To acquire the data concerning the tolerable upper intake level which prevents health problems from an excessive intake of pantothenic acid, an animal experiment was done. Rats of the Wistar strain (male, 3 wk old) were fed on a diet which contains 0%, 0.0016% (control group), 1%, or 3% calcium pantothenate for 29 d. The amount of weight increase, the food intake, and the organ weights were measured, as well as the pantothenic acid contents in urine, the liver and blood. Moreover, to learn the influence of excessive pantothenic acid on other water-soluble vitamin metabolism, thiamin, riboflavin, a vitamin B6 catabolite, the niacin catabolites, and ascorbic acid in urine were measured. As for the 3% addition group, enlargement of the testis, diarrhea, and hair damage were observed, and the amount of weight increase and the food intake were less than those of the control group. However, abnormality was not seen in the 1% addition group. The amount of pantothenic acid in urine, the liver, and blood showed a high correlation with intake level of pantothenic acid. It was only for 4-pyridoxic acid, a vitamin B6 catabolite, in urine that a remarkable difference was observed against the control group. Moreover, the (2-Py+4-Py)/MNA excretion ratio for these metabolites of the nicotinamide also indicated a low value in the 3% pantothenic acid group. As for the calcium pantothenate, it was found that the 3 % level in the diet was the lowest-observed-adverse-effect-level (LOAEL) and the 1% level was the noobserved-adverse-effect-level (NOAEL).

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α-Tocopheryl succinate (TS), which is known to induce apoptosis selectively in cancer cells, has attracted attention as a chemotherapeutic agent. Recently, we found that α-tocopheryl malonate (TM) and α-tocopheryl oxalate (TO), among the α-tocopheryl esters tested, have high apoptogenic activity as well as TS. In this study, we investigated the characteristics of their cytotoxicity on normal cells and cancer cells in vitro, and their anticancer effects on mice inoculated with melanoma B16-F1 cells in vivo. The order of in vitro cytotoxicity was TO≥TM>>TS in all cell lines examined. Addition of exogenous superoxide dismutase (SOD) and the antioxidant N-acetyl cysteine (NAC) inhibited TS and TM but not TO-induced cell deaths. A selective cytotoxic effect on cancer cells was observed with TS but not with TM or TO.c-Jun N-terminal kinase (JNK) inhibitor II prevented cell death induced by TS but did not prevent cell deaths induced either by TM or TO. Intravenous administration of vesiculated TS and TM to mice inoculated with melanoma B l 6-Fl cells prevented tumor growth and enhanced the mean survival time, but TO administration killed the mice due to its acute high toxicity. From these results, we discussed the characteristics of their selective cytotoxicity toward tumor cells in vitro and anti-cancer effects in vivo.

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It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H₂O₂. This H₂O₂-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with Lascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity, had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added, it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.

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The Ussing chamber is often used for in vitro investigations into the transport physiology of epithelia including the small intestine. However, the morphological and functional integrity of the small intestine incubated in an Ussing chamber has been largely ignored. The present study attempted to compare the mucosal injury among different segments of the mouse small intestine when incubated in an Ussing chamber. To assess the functional damage, we measured changes in the transmural potential difference (PD) evoked by adding glucose to the mucosal solution and forskolin to the mucosal and serosal solutions. Morphological deterioration was assessed histologically. The villi in the duodenum and proximal jejunum were morphologically damaged by 2h of incubation in an Ussing chamber and almost completely destroyed within 4h, while crypts remained intact. The villi were moderately damaged in the distal jejunum. In contrast, the integrity of the villi and crypts was maintained in the ileum for 4h. The basal PD and forskolin-induced PD were maintained up to 4h of incubation in all segments. On the other hand, the glucoseinduced PD was not apparent in the duodenum at Oh, and was gradually suppressed to zero in the proximal jejunum by 4h, although the glucose-induced PD was maintained for 4h in the ileum. The loss of villous epithelial integrity was correlated with the disappearance of the glucose-induced PD, while both the basal and forskolin-induced PD were retained, even in the tissue with disrupted villi. It is concluded that the mucosa of the proximal small intestine was rapidly injured in the Ussing chamber, particularly in the villi, while the integrity of the mucosa of the distal small intestine was maintained for 4 h. The glucose-induced PD, but not the basal or forskolin-induced PD, can be used as a marker of the villous integrity.

