Method for quantifying NSAIDs and clofibric acid in aqueous samples, lumpfish (Cyclopterus lumpus) roe, and zebrafish (Danio rerio) eleutheroembryos and evaluation of their bioconcentration in zebrafish eleutheroembryos.

Pharmaceuticals such as nonsteroidal anti-inflammatory drugs (NSAIDs) and lipid regulators are being repeatedly detected at low concentrations (pg · mL-1-ng · mL-1) in the environment. A large fraction of these compounds are ionizable. Ionized compounds show different physico-chemical properties and environmental behavior in comparison to their neutral analogs; as a consequence, the quantification methods currently available, based on the neutral molecules, might not be suitable to detect the corresponding ionized compounds. To overcome this problem, we developed a specific analytical method to quantify NSAIDs and lipid regulators (i.e., ibuprofen, diclofenac, naproxen, and clofibric acid) and their ionized compounds. This method is based on three steps: (1) the extraction of the organic compounds with an organic solvent assisted with an ultrasonic probe, (2) the cleaning of the extracts with a dispersive SPE with C18, and (3) the determination of the chemical compounds by GC-MS (prior derivatization of the analytes). We demonstrated that the proposed method can successfully quantify the pharmaceuticals and their ionized compounds in aqueous samples, lumpfish eggs, and zebrafish eleutheroembryos. Additionally, it allows the extraction and the cleanup of extracts from small samples (0.010 g of wet weight in pools of 20 larvae) and complex matrixes (due to high lipid content) and can be used as a basis for bioaccumulation assays performed with zebrafish eleutheroembryos in alternative to OECD test 305.

A novel flow injection chemiluminescence determination of Cr(VI) with Dichlorotris(1,10-phenanthroline)ruthenium(II).

A selective novel reverse flow injection system with chemiluminescence detection (rFI-CL) for the determination of Cr(VI) in presence of Cr(III) with Dichlorotris (1,10-phenanthroline)ruthenium(II), (Ru(phen)(3)Cl(2)), is described in this work. This new method is based on the oxidation capacity of Cr(VI) in H(2)SO(4) media. First, the Ruthenium(II) complex is oxidized to Ruthenium(III) complex by Cr(VI) and afterwards it is reduced to the excited state of the Ruthenium(II) complex by a sodium oxalate solution, emitting light inside the detector. The intensity of chemiluminescence (CL) is proportional to the concentration of Cr(VI) and, under optimum conditions, it can be determined over the range of 3-300 µg L(-1) with a detection limit of 0.9 µg L(-1). The RSD was 8.4% and 1.5% at 5 and 50 µg L(-1), respectively. For the rFI-CL method various analytical parameters were optimized: flow rate (1 mL min(-1)), H(2)SO(4) carrier concentration (20% w/V), Ru(phen)(3)Cl(2) concentration (5mM) and sodium oxalate concentration (0.1M). The effect of Cr(III), Fe(III), Al(III), Cd(II), Zn(II), Hg(II), Pb(II), Ca(II) and Mg(II), was studied. The method is highly sensitive and selective, allowing a fast, on-line determination of Cr(VI) in the presence of Cr(III). Finally, the method was tested in four different water samples (tap, reservoir, well and mineral), with good recovery percentage.

Speciation of nickel by HPLC-UV/MS in pea nodules.

A new and sensitive methodology based on normal phase HPLC has been developed for the speciation of nickel in low-complexity plant extracts. The method combines a silica stationary phase column, a 9:1 (v/v) hexane:ethanol mixture as mobile phase, and the detection of nickel complexes by either UV or MS. The developed methodology was applied to the speciation of nickel complexes in the cytoplasm of pea root nodules. Results obtained indicate that nickel citrate and nickel malate accounts for 99% of nickel present in pea nodule cytoplasm fraction. The low detection limit of the method (<0.2 nM) enables nickel speciation in non-hyperaccumulator plants.

Development of a microalgal PAM test method for Cu(II) in waters: comparison of using spectrofluorometry.

Test methods are needed to monitor Cu concentrations in reservoirs and water supplies. Dictyosphaerium chlorelloides (Chlorophyta) cells were immobilized in a silicate sol-gel and the toxic effects of Cu(II) were examined using different techniques: fluorescence measurements (using a spectrofluorometer with an optic fiber coupled to a flow cell or a 96-well-plate reader) or by Pulse Amplitude Modulation (PAM) parameters using a portable instrument and the pulse saturation method. Fm' and qN were the most sensitive indicator parameters when performing Cu analysis in water. D. chlorelloides PAM biosensor presented a detection limit of 0.6 mg l(-1) for Cu(II), within the limits to establish if Cu concentrations exceeded regulatory levels. Moreover, a 1.9 mg Cu l(-1) (30 microM) resistant strain of the D. chlorelloides microalgae was produced in order to obtain more selectivity on the metal determination.

