A Molecular Imprinted Polymer as a Flow-Through Optical Sensor for Oxazepam.

A flow-through optosensing system for oxazepam recognition with fluorescence detection was performed by means of a molecular imprinted polymer based on its acid hydrolysis product, 2-amino-5-chlorobenzophenone. The synthesis was conducted via a noncovalent imprinting methodology, using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a cross-linking agent. Hydrolysis (types and concentration of acids), polymer retention capacity, binding properties, and elution (selectivity and reversibility) conditions were optimized. The selected molecular imprinted polymer had a molar ratio composition of 1 : 6 : 45 (template : functional monomer : cross-linker). The proposed method was applied to the determination of oxazepam in a pharmaceutical formulation. External standard calibration, standard additions calibration, and Youden's calibration were carried out in order to evaluate constant and proportional errors due to the matrix. The developed metabolite-based recognition system for benzodiazepines is an innovative procedure that could be followed in routine and quality control assays.

Binding of several benzodiazepines to bovine serum albumin: Fluorescence study.

The interactions of lorazepam, oxazepam and bromazepam with bovine serum albumin (BSA) were studied by fluorescence spectrometry. The Stern-Volmer quenching constants and corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. The binding constants and the number of binding sites were also investigated. The distances between the donor (BSA) and the acceptors (benzodiazepines) were obtained according to fluorescence resonance energy transfer and conformational changes of BSA were observed from synchronous fluorescence spectra.

Modificaciones de las lipoproteínas de baja densidad en pacientes diabéticos / Modification of low-density lipoproteins in diabetic patients

En los pacientes diabéticos, las lipoproteínas presentan frecuentemente cambios cualitativos y cuantitativos en su composición y varios pasos del metabolismo están alterados. Estas anormalidades contribuyen al incremento del riesgo de enfermedad cardiovascular y se la conoce como "dislipemia aterogénica". En este trabajo se estudiaron en una población de pacientes diabéticos las modificaciones fisicoquímicas por glicación de sus lipoproteínas de baja densidad (LDL), los resultados se compararon con una población control y con ensayos de glicación in vitro. Las LDL se aislaron por precipitación selectiva y las modificaciones se evaluaron por el incremento de fructosamina, el consumo de los residuos e-amino de lisina, guanidinio de arginina y la disminución de la fluorescencia del grupo indol del triptofano. Los procedimientos seleccionados resultan accesibles al laboratorio clínico. Los valores medios para todos los analitos medidos fueron significativamente diferentes de los de la población control (p0,0001); de la comparación del ensayo in vitro pudo deducirse que las alteraciones de los residuos de arginina serían un marcador temprano de las modificaciones por glicación, en tanto que las producidas en los residuos de lisina y triptofano representarían indicadores de las alteraciones de mediano plazo. Vale considerar la utilidad de evaluar simultáneamente en una misma molécula, indicadores de control glucémico tanto de corto como de mediano plazo, teniendo en cuenta su relevancia en la fisiopatología de la ateroesclerosis.

In diabetic patients, lipoproteins usually show qualitative and quantitative changes in their composition and as a consequence, severa! metabolic pathways are altered. These alterations contribute to an increase in the risk of cardiovascular disease, also known as "atherogenic dyslipidemia". The present workstudied the physicochemical modifications by glycation of low density lipoproteins (LDL) in a population of diabetic patients. The results were compared with a control population and with the results obtained from an in vitro glycation assay. LDL were isolated byselective precipitation and the modifications were assessed by the increase in fructosamine level, the decrease of e-amino group of lysine, guanidinio of arginine and indol fluorescence of tryptophan residues. The select methods are available for clinical laboratories. The mean valúes for all the measured analytes were significantly different from those obtained for the control population (p< 0.0001). Taking into account the results of the in vitro kinetlcs assays, it can be assumed that the modifications of the arginine residues would be an early glycation marker while the changes in the lysine and triptophan residues may be considered middle term alterations. It is worth remarking the usefulness of evaluating, early and middle term ¡ndicators of glycaemic control simultaneously in the same molecule. These findings are relevant for the pathophysiology of atherogenesis.

Simultaneous determination of carbaryl and 1-naphthol by first-derivative synchronous non-protected room temperature phosphorescence.

