Selenium speciation in enriched vegetables

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Review

Selenium speciation in enriched vegetables

Krystyna Pyrzynska *

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

a r t i c l e i n f o

Article history:Received 9 June 2008Received in revised form 5 September 2008Accepted 8 November 2008

Keywords:Selenium speciesEnriched vegetablesExtractionHPLC

a b s t r a c t

The ability of several plants to accumulate and transform inorganic forms of selenium into bioactiveorganic compounds has important implications for human nutrition and health. Se-enriched Allium groupvegetables such as garlic, onion and ramps have been mainly the subject of several studies in the recentyears. Apart from the total Se uptake, enrichment treatments normally undergo certain metabolicchanges that determine the final product as well as its translocation and accumulation in different planttissues. For this reason, it is important to find which form of selenium should be used for supplementa-tion to obtain a high content of this element in the final plant. Moreover, its distribution in different partsof plants as well as characterisation and quantification of individual species becomes an issue. Thisreview gives a brief, critical overview of the studies carried out to characterise selenium species producedby different enriched vegetables. The use of different extraction and clean-up methodologies will be dis-cussed in conjunction with different selenium enrichment procedures.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11832. Procedures for selenium speciation analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184

2.1. Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11842.2. Clean-up procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186

3. Uptake of selenium by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11863.1. Total selenium uptake. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11863.2. Transformation and distribution of selenium species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188

4. Bioavailability of selenium from enriched vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11885. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189

1. Introduction

Selenium is an important element from environmental and bio-logical point of view being essential in a very narrow concentrationrange, while outside this range deficiency or toxicity occurs (Ellis &Salt, 2003; Sager, 2006). It has been recognised as an integral com-ponent of different enzymes such as gluthathione peroxidase andthioredoxin reductase, which participate in the antioxidant protec-tion of cells (Birringer, Pilawa, & Flohc, 2002). Moreover, several

studies have suggested that some organic forms of selenium couldshow anticarcinogenic properties against certain types of cancer(Clark et al., 1996; Rayman, 2005; Shen et al., 2006). Diet with lessthan 0.1 lg Se g�1 results in deficiency, while regular consumptionof food containing more than 1 lg Se g�1 results in toxicity(Whanger, 2002). On the basis of this estimate, daily selenium in-takes for humans that vary according to the country or region, ageand sex have been recommended and recently compiled by Ray-man (2004).

Primary selenium compounds of interest in plants include inor-ganic species (selenite Se(IV) and selenate Se(VI)), simple organicspecies (methylselenol, dimethylselenide, diethylselenide and

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dimethyloselenooxide), amino acids and low molecular speciessuch as selenomethionine (SeMet), selenocysteine (SeCys), seleno-cystine (SeCys2), selenohomocysteine (SeHoCys2), Se-methylsele-nocysteine (MeSeCys), Se-methylselenomethionine (MeSeMet),Se-allylselenocysteine (AllSeCys), Se-propylselenocysteine (PrSe-Cys), c-glutamyl-Se-methylselenocysteine c-Glu-MeSeCys), Se-adenosylselenohomocysteine, selenoglutatione and other com-pounds such as selenoproteins or selenoenzymes.

In the world, deficiency of selenium in diet is more commonthan its abundance and nutritional supplements have been recom-mended to increase daily Se intake. There is a growing market onthe development of selenium-enriched nutritional supplementsand selenised yeast (generally, Saccharomyces cerevisiae) has beenwidely investigated (Chassaigne, Chery, Bordin, & Rodriguez,2002; Mounicou, McSheeby, Szpunar, Potin-Gautier, & Lobinski,2002; Uden et al., 2004; Połatajko et al., 2004). The majority ofselenium present in selenised yeast samples (about 70%) is in theform of selenomethionine. Some studies have revealed that thiscompound tends to accumulate in tissues, and this represents itsmain limitation for cancer inhibition (Ip, Thompson, Zhu, &Gauther, 2000b; Ip et al., 2000a). Recent studies have shown thatthe amount of selenium in over-the-counter supplements can belower than advertised and large discrepancies are encounteredbetween declared selenium species and those found by analysis(Gosetti et al., 2007; Stibij, Smarkolj, & Krbavcic, 2005).

The ability of several plants to accumulate and transform inor-ganic forms of selenium into bioactive organic compounds hasimportant implications for human nutrition and health (Ellis & Salt,2003; Zayed, Lytle, & Terry, 1998). Some plants when grown inselenium-rich soils may accumulate large amounts of selenium(up to thousands of mg/kg dry mass) without showing symptomsto toxicity and they could be applied for phytoremediation of pol-luted soils (Terry, Zayed, de Souza, & Tarun, 2000). A member ofthe Brassicaceae family, Indian mustard (Brassica juncea) is one ofthe most studied plants in terms of Se accumulation because ofits fast growing cycle and high biomass (Kahakachchi, Boakye,Uden, & Tyson, 2004; Montes-Bayón et al., 2002a; Mounicouet al., 2006). The resistance to excessive Se has been related tothe formation of organo-selenium compounds that cannot beincorporated into proteins and also the ability of these plants toconvert selenium into volatile species (Terry et al., 2000).

