Correlation between of small dense low‐density lipoprotein cholesterol with acute cerebral infarction and carotid atherosclerotic plaque stability

Background Acute cerebral infarction (ACI) is seriously harmful to human health worldwide. However, at present, the risk of disease onset is still not accurately predicted for some people. Methods Five hundred and nineteen patients with ACI and 300 healthy controls were included in this study. We divided the patients into three groups according to the results of cervical artery contrast‐enhanced ultrasound. Ninety‐five patients were in the CAS without plaque group, 108 patients were in the stable plaque group, and 316 patients were in the unstable plaque group. TC, TG, HDL‐C, LDL‐C, and sdLDL‐C were measured in all subjects. Results The level of small dense low‐density lipoprotein cholesterol (sdLDL‐C) in the ACI group was significantly higher than that in the control group (P < 0.001). Logistic regression analysis showed that sdLDL‐C was an independent risk factor for ACI (OR = 1.067, 95% CI: 1.041‐1.093, P < 0.001); serum sdLDL‐C was significantly higher in the unstable plaque group than in the stable plaque group and plaque‐free group (P < 0.05, P < 0.001); serum sdLDL‐C was also higher in the stable plaque group than the plaque‐free group (P < 0.001). Logistic regression analysis showed that sdLDL‐C was an independent risk factor for unstable carotid plaques (OR = 1.053, 95% CI: 1.038‐1.068, P < 0.001); Spearman correlation analysis showed that sdLDL‐C test results were positively correlated with carotid plaque stability (r = 0.363, P < 0.001). Conclusion Small dense low‐density lipoprotein cholesterol is an independent risk factor for the onset of ACI and may be an early serum marker for this disease.

traditional risk factors can only partially predict the risk of ACI, and the risk of morbidity in a considerable number of people cannot be accurately predicted.
Low-density lipoprotein cholesterol is heterogeneous and can be divided into high cholesterol content, large particle size (peak diameter >25.8 nm) LDL-C A; low cholesterol content, small particle size (diameter peak <25.8 nm) LDL-C B; and small dense low-density lipoprotein cholesterol (sdLDL-C). 4 Recent studies have confirmed that sdLDL-C has stronger atherosclerosis ability than LDL-C and has been included in the recently reported important cardiovascular and cerebrovascular disease risk factors by the American Cholesterol Education Program Adult Treatment Group. 5 sdLDL-C is associated with the number of atherosclerotic plaques and with carotid stenosis caused by atherosclerotic plaque. 6,7 Studies by Alberto Zambon 8 have suggested that sdLDL-C has a significant effect on carotid plaque cell composition.
Despite the significant role of sdLDL-C in atherosclerosis, 6 whether this relation is consistent with the existing research conclusions, whether sdLDL-C is related to the stability of CAS plaque, and whether sdLDL-C can better predict the risk of ACI have been under studied. Therefore, this study evaluated patients diagnosed with ACI in our hospital and measured the serum sdLDL-C level. The aims were as follows: (a) to observe the relationship between serum sdLDL-C level and ACI by observing the level of sdLDL-C in patients with normal LDL-C levels. (b) To determine whether serum sdLDL-C can be used as a serum marker for predicting ACI by verifying whether there is a correlation between serum sdLDL-C levels and CAS plaques with different levels of stability.

| Intravascular ultrasound imaging and analysis
Carotid ultrasound examination method: Siemens Acuson S2000 color Doppler ultrasound diagnostic instrument with 914 probe frequency of 4.0~9.0 MHz. The subject was placed in the supine position. The carotid artery was cut at 2 cm above the carotid sinus level and at 1.5 cm from the carotid sinus level, and a longitudinal incision was performed. The arterial bifurcation, internal carotid artery, and external carotid artery were scanned, and the carotid intima-media thickness (IMT) was measured.
Plaques are focal structures of at least 0.5 mm or 50% of the surrounding IMT value that are encroaching into the arterial lumen or focal structures that demonstrate a thickness >1.5 mm as measured from the intima-lumen interface to the media-adventitia interface; otherwise, structures were not considered as plaques. 9 Contrast-enhanced ultrasound (CEUS) examination method: After the target plaque is found by standard ultrasound examination, the image is adjusted and partially magnified to clearly show the plaque morphology while the patient is calmly breathing. The ultrasound contrast agent is produced by Bracco International BV. Sonovi (injection sulfur hexafluoride microbubble 59 mg lyophilized powder) dissolved in 5 mL of physiological saline (0.9%) and shaken well (microbubble concentration 5 mg/mL); 2 mL of contrast agent was initially administered in the median vein of the elbow, and then, 5 mL of physiological saline (0.9%) was injected. The timing of contrast injection was preplanned and noted, and the image was collected. Contrastenhanced neovascularization was evaluated as follows: grade 0: no enhancement in the plaque; grade 1: contrast agent microbubbles appear at only the base or middle of the plaque along the direction of the plaque thickness; and grade 2: contrast agent microbubbles appear near the intima along the direction of plaque thickness. 10 The stability of the plaque is defined by the extent of neovascularization in the plaque and the extent of plaque-induced stenosis as suggested by the carotid ultrasound contrast. 11 Neovascularization in the plaque with a grade of 0-1 and stenosis of <50% indicate a stable plaque; neovascularization in the plaque with a grade of 2 or stenosis of 50%-99% indicates an unstable plaque. 12 | 3 of 8

