Nutritional Availability of Methylated Selenometabolites in Gut Microbiota, Dimethyldiselenide and Dimethylselenide, in Rats

Abstract

Selenium (Se) is an essential micronutrient for animals. Various chemical forms of Se exist in nature, each with distinct physiological, nutritional, and toxicological properties. In this study, we aimed to determine whether dimethyldiselenide (DMDSe, a monomethylated Se (MMSe) compound) and dimethylselenide (DMSe, a dimethylated Se compound), known gut bacterial metabolites, could serve as Se sources in rats. DMDSe could be utilized for selenoprotein biosynthesis and was metabolized into urinary selenometabolites. By contrast, DMSe was not utilized for selenoprotein biosynthesis but was further methylated to trimethylselenonium ion (TMSe), one of the urinary Se metabolites. Our findings indicate that dimethylated Se is not readily available as an Se source in rats, unlike MMSe. Selenoprotein biosynthesis requires selenide, an unmethylated form of Se, in the metabolic pathway. Our observations support the hypothesis that demethylation occurs on MMSe as a reversible methylation step but not on dimethylated Se. This suggests that the second methylation step is crucial for inactivating Se and plays a significant role in metabolism to maintain Se homeostasis in animals. Gut microbiota, which can synthesize both DMDSe and DMSe, may contribute to host Se metabolism through methylation processes.

INTRODUCTION

Selenium (Se) is an essential dietary trace element for humans and animals. Se plays crucial roles within the body as selenocysteine (SeCys) in selenoproteins like glutathione peroxidase and thioredoxin reductase. The biosynthesis of selenoproteins is tightly regulated by a well-established machinery involving the UGA codon and a selenocysteine insertion sequence.¹⁾ SeCys, incorporated into a primary structure of selenoproteins, is derived from selenide (Se²⁻), an intermediate metabolite from dietary Se sources.²⁾ Various selenocompounds exist in nature, and animals can ingest many of these as Se sources. These compounds are typically metabolized once into selenide to be utilized for selenoprotein synthesis.

Major selenocompounds reported in previous studies include inorganic salts like selenite and selenate, which are present in the environment. Selenomethionine (SeMet) is found in plants and yeast^3,4) Other selenoamino acids, such as Se-methylselenocysteine (MeSeCys), selenohomolanthionine, and γ-glutamyl-Se-methylselenocysteine, have also been reported in plants.^5,6) 1β-Methylseleno-N-acetyl-d-galactosamine (SeSugar)⁷⁾ and trimethylselenonium ion (TMSe)⁸⁾ are urinary metabolites in animals, and selenocyanate has been identified as a metabolite in animal cells.⁹⁾ The biosynthesis of elemental Se as nanoparticles (Se-NPs) has also been reported.¹⁰⁾ These selenocompounds have been evaluated for their nutritional value as Se sources^11,12) Selenoamino acids and SeSugar can be efficiently biotransformed into selenoproteins from their original forms via selenide, whereas TMSe cannot. Recently, we reported that TMSe, a urinary selenometabolite, is not utilized for the biosynthesis of serum selenoproteins, such as extracellular glutathione peroxidase 3 (GPX3) and selenoprotein P (SELENOP).¹³⁾ In addition to animal biotransformation, gut microbiota also contributes to Se metabolism in the host.¹⁴⁾ We demonstrated that gut microbiota can modify the nutritional availability of SeSugar, even though intravenously administered SeSugar is not utilized for serum selenoprotein biosynthesis.¹⁵⁾ Gut bacteria have been shown to produce volatile methylated selenocompounds, such as dimethyldiselenide (DMDSe, a monomethylated Se (MMSe) compound) and dimethylselenide (DMSe, a dimethylated Se compound), from selenite, SeMet, and MeSeCys.^16,17) However, the nutritional availability of these volatile selenocompounds remains unclear. In this study, we investigated the distribution and excretion of DMDSe and DMSe in an animal body to assess their potential as a dietary Se source produced by gut microbiota. These findings are expected to provide new insights into the interplay between gut microbiota and the host animal in the context of Se availability.

MATERIALS AND METHODS

Materials

DMDSe was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), while DMSe was obtained from Alfa Aesar (Haverhill, MA, U.S.A.). Se-methylseleno-l-cysteine (MeSeCys) was purchased from Nacalai Tesque (Kyoto, Japan). Ammonium acetate and Trizma® hydrochloride solution (1 M, pH 7.4) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.), and nitric acid was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The chemical structures of the selenocompounds discussed in this study are shown in Fig. 1.

