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Characterization, biological interactions and in-vivo detection of selenotrisulfide derivatives of glutathion, cysteine and homocysteine by HPLC-ICP-MS{ Paula Braga,a Marıá Montes-Bayoń,a Jesu´s Alvarez,b J. Manuel Lo´pezb and Alfredo Sanz-Medel*a a

Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Juliań Claverıá 8, 33006 Oviedo, Spain. E-mail: [email protected] b Department of Morphology and Cellular Biology, Faculty of Medicine, University of Oviedo, C/Juliań Claverıá 6, 33006 Oviedo, Spain Received 18th February 2004, Accepted 5th May 2004 First published as an Advance Article on the web 9th August 2004

Thiol-containing compounds such as glutathione, cysteine and homocysteine react with selenite under specific conditions to form selenotrisulfides of significance in cellular systems. This study describes the synthesis and mass spectrometric characterization of selenotrisulfides of glutathione (GSH), cysteine (Cys) and homocysteine (HCys) by electrospray-MS (ESI-MS). The synthesized species are separated by reversed phase-ion pairing HPLC and detected by selective monitoring of Se (m/z 77, 78 and 80) using a collision/reaction cell-inductively coupled plasma-mass spectrometer (CRC-ICP-MS). The cell is pressurized using H2 as reaction gas at a flow of 3.5 ml min21 for minimizing Ar polyatomic ions affecting m/z 78 and 80. Separation of the species is performed in a C8 column with a mobile phase containing 12% methanol and 0.05% heptafluorobutyric acid (HFBA). Using this hyphenated system (HPLC-ICP-MS) LDs below 1 ppb were attained for all the species under study. HPLC-ICP-MS was used also as a tool to study the presence of an excess of GSH and the pH influence on the stability of the Se–glutathion conjugate (GS–Se–SG) as model. Similarly, GS-Se-SG is evaluated as a possible substrate for the enzyme glutathione reductase (GR) in the presence of NADPH. The isolation and HPLC-ICP-MS detection of the synthesized selenotrisulfides in rat liver cytosols of exposed animals will be also discussed.

DOI: 10.1039/b402478h

Introduction

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Selenium (Se) is an essential element, with important physiological functions in the organism, that is obtained from dietary sources including cereals and vegetables.1,2 However, Se bioavailability, defined as the proportion of the ingested nutrient that is used for normal physiological functions or storage, depends on the dosage as well as on the species supplemented.3,4 In this regard, Se-methionine (Se-met) has shown itself to be absorbed and retained more efficiently than inorganic selenate or selenite. This could be ascribed to the possible non-specific incorporation of Se-met into proteins due to the inability of tRNAMet to discriminate between methionine and Se-methionine.2 In the case of the inorganic species (SeIV and SeVI), a different metabolic pathway has been proposed for Se assimilation in analogy to sulfur metabolism. In the proposed mechanism of incorporation, SeVI is presumed to be activated by ATP sulfurylase to adenosine phosphoselenate, which is then reduced to selenite in an enzymatic pathway.5 However, despite the fact that activation of ATP sulfurylase in the presence of SeVI has been observed, none of the P–Se containing species have ever been isolated or characterized. SeIV, on the other hand, reacts with sulfydryl compounds to yield selenotrisulfides, which were first described by Painter.6 Initial in-vitro studies carried out by Ganther7 demonstrated that using reduced glutathione (GSH), a tripeptide present in most animal tissues, it was possible to isolate the GS–Se–SG conjugate. The reaction took place in a molar ratio of 4 : 1 (GSH : Se) under acidic conditions and the product undergoes decomposition at higher pH values. Since glutathione is the { Presented at the 2004 Winter Conference on Plasma Spectrochemistry, Fort Lauderdale, FL, USA, January 5–10, 2004. J. Anal. At. Spectrom., 2004, 19, 1128–1133

