Coronary heart disease (CHD) is a multifactorial disease having environmental and genetic components. Among the risk factors identified by epidemiological studies, low plasma high-density lipoprotein cholesterol (HDL-C) concentration is one of the strongest.1 For any given low-density lipoprotein (LDL) concentration, the HDL-C concentration is inversely correlated with the risk of CHD and stroke.2.3 But total plasma HDL-C level is a crude measure of their protective effects, as numerous studies have revealed differences between HDL subclasses and properties such as reverse cholesterol transport (RCT), anti-inflammatory and antioxidant properties.4 Subfractions within the HDL particle range are of great interest in research to identify the precise mechanisms by which HDL may exert protective effects against atherosclerosis. Apolipoprotein A-I (apoA-I) is the main and active component of HDL particles, and population studies have shown a highly consistent, inverse correlation between plasma concentrations of apoA-I and CHD risk in human.5
Serum amyloid A (SAA) is a 12-kD acute-phase protein, circulating level of which can be induced up to 1000-fold. Similar to high-sensitive C-reactive protein (hsCRP), SAA is synthesized in the liver in response to infection, inflammation, injury, or stress.6 HDL is the major carrier of SAA in human, while SAA does not exist in a free form and associates with non-HDL lipoproteins in the absence of HDL.7 During the acute-phase reaction, SAA is secreted as the predominant apolipoprotein on plasma HDL-C particles, where it is thought to replace apoA-I and alter HDL-mediated cholesterol delivery to cells and increased selective CE uptake by macrophages.8 Due to its wider dynamic range and more rapid response, SAA has led some to suggest that it may be a better marker of disease activity.9,10 Prospective studies have demonstrated that hsCRP predicts future cardiovascular disease risk,11 however, the association between the SAA level and the extent of coronary stenosis in patients with CHD remains controversial.
This cross-section study was designed to investigate the association of two HDL-C associated factors apoA-I and SAA with the presence and extent of CHD assessed by coronary angiography in a population of Chinese patients with non-diabetic CHD.
Two hundred and twenty-four (158 males and 66 females) were selected from those who admitted to Fu Wai Cardiovascular Disease Hospital from November 2008 to June 2009. All patients underwent coronary angiography. Patients with lesions of less than 50% luminal narrowing were defined as nonsignificant stenosis or 0-vessel disease (n=42); luminal narrowing of 50% or more of at least one major coronary artery was defined as a significant lesion (n=184) and the patients were referred to as having single-vessel disease group (SVD, n=69), double-vessel disease group (DVD, n=54) or three or more diseased vessels (multivessel) group (MVD, n=61).
Patients with diabetes, acute myocardial infarction, or who had experienced surgery or trauma in the 12 weeks preceding admission and those taking lipid-lowering medications were excluded. Patients with renal and hepatic insufficiency, chronic inflammatory disease, thyroid dysfunction, cerebrovascular accident, significant weight loss and immobilization were also excluded.
The study was planned according to the ethical guidelines of the Declaration of Helsinki. The study protocol was approved by the local Institutional Review Committee. Informed consent was obtained from each subject enrolled into the study.
Hypertension was defined when a patient was taking antihypertensive drugs on admission, or if systolic or diastolic blood pressure was ≥140 mmHg or ≥90 mmHg, respectively, upon examination.
Diagnosis of diabetes mellitus was made in accordance with the criteria of the National Diabetes Data Group, or if patients were on hypoglycemic medication upon admission.
Family history of CHD was defined as CHD (angina and/or myocardial infarction) diagnosed in first degree relatives below the age of 55 years.
Body mass index (BMI) was calculated by dividing weight (kg) by the square of height (m2).
A smoker was defined as someone who regularly smoked 5 or more cigarettes a day. Patients who had stopped smoking for more than 10 years before disease onset were classified as nonsmokers.
