Selenium Bioaccessibility and Speciation in Selenium-Enriched Lettuce

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Selenium bioaccessibility and speciation in selenium-enriched lettuce: investigation of the selenocompounds liberated after in vitro simulated human digestion using two-dimensional HPLC-ICP-MS Emanueli DO NASCIMENTO DA SILVA, Federica AURELI, Marilena D'AMATO, Andrea RAGGI, Solange CADORE, and Francesco CUBADDA J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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Journal of Agricultural and Food Chemistry

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Title

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Selenium bioaccessibility and speciation in selenium-enriched lettuce:

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investigation of the selenocompounds liberated after in vitro simulated human

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digestion using two-dimensional HPLC-ICP-MS

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Running title

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Selenium bioaccessibility and speciation in selenium-enriched lettuce

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Authors

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Emanueli DO NASCIMENTO DA SILVAa, Federica AURELIb, Marilena D’AMATOb, Andrea RAGGIb,

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Solange CADOREa, Francesco CUBADDAb*

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Affiliations

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a

Institute of Chemistry, University of Campinas, CEP 6154, 13083-970, Campinas, SP, Brazil

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b

Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità-

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Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy.

* Corresponding author

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Viale Regina Elena 299, 00161 Rome, Italy

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e-mail: [email protected]

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phone +39 06 49906024

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fax +39 06 49902540

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1 ACS Paragon Plus Environment


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Abstract

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The evaluation of selenium-enriched vegetables as potential dietary sources of selenium, an

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essential element for humans, requires an assessment of the plant accumulation ability as well as of

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the bioaccessibility and speciation of the accumulated selenium, which influence its biological

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effects in humans. Lettuce hydroponically grown at three selenite (SeVI)/selenate (SeIV)

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amendment levels was characterized accordingly. Selenium accumulation in lettuce leaves was

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greatest with Se(VI) amendment, whereas bioaccessibility was 70% on average in both cases.

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Selenium speciation in gastrointestinal hydrolysates, characterized by anion and cation exchange

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HPLC-ICP-MS, showed that Se(IV) was largely biotransformed into organoselenium metabolites,

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with selenomethionine accounting for 1/3 of the total detected species, whereas Se(VI) was

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incorporated as such in the edible portion of the plant, with only a small fraction (~20%) converted

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into organic species. Taking into account both nutritional quality and safety, the Se(IV)-enriched

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lettuce appeared more favourable as potential selenium source for human consumption.

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Keywords: selenium, food, speciation, in vitro simulated digestion, biofortification, human health

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1 Introduction

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Selenium is an essential element in humans, being needed as L-selenocysteine (SeCys) for the

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synthesis of selenoproteins and found as such in the active centre of a number of selenoprotein

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enzymes.1 Iodothyronine deiodinases, glutathione peroxidases (GPxs), thioredoxin reductases, and

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selenoprotein P (SEPP1) are important selenoproteins that have a variety of functions, including

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antioxidant effects, T-cell immunity, thyroid hormone metabolism, selenium homeostasis and

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transport, and skeletal and cardiac muscle metabolism.2 Insufficient or sup-optimal selenium intakes

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may cause a range of detrimental effects on human health and the relationships between selenium

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intake/status and various health outcomes, e.g. gastrointestinal and prostate cancer, cardiovascular

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disease, diabetes, male fertility, have been intensively investigated in the last years. Evidence to

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date suggests that selenium undernutrition may play an important role in some of these conditions

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but since the range of intake separating deficiency and toxicity is narrow, care has to be taken in

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identifying the optimal range for health and avoiding selenium overexposure.1,2

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Dietary reference intakes set on the basis of the optimization of plasma GPx3 activity are

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typically around 55 µg/day,3 whereas higher dietary intakes are necessary for the levelling off of

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plasma SEPP1 concentration and an adequate intake of 70 µg/day for adults was set by EFSA using

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this criterion.4 The selenium content of staple foods such as grains and vegetables depends on the

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selenium content of the soil as well as on its geochemical characteristics, which modulate selenium

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phytoavailability. The amount of selenium in the diet largely depends on where crops are cultivated,

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the soil/fodder to which animals are exposed, and the actual foods consumed. Low-selenium areas

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are present in a number of countries worldwide and therefore a large proportion of the human

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population is thought to have sub-optimal to insufficient selenium intakes.3,5

