Present status of the arrangements for Dietary Reference Intakes for Japanese(DRIs)were introduced. Technical problems in the determination of the trace elements including unstable food compositions in food were pointed out. Scientific problems of there process in the arrangements were discussed. Future works to arrange DRI with scientific evidences were proposed.

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In 2002, we found zinc(Zn)deficiencies in many of the patients visiting our small public clinic in Kitamimaki, a village in Central Japan's Nagano Prefecture. To learn more about the extent of this potential public health problem, we conducted three surveys to study the Zn levels of residents living in the area. These studies were the Kitamimaki Study(n=1431 participants)at Kitamimaki Village in 2003, the Tomi Study(n=1773)at Tomi City in 2005, and the Nagano Study(n=851)at seven clinics widely scattered throughout Nagano Prefecture from November 2005 through March 2006. All these surveys showed that mean Zn levels of the residents were lower than the prevailing, commonly accepted "normal" value for people in Japan. Residents' Zn levels were also lower than those of subjects studied in the 1985 Center for Disease Control NHANES II health survey, in which serum Zn levels were examined from 1976 through 1980 in the United States of America. These findings suggest that we must pay greater attention to Zn deficiency, especially among Nagano Prefecture's elderly, and lead us to speculate that this health threatening tendency may be found on a larger scale nationwide. We discuss possible causes of Zn deficiency, including zinc content in various types of food, eating habits, local soil composition, food additives, medicines, and other factors that may lower the serum zinc level of subjects.

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The main purpose of this study is to estimate adequate intake of copper(Cu)in inpatients of both sexes. Elderly inpatients with enteral nutrition through either nasogastric tube or gastric fistula(156 subjects : 62 men, 94 women)received 4 kinds of Cu amounts : daily Cu intakes of averaged 0.1 mg for 145 days in 60 cases, of 0.388 mg for 101 days in 22, of 0.691 mg for 105 days in 23, and of 1.36 mg for 76 days in 10. Another 9 cases received L-8 liquid food for 24 months, which contained 0.8 mg of Cu per 1,000 kcal. Of those Cu intakes 0.1 mg resulted in hypocupremia, and over 0.691 mg of Cu intakes contributed to normal levels of serum Cu. Elderly inpatients with hospital diets(315 cases : 112 men, 203 women)showed normal values of serum Cu for averaged 36.4 to 52.9 months, who received daily averaged 0.88 to 1.03 mg of Cu. From above findings 0.7 to 1.0 mg of Cu were estimated to be adequate.

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There is a possibility that long term enteral nutrition will cause trace element deficiency. On "6^th revision" , contents of trace element was adopted for the first time. Cu1. 4-1.6, Zn10-11mg, Se40-50µg per day. Our research showed the result that Cu0.5-1.0mg, Zn10-15mg, Se50 per 1000 kcal are enough to maintain serum level." Dietary Reference Intakes(DRI)for Japanese, 2005 "recommends that Cu0.7, Zn7mg, Se25-30 per day. 2005 is a standard for normal person. Trace elements such as Zn, Se will not enough for patients under long term enteral nutrition, if we follow this standard. So, we need to make another standard.

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In Japan, patients with taste disorder are increasing. The incidence of taste disorder due to diet(insufficient intake of zinc)is especially high. For diagnosis of zinc deficiency, we have been using the measurement of serum zinc usually. However, we have not yet had the definite cut off value until now. Therefore, we would like to propose the value less than 80µg/dl as clinical diagnosis value of zinc deficiency, referring to the many reports and the conclusion of Yokoi's study(Association between plasma zinc concentrations and zinc kinetic parameters in pre-menopausal women. ).

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Both the deficiency and the excess of trace elements can severely damage of the central nervous system. In particular, recent studies have suggested the implication of aluminum and zinc in the pathogenesis of senile-type dementia including Alzheimer's disease and vascular dementia. We investigated the neurotoxicity of aluminum on primary cultured cerebrocortical neurons and found several abnormal changes similar to those observed in Alzheimer's disease. Furthermore, we found that zinc caused the death of cultured neurons, and investigated the underlying molecular mechanism. We also found that disruption of Ca homeostasis may underlie the molecular mechanism of neurotoxicity induced by aluminum or zinc. Our results indicate the significance of trace elements in the brain function and suggest their implications in the pathogenesis of senile-type dementia, including Alzheimer's disease and vascular type dementia.