Blood specimens were collected from all subjects before the day of catheterization (after overnight fasting for at least 12 hours), prior to the injection of any contrast materials or heparin. Serum was isolated by centrifugation (3000 r/min, 10 minutes, 4°C) and frozen at -80°C for later measurement of apoA-I and SAA. Total cholesterol (TC) and triglycerides were assayed by routine enzymatic methods (GPO-PAP) using a Beckman DxC800 analyzer (Fullerton, USA). HDL-C and LDL-C were measured using the chemical modification and selective melting kit (Kyowa Medex, Tokyo, Japan). Serum uric acid (UA) concentrations were measured using a synchron system analyzer (Beckman Coulter, Fullerton). Serum concentrations of hsCRP were measured using the particle-enhanced immuno-turbidimetric kit (Orion Diagnostica, Espoo, Finland) and a Beckman Image system. ApoA-I concentrations were quantified by an enzyme-linked immunosorbent assay (ELISA) kit (Assaypro, USA). SAA concentrations were also measured by ELISA (BioSource International, Camarillo, USA).
Selective coronary angiography was performed with the technique of Judkins. Coronary angiograms were visually assessed by two independent observers blinded to the identity and clinical characteristics of the patients. The four major coronary arteries and their main secondary branches were considered separately, that is, left main coronary artery (LM), left anterior descending artery (LAD), circumflex artery (LCX) and right coronary artery (RCA). The presence of a collateral circulation and the value of the ejection fraction of the left ventricle were not entered into the final analysis.
The Gensini score was used to assess the severity of CHD: it graded narrowing of the lumen of the coronary artery and scored it as 1 for 1%-25% narrowing, 2 for 26%-50% narrowing, 4 for 51%-75%, 8 for 76%-90%, 16 for 91%-99% and 32 for a completely occluded artery. This score was then multiplied by a factor according to the importance of the coronary artery. The multiplication factor for a LM lesion was 5; it was 2.5 for proximal LAD and LCX lesions, 1.5 for a mid-LAD lesion, and 1 for distal LAD, mid/distal LCX and right coronary artery lesions. The multiplication factor for any other branch was 0.5.
Data were analyzed using SPSS statistical software (version 13, SPSS Inc., USA). The results of normal distribution quantitative variables are presented as the mean ± standard deviation (SD) and the results of qualitative variables as percentages. To compare quantitative and qualitative variables between patients with and without CHD group, t test and chi-square test were used, respectively. TG, hsCRP and SAA did not show a normal distribution and were expressed as median (interquartile range). The significance of differences between the medians was determined by a non-parametric method (Mann-Whitney U and Kruskal-Wallis H tests). To elucidate the association between the extent of CHD and the risk factors, patients were categorized according to the number of stenosed vessels.
Binary Logistic regression analysis was used to seek possible independent associations between the different variables and the presence/absence of CHD. Adjustment was performed to correct the influence for demographic (BMI, hypertension, family history of CHD, smoking status), lipid factors (TC, LDL-C, HDL-C, triglycerides, apoA-I and lipoprotein(a)) as well as inflammation related factors (hsCRP, SAA and UA). Confounding variables were entered as: BMI (0: <26, 1: >26), smoking status (0: nonsmokers, 1: smokers), hypertension (0: absent, 1: present), family history of CHD (0: absent, 1: present), high TC (0: <4.78 mmol/L, 1: >4.78 mmol/L), high triglycerides (0: <1.76 mmol/L, 1: >1.76 mmol/L), high LDL (0: <3.40 mmol/L, 1: >3.40 mmol/L), low HDL (0: <0.91 mmol/L, 1: >0.91 mmol/L), low apoA-I (0: <0.65 mg/L, 1: >0.65 mg/L), high LP(a) (0: <300 mg/L, 1: >300 mg/L), high hsCRP (0: <3.0 mg/L, 1: >3.0 mg/L), high SAA (0: ≤10.0 mg/L, 1: >10.0 mg/L), high UA (0: ≤416 μmol/L, 1: >416 μmol/L). In addition, multivariate linear regression analysis was performed to examine the effect of the parameters on the extent of CHD. Odds ratios (OR) were calculated after adjusting for demographic factors, lipid factors as well as inflammation related factors. For each OR, two-tailed probability values and 95% confidence intervals (CI) were estimated. Differences were considered to be statistically significant when P <0.05.
The demographic characteristics of patients with and without CHD group are summarized in Table 1. There were no significant differences in sex distribution, age and BMI between the patients with and without CHD. Similarly, there were no significant differences in hypertension, smoking and family history of CHD between the two groups.
