Atherosclerosis is the most important contributor to increasing burden of coronary artery disease (CAD). Growing evidence suggests that the ratios of Apo B/Apo A-I and Lp(a) are better indexes for risk assessment of CAD. Elevated plasma levels of lipoprotein(a) in humans represent a major in-herited risk factor for atherosclerosis. Thus, a study was performed to determine the association betwwen serum Apo B, Apo A-I, and lipoprotein(a) levels, and severity of CAD in patients with CAD confirmed on coronary angiography findings. An analytical case control study was carried out with 85 patients (58 males and 27 females) 40 - 60 years of age confirmed as having CAD on coronary angiography and 85 age and sex matched healthy volunteers as controls. Serum samples were an-alyzed for Apo A-1 LDL, Apo B, Apo A-I, and lipoprotein(a) concentration and the severity of CAD was assessed using coronary angiography scoring method. Patients with CAD had significantly high serum LDL-C, Apo B and Lp(a) levels compared to control subjects. However, serum Apo A-I level did not show a significant difference between two groups. Subjects with a positive family history of CAD with increased serum Lp(a) ≥ 17.3 mg/dL have high risk for development of CAD. Present study suggests that serum Lp(a) cut-off value of 17.3 mg/dL may be an important predictor in ruling out major vessel disease and luminal narrowing by atheroma.
Atherosclerosis is the most important contributor to increasing burden of coronary artery disease (CAD). Although the precise mechanisms involved in the pathogenesis are not clear, it has been postulated that the reactive oxygen species (ROS) formation in vascular endothelial cells can promote conversion of low density lipoprotein cholesterol (LDL-C) to atherogenic oxidized LDL (oxLDL). This oxLDL and the subsequent generation of lipidperoxidase accelerate the development of atherosclerotic plaque [
Several mechanisms by which high density lipoprotein (HDL) protects against development of CAD have now been identified. The reverse cholesterol transport (RCT) is one of the well established antiatherogenic functions of HDL. This cardioprotective effect of HDL is largely due to Apo A-I mediated cellular cholesterol efflux, Lecithinecholesterol acyl transferase (LCAT) mediated maturation of HDL particles and several antioxidative processes. Thus, the Apo A-I is the main structural protein of antiatherogenic lipoprotein which is more important than estimation of total HDL content for biochemical pathways that make HDL antiatherogenic. However, these studies did not address the crucial question of whether the Apo A-I levels predicted the severity of CAD which was independent of traditional lipid based variables [
The ratio of LDL/HDL cholesterol conventionally represents the balance of pro atherogenic and anti-athero- genic lipids. However, growing evidence supports the idea that the ratios of Apo B/Apo A-I and Lp(a) are better indexes for risk assessment of CAD [
Epidemiologic studies have identified Lp(a) as a risk factor for atherosclerotic diseases such as coronary artery disease and cerebrovascular accidents. The structure of lipoprotein(a) is similar to plasminogen and tPA (tissue plasminogen activator) and it competes with plasminogen for its binding site, leading to reduced fibrinolysis. Also, because Lp(a) stimulates secretion of PAI-1, it leads to thrombogenesis. Lp(a) also carries cholesterol and thus contributes to atherosclerosis [
The relationship between lipoprotein levels and the degree of coronary atherosclerosis is less consistent and depends on the method of assessment on patient. Sullivan et al. [
An analytical case control study was carried out with 85 patients (58 males and 27 females) 40 - 60 years of age confirmed as having CAD on coronary angiography findings at Cardiology Unit, National Hospital and Nawaloka Hospitals PLC, Colombo Sri Lanka during 2013 and 2014. Subjects with a history of cardiovascular, renal, hepatic disease, malignancy and on hypolipidemic therapy and oral anticoagulants that could interfere with the present study were excluded from the study. A total of 85 age and sex matched healthy volunteers who had normal exercise ECG and estimated Glomerular Filtration Rate (eGFR) more than 60 ml/min/1.73 m2 attending a routine health screening program at Family Health Care Centre, University of Sri Jayewardenepura, Nugegoda, Sri Lanka were recruited as controls to compare the lipoprotein parameters with patients. The sample size of the study was calculated for a matched case control study with a power of 80%; ratio of cases to controls 1:1; exposure in controls 30%; expected odds ratio of 2.6 and an alpha error of 5%.
