During the last 10 years, we showed that prostaglandins (PG) Dz and E2 are the major sleep regulating substances in the brain, each promoting sleep and wakefulness. Recently we discovered that various inorganic quadrivalent selenium compounds are potent, specific and .non-competitive inhibitors of brain prostaglandin D synthase. Ki is about 10μM. Hexavalent compounds and organic selenium compounds are not inhibitory. Other enzymes in the arachidonate cascade system are not inhibited. This inhibition is reversible and can be reversed by sulfhydryl compounds such as glutathione or DTT. When one of these inorganic quadrivalent selenium compounds was infused into the third ventricle of a freely moving rat, sleep was inhibited time-and dose-dependently and reversibly, indicating that PGD synthase is the key enzyme in sleep regulation and that PGD₂ is the endogenous sleep-regulating substance in the rat under physiological conditions.

Because the sleep pattern of rats is somewhat different from that of primates, we further extended these studies to the Macaca mulatta, that is, the Rhesusmonkey. More recently, we found that intraventricular infusion of prostaglandin E₂ reduces the amount of diurnal sleep of rats, or increases the amount of wakefulness.

There are many reports that oligopeptides effect on the lipaase activity or fatty acid synthesis. In this report ,we studied about the influence of oligopeptides on the lipid metabolism and the total body fat by the continuous administration of oligopeptide to 21 lipid pathobolism patients and 21 healthy controls. Tablets of oligopeptide were administered orally to each group for 3 months (200mg oligopetide par 1 tablet, 10 tablets par day) . Blood test, measurement of body weight and total body fat were carried out once a month. The body weight and total body fat decreased in lipid pathobolism patients and healthy control subjects. We conclude that the intake of oligopeptide might be available for the lipid metabolim.

A zinc deficiency induces a severe immunodeficiency and alopecia. The mechanism of zinc deficiency-induced immunodeficiency was examined by analyzing, the thymus atrophy and the changes in the membrane surface antigens of T cells of zinc-deficient SPF Wistar-derived rats. Zinc deficiency induced severe and reversible thymus atrophy. The zinc-deficient thymocytes revealed significant increases in the percentage of CD4 antigen cells (rat T helper cells and macrophages) , CDS antigen cells (rat T Suppressor / cytotoxic cells) and CD4 & CD8 double-negative cells, and decreases in the percentage of CD4 & CD8 double-positive cells (undifferentiated cells) and Thy 1, 1 antigen cells (total T cells) . In addition, significant decreases in the percentage of The 1, 1 antigen cells, slight decreases in the percentage of α / β (T cell receptor) antigen cells and CD2 antigen cells (E rosette forming cells), and a significant increase in the percentage of CD4 & CD8 double-positive cells were observed in the peripheral T cells of the zinc-deficient rats. Furthermore, thymulin activity decreased significantly in the serum of zinc-deficient rats. These findings indicate that the immunodeficiency induced by zinc deficiency is manifested as T cell dysfunction which is caused by not only quantitative changes in the T cells such as thymus atrophy, but also qualitative changes in the T cells such as changes of their membrane surface antigens, that is, impairment of the process of differentiation and maturation of T cells in the thymus. The mechanism of zinc deficiency-induced alopecia was examined by analyzing the movement of trace elements in the hair and fatty acids in the skin of zinc-deficient SPF Wistar-derived rats. Zinc deficiency induced severe and reversible alopecia. With the progress of falling-out of the hair (or with the decrease in the concentration of zinc in the hair), a marked tendency for accumulation of manganese and calcium into the hair was seen, and a decrease in the concentration of linoleic acid and a increase in the concentration of palmitic acid in the skin were seen. The relations of manganese and unsaturated fatty acids such as linoleic acids to alopecia are now under investigation.

