小椋 康光

Copper (Cu) is one of the essential metals and its homeostasis is strictly regulated. Metallothionein (NIT) is induced by excess Cu to mask the Cu toxicity. Although the role of NIT in Cu toxicity has been explained in terms of Cu sequestration, its role under Cu-deficient conditions is not known. This study was carried out to determine the role of NIT in Cu depletion by a Cu(I)-specific chelator, bathocuproine sulfonate (BCS), in cultured cells established from MT-knockout mouse and its wild type. Viability was decreased more severely in MT-null cells than in wild-type cells by BCS treatment. The expression levels of both NIT isoforms were increased by BCS treatment in wild-type cells. Thus, NIT was shown to be induced under Cu-deficient conditions to maintain the activities of intracellular cuproenzymes such as cytochrome c oxidase and Cu,zinc-superoxide dismutase. (c) 2006 Elsevier Inc. All rights reserved.

Liquid chromatography (LC) hyphenated with both elemental and molecular mass spectrometry has been used for Se speciation in Se-enriched garlic. Different species were separated by ion-pair liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) after hot-water extraction. They were identified by on-line reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (RPLC-ESI-MS-MS). Se-methionine and Se-methylselenocysteine were determined by monitoring their product ions. Another compound, gamma-glutamyl-Se-methylselenocysteine, shown to be the most abundant form of Se in the garlic, was determined without any additional sample pre-treatment after extraction and without the need for a synthesized standard. Product ions for this dipeptide were detected by LC-ESI-MS-MS for three isotopes of Se:Se-78, Se-80: and Se-82. The method was extended to the species extracted during in-vitro gastrointestinal digestion. Because both Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine have anticarcinogenic properties, their extractability and stability during human digestion are very important. Garlic was also treated with saliva, to enable detection and analysis of species extracted during mastication. Detailed information on the extractability of selenium species by both simulated gastric and intestinal fluid are given, and variation of the distribution of Se among the different species with time is discussed. Although the main species in garlic is the dipeptide gamma-glutamyl-Se-methylselenocysteine, Se-methylselenocysteine is the main compound present in the extracts after treatment with gastrointestinal fluids. Two more, so far unknown compounds were observed in the chromatogram. The extracted species and their transformations were analysed by combining LC-ICP-MS and LC-ESI-MS-MS. In both the simulated gastric and intestinal digests, Se-methionine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine could be determined by LC-ESI-MS-MS by measuring their typical product ions.

Inductively coupled plasma-mass spectrometry (ICP-MS) equipped with a reaction/collision cell has been successfully used for dissociating polyatomic interferences. Hydrogen (H-2) is one of the most effective and frequently used reaction/collision gases. However, the H-2 reaction produces interference in the detection of selenium (Se) in bromine (Br)-containing samples, such as extracellular fluids. In this study, deuterium (D-2) was evaluated for possible use as a reaction gas instead of H-2. Although Se concentration in serum and urine was over-estimated in the H-2 reaction mode, it was determined accurately in the D-2 one. In speciation analyses, the background counts at m/z 77, 78 and 80 were reduced and the signal-to-noise (S/N) ratios were improved by either the H-2 or the D-2 reaction. The (BrH+)-Br-79-H-1 and (BrH+)-Br-81-H-1 interferences appearing at m/z 80 and 82, respectively, were decreased by changing from the H, reaction mode to the D-2 one. Thus, D-2 was effective in dissociating polyatomic interferences and removing Br interferences during Se determination and speciation, suggesting that the D-2 reaction mode is useful for selenometallomics, particularly in samples containing Br, such as serum, urine and cell culture medium. (c) 2005 Elsevier B.V. All rights reserved.

Once selenium (Se) is absorbed by the body, it is excreted mostly into the urine and the major metabolite is 1 beta-methylseleno-N-acetyl-D-galactosamine (selenosugar) within the required to low-toxic range. Selenosugar plateaus with a dose higher than 2.0 mu g Se/ml water or g diet, and trimethylselenonium (TMSe) starts to increase, indicating that TMSe can be a biomarker of excessive and toxic doses of Se. Here, we show dose-related changes in the two urinary Se metabolites to clarify the relationship between the dose and urinary metabolites by feeding selenite to rats. It was also examined whether the metabolites are related to age, and further whether a possible exogenous Source of the N-acetyl-D-galactosamine moiety, chondroitin 4-sulfate, affects the urinary metabolites. Selenite in drinking water was fed ad libitum to male Wistar rats of 36 and 5 weeks of age, and the concentrations of Se ill the urine and organs were determined together with speciation of the urinary Se metabolites. In young rats, selenosugar was always the major urinary metabolite and TMSe increased with a dose higher than 2.0 mu g Se/ml drinking water. On the other hand, in adult rats, TMSe increased only marginally despite that the rats suffered much more greatly from the Se toxicity suggesting that TMSe cannot be a biomarker of Se toxicity. The results suggest that sources of the sugar moiety of selenosugar are more abundant in adult rats than in young rats. Chondroitin 4-sulfate did not affect the ratio of the two urinary metabolites, suggesting that the sugar source is of endogenous origin and that it increases with age. (c) 2004 Elsevier Inc. All rights reserved.

The present study revealed the mechanism underlying the nuclear trafficking of metallothionein (MT). Nuclear localization of MT in digitonin-permeabilized BALB 3T3 cells was enhanced in the presence of a cytosolic factor added as a rat red blood cell lysate by oxidation with H2O2 in a dose-dependent manner, but inhibited with excess glutathione. A cytosolic partner was assumed to bind MT and retain it in the cytoplasm, and its oxidation can mobilize MT to the nuclei on cellular oxidation. Pre-treatment of nuclei with H2O2 did not enhance the localization, and MT that had been localized in the nuclei was washed out, indicating that MT is in the nuclei as a result of a higher rate of uptake by the nuclei than the rate of diffusion from the nuclei. Nuclear localization of lysozyme and nuclear localization signal (NLS)-bearing allophycocyanin were not enhanced by the oxidation in the presence of cytosolic factor, suggesting that the nuclear traffic occurring on oxidation is specific to MT. Moreover, when cells were arrested the cell cycle at the S phase, MT was localized in the nuclei in response to coincidental generation of a feeble reactive oxygen species (ROS). These observations suggest that MT comes localized in the nuclei on the sensing of intracellular oxidation, whereby a cytosolic partner specific to MT comes oxidized as a cargo system, MT being localized as a result of enhanced uptake in the nuclei and re-localized in the cytoplasm diffusely. Nuclear MT was proposed to protect the nuclei from the oxidation occurring with progression of the cell cycle. (C) 2004 Wiley-Liss, Inc.

