could be partially due
57
to elevated postprandial plasma TG resulting in competition for lipolysis between
chylomicrons and VLDL (I).
Plasma VLDL (the precursor of plasma LDL) is secreted by the liver in response
to a caloric load. Epidemiological studies have shown correlations between both
preprandial and postprandial hypertriglyceridemia and CHD (I). The apoprotein
\
characteristic of VLDL (apo-C and apo-B) have been found in human arterial lesions. In
normal subjects, the interaction of VLDL with lipoprotein lipase appears to give rise to
intermediate density lipoproteins (IDL) that are degraded to LDL. When normal rabbits
were given a single meal with 500 mg of cholesterol, the VLDL cholesterol began to rise
within the first few hours. The VLDL level did not return to baseline for 24 hours and a
second cholesterol-containing meal caused an additional rapid upswing in VLDL
cholesterol levels (I). Because of this initial and rapid rise in VLDL cholesterol it may
have been insightful to have performed a VLDL oxidation procedure along with the LDL.
This indicates that VLDL oxidation may be a critical parameter to assess in future
studies.
In our analysis no difference in the susceptibility of postprandial LDL particles to.
oxidation was observed following the ingestion of a high saturated fat meal. It is
plausible that our acute study didn’t elicit a sufficient transfer of fatty acids, vitamins, and
isoflavones between the lipoproteins and plasma to result in changes in the resistance of
the LDL particles to oxidation within the timescale examined (4).
Time Frame of the Study
The length of time, short term vs. long term (>1 day) that a study is executed may
be a major determination of the results that were found in the current study. It is apparent
through the literature that numerous longer-term studies (>1 day in length) have shown
significant results in the ability of soy to decrease the incidence of the LDL oxidative
event (3,4, 16).
To illustrate this, Wiseman et al. (39) recruited 24 healthy men and women for a
17-day study comparing a high isoflavone food (56 mg aglycone isoflavones) and a low
isoflavone food (1.9 mg aglycone isoflavones) with a 25 day washout between
treatments. The results showed a significantly longer lag time (p=0.017) for copper
induced LDL oxidation after the high isoflavone treatment (48 ± 2.4 min) vs. the low
isoflavone treatment (44 ±1.9 min) (39).
Similarly, Tikkanen et al. (16) recruited six healthy volunteers to receive 3 soy
bars (19 mg aglycone isoflavones) for 2 weeks. Compared with the values when the
participants were not consuming soy, lag phases of LDL oxidation were prolonged by a
mean of 20 min (p<0.02) during soy intake, indicating a reduced susceptibility to
oxidation for the 2 week duration of the study (16).
The literature supports the effect of the longer term feeding of a soy based diet to
observe a reduction in oxidative stress, however, short term studies have not yet
determined this relationship to such a great extent. Therefore in future studies of this
kind, a study period of greater than I day should be implemented in an attempt to elicit a
reduced susceptibility of LDL to oxidation.
59
Individual Variability
The measure of individual variability between the subjects in the current study
was observed in two visual patterns. Individuals exhibiting a pattern A response (46.7%)
had a greater lag time (whether positive or negative) when fed the soy protein vs. the
placebo. Individuals defined in the pattern B category (53.3%) had a greater lag time
(whether positive or negative) when fed the milk protein vs. the soy.
Cohn et al. (3) carried out a study that illustrates this individual variability when
incorporating a high fat vs. a low fat meal. The purpose of this study was to determine the
response of plasma triglycerides and cholesterol between subjects fed high-fat meals.
Subjects were fed a fat meal containing 1.0 g of fat/kg body weight and 7.0 mg/kg of
cholesterol, the total of which contained 53% fat, 23.5% protein, and 23.5% carbohydrate
(the total calorie amount was not noted). The diet was comprised of soybean oil (an oil
high in predominately PUFA) (n=12) and soybean oil plus cream (high in SFA) (n=10).
Plasma triglyceride and cholesterol concentrations were measured at hourly intervals for
12 hours after the fat meal. The mean plasma cholesterol concentration for the group as a
whole did not change significantly after the fat meal. The mean data disguises the fact
that some subjects had significant increases in plasma cholesterol, while a similar number
had a consistent decrease. Seven subjects had a significant rise in plasma cholesterol
concentration (6.0 ± 2.1% increase), while 10 subjects had a significant decrease (7.1 ±
1.2%); five subjects had no significant change. Results also showed a mean plasma
triglyceride concentration significant rise within I hour (100 mg/dl at baseline to 130
mg/dl at hour I; pO.OOl). It peaked at hour 4 (240 mg/dl; p<0.001)) and remained
60
significantly elevated for 9 hours after the fat meal (150 mg/dl; p<0.01). Individual
variability in postprandial plasma triglyceridemia was associated with the variable
changes in plasma cholesterolemia (r=0.64) (3).
This study observed that varied
responses are given in the incorporation of different fat meals.
As was previously described, the interindividual variability in our study was quite
varied. Therefore, it is advantageous to question why there was so much variability and
test for these reasons. Analyzing plasma TG for every time point for each participant
would yield further insight into individual variability of the population, and potentially be
able to further group the subjects into more stringent categories dependent on both their
TG response to the fat meal as well as their response to the treatment.
Subject Antioxidant Status and Calorie Intake
Interindividual differences in pre-existing plasma lipid peroxide and antioxidant
levels may have been a factor influencing LDL oxidizability (36) and contribute to the
variations seen in subject responses. Because of this possibility, it may be necessary to
determine individual antioxidant status prior to beginning the study protocol.
Circulating LDL is protected against oxidation by relatively high concentrations
of water-soluble antioxidants in plasma. The oxidation of LDL mainly occurs in the
artery wall suggesting the lipophilic antioxidants in LDL could be important. These lipid
soluble scavengers of peroxyl radicals could be spread throughout the surface
phospholipid layer of LDL particles. The LDL is known to carry many natural
antioxidants with vitamin E (mainly ot tocopherol) being the major endogenous
61
antioxidant contained in LDL particles (contributing approximately 30% to the variation
in lag time) (63).
Copper-induced oxidation most likely starts from the PUFA in the surface
phospholipids layer and then propagates to the core. In theory, the prolongation of the
lag phase during soy intake could be caused by LDL-bound antioxidants, such as
genistein and daidzein, acting as peroxyl radical scavengers (16). Thus it becomes
apparent that other antioxidants present in the LDL molecule (besides those coming from
soy itself) contribute to the variation in lag time.
We are also concerned as to whether the varied calorie intake of the subjects for
the three days prior to the study was also due, in part, to the nonsignificance of the results
found. From analysis of the three day diet records, it is apparent that calorie intake of our
population of subjects was quite varied (range 1654 kcal to 4572 kcal). It may be
possible that due to this varied intake as well as the type of foods eaten (e.g. one having a
diet in excess of SFA vs. another not having this excess) could possibly affect the results
of the challenge meal and the soy shake. In future studies it may be beneficial to include
an identical background diet for all Subjects for an amount of time before implementation
of the test diets in order to keep all subjects as near to the same as possible.
Pre-screening All Subjects to Determine Which Were Equol Producers
Genistein and daidzein are both naturally occurring components of soy food
products, whereas equol is derived from daidzein from the action of the gut microflora
(64). Considerable attention has been focused recently on the antioxidant properties of
62
genistein, the isoflavone that is present in greatest concentrations in soy foods. However,
daidzein is more bioavailable and equol is the most potent antioxidant isoflavone. In
addition, the precursor/product relationship of daidzein and equol (see Figure 24) is of
special interest due to the differential metabolism of daidzein in humans. Thirty percent
of individuals produce equol as reflected by a 150-900 fold higher concentration of equol
in the plasma or urine (64). Those individuals with higher equol levels are characterized
as “responders” (those who do convert daidzein to equol) vs. “nonresponders” (those who
did not convert daidzein to equol).
G u t M ic r o flo r a
D a id z e in
Figure 24. Structure of Daidzein and Equol
Since the conversion of daidzein to equol occurs by the action of microflora, it
has been proposed that the type of bacteria found in the colon of the ‘responders’ is
different from the ‘non-responders’. Moreover, it has been reported that an individual is
consistently either a ‘responder’ or ‘non-responder’, suggesting that the reason for this
individual variability may be intrinsic or perhaps even genetically determined (64).
Hence, it would be worthwhile in future studies to examine this parameter to elicit if the
lack of significance was, in part, due to the subject’s status as responders or non­
responders.
63
Setchell et al. (65) measured plasma equol in subjects fed daidzein (aglycone
form) or daidzin (glycoside form). Equol was found in the plasma of only three of the
nine subjects (33%). Those individuals with the physiological ability to convert daidzin
to daidzein, and from daidzein to equol were referred to as “converters”. Additionally the
plasma profiles of the subjects participating in this study showed that after a single
ingestion it takes at least 6-8 hours before equol appears in substantial amounts in the
plasma (65). Considering this time point and the 6-hour time point analyzed in the
current study, equol production may have not attained its full potential in potential
converters, thus not yet achieving its full antioxidative ability.
However, from a previous study done in our lab it was determined that in twice
the time of our study (12 hours) no significance was found on LDL oxidation parameters
(lag time, propagation rate, or initial absorbance) (36). Had the subjects been converters,
we would have expected a significant increase in lag time at the time that equol was in
greatest concentrations.
Inappropriate Method of Assessment
Several methods have been developed to measure the oxidative stress of a
biological sample. Our use of the ex vivo LDL-oxidation technique using copper as a
pro-oxidant has raised some questions as to its independent accuracy as a marker of
oxidative stress as it pertains to our study design. It is possible that the water-soluble
antioxidants studied in this experiment (isoflavones) are essentially stripped from their
aqueous environment upon using this method, and are therefore not able to elicit their
64
effect. Therefore it is essential that other methods be used in conjunction with this
technique.
The use of the total radical trapping parameter (TRAP) assay has been the most
widely used method for measuring total antioxidant capacity of plasma or serum during
the last decade. The TRAP assay uses peroxyl radicals generated from 2,2'-azobis (2amidinopropane) dihydrochloride and peroxidizable materials to the plasma. The
oxidation is then monitored by measuring the oxygen consumed during the reaction.
During an induction period, this oxidation is inhibited by the antioxidants in the plasma.
The length of the induction period (lag phase) is compared to that of an internal standard,
and then quantitatively related to the antioxidant capacity of the plasma. A major
concern with the TRAP assay is the oxygen electrode end point; an oxygen electrode will
not maintain its stability over the period of time required to carry out the oxidative
process, therefore not making this a sensitive marker of oxidative stress (63).
Determining urine or plasma isoprostanes are another possible method that could
be included in a study design to increase reliability. Isoprostanes are produced from
oxidative modification of PUFA via a free radical-catalyzed mechanism. Isoprostanes
are generated initially at the site of a free radical attack, of the PUFA arachidonate, in cell
membranes; they then circulate in plasma and are excreted in the urine. Evidence has
accumulated to suggest that measurement of isoprostanes can provide a sensitive and
specific assessment of lipid peroxidation both in vitro and in vivo. However, plasma
isoprostanes are measured via a very expensive and elaborate technique, with urine being
collected typically in 24 hour samples (66).
65
Another potentially advantageous analysis would be measuring one of the
byproducts of lipid peroxidation, malondialdehyde (MDA) through a method known as
TEARS. It has been shown that modification of protein with MDA changes antigenicity,
function, and turnover kinetics of various proteins and has also been implicated as a
pathogenic mechanism of atherosclerosis (67). This method could thus possibly be
feasible for future studies.
Because of the complications of specific and singular analyses no single
measurement of antioxidant status is completely sufficient for all purposes, but rather a
“battery” of measurements may be necessary to adequately assess oxidative stress in
biological systems (63). In this regard LDL oxidation would have been an ample tool in
the evaluation of LDL oxidation if it were used in conjunction with another method of
analysis (TRAP, isoprostanes, or MDA) implemented into the study design. Therefore
future studies in which LDL-oxidation is used should also include other methods of
oxidation to complement this protocol.
Those in a High Risk Population
It is also possible that our subject population of normocholesterolemic individuals
may have been an inappropriate group to examine. To illustrate this point, it is helpful to
look at the research that has predominantly shown beneficial effects of soy in individuals
with moderate to severe hypercholesterolemia (258 to 5335 mg/dL) but no significant
effect in individuals with normal cholesterol levels ( r200 mg/dL) (10, 15).
66
Jenkins et al. (9) studied a population of 25 hyperlipidemia subjects (15 men and
10 postmenopausal women) for 6 weeks on a breakfast cereal containing soy (providing
36 g of soy protein/d and 168 mg of isoflavones/d) and a control diet (of a commercial
breakfast cereal product containing no isoflavones). Total conjugated dienes were
significantly reduced on the soy diet compared with the control (9.2% ± 4.3%, p=0.42),
and the ratio of conjugated dienes to cholesterol in the LDL fraction was also reduced
(8.7% ± 4.2%, p=0.050) following the three-weeks of soy consumption. This study
indicates a beneficial effect of soy in reducing indices of LDL oxidation in the
hyperlipidemic individual (9). These results possibly indicate that had we studied a highrisk population, such as hyperlipidemics, we may have also seen a beneficial effect for
soy isoflavones in the reduction of oxidative stress.
A study that is perhaps even more relevant to this argument is the meta-analysis
by Anderson et al. (32). In this analysis of 38 studies, one of the parameters that were
specifically examined was the response to soy in relation to an individual’s cholesterol
concentration. Subjects with normal cholesterol levels ( ^OO mg/dL) had nonsignificant
reductions in serum cholesterol and of LDL cholesterol while receiving the soy protein
diet of 3.3% and 7.7%, respectively. Those with mild hypercholesterolemia (200-255
mg/dL) had nonsignificant reductions in serum cholesterol of 4.4%, and LDL cholesterol
of 6.8%. Subjects with moderate hypercholesterolemia (259-333 mg/dL), had significant
decreases in serum cholesterol of 7.4%, and also in LDL cholesterol of 9.8%, yet subjects
with severe hypercholesterolemia (S 3 5 mg/dL), had significant reductions in serum
cholesterol of 19.6%, and also in LDL cholesterol of 24.0% (32). It is apparent that
67
those subjects with higher cholesterol levels (moderate to severe hypercholesterolemia)
upon initiation of the study had a greater response to the soy treatment than those with
lower cholesterol levels (normal to mild hypercholesterolemia).
From the studies presented, it appears that hypercholesterolemic individuals have
the best response to soy treatment through its ability to decrease LDL as well as the
oxidation of LDL. Because this high-risk population experiences a greater response to
soy treatment, an appropriate question would be whether other high risk groups would
also be responders to soy treatment, such as smokers and those with diabetes mellitus
(DM). Cigarette smoking has been clearly identified as an independent, major risk factor
of atherosclerosis and CHD (68). Since cigarette smoke generates abundant free radicals,
it has been suggested that lipid peroxidation in LDL is also increased in this population
(68). It is also been established that diabetes is associated with increased morbidity and
mortality resulting from CHD (69).
