A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species

Noninvasive fecal glucocorticoid analysis has tremendous potential as a means of assessing stress associated with environmental disturbance in wildlife. However, interspecific variation in excreted glucocorticoid metabolites requires careful selection of the antibody used in their quantification. We compared four antibodies for detecting the major fecal cortisol metabolites in yellow baboons following (3)H cortisol administration, ACTH challenge, and HPLC separation of fecal glucocorticoid metabolites. The most effective antibody (ICN corticosterone RIA; Cat. No. 07-120102) demonstrated relatively high cross-reactivities to the major cortisol metabolites present in feces during peak excretion, following both radiolabel infusion and ACTH challenge. This same antibody also detected increased fecal glucocorticoid metabolites after ACTH administration in the African elephant, black rhinoceros, Roosevelt elk, gerenuk, scimitar-horned oryx, Alaskan sea otter, Malayan sun bear, cheetah, clouded leopard, longtailed macaque, and northern spotted owl. Results suggest that (1) fecal glucocorticoid assays reliably detect endogenous changes in adrenal activity of a diverse array of species and (2) where comparisons were made, the ICN corticosterone antibody generally was superior to other antibodies for measuring glucocorticoid metabolites in feces.     

Comparative aspects of the metabolism and excretion of cortisol in three individual nonhuman primates

A radiometabolism study is described to provide the first comparative data on the time course, route, and characteristics of excreted [3H]cortisol metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macacafascicularis), and the chimpanzee (Pan troglodytes). A low dose (40-100 microCi) of 3H-labeled cortisol was administered intravenously to one adult male of each species and the excreta collected over a 5-day period postinjection. The major proportion of radioactivity was excreted in the urine (>80%). Peak radioactivity in urine was recovered within 5.5 h following injection in all three species, while in the feces peak levels of radioactivity were recovered within 26 h postinjection. In all three species, urinary metabolites were primarily excreted as conjugates (61-87%), whereas the percentage of conjugated metabolites in feces was 50% or less. The number and relative abundance of urinary and fecal [3H]cortisol metabolites were determined by reverse-phase high-performance liquid chromatography (HPLC) and immunoreactivity of the radioactivity peaks was assessed by screening HPLC fractions with established cortisol, corticosterone, and 11-oxoetiocholanolone enzyme immunoassays (EIA), the latter being a group-specific assay for measuring 11,17-dioxoandrostanes. HPLC separation of urinary and fecal extracts revealed multiple peaks of radioactivity, several of which were common to all three species. The relative proportion of these peaks, however, differed considerably among species and between urine and feces. HPLC indicated that native cortisol was a major urinary excretory product in the marmoset, while comparatively small amounts were present in the urine of the macaque and chimpanzee. In contrast, in feces, cortisol was only detected in low amounts in the marmoset and was virtually absent in the macaque and chimpanzee. In all three species, one of the major radioactivity peaks showed a retention time comparable to 11-oxoetiocholanolone and high immunoreactivity in the 11-oxoetiocholanolone EIA. The measurement of urinary- and/or fecal-immunoreactive 11,17-dioxoandrostanes is therefore implicated for noninvasive assessment of adrenal function in Old World monkeys, New World monkeys, and great apes.     

Characterization of urinary and fecal metabolites of testosterone and their measurement for assessing gonadal endocrine function in male nonhuman primates