Blood samples (5 ml) were drawn after overnight fast (8 - 10 hrs) from antecubital vein, from both patients and controls. Clotted blood samples were centrifuged for 10 minutes at 3500 rpm to separate serum and stored at −20˚C pending analysis. LDL, Apo B, Apo A-I, and lipoprotein(a) activity was measured by using Biosystems S.A (Barcelona, Spain) assay kits on Konelab 20XT Clinical Chemistry Analyzer (Thermo, Finland).
Assay principle of LDL based on solubility of cholesterol from HDL, very low density lipoproteins (VLDL) and chylomicrons. The cholesterol esters are broken down by cholesterol esterase and cholesterol oxidase in a non-colour forming reaction. Then, solubilizes cholesterol from LDL in the sample and ultimately produces H2O2. The LDL cholesterol is then spectrophotometrically measured at 540 nm wavelength by using peroxidase [
Apo A-1 in the sample precipitates in the presence of imidazole buffer, sodium azide and Goat anti-human Apo A-1 antibodies. The light scattering of the antigen-antibody complexes is proportional to the Apo A-1 concentration and can be measured by turbidimetrically at 340 nm wavelength [
The Lp(a) level measure by reaction gives an insoluble aggregate that causes turbidity in presence of glycine buffer, sodium azide and anti-human lipoprotein(a) antibodies. The degree of turbidity can be measured optically at 600 nm wavelength and it is proportional to the amount of Lp(a) in the sample.
Coronary angiography reports and the compact disc recordings of angiograms were independently reviewed by an interventional cardiologist, who was blinded to the patients’ clinical and laboratory findings. Angiogram findings were then evaluated using three different score systems―vessel score, stenosis score, and extent score. The angiograms were scored according to a method described by Sullivan et al. in 1990 [
This was calculated as the number of vessels with a significant stenosis (70% or greater reduction in lumen diameter). Depending on the number of vessels involved, vessel score ranged from 0 to 3. The left main coronary artery stenosis was scored as single vessel disease.
The stenosis score was calculated by a modified Gensini score as described by Reardon et al. in 1985 [
The extent score was calculated according to the method described by Sullivan et al. in 1990 which indicates the proportional of the coronary artery tree involved by angiogrphycally detectable atheroma. The proportion of each vessel involved by atheroma, identified as luminal irregulartity was multiplied by a factor for each vessel: left main artery, 5; left anterior descending artery, 20; main diagonal branch, 10; first septal perforate, 5; left circumflex artery, 20; obtuse marginal and postero-lateral vessels,10; right coronary artery, 20; and main posterior descending branch,10. When the major lateral wall branch was a large obtuse marginal on intermediate vessels, this was given a factor of 20 and the left circumflex artery a factor of 10. When a vessel was occluded and the distal vessel not fully visualized by collateral flow, the proportion of vessel not visualized was given the mean extent score of the remaining vessels. The scores for each vessel or branch were added to give a total score out of 100, being the percentage of the coronary intimal surface area involved by atheroma.
Reference intervals for LDL, Apo B, Apo A-I, and Lp(a) levels in control subjects were determined using 95% confidence intervals (Cl’s). Owing to the skewed distribution of LDL, Apo B, Apo A-I, and Lipoprotein(a) levels, logarithmic transformations of these data were performed using SPSS software version 16.0 (Chicago, Illnois).
Continuous variables were analyzed using independent sample t test, Analysis of variance (ANOVA) and Spearman correlations were calculated to determine the correlation between the coronary angiogram scores and LDL, Apo B, Apo A-I, and Lp(a) levels. Furthermore, categorical variables were presented as absolute frequencies and percentages, whilst analyzed using chi square, Fisher’s exact test, where appropriate. Multivariate logistic regression was performed and Odds Ratio was calculated to assess risk of CAD in population. A p value ≤ 0.05 was considered statistically significant. The accuracy of detecting the severity of CAD in patients using LDL, Apo B, Apo A-I, and lipoprotein(a) levels were determined by measuring the area under the Receiver Operating Characteristics (ROC) curve, 95% confidence interval, sensitivity, specificity and positive and negative predictive values and likelihood ratio based on the coronary angiography findings.
Informed written consent from all participants was obtained prior to enrolment to the study. The study protocol was approved by the Ethics Review Committee of the Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka and the experiment was conducted in conformity with guideline of Helsinki declaration. An interview-administered questionnaire was made for all subjects recruited in the study. Informed written consent was obtained from all participants.
