Serum fasting lipid profile and lipoprotein(a) in northern keralite patients with myocardial infarction

Abstract               Coronary artery disease isrising as a major health problem in Kerala. The presentstudy was aimed to verify whether there is increase in the concentration of Lipoprotein (a) [Lp(a)] and fasting serum lipid profile in Northern Keralite Myocardial infarction(MI) patients. Fasting blood samples were taken from 606 patients and their total cholesterol, LDL cholesterol, VLDL cholesterol, HDL cholesterol, triglycerides and serum Lp(a) were determined. The control group consisted of 615 age matched healthy individuals. The mean serum levels of Lp(a) levels were found significantly higher in patients with MI (p<0.0001) as compared to the controls. The values were 24.15 ± 0.75 and 8.6 ± 0.34 mg/dl in the test and control groups respectively. The mean values of serum cholesterol were increased significantly (p<0.0001) in test group (205 ± 1.86 mg %) when compared to the control (174 ± 0.74 mg %).Similarly, the mean values of serum LDL cholesterol were increased significantly (p<0.0001) in test group (137 ± 1.68 mg %) when compared to the control (108 ± .69 mg %).Whereas, the mean values of serum HDL cholesterol were decreased significantly (p<0.030)in test group (42 ± 0.38 mg %) when compared to the control group (45 ± 0.39 mg %). Besides, the mean values of serum triglycerides (TG) were increased significantly (p<0.0001) in test group (132 ± 3.31 mg %) when compared to the healthy control subjects (109 ±1.20 mg %). The results of this study suggest that high level of Lp(a), LDL, TG and low levels of HDL have a distinctive association with MI, independent of other common coronary risk factors. Henceforth, Lp(a) level in serum appears to be a promising marker for diagnosis of coronary artery diseases.

Key Word:LDL, HDL, Triglycerides, Coronary artery disease

INTRODUCTION

Myocardial infarction (MI) is the most important concerns of coronary artery disease. MI occurs when blood flow to an area of the cardiac muscle is suddenly blocked, leading to ischemia and death of myocardial tissues.This occlusion (blockage) is most commonly due to atherosclerotic plaque, which is an unstable collection of lipids (cholesterol and fatty acids) and white blood cells (especially macrophages) in the wall of an artery. The myocardium becomes inflamed and necrotic at the point of obstruction. The damaged area quickly loses its ability to contract and conduct electrical impulses and oxygen supplies are depleted. This type of damage is irreversible and the area of necrosis is eventually replaced by fibrous scar tissues¹. Atherosclerosis of the coronary arteries commonly causes myocardial infarction (MI) and angina pectoris. Atherosclerosis remains the major cause of death and premature disability in developed societies. An integrated view of experimental results in animals and studies of human atherosclerosis suggests that the "fatty streak" represents the initial lesion of atherosclerosis. These early lesions most often seem to arise from focal increases in the content of lipoproteins within the regions of the intima. This accumulation of lipoprotein particles may not only result simply from increased permeability, or "leakiness," of the overlying endothelium, but alsothese lipoproteins bind to constituents of the extracellular matrix.It is increasing the residence time of the lipid-rich particles within the arterial wall.Lipoproteins that accumulate in the extracellular space of the intima of arteries often associate with glycosaminoglycans of the arterial extracellular matrix, an interaction that may slow the egress of these lipid-rich particles from the intima. Lipoprotein particles in the extracellular space of the intima, particularly those retained by binding to matrix macromolecules, may undergo oxidative modifications. Considerable evidence supports a pathogenic role for products of oxidized lipoproteins in atherogenesis².In the past few years, much consideration has been engrossed on serum Lipoprotein (a), mainly because of their strong association with coronary artery disease. Lp(a) is an altered form of LDL that contains the apolipoprotein B-100 portion of LDL linked to apolipoprotein A.Lp (a) levels are associated with coronary and cerebrovascular disease risk, independent of total cholesterol or LDL levels. Lp (a) may modulate fibrinolysis, and individuals with elevated Lp (a) levels have increased CHD risk³.The age-adjusted CAD (coronary artery disease) mortality rates per 100,000 are 382 for men and 128 for women in Kerala.These CAD rates in Kerala are higher than those of industrialized countries and 3 to 6 times higher than Japanese and rural Chinese. CAD in Kerala is premature and malignant resulting in death at a very young age. Approximately 60% of CAD deaths in men and 40% of CAD deaths in women occur before the age of 65 years [4]. The average age of a first heart attack decreased by at least 10 years in Kerala, in sharp contrast to a 20 year increase in many western countries.In the 1960s and 70s, heart attack in the very young (before the age of 40) was very uncommon in Kerala.Heart attack rate among men in this age group increased 40-fold by 1990 with at least 20% heart attacks occurring before age 40 and 50% before age 50^5-6.The current study was intended to verify whether there is increase in the concentration of Lp(a) and other serum lipids in Northern Keralite MI patients, without having any traditional risk factors except for positive family history and to compare the results with that of age matched healthy controls.

