Helle Rüsz Hansen

Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites

• Inorganic arsenic (As(i) ) in rice (Oryza sativa) grains is a possible threat to human health, with risk being strongly linked to total dietary rice consumption and consumed rice As(i) content. This study aimed to identify the range and stability of genetic variation in grain arsenic (As) in rice. • Six field trials were conducted (one each in Bangladesh and China, two in Arkansas, USA over 2 yr, and two in Texas, USA comparing flooded and nonflood treatments) on a large number of common rice cultivars (c. 300) representing genetic diversity among international rice cultivars. • Within each field there was a 3-34 fold range in grain As concentration which varied between rice subpopulations. Importantly, As(i) correlated strongly with total As among a subset of 40 cultivars harvested in Bangladesh and China. • Genetic variation at all field sites was a large determining factor for grain As concentration, indicating that cultivars low in grain As could be developed through breeding. The temperate japonicas exhibited lower grain As compared with other subpopulations. Effects for year, location and flooding management were also statistically significant, suggesting that breeding strategies must take into account environmental factors.

Detection of antimony species in citrus juices and drinking water stored in PET containers

In this paper we report on the presence of inorganic Sb(III), a novel Sb(V)–citrate complex and a species tentatively identified as being non-complexed inorganic Sb(V) in citrus fruit juices stored in PET bottles.

Complementary use of molecular and element-specific mass spectrometry for identification of selenium compounds related to human selenium metabolism

The aim of this paper is to give an overview of analytical data on the identification of selenium compounds in biological samples with relevance for selenium metabolism. Only studies applying the combination of element-specific inductively coupled plasma mass spectrometry as well as molecular electrospray mass spectrometry detection have been included. Hence, selenium compounds are only considered identified if molecular mass spectra obtained by analysis of the authentic biological sample have been provided. Selenium compounds identified in selenium-accumulating plants and yeast are included, as extracts from such plants and yeast have been widely used for examination of the cancer-preventive effect of selenium in cell lines, animal models and human intervention trials. Hence, these selenium compounds are available for absorption and further metabolism. Identification of selenium metabolites in simulated gastric and intestinal juice, intestinal epithelial tissue, liver and urine is described. Hence, selenium metabolites identified in relation to absorption, metabolism and excretion are included.

Arsenic accumulation and speciation analysis in wool from sheep exposed to arsenosugars

Wool or hair fibre is a metabolically dead material after it has left the epidermis. During growth the fibre in the root is a metabolically very active organ, which is highly influenced by the health status of the living being. Arsenic is one of the elements that is easily taken up by the cells of the root and stored in the fibre afterwards. Here we show that arsenic can quantitatively be extracted by boiling the wool fibre or hair in water. The high intake of arsenic species by the sheep of North Ronaldsay (the seaweed-eating sheep) leads to a high arsenic concentration in wool (mean 5.2+/-2.3 mug g(-1)). The wool of lambs of these sheep, which are not exposed to seaweed, contains about 10 times less arsenic, which is still elevated compared to uncontaminated wool. The arsenic species identified in wool extract are arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)) and monomethylarsonious acid (MMA(III)) as minor species. The major species is dimethylated arsenic DMA in its tri- and pentavalent form (dimethylarsinous acid (DMA(III)) and dimethylarsinic acid (DMA(V))) accounting for 85% of the specified arsenic in the wool which reflects the amount of dimethylated species (i.e. the arsenoribofuranosides) taken up by seaweed being the main food source of the sheep. However, there are unknown arsenic species in the extract, which are not eluting from a strong anion exchange column. In vitro incubation experiments with this kind of wool showed that it has reducing properties but no demethylation was recorded. The absorption ability of the wool for methylated arsenic species is negligible, while inorganic arsenic is easier to be absorbed in the fibre (11-17%). This means that the species integrity is only guaranteed in terms of the degree of methylation but not in terms of their redox status.

New arsenosugar metabolite determined in urine by parallel use of HPLC-ICP-MS and HPLC-ESI-MS

Here we report a new arsenic metabolite found in sheep’s urine after arsenosugar ingestion identified by the parallel use of HPLC-ICP-MS and HPLC-ESI-MS. The two methods together provide an excellent tool for identification of novel arsenic compounds in body fluids and extracts. Besides the previously reported major arsenic metabolite dimethylarsinic acid (DMA(V)) and the minor dimethylarsinoylethanol (DMAE), here we identify the second most abundant metabolite as dimethylarsinoylacetate (DMAA). The pH dependent retention behaviour of all three arsenic compounds was studied on an anion-exchange column in the pH range 3–8.5. This is the first time DMAA has been reported as a metabolite in urine after arsenosugar ingestion.

The use of inductively coupled plasma mass spectrometry as a detector in drug metabolism studies

Background: The inherent properties of element selectivity combined with high sensitivity and structure independent response, make inductively coupled plasma mass spectrometry (ICP-MS) an interesting alternative detection technique in drug metabolism studies. Objective: The application of online separation with ICP-MS detection in drug metabolism studies is reviewed with focus on the merits and demerits of this detection technique. The prerequisite for inclusion in this review is that the study involves a separation technique hyphenated online to the ICP-MS detection. Result/conclusion: ICP-MS detection is found to be advantageous for analysis of all drug substances detectable by ICP-MS compared to radiochemical detection. Detectable drugs are limited to halogen-, sulfur-, metal- and metalloid-containing compounds. The drawback of interference from endogenous compounds on quantitative mass balance estimations of non-metal drugs is addressed. The potential of determining the stoichiometry in metallo-drug biomolecule interactions is pointed out by presenting examples of simultaneous monitoring of metals in metallo-drugs and intrinsic ICP-MS detectable elements in biomolecules. It is concluded that ICP-MS detection is an indispensable technique in drug metabolism studies of metallo-drugs, although the applicability for traditional drugs is limited.