There are three separate pools of brain zinc, i. e., free zinc, vesicular zinc, and protein-bound zinc. Each of them has physiological important roles. We suspected that behavioral disoders were induced by some change of brain zinc concentration during zinc deficiency. Twenty-seven male wister-strain rats (four weeks age) were divided into three groups: zinc deficient (ZD), pair fed (PF), and ad libtum (AD). Zinc concentrations in diets were 1mg/kg (ZD) and 30mg/kg (PF, AD) . After three weeks of dietary period, the hippocampus, the cerebellum and the striatum were collected, and whole zinc concentrations in these tissues were measured by atomic absorption spectrophotometer.

Moreover, homegenized hippocampus was devided two fractions ( >5kDa and < 5kDa) by the dialitic filter, and zinc concentrations in these filtrate (<5kDa)ware measured in the same way. Zinc deficiency remarkably decreased the body weight gain and feed intake. In addision, plasma zinc concentration was lowered in the zinc deficient rats. Though such obvious conditions occured in the zinc deficient animals, zinc concentration in brains and ultrafiltable zinc in the hippocampus were not changed.

We examined zinc status in relation to thyroid hormone in disabled persons. Out of 134 patients, thirteen patients revealed low levels of serum free T₃ and T₃, elevated levels of serum reverse T₃ and elevated reaction of TSH after TRH injection. The subjects also showed increase in urinary zinc excretion, moderately zinc deficiency evaluated by total body zinc clearance and moderate to severe brain atrophy estimated by computed tomographic scan. After supplementation of zinc sulphate 5-10 mg per kg body weight to 13 disabled patients for 12 months, it was shown th.at levels of serum free T₃ and T₃ increased accompanied by decrease in serum reverse T₃ and TSH after TRH injection.

Serum concentration of selenium did not change in patients during zinc supplementation. The findings of the present study suggest that zinc may be related to the active mechanism of Type II deiodinase which appears to carry out only 5’-deiodination.

To clarify the mechanism of oxidative stress in skeletal muscle atrophied by immobilization, we measured the activities of antioxidant enzymes and xanthine oxidase (XOD) in a typical slow red muscle, the soleus. Male Wistar rats (14 wk old ) whose one ankle joints were immobilized in the fully extended position were killed after 4, 8, and 12 days. The activities of Mn-containing superoxide dismutase (Mn-SOD), Cu, Zn-containing superoxide dismutase (Cu, Zn-SOD), Se-dependent glutathione peroxidase (Se-GSHPx), glutathione S-transferase, catalase, glutathione reductase were measured spectrophotometrically. The XOD activity and the concentrations of hypoxanthine, xanthine, and urate were measured using a high performance liquid chromatography. Increased Cu, Zn-SOD and decreased Mn-SOD in atrophy might reflect increased generation of superoxide anions in the cytoplasm rather than in the mitochondria. The source of superoxide anions in the cytoplasm might be the increased superoxide-producing XOD. Enhanced generation of superoxide anions and increased Cu, Zn-SOD activity in atrophy suggested the enhanced generation of hydrogenvperoxide in the cytoplasm. Owing to the unchanged activity of Se-GSHPx and the unchanged or slightly increased activity of catalase in atrophy, the ability to degrade hydrogen peroxide might not increase so much. Hence, hydrogen peroxide is expected to be increased in atrophy. Because there is also an increase of iron in the microsome of atrophied muscles, the production of hydroxyl radicals, the most aggeressive of radicals, might consequently be elevated.

We have been reported that the effects on neuroreceptor and metabolic analysis of vanadate for the correlation between neurological disorder and vanadium. This report is of biodistribution studies on the location of ascorbic acid in these animals. A solution of sodium ortovanadate (Na₃ VO₄, 100ppm) was administered into Wistar rats as a drinking water for 10 months. In serum, liver, spleen, kidney and adrenal, the concentration of ascorbic acid were unchanged. The level of ascorbic acid was decreased in brain only with longterm drinking of vanadate. A reduction of ascorbic acid in each part of brain was different.