Lipid parameters of patients and controls are given in
Multivariate logistic regression analysis was performed to eliminate the influences of confounding factors for CAD. Among the risk factors assessed, patients with a family history of premature heart disease showed a significant association with CAD when compared to controls [OR 7.13 (95% CI 2.24 - 22.68), p = 0.001]. Furthermore, results also revealed that patients with CAD had significantly high serum Apo B [1.03 (95% CI 1.01 - 1.06) p = 0.006] and Lp(a) [1.02 (95% CI 0.98 - 1.07) p = 0.009] levels compared to control subjects. However, when considering the correlation coefficient values associated with vessel and stenosis scores, it was more evident that the Lp(a) would be the better indicator of assessing the severity of CAD in patients. Thus, the positive family history of CAD and increased serum Lp(a) levels appeared to be the most significant predictors for development of CAD (
Severity of coronary artery disease (CAD) was assessed by the vessels score, stenosis score, and extent score by perusing the coronary angiogram reports showed a mean vessel score of 1.67 out of a maximum of 3 (95% CI 1.46 - 1.87), which indicates that most of the patients in the study population had involvement of at least two out of the main three coronary vessels (
The mean stenosis score of the patients was 8.09 out of a theoretical maximum of 32 (95% CI 6.87 - 9.30), indicating that more than one third of the collective vessel diameter of the segments studied was having significant stenosis 50.96% out of a theoretical maximum of 100% (95% CI 45.82 - 56.11). Thus overall, results indicate that the severity of CAD was higher in this study population (
Mean | SD | Minimum | Maximum | p value* | |
---|---|---|---|---|---|
Apolipoprotein A1 (mg/dl) | |||||
Patients | 110.6 | 20.8 | 71 | 181 | 0.962 |
Controls | 110.8 | 18.3 | 79 | 169 | |
LDL (mg/dl) | |||||
Patients | 85.1 | 34.3 | 19.4 | 154.6 | 0.003** |
Controls | 68.0 | 25.0 | 25.6 | 129.1 | |
Apolipoprotein B (mg/dl) | |||||
Patients | 95.2 | 25.3 | 41.5 | 152.2 | 0.025** |
Controls | 74.7 | 23.0 | 37.8 | 143.7 | |
Lipoprotein(a) (mg/dl) | |||||
Patients | 23.8 | 12.7 | 10.4 | 64.3 | 0.001** |
Controls | 19.2 | 7.9 | 7.2 | 39.2 |
*Independent sample t test comparing Patients and controls. **Significant at p ≤ 0.05.
Variable | Odds ratio | 95% confidence interval | p value | |
---|---|---|---|---|
Age (Years)# | 1. 01 | 0.96 | −1.06 | 0.526 |
Gender | 0.86 | 0.28 | −2.58 | 0.790 |
Positive Family history of CAD | 7.13 | 2.24 | −22.68 | 0. 001* |
Apo A1 | 0.988 | 0.96 | −1.01 | 0.352 |
LDL | 1. 00 | 0.98 | −1.02 | 0.838 |
Apo B | 1. 03 | 1. 01 | −1.06 | 0. 006* |
Lp(a) | 1. 02 | 0.98 | −1.07 | 0.009* |
#Consider as continuous variable; *Significant at p ≤ 0.05.
Mean | SD | 95% CI | |
---|---|---|---|
Vessel score | 1.67 | 0.76 | 1.46 - 1.87 |
Stenosis score | 8.09 | 4.51 | 6.87 - 9.30 |
Extent score | 50.96 | 19.39 | 45.82 - 56.11 |
The frequency distribution of three severity scoring systems in patients with CAD is given in
The diagnostic accuracy of lipoproteins for CAD based was determined by measuring the area under the ROC
Patients (n) | % | ||
---|---|---|---|
Vessel score | Single vessel disease | 37 | 43 |
Double vessel disease | 27 | 32 | |
Triple vessel disease | 21 | 25 | |
Stenosis score | (1 - 7) Mild (8 - 15) Moderate (16≤) Severe | 44 23 18 | 52 27 21 |
Extent score | (≥25%) Mild (26% - 49%) Moderate (≥50%) Severe | 13 32 40 | 15 38 47 |
curve (AUC), 95% confidence interval, sensitivity (SE%), specificity (SP%), positive predictive value (PP%), negative predictive values (NP%), Positive likelihood ratio (PLR) and negative likelihood ratio (NLR) (
