Agostinho A. Almeida

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).

Iron levels in the human brain: a post-mortem study of anatomical region differences and age-related changes.

The link between brain iron homeostasis and neurodegenerative disease has been the subject of extensive research. There is increasing evidence of iron accumulation during ageing, and altered iron levels in some specific brain regions in neurodegenerative disease patients have been reported. Using graphite furnace atomic absorption spectrometry after microwave-assisted acid digestion of the samples, iron levels were determined in 14 different areas of the human brain [frontal cortex, superior and middle temporal, caudate nucleus, putamen, globus pallidus, cingulated gyrus, hippocampus, inferior parietal lobule, visual cortex of the occipital lobe, midbrain, pons (locus coeruleus), medulla and cerebellum (dentate nucleus)] of n=42 adult individuals (71±12 years old, range: 53-101 years old) with no known history or evidence of neurodegenerative, neurological or psychiatric disorders. It was found that the iron distribution in the adult human brain is quite heterogeneous. The highest levels were found in the putamen (mean±SD, range: 855±295μg/g, 304-1628μg/g) and globus pallidus (739±390μg/g, 225-1870μg/g), and the lowest levels were observed in the pons (98±43μg/g, 11-253μg/g) and medulla (56±25μg/g, 13-115μg/g). Globally, iron levels proved to be age-related. The positive correlation between iron levels and age was most significant in the basal ganglia (caudate nucleus, putamen and globus pallidus). Compared with the age-matched control group, altered iron levels were observed in specific brain areas of one Parkinsons disease patient (the basal ganglia) and two Alzheimers disease patients (the hippocampus).

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.

Phytostabilization of nickel by the zinc and cadmium hyperaccumulator Solanum nigrum L. Are metallothioneins involved

Some heavy metals (HM) are highly reactive and consequently can be toxic to living cells when present at high levels. Consequently, strategies for reducing HM toxicity in the environmental must be undertaken. This work focused on evaluating the Nickel (Ni) accumulation potential of the hyperaccumulator Solanum nigrum L., and the participation of metallothioneins (MT) in the plant Ni homeostasis. Metallothioneins (MT) are gene-encoded metal chelators that participate in the transport, sequestration and storage of metals. After different periods of exposure to different Ni concentrations, plant biometric and biochemical parameters were accessed to determine the effects caused by this pollutant. Semi-quantitative RT-PCR reactions were performed to investigate the specific accumulation of MT-related transcripts throughout the plant and in response to Ni exposure. The data obtained revealed that Ni induced toxicity symptoms and accumulated mostly in roots, where it caused membrane damage in the shock-treated plants, with a parallel increase of free proline content, suggesting that proline participates in protecting root cells from oxidative stress. The MT-specific mRNA accumulation analysis showed that MT2a- and MT2d-encoding genes are constitutively active, that Ni stimulated their transcript accumulation, and also that Ni induced the de novo accumulation of MT2c- and MT3-related transcripts in shoots, exerting no influence on MT1 mRNA accumulation. These results strongly suggest the involvement of MT2a, MT2c, MT2d and MT3 in S. nigrum Ni homeostasis and detoxification, this way contributing to the clarification of the roles the various types of MTs play in metal homeostasis and detoxification in plants.

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.

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 μg/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 + b1X1 + b2X2 + b3X3, where X1 = ln m (Sb), X2 = ln m(Ba) and X3 = 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.

Ion chromatographic determination of iodide in urine and serum using a tubular ion-selective electrode based on a homogeneous crystalline membrane

The use of a laboratory-made iodide ion-selective electrode with tubular configuration and based on a crystalline membrane (AgI/Ag2S) as the detector for ion chromatographic determination of iodide in urine and serum is described. A CIS reversed-phase column was coated withN-cetylpyridinium chloride to prepare a low-exchange-capacity analytical column and with hexadecyltrimethylammonium bromide to prepare a concentrator pre-column. A 2.0 ml min−1 flow rate of deionized water and 0.1 mol 1−1 KNO3 solution was used for the pre-concentration and for the chromatographic separation, respectively. For optimum performance of the detector a background level of iodide was added into the column effluent. A linear relationship (r = 0.9997) between tubular electrode potential (as peak height) and iodide concentration in the range 5–400 μg 1−1 and a detection limit of 1.47 μg 1−1 were obtained. The method shows good reproducibility for both peak height (2.2% RSD) and retention time (1.3% RSD). Recoveries on its application to the samples were 93.0–100.9% for urine and 91.4–106.0% for serum.

FH8--a small EF-hand protein from Fasciola hepatica.

Vaccine and drug development for fasciolasis rely on a thorough understanding of the mechanisms involved in parasite–host interactions. FH8 is an 8 kDa protein secreted by the parasite Fasciola hepatica in the early stages of infection. Sequence analysis revealed that FH8 has two EF‐hand Ca2+‐binding motifs, and our experimental data show that the protein binds Ca2+ and that this induces conformational alterations, thus causing it to behave like a sensor protein. Moreover, FH8 displays low affinity for Ca2+ (Kobs = 104 m−1) and is highly stable in its apo and Ca2+‐loaded states. Homology models were built for FH8 in both states. It has only one globular domain, with two binding sites and appropriate groups in the positions for coordination of the metal ions. However, an unusually high content of positively charged amino acids in one of the binding sites, when compared with the prototypical sensor proteins, potentially affects the protein’s affinity for Ca2+. The only Cys present in FH8, conserved in the homologous proteins of other helminth parasites, is located on the surface, allowing the formation of dimers, detected on SDS gels. These findings reflect specificities of FH8, which are most probably related to its roles both in the parasite and in the host.

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.

Determination of lead and aluminium in port wine by electrothermal atomic absorption spectrometry

Methods for the determination of lead and aluminium in port wine by electrothermal atomic absorption spectrometry based on the stabilized temperature platform furnace concept are described. Except for the dilution step, no sample preparation was required, thereby enabling the direct determination of the elements with little chance of contamination. The determination were performed in the linear range 1.9–100 µg l–1 for lead and 1.6–50 µg l–1 for aluminium in the port wine samples, after appropriate dilution (usually 1 + 9 for lead and 1 + 24 for aluminium). These correspond to detection limits of 19 and 40 µg l–1 for lead and aluminium, respectively, in undiluted samples. The precision was better than 4%(at a concentration level of about 25 µg l–1) for lead and 3%(at a concentration level of about 35 µg l–1) for aluminium. The comparison of the results obtained for 18 port wine samples by direct interpolation of a calibration graph prepared with aqueous standard solutions and by the standard additions method produced shifts that were always less than 10% for lead and 8% for aluminium. The simplicity and rapidity of each analysis (a graphite furnace programme time of 159 s for lead and 85 s for aluminium) makes this method most attractive for routine analysis.