LC-ICP-MS and LC-ESI-(MS)ⁿ identification of Se-methylselenocysteine and selenomethionine as metabolites of methylseleninic acid in rat hepatocytes

Abstract

The metabolism of methylseleninic acid in isolated rat hepatocytes was investigated. Selenium containing metabolites excreted from the cells were detected in the supernatant of the incubation sample by LC-ICP-MS. After pre-treatment of the supernatant by preparative chromatography and pre-concentration by lyophilisation, a major metabolite was identified by molecular mass spectrometry as Se-methylselenocysteine by LC-ESI-MS, MS² and MS³ and a minor metabolite was identified as selenomethionine by LC-ESI-MS² and MS³ and LC-ESI-MS²(SRM). This is the first time these metabolites have been identified in hepatocytes. Complementary data from ion trap and triple quadrupole MS instruments provided solid proof of metabolite identities. A time course study showed that S-(methylseleno)cysteine and S-(methylseleno)glutathione were intermediates in the formation of the major metabolite. It is questioned if methylseleninic acid is a relevant model compound for methylated Se-amino acids in vitro.

---

Introduction

Clinical trials and epidemiological studies in humans have shown that selenium is a cancer protective agent.^1,2 This has led to an intensive research effort in order to identify the active species involved in the cancer protective mechanism. Based on an in vivo experiment in a chemically induced rat mammary tumour model, Ip and Ganther suggested that partially methylated selenium species may be directly involved^3,4 in selenium bioactivation. Methylselenol (MeSeH) has been suggested as a key metabolite in selenium cancer chemoprevention.⁵ However, it has never been isolated nor identified, probably because it is far too reactive and volatile to be handled as is. Se-methylselenocysteine (Se-MeSeCys) and methylseleninic acid (MeSeA) are suggested precursors for MeSeH formation. Se-MeSeCys is believed to produce MeSeH when cleaved by the β-lyase enzymes and MeSeA is believed to produce MeSeH by a series of reductions by thiols such as cysteine and glutathione.⁶ Hence, MeSeA has been widely used in cancer research as a precursor for MeSeH in studies performed both in vitro and in vivo.^6–11 To assess the relevance of using MeSeA as a model compound, it is important to know if it is metabolized in a similar way to nutritionally available selenium species.

The analytical challenge in metabolism studies is related to the often very complex matrices of the samples and the low concentration of the metabolites of interest. Although coupling of LC to ICP-MS is not entirely straight forward,¹² the combination of efficient separation of sample components and the good sensitivity offered by the element specific ICP-MS detector provides an excellent tool for detection of selenium containing compounds. Molecular MS provides the structural information that is inherently lost upon LC-ICP-MS analysis. It is the analytical approach of choice for elemental speciation to detect species by elemental analysis followed by structural identification of the detected species by molecular MS.¹³ The use of LC-ICP-MS in selenium speciation analysis was reviewed by B'Hymer and Caruso¹⁴ and a thorough review of the use of molecular MS in speciation analysis was written by Rosenberg.¹⁵ Selenium compounds related to selenium metabolism identified by the complementary use of element- and molecular specific mass spectrometry analysis were recently reviewed.¹⁶

The metabolism of selenium is far from completely elucidated. A selenium metabolism model was originally proposed by Ganther¹⁷ and is generally accepted although all steps have not been verified. The model describes the metabolism of two seleno-compounds: selenomethionine (SeMet)—the main selenium compound in plants and selenized yeast—and selenite, which is not present in food in larger amounts but is the seleno-compound in many nutritional preparations. In this model, all selenium species are metabolized into hydrogenselenide, which is considered a common intermediary metabolite for Se-methylation and further incorporation of selenium into Se-amino acids and proteins or for excretion. The end product of selenium metabolism—the main urinary metabolite, regardless of ingested selenium species—has been identified as a selenosugar in rats¹⁸ as well as in humans.¹⁹ Dimethylselenide has been identified in human breath as the main volatile end product of selenium metabolism.^20,21 The metabolism of other organic selenium species such as Se-MeSeCys present in significant amounts in vegetables in particular broccoli, onion and garlic also need to be included in the selenium metabolism model. How the suggested common intermediary hydrogenselenide and methylselenol pools from different selenium species are derived is still not completely resolved.

