The Value of Small Dense Low-Density Lipoprotein Cholesterol in Predicting the Risk and Severity of Coronary Heart Disease in Patients with Type 2 Diabetes Mellitus


 Background: Elevated small density low – density lipoprotein cholesterol (sdLDL-C) particles are hallmarks of atherogenic dyslipidemia in patients with type 2 diabetes mellitus (T2DM), which is hypothesized to drive atherosclerotic risk. The present study aims to investigate the association between serum sdLDL-C level and the presence and severity of coronary heart disease (CHD). Method: A total of 3684 consecutive patients with T2DM who received selective coronary angiography (CAG) were enrolled. The patients were subsequently divided into CHD and non-CHD groups according to results of CAG. The severity of CHD was evaluated by the number of stenotic, and the Gensini Score (GS).The patients were subsequently divided into four groups by the quartiles of sdLDL-C and evaluate the risk and severity of sdLDL-C and CHD.Results: The sdLDL-C levels in CHD group were significantly higher than non-CHD group [0.80 (0.49) mmol/L vs 0.70 (0.30) mmol/L, P < 0.001]. The results from CHD subgroup analysis indicated that the patients with multiple-vessel disease and high GS had higher sdLDL-C levels compared with those in their matched subgroups (P < 0.05). By adjusting the confounding factors and analyzing by the multiple logistic regression, sdLDL-C had independent correlation with the presence and severity of CHD [CHD: OR = 2.757, 95%, CI = 1.662 - 5.364, P = 0.023; multiple-vessel disease: OR = 3.788, 95%, CI = 1.866 - 7.685, P = 0.026; High GS: OR = 3.054, 95%, CI = 1.944 - 5.699, P = 0.022].Conclusion: In T2DM patients, the increase of sdLDL-C is related to the increase the prevalence and the severity of CHD. After adjustment for other risk factors, sdLDL-C was found to be an independent risk factor for CHD. Therefore, measuring sdLDL-C may allow for T2DM patients to predict the presence and severity of CHD.


Introduction
Coronary heart disease (CHD) is one of the most common diseases in the world and is associated with very high rates of morbidity and mortality [1][2][3]. Over the past decades, a large number of studies have examined the modi able risk factors such as diabetes, hypertension, dyslipidemia, and smoking for the early evaluation of cardiovascular risks [4,5]. Type 2 diabetes mellitus (T2DM) is a common metabolic disorder characterized by hyperglycemia and insulin resistance [6]. Dyslipidaemia in T2DM may exist alone or associate with metabolic syndrome, and thus increases cardiovascular risk [7,8]. The typical pattern of diabetic dyslipidaemia consists of elevated low density lipoprotein cholesterol (LDL-C) triglycerides, low high density lipoprotein cholesterol (HDL-C) [9].
Plasma LDL-C is composed of a series of granules with different sizes, densities and chemical compositions [10]. In general, LDL-C with smaller particles and higher density is named as small dense low density lipoprotein cholesterol (sdLDL-C). On the contrary LDL-C with larger particles and smaller density is de ned as large and light LDL-C; and the subcomponent between them is medium density LDL-C. Compared with LDL-C, sdLDL-C is thought to be more atherogenic [11,12]. Evidences from basic and clinical studies have shown that sdLDL-C is a pro-atherogenic risk factor for atherosclerosis and elevated levels of sdLDL-C may promote the development of CHD [13,14]. However, the role of sdLDL-C in stable CHD patients, especially in patients with special disease status such as T2DM, has not been fully determined. Therefore, the aim of this study is to determine the relationship between sdLDL-C and the presence and severity of coronary heart disease in Chinese patients with T2DM.

