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Association of low-density lipoprotein particle size and ratio of different lipoproteins and apolipoproteins with coronary heart disease

      Summary

      Background

      Worldwide coronary heart disease (CHD) is estimated to be the leading cause of death. Current knowledge about prevention of CHD is mainly derived from developed countries. Therefore, this study aimed to find out the association of CHD with ratios of different lipoproteins and apolipoproteins, LDL particle size, as well as different traditional risk factors in Asian Indian population in Eastern part of India.

      Methods

      Case–control study of 100 patients with CHD and 98 healthy controls were age and sex matched. After clinical evaluation, blood samples were collected for biochemical assays.

      Results

      Multivariate logistic regression analysis found apoB (OR 2.96; 95% CI 1.02–8.54), apoB/HDL-c (OR 4.14; 95% CI 1.33–12.83), nonHDL-c (OR 5.41; 95% CI 2.08–14.10), apoB/apoAI (OR 6.64; 95% CI 2.37–18.57), and LDL particle size (9.59; 95% CI 2.92–31.54) were independently associated with CHD. Area under the ROC curves derived from the model (AUROC 0.947; 95% CI 0.916–0.977) are significantly higher than any other variables.

      Conclusions

      Findings from the multivariate analysis, apoB, apoB/HDL-c, nonHDL-c, apoB/apoAI, and LDL particle size are potent indicators and useful for diagnosis of predisposed CHD.

      Keywords

      Introduction

      Coronary heart diseases (CHD) account for a large proportion of all deaths and disability worldwide. Coronary artery disease (CAD) [
      • Ailhaud G.
      ] and ischemic heart disease [
      • Lamarshe B.
      • Després J.P.
      • Moorjani S.
      • Cantin B.
      • Degenais G.R.
      • Lupien J.R.
      Prevalence of dyslipidemic phenotypes in ischemic heart disease.
      ] are the synonyms of CHD. The prevalence of CHD is known to be high among people of Asian Indian origin. Indians settled in the USA have a four fold higher prevalence of CHD than Caucasian Americans and six fold higher hospitalizations than Chinese Americans [
      • Enas E.A.
      Coronary artery disease epidemic in Indians: a cause for alarm and call for action.
      ]. Rates are rising in India, and by 2015 CHD has been predicted to rank first among the causes of death in the Indian population [
      • Reddy K.S.
      Cardiovascular disease in India.
      ]. The high tendency to develop premature and accelerated CHD in Asian Indians is only partially explained by the presence of conventional risk factors such as insulin resistance and abdominal obesity [
      • Misra A.
      • Vikram N.K.
      Insulin resistance syndrome (metabolic syndrome) and Asian Indian.
      ], etc. Among the conventional risk factors, elevated levels of serum total cholesterol (TC) [
      • Stamler J.
      • Daviglus M.L.
      • Garside D.B.
      • Dyer A.R.
      • Greenland P.
      • Neaton J.D.
      Relationship of baseline serum cholesterol levels in 3 large cohorts of younger men to long-term coronary, cardiovascular and all cause mortality and to longevity.
      ], low density lipoprotein cholesterol (LDL-c) [
      • Srinivasan S.R.
      • Ehnholm C.
      • Wattignecy W.A.
      • Bao W.
      • Berenson G.S.
      The relation of apolipoprotein E polymorphism to multiple cardiovascular risks in children: the Bogalusa Heart study.
      ], triglycerides (TG) [
      • Wiklund O.
      • Angelin B.
      • Olofsson S.-O.
      • Ericson M.
      • Fager G.
      • Berglund L.
      • et al.
      Apolipoprotein (a) and ischemic heart disease in familial hypercholesterolemia.
      ,
      • Satoh H.
      • Nishino T.
      • Tomita K.
      • Tsutsui H.
      Fasting triglyceride is a significant risk factor for coronary artery disease in middle aged Japanese man—result from 10 year cohort study.
      ], apolipoprotein B (apoB) [
      • Larmache B.
      • Lemieux I.
      • Després J.P.
      The small, dense LDL phenotype and the risk of coronary heart disease: epidemiology, pathophysiology and therapeutic aspects.
      ], and lower levels of high density lipoprotein cholesterol (HDL-c) [
      • Stampfer M.J.
      • Sacks F.M.
      • Salvini S.
      • Willent W.C.
      • Hennekens C.H.
      A prospective study of cholesterol, apolipoproteins and the risk of myocardial infarction.
      ,
      • Sun Y.H.
      • Yang Y.J.
      • Pei W.D.
      • Wu Y.J.
      • Gao R.L.
      Patients with low high-density lipoprotein-cholesterol or smoking are more likely to develop myocardial infarction among subjects with a visible lesion or stenosis in coronary artery.
      ] and apolipoprotein AI (apoAI) [
      • Chan L.
      Apolipoprotein B, the major protein component of triglyceride rich and low density lipoproteins.
      ] are the well known and established biomarker in CHD. Small dense LDL is considered to promote atherosclerosis in CHD because of its low affinity for LDL receptors [
      • Galeano N.F.
      • Al-Haideri M.
      • Keyserman F.
      • Rumsey S.C.
      • Deckelbaum R.J.
      Small dense low density lipoprotein has increased affinity for LDL receptor independent cell surface binding sites: a potential mechanism for increased atherogenicity.
      ] and susceptibility to oxidative modification [
      • Kondo A.
      • Muranaka Y.
      • Ohta I.
      • Notsu K.
      • Manabe M.
      • Kotani K.
      • et al.
      Relationship between triglyceride concentration and LDL size evaluated by malondialdehyde-modified LDL.
      ].
      Since the number of deaths in the Indian population due to CHD have drastically increased, it is not surprising that the medical fraternity is now focusing more on finding preventive measures. Current knowledge about the prevention of CHD and cardiovascular disease is mainly derived from studies done in populations of European and American origin. As far as the eastern part of India is concerned, there was no study of different apolipoproteins levels, assessment of LDL particle size, and some ratios of lipoproteins and apolipoproteins with the conventional risk factors in blood, which are connected with CHD.
      We have, therefore, attempted to find out the association of CHD with LDL particle size, ratios of different lipoproteins and apolipoproteins, as well as different traditional risk factors and the diagnostic performance of this newly proposed biomarker in CHD compared against known lipid markers. Hence this investigation was undertaken to determine whether the ratios of different lipoproteins and apolipoproteins are associated with CHD and also whether apoB/apoAI ratio is better than the cholesterol ratios to predict CHD. So it is very important to study the newly arisen biomarker along with the conventional risk factors among Asian Indian population in the eastern part of India.