As vanadium has a wide broad of redox potential, which depend on oxidation states and differ from the potentials of iron or copper ions, various physiological roles have been expected. For understanding the physiological and pharmacological roles of vanadium compounds, both distributions of vanadium, determined by neutron activation analysis (NAA) method and kinetics of vanadium, estimated by blood cir- culation monitoring electron spin resonance (BCM-ESR) method were examined. When rats were treated with vanadyl sulfate (+4), vanadium was well incorporated in both normal and STZ-diabetic rats, where the significant differences were not observed. In subcellular fractions of the liver of rats treated with vanadyl sulfate (+4)or sodium vanadate (+5),vanadium concentrations of nuclear fractions de- creased and those of mitochondria and microsomal fractions increased as the administered vanadium was elevated. Vanadium concentrations in nuclear fractions were higher in the rats treated with vanadyl sul- fate than those with sodium vanadate. When rats were given vanadyl sulfate by i. v. injections, ESR spec- tra with a hyperfine structure of eight line signals due to vanadyl ions appeared immediately after the in- jection and decreased slowly. This BCM-ESR method can be applied for the real time analysis of not only blood vanadium concentrations but metal ions in the paramagnetic states.

The present study was aimed to examine the effects of dietary deficiency of selenium (Se) and/ or diet- ary excess of iron (Fe) on the iron levels in serum and various tissues of rats. The animals were fed either of following diets : Se-deficient/Fe-adequate (0. 0035% Fe as ferric citrate) , Se-adequate (0.1ppm Se as sodium selenite) /Fe adequate, Se-deficient/Fe-excess (0.25% carbonyl Fe) and, Se-adequate/Fe-excess. Rats were killed after 30 weeks of feeding period and the levels of Fe and Se in serum, liver, kidney, spleen, heart and lung were determined by an inductively coupled argon emission spectrophotometer. Hematological parameters were obtained by usual procedures. Within Fe-adequate groups the Fe levels were significantly higher in serum and most of analyzed tissues of Se-deficient rats than those of Se-adequate rats. The conclusion derived is that excess dietary Fe did not augment the conditions produced by Se deficiency. These results confirm the previously held notion that Se deficiency increases Fe levels in serum and other tissues because of Fe liberated by promoted hemolysis.

To assess the selenium (Se) status of Japanese, ten 5-day balance study for Se were carried out in 60 healthy female subjects aged 20-22 years. Experimental diets were composed of ordinary foods and were typical of Japanese usual dietary habits. The same menu was used in each experiment during the 7 experimental days. Each experimental diet was characterized by protein source in the diets. Bread in the diets in Exp. 1-9 was substituted for rice in the diet in Exp. 10. The overall average with SD of dietary Se intake was 89±25μg/ day, ranged from 4 to 141 μ g/ day. Apparent absorption, expressed as ratio of fecal excretion to intake, was 74±11% . Se balances showed positive except Exp. 10. Se retention was 18 ±23μ g/ day. Se intake was significantly and positively correlated with urinary excretion (r=0.376), urinary and fecal excretion (r=0.556) , and Se balance (r=0.834). Regression of Se balance on dietary Se intake indicated that these subjects needed 52μg of dietary Se per day to stay in babance. When balance and intake were adjusted for body weight, they needed 1μg of selenium per kg of body weight to maintain balance.

Serum samples were collected from 167 Japanese rural women (aged 35-81 years) in 1990 and from the same subjects in 1992. Atherogenic index [=(total cholesterol - HDL-cholesterol) /HDL-cholesterol] was a significant increasing factor of the serum selenium (Se) concentration in 1990 for women aged above 60 years by multiple regression analyses, which was consistent with our preliminary findings for coastal inhabitants of Fukui Pref. in 1986. Furthermore, the change in atherogenic cholesterol concentrations (total cholesterol - HDL-cholesterol) between the sampling years was a significant increasing factor of the change in serum Se concentrations for all subjects. Thus, our results suggest that serum Se concentration can increase as atherogenic cholesterol concentration increases for Japanese women and such a positive relationship may reflect some role of serum selenium in protecting atherogenic lipoproteins from oxidation.

