Thiaminase I from the seawater fish Fisturalia petimba was characterized, and pI polymorphism of the enzyme was first described, together with the active subfragment of thiaminase I. The liver of F. petimba contained thiaminase I of at least three different pIs and the major fraction exhibited pI 5.7. The most evident difference among pI isozymes was the size of the active subfragments into which they were dissociated. pI 5.7 enzyme dissociated into subfragments of 25 kDa, while pI 7-9 enzymes dissociated into approx. 22 kDa. The reaction rate measured by pyridine as the second substrate was three times higher in pI 7-9 enzymes compared with pI 5.7 enzyme. The degree of cadmium inhibition, when aniline was the co-substrate, also showed obvious differences between pI isozymes. When the major pI fraction was further purified by the difference in hydrophobicity, a smaller active fragment of approx. 22 kDa appeared, indicating the possibility that the difference in the size of active subfragment between isozymes is a result of partial fragmentation. The pI 5.7 enzyme was purified 250 times and the size of the most purified preparation was found to be 106 kDa by gel filtration analysis. The purified preparation gave an active 25 kDa subfragment by SDS-PAGE, together with a 15 kDa non-active subfragment. The enzyme was, thus, inferred to contain active subfragments together with the 15 kDa non-active fragments. Amino acid sequencing of the 25 kDa active subfragment revealed, together with the fully processed N-terminal sequence, two N-terminal peptides with extra Pro-Ser and Gly-Pro-Ser attached to it, and the NCBI non-redundant database did not show significant similarity to other known proteins. On the other hand, the molecular mass of the holoenzyme from the viscera of the seawater fish Engraulis japonica was estimated to be approx. 100 kDa by gel filtration chromatography. An SDS-PAGE analysis revealed that it contained an active subfragment of 22 kDa.

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Dietary intake, either through specific nutrients or representative food groups, can influence obesity-related oxidative stress markers. This study evaluated the potential associations between vitamin A intake and several anthropometrical, biochemical and dietary features in healthy young adults, emphasizing the putative relationships between total antioxidant consumption and vitamin A intake. This translational research enrolled 61 healthy young adults aged 18-22 y. Anthropometrical and blood pressure measurements, blood samples and nutritional intake data were collected. After adjusting for total energy intake, vitamin A intake showed a negative correlation with several adiposity measurements. Furthermore, vitamin A consumption was positively associated with serum total cholesterol as well as with the intake of antioxidant foodstuffs. So, vitamin A intake seems to be related, not only with the total antioxidant intake, but also with several anthropometrical and biochemical measurements linked to metabolic syndrome manifestations and other features related to oxidative stress in healthy young adults.

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The aim of the present study was to determine if folate alters Notch signaling and cell proliferation in neural stem cells (NSCs). NSCs were isolated from neonatal rats and grown in serum-free suspension culture. The cells were identified as NSCs by their expression of immunoreactive nestin. Individual cultures were assigned to one of three treatment groups: vehicle control, low-dose folate group (Folate-L, liquid media contained 4 mg/L folate), or high-dose folate group (Folate-H, liquid media contained 40 mg/L folate). Proliferating cells were identified by labeling with 5-bromo-2'-deoxyuridine (BrdU). Cell proliferation was quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Gene expression of components of the Notch signaling system (Notch1, Hes1 and Mash1) was quantified by real-time polymerase chain reaction (PCR) assay. We observed that Nestin-positive NSCs grew as neurospheres in the serum-free suspension cultures. Folate increased the rate of cell proliferation compared to vehicle control (p<0.05). During cell proliferation, folate also increased Notch1 and Hes1 expression and decreased Mash1 expression compared to vehicle control (p<0.05). These results suggest that NSCs cultured from neonatal rats respond to folate with altered Notch signaling and increased cell proliferation.

