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Study of Lipid Profile in Coronary Heart Disease patients in Libya

Rambabu Kondredddy5, Ali Chenak1,Uma Shankar Akula4, Addison garabet1, Shakila Srikumar3, Abdalla M Jarari2, Jagannadha Rao Peela3*
1 Department of Biochemistry,Faculty of Medicine, SIRT University., Sirt, Libya, 2 Department of Biochemistry, Faculty of Medicine, Benghazi University., Benghazi, Libya. 3 Quest International University Perak, Ipoh, Malaysia, 4 Department of Biochemistry, Faculty of Medicine, AIMST University, Bedong, Malaysia, 5 Department of Biochemistry, Mamata medical college, Khammam, India Correspondence: * Assistant Professor, Department of Biochemistry, Faculty of Medicine, Quest International University Perak, 30250 Ipoh,Perak, Malaysia.

 eela.jagannadha@qiup. p edu.my   peelajagannadh@ rediffmail.com. Ph: +60105010039

Abstract

Introduction: Coronary Heart Disease (CHD) is widely prevalent across the globe
and significantly high level of Cholesterol in circulation is a single major risk factor associated with Coronary Heart Disease. There have been numerous studies confirming the association of hyperlipidemias with Coronary Heart Disease in most of the Western and Asian countries of the world, where this disease imposes high personal, social and economic burden. This study conducted on a small population in Libya revalidates this finding.

Objective: The current study has been conducted for a period extending from
2007 to 2009 on a small population of patients from cardiac unit in the general hospital attached to a medical university in the city of Sirt, Libya, to estimate the predictive value and revalidate an association of abnormal lipid profile with Coronary Heart Disease in this area. Not many studies concerning this association of lipid parameters with CHD had been conducted in Libya. Hence this pilot study was initiated in a small group with an intention of continuing in a larger population to determine the normal and hence abnormal ranges in this particular region and further on, extending the work on evaluating genetic variations in this Mediterranean area.

Materials and Methods: The current study is a case-control pilot study; conducted on a total of 93 patients diagnosed with Coronary Heart Disease (history of angina or surviving myocardial infarction) with or without diabetes mellitus and hypertension, from a coronary care unit of Ibn Sina teaching hospital and from the Clinic of Cardiology in Sirt Central Polyclinic. The study extended for a period of two years from 2007 up to 2009. 167 clinically healthy subjects served as population-based controls. The age of the cases as well as the controls ranged from 41 to 80 years. Fasting samples of blood were collected both from cases and healthy controls and sent to the laboratory for processing and performance of the lipid profiles in batches. Lipid profiling included Total Cholesterol (TC), Low Density Lipoprotein-Cholesterol (LDLC), High Density Lipoprotein-Cholesterol (HDLC), Triacylglycerols (TAG) and calculating Very Low Density Lipoprotein-Cholesterol (VLDLC).

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Results: Lipid profile of patients with Coronary Heart Disease showed significant
variation from that of the control group. The Total Cholesterol (p<0.05) and Low

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Density Lipoprotein-Cholesterol (p<0.05) were significantly higher in the cases, whereas the High Density Lipoprotein-Cholesterol had significantly lowered (p<0.05) from that of the controls. Serum Triacylglycerol was raised among cases than that of the controls but was not significant statistically. The Total Cholesterol to High density lipoprotein-Cholesterol and Low density lipoprotein-Cholesterol to High density lipoprotein-Cholesterol ratios also were significantly higher in cases than in controls, whereas the rise in Triacylglycerol to High Density Lipoprotein-Cholesterol ratio was not found to be significant.

Conclusion: The findings of the present study indicated high serum cholesterol as an important single
risk factor for Coronary Heart Disease and an increase in the levels of LDL-Cholesterol and a decrease in HDL-Cholesterol add further value to the association observed. Levels of the blood lipids are lower even among the patients is an interesting finding in this study, despite there being a variation between the cases and the controls. Hence a need to extend the study on a larger population to determine the normal range in this region and an urgent need for appropriate interventions to maintain the serum total cholesterol and LDL-C levels within a prescribed normal range.

Key words: Cholesterol, Lipoproteins, Triacylglycerol, Coronary Heart Disease, Sirt, Libya.