Effect of administration with 6-propyl-2-thiouracil (PTU) to selenium (Se)-deficient or-adequate rats on the urinary ketone body excretion was studied. Rats (50 to 60g) were fed on Torula yeast-based Se deficient diet (Se content, <0.01μg/g) or the diet s叩 plementedwith sodium selenite (0.1μg Se/ g) . The rats were given 0.05% PTU solution or deionized water as a drinking water. After the feeding for 6 weeks, the PTU-loading made a severe inhibition in the growth. The rats fed the Se-deficient diet showed remarkably low hepatic Se contents and glutathione peroxidase activities compared to the Se-adequate rats irrespective of the PTU-loading. Serum thyroid hormone levels were remarkably de- creased in the PTU-received rats. In the rats without PTU, the Se deficiency made significantly higher serum T4 and lower T3 levels. Urinary ketone body excretion during the fasting was significantly higher in the Se-deficient rats than in the adequate rats irrespective of serun thyroid hormone levels. These re- sults indicate that the increase of urinary ketone body excretion in the Se-deficient rats is independent to the seurm thyroid hormone levels.

Studies were perfeormed to clarify the effects of methionine and/ or selenium deficiency and different forms of selenium (sodium selenate and selenomethionine) on selenium metabolism and the enzymatic activities of type I iodothyronine 5’deiodinase and glutathione peroxidase, Fourty-two weanling male Wistar rats were divided into six groups and pairing fed the respective diets for 4 wk. The results showed that methionine-deficiency interfered with selenate absorption which induced lower selenium con- centration, type I 5’ deiodinase and glutathione peroxidase activities in liver whereas did not affect selenomethionine absorption. Glutathione of liver was decreased in methionine-deficiency which may account for the lower absorption of selenate in methionine-deficiency. Selenium concentrations and activities of type I 5’ deiodinase and glutathione peroxidase were decreased in selenium-deficiency. It is suggested that methionine-deficiency diminishes selenoenzymatic activities not only glutathione peroxidase but type I 5’deiodinase which may be due to the bad absorption or availability of seleuium.

Selenium analogue of glutathione (glutaselenone) catalyzes a glutathione peroxidase-like reaction. We found that glutaselenone and glutathione form a selenosulfide conjugate intermediate in the presence of hydrogen peroxide. We synthesized glutaselenone diselenide and seleninic acid : they were converted to the selenosulfide in the presence of glutathione and hydrogen peroxide. Therefore, it appeared that the selenosulfide is essentially the catalyst in reduction of peroxide and oxidation of glutathione.

Glutathione peroxidase-like activity is usually determined by the glutathione reductase method (GR-method). Four diastereomers of glutaselenone were different in catalytic activity from one another, when the activity was determined by GR-method. However, their activities were essencially the same when determined by the o-phthalaldehyde method (OPA-method) . GR acts on glutaselenone diselenide and selenosulufide, and the discrepancy can be attributed to the action of GR among these selenium con- teining peptides.

The calcium-binding protein (CaBP) has been purified from rat kidney.

The purified CaBP was about 28000 molecular weight and was detected in kidney and brain. The tissue distribution and the content of CaBP were determined in normal and vitamin B₆ (B₆) deficient rats. In rat kidney, CaBP was present at the distal tubules and located in nuclei and cytoplasm. There was no difference in CaBP distribution between two groups. However, the content of CaBP in B₆ deficient rats was significantly lower than that in normal rats. The content of serum calcium (Ca) in Ca deficient rats was significantly lower than that in Ca administrated rats. On the other hand, the content of renal CaBP in the former was significantly higher than that in the latter. Thus the content of renal CaBP was inversely proportional to the conetent of serum Ca.

We inferred that the decrease of the content of the renal CaBP was at least one factor in the formation of renal calculi observed in B₆ deficient rats.

The relationship between the physical performance and the vertebral bone mineral density was studied in 138 (70 boys and 68 girls) normal children aged six to thirteen. The physical performance evaluated by the Sports activity and Bone mineral density of the lumbar vertebrae (L2-4BMD) was determined in the anteroposterior direction by the dual energy X-ray absorptiometry method (DEXA, QDR-1000, Hologic) The reported incidence of fracture varies ; in general, it is highest among 12-year-old children, followed by 11-year-old children. Bone mineral density of the lumber vertebrae increased with height, weight, and age. The grip strength correlated significantly with the vertebral BMD. In the age of 11 and 12 years, vertebral BMD was significant higher in girls than in boys (p<0.05). The deleterious effects of immobilization and inactivity on bone are wellknown. Our data suggest that a positive relationship between physical activity and bone mineral density in lumbar vertebrae.

