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Aug 31, 2009 - Speciation Analysis of Selenium Metabolites in Urine and Breath by HPLC- and GC-Inductively Coupled Plasma-MS after Administration of S...
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Chem. Res. Toxicol. 2009, 22, 1795–1801

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Speciation Analysis of Selenium Metabolites in Urine and Breath by HPLC- and GC-Inductively Coupled Plasma-MS after Administration of Selenomethionine and Methylselenocysteine to Rats Yuki Ohta,† Yayoi Kobayashi,‡ Sakae Konishi,† and Seishiro Hirano*,§ Graduate School of Pharmaceutical Sciences, Chiba UniVersity, Chuo, Chiba 260-8675 Japan, Molecular and Cellular Toxicology Section, EnVironmental Health Sciences DiVision, and EnVironmental Nanotoxicology Section, Research Center for EnVironmental Risk, National Institute for EnVironmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan ReceiVed June 18, 2009

Selenium is an essential trace element found in vegetables as selenomethionine (SeMet) and methylselenocysteine (MeSeCys). In the present study, we used stable isotopes of Se to investigate differences between how SeMet and MeSeCys are metabolized, using methylseleninic acid (MSA) as a reference methylselenol source. A mixture containing 76Se-SeMet, 77Se-MeSeCys, and 82Se-MSA (each 25 µg Se/kg b.w.) was orally administered to rats, and then, speciation analyses of Se in urine and exhaled gas were conducted using HPLC-inductively coupled plasma (ICP)-MS and GC-ICP-MS, respectively. The proportions of 76Se-, 77Se-, and 82Se-selenosugar (Se-sugar) to total urinary Se metabolites originating from each tracer were very similar, while the proportion of 77Se-tirmethylselenonium (TMSe) was much less than that of76Se- and 82Se-TMSe in urine, suggesting that77Se-SeMet is less efficiently metabolized to TMSe. Similarly, there was significantly less 77Se-dimethylselenide (DMSe) originating from 77SeSeMet than76Se- and 82Se-DMSe originating from 76Se-MeSeCys and 82Se-MSA in exhaled gas. It is generally accepted that DMSe and TMSe are metabolites of methylselenol, a putative metabolic intermediate in Se metabolism. Methylselenol is believed to be responsible for the cancer chemoprevention effects of Se. These results suggest that MeSeCys is converted to methylselenol more efficiently than is SeMet and that urinary TMSe and exhaled DMSe might be useful biomarkers for the generation of cancer chemopreventive forms of Se. Introduction Selenium is an essential trace element for humans and animals and exhibits its physiological effect through selenocysteine (SeCys) synthesized from selenide (1-3). However, Se is also recognized as a toxic metalloid, with little difference between its effective and toxic concentrations (4). The toxicity of Se varies with its chemical form (5, 6), so special attention should be paid to the chemical form of dietary Se. Dietary Se is believed to exhibit cancer chemoprevention effects, especially toward prostate cancer, by generating methylselenol, a putative Se active metabolic intermediate (7-11). It is believed that selenomethionine (SeMet) and methylseleninic acid (MSA) generate methylselenol by enzymatic reaction with γ-lyase (5, 12) and chemical reaction with reductants (13, 14) (e.g., GSH), respectively. MSA is commonly used for investigating the mechanisms underlying the cancer chemoprevention effect of Se during in vitro experiments. Several SeMet-containing supplements are commercially available and have been studied in clinical trials for their anticancer effects (15-17). On the other hand, methylselenocysteine (MeSeCys) is a natural compound that is also metabolized to methylselenol by enzymatic reaction * To whom correspondence should be addressed. Tel: +81 29-850-2512. Fax: +81 29-850-2512. E-mail: [email protected]. † Chiba University. ‡ Environmental Health Sciences Division, National Institute for Environmental Studies. § Research Center for Environmental Risk, National Institute for Environmental Studies.