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The advent of miniaturized HPLC techniques, i.e., narrow bore, capillary, and nano HPLC, hyphenated with an inductively coupled plasma-mass spectrometer(ICP-MS)in speciation is expected to pave the way to new applications in metallome analyses. In this review, the author introduces recent applications of narrow bore and capillary HPLC-ICP-MS to speciation of bio-trace elements. In particular, our recent results using these miniaturized techniques were introduced. First, the application of the narrow bore system to minute amounts of tissue supernatants from mouse neonates bearing a mutation in a Cu-regulating gene was discussed. Second, combination of the gene modification technique and the miniaturized hyphenated technique were introduced, i.e., the application of on-line two-dimensional(2D)capillary HPLC-ICP-MS to a sample obtained from gene-modified cells was shown. The isoform-specific suppression of metallothioneins(MTs)induced by RNA interference was observed with the capillary system. On-line 2D capillary HPLC-ICP-MS showed some advantages over reverse transcription polymerase chain reaction in the specific detection of MT isoforms. New biological applications of the miniaturized hyphenated techniques combined with the gene modification technique are expected to contribute significantly to metallomics research.

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At first in this review, a brief history from the discovery of nuclear fission to the development of nuclear reactors is given. And then, nuclear fission and neutron capture reactions which occur in nuclear reactors are explained by stressing on nuclear transformations. The structure of nuclear power reactors, mainly pressurized water reactors(PWR)is briefly described. In the main part of this review, advantageous points in nuclear power for electric production are demonstrated. And then, several dangerous points in nuclear power are shown by a table and countermeasures against them are also given. Thereafter, the safety and the reliability of nuclear power reactors are defined and research activities how to improve them are explained. In the last part, introduced are several examples of related research works being carried out at the Institute of Nuclear Safety System, Inc. (INSS)as follows:(1)Recent activities of collecting and analyzing adverse events occurred in nuclear power plants in the world and of giving related proposals for improvement to all electric power companies in Japan, which possess PWRs. (2)Framework of research activities for countermeasures against ageing of nuclear power plants. (3)Two examples of measured results, atomic mappings of impurity elements in a specimen of reactor vessel steel after neutron irradiation and at a tip of stress corrosion crack in stainless steel which is mainly used for the main part of the primary water system of PWR. Both microscopic analyses are performed to elucidate the mechanisms of neutron irradiation embrittlement and stress corrosion cracking. (4)Analysis of severe accidents of PWR plants for the preparedness of nuclear disasters. Application of the calculated results to the exercise of nuclear disasters.

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Factors influencing the inhibition by oxalic acid and its salts(oxalates)of a copper-containing enzyme, tyrosinase, were studied by varying the pH of the reaction mixtures and preincubating the enzyme with the inhibitors under different conditions of the time, pH and temperature. The alleviative effects by long-chain fatty acids, which are known as endogenous activators of the normally-latent enzyme in some organisms, were also examined. The inhibition rate for the tyrosinase activity by oxalates of the same concentration was elevated along with a reduction in the pH values from neutral to mildly acidic. The preincubation treatments time-dependently inactivated the enzyme, and the inhibition was further enhanced by freezing the reaction mixtures at -20°Cat nearly neutral pH. The addition to the reaction mixtures of long-chain fatty acids and their salts significantly alleviated the inhibition, and the more hydrophilic compounds, which can be applied to aqueous media at higher concentrations, exhibited more remarkable effects. These results suggest that the inhibition of tyrosinase by oxalates is influenced by various factors, which may stabilize or remove the chelate linkage between the inhibitors and the functional copper ions in the enzyme molecule.

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This report demonstrates that infants and children have suffered from high aluminum accumulation, based on the evidence data of 1,720 children. For estimating its body burden, the scalp hair aluminum concentrations were measured with inductively coupled mass spectrometry(ICP-MS). The geometric mean of hair aluminum concentration was 10.4 and 11.1 µg/g(ppm)for boys and 11.8 and 10.5 ppm for girls, in the groups aged 0-3 and 4-9 years, respectively. These levels were high-significantly(p < 0.0000)and nearly 3-fold higher than those in the adults(3.8 and 4.0 ppm for male and female). Ten children exhibited higher aluminum levels exceeding 50 ppm, and the highest level observed was 116.8 ppm in a five-year-old boy. This wide-used metal should be monitored as toxic element risky to the young generation and its adverse effects remain to be clarified.