      Methods

      Population description and sample collection

      All subjects are Indian adults (1) CHD group: 100 patients (86 male, 14 female) with typical angina and electrocardiographic study, tread mill test, stress echo and echocardiographic evidence of ischemia or infarction, aged between 45 and 65 years old. (2) Control group: 98 healthy age and sex-matched subjects (86 male, 12 female) aged between 45 and 65 years. The controls comprised the spouses, neighbors, and people from same work place of the patients, with the same socio-cultural background, in whom the clinical history, the objective search for signals of CHD, and the electrocardiographic as well as echocardiographic examination did not suggest the presence of that disease. All patients and controls with ancestral origin were from the eastern part of India. The present study was conducted during the period of October 2006–February 2007 at the out-patients department and also with in-patients of NRS Medical College & Hospital, Kolkata. The subjects of the present study were part of a health examination between the Immunotechnology Section, Bose Institute, Kolkata and the Department of Cardiology, NRS Medical College & Hospital, Kolkata. The institutional ethical committee approved the study protocol. Informed consent was obtained from the participants. The cardiologist completed a clinical questionnaire for each subject. Venous blood samples (5 ml) were collected in the morning at the point of medical check-up into a sterile tube after a 12-h overnight fast and also before the patient took any lipid-lowering drugs. Any patients or controls found to have taken any lipid-lowering drugs were excluded from the study. Serum was harvested on the day of sample collection by centrifugation at 3000 rpm for 10 min at room temperature using tabletop centrifuge (Remi Pvt. Ltd., Mumbai, India). Subsequently, the serum was divided into aliquots for determination of lipids, glucose, apoA1, apoB, and apoE analysis.

      Questionnaires and clinical characteristics

      Questionnaires were distributed at the time of the medical check-up. Participants were questioned about smoking and about any use of lipid-lowering medication. They were questioned about the amount of alcoholic beverage (country liquor) drunk per day as well as number of cigarettes, or “biri” (a type of local cigarette) smoked per day. Information about personal and family history of cardiovascular disease, and risk factors (hypertension) was obtained. Waist circumference was measured just above the naval over light clothing, using unstreatched tape meter, without any pressure to body surface, and was recorded to the nearest 0.1 cm.

      Blood biochemistry

      Glucose was determined by using GOD-POD reagent (Merck Ltd., Mumbai, India). TC was determined enzymatically using CHOD/POD-Phosphotungstate reagent. HDL-c was determined with CHOD/POD-Phosphotungstate reagent (Accurex Biomedical Pvt. Ltd., Mumbai, India), after precipitation with phosphotungstic acid. TG was determined using GOP-POD reagent (Accurex Biomedical). Besides values of LDL-c was estimated using the formulae LDL-c = TC − (HDL-c + FTG/5) [
      • Varley H.
      • Gowenlock A.H.
      • Bell M.
      Practical clinical biochemistry.
      ]. Determination of LDL particle size is based on the method of Krauss and Burke using gradient polyacrylamide gel electrophoresis [
      • Krauss R.M.
      • Burke D.J.
      Identification of multiple subclass of plasma low density lipoproteins in normal humans.
      ]. Apolipoprotein AI, B, and E were determined by immunoturbidimetry method using auto N “DAIICHI” reagent and for standard measurement using Apo auto N “DAIICHI” calibrator. For these measurements we used Microlab 2000 semi-auto analyzer (Merck Ltd.).

