An ultra-sensitive liquid chromatography-fluorescence detection method for determining hCE1 activity and evaluation of hCE1 as a biomarker of liver injury.

Human carboxylesterase 1 (hCE1) is highly expressed in the liver and can be released into the bloodstream upon hepatocellular injury, suggesting its potential as a biomarker for liver damage. However, current methods for measuring hCE1 activity suffer from poor selectivity and insufficient sensitivity and are easily interfered with by serum albumin (HSA), butyrocholinesterase (BChE), and paraoxonase 1 (PON1). In this study, a novel fluorescent probe, NCMNe, was designed and synthesized, and an ultrasensitive liquid chromatography-fluorescence detection (LC-FD) method was established for quantifying plasma hCE1 activity and evaluating its feasibility as a biomarker of liver injury. Specificity studies showed that NCMNe was selectively hydrolyzed by hCE1 without interference from HSA, PON1, or BChE. The developed LC-FD method achieved a lower limit of quantification of 0.0001 μM, representing the most sensitive method reported to date for hCE1 activity determination. Clinical sample analysis revealed that plasma hCE1 activity in patients with hepatitis B was significantly higher than that in healthy controls and was strongly correlated with aspartate transaminase (AST)and alanine transaminase (ALT). Receiver operating characteristic (ROC) curve analysis demonstrated that plasma hCE1 activity measurement was effective for detecting liver pathology (AUC = 0.901, p < 0.0001), with diagnostic performance comparable to that of ALT and AST. Collectively, the proposed LC-FD method enables highly sensitive and selective determination of plasma hCE1 activity and supports hCE1 as a promising serum biomarker for liver injury, providing a new approach for early diagnosis and disease assessment.