The applicability of non-protected room temperature phosphorescence (NP-RTP) in real samples was demonstrated in the present work. In this methodology, only two reagents, potassium iodide and sodium sulfite, were used to obtain phosphorescent signals. Overlapping of the phosphorescence spectra was resolved by using first-derivative synchronous phosphorimetry. The synchronous first-derivative spectra of carbaryl and 1-naphthol in the mixture were completely separated by changing the synchronous wavelength interval; with 240 nm the first-derivative spectra of carbaryl were recorded, while with 200 nm those of 1-naphthol appeared. The intensities in the spectra were proportional to the concentration of carbaryl and 1-naphthol. The calibration graphs were linear up to at least 1.1 x 10(-5) mol L(-1) for carbaryl and 1.3 x 10(-5) mol L(-1) for 1-naphthol, and the correlation coefficients were 0.9971 and 0.9932, respectively. Carbaryl and 1-naphthol were successfully determined by the proposed method in a hydrolyzed sample of a commercial formulation.

Biological effects of a complex of vanadium(V) with salicylaldehyde semicarbazone in osteoblasts in culture: mechanism of action.

Vanadium compounds display important pharmacological actions in vivo and in vitro systems. Semicarbazones are versatile ligands with therapeutic effects. Herein, we report the effects of V(V)O(2)(salicylaldehydesemicarbazone) (V(V)-Salsem) on two osteoblast cell lines in culture (MC3T3-E1 and UMR106). V(V)-Salsem inhibited cell proliferation in a dose response manner. At 100muM, the complex caused an inhibition of ca. 48% and 38% for the normal and the tumoral osteoblasts, respectively (p<0.001). This inhibition could be partially reversed to 35% and 28% by NAC (N-acetylcysteine) and a mixture of vitamins E and C. Changes in cell proliferation correlated with morphological alterations and the disruption of actin cytoskeleton fibers. The complex also enhanced the level of ROS (reactive oxygen species) up to ca. 100% over basal in both cell lines. Activation of ERK signalling cascade was also observed. These events led to apoptosis (up to 44% in MC3T3-E1 and 33% in UMR106 cells). Scavengers of ROS and inhibitors of ERK cascade allowed to elucidate the mechanisms involved in the cytotoxicity. In conclusion, V(V)-Salsem displayed cytotoxic effects on osteoblasts in culture through the production of free radicals and the activation of ERK cascade. These mechanisms triggered the apoptotic events that conveyed to cell death.

Determination of atenolol by the micelle-stabilized room-temperature phosphorescence methodology.

A micellar-stabilized room-temperature phosphorescence (MS-RTP) method for the determination of atenolol has been developed in micellar solutions of sodium dodecylsulphate (SDS) in the presence of thallium(I) as a heavy atom and sodium sulphite as an oxygen scavenger. The effects of thallium(I) nitrate, SDS and sodium sulphite concentrations on atenolol MS-RTP intensity were studied. Optimized conditions to obtain maximum sensitivity were 0.015 mol/L thallium(I) nitrate, 0.1 mol/L SDS and 0.0075 mol/L sodium sulphite. The maximum phosphorescence signal was completely developed in 10 min and the intensity was measured at lambda(ex) = 272 nm and lambda(em) = 412 nm. The linear range of application obtained was 2.01-16.00 microg/mL. The detection limit estimated from the least-squares regression analysis was 0.86 microg/mL and the relative standard deviation of 10 replicates was 1.7%. The proposed method was applied to the determination of atenolol in a pharmaceutical formulation. The quantitation was carried out by means of standard calibration, standard-additions calibration and Youden calibration. These three experiments were necessary to evaluate the presence of constant and proportional errors due to the matrix.

Alendronate induces anti-migratory effects and inhibition of neutral phosphatases in UMR106 osteosarcoma cells.

Bisphosphonates are nonhydrolysable pyrophosphate analogues that prevent bone loss in several types of cancer. However, the mechanisms of anticancer action of bisphosphonates are not completely known. We have previously shown that nitrogen-containing bisphosphonates directly inhibit alkaline phosphatase of UMR106 rat osteosarcoma cells. In this study, we evaluated the effects of alendronate on the migration of UMR106 osteosarcoma using a model of multicellular cell spheroids, as well as the alendronate effect on neutral phosphatases. Alendronate significantly inhibited the migration of osteoblasts in a dose-dependent manner (10(-6)-10(-4) M). This effect was also dependent on calcium availability. The spheroid morphology and distribution of actin fibers were also affected by alendronate treatment. Alendronate dose-dependently inhibited neutral phosphatase activity in cell-free osteoblastic extracts as well as in osteoblasts in culture. Our results show that alendronate inhibits cell migration through mechanisms dependent on calcium, and that seem to involve inhibition of phosphotyrosine-neutral-phosphatases and disassembly of actin stress fibers.