In a previous study, the metabolism of MeSeA in intestinal epithelial cell homogenates was investigated. MeSeA reacts with cysteine and glutathione from the epithelial cell homogenates to form S-(methylseleno)cysteine and S-(methylseleno)glutathione. The former was identified by LC-ESI-MS and the latter by chromatographic retention time matching with a standard.²² Infante et al. incubated a human lymphoma B-cell line with MeSeA. Three metabolites were identified in the cell extracts by retention time matching with standards as Se-MeSeCys, SeMet and γ-glutamyl-methylselenocysteine, respectively. The identity of the major metabolite, Se-MeSeCys, was verified by LC-ESI-MS²(SRM). Also the volatile species, dimethylselenide and dimethyldiselenide, were identified by GC-MS in the headspace of the incubated cell line.²³

The liver is the major organ of metabolism and ingested species will pass the liver via the portal vein upon absorption before entering the systemic circulation. As hepatocytes contain the full complement of enzymes and cofactors they are physiologically relevant for in vitro metabolism studies.²⁴ Some studies of hepatocytes treated with selenium have been published. Most studies involve selenium as selenite and the focus of the studies have been selenite toxicology rather than speciation of metabolites.^25–28 Both Park and Whanger²⁹ and Ståhl et al.³⁰ observed volatilization of selenium from rat hepatocytes treated with selenite although not to the same extend. Ståhl et al. indirectly identified the volatile species as dimethylselenide. Further speciation of selenium in soluble and insoluble cell fractions was not performed in either study. The objective of the present study was to investigate metabolism of MeSeA in isolated rat hepatocytes by speciation of selenium containing metabolites.

Experimental

Incubations

Hepatocytes were isolated from Sprague Dawley rats (150–200 g) obtained from Charles River Laboratories (Sulzfeld, Germany). The isolation was performed according to the two-step perfusion model described by LeCluyse et al.³¹ and cryopreserved as described by Le Cam et al.³² Isolated hepatocytes were suspended in Dulbecco's modified Eagle's medium containing 10% dimethyl sulfoxide. The suspension was immediately frozen at −20 °C for 20 min followed by 1 h storage at −80 °C before storage in liquid nitrogen. Upon thawing, cryopreserved hepatocytes were suspended in Dulbecco's modified Eagle's medium and incubated for 0, 15, 30, 60 min or 4 h with 5, 10 or 100 µM MeSeA. Cell viability was determined by the trypan blue exclusion method. At the end of the incubation periode, the cells were separated from the medium by centrifugation at 4000g for 5 min. The incubation medium was analyzed directly, while the cell pellet was treated with acetonitrile. Acetonitrile extract was evaporated under a N₂ stream. The residue was sonicated after addition of a solution of 20 mmol L⁻¹ ammonium acetate in 2% methanol. This fraction was separated by centrifugation 1000g for 5 min and the supernatant was analyzed. The remaining pellet was solubilised in 5% sodium dodecyl phosphate by sonification prior to total selenium analysis. All samples were stored at −20 °C until analysis.

Analytical chromatography

All analytical chromatography was performed with a Gemini C18, 5 µm, 110 Å, 2 mm id × 250 mm (Phenomenex, SupWare, Denmark) column at flow rate 200 µL min⁻¹. LC-ICP-MS analyses were performed at room temperature with 20 mol L⁻¹ ammoniumacetate in 2% methanol in addition to the system described for LC-ESI-MS analysis. 15 µL sample aliquots were injected. Time course study and assessment of apolar compounds were analyzed by gradient elution with mobile phases (A) 0.1% formic acid in 2% methanol and (B) 0,1% formic acid in 50% methanol. The linear gradient was 0–100% mobile phase B in 5 min followed by re-equilibration of the column in 100% mobile phase A. LC-ESI-MS analyses were performed at room temperature (about 25 °C) with 0.1% formic acid in 2% methanol. 4–10 µL sample aliquots were injected.