Introduction
Coronary heart disease (CHD) is one of the most important causes of morbidity and mortality in developed countries.1, and the burden of CHD is now increasing in developing countries also2. By the year 2020, World Health Organization (WHO) is predicting more than 11.1 million deaths from CHD. It is projected that the annual number of deaths due to cardiovascular disease will increase from 17 million in 2008 to 25 million in 2030.3 Approximately out of 14 million Americans affected with coronary heart disease(CHD), 1.5 million develop Acute Myocardial Infarction (AMI), and 500,000 of these individuals die every year.4 The epidemic of CHD in Sub Saharan African countries(SSA) is driven by multiple factors working collectively. Lifestyle factors such as diet and smoking contribute to the increasing rates of CHD in SSA. Some lifestyle factors are considered gendered in that some are more common in women and others in men. For instance, obesity is a predominant risk factor for women when compared to that for men, but smoking still remains a risk factor for men6. CHD requires an integrated approach to the reduction of its risk factors. Identification and management of risk factors are essential for preventing CHD in asymptomatic individuals mainly over 40 years of age as primary prevention, and for preventing recurrent events in patients with established disease as secondary prevention7. Risk factors management should be conceived as prevention or treatment of the atherosclerotic disease process itself.8 CHD risk factors are modifiable and unmodifiable; the presence of unmodifiable risk factors may necessitate more intense management of modifiable risk factors.9 In the last thirty years, considerable advances have been achieved in the determination and improvement of CHD

risk factors including diabetes10. Lipids and lipoproteins have become increasingly important in clinical practice, primarily because of their association with CHD, in case of their abnormalities known as dyslipidemia, and became the major risk factor for the development of this disease, according to epidemiological studies, especially in affluent countries where fat consumption is high.11 Although there are several studies on the incidence and prevalence of CHD in most countries of the world, data from Libya and other Arab countries are generally minimal as compared to other countries. Hence, this study was carried out to estimate the predictive value and the correlation of lipid profile with CHD in a population from Sirt city, a small town in North Central Libya, bordering the Mediterranean Sea. People here enjoy a Mediterranean climate, and consume a traditional Mediterranean diet rich in olive and corn oil and camel and sheep meat.

Materials and methods

Subjects
The present study is a case-control study carried out on a total of 93 CHD patients (history of angina or surviving myocardial infarction) with or without DM and HTN, admitted and diagnosed in coronary care unit of IBN SINA Teaching Hospital and Clinic of Cardiology in Sirt Central Polyclinic. CHD cases with liver impairment, renal disease or thyroid disease were not included in the study. 167 clinically healthy subjects aged 41 to 80 years who served as population-based controls were chosen. This study was initiated after obtaining consent from the patients and healthy volunteers and after a prior approval by the Sirt university ethics committee.

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Methods
Blood from healthy controls was drawn after an overnight fast (for 12-14 hours) with only water being allowed during the last 8 hours. Each participant was instructed to be on their regular diet for the previous week. Blood from the patients diagnosed with CHD was collected the next morning of their admission. Blood samples collected in anticoagulant coated tubes were centrifuged after 30 minutes and the plasma was separated and frozen at -20C; lipid profile was performed within one week for each group of samples, after running the controls for confirmation of the accuracy of each test, according to the procedures provided with Biocon kits.

Methods
Total cholesterol (TC) was estimated using the Cholesterol Oxidase Phenol 4-Aminoantipyrine peroxidase (CHOD-PAP) method.12,13,14 Triacylglycerols (TAG) levels were measured by GPO-PAP enzymatic method12,15 High density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were estimated using a two-step procedure: (i) precipitation and (ii) enzymatic determination.12,16,17 The National Cholesterol Education Panel Adult Treatment Panel III (NCEP ATP III) classification of these parameters is given in Table 1.18

Results
Table 1: ATP III Classification of Total, LDL, and HDL Cholesterol and Triacylglycerol.
Cholesterol and triacylglycerols, mg/dl Total cholesterol <200 200-239 >240 LDL cholesterol <100 100-129 130-159 160-189 >190 HDL cholesterol <40 >60 Triacylglycerols <150 150-199 200-500 >500 Descriptor Desirable Borderline high High Optimal Near optimal/above optimal Borderline high High Very high Low High Normal Borderline high High Very high