At location with low selenium content in soils, the most effec-tive way to increase the amount of selenium in cultivated cropsis to use Se-enriched fertilisers, spray the crops with selenium saltsor treat the seeds with an aqueous selenium (Carvalho, Gallardo-Williams, Benson, & Martin, 2003; Gupta & Gupta, 2002; Slekovec& Goessler, 2005). Some problems are discussed with the seleniumfertilisation practice such as uncontrolled over-uptake, whichmight result in possible environmental problems (Makela et al.,1995). However, growing plants enriched with selenium could bean effective way to reduce dietary deficiencies and increase healthbenefits. Thus, it is important to find plants capable of toleratingand transforming selenium into bioactive compounds. Alliumgroup vegetables such as garlic, onion, broccoli and ramps havebeen mainly the subject of several studies. Major Se-species re-ported in these selenised plants are Se-methylselenocysteine andderivatives such as c-glutamyl-Se-methylselenocysteine, whichare known to be more effective inhibitors of tumor formation.

Some chemical forms of selenium are more readily available toplants than others. Apart from the total Se uptake, enrichmenttreatments normally undergo certain metabolic changes thatdetermine the final product as well as its translocation and accu-mulation in different plant tissues. For this reason, it is importantto find which form of selenium should be used for supplementa-tion to obtain high content of this element in the final plant. More-over, its distribution in different parts of plants as well as

characterisation and quantification of individual species becomesan issue.

The present work summarises the studies carried out to charac-terise selenium species produced by different enriched vegetables.The use of different extraction and clean-up methodologies will bediscussed in conjunction with different selenium enrichment pro-cedures. A brief description concerning total Se incorporation aswell as transportation and distribution of its species is also pre-sented herein.

2. Procedures for selenium speciation analysis

The increasing role of selenium in food and fodder supplemen-tation has resulted in a need for reliable and validated analyticalprocedures for identification and quantification of different sele-nium species. The comprehensive reviews concerning the analyti-cal methods applied for selenium speciation have been recentlypublished (B‘Hymer & Caruso, 2006; Dumont, Vanhaecke, & Corn-elis, 2006; Infante, Hearn, & Catterick, 2005; Montes-Bayón, Grant,Meija, & Caruso, 2002b; Połatajko, Jakubowski, & Szpunar, 2006).

The most frequently used technique for selenium speciationanalysis is today chromatographic or electrophoretic separationcoupled to sensitive inductively coupled plasma mass spectrome-try (ICP-MS) detection (Lobinski, Edmonds, Suzuki, & Uden, 2000;Pyrzynska, 2001). The addition of methane gas to the ICP provedto be an efficient way of loading the plasma with carbon, resultingin an improved detection limit down to 0.35 and 0.49 ng/kg for sel-enite and selenomethionine, respectively (Wartburton & Geonage-Infante, 2007). Liquid chromatography (LC) operating in differentmodes (size-exclusion, ion-exchange and reversed-phase or re-versed-phase ion-pair) has the advantage of performing separa-tions of nonvolatile selenium species and has generally betterversatility than gas chromatography, which may require a derivat-isation step before analysis. Ultra performance LC allows separa-tions on column materials at high pressure up to 108 Pa usingparticle diameters of 1.7 lm, which increases the efficiency, theresolution and the speed of the separation (Bendahl, Stürup, Gam-melgaard, & Hansen, 2005).

The most common approach for the determination of volatileselenium species involves a preconcentration step by cryogenictrapping, followed either by thermal desorption or by extractionusing an organic solvent, prior to separation by gas chromatogra-phy (Pecheyran, Quentel, Lacuyer, & Donard, 1998; Yang, Mester,& Sturgeon, 2004). Recently, solid-phase microextraction was pro-posed as an attractive method for sample preparation compatiblewith gas chromatography and ICP-MS (Vonderheide, Montes-Ba-yon, & Caruso, 2002) or MIP-OES (Dietz, Landaluze, Ximcnez-Embún, Madrid-Albarrán, & Cámara, 2004) detection.

The identification of the separated species is based mainly onthe comparison of retention times to those of chemical standardsand/or by standard addition method. However, the commercialstandards are not available for all selenocompounds. This problemcan be solved by the synthesis of species expected to be found inthe analysed samples or by isolation of the purified compoundsfrom the sample for their further identification by electrospray ormatrix-assisted laser desorption ionisation mass spectrometry(Encinar et al., 2003; Połatajko et al., 2006).

2.1. Extraction

Sample pretreatment is necessary to convert the original sam-ple to a form that can be analysed. The extraction efficiency mustbe considered in parallel to minimising changes in species nature,thus special care is needed to prevent possible losses and/or spe-cies inconversion. The speciation results are affected by the choice

1184 K. Pyrzynska / Food Chemistry 114 (2009) 1183–1191


of pretreatment procedure. Common techniques include differentleaching and extraction modes, enzymatic hydrolysis and volatili-sation with or without species preconcentration (Gómez-Ariza,Morales, Sanchez-Rodas, & Velasco, 2001; Wrobel, Wrobel, &Caruso, 2005). Enzymatic hydrolysis, when assisted by ultra-sounds, has provided a dramatic reduction of the extraction timewithout negligible species degradation (Capelo, Ximenez-Embun,Madrid-Albarran, & Camara, 2004; Montes-Bayón, Diaz-Molet,Gonzalez, & Sanz-Medel, 2006).

