Charlotte Gabel-Jensen

Selectivity in microemulsion electrokinetic chromatography.

Microemulsion electrokinetic chromatography (MEEKC) is a most promising separation technique providing good selectivity and high separation efficiency of anionic, cationic as well as neutral solutes. In MEEKC lipophilic organic solvents dispersed as tiny droplets in an aqueous buffer by the use of surfactants provide a pseudo-stationary phase to which the solutes may have an affinity either to the surface or they may even partition into the droplets. When the droplets are charged, typically negatively, they will migrate opposite to the electroosmotic flow and hence separation of neutral solutes may take place. In the present paper focus has been set on how to change selectivity in MEEKC. Changes in the nature of surfactant as well as in pH have been shown to be powerful tools in changing the selectivity. The type of lipophilic organic phase is of less importance for the separation of fairly lipophilic solutes. Also changes in the temperature surrounding the capillary may alter the selectivity.

Microemulsion electrokinetic chromatography – or solvent-modified micellar electrokinetic chromatography?

An overview of the microemulsion electrokinetic chromatography technique and its fields of applications in analytical chemistry are given. The separation mechanisms involved are discussed and the technique is compared to solvent-modified micellar electrokinetic chromatography.

Separation of neutral compounds by microemulsion electrokinetic chromatography: Fundamental studies on selectivity

The selectivity of microemulsion electrokinetic chromatography (MEEKC) was studied utilizing some uncharged model compounds like aromatic amides, steroids, and esters of nicotinic acid. The cosurfactant of the microemulsion was found to be the most important factor affecting the selectivity, and alteration between 6.6% of 1‐propanol, 1‐butanol, tetrahydrofuran, and 2‐ethoxyethanol caused several substantial changes in the migration order. In addition, the nature of the surfactant was found to significantly affect the selectivity. In this case, changes in order of migration was observed by replacement of half the content of sodium dodecyl sulfate (SDS) with either sodium dioctyl sulfosuccinate (SDOSS), 3‐(N,N‐dimethylmyristylammonio) propanesulfonate (MAPS), polyoxyethylene sorbitan monolaurate (Tween 21), and polyoxyethylene 23 lauryl ether (Brij 35). MEEKC was also accomplished with 3.3% of the anionic surfactant sodium cholate and with the cationic surfactant N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMA). Both provided substantial differences in selectivity as compared to the SDS‐based systems. With SDS as surfacant, the concentration was varied within 1.0–4.5%. Minor selectivity changes were observed as the concentration of the surfacant was reduced, but the major effect was a reduction in the total migration time. The organic solvent of the microemulsion droplets was found only to have minor impact on the selectivity.

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.

Surveying selenium speciation from soil to cell—forms and transformations

The aim of this review is to present and evaluate the present knowledge of which selenium species are available to the general population in the form of food and common supplements and how these species are metabolized in mammals. The overview of the selenium sources takes a horizontal approach, which encompasses identification of new metabolites in yeast and food of plant and animal origin, whereas the survey of the mammalian metabolism takes a horizontal as well as a vertical approach. The vertical approach encompasses studies on dynamic conversions of selenium compounds within cells, tissues or whole organisms. New and improved sample preparation, separation and detection methods are evaluated from an analytical chemical perspective to cover the progress in horizontal speciation, whereas the analytical methods for the vertical speciation and the interpretations of the results are evaluated from a biological angle as well.

Comparison of microemulsion electrokinetic chromatography and solvent‐modified micellar electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) was investigated with the goal of elucidating the influence exerted by the amount of lipophilic organic phase and the amount of co-surfactant on the migration of a number of neutral test solutes. Furthermore, MEEKC was compared to solvent-modified micellar electrokinetic chromatography (MEKC). The organic solvents methanol, ethanol, 1-propanol, 1-butanol, acetonitrile, tetrahydrofuran, and 2-ethoxyethanol serving as co-surfactants in MEEKC were used in similar concentrations in MEKC systems in order to compare the selectivity and separation efficiency of the two kinds of system. The solvent-modified MEKC technique proved to be very similar to MEEKC in performance with respect to both separation selectivity and efficiency of separation.

