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Fish Sci (2011) 77:679–686 DOI 10.1007/s12562-011-0360-9

ORIGINAL ARTICLE

Food Science and Technology

Selenoneine, total selenium, and total mercury content in the muscle of fishes Yumiko Yamashita • Heidi Amlund • Tamami Suzuki • Tatsuro Hara • Mohammed Anwar Hossain • Takeshi Yabu Ken Touhata • Michiaki Yamashita



Received: 26 November 2010 / Accepted: 14 March 2011 / Published online: 3 June 2011 Ó The Japanese Society of Fisheries Science 2011

Abstract Levels of the selenium-containing imidazole compound selenoneine and overall organic selenium were measured in the muscle of fishes by speciation analysis. The method involves monitoring 82Se levels by liquid chromatography inductively coupled plasma mass spectroscopy using a gel filtration column. Selenoneine levels were found to be highest in swordfish muscle (concentration 2.8 nmol/g tissue). The selenoneine contents of bigeye tuna, Pacific bluefin tuna, albacore, yellowfin tuna, and alfonsino muscle were 1.3–2.6 nmol/g tissue. In muscle of these fishes, most organic selenium (9–42%) was present as selenoneine. In other fish species, such as Pacific sardine, greeneye, skipjack, Pacific mackerel, horse mackerel, red sea bream, and Japanese barracuda, selenoneine levels were 0.1–1.4 nmol/g tissue, accounting for 3–34% of organic selenium. In contrast, muscle of Japanese conger, Japanese anchovy, chum salmon, Pacific saury, white croaker, and marbled sole contained levels of selenoneine below the level of detection (\0.05 nmol/g tissue). Mercury and selenium contents were 0.01–5.12 nmol/g tissue and 1.4–19.1 nmol/g tissue. The Se-to-Hg molar ratio varied from species to species, ranging from 1 for swordfish to 217 for marbled sole.

Y. Yamashita (&)  T. Suzuki  T. Hara  M. A. Hossain  T. Yabu  K. Touhata  M. Yamashita National Research Institute of Fisheries Science, Kanazawa, Yokohama, Kanagawa 236-8648, Japan e-mail: [email protected] H. Amlund National Institute of Nutrition and Seafood Research (NIFES), P.O. Box 2029, Nordnes, 5817 Bergen, Norway

Keywords Selenoneine  Selenium  Mercury  Food safety  Muscle  Fish  Seafood

Introduction A novel selenium-containing compound, 2-selenyl-Na,Na, Na-trimethyl-L-histidine (selenoneine), was identified as the predominant chemical form of organic selenium in blood and other tissues of bluefin tuna [1, 2]. This selenium compound contains an imidazole ring with a unique selenoketone group and shows strong free-radical scavenging activity [1, 2]. Therefore, dietary intake of selenoneine in fish may enhance free-radical detoxification functions. Indeed, dietary intake of selenoneine through fish consumption is thought to be important for enhancing antioxidant effects in tissues and cells. Dietary selenium intake is postulated to protect against mercury toxicity [3–11]. Increasing blood Hg-to-Se ratios are indicative of increasing risk or harm. Thus, assessment of methylmercury (MeHg) exposure should involve evaluation of blood Hg-to-Se ratios, rather than mercury levels alone. Supplementation with nutritionally relevant amounts of selenium counteracts MeHg toxicity [3–11]. An accurate index of the risk of fish consumption is thought to be best provided by measuring not only MeHg exposure [7] but also levels of selenium and other factors that benefit health and reduce MeHg toxicity. Thus, selenoneine in seafood may represent the most significant dietary source of antioxidant-active selenium. In a previous report, levels of selenoneine in the blood and other tissues of bluefin tuna were found to be high. In this study we compared the selenoneine, total selenium, and total mercury contents of muscle of various fish species commonly consumed in Japan.

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Fig. 1 Speciation analysis of organic selenium in the muscle of fishes by LC–ICP–MS. Water-soluble selenium compounds in fish muscle were speciated though LC–ICP–MS analysis. Sample (0.1 g) was homogenized in 5 vol. water, and 20 ll supernatant was analyzed by LC–ICP–MS after two- to fourfold dilution in mobile phase (0.1 M ammonium formate containing 0.1% Igepal CA630). An Ultrahydrogel 120 (7.8 9 250 mm) column equilibrated with 100 mM ammonium formate buffer (flow rate 1 ml/min) was used. Asterisk indicates selenoproteins including GPx eluted near the void volume of the column. Cross indicates elution of unidentified organic selenium. Arrow elution of selenoneine