Usually, the freeze-dried or dried and homogenised plant mate-rial has been analyzed. For extraction of high molecular weightfraction (apparent MW >10 kDa) in vegetables sodium hydroxidesolution is used (Shah, Kannamkumarath, Wuilloud, Wuilloud, &Caruso, 2004; Wrobel et al., 2004). Typical pretreatments for lowmolecular weight compounds involved hot-water extraction (Au-ger, Yang, Arnault, Pannier, & Potin-Gautier, 2004; Dumont et al.,2006; Ge et al., 1996; Kotrebai, Birringer, Tyson, Block, & Uden,1999; Kotrebai, Birringer, Tyson, Block, & Uden, 2000; Mazej, Fal-noga, Veber, & Stibilj, 2006; McSheeby et al., 2000; Ogra, Ishiwata,Iwashita, & Suzuki, 2005; Ogra et al., 2007; Pedrero, Madrid, &Camára, 2006; Whanger, Ip, Polan, Uden, & Welbaum, 2000) orenzymatic digestion with protease, proteinase or driselase at roomtemperature (Cankur, Yathavakilla, & Caruso, 2006; Ferri, Favero, &Frasconi, 2007; Ge et al., 1996; Ip et al., 2000a; Kotrebai et al.,1999; Kotrebai et al., 2000; Kápolina, Shah, Caruso, & Fedor,2007; Larsen et al., 2006; Mazej et al., 2006; Montes-Bayón et al.,2006; Pedrero et al., 2006; Shah et al., 2004; Smrekolj, Stibilj, Kreft,& Kápolna, 2005; Whanger et al., 2000). The use of proteolytic en-zymes ensured the liberation of Se species contained in peptides orproteins. Extraction using an aqueous solution of enzyme-deacti-vating hydroxylamine hydrochloride counteracted the possibledegradation of selenium labile species by enzymes (such as alliin-ase) that occur naturally in garlic (Larsen et al., 2006). Methanol(Ge et al., 1996; Slekovec & Goessler, 2005) or ethanol (Wrobelet al., 2004) in the presence of acid or HCl solution alone (Kotrebaiet al., 1999; Montes-Bayón et al., 2006; Shah et al., 2004) as well asa water–chloroform–methanol mixture (Kápolina et al., 2007;Wrobel et al., 2004) has been also used for the extraction of sele-nium compounds from bulk plant material.

Extraction efficiency depends upon the nature of the sampleand the extraction conditions. The reported solubilisation effi-ciency of selenium species usually approaches 100% for enzymatictreatment and was lower (75–90%) for hot-water extraction fromAllium vegetables. Besides being more efficient, the enzymatictreatments liberated more SeMet (Larsen et al., 2006). However,for some plant materials much lower efficiency of water extractionwas achieved; 68.5% of total Se in the selenised Japanese pungentradish (Ogra et al., 2007), 67% in chicory leaves (Mazej et al., 2006)and 55% in pumpkin (Smrekolj et al., 2005) were found in water-soluble forms. Higher recovery of selenium than in water was ob-tained with Tris–HCl buffer, but problems arise when extracts wereconcentrated before chromatographic separation of Se species(Mazej et al., 2006). With the perchloric acid–ethanol mixture(8:2) only �30% of total selenium content could be extracted fromchive samples, while the proteolytic enzymes increased the recov-ery up to 70%.

The findings of Roberge, Borgerding, and Finley (2003) suggestthat the results of selenium speciation in plants are strongly de-pended on the extraction conditions employed and support a cur-rent debate on the extent to which chemical preparation influencesthe distribution of selenium over its different inorganic and organicforms. Nine different buffering systems in the pH range of 1–9were studied for the extraction of selenium compounds from en-riched broccoli (Roberge et al., 2003). In general, buffered extrac-tions removed more selenium from the plant in comparison withnonbuffered water medium (Fig. 1A). However, they did not affect

the amount that was extracted into the final aqueous phase(Fig. 1B). Mass balance presented in Fig. 1C indicated that the useof nonbuffered aqueous phase resulted in a more quantitativeextraction, whereas ascorbate, phosphate or glycine/HCl buffersaccounted for less than 70% of the total Se. The remaining seleniumwas either adsorbed on the walls of the extraction vessel and thestir bar or volatilised and lost to the atmosphere. Additionally,spiked selenium standards were lost to the different extents fromthe extracting solutions; when MeSeCys was spiked into a gly-cine/HCl buffer at pH 3.0 only about 60% of Se was recovered, whilein the same buffer 98% of Se from a selenocystine spike was deter-mined. The investigations whether spiked selenium compoundsmay be lost by sorption to matrix solids reached the conclusionsthat each buffer system should be assessed individually becauseselenium species and used buffers each has their own interactionswith the matrix. In the case of broccoli, a pH 7 phosphate bufferwas recommended.