Effect of casei叩 hosphopeptide (CPP) on mineral metabolism was investigated in rats.Animals were fed a control diet (CPP 0%) or CPP diets (CPP 0.1, 0.3, 0.5%). All diets contained the same amounts of calcium (0.4% ) , phosphorus (0.8% ) and zinc (10.5mg/kg). Though an increase in the calcium retention was not observed, the deposition of calcium in a femur was greater in the CPP diet groups than those in the control diet group. Apparent absorption and retention of zinc were greater in two of the CPP diet groups (0.3, 0.5%). Apparent absorption and retention of iron were greater in the 0.5% CPP diet group. Apparent absorption and retention of copper were greater in the all CPP diet groups. These results suggest that CPP supplementation may have a significant effect on the absorption of trace elements.

We administered copper and other chelating or reducing compounds directly into the air sacs of 14-day-old fertile eggs and after 5 days of incubation we examined embryonic growth, mortality and hepatic minerals of chick embryos. Adminstration of EDTA-2Na with copper (II) stimulated copper absorption and hepatic copper accumulation while that of tetrathiomolybdate with copper completely suppressed them. Although other agents such as deferoxamine mesylate, bathophenanthroline sulfonic acid, L-ascorbic acid and D-penicillamine conld alleviate the copper toxicity to some extent, they were not so effective as EDTA or thiomolybdate in either stimulatory or suppressive ways for the hepatic copper accumulation. Examination of the hepatic minerals (Cu, Zn, fe) revealed that the treatment group which had a higher death rate from excess copper absorption showed significantly higer tissue iron deposition probably due to the occurrence of hemolysis. These results suggest that excess copper absorption increases tissue iron deposition and that both free copper and free iron contribute to produce harmful radical species for membrane lipid peroxidation.

To carify the effect of aging on mineral status in female mice, mineral concentrations in tissues were determined. Five female mice ( 2, 6 , 10 months old) were fed on commercial diet. Iron, zinc, copper, calcium and magnesium concentrations in blood, liver, kidney, heart, brain, lung and spleen of these mice were determined by a flame atomic absorption spectrophotometer. Iron concentrations in liver, kidney, heart, brain and spleen were increased with age. Zinc concentrations in heart and lung were decreased with age. It is noteworthy that the copper concentration in brain of 10 month old mice was markedly higher as compared with younger mice. Calcium concentration in kidney of 10 month old mice was higher than 2 and 6 months old.

The effect of tin deficiency on the growth, iron and copper concentrations in tissues, and lipid parameters of plasma was studied. Tin, iron and copper cocentrations in tissues, and triglycerides, total cholesterol and total phospholipids of plasma were measured in rats fed a tin-adequate diet (2 mg tin/kg diet) or a tin-deficient diet (0.02 mg diet) Dietary tin deficiency decreased body weight gain and food efficiency. When rats fed a tin-deficient diet were compared with rats fed a tin-adequate diet, iron concentrations were highr in spleen and kidney, but lower in muscle ; copper concentrations were lower in heart and tibia ; triglycerides in plasma were higher, but total cholesterol and total phopholipids in plasma were lower. These results suggest that tin deficiency evokes ineffective utilization of iron in muscle, decreased copper retention and abnormalities in plasma lipid parameters.

