Validation of a radioimmunoassay for the determination of total corticosterone in rat plasma

Abstract

A radioimmunoassay (RIA) for total corticosterone (CORT) in rat plasma requiring a plasma volume of 2 μl was established. The importance of inactivating plasma corticosteroid-binding globulin (CBG), denatured by heat, before measuring CORT was shown. The method was evaluated and shown to be within analytical and statistical controls, with a recovery not significantly different from 100%. The limit of detection (LD) was 0.2 μg dl −1. Intra-assay coefficients of variation (CVs) were 7.2% for plasma samples at 33.5 μg dl −1 CORT ( n=10) and 8.5% at 0.75 μg dl −1 CORT ( n=18). Inter-assay CVs were 6.8% at 23.1 μg dl −1 CORT ( n=8) and 5.4% at 8.9 μg dl −1 CORT ( n=8). Validation of the method was performed using a group of male rats (Mol:WIST Han; M & B A/S, Denmark; n=12), housed under conventional environmental conditions from the age of 4 weeks. The rats were 3 and 7/2 months old when they underwent the blood sampling procedure. The result showed that the method had a capacity to detect CORT concentrations comparable with previous reported basal concentrations. Finally, the possible stress inducing effect of the blood sampling procedure was examined using two groups of male rats housed under either conventional or enriched environmental conditions. The result indicated that conventional environment housing induces slightly stressed animals compared to enriched housing. Enriched housing may provide an environment that makes it possible for rats to compensate for a stressful situation, i.e., the blood sampling procedure.