TC and LDL-C levels showed a significant increase while HDL-C was significantly decreased among the cases. Mean and standard deviation values of TC in the cases was 195.4421.58 mg/dL (MeanSD) vs. 189.4210.45 mg/dL in the controls (p<0.05). LDL-C level in the cases was 127.4213.10 mg/dL vs. 117.677.10 mg/dL in the controls (p<0.05). HDL-C level in the cases was 34.841.66 mg/dL compared to 39.282.94 mg/dL in the controls (p<0.05). TGs did not show a significant increase in cases (164.7828.76 mg/dL), compared to the controls (165.4115.12 mg/dL). The TC/HDL-C ratio and LDLC/HDL-C ratio were significantly high in cases but the ratio between TAGs and HDL-C was not significantly high. In the present study, the incidence of CHD was more among males; especially those in the age group of 51 to 60 years (Fig. 1). Lipid profile of patients in CHD was significantly different from that of the controls; though within the desirable levels ( Table 1). TC (p<0.05) and LDL-C (p<0.05) were significantly higher while the HDL-C was significantly lower (p<0.05) in cases in comparison with that of controls (Fig. 2). TAG was raised among patients with CHD than that of the controls but not significant statistically. The TC to HDL-C and LDL-C to HDL-C ratios were significantly higher in CHD patients than controls, whereas the rise in TAG to HDL-C ratio was not significant (Fig. 2). The results of total cases and controls are summarized in Table.2 and the age-variable lipid profiles in males and females are described in tables 3 and 4.

ATP, Adult Treatment Panel; HDL, high-density lipoprotein; LDL, low-density lipoprotein. * Primary target of therapy. Source: National Cholesterol Education Panel Adult Treatment Panel III Expert Panel (including PE McBride):Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Circulation 2002;106:3143-342. Statistical methods in this study was done by using Microsoft excel and Graph Pad software, GraphPad inc, USA.

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Fig. 1. Age related incidence of CHD.

Fig. 2. Lipid profile in CHD.

Table.2 - Mean and standard deviation values of total cases and controls
Tc (mg/dl) Controls Cases P-value 189.4210.45 195.4421.58 <0.05 Tag (mg/dl) 165.4115.12 164.7828.76 Ns Hdl( mg/dl) 39.282.94 34.841.66 <0.05 Ldl( mg/dl) 117.677.10 127.4213.10 <0.05

TC: Total Cholesterol, TAG: triacylglycerol, HDL: High density Lipoproteins, LDL: Low density lipoproteins, NS: not significant.

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Table.3 Males.
TC (mg/dl) Age:41-50 CONTROLS CASES P-VALUE Age:51-60 CONTROLS CONTROLS P-VALUE Age:61-70 CONTROLS CASES P-VALUE Age:71-80 CONTROLS CASES P-VALUE 175.1226.95 172.8224.25 0.843 152.919.97 133.837.34 0.17 34.846.6 33.45.78 0.642 111.816.87 113.226.02 0.8881 0.6084 0.6079 0.4867 198.0819.94 166.5828.88 0.0051 174.6631.59 134.646.15 0.994 37.894.15 33.515.71 0.042 122.4117.02 116.1622.13 0.446 0.503 0.427 0.0318 119.2517.08 206.8121.15 0.0293 169.7524.91 192.8716.74 0.0042 38.435.63 34.125.71 0.0397 121.520.3 126.6218.15 0.457 0.0001 0.0046 0.0032 179.1428.07 199.2813.56 0.0229 155.7843.77 161.3543.4 0.741 39.145.89 35.714.33 0.085 112.3521.94 127.0711.72 0.0265 0.0401 0.177 0.0003 TAG(mg/dl) HDL(mg/dl) LDL(mg/dl) TC/HDL TAG/HDL LDL/HDL

TC: Total Cholesterol, TAG: triacylglycerol, HDL: High density Lipoproteins, LDL: Low density lipoproteins,

Table.4 Females.
TC (mg/dl) Age: 41-50 CONTROLS CASES P-VALUE Age:51-60 CONTROLS CONTROLS P-VALUE Age:61-70 CONTROLS CASES P-VALUE Age:71-80 CONTROLS CASES P-VALUE 201.7218.88 172.7231.63 0.023 161.6331.8 137.0943.99 0.168 38.633.88 33.276.94 0.047 12814.78 111.8123.16 0.0187 0.814 0.02 0.948 19914.14 222.225.86 0.0228 175.536.71 196.628.37 0.1178 38.35.1 34..56.15 0.1499 123.213.01 146.324.76 0.0178 .0001 0.034 0.0143 178.429.11 208.423.43 0.0164 190.344.09 19842.18 0.694 42.86.32 36.25.61 0.0238 107.335.08 139.418.96 0.0203 .0001 0.153 0.0004 192.725.49 214.720.67 0.0482 142.843.51 161.420.06 0.235 44.27.09 384.57 0.0321 114.815 138.818.34 0.0049 0.002 0.167 0.0001 TAG(mg/dl) HDL(mg/dl) LDL(mg/dl) TC/HDL TAG/HDL LDL/HDL

