Nutritional supplementation and enhanced antioxidant function by dietary intake of selenoneine and other selenium compounds in red seabream Pagrus major

doi:10.21203/rs.3.rs-2119915/v1

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Nutritional supplementation and enhanced antioxidant function by dietary intake of selenoneine and other selenium compounds in red seabream Pagrus major

https://doi.org/10.21203/rs.3.rs-2119915/v1

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published 18 Jul, 2023

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Selenoneine, 2-selenyl-N_α, N_α, N_α-trimethyl-_L-histidine, is the major organic selenium compound in marine fish. To characterize biological antioxidant function of selenoneine in fish, the accumulation of selenoneine and other selenium compounds, i. e., sodium selenite and selenomethionine, in the muscle and other tissues of red seabream. We reared red seabream by feeding of 1% dry pellet containing of sodium selenite, selenomethionine, or selenoneine of body weight twice a day for 4 weeks. After that, we replaced to 1% of normal commercial dry pellet of body weight twice a day for 1 week from the selenium supplementation, and tissue distribution of total selenium was determined. Selenium supplementation with selenoneine, selenomethionine, and sodium selenite enhanced selenium accumulation in the white muscle, kidney, and hepatopancreas in comparison with the control group. By the dietary intake of selenoneine, total selenium concentrations were increased in the white muscle, heart, kidney, spleen, hepatopancreas, brain, and blood in a dose-dependent manner during the trials after 2 weeks. Dietary intake of selenoneine as well as sodium selenite and selenomethionine reduced ORP.

Selenium

Selenoneine

Intake

Antioxidant

Fish

Selenium is an essential micronutrient for animals (Combs and Combs 1986; Himeno and Imura 2000; Imura and Naganuma 1991). Selenium has several physiological functions as selenocysteine for the redox enzymes, such as, glutathione peroxidase (Brigelius-Flohé 1999; Rotruck et al. 1973), thioredoxin reductase (Mustacich and Powis 2000), thyroid hormone deiodinases (Arthur et al. 1993), and selenoprotein P (Burk and Hill 1994). The antioxidant function of selenium plays protective roles in 50 human disease, including prostate, lung, and intestine/colon cancers, immunodeficiency and heart diseases (Ghose et al. 2001; Kiremidjian-Schumacher and Roy 2001; Salonen et al. 1982). Selenium deficiency caused myopathy, exudative diathesis, and pancreatic degeneration to animals and birds (Combs 1986). Muscle dystrophy by selenium deficiency was also reported in carp and rainbow trout (Bell et al. 1984; Combs 1986; Gatlin and Wilson 1984; Poston et al. 1976). “Burnt meat” is a problem in aquaculture and fisheries of pelagic ocean fish, such as tuna, yellowtail, and mackerel in Japan (Yamashita, 2014; Yamashita & Yamashita, 2015). The meat appears “cooked” although raw, and the flesh lacks the typical bright red color of meat. The burnt meat has a more watery, softer texture, and is often seen in fish under stress conditions, for example those caught during the summer spawning period when the water temperature is high. The oxidative stress is caused by selenium deficiency and hypoxia. After catching, extensive apoptosis and autophagy occur in the white muscle, and hemolysis occurs in fish that contain blood selenium levels of less than 1 µg/g tissue. The antioxidant effects of selenoneine may be essential in enabling fish to adapt and survive in low-oxygen marine environments. In rats, selenium deficiency was shown to induce hemolysis (Rotruck et al. 1972). White muscle disease in selenium-deficient animals shows similar features (Combs 1986). Selenium levels were closely related to the DPPH radical scavenging activity against red blood cells in the blood of bluefin tuna (Yamashita, 2014). Therefore, selenium supplementation in cultured fish might reduce the burnt meat problem in aquaculture, and the nutritional selenium requirement of ocean fish species should be evaluated.

Selenoneine, 2-selenyl-N_α, N_α, N_α-trimethyl-_L-histidine, is a novel selenium containing compound found in the blood and other tissues of fish (Yamashita and Yamashita 2010; Yamashita et al. 2010; Yamashita et al. 2011; Yamashita et al. 2013a) and is a major organic selenium compound in fish as well as selenoproteins (Yamashita et al. 2013a). Selenoneine is taken up into cell by the specific organic cation/carnitine transporter-1 (OCTN1) (Yamashita et al. 2010) and binds to the heme of hemoglobin and myoglobin to prevent autooxidation of iron ions (Yamashita and Yamashita 2010). For industrial application of selenium, selenoneine is produced from fisheries waste and be used for functional foods and feed. Although the antioxidant function of selenoneine is expected, there are no studies on oral administration to fish.