Study population
One thousand eight hundred and forty-two Chinese Han patients (2152 males, 1532 females) from Peking University International Hospital and Peking University People's Hospital were sequentially enrolled between October 2015 to October 2019. All patients who were diagnosed with T2DM, which was de ned according to the American Diabetes Association (ADA) criteria: 1) self-reported history of T2DM, 2) under current treatment of insulin or oral hypoglycemic medicine, 3) repeated fasting plasma glucose (FPG) 7.0 mmol/L, 4) glycated hemoglobin A1c (HbA1c) 6.5%.
The diagnostic criteria for patients with CHD are coronary angiography (CAG) performed in our institution and at least one major coronary artery occlusion or stenosis is found to be more than 75% and the severity of CHD was evaluated by Gensini score (GS ) system. The diabetic patients were divided into non-CHD group and CHD group based on the results of CAG. The diabetic patients with CHD were classi ed into the GS tertiles: low GS (GS ≤ 25), intermediate GS (GS: 26-40) and high GS (GS ≥ 41).
Exclusion criteria included: 1) any known in ammatory or infectious disease, or con rmed or suspected cancer, 2) acute coronary syndrome within the previous six months, 3) percutaneous coronary intervention within the previous three months, 4) history of coronary artery bypass operation, 5) chronic heart failure, cardiomyopathy, valvular heart disease, 6) pulmonary heart disease, or 7) severe liver and kidney dysfunction.

Conventional clinical and laboratory indicator tests
Blood samples were collected in the morning after overnight fasting for at least 12 h. All measurements were performed within 6 h. FBG, homocysteine (HCY), hypersensitive C-reactive protein (hs-CRP), serum lipid pro les, including triglycerides (TG), total cholesterol (TC), LDL-C and HDL-C were determined on a Beckman AU5832 analyzer (Beckman Coulter Inc., USA). Apolipoproteins A-1 (apoA1), and B (apoB) were measured by immunoturbidimetry (Daiichi Pure Chemicals Co., Ltd., Tokyo). Lp(a) in the serum samples was measured using latex enhanced immunoturbidimetry Lp(a) kit (Roche Inc., Germany). Direct quantitative determination of sdLDL-C assay was done using sdLDL-C reagent kits (Denka Seiken Co., Ltd. Japan). Hemoglobin A1c (HbA1c) was measured using high-performance liquid chromatography technology (Trinity Biotech Inc., USA).

Statistical analyses
The distributions of all quantitative variables were analysed using the one-sample Kolmogorov-Smirnov test. Normally distributed data are reported as means ± standard deviations, and the differences between various groups were compared using the Analysis of Variance. Non-normally distributed continuous data were reported as medians (inter-quartile ranges), and the differences between various groups were compared using the Kruskal-Wallis test. Categorical data was presented as percentage (%) and compared by Chisquare test. Mantel-Haenszel test for linear trend was used to detect whether sdLDL-C levels was positively correlated with the CHD. The association of sdLDL-C with the presence and severity of CHD was analyzed by multivariate logistic regression adjusted for age, gender, BMI, glucose, HbA1c, ApoB, ApoA1, TC, TG, HDL-C, LDL-C, Lp(a), hs-CRP, and HCY. All data analyses were performed using SPSS version 22.0 for Windows (IBM Corp., USA). P < 0.05 was considered to be statistically signi cant.