most abundant biological thiol in cells (7.5 mM in hepatocytes and 3.0 mM in red blood cells), the formation of the conjugate GS–Se–SG has been proposed by some authors as intermediate in the reduction of SeIV in-vivo.8 In this sense, the reactions of selenite with various low molecular weight thiols like Cys to form selenotrisulfides, followed by enzymatic reactions with flavine-dependent disulfide reductases like glutathione reductase7 (GR) or thioredoxin reductase,9 might feed into the pathway for selenocysteine formation, the key compound for Se–protein biosynthesis.1,10 Furthermore, GS–Se–SG has shown anticarcinogenic activity through the inhibition of cell proliferation and apoptosis of tumoral cells.11,12 The last seems to be directly correlated to the production of the superoxide anion (O2?2) as a consequence of H2Se formation through the stepwise reduction of GS–Se–SG forming GS–Se–H (see Fig. 1).11 Initial in-vivo studies by Gyurasics et al.13 (injecting radioactive 75Se into rats) described the formation of a Se-trisulfide

Fig. 1 Schematic illustration showing the metabolic reduction of SeIV with GSH. SeIV is a precursor of methylselenol through the formation of hydrogen selenide.

This journal is ß The Royal Society of Chemistry 2004

in the bile of the exposed animals. However, the lack of selectivity among Se-trisulfides in the chromatographic separation did not allow the unequivocal characterization of the species. Recently, Lindemann et al.14 found evidence of the presence of GS–Se–SG in SeIV enriched Saccharomyces cerevisiae by mass spectrometric techniques (HPLC-ICP-MS and ESI-Q-TOF) and confirmed its structure by collisionally induced dissociation. In the light of this lack of evidence for Se-trisulfide complex formation in living organisms, the main goal of this study is the identification of the Se-trisulfides formed during SeIV metabolism in rat liver cytosol. For this purpose, the initial synthesis and characterization by ESI-MS of the different Se-conjugates likely to be present in biological systems has been performed. Once synthesized, those species have to be chromatographically separated since some of them could be present simultaneously in the biological systems. Thus a selective chromatographic method for the separation of the different conjugates has to be developed. The reactivity characteristics of GS–Se–SG in a biological system are evaluated in terms of stability with pH, decomposition in the presence of excess of GSH and interaction with enzymes present in the corresponding biological media (e.g., GR); they are investigated using elemental speciation of Se by HPLC-ICP-MS. The presence of the selenotrisulfides in such mammalian tissues (rat liver cytosols) as SeIV injected animals are finally studied using the developed methodology.

Experimental

Chemicals SeIV for the synthesis of the Se-trisulfides was obtained by dilution of the standard of H2SeO3 (1000 mg ml21) from Merck (Darmstadt, Germany) in 0.1 M HCl (Merck) to a final concentration of 100 mg ml21 (as Se). Reduced glutathione, cysteine and homocysteine were obtained from Fluka (GSH) and Aldrich, respectively (Sigma Chemical Company, St. Louis, MO, USA). These compounds were diluted in Milli-Q water (18 MV cm) (Millipore Co., Bedford, MA, USA) previously de-oxygenated by flushing He through for 15 min at a final concentration of 0.1 M (as compound). The mobile phase for HPLC contains 0.05% heptafluorobutyric acid (HFBA) from Sigma and 12% MeOH (Merck) (pH ~ 3) and the chromatographic flow is 1 ml min21 in the reversed phase system. For SEC, 0.7 ml min21 of 50 mM Tris-HCl buffer (pH ~ 7) was used as mobile phase. Tris(hydroxymethyl)aminomethane (TRIS) for sample homogenization, phenylmethane sulfonyl fluoride (PMSF), used as proteinase inhibitor, GR from yeast and NADPH were purchased from Aldrich. Procedures