      Statistical analysis

      After the completion of each experiment, the data were recorded on pre-designed proforma and managed with Microsoft Excel software. Data entry was double checked for any human error. All calculations were performed using SPSS version 10.0 software package for windows and MedCalc software were used for comparison between the area under the receiver operating characteristic curve. Data are presented as means (S.D.) and percentage of the population studied. Student's t-test (for parametric variables) and Mann–Whitney U-test (for non-parametric variables) were used where it was applicable to estimate the significance of difference between two groups. Receivers operating characteristic (ROC) curves were used to determine the optimal cut-off values of these ratios of lipoprotein and apolipoproteins. The points of convergence of sensitivity and specificity determined the optimal cut-off points for these risk factors. Then all the continuous variables were dichotomized. Independent indicators for the presence of CHD were also selected by a forward stepwise conditional logistic regression analysis. In this method, the lipid and non-lipid parameters were selected into the model in the order of statistical significance. For all odds ratios, we calculated 95% confidence intervals (CIs) of each variable and association study was done with Pearson χ2-test. Association of variables having p < 0.05 was considered as statistical significance. All statistical tests of hypothesis are two sided.

      Results

      We studied a total of 198 subjects, of which 100 patients had CHD [86 male (86%) and 14 female (14%)], mean (S.D.) age 54.80 (8.60) years and 98 age and sex-matched healthy controls [86 male (87.75%) and 12 female (12.25%)] mean (S.D.) age 55.54 (9.73) (Table 1). Smoking, alcohol consumption, hypertension and increased waist circumference were highly prevalent in the group with CHD as compared to control group (Table 1). The mean value of TC and LDL-c were significantly higher in CHD patients compared with controls, but the mean value of HDL-c was significantly lower in CHD patients than controls. We also observed that the mean value of TG and blood glucose levels was higher in CHD patient than the control group (Table 2). Whereas the mean ratio of LDL-c/HDL-c, TC/HDL-c, TG/HDL-c and nonHDL-c/HDL-c were higher among CHD patients compared to controls and all the results were statistically highly significant (Table 2). Among the apolipoproteins, mean value of apoB was higher in patients compared to controls and value of apoAI was just reverse of it and both the values were statistically highly significant, on the other hand mean value of apoE was similar in patients and controls and the value was statistically insignificant. But the ratio of apoB/apoAI was highly significant and the mean value was higher in CHD patients compared to the control group (Table 2). Among the studied ratios, some are, e.g. apo B/HDL-c shown to be statistically significant. On the contrary LDL-c/apo B, HDL-c/apoA1 ratios are lower in CHD patients but are statistically insignificant.
      Table 1Characteristic of Asian Indian population in Eastern part of India in the case and control group
      VariableCase (N = 100)Control (N = 98)p-Value
      Mann–Whitney test for comparisons between the case and control group.
      Age, year (mean, S.D.)54.80 (8.60)55.54 (9.73)Matched
      Sex (n, %)Matched
       Male86 (86%)86 (87.75%)
       Female14 (14%)12 (12.25%)
      Smoking0.000
       Yes63 (63%)27 (27.55%)
       No27 (27%)71 (72.45%)
