A Critical Review of Analytical Methods in Pharmaceutical Matrices for Determination of Corticosteroids.

Corticosteroids are a class of hormones released by the adrenal cortex, which includes glucocorticoids and mineralocorticoids. Glucocorticoids have an important role in the metabolism of carbohydrates, proteins and calcium and effective anti-inflammatory and immunosuppressive activity. Due to their intense immunomodulatory and anti-inflammatory activity, glucocorticoids are used in the treatment of various inflammatory, malignant, allergic conditions such as rhinitis, asthma, dermatological, rheumatic, ophthalmic and neurological diseases, as well as after organ transplants. They are the most widely prescribed drugs in the world. The objective of this review is to provide an overview of the analytical methods in pharmaceutical matrices for determination of corticosteroids. In this study, the predominance of liquid chromatography methods for the analysis of corticosteroids from pharmaceutical products is evident for both liquid and semisolid dosage forms as well as for solids. The same can be said for topical, oral and parenteral formulations. Methods such as spectrophotometry are also used, but given the advantages of chromatographic methods such as better selectivity and sensitivity, they have become the choice for analysis of these drugs, however, most methods still do not meet the credentials of "green chemistry."

Steroid Profiling and Immunohistochemistry for Subtyping and Outcome Prediction in Primary Aldosteronism-a Review.

PURPOSE OF REVIEW: Steroid profiling and immunohistochemistry are both promising new tools used to improve diagnostic accuracy in the work-up of primary aldosteronism (PA) and to predict treatment outcomes. Herein, we review the recent literature and present an outlook to the future of diagnostics and therapeutic decision-making in patients with PA. RECENT FINDING: PA is the most common endocrine cause of arterial hypertension and unilateral forms of the disease are potentially curable by surgical resection of the overactive adrenal. Recent studies have shown that adrenal steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be helpful for subtyping unilateral and bilateral forms of PA, classifying patients with a unilateral aldosterone-producing adenoma (APA) according to the presence of driver mutations of aldosterone production in APAs, and potentially predicting the outcomes of surgical treatment for unilateral PA. Following adrenalectomy, immunohistochemistry of aldosterone synthase (CYP11B2) in resected adrenals is a new tool to analyze "functional" histopathology and may be an indicator of biochemical outcomes after surgery. Biochemical and clinical outcomes of therapy in PA vary widely among patients. Peripheral venous steroid profiling at baseline could improve diagnostic accuracy and help in surgical decision-making in cases of a suspected APA; results of "functional" histopathology could help determine which patients are likely to need close post-surgical follow-up for persistent aldosteronism.

Determination of two progestin metabolites (17α-hydroxypregnanolone and pregnanediol) and different classes of steroids (androgens, estrogens, corticosteroids, progestins) in rivers and wastewaters by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

A highly sensitive and robust method was developed for routine analysis of two progestin metabolites, 17α-hydroxypregnanolone (17OH-Δ5P) and pregnanediol (PD), and 31 other natural and synthetic steroids and related metabolites (estrogens, androgens, corticosteroids, progestins) in river water, as well as influents and effluents of municipal wastewater treatment plants (WWTP) using HPLC-MS/MS combined with solid-phase extraction. For the various matrixes considered, the optimized method showed satisfactory performance with recoveries of 70-120% for most of target steroids. The method detection limits (MDLs) ranged from 0.01 to 3ng/L for river water, 0.02 to 10ng/L for WWTP effluents, and 0.1 to 40ng/L for influents with good linearity and reproducibility. The developed method was successfully applied for the analysis of steroids in rivers and WWTP influent and effluents. WWTP influents concentrations of 17OH-Δ5P and PD were 51-256ng/L and up to 400ng/L, respectively, along with androstenedione (concentration range: 38-220ng/L), testosterone (11-26ng/L), estrone (2.3-37ng/L), 17ß-estradiol (N.D.-8.7ng/L), 17α-hydroxyprogesterone (N.D.-66ng/L), medroxyprogesterone acetate (N.D.-5.3ng/L), and progesterone (2.0-22ng/L), while only androstenedione (ADD), estrone (E1), and estriol (E3) were detected in effluent with concentrations ranging up to 1.7ng/L, 0.90ng/L and 0.8ng/L, respectively. In river water samples, only ADD and E1 were detected with concentrations up to 1.0ng/L and 0.91ng/L. Our procedure represents the first method for analyzing 17OH-Δ5P and PD in environmental samples along with a large series of steroids.