Multi-mode gel filtration HPLC columns of capillary size coupled with ICP-MS were evaluated as to the separation of naturally occurring selenocompounds. The major selenocompounds found in selenized garlic, i.e., Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine, were separated on the narrower column (GS320A-M5D, 0.5 mm i.d. x 150 mm). The selenometabolites found in the urine of a selenium-toxicosis rat, i.e., selenosugar (1-beta-methylseleno-N-acetyl-D-galactosamine) and trimethylselenonium, were also separated on the same column. On the other hand, the selenoamino acids, i.e., Se-methylselenocysteine and selenomethionine, were separated on the larger column (GS320A-M8E, 0.8 mm i.d. x 250 mm). These samples were introduced directly into the capillary columns without any pretreatment. The capillary HPLC separated these selenocompounds into distinct peaks with satisfactory sensitivity and a better S/N ratio than conventional HPLC despite that the sample volume was reduced to 1/200 ( 100 nl).

Cox17p is cloned from yeast as a chaperone to deliver copper to the mitochondria of assembly for cytochrome c oxidase (CCO). In mammals, CCO is a key enzyme for cellular respiration and a defect in its function is associated with severe neonatal or infantile lactic acidosis and early death. Recently, we found that Cox17p is not only required for mitochondrial oxidative phosphorylation but also is essential for embryonic growth and development in COX1 7 gene-deficient mice. To investigate its biochemical features, recombinant human Cox17p was overexpressed and purified without a purification tag. It specifically binds Cu(1) at a molar copper content of 3.3 +/- 0.04 under reduced conditions and significantly activates the mitochondrial CCO in vitro. Although the CuCox17p complex was maintained between pH values from 5.0 to 7.7, Cu was completely released from Cox17p at pH 8.0. An acute exposure of excess amount of copper ion to mouse cells resulted in a significant reduction of Cox17p mRNA expression, whereas copper starvation maintained the Cox17p transcription level. These results suggest that the stringent selectivity of Cox17p for copper is required for CCO activation to prevent copper overload, or promote the Supply of copper. (C) 2004 Elsevier Inc. All rights reserved.

The observed toxicity of arsenic is highly dependent on animal species and differences in metabolism. Rats are one of the most tolerant species, and the metabolic pathway is quite different in some aspects from those of other mammals. The distinct metabolic pathway including the preferential accumulation in red blood cells (RBCs) has been explained, whereby allowing an effective use of rats as an animal model for the arsenic metabolism. In the present study, distributions of arsenic among organs/tissues/body fluids and their chemical forms were studied after intravenous injection of arsenic in the forms of dimethylarsinic (DMA(V)) and monomethyl arsonic acids (MMA(V)) to rats. DMA(V) and MMA(V) were mostly excreted into urine immediately after the injection as the intact forms, and both forms were taken up less effectively by organs/tissues than arsenite. The methylated arsenics distributed in organs/tissues were excreted directly into urine and excreted before being redistributed in RBCs. DMA(V) and MMA(V) taken up by the liver were transformed to metabolites not yet identified, accumulated transiently in the liver, and then they disappeared from the liver. The unidentified metabolites were assumed to be transformed from dimethylarsinic acid (DMA(III)) following the consecutive metabolic reactions [MMA(V) --> monomethylarsonous acid (MMA(III)) --> DMA(V) --> DMA(III)]. The unidentified metabolites were excreted not into the bile but into the bloodstream. Injections of DMA(V) and MMA(V) induced a biliary excretion of arsenic but only at 0.2-0.3% of the dose, the arsenic in the bile being their intact free fortris. (C) 2004 Elsevier Inc. All rights reserved.

Speciation of arsenicals in biological samples is an essential tool to gain insight into its distribution in tissues and its species-specific toxicity to target organs. Biological samples (urine, hair, fingernail) examined in the present study were collected from 41 people of West Bengal, India, who were drinking arsenic (As)-contaminated water, whereas 25 blood and urine samples were collected from a population who stopped drinking As contaminated water 2 years before the blood collection. Speciation of arsenicals in urine, water-methanol extract of freeze-dried red blood cells (RBCs), trichloroacetic acid treated plasma, and water extract of hair and fingernail was carried out by high-performance liquid chromatography (HPLC)-inductively coupled argon plasma mass spectrometry (ICP MS). Urine contained arsenobetaine (AsB, 1.0%), arsenite (iAs(III), 11.3), arsenate (iAs(V) 10.1), monomethylarsonous acid (MMA(III), 6.6), monomethylarsonic acid (MMA(V), 10.5), dimethylarsinous acid (DMA(III), 13.0), and dimethylarsinic acid (DMA(V), 47.5); fingernail contained iAs(III) (62.4%), iAs(V) (20.2), MMA(V) (5.7), DMA(III) (8.9), and DMA(V) (2.8); hair contained iAs(III) (58.9%), iAs(V) (34.8), MMA(V) (2.9), and DMA(V) (3.4); RBCs contained AsB (22.5%) and DMA(V) (77.5); and blood plasma contained AsB (16.7%), iAs(III) (21.1), MMA(V) (27.1), and DMA(V) (35.1). MMA(III), DMA(III), and iAs(V) were not found in any plasma and RBCs samples, but urine contained all of them. Arsenic in urine, fingernails, and hair are positively correlated with water As, suggesting that any of these measurements could be considered as a biomarker to As exposure. Status of urine and exogenous contamination of hair urgently need speciation of As in these samples, but speciation of As in nail is related to its total As (tAs) concentration. Therefore, total As concentrations of nails could be considered as biomarker to As exposure in the endemic areas. (C) 2004 Elsevier Inc. All rights reserved.

