Int. Med J Vol. 6 No 2 December 2007

Long-Term Exercise Effect on Plasma and Aorta Homocysteine and 15-F₂₁-Isoprostane Concentrations in the High Cholesterol-Induced Atherosclerosis in Rabbits.

Mohsen. Alipour¹ Ph. D, Davood Sohrabi² Ph. D, Ali Awsat Mellati³ Ph. D, Mustafa Mohammadi⁴ Ph. D.

¹Department of Physiology, Zanjan University of Medical Sciences, Zanjan, Iran

^1: E-mail: alipourmohsen@yahoo.com    

²Department of Histology, Zanjan University of Medical Sciences, Zanjan, Iran

E-mail: sohrabidavood@yahoo.com

³Depatment of Biochemistry, Zanjan University of Medical Sciences, Zanjan, Iran

E-mail: mellatiA@yahoo.com

⁴Department of Physiology, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

E-mail: mohammadin@yahoo.com

ABSTRACT

Introduction: Diet and exercise can affect atherosclerotic risk factors. The present study was designed to examine the combined effect of long-term exercise and high cholesterol diet on malondialdehyde, 15-F₂₁-isoprostane and total homocysteine in atherosclerotic rabbit model. Methods: 56 male Dutch rabbits were divided into four groups: The normal diet control (NC), normal diet with exercise (NE), high-cholesterol diet control (HC) and high cholesterol diet with exercise (HE). Both NE and HE groups ran on a treadmill at 0.88 km/h for 10–90 min/day, 5 day/week, and 12 weeks in total. At the end of exercise protocol, blood samples were drawn, thoracic aorta was isolated and the mentioned parameters were measured using commercially available enzyme immunoassay kits. Results: The HE group showed reduction in atherosclerotic lesions in aorta along with reduction in plasma cholesterol profile. The HE group showed significantly reduced plasma and aorta concentrations of 15-F₂₁ and homocysteine as compared to NC. Malondialdehyde levels did not show a significant difference in response to exercise and/ or high cholesterol diet feeding. Conclusion: Our results suggest that elevated homocysteine level by high dietary cholesterol induced-oxidative stress can be considered as one of the multiple risk factors in the development of atherosclerosis. In addition, exercise may play a role in the reduction of plasma and aorta homocysteine and 15-F₂₁-isoprostane which could be a key for prevention and attenuation of atherosclerosis.

Key words: Exercise, Homocysteine, Lipid peroxidation, 15-F_21-isoprostane, Atherosclerosis

INTRODUCTION

Cardiovascular diseases (CVD) are a major public health problem in many countries and their background often begins with atherosclerosis ¹. Conventional risk factors for atherosclerosis account for approximately 50% of all cases. Evidence now indicates that homocysteine, which occurs in approximately 5 to 7% of the general population, is currently regarded as an independent and modifiable risk factor for atherosclerosis ². Homocysteine is a sulfur-containing amino acid that is not used for the synthesis of proteins and is produced by the intracellular demethylation of methionine in the methylation processes. In any condition which homocysteine production is high or its metabolism is impaired, homocysteine accumulates in the cell and is exported to the extracellular fluids ³. One mechanism by which increased homocysteine has been imposed to influence its pathological effects is by promoting increased oxidative stress ^3-5. An increase in oxidative stress in the cells may be due to physical activity. On the other hand, homocysteine may be considered among the glutathione (GSH) precursors released in the blood to counteract exercise-induced oxidative stress ⁶.  Exercise has been reported to reduce the incidence of CVD ⁷. While the effects of exercise on the traditional risk factors of atherosclerosis are well documented, few studies have addressed the potential for exercise to modify the recently recognized CVD risk factor, Hcy, in plasma and aorta in animal models.

The most widely used assay for lipid peroxidation involves the measurement of malondialdehyde (MDA) - thiobarbituric acid (TBA) adducts due to its simplicity ^{8, 9}. The effect of exercise-induced oxidative stress on lipid peroxidation based on the measurement of plasma and tissues MDA as an indirect measure of oxidative stress are mostly controversial ^{8, 9, 10}.

Isoprostanes are produced during peroxidation of membrane lipids by free radicals and reactive oxygen species. In the past 10 years, dozens of disease states and experimental conditions with diverse etiologies have been shown to be associated with marked increases in urinary, plasma and tissue levels of isoprostanes. In fact, isoprostanes may mediate many of the features of the disease states for which they are used as indicators ¹¹. 15-F₂₁-isoprostane is a prostaglandin isomer synthesized in vivo, dependently or independently of the activity of cyclo-oxygenase ^11,12. To our knowledge, a few studies concerned with the effect of exercise on plasma and aorta15-F₂₁-isoprostane have been reported ^13-15.