catalyzed by β-lyase (10, 18). However, details of the metabolism of MeSeCys remain to be elucidated. Selenium is metabolized by serial biological methylation reactions [selenide; HSe- f methylselenol; CH3SeH f dimethylselenide (DMSe); (CH3)2Se f trimethylselenonium (TMSe); (CH3)3Se+] (19, 20) similar to other metalloids (e.g., arsenic and tellurium) (21, 22). It has been shown that SeMet is catabolized to methylselenol by γ-lyase and then further metabolized and excreted as DMSe and TMSe (23). Indeed, TMSe and DMSe were reported to be excreted into urine and exhaled air as major metabolites of Se after high-dose ingestion of SeMet (23). However, only recently has it been shown that low concentrations of Se metabolites can be measured quantitatively, and selenosugar (Se-sugar), probably synthesized from selenide, was found to be a major urinary metabolite of Se instead of TMSe (24-26). Highly sensitive speciation analysis for metals and several metalloids including Se is possible using HPLC and GCinductively coupled plasma (ICP)-MS. However, the sensitivity of ICP-MS for Se is lower than for other elements. The low sensitivity for Se in MS is partly due to several interferences on major Se isotopes (27) and the presence of many stable isotopes [74Se (0.89%), 76Se (9.36%), 77Se (7.63%), 78Se (23.4%), 80 Se (49.6%), and 82Se (8.73%)]. Therefore, using tracers enriched with a stable isotope should enhance the sensitivity of HPLC- and GC-ICP-MS measurements for Se, in addition to being generally advantageous for metabolic studies of other essential elements (28).

10.1021/tx900202m CCC: $40.75  2009 American Chemical Society Published on Web 08/31/2009

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The blood glutathione peroxidase 3 (GPX3) activity has been used as a biomarker of Se deficiency states (3), since GPX3 contains SeCys. However, GPX3 activity may not be a good biomarker for monitoring the cancer chemoprevention effect of Se since GPX3 is synthesized via selenide and not methylselenol. Therefore, the development of a new biomarker is important for estimating methylselenol levels in biological systems. Unfortunately, there is no satisfactory method for measuring methylselenol directly and quantitatively because of its high reactivity. DMSe and TMSe are known to be methylated metabolites of methylselenol; therefore, these methylated Se compounds are potentially good biomarkers for methylselenol. To our knowledge, the excretion of DMSe and TMSe has never been determined simultaneously under subtoxic conditions. We therefore investigated the excretion of DMSe and TMSe after administration of 77Se-SeMet and 76Se-MeSeCys, which are the presumed precursors of methylselenol.

Ohta et al. Scheme 1. Schematic Illustration of the Collection of Exhaled DMSe

Experimental Procedures Reagents. The elemental forms of 76Se (99.8% enriched), 77Se (99.9%), and 82Se (99.9%) were purchased from Isoflex USA (San Francisco, CA). D2 gas (99.9%) was purchased from Aldrich (Miamisburg, OH). Methyl lithium dissolved in diethyl ether at a concentration of 1.0 mol/L was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). A selenium standard solution (1000 µg/ mL) for ICP-MS was purchased from SPEX CertiPrep (Metuchen, NJ). DMSe and TMSe were purchased from Tri Chemical Laboratories Inc. (Yamanashi, Japan). All other reagents were analytical grade. Purified water (18.2 MΩ cm) from Milli-Q SP (Millipore, Bedford, MA) was used throughout the experiment. 82 Se-MSA. 82Se-MSA was synthesized as reported elsewhere from the isotopically enriched Se powder. Their chemical and isotopic purities were confirmed by HPLC-ICP-MS (29). Se-Sugar. Elemental selenium was suspended in dry tetrahydrofuran (1 mL) under a nitrogen atmosphere, after which methyl lithium was slowly added. The resulting lithium methylselenide was reacted with 1R-chlorogalactosamine tetraacetate to produce 1βmethylselenogalactosamine tetraacetate. The acetyl groups were removed with a catalytic amount of sodium methoxide to obtain Se-sugar, as reported elsewhere (30). The structure and purity of the Se-sugar were confirmed by fast atom bombardment (FAB)MS and NMR spectroscopy, together with HPLC-ICP-MS. 76 Se-MeSeCys. 76Se-selenium (elemental form, 7.6 mg) was suspended in dry THF (1 mL) under an atmosphere of nitrogen, and then, CH3Li (1 M THF solution, 2.7 mL) was added under stirring conditions. Then, 3-chloro-L-alanine hydrochloride (43 mg) dissolved in dry ethanol (0.5 mL) was added slowly to the solution. After it was refluxed for 2 h, the reaction mixture was evaporated in vacuo, and the residue was dissolved in 1 M HCl (1.5 mL) and washed twice with 2 mL of diethylether. The aqueous layer was neutralized with 1 M NaOH, and then, the crude product was purified on an ODS column to obtain 76Se-MeSeCys (yield estimated as selenium, 12%). The chemical and isotopic purities were confirmed by HPCL-ICP-MS (25). 77 Se-SeMet. 77Se-selenium (elemental form, 7.7 mg) was suspended in dry THF (1 mL) under an atmosphere of nitrogen, and then, CH3Li (1.13 M THF solution, 2.1 mL) was added. The reaction proceeded at room temperature for 5 min under stirring conditions. (S)-(+)-2-Amino-4-bromo-bytyric acid hydrobromide (71 mg, MW ) 262.93) dissolved in dry ethanol (0.5 mL) was gradually added to the reaction mixtur, and the solution was refluxed for 2 h. The reaction mixture was evaporated in vacuo, and the residue was dissolved in an aqueous solution of 1 M HCl (1.5 mL). 77 Se-SeMet was purified on an ODS column (estimated yield as selenium, 50%). The chemical and isotopic purities were confirmed by HPLC-ICP-MS (25). Animal Experiments. Five week old male Wistar rats were purchased from Clea Japan (Tokyo, Japan). Animals were maintained in stainless wire cages at 22 ( 2 °C and 50-55% relative

