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A new approach using paper spray ionization mass spectrometry (PSI-MS) for the analysis of steroid hormones in wastewater samples has been demonstrated. Triangular papers containing paraffin barriers as microfluidic channels were used to direct the sample solution to the paper tip, preventing the sample from spreading over the corners of the paper. The method was used to analyze the hormones levonorgestrel and algestone acetophenide in industrial wastewaters. Analytical curves presented a correlation coefficient (R2) above 0.99. Limits of quantification were below 2.3 ppm and limits of detection below 0.7 ppm. Values of precision (coefficient of variation) and accuracy (relative error) were less than 15% for all analyses. Recovery results ranged from 82% to 102%. Levonorgestrel was also analyzed by high-performance liquid chromatography coupled to mass spectrometry in order to compare the analytical performance with PSI-MS. No statistically significant differences were found between both methods. This study demonstrates the usefulness of PSI-MS for rapid analysis of hormones in industrial wastewater samples and also indicates its potential to be employed as a simple and reliable analytical method in environmental sciences.

Stigmasterol is a phytosterol contained in Kraft mill effluent that is able to increase over 100% after aerobic biological treatment. This compound can act as an endocrine disrupter as its structure is similar to that of cholesterol. The aim of this study was to evaluate the removal of stigmasterol from Kraft mill effluents treated by a moving bed biofilm reactor (MBBR) with steroidal metabolite detection. The MBBR was operated for 145 days, with a hydraulic retention time of 2 days. Stigmasterol and steroidal metabolites were detected by gas chromatography with a flame ionization detector during MBBR operation. The results show that the MBBR removed 87.4% of biological oxygen demand (BOD5), 61.5% of chemical oxygen demand (COD), 24.5% of phenol and 31.5% of lignin, expressed in average values. The MBBR system successfully removed 100% of the stigmasterol contained in the influent (33 µg L(-1)) after 5 weeks of operation. In that case, the organic load rate was 0.343 kg COD m(-3) d(-1). Furthermore, different steroidal compounds (e.g., testosterone propionate, stigmast-4-en-3-one, 5α-pregnan-12-one-20α-hydroxy, 5α-pregnane-3,11,20-trione and 3α-hydroxy-5α-androstane-11,17-dione were detected in the Kraft mill effluent as potential products of phytosterol biotransformation.

Background: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. Methods: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. Results: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. Conclusions: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.

Laboratory tests have been used to assess the regulatory and research questions related to the effects of pulp mill effluents on aquatic biota. Acute, short-term laboratory tests have clearly shown the improvement in final effluent quality following installation of secondary treatment at Canadian pulp mills. In an effort to predict and investigate impacts on wild fish, laboratory bioassays were developed to examine sublethal endpoints: induction of hepatic mixed function oxygenase activity and reduction of sex steroid concentrations. These laboratory assays have been used to assess whole effluents, specific chemicals, and components of pulp mill processes, and to discriminate between historical and present-day effluent discharges. These tests have shown that induction of mixed-function oxygenase activity and reduction of sex steroid concentrations are produced by effluents from a variety of mill types, with and without chlorine bleaching, in hardwood and softwood pulping facilities, and before and after effluent treatment. These short-term bioassays have enabled reductions in sex steroid concentrations to be linked to mill process streams, and have provided information on effective waste stream treatment. Longer term, life-cycle fish bioassays have shown that chronic exposure to pulp mill effluents commonly results in growth enhancement, liver enlargement, and decreases in gonad size, secondary sex characteristics, and fecundity. These long-term laboratory exposures are able to mimic the most commonly observed alterations of wild fish exposed to pulp mill effluents: increases in condition factors, increases in liver-somatic indices, and decreases in gonadosomatic indices. This pattern of response is a combination of nutrient enrichment with metabolic disruption. The most sensitive and biologically meaningful endpoint is decreased reproduction in fish life-cycle exposures. As the laboratory tests move forward into the next decade, attention will focus on the reproductive endpoints and on the possibility of shortening the fish bioassays while still maintaining sensitivity and biological relevance.