Two unidentified arsenic metabolites were detected in the liver of rats on a gel filtration column by HPLC inductively coupled argon plasma mass spectrometry after an injection of dimethylarsinic (DMA(V)), dimethylarsinous (DMA(III)), monomethylarsonic (MMA(V)), or monomethylarsonous (MMA(III)) acid. The same arsenicals were also produced in vitro by incubation of DMA(III) in the liver supernatant but not by DMA(V). The two arsenic metabolites eluted at the same retention times as those of the two arsenicals prepared by reaction of DMA(V) with either thiosulfate plus disulfite or hydrogen sulfide or sodium sulfide plus sulfuric acid. The faster and slower eluting products on a gel filtration column were assigned as dimethyldithioarsinic acid (dimethylarsinodithioic acid) (DMTA(V)) and dimethylthioarsinous acid (DMTA(III)) from mass spectrometric data at m/z = 170 and 138 by electrospray ionization mass spectrometry with negative and positive ion modes, respectively. They were prepared selectively by reacting DMA(V) with hydrogen sulfide or sodium sulfide plus sulfuric acid under different reaction conditions. DMA(III) but not DMA(V) was transformed to DMTA(III) and DMTA(V) in the presence of sodium sulfide in vitro, suggesting that DMA(V) is reduced to DMA(III) with hydrogen sulfide, thiolated to DMTA(III), and then further thiolated oxidatively to DMTA(V). Metabolically, it is assumed that DMA(III) is transformed to DMTA(III) in the presence of sulfide ions, and then, DMTA(III) is oxidatively thiolated to DMTA(V). As the chemical species produced by reduction with the Reay and Asher method are DMTA(III) and DMTA(V), and different from DMA(III), the studies carried out with DMA(III) with the Reay and Asher method have to be reexamined.

A possible appearance of reactive oxygen species (ROS) with the normal cell cycle was studied to find how ROS are generated in cells in relation to the cell cycle. The production of ROS in relation to the cell cycle was examined by determining the changes in intracellular ROS concentrations at different phases of the cell cycle by culturing BALB 3T3 cells in the presence and absence of aphidicolin. The amoupts of intracellular ROS and the cell population at specific phases (S and G2/M) were determined as the fluorescence of dichlorodihydrofluorescein and propidium iodide taken up simultaneously by the cells, respectively, by flow cytometry. Although intracellular ROS remained at the control levels when the cell growth was arrested with aphidicolin at the G I phase, they increased when the arrest was released to result in the increase of the cell population at the S phase. Furthermore, ROS was shown to disturb/ stop the cell cycle by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell cycle was regulated through oxidative stress by exposure to hydrogen peroxide and glutathione ethyl ester. The cell cycle was prevented more sensitively in metallothionein-null cells than in the wild type cells. Based on the present observations, we proposed for the first time that ROS are generated synchronously with the normal cell cycle, and that they have to be controlled at certain level for normal progress of the cell cycle. (C) 2004 Published by Elsevier Inc.

Nail and hair are rich in fibrous proteins, i.e., alpha-keratins that contain abundant cysteine residues (up to 22% in nail and 10-14% in hair). Although they are metabolically dead materials in the epidermis, the roots are highly influenced by the health status of the living beings and their analyses are used as a tool to monitor occupational and environmental exposure to toxic elements. The aims of the present study are to speciate arsenicals in human nail and hair and also to judge whether they should be used as a biomarker to arsenic (As) exposure and/or toxicity. All human fingernail and hair samples (n = 47) were collected from the As-affected area of West Bengal, India. Speciation of arsenicals in water extracts of fingernails and hair at 90degreesC was carried out by HPLC-inductively coupled argon plasma mass spectrometer (ICP MS). Fingernails contained iAs(III) (58.6%), iAs(V) (21.5), MMA(V) (7.7), DMA(III) (9.2), and DMA(V) (3.0), and hair contained iAs(III) (60.9%), iAs(V) (33.2), MMA(V) (2.2), and DMA(V) (3.6). Fingernails contained DMA(III) but hair did not. The higher percentage of iAs(III) both in fingernails and hair than that of iAs(V) suggests more affinity of iAs(III) to keratin. Although all arsenicals in fingernails and hair correlate to As exposure positively, As speciation in fingernails seems to be more correlated with arsenism than that in hair. Exogenous contamination is a confounding factor for hair to consider it as a biomarker, whereas this is mostly absent in fingernails, which recommends it to be a better biomarker to arsenic exposure. DMA(III). content in fingernails and DMA(V) contents in both fingernails and hair could be the biomarker to As exposure. (C) 2003 Elsevier Science (USA). All rights reserved.

In a series of experiments, the effects of soy protein isolate (SPI), defatted soy (DFS) or SPI supplemented with L-methionine (SPIM) were examined in the Long-Evans rat with a cinnamon coat color (LEC rat), a model animal of Wilson's disease with a hereditary defect in the Atp7b gene resulting in defective copper metabolism and copper accumulation in hepatocytes. Milk casein in the control AIN-93G diet (20 g/100 g) was totally or 60% replaced by the soy products, SPI, DFS or SPIM (L-Met added to be equal to that in the control diet) beginning when rats were 6 wk old. Copper and iron concentrations in SPI and DFS were measured and the concentrations of these metals in the salt mix were adjusted so that test and the control diets had the same final concentrations. Food intake did not differ among groups. Rats were euthanized when they became moribund with jaundice. Survival time in the SPI diet group was shorter (14.0 +/- 0.8 wk) than in the control group (19.1 +/- 1.7 wk) (P < 0.001), and that in the DFS diet group was intermediate (16.0 +/- 1.7 wk). Survival time in the SPIM diet group did not differ from that of the SPI diet group. Copper concentrations in the livers of rats in the SPI and SPIM diet groups were similar to80% higher than in rats fed the control diet. Liver iron concentrations did not differ among the groups. The results, including histological analyses, indicate that SPI enhances copper uptake into the liver cells and promotes liver cell damage in LEC rats. However, this did not occur in the livers of F344 rats with wild-type Atp7b. Recommendations to individuals suffering from Wilson's disease to avoid consuming soy protein may be warranted.

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age A six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats. (C) 2003 Elsevier Science (USA). All rights reserved.