MATERIALS AND METHODS

A total of 1221 (n = 1221) subjects were included in this study. In which, 606 cases were encompassed in test group and 615were age and sex matched controls.In test group there were 528 (87.1%) males and 78 (12.9 %) females.Similarly in control group 535 (87.0 %) were males and 80 (13.0%) were females.Thus a total of 1221 subjects, of which 87.0% were males and 13.0% were females. Diagnosis of myocardial infarction was confirmed clinically, electrocardiographically and biochemically (elevated cardiac enzymes) were selected for the study. Subject groups with previous history of MI, stroke, heart disease, liver disease, blood disorders, type 1 diabetes, and other co-morbid illnesses were excluded. Also, patients with risk factors other than smoking, diabetes, hypertension, and dyslipidemia were excluded, since they are not major concern in Indian population. Apparently healthy subjects without any previous history of chest pain or myocardial infarction (MI) served as controls.A written consent was obtained from all the subjects or their relatives who accompanied them after explaining the details of the research work.5 mL fasting venous blood was collected from each subjects and of which 1 mL was collected in fluoride oxalate bottle, 1mL in buffered sodium citrate (3.2%) containing bottle and the remaining 3 mL in plain dry tube. Plasma separated from anticoagulated blood samples were used for the estimation of glucose and fibrinogen. The blood collected in dry tube was allowed to clot and serum was separated.This serum sample was used for all the biochemical investigations in this study.As far as possible the investigations are carried out on the same day of collection or the samples were stored at -70^oC. All the reagents and test kits for the quantitative estimation were obtained from Merck group India and Transasia biomedical company, Mumbai.

Using the serum sample, the following biochemical investigations were done:

1. Cholesterol by cholesterol oxidase-peroxidase method,
2. LDL cholesterol by turbidimetric immunoassay,
3. HDL cholesterol by turbidimetric immunoassay,
4. Triglycerides by glycerol phosphate oxidase (GPO) – Trinder method,
5. Lipoprotein (a) by multipoint calibration with fixed time mode.

Statistical Analysis: All values of the results are expressed as the mean + standard deviation. Statistical analysis was performed using the software origin-8 (Origin LAB, Origin Lab Corp., MA, USA). p value of <0.05 was considered significant and <0.01 as highly significant.

RESULT

Lipid profile: Fasting serum lipid profile was analyzed in test groups and control subjects (Fig. 1). The lipid profile consists of a group of investigations which includes cholesterol fractions (Total cholesterol, HDL cholesterol and LDL cholesterol) and triglycerides. The mean values of serum cholesterol were increased in test group when compared to the control group (healthy group with no history of myocardial infarction). The mean values were 205 ± 1.86mg% for test group and 174 ± 0.74 mg% for the control group.When test group was compared with the control group (healthy group), the mean values were found to be highly significant (P<0.0001). Similarly, the mean values of serumLDL cholesterol were increased in test group when compared to the control group.The mean values were 137 ± 1.68 mg% for test group and 108 ± .69 mg% for control group.When test group was compared with the control group, the mean values were found to be highly significant (P<0.0001).Whereas, the mean values of serumHDLcholesterol were decreased in test group when compared to the control group (healthy group with no history of myocardial infarction).The mean values were 42 ± 0.38 mg% for test group and 45 ± 0.39 mg% for the control group. When test group was compared with the control group, the mean values were found to be significantly different (P<0.030).Besides, the mean values of serum triglycerides were increased in test group when compared to the control group. The values were 132 ± 3.31 mg% for test group and 109 ±1.20mg% for the control group.When test group was compared with the control group, the mean values were found to be highly significant (p<0.0001).

Figure 1:Fasting serum lipid profile in test and control groups

Lipoprotein (a): Mean serum levels of Lipoprotein (a) values were significantly higher in MI patients.The mean lipoprotein (a) values were 24.15 ± 0.75, 8.6 ± 0.34 mg/dl in the test group and the control group respectively. When test group was compared with the control group, the mean values were found to be highly significant (P<0.0001) (Fig. 2).