Hormonal and behavioral variation in pied tamarins housed in different management conditions.

Pied tamarins are an endangered Amazonian primate that has limited breeding success in zoos. Unfortunately, little is known about their reproductive biology and adrenocortical activity. Objectives were: (1) determine if fecal hormones could be utilized to monitor gonadal and adrenocortical activity; (2) characterize male and female gonadal and adrenocortical hormones; and (3) determine if there were differences between adrenocortical activity and behavior in a nonbreeding, on-exhibit (NB-ON) pair compared to a breeding, off-exhibit (B-OFF) pair. Fecal samples were collected from four (two males; two females) individuals. Hormones were analyzed for fecal progesterone (FPM), androgen (FAM), and glucocorticoid (FGM) metabolites by enzyme immunoassay. Behavioral observations were conducted for 6 months. Data were collected on instantaneous behavior, location, and all occurrences of intraspecific behaviors. Fecal progesterone metabolites were validated by pregnancy (mean ± SE, pregnant: 28.47 ± 1.60 µg/g; nonpregnant: 8.63 ± 0.89 µg/g). Fecal androgen metabolites were higher (T = 31,971, P < 0.05) in the B-OFF male (863.66 ± 46.30 µg/g) than the NB-ON male (838.63 ± 60.70 µg/g). Fecal glucocorticoid metabolites were validated by response to veterinary procedure with elevated values (7.31 ± 1.48 µg/g) seven times the baseline (0.37 ± 0.04 µg/g) at 24-hr postphysical. Females had higher baseline FGM than the males (P < 0.05). Baseline FGM were higher (P < 0.05) in the NB-ON female (0.93 ± 0.03 µg/g) compared to the B-OFF female (0.38 ± 0.02 µg/g). Similarly, the NB-ON male's FGM baseline (0.71 ± 0.03 µg/g) were higher (P < 0.05) than the B-OFF male (0.21 ± 0.01 µg/g). Behavioral data revealed stereotypical behaviors in the NB-ON pair but no stereotypical behaviors in the B-OFF pair. Fecal hormone monitoring and behavioral analysis may provide insight on the limited breeding success of pied tamarins in zoos.

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas.

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September-November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March-August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat.

Steroid profiling by gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry for adrenal diseases.

The ability to measure steroid hormone concentrations in blood and urine specimens is central to the diagnosis and proper treatment of adrenal diseases. The traditional approach has been to assay each steroid hormone, precursor, or metabolite using individual aliquots of serum, each with a separate immunoassay. For complex diseases, such as congenital adrenal hyperplasia and adrenocortical cancer, in which the assay of several steroids is essential for management, this approach is time consuming and costly, in addition to using large amounts of serum. Gas chromatography/mass spectrometry profiling of steroid metabolites in urine has been employed for many years but only in a small number of specialized laboratories and suffers from slow throughput. The advent of commercial high-performance liquid chromatography instruments coupled to tandem mass spectrometers offers the potential for medium- to high-throughput profiling of serum steroids using small quantities of sample. Here, we review the physical principles of mass spectrometry, the instrumentation used for these techniques, the terminology used in this field and applications to steroid analysis.

Simultaneous determination of corticosteroids, androgens, and progesterone in river water by liquid chromatography-tandem mass spectrometry.