Fig. 1. Chemical Structures of the Studied Selenocompounds

Animal Care

Four-week-old specific-pathogen-free (SPF) male Wistar rats were purchased from Japan SLC (Shizuoka, Japan). They were housed in a humidity-controlled room at 25 ± 2°C with a 12-h light-dark cycle and fed a commercial diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. After a 1-week acclimation period, the rats were switched to an Se-deficient diet (Oriental Yeast Co., Ltd.) and Milli-Q water (18.3 MΩ cm). MF and the deficient diets contained Se at approx. 300 ng/g and <5 ng Se/g.¹²⁾ Rats fed the Se-deficient diet and the Milli-Q water for 3 weeks were designated as Se-deficient.¹³⁾ Rats continuously fed the commercial diet and tap water served as the positive control. All animal experiments were carried out in accordance with the Guidelines of the Animal Investigation Committee, Chiba University, Japan.

Se Speciation in Rat Serum

Animal experiments were conducted as previously described.¹³⁾ Se-deficient rats were intravenously administered MeSeCys, DMDSe, or DMSe in saline at a dosage of 10 μg Se/200 g b.w. once daily for 2 consecutive days. The dose was adapted according to our previous study to evaluate the nutritional availability of Se compounds.¹³⁾ DMDSe or DMSe was once dissolved in a small volume of ethanol, and then, the ethanol solution was diluted with saline at a concentration of 50 μg Se/mL. The solution was prepared just before the administration. Saline-treated rats served as the negative controls. All rats were euthanized 24 h after the final administration. Non-heparinized blood was collected and centrifuged at 1500× g for 20 min to isolate serum. The serum was subsequently stored at −25°C until further analysis. A 20 μL aliquot of the obtained serum was applied to an inductively coupled plasma (ICP)-MS/MS (Agilent 8800, Agilent Technologies, Hachioji, Japan) coupled with an HPLC (LC-ICP-MS) to analyze the distribution of Se in the serum. The HPLC system (Prominence, Shimadzu, Kyoto, Japan) was equipped with an online degasser, an HPLC pump, a Rheodyne six-port injector, and a multi-mode size exclusion column (Shodex GS-520HQ, exclusion size >300000 Da, 7.5 i.d. × 300 mm with a guard column; Showa Denko, Tokyo, Japan). The column was eluted with 50 mM Tris–HCl buffer (pH 7.4) at a flow rate of 0.6 mL/min. The eluate was directly introduced into the nebulizer tube of the ICP-MS/MS instrument. Se was detected in the O₂ mass shift mode by monitoring m/z 80 (Q1) and m/z 96 (Q3) for ⁸⁰Se and ⁸⁰Se¹⁶O, respectively. The ICP-MS/MS operating conditions included an O₂ gas flow rate of 0.3 mL/min. Serum selenoprotein levels were calculated from each peak area.

Se Quantification in Rat Organs

Livers and kidneys were collected from the rats mentioned above. These organs were stored at −25°C until further analysis. A 0.25 g portion of each organ was digested with 1 mL of concentrated nitric acid in a perfluoro alkoxy alkane (PFA) test tube. The PFA tubes were then placed in a microwave digestion system (P-25 series, SAN-AI Kagaku Co., Ltd., Nagoya, Japan) and heated at 300 W for 1 min, twice. Se concentration was finally determined using ICP-MS/MS.

To assess the accuracy of our method, we analyzed a certified reference material (NMIJ CRM 7402-a No.539 cod fish tissue). The measured Se concentration of 1.85 ± 0.18 mg/kg was within the range of the certified value (1.8 ± 0.2 mg/kg), demonstrating the reliability of the technique.

Se Speciation in Rat Urine

Urine samples were collected every 24 h from before and after Se administration. Samples were stored at −25°C and filtered through a 0.45 μm syringe filter prior to Se speciation. A 20 μL aliquot of urine was injected into an LC-ICP-MS/MS system equipped with a multi-mode size exclusion column (Shodex GS-320HQ, exclusion size >40000 Da, 7.5 i.d. × 300 mm with a guard column; Showa Denko) for the separation and detection of Se species. The column was eluted with 50 mM ammonium acetate buffer (pH 6.5) at a flow rate of 0.6 mL/min. The eluate was directly introduced into the nebulizer tube of the ICP-MS/MS instrument. Se was detected in the O₂ mass shift mode by monitoring m/z 80 (Q1) and m/z 96 (Q3) for ⁸⁰Se and ⁸⁰Se¹⁶O, respectively. The ICP-MS/MS operating conditions included an O₂ gas flow rate of 0.3 mL/min.