Seghrouchni et al. (69) studied in vitro susceptibility to the oxidation of LDL
isolated from the plasma of three groups of diabetics (type I, type 2, and insulin
dependent type 2) by measuring the total fatty acids, MDA, and a-tocopherol in oxidized
and native LDL. Blood samples were obtained after fasting conditions from 100 patients
with DM. The results showed that diabetics have a significantly lower lag time (59.21 ±
15.98 min type I; 56.64 ± 16.17 min type 2; and 56.10 ± 17.10 min for insulin-dependent
type 2, p<0.05) as compared to a normal DM free control group (66.25 ± 6.13 min) (69).
The current study has provided information on the postprandial effects of a high
SF meal administered with a milk protein shake vs. a soy protein shake on LDL oxidation
68
parameters (lag time, propagation rate, and initial absorbance). The studies discussed,
however, provide further insight to a more detailed design. Perhaps a study in which a
high risk population was given a high PUFA meal alongside a treatment shake (soy vs.
milk) for 2 days while measuring lipid parameters (TG, LDL, VLDL, HDL, and TC),
VLDL-LDL and LDL oxidation. It may also be beneficial to control for individual
antioxidant status prior to the study, as well as to screen for those individuals who are
equol producers. This protocol may serve to demonstrate the protective effect of soy on
the oxidative modification of LDL.
69
CHAPTER 6
SUMMARY
Coronary heart disease, one of the most common and serious forms of
cardiovascular disease, is a major public health concern because it causes more deaths in
the U.S. than any other disease. Zilversmit et al. (I) was the first to determine that
atherosclerosis is a postprandial phenomenon with lipoprotein particles in the fed state
contributing to atherosclerosis. Because of our society’s consumption of regular meals
(typically high in fat) throughout a day, we exist primarily in a postprandial state.
Zilversmit also maintained the view that lipids accumulated as the result not only of
abnormally high concentrations of LDL in the blood plasma, but also as a consequence of
the normal process of lipid absorption and transport. This process has the potential to be
pathogenic in persons who consume a diet rich in fat and cholesterol (I). Therefore
nutritional practices are of great concern in regard to studying the process of
atherosclerosis.
The specific ability of soy and its components to reduce total cholesterol, LDL
cholesterol, and TG concentrations was documented in 1995 when Anderson et al. (32)
conducted a meta-analysis from 38 human studies to show that the combination of the
lipid lowering effects of soy as well as the antioxidant properties of soy isoflavones could
contribute to a reduction in LDL oxidation.
Following the work by Anderson and his colleagues, on October 26, 1999, the
FDA authorized the use of health claims regarding soy protein in reducing the risk of
70
CHD on labeling of foods containing soy protein. This final rule was based on the FDA's
conclusion that foods containing soy protein included in a diet low in saturated fat and
cholesterol may reduce the risk of CHD by lowering blood cholesterol levels (8).
The purpose of our study was to determine if a difference exists between 39 g soy
protein (85 mg isoflavones) and 39.9 g milk protein (0 mg isoflavones) when consumed
in combination with a high-fat diet to determine their relative protection against
postprandial copper-induced LDL oxidation. No significant differences were observed
(p>0.05) between the protein type (soy vs. milk), time (baseline, 2, 4, 6), or interaction
between the factors on LDL oxidation (lag time, propagation rate, or initial absorbance)
measurements.
The inability of our study to produce any significant results could possibly be due
to the I) the implementation of a high SFA meal vs. a high PUFA meal; 2) time frame of
the study (short term vs. long term); 3) the individual variability between the subjects; 4)
the antioxidant status and calorie intake of the subjects before study; 5) failure to pre­
screen all subjects to determine which were equol producers; 6) inappropriate method of
assessment; and 7) the use of normocholesterolemic men vs. using hypercholesterolemic
men or those in a high risk population (as described in the discussion section of this
paper). Although any of these factors alone is probably not entirely responsible for the
insignificance of the results all should be seriously considered when undertaking any
further studies of this kind.
Although no statistical significance was seen between the soy and milk protein
treatments of this study or the time points analyzed on the parameters of lag time,
71
propagation rate, and initial absorbance, valuable information can be provided. Through
examining individual patterns of the subjects participating, and taking into consideration
the points discussed to further research in this area we now are aware of possible
directions to base future studies considering the recommendations presented in this paper.
Atherosclerosis continues to be the major debilitating disease, of our era. It is
promising that through the progress of research, such as the study we have conducted,
that someday we will have intimate knowledge to be able to produce nutritional practices
to impede the process of atherosclerosis.
72
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73
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APPENDICES
80
APPENDIX A
MEDICAL HISTORY QUESTIONNAIRE
81
Medical History Questionnaire
Please answer the following questions honestly and to the best of your knowledge. All
information provided here is completely confidential. Please ask for clarification if
needed.
Subject Nameii_____________________
(Last)
" (First)
(MI)
Phone Number we can reach you at:________________ ________
Address:
Sex:
M
Weight:
F
Age:_____yrs_____mo
lbs
Height:
Date of Birth
. f t
State of birth:
Do you smoke cigarettes?
Yes
Are you an ex-smoker?
Yes
If so, how long ago did you quit?
No
No
Race (circle):
1. American Indian or Alaska Native
2. African American
3. Caucasian
4. Asian
5. Hispanic
6. Other (specify):_______________
Marital Status (circle one):
1. single
2. married
3. divbrced/separated
4. widowed
Medical History (circle any, and give age at diagnosis):
Age
1. Diabetes
2. Thyroid Disease
82
3. Cirrhosis
____
4. Hepatitis
____
5. Gall Stones
____
6. Kidney Stones
^___
7. Nephritis
____
8. Cancer (specify)
____
9. High Blood Pressure
____
10. Angina
____
11. Allergies (specify)
____
12. Goiter
____
13. Cardiovascular Disease
____
14. Depression requiring medication____
15. ' Insomnia requiring medication __
Drug History:
1. Do you currently take any medications on a regular basis?___
If yes, please specify_______________________ _________
2. Have you taken medication regularly in the past?__________
If yes, please specify________________________________
3. Do you currently take vitamin supplements on a regular basis?
If yes, please specify_________ ___________________ ___
Have you in the past? ____________
If so, how long ago?_______________
4. Do you currently take herbal supplements on a regular basis? _
If yes, please specify________________________________
Have you in the past?_____________
If so, how long ago?_______________
83
Diet History:
a. Are you currently on a diet to lose weight?
Yes
No
i. If yes, please explain________^___________________
b. Are you a vegetarian?
Yes
No
c. If yes, circle one of the following
i. Lacto-ovo (consume milk, milk products, and eggs)
ii. Ovo (consume eggs but no milk or milk products)
iii Lacto (consume milk and milk products but no eggs)
iv. Vegan (consume no animal products
d. Please specify any food allergies (soy, milk, peanut, etc):________________ __
e. How many alcoholic drinks (12 oz beer, 6 oz wine or 1.5 oz distilled alcohol) do you
typically consume (circle one)?
i. 0-1 drinks/day
ii. 1-2 drinks/day
iii. >2 drinks/day
If yes, how many days/week do you consume >2 drinks/day?________
f. Do you consume soy on a regular basis?
Yes
No
g. If yes, how often?___________________
h. Which soy products do you typically eat (please circle)? How often (servings/day,
week, month, year)?
i. Tofu (1/2 cup = I serving)
______________ _____
ii. Soy milk (I cup = I serving)__________ ________
iii. Soy nuts (1/4 cup = I serving)________ _________
iv. Soy protein powder concentrate (1/4 cup = I serving)________________
v. Soy bar (i.e. Luna Bars, Genisoy Bars)____________ ■
vi. Soybeans (1/2 cup = I serving)________________ _____
vii. Soy burgers (i.e. Garden Burger, Boca Burger)_______________ ___
84
viii. Tempeh (1/2 cup = I serving)______________________
Portion sizes from the 2000 Soyfoods Guide
Video Rental suggestions for days spent at the Nutrition Research Lab:
Please list dates (preferably weekend days) that would be best for you to spend 6 hours at
the Nutrition Research Lab:
Please list any weekend for which you would be unable to spend 6 hours at the Nutrition
Research Lab:
85
APPENDIX B
HUMAN SUBJECTS CONSENT FORM
86
SUBJECT CONSENT FORM
FOR
PARTICIPATION IN HUMAN RESEARCH
MONTANA STATE UNIVERSITY
PROJECT TITLE: Effects of soy isoflavone consumption following a high-fat or highcarbohydrate meal on the oxidative resistance of low-density lipoprotein in healthy,
young men.
PRINCIPLE INVESTIGATOR: Christina Gayer Campbell, PhD, RD
Assistant Professor, Nutrition
Department of Health and Human Development
Herrick Hall, Room 20, Montana State University
Bozeman, MT 59717-3540
406-994-5002, ccampbel@montana.edu
PURPOSE AND RATIONALE OF THE STUDY:
A considerable amount of research has been done to investigate the potential benefits of
soy on cardiovascular disease (CVD). The research has shown that most people would
benefit from the incorporation of soy into their diets. Interventions lasting 1-2 months
have been able to demonstrate a relationship between soy consumption and a reduction in
the modification to the low-density lipoprotein (LDL), also known as the “bad
cholesterol” that may promote the development of CVD. Past research has mainly
focused on measuring the “bad cholesterol” during the lasted state, yet evidence suggests
that LDL is more susceptible to modification following the consumption of food. The
relationship between meal composition and measures of LDL modification following
food consumption may provide useful insight into the prevention of CVD.
The purpose of this study is to determine if the effects of a meal containing soy protein
and soy’s natural antioxidants, isoflavone, versus milk protein (no isoflavones) can
minimize measures of LDL modification when the soy meal is consumed along with a
high-fat or high-carbohydrate (CHO) meal, both of which have been shown to increase
the concentration of circulating lipids in the blood.
You are being asked to participate because you are a healthy, non-vegetarian, 20-40 year
old individual living in the communities surrounding Montana State University who has
shown interest in our study by responding to our recruiting flyer. You have been
identified as a possible participant based on several criteria including: no regular use of a
vitamin or mineral supplement in the last 3 months, no regular consumption of soy
protein (> 2 servings of 6.25g/day), no known existence of metabolic disorders (thyroid
disease, diabetes, liver or renal disease), non-use of oral prescription medications
(including antibiotics), non-obese (BMK30kg/m2), no cigarette smoking within the last 3
87
years, absence of certain food allergies (soy, milk, peanuts), no strict vegetarian status
and no regular alcohol consumption (regular = >2 drinks/d for men; I drink = 12 oz beer,
6 oz wine or 1.5 oz distilled alcohol):
STUDY OUTLINE
If you agree to participate, you will be asked to complete the following:
You will be required to come into the Nutrition Research Lab (NRL) at Herrick Hall on
the campus of Montana State University on three separate occasions. During the 1st visit,
we will draw a lasted blood sample for a preliminary screening to determine your levels
of total cholesterol, high-density lipoprotein and triglycerides. If your levels are within
normal limits, you will be eligible to participate in the study. On the other two occasions,
you will be asked to report to the NRL in a fasted state (no food or beverage, except
water, for 10 hours) and to have refrained from any moderate to strenuous physical
activity or alcohol consumption for at least 24 hours prior to the study date. You will be
required to bring completed 3-day weighed (requires that subjects weigh all food and
beverage) diet records for the days just prior to the study date. A dietetics student that
has been trained on giving dietary analysis instructions will give you detailed instructions
on how to properly complete the forms and tips on accurately weighing food. You will
be provided with a dietary scale, at no cost to you, for use during the study to facilitate
the process. You may perceive this to be a tedious process, however it is the most
accurate means of collecting dietary intake information.
On each of the of the feeding study days, you will enter the NRL at 0630 h and stay until
1300 h. After the measurement of body weight and height, an individual trained in
drawing blood from the forearm will take a blood sample. Following the collection of
the fasted blood, you will be given 20 minutes to consume the test meal. The meal
(shake and muffin) will provide approximately 850 calories, which is equivalent to one
third of your daily calories. You will randomly be assigned to one of two groups; Group
A, the high-fat meal or B, the high-CHO meal. Within your group, you will randomly be
assigned to receive meal I or 2 first. On your subsequent visit to the NRL, you will
receive the meal you have not yet had. The study is a double blind crossover, which
means that neither you nor the research team knows which meal contains the soy or the
milk protein. Utilizing this research design helps to minimize the placebo effect.
During the study period you will remain in Herrick Hall; a television and videos will be
available for you to watch. Blood will be collected from your forearm at 2, 4 and 6 hours
following the consumption of the test meal for a total of 72 mL of blood. You will not be
allowed to eat or drink anything except water for the six hours following the test meal.
88
Group A
Test Meal I
High fat muffin
Protein shake
40 g soy protein (80 mg isoflavones)
isoflavones)
I banana
12-16 oz water
20% protein, 40% fat, 40% CHO
Group B
Test Meal I
High CHO muffin
Protein shake
40 g soy protein (80 mg
Test Meal 2
High fat muffin
Protein shake
40 g milk protein (0 mg isoflavones)
isoflavones)
I banana
12-16 oz water
20% protein, 40% fat, 40% CHO
Test Meal 2
High CHO muffin
Protein shake
40 g milk protein (0 mg
I banana
12-16 oz water
20% protein, 20% fat, 60% CHO
I banana
12-16 oz water
20% protein, 20% fat, 60% CHO
RISKS:
Approximately I tablespoon of blood will be removed by putting a needle in your vein on
4 occasions. This is the standard medical method used to obtain blood for tests. There is
momentary pain at the time the needle is inserted into the vein, but other discomfort
should be minimal. In about 10% of the cases there is a small amount of bleeding under
the skin, which will produce a bruise. This risk of infection is less than I in 1,000.
Subjects may experience gastrointestinal distress as a result of protein shake
consumption.
Soy protein does not pose any unusual risks of allergenic responses. Soy protein is less
allergenic than cow’s milk. There are, of course, certain people who may be allergic to
soy. Allergy to soy is reported as approximately 0.5 percent incidence in the adult
population,
BENEFITS:
All participants will be provided with a summary and explanation of their results from the
study including the lipid panel profile and dietary intake analysis.
FUNDING:
This is currently not a funded project.
89
CONFIDENTIALITY:
The data obtained from the study will be regarded as privileged and confidential. Your
privacy will be maintained in any future analysis and/or presentation of the data with the
use of coded identification for each participant’s data. All data will be stored in a locked
file cabinet with access only by the principal investigator. Additionally, any data entered
into the computer will be available with restricted password only.
FREEDOM OF CONSENT:
Participation in this study is completely voluntary. You may withdraw consent in person
with the principal investigator, Dr. Christina Campbell, at any time. In the event of any
physical injury occurring in connection with the study, Montana State University will not
provide any special compensation or any medical treatment. We will advise and assist
the participant in receiving medical treatment. Additionally, Montana State University
will not be held responsible for injury or accidents that may occur when traveling to and
from campus.
