Direct radioimmunoassays for plasma corticosterone and aldosterone in frog. I. Validation of the methods and evidence for daily rhythms in a natural environment

Abstract

Two radioimmunoassay techniques for direct measurement of frog plasma concentrations of corticosterone after prior ethanol extraction for deproteinization, and of aldosterone without prior extraction, are described. Specific antibodies against corticosterone 21-hemisuccinate and aldosterone 18,21-diacetate-3-carboxymethoxime derivatives conjugated to bovine serum albumin are raised in rabbits. The sensitivity threshold of the assays allows the assessment of corticosterone in 10-μl and aldosterone in 5-μl samples of plasma. Sephadex LH-20 chromatography demonstrates the validity of both methods. The intra- and interassay reproducibilities and the accuracy of each assay have been studied. The conditions making it possible to reduce aldosterone fluctuations during blood taking have been ascertained. Using these techniques corticosterone and aldosterone concentrations have been assessed in the plasma of 214 frogs caught in their natural habitat at 4-hr intervals during a 40-hr period in mid-June. The existence of synchronous and reproducible 24-hr rhythms of corticosterone and aldosterone plasma levels has been demonstrated. High concentrations of both corticosteroids are recorded during the night and low concentrations are recorded during daylight. The amplitude of corticosterone fluctuations is 3.5-fold greater than that of aldosterone fluctuations. Corticosteroid rhythms are compared to activity phases of frogs during the day at this period of the year.