The assessment of steroidal hormones in the environment requires sensitive and selective analytical techniques suitable for sample matrices. This paper reports a simple method to analyze simultaneously six corticosteroids (triamcinolone, cortisol, dexamethasone, flumethasone, prednisolone, triamcinolone acetonide), four androgens (boldenone, epitestosterone, methyltestosterone, nortestosterone), and progesterone in river and drinking water sources. The developed method is based on a single solid-phase extraction (SPE) followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) with atmospheric pressure chemical ionization (APCI). The main advantage of this method over other methods includes the use of a single SPE with a low volume cartridge for sample preparation, separation of steroids on alkyl-amide stationary phase with no matrix interferences by LC-MS/MS analysis, and simultaneous analysis of more than two groups of steroids. The method was characterized by generally good performance, analyzing three groups of steroids using 100 and 1000mL samples with average recovery ranges of 80-109% and 68-126%, respectively at a level of 1ngL(-1). The limit of detection (LOD) ranged from 0.06 to 0.2 and 0.01-0.21ngL(-1) for 100mL and 1000mL samples volumes, respectively. Sixty samples of Danube River and drinking water sources from different regions of Hungary were collected to analyze target steroids in two sampling periods in 2008 and 2009. Steroids, except cortisol, dexamethasone, flumethasone, prednisolone, epitestosterone and progesterone were below detection limits. Endogenous steroids (cortisol, epitestosterone, progesterone) were present in the concentration range of 0.08-2.67ngL(-1) while synthetic corticosteroids (dexamethasone, flumethasone, and prednisolone) varied from 0.064 to 1.43ngL(-1). Steroids were present in river water, except progesterone, which was present only in ground water. Levels of steroids are compared with other rivers in the world and were briefly discussed.

Crescimento e composição corporal em crianças e adolescentes com síndrome nefrótica córtico-dependente / Growth and body composition in children and adolescents with steroid-dependent nephrotic syndrome

Objetivo: Avaliar crescimento e composição corporal em crianças e adolescentes com Síndrome Nefrótica Córtico-Dependente (SNCD). Material e Métodos: Foram incluídos todos os pacientes de 5 a 18 anos, em acompanhamento por pelo menos dois anos, com diagnóstico de SNCD. Foram coletados dados referentes a: tempo de tratamento, valores consecutivos do colesterol, albuminemia, proteinemia total, dose de uso corticoide e peso, estatura e idade da primeira consulta. As avaliações antropométricas dobra cutãnea tricipital e subescapular, índice de massa corpórea, circunferência da cintura e z-escore de estatura/idade foram realizadas durante as consultas de rotina e realizadas somente quando se considerou a criança sem edema clinicamente visível. Estatística não paramétrica com p<0,05. Resultados: Foram estudados 18 pacientes, 11 do sexo masculino (61,1%), idade entre 6 e 16 anos (12,2 +-2,98), tempo médio de tratamento de 6,75 +- 3,75 anos. os valores iniciais do z-escore foram significativamente maiores do que os finais (-0,69 +- 0,80 e de -2,07 +- 1,61; p=0,003). A evolução individual do z-escore mostrou que houve diminuição em 14 (-1,37 +- 1,55) e manutenção dos valores em quatro pacientes. Comparando-se vários parâmetros que poderiam ser responsáveis pela diferença de evolução, somente a proteinúria residual foi significativamente diferente. A medida da circunferência muscular do braço foi significativamente menor no grupo com perda de z-escore. Conclusões: Foi observado na maioria dos pacientes, déficit de estatura e diminuição da massa magra, provavelmente associados à gravidade do quadro nefrótico, que necessitou de doses elevadas e prolongadas de corticóide.

Objective: To evaluate growth and body composition in children and adolescents with steroid-dependent nephrotic syndrome (SNCD). Methods: We included all patients 5-18 years, accompanied by at least two years, diagnosed with SNCD. Data were collected regarding the following: duration of treatment, consecutive values of cholesterol, albumin, total serum protein, dose of corticosteroid use and weight, height and age at first consultation. The anthropometric assessments triceps and subscapular, body mass index, waist circumference, and z-score height / age were performed during routine consultations and carried out only when they saw the child without edema clinically visible. Nonparametric statistical p <0.05. Results: We studied 18 patients, 11 male (61.1%), aged 6 to 16 years (12.2 + -2.98), mean treatment time of 6.75 + - 3.75 years. the initial values of z-scores were significantly higher than the final (-0.69 + - 0.80 and -2.07 + - 1.61, p = 0.003). The individual evolution of z-scores showed that there was a decrease in 14 (-1.37 + - 1.55) and maintenance of values in four patients. Comparing the various parameters that could account for the difference in evolution, only the residual proteinuria was significantly different. The measurement of arm muscle circumference was significantly lower in the group with loss of z-scores. Conclusions: We found in most patients, stunting and reduction in lean body mass, probably associated with the severity of the nephrotic, which required high doses and prolonged corticosteroid.

