Human and murine steroid 5ß-reductases (AKR1D1 and AKR1D4): insights into the role of the catalytic glutamic acid.

Mammalian steroid 5ß-reductases belong to the Aldo-Keto Reductase 1D sub-family and are essential for the formation of A-ring 5ß-reduced steroids. Steroid 5ß-reduction is required for the biosynthesis of bile-acids and the metabolism of all steroid hormones that contain a Δ4-3-ketosteroid functionally to yield the 5ß-reduced metabolites. In mammalian AKR1D enzymes the conserved catalytic tetrad found in all AKRs (Y55, H117, K84 and D50) has changed in that the conserved H117 is replaced with a glutamic acid (E120). E120 may act as a "superacid" to facilitate enolization of the Δ4-ketosteroid. In addition, the absence of the bulky imidazole side chain of histidine in E120 permits the steroid to penetrate deeper into the active site so that hydride transfer can occur to the steroid C5 position. In murine steroid 5ß-reductase AKR1D4, we find that there is a long-form, with an 18 amino-acid extension at the N-terminus (AKR1D4L) and a short-form (AKR1D4S), where the latter is recognized as AKR1D4 by the major data-bases. Both enzymes were purified to homogeneity and product profiling was performed. With progesterone and cortisol, AKR1D4L and AKR1D4S catalyzed smooth conversion to the 5ß-dihydrosteroids. However, with Δ4-androstene-3,17-dione as substrate, a mixture of products was observed which included, 5ß-androstane-3,17-dione (expected) but 3α-hydroxy-5ß- androstan-17-one was also formed. The latter compound was distinguished from its isomeric 3ß-hydroxy-5ß-androstan-17-one by forming picolinic acid derivatives followed by LC-MS. These data show that AKR1D4L and AKR1D4S also act as 3α-hydroxysteroid dehydrogenases when presented with Δ4-androstene-3,17-dione and suggest that E120 alters the position the steroid to enable a correct trajectory for hydride transfer and may not act as a "superacid".

Effect of Heat Stress on Concentrations of Faecal Cortisol Metabolites in Dairy Cows.

The negative impact of heat stress on health and productivity of dairy cows is well known. Heat stress can be quantified with the temperature-humidity index (THI) and is defined as a THI ≥ 72. Additionally, animal welfare is affected in cows living under heat stress conditions. Finding a way to quantify heat stress in dairy cows has been of increasing interest over the past decades. Therefore, the objective of this study was to evaluate concentrations of faecal glucocorticoid metabolites [i.e. 11,17-dioxoandrostanes (11,17-DOA)] as an indirect stress parameter in dairy cows without heat stress (DOA 0), with heat stress on a single day (acute heat stress, DOA 1) or with more than a single day of heat stress (chronic heat stress, DOA 2). Cows were housed in five farms under moderate European climates. Two statistical approaches (approach 1 and approach 2) were assessed. Using approach 1, concentrations of faecal 11,17-DOA were compared among DOA 0, DOA 1 and DOA 2 samples regardless of their origin (i.e. cow, unpaired comparison with a one-way anova). Using approach 2, a cow was considered as its own control; that is 11,17-DOA was treated as a cow-specific factor and only paired samples were included in the analysis for this approach (paired comparison with t-tests). In approach 1 (p = 0.006) and approach 2 (p = 0.038), 11,17-DOA values of cows under acute heat stress were higher compared to those of cows without heat stress. Our results also indicate that acute heat stress has to be considered as a confounder in studies measuring faecal glucocorticoid metabolites in cows to evaluate other stressful situations.

Matrix effect during the membrane-assisted solvent extraction coupled to liquid chromatography tandem mass spectrometry for the determination of a variety of endocrine disrupting compounds in wastewater.