      Hypertension0.000
       Yes66 (66%)13 (13.27%)
       No34 (34%)85 (86.73%)
      Alcohol0.000
       Yes27 (27%)12 (12.25%)
       No73 (73%)86 (87.75%)
      Waist circumference (cm)88.75 (7.15)85.42 (5.17)0.000
      a Mann–Whitney test for comparisons between the case and control group.
      Table 2The demographic information in CHD group and control groups (mean, S.D.)
      VariablesCHD (n = 100)Control (n = 98)p-Value
      Non paired Student's t-test for comparisons between the mean value of case and control groups.
      Glucose (mg/dl)90.91 (29.89)81.06 (18.46)0.006
      Total cholesterol (mg/dl)191.37 (36.42)161.11 (32.11)0.000
      LDL-c (mg/dl)124.45 (33.28)99.97 (28.22)0.000
      HDL-c (mg/dl)29.03 (6.33)34.18 (7.66)0.000
      NonHDL-c (mg/dl)162.17 (36.09)126.93 (31.34)0.000
      TG (mg/dl)188.61 (52.38)134.77 (48.04)0.000
      apoA1 (mg/dl)119.69 (21.90)129.79 (22.49)0.002
      apoB (mg/dl)114.74 (16.14)89.06 (15.88)0.000
      apoE (mg/dl)4.14 (0.72)4.11 (0.70)0.769
      LDL-c/HDL-c4.44 (1.42)3.10 (1.45)0.000
      TC/HDL-c6.80 (1.73)4.94 (1.71)0.000
      TG/HDL-c6.78 (2.90)4.19 (1.98)0.000
      apoB/ApoA10.99 (0.24)0.69 (0.13)0.000
      apoB/HDL-c4.13 (1.02)2.73 (0.82)0.000
      LDL-c/apoB1.09 (0.29)1.15(0.39)0.213
      HDL-c/apoAI0.25 (0.07)0.27 (0.07)0.056
      NonHDL-c/HDL-c5.80 (1.73)3.94 (1.71)0.000
      LDL particle size (nm)24.60 (1.02)25.59 (0.67)0.000
      a Non paired Student's t-test for comparisons between the mean value of case and control groups.
      Diagnostic implications of ratios of different lipoproteins and apolipoproteins against the lipids and apolipoprotein markers were assessed by ROC curves analysis. The analysis demonstrated that apoB (AUROC 0.885), apoB/apoAI (AUROC 0.882), and apoB/HDL-c (AUROC 0.882) were shown to have superior discriminative ability for CHD against the other parameters (Table 3). There was no statistically significant difference found in AUROC between these three variables. In Table 4 optimal cut-off value, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of different parameters are shown.
      Table 3Area under the receiver operating characteristic curve (AUROC) for different variables
      VariablesAUROCStandard errorp-Value95% CI
      Glucose0.5910.0420.0290.508–0.673
      TC0.7670.0350.0000.699–0.836
      LDL-c0.7430.0360.0000.673–0.814
      HDL-c0.7210.0370.0000.577–0.864
      NonHDL-c0.8000.0330.0000.735–0.865
      TG0.7980.0320.0000.737–0.860
      apoAI0.6230.0400.0030.545–0.700
      apoB0.8850.0230.0000.840–0.931
      apoE0.5190.0420.6440.437–0.601
      LDL-c/HDL-c0.8060.0320.0000.743–0.868
      TC/HDL-c0.8350.0300.0000.777–0.893
      TG/HDL-c0.8350.0290.0000.777–0.893
      NonHDL-c/HDL-c0.8350.0300.0000.777–0.893
      apoB/apoAI0.8820.0230.0000.838–0.927
      apoB/HDL-c0.8820.0250.0000.833–0.931
      LDL-c/apoB0.5040.0420.9140.423–0.586
      HDL-c/apoAI0.5990.0400.0160.520–0.678
      LDL particle size0.8600.0280.0000.806–0.914
      Table 4Optimal cut-off value, sensitivity, specificity, and positive predictive values (PPV) and negative predictive values (NPV) of different variables
      VariablesCut-offSensitivitySpecificityPPVNPV
      WC (cm)850.6800.5310.5960.620
      Glucose (mg/dl)1000.3130.8770.7200.558
      TC (mg/dl)1750.7240.7550.7470.732
      LDL-c (mg/dl)1080.6940.6840.6870.691
      HDL-c (mg/dl)300.6260.7240.6970.657
      NonHDL-c (mg/dl)1400.7750.7650.7670.773
      TG (mg/dl)1500.7860.6730.7060.758
      apoAI (mg/dl)1210.5000.6730.6100.569
      apoB (mg/dl)1000.8300.7340.7610.808
      apoE (mg/dl)3.950.4900.4790.4900.479
