Abstract
Thank you for giving us the opportunity to reply to the comment by Hau and co-workers on our recent paper dealing with different ways of expressing measures of faecal glucocorticoid metabolites in laboratory rats. 1
We welcome discussion on this important topic, as we believe non-invasive methods are a helpful tool for evaluating adrenocortical activity in a variety of species and research fields. Our group pioneered (e.g. Palme and Möstl 2 ) the measurement of faecal cortisol and corticosterone metabolites (FCM) by radiometabolism experiments as well as by the development and validation of group-specific enzyme immunoassays (reviewed in Palme et al. 3 and Möstl et al. 4 ). In all our publications, however, we have always stressed that the application of this method is by no means simple and straightforward. Particularly, we have very much highlighted the importance of rigorous validation (including a physiological validation) before this method can be used for analysing samples of a given species. 5,6
In their comment Hau et al. claim that it is more accurate to give absolute amounts of excreted FCM. Their statement is based on the assumption of a constant bile flow: concentrations of FCM are thus expected to vary according to the quantity and motility of gut contents and the amounts of faeces excreted by the animal. We feel that this description of the excretion of steroids via the gut is oversimplified. First, bile flow is influenced by many factors (e.g. existence of a gall bladder, food quality and amount 7 or anaesthesia 8 ) and thus should not be assumed to be constant. Second, factors other than the rate of bile flow are involved, such as enterohepatic recirculation and different clearance rates of steroid hormones due to the different amounts and natures of food consumed. The latter is particularly important for the present discussion, as a recently published paper provides further evidence (see also our discussion in Lepschy et al. 1 ) that more bulky material in the gut results in increased clearance rates and thus excretion of steroids. 9
In our recent paper 1 we addressed two important issues concerning the excretion of FCM: (i) the expression of results of FCM measurements (absolute amounts versus concentrations) and (ii) the influence of the intervals set for sampling the faeces. These factors should not be confused. Furthermore, we firmly believe that such issues should be clarified on the basis of experimental data. Our validation experiments in rats of both sexes, 1,10 including stimulation and suppression of the adrenal cortex, and two control experiments formed the basis for deciding on the details of the procedure to be used. Our data imply that both measures are highly correlated. However, FCM concentrations reflected adrenocortical activity as good as or, at least in the case of the adrenocorticotropic hormone (ACTH) challenge in females, even better than the absolute amounts of excreted FCM.
We noted that the loss of faecal matter hinders the display of results as absolute amounts. However, we are not persuaded that we are intentionally or unintentionally encouraging other researchers to collect faecal samples in a sloppy manner or incompletely or that we are misleading colleagues to embark on studies to measure FCM without due care and attention. In fact, we repeatedly ask for a careful and sound physiological validation (at least an ACTH challenge) of a newly established method and state that ‘the proper and reliable measurement of this parameter is not straightforward and has certain requirements’. 1 The requirements include a carefully planned and rigorously conducted sampling regimen. Frequent sampling is essential to detect, for example, short-term increases in adrenocortical activity (acute stress 6 ). In addition, sampling of single faecal pellets over an extended period of time from a large number of animals may be used to evaluate, for example, the effects of human disturbance on wildlife populations (chronic stress 11 ). We encourage interested researchers to read our papers and find more detailed discussions of important issues (ref. 1 but also refs. 3–6) and are confident that they will not be misguided. We are of course happy to answer questions regarding our methodology and to make our enzyme immunoassays available to other researchers as well.