Expression of ion transport proteins and routine metabolism in juveniles of tropical gar (Atractosteus tropicus) exposed to ammonia.

Tropical gar (Atractosteus tropicus) thrives in aquatic habitats with high levels of total nitrogen (TAN) and unionized ammonia (NH3). However, the tolerance of TAN and NH3, the excretion mechanisms involved, and the effects of these chemicals on routine metabolism are still unknown. Therefore, our objectives were to assess the acute toxicity of TAN and NH3 in A. tropicus juveniles after a 96-h exposure (LC50-96 h) to NH4Cl and after chronic exposure to two concentrations (15% and 30% of LC50-96 h TAN) for 12 days, as well as to evaluate the transcriptional effects associated with Rhesus proteins (rhag, rhbg, rhcg) and ion transporters (NHE, NKA, NKCC, and CFTR) in gills and skin; and to determine the effects of TAN and NH3 on routine metabolism through oxygen consumption (µM g-1 h-1) and gill ventilation frequency (beats min-1). LC50-96 h values were 100.20 ± 11.21 mg/L for TAN and 3.756 ± 0.259 mg/L for NH3. The genes encoding Rhesus proteins and ion transporters in gills and skin showed a differential expression according to TAN concentrations and exposure time. Oxygen consumption on day 12 showed significant differences between treatments with 15% and 30% TAN. Gill ventilation frequency on day 12 was higher in fish exposed to 30% TAN. In conclusion, A. tropicus juveniles are highly tolerant to TAN, showing upregulation of the genes involved in TAN excretion through gills and skin, which affects routine oxygen consumption and energetic cost. These findings are relevant for understanding adaptations in the physiological response of a tropical ancestral air-breathing fish.

One Health Approach: An Overview of Q Fever in Livestock, Wildlife and Humans in Asturias (Northwestern Spain).

This study aimed to investigate the seroprevalence of C. burnetii in domestic ruminants, wild ungulates, as well as the current situation of Q fever in humans in a small region in northwestern Spain where a close contact at the wildlife-livestock-human interface exists, and information on C. burnetii infection is scarce. Seroprevalence of C. burnetii was 8.4% in sheep, 18.4% in cattle, and 24.4% in goats. Real-time PCR analysis of environmental samples collected in 25 livestock farms detected Coxiella DNA in dust and/or aerosols collected in 20 of them. Analysis of sera from 327 wild ungulates revealed lower seroprevalence than that found in domestic ruminants, with 8.4% of Iberian red deer, 7.3% chamois, 6.9% fallow deer, 5.5% European wild boar and 3.5% of roe deer harboring antibodies to C. burnetii. Exposure to the pathogen in humans was determined by IFAT analysis of 1312 blood samples collected from patients admitted at healthcare centers with Q fever compatible symptoms, such as fever and/or pneumonia. Results showed that 15.9% of the patients had IFAT titers ≥ 1/128 suggestive of probable acute infection. This study is an example of a One Health approach with medical and veterinary institutions involved in investigating zoonotic diseases.

Expression of long-chain polyunsaturated fatty acids biosynthesis genes during the early life-cycle stages of the tropical gar Atractosteus tropicus.

Long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), including eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), are essential in multiple physiological processes, especially during early development of vertebrates. LC-PUFA biosynthesis is achieved by two key families of enzymes, fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl). The present study determined the expression patterns of genes encoding desaturases (fads1 and fads2) and elongases (elovl2 and elovl5) involved in the LC-PUFA biosynthesis during early life-stages of the tropical gar Atractosteus tropicus. We further analyzed the fatty acid profiles during early development of A. tropicus to evaluate the impact of Fads and Elovl enzymatic activities. Specific oligonucleotides were designed from A. tropicus transcriptome to perform qPCR (quantitative polymerase chain reaction) on embryonic and larval stages, along with several organs (intestine, white muscle, brain, liver, heart, mesenteric adipose, kidney, gill, swim bladder, stomach, and spleen) collected from juvenile specimens. Fatty acid content of feeds and embryonic and larval stages were analyzed. Results show that fads1, fads2, elovl2 and elovl5 expression was detected from embryonic stages with expression peaks from day 15 post hatching, which could be related to transcriptional and dietary factors. Moreover, fads1, fads2 and elovl2 showed a higher expression in intestine, while elovl5 showed a higher expression in liver, suggesting that the tropical gar activates its LC-PUFA biosynthetic machinery to produce ARA, EPA and DHA to satisfy physiological demands at crucial developmental milestones during early development.