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L-selenomethionine (SeMet) is the predominant selenium species in almost all food sources

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whereas SeCys is the main form of selenium in mammalian proteins and is typically found in foods

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of animal origin.6 SeMet may unspecifically replace methionine residues in proteins, with the 3 ACS Paragon Plus Environment


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resulting proteins being referred to as selenium-containing proteins, whereas SeCys constitutes a

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specific amino acid residue in selenoproteins.1,3 Selenites (SeO32–, Se(IV)) and selenates (SeO42–,

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Se(VI)), the most common selenium inorganic compounds, are found in water and, as minor

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species, in most foods.1,7 In plants of the Brassica genus (e.g. broccoli) and the Allium genus (e.g.

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onion, garlic), the non-protein selenoaminoacids Se-methylselenocysteine (MeSeCys) and γ-

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glutamyl-Se-methylselenocysteine (γ-Glu-MeSeCys) are present and become the major species

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when these vegetables incorporate large amount of selenium.8,9

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Dietary selenium is generally well absorbed, but retention is higher with organic compounds

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compared to inorganic ones.6 Se(VI) is better absorbed than Se(IV), but a significant fraction is lost

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in the urine, whereas Se(IV) is better retained than Se(VI).10 Upon absorption, SeCys, Se(IV) and

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Se(VI) are available for the synthesis of selenoproteins whereas SeMet can substitute for

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methionine in proteins where it can act as a selenium store, entering the functional selenium body

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pool to be converted to SeCys upon turnover of tissue proteins.4 At dietary levels leading to

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excessive intakes, toxic effects of selenium are also species-related, with inorganic selenium – and

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especially Se(IV) – being more toxic than SeMet and most organic selenocompounds.6,11 In

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summary, the beneficial or toxic effects of selenium are not only dose-dependent, but also related to

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the chemical form of the element and its bioavailability. Therefore selenium speciation is important

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to have an insight into the bioavailability of dietary selenium and the relationships between intake,

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selenium status and health outcomes.

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Even though no selenium requirement has been shown and selenium essentiality is still

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controversial for higher plants, plants readily take up and assimilate selenium using sulfur

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transporters and biochemical pathways. Importantly, plant foods are the main source of dietary

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selenium and thus plant selenium metabolism is key for selenium nutrition of humans.12-18 A

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number of studies investigated the extent of selenium uptake and assimilation when different plant

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species are selenium-enriched, focusing on the conversion of inorganic selenium in soil/growth


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medium into organoselenium compounds biosynthesized in plant tissues. Although Se-enrichment

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of lettuce (Lactuca sativa L.) has been widely studied in either soil or hydroponic growing

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conditions,19-28 limited and conflicting information is available on the selenium species found in the

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edible portion of the plant.29-30 Furthermore, no information exists on selenium bioaccessibility in

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selenium-enriched lettuce, i.e. on the selenium fraction in lettuce leaves that is solubilized after

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human gastrointestinal digestion and becomes available for intestinal absorption.11-31

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The aim of this study was to investigate the extent of selenium accumulation and selenium

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bioaccessibility in lettuce hydroponically grown on Se(VI) and Se(VI) enriched substrates, and

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characterize the selenium species released after in vitro simulated human digestion by HPLC-ICP-

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MS. Since the use of a single chromatographic principle in HPLC-ICP-MS determination of the

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released selenocompounds is at risk of misidentification due to the possible coelution of some of

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them, we applied a two-dimensional approach using both anion and cation exchange HPLC-ICP-

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MS.

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2 Material and methods

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2.1 Instrumentation

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Chromatographic separations were performed using an HPLC system consisting of a Perkin-

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Elmer Series 200 LC binary pump, an autosampler, and a column thermostat. The outlet of the

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HPLC column was directly connected via PEEK capillary tubing to the nebulizer of the ICP-MS

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(Nexion 350D, Perkin-Elmer, USA), which served as the selenium specific detector for both total

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and speciation analysis. The ICP-MS instrument was equipped with a quartz concentric nebulizer

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and a quartz cyclonic spray chamber. Chromatographic data were collected, stored, and processed

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using the Perkin-Elmer software Chromera version 4.1.0.



