Simultaneous Radioimmunoassay for the Measurement of Adenosine 3′,5′-Monophosphate and Guanosine 3′,5′-Monophosphate

Abstract

To facilitate studies of the physiologic roles of guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP), the radioimmunoassay techniques for the measurement of the cyclic nucleotides have been modified to allow measurement of both cyclic AMP and cyclic GMP in the same sample. The system employs antibodies with high specificity for these cyclic nucleotides, and both an I-labeled cyclic GMP derivative and an 1 3 Ilabeled cyclic AMP derivative as markers. Samples are automatically counted on a dual channel gamma spectrophotometer equipped with a punched paper tape printout; the nucleotide concentrations may be determined from hand-drawn standard curves or calculated by computer. Values for tissue and fluid concentrations of the cyclic nucleotides obtained by this double assay procedure are identical with the values obtained by the individual radioimmunoassays; the precision in the simultaneous assays was also comparable to that of the single assays. (Endocrinology 90: 330, 1972) A DENOSINE 3',5'-monophosphate (cyclic -^* AMP) plays an important role as the mediator of the effects of many hormones in a wide variety of tissues (1). Guanosine 3',5'monophosphate (cyclic GMP), like cyclic AMP, is also found in most animal tissues and body fluids. The biological role of cyclic GMP has not yet been established; however, in certain instances this nucleotide has effects distinct from those of cyclic AMP (2,3). Furthermore, its intracellular level is influenced by different factors (4-7), and it is thought that cyclic GMP may also function as an intracellular regulatory agent. Received June 14, 1971. * Dr. Steiner is an Established Investigator of the American Heart Association. Recently, Steiner and his associates have introduced precise, specific and sensitive radioimmunoassay techniques for the measurement of cyclic AMP, cyclic GMP, and other cyclic nucleotides (8,9). The assays are based on the competition between the cyclic nucleotide and an iodinated derivative for binding sites on antibody which had been prepared by immunizing rabbits with a 2'-O-succinyl cyclic nucleotide derivative conjugated to a large protein. Free and antibodybound marker are separated by ammonium sulfate precipitation. The specificity of the assays is such that preliminary purification steps are not necessary, and large numbers of samples may be processed conveniently and accurately. In order to further facilitate determinations of cyclic AMP and cyclic GMP, the radioimmunoassay system has been adapted to measure January 1972 NOTES AND COMMENTS 331 both of these compounds simultaneously. The tyrosinated derivatives of cyclic AMP and cyclic GMP are labeled with I and I respectively, and added to assay tubes which contain antibodies for cyclic AMP and cyclic GMP. After incubation, the antibodies are precipitated with (NH4)2SO4 and the I and I in the precipitate are counted in a dual channel gamma spectrophotometer. The principle employed is similar to that introduced by Morgan for simultaneous radioimmunoassay of insulin and growth hormone (11).