Seafood is a major selenium source in Japan. Red seabream Pagrus major is one of the most popular marine fish and its farming is active in Japan (Imai, 2005). Although the color of the body surface is important for red seabream, cultured red seabream may turn black by oxidative stress such as sunlight. Selenoneine suppresses melanine synthesis by inhibiting tyrosinase (Seko et al. 2019). Hence, selenoneine may improve blackening of cultured red seabream.

In this study, we compared the accumulation and antioxidant function of selenoneine and other selenium compounds, i. e., sodium selenite and selenomethionine, in the muscle and other tissues of red seabream.

Materials

Sodium selenite was purchased from Wako Pure Chemical Industries (Osaka, Japan). Seleno-L-methionine was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Selenoneine was purified from internal organs of skipjack tuna and blood of tuna at the National Fisheries University according to methods described previously (Yamashita and Yamashita 2010).

Preparation of Feed The feed was purchased from Hayashikane Sangyo (Yamaguchi, Japan). Sodium selenite, selenomethionine, or selenoneine solution were sprayed on feed and then dried in vacuum. The final selenium concentration of each feed is shown in Table S1.

Fish

Red seabream fry was obtained from private seed and seedling production company (Oita, Japan) and cultured for seven months up to average body weight of 250 g. We reared red seabream by feeding of 1% dry pellet containing of sodium selenite, selenomethionine, or selenoneine of body weight twice a day for 4 weeks. After that, we replaced to 1% of normal commercial dry pellet of body weight twice a day for 1 week from the selenium supplementation. Average water temperature was 20.9 ℃ (19.0-23.9 ℃) and average dissolved oxygen was 7.41 mg/L. The body weight change of red seabream for 5 weeks is shown in Table S2.

Selenium Concentration Measurement

Total selenium concentration was determined by fluorometric assay with 2,3-diamino-naphthalene (DAN), after digestion in 1.5 mL of nitric acid and perchloric acid (1:2 in volume) at 200–220 ℃ (Watkinson 1966; Yamashita and Yamashita 2010).

Selenoneine Concentration Measurement

Selenoneine was separated by chromatography using a Shodex GF-310 column (4.5 mm×150 mm; Showa Denko, Tokyo, Japan) equilibrated with 100 mM ammonium formate buffer containing 0.1% (w/v) Igepal CA-630 (MP Biomedicals, CA, USA). The injection volume was at 10 µL. The mobile phase delivered at 0.5 mL/min isocratically. ⁸²Se was detected with high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS; Agilent 1100 Series and Agilent 7500 Series, Agilent Technology) (Yamashita and Yamashita 2010). During separation, selenoneine was eluted at a retention time of 200 second, and the selenium concentration was determined using selenoneine as a standard.

ORP Measurement

ORP was measured with ORP electrode (9300, Horiba, Kyoto, Tokyo). The electrode was pierced into muscle and pressed gently until the potential stabilized for 60 seconds.

Glutathione peroxidase (GPx) activity

GPx activity was examined by monitoring the oxidation of NADPH in the presence of GSH reductase, which catalyzes the reduction of oxidized GSH formed by GPx (Rotruck et al. 1973; Yamashita et al. 2012). The activity measured at 37 ℃ in a solution containing 50 mM sodium phosphate buffer (ph 7.0), 2.5 mM EDTA, 1 mM sodium azide, 0.5 mM reduced glutathione, 0.005 U/mol glutathione reductase, 0.4 mM hydrogen peroxide, 0.15 mM NADPH. Solution fluorescence was measured with a fluorometer at 460 nm with an excitation wavelength of 355 nm. One enzyme unit of GPx activity is defined as 1 µmol of NADPH oxidized per minute at 37 ℃.

Uptake factor

Uptake factor is defined as a ratio of selenoneine concentration in muscle or blood to those in feed, and is the same as the biomagnification factor (Yamada et al. 1994). Thus, uptake factor is an indicator showing the degree of accumulation of selenoneine in muscle or blood by dietary intake of feed. uptake factor were calculated by the following equation on the equilibrium period of the dietary exposure experiments:

uptake factor = ((Cen - Cb) / CF) × 10^− 3

where Cen is selenoneine concentration in muscle or blood of fish fed with selenoneine-contained feed (ng/kg), Cb is selenoneine concentration in muscle or blood of the control fish (ng/kg wet), and CF is the selenoneine concentration in the contained feed (µg/kg).