A high sensitive high-performance liquid chromatographic method for determination of pmol levels glutathione (GSH) and glutathione disulfide (GSSG) in biological samples is described. GSH and GSSG were separated isocratically on a reversed-phase column (ODS column). The mobile phase consisted of 50mM sodium phosphate buffer (pH 3.0). After chromatographic separation, GSH and GSSG were converted to fluorescent derivatives by post-label reaction with o-phthalaldehyde under the alkaline condition. A 0.5pmol quantity of GSH and GSSG can be determined and the analytical recoveries were found to be almost 100%. The procedure is rapid, completed within 15 min, and it has. been successfully applied to various biological samples.

cDNA clones, rCDO-39 and hCDO-1 , were isolated and characterized. rCDO-39, encoding the rat liver CDO contained a 1,458 b insert. hCDO-1, encoding the human liver CDO contained a 1,516 b insert. Both cDNAs encoded the polypeptides of 200 amino acids. Sequence comparison revealed a 91% homology in the amino acids between rat and human CDO. A potential iron binding domain was observed in the amino acid sequences of rat and human CDO. Significant expression of CDO gene was observed in liver, kidney, lung and brain but not in heart and skeletal muscle.

Recently, farmers are likely to feed low vitamin A diets to beef cattle in Japan, because of the improvement of carcass quality. However, the relationship between vitamin A status and fat accumulation in animal is unknown. This study was conducted to elucidate the effect of vitamin A deficiency on fat metabolism in rats.

Forty-nine female rats of Wistar strain ( 4 weeks of age) were utilized in this study. They were fed vitamin A deficient diet during 40 weeks after the feeding of vitamin A adequate diet up to 10 weeks of age. They were sacrificed at 30, 42 and 50 weeks of age in the fed state. Blood serum, liver and abdominal fat were collected for the determinaton of lipid levels and fatty acid composition.

Rats fed vitamin A deficient diet exhibited an increase in serum triacylglycerol (TG) at 30 weeks of age, whereas further feeding of deficient diet reduced serum and hepatic TG levels. Serum total cholesterol, free fatty acid (FFA) and glucose levels were found to be reduced during the experimental period, although not so severely as serum TG level. In contrast to the findings in serum and liver, lipid characteristics of abdominal fat in rats given vitamin A deficient diet were not different from control with the exception of FFA content at 50 weeks of age. These results suggest that TG rich lipoprotein secretion from liver is depressed when hepatic vitamin A store is completely exhausted in the dificient animals.

The effects of acute ethanol on rat brain gangliosides were investigated. 3g/kg of ethanol was administered intraperitoneally and the total amounts of N-acetylneuraminic acid (NANA) decreased only in serum, but not in brain regions at two hours after injecton. Time course of amounts of total and gangliosidic NANA were determined, and the decrease of total NANA were only at 1 hr in cerebral cortex and returned to basal lebel at 4 hours. The amounts of gangliosidic NANA did not alter significantly. We investigated cleavage of NANA by using ³H labeled precursor of it, N-acetylmannosamine,-[N-mannosamine6H (N)]-. 48 hours after microinjection of precursor, dialysates were collected by microdialysis technique and were separated by HPLC system, and the radioactivity of NANA fraction were measured by liquid scintillation counter. Acute ethanol increased the liberation of NANA radioactivities by neuraminidase in striatum. Therefore, we speculated that the effect of acute ethanol is a conformational change of gangliosides or their surrounding areas.

We reported that acute and chronic caffeine administration affected neurotransmitters in rat brain striatum.

We have measured extracellular acetvlcholine (ACh) to investigate acute effect of caffeine in rat hippocampus and chronic effect of caffeine by changing chronic period in rat striatum. The concetration of ACh in the rat hippocampus was increased by the acute administration of caffeine (50 or 100mg/kg ; i. p.) whereas chronic administration of caffeine (for a period of 1, 3, 5, or 7 days in a dose of 50 or 100mg/kg ; i. p.) decreased the concentration of ACh. The similar effect was observed to the previous study.

We have synthesized ¹¹C-labeled caffeines and examined the detail brain distribution of caffeine. Although the ¹¹C-labeled caffeines distributed in the whole brain, considerably higher accumulation in the thalamus and hypothalamus was observed. The differennce in the distribution of ¹¹C-labeled caffeines was due to their higher specific activity than commerially available caffeines which give a uniform distribution.

These results suggest that the acute and chronic administrations of caffeine cause contrast, effect on in vivo extracellular ACh concentration.