Inhibition of ornithine decarboxylase restores hypoxic pulmonary vasoconstriction in endotoxemic mice.

Endotoxemia impairs hypoxic pulmonary vasoconstriction which leads to systemic hypoxemia. This derogation is attributable to increased activity of nitric oxide synthase 2 and arginase metabolism. Gene expression analysis has shown increased expression of ornithine decarboxylase in lungs of endotoxemic mice, a downstream enzyme of arginase metabolism. The aim of this study was to investigate whether inhibition of ornithine decarboxylase increases hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice. Mice received lipopolysaccharides or saline intraperitoneal, and hypoxic pulmonary vasoconstriction was measured using an isolated perfused mouse lung model. Additional mice with and without endotoxemia were pretreated with the ornithine decarboxylase-inhibitor difluoromethylornithine before examination of hypoxic pulmonary vasoconstriction. Hypoxic pulmonary vasoconstriction was defined as the difference of pulmonary arterial pressure between normoxic and hypoxic ventilation. In addition, lung tissue was analyzed using real-time quantitative polymerase chain reaction, Western blot and immunohistochemistry. Lipopolysaccharides caused an up-regulation of ornithine decarboxylase mRNA level (2.73 ± 0.19-fold increase, p < 0.05) as well as ornithine decarboxylase protein level (4.05 ± 0.37-fold increase, p < 0.05). Endotoxemia attenuated hypoxic pulmonary vasoconstriction when compared with untreated control mice (26.3 ± 9.7% vs. 67.0 ± 17.5%). Difluoromethylornithine (20, 100, 500 mg kg-1 body weight intraperitoneal) restored hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice in a dose-dependent way (25.8 ± 9.9%, 57.3 ± 17.2%, 62.3 ± 12.4%) and decreased hypoxic pulmonary vasoconstriction in control mice (53.6 ± 13.6%, 40.0 ± 14.9%, 35.9 ± 12.4%). These results show that endotoxemia induces ornithine decarboxylase expression and suggest that ornithine decarboxylase contributes to the endotoxemia-induced impairment of hypoxic pulmonary vasoconstriction. Inhibition of ornithine decarboxylase might be a target in the therapy of diseases with inflammation impaired hypoxic pulmonary vasoconstriction, like the sepsis-associated acute respiratory distress syndrome (ARDS).

Effects of pharmaceuticals used to treat salmon lice on non-target species: Evidence from a systematic review.

Aquaculture is currently one of the best prospects to help meet the growing need for protein in the human diet. However, aquaculture development and production result in consequences for the environment and also impact other productive activities. Salmon and trout cage culture has required the use of large quantities of pharmaceuticals in order to control outbreaks and the persistence of different pathogens, including sea lice (parasitic copepods), which cause economic losses of around 0.39 €â€¯Kg-1 of salmon produced. The pharmaceuticals currently used for the control of sea lice (cypermethrin, deltamethrin, azamethiphos, hydrogen peroxide) are applied by in situ immersion treatments, enclosing net pens using tarpaulin and then bathing fish with the pharmaceutical. After treatment the pharmaceuticals are released into the surrounding environment, exposing non-target species. Although the effects of such pharmaceutical exposure has been studied in some species, to date a systematic and exhaustive review of these potential effects has not yet been performed. In this study, an exhaustive review of the literature evaluating lethal and sub-lethal effects of anti-sea lice pharmaceuticals on non-target crustaceans and bivalves was performed, in order to assess the extent of the effects, toxicity, variables affecting such toxicity and identify potential synergistic effects previously unexplored. Our results show clear negative effects at concentrations lower than those used in treatments against sea lice in all of the species studied. Likewise, this study demonstrates knowledge gaps that need to be addressed in order to improve our understanding of the effects of these pharmaceuticals on non-target species, ecosystems in general and other productive activities.

Analysis of ethyl glucuronide in human serum by capillary electrophoresis with sample self-stacking and indirect detection.

Ethyl glucuronide (EtG), a metabolite of ethanol, is a marker of recent alcohol consumption. In the past few years, its analysis in body fluids has attracted considerable attention because it closes a gap between short time and long time alcohol markers such as ethanol and carbohydrate-deficient transferrin, respectively. The capillary zone electrophoresis (CZE) analysis of EtG in model mixtures and human serum is reported using uncoated and coated fused-silica capillaries together with acidic buffers in the pH range between 3.2 and 4.4 and indirect detection. In these approaches, separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) is based upon transient isotachophoretic stacking referred to as sample self-stacking. The selection of a favorable bufferco-ion and pH is shown to be crucial for optimized sensitivity. Abuffercomposed of 10 mM nicotinic acid and epsilon-aminocaproic acid (pH 4.3) is demonstrated to provide a detection limit for EtG in serum of 0.1 microg/ml, a value that is relevant for clinical and forensic purposes.

