Influence of Dietary Selenium Species on Selenoamino Acid Levels in

Jul 10, 2015 - feeding trial was performed with rainbow trout fry using either a plant-based or a fish meal-based diet. Se yeast and selenite were use...
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Influence of Dietary Selenium Species on Selenoamino Acid Levels in Rainbow Trout Simon Godin,*,† Stéphanie Fontagné-Dicharry,§ Maïté Bueno,† Philippe Tacon,# Philip Antony Jesu Prabhu,§ Sachi Kaushik,§ Françoise Médale,§ and Brice Bouyssiere† †

LCABIE, UMR 5254 CNRS, 2 Avenue Pierre Angot, F-64053 Pau Cedex 09, France INRA, UR1067 NuMéA, Route Départementale 918, F-64310 Saint-Pée-sur-Nivelle, France # Lesaffre Feed Additives, 137 Rue Gabriel Péri, F-59700 Marcq-en-Barœul, France §

ABSTRACT: Two forms of selenium (Se) supplementation of fish feeds were compared in two different basal diets. A 12-week feeding trial was performed with rainbow trout fry using either a plant-based or a fish meal-based diet. Se yeast and selenite were used for Se supplementation. Total Se and Se speciation were determined in both diets and whole body of trout fry using inductively coupled plasma mass spectrometry (ICP MS) and high-performance liquid chromatography (HPLC). The two selenoamino acids, selenomethionine (SeMet) and selenocysteine (SeCys), were determined in whole body of fry after enzymatic digestion using protease type XIV with a prior derivatization step in the case of SeCys. The plant-based basal diet was found to have a much lower total Se than the fish meal-based basal diet with concentrations of 496 and 1222 μg(Se) kg−1, respectively. Dietary Se yeast had a higher ability to raise whole body Se compared to selenite. SeMet concentration in the fry was increased only in the case of Se yeast supplementation, whereas SeCys levels were similar at the end of the feeding trial for both Se supplemented forms. The results show that the fate of dietary Se in fry is highly dependent on the form brought through supplementation and that a plant-based diet clearly benefits from Se supplementation. KEYWORDS: selenium speciation, selenomethionine, selenocysteine, supplementation, fish

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INTRODUCTION The recent increase in worldwide fish consumption has been accompanied by a significant rise of the aquaculture contribution. Over the past decade, the input of aquaculture in global fish production has increased from approximately 30 to 50%,1 with a consequent rise in demand for formulated aquaculture feeds. Traditionally, the two major ingredients in aquaculture feeds are fish meal (FM) and fish oil (FO), which are produced from reduction of lower value whole small fish or undersized commercial food-fish species, as well as from processing wastes (this last part representing on average 33% in Europe). In 2012, it was estimated that 14% of global captures were destined for nonfood uses such as FM and FO production.2 Given the growing demand for feeds for farmed fish and the limited availability of capture fisheries, there is a strong emphasis on maximizing the use of locally available feedgrade ingredient sources and moving away from the use of potential food-grade feed resources.3 Thus, the current situation has raised two major issues: first, an economic issue because the price of fish meal has tremendously increased over recent years and, second, an environmental issue due to overfishing risks associated with fish meal production.4,5 To develop a more sustainable aquaculture, there is currently a trend toward the development of low FM−FO feeds for almost all species of farmed fish and shrimp. These feeds are based on plant ingredients and allow therefore a significant decrease in the use of fish meal.6 However, there are concerns regarding micronutrient levels in such feeds based on plant protein sources.7,8 Among these micronutrients, selenium (Se) is of major importance due to its implication in redox status © 2015 American Chemical Society

regulation of cells. In fact, Se is an essential element to all animals; it forms the active center of many selenoenzymes, such as glutathione peroxidase (GPx) or thioredoxin reductase (TRx), which are principally in charge of protection against oxidative damage.9,10 In biological systems, Se can exist under many chemical forms, which were previously described,9 but is usually predominantly found bound to protein in animals.11,12 Selenomethionine (SeMet) and selenocysteine (SeCys) are the two forms that are incorporated into proteins. However, it must be stressed that incorporation of SeMet is said to be nonspecific and can occur in lieu of methionine in all general proteins, whereas the incorporation of SeCys is encoded in the genome by a specific codon and is sometimes even referred to as the 21st amino acid. Therefore, proteins that contain SeCys are called selenoproteins, whereas proteins that have only SeMet residues in their sequence are called selenium-containing proteins.10 Finally, selenium also shows an ambivalent behavior because a toxicity situation can occur if intake reaches too high levels.13 Se supplementation of feeds is often made either with an inorganic form such as sodium selenite (SeIV) or with Se yeast, which is known to contain mostly organic forms of selenium, SeMet being usually the major Se compound. 14 Se supplementation of fish feeds has been already investigated in several studies,15−17 but criteria taken into consideration to Received: Revised: Accepted: Published: 6484