Instrumentation The ESI-MS system is a HP 1100 Series LC/MSC by Agilent (Agilent Technologies, Waldbronn, Germany) with a quadrupole mass analyzer and API-Electrospray 61948 A as sample introduction system. Selenium was monitored on-line by ICPMS using a system from Agilent, Model 7500c (Agilent Technologies, Tokyo, Japan), which enables the reduction of spectral interferences by the addition of helium/hydrogen in a collision/reaction cell. Optimized operating conditions are summarized in Table 1. A Shimadzu HPLC pump was used as the solvent delivery system (Shimadzu LC-10AD, Shimadzu Corporation, Kyoto, Japan) and injections were made using an injection valve (Model 7125) with a 100 ml injection loop (Rheodyne, Cotati, CA, USA). Separation of the Se-trisulfides was carried out in a reversed phase chromatographic column Luna C8 (2) 5 mm (250 6 4.6 mm) especially designed to run at acidic pH (Phenomenex, Table 1 Final operating conditions for ICP-MS, ESI-MS and reversed phase HPLC ICP-MS parameters— Forward power External flow Internal flow Carrier gas flow Selected isotopes Dwell time Shield torch Collision/reaction gas Flow ES-MS parameters— Capillary voltage/kV Cone voltage/V Nebulizing gas HPLC parameters— Reversed-phase ion pairing Column Mobile phase Flow rate Injection volume

CA, USA). The column was connected directly to the ICP-MS nebulizer through orange PEEK1 tubing. The size exclusion chromatographic column was a Superdex 200 HR 10/30 (Pharmacia Biotech, Uppsala, Sweden) with an optimum separation range of 10–600 kDa. The centrifuge employed for sample treatment was a Biofuge stratos Heraeus (Kendro Instruments, Hanau, Germany).

1500 W 15 L min21 1.0 L min21 1.12 L min21 77 Se, 78Se, 80Se 0.2 On H2 3.5 ml min21 3 30 N2 C8 (2) (4.6 6 250 mm) 0.05% HFBA and 12% MeOH 1 ml min21 100 ml

Synthesis of the Se-trisulfides. The reactions are only slightly different from that proposed by Ganther et al.7 for SeIV and GSH. In brief, 1 ml of selenite (100 mg ml21) is mixed with 50 ml of 0.1 M ice cold GSH solution made up in de-oxygenated water. The mixture was kept in ice for 15 min under a N2 stream to avoid oxidation of the sulfides and then stored at 220 uC. An aliquot of this solution was diluted 100-fold before HPLC analysis. Similar incubation was performed using Cys, HCys and a mixture of Cys–GSH to prepare the analogous selenotrisulfides which might be also formed in-vivo. Enzyme assays. The reaction between GS–Se–SG and GR from yeast (0.2 units of the enzyme) was performed in 50 mM Tris-HCl (pH 5, 6 and 7.5), 1 mM EDTA and 200 mM NADPH. The reaction takes place for 5 min at room temperature and the peak area of the remaining GS–Se–SG is quantified. Sample treatment. Female Wistar rats (5 weeks old) were fed a standard diet plus tap water. Rats were injected intraperitoneal once with a SeIV solution containing 20 mg L21 Se and at a dose of 3 ml kg21 body weight. After 0.5, 1, 2 and 4 hours the livers were excised and washed with 50 mM TRIS-HCl (pH 6) and then homogenized in 10 ml of the same buffer with a Polytron homogenizer (Politron1 PT 3000 Kinematica AG, Littau, Lucerne, Switzerland). The homogenates were centrifuged with a centrifuge at 20 000 g for 30 min at 4 uC. Total Se determination in the supernatant (diluted 1 : 10) was performed by external calibration in the ICP-MS using Ge (10 ng ml21) as internal standard. For speciation, the supernatant was treated once with 2 ml of 100% methanol and centrifuged for 15 min and the precipitate re-extracted with 80% methanol for another 15 min as described by Gyurasics et al.13 The methanolic extracts were then combined and pre-concentrated under a N2 stream. Glutathion reductase activity measurements. GR catalyzes the reduction of GSSG (or GS–Se–SG) by oxidation of J. Anal. At. Spectrom., 2004, 19, 1128–1133

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NADPH to obtain NADP1 and GSH. The activity of the enzyme is measured by monitoring the decrease in absorbance at 340 nm due to the oxidation of NADPH using a spectrophotometer and determined with a millimolar extinction coefficient of 6.22. The absorbance of the reference cell was automatically subtracted. The results are expressed as nmol NADPH per mg of protein. Total protein content in the liver tissue is calculated by Bradford assay using bovine albumin as standard.