Preparative chromatography

The cell medium sample from incubation of 100 µmol L⁻¹ for 4 h was subjected to preparative chromatography. The metabolites were separated and fractionated by reversed phase liquid chromatography. The column was a GraceSmart C18, 5 µm, 110 Å, 4,6 mm id × 250 mm (Grace, Deerfield, IL, USA). Elution was isocratic with 20 mmol L⁻¹ ammoniumacetate in 2% methanol at a flow rate of 1 mL min⁻¹. 100 µL sample was injected. The relevant fractions were lyophilized and dissolved in 0.1% formic acid in 2% methanol for LC-ESI-MS analysis.

ICP-MS

The ICP-MS was a PE Sciex Elan 6000 (Perkin Elmer, Norwalk, CT, USA) equipped with a Micro Mist glass concentric nebulizer (Glass Expansion, Romainmontier, Switzerland) and a PC³ cyclonic spraychamber (Elemental Scientific Inc., Omaha, NE, USA). When the mobile phase methanol concentration was above 5%, the spray chamber was operated at 4 °C. The sample uptake rate was 200 µL min⁻¹. ICP-MS sampler and skimmer cones were made of platinum. The plasma and auxiliary gas flow rates were 15 L min⁻¹ and 1.2 L min⁻¹, respectively. The nebulizer gas flow, lens voltage and ICP RF power were optimized regularly with a solution of 100 µg Se L⁻¹ in mobile phase. The data requisition settings were: dwell-time 500 ms, sweeps per reading 1 and readings per replicate were varied corresponding to chromatographic runtime. ⁷⁷Se, ⁷⁸Se and ⁸²Se isotopes were monitored.

LC-ESI-MS (ion trap)

The ESI-MS ion trap detector was a G2445 LC/MSD Trap equipped with an API-electrospray interface (Agilent) controlled by LC/MSD Trap software (Bruker Daltronics Inc.) used for data acquisition and processing. The ESI-MS was coupled to a LC system consisting of a G1322A degasser, a G1312A binary pump, a G1315B diode array detector, a G1316A column compartment and a G1313A autosampler (all from Agilent). The electrospray was produced in the positive ionization mode and the mass spectrometer was optimised by direct infusion of 1 mg L⁻¹Se-MeSeCys and selenomethionine standards at 20 µL min⁻¹. The optimised ESI Ion Trap MS parameters were; capillary voltage: 3000 V (Se-MeSeCys) and 3500 V (SeMet), nebuliser pressure: 30 psi, drying temperature: 350 °C, drying gas flow: 10 L min⁻¹ and the trap drive was 25 (arbitrary unit). All MSⁿ experiments were performed with an isolation width of 1.0 mass unit and the fragmentation amplitude was 0.6 (arbitrary units).

LC-ESI-MS (triple quadrupole)

The ESI MS triple quadrupole detector was a Thermo Finnigan TSQ Quantum Utra AM triple quadrupole mass spectrometer with a ESI interface coupled to a Thermo Finnigan Surveyor LC system (Thermo Fisher Scientific, Waltham, MA, USA). The mass spectrometer was optimized by 25 µL min⁻¹ direct infusion of a 1 mg L⁻¹ selenomethionine standard. The optimized ESI triple quadrupole MS parameters were; spray voltage; 4000 V, sheath gas pressure: 55 psi, capillary temperature: 350 °C, auxiliary gas pressure: 8 psi. The instrument was used in the selected reaction monitoring (SRM) mode. The scan width was 0.5 mass units and the collision energy was 40 (m/z 198/196 to 109/107) and 28 (198/196 to 152/150 or 135/133).