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The effects of amla on low-density lipoprotein (LDL) oxidation and cholesterol levels were investigated in vitro and in vivo using C²⁺-induced LDL oxidation and choles terol-fed rats. SunAmla and ethyl acetate (EtOAc) extract of amla significantly inhibited thiobarbituric acid (TBA)-reactive substance level in the Cu²⁺-induced LDL oxidation and the effects were stronger than those of probucol. In addition, the administration of SunAmla (at a dose of 20 or 40mg/kg body weight/d) or EtOAc extract of amla (at a dose of 10 or 20mg/kg body weight/d) for 20 d to rats fed 1% cholesterol diet significantly reduced total, free and LDL-cholesterol levels in a dose-dependent manner, and EtOAc extract of amla exhibited more potent serum cholesterol-lowering effect than SunAmla in the same amount. Furthermore, the oxidized LDL level in serum was markedly elevated in choles terol-fed control rats as compared with normal rats, while it was significantly decreased by the administration of SunAmla or EtOAc extract of amla, Moreover, the serum TBA-reactive substance level was also significantly decreased after oral administration of SunAmla or EtOAc extract of amla. These results suggest that amla may be effective for hypercholester olemia and prevention of atherosclerosis.

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Eicosapentaenoic acid (EPA) has been shown to exert anti-inflammatory actions. To evaluate the effects of EPA on chronic hepatitis C, we administered EPA ethyl ester capsules to patients receiving the combination therapy of interferon αa-2b and ribavirin. EPA (1, 800mg/d) was supplemented in combination with vitamin E (300mg/d) and C (600mg/d) to 5 chronic hepatitis C patients (EPA group). Five patients were administered vitamin E and C but not EPA (control group). Blood samples were obtained before and after 4, 8, 12 and 24 wk of therapy and analyzed for fatty acid compositions of erythrocyte and plasma and serum 8-hydroxy-2'-deoxyguanosine. EPA in erythrocyte membrane rose to 3 fold the basal level in the EPA group, while it decreased significantly in the control group after 24 wk of therapy. Lymphocyte counts in the EPA group increased to 120.8±25.4% after 4 wk of therapy and maintained the basal level throughout therapy, whereas the counts decreased significantly in controls. The serum alanine aminotransferase level was improved significantly in the EPA group. Changes in lymphocyte counts following 24 wk of therapy correlated with the EPA level in erythrocyte. The serum 8-hydroxy-2'-deoxyguanosine level at 24 wk in the EPA group was significantly lower than that in controls. These observations may suggest the beneficial effect of EPA supplementation in the treatment of chronic hepatitis C patients.

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We have previously shown, through evidence that the expression of calbindinD9k protein shows a dose-dependent change in the intestine, that calbindin-D9k plays a role in the ability of a fructooligosaccharide (FOS) diet to increase calcium absorption. This study shows that the regulation of calbindin-D9k expression occurs at the transcriptional level in a segment-specific manner, decreasing in the proximal intestine and increasing in the colorectal segment. To determine the transcriptional regulation of the FOS diet on calbindinD9k expression, two transcription factors, vitamin D receptor (VDR) and cdx-2, were analyzed during 1 0 d feeding of the FOS diet. The mRNA expression of VDR and cdx-2 was influenced by the FOS diet and showed a segment-specific change. In the proximal small intestine, there was a significant correlation between the changes in both mRNAs (r=0.69, p<0.01), while the expression of calbindin-D9k correlated neither with VDR nor with cdx2. This means that the transcriptional change induced by the FOS diet was not regulated by VDR and cdx-2. In the colorectal segment, there were significant correlations between gene expressions of calbindin-D9k vs. VDR, r=0.73, p<0.01 and calbindin-D9k vs. cdx-2, r=0.52, p<0.05. These results suggested that both transcription factors, VDR and cdx-2, were involved in the regulation of calbindin-D9k gene expression in the colorectal segment during the process through which the FOS diet enhanced calcium absorption.