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2.2 Reagents and standards

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Deionized water obtained by a Milli-Q Element System (Millipore, France) was used

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throughout the work. Nitric acid 68% v/v (Carlo Erba Reagenti, Italy) and hydrogen peroxide 31%

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v/v (Merck KGaA, Germany), both ultrapure grade, were used for oxidative digestion of samples

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and gastrointestinal hydrolysates. Calibrants and the internal standard solutions used for total

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selenium analysis were obtained from standard certified solutions with a content of 1 mg mL-1

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(High Purity Standard, USA), by dilution with acidified (HNO3) deionized water as necessary. For

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speciation analysis, 1 mg mL-1 stock solutions, expressed as selenium, were prepared by dissolving

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in water adequate amounts of selenious acid [Se(IV)], selenic acid [Se(VI)], SeMet, L-selenocystine

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(SeCys2), and MeSeCys (all from Sigma–Aldrich, USA), methyl-selenomethionine (MeSeMet), and

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γ-glutamyl-Se-methylselenocysteine

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Selenohomolanthionine (SeHLan) was kindly provided by Prof. Yasumitsu Ogra. Selenomethionine

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selenoxide (SeOMet) was prepared according to Michalska-Kacymirow et al.32 Standard stock

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solutions were stored at 4 ºC and the exact concentrations were ascertained by ICP-MS analysis.

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The purity of the standards was checked by HPLC-ICP-MS and no species interconversion was

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found. Analytical-grade ammonium acetate, pyridine, formic acid 98% (Merck KgaA, Darmstadt,

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Germany), and methanol (J.T. Baker, Deventer, The Netherlands) were used for the preparation of

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the chromatographic mobile phases. Mobile phases were filtered through a Millipore Express Plus

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0.22 µm membrane. Porcine enzymes (pepsin, pancreatin), α-amylase from Bacillus subtilis, bile

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salts, KCl, and MgCl2(H2O)6, (all from Sigma–Aldrich, USA), CaCl2, (Baker Instra Analyzed,

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Avantor Material, The Netherlands), NaCl, (NH4)CO3, KH2PO4, NaHCO3 (all from Merck KGaA,

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Germany) were used in simulated gastrointestinal digestion. Ultrapure grade 37% v/v hydrochloric

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acid (Carlo Erba Reagenti, Italy) and sodium hydroxide (Sigma–Aldrich, USA) were used to adjust

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pH.

(γ-Glu-MeSeCys)

(PharmaSe,


Lubbock,

TX).

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2.3 Procedures

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2.3.1 Selenium-enrichment of lettuce

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The greenhouse experiments were conducted at the School of Agricultural Engineering of the

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University of Campinas. Seedlings of red leaf lettuce (Lactuca sativa L., cv. "Veneza Roxa") were

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grown hydroponically. Each hydroponic system had four channels, each connected to 10-L vessels

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of nutrient solution, and comprised 3-4 control or Se-enriched plants. The composition of the

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nutrient solution (Hidrogood, Brazil) is shown in Table S1 (Supporting information). Nutrient

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solutions were prepared so as to contain 0, 10, 25 and 40 µmol Se L-1 of either sodium selenate

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(Na2SeO4) or sodium selenite (Na2SeO3) (Sigma-Aldrich, USA), according to the design shown in

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Table S2 (Supporting information). The solutions were constantly aerated and the pH was

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monitored daily and adjusted between 5.5-6.5 with 6 mol L-1 NaOH or 6 mol L-1 HCl, as necessary.

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The conductivity was maintained in the range 2.5-3.5 mS cm-1; when the value was out of this range

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the solution was replaced. After 28 days, the plants were harvested, washed with tap and deionized

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water, and dried in an oven at 60 °C for 72 h.

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2.3.2 In vitro simulated gastrointestinal digestion

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Selenium bioaccessibility was assessed using a standardised static in vitro method simulating

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human gastric and gastrointestinal digestion.33 Table S3 (Supporting information) summarizes the

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composition of the gastrointestinal fluids used in this study and Figure S1 (Supporting information)

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shows the overall scheme of the in vitro digestion experiments. Briefly, each batch of lettuce

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samples was incubated for 2 minutes at 37 ºC with saliva in a mixing water bath (GFL 1083,

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Gesellschaft für Labortechnik mbH, Burgwedel, Germany), then the gastric juice was added and the

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samples were kept in the mixing water bath for 2 h. Another batch of samples was submitted in

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parallel to the same procedure, but after the gastric digestion the intestinal juice was added and the

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samples were incubated for other 2 h. The gastric and the gastrointestinal hydrolysates were

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centrifuged at 10,000 rpm for 25 min at 4 ºC. The supernatant was then collected, filtered through



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0.45 µm membranes, divided into aliquots and stored at -80 ºC until analysis. Procedural blanks

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were run in parallel in order to check the presence of selenium in the reagents. The in vitro

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enzymolysis procedure was carried out in triplicate for each lettuce sample.