Metabolic pathway for arsenic was studied in Eisai hyperbilirubinemic (EHB) rats to estimate the participation of hepato-enteric circulation during the transformation and distribution. Arsenite was injected intravenously into male EHB rats of 8 weeks of age at a single dose of 0.5 mg As/kg body weight, and time-dependent changes in the concentration of arsenic in organs and body fluids were determined. Arsenic disappeared from the bloodstream within 10 min after the injection, and appeared in the liver (40% of the dose), kidneys, lungs, spleen, testes, skin (in total 15%) and urine (10%). The rest (35%) was not detected in any of these organs/tissues/body fluids. However, arsenic accumulating in the erythrocytes amounted to 90% of the dose after 6 h, indicating that the hepato-enteric circulation can explain only a half of the missing 70% of the dose in normal rats in our previous study. Although the arsenite once distributed in the liver was redistributed in the erythrocytes by, 1 h, it took 6 h for the missing 35% to reappear in the erythrocytes, suggesting that the missing arsenic be transformed more slowly in the hidden organ. Although the concentration was low, the whole arsenic in the muscle was calculated to amount approximately to the missing one (31%). Arsenite injected intravenously into normal rats was estimated to distribute at first in the liver (40%), muscle (35%), other organs (15%) and urine (10%) in its original form in an organ-specific manner, and then redistributed in the erythrocytes after being transformed to dimethylated arsenic with different reduction/methylation efficiency, the liver being most active in the transformation and in the excretion into the hepato-enteric circulation.

In addition to the major urinary selenometabolite, 1beta-methylseleno-N-acetyl-D-galactosamine (selenosugar), several minor selenometabolites were also detected in rat urine by high performance liquid chromatography inductively coupled argon plasma mass spectrometry. One of them, in a partially purified urine sample, was generated from selenosugar under aerobic conditions and also transformed through oxidation by H2O2. It was identified as monomethylseleninic acid. Therefore, this urinary selenocompound should not be regarded as a naturally occurring metabolite but as a selenosugar artifact. Moreover, an additional selenometabolite was detected in the same urine sample. Although this selenometabolite has not been identified yet, it seems to contain monomethylselenyl group(s) because of its transformation to monomethylseleninic acid through oxidation. Thus, urine samples must be handled with care so as not to allow oxidation by air, to prevent transformation to artificial selenocompounds.

Essential micronutrient selenium is excreted into the urine and/or expired after being transformed to methylated metabolites. Monomethylated selenium is excreted into the urine in response to a supply within the required to low-toxic range, whereas tri- and dimethylated selenium increase with excessive supply at a toxic dose. Here we show that the major urinary selenium metabolite within the required to low-toxic range is a selenosugar. The structure of 1beta-methylseleno-N-acetyl-D-galactosamine was deduced from the spectroscopic data and confirmed by chemical synthesis. This metabolite was also detected in the liver, and an additional metabolite increased with inhibition of methylation. The latter metabolite was again a selenosugar conjugated with glutathione instead of a methyl group and was assumed to be a precursor for methylation to the former metabolite. A metabolic pathway for the urinary excretion of selenium, i.e., from the glutathione-S-conjugated selenosugar to the methylated one, was proposed. Urinary monomethylated (selenosugar) and trimethylated selenium can be used as specific indices that increase within the required to low-toxic range and with a distinct toxic dose, respectively.

In an animal model of Wilson disease, Long-Evans rats with cinnamon-colored coat (LEC rats), copper (Cu) accumulates in the liver with age up to the onset of acute hepatitis owing to a hereditary defective transporter for the efflux of Cu, ATP7B. The plasma Cu concentration is low in LEC rats because of the excretion of apo-ceruloplasmin (apo-Cp). However, toward and after the onset of chronic hepatitis, plasma Cu concentration increases in the form of holo-Cp, while the liver Cu concentration is maintained at a constant level without the occurrence of fulminant hepatitis. In the present study, the material balance of Cu was studied in LEC rats with chronic hepatitis in order to elucidate the mechanisms underlying the increase of holo-Cp in plasma and the maintenance of Cu at a constant level in the liver. The relationship between the Cu concentration and ferroxidase activity of Cp was analyzed in the plasma of LEC rats of different ages and of Wistar rats fed a Cu-deficient diet for different durations. Cu was suggested to be delivered to Cp in an all-or-nothing manner, resulting in the excretion of fully Cu-occupied holo-Cp (Cu-6-Cp) or totally Cu-unoccupied Cu-0-Cp (apo-Cp), but not partially Cu-occupied Cu-n-Cp (where n = 1-5). The increase of holo-Cp in acute and chronic hepatitis in LEC rats was explained by the delivery of Cu, accumulating in the non-metallothionein-bound form, to Cp outside the Golgi apparatus of the liver. The plasma Cu concentration and ferroxidase activity were proposed to be specific indicators of the appearance of non-metallothionein-bound Cu in the liver of LEC rats.

Inorganic arsenic is metabolized by consecutive reduction and methylation reactions to dimethylated arsenic (DMA), and then excreted into the urine mostly in the form of DMA. Therefore, arsenic metabolites in the body fluids and organs/tissues are present in the form of inorganic (arsenite and arsenate) and methylated arsenics (MMA and DMA). Although pentavalent arsenics can be present mostly in the form of free ions, trivalent ones may be present more in the forms conjugated with thiol groups of glutathione (GSH) or proteins. Arsenic in the body fluids (plasma, bile and urine) is present in the soluble forms and can be speciated on ion exchange columns by HPLC with on-line detection by an inductively coupled argon plasma-mass spectrometer (ICP-MS), Free forms of arsenite, arsenate, and monomethylarsonous, mono methylarsonic, dimethylarsinous and dimethylarsinic acids in the body fluids have been demonstrated to be speciated simultaneously within 10 min or so on both anion and cation exchange columns together with arsenobetaine (AsB) and arsenocholine (AsC). Trivalent arsenics conjugated with GSH were eluted in intact forms on an anion exchange column but were liberated into free forms on a cation exchange column. Thus, free and GSH-conjugated arsenic metabolites in the bile and urine have been speciated simultaneously on ion exchange columns by HPLC-ICP-MS. (C) 2002 Elsevier Science B.V. All rights reserved.

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and -electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-β-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine. © 2002 Elsevier Science B.V. All rights reserved.

