Metabolic pathway for selenium in the body: speciation by HPLC-ICP MS with enriched Se.

Selenium (Se) is an ultramicro essential nutrient and both inorganic (selenite and selenate) and organic (selenocysteine and selenomethionine) forms of Se can be used as nutritional sources. Metabolic pathways for Se in the body were studied for selenite and selenate, with the use of enriched 82Se, by speciation with separation by gel filtration HPLC and detection by element-specific mass spectrometry with ionization with inductively coupled argon plasma (HPLC-ICP MS). The concentrations of 82Se in organs and body fluids and the distributions of their constituents depending on the dose and time after the intravenous administration of 82Se-selenite and -selenate to rats were determined. Selenite was taken up by red blood cells within several minutes, reduced to selenide by glutathione, and then transported to the plasma, bound selectively to albumin and transferred to the liver. Contrary to selenite, intact selenate was either taken up directly by the liver or excreted into the urine. The 82Se of selenite origin and that of selenate origin were detected in the forms of the two Se peak materials in the liver, A and B. The former one was methylated to the latter in vivo and in vitro. The latter one was identical with the major urinary metabolite and it was identified as Se-methyl-N-acetyl-selenohexosamine (selenosugar). The chemical species-specific metabolic pathway for Se was explained by the metabolic regulation through selenide as the assumed common intermediate for the inorganic and organic Se sources and as the checkpoint metabolite between utilization for the selenoprotein synthesis and methylation for the excretion of Se.

Glutathione-conjugated arsenics in the potential hepato-enteric circulation in rats.

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 [CH(3)As(III)(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 [CH(3)As(III)(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.

Animal species difference in the uptake of dimethylarsinous acid (DMA(III)) by red blood cells.

The animal species difference in the metabolism of arsenic was studied from the viewpoint of the mechanism underlying its distribution in the form of dimethylated arsenic in red blood cells (RBCs). Dimethylarsinic (DMA(V)) and dimethylarsinous (DMA(III)) acids were incubated with rat, hamster, mouse, and human RBCs, and the uptake rates and chemical forms of arsenic were determined. Although DMA(V) was practically not or taken up slowly by RBCs of all the present animal species, DMA(III) was taken efficiently in the order of rat > hamster > human, RBCs of mice taking it up less efficiently and with a different pattern from the former three animals. Further, although DMA(III) taken up by rat RBCs was retained, that by hamster ones was effluxed in the form of DMA(V). The uptake of DMA(III) and efflux of DMA(V) took place much more slowly in human RBCs than rat and hamster ones. The uptake of DMA(III) by RBCs was inhibited on the oxidation of glutathione with diamide. Incubation of DMA(III), but not of DMA(V), with a hemolysate produced a high molecular weight complex, which increases in the presence of glutathione, suggesting that DMA(III) taken up by RBCs is retained through the formation of a complex with protein(s) specific to animal species, and effluxed from RBCs after being oxidized to DMA(V). These results indicate that DMA is taken up by RBCs in the form of DMA(III), and that the uptake and efflux rates are dependent on the animal species, the effluxed arsenic being DMA(V). The present results suggest that the uptake of DMA by RBCs is an additional contributing factor to the animal species difference in the metabolism of arsenic in addition to the reduction and methylation capacity in the liver.

Speciation and metabolism of selenium injected with 82Se-enriched selenite and selenate in rats.

Selenate and selenite injected intravenously into rats were speciated by the HPLC-ICP MS method with use of an enriched stable isotope as the tracer. In dose-relation experiments, 82Se-enriched selenate or selenite was injected intravenously into male Wistar rats of 8 weeks of age (three rats/group) at single doses of 10, 25, 50, 100 and 200 microg/kg body weight for the selenate group, and 2, 5, 10, 25 and 50 microg/kg body weight for the selenite group. The animals were sacrificed 1 or 24 h later, and the concentrations and distributions of 82Se in the liver, kidneys, serum, and urine remaining in the bladder or 24-h urine were determined. In time-course experiments, 82Se-enriched selenate and selenite were injected at doses of 50 and 10 microg/kg body weight, respectively, and the animals were sacrificed 5, 15, 30, 60 and 180 min later. It was suggested that selenate is directly taken up by the liver with an efficiency of approximately 1/2 compared with selenite, the latter being taken up by the liver after being metabolized to selenide in red blood cells. Although selenate and selenite were metabolized differently in the bloodstream, and also a part of only selenate was excreted directly into the urine, the 82Se taken up by the liver was shown to be metabolized in a manner indistinguishable between selenate and selenite. 82Se of selenite origin but not of selenate origin was suggested to undergo redox reaction in the bloodstream. These results suggest that although parenteral selenate is utilized less efficiently by the body, it is utilized in the liver in a similar manner to selenite much more safely.

Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal, India.

A speciation technique for arsenic has been developed using an anion-exchange high-performance liquid chromatography/inductively coupled argon plasma mass spectrometer (HPLC/ICP MS). Under optimized conditions, eight arsenic species [arsenocholine, arsenobetaine, dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), arsenite (As(III)), and arsenate (As(V))] can be separated with isocratic elution within 10 min. The detection limit of arsenic compounds was 0.14-0.33 microg/L. To validate the method, Standard Reference Material in freeze-dried urine, SRM-2670, containing both normal and elevated levels of arsenic was analyzed. The method was applied to determine arsenic species in urine samples from three arsenic-affected districts of West Bengal, India. Both DMA(III) and MMA(III) were detected directly (i.e., without any prechemical treatment) for the first time in the urine of some humans exposed to inorganic arsenic through their drinking water. Of 428 subjects, MMA(III) was found in 48% and DMA(III) in 72%. Our results indicate the following. (1) Since MMA(III) and DMA(III) are more toxic than inorganic arsenic, it is essential to re-evaluate the hypothesis that methylation is the detoxification pathway for inorganic arsenic. (2) Since MMA(V) reductase with glutathione (GSH) is responsible for conversion of MMA(V) to MMA(III) in vivo, is DMA(V) reductase with GSH responsible for conversion of DMA(V) to DMA(III) in vivo? (3) Since DMA(III) forms iron-dependent reactive oxygen species (ROS) which causes DNA damage in vivo, DMA(III) may be responsible for arsenic carcinogenesis in human.

Exchange of endogenous selenium for dietary selenium as 82Se-enriched selenite in brain, liver, kidneys and testes.

Male Wistar rats were fed a diet containing selenium (Se) in the form of 82Se-enriched selenite at the adequate concentration of 0.2 microg Se/g diet, i.e. a Se-deficient diet (<0.03 microg Se/g) fortified with 82Se-enriched selenite, from 5 weeks of age for 20 days, and the systemic disposition of the labelled Se and exchange of endogenous naturally occurring Se for the labelled Se were monitored in four organs. Features characteristic of each organ in terms of Se metabolism were revealed by plotting the disposition of 82Se and exchange of endogenous Se for 82Se against the number of days of feeding 82Se-selenite. Labelled Se amounted to 83.7, 80.8, 73.2 and 41.9% of the total Se in the liver, kidneys, testes and brain, respectively, after feeding 82Se-selenite for 20 days, suggesting that the disposition and exchange were most efficient in the liver but least efficient in the brain. However, when the weight gain of the four organs during the feeding period was taken into consideration, the apparent higher exchange was concluded to be caused by weight gain, i.e., more efficient uptake of the labelled Se by proliferating cells than non-proliferating cells in the liver, kidneys and testes. On the other hand, the uptake and exchange in non-proliferating cells were greater in the brain than in the other organs, especially in the late observation period. The relative metabolic turnover rates of selenoproteins were shown to be easy to determine from the relative exchange rates of endogenous Se for exogenous Se in the distribution profiles of Se obtained by the HPLC-ICP MS method.

[Biological regulation of copper and selective removal of copper: therapy for Wilson disease and its molecular mechanism].