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2.3.3 Total selenium analysis

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For total selenium analysis, samples were submitted to microwave-assisted oxidative

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digestion by means of an Ultrawave single reaction chamber (Milestone, Sorisole, Italy). For lettuce

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samples, approximately 0.15 g of dried sample weighed into a Teflon® flask were added with 3 mL

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of HNO3 and 1 mL of deionized water. Total selenium solubilized by gastric and gastrointestinal

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digestion was also determined by ICP-MS. Aliquots of extracts (2 mL) were added with 1 mL of

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HNO3, and submitted to the same digestion procedure as above. The digested samples were made

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up to 20 mL with deionized water prior analysis. Measurements were carried out using

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quantification. The limits of detection and quantification were 0.06 and 0.21 µg L-1, respectively.

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The accuracy of total selenium determinations, as assessed through analysis of the reference

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material in cabbage matrix IAEA 359 (International Atomic Energy Agency, Vienna, Austria), was

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satisfactory with no statistical difference between found values (0.12 µg g-1, 95% C.I. = 0.113-0.133

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µg g-1) and information values (0.12 µg g-1, 95% C.I. = 0.109-0.131 µg g-1).

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2.3.4 Selenium speciation analysis

Se as analytical masses with

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Se and

Ge as internal standard, and the method of standard additions for

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The selenium species liberated by simulated gastrointestinal digestion were characterized by

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anion exchange and cation exchange HPLC-ICP-MS. Selenocompounds in extracts were identified

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by retention time matching with the standard substances spiked to the sample extracts. Quantitative

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calculations were based on peak areas using external calibration or the method of standard additions

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as appropriate, depending on sample dilution. For anion exchange separation a PRP-X100 column

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(4.6·x 250 mm, 10 µm) equipped with a guard-column was used; the eluents were 5 mM acetate

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buffer (pH 4.7) (A) and 150 mM acetate buffer (pH 4.7) (B), and gradient elution at 1.0 mL min-1 8 ACS Paragon Plus Environment

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(0-4 min-100% A, 4-6 min-from 100% A to 15% A, 6-30 min-85% B and 15% A) was used. Fifty

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µL of the filtered hydrolisates were injected. For cation exchange separation a Chrompack

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IonoSpher-5C column (3.0·x 100 mm, 5 µm) equipped with guard-column was used; eluent A, i.e.

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3% (v/v) MeOH at pH 3.0, and eluent B, i.e. 10 mM pyridinium formate with 3% (v/v) MeOH at

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pH 3.0 - were used at 1 mL/min according to the gradient programme 0.1-3.5 min-92.5% A/7.5% B,

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3.5-13.5 min-72% A/28% B, 13.5-16.5 min-72% A/28% B, 16.5-28 min-92.5% A/7.5% B. Twenty

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µL of the filtered hydrolysates were injected for measurements. The chromatographic purity of the

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anion exchange peak eluting at 2.7 min was assessed by orthogonal chromatography, i.e. collecting

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the fraction eluting from 1.5 to 4.5 minutes and analysing it by cation exchange HPLC-ICP-MS

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with the same conditions as above.

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3 Results and discussion

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3.1 Selenium accumulation in lettuce

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There was no observable toxic effect of selenium application on lettuce plants in the

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conditions of this study. In particular, there was no statistically significant influence of the selenium

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form and applied concentration on plant biomass (ANOVA, p > 0.05).

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Selenium accumulation in lettuce leaves as a function of applied selenium was quite different

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when the latter was provided in the form of Se(IV) compared to Se(VI) (Figure 1). The selenium

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concentrations in the edible portion of the plant resulting from the treatment with Se(VI) were much

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higher (Table 1). The dissimilar selenium deposition observed in lettuce leaf tissue is consistent

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with the different absorption and assimilation of Se(IV) and Se(VI) in higher plants. Se(IV) is

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rapidly converted to organic forms, which have low mobility in the xylem and are incorporated as

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selenoaminoacids into root proteins; in contrast, Se(VI) in roots is not easily converted to organic

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forms and being much more mobile in the xylem it is easily transported to the aerial part of the



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plant.13,15-18 This is confirmed by the high Se(VI) to total selenium ratio found in leaves of Se(VI)-

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enriched plants (vide infra).

