Protease activity has been detected in various species of scorpion venoms (Morgenstern et al., 2011; Seyedian et al., 2010). However, little information about their primary structure has been available. In our study, we were unable to find gelatinase activity in the venoms analysed. In an early study from Almeida et al. (2002), a gelatinase activity associated with serine proteases was observed in venoms from T.

serrulatus and T. bahiensis. In addition, a gelatinase activity attributed to the presence of a metalloproteinase was recently observed in the venom of Hemiscorpius lepturus, a scorpion found in Iran ( check details Seyedian et al., 2010). These discrepancies might be due to the sensitivity of the methods of measurement or to intraspecific/interspecific variations in venom composition. A FRET substrate, a dynorphin analogue peptide, was used in our proteolytic studies. Using this fluorometric method, it was possible to demonstrate that the Tityus spp. venoms studied were able to hydrolyse the substrate (Abz-FLRRV-EDDnp), with optimal hydrolysis efficiency Anti-infection Compound Library cell line at pH 8.5 and 10. Under these conditions,

venom from T. bahiensis demonstrated more than two times greater proteolytic activity compared to venom from T. serrulatus and T. stigmurus. Furthermore, the proteolytic activity was completely inhibited by the metalloproteinase inhibitor 1,10-phenanthroline Sitaxentan but not by PMSF, a serine protease inhibitor. The first metalloproteinase from the venom of T. serrulatus was recently identified and characterised ( Fletcher et al., 2010). This enzyme, named antarease, exhibits action on the protein vesicle-associated membrane proteins 2 and 8 (VAMP2 and VAMP8), also known as synaptobrevins. Antarease has a molecular mass of 25.5 kDa. The cleavage

sites in VAMP2 were identified as L//KRK//Y and those in VAMP8 as A//RK//F. The antarease VAMP2 cleavage site is similar to that of the metalloproteinase cleavage site of dynorphin 1-13 (L//RR) from T. serrulatus, T. bahiensis and T. stigmurus venoms found in this study. This result suggests that dynorphin-cleaving metalloproteinases detected in T. serrulatus, T. bahiensis and T. stigmurus venoms might be antarease-like molecules. Further studies will be performed to purify and characterise the dynorphin-cleaving metalloproteinases from Tityus spp. venoms. The dynorphin-degrading capacity of Tityus spp. venoms, resulting in the generation of the biologically active peptide leu-enkephalin, might be implicated in the hypotension and bradycardia symptoms ( Feldman et al., 1996), as observed in patients stung by Tityus scorpions.

, 1965). At the Maneroo Platform, two major faults were recognised (Westland Structure and Stormhill Fault). Both structures trend northerly and vertical displacements of up to 300 m have been registered according to Vine et al. (1965) but displacement was later amended

to 640 m by Ransley and Smerdon Carfilzomib mw (2012). The differences in the displacement registered in these structures are discussed in Section 4.1.2. The Dariven Fault and Maranthona Monocline (Van Heeswijck, 2010) are also recognised in the area to the east of the Hulton-Rand Structure, but there is little information about relative movement. During the deposition of the Eromanga Basin, this area was tectonically inactive and the faulting, folding and uplift of the basin units is considered to be post-depositional. Uplift was recorded

in the eastern part of basin, including uplift of the Koburra Trough, with associated erosion leaving the selleck Galilee Basin exposed in this area (Shaw, 1991). In the current study, the faults classified as regional structures cross the entire stratigraphic sequence from the basement to the surface. In addition, there is also another type of fault, classified as local faults that cross only part of the stratigraphic sequence and are not visible at the surface. The GAB is one of the major hydrogeological features of Australia, and is comprised of the sedimentary Clarence-Moreton, Eromanga, Surat and Carpentaria basins, Ketotifen and parts of the Bowen and Galilee basins. The confined aquifers of the GAB are bounded by the Rewan Formation at the base, and the Winton Formation at the top