Besides the maximum efficiency, the stability of seleniumspecies during extraction process should be taken into accountwhen choosing extraction conditions. Reported conversion of

Fig. 1. Extraction efficiency of selenium from broccoli. (A) percentage of Seremaining in the solid after the extraction; (B) percentage of Se found in theextracting solution; and (C) total Se accounted for the postextraction liquid and thesolid. Ascorbate and glycine/HCl buffers were initially set at pH 3.0, phosphatebuffer was set at pH 7.0 (Roberge, Borgerding & Finley, 2003). (Copyright AmericanChemical Society. Reproduced with permission).

K. Pyrzynska / Food Chemistry 114 (2009) 1183–1191 1185


selenoamino acids includes oxidation and degradation by naturallyoccurring enzymes. Mazej et al. (2006) observed several unknownoxidation products in a test experiment when SeMet was treatedwith a 2% solution of H2O2 overnight at room temperature. Afterheating with H2O2 at 80 0 C for 50 min, Se(IV) was the main prod-uct. Under both conditions the original SeMet was not detected.Similar observations were reported by Uden et al. (2004) andGammelgaard, Cornett, Olsen, Bendahl, and Hansen (2003). An-other important factor is the stability of selenium species in asupernatant after enzyme hydrolysis. Solutions of SeMet were sta-ble for at least 2 weeks at �20 �C, while in the presence of proteaseXIV (incubation for 24 h at 37 �C and storage at �20 �C) two addi-tional products besides SeMet were observed (Pedrero et al., 2006).These results differ from those of Smrekolj et al. (2005) which re-ported that the supernatants of enzymatic extract stored at �18 �Cin plastic tubes for 5 months showed no significant difference ofselenium content in the form of selenomethionine. From the otherhand, Dumont et al. (2004) suggested that the transformation ofSeMet is not the consequence of the action of protease but ismainly caused by the yeast matrix.

The most effective way to avoid these problems is to performthe measurements in vivo, it means on living plants without theneed for any form of sample pretreatment and even the necessityfor cutting plant tissues into pieces. X-ray absorption near-edgestructure spectroscopy, one of the few techniques which allow per-forming nondestructive speciation analysis of solid material, hasbeen applied for the direct selenium speciation at selected positionwithin the roots and leaves of Se-exposed onions (Bulska et al.,2006).

2.2. Clean-up procedures

The common use of ICP-MS as a selective selenium detector al-lows the screening of Se species after chromatographic analysis aswell as their relative abundance. However, other constituents of asample might also co-elute with the target analytes modifyingretention times and peak shapes, thus affecting the separationand identification. This is particularly important when the selectedfractions are collected for the identification of selenium species bymolecular mass spectroscopy.

The most common approaches for the pretreatment of biologi-cal samples prior to analysis deal with the use of C18 cartridges(Montes-Bayón et al., 2002b; Roberge et al., 2003) or ion-exchangeresins (Montes-Bayón et al., 2002b; Wrobel et al., 2004). Anion-ex-change resin Dowex 1 � 8 (in the acetate form) was used forremoving of inorganic selenium species from the extract (Wrobelet al., 2004), while ion-exchanger Dowex 50W � 4 for the isolationof selenoamino compounds (Montes-Bayón et al., 2002b). Themain limitation of such treatment is the lability of certain species,as it was observed for Se-methylselenocysteine.

The extract coming from the enzymatic hydrolysis was clean-upby passing through a 3 kDa ultracentrifugation membrane (Mon-tes-Bayón et al., 2006) or through a 5 kDa molecular weight cutofffilters (Kápolina et al., 2007).

3. Uptake of selenium by plants

Selenium is present in soil (naturally or due to anthropogenicactivities) and can enter the food chain through the plants. Someplants have the ability not only to grow in the presence of elevatedlevels of selenium, but also to accumulate its species. One of theaccumulation mechanisms for selenium tolerant plants is the for-mation of its organic compounds that cannot be incorporated intoproteins, thereby avoiding toxicity. Uptake and accumulation ofselenium by plants are determined by its concentration and chem-

ical forms, the presence of competing ions and the affinity of a par-ticular plant to absorb and metabolise selenium (Terry et al., 2000;Zayed et al., 1998). Selenite readily competes with the uptake ofsulfate and it has been proposed that both anions are taken upvia a sulfate transporter in the root plasma membrane (Terryet al., 2000). Se(VI) is reduced to Se(IV), which in turn is reducedto selenide, involving reduced glutathione in the process (Dumontet al., 2006). The selenide is then transformed to selenoaminoacids, such as SeCys and SeMet. Selenomethionine can be furthermetabolised to Se-adenosyl-SeMet, which in turn is converted toSe-methylselenocysteine and c-glutamyl-Se-methylselenocys-teine. Some plants can take up high quantities of Se from soiland then transform it through several biochemical steps into vola-tile species, mainly dimethylselenide, in the phytovolatilisationprocess.

