Assessment of urine and fecal testosterone metabolite excretion in Chinchilla lanigera males

Endemic chinchilla (Chinchilla spp.) populations are nearly extinct in the wild (South America). In captive animals (Chinchilla lanigera and C. brevicaudata), reproduction is characterized by poor fertility and limited by seasonal breeding patterns. Techniques applied for studying male reproductive physiology in these species are often invasive and stressful (i.e. repeated blood sampling for sexual steroids analysis). To evaluate endocrine testicular function, the present experiments were designed to (a) determine the main route of testosterone excretion (14C-testosterone infusion in four males); (b) validate urine and fecal testosterone metabolite measurements (HPLC was used to separate metabolites and immunoreactivity was assessed in all metabolites using a commercial testosterone radioimmunoassay, and parallelism, accuracy and precision tests were conducted to validate the immunoassay); and (c) investigate the biological relevance of the techniques applied (quantification of testosterone metabolite excretion into urine and feces from five males injected with hCG and comparison between 10 males and 10 females). Radiolabelled metabolites of 14C-testosterone were excreted, 84.7+/-4.2 % in urine and 15.2+/-3.9 % in feces. A total of 82.7+/-4.2% of urinary and 45.7+/-13.6% of fecal radioactivity was excreted over the first 24 h period post-infusion (metabolite concentration peaked at 8.2+/-2.5 h and 22.0+/-7.0 h, respectively). Several urinary and fecal androgen metabolites were separated by HPLC but only fecal metabolites were associated with native testosterone; however, there was immunoreactivity in more than one metabolite derived from 14C-testosterone. After hCG administration, an increase in androgen metabolite excretion was observed (p<0.05). Males excreted greater amounts daily of urinary androgen metabolites as compared with females (p<0.05); this difference was not evident in feces. Results of the present study indicate that the procedure used is a reliable and non-invasive method to repeatedly monitor variations in testicular endocrine activity in this species. It can be a useful tool that would help ensure the survival of the wild populations as well as to provide the basis for a more efficient use by the fur industry.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: VIII. Correspondence of urinary and plasma steroids in the llama (Lama glama) during nonconceptive and conceptive cycles

Ovarian function was evaluated in mature female llamas (n = 2) during seven ovulations in 2 conceptive and 5 nonconceptive ovarian cycles by measuring urinary and plasma hormone concentrations. Ovulation was induced by three different methods; administration of gonadotropin-releasing hormone (GnRH), copulation with a vasectomized male and copulation with an intact male. Plasma estradiol and progesterone concentrations, and urinary concentrations of estrogen conjugates and two progesterone metabolites, pregnanediol-3-glucuronide (PdG), and immunoreactive (iPdG), concentrations were compared to determine their value in monitoring ovarian function. Estrogen concentrations in urine corresponded to estradiol levels in plasma and accurately reflected changes in follicular activity when evaluated over several daily samples. Plasma progesterone and urinary iPdG were reliable indicators of luteal function. These data represent the first comparison of blood and urinary hormone measurements for monitoring the complete ovarian cycle of an ungulate, and demonstrates that either can be used to assess changes in ovarian activity in this species.     

Urinary corticosteroid excretion patterns in the okapi (Okapia johnstoni)

Stress is known to alter a variety of biological processes, including behavior and reproduction. It is therefore important to understand the stress levels of animals in captivity, especially those for whom captive breeding is a priority, such as the okapi. Levels of stress hormones can be measured from samples collected noninvasively, such as urine or feces, which are preferable with nondomestic species for whom drawing blood might in itself be a considerable stressor. To understand the excretion of cortisol in urine in the okapi, four (1.3) animals were subject to three injections: saline, 200 IU of an adrenocorticotropic hormone (ACTH) analogue, and 300 IU of the analogue. Their 24‐hr urinary corticosteroid levels were compared with 4 baseline days. Injection with the ACTH analogue significantly increased the urinary corticosteroid levels compared with saline injections and baseline. Eight (3.5) okapi were then observed for 24 hr per day for 5 days to determine their normal patterns of corticosteroid production. The mean corticosteroid levels varied significantly by individual. A significant circadian pattern in urinary corticosteroid was apparent independent of individual or gender, with cortisol rising during the daylight hours and decreasing again at night. Zoo Biol 27:381–393, 2008. © 2008 Wiley‐Liss, Inc.     