Membrane-assisted solvent extraction (MASE) coupled to liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) was studied for the determination of a variety of emerging and priority compounds in wastewater. Among the target analytes studied certain hormones (estrone (E1), 17ß-estradiol (E2), androsterone (ADT), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES), equilin (EQ), testosterone (TT), mestranol (MeEE2), 19-norethisterone (NT), progesterone (PG) and equilenin (EQN)), alkylphenols (APs) (4-tert-octylphenol (4tOP), nonylphenol technical mixture (NPs) and 4n-octylphenol (4nOP)) and BPA were included. The work was primarily focused in the LC-MS/MS detection step, both in terms of variable optimization and with respect to the matrix effect study. Both, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were assessed both in the negative and positive mode, including the optimization of MS/MS operating conditions. The best results were obtained, in most of the cases, for ESI using 0.05% ammonium hydroxide as buffer solution in the mobile phase, composed with methanol and water. Under optimum detection conditions, matrix effect during the detection step was thoroughly studied. Dilution, correction with deuterated analogues and clean-up of the extracts were evaluated for matrix effect correction. Clean-up with Florisil together with correction with deuterated analogues provided the most satisfactory results, with apparent recoveries in the 57-136% range and method detection limits in the low ngL(-1) level for most of the analytes. For further validation of the method, two separated extraction procedures, the above mentioned MASE, and conventional solid phase extraction (SPE) were compared during the analysis of real samples and comparable results were successfully obtained for E1, E2, EE2, DES, NT, TT, EQ, PG, BPA, ADT, 4nOP, 4tOP, NPs and EQN.

Nuclear and mitochondrial multilocus phylogeny and survey of alkaloid content in true salamanders of the genus Salamandra (Salamandridae).

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae.

Measurement of fecal glucocorticoid metabolites and evaluation of udder characteristics to estimate stress after sudden dry-off in dairy cows with different milk yields.

Sudden dry-off is an established management practice in the dairy industry. But milk yield has been increasing continuously during the last decades. There is no information whether the dry-off procedure, which often results in swollen and firm udders, causes stress, particularly in high-producing dairy cows. Therefore, we evaluated the effect of a sudden dry-off on extramammary udder pressure and the concentration of fecal glucocorticoid metabolites (i.e., 11,17-dioxoandrostane, 11,17-DOA) as an indirect stress parameter. Measurements were carried out within the last week before dry-off and until 9d after dry-off considering 3 groups of milk yield (i.e., low: <15 kg/d, medium: 15-20 kg/d, and high: >20 kg/d). Udder pressure increased in all yield groups after dry-off, peaked at d 2 after dry-off and decreased afterwards. Pressures were highest in high-yielding cows and lowest in low-yielding cows. But only in high-yielding cows was udder pressure after dry-off higher than before dry-off. Baseline 11,17-DOA concentrations depended on milk yield. They were highest in low-yielding (121.7 ± 33.3 ng/g) and lowest in high-yielding cows (71.1 ± 30.0 ng/g). After dry-off, 11,17-DOA increased in all yield groups and peaked at d 3. Whereas in medium- and high-yielding cows 11,17-DOA levels differed significantly from their respective baseline during the whole 9-d measuring period, low-yielding cows showed elevated 11,17-DOA levels only on d 3 after dry-off. However, especially the increase in 11,17-DOA after dry-off between the 3 yield groups was considerably different. Mean 11,17-DOA increase from baseline to d 3 was highest in high-yielding cows (129.1%) and considerably lower in low-yielding cows (40.1%). The highest fecal 11,17-DOA concentrations were measured on d 3 after dry-off, indicating that the stress was most intense on d 2, which is due to an 18-h time lag; at about the same time, udder pressure peaked. Our results showed a negligible effect of a sudden dry-off on low-yielding cows. High-yielding cows, however, faced high extramammary pressures and increased glucocorticoid production. Considering animal welfare aspects, a review of the current dry-off strategies might be warranted.

Simultaneous determination of estrogenic and androgenic hormones in water by isotope dilution gas chromatography-tandem mass spectrometry.

