Determination of picomole quantities of acetylcholine and choline in physiologic salt solutions

✍ Scribed by Mark L. Gilberstadt; James A. Russell

• Publisher: Elsevier Science
• Year: 1984
• Tongue: English
• Weight: 611 KB
• Volume: 138
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(84)90772-3

✦ Synopsis

An assay capable of detecting tens-of-picomole quantities of choline and acetylcholine in milliliter volumes of a physiological salt solution has been developed. Silica column chromatography was used to bind and separate IO-3000 pmol ['4C]choline and [?Z]acetylcholine standards made up in 3 ml of a bicarbonate-buffered Krebs-Ringer solution. The silica columns bound 95-98% of both choline and acetylcholine. Of the bound choline 84-87% was eluted in 1.5 ml of 0.075 N HCl, whereas 95-98% of the bound acetylcholine was eluted in a subsequent wash with 1.5 ml of 0.030 N HCl in 10% 2-butanone. Vacuum centrifugation of the eluants yielded small white pellets with losses of choline and acetylcholine of only 1%. Dried pellets of unlabeled choline and acetylcholine standards were assayed radioenzymatically using [y-32P]ATP, choline kinase, and acetylcholinesterase. The net disintegrations per minute of choline["P]phosphate product was proportional to both the acetylcholine (lo-3000 pmol) and choline (30-3000 pmol) standards. The "limit sensitivity" was 8.5 pmol for acetylcholine and 11.4 pmol for choline. Cross-contamination of the choline assay by acetylcholine averaged 1.3%, whereas contamination of the acetylcholine assay by choline averaged 3.1%.