Distribution and time course of corticosterone excretion in faeces and urine of female mice with varying systemic concentrations

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Abstract

Quantification of corticosterone metabolites excreted in faeces and urine is increasingly being used for assessment of preceding corticosterone concentrations in the circulation. This is a promising approach to non-invasive stress assessment in laboratory rodents. It is however unknown whether the proportions of corticosterone metabolites excreted in faeces and urine may differ, depending on the concentration of corticosterone in blood. This uncertainty undermines the applicability of urinary and faecal corticosterone metabolite measurements as biomarkers for stress. Therefore, the terminal distribution and time course of corticosterone excretion, after intravenous injection of varying corticosterone concentrations, was investigated in female mice. Female BALB/c mice excreted 60% of all corticosterone in the urine with an approximate delay of 5 h from tail vein administration. The remaining 40% were excreted in faeces, with an approximate delay of 9 h from administration. The faecal/urinary excretion ratio, as well as time course of excretion, remained unaltered by administration of various doses of corticosterone covering the entire physiological range of serum corticosterone. Although currently untested for other strains of mice and species of animals, these findings add credence to the utility of faecal and urinary corticosterone as non-invasive biomarkers for physiological stress.

Introduction

In striving towards more accurate and continued ethically defensible in vivo biomedical research it is of great value to be able to assess stress levels, non-invasively, in laboratory animals. The ultimate aim with stress level assessments is the complete elimination of avoidable stress and pain in animal experimentation. The most popular animal species in biomedical research in Europe is, by far, the laboratory mouse (Commission to the Council and the European Parliament, 2007), and this is probably also the case worldwide (Taylor et al., 2008). The most common biomarker used to evaluate acute stress response in mice is serum corticosterone. Corticosterone (CORT) is an effector hormone of the hypothalamic–pituitary–adrenal (HPA) axis, and is released as the HPA axis is activated in response to a stressor. Blood sampling, and the associated physical restraint of the animal, is however a stressful procedure and serum CORT is therefore affected (Abelson et al., 2005, Gartner et al., 1980, Vachon and Moreau, 2001). Furthermore, serum CORT only provides a snapshot of a parameter which varies greatly over time and between individuals (Atkinson et al., 2006). For these reasons, measuring corticosterone and corticosterone metabolites (CM) in excreta has become a widely accepted alternative (Bamberg et al., 2001, Möstl and Palme, 2002), providing a non-invasive method for assessing stress levels. By measuring CM in faeces or a pooled sample of urine an average CORT turnover over a longer time period can be established (Palme et al., 2005).

An implicit assumption in comparing excreted CM levels to determine relative stress levels in animals is that the time course and distribution of CM in the two terminal compartments, urine and faeces, remain unaffected by varying concentrations of circulating CORT. This is an assumption that has never been tested. Considering that both time course and terminal distribution – i.e. excretion in faeces and urine – of a number of endogenous (e.g. folate (Gregory et al., 1998)), as well as exogenous (e.g. mercury (Morcillo and Santamaria, 1995)), compounds are highly variable, and affected by the systemic concentration, it is vital to test whether this is also the case for corticosterone.

The aim of the present study was to investigate the time course and terminal distribution of CM following tail vein injection of mice with varying doses of CORT, where a subset had been radioactively labelled. This provides valuable information on whether faecal and urinary CM can be used as independent, but equally applicable, measures of preceding serum CORT concentrations.

Section snippets

Animals

Thirty-six female, 7–9 weeks old, BALB/c mice (Taconic, Ry, Denmark) with a FELASA approved health status, each weighing approximately 20 g, were group housed and acclimatised for 2 weeks prior to the experiment. The animals were subjected to standard housing conditions throughout the experiment: Diurnal rhythm was regulated through a 12 h light/12 h dark cycle (lights on from 6:00), temperature was kept at 20–22 °C, with a relative humidity of 30–60%. Food pellets (Altromin 1319; Brogaarden,

Time course

The recovered 3H activity resulted in corrected doses covering a range of 0–51 μg/kg bw. No two experimental units received the same dose. In two of the experimental units, there was cross-contamination between two of the urine samples (from the same experimental unit) prior to the scintillation analysis. No mean residence time could therefore be calculated for these units, but a CM distribution between the terminal compartments could still be established.

Fig. 1a and b present the mean residence

Discussion

The concentration–time profiles for excreted 3H-CORT, as well as the final measurements, supported our and others’ (Touma et al., 2003) previous findings, where all of the injected CORT is excreted within 26 h. For female BALB/c mice the present results demonstrate that 60% of any injected CORT is excreted in the urine with a delay of, on average, 5 h. The remaining 40% is excreted in the faeces nearly 9 h after entering the blood stream. The faecal/urinary ratio of CM coincided well with previous

Acknowledgments

This study received generous funding from the Danish Research Council. The authors thank Trine Marie Nielsen, Niels Grunnet and Lis Frandsen for expert technical assistance.

References (21)

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