Quantification of the metal distribution in metallothioneins of the human liver by HPLC coupled with ICP-AES

Fractions containing metallothioneins (MT’s), extracted from the liver cytosol of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for differences in the trace-element binding pattern. After the extraction of supernatants from the tissue samples, membrane ultrafiltration of the cytosolic solution was carried out to separate all high-molecular proteins with molecular weights >100 kDa. This procedure retains the metal content of the MT’s in its initial form, in contrast to the often-used heat treatment of samples, which changes the copper distribution significantly. The MT’s themself were isolated using size exclusion and anion exchange chromatography. Their metal content was determined simultaneously on-line by combination with an ICP-AES as element detector. Calibration of the procedure was performed by means of a column by-pass-injection of elemental standards into the separation system. The MT content in the samples was calculated using the determined metal concentrations and the generally accepted metal/protein ratios for Cu (12:1), Cd (7:1) and Zn (7:1). These values were compared with values resulting from a ¹⁰⁹Cd-saturation-assay. When various liver samples of different pathogenesis were compared, the highest level of Cu-MT was found in primary biliary cirrhosis.     

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.     

Second derivative synchronous fluorescence spectroscopy for the simultaneous determination of metoclopramide and pyridoxine in syrup and human plasma

A rapid, simple, and highly sensitive second derivative synchronous fluorometric method has been developed for the simultaneous determination of metoclopramide (MT) and pyridoxine (PY) in a binary mixture. The method is based on measurement of the native fluorescence of these drugs at delta lambda = 80 nm in methanol. The different experimental parameters affecting the native fluorescence of the drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the ranges of 0.02-0.4 and 0.1-2 microg/mL for MT and PY, respectively. The limits of detection were 0.003 and 0.007 microg/mL and the limits of quantification were 0.008 and 0.02 microg/mL for MT and PY, respectively. The proposed method was successfully applied to the determination of MT and PY in synthetic mixtures and in commercial syrup. The results were in good agreement with those obtained with a reported method. The high sensitivity attained by the proposed method allowed the determination of MT in spiked and real human plasma samples. The mean percent recoveries of MT from spiked and real human plasma (n = 3) were 93.72 +/- 3.15 and 89.72 +/- 2.19 respectively.     

HPLC-based method using sample pre-column clean-up for the determination of methanethiol and ethanethiol in parenteral amino acid solutions

A method has been developed for the chromatographic determination of methanethiol (MT) and ethanethiol (ET) as contaminants in amino acid parenteral nutrition (PN) solutions. The clean-up of the samples before chromatographic analysis was investigated by solid-phase extraction (SPE) on pre-columns filled with polyethylene powder (PE), aluminium oxide (AlOx), silica (SiOx), or polyurethane foam (PUF) as adsorbents. The thiols were more efficiently separated from the matrices by SPE on PUF pre-columns. Simultaneous derivatization and elution with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) enabled further discrimination between MT and ET by reversed-phase HPLC with spectrophotometric detection. The retention times for the derivatized MT and ET species were 12.5 and 23.0 min, respectively. Recoveries from spiked PN samples were calculated to be approximately 90%, and the MT and ET content of commercial PN solutions was determined using the methodology described. Detection limits of 15 and 10 microg L(-1) were calculated for MT and ET, respectively.     

HPLC-based method using sample pre-column clean-up for the determination of methanethiol and ethanethiol in parenteral amino acid solutions

A method has been developed for the chromatographic determination of methanethiol (MT) and ethanethiol (ET) as contaminants in amino acid parenteral nutrition (PN) solutions. The clean-up of the samples before chromatographic analysis was investigated by solid-phase extraction (SPE) on pre-columns filled with polyethylene powder (PE), aluminium oxide (AlOx), silica (SiOx), or polyurethane foam (PUF) as adsorbents. The thiols were more efficiently separated from the matrices by SPE on PUF pre-columns. Simultaneous derivatization and elution with DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) enabled further discrimination between MT and ET by reversed-phase HPLC with spectrophotometric detection. The retention times for the derivatized MT and ET species were 12.5 and 23.0 min, respectively. Recoveries from spiked PN samples were calculated to be approximately 90%, and the MT and ET content of commercial PN solutions was determined using the methodology described. Detection limits of 15 and 10 μg L^–1 were calculated for MT and ET, respectively.     

