A simple method for detecting overlap of nanogram quantities of choline onto the acetylcholine band in paper electrophoresis
✍ Scribed by H. Ladinsky; S. Consolo; S.R. Bareggi
- Publisher
- Elsevier Science
- Year
- 1972
- Tongue
- English
- Weight
- 357 KB
- Volume
- 50
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
One of the major problems in estimating acetylcholine and choline in tissue samples is the separation of these two substances. Feigenson and Saelens (1) and Saelens, Allen, and Simke (2) used the paper electrophoretic method of Potter and Murphy (3) to separate these two amines while devising their radiochemical method for acetylcholine and choline.
According to Feigenson and Saelens (1)) the acetylcholine and choline bands are extremely close to each other and 0.5% overlap of choline onto the acetylcholine band has been observed.
Recent findings indicate that in mouse and rat brain there is about a 4:l molar ratio of choline to acetylcholine (4-6), while in mouse cerebellum the molar ratio is 24: 1 (unpublished results). The large difference in concentration between these two amines in cerebellum makes it critical that they be quantitatively separated before analysis, or that the percentage overlap be known, since even 0.5% overlap of choline onto the acetylcholine band would result in a 10-12s error in the acetylcholine concentration found.
This report describes a simple method for determining the degree of choline overlap in the same sample in which acetylcholine and choline are determined by the method to Saelens, Allen, and Simke (2) using tritiated choline of high specific activity as a tracer and double-isotope technique.
METHODS
Assay of Acetylcholine and Choline
Female Albino-Swiss mice (20 gm body weight) were sacrificed by a 3 set immersion in liquid nitrogen. Whole brains or cerebella were removed from the cranium within 30 set, frozen in liquid nitrogen, and pulverized in a specially designed mortar and pestle (7). 460