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To investigate how aging alters B-group vitamin metabolism, rats were fed with niacin-free 20% casein diet from 3 to 80 wk old, and the urinary excretions of the B group vitamins were periodically measured. The blood and liver B-group vitamin levels in 80-wk-old rats were also compared with those in 8-wk-old rats. The urinary excretion of thiamin, riboflavin, vitamin B₆ metabolite 4-pyridoxic acid, pantothenic acid, folic acid and biotin were not altered during 540 d. The urinary vitamin B₁₂ increased by 8-fold at 29 wk old, and further increased at 80 wk old. Conversion of nicotinamide from tryptophan gradually decreased to 60% from 29 to 48 wk old. Plasma PLP, vitamin B₁₂ and folate levels in 80-wk-old rats were lower than those in 8-wk-old rats, consistent with lower liver vitamin B₆ and folate levels in aged rats. Plasma and liver biotin levels in aged rats were higher than those in young rats. Other B-group vitamins such as vitamin B₁, vitamin B₂, niacin and pantothenic acid levels in blood and liver from aged rats were same as those from young rats. Alteration of vitamin B₆ metabolism in particular is similar to the observations in eldery humans reported previously. Our findings suggest that aged rats can be useful models to investigate aging-related B-group vitamin metabolism.

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The effects of oleuropein, a phenolic compound in extra virgin olive oil (EV-olive oil), on triglyceride metabolism were investigated by measuring the degree of thermogenesis in interscapular brown adipose tissue (IBAT), and noradrenaline and adrenaline secretions in rats. In Experiment 1, rats were given a high-fat diet (control diet) with the oleuropein supplementation of 1, 2 or 4 mg/kg of diet (0.1, 0.2 or 0.4% oleuropein diet, respectively). After 28 d of feeding, body weight, perirenal adipose tissue, epididymal fat pad, and plasma triglyceride, free fatty acid and total cholesterol concentrations were reduced by the 0.1, 0.2 or 0.4% oleuropein diet and were significantly lowest in rats fed the 0.4% oleuropein diet, as compared with those of rats fed with the control diet. The content of uncoupling protein 1 (UCP1) in IBAT and urinary noradrenaline and adrenaline excretions were significantly higher in rats fed the 0.1 or 0.2% oleuropein diet, as compared with those of rats fed with the control diet, although there were no significant differences in rats fed the 0.4% oleuropein diet. In Experiment 2, the effects of oleuropein on noradrenaline and adrenaline secretion were evaluated. The intravenous administration of oleuropein and oleuropein aglycone significantly increased plasma noradrenaline and adrenaline concentrations. Furthermore, oleuropein aglycone induced the secretions of noradrenaline and adrenaline about ten fold more potently than oleuropein. These results suggest that the phenolic compound oleuropein in EV-olive oil enhances thermogenesis by increasing the UCP1 content in IBAT and noradrenaline and adrenaline secretions in rats.

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Malnutrition modifies resistance to infection by impairing a number of physiological processes including hematopoesis and the immune response. In this study, we examined the production of Interleukin-4 (IL-4) and IL-10 in response to lipopolysaccharide (LPS) and also evaluated the cellularity of the blood, bone marrow, and spleen in a mouse model of protein-energy malnutrition. Two-month-old male Swiss mice were subjected to protein-energy malnutrition (PEM) with a low-protein diet (4%) as compared to the control diet (20%). When the experimental group lost approximately 20% of their original body weight, the animals from both groups received 1.25 μg of LPS intravenously. The cells in the blood, bone marrow, and spleen were counted, and circulating levels of IL-4 and IL-10 were evaluated in animals stimulated with LPS. Cells from the spleen, bone marrow, and peritoneal cavity of non-inoculated animals were collected for culture to evaluate the production of IL-4 and IL-10 after stimulating these cells with 1.25 μg of LPS in vitro. Malnourished animals presented leucopenia and a severe reduction in bone marrow, spleen, and peritoneal cavity cellularity before and after stimulus with LPS. The circulating levels of IL-10 were increased in malnourished animals inoculated with LPS when compared to control animals, although the levels of IL-4 did not differ. In cells cultured with LPS, we observed high levels of IL-10 in the bone marrow cells of malnourished animals. These findings suggest that malnourished mice present a deficient immune response to LPS. These alterations may be partly responsible for the immunodeficiency observed in these malnourished mice.