Effects of redox conditions and zinc(II) ions on metallothionein aggregation revealed by chip capillary electrophoresis

Metallothioneins (MTs) belong to cysteine-rich proteins with unique higher structure. One of the most known MT's functions is metals detoxification and maintaining their homeostasis in a cell. Structure of MT with naturally occurred zinc(II) ions can be affected by concentration of metal ions as well as redox milieu inside a cell, however the exact explanation and biochemical effects of the structural changes are still missing. In this study we used capillary electrophoresis on chip coupled with fluorescence detection to determine structural changes of MT with increasing concentration of zinc(II) ions and under various redox conditions. To investigate the structural-dependent effects, reduced and/or oxidized apo-MT (MT without natural occurred metal ion) was prepared. Zinc binding into reduced and/or oxidized apo-MT was compared. MT was incubated with 0, 5, 15, 25, 50 and 100 μM ZnCl₂ for 1 h in 37 °C. Formation of MT aggregates with increasing zinc concentration was observed by spectrophotometry, chip capillary electrophoresis, and SDS-PAGE. We found out that reduced MT forms aggregates more readily compared to oxidized MT. Using the chip capillary electrophoresis allowed us relative quantification of MT aggregation as a decrease in the area of the signal corresponding to the monomer form of MT (Mw 15 kDa, migration time 26.5 s) and its ratio to total signal (sum of all signals measured by the electrophoresis). The dependences had an exponential character with equation y = 2.4 × e^−0.01x, R² = 0.945 for 15 kDa peak area and y = 0.11 × e^−0.01x, R² = 0.938 for decrease of 15 kDa peak area ratio to the total signal. Zn–MT interaction was 30% faster during the first 15 min and 50% faster during the whole experiment for reduced MT. It can be concluded that formation of MT aggregates is dependent on redox state and Zn(II) concentration.     

Speciation analysis of platinum antitumoral drugs in impacted tissues

Chemical compounds containing platinum have been employed since 1978 as drugs to beat certain type of tumours. Nevertheless, besides of their exceptional antitumoral properties, these drugs also have important deleterious side effects, such as, nephrotoxicity and ototoxicity.A study of Pt accumulation and a speciation analysis has been performed by ICP-MS in samples from kidney and inner ear in a controlled population of Wistar rats treated with, either, cisplatin, carboplatin or oxaliplatin. The results on Pt accumulation point out to drug structure and not only to Pt content as the responsible for the alteration of organ functionality.Speciation studies in the samples from kidney and inner ear were performed coupling two-dimensional liquid chromatography (2D-LC) to ICP-MS. Size exclusion (SEC) and anion exchange fast protein liquid chromatography (FPLC) was employed for 2D orthogonal separation. After these separations, free drug peaks were not observed in any of the samples.The binding of Pt to biomolecules was demonstrated by SEC and, independently of the drug used, Pt eluted as two main bands with molecular weights of 12 kDa and 25-65 kDa for inner ear samples, and as two different bands with 20 kDa and 50-60 kDa in the samples from kidney. However, the relative band intensity presented important differences for the three drugs. Using the same chromatographic conditions, it was shown that a metallothionein (MT) standard eluted in the same position as some of the cytosolic Pt-biomolecules.High Pt-containing fractions eluting from the SEC column were analysed by anion exchange FPLC after a preconcentration step. Among the different preconcentration methods tested, sample focusing on the head of the FPLC column shows main advantages. In this way, the separation by 2D chromatography of the high molecular Pt-species has been considerably improved.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

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.     

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.     

Electrochemical methods for quantification and characterization of metallothioneins induced in Mytilus galloprovincialis

The quantification of the purified metallothionein (MT) component, isolated from the digestive gland of cadmium-exposed Mytilus galloprovincialis, is described based on the analysis of Cd(II) and SH-groups content, applying electrochemical methods. Advantages and disadvantages of the Brdicka procedure for the determination of the MT content is discussed. The saturation of binding positions of purified MT with Cd(2+) ions can be directly followed voltammetrically. Irrespective of the MT concentration, the saturation with Cd(2+) of in vivo induced mussel MT is achieved at a molar ratio of 5. Cd(2+) ions are rapidly displaced from the Cd-Th complex after the addition of Pb(2+) ions, which indicates the kinetically labile type of the complex.     