Determination of benzimidazole fungicides in water samples by on-line MISPE-HPLC.

An analytical methodology based on an on-line sample enrichment of water samples by means of an imprinted polymer, and the separation of benzimidazole compounds within a C(18) column by ion-pair reversed-phase liquid chromatography, has been developed. The molecularly imprinted polymer has been synthesized by precipitation polymerization using thiabendazole as template molecule, methacrylic acid as functional monomer, and divinylbenzene as cross-linker. Initial experiments carried out by solid-phase extraction on cartridges demonstrated a clear imprint effect for thiabendazole, as well as the ability of the imprinted polymer to selectively rebind several benzimidazole compounds. The developed methodology has been applied to the quantification of thiabendazole, carbendazim, and benomyl in river, tap, and well water samples within a single analytical run at concentration levels below the legislated maximum concentration levels. In this sense, detection limits of 2.3-5.7 ng.L(-1) have been obtained for the analysis of benzimidazole fungicides in different water matrices. Recoveries obtained for the determination of benzimidazole fungicides in spiked samples ranged from 87% to 95%, with RSD below 5% in all cases.

Estudio anatómico de la transferencia de los nervios accesorio y toracodorsal al nervio cubital en el gato / Anatomic study of spinal accesory and thoracodorsal nerves transfer to ulnar nerve in cats

Las lesiones del plexo braquial son una de las patologías más graves y con mayor número de secuelas del miembro superior. En el momento actual las transferencias nerviosas se encuentran en primera línea del armamento terapéutico para reconstruir funciones proximales del miembro superior. En el estudio que presentamos se realizaron 20 transferencias nerviosas al nervio cubital del gato común, tomando bien el nervio accesorio del espinal (10 casos) o bien el nervio toracodorsal (10 casos). Como grupo controles utilizó el lado contralateral al intervenido. Durante el año siguiente, se evaluó la reinervación mediante estudios electromiográficos, histológicos de nervio y músculo, así como histoquímicos de médula espinal. Tras el análisis de los resultados encontramos que las motoneuronas de ambos nervios donantes son capaces de conseguir reinervaciones parciales del territorio cubital (AU)

A brachial plexus injury is one of the most severe pathologies of the upper limb, and also has severe sequels. In the actual state of the art, nerve transfers are being used as first line of therapeutic approach in the reconstruction of proximal functions of the upper limb. In this study 20 nerve transfers were made to the ulnar nerve of the cat, using the spinal accessory nerve (10 cases) or the thoracodorsal nerve (10cases). The opposite side was used as control. During next year, reinnervation was assessed by electromyography, nerve and muscle histology and histochemical evaluation of the spinal cord. We found that motoneurons of both donor nerves are able to make partial reinervation of the ulnar nerve territory (AU)

Anatomical and functional connectivity of the transected ulnar nerve after accessory nerve neurotization in the cat.

AIM: The objective of this experimental study was to test the capacity of accessory nerve motoneurons to innervate muscles of the ulnar nerve territory after direct anastomosis. METHODS: This study used 22 cats in two groups: experimental group (15 cats) and control group (7 cats). The first one was followed during twelve months using electromyographic records every two months postsurgery; muscle and nerve histological assessment and counting horseradish peroxidase-labeled motoneurons. RESULTS: Our results showed that reinnervation was achieved in 12/15 nerves. The number of HRP labelled medullar motoneurons after anastomosis showed a significant statistic difference with a simple ulnar nerve transection; there was no significant statistic difference in labelling between the group with an anastomosis and the one with a simple accessory nerve transection. CONCLUSIONS: Direct anastomosis between the spinal accessory nerve and the ulnar nerve is achievable and thus, the accessory spinal nerve is another possible choice for correcting the motor deficit arising from lower brachial plexus avulsion, but the limited number of motoneurons would only allow partial reinnervation..

Semi-covalent imprinted polymer using propazine methacrylate as template molecule for the clean-up of triazines in soil and vegetable samples.

