The purpose of this study is to find a method of cooking natto that prevents the appearance of high-plasma vitamin K concentrations after the consumption of natto, so that patients taking warfarin can benefit from eating natto. Five cooking methods were examined to determine which could most effectively decrease the count of the living Bacillus subtilis in natto. Volunteers ate natto or treated natto, and their plasma vitamin K level was measured at 5, 8, 24 and 48 h thereafter. One gram of natto contained 9.7±0.1 Log cfu/mL of Bacillus subtilis. Boiling significantly reduced the Bacillus subtilis count to 5.1±0.3 Log cfu/mL, and concomitantly reduced the content of menaquinone-7 (MK-7), which is a form of vitamin K synthesized by Bacillus subtilis, from 660.40±65.32 ng/mL to 78.50±11.12 ng/mL. Untreated natto increased the MK-7 concentration in blood from 1.86±1.51 ng/mL to 14.54±4.12 ng/mL at 5 h after intake, and the MK-7 concentration remained elevated at 8, 24 and 48 h (7.29±2.20, 6.97±2.60, and 5.37±1.94 ng/mL, respectively). In contrast, boiled natto increased plasma MK-7 only mildly (from 1.61±1.11 to 4.02±0.82 ng/mL at 5 h) and the concentration remained relatively stable up to 48 h (3.46±0.83, 4.22±1.51 and 2.77±0.75 ng/mL at 8, 24 and 48 h, respectively). In conclusion, boiled natto did not cause a marked increase in the plasma concentration of vitamin K in subjects who consumed it. Thus, patients on warfarin may be able to eat boiled natto without ill effects.

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The objective of this study was to clarify the relationship between the accumulation of S-adenosylhomocysteine (SAH) and the change in the SAH hydrolase activity in vitamin B₆ (B₆). Male Wistar rats were fed a control diet (control and pair-fed groups) or B₆-free diet (B₆-deficient group) for 5 wk. Although the SAH-synthetic activity of SAH hydrolase significantly increased in the B₆-deficient group, SAH-hydrolytic activity of SAH hydrolase showed no significant difference in the liver among the three groups. On the other hand, SAH hydrolase mRNA in the liver did not show any significant change. Thus, the accumulation of SAH would be due to the increased SAH-synthetic activity of SAH hydrolase. The disturbed methionine metabolism by B₆-deficiency, such as a significant increase of plasma homocysteine, might induce the activation of SAH hydrolase in the direction of SAH synthesis.

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The purpose of the present study was to examine the effect of pretreatment with risedronate and/or vitamin K₂ and treatment continuation with reduced dosing frequency of the drugs on the early cancellous bone loss induced by ovariectomy (OVX) in rats. Eighty female Sprague-Dawley rats, 4 mo of age, were randomized by the stratified weight method into eight groups (n=10 in each group); rats subjected to OVX, but not sham-operated rats, were treated with vehicle, risedronate, vitamin K₂ (menatetrenone), or risedronate+vitamin K₂ for 4 wk before the surgery, and the treatment was either discontinued (pretreatment groups) or continued after the surgery (treatment continuation groups) for 2 wk. Sham-operated rats (controls) were treated with the vehicle throughout the experimental period. During the 4 wk prior to the surgery (pretreatment), risedronate and vitamin K₂ were administered five times a week either subcutaneously at a dose of 2.5 μg/kg body weight (risedronate) or orally at the dose of 30 mg/kg body weight (vitamin K₂). During the 2 wk after the surgery (treatment continuation), the dosing frequency of the drugs was reduced to twice a week. Risedronate and vitamin K₂ had an anti-resorptive effect on the bone. Pretreatment with risedronate alone, but not vitamin K₂ alone, prevented the loss of the cancellous bone volume/total volume (BV/TV) of the proximal tibial metaphysis after OVX. Treatment continuation with vitamin K₂ alone prevented the loss of the cancellous BV/TV after OVX, while treatment continuation with risedronate alone increased the cancellous BV/TV to beyond the values in controls. Pretreatment with risedronate+vitamin K₂ had a more beneficial effect in increasing the cancellous bone mass than pretreatment with risedronate alone. Treatment continuation with risedronate and/or vitamin K₂ appeared to have a more beneficial effect in increasing the cancellous bone mass than the respective pretreatment. Neither the total tissue area nor the cortical area of the tibial diaphysis was affected by any treatment. The present study demonstrated that pretreatment with risedronate had a beneficial effect on the early cancellous bone loss after OVX in rats, with a more beneficial effect when combined with vitamin K₂. Moreover, even though the dosing frequency of the drugs was reduced after OVX, treatment continuation appeared to be more beneficial than pretreatment for increasing the cancellous bone mass.