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Materials and methods Materials Fish samples of Japanese conger (average body weight 187 g), Pacific sardine (average body weight 121 g), Japanese anchovy (average body weight 8.4 g), Japanese barracuda (average body weight 93 g), chum salmon (average half-fillet weight 1430 g), greeneye (average body weight 19 g), Pacific saury (average body weight 191 g), alfonsino (average body weight 1.97 kg), red sea bream (average body weight 403 g), white croaker (average body weight 469 g), horse mackerel (average body weight 100 g), Pacific mackerel (average body weight 427 g), skipjack (average body weight 3.39 kg), and marbled sole (average body weight 366 g) were

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collected from fish obtained at a local market in Yokohama and stored frozen at -80°C until use. yellowfin tuna, albacore, bigeye tuna, and swordfish were obtained as fillet (weight *300 g). Pacific bluefin tuna were the same as the samples in the previous paper [1]. White muscle were cut from the dorsal muscle at the center of body axis and used for chemical analysis. Glutathione peroxidase (GPx) from bovine erythrocyte was purchased from Sigma–Aldrich (St Louis, MI, USA). Methylmercury chloride and 2,3-diaminonaphthalene were obtained from Tokyo Kasei (Tokyo, Japan). Selenium concentration measurement To measure total selenium levels, each sample (0.1–0.2 g) was digested at 200–220°C in 1 ml of a 1:2 mixture of

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nitric acid and perchloric acid. Selenium concentration was measured using a fluorometric assay employing 2,3-diaminonaphthalene [12]. For selenium speciation analysis, chromatographic separation was performed using a 712p high-performance liquid chromatography (HPLC) pump (GL-Sciences, Tokyo, Japan) in conjunction with an Ultrahydrogel 120 (7.8 9 250 mm; Nihon Waters, Tokyo, Japan) analytic column equilibrated with 0.1 M ammonium formate buffer containing 0.1% (w/v) Igepal CA-630 (Sigma-Aldrich Japan, Tokyo, Japan) according to the method described previously [1]. Each sample (0.1 g) was homogenized in 0.5 ml water, and 20 ll supernatant was analyzed after

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two- to fourfold dilution in the mobile phase. The injection volume was fixed at 20 ll and the mobile phase delivered isocratically at rate of 1 ml/min. Selenium was detected through online liquid chromatography inductively coupled plasma mass spectrometry (LC–ICP–MS), performed using an ELAN DRC II mass spectrometer (PerkinElmer, Waltham, MA, USA) in conjunction with a concentric quartz nebulizer (WE02-4371) and a quartz sample injector (2 mm id). Using this setup, 82Se levels were monitored. Plasma and auxiliary argon gas flow rates were 17 and 1.3 l/min, respectively. The nebulization argon gas flow rate was 1.02 l/min. The radiofrequency power was 1300 W. During separation, GPx and selenoneine were

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Fig. 1 continued

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eluted at retention times of 5.4 and 10.1 min, respectively, and the selenium concentration was determined [12] using the respective compounds as standards.

Statistical analysis Statistical analysis was carried out using Graphpad PRISMTM 5.03 (Graphpad Software Inc., USA).

Mercury concentration determination Total mercury levels were determined by flameless atomic absorption spectrometry at 253.7 nm using an HG-310 mercury analyzer (Hiranuma, Tokyo, Japan) according to the manufacturer’s instructions after digestion of the sample material (0.1–0.5 g) with 2 ml of 1:2:1 mixture of nitric acid/perchloric acid/sulfuric acid and dilution in water to 25–125 ml.

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Results Tissue levels of selenoneine were determined by a speciation analysis method used for detection of organic selenium in animal tissues. This method involves monitoring 82Se levels by LC–ICP–MS using a gel permeation chromatography (GPC) column (Fig. 1) [1]. Levels of

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selenoneine and other organic selenium compounds were determined in muscle of various fishes (Table 1; Fig. 1). Swordfish muscle contained the highest level of selenoneine (2.8 nmol/g tissue) among the fishes examined in this study. The selenoneine contents of muscle from tuna species, such as Pacific bluefin tuna, bigeye tuna, yellowfin tuna, and albacore, were 1.6–2.6 nmol/g tissue, indicating that most of the selenium (9–42%) was present as selenoneine. In other fish species, such as Pacific sardine, greeneye, alfonsino, skipjack, Pacific mackerel, horse mackerel, red sea bream, and Japanese barracuda, selenoneine levels ranged between 0.1 and 1.4 nmol/g tissue. In contrast, muscle of Japanese conger, Japanese anchovy, chum salmon, Pacific saury, white croaker, marbled sole, and Japanese flounder contained undetectable levels of selenoneine (\0.05 nmol/g tissue). Collectively, these results show that selenoneine is found in tissue of various fishes and is present at especially high levels in muscle of tuna species. Furthermore, they

indicate that selenoneine is the predominant form of organic selenium in fish tissues. To calculate the Se-to-Hg molar ratio in fish muscles, we also determined total mercury contents in each same fish sample (Table 1). The predatory fish species, such as alfonsino, tuna, swordfish, and skipjack, contained higher levels of total mercury ranging between 0.65 and 6.32 nmol/g tissue, and other fish species contained less than 0.53 nmol/g tissue. The Se-to-Hg molar ratio was estimated to range from 1 for swordfish to 217 for Japanese anchovy (Table 1).

