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This paper describes a sensitive and specific enzymatic-radioisotopic method for determining plasma choline. Assays may be performed without prior extraction of the tissue. Plasma is first heated to destroy enzymes that would otherwise produce free choline from that which is normally bound. The free choline in plasma is then converted to phosphorylcholine [
32P], in the presence of ATP-γ-
32P, in a reaction catalyzed by choline kinase. Phosphorylcholine [
32P], isolated by ion-exchange chromatography, is measured as an index of the concentration of free choline. The concentration of plasma choline in man and in several species of laboratory animals was determined, and found to range from 5.5 nmoles/ml in dogs to 15.4 nmoles/ml in guinea pigs. The concentration of free choline in plasma of adult rats raised on a choline-deficient diet was half that of littermate controls raised on a control diet supplemented with free choline.