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We studied the effects of varying the protein and oil contents in the diet and three different feeding methods on the utilization of dietary calcium (Ca) in female Fischer rats. The experimental diets were based on the AIN-76 diet. Rats were fed one of nine experimental diets containing different levels of protein and oil. The experimental diets contained 10, 20 or 40% of protein (milk casein) and 5, 10 or 20% of soybean oil. The three meal feeding methods were ad libitum feeding, pair feeding and adjustable feeding. The experimental diets that were given by prepared feeding were supplemented with a mineral and vitamin solution. The effects of the protein and oil contents in the diet were analyzed by two-way ANOVA. Among rats that were fed ad libitum, the oil content in the diet affected the degree of mineral and vitamin intake. Among the rats fed ad libitum, the oil content in the diet had a significant effect on the level of Ca intake, although it did not have a significant effect on the level of energy intake. For pair feeding, the feeding volume was limited in each group; therefore, there was a significant difference in energy intake and there was no significant difference in Ca intake among each diet group. For adjustable feeding, there was considerable mineral and vitamin intake, which effected the reduction of feeding volume depending on the oil volume in the diet. There were no significant differences in the energy, Ca and other mineral and vitamin intakes among the nine groups that were fed using adjustable feeding. In analyzing two-way ANOVA, in which the parameters were the contents of protein and oil in the experimental diets, there were differences in Ca utilization among rats that were fed using the three feeding methods. This result was shown to reflect on the difference as energy, mineral including Ca, and vitamin intake. However, the protein content in the diet had a significant effect on urinary Ca excretion in all three feeding methods. In addition, it was clear that the intake of protein and oil affected kidney calcification in all three feeding methods. The AIN Experimental Diet Committee reported that kidney calcification was found among rats that were fed the AIN-76 diet and that one of the causes of kidney calcification was the Ca/P ratio in this diet. The protein and oil contents in the diets had significant effects on the degree of kidney calcification among rats that were fed by pair feeding or adjustable feeding. The results of this study suggest that the protein and oil contents in the diet play an important role in kidney calcification.

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Genistein is a phytoestrogen contained at high levels in soy products and has been shown to regulate immunoresponse. In this study, we evaluated the effects of genistein on the production of cytokines from antigen (Ag)-specific T cells using DO11.10 transgenic mice because the direct effect of genistein on Ag-specific cytokine production has not been elucidated. The oral administration of 20 mg/kg genistein increased IFN-γ and IL-4 production from DO11.10⁺ T cells in response to ovalbumin (OVA)_323-339 peptide in female DO11.10 mice. Analysis of intracellular cytokine synthesis revealed that the percentages of cytokine-producing cells in the control and genistein-treated groups were not different, indicating that increased cytokine production occurred at the single-cell level. In contrast to the female mice, genistein did not increase cytokine production in male mice, suggesting that the effect of genistein on cytokine production is gender-dependent.

