This investigation was directed towards finding the need of vitamin D for fish. The freshwater column feeder fish Labeo rohita (Rora) was used for the study. Early fry stage fish were divided into four experimental groups of 350 each: two groups were kept in natural light, while the other two were maintained under total darkness. One each of the light and dark-grown groups was supplied dietary vitamin D₃ [1, 650 i.u/kg diet], whereas the other groups were fed a vitamin D-deficient diet for six months. The known vitamin D-related functions and growth parameters were studied in these four experimental groups of fish. The results showed that fish reared on vitamin D-deficient diet and in dark did not have even traces of liver vitamin D, indicating a state of vitamin D deficiency in these fish. No significant differences were observed in percent bone to body weight or dry matter of the vitamin D-deficient/ supplemented groups of fish grown in light/dark. Further, it was also observed that there were no significant changes in bone and carcass ash or calcium and phosphorus content in response to vitamin D3 sup-plementation as compared to the groups which did not receive vitamin D3 (grown both in light and dark). Also, there was little change in several other parameters like carcass protein and lipid, mortality rates, hepatosomatic index, and feed efficiency between the vitamin D-deficient/ supplemented groups of fish. Thus, these findings suggest that vitamin D may not be an essential nutrient for Rora (Labeo rohita) as a representa-tive of freshwater fish.

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we used the sucrose preference test and taste nerve recording to investigate the effect of dietary biotin on the abnormal sucrose taste sensitivity and preferences seen during the course of diabetes mellitus. For this, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats. The chorda tympani nerve (CT nerve) response to sucrose (>1M) was of greater relative magnitude in OLETF rats than in non-diabetic control (Long-Evans Tokushima Lean, LETO) rats, but the responses to other basic taste stimuli (such as HCI, quinine-HC1 and L-glutamic acid) did not differ between the two groups. In behavioral experiments using a two-bottle preference test, solution intake for sucrose (>50mM) was higher in OLETF rats than in LETO rats. The neural responses to sucrose (1.5-2M) in OLETF rats were lower when given a biotin-high diet (BH-OLETF) than when given a biotin-basal diet (BB-OLETF), but this was not true of the other basic tastes. However, there were no significant differences between BH-OLETF and BB-OLETF rats in terms of sucrose solution intake. These findings suggest that the enhanced sugar sensitivity observed in OLETF rats is probably the result of a genetic difference between OLETF and LETO rats, though the discrepancy can be modified by the dietary biotin level.

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In order to elucidate the effect of L-ascorbic acid (AsA) on the formation of pyridinoline, a mature crosslink of collagen, its content in cartilage collagen of guinea pigs supplemented with and without AsA in the growing process (4-8 weeks of age) and in the period of maturity (10-14 weeks of age) was examined. The AsA-deficient animals, for four weeks during the growing process, had a significantly higher content of pyridinoline in their collagen than the AsA-supplemented group, indicating that the depletion of AsA induced increasing contents of pyridinoline. On the other hand, in the period of maturity, the pyridinoline content in the collagen decreased with age, whereas no difference between AsA-deficient and -supplemented groups was observed. Based on these results, it is assumed that AsA affects the formation of pyridinoline, especially in the growing period.

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The effect of vitamin E on the contact sensitization responses induced in mice by 2, 4-dinitrochlorobenzene (DNCB) was studied. Mice were fed a vitamin E-adequate or a vitamin E-deficient diet for 5 weeks. The amounts of thiobarbituric acid-reactive substances in the spleens and draining auricular lymph nodes of mice were decreased by dietary vitamin E. Dietary vitamin E prevented lipid peroxidation in the spleens and lymph nodes of mice. Contact sensitization develops in two phases, induction (sensitization) and elicitation. Following sensitization to DNCB on ears, draining lymph node responses, i.e., lymph node weight, total lymph node cell number and in vitro lymph node cell proliferation as assessed by [³H]methyl thymidine incorporation, were examined. These responses, activated by DNCB, were lower in the mice fed a vitamin E-deficient diet as compared with those of the mice fed a vitamin E-adequate diet. In the elicitation phase, lymphocytes from sensitized mice respond to the antigen and blastogenate in vitro. The blastogenesis of spleen lymphocytes in the DNCB-sensitized mice was decreased by vitamin E deficiency, which was enhanced by exogenously adding vitamin E. It was found that vitamin E deficiency decreases the contact sensitization responses to DNCB in mice, but responses were restored by exogenous vitamin E. In conclusion, vitamin E may participate in the lymphocyte responses to contact allergens through scavenging reactive oxygen species.