Objectives: To study myopathies with serum antibodies to the signal recognition particle (SRP), an unusual, myositis specific antibody associated syndrome that has not been well characterised pathologically. Methods: Clinical, laboratory, and myopathological features were evaluated in seven consecutive patients with a myopathy and serum anti-SRP antibodies, identified over three years. The anti-SRP myopathy was compared with myopathology in other types of inflammatory and immune myopathies. Results: The patients with anti-SRP antibodies developed weakness at ages ranging from 32 to 70 years. Onset was seasonal (August to January). Weakness became severe and disability developed rapidly over a period of months. Muscle pain and fatigue were present in some patients. No patient had a dermatomyositis-like rash. Serum creatine kinase was very high (3000 to 25 000 IU/l). Muscle biopsies showed an active myopathy, including muscle fibre necrosis and regeneration. There was prominent endomysial fibrosis, but little or no inflammation. Endomysial capillaries were enlarged, reduced in number, and associated with deposits of the terminal components of complement (C5b-9, membrane attack complex). Strength improved in several patients after corticosteroid treatment. Conclusions: Myopathies associated with anti-SRP antibodies may produce severe and rapidly progressive weakness and disability. Muscle biopsies show active myopathy with pathological changes in endomysial capillaries but little inflammation. Corticosteroid treatment early in the course of the illness is often followed by improvement in strength. In patients with rapidly progressive myopathies and a high serum creatine kinase but little inflammation on muscle biopsy, measurement of anti-SRP antibodies and pathological examination of muscle, including evaluation of endomysial capillaries, may provide useful information on diagnosis and treatment.

This study compared the use of sentinel species- and community-based field approaches for assessing the biological status of fish living in a river receiving pulp and paper mill effluents. Three approaches were compared. Two approaches used sentinel species. One of these involved an internal/external examination of the fish that leads to the calculation of a fish health assessment index (HAI) and the other involved biochemical measurements of hepatic mixed function oxidase (MFO) activity and plasma steroid levels. The third approach characterized the fish community structure according to an index of biotic integrity (IBI). The comparison focused on how the methods respond to the hypothesis that recent process modifications/effluent treatment changes, resulting in demonstrable improvements in effluent quality, have beneficial effects on fish. Neither of the approaches using sentinel fish indicated clear mill-related influences either before or after the process modifications/effluent treatment changes. There was no evidence of depressed plasma steroids and increased MFO activity in fish frequently associated with mill effluent exposure in previous studies. While the HAI was higher at stations downstream from two mills, this could not be linked to effluent exposure alone. In contrast, the study of community structure showed a substantial improvement in fish assemblages at all the mill sites.

Instituto de Recursos Naturales y Agrobiología, CSIC, P.O. Box 1052, 41080 Seville, Spain, The chemical composition of lignin and lipids of bast fibers from industrial hemp (Cannabis sativa) used for high-quality paper pulp production was studied. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) of fibers showed a lignin with a p-hydroxyphenyl:guaiacyl:syringyl unit (H: G:S) molar proportion of 13:53:34 (S/G ratio of 0.64). p-Hydroxycinnamic acids, namely, p-coumaric and ferulic acids, were found in only trace amounts. Among the lipids, the main compounds identified by GC/MS of the hemp fibers extracts were series of n-alkanes, free and esterified sterols and triterpenols, waxes, and long-chain n-fatty acids. Other compounds such as n-aldehydes, n-fatty alcohols, steroid hydrocarbons, and steroid and triterpenoid ketones as well as steryl glycosides were also found., This study has been supported by the Spanish Ministerio de Ciencia y Tecnología (MCyT) and FEDER funds (Projects AGL2002-00393).