Results and discussion Synthesis and chromatographic separation of Se-trisulfides Studies on the metabolism of SeIV have shown that this species undergoes reductions in animals through reaction with thiol compounds as cysteine, glutathione or homocysteine.1 The most abundant biological thiols in mammals are the tripeptide glutathione (L-c-glutamil-L-cysteinylglycine) and L-cysteine. Two human cell types have particularly high intracellular GSH concentrations: red blood cells (3 mM GSH) and hepatocytes (7.5 mM GSH). Cysteine is also commonly found in mammalian cells, although in concentrations about one order of magnitude lower than that of GSH. Therefore, complexes of the type Cys–Se–Cys, GS–Se–SG and GS–Se–Cys are expected to be formed in most cellular systems. Homocysteine, on the other hand, a scarcer amino acid (5–15 mM in plasma) also involved in the methionine mechanism,15 exists as the free amino acid in blood and tissues and could also possibly react with SeIV. Fig. 2 (A, B and C) shows the observed ESI-MS spectra corresponding to the formation of the Se-trisulfides of GSH, Cys and HCys, respectively, synthesized as described in the procedures section using a 4 : 1 molar ratio thiol/selenium (considered optimum for GSH in previous studies). In all cases, it is possible to observe the isotope pattern of Se in the corresponding protonated molecular ions (at m/z 692.9, 348.8 and 320.9, respectively). Observing the whole mass spectrum corresponding to the synthesized GS–Se–SG complex (Fig. 2(A)) it is also possible to identify the dimer GS–SG as a reaction product (protonated molecular ion m/z 613.0) and the unreacted GSH (protonated molecular ion m/z 308). However, no other Se-containing species seemed to be produced as a result of our synthesis experiments. Similar results can be observed in the synthesis of

the Cys and HCys conjugates. In both cases, the Cys–Cys and HCys–HCys protonated molecular ions were observed. When the most abundant biological thiols (GSH and Cys) were mixed with SeIV the Se-trisulfide combination of GS–Se–Cys was observed along with GS–Se–SG and Cys–Se–Cys. GS–Se–Cys shows a protonated molecular ion of 506.8 and exhibits the Se isotope pattern. Since all these thiols can be present simultaneously in biological systems, it was necessary to develop a chromatographic method for the separation of the different conjugates. In this sense, ion-pairing reversed phase HPLC has proved to be very adequate for the separation of several Se species present in natural products using perfluorinated ion-pairing reagents as counter-ions.16,17 Thus, HFBA was selected as the counter ion fluorinated agent with an optimum concentration of 0.05% (pH 3) and 12% final concentration of MeOH. Using these separation conditions and ICP-MS as Se selective detector, and monitoring m/z 77, 78, 80, the chromatograms shown in Fig. 3 were obtained at the three masses. Final operating conditions required 3.5 ml min21 H2 as cell gas to decrease the interference 40 Ar40Ar1 over 80Se to about 170 cps. SeIV (spiked in the mixture after synthesizing the Se-trisulfides) eluted in the void volume while Cys–Se–Cys (tr 6.5 min), GS–Se–Cys (tr 13.5 min)

Fig. 3 Chromatographic separation of the SeIV conjugates with GSH and Cys using ICP-MS detection (100 ppb as Se per compound). The thin, continuous trace corresponds to 77Se, the broken line to 78Se and the continuous thick trace corresponds to 80Se. The mobile phase contained 0.05% HFBA and 12% MeOH. The inset shows the mass spectra (ESI-MS) of the mixed trisulfide of GS–Se–Cys.

