Serum regucalcin is a useful indicator of liver injury severity in patients with hepatitis B virus-related liver diseases

Abstract

Regucalcin is a soluble protein that is principally expressed in hepatocytes. Studies of regucalcin have mainly been conducted in animals due to a lack of commercially available kits. We aimed to develop an enzyme-linked immunosorbent assay (ELISA) to quantify serum regucalcin in patients with hepatitis B virus (HBV)-related disease. High-titer monoclonal antibodies and a polyclonal antibody to regucalcin were produced, a double-antibody sandwich ELISA method was established, and serum regucalcin was determined in 47 chronic hepatitis B (CHB) patients, 91 HBV-related acute-on-chronic liver failure (HBV-ACLF) patients, and 33 healthy controls. The ELISA demonstrated an appropriate linear range, and high levels of reproducibility, sensitivity, specificity, accuracy, and stability. The median serum regucalcin concentrations in HBV-ACLF and CHB patients were 5.46 and 3.76 ng/mL, respectively (P<0.01), which were much higher than in healthy controls (1.72 ng/mL, both P<0.01). For the differentiation of CHB patients and healthy controls, the area under curve (AUC) was 0.86 with a cut-off of 2.42 ng/mL, 85.7% sensitivity, and 78.8% specificity. In contrast, the AUC of alanine aminotransferase (ALT) was lower (AUC=0.80, P=0.01). To differentiate ACLF from CHB, the AUC was 0.72 with a cut-off of 4.26 ng/mL, 77.0% sensitivity, and 61.2% specificity while the AUC of ALT was 0.41 (P=0.07). Thus, we have developed an ELISA that is suitable for measuring serum regucalcin and have shown that serum regucalcin increased with the severity of liver injury due to HBV-related diseases, such that it appears to be more useful than ALT as a marker of liver injury.

Figures

Figure 1. Standard curve for regucalcin constructed using absorbance (optical density, OD) values and serum regucalcin concentrations.

Figure 2. Serum regucalcin concentrations in heathy controls, and chronic hepatitis B (CHB) and acute-on-chronic liver failure (ACLF) patients. The top and bottom of each box represent the 25th and 75th percentiles. The line through the box is the median and the error bars represent the 5th and 95th percentiles. Statistical analysis was done with the Mann-Whitney test.

Figure 3. Receiver operating characteristic (ROC) curves for regucalcin (RGN) and alanine aminotransferase (ALT) in chronic hepatitis B (CHB) (A) and acute-on-chronic liver failure (ACLF) (B) patients. A, The area under the curve (AUC) for RGN was 0.86 with a cut-off of 2.42 ng/mL. The sensitivity and specificity were 85.7 and 78.8%, respectively. The AUC for ALT was 0.80 (95%CI: 0.70–0.89, P<0.01) with a cut-off of 28.30 U/L (sensitivity: 0.62 and specificity: 0.91), which is lower than the AUC for RGN (P=0.01). B, The AUC for RGN was 0.72 with a cut-off of 4.26 ng/mL. The sensitivity and specificity were 77.0 and 61.2%, respectively. In contrast, the AUC of ALT was only 0.41 (95%CI: 0.30–0.51, P=0.07) (P<0.01).