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The effects of orally ingesting 500 mL of four kinds of test solutions that were isotonic to physiological saline (i.e., containing sodium (Na) and potassium (K) as chloride with Na/K molar ratios of 1, 2, 3 and 4) on urine flow, Na and K excretions from Japanese male students were investigated. The subjects assembled at the National Institute of Nutrition the day before the oral ingestions, which were conducted three times for each subject. They were permitted to eat freely until 8:00 pm, and then the intake of food and drinks except tap water was prohibited until 10:00 pm. After that, no food or drinks except the test solution used, were permitted to be ingested until the end of the final collection of urine. Subjects were woken up at 7:00 am the next morning, at which time they emptied their bladder. At 8:00 am, after sampling control urine before ingestion, they ingested 500 mL of water, the physiological saline or one of the test solutions. Urine was collected every 30 min for 4 h after ingestion. Urine flow was significantly higher for those who drank solutions Na/K=1 and 2 than those who drank the saline (paired-t test), but that for those who drank solutions Na/K=3 and 4 was not significantly higher. For those who drank solution Na/k=2, urine Na was significantly higher than that of those who drank the saline 30 to 60 min after ingestion. An increase in urine flow after K ingestion may be an essential factor for K-induced Na uresis.

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This study determined the effect of long-term administration of 4^G-β-D-galactosylsucrose (lactosucrose; LS) on intestinal calcium absorption. In a randomized, single-blind, parallel-group study, LS (n=9, 6.0 g twice daily) or a placebo (maltose; n=8, 6.0 g twice daily) was administered to healthy young women for 92 wk; the study also included a 4-wk post-administration period. All participants completed the study. Dietary nutrient intake; fecal weight, pH, and moisture content; fecal concentrations of short-chain fatty acids (SCFA), putrefactive products, ammonia, and minerals (calcium, magnesium, phosphorus, and iron); and serum calcium and osteocalcin concentrations were measured every 24 wk. Urinary pyridinoline (PYR) and deoxypyridinoline (DPD), and urinary calcium excretion were measured every 12 wk. Significant effects of oligosaccharide treatment, time, and the interaction between oligosaccharide treatment and time were observed for fecal pH, SCFA, ammonia, and putrefactive product values (p<0.05). Fecal pH, ammonia, and putrefactive product values decreased in the LS group, and the fecal SCFA concentration significantly increased during the administration period; these changes were not observed 4 wk post-administration. To examine the mineral balance of calcium, magnesium, and phosphorus in detail, all the participants completed a 6-d mineral balance study, sometime between week 56 and 60 of the longer study. During the mineral balance study, the daily calcium intake was set at 400 mg; all feces and urine were collected each day for 6 d after an 8-d acclimation period. In the balance study, fecal calcium excretion was significantly lower in the LS group than in the placebo group (p<0.05), and apparent calcium absorption and retention, apparent magnesium and phosphorus absorption, and magnesium retention were significantly higher in the LS group than in the placebo group (p<0.05). Our results suggest that the administration of LS produces a long-term enhancement of intestinal calcium absorption in healthy young women with lower than recommended calcium intakes.

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It is postulated that dietary carbohydrates and thyroid hormones are major regulators for expression of the lactase/phlorizin hydrolase (LPH) gene in rat jejunum. In this study, we investigated the effects of thyroid hormones and dietary sucrose on LPH gene expression and lactase activity in starved rats. Firstly, animals at 8 wk of age were fed a low-starch diet (5.5% energy as cornstarch) or high-starch diet (71% energy as cornstarch) for 7 d (experiment 1). The mRNA level of LPH as well as lactase activity significantly decreased in rats fed the low-starch diet as compared to those fed the high-starch diet. To investigate the effects of thyroid hormone status, the animals previously fed the low-starch diet were starved for 3 d, and half of the animals were given intraperitoneal (i.p.) injections of 20 μg/100 g body weight triiodothyronine (T₃) twice daily (experiment 2). The LPH mRNA level and lactase activity were elevated by starvation for 3 d, but they were repressed by the injection of T₃ during starvation. To investigate the effects of dietary sucrose in starved rats, they were force-fed a sucrose diet for 6 h (experiment 3). The LPH gene expression and lactase activity were up-regulated by force-feeding a sucrose diet, only when the animals were kept in euthyroid status by daily T₃ administrations. In contrast, the sucrase-isomaltase mRNA levels and sucrase activity were unaffected by force-feeding the sucrose diet for both T₃-treated and untreated starved rats. Our work suggests that dietary sucrose is capable of enhancing lactase gene expression in starved rats when they have a sustainable thyroid hormone level.

