Metabolism of selenite to selenosugar and trimethylselenonium in vivo: tissue dependency and requirement for S-adenosylmethionine-dependent methylation.

Impaired S-adenosylmethionine (SAM)-dependent transmethylation and methylation capacity feature in diseases related to obesity or aging, and selenium (Se) metabolism is altered in these states. We tested the hypothesis that SAM metabolism is required for methylation and excretion of Se in a rat model. Four hours after selenite and periodate-oxidized adenosine (POA; an inhibitor of SAM metabolism) were administered, circulating markers of single-carbon status were unchanged, except for decreased circulating phosphatidylcholine (P<.05). In contrast, liver and kidney SAM and S-adenosylhomocysteine were elevated (P<.05 for all). Concentrations of total Se were significantly elevated in both liver (P<.001) and kidney (P<.01), however the degree of accumulation in liver was significantly greater than that of kidney (P<.05). Red blood cell Se levels were decreased (P=.01). Trimethylselenonium levels were decreased in liver and kidney (P=.001 for both tissues) and Se-methyl-N-acetylselenohexosamine selenosugar was decreased in liver (P=.001). Urinary output of both trimethylselenonium (P=.001) and selenosugar (P=.01) was decreased as well. Trimethylselenonium production is more inhibited by POA than is selenosugar production (P<.05). This work indicates that low molecular weight Se metabolism requires SAM-dependent methylation, and disrupting the conversion of SAM to S-adenosylhomocysteine prevents conversion of selenite and intermediate metabolites to final excretory forms, suggesting implications for selenium supplementation under conditions where transmethylation is suboptimal, such as in the case of obese or aging individuals.

Internal correction of spectral interferences and mass bias for selenium metabolism studies using enriched stable isotopes in combination with multiple linear regression.

The analytical methodology for the in vivo study of selenium metabolism using two enriched selenium isotopes has been modified, allowing for the internal correction of spectral interferences and mass bias both for total selenium and speciation analysis. The method is based on the combination of an already described dual-isotope procedure with a new data treatment strategy based on multiple linear regression. A metabolic enriched isotope ((77)Se) is given orally to the test subject and a second isotope ((74)Se) is employed for quantification. In our approach, all possible polyatomic interferences occurring in the measurement of the isotope composition of selenium by collision cell quadrupole ICP-MS are taken into account and their relative contribution calculated by multiple linear regression after minimisation of the residuals. As a result, all spectral interferences and mass bias are corrected internally allowing the fast and independent quantification of natural abundance selenium ((nat)Se) and enriched (77)Se. In this sense, the calculation of the tracer/tracee ratio in each sample is straightforward. The method has been applied to study the time-related tissue incorporation of (77)Se in male Wistar rats while maintaining the (nat)Se steady-state conditions. Additionally, metabolically relevant information such as selenoprotein synthesis and selenium elimination in urine could be studied using the proposed methodology. In this case, serum proteins were separated by affinity chromatography while reverse phase was employed for urine metabolites. In both cases, (74)Se was used as a post-column isotope dilution spike. The application of multiple linear regression to the whole chromatogram allowed us to calculate the contribution of bromine hydride, selenium hydride, argon polyatomics and mass bias on the observed selenium isotope patterns. By minimising the square sum of residuals for the whole chromatogram, internal correction of spectral interferences and mass bias could be accomplished. As a result, the tracer/tracee ratio could be calculated for each selenium-containing species and a time relationship for synthesis and degradation established. Both selenite and selenized yeast labelled with (77)Se were employed for comparative purposes.

Investigation of the selenium metabolism in cancer cell lines.

The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 µM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

Formation of methylselenol, dimethylselenide and dimethyldiselenide in in vitro metabolism models determined by headspace GC-MS.

The aim of this study was to identify the presence of MeSeH in metabolic reactions. An analytical method based on direct headspace GC-MS, eliminating loss of volatile species during sample pretreatment procedures, was developed for this purpose. The in vitro conversion of selenium compounds to the volatile species methylselenol, MeSeH, dimethyl selenide, DMeSe and dimethyl diselenide, DMeDSe was investigated. The analytical method was evaluated by means of standards of dimethyl diselenide, dimethyl selenide. The corresponding sulfides were found unsuitable as internal standards as they interacted with the selenides. The limit of detection was 0.25 µmol L(-1) (20 µg L(-1)) for the selenide as well as the diselenide. Formation of MeSeH was not observed in significant amount when selenomethionine was incubated with the enzyme l-methionine-γ-lyase; instead large amounts of DMeDSe were formed. In aqueous solution, methylseleninic acid, MeSeA reacted spontaneously with glutathione, GSH to form DMeDSe. In strongly reducing environments, however, MeSeH was also observed. When the formed MeSeH was trapped with iodoacetic acid, no DMeDSe was detected indicating that DMeDSe formation was due to spontaneous oxidation of MeSeH. These findings imply that DMeDSe may be a marker for the production of MeSeH in in vitro models. When MeSeA, Se-methylselenocysteine, Se-MeSeCys and SeMet were incubated with Jurkat cells, DMeDSe formation was only observed in the case of MeSeA. Trace amounts of DMeSe was observed in the vial with MeSeA as well as Se-MeSeCys. When DMeSe and DMeDSe were added to plasma, the sensitivity of only DMeDSe decreased significantly, implicating that DMeDSe underwent a reaction with plasma hindering the volatilization. This emphasizes that results from in vitro selenium metabolism studies may not be uncritically interpreted as consistent with the in vivo reality.

A method for analysis of dimethyl selenide and dimethyl diselenide by LC-ICP-DRC-MS.

The aim of this work was to develop a simple and fast high performance liquid chromatography-inductively coupled argon plasma (ICP) mass spectrometry (MS) method capable of separating and detecting the two volatile selenium species dimethyl selenide (DMeSe) and dimethyl diselenide (DMeDSe) in biological samples. Dimethyl selenide and dimethyl diselenide were separated on a short reversed phase column using an eluent containing 40% methanol and detected by dynamic reaction cell ICP-MS monitoring the (80)Se isotope. The limit of detection was 8 nM for both species (corresponding to 0.6 and 1.3 µg Se/L for DMeDSe and DMeSe, respectively). Both compounds exhibited a linear signal-concentration relationship in the investigated concentration range of 0.1-1 µM with a precision on the determinations better than 3%. The method was applied for analysis of samples from cancer cell lines incubated with methylseleninic acid, selenomethionine, Se-methylselenocysteine, and sodium selenite. DMeDSe were detected in some samples. The method offers a simple and fast analysis of DMeDSe and DMeSe using standard liquid chromatography coupled with ICP-MS equipment and interfacing.