Metallothionein (MT) protects the body from both harmful non-essential and excessive essential metals. Copper (Cu) is an essential metal, and its concentration in the body is regulated at a constant level between excess and deficient ones. Cu accumulating in the livers of Wilson disease patients and its animal model, Long-Evans rats with a cinnamon-like coat color (LEC) rats, is in the form of Cu,Zn-MT, MT being an antioxidant. Contrary to the efficient production of MT in response to excessive accumulation of Cu in LEC rats, Cu-binding to MT only occurs marginally under normal conditions. However, the present study revealed that Cu binds to MT more with a severe Cu-deficiency. Namely, male C57BL/6J mice were fed a Cu-deficient diet (0.037 mg Cu/g) and deionized water containing trientine, and then the concentration and distribution of Cu were determined. It was suggested that the cessation of biliary excretion and limitation of the Cu supply to ceruloplasmin are the first responses on feeding of a Cu-deficient diet, followed by an increase in Cu-MT with maintenance of the Cu concentration in the liver. These results suggest that MT causes the recruitment of Cu in a Cu-deficient environment by sequestering Cu from degraded Cu-enzymes and delivering it to Cu chaperones. (C)2002 Elsevier Science B.V. All rights reserved.

The metabolic pathways for arsenic were precisely studied by determining the metabolic balance and chemical species of arsenic to gain an insight into the mechanisms underlying the animal species difference in the metabolism and preferential accumulation of arsenic in red blood cells (RBCs) in rats. Male Wistar rats were injected intravenously with a single dose of arsenite (iAs(III)) at 2.0 mg of As/kg of body weight, and then the time-dependent changes in the concentrations of arsenic in organs and body fluids were determined. Furthermore, arsenic in the bile was analyzed on anion and cation exchange columns by high-performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS). The metabolic balance and speciation studies revealed that arsenic is potentially transferred to the hepato-enteric circulation through excretion from the liver in a form conjugated with glutathione (GSH). iAs(III) is methylated to mono (MMA)- and dimethylated (DMA) arsenics in the liver during circulation in the conjugated form [iAs(III)(GS)(3)], and a part of MMA is excreted into the bile in the forms of MMA(III) and MMA(V), the former being mostly in the conjugated form [CH3AsIII(GS)(2)], and the latter being in the nonconjugated free form. DMA(III) and DMA(V) were not detected in the bile. In the urine, arsenic was detected in the forms of iAs(III), arsenate, MMA(V), and DMA(V), iAs(III) being the major arsenic in the first 6-h-urine, and DMA(V) being increased in the second 6-h-urine. The present metabolic balance and speciation study suggests that iAs(III) is methylated in the liver during its hepato-enteric circulation through the formation of the GSH-cojugated form [iAs(III)(GS)(3)], and MMA(III) and MMA(V) are partly excreted into the bile, the former being in the conjugated form [CH3AsIII(GS)(2)]. DMA is not excreted into the bile but into the bloodstream, accumulating in RBCs, and then excreted into the urine mostly in the form of DMA(V) in rats.

Transcription of mammalian metallothionein (MT) genes is activated by heavy metals via multiple copies of a cis-acting DNA element, the metal-responsive element (MRE), Our previous studies have shown that certain MREs of the human MT-IIA gene (MREb, MREc, MREd, and MREf) are less active than the others (MREa, MREe, and MREg). Gel shift analysis of HeLa cell nuclear proteins revealed that whereas the active MREs strongly bind the transcription factor MTF-1 essential for metal regulation, the less active MREs bind another distinct protein, MREb-BF. This protein recognizes the GC-rich region of MREb rather than the MRE core required for MTF-1 binding. All the MREs recognized by MREb-BF contain the CGCCC and/or CACCC motif, suggesting that the MREb-BF-MRE complex contains Sp1 or related proteins. Supershift analysis using antibodies against Sp1 family proteins as well as gel shift analysis using the recombinant Sp1 demonstrated that Sp1 represents the majority of MREb-BF activity. An MREb mutant with reduced affinity to Sp1 mediated zinc-inducible transcription much more actively than the wild-type MREb, Furthermore, when placed in the native promoter, this mutant MREb raised the overall promoter activity. These results strongly suggest that Sp1 acts as a negative regulator of transcription mediated by specific MREs.

Metabolic, nutritional and toxicological aspects of selenium (Se) were studied using a hyphenated technique. Se in biological samples was separated by HPLC and Se in the eluate was detected in-line by mass spectrometry with ionization by inductively coupled argon plasma (HPLC-ICP MS). The distributions of Se in the soluble fractions of various organs and body fluids were determined after ingestion or injection of naturally occurring Se or Se enriched with a stable isotope in the form of selenite, selenate or selenomethionine. Metabolic pathways specific to each Se species were discussed based on the results of speciation of each Se metabolite. Selenite in the bloodstream was taken up by red blood cells and reduced to selenide, and then the reduced form of Se (selenide) was transported to the plasma, where it was bound selectively to albumin and was then transported to the liver. On the other hand, intravenous selenate was taken up directly by the liver. Excess Se derived from any nutritional Se species is mainly excreted in the urine after being methylated in the liver. The mechanisms underlying the interaction between Se and mercuric ions in the bloodstream were explained by the formation of a ternary complex, {(HgSe)(n)}(m)-selenoprotein P (n is the number of (HgSe) complexes and in is the number of the binding sites for the (HgSe)(n) complex on selenoprotein P). The complex between Hg and Se in the bloodstream was thus explained by the interaction between their specific chemical species in each metabolic pathway. The sensitivity of the HPLC-ICP MS method was enhanced with the use of enriched stable isotopes by the simultaneous detection and speciation of both endogenous and exogenous Se.

Although metallothionein (MT) was first characterized as a cytoplasmic protein, it is now known to be localized in the nucleus depending on various cellular events, such as cell proliferation. The suggested roles of karyophilic MT are: to 1) regulate the biological pool of the essential metals zinc (Zn) and copper (Cu), and especially to supply Zn to Zn-requiring enzymes/transcription factors through activated cell proliferation, and 2) to protect DNA from oxidative stress including those caused by antitumor agents. Translocation of MT to the nucleus might be mediated, depending on cellular events, by a structural change in MT itself or through the appearance of nuclear binding proteins. Supporting the former possibility, MT is known to have some structural features, namely, highly conserved lysyl residues, which are anticipated to act as nuclear localization signal (NLS). In addition, concomitant appearance of non-acetylated MT, without post-translational acetylation, and nuclear localization of MT, have been reported. Supporting the latter possibility, MT-partner proteins might participate in the nuclear trafficking of MT (i.e., an MT-nuclear translocator or a nuclear chaperone of MT). We now provide an overview of the current knowledge on both mechanisms.