      LDL-c/HDL-c3.400.7650.7040.7210.750
      TC/HDL-c5.300.8060.7240.7450.789
      TG/HDL-c4.910.8160.7450.7620.802
      NonHDL-c/HDL-c4.420.8060.7760.7820.800
      apoB/apoA10.800.7900.7350.7520.774
      apoB/HDL-c3.070.8790.6940.7430.850
      LDL-c/ApoB1.040.5200.4690.4950.494
      HDL-c/apoAI0.240.5660.5740.5740.566
      LDL particle size (nm)25.50.6630.9100.8800.739
      Among the categorical variables, hypertension had 12 times higher odds ratio in patients than controls and increased risk for CHD. The data in Table 5 indicate the risk of CHD of conventional risk factors along with the different lipoprotein and apolipoprotein ratios and LDL particle size. In the present study it was observed that those with LDL particle size less than 25.5 nm have risks of CHD 20 times higher than those with LDL particle size more than 25.5 nm. The risk for the ratio of apoB/HDL-c, nonHDL-c/HDL-c, TG/HDL-c, TC/HDL-c, apoB/apoAI and LDL-c/HDL-c are 16 times, 14 times, 12 times, 10 times, 10 times and 7 times higher in disease than control group, respectively. But the odds ratios of LDL-c/apoB and HDL-c/apoAI are not significant. Among the conventional risk factors the risk of apoB and nonHDL-c are 13 and 11 times higher in CHD patients than controls.
      Table 5Univariate logistic regression analysis of different markers in CHD
      VariablesOdds ratio95% CIp-Value
      Smoking4.472.45–8.160.000
      WC2.401.34–4.280.003
      Hypertension12.696.20–25.950.000
      Alcohol intake2.651.25–5.600.009
      Glucose3.261.56–6.830.001
      TC8.104.27–15.360.000
      LDL-c4.892.67–8.960.000
      HDL-c4.402.41–8.040.000
      NonHDL-c11.265.78–21.920.000
      TG7.563.98–14.350.000
      apoAI2.061.15–3.660.013
      apoB13.526.79–26.900.000
      apoE0.8800.50–1.540.669
      LDL-c/HDL-c7.754.10–14.670.000
      TC/HDL-c10.935.60–21.340.000
      TG/HDL-c12.976.54–25.710.000
      NonHDL-c/HDL-c14.367.20–28.630.000
      apoB/apoA110.415.39–20.110.000
      apoB/HDL-c16.437.83–34.470.000
      LDL-c/apoB0.9600.541–1.6820.886
      HDL-c/apoAI1.730.981–3.500.054
      LDL particle size20.859.32–46.630.000
      Regression models were used to predict the value of a response variable using the dichotomized explanatory variables. From the univariate logistic regression analysis 15 potential clinical predictors of CHD were evaluated (Table 5). But in the final predictions model (Fig. 1) only five of these factors: the presence of nonHDL-c (OR 5.41; p = 0.001), apoB (OR 2.96; p = 0.045), apoB/apoAI (OR 6.64; p = 0.000), apoB/HDL-c (OR 4.41; p = 0.014) and LDL particle size (OR 9.59; p = 0.000) were found (Table 6). Area under the ROC curves derived from the multivariate model (AUROC 0.947; 95% CI 0.916–0.977) were statistically significantly higher than nonHDL-c (p < 0.001), apoB (p = 0.019), apoB/apoAI (p = 0.009), apoB/HDL-c (p = 0.009) and LDL particle size (p = 0.001).
      Figure thumbnail gr1
      Figure 1Receiver-operating characteristic curve for the prediction model of coronary heart disease. The ROC curve was drawn from the equation that was calculated by logistic regression and is as follows: disease [yes or no] = 1.085 apoB + 1.421 apoB/HDL-c + 1.689 nonHDL-c + 1.849 apoB/apoAI + 2.262 LDL particle size − 11.702.
      Table 6Multivariate logistic regression analysis
      Independent variablesRegression coefficientOdds ratio95% CIp-Value
      apoB1.0852.961.02–8.540.045
      apB/HDL-c1.4214.141.33–12.830.014
      NonHDL-c1.6895.412.08–14.100.000
      apoB/apoAI1.8946.642.37–18.570.000
      LDL particle size2.2629.592.92–31.540.000