Effects of pentoxifylline on TNF-alpha and lung histopathology in HCL-induced lung injury.

OBJECTIVE: To evaluate the effects of pentoxifylline on hydrochloric acid-induced lung lesions in rats subjected to mechanical ventilation. METHODS: Twenty male, adult Wistar-EPM-1 rats were anesthetized and randomly grouped (n=5 animals per group) as follows: control-MV (mechanical ventilation, MV group); bilateral instillation of HCl (HCl group); bilateral instillation of HCl followed by pentoxifylline (50 mg/kg bw) infusion (HCl+PTX group) and pentoxifylline infusion followed by bilateral instillation of HCl (PTX+HCl group). At 20, 30, 90 and 180 min after treatments, the blood partial pressures of CO2 and O2 were measured. The animals were euthanized, and bronchoalveolar lavages were taken to determine the contents of total proteins, corticosterone [corrected] and TNF-alpha. Samples of lung tissue were used for histomorphometric studies and determining the wet-to-dry (W/D) lung weight ratio. RESULTS: In the MV group, rats had alveolar septal congestion, and, in the HCl group, a remarkable recruitment of neutrophils and macrophages into the alveoli was noticed; these events were reduced in the animals with PTX+HCl. The partial pressure of oxygen increased in PTX+HCl animals (121+/-5 mmHg) as compared with the HCl (62+/-6 mmHg) and HCl+PTX (67+/-3 mmHg) groups within 30 minutes. TNF-alpha levels in bronchoalveolar lavage were significantly higher in the HCl group (458+/-50 pg/mL), reduced in the HCl+PTX group (329+/-45 pg/mL) and lowest in the PTX+HCl group (229+/-41 pg/mL). The levels of corticosterone [corrected] in bronchoalveolar lavage were significantly lower in the HCl (8+/-1.3 ng/mL) and HCl+PTX group (16+/-2 ng/mL) and were highest in the PTX+HCl (27+/-1.9 ng/mL). CONCLUSION: Pretreatment with PTX improves oxygenation, reduces TNF-alpha concentration and increases the concentration of corticosterone [corrected] in bronchoalveolar lavage upon lung lesion induced by HCl.

Validação laboratorial e fisiológica de conjunto comercial para a quantificação de corticóides fecais em chimpanzé (Pan troglodytes) e orangotango (Pongo pygmaeus), cativos e submetidos a enriquecimentos ambientais / Laboratorial and physiological validation of comercial kits for quantification of fecal corticoids of captive chimpanzees (Pan troglodytes) and orangutangs (Pongo pygmaeus) under environmental enriched conditions

Neste trabalho foi realizado estudo comparativo dos níveis de corticóides fecais (CF) de chimpanzé (Pan troglodytes) e orangotango(Pongo pygmaeus). Foram analisadas amostras coletadas em duas fases distintas, relacionadas com a introdução de técnicas de enriquecimento ambiental, a saber: Base (antes da introdução) e Habituação(imediatamente após). Realizamos as validações do conjunto comercial para radioimunoensaio ImmunuChem™ Double Antibody Corticosterone da MP Biomedicals, para mensuração de CF. A validação laboratorial dos conjuntos diagnósticos para uso em extrato fecal de primatas foi realizada pelo método de paralelismo, no qual, para cada espécie, concentrações conhecidas de corticosterona foram adicionadas a um pool de extratos fecais, sendo estas amostras analisadas em seguida. As inclinações das curvas obtidas nestes ensaios e da curva padrão do ensaio foram então comparadas. Os resultados obtidos para chimpanzé e orangotango, foram respectivamente, Y=17,23+1,31*X;R...