The use of deuterium-labeled cortisol for in vivo evaluation of renal 11beta-HSD activity in man: urinary excretion of cortisol, cortisone and their A-ring reduced metabolites

This study describes a new approach using stable isotope methodology in evaluating 11beta-HSD activities in vivo based on urinary excretion of cortisol, cortisone, and their A-ring reduced metabolites. The method involved the measurement of deuterium-labeled cortisol and its deuterium-labeled metabolites by GC/MS simultaneously with endogenous cortisol, cortisone, and their A-ring reduced metabolites after oral administration of deuterium-labeled cortisol to normal human subjects. This stable isotope approach offered unique advantages in assessing the appropriateness of measuring unconjugated and total (unconjugated + conjugated) cortisol, cortisone, and their A-ring reduced metabolites in urine as indices of renal 11beta-HSD2 activity in man. Our results strongly support that the measurement of urinary unconjugated cortisol and cortisone is a significant advance in assessing 11beta-HSD2 activity.     

Seasonal dynamics of agonistic behavior and hormones in an ex situ all‐male colony of large flying foxes

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all‐male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all‐male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all‐male colony of 12 large flying foxes at Disney's Animal Kingdom^®. We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all‐occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open‐mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.     

Cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids

Three domestic cats were given i.m. injections of ³H-cortisol to determine the time course and relative proportion of excreted ³H-cortisol metabolites into urine and feces. Most urinary radioactivity was detected in the first sample collected at 3.9 ± 2.5 hr postinjection and accounted for 13.9 ± 2.1% of the total radioactivity recovered. High performance liquid chromatography (HPLC) detected four urinary metabolites, one of which (13.7% urinary radioactivity) eluted with the ³H-cortisol reference tracer and was quantifiable using a commercial cortisol radioimmunoassay (RIA). The majority of cortisol metabolites in feces (85.9 ± 2.1%) was excreted at 22.3 ± 6.2 hr. HPLC analysis detected several fecal metabolites consisting primarily of nonhydolyzable water-soluble forms, none of which eluted with ³H-cortisol or ³H-corticosterone reference tracers. No immunoreactivity was detected in HPLC-separated fecal eluates using the cortisol RIA; however, two of the more polar metabolites were quantifiable using a commerical cortisosterone RIA. The physiological relevance of the immunoreactive fecal metabolites was determined in four domestic cats given an adrenocorticotropin (ACTH) challenge. Increased serum cortisol concentrations were detected within 30 min of ACTH injection, which was maintained for at least 6 hr. A corresponding increase in fecal cortisol metabolite concentrations (ranging from 238% to 826% over individual baseline values) was observed 24–48 hr later. These data indicate that adrenocortical activity can be monitored nonivasively in the cat by measuring cortisol metabolites excreted in feces. This procedure is a potentially valuable tool for endangered felid management to help evaluate responses to physiological and psychological stressors associated with environmental conditions and husbandry practices. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Noninvasive monitoring of fecal cortisol metabolites in the eastern chipmunk (Tamias striatus): validation and comparison of two enzyme immunoassays

