Evaluation of small dense low‐density lipoprotein concentration for predicting the risk of acute coronary syndrome in Chinese population

Abstract Background Acute coronary syndrome (ACS) is the leading cause of death in developing and developed countries, yet assessing the risk of its development remains challenging. Several lines of evidence indicate that small, dense low‐density lipoproteins (sd‐LDL) are associated with increased cardiovascular disease risk. We aim to evaluate sd‐LDL concentration for predicting the risk of ACS in Chinese population. Methods Baseline characteristics of 121 patients with ACS and 172 healthy controls were obtained. Plasma sd‐LDL‐C was measured using homogeneous assay, and the proportion of sd‐LDL‐C in LDL‐C was detected. Results There was gender and age effect on the sd‐LDL‐C concentration and sd‐LDL‐C/LDL‐C ratio among healthy subjects. Elevated sd‐LDL‐C concentrations and sd‐LDL‐C/LDL‐C ratio were observed in ACS patients with unstable angina pectoris (UAP), non–ST‐segment elevation myocardial infarction (STEMI), and ST‐segment elevation myocardial infarction (NSTEMI) compared with healthy controls (P < .05); however, there were no differences among ACS groups. According to Pearson's correlation coefficient analyses, sd‐LDL‐C concentration and sd‐LDL‐C/LDL‐C ratio were positively correlated with triglyceride (TG) and LDL‐C concentrations (P < .05) and negatively correlated with high‐density lipoprotein (HDL) concentration (P < .05). Based on the receiver operating characteristic (ROC) curves, the cutoff values of sd‐LDL‐C and sd‐LDL‐C/LDL‐C ratio for the prediction of ACS were 1.06 mmol/L and 34.55%, respectively. Multivariate logistic regression analysis demonstrated that the sd‐LDL‐C/LDL‐C ratio, but not sd‐LDL‐C concentration, was significantly associated with ACS events [OR (95% CI): 1.24, 1.11‐1.38, P < .001]. Conclusions The sd‐LDL‐C/LDL‐C ratio may be associated with an increased risk of developing ACS in Chinese population.


| INTRODUC TI ON
Acute coronary syndrome (ACS) is a more severe type of coronary artery disease (CAD), which contributed to a major cause of mortality and morbidity in developed and developing countries. This syndrome consists of unstable angina pectoris (UAP), non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI). 1 There is accumulating evidence that ACS is related to recent activation of the immune-mediated inflammatory process associated with atherosclerotic plaques. 2 Low-density lipoprotein (LDL) is the principal cholesterol-carrying lipoprotein in human plasma and, as such, contributes significantly to the morbidity and mortality of CAD. 3 Therefore, European Society of Cardiology (ESC) guidelines for preventing ACS recommend decreasing low-density lipoprotein cholesterol (LDL-C) to a target level of <1.8 mmol/L. 4 Large prospective cohort studies comparing LDL particle number with LDL-C level have demonstrated that the LDL-C particle number was more strongly associated with the risk of CAD than the LDL-C level. 5,6 Therefore, evaluation of LDL levels cannot identify all individuals with incident CAD, as many CAD events occur in subjects with normal LDL levels. 7 Low-density lipoprotein is composed of heterogeneous particles that differ in density, size, and chemical composition. LDL particles are divided into 2 distinct phenotypes: pattern A, with a higher proportion of large, more buoyant LDL particles (lb-LDL), and pattern B, with a predominance of small dense LDL (sd-LDL) particles. 8

