Beyl Hannah E, Jimeno Blanca, Lynn Sharon E, Breuner Creagh W
The Wildlife Biology Program, The University of Montana, 32 Campus Drive, HS 104, Missoula, MT 59801, United States.
Organismal Biology, Ecology, and Evolution, The University of Montana. 32 Campus Drive, HS 104, Missoula, MT 59801, United States.
Gen Comp Endocrinol. 2021 Sep 1;310:113810. doi: 10.1016/j.ygcen.2021.113810. Epub 2021 May 6.
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 (K; 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 K's calculated at 41 °C to existing data sets to examine how the change in K affects free corticosterone estimates and data interpretation. K'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.
糖皮质激素常被检测以评估生物体如何在生理上应对环境挑战。在血浆中，糖皮质激素以两种形式循环：与皮质类固醇结合球蛋白（CBG）结合或未结合（游离）。检测CBG能让我们估算血浆样本中游离糖皮质激素的含量。然而，游离糖皮质激素的估算受检测CBG时所用的测定温度影响，温度越低，特异性结合最大化，但可能低估糖皮质激素对CBG的亲和力。在此，我们测试了与传统的4℃孵育温度相比，生物学相关的孵育温度（41℃）如何改变解离常数（K；用于估算游离糖皮质激素水平），涉及四种常见的鸟类。然后，我们将在41℃计算出的新K值应用于现有数据集，以研究K值的变化如何影响游离皮质酮的估算及数据解读。在较高的孵育温度下，K值通常更高（对皮质酮的亲和力更低），这导致所有四个物种中估算的游离皮质酮水平更高，但亚种间存在差异。游离皮质酮水平的这种增加并没有定性地改变先前报道的统计关系，但确实影响了方差和α（P）值。我们建议未来的检测在生物学相关温度下进行，以便更准确地估算体内游离皮质酮水平，并增加检测游离皮质酮生物学模式的机会，而这些模式可能无法通过倾向于低估游离皮质酮水平的经典方法揭示出来。
