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

Abstract

The development of methods to quantify hormones from non-invasively collected samples such as urine or feces has facilitated endocrinology research on wild-living animals. To ensure that hormone measurements are biologically meaningful, method validations are highly recommended for each new species or sample matrix. Our aim was to validate three commonly used enzyme immunoassays (EIA), one for analysis of cortisol and two for analysis of testosterone, to assess adrenocortical and gonadal activity, respectively, from the urine of male Barbary macaques. We compared EIA and liquid chromatography-mass spectrometry (LC-MS) results to determine if the EIA measurements truly reflect levels of the target hormone and to determine if cross-reactivities with other steroids were potentially confounding results. Furthermore, we conducted a biological validation of testosterone to ensure that both EIA and LC-MS were able to capture physiologically meaningful differences in hormone levels. We found that cortisol measured by EIA correlated strongly with cortisol measured by LC-MS in both adult and immature males, without the need for deconjugation of steroids in the urine. Both testosterone EIAs correlated strongly with LC-MS in adult males, but only if steroids in the urine were deconjugated by enzymatic hydrolysis prior to analysis. However, in immature males, EIA and LC-MS results did not correlate significantly. Further correlation analyses suggest this is likely due to cross-reactivity of the testosterone antibody with other adrenal steroids such as cortisol, DHEA, and likely others, which are present at much higher concentrations relative to testosterone in immature males. Testosterone levels were significantly higher in adult compared to immature males as measured by LC-MS but not as measured by EIA. Taken together, our results suggest that the testosterone EIAs are suitable to assess gonadal activity in adult but not immature males, and only if a hydrolysis of the urine is conducted prior to analysis.

Testosterone and cortisol are negatively associated with ritualized bonding behavior in male macaques

“…The measurement of immunoreactive cortisol (iuC) and testosterone (iuT) from the urine of male 203 Barbary macaques has been previously validated (Rincon et al, 2019). Furthermore, we found a 204 negative correlation between cortisol and time of day, thus further biologically validating our 205 cortisol measurement (Table 3).…”

“…Extracts were then transferred into 2 ml eppendorf safe-lock tubes and stored at -20°C until analysis for testosterone concentrations. The efficiency of the combined hydrolysis and extraction procedure was assessed using internal controls of testosterone glucuronide run together with each set of samples as previously described (Rincon et al, 2019). The combined hydrolysis/extraction efficiency was 62.1 ± 4.2% mean ± SD (range: 50-70%; N = 36).…”

“…We measured iuT from samples following extraction and hydrolysis (described above), and iuC from unprocessed diluted urine by using enzyme immunoassays as described in detail in Rincon et al (2019). For the testosterone assay (Palme and Möstl, 1994), sensitivity at 90% binding was 0.3 pg; intra-assay CVs of high and low value quality controls were <10%, while figures for inter-assay CVs were 13.2% (high) and 19.5% (low).…”

Methodological confounds of measuring urinary oxidative stress in wild animals

“…Environmental contamination of samples can be introduced through a variety of ways, for example, mixing with soil, feces, or contact with vegetation. Contamination of samples through contact with vegetation is one of the most difficult sources of contamination to avoid because the collection of urine directly from the stream of a wild animal is not always possible and samples are regularly collected from the surface of vegetation (Fedurek et al, 2016; Higham et al, 2011; Rincon et al, 2019; Surbeck et al, 2012; Thompson González et al, 2020). Although the potential effects of leaf‐borne contaminants on urinary biomarker measurement have been successfully ruled out for some metabolites (testosterone and creatinine: Muller & Wrangham, 2004; Marshall & Hohmann, 2005; and estrone conjugates: Knott, 2005), this is yet to be confirmed for OS markers.…”