Mudassir Ahmad Khan ( Department of Biochemistry, Basic Medical Sciences Institute, Jinnah Post Graduate Medical Centre, Karachi. )
Abdul Baseer ( Department of Biochemistry, Basic Medical Sciences Institute, Jinnah Post Graduate Medical Centre, Karachi. )
Objective: To assess and compare the status of lipid peroxidation, both in control subjects and in coronary heart disease patients.
Methods: Serum total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and malondialdehyde levels were determined in 46 patients with coronary heart disease and 50 age matched control healthy subjects. 29 male coronary heart disease patients were divided into smoker (n=19) and nonsmoker (n=l0) groups, to observe the effect of smoking on lipid peroxidation in coronary heart disease patients.
Results: Malondialdehyde and lipid parameters were found significantly high (P< 0.001) with the exception of high-density lipoprotein cholesterol which was significantly low (P< 0.001) in coronary heart disease patients. Smokers with coronary heart disease showed significantly increased (P< 0.025) malondialdehyde levels as compared to nonsmokers with coronary heart disease.
Conclusion: Elevated serum levels of malondialdehyde indicate increase in the level of production of oxygen free radicals, suggesting their possible role in atherogenesis, leading to coronary heart disease (JPMA 50:261, 2000).
Free radicals are reactive chemical species. All of the major class of biomolecules may be attacked by free radicals, but lipids are probably the most susceptible. Cell membranes are rich source of polyunsaturated fatty acids, and are readily attacked by oxidizing radicals. The oxidative destruction of polyunsaturated fatty acids is known as lipid peroxidation. It proceeds as a self--perpetuating chain reaction1,2. Free radical species derived from oxygen are named as reactive oxygen species (ROS)3 and the damage inflicted by these ROS is referred to as oxidative stress4,5. Oxidant stress is known to increase the production of free radicals. Both the oxidative stress and the production of ROS intracellularly, have been implicated in the pathogenesis of a variety of disease states6,7, like cancer and cardiovascular disease7.
Coronary heart disease (CHD) is a health problem of major proportion of population, and is a serious threat to life8. It is the single leading cause of death for adults worldwide, and is expected to be the leading cause of morbidity and mortality in the western world well into the 21st century9. CHD is the most common cause of lethal atherosclerotic disease8. The most important risk factors for CHD include hypertension, hypercholesterolaemia, cigarette smoking10,11, diabetes mellitus and high-fat diet11. Increased serum cholesterol, low-density lipoprotein cholesterol (LDL-c) and low high-density lipoprotein cholesterol (HDL-c) concentrations are associated with increased risk of premature atherosclerosis12,13, During past few years considerable amount of evidence, has accumulated indicating the crucial and causative role of free radicals in the pathogenesis of atheroscIerosis14,15. While the mechanisms for atherosclerosis are not completely understood, it is suggested that16, free radical modification of low-density lipoprotein (LDL) within the arterial wall renders it more atherogenic, and has been implicated as an early step in atherogenesis16-20. According to oxidation theory21, oxidation of LDL assists in the formation of foam cells, and contributes to various proatherogenic processes21,22
Oxidative activity of free radicals can be determined by measuring their oxidative products in a biological system23. Malondialdehyde (MDA) is the breakdown product of lipid peroxidation and its assessment is considered as a reliable marker of oxidative damage24-26. The purpose of our study was to assess and compare the level of MDA in patients with CHD and in control healthy subjects.
Patients and Methods
This study included 46 patients (32-61 years) with CHD and 50 control healthy subjects (32-56 years). Control subjects were non-smokers, having no cardiovascular, or any other disease. Diagnosis of CHD was based on documented evidence of attack of myocardial infarction (recent or past) or documented evidence of angina with subsequent sudden death or congestive heart failure27. The patients with CHD having had first attack of myocardial infarction with in the last week were included. Patients receiving lipid lowering drug therapy were excluded.
Male to female ratio was 28:22 for control and 29:17 for CHD groups respectively. The male patients having CHD were divided into two groups on the basis of smoking. The first group (n=l0) included CHD patients with no history of smoking in the past or present, whereas the second group (n=19) comprised of CHD patients who were smokers, with a history of smoking 20-25 cigarettes per day for the last 10-30 years. All female CHD patients were non¬smokers.
