We have developed the multitracer technique using the RIKEN Ring Cyclotron, which can accelerate heavy ions up to 135MeV /nucleon. Irradiation with such heavy ions generates a wide range of radioisotopes in targets mainly due to nuclear fragmentation. Our idea was to use these radioisotopes together as a multitracer in order to trace the behavior of many elements simultaneously in various systems. Radiochemical procedures for preparation of carrier- and salt-free multitracers were established. So far, more than fifty elements can be traced using this technique. The multitracers are now used to investigate the behavior of various elements in chemistry, biochemistry and biology in cooperation with laboratories in universities and research institutes. Especially, uptake, transfer and metabolism of trace elements in animal bodies are now extensively studied using the multitracer technique. Several examples of such studeis are presented.

In 1936, Ida Noddack proposed a hypothesis called “All Present Theory of the Elements“, in which she predicted the presence of all elements in geological samples (minerals and rocks). Based on the Noddack’s hypothesis, the present authors are proposing a new concept of “Extended All Present Theory of the Elements”, which indicates that the All Present Theory should be extended to all the biological systems including human beings. In order to elucidate or prove the Extended All Present Theory of the Elements, the experimental results for the determination of all rare earth elements in human blood serum, and multi-element determination of major-to-ultratrace elements in black and green tea leaves were introduced together with the chemical speciation of trace elements in black-tea infusion. Finally, the correlation of elemental concentrations between human blood serum and seawater was discussed from the viewpoints of chemical and biological evolution of the life in seawater. It should be stressed here that the recent research for trace elements has been supported by the development of high-sensitive and multi-element analytical methods, especially ICP-MS (inductively coupled plasma mass spectrometry)

Firstly, the antioxidative activities of the ten kinds of plants extracts were evaluated by three methods. The conker (Aesculus turbinata Blume.), the barilla plant (Salsola Komarovi Iijin.) and the purslane (Potulaca oleracea L.) extract was found to be a strong antioxidant when tested by the TBA method using deoxyribose as the substrate, whereasthe Colorimetric assay using Methyl indole as a reagent and linolenic acid as the substrate indicated that the activity of athers. The seedvessel of a conker, the barilla plant (Salsola Komarovi Iijin) and the purslane (Potulaca oleracea L.) extracts were showed extremaly strong ・OH radical scavenging activity when tested by the ESR method.

Secondly, the effect of Salsola Komarovi Iijin. and Potulaca oleracea L. extracts studied in the overiectmized rat model of osteoporosis.

Twelve week old female SD rats were randomly assigned to following four groups, i.e., sham operated (Sham); over mized (OVX); OVX and fed a diet containing 1. 5% Salsola Komarovi Iijin. extracts (OVX + SK); OVX and fed a diet containing 1. 5% Potulaca oleracea L. extracts (OVX + PO).

In conclusion, the OVX and OVX + SK group were to be sthenia both deossification or osteogenesis.

Series of research in our laboratory has shown that restoration of morphology and motility of TBTCl-intoxicated Euglena gracilis Z should be resulting from trace minerals in the processed aqueous solution. To examine the detoxication effect of mineral-encaged zeolite on the impaired cells, we used the bioassay system using Euglena gracilis Z as the model organism and TBTCl as the model xenobiotic. Under the polluted environment, Euglena cells changes their shape to spherical form from spindle form. By taking advantage of this unique character, we examined the different types of zeolites which encaged different minerals. TBTCl-intoxicated Euglena cells were separately washed with the water species processed by zeolite, then incubated in the processed water for up to 3 hours. The restoration of motility of the cell was estimated by observing the motile cell number using a video-microscopy system. A remarkable recovery of cell motility was observed with the incubation system using Fe, Mn and Zn encaging zeolite. However, they did not show any recovery effect when they were treated with a chelator, Chelex-100.

We examined the susceptibility of liver chromatin of mice at prenatally 16-days (fetus) and postnatally 19~23-weeks (adult) for hydroxyl radical (・OH). DNA strand breakages in both fetal and adult liver nuclei by Cu (II) -H₂O₂ system were shown to be more extensive that that by Fe (III) -H₂O₂ system under the same experimental conditions. DNA damage in nuclei also showed higher rate and extent in fetal liver than that of adult, especially for oxidation by Cu (II) -H₂O₂ system. The reduced glutathione (GSH), N-acetylcysteine (NAC) and ascorbic acid (ASC) showed the promotive effects on DNA damages by ・OH, whereas cysteine (CYS) showed the antioxidative effect. In fetal liver, 5-methylcytosine (5mC) contents in chromatin were less than that of adult, suggesting the active chromatin structure in fetal liver nuclei. The increase of DNA damage by ・OH in fetal liver nuclei which accompanied the expansion of chromatin, indicated the importance of chromatin compaction for the oxygen free radical injury. The results also suggested that Cu (II) and Fe (III) ions act at different nuclei loci, and that Cu (II) ion binds to unmethylated sites on DNA constructing the nuclear matrix.