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This experiment was conducted to study the effects of vitamin E on growth inhibition and lipid peroxidation in rats treated with different levels of corticosterone (CTC). Rats (Sprague-Dawley strain, 5 weeks of age) were divided into two groups: control group receiving a basal diet containing 60 mg DL-α-tocopheryl acetate/kg diet, and vitamin E group receiving the same diet supplemented with 5, 000 mg tocopherol. After 6 days, rats of both diet groups were further divided 'into three groups by dose levels of CTC treatment (0, 25, and 100mg CTC/kg body weight/d). CTC was administered to the rats by subcutaneous injection for 4 d. Growth was dose-dependently inhibited by the CTC treatment. Feeding the vitamin E diet significantly (p<0.05) improved growth retardation. Feed efficiency was lowered by CTC treatment, while this was significantly (p<0.05) minimized by feeding the vitamin E diet. Lipid peroxidation (TBARS) in the liver was elevated by the CTC treatment (p<0.001) when the rats were fed the basal diet. The increment in TBARS was significantly (p<0.001) reduced by vitamin E. The activities of glutathione S-transferase (GST) and superoxide dismutase (SOD) were significantly reduced by the CTC treatment in a dose-dependent manner in both dietary groups. Feeding vitamin E significantly (p<0.001) improved the reduction in GST activity. The SOD activity showed some tendency. The present results demonstrate the effectiveness of vitamin E in improving growth retardation in glucocorticoid-treated rats and suggest that reductions in increased lipid peroxidation due to CTC may be an important factor of the action of vitamin E.

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The recovering effect of betaine (derivative of choline) on carbon tetrachloride (CCl₄)-injured liver was investigated by oral and intraperitoneal administration of betaine at 24h after acute CCl₄ in-toxication. The effect of betaine was estimated by the activity of alanine aminotransferase (ALT) released into the serum, histological score, and the incorporation of S-phase indicator (5-bromo-2-deoxyuridine/5-fluoro-2-deoxyuridine; BrdU/FdU). A significant decrease in the activity of serum ALT was observed by the intraperitoneal (3mg/kg body) or oral (15mg/kg body) administration of betaine. Furthermore, an increase in the uptake of BrdU into the hepatocyte nuclear DNA and a reduction in liver necrosis after the oral treatment of betaine (15mg/kg body) was observed. From these results, the administration of betaine showed a significant effect in recovering CCl₄-injured liver.

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This study was designed to investigate the modulatory effect of dietary soybean protein on the skeleton of an ovariectomized rat model with postmenopausal osteoporosis. Thirty-two female Sprague-Dawley rats were weight matched and divided into the following four experimental groups: Soy group, ovariectomized and fed soy protein diet; Estrogen group, ovariectomized, fed casein diet and injected with estrogen; Casein group, ovariectomized and fed casein diet; and Sham group, sham-operated and fed casein diet. The diets and estrogen were started two weeks after surgery, and continued for four weeks. Rats in the Sham, Soy and Estrogen groups had significantly higher (p<0.05) femur and tibia ash content than those in the Casein group. Accordingly, the calcium content of the tibia and femur were also significantly higher (p<0.05) in the Soy, Estrogen and Sham groups as compared to the Casein group. Serum total and bone-type alkaline phosphatase levels were both significantly lower (p<0.05) in the Estrogen and Sham groups in relation to the Soy and Casein groups. This study demonstrated that a 22% soybean protein diet could be just as effective as daily estrogen administration in suppressing bone loss due to ovariectomy. However, unlike estrogen, soy protein diet did not have any uterotrophic effect and did not decrease the markers of bone turnover measured, suggesting a possible difference in the mechanism of action.

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Total body bone mineral density was measured by dual energy X-ray absorptiometry in 52 children who were very low birth weight (VLBW) infants without cerebral palsy and mental retardation (postconceptional age, from 10 mo to 6 y and 6 mo). VLBW infants in this study seemed to show compensatory acceleration of total body bone development, catching up with the control group during early childhood. However, in VLBW infants with at least one of the three factors such as total parenteral nutrition for 1 week or more, assisted ventilation for 1 week or more, or oxygen therapy for 28 d or more in their early stage after birth, adequate mineral supplementation might be especially important for long-term bone development.