TC: Total Cholesterol, TAG: triacylglycerol, HDL: High density Lipoproteins, LDL: Low density lipoproteins,

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Discussion
In developed countries like the United States, although there has been a very significant decrease in the incidence of vascular diseases like CHD1, cerebrovascular disease, and peripheral vascular diseases, yet CHD remains to be the major cause of death. The major risk factors are elevated LDL-C, reduced HDL-C, smoking, hypertension, insulin resistance with or without overt diabetes mellitus, age, and family history of premature CHD. Modifiable risk factors account for 85% of the elevated CHD risk, of which the most important is plasma cholesterol.19,20 TC levels of <160 mg/dl is able to decrease CHD risk, even if other risk factors are present. The key role of cholesterol in CHD has given rise to the universally accepted cholesterol-diet-CHD hypothesis. According to this hypothesis, increased plasma cholesterol concentrations increase the risk of CHD and decreasing plasma cholesterol levels decreases the risk of CHD.21 The Multiple Risk Factor Intervention Trial (MRFIT) showed that there is an increased risk at levels >200 mg/dL.22 The Seven Countries Study also demonstrated that elevated plasma cholesterol levels increased the incidence of CHD.23 The Framingham study clearly demonstrated the association of elevated cholesterol with CHD.24 Epidemiologic studies have linked the intake of high levels of dietary fat rich in cholesterol and saturated fats, with increased plasma cholesterol levels.25,26 Therefore, restriction of saturated fat and cholesterol is the cornerstone of dietary therapy to reduce elevated blood cholesterol levels. In the present study CHD incidence was more among males, especially those in the age group 51- 60 years. Among females, there was no difference in the age based incidence, and the incidence was lower than that in males except in the 71-80 age groups, where it was slightly higher among females. Despite the extensive literature on the relationship of lipid and lipoprotein particles to CHD incidence, there has been controversial evidence on the specific association of TAG with CHD. The Framingham study demonstrated that TAGs are independently related in women at all ages but missing statistical significance in the multivariate studies in men. According to two meta-analyses, TAGs were independent risk factors for CHD, even after adjustment with HDL-C, which is strongly and inversely correlated with TAGs.27. In the present study, although there was an increase in the triacylglycerol levels in the cases compared to the controls; yet the increase was not statistically significant. When an age and gender-wise analysis was done, it was found to be statistically significant only in men in the 51-60 years age group. In spite of the ambiguous evidence, it is considered that elevated TAG increases the risk

of CHD according to international guidelines. In European and US guidelines, Hypertriacylglycerolemia in combination with low HDL-C levels are considered as at risk when deciding on therapy28. Both basic science and clinical studies support the inverse relationship between HDL-C levels and atherosclerosis. HDL enhances the reverse cholesterol transport and has antioxidative, anti-inflammatory, antithrombotic, and vasoprotective effects.29 Studies have also consistently demonstrated that HDL-C is inversely associated with the risk of CHD.30 Thus, an increase in HDL-C is linearly associated with a reduction in cardiovascular risk. In line with these findings, present study demonstrated a significant decrease in the HDL-C levels in patients with CHD when compared to controls. Additionally, this decrease was significant both in males and females in all age groups, except in the age group 71-80 in males and 61-70 in females. Clinical trials of HDL-C-modifying therapies have reported that an increase in HDL-C was associated with a reduction in adverse cardiovascular events.31 In the Bezafibrate Infarction Prevention trial, every 5 mg/dl increase in HDL-C was associated with a 27% reduction in risk of cardiac mortality.32 Another recent prospective cohort study of apparently healthy male physicians demonstrated that an increase in HDL-C of 12.5 mg/dl over 14 years was associated with a 57% lesser risk of developing CHD.33 However, according to major clinical guidelines, HDL-C is a secondary target in CHD prevention. Current guidelines from the Adult Treatment Panel III emphasize on targeting primarily LDL-C, secondarily nonHDL-C, and then HDL-C.34 According to the American Diabetes Association, HDL-C should be considered a secondary target along with TAG, with a goal of HDL-C levels >40 mg/dL.35 The recent American Heart Association / National Heart, Lung, and Blood Institute scientific statement proposes that HDL-C should be a tertiary target, following LDL-C and TAG, with goals of HDL-C levels >40 mg/dl in men and >50 mg/dl in women.36 The importance of LDL-C in the pathogenesis of CHD is well documented, and so is the benefit of lowering LDL in highrisk patients.37 This study demonstrated a significant increase in LDL-C in the CHD group. In women, the increase was statistically significant in all age groups, but it was statistically significant in males only in the 41-50 age groups. The National Cholesterol Education Program (NCEP) recommends an LDL-C goal of <100 mg/dl in patients with established CHD and in those who are CHD risk-equivalent. Aggressive LDL-C reduction is associated with less atherosclerosis progression, lower rates of revascularization, and fewer ischemic events compared with moderate LDL-C reduction or conventional treatment.38 Considering that an increase in both TC and a decrease in HDL-C is associated with an increase in the risk of CHD, a ratio of TC/HDL-C ratio is also important in assessing the Copyright iMedPub