Please feel free to ask any questions or express your concerns regarding this study. The
investigator will attempt to answer all of your questions. Contact Dr. Christina Campbell
at 994-5002.
Please address any questions relating to the rights of human subjects to Mark Quinn,
Chair, Human Subjects Committee, 994-5721.
AUTHORIZATION:
If over 18 years of age:
I have read the above and understand the discomforts, inconveniences and risk of this
study. I ,________________________________(your name), agree to participate in the
project. I understand that I may later refuse to participate, and that I may withdraw from
the study at any time. I have received a copy of this consent form for my own records.
Signed:___
Witness:__
Investigator:
Date:
APPENDIX C
DIET RECORD DIRECTIONS AND FORM
91
Directions for
3-Day Weighed Diet Records
°
Please weigh and record all foods and beverages consumed for the three days
prior to reporting to the Nutrition Research Lab for a feeding study day (i.e. Ifyou
will be reporting to the Lab on a Sunday, please record for Thursday, Friday and
Saturday).
°
Keep your food record current. List foods immediately after they are weighed.
Do not wait until the end of the day to record entries.
o Please print all entries.
o Be as specific as possible when describing the food or beverage:
o Include the method of preparation used (boiled, baked, broiled, fried,
grilled, steamed, raw, etc); example: chicken breast, skinless, broiled
o Include a well detailed description of the food item (fresh, canned, packed
in heavy or light syrup, packed in water or oil, skinless, boneless, cut of
meat,, brand name); examples: peaches in heavy syrup, tuna in oil, broiled
T-bone steak, microwave heated canned corn
o Include label with the nutritional information for any unusual items or if
unsure how to record
°
Include the name of restaurant if eating out
o Report only the portion of the food that was actually eaten; example: T-bone
steak, grilled -100 g (do not include the weight o f the bone)
°
Record amounts in either grams or ounces (wt)
o Remember to record condiments (ketchup, soy sauce, mustard, ranch dressing,
etc) as well as any fats used in cooking (oils, butter, margarine, etc), it is
acceptable to measure these (Tbsp, tsp, etc)
o And finally, try not to alter your normal diet during the period that you keep this
record
o Please bring the scale and diet records to the Nutrition Research Lab with you on
your scheduled blood draw day.. .THANK YOU!!!
3-Day Weighed Food Record Form
Name:________________
Subject ID#
_____
Date Recorded:__ _______
Day: M T W Th F Sa Su
Typical Day?
_____
T im e
D e s c rip tio n o f F o o d Ite m E a te n & C o o k in g M e th o d
W e ig h t
93
APPENDIX D
PHYSICAL ACTIVITY LOG
94
-
29’ No' 6 S upplem ent
Official Jo u rn al of Ihe American College o f S p o rts Medicine
Activity Codes for tUe Bouchard Three Day Physical Activity Record
Category
of activity
I
2
3
4
5
6
7
8
Example of activity for each category
Lying, down:
- sleeping
‘ - resting in bed
Sealed:
- listening in class
- eating
- writing by hand or typing
- reading
- listening to Uie radio or T.V.
- taking a bath.
Standing; light activity:
- wasliing oneself
- shaving
- combing hair
- dusting
- cooking
(jetting dressed
Taking a shower
Driving a car
Taking a walk (strolling)
Light manual work:
- housework (wasliing
- carpentry
windows, sweeping etc.)
- masonry
- tailor
- driving a farm
- baker
tractor
- printer
- cleaning trees
- brewer
- working in the
- cobbler
chemical or
- mechanic
eleclrio.industries
- electrician
- feeding animals
- painter
on a farm
- lab-work
- doing the bed
Riding a moped
Moderately quick walking
(going to school, shopping)
Light sport or leisure activities:
- light canoeing
- archery
- volleyball
- ninepins
- table tennis
- croquet
- baseball (except the pitcher)
- sailing
- golf
- cycling (leisure)
- rowing
Moderate manual work:
- machine operating (building industry)
- repairing a fence
- loading bags or boxes
- plantation work
- forest work (machine sawing and log handling)
- mine work
- shoveling snow
Moderate sport or leisure activities:
- baseball (pitcher)
- horseback riding
- badminton
- Alpine skiing
- canoeing
- cross-country
- cycling (race bike)
skiing (leisure)
- dancing
- swimming
- tennis
- gymnastics
- jogging (slow running)
- brisk walking
Approximate energy
expenditure
(kcal/kg/15 min)
0.26
0.38
0.57
0.70
0.83
1.20
1.40
1.0
S2]
95
S22
Official Journal of;the American College o f S p o rts Medicine
Categoryof activity
9
MEDICINE AND SCIENCE IN SPORTS AND EXERCISE
Example of activity for each category'
Approximate, energy
expenditure
(kcal/icg/lS min)
Intense .marmai work:
felling, a tree with ah ax
- ,sawing with a hand-saw.
- working, with a pitchfork (bn a, faith)
- cutting, tree branches
1.95
Intense sport or leisure.activities:
- running in arace.
- boxing
- mountain-climbing
- squash
- cross-country skiing
- ice hockey
- basketball
- football
-racquclba'll
96
S20
Official Journal of the American College of S p o rts M edicine
MEDICINE'AND SCIENCE IN SPORTS. AMD EXERCISE
BOUCHARD THREE P A Y PHYSICAL ACTIVITY RECORD
Dav I
Date:______/____ J ______
day
month year
-sMinute
0-15
Last name:
First name:
H our\
0
I
2
3
4
5
6
7
In each box, write Uie number which
8
corresponds to the activity which you
9
have carried out during this 15 minute
10
period. Please consult the activity card
11
that follows to establish the proper
12
coding. If an activity is carried out over
13
a long period (e.g. sleeping) you can
14
draw a continuous line in the rectangular
15
boxes which follow until such a time
16
When there is a change in activity.
17
To understand this better, we suggest that
18.
you take a,look at the example that
19
follows.
20
21
22
23
16-30
31-45
46-60 ,
APPENDIX E
CHALLENGE MEAL DIET ANALYSIS
98
HS Client Diet Record Nutrient Analysis
F ir s t:
M id d le :
L a s t:
C om oanv:
I d e n tif ic a tio n N u m b e r :
D a te o f B irth :
H e io h t:
Muffin a n d S h a k e
T o ta l D a y s :
I
A v g . D aily K c a ls : 955.741
T o ta l F o o d s :
D ie t N a m e :
Muffin a n d S h a k e
W e io h t:
3
New Diet Record
P e r c e n t a g e o f K c a ls
P ro tein
C a rb o h y d ra te
F at, total
A lcohol
1 8 .3 %
4 1 .0 %
4 0 .7 %
0 .0 %
E xchanges
Fat
Fruit
N u trie n t
W eig h t
K ilocalories
P ro tein
C a rb o h y d ra te
F at, T otal
A lcohol
C h o le ste ro l
S a tu r a te d F at
M o n o u n s a tu ra te d F at
P o ly u n s a tu ra te d F at
M FA 18:1, O leic
P F A 18:2, Linoleic
P F A 18:3, Linolenic
P F A 2 0 :5 , EPA
P F A 2 2 :6 , DHA
S o d iu m
P o ta s s iu m
V itam in A (R E )
V itam in A (IU)
B e ta - C a ro te n e
A lp h a -C a ro te n e
L utein ( + Z e a x a n th in )
B eta-C ry p to x an th in
Lycopene
V itam in C
C alcium
Iron
V itam in D (ug)
V itam in D (IU)
V itam in E (m g)
V itam in E (IU)
A lp h a -T o c o p h e ro l
T hiam in
Riboflavin
N iacin
P yridoxine (V itam in B6)
F o la te
C o b a la m in (V itam in B I 2)
Biotin
P a n to th e n ic Acid
V itam in K
P h o s p h o ru s
Iodine
M onday. April 2 1 , 2 0 0 3
0 .0 0
1.50
V a lu e
U n it
3 1 2 .4 6 8
9 5 5 .7 4 1
4 4 .6 6 8
1 0 0 .0 3 3
4 4 .0 6 5
g
Kcal
0.000
38 802
2 6 .4 3 0
6.751
6 .7 0 4
6 .1 3 0
6 .3 3 4
0 .2 4 3
0.000
0.000
8 9 5 .4 5 5
1 0 9 5 .8 5 6
132 672
1 6 1 2 .9 1 6
4 5 .8 9 8
9 .8 0 0
7 .2 0 0
g
mg
g
g
g
g
g
g
g
g
mg
mg
RE
IU
Mg
0.000
0.000
1 0.1 9 7
1 5 0 2 .2 8 0
5 .5 0 2
mg
mg
mg
0 .1 9 6
2 0 7 .8 2 4
3.281
4 .8 8 9
2 416
0.121
1 .0 8 0
1.4 2 3
0 .6 0 0
1 2 3 .5 2 6
1.8 5 6
5 .4 4 5
0 .3 6 4
1 2 .9 5 3
1 0 7 8 .1 3 8
Mg
I
%
g
g
g
Mg
Mg
Mg
Mg
0.000
G oal
IU
mg
IU
mg
mg
mg
mg
mg
Mg
Mg
Mg
mg
Mg
mg
Mg
Firsl DataBank Nutritionist Pro1"
99
fiS Client Diet Record Nutrient Analysis
F ir s t:
M id d le :
L a s t:
C om oanv:
I d e n tif ic a tio n N u m b e r :
D a te o f B irth :
H e lo h t:
Muffin a n d S h a k e
N u trie n t
M a g n e siu m
Z inc
C opper
M an g an ese
S e le n iu m
F luoride
C h ro m iu m
M oly b d en u m
D ietary F iber, T otal
S o lu b le F ib e r
In so lu b le F ib er
C ru d e F ib e r
S u g a r, T otal
G lu c o s e
G a la c to s e
F ru c to s e
S u c ro se
L a c to s e
M a lto se
S u g a rA Ic o h o I
O th e r C a rb o h y d ra te
T ry p to p h a n
T h re o n in e
Is o le u cin e
L eu c in e
L ysine
M eth io n in e
C y stin e
P h e n y la la n in e
T y ro sin e
V aline
A rginine
H istidine
A lan in e
A sp a rtic A d d
G lu ta m ic Acid
G lycine
P roline
S e rin e
M oisture
A sh
C affe in e
M o n d ay , April 21 , 2 0 0 3
V a lu e
1 5 6 .7 1 8
0 .3 4 2
0 .1 5 8
0 .3 2 7
6 .8 9 1
5 5 .9 0 1
0 .0 0 9
1 .9 6 9
4 .1 2 7
0 .5 0 0
1 202
0 .6 9 5
5 5 .9 6 9
5 .0 1 9
0 .0 0 0
4 .1 4 3
1 2 .3 7 0
0 .0 0 0
0 .0 1 6
U n it
W e ia h t:
%
mg
mg
mg
mg
pg
pg
mg
pg
g
g
g
g
g
g
g
g
g
g
g
4 3 .6 9 8
135862
1 5 9 .4 2 0
2 7 6 .5 8 1
1 8 6 .3 7 2
8 3 .2 2 7
7 7 .1 2 6
1 7 8 .2 8 4
mg
mg
mg
mg
mg
mg
mg
mg
1 1 7 .5 5 7
1 9 0 .7 1 0
1 7 3 .4 3 0
1 3 9 .1 3 3
163231
3 2 6 .1 0 9
6 2 5 .9 8 6
1 2 6 .4 2 7
2 0 9 .2 8 7
2 0 6 .6 0 4
1 08.3 2 1
2 .6 3 9
0 .0 0 0
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
g
g
mg
2
G oal
Muffin a n d S h a k e
First DataBank Nutritionist Pro™
100
APPENDIX F
THE SOLAE COMPANY
101
R O r B ox 88940
S L L o u is, M O 6 3 1 8 8
P h o n e : 3 1 4 -9 8 2 - 1 2 6 8
F a x : 3 1 4 -9 8 2 -3 9 8 0
E m ail: b ro w n J I@ so la e .c o m
Dr. Christina Campbell
Montana State University
20 Herriok Hall
Bozeman MT 59717
406-994-5002
April 1 , 2003
Dear Dr. Campbell:
The undersigned acknow ledges that the powdered Nutritional beverages, manufactured by Solae
L.L.C, shipped to you with the designations Sample 858 and 461 contain th e following
ingredients:
S am p le 8 58 - The Soy Vanilla Nutritional Beverage Powder ( f o rm u la te d t o
d e liv e r
20
g s o y p ro te in w ith
2 .0 m g i s o f i a v o n e s ’ / g o f s o y p ro te in ) V a g ly c o n e w e ig h t
Ingredients: Isolated s o y protein, fructose, sucrose, maitodextrin, potassium citrate, artificiai
flavor and guar gum. V itam ins & Minerals: Calcium phosphate, m agnesium phosphate,
riboflavin, vitamin A paimitate, folic acid, vitamin D3 and vitamin B i 2.
S am p le 461 - T h e Placebo Vanilla Nutritional B everage Powder
Ingredients: Milk protein isolate, fructose, sucrose, maitodextrin, potassium citrate, artificiai
flavor and guar gum. V itam ins & Minerals: Calcium phosphate,, m agnesium phosphate,
riboflavin, vitamin A paimitate, folic acid, vitamin D3 and vitamin BI 2.
The U.S. Food and Drug Administration’s Regulations (21 G F R 101.100 (d)(2)) require that a
copy of this agreem ent be kept on file for two (2) years beyond the final shipm ent or delivery of
the above-referenced products. This document should be available for Inspection, upon request,
by any officer or em ployee of the FDA.
