Structural changes in metallothionein isoforms revealed by capillary electrophoresis and Brdicka reaction

Metallothionein (MT) as a potential cancer marker is at the center of interest and its properties, functions and behavior under various conditions is intensively studied. In the present study, two major mammalian MT isoforms (MT‐1 and MT‐2) were separated using capillary electrophoresis (CE) coupled with UV detector in order to describe their basic behavior. Under the optimized conditions, the separation of both isoforms was enabled as well as estimation of detection limits as subunits and units of ng per μL for MT‐2 and MT‐1, respectively. Further, the effects of thermal treatment and the presence of denaturing agent such as urea on MT‐1 and MT‐2 isoforms were studied by CE‐UV. Thermal treatment caused an increase in the signals of both isoforms. A new parameter called precipitation rate has been defined based on this finding. This parameter can be expressed as a slope of the linear regression of the time dependency curve recalculated on the MT concentration. The thermal precipitation rate for MT‐1 and MT‐2 was determined as 1.1 and 0.9 ng of MT/min, respectively. The chemical precipitation rate calculated from the linear regression for both isoforms provided the same value of 0.25 ng of MT/min. The results were confirmed by manual spectrometric measurements and by differential pulse voltammetry Brdicka reaction. Based on these results, a model of MT behavior under the conditions studied was suggested.     

Differential ESI-MS behaviour of highly similar metallothioneins

ESI-MS can only be accepted as a quantification method when using standards with a high resemblance to the analyte(s). Unfortunately, this is usually not applicable to metallothioneins (MTs), a superfamily of singular metal-binding cysteine-rich proteins, present in all living organisms, since the absence of suitable reference material due to the high diversity among metal-MT species precludes their quantification by molecular mass spectrometry. Even thus, it is widely assumed that the intensities of the ESI-MS peaks of similar species are directly correlated with their relative concentration in the sample, and this has been extended to the determination of different MT proteins coexisting in a sample.Practically all organisms contain several MT isoforms, some of them exhibiting highly similar sequences, with conserved coordinating Cys residues. For the current analysis, we used as a model system the MT isoforms of two terrestrial snails (Helix pomatia and Cornu aspersum). Hence, distinct samples were prepared by mixing, at different molar ratios, the recombinant HpCuMT and HpCdMT isoforms from H. pomatia, or the recombinant CaCuMT, CaCdMT and CaCdCuMT isoforms from C. aspersum, and they were analyzed by ESI-MS both at neutral pH (for Zn-loaded MT forms) and at acidic pH (for the corresponding apo-forms). The results here presented reveal that the ESI-MS peak intensity of a single MT species strongly depends on its sensitivity to be ionized, and thus, on the presence or absence of metal ions bound. Furthermore, our data demonstrate that very similar MT isoforms of the same organism with similar pI (ranging from 7.9 to 8.3) can show a clear different sensitivity to ES ionization, something that cannot be readily predicted only by consideration of their amino acid content. In conclusion, even in this optimum case, deductions about quantity features of MT samples drawn from ESI-MS measurements should be carefully considered.     

Anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma-isotope dilution-time-of-flight mass spectrometry for speciation analysis of metal complexes with metallothionein isoforms in gibel carp (Carassius auratus gibelio) exposed to environmental metal pollution

The capability of post-column isotope dilution (ID) combined with anion-exchange HPLC-ICP-time-of-flight (TOF)-MS was for the first time investigated for environmental quality assessment through metal speciation analysis of metallothionein (MT) isoforms in cytosols of gibel carp (Carassius auratus gibelio), used as biomarkers for environmental metal exposure. A full spectral scanning of the biological sample (with 50 microl injection volume) using ICP-TOF-MS in transient mode allowed fast multi-isotope screening of cytosolic metal-containing fractions and to investigate the presence of matrix-induced interferences. The MT cytosolic fraction of liver and kidney of the carp, sampled at three different sampling sites in Belgium, was partially purified using size-exclusion (SE) HPLC. Quantification of the elements Cd (toxic) and Zn and Cu (essential) associated with MT isoforms in this fraction was addressed using an hybrid approach based on post-column addition of the enriched isotopes 65Cu, 67Zn, 106Cd and monitoring on-line the isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd by ICP-MS with a time of flight instrument, which was coupled to anion-exchange HPLC. With this separation method, baseline separation of up to five MT isoforms, which is required for quantitative metal speciation by HPLC-ICP-IDMS, was achieved within a run of 15 min. The MT fraction of the cytosols was also analysed for the total metal content using IDMS with size-exclusion HPLC-ICP-MS and species-unspecific calibration. Results showed significant differences between speciation results and total MT concentrations of control fish and fish from the most contaminated sampling sites, revealing the potential of gibel carp MT for sequestering excess intracellular free-ions (essential and toxic elements) and for its protection against metal toxicity. Preferences for metal sequestration of metal complexes with MT isoforms were also found to be tissue-specific: excess of Cd was found preferably bound to a major MT isoform (tR = 8.0 min) in kidney, whereas excess intracellular Zn appeared to be mostly sequestered by four MT isoforms (tR=7.3, 8.0, 12.2 and 14.4 min) in liver, the MT form with tR = 8.0 min being the main Zn scavenger form. Such kind of quantitative speciation information on the preferences of MT isoforms in different fish organs for sequestering heavy metals, reported here for the first time, is important to elucidate the role of isoform-specific induction of vertebrate fish MT in metal detoxification and the use of MT as biomarker.     