      Discussion

      The exact etiology of CHD is unknown; a large number of risk factors are known to be associated with CHD. Obesity [
      • Matsuzawa Y.
      • Nakamura T.
      • Shimomura I.
      • Kotani K.
      ], hypertension [
      • McInnes G.T.
      Hypertension and coronary artery disease: cause and effect.
      ], smoking [
      • Siekmeier R.
      • Wülfroth P.
      • Wieland H.
      • Gross W.
      • März W.
      Low density lipoprotein susceptibility to in vitro oxidation in healthy smokers and nonsmokers.
      ,
      • Hozawa A.
      • Folsom A.R.
      • Sharrett A.R.
      • Payne T.J.
      • Chambless L.E.
      Does the impact of smoking on coronary heart disease differ by low-density lipoprotein cholesterol level? The Atherosclerosis Risk in Communities (ARIC) Study.
      ], family history [
      • Carmena R.
      • Lussier-Cacan S.
      • Roy M.
      • Minnic A.
      • Lingenhel A.
      • Kronenberg F.
      • et al.
      Lp(a) levels and atherosclerotic vascular disease in a sample of patients with familial hypercholesterolemia sharing the same gene defect.
      ], diabetes mellitus [
      • Lewis G.F.
      • Steiner G.
      Hypertriglyceridemia and its metabolic consequences as a risk factor for atherosclerotic cardiovascular disease in non-insulin-dependent diabetes mellitus.
      ], and plasma lipoprotein abnormalities [
      • Lewis G.F.
      • Steiner G.
      Hypertriglyceridemia and its metabolic consequences as a risk factor for atherosclerotic cardiovascular disease in non-insulin-dependent diabetes mellitus.
      ] are the conventional risk factors of this disease. But in our knowledge, limited studies have been undertaken to investigate the relationship between the ratios of different lipoproteins and apolipoproteins and LDL particle size with CHD in the Asian Indian population in eastern part of India.
      The aim of this study was to identify patients with early CHD associated with the conventional risk factors and some of the new risk biochemical markers, including apolipoprotein variables, LDL particle size, in addition to ratios of different lipoproteins and apolipoproteins. These ratios are chosen because human physiological systems need all the lipoproteins as well as apolipoproteins but it has certain limits. When these limits cross, the systems face a lot of problems. So the human physiological systems need to balance all the good and bad things. However, here we considered ratios of different lipoproteins and apolipoproteins levels such as LDL-c/HDL-c, TC/HDL-c, TG/HDL-c, nonHDL-c/HDL-c, apoB/apoAI, apoB/HDL-c, LDL-c/apoB and HDL-c/apoAI are important factors for diagnosis of premature CHD.
      The relevant findings in early CHD were as follows: hypertension, waist circumference, alcohol consumption and smoking were statistically highly significant with the CHD. Abnormal levels of lipids and apolipoproteins characterize the patients with early CHD who had a type of dyslipidemia with an increase in atherogenic lipoproteins and apoB, lower levels of HDL-c and apoAI. According to Masunaga et al. moderate drinking reduces the incidence of cardiovascular events [
      • Masunaga N.
      • Kimura A.
      • Miyatak M.
      • Nishioka N.
      • Hirano Y.
      • Hayashi T.
      • et al.
      Effects of alcohol consumption on cardiovascular events in male patients with healed myocardial infarction.
      ], but in our study we found that 27% and 12.25% among total number of patients and controls, respectively, were drinking country liquor per day, and the data show alcohol intake is significantly associated with CHD (Table 1). A study by Stamler et al. found a strong association between serum cholesterol, CAD and cardiovascular death [
      • Stamler J.
      • Daviglus M.L.
      • Garside D.B.
      • Dyer A.R.
      • Greenland P.
      • Neaton J.D.
      Relationship of baseline serum cholesterol levels in 3 large cohorts of younger men to long-term coronary, cardiovascular and all cause mortality and to longevity.
      ]. Gandhi reported that mean serum TC level was 160 mg/dl in males and 150 mg/dl in females [
      • Gandhi B.M.
      Lipoprotein composition of normal healthy subjects in Northern India.
      ]. Our study shows that mean TC in patients is 191.37 mg/dl and the control group is 161.11 mg/dl. LDL-c is the strongest predictor of CHD and some studies show that the disease is closely correlated with high concentrations of TC and LDL-c [
      • Srinivasan S.R.
      • Ehnholm C.
      • Wattignecy W.A.
      • Bao W.
      • Berenson G.S.
      The relation of apolipoprotein E polymorphism to multiple cardiovascular risks in children: the Bogalusa Heart study.
      ]. Natio reported a high incidence of atherosclerosis and CHD in subjects with LDL-c above 130 mg/dl [
      • Natio H.K.
      Coronary artery disease & disorders of lipid metabolism.
      ]. In our study we have observed that an elevated level of LDL-c is associated with CHD. The high concentrations of TC and LDL-c in patients means that they are 8 and 4 times more at risk than those with normal concentrations, respectively. Numerous epidemiological studies from North America and Europe have conclusively demonstrated that high levels of HDL-c protect against CHD [
      • Stampfer M.J.
      • Sacks F.M.
      • Salvini S.
      • Willent W.C.
      • Hennekens C.H.
      A prospective study of cholesterol, apolipoproteins and the risk of myocardial infarction.
      ]. According to Stain and Myers HDL-c below 35 mg/dl was associated with atherosclerosis and CHD [
