H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection.

During infections, the host redistributes iron in order to starve pathogens from this nutrient. Several proteins are involved in iron absorption, transport, and storage. Ferritin is the most important iron storage protein. It is composed of variable proportions of two peptides, the L- and H-ferritins (FTL and FTH). We previously showed that macrophages increase their expression of FTH1 when they are infected in vitro with Mycobacterium avium, without a significant increase in FTL. In this work, we investigated the role of macrophage FTH1 in M. avium infection in vivo. We found that mice deficient in FTH1 in myeloid cells are more resistant to M. avium infection, presenting lower bacterial loads and lower levels of proinflammatory cytokines than wild-type littermates, due to the lower levels of available iron in the tissues. Importantly, we also found that FTH1 produced by myeloid cells in response to infection may be found in circulation and that it plays a key role in iron redistribution. Specifically, in the absence of FTH1 in myeloid cells, increased expression of ferroportin is observed in liver granulomas and increased iron accumulation occurs in hepatocytes. These results highlight the importance of FTH1 expression in myeloid cells for iron redistribution during infection.

Effect of Zinc Source and Exogenous Enzymes Supplementation on Zinc Status in Dogs Fed High Phytate Diets.

Zinc is an essential element, a cofactor of many enzymes, and performs catalytic, structural and regulatory functions. Once in the gastrointestinal tract, zinc can interact with food constituents. Phytic acid, the major phosphorus storage in plants, limits zinc availability from animal feeds due to the formation of insoluble complexes with phytates. This study tested the effect of supplemental zinc source (zinc sulfate and a chelate zinc proteinate) and the addition of exogenous enzymes from a solid-state fermentation product of Aspergillus niger to a high phytate diet. The study was designed according to three Latin Squares 4 × 4 with a 2 × 2 factorial arrangement of treatments, with four periods, four diets, and 12 young adult Beagles. Periods lasted 5 weeks each. Diets were supplemented with 75 mg/kg of zinc sulfate (IZ) or zinc proteinate (OZ), and without or with 200 mg/kg of exogenous enzymes (IZ+, OZ+). Results showed that zinc proteinate increased the bioavailability of phosphorus, yet the zinc biomarkers remained unaffected by the zinc source, with the exception of lymphocyte subsets that benefit from zinc proteinate. The use of exogenous enzymes did not affect zinc availability nor nutrient and energy digestibility.

Flow-Based Dynamic Approach to Assess Bioaccessible Zinc in Dry Dog Food Samples.

This work proposes a simple and easy-to-use flow-through system for the implementation of dynamic extractions, aiming at the evaluation of bioaccessible zinc and the characterization of leaching kinetics in dry dog food samples. The kinetic profile of Zn extraction was determined by flame atomic absorption spectroscopy and the results were fitted in an exponential function (R2 > 0.960) compatible with a two first-order reactions model. Values of fast leachable Zn ranged from 83 ± 1 to 313 ± 5 mg of Zn per kg of sample, with associated rate constants ranging from 0.162 ± 0.004 to 0.290 ± 0.014 min-1. Similar results were observed compared to the static batch extraction. The percentage of bioaccessible Zn ranged from 49.0 to 70.0%, with an average value of 58.2% in relation to total Zn content. Principal component analysis regarding the variables fast leachable Zn, associated rate constant, total Zn, and market segment, has shown that 84.6% of variance is explained by two components, where the second component (24.0%) presented loadings only for the fast leachable Zn and associated rate constant. The proposed method is suitable for the fast evaluation (<1 h) of leaching kinetics and bioaccessibility in dry dog food.

H-Ferritin is essential for macrophages' capacity to store or detoxify exogenously added iron.

Macrophages are central cells both in the immune response and in iron homeostasis. Iron is both essential and potentially toxic. Therefore, iron acquisition, transport, storage, and release are tightly regulated, by several important proteins. Cytosolic ferritin is an iron storage protein composed of 24 subunits of either the L- or the H-type chains. H-ferritin differs from L-ferritin in the capacity to oxidize Fe2+ to Fe3+. In this work, we investigated the role played by H-ferritin in the macrophages' ability to respond to immune stimuli and to deal with exogenously added iron. We used mice with a conditional deletion of the H-ferritin gene in the myeloid lineage to obtain bone marrow-derived macrophages. These macrophages had normal viability and gene expression under basal culture conditions. However, when treated with interferon-gamma and lipopolysaccharide they had a lower activation of Nitric Oxide Synthase 2. Furthermore, H-ferritin-deficient macrophages had a higher sensitivity to iron-induced toxicity. This sensitivity was associated with a lower intracellular iron accumulation but a higher production of reactive oxygen species. These data indicate that H-ferritin modulates macrophage response to immune stimuli and that it plays an essential role in protection against iron-induced oxidative stress and cell death.