A semi-covalent imprinted polymer was prepared by precipitation polymerisation using propazine methacrylate as template molecule, ethylene glycol dimethacrylate as cross-linker and toluene as porogen. After removal of propazine by basic hydrolysis of the covalent bond, the optimum loading, washing and elution conditions for the solid-phase extraction of the selected triazines were established. The binding sites present in the polymeric matrix were characterised by fitting the experimental results of several rebinding studies to the Langmuir-Freundlich isotherm. Subsequently, an analytical methodology based on molecularly imprinted solid-phase extraction (MISPE) was developed for the determination of several triazinic herbicides in soil and vegetable samples. Following this procedure, a good degree of clean-up of the sample extracts was easily achieved, allowing the HPLC-UV determination of selected triazines in complex samples at low concentration levels.

Single fiber electromyography (SFEMG) in mitochondrial diseases (MD).

Conventional EMG, nerve conduction studies and SFEMG were performed in 18 patients with various phenotypes of MD. 14 cases showed findings consistent with mild myopathy, 2 patients signs of sensory-motor axonal neuropathy and 2 cases a mixture of myopathy and axonal neuropathy. Motor unit fiber density was mild increased in 8 out of 13 tested cases. Jitter was abnormal in 10 out of 18 tested patients. Jitter abnormalities were not related to myopathic or neurogenic features in the EMG study, and may be observed in muscles without clinical weakness. The results suggest the existence of neuromuscular transmission disturbances in patients with MD.

Characterisation and quality assessment of binding sites on a propazine-imprinted polymer prepared by precipitation polymerisation.

In this paper, the Langmuir-Freundlich isotherm (LF) is used to characterise a propazine-imprinted polymer obtained by precipitation polymerisation (MIP-P). Different rebinding studies were carried out allowing to explain the different interactions taking place between the molecularly imprinted polymer and six triazinic herbicides (desisopropylatrazine, desethylatrazine, simazine, atrazine, propazine and prometryn). The LF fitting parameters obtained (total number of binding sites, heterogeneity index and mean binding affinity) were compared to those obtained in a previous work for a propazine-imprinted polymer prepared by bulk polymerisation (MIP-B). From that study, it was concluded that precipitation polymerisation yielded polymers with a more homogeneous binding site distribution and higher affinity constants.

Permanently oriented antibody immobilization for digoxin determination with a flow-through fluoroimmunosensor.

This paper reports a new flow-through fluoroimmunosensor, the function of which is based on antibodies immobilized on an inmunoreactor of controlled-pore glass (CPG), for determination of digoxin, used in the treatment of congestive heart failure and artery disease. The immunosensor has a detection limit of 1.20 microg L(-1) and provides high reproducibility (RSD=4.5% for a concentration of 0.0025 mg L(-1), and RSD=6.7% for 0.01 mg L(-1)). The optimum working concentration range was found to be 1.2 x 10(-3)-4.0 x 10(-2) mg L(-1). The lifetime of the immunosensor was about 50 immunoassays; if stored unused its lifetime can be extended to three months. A sample speed of about 10-12 samples per hour can be attained. Possible interference from substances with structures similar to digoxin (morphine, heroin, tebaine, codeine, pentazocine and narcotine) was investigated. No cross-reactivity was seen at the highest digoxin: interferent ratio studied (1:100). The proposed fluoroimmunosensor was successfully used to determine digoxin concentrations in human serum samples.

Clean-up of triazines in vegetable extracts by molecularly-imprinted solid-phase extraction using a propazine-imprinted polymer.

An analytical methodology based on a molecularly imprinted solid-phase extraction (MISPE) procedure was developed for the determination of several triazines (atrazine, simazine, desethylatrazine (DEA), desisopropylatrazine (DIA), and propazine) in vegetable samples. A methacrylic acid-based imprinted polymer was prepared by precipitation polymerisation using propazine as template and toluene as porogen. After removal of the template by Soxhlet extraction, the optimum loading, washing, and elution conditions for MISPE of the selected triazines were established. The optimised MISPE procedure was applied to the extraction of the selected triazines in pea, potato, and corn sample extracts and a high degree of clean-up was obtained. However, some remaining interferences, non-specifically and strongly bound to the polymeric matrix, appeared in the chromatogram, preventing quantification of DIA in potatoes and DIA, DEA, and propazine in corn samples. Thus, a new clean-up protocol based on the use of a non-imprinted polymer for removal of these interferences prior to the MISPE step was developed. By following the new two-step MISPE procedure, the matrix compounds were almost completely removed, allowing the determination of all the triazines selected at concentration levels below the established maximum residue limits, making the developed procedure suitable for monitoring these analytes in vegetable samples.