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Several studies observed that lettuce leaves readily accumulate selenium - and at a greater

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degree when supplied as Se(VI) compared to Se(IV) - as found in the present study.21,23-26 In

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comparative studies, lettuce was found to achieve higher selenium concentrations in the edible

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portion than other plants (e.g. radish, tomato, strawberry) under the same growing and

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biofortification conditions; for other species (e.g. chicory and cucumber) the selenium levels were

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comparable.19-20,22 An evaluation of 30 diverse accessions of lettuce for their capacity to accumulate

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selenium showed an over twofold change in total selenium levels between cultivars, highlighting

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useful variability in lettuce germplasm for optimal biofortification.25 Selenium accumulation, for

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Se(VI) treatment, appeared to be associated with an altered expression of genes involved in

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selenium/sulphur uptake and assimilation, whereas the stimulating effect on plant growth found in

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some cases correlated with the activities of antioxidant enzymes.25

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3.2 Selenium bioaccessibility in lettuce

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The fraction of leaf selenium that was solubilized after incubation with simulated salivary

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fluid and simulated gastric fluid (succession of the oral and gastric phases of human digestion) is

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referred to as bioaccessible selenium after gastric digestion in this work. Se(IV)-enriched lettuce

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had a mean gastric bioaccessibility of 32% (s.d. 2%, min-max 31-34%) whereas for Se(VI)-

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enriched lettuce the fraction solubilized was 50% on average (s.d. 8%, min-max 43-58%), and it

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was positively associated (r=0.978) with the magnitude of Se(VI) application (Table 1). In the non-

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enriched lettuce, the fraction of leaf selenium solubilized amounts to an intermediate value of 42%

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(s.d. 9%).

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After completion of the simulated human digestion with the intestinal phase, Se(IV)-enriched

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lettuce had a gastrointestinal bioaccessibility of 68% on average (s.d. 3%, min-max 65-71%), which


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was negatively associated (r=-0.997) with the magnitude of Se(IV) application, whereas for Se(VI)-

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enriched lettuce the fraction solubilized is 72% on average (s.d. 6%, min-max 66-76%), and it is

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greater (76%) for the two higher levels of Se(VI) application (Table 1).

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The higher gastric bioaccessibility of selenium in Se(VI)-enriched plants is consistent with a

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selenium speciation dominated by Se(VI), which is easily solubilized in the conditions of human

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stomach digestion. On the other hand, if a substantial proportion of the total selenium is present as

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protein-bound SeMet, the initial proteolytic hydrolysis promoted by pepsin in the gastric phase is

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not sufficient for the release of the selenoaminoacid that requires the cleavage of peptide bonds

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occurring during intestinal proteolysis. The latter is the case of Se(IV)-enriched plants (vide infra).

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Irrespectively of the type of supplementation, the supplementation level and the resulting leaf

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selenium concentration and speciation, the gastrointestinal bioaccessibility of selenium in Se-

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enriched lettuce was satisfactory, i.e. 70% on average compared to 62% of the non-enriched lettuce.

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This value is in the range of the levels commonly observed for selenium bioavailability (in vivo) or

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bioaccessibility (in vitro) in plant sources.10-11,31

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3.3 Speciation of bioaccessible selenium in lettuce

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Figure 2 shows the anion exchange HPLC separation with ICP-MS detection of a mixture of 9

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selenium standards. Cation exchange HPLC separation with ICP-MS detection was used as an

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alternative chromatographic system and Figure 3 shows the HPLC-ICP-MS chromatogram of 9

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selenium compounds of interest.

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Analysis of the gastrointestinal hydrolysates by HPLC-ICP-MS was carried out to

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characterize the bioaccessible selenium chemical species, i.e. the actual selenium compounds that

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are released after simulated human gastrointestinal digestion and become available for intestinal

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absorption. Since the retention time of some species was affected by the sample matrix, correct

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assignment of identity as well as quantification were based on coelution with spiked authentic



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standards. In addition, the samples were analysed at different dilutions and in all cases species

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identification was substantiated by spiking the sample with the authentic standard substance.