Statistical Analysis

All experiments were conducted in triplicate, and the results are expressed as means ± S.D. Statistical analyses were conducted with OriginPro ver. 2021b (Northampton, MA, U.S.A.). Statistical significance was determined by one-way ANOVA followed by Tukey’s test. A p-value of less than 0.05 was considered statistically significant. N.D. data were substituted as imputation with half the detection limit.

RESULTS

Evaluation of Se Nutritional Availability

Two serum selenoproteins, GPX3 and selenoprotein P (SELENOP), were identified and quantified by LC-ICP-MS with retention times of 10.8 and 13.4 min, respectively¹⁸⁾ (Fig. 2a). The nutritional availability of selenocompounds was assessed by measuring the recovery of these serum proteins, which served as biomarkers of the Se status. For example, selenoprotein P plays various pivotal roles.¹⁹⁾ Serum selenoprotein levels were measured in Se-deficient rats and Se-sufficient rats (fed a commercial diet) to establish the baseline levels. The Se-sufficient level was set as 100% for comparison. The administration of MeSeCys resulted in a significant recovery of GPX3 and SELENOP, reaching 67.0 ± 14.3 and 71.8 ± 19.0%, respectively. DMDSe exhibited similar recovery rates to MeSeCys for GPX3 (61.2 ± 0.8%) and SELENOP (53.2 ± 1.7%). By contrast, DMSe administration did not significantly improve the serum selenoprotein levels compared with the Se-deficient control, with GPX3 and SELENOP recovery rates of 27.5 ± 3.2 and 24.1 ± 0.5%, respectively (Figs. 2b and 2c).

Fig. 2. Evaluation of Selenocompound Nutritional Availability

(a) Speciation profile of Se in rat serum obtained by LC-ICP-MS. (b, c) Recovery of extracellular glutathione peroxidase 3 (GPX3) and selenoprotein P (SELENOP) in rats following selenocompound administration. Data are presented as mean ± standard deviation (S.D.). Significant differences are indicated by different letters (Tukey’s test, p < 0.05).

The organ selenium levels in the Se-deficient and Se-adequate rats under these experimental conditions are shown in our previous study,¹²⁾ and the effects of Se-supplementation are also shown in the study. Liver and kidney Se concentrations were significantly higher in MeSeCys-treated rats (liver: 613.1 ± 63.3 ng/g, kidney: 4647.5 ± 135.7 ng/g) than in Se-deficient rats (liver: 227.2 ± 15.7 ng/g, kidney: 2920.4 ± 183.5 ng/g) (Fig. 3). DMDSe administration significantly increased Se levels in both the liver (456.3 ± 73.7 ng/g) and the kidney (3862.9 ± 71.5 ng/g). By contrast, DMSe administration did not significantly increase Se levels in either organ (liver: 180.3 ± 10.7 ng/g, kidney: 2408.0 ± 166.6 ng/g).

Fig. 3. Se Concentration in (a) Liver and (b) Kidney Tissues of Rats Following Selenocompound Administration

Data are presented as mean ± S.D. Different letters indicate significant differences (Tukey’s test, p < 0.05).

Urinary Excretion of Selenocompounds

LC-ICP-MS analysis revealed the presence of two urinary selenometabolites, SeSugar and TMSe, with retention times of 20.2 and 22.2 min, respectively (Fig. 4a). In our previous study performed under the same condition as this study, no apparent peaks were detected in the urine of Se-deficient rat by LC-ICP-MS.¹³⁾ While both SeSugar and TMSe were detected in the urine of DMDSe-treated rats, the levels were lower than those observed after MeSeCys administration. By contrast, only TMSe was detected in the urine of DMSe-treated rats (Figs. 4b and 4c).

Fig. 4. Urinary Excretion of Se Metabolites

(a) Speciation profile of urinary selenometabolites by LC-ICP-MS. (b, c) Relative concentrations of SeSugar and TMSe in rat urine following selenocompound administration. Data are presented as mean ± S.D. Significant differences are indicated by different letters (Tukey’s test with imputation substituting half the detection limit for N.D., p < 0.05).