(Fig. 3), but the complete rock sequence is not present across the entire GAB (Habermehl, 1980 and Habermehl, 2001). GAB aquifers in the study area include: the Clematis Group, Hutton, Adori and Hooray sandstones, Cadna-owie Formation (and their equivalents), the Mackunda and Winton formations (Fig. 3). The major confining beds in the study area are the Rewan Group, Moolayember, Birkhead, Westbourne, Wallumbilla and Toolebuc formations and their equivalents, as well as the Allaru Mudstone and parts of the Mackunda and Winton formations (Habermehl, 1980, Reyenga et al., 1998 and Habermehl, 2001; Fig. 3). The confined aquifers can be divided into two groups based on their potentiometric surfaces: (1) Lower Cretaceous-Jurassic sequence, also known as the artesian group; and Groundwater flow directions throughout the GAB are variable, with major flow towards the south and southwest, but in the northern GAB locally towards the west and north (Habermehl, 1983). In the area of the 3D geological model domain of this study, groundwater flow is largely towards the west based on the potentiometric map of the Hooray Sandstone and Cadna-owie Formation (Radke et al., 2000). This current study develops a 3D geological/hydrogeological model using GoCAD software (Paradigm Geophysical Pty Ltd., version 2009.

The AUC of each risk assessor for fracture at follow-up was modeled by univariate logistic regression on the risk assessor as only explanatory variable. In order to adjust for censored women and take time to event (fracture) into consideration, we estimated the Harrell’s C index by Cox regression modeling. Harrell’s C is analog to AUC in a survival setting. Standard errors robust for cross validation were achieved by the Jack

knife-method. www.selleckchem.com/products/Etopophos.html Tool assessors with AUC statistics of 0.50 do not perform better than chance alone, while tools with higher AUC statistics perform better than chance. We compared AUC statistics between FRAX® and simpler tools using the “roccomp” procedure in STATA. Finally, the population was divided into quartiles based on fracture risk as predicted by each tool and compared the observed fracture rates across the quartiles. Agreement as to how well each tool assigned the women to risk quartiles was tested using weighted kappa statistic. All analyses were conducted using STATA 12. As previously reported [24], the respondent rate to the questionnaire was 84%. A total of 334 questionnaires were blank or had several missing items and were excluded leaving 3860 complete questionnaires. We further Ganetespib excluded, 246 women diagnosed

with and treated for osteoporosis, leaving 3614 women for analysis. The follow-up period ranged from March 2009 to April 2012. Mean follow up time in the total cohort was 36 months (range 30 to 37 months) and the total follow-up comprised 10,385 person-years. During follow-up, 156 (4%) women suffered “major osteoporotic fractures”, 225 (6%) women sustained an “osteoporotic fracture”, 174 women died and 6 were lost to follow-up. The Kaplan–Meier ROS1 plots of cumulative incidence

of major osteoporotic fracture are shown in Fig. 1. The 3 year cumulative “major osteoporosis fracture” estimates for all the tools were similar and ranged at high risk of fracture from 8% in the FRAX® curve to approximate 6% for the SCORE tool. Nearly identical curves were seen in competing-risks regression (data not shown). Baseline characteristics of the study population overall and stratified according to incident fractures are shown in Table 2. The mean age of the women was 64 ± 13 years and mean BMI was 26 ± 5 kg/m2. Women with incident fractures were older (mean age 73 ± 11 versus 63 ± 13 years, p = 0.001), had more frequent history of fractures (22% versus 9%, p < 0.001) and history of falls during the previous 12 months (14% versus 6%, p < 0.001), had diseases more often related to secondary osteoporosis (26% versus 18%, p = 0.011), and had less frequently used estrogen currently (3% versus 11%, p = 0.001). ROC curve analysis was used to assess the discrimination between the tools. AUC values were very similar (0.703 to 0.722) with no significant differences (p = 0.86) in the AUC values between FRAX® and the more simple tools (Table 3).

LiRecDT1-GFP binding was evaluated as described above, except that B16-F10 cells (0.5 × 103 cells) were incubated

with 10 μg/mL of the recombinant fluorescent toxin (5 h, 37 °C). Non-specific binding of GFP alone to the cells was evaluated as a negative control. For binding competition assays, the fluorescence protocol was the same as described above, except that B16-F10 cells were previously incubated with an excess of LiRecDT1 (100 μg/mL) for 1 h at 37 °C 5-FU in vitro and then with 10 μg/mL LiRecDT1-GFP. The samples were observed using a Zeiss Axio Observer.Z1 inverted microscope (Carl Zeiss, Germany). Single images were obtained using a 63× oil lens for differential interface contrast (DIC) microscopy and a monochromatic camera (AxioCam HRm, Carl Zeiss) to examine fluorescence intensity. Finally, AxioVision LE software was used for image processing and morphometric measurements in the Zeiss image format compound screening assay (ZVI). B16-F10 cells (1 × 108 cells/mL) were prepared in Ringer’s Solution (122.5 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 10 mM HEPES, 11 mM glucose, 1 mM NaH2PO4, pH 7.4) containing 5 mM CaCl2 and treated according to Kaestner et al. (2006) and Haase et al. (2009). B16-F10 cells were loaded with Fluo-4 AM (10 μM) in buffer with Pluronic F-127 (0.01%) for 30 min at 37 °C. This indicator exhibits high-affinity binding to Ca2+ (Kd = 345 nM)