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Anion exchange HPLC-ICP-MS revealed that at least twelve chemical species were present in

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the case of Se(IV)-enriched lettuce (Figure 4). The inorganic species (Se(IV) and Se(VI)) are a very

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minor fraction of the total chromatographed selenium and they always account for ≤7% of the sum

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of the eluting species (Table 2). More than 90% of the selenium eluted from the column is thus

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represented by organic compounds, with two of them featuring as major species and constituting

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~60% of the sum of the species. One of these two species (retention time 6.9 min) was identified as

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SeMet. Apart from SeMet, none of the other organic compounds in Figure 2 matched any of the

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unknown peaks in Figure 4. It has to be noticed that nearly 40% of the selenium solubilized by in

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vitro gastrointestinal digestion does not elute from the column, which highlights the presence of

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strongly retained compounds most likely of organic nature.

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In order to characterize more closely the different organoselenium species present, cation

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exchange HPLC-ICP-MS was used as an additional, complementary analytical approach (Figure 5).

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An excellent agreement was found for SeMet, which again was found to account for one third of the

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selenium species eluted (Table 3). Spiking experiments with SeCys2 showed coelution with the

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peak at 10.5 min, thus providing evidence of the presence of this species in sample extracts.

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A specific attention was paid to the possible presence of MeSeCys, which was reported as the

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major organic species in lettuce in a previous study.30 However, in spiking experiments, none of the

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several organic compounds present coeluted with the MeSeCys standard, thus demonstrating that

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this species was absent. The occurrence of SeHLan, a non-protein selenoaminoacid produced by

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plants via the sulfur assimilation pathway and identified for the first time in selenium-enriched

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Japanese pungent radish,34 was also excluded based on spiking experiments. Co-chromatography of

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the γ-Glu-MeSeCys standard spiked in the sample extracts showed that this species was absent as

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well. 12 ACS Paragon Plus Environment

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An attempt was then made to identify the organoselenium compounds present in the anion

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exchange fraction eluting between 1.5-4.5 min, which was collected and injected in cation exchange

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HPLC-ICP-MS. The fraction contained a species eluting in the void and one at ~8 min, which

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however did not coelute with any of the available standards and, in particular, SeOMet. This species

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was a potential candidate for the identity of the anion exchange peak at 2.7 min as it is easily

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formed by SeMet oxidation and thus it has been widely found in enzymatic extracts of plant

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materials and selenized yeast.35-38

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Unfortunately, post-column recovery did not improve with cation exchange chromatography,

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confirming that reactive compounds that are retained by the column are largely present in the

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gastrointestinal hydrolysate of Se(IV)-enriched lettuce. In summary, SeMet was confirmed to

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account for about one third of the sum of the species; taking into account post-column recovery, this

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translates in not less than 20% of the bioaccessible selenium after gastrointestinal digestion.

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In gastrointestinal extracts of Se(VI)-enriched lettuce, Se(VI) was found to be the

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predominant species (Figure 6), accounting for 64-80% of the eluting selenium in anion exchange

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chromatography (Table 2). Cation exchange chromatography, where Se(VI) elutes in the void

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(along with some of the early-eluting species in anion exchange chromatography), showed good

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quantitative agreement (Table 3). Both chromatographic approaches gave consistent results for

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SeMet, which account for 11-12% of the sum of the species in lettuce grown at the lowest applied

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dose of Se(VI) and decreases to 4-5% at the highest dose. No MeSeCys was found to be present in

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extracts of Se(VI)-enriched lettuce, and the same hold true for the other organic species in Fig. 3.

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Taken together, these results confirms that lettuce plants are able to metabolize the supplied

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Se(IV) to SeMet and other organic compounds. Upon Se(VI) amendment, the compound is

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efficiently translocated to the aerial part of the plant mostly unchanged. The more extensive

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biotransformation to organic forms of Se(IV) compared to Se(VI) is in line with the available

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evidence.13,15-18 13 ACS Paragon Plus Environment


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From the standpoint of both nutritional quality and safety, the Se(IV)-enriched lettuce

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produced at the lowest fortification levels investigated in this study appears more favourable as

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potential selenium source for human consumption. Taking the 25 µM sample as an example, it

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provides 1.6 µg g-1 fresh weight of bioaccessible selenium (dry to fresh weight conversion factor is

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19.8), equivalent to 135 µg for a 100 g portion of lettuce, consisting of

Selenium Bioaccessibility and Speciation in Selenium-Enriched Lettuce

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