Figure 2: Lipoprotein (a) in MI patients and healthy controls

DISCUSION

In this study we have observed that serum total cholesterol, triglyceride and LDL cholesterol were increased significantly in MI patients when compared with that of control subjects.In the case of HDL cholesterol, the condition was reversed.Serum cholesterol was an independent risk factor for acute myocardial infarction².Moreover, there was a multiplicative effect between cholesterol and other major risk factors on the relative risk of acute myocardial infarction⁷.High total cholesterol, high LDL cholesterol, high triglycerides, low HDL-cholesterol playing important role in Coronary Artery Disease (CAD) causation, and importance has been shown in the oxidized fraction of LDL as one of the risk factors. The main mechanisms by which LDL particles act as a risk factor have been shown by Brown and Goldstein⁸.They showed a receptor mediated uptake of LDL-cholesterol by cells subsequent to incorporation of cholesterol in cells. A study of Holvoet et al⁹ have shown that CAD patients had a higher level of circulating oxidized LDL as compared to controls and the sensitivity of this circulating oxidized LDL was 76% for cases of CAD.Many researchers reported that the prevalence of CAD among South Indians is increasing rapidly. Urgent steps are needed to modify lifestyle by increasing physical activity, modifying diet and perhaps making aggressive use of statins as part of the preventive strategy to reduce risk factors and thus, the burden of CAD in this population¹⁰. In present study the mean value of serum triglyceride were increased in test group when compared to the control group. A major number of our study group were diabetic or alcohol users or habituated with unhealthy diet. Diabetes, unhealthy diet, excess alcohol consumption etc. are certain reasons for hypertriglyceridemia. In our study we observed 30.2% of patients with abnormal serum triglyceride level. Austin et al.,¹¹ have shown high risk of CAD in patients with increased serum triglyceride levels. In a meta-analysis of 17 population-based prospective studies, increased plasma triglyceride levels were associated with increased coronary disease risk in both men and women, after adjustment for HDL cholesterol and other risk factors^12.Hiroyasu Iso et al.^,13 reported that serum triglycerides predict the risk of coronary heart disease, independent of total cholesterol and HDL cholesterol, among Japanese men and women who possess low mean values of total cholesterol by Western standards.In the present study,mean values of serumHDL cholesterol were decreased in test group when compared to the control group(P<0.030).The HDL particles induce the removal of cholesterol from cells, including those in atherosclerotic plaques, and carry them to the liver, by reverse cholesterol transport.The association between reduced HDL cholesterol levels and increased risk of heart disease is, well established; independently of triglyceride level and other risk factors¹⁴. But the mechanisms by which HDL confer protection from atherosclerosis include more than just reverse cholesterol transport.HDL particles seem to have anti-inflammatory and antioxidant properties, inhibiting the oxidation of LDL cholesterol and the expression of cellular adhesion molecules and monocyte recruitment.The HDL may also reduce the risk of thrombosis by inhibiting platelet activation and aggregation. Roe et al.,¹⁵ reported thatalmost one-fifth of patients with acute coronary syndromes (ACS) have very low HDL levels (10-29 mg/dL), a finding that adds incrementally to a greater burden of atherosclerosis and a higher risk of mortality; 35% had low HDL levels (30-39 mg/dL), 39% had normal HDL levels (40-59 mg/dL), and 9.0% had high HDL levels (60-100 mg/dL).Consequently, strategies for mitigating the adverse prognosis associated with very low HDL levels warrant further exploration in patients with ACS. Watkins et al¹⁶has shown a decrease in the risk of CAD by 2.3% with every 1 mg/dl rise in serum HDL-cholesterol.A study conducted by American Medical Association¹⁷ had shown increased risk of CAD in patients with total cholesterol - HDL cholesterol ratio > 4.However, in our study the value of fasting Lipid Profile ofmajority of the test subject were normal while comparing with that of western population.According to the western standards serum total cholesterol level of 36.1% of our test subjects were within normal limit.LDL cholesterol level of 55.9% MI patients, serum triglyceride level of 69.8% MI patients and HDL cholesterol level of 92.1% MI patients in this study were normal when compared to western population.From the present study we pointed out the importance to develop the reference range in Indian scenario. Many physicians still follow the reference range according to the western standards. Although South Asians have levels of LDL cholesterol comparable to other populations, LDL particle size tends to be smaller¹⁸. Small LDL particles, through increased susceptibility to oxidation, are more atherogenic than larger particles.HDL particle size, in addition to the actual level of HDL cholesterol, also appears to be an important predictor of CHD risk.South Asians not only have lower HDL levels but also have a higher concentration of small, less-protective HDL particles¹⁹.Asian Indian males have a higher prevalence of low HDL2b than non-Asian Indians, which suggests impaired reverse cholesterol transport. This finding was also observed in Asian Indian men with apparently normal HDL values²⁰.A study of Ram