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Concentrations of total arsenic and individual arsenic compounds were determined in hawksbill turtles and green turtles to characterize their arsenic accumulation. Results of tissue distribution of total arsenic showed that the levels were the highest in muscle of both species. Concentrations of total arsenic in tissues of hawksbill turtles were significantly higher than those of green turtles and were comparable to lower-order marine animals. Chemical speciation of arsenic revealed that arsenobetaine was the major arsenic compound in these two species. Relatively high concentrations of non-extracted arsenic were observed in liver of both turtles. Interestingly, high concentration of trimethylarsine oxide was detected in hawksbill turtles. Concentrations of arsenobetaine in the tissues of hawksbill turtles and green turtles were much higher than those in their respective stomach contents. This implies that sea turtles may effectively bioaccumulate arsenobetaine from their prey.

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Immunotoxicity is one of toxic effects of dibutyltin(DBT)compounds. In our previous study, the inhibitory effects of DBT on the productions of tumor necrosis a(TNFα)and interleukin-1β in a murine macrophage cell line, J774.1, were demonstrated. In this study, the effects of DBT dichloride from 0.125 µM to 2.0 µM on the cell viability and productions of cytokines including TNFα, IL-10 and IL-12p40 were investigated. J774.1 cells were exposed to DBT dichloride at 0, 0.125, 0.25, 0.5, 1.0, 1.5 or 2.0 µM. After 18 hours, lipopolysaccharide was added to each well. Total RNA was extracted from the cells after an additional 6 hours of incubation. Real-time PCR was used to analyze the mRNA expression for TNFα, IL-10, IL-12p40 and glycelaldehyde-3-phosphate dehydrogenase(GAPDH)in J774.1 cells. The cell viabilities were determined and the supernatants were sampled after additional 24 hours of incubation. The concentrations of TNFα, IL-10 and IL-12p40 in the supernatant were determined by ELISA. The mean cell viabilities in the groups exposed to DBT at 0.5 µM and over were significantly lower than that of the control. The mean mRNA expression of IL-12p40 in the 0.25 µM group was significantly higher than that in the control group. The mean concentrations of TNFα in the supernatant in the 1.0, 1.5 and 2.0 µM groups were significantly lower than that of the control, and that in 0.125, 0.25 and 0.5 µM groups were significantly higher. The mean concentration of IL-12p40 in the 0.25 µM group was significantly higher than that of the control and that in the 1.0, 1.5 and 2.0 µM groups were significantly lower. There were no significant differences in mRNA expression and protein level for IL-10. DBT is toxic to macrophages, and the inhibition of the production of cytokines by DBT does not occur for all cytokines.

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Wilson disease is an autosomal recessive disorder of copper metabolism characterized by hepatic and/or neurological manifestations. This biochemical features of this disease are low serum ceruloplamine levels and high urinary copper excretion. Early diagnosis is very important to improve the prognosis of this disease. However, some patients revealed atypical biochemical findings. This study presents the efficacy of D-penicillamine challenge test for diagnosis of Wilson disease. Five patients and five normal controls were loaded 20mg/kg of D-penicillamine. Urinary copper / body weight(kg)ratio and/or urinary copper / creatinine ratio showed significant difference between Wilson disease patients and controls. The D-penicillamine challenge test will be useful for diagnosis of Wilson disease.

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Selenophosphate, a key intermediate in selenium metabolism, is synthesized from selenide and ATP by selenophosphate synthetase(SPS). Mammals have two SPS isozymes known as SPS1 and SPS2. Mouse SPS2 contains a catalytically important selenocysteine residue at position 63 of the polypeptide chain. In contrast, SPS1 has a Thr-29 in the position that corresponds to Sec-63 of SPS2. SPS1 and Sec63Cys mutant SPS2 were purified to homogeneity and characterized. SPS2(Sec63Cys)catalyzed the selenide-dependent synthesis of selenophosphate, but SPS1 showed no activity. However, the Thr29Cys mutant of SPS1 exhibited SPS activity. These findings raise the intriguing possibility that SPS1 may exert its catalytic function in the presence of thiols in the vicinity of position 29.