February 9, 2015 April 30, 2015 June 18, 2015 July 10, 2015 DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492

Article

Journal of Agricultural and Food Chemistry evaluate the effects of such supplementation on Se metabolism differ depending on studies. Schram et al.17 determined total Se and Se speciation in muscle, Kücu̧ ̈kbay et al.15 measured total Se in both muscle and serum, as well as GPx activity in serum, and Rider et al.16 quantified total Se in whole body of fish and also determined hepatic GPx and TRx activities. The two latter studies concluded on a beneficial effect of Se supplementation and on the superiority of the organic form of Se supplementation over supplementation with selenite. In the present study, Se form supplementation was compared in two different types of feeds, based on either fish meal and fish oil (base M0) or plant ingredients and vegetable oils (V0). To be relevant to European regulation limiting Se supplementation at +200 μg kg−1 organic Se, supplementation levels were kept rather low (+300 μg kg−1) compared to what is usually found in such studies (maximum supplementation levels were, respectively, +6650 and +6670 μg kg−1 in the studies of Schram et al.17 and Rider et al.16). Also, taking into consideration that Se requirement/utilization might differ depending on the age of trout,18 the study was undertaken with first-feeding swim-up fry of rainbow trout, whereas previously mentioned studies were undertaken with juvenile fish (20 g body mass). Given the uneven distribution of Se in the tissues/organs of fish,19 it was chosen to measure total Se in the whole body. The objective of the study was hence to evaluate, on the basis of speciation analysis results, whether a plant-based diet may induce a different utilization and fate of Se in rainbow trout compared to the traditional FM-based diet. In addition, the potential benefit of a Se supplementation was investigated, together with the suitability of the chemical form used for supplementation.

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Table 1. Main Characteristics of the Diets Used in the Feeding Trial diet

basal ingredient

M0 MSeIV MSeMet

fish meal and fish oil

P0 PSeIV PSeMet

plants

supplementation form (amount) Na2SeO3 (300 μg(Se) kg−1) Selsaf (300 μg(Se) kg−1)

Na2SeO3 (300 μg(Se) kg−1) Selsaf (300 μg(Se) kg−1)

selenite) or an organic form of Se (Se-enriched yeast Selsaf (Lesaffre, Marcq en Baroeul, France), containing 2200 ± 200 μg kg−1 Se with the following composition: SeMet, 63%; SeIV, 98%), seleno-DL-methionine (>99%), and seleno-DLcystine, all purchased from Sigma-Aldrich. Carbamidomethylated selenocysteine (SeCys-CAM) standard was prepared according to the method of Dernovics et al.;20 briefly, selenocystine in TRIS-HCl buffer was reduced using dithiothreitol and carbamidomethylation was carried out by the addition of iodoacetamide; the excess of resulting iodoacetamide was further destroyed using dithiothreitol. Instrumentation. Total Se measurements were carried out on an ICP QMS equipped with a collision/reaction cell (Agilent 7500ce, Agilent Technologies, Wilmington, DE, USA). H2 was used as reaction gas; flow was kept close to 4 mL min−1 and optimized daily to achieve proper background signal.21 Chromatographic separations were performed using an 1100 series HPLC pump (Agilent Technologies) with anion-exchange (PRP-X100, 10 μm, 4.1 × 250 mm; Hamilton Co., Reno, NV, USA) and reversed phase (C8 Altima column, 5 μm 150 × 4.6 mm; Alltech, Deerfield, IL, USA) HPLC columns. HPLCICP QMS analyses were achieved by connecting directly the exit of the HPLC column to the Meinhard nebulizer (Glass Expansion, Romainmotier, Switzerland) of the ICP QMS. The lyophilizer was a model LP3 (Jouan, Saint-Herblain, France). Feeding Trial and Sample Collection. A 12-week feeding trial was carried out using either FM- or plant-based diets supplemented with two different forms of Se (six diets in total, two basal diets, M0 and P0, and four supplemented diets, MSeIV, MSeMet, PSeIV, and PSeMet), as summarized in Table 1. Basically, diets consisted of two groups, one based on marine ingredients (74%) and the other based on plant ingredients (88%). Each group of diets contained one control diet and two diets supplemented with either an inorganic (sodium 6485

DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492

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Journal of Agricultural and Food Chemistry Table 3. Total Selenium Concentrations in Diets and Mass Balance Verification for Sodium Hydroxide and Protease Extractiona sodium hydroxide extraction

protease extraction

diet

total Se (μg kg−1)

total Se in extractb (μg kg−1)

total Se in residuec (μg kg−1)

mass balance (%)

total Se in extractb (μg kg−1)

total Se in residuec (μg kg−1)

mass balance (%)

M0 MSeIV MSeMet

1222 ± 19 1648 ± 36 1703 ± 8

688 ± 9 965 ± 28 875 ± 7

338 ± 9 349 ± 8 438 ± 2

84 ± 2 80 ± 3 77 ± 1

1003 ± 23 1259 ± 16 1318 ± 8

183 ± 10 242 ± 15 159 ± 2

97 ± 3 91 ± 3 87 ± 1

P0 PSeIV PSeMet

496 ± 26 915 ± 12 871 ± 3

352 ± 26 675 ± 7 547 ± 10

57 ± 8 42 ± 1 109 ± 2

82 ± 9 78 ± 2 75 ± 2

360 ± 9 741 ± 27 644 ± 11

65 ± 10 102 ± 8 91 ± 13

86 ± 6 92 ± 4 84 ± 3

Values are the mean ± standard error of the mean, n = 3. bCalculated as (total Se in the extract × mass of extract)/(mass of diet). cCalculated as (total Se in the residue × mass of residue)/(mass of diet).

a

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by external calibration using m/z 78. In the case of fish, internal calibration was used with gallium as internal standard. Inorganic Selenium Species Determination in Diets. A 100mg portion of feed was extracted with 5 mL of a 0.1 M sodium hydroxide solution, and the mixture was mechanically shaken with an elliptic table during 20 h at room temperature. After centrifugation (2600g, 10 min), supernatant was collected and diluted 2 times with HPLC mobile phase. Inorganic selenium species were then separated and detected by HPLC-ICP QMS. A 100-μL aliquot was injected on the anion-exchange chromatography column and eluted with 5 mM ammonium citrate solution (pH 5.3) containing 3% of methanol. Quantification of selenium species was achieved according to the method of standard additions. Selenoamino Acid Determination. SeMet and SeCys determination was done on the basis of the work of Bierla et al.22 For SeMet, 100 mg of sample (feeds or fish) was weighed in a polypropylene tube, and 5 mL of a solution of 0.1 M TRIS-HCl, pH 7.5, containing 10 mg of protease type XIV Streptomyces griseous was added. This mixture was incubated for 20 h at 37 °C and centrifuged (2600g, 10 min). The supernatant was collected and incubated twice with 10 mg of protease. Finally, the supernatant was diluted 2-fold, and 100 μL was injected on the previously mentioned anion-exchange column coupled to ICP QMS. SeCys concentration was determined only in fish samples, and a preliminary derivatization step was carried out using the following procedure. Two milliliters of a 7 M urea solution (in 0.1 M TRIS-HCl, pH 7.5) was added to 100 mg of fish, and the mixture was sonicated for 10 min using an ultrasound probe (VC 505 fitted with a tapered microtip, Sonics & Materials, Newtown, CT, USA) to denature proteins. Then, 30 μL of a dithiothreitol solution at 0.2 M (in 0.1 M TRIS-HCl, pH 7.5) was added, and the mixture was shaken during 1 h. Forty microliters of a 0.5 M iodoacetamide solution (in 0.1 M TRISHCl, pH 7.5) was added and the mixture shaken for an additional hour in the dark. Dithiothreitol (375 μL, 0.2 M) was finally added to destroy iodoacetamide excess. Fourteen milliliters of 0.1 M TRIS-HCl was added to decrease urea concentration, and proteolytic digestion was performed with protease as previously described. Sample was then frozen and freeze-dried, the residue was redissolved in 1.2 mL of MilliQ water, and a 100 μL aliquot was injected on a Superdex Peptide column (300 × 10 mm, Pharmacia, Uppsala, Sweden) eluted with a 10 mM TRIS-HCl, pH 7.5, solution. The low molecular weight fraction was collected (25−35 min) and freeze-dried, and the obtained residue was redissolved and injected on a C8 and eluted with a 0.1% heptafluorobutyric acid solution containing 5% of methanol. For both SeMet and SeCys, m/z 77, 78, 79, and 80 were monitored, and quantification was achieved by using the method of standard additions. Statistics. Statistical analyses were carried out using the software Statbox (Grimmer Logiciels, Paris, France). Total selenium, SeMet, and SeCys concentrations were analyzed using one-way ANOVA and Student−Newman−Keuls post hoc test (p < 0.05) to test the effect of the supplementation form.