A rapid gas chromatography-tandem mass spectrometry (GC-MS/MS) analytical method was developed for the simultaneous analysis of 7 estrogenic hormones (17α-estradiol, 17ß-estradiol, estrone, mestranol, 17α-ethynylestradiol, levonorgestrel, estriol) and 5 androgenic hormones (testosterone, androsterone, etiocholanolone, dihydrotestosterone, androstenedione) in aqueous matrices. This method is unique in its inclusion of all 12 of these estrogens and androgens and is of particular value due to its very short chromatographic run time of 15 min. The use of isotope dilution for all analytes ensures the accurate quantification, accounting for analytical variabilities that may be introduced during sample processing and instrumental analysis. Direct isotopically labelled analogues were used for 8 of the 12 hormones and satisfactory isotope standards were identified for the remaining 4 hormones. Method detection levels (MDLs) were determined to describe analyte concentrations sufficient to provide a signal with 99% certainty of detection. The established MDLs for most analytes were 1-5 ngL(-1) in a variety of aqueous matrices. However, slightly higher MDLs were observed for etiocholanolone, androstenedione, testosterone, levonorgestrel and dihydrotestosterone in some aqueous matrices. Sample matrices were observed to have only a minor impact on MDLs and the method validation confirmed satisfactory method stability over intra-day and inter-day analyses of surface water and tertiary treated effluent samples.

Application of high-performance liquid chromatography with amperometric and coulometric detection to the analysis of SZ1677, a new neuromuscular blocking agent, and its two derivatives.

A HPLC method with amperometric and coulometric detection for the determination of SZ1677 and its two derivatives SZ1676 and SZ1823 has been developed. This active substance is under development (clinical trial) and there are no analytical methods published for the determination of SZ1677 thus far. The limit of quantitation for SZ1677 was 25 and 100 ng ml(-1) by the coulometric and amperometric detection, respectively. The elaborate method for the simultaneous analysis of SZ1677 and its two derivatives proved to be fast, precise, accurate and sensitive.

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis).

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.

Steroid hormone related male biased parasitism in chamois, Rupicapra rupicapra rupicapra.

Parasites are linked with their host in a trophic interaction with implications for both hosts and parasites. Interaction stretches from the host's immune response to the structuring of communities and the evolution of biodiversity. As in many species sex determines life history strategy, response to parasites may be sex-specific. Males of vertebrate species tend to exhibit higher rates of parasites than females. Sex-associated hormones may influence immunocompetence and are hypothesised to lead to this bias. In a field study, we tested the prediction of male biased parasitism (MBP) in free ranging chamois (Rupicapra rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminths. We further investigated sex differences in faecal androgen (testosterone and epiandrosterone), cortisol and oestrogen metabolites using enzyme immunoassays (EIA) to evaluate the impact of these hormones on sex dependent parasite susceptibility. Non-invasive methods were used and the study was conducted throughout a year to detect seasonal patterns. Hormone levels and parasite counts varied significantly throughout the year. Male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. The hypothesis of MBP originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. The faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. Our study shows that sex differences in steroid levels play an important role to explain MBP, although they alone cannot fully explain the phenomenon.

Mass spectrometric analysis of androstan-17beta-ol-3-one and androstadiene-17beta-ol-3-one isomers.

Mass spectrometric identification and characterization of steroids using electrospray ionization and tandem mass spectrometry has advantages in drug testing and doping control analysis attributable to limitations of gas chromatography followed by electron ionization mass spectrometry. Steroids with an androstadiene-17beta-ol-3-one nucleus and double bonds located either at C-1 and C-4, C-4 and C-9, or C-4 and C-6 were used to determine characteristic fragmentation pathways. Diagnostic dissociation routes are proposed using deuterium labeling, MS3 experiments, and analyses of structurally closely related compounds. Steroids such as boldenone (androst-1,4-diene-17beta-ol-3-one) produced characteristic product ions at m/z 121, 135, and 147. Compounds with double bonds at C-4 and C-9 generated abundant product ions at m/z 145 and 147. Conjugated double bonds at C-4 and C-6 gave rise to an intense and characteristic signal at m/z 133. Stereochemical differentiation between 5alpha- and 5beta-isomers of androstan-17beta-ol-3-ones was possible because of significant differences in relative abundance of product ions generated by elimination of acetone from alpha,beta-saturated 3-keto steroids.