Volcanic ashes as a source for nitrated and oxygenated polycyclic aromatic hydrocarbon pollution.

In this study, a novel, simple, and highly sensitive analytical procedure for the quantitative evaluation of oxygenated and nitrated polycyclic aromatic hydrocarbons in volcanic ash samples based on dispersive solid-liquid microextraction (DSLME) coupled to ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Diverse chemometric tools were applied to optimize DSLME working conditions. Thus, a linear calibration curve for all the target analytes in the concentration range from 0.01 to 100 µg g-1 (r2 > 0.994) was obtained. The limits of detection for all the compounds were between 14.6 and 56.0 pg g-1, with high reproducibility (relative standard deviation (RSD) was below 8.1% for all the analytes). Additionally, recoveries ranged from 94.2 to 100%. The applicability of the method was evaluated and the feasibility of the existence of nitrated and oxygenated-PAHs in volcanic ashes at ultra-trace levels was demonstrated, which reveals an unknown source of distribution of these pollutants to the environment. Graphical Abstract.

Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.(AU)

El pez pipa (Microphis brachyurus) es un organismo dulceacuícola con alto potencial económico para la acuarofilia; sin embargo, es necesario implementar su cultivo a través de estudios de fisiología digestiva. Se evaluó el efecto del pH, temperatura e inhibidores sobre las actividades enzimáticas de proteasas ácidas y alcalinas. El pH óptimo de proteasas estomacales es de 2, mientras que el de proteases intestinales es de 10. La temperatura óptima de proteasas ácidas es de 35 ºC y las alcalinas de 45 ºC. La estabilidad térmica para proteasas ácidas y alcalinas es a los 35 ºC (más de 100% de actividad residual). La estabilidad a los diferentes pH de las proteasas ácidas es en 2 (50 % de la actividad residual), mientras que para las proteasas alcalinas es en 10 (90 % de la actividad residual). Las proteasas ácidas fueron inhibidas en 80% con pepstatina A y las proteasas alcalinas fueron altamente inhibidas con TLCK para tripsina (75%) y TPCK quimitripsina (80%). Finalmente, las metaloproteasas fueron inactivadas con EDTA en 70%. En conclusión, M. brachyurus tiene una buena capacidad digestiva al degradar una amplia variedad de proteinas debido a su alta actividad proteolítica.(AU)

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.

A comparative evaluation of different ionic liquids for arsenic species separation and determination in wine varietals by liquid chromatography - hydride generation atomic fluorescence spectrometry.

The application of different ionic liquids (ILs) as modifiers for chromatographic separation and determination of arsenite [As(III)], arsenate [As(V)], dimethylarsonic acid (DMA) and monomethylarsonic acid (MMA) species in wine samples, by reversed-phase high performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry detection (RP-HPLC-HG-AFS) was studied in this work. Several factors influencing the chromatographic separation of the As species, such as pH of the mobile phase, buffer solution concentration, buffer type, IL concentration and length of alkyl groups in ILs were evaluated. The complete separation of As species was achieved using a C18 column in isocratic mode with a mobile phase composed of 0.5% (v/v) 1-octyl-3-methylimidazolium chloride ([C8mim]Cl) and 5% (v/v) methanol at pH 8.5. A multivariate methodology was used to optimize the variables involved in AFS detection of As species after they were separated by HPLC. The ILs showed remarkable performance for the separation of As species, which was obtained within 18min with a resolution higher than 0.83. The limits of detection for As(III), As(V), MMA and DMA were 0.81, 0.89, 0.62 and 1.00µg As L(-1). The proposed method was applied for As speciation analysis in white and red wine samples originated from different grape varieties.

Single-step solubilization of milk samples with N,N-dimethylformamide for inductively coupled plasma-mass spectrometry analysis and classification based on their elemental composition.