humidity, with a light/dark cycle of 12/12 h for 2-3 days before the experiment. They were fed a standard diet (CE-2; CLEA Japan) and tap water ad libitum. All animal experiments were carried out according to the “Principles of Laboratory Animal Care” (NIH version, revised 1996) and the guidelines of the Ethics Committee for Experimental Animals of the National Institute for Environmental Studies. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg b.w.). The trachea was exposed and cannulated with a Y-piece (Harvard, Boston, MA) connected to an artificial ventilator (model SN-480-7, Sinano, Tokyo, Japan) (Scheme 1). To sustain anesthesia, sodium pentobarbital was infused into the femoral vein at 97.2 µg/min by a syringe controller (BASi, West Lafayette, IN). The tidal volume (1.0 mL) and the respiratory rate (65 breaths/min) had been determined based on optimal cardiac rate (110-130 beats/min) before the experiment was initiated. A mixture of 76Se-MeSeCys, 77 Se-SeMet, and 82Se-MSA was orally administered to three rats (25 µg Se/kg b.w. for each Se compound), and then, mechanical ventilation was performed to collect DMSe in the exhaled gas under the following conditions. Blood pressure was directly measured by cannulating the abdominal aorta with a MLT 844 physiological pressure transducer (ADInstruments, CO Springs, CO) connected to a BRIDG Amp and PowerLab/8sp (ADInstruments). The cardiac rate was calculated from the interval time of systolic and diastolic blood pressure using Scope software (ADInstruments) in the preliminary test. The body temperature and heart beat were monitored by a thermometer and a photoplethysmograph PT-300 (Fukuda, Tokyo, Japan) while collecting the exhaled gas. The exhaled gas was concentrated in a cold trap at -78 °C (dry ice/ acetone slush), and the recovery of DMSe was determined. A tightly sealed vessel containing 1.0 mL of 100 ng Se/mL DMSe aqueous was connected to the mechanical ventilator instead of the rat. DMSe was purged by mechanical ventilation with fresh air at 40 °C for 1 h. Volatilized DMSe was collected in the cold trap and then diluted with 140 µL of ice cold Milli-Q water. Urine was obtained from the urinary bladder 6 h after administration of the test compound using a syringe. Measurement of Total Se. Total Se concentrations in urine were measured by ICP-MS after digestion with 30% nitric acid and 15% hydrogen peroxide. NIST SRM 1577b bovine liver was used for analytical quality assurance, and the difference between the measured value for the total Se concentration and the certified value (0.73 ( 0.06 µg/g) was within (4.0%. HPLC-ICP-MS and GC-ICP-MS. HPLC (Agilent 1200 system) or GC (Agilent 7890A) were connected to ICP-MS (Agilent 7500cs)