The aims of the present study were (i) to provide basic comparative data on the time course, route, and characteristics of excreted [14C]testosterone (T) metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macaca fascicularis) and the chimpanzee (Pan troglodytes) and (ii) to use this information to help validate the measurement of urinary and fecal testosterone metabolites for assessing androgen status in Anthropoid primates. Radiolabeled 14C-T (10-30 microCi) was injected intravenously into one adult male of each species and the excreta collected over the next 5 days. Peak radioactivity in urine was detected within 2h and accounted for 67% (Mf), 80% (Cj) and 91% (Pt) of the total radioactivity recovered. The time course of excretion of radioactivity in feces showed a higher variation between species (4-26 h to peak values). In all three species, the majority (>90%) of urinary metabolites were excreted as conjugates whereas the proportion of conjugated metabolites in feces was substantially lower and more variable. High pressure liquid chromatography (HPLC) analysis of urinary and fecal extracts revealed multiple peaks of radioactivity in all three individuals, but each with a distinctive pattern. Native T was excreted in only small amounts into the urine, whereas it was virtually absent in the feces of all three individuals. Three C17 group-specific enzymeimmunoassays using antisera against testosterone, 5alpha-androstane-17alpha-ol-3-one and androsterone were evaluated for their ability to discriminate immunoreactive androgen levels between intact males, castrated males and females based on measurements in urine and feces. In the marmoset, all assays (except for T in feces) clearly discriminated between test groups; in the chimpanzee significantly higher levels of androgen immunoreactivity in intact versus castrated males were measured in urine, but not feces. In the macaque, only the 5alpha-androstanolone measurement in feces discriminated between groups. Data on the results of a radiometabolism study using 3H-DHEA (a weak adrenal androgen) in a long-tailed macaque suggested that co-measurement of metabolites derived from T and DHEA in the assays tested might explain the difficulties in discriminating gonadal status in the two Old World primate species. Collectively, the data show that T metabolism in primates is highly complex and that no single method for noninvasive assessment of androgen status can be used for application across species. The importance of a proper validation of the methodology for each species is emphasised.     

A longitudinal study of LH, gonadal and adrenal steroids in four intact Asian bull elephants (Elephas maximus) and one castrate African bull (Loxodonta africana) during musth and non-musth periods

During their annual musth cycle, adult African and Asian bull elephants have increased gonadal androgens (testosterone [T], dihydrotestosterone [DHT], androstenedione [A4]). Because musth is a physiologically and psychologically stressful time, this study was conducted to investigate whether the adrenal glands (stimulated by stress) increase production of both glucocorticoids and androgens during musth. Weekly serum samples were taken for 11-15 months from four intact adult Asian bull elephants, and from a castrate African bull elephant who exhibits musth. Testosterone, androstenediol (A5), A4, luteinizing hormone (LH), cortisol, and dehydroepiandrosterone (DHEA) were measured in each sample. In three of the four intact bulls, all hormones measured increased during musth. Adrenal androgens were strongly correlated with LH and testicular androgens, though not to cortisol. None of the hormones measured in the castrate bull increased during his musth cycles. While the significance of adrenal activity in the elephant during musth has yet to be determined, this study provides evidence that the adrenal gland actively produces both glucocorticoids and androgens during musth in the Asian elephant.     

Quantitative estimation of absorption and degradation of a carnitine supplement by human adults

Results of kinetic and pharmacokinetic studies have suggested that dietary carnitine supplements are not totally absorbed, and are in part degraded in the gastrointestinal tract of humans. To determine the metabolic fate of dietary carnitine supplements in humans, we administered orally a tracer dose of [methyl-3H]L-carnitine with a meal to five normal adult males, who had been adapted to a high-carnitine diet plus carnitine supplement (2 g/d) for 14 days. Appearance of [methyl-3H]L-carnitine and metabolites in serum, and urinary and fecal excretion of radiolabeled carnitine and metabolites was monitored for 5 to 11 days following administration of the test dose. Maximum concentration of [methyl-3H]L-carnitine in serum occurred at 2.0 to 4.5 hours after administration of the tracer, indicating relatively slow absorption from the intestinal lumen. Total radioactive metabolites excreted in urine and feces ranged from 47% to 55% of the ingested tracer. Major metabolites found were [3H]trimethylamine N-oxide (8% to 49% of the administered dose; excreted primarily in urine) and [3H]gamma-butyrobetaine (0.44% to 45% of the administered dose; excreted primarily in feces). Urinary excretion of total carnitine was 16% to 23% of intake. Fecal excretion of total carnitine was negligible (less than 2% of total carnitine excretion).     

Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses

Measurement of glucocorticoid metabolites in feces has become an accepted method for the noninvasive evaluation of adrenocortical activity. The objective of this study was to determine if a simple cortisol enzyme immunoassay (EIA) was suitable for monitoring adrenocortical activity in a variety of carnivore species. Performance of the cortisol EIA was gauged by comparison to a corticosterone radioimmunoassay (RIA) that has been used for measuring glucocorticoid metabolites in feces of numerous species. Tests for parallelism and extraction efficiency were used to compare the cortisol EIA and corticosterone RIA across eight species of carnivores (Himalayan black bear, sloth bear, domestic cat, cheetah, clouded leopard, black-footed ferret, slender-tailed meerkat, and red wolf). The biological relevance of immunoreactive glucocorticoid metabolites in feces was established for at least one species of each Carnivora family studied with an adrenocorticotropic hormone (ACTH) challenge. High performance liquid chromatography (HPLC) analysis of fecal extracts for each species revealed (1) the presence of multiple immunoreactive glucocorticoid metabolites in feces, but (2) the two immunoassays measured different metabolites, and (3) there were differences across species in the number and polarities of metabolites identified between assay systems. ACTH challenge studies revealed increases in fecal metabolite concentrations measured by the cortisol EIA and corticosterone RIA of approximately 228-1145% and approximately 231-4150% above pre-treatment baseline, respectively, within 1-2 days of injection. Concentrations of fecal glucocorticoid metabolites measured by the cortisol EIA and corticosterone RIA during longitudinal evaluation (i.e., >50 days) of several species were significantly correlated (P<0.0025, correlation coefficient range 0.383-0.975). Adrenocortical responses to physical and psychological stressors during longitudinal evaluations varied with the type of stimulus, between episodes of the same stimulus, and among species. Significant elevations of glucocorticoid metabolites were observed following some potentially stressful situations [anesthesia (2 of 3 subjects), restraint and saline injection (2 of 2 subjects), restraint and blood sampling (2 of 6 episodes), medical treatment (1 of 1 subject)], but not in all cases [e.g., gonadotropin injection (n=4), physical restraint only (n=1), mate introduction/breeding (n=1), social tension (n=1), construction (n=2) or relocation (n=1)]. Results reinforced the importance of an adequate baseline period of fecal sampling and frequent collections to assess adrenocortical status. The corticosterone RIA detected greater adrenocortical responses to exogenous ACTH and stressful exogenous stimuli in the Himalayan black bear, domestic cat (female), cheetah, clouded leopard, slender-tailed meerkat, and red wolf, whereas the cortisol EIA proved superior to resolving adrenocortical responses in the black-footed ferret and domestic cat (male). Overall results suggest the cortisol EIA tested in this study offers a practical method for laboratories restricted in the usage of radioisotopes (e.g., zoological institutions and field facilities) to integrate noninvasive monitoring of adrenocortical activity into studies of carnivore behavior and physiology.     

The in vivo metabolism of cortisol and corticosterone by the macaque monkey (Macaca fascicularis).

(4-14C) Cortisol was administered intramuscularly to one adult female macaque monkey, MF3 (Macaca fascicularis). To adult female macaque monkey, MF4, (4-14C)corticosterone was administered intramuscularly. Urine samples were collected and the metabolites excreted identified using gas chromatography, radio-gas chromatography and gas chromatography-mass spectrometry. The principal metabolites of cortisol were identified as glucuronide conjugates of 11-oxygenated-17-oxosteroids. The excretion of tetrahydrocortisol and tetrahydrocortisone relative to the other corticosteroid metabolites was low compared with that of man. Two compounds, 3 beta-cortol and 3 beta-cortolone not normally present in human urine were identified in the urine from this species. The prinicpal metabolites of corticosterone were glucuronide conjugates of hexahydroCompound A and hexahydrocorticosterone. Two unidentified radioactive compounds were also present.     

Noninvasive fecal monitoring of glucocorticoids in spotted hyenas, Crocuta crocuta.