Therefore, the present study was designed to examine the effects of long-term exercise and/ or high cholesterol diet on MDA, 15-F₂₁-isoprostane and total homocysteine in thoracic aorta and plasma in experimental atherosclerotic rabbit model. We also hypothesized that high cholesterol diet would increase concentrations of mentioned parameters and we aim to determine whether these variations could be attenuated by long-term exercise.

MATERIALS AND METHODS

Animals and Diet

Fifty six male Dutch white rabbits (1.3 kg at the beginning) were divided into four groups: The normal diet control (NC) group, normal diet with exercise (NE) group, high-cholesterol diet control (HC) group and high-cholesterol diet with exercise (HE) group. The control groups were fed with normal rabbit chow, whereas the high cholesterol diet groups were fed with high cholesterol diet (2%). All animals were housed in an environmentally controlled room.

The exercise protocol was the same as that has been used previously ¹⁶. After 1 week of familiarization, the exercise training groups (HE and NE) ran on a leveled treadmill (Danish Yakhteh Co, Tabriz, Iran) at a speed of 15 m/min for 10 minutes per day for the first week. The running time was extended 5-10 min each week until they could run for 80 minutes per day. They underwent exercises for 5 days per week for a total of 12 weeks. Intensity of this exercise is approximately 70% of their maximal exercise capacity ¹⁷. In contrast, the sedentary groups (NC and HC) were placed on the treadmill for 10 minutes each day without receiving any exercise training. Rabbits were anesthetized at the end of experiments by injecting ketamine (25mg/kg, i.v.) and sodium pentobarbital (20 mg/kg, i.v.) via the margin ear vein. To avoid the acute effect of exercise, animals were sacrificed 48 h after exercise. Blood samples were drawn from the inferior vena cava and were stored in tubes for determination of plasma cholesterol profile, plasma and aorta concentrations of MDA, 15-F₂₁-isoprostane and total homocysteine (tHcy).

Histological Study of Thoracic aorta

Thoracic aorta was immediately isolated and was placed in formalin 10%. Briefly, after tissue processing steps, several serial sections of blood segments (6µm thick) were stained by standard hematoxylin-eosin and studied by light microscopy. Atherosclerotic lesions were assessed on a scale from 0 to 5. A segment of vessel that did not have visible lesion was given a score of 0, and a segment that was completely covered by atherosclerotic lesion was given a score of 5_.The area of thickened intima was assessed and calculated statistically among animals and was expressed as the percentage of luminal area of the vessel ring ¹⁸.

Assays

a) Plasma cholesterol profile. Plasma cholesterol profile including low density lipoprotein (LDL-C), high density lipoprotein HDL-C), very low density lipoprotein (VLDL-C), triglyceride (TG) and total cholesterol was determined using automatic analyzer (Abbott Alcyon 300, USA). Intra-assay and inter-assay CV% was less than10% in all of the cholesterol fractions. b) Plasma MDA. The amount of MDA was determined by the TBA (Thiobarbituric acid) assay. All reagents that were used in this assay were obtained from Merck (Darmstadt Germany). Briefly, 0.50 ml of plasma was added to 3ml of 1% phosphoric acid, 1 ml of 0.60% TBA, and 0.15ml of 0.20% butylated hydroxytoluene (BHT) in 95% methanol. The samples were heated in a boiling water bath for 45 minutes, cooled and 4 ml of 1- butanol was added. Thereafter butanol phase was separated by centrifugation at 3000 rpm for 10 minutes and absorbance was measured at 532 nm. The concentration of MDA was expressed as μM ¹⁹. c)  Plasma 15-F₂₁-isoprostane. The concentrations of 15-F₂₁-isoprostane were measured using commercially available enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI, USA). The procedure for the EIA was according to the instruction provided by the manufacturer. The sample volume that was used was 50 µl. Absorbance was measured at a wavelength of 405 nm using enzyme-linked immunosorbent assay (ELISA) reader (STAT FAX 2100, USA). The concentration of 15-F21-isoprostane was calculated from a semi-logarithmic standard curve of standard samples vs. %B/B₀ (ratio of the absorbance of a particular sample or standard well to that of the maximum binding (B₀) well), and data was presented as pg/ml. The intra-assay coefficient of variation was less than 9%. The sensitivity and specificity of the15-F₂₁-isoprostane assay were 5 pg/ml and 100%, respectively, (Cayman Chemical, Ann Arbor, MI, USA).  d) Plasma total homocysteine (tHcy). THcy levels were measured using Axis homocysteine EIA kit Axis-shield AS, Germany). The procedure for the EIA was according to the guidelines provided with the kit. The sample volume that was used was 50 µl. Absorbance was measured at a wavelength of 450 nm using ELISA reader (STAT FAX 2100, USA). The concentration of tHcy was calculated from a semi-logarithmic standard curve of standard samples vs. absorbance (450). The intra-assay coefficient of variation was <10%. The sensitivity of the tHcy was 2.0 µM.