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This study was undertaken to examine the relationships between zinc parameters (serum alkaline phosphatase activity, and zinc concentration in serum and femur) and either body weight gain, protein intake or survival time in zinc deficient rats. Demineralized soy protein (DP) and alkali-treated soy protein (AP) were used as dietary protein sources in order to make zinc deficient diets (<0.5mg zinc/kg diet). The sane diets (DP and AP diets) supplemented with lysine or cystine were also given freely to rats. Moreover, in other experiments, the above relationships were examined. During the early stages, a significant negative correlation between body weight gain and survival time in zinc-deficient rats was demonstrated. The significant negative correlation coefficient between protein intake and survival time increased gradually, and the maximum value of this correlation coefficient was higher than that between body weight gain and survival time. The significant correlations between protein intake and femur zinc concentration were recognized throughout the longer periods. These results show that the number of zinc-deficient rats which survive decreases as protein intake increases throughout the feeding period, that the influence of protein intake is greater than that of body weight in early stages, and that the zinc requirements of rats rise with the increase in protein intake.

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We investigated the effect of short-term feeding of conjugated linoleic acid (CLA) on adipose tissue weights, liver weight, hepatic lipid metabolism, and serum lipoprotein profiles in C57BL/6J mice. Mice were fed semi-synthetic diets containing either 6% high-linoleic safflower oil (HL-SAF) or 4% HL-SAF+2% CLA for 1 wk. Short-term feeding of CLA showed an anti-obesity effect without inducing hepatomegaly in mice. In addition to the decline of hepatic triglyceride concentration, significant inhibition of Δ9 desaturation of fatty acid in the total liver lipids was found in CLA-fed mice. The CLA diet significantly increased the activities of peroxisomal β-oxidation and decreased the activities of diacylglycerol acyltransferase, a triglyceride synthesis-related enzyme, in the liver. Moreover, serum lipoprotein profiles of CLA-fed mice showed preferable changes in the atherogenic indices. However, serum leptin and adiponectin were drastically decreased by CLA feeding, suggesting that prolonged administration of CLA would induce further decrease of serum adipocytokine levels, which may be a cause of lipodystrophy in mice. These results show that shortterm feeding of CLA does not induce adverse effect in C57BL/6J mice.

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Xylooligosaccharides (XOS) are mainly composed of two or three xylose units with β-1, 4 linkages. They are obtained by hemicellulose hydrolysis, which is relatively abundant in the cell walls of grains. XOS increases the number of intestinal Bifidobacterium in humans, and maintains the fecal water content within the normal range. To examine the effect of XOS intake on severe constipation in pregnancy, which is predominant in the third trimester, thirty constipated pregnant women were treated with 4.2 g XOS daily for 4 wk. During the study, the clinical efficacy was assessed using a daily diary. The subjects indicated the number of stools and the clinical symptom scores. Twenty-nine subjects completed the study. The mean number of stools was 1.1±0.4 in the pre-treatment week, and increased in weeks 1-4 of XOS administrationto 5.3±2.1, 5.9±2.5, 6.2±2.2 and 6.7±1.9, respectively. At the end of the study, 2 7 subjects could defecate spontaneously. The occurrence of very loose or very hard stools decreased and the stool consistency normalized. The stool color changed from dark to yellowish brown. No side effects were observed. XOS intake was highly effective for the reduction of severe constipation in pregnant women without adverse effects.