According to ROC given in vessel score the AUCs for Lp(a) was 63.9% with a detectable cutoff values of 17.3 mg/dl keeping acceptable sensitivity (79%) and positive and negative predictive values of 57% and 65% suggesting that Lp(a) appeared to be an important predictive marker of ruling out CAD in the present study population (
When considering the stenosis score the Lp(a) cut off value of 17.3 mg/dl showed a high sensitivity and negative predictive values (Se 71%, NPV 75%, AUC = 0.572) compared to other makers suggesting that the Lp(a) may have a value in ruling out major vessel disease and luminal narrowing by atheroma (
However, Lp(a) has shown a moderate but significant sensitivity and negative predictive values of 70% and 55% respectively (AUC 0.504) with a moderate accuracy for predicting severity of CAD for extent score compared to stenosis score (
The traditional risk factors like hypertension, dyslipidemia, diabetes mellitus, obesity, smoking, family history of heart disease etc. have been long recognized as the major risk factors for development of CAD [
The atherogenic dyslipidemic profile appears to accelerate the progression of atherosclerosis. Thus, measurement of different forms of apolipoprotein may improve cardiovascular risk prediction [
Recently, several meta-analyses have provided evidence on the association between the atherogenic lipoproteins and the severity of CAD. However, these studies have reported inconsistent results due to differences in the methods of assessment of atherosclerosis burden [
Cut-off values | (%) SE 95%CI | (%) SP 95%CI | (%) PPV 95%CI | (%) NPV 95%CI | PLR 95% CI | NLR 95% CI | AUC 95% CI | p value |
---|---|---|---|---|---|---|---|---|
Vessel score | ||||||||
Apo A-1 112.5 (mg/dl) | 64 44 - 81 | 55 35 - 74 | 58 40 - 76 | 62 41 - 80 | 1.43 0.8 - 2.4 | 0.65 0.4 - 1.2 | 0.370 0.223 - 0.517 | 0.092 |
LDL-C 73.2 (mg/dl) | 72 51 - 87 | 31 15 - 51 | 50 34 - 66 | 53 28 - 77 | 1.04 0.7 - 1.4 | 0.92 0.4 - 2.05 | 0.555 0.403 - 0.707 | 0.478 |
Apo B 68.1 (mg/dl) | 79 59 - 92 | 7 1 - 23 | 45 31 - 60 | 25 4 - 65 | 0.84 0.7 - 1.0 | 3.11 0.7 - 14.1 | 0.601 0.446 - 0.756 | 0.191 |
Lp (a) 17.3 (mg/dl) | 75 55 - 89 | 45 26 - 64 | 57 40 - 73 | 65 41 - 85 | 1.36 0.9 - 2.0 | 0.56 0.3 - 1.2 | 0.639 0.494 - 0.784 | 0.071 |
ApoB/ApoA1 0.61 | 79 59 - 92 | 14 4 - 32 | 47 32 - 62 | 40 12 - 74 | 0.91 0.7 - 1.2 | 1.55 0.5 - 4.9 | 0.634 0.482 - 0.786 | 0.082 |
Stenosis score | 0.153 | |||||||
Apo A-1 112.5 (mg/dl) | 58 34 - 78 | 52 36 - 69 | 39 22 - 58 | 70 51 - 85 | 1.20 0.7 - 1.9 | 0.82 0.5 - 1.5 | 0.379 0.227 - 0.532 | |
LDL-C 73.2 (mg/dl) | 71 44 - 90 | 30 17 - 47 | 30 17 - 47 | 71 44 - 90 | 1.01 0.7 - 1.4 | 0.98 0.4 - 2.4 | 0.494 0.315 - 0.674 | 0.944 |
Apo B 68.1 (mg/dl) | 77 50 - 93 | 10 3 - 24 | 27 15 - 41 | 50 16 - 84 | 0.85 0.6 - 1.1 | 2.35 0.7 - 8.3 | 0.542 0.356 - 0.728 | 0.619 |
Lp (a) 17.3 (mg/dl) | 71 44 - 90 | 38 23 - 54 | 32 18 - 50 | 75 51 - 91 | 1.13 0.8 - 1.7 | 0.78 0.3 - 1.8 | 0.572 0.395 - 0.749 | 0.393 |
ApoB/ApoA1 0.61 | 77 50 - 93 | 15 6 - 30 | 28 16 - 43 | 60 26 - 88 | 0.90 0.7 - 1.2 | 1.57 0.5 - 4.9 | 0.607 0.423 - 0.792 | 0.203 |
Extent score | 0.167 | |||||||
Apo A-1 112.5 (mg/dl) | 60 41 - 77 | 52 32 - 71 | 58 39 - 75 | 54 33 - 73 | 1.25 0.8 - 2.0 | 0.77 0.4 - 1.4 | 0.393 0.243 - 0.544 | |
LDL-C 73.2 (mg/dl) | 67 47 - 83 | 26 11 - 46 | 50 34 - 66 | 41 19 - 67 | 0.90 0.6 - 1.3 | 1.29 0.6 - 2.90 | 0.487 0.293 - 0.597 | 0.867 |
Apo B 68.1 (mg/dl) | 77 58 - 90 | 4 0.6 - 19 | 47 33 - 62 | 13 2 - 53 | 0.80 0.6 - 1.0 | 6.3 0.8 - 47.9 | 0.445 0.293 - 0.597 | 0.477 |