A single-step procedure for trace elements analysis of milk samples is presented. Solubilization with small amounts of dymethylformamide (DMF) was assayed prior to inductively coupled plasma mass spectrometry (ICPMS) detection with a high efficiency sample introduction system. All main instrumental conditions were optimized in order to readily introduce the samples without matrix elimination. In order to assess and mitigate matrix effects in the determination of As, Cd, Co, Cu, Eu, Ga, Gd, Ge, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sm, S, Sr, Ta, Tb, V, Zn, and Zr, matrix matching calibration with (103)Rh as internal standard (IS) was performed. The obtained limits of detection were between 0.68 (Tb) and 30 (Zn) µg L(-1). For accuracy verification, certified Skim milk powder reference material (BCR 063R) was employed. The developed method was applied to trace elements analysis of commercially available milks. Principal components analysis was used to correlate the content of trace metals with the kind of milk, obtaining a classification according to adults, baby or baby fortified milks. The outcomes highlight a simple and fast approach that could be trustworthy for routine analysis, quality control and traceability of milks.

Vapor generation - atomic spectrometric techniques. Expanding frontiers through specific-species preconcentration. A review.

We review recent progress in preconcentration strategies associated to vapor generation techniques coupled to atomic spectrometric (VGT-AS) for specific chemical species detection. This discussion focuses on the central role of different preconcentration approaches, both before and after VG process. The former was based on the classical solid phase and liquid-liquid extraction procedures which, aided by automation and miniaturization strategies, have strengthened the role of VGT-AS in several research fields including environmental, clinical, and others. We then examine some of the new vapor trapping strategies (atom-trapping, hydride trapping, cryotrapping) that entail improvements in selectivity through interference elimination, but also they allow reaching ultra-low detection limits for a large number of chemical species generated in conventional VG systems, including complete separation of several species of the same element. This review covers more than 100 bibliographic references from 2009 up to date, found in SCOPUS database and in individual searches in specific journals. We finally conclude by giving some outlook on future directions of this field.

Determination of thimerosal in pharmaceutical industry effluents and river waters by HPLC coupled to atomic fluorescence spectrometry through post-column UV-assisted vapor generation.

A high performance liquid chromatography coupled with atomic fluorescence spectrometry method for the determination of thimerosal (sodium ethylmercury thiosalicylate, C9H9HgNaO2S), ethylmercury, and inorganic mercury is proposed. Mercury vapor is generated by the post-column reduction of mercury species in formic acid media using UV-radiation. Thimerosal is quantitatively converted to Hg(II) followed by the reduction of Hg(II) to Hg(0). This method is applied to the determination of thimerosal (THM), ethylmercury (EtHg) and inorganic Hg in samples of a pharmaceutical industry effluent, and in waters of the San Luis River situated in the west side of San Luis city (Middle West, Argentine) where the effluents are dumped. The limit of detections, calculated on the basis of the 3σ criterion, where 0.09, 0.09 and 0.07 µg L(-1) for THM, EtHg(II) and for Hg(II), respectively. Linearity was attained from levels close to the detection limit up to at least 100 µg L(-1).

Characterization of Hg-phytochelatins complexes in vines (Vitis vinifera cv Malbec) as defense mechanism against metal stress.

An approach to understand vines (Vitis vinifera) defense mechanism against heavy metal stress by isolation and determination of Hg-phytochelatins (PCs) complexes was performed. PCs are important molecules involved in the control of metal concentration in plants. PCs complex toxic metals through -SH groups and stores them inside cells vacuole avoiding any toxic effect of free metals in the cytosol. The Hg-PCs identification was achieved by determination of Hg and S as hetero-tagged atoms. A method involving two-dimensional chromatographic analysis coupled to atomic spectrometry and confirmation by tandem mass spectrometry is proposed. An approach involving size exclusion chromatography coupled to inductively coupled plasma mass spectrometry on roots, stems, and leaves extracts describing Hg distribution according to molecular weight and sulfur associations is proposed for the first time. Medium-low molecular weight Hg-S associations of 29-100 kDa were found, suggesting PCs presence. A second approach employing reversed-phase chromatography coupled to atomic fluorescence spectrometry analysis allowed the determination of Hg-PCs complexes within the mentioned fractions. Chromatograms showed Hg-PC2, Hg-PC3 and Hg-PC4 presence only in roots. Hg-PCs presence in roots was confirmed by ESI-MS/MS analysis.