The biochemical characteristics of patients with and without CHD group are summarized in Table 2. There was no significant difference in serum levels of triglyceride, TC, LDL-C and lipoprotein(a) between the two groups. The concentrations of hsCRP (P <0.01) and SAA (P <0.05) were significantly higher in the CHD groups, while concentrations of HDL-C (P <0.05) and the apoA-I (P ≤0.001) were both significantly lower in CHD patients compared to the non-CHD group.
Comparisons between the means of the four groups subdivided on the basis of stenosed vessels (0-VD, SVD, DVD and MVD group) are given in Table 3. Nonstatistically significant differences were seen for triglyceride, TC, LDL-C, lipoprotein(a), and UA. Mean values of HDL-C were significantly lower in the SVD and MVD than in those of the 0-VD group, but no statistically significant difference among the stenosed groups (P <0.05). The apoA-I concentrations were lower in the group with SVD (0.69 mg/L), DVD (0.63 mg/L) and MVD (0.61 mg/L) than in the group with 0-VD (0.76 mg/L), and the differences were significant (P <0.05); the concentrations of apoA-I decreased with the increase in vascular damage, but the difference did not reach statistical significance between DVD vs. MVD and SVD vs. DVD. The hsCRP and SAA levels tended to increase depending on the numbers of >50% stenosed vessels. The mean value of SAA was significantly higher in the DVD and MVD groups than in the 0-VD and SVD groups, but no statistically significant difference between the SVD and 0-VD groups. The median of hsCRP were 1.08 mg/L in 0-VD, 1.30 mg/L in SVD, 1.68 mg/L in DVD, 3.10 mg/L in MVD, and the differences among the four groups were significant (P <0.05).
We next performed binary Logistic regression analysis to determine whether the concentration of HDL-C, apoA-I and SAA had any potential for prediction of presence of CHD (Table 4). When HDL-C, apoA-I and SAA were entered separately, the unadjusted OR (CI) for HDL-C was 0.188 (0.048-0.734, P=0.016), OR (CI) for apoA-I was 0.093 (0.990-0.997, P=0.000), OR (CI) for SAA was 2.571 (1.029-6.424, P <0.05). The models were built adjusting for demographic, lipid and inflammatory related risk factors separately, and the association between SAA, HDL-C and CHD was lost after adjusting the other risk Factors. In contrast, the association between reduction of apoA-I and CHD remained strong, regardless of the confounding variables.
To establish the independent value of serum SAA and apoA-I in predicting the extent of CHD, we performed multivariate linear regression analysis (Table 5). The present analysis revealed that apoA-I and SAA level were predictors of the extent of CHD (P=0.020 for SAA, P=0.000 for apoA-I), while the concentration of HDL-C had no potential for the prediction of the extent of CHD (P=0.550). To find out how apoA-I and SAA behaved as predictors of CHD extent in the presence of other potential predictors, we included separately demographic, lipid as well as inflammation related factors in this model. Even after combination with risk factors, the associations between SAA, apoA-I and the extent of CHD remained strong, both SAA and apoA-I were independent predictors for extent of CHD.
Population studies have consistently indicated that HDL-C levels are a strong, independent inverse predictor of cardiovascular disease.1-3“ However, the role that reduced HDL-C played in development of CHD was controversial, for instance in the Framingham study, it was found that approximately 44% of CHD events in men and 43% in women occurred in persons with normal HDL cholesterol levels.12,13 In our study, we found significant difference in the mean values of the HDL-C (P ≤0.01) between the CHD and non-CHD group. After subdividing the patients on the basis of the number of stenosed vessels, we found no statistically significant difference of HDL-C with the increase of stenosed vessels. Logistic regression analysis revealed that concentration of HDL-C had no potential for the prediction of the presence and extent of CHD.