A comparative study of fecal corticoids (FC) concentrations was carried out with chimpanzees (Pan troglodytes) e orangutans (Pongo pygmaeus). Fecal samples were collected before (Basal) and just after (Habituation)enrichment introduction and analyzed. We performed biochemical and physiological validations of the ImmunuChem™ Double Antibody Corticosterone kit for radioimmunoassay from MP Biomedicals for quantifying FC concentrations. To establish the biochemical validity of our assay we performed parallelism assays in which pooled fecal extracts from both species were spiked with known quantities of corticosterone standard and the slopes of the curves obtained with these samples and the standard curves of the kits were compared. The correlation coefficients were R

Effects of pentoxifylline on TNF-alpha and lung histopathology in HCL-induced lung injury

OBJECTIVE: To evaluate the effects of pentoxifylline on hydrochloric acid-induced lung lesions in rats subjected to mechanical ventilation. METHODS: Twenty male, adult Wistar-EPM-1 rats were anesthetized and randomly grouped (n=5 animals per group) as follows: control-MV (mechanical ventilation, MV group); bilateral instillation of HCl (HCl group); bilateral instillation of HCl followed by pentoxifylline (50 mg/kg bw) infusion (HCl+PTX group) and pentoxifylline infusion followed by bilateral instillation of HCl (PTX+HCl group). At 20, 30, 90 and 180 min after treatments, the blood partial pressures of CO2 and O2 were measured. The animals were euthanized, and bronchoalveolar lavages were taken to determine the contents of total proteins, corticosteroid and TNF-alpha. Samples of lung tissue were used for histomorphometric studies and determining the wet-to-dry (W/D) lung weight ratio. RESULTS: In the MV group, rats had alveolar septal congestion, and, in the HCl group, a remarkable recruitment of neutrophils and macrophages into the alveoli was noticed; these events were reduced in the animals with PTX+HCl. The partial pressure of oxygen increased in PTX+HCl animals (121±5 mmHg) as compared with the HCl (62±6 mmHg) and HCl+PTX (67±3 mmHg) groups within 30 minutes. TNF-alpha levels in bronchoalveolar lavage were significantly higher in the HCl group (458±50 pg/mL), reduced in the HCl+PTX group (329±45 pg/mL) and lowest in the PTX+HCl group (229±41 pg/mL). The levels of corticosteroid in bronchoalveolar lavage were significantly lower in the HCl (8±1.3 ng/mL) and HCl+PTX group (16±2 ng/mL) and were highest in the PTX+HCl (27±1.9 ng/mL). CONCLUSION: Pretreatment with PTX improves oxygenation, reduces TNF-alpha concentration and increases the concentration of corticosteroid in bronchoalveolar lavage upon lung lesion induced by HCl.

Expression of molecular equivalent of hypothalamic-pituitary-adrenal axis in adult retinal pigment epithelium.

We have investigated expression of molecular elements of the hypothalamic-pituitary-adrenal (HPA) axis in the human retinal pigment epithelium (RPE) cells. The presence of corticotropin-releasing factor (CRF); urocortins I, II and III; CRF receptor type 1 (CRFR1); POMC and prohormone convertases 1 and 2 (PC1 and PC2) mRNAs were shown by RT-PCR; the protein products were detected by ELISA, western blot or immunocytochemical methods in an ARPE-19 cell line derived from an adult human donor. CRFR2 was below the level of detectability. The CRFR1 was functional as evidenced by CRF stimulation of cAMP and inositol triphosphate production as well as by ligand induction of transcriptional activity of inducible cis-elements cAMP responsive element (CRE), activator protein 1 responsive element (AP-1) and POMC promoter) in ARPE-19 using luciferase reporter assay. Immunoreactivities representative of CRF, pre-urocortin, CRFR1 receptor and ACTH were also detected in mouse retina by in situ immunocytochemistry. Finally, using RT-PCR, we detected expression of genes encoding four key enzymes participating in steroids synthesis (CYP11A1, CYP11B1, CYP17 and CYP21A2) and showed transformation of progesterone into cortisol-immunoreactivity in cultured ARPE-19 cells. Therefore, we suggest that ocular tissue expresses CRF-driven signalling system that follows organisational structure of the HPA axis.

Multi-analyte approach for the determination of ng L(-1) levels of steroid hormones in unidentified aqueous samples.