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Previously, this author reported that the fermentation of quinoa with Rhizopus oligosporeus increased antioxidant activity, and the antioxidant activity of the 80% methanol extract of the fermented quinoa (Q-tempeh) was higher than the other extracts with n-hexane and water in vitro. In this paper, to clarify a beneficial effect of the fermentation of quinoa with R, oligosporus, the antioxidant activity of 80% methanol extract of Q-tempeh was investigated in rats ex vivo and in vivo. In the ex vivo experiment, the 80% methanol extract from Q-tempeh increased both activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver, and accelerated the production of 12-hydroxyeicosatetraenoic acid (12-HETE) in the lung. In rats fed vitamin E-free diets with 80% methanol extract of Q-tempeh, the a-tocopherol concentration, thiobarbituric acid-reactive substance (TBARS) value, and activities of GSH-Px and SOD in serum showed a similar concentration to those of the control rats fed a vitamin E-supplemented diet. However, the hepatic GSH-Px and SOD activities were higher than those in the control rats. On the other hand, in rats fed a vitamin E-free diet with the 80% methanol extract of quinoa, the serum a-tocopherol level was lower, and both TBARS values of serum and liver were higher than those in the control rats. From these results, the 80% methanol extract of R-tempeh was inferred to be an active superoxide scavenger and peroxide reducer in vivo.

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We examined the effects of Amylomyces rouxii, which is a mold found in some fermented foods in Indonesia, on serum cholesterol and hepatic LDL receptor mRNA in rats. Rats were fed a 0.5% cholesterol-enriched diet with (A. rouxii group) or without (control group) 30g/kg A. rouxii for 4 wk. There were no significant differences in the body weight, food intake or liver weight among the groups. However, the weight of the cecum in the A. rouxii-fed group was significantly higher than that in the control group. The cecal pH in the A. rouxii-fed group was significantly lower than that in the control group. Cecal acetic acid, propionic acid and total SCFA concentrations in the A. rouxii-fed group were significantly higher than those in the control group. The serum total cholesterol and VLDL+intermediate density lipoprotein (IDL)+LDL-cholesterol concentrations in the control group were significantly higher than those in the A. rouxii-fed group at the end of the 4-wk feeding period. There were no significant differences in the HDL-cholesterol or triglyceride concentrations between the groups. The hepatic LDL receptor and cholesterol 7α-hydroxylase mRNA levels in the A. rouxii-fed group were significantly higher than those in the control group. The results of this study demonstrate that feeding of A. rouxii lowers the serum total cholesterol level by enhancement of the cecal SCFA concentration and the hepatic LDL receptor mRNA.

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To examine the serum triglyceride (TG)-lowering effect of a soluble hesperidin derivative, glucosyl hesperidin (G-hesperidin), and its mechanisms, we carried out a G-hesperidin administration test in hypertriglyceridemic subjects. G-Hesperidin was administered to the subjects at 500 mg/d for 24 wk. In this study, the subjects were classified into highTG type (TG>150 mg/dL), borderline-TG type (TG 110-150mg/dL) and normal-TG type (TG<110 mg/dL) on the basis of their initial serum TG values. Among these phenotypes, serum TG level significantly decreased in the high-TG type during the G-hesperidin administration period. It was also observed that elevated values of serum remnant-like particle cholesterol (RLP-C), apolipoprotein (apo) B, apo C-II, apo C-III and apo E occurred in the high-TG type and that these serum levels were significantly reduced by G-hesperidin administration. Moreover, polyacrylamide gel electrophoresis analysis of serum lipoproteins revealed that the very low-density lipoprotein (VLDL)/low-density lipoprotein (LDL) ratio and LDL migration index of the high-TG type were remarkably higher than those of the other phenotypes but that their high values were significantly reduced by the administration. These results indicate that G-hesperidin preferentially lowers serum TG in hypertriglyceridemic subjects and that this effect is possibly caused by the improvement of VLDL metabolic abnormality, leading to the reduction of small dense LDL.