RESULTS AND DISCUSSION Optimization of Sample Preparation Conditions. Several extracting solutions were evaluated for their extraction efficiencies and compatibility with Se species preservation. For inorganic Se species in the diets, tested extractants were water, 50 mM TRIS-HCl buffer, pH 7.5,17,23 and 0.1 M sodium hydroxide solution.24 Water and TRIS buffer gave extraction yields of total Se below 20% with a rather large relative standard deviation (36−49%). Sodium hydroxide extraction yields were above 45% with more consistent results (RSD ranged from 6 to 27%). Moreover, the stability of Se species in NaOH extracting solution has been previously demonstrated;25 it was therefore chosen for analysis of inorganic species in diets. Determination of SeMet in diets was optimized with regard to the enzymatic digestion. With a single addition of 10 mg of protease XIV, it was found that SeMet concentration obtained in the extract was stable after 20 h of extraction, and extending digestion time to 48 h did not improve results. Defatting the sample with n-heptane prior to enzymatic digestion did not increase SeMet concentration in the extract. The use of an ultrasound probe26 instead of an elliptic table was unsuccessful in improving protein digestion and did not increase extraction yields. Larger amounts of protease XIV (up to 20 mg) as a single addition were also ineffective; however, a second addition of 10 mg of protease in the supernatant resulting from a first digestion clearly improved SeMet concentration in the final extract. Finally, two further additions of 10 mg of protease were used, resulting in an increase of SeMet concentration in the final extract of around 50% compared to single-step digestion. Se mass balances, determined by measuring total Se in both extract and residue after both extractions, are shown in Table 3. The lower mass balance values obtained in the case of NaOH extraction could be due to the difficulty of collecting residue after extraction because it took in this case the consistency of a very dense slurry. For SeCys determination in fry, higher (2 and 4 times more) and lower (0.5 and 10 times less) concentrations of derivatization reagents were tested. Although the chromatographic peak height of selenocysteine was not affected with 2 and 4 times the conditions described above, several new minor Se-containing peaks were observed with higher reagent concentrations. It appeared that these new peaks were most likely derivatization products of SeMet and made the chromatogram more complex and not well resolved. Lower concentrations of derivatizing reagents did not increase SeCys peak height, which indicates that no overderivatization occurred in the conditions described above. 6486

DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492

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Journal of Agricultural and Food Chemistry Table 4. SeIV, SeVI, and SeMet Concentrations Determined in Dietsa

a

diet

SeIV (μg(Se) kg−1)

M0 MSeIV MSeMet

48 ± 5 105 ± 6 53 ± 6

P0 PSeIV PSeMet

55 ± 10 115 ± 14 55 ± 4

SeVI (μg(Se) kg−1)

SeMet (μg(Se) kg−1)

total calcd (μg(Se) kg−1)

identification (%)

ND ND ND

288 ± 15 290 ± 3 390 ± 8

336 ± 19 395 ± 8 343 ± 13

28 ± 2 24 ± 1 26 ± 1

10 ± 2 11 ± 2 12 ± 1

98 ± 7 95 ± 2 196 ± 6

163 ± 17 221 ± 15 263 ± 9

33 ± 4 24 ± 2 31 ± 1

b

Values are the mean ± standard error of the mean, n = 3. bNot detected.