Lp (a) 17.3 (mg/dl) | 70 51 - 85 | 41 22 - 61 | 57 40 - 73 | 55 32 - 77 | 1.18 0.8 - 1.8 | 0.74 0.3 - 1.5 | 0.504 0.351 - 0.656 | 0.962 |
ApoB/ApoA1 0.61 | 77 58 - 90 | 11 3 - 29 | 49 34 - 64 | 30 7 - 65 | 0.86 0.7 - 1.1 | 2.10 0.6 - 7.32 | 0.491 0.337 - 0.646 | 0.911 |
SE―Sensitivity; SP―Specificity; PPV―Positive predictive value; NPV―Negative predictive value; PLR―Positive likelihood ratio; NLR―Negative likelihood ratio; AUC―Area under curve; CI―Confidence interval.
to investigate the association of lipoprotein parameters and the severity of CAD based on vessel, stenosis and extent score based on coronary angiography findings.
According to coronary angiography evaluation, it was observed that Lp(a) showed a best cutoff value of 17.3 mg/dL with significant AUC (0.69) and sensitivity and negative predictive value of 75% and 65% respectively, with the vessel score compared to LDL, Apo B, Apo A-I and even with the Apo B/Apo A-I ratio. In addition the Lp(a) also showed a high sensitivity (71%) and a negative predictive value (75%) with the stenosis score indicating that Lp(a) may have a clinical importance in ruling out major vessel disease and luminal narrowing by coronary atherosclerosis. However, when considering the extent score, Lp(a) showed a moderate accuracy with a sensitivity and negative predictive values of 70% and 55% respectively. As extent score quantify the proportion length of coronary endothelial surface area occluded by atherosclerosis, the extent score can be used as a strong and independent predictor for CAD. Thus, Lp(a) may have a moderate potential value in predicting the extent score. However, when considering the three coronary angiography scores, the serum Lp(a) cut-off value of 17.3 mg/dL has a potential in ruling out the severity of CAD. Thus, the serum Lp(a) may be an important marker for screening patients with strong risk associated factors for CAD compared to LDL, Apo B and Apo A-I. Very few studies have reported cut-off values of Lp(a) established based on coronary angiography [
Serum Lp(a) levels showed a significant association with the severity of CAD assessed by vessel, stenosis and extent scores which place emphasis on the number of coronary vessels involved and their luminal narrowing. Individuals possessing a cut-off value of ≥17.3 mg/dL for Lp(a) with positive family history of premature heart diseases appear to be more susceptible for CAD. However, more patient numbers need the past clinical database on prevalence of CAD. Thus, lager sample numbers may be needed as far as the cutoff values are concerned in predicting the accuracy for CAD.
This research was funded by University of Sri Jayewardenepura, Nugegoda, Sri Lanka (Grant No. ASP/06/ PR/MED/2012/19). The authors thank: (i) The staff of the Cardiology Unit, Nawaloka, Hospital plc, Colombo and the National Hospital Colombo, Sri Lanka for providing facilities for selection of patients; (ii) Family Health Care Centre, Dept. of Family Medicine, University of Sri Jayewardenepura, Sri Lanka for assisting selection of volunteers as control subjects; (iii) Dr. W.A.A. Wijayasiri, Head of Dept. of Community Medicine, University of Sri Jayewardenepura, Sri Lanka for the assistance with data analysis.
Pradeep RasikaPerera,DinushkaWickramasinghe,HemanthaPeiris,LalChandrasena,VajiraSenaratne, (2015) Association between LDL, Apolipoprotein-B Apolipoprotein A-I and Lipoprotein(a) and Severity of Coronary Artery Disease Based on Coronary Angiography. Journal of Biosciences and Medicines,03,53-61. doi: 10.4236/jbm.2015.38006