Statistical Analysis

The results are expressed as means ± standard error. Data were analyzed by Dunnett’s multiple comparison test to identify any significant differences (P < 0.05).

Total Selenium Concentration

As shown in Table S1, we used commercially available dry pellet as the control diet, and dry pellets containing selenium compounds, such as sodium selenite, selenomethionine, and selenoneine at 1 mg Se/kg or 2 mg Se/kg for feeding experiments.

We measured total selenium concentration in the tissues, such as, white muscle, heart, kidney, spleen, hepatopancreas, brain, and blood cell (Table S3) for each feeding group. In the group of the sodium selenite at 1 mg Se/kg and 2 mg Se/kg, total selenium was accumulated in white muscle, kidney, and hepatopancreas in compared with the control group. In the group of the selenomethionine at 1 mg Se/kg and 2 mg Se/kg, total selenium was accumulated in white muscle and heart. In the group of the selenoneine at 1 mg Se/kg, total selenium was accumulated in white muscle, kidney, spleen, hepatopancreas, brain, and blood cell. In the group of the selenoneine at 2 mg Se/kg, total selenium was accumulated in white muscle, heart, kidney, spleen, hepatopancreas, brain, and blood cell.

Selenoneine Concentrations

Selenoneine concentrations were determined in the white muscle and blood cell by monitoring ⁸²Se levels by HPLC-ICP-MS (Table 1). In the control group, selenoneine levels were 0.018 ± 0.001 mg Se/kg in the white muscle and 2.38 ± 0.71 mg Se/kg in the blood cells. In the fish fed with selenoneine at 1 mg Se/kg and 2 mg Se/kg, selenoneine was accumulated in both white muscle and blood cell. Selenonine levels were 4.72 ± 1.06 mg Se/kg in the blood cells and 0.025 ± 0.003 mg Se/kg in the white muscle of fish fed with 1 mg Se/kg, and 10.29 ± 3.14 mg Se/kg in the blood cells and 0.030 ± 0.007 mg Se/kg of fish fed with 2 mg Se/kg. These selenoneine concentrations accounted for more than 63% of total selenium in blood cell and for 4.6-7.0% of selenium in white muscle. In contrast, the fish fed with other selenium compounds, sodium selenite and selenomethionine, contained small amount of selenoneine similar to the control fish.

Table 1

Selenoneine concentration in white muscle and blood cell in the fish cultured for 4 weeks.
	Selenoneine concentration (mg Se/kg)
tissue	control	sodium selenite			selenomethionine		selenoneine
	control	1 mg Se/kg		2 mg Se/kg	1 mg Se/kg	2 mg Se/kg	1 mg Se/kg	2 mg Se/kg
white muscle	0.018 ± 0.001		0.019 ± 0.002	0.020 ± 0.003	0.019 ± 0.003	0.019 ± 0.003	0.025 ± 0.003^*	0.030 ± 0.007^*
blood cell	2.38 ± 0.71		1.98 ± 0.39	1.91 ± 0.43	1.64 ± 0.34	1.78 ± 0.34	4.72 ± 1.06^*	10.29 ± 3.14^*
*Asterisks indicate statistical significance in compared with the control group (p < 0.05, ANOVA).

We evaluated the uptake factor and the half life from the concentrations of selenoneine in the feed, white muscle and blood of the fish cultured for 4 weeks and after replaced from selenoneine-feed to control feed for additional one week. The uptake factor of selenoneine from the artificial feed containing selenoneine at 0.1-2 ppm Se was calculated to be 0.0062 in the white muscle and 4.0 in the blood, indicating that tue uptake in the blood was higher than that in white muscle. In addition, the half life of selenoneine from the blood and white muscle were estimated to be 60 days in the white muscle and 30 days in the blood (Table 2).

Table 2

Selenoneine concentration in white muscle and feed, and uptake factor in selenoneine 1 ppm and 2 ppm administration group
	control	selenoneine 1 mg Se/kg	selenoneine 2 mg Se/kg
Total Se concentration in feed (mg Se/kg)	1.9	3.0	4.1
Selenoneine concentration in feed (mg Se/kg)	0.1	1.1	2.2
Selenoneine concentration in white muscle (µg Se/kg)	17.7 ± 1.1	25.4 ± 3.2	30.0 ± 6.6
uptake factor		7.2 ± 2.7	5.6 ± 2.7

Orp

ORP in muscle was analyzed to evaluate the redox status in fish (Fig. 1). All of selenium administration groups decrease ORP at 2 weeks compared to control, but not 4 weeks and 5 weeks. Correlation coefficient between total selenium content and ORP in white muscle was − 0.46 (Fig. 2), and the t-test showed a significant (P = 0.032).