Capillary zone electrophoresis in phosphate buffer--known or unknown?

It has been shown recently that the analysis records in capillary electrophoresis may involve regions with extremely strong electromigration dispersion of peaks. Such a fundamental effect is due to the existence of more centers of symmetry in a given electrolyte system. This paper shows that even such a simple and frequently used electrolyte system as phosphate buffer may exhibit more than one center of symmetry. By using the peak shape diagram approach we have revealed that neutral and alkaline phosphate buffers have two centers of symmetry and one center of extreme dispersion. Model experiments confirmed this new important discovery.

Why robust background electrolytes containing multivalent ionic species can fail in capillary zone electrophoresis.

In this paper it is demonstrated that system peaks are induced by multivalent weak ionic species in background electrolytes. Their existence is derived from SystCharts and from Peak Shape Diagrams and the theory is confirmed experimentally. If analytes are present in a sample, with mobilities approximately equal to the mobility of a system peak, they interact, resulting in a strong increase of electromigration dispersion. This leads to strong peak broadening, peak deformation and a loss of resolution. Typical background electrolytes containing multivalent ionic species, e.g. phosphate and phthalate buffers, often reported to be robust electrolytes, are therefore not always universally applicable and can fail for the application of specific analytes. This paper reports a systematic study of the above phenomena and shows both theoretical and experimental results for background electrolytes containing phosphoric acid and phthalic acid.

Innovative developments in isotachophoresis (displacement electrophoresis).

This Mini Review is aimed at characterizing the innovative developments in isotachophoresis (ITP) during the past few years, discussing in turn new theoretical, analytical, preparative and applicative aspects of this unique separation method. Examples given from our laboratory include the study of the detailed dynamics of the ITP separation of four components by computer simulation and experimental validation in a capillary-type instrument with multiple sensors along the separation trough; the anionic ITP analysis in presence of a strong cathodic electroosmotic flow using an open-tubular fused-silica capillary with on-column multiwavelength detection, and the fractionation of proteins in a screen-segmented, rotating column as well as by recycling ITP.

New aspects of buffering with multivalent weak acids in capillary zone electrophoresis: pros and cons of the phosphate buffer.

Phosphate buffer is frequently used as background electrolyte in capillary electrophoresis. It can cover a broad range of pH due to the three dissociation constants (pK1 = 2.0, pK2 = 7.2, and pK3 = 11.0) of phosphoric acid and because it is UV-light transparent. This contribution brings a theoretical study of the analytical separation performance (sample window, regions of peak symmetry, regions of fronting and tailing peaks) of phosphate buffer, serving as a model of buffering with multivalent weak acids. The study is based on the use of peak shape diagrams and covers the pH range 2.55-11.43. New important general knowledge has been revealed that single multivalent weak acids mimic the performance of background electrolytes with multiple coanionic species for anionic analyses. It is shown that simple phosphate buffers prepared by mixing phosphoric acid and potassium hydroxide may exhibit up to two regions of symmetry, of fronting as well as of tailing zones, on the mobility scale inside the sample window. Moreover they may exhibit a "schizophrenic" region of enormous electromigration dispersion.

Recent progress in capillary isotachophoresis.

This article is a continuation of previous reviews and summarizes the progress of analytical capillary isotachophoresis in the years 1997-1999. Papers reviewed include theoretical and methodological aspects as well as analytical applications. Included are also papers using isotachophoresis and/or isotachophoretic principles as part of multidimensional separation schemes.

Theory of zone separation in isotachophoresis: a diffusional approach.