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Bacterial selenocysteine synthase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of seryl-tRNA^Sec to selenocysteyl-tRNA^Sec for selenoprotein biosynthesis. Human selenocysteine synthase(SecS),originally annotated as SLA/LP, was previously reported to operate in selenocysteyl-tRNA^Sec synthesis, but the mechanism of conversion from Ser-tRNA^Sec by the eukaryotic enzyme remained unresolved. Herein, the human cDNA encoding SecS has been cloned and overexpressed in Escherichia coli. SecS was co-purified with E. coli tRNAs, which was revealed to contain tRNA^Sec by PCR analysis. The purified enzyme exhibited a UV-visible absorption maximum at 420 nm characteristic of pyridoxal 5'-phosphate-dependent enzymes. In vitro selenocysteyl-tRNA^Sec synthesis assay suggests that the formation of phosphoseryl-tRNA^Sec is essential for human seryl-tRNA^Sec, but not archaeal seryl-tRNA^Sec to be converted to selenocysteyl-tRNA^Sec by human SecS.

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Selenium, an essential trace element, is co-translationally incorporated into selenoproteins as the 21st amino acid selenocysteine. Selenite serves as an inorganic selenium source for selenoprotein biosynthesis through reduction to selenide, which is converted to selenophosphate, the essential selenium donor in selenocysteyl-tRNA^[Ser]Sec synthesis. However, the pathways for selenite reduction in mammalian cells have not yet been clarified. Based on metabolic labeling with [⁷⁵Se]selenite and RNA silencing studies, we here present evidence that thioredoxin reductase 1, but not thioredoxin, is crucial for selenite utilization to form selenoproteins in HeLa cells. We suggest that thioredoxin reductase 1 plays a role as a selenite-reducing enzyme in vivo.

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DNA damages induced by sodium selenite in the presence of reduced glutathione(GSH)were analyzed by agarose gel electrophoresis. Plasmid DNA was incubated with selenite and glutathione in Tris-borate-EDTA(TBE)buffer(pH8.2)at 37°C. The DNA remained supercoiled form when it was incubated alone, with selenite, or with GSH for 2 hours. DNA with open circular form was observed when it was incubated in the presence of both selenite and GSH, and the electrophoretic band strength corresponding to open circular form was highest when GSH was present 100 times excess moles of selenite. The open circular DNA increased with time of incubation at least up to 4 hours under the same conditions, though the increase was not marked in 2-4 hours. The open circular DNA was not observed just after the start of incubation. The results indicated that a single strand break of DNA chain occurs when plasmid DNA is incubated in the presence of both selenite and GSH for more than 1 hour forming an open circular DNA, the amount of which was dependent on the molar ratio of selenite and GSH. It was suggested that excess amount of GSH accelerated the breakdown of initially formed selenodiglutathione(GSSeSG)and the production of superoxide anion or other reactive species, and DNA may have been damaged by these species. The reaction between sodium selenite and GSH in TBE buffer was analyzed by HPLC. The peaks corresponding to GSH, oxidized glutathione(GSSG),and GSSeSG were observed in chromatograms. The peak area of GSH decreased and that of GSSG increased with the time of reaction. However, the time profile of peak area of GSSeSG depended on the molar ratio of selenite and GSH in the reaction mixture; the peak area increased with time when selenite:GSH=1:100 and it decreased when selenite:GSH=1:1, 1:4, and 1:10. Thus, the extent of single strand break of DNA was highest under the conditions with highest GSSeSG production. These results suggested that GSSeSG may possibly be a compound that catalyzes single strand breaks of DNA.

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Dietary intakes of six trace elements, bismuth(Bi),cadmium(Cd),cobalt(Co),chromium(Cr),lead(Pb),and thallium(Tl)in Ukrainian subjects were estimated in relation to the health effects on habitants after the Chernobyl accident. Two hundred and sixty-eight diet samples were collected from twenty-five oblasts(regions)using a duplicate portion method. The elements were determined by inductively coupled plasma mass spectrometry(ICP-MS). For Ukrainians, median daily intakes of Bi, Cd, Co, Cr, Pb and Tl were 0.368, 7.96, 9.71, 113, 33.1 and 0.372 µg per person, respectively. The intakes of Bi, Cd, Co, Pb, and Tl in Ukrainians were lower than worldwide reported values, while that of Cr was higher.

