Development And Application Of COMT Activity Evaluation Method

COMT is responsible for the metabolic inactivation of various catecholamine neurotransmitters(such as dopamine)in the human body,thereby regulating various behaviors such as emotions,feelings and cognitive functions.A large number of studies have shown that COMT is closely related to the occurrence and development of various psychiatric and neurological diseases such as schizophrenia and Parkinson’s disease,and is the therapeutic target of Parkinson’s disease and other diseases.It quantitatively evaluates the COMT activity of complex biological systems for related diseases.Early diagnosis and the development of therapeutic drugs are of great significance.However,the industry has been short of efficient and highly sensitive detection methods for COMT activity in complex systems.To this end,the first two-photon COMT fluorescent substrate(3-BTD)independently developed by the subject in the previous period is the core.For different research objects and experimental purposes,we constructed the high-throughput detection method of COMT active based on the microplate reader,and the high-sensitivity detection method of COMT activity based on liquid phase-fluorescence(LC-FD).On this basis,the application of the species difference evaluation of COMT activity and the quantitative evaluation of COMT activity in individual erythrocyte were carried out.The specific research contents and results are as follows:(1)The construction of a COMT activity fluorescence detection method based on the high-throughput detection of the microplate reader and the study of the differences in COMT activity species.First,with COMT fluorescent substrate(3-BTD)as the core,with the help of the in vitro COMT metabolism research technology system,a high-efficiency COMT activity evaluation method was constructed on the microplate reader to complete the incubation conditions optimization,sample pretreatment,and detection process related to COMT detection.And the optimization of quantitative methods,as well as the verification by liquid phase methods to ensure the accuracy and reliability of the evaluation methods.Secondly,this method was used to study the interspecies differences of COMT in the liver S9 of humans and seven experimental animals(including monkeys,dogs,mice,rats,minipigs,guinea pigs and New Zealand rabbits).The results show that all liver S9 from different animals can catalyze 3-BTD methylation,but the reaction rates show significant differences.In addition,different effects of tolcapone(positive control of COMT inhibitor)on 3-BTD methylation invarious species were observed.The apparent kinetic parameters and intrinsic clearance(Clint)of 3-BTD methylation in liver S9 of different animals were determined.The catalytic efficiency was RLS9> DLS9 ≈ PLS9> MLS9> CyLS9>RaLS9> GpLS9> HLS9.The above findings are of great significance for the development of targeted COMT therapeutic drugs and for exploring the biological functions of COMT in different animal models.(2)High-sensitivity analysis method for COMT activity based on LC-FD detection and evaluation of COMT activity in individual erythrocyte was constructed.In order to solve the requirements of high stability and high sensitivity in the analysis of clinical samples,this subject has developed a liquid phase fluorescence(LC-FD)with high selectivity,high stability,and high sensitivity,taking the COMT fluorescent substrate(3-BTD)as the core.The detection method is used for the highly sensitive detection of COMT activity in complex biological systems such as erythrocyte and cell preparations.The results show that COMT activity in different erythrocyte has5-fold difference.This detection method has great potential in accurately measuring the activity of COMT in complex biological samples,which is helpful to carry out in-depth research on the biological function of human COMT,the occurrence and development mechanism,diagnosis and treatment of related diseases.In summary,the study is based on the practical problems faced by the current COMT activity detection.The first two-photon COMT fluorescent substrate(3-BTD)independently developed in the previous period is the core.For different research objects and experimental purposes,the COMT activity fluorescence detection method based on the high-throughput detection of the microplate reader and the liquid-fluorescence(LC-FD)COMT activity high sensitivity detection method were constructed,and the species differences of COMT activity were carried out applied research such as evaluation and quantitative evaluation of COMT activity in individual erythrocyte.This study not only provides an effective method and experimental basis for the detection of COMT activity in complex systems,but also provides new methods for the development of COMT-targeted therapeutic drugs and the exploration of the biological function of COMT and the development mechanism of related diseases.