Surface activity, lipid profiles and their implications in cervical cancer.

BACKGROUND: The profiles of lipids in normal and cancerous tissues may differ revealing information about cancer development and progression. Lipids being surface active, changes in lipid profiles can manifest as altered surface activity profiles. Langmuir monolayers offer a convenient model for evaluating surface activity of biological membranes. AIMS: The aims of this study were to quantify phospholipids and their effects on surface activity of normal and cancerous human cervical tissues as well as to evaluate the role of phosphatidylcholine (PC) and sphingomyelin (SM) in cervical cancer using Langmuir monolayers. METHODS AND MATERIALS: Lipid quantification was done using thin layer chromatography and phosphorus assay. Surface activity was evaluated using Langmuir monolayers. Monolayers were formed on the surface of deionized water by spreading tissue organic phase corresponding to 1 mg of tissue and studying their surface pressure-area isotherms at body temperature. The PC and SM contents of cancerous human cervical tissues were higher than those of the normal human cervical tissues. Role of PC and SM were evaluated by adding varying amounts of these lipids to normal cervical pooled organic phase. STATISTICAL ANALYSIS: Student's t-test (p < 0.05) and one-way analysis of variance (ANOVA) was used. RESULTS: Our results reveals that the phosphatidylglycerol level in cancerous cervical tissue was nearly five folds higher than that in normal cervical tissue. Also PC and sphingomyelin SM were found to be the major phospholipid components in cancerous and normal cervical tissues respectively. The addition of either 1.5 microg DPPC or 0.5 microg SM /mg of tissue to the normal organic phase changed its surface activity profile to that of the cancerous tissues. Statistically significant surface activity parameters showed that PC and SM have remarkable roles in shifting the normal cervical lipophilic surface activity towards that of cancerous lipophilic component. CONCLUSION: The Langmuir monolayer technique was sensitive to detect changes in tensiometric profiles of cervical cancers and these could be modulated by alterations in phosphatidylcholine and sphingomyelin levels. Therapeutic strategies may be designed to modulate these tensiometric profiles and lipid constituents of cancerous tissues.

Anabolic steroids in sport: biochemical, clinical and analytical perspectives.

International Olympic Committee accredited laboratories play a key role in upholding the principle of fair play and innate ability, as desired by the majority of sports competitors and spectators. Not only does doping damage the image of sport, but it can also be harmful to the individual. The great majority of samples test negative but, when an adverse finding is declared, the analytical data must be of a sufficiently high standard to withstand legal challenges by third parties. The most widely misused performance-enhancing drugs are the anabolic-androgenic steroids, commonly referred to as 'anabolic steroids'. This review attempts to address the complex issues concerning anabolic steroids in sport by considering the clinical, biochemical and analytical perspectives.

Fecal glucocorticoid metabolites as a measure of adrenal activity in dairy cattle.

The feasibility of monitoring acute adrenal activity in New Zealand dairy cattle by measuring fecal glucocorticoid metabolites was investigated. Fecal glucocorticoid measurement has potential as an indicator of adrenal activity and animal stress because sampling is relatively noninvasive, does not interfere with the stress response itself, and permits on-farm monitoring. Fecal samples were collected from dairy cattle following ACTH challenge and exposure to stressors (novel environment, transport). Two immunoassays (11,17-dioxoandrostane enzymeimmunoassay and ICN corticosterone radioimmunoassay) were compared. Both assays detected increased immunoreactive fecal glucocorticoid metabolites following acute adrenal activity and the temporal relationship between plasma corticosteroids and fecal metabolite excretion was determined. The time to peak excretion was closely related to the transit time of digesta passing between the bile duct and the rectum and was affected by seasonal changes in feed intake and pasture digestibility. We conclude that measurement of glucocorticoid metabolites reliably indicates acute adrenal activity in dairy cattle and in combination with other physiological and behavioral measures has potential for monitoring health and welfare in dairy cattle. (c) 2002 Elsevier Science (USA).