Total selenium determination by flow injection analysis

Total selenium was determined by flow injection analysis. The mobile phase was 20 mmol L⁻¹ ammoniumacetate in 2% methanol. The flow rate was 200 µL min⁻¹ and the injected volume was 5 µL. The signal was assessed as the mean peak area of three replicate injections. Quantities were determined by the standard addition method at five concentration levels. The working standard solutions used for spiking were prepared from a PE Pure Atomic Spectroscopy Standard (Perkin Elmer, Norwalk, CT, USA) containing 1.000 mg L⁻¹ Se. The method was validated against a certified serum control containing 83 µg L⁻¹ (77–89 µg L⁻¹) Se (Seronorm Level I, SERO AS, Billingstad, Norway).

Results and discussion

Incubation of hepatocytes with methylseleninic acid

The hepatocytes (post-thawed viabilities from 62 to 63%) were incubated with small amounts of MeSeA (5 µM and 10 µM). The metabolite pattern was similar when the hepatocytes were incubated with 5 µM and 10 µM MeSeA. The cellular suspensions were divided in three fractions after incubation: Cell medium (extracellular/exctreted metabolites), lysate (intracellular metabolites) and insoluble fraction (membrane/protein bound/associated metabolites). In cell medium, two metabolites (M1 and M2) were detected by LC-ICP-MS analysis with retention time 4.1 min and 6.0 respectively (Fig. 1). MeSeA eluted after 3.3 min. Chromatographic analysis of cell lysates and the insoluble fraction was not performed. Metabolites M1 and M2 co-eluted with Se-MeSeCys and SeMet in two different chromatographic systems, which indicated the identity of these metabolites. To obtain metabolites in a concentration allowing identification by molecular mass spectrometry, the hepatocytes were incubated with a large amount of MeSeA (100 µM). This concentration is far above any physiological relevance, but the metabolism pattern was qualitatively similar to that obtained with lower concentrations of MeSeA. Furthermore, two metabolites (M1 and M3) were detected in the lysates, eluting at 4.1 min and 4.7 min respectively. Additionally, viability of hepatocytes was not significantly altered upon treatment with the high dose of MeSeA. To assess if any apolar metabolites were present in the cell medium the sample was subjected to gradient analysis (results not shown). No further metabolites were detected. The column recovery in this experiment was 97%.

Fig. 1 LC-ICP(⁸²Se)-MS chromatograms of hepatocytes incubated with MeSeA for 4 h; (a) cell medium (10 µmol L⁻¹ MeSeA), (b) cell medium (100 µmol L⁻¹) and (c) cell lysate (100 µmol L⁻¹). Metabolites were eluted with 20 mmol L⁻¹ ammonium acetate in 2% methanol.

Identification of M1

The optimized ESI-MS (full scan, MS² and MS³) methods were coupled with the chromatographic system used for LC-ESI-MS analysis. In this chromatographic system M1 eluted at 4.1 min as well. Mass spectra were extracted in retention time interval 4.1 to 4.3 min (Fig. 2). LC-ESI-MS resulted in a spectrum with the characteristic selenium isotope pattern at m/z 184 corresponding to ⁸⁰Se. Isolation and fragmentation of the parent ion (m/z 184) led to formation of an intense ion at m/z 167, corresponding to loss of ammonia ([M − NH₃]⁺). This ion was isolated and further fragmented in a MS³ experiment that resulted in fragment ions at m/z 149 [(M − NH₃) − H₂O]⁺, 139 [(M − HN₃) − CO]⁺, 123 [(M − NH₃) − COO]⁺ and 95 [CH₃Se]⁺ (Table 1). This fragmentation pattern was identical to the fragmentation pattern of a 1 mg L⁻¹Se-MeSeCys standard. Similar MS² and MS³ experiments of parent ion m/z 182 for the ⁷⁸Se compound also verified the extracted full scan selenium isotope pattern.