Zinc and vanadium have been reported to possess insulin-mimetic activity on various types of cells. 3T3-L1 preadipocyte which is derived from mouse can differentiate into adipocyte by several stimulators including insulin. We examined the stimulative effects of zinc and vanadium on the differentiation of 3T3-L1 preadipocyte. The supplementation of zinc during day 0-2 after confluence increased GPDH activity (a parameter of adipocyte differentiation) on day 8. The addition of zinc to 3T3-L1 cells for day 2-8 also enhanced GPDH activity. However no effect was observed when zinc was supplemented to fully differentiated 3T3-L1 adipocytes. Vanadium also increased GPDH activity in dose dependent manner when added for day 2-8.

By using cultured mouse fibroblast cells transformed with Simian virus 40 which bears the putative oncoprotein-oncosuppressor system, the rice bran was shown to contain anti-cancer substances which are selectively cytotoxic to the SV40-transformed cells but not to the non-transformed counterpart cells. Active substances were found in a small molecular fraction between the molecular weights of 500 and 1,000 dalton, as revealed by molecular sieve chromatography on a Bio-gel P-2 column. LD50 and LD90 value of the active fraction was 1.0mg/ml and 3.0mg/ml to SV40-transformed cell, respectively, wheras at these concentrations nearly no cytotoxicity was observed to nontransformed cells.

The biodistribution of the labelled sialoglycoconjugates, ¹⁴C-sialic acid, ¹⁴C-sialyllactose and ¹⁴C-ganglioside (GM3) and their transference into the brain of rats were studied. The intravenously administrated ¹⁴C-sialic acid and ¹⁴C-sialyllactose were excreted immediately from the kidney whereas ¹⁴C-ganglioside (GM3) was accumulated in the liver. By oral administration, ¹⁴C-sialyllactose and ¹⁴C-ganglioside (GM3) were accumulated in the salivaly gland, spleen and brain. The oral administered ¹⁴C-sialyllactose was shown higher transference in to the brain than the case of intravenous administration. In the case of ¹⁴C-ganglioside (GM3), some was gradually metabolized to GM2, GD3 and GD2 the remained in brain.

It was clear that the orally administered ganglioside (GM3) was transferred into the brain, and was accumulated itself there and some metabolized.

In the present study, we examined the expression of endothelin (ET) -1 and a role of angiotensin II in glomeruli from rats with unilateral ureteral obstruction (UUO) fed a zinc (Zn) deficiency or a standard diet. The expression of ET-1 was substantially potentiated in glomeruli of the obstructed kidney (OK) from rats with UUO fed a Zn deficiency v.s. a standard diet. No expression of ET-1, however, was observed in glomeruli of the contralateral, non-obstructed kidney from rats with UUO fed a Zn deficiency or a standard diet. Prior administration of the angiotensin converting enzyme inhibitor, enalapril markedly decreased the enhanced expression of ET-1 observed in glomeruli of the OK from rats with UUO fed a Zn deficiency diet. Indeed, the renin-angiotensin system (RAS) is known to be activated after the onset of ureteral obstruction. These findings suggest that Zn deficiency intensifies the expression of ET-1 in glomeruli of the OK from rats with UUO by further enhancing the RAS upregulated after the onset of ureteral obstruction.

Zinc is a very important nutrient in a normal reproductive function and embryonic development of mammals. It is well-known that the oyster extract contains a large amount of zinc. We studied the effects of oyster extract on reproductive function, embryonic development and serum levels of zinc in zinc-deficient mice. Zinc deficiency during pregnancy induced the decrease in successful pregnancy rate, maternal weight gain, the number of live fetuses and fetal body weight in mouse dams. Also zinc deficiency caused a number of external malformations and growth retardation in fetuses. However, the supplement of oyster extract prevented the induction of reproductive and embryonic defects in zinc-deficient mice. There were significantly lower serum concentration of zinc in pregnant dams fed zinc-deficient diet. However, the serum zinc concentration attained the normal level in the oyster extract-supplemented group. No difference in the concentration of serum zinc. was observed between the oyster extract-and zinc carbonate-supplemented groups. From these findings, it is suggested that the oyster extract may be a useful diet which can prevent the reproductive and embryonic defects in the zinc-deficient status.