The aim of this study was to validate a method for measuring glucocorticoids noninvasively in feces of spotted hyenas (Crocuta crocuta). Three established enzyme immunoassays (EIA) for cortisol, corticosterone, and 11-oxoetiocholanolone were tested, but proved unsatisfactory. A new EIA using another corticosterone antibody was established and was used for all subsequent analyses; this EIA was validated by demonstrating parallelism between serial dilutions of spotted hyena fecal extracts and dilutions of standard corticosterone and by the recovery of corticosterone added to fecal extracts. High-performance liquid chromatography (HPLC) fractions analyzed by EIA showed various immunoreactive substances with polarities of unconjugated steroids. The physiological relevance of fecal glucocorticoid metabolites was further validated by demonstrating that (1) injection of exogenous ACTH to four males and two females led to a significant increase in fecal glucocorticoid metabolites within 24-50 h, (2) the translocation of a male spotted hyena to a new enclosure resulted in a fivefold increase compared to baseline concentrations, and (3) agonistic social interactions and physical conflict resulted in large increases of fecal glucocorticoid metabolites in both protagonists. Fecal steroid assessment is therefore of use in monitoring adrenal activity in spotted hyenas.     

The characterization of polar corticosteroids in the urine of the macaque monkey (macaca fascicularis) and the baboon (papio hamadryas).

Computerised gas chromatography-mass spectrometry was employed in the identification of polar corticosteroid metabolites excreted in the urine from the macaque monkey (Macaca fascicularis) and the baboon (Papio hamadryas). The following steroids were identified in significant amounts in the urine from both species: 3alpha,17alpha,20alpha, 21-tetrahydroxy-5beta-pregnan-11-one; 3alpha,17alpha,20beta,21-tetrahydroxy-5beta-pregnan-11-one; 5beta-pregnane-3alpha,11beta,17alpha,20alpha,21-pentol; 5beta-pregnane-3alpha,11beta,17alpha,20beta-pentol; 5alpha-pregnane-3beta,11beta,17alpha,20beta,21-pentol. 11beta,17alpha,21-Trihydroxy-4-pregnene-3,20-dione (cortisol), 11beta,17alpha,20beta,21-tetrahydroxy-4-pregnen-3-one and 11beta,17alpha,20beta,21-tetrahydroxy-5xi-pregnan-3-one were identified in macaque monkey urine. Two steroids, 17alpha,20beta,21-trihydroxy-4-pregnane-3,11-dione and 17alpha,20alpha,21-trihydroxy-4-pregnene-3,11-dione were excreted as major C21 metabolites in the baboon but were not identified in the urine from the macaque monkey. 3beta-Hydroxy-5alpha-pregnane metabolites were identified in the urine from both species. All these steroids were excreted conjugated to glucuronic acid, evidenced by their recovery after hydrolysis with beta-glucuronidase enzyme. An efficient 20beta-reduction of corticosteroids in both species is apparent, and the excretion pattern of polar steroid metabolites in the two species was shown to be similar.     

Understanding the role of glucocorticoids in obesity: tissue-specific alterations of corticosterone metabolism in obese Zucker rats

The role of glucocorticoids in obesity is poorly understood. Observations in obese men suggest enhanced inactivation of cortisol by 5alpha-reductase and altered reactivation of cortisone to cortisol by 11betahydroxysteroid dehydrogenase type 1 (11betaHSD1). These changes in glucocorticoid metabolism may influence corticosteroid receptor activation and feedback regulation of the hypothalamic-pituitary-adrenal axis (HPA). We have compared corticosterone metabolism in vivo and in vitro in male obese and lean Zucker rats, aged 9 weeks (n = 8/group). Steroids were measured in 72-h urine and 0900 h trunk blood samples. 5alpha-Reductase type 1 and 11betaHSD activities were assessed in dissected tissues. Obese animals were hypercorticosteronemic and excreted more total corticosterone metabolites (2264+/-623 vs. 388+/-144 ng/72 h; P = 0.003), with a greater proportion being 5alpha-reduced or 11-oxidized. 11-Dehydrocorticosterone was also elevated in plasma (73+/-9 vs. 18+/-2 nM; P = 0.001) and urine (408+/-111 vs. <28 ng/72 h; P = 0.01). In liver of obese rats, 5alpha-reductase type 1 activity was greater (20.6+/-2.7% vs. 14.1+/-1.5%; P<0.04), but 11betaHSD1 activity (maximum velocity, 3.43+/-0.56 vs. 6.57+/-1.13 nmol/min/mg protein; P = 0.01) and messenger RNA levels (0.56+/-0.08 vs. 1.03+/-0.15; P = 0.02) were lower. In contrast, in obese rats, 11betaHSD1 activity was not different in skeletal muscle and sc fat and was higher in omental fat(36.4+/-6.2 vs. 19.2+/-6.6; P = 0.01), whereas 11betaHSD2 activity was higher in kidney (16.7+/-0.6% vs. 11.3+/-1.5%; p = 0.01). We conclude that greater inactivation of glucocorticoids by 5alpha-reductase in liver and 11betaHSD2 in kidney combined with impaired reactivation of glucocorticoids by 11betaHSD1 in liver may increase the MCR of glucocorticoids and decrease local glucocorticoid concentrations at these sites. By contrast, enhanced 11betaHSD1 in omental adipose tissue may increase local glucocorticoid receptor activation and promote obesity.     