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We examined the changes in serum, liver, kidney, brain, and muscle L-tryptophan (Trp) levels in rats with puromycin aminonucleoside (PAN) nephrosis following oral Trp administration in order to elucidate the fate of elevated Trp in the blood of PAN nephrotic rats with oral Trp administration in their body. Before administration, nephrotic rats had higher liver and kidney Trp contents and lower serum Trp concentration than nonnephrotic rats. At 15min after oral administration of Trp (100μmol/kg body weight), PAN nephrotic rats had a much higher amount of liver Trp than non-nephrotic rats but there were no differences in increased amounts of serum, kidney, brain, and muscle Trp between the two groups. At 30min after oral Trp administration, PAN nephrotic rats had a lower increased amount of serum Trp than non-nephrotic rats and had no increase in liver Trp content, although the increased amounts of Trp in other tissues were similar in both groups. At 30 min after oral Trp administration, liver tryptophan 2, 3-dioxygenase activity increased approximately 2 fold in PAN nephrotic rats. These results indicate that elevated Trp in the blood of PAN nephrotic rats with oral Trp administration is transported mainly into the liver rapidly and then the transported Trp is actively metabolized by activated tryptophan 2, 3dioxygenase in the tissue.

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Intake of sulfated polysaccharides, such as fucoidan or λ-carrageenan extracted from seaweeds, has been shown to enhance immune responses, resulting in inhibition of tumor growth. However, little is known about the mechanisms by which these sulfated compounds mediate the enhancement. In the present study, we examined the effect of sulfated polysaccharides from seaweeds on esterase activity of a lymphocyte tryptase, granzyme A (GzmA), which is believed to induce the production of cytokines in a variety of cells. Inclusion of fucoidan (from Fucus vesiculosus) or λ-carrageenan (from Gigartina aciculaire and Gigartina) in the reaction mixture increased the hydrolysis of Nα-benzyloxy-L-lysine thiobenzyl ester (BLT) by a recombinant rat GzmA in a concentration-dependent manner. Heparin, a sulfated polysaccharide from animal tissues, also increase the BLT hydrolysis, but the effect was less remarkable than those of the polysaccharides from the seaweeds. Hanes-Woolf analysis revealed that the enhancements in the presence of these sulfated compounds from the seaweeds were attributed to the increases in the affinity of the enzyme toward the substrate but not to those in the turnover rate. Chondroitin sulfate A, a sulfated polysaccharide found in animal and plant tissues, showed no positive effect on the hydrolysis. In the present paper, we propose that the enhancement of immune responses by intake of the sulfated polysaccharides from seaweeds can be partially accounted for by their direct effects on GzmA.

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To examine the selenium (Se) status of rats intermittently supplemented with Se, we measured tissue Se contents and glutathione peroxidase (GPx) activities in rats fed a Se-deficient diet intermittently supplemented with selenate. In experiment 1, four groups of male 4-wk-old Wistar rats were fed a Torula yeast-based Se-deficient diet (Se content, <0.01μg/g) for 2 8 d. During the experimental period, the diet of each group was supplemented with sodium selenate (0.17μg Se/g) for 0, 1, 2 or 7 d/wk. The tissue Se contents and GPx activities both increased gradually with an increase in frequency of the selenate supplementation, and significant linear regressions were observed between the frequency and these Se indices. In particular, the correlation coefficient in the liver and plasma indices was nearly equal to a value of 1.0. In experiment 2, three groups of rats were fed the Se-deficient basal diet for 2 8 d. Among these, one group was daily supplemented with sodium selenate to the Se-deficient diet at a level of 0.17μg Se/g, and another group was intermittently supplemented with the selenate at a level of 1.19μg Se/g for 1 d/wk. The tissue Se contents and GPx activities both were increased by the selenate supplementation and no significant difference was observed between daily and weekly supplementation in the Se indices except in erythrocyte Se. These results indicate that Se status in the growth period is dependent on total Se intake in this period and that weekly intermittent supplementation with Se can maintain adequate Se status.

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