and shows a large increase in fluorescence intensity in response to Ca2+ binding (>100 fold). Subsequently, the cells were washed SPTBN5 twice with Ringer’s Solution and equilibrated for de-esterification for 30 min at room temperature. Then, the cells were incubated with 25 μg/mL recombinant phospholipase-D (LiRecDT1) for 5, 15, 30, 45, 60 or 90 min.

Cells incubated under the same laboratory conditions but in the absence of phospholipase-D for 90 min were used as a control. Following this reaction, the cells were transferred to Black 96-well plates at a density of 1 × 106 cells/well in a total volume of 200 μL, and the resulting fluorescence was recorded on a Tecan Infinite M200 spectrofluorometer (Tecan) using an excitation wavelength of 485 nm and measuring emission at 535 nm. Additionally, Fluo-4 dye-loaded B16-F10 cells were allowed to settle onto coverslips, and images of calcium-dependent fluorescence were obtained using an Axio Observer.Z1 inverted microscope Zeiss (Carl Zeiss, Germany). Fluo-4 AM was excited at 488 nm, with emission detected using an LP 505 nm filter (green channel). Single images were obtained using a 63× oil lens for differential interface contrast (DIC) microscopy and a monochromatic camera (AxioCam HRm, Zeiss, Carl Zeiss, Germany) to measure the fluorescence intensity. Finally, AxioVision LE software was used for image processing and to perform morphometric measurements in the Zeiss image format (ZVI).

Kept at a safe distance, however, excitement about the discovery was infectious but shouts of unbridled joy accompanied the huge “whoomphs” when the devices were exploded in situ. It is now almost 70 years since World War II ended but a television programme on the impending homecoming from Afghanistan of the Royal Marines, who were filmed packing up

their ordinance for repatriation, made me think again about the disposal of such weaponry in the past – a subject that seems to have dropped out of common, and scientific, concern. A simple click here search quickly provided a few interesting and, apparently, mostly forgotten facts. After World War II, the United States and other European countries dumped 300,000 tonnes of conventional and chemical munitions into the ocean. This figure, however, incredible as it is, pales when one learns that in Europe alone, in excess of one million tonnes of munitions were dumped in Beaufort’s Dyke, in the Irish Sea, some 168,000 tonnes in the Skagerrak (Denmark) and some 300,000 tonnes in the North Sea. There are actually 148 individual selleck dump sites spreading south

from Iceland to Gibralter, most along the coast of France, and they contain conventional explosives such as bombs, grenades, torpedoes and mines, but also chemical munitions containing phosgene and mustard gases as well as the nerve gases, lewisite, sarin and tabun. The example of Great Britain’s biggest dump site provides an example of the scale of the problem. Beaufort’s Dyke is a deep (∼200 m) trench located between Scotland and Northern Ireland in the Irish Sea. It is 50 km long and 3.5 km wide. In 1995, following the discovery of incendiary devices along the coastline of the Firth of Clyde, some of which self-ignited as they dried, the Fisheries Research Services of the Scottish Buspirone HCl Executive conducted an acoustic survey of the dyke to determine the distribution and density of the munitions. The survey also obtained seabed, shellfish and fish samples for analyses of contaminants. The survey showed that the munitions were spread

far and wide across the seabed, but that there was no identifiable chemical contamination of either the seabed or the fishery resources. In 2004, however, a local councillor from Northern Ireland, reported in a BBC programme on the subject that incendiary bombs drift onto the shores (of Northern Ireland) each winter with ‘hundreds upon hundreds of these things getting washed up in a matter of days’. He added that. ‘a couple of young boys here locally got burns off them, and another [boy] in Scotland was burnt’. A former Royal Navy diver specialising in bomb and mine clearance offered the opinion that the oldest munitions in the Dyke were losing their ability to withstand corrosion and that there are (possibly) two or three sporadic spontaneous undersea explosions each month.

