In vivo bioavailability of selenium in enriched Pleurotus ostreatus mushrooms

Abstract

The in vivobioavailability of Se was investigated in enriched Pleurotus ostreatus mushrooms. A bioavailability study was performed using 64 Wistar male rats separated in 8 groups and fed with different diets: without Se, with mushrooms without Se, with enriched mushrooms containing 0.15, 0.30 or 0.45 mg kg⁻¹Se and a normal diet containing 0.15 mg kg⁻¹ of Se using sodium selenate. The experiment was performed in two periods: depletion (14 days) and repletion (21 days), according to the Association of Official Analytical Chemists. After five weeks, the rats were sacrificed under carbon dioxide, and blood was drawn by heart puncture. Blood plasma was separated by centrifugation. The total Se concentration in the plasma of rats fed with enriched mushrooms was higher than in rats fed with a normal diet containing sodium selenate. The plasma protein profiles were obtained using size exclusion chromatography (SEC) and UV detectors. Aliquots of effluents (0.5 mL per minute) were collected throughout in the end of the chomatographic column. However, Se was determined by graphite furnace atomic absorption spectrometry (GF AAS) only in the aliquots where proteins were detected by SEC-UV. The plasma protein profile of rats fed with different diets was similar. The highest Se concentration was observed in a peptide presenting 8 kDa. Furthermore, the higher Se concentration in this peptide was obtained for rats fed with a diet using enriched mushrooms (7 μg L⁻¹Se) compared to other diets (2–5 μg L⁻¹Se). These results showed that Se-enriched mushrooms can be considered as an alternative Se food source for humans, due to their high bioavailability .

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1. Introduction

Mushrooms are edible products with high protein and mineral content, but low carbohydrate and fat values. Mushrooms provide relatively high concentrations of essential elements, such as Se.¹ The extensive human health benefits of dietary selenium (Se) are well established and include its antioxidant properties and positive contributions to normal thyroid and immune function and fertility.^2–4 A recommended dietary allowance (RDA) has been established for 55 μg d⁻¹Se for adults.³

The occurrence of several metals and metalloids in wild growing and cultivated mushrooms has been extensively studied aiming the possible use of these species as environmental pollution bioindicators, to optimize commercial cultivation processes, to investigate accumulation and distribution, to elucidate the mechanisms responsible for elemental uptake, and to evaluate the use of edible mushrooms as dietary sources of bioelements.⁵

The enrichment of mushrooms or other foods⁶ with Se allows the production of one functional food with high economic value. Functional foods are fortified, enhanced or enriched with certain nutrients for the purpose of increasing their health benefits.⁷ The knowledge of compositional, nutritional and functional properties of foods is fundamental for defining their quality. In general, nutritional properties are characterised by both the abundance and bioavailability of essential nutrients.

The bioavailability is defined as the degree to which a nutrient, toxin, or other substances become available for body use or deposition after exposure.⁸ When the exposure of the substance is oral, bioavailability generally includes absorption, body utilization, and/or deposition.⁶ In general, in vivo and in vitro methods can be used to estimate Sebioavailability . In vitro systems usually consist of the simulated gastrointestinal digestion, followed by measurement of the dialyzable mineral fraction across a semi permeable membrane.^9–11 On the other hand, the in vivo methods involve the use of animals or human beings. In this case, the interaction of Se with other compounds presented in the organism influences its bioavailability .^9–11