IFN-γ-Dependent Reduction of Erythrocyte Life Span Leads to Anemia during Mycobacterial Infection.

Anemia is a frequent and challenging complication of mycobacterial infections. We used a model of disseminated Mycobacterium avium infection in mice to investigate the mechanisms of mycobacteria-induced anemia. We found increased formation of RBC in the bone marrow and spleen of infected mice. Infection induced reticulocytosis and the premature egress of immature progenitors to the systemic circulation in an IFN-γ (IFNG)-dependent way. The newly formed RBC had reduced CD47 surface expression and a reduced life span and were phagocytosed in the liver of infected mice, increasing iron recycling in this organ. The increased engulfment and degradation of RBC was independent of IFNG sensing by macrophages. Together, our findings demonstrate that mycobacterial infection alters the formation of erythrocytes, leading to their accelerated removal from circulation and hemolytic anemia. This comprehensive elucidation of the mechanisms underlying mycobacteria-induced anemia has important implications for its efficient clinical management.

Mineral Composition of Dry Dog Foods: Impact on Nutrition and Potential Toxicity.

Detailed mineral profile of a selection of commercially available complete dry dog foods was determined using ICP-MS (Se, Cu, Mn, Zn, and nonessential trace elements), flame photometry (Na and K) and atomic and molecular spectrophotometry (Ca, P, Mg, and Fe). The contribution of ingredients to the mineral composition was correlated to the food market segment. Results showed an oversupply of essential elements due to the energy density effect on feed intake. Additives contributed from 40.8 to 55.1% to the total trace elements contents. With the exception of Se, all trace elements were supplied above the nutritional requirements of adult dogs. Legal limits of Cu, Se, and Zn were surpassed. The content of nonessential trace elements included values in the range of nanograms to micrograms per kg, without surpassing safe upper limits. This work brings awareness to the need to find supplementation strategies that ensure nutritional adequacy and avoid waste.

Nutritional, chemical and antioxidant/pro-oxidant profiles of silverskin, a coffee roasting by-product.

Coffee silverskin (a coffee roasting by-product) contains high amounts of dietary fibre (49% insoluble and 7% soluble) and protein (19%). Potassium (∼5g/100g), magnesium (2g/100g) and calcium (0.6g/100g) are the major macrominerals. The vitamin E profile of silverskin comprises α-tocopherol, ß-tocopherol, É£-tocopherol, δ-tocopherol, ß-tocotrienol, É£-tocotrienol, and δ-tocotrienol. The fatty acid profile is mainly saturated (C16:0 and C22:0), but the total amount of fat is low (2.4%). Caffeine (1.25g/100g), chlorogenic acid (246mg/100g), and 5-hydroxymethylfurfural (5.68mg/100g) are also present in silverskin. Total phenolics and flavonoids are partially responsible for the in vitro antioxidant activity. Silverskin extracts protected erythrocytes from oxidative AAPH- and H2O2-induced hemolysis, but at high concentrations a pro-oxidant effect on erythrocyte morphology was observed.

Trace elements in wild edible Aplysia species: Relationship with the desaturation-elongation indexes of fatty acids.

The profile of essential and non-essential elements was traced in the edible sea hares Aplysia depilans Gmelin, Aplysia fasciata Poiret and Aplysia punctata Cuvier. Manganese (Mn), iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) were identified as the major essential elements. Risk assessment evidenced that the levels of cadmium (Cd) and lead (Pb) did not exceed the maximum limit value established by the European Regulation, the contents of chromium (Cr), nickel (Ni) and arsenic (As) being also below the levels established by the FDA guide. A correlation between trace elements levels and desaturation-elongation indexes of fatty acids was found. While Cd, Se and molybdenum (Mo) seem to promote the desaturation-elongation process involved on the production of C20:4n-6c, Ni, Cr and Fe may potentiate the conversion of C18:3n-3c to C20:5n-3c. Furthermore, cobalt (Co), Ni and Cu appear to decrease Δ9 desaturation index. Besides the suggested biosynthetic switch modulated by trace elements, the nutritional value of the species is further strengthened.