Multielemental analysis in vegetable edible oils by inductively coupled plasma mass spectrometry after solubilisation with tetramethylammonium hydroxide.

Trace metals have negative effects on the oxidative stability of edible oils and they are important because of possibility for oils characterisation. A single-step procedure for trace elemental analysis of edible oils is presented. To this aim, a solubilisation with tetramethylammonium hydroxide (TMAH) was assayed prior to inductively coupled plasma mass spectrometry detection. Small amounts of TMAH were used, resulting in high elemental concentrations. This method was applied to edible oils commercially available in Argentine. Elements present in small amounts (Cu, Ge, Mn, Mo, Ni, Sb, Sr, Ti, and V) were determined in olive, corn, almond and sunflower oils. The limits of detection were between 0.004 µg g(-1) for Mn and Sr, and 0.32 µg g(-1) for Sb. Principal components analysis was used to correlate the content of trace metals with the type of oils. The two first principal components retained 91.6% of the variability of the system. This is a relatively simple and safe procedure, and could be an attractive alternative for quality control, traceability and routine analysis of edible oils.

Determination of scandium in acid mine drainage by ICP-OES with flow injection on-line preconcentration using oxidized multiwalled carbon nanotubes.

An on-line scandium preconcentration and determination system implemented with inductively coupled plasma optical emission spectrometry associated with flow injection was studied. Trace amounts of scandium were preconcentrated by sorption on a minicolumn packed with oxidized multiwalled carbon nanotubes, at pH 1.5. The retained analyte was removed from the minicolumn with 30% (v/v) nitric acid. A total enrichment factor of 225-fold was obtained within a preconcentration time of 300 s (for a 25 mL sample volume). The overall time required for preconcentration and elution of 25 mL of sample was about 6 min; the throughput was about 10 samples per hour. The value of the detection limit was 4 ng L(-1) and the precision for 10 replicate determinations at 100 ng L(-1) Sc level was 5% relative standard deviation, calculated from the peak heights obtained. The calibration graph using the preconcentration system was linear with a correlation coefficient of 0.9996 at levels near the detection limits up to at least 10 mg L(-1). After optimization, the method was successfully applied to the determination of Sc in an acid drainage from an abandoned mine located in the province of San Luis, Argentina.

Rapid and sensitive HILIC-MS/MS analysis of carnitine and acetylcarnitine in biological fluids.

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

Risk assessment on irrigation of Vitis vinifera L. cv Malbec with Hg contaminated waters.

Concerns regard watering crops with Hg contaminated waters have arisen worldwide recently. In these sense Hg uptake by Vitis vinifera L. cv. Malbec was evaluated under greenhouse conditions by the administration of Hg(2+) for 4 days through irrigation water (short-term administration). Vines uptake Hg translocating it from roots through stems to leaves. Roots accumulated the higher Hg concentration. Hg in stems and leaves was accumulated mostly as organic Hg, bind to different moieties. Size exclusion chromatography (SEC) and ion pair chromatography (IPC) were employed to reach insights into these ligands. Hg is distributed mainly in high molecular weight fractions of 669 kDa in vine plants. In stems and leaves, Hg-S associations were found in 669 and 66 kDa fractions. Hg-S association at 66 kDa suggests a possible protein or peptide binding affecting vines normal physiology. Since Hg contamination through organomercurials is more harmful than Hg(2+) itself, methyl mercury, dimethyl mercury, and phenyl mercury, more toxic Hg species were evaluated with negative results.

Method development for Cd and Hg determination in biodiesel by electrothermal atomic absorption spectrometry with emulsion sample introduction.