Fig. 2 Mass spectra obtained by ESI-MS of the synthesized conjugates of SeIV with (A) glutathion (GSH), (B) homocysteine (HCys) and (C) cysteine (Cys). 1130

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Table 2 Analytical performance characteristics and retention time of the species Species

Cys–Se–Cys

Cys–Se–SG

GS–Se–SG

Retention time/min 6.5 13.5 32.5 0.5 0.7 0.9 RSDrett (%)a 0.1 0.2 1 LOD/ng ml21b b 6.3 6.7 7.5 RSD (%) 1 321 507 693 ESI-MS (M 1 H) a Calculated for three manual injections and estimated as peak height. b As 80Se and estimated as peak height.

and GS–Se–SG (tr 32.5 min) were clearly separated (see Fig. 3). The inset shows, as an example, the mass spectrum of the conjugate GS–Se–Cys obtained by ESI-MS at m/z 506.8. The limits of detection of the Se species in the HPLC-ICPMS system ranged from 0.1 ppb for Cys–Se–Cys to 1 ppb for GS–Se–SG as Se (peak height) with a precision of about 6% for three manual injections of each of the compounds, as can be observed in Table 2. As individual injections of the different synthesized species showed no other Se signal than that one ascribed to the desired compound, final calculation of DLs was carried out assuming 100% Se going to the corresponding Se– trisulfide. When Cys and GSH are mixed with SeIV, the peak area distribution among species is 15 : 44 : 41 (Cys–Se–Cys/ Cys–Se–Glu/Glu–Se–Glu). Considering this, a formal DL for the mixed trisulfide (GS–Se–Cys) is 0.2 ppb as Se (as peak height). Stability of Se-trisulfides at varying pH using GS–Se–SG as model Previous reports on GS–Se–SG stability showed that this species seems to be relatively unstable at physiological (neutral) pH.7,18 However, most of such studies have been carried out by measuring volatile species formation (H2Se) rather than by selective monitoring of the GS–Se–SG species itself. Therefore, different aliquots of the latter complex were diluted using MilliQ water and Tris-buffer, to obtain final pH values of 3.5, 6 and 7.5. Fig. 4 shows the chromatograms for GS–Se–SG at the three pH values and, as can be seen, significant decomposition takes place at pH 6 (the peak area is about 60 ¡ 5% of that seen at pH 3.5) accompanied by the appearance of new unknown Se containing species. This is even more dramatic at pH 7.5, where the peak area of the compound is approximately 10 ¡ 2 % of the initial Se–diglutathione peak area, as can be seen in the inset. Therefore, Se–trisulfides speciation should secure slightly acidic conditions in order maintain such species integrity. Within the cell, the integrity of GS–S–GS could be preserved

Fig. 4 Chromatographic profile of GS–Se–SG obtained by HPLCICP-MS (80Se) at different pH values. In all cases the synthesis is carried out in acid conditions and the final product diluted in Milli-Q (final pH 3.5) and Tris-HCl (final pH 6 and 7.5, respectively). The inset shows the peak area decrease versus pH.

