Assay temperature affects corticosteroid-binding globulin and free corticosterone estimates across species

Hannah E. Beyl, Blanca Jimeno, Sharon E. Lynn, Creagh W. Breuner

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1 Scopus citations


Glucocorticoid hormones are often measured to assess how organisms physiologically respond to challenges in their environment. In plasma, glucocorticoids circulate in two forms: bound to corticosteroid-binding globulins (CBG) or unbound (free). Measuring CBG allows us to estimate the amount of free glucocorticoids present in a plasma sample. However, free glucocorticoid estimates are affected by the assay temperature used when measuring CBG, with colder temperatures maximizing specific binding but likely underestimating glucocorticoid's affinity for CBG. Here, we test how a biologically relevant incubation temperature (41 °C) changes the disassociation constant (Kd; used to estimate free glucocorticoid levels) when compared to the traditional 4 °C incubation temperature, across four commonly studied avian species. We then apply the new Kd’s calculated at 41 °C to existing data sets to examine how the change in Kd affects free corticosterone estimates and data interpretation. Kd’s were generally higher (lower affinity for CORT) at warmer incubation temperatures which resulted in higher levels of estimated free CORT in all four species but differed among subspecies. This increase in free CORT levels did not qualitatively change previously reported statistical relationships, but did affect variance and alpha (P) values. We suggest that future assays be run at biologically relevant temperatures for more accurate estimates of free CORT levels in vivo and to increase the chances of detecting biological patterns of free-CORT that may not be revealed with the classic methodology that tends to underestimate free CORT levels.

Original languageEnglish
Article number113810
JournalGeneral and Comparative Endocrinology
StatePublished - Sep 1 2021


We would like to thank Oliver Love for use of his European Starling data set. We thank Libby Natola and Quinn McCallum at UBC for supplying Z. l. pugetensis plasma. We thank the Breuner Lab members who helped with early feedback and comments on this research.

FundersFunder number
University of British Columbia


    • Birds
    • Corticosteroid-binding globulin
    • Disassociation constant
    • Free corticosterone
    • Temperature


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