Copper (Cu) is an essential trace element and constitutes the active center of the redox Cu enzymes such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD), ceruloplasmin and cytochrome c oxidase. Among hereditary diseases due to a defect in the metabolism of Cu, Menkes disease (caused by a Cu deficiency) and Wilson disease (caused by the excessive accumulation of Cu) have been shown to be caused by the mutation of genes encoding Cu-binding ATPase for the efflux of Cu, ATP7A and ATP7B, respectively. Following the identification of these causative genes, intracellular Cu transporters (Cu chaperones) specific for the Golgi apparatus, mitochondria and Cu, Zn-SOD were discovered, and these findings have facilitated the study of the underlying mechanisms of the biological regulation of Cu. Apart from these physiological and biochemical studies, toxicological studies have elucidated the underlying mechanisms of the occurrence of acute hepatitis caused by the accumulation of Cu accumulating in the liver of an animal model for Wilson disease, LEC rats. In these toxicological studies, two biological aspects of metallothionein (MT), i.e., antioxidant and prooxidant depending on the Cu/Zn ratio in Cu-containing MT have been proposed. The present article overviews the recent findings on the biological regulation of Cu and on the toxicological aspect of Cu. It is known that Cu forms a stable ternary complex with molybdenum and sulfur under reductive conditions in the body. On the basis of this observation, tetrathiomolybdate (TTM) has been applied to remove Cu from the liver of Long-Evans rats with a cinnamon-like coat color (LEC) rats. Precise mechanisms underlying the complex formation between Cu bound to MT and TTM were presented, and an appropriate protocol for the chelation therapy was also proposed together with the mechanisms underlying the occurrence of side-effects.

Nuclear trafficking of metallothionein: possible mechanisms and current knowledge.

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.

Increase in mutation frequency in lung of Big Blue rat by exposure to diesel exhaust.

Exposure to diesel exhaust (DE) is known to cause lung tumors in rats. To clarify the mutagenicity of DE, we estimated mutant frequency (MF) and determined the mutation spectra in rat lung after exposure to DE using lambda/lacI transgenic rats (Big Blue system). Male Big Blue rats (6 weeks old) were exposed for 4 weeks to 1 or 6 mg/m(3) DE, which contains suspended particulate matter. Control rats were maintained in filtered clean air. After exposure to 6 mg/m(3) DE, MF in lung was 4.8-fold higher than in control rats (P < 0.01), but no increase in MF was observed in rats exposed to 1 mg/m(3) DE. Sixty-nine mutants were identified after exposure to 6 mg/m(3) DE. The major mutations were A:T-->G:C (18 mutations) and G:C-->A:T (19 mutations) transitions. Remarkably, G-->T transversion of the lacI gene at site 221 was a hot-spot induced by exposure to DE, and there were complex mutations in which multiple mutations occurred in a single mutant, especially in the rats exposed to 6 mg/m(3) DE. DNA adducts formed by DE were analyzed using a (32)P-post-label TLC method and the amount of 8-hydroxydeoxyguanosine (8-OHdG) was measured using HPLC. Relative adduct level and amount of 8-OHdG were significantly increased in the rats exposed to 6 mg/m(3) DE compared with the controls (3.0- and 2.2-fold, respectively; P < 0.01). The level of cytochrome P450 1A1 mRNA was shown by northern blot analysis to be significantly increased in the lungs of rats exposed to 6 mg/m(3) DE (5.5-fold; P < 0.01). These results indicate that DE causes lesions in genomic DNA and acts as a mutagen in rat lung.

Metabolic fate of the insoluble copper/tetrathiomolybdate complex formed in the liver of LEC rats with excess tetrathiomolybdate.

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, can be removed from the MT with tetrathiomolybdate (TTM). However, the insoluble Cu/TTM complex formed with excess TTM is known to be deposited in the liver. The metabolic fate of the insoluble Cu/TTM complex was investigated in the present study. LEC rats were injected with TTM at the dose of 10 mg/kg body weight for 8 consecutive days and were fed with a standard or low Cu diet for 14 days after the last injection. About 95% of the Cu in the liver became insoluble together with Mo. The concentration of Cu in the liver supernatants of rats fed with the standard diet increased significantly compared with that in rats dissected 24 h after the last injection (control rats), while the concentration in rats fed with the low Cu diet remained at a comparable level to that in the controls. The rate of Cu accumulation in the livers of rats fed with the standard diet did not differ before and after the treatment, suggesting that there was no rebound effect by treatment with TTM. These results suggest that the insoluble Cu/TTM complex is resolubilized in the liver, and that the solubilized complex is excreted into the bile and blood, i.e., the insoluble Cu/TTM complex is not the source of Cu re-accumulation in the form bound to MT in the liver after TTM treatment. It was concluded that, once Cu is complexed with TTM, the metal is excreted either immediately in the soluble form or slowly in the insoluble form into the bile and blood.