Comparative endocrinology of testicular, adrenal and thyroid function in captive Asian and African elephant bulls

Concentrations of serum testosterone, cortisol, thyroxine (free and total T4), triiodothyronine (free and total T3) and thyroid stimulating hormone (TSH) were measured to assess adrenal and thyroid function as they relate to testicular activity and musth in captive elephants. Blood samples were collected approximately weekly from Asian (n=8) and African (n=12) bulls at seven facilities for periods of 4 months to 9.5 years. Age ranges at study onset were 8-50 years for Asian and 10-21 years for African elephants. Based on keeper logs, seven Asian and three African bulls exhibited behavioral and/or physical (temporal gland secretion, TGS, or urine dribbling, UD) signs of musth, which lasted 2.8+/-2.5 months in duration. Serum testosterone was elevated during musth, with concentrations often exceeding 100 ng/ml. Patterns of testosterone secretion and musth varied among bulls with no evidence of seasonality (P>0.05). Only three bulls at one facility exhibited classic, well-defined yearly musth cycles. Others exhibited more irregular cycles, with musth symptoms often occurring more than once a year. A number of bulls (1 Asian, 9 African) had consistently low testosterone (<10 ng/ml) and never exhibited significant TGS or UD. At facilities with multiple bulls (n=3), testosterone concentrations were highest in the oldest, most dominant male. There were positive correlations between testosterone and cortisol for six of seven Asian and all three African males that exhibited musth (range, r=0.23-0.52; P<0.05), but no significant correlations for bulls that did not (P>0.05). For the three bulls that exhibited yearly musth cycles, TSH was positively correlated (range, r=0.22-0.28; P<0.05) and thyroid hormones (T3, T4) were negatively correlated (range, r=-0.25 to -0.47; P<0.05) to testosterone secretion. In the remaining bulls, there were no clear relationships between thyroid activity and musth status. Overall mean testosterone and cortisol concentrations increased with age for all bulls combined, whereas thyroid activity declined. In summary, a number of bulls did not exhibit musth despite being of adequate physical maturity. Cortisol and testosterone were correlated in most bulls exhibiting musth, indicating a possible role for the adrenal gland in modulating or facilitating downstream responses. Data were generally inconclusive as to a role for thyroid hormones in male reproduction, but the finding of discrete patterns in bulls showing clear testosterone cycles suggests they may facilitate expression or control of musth in some individuals.     

Effects of prenatal stress on hypothalamic–pituitary–adrenal (HPA) axis function over two generations of guinea pigs (Cavia aperea f. porcellus)

