Faecal glucocorticoid metabolites: how to express yourself - comparison of absolute amounts versus concentrations in samples from a study in laboratory rats

During the last two decades, measurement of faecal cortisol or corticosterone metabolites (FCM) has become one of the most important tools to non-invasively monitor stress in animals. However, to reliably assess an animal's adrenocortical activity, a careful validation of this technique for each species and sex investigated is obligatory. Usually results in these validation studies and in subsequent applications are expressed as concentration (FCM(conc)). Nevertheless, some authors express their results as absolute amounts (FCM(abs)) and claim this to be more accurate. A physiological validation to prove this assumption, however, is still missing as well as information about the influence of the intervals set for faecal sampling, although the chosen intervals might play an important role. Since FCM(conc) and FCM(abs) may differ and therefore lead to different conclusions, our study aimed to gain fundamental and scientifically valid information about these parameters by re-analysing a set of data obtained in a study on laboratory rats. The data basis used was derived from four validation experiments performed in male and female rats: an adrenocorticotrophic hormone challenge test, a dexamethasone (Dex) suppression test, an investigation of the diurnal variation (DV) of glucocorticoids and the stress response in reaction to the injection procedure itself (for details see Lepschy et al. Non-invasive measurement of adrenocortical activity in male and female rats. Lab Anim 2007;41:372-87). Faecal samples were collected in short time intervals and the exact amount of faeces voided during each sampling interval was documented. Throughout all performed tests strong positive correlations between FCM(conc) and FCM(abs) were found (median of r(s) > 0.72). In males, for all calculated sampling intervals (4, 8 and 12 h) pharmacological stimulation, suppression and the DV of adrenocortical activity were reflected accurately using both FCM(conc) and FCM(abs). In females, suppression of FCM by Dex was also clearly reflected in both systems. However, pharmacological stimulation was only reflected accurately by means of FCM(conc), which clearly limits the usability of FCM(abs). Thus, using the data of physiological validation experiments, we clearly demonstrate for the first time advantages and disadvantages of presenting results as FCM(conc) or FCM(abs). Based on our findings in laboratory animals such as rats, giving results as FCM(conc) seems to be more appropriate and FCM(abs) - if at all - might only be used as an addition.