We calculated free CORT levels using the mass-action-based
equation by Barsano and Baumann (Barsano and Baumann, 1989):
In this equation, Hfree is the free hormone level, Htotal is the total
hormone level, Bmax is the total binding capacity for CBG, and Ka
(nmoll–1) is the association constant and is equal to 1/Kd, where Kd
is the affinity of CORT for CBG. The affinity of CORT for CBG
was determined from equilibrium saturation binding analysis on
pooled plasma samples from Wada et al. (Wada et al., 2007).
Individual CBG capacity estimates were ~83% of Bmax so capacity
values were increased to 100% for free CORT calculations.
We compared nestling CORT physiology by examining the
amount of total and free CORT circulating before restraint stress
(baseline CORT) and after 15 and 30min of stress exposure.
Additionally, we examined the amount of total and free CORT
released during 30min of stress exposure (total integrated CORT).
Total integrated CORT represents the total amount of CORT that
target tissues are exposed to during an acute stressor.