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Lipids extracted from tsao-ko were separated into three fractions with silica gel column chromatography and fed to mice (3 mo old) for 90 d to clarify their inhibitory activity on digestive enzyme activity. The diets contained the following: control—no tsao-ko, 0.05% total lipid of tsao-ko (TL), 0.0109% chloroform fraction (CF), 0.0245% acetone fraction (AF), or 0.00365% methanol fraction (MeF). Although CF and AF slightly inhibited the activities of α-glucosidase, α-amylase, and lipase, intakes of these fractions had little influence on plasma and liver lipid concentrations when compared with the control diet. MeF did not inhibit α-glucosidase but had DPPH radical scavenging activity and the mice fed this fraction had the most marked reduction in plasma glucose and TBARS concentrations compared with the other diet groups. These results suggest that the fat-soluble polar components of tsao-ko contain an active component that might be associated with decreased plasma glucose and TBARS concentrations in mice.

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Objective: To investigate dietary quality among Japanese adults with a high vegetable diet, to consider dietary recommendation for vegetable intake. Design and setting: In the cross-sectional study of the National Health and Nutrition Survey 2003, we conducted the secondary analyses. The food-weighing method in one-day assessed the dietary intake. From 11,630 subjects, 2,305 men and 2,312 non-pregnant/lactating women, aged 20-69 y, and with an energy intake between 1,500 and 3,712 kcal were selected. Associations between vegetable, nutrient-density, and food intake were analyzed according to tertile cutoff: low vegetable diet (LVD), medium vegetable diet (MVD), or high vegetable diet (HVD). Differences across subgroups were tested after age adjustment. Results: Mean vegetable intakes were 309 g for men and 318 g for women. Only 35% of Japanese met the vegetable intake (VI) recommendation of ≥350 g/d. VI had a positive association with age. Men 20-29 y-old and women 30-39 y-old were the subjects with the lowest VI. HVD subjects had higher intake for most food groups, whereas wheat in men; and wheat, sweets, and alcohol in women were negatively associated with VI. Main sources of energy for men and women with HVD were rice, wheat, and meat. HVD also had higher micronutrient-density. Conclusion: These analyses demonstrated the beneficial effects of HVD on dietary quality in the population studied. We concluded recommendations for adequate vegetable intake are expected to improve diet quality among Japanese adults, especially for the group aged 20-39.

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The aim of present study was to assess the association between serum zinc and oxidant/antioxidant status in children with protein energy malnutrition. Serum zinc, total antioxidant capacity and malondialdehyde were measured spectrophotometrically in 100 children (6 mo to 5 y); out of these, 50 children were malnourished and 50 children served as controls. Serum zinc levels were found to be significantly low in the malnourished (p<0.001). Serum zinc levels in Grade I and Grade II malnourished were 82.7 and 67.7 μg/dL respectively and in Grade III and IV combined was 53.2 μg/dL as compared to 109.5 μg/dL in the control group. These levels were significantly lower in children who had skin lesions than in those without such lesions (p<0.001). Total antioxidant capacity was found to be significantly lowered in malnourished children (Grade I=1.3 mmol/L, Grade II=1.1 mmol/L, Grade III and IV=0.5 mmol/L) as compared to 2.0 mmol/L in the control group (p<0.001). The malondialdehyde concentration in malnourished children was significantly higher (p<0.001) (Grade I=1.6 nmol/mL, Grade II=1.9 nmol/mL, Grade III and IV=2.9 nmol/mL) as compared to 1.3 nmol/mL in controls. Total antioxidant capacity and hypoalbuminaemia were also correlated positively with low serum zinc level. Serum trace element deficiency leading to depleted antioxidant protection may be a contributing factor to the pathophysiology of protein energy malnutrition and replacement of these elements in the management of this condition might be important.

