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.     

Selenomethionine content of candidate reference materials.

Selenium has been identified as an antioxidant of importance in the diet. Accurate determination of its chemical forms depends on the availability of suitable reference materials (RMs). Two candidate reference materials for determination of selenomethionine (Semet) in food-related materials, a standard wheat gluten sample (NIST RM 8418 Wheat Gluten) and a commercial selenium enriched yeast, have been examined by use of a gas chromatography-isotope dilution mass spectrometry (IDMS) procedure, after treatment of the matrix with 0.1 mol L(-1) hydrochloric acid containing stannous chloride, addition of CNBr, and extraction with chloroform. This procedure results in cleavage of the CH3Se group to form volatile CH3SeCN. Addition of isotopically enriched 74Semet to an analytical sample enables estimation of the naturally occurring protein-bound 80Semet by IDMS without a protein-digestion process. We found that the Wheat Gluten RM contains a significant amount of Semet as a portion of its assigned value of 2.58 microg Se(total g(-1). Commercial selenium yeast tablets are labeled as containing an elevated level of "organic selenium", usually as Semet. The sample we investigated contained 210 microg Se(total) g(-1) sample as determined separately by IDMS, measuring elemental selenium after digestion. 73% of this total (153 +/- 21 microg Se(semet) g(-1); n = 23) was present as Semet. Thus, these two materials contain significant amounts of their total selenium content as Semet and would be good candidates for further study and characterization as reference materials for determining this important food component. The CNBr reaction used will also enable the determination of Se-(methyl)selenocysteine, the biological role of which is of recent interest. In addition to matrix RMs for Semet, it is important to have standard materials of the pure substance. We have examined a sample of a candidate standard material of selenomethionine being prepared by the USP. It was confirmed that this material is pure selenomethionine.     

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).     

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).     

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%).     

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.     

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.     

Determination of selenium status using the nail biologic monitor in a canine model

Toenails and fingernails are routinely used to estimate selenium status in epidemiological studies; however, literature validating nail selenium concentration as a surrogate for critical organs is limited. In this study diets of intact male dogs were selenium supplemented at two physiological levels (3 and 6 μg/kg/day) in two different forms, selenomethionine and selenium-enriched bioformed yeast. The selenium-adequate basal diet consumed by the treatment and control groups during the 4-week run-in period and throughout the trial contained 0.3 ppm selenium. After 7 months the dogs in the two treatment groups and the control group were euthanized. Representative tissue samples from prostate, brain, liver, heart and skeletal muscle were collected, rinsed and frozen. Toenail clippings from multiple toes were also collected. Selenium was determined by neutron activation analysis using Se77m (half life = 17.4 s) at the University of Missouri Research Reactor Center. NIST SRM 1577, Bovine Liver was analyzed as a quality control. The analysts were blinded to control and treatment group assignments. As expected, tissue selenium levels increased proportionally with supplementation. A slightly greater increase in tissue selenium was observed for the purified selenomethionine compared to the bioformed yeast; however this trend was significant only for brain tissue. Toenail selenium concentrations and tissue selenium were highly correlated (p < 0.003) with Pearson coefficients of 0.759 (skeletal muscle), 0.745 (heart), 0.729 (brain), 0.723 (prostate), and 0.632 (liver). The toenail biologic monitor accurately assesses selenium status in skeletal muscle, heart, brain, prostate, and liver in the canine model.     

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.     

Selenomethionine content of candidate reference materials

Selenium has been identified as an antioxidant of importance in the diet. Accurate determination of its chemical forms depends on the availability of suitable reference materials (RMs). Two candidate reference materials for determination of selenomethionine (Semet) in food-related materials, a standard wheat gluten sample (NIST RM 8418 Wheat Gluten) and a commercial selenium enriched yeast, have been examined by use of a gas chromatography–isotope dilution mass spectrometry (IDMS) procedure, after treatment of the matrix with 0.1 mol L^–1 hydrochloric acid containing stannous chloride, addition of CNBr, and extraction with chloroform. This procedure results in cleavage of the CH₃Se group to form volatile CH₃SeCN. Addition of isotopically enriched ⁷⁴Semet to an analytical sample enables estimation of the naturally occurring protein-bound ⁸⁰Semet by IDMS without a protein-digestion process. We found that the Wheat Gluten RM contains a significant amount of Semet as a portion of its assigned value of 2.58 μg Se_total g^–1. Commercial selenium yeast tablets are labeled as containing an elevated level of “organic selenium”, usually as Semet. The sample we investigated contained 210 μg Se_total g^–1 sample as determined separately by IDMS, measuring elemental selenium after digestion. 73% of this total (153±21 μg Se_Semet g^–1; n = 23) was present as Semet. Thus, these two materials contain significant amounts of their total selenium content as Semet and would be good candidates for further study and characterization as reference materials for determining this important food component. The CNBr reaction used will also enable the determination of Se-(methyl)selenocysteine, the biological role of which is of recent interest. In addition to matrix RMs for Semet, it is important to have standard materials of the pure substance. We have examined a sample of a candidate standard material of selenomethionine being prepared by the USP. It was confirmed that this material is pure selenomethionine. Received: 13 December 2000 / Revised: 5 March 2001 / Accepted: 12 March 2001     