Monitoring fecal glucocorticoid metabolites in wild animals, using enzyme immunoassays, enables the study of endocrinological patterns relevant to ecology and evolution. While some researchers use antibodies against the parent hormone (which is typically absent from fecal samples), others advocate the use of antibodies designed to detect glucocorticoid metabolites. We validated two assays to monitor fecal cortisol metabolites in the eastern chipmunk (Tamias striatus). We compared an antibody produced against cortisol and one produced against 5α-pregnane-3β, 11β, 21-triol-20-one using a radiometabolism study and an injection with adrenocorticotropic hormone (ACTH). Most cortisol metabolites were excreted in the urine (～83%). Peak excretion in the feces occurred 8 h after injection. Both assays detected an increase in fecal cortisol metabolite levels after injection of ACTH. Males, but not females, exhibited a circadian variation in metabolite levels. The sexes did not exhibit any difference over the time course and route of excretion or the relative increase in fecal cortisol metabolite levels after ACTH injection. The cortisol assay displayed higher reactivity to ACTH injection relative to baseline than did the metabolite assay. While both antibodies gave comparable results, the cortisol antibody was more sensitive to changes in plasma cortisol levels in eastern chipmunks.     

Study of natural and artificial corticosteroid phase II metabolites in bovine urine using HPLC-MS/MS

Corticosteroid compounds are widely used therapeutically for their anti-inflammatory properties and sometimes as growth promoters in food producing animals. In the field of drug residue analysis, knowledge of the main metabolic pathways of target analytes improves the efficiency of the corresponding control. Thus, phase II metabolism of corticosteroids, for which very little literature is available, was investigated in cattle. An LC-MS/MS detection method was developed for five commercially available conjugated corticosteroids, permitting direct monitoring during the development of their separation on anion exchange SPE. This separation method is further applicable to other potential urinary conjugated corticosteroids. Because our purpose was not to identify all the existing corticosteroid phase II metabolites, but to obtain their total relative proportions, enzymatic hydrolysis was optimized and performed on each separated fraction (glucuronides and sulfates). Finally, the phase II metabolic profiles of natural and artificial corticosteroids in bovine urine were studied and compared. LC-MS/MS detection with negative electrospray ionization appeared efficient for both glucuronide and sulfate conjugated corticosteroids, and quaternary ammonium stationary phase permitted their effective separation. The experimental design used for optimization of the enzymatic hydrolysis with a purified Helix pomatia preparation demonstrated optimal values for pH 5.2, temperature of 50 degrees C and incubation duration of 4h. Results on bovine urine samples collected on two animals before and after dexamethasone administration showed important differences regarding the proportion of total conjugated forms between endogenous cortisol, endogenous tetrahydrocortisol, and exogenous dexamethasone. This proportion appeared significantly higher for tetrahydrocortisol (40-65%) than cortisol (2-8%) or dexamethasone (4-27%). This innovative methodology demonstrates the suitability of anion exchange SPE and LC-MS/MS for the study of steroid hormones phase II metabolism, and appears promising to investigate metabolic profile differences linked to the hormone administration mode or origin, with direct application in the field of doping controls.     

Cortisol metabolism and excretion in the isolated perfused rat kidney

The isolated perfused rat kidney allows a simultaneous kinetic study of both the renal metabolism and the urinary excretion of cortisol and its metabolites in the rat. In this system, cortisol was completely metabolized within 120 minutes. The main renal metabolites of cortisol (cortisone, 20 reduced cortisol and 20 reduced cortisone) were found in the recirculating perfusate and in urine. The formation of these metabolites was quantitatively evaluated and compared to a theoretical model.     