| Blood sampling and measurement of lipids
A total of 5 mL of venous blood were collected after fasting (>12 hours) to test the total cholesterol (TC), triglyceride (TG), LDL-C, high-density lipoprotein cholesterol (HDL-C), and sdLDL-C levels. Venous blood was collected using a procoagulant blood collection tube with an inert separation gel at the bottom (Liuyang Sanli Medical Technology Development, Inc). After blood collection, the tube was quickly inverted and mixed. After standing for a period of time, the serum was separated by centrifugation at 1700 g for 10 minutes and immediately detected on the machine.
The above items were tested using a Beckman Coulter AU5800 machine (Beckman Coulter, Inc, Brea, CA). sdLDL-C was detected by the peroxidase method, and the reagents were all from Beijing Jiuqiang Biotechnologies, Inc Quality control was performed using the company's quality control products.

| Statistical analysis
Statistical analysis was performed using IBM SPSS statistical software version 17.0 (SPSS Inc, Chicago, IL. A 2-sided P-value of 0.05 was considered significant. The Kolmogorov-Smirnov test was selected to assess the normality of the calculated parameters. Measurement data are shown as the mean ± standard deviation (SD). Student's t test was used for comparison between the two groups, one-way ANOVA was used for comparison between multiple sample means, and pairwise comparisons were performed using Bonferroni's t test. The chi-squared test was used for categorical variables. Independent risk factor analysis was performed using logistic regression. Correlation analysis between sdLDL-C and TG and between sdLDL-C and CAS stability in ACI patients was performed by Pearson correlation analysis, Spearman correlation analysis, and partial correlation analysis. The diagnostic efficacy of unstable plaques in ACI patients with CAS was verified using the receiver operating characteristic (ROC) curve.

| Comparison of baseline data between the ACI group and the control group
A total of 519 patients in the ACI group and 300 age-and sexmatched patients were selected. The proportion of patients with hypertension and hyperlipidemia and the TC, TG, LDL-C, HDL-C, and sdLDL-C levels in the ACI group were significantly different from those in the control group (P < 0.01, Table 1).

| Correlation analysis between sdLDL-C and TG
Pearson correlation analysis and partial correlation analysis were used to evaluate the correlation between sdLDL-C and TG and between TC and LDL-C. It was found that sdLDL-C was positively correlated with TG (P < 0.001), as shown in Table 2 and Figure 1.

| High sdLDL-C/LDL-C predicts onset of ACI even with a normal LDL-C level
The sdLDL-C level was higher in the high LDL-C group of patients with ACI than in the normal LDL-C group (P < 0.05) ( Table 3).
There was no significant difference in sex, age, alcohol use, smoking status, and TC, TG, LDL-C, and HDL-C level between the ACI group with normal LDL-C levels and the control group (P > 0.05). There was a statistically significant difference in the ratio of sdLDL-C to LDL-C and the level of serum sdLDL-C between the ACI group with normal LDL-C levels and the control group (P < 0.05, Table 4).

| sdLDL-C level and ACI events
Logistic regression was used to determine whether sdLDL-C was an independent variable. The results showed that sdLDL-C level was an independent risk factor for ACI (P < 0.001) (

| Comparison of baseline data among ACI patients without plaques and those with stable plaques and unstable plaques
A total of 519 patients with ACI were selected. The proportion of plaque-free patients in the three groups was 18.3%, the stable plaque group accounted for 20.8% of patients, the unstable plaque group accounted for 60.9% of patients, and the plaque groups (stable plaque  TG between the two groups (P > 0.05). The age of the unstable plaque group and the age of the stable plaque group were significantly different from that of the plaque-free group (P < 0.05). There was no significant difference between the serum TC and LDL-C levels in the stable plaque groups and the unstable plaque group, which were significantly higher than those in the plaque-free group (P < 0.05, P < 0.001). The serum HDL-C and sdLDL-C levels in the unstable plaque group were higher than those in the other two groups, and the difference was statistically significant (P < 0.05, P < 0.001) ( Table 6).  Table 7 shows that the sdLDL-C level gradually increases with changes in plaque properties and is an independent risk factor for unstable plaques in ACI patients. Spearman correlation analysis showed a positive correlation between sdLDL-C levels and changes in arterial plaque properties (Table 8, Figure 2). TA B L E 6 Comparison of baseline data of three groups with CAS plaque stability in patients with ACI