A novel method for analysis of biodiesel by electrothermal atomic absorption spectrometry is described. This analytical strategy involves sample preparation as emulsions for routine and reliable determination of Cd and Hg. Several experimental conditions were investigated, including emulsion stability and composition, furnace temperature program and matrix modification. Different calibration strategies were also evaluated, being the analyte addition method preferred both for Cd and Hg. The accuracy was verified through comparison with an acid digestion in a microwave closed system. The injection repeatability was evaluated as the average relative standard deviation (R.S.D %) for five successive firings and was better than 4.4% for Cd and 5.4% Hg respectively. The detection limits, evaluated by the 3σ concept of calculation (n=10), were of 10.2 µg kg(-1) (0.9 µg L(-1)) for Hg and 0.3 µg kg(-1) (0.04 µg L(-1)) for Cd. This method was successfully applied to the determination of Cd and Hg in biodiesel samples obtained from local vendors.

Determination of chemical oxygen demand by a flow injection method based on microwave digestion and chromium speciation coupled to inductively coupled plasma optical emission spectrometry.

The present paper evaluates the applicability of a new FIA system for COD determination. The new system, flow injection microwave solid phase extraction by means of activated carbon (FI-MW-SPE), consists of a digestion circuit, placed in a home MW oven, coupled to an ICP-OES spectrophotometer. Doehlert experimental design was used to speed up the optimization of different experimental variables studied for assisted digestion methods. The method provided a high throughput of about 18 samples h(-1). To assess the accuracy of analytical methods linear regression, elliptic joint confidence region (EJCR) was used. A large linear range of 2.78-850 mg O(2) L(-1) with an excellent detection limit of 0.94 mg O(2) L(-1) was obtained. The interference by high chloride concentration was studied, and values below 3000 mg Cl(-) ions L(-1), allowed the estimation of COD load without any masking agents. COD values for various types of wastewater samples were correlated with those obtained by standard manual methods. Moreover, interferences due to matrix nature are absent; since matrix is washed out of the column before Cr (III) is eluted. This method reduces the time, reagent volume, hazardous emission, external contamination, with a good reproducibility and accuracy.

Multivariate factorial analysis to design a robust batch leaching test to assess the volcanic ash geochemical hazard.

A method to obtain robust information on short term leaching behaviour of volcanic ashes has been developed independently on the sample age. A mixed factorial design (MFD) was employed as a multivariate strategy for the evaluation of the effects of selected control factors and their interactions (amount of sample (A), contact time (B), and liquid to solid ratio or L/S (C)) on the leaching process of selected metals (Na, K, Mg, Ca, Si, Al, V, Mn, Fe, and Co) and anions (Cl(-) and SO(4)(2-)). Box plots of the data acquired were used to evaluate the reproducibility achieved at different experimental conditions. Both the amount of sample (A) and leaching time (B) had a significant effect on the element stripping whereas the L/S ratio influenced only few elements. The lowest dispersion values have been observed when 1.0 g was leached with an L/S ratio equal to 10, shaking during 4 h. The entire method is completed within few hours, and it is simple, feasible and reliable in laboratory conditions.

Monitoring of phenolic compounds for the quality control of Melissa officinalis products by capillary electrophoresis.

INTRODUCTION: Official assays for the quality control of Melissa officinalis L. (Lamiaceae) leaves establish the quantification of total hydroxycinnamic derivatives expressed as rosmarinic acid. OBJECTIVE: The goal of this work was to develop a simple, fast and reliable method for monitoring the phenolic composition in herbs from the Lamiaceae family and for rapidly detecting M. officinalis adulteration or substitution in commercial medicinal samples in Argentina. METHODOLOGY: A capillary zone electrophoresis (CZE) method was performed under the following conditions: the background electrolyte (BGE) consisted of 20 m m sodium tetraborate buffer, pH 9.2; the applied voltage was 25 kV; the capillary and sample temperatures were kept at 25 °C; the hydrodynamic mode was selected for the sample injection (3.45 kPa during 5 s). RESULTS: A CZE method that achieved the separation and simultaneous determination of eight related phenolic compounds in less than 11 min was optimised for application to control quality analysis of M. officinalis-based products. The method was validated according to the US Federal Drug Agency requirements and offers advantages in terms of analysis time, cost and operation. CONCLUSIONS: The proposed methodology can be applied to the standardisation and quality control of plant material and phytopharmaceutical products derived from the Lamiaceae family, as indicated by the results obtained in the analysis of commercial medicinal products in Argentina.