Preparation of thoracic aorta fractions

Thoracic aortas were isolated, cleaned of gross adventitial tissue, blotted dry and weighed. They were then homogenized in 5-10 volumes of 50 mmol/L phosphate buffer (PH 7.4) at 4˚C for 30 seconds using a homogenizer. The homogenate was filtered through cheese cloth and the filtrate was centrifuged at 1500g for 10 min. The resulting supernatants were used for measurement of MDA, 15-F₂₁-isoprostane and tHcy ²⁰.

Statistical Analysis:

Data was expressed as means ±SD; statistical computations were calculated using SPSS 10 for windows software (SPSS INC, Chicago, IL, USA). Sample size (animal numbers) was indicated by n (n=14 rabbit for each group). Result among four groups was analyzed by ANOVA and further by Tukey HSD as post hoc test. Student- t-test was used to compare the differences of two groups and P values considered at significant at 0.05.

RESULTS

Histological Examination Results

The HC group revealed atherosclerotic lesions and thickening of the intima in thoracic aorta as compared to NC group (3.9 ± 0 .17 vs. 0, p<0.05, 78% vs.0%). After twelve weeks of exercise atherosclerotic lesions was significantly reduced in HE as compared to HC group (2.3 ± 0.30 vs. 3.9 ± 0 .17, p<0.05, 46% vs.78%). There was no significant lesion in HE and NC group noted.

Plasma Cholesterol Profile Results

HC group showed significantly increased plasma total cholesterol, LDL- C, HDL-C, VLDL-C, and TG (P<0.05). There were significantly reduced diet-increased plasma levels of VLDL-C, LDL-C and TG in HE group as compared to HC. (P<0.05). Plasma total cholesterol was reduced significantly only in NE group. There was no significant reduction of VLDL-C in NE as compared to NC (Table 1). There were a positive correlation among plasma cholesterol, tHcy and 15-F₂₁-isoprostane in exercised groups compared with control (0.61-0.76)

Results of Plasma Total homocysteine, 15-F₂₁-isoprostane and MDA

HC group showed significant increase in the levels of plasma and thoracic aorta tHcy, and MDA (P<0.5) (Table 2). There were no significant changes in 15-F₂₁-isoprostane level in both aorta and plasma in HC group as compared to NC. NE group showed significant reduction in diet-increased plasma and aorta levels of tHcy and 15-F₂₁-isoprostane as compared to NC group (P<0.05, Table 2). There were no significant differences the plasma and aorta levels of tHcy and 15-F₂₁-isoprostane in HE as compared to NE (Table 3).There were a significant correlation between the changes of tHcy in aorta and plasma (r =0.68). Pattern of the changes in15-F₂₁-isoprostane concentrations in aorta and plasma also were the same (r = 0.77). Coefficient correlation about the decrease of tHcy and 15-F₂₁-isoprostane concentrations in the groups which were fed with normal rabbit chow with or without exercise was 0.59 and 0.66 respectively.   MDA concentrations did not show significant difference in response to exercise and/ or high cholesterol diet feeding in plasma and aorta (Table 2, 3, r = 0.76).

DISCUSSION

Our results demonstrated that 12 weeks of high cholesterol diet feeding led to the development of atherosclerosis in the thoracic aorta associated with hypercholesterolemia and significant elevation of plasma and aorta tHcy concentrations. 12 weeks of moderate exercise decreased atherogenic diet-induced atherosclerotic lesions in the aorta along with a significant reduction of plasma and aorta concentrations of tHcy and 15-F₂₁-isoprostane in normal animals.

We have previously reported that plasma MDA concentration was increased after 8 weeks of exercise and /or high cholesterol diet feeding ¹⁶. In this study MDA levels did not show significant changes in response to 12 weeks of exercise and /or high cholesterol diet feeding. There are inconsistent and conflicting results about the effects of exercise on MDA levels as a marker of oxidative damage^{10, 19, 21-23}. Our present study is in agreement with some of them^{9, 23}. The inconsistency among the results of studies may be reflection of differences in exercise intensity and duration, type of study or training or method used for assessment.

