Andrea Raab

Metal Chelation and Inhibition of Bacterial Growth in Tissue Abscesses

Bacterial infection often results in the formation of tissue abscesses, which represent the primary site of interaction between invading bacteria and the innate immune system. We identify the host protein calprotectin as a neutrophil-dependent factor expressed inside Staphylococcus aureus abscesses. Neutrophil-derived calprotectin inhibited S. aureus growth through chelation of nutrient Mn2+ and Zn2+: an activity that results in reprogramming of the bacterial transcriptome. The abscesses of mice lacking calprotectin were enriched in metal, and staphylococcal proliferation was enhanced in these metal-rich abscesses. These results demonstrate that calprotectin is a critical factor in the innate immune response to infection and define metal chelation as a strategy for inhibiting microbial growth inside abscessed tissue.

Mechanisms of Arsenic Hyperaccumulation in Pteris vittata. Uptake Kinetics, Interactions with Phosphate, and Arsenic Speciation

The mechanisms of arsenic (As) hyperaccumulation inPteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg−1 dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).

The Nature of Arsenic-Phytochelatin Complexes in Holcus lanatus and Pteris cretica

We have developed a method to extract and separate phytochelatins (PCs)—metal(loid) complexes using parallel metal(loid)-specific (inductively coupled plasma-mass spectrometry) and organic-specific (electrospray ionization-mass spectrometry) detection systems—and use it here to ascertain the nature of arsenic (As)-PC complexes in plant extracts. This study is the first unequivocal report, to our knowledge, of PC complex coordination chemistry in plant extracts for any metal or metalloid ion. The As-tolerant grass Holcus lanatus and the As hyperaccumulator Pteris cretica were used as model plants. In an in vitro experiment using a mixture of reduced glutathione (GS), PC2, and PC3, As preferred the formation of the arsenite [As(III)]-PC3 complex over GS-As(III)-PC2, As(III)-(GS)3, As(III)-PC2, or As(III)-(PC2)2 (GS: glutathione bound to arsenic via sulphur of cysteine). In H. lanatus, the As(III)-PC3 complex was the dominant complex, although reduced glutathione, PC2, and PC3 were found in the extract. P. cretica only synthesizes PC2 and forms dominantly the GS-As(III)-PC2 complex. This is the first evidence, to our knowledge, for the existence of mixed glutathione-PC-metal(loid) complexes in plant tissues or in vitro. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica.

Uptake and translocation of inorganic and methylated arsenic species by plants

Environmental context. The molecular occurrence of arsenic in soils can vary as a result of soil conditions and/or application of arsenic-containing herbicides or fertiliser. Although large amounts of As-containing herbicides are used for different crops, there is still a lack of understanding as to how the molecular form of As determines the uptake of arsenic into plants and, in particular, the translocation into shoot and grain. Abstract. The uptake and translocation into shoots of arsenate, methylarsonate (MA), and dimethylarsinate (DMA) by 46 different plant species were studied. The plants (n = 3 per As species) were exposed for 24 h to 1 mg of As per litre under identical conditions. Total arsenic was measured in the roots and the shoots by acid digestion and inductively coupled plasma mass spectrometry from which, besides total As values, root absorption factors and shoot-to-root transfer factors were calculated. As uptake into the root for the different plant species ranged from 1.2 to 95 (μg of As per g of dry weight) for AsV, from 0.9 to 44 for MAV and from 0.8 to 13 for DMAV, whereas in shoots the As concentration ranged from 0.10 to 17 for AsV, 0.1 to 13 for MAV, and 0.2 to 17 for DMAV. The mean root absorption factor for AsV (1.2 to 95%) was five times higher than for DMAV (0.8 to 13%) and 2.5 times higher than for MAV (0.9 to 44%). Although the uptake of arsenic in the form of AsV was significantly higher than that of MAV and DMAV, the translocation of the methylated species was more efficient in most plant species studied. Thus, an exposure of plants to DMAV or MAV can result in higher arsenic concentrations in the shoots than when exposed to AsV. Shoot-to-root transfer factors (TFs) for all plants varied with plant and arsenic species. While AsV had a median TF of 0.09, the TF of DMAV was nearly a factor of 10 higher (0.81). The median TF for MAV was in between (0.30). Although the TF for MAV correlates well with the TF for DMAV, the plants can be separated into two groups according to their TF of DMAV in relation to their TF of AsV. One group can immobilise DMAV in the roots, while the other group translocates DMAV very efficiently into the shoot. The reason for this is as yet unknown.

