Accumulation of radioactivity after repeated infusion of 3H-adrenaline and 3H-noradrenaline in the rat as a model animal.

Besides enzymatic inactivation, catecholamines bind non-enzymatically and irreversible to proteins. The physiological impact of these catecholamine adducts is still unclear. We therefore collected basic data about the distribution of catecholamine adducts in the rat after repeated intravenous administration of (3)H-adrenaline and (3)H-noradrenaline. In all animals radioactivity in blood increased until the last injection on Day 7 and decreased then slowly close to background values (plasma) or remained higher (erythrocytes). In all sampled tissues radioactivity could be found, but only in hair high amounts remained present even after 3 weeks. Half-life of rat serum albumin loaded with (3)H-adrenaline or (3)H-noradrenaline was not altered. This study provides basic knowledge about the distribution of catecholamines or their adducts, but physiological effects could not be demonstrated. However, for the first time deposition and accumulation of catecholamines (adducts) in the hair could be proven, suggesting that hair might be used for evaluating long term stress.

The influence of trilostane on steroid hormone metabolism in canine adrenal glands and corpora lutea-an in vitro study.

Trilostane is widely used to treat hyperadrenocorticism in dogs. Trilostane competitively inhibits the enzyme 3-beta hydroxysteroid dehydrogenase (3ß-HSD), which converts pregnenolone (P5) to progesterone (P4) and dehydroepiandrosterone (DHEA) to androstendione (A4). Although trilostane is frequently used in dogs, the molecular mechanism underlying its effect on canine steroid hormone biosynthesis is still an enigma. Multiple enzymes of 3ß-HSD have been found in humans, rats and mice and their presence might explain the contradictory results of studies on the effectiveness of trilostane. We therefore investigated the influence of trilostane on steroid hormone metabolism in dogs by means of an in vitro model. Canine adrenal glands from freshly euthanized dogs and corpora lutea (CL) were incubated with increasing doses of trilostane. Tritiated P5 or DHEA were used as substrates. The resulting radioactive metabolites were extracted, separated by thin layer chromatography and visualized by autoradiography. A wide variety of radioactive metabolites were formed in the adrenal glands and in the CL, indicating high metabolic activity in both tissues. In the adrenal cortex, trilostane influences the P5 metabolism in a dose- and time-dependent manner, while DHEA metabolism and metabolism of both hormones in the CL were unaffected. The results indicate for the first time that there might be more than one enzyme of 3ß-HSD present in dogs and that trilostane selectively inhibits P5 conversion to P4 only in the adrenal gland.

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.

Excretion of catecholamines in rats, mice and chicken.

Stress assessment favours methods, which do not interfere with an animal's endocrine status. To develop such non-invasive methods, detailed knowledge about the excretion of hormone metabolites in the faeces and urine is necessary. Our study was therefore designed to generate basic information about catecholamine excretion in rats, mice and chickens. After administration of (3)H-epinephrine or (3)H-norepinephrine to male and female rats, mice and chickens, all voided excreta were collected for 4 weeks, 3 weeks or for 10 days, respectively. Peak concentrations of radioactivity appeared in one of the first urinary samples of mice and rats and in the first droppings in chickens 0.2-7.2 h after injection. In rats, between 77.3 and 95.6% of the recovered catecholamine metabolites were found in the urine, while in mice, a mean of 76.3% were excreted in the urine. Peak concentrations in the faeces were found 7.4 h post injection in mice, and after about 16.4 h in rats (means). Our study provides valuable data about the route and the profile of catecholamine excretion in three frequently used species of laboratory animals. This represents the first step in the development of a reliable, non-invasive quantification of epinephrine and norepinephrine to monitor sympatho-adrenomedullary activity, although promising results for the development of a non-invasive method were found only for the chicken.

Non-invasive measurement of adrenocortical activity in male and female rats.

Rats are widely used in biomedical research as animal models for human diseases. However, due to their small body size, blood sampling is complicated and invasive and thereby can seriously interfere with endocrine functions and possibly compromise the animals' welfare. Therefore, a non-invasive technique to monitor stress hormones in these animals is highly desired. Our study aimed to gain general information about corticosterone metabolism and excretion and to validate a 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay (EIA) to reliably measure faecal corticosterone metabolites (CMs) in laboratory rats. In total, 18 rats were administered 2.3 MBq of (3)H-corticosterone intravenously and per os, respectively (intravenous: 6 males and 6 females; per os: 3 males and 3 females). Subsequently, all voided excreta were frequently collected for five days. About 75+/-9% of the recovered CMs were found in the faeces. Peak concentrations of radiolabelled steroids appeared in the urine after 1.7+/-0.6 h in males and after 6.0+/-3.5 h in females. In faeces, maxima were observed after 14.7+/-2.4 h in both sexes. In principle, the time course and delay for both routes of administration (intravenous or per os) were the same, except for a delay of peak concentrations in urine (4.5+/-2.1 h) in per os administered males. Using high-performance liquid chromatography (HPLC), faecal (3)H-CMs were characterized and differences were found between the sexes. In both sexes, corticosterone was extensively metabolized, but while males showed only minor variations in their CM patterns, those of females differed largely between individuals. To validate the mentioned EIA, we investigated the diurnal variation (DV) of glucocorticoids as well as effects of the injection procedure itself and conducted an adrenocorticotropic hormone challenge test and a dexamethasone suppression test, using six male and six female rats each. Our results demonstrated that pharmacological stimulation, suppression and DV of adrenocortical activity were accurately reflected by means of CM measurement in faeces. By successful physiological validation, we proved for the first time the suitability of an immunoassay to non-invasively monitor adrenocortical activity in rats of both sexes. This method opens up new perspectives for biomedical and pharmacological investigations as well as for animal welfare related issues.