HDL-C particles can vary substantially in size, density, composition, and functional properties, potentially affecting their relationship to atherosclerosis.14,15 Van der Steeg et al16 indicated very high HDL-C (>1.8 mmol/L), and large size HDL particles, may confer a two-fold increase in cardiovascular risk. By contrast, higher apoA-I remained an independent, negative predictor of cardiovascular risk,16 which suggested that the intrinsic properties of HDL-C particles, rather than low HDL-C levels per se, were determinants of the role of HDL-C on CHD, and the cardioprotective effect of the HDL system may relate chiefly to the apoA-I content of HDL particles. In our study, the apoA-I of CHD patients were significantly lower than patients without CHD (P ≤0.01). The concentrations of apoA-I decreased with the increase in vascular damage, but the difference did not reach statistical significance. Binary Logistic regression analysis revealed that there was a strong association between the reduced apoA-I concentrations and prevalence of CHD, and the association was independent of other risk factors associated with CHD. Similarly, multiple linear regression analysis showed a strong independent association between apoA-I levels and the extent of CHD. Quantification of apoA-I as an independent predictor may provide a valuable tool to assess the presence and the extent of CHD.
The role of immune system and inflammatory pathway in the development of atherosclerotic disease was well established, and during the acute phase as well as chronic inflammation, plasma levels and apolipoprotein content of HDL can be significantly altered (depletion in apoA-I and increase in SAA), leading to attenuated anti-inflammatory activities of HDL.17 Systemic markers of inflammation appear useful for indicating elevated cardiovascular disease risk.18 Similar to hsCRP, elevated plasma levels of SAA have been reported to represent a CHD risk factor.10 However, the current evidence is insufficient because of the relatively few and controversial studies targeting associations between the extent of CHD and inflammatory markers.19,20 In our study, the mean values of the SAA and hsCRP in CHD group were significantly higher than non-CHD group (P <0.01). The concentration of hsCRP and SAA was increased with the increase in vascular damage. Binary Logistic regression analysis revealed the relationship between SAA and presence of CHD disappeared when lipid related factors were considered. Our results confirmed the previous finding that SAA was a weak discriminative power for detecting CHD in a working population.21 Multiple linear regression analysis showed a strong independent association between circulation SAA levels and the extent of CHD. This result also provides evidence that the association between SAA and the extent of CHD was not affected by the potential confounding effects of other clinical, biochemical features of the study participants.
Controversial results of the association of SAA with the extent and severity of CHD are in accordance with the unclear explanation of the exact role of inflammatory markers in the atherosclerosis process. It has been discussed whether hsCRP is only a marker of chronic inflammation without an independent role in the development of disease, or it is an active component in the development of disease and tissue damage.22
Laboratory studies have demonstrated enrichment of HDL with SAA impairs cholesterol efflux properties of HDL, promoted anti-inflammatory activity of HDL became deficient and even transformed into pro-inflammatory action under conditions favoring development of atherosclerosis.23 Johnson et al10 reported a moderate independent relationship between SAA and cardiovascular events. These results further supported the role of inflammation in the pathophysiology of destabilization of vulnerable coronary artery atherosclerotic plaques.
Some limitations of our study should be noted. Firstly, the study sample size was relatively small which could affect the results. Secondly, when we performed binary Logistic regression analysis to determine whether the SAA had any potential for the prediction of presence of CHD, we took patients with normal coronary angiography or stenosis <50% as control group, but only few of them were without any stenosis at all, the SAA level of these patients maybe higher than healthy controls which could limit the generalization of our results. Thirdly, the present study was not designed to evaluate the exact mechanism of the associations among SAA, HDL-C and angiographic CHD, so we could not determine whether the association between SAA and the extent of CHD depended on the changes in HDL-C.
In summary, the present study suggests that total plasma HDL-C level is not a perfect biomarker for CHD. The increased SAA in HDL could represent the inflammatory markers of the extent of coronary stenosis in patients with CHD. In contrast to SAA, the level of apoA-I was also associated with the presence of CHD, indicating that this parameter is not only a marker of CHD presence but also a quantitative indicator of CHD extent. In short, determining the change apolipoprotein content within HDL particle is a more accurate and effective method to evaluate the impact of HDL on CHD.
1. Miller GJ, Miller NE. Plasma high-density lipoprotein concentration and the development of ischaemic heart disease. Lancet 1975; 1: 16-19.
2. Assmann G, Schulte H, von Eckardstein A, Huang Y. High-density lipoprotein cholesterol as a predictor of coronary heart disease
risk: the PROCAM experience and pathophysiological implications for reverse cholesterol transport. Atherosclerosis 1996; 124 Suppl: s11-s20.
3. Castelli WP, Garrison RJ, Wilson PW, Kannel WB, Abbott RD, Kalousdian S. Incidence of coronary heart disease
and lipoprotein cholesterol levels. The Framingham Study. JAMA 1986; 256: 2835-2838.