Table 1 Extracted spectra at retention time 4.1 to 4.3 min

	Fragment ions (m/z)
	Standard	Sample
MS^2
Isolated mass 184	166.9	166.9
Isolated mass 182	164.9	164.9
MS^3
Isolated masses 184/167	149.0	148.9
	138.9	138.9
	122.9	122.8
	95.0	95.1
Isolated masses 182/165	146.9	146.9
	136.9	137.0
	120.9	121.0
	92.9	93.2

---

Fig. 2 LC-ESI-MS (ion trap) spectra (4.1 to 4.3 min) of isolated and pre-concentrated metabolite M1. The mobile phase was 0.1% formic acid in 2% methanol.

Identification of M2

Due to limited formation of metabolite M2 and limited amounts of incubation medium, the identity of this metabolite was established by two different molecular mass spectrometry approaches.

Full scan LC-ESI-MS² and LC-ESI-MS³ experiments were performed by use of an ion-trap instrument. Extracted spectra in MS² mode for a 1 mg L⁻¹ selenomethionine standard and the purified and concentrated metabolite M2 were similar (Fig. 3). Isolation and fragmentation of the parent ion (m/z 198.0) led to formation of an intense ion at m/z 180.9, corresponding to the loss of ammonia ([M − NH₃]⁺). A minor fragment ion was observed at m/z 151.9 corresponding to parallel loss of formic acid ([M − HCOOH]⁺). The major fragment ion (m/z 180.9) was isolated and further fragmented by LC-ESI-MS³ analyses. In the MS³ mode, the extracted spectrum for M2 showed two fragment ions at m/z 153.0 ([(M − NH₃) − CO]⁺) and 109.1, corresponding to cleavage of the bond in α position to the Se atom ([CH₃–Se–CH₂]⁺). The fragment ion at m/z 135.0 ([(M − NH₃) − HCOOH]⁺), observed with SeMet standard, did not appear. The signal for the standard was approximately 10⁴ times more intense than the signals for the metabolite. The low intensity for the sample was due to low concentration of the metabolite and probably also due to ion suppression in the biological sample. Although preparative chromatography was performed, it still contained salt and other interfering species.

Fig. 3 LC-ESI-MS (ion trap) spectra (5.1 to 5.3 min) of (a) selenomethionine and (b) isolated and pre-concentrated metabolite M2. The mobile phase was 0.1% formic acid in 2% methanol.

Based on LC-ESI-MS² full scan spectra of a 1 mg L⁻¹ selenomethionine standard, three specific transitions from the parent ion to the fragment ions (obtained by collision induced dissociation) were selected for selected reaction monitoring (SRM) mode analysis (triple quadrupole instrument). Fig. 4 shows the SRM chromatograms of the selected transitions for the isolated and concentrated metabolite M2 and a SeMet standard of similar concentration. In all SRM experiments, transitions for parent ion corresponding to both ⁸⁰Se and ⁷⁸Se were monitored. The chromatographic peak at retention time 4.9 min was observed for all selected transitions. Although the same chromatographic setup (identical column and mobile phase) was used, the retention time of SeMet was slightly displaced due to different void volumes of the LC/MSD Trap and the surveyor/TSQ Quantum instruments.

Fig. 4 LC-ESI-MS(SRM) (triple quadrupole) chromatograms of 750 ppb selenomethionine (grey) and of isolated and pre-concentrated metabolite M2 (black). The mobile phase was 0.1% formic acid in 2% methanol.