AGREED TO AND ACCEPTED BY:
A representative of S oIae study number 506 titled: “The postprandial effect of so y isoflavone
consumption following a high-fat or high-carbohydrate m eal on oxidative resistance of .low-density
lipoprotein in healthy, young men"
(UATITlE:
u •
Surmrary of product data for S tudy $06 (Campbet!/Montana State Unlv)
S tu d y title:
T h e p o s tp ra n d ia l e ffe c t of so y fecflav o n e c o n s u m p tio n fottowing a h ig h -fat o r
h ig h -c a rb o h y d ra te m e a l o n oxidative re s is ta n c e o f lo w -d e n sity lipoprotein in h ealth y , y o u n g m e n
P ro d o * :
S a m p le 8 5 6 - S oy V anilla Nutrftkm al B e v e ra g e P o w d e r (fo rm u la te d to d eliver 2 0 g s o y p ro tein w ith 2 .0 m g feofiavones, ag fy co n e w eig h t
S a m p le 461 - Milk P la c e b o V a n ila N utritional B e v e ra g e P o w d e r
P ro d u c t
S a m p le *
L o t#
P r o te in
I s o f la v o n e s (a g io )
I s o f U v o n e s (a g io )
g /d a y
m g /4 4 g s e r v
m g 'd a y
1 9 .6
3 9 .0
42 7
8 5 .4
19 .9
3 9 .9
P r o te in
glte g
A f 1.2 Cl VA TDCA 44
658
AFTMP C l VA 7 0 C A 44
461
G 2 6 5 -0
G 2 6 6 -0
eerv
Ig of s o y p rotein)
103
The Solae Company
P.O. BOX 88940
ST. LOUIS, MO 63188
AFTMP Cl VA 70CA 44
Lot # G266-0
Microbiology
Composition
P r o t e i n (% )
4 5 .3
A e r o b i c P l a t e C o u n t ( /g )
C a lc iu m ( m g /1 0 0 g )
1720
C o n fo rm c o u n t (M P N /g )
<3
A s h (% )
7 .3 3
E . c o li ( M P N /g )
<3
F a t , a d d h y d r o l y s i s (% )
M o ld (/g )
0 .6 5 4
M o i s t u r e (% )
2 .8 6
S a lm o n e lla (/3 7 5 g )
P h o s p h o r u s ( m g / 1 OO g)
1180
Y e a s t (/g )
P o t a s s i u m ( m g / 1 O O g)
718
S o d i u m ( m g /1 OOg)
250
fl
<10
I
<10
N e g a tiv e
<10
Isoflavone Analysis
All F o r m s *
I
rn g /g
m g /g p ro d u c t
I p ro te in
a s is
a s is
G e n is te in - c o n ta in in g c o m p o u n d s
G e n is te in
D a id z e ln - c o n ta in in g c o m p o u n d s
D a id z e in
G ly c ite in -c o n ta in in g c o m p o u n d s
TO T A L IS O F L A V O N E S
'Aglycones1Glycosides, Glycoside e ste rs
I
m g /g p ro d u c t
A g ly c o n e C o m p o n e n ts
G ly d te in
T o ta l A g ly c o n e C o m p o n e n ts
I
I
m g /g
p ro te in
104
AF1.2 Cl VA70CA 44
Lot # G265-0
Microbiology
Composition
P r o te i n (% )
4 4 .3
A e r o b ic P l a t e C o u n t (/g )
C a lc iu m (m g /1 OOg)
1630
C o lif o r m c o u n t ( M P N /g )
A s h (% )
6 .6 8
E . c o li (M P N /g )
F a t , a d d h y d r o ly s is (% )
2 .3 5
M o ld (/g )
M o is tu r e (% )
2 .3 7
S a l m o n e l l a ( /3 7 5 g )
P h o s p h o r u s (m g /1 OOg)
1290
Y e a s t (/g )
P o t a s s i u m (m g /1 OOg)
1450
S o d iu m (m g /1 OOg)
<10
n r
i
i
i
<3
<3
<10
N e g a tiv e
<10
418
Isoflavone Analysis
All F o r m s '
m g /g p r o d u c t
a s is
m g /g
p r o te in
A g ly c o n e C o m p o n e n t s
a s is
m g /g
p ro te in
m g /g p r o d u c t
G e n is te in - c o n ta in in g c o m p o u n d s
0 .8 7
1 .9 6
G e n is te in
0 .5 1
1 .1 5
D a 'id z e in -c o n ta in in g c o m p o u n d s
0 .6 5
1 .4 7
D a id z e in
0 .3 8
0 .8 6
G ly c ite in - c o n ta in in g c o m p o u n d s
0 .1 4
0 .3 1
G ly c ite in
0 .0 8
0 .1 9
T O T A L IS O F L A V O N E S
1 .6 6
3 .7 4
T o ta l A g ly c o n e C o m p o n e n t s
0 .9 7
2 .2 0
'Agtycones, Glycosides, Glycoside esters
105
APPENDIX G
LDL OXIDATION PROCEDURE
106
LDL Oxidation Procedure
Notes: Gloves should be worn throughout the procedure (when in contact with
bodily fluids).
All solutions are prepared fresh daily unless otherwise stated.
I. Plasma Preparation
1. Collect blood samples in 6ml Vacuette Blood Collecting Tubes (1-2) with EDTA
(purple top) by venipuncture
2. Place tubes into rotor, being sure that they are balanced, and check to ensure that
the nut on top is tight and that the rotor is secure.
3. Close the lid on the Marathon 21000R centrifuge and set parameters at 2500 rpm,
16°C for IOminutes and Start.
4. Aspirate off plasma with Pasteur pipet and place into pcentrifuge tubes, about I
ml in each tube (2-3 tubes)
5. Place in -SO0C Revco freezer for storage (be sure to record in log where samples
are place, box & shelf) or continue on with isolation immediately
II. 3 days before:
Turn on incubator to achieve constant 37°C - KEEP TRACK OF TEMPS!
IV. Night before:
1. Set samples out (10 max) in freezer.for the time points and samples you will
be working with tomorrow
2. Make NaCla (2 solutions) and PBS spin these to mix and set at room temp this only needs to be done once per week (Takes -20-30 minutes to prepare
samples)
a. Prepare 0.9% NaCl solution (weigh 900mg NaCl and IOOmg EDTA in
a piece of weighing paper, transfer to a 125ml erlenmeyer flask with
the aid of 100ml of deionized (DI) water, add a small stir bar, stir on a
magnetic stir plate to dissolve); need 1A to I ml per sample
b. Prepare the 16.7% NaCl solution (weigh 16.7g NaCl and IOOmg
EDTA on a piece of weighing paper, transfer to a 125ml erlenmeyer
flask with the aid of 100ml of DI water, add a small stir bar, stir on a
magnetic stir plate to dissolve, this solution may take a long time to
dissolve); need 1A to I ml per sample [soln good for I week]. Will
take about an hour to dissolve
c. Prepare PBS buffer (St. Lukes procedure): 22.8mg (.228 g) Sodium
Phosphate Monobasic + 115mg (1.15 g) Sodium Phosphate Dibasic +
935mg (9.35 g) Sodium Chloride + 90ml DI water in a 100 ml
volumetric flask, add stir bar and stir on magnetic stir plate to dissolve,
107
pH 7.4 (adjust with NaOH or HCl if necessary), add DI water to
volume (*100 ml of PBS is required for each 1-2 samples) [soln
good for I week]. Use 1000 ml beaker and MULTIPLY ALL BY 10
TO MAKE I L.
V. Day of;
1. Remove samples from -SO0C freezer (if necessary) and allow to defrost. To
defrost the samples takes about 15-20 minutes (Note: it helps to defrost
samples quicker if you hold them in your hand!!)
2. Turn on centrifuge in comp lab
3. Label first set of Beckman tubes for VLDL and LDL isolation step
4. Spin to mix PBS and NaCl2 mixtures - if needed
5. Turn on plate shaker, plate reader, and computer
VI. Isolation (ref. Lipoprotein Separations Using TL-100 Tabletop Ultracentrifuge, H.K.
Naito) Takes approximately 6 hours
1. To Beckman Centrifuge Tubes (Polyallomer, 7/16x1 3/8 in; reorder # 347287)
add 500/d of the 0.9% NaCl solution and 500/d of plasma (EDTA); repeat this
step into a second centrifuge tube for each sample (to do in duplicate)
2. Centrifuge (Door-Enter/Display-Start if on correct program) at 100,000 rpm for
2.5 hours at 16°C in the Beckman Optima TLX Ultracentrifuge with a Beckman
TLA 120.2 Rotor (S.N. 94U635; 120K RPM) [Door (to open),Enter/Display,
Start (if on correct program)]
3. When step 2 has been completed, remove centrifuge tubes (view with a black
background, the top layer should be cloudy/white) and slice with the Beckman
Centritube Sheer at approximately the 500 /d mark (determine this by pipetting
5OOul on 0.9% NaCl into an empty centrifuge tube and using that tube to set up
the sheer), discard the top layer (VLDL layer) in a biohazards box and transfer the
bottom layer (LDL and HDL layer) to a new centrifuge tube with a Pasteur pipet
(mix the viscous bottom by carefully drawing it up into the pipet and expelling it,
repeat 3-4 times), pipet in 500 fiL of the 16.7% NaCl solution. Wash sheer with
water.
4. Centrifuge again as in step 2 of Isolation
5. Label microcentrifuge tubes for Isolated LDL, Desalted LDL, and Adjusted LDL.
6. Wash centrifuge sheer with KimWipe and water
7. When last centrifuge has approximately 20 mintues take the caps off of EconoPac columns
8. When step 4 has been completed (view centrifuge tube with a white background,
it should have a yellow/gold top layer), slice centrifuge tube and remove top
500/d (LDL layer), make a composite of the 2 centrifuge tube’s top layers for
each sample and place in a microcentrifuge tube and cover, discard the bottom
108
layer in a biohazards box; The LDL has now been isolated. Wash centrifuge
sheer with water and KimWipe
9. MIX the isolated LDL thoroughly!
While samples are spinning (either during spin I or 2):
I . Prepare Working Reagent (WR) for the protein assay: using the Pierce BCA
Protein Assay Kit (23225) mix 50 parts of BCA reagent A with I part of BCA
reagent B keeping in mind that 200/d is required per well used (solution can
be stored for I day at room temperature)
a. Ex: 8 tubes of standard + blank (both in duplicate) =18
10 ml reagent A = 10000 ql = 200 pi reagent B (Seal with parafilm)
50
2. Prepare standard solutions with bovine serum albumin (BSA) (in hands box)
standard provided according to Pierce table I by pipeting into separate
microcentrifuge tubes and mixing :
Label
Concentration fue/ml)
Volume BSA
Stock
2000
3OOpl stock
A
1500
3OOpl stock
B
1000
IOOpl stock
C
750
175pl A
D
500
IOOplB
E
250
IOOplD
F
125
IOOplE
G
50
IOOplF
* Remember to mix THOURGHLY!!!
* Empty glass contents into pcent tube (this is the stock)
Volume PBS ful)
0
100
100
175
100
100
100
400
VII. Desalting (ref.Econo-Pac 10DG Columns Manual #732-2010; Palomaki et al,
Journal of Lipid Research v39, 1998, 1430-7))
1. Add < 3ml of isolated LDL to the column (350-450/d), allow to enter column
2. Add PBS to achieve a total volume of 3ml (3000 pi):
3ml - volume of isolated LDL = volume of PB S to be added to the column
Ex: 3000 - 450 = 2550 pi of PBS to be added to the column
3. Use a 10ml graduated cylinder to measure the first 3ml of effluent and discard
(solution should stop flowing at about this point)
4. Add [1.5 X volume of isolated LDL] to elute the higher molecular weight
components, collect in test tube and cover, MIX
Ex: 1.5x450 = 675 pi PBS
5. STORE COLUMN IN PBS, REPLACE CAP AND TIP, PUT SAMPLES IN
FRIG
6. Turn on plate reader and allow to warm up (~30 minutes)
109
I x - Protein Concentration (ref. Pierce BCA Protein Assay Kit 23225, microwell plate
protocol)
I. Pipet 25/xl of each of the standard solutions from step I (stock-G), sample
solutions(isolated and desalted LDL) and blank solutions (PBS) into separate
wells of a 96 well Nunc microwell plate; Prepare in duplicate (2 wells for
each!) Ex:
I
A PBS
B “
C Stock
D “
E A
F “
G B
H“
2. Add 200jttl of WR (from step 8b of Isolation)to each well
3. Shake plate for 30 seconds on a plate shaker
4. Cover plate with a plate sealer (Seal Plate NonSterile 100-THER-PLT, Excel
scientific 760-249-6371)
5. Incubate for 30 minutes at 37°C in plate shaker/incubator
6. Prepare Cupric Chloride Dihydrate solution (weigh 85.2mg [.0852 g] on
weighing paper and transfer into a 100ml volumetric flask with the aid of DI
water, bring solution to 100ml volume with DI water and shake to dissolve
(stock solution), dilute I ml of this stock solution plus 9 ml DI water and mix
(this is the Cupric Chloride Dihydrate solution for the LDL oxidation, part V)
.PREPARE DAILY
7. Allow plate to cool to room temperature
8. KC Junior: START - PROGRAM - KC JR - OK
9. Read absorbance at 562 nm on KC Junior plate reader (p. 6-10 of User’s
Guide)
10. Open existing protocol for protein assay by going to Protocol, then Open
Protocol, then selecting BCAprotein
11. Once in the BCAprotein (ST-protein )protocol, open the Modify Protocol, go
to Read Method and check to make sure that the primary wavelength is
correct and that the plate geometry encompasses the wells that you need to
read (if not go to Read and change the first and last locations or click to have
• it read the full plate)
12. In the Modify Protocol screen, go to template and enter in your Well ID’s; the
concentrations of the standard solutions can also be entered at this time (or
after the read)
13. Click OK to exit the Modify Protocol Screen
no
14. Click Read Plate to initiate reading (you can enter a results ID at this time or
at a later time), the dialog will prompt you to place the plate on the reader and
press OK
15. When the read is Complete, results are automatically displayed
16. Want a R2 = .99 or .98
17. Print Std. Curve and Results
18. To save results, select Results/Save Results (enter the results ID at this time if
it was not done earlier), click OK to save results
X. LDL oxidation (ref. Esterbauer et al, St.Lukes protocol)
1. Using the results of the protein assay, dilute (with PBS) the samples of isolated
and desalted LDL to achieve a concentration of 0.10 mg protein/ml tor I PO
jug/mf) from column report:
Calculation of Dilution (using 200/11 of the isolated, desalted sample):
/A PBS = sample concentration in /xg/ml x 200ul sample
100 /tg/ml
o
- 200 jil sample
Change the # of fil sample in both places, if 200 /d is not the desired
amount
o Change final concentration if 100 p,g/ml is not desired
° Use the sample concentration from the protein assay
o Note: 1000 /ig = I mg
Ex: D l =353.6; D2 = 242.1
(353.6x2001 - 200 = 507 pi PBS for Dl
(100)
(242.1 x 2001 - 200 = 284 pi PBS for D2
(100)
2. Dilute as calculated above into microcentrifuge tubes and mix : 200 pi sample +
_pl PBS to dilute. Label these new tubes with an A
3. Read on KC Junior plate reader using existing LDL oxidation (ST-LDLox)
protocol at a wavelength of 234 nm and at a temperature of 37°C
a. Open existing protocol for LDL oxidation by going to Protocol, then Open
Protocol, then selecting LDL Ox.
b. Once in the LDL Ox protocol, open the Modify Protocol, go to Read
Method and check to make sure that the primary wavelength is correct and
that the plate geometry encompasses the wells that you need to read (if not
go to Read and change the first and last locations or click to have it read
the full plate)
c. In the Modify Protocol screen, go to template and enter in your Well ID’s
d. Click OK to exit the Modify Protocol Screen
Ill
e. Click Read Plate to initiate reading (you can enter a results ID at this time
or at a later time), the dialog will prompt you to place the plate on the
reader and press OK
4. Pipet 100/d of each LDL sample solution (isolated, desalted and adjusted for
protein concentration) into separate wells of a Costar 96 well UV flat bottom plate
(370/d, 3635); prepare in duplicate. DO QUICKLY!
5. Pipet 10/d of the Cupric Chloride Dihydrate solution (step 8d of Isolation) into
each well; seal with plate sealer. DO QUICKLY!