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Purpose: Very low birth weight infants have high nutritional needs because nutrient storage in their bodies is very limited and their growth is rapid. Because they differ from full-term infants, their need for minerals and trace elements may also differ. During the neonatal stage, commercial supplements to breast milk and milk-based formulas have been used to feed very low birth weight infants. However, whether the trace element concentrations of these products are appropriate for very low birth weight infants is uncertain. In this study, we analyze the concentrations of trace elements in the serum of low birth weight infants and the breast milk of their mothers. Methods: Serum samples from eleven low birth weight infants, 708 to 1768 g birth weight, were collected until 30 weeks after the birth, and breast milk samples were also collected on 1 to 5 times. Calcium, magnesium, and essential trace elements(iron, zinc, copper, selenium, rubidium)in the serum and breast milk were measured by plasma-mass spectrometry(ICP-MS). Results and conclusions: Concentrations of iron, zinc, copper, selenium, and rubidium were significantly lower in the serum of low birth weight infants as compared to full-term infants. Serum calcium and magnesium concentrations did not differ from those of full-term infants. Although iron, zinc, and selenium concentrations were decreased during the weeks after one's birth, individual differences were large. Trace element concentrations in the breast milk of mothers of very low birth weight infants did not differ from those in the breast milk of mothers of full-term infants, and no correlations with concentrations in the serum of very low birth weight infants were observed. These findings suggest that very low birth weight infants may be deficient in iron, zinc, copper, selenium, and rubidium. They may need additional sources of these trace elements.

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Harmful systemic effects were studied 1 hour after acute sub-lethal exposure to hydrofluoric acid(HFA)in previous study. This study was designed to investigate the early dynamic state of F in blood and urine in rats as a model of accidental occupational exposure to HFA. It was also designed to determine the relationship between the kinetics and harmful effects on kidney. To evaluate the kinetics of F exposure and metabolism, rats received a single intravenous injection of HFA(3.2, 6.4, or 9.6(LD₅)mg/kg)or saline. The each volume was 1 ml and the concentrations of HFA were 0.1, 0.2, 0.3%, respectively. Serum ionized fluoride(F)concentrations were determined 0, 5, 10, 30, 60, 120, and 300. Calculating the elimination data of F from serum required two-compartment modeling. Urine was collected for 300 min. urinary parameters were measured to consider the renal injury. T_1/2β, AUC_0→300 were significantly in 0.3% group longer than 0.1 and 0.2% groups. The total body clearance, V₁, V₂, V_ss, K₁₀ were significantly lower in 0.3% group than 0.1% groups. The pharmacokinetic parameters indicated the retention of F in blood. NAG/Cr and glucose were increased and F clearance, urine volume and excretion of electrolytes were decreased in 0.3% group compared with other groups. Deceased urinary F showed the disorder of F excretion from kidney. Therefore, the metabolism of F was markedly delayed in 0.3%. The kidney is main excretory organ, and also the target organ of F. The glomerular dysfunction and renal tubular injury were caused by HFA exposure. Abnormalities in F kinetics could result from renal dysfunction at a sub-lethal toxic dose. In previous study, the same dose of HFA could also cause electrolyte abnormalities and metabolic acidosis. In conclusion, metabolism of F was mainly disordered by glomerular dysfunction.

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Chromium(Cr)is an essential trace element for normal carbohydrate metabolism, and its deficiency in animals can cause a diabetic-like state. Human and experimental animal studies suggest that urinary Cr excretion is increased in diabetic patients. To investigate whether hyperglycemia-induced elevation of urinary Cr excretion at relatively early stages of diabetes reduces tissue Cr storage, we assessed Cr excretion and Cr distribution in streptozotocin(STZ)-induced diabetic mice. Male C57BL mice were randomly assigned to STZ or control groups. STZ mice were injected with STZ to induce insulin-dependent diabetes(day 0)and 24-hour urinary collections were taken 3, 4 and 5 days after the STZ treatment. An inductively coupled plasma mass spectrometer equipped with a dynamic reaction cell was used for the determination of Cr in urine, plasma and tissue samples. The urinary Cr concentration and urine volume in the STZ-treated mice on day 5 after STZ injection was approximately∼3 times and 7 times higher than those of control. STZ-induced diabetic mice excreted 35-fold Cr compared to control mice, during 5 days after STZ injection. The renal Cr concentrations in STZ-induced diabetic mice were significantly lower than those in controls(p < 0.05). These results suggested that hyperglycemia by STZ increased urinary Cr output and caused reduction of renal Cr concentration.

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