Elemental speciation at the molecular level has been successfully performed by conventional proteomics approaches with the separation by size exclusion chromatography coupled to ultraviolet and inductively coupled plasma mass spectrometry (SEC-UV-ICP-MS), and identification by ion chromatographic-associated electrospray mass spectrometry (ESI-MS/MS) and matrix assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS).^12,13Atomic absorption spectrometry (AAS), inductively coupled optical emission spectrometry (ICP OES) and inductively coupled plasma mass spectrometry (ICP-MS) have been applied for the elemental determination.¹⁴ The ICP-MS coupled to SEC is more frequently used due to the possibility of on-line hyphenation and it is widely appreciated for its isotope specificity, versatility and high sensitivity.^12–15Graphite furnace atomic absorption spectrometry (GF AAS) has been used for trace and ultra trace element determinations in several materials. Unfortunately, the sequential nature of the drying and washing steps prior to atomisation makes it difficult to couple to the continuous flow of the chromatographic effluent.¹⁵ In spite of this disadvantage, procedures based on fraction collection and posterior off-line analyses by GF AAS detection have been successfully applied to accomplish speciation.¹⁵ Although on-line approaches by ICP-MS coupled to SEC are more elegant and faster in metal speciation and metallomics, the attested high sensitivity, direct analysis capability and low interference level of GF AAS may make it one real alternative for the off-line coupling to SEC.

Regarding the nutritional importance of the Se, the main objective of this paper is to evaluate the bioavailability of this element in enriched mushrooms (Pleurotus ostreatus) using an in vivo method involving graphite furnace atomic absorption spectrometry (GF AAS) for total Se determination and size exclusion chromatography (SEC) coupled off-line with GF AAS for selenoprotein identification.

2. Experimental

2.1 Instrumental

A SIMAA-6000 graphite furnace atomic absorption spectrometer with a longitudinal Zeeman-effect background correction system, Echelle optical arrangement, solid state detector and standard THGA tube with pyrolitically coated integrated platform (Perkin-Elmer, Norwalk, CT) was used for the Se determinations. The spectrometer was operated using an electrodeless discharge lamp. Solutions were delivered into the graphite tube by means of an AS-72 autosampler. The setup of the instrumental conditions was: λ = 190 nm, current lamp = 290 mA, and bandpass = 0.7 nm). The heating program is shown in Table 1.

Table 1 Heating program to Se determination by GF AAS

Step	T/°C	Ramp/s	Hold/s	Ar flow rate/mL min^−1
a Injection temperature: 30 °C; Pipette speed: 100%
Drying I	100	10	15	250
Drying II	130	10	20	250
Pyrolysis	1200	100	20	250
Atomization	2300	0	5	0
Cleaning	2500	1	2	250

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The digestion of mushroom samples was carried out in a closed vessel microwave oven, model Microwave 3000 (Anton Paar, Graz, Austria).

A chromatographic system (CBM-20A, Shimatzu, Japan) equipped with degassing, piston pump, UV-Visdetector and an autosampler with 110 sampling positions was used to separate molecular weight compounds by SEC. The Superose 12 10/300 GL (10–300 kDa) (Amersham Biosciences, Sweden) was used as the stationary phase and 0.2 mol L⁻¹ Tris/HCl (pH = 7.5) as the mobile phase. The column was percolated with 36 mL of buffer and aliquots of 0.5 mL were collected every 1 min. The flow rate, injection volume and UV wavelength parameters were 0.5 mL min⁻¹, 200 μL and 280 nm, respectively. A reference mixture of ribonuclease A (∼13.7 kDa), aprotinin (∼6.5 kDa), aldolase (∼158 kDa) carbonic anhydrase (∼29 kDa), ferritin (∼440 kDa), ovalbumin (∼43 kDa) and conalbumin (∼75 kDa) (GE Healthcare, Piscataway, USA) was used to calibrate the column. A solution of 1.0 mg mL⁻¹ of Blue dextran 2000 was used to obtain the column void volume (V_o).

2.2 Reagents

All solutions were prepared from analytical reagent grade chemicals using high-purity deionized water obtained from a Milli-Q water purification system (Millipore, Belford, USA). Se analytical solution (Na₂SeO₃) of 1000 mg L⁻¹ was used for mushroom enrichment and in its determination by GF AAS. In the last case, a volume of 10 μL of chemical modifier solution of 5 μg Pd and 3 μg Mg was co-injected with 10 μL of samples or analytical solutions into the graphite furnace.

The acid digestion of mushrooms was done using 65% v/v HNO₃ and 30% w/w H₂O₂ from Merck (Darmstadt, Germany). A buffer solution of 0.2 mol L⁻¹ Tris/HCl was prepared by dissolving Tris(hydroxymethyl)aminomethane (USB Corporation) in deionized water and adjusting the pH to 7.5 with HCl (Merck).