Screening of OATP1B1/3 and OCT1 inhibitors in cryopreserved hepatocytes in suspension

Drug-drug interactions involving hepatic drug transporters may have clinical consequences and jeopardize development of promising drug candidates. Organic anion transporting polypeptides (OATP/Oatp) and the organic cation transporters (OCT/Oct) are among the most important transporters involved in xenobiotic uptake in the liver. In the present study, 179 molecules have been tested as inhibitors of the uptake of estradiol-17betaD-glucuronide (E(2)17betaG), substrate of OATP1B1/3 (rOatp), or 1-methyl-4-phenylpyridinium (MPP+), substrate of OCT1 (rOct1), into suspended cryopreserved hepatocytes from humans and rats. Uptake was assessed in 96-well plates by measuring intracellular accumulation of radioactive substrate in hepatocytes in presence or absence of inhibitor. In rat hepatocytes 140 compounds were identified as inhibitors (inhibition at 20 microM > or = 30%) of E(2)17betaG uptake and 77 compounds inhibitors of MPP+ uptake. The most potent inhibitors of rOatp and rOct1 were dantrolene sodium (K(i)=2 +/- 9 microM) and bepridil (K(i)=14 +/- 2 microM), respectively. In human hepatocytes, the most potent inhibitors of E(2)17betaG and MPP+ uptake were capsazepine (K(i)=14 +/- 5 microM) and cyproheptadine (K(i)=19+/-3 microM), respectively. Structure-activity relationship (SAR) analysis of all tested compounds suggested that lipophilicity, polarity, pK(a) and the number of hydrogen bond donors and acceptors play a role in their interaction with the transporters investigated. The method used here is a simple tool to screen large number of compounds as inhibitors of the uptake of substrates into suspended hepatocytes.

Comparison of two CE-ICP-MS interfaces and quantitative measurements of carboplatin in plasma samples using an internal standard

Two commercially available CE-ICP-MS interfaces were investigated with respect to precision, limits of detection and ease-of-use. Our results demonstrate that the CEI-100 interface was more suited for the detection of carboplatin in our experimental set-up. A relative detection limit of 21 µg Pt L−1 corresponding to an absolute detection limit of 0.1 fg Pt was found for the CEI-100 interface. The quantitative aspects of CE-ICP-MS were addressed and a method was developed using sodium diatrizoate as an internal standard added to the samples to correct for changes in sensitivity between runs. A significant improvement on the repeatability was obtained. The method was applied for the determination of free carboplatin in plasma incubations. The percentage of free carboplatin was calculated both based on the calibration curve and based on the fraction of free carboplatin relative to the total platinum area. Significantly different results were obtained using the two methods, probably due to adsorption of the plasma proteins to the incubation vial or to the capillary or due to formation of carboplatin adducts in concentrations below the limit of detection. This indicates a need for critical evaluation of the quantitative CE-ICP-MS measurements used for kinetic profiling.

Reduction of Sb(V) in a Human Macrophage Cell Line Measured by HPLC-ICP-MS

Drugs based on pentavalent antimony are first-line treatment of the parasite disease leishmaniasis. It is generally believed that Sb(V) acts as a prodrug, which is activated by reduction to Sb(III); however, the site of reduction is not known. It has been hypothesised that the reduction takes place in the parasites’ host cells, the macrophages. In this study, the human macrophage cell line Mono Mac 6 was exposed to Sb(V) in form of the drug sodium stibogluconate (Pentostam™). Cell extracts were analysed for Sb species by high-performance liquid chromatography with inductively coupled plasma-mass spectrometry detection. We found that Sb(V) is actually reduced to Sb(III) in the macrophages; up to 23% of the intracellular Sb was found as Sb(III). Transfer of the cells to Sb-free medium rapidly decreased their Sb(V) and Sb(III) content. Induction of the cell’s production of reactive oxygen species did not have any marked effect on the intracellular amounts of Sb(III).

Analytical techniques and methods used for antimony speciation analysis in biological matrices

Reports of antimony speciation analysis in biological matrices are so far limited. More specifically, only inorganic antimony (V) and antimony (III), mono-, di- and tri-methylantimony species, along with four complexes of SbV with lactate and citrate ligands, and two complexes of SbIII with glutathione have been so far identified. The limited progress may partly be attributed to the lack of suitable analytical methodologies. The main problems and challenges faced are (1) to achieve quantitative Sb extraction from solid biological materials, (2) species instability and their preservation during sample processing and analysis, (3) low chromatographic recovery of Sb from chromatographic columns with increased potential for species conversion when using stronger eluting mobile phases, and (4) trace level Sb species present at too low concentrations for molecular mass spectrometric identification. In the present article the hitherto used analytical methods and techniques are reviewed, with emphasis on identifying their main limitations, as well as highlighting recent breakthroughs. Re-assessing some of our current practises seems appropriate if we want to further advance the Sb speciation field, especially concerning the identification of Sb-complexes. Because of the generally low extractability, future antimony speciation studies may have to also rely on solid state analytical methods. In the case of sufficient extractability further advances in new separation methods are urgently required in order to achieve high column recoveries and maintain intact Sb species for further detection.