MeSeCys Is a Natural Methylselenol-Generating Agent Table 1. HPLC- and GC-ICP-MS Parametersa parameters

HPLC

GC

RF power carrier gas make-up gas extraction lens Einzel 1,3 Einzel 2 cell input cell output plate bias D2 gas octapole bias QP bias

1600 W 0.81 L/min 0.35 L/min 3.5 V -100 V 15 V -24 V -12 V -45 V 2.2 mL -16 V -15 V

900 W 1.3 L/min 0.0 L/min -130 V -160 V 23.5 V -10 V -10 V -30 V 2.0 mL -10 V -8 V

Chem. Res. Toxicol., Vol. 22, No. 11, 2009 1797 If the intensity of m/z 78 was larger than the detection limit (the intensity at three times standard deviation of background), then corrected m/z X ) intensity of m/z X - (intensity of m/z 78 × natural abundance ratio of xSe/natural abundance ratio of 78Se) where X is the mass number of Se. Determinations of Se-sugar, TMSe, and DMSe concentrations were performed with a calibration curve based on the peak area. Data are presented as means ( SDs. Statistical analysis of data was performed using Kruskal-Wallis test followed by Scheff’s multiple comparison. The level of significance was set at p < 0.05.

Results

a

The HPLC and GC-ICP-MS parameters were tuned with 100 ng Se/ mL selenite and He gas containing 95.8 ppm Xe, respectively.

through a concentric nebulizer or a GC-ICP-MS transfer and injection line (all instruments: Agilent Technologies, Tokyo, Japan). Urine samples (20 µL) were injected into an HPLC equipped with a gel filtration column (GS-320HQ, Shodex, Tokyo, Japan) and eluted with 50 mM ammonium acetate (pH 6.8) at a flow rate of 0.5 mL/min. The trapped exhaled gas samples (4 µL, split mode 1:1) were analyzed by a GC equipped with a DB-620 column (30 m × 320 µm × 1.8 mm, Agilent) at a carrier gas flow rate of 4 mL He/min [column oven: 40 °C (3 min), 10 °C/min to 80 °C (0 min), and 40 °C/min to 200 °C (5 min)]. DMSe standard solution was prepared by dissolving DMSe into cold water using chilled glassware. The ICP-MS measurement conditions are summarized in Table 1. Data Analysis and Statistics. Data analysis and statistics were run on Mathematica 5.2 (Wolfram Research, IL) and R (31). Mass bias and SeD+ interference were corrected using selenite (for HPLCICP-MS) and DMSe (for GC-ICP-MS) (i.e., the variation in sensitivity among m/z 76, 77, 78, and 82 was corrected.). Because 76 Se, 77Se, and 82Se were present in the rat samples from natural sources before the administration of the 76Se, 77Se, and 82Se tracers, signal intensities corresponding to endogenous 76Se, 77Se, and 82Se were adjusted using the following equation.

76

A mixture of Se-SeMet, 77Se-MeSeCys, and 82Se-MSA was orally administered to rats, and then, the rats were mechanically ventilated to collect volatile Se compounds in the exhaled gas. None of the rats died during gas collection. Expired gas was collected in a cold trap (-78 °C) connected to a mechanical ventilator. The recovery of DMSe in the cold trap was determined to be 80.2 ( 7.1%, when DMSe aqueous, instead of the rat, was purged by mechanical ventilation. When exhaled gas (tidal volume 1 mL, 65 breaths/min) was trapped in 1 h increments, almost the same amount of ice (139.7 ( 2.0 mg) was formed each time due to moisture in the exhaled gas. The ice was slowly melted before injection into the GC-ICP-MS. Exhaled Se was speciated by GC-ICP-MS (Figure 1). The detection limit of DMSe by GC-ICP-MS (the concentration that gave an intensity at three times the SD of background) was 0.033 ( 0.002 ng Se when a 4 µL sample was injected. DMSe originating from endogenous Se (78Se), 76Se-MeSeCys, and 82SeMSA was detected in the first 1 h of expired gas sample. On the other hand, DMSe originating from 77Se-SeMet was not detected during this time period. It is noteworthy that endogenous Se was not detected as DMSe in exhaled gas prior to administration of the test samples (Figure 1, untreated). Exhala-

Figure 1. GC-ICP-MS profiles of labeled Se in cold-trapped exhaled gas. DMSe originating from (a′) endogenous Se (78Se) without the administration of the Se mixture (untreated) and (a) endogenous Se (78Se), (b) 76Se-MeSeCys, (c) 77Se-SeMet, and (d) 82Se-MSA with the administration of the Se mixture (treated), detected at a retention time of 2.98 min. Exhaled gas was trapped for 1 h from an untreated rat (0-1 h after the administration of Se-free water) and a treated rat (0-1, 1-2, and 2-3 h after the administration of the test sample).