Indirect fluorometric determination of diclofenac sodium.

A simple and easy method of analysis for diclofenac sodium is reported. A spectrofluorometric method for the microdetermination of diclofenac sodium has been developed through its reaction with cerium(IV) in an acidic solution and measurement of the fluorescence of the Ce(III) ions produced. Under the optimum experimental conditions for the oxidation reaction, 1.0 M H2SO4 with 90 min of heating time (100 degrees C), the range of application is 124.3-600 ng mL(-1) and the limit of detection is 72.7 ng mL(-1). The proposed method was applied to the determination of diclofenac sodium in pharmaceutical tablets. The results of the analysis show a good agreement with those obtained by the official USP 27 HPLC method.

Osteogenic activity of vanadyl(IV)-ascorbate complex: evaluation of its mechanism of action.

We have previously shown that different vanadium(IV) complexes regulate osteoblastic growth. Since vanadium compounds are accumulated in vivo in bone, they may affect bone turnover. The development of vanadium complexes with different ligands could be an alternative strategy of use in skeletal tissue engineering. In this study, we have investigated the osteogenic properties of a vanadyl(IV)-ascorbate (VOAsc) complex, as well as its possible mechanisms of action, on two osteoblastic cell lines in culture. VOAsc (2.5-25 microM) significantly stimulated osteoblastic proliferation (113-125% basal, p<0.01) in UMR106 cells, but not in the MC3T3E1 cell line. VOAsc (5-100 micrioM) dose-dependently stimulated type-I collagen production (107-156% basal) in osteoblasts. After 3 weeks of culture, 5-25 microM VOAsc increased the formation of nodules of mineralization in MC3T3E1 cells (7.7-20-fold control, p<0.001). VOAsc (50-100 microM) significantly stimulated apoptosis in both cell lines (170-230% basal, p<0.02-0.002), but did not affect reactive oxygen species production. The complex inhibited alkaline and neutral phosphatases from osteoblastic extracts with semi-maximal effect at 10 microM doses. VOAsc induced the activation and redistribution of P-ERK in a time- and dose-dependent manner. Inhibitors of the mitogen activated protein kinases (MAPK) pathway (PD98059 and UO126) partially blocked the VOAsc-enhanced osteoblastic proliferation and collagen production. In addition, wortmanin, a PI-3-K inhibitor and type-L channel blocker nifedipine also partially abrogated these effects of VOAsc on osteoblasts. Our in vitro results suggest that this vanadyl(IV)-ascorbate complex could be a useful pharmacological tool for bone tissue regeneration.

Biosensors of inorganic lead exposure and effect in an adult amphibian.

Lead (Pb) is a ubiquitous environmental pollutant, widely distributed, representing a high toxicological and ecotoxicological risk. Several morphological, functional, and biochemical parameters have been proposed as biomarkers of effect and exposure to Pb. The information related to adverse effects of Pb is not abundant for adult amphibians. These animals are of interest, because during their development they move from aquatic to terrestrial habitats, which may be polluted by the metal since they are receptors of products generated by anthropogenic activities. Previous studies carried out on the adult South American toad Bufo arenarum (Amphibia, Anura) showed that it has a high tolerance to lead and studied the effect of sublethal doses of the metal on the erythrocyte osmotic fragility and delta-ALAD activity. It was also shown that after a single injection of Pb, a significant increase in the number of reticulocytes was produced, suggesting the suitability of those cell counts as a biomarker of exposure to the metal; its impact on the immune system of the toads was also studied. In this work we extend our early studies on the same species evaluating the chronic effect of sublethal Pb (equivalent to 5.6% of the 120-h LD-50) on free erythrocyte protoporphyrin (FEP) and blood Pb and delta-ALAD activity; blood lead was positively associated with a significant decrease in the enzyme activity and to an increase in the FEP level. Pb concentration in target organs (liver, spleen, femur, and kidney) and the total cumulated amount as well as its impact over the mass of those organs were also determined. In addition, the magnitude of the possible depuration through urine and intestine was evaluated. Our results showed that FEP, delta-ALAD, and blood Pb are reliable biosensors of chronic metal intoxication, the former being the marker with the highest sensitivity.