6. Run KC Junior
a. When the read is complete, results are automatically displayed
b. To save results, select Results/Save Results (enter the results ID at this
time if it was not done earlier), click OK to save results
c. The slope of the propagation phase can be determined by: selecting Open
Results, selecting Kinetic Curve under the Kinetic View Options, clicking
on the well of interest to enlarge, determining the points included in the
propagation phase and clicking OK to close. Then, click the Calculations
Options to open the Kinetic Calculations dialog, the first and last read
points can then be changed to encompass the points of interest only, the
slope will automatically recalculate based on the new range
d. Protocol - save protocols
7. Write down temperature can leave @ read 3, keep checking and logging temp
VI. Data;
^
1. Enter values into Excel program via KCjr->Excel Columns Directions (attached)
2. To Copy the column:
a. Select cells and copy to clipboard
b. Open in “lag & prop #2” template in Excel and paste (click on first cell
and paste)
3. Record lag time and propagation rate; print data and graphs
a. Lag phase and propagation phase duration
i. Longer = better
b. Maximal oxidation rate (measured within propagation phase)/maximum
dienes
112
APPENDIX H
STATISTICAL DATA
113
General Linear Model
Withln-SubJects Factors
Measure: MEASUREJ
TIMEPTS
1
2
3
4
TRTMNTS
1
2
1
2
1
2
1
2
Dependent
, Variable
T0BLK858
T0BLK46.1
T2BLK858 ,
T28LK46.1
T4BLK858:
T4BLK481
T6BLK858
T6BLK461
Multivariate Testsb
Effect
TlMEPTS
TRTMNTS
TIMEPTS ‘
TRTMNTS
Pillai's Trace
Wilks' Lambda
Hotellingls Trace
Roy’s. Largest Root
Pillai's Trace
Wilks' Lambda
Hotelling's Trace
Roy’s largest Root
Pillai's Trace
Wilks' Lambda
Hotelling's-Trace
Roy's Largest Root
Value
,042
,958 I
,044
.044
.020
.980
.020
,020
.266
.734
.36.3
.363.,
F
.176'
.1.76'
.176''
,176'
.281a
281*
28 1 '
261*
1.450*
1.450'
1.450=
1.450*
a, Exact statistic
b,
"
Design: Intercept
Within Subjects Design: TIMEPTS+TRTMNTS+TIMEPTS'TRTMNTS
Hypothesi
sdf
3.000
3:000
3,000
3.000
1.000
1.000
1.000
1.000.
3.000
3,000
3.000
3.000
Error df
12; 000
12.000
1'2,0Q0
12:000 1
14.000
1.4.000
14.000
14.000
12.000.
12.000
12.000
12.000
Sig..
.911
.911
.911'
,911
.604
.604
.604
,604
.277
.277
.277
277
114
M a u c h ly 's T e s t O f S p h e r I c i t y b
Measure; MEASUREJ-
Epsilona
Within Subjects Effect
TIMEPTS
TRTMNTS
'TIMEPTS * TRTMNTS
Mauchly's
W
.450
1,000
Approx,
Ghi-Squar
e
10.149
.000
.591
-6.693
50
Graerihou
se-Gelsse
Sig.—’-px
T
Z .072 )
.650
1.000
5
)
df
,732
Huynh-Fet
. dt
.754
1:000
.873
Lower-bou
•nd
,333
1,000,
.333
115
T e s t s o f W ith in - S u b J e c ts E f f e c t s
Measure: MEASUREJ
Source
TIMEPTS
Error(TIMEPTS)
TRTMNTS
- Error(TRTMNTS)
' TIMEPTS *
TRTMNTS
Error(TIMEPTS‘TRT
MNTS)
Type III
Sum of
Smiates
Sphericity Assumed ( . 3.855E-Q4
)
Greenhouse-Geisser
3.865E-04
Huynh-Feldt
3.865E-04
Lower-bound
s.ms.E&t
Sphericity Assumed CJ,,798E-12>
Greenhouse-Geisser
1.798E-02
HUynh-FeIdt
1.798E-02
Lower-bound
1.798E-02
SjDhericity Assumed ( ''1.200E-04';
»
Greenhouse-Gelsser
Huynh-Feldt
t.200E-04
Lower-bound
I JilOE=Qd ■
SphericityAssumed <^.984E-03j
Greehhouse-Geisser
5.984E-03
Huynh-Feldf
S.984E-03
Lower-bound
5.S84E-03
Sphericity Assumed
OlOSE^OSj
Greenhouse-Gelsser
1.108E-03 .
Huynh-Feldt
1.108E-03
Lower-bound
J.J.08E=03
SphericityAssUmed C,
GreenhoUse-Geisser
1.257-E-02
HuynIrFeldt
1.257E-02
Lower-bound
1.257E-02
JcSSTE1O^
Mean
Square
^T:288E-04>
1:951 1.981E-04
2.263 1.708E-04
1.000 3.865E-04
df
cu;
CdC
27.316
31.682
14.000
CC
1.000
1.000
1.000
CC
14:000
14.000
14.000
F_._
Sig._
)
.301
.301
.301
.737
.768.592
C O
.281
.281
.281
604
604:
1.234
1.234
1.234
,309
,285
6.582E-04
5.675E-04
1.284E-03
1.200E-04
1.200E-0412C.0E44
%274E-0_4)
4.274E-04
4.274E-04
4.2Z4E=04
5.047E-04
2.619 - 4.229E-04
1.000 1.108M3
C O ^9935-34,
30,726 4.O91E-04
36:669 3.428E-04
14.000 8.978E-04.
116
T e s t s o f W i th in - S u b je c ts C o n t r a s t s
M e a s u re : M E A S U R E _1
S o u rce
T IM E P T S
T IM E P T S
L in e a r
E rrd r(T IM E P T S )
TRTM N TS
Txp'e III
S u m of
S q u a re s
2.-12 4 E - 0 4 ,
Q u a 'd fa tlc
1 :6 5 7 E -0 4 .
C u b ic
'8.402E-Cii3'
L in e a r
' 7.2-1 SE -03,
M ean
S q u a re .
,df
2 .1 2 4 E - 0 4 ,
1
1 .6 5 7 E -0 4 .
.281
I : 8 .4 3 2 E -C 3 '
145 .1 5 7 6 - 0 4
.0 4 7
Q u a d ra tic
8.25 5 E -:0 3
14
5 .8 9 7 E - 0 4
C u b ic
2 .5 0 4 E -0 3 '
. 1.2Q,0E-04
4 .2 7 4 E -0 4 .
TRTM NTS
L in ea r
1 .2 Q 0 E -0 4
14
1
E rrd r(T R T M N T S )
L in ea r
-5 .9 8 4 E -0 3
14'
S iq.
.531
.4 1 2
. .604;
.8 3 2
1 .7 8 9 E r 0 4 "
.281
„6 0 4
.4 0 0
T IM E P T S *■
L in e a r
L in ea r
4 .1 3 3 E - 0 4
1
4 . 1 3 3 E -0 4
.7 5 4
T R l MN I S
Q u a d ra tic
L in ea r
6M 6 '5 E -0 4
'1
'6 .1 6 5 E - 0 4
3 .4 2 3
.0 8 6
C ubic-
L in ea r
7 .7 7 6 E -0 5
.4 5 8
.5 1 0
' 7 .7 7 6 E -0 5
E rro r(T IM E P T S * T R T
L in e a r
M NTS)
;
1
. L in ea r
7 .67 1 E -0 3
14
Q u a d r a tic
L in ea r
2 .5 2 2 E -0 3
14. ' 1 .8 0 .1 5 -0 4
C u b ic
L in e a r
2 .3 7 6 E -0 3
14
5 .4 .7 9 5 -0 4
1 .6 9 7 E -.0 4
T e s ts o f B e tw e e n -S u b je c ts E ffe c ts
.
F
1
M e a s u re : M ,EASU RE_1
T r a n s fo r m e d V a ria b le ; A v e ra g e
i S o u rce
: I n te rc e p t
E rro r
T y p e III
S u m of
S q u a re s
df
!
M ea n
S q u a re
.5 9 7
1
.597'
4 .9 5 4 E -0 2
1:4
. 3 .5 3 9 E -0 3
F
1 6 8 .7 4 2
S ig .
.0 0 0
1
General Linear Model
V V ith in -S u b je c ts F a c t p r s
M e a s u re : M E A S U R E J
T IM E P T S
1
2
.3
D e p e n d e n t.
V a ria b le
TRTM NTS
1
T2848PRD
2
T 2 4 6 IP R D
1
Y 4848PR D
2
T4461PRD
1
T6848PRD
2
T6461PR D
D e s c r ip ti v e S t a t i s t i c s
M ean
S td .
D e v iatio n
N
: T2848PR df
6 .'8 3 3 E -0 5
'5 .7 5 9 6 - 0 4 '
15
T2461PR df
-1 .4 8 E -0 4
4 .6 7 2 E - 0 4
15
T4848PRdf
-2 .6 7 E -0 5 '
5.877,E -0 4
15
T4461PR df
-2 .1 3 E -0 4
3 .5 6 2 E -0 4
15
T6848PRdf
-9 .3 3 E -0 5
4 .3 4 9 E -0 4
1.5
T6461PRdf
-1 .0 3 E -0 4
6 .5 1 8 E -0 4
15
118
M u ltiv a r ia te T e s f s b
E ffect
T IM E P T S
TRTM NTS
T lM E P T S *
TRTM NTS
H y p o th e si
sdf
F
. V alu e
P illai's T r a c e
.0 4 8
,3 2 8 °
2 :0 0 0
,E h ro rd f
1 3 .0 0 0
W ilks' L a m b d a
.9 5 2
.3 2 8 °
2 .0 0 0 '
1 3 ,0 0 0
H o tellin g 's T r a c e
.051
,3 2 8 ° '
2 .0 0 0
1 3 ,0 0 0
R o y ls L a rg e s t R o o t
.051
.3 2 8 °
2 .0 0 0
1 3 .0 0 0
S lg-i
.m
P illai's T r a c e
.0 6 9
1 .0 8 9 °
1 .0 0 0
1 4 ,0 0 0
.32'S
W ilk s' L a m b d a
.931
1 .0 3 9 °
1 .0 0 0
1 4 .0 0 0
.sil
.3 #
H o te llin g 's T ra c e
.0 7 4
1 .0 3 9 °
1 ,0 0 0
1 4 .0 0 0
R o y 's L a rg e s t R o o t
.07 4
1 .0 3 9 °
1 .0 0 0
1 4 .0 0 0
P illai's T r a c e
.0 9 8
.7 0 6 °
2 .0 0 0
1 3 ,0 0 0
m
W ilks' L a m b d a
.9 0 2
.7 0 6 °
2 .0 0 0
1 3 .0 0 0
'Ir
H o te llin g 's T r a c e
.1 0 9
.7 0 6 °
2 .0 0 0
1 3 .0 0 0
R o y 's L a rg e s t R o o t
.1 0 9
.706=
2 .0 0 0
1 3 .0 0 0
,d 1
ili
a . E x a c t s ta tis tic
b.
D e sig n : In te rc e p t
W ithin S u b je c ts D e sig n : T IM E P T S + T R T M N T S + T IM E P T S T R T M N T S
M a u c h ly 's T e s t o f S p h e r i c I t y b
M e a s u re : M E A S U R E _1
E p s ilo n a
W ithin S u b je c ts E ffect
T IM E P T S
TRTM NTS
T IM E P T S ‘ TRTM N TS
M a u c h ly 's
W
.8 5 5
1 .0 0 0
.9 7 7
A pprox,
C h i-S q u a r
e
2 :0 3 8
• G re e n h o U
s e - G e ls s e
— ^
r
■df
2
.0 0 0 ,
0.
.3 0 4
2
SjgT
Z .3 5 8 )
1
H u y n h -F e l
dt,
L o w er-b o u
nd
.8 7 3
.9 8 7
.500'
1 .0 0 0
1 .0 0 0
1 .000
.9 7 7
1 .0 0 0
.5 0 0
119
T e s t s o f 'W i th in - S u b je c ts E f f e c t s
M e a s u re : M E A S U R E J
T y p e III
Sum of
^ S q u a re s
S o u rce
YlMfeP T S
S p h e ric ity A s s u m e d
-1.027E-0Y )
G r e e n 'h o u s e - G e is s e r
H u y n h -F e ld t
L o w e r-b o u n d
E rro r(T IM E P T S )
S p h e r ic ity A s s u m e d
Y sF
1 .0 2 7 E -0 7
1.975
..1 .0 2 7 E -0 7
I . OtiO1
M ea n
—S q u a r e
ms
1..-027.E-Q7
.2 6 9 '
.6 1 2
■5.346E-06
2 4 .4 5 2
5 .3 4 6 E -0 6
5 .3 1 6 E l0 6 .
2 7 .6 4 8
1 ,9 3 4 E -0 7
1 4 ,0 0 0
3 S j.9 E ^ Q I
i U jllE rO T )
.7 3 7
.7 6 3
H u y n h -F e ld t
S p h e ric ity A s s u m e d
S iq,
.2 6 9 -
< = 2 8 : (le o e g a z ;
.
F
5 .2 0 2 E -0 8 .
G re e n h o u se -G e lss e r
2 . 1 8 6 E -0 7
Cu l o s I 1
C 32& ,
G r e e n h o u s e - G e is s e r
4 .2 7 1 E -0 7
P S 5
4.2-71 E -0 7
1 .0 3 9
.3 2 5
H u y n h -F e ld t
4 .2 7 1 E -0 7
1 .0 0 0 '
4 .2 7 1 E -0 7
1 .0 3 9
.3 2 5 ■
,.42-7-1 E -0 7
1 .0 0 0
4 .2 7 J .M 7
1 .0 3 9
.3 2 5 .
1 4 .0 0 0
4 .1 1 2 E -0 7
1 4 .0 0 0
4 .1 1 2 E -0 7
1 4 .0 0 0
A1I lZ f c O J
L o w e r-b o u n d
E rro r(T R T M N T S )
,T 027& 07
f r5 .3 4 6 E .-fi6
L o w e r-b o u n d
TRTM NTS
df
S p h e ric ity A s s u m e d
,
G r e e n h o u s e - G e is s e r
H u y n h -F e ld t
Lpw er-bdurici
5 .7 5 7 E -0 6
5.75.7EiD 6
c P -8 7 -1 E - O f )
CS:
TIM EPTS"*
S p h e r ic ity A s s u m e d
TRTM NTS
G re e n h o U s e - G e is s e r
1 .871 E -0 7
1.955
H u y n h -F e ld t
1.871 E -0 7
L o w e r-b o u n d
j-B 71.E -Q .7_
2.000
1.000
!
