La entropía espectral en la monitorización de la profundidad de la anestesia / Spectral entropy in monitoring anesthetic depth

La posibilidad de monitorizar la respuesta cerebral a los hipnóticos en cada una de las fases de la anestesia general, en la que interactúan además estímulos nociceptivos y hemodinámicos, es un tema objeto de intensa investigación desde hace muchos años. Los monitores de profundidad de la anestesia de los que disponemos en la actualidad utilizan el procesamiento del registro electroencefalográfico mediante diferentes algoritmos, algunos no conocidos en su totalidad, para, de una forma simplificada, obtener un parámetro numérico que se aproxima al estado de la actividad cerebral en cada momento. En esta revisión descriptiva se evalúa la capacidad de la entropía espectral de reflejar adecuadamente el comportamiento eléctrico cerebral en respuesta a los hipnóticos y al efecto de los estímulos nociceptivos de diferente intensidad que tienen lugar durante una intervención quirúrgica (AU)

Monitoring the brain response to hypnotics in general anesthesia, with the nociceptive and hemodynamic stimulus interaction, has been a subject of intense investigation for many years. Nowadays, monitors of depth of anesthesia are based in processed electroencephalogram by different algorithms, some of them unknown, to obtain a simplified numeric parameter approximate to brain activity state in each moment. In this review we evaluate if spectral entropy suitably reflects the brain electric behavior in response to hypnotics and the different intensity nociceptive stimulus effect during a surgical procedure (AU)

Spectral entropy in monitoring anesthetic depth. / La entropía espectral en la monitorización de la profundidad de la anestesia.

Monitoring the brain response to hypnotics in general anesthesia, with the nociceptive and hemodynamic stimulus interaction, has been a subject of intense investigation for many years. Nowadays, monitors of depth of anesthesia are based in processed electroencephalogram by different algorithms, some of them unknown, to obtain a simplified numeric parameter approximate to brain activity state in each moment. In this review we evaluate if spectral entropy suitably reflects the brain electric behavior in response to hypnotics and the different intensity nociceptive stimulus effect during a surgical procedure.

Development of a new magnetic beads-based immunoprecipitation strategy for proteomics analysis.

Sample pre-treatment is a critical step for an efficient and reliable analysis and it is highly dependent on the complexity of the matrix. This work shows an example of application of an immunoprecipitation approach using a new magnetic beads-based format, which allows a selective/specific extraction of potential biomarkers from metastatic prostate cancer. Results obtained on the development of this method, and its application for the extraction and pre-concentration of certain biomarkers present in metastatic cell lines of prostate cancer, are presented and discussed. It is concluded that the efficiency of the immunoprecipitation step is clearly compromised by the crosslinking conditions and it is highly dependent on the specificity of selected antibodies. The epoxy magnetic beads used in this work allowed an effective crosslinking of the antibodies contributing to an increased efficiency of the immunoprecipitation step. The optimized conditions for the application of these epoxy magnetic beads for the immunoprecipitation of anti-TUBA3C in metastatic prostate cancer cell line (PC3) are discussed here, as an example of application of the immnuprecipitation approach developed, which resulted in a very efficient tool for a specific extraction and pre-concentration of the targeted protein and, therefore, contributing to the efficiency of further analysis.

Determination of yessotoxins and pectenotoxins in shellfish by capillary electrophoresis-electrospray ionization-mass spectrometry.

Conditions for the determination of lipophilic marine toxins, such as yessotoxins and pectenotoxins (PTX)-6, were investigated with capillary electrophoresis coupled to mass spectrometry (MS) with an electrospray ionization source. After optimization, a simple and MS compatible alkaline volatile buffer solution of ammonium acetate was selected as background electrolyte, with isopropanol/water (80/20, v/v) sheath liquid modified with ammonium acetate used at the electrospray ionization (ESI) source. Previously to capillary electrophoresis (CE) separations, the application of an on-line sample pre-concentration approach based on field-amplified sample stacking was accomplished to increase sensitivity. As a result, the limits of detection provided by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) were 0.02 microg ml(-1) (0.01 microg g(-1)), which corresponded to 1.25 pg for yessotoxin and 0.25 microg ml(-1) (0.13 microg g(-1) and 13.25 pg on capillary) for PTX-6. Accuracy tests showed 97.7% recovery from spiked blank mussel samples that showed no significant matrix influence running under optimal conditions. Intermediate precision was close to 4% relative standard deviation (RSD) for the migration time, and an RSD of 7.5% for peak areas. The method was successfully applied to naturally contaminated seafood samples in which yessotoxins and pectenotoxins-6 were clearly determined. This work demonstrated the potential of CE-ESI-MS to be applied for a sensitive determination of lipophilic toxins from the marine environment as alternative to liquid chromatography-electrospray ionization-single quadrupole mass spectrometry (LC-ESI-MS) for this purpose.