Technological advances have meant that the data have a very high resolution and are very reliable. Our findings show that temperature is subject to both seasonal and long-term variations. A phase shift of the annual temperature signal was observed in the layer above the halocline, where ocean-atmosphere interaction occurs. This could be due to wind mixing,

which modifies the temperature of the upper layer, but only at a depth of about 30–40 m. Convection could also be an important Ganetespib in vitro process in the transmission of the signal to the lower layers. The amplitude decreases with depth, which smoothes the seasonal function out. For the whole period of 1900–1980, the water temperature in all basins has shown a positive trend (Lepperänta & Myrberg 2009). The increase in the surface layer has been of the order of 0.5°C during the last 100 years. The reason is not yet exactly clear, but it is evidently associated with a similar rise in the

atmospheric surface layer temperature in the region. Since the 1960s, a reverse trend can be observed (BD is an exception), especially strong in the period 1977–1989 (Cyberska 1994). The present results show that in 1998–2010 there was a positive trend, exceptionally strong at the surface (0.11°C year−1) and in the near-bottom layer (0.16°C year−1). The rise in the water temperature in the near-bottom and transition layers could be due to the increasing impact of small and medium-sized baroclinic Roxadustat solubility dmso inflows

(Matthäus & Franck 1992) and to the reduced occurrence of large barotropic inflows, as reported recently by Feistel et al. (2006) and Mohrholz et al. (2006). The previous decrease in salinity in 1977–1989 (Cyberska 1994) was due to long-term Protein tyrosine phosphatase stagnation and occurred after large inflows between 1975–1976 and 1976–1977. This study shows that in 1998–2010, the salinity increased throughout the water column (Figure 8). This could have been caused by an increase in the frequency of small and medium-sized inflows. This study is important because it extends existing time series of temperature and salinity. The above analysis shows the changes in temperature and salinity that have occurred over the last 12 years in the entire cross-section. The series of measurement is too short to be used to predict future changes. To be able to do this, the time-scale will have to be prolonged. The future work of the authors will be extended by modelling results and available in situ measurements. A combination of these tools should enable temperature and salinity changes to be determined with precision. “
“The Volume Scattering Functions (VSF), a topic of interest to marine optics researchers for several decades, are still the least-known optical properties of sea water.

Animals were deeply anesthetized with ketamine and submitted to neurophysiological evaluation by electromyography of the mandibular branch of the facial nerve aiming at obtaining selleck screening library compound muscle action potentials (CMAPs). Outcome variables were the CMAP amplitude and latency values. To obtain the CMAPs, we used a portable electromyography system (Neuro-MEP-Micro®, Neurosoft, Dhaka, Bangladesh) connected to a battery-operated Pavilion dv5C portable personal computer (Hewlett-Packard). The Neuro-MEP.NET software (version 2.4.23.0, Neurosoft) was employed to assess the CMAP data obtained under the following configuration of the electromyography

system: 10-Hz high-pass filter, 10-kHz low-pass filter, notch filter off, 60 mV of leading edge signal, and 10-kHz of sampling rate. The electromyography protocol has been established specifically for

evaluation of the rat facial nerve and described in detail by Salomone et al. (2012). Histomorphometric analyses were performed blindly six weeks after surgical procedure, and this method was well established by Costa et al., 2006, Costa et al., 2007 and Costa et al., 2012. After sacrifice, the surgically repaired portion of the facial nerve was cut into four parts, two distally and two proximally related to the graft. One pair of proximal (middle learn more of the autografting) and distal (3 mm distal to autografting) sections was fixed in 2% glutaraldehyde and 1% paraformaldehyde in 0.0031 M phosphate buffer, pH 7.3. After 60 min. in solution A, the tissue was postfixed for 2 h in 2% osmium tetroxide in phosphate buffer, dehydrated in ethanol, infiltrated