C g ze>
.’8 7 6
,4 2 6
9 .3 5 3 E -0 8
.8 7 6
.4 2 8
! jU 8 J 4 E .0 Z
.8 7 6
.3 6 5
D ^ 2 8 ] ^ 1 .0 6 8 E - 0 7 ' )
E rro rL T IM E P T S T R T
S p h e ric ity A s s u m e d
W hlTS)
G r e e n h o u s e - G e is s e r
2 .9 9 0 E -0 6
H u y n h -F e ld t
2 .9 9 0 E -0 6
2 8 .0 0 0
1 .0 6 8 E -0 7
L o w e r-b o u n d
2 .9 9 0 E -0 6
1 4 .0 0 0
2 .1 3 6 E -0 7
-2 7 .3 6 7
sT T O S -Z fcS T
120
T e s t s d f 'V V ith ln -S u b Jec ts C o n t r a s t s
M e a s u re : M E A S U R E ,.!
S o u rce
T IM E P T S
T IM E P T S
L in e a r
TRTM NTS
■ Q uadratic
E rro r(T IM E P T S )
T y p e III.
S u m of
S q u a re s ..
M ean
S q u a re
df
F
Slqt
'5.1 OA1E-OS i
1
5 .4 0 4 E -0 8
'. 1 9 6 . 1
5 .!6 '8 E -:0 8'
I
.5 .!6 8 E -0 8
.4 2 3
.6 6 4
.526
: 2 .5 9 8 E -0 7
!.0 3 9 ’
.325
L in ea r
1 3 .6 3 8 E -0 6
!'4
Q u a d ra tig
: !J O O E -O S .
1'4-
!.2 2 0 E -Q 7
TRTM NTS
L in ea r
4.2 7 1 .E -0 7
1
4.27.!'E-0%
E rrd r(T R T M N T S )
L in ea r
5 .7 5 7 E -0 6
U
. 4 U 4 2 E -0 7
L in e a r
Q u a d ra tic
L in ea r
L in ea r
T 6 0 2 E -0 7
I
!.6 0 2 E - 0 7
! .3 6 4
2 .6 8 9 E -0 8
1
2 .6 8 9 E -0 8
_ .2 8 0 '
E rror(T IM E P T S *T R T
L in e a r
L in ea r
!,6 4 4 E - 0 6 '
!:4,
MN* S j
Q u a d ra tic - L in ea r
1 .3 4 6 E -0 6 :
!4
T IM E P T S *
I R l MN I S
T e s ts o f B e tw e e n -S u b je c ts E ffe c ts
M e a s u re : M E A S U R E ,.!
T ra n s fo rm e d V a ria b le : A v e ra g e
S o u rce
T ypetlll
S um .'of
S d u a re s
, In te rc e p t
6 .6 7 4 E -0 7
.
E rro r
-
M ea n
S q u a re
df
I 1 ■ 6.674E -07
8 .8 ! 4 E - 0 6
.
!4 .
6,2 9 5 E r.0 7
Estimated Marginal Means
G ra n d M ean
M e a s u re : M E A S U R E .,!'
9 5 % C o n fid e n c e In terv al ,
M ean
-8 .6 ! l'E -0 5
S td . E rro r I
.0 0 0
'L o w er
B ound.
U pper
B ound
-2 .6 5 5 E -0 4
. 9 .3 2 7 E r0 5 -
F
! .0 6 0
S ig.
.321 '
! .! J 4 E - 0 7 .9 :6 !d E -.0 8
.2 6 2
.6 0 5 '
General Linear Model
W ith in -S U b J e c ts F a c t o r s
M e a s u re : MEASURE__1
T IM E P IS I
2
3
trtm nts
D ependent
V a ria b le
1
T2848LTD
2
T2461L TD
1
T4848L TD
Z
T4461L TD
1
I6 8 4 8 L T D
2
T6461LTD
'
D e s c rip tiv e S ta tis tic s
M ean
S td .
. D ev iatio n
N
T2848L Tdf
-2 .3 7 6 7
4 ,8 4 1 7
15
T 2 4 6 IL T d f
-.■5367
8 .0 6 7 3
15
15
T4848LTdf
7 2.09 3 3
4 .0 7 5 3
T 4 4 8 1 LTdf
- 1 .3 3 6 7
1 1 .3 6 4 3
15
T6848L Tdf
-1 .5 5 6 7
5 .1 7 2 9
15
T6461L Tdf
-.9 3 6 7
7 .3 0 2 4
15
122
M u ltiv a ria te T e s t s b
E ffect
T IM E P T S
TRTM NTS
T IM E P T S *
TRTM NTS
V a lu e
P illai's T ra c e
F
.0 2 3
H y p o th e si
.s d f
.1 559 I
2 .0 0 0
E rro r df
1 3 .0 0 0
W ilks' L a m b d a
.977
.1 55«
2 .0 0 0
H o te llin g 's T r a c e
.0 2 4
.1 5 5 "
2 .0 0 0
1 3 ,0 0 0
R o y 's L a rg e s t R o o t
.0 2 4
.1 5 5 "
2 .0 0 0
1 3 .0 0 0
S Ig l l K
■ #
■
'
1 3 .0 0 0
Pillai's T r a c e
.0 1 5
.2 1 9 "
1 :0 0 0
1'4:000
W ilks' L a m b d a
.98 5
.219=
1 .0 0 0
1 4 .0 0 0
.6 #
H o te llin g 's T r a c e
.ore
.2 1 9 "
1 .0 0 0
1 4 .0 0 0
R o y 's; L a rg e s t R o o t
.0 1 6
.219=
1 .0 0 0
1 4 .0 0 0
Pillai's T r a c e
.0 6 9
.4 8 1 "
2 .0 0 0
1 3 .0 0 0
.eib
W ilk s' L a m b d a
.931
.4 8 1 "'
2 .0 0 0
1 3 .0 0 0
.6 $
H o te llin g 's T r a c e
.0 7 4
.4 8 1 "
2 .0 0 0
1 3 .0 0 0
.6 2 9
R o y 's L a rg e s t R o o t
.0 7 4
.481=
2 .0 0 0
1 3 .0 0 0
.elb
mi
A
a , E x a c t s ta tis tic
b.
D e sig n : In te rc e p t
W ithin S u b je c ts D e sig n : T IM E P T S + T R T M N T S + T IM E P T S 'T R T M N T S
M a u c tily 's T e s t O f S p h e r I c i t y b
M e a s u re : M E A S U R E _1
E p silo n "
W ithin S u b je c ts E ffe ct
T IM E P T S
TRTM NTS
T IM E P T S "T R T M N T S
A pp ro x .
C h i-S q u a r
e
.451
10.346.
2
G re e n h o u
s e - G e is s e
r
S ig 1— ^
/
.0 0 6 )
.6 4 6
1 .0 0 0
.0 0 0
0
1 .0 0 0
.372
1 2 .8 4 0
2
M a u c h ly 's
W
df
j/^ 0 0 2 ^ )
.6 1 4 '
H u y n h -F e l
dt
.6 8 3
, L o w er-b o u
nd
.500
1 ,0 0 0
1 .0 0 0
.6 4 3
.5 0 0
123
T e s t s O f W ith I n - S u b je c ts E f f e c t s
M e a s u re : M E A S U R E _1
T y p e III
S u m of
S q u a re s
S o u rce
lTlM fePTS
S p h e ric ity A s s u m e d
3 .3 0 2
G r e e n h o u s e - G e is s e r
3#%
M ean
S q u a re
df
1.651
.0 5 8
1.291
2 .5 5 7
.0 5 8 '
H u y n h -F e ld t
L o w e r-b o u n d
E rro r(T IM E P T S )
S p h e ric ity A s s u m e d
G r e e n h o u s e - G e is s e r
H u y n h -F e ld t
L o w e r-b o u n d
TRTM NTS
3 .3 0 2
1 .0 0 0
28
2 8 .2 7 7
1 8 .0 7 8
_4& Z96
7 9 1 .7 5 1
S p h e ric ity A s s u m e d
G r e e n h o u s e - G e is s e r
L o w e r-b o u n d
G r e e n h o u s e - G e ls s e r
H u y n h -F e ld t
L o w e r-b o u n d
T IM E P T S 1
S p h e ric ity A s s u m e d ,
TRTM NTS
G r e e n h o u s e - G e ls s e r
E rro r(T lM E P T S * T R T
S p h e ric ity A s s u m e d
M N T S)
G r e e n h o u s e - G d is s e r
.
5 6 .5 5 4
1
2 5 .8 6 7
.2 1 9
.6 4 7
__ 2 5 .fi6 7
1 .0 0 0
2 ff8 6 7
.2 1 9
.6 4 7
1 .0 0 0
2 5 .8 6 7
.2 1 9
.6.47
14
1 1 7 .9 6 5
• ,1 5 2
.8 6 0
c m : C & gg)
1 6 5 1 .5 1 3
uaoo
.„ 1 6 5 - 1 ^ 1 3
C
. 1651.51-3
1 4 .0 0 0 ,
6 .7 0 2
J L tZ A S L
)■
1 1 7 .9 6 5
3.351.
.1 5 2
)
. .q g >
6 .7 0 2
6 1 6 :8 7 9
28
J jS iffZ S
1 7 .2 0 4
6 1 6 .8 7 9
1 '4.000
2 2 .0 3 1
H u y n h -F e ld t
L o w e r-b o u n d
46=
14;Q 00
H u y n h -F e id t
L o w e r-b o u n d
.8 7 0
2 5 .8 6 7
2 5 .8 6 7
S p h e ric ity A s s u m e d
3 .3 0 2
S ip .
.943-
1 cS H D C j iM
H u y n h -F e ld t
E rror(T R T M N T S )
c a
"791.75-1
7SU §1
C T y g iT S iv
F
2
eVfit-
Cm.7 0 2
124
T e s t s o f W ith ln - S u b je c ts C o n t r a s t s
M e a s u re : M E A S U R E J .
S o u rce
T IM E P T S
T IM E P T S
L in e a r
T y p e III
S u m of
S q u a r e s ,.
TR T M N T S
.6 6 2
Q u a d ra tic
E fro r(T IM E P T S )
.
L lg e a f
.24.9.982
5 4 1 .7 5 9
JR T M N T S '
L in ea r
'E rror(T R T M N T S )
L in ea r
'
1 6 5 1 .5 1 3
i
5 .5 ,8 2
T IM E P T S *
L in e a r
L in e a r
TRTM NTS
Q u a d ra tic
L in ea r
Error(,TIM E PTS *TR T
M N T 5)
, L in e a r
.
1 ■
SigifO
-Q u a d ra tic
.
M ean
S q u a re
df
I
.
2 5 ,8 6 7
1 .1 2 0 '
..
I ,
'1
5 :5 8 2
..9 5 5 :
..345'
1 .1 2 0
,0 2 9
.8 6 7 "
5 :8 4 3
L in e a r
5 3 5 .0 7 7
1:4
T r a n s fo r m e d V a ria b le : A v e ra g e
E rro r
1 9 5 .2 1 7 '
1
195.2.17
1 3 4 0 .9 2 7
14
9 5 ,781
Estimated Marginal Means
G ra n d M ean
M e a s u re : M E A S U R E J
9 5 % C o n fid e n c e In terv al
M ean
-1.-473
■Std. E rro r
1032
lo w e r
B ound
-3 :6 8 5 ,
.
U pper
B ound’
.7 4 0
F.
2 .0 3 8 ■
.6 4 7
1 1 7 .9 6 5 ,
14
M ean
•S q u a re
.21.9.
. 2 5 .8 6 7 '
14-
.81,,802
df
.798
1 7 .8 5 7
T e s ts o f B e tw e e n - S u b je c ts E ffe c ts
S o u rce
In te rc e p t
.0 6 8
3 8 .6 9 7 :
M e a s u re : M E A S U R E _1
T y p e III
S u m of
S q u a re s
.8 5 0
2 .6 4 0
1.4
lin e a r
Q U ad ratib
S ip.
.0 3 7
14 '
I -.
F
.6 6 2
S ig ..
.1 7 5
I
3 8 :2 2 0 .
General Linear IVlodeI
W lth I n - S u b je d ts F a c t o r s
M e a s u re : M E A S U R E D
T IM E P T S
1
2
3
4
'
TR T M T S
1
D ependent
V a ria b le
T0848PR
2
T0461PR
1
T2648PR
2
T 2461.P R
T
T4848PR
2
T446TPR
1
T6848PR
2
T6461PR
'
D e s c r ip ti v e S t a t i s t i c s
M ea n
6 td .
D eviation
T0848PR
4 ,1 OZErOS-
6:'9'38E -04
T046.1 P R
4 .1 0 8 E -0 3
' 6 .8 5 0 E -0 4
N
18'
15
T2848PR
4."1'70E-03
'8 .0 4 2 2 -0 4
15
T2461PR
3 .9 6 Q E -0 3
8 .2 5 2 E -0 4
15
T4848PR
4 .0 7 5 E -0 3 -
8 .1 9 5 E -0 4
15
T4461PR
3 .8 9 5 E -0 3 .
6 .8 2 8 E -0 4
15
T6848PR
' 4 .0 0 8 E -0 3
5 .6 4 3 E -0 4
T6
T6461 PR
4 .0 0 5 E -0 3 .
7 .3 T 0 E r0 4
15
126
M u ltiv a ria te T e s t s b
E ffect
I I iV If c r
V alu e
S
TRTM TS
TIM EPTS-*
TK T M T S
H y p o th e si
Sdf
F
E rro r df
S iq.
P illars T ra c e
.1 6 3
.777=
3 .0 0 0
1 2 ,0 0 0
.5 2 9
W ilks' L a m b d a
.8 3 7
.7 7 7 a
3 .0 0 0
1 2 :0 0 0
.5 2 9
H o tellin g 's T ra c e
.1 9 4
.7 7 7 a
3 .0 0 0
1 2 .0 0 0
.5 2 9
R o y 's L a rg e s t R o o t
.194
.717*
3 .0 0 0
1 2 .0 0 0
.5 2 9
P illai's T r a c e
.041
.5 9 7 a
1 .0 0 0
1 4 .0 0 0
.4 5 2
W ilks' L a m b d a
.959
1 .0 0 0
1 4 .0 0 0
.4 5 2
1 .0 0 0
1 4 .0 0 0
.4 5 2
1 .0 0 0
1 4 .0 0 0
.4 5 2
H o tellin g 's T r a c e
.043
.557=
.597=
R o y 's L a rg e s t R o o t
.0 4 3
.5 9 7 a
Pillai's T ra c e
.202
T .0 1 2 9
3 .0 0 0
1 2 .0 0 0
.421
W ilks' L a m b d a
.7 9 8
1 .0 1 2 a
3 .0 0 0
1 2 .0 0 0 .
.421
H o tellin g 's T r a c e
.253
1.01.2®
3 .0 0 0
1 2 .0 0 0
.421
R o y 's L a rg e s t R o o t
.253'
1.01.2®
3 .0 0 0
1 2 .0 0 0
.421
b.
D e sig n : In te rc e p t
Within Subjects Design: TIMEPTS+TRTMTS+TIMEPTS'TRTMTS
M a u c h ly 's T e s t O f S p h e r I c it y b
M e a s u re : M E A S U R E J
Epsilon®
W ithin S u b je c ts E ffect
T IM E P T S
TRTM TS
T IM E P T S * T R T M T S
M au c h ly 's
W
A pprox.