Discussion Levels of selenoneine and other organic selenium compounds were determined in white muscle of several species of fish. In addition, total selenium and mercury levels were assayed in the same samples. High levels of selenoneine

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Table 1 Contents of selenoneine, total selenium, and total mercury in the muscle of fishes Species

Selenoneine, nmol/g

Selenium, nmol/g (mg/kg)

Mercury, nmol/g (mg/kg)

Molar ratio of Se to Hg

Japanese conger

ND

2.5 ± 1.1 (0.20)

0.17 ± 0.05 (0.03)

16

1.4 ± 0.6

4.1 ± 0.9 (0.33)

0.10 ± 0.0 (0.02)

42

ND

3.1 ± 1.8 (0.25)

0.05 ± 0.03 (0.01)

96

Japanese barracuda Sphyraena japonica

0.1 ± 0.1

1.8 ± 0.2 (0.14)

0.04 ± 0.02 (0.01)

59

Chum salmon

ND

3.9 ± 0.3 (0.30)

0.09 ± 0.03 (0.02)

49

1.4 ± 0.5

5.3 ± 0.6 (0.43)

0.17 ± 0.07 (0.03)

28

ND

2.6 ± 0.4 (0.21)

0.30 ± 0.08 (0.06)

9

1.3 ± 0.5

18.1 ± 12.5 (1.44)

6.32 ± 2.43 (1.28)

3

0.4 ± 0.1

3.7 ± 0.3 (0.29)

0.17 ± 0.04 (0.03)

24

ND

3.6 ± 1.0 (0.28)

0.53 ± 0.32 (0.11)

7

Horse mackerel

0.5 ± 0.2

5.0 ± 1.4 (0.40)

0.15 ± 0.0 (0.03)

34

Trachurus japonicus Pacific mackerel

0.6 ± 0.2

3.4 ± 0.6 (0.27)

0.09 ± 0.02 (0.02)

39

1.0 ± 0.6

6.2 ± 3.4 (0.49)

0.66 ± 0.08 (0.13)

9

1.6 ± 0.5

9.4 ± 3.91 (0.76)

1.92 ± 1.22 (0.39)

10

1.7 ± 0.3

19.1 ± 8.3 (1.51)

1.12 ± 0.80 (0.23)

20

2.4 ± 0.3a

7.4 ± 1.1 (0.6)

0.84 ± 0.06 (0.17)

9

2.6 ± 1.8

14.1 ± 6.6 (1.13)

2.20 ± 0.24 (0.45)

7

2.8 ± 0.6

6.6 ± 1.5 (0.53)

5.12 ± 1.54 (1.03)

1

ND

1.4 ± 0.0 (0.11)

0.01 ± 0.0 (0.002)

217

Conger myriaster Pacific sardine Sardinops melanostictus Japanese anchovy Engraulis japonica

Oncorhynchus keta Greeneye Chlorophthalmus albatrossis Pacific saury Cololabis saira Alfonsino Beryx splendens Red sea bream Pagrus major White croaker Pennahia argentata

Scomber japonicus Skipjack Euthynnus pelamis Yellowfin tuna Thunnus albacores Albacore Thunnus alalunga Pacific bluefin tuna Thunnus orientalis Bigeye tuna Thunnus obesus Swordfish Xiphias gladius Marbled sole Pleuronectes yokohamae Each value indicates the mean ± SD of three individuals, except for alfonsino and skipjack where five and four individuals were included ND lower than detection limit at 0.05 nmol of Se/g a

Selenoneine content has been reported in the previous paper [1]

(1.3–2.8 nmol/g tissue) were found in muscle of swordfish, tuna, and alfonsino that often contained high levels of MeHg at concentration of over 0.5 lg Hg/g tissue and are listed in fish consumption advisories from the Japanese government. In muscle of these fishes, a large proportion of

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the organic selenium was present as selenoneine. While muscle of most of the other fishes also contained selenoneine, levels of selenoneine in Japanese conger, Japanese anchovy, chum salmon, Pacific saury, white croaker, and marbled sole were below the level of detection

