School of Life Science and Medicine, Dalian University of Technology, Panjin 124221, China; Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
School of Life Science and Medicine, Dalian University of Technology, Panjin 124221, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 10;1157:122333. doi: 10.1016/j.jchromb.2020.122333. Epub 2020 Aug 16.
Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.
儿茶酚-O-甲基转移酶（COMT）是人类中主要的药物代谢酶。由于其在催化内源性儿茶酚胺和儿茶酚雌激素的代谢失活中起着重要作用，COMT 的表达直接与各种精神疾病和癌症有关。然而，缺乏一种精确测量生物样品中 COMT 活性的实用方法。在本研究中，我们建立了一种基于荧光检测的液相色谱-荧光检测（LC-FD）方法，该方法基于荧光探针 3-BTD 的甲基化产物 3-BTMD 的荧光检测，用于灵敏地定量人红细胞和细胞匀浆中的 COMT 活性。建立的 LC-FD 方法进行了选择性、灵敏度、线性范围、精密度和准确度、回收率、生物基质效应和稳定性的验证。该方法对 3-BTMD（COMT 对 3-BTD 的甲基化产物）的定量下限为 0.0083 nM，比以前发表的方法低近 10 倍。该方法具有良好的精密度，日内和日间相对标准偏差（RSD）低于 5%。此外，该方法具有出色的抗干扰能力，内源性物质对 3-BTMD 的荧光检测没有影响。该方法通过成功应用于测量个体人红细胞（n=13）和来自四种不同人细胞系的细胞匀浆中的 COMT 活性，证明了其实际应用。我们的结果表明，该方法将在准确测定生物样品中 COMT 的天然活性方面具有重要价值，这有利于全面了解 COMT 在生理和病理条件下的作用。
