科研之友
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摘要
Background: Cholinergic components in non-neuronal tissues and cells are proposed as important in maintaining cellular proliferation and immune homeostasis. However, direct quantification of non neuronal acetylcholine (ACh) in cells has been inefficient. Therefore, we developed a stable method for determination of intracellular ACh. @@@ Methods: We used ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with Waters CORTECS chromatographic columns to measure the intracellular ACh in human peripheral blood mononuclear cells (PBMCs). The presence of ACh was validated by RT-qPCR of mRNAs of cholinergic components. @@@ Results: This method successfully separated ACh from iso-ACh with highly sensitive precursor/product ion transitions and allowed a faster chromatography run time within 3.5 min. The detection limit of intracellular ACh was 0.005 ng/10(6) PBMCs. Intra-assay and inter-assay coefficient variations were all <7.0%. Mean recoveries in the samples were between 93.20% and 104.73%. Here, intracellular ACh was stable under multiple storage conditions, partly attributable to mutually stable relationships among cholinergic components. This method was successfully applied in a stroke study and revealed activation of the stroke-induced cholinergic anti-inflammatory pathway. @@@ Conclusions: This method allows direct determination of intracellular ACh in various tissues. Evidences of cholinergic activity linking both the nervous system and non-neuronal system can be profiled by assessing ACh.
- 出版日期2016-6-1
- 单位天津市疾病预防控制中心; 天津医科大学; 天津市环湖医院
