Hair cortisol analyses in different mammal species: choosing the wrong assay may lead to erroneous results.

Katarina Jewgenow,Martin Dehnhard,Alexandre Azevedo,Mareen Albrecht,Clemens Kirschbaum,Ben Dantzer

doi:10.1093/conphys/coaa009

Katarina Jewgenow, Martin Dehnhard + Show 4 more

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https://doi.org/10.1093/conphys/coaa009

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Abstract

Wild animals are faced with a broad range of environmental stressors and research is needed to better understand their effect on populations. Hormone analysis based on enzyme immunoassays (EIAs) can provide valuable information on adrenocortical activity (stress), and assessment of cortisol in hair may allow the quantification of cortisol production. To validate hair hormone analysis, we compared two EIAs based on antibodies against cortisol-3-CMO-BSA and cortisol-21-HS-BSA for hair glucocorticoid (hGC) measurements in Egyptian mongoose, Iberian lynx, Alpine marmot, Asiatic black bear, spotted hyena and cheetah, with results obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Both EIAs were also characterized by HPLC immunograms. Our results revealed that the cortisol-21-HS EIA measured 2.3- to 12-fold higher hGC concentrations than the cortisol-3-CMO assay. In dependence of the species, high-performance liquid chromatography (HPLC) immunograms showed that up to 70% of immunoreactivities determined by the cortisol-21-HS constituted of unknown unpolar compounds leading to an overestimation of hGC. The cortisol-3-CMO EIA expressed a better specificity, with 32.1-67.4% of immunoreactivity represented by cortisol and cortisone. The LC-MS/MS analyses (gold standard) revealed that the cortisol-3-CMO EIA also resulted in an (up to 3-fold) overestimation of hGC, but EIA results were correlated with LC-MS/MS in the mongoose, the lynx, the spotted hyena and the marmot. No correlation was obtained for Asiatic black bears. As a result of our study, we strongly recommend to test any cortisol EIA for its specificity towards extracted hair components. In all analyzed species, except the Asiatic black bear, cortisone and cortisol were simultaneously present in hair extracts; consequently, an appropriate EIA should cross-react to these two glucocorticoid hormones and express negligible affinity towards substances with less polarity than corticosterone. Choosing the wrong EIA for hGC analyses may lead to overestimations of hGC or-in the worst case-to results that do not mirror real adrenocortical activity.

Highlights

Wild animals are faced with a broad range of environmental stressors, and one physiological process helping vertebrates to cope with any challenge is the stimulation of the hypothalamic pituitary axis (HPA) leading to the release of glucocorticoid (GC; corticosterone/cortisol) hormones
Hair has been recognized as a biomaterial that accumulates GC hormones (Davenport et al, 2006; Kirschbaum et al, 2009; Macbeth et al, 2010) and is supposed to reflect average blood serum level
Authentic hormones are present within the inert hair matrix as shown for human and many other species, the precise mechanisms by which steroid hormones are incorporated into hair are still not fully understood (Ito et al, 2005)

Summary

Introduction

Wild animals are faced with a broad range of environmental stressors, and one physiological process helping vertebrates to cope with any challenge is the stimulation of the hypothalamic pituitary axis (HPA) leading to the release of glucocorticoid (GC; corticosterone/cortisol) hormones These metabolic hormones are involved in the maintenance of energetic balance and act as key components of the acute stress response. In case of acute stress events, increased levels of GC are essential to shift energy towards the functions demanded to rapidly cope with the stressor (amongst others dominance behaviour, intra- and inter-species competition, escape from predators and human–wildlife conflicts) (McEwen et al, 2003) These changes are of limited duration, and thereafter GC levels return to baseline, but if adverse environmental conditions act permanently, continuously elevated GC secretion may cause deleterious consequences by altering individual fitness, immune function and reproductive success (Terio et al, 1999; Tilbrook et al, 1999; Ludwig et al, 2019) and eventually reduce survival (Wingfield et al, 2003). The possible impact of an environmental stressor, either of anthropogenic or of natural origin on an animal’s fitness, can only be detected if physiological baselines are known and reliable measurements of GC are performed on a regular and large-scale basis

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