sd-LDL
particles are considered to be highly atherogenic as a result of higher penetration into the arterial wall, lower binding affinity for the LDL receptor, prolonged plasma half-life, and lower resistance to oxidative stress compared with buoyant LDL. 9 Using LDL-C to evaluate cholesterol-related CAD risk will underestimate actual risk in individuals who have optimal LDL-C levels but high levels of sd-LDL-C. 10 Due to the atherogenic properties of sd-LDL, its measurement may be useful for risk assessment.
Small dense low-density lipoprotein has been found to be associated with increased risk of cardiovascular disease in cross-sectional studies and prospective observational studies using traditional measurement methods. [11][12][13][14][15] However, to date, few studies have explored the role of sd-LDL-C in the diagnosis and treatment of ACS.
Fukushima et al 16 found that the sd-LDL-C concentration was significantly higher in patients with ACS compared with controls, especially those with metabolic syndrome, in addition, the reduction in sd-LDL-C by atorvastatin was much more greater than LDL-C in patients with ACS, which indicated that sd-LDL-C is a superior therapeutic marker of statin treatment in patients with ACS. In general, Chinese patients typically have lower baseline levels of LDL-C than the patients of western countries. 17 Because of the complication of pathophysiological processes of atherothrombosis and racial differences, whether sd-LDL-C concentration or the sd-LDL-C/LDL-C ratio is a better predictor of ACS risk than LDL-C concentration or other traditional cardiovascular risk factors in Chinese population remains unknown.

| Study population
This study enrolled one hundred and seventy-two healthy subjects who were randomly selected from consecutive subjects visiting the This study was carried out in accordance with the principles of the Declaration of Helsinki and was approved by the ethics committees of Guangzhou First People's Hospital.

| Baseline examination
Fasting blood samples were obtained by venipuncture after the participants had fasted for at least 10 hours and centrifuged immediately.
The diagnosis of hypertension was based on a history of hypertension, systolic blood pressure above 140 mm Hg, or diastolic blood pressure above 90 mm Hg. The average of the second and third measurements of blood pressure was used for analysis. Diabetes mellitus was defined as a fasting glucose level ≥7.0 mmol/L (126 mg/dL) or a self-reported acute coronary syndrome, Chinese population, risk factor, small dense LDL history of physician-diagnosed diabetes or treatment for diabetes.
Information on smoking status and other medical histories of the patients were obtained through surveys by a well-trained nurse. For this study, we used archived plasma samples of healthy controls and patients with ACS that had been frozen at −80°C and never previously thawed. The determination of sd-LDL-C concentrations was performed by a fully automated homogeneous method (Denka Seiken Co., Ltd.) in an automated biochemistry analyzer (AU5800;

| Laboratory measurements
Beckman Coulter) as previously described. 20 The ratio of sd-LDL-C/LDL-C was calculated as measured sd-LDL-C (mmol/L)/ LDL-C (mmol/L). 18 All the laboratory measurements of patients with ACS and healthy subjects were under no treatment after admission.

| Statistical analysis
Statistical analyses were conducted with SPSSII for Windows (19.0).
Continuous data are presented as the mean ± SD and compared with Student's t test. Correlation analyses for non-parametric (Spearman's Rho) data were performed to establish relationships between sd-LDL-C concentration or the sd-LDL-C/LDL-C ratio and traditional cardiovascular risk factors. A one-way analysis of variance (ANOVA) and Fisher's protected least significant difference were used to compare the mean values of sd-LDL-C and the sd-LDL-C/LDL-C ratio among groups. Receiver operating characteristic (ROC) curve analysis was used to determine the ability of sd-LDL-C concentration, sd-LDL-C/LDL-C ratio, and LDL-C concentration to predict ACS events.
To examine the association of sd-LDL-C and the sd-LDL-C/LDL-C ratio with ACS events, we performed a multivariate logistic regression analysis; all odds ratios (ORs) are presented with their 95% confidence intervals (CIs). P < .05 was considered statistically significant.

| Baseline characteristics
Baseline characteristics of all patients with ACS and healthy subjects at admission to the hospital are provided in Table 1. The mean ages of the patients in the UAP, STEMI, and NSTEMI groups were 65.70, 64.44, and 63.93 years, respectively. All the study participants were Chinese, and over 60% was male.

| sd-LDL-C concentrations and sd-LDL-C/LDL-C ratios in healthy subjects
The precision and accuracy of the automated homogeneous assay were evaluated by analyzing 20 replicates of quantitation standard which showed similar diagnostic performance as previous study. 19 One hundred and seventy-two subjects were classified into the male and female groups. The age of the subjects ranged from 22 to 76 years old, and the mean age was 42.3 ± 16.7 years. To determine whether there was an age or a gender difference, we first compared the sd-LDL-C concentration and sd-LDL-C/LDL-C ratio between females and males ( Figure 1A,B) and then separated the healthy female and male participants into five age groups ( Figure 1C,D). and .001, respectively). Additionally, the sd-LDL-C/LDL-C ratio in the above-60-years age group was significantly higher than that in the 30-to 39-and 40-to 49-year age groups (27.64 ± 7.40 and 28.00 ± 7.20 vs 30.48 ± 9.01%; P = .010 and .031, respectively).