兔肝金属硫蛋白亚型异构体的高效液相色谱分离与ESI-MS,MALDI-TOF-MS鉴定

由于金属硫蛋白(MT)基因的多态性,决定不同亚型的MT异构体的存在,MT亚型异构体的结构是MT功能研究的基础.通过离子交换柱可将MT分成MT-1和MT-2两个异构体,用不同条件的反相高效液相色谱(RP-HPLC)可将MT-1和MT-2分成不同的亚型异构体,并利用MALDI-TOFMS和LC-ESI-MS对比确定了它们的分子量.结果表明,兔肝MT在不同的pH条件下分离得到不同分子量的亚型异构体.在酸性条件下,MT-1可分为2个主要亚型异构体,分子量分别为6149.0和6244.5,而MT-2主要分为3个亚型异构体,分子量分别为6149.0,6244.0和6127.0.MT-1和MT-2有2个亚型异构体分子量相同的异构体存在.在酸性条件下,MT-1的2个异构体及MT-2分子量为6127的亚型异构体可稳定存在.     

Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH

A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I.     

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 μm I.D. columns give better separations than 50 or 75 μm I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C₁ or C₁₈ hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress. Analysis of sheep liver extracts using optimised conditions showed the expression of at least 4 MT isoforms in response to Zn injection and 3 of these forms were evident in extracts from untreated sheep. We therefore conclude that MECC is a suitable method for MT isoform analysis.     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

Metallothioneins from horse kidney studied by separation with capillary zone electrophoresis below and above the isoelectric points

Capillary zone electrophoresis (CZE) was used to study metallothionein (MT) isoforms and non-MT components in the horse kidney MT preparation produced by Sigma. This technique was found to be well suited for studies of such forms. The non-MTs are heat resistant, they do not bind metal, but comigrate with MT in the various systems generally used for MT isolations. These forms may cause discrepancies between claimed MT content and sample weight as well as confusion in MT identification. The functional metal binding ability in the Sigma sample was measured, and the elution profile from an anion exchange column, commonly used for separation of tissue MTs, was determined in order to ascertain the MT-I isoform content. Optimum conditions for the separation of the MT isoforms have been studied in the polyacrylamide coated capillary at pHs below and above the isoelectric points (pIs) in various buffer systems. Evidence is put forward that MT forms oligomers or aggregates in the metal binding situation at pHs above pI. Our results may indicate that the oligomerized MT-IA form binds additional Cd atoms than the expected number of seven per monomer.     

Narrow-bore HPLC–ICP–MS for speciation of copper in mutant mouse neonates bearing a defect in Cu metabolism

Minute amounts of tissue supernatants from mouse neonates bearing a mutation in the copper (Cu)-transporter gene, Atp7a, were injected into narrow-bore HPLC coupled with an inductively coupled plasma–mass spectrometer (ICP–MS) to examine Cu metabolism. In the 14-day-old mutant neonates, Cu accumulated in the intestine in the metallothionein (MT)-bound form, and mRNA expression of the two MT isoforms was increased. Meanwhile, Cu in the MT-bound form (Cu-MT) was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. These results suggest that Cu is not secreted by intestinal microvillus cells into bloodstream due to the defect of Atp7a, and systemic depletion of Cu occurred. On the other hand, in the kidneys of mutant mice, Cu accumulated in the MT-bound form despite the fact that mRNA expression of the two MT isoforms was low. Part of Cu-MT in microvillus cells may be released into bloodstream at turnover and be preferably taken up by the kidneys. Consequently, the mRNA expression of MT isoforms was not always coincident with the amounts of MT proteins binding Cu, and narrow bore HPLC–ICP–MS used for MT protein determination is a complementary technique to real-time RT-PCR used for MT mRNA determination in Cu speciation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.     