Background: Steroid administration is beneficial in Duchenne muscular dystrophy (DMD), but the response, incidence, and the severity of side effects are variable. Aims: To investigate whether glucocorticoid receptor ( GRL ) gene polymorphisms may be responsible for glucocorticoid sensitivity in DMD. Methods: Forty eight DMD patients treated either with prednisone or deflazacort were subjected to genetic analyses of the GRL gene. Results: Mutation studies revealed an heterozygous A to G mutation at GRL cDNA position 1220 in three DMD patients resulting in an asparagine to serine amino acid change at amino acid position 363 (N363S). The N363S carrier DMD patients showed a trend towards a later age at loss of ambulation in comparison with non-carrier patients. Conclusions: These data suggest that the N363S GRL polymorphism may be implicated in the long term response to glucocorticoids.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, Spain; E-mail address: delrio@irnase.csic.es (J.C. del Río), The chemical composition of lignin and lipids of bast fibers from kenaf (Hibiscus cannabinus) used for high-quality paper pulp production was studied. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) of fibers showed a lignin with high syringyl:guaiacyl ratio (5.4) and minor amounts of p-hydroxyphenyl units. Simultaneously, sinapyl and coniferyl acetates were also identified, indicating that this lignin is partially acetylated. p-Hydroxycinnamic acids were only found in trace amounts. The main lipids identified by GC/MS of extracts from kenaf fibers were series of long chain n-fatty acids, waxes, n-alkanes and n-fatty alcohols. Free and esterified sterols and triterpenols, steroid hydrocarbons, steroid and triterpenoid ketones, as well as steryl glycosides were also found. Finally, the fate of the main constituents of kenaf fibers in alkaline pulping was also investigated., This study has been supported by the Spanish Ministerio de Ciencia y Tecnología (MCyT) and FEDER funds (project AGL2002-00393). A.G. acknowledges a "Ramón y Cajal" contract of the Spanish MCyT. We thank CELESA (Tortosa, Spain) for providing the kenaf and pulp samples, S. Camarero (CIB, CSIC, Madrid) for Klason lignin and hemicellulose analyses and A. T. Martínez (CIB, CSIC, Madrid) for critical reading of the manuscript.

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed on the day of pro-oestrus at 11.00, 14.00, 16.00, 2.00, 22.00 and 24.00 h. The isolated preovulatory follicles were placed individually on a small triple disc of filter paper. They were cut in half and the flowing out follicular fluid (FF) was allowed to soak into the underlying filter paper disc, which was subsequently placed at the bottom of incubation chamber. It was rinsed with the incubation medium and this medium, containing FF, was collected. The remaining follicular wall was incubated for 3h. Some of the incubation media were supplemented with testosterone (T). The steroid content was estimated by radioimmunoassay. The secretion of estradiol (E) by follicular wall was high until 20.00 h while that of androgens until 22.00 h, and both declined thereafter. Relatively low progesterone (P) release rose sharply at 22.00 h and this hormone became the main steroid secreted at 24.00 h. The addition of T always enhanced the release of estradiol. Follicular fluid contained very little estradiol, while prevailing steroids were androgens. Decrease of androgen concentrations was found only at 24.00 h. Progesterone level, much lower than that of androgens, started to rise at 20.00 h and P was the main steroid present in FF at 24.00 h. The present results suggest an existence of selective passage of steroids into FF and show a presence of androgenic milieu surrounding cumulus oophorus in preovulatory FF. The results also indicate that the aromatase activity is sustained until ovulation and stress the role of the P rise observed in the secretion rate of follicular wall and FF content just before ovulation.

The objective of this study was to characterize the effects of pulp mill effluent on the properties of the sex steroid binding protein (SBP) in the plasma of white sucker (Catostomus commersoni) and longnose sucker (C. catostomus). SBPs which specifically bind estradiol and testosterone with high affinity (k(D) approximately 3 nM) and low capacity (B(max) approximately 73-81 nM) were identified in both species. Subsequent studies determined if the properties of the SBP in white sucker exposed to bleached kraft mill effluent (BKME) at Terrace Bay, ON, and in longnose sucker exposed to BKME at Grande Prairie, AB. differed from appropriate reference fish. There were no effects of BKME exposure on the binding affinity (k(D)) of the SBP in either species, but there was a significant increase in the binding capacity (B(max)) of longnose sucker SBP exposed to BKME. The livers of nai;ve white sucker exposed to effluent at Terrace Bay or a bleached sulfite/groundwood mill in Edmundston, NB, rapidly accumulated compounds of differing hydrophobicity that bound to both the white sucker and goldfish (Carassius auratus) SBP. Conversely, there was reduced accumulation of SBP ligands in the bile of effluent-exposed fish. We have demonstrated that constituents present within pulp mill effluent bind to both the white sucker and goldfish SBP, and that native species residing downstream of pulp mill effluents may experience modifications in the properties of their SBP.