in propilene oxide and included in Epoxi® resin (Burlington, VT) until polymerization. Transversal, 1-μm sections were made and stained with 1% toluidine blue. Histological observations were carried out using light microscopy (Nikon Eclipse E 600, Nikon, Japan). The slides were photographed with a digital camera (Nikon Coolpix E 955, Nikon, Japan), and cell measurement taken (Sigma Scan Pro 5.0 software, SPSS Science). Qualitative analyses were performed according to general nerve architecture, pattern of tissue organization and myelination. For quantitative analyses of distal portion of the facial nerve, axons were counted in C-X-C chemokine receptor type 7 (CXCR-7) a partial area of 9.000 μm2 in three random microscopic fields for every fiber displaying its center within it. Total axon density was obtained by the ratio between total axon number and area. The shortest external diameter (including the myelin sheath) of all axons within a partial, randomly selected area (3.000 μm2) of the transversal section of the nerve was measured to evaluate the maturation of myelinated fibers (Mayhew and Sharma, 1984). The second pair of proximal and distal sections was fixed in 4% paraformaldehyde in phosphate-buffered saline.

M.); differences were considered significant when p ≤ 0.05. All analyses were performed by using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA — SPSS version 15.0) software, and GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA — version 4.02) software. The present work was supported by grants from Conselho http://www.selleckchem.com/products/BKM-120.html Nacional de Desenvolvimento Científico e Tecnológico (CNPq — Brazil), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES) and Rede Instituto Brasileiro de Neurociências (IBN-net) — 01.06.0842-00. We thank Mr. Steve Niedermeier for language revision. Last but not least, we thank all colleagues for technical assistance. “
“Hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome (OMIM 238970) is an autosomal recessive disorder due to mutation in the gene that encodes the mitochondrial ornithine (Orn) transporter ORNT1 (SLC25A15) ( Camacho et al., 1999, Fell et al., 1974, Korman et al., 2004, Tessa et al., 2009 and Valle and Simell, 2001). The inability to import Orn from the cytosol check details into the mitochondria results in intramitochondrial Orn deficiency and a functional impairment of the urea cycle at the level of ornithine transcarbamylase, with consequent hyperammonemia. The defect also gives rise to cytoplasmatic accumulation of

Orn resulting in hyperornithinemia. In the absence of intramitochondrial Orn, accumulating carbamoyl phosphate may condense with lysine to form homocitrulline (Hcit) leading to homocitrullinuria ( Valle and Simell, 2001). The clinical features of neurological symptoms in HHH syndrome are very peculiar since, besides some unspecific signs similar to the others urea cycle defects (hypotonia, seizures, ataxia,

coma, etc.), patients exhibit a pyramidal syndrome with progressive spastic paraplegia. Neuropathological findings include multiple, nonspecific T2 hyperintense foci in occipital, parietal and frontal white matter, with subcortical and cortical atrophy associated with swelling typically seen in demyelinating diseases (Al-Hassnan et al., 2008). It should be stressed that among the urea cycle defects, pyramidal dysfunction is also present in argininemia and therefore both disorders share a common characteristic clinical picture (Valle and Simell, 2001). The mechanisms Inositol monophosphatase 1 of central nervous system (CNS) impairment in HHH syndrome are poorly known (Palmieri, 2008 and Salvi et al., 2001), although it has been hypothesized that the neurologic damage presented by the patients are probably secondary to the episodic hyperammonemia. However, chronic accumulation of Orn, Hcit and other metabolic factors cannot be ruled out as contributing causes of the neurological symptoms and brain abnormalities seen in these patients, especially during crises of metabolic decompensation, in which the concentrations of these metabolites dramatically increase.

The most significant threats to seagrass in the BHS are deforestation and coastal development causing increased turbidity and sedimentation from runoff, as well as reclamation of shallow coastal habitats that smothers seagrass beds. The BHS boasts the highest diversity of corals, reef fishes and stomatopods in the world (Veron et al., 2009, Huffard et al., 2009, Allen and Erdmann, 2009 and Allen and Erdmann, 2012). Surveys have recorded over 577 described species of scleractinian corals (75% of the world’s total), with individual reefs hosting up to 280 species per hectare Omipalisib (Veron et al., 2009 and Wallace et al., 2011). An additional 25–40 undescribed coral species have

also been collected, such that the total scleractinian diversity in the BHS is expected to exceed 600 species once taxonomic work is completed on these