Noninvasive monitoring of adrenocortical activity in roe deer (Capreolus capreolus) by measurement of fecal cortisol metabolites.

A method for measuring glucocorticoids noninvasively in feces of roe deer was established and validated. The enzyme immunoassay (EIA) measures 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites. Such measurement avoids blood sampling and reflects a dampened pattern of diurnal glucocorticoid secretion, providing an integrated measure of adrenocortical activity. After high-performance liquid chromatography, the presence of at least three different immunoreactive 11,17-DOA in the feces of roe deer was demonstrated. The physiological relevance of these fecal cortisol metabolites to adrenocortical activity was evaluated with an adrenocorticotropic hormone challenge test: cortisol metabolite concentrations exceeded pretreatment levels (31-78 ng/g) up to 13-fold (183-944 ng/g) within 8-23 h. Starting from basal levels between 13 and 71 ng/g, a suppression of adrenocortical activity after dexamethasone administration, indicated by metabolite levels close to the detection limit, was obtained 36-81 h after treatment, whereas unmetabolized dexamethasone was detectable in feces 12 h after its injection. Fecal glucocorticoid metabolite assessment via EIA is therefore of use in the monitoring of adrenocortical activity in roe deer. In a second experiment, capture, veterinary treatment, and transportation of animals were used as experimental stresses. This resulted in a 7.5-fold increase of fecal metabolites (1200 +/- 880 ng/g, mean +/- SD) compared to baseline concentrations. The administration of a long-acting tranquilizer (LAT), designed to minimize the physiological stress response, 2 days prior to a similar stress event led to a reduced stress response, resulting in only a 4-fold increase of fecal metabolites (650 +/- 280 ng/g; mean +/- SD). Therefore, LATs should be further investigated for their effectiveness in reducing stress responses in zoo and wild animals, e.g., when translocations are necessary.

Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock.

After 14C-labelled cortisol infusion in ponies and pigs, faecal samples were collected. Extraction of 0.5 g faeces with 5 ml 80-90% methanol yielded the highest radioactivity in the supernatant. Most of the metabolites were ether soluble. After high performance liquid chromatography (HPLC), the presence of immunoreactive metabolites was demonstrated by measuring each HPLC fraction using enzyme immunoassays for cortisol, corticosterone and 11-oxoaetiocholanolone. Only the assay for 11-oxoaetiocholanolone revealed peaks with co-eluting radioactivity. For biological validation of the test system, adrenocorticotrophic hormone (ACTH) and dexamethasone were injected intravenously successively in both species (n = 6). Cortisol concentration in blood and the 11-oxoaetiocholanolone immunoreactive substances in faeces were determined. In horse faeces, basal values of 2.3-35.2 nmol/kg were measured. After ACTH administration, an increase (more than 200% above basal values) of these metabolites was seen about 1 day after ACTH administration. After dexamethasone injection the levels decreased, reaching minimum concentrations 2 days after administration. In pigs, an increase in these metabolites was measured in only three animals after ACTH; dexamethasone did not cause a decrease. The stability of the samples after defecation was tested by storing samples from cows, horses and pigs at room temperature. It was shown that there was a significant increase in the concentration of measured cortisol metabolites in bovine, equine and porcine faeces after storage for 1 h, 4 h and 24 h, respectively. In frozen samples this effect was diminished after thawing samples at 40 degrees C; thawing the samples at 95 degrees C prevented an increase in immunoreactive substances.