Recent studies have focused on 15-F₂₁-isoprostane as an index of lipid peroxidation. 15-F₂₁-isoprostane is a specific, chemically stable compound and quantitative marker of oxidative stress and its quantification has been suggested to be a reliable measure of oxidative injury and may provide a reliable marker of free radical-induced lipid peroxidation in vivo ^{11, 12, 24}. Assessment of both MDA and 15-F₂₁-isoprostane may provide complementary information and at the same time improve specificity ⁸. In contrast to plasma MDA levels that were considerably increased by high cholesterol diet, however, plasma15-F₂₁-isoprostane levels were non-significantly affected by high cholesterol diet feeding and this is supported by other studies that have reported that the levels of 15-F₂₁-isoprostane is unaffected by lipid content of diet ^12,24. Information about the effects of exercise on isoprostanes especially in animal models is insufficient and mostly controversial ^13,14,25,26. To our knowledge no study has been published regarding the effect of exercise on the vessel content of the isoprostanes. For the first time, we measured the changes of 15-F₂₁-isoprostane and tHcy concentrations in thoracic aorta of rabbits. Their pattern of variations was similar to the plasma but their amounts were less than plasma. Elevation of plasma and aorta 15-F₂₁-isoprostane and MDA concentrations in our results may be attributable to high sensitivity of rabbit to free radical production by high cholesterol diet feeding. Reduction of 15-F₂₁-isoprostane in normal exercised animals in our work, suggests amelioration of oxidative stress and endothelium dysfunction by exercise intervention that may be related to an increase in NO availability. Increase in NO could be due to either an increase in NO production or a decrease in NO sequestration by reactive oxygen species and hence reduced scavenging NO ^{27, 28}. The enhancement of antioxidant enzymes, total antioxidant capacity and possibly ongoing oxidative clearance of oxidized-lipids and lipoproteins, in our previous study ¹⁶ may contribute in the reduction of lipid peroxidation and decrease in tHcy concentration by exercise in the present work.

We observed that high cholesterol diet feeding induced hypercholesterolemia that was correlated with the elevation of tHcy concentrations in plasma and thoracic aorta.  As well as 15-F₂₁-isoprostane changes were positively correlated with tHcy in NE and HE groups. Although hyperhomocysteinemia has been classified as a risk factor for CVD, its role in the development of atherosclerosis has been disputed. Some epidemiological studies have reported either a weak or no correlation between plasma homocysteine and the extent of coronary atherosclerosis. On the other hand, in vitro studies have shown that homocysteine stimulates cholesterol production in hepatocytes ²⁹, and there is a possibility in our study that, plasma and aorta homocysteine levels may be elevated by cholesterol intervention. In the other word, cholesterol and homocysteine metabolism may interrelated, and homocysteine may act in conjunction with other risk factors, such as cholesterol, to initiation or development of atherosclerosis ^{1, 29, 30}. One mechanism by which increased homocysteine has been proposed to influence its pathological effects is by promoting increased oxidative stress. Oaother proposed mechanism is that because homocysteine is a thiol, it can undergo autooxidation and oxidation with other thiols. The resulting ROS, hydrogen peroxide and superoxide anion radical generate oxidative stress ^{3, 31}. Lowering plasma homocysteine has been shown to slow the progression of subclinical atherosclerosis and also improves endothelial function in men with coronary artery disease. It may therefore seem reasonable to speculate that the fall in homocysteine with regular exercise, as seen in our study, may be one of many mediating factors leading to the beneficial effects of exercise on endothelial function ³². Generally, equivocal results have been obtained from studies examining the effects of acute and chronic exercise on tHcy ^{1,30,32,33,34,35,37}. Contrary to our results; human studies have shown that 6 months of exercise training increases serum tHcy concentrations ³⁰. According to other human studies, physical activity is probably not or weakly inversely associated with the tHcy concentration ¹. Elevation of tHcy in the blood of horses has also been found in response to submaximal physical exercise ⁶. These inconsistencies among the results of studies may be reflection of differences in exercise intensity and duration, type of study or training or method used for assessment and also maximal oxygen uptake. In our study, the type of exercise selected was a regular, moderate and progressive. There is a many of data including increased aerobic metabolism during exercise as a source of oxidative stress, demonstrating a pivotal role for oxidation-sensitive mechanisms in endothelial dysfunction and atherosclerosis ²⁸. It seems, in contrast to high frequent and high intensity exercise, low or moderate intensity exercise and higher duration may result in the reduction of homocysteine. In the other word, potential difference in the ability of exercise to modulate CVD risk factors including homocysteine depends on type of exercise and other connected factors or risk factors ³⁰. However, progressive adaptation to exercise may decrease systemic oxidative stress, thereby improving vascular function ²⁸. These protective mechanisms could include increased antioxidant defenses that we observed in our previous study ¹⁶, reduced basal production of oxidants, and reduction of radical leak during oxidative phosphorylation. The mild oxidative stress induced by graduated exercise and protective factors such as NO and antioxidants that are favorably modified by exercise could be responsible for its beneficial effects on the reduction of tHcy concentration, vascular dysfunction and atherosclerosis. In the present study, although pattern of the changes of tHcy concentration by exercise in the rabbits on an atherogenic high cholesterol diet was similar to we observed in normal rabbits, but its non-significant reduction may be attributable to additional oxidative stress imposed on the tissues, decreased intrinsic antioxidant defense capacity, limited bioavailability of NO as well as altered blood vessel elasticity by high cholesterol diet ^{28, 38, 39}.

