Abstract 4352077: A novel method for measuring HDL-bound unconjugated bilirubin using an eel fluorescent protein reveals its association with reduced coronary artery disease

Background: Owing to its antioxidant activity, unconjugated bilirubin (UCB) has been shown to have protective effects against oxidative stress-mediated diseases, including coronary artery disease (CAD). An original study on UnaG, a fluorescent protein that emits green fluorescence upon binding to UCB, has revealed the presence of bilirubin in high-density lipoprotein (HDL) particles. However, quantifying UCB in HDL particles has been hampered by the overwhelming amount of albumin-bound UCB in blood samples. Accordingly, the relationship between HDL-bound UCB levels and CAD recurrence has remained unclear. Hypothesis: Higher HDL-bound UCB levels are associated with reduced CAD recurrence. Therefore, accurate quantification of HDL-bound UCB may be a predictive biomarker for CAD recurrence. Methods: We have developed an efficient technique for preparing albumin-free HDL from serum using the dextran sulfate precipitation method. We utilized apoUnaG to establish a direct fluorometric method for quantifying UCB in HDL samples. We measured HDL-bound UCB levels in blood samples from 100 patients who had undergone percutaneous coronary intervention. A comparison was made between the presence and absence of revascularization. Results: SDS-PAGE analysis and biochemical tests revealed that the HDL fraction for UCB assay was completely devoid of albumin. In addition, HDL cholesterol levels in the HDL fraction were strongly correlated with those in serum, validating the recovery efficiency of our method for HDL purification. apoUnaG was able to quantify HDL-bound UCB in serum samples from CAD patients, with measured UCB levels ranging from 0.00781 μM to 0.5 μM. Remarkably, HDL-bound UCB levels were significantly lower in the recurrence group than in the non-recurrence group {median (IQR): 0.0531 (0.0285–0.126) and 0.0911 (0.0495–0.171), respectively; p = 0.02}. In contrast, no significant differences were observed in the serum levels of UCB and HDL-cholesterol between the two groups. Conclusion: The combination of the classic dextran sulfate precipitation method and the recently discovered eel fluorescent protein is a simple yet reproducible technique for quantifying UCB in HDL fractions. High levels of HDL-bound UCB were associated with a reduced risk of CAD recurrence.