Identification and quantification of metallothionein isoforms and superoxide dismutase in spiked liver extracts using HPLC-ESI-MS offline coupling and HPLC-ICP-MS online coupling

A two-dimensional chromatographic method for the characterization of metallothionein isoforms (MT) and superoxide dismutase (SOD) in spiked liver extracts was developed for the optimization of extraction procedures from liver samples. Element-specific detection (ICP-MS) and molecule-specific detection (ESI-MS) were applied for maximum species information. A special focus was laid on the quantitative data evaluation (species stoichiometry, calibration with and without matrix, recovery), which is neglected in most MT/SOD publications with hyphenated techniques. Linearity, precision (residual standard deviation of calibration curves <10%), and detection limits (<0.6 mg L(-1) for MT isoforms and 13 mg L(-1) for SOD) prove the suitability of the method for quantification. An alternative quantification is proposed for the extension towards other lesser or even unknown trace element species, especially the native porcine MT and SOD.     

Metal speciation of metallothionein in white sea catfish, Netuma barba, and pearl cichlid, Geophagus brasiliensis, by orthogonal liquid chromatography coupled to ICP-MS detection

Metal speciation analysis in MTs was carried out in two tropical fish species of Brazil, the freshwater fish pearl cichlid (Geophagus brasiliensis) and the marine fish white sea catfish (Netuma barba), that are presently used to monitor the effects of heavy metal pollution in aquatic ecosystems in Brazil. In order to obtain the MT fraction, liver cytosols from both fish species where subjected to size exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. That MT fraction was then separated by anion-exchange (AE)-FPLC, whose optimal chromatographic conditions were optimized for the separation of the different hepatic MT isoforms existing in both fish species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with the ICP-MS instrument. The analytical results showed that MTs of these fish species, unknown so far, exhibited unique characteristics in comparison with standard MTs and other fish liver MTs. In fact, MT isoforms of N. barba turned out to be very anionic, as indicated by their high retention in the Mono Q column and the strong ionic strength required to separate them. As for G. brasiliensis, cadmium was exclusively present in only one of the peaks of the MT isoforms showing a unique metal-binding behavior for MT in this fish species. The differences between the MTs among these species and the different association of metals in particular MT isoforms display the importance of the metal speciation analysis in these proteins prior to its use as bioindicators.     

Characterization of the metal composition of metallothionein isoforms using reversed-phase high-performance liquid chromatography with atomic absorption spectrophotometric detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate metallothionein (MT) isoforms and on-line atomic absorption spectrophotometric (AAS) detection was used to quantitatively determine their metal content. With this coupled system (HPLC-AAS), it was possible to determine the zinc, cadmium and copper content of individual horse kidney MT isoforms. When rabbit liver MT and the purified isoforms (MT-1 and MT-2) were subjected to RP-HPLC and the zinc-containing peaks of the MT sample to MT-1 or MT-2. HPLC-AAS was used to identify zinc-induced MT in heat-treated cytosol from turkey hen liver, thereby demonstrating its application to the analysis of crude tissue extracts. A standard curve was established using turkey liver MT for the quantitative determination of the zinc content of MT isoforms. There was excellent linear correlation between the micrograms of zinc bound to MT injected onto the column (ranging from 0.34 to 3.43 micrograms of MT-bond zinc) and the integrated peak area of the atomic absorbance for zinc. Using this standard curve, it was possible to quantitate the amount of MT-bound zinc in cytosol extracts of cultured turkey embryo hepatocytes exposed to varying levels of supplemental zinc in the culture medium.     

Purification of metallothionein proteins from the crab, Portunus pelagicus-selectivity of hydrophobic interaction chromatography

Both hydrophobic interaction chromatography (HIC) and ion-exchange chromatography (IEC) are commonly used for the purification of proteins as their mild elution conditions preclude the dangers of denaturation. Of the two methods, IEC which fractionates proteins on the basis of charge differences, exhibits a higher selectivity. In the case of the metallothionein (MT) proteins, the charge states of the proteins are very susceptible to change due to oxidation of the abundant thiol groups. This complicates fractionation with IEC. Separation using HIC, on the other hand, is based on the exposed hydrophobic groups, which remain relatively intact if the tertiary structure of the proteins is not disrupted. In this work, the successful isolation of two MT isoforms from the tropical crab species, Portunus pelagicus, using HIC serves to demonstrate the high selectivity of this technique, in addition to the fact that it is indifferent to the state of oxidation of the MT proteins during the purification procedure.     

