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.     

金属硫蛋白MT-2a与Cd(Ⅱ)和Cu(Ⅰ)络合的电喷雾质谱表征

采用电喷雾质谱法(ESI-MS)研究金属硫蛋白(MT,存在α和β两种结构域)-2a与金属离子Cd和Cu的络合作用.MT-2a由反相色谱分离纯化制得,并以电喷雾质谱鉴别.通过ESI-MS考察MT-2a与不同量的Cd和Cu的络合,结果表明,Cd能够优先与MT的α结构域络合,形成M2+4S11结构,并且该络合过程存在着明显的协同效应;Cd与MT的β结构域的络合形式为M2+4S9,其呈现出明显的随机松散络合特性;Cu优先与MT的β结构域络合,其络合形式由Cu4逐渐过渡到更高结合态;在Cu含量较高的条件下,Cu与MT呈现出多种络合方式.     

Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs’ polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.     

Differences in trace element concentrations between Alzheimer and “normal” human brain tissue using instrumental neutron activation analysis (INAA)

Brain samples obtained from the Netherlands Brain Bank were taken fromthe superior frontal gyrus, superior parietal gyrus and medial temporal gyrusof 'normal' and Alzheimer's disease subjects in order todetermine elemental concentrations and compare elemental composition. Brainsamples from the cortex were taken from 18 subjects, eight 'normals'(6 males and 2 females) and eleven with Alzheimer's disease, (1 maleand 10 females) and the following elemental concentrations, Na, K, Fe, Zn,Se, Br, Rb, Ag, Cs, Ba, and Eu were determined by instrumental neutron activationanalysis (INAA). The element which showed the greatest difference was Br,which was found to be significantly elevated in the cortex of Alzheimer'sdisease brains as compared to the 'normals' at significance (p<0.001).     

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.     

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.     

Separation and characterization of rabbit liver apothioneins by capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry

The present study establishes a method for the separation and characterization of rabbit liver metallothionein (MT) subisoforms by capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry (CE-ESI-TOF-MS) via a sheath-flow interface. Directly coupled-CE-MS enables the extraction of specific molecular weight information and thereby facilitates the identification of peaks when no reference materials are available, as in the case of MT subisoforms. The analysis described here revealed the presence of the apothioneins MT-1a, MT-2d, and MT-2e, belonging to MT-I sample, and MT-2a, MT-2b, and MT-2c, belonging to MT-II. Several non-N-acetylated forms were also detected as traces appearing with their respective acetylated forms in both samples. Similar results were found when MALDI-TOF experiments were performed, identifying all the sequenced rabbit liver MTs as apo-MT-forms, as in the CE-ESI-MS coupling.     

Mutations in mitochondrial-encoded cytochrome c oxidase subunits I, II, and III genes detected in Alzheimer's disease using single-strand conformation polymorphism

A "mitochondrial hypothesis" of late onset Alzheimer's disease (AD) has been proposed. Biochemical studies indicate that there is a significant decrease in cytochrome oxidase (CO) activity as well as perturbed CO I and CO III mRNA levels in platelets and brain tissue from Alzheimer's patients. Using the electrophoretic mutation detection technique SSCP and DNA sequencing, we have identified 20 point mutations in the mitochondrial-encoded CO subunits (CO I, II, and III) in AD and age-matched control brain samples. Eight of the mutations are new variants of the mitochondrial genome. The efficiency of SSCP in detecting mutations in the CO subunits was estimated to be 80% when compared to dideoxy sequencing. One of the mutations (at position 9,861) results in a phenylalanine-->leucine substitution at a highly conserved residue in CO III. CO activity was reduced by an average of 35% in all AD brains compared to age-matched control samples, which agrees with previous reports. CO activity in one of the AD brain samples carrying the 9,861 mutation decreased by 80% relative to control brain samples, suggesting that the phenotypic expression of this mutation may result in reduced CO activity and compromised mitochondrial function.     