Fibre fortification of wheat bread: impact on mineral composition and bioaccessibility.

In this work, wheat bread was fortified with fibre enriched extracts recovered from agroindustry by-products, namely, elderberry skin, pulp and seeds (EE); orange peel (OE); pomegranate peel and interior membranes (PE); and spent yeast (YE). The impact of this fortification on the total and bioaccessible mineral composition of wheat breads, estimated mineral daily intake, and the relationship between bioaccessibility and dietary fibre was evaluated. Fortification with OE, EE, and PE improved the content of essential minerals in bread when compared to control bread. The exception was bread fortified with YE, which presented a mineral content similar to control bread, but its mineral bioaccessibility was significantly higher than in all the other bread formulations. The opposite was observed for PE bread, which presented a significant reduction of bioaccessible minerals. We concluded that the origin of the fibre rich extract must be carefully selected, to avoid potential negative impact on mineral bioaccessibility.

Anthropogenic pressure in a Portuguese river: Endocrine-disrupting compounds, trace elements and nutrients.

Natural organic compounds such as phytoestrogens and phytosterols found in various plants, as well as mycotoxins produced by fungi, can be found in aquatic environments. The aim of this study was to investigate the occurrence of three different classes of natural estrogenic compounds, i.e., phytoestrogens, phytosterols and mycotoxins, in estuarine water samples from the Ave River estuary. For that, water samples were collected at five sampling points distributed along the estuary at low tide, during 1 year, processed by solid-phase extraction (SPE) and analyzed by gas chromatography coupled to mass spectrometry (GC-MS). To correlate the presence of phytoestrogens and phytosterols in the estuarine water, local flora was collected on riverside. Trace elements content and physicochemical parameters such as nutrients and dissolved oxygen were also determined seasonally at each sampling point, to give insights for the evaluation of water quality and anthropogenic pressure. Both phytoestrogens and phytosterols showed a seasonal variation, with the highest values observed in spring and summer and the lowest in winter. Daidzein (DAID) was found up to 404.0 ng L(-1) in spring and coumestrol (COUM) was found up to 165.0 ng L(-1) in summer. The mycotoxin deoxynivalenol (DON) was ubiquitously determined with values ranging from 59.5 to 642.4 ng L(-1). Nutrients and metals distribution and concentration varied among sampling stations and seasons. This study revealed for the first time the presence of mycotoxins, various classes of phytoestrogens and stigmasterol (STG) in estuarine water from the Ave River (Portugal), and the evaluation of the water quality confirmed that this estuary is still highly impacted by anthropogenic activities.

Assessment of metal(loid)s phytoavailability in intensive agricultural soils by the application of single extractions to rhizosphere soil.

The influence of soil properties on the phytoavailability of metal(loid)s in a soil-plant system was evaluated. The content of extractable metal(loid)s obtained by using different extraction methods was also compared. To perform this study, a test plant (Lactuca sativa) and rhizosphere soil were sampled at 5 different time points (2, 4, 6, 8 and 10 weeks of plant growth). Four extraction methods (Mehlich 3, DTPA, NH4NO3 and CaCl2) were used. Significant positive correlations between the soil extractable content and lettuce shoot content were obtained for several metal(loid)s. The extraction with NH4NO3 showed the higher number of strong positive correlations indicating the suitability of this method to estimate metal(loid)s phytoavailability. The soil CEC, OM, pH, texture and oxides content significantly influenced the distribution of metal(loid)s between the phytoavailable and non-phytoavailable fractions. A reliable prediction model for Cr, V, Ni, As, Pb, Co, Cd, and Sb phytoavailability was obtained considering the amount of metal(loid) extracted by the NH4NO3 method and the main soil properties. This work shows that the analysis of rhizosphere soil by single extractions methods is a reliable approach to estimate metal(loid)s phytoavailability.

