Sample preparation and HPLC separation approaches to speciation analysis of selenium in yeast by ICP-MS Corinne Casiot, Joanna Szpunar, Ryszard £obin�ski and Martine Potin-Gautier* Laboratoire de Chimie Analytique, Bio-inorganique et Environnement, CNRS EP132, Universite� de Pau et de Pays de l’Adour, He�lioparc, 2, av. Pr. Angot, 64 053 Pau Ce� de�x 9, France Received 18th November 1998, Accepted 8th February 1999 Eight solid–liquid extraction procedures were evaluated for the recovery of selenium species from yeast.Speciation of Se in the extracts was characterized by diVerent types of HPLC, including size-exclusion, anion-exchange and reversed-phase chromatography with ICP-MS detection. The results obtained depended critically on the sample preparation procedure used. Leaching with water and with methanol led only to 10–20% recoveries of Se, split into eight compounds, among which Se(IV) and selenomethionine could be identified. Leaching with pectinolytic enzymes released an additional 20% of selenomethionine. Leaching with sodium dodecyl sulfate solution allowed the solubilization of a selenoprotein that accounted for ca. 30% of the total Se present. Leaching with proteolytic enzymes led to recoveries of Se above 85%, the majority as selenomethionine. Hydrolysis of the yeast with tetramethylammonium hydroxide solubilized the sample completely but the Se species present were entirely degraded to selenomethionine and inorganic selenium.A sequential leaching procedure is proposed for the evaluation of selenium speciation in yeast without the need for a coupled technique. selenium compounds, including selenocysteine, selenomethion- Introduction ine, methylselenocysteine and inorganic forms. The use of Selenium is an essential micronutrient for living organisms but enzymic hydrolysis led Gilon et al.5 to the conclusion that may be toxic in specific forms [e.g., Se(VI)] and at higher more than 80% of Se in the yeast was present in three forms: concentrations.1 It is a component of the human enzyme inorganic selenium, selenocysteine and selenomethionine.glutathione peroxidase and has been reported to show a The objective of this work was to develop and to evaluate protective eVect against cancer.2,3 The observation of statisti- critically diVerent leaching/extraction procedures for the recovcally significant reductions in cancer mortality and cancer ery of selenium species from yeast.Size-exclusion chromatograincidence in a population consuming yeast-derived nutritional phy with ICP-MS was developed to monitor the speciation supplements by Clark et al.4 induced a surge of interest in Se- of selenium in the diVerent extracts and was compared enriched yeast. Since the bioavailability and the toxicity of Se with anion-exchange and reversed-phase chromatography. are closely correlated with its chemical form, this interest has Attempts were made to optimize a sequential extraction protobeen translated into the quest for information on selenium col in such a way that particular classes of selenium species speciation in yeast.5–9 are selectively recovered with the consecutive extraction In terms of analytical approaches, the direct coupling of solutions. high-performance liquid chromatography (HPLC) with ICPMS is now an established approach to the determination of Experimental Se species.10–13 Separation modes used included ion-exchange (anion and cation) and anion-pairing reversed-phase chroma- Apparatus tography (for an exhaustive list of references, see refs. 7 and The ICP-MS instrument used was an HP4500 (Yokogawa 14. The choice of species studied [Se(IV), Se(VI), selenome- Analytical Systems, Tokyo, Japan). The sample introduction thionine and selenocysteine] has been mainly dictated by the system used included a Scott double-pass spray chamber fitted commercial availability of standards, with the exception of with a Babington nebulizer. For chromatographic experiments some recent studies in which a large number of synthetic aModel 9012 HPLC pump (Varian Chromatography Systems, organoselenium compounds were used.6,7 The majority of Walnut Creek, CA, USA) was used as the sample delivery studies have focused on the development of coupled