A sensitive and specific radioenzymatic assay for the simultaneous determination of choline and phosphatidylcholine.
A radioenzymatic procedure for the simultaneous measurement of picomol quantities of phosphatidylcholine and choline is described. Phospholipase-D from Streptomyces chromofuscus hydrolyzes phosphatidylcholine to choline and phosphatidic acid. Choline kinase in the presence of [32P]ATP phosphorylates the choline to form [32P]phosphorylcholine. The phosphorylcholine, isolated by ion exchange chromatography, is measured by scintillation spectroscopy. Using a chloroform: methanol separation, phosphatidylcholine and choline can be measured from the same sample. Different sources of phospholipase-D were compared for their ability to hydrolyze phosphatidylcholine to choline and phosphatidic acid. Phospholipase-D from Streptomyces chromofuscus was found to result in almost complete hydrolysis. As a measure of specificity, lysophosphatidylcholine and sphingomyelin were assayed using this method and were found to result in, at most, only 2% of the amount of phosphorylcholine produced compared to phosphatidylcholine. This procedure allows for the simultaneous measurement of choline and phosphatidylcholine in brain and serum samples with a very high degree of sensitivity and specificity.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002794:Choline / Q000378:metabolism*; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D; D010761:Phosphorus Radioisotopes
D010713:Phosphatidylcholines; D010761:Phosphorus Radioisotopes; D010739:Phospholipase D; D002794:Choline