Changes in macrominerals, trace elements and pigments content during lettuce (Lactuca sativa L.) growth: influence of soil composition.

Changes in macrominerals, trace elements and photosynthetic pigments were monitored at 5 stages of lettuce growth. Plants were grown in three experimental agriculture greenhouse fields (A1, A2 and A3). Soil composition was also monitored to understand its influence on lettuce composition. In general, the content of macrominerals, trace elements, chlorophylls and carotenoids decreased during lettuce growth and consequently, high nutritional value was observed at younger stages. A2 lettuces showed an increase of Fe, Al, Cr, V and Pb due to the different soil physicochemical parameters. Multiple linear regression analysis with stepwise variable selection, indicated that soil characteristics, namely, pH(CaCl2) for Fe and Cr, silt and fine-sand for Al and V, OM for Al and Pb, coarse-sand and CEC for Cr, had a key role determining element bioavailability and plant mineral content. Thus, lettuce nutritional value was strongly dependent of growth stage and soil characteristics.

Staphylococcus epidermidis biofilms with higher proportions of dormant bacteria induce a lower activation of murine macrophages.

Staphylococcus epidermidis is an opportunistic pathogen and, due to its ability to establish biofilms, is a leading causative agent of indwelling medical device-associated infection. The presence of high amounts of dormant bacteria is a hallmark of biofilms, making them more tolerant to antimicrobials and to the host immune response. We observed that S. epidermidis biofilms grown in excess glucose accumulated high amounts of viable but non-culturable (VBNC) bacteria, as assessed by their low ratio of culturable bacteria over the number of viable bacteria. This effect, which was a consequence of the accumulation of acidic compounds due to glucose metabolism, was counteracted by high extracellular levels of calcium and magnesium added to the culture medium allowing modulation of the proportions of VBNC bacteria within S. epidermidis biofilms. Using bacterial inocula obtained from biofilms with high and low proportions of VBNC bacteria, their stimulatory effect on murine macrophages was evaluated in vitro and in vivo. The inoculum enriched in VBNC bacteria induced in vitro a lower production of tumour necrosis factor alpha, interleukin-1 and interleukin-6 by bone-marrow-derived murine macrophages and, in vivo, a lower stimulatory effect on peritoneal macrophages, assessed by increased surface expression of Gr1 and major histocompatibility complex class II molecules. Overall, these results show that environmental conditions, such as pH and extracellular levels of calcium and magnesium, can induce dormancy in S. epidermidis biofilms. Moreover, they show that bacterial suspensions enriched in dormant cells are less inflammatory, suggesting that dormancy can contribute to the immune evasion of biofilms.

A reflectance flow-through thionine sol-gel sensor for the determination of Se(IV).

In this work, a reversible sensor to assess the total Se(IV) content in samples is described. Pre-activated glass slides were spin-coated with 100 microL of a 20-h aged sol-gel mixture of 1 mL of tetramethoxysilane, 305 microL of 50 mmol L(-1) HCl and 2.0 mg of thionine. The flow-cell consisted of one of those slides as a window, and was filled with beads of a polystyrene anionic exchange resin to retain Se(IV) in the form of selenite ions. A reflectance transduction scheme at a wavelength of 596 nm was adopted. The cell was coupled to a multicommutation flow system where a programmed volume of a sample solution and 373 microL of 0.4 mmol L(-1) iodide in a 1.6 mol L(-1) HCl solution were sequentially inserted into the cell. The iodine produced from the reaction of retained Se(IV) with iodide bleached the blue color of thionine. Considering a sample volume of 2.30 mL, with which the preconcentration step was minimized, a linear dynamic working range between 1.5 to 20 microg mL(-1) and a detection limit of 0.29 microg mL(-1) were obtained. The sensor enabled us to perform approximately 200 assays, and provided results similar to those of electrothermal atomic absorption spectrometry.

Trace elements in human milk: correlation with blood levels, inter-element correlations and changes in concentration during the first month of lactation.