Separation and identification of the selenium-sulfur amino acid S-(methylseleno)cysteine in intestinal epithelial cell homogenates by LC-ICP-MS and LC-ESI-MS after incubation with methylseleninic acid

The metabolism of methylseleninic acid, MeSeA in the gastro-intestinal tract was studied by incubation of MeSeA with homogenized intestinal epithelial cells from pigs. The major selenium-containing metabolite was identified by analysis of the supernatant of the incubated cells by LC-ICP-MS. The identity of the compound was established by LC-ESI-MS/MS after purification by preparative chromatography. The metabolite appeared to be a selenium-sulfur amino acid, S-(methylseleno)cysteine (CH3Se–S–CH2CH(NH2)COOH). The selenium-sulfur ratio was confirmed to be 1:1 by simultaneous monitoring of selenium and sulfur as the oxides by LC-ICP-MS. Analysis of a synthesized standard of S-(methylseleno)cysteine by LC-ESI-MS resulted in the same mass spectrum with the same fragmentation pattern as the isolated metabolite. The formation of this selenium compound did not require enzymatic systems but only the presence of cysteine. The presence of this selenium compound in mammalian cell models after addition of MeSeA has not previously been reported.

Selenium metabolism in hepatocytes incubated with selenite, selenate, selenomethionine, Se-methylselenocysteine and methylseleninc acid and analysed by LC-ICP-MS

The aim of the present work was to estimate the amounts of selenium metabolites produced in a liver cell model. Five different selenium compounds comprising the inorganic selenium species selenate and selenite, methylseleninic acid (MeSeA) and the selenoamino acids, selenomethionine (SeMet) and Se-methylselenocysteine (Se-MeSeCys) were incubated in rat hepatocytes in amounts of 5 μM for 4 h. The distribution of selenium between media, lysate and insoluble fractions was determined by means of flow injection ICP-MS. Speciation analysis of growth media and lysate samples by LC-ICP-MS showed only limited metabolism of selenate, SeMet, and Se-MeSeCys, while MeSeA and selenite were extensively metabolized. Small molecule metabolites constituted less than 5% of the applied amount and some of the transformed selenium was detected in the protein fraction of the samples although all Se could not be accounted for. This result indicates that more important metabolism pathways may be related to protein bound selenium.

LC-ICP-MS and LC-ESI-(MS)n identification of Se-methylselenocysteine and selenomethionine as metabolites of methylseleninic acid in rat hepatocytes

The metabolism of methylseleninic acid in isolated rat hepatocytes was investigated. Selenium containing metabolites excreted from the cells were detected in the supernatant of the incubation sample by LC-ICP-MS. After pre-treatment of the supernatant by preparative chromatography and pre-concentration by lyophilisation, a major metabolite was identified by molecular mass spectrometry as Se-methylselenocysteine by LC-ESI-MS, MS2 and MS3 and a minor metabolite was identified as selenomethionine by LC-ESI-MS2 and MS3 and LC-ESI-MS2(SRM). This is the first time these metabolites have been identified in hepatocytes. Complementary data from ion trap and triple quadrupole MS instruments provided solid proof of metabolite identities. A time course study showed that S-(methylseleno)cysteine and S-(methylseleno)glutathione were intermediates in the formation of the major metabolite. It is questioned if methylseleninic acid is a relevant model compound for methylated Se-amino acids in vitro.

The Selenium Metabolite Methylselenol Regulates the Expression of Ligands that Trigger Immune Activation through the Lymphocyte Receptor NKG2D

Background: Immune activation through a balanced cell surface expression of human NKG2D ligands is crucial for the elimination of diseased cells. Results: Methylselenol induces the expression of the NKG2D ligands MICA/B but specifically inhibits ULBP2 protein expression. Conclusion: Methylselenol regulates NKG2D ligand expression on transcriptional and posttranscriptional levels. Significance: Methylselenol is the first identified metabolite that diversely regulates NKG2D ligands, and, therefore, its implementation could improve NKG2D-based immune therapy. For decades, selenium research has been focused on the identification of active metabolites, which are crucial for selenium chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include increased formation of reactive oxygen species, induction of DNA damage, triggering of apoptosis, and inhibition of angiogenesis. Here we reveal that CH3SeH modulates the cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways that occur early during malignant transformation and enable the recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional levels. CH3SeH induced the transcription of MHC class I polypeptide-related sequence MICA/B and ULBP2 mRNA. However, the induction of cell surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH regulation of NKG2D ligands. A balanced cell surface expression of NKG2D ligands is considered to be an innate barrier against tumor development. Therefore, our work indicates that the application of selenium compounds that are metabolized to CH3SeH could improve NKG2D-based immune therapy.