Effect of PSP toxins in white leg shrimp Litopenaeus vannamei Boone, 1931.

Cultured shrimp are often exposed to different toxic products during rearing practices that may affect survival and quality of the product. An evaluation of the effects of paralytic shellfish toxins (PSP) from species of Gymnodinium catenatum in white leg shrimp (Litopenaeus vannamei) has been carried out in this study. Death was observed at doses > 5.0 MU (equivalent to 1.1 mug/g of STX), while lower doses provoked paralysis of pereiopods, disequilibrium, and abdominal spasms in the animals. Target organs such as the heart and brain were severely damaged, with cohesion loss and cell density reduction evidenced by histological analysis. Hence, pond productivity and quality of the harvested organisms may be affected by PSP toxins. This is the 1st report on the effect of PSP toxins from G. catenatum in white eg shrimp.

HPLC and HPCE analysis of microcystins RR, LR and YR present in cyanobacteria and water by using immunoaffinity extraction.

Microcystins, which have their origin in species of cyanobacteria present in freshwaters, have recently been found to be important contaminants of the aquatic environment at trace levels. HPLC and HPCE with UV detection have been applied in the determination of such toxic compounds. Immunoaffinity chromatography for the selective extraction and clean-up of microcystins has been successfully applied to different matrices. Simple protocols for unambiguous determination of these toxins are presented and the immunoaffinity clean-up is compared with conventionally used solid phase extraction procedures. The development and optimisation of an on-line preconcentration procedure based on field amplified sample stacking for the analysis of microcystins by HPCE in the micellar electrokinetic chromatography mode is also described, using borate buffer with the anionic surfactant SDS, as separation electrolyte. Results indicate that sub-nanogram/gram content of microcystins can be detected in water samples, while sub-microgram/gram concentrations can be determined in algae samples.

Effect of pH on the oxidation of paralytic shellfish poisoning toxins for analysis by liquid chromatography.

The effect of pH on the oxidation of individual PSP toxins using both periodate and peroxide oxidations was studied. It was found that the optimum pH for individual toxins varied considerably. For periodate oxidations, pH 8.2 produced the maximum yield of fluorescent products for neosaxitoxin and GTX1/GTX4 while the non-hydroxylated toxins (saxitoxin, GTX2/GTX3, decarbamoyl saxitoxin, GTX5) showed optimum pHs from about pH 10-11.5. Neosaxitoxin and GTX1/GTX4 did not produce significant fluorescent oxidation products with peroxide oxidation at any of the pHs studied (pH 8.2-12.8). The non-hydroxylated toxins all showed optimum pHs above pH 12 with peroxide oxidation. Yields of fluorescent products of these toxins decreased substantially at pHs below pH 12. Neosaxitoxin and GTX1/GTX4 each produced three product peaks at pH 8.2 with periodate oxidation. There was no pH where these toxins produced predominantly a single oxidation product. Decarbamoyl saxitoxin always produced two oxidation products with both oxidation reactions at the pHs studied. However, the relative yields of the products changed with pH. At low pH the second eluting product predominated, while at higher pH values the first eluting product predominated. This pattern was observed for both oxidation reactions. The other non-hydroxylated toxins produced mainly single unique products with both oxidation reactions over the pH range studied. No single pH was found optimum for the oxidation of both hydroxylated and non-hydroxylated toxins without a significant compromise in yield of oxidation products. This has implications for the post column oxidation liquid chromatographic methods, since small changes in pH of the post column oxidant can both positively and negatively affect the yields of oxidation products of toxin mixtures leading to increased error in the subsequent quantitation of these compounds.

New fluorimetric method of liquid chromatography for the determination of the neurotoxin domoic acid in seafood and marine phytoplankton.

Domoic acid (DA) is a neurotoxic amino acid that is responsible for the human toxic syndrome, amnesic shellfish poisoning (ASP). A new rapid, sensitive liquid chromatographic (LC) method has been developed for the determination of DA in various marine samples. DA in marine biological materials was derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and analysed using isocratic reversed-phase LC with fluorimetric detection. The calibration, based on standard DA solutions, was linear in the range 0.04-2 microg/ml (r2=0.998) and the detection limit (3:1, signal/noise) was better than 1 ng/ml. Using the certified reference material (MUS-1B), recoveries of DA from shellfish tissue were >95% (n=5). When a strong anion exchange SPE cartridge was used for sample clean-up the detection limit was 6 ng DA/g mussel tissue. Good reproducibility was achieved with RSD values ranging from 3% for 8 microg DA/g (n=5), to 5% for 0.04 microg DA/g (n=5). This new method was successfully applied to the determination of DA in naturally contaminated shellfish and in marine phytoplankton cultures of Pseudonitzschia sp.

Further studies on the analysis of DSP toxin profiles in galician mussels.