Prenatal stress can alter hypothalamic-pituitary-adrenal axis function with potential consequences for later life. The aim of our study was to examine in guinea pigs (Cavia aperea f. porcellus) the effects of stress experienced during F0 pregnancy on glucocorticoid levels in plasma and feces, as well as challenge performance, in F1 offspring (n=44) and fecal glucocorticoid levels in F2 offspring (n=67). F1 animals were either born to F0 dams that had been stressed with strobe light during early to mid pregnancy, resulting in a short term increase but long-term down-regulation of maternal glucocorticoid levels, or to undisturbed F0 dams. The same stressor was used as a challenge for F1 offspring at age 26 days and around 100 days. Basal plasma cortisol concentrations during early F1 development, as well as overall glucocorticoid levels at challenge tests, were lower in F1 animals that were prenatally stressed than in control animals. Fecal cortisol metabolites were initially at lower levels in prenatally stressed F1 animals, relative to control animals, but shifted to higher levels around day 68, with an additional sex difference. Effects were also seen in the F2 generation, as male but not female offspring of prenatally stressed F1 animals had significantly higher levels of cortisol metabolites in feces after weaning. We conclude that stress exposure of F0 dams resulted in lower basal glucocorticoid levels in F1 offspring during the pre-pubertal phase and during stress exposure, but higher glucocorticoid levels in post-adolescent F1 animals. Also in males of F2 generation effects of stress exposure of F0 dams were detected.     

Fecal cortisol metabolite levels in free-ranging North American red squirrels: Assay validation and the effects of reproductive condition

Patterns in stress hormone (glucocorticoid: GC) levels and their relationship to reproductive condition in natural populations are rarely investigated. In this study, we (1) validate an enzyme-immunoassay to measure fecal cortisol metabolite (FCM) levels in North American red squirrels (Tamiasciurus hudsonicus), and (2) examine relationships between FCM levels and reproductive condition in a free-ranging red squirrel population. Injected radiolabeled cortisol was entirely metabolized and excreted in both the urine (mean ± SE; 70.3 ± 0.02%) and feces (29.7 ± 0.02%), with a lag time to peak excretion in the feces of 10.9 ± 2.3 h. Our antibody reacted with several cortisol metabolites, and an adrenocorticotropic injection significantly increased FCM levels above baseline levels at 8 h post-injection. Relative to baseline levels, manipulation by handling also tended to increase FCM levels at 8 h post-manipulation, but this difference was not significant. FCM levels did not differ significantly between samples frozen immediately and 5 h after collection. Reproductive condition significantly affected FCM levels in free-ranging females (pregnant > lactating > post-lactating > non-breeding) but not males (scrotal testes vs. abdominal testes). Among females with known parturition dates, FCM levels increased during gestation, peaked at parturition, and declined during lactation. The difference between pregnant and lactating females was therefore dependent upon when the fecal samples were obtained during these periods, suggesting caution in categorizing reproductive stages. This study demonstrates the utility of fecal hormone metabolite assays to document patterns of glucocorticoid levels in free-ranging animals.     

Treatment of familial hyperaldosteronism type I: only partial suppression of adrenocorticotropin required to correct hypertension

In familial hyperaldosteronism type I, inheritance of a hybrid 11beta-hydroxylase/aldosterone synthase gene leads to ACTH-regulated overproduction of aldosterone (causing hypertension) and of "hybrid" steroids, 18-hydroxy- and 18-oxo-cortisol. To determine whether complete suppression of the hybrid gene is necessary to normalize blood pressure, we sought evidence of persisting expression in eight patients who were rendered normotensive for 1.3-4.5 yr by glucocorticoid treatment. At the time of the study, six patients were receiving dexamethasone (0.125-0.25 mg/day) and two patients were taking prednisolone (2.5 or 5 mg/day). Urinary 18-oxo-cortisol levels during treatment demonstrated close correlation with mean "day curve" (blood collected every 2 h for 24 h) cortisol (r = 0.74), consistent with regulation by ACTH. Although urinary 18-oxo-cortisol levels were lower during than before treatment (mean 12.6 +/- 2.4 SEM vs. 35.0 +/- 5.6 nmol/mmol creatinine; P < 0.01), they remained above normal (0.8-5.2 nmol/mmol creatinine) in all eight patients. Although mean upright plasma potassium levels during treatment were higher, aldosterone levels lower, PRA levels higher, and aldosterone to PRA ratios lower than before treatment, PRA levels were uncorrected (< 13 pmol/L x min) and aldosterone to PRA ratios were uncorrected (>65) during treatment in four patients. For each of the eight patients, day curve aldosterone levels during treatment correlated more tightly with cortisol (mean r for the eight patients, 0.87 +/- 0.05 SEM) than with PRA (mean r = 0.36 +/- 0.10 SEM). Hence, control of hypertension by glucocorticoid treatment was associated, in all patients, with only partial suppression of ACTH-regulated hybrid steroid and aldosterone production. Normalization of urinary hybrid steroid levels and abolition of ACTH-regulated aldosterone production is not a requisite for hypertension control and, if used as a treatment goal, may unnecessarily increase the risk of Cushingoid side effects.     