The qualitative characteristics of isotachophoresis differ substantially from "classical" separation methods such as chromatography or zone electrophoresis. Self-sharpening zone boundaries and step-like concentration profiles are the most specific features of this method, which does not allow the description of the isotachophoretic separation in usual "chromatographic" terms, such as resolution or number of theoretical plates. A theory is presented in this paper, combining the usual isotachophoretic separation characteristics with the theory of isotachoporetic zone boundary, which is the only element of the isotachophoretic system with dispersional properties. This allows us to consider situations near the limits of the isotachophoretic method as far as both selectivity and sample amount (i.e., zone size) are concerned. Based on a simplified expression of the concentration profiles across the isotachophoretic zone boundary, separation and separation limits are described and discussed in terms of resolution, selectivity and zone capacity. Equations are derived showing the relationships between resolution, boundary width, selectivity, sample amount, and column and electrolyte conditions. A simple phenomenological equation is presented, expressing isotachophoretic resolution as a function of only sample amount (or sample zone length) and boundary width. A thermodynamic form of this equation is derived, which is shown to be similar to such an expression for resolution in zone electrophoresis. In both cases resolution is a function of sample selectivity, electric field strength and column length. A simple theoretical model for zone capacity is presented, making it possible to estimate the separation performance of isotachophoretic systems. Based on the presented theory, parallels between isotachophoresis and zone electrophoresis are discussed and both methods are compared.

Recent application and developments of capillary isotachophoresis.

Capillary isotachophoresis is a powerful electromigration separation method with a pronounced capability to concentrate trace components in diluted samples. At present, capillary isotachophoresis is utilized predominantly as the first step in on-line combination with capillary zone electrophoresis. This article is a continuation of previous reviews and summarizes the results published during 1993-1996.

Isotachophoresis of proteins in uncoated open-tubular fused-silica capillaries with a simple approach for column conditioning.

The isotachophoretic determination of proteins in uncoated open-tubular fused-silica capillaries of 50 and 75 microns I.C. with on-column multi-wavelength detection is reported. Small amounts of hydroxypropylmethylcellulose added to the leader provide an efficient method of dynamic column conditioning which allows the high-resolution isotachophoretic determination of most proteins to be performed in the presence of an electro-osmotic flow. Different approaches for cationic and anionic analyses are discussed and illustrated with selected examples.

Fractionation of human serum proteins by capillary and recycling isotachophoresis.

The fractionation of human serum proteins using capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) in presence of low molecular mass spacer compounds is reported. Anionic CITP was performed in an instrument equipped with a Teflon capillary of 0.5 mm ID as well as in an apparatus which features an open-tubular fused-silica capillary of 75 microns ID. RITP was performed in a recycling fast flow focusing apparatus in which fluid flows rapidly through a narrow, rectangular cell and the effluent from each outlet port is reinjected into the electrophoresis chamber through the corresponding input port. Typically, 1 mL serum was processed batchwise within about 2.5 h prior to collection of 30 fractions of about 4 mL each. The fractions were analyzed separately for conductivity, pH and UV absorbance and selected fractions were characterized by an immunoassay for transferrin, as well as by gel isoelectric focusing, two-dimensional gel electrophoresis, CITP and capillary zone electrophoresis. The search for suitable electrolyte systems and spacers was executed by CITP and by computer simulation. For simple configurations, separations predicted by simulation are shown to qualitatively agree with fractionation performed by CITP and RITP. Configurations producing three protein subgroups, the first containing mainly albumin, the second transferrin and the third the globulins, are discussed.

Purification of ovalbumin and lysozyme from a commercial product by recycling isotachophoresis.

The aim of this work was to test the suitability of using recycling isotachophoresis (RITP) for the purification of ovalbumin (OVA) and/or lysozyme (LYSO) from a commercial OVA product containing LYSO and conalbumin (CAL) as major proteinaceous impurities. The search for suitable electrolyte systems and spacers was carried out by capillary isotachophoresis. RITP was performed in a recycling free-flow focusing apparatus in the batch mode with immobilization of the advancing zone structure via a controlled counterflow. Typically 700 mg of the commercial product were processed within 2 h. Enhancement of the sample load was achieved by a feed of sample under counterflow control. The collected fractions were analysed separately for conductivity, pH and ultraviolet absorption, and selected fractions were characterized by analytical capillary electrophoretic methods. All three proteins could be separated and fractionated using suitable spacers. Depending on the chosen conditions either OVA or LYSO could be purified in amounts larger than milligrams per hour (OVA 300 mg/h; LYSO 10 mg/h). The instability of CAL in solution prevented its isolation in the investigated configurations.

Identification of peaks in capillary zone electrophoresis based on actual mobilities.

A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.

Sample self-stacking and sample stacking in zone electrophoresis with major sample components of like charge: general model and scheme of possible modes.