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(\0.05 nmol/g tissue). We are now trying to determine low levels (\0.05 nmol/g tissue) of selenoneine in the tissue samples by optimization of the extraction method. Selenoprotein P [13], glutathione peroxidases [13, 14], and selenosugars [15, 16] were reported to be distributed in animal and fish tissues. Further studies are also required to identify other unknown selenium compounds, such as selenoproteins, selenosugars, and insoluble organic selenium, and their metabolic pathways in fish tissues. Previous studies [1, 2] revealed that selenoneine is an abundant selenium compound that is widely distributed in animal tissues, including blood, hepatopancreas, spleen, heart, and skeletal muscle of tuna; mackerel and tilapia blood; porcine kidney; chicken gizzard, liver, and heart; and squid hepatopancreas. Tuna and mackerel blood contained high levels of selenoneine (430–437 nmol/g tissue) [1]. Selenoneine may be an important member of the redox cycle in animal cells [1, 2]. GPx and other selenoproteins, whose expression is induced by selenium intake, are thought to enhance antioxidant activity in animal tissues and cells [17–23]. Selenoneine itself may play a key role as a strong free-radical scavenger in a variety of physiological and nutritional processes [1, 2]. In the present study, selenoneine was found to be present at high levels ([1 nmol/g tissue) in muscle of certain fish species. Our data (unpublished) suggest that it functions as an antioxidant in muscle of large predatory fish. Tuna’s ‘‘burnt meat’’ courses a problem on the tuna cultured in Japan [2, 17]. This phenomenon, in which raw meat becomes as though ‘‘cooked,’’ lacking a bright red meat color and having a more watery, softer texture, is often seen in tuna and mackerel under conditions of stress, for example, when fish are caught during the spawning period in summer [2, 17]. Oxidative stress is caused by selenium deficiency and hypoxia [2, 17]. After catching, extensive apoptosis and autophagy occur in white muscle, and hemolysis occurs in fish that contain low selenium levels (blood concentration \1 lg/g tissue). The antioxidant effects of selenoneine are essential in enabling fish to adapt to and survive low-oxygen marine environments. In rats, selenium deficiency was found to induce hemolysis [21, 22]. White muscle disease, which occurs in seleniumdeficient animals, shows similar features [23]. The results of selenoproteome analysis revealed that evolution from fish to mammals was accompanied by decreased use of selenocysteine, as a result of selenocysteine/cysteine transitions [24]. Moreover, selenium intake in tuna and other predatory fishes may differ from that in terrestrial animals. Dietary selenium is postulated to protect against mercury toxicity and to reduce mercury accumulation [25]. According to a previous study [26], consistently high levels of total mercury were found in alfonsino (0.78 lg Hg/g tissue), Atlantic bluefin tuna (0.42 lg Hg/g tissue), Pacific

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bluefin tuna (0.59 lg Hg/g tissue), bigeye tuna (0.98 lg Hg/g tissue), blue marlin (0.56 lg Hg/g tissue), striped marlin (0.51 lg Hg/g tissue), and swordfish (0.47 lg Hg/g tissue). Other fish species had lower levels of total mercury or MeHg in muscle than the maximum permitted level of mercury in fish in Japan (0.4 lg Hg/g tissue) [26]. When we analyzed the relationship between body weight and total mercury content in muscle of all fish individuals used in this study except for salmon and tuna, mercury contents were significantly correlated with body weight (R2 = 0.2864, P = 0.0003), and total selenium contents were also correlated with body weight (R2 = 0.1952, P = 0.0038). In addition, selenoneine contents (R2 = 0.2039, P = 0.003) and total selenium contents (R2 = 0.4416, P \ 0.0001) of all fish individuals used in this study were correlated to total mercury. The Seto-Hg molar ratio was estimated to be 3.0, and varied from species to species, ranging from 1 for swordfish to 217 for marbled sole (Table 1). Such ratios may be important for estimating the MeHg risks associated with fish consumption. Mercury accumulation in muscle may be responsible for the selenium metabolism. Recently we elucidated that selenoneine is an essential molecule in the MeHg detoxification pathway. Selenoneine was found to accelerate excretion and demethylation of MeHg by secretory extracellular lysosomal vesicle formation via specific organic cation/carnitine transporter OCTN1 (Yamashita et al., unpublished). Previously, animal trials of feeding with both MeHg (0.5 or 50 lmol/kg in diet) and sodium selenite showed that the toxicity of MeHg was reduced by selenium intake for Se-to-Hg molar ratio above 0.2 [9, 11]. Thus, the range of Se-to-Hg molar ratio from 1 to 217 found in the muscle of fishes in this study is thought to represent normal physiological states from the viewpoint of MeHg bioaccumulation and metabolism. The health risks and health benefits should be estimated by comparing the physiological and nutritional effects of muscle of different fishes with different Se-to-Hg molar ratios in animal studies and human trials. Acknowledgments This work was supported in part by grants from the Japan Society for the Promotion of Science, the Fisheries Research Agency, and the Ministry of Agriculture, Forestry, and Fisheries of Japan.

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