2.3 Enriched mushrooms

Coffee rusk was used in the growing of the mushrooms. This rusk was boiled for 2 h and centrifuged at 1800 rpm for 5 min. A mass of approximately 1.5 kg was inserted into the polypropylene bags and autoclaved for 2 h. This procedure was executed 3 times in each 48 h period. After this, 5 mL of sodium selenite solution (25.4 mg kg⁻¹) was added in the coffee rusk with the mushrooms. The incubation period was 15 days at 25 °C and the fructification time was 39 days at 20 °C with 90% air humidity. The non-enriched mushrooms were also cultivated. The mushroom samples were dried at 45 °C until constant weight and triturated in a knives mill.

2.4 Se determination in mushrooms

The ground mushrooms were subjected to acid digestion in a microwave oven, using diluted oxidant mixture (2.0 ml HNO₃ + 1.0 mL H₂O₂ + 3.0 mL H₂O). The microwave heating program presented four steps (Temperature/ºC; ramp/min; hold/min): 1 (140; 5; 1), 2 (180; 4; 5), 3 (200; 4; 10), 4 (0, 0, 20). The final volume was 15 mL. Se concentration in the digested samples was determined by GF AAS.

2.5 Bioavailability study

2.5.1 Animals and diets. Sixty-four male Wistar rats weighing between 111 and 167 g were individually kept in stainless steel cages at 18–24 °C and 12-hour light and dark cycles. The experiment was performed in two periods: depletion (14 days) and repletion (21 days), according to the Association of Official Analytical Chemists.¹⁶ This study was reviewed and approved by the Committee of Postgraduate Programme at the Federal University of Viçosa (MG-Brazil).

In the period of depletion, the rats were fed with deionized water and a control diet (AIN-93G diet¹⁶ without Se). In the period of repletion, the animals were divided into eight groups: G1 control diet; G2, G3 and G4 control diet + 0.46, 0.93 and 1.40 g of enriched mushrooms, respectively; G5, G6 and G7 control diet + 0.46, 0.93 and 1.40 g of non-enriched mushrooms, respectively; and G8 control diet + sodium selenite (0.15 mg kg⁻¹). The Se concentrations in the enriched mushrooms and in a control diet of rats (0.15 mg kg⁻¹) were considered for determination of mushroom masses added in the rat diets.¹⁷

After the repletion period, the animals were killed under carbon dioxide, and blood was drawn by heart puncture and mixed with heparin to prevent blood coagulation. The blood of the eight rats of each group was mixed. Next, this mixture was submitted to centrifugation (1000 rpm, 4 °C and 10 min) for plasma separation. In this sample, total Se concentration and association to proteins were determined by GF AAS and SEC-UV off-line, respectively.

2.5.2 Proteins and Se in the plasma. The total concentration of Se in the plasma was done according procedure proposed in the literature.¹⁸ However, different and appropriated dilution (10–20 times) was adopted in this work. For the SEC separation a volume of 100 μL of the diluted plasma (2 times) was injected into the column. The molecular spectra were continuously monitored by UV detector.

Aliquots of effluents (0.5 mL per minute) were collected throughout in the end of the chomatographic column. However, Se was determined by GF AAS only in the aliquots where proteins were detected by SEC-UV. The concentration of Se in each fraction was obtained discounting the blank values eluted in the same elution volume. Aliquots of 10 μL of effluent were co-injected into the graphite tube with 10 μL of chemical modifier (5 μg Pd + 3 μg Mg). The calibrations were done with successive dilutions of analytical-grade Tritisol® solutions of 1000 mg L⁻¹ of Se in 0.2 mol L⁻¹ of Tris-HCl (pH = 7.5). The linear ranges were 20–80 μg L⁻¹ of Se. The plasma samples submitted to chromatographic separation were G1, G2, G6, and G8.