GPx activity

GPx activity in hepatopancreas of the fish at 4 weeks were compared with the activities of the selenium supplemented groups (Fig. 3). There was no apparent difference in the activities among the different groups.

A feeding experiment of red seabream with feeds containing of sodium selenite, selenomethionine, or selenoneine was carried out for 4 weeks. After selenium supplementation, we replaced to normal commercial dry pellet for 1 week from the selenium supplementation, and tissue distribution of total selenium was determined. Selenium supplementation with selenoneine, selenomethionine, and sodium selenite enhanced selenium accumulation in the white muscle, kidney, and hepatopancreas in comparison with the control group. By the dietary intake of selenoneine, total selenium concentrations were increased in the white muscle, heart, kidney, spleen, hepatopancreas, brain, and blood in a dose-dependent manner during the trials after 2 weeks. Dietary intake of selenoneine as well as sodium selenite and selenomethionine reduced ORP.

Levels of selenium were determined in white muscle, heart, kidney, spleen, hepatopancreas, brain, and blood cell at 2 weeks, 4 weeks, and 5weeks. Levels of selenoneine were determined in white muscle and blood cell at 2weeks. Dietary intake of sodium selenite or selenomethionine resulted in accumulation selenium in some tissue while dietary intake of selenoneine resulted in accumulation selenium in all tissue we measured in this study. Especially, high levels of selenoneine accumulated in blood cell. It is suggested that selenium accumulation mechanism differs for each selenium compound and selenoneine accumulates in each tissue through blood cell.

Selenoneine has antioxidant function (Yamashita and Yamashita 2010) and methylmercury (MeHg) detoxification (Yamashita et.al. 2013b). In a zebrafish toxicity assay, selenoneine demethylated MeHg and then the demethylated MeHg is taken up into secretory extracellur lysosomal vesicles and discharged through OCTN1 (Yamashita et.al. 2013b). Yamashita et.al. also reported red blood cells express the OCTN1 and accumulated selenoneine (Yamashita et.al. 2013a). Therefore, the reason why selenium accumulates in blood cells at a high level is that it exerts an antioxidant and mercury detoxifying functions on blood cell which have a high risk of oxidation and contain a large amount of methylmercury.

The ratio of selenium used in selenoneine in white muscle of wild red seabream was about 10% (Yamashita et.al. 2011). In this study, that of the control and selenoneine 2 ppm administration groups were about 5% and about 7.0%, respectively. This result indicates that dietary intake of selenoneine increase the ratio of selenoneine and enhance nutritional status.

Selenoneine administration groups reduced ORP as well as sodium selenite and selenomethionine at 2 weeks. ORP in the control group at 4 and 5 weeks decreased, but it may be due to selenium originally in the commercial dry pellet. Our unpublished research showed that dietary intake of selenoneine in yellowtail reduced ORP and approached the ORP of wild fish. Measurement of ORP can be used for evaluation of antioxidant activity in the tissues by dietary intake of selenium in vivo.

In conclusion, we showed that selenoneine accumulated in the body and enhanced antioxidant function by dietary intake of selenoneine. To utilize selenoneine in food and aquaculture, we should demonstrate biological function in vivo and required amount of selenoneine for each fish species.

ORP, oxidation-reduction potential; HPLC-ICP-MS, high performance liquid chromatography inductively coupled plasma mass spectrometry; GPx, glutathione peroxidase; GPC, gel permeation chromatography; OCTN1, organic cations/carnitine transporter-1; Se, selenium; MeHg, methylmercury

Conflicts of interest/Competing interests

The authors have no relevant financial or non-financial interests to disclose. The authors have no conflicts of interest to declare that are relevant to the content of this article.

Funding declaration

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability statement

All data generated or analysed during this study are included in this published article.

Author contributions statement

Y. Shimokawa, K. Abe, M. Ohura, M. Yamamoto, H. Ando, and T. Tohfuku prepared fish feed, reared fish, and assayed selenium and other chemical components. M. Kondo supervised rearing the fish. Y. Shimokawa and M. Yamashita wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.

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No competing interests reported.

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Nutritional supplementation and enhanced antioxidant function by dietary intake of selenoneine and other selenium compounds in red seabream Pagrus major

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Abstract

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Orp

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