Glycosylation Of Diabetic Patients With Coronary Atherosclerotic Heart Disease In Patients With High-density Lipoprotein Cholesterol Function In Research

High density lipoprotein(HDL) promotes reverse cholesterol transport(RCT),thus plays important roles in many antiatherogenic pathways.In addition to serum levels, the component and structure of HDL significantly affect its functions.Physically or chemically modified HDL brought forward a series of function changing,can even turn into proatherogenic proteins.Dyslipidemia is a common finding in type 2 diabetes patients,along with increased risk of artherosclerosis.Serum proteins of patients with type 2 diabetes can carry on nonenzymatic glycation,but controversial opinions still exist on whether the functional defects of HDL correlated with it glycation levels.Understanding the distribution of glycation levels of HDL,as well as its impact on HDLs' functions,may contributes to the treatment and prevention of artheriosclerosis in type 2 diabetes.We extracted HDL from diabetic patients' serum samples,as well as from healthy subjects' samples for in vitro glycation,and compared HDL mediated intracellular cholesterol efflux and anti-oxidative function between groups.Our aim was to identify the relationships between HDL glycation and its anti-oxidative function or efflux capacity by different means,as well as to examine the impact of statins on HDL glycation and function.OBJECTIVE:To discuss the mechanism of intracellular accumulation of cholesterol in diabetic patients with or without coronary artery disease(CAD),by evaluating the effects of glycation of HDL on its efflux capacity and anti-oxidative function,as well as to establish a proper method for determination of glycation of HDL,and explore the role of statins on functions of HDL associated with glycation and diabetes.METHOD:1.Serum was collected from 19 CAD patients,with or without type 2 diabetes,as well as 11 healthy age-matched subjects;2.HDL was isolated by ultracentrifugation,from serum of both healthy subjects' and patients' samples;3.Mixed HDL from healthy subjects was incubated with glucose of different levels, before further experiment with patient samples;4.Glycation levels of HDL was evaluated by measuring fructosamine or trinitrophene sulphonic acid(TNBS) method.The applicability of each method was evaluated; 5.HDL mediated of cholesterol efflux from human umbilical vein epithelial cells(HUVECs) or HepG2 cells mediated by each HDL sample was measured with ¹⁴C-cholesteryl oleate labeled LDL in vitro,and the correlation with glycation level was analyzed;6.Anti-oxidative fuction was evaluated by detecting the optical density(OD,234nm) and calculating the lag time(Tl_ag) of oxidation of the LDL incubated with HDL samples,with oxidative stress provided by Cu²⁺.Correlations between anti-oxidative functions and glycation levels were analyzed;7.Effects of statins on glycation level and functions of HDL were analyzed.RESULT:1.The concentration and glycation levels of HDL in our study went beyond the sensitivity of the measurement with fructosamine method;2.According to the result of TNBS testing,the level of HDL glycation in diabetic patients was significantly higher than that in non-diabetic ones(1.0172 vs.0.8067, p=0.017).HDL glycation levels were not correlated with HbA1c or free blood glucose levels(p=0.364 and 0.388,respectively);3.The rates of efflux mediated by HDL glycated in vitro in HUEVC or HepG2 cells were not correlated with glycation levels of HDL;4.Among diabetic and/or CAD patients,no significant differences were observed between efflux rates among groups with high HDL glycation levels and low levels(11.38%vs.12.09%,p=0.313);5.The Lag time of LDL oxidation in the presence of HDL was correlated HDL glycation levels(Pearson's Correlation Coefficient -0.504,p=0.022),and T_lag was significantly lower in the group with higher HDL glycation levels(90.37min vs. 102.46min,p=0.022);6.OD_max of LDL oxidation were not significantly differed between groups with higher and lower HDL glycation levels;7.The use of statins didn't affect the level of HDL glycation(0.9823 vs.1.0730, p=0.395),however,it significantly increased cholesterol efflux in HUVECs mediated by HDL from diabetic patients(12.23%vs.10.28%,p=0.024),as well as prolonged the lag time of LDL oxidation in the presence of HDL from patients(100.68min vs. 95.03min,p=0.020).CONCLUSION: 1.The TNBS method can sufficiently detect glycation levels of HDL isolated by ultracentrifuge.The fructosamine method has a lower sensitivity,and is therefore,not capable for glycation level determination of ultracentrifugally isolated HDL;2.In compare with non-diabetic subjects,diabetic patients have HDL with higher glycation level.The HbA1c and free blood glucose levels can not sufficiently predict HDL glycation levels in diabetic patients.The use of statins doesn't lower HDL glycation level;3.HDLs from diabetic patients have defected ability in inducing cholesterol efflux, which can be reverted by the use of statins.Glycation doesn't directly impair the cholesterol efflux capacity of HDL.4.Glycation may be one of the factors causing defect anti-oxidative functions of HDLs in diabetic patients.The improvement of HDL's anti-oxidative functions by statins is independent to the state of HDL glycation.