Since the 1970s, many analytical methods for the detection of illegal growth promoters, such as thyreostats, anabolics, beta-agonists and corticosteroids have been developed for a wide range of matrices of animal origin, including meat, fat, organ tissue, urine and faeces. The aim of this study was to develop an analytical method for the determination of ng L(-1) levels of estrogens, gestagens, androgens (EGAs) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements), commercially available on the 'black' market. For this, extraction was performed with Bakerbond C18 speedisk, a technique commonly used in environmental analysis. After fractionation, four fractions were collected using a methanol:water gradient program. Gas chromatography coupled to electron impact multiple mass spectrometry (GC-EI-MS2) screening for the EGAs was carried out on the derivatized extracts. For the detection of corticosteroids, gas chromatography coupled to negative chemical ionization mass spectrometry (GC-NCI-MS) was used after oxidation of the extracts. Confirmation was done by liquid chromatography coupled to electrospray ionization multiple mass spectrometry (LC-ESI-MS2). The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ng L(-1) level. This study demonstrated the occurrence of both androgens and corticosteroids in different commercial aqueous samples.

High-performance liquid chromatography for hormone assay.

High-performance liquid chromatography (HPLC) is a refinement of traditional column chromatographic techniques. The speed of analysis and the resolution are increased with new column-packing materials and eluant pumped through the column at high pressure. The potential for achieving measurements of hormones in small volumes of plasma or urine is limited, both in normal and pathological situations. Using HPLC with ultraviolet absorption, the detection limit is only nanogram amounts of hormones per milliliter of blood serum. The applications of the technique to specific hormones from recent and older literature will be used throughout this chapter to illustrate aspects of the technology.

Gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry (GC-MS) has been used in hormone assays particularly for steroids in biological fluids. The combination of GC with MS exploits the high-resolving power of gas chromatography to separate closely related molecules, and the ability of the MS to provide precise data for identification and quantification of the separated substances. GC-MS is a very powerful technique for analysis with specificity of hormones in biological fluids. The general principles of GC-MS are described in this chapter along with some examples that illustrate specific applications of hormone analysis.

Uso antenatal de corticosteróide e evolução clínica de recém-nascidos pré-termo / Antenatal corticosteroid use and clinical evolution of preterm newborn infants

O objetivo é descrever a freqüência de utilização de corticosteróide antenatal e a evolução clínica dos recém-nascidos pré-termo. Estudo observacional prospectivo tipo coorte de todos os neonatos com idade gestacional entre 23 e 34 semanas nacidos na Rede Brasileira de Pesquisas Neonatais entre agosto e dezembro de 2001. Os prontuários médicos foram revistos, as mães entrevistadas e os pré-termos acompanhados. A análise dos dados foi realizada com o teste do qui-quadrado, t de Student, Mann-Whitney, ANOVA e regressão logística múltipla, com nível de significância de 5%.

Non-invasive faecal steroid monitoring of ovarian and adrenal activity in farmed blue fox (Alopex lagopus) females during late pregnancy, parturition and lactation onset.

In the semi-domesticated blue fox, handling stress may influence reproductive performance and increase perinatal pup loss. Ovarian and adrenal steroids were analysed in faecal samples collected from mid-gestation through the first week of lactation in 40 female blue foxes to characterize hormone patterns during this important reproductive period. Daily faecal samples were collected from 40 foxes during 30 pregnancies, one late abortion and nine bred-matched non-pregnancies. Mean concentrations of faecal progestagens over the 10 days before birth were significantly higher in pregnant compared to non-pregnant females (51+/-1.50 microg/g versus 36+/-3.72 microg/g, respectively; P < 0.01). From 10 to 3 days before whelping, total faecal oestrogen concentrations also were higher (P < 0.01) in pregnant (1082+/-41.69 ng/g) than non-pregnant (628+/-72.43 ng/g) foxes, before declining to non-pregnant values (402+/-24.88 ng/g) after parturition. Overall mean faecal corticoid concentrations from 3 to 20 days before whelping differed between pregnant and non-pregnant foxes (128+/-3.11 ng/g versus 103+/-5.86 ng/g, respectively; P < 0.01). Furthermore, in pregnant foxes, corticoid excretion increased further from 2 days before to 3 days after whelping (216+/-13.71 ng/g; P < 0.01). Thereafter, corticoid concentrations were similar between pregnant and non-pregnant females (P > 0.05). In sum, the faecal steroid hormone patterns for oestrogens and progestagens were similar to those previously obtained by analyses of fox serum hormones, with both steroids being higher in pregnant than non-pregnant foxes at the end of gestation. The elevation in corticoid concentrations in pregnant females suggests that adrenal activation is involved in the initiation of parturition in the blue fox. Thus, faecal steroid analyses can be used to monitor ovarian activity during pregnancy and pseudopregnancy in farmed blue fox females.