      • Stain E.A.
      • Myers G.L.
      Lipids, apolipoproteins and lipoproteins.
      ]. Another group showed that low HDL-c or smoking is more likely to result in myocardial infarction [
      • Sun Y.H.
      • Yang Y.J.
      • Pie W.D.
      • Wu Y.J.
      • Gao R.L.
      Patients with low high-density lipoprotein cholesterol or smoking are more likely to develop myocardial infarction among subjects with a visible lesion or stenosis in coronary artery.
      ]. But in our observation we have found that below 30 mg/dl HDL-c concentrations is a risk for CHD in the eastern part of the Indian population. According to Wiklund et al. TG level above 200 mg/dl is a risk for CHD [
      • Wiklund O.
      • Angelin B.
      • Olofsson S.-O.
      • Ericson M.
      • Fager G.
      • Berglund L.
      • et al.
      Apolipoprotein (a) and ischemic heart disease in familial hypercholesterolemia.
      ]. In our study, hypertriglyceridemia alone was not observed in the group with early CHD. However, the ratio of LDL-c to HDL-c or TC to HDL-c is accepted as an extremely important indicator of atherogenesis [
      • Stampfer M.J.
      • Sacks F.M.
      • Salvini S.
      • Willent W.C.
      • Hennekens C.H.
      A prospective study of cholesterol, apolipoproteins and the risk of myocardial infarction.
      ]. In consideration of these, we also studied the relationship of these ratios with CHD, and found that these ratios are highly significantly associated with CHD, as found by other groups [
      • Panagiotakos D.B.
      • Pitsavos C.
      • Skoumas J.
      • Chrysohoou C.
      • Toutouza M.
      • Stefanadis C.I.
      • et al.
      Importance of LDL/HDL cholesterol ratio as a predictor for coronary heart disease events in patients with heterozygous familial hypercholesterolaemia: a 15-year follow-up (1987–2002).
      ]. The American Diabetes Association (64th Scientific Session, 2004, Orlando, FL, USA) proposed apoB/HDL-c ratio as the predictor of atherosclerotic disease. We observed that the ratio of apoB/HDL-c and HDL-c is strongly associated with CHD risk among Asian Indian populations in the eastern part of India. According to Maruyama et al. concentration of small dense LDL is positively associated with TG/HDL-c ratio [
      • Maruyama C.
      • Imamura K.
      • Teramoto T.
      Assessment of LDL Particle Size by Triglyceride/HDL Cholesterol Ratio in Non-diabetic Healthy Subjects without Prominent Hyperlipidemia.
      ]. In our study, we observed that the ratio of TG to HDL-c is higher in CHD patients compared to the control group. Several studies have related the higher level of apoB and lower level of apoAI to the early occurrence of CHD. We observed that apoAI concentration is lower and apoB concentration is higher in the CHD group than controls as found in other studies [
      • Chan L.
      Apolipoprotein B, the major protein component of triglyceride rich and low density lipoproteins.
      ]. A large number of epidemiological studies have identified small dense LDL as an independent risk factor for CHD [
      • Krauss R.M.
      Heterogenicity of plasma low-density lipoproteins and atherosclerosis risk.
      ] which is often associated with both hypertriglyceridemia and low HDL-c. However, several reports have shown that a higher LDL-c/apoB ratio identifies subjects with predominantly large buoyant LDL particles, whereas a lower value will point to predominantly small dense LDL particles [
      • Wagner A.M.
      • Jorba D.
      • Rigla M.
      • Alonso E.
      • Ordonez-Llanos J.
      • Perez A.
      LDL cholesterol/apolipoprotein B ratio is a good predictor of LDL phenotype B in type 2 diabetes.
      ,
      • Sniderman A.D.
      • Lamarche B.
      • Tilley J.
      • Secombe D.W.
      • Forhlich J.
      Hypertriglyceridemia hyperapo B in type 2 diabetes.
      ,
      • Sniderman A.D.
      • Dagenasis G.R.
      • Cantin B.
      • Despres J.P.
      • Lamarch B.
      High lipoprotein B with low high density lipoprotein cholesterol and normal plasma triglycerides and cholesterol.
      ,
      • Sniderman A.D.
      • St. Pierre A.
      • Cantin B.
      • Dagenais G.R.
      • Deprés J.P.
      • Lamarche B.
      Concordance/discordance between plasma apolipoprotein B levels and the cholesterol indexes of atherosclerotic risk.
      ]. Increase in serum apoB may suggest a large number of LDL particles in blood circulation [
      • Larmache B.
      • Lemieux I.
      • Després J.P.
      The small, dense LDL phenotype and the risk of coronary heart disease: epidemiology, pathophysiology and therapeutic aspects.
      ]. We observed that in patients, increased LDL-c is associated with increased apoB levels. LDL-c to apoB ratio was less than 1.2 in CHD patients found by one group [
      • Enas E.A.
      • Senthilkumar A.
      Coronary artery disease in Asian Indians: an update and review.
      ], but in our study we observed that in both CHD patients and controls the ratio of LDL-c to apoB is less than 1.2 though the concentration is higher in control groups than CHD patients. The apoB/apoAI ratio represents the balance of proatherogenic and antiatherogenic lipoproteins and also identifies the lipoprotein related risk of vascular disease [
      • Sniderman A.D.
      • Jungner I.
      • Holme I.
      • Aastveit A.
      • Walldius G.
      Errors that result from using the TC/HDL-c ratio rather than the apoB/apoA-I ratio to identify the lipoprotein related risk of vascular disease.