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We compared the effects of different n-3 polyunsaturated fatty acids (PUFA) on platelet aggregation and lipid metabolism in rats. a-Linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosa-hexaenoic acid (DHA) were used as n-3 PUFA sources. The rats were fed diets containing 10% lipids (polyunsaturated/saturated fatty acid (P/S) ratio=1.0; n-3/n-6=0.02 for the control group, 0.2 for the test groups) for two weeks. The platelet counts, platelet aggregation, and production of thromboxane A2 (TXA2), plasma total cholesterol (TC) and triacylglyc-erols (TG) were not different between the ALA group and the control group, but showed a decreasing tendency for the EPA group and significant decreases for the DHA group. The production of prostacyclin in the aorta was significantly decreased in all of the n-3 PUFA groups when compared with that in the control group. Liver TC and TG concentrations were significantly decreased in the DHA group when compared with those in the control group. Based on the above, it is assumed that the physiological action exerted by n-3 PUFA differs by type and that DHA is a more effective n-3 PUFA, both for suppressing platelet aggregation and for modulating lipid metabolism in the plasma and liver of rats.

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In this study, lysozyme was modified by glucose in the dry state (50°C, RH 75%) and the peptide patterns were investigated using HPLC after hydrolyzing by the pepsin-pancreatin system. Native or modified lysozyme was also administered to rats to clarify digestibility and absorbability in vivo. The digestibility of lysozyme modified by glucose decreased depending on reaction time. In vitro, when lysine residue of lysozyme was modified by glucose at a level of about 50%, the generated peptide patterns with molecular weights (MW) below 3, 000 Da were similar to that of native lysozyme, and the yields of peptides from modified lysozyme were lower than those of native lysozyme. However, there are some differences between the hydrolysate patterns of native and modified lysozymes of MW over 10, 000 Da, and the yields of these peptides from modified lysozyme are higher than those from native lysozyme. In vivo, the percentage of 30 d-modified lysozyme remaining in rat digestive tracts after 90 min of administration was 11% of the dosage as compared with 0.4% for native lysozyme. However, the digestive peptide patterns of native or modified lysozymes in the rat small intestine were also similar to those of the control. Consequently, it is estimated that modified lysozyme was digested as easily as native lysozyme when the degree of modification of lysine residue by glucose was about 60% even in vivo.

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This study was conducted to examine the influence of dietary fat on the metabolism and excretion of hexachlorobenzene (HCB), a ubiquitous food contaminant which is metabolized at a low rate. Three groups of rats were fed semi-purified diets containing 10 g/100 g of either soybean oil, lard or fish oil for 2 wk and then given a single dose of HCB by intragastric gavage. The concentrations of HCB and pentachlorophenol (PCP), a major metabolite of HCB, were monitored in the blood for 5 d. Fecal excretion of HCB did not differ among the three groups, indicating no difference in HCB retained in the body among the groups. Concen-trations of HCB in blood, liver and brain samples from the lard and fish oil groups, the members of which had a low fat tissue mass, were con-sistently higher as compared with those in samples from the soybean oil group. The concentration of PCP and the PCP/HCB ratio in the blood were higher in the fish oil group than in the other groups. In addition, the amount of PCP excreted in urine was highest in the fish oil group. The hepatic cytochrome P-450 content in the fish oil group was higher than that in the other groups. These findings indicate that feeding fish oil to rats accelerated HCB metabolism. An increase in hepatic HCB concentration due to a small fat tissue mass and high hepatic cytochrome P-450 content may have played a role in accelerating HCB metabolism in the fish oil group.