Further studies on mussel samples from Galicia, Spain, have revealed the presence of okadaic acid (OA), dinophysistoxin 2 (DTX2), and the fatty acid acyl esters of both of these toxins as the "DTX3" complex. Measurements were performed with an improved in situ method for the formation of 9-anthryldiazomethane (ADAM) derivatives followed by liquid chromatography with fluorescence detection. Base hydrolysis of DTX3 toxins gave free OA and DTX2, which were determined following ADAM derivatization. Results were confirmed by liquid chromatography/mass spectrometry analyses, and in most of the samples, free DTX2 was the most abundant toxin. However, the OA/DTX2 ratio in the DTX3 conjugated form was different, with OA being the most abundant in all cases. This difference could be due to different rates of metabolism of OA and DTX2 to the acyl esters or due to contamination of the shellfish by the two toxins at different points in time, resulting in less acyl ester formation for one toxin versus the other. The second possibility would be reasonable if two different source organisms were producing the toxins.

Capillary electrophoresis with diode array detection as an alternative analytical method for paralytic and amnesic shellfish toxins.

In recent years the marine environment has been seriously damaged by the presence of several toxic phytoplanktonic species, such as dinoflagellates and other toxic algae, which contaminate shellfish and other marine products. Amnesic and paralytic shellfish toxins are examples of these contaminants. The search for sensitive methodologies for the analysis of such compounds is one of the aims of researchers working in the marine environment. High-performance liquid chromatographic methods have been used for this purpose, allowing the detection of very low levels of these toxins. Recently, capillary electrophoresis (CE) has been used as an alternative for the separation and analysis of these compounds. In this paper, we report the optimization of CE procedures for their analysis. Due to the complexity of the matrix, clean-up procedures are required for removing interferences which affect the electrophoretic resolution. The influences of electrophoretic parameters such as voltage, buffer concentrations and organic modifiers, were studied in order to optimize the electrophoretic system to achieve high resolution as well as an accurate quantitation. Extraction and other steps such as clean-up of samples prior to the electrophoretic analysis have been also studied. Different buffers and organic modifiers were used in order to improve the separation of the toxic components, and consequently to obtain accurate quantitative information about the amount of toxins present in the contaminated samples.

Improved method for preparation and use of 9-anthryldiazomethane for derivatization of hydroxycarboxylic acids. Application to diarrhetic shellfish poisoning toxins.

Application of a method for the "in situ" generation of 9-anthryldiazomethane (ADAM) to the derivatization of the carboxyl function in diarrhetic shellfish poisoning (DSP) toxins revealed the formation of artifact products. Using liquid chromatography-mass spectrometry, it was determined that these artifacts were due to base-catalyzed reactions between the solvent, ethyl acetate, and the hydroxyl groups of the analyte to produce O-acetylated ADAM derivatives. Using a new formulation, with tetrahydrofuran as solvent, it was possible to eliminate these artifact reactions. Various reaction parameters have also been re-optimized to ensure quantitative derivatizations. An assessment method was developed that was useful not only for optimizing reaction parameters, but also for evaluating the reagent potency before use on important samples. Finally, application of the method to the determination of DSP toxins in plankton and mussel tissue was demonstrated.

High-performance liquid chromatographic methods for determination of marine biotoxins.

Paralysing and diarrhetic shellfish poisonings (PSP and DSP) are important intoxications caused by the consumption of shellfish, mainly bivalve molluscs, contaminated by certain species of toxic dinoflagellates present in the marine phytoplankton. Their appearance and massive reproduction take place in some periods of the year, causing the phenomenon commonly known as 'red tide'. This causes significant problems to health and to the economy of the Galician region. The AOAC mouse bioassay is the most commonly used method of analysis for these toxic compounds, being the official method in most countries. Owing to the lack of sensitivity and selectivity of the biological assay, HPLC methods were developed as an alternative methodology. In this paper work carried out on the improvement of the chromatographic conditions in order to achieve accurate information about the PSP and DSP compounds present in the studied samples is reported.

Simultaneous occurrence of diarrhetic and paralytic shellfish poisoning toxins in Spanish mussels in 1993.

Mussel aquaculture is an important industry for the Galician Rias, located in northwestern Atlantic coast of Spain. Since 1976 this region has been seriously affected by incidents of paralytic and diarrhetic shellfish poisoning (PSP and DSP). A particularly bad episode occurred in 1993, when the toxic event lasted for an unusually long period. Many people were stricken ill with unusual symptoms. In this paper we report on the chemical analysis of toxic 1993 mussel samples, using the techniques of liquid chromatography and capillary electrophoresis coupled with mass spectrometry. These analyses revealed a very complex toxin profile, with both PSP and DSP toxins present. Two DSP toxins, okadaic acid and DTX2, were observed, while the primary PSP toxins were B1 and the decarbamoylated derivatives of saxitoxin, GTX2 and GTX3. Small amounts of saxitoxin and other as yet unidentified PSP toxins were observed.