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The purpose of this study was to evaluate the effect of xylooligosaccharide (XOS) on the blood sugar, lipids and oxidative status in type 2 diabetes mellitus (DM). A total of 26 outpatient subjects of Taichung Veterans General Hospital, Taiwan, with HbA1c levels between 7.0 and 10.0% and triglyceride <400 mg/dL were enrolled in the present study. Subjects were supplemented with 4 g/d XOS (n=12) or a placebo (n=14) for 8 wk in a randomized double-blind clinical design. The results showed that the anthropometric values and nutrient intakes did not change during the experimental period. XOS supplementation not only reduced the glucose, HbA1c and fructosamine concentrations, but also decreased the levels of total cholesterol, low density lipoprotein (LDL) cholesterol, oxidized low density lipoprotein (ox-LDL) and apolipoprotein B. The activity of catalase of the erythrocyte sample decreased in the XOS group, but not the activities of superoxide dismutase and glutathione peroxidase. In conclusion, the dietary supplementation with XOS for 8 wk was effective in improving the blood sugar and lipids in type 2 diabetes, indicating that XOS-containing diets might be beneficial to DM subjects.

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The aim of this study was to test the hypothesis that genistein protects vascular endothelial cells against the pro-atherosclerotic stressor, oxidized low-density lipoprotein (ox-LDL), by inducing antioxidant enzymes and preventing apoptosis. Human umbilical cord-derived endothelial cells (ECV 304) were incubated with genistein (10-100 μmol/L), the radical scavenging antioxidant vitamin E (α-tocopherol, 50 μmol/L), or vehicle for 24 h and then were incubated with ox-LDL for an additional 24 h. Subsequently, antioxidant enzyme activities, lipid peroxidation, adhesion to monocytes, cell morphology, viability and apoptotic index were assessed. Ox-LDL decreased superoxide dismutase and glutathione peroxidase activities in endothelial cells and caused lipid peroxidation, adhesion to monocytes, morphological injury and apoptosis (p<0.05). These effects were prevented by vitamin E and dose-dependently by genistein (p<0.05). Further, this effect of genistein is associated with maintenance of antioxidant enzyme activities and inhibition of lipid peroxidation.

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Segment membranes from 4 citrus species selected from 4 sections were treated with water to obtain polysaccharides containing pectin. The extracts, which inhibited pancreatic lipase activity in a concentration-dependent manner, were divided into high molecular weight fractions [molecular weight (M.W.) >300,000], which inhibited the activity strongly, and low molecular weight fractions (M.W. <300,000), which did not show such strong inhibition. The high molecular weight fractions were composed mainly of a characteristic sugar of pectin, namely, galacturonic acid. A galacturonic acid-rich fraction purified by anion exchange chromatography from a water extract also strongly inhibited the activity. The inhibitory activity of the high molecular weight fraction was much stronger than that of commercial citrus pectin. The results suggest that pectin from segment membranes of citrus fruits might be useful as a functional food, especially as a fat-reducing material.

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Chestnut astringent skin (CAS) extract inhibited pancreatic α-amylase and intestinal α-glucosidase in a concentration-dependent manner with the 50% inhibition concentration (IC₅₀) for amylase, maltase and sucrase being 7.5, 650 and 390 μg/mL, respectively. We have investigated the effect of CAS extract on carbohydrate absorption in normal rats. Oral administration of CAS extract to rats fed cornstarch (2 g/kg body weight) significantly suppressed the increase of blood glucose levels and the area under the curve (AUC). Administration of CAS extract to rats fed maltose or sucrose delayed the increase of blood glucose level and slightly suppressed AUC, but not significantly. Administration of CAS extract to rats fed glucose did not affect the increase in blood glucose level or AUC. Similar results were observed with type-2 diabetic model rats (GK/jcl). To test the effect of CAS extract on diabetes, type 2 diabetic model mice (db/db mice) were fed a standard laboratory diet containing 1 or 2% CAS extract. CAS extract prevented increases in body weight and fasting blood glucose concentration. These data suggest that CAS extract has an anti-diabetic function in type 2 diabetic mice that mainly functions through inhibition of α-amylase.

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