Bioquímica del líquido quístico mamario, III: niveles del factor de crecimiento epidérmico, gonadotrofina coriónica y andrógenos conjugados / Biochemistry of breast cyst fluids, III: levels of NA, K, androgens, epidermal growth factor and human chorionic gonadotropin

En la última década se ha implementado una serie de análisis bioquímicos que permiten identificar varios tipos de líquidos quísticos (LQs). En el presente trabajo se confirma la presencia de polipéptidos y esteroides conjugados -como el factor de crecimiento epidérmico (FCE), el sulfato de dehidroepiandrosterona (S-DHEA) y el androstano-3O, 17ß-diol glucurónido (3O-Adiol G)- a veces en concentraciones muy elevadas con respecto a los niveles encontrados simultáneamente en el plasma circulante. Como contraste, la concentración del cortisol apenas alcanza a un 20 por ciento del normalmente hallado en el plasma. Se demuestra además que la concentración intraquística del 3O-Adiol G se correlaciona positiva y significativamente con la del S-DHEA (r = 0,8744, p < 0,0001) y con el FCE (r = 0,8949, p < 0,0001), con amplia variabilidad en los resultados. Se establece también una correlación negativa entre el 3O-Adiol G y el cociente Na/K (r = - 0,6592, p = 0,0001). Por último, se determinan los niveles de la gonadotrofina coriónica (hCG), utilizando un sistema automatizado de quimioluminiscencia, demostrándose que esta glicoproteína se encuentra en cantidades determinables (> 1,1 mUI/ml) en el 73,8 por ciento de los LQs analizados. En el 57,4 por ciento los niveles superan a los encontrados normalmente en el plasma que oscilan entre < 1,1 mUl/ml y 5,5 mUl/ml. En un 4,9 por ciento las concentraciones resultan significativamente elevadas, alcanzando hasta las 1.000 mUl/ml. Se demuestra una correlación negativa con alta significación estadística entre los valores normalizados de la hCG con los niveles del S-DHEA, del 3O-Adiol G y del FCE y una correlación positiva con el cociente NA/K. Se discute la posibilidad de que el FCE, los esteroides conjugados y la hCG puedan ser sintetizados de novo en el tejido epitelial que recubre las paredes del quiste (AU)

Bioquímica del líquido quístico mamario, III: niveles del factor de crecimiento epidérmico, gonadotrofina coriónica y andrógenos conjugados / Biochemistry of breast cyst fluids, III: levels of NA, K, androgens, epidermal growth factor and human chorionic gonadotropin

En la última década se ha implementado una serie de análisis bioquímicos que permiten identificar varios tipos de líquidos quísticos (LQs). En el presente trabajo se confirma la presencia de polipéptidos y esteroides conjugados -como el factor de crecimiento epidérmico (FCE), el sulfato de dehidroepiandrosterona (S-DHEA) y el androstano-3Ó, 17ß-diol glucurónido (3Ó-Adiol G)- a veces en concentraciones muy elevadas con respecto a los niveles encontrados simultáneamente en el plasma circulante. Como contraste, la concentración del cortisol apenas alcanza a un 20 por ciento del normalmente hallado en el plasma. Se demuestra además que la concentración intraquística del 3Ó-Adiol G se correlaciona positiva y significativamente con la del S-DHEA (r = 0,8744, p < 0,0001) y con el FCE (r = 0,8949, p < 0,0001), con amplia variabilidad en los resultados. Se establece también una correlación negativa entre el 3Ó-Adiol G y el cociente Na/K (r = - 0,6592, p = 0,0001). Por último, se determinan los niveles de la gonadotrofina coriónica (hCG), utilizando un sistema automatizado de quimioluminiscencia, demostrándose que esta glicoproteína se encuentra en cantidades determinables (> 1,1 mUI/ml) en el 73,8 por ciento de los LQs analizados. En el 57,4 por ciento los niveles superan a los encontrados normalmente en el plasma que oscilan entre < 1,1 mUl/ml y 5,5 mUl/ml. En un 4,9 por ciento las concentraciones resultan significativamente elevadas, alcanzando hasta las 1.000 mUl/ml. Se demuestra una correlación negativa con alta significación estadística entre los valores normalizados de la hCG con los niveles del S-DHEA, del 3Ó-Adiol G y del FCE y una correlación positiva con el cociente NA/K. Se discute la posibilidad de que el FCE, los esteroides conjugados y la hCG puedan ser sintetizados de novo en el tejido epitelial que recubre las paredes del quiste

Reversed-phase high-performance liquid chromatography of the cardiac glycoside LNF-209 with refractive index detection.