The uptake and the distribution of radioactive trace elements in Se-deficient rats were examined by the multitracer technique, which can be used to evaluate the behavior of many elements under the same experimental condition. The uptake of Se was larger in the brain, spleen, and testicles of the Se-deficient rats than in those of the normal ones. The uptake of As, Fe, and Sc was larger in the liver of Se-deficient rats than in that of normal ones. In the bone, the uptake of Zr of Se-defici0.ent rats was larger than that of normal ones. Selenium is known to be in a competitive or synergetic relationship with several metals. From the present result on Sc and Zr; it was newly elucidated that there is also some interaction between those elements and Se.

Contents of 12 trace elements (Na, Fe, Al, Zn, Mn, Cu, Ni, Cr, Se, As, Hg, Pb) in 30 black tea infusion samples were measured by inductively coupled plasma spectrometry or inductively coupled plasma mass spectrometry. The 30 tea samples consisted of Indian tea (9 samples), Ceylon tea (5 samples), Chinese tea (6 samples), Taiwan tea (3 samples), African tea (4 samples) and others (3 samples). The content of each element was as follows (mean±SD):Na, 2.19±2.33ppm; Fe, 0.077±0.036ppm; Mn, 3.83±2.31; Zn, 0.249±0.113 ppm; Al, 3.44±2.18ppm; Ni, 0.040±0.020 ppm; Cr, 0.002 ± 0.003 ppm; Cu, 0.042± 0.023 ppm; As, 1.52±2.31ppb; Hg, 0.027±0.021ppb; Se, 0.55±0.51ppb; Pb, 3.38±3.26 ppb. A regional variation was observed in Na and As contents; African tea showed higher Na contents and Chinese tea showed higher As values than other samples. Factorial analysis showed that the 12 elements were classified 3 groups; elements to belong to the same group showed a similar variation in their concentrations in the tea infusion each other. One group consisted of Mn, Zn, Al, Ni, Cr and Cu, and another group consisted of Fe, Se, As, Hg and Pb. Variation of Na was independent of those of other elements.

Cholesterol was heated at various temperature for various periods of time with or without metal salt and/or fatty acid. Cholesterol was degraded depending on the temperature and heating periods of time, but the amounts of cholesterol oxides produced were very little. In addition, the species and the molar ratio of cholesterol oxides produced were almost the same pattern. Then, almost cholesterol might be degraded into much more small molecular substances. Fatty acids, especially polyunsaturated fatty acids, accelerated the degradation of cholesterol. However, large amounts of polyunsaturated fatty acids depressed the oxidative degradation of cholesterol. A small amount of copper salt, CuCl and CuCl₂, acceleated the degradation of cholesterol especially in the presence of fatty acid, but large amounts of copper salts depressed the oxidation. In the case of iron salts, FeCl₂ and FeCl₃, oxidative degradation of cholesterol was accelerated depending on the amounts of iron salts added to the reaction mixture.

Metal-catalyzed oxidative inactivation of AMP deaminase was applied to the structure-activity relationship studies of antioxidants in permeabilized yeast cells. AMP deaminase was readily inactivated by hydrogen peroxide plus reduced copper, and the inactivation may be due to the hydroxyl radical generated through the Fenton reaction at the copper-binding sites of the enzyme. Flavonol and flavone with both 2, 3-double bond and 4-carbonyl group showed a protective effect on the copper-mediated inactivation of AMP deaminase. Baicalein representative of flavone without hydroxyl group at 3-position showed the most potent protective effect on the AMP deaminase. Flavonols, which has hydroxyl group at 3-position, also protected AMP deaminase to a lesser extent, but glycosylation of 3-hydroxyl group of flavonol nullified this protection. Flavanone and flavanol with saturated 2, 3-bond and isoflavone with phenol group at 3-position showed little or no protection of the enzyme.

Protective effect of flavonoids on the oxidative inactivation of AMP deaminase was closely correlated with the inhibition of the formation of thiobarbituric acid-reactive substances as an index of lipid peroxidation. Antioxidant action of flavonoid is mainly depends on the 2, 3-carbon-carbon double bond in conjugation with a 4-oxo group, which participates in the scavenging oxygen radicals and in the chelation of transition metals. This method may be useful for evaluating the antioxidant action of biological materials.

In 1925, Kynurenin has discovered by Matsuoka and Yoshimatsu and was named as Kynurenin by Prof. Yashiro Kotake.

Since then, these metabolites have experimented by Yashiro Kotake, Yahito Kotake, Y azo Kotake, K Ichihara and Osamu Hayaishi et al.