Blood samples were collected after an overnight fast and sera separated and stored at -20°C till analysis. Serum total cholesterol, triglycerides and HDL-c were measured by enzymatic calorimeteric method using the kits supplied by Bio-Systems, Spain. LDL-c was calculated by Friedwald formula28.
Malondialdehyde content of samples was determined by the thiobarbituric acid (TBA) activity by using the method recommended by Buege and Aust29. MDA of the serum sample reacts with TBA to form a coloured pigment, the absorption of which is measured by spectrophotometer at 535nm.
The results are expressed as mean±S.E.M. and all statistical calculations were made by applying paired Student’s t-test with the significance level at P<0.05.
The serum lipid profile and MDA levels were measured in control subjects and in patients having CHD. The control and CHD groups were age-matched, with mean and S.E.M. of 45.08±0.98 years (control group) and 46.87±1.14 years (CHD group). The body mass index (BMI), and the serum levels of MDA, cholesterol, triglycerides and LDL-c were significantly high (P< 0.001), whereas the levels of HDL-c were significantly low (P< 0.00) in CHD patients as compared to the control subjects (Table 1).
The CHD patients who were non-smokers and the group of CHD patients who were smokers, when compared showed no statistically significant difference in all the lipid#
P value non-significant as compared to control subjects.
P< 0.001 as compared to control subjects.
Table 2. Serum lipid and Malondialdehyde concentrations in CHD patients (Both non-smokers and smokers).
Table 1. Serum lipid and Malondialdehyde concentrations in control and CHD patients.
= P value non-significant as compared to CUD patients who were non¬smokers.
t = P< 0.025 as compared to CHD patients who were non-smokers.
parameters, except for the MDA levels, where a significant increase (P< 0.025) was observed in CHD patients who were smokers as compared to the CHD patients who were non-smokers (Table 2).
Coronary heart disease (CHD) is caused by long-term deposition of lipids in coronary arteries, which lead to atherosclerosis and necrosis of the heart tissue3°. The basic lesion of atherosclerosis is the intimal plaque. The development of a plaque involves, accumulation of lipid (both within macrophages and free in the tissues), proliferation of smooth muscle cells, and the formation of collagen. All these processes lead to the formation of a plaque, which consists of a core of extracellular lipid contained in a fibrous collagenous capsule. Disruption of the atherosclerotic plaque and the superimposed formation of platelet-rich thrombus, produces subtotal or total occlusion of coronary circulation. This leads to myocardial ischaemia31. Rapid restoration of the blood flow to the ischaemic myocardium lessens cardiac damage and improves the early and long-term morbidity and mortality32. Myocardial ischaemia can result from a number of causes, as atherosclerosis or thromboembolism, or can be produced during surgical interventions such as, percutaneous trans lum ina coronary angiop lasty, coronary artery bypass or transplantation. Whatever is the source of ischaemia, consequences are always the same, i.e., lack of oxygen to the myocardium and a lack of suitable substrate for metabolism33. Oxygen free radicals have been implicated in cardiac ischaemic injury. These free radicals (superoxide anions and hydroxyl radicals) are produced in the body by reduction of oxygen. In normal circumstances they are removed by the different scavenger systems present in blood and tissues. In case of myocardial ischaemia, which can lead to myocardial infarction, excessive free radicals may be generated34. Pucheu et al35 suggested that measurement of malondialdehyde is a good marker of radical stress during reperfusion of the ischaemic myocardium, and also showed significantly increased malondialdehyde concentrations in group of CHD patients who were subjected to intravenous thrombolysis than those who had not been subjected to thrombolysis. Oen and colleague’s36 showed significantly increased levels of lipid peroxides in patients suffering from coronary heart disease as compared to the control subjects. Belch et a and Dincic et al37 showed evidence -of increased free radical activity in patients with myocardial ischaemia than the control subjects. Our results also show significantly increased concentrations of malondialdehyde, as an index of lipid peroxidation, in CHD patients.