The main fragment produced in the triple quadrupole instrument was m/z 109.0 whereas the main fragment produced in the ion trap (MS² mode) was m/z 180.9. The m/z 109.0 fragment ion was only observed in the MS³ mode on the ion trap instrument. As these significant different observations were done for both the unknown species M2 and a SeMet standard the evidence of the identity of the metabolite is improved. In lymphoma B-cells incubated with MeSeA by Infante et al²³ SeMet was also detected. The identity of SeMet was established by chromatographic co-elution. As it traditionally has been accepted that animals (including humans) were not capable of synthesizing SeMet but exclusively obtained this amino acid via food or nutritional supplements, this observation was very surprising. Although MeSeA is not a constituent of food and therefore not a part of the human selenium intake, the observation opens the question whether SeMet might be biosynthesized from other nutritionally available selenium compounds as well.

The recovery of the dosed selenium was determined by flow injection analysis. The total selenium determination was validated against a certified serum control, as no reference material for hepatocytes is available. The accuracy was 93% of the certified Se amount and within the 95% confidence interval provided by the manufacturer. The relative standard deviation was 6% (n = 4) and the mean quantity was 78 µg L⁻¹. In five incubation vials, 22–27% of the dosed selenium incubated was recovered; 14–16% was found in cell medium, less than 1% in cell lysate and 7–12% in the insoluble fraction. This indicates that about 75% of the dosed selenium was lost, probably due to formation of volatile species. This is in agreement with the findings of Infante et al.²³ who detected volatile selenium species in the head space of lymphoma B-cells incubated with MeSeA, although no quantities were reported. Hence, formation of volatile selenium species by the hepatocytes is plausible. The presence of volatile species may severely compromise the accuracy of the quantitative determination. Juresa et al.³³ observed a large LC-ICP-MS peak for dimethyldiselenide in urine even though it was only present in trace amounts. The same group thoroughly investigated the consequences for total selenium determination in samples containing volatile selenium species.³⁴ They found up to 45 times the nominal amount of dimethylselenide and dimethyldiselenide by flow injection ICP-MS analysis. As a consequence of the over-estimation of volatile selenium species, it could be argued that the true recovery of the dosed selenium may be even less than 22–27%. However, it seems very likely that the volatile species are lost from the samples during incubation and further sample processing i.e. during evaporation of cell lysates and insoluble fractions. This is in concordance with Juresa et al.³³ also observed loss of volatile selenium from urine samples upon storage and handling.

A short time course study with low concentration of MeSeA (10 µM) was performed. Cell medium samples were analyzed with gradient elution (Fig. 5). MeSeA was instantaneously metabolized and formation of Se-MeSeCys was detected at the first time point (15 min). The amount of Se-MeSeCys increased over the one hour incubation period. SeMet at retention time 4.9 min, was not detected. A selenium species was detected at retention time 6.0 min. This peak decreased in size over time and it was not detected after 60 min incubation. An additional selenium species was detected at limit of detection levels at retention time 12.2 min. The metabolites, that were only detected in the short time incubations of the time course, coeluted with synthesized S-(methylseleno)cysteine (S-(MeSe)Cys) and S-(methylseleno)glutathione (S-(MeSe)SG) respectively (Fig. 5a). As the S–Se metabolites were only observed in limited amounts it was not possible to prove their identity. Formation of these S–Se species would be expected as they are formed spontaneously upon reaction of MeSeA with cysteine and glutathione²² that are present in cells. Furthermore, S-(MeSe)Cys and S-(MeSe)SG have been identified in other biological samples as well.^22,35 Sinha et al.⁶ proposed MeSeA metabolism to be subject to a series of reductions by glutathione to S-(MeSe)SG and further to MeSeH. MeSeH then undergoes oxidation or methylation to form the volatile species dimethyldiselenide (MeSe-SeMe) or dimethylselenide (MeSeMe), respectively. As mentioned earlier, the formation of these volatile metabolites of MeSeA has been verified by GC-MS by Infante et al.²³

Fig. 5 LC-ICP(⁸²Se)-MS chromatograms for time course study. (a) Incubation stopped immediately upon addition of MeSeA (black) including the sample spiked with S-(methylseleno)cysteine (S-(MeSe)Cys) and S-(methylseleno)glutathione (S-(MeSe)SG) (grey), (b–d) incubations stopped after 15, 30 and 60 min. The metabolites were eluted with a linear gradient of 0.1% formic acid in 2 to 50% methanol over 5 min followed by column equilibration in 0.1% formic acid in 2% methanol.