B Singhaet al²¹ in Indian population shows that Serum cholesterol level was directly related to prevalence of coronary artery disease even in those with low cholesterol concentration (<5.18 mmol/l) and it is possible that some Indian populations may benefit by increased physical activity and decline in serum cholesterol below the range of desired serum cholesterol in developed countries.So the major concern of our observation is that subjects who maintain desirable cholesterol concentration as per western standards also are targets for myocardial infarction.In the circumstances, further study is needed to implement an ideal normal range of serum or plasma lipid of our population.Also analysis of other risk factors that are non-conventional and newly emerging will be of immense importance in the eventual assessment of the risk status. Lp (a) is an LDL like particle which has apolipoprotein (a) attached to apolipoprotein B molecule via a disulphide bond. There are 34 different Lp (a) isoforms depending on the size of apo (a) polymorphism. Plasma Lp (a) levels are highly hereditary and a stable lifelong level is attained by age two.Ninety percent of the variation in plasma levels is accounted by the apo (a) gene and 70 % by the size of apo (a) isoforms ²².Lp (a) may contribute to the development of not only atherosclerosis like LDL, but alsothrombosis and thus MI ²³.Mechanism of pathogenicity of excess Lp (a) include enhanced thrombogenesis and impaired fibrinolysis by competing with plasminogen, inhibition of transforming growth factor B, destabilization of plaque, increased smooth muscle cell proliferation and migration, formation of occlusive thrombus, impaired formation of collateral vessels, enhanced oxidation uptake and retention of LDL cholesterol and up regulation of expression of the plasminogen activator inhibitor²⁴In the present study we observed that Lp (a) level in test group was highly significant while comparing with healthy control group (P<0.0001).Lima et al³ determined serum levels of Lp (a) and lipid profile of a group of individuals submitted to coronary angiography, with the aim of establishing the possible correlation between these parameters and the severity of coronary artery disease.The value obtained in the test and control groups for Lp(a) indicate a progressive increase in the serum levels of this parameter according to the severity of coronary atheromatosis. Similar findings have been reported by Uusimaa et al²⁵ that the serum Lp(a) level is associated with the angiographic severity of CAD.Atherosclerotic plaques, but not normal human arteries contain Lp (a),and results from in vitro and animal studies have implicated Lp (a) in foam-cell formation, smooth muscle–cell proliferation, and plaque inflammation and instability^26-27.Lp (a) can cross the endothelial barrier between plasma and the arterial intima and may be trapped within the arterial intima, particularly at sites of injury. Thus, Lp (a) can deliver cholesterol to atherosclerotic plaques like LDL. Furthermore, Lp (a) promotes thrombosis partly through competitive inhibition of plasmin generation and through inactivation of tissue factor pathway inhibitor, a potent inhibitor of the tissue factor–mediated coagulation cascade.In present study we found that the clinicians here in north Kerala were not considering the Lp (a) value at all and they were giving importance to FLP values for the treatment of MI patients. But it is to be included in routine analysis for better management of CAD. Geethanjali et al²⁸ studied the Plasma lipoprotein (a) levels in south Indian patients and reported that it is significantly elevated in patients with coronary artery disease as compared to controls. Zorio et al²⁹ reported that Lp(a) levels are markers of early MI and that Lp(a) levels >30 mg/dL are associated with severe patterns of coronary atherosclerosis. Extreme lipoprotein (a) levels predict a three to four fold increase in risk of MI in the general population and absolute ten year risks of 20% and 35% in high-risk women and men. However, Nascetti et al³⁰ did not observe any change in Lp (a) levels in cardiovascular disease patients and they concluded that Lp (a) should not be considered as an independent risk factor in CVD patients. So both the prothrombic and atherogenic mechanisms of Lp (a) were providing more defined indications for the determination of Lp(a) values in clinical practice.Here, there was enough evidence to support the introduction of routine assessment of Lp(a) levels in clinical laboratories in the monitoring of patients at risk for coronary artery disease.

CONCLUSION

The results of this study suggest that high level of Lp(a), LDL, TG and low levels of HDL have a distinctive association with MI, independent of other common coronary risk factors. In past few decades the combat to reduce the incidence of coronary artery disease has directed the researchers to look for various clinical markers, which would aid early diagnosis of the diseases. Our data also support that regardless of othertraditional risk factors, increase in Lp(a) is an independent risk factor for MIpatients with positive family history. Thus elevated levels of Lp(a) may assist as an important criterion to identify the individuals who need to undergo Lp(a)lowering treatment, thereby preventing them from coronary artery diseases. Thus, the Lp(a) level in serum appears to be a promising marker for diagnosis and treatment of coronary artery diseases in Kerala population as well.

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