The syndrome of apparent mineralocorticoid excess: its association with 11 beta-dehydrogenase and 5 beta-reductase deficiency and some consequences for corticosteroid metabolism

We describe the metabolism of cortisol (F) in three children, two of them siblings, with apparent mineralocorticoid excess (AME). As with prior patients with AME, oxidation of F to cortisone (E) was impaired, but reduction of E to F was not. We propose that this metabolic defect is caused by deficient 11-dehydrogenase associated with unimpaired 11-reductase. The following supporting observations were made: urinary C21 11-hydroxy metabolites exceeded C21 11-oxo metabolites: ratio of urinary cortols to cortolones, 6.6 +/- 2.8 (+/- SD; normal, 0.47); tetrahydrocortisol (THF) and alloTHF to tetrahydrocortisone, 14.6 +/- 5.6 (normal, approximately 1); normal subjects oxidized [11 alpha-3H]F with transfer of 3H to water; the patients did not; 11-hydroxy, but not 11-oxo, C19 steroids were excreted into the urine; and fibroblasts from patients had 5 times more 11-reductase activity than normal subjects, though fibroblasts from neither group had 11-dehydrogenase activity. Other defects of cortisol metabolism not directly associated with 11-dehydrogenase deficiency were found: impaired conversion of tetrahydro to hexahydro neutral steroids, indicating defective reductive metabolism of the side chain; depressed F production rate and increased half-life of circulating F, resulting in normal blood levels of F; increased excretion of unconjugated F metabolites; and decreased excretion of THF relative to alloTHF, consistent with a 5 beta-reductase defect. Excretion of acidic metabolites of F (cortoic acids) was within the normal range. However, little or no 20 beta-hydroxy acids were excreted, while the level of urinary 20 alpha-hydroxy acids was increased. The 11-hydroxy to 11-oxo ratio of acid metabolites was similar to values in normal subjects. The proportion of cortoic acids relative to neutral hexahydro metabolites was increased (0.37 to 1.27 in patients; 22 in normal subjects). We conclude that children with AME have multiple defects in the conversion of F to neutral metabolites, while metabolism to cortoic acids was less extensively affected. How the defects in cortisol metabolism and the symptoms of AME are related remains to be determined.     

Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice

Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.     

LC-MS analysis of androgen metabolites in serum and urine from east African chimpanzees (Pan troglodytes schweinfurthii)

Testosterone regulates a wide variety of behavioral and physiological traits in male vertebrates. It influences reproductive and aggressive behaviors and is used as a marker of gonadal activity. While testosterone is the primary biologically active male gonadal steroid in the blood, it is metabolized into a variety of related steroids when excreted via urine and feces. To monitor endocrinological profiles studies on wild-living animals primarily rely on non-invasively collected samples such as urine or feces. Since a number of androgen metabolites that are found in high concentrations in these matrices do not stem exclusively from gonadal production, but are also produced by the adrenal cortex, the metabolism and excretion pattern of testosterone and its characteristic metabolites have to be investigated. Here, we compare the levels of 11 androgens and their metabolites in serum and urine (after hydrolytic/solvolytic cleavage of conjugates) from female, and intact and castrated male chimpanzees to investigate whether they were of testicular or adrenal origin. For serum, significant differences in concentrations were found only for native testosterone. For urine, testosterone concentrations showed the largest differences between intact and castrated males, and intact males and females, while no differences were seen between females and castrated males. Epitestosterone levels revealed the same pattern. These differences in urinary concentrations could also be seen for 5α-androstane-3α,17β-diol (androstanediol), and less clearly for 5α-dihydrotestosterone (5α-DHT), etiocholanolone, and androsterone. In urine of males, significant correlations were found between the levels of testosterone and 5α-androstane-3α,17β-diol, as well as between testosterone and epitestosterone. Therefore, the clearest urinary markers of gonadal activity in male chimpanzees seems to be testosterone itself.     