Total Selenium in Fish Feeds. The comparison of the two basal diets, M0 and P0, shows that total Se concentration was much higher in the FM-based diet (Table 3). This is not surprising considering the fact that Se concentrations in animal tissues are generally higher than in plants;27 thus, these concentrations reflect the Se content of the raw materials used for the production of these feeds. This also highlights the fact that plant-based diets may require a Se supplementation to compete with marine diets regarding total selenium content. In addition, these results confirmed that very similar Se levels were reached for both Se supplementations in each diet group. The Se concentrations found in the marine ingredient-based diets were in the same range as data from the literature.17,28,29 For all feeds used in this experiment, the Se levels were high enough to avoid deficiency situation (3000 μg kg−1 of Se dry feed).30 Selenium Speciation in Fish Feeds. The concentration of SeIV in diet MSeIV was around twice those found in diets M0 and MSeMet (Table 4). Similarly, the concentration of SeIV in diet PSeIV was around 2 times higher compared to diets P0 or PSeMet. In both marine- and plant-based diets, the increase in SeIV concentration resulting from sodium selenite supplementation was close to 60 μg kg−1 (respectively, +57 and +60 μg kg−1 for diets MSeIV and PSeIV). An example of a chromatogram obtained in HPLC-ICP QMS for a sodium hydroxide extract of diet is given in Figure 1a; it can be observed that without protease, the amount of free SeMet is very low. For SeMet concentrations, no differences were observed for diets M0 and MSeIV, whereas the SeMet level of diet MSeMet was increased by 102 μg kg−1. For plant-based diets, a very similar increase (98 μg kg−1) was found in diet PSeMet compared to diets P0 and PSeIV. Figure 1b shows an example chromatogram obtained for a protease extract, where it can be noted that inorganic selenium species are not well extracted in the pH condition of proteolytic digestion. All in all, the results of speciation analysis confirmed the supplementation desired in the experimental design, although no quantitative recovery of initial supplementation could be achieved. This could be explained by a potential nonreversible adsorption of Se on dietary ingredients during the feed preparation process (extrusion), which involves both heating and pressure. Nevertheless, supplementation levels for SeIV and SeMet were approximately the same for both plant and marine diets. SeVI was detected only in plant-based diets, which is in agreement with the fact that the main Se source for plants are selenium species found in soils, which are most often inorganic species such as selenite and selenate,31 and the previously observed low metabolization of SeVI in plants.32 The three plant-based diets also present the same SeVI level, which indicates that no species conversion of Se used for

Figure 1. Example anion-exchange chromatograms (HPLC-ICP QMS) for diet (PSeMet) extracts: (a) sodium hydroxide extract; (b) protease extract. UK1 and UK2, unknown Se-containing peaks.

supplementation occurred during diet preparation. Although SeMet concentration found in the basal marine diet was around 3 times higher than in the basal plant diet, it should be noted that the SeMet/total Se ratios, 24 and 20%, respectively, for M0 and P0, were relatively close for these two kinds of feeds. These percentages appear to be rather low compared to values that can be found in the literature for the individual constituents of these diets; it is likely that the feed preparation process mentioned above (extrusion) may have induced transformations in the constituents of diets and thus made the proteolytic digestion not as efficient as the one observed for these constituents when analyzed separately. This is furthermore supported by several observations of the low digestibility of Se from fish meal.33,34 The percentage of Se identification in diets 6487

DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492

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Journal of Agricultural and Food Chemistry

form,16 which suggests that Se supplementation is more effective with plant-based diets compared to diets based on fish meal. It can be assumed that this comes from the different speciation of selenium that can be found in diets depending on the basal ingredients; however, due to the low percentage of identification of selenium obtained in diets in this work, this cannot be firmly stated at this point. As previously observed,17 a good correlation (R2 = 0.97) was found between total Se in diets and total Se in the whole body of fry (Figure 2). The

ranged from 28 to 33%, these results are similar to those previously reported by Schram et al.17 This low identification percentage was obtained despite relatively good extraction yields obtained for sodium hydroxide and protease extraction (Table 5). Identification in sodium hydroxide extracts was very Table 5. Extraction Yields and Percentage of Identification in Extracts for Diet Speciation Analysisa

a

diet

NaOH extraction yield (%)

identification in NaOH extract (%)

protease extraction yield (%)

identification in protease extract (%)

M0 MSeIV MSeMet

56 ± 2 58 ± 2 51 ± 1

7±1 11 ± 1 6±1

82 ± 3 76 ± 2 77 ± 1

29 ± 2 23 ± 1 30 ± 1

P0 PSeIV PSeMet

71 ± 7 74 ± 2 63 ± 2

18 ± 4 19 ± 2 12 ± 1

73 ± 5 81 ± 3 74 ± 2

27 ± 2 13 ± 1 30 ± 1

Values are the mean ± standard error of the mean, n = 3.