Study population characteristics
A total of 3684 participants were enrolled in this study, including 2220 CHD patients and 1464 non-CHD patients. Table 1 shows clinical characteristics and risk factors of all participants, including age, gender, glucose, HbA1c, blood-lipid indicators, Lp(a), hs-CRP, HCY and sdLDL-C. Levels of ApoB, LDL-C, hs-CRP, HCY, and sdLDL-C in CHD group were higher than those in non-CHD group. The CHD group had a signi cantly lower apoA1 and HDL-C levels compared with non-CHD group. There were no signi cant differences of other variables between the two groups (P > 0.05). Relation of sdLDL-C levels and severity of CHD Based on the CAG results of each individual, the diabetic patients with CHD were further divided into the following subgroups including multiple-vessel disease group, and high GS group for the purpose of intensively evaluating the relation of sdLDL-C to the severity of CHD (Table 2 and Table 3).  The patients with diabetes and CHD were then classi ed into single-vessel (n = 576), two-vessel (n = 656), and multiple-vessel disease (n = 988) groups (Table 2) (Table 3). It can be seen from the results that the serum sdLDL-C levels in the high GS group was signi cantly higher than that in the other two groups [0.71 (0.29) mmol/L vs. 0.80 (0.27) mmol/L vs. 0.91 (0.53) mmol/L, P < 0.001].
After dividing diabetic patients with CHD into four groups according to quartiles of sdLDL-C levels, The results show that there is a linear correlation between the level of sdLDL-C and the multiple-vessel disease group and the high GS group. With the increase of serum sdLDL-C, the degree of CHD obstruction is more serious. (Table 4) (P < 0.001).  For exploring the role of sdLDL-C in CHD, univariate and multivariate regression analyses were also performed in our study. All participants were divided into four quartiles of sdLDL-C levels, and the present and severity of CHD in individuals with different sdLDL-C levels was assessed. Through univariate logistic regression, sdLDL-C levels were associated with the presence and severity of CHD (CHD group vs. Discussion sdLDL-C is a distinct LDL-C subclass, which is associated with raised TG and decreased HDL-C levels in adiposity and diabetes, playing a distinct metabolic role in atherosclerosis [15][16][17][18]. In this study including patients with T2DM, we found that there was an independent association between sdLDL-C and CHD.
Brie y, the patients with more severe coronary stenosis had higher sdLDL-C levels and sdLDL-C was independently associated with the presence and severity of angiography-proven CHD presented as GS multiple-vessel disease in patients with T2DM. These ndings provide evidence that the measurement of serum sdLDL-C is clinically valuable for estimating the future onset of CHD beyond the prede ned cardiovascular risks [19,20].
Many studies have con rmed the high concentrations of sdLDL-C may be a risk factor for CHD [21][22][23][24]. Several studies in different populations using various sdLDL-C measurements suggested that the sdLDL-C was related to the carotid intima thickness and in association with progression of carotid artery plaque [25]. In an 11-year follow-up study involving 11419 men and women, 1158 participants who developed CHD showed average sdLDL-C concentrations of 43.5 mg/dL [26]. Moreover, the increased sdLDL-C concentrations were correlated with a higher propensity for diabetes, arterial hypertension, increase in body mass index (BMI) and hs-CRP values. In the Multi-Ethnic Study of Atherosclerosis (MESA), a higher concentration of sdLDL-C was associated with the development of CHD among overall study participants, but the association was not statistically signi cant among patients with T2DM [27].
Our result was inconsistent with this nding. The reasons for this discrepancy are not entirely clear, but the con ict between the results of the study may be caused by a variety of confounding factors, such as different population characteristics, study design, disease status, confounding variables, or sdLDL-C measurement method. Therefore, the inconsistent effect of sdLDL-C on CHD in patients with T2DM may also mean that the role of sdLDL-C in heart metabolic diseases is complex and needs to be studied carefully [28]. That's why we used a large Chinese cohort for this study. In our study, we found that the level of sdLDL-C in T2DM patients was related to the presence of angiographically con rmed CHD. In addition, in order to further study the relationship between sdLDL-C and CHD severity in diabetic patients, we used GS system to conduct multiple subgroup analysis.
There are several limitations in the present study. Firstly, there are only two centers for this study, which might result in selective biases. Secondly, we were able to measure the serum sdLDL-C level and other risk factors only once at baseline. We were also unable to obtain information about medical treatment during the follow-up period. Finally, although there is statistical signi cance between the level of sdLDL-C and CHD in patients with T2DM, CHD is a complex disease with many pathogenic factors, which also requires more research to con rm our ndings.

Conclusion
In conclusion, our study indicates that the increase of sdLDL-C is an independent predictor of CHD, and is related to the severity of CHD. These ndings suggest that sdLDL-C is a crucial biomarker for the prediction of the occurrence and severity of CHD complicated with T2DM.