A theoretical study is presented of zone electrophoretic behavior of samples that contain one or more minor analytes and at least one major ionogenic component of like charge. Based on a simple model comprising weak univalent anionic electrolytes, conditions are derived under which analytes are temporarily focused isotachophoretically into very narrow zones by a sample self-stacking effect provided by the major sample components. Requirements for minimal/maximal mobility and a background coion concentration dependent minimal concentration of a major sample component (stacker) are presented. For systems in which sample self-stacking does not apply, an expression for the concentrating factor is derived that involves the effects of both nonselective (classical) and selective sample stacking, the latter being a consequence of electrophoretic separation of the minor analyte from the major component. The theory derived is discussed with selected model examples by using both numerical calculation and computer simulation.

Chloroperoxidase generation of singlet Delta molecular oxygen observed directly by spectroscopy in the 1- to 1.6-mum region.

The (0,0) (1)Delta(g) --> (3)Sigma(-) (g) singlet molecular oxygen chemiluminescence emission from a biological reaction system, chloroperoxidase (EC 1.11.1.10) acting on hydrogen peroxide at low pH (phosphate buffer, pH 2.85) with Cl(-) as a cosubstrate, was recorded with an ultrasensitive IR spectrometer. The strong chemiluminescence emission peak observed at 1.30 mum provides clear evidence of the enzymatic generation of (excited) singlet oxygen from peroxide in this system.

System peaks in capillary zone electrophoresis. 3. Practical rules for predicting the existence of system peaks in capillary zone electrophoresis of anions using indirect spectrophotometric detection.

A theoretical and experimental study of the existence and evolution of system peaks in capillary zone electrophoresis (CZE) with indirect spectrophotometric detection is presented with respect to the effect of the number of coions present in the background electrolyte (BGE). It is shown that in BGEs having only one coion (i.e., the UV-absorbing probe anion), the sample produces only negative peaks due to each analyte anion and no system peaks, with the number of sample peaks corresponding to the number of analytes present in the sample injected. In BGEs containing two coions, a sample with one analyte anion produces one negative indirect detection peak and one system peak. The transition between BGEs having one coion and those with two coions has also been studied and it has been shown that an addition of ca. 5% of the second coion to a single coion BGE causes the resulting BGE to behave macroscopically as a regular two-coion BGE. A descriptive model is proposed, based on transient isotachophoresis (transient stacking) of the sample species and of the coion from the BGE which has the closest mobility to the sample ion. This model explains qualitatively the formation and evolution of the sample peak (containing the sample species and being detected by indirect detection due to displacement of the UV-absorbing probe in its zone) and the system peak (containing no sample species and being a vacancy in the continuum of coions of the BGE). It is shown that the system peak may be positive or negative as it corresponds to the situation where the vacancy of one component of the BGE results in an enhanced concentration of the other component. It has been demonstrated that the system peak is created by a vacancy of that component of the BGE which has the greatest difference in mobility relative to that of the sample species. On indirect detection in BGEs containing two coions the sample displaces predominantly the BGE coion which has a mobility closest to that of the analyte anion. In systems with BGEs containing two coions, a sample having n analytes produces n sample peaks and one system peak, the sign and magnitude of which are dependent on the sum of the UV absorbances of the analytes involved. The effect of bicarbonate from atmospheric CO2 has also been studied and it has been shown that weakly alkaline BGEs with a single anionic UV-absorbing coion, such as those currently used for anionic analyses with indirect detection, may suffer from the presence of system peaks due to bicarbonate.

"Schizophrenic" behavior of zones in capillary zone electrophoresis: explanation of an old problem.

Background electrolyte (BGE) systems with two coions are frequently used in capillary zone electrophoresis (CZE), especially in cases where indirect optical detection is employed. This study investigates the behavior of analytes, which possess mobilities intermediate to those of the BGE coions used. Besides the expected behavior, where the analytes provide either tailing or fronting zones, unusual behavior with extraordinary zone broadening is also observed in some cases. The explanation for this effect is that binary BGE systems involve, as a physico-chemical rule, a region where the analytes are forced by one coion to give tailing zones and simultaneously by the other coion to give fronting zones. The result of this "schizophrenic" situation is extraordinary zone broadening and deterioration of the detection record. A series of experiments is presented showing in a telling way the electromigration behavior of the discussed type of zones as well as the ways to remedy the deterioration of the peak shape by a mere slight changing of the quantitative composition of the BGE used.