Steroid metabolism and validation of noninvasive endocrine monitoring in the African wild dog (Lycaon pictus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for African wild dogs (Lycaon pictus) and to establish physiological validity of these methods by evaluating longitudinal reproductive-endocrine profiles in captive individuals. To determine the primary excretory by-products of ovarian steroid metabolism, [¹⁴C]-progesterone and [³H]-estradiol were co-administered to a female and all excreta were collected for 80 hr postinjection. Radiolabel excretion peaked ≤ 18 hr postinfusion, and progesterone and estradiol metabolites were excreted in almost equivalent proportions in urine (39.7 and 41.1%, respectively) and feces (60.3 and 58.9%, respectively). Most of the urinary metabolites were conjugated (estradiol, 94.3 ± 0.3%; progesterone, 90.4 ± 0.5%), so that immunoassays for pregnanediol-3α-glucuronide (PdG) and estrogen conjugates (EC) were effective for assessing steroid metabolites. Two immunoreactive estrogens (estradiol and estrone) and at least one immunoreactive progesterone metabolite (3α-hydroxy-5α, pregnan-20-one) were detected in feces. Urine and fecal samples were collected (1–3 times per week) for 1.5 yr from one adult female and two adult males to assess longitudinal steroid metabolite excretion. Overall correlation of urinary PdG to matched, same-day fecal progesterone metabolites immunoreactivity was 0.38 (n = 71, P < 0.05). Similarly, urinary EC was correlated (P < 0.05) with same-day fecal estrogen immunoreactivity (r = 0.49, n = 71). During pregnancy and nonpregnant cycles, copulation occurred at the time of peak (or declining) estrogen metabolites and increasing progesterone metabolites concentrations. Estrus duration was 6–9 days and gestation lasted 69 days with parturition occurring coincident with a drop in progesterone metabolites. Males exhibited seasonal trends in fecal testosterone excretion with maximal concentrations from July to September coincident with peak mating activity. Although these limited longitudinal hormone profiles should be interpreted cautiously, noninvasive gonadal steroid monitoring suggests that: (1) both female and male wild dogs may exhibit reproductive seasonality in North America, (2) females are monoestrous, and (3) peak testicular activity occurs between August and October coincident with mating behavior. From a conservation perspective, noninvasive endocrine monitoring techniques should be useful for augmenting captive breeding programs, as well as for developing an improved understanding of the physiological mechanisms underlying reproductive suppression in response to social and ecological pressures. Zoo Biol 16:533–548, 1997. © 1997 Wiley-Liss, Inc.     

A non-invasive technique for analyzing fecal cortisol metabolites in snowshoe hares (<Emphasis Type="Italic">Lepus americanus</Emphasis>)

To develop non-invasive techniques for monitoring steroid stress hormones in the feces of free-living animals, extensive knowledge of their metabolism and excretion is essential. Here, we conducted four studies to validate the use of an enzyme immunoassay for monitoring fecal cortisol metabolites in snowshoe hares (Lepus americanus). First, we injected 11 hares with radioactive cortisol and collected all voided urine and feces for 4 days. Radioactive metabolites were recovered predominantly in the urine (59%), with only 8% recovered in the feces. Peak radioactivity was detected an average of 3.5 and 5.7 h after injection in the urine and feces, respectively. Second, we investigated diurnal rhythms in fecal cortisol metabolites by measuring recovered radioactivity 2 days after the radioactive cortisol injection. The total amount of radioactivity recovered showed a strong diurnal rhythm, but the amount of radioactivity excreted per gram of feces did not, remaining constant. Third, we injected hares with dexamethasone to suppress fecal cortisol metabolites and 2 days later with adrenocorticotropic hormone to increase fecal cortisol metabolites. Dexamethasone decreased fecal cortisol metabolites concentrations by 61% and adrenocorticotropic hormone increased them by 1,000%, 8–12 h after injection. Fourth, we exposed hares to a simulated predator (dog). This increased the fecal cortisol metabolites concentrations by 175% compared with baseline concentrations 8–12 h after exposure. Thus, this enzyme immunoassay provides a robust foundation for non-invasive field studies of stress in hares.     