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risk for CHD. According to the Framingham Heart Study, the TC/HDL-C ratio is another powerful predictor at all ages in women and is the only lipid predictor independently related to CHD in men 65 to 80 years old.24 In this study, there was an overall significant increase in the TC/HDL-C; it was extremely significant (p<0.0001) in men of 51-60 age group and in women between 51-70 years of age. In another study of patients with heterozygous familial hypercholesterolemia subjects, plasma HDL cholesterol values and TC/HDL ratios were found to be two important coronary risk factors. Hence, the authors opined that treatment of familial hypercholesterolemia should focus not only on lowering total and LDL cholesterol levels, but also on increasing HDL cholesterol values for coronary heart disease prevention.39 A high TAG level and low HDL-C are basic characteristics of insulin resistance and the metabolic syndrome, which is strongly associated with CHD.40 TAG/HDL-C ratio, is a relatively new lipoprotein index that indicates the presence of small dense LDL particles and could serve as a good predictor of CHD. The TAG/HDL-C ratio indicates the relative size of LDL particles and therefore, their atherogenic potential. A low TAG/HDL-C ratio indicates the presence of primarily large, non-atherogenic LDL particles, while a high TAG/HDL-C ratio indicates a larger proportion of small, dense pro-atherogenic LDL particles41. With an increase in the TG/HDL-C ratio and TC/HDL ratio, HDL particles are shifted toward smaller sizes, implying that the maturation of HDL-C is hampered, resulting in an increased risk of CHD.42 In the present study, the TAG/ HDL-C ratio was increased in the cases but was not significant statistically. The association was very significant only among males in the age group of 51-60 years. The LDL-C/HDL-C ratio is a valuable and a standard tool to evaluate CVD risk in all populations43. In a study evaluating the prognostic significance of several risk factors on the outcome of CHD in 639 cardiovascular disease-free subjects with heterozygous familial hypercholesterolemia (FH), it was found that a one-unit difference in LDL-C/HDL-C ratio was associated with a 17% higher risk. This study shows that LDL-C levels eight-times more than HDL-C predicts an adverse CHD event, in patients with FH.44 This study showed an overall significant increase in the LDL-C/HDL-C ratio. When an age and gender-wise analysis was done, a significant increase in this parameter was seen in both men and women in the CHD group across all age groups except those aged between 71 and 80 years.

Conclusion
Coronary heart disease is a major public health problem globally, being an important cause of morbidity and mortality. Plasma cholesterol levels have been implicated in the causation of CHD. The findings from this study revalidate this scientific observation. We observed that there was a significant rise in the levels of TC and LDL-C even among patients who had recovered from CHD when compared to the controls. Triacyliglycerol level was also increased among the CHD group from that of the control group, but was not statistically significant. HDL-C was significantly reduced in the CHD group compared to controls. The TC/HDL-C and LDL/HDL-C ratios were significantly increased in the CHD group; whereas TAG/ HDL-C ratio was not significantly elevated. These findings in our study indicate that CHD is associated with an increase in the TC and LDL-C and and a decrease in HDL-C levels when compared with the controls despite the levels being within the desired levels as per the Adult Treatment Panel III ( Table 1). In this study, the highest incidence of CHD was observed in males between 51-60 years of age. It is therefore important to focus on reduction of cholesterol levels in the Libyan population much more than the levels observed in populations studied elsewhere. Lifestyle measures like consumption of a proper diet along with regular exercise; and if required, cholesterol-lowering therapy (e.g. statins) should be initiated in susceptible populations even though the levels may be well within the internationally desired levels ( Table 1) The present study aims at the creation of public awareness about the risk factors for cardiac disease, regular screening and necessary interventions. Readers would not only have a general idea about the risk factors for cardiac disease, but would also appreciate the significance of conducting lipid profile, a clinical chemistry laboratory investigation as a screening procedure and understand that the range of normal values can vary regionally. The present study was not funded and supported by any agency and hence there is no conflict of interest.

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