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Figure 2. Quantitative determination of DMSe originating from endogenous Se (78Se) and exogenous 76Se-MeSeCys, 77Se-SeMet, and 82 Se-MSA in the exhaled gas of rats. Exhaled gas with artificial ventilation was trapped in 1 h increments up to 3 h after oral administration of the test sample. *Significantly different from 77SeSeMet (n ) 3). Figure 5. Concentrations of Se-sugar and TMSe in urine (a) and percentage of Se-sugar and TMSe to total urinary metabolites originating from each labeled Se (b) after the administration of selenocompounds. *Significantly different from 77Se-SeMet (n ) 3).

Figure 3. Excretion of labeled Se in urine (6 h) after the administration of 76Se-MeSeCys, 77Se-SeMet, and 82Se-MSA. *Significantly different from 77Se-SeMet (n ) 3).

tion of DMSe peaked 1-2 h after the administration of all of the selenium compounds, although only a small amount of 77Se-

DMSe originating from 77Se-SeMet was detected in this time period. No volatile Se compound except DMSe was observed in the exhaled gas. The amounts of Se exhaled for 3 h after sample administration were 0.60 ( 0.31, 1.49 ( 0.19, 0.21 ( 0.13, and 2.38 ( 0.61 ng Se for endogenous (78Se), 76Se-, 77Se-, and 82Se-DMSe, respectively (Figure 2). Therefore, the percentage of DMSe exhaled for 3 h following the administration of each original Se compound was 82Se-MSA, 0.062% > 76SeMeSeCys, 0.039% . 77Se-SeMet, 0.005%. Urinary excretion of Se is shown in Figure 3. Although all Se tracers were metabolized and excreted into the urine, the order of the urinary excretion rate was 76Se-MeSeCys > 82SeMSA > 77Se-SeMet. SeMet is known to be incorporated into proteins as a methionine analogue, but MSA and MeSeCys are not directly incorporated. Thus, SeMet is presumed to be more slowly excreted as compared to the other two Se compounds. The chemical forms of the selenium metabolites in the urine were determined by HPLC-ICP-MS (Figure 4). The major

Figure 4. Elution profiles of labeled Se in urine. Elution profiles of Se-sugar and TMSe were shown in the inset (m/z 78, natural abundance).

MeSeCys Is a Natural Methylselenol-Generating Agent

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Scheme 2. Proposed Metabolic Pathway for Se Compoundsa

a

MeSeCys and MSA generate methylselenol efficiently than SeMet. Accordingly, larger amounts of DMSe and TMSe generated from MeSeCys and MSA were excreted into exhaled gas and urine when MeSeCys and MSA were administered. Solid and dotted arrows indicate major and minor routes of metabolic pathways, respectively.

urinary metabolite was Se-sugar, with a smaller amount of TMSe, for all of the administered Se compounds. It is interesting to note that the urinary excretion of 78Se-selenosugar (endogenous) was also increased. One possible explanation for this phenomenon is that the administered 76,77,82Se were utilized, and endogenous Se (78Se was monitored) was excreted as the excess in the form of Se-sugar. DMSe in the exhaled gas was not detected in the untreated, but Se-sugar was excreted into the urine before sample administration (Figure 4, untreated). The peaks with retention times of 15-20 min were not identified in the present study. Percentages of urinary excretion of Se-sugar and TMSe are shown in Figure 5. Excretion of Se-sugar originating from Se tracers in the urine did not vary largely among the Se compounds (63.3-69.3%). On the other hand, the percentage of TMSe originating from 82Se-MSA and 76SeMeSeCys was much higher than that originating from 77SeSeMet.