3. Results and discussion

3.1 Se determination in the enriched mushrooms

The total Se concentration in the enrichment mushrooms was 261 ± 38 μg g⁻¹ (n = 3). The enrichment using 25.4 mg kg⁻¹ of sodium selenite resulted in the 19% (w/w) of absorption by mushrooms. In this situation, the physical characteristics and collected numbers were similar to those cultivated without Se addition. In the non-enriched mushrooms, low concentrations of Se was found, ranging from 0.12 to 0.96 μg g⁻¹. In general, some mushrooms species are capable of accumulating high Se concentrations, such as Agaricus bisporus, Boletus edulis, B. pinicola, B. aestivalis and Xerocomus badius.^19,20 According to Munoz et al.,²¹ the incorporation of Se from the growth medium to the mycelia of Pleurotus ostreatus was observed with relative distribution between the cytosol plus cell membranes fraction and the ell walls fraction of about 44 and 56%, respectively.

3.2 Se determination in the rat plasma

The total concentration in the plasma of rats fed with enriched mushrooms (G2, G3 and G4) was higher than in the others situations (G1, G5, G6, G7 and G8), as shown in Table 2. The different masses of the enriched (G2, G3 and G4) and non-enriched (G4, G5 and G6) mushrooms added in the diet of rats did not influence the Se absorption, because it obtained similar concentrations in each group. It is important to point out that the Sebioavailability in the enriched mushrooms was higher in rats fed with sodium selenite. This result suggested that in the enriched mushrooms the Se can be found in the organic forms, mainly selenoproteins, because these species are more bioavailable than inorganic forms.^22,23

Table 2 Se concentration in plasma of rats

Groups	Se concentration/μg L^−1 ± standard deviation (n = 3)
G1	269 ± 60
G2	680 ± 58
G3	731 ± 88
G4	723 ± 78
G5	295 ± 74
G6	291 ± 67
G7	287 ± 39
G8	513 ± 69

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3.3 Proteins plasma profile

The SEC profiles for the diluted plasma (2 times) using UV detection (280 nm) are depicted in Fig. 1. The relation between eluted volume and the logarithm of molecular weight was linear in the range of 6.5 to 440 kDa. The equation calibration curve was K_av = 0.4933 − 0.2583 × log MW (R² = 0.9799), where K_av is the partition coefficient, calculated by K_av = (V_elution − V_o)/(V_total − V_o). The linear response was lost for all species eluted in volumes below void volume (9.0 mL) and above 25 mL (V_total). It is important to detach that chromatographic run time was extended to 72 min (36 mL) to ensure the elution of all fractions.

Fig. 1 SEC-UV chromatogram of rat plasma of different groups: G1 (A), G2 (B), G6 (C), and G8 (D).

Independently of the rat’s alimentation, the protein profiles were similar. However, the low analytical signals of proteins were obtained in the plasma of rats fed with mushrooms (G2 and G6), Fig. 1. The analytical signals showed the prevalence of four species of high molecular weight (HMW) between 80 and 8 kDa. The highest Se concentration was found in the peptide of, approximately, 8 kDa.

Low molecular weight (LMW) compounds were found in all plasma samples (<6.5 kDa). However, the molecular weights could not be determined because the elution was outside the calibration range of the Superose 12. These compounds may be oligopeptides (2–10 amino acid residues)²⁴ or other compounds that can cause interference at 280 nm.²⁵

3.4 SEC with off-line coupled GF AAS

Selenium in the aliquots of the chromatographic effluents (0.5 mL per minute) of the plasma samples (G1, G2, G6 and G8) was determined by GF AAS in each protein fraction detected by SEC-UV. In the G1, G2 and G8 groups were found high concentration of Se associated to the peptide of, approximately, 8 kDa (2.8 ± 0.9 μg L⁻¹; 6.9 ± 0.8 μg L⁻¹; and 5.3 ± 1.1 μg L⁻¹, respectively). The rats of the G2 (enriched mushrooms) and G8 (sodium selenite) groups were fed with Se concentration very close to 0.15 mg kg⁻¹. The difference is in the Se forms. In mushrooms it is provable that Se is associated to proteins and not free as inorganic species. In both groups Se was found in higher concentrations when associated to peptides of, approximately, 8 kDa than in others peptides. It is important to point out that the Se concentration was similar in the peptide of 8 kDa. In light of this information it is possible to observe that Se forms (inorganic or associated to proteins) can not influence the absorption of Se by rats and its incorporation in the plasma peptide, mainly of 8 kDa