techsystem.All the connections were made of PEEK tubing niques; applications to real samples have been scarce. (0.17 mm id). Injections were made using a Model 7725 Speciation analysis of yeast by HPLC-ICP-MS requires that injection valve (Rheodyne, Cotati, CA, USA) with a 100 ml the endogenous Se species present in the yeast are extracted injection loop.Chromatographic results were processed using without modification of their chemical form or disturbance to Chromatosoftware (Hewlett-Packard, Avondale, PA, USA). the equilibrium between the various species present. Previous Quantifications were performed in the peak area mode.studies of selenium speciation in yeast either concerned the water soluble fraction only (containing ca. 10% of the total Chromatographic columns selenium)6–8 or were aimed at the maximization of the Se recovery by degrading the species originally present with a Size-exclusion chromatographic separations were performed mixture of proteolytic enzymes.5 The results of these pro- on a 300×10 mm id, 13 mm Superdex-200 HR 10/30 SEC cedures have been contradictory. By leaching with an aqueous column (Pharmacia Biotech, Uppsala, Sweden) with an exclusion limit of 1300 kDa and an eVective separation range solution, Bird et al.6,7 reported the presence of more than 20 J.Anal. At. Spectrom., 1999, 14, 645–650 645between 10 and 600 kDa. Anion-exchange and reversed-phase HPLC-ICP-MS conditions chromatographic separations were performed on a The chromatographic separation conditions are summarized 250×4.1 mm id, 10 mm Hamilton PRP-X100 column in Table 1.The ICP-MS measurement conditions, given in (Hamilton, Reno, NV, USA) and a 150×4.6 mm id, 5 mm Table 2, were optimized daily using a standard built-in software Inertsil ODS-2 column (Interchim, Montluc�on, France), procedure for injection of a 10 mg l-1 solution containing Li, respectively. Y, Ce and Tl into a flow of the chromatographic mobile phase at the flow rate given in Table 1. Three major selenium Standards and samples isotopes, 77Se, 78Se and 82Se (with relative abundances of 7.63, 23.78 and 8.73%, respectively), were monitored.DL-Selenocysteine, DL-selenomethionine and selenoethionine were purchased from Sigma (St. Quentin Fallavier, France) Results and discussion and were used without further purification (90% purity for selenocystine). Stock standard solutions containing 1 mg ml-1 Experimental conditions reported in the literature for anionof each compound in de-ionized water (Millipore, Bedford, exchange (AEC)5 and reversed-phase (RPC)6,7 chromatogra- MA, USA; 18 MV) were stored in the dark at 4 °C.phy of selenium species were used. In order to detect the Hydrochloric acid (3%) was used to dissolve selenocysteine. presence of high molecular mass compounds within the same Working standard solutions were prepared daily by dilution run, size-exclusion chromatography (SEC) was optimized. The in de-ionized water. SEC-ICP-MS coupling has been a method of choice for the A sample of industrially produced selenium enriched yeast characterization of high and medium molecular mass metal was used.Saccharomyces cerevisiae was grown in the presence (metalloid) compounds in biological samples.15 Despite the of sodium selenite, from which it naturally synthesizes organic fact that, in theory, the separation should be based on the seleno compounds. It was then pasteurized and dried (to avoid analyte molecular mass, secondary adsorption and ionfurther growth and facilitate handling). exchange eVects apparently play an important role in SEC and the separation of small compounds with similar molecular Reagents masses can be achieved in addition to the possibility of the unambiguous identification of the presence of selenoproteins.Analytical reagent grade sodium dodecyl sulfate (SDS), tetra- The signal intensity ratios measured were: 78Se577Se, methylammonium hydroxide (TMAH) (2in 3.0±0.2, 82Se577Se 0.98±0.07 and 78Se582Se 3.0±0.2 in the water), phenylmethylsulfonyl fluoride (PMSF), methanol, range 1040–1400 s in SEC-ICP-MS.Comparison with the ammonium mono- and dihydrogenphosphate and tris(hydtheoretical values of 3.1, 1.1 and 2.7, respectively, indicates roxymethyl )aminomethane (TRIS) were purchased from that no significant interferences occurred. Aldrich (St. Quentin Fallavier, France). The enzymes Driselase Fig. 1(a), (b) and (c) show the chromatographic profiles and pronase E (protease XIV type) were obtained from obtained by SEC-ICP-MS, AEC-ICP-MS and RPC-ICP-MS, Aldrich.Milli-Q water (18 MV) (Millipore) was used through- respectively, for supernatants obtained by each of the sample out. BuVers were prepared by dissolving the appropriate preparation procedures. The results of procedures A–C ( leach- amount of salts in de-ionized water and the pH was adjusted ing with diVerent aqueous solutions) leading to similar chroma- by dropwise addition of 30% ammonia solution or HCl.The tographic profiles are presented in one panel. The same eluents were filtered (0.45 mm) and de-gassed before use. concerns leaching with the SDS solution with and without the addition of PMSF (procedures E and F). Procedures The recovery of Se in the sample preparation procedures was determined by SEC-HPLC using the peak area quantifi- Eight sample preparation (solid–liquid extraction) procedures cation mode. In all cases except the TMAH digestion (prowere used. For each of them a sample of 200 mg of yeast was cedure H), solid residues were observed after extraction.The placed in a centrifuge tube followed by the addition of the SEC-ICP-MS results obtained for this procedure matched the following reagents: value obtained by flow injection analyses and was taken as A: 5 ml of hot water (85–90 °C); the sample was stirred 100%. Table 2 summarizes the recoveries and relative abun- for 1 h. dances of particular chromatographic signals in the B: 5 ml of 10% MeOH in 0.2 M HCl; the sample was chromatograms.sonicated for 1 h. C: 5 ml of 30 mM TRIS-HCl buVer (pH 7) and 0.1 mM Leaching with aqueous solutions PMSF; the sample was sonicated for 1 h. D: 5 ml of 4% Driselase in 30 mM TRIS-HCl buVer (pH 7) The results obtained by extraction with hot water are almost identical with those obtained with methanol–HCl and with a in the presence of 1 mM PMSF; the sample was incubated for 1 h at room temperature. neutral buVer (TRIS–HCl, pH 7.0) [Fig. 1(a)–(c) top panel ], which is in agreement with an earlier report.7 Only ca. 10% of E: 5 ml of 30 mM TRIS-HCl buVer (pH 7) containing 4% SDS. The sample was homogenized and incubated for 1 h. the selenium present in the sample is extracted. The compounds recovered include the weakly bound and water-soluble selenite, F: 5 ml of 20 mM ammonium phosphate buVer (pH 7.4), 0.15 M NaCl, 0.1 mM PMSF, 1 mM EDTA, 5% SDS; the sample selenoamino acids and possibly trimethylselenonium and selenoglutathione.Injection of standards and spiking experiments was stirred for 1 h. G: 5 ml of phosphate buVer (pH 7.5) containing 20 mg of indicated the presence of selenomethionine and Se(IV) in the low molecular mass elution volume range of the column, pronase and 10 mg of lipase; the sample was incubated for 16 h at 37 °C. which was further confirmed by orthogonal (AEC and RPC) separation chromatographic techniques. The best resolution H: 5 ml of 25% TMAH solution in hot (60 °C) water; the sample was incubated for 4 h.was achieved by anion-exchange chromatography, which allows the discrimination of seven well resolved signals. The After the sample preparation procedure the sample was centrifuged using a Hettich Universal 16 centrifuge at 4000 retention times of two of them match those of Se(IV) and selenomethionine. Selenocysteine would elute in (or close to) min-1 for 30 min. An aliquot of the supernatant was filtered through a 0.45 mm filter and injected on to the chromato- the void in anion-exchange and reversed-phase chromatography and its positive identification in a real sample by these graphic column. 