Using inductively coupled plasma mass spectrometry (ICP-MS) based analytical procedures, the concentration of several trace elements (Mn, As, Pb, Co, Ni, Cu, Zn and Se) was determined in human milk samples collected from a group of healthy lactating Portuguese women (n=44), both on the 2nd day postpartum (i.e., colostrum; n=34) and at 1 month postpartum (i.e., mature milk; n=19). Blood samples (n=44), collected on the 2nd day after parturition, were also analyzed for the same trace elements. No major correlations were observed between the levels of the analyzed trace elements in blood and colostrum samples. All the studied elements, except for Co, Pb and Ni, showed a significant trend for a decrease in concentration in milk during the first month of lactation. This trend was more pronounced for Zn and Se, whose levels decreased to approximately 23% and 44% of their initial mean concentration, respectively. With the exception of Co (r=0.607) and Zn (r=0.487), no significant correlations were observed when comparing the levels of each trace element between samples of colostrum and mature milk. Several inter-element correlations were found within each type of milk sample. The most significant were: (i) Se vs Cu (r=0.828) and Se vs Co (r=0.605) in colostrum samples and (ii) Ni vs Pb (r=0.756), Ni vs Mn (r=0.743) and Se vs Co (r=0.714) in mature milk samples. An inverse correlation between Zn and Se was also found in both types of milk sample; however, it only reached statistical significance for mature milk (r=-0.624).

Determination of Rh, Pd and Pt in urine samples using a pre-concentration sequential injection analysis system coupled to a quadrupole-inductively coupled plasma-mass spectrometer.

The proposed flow system was developed in order to minimize the drawbacks related to the PGEs determination by quadrupole-inductively coupled plasma-mass spectrometry (Q-ICP-MS). It was intended not only to lower the limits of detection (LODs) but also to eliminate the interferences originating from some atomic and molecular ions produced in the argon plasma. This was accomplished by means of an on-line sample clean-up/pre-concentration step, using a chelating resin (Metalfixtrade mark Chelaminetrade mark) in which Rh, Pd and Pt were preferably retained when compared with the interfering species. The results obtained by using the developed flow system in the analysis of urine samples are presented. With a sampling rate of 9 samples h(-1) (i.e., 27 determinations) and a sample consumption of ca. 10 mL, the developed flow system allowed linear calibration plots up to 100 ngL(-1) with detection limits of 1.2 ngL(-1) (Rh), 0.4 ngL(-1) (Pd) and 0.9 ngL(-1) (Pt). Repeatability studies showed good precision (R.S.D.%, n=5): 3.7% (Rh); 2.6% (Pd) and 2.4% (Pt), for 10 ngL(-1); 2.4% (Rh); 1.4% (Pd) and 1.9% (Pt), for 50 ngL(-1); and 1.3% (Rh); 0.58% (Pd) and 0.62% (Pt), for 100 ngL(-1). By spiking human urine samples, recovery tests were performed, and the values obtained ranged between 89% and 105% (Rh); 90% and 104% (Pd); and 93% and 105% (Pt).

Firing distance estimation through the analysis of the gunshot residue deposit pattern around the bullet entrance hole by inductively coupled plasma-mass spectrometry: an experimental study.

The use of inductively coupled plasma-mass spectrometry (ICP-MS) in the study of gunshot residues (GSR) is relatively recent, and only a few studies have been published on the subject. In the present paper, this instrumental technique has been used to study the deposit pattern of the GSR around the bullet entrance hole, through the analysis of antimony (Sb), barium (Ba), and lead (Pb). The data obtained were used to establish a mathematical model for estimating the firing distance. Test shots using a 6.35-mm pistol were made against a target of cotton tissue, and the amounts of Sb, Ba, and Pb deposited in quadrangular pieces of the target, cut from 4 radial positions, were determined by ICP-MS. In these experimental conditions, it was possible to estimate the firing distance on the interval [20-80] cm. The best linear correlation between ln m and d, where m is the mass of Sb, Ba, or Pb in the samples, expressed in mug/g of target tissue, and d the firing distance, was obtained at radial distances between 3.5 cm and 4.5 cm from the entrance hole. The best regression curve which adjusted to the data was a linear multiple regression between the firing distance and the logarithm of the mass of each element: d = a + b(1)X(1) + b(2)X(2) + b(3)X(3), where X(1) = ln m (Sb), X(2) = ln m(Ba) and X(3) = ln m (Pb). The accuracy of firing distance estimation using only 1 or 2 elements was not significantly different from the one obtained with the 3 elements.