In the G6 group (non-enriched mushrooms), Se concentration was similar to the concentration found in the plasma of rats fed with the control diet without Se and mushrooms (G1 group), Table 2. However, for the G6 group Se concentration in all compounds separated by chromatographic techniques was below the detection limit (0.7 μg L⁻¹). On the other hand, in the G1 group with non-enriched diets (without Se and mushrooms), Se was found associated to two molecules of 0.49 and 8 kDa. This difference can be due to the addition of non-enriched mushrooms (G6) in the diet, inhibiting the absorption and association of Se to peptides of 8 kDa. Nevertheless more studies about this effect are necessary to construct consistent conclusions.

It was found that Se associated to species of LMW (0.49 kDa) in the G1 (1.4 ± 0.8 μg L⁻¹) and G2 (1.1 ± 0.8 μg L⁻¹), was also in species of HMW (14.7 kDa) in the G8 (1.7± 0.8 μg L⁻¹).

Based on the masses of Se (0.03 μg for G1, 0.07 μg for G2, and 0.06 μg for G8) injected into the size exclusion column, the recoveries of Se obtained from the sum of all effluent portions were 7, 6, and 6%, respectively. Due to the low percentage of Se in the proteins, it is possible that presence of Se was in others forms, such as ionic species.

In general, in plasma the main selenoproteins were glutathione peroxidases (19%) and selenoprotein P (53%).^26–28 In these species, Se is associated to cysteine.^29,30 Selenoprotein P is an extracellularprotein that contains over 60% of the Se in rat plasma.³¹ There are multiple forms of rat selenoprotein P present in plasma, five forms were characterized, being two isoforms of 45 kDa and three of 57 kDa.³⁰ Although the selenoprotein P is more abundant in rats, this selenoprotein was not detected in this work.

The Sebioavailability has a high enough variability, principally due to the different chemical forms and factors previously indicated that exist in foods.³¹ Studies in mushrooms revealed the presence of selenomethionine.^20,32 In enriched mushrooms the association of Se to compounds of LMW (<10 kDa) was observed,^33,34 as shown in this work. It is important to point out that the growth of mushrooms in the presence of high concentrations of Se resulted in the incorporation of this element into the proteins, mainly of 8 kDa, indicating the Se availability for absorption and use of biological functions of rats.¹⁹

4. Conclusion

In enriched mushrooms, the high Sebioavailability was verified using in vivo methods. The enriched mushroom consumed by the rats showed that Se can be more bioavailable in one form, since that the Se concentration in the plasmas of rats fed with enriched mushroom (G2–G4) was higher than in rats fed with sodium selenite in the diets (G8). By comparing the protein profiles obtained by SEC-UV there were no differences observed in the distribution of the plasma proteins of rats fed with different diets, including the diets with the addition of enriched mushrooms.

For G1, G2 and G8 rat groups, high Se concentration was found in the molecular weight peptide of 8 kDa. The groups G2 and G8 that were fed with diets containing enriched mushroom or sodium selenite, respectively, presented the same Se concentration in the peptide of 8 kDa, revealing that Se forms did not affect the absorption and incorporation of this element in these peptides. The G1 group (rats fed with diets without Se and mushrooms) was found Se in lower concentrations than in the G2 and G8 groups. In the G6 group (rats fed with the diet including non-enriched mushrooms) association of Se to the peptide of 8 kDa was not found, as was observed in the other groups (G1, G2 and G8). For this, the addition of mushroom into the conventional diets of rats can have promoted the inhibition of the Se absorption by the peptides.

Acknowledgements

The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support, researchships and fellowship provided.

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