Discussion MSA and SeMet are commonly used as methylselenolgenerating agents for in vitro (7, 8) and in vivo (15, 32) experiments, respectively. MSA generates methylselenol by chemical reaction with reductants (e.g., GSH), while SeMet generates methylselenol by enzymatic reaction with γ-lyase. MeSeCys has also been shown to generate methylselenol by enzymatic reaction with β-lyase. Methylselenol, a putative intermediate in Se metabolism, has not previously been directly detected; however, its further methylated forms, DMSe and TMSe, are relatively stable. Therefore, we quantitatively measured DMSe in exhaled gas and TMSe in urine simultaneously to estimate the efficiency of methylselenol generation from Se compounds. DMSe is a volatile Se compound (bp 58 °C). Even though aqueous samples containing DMSe were treated at low temperatures, there seemed to be some loss of DMSe (e.g., when the same ice-cold DMSe aqueous sample was measured repeatedly by GC-ICP-MS, the peak intensity gradually decreased). Hence, metabolic differences among SeMet, MeSeCys, and MSA were compared using tracers labeled with different

stable isotopes (76Se-SeMet, 77Se-MeSeCys, and 82Se-MSA). Given that some 76Se-, 77Se-, and 82Se-DMSe was lost due to volatilization from the trapped exhaled gas, the ratio of 76Se-, 77 Se-, and 82Se-DMSe should not have been changed. A larger amount of 76Se-MeSeCys and 82Se-MSA was excreted as DMSe in the exhaled gas as compared to SeMet. Similarly, MSA and MeSeCys were more efficiently excreted as TMSe into urine as compared to SeMet. SeMet was probably not efficiently converted to DMSe and TMSe because SeMet is not only incorporated into proteins as a methionine analogue (5, 33) but is also metabolized to selenide rather than methylselenol by trans-selenation pathways, which are similar to trans-sulfuration pathways (5, 33). It has been reported that SeMet is excreted as DMSe due to direct methylation of SeMet to Se-methyl selenomethinine by a unique methylation pathway in Se-accumulating plants, although this pathway has not been observed in mammals (34). There might be another metabolic pathway that generates DMSe by direct methylation of MeSeCys to Se-dimethyl selenocysteine in mammals. Indeed, when Sedimethyl selenocysteine was administered to rats, a large amount of DMSe was excreted (23). Although we do not deny involvement of the MeSeCys-methylation metabolic pathway (i.e., MeSeCys f Se-dimethyl selenocysteine f DMSe), this pathway cannot play a key role, if any, because Se-dimethyl selenocysteine has not been detected in mammals or plants. MeSeCys generates methylselenol, a Se compound with putative anticancer activity (35-37), more efficiently than SeMet, as estimated by excretion of TMSe and DMSe in exhaled gas and urine. Therefore, MeSeCys could be useful as an alternative to SeMet, a currently used methylselenol-generating agent. It is worth noting that MeSeCys was excreted into urine as Se-sugar as efficiently as SeMet. SeMet may be converted to Se-sugar through not only the γ-lyase pathway but also via a transselenation pathway, which does not involve methylselenol. On the other hand, MeSeCys may be converted to Se-sugar mainly through the β-lyase pathway where methylselenol is generated (Scheme 2). These results suggest that β-lyase is more active than γ-lyase. SeMet was adopted into the Selenium and Vitamin E Cancer Prevention Trial (SELECT), one of the largest prostate

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cancer prevention trials. Although selenized yeast containing SeMet as a major selenium species reportedly prevents prostate cancer, the SELECT study suggests that SeMet at a daily dose level of 200 µg is not effective for cancer prevention (38). Selenized yeast contains not only SeMet but also other more active Se substances like γ-glutamyl-MeSeCys. Other methylated selenium compounds such as γ-glutamyl-MeSeCys merit evaluation (39). The present study suggests that low-dose Se supplementation with MeSeCys could decrease the incidence of prostate cancer by generating methylselenol more efficiently than SeMet. In conclusion, the excretion of DMSe and TMSe is clearly linked with the ingested chemical form of Se. Accordingly, urinary TMSe or exhaled DMSe can be reliable biomarkers of the generation of cancer preventive methylselenol following the ingestion of Se compounds.

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