Fecal and urinary excretion of six iodothyronines in the rat

Fecal and urinary excretion rates of six iodothyronines were assessed in the rat maintained under normal steady state physiological conditions, to gain a more comprehensive understanding of the mechanisms of control of normal thyroid hormone economy and metabolism. Groups of young adult male rats were injected with trace doses of T4, T3, rT3, 3,3'-diiodothyronine (T2), 3',5'-T2, or 3'-monoiodothyronine, each labeled with 125I, and feces and urine were collected separately for up to 10 days. Pooled fecal pellets were homogenized in saline, extracted in ethanol, evaporated under vacuum, and reconstituted in NaOH. Fecal extracts and urine were chromatographed on Sephadex G25 columns under conditions providing quantitative separations of components of interest. A new technique was also developed, based on a model of the in vitro extraction and measurement process, to correct chromatographic results for possible variable recoveries and possible artifactious degradation of radioactively labeled components. No iodothyronines or their conjugates were excreted in urine; all radioactivity was in the form of iodide. In feces, about 30% of the [125I]T3 injected was excreted as T3; and 24% of the [125I]T4 injected was excreted as T4, plus 4% as T3. Together, these results imply that about 24% of endogenous T4 production is excreted as T4 and 76% is irreversibly metabolized; and for T3, about 30% of endogenous T3 production is excreted as T3 and 70% is degraded. For the nonhormonal iodothyronines, about 6% of injected monoiodothyronine, 3% of injected 3',5'-T2, 2% of injected 3,3'-T2, and less than 1% of injected rT3 were excreted in feces as such, indicating that these substances are nearly completely deiodinated in vivo. Very little (1-7%) iodide was excreted as such in feces, which also were devoid of measurable conjugates. An open question is whether the substantial wastage of thyroid hormones in feces represents poor hormone economy in the usually accepted sense or a functional property of overall thyroid hormone regulation.     

Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. II. excretion of metabolites

Ecdysteroid (Ecd) excretion patterns were followed during the molt cycle of adult male and female lobsters. Homarus americanus. Urine was the major route of Ecd elimination, amounting to greater than or equal to 96% of the excreted radioimmunoassay activity for all molt stages. The other identified route of Ecd elimination from the hemolymph was the feces, which accounted for the remaining 4% of the total Ecd excretion. High polarity metabolites (HP), including 20,26-dihydroxyecdysone (2026E) and 20-hydroxyecdysonoic acid (20EA), were the major types of Ecds found in the urine. Other urinary Ecd components included 20-hydroxyecdysone (20E), ecdysone (E), and ponasterone A (P). The major portion of urinary HP was composed of conjugates of 2026E, 20E, E, P, and other unidentified metabolites. The fecal Ecds were predominately HP and apolar metabolites. Apolar fecal Ecds were hydrolyzable to release 20EA, 2026E, 20E, E, P, and other metabolites. By means of intubation, [3H]E was placed directly into the cardiac stomach of lobsters. The gut pathway formed an apolar conjugate of [3H]E which was found exclusively in the feces. Lobsters are therefore capable of excreting ingested Ecds without absorption.     

The Metabolism of 14 C-Disulfiram by the Rat

After administration of ¹⁴C-disulfiram to rats by stomach tube, we found that 87% of the radioactivity was excreted in urine and 7% in feces. Greater than 80% of the radioactivity was excreted by 48 hr. Small but measurable radioactivity was excreted in urine up to 144 hr after administration. Total recovery of radioactivity at 144 hr was 95% of the ingested dose with less than 1% in organs, blood, and carcass; the remainder was in urine and feces. Studies on specific radioactivity showed that diethylamine, a major urinary metabolite of disulfiram, is excreted in the urine undiluted with endogenous diethylamine. Pretreatment of rats with unlabeled disulfiram leads to a more rapid catabolism of the radioactive drug and more rapid excretion of radioactivity in the urine. Further, pretreatment appears to induce formation of a glucuronide conjugate of a disulfiram metabolite.