Abstract
Thank you for giving us the opportunity to comment on a recent paper in Laboratory Animals: Faecal glucocorticoid metabolites: how to express yourself – comparison of absolute amounts versus concentrations in samples from a study in laboratory rats. 1
We have for more than 10 years used faecal excretion of corticosteroids as a measure of preceding stress in laboratory animals. Corticosteroids in the circulation are excreted in urine and faeces. In urine, corticosteroids can be quantified and concentrations adjusted, using creatinine concentrations to make allowance for irrelevant variations in urine volume.
The corticosteroids excreted in faeces enter the gastrointestinal (GI) tract predominantly via bile and the regulation of bile flow into the GI tract in man and across species – with or without gall bladder – is poorly understood. It is, however, well known that bile flow into the alimentary tract is not constant nor directly coupled to – or coordinated with – the flow of intestinal contents. 2,3 Thus, bile with corticosteroids enters the GI tract regardless of whether there is no flow in the GI tract (as during invasive surgery) or whether the gut is full of fast flowing more or less bulky materials.
Consequently, concentrations of corticosteroids in faeces vary, not just according to the amounts entering the bile from the blood but also according to the quantity and motility of gut contents and thus amounts of faeces voided by the animal. 4,5 If – for example – an animal has been subjected to surgery, it is not uncommon that it voids smaller amounts of faeces in the early stages after operation compared with unoperated controls, obviously with high concentrations of corticosteroids whether the animal has been stressed or not. 6
We have documented, e.g. in rats, 7 poultry, 8 pigs 9 and monkeys, 10 that in order to obtain reliable and biologically meaningful results it is – not surprisingly – important to collect all faeces and quantify total corticosteroid excretion per kilogram body weight per time unit.
Conservation and wildlife biologists may not have the possibility to investigate hormonal changes in faeces in detail, and have to make do with samples, but this is not the case for biomedical scientists working with laboratory animals.
Measuring all molecules in a very heterogeneous biological compartment is more accurate than base assumptions on samples obtained from the compartment. We are therefore concerned that the paper by Lepschy and co-workers, perhaps unintentionally, may misguide colleagues with little endocrinology background to – based on this article – embark on studies measuring corticosterone concentrations in faecal samples as measures of preceding stress in animals. This would result in analyses that would contain more false-positive and false-negative results than if they went through the trouble and collected all faeces in the relevant time windows and quantified corticosteroid excretion as number of molecules excreted per time unit per kilogram body weight.