Quantification of the metal distribution in metallothioneins of the human liver by HPLC coupled with ICP-AES

Fractions containing metallothioneins (MT's), extracted from the liver cytosol of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for differences in the trace-element binding pattern. After the extraction of supernatants from the tissue samples, membrane ultrafiltration of the cytosolic solution was carried out to separate all high-molecular proteins with molecular weights >100 kDa. This procedure retains the metal content of the MT's in its initial form, in contrast to the often-used heat treatment of samples, which changes the copper distribution significantly. The MT's themself were isolated using size exclusion and anion exchange chromatography. Their metal content was determined simultaneously on-line by combination with an ICP-AES as element detector. Calibration of the procedure was performed by means of a column by-pass-injection of elemental standards into the separation system. The MT content in the samples was calculated using the determined metal concentrations and the generally accepted metal/protein ratios for Cu (12:1), Cd (7:1) and Zn (7:1). These values were compared with values resulting from a 109Cd-saturation-assay. When various liver samples of different pathogenesis were compared, the highest level of Cu-MT was found in primary biliary cirrhosis.     

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.     

Poly-N-Isopropylacrylamide Hydrogel Based Fluoroimmunoassay of Methyltestosterone

IntroductionMethyltestosterone(MT)isasynthesizedsteroid.Athletesusesyntheticsteroidstoincreasetheirsportgrade.This,fromtheviewofethics,violatedtheoriginalrulesofsport.Therefore,themonitoringofthedopeabusehasbecomemoreandmoreimportantworldwide.Sofar.themonitoringiscommonlycarriedoutbychromatographicseparationfollowedbymassspectrometricanalysis.Thetargetsamplesarenormallyurinel~2.Themonitoringbasedonbloodsampleismainlyusedasacomplementarymethodtoovercometheshortcomingfromtheanalysisusingurines…     

甘露聚糖肽的结构鉴定

采用离子交换柱层析和凝胶柱层析从甘露聚糖肽原料中分离纯化得到4种均一糖肽MT1-A,MT1-B,MT2-A和MT2-B;通过单糖组成分析、甲基化分析、氢核磁共振谱(~1H NMR)、红外光谱(IR)和氨基酸组成分析等手段对均一糖肽MT1-A,MT1-B和MT2-B的结构进行了鉴定.结果表明,3种均一糖肽的单糖组成、糖残基的连接方式与MT2-A相同,但是分子量以及氨基酸总量有所不同.测定了不同批次甘露聚糖肽原料的~1H NMR谱,以MT2-A的~1H NMR谱图为标准谱,以异头氢区域为鉴定区域,通过计算不同批次甘露聚糖肽原料以及其它3种均一糖肽与MT2-A的~1H NMR谱的相关系数,定量评价了不同批次甘露聚糖肽原料与4种均一糖肽的相似程度.结果表明,甘露聚糖肽是由糖链结构一致、分子量和氨基酸含量不同的几种均一糖肽构成的混合物.     

Preparative separation and determination of matrine from the Chinese medicinal plant <Emphasis Type="Italic"> Sophora flavescens </Emphasis>Ait by molecularly imprinted solid-phase extraction

Molecularly imprinted microspheres (MIMs) were synthesized by micro-suspension polymerization using matrine (MT) as template. The MIMs were employed for solid-phase extraction (SPE) and as chromatographic stationary phase for the determination of MT from the Chinese medicinal plant Sophora flavescens. The effects of the various eluents, their concentrations and volumes on the retention behavior were investigated. The selectivity and capacity of the imprinted microspheres against MT was also discussed. The results showed that the MIMs exhibited stronger specific affinity to MT than to oxymatrine (OMT). Methanol-water (3:7, v/v) was used for washing impurities from the MIMs-SPE cartridge loaded with the herb extracts, while methanol-glacial acetic acid (9:1, v/v) was used for eluting MT. The maximum load of MT and the recovery of MIMs cartridge towards MT were 38.7 microg g(-1) and 71.4%, respectively. The method developed might be used to separate and extract effective constituents from Chinese medicinal plants on a large scale.     