4. Van Lenten BJ, Navab M, Shih D, Fogelman AM, Lusis AJ. The role of high-density lipoproteins in oxidation and inflammation. Trends Cardio vasc Med 2001; 11: 155-161.
5. Boden WE. High-density lipoprotein cholesterol as an independent risk factor in cardiovascular disease: assessing the data from Framingham to the Veterans Affairs High-Density Lipoprotein Intervention Trial. Am J Cardiol 2000; 86: 19-22.
6. Malle E, Steinmetz A, Raynes JG. Serum amyloid A (SAA): an acute phase protein and apolipoprotein. Atherosclerosis 1993; 102: 131-146.
7. Cabana VG, Feng N, Reardon CA, Getz GS, Webb NR, Beer FC, et al. Influence of apoA-I and apoE on the formation of serum amyloid A-containing lipoproteins in vivo
and in vitro.
J Lipid Res 2004; 45: 317-325.
8. Artl A, Marsche G, Lestavel S, Sattler W, Malle E. Role of serum amyloid A during metabolism of acute-phase HDL by macrophages. Arterioscler Thromb Vasc Biol 2000; 20: 763-772.
9. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999; 340: 448-454.
10. Johnson BD, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, Shaw LJ, et al. Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women: the National Heart, Lung and Blood Institue-Sponsored Women's Ischemia Syndrome Evaluation (WISE). Circulation 2004; 109: 726-732.
11. Memon L, Kalimanovska VS, Stanojevic NB, Ostric DK, Ivanovic DK, Spasic S, et al. Association of C-reactive protein with the presence and extent of angiographically verified coronary artery disease. Tohoku J Exp Med 2006; 209: 197-206.
12. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High density lipoprotein as a protective factor against coronary heart disease
. Am J Med 1977; 62: 707-714.
13. Ansell BJ, Navab M, Hama S, Kamranpour N, Fonarow G, Hough G, et al. Inflammatory/anti-inflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment. Circulation 2003; 108: 2751-2756.
14. Ansell BJ, Watson KE, Fogelman AM, Navab M, Fonarow GC. High-density lipoprotein function: recent advances. J Am Coll Cardiol 2005; 46: 1792-1798.
15. Ansell BJ, Fonarow GC, Fogelman AM. High density lipoprotein: is it always atheroprotective? Curr Atheroscler Rep 2006; 8: 405-411.
16. van der Steeg WA, Holme I, Boekholdt SM, Larsen ML, Lindahl C, Stroes EG, et al. High-density lipoprotein cholesterol, high-density lipoprotein particle size, and apolipoprotein A-I: significance for cardiovascular risk: The IDEAL and EPIC-Norfolk studies. J Am Coll Cardiol 2008; 51: 634-642.
17. Kontush A, Chapman MJ. Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev 2006; 58: 342-374.
18. Memon L, Kalimanovska VA, Stanojevic NB, Ostric DK. Association of C-reactive protein with the presence and extent of angiographically verified coronary artery disease. Tohoku J Exp Med 2006; 209: 197-206.
19. Sano TM, Tanaka AM, Namba MM, Nishibori YM, Nishida YM, Kawarabayashi TM, et al. C-reactive protein and lesion morphology in patients with acute myocardial infarction. Circulation 2003; 108: 282-285.
20. Rifai N, Joubran R, Yu H, Asmi M, Jouma M. Inflammatory markers in men with angiographically documented coronary heart disease
. Clin Chem 1999; 45: 1967-1973.
21. Delanghe JR, Langlois MR, Bacquer DD, Mak R, Capel P, Renterghem LV. Discriminative value of serum amyloid A and other acute-phase proteins for coronary heart disease
. Atherosclerosis 2002; 160: 471-476.
22. Chait A, Han CY, Oram JF, Heinecke JW. Thematic review series: the immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease? J Lipid Res 2005; 46: 389-403.
23. Navab M, Berliner JA, Subbanagounder G, Hama S, Lusis AJ, Castellani LW, et al. HDL and the inflammatory response induced by LDL-derived oxidized phospholipids. Arterioscler Thromb Vasc Biol 2001; 21: 481-488.