Cheryl Adkins, Dr. James Turk
University of Missouri-Columbia, College of Veterinary Medicine
The University of Missouri Veterinary Research Scholars Program was supported by funds from Merck-Merial, Pfizer and the MU College of Veterinary Medicine,
and this project was in part funded by NHLBI PO2 HL52490.
2. Introduction
4. Materials & Methods
6. Conclusion
References
1.  Griendling, Kathy K., FitzGerald, Garret A.  “Oxidative Stress and Cardiovascular Injury Part I:
  Basic Mechanisms and In Vivo Monitoring of ROS”  Circulation 2003; 108:1912-1916
2.  The Merck Manual. Sec. 16, Ch. 201, Arteriosclerosis.
3.Cowther, Mark A. The Pathogenesis of Atherosclerosis. Hematology Jan 2005:436-441.
4.Turk and Laughlin. Physical Activity and Atherosclerosis. Which animal model? Invited Review Can J Appl Physiol 29: 657-683, 2004.
5.
5.
** Chart taken from Cherubini A, Ruggiero C, Polidori MC, Mecocci P. “Potential Markers of Oxidative Stress in Stroke” Free Radic Biol Med 2005, Oct 1;39(7):841-52.
1. Abstract
The ZeptoMetrix Corporation TBARS Assay Kit was used to determine lipid oxidation.  This kit measures MDA adduct with thiobarbituric acid, a 1:2 ratio.  Frozen serum, stored at -70◦C was used in the experiment. The results were read with a spectrophotometer set at 532 nm.
3. Pathogenesis of Atherosclerosis
5. Results
Cardiovascular disease is the leading cause of death in the United States.  Atherosclerosis is the major contributor to cardiovascular morbidity and mortality.  Oxidative damage to LDL by free radicals is believed to contribute to the development of atherosclerosis.  Free radicals are formed from naturally occurring reactive oxygen species (ROS) in the body, such as superoxide (O2-), nitric oxide (NO), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-).  In the presence of O2- ,NO produced by normal endothelium becomes ONOO-.  ONOO- not only causes a decrease in cGMP leading to damage of the vessels, but it is also mediates further lipid peroxidation.  When plasma LDL sustains oxidative damage it enhances the effects of oxidized LDL in the arterial wall leading to the accumulation of ‘foamy macrophages’ seen with atherosclerosis.  Oxidative damage occurs not only to LDL, but also to a variety of other plasma and tissue constituents; including lipid hydroperoxides and aldehydes1.  These 2 thiobarbituric acid reactive substances (TBARS) increase with oxidative stress.  The purpose of this study is to quantify the amount of ROS using TBARS in 4 groups (N=8 each) of pigs: normal fat sedentary (NFSed), normal fat exercised (NFEx), high fat cholesterol sedentary (HFCSed), and high fat cholesterol exercised (HFCEx).  Results indicate that TBARs in plasma of pigs fed HFC diet are greater than in pigs fed NF diet.  Plasma TBARs in HFCEx were significantly greater than in NFSed or NFEx pigs.
Isoprostanes
MDA (A TBARS)
Atherosclerosis and complications stemming from it are the leading cause of death among Americans.  Atherosclerosis is defined by describing its clinical pathologic appearance as irregular subintimal thickenings of medium and large arteries (atheromatous plaques) consisting of cholesterol and lipids.2 Arteries not only become narrowed due to the thickening of the intima, decreasing blood flow, but they also fail to respond to dilate; decreasing blood flow.  Many risk factors for atherosclerosis have been identified including but not limited to: hyperlipidemia, hypertension, diabetes, smoking, obesity, and physical inactivity3.
Many theories exist on how atherosclerosis begins (inflammation, infection, lipid metabolism abnormalities), but all agree that lipid accumulation in macrophages
(foam cells) is the defining identification of atherosclerosis.  Lipid oxidation and accumulation must occur, whether it is the initiating event, occurs somewhere in the middle, or if it is the end stage of the pathway for the atheromatous plaque to form.
This study will demonstrate that high fat and cholesterol diets will cause TBARS levels to increase.
•TBARS were significantly higher in pigs fed high fat and cholesterol diet.
•There is a correlation between an increase in TBARs and an increase in LDL.
•There is more lipid oxidation in High Fat fed pigs leading to an increased risk of developing atherosclerosis.
Injury to endothelium
Leukocytes adhere to endothelium
Leukocytes recruit monocytes
Monocytes accumulate
 in sub-endothelial
 space
Elevation in
Isoprostanes and
 MDA at the same
time as elevation
in LDL
LDL susceptible
to modification
and oxidation
Modified LDL chemotactic to monocyctes
Foam cells accumulate to form a fatty streak
Fatty steak and fibrous plaque enlarge and bulge into lumen
Subendothelium exposed
Subendothelium tears
Platelets aggregate and thrombi form
Elevation in plasma LDL
Lipid accumulation in macrophages (foam cells)
Migration of monocytes into the intima and become macrophages
Lipid Radicals L*
Peroxyl Radicals LOO*
Conjugated Dienic Lipid
Hydroperoxides
Peroxyl Radical
Decomposition Products
Aldehydes
HNE
Dienals
Alkanes
Pentane
Ethane
Conjugated Dienes
Alkoxyl Radical    LO*
**
NF
HF
N=16
N=16
P < 0.02
Thiobarbituric Acid Reactive Substances in Hypercholesterolemic
Pigs Fed Coconut, Corn Oil, and Cholesterol
Graph A Comparison of lipid oxidation of serum measured by TBARS between the NFSed, NFEx, HFSed, HFEx groups of Yucatan pigs used in this sturdy. P=0.002
HFM9 & HFM 10
HFM9 & HFM10 delta TBARS
NFSed
HFSed
NFEx
HFEx
N=8
N=8
N=8
N=8
P = 0.12
Graph B. Comparison of NFSed, NFEx, HFSed, HFEx delta TBARS (mean, interquartile range) in serum. P=0.12
HFM 9 & HFM 10 Normal Fat Fed & High Fat Fed Groups
Graph C.  Lipid oxidation of serum between sedentary and exercised Normal Fat fed pigs and sedentary and exercised High Fat fed pigs by measuring TBARS. P <0.001
delta TBARS of HFM9 & HFM10 Normal Fat & High Fat Diets
Graph D. Comparison of delta TBARS between sedentary and exercised Normal Fat fed pigs and sedentary and exercised High Fat fed pigs.
LDL and TBARS of HFM9 & HFM 10 Pigs
Graph E. Plasma LDL levels of Normal Fat and High Fat diets in correlation to serum TBARS levels. r=0.51, P=0.02
Figure 6 from Reference # 4.  A: Cross section of common carotid artery stained with oil-red-o demonstrates accumulation of small numbers of lipid-laden (red droplets) cells within the intima, Stary stage I; B: Cross section of common carotid artery stained with Verhoeff’s elastic method demonstrates multiple layers of lipid-laden foam cells within the intima, Stary stage II; C: Cross section of common carotid artery stained with Verhoeff’s elastic method demonstrates central cavitation of the lipid-laden foam cells associated with extraction of extracellular lipid pools during tissue processing, Stary stage III; D: Cross section of common carotid artery stained with Alizarin red for mineral central multifocal mineralization (arrow) in association with lipid-laden foam cells, Stary stage IV.