Analysis of metallothioneins by means of capillary electrophoresis coupled to electrospray mass spectrometry with sheathless interfacing

Capillary electrophoresis (CE) coupled to electrospray mass spectrometry via sheathless interfacing has been applied to the analysis of mammalian metallothionein (MT) extracts. In a rabbit-liver extract, four (MT-2C, MT-2A, MT-2D and MT-2E) out of six known MT sub-isoforms were unambiguously identified under three CE-resolved peaks. A fourth peak was found to contain MT-1A and/or MT-2B, whose molecular masses differ by only 1 Da. Traces of non-N-acetylated MT-2D and MT-2E were observed in a fifth, minor peak. In a rat-liver extract, both MT-1 and MT-2 were resolved and identified. Non-N-acetylated MT-2 was also identified in a resolved, minor peak. Minimum detectable amounts of MTs have been estimated to be approximately 0.6 fmol per sub-isoform.     

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.     

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.     

Optimisation of extraction procedures for metallothionein-isoforms and superoxide dismutase from liver samples using spiking experiments

The speciation of trace element species in solid matrices like liver samples is still problematic due to two reasons. On the one hand direct methods with sufficient selectivity and sensitivity are currently not available. Therefore extraction procedures have to be applied which are often problematic in respect to species stability. On the other hand there are no reference materials with known amounts of metal proteins like metallothionein-isoforms (MT) and superoxide dismutase (SOD) for quality control. So the aim of this study was to develop and optimise procedures for the species-preserving extraction of the model compounds MT and SOD from liver samples. Spiking experiments were performed to overcome the lack of appropriate reference materials. In a first step the stability of the model species without liver matrix was investigated by the variation of several extraction parameters. The extractant and exposure to ultrasonic energy especially had a great influence on the recovery of the species while temperature, buffer concentration and atmospheric conditions were less critical. In a second step spiked liver samples were extracted with a selection of procedures taken from the literature. Most of these methods provided recoveries between 70% and 100%. Additionally the buffer concentration and the extractant-to-liver ratio were varied for optimisation. The metal balance of an extraction showed recoveries of 81% for Cd, 94% for Cu and 87% for Zn.     

Near-infrared Fluorescence Spectroscopy Detects Alzheimer's Disease In Vitro

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.     

Increased gamma-aminobutyrate aminotransferase activity in brain of patients with Alzheimer's disease

In order to search for more proximal factors in the pathogenesis of Alzheimer's disease, we studied the activities of various enzyme in the brains of patients, as well as control cases, by postmortem autopsy. In addition to the findings already known, such as the increase in prolyl endopeptidase (post-proline cleaving enzyme, PPCE) activity and the decrease in kallikrein activity, we found, anew, an increase in aminobutyrate aminotransferase (GABA-T) activity in the Alzheimer brain. This may be an important impetus for the reduction of gamma-aminobutyric acid (GABA) in the brain, one of the neurotransmitters. It has to be determined whether the former two abnormalities offer a background for such an abnormality of the neurotransmitter.     

Rapid analysis for cadmium metallothionein complexes by HPLC using microparticulate stationary phases

Different columns with microparticulate (1.5 and 2 μm) stationary phases were investigated for the analysis of the polymorphism of mammalian metallothionein (MT) by reversed-phase HPLC. When a non-porous 1.5 μm stationary phase was used, the duration of the chromatographic run was reduced 10-fold (in comparison with the conventional 5 μm packing) without any loss in resolution. The method was applied to the analysis of MT-1 and MT-2 preparations from rabbit liver.     