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was removed with ammonium tetrathiomolybdate (TTM), and the fate of the Cu complexed with TTM and mobilized from the liver was determined. TTM was injected intravenously as a single dose of 2, 10 or 50 mg TTM/kg body weight into LEC and Wistar (normal Cu metabolism) rats, and then the concentrations of Cu and molybdenum (Mo) in the bile and plasma were monitored with time after the injection. In Wistar rats, most of the Mo was excreted into the urine, only a small quantity being excreted into the bile, while Cu excreted into the urine decreased. However, in LEC rats, Cu:and Mo were excreted into the bile and blood, and the bile is recognized for the first time as the major route of excretion. The Cu excreted into both the bile and plasma was accompanied by an equimolar amount of Mo. The relative ratio of the amounts of Cu excreted into the bile and plasma was 40/60 for the low and high dose groups, and 70/30 for the medium dose group. The systemic dispositions of the Cu mobilized from the liver and the Mo complexed with the Cu were also determined for the kidneys, spleen and brain together with their urinal excretion. Although Mo in the three organs and Cu in the kidneys and spleen were increased or showed a tendency to increase, Cu in the brain was not increased at all doses of TTM. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Mammalian metallothionein genes are transcriptionally regulated by heavy metals through cis-acting metal responsive elements (MREs). The MRE-binding transcription factor-1 (MTF-1), a protein containing six C2H2-type Zn fingers, is essential for MRE-mediated transcriptional activation. DNA binding of MTF-1 is known to be stimulated by Zn in vitro, but the binding was also largely influenced by redox conditions, suggesting that redox signals could modulate MTF-1 activity. To locate the functional domain required for Zn regulation, several deletion mutants of human MTF-1b, a newly cloned transcriptionally active MTF-1 variant, were characterized. This analysis showed that the N-terminal region and Zn-finger domain play roles in metal response. Functional roles of individual Zn fingers were estimated by co-transfection assays by using an MRE-driven reporter gene and vectors that express MTF-1b mutants each carrying one defective finger. Mutations in the N-terminal four fingers dramatically reduced the transcriptional activity, and at least for three of them the transcriptional defect was due to reduced DNA binding. These results indicate that the six Zn fingers are not functionally equivalent, probably sharing distinct roles such as direct DNA recognition and regulatory functions. (C) 2000 Wiley-Liss, Inc.

Heavy metals are expected to affect the biological activity of various metal-containing proteins, including transcriptional regulators. We studied the effects of several heavy metal ions on the DNA-binding activity of a Zn- finger transcription factor, Sp1. With respect to both DNA elements through which Sp1 acts positively and negatively, Cd2+ inhibited DNA-binding of Sp1 at 20 μM or higher, while the toxic effect of Zn2+ was obvious only at more than 200 μM. Inhibition was also apparent for Cu2+ but less remarkable for Hg2+. The inhibition by Cd2+ was relieved by the addition of Zn2+ at much lower concentrations than that of C2+. These results suggest that the toxic potential of heavy metals could be largely influenced by the intracellular Zn2+ concentration.

A zinc (Zn)-binding protein that is present specifically in the livers of male adult rats was detected by HPLC with in-line detection by mass spectrometry (ICP MS). The Zn-binding protein was purified on Sephadex G-75 and G3000SW HPLC columns, and was identified as carbonic anhydrase III (CAIII) based on the amino acid sequence of a peptide obtained on lysyl endopeptidase digestion. CAIII is expressed as one of the major Zn-binding proteins in the livers of male rats in an age-dependent manner, a comparable amount of Zn to that of copper, Zn-superoxide dismutase (Cu,Zn-SOD) being bound to CAIII at 8 weeks of age. Castration at 4 or 8 weeks of age was shown to reduce Zn bound to CAIII to 47.5% of the sham-operated control level, suggesting that the sex-dependent expression of CAIII is partly regulated by a sex hormone, androgen. The concentration of CAIII in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was also estimated as Zn bound to CAIII and shown to be lower than that in Wistar rats before the onset of hepatitis. The concentration of CAIII was decreased specifically by repeated injections of cupric ions without the Cu,Zn-SOD concentration being affected. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Tetrathiomolybdate (TTM) can be used as a specific chelator to remove copper (Cu) accumulating in the form bound to metallothionein (MT) in the livers of Wilson disease patients and Long-Evans rats with a cinnamon-like coat color (LEC rats). However, an adverse effect, hepatotoxicity, was observed occasionally on its clinical application. The mechanism underlying the adverse effect of TTM has been studied in comparison with dithiomolybdate (DTM), and a safer and more effective therapy by TTM was proposed based on the mechanism. The activity of glutamic-pyruvic transaminase (GPT) in serum was shown to increase significantly on the treatment of Wistar rats with sulfide produced through hydrolytic degradation of TTM and DTM, the latter being more easily degraded. The hydrolytic degradation of TTM was enhanced under acidic conditions. Cu in Cu-containing enzymes such as Cu,Zn-superoxide dismutase (SOD) in liver and ceruloplasmin (Cp) in plasma was decreased by excessive thiomolybdates, the Cu being found in the plasma in the form of a Cu/thiomolybdate/albumin complex. The decreased amounts of Cu in SOD and Cp were explained by the sequestration of Cu from their chaperones by thiomolybdates rather than the direct removal of Cu from the enzymes. Although both TTM and DTM remove Cu from MT, DTM is not appropriate as a therapeutic agent for Wilson disease due to its easy hydrolysis and production of sulfide. (C) 1999 Elsevier Science Inc. All rights reserved.