| Identification of CAS with unstable plaques in patients with ACI with the receiver operating characteristic (ROC) curve of sdLDL-C
With unstable plaque as the state variable and sdLDL-C as the test variable, a ROC curve was obtained, and the area was 0.695;

| D ISCUSS I ON
Cerebral infarction is the result of multiple factors. Common factors such as age, sex, hypertension, diabetes, elevated TC and LDL-C levels, decreased HDL-C level, smoking, and family history have prognostic value, especially LDL-C elevation, which is considered the most important traditional risk factor for cerebral infarction. CAS is the pathological basis of ACI, and sdLDL-C, one of the subcomponents of LDL-C, is more potent than coronary atherosclerosis. 13 The results of this study show that the level of sdLDL-C in the ACI group was significantly higher than that in the control group (P < 0.001); further studies found that the level of sdLDL-C in the unstable plaque group was significantly higher than that in the stable plaque group and the plaque-free group (P < 0.05, P < 0.001).
Based on the mechanism by which sdLDL-C induces atherosclerosis, it can be concluded that sdLDL-C carries a higher risk of coronary heart disease than LDL-C. Several large studies have evaluated the relationship between LDL-C particle size and coronary heart disease.
Sakai K et al studied sdLDL-C levels in 345 Japanese men ≥65 years of age with stable coronary artery disease. sdLDL-C is a more effective secondary biomarker for cardiovascular events than LDL-C. 14 The highest level of sdLDL-C was reported by a project funded by the National Cardiopulmonary Hematology Institute, which had used a new automated detection method for small-density cholesterol in the determination of coronary heart disease and atherosclerosis risk.
Individuals in the higher quartile groups had a higher risk of coronary heart disease than individuals in the lowest quartile of LDL-C levels. 15 In addition, the National Human Genome Research Institute project and the National Institutes of Health project-supported study, "Small, dense low-density lipoprotein cholesterol predicts the risk of coronary heart disease based on the risk of atherosclerosis (ARIC) in community populations," found that sdLDL-C is significantly associated with the occurrence of coronary heart disease. 16 To date, there have been few studies on the relationship between sdLDL-C and ACI.
The relationship between sdLDL-C and carotid plaque stability has not yet been reported, and the pathological basis of ACI is the same as that of coronary heart disease. We considered whether sdLDL-C can be used as an independent risk factor for ACI. The results of logistic regression analysis showed that sdLDL-C levels were positively correlated with ACI. After adjusting for other traditional risk factors, sdLDL-C was an independent risk factor for ACI, suggesting that sdLDL-C may be a potential biomarker for predicting the occurrence of cerebrovascular events.
The occurrence of cerebral infarction is closely related to the stability of CAS plaques, so accurate prediction of plaque properties It is traditionally thought that LDL-C can predict risk in ACI patients, but LDL-C levels are in the normal range in some ACI patients. The results of this study found that the level of sdLDL-C in the high LDL-C group of ACI patients was indeed higher than that in the normal LDL-C group (P < 0.05). The levels of sdLDL-C and the ratio of sdLDL-C to LDL-C were significantly different between the ACI group and the control group when the level of LDL-C is normal (P < 0.05). The LDL-C level alone does not accurately reflect risk in ACI patients; even in those with normal LDL-C levels, the risk of ACI cannot be ruled out. sdLDL-C level and the ratio of sdLDL-C to LDL-C in the ACI group with normal LDL-C levels were higher than those in the control group with normal LDL-C levels. This result may suggest that in patients with normal LDL-C levels, we can determine the risk of ACI by the level of sdLDL-C and the ratio of sdLDL-C to LDL-C.
It has been reported in the literature that TG can regulate the particle size of sdLDL-C. 18 Indeed, the sdLDL-C size was significantly larger in the high TG group than in the low TG group (P < 0.001), and Pearson correlation analysis showed a positive correlation between sdLDL-C and TG, which is consistent with the findings reported by Rizzo. 18 It has been proven that sdLDL-C production is closely related to TG.
This finding is even more meaningful considering that dyslipidemia in China is mainly attributed to hypertriglyceridemia. Furthermore, we explored the correlation between TG and CAS, and the results showed that TG is correlated with stable plaques. There was no significant difference in the grouping (P > 0.05), but there was a significant difference in sdLDL-C level (P < 0.001), which again showed that sdLDL-C was superior to traditional factors in predicting ACI.
In summary, this study analyzed the association of sdLDL-C level with ACI and CAS plaque stability in patients with ACI. The results showed that sdLDL-C is not only an independent risk factor for unstable plaques but also positively correlated with CAS plaque stability. This finding indicates that the level of serum sdLDL-C can help clinicians identify high-risk patients so that timely prevention measures can be taken.