The lipidic organic fraction of 48 sewage sludges that originated from food-processing, paper-mill and domestic (urban, small urban, and rural) wastewater-treatment plants of the Lorraine region (Northeast of France) was characterised by gas chromatography coupled with mass spectrometer (GC-MS). This study enables us to define an average organic composition typical of each group of sewage sludges. Linear alkyl benzenes (LABs) are only present in domestic sludges, sterols in food-processing and domestic sludges. Paper-mill sludges are characterised by the specific distribution of n-alkanes. Besides, all the domestic sludges, whatever the size of the wastewater catchment, are characterised by the same distribution of polar compounds. Differences can be evidenced in the distribution of the aliphatic compounds of some domestic sewage sludges and are attributed to the important contribution of petroleum products in their sewer system. Moreover, this study highlights the correlation between abundance of polycyclic aromatic hydrocarbons (PAHs) in the aromatic fraction of some sewage sludges and a distribution of n-alkanes characteristic of heavy petroleum products into the corresponding aliphatic fraction. This might be a clue for the determination of punctual sources of PAHs.

Based on our method for the qualitative test for steriod hormones by paper chromatography Fujisaki, et al.(1953) the quantitative determinations of several steroids were investigated. It was found that within a certain range of the amount the logarithms of each spot area of estrone, α-estradiol, dehydroepiandrosterone, Δ5-androstendiol, methyltestosterone, mcthylandrostendiol and androstane-17β-ol-3-one were directly proportional to the logarithms of the amount of the hormone, thus rendering the quantitative assay possible.

Three new peptides with high steroidogenic activity in vivo have been isolated from porcine pituitary extracts. Similar in molecular weight to porcine ACTH, they differ from the usual amino acid composition in molar ratios of aspartic acid, threonine, serine, glycine, valine, methionine and phenylalanine.

A wide range of Δ 4 - and Δ 1 , 4 -3-ketosteroids, from weakly polar to strongly polar, may be chromatographed and detected on cellulose triaacetate paper, with benzene—methanol—water mixtures as developing solvents. Three systems have been worked out. In the first a one-phase mixture of benzene, methanol and water is used as the mobile phase and the paper is not accommodated. A “reversed phase” type of chromatogram is obtained, i.e. the more polar compounds have higher R F values than the less polar ones, as is usual for chromatography on acetylated paper. If, howerver, two-phase mixtures of benzene, methanol and water are used, as was the case in the other two methods, “reversed phases”, as well as “convetional” type chomatograms may be obtained. This depeds upon whether the lower or the upper layer is used as the mobile phase, the other layer being used for accommodation of the paper. Perhaps the first-mentioned method should, in contrast to the other two, be regarded as adsorption chromatography, rather than as partition chromatography. Well-reproducible R F values are obtained and, especially with the two-phase mixtures, compact spots are found. Detection methods and R F dta are given for various Δ 4 - and Δ 1 , 4 -3- ketosteroids, ranging from C 18 to C 27 compounds and including sex hormones and adrenoccortical hormones.

Es wird eine einfache mikroanalytische Technik zur Ausfuhrung von Reaktionen mit fluchtigen Reagenzien beschrieben.

A system of paper chromatography has been presented in which the stationary phase is a high-boiling hydrocarbon mixture (odorless kerosene) and the mobile phase is 60% aq. propanol. This system is not only useful for separation and identification of the cholesterol class of compounds but may be conveniently used to free tissue extracts of so-called fat. The use of the system in preparative chromatography has been discussed. Special application of two color reagents, phosphomolybdic acid and phosphotungstic acid, has been described. Proper use of the former can make it fairly specific for some steroids with secondary alcohol functions. Phosphotungstic acid was shown to give a characteristic pink color with many steroids having Δ5, 3β-ol functions if warmed gently lavender a vivid lavender if heated more strongly. Certain steroids with allylic alcohol functions were also very chromogenic but with varying results. Precautions and limitations in the use of the reagents were discussed.