In this report, we display the action of taurine in diabetic rats through Kynureninase activity of rat liver.

And we discuss about the interrelationship between the structure of Kynureninase protein and cystathionine synthase or lyase, and also about the difference aganinst decarboxylase (5-OH Trp decarboxylase, DOPA decarboxylase and His decarboxylase protein related Zn²⁺ ion).

The effects of oyster extract on tumor cells were studied using three cell lines (mice myeloma cell and A31 and SV-T2 cells originated from Balb/c 3T3 cell) and mice inoculated with P388 leukemia. Hot water extract was cytostatic against A31 cell ,and SV-T2 cell, but not against myeloma cell. And the high molecular weight fraction inhibited slightly the proliferation of myeloma cell and SV-T2 cell, but not of A31 cell. The Low molecular weight fraction were cytostatic against these three cells. On mice With P388 leukemia, the high and low molecular weight fractions were more effective in prolonging life than hot water extract.

In this study, we investigated about the effects of the oyster extract on the metabolism of the glucide and lipid in exercised rats. The oyster extract was prepared from Oyster ( Crassostrea gigas).

Wistar male rats of 4 weeks age were divided into 3 groups: (1) the normal diet with exercise (T-group), (2) the normal diet without exercise (C-group), and (3) the oyster extract added diet without exercise (O-group). After 4 weeks under these conditions, each group rats were forced to exercise. After the exercise, we analyzed triglyceride (TG), total cholesterol (TC), blood sugar level, lactate dehydrogenase (LDH), lactic acid, and free fatty acid in plasma, and also glycogen, TG, TC in liver.

The change of blood sugar level, LDH and lactic acid in T-group was less than those of the other groups. And the change of blood sugar level, LDH and lactic acid in O-group was less than that of C-group.

To examine the effect of zinc (Zn) deficiency, we investigate the acceleration of apoptosis in Zn-deficient rat. Six-week-old male Sprague-Dawley (SD) rats were fed on diets Supplemented 0 or 0.02% Zn. After 90 days from administration of the Zn diet, detection of apoptotic cells in skin, thymus and kidney was carried out using TUNEL (TdT-mediated dUTP-biotin nick end labeling) method. When compared to rat on 0.02% Zn diet, the number of TUNEL-positive cells in the thymus and skin was higher in the rat on 0% Zn diet. Furthermore, TUNEL-positive cells were detected in the renal cortex on the 0% Zn diet, however, none were detected in the rat on the O. 02% Zn diet. These findings suggest that Zn deficiency may affect the cell cycle.

Long-Evans Cinnamon (LEC) rats develop spontaneous hepatic injury. We obtained evidence which shows unusual accumulation of copper (Cu) in the livers of LEC rats, followed by the finding of copper-metallothionein (Cu-MT) induction. On the other hand, Ebara et al. have found that copper is accumulated in the human liver suffered from hepatoma. Thus, we measured MT and 4 metals (Cu, Zn, Fe, and Mn) and compared the Cu-MT levels in the livers of LEC rats and human suffered from hepatoma. The levels of MT and Cu in the LEC rat liver remarkably increased with age. The MT levels in human liver were evaluated as logarithmic concentration ratio [log ((concentration in tumor site) / (concentration in juxta-tumorsite))] was decreased with the tumor size, but no changes in the Cu levels were found. Then, we separated the MT in human livers by gel filtration on Superose 12. In tumor site of the human liver, MT was determined as Cu-MT, whereas in juxta-tumor site, the presence of Cu, Zn-MT was suggested. These results indicate that MT is induced as Cu-MT in the early stage of hepatoma, but it decreases in the development of hepatoma. Thus, we conclude that the development of hepatoma in LEC rats and human might be understood with respect of the induction of Cu-MT but both rates for the formation and degradation of MT are different between LEC rat and human hepatoma.

Biological functions of vanadium have been investigated by many researchers. Especially, the insulin-like effect of vanadium is the most remarkable. Recently, vanadyl ion (+4 oxidation state of vanadium) and its complexes have been shown to normalize the blood glucose levels of streptozotocin-induced diabetic rats (STZ-rats). We have examined to find more effective vanadyl complexes with insulin-mimetic activity and prepared less toxic complexes with several types of coordination mode. Among them, vanadyl-methylpicolinate complex (VO-MPA) was found to exhibit higher insulin- mimetic activities and less toxic than other complexes as evaluated by both in vitro and in vivo experiments. Electron spin resonance (ESR) is available to measure the paramagnetic compounds in biological samples. We have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze the ESR signals due to paramagnetic metal ions and stable radicals in real time. In the present pharmacokinetic investigation to elucidate the relationship between the blood glucose normalizing effect and global disposition of vanadium, we report first the experiments, in which the BCM-ESR method was applied to evaluate the disposition of vanadyl states in circulating blood after intravenous administration to male Wistar rats. ESR spectra due to vanadyl state were obtained in circulating blood, and pharmacokinetic parameters for them were estimated by using the compartment model. The results of the experiments indicated that vanadium considerably distributed to peripheral tissues and eliminated from the body through urine, and the accumulation of vanadium in the tissue was enhanced by VO-MPA treatment. The long-acting property of VO-MPA was suggested by the incorporation and accumulation of vanadium in the bone. We conclude that the tissue distribution of vanadium is closely related with the antidiabetic activity of the vanadyl complex.

