Analytical strategies for the early quality and safety assurance in the global feed chain. Approaches for nitrogen adulterants in soybean meal and mineral and transformer oils in vegetable oils.

In the past decade, several major food safety crises originated from problems with feed. Consequently, there is an urgent need for early detection of fraudulent adulteration and contamination in the feed chain. Strategies are presented for two specific cases, viz. adulterations of (i) soybean meal with melamine and other types of adulterants/contaminants and (ii) vegetable oils with mineral oil, transformer oil or other oils. These strategies comprise screening at the feed mill or port of entry with non-destructive spectroscopic methods (NIRS and Raman), followed by post-screening and confirmation in the laboratory with MS-based methods. The spectroscopic techniques are suitable for on-site and on-line applications. Currently they are suited to detect fraudulent adulteration at relatively high levels but not to detect low level contamination. The potential use of the strategies for non-targeted analysis is demonstrated.

A mesocosm analytical study on the impact of freshwater mussel (Lamellidens marginalis Lamarck) mediated bioturbation and biodeposition on some ecological factors of a freshwater lake

The biotic potential of the benthic filter feeding freshwater bivalve mollusc Lamellidens marginalis (Lamarck) influencing the nutrient dynamics of the bottom sediments of the lake by means of biodeposition and bioturbation activities were analysed using a lake mesocosm experiment. Five control as well as experimental mesocosms was maintained up to 60 days (d). The factors studied included the percentage of water content of the sediment, percentage of total nitrogen, percentage of organic matter along with the total phosphorus and humic acid content. While total phosphorus and humic acid content of the experimental mesocosoms showed gradual and significant increases from 30d of the experiment to reach the maximum levels after 60d, the percentage of organic matter registered significant increases right from 15d onwards and reached the maximum values after 60d. On the other hand, while the percentage of water content of the sediments of the experimental mesocosoms increased only up to 30d experiment, percentage of nitrogen was increased during the first half and at the fag end of the experiment. All the investigated ecological factors were found to be significantly influenced by the presence of L. marginalis in the experimental mesocosms. The study indicated that the mussel influence the nutrient dynamics of the inhabitant ecosystem through the processes of excretion, biodeposition of pseudofaeces and faeces, along with the bioturbation of the sediments brought about by their ploughing movements. KEYWORDS: freshwater mussel, Lamellidens marginalis, bioturbation, biodeposition, mesocosms.

Biomarkers of Nitrosative Stress: Development and validation of a new analytical method for 3-Nitrotyrosine quantification

Background: The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT). 3-NT levels in biological samples have been associated with numerous physiological and pathological conditions. For this reason, several attempts have been made in order to develop methods that accurately quantify 3-NT in biological samples. Regarding chromatographic methods, they seem to be very accurate, showing very good sensibility and specificity. However, accurate quantification of this molecule, which is present at very low concentrations both at physiological and pathological states, is always a complex task and a target of intense research. Objectives: We aimed to develop a simple, rapid, low-cost and sensitive 3-NT quantification method for use in medical laboratories as an additional tool for diagnosis and/or treatment monitoring of a wide range of pathologies. We also aimed to evaluate the performance of the HPLC-based method developed here in a wide range of biological matrices. Material and methods: All experiments were performed on a Hitachi LaChrom Elite® HPLC system and separation was carried out using a Lichrocart® 250-4 Lichrospher 100 RP-18 (5μm) column. The method was further validated according to ICH guidelines. The biological matrices tested were serum, whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions. Results: From all the protocols tested, the best results were obtained using 0.5% CH3COOH:MeOH:H2O (15:15:70) as the mobile phase, with detection at wavelengths 215, 276 and 356 nm, at 25ºC, and using a flow rate of 1 mL/min. By using this protocol, it was possible to obtain a linear calibration curve (correlation coefficient = 1), limits of detection and quantification in the order of ng/mL, and a short analysis time (<15 minutes per sample). Additionally, the developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356 nm. Conclusion: The method described in this study, which was successfully developed and validated for 3-NT quantification, is simple, cheap and fast, rendering it suitable for analysis in a wide range of biological matrices.

Development of a new analytical approach based on cellulose membrane and chelator for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed in this study, based on a system equipped with a cellulose membrane and a tetraethylenepentamine hexaacetate chelator (MD-TEPHA) for in situ characterization of the lability of metal species in aquatic systems. To this end, the DM-TEPHA system was prepared by adding TEPHA chelator to cellulose bags pre-purified with 1.0 mol L-1 of HCl and NaOH solutions. After the MD-TEPHA system was sealed, it was examined in the laboratory to evaluate the influence of complexation time (0-24 h), pH (3.0, 4.0, 5.0, 6.0 and 7.0), metal ions (Cu, Cd, Fe, Mn and Ni) and concentration of organic matter (15, 30 and 60 mg L-1) on the relative lability of metal species by TEPHA chelator. The results showed that Fe and Cu metals were complexed more slowly by TEPHA chelator in the MD-TEPHA system than were Cd, Ni and Mn in all pH used. It was also found that the pH strongly influences the process of metal complexation by the MD-TEPHA system. At all the pH levels, Cd, Mn and Ni showed greater complexation with TEPHA chelator (recovery of about 95-75%) than did Cu and Fe metals. Time also affects the lability of metal species complexed by aquatic humic substances (AHS); while Cd, Ni and Mn showed a faster kinetics, reaching equilibrium after about 100 min, and Cu and Fe approached equilibrium after 400 min. Increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems. (c) 2006 Elsevier B.V. All rights reserved.

