Investigation of the recovery of selenomethionine from selenized yeast by two-dimensional LC–ICP MS

Determination of selenomethionine in selenized yeast by HPLC–ICP MS has been revisited with the focus on recovery of this amino acid during the proteolytic digestion and chromatography steps. Recovery of the extracted selenium from an anion-exchange column was 100% but selenomethionine quantified by the method of standard additions accounted only for 67% of the selenium injected. Analysis (by size-exclusion LC–ICP MS) of the eluate collected before and after the selenomethionine peak showed the presence of oxidized selenomethionine (ca. 3%) and selenomethionine likely to be unspecifically associated with the biological matrix continuum (ca. 11%). This finding was validated by two-dimensional LC–ICP MS using a different elution order, i.e. size-exclusion anion-exchange. The approach developed enabled demonstration that more than 80% of selenium in the selenized yeast is actually present in the form of selenomethionine and suggests that many results reported elsewhere for the concentration of this vital amino acid in selenized yeast may be negatively biased. The research also provided insight into speciation of selenium in the solid residue after proteolytic extraction but the additional amount of selenomethionine recovered was negligible (<1.5%).     

Selenium distribution in tissues and monitor materials after long-term selenium supplementation investigated by neutron activation analysis

The effects of long-term selenium supplementation on the selenium body status were investigated in humans and rats. Selenium was determined in human muscle biopsies and monitor materials and in rat tissues by neutron activation analysis. The results showed that the body selenium load is raised by additional supply of selenomethionine or selenomethionine-containing yeast but not proportionally to the intake. The surplus selenium can serve as an endogenous source to maintain the selenoprotein levels during insufficient supply. Highly significant correlations between the muscle selenium concentrations and those in blood, blood fractions, hair and nails indicate that the selenium status can be assessed by analysis of these monitor materials.     

Determination of selenoamino acids by gas chromatography-mass spectrometry

The application of the recently introduced ethylchloroformate derivatization method for the separation and determination of selenomethionine and selenocystein in selenium-enriched yeast and yeast-free tablets by means of a gas chromatography-mass spectrometry (GC-MS) system has been studied. The efficiency of three methods for the extraction of selenomethionine from the tablets were compared. Total selenium content of the same tablets were measured using inductively coupled plasma (ICP)-MS and it was found that in the selenized yeast tablets about 80% of the total selenium is present as selenomethionine. The results were in agreement with the values in the labels and with the literature. The accuracy of the total selenium analysis was controlled by the analysis of a reference material.     

Selenium status and cancer mortality in subjects residing in four Canadian provinces

The objective of this study was to measure selenium status in maleand female Canadian subjects relative to cancer mortality in their respectiveprovinces. Toenail specimens from 755 subjects, 377 males and 378 females,living in Vancouver (186), Edmonton (188), Toronto (197) and Montreal (184)were analyzed by instrumental neutron activation analysis giving means of0.968±0.177, 0.950±0.148, 0.932±0.135 and 0.896±0.127ppm Se, respectively. The effect of selenium determinants such as gender,selenium supplementation and smoking on selenium status is presented. Detailsof the observed inverse relationship of selenium status and cancer mortalityare discussed.     

Analysis of the selenium species distribution in cow blood by size exclusion liquid chromatography–inductively coupled plasma collision cell mass spectrometry (SEC–ICPccMS)

A method for performing rapid semiquantitative screening of the distribution of Se species in the blood of cows fed with a diet enriched in selenized yeast was optimized. The method was based on direct injection of a blood sample onto a high resolution size exclusion chromatographic column and fractionation of the selenium species. Selenium was detected on-line by ICP-MS with a collision cell. The concentrations of selenized haemoglobin and free selenomethionine were estimated using the chromatogram. The method was applied to a study involving 15 control and 15 treated dairy cows at four different supplementation time points. The increase in the selenomethionine and selenized haemoglobin was a linear function of the total selenium concentration. A threshold value of 600 ng ml(-1) of total Se was established beyond which selenomethionine could not be incorporated into the protein. No inorganic selenium was found to be present. The total selenium in cow blood correlated well with that in milk. The selenium supplementation did not change the protein distribution profiles for other essential elements (Cu, Fe, Mn, Zn).     