An alternative interface for CE-ICP-MS cadmium speciation in metallothioneins based on volatile species generation

An alternative CE-ICP-MS interface based on volatile species generation (VSG) is here developed, evaluated and compared to the conventional sample introduction systems via nebulisation. For this purpose, the speciation of Cd-metallothioneins (MTs) in rabbit liver is taken as a model. Cd, bound to the different MT isoforms previously separated by CE, is transformed into volatile species at the exit of the capillary and on-line detected by ICP-MS. Optimum conditions for Cd VSG have been investigated in a flow injection device, using NaBH₄ as hydrogenation reagent in a HCl medium containing cobalt and thiourea as catalysts. Sample volume injected, CE separation voltage and reagents flows have been optimised. Analytical performance characteristics of the CE-VSG-ICP-(Q)MS coupling developed were evaluated, in terms of repeatability and linearity of response, using standard rabbit liver metallothionein isoforms (MT1 and MT2). Detection limits for Cd-MTs turned out to be almost one order of magnitude better than those derived from using a conventional Babington nebuliser-based interface. Compared to a MicroMist-based interface detection limits resulted to be similar, but the observed peak height was eight times higher using the VSG interface, indicating the enhanced analyte transport efficiency derived from VSG sample introduction systems.     

金属硫蛋白的色谱/电喷雾电离质谱联用表征方法

通过反相液相色谱(RPLC)与电喷雾电离质谱(ESI-MS)的联用技术,对镉诱导金属硫蛋白标准物质MT-1和MT-2的结构进行表征分析。采用Vydac C8 反相色谱柱(250 mm×2.1 mm i.d., 5 μm, 30 nm),流动相A为pH 6.0的5 mmol/L乙酸铵水溶液,流动相B为pH 6.0的5 mmol/L乙酸铵的甲醇-水(体积比为1∶1)溶液,流动相流速为0.20 mL/min,在40 min内流动相B的体积分数从10%增加到37.5%进行梯度洗脱。分别用紫外(UV)和ESI-M     

Characterization and quantification of metallothionein isoforms by capillary electrophoresis–inductively coupled plasma–isotope-dilution mass spectrometry

In a new approach to the characterization and quantification of metallothionein isoforms an on-line isotope-dilution method in combination with the coupling of capillary electrophoresis (CE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) is reported. Metallothionein (MT) isoforms are separated by CE and the elements Cu, Zn, Cd, and S are detected simultaneously by use of ICP-SFMS in the medium resolution mode. On-line isotope dilution is performed by continuous introduction of an isotopically enriched, species-unspecific spike solution after the separation step. MT from rabbit liver and a further purified MT-1 isoform were quantified by determination of sulfur, and the stoichiometric compositions of the metalloprotein complexes are characterized by determination of their sulfur-to-metal ratios.     

Determination of dietary cadmium-induced metallothioneins in rabbit kidneys and cadmium in metallothioneins by anion-exchange high-performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry.

A rapid method is described for the determination of dietary cadmium-induced metallothioneins (MTs) in rabbit kidneys by anion-exchange high-performance liquid chromatography. Rabbit kidney MT-I and MT-II were eluted at ca. 15.0 and 18.8 min, respectively, from a DEAE-5PW anion-exchange column with a Tris-HCl buffer (0.01-0.25 M, pH 8.6) and detected by ultraviolet absorbance at 254 nm. A standard calibration curve was constructed using purified standard MT isoforms, which demonstrated an excellent linear correlation between UV absorbance peak heights and the amounts of MT isoforms. Feeding a dose of cadmium for some days resulted in an increase in MT concentrations in rabbit kidneys, but not in the livers. The cadmium concentrations in MT-I and MT-II elutions were determined by graphite furnace atomic absorption spectrometry. MT-I and MT-II showed some differences associated with the oral intake of cadmium. Dietary cadmium also caused zinc to accumulate in kidneys to some extent. The effects of dietary oleic acid on the synthesis of MTs were also studied. Based on the method of standard additions, the recovery of MTs exceeded 93% and replicated injection of samples yielded a relative standard deviation of 2.4% at an MT level of 280 micrograms/g.     

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris-HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM beta-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg(-1). In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.     

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.