Estimation of immunoreactive testicular androgen metabolites in the urine of saddle-back tamarins

Hydrolysis, extraction, and radioimmunoassay techniques for the estimation of excreted testosterone metabolites in the urine of saddle-back tamarins have been validated and are described. The steroids measured with the testosterone antiserum used are mostly present as glucuronides and sulfates. Immunoreactivity in high-performance liquid chromatography (HPLC) fractions of urinary extracts and in a standard mixture of cortisol, testosterone, and dihydrotestosterone (DHT) were compared. The fractions with the same retention data as testosterone accounted for the major part of the immunoreactivity. Several other immunoreactive compounds of unknown identity were present in low concentrations. These results suggest that testosterone conjugates are the major steroid metabolites measured with this method in Saguinus fuscicollis. Urinary testosterone levels of castrated males were much lower than those of intact males. Testosterone treatment of castrated males resulted in a temporary superphysiological increase in the levels of urinary testosterone and in an individually variable increase in the levels of the minor immunoreactive compounds. These results suggest that estimation of testosterone metabolite levels in urine is a valid method for the assessment of testicular activity in Saguinus fuscicollis.     

Adrenal activity and anxiety-like behavior in fur-chewing chinchillas (Chinchilla lanigera)

Due to its complexity, in combination with a lack of scientific reports, fur-chewing became one of the most challenging behavioral problems common to captive chinchillas. In the last years, the hypothesis that fur-chewing is an abnormal repetitive behavior and that stress plays a role in its development and performance has arisen. Here, we investigated whether a relationship existed between the expression and intensity of fur-chewing behavior, elevated urinary cortisol excretion and anxiety-related behaviors. Specifically, we evaluated the following parameters in behaviorally normal and fur-chewing animals of both sexes: (1) mean concentrations of urinary cortisol metabolites and (2) anxiety-like behavior in an elevated plus-maze test. Urinary cortisol metabolites were higher only in females that expressed the most severe form of the fur-chewing behavior (P≤0.05). Likewise, only fur-chewing females exhibited increased (P≤0.05) anxiety-like behaviors associated with the elevated plus-maze test. Overall, these data provided additional evidence to support the concept that fur-chewing is a manifestation of physiological stress in chinchilla, and that a female sex bias exists in the development of this abnormal behavior.     

The corticosteroid metabolic profile of the mouse

Data are presented on the urinary corticosteroid metabolic profile of the mouse strain 129/svJ. Through the use of GC/MS we have characterized, or tentatively identified corticosterone (Kendall's compound B) metabolites of both the 11beta-hydroxy and 11-carbonyl (compound A) series in urine. Full mass spectra of the methyloxime-trimethylether derivatives of 15 metabolites are included in the paper as an aid to other researchers in the field. Metabolites ranged in polarity from tetrahydrocorticosterone (THB) to dihydroxy-corticosterone with dominance of highly polar steroids. We found that prior to excretion corticosterone can undergo oxidation at position 11beta, reduction at position 20 and A-ring reduction. Metabolites retaining the 3-oxo-4-ene structure can be hydroxylated at position 6beta- as well as at an unidentified position, probably 16alpha-. Saturated steroids can be hydroxylated at positions 1beta-, 6alpha-, 15alpha- and 16alpha. A pair of hydroxy-20-dihydro-corticosterone metabolites (OH-DHB) were the most important excretory products accounting for about 40% of the total. One metabolite of this type was identified as 6beta-hydroxy-DHB; the other, of similar quantitative importance was probably 16alpha-hydroxy-DHB. The ratio of metabolites of corticosterone (B) to those of 11-dehydro-corticosterone (A) was greater than 9:1, considerably higher than that for the equivalent "human" ratio of 1:1 for cortisol to cortisone metabolites. Results from this study allowed the evaluation of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in mice with deleted glucose-6-phosphate transporter (G6PT). These mice had attenuated back-conversion of A to B resulting in an increased ratio of A-metabolites to B-metabolites [Walker EA, Ahmed A, Lavery GG, Tomlinson JW, Kim SY, Cooper MS, Stewart PM, 11beta-Hydroxysteroid dehydrogenase type 1 regulation by intracellular glucose-6-phosphate, provides evidence for a novel link between glucose metabolism and HPA axis function. J Biol Chem 2007;282:27030-6]. We believe this study is currently the most comprehensive on the urinary steroid metabolic profile of the mouse. Quantitatively less steroid is excreted in urine than in feces by this species but urine analysis is more straightforward and the hepatic metabolites are less subject to microbial degradation than if feces was analyzed.     