Cigarette smoking adversely affects the lipid profile, leading to lower levels of HDL-c and higher levels of LDL¬c and triglycerides. Production of oxygen free radicals is increased with smoking, which may play a role in atherosclerosis, leading to CHD. In general, smokers have about twice the risk of developing CHD as do nonsmoker’s38. Wang and associates39 studied healthy men (smokers and nonsmokers) and showed increased level of oxygen free radicals in smokers as compared to nonsmokers. We studied smoker and nonsmoker patients with CHD, and found significantly elevated level of oxygen free radicals in smokers with CHD than nonsmoker CHD patients.
Wu et al30 reported no significant differences in total cholesterol, triglycerides and LDL-c concentrations in both control and CHD patients. However, he found lower levels of HDL-c in patients with CHD. Hargreaves and co-workers40 found no relationship between serum total cholesterol concentration and CHD. He also showed no significant change in the total cholesterol levels of subjects with and without CHD whereas, the HDL-c concentrations were significantly lower and LDL-c concentrations were significantly higher in patients with CHD as compared to control subjects. He pointed out that both increased LDL-c and decreased HDL-c contribute to the development of Cl-ID. Sandkamp and colleagues41 reported significantly elevated levels of total cholesterol, LDL-c and decreased levels of HDL-c in patients with CHD as compared to control subjects. Our results also show significantly increased concentrations of total cholesterol, LDL-c and significantly decreased concentrations of HDL-c in CHD patients than in control subjects.
In conclusion, our study demonstrates a significant relationship between elevated level of malondialdehyde and CHD. Elevated levels of malondialdehyde indicate increase in the level of production of oxygen free radicals, suggesting their possible role in atherogenesis, leading to coronary heart disease.
1.Cheeseman KH, Slater TF. An introduction to flee radical biochemistry. Br. Med. Bull., l993;49:481-93.
2.Chancerelle Y. Kergonou JF. immunologic relevance of inalonic dialdehyde, Ann. Pharm. Fr., 1995;53:241-50. bstract].
3.Mugge A. The role of reactive oxygen species in atherosclerosis. Z Kardiol., 1998;87:851-864.
4.Seven A, Seymen 0, Hatemi S. et al. Lipid peroxidation and vitamin E supplementation in experimental hyperthyroidism. Clin. Chem.,1996:42:1118-19.
5.Sics H. Oxidative stress: Oxidants and antioxidants. Exp. Physiol.,1997;82:291-95.
6.Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ. Res., 1999:85:753-66.
7.Gey KF. Prospects for the prevention of free radical disease, regarding cancer and cardiovascular disease. Br. Med. Bull,, 1993;49:679-99.
8.Kannel WB. Overview of atherosclerosis. Clin. Ther., l998;20(Suppl. B):B2-Bl7
9.Lloyd-Jones DM, larson MG, l3eiser A, et al. Lifetime risk of developing coronary heart disease. Lancet, 1999;353:89-92.
10.Fuller ill, Shipley MJ, Rose G. et al. Mortality from coronary heart disease and stroke in relation to degree ofglycaemia the Whitehall Study. Br. Med. J.,1983;287:867-70.
11.Vogel RA. Coronary risk factors. endothelial function, and atherosclerosis: A review. Clin. Cardiol., 1997;20:426-32.
12.Shestov DB, Deev AD, Klinov AV, et al. Increased risk of coronary heart disease death in men with low-density lipoprotein cholesterol in the Russian Lipid Research Clinics, prevalence follow-up study. Circulation, 1993:88:846-53.
13.Burke AP, Farb A, Malcom GT, et al. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N. EngI. J. Med., 1997;336:1276-82.
14.Liu K, Cuddy E, Pierce GN. Oxidative status of lipoproteins in coronary disease patients. Am, Heart J., 1992;123:285-90.
15.Esterbauer H, Wag G, PuhI H. Lipid peroxidation and its role in atherosclerosis. Br. Med, Bu l., 1993:49:566-76.
16.Hoffman RM, Garewal HS. Antioxidants and coronary artery disease prevention. In: Antioxidants and disease prevention, Garewal HS. eds. CRC Press L1.C, New York, 1997, pp. 13 1-47.