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The antioxidative substances contained in cacao liquor, which is one of the major ingredients of chocolate, were separated by column chromatography and high-performance liquid chromatography. Three major compounds were purified and two of them were identified by ¹H, ¹³C NMR and mass spectra as (-)-epicatechin (EC) and (+)-catechin (CA). Their antioxidative activity was measured by monitoring the peroxide value of linoleic acid and the thiobarbituric acid-reactive substance values of erythrocyte ghost membranes and microsomes. EC and CA had strong antioxidative effects in all three methods, but one unidentified peak was found to be less effective. Additionally, we ana-lyzed the polyphenol concentration of cacao liquor extractions produced in several countries. The total polyphenol concentration was 7.0 to 13.0%, catechin concentration was 0.31 to 0.49%, and epicatechin con-centration was 0.35 to 1.68% in the extractions. It is believed that cho-colate is stable against oxidative deterioration on account of the presence of these polyphenolic compounds, and it is also expected to have a pro-tective role against lipid peroxidation in living systems.

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A thiamin-binding protein from buckwheat (Fagopyrum esculentum Moench) seeds gave two bands of 56- and 50-kDa in the absence of 2-mercaptoethanol and a single band of 25-kDa in the presence of 2-mercaptoethanol on sodium dodecylsulfate gel electrophoresis. These results indicate that the protein consists of polypeptides linked by disulfide bond(s). The protein isolated from buckwheat seeds did not have immunological homology with the thiamin-binding proteins from rice seeds and sesame seeds. However, the binding of the protein to thiamin was inhibited by the modification of the carboxyl residues in the protein as well as that of the thiamin-binding protein from rice seeds. These results suggest that the thiamin-binding protein from buckwheat seeds differs from those from rice seeds and sesame seeds as to subunit structure or immunological properties, but resembles them in the mechanism of binding thiamin.

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When an anion-exchange resin column (Plasorba BR-350) was used for the treatment of unconjugated hyperbilirubinemia, we found an unexpected decrease in plasma retinol (vitamin A) concentrations in a patient with type I Crigler-Najjar syndrome. The purpose of our study was to investigate the mechanism of this decrease in plasma retinol. When the patient's serum bilirubin exceeded the bilirubin binding capacity of 14.7, umol bilirubin/g serum albumin (i.e., 720μmol/L of bilirubin), abrupt deterioration of the patient's neurologic status (suppression in his gait and speech) occurred, so the need to apply plasmapheresis to reduce the unconjugated bilirubin was indicated. Blood was drawn from the radial artery at a flow rate of 160 mL/min and pumped into a membrane plasma separator at a rate of 40 mL/min. The plasma was passed through the bilirubin adsorbent column and returned to the venous blood line of the plasma separator. Plasma samples were taken at the inlet and outlet of the bilirubin adsorbent column before and after treatment. The con-centration of unconjugated bilirubin in plasma was effectively reduced by the perfusion, but plasma retinol was coincidentally decreased by the perfusion to vitamin A deficiency levels. The patient's plasma retinol was 2, 127 nmol/L at the beginning of therapy and decreased to 1, 492 nmol/L after repeated adsorption treatments. As the amounts of decrease in retinol (912±123 nmol/L) after the perfusion were almost equal to those in retinol-binding protein (1, 010±192 nmol/L), retinol may have been removed as a form of holo retinol-binding protein. Decreases in retinol and retinol-binding protein levels were also observed in low-density lipoprotein (LDL) apheresis with a dextran sulfate column (i.e., a cation-exchange resin column). In the patient with Crigler-Najjar syndrome, retinol taken dietarily was removed by plasmapheresis. However, the patient manifested no clinical symptoms associated with vitamin A deficiency, since his liver storage of retinol could supply the loss caused by plasmapheresis treatment. We should measure plasma retinol concentrations to evaluate the loss of retinol during plasmapheresis treatment coupled with an anion-exchange resin column.

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The influence of black tea polyphenols on plasma lipid levels was investigated in rats fed a 15% lard and 1% cholesterol diet. The diet was supplemented with 1% black tea polyphenols extracted and con-densed from black tea. Rats fed the lard-cholesterol diet showed an in-crease in plasma cholesterol and liver lipids compared to rats fed a basal diet. The supplementation of black tea polyphenols in this lard-cholesterol diet decreased the lipid levels in the plasma and increased the fecal excretion of total lipids and cholesterol. On the other hands, 1% supplementation of either instant black tea with a 20% polyphenol content or 0.2% supplementation of EGCg in the lard-cholesterol diet had no effect on plasma cholesterol and phospholipid levels. These results suggest that a high dose of black tea polyphenols exerts a hypocholesterolemic effect in cholesterol-fed rats.

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