Several authors (Mazej et al., 2006; Shah et al., 2004; Smrekoljet al., 2005; Whanger et al., 2000) have reported that different veg-etables grown in selenium-enriched culture media did not appar-ently exhibit the symptoms of toxicity. However, the growth ofonion roots in a standard medium containing inorganic seleniumcompounds was inhibited with respect to the control plants; themore pronounced effect was observed in the presence of Se(VI)(Wrobel et al., 2004). Also a systematic growth decrease of onionleaves was observed. Carvalho et al. (2003) reported that for the to-mato plants there was a significant difference between the controlplants and those treated with a high portion of Se organic com-pounds with respect to the amount of fruit produced. On the con-trary, Pedrero et al. (2006) observed a reduction of about 25% in thegrowth of radish roots exposed to Se(IV) when compared withplants grown in the presence of Se(VI). Dill plants supplementedwith Se(IV) produced 10–15% less overall yield than control sample(Cankur et al., 2006). Similar results have also been observed withother plants, such as lupine, Indian mustard and sunflower (Ximè-nez-Embún, Alonso, Madrid-Albarrán, & Camára, 2004).

Although Se has not been classified as an essential nutrient forplant, its beneficial effects on metabolism and plant growth havebeen shown recently. Selenite application was reported to increasethe concentration of soluble sugars and caffeine in coffee leaves(Mazzafera, 1998), to enhance the quality and yield of green tea(Hu, Xu, & Pang, 2003), to promote lettuce growth, especially underUV-stress conditions (Xue, Hartikainen, & Piironen, 2001). At lowlevels, selenate improved the processing quality of potato tubersby diminishing and retarding their raw darkening (Turakainen,Hartikainen, Ekholm, & Seppänen, 2006).

3.1. Total selenium uptake

The antioxidant and anticarcinogenic properties attributed tosome selenocompounds justify the increasing interest in growingselenium-enriched vegetables, which represents an importantsource of this element in the human diet. The highest chemopre-ventive activity was observed for vegetables from Allium groupsuch as garlic, onion and ramp (Ip et al., 2000a; Whanger et al.,2000), thus these plants are mostly used to study the uptake, incor-poration and distribution of selenium.

Various increases of selenium content in the growth mixture aswell as time of exposition to selenium generally resulted in propor-tional increases of the Se content in vegetables (Mazej et al., 2006;Pedrero, Elvira, Camára, & Madrid, 2007; Shah et al., 2004; Whang-er et al., 2000). Larsen et al. (2006) reported that the addition ofsymbiotic fungi (mycorrhiza) to the natural soil increased the sele-nium uptake by garlic 10-fold to 15 lg/g (dry mass). Fertilisationwith Se(VI) increased the Se content strongly to several hundredsof lg/g and the addition of mycorrhiza in combination with sele-nite fertilisation had an additional but less pronounced positiveeffect.



The transportation of selenium from root to shoot after uptakeis highly dependent on its form present in the growth medium. Astudy by Zayed et al. (1998) demonstrated that Se(VI) is muchmore easily transported than Se(IV) or any organic species suchas selenomethionine. It was explained that selenite is poorly trans-located to shoots and is rapidly converted to organic forms such asSeMet, which is retained in the roots, while selenate uses the sul-fate path through the plants. Indeed, higher selenium levels werefound in onion leaves (601 lg/g) as compared to bulbs (51.3 lg/g) in the samples enriched with selenate (Wrobel et al., 2004).Onion exposed to Se(IV) had lower selenium content in its leavesand bulbs, 154 lg/g and 15.6 lg/g, respectively. Similar observa-tions were reported for broccoli cultivated in the presence ofSe(IV); Se content in roots and florets was very similar while inshoots the concentration was about 90% inferior (Pedrero et al.,2007).

Detailed in vivo investigations of the distribution of variousselenium species in virtual cross sections of onion root tips andgreen leaf showed that selenium transport takes place via different

mechanisms (Bulska et al., 2006). For onions exposed to Se(IV),selenium infiltrate the cytoplasm through the cell membrane(symplastic transport) where it is reduced to Se(�II), most proba-bly to MeSeCys. This transformation could be considered as adefensive inactivation mechanism of the cell against a toxic chem-ical species. Selenium taken up by onion roots as Se(VI) is mostlytransported by an apoplastic mechanism. This involves the inerttransport via the microcapillaries in the root peripheral tissues to-ward the other parts without the infiltration of selenium com-pounds inside the cell and without being transformed into otherchemical forms. Because it is still transported to the onion leaves,a part of Se(VI) undergoes reduction and metabolised into nontoxicamino acid forms.

Some plants such as radish grown in the presence of Se(IV) andSe(VI) accumulated similar quantities of total selenium, showing acertain independence of the chemical form present in the culturemedia (Pedrero et al., 2006). However, the selenium speciation re-sults presented in Table 1 show that the distribution of this ele-ment is remarkably different depending on the selenium media.

Table 1Distribution of selenium species in Se-enriched vegetables.

Plant Se addition Total Sea

(lg/g)Selenium species (%)b Refs.

