Non-HPLC separation of water-soluble choline metabolites by two-dimensional high voltage electrophoresis and thin layer chromatography

In cholinergic neurons choline is directed to three main pathways; (1) conv ersion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-cho line) for the synthesis of phosphatidylcholine, (2) acylation to the neurot ransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolit es is important for a better understanding of the regulation of these pathw ays in neurons. A non-HPLC method for the simultaneous separation of five c holine metabolites found in neurons is described. High Voltage electrophore sis (HVE) was combined with thin layer chromatography (TLC) to separate cho line, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in ra diotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl-H-3]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some 'tailing' by PCh significantly elevated the radioactivit y measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followe d by TLC in the second dimension. This two-dimensional approach was consist ently reproducible and achieved excellent resolution of all five metabolite s. In addition, this technique also resolved a sixth choline-containing met abolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidy lcholine. (C) 1999 Elsevier Science B.V. All rights reserved.