LNF-209 is a glycoside, similar to digoxin, which has potential for use in the treatment of congestive heart failure. However, unlike digoxin it exhibits virtually no useful UV absorption spectra, making detection difficult. One means of detection is the refractive index detector, but like most bulk property detectors it has certain limitations. Its sensitivity is limited and it is sensitive to small changes in a number of parameters, such as temperature, mobile phase composition, and flow-rate. These parameters must be closely controlled to obtain a stable baseline. This paper describes the steps taken to control the system and the development and validation of an assay for LNF-209 in dosing solutions. The method developed is capable of quantitating LNF-209 in solutions of sterile water and 5% dextrose at concentrations ranging from 8 to 6000 micrograms/ml. The method is linear over this range and quantitative recovery is obtained. The overall average relative standard deviation for replicate analysis of several samples at various concentrations assayed over two days was 2.3%.

Structures and concentrations of fifteen different steroid sulfates in human breast cyst fluids.

By applying capillary gas chromatography (GC) and gas chromatography mass spectrometry (GC-MS), a simultaneous quantitation of all important steroid sulfates present in a number of breast cyst fluids, has been obtained. The fact that prevailing androgen sulfate structures in the cyst fluids are different from those in blood suggests at least intracystic metabolism of blood-born precursors. Particularly greater amounts of 5 alpha-reduced steroids are found in breast cysts. 5 alpha-Androstane-3 alpha,17 beta-diol is a major androgen sulfate of breast cyst fluids, its concentration being some 2000-fold that of blood. After prolonged topical application of progesterone on the breast, an accumulation of the sulfates of several pregnanediol isomers could be observed.

Selective reactions in the analytical characterisation of steroids by gas chromatography-mass spectrometry.

The analytical characterization, by GC-MS, of individual compounds in mixtures of steroids, such as occur frequently in biological extracts, is difficult because of the close similarities in structure and properties of many components. The improved separating power of capillary (open-tubular) columns alleviates the problem, but does not solve it fully: for example, the coincidence of retention times of two different compounds may still be virtually complete. Comparative analyses on two distinctively different phases afford one valuable application of selectivity, but may not always be feasible when costly columns are required. Comparative analyses of the sample, before and after effecting its modification by well-defined reactions, are inexpensive and are particularly when selective transformations are used. The use of the microbial enzyme cholesterol oxidase as a selective oxidant for 3 beta-hydroxysteroids (chiefly limited to 4-ene, 5-ene and 5 alpha-types) is illustrated for a model mixture of androstanols related to the boar pheromone (5 alpha, 16-androsten-3 alpha-ol). Retention regularities and changes in mass spectra enhance the reliability of identifications. An exploratory application of cholesterol oxidase in the analysis of minor "polar" sterols in human serum is reported. Most of the known minor sterols are good substrates for the enzyme, and their transformation products yield distinctive GC-MS data, as exemplified for the 7 alpha- and 7 beta-hydroxycholesterols. Another convenient and versatile selective reagent is methaneboronic acid, which yields cyclic esters of suitably constituted diols. These derivatives have shorter retention times (on "non-polar" phases) than the di-TMS ethers, chiefly by virtue of their much lower molecular weights. The mass spectra of cyclic boronates generally show clear molecular ions, also fragmentations that complement the information obtainable from the di-TMS ethers. These features are illustrated for a group of diols and triols of the 5 alpha-pregnane series.