Se(IV) Se(VI) SeMet SeCys2 MeSeCys c-Glu-MeSeCys

Garlic (Allium sativum) Na2SeO4 + myccorhiza (50 mg/kg, 4weeks)

969 Enzymatic/water extraction Larsen et al.(2006)– –/9c 2/1c – 3/5c 64/62c

296 Enzymatic/water extraction Ip et al. (2000a)– 2c 13c 0.5c 3c 73c/85c

BaSeO3 + BaSeO4 (500 mg/m3 of each,8 months)

96 Water extraction Dumont et al.(2006)– – 15.5d 6.0d 28.8d 49.7d

Onion (Allium cepa) HClO4–ethanol extraction Wrobel et al.(2004)Na2SeO3 154 – – 0.3 0.5e 4.0 –

Na2SeO4 (15 mg/kg, 8 days) 601 – – 0.2 0.1e 1.9 –Green onion (Allium

fistulosum)Na2SeO3 (15 mg/kg, 4 months) 30.3 Enzymatic extraction/HCl hydrolysis Shah et al.

(2004)+/� �/� +/� +/� �/+ �/+Ramp (Allium tricoccum) Na2SeO4 (30 mg/l) 252 – 42 – – 35 1.4 Whanger et al.

(2000)Shallot (Allium

ascalonicum)BaSeO3 + BaSeO4 (500 mg/m3 of each,8 months)

226.8 Water extraction Ogra et al.(2005)– 28 – – 5.4 66

Broccoli (Brassicaoleracea)

Na2SeO3 (1 mg/l, 40 days) 27f Enzymatic extraction Pedrero et al.(2007)7 – 11.7 – 45.6 –

Enzymatic extraction Kotrebai et al.(2000)Garlic 68 – 1 18 0.5 2.5 68

235 – 1.5 17 0.5 3 701355 – 4 13 – 60 8

Ramp 48 – 1 21 – 34 3524 – 22 5 – 44 1.5

Onion 96 – 10 5 1 1 63140 – 33 10 – 5 35

Chives (Alliumschoenpprasum)

HClO4–ethanol extraction/enzymatic extraction Kápolina et al.(2007)Na2SeO3 222 �/3 21/5 �/5 40/42 28/36 –

Na2SeO4 613 �/� 81/51 �/� 5/2 3/20 –SeMet (10 mg/l, 14 days) 265 �/1 5/� �/3 35/37 46/48 –

Chicory (Cichoriumintybus)

Na2SeO4 (7 mg/l, 41 days) 480 Enzymatic/water extraction Mazej et al.(2006)0.1/0.05 63/63 8.1/0 <LOD 0.7/

traces–

Radish (Raphanussatinus)

Enzymatic extraction Ogra et al.(2007)Na2SeO3 112 – 0.9 16.1 5.3 74.1 –

Na2SeO4 (5 mg/kg, 40 days) 120 – 56.7 16.7 15.8 5.8 –Dill (Anethum

gravealnes)Na2SeO3 (10 mg/l, 14 days) 10.3 � � + <3 13 – Cankur et al.

(2006)Pumpkin (Cucurbia

pepo)Enzymaticextraction

Smrekolj et al.(2005)

Na2SeO4 1.1 – – 85 – – –

+ detected but not quantifieda Dry weight.b Relative to total Se in a sample.c Based on total chromatographed selenium.d Relative to total Se in the extract.e Se-cysteine.f Fresh weight.



Most of the selenium supplemented by foliar aspiration over theplants was accumulated in the upper vegetative parts, with thehighest Se concentration in onion and radish leaves, followed bygarlic leaves and cabbage (Slekovec & Goessler, 2005).

3.2. Transformation and distribution of selenium species

As the highest chemopreventive activity was observed for Se-alk(en)ylselenocysteines and their c-glutamyl derivatives (Blocket al., 2001), the efficiency of Se-enrichment procedure, its distri-bution in different parts of plants as well as characterisation andquantification of individual species becomes an issue. Estimationof diets for selenium adequacy requires information not only onthe total Se content but also on the amount of its species accessiblein a sample. The efforts made for characterisation of selenium spe-cies can be divided in the analysis of the macromolecules (peptidesand proteins) and that of small molecules (amino acids, selenateand selenate).

Size-exclusion chromatography coupled to ICP-MS detectionhas been used to study the distribution of selenium in covalentlybound high molecular weight biomolecules (Kápolina et al.,2007; Shah et al., 2004; Wrobel et al., 2004). It was found thatthe incorporation of selenium to the HMW fraction was more pro-nounced in the onion leaves relative to the bulbs (Wrobel et al.,2004). A higher contribution of HMW compounds was observedin the samples with Se(IV) enrichment (33% in leaves, 26% in bulbs)than with Se(VI) (3% and 5%, respectively). In a similar way, supple-mentation of chive plant with Se(VI) leads to more pronouncedincorporation in LMW selenium species when compared to Se(IV)and SeMet (Kápolina et al., 2007).