DISCUSSION

In this study, we observed that MeSeCys, a known bioavailable form of Se, was efficiently utilized for the biosynthesis of selenoproteins and the production of urinary selenometabolites (Figs. 2–4), corroborating previous findings.¹³⁾ Our previous studies have demonstrated that the recovery of serum selenoproteins, as measured by LC-ICP-MS, is a reliable indicator of the nutritional availability of various selenocompounds.^11–13,15) DMDSe was also found to be an Se source for selenoprotein synthesis (Fig. 2c). The incorporation of DMDSe into selenoproteins suggests that it undergoes demethylation to selenide, a crucial common intermediate in the Se metabolic pathway. Similarly, the biosynthesis of SeSugar, a major urinary selenometabolite, likely involves selenide as a common intermediate with selenoproteins.²⁾ The production of SeSugar from DMDSe, along with other Se sources, provides evidence for the demethylation of MMSe such as DMDSe (Fig. 4b). However, DMDSe was less efficiently utilized than MeSeCys for selenoprotein synthesis (Fig. 2c). The lower DMDSe was also incorporated into selenoproteins in organs (Fig. 3b). These may be attributed to its volatile nature. DMDSe may be readily exhaled like DMSe,²⁰⁾ potentially limiting its bioavailability for selenoprotein synthesis and urinary metabolite formation.

Unlike MeSeCys and DMDSe, DMSe exhibited a unique metabolic profile in rats. DMSe was not incorporated into serum selenoproteins or metabolized into urinary SeSugar (Figs. 2b, 2c, and 4b), suggesting it is not readily utilized as an Se source. This metabolic inertness of DMSe is consistent with a previous study reporting low toxicity following high-dose inhalation of DMSe.²¹⁾ Methylselenol (MeSeH) can be generated through various metabolic pathways, including the methylation of selenide, the β-elimination of MeSeCys, and the simple reduction of DMDSe. Selenide, MeSeCys, and DMDSe are considered Se sources for the biosynthesis of selenoproteins and the production of urinary SeSugar. The observation that DMDSe can be utilized for selenoprotein synthesis suggests that the monomethylation of selenide is a reversible process. However, the lack of utilization of DMSe indicates that dimethylation may be an irreversible step in Se metabolism. This second methylation step could serve as a mechanism to contribute to the excretion of the surplus selenide for Se homeostasis, together with further methylation to produce TMSe, which is excreted into the urine when surplus amounts of Se are ingested.²²⁾ TMSe has been reported to be nutritionally unavailable,^11,13) indicating the metabolism of nutritional Se to TMSe via DMSe was the detoxication to form nutritional inert Se in animals. Our findings indicate that DMSe is efficiently metabolized to TMSe, even in Se-deficient rats. The second methylation step appears as a key step for removing excess Se from the metabolic pool. By contrast, the third methylation step, leading to the formation of TMSe, may be a subsidiary detoxification mechanism. However, the amount of DMSe in the breath was not determined in this study because it is practically difficult to quantitatively determine it.

The monomethylated and dimethylated selenocompounds, DMDSe and DMSe, are known metabolites of gut bacteria. The differential utilization of these compounds suggests that their biosynthesis in the gut has specific biological implications. Remarkably, the production of DMSe by gut bacteria may contribute to the regulation of ingested-Se utilization in the host organism. This biotransformation may represent a mechanism for maintaining Se homeostasis. The diversity of gut microbiota can influence the types of Se metabolites produced, leading to a diverse range of Se metabolic pathways in the host. Further research is needed to explore the specific role of gut microbiota in shaping Se metabolism and its impact on host health.

In conclusion, we investigated the metabolism and the nutritional availability of gut bacterial metabolites, DMDSe and DMSe, in rats. DMDSe, a monomethylated Se compound, was utilized for the biosynthesis of selenoproteins and urinary selenometabolites. By contrast, DMSe, the dimethylated compound, was primarily methylated into TMSe, a non-bioactive form of Se, which is readily excreted in urine. This suggests that the second methylation step, leading to the formation of DMSe, is a critical metabolic pathway for the excretion of excess Se. The subsequent methylation of DMSe to TMSe further facilitates the elimination of Se from the body. Consequently, the second methylation is the crossroads in the homeostasis of Se, that is, its utilization and excretion in animals. Further research is needed to fully elucidate the mechanisms underlying these processes and their implications for host health.

