Measuring fecal testosterone in females and fecal estrogens in males: Comparison of RIA and LC/MS/MS methods for wild baboons (Papio cynocephalus)

Abstract

The development of non-invasive methods, particularly fecal determination, has made possible the assessment of hormone concentrations in wild animal populations. However, measuring fecal metabolites needs careful validation for each species and for each sex. We investigated whether radioimmunoassays (RIAs) previously used to measure fecal testosterone (fT) in male baboons and fecal estrogens (fE) in female baboons were well suited to measure these hormones in the opposite sex. We compared fE and fT concentrations determined by RIA to those measured by liquid chromatography combined with triple quadropole mass spectrometry (LC/MS/MS), a highly specific method. Additionally, we conducted a biological validation to assure that the measurements of fecal concentrations reflected physiological levels of the hormone of interest. Several tests produced expected results that led us to conclude that our RIAs can reliably measure fT and fE in both sexes, and that within-sex comparisons of these measures are valid: (i) fTRIA were significantly correlated to fTLC/MS/MS for both sexes; (ii) fTRIA were higher in adult than in immature males; (iii) fTRIA were higher in pregnant than non-pregnant females; (iv) fERIA were correlated with 17β-estradiol (fE2) and with estrone (fE1) determined by LC/MS/MS in pregnant females; (v) fERIA were significantly correlated with fE2 in non-pregnant females and nearly significantly correlated in males; (vi) fERIA were higher in adult males than in immature males. fERIA were higher in females than in males, as predicted, but unexpectedly, fTRIA were higher in females than in males, suggesting a difference in steroid metabolism in the two sexes; consequently, we conclude that while within-sex comparisons are valid, fTRIA should not be used for intersexual comparisons. Our results should open the field to important additional studies, as to date the roles of testosterone in females and estrogens in males have been little investigated.

The Evolution of Mechanisms Driving the Stomatal Response to Vapor Pressure Deficit

“…This provides, to our knowledge, the first quantitative evidence that increases in foliar ABA levels in response to changes in VPD in angiosperms can exceed levels that will cause a similar degree of stomatal closure in the absence of water stress, and complements conclusions reached by the qualitative assessments of ABA signaling and synthesis mutants: namely, that ABA levels play a major role in delivering stomatal responses to VPD in angiosperms (Xie et al, 2006). Many of the past methods, especially the popular immunoassay methods, used to quantify foliar ABA levels (WalkerSimmons et al, 2000) and other hormones, from foliar auxin levels (Cohen et al, 1987) to steroids in baboon feces (Gesquiere et al, 2014), unless rigorously standardized against physiocochemical methods, do not possess the resolution required to detect small but functionally significant increases in hormone levels, shown here to occur in angiosperm leaves during exposure to high VPD. This limitation in resolution may explain previous observations that ABA levels only noticeably increase when plants are exposed to water stress sufficient enough to cause turgor loss (Pierce and Raschke, 1980;Trejo and Davies, 1991;Wilkinson and Davies, 1997) and the paucity of data showing rapid ABA accumulation in leaves during an increasing VPD transition.…”

Measuring urinary cortisol and testosterone levels in male Barbary macaques: A comparison of EIA and LC-MS

“…Liquid chromatography-mass spectrometry (LC-MS) can also be used as a tool to validate immunoassay measurements (Gesquiere et al, 2014;Habumuremyi et al, 2014;Preis et al, 2011). In contrast to immunoassays, LC-MS allows for highly specific measurements of hormones in samples based on their molecular weight and charge (Cross and Hornshaw, 2016; without any confounding effects of antibody cross reactivity.…”

“…Therefore, comparing immunoassay measurements to LC-MS is a useful way to deduce if measurements from an immunoassay indeed reflect the concentration of the target hormone and which cross-reacting metabolites may be potentially confounding results (Gesquiere et al, 2014;Habumuremyi et al, 2014;Preis et al, 2011). Both immunoassays and LC-MS may be applied to non-invasively collected samples and the comparison of their measurements offer a useful alternative when more invasive validation methods are not feasible or desirable.…”

Hormonal correlates of natal dispersal and rank attainment in wild male baboons

“…For fT assays, we have used different kits over the years, all of which produce highly correlated results. These kits have included the kit from Equate (SolidPhase, Portland, ME; See Lynch et al, 2003 for validation), the kit from Diagnostic Systems Laboratories (Beckman Coulter, Webster TX; Beehner et al, 2009; Gesquiere et al, 2011), and the kit from Pantex (Santa Monica, CA; Gesquiere et al, 2014). To render the fT concentrations directly comparable across kits, a correction factor was applied to the fT from the different kits (details in Gesquiere et al, 2011).…”