In conclusion, our findings showed that 12 weeks of high cholesterol diet feeding induced atherosclerotic lesions in thoracic aorta that was associated with hypercholesterolemia and significant elevation of plasma and aorta tHcy, MDA and 15-F₂₁-isoprostane concentrations. 15-F₂₁-isoprostane may be considered as a reliable measure of oxidative injury than MDA and may provide a reliable marker of lipid peroxidation in vivo.  Although tHcy and 15-F₂₁-isoprostane levels were within the normal range in all groups, we found that 12 weeks of moderate and graduated exercise decreased atherogenic diet-induced atherosclerotic lesions in the aorta along with reduction of plasma and aorta concentrations of tHcy and 15-F₂₁-isoprostane in the normal animals that may be attributable to amelioration of antioxidant defenses and NO bioavailability. The effect of exercise on the mentioned parameters is limited in the rabbits on an atherogenic high cholesterol diet, possibly in part because of alterations in the ability to adapt to exercise–induced oxidative stress in high cholesterol diet. Therefore, based on our results, elevated homocysteine level by high dietary cholesterol induced-oxidative stress can be considered as one of the multiple risk factors in the development of atherosclerosis, and exercise may play a possible role in the reduction of plasma and aorta homocysteine and 15-F₂₁-isoprostane that may be a key for prevention and attenuation of atherosclerosis.

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 Tables

Table1: Comparison of the plasma cholesterol profile changes (mg/dl) among four groups of rabbits under long-term exercise and /or high cholesterol diet.Groups                   

Data are expressed as mean ± SD (n=14) for each group.

Differences of p<0.05 were considered significant.         

† NE vs. NC                                              * HC vs. NC and NE

● HE vs. NC and NE                                   # HE vs. HC                                       

Abbreviations: NC, normal diet control; NE, normal diet with exercise; HC,   High cholesterol diet control; HE, high cholesterol diet with exercise. LDL, Low density    lipoprotein-cholesterol HDL-C, high density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; TG, triglyceride                                                                                                                                                       

Table 2: Influence of 12-week exercise and /or high cholesterol diet on plasma Malondialdehyde (MDA), 15-F₂₁-isoprostane and total homocysteine (tHcy). Groups

Data are expressed as mean ± SD (n=14) for each group.

 Differences of p<0.05 were considered significant.

† NE vs. NC                                              * HC vs. NE         

 ● HE vs. NE                                              $ HC vs. NC

Abbreviations: NC, normal diet control; NE, normal diet with exercise; HC, High cholesterol diet control; HE, high cholesterol diet with exercise. MDA, Malondialdehyde; tHcy, total homocysteine.

Table 3: Influence of 12-week exercise and /or high cholesterol diet on thoracic aorta Malondialdehyde (MDA), 15-F₂₁-isoprostane and total homocysteine (tHcy). Groups

Data are expressed as mean ± SD (n=14) for each group.

 Differences of p<0.05 were considered significant.

† NE vs. NC                                              * HC vs. NE         

 ● HE vs. NE                                              $ HC vs. NC

Abbreviations: NC, normal diet control; NE, normal diet with exercise; HC, High cholesterol diet control; HE, high cholesterol diet with exercise. MDA, Malondialdehyde; tHcy, tota|/homo><t> nehr>

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