A reservable antiadhesion barrier gel reduces the perineural adhesions in rats after anastomosis.

AIM: The objective of this experimental study was to assess the effectiveness of ADCON T/N in reducing perinerural adhesions when applied around a previously exposed and then anastomosed peripheral nerve in a rat model. METHODS: Sixteen adult 8-month-old male Wistar rats were used for this study. After the ulnar nerve was exposed bilaterally, the nerves were sectioned and anastomosised. ADCON T/N gel was applied around the nerve suture on 1 limb, while the contralateral limb was left untreated (sham operated control). Treatments were administered according to a randomised schedule revealed to the operator only after the perineural adhesions observed during the 2nd operation had been dissected and complete haemostasis achieved. In other group of 4 Wistar rats, the ulnar nerve was sectioned and the 2 ends connected with a 5 mm silastic tube that was placed bilaterally in each rat but on only one side, chosen randomly, was the nerve treated with ADCON TN and placed within the tube on that side. RESULTS: Three months after the 1st operation, the same nerves were exposed again in all animals under general anesthesia before microsurgical external neurolysis. The neurolysis sites in 16 rats were evaluated by blinded surgical dissection immediately before death. Parameters examined included: quality of wound healing, possible adverse effects, presence of residual implant material and perineural adhesions. Perineural adhesions around the ulnar nerve were visually scored using a 4-point qualitative scale (Mean 2.81 without gel, and 1.18 with ADCON T/N. p=0.017). There was no evidence of persistence of residual implant material in the ADCON T/N treated sites. No significant difference between the average density of myelinated axons was observed in either group. All ADCON T/N treated rats (4 animals) showed axonal growth through the tube connecting the 2 ends. CONCLUSION: ADCON-TN decreased fibrosis around the nerve anastomosis sites and did not impede growth between the severed ends of the axons. If reoperation becomes necessary, a reduction of fibrosis would facilitate access to the old site and decrease the risk of nerve lesions.

Molecular recognition in a propazine-imprinted polymer and its application to the determination of triazines in environmental samples.

An analytical methodology for the determination of triazines in environmental samples incorporating a molecularly imprinted solid-phase extraction (MISPE) process using a propazine-imprinted polymer was developed. Two different polymers were prepared using acetonitrile or toluene as porogen, and their optimum loading, washing, and elution conditions were established. Although both polymers were able to recognize several chlorotriazines (propazine, atrazine, simazine, desethylatrazine, and desisopropylatrazine), the polymer prepared in toluene showed the best performance and was also capable of recognizing a methylthiotriazine (prometryn). A binding study carried out in this polymer demonstrated that it possesses heterogeneous binding sites with different binding abilities. From this study, it was also concluded that desethylatrazine and desisopropylatrazine displace the other triazines at high concentrations, including the template molecule. The accuracy and selectivity of the MISPE process developed was verified using a certified reference material for drinking water containing atrazine and simazine among other commonly used pesticides. Finally, the MISPE procedure was successfully applied to the cleanup of drinking and groundwater, soil, and corn sample extracts, and the triazines were determined by micellar electrokinetic chromatography.

Trace-level determination of triazines and several degradation products in environmental waters by disk solid-phase extraction and micellar electrokinetic chromatography.

An analytical method combining disk solid-phase extraction with micellar electrokinetic chromatography has been developed for the determination of atrazine, simazine, hydroxyatrazine, deisopropylatrazine, deethylatrazine, propazine and prometryn in water samples. The influence of the buffer and sodium dodecyl sulfate (SDS) concentration, pH and organic modifier on the separation has been studied. Baseline separation of the seven triazines was achieved under the following conditions: 10 mM borate buffer, 60 mM SDS, 20% methanol and pH 9.2. C18-bonded silica and poly(styrene-divinylbenzene) (PS-DVB) disks were evaluated for solid-phase extraction of the selected pesticides (11 of water sample). Using two PS-DVB disks, quantitative recoveries were obtained for all pesticides tested. The method was successfully applied for the determination of the seven triazines in drinking and well water at the 0.1 microg l(-1) and 0.5 microg l(-1) concentration levels, respectively. The detection limits for these analytes using the proposed analytical method were within the 0.02-0.06 microg l(-1) range in drinking water and the 0.06-0.30 microg l(-1) range in well water.

Development of a fluoroimmunosensor for theophylline using immobilised antibody.