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.     

Assessment of female reproductive status in captive-housed Hanuman langurs (Presbytis entellus) by measurement of urinary and fecal steroid excretion patterns

The study reports on the use of urinary and fecal hormone measurements for monitoring female reproductive status in captive-housed Hanuman langurs (Presbytis entellus). Matched urine and fecal samples collected throughout 7 complete menstrual cycles of two females, and during part of one pregnancy in a third female were analyzed. Estrone conjugates (E1C) and immunoreactive pregnanediol glucuronide (PdG) in urine and immunoreactive estradiol (E2), progesterone (P4), pregnanediol (Pd) and 20α-hydroxyprogesterone (20αOHP) in feces were measured by enzymeimmunoassay. E1C and PdG in urine were excreted in a cyclic pattern with E1C levels increasing 3- to 4-fold during the follicular phase to reach preovulatory peak values 2 days before a defined rise in PdG concentrations. Cycle lengths ranged between 20 and 34 days comprising a variable follicular phase of 7–21 days and a more consistent luteal phase of 12–14 days. High pressure liquid chromatography (HPLC) analysis of fecal extracts confirmed the presence of all fecal hormones measured, but indicated large amounts of additional immunoreactivity in the three progestin assays. The patterns of excretion of fecal E2 and all three fecal progestins corresponded well with those of steroid metabolites in urine in showing a clear and well defined follicular phase E2 rise followed by a luteal phase progestin increase. Measurement of 20αOHP immunoreactivity revealed the most stable baseline and the highest follicular/luteal phase differential. Levels of all hormones were clearly elevated during pregnancy although urinary E1C and PdG showed a more pronounced increase compared to fecal metabolites. The results indicate that urinary and fecal hormone analysis can be applied to noninvasive monitoring of reproductive status in the Hanuman langur. © 1995 Wiley-Liss, Inc.     

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C₁₈ cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.     

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.     

Investigation of the origin of prednisolone in cow urine

After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. Because the majority of positive samples were detected at the slaughterhouse, researchers hypothesised that, together with increased cortisol and cortisone, a small quantity of prednisolone could be produced by the cows in stressful situations.In the present study, three dairy cows underwent intramuscular treatments with tetracosactide hexaacetate, a synthetic analogue of adrenocorticotropic hormone, to simulate stress. The animals were slaughtered at the end of the study. The results indicated that prednisolone could be detected occasionally in the non-stressful state, but was consistently found in the urine of stressed cows (concentrations ranged from 1.01 to 4.08 ng/mL). To confirm the stress condition, urinary cortisol and cortisone were also detected at high concentrations in the urine, typically at concentrations of hundreds of nanograms per millilitre.The results of this preliminary study did not reveal the metabolic pathway responsible for prednisolone but suggested that this corticosteroid could be produced endogenously.     

Hemoglobin affinity in Andean rodents

Blood hemoglobin oxygen affinity (P50) was measured in three Andean species and in the laboratory rat (control), all raised near sea level. Chinchilla lanigera (Molina, 1792) has an altitudinal habitat range from low Andean slopes up to 3000 m., while Chinchilla brevicaudata (Waterhouse, 1848) has an altitudinal range from 3000 to 5000 m. The laboratory type guinea pig, wild type guinea pig (Cavia porcellus), (Waterhouse, 1748), and laboratory rat (Rattus norvegicus) were also raised at sea level. The Andean species had high hemoglobin oxygen affinities (low P50) compared with the rat. Chinchilla brevicaudata had a higher affinity than Chinchilla lanigera. The wild type guinea pig had a higher affinity than the laboratory type. As has been shown in other species, this is another example of an inverse correlation between the altitude level and the P50 values. This is the first hemoglobin oxygen affinity study in Chinchilla brevicaudata.