It is now clearly established that Se-MeSeCys and SeMet are derived from the metabolism of MeSeA in isolated rat hepatocytes. Hence, this observation should be included in metabolism models. As dimethylselenide and dimethyldiselenide are considered end products of selenium metabolism, there is reason to believe that MeSeA is metabolized through different pathways - one that leads to formation of volatile species and pathways that lead to formation of Se-MeSeCys and SeMet and probably other not yet identified selenium species (Fig. 6). Selenoproteins and selenosugars are included in the metabolic pathways of MeSeA, as Suzuki et al. observed selenium of isotopic labeled MeSeA origin in selenoproteins (extracellular glutathione peroxidase and selenoprotein P) and selenosugar in the rat.^36,37

Fig. 6 Proposed metabolic scheme for MeSeA, a modified version of the model proposed by Sinha et al.⁶ Structures of all species in the figure were reported in a recent review by Gammelgaard et al.¹⁶

MeSeA is reduced via the thiols cysteine and glutathione resulting in MeSeH and the oxidized forms of the thiols, whereas Se-MeSeCys is believed to be metabolized by the enzyme β-lyase with the end products MeSeH and alanine. Thus, the serial reduction of MeSeA bares more resemblance to the initial metabolism of selenite (Fig. 6) than to the enzymatic cleavage of Se-MeSeCys. This, combined with the fact that Se-MeSeCys is a metabolite of MeSeA in both healthy and cancer cells indicate that MeSeA may not be a relevant model compound for methylated Se-amino acids in in vitro studies.