Identification and determination of aesculin and aesculetin in ash barks by capillary zone electrophoresis

Summary A capillary zone electrophoresis method for identification and determination of aesculin and aesculetin has been established using borate-phosphate buffer containing 30% ethanol with on-column UV detection. A detailed investigation of the influence of changes in borate concentration, pH, applied voltage, temperature and organic modifier was then carried out. For both aesculin and aesculetin, a linear plot of migration time (MT) against borate concentration was obtained, and ln[measured peak area (MA)] and lnMT both gave linear plots against ln(applied voltage) with correlation coefficient r>0.999, which also resulted in a linear correlation between MA and MT (r≥0.9998) under varied voltage. Ethanol as organic modifier to the background electrolytes helped in separating aesculin and aesculetin from other components in ash barks. The reproducibility with relative standard deviation in MT and in normalized peak area(NA) and linearity based on NA against concentration were evaluated. Finally, the method was successfully applied to monitor the quality of different ash barks and to compare the effect of sample preparation on content of bioactive components in ash bark. Results indicate that CZE promises to be applicable to quality control of traditional Chinese medicines containing aesculin and aesculetin.     

Nanogapped impedimetric immunosensor for the detection of 16 kDa heat shock protein against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The 16 kDa heat shock protein (16 kDa HSP) against Mycobacterium tuberculosis (MT), expressed during the growth phase of MT, is a potential target in diagnostic tests for tuberculosis (TB). We describe here a method for impedimetric determination of the antigen by using a nanogapped dielectric surface consisting of a silver support coated with a thin finger-shaped coating made from zinc oxide and gold and patterned through a lift-off process. The electrode was characterized by scanning electron microscopy, field emission scanning electron microscopy, atomic force microscopy, and energy-dispersive X-ray spectroscopy. Surface chemical functionalization and immobilization of antibody against the 16 kDa HSP was evidenced by FTIR. In order to improve the detection limit, the antigen was conjugated to 10 nm gold nanoparticles. The resulting biosensor is capable of detecting the 16 kDa HSP in concentrations as low as 100 fM. The method covers a wide analytical range that extends from 100 fM to 1 nM.

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Graphical abstract Schematic presentation of the nanogapped impedimetric immunosensor for the diagnosis of tuberculosis

     

Simultaneous determination and pharmacokinetic study of oxymatrine and matrine in beagle dog plasma after oral administration of Kushen formula granule, oxymatrine and matrine by LC-MS/MS

A rapid, specific and sensitive LC-MS/MS method was developed for the determination of oxymatrine (OMT) and matrine (MT) in beagle dog plasma. The method was applied to study the pharmacokinetics of OMT and MT after oral administration of OMT, MT and Kushen formula granule (KFG) containing equivalent amounts of OMT and MT in a three-period crossover design. The analysis was carried out on an Acquity UPLC BEH C(18) column by linear gradient elution with 0.01% acetic acid-water-methanol as mobile phase. Detection was by positive ion electrospray ionization (ESI) mass spectrometry with multiple-reaction monitoring (MRM). Linear calibration curves were both obtained over the concentration range 15-2000 ng/mL, with a limit of quantification of 15 ng/mL. The matrix effect was minimized. The intra- and inter-day precisions (RSDs) were less than 12.4 and 14.7%, respectively, and the accuracy (RE) was from -2.1 to 2.7%. The validated method was used to determine the concentration-time profiles of OMT and MT. The results indicated that the absorption of OMT and MT after oral administration of KFG was significantly greater than that after oral administration of pure components.     

Electrochemical methods for quantification and characterization of metallothioneins induced in Mytilus galloprovincialis

The quantification of the purified metallothionein (MT) component, isolated from the digestive gland of cadmium-exposed Mytilus galloprovincialis, is described based on the analysis of Cd(II) and SH-groups content, applying electrochemical methods. Advantages and disadvantages of the Brdika procedure for the determination of the MT content is discussed. The saturation of binding positions of purified MT with Cd²⁺ ions can be directly followed voltammetrically. Irrespective of the MT concentration, the saturation with Cd²⁺ of in vivo induced mussel MT is achieved at a molar ratio of 5. Cd²⁺ ions are rapidly displaced from the Cd-Th complex after the addition of Pb²⁺ ions, which indicates the kinetically labile type of the complex.