646 J. Anal. At. Spectrom., 1999, 14, 645–650Table 1 Experimental chromatographic conditions Separation mode Parameter Size-exclusion Anion-exchange Ion-pair reversed phase Superdex-200 Hamilton PRP-X100 Inertsil ODS-2 Analytical column Eluent 30 mM TRIS–HCl buVer (pH 7.0) Ammonium phosphate buVer; 0.1% TFA in 2% MeOH (A) 6.25 mM (pH 5.5); (B) 25 mM (pH 11) Eluent flow rate/ml min-1 1.0 1.4 1.0 Elution programme Isocratic Gradient: 0–9 min, 100% A; Isocratic 9–10 min, 100% A to 100% B; 10–20 min, 100% B Injection volume/ml 100 100 100 Run time/min 40 20 40 Table 2 Experimental ICP-MS conditions Leaching with non-proteolytic (pectinolytic) enzymes The first attempt was to release selectively any selenium present Nebulizer Babington Rf power 1350 W in the sample that is not protein bound.The mixture of Nebulizer gas (argon) flow rate 1.1 l min-1 enzymes of choice should therefore be devoid of proteolytic Lens voltages— activity. Driselase, a commercial enzyme preparation contain- Extraction lenses Extract 1: -120 V ing laminarinase, xylanase and cellulase that is used to destroy Extract 2: -115 V the cell walls of fungi,16 was chosen. An attack with this Einzel lenses Einzel 1, 3: -100 V enzyme would therefore release the selenium compounds Einzel 2: -6 V Omega lenses Bias: -40 V trapped in the cell walls, either physico-mechanically or chemi- (+): 3 V cally as coordination complexes with plant cell components.(-): -26 V The chromatograms shown in the panel next to the top in Acquisition parameters— Fig. 1(a)–(c) vary distinctly from those obtained after leaching Data acquisition mode Time resolved analysis with hot water. The solid residue after leaching and centrifu- Intergration time 100 ms gation is smaller that in the case of water and methanol Number of replicates 1 Isotopes monitored 77Se, 78Se, 82Se extractions.The amount of selenium released with Driselase is more than twice that recovered without the enzyme. The retention time of the dominant species matches that of selenomethionine in the size-exclusion and anion-exchange chromatograms but not in the reversed-phase chromatogram. The Se techniques is impossible. The diVerence between the elution volumes of the selenoamino acids studied by SEC (chromato- eluting at the exclusion volume of the column is accompanied by a small signal corresponding to a molecular mass of grams not shown) is too small to allow their positive identification. 20–30 kDa, which is likely to be a polysaccharide liberated from the yeast cell walls. Another important peculiarity of this The results obtained are in agreement with the two literature reports published so far. An intense unidentified compound chromatogram is the absence of the peak that elutes last (1320 s) in the case of water extraction.This suggest that accounting for ca. 35% of extracted selenium (for the hot water and MeOH–HCl extractions, procedures A and B) or Driselase is responsible for the degradation of this compound. 45% (for the extraction with neutral buVer, procedure C) of the water-soluble Se has also been observed elsewhere.8 Leaching with SDS The amount of selenium recovered in a second extraction was below 5% of that extracted with the first portion of the Yeast is known for its high protein content, which is approximately 40% dry mass.17 Because of the similarity of selenium leaching solution.In the analysed sample 90% of Se is present in the form of species that are insoluble in water. EVorts were to sulfur, selenoamino acids can also be metabolized into proteins. Most studies on selenium-containing proteins have therefore made to study the speciation of selenium in that fraction. dealt with human and animal tissues, whereas information on Table 3 Extraction yields and distribution of selenium species in size-exclusion chromatography (total Se concentration: 1183±41 mg g-1) Concentration of major species (as Se) /mg g-1 (% of the extracted Se) Unknown low Procedure Selenium Extraction yield High molecular Selenoamino molecular mass used extracted/mg g-1 (% of total Se) mass fraction Inorganic Se acids compound A 118.3 10 7.1 (6) 41.4 (35) 9.5 (8) 41.4 (35) B 153.8 13 6.2 (4) 52.3 (34) 69.2 (45) C 118.3 10 7.1 (6) 40.2 (34) 10.6 (9) 41.4 (35) D 319.4 27 22.4 (7) 89.4 (28) 143.7 (45) 54.3 (17) E 496.9 42 362.7 (73) 19.9 (4) 9.9 (2) 49.7 (10) F 437.7 37 319.5 (73) 21.9 (5) 13.1 (3) 30.6 (7) G 1041 88 10.4 (1) 187.4 (18) 780.8 (75) 41.6 (4) H 1180 100 — 1147 (97) J.Anal. At. Spectrom., 1999, 14, 645–650 647Fig. 1 HPLC with ICP-MS detection of leachates containing selenium compounds using diVerent sample preparation procedures: (a) sizeexclusion