Participants and biochemical parameters
The characteristics of the patients and healthy subjects and their biochemical parameters are summarized in the Table 1 and 2 respectively. All ACS patients included in the study had ST segment elevation myocardial infarction (STEMI). There was a significant difference in the mean age of control group and ACS group (p <0.001). There were more male subjects in ACS group than in control group. The ACS group had higher proportion of participants with diabetes and hypertension. They also had a higher percentage of smokers (p <0.0001).
Patients presenting with ACS had significantly lower HDL-C (45.4 ± 9.6 vs 40.2 ± 9.6 mg/dl, p <0.001) and apolipoprotein A-I (142.1 ± 43.3 vs 112.5 ±27.3 mg/dl, p <0.001) levels as compared to control subjects. ACS patients had significantly higher apo B levels as compared to controls. No significant difference was observed in total cholesterol (TC), LDL-C, very low density lipoprotein cholesterol (VLDL-C) and triglycerides (TG) between two groups overall.
HDL functions
We observed that ACS patients had significantly lower HDL cholesterol efflux capacity, 0.88 ± 0.15 Arbitrary Units (AU) in ACS and 1.02 ±0.16 AU in control subjects (p<0.001) (Fig. 1a). Arylesterase activity of PON1 was significantly lower in ACS patients (112.0 ± 36.3 vs 82.1 ± 27.9 U/mL, p<0.001) (Fig. 1b). Paraoxonase activity of PON1 was significantly reduced in ACSpatients(105.1 ± 56.4 U/mL in controls and 69.1 ± 38.8 U/mL in ACS subjects)(Fig.1c).
Smokers with ACS had significantly lower arylesterase activity compared to non-smokers in ACS group (76.7 vs 88.7 U/mL, p=0.008). CEC was lower in smokers (0.87 ± 0.15 AU) than in non-smokers (0.89 ± 0.14 AU) within the ACS group but the difference was not statistically significant. HDL functions were also found to be significantly lower in male and female ACS subjects compared to male and female controls respectively (Table 3).
HDL functions in ACS patients at follow-up
The follow-up samples from 100 patients were available. All ACS patients enrolled in the study were statin naïve and were on high dose statin after they were diagnosed with ACS. 45 were lost to follow-up and 5 had a second event of ACS or had died. A significant decrease in body mass index was observed after six months follow up (25.69 ± 3.9 vs 24.7 ± 4.0 kg/m2, p<0.001). In the study, out of 100 ACS patients who were followed up, 62 were smokers at presentation and out of these 62, 37 quit smoking by the end of follow up.
Change in lipid profile after therapy is summarized in Table 2. A significant reduction was observed in total cholesterol, VLDL cholesterol and LDL-C levels after therapy. HDL-C also decreased after treatment, but the decrease was not significant; however, there was a significant increase in apolipoprotein A-I levels. Triglyceride levels also decreased after therapy, but the decrease was not significant. Apolipoprotein B levels were reduced after six months. The patients at follow-up had significantly lower total cholesterol, apo B and LDL-C levels compared to control subjects. They also had significantly lower apo A-I levels and HDL-C levels.
We observed a significant increase in cholesterol efflux capacity at follow up after six months in ACS patients (0.89 ± 0.14 vs 0.98 ± 0.17 AU; p<0.001). The efflux capacity of ACS follow-up and control subjects were found to be similar (Fig. 1a). A significant improvement in PON1 arylesterase (Baseline: 85.4 ± 30.2; Follow up: 101.8 ± 30.6 U/mL; p<0.001) (Fig. 1b) and paraoxonase activity (Baseline: 69.1 ± 42.; Follow-up 81.1 ±45.5 U/mL; p<0.001) (Fig. 1c) was observed after six months of therapy. Although there was marked increase in paraoxonase and arylesterase activity of ACS patients after statin therapy, it was still significantlylower compared to control subjects.
Correlation between HDL function and apolipoprotein A-I
Linear regression analyses showed a significant positive correlation between cholesterol efflux capacity and apolipoprotein A-I (r = 0.39. p = 0.001), and a similar correlation was observed in control (r = 0.29, p = 0.0079), ACS baseline (r = 0.3, p = 0.0013) (Fig. 2a) and ACS follow-up (r = 0.26, p = 0.01) groups separately (Fig. 2d). However, no correlation was observed between CEC and HDL-C levels in control (r = 0.02, p = 0.82) or ACS group (acute phase: r = 0.07, p = 0.47)
A strong positive correlation was observed between arylesterase activity and the levels of apolipoprotein A-I in controls (r = 0.3, p = 0.0012), ACS baseline (r = 0.19, p = 0.019) (Fig. 2b) and ACS follow-up groups (r = 0.26, p = 0.007). Fig. 2e). We did not observe any correlation between paraoxonase activity and apolipoprotein A-I levels and HDL- C levels (Fig. 2c)
hs-CRP levels were measured as an index of inflammation in acute coronary syndrome subjects. Cholesterol efflux capacity showed an inverse correlation with hs-CRP levels at follow-up (r = -0.2, p = 0.03) and the same trend was also observed in the baseline ACS data (r = -0.2, p = 0.03) (Fig. 2f).
Association of HDL functions with acute coronary syndrome
Logistic regression was performed to analyze the association of apolipoprotein A-I, cholesterol efflux capacity and PON1 activity of HDL with the odds of having ACS (Fig. 3). Apolipoprotein A-I, Cholesterol efflux capacity, PON1 paraoxonase and arylesterase activities were seen to have a protective effect (Model 1). The effect of apolipoprotein A-I, efflux capacity and antioxidative activity remained significant even after adjustment for age, gender, BMI and other cardiovascular risk factors like smoking, diabetes, hypertension and LDL-C (Model 2) (Fig. 3). Higher cholesterol efflux capacity (odds ratio per 1-SD increase: 0.49; 95% confidence interval: 0.29-0.8; p = 0.006), PON1 paraoxonase activity (odds ratio per 1-SD increase: 0.44; 95% confidence interval: 0.28-0.66; p = 0.0002) and arylesterase activity (odds ratio per 1-SD: 0.50; 95% confidence interval: 0.34-0.72; p = 0.003) were associated with lower odds of development of ACS even after additional adjustment for HDL-C levels (Model 3).
We then assessed interactions between HDL functions (cholesterol efflux capacity and arylesterase activity, cholesterol efflux capacity and paraoxonase activity) to predict the probability of ACS. This interaction is meant to represent how the effect of CEC on the predicted probability of ACS differs across levels of arylesterase activity and vice versa (Fig. 4a). Similarly, the effect of CEC on the predicted probability of ACS at different levels of PON was also analyzed as in Fig. 4b. We calculated the predicted probability of acute coronary syndrome for all combinations of cholesterol efflux capacity ranging from 0.5 to 1.5 with an increment of 0.5, and arylesterase activity, ranging from 20 to 210 U/mL (increment 50). Similarly, we also analyzed the interaction of CEC with paraoxonase activity (ranging from 40 to 240 U/mL with an increment of 10) We observed that an individual with cholesterol efflux capacity (CEC) 1 A.U. and arylesterase activity (ARE) 100 U/mL has a 50% chance of having acute coronary syndrome (ACS) (p <0.001), while an individual with CEC 1 A.U. and ARE 190 U/mL has a 10% chance of having ACS (p = 0.06). We found that the interaction between two HDL functions was significant for lower values of cholesterol efflux capacity and PON1 activity (arylesterase activity and paraoxonase activity) but was not significant for low cholesterol efflux values and high arylesterase or paraoxonase values.