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

• Published in: General and comparative endocrinology Vol. 310; p. 113810
• Main Authors: Beyl, Hannah E., Jimeno, Blanca, Lynn, Sharon E., Breuner, Creagh W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-09-2021
• Subjects: Animals; Birds; Birds - metabolism; Corticosteroid-binding globulin; Corticosterone; Disassociation constant; Free corticosterone; Temperature; Transcortin - metabolism; Temperature; Disassociation constant; Corticosteroid-binding globulin; Birds; Kd; CBG; Free corticosterone
• ISSN: 0016-6480; 1095-6840
• DOI: 10.1016/j.ygcen.2021.113810

•Corticosteroid-binding globulin assay incubation temperature affects resulting free corticosterone estimates.•Disassociation constants increased in four species when incubated at 41 °C (average avian body temperature) compared to 4 °C (historical assay incubation temperature).•Biologically relevant patterns did not change with an increase in temperature but amount of free corticosterone does.•Recommend future corticosteroid-binding assays utilize a biologically relevant temperature for accurate representation of free corticosterone in vivo.
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.