chromatography; (b) anion-exchange chromatography; and (c) reversed-phase chromatography.Capital letters refer to the procedures described in the Procedures section. Data are shown for 82Se. fungi and plants is scarce.18–21 About 80% of Se incorporated The size-exclusion ICP-MS trace [Fig. 1(a), middle panel ] shows an intense signal corresponding to a high molecular in yeast was found associated with high molecular mass compounds (cell walls, mitochondria, microsomes, proteins mass compound (molecular mass >100 kDa) that accounts for ca. 30% of the initial Se present in the sample. The and nucleic acids) or present in water-soluble proteins.17 SDS polyacrylamide gel electrophoresis (PAGE) combined with chromatographic pattern of the low molecular mass fraction (passing through a 10 kDa cut-oV filter) is undistorted by the neutron activation analysis allowed the conclusion that Se in the seeds of coco de mono (Lecythis ollaria) was present in SDS leaching.The amounts of selenium recovered in a second and third extraction were ca. 22% and 6%, respectively, in extremely selenium-rich proteins with molecular masses below 20 kDa.18 A procedure aimed at the recovery of the Se- comparison with that extracted with the first portion of the leaching solution. containing protein fraction was therefore attempted. SDS is widely used to denature proteins and, by forming The data in Table 2 indicate that even after repeated leaching with the SDS solution, 60% of selenium present in the sample ion pairs, to render them water soluble.This reagent has been employed for the recovery of selenoproteins from mammalian still remains behind in the solid residue.We therefore attempted to recover it by partial degradation of the insoluble compound, tissues.22,23 The extraction is typically carried out at 4 °C in the presence of PMSF to retard the naturally present proteo- probably protein. An enzymatic attack with a mixture of proteolytic enzymes as described by Gilon et al.5 was the lytic activity of enzymes liberated during the treatment. Fig. 1(a) (middle panel ) shows no diVerence in the mor- method of choice. phology of a yeast extract chromatogram, regardless of whether the leaching with the 4% SDS aqueous solution was Release of Se from yeast by proteolysis of selenoproteins done in the ice-bath with PMSF (protease inhibitor) or at room temperature without the reagent.This is probably The recovery of selenium compounds with a mixture of proteolytic enzymes has often been used to ensure the quanti- because the enzymes in the dry sample analyzed had already lost their activity or that the selenium compound recovered is tative recovery of Se from biological samples.5,8,9,24 Protease is able to break the peptic bonds of proteins present in the not a protein so it is resistant to the degradation by proteolytic enzymes. sample, so information concerning the original selenium- 648 J.Anal. At. Spectrom., 1999, 14, 645–650Sequential extraction of Se species from yeast The results obtained above suggest the possibility of developing a sequential leaching procedure that is able to provide information on the speciation of selenium in nutritional yeast supplements without the need for a chromatographic separation. Fig. 2 shows chromatograms of sequential extracts of a selenized yeast sample using diVerent leaching agents (in contrast to Fig. 1, now it was the residue after one leaching procedure that was subjected to leaching with another reagent). The three characteristic leaching reagents to be applied sequentially included (1) hot water [Fig. 2(a)] to extract water-soluble fraction, (2) Driselase [Fig. 2(b)] to release selenium present in cell and (3) SDS [Fig. 2(c)] to give the protein-soluble fraction. Considering the limited information available on the identity of chromatographic signals, the determination of total selenium in the sequential leachates gives as much information as HPLC-ICP-MS.Conclusions Size-exclusion chromatography with ICP-MS detection is a useful technique for screening selenium species in yeast extracts since it combines the satisfactory resolution of the small watersoluble Se species with the possibility of monitoring the high molecular mass