Acknowledgments

This work was partly supported by JSPS KAKENHI Grant Nos. JP23K13901, 24H00749, and 24K21304.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1)  Davis CD, Tsuji PA, Milner JA. Selenoproteins and cancer prevention. Annu. Rev. Nutr., 32, 73–95 (2012).
• 2)  Suzuki KT. Metabolomics of selenium: Se metabolites based on speciation studies. J. Health Sci., 51, 107–114 (2005).
• 3)  Stadlober M, Sager M, Irgolic KJ. Effects of selenate supplemented fertilisation on the selenium level of cereals—identification and quantification of selenium compounds by HPLC-ICP–MS. Food Chem., 73, 357–366 (2001).
• 4)  Amoako PO, Kahakachchi CL, Dodova EN, Uden PC, Tyson JF. Speciation, quantification and stability of selenomethionine, S-(methylseleno)cysteine and selenomethionine Se-oxide in yeast-based nutritional supplements. J. Anal. At. Spectrom., 22, 938–946 (2007).
• 5)  Ogra Y, Kitaguchi T, Ishiwata K, Suzuki N, Iwashita Y, Suzuki KT. Identification of selenohomolanthionine in selenium-enriched Japanese pungent radish. J. Anal. At. Spectrom., 22, 1390–1396 (2007).
• 6)  Kotrebai M, Birringer M, Tyson JF, Block E, Uden PC. Selenium speciation in enriched and natural samples by HPLC-ICP-MS and HPLC-ESI-MS with perfluorinated carboxylic acid ion-pairing agents. Analyst, 125, 71–78 (2000).
• 7)  Ogra Y, Ishiwata K, Takayama H, Aimi N, Suzuki KT. Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography-inductively coupled plasma mass spectrometry and –electrospray ionization tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 767, 301–312 (2002).
• 8)  Byard JL. Trimethyl selenide. A urinary metabolite of selenite. Arch. Biochem. Biophys., 130, 556–560 (1969).
• 9)  Anan Y, Kimura M, Hayashi M, Koike R, Ogra Y. Detoxification of selenite to form selenocyanate in mammalian cells. Chem. Res. Toxicol., 28, 1803–1814 (2015).
• 10)  Shirsat S, Kadam A, Naushad M, Mane RS. Selenium nanostructures: microbial synthesis and applications. RSC Adv., 5, 92799–92811 (2015).
• 11)  Takahashi K, Suzuki N, Ogra Y. Bioavailability comparison of nine bioselenocompounds in vitro and in vivo. Int. J. Mol. Sci., 18, 506 (2017).
• 12)  Takahashi K, Ochi A, Mihara H, Ogra Y. Comparison of nutritional availability of biogenic selenium nanoparticles and chemically synthesized selenium nanoparticles. Biol. Trace Elem. Res., 201, 4861–4869 (2023).
• 13)  Takahashi K, Suzuki N, Ogra Y. Effect of administration route and dose on metabolism of nine bioselenocompounds. J. Trace Elem. Med. Biol., 49, 113–118 (2018).
• 14)  Ferreira RLU, Sena-Evangelista KCM, de Azevedo EP, Pinheiro FI, Cobucci RN, Pedrosa LFC. Selenium in human health and gut microflora: bioavailability of selenocompounds and relationship with diseases. Front. Nutr., 8, 685317 (2021).
• 15)  Takahashi K, Suzuki N, Ogra Y. Effect of gut microflora on nutritional availability of selenium. Food Chem., 319, 126537 (2020).
• 16)  Takahashi K, Horiai S, Yamagishi Y, Nagasawa S, Iwase H, Ogra Y. Biotransformation of se-methylselenocysteine into volatile selenocompounds by bacteria isolated from rat gut microflora. J. Funct. Foods, 110, 105859 (2023).
• 17)  Krittaphol W, McDowell A, Thomson CD, Mikov M, Fawcett JP. Biotransformation of l-Selenomethionine and selenite in rat gut contents. Biol. Trace Elem. Res., 139, 188–196 (2011).
• 18)  Anan Y, Hatakeyama Y, Tokumoto M, Ogra Y. Chromatographic behavior of selenoproteins in rat serum detected by inductively coupled plasma mass spectrometry. Anal. Sci., 29, 787–792 (2013).
• 19)  Tsutsumi R, Saito Y. Selenoprotein P; P for plasma, prognosis, prophylaxis, and more. Biol. Pharm. Bull., 43, 366–374 (2020).
• 20)  Kremer D, Ilgen G, Feldmann J. GC-ICP-MS determination of dimethylselenide in human breath after ingestion of 77Se-enriched selenite: Monitoring of in-vivo methylation of selenium. Anal. Bioanal. Chem., 383, 509–515 (2005).
• 21)  Al-Bayati MA, Raabe OG, Teague SV. Effect of inhaled dimethylselenide in the Fischer 344 male rat. J. Toxicol. Environ. Health, 37, 549–557 (1992).
• 22)  Suzuki KT, Kurasaki K, Okazaki N, Ogra Y. Selenosugar and trimethylselenonium among urinary Se metabolites: dose- and age-related changes. Toxicol. Appl. Pharmacol., 206, 1–8 (2005).