Leaching of Se compounds from the proteins is mainly done byusing enzymes and to a lesser extent by means of acid digestion. Itwas concluded that Se-enriched garlic does not accumulate thiselement in the form of selenoproteins since enzymatic treatmentdid not provide extraction yields significantly higher than thoseobserved with 0.1 M HCl or ammonium acetate at pH 5.6 and theproteolytic sample pretreatment did not cleave the c-peptide bondof c-Glu-MeSeCys to form MeSeCys (Capelo et al., 2004). Addition-ally, the results showed that neither enrichment with Se(VI) northe addition of mycorrhiza to the soil affected the relative abun-dance of these two selenium species (Larsen et al., 2006).

Se-methylselenocysteine was shown to be the major Se aminoacid in Allium vegetables grown on Se-enriched soils or culturedhydroponically. Also the minor amounts of SeCys2 and SeMet werefound. They were identified based on retention time matching withcommercially available or in-house synthesised selenium stan-dards. The presence of c-Glu-MeSeCys in plant extracts was con-firmed by ESI-MS–MS based on the isotopic pattern of Se and theproduct ions (Ip et al., 2000a; Larsen et al., 2006; Ogra et al.,2005; Shah et al., 2004). Analysis of natural samples without sele-nium treatment showed the presence of the same compounds ob-served as the principal species of Se-enriched vegetables (Kotrebaiet al., 2000).

Total selenium content affects distribution of this element inthe samples of the same type (Table 1). The absolute content of Se-Met and c-Glu-MeSeCys increased as the total selenium contentincreased in garlic samples (Kotrebai et al., 2000). However, the in-crease of c-Glu-MeSeCys was observed only to the point when to-tal Se was about 300 lg/g. It was calculated that at concentrationsbelow 333 lg/g selenium is incorporated mainly into c-Glu-MeSe-Cys and SeMet but above this value MeSeCys is the major productof the selenium conversion processes. The authors assumed that c-Glu-MeSeCys serves as a carrier of MeSeCys, which is furthermetabolised to methylselenol by the action of b-lyase enzyme. Itwas also suggested that the endogenous production of monome-thylated selenium could be a critical factor in selenium chemopre-

vention (Ip et al., 2000a). Chiral speciation revealed the majorenantiomers as L-MeSeCys and L-SeMet (Kápolina et al., 2007).

The distribution of selenium among its species is remarkablydifferent depending on the selenium media used for plant treat-ment (Table 1). A notable amount of inorganic selenium was foundin Se(VI)-enriched chive (Kápolina et al., 2007), ramp (Whangeret al., 2000) as well as chicory (Pedrero et al., 2006) samples. Whensupplementation was done with Se(IV) and SeMet, the metabolismof selenium species in chive leads to the formation of more organicforms (Kápolina et al., 2007). In radish plants cultivated in Se(IV)media, about 95% of selenium content was transformed in the or-ganic species (Pedrero et al., 2006). In contrast, for those vegetablesgrown in selenate media, only 38% of Se was found to be present asorganic forms, whereas remaining was Se(VI). A pungent variant ofJapanese radish (Raphanus sativus L. ev. ‘‘Yukibijin”) enriched withSe by sprinkling with barium selenate and barium selenite becomea low accumulator (30 lg Se/g) (Ogra et al., 2007). The content ofMeSeCys relative to total Se was 10–12% and no other methylatedselenoamino acid derivatives were detected. However, a uniquecompound – selenohomolanthionine, probably formed from sele-nohomocysteine by cystathione c-synthase was detected in thewater extract. It could be one of the possible unknown Se com-pounds in various selenised plants reported in the literature.

4. Bioavailability of selenium from enriched vegetables

An adequate dietary intake of selenium would not necessarilymean that human body could adsorb the whole amount ingested.During ingestion, food components are exposed to different condi-tions (e.g., change in pH between the gastric and the intestinaltrack), which may affect the distribution of initial species. To pre-dict the bioaccessibility of organic and inorganic selenium formsfrom Se-enriched vegetables it is necessary to investigate seleniumspeciation in gastrointestinal model, which involve gastric diges-tion for the simulation of the stomach and a subsequent intestinaldigestion step.

The soluble Se fractions after simulated gastric and gastrointes-tinal digestion of radish enriched with either Se(IV) or Se(VI) wereabout 70% in gastric extracts and increased to 90% and 100% forplants exposed to selenate and selenite, respectively (Pedreroet al., 2006). The distribution of selenium compounds in both sim-ulated extracts was very similar. The species identified grown inthe presence of Se(IV) were SeCys2, SeMet and MeSeCys, the latterbeing the major compound of the sample (Fig. 2A). In contrast, inradish enriched with selenate, 60% of the selenium was presentin inorganic form, and also SeCys2, MeSeCys and SeMet were iden-tified (Fig. 2B). These results suggest that radish plants grown inselenite media could be a good source of selenium in human dietssince practically all the Se content was identified as selenoaminoacids, from which 75% of it was found to be Se-methylselenocysteine.

Simulation of the human digestion from green onion and chive,both supplemented with Se(VI), gave the extraction yield of 80–90%, however, Se was only in the form of selenate (Kápolna & Fodor,2007). It was assumed that the degradation of organic Se took placeduring the digestion process, since the content of inorganic sele-nium species significantly increased compared to results obtainedfor proteolytic extraction and the main initial Se forms – MeSeCysand SeCys2 – could not even be detected in the gastrointestinalextract. Two oxidation processes were postulated: oxidation ofSe(IV) to Se(VI) and selenomethionine to SeOMet as the effect ofthe significant pH change between gastric and the intestinal tracks(Kápolna & Fodor, 2007).