A flowthrough theophylline fluoroimmunosensor with an antibody covalently immobilised on a solid support has been developed. The immobilisation technique proposed in this paper used Protein-A on control pore glass (Protein A-CPG) in an immunoreactor and dimethylsuberimidate as a cross-linking agent. Several supports and cross-linking reagents were tested in order to obtain oriented immobilisation and thus efficiency of the immunological reaction and reusability of the immunosensor. The immunosensor performance characteristics were established. The precision expressed as RSD, was 1.6%; the detection limit was 3 mug l(-1); the immunoreactor lifetime was established in 80 assays and there were no interferences with structurally similar compounds such as aminophylline, dihydroxypropyltheophylline and caffeine in the determination of the analyte. This fluoroimmunosensor was applied to determine theophylline in human serum samples from patients of the Puerta de Hierro Hospital in Madrid. The results obtained show that there are no significant differences between the proposed immunosensor and the high-pressure liquid chromatographic method with UV detection used by the Hospital, thus demonstrating the validity of the method.

Anatomical and functional connectivity of the transected ulnar nerve after intercostal neurotization in cats.

OBJECT: Acute transfer of three intercostal nerves to the ulnar nerve was performed in cats for histological and clinical evaluation of a distal muscle reinnervation. METHODS: Infraclavicular intercostal-ulnar communications were created after dividing the motor branches of the upper intercostal nerves in 14 adult cats. Reinnervation of distal forelimb muscles in the ulnar territory was assessed by electromyographic (EMG) studies and motor function rating each month until 18 months postsurgery. In five of these treated animals, and in tour controls, horseradish peroxidase (HRP) was applied to the ulnar or intercostal nerves to study the amount and distribution of retrograde motor neuron labeling in the spinal cord. Also, samples of reinnervated muscles and neurotized ulnar nerves were processed to assess regeneration. Simple ulnar transection without reconstruction led to permanent atrophy of ulnar muscles, lack of recovery according to EMG or clinical studies, and disappearance of the ulnar motor neuron pool. In contrast, ulnar neurotization with the intercostal nerves led to a high rate of functional recovery, which began 5 months postsurgery, and progressed from muscle activity synchronized with ventilatory movements to spontaneous movements that were independent of respiration. This recovery was accompanied by substantial retrograde labeling of intercostal motor neurons after HRP application in the ulnar nerve. Cell counts showed that practically the whole motor neuron pool of the involved intercostal nerves contributed to reinnervation of the transected ulnar nerve. CONCLUSIONS: These findings demonstrate that the use of intercostal nerves to neurotize long brachial plexus nerves can achieve long-lasting and successful reinnervation of distal forelimb muscles.

Flow-through fluorescence immunosensor for atrazine determination.

A new flow-through fluoroimmunosensor for atrazine determination based on the use of protein A immobilized on controlled pore glass as immunoreactor is reported. The support, placed in the optical path of the flow cell, allows the 'in situ' quantification of atrazine by on-line antigen-antibody binding upon successive injections of both substances. The immunosensor has a detection limit of 2.1 mug l(-1), a sample speed of about 10 samples per hour, and provides high reproducibility both within-day (3.2% for 5 mug l(-1) and 2.2% for 30 mug l(-1)) and between days. The optimum working concentration range was 2.1-50 mug l(-1). Possible interferences of other triazines like simazine, desethylatrazine (DEA) and desisopropylatrazine (DIA) were evaluated. Simazine and DIA were not cross-reactive; however, the cross-reactivity for DEA was CR=7.7%. The proposed immunosensor was successfully applied to the determination of atrazine in drinking water and citrus fruits.

Evaluation of congenital dysautonomia other than Riley-Day syndrome.

We report on four children, from different families, who suffer from a congenital autonomic disorder, presumably inherited. Three of them have a sensory neuropathy but do not fit any described hereditary sensory and autonomic neuropathy. All four were examined along with some of their immediate family members. We assessed the cardiovagal, sympathetic adrenergic and sympathetic cholinergic functions with a battery of non-invasive tests. Results demonstrated that sudomotor and cardiovascular orthostatic regulation exhibited the greatest abnormalities, pointing to a predominant impairment of sympathetic components, both cholinergic and adrenergic. The overall examination showed a heterogeneous group of congenital dysautonomia, exclusive of Riley-Day or other recognized hereditary sensory and autonomic neuropathies. We emphasize the importance of studying whole family groups to diagnose subclinical impairment and to provide correct genetic counselling.