fraction. The most critical step in speciation analysis for selenium compounds in yeast is the extraction of the intact species from a sample.Basic information on speciation of Se in yeast can apparently be obtained without the need for a coupled technique by sequential leaching with carefully designed reagents. The study reveals the existence of various Se species in yeast, which should be isolated and purified for identification by, e.g., electrospray MS.27 Fig. 2 Size-exclusion chromatograms with ICP-MS detection obtained References for leachates using the sequential extraction approach: (a) leaching 1 M.S. Alaejos and C. D. Romero, Chem. Rev., 1995, 95, 227. with hot water; (b) leaching of the solid residue after (a) with a 2 T. C. Stadtman, J. Biol. Chem., 1991, 266, 257. solution of Driselase; (c) leaching of the solid residue after (b) with a 3 F. Dubois and F. Belleville, Pathol. Biol., 1988, 36, 1017. solution of SDS. Data are shown for 82Se. 4 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, Jr., H. K. Park, B. B. Sanders, Jr., C. L. Smith and J. R. Taylor, J. Am. Med. species proteins is lost. Indeed, the chromatograms obtained Assoc., 1996, 276, 1957. 5 N. Gilon, A. Astruc, M. Astruc and M. Potin-Gautier, Appl. for a leachate of the yeast sample with proteolytic enzymes Organomet. Chem., 1995, 9, 623. [Fig. 1(a)–(c), panel next to the bottom] indicate the presence 6 S. M. Bird, P.C. Uden, J. F. Tyson, E. Block and E. Denoyer, J. of a single intense peak. This peak was identified also by Anal. At. Spectrom., 1997, 12, 785. anion-exchange chromatography as selenomethionine. 7 S. M. Bird, H.-H. Ge, P. C. Uden, J. F. Tyson, E. Block and E. The proteolytic hydrolysis leads to the solubilization of Denoyer, J. Chromatogr., 1997, 789, 349. almost 90% of the selenium initially present in yeast. 8 J. Zheng, W. Go� ssler and W. Kosmus, Trace Elem. Electrol., 1998, 15, 70. 9 R.Mun� oz Olivas, O. F. X. Donard, N. Gilon and M. Potin- Quantitative recovery of Se from yeast Gautier, J. Anal. At. Spectrom., 1996, 11, 1171. 10 R. Mun�oz Olivas, O. F. X. Donard, C. Camara and Ph. All the procedures investigated above left a small solid residue Quevauvillier, Anal. Chim. Acta, 1994, 286, 357. after centrifugation of the supernatant. This residue always 11 K. Pyrzyn� ska, Chem. Anal. (Warsaw), 1995, 40, 677. contained some selenium. We therefore attempted to solubilize 12 G.Ko� lbl, K. Kalcher, K. J. Irgolic and R. J. Magee, Appl. the yeast sample completely to recover all of the Se. Two Organomet. Chem., 1992, 7, 443. approaches were considered: acid protein hydrolysis with HCl 13 X. Dauchy, M. Potin-Gautier and M. Astruc, Fresenius’ J. Anal. Chem., 1994, 348, 792. at 110 °C, which was reported earlier to degrade the Se 14 W. Go� ssler, C. Ku� hnelt, C. Schlagenhaufen, K. Kalcher, M. compounds in yeast,25 and alkaline hydrolysis using TMAH, Abegaz and K.Irgolic, J. Chromatogr., 1997, 789, 233. which has been used to solubilize biological tissues allowing 15 A. Makarov and J. Szpunar, Analusis, 1998, 26, M44. the quantitative recovery of intact organometallic species.rin and Z. Gunata, Ele�ments d’Oenologie, ed. C. Flanzy, The latter approach was investigated in more detail. Lavoisier, Paris, 1998. Chromatograms obtained after the hydrolysis of a yeast 17 M. Korhola, A. Vainio and K. Edelmann, Ann. Clin. Res., 1986, 18, 65. sample with TMAH are shown in Fig. 1(a)–(c) (bottom 18 C. Hammel, A. Kyriakopoulos, D. Behne, D. Gawlik and P. panel ). No solid residue is left after the solubilization. The Bratter, J. Trace Elem. Med. Biol., 1996, 10, 96. total peak area accounts for 97% of the total Se present. The 19 M. A. Beilstein, P. D. Whanger and G. Q. Yang, Biomed. Environ. morphology of the chromatograms obtained using the diVerent Sci., 1991, 4, 392. separation mechanisms indicates the degradation of the Se 20 T. W.-M. Fan, A. N. Lane and R. M. Higashi, Environ. Sci. species initially present to inorganic selenium [probably Technol., 1997, 31, 569. 21 Z. Ouyang, J. A.Wu and L. Q. Xie, Anal. Biochem., 1989, 178, 77. Se(VI)]. Some traces of selenoamino acids are still left. J. Anal. At. 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