From brain to food: analysis of phosphatidylcholins, lyso-phosphatidylcholins and phosphatidylcholin-plasmalogens derivates in Alzheimer's disease human post mortem brains and mice model via mass spectrometry

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by extracellular senile plaques mainly consisting of Aβ, a 40-42 amino acid long peptide, and intracellular neurofibrillary tangles, accompanied by an excessive loss of synapses. Recently evidence accumulated that nutrition, especially polyunsaturated fatty acids, influences AD pathogenesis. Especially mid-life food habits with the consumption of specific fatty acids (FA) appear to influence the disease risk. The timely separation between food intake and disease makes a direct correlation with detailed analysis of eating habits combined with accurate food analysis nearly unattainable. A possible solution to circumvent these difficulties is to investigate the FA composition in human post mortem brain. In this study we focused on the main phospholipids phosphatidylcholin (PC), phosphatidylcholin-plasmalogen (PC-PL) and lyso-phosphatidylcholin (lyso-PC) in AD brains compared to control brains. Frontal cortices, temporal cortices and cerebellum of 30 AD (mean 78 years) and 14 control aged matched brains (mean 77.4 years) as well as APP transgenic mice compared to control mice were analyzed using an AB Sciex 4000 Qtrap mass spectrometer utilizing a FIA MS/MS method. PC, PC-PL and lyso-PC metabolites were analyzed in respect to saturation level and FA composition. As expected, the majority of the lipid species showed no significant differences, but interestingly a few species revealed a highly significant reduction in AD brains. These FAs are potential candidates for further food analysis in respect to AD pathology. Additionally, we show that the method applied with multiple reaction monitoring (MRM) used for this study is suitable for semi quantitative analysis of small amounts (10 μl) of brain tissue.     

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     

Determination of rare earth and other trace element abundances in human kidney stones and brain tissue by instrumental neutron activation analysis

We have used INAA to analyze more than 30 minor and trace elements in 10 human kidney stones (phosphate and oxalate types). In addition we also analyzed human brain tissue samples for trace elements by INAA to determine the limitations of the method. Samples were taken from the temporal and frontal cortex of the brain of a patient that suffered from dialysis encephalopathy (where an increased Al content is expected), as well as a number of control samples. Trace elements were analyzed to study possible compositional differences other than the Al content. The analyses were done using large volume HPGe detectors; because of the low abundances, accuracy and precision vary between 3–80% for individual elements. We found a difference between the rare earth element (REE) patterns for apatite and oxalate kidney stones, and a fractionation compared to typical REE contents in plants. For the brain samples there is evidence for differences between the dialysis patient and the control samples not only for Al, but also for some other elements including the REEs, but most differences are minimal, and may not be significant.     

INNA for interelement correlations in rats after mercuric chloride exposure

In an animal model study, we exposed rats to mercuric chloride through drinking water continuously for eight to ten months. A group of these rats were then taken off mercuric chloride water and fed distilled water. A control group of rats was given distilled water. Rat brain, spinal cord, and kidney were analyzed to determine Hg and nine other elements by INAA. Significant imbalances were detected among the groups. Most of the mercury (Hg) was found to be eliminated from the tissues studied within the first thirty days. Implications of the data are discussed in light of observed trace element imbalances in amyotrophic lateral sclerosis and Alzheimer's disease.     

Alteration of biological samples in speciation analysis of metalloproteins

For investigations of metalloproteins by speciation analysis, the integrity of the protein–metal complexes before and during separation is crucial. Knowledge about potential alterations of the samples is thus essential to avoid misinterpretations of the analytical results. Chromatographic element profiles of different cytosolic samples from animal tissues were measured repeatedly to estimate the sample stability. The dependence of the signals on the dwell time of the sample in an autosampling device at 4 °C for a period of 10 h was observed. Alterations in the element content of different metal-containing fractions were quantified by means of recovery values. Some metalloprotein fractions (e.g. ≈27-kDa arsenic, ≈27-kDa iron and different zinc fractions) were stable or only minor alterations were observed and for their investigation an autosampling device is therefore suitable. However, most of the other metalloprotein fractions, especially nickel-containing proteins, showed major alterations: these samples should therefore be analysed immediately after preparation or directly after thawing. Figure Chromatographic manganese-profiles of 11 repeated SEC-ICP-MS-separations of rat brain cytosol. The first sample at time 0 h was the run immediately started after thawing of the prepared cytosol; the other samples were measured hourly, taken from the same sample vial. In addition to the time axis the estimated molecular mass axis is plotted Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.