Development of new analytical procedures of isotope geochemistry by mass spectrometry. Application to the study of high-pressure metamorphic rocks from the Iberian Massif

319 p.

Levels of dioxin-like PCBs in low-volume serum samples of male patients attending fertility clinics

An accurate and easy method for the extraction, cleanup, and HRGC-HRMS analysis of dioxin-like PCBs (DL-PCBs) in low-volume serum samples (1 mL) was developed. Serum samples were extracted several times using n-hexane and purified by acid washing. Recovery rates of labeled congeners ranged from 70 to 110 % and the limits of detection were below 1 pg/g on lipid basis. Although human studies are limited and contradictory, several studies have shown that DL-PCBs can have adverse effects on the male reproductive system. In this way, the present method was applied to 21 serum samples of male patients attending fertility clinics. The total levels obtained for the patients ranged from 6.90 to 84.1 pg WHO-TEQ/g lipid, with a mean value of 20.3 pg WHO-TEQ/g lipid. The predominant PCBs (the sum of PCB 118, 156, and 105) contributed 67 % to the mean concentration of total DL-PCBs in the samples analyzed.

Progress in analytical approaches integrating Livestock and Biodiversity to identify HNV Montados

The World Congress Silvo-Pastoral Systems 2016 aims to gather researchers from different disciplines, practitioners and policy makers at different governance levels that deal with the management and sustainability of silvo-pastoral systems. In this way the congress will create a fertile context to progress through interdisciplinarity research approaches that can help translate scientific knowledge into new adaptive management solutions, and thus bridge from science to practice. The aim is also to gather and compare knowledge from silvo-pastoral systems around the world, which share drought as a limiting factor, so that they can be discussed and evaluated.

Analytical approaches for the measurement of sulfide in biological and pharmaceutical fields

Hydrogen sulfide (H2S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H2S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of various diseases. However, H2S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H2S level. Moreover, quantitative validation has still not been described based on a survey of previously published works. In this study, we developed and validated an improved analytical protocol for the MBB RP-HPLC method. Main parameters like MBB concentration, temperature, reaction time, and sample handling were optimized, and the calibration method was further validated using leave-one-out cross-validation (CV) and tested in a clinical setting. The method shows high sensitivity and allows the quantification of H2S species, with a limit of detection (LOD) of 0.5 µM and a limit of quantification (LOQ) of 0.9 µM. Additionally, this model was successfully applied in measurements of H2S levels in the serum of patients subjected to inhalation with vapors rich in H2S. In addition, a properly procedure was established for H2S release with the modified MBB HPLC-FLD method. The proposed analytical approach demonstrated the slow-release kinetics of H2S from the multilayer Silk-Fibroin scaffolds with the combination of different H2S donor’s concentration with respect to the weight of PLGA nanofiber. In the end, some efforts were made on sulfide measurements by using size exclusion chromatography fluorescence/ultraviolet detection and inductively coupled plasma-mass spectrometry (SEC-FLD/UV-ICP/MS). It’s intended as a preliminary study in order to define the feasibility of a separation-detection-quantification platform to analyze biological samples and quantify sulfur species.

Nanomolar Levels Of Pahs In Extracts From Urban Air Induce Mapk Signaling In Hepg2 Cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Polyphenol Extraction Optimisation From Ceylon Gooseberry (dovyalis Hebecarpa) Pulp.

Originally from Asia, Dovyalis hebecarpa is a dark purple/red exotic berry now also produced in Brazil. However, no reports were found in the literature about phenolic extraction or characterisation of this berry. In this study we evaluate the extraction optimisation of anthocyanins and total phenolics in D. hebecarpa berries aiming at the development of a simple and mild analytical technique. Multivariate analysis was used to optimise the extraction variables (ethanol:water:acetone solvent proportions, times, and acid concentrations) at different levels. Acetone/water (20/80 v/v) gave the highest anthocyanin extraction yield, but pure water and different proportions of acetone/water or acetone/ethanol/water (with >50% of water) were also effective. Neither acid concentration nor time had a significant effect on extraction efficiency allowing to fix the recommended parameters at the lowest values tested (0.35% formic acid v/v, and 17.6 min). Under optimised conditions, extraction efficiencies were increased by 31.5% and 11% for anthocyanin and total phenolics, respectively as compared to traditional methods that use more solvent and time. Thus, the optimised methodology increased yields being less hazardous and time consuming than traditional methods. Finally, freeze-dried D. hebecarpa showed high content of target phytochemicals (319 mg/100g and 1,421 mg/100g of total anthocyanin and total phenolic content, respectively).