Complexation of Arsenite with Phytochelatins Reduces Arsenite Efflux and Translocation from Roots to Shoots in Arabidopsis

Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

Inorganic arsenic levels in baby rice are of concern

Inorganic arsenic is a chronic exposure carcinogen. Analysis of UK baby rice revealed a median inorganic arsenic content (n=17) of 0.11 mg/kg. By plotting inorganic arsenic against total arsenic, it was found that inorganic concentrations increased linearly up to 0.25 mg/kg total arsenic, then plateaued at 0.16 mg/kg at higher total arsenic concentrations. Inorganic arsenic intake by babies (4-12 months) was considered with respect to current dietary ingestion regulations. It was found that 35% of the baby rice samples analysed would be illegal for sale in China which has regulatory limit of 0.15 mg/kg inorganic arsenic. EU and US food regulations on arsenic are non-existent. When baby inorganic arsenic intake from rice was considered, median consumption (expressed as microg/kg/d) was higher than drinking water maximum exposures predicted for adults in these regions when water intake was expressed on a bodyweight basis.

Survey of arsenic and its speciation in rice products such as breakfast cereals, rice crackers and Japanese rice condiments.

Rice has been demonstrated to be one of the major contributors to arsenic (As) in human diets in addition to drinking water, but little is known about rice products as an additional source of As exposure. Rice products were analyzed for total As and a subset of samples were measured for arsenic speciation using high performance liquid chromatography interfaced with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). A wide range of rice products had total and inorganic arsenic levels that typified those found in rice grain including, crisped rice, puffed rice, rice crackers, rice noodles and a range of Japanese rice condiments as well as rice products targeted at the macrobiotic, vegan, lactose intolerant and gluten intolerance food market. Most As in rice products are inorganic As (75.2-90.1%). This study provides a wider appreciation of how inorganic arsenic derived from rice products enters the human diet.

Arsenic–glutathione complexes—their stability in solution and during separation by different HPLC modes

Complexes of arsenic compounds and glutathione are believed to play an essential part in the metabolism and transport of inorganic arsenic and its methylated species. Up to now, the evidence of their presence is mostly indirect. We studied the stability and chromatographic behaviour of glutathione complexes with trivalent arsenic: i.e. AsIII(GS)3, MAIII(GS)2 and DMAIII(GS) under different conditions. Standard ion chromatography using PRP X-100 and carbonate or formic acid buffer disintegrated the complexes, while all three complexes are stable and separable by reversed phase chromatography (0.1% formic acid/acetonitrile gradient). AsIII(GS)3 and MAIII(GS)2 were more stable than DMAIII(GS), which even under optimal conditions tended to degrade on the column at 25 °C. Chromatography at 6 °C can retain the integrity of the samples. These results shed more light on the interpretation of a vast number of previously published arsenic speciation studies, which have used chromatographic separation techniques with the assumption that the integrity of the arsenic species is guaranteed.

Cooking rice in a high water to rice ratio reduces inorganic arsenic content

Total arsenic and arsenic speciation was performed on different rice types (basmati, long-grain, polished ([white] and wholegrain [brown]) that had undergone various forms of cooking. The effect of rinse washing, low volume (2.5 : 1 water : rice) and high volume (6 : 1 water : rice) cooking, as well as steaming, were investigated. Rinse washing was effective at removing circa. 10% of the total and inorganic arsenic from basmati rice, but was less effective for other rice types. While steaming reduced total and inorganic arsenic rice content, it did not do so consistently across all rice types investigated. Low volume water cooking did not remove arsenic. High volume water : rice cooking did effectively remove both total and inorganic arsenic for the long-grain and basmati rice (parboiled was not investigated in high volume cooking water experiment), by 35% and 45% for total and inorganic arsenic content, respectively, compared to uncooked (raw) rice. To reduce arsenic content of cooked rice, specifically the inorganic component, rinse washing and high volume of cooking water are effective.

Speciation as an analytical aid in trace element research in infant nutrition

The aim of this work was twofold: to study the binding pattern of trace elements in formulas as compared with breast milk and the relationship between trace elements in breast milk and in maternal dietary intake. To investigate the binding form of trace elements in these nutritive fluids, methods for protein separation were combined with methods for trace element determination in the eluted fractions. HPLC and ICP-AES or ICP-MS were coupled on-line for the simultaneous speciation of elements of nutritional interest, viz., Ca, K, Mg, P, S, Co, Cu, Fe, I, Mn, Mo, Se and Zn, and also the heavy metals Cd and Pb in both human mild whey and formulas. In order to minimize interactions between the labile metal protein complexes and the column material, size-exclusion chromatography was used for protein separation. The binding pattern of trace elements in formulas is significantly different from that in breast milk and depends on its main component (cows milk or soy), its processing (hydrolysis) and the chemical form (inorganic) of the added compounds. For example, compared with breast-fed infants the iron supply of formula-fed infants is much higher (up to 20-fold); in addition, the binding forms of Fe are very different in the two fluids. This has to be evaluated with respect to interactions with other elements during intestinal uptake. The investigation of breast milk samples from different regions of the world showed comparable shapes for teh elution profiles and for Mo and Se a dependence on the regional maternal dietary intake. Speciation studies carried out on breast milk samples as a function of the selenium content showed significant changes in the zinc-binding pattern. In particular, citrate (as a zinc-binding component) was found to decrease with increasing dietary selenium intake of the mother.