17.Jialal I. Evolving lipoprotein risk factors: lipoprotein (a) and oxidized low-density lipoprotein. Clin. Chem., 1998;44:1827-32.
18.Mosca L, Rubenfire M. Tarshis T, et al. Clinical predictors of oxidized low-density Iipoprotein in patients with coronary heart disease. Am. J. Cardiol.,1997;80:825-30.
19.Witting P. Pettersson K, Ostlund-Lindqvist A. et al. Dissociation of atherogenesis from aortic accumulation of lipid hvdro(pcr)xides in Watanabe heritable hyperlipidemic rabbits. J. Clin. Invest., 1999;104:213-20.
20.Heinecke JW. Is lipid peroxidation relevant to atherogcnesis? J. Clin. Invest., 1999: 104: 135-36.
21.Steinberg D, Parthasarathy S. Carew TE. et al. Beyond cholesterol: modifications of low-density lipoprotein that increase its athcrogenicitv. N. EngI. J. Med., 1989;320:915-24.
22.Upston JM, Terentis AC, Stocker R. Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiathcrogcnic supplement. FASEB. J., 1999:13:977-94.
23.Wang W, Pang CCP, Rogers MS. et al. Lipid peroxidation in cord blood at birth. Am. J. Obstet. Gynecol., 1996;174:62-65.
24.Kose K, Dogan P. Lipoperoxidation induced by hydrogen peroxide in human erythrocyte membranes-I. Protective effect of Ginkgo Biloba Extract (EGb 761). J. Intern. Med. Res., 1995:23:1-8.
25.Halliwell B. Oxidants and human disease: some new concepts. FASEB J., 1987:1:358-64.
26.Stossel TP, Mason RJ, Smith AL. Lipid peroxidation by human blood phagocytes. J. Clin. Invest., 1974;54:638-45.
27.Krolcwski AS. Kosinski EJ, Warram JH, et al. Magnitude and determinants of coronary artery disease in juvenile-onset insulin dependent diabetes mellitus. Am. J. Cardiol., 1987;59:750-55.
28.Friedewald WT. Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of preparative ultraccntrifuge. Clin. Chem.. 1972;18:499-502.
29.Valenzuela A. The biological significance of malondialdehvde determination in the assessment of tissue oxidative stiess. Life Sci., 1991:48:301-309.
30.Wu JH, Kao J, Wen M, et al. Coronary artery disease risk predicted by plasma concentrations of high-density lipoprotein cholesterol, apolipoprotcin Al, apolipoprotein B, and lipoprotein (a) in a general Chinese population. Clin. Chem.. 1993:39:209-12.
31.Davies MJ. The role of plaque pathology in coronary thrombosis. Clin. Cardiol., 1997:20(Suppl. I. Intn’ I):12-17.
32.Rozenman Y, Gotsman MS. The earliest diagnosis of acute myocardial infarction. Annu. Rev. Med., 1994;45:31-44.
33.Flitter WD, Free radicals and myocardial reperfusion injury. Br. Med. Bull., 1993:49:545-55.
34.Belch JJF, Bridges AB, Scott N, et al. Oxygen free radicals and congestive heart failure. Br. llcart J., 1991;65:254-58.
35.Pucheu S. Coudray C, Vanzetto G, et al. Assessment of radical activity during the acute phase of myocardial infarction following fibrinolysis: utility of assaying plasma malondialdehyde. Free Radic. Biol. Med., 1995;19:873-81.
36.Oen LII, Ulomo H, Suyatna F, et al. Plasma lipid peroxides in coronary heart disease. Int. J. Clin. Phannacol. ‘flier. Toxicol., 1992;30:77-80. Abstract).
37.Dincic D, Jovic P, Obradovic S, et al. Lipid peroxidation intensity and lipid status parameters in the estimation of the severity of ischaemic heart disease. Vojnosanit. Pregl., 1998;55:359-67, l Abstracti.
38.Parinley WW. Nonlipoprotein risk factors for coronary heari disease: evaluation and management. Am. J. Med., 1997;102:7-14.