Treadmill Training Increases Sirt-1 And Pgc-1 α Protein Levels And Ampk Phosphorylation In Quadriceps Of Middle-aged Rats In An Intensity-dependent Manner.

The present study investigated the effects of running at 0.8 or 1.2 km/h on inflammatory proteins (i.e., protein levels of TNF- α , IL-1 β , and NF- κ B) and metabolic proteins (i.e., protein levels of SIRT-1 and PGC-1 α , and AMPK phosphorylation) in quadriceps of rats. Male Wistar rats at 3 (young) and 18 months (middle-aged rats) of age were divided into nonexercised (NE) and exercised at 0.8 or 1.2 km/h. The rats were trained on treadmill, 50 min per day, 5 days per week, during 8 weeks. Forty-eight hours after the last training session, muscles were removed, homogenized, and analyzed using biochemical and western blot techniques. Our results showed that: (a) running at 0.8 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with NE rats; (b) these responses were lower for the inflammatory proteins and higher for the metabolic proteins in young rats compared with middle-aged rats; (c) running at 1.2 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with 0.8 km/h; (d) these responses were similar between young and middle-aged rats when trained at 1.2 km. In summary, the age-related increases in inflammatory proteins, and the age-related declines in metabolic proteins can be reversed and largely improved by treadmill training.

The Influence Of Altered Occlusion On Pro-inflammatory Cytokine Levels In The Tmj Synovial Tissues Of Rats.

The aim of this study was to evaluate whether altered occlusion affects both the condylar cartilage thickness and the cytokine levels of the TMJs of rats. Thirty adult-male rats (n=30) were randomly assigned to three experimental conditions: a control group that underwent sham operations with unaltered occlusion; an FPDM group that underwent functional posterior displacement of the mandible that was induced by an incisor guiding appliance; and an iOVD group in which the increased occlusal vertical dimension was induced in the molars. The rats were subjected to the FPDM or iOVD model for 14 days and then killed. Both the right and left TMJs were removed and randomly assigned to examination with staining or immunoassay techniques. Toluidine blue staining was used to measure the thicknesses of the four layers of the articular cartilage (i.e., the fibrous, proliferating, mature, and hypertrophic layers). ELISA assays were used to assess the concentrations of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, and tumour necrosis factor (TNF-α). The measurements of the articular cartilage layers and cytokine concentrations were analyzed with ANOVA and Tukey's tests and Kruskal-Wallis and Dunn tests, respectively (α=5%). The thickness of articular cartilage in the FPDM group (0.3±0.03mm) was significantly greater than those of the control (0.2±0.01mm) and iOVD (0.25±0.03mm) groups. No significant difference was observed between the control and iOVD groups. The four articular cartilage layers were thicker in the FPDM group than in the control and iOVD groups, and the latter two groups did not differ one from each other. Both the FPDM and iOVD groups exhibited higher cytokine levels than did the control (p<0.05) group. Compared to the FPDM group, the iOVD group exhibited significantly higher levels of IL-1β and TNF-α. Both models induced inflammation in the TMJ and caused significant structural changes in the TMJ and surrounding tissues.

A Modified Acidic Approach For Dna Extraction From Plant Species Containing High Levels Of Secondary Metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Determination Of Flavanones In Orange Juices Obtained From Different Sources By Hplc/dad.

Flavanones (hesperidin, naringenin, naringin, and poncirin) in industrial, hand-squeezed orange juices and from fresh-in-squeeze machines orange juices were determined by HPLC/DAD analysis using a previously described liquid-liquid extraction method. Method validation including the accuracy was performed by using recovery tests. Samples (36) collected from different Brazilian locations and brands were analyzed. Concentrations were determined using an external standard curve. The limits of detection (LOD) and the limits of quantification (LOQ) calculated were 0.0037, 1.87, 0.0147, and 0.0066 mg 100 g(-1) and 0.0089, 7.84, 0.0302, and 0.0200 mg 100 g(-1) for naringin, hesperidin, poncirin, and naringenin, respectively. The results demonstrated that hesperidin was present at the highest concentration levels, especially in the industrial orange juices. Its average content and concentration range were 69.85 and 18.80-139.00 mg 100 g(-1). The other flavanones showed the lowest concentration levels. The average contents and concentration ranges found were 0.019, 0.01-0.30, and 0.12 and 0.1-0.17, 0.13, and 0.01-0.36 mg 100 g(-1), respectively. The results were also evaluated using the principal component analysis (PCA) multivariate analysis technique which showed that poncirin, naringenin, and naringin were the principal elements that contributed to the variability in the sample concentrations.

Reduced Plasma Angiotensin Ii Levels Are Reversed By Hydroxyurea Treatment In Mice With Sickle Cell Disease.

Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.