References

1. L. C. Clark, G. F. Combs, Jr, B. W. Turnbull, E. H. Slate, D. K. Chalker, J. Chow, L. S. Davis, R. A. Glover, G. F. Graham, E. G. Gross, A. Krongrad, J. L. Lesher, H. K. Park, B. B. Sanders, C. L. Smith and J. R. Taylor, J. Am. Med. Assoc., 1996, 276, 1957–1963.
2. P. D. Whanger, Br. J. Nutr., 2004, 91, 11–28.
3. C. Ip and H. E. Ganther, Cancer Res., 1990, 50, 1206–1211.
4. C. Ip, C. Hayes, R. M. Budnick and H. E. Ganther, Cancer Res., 1991, 51, 595–600.
5. C. Ip, Y. Dong and H. E. Ganther, Cancer Metastasis Rev., 2002, 21, 281–289.
6. R. Sinha, E. Unni, H. E. Ganther and D. Medina, Biochem. Pharmacol., 2001, 61, 311–317.
7. Y. Dong, H. E. Ganther, C. Stewart and C. Ip, Cancer Res., 2002, 62, 708–714.
8. H. Zhao, M. L. Whitfield, T. Xu, D. Botstein and J. D. Brooks, Mol. Biol. Cell, 2004, 15, 506–519.
9. Y. Dong, H. Zhang, L. Hawthorn, H. E. Ganther and C. Ip, Cancer Res., 2003, 63, 52–59.
10. H. M. Shen, W. X. Ding and C. N. Ong, Free Radical Biol. Med., 2002, 33, 552–561.
11. C. Ip, H. J. Thompson, Z. Zhu and H. E. Ganther, Cancer Res., 2000, 60, 2882–2886.
12. M. Montes-Bayon, K. DeNicola and J. A. Caruso, J. Chromatogr., A, 2003, 1000, 457–476.
13. J. Szpunar and R. Lobinski, Anal. Bioanal. Chem., 2002, 373, 404–411.
14. C. B'Hymer and J. A. Caruso, J. Chromatogr., A, 2006, 1114, 1–20.
15. E. Rosenberg, J. Chromatogr., A, 2003, 1000, 841–889.
16. B. Gammelgaard, C. Gabel-Jensen, S. Sturup and H. R. Hansen, Anal. Bioanal. Chem., 2008, 390, 1691–1706.
17. H. E. Ganther, in Trace Element - Analytical Chemistry in Medicine and Biology, ed. Peter Brätter and Peter Schramel, Walter de Gruyter & Co., Berlin, 1984, pp. 3–24.
18. Y. Ogra, K. Ishiwata, H. Takayama, N. Aimi and K. T. Suzuki, J. Chromatogr., B, 2002, 767, 301–312.
19. B. Gammelgaard, K. G. Madsen, J. Bjerrum, L. Bendahl, O. Jons, J. Olsen and U. Sidenius, J. Anal. At. Spectrom., 2003, 18, 65–70.
20. D. Kremer, G. Ilgen and J. Feldmann, Anal. Bioanal. Chem., 2005, 383, 509–515.
21. X. J. Cai, E. Block, P. C. Uden, B. D. Quimby and J. J. Sullivan, J. Agric. Food Chem., 1995, 43, 1751–1753.
22. C. Gabel-Jensen, K. Lunoe, K. G. Madsen, J. Bendix, Claus Cornett, S. Sturup, H. R. Hansen and B. Gammelgaard, J. Anal. At. Spectrom., 2008, 23, 727–732.
23. H. G. Infante, S. P. Joel, E. Warburton, C. Hopley, R. Hearn and S. Juliger, J. Anal. At. Spectrom., 2007, 22, 888–896.
24. L. Jia and X. D. Liu, Curr. Drug Metab., 2007, 8, 822–829.
25. I. Anundi, J. Hogberg and A. Stahl, Arch. Toxicol., 1982, 50, 113–123.
26. M. Weiller, M. Latta, M. Kresse, R. Lucas and A. Wendel, Toxicology, 2004, 201, 21–30.
27. I. Anundi, A. Stahl and J. Hogberg, Chem.-Biol. Interact., 1984, 50, 277–288.
28. J. Hogberg, T. Ekstrom, I. Anundi and A. Kristoferson, Toxicology, 1980, 17, 113–118.
29. Y. C. Park and P. D. Whanger, Toxicology, 1995, 100, 151–162.
30. A. Stahl, I. Anundi and J. Hogberg, Biochem. Pharmacol., 1984, 33, 1111–1117.
31. E. LeCluyse, P. Bullock, A. Parkinson and J. Hochman, in Models for Assessing Drug Absorption and Metabolism, ed. R. T. Borchard, P. L. Smith, and G. Wilson,Plenum Press, New York, 1996, pp. 121–160.
32. Cam A. Le, A. Guillouzo and P. Freychet, Exp. Cell Res., 1976, 98, 382–395.
33. D. Juresa, J. Darrouzes, N. Kienzl, M. Bueno, F. Pannier, M. Potin-Gautier, K. A. Francesconi and D. Kuehnelt, J. Anal. At. Spectrom., 2006, 21, 684–690.
34. D. Juresa, D. Kuehnelt and K. A. Francesconi, Anal. Chem., 2006, 78, 8569–8574.
35. P. O. Amoako, C. L. Kahakachchi, E. N. Dodova, P. C. Uden and J. F. Tyson, J. Anal. At. Spectrom., 2007, 22, 938–946.
36. K. T. Suzuki, C. Doi and N. Suzuki, Toxicol. Appl. Pharmacol., 2006, 217, 185–195.
37. K. T. Suzuki, Y. Ohta and N. Suzuki, Toxicol. Appl. Pharmacol., 2006, 217, 51–62.

---