Speciation and bioavailability of selenium in yeast-based intervention agents used in cancer chemoprevention studies

This study investigated the speciation and bioavailability of selenium in yeast-based intervention agents from multiple manufacturers from several time points. Sources of selenized yeast included Nutrition 21 (San Diego, CA), which supplied the Nutritional Prevention of Cancer (NPC) Trial from 1981-1996; Cypress Systems (Fresno, CA; 1997-1999); and Pharma Nord (Vejle, Denmark; 1999-2000), which supplied the Prevention of Cancer by Intervention by Selenium (PRECISE) Trial pilot studies. The low-molecular-selenium species were liberated from the samples by proteolytic hydrolysis followed by separation by ion exchange liquid chromatography and detection by inductively coupled plasma-mass spectrometry. The results for the NPC tablets showed that selenomethionine, together with 3 unidentified selenium compounds, were predominant in the sample hydrolysates. The relative amounts of the 4 selenium species varied (p < 0.05) among several of the 7 tablet batches used during the course of the NPC Trial. In comparison, 5 batches of more recently produced selenized yeasts, which were used as a source of selenium in the PRECISE and other trials, contained less of the unknown compounds and more selenomethionine at 54-60% of the total selenium in the yeasts. One batch of yeast, however (from 1985), which originated from the same producer as the yeast used in the NPC tablets, contained only 27% of selenium in the sample as selenomethionine. Human subjects receiving 200 microg selenium/day in the UK PRECISE Pilot Trial showed a higher concentration (p < 0.01) and higher increase from baseline in plasma selenium than did the same dosage used in the NPC Trial. Differences in intake, speciation, or bioavailability of selenium from the yeast-based supplements in the population groups studied may explain this. Furthermore, the selenium concentration in whole blood from the Danish PRECISE Pilot Trial was higher (p < 0.001) than that obtained with synthetic L-selenomethionine in a comparable group of Danes, both groups having been treated with 300 microg selenium/day.     

Selenoamino acid speciation using HPLC–ETAAS following an enzymic hydrolysis of selenoprotein

A high-pressure liquid chromatography–electrothermal atomic absorption spectroscopy (HPLCETAAS) hyphenated technique was used for the determination of seleno compounds present in a selenium-enriched yeast. Conditions were optimized for the separation and quantification of the selenoamino acids, selenocystine and selenomethionine, in the presence of other compounds. The separation was achieved by ion-pairing chromatography using sodium heptanesulphonate as the anionic counterion. On-line detection was carried out using electrothermal atomic absorption with palladium(II) as a matrix modifier. Different extraction procedures were tested on a seleniumenriched yeast. A 92% recovery of the total selenium present in the material was obtained. Attempts to evaluate selenium speciation were carried out; selenomethionine and selenocystine were identified as the major components (42% and 35% respectively).     

Identification of selenomethionine in selenized yeast using two-dimensional liquid chromatography-mass spectrometry based proteomic analysis

Selenium-enriched yeast has been commonly used as a nutritional supplement. Here we describe a protocol used to investigate the metabolic fate of inorganic selenium in yeast. We provide definitive, mass spectrometry based evidence for the non-specific incorporation of selenomethionine in the yeast proteome involving the replacement of about 30% of all methionine with selenomethionine.     

TISSUE SELENIUM LEVELS AND GROWTH RESPONSES OF MICE FED SELENOMETHIONINE,SE-METHYLSELENOCYSTEINE OR SODIUM SELENITE

Abstract

Mice fed diets containing selenomethionine at a level of 20 ppm selenium and raised to 30 ppm selenium at 3 weeks on experiment showed (1) delayed response to selenium toxicity, (2) slow recovery from the toxicity after removal of selenium from the diet and (3) relatively high deposition and retention of tissue selenium. These data suggest that selenomethonine initially becomes incorporated in to the primary structure of proteins and as such is not particularly toxic. However, upon its slow removal from protein, selenomethionine becomes toxic by forming selenium IV compounds through a pathway similar to that followed by methionine.

Mice fed diets containing sodium selenite or Se-methylselenocysteine at the same level of selenium as the selenomethionine diet showed (1) immediate response to selenium toxicity (2) rapid recovery from the toxicity after removal of selenium from the diet and (3) relatively low deposition and relatively rapid depletion of tissue selenium. These data suggest that sodium selenite and Se-methylselenocysteine ultimately follow similar metabolic pathways and do not become part of the primary structure of proteins. A possible metabolic route for Se-methylselenocysteine is that it is oxidized to toxic selenium IV compounds through an oxidative pathway similar to that followed by S-methylcysteine.     

Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast.     

Selenium Occurrence,Distribution and Speciation in the Cockle Anadara trapezia and the Mullet Mugil cephalus

Data on the factors affecting the accumulation of selenium in the cockle Anadara trapezia and mullet Mugil cephalus are presented, together with the distribution and speciation of selenium in tissues. Selenium concentration in whole cockles showed a small but significant decrease with weight. No further decrease in selenium concentration was apparent once an organism reached 0.25 g dry weight. Selenium concentration in cockles was not dependent on sex. The tissue distribution of selenium concentration in cockles was in the order gill>intestine>adductor>mantle >foot. Selenium concentrations in liver tissues of mullet increased with the whole weight of the fish. In contrast, selenium concentrations in muscle, stomach, heart and kidney tissues were fairly low and constant in fish weighing less than 200 g (20 cm in length). Fish of greater weight and size (>250 g and >30 cm) had higher and more variable selenium concentrations. No differences in selenium concentration between male and female fish occurred; however, the sex of many of the fish could not be distinguished. The tissue distribution of selenium concentration in mullet was in the order liver>stomach>heart>muscle>kidney. Most of the selenium recovered from both the cockle tissues and the mullet muscle tissues was found to be associated with proteins and to be present as selenocysteine. A conceptual model is presented for selenium transformations in marine organisms based on the formation of selenoamino-acids and subsequent incorporation into proteins. © 1997 by John Wiley & Sons, Ltd.     

Effect of selenium in organic and inorganic form on liver,kidney, brain and muscle of <Emphasis Type="Italic">Wistar</Emphasis> rats

Selenium is a micronutrient, localized in the active sites of enzymes such as glutathione peroxidase and thioredoxin reductase, and participating together with these enzymes in an antioxidant defence system of organisms against free radicals. Administration of selenium is necessary for maintaining oxidative homeostasis. The present experiment is aimed at investigation of selenium impact on basal metabolic processes and selected antioxidants in a Wistar rat model, fed selenium in organic and inorganic forms. Liver, kidney, brain and muscle were sampled during a month-long feeding with four different doses of selenium (0.075 mg or 1.5 mg of inorganic and/or organic selenium per kg of feed). We found a significant reduction in glutathione level in liver tissue regardless of the form of the administered selenium. On the other hand, selenium caused a decreased glutathione reductase level in the liver and metallothionein level in the liver, kidney and muscle.      

硒酵母中有机硒及硒代氨基酸含量的测定方法

报道了人工培养硒酵母中有机硒及硒代胱氨酸（ＳｅＣｙｓ）和硒代蛋氨酸（ＳｅＭｅｔ）含量的测定方法。采用透析处理法使硒酵母中的无机硒和有机硒得以分离,并采用催化分光光度法测定了硒酵母中有机硒的含量;采用氨基酸自动分析仪测定了硒酵母中ＳｅＣｙｓ和ＳｅＭｅｔ的含量。     

Methodological advances for selenium speciation analysis in yeast

Advances in analytical methodology for speciation of selenium in selenized-yeast food supplements were discussed on the basis of the recent developments in the authors’ laboratory. Particular attention was given to the sample preparation with regard to the fractionation of selenium into different classes of chemical species, the high resolution fractionation of selenium from yeast water extracts by size-exclusion chromatography and characterization of the water soluble protein fraction by combined matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry (TOF MS) and electrospray quadrupole-TOF tandem MS. The true speciation of protein-incorporated selenium (down to individual proteins characterized by a unique aminoacid sequence) was discussed using an example of a family of selenium-containing proteins formed in yeast by the substitution of methionine residues by selenomethionine in a salt stress-induced protein.     

Toenail selenium level among healthy residents of two Polish Districts

The goal of this study was to evaluate the selenium mass fraction in toenail clippings taken from random inhabitants living in various areas of the Pomeranian (Northern Poland) and Lubuskie (Western Poland) Districts. Toenail clippings were analyzed by instrumental neutron activation analysis (INAA) giving means of 0.57±0.10 and 0.60±0.16 mg·kg⁻¹ for the two areas, respectively, but the difference was statistically not significant. In additional, it was found that gender, age, body mass index (BMI), smoking, and selenium supplementation are factors with apparent effects to the selenium levels in toenail clippings.     