Dumont, Ogra, Vanhaecke, Suzuki, and Cornelis (2006) studiedthe extracts of Se-enriched garlic treated with simulated gastric



fluid in the time-related experiments for periods up to 24 h. Thesame Se profiles were observed and the main species eluted atthe retention time of SeMet, SeCys2, MeSeCys and c-Glu-MeSeCys.During prolonged time the amount of c-Glu-MeSeCys was de-creased and the content of MeSeCys increased, thus, it might bethat during this treatment the dipeptide loses its glutamyl moietyforming the resulting selenoamino acid. The same compounds andtrends were observed when garlic was subjected to in vivo intesti-nal digestion for different times. Additionally, the content of SeMetwas increased with longer digestion time. Garlic samples were alsotreated with saliva, to enable detection and analysis of Se speciesextracted during mastication (Dumont, Ogra, et al., 2006).Although the main species corresponded to those observed in thehot-water extract, their relative distribution was different – MeSe-Cys, SeMet, SeCys2 and c-Glu-MeSeCys accounting, respectively,for 40.6%, 10.0%, 5.1% and 37.1% of the Se. It should be noted thatseveral unidentified selenium compounds were also reported ingastrointestinal fluids (Dumont et al., 2006; Kápolna & Fodor,2007; Pedrero et al., 2006). One possible reason for a variety of un-known species could be limited cleavage specificity of the enzymespresent in a digestion medium (Wrobel, Kannamkumarath, Wro-bel, & Caruso, 2003).

5. Conclusion

The antioxidant and anticarcinogenic properties attributed tosome selenocompounds justify the increasing interest in growingSe-enriched vegetables, which represents an important source ofthis element in the human diet. Allium vegetables such as garlic,onion or chive are consumed everywhere and sufficient enrich-ment of these plants with selenium could provide a good sourceof supplementation, using them as seasoning in our diet. However,the degree of enrichment with selenium and – equally important –its distribution among the naturally synthesised molecular speciesmust be investigated in order to provide the necessary knowledgeto evaluate their disease-preventive potential.

Generally, total selenium measurements indicated that supple-mentation with Se(VI) of vegetables during their growing periodresulted in the highest total Se level as compared with the enrich-ment with Se(IV) or SeMet. However, the speciation studies re-vealed that a significant part of the total selenium was notmetabolised in the sample and resulted in accumulation as inor-ganic forms (Kápolina et al., 2007; Mazej et al., 2006; Ogra et al.,2005; Ogra et al., 2007; Whanger et al., 2000). Detailed studies ofthe distribution of Se species showed that selenium uptake andtransport takes place via different mechanisms, depending of thenature of the compounds originally taken up (Bulska et al., 2006).Better incorporation of selenium to organic species was observedfor the plants grown in the presence of selenite (Kápolina et al.,

2007; Ogra et al., 2007; Pedrero et al., 2007; Shah et al., 2004; Wro-bel et al., 2004), where this element is metabolised into a selenoa-mino acid form immediately after uptake within the entire crosssection of the root. This transformation could be considered as adefensive inactivation mechanism of the plant cell against a toxicchemical species. The major compounds found in the plant sam-ples enriched with Se(IV) were MeSeCys, SeMet, SeCys2 and c-Glu-MeSeCys (Table 1). Accumulation of these organic forms, par-ticularly MeSeCys and c-Glu-MeSeCys, may also be increased aftergenetic modification, which has been shown for the Indian mus-tard (Brassica juncea) (Van Huysen, Terry, & Pilon-Smits, 2004). Insimulated gastric and intestinal fluids, the same selenium specieswere observed as in the fresh plants of garlic (Dumont, Ogra,et al., 2006) and chive (Pedrero et al., 2006), particularly in vegeta-bles grown in selenite media, with MeSeCys as the principal com-ponent. These results suggest that these plants could be a goodsource in human diets of selenium species, which can enhance can-cer prevention.

In cultivation of Se-enriched vegetables, the amount of sele-nium left in the soil after supplementation should be also takeninto consideration. Selenite ions are less available in comparisonto selenate because of their high affinity to sorption sites of sedi-ment and soil constituents. Leaching of Se(IV) from soil by runoffis much lower as well as the possibility for pollution of both sur-face and wastewater. Thus, the amount of selenium left in the soilafter supplementation with Se(IV) would not be toxic when newseeds were planted.

One of the main topics of interest in the future is probably theinvestigation of the metabolism of MeSeCys and c-Glu-MeSeCys.These compounds might follow a different pathway in human bodyand are of specific interest in cancer research. Another topic of con-cern is the stability and distribution of selenium species in plantsafter different food treatments (e.g., cooking, drying and heating).Selenium speciation studies in the boiled vegetables need atten-tion in order to evaluate the effect of the cooking process. Wewould like to know whether these plants can be consumed raweither in fresh salads or in soups.

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Selenium speciation in enriched vegetables