Excretion of copper complexed with thiomolybdate into the bile and blood in LEC rats.

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.

Upregulation of heme oxygenase gene expression in rat lung epithelial cells following exposure to cadmium.

Inhalation exposure to cadmium (Cd) is associated with inflammatory lesion in the lung. In the present study we have investigated cytotoxic effects of Cd on immortalized rat lung epithelial cells (SV40-T2) and gene expression in those cells following in vitro exposure to sublethal concentrations of cadmium chloride (CdCl(2)). The polymerase chain reaction (PCR)-based subtraction method was used to find differentially expressed genes between control and Cd-exposed SV40-T2 cells. The most prominent cDNA on an agarose gel was identical to a fragment of rat heme oxygenase (HO) gene. Northern blot analysis indicated that the level of HO mRNA expression in SV40-T2 cells was increased to 38-fold of the control value following exposure to 2.5 microM CdCl(2) for 4 h. These results suggest that HO gene expression is one of the most sensitive biomarkers for acute exposure to Cd in the lung.

Comparative mechanism and toxicity of tetra- and dithiomolybdates in the removal of copper.

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.

Copper increases in both plasma and red blood cells at the onset of acute hepatitis in LEC rats.

Ceruloplasmin is excreted mostly in the apo-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.

Identification, by cDNA microarray, of A-raf and proliferating cell nuclear antigen as genes induced in rat lung by exposure to diesel exhaust.

Diesel exhaust particles (DEP) contain various carcinogens and mutagens, and chronic exposure to diesel exhaust (DE) induces pulmonary cancer in experimental animals. However, the oncogenes involved in pulmonary carcinogenesis have not been identified. After F344 rats were exposed to DE containing 6 mg/m3 DEP for 4 weeks, oncogenes and related genes expressed in their lungs were surveyed using a new technique, cDNA microarray, and the results were confirmed by northern blot analysis. Expression of A-raf and proliferating cell nuclear antigen (PCNA) mRNAs was induced in rat lung by exposure to DE. These results suggest that A-raf and PCNA might contribute to pulmonary carcinogenesis in rats.

Biological significance of non-acetylated metallothionein.

The biological significance of non-acetylated metallothionein (MT) was investigated from the viewpoint of N(alpha)-acetylation after induction of MT synthesis by metallic and non-metallic inducers, by partial hepatectomy and under physiological conditions. N(alpha)-Acetylated and non-acetylated forms of MT-2 in liver supernatants and plasma were detected by the tandem size-exclusion and anion-exchange HPLC columns with in-line detection by mass spectrometry. The non-acetylated isoform of MT-2 (MT-2') was present at a comparable level to the N(alpha)-acetylated form of MT-2 (MT-2) at an early stage after induction by not only zinc but also cadmium, and by partial hepatectomy in the livers of rats. Plasma MT-2 in neonatal rats was similar to liver MT-2 in the composition of N(alpha)-acetylated and non-acetylated forms, suggesting that there are no differences in the roles of N(alpha)-acetylation of MT in the extracellular trafficking of MT. The column switching HPLC method with in-line detection by inductively coupled argon plasma mass spectrometry (ICP-MS) was shown to be a sensitive and powerful method to detect MT proteins at not only isoform level but also at acetylated and non-acetylated form levels.

Speciation of metabolites of selenate in rats by HPLC-ICP-MS.

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 HPLC-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.

Incorporation of selenium into selenoprotein P and extracellular glutathione peroxidase: HPLC-ICPMS data with enriched selenite.

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, [82Se]selenite. [82Se]selenite was injected intravenously into rats at a dose of 25 micrograms 82Se kg-1 body weight, and the concentrations of labeled 82Se and naturally occurring 77Se 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) 82Se and endogenous (naturally occurring) 77Se 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.

Identification of the zinc-binding protein specifically present in male rat liver as carbonic anhydrase III.

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.