| Association of sd-LDL-C and the sd-LDL-C/ LDL-C ratio with cardiovascular risk factors
The correlations of the sd-LDL-C concentration and sd-LDL-C/LDL-C ratio with various traditional cardiovascular risk factors are shown in Table 2. Strong positive correlations of sd-LDL-C concentration and the sd-LDL-C/LDL-C ratio with lipid risk factors, such as log triglycerides (P < .001 and P < .001, respectively) and LDL-C (P = .003 and F I G U R E 1 The concentration of sd-LDL-C and the sd-LDL-C/LDL-C ratio in healthy subject. A, B, Gender difference between healthy males and females at the concentration of sd-LDL-C and the sd-LDL-C/LDL-C ratio. C, D, Age difference between healthy males and females at the concentration of sd-LDL-C and the sd-LDL-C/LDL-C ratio P < .001, respectively), were observed. In addition, HDL-C concentration showed a negative correlation with sd-LDL-C concentration and the sd-LDL-C/LDL-C ratio (P = .013 and P < .001, respectively).
Additionally, hs-cTnI was not significantly correlated with sd-LDL-C concentration and the sd-LDL-C/LDL-C ratio.

| sd-LDL-C concentrations and ACS events
To eliminate the effects of gender and age, we selected the group of above 50 years old of healthy subjects as normal control and di-  (Table 3).

| DISCUSS IONS
In the present study, we investigated the relationship between plasma concentrations of sd-LDL-C and risk of ACS using a newly developed automated homogeneous sd-LDL-C assay. Our results showed that elevated concentrations of sd-LDL-C and elevated sd-LDL-C/LDL-C ratios were strongly associated with ACS.
Small dense low-density lipoprotein is traditionally measured by ultracentrifugation 21   Note: Model 1 was adjusted for age, men, sd-LDL-C, and sd-LDL-C/LDL-C. Model 2 was adjusted for age, men, sd-LDL-C, sd-LDL-C/LDL-C, and LDL-C. Model 3 was adjusted for age, men, sd-LDL-C, sd-LDL-C/LDL-C, and HDL-C. *P < .05. **P < .01. ***P < .001. that a high sd-LDL-C/LDL-C ratio was associated with a 1.25-fold higher risk of ACS (P < .001), while sd-LDL-C concentration was not significantly associated with ACS (P > .05). Therefore, a high sd-LDL-C/LDL-C ratio might be a more important risk factor for cardiovascular events among patients with ACS than a high sd-LDL-C concentration.
Several previous studies have assessed the associations of sd-LDL-C with traditional cardiovascular risk factors. 3 In agreement with previous studies, we also found that plasma sd-LDL-C concentrations and the sd-LDL-C/LDL-C ratio were positively correlated with plasma TG and LDL-C concentrations; however, sd-LDL-C concentrations and sd-LDL-C/LDL-C ratios were negatively correlated with HDL-C concentration. Thus, these data suggest that sd-LDL-C is adversely associated with cardiovascular lipid risk factors and that increased concentrations of sd-LDL-C and high sd-LDL-C/LDL-C ratios may be associated with metabolic disorders.
Our data suggest that the sd-LDL-C/LDL-C ratio is a promising novel parameter for the assessment of ACS risk. The advantage of the automated homogeneous assay for determining sd-LCL-C content is excellent reproducibility, which makes this assay much more user-friendly and more applicable than traditional methods.
However, the present study also some limitations: First, the cohort was relatively small; therefore, additional large cohort studies are required to clarify the predictive value of the sd-LDL-C/LDL-C ratio, as measured by the homogeneous assay, in subjects with ACS.
Second, the ACS study population represented a heterogeneous cohort with regards to gender. Third, the plasma we used in our study was previously stored at −80°C; therefore, whether the results of this study can be extended to fresh plasma samples remains to be determined.