Determination of Cyclic Adenosine 3′,5′-Monophosphate in Urine

Abstract

A modification of Aurbach & Houston's enzymic method for measuring cAMP is presented. The procedure is relatively simple and in several respects new. Urinary cAMP is separated from other nucleotides and phosphate by ZnS0₄-Ba(OH)₂ precipitation and column chromatography. The eluate is concentrated by evaporation. Recovery at this stage is 60-82%. The cAMP from urine and the standards are dissolved in a reaction mixture and converted to 5-AMP with cyclic 3′,5′-nucleotide phosphodiesterase (PDE) and further to ATP with adenylate kinase and pyruvate kinase. The ATP formed is labelled with ³²P by an exchange reaction catalysed by glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase. The remaining ³²P used to count the [32P]ATP in the aqueous phase. Daily human urinary cAMP excretion is 3380 ±836 nmol (S.D.). After an injection of 100 USP units of parathormone intravenously into a patient with idiopathic hypoparathyroidism, urinary cAMP excretion increased 40-fold above the basal concentration within 30 min. Drinking of coffee or water did not affect cAMP excretion. The limit of detection of the method is 170 pmol of cAMP, and the variation coefficient for urine ranges from 7 to 10%. When the enzymic cAMP method was compared with a radioimmunological procedure, the correlation coefficient was found to be 0.98.

Key Words:

• Cyclic adenosine 3′
• 5′-monophosphate (cAMP)
• enzymic cAMP assay
• urinary cAMP