Determination of corticosteroids in tissue samples by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of corticosterone and 11-dehydrocorticosterone (11-DHC) levels in KKA(y) mouse liver and adipose tissue, and hydrocortisone and cortisone levels in human adipose tissue has been developed. The corticosteroids were extracted from liver tissue with methanol/water and ethyl acetate for adipose tissue samples. Corticosterone and 11-DHC were separated with a methanol gradient and hydrocortisone and cortisone with an acetonitrile gradient containing trifluoroacetic acid on a reversed-phase column within 15 min. The corticosteroids were detected after electrospray ionization in positive mode with multiple reaction monitoring (MRM). The limits of quantification (LOQ) were estimated to be 15 nmol/kg liver and 1.6 nmol/kg adipose tissue for corticosterone and 5.4 nmol/kg liver and 0.92 nmol/kg adipose tissue for 11-DHC. The LOQ was estimated to be 0.2 nmol/kg adipose tissue for hydrocortisone and 0.4 nmol/kg adipose tissue for cortisone. The limits of detection (LOD) at 3 times S/N were estimated to be 0.07 nmol/kg adipose tissue for hydrocortisone 0.1 nmol/kg adipose tissue for cortisone. The variation of endogenous levels in KKA(y) mouse from different animals (CV%) was high with mean liver tissue levels of 117+/-25 (S.D.)nmol/kg for corticosterone and 62+/-19 (S.D.)nmol/kg for 11-DHC (n=5) and adipose tissue levels of 39+/-20 (S.D.)nmol/kg for corticosterone and 2.4+/-0.9 (S.D.)nmol/kg for 11-DHC (n=9). Endogenous levels in human biopsy samples from adipose tissue were 12+/-7.0 (S.D.)nmol/kg for hydrocortisone and 3.0+/-1.6 (S.D.)nmol/kg for cortisone (n=16). The new LC-MS/MS methods showed sufficient sensitivity and selectivity for determination of endogenous levels of corticosteroids in both KKA(y) mouse liver and adipose tissue samples and human adipose tissue samples. The selectivity of the methods was verified by analysis of two different productions from each analyte.

The relationship between vaginal electrical impedence and hormone profiles during pregnancy and parturition of a white rhinoceros (Ceratotherium simum simum).

In this study, an attempt was made to use vaginal electrical impedance to predict calving in a female white rhinoceros (Ceratotherium simum simum) and to determine the relationship between vaginal electrical impedance and hormonal profiles during pregnancy. The principle behind vaginal electrical impedance is that a change in the ionic balance of vaginal and cervical mucus occurs in response to changes in reproductive hormones. Three times weekly vaginal electrical impedance readings and fecal samples were collected from midgestation to calving (a 6-mo period). The extracted fecal samples were analyzed for immunoreactive estrogens, progestagens, and corticoids by RIA. Vaginal electrical impedance readings did not decrease before calving but remained consistent throughout the last 140 days of pregnancy. Fecal progestagens in the white rhinoceros decreased between day 17 and day 1 before calving, whereas estrogens increased between 4 and 2 mo before calving, with an additional increase occurring 1 mo before calving. Fecal corticoids increased 5 mo before calving, slowly declined, and increased again within 3 wk before calving. A decline in vaginal electrical impedance was noted 168 days before calving and remained at low levels for 4 wk. At the time of this decrease, the female became aggressive toward the male and began lactating. Fecal progestagens and estrogens did not change during this time; however, fecal corticoids increased as vaginal electrical impedance readings returned to normal along with her behavior and cessation of lactation. In summary, the use of vaginal electrical impedance could not predict parturition in the white rhinoceros. However, an anomaly occurred during pregnancy that was supported by vaginal electrical impedance readings, a change in female behavior, premature lactation, and a subsequent increase in fecal corticoids. The etiology of this physiological anomaly is unknown, yet it did not compromise pregnancy.