The standard protocol was modified to increase assay sensitivity and to account for the different sample volumes and type. Additional standards were included, and all reagent volumes in the assay protocol were halved, except for the [125I]cortisol tracer, which was reduced to 40%. To control for differences in sample composition (YSF, allantoic fluid, plasma, culture media, and tissue homogenate) and volume, a double-extraction step was performed as follows. nluid samples, and distilled water (dH2O) to make up 200 ^l, were vortexed in 12 X 75-mm glass tubes (30-sec bursts every 5 min for 15 min) with 1 ml dichloromethane (CH2Cl2). Adrenal homogenates were similarly treated but were vortexed over a 60-min period. The dichloromethane was drawn off from beneath the aqueous samples, transferred to clean tubes, and evaporated to dryness. The aqueous fractions were then extracted a second time, and the solvent was dried down in tubes from the first extraction. The dried extraction tubes were used as sample tubes in the assay protocol. proventil inhaler
The efficiency of the extraction procedure was 91% and 99%, as determined by recovery of [3H]cortisol from tam-mar YSF and adult plasma, respectively. The sensitivity of the assay, defined as the lowest concentration that differed from the zero standard by more than twice the standard deviation, was below 25 pg of cortisol per tube. Accuracy was determined by assay of adult plasma to which known amounts of cortisol had been added and was within 13.8%. The interassay coefficient of variation (CV) for YSF (n = 5 assays) and adult plasma (n = 5 assays) samples containing 53.7 ± 3.6 (mean ± SD) and 111.3 ± 26.4 ng of cortisol per ml were 6.7% and 14.7%, respectively. The intraassay CV for YSF (n = 4 tubes) and adult plasma (n = 4 tubes) samples containing 56.4 ± 5.9 and 106.1 ± 9.2 ng of cortisol per milliliter were 10.5% and 8.7%, respectively. Parallelism was confirmed by assaying increasing volumes of both a nonzero and zero YSF sample. Extraction blanks were consistently below assay sensitivity.
Statistical analysis was performed using SYSTAT (SPSS Inc., Chicago, IL), and data are graphed as mean ± standard error of the mean (SEM); p values of less than 0.05 were considered significant.