Selenium speciation in animal tissues after enzymatic digestion by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry

A procedure is described for the enzymatic digestion of tuna and mussel samples that allows the determination of selenium species by high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry. The species were extracted by two-step enzymatic hydrolysis with a non-specific protease (subtilisin). The selenium species were separated on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions, namely phosphate buffers at pH 2.8 and pH 6.0 as mobile phases. The method determines organic (trimethylselenonium, selenocystine, selenomethionine and selenoethionine) and inorganic selenium species (selenite and selenate), but only organic selenium species were found in the samples. The sum of identified selenium species in the sample was about 30% of the total selenium present in the enzymatic extract despite the fact that recoveries of total hydrolysed selenium were 93-102%. Trimethylselenonium ion and selenomethionine were found in both tuna and mussel samples and an unknown selenium species was also found in tuna samples.     

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.     

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.     

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。     

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。     

The determination of selenomethionine in selenium yeast by cyanogen bromide gas chromatography

A new method for the determination of bonded selenomethionine in selenium yeast by gas chromatography is established. Bonded selenomethionine is rapidly and precisely determined by measuring CH3SeCN released during the reaction of selenoprotein with CNBr (CNBr-GC method). Conditions for the reaction and chromatography are described. The results of CNBr-GC are compared with those obtained by acid hydrolysis/ion exchange chromatography. This new method has the advantage of being accurate, sensitive and selective.     

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     

Selenium species determination in selenium-enriched pumpkin (Cucurbita pepo L.) seeds by HPLC-UV-HG-AFS.

Pumpkins were treated by spraying the leaves in the flowering period with a water solution containing 1.5 mg Se per liter in the form of Na2SeO4. The average total selenium content of seeds was found to be 0.19 microg g(-1) in nontreated pumpkins and 1.1 microg g(-1) in exposed ones. For speciation analysis, enzymatic hydrolysis with different amounts of Protease XIV was carried out. Under optimal conditions of enzymatic hydrolysis, 90% of the total selenium was found in soluble forms. Separation of species was performed using HPLC on anion and cation exchange columns and for detection UVHG-AFS was applied. In enzymatic hydrolysis extracts, the main fraction of selenium was bound as selenomethionine (SeMet), representing on average of 81 +/- 8% of the total Se content in the sample.     

Determination of Selenium Species in Cordyceps militaris by High-performance Liquid Chromatography Coupled to Hydride Generation Atomic Fluorescence Spectrometry

The purpose of this work is to develop a high-efficiency extraction method for determining the selenium species in Cordyceps militaris. Six extraction solutions, including hot water, HCl, methanol–water, ammonium acetate, protease XIV, and protease K, combined with ultrasound-assisted extraction, were utilized in the measurements. The selenium species in the extracts were separated and characterized by high-performance liquid chromatography. Their concentrations were subsequently determined by hydride generation atomic fluorescence spectrometry. The 25?mM ammonium acetate was selected as the extraction solution due to its advantages in cost and efficiency. Validation was performed, and the selenium species recoveries were 69–97% for selenocystine, selenite, selenomethionine, and selenate with good linearity and precision. The major selenium species in C. militaris were selenocystine and selenomethionine that accounted for almost 73.1?±?1.6% of the total selenium.     

液相色谱-双通道原子荧光检测联用法同时测定砷和硒的形态

建立了一种利用高效液相色谱-双通道原子荧光检测联用同时进行砷和硒形态分析的方法。以10 mmol/L NH4H2PO4溶液(pH 5.6)(添加2.5%(体积分数)的甲醇)为流动相,在12 min内同时分离了三价砷(As(III))、一甲基砷(MMA)、二甲基砷(DMA)、五价砷(As(V))、硒代胱氨酸(SeCys)、硒代蛋氨酸(SeMet)和四价硒［Se(IV)］等化合物。As(III)、DMA、MMA、As(V)、SeCys、SeMet和Se(IV)的检出限分别为1,3,2,3,4,18和3 μg/L (进样量为200 μL),5次测定的相对标准偏差为1.9%～6.1%(As 100 μg/L, Se 300 μg/L)。应用该方法对人体尿样及硒酵母片中砷和硒的形态进行了分析,目标物在尿样中的加标回收率为83%～108%,在硒酵母片中的加标回收率为88%～105%。实验结果表明,该方法可用于尿样及药品中砷和硒形态的日常分析。该方法减少了样品的分析时间和试剂用量,降低了工作强度,提高了工作效率。