in certain organelles such as lysosomes that exhibit low pH values (around pH 5.0). Moreover, lysosomes have been reported to be rich in glutathion, enhancing the formation of the compound. In this regard, there are two different ways by which small molecules can enter the cell: the first involves the pass across the plasma membrane leading directly to the cystosol (physiological pH). The second involves the so called ‘‘endocytic pathway’’. Many small molecules (including cholesterol) enter the cell via this pathway, and this could be also the case with the GS–Se–SG conjugates. The endocytosed molecules are then delivered into the endosomes (which inner part is at pH 6) that later led to the lysosomes characterized by lower pH. Enzymatic and non-enzymatic reduction of Se-diglutathion It has been documented by several authors1,7,19 that GS–Se–SG can be further reduced by GSH to H2Se via GS–Se–H formation as follows: The first non-enzymatic reaction between glutathione and selenous acid produces GS–Se–SG as a stable product. Once attached to the glutathione moiety, selenium could undergo further reduction by an excess of GSH to a labile intermediate called selenopersulfide (GS–Se–H). On the other hand, GS–Se–SG is also a substrate for the action of certain enzymes such as glutathione reductase7 (GR) or thioredoxin reductase9 (TrxR), present in most mammalian tissues. In fact, this has been proposed as a mechanistic explanation for some of the physiological actions of selenium compounds on cell proliferation.2 Since both enzymes catalyze the reduction of Se–diglutathion by oxidation of NADPH to NADP1 in living systems while excess of GSH is also expected in most mammalian cells, both bio- and chemical reduction mechanisms might take place in-vivo. Therefore, an in-vitro study has been performed using Yeast GR (as model enzyme) to investigate chemical and biochemical GS–Se–SG reduction pathways. Chemical reduction. In the case of the addition of an excess of GSH, two different reagent concentrations (2- and 4-times the stoichiometric amount of GSH) were tested and the effect on the GS–Se–SG peak area was evaluated. The addition of 2-fold excess concentration of GSH resulted in a decrease of the peak area of GS–Se–SG of about 35% with respect to that obtained for the stoichiometric ratio (4 : 1). However, no further significant suppression on the GS–Se–SG signal was observed when adding 4-times the initial concentration of GSH, in agreement with results obtained by other authors.7 Therefore, the presence of an important excess of GSH in most cellular systems is not going to limit significantly the detection of GS–Se–SG in such systems. Biochemical reduction. The effect of GR in the reaction media was also studied. The most important role of this cystosolic enzyme is to maintain the level of intracellular GSH by reduction of GS–SG, which is further employed in the elimination of peroxide radicals through the action of another enzyme, glutathione peroxidase (GPx).20 GR shows optimum activity at pH 7. Unfortunately, in these conditions, an important decomposition of the synthesized GS–Se–SG occurs, as described in the previous section. Therefore, in this case, a more careful study of the GS–Se–SG peak area versus pH in the presence of the enzyme was required. To test the ability of GR to reduce GS–Se–SG, reaction mixtures containing 100mM Tris buffer (pH 5, 6 and 7), 1 mM EDTA, 0.2 mM NADPH and 0.1 mM GS–Se–SG were supplemented with yeast GR and left to react for 5 min. Results showed that the enzyme still exhibits activity at pH 6 while decomposition of the GS–Se–SG species is not extensive. Fig. 5 shows the chromatographic profile observed for the synthesized J. Anal. At. Spectrom., 2004, 19, 1128–1133

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Fig. 5 Reduction of GS–Se–SG by yeast GR at pH 6. The initial reduction of the peak area of the compound due to the pH is even more dramatic when the enzyme is present in the reaction. The inset shows the full scale chromatogram.

GS–Se–SG at pH 6 with and without enzyme: a signal decrease of approximately 50% of the initial peak area in the presence of GR is apparent. The inset corresponds to the full scale chromatogram showing a high intensity Se-containing species which elutes on the void volume. This signal could be ascribed to the formation of volatile H2Se, which is highly increased in the presence of GR, both in aerobic and anaerobic conditions. At pH 7.5 in the presence of GR in the reaction medium, no detectable signal of GS–Se–SG could be observed in the chromatogram (after the 5 min of reaction), pointing out the efficient catalytic reduction of this species by the enzyme.

Presence of Se-trisulfides in rat liver cytosol In order to investigate if GS–Se–SG was detectable by the proposed hybrid methodology in real samples of rat liver cytosol, Wistar female rats were injected (intraperitoneal) with 3 ml kg21 of SeIV and sacrificed after different exposure times (0.5. 1, 2 and 4 h). The liver was subsequently extracted for total Se determination before further speciation experiments. The homogenization of the tissues was performed in Tris-HCl buffered at pH 6 in order to minimize protein precipitation and simultaneously preserve, as much as possible, the integrity of the sought species unstable at physiological pH (Fig. 4). In agreement with Suzuki et. al.21 we observed that Se concentration in the liver tissue increased with time after injection, reaching a maximum of 394 ¡ 45 ng g21 Se (in the tissue) after 2 h (average of three individuals). Then, Se concentration decreased in the liver tissue, probably due to its metabolism and/or excretion in urine. Therefore, 2 h after injection was selected for GS–Se–SG speciation experiments. In order to study the Se distribution among different compounds present in the liver tissue, initial SEC-ICP-MS experiments were performed in the homogenized tissue. Fig. 6 shows the chromatographic profile (34S and 80Se) obtained by size exclusion chromatography (SEC) of the homogenized liver tissue using 50 mM Tris-HCl (pH 6) as the mobile phase at 0.7 ml min21. Several high molecular weight Se/S containing species, presumably proteins, can be observed (retention times ranged from 12 to 28 min) with molecular weights ranging from 500 to 50 KDa. Also, there is an important narrow peak corresponding to some low molecular weight Se/S containing species in the region where the used column does not show specific retention (30–35 min). The addition of SeIV to that homogenized tissue followed by immediate SEC separation showed most Se to be in the low molecular weight fraction, as expected, and very little incorporated into the proteins (see Fig. 6). These findings agree with the in-vitro experiments reported by Suzuki et al.22 spiking SeIV in red blood cells where 1132


