Enzymic analysis of cyclic 3', 5'-AMP in mammalian tissues and urine

The details are presented for the analysis of 3′,5′ cyclic adenosine monophosphate (3′5′CAMP) in milligram amounts of mammalian tissues (muscles, liver, brain, and kidney) and in microliter samples of urine. An examination of the sources of difficulty and how they are effectively handled is also included. In the determination of tissue 3′5′CAMP the cyclic nucleotide is first separated from 5′-nucleoside mono-, di-, and triphosphates by cellulose thin-layer chromatography following Ba(OH)₂-ZnSO₄ precipitation of extracts. After quantitative recovery 3′,5′CAMP is converted to 5′ AMP and subsequently to ATP by the actions of phosphodiesterase, myokinase, and pyruvate kinase. Enzymic cycling with the hexokinase-pyruvate kinase system is then used to produce a proportional concentration of G-6-P equivalent to several thousand fold the ATP concentration and the G-6-P measured fluorometrically. Cyclic adenylate in urine samples is determined directly without prior separation from any urinary components. Examples are presented of the analytical procedures applied to the measurement of 3′5′CAMP levels in tissues and urine after various experimental treatments. These include the effects of epinephrine in skeletal muscle in vitro and in vivo, of adrenalectomy and hydrocortisone in liver, of ischemia in brain, and of hypertonic infusion on urinary excretion of 3′5′CAMP.