Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C₁₈ reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C₁₈ ODS Hypersil analytical column (5 μm particle size; 250 mm × 4.6 mm I.D.) using an acetonitrile—water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N′,N′-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75–400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

Subcellular Distribution of 3α-Hydroxysteroid Dehydrogenase and Antiestrogen Action on Androgen-Metabolizing Enzymes in Rat Pituitary Gland

Abstract: Gonadectomy of male rats led to a threefold increase of 3α-hydroxysteroid dehydrogenase (3α-HSDH) activity in pituitary homogenates that could be completely reversed by chronic administration of estradiol or 5α-dihydrotestosterone (DHT). 3α-HSDH was found to be distributed mainly between the 10,000 g and 100,000 g sediments from whole homogenates. The microsomal enzyme activity showed a substantial specificity for NADH whereas the cytosolic enzyme (100,000 g supernatant) demonstrated a slight preference for NADPH. The changes in V _max found in homogenates following gonadectomy and gonadal steroid administration reflected changes in NADH- linked activity of the microsomal, but not the cytosolic enzyme. Estradiol-induced suppression of NADH-linked 3α-HSDH activity in pituitary homogenates from gonadectomized rats of either sex was accompanied by a similar suppression of NADPH-linked 5α-reductase activity and a marked decrease of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release. In the ovariectomized rat chronic administration of nonsteroidal antiestrogens had strong estrogenic effects on 3α-HSDH activity and LH release, but not on 5α-reductase activity and FSH release. In the gonadectomized male rat, which was much less sensitive to intrinsic estrogenicity of the antiestrogens tested, nafoxidine completely blocked estradiol-induced suppression of 5α-reductase activity and FSH release and partially antagonized suppression of LH release. The trans -isomeric, substituted triphenylethylenes, tamoxifen, and enclomiphene, as well as nitromifene (mixture of trans and cis isomers) were able partially to counteract estradiol-induced suppression of 5α-reductase, but not 3α-HSDH activity. It is concluded that estradiol action on pituitary 5α-reductase, but not 3α-HSDH activity, involves an estrogen receptor mechanism.     

Immunohistochemical localization of secretory proteins in the endometrial epithelium of the rabbit

Summary Proteins of uterine fluid and lung homogenates of the rabbit were separated by gel and ion exchange chromatography. Purified protein fractions were used for immunisation and antiserum production. By means of several absorptions, six monospecific antisera against uteroglobin and five other proteins were obtained. Using immunohistochemistry, four of them could be localised in the uterine epithelium from oestrus and the first and the seventh day post coitum, and also in the blastocyst. The present study indicates the involvement of different endometrial cells in the synthesis and release of the various proteins of uterine secretion.Supported by grant Ki 154/6-7 from the Deutsche Forschungsgemeinschaft     

Immunological study of the tissue expression of D-beta-hydroxybutyrate dehydrogenase, a ketone body-converting enzyme

In rats, as in most mammal, ketone bodies are mainly produced in liver while they are metabolized in extrahepatic tissues. The expression of mitochondrial membrane-bound D-beta-hydroxybutyrate dehydrogenase (BDH), a ketone body-converting enzyme, has been estimated by two immunological techniques: immunohistofluorescence and Western blotting. The in situ labeling with anti-BDH antibody shows that the enzyme is expressed differently among the organs. Furthermore, within a given organ there are strong differences according to the cell type. The quantification of the enzyme by immunoblotting reveals that liver mitochondria have the highest content (more than 3% in protein mass). This content is 3,5 and 10 times lower in kidney, heart and brain mitochondria, respectively. Parallel D-beta-hydroxybutyrate dehydrogenase activity measurements on isolated mitochondria show differences in molecular activity of this enzyme according to the tissue origin. Due to the phospholipid requirement of this enzyme these differences in molecular activity are related to specific membrane lipid composition.     

Phylogenetic position of Gluconobacter species as a coherent cluster separated from all Acetobacter species on the basis of 16S ribosomal RNA sequences

Abstract The 16S rRNA sequences from the Gluconobacter species G. asaii G. cerinus and G. frateurii were determined and compared with homologous sequences from published databases and sequences of G. oxydans and Acetobacter species previously described [Sievers M., Ludwig W. and Teuber M. (1994) System. Appl. Microbiol. 17, 189–196]. The Gluconobacter species have unique 16S rRNA sequences and exhibit sequence similarity values of 97.4 to 99.1%, corresponding to 36 to 14 base differences. The phylogenetic tree inferring methods (distance matrix, maximum parsimony and maximum likelihood) show that the species of Gluconobacter form a coherent, closely related cluster. Based on the distance matrix method including Rhodopila globiformis as an outgroup reference organism, Gluconobacter is well separated from Acetobacter .     

Multiple Stressors in Agricultural Streams: A Mesocosm Study of Interactions among Raised Water Temperature,Sediment Addition and Nutrient Enrichment

Changes to land use affect streams through nutrient enrichment, increased inputs of sediment and, where riparian vegetation has been removed, raised water temperature. We manipulated all three stressors in experimental streamside channels for 30 days and determined the individual and pair-wise combined effects on benthic invertebrate and algal communities and on leaf decay, a measure of ecosystem functioning. We added nutrients (phosphorus+nitrogen; high, intermediate, natural) and/or sediment (grain size 0.2 mm; high, intermediate, natural) to 18 channels supplied with water from a nearby stream. Temperature was increased by 1.4°C in half the channels, simulating the loss of upstream and adjacent riparian shade. Sediment affected 93% of all biological response variables (either as an individual effect or via an interaction with another stressor) generally in a negative manner, while nutrient enrichment affected 59% (mostly positive) and raised temperature 59% (mostly positive). More of the algal components of the community responded to stressors acting individually than did invertebrate components, whereas pair-wise stressor interactions were more common in the invertebrate community. Stressors interacted often and in a complex manner, with interactions between sediment and temperature most common. Thus, the negative impact of high sediment on taxon richness of both algae and invertebrates was stronger at raised temperature, further reducing biodiversity. In addition, the decay rate of leaf material (strength loss) accelerated with nutrient enrichment at ambient but not at raised temperature. A key implication of our findings for resource managers is that the removal of riparian shading from streams already subjected to high sediment inputs, or land-use changes that increase erosion or nutrient runoff in a landscape without riparian buffers, may have unexpected effects on stream health. We highlight the likely importance of intact or restored buffer strips, both in reducing sediment input and in maintaining cooler water temperatures.     

Rhodamine phalloidin F-actin: critical concentration versus tensile strength.

The mechanic and elastic properties of rhodamine phalloidin F-actin were investigated as a function of the ionic strength and in the absence of Mg2+. By increasing ionic strength from 3 to 19 mM, critical concentration decreased from 146 to 36 nM and the yield strength increased from 5.6 pN to 28.6 pN. At the ionic strength of 12-13 mM, the elastic modulus by stretching increased by 330-430 kP. nm-1 up to the break point, where it was 38-44.2 MP. The work required to break the filament, 403-439 kJ.M-1 provides an estimate of the free energy of annealing of rhodamine phalloidin F-actin, the annealing constant being 2.8 x 1074 M-1.