Department of Clinical Biochemistry, Institut de Recerca Sant Joan de Déu (IRSJD), Barcelona, Spain.
Centre for Research in Rare Diseases (CIBERER-ISCIII), Barcelona, Spain.
Nat Protoc. 2017 Nov;12(11):2359-2375. doi: 10.1038/nprot.2017.103. Epub 2017 Oct 19.
The presence of monoamines and their cofactors (the pterins and vitamin B (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.
人脑脊液（CSF）中存在单胺及其辅因子（蝶呤和维生素 B（吡哆醛磷酸（PLP））），可作为大脑中多巴胺和 5-羟色胺生物合成和周转的指标。此外，这些分子的 CSF 水平异常与各种神经疾病有关，包括导致多巴胺和 5-羟色胺缺乏的遗传疾病。在这里，我们提供了一套定量高效液相色谱（HPLC）方法来确定 CSF 中单胺及其辅因子的水平。该方案描述了用于分析不同分子的 CSF 样品制备、HPLC 校准和分析以及数据定量和解释的逐步程序。与血浆/组织/血液样本不同，CSF 需要最少的样品制备：在该方案中，只有 PLP 的分析需要与三氯乙酸混合以释放与蛋白质结合的维生素，离心，并将上清液与磷酸缓冲液和氰化钠混合在碱性条件下进行衍生化。单胺通过 HPLC 与库仑电化学检测（ED）分析，蝶呤通过 HPLC 与偶联库仑电化学和荧光检测分析，PLP 通过 HPLC 与荧光检测分析。所有化合物的定量均通过外部校准程序实现，在每次运行中分析内部质量控制和标准品。我们预计通过测量人 CSF 和其他生物样本中的这些化合物，将促进对多巴胺和 5-羟色胺紊乱的研究。不同程序的估计时间主要取决于电化学检测器的稳定时间。建议过夜稳定该检测器，并且在该步骤之后，预分析平衡很少超过 3 小时。