low, especially in FM-based diets, which is very likely due to Se being mainly present in proteins in these extracts and therefore not available for detection in this form. In the case of protease extracts, identification was close to 30% except for diet PSeIV, for which identification was 15%; this is relevant with SeIV being the major species found in this diet and the fact that inorganic species were not measured in these extracts because measured concentrations were found to be not repeatable under these pH conditions. A low column recovery was finally found to be responsible for the quite low percentage of identification in protease extracts. Total Se in Whole Body of Fry. Total Se was determined in initial fry at the beginning of the feeding trial and was found to be 227 ± 2 μg kg−1. Total Se levels in fry at the end of the trial are shown in Table 6; whole body Se was significantly (p < 0.05) increased for all groups fed the Se-supplemented diets. Increases of, respectively, +39 and +64 μg kg−1 were found for diets MSeIV and MSeMet compared to the basal diet M0. For the plant diets, increases of, respectively, +61 and +86 μg kg−1 were found for diets PSeIV and PSeMet compared to the basal diet P0. These results indicate that supplementation with Se yeast has a higher capacity to raise the whole body Se level in comparison to sodium selenite. This is in agreement with previously reported data by Rider et al. in a similar feeding trial using juvenile trouts and higher supplementation levels.16 In addition, the increases observed were higher in the case of plant-based diets, and it was shown that dose response was almost linear until 2000 μg kg−1 regardless of supplementation

Figure 2. Linear regression between total Se in diets and total Se in whole body of fry: (◆) plant-based diets; (×) fish meal-based diets. Horizontal and vertical error bars are standard error of the mean.

observation that whole body total Se was lower than initial state for fish fed diets P0 and PSeIV does not necessarily imply that these diets were Se deficient but emphasizes the necessity to supplement plant-based diets. Whole Body Selenomethionine and Selenocysteine Concentration in Fry. A typical chromatogram obtained in HPLC-ICP QMS for a proteolytic digest of fry is given in Figure 3. SeMet concentrations were statistically not changed with selenite supplementation of the basal diet for both marine and plant diets (Table 6), which is in agreement with the suggested metabolic pathway of Se in animals in which SeMet cannot be synthesized from selenite.35 On the other hand, the two diets supplemented with Se yeast led to increased SeMet concentrations in fry, although this increase was significant only in the case of diet PSeMet (p < 0.05). There was a good correlation between SeMet concentrations in diets and SeMet levels in whole body of fry (Figure 4); this concurs with the conclusion of Whanger et al. on the biological significance of SeMet,36 this selenoamino acid being mostly intact after ingestion, especially in the case of diets containing relatively

Table 6. Total Se, SeMet, and SeCys Concentrations, Ratio of SeMet and SeCys over Total Se, and Percentage of Identification in Whole Body Fry at the End of the Feeding Triala diet

total Se (μg(Se) kg−1, fw) α

SeMet (μg(Se) kg−1, fw)

M0 MSeIV MSeMet

277 ± 3 316 β ± 3 341 γ ± 4

123 127 149

P0 PSeIV PSeMet

138 δ ± 1 199 ε ± 2 224 η ± 6

78 75 111

α α α

β β α

SeCys (μg(Se) kg−1, fw)

± 13 ± 10 ± 16

SeMet/total Se αβ

SeCys/total Se αβ

identificationb

±1 ±8 ±3

44 ± 8 40 β ± 6 44 αβ ± 8

26 ± 1 22 αβ ± 4 20 β ± 1

71 ± 9 62 ± 10 64 ± 9

47 β ± 4 59 αβ ± 4 59 αβ ± 4

57 αβ ± 5 38 β ± 3 50 αβ ± 4

34 γ ± 5 30 αγ ± 4 26 αβ ± 3

91 ± 10 67 ± 6 76 ± 7

73 70 69

±4 ±3 ±3

α α α

Values are the mean ± standard error of the mean, n = 3. Within columns, values with unlike Greek letters (α, β, γ, δ, ε, η) are significantly different (one-way ANOVA and Student−Newman−Keuls post hoc test, p < 0.05). bNot statistically processed. a

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DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492

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Journal of Agricultural and Food Chemistry

Figure 3. Typical anion-exchange chromatogram (HPLC-ICP QMS) for selenomethionine analysis in protease extract of fry. UK1 and UK2, unknowns.

Figure 5. Typical reversed-phase chromatogram (HPLC-ICP QMS) for selenocysteine analysis in protease extract of fry after derivatization. SeCysCAM, carbamidomethyl-selenocysteine; UK, unknown.

