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

General and Comparative Endocrinology
Hannah E BeylC W Breuner

Abstract

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 p...Continue Reading

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