Ceruloplasmin is excreted mostly in the ape-form in Wilson's disease patients and Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model for Wilson's disease, and hence the concentration of Cu in the plasma is low. However, it increases toward and at the onset of acute hepatitis in LEC rats, the increased Cu in the plasma being bound to ceruloplasmin, metallothionein and albumin. Changes in the concentration of Cu in red blood cells (RBCs) were monitored with age for the first time together with that in the plasma in LEC rats. Cu in the RBCs was found to increase to a 5-7 times higher level than that in the plasma toward the onset and peaked at the onset, the pattern being similar to that in the plasma. The source of the Cu increase in the RBCs was discussed, and it was assumed that the so-called free Cu ions that leak from the damaged hepatocytes are bound to albumin and/or taken up by the RBCs.

Multiple copies of a cis-acting DNA element, metal-responsive element (MRE) are required for heavy metal-induced transcriptional activation of mammalian metallothionein genes. To approach the regulatory mechanism mediated by these multiple elements, we studied the properties of seven MREs located upstream of the human metallothionein-IIA (hMT-IIA) gene in detail. Transfection assays of reporter gene constructs each containing one of these MREs as the promoter element revealed that only four MREs can mediate zinc response. With respect to the distribution of active MREs over the promoter region, the hMT-IIA gene is largely different from the mouse metallothionein-1 gene, suggesting that MRE arrangement is not an important factor for metal regulation. Experiments using various model promoters showed that multiple MRE copies act highly synergistically, supporting the biological significance of the multiplicity. Only the four active MREs efficiently bound the purified transcription factor human MTF-1, and MRE mutants defective in binding this protein lost the ability to support zinc-induced reporter gene expression, strongly suggesting that the direct interaction between human MTF-1 and a set of the selected MREs plays the major role in heavy metal regulation. In protein/DNA binding reactions in vitro, the purified human MTF-1 was activated by zinc but not by other metallothionein-inducing heavy metals, supporting the idea that zinc is the direct modulator of human MTF-1.

The metabolic turnover of selenoprotein P (Sel P) and extracellular glutathione peroxidase (eGPx) in plasma was examined by HPLC-ICPMS, with the use of enriched Se, [Se-82]selenite. [Se-82]selenite was injected intravenously into rats at a dose of 25 mu g Se-82 kg(-1) body weight, and the concentrations of labeled Se-82 and naturally occurring Se-77 in the serum, liver and kidneys were determined in samples obtained 1, 3, 6, 9, 12, 18 and 24 h after the injection. The distributions of both exogenous (labeled) Se-82 and endogenous (naturally occurring) Se-77 in serum, and supernatant fractions of the liver and kidneys were determined on a gel filtration column by HPLC-ICPMS. This dose was shown not to affect the constitutive levels of cellular GPx (cGPx), eGPx and Sel P. The labeled Se in Sel P increased soon after the injection, peaked at 6-9 h and then decreased, whereas that in eGPx continued increasing after 6 h post-injection and then throughout the remaining observation period in the bloodstream. These observations demonstrated the rapid and efficient incorporation of Se into Sel P in the liver and excretion into the plasma followed by the slow and steady incorporation of Se into eGPx in the kidneys and excretion into the plasma, with a minimal response of cGPx to selenite injection.

The metabolic pathway for and metabolites of selenium (Se) administered intravenously to rats in the form of selenate at a dose of 0.3 mg Se kg-1 body weight were studied by speciating Se in the bloodstream, liver and urine by KPLC-inductively coupled argon plasma mass spectrometry. Selenate was not taken up by red blood cells (RBCs) and disappeared from the bloodstream much faster than selenite, without any change in its chemical form before it disappeared from the plasma. Selenium excreted into the urine after the administration of selenate showed different patterns from those of selenite in both amounts and chemical forms. With the selenate group, the concentration of Se in urine was highest at 0-6 h and the chemical species of Se was selenate at 0-6 h thereafter a monomethylselenol-related Se compound (MMSe*) and trimethylselenonium ions (TMSe) appeared, selenate not being excreted after 6 h. On the other hand. in the selenite group, the concentration of Se peaked at 6-12 h, and the chemical species of Se were MMSe* and TMSe. Selenate was reduced in vitro on incubation in either a liver homogenate or supernatant fraction, although much more slowly than in the whole body. Selenate was not reduced by glutathione or dithiothreitol. The results suggest that in contrast to selenite, which is taken up by and reduced in RBCs, and then transferred to the liver, approximately 20% of the selenate administered to rats was excreted into the urine without any change in its chemical form with the present dose, and the major portion of selenate was taken up by the liver, reduced and then utilized for the synthesis of selenoproteins or excreted into the urine after being methylated.

The uptake of tetrathiomolybdate (TTM) by the liver and the removal of copper (Cu) accumulating in the liver in a form bound to metallothionein (MT) by TTM were studied in Long-Evans cinnamon (LEC) rats, an animal model of Wilson disease, in order to develop better treatments for the disease and Cu toxicity. Although molybdenum (Mo) was incorporated in a dose-dependent manner into the livers of both LEC and Long-Evans agouti (LEA) rats, the original strain of LEC rats used as a reference animal, the uptake into the liver of LEC rats was 13 times higher than that in LEA rats. The concentration of Mo in the soluble fraction plateaued and it was distributed more in the insoluble fraction with a higher dose in LEC rats. The concentration of Cu in the whole livers of LEC rats was decreased by TTM in a dose-dependent manner only at lower doses. However, the concentration of Cu in the soluble fraction continued to decrease with the dose of TTM. The results can be explained in terms of complex formation. Namely, TTM forms a complex with Cu, tentatively referred to a Cu/TTM complex, that can be effluxed into the bloodstream, and then binds selectively to albumin when the dose of TTM is low. On the other hand, TTM forms an insoluble complex, named as a Cu/TTM polymer that is precipitated in the liver when the dose is high. The results further indicate that TTM taken up by a cell is immobilized in the cell through the dose-dependent formation of a complex containing Cu, Mo and sulfur (S), which causes further uptake of TTM. TTM injected into rats or incubated in vitro with serum does not remove Cu from ceruloplasmin. TTM is, thus, suggested to target a cell accumulating excess Cu as Cu-MT, and to remove Cu selectively without interacting with Cu in Cu-enzymes. The results indicate that TTM is taken up by the liver depending on the amount of Cu accumulating in the form of MT, and then Cu is effluxed together with Mo in the form of Cu/TTM complex into the bloodstream. (C) 1998 Elsevier Science Inc. All rights reserved.