SeMet/total Se ratio between fry fed diets P0 and PSeMet, suggesting that the addition of Se yeast to P0 did not change the proportion of Se in the diet available for incorporation as SeMet in fry proteins. For fry fed FM-based diets, SeMet/total Se ratio was not significantly different for any diets, which could be due to the fact that the proportion of added Se through supplementation in these diets was much lower compared to plant diets (around +40% in FM-based diets versus +80% in plant-based diets), minimizing the effect of the supplementation. The SeCys/total Se ratio was found to be significantly (p < 0.05) higher in fry fed diet P0 compared with the ratio for fry fed diet M0, which indicates that Se from diet P0 is mainly transformed into selenoproteins and that the storage of Se in other forms is limited for this diet compared to other dietary treatments. Also, although no difference was found for this ratio between fry fed diets P0 and PSeIV, fry fed diet P0 were found to have a significantly (p < 0.05) higher SeCys/total Se ratio than fry fed the PSeMet diet; this seems to indicate that the major fate for added Se yeast was not SeCys synthesis. Nevertheless, no significant difference was found for SeCys/ total Se ratio between fry fed diets PSeIV and PSeMet or between fry fed diets MSeIV and MSeMet, whereas all supplemented diets, either FM- or plant-based, increased total Se levels in fry. This confirms the similar abilities of supplemented Se yeast and selenite to be metabolized into SeCys. Overall, these results indicate that the superiority of Se yeast in increasing Se levels of fry seems to lie in its ability to increase more efficiently SeMet levels. However, according to SeCys determination performed in this study, supplementation with selenite appears to be able to compete with Se yeast when it comes to raising selenoprotein expression. To our knowledge, such an observation has not been made in fish until now, but these results are in agreement with observations made in humans in a study performed by Thomson et al.,38 where selenate and Se yeast supplementations of 32 New Zealand women were compared. This work demonstrated that higher total Se values in blood were obtained with Se yeast supplementation, but very close glutathione peroxidase activities were achieved with both types of supplementations. This is also in agreement with the fact that SeCys is considered to be the major selenium species formed after selenite

Figure 4. Linear regression between selenomethionine concentrations in diets and whole body of fry fry: (◆) plant-based diets; (×) fish meal-based diets. Horizontal and vertical error bars are standard error of the mean.

low methionine levels such as those based on plant ingredients.37 With regard to SeCys, the three marine diets lead to the same SeCys concentration at the end of trial, suggesting that Se requirements for selenoprotein expression were already fulfilled with the basal diet M0. Conversely, plant diet P0 led to SeCys concentration in fry significantly (p < 0.05) lower than in the case of fry fed the FM-based diets. Both selenite and Se yeast supplementations of the basal diet P0 restored SeCys concentrations to levels similar to those obtained with the marine diets. Furthermore, there was no significant difference between SeCys levels reached for PSeIV and PSeMet diets. An example chromatogram obtained in reversed phase chromatography for a proteolytic extract of fry after SeCys derivatization is shown in Figure 5. The ratios of SeMet and SeCys concentrations over total Se one were also calculated in fry to roughly estimate the selenium utilization of diets from the speciation point of view. Fry fed diet P0 showed ratios of SeMet/total Se significantly (p < 0.05) higher than those fed diet PSeIV, indicating that the major fate of the added selenite in PSeIV was not SeMet production. This is consistent with the inability of selenite to be metabolized in SeMet. Conversely, no significant difference was found for 6489

DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492

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Journal of Agricultural and Food Chemistry supplementation.36 The percentage of identification of Se species was higher in fry fed plant-based diets, possibly due to a more complex selenium mixture present in the diets based on fish meal as a consequence of the more developed Se metabolism in fish in comparison to plants. Also, Se identification percentages in whole body were lower in fry fed Se-supplemented diets compared to fry fed basal diets. This may indicate that the part of Se brought through supplementation was transformed to selenium species other than SeMet or SeCys. Another explanation could be that degradation products of these two selenoamino acid were not determined in this study, and a buildup of these species such as presumably selenosugars35,39 would have led to a drop in identification of Se in whole body of fry. To our knowledge, such aspects of Se supplementation of fish were until now rather overlooked, principally due to the fact that conclusions of studies were mainly based on total Se observations. Moreover, Se utilization is often assessed through measurements of GPx or TRx activities.40 Fish have one of the largest selenoproteomes known, with 30−37 selenoproteins versus 25 in humans, and several selenoproteins are specific to fish.41 Besides, SeCys residues are more numerous in fish selenoproteins in comparison with those known in mammals; for instance, selenoprotein P (selP), which is principally believed to act as SeCys transporter,42 contains 16−17 SeCys residues in fish, whereas it ranges from 7 to 15 in mammals.41 Thus, SeCys determination may allow for full coverage of the selenoproteome assessment providing complementary information together with the measurement of activities of selenoenzymes. On the other hand, selenoproteins cannot be differentiated through SeCys determination, which can be troublesome when Se utilization is evaluated, taking into consideration that some selenoproteins are more essential than others and that selenium intake can affect selenoprotein expression pattern.43 Also, information obtained through SeMet determination should not be disregarded because the transformation of SeMet into SeCys was found to be rather limited in rats; Waschulewski et al.37 showed that after ingestion of SeMet incorporated in a low methionine containing diet during 21 days, GPx activity dropped close to 50% compared to the initial state. Switching to a Se-deficient diet, GPx activity was increased for a short time and then kept dropping while total Se in tissue also decreased; this short time increase was significantly affected by methionine content of the diet. Therefore, the availability of tissue SeMet for GPx synthesis seems to be related to protein turnover and was found to be quite low. SeMet determination could consequently allow determination of the part of incorporated selenium that is not directly available to the considered organism. Hence, due to the particular metabolism of selenium, attention should be paid to the utilization of this essential element by organisms when the forms of supplementation are compared. In this respect, selenium speciation analysis can provide useful information that cannot be retrieved from total Se analysis. To conclude, the results obtained in this work showed the usefulness of performing selenium speciation in nutrition studies and the gain in terms of information on metabolic utilization of Se that can be collected. It was shown that the form of Se supplementation can lead to differences in incorporation of Se in both qualitative and quantitative terms. In agreement with previously published data, Se yeast and therefore organic Se showed an undisputably superior ability to increase Se levels in rainbow trout fry compared to inorganic

Se. Speciation analyses showed that this is a consequence of the inability of selenite to be incorporated into general proteins under SeMet form and is a clear advantage of Se yeast when the aim is to produce Se-enriched food. On the other hand, the essentiality of Se as a micronutrient is related to its incorporation into selenoproteins, and results obtained here showed that inorganic Se has the ability to fortify this selenoprotein synthesis as organic Se does when supplied in similar amounts. Thus, the determination of SeMet and SeCys provides further knowledge in addition to monitoring selenoenzymes. Although the results in this study did not clearly indicate whether the nonsupplemented plant-based diet led to a Se deficiency situation in fish or not, selenoprotein levels were much lower in this diet compared to the one obtained with the traditional nonsupplemented fish meal-based one, and the increase in SeCys level induced by supplementation appears as a clear benefit for the plant-based diet.

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AUTHOR INFORMATION

Corresponding Author

*(S.G.) Phone: + 33 559407762. Fax: + 33 559407674. E-mail: [email protected]. Funding

This work was supported by a grant from the French Ministry of Higher Education and Research. Financial support was also p r o v i d e d b y t h e C o n s e i l R é g i o n a l d ’ A q u i t a i n e (20071303002PFM). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS F. Terrier, F. Sandres, and Y. Hontang are acknowledged for the preparation of diets and care of fish.

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ABBREVIATIONS USED FM, fish meal; FO, fish oil; Se, selenium; GPx, glutathione peroxidase; TRx, thioredoxin reductase; SeMet, selenomethionine; SeCys, selenocysteine; SeIV, selenite; SeCys-CAM, carbamidomethylated sele no cysteine; TRIS, tr is(hydroxymethyl)aminomethane; ICP QMS, inductively coupled plasma quadrupole mass spectrometry; HPLC, highperformance liquid chromatography; M0, marine ingredients basal diet; P0, plant ingredients basal diet; MSeIV, M0 supplemented with 300 μg kg−1 of sodium selenite; MSeMet, M0 supplemented with 300 μg kg−1 of Se as Se yeast; PSeIV, P0 supplemented with 300 μg kg−1 of sodium selenite; PSeMet, M0 supplemented with 300 μg kg−1 of Se as Se yeast; selP, selenoprotein P

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REFERENCES

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DOI: 10.1021/acs.jafc.5b00768 J. Agric. Food Chem. 2015, 63, 6484−6492