collections (L. DeVantier and E. Turak, personal communication). Within the BHS the highest diversity of corals selleck inhibitor has been recorded in Raja Ampat, with 553 known species (Veron et al., 2009). Two rapid ecological assessments conducted in 2001 and 2002 in Raja Ampat also recorded 41 of the 90 Alcyonacean (soft coral) genera and 699 mollusc species (McKenna et al., 2002 and Donnelly et al., 2003), while more recent studies have documented 57 reef-associated stomatopod species in the BHS, four of which are considered endemic to the region (Huffard et al., 2009). Corals have been found to 160m depth in Raja Ampat, though those beyond the reaches of SCUBA remain uncharacterized (B. Robison, unpublished data). Similarly, intensive survey work around the BHS over the last decade has recorded 1638 species of coral reef fishes comprising 476 genera and 117 families (Allen and Erdmann, 2009 and Allen and Erdmann, 2012). Within the BHS, the highest diversities have been recorded in Raja Ampat (1437 spp.), the Fakfak-Kaimana coast (1005 spp.) and Cendrawasih Bay (965 spp.). Allen and Erdmann (2009) reported a total of 26 endemic reef fish species (from 14 families) in the BHS, though

more recent surveys have now increased this total to 41 (Dimara et al., 2010 and Allen and Erdmann, 2012). The factors that contribute to local endemism are selleck thought to be in part associated with the geological history of the region. For example, there is evidence that Cendrawasih Bay was isolated for a substantial period over the past 5 million years, resulting in high local endemism (11 endemic reef fishes and 18 endemic reef-building corals currently recognized), and significant genetic divergence of many marine invertebrate populations in the Bay (DeBoer et al., 2008, Crandall et al., 2008, Wallace et al., 2011 and Allen and Erdmann, 2012). The main reef types found in the region are fringing and patch reefs, and to a lesser extent seamounts, atolls and barrier reefs (Fig. 6; McKenna et al., 2002, WWF, 2003 and Donnelly et al.

, 2005 and Shah et al., 2008). The same holds true for the integrity test BLUE which utilizes the absorption Selleckchem GPCR Compound Library of methylene blue as a measure for barrier functionality. In contrast to TWF, TEER, TEWL and BLUE the integrity test ISTD supplies information of the barrier function over the whole experimental period and avoids the elongation of the

test period. But the presence of an additional compound in the donor may influence the absorption characteristic of the test compound because of changes in solubility or saturation levels of the test compound and effects of the solvent on the barrier system (Barry, 1987 and Dugard and Scott, 1986). Due to this influence the inertness of an ISTD must be proven. 3H-sucrose and phenol red have been used as ISTD in the past, but systematic validation and provision of a sufficient dataset is still missing (Balaguer et al., 2006, Pendlington et al., 1997 and Walters et al., 1997). The purpose of the current work was to investigate the suitability of different skin integrity tests to differentiate impaired and intact human skin. Based on the absorption results of four test compounds (testosterone, caffeine, 2-ethyl-4-chlorophenoxyacetic acid (MCPA) and 2-methyl-4-chlorophenoxyacetyl ethylhexylester (MCPA-EHE)) through human and generally

more permeable reconstructed human skin (StrataTest®), the common limit values for the standard integrity methods TEER, TWF and TEWL were Selleckchem Selumetinib assessed. Additionally, results of five skin integrity tests (TEER, TWF, TEWL, ISTD and BLUE) were correlated to absorption results derived with human skin or reconstructed human skin to evaluate their ability to explain minor differences in barrier function. Full-thickness and dermatomed human skin samples were applied to check for a possible effect of the skin preparation. Due to a lower donor dependency, rat skin was used in addition and chosen for a special experiment in which skin samples were systematically damaged to different grades before Methamphetamine use. As model ISTD 3H-testosterone

was chosen. It was applied in parallel to test compound 14C-MCPA. For human skin experiments two further well-investigated reference compounds with different physico-chemical properties were applied as ISTDs (3H-caffeine and 3H-mannitol) (OECD, 2004a, Peck et al., 1995, Schäfer-Korting et al., 2008 and van de Sandt et al., 2004) to get an insight on the effect of ISTD selection. Additional experiments were conducted to check for effects of the present ISTDs on the analytics and absorption characteristics of the test compound. MCPA-2EHE, MCPA, dimethylamine (DMA; 60%), silicone antifoam emulsion (SRE) and ethylenediaminetetraacetic acid (EDTA) were provided by AH Marks and Co, Wyke, Bradford, Great Britain. Testosterone, caffeine, ethanol and methylene blue were purchased from Sigma Aldrich, St.