An improved dispersed adrenal cell assay for corticotropin in rat plasma

The present study was designed to improve the dispersed adrenal cell technique for determining adrenocorticotrophic hormone (ACTH) concentrations in small amounts of rat plasma. Priming with ACTH, incubation with methyl-isobutylxanthine, or dexamethasone pre-treatment were employed as modifications. Of these, only dexamethasone pre-treatment increased the sensitivity of the assay. The adrenal fragments obtained from 10-12 adult male rats pre-treated with dexamethasone (100 micrograms/kg B.W.) one hour before sacrifice, were digested with collagenase and deoxyribonuclease solution for 30 minutes. The dispersed cells were collected by centrifugation and resuspended in Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin. Aliquots of cell suspension (3-4 X 10(4)/tube) were incubated with various doses of ACTH1-24 or the eluate of plasma samples at 37 degrees C for 2 hours in an atmosphere of 95% O2/5% CO2 in a Dubnoff shaker. The quantity of corticosterone produced was measured fluorimetrically. The assay is precise (lambda = 0.06), extremely sensitive (10 fg/tube), and convenient. One skilled technician can handle 15 to 20 plasma samples per day using 10 rats as the source of assay cells. ACTH can be measured in as little as 10-50 microliters of eluate.