用离子抑制色谱法分析二（2,2,6,6—四甲基—4—哌啶基）马来酸酯合？…

建立了用离子抑制色谱法分析二（２,２,６,６－四甲基－４－哌啶基）马来酸酯合成反应液的方法。平均回收率为９８．８％,相对标准偏差为０．５６％,测量的平均相对偏差不大于５．０％,方法简单,快速,可用于工艺条件的选择和质量检测。     

Development of new analytical methods for selenium speciation in selenium-enriched yeast material

A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material. The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns. It showed that water-soluble selenium is present in several fractions with a large mass distribution. Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments. The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis. Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography. The study revealed the existence of various unidentified Se species in yeast material. The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS. This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands.     

Effect of sample preparation methods on the D,L-enantiomer ratio of extracted selenomethionine

Effects of the two most widespread sample preparation techniques on the d,l-enantiomer ratio of extracted selenomethionine were monitored through the analysis of the certified reference material selenium-enriched yeast and the isolated protein fraction of high selenium monkeypot nut. The extracted selenomethionine (SeMet) fractions were orthogonally cleaned up with anion exchange chromatography before carrying out the enantiomer-specific detection to increase the robustness and the efficiency of the subsequent o-phthal-aldehyde and n-isobutyril-cysteine-based derivatisation process and reversed phase-high-performance liquid chromatography-inductively coupled plasma mass spectroscopy (ICP-MS) detection. The two techniques, namely methanesulphonic acid (MSA) based digestion and proteolytic digestion with protease XIV, resulted in significantly different ratio of d,l-selenomethionine with the final results of 2.2–2.7% and 0.5–0.6% of d-SeMet, respectively. The study revealed significant differences in the ICP-MS-related sensitivity of the derivatised selenomethionine enantiomers, which calls attention to the quantification of this selenoamino acid after MSA hydrolysis.     

Updated estimates of the selenomethionine content of NIST wheat reference materials by GC–IDMS

Updated estimates of the selenomethionine content of four NIST wheat reference materials have been obtained by use of a revised gas chromatography–stable-isotope dilution mass spectrometric method. The revised method makes use of digestion with methanesulfonic acid, which enables more complete recovery of endogenous selenomethionine than was previously achieved by overnight denaturing treatment in 0.1 mol L⁻¹ HCl. The NIST wheat reference materials each contain approximately 55% of their total Se content as selenomethionine. Information about forms of Se in reference materials adds value to these materials in Se speciation studies. Estimates of selenomethionine content are also provided for other wheat samples, including several grown under conditions of exposure to high Se levels. These samples also contain approximately 55% of their total Se content as selenomethionine. The consistent level of 55% of total selenium occurring in the form of selenomethionine when the total selenium content varies by a factor of 500 is suggestive of an active mechanism of incorporation of selenium into wheat grain. Figure Selenomethionine content of wheat samples     

Determination of selenomethionine, selenocysteine, and inorganic selenium in eggs by HPLC–inductively coupled plasma mass spectrometry

A method for the simultaneous determination of selenomethionine (SeMet), selenocysteine (SeCys), and selenite [Se(IV)] in chicken eggs was developed. A sample preparation protocol including defatting, protein denaturation, and carbamidomethylation was optimized in order to achieve complete protein digestion and to avoid SeCys losses. Quantification was carried out by reversed-phase HPLC–inductively coupled plasma mass spectrometry (ICP MS) after quantitative isolation of the selenium-containing fraction by size-exclusion liquid chromatography. The detection limits were 0.06, 0.003, and 0.01 μg g⁻¹ (dry weight) for SeCys, Se(IV) and SeMet, respectively, and the precision was 5–10%. The end products of carbamidomethylation of the different selenium species were identified for the first time by electrospray QTOF MS after custom-designed 2D HPLC purification. Differences in selenium speciation in egg yolk and white were highlighted, the yolk containing more SeCys and the white more SeMet. An insight into selenium bioaccessibility in eggs was obtained by digestion with simulated gastric and gastrointestinal juices and size-exclusion HPLC-ICP MS.