In addition to the usual two rat metallothionein (MT) isoforms (MT-I and MT-II), a third isoform of metallothionein (MT-II') is known to be induced by zinc in the liver of rats and mice, and by epidermal growth factors in cultured cells. Although the third isoform has been suggested to be similar and related to MT-II based on its behavior on size-exclusion and ion exchange HPLC columns, further characterization has not been performed. MT-II' was identified in the present study as the unacetylated isoform of MT-II based on mass spectrometric data obtained by matrix assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOFMS) (i.e., MT-II' was 42 Da smaller than MT-II in its molecular mass). Chromatographic properties of MT-II' were consistent with this species being an unacetylated isoform of MT-II arising as a result on lack of acetylation rather than a post- translational deacetylation event (i.e., an isoform of MT-II not co- translationally acetylated, based on the change in its composition relative to MT-II and MT-I after induction of MT expression). Although MT-I' was not separated under the present conditions, the MT-I fraction gave a mass peak corresponding to a species 42 Da smaller than MT-I. This suggested that non- acetylated isoforms of both MT-I and MT-II were present.

Mutant Long-Evans rats with a cinnamon coat-color (LEG rats) have been established as an animal model for Wilson disease, a genetic disorder of copper (Cu) metabolism. Systemic disposition of molybdenum (Mo) and altered distributions of Cu were compared in eight organs between LEC rats and Wistar rats (normal) at different times after a single intraperitoneal injection of tetrathiomolybdate (TTM) for chelation therapy. Excretion through urine and feces was also examined. Hepatic disposition of Mo was dramatically increased in LEC rats, suggesting that the interaction of TTM with Cu results in enhanced uptake of Mo. Concentrations of Mo and Cu decreased in the liver of LEC rats over time, whereas those in the spleen increased. Although the concentration of Mo taken up by the kidney decreased over time after an initial increase in both rats, Cu concentration increased over time. Cu was not redistributed to the brain. Excretion of Mo through urine was decreased and that into feces was increased in LEC rats compared with those in Wistar rats. These results indicate that TTM is taken up by the liver depending on the Cu content, and the Cu and Mo removed from the liver are mostly excreted through feces. Redistribution of Cu was observed in the spleen and kidneys, but not in the brain.

Chelation therapy with tetrathiomolybdate (TTM) was applied to Long-Evans rats with a cinnamon coat-color (LEG rats), an animal model for Wilson disease, to remove copper (Cu) accumulated in the liver in a form bound to metallothionein (MT). Changes in molybdenum (Mo) and Cu concentrations and their biological forms in serum of LEC rats determined at different times after a single intraperitoneal injection were compared with those of Wistar (normal) rats. The change in Mo concentration in serum of normal rats was mono-phasic, whereas in LEC rats it was bi-phasic. The phase in normal rats and the first phase in LEC rats appeared to reflect the process of uptake and disappearance of TTM in the livers of Wistar and LEC rats. On the other hand, the second phase in LEC rats paralleled the changes of Cu and appeared to reflect the complex formation (Cu/thiomolybdate complex) between Mo and Cu accumulated in the liver, The complex was specifically bound to albumin as determined by high performance liquid chromatography with inductively coupled plasma-mass spectrometry (HPLC/ICP-MS). The results suggested that the changes in the Mo concentration in serum reflected the amount of Cu in the liver.

Copper(Cu) accumulating in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color) is bound to metallothionein (MT). Mechanisms for the removal of Cu by tetrathiomolybdate (TTM) were studied by the high performance liquid chromatography/inductively coupled plasma - mass spectrometry (HPLC/ICP-MS) method. MT containing Cu and cadmium (Cd) (Cu,Cd-MT) was reacted with TTM at a molar ratio of TTM/Cu = 0.5. A complex containing Cu, Cd and molybdenum (Mo) was formed and migrated to a position corresponding to an MT dimer on a gel filtration column. This complex designated previously as a dimer of MT through -S-Cu-S- bridge was revised to be a complex formed between MT and TTM through (MT)-S-Cu-S-(TTM) bridge with differing numbers of TTM bound to MT.

Tetrathiomolybdate (TTM) removes copper (Cu) accumulating in a form bound to metallothionein(MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color). The first step in the removal of Cu from Cu-MT has been shown to form a complex between MT and TTM through (MT)-S-Cu-S-(TTM) bridges (referred to as MT/TTM complex). Additional TTM was demonstrated to remove Cu from MT/TTM complex as the second step to form Cu/TTM complex by liberating MT. The Cu/TTM complex binds specifically to albumin in serum and to high molecular weight proteins in the absence of albumin, and is assumed to be a form of Cu for efflux by the treatment with TTM.

Tetrathiomolybdate (TTM) removes copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color). The first step in the removal of Cu from Cu-MT has been shown to form a complex between MT and TTM through (MT)-S-Cu-S-(TTM) bridges (referred to as MT/TTM complex). Additional TTM was demonstrated to remove Cu from MT/TTM complex as the second step to form Cu/TTM complex by liberating MT. The Cu/TTM complex binds specifically to albumin in serum and to high molecular weight proteins in the absence of albumin, and is assumed to be a form of Cu for efflux by the treatment with TTM.

Copper (Cu) accumulating in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color) is bound to metallothionein (MT). Mechanisms for the removal of Cu by tetrathiomolybdate (TTM) were studied by the high performance liquid chromatography/inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) method. MT containing Cu and cadmium (Cd) (Cu,Cd- MT) was reacted with TTM at a molar ratio of TTM/Cu = 0.5. A complex containing Cu, Cd and molybdenum (Mo) was formed and migrated to a position corresponding to an MT dimer on a gel filtration column. This complex designated previously as a dimer of MT through -S-Cu-S- bridge was revised to be a complex formed between MT and TTM through (MT)-S-Cu-S-(TTM) bridge with differing numbers of TTM bound to MT.

Simultaneous speciation of selenium and sulfur species in selenized odorless garlic (Allium sativum L. Shiro) and shallot (Allium ascalonicum) by HPLC-inductively coupled plasma - (octopole reaction system) - mass spectrometry and electrospray ionization 窶・tandem mass spectrometry.