      ]. However, the ratio of apoB/apoAI was significantly higher in CHD patients than controls, and it is associated with the disease. According to Sposito et al. HDL-c to apoAI ratio was significantly lower in a liver failure group than controls [
      • Sposito A.C.
      • Vinagre C.G.
      • Pandullo F.L.
      • Mies S.
      • Raia S.
      • Ranires J.A.F.
      Apolipoprotein and lipid abnormalities in chronic liver failure.
      ]. In our study, we also found that HDL-c to apoAI ratio is lower in CHD patients than controls, but the difference is not statistically significant.
      The ROC curve, which is defined as a plot of test sensitivity versus its 1-specificity was used to describe and compare the performance of the diagnostic test. The AUROC for apoB/apoAI, apoB/HDL-c, LDL particle size, TC/HDL-c and nonHDL-c/HDL-c are greater than other biochemical markers (except apoB) suggesting that these may provide a better discriminating test for CHD. And also the difference between the AUROC for apoB and apoB/apoAI (p = 0.832), apoB/HDL-c (p = 0.832), LDL particle size (p = 0.516), TC/HDL-c (p = 0.118) and nonHDL-c/HDL-c (p = 0.118) were not statistically significant. But from these ratios apoB to apoAI ratio is equivalent or better predictive than other lipoprotein and apolipoprotein ratios for screening of CHD? The results were evaluated by ROC curves. Table 3 shows that the ratio of apoB to apoAI and apoB to HDL-c are the best discriminators on the basis of ROC curve analysis. Though at a cut-off point sensitivity of apoB/HDL-c showed 11.26% higher rate than apoB/apoAI (Table 4) and in Table 5, the odds ratio is 16.43 for ratio of apoB to HDL-c and 10.41 for ratio of apoB to apoAI that defines a 57% difference. So, from the above analysis it is concluded that apoB to apoAI ratio is a good predictive marker for CHD, but also apoB to HDL-c ratio is a good one and data from Table 4, Table 5 support the apoB to HDL-c ratio as clinically superior to apo B/apoAI ratio.
      However, there are strong correlations between each marker and therefore, we performed a multivariate logistic regression analysis and found that apoB, apoB/HDL-c, nonHDL-c, apoB/apoAI and LDL particle size are contributed in this model. The Hosmer–Lemeshow test (p = 0.561) result indicated that the number of CHD patients is not significantly different from those predicted by the model, and that the overall model fit is good. The model using nonHDL-c, apoB, apoB/apoAI, apoB/HDL-c and LDL particle size to predict the probability of being a case or control in the study sample categorized 89 of 100 cases (89%) and 86 of 98 controls (87.75%) similarly. AUROC derived from the model was statistically significantly higher from any other variables. So, this model (Fig. 1) is the best discriminator for CHD. We included patients with acute myocardial infarction (AMI), unstable angina and stable angina in our study. We classified the cases of AMI considering WHO and American Heart Association criteria [
      • Luepker R.V.
      • Apple F.S.
      • Christenson R.H.
      • Crow R.S.
      • Fortmann S.P.
      • Goff D.
      • et al.
      Case definitions for acute coronary heart disease in epidemiology and clinical research studies: a statement from the AHA Council on Epidemiology and Prevention; AHA Statistics Committee; World Heart Federation Council on Epidemiology and Prevention; the European Society of Cardiology Working Group on Epidemiology and Prevention; Centers for Disease Control and Prevention; and the National Heart, Lung, and Blood Institute.
      ]; unstable angina consistent with Braunwald clinical classification [
      • Hamm C.W.
      • Braunwald E.
      A classification of unstable angina revisited.
      ]. The patients with stable angina were determined by clinical history, ECG changes and corroborative evidence of the treadmill test, as well as stress echocardiography. Atherosclerosis and unstable plaque is the major contributory factor for CAD. We have a limitation in our study regarding the estimation of the extent and burden of atherosclerosis by doing coronary angiography and multidetector row computed tomography.
      Several strengths are present in our study. First, the case–control study has several advantages over other designs, especially, a cohort study. Second, fasting bloods were collected enabling valid determination of HDL-c, LDL-c, TG, TC, glucose and apolipoproteins. Third, our study included several risk factors that have previously not been assessed with conventional risk factors, which might be the best marker of the balance of atherogenic and antiatherogenic particles. In conclusion, since in the world, there is vast ethnic and cultural heterogeneity, future investigations should be undertaken on other populations to determine the relative role of our regression equation (Fig. 1) based on nonHDL-c, apoB, apoB/apoAI, apoB/HDL-c and LDL particle size, which may be useful as markers for diagnosis of predisposition to CHD in a population.

      Acknowledgments

      The authors are grateful to Dr Monoj Kar of the Department of Biochemistry, N.R.S. Medical College & Hospital, Kolkata, India, for allowing us to use the semi-auto analyzer and also thankful to the staff of the Department of Cardiology, of the same medical college for their cooperation during the sample collection. We are also thankful to the Department of Information Centre, Indian Institute of Chemical Biology, Kolkata, for help in analysis of the data. We acknowledge Mr. Ranjit Kumar Das for his kind help in this work.

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