C h i-S q u a r
e
df -
.7 3 8
3 .8 7 4
5
1 .0 0 0
.0 0 0
0
.751
3 .6 4 2
5
S jg r —
Z .5 6 9
G re e n h o u
s e -G e is s e
■\
r
J
\
Z
.6 0 3
)
354"
H u y n h -F e l
dt
L o w er-b o u
nd
1 .0 0 0
.3 3 3
1 .0 0 0
1 .0 0 0
1 .0 0 0
.8 6 9
1.00(5
.3 3 3
127
T e s ts o f W ith ln -S u b je tts E ffe c ts
,M e a s u re : ly E A S U R E _ 1
t y p e III
S u m of
^ S q u a re _ s
S o u rce
T IM E P T S
S p h e ric ity A s s u m e d
L o w e r-b o u n d
TRTM TS
E rro r(T R T M T S )
T IM E P T S * T R T M T S
S p h e ric ity A s s u m e d
'2 .5 6 3 '
3 .0 0 0
1 :o q o .
C- 7 . 5 5 0 E-C o^
iW
t
2,SflGEWQZ
.5 0 0
rE .7 9 S E -.O Z /
7.55C E -C 5, '
3 5 .8 8 7
2 .1 0 4 E - 0 7
4 2 .0 0 0
1 .7 9 8 E -0 7
L o w e r-b o u n d
7 .5 5 0 E ip 6
1 4 .0 0 0
5 ,3 2 3 6 5 7
r2 .8 0 3 > a y
G re e n h o u s e -G e is s e r
,'500
.5 0 0
7 .5 5 0 E -0 6 .
S p h e ric ity A s s u m e d
Sip.
<326
" 2 .8 0 3 E - 0 7
1 .0 0 0
2 .8 0 3 E -0 7
.5 9 7
H u y n h -F e ld t
2 .8 0 3 E - 0 7
1 .0 0 0
2 .8 0 3 E - 0 7
.5 9 7
L o w e r-b o u n d
1 .0 0 0
§ ,8 0 3 E=OZ .
.5 9 7
S p h e ric ity A s s u m e d '
2,803E=.Q7
< -6 ,5 7 0 E -0 B > ’
G re e n h o u s e -G e is s e r
6 .SJO ErO S
1 4 .0 0 0
4 :6 9 3 E -0 7
H u y n h -F e ld t
6 .5 7 0 E - 0 6
1 4 .0 0 0
4 .6 9 3 E -0 7
L o w e r-b o u n d
6 t§ZQ.E-06
14.00.0
45.93E-Q 7L
S p h e ric ity A s s u m e d
L o w e r-b o u n d
j '2 .9 3 8 E - 0 2 )
C m,
C l : S ia js iE ^ B /
2 '.9 3 8 E -0 7
2 .6 0 8
1 .1 2 7 E -0 7
2 .9 3 8 E -0 7
3 .0 3 3
9 .7 9 4 E -0 8 '
i 2 J38E = S 7
1 : 0 0 0 , : S -O S B E ^ Z
S p h e ric ity A s s u m e d
4 .4 2 9 E - 0 6
H u y n h -F e ld t
6 ^ -lS i g 5
4 .4 2 9 E - 0 6
4 2 .0 0 0
1 .0 5 5 E -0 7
L o w e r-b o u n d
4 .4 2 9 E r 0 6
1 4 .0 0 0
3 .1 6 4 E -0 7
G re e n h o u s e -G e ls s e r
%
I
CSXL
8 .9 8 5 E -0 8 ,
H u y n h -F e ld t
G re e n h o u s e -G e is s e r
'
F
G r e e r ih o u s e - G e is s e r
.H u y n h .-F eld t
E rro r(T lM E P T S * T R T M T
M ea n
S auace
~ 2 i6 9 6 E - Q 7 t
G r e e n h o u s e - G e is s e r , -2 :6 9 6 - ^ 9 7
H u y n h -F e ld t
2 .6 9 6 E - 0 7
E rro r(T IM E P T S )
df
.9 2 9
,9 2 9 ,
.9 2 9
a
-
128
T e s t s o f W ith ln - S u b je c ts C o n t r a s t s
M e a s u re : M E A SU R E _1
S o u rce
IlI M b P I S
T IM E P T S
L in e a r
TR TM TS
Q u a d ra tic
E rror(T IM E P T S )
T y p e III
S u m of
S q u a re s
M ean
S q u a re
df
2 .1 0 9 E -O 7
1 .2 3 3
2 .8 5 2 E -0 8
1
2 .8 5 2 E -0 8
.1 3 0
.7 2 3
.2 0 2
.6 6 0
.5 9 7
.4 5 2
1 .0 0 0
C u b ic
3 ,0 1 0 E -0 8
L in ea r
, 2 .3 9 5 E -0 6
.
1
3 .0 1 0 E -0 8
14
1 .7 1 1 E -0 7
2 .1 8 8 E -0 7
Q u a d ra tic
3 ,6 6 3 E -0 6
14
C u b ic
2 .0 9 1 E -06
14
1 .4 9 4 E -0 7
L in ea r
2 .8 0 3 E -0 7
1
2 .8 0 3 E -0 7
E rror(T R T M T S )
L in ea r "
6 .5 7 0 E -0 6
14
4 .6 9 3 E -0 7
E rro r(T IM E P T S ‘T R T M T
L in ea r
.0 0 0
1
.OOQ
.0 0 0
L in ea r
2 .9 0 1 E -0 7
1
2 .901 E -0 7
2 .8 2 6
.1 1 5
C u b ic
L in ea r
.3 .7 5 0 E -0 9
1
3 .7 5 0 E -0 9
.0 5 3
.821
L in ea r
L in ear
2 .0 0 0 E -0 6
14
1 .4 2 8 E -0 7
Q u a d ra tic
L in ear
t.4 3 7 E - 0 6
14 .
1 .0 2 7 E -0 7
C u b ic
L in ea r
9 .9 2 3 E -0 7
14
7.0 8 8 E -O 8
.
T ra n s fo r m e d V a ria b le :-A v e ra g e
1 .9 5 9 E -0 3
E rro r
4 .122E -Q 5
.286
L in e a r
T e s t s Qf B e f w e e n - S u b j e c t s E f f e c t s
T y p e 111
Sum of
S q u a re s
'
Q u a d ra tic
M e a s u re : M E A SU R E _1
S o u rce
In te rc e p t
Siq.
1
TRTM TS
T IM E P T S ' TRTM TS
F
•2:T09E-07
M ean
S q u a re
df
1'
1 .9 5 9 E -0 3
14 ■, 2 .9 4 4 E -0 6
Estimated Marginal Means
F
6 6 5 .4 1 0
S iq ,
.0 0 0
129
1. G ra n d M ean
M e a s u re : M E A S U R E _1
9 5 % C o n fid e n c e Interval
M ean
S td ’. E rro r
4 .0 4 d E rO 3
.0 0 0
L ow er
Bound-
U pper
Bound
3 .7 0 4 E -0 3
4 .3 7 6 E -0 3
.
2. TIMEPTS
E s tim a te s
M e a s u re : M EA S'U R E _T
95.% .C o n fid e n c e In terv al
L ow er
Bound
U pper
B ound
.0 0 0
3.753E -G 3
4 .4 5 7 E - 0 3
,0 0 0
3 .6 3 6 E -0 3 ,
4 .4 9 4 E -0 3
3 .9 8 5 E -0 3
.0 0 0
3 .6 0 2 E -0 3
4 .3 6 8 E -0 3
4 .0 0 7 E -0 3
.0 0 0
3 .7 2 4 E -0 3
4 .2 8 9 E -0 3 .
T IM E P T S
1
4 .1 0 5 E -0 3
2
4 :0 6 5 E -0 3
3
4
M ean
S td . E rro r
130
P a lr y / is e C o m p a r i s o n s
M e a s u re : M E A SU R 'E „1
(I) T IM E P T S
1
M ea n
D ifferen c e
(I-J)
4 ,0 0 0 E -0 S
(J ) T IM E P T S
2
3
2
3
4
9 5 % C o n fid e n c e In terv al
. f o r D ifferen ce^
S td . E rro r
1 .2 0 0 E -0 4 ,
S ig ,a
L o w er
Bound
U pper
Bound
.0 0 0
.7 4 4
- 2 .1 7 5 E -0 4
2 .9 7 5 E -0 4 .
.0 0 0
.1 7 4
-5 .9 8 0 E -0 5
2 ,9 9 8 & -0 4 .
4
9 .8 3 3 E -0 5
.0 0 0 ,
.3 9 0 ' - 1 .3 9 3 E - 0 4 ,'
3.360E%0'4
1
- 4 .OOOE-OS
.0 0 0
- 2 .9 7 5 E -0 4
2 .1 7 5 E -0 4
3
8-O0OE-O5
.0 0 0
.7 4 4
.4 1 5
- 1 .2 4 5 E - 0 4
2 .8 4 5 E - 0 4
.0 0 0
.6 6 4
-2 .2 4 O E -0 4
.0 0 0
.174:
-2 .9 9 8 E - 0 4
3 .4 0 6 E -0 4
‘ 5 i9 8 0 E -0 5
4
5 .8 3 3 E -0 5
1
-1 ..2 0 0 E -0 4
.
2.
-SlOOOE-OS'
.ooo
.4 1 5
- 2 .8 4 5 E -0 4
4
-2 .T 6 7 E -0 5
.00Q.
.'845
' T2 :5 4 4 E -0 4
1.2 4 5 E -C 4
2 .1 t1 :E ,0 4 '
1
-9 /8 3 3 E -0 5
.0 0 0
.3 9 0
-3 .3 6 0 E -0 4
.1.3 9 3 E -0 4
2
-5 .8 3 3 E -0 5
.0 0 0
.6 6 4
-3 .4 0 6 E -0 4
2 .2 4 0 E - 0 4 '
3
2..1 6 7 E -0 5
.0 0 0 .
.8 4 5
t2..1,1:1E-04
2 .5 4 4 E f0 4
B a s e d o n e stim a te d , m a rg in a l ,m e a n s
a . A d ju s tm e n t fo r m ultiple c o m p a ris o n s : L e a s t S ig n ific an t D ifferen c e (e q u iv a le n t to n o a d ju s tm e n ts ) .
M u ltiv a ria te T e s t s
V a lu e
.
H y p o th e si
. s df
F
1 E rro r d f
S ip .
Pillai's tr a c e
.1 6 3
•7 7 7 a
3 .0 0 0
1 2 .0 0 0
529
W ilk s' la m b d a
.8 3 7 '
.7 7 7 a
3 .0 0 0
1 2 .0 0 0
.5 2 9
H otelllng^s .trace,
.1 9 4
,7 7 7 a
3 .0 0 0
1 2 :0 0 0
529
R o y 's la r g e s t.r o o t
.1 9 4
.777=
3.-000
1 2 .0 0 0
. .5 2 9
131
E s tim a te s
M e a s u re : M E A S U R E _1
9 5 % C o n fid e n c e In terv al
Low er
Bound
U pper
B ound
TRTM TS
1
4 .0 8 9 E -0 3
.0 0 0
3 .7 2 6 E -0 3
4 4 5 2 E -0 3
2
3 .9 9 2 E -0 3
.0 0 0
3 .6 3 2 E -0 3
4 .3 5 2 E - 0 3
M ean
S td . E rro r
P a irw is e C o m p a ris o n s
M e a s u re : M E A S U R E _1
:95% C o n fid e n c e In terv al
f o r D iffe re n c e 3
M ea n
D ifferen c e
(I) T R T M T S '
I
(J ) T R T M T S
2
2
1
(I-J)
9 :6 6 7 E -0 5
S td , E rro r
.0 0 0
1 - 9 .6 6 7 E :0 5
.0 0 0
Low er
B ound
U pper
Bound
.4 5 2
-1 ..7 1 6 E -0 4
3 .6 4 9 E -0 4
.4 5 2
-3 .6 4 9 E - 0 4
1 .7 1 6 E -0 4
S ig .B
B a s e d .on e s tim a te d m a rg in a l m e a n s
a . A d ju s tm e n t fo r m ultiple c o m p a ris o n s : L e a s t S ig n ific an t D ifferen c e ( e q u iv a le n t to n o a d ju s tm e n ts ).
M u ltiv a r ia te T e s t s
V a lu e
F
PHIal's tra c e
.041
.5 9 7 a
W ilks' la m b d a
.9 5 9
,5 9 7 3
H o te llin g 's tra c e
.0 4 3
R o y 's l a r g e s t ro o t
.0 4 3
H y p o th e si
sdf
1 .0 0 0
E rro rd f
S ig .
1 4 .0 0 0 '
.4 5 2
1 .0 0 0
1 4 .0 0 0
.4 5 2
.5 9 7 a
1 .0 0 0
1 4 .0 0 0
.4 5 2
,5 9 7 a
1 .0 0 0
1 4 .0 0 0
.4 5 2
■
132
4 . T IM E P T S * T R T M T S
M e a s u re : M E A S U R E J
9 5 % C o n f id e n c e In te rv a l ••
T IM E P T S
1
L o w er
Bound
U pper
B ound
.0 0 0
3 .7 1 7 E -0 3
4 .4 8 6 E - 0 3
.0 0 0
3 .7 2 9 E -0 3 . '
4 .4 8 8 E 70 3
,0 0 0
3 .7 2 5 E - 0 3 , 4 .6 1 '5 E -0 3
TRTM TS
I
4 . 1 0 2 E -0 3
2
4 .1 0 B E -0 3
.2
I
4.1-70E -03
2
1
3 .9 6 0 E -0 3
.0 0 0
3 .5 0 3 E -0 3
4 .4 1 7 E - 0 3
'3
4 .0 7 5 E -0 3 .
.0 0 0
3 :6 2 1 E -0 3
4 .5 2 9 E - 0 3
,
4
. 2
1
3 .8 .9 5 E -0 3
.0 0 0 1 3 ..5 1 7 E -0 3
4 .2 7 3 E - 0 3
4 .0 0 8 E -0 3 '
.0 0 0
2
4 .0 0 5 E -0 3
>
M ean-
S td . E rro r
.
3 :6 9 6 E -0 3
4 .3 2 1 E -0 3
.0 0 0 : 3 .6 0 0 E -0 3
4 .4 1 0 E - 0 3
General Linear Model
W ith in -S u b je c ts F a c to r s
M e a s u r e : M E A S U R E _1
T IM E P T S
1
2
'
3
4.
D ependent
V a ria b le
TRTM TS
1
T0848L T
2
T0461L T
1
T2848LT
2
T246.1.LT
1
T4848L T
2
T 446T L T
I
T6848LT
2
T 6 46T L T
D e s c rip tiv e S ta tis tic s
S td .
D e v iatio n
T0848L T
M ean
6 8 ,9 7 0 0
T0461L T
6 8 .7 3 0 0
13.9271.