In this study, we examined the in vitro cytotoxic effects of an organic arsenic compound containing in seafood, trimethyl (2-hydroxyethyl) -arsonium cation, namely arsenocholine (AsCho), on murine splenocytes, thymocytes and bone marrow (BM) cells comparing with those of an inorganic arsenical, sodium arsenite using synthesized pure material. Sodium arsenite showed strong cytotoxicity on all of these cells, and it’s IC₅₀ was 5-6 μM. In contrast, AsCho had no cytotoxicity on these cells even at concentrations over 10 mM, and it slightly enhanced the viability of BM cells at doses over 100 μM. It is interesting that this unique biological effect was found in AsCho, an organic arsenic compound containing in some marine animals which are daily ingested as seafood in many countries.

Eleven primiparous and ten multiparous Holstein Friesian cows and their calves were studied to evaluate vitamin E status of prepartum and postpartum period. Plasma α-tocopherol concentrations of multiparous cows were higher than those of primiparous cows. Multiparous cows had larger decrease and increase in plasma α-tocopherol and total lipid concentrations round about the time of parturition than primiparous cows. Alpha-tocopherol concentrations in colostrum of multiparous cows were twice high as those of primiparous cows. Plasma α-tocopherol concentrations of calves from multiparous cows were higher than those of primiparous cows on 5 days of the age. Plasma α-tocopherol concentrations of calves started to decrease on 5 or 15 days of the age and showed 23 μg/ dl on 90 days of the age.

The results of this study indicated that higher plasma α-tocopherol levels of multiparous cows than primiparous cows afford their calves higher plasma α-tocopherol levels immediately after birth because of higherα -tocopherol concentrations in the colostrum. Plasma α-tocopherol concentrations of calves decrease as the calves become dependent upon calf starter with low α-tocopherol concentration.

For the use of Kampa medicines in treating anemia during pregnancy, a total of 90 patients participated in this investigation. The patients were divided into Groups A, B, and C. Group A had 38 patients taking Toki-shakuyaku-san and Kojin, which contained relatively high Cu, Zn, and Ca for a moderate constitution, Group B had 17 patients taking Ryulan-shakan-to, which contained relatively high Cu, Fe and Mg concentrations for a robust constitution, and Group C had 35 patients taking kami-kihi-to, which contained relatively low minerals for a deficient constitution.

For the Hb increases in Group A following medications the increases in Group A were the most, and the Group B were the next, with a relatively lesser for the Group C.

Kampo medicines, which were effective for the treatment of anemia during pregnancy, were found to contain relatively high Cu, Zn, Fe and Ca.

Anemia and food intake were examined on 14 female professional volleyball players (22.1±2.9 years) and 20 female health nursing school students (23.0±2.1 years). Daily total energy intake of the players was estimated to be more than 3600kcal. Because of no change of body weight for the last 1 year, this high energy intake would equilibrate their energy consumption. Intake of all other nutrients were remarkably higher in the players than the student. Adjusted value of protein intake of the players was comparable to that of the students and exceeded the recommended dietary allowance. Higher value of adjusted iron intake was observed in the players than the students. Compared to the students, the players showed higher serum LDH and CPK activities and lower Hb and serum iron/total iron binding capacity ratio. Serum haptoglobin was significantly lower in the players than the students, it suggested the hemolysis was caused by exercise. These results suggest that strenuous exercise such as professional volleyball play can result in a decrease of Hb and iron status.

N-(9-Acridinyl) maleimide (NAM) fluorometry was applied to low molecular thiol (SH) and disulfide (SS) groups in the pacific saury (Coloalabis saira) and the oyster (Crassostrea gigas) . The low molecular SH was measured in buffer (pH 8.80) at room temperature after standing for one hour. The method was preliminary applied to check the change of low molecular SH and low molecular SS in the pacific saury and the oyster during the preservation at 4°C and the storage refrigeration.