DETERMINATION OF PICOMOLE QUANTITIES OF ACETYLCHOLINE AND CHOLINE IN PHYSIOLOGIC SALT-SOLUTIONS
DETERMINATION OF PICOMOLE QUANTITIES OF ACETYLCHOLINE AND CHOLINE IN PHYSIOLOGIC SALT-SOLUTIONS
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DOI：
10.1016/0003-2697(84)90772-3
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发表时间：
1984-01-01
影响因子：
2.9
通讯作者：
RUSSELL, JA
中科院分区：
文献类型：
--
作者：
GILBERSTADT, ML;RUSSELL, JA
开发了一种能够检测以毫升体积的生理盐溶液为单位的数十皮摩尔量的胆碱和乙酰胆碱的测定法。使用二氧化硅柱色谱法结合并分离在3 ml碳酸氢盐缓冲的Krebs-Ringer溶液中配制的10-3000 pmol [14 C]胆碱和[14 C]乙酰胆碱标准品。硅胶柱结合95-98%的胆碱和乙酰胆碱。结合的胆碱的84-87%在1.5 ml 0.075 N HCl中洗脱，而95-98%的结合的乙酰胆碱在随后用1.5 ml 0.030 N HCl的10%2-丁酮溶液洗涤中洗脱。洗脱液的真空离心产生小的白色沉淀，胆碱和乙酰胆碱的损失仅为1%。未标记胆碱和乙酰胆碱标准品的干燥沉淀用[γ-氨基]放射酶法测定。32 P]ATP、胆碱激酶和乙酰胆碱酯酶。磷酸胆碱[32 P]产物的每分钟净衰变（dpm）与乙酰胆碱（10-3000 pmol）和胆碱（30-3000 pmol）标准品均成比例。乙酰胆碱的极限灵敏度为8.5 pmol，胆碱为11.4 pmol。乙酰胆碱对胆碱测定的交叉污染平均为1.3%，而胆碱对乙酰胆碱测定的污染平均为3.1%。
An assay capable of detecting tens-of-picomole quantities of choline and acetylcholine in milliliter volumes of a physiological salt solution was developed. Silica column chromatography was used to bind and separate 10-3000 pmol [14C]choline and [14C]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 [.gamma.-32P]ATP, choline kinase and acetylcholinesterase. The net disintegrations per minute (dpm) of choline [32P]phosphate product was proportional to both the acetylcholine (10-3000 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%.