15 '
T2848L T
'6 6 .5 9 3 3
1 1 .8 2 0 5
15
T2461LT
6 7 .5 9 3 3
1 2 .6 3 6 3
15 !
T 4848LT
6 6 ,8 7 6 7
1 2 .5 9 9 9
15
T4461L T
6 6 .7 9 3 3 -
1 7 .3 4 6 8
T5.
6 7 ,4 1 3 3
1 2 .8 3 0 6
15
6 7 .1 9 3 3
1 2 .4 1 5 9
15
T6848LT
T6461L T
■
1 0 .8 1 9 2
N
15
134
M u ltiv a ria te T e s ts P
E ffect
T lM E P T S
V alue
.1 5 3 '
PiIIaPs T ra c e
W ilks' L am b d a
H y p o th e si
s df
'3 .0 0 0
E rro r d f
1 2 .0 0 0
Sip.
.5 5 8 .
.722»;
3 .0 0 0
1 2 .0 0 0
.5 5 8
.722*'
3 .0 0 0
1 2 .0 0 0
.5 5 8
R o y 's L a rg e s t R o o t
.1 8 0 .
.7 2 2 »
•3.000
1 2 .0 0 0
.5 5 8 ,
Pillai's T ra c e
.000,
.OOOa
1 .0 0 0
1 4 .0 0 0
.9 8 3
W ilks' L am b d a
H otelling's T ra c e
TIM E PT S *
TR TM TS
.722*
.847
.1 8 0
H otelling's T ra c e
TR T M T S
F
1.0 0 0
.0 0 0 a
1:0 0 0
1 4 .0 0 0
.9 8 3
.000
.0 0 0 a
1.000,
1 4 .0 0 0
.9 8 3
.9 8 3 ,
R o y 's L a rg e s t R o o t
.000
.0 0 0 a
1 .0 0 0
1 4 .0 0 0
Pillai's T ra c e
.081
.3 5 2 a
3 .0 0 0
1 2 .0 0 0 '
.7 8 8
W ilks' L am b d a
.919
.362»
3 .0 0 0
12:000
.7 8 8
H otelling's T r a c e
.088
.3 5 2 a
3 .0 0 0
1 2 .0 0 0
.7 8 8
R o y 's L a rg e s t R o o t
.088
.362»
3 .0 0 0
1 2 .0 0 0
.788
a. E x a c t s ta tis tic
b.
D esig n ; In te rc e p t
W ithin S u b je c ts D esig n : T IM E PT S + T R T M T S + T IM E P T S 'T R T M T S
M a u c h ly 's T e s t o f S p h e r ic ity 6
M e a s u re : M EA SU R E_1
E p sllo n a
W ithin, S u b je c ts Effect,
TIM E PT S
TR T M T S
T IM E P T S * TR TM TS
M au ch ly 's
W
A pprok.
C h l-S q u a r
e
.0 3 6
/G re 'e n h o u .
s e - G e |s s e
\
r
|
.645
.0 2 4
'
,s is T - ^
. df
.390
1 1 .970
5
1 .0 0 0
.0 0 0
0
.3 6 0
.1-2.989
5;
Z
/
I
H u y n h -F e l.
dt
.7 4 6
L o w er-b o u '
nd*
.333
UOO
1 .0 0 0
1 .000
^% 734
.8 7 7
.333
135
T e s t s o f W lth ln - S u b je c ts E f f e c t s
M e a s u re : M E A S U R E _ 1 '
T y p e III
S u m of
S q u a re s
5 2 .1 0 6
S o u rce
I iM b K iti
S p h e ric ity A s s u m e d
, -
G re e n h o u s e = G a is s e r
^ — tju y n h - F e ld t
J )
£
L o w e r-b o u n d
I R I M TS
5 2 .1 0 6
1 .0 0 0
1 1 2 6 .9 8 3
42
1 1 2 6 .9 8 3
^gE O S ds
i 1 2 6 .9 8 3 )
Q M s y v
1 4 .0 0 0
1 1 2 6 .9 8 3
G .r e e n h o u s e - G e is s e r
i
<
L o w e r-b o u n d
E rror(T R T M T S )
S p h e ric ity A s s u m e d
1 1 3 9 .4 5 7
G r e e n h o u s e - G e is s e r
1 1 .39.457
H u y n h -F e ld t
L o w e r-b o u n d
T IM E P T S * T R T M T S
1 1 3 9 .4 5 7
1
3 .8 5 2 E -0 2
.00.0
3 .8 5 2 E -0 2
.0 0 0
S
m
:
3 .8 5 2 E -0 2
14
c
a
8 1 .3 9 0
4 .3 9 0
5 .9 7 9
t r r o r ( H M E P T S 'T R T M T
S p h e ric ity A s s u m e d
sI
G r e e n h o u s e - G e ls s e f
H u y n h -F e ld t
L o w e r-b o u n d
1 ,0 0 0
1 3 .1 6 9
1 0 2 9 .7 5 7
42
2 4 .5 1 8
1929:7.57
3 9 5 .3 7
5 3 5 .9 4
1 4 .0 0 0
d z a g )7 3 .5 5 4
J lO g a .,7 5 7
1 0 2 9 .7 5 7
a g : I
.983
8 1 .3 9 0
C l t D P O i, )
1 4 .0 0 0
1 3 JJB L
m :
1 3 .1 6 9
,1 :
.
8 1 .3 9 0
1 3 .1 6 9
m
4:
%
)
8 0 .4 9 9
1 .0 0 0
3 m
1 .0 0 0
=
4 1 .6 1 0
<
G r e e n h o u s e - G e is s e r
H u y n h -F e ld t
.
C
.6 4 7
2 6 :8 3 3
S p h e ric ity A s s u m e d
L o w e r-b o u n d
.6 4 7
^26^134
S p h e ric ity A s s u m e d
H u y n h -F e ld t
F
. Q g M 06
G r e e n h o u s e - G e is s e r
H u y n h -F e ld t
1 7 .3 6 9
5 2 ..1 0 6 _
L o w e r-b o u n d
S p h e ric ity A s s u m e d
L rro r(H M E P T S )
M ea n
S q u a re
df
.1 7 9
.1 7 9
.ifs
\k>
136
T e s ts, o f W ith ln -S u b je c ts C o n tr a s ts
M e a s u r e : M EA S.U RE _1
S o u rce
T IM E P T S
.
_
T IM E P T S
L in e a r
T y p e III
Sum of
S q u a re s
TRTM TS
Q u a d ra tic
2 7 .7 9 2
1
2 7 .7 9 2
2 .T 8 5 ,
1
.3 3 4
3 8 6 .9 2 5
14
1 7 8 .0 5 8
14
1 2 .7 1 8
C u b ic
5 6 2 .0 0 0
14
4 0 .1 4 3
'1
3 .8 5 2 E -0 2
14
8 1 .3 9 0
L in ea r
-
3 .8 .5 2 E -0 2
1 1 3 9 .4 5 7 I
E rro r
.16.1
.9 2 9
2 7 .6 3 7 '
.0 0 0
.
.9 8 3
L in e a r
1
.2 2 6
..0.1.1
.9 1 7
L in e a r
7 :3 2 5
\
7 .3 2 6
.4 5 0
,51.3
C u b ic
L in e a r
5 :6 1 6
1
5 /6 1 6
J 50
.7 0 4
L in e a r
L in e a r
2 7 8 .9 8 2
14
1 9 :9 2 7
Q u a d ra tic
L in ea r
'C u b ic
L in ea r
.2 2 6 ,
2 2 8 :1 0 7
1.4
1 6 :2 9 3
5 2 2 ,6 6 9
14
37.333
M e a s u re : M E A S U R E _ 1
,
.0 0 8
Q u a d r a tic
T r a n s f o r m e d V a ria b le ; A v e ra g e
S o u rce
: In te rc e p t
.3 6 7 .
,
L in e a r
T e s ts o f B e tw e e n -S u b J e c ts E ffe c ts
T y p e III
Sum of
S q u a re s
Siq.
.8 6 8
L in e a r
L in e a r
F
'2 3 .9 8 0 '
Q u a d ra tic
TRTM TS
E rro r(T IM E P T S ‘ T R T M T
M ea n
S q u a re
1
.334'
E rro r(T R T M T S )
T IM E P T S * T R T M T S
.
2 3 .9 8 0 .
C u b ic
E rro r(T IM E P T S )
df
M ean
,S q u a re
df
5 4 5 8 6 6 ,1 1
1
/5 4 5 8 6 6 .1 1
1 6 1 4 7 .9 4 5
14
1 1 5 3 :4 2 5
Estimated: Marginal Meians
F
4 7 3 :2 5 7
S lg .
.0 0 0 '
137
I. Grand Mean
M easu re,; M E A S U R E J
95-%- C o n fid e n c e Interval'
M ean.
! 'S td. E rro r
6 7 .4 4 5 , . ‘ .3 .7 0 0
L ow er
.,B o u n d
60; 7 9 6
U pper
,B ound
7 4 :0 9 5
2. TIIWEPTS
Estim ates
M e a s u re : M E A S U R E J
9 5 % C o n fid e n c e In terv al
T IM E P T S
I
M ea n
S td . E rro r
L ow er
B ound .
L ipper
B ound
2
6 8 :5 5 0
2 :9 6 2
6 2 .1 9 7
7 4 .9 0 3
6 7 :0 9 3
'3 .0 8 6
6 0 :4 7 5
7 3 :7 1 1 1
3
6 6 .8 3 5
3 .5 6 7 '
5 9 .1 8 5
7 4 .4 8 5
4
6 7 .3 0 3
3 .1 6 0 -
6 0 .4 8 2 '
7 4 .1 2 5 .
138
Pairwise Comparisons
M e a s u rti: M E A S U R E _1
95% . C o n fid e n c e In terv al
f o r D ifferen c e 3
M ean
D ifferen c e
(I) T lM E P T S
1
2
(J ) T IM E P T S
2
(I-J)
1 .4 5 7
S td . E rro r
3,
1 .7 1 5
1 .6 0 8
4
1 .2 4 7
1
-1 .4 5 7
3
.2 5 8
-.210
4
1
2
3
4
U pper
B ound
.2 1 5
-.9 4 7
3 .8 6 0
.3 0 4
- 1 .7 3 3
5 .1 6 3
3 ,6 3 2
1.112
1.121
.281
- 1 .1 3 9
.2 1 5
- 3 .8 6 0
.9 4 7
1 .8 1 2
.8 8 9
- 3 .6 2 7
4 .1 4 4
2 .1 3 0
1.091
.8 5 0
- 2 .5 5 0
1 .6 0 8
.3 0 4
- 5 .1 6 3
1 .7 3 3
-.2 5 8
1 .81 2
.8 8 9
- 4 .1 4 4
'3 .6 2 7
1 .8 6 5
-1 .2 4 7
.210
3
L o w er
Bound
-1 .7 1 5
-.4 6 8
1
2
4
1.121
S ig .a
.4 6 8
1.085
1.112
.6 7 3
-2 .8 0 1
.281
- 3 .6 3 2
1 .1 3 0
1.091
.8 5 0
- 2 .1 3 0
2 .5 5 0
1 .0 8 8
.6 7 3
„ 1 .8 6 5
2 .8 0 1
B a s e d o n e s ti m a te d 1m a rg in a l m e a n s
a. A d ju s tm e h tf o r m ultiple c o m p a ris o n s : L e a s t S ig n ifican t D ifferen c e (e q u iv a le n t to n o a d ju s tm e n ts ).
M u ltiv a ria te T e s t s
PiIIaI1S tr a c e
V a lu e
.1 5 3
H y p o th e si
s .d f
F
.7 2 2 a
3 .0 0 0
WIIKs' la m b d a
.8 4 7
.7 2 2 "
3 ,0 0 0
H o te llin g 's tr a c e
.1 8 0
.722"
3 .0 0 0
R o y 's l a r g e s t ro o t
.1 8 0
.7 2 2 a
3 .0 0 0
E rro rd f .
12.000
12.000
12.000
12.000
Big.
.5 5 8
.5 5 8 .
.5 5 8
.5 5 8
139
Estim ates
M e a s u re : M E A S U R E _1
9 5 % C o n fid e n c e In terv al
TRTM TS
I
M ean
6 7 .4 6 3
2
S td . E rro r
3.031
L o w er
B ound
6 0 :9 6 3
3 .3 7 6
6 0 .1 8 8
6 7 .4 2 8 ,
U pper
Bound
7 3 .9 6 4
7 4 .6 6 7
Pairwise. Com parisons
M e a s u re : M E A S U R E _1
9 5 % C o n fid e n c e In terv al
fo r D ifferen c e 0
M ea n
D ifferen c e
(I)-T R T M T S
'I
2
. .
(J ) T R T M T S
2
I
(I-J)
3 .5 8 3 E -0 2 „
S td . E rro r
-3 .5 8 3 E -0 2
S ig .0
L o w er
B ound
U pper
B ound
1 .6 4 7
.9 8 3
-3 .4 9 7
3 .5 6 9
1 .6 4 7
.9 8 3
-3 .5 6 9
3 .4 9 7
B a s e d o n e s tim a te d m a rg in a l m e a n s
a . A d J u s tm e n tfo r m ultiple c o m p a ris o n s : L e a s t S ig n ific an t D ifferen c e (e q u iv a le n t to n o a d ju s tm e n ts ):
Multivariate Tests
•
' P illai's tra c e
W ilks' la m b d a
H o te llin g 's tra c e
R o y 's l a r g e s t ro o t
,
V a lu e
.000
1.000
.000
.000 .
H y p o th e sl
sdf
F
.OOOa
.OOOa
.OOOa
.OOOa -
1.000
1.0 0 0 '
1.000
1.000
E rro r d f
1 4 .0 0 0
S ig.
.9 8 3
1 4 .0 0 0
.9 8 3 -
1 4 .0 0 0
.9 8 3
. 1 4 .0 0 0
.9 8 3 .
140
4'. T IM E P T S * T R T M T S
M e a s u re : M E A S U R E _1
9 5 % C o n fid e n c e In terv al
T IM E P T S
I
2
3
4
TRTM TS
I
2
1
2
1
2
I
2
L o w er
Bound
U pper
Bound
M ean
6 8 .9 7 0
S td . E rro r
2 .7 9 4
6 2 .9 7 9
6 8 .1 3 0
3 .5 9 6
6 0 :4 1 7
7 5 .8 4 3
6 6 .5 9 3
3 .0 5 2
6 0 .0 4 7
7 3 .1 3 9
7 4 .5 9 1
7 4 .9 6 1
6 7 .5 9 3
3.26.3
6 0 .5 9 6
6 6 .8 7 7
3 .2 5 3
'5 9 :8 9 9
7 3 .8 5 4
6 6 ,7 9 3
.4.479
5 7 .1 8 7 .
7 6 .4 0 0
6 7 .4 1 3
3 .3 1 3
6 0 .3 0 8
6 7 ,1 9 3 ,
3 .2 0 6
6 0 .3 1 8 ■
7 4 .5 1 9
. 7 4 .0 6 9