Fig. 6 Size exclusion chromatography-ICP-MS of the rat liver cystosol exposed to SeIV. Selenium traces (80Se) (a and b) correspond to the homogenized tissue (a) and to the same tissue plus a spike of 20 ppb Se (b). The sulfur trace (34S) belongs to the non-spiked tissue.

a fast SeIV reduction followed by partial unspecific incorporation into proteins was observed. Once the liver was processed (after protein precipitation), as described in Procedures, the separation of the sought species was performed on the C8 column. The chromatographic profile observed is given in Fig. 7 for two different samples on two different days. In the 80Se channel, it is possible to observe the presence of some inorganic species eluting in the void volume, as well as some other unknown species eluting at about 9 min containing S (not shown) and Se. It is also possible to observe the presence of Cys–Se–Cys at 6.5 min and traces of GS–Se–SG at about 32 min by matching retention times with the standards (see Fig. 3). These chromatographic results are reproducible among individuals, as can be seen in Fig. 7. Further spiking of synthesized GS–Se–SG to the real tissue sample provided confirmation of the presence of the sought species by a noticeable increase of the peak area at 32 min indicating the in-vivo presence of such a complex. However, since only low levels of the expected trisulfides

Fig. 7 Reversed phase chromatography-ICP-MS chromatograms of two different rat liver cytosols after being processed to obtain the Se metabolites of interest. It is possible to observe the presence of the Cys– Se–Cys and traces of GS–Se–SG and Cys–Se–SG. The upper chromatogram is off-axes for clarity.

were observed, we resorted to the measurement of the activity of the Glutathion Reductase in the liver. Such experiment could provide an estimation of the possible increase of the enzymatic activity due to the presence of higher concentration of Se in the tissues. Unfortunately, the obtained results (referred as activity per mg of protein in the liver tissue) were not significantly different from those observed for the control animals (8.7 ¡ 1.1 versus 10.6 ¡0.06 in the control). In other words, a higher GR enzymatic activity does not seem to be the only mechanism for the observed low GS-Se-SG levels. Complex consumption could be ascribed also to a rapid metabolization of this species which has exhibited an absorption rate ten times higher than that of the Se (IV) in mammalian tissues.23

Conclusions Synthesis, characterization by ESI-MS and chromatographic separation of several Se-trisulfides has been accomplished. The separation of such species followed by specific Se detection by ICP-MS has provided an extremely valuable tool to study the great reactivity and the in-vitro physiological interactions of these Se/S compounds. The reaction of SeIV with SH groups has proved to be strongly pH dependent and the reaction products (e.g., GS–Se–SG) are known to be substrates for the action of some flavine-dependent enzymes like glutathion reductase (with fast reaction kinetics). Then, isolation and characterization of the selenotrisulfide species in in-vivo systems, such as rat liver cystosols, could be accomplished by using the developed hybrid HPLC-ICP-MS techniques. In any case, the observed selenotrisulfide levels are too low for characterization of the species by ESI-MS in the tissue. This experiment would probably require the inhibition of the activity of such enzymes in order to secure S/Se species levels in the tissue high enough for electrospray characterization.

Acknowledgements The authors thank Juris Meija for valuable discussions and the Ministry of Science and Technology of the Spanish Government for financial support under the Project

MCT-00-BQU-0468, Ramon y Cajal contract of M. MontesBayoń. Financial support of J. Alvarez by the IUOPA is also acknowledged. Also thanks are expressed to the Regional Government of Asturias for financial support under the project PR01-GE8.

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