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The present study was designed to investigate the effect of aging and β₃-adrenergic-receptor (β₃-AR) polymorphism on the thermic effect of meal (TEM) and sympathetic nervous system (SNS) response to a high-fat meal in 13 boys, 12 young men, and 11 middle-aged men. SNS activity was assessed via power spectral analysis of heart rate variability. Significantly higher very-low-frequency (VLF) components associated with thermogenic SNS activity and energy expenditure per lean body mass (EE_LBM) were observed in boys during the pre- and postprandial periods. There were no significant differences in VLF and EE_LBM in the preprandial period between the young and middle-aged men. After feeding, however, the middle-aged men showed a significantly lower TEM (% test-meal energy) and VLF compared to the young men. A multiple regression analysis revealed that age was the only significant variable contributing to both TEM and VLF, but β₃-AR polymorphism and percentage of body fat were not statistically significant. In conclusion, age likely has a greater influence on TEM and SNS thermoregulation than genetic factors such as β₃-AR polymorphism, suggesting that this age-related decrease in thermogenic response may be involved in the development of obesity among middle-aged men.

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The estimation of total daily energy expenditure (TEE) or the energy needs of athletes is important for maintaining a proper energy balance during training. This study was conducted to measure the TEE of Japanese middle- and long-distance runners using a heart-rate monitoring method (the HR method), and to ascertain the impact of three key energy expenditure (EE) components occurring within the day; sleep, exercise (training) and inactivity (other daily habitual activities). Additionally, energy balance was evaluated by comparing TEE with energy intake (EI). The mean TEE of six male athletes aged 19-21 y was 4,514±739 kcal/d and their mean EI was 3,784±91 kcal/d during the training period of 11 d. This negative energy balance led to a significant decline in body weight (p<0.01), indicating that proper care should be taken to balance energy during training. The mean daily EEs during sleep, inactivity, and exercise were 536±46 (12.0% of TEE), 1,722±145 (38.7%), and 2,255±658 (49.3%) kcal/d, respectively. There was a significant correlation between TEE and exercise EE (p<0.01) in the athletes. The TEE of the endurance athletes was higher than expected and significantly affected by exercise EE, indicating that the TEE of athletes, in general, must be carefully evaluated before incorporating nutritional support for them.

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The purpose of this study was to clarify the effects of nutrients on the gonadal development of male rats kept under constant darkness as a model of disturbed daily rhythm. This experimental protocol was designed based on the L₁₆(2¹⁵)-type orthogonal array, which can examine six factors. Five minerals (calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), and zinc (Zn)) were selected as experimental factors, and the dietary content of these minerals was normal (AIN-76 diet) or 1/3.5 of the normal content. Lighting conditions (constant darkness or normal lighting) were also added as a factor. Four-week-old rats (Fischer 344 strain) were kept under constant darkness or normal lighting (12-h light/dark cycle) for 4 wk. After 4 wk, the gonadal weights and serum testosterone content were evaluated. The lighting condition, Ca, Mg, and Na, and the interactions between the lighting condition and Ca, and Mg and Na were observed to affect the testes weight. Among the constant darkness groups (D-groups), the highest value for testes weight was observed under the normal-Ca, normal-Mg, and normal-Na diet, and the lowest value was observed under the low-Ca, normal-Mg, and low-Na diet. Among the normal lighting groups (N-groups), the highest value for testes weight was observed under the low-Ca, normal-Mg, and normal-Na diet, and the lowest value was observed under the normal-Ca, normal-Mg, and low-Na diet. Among the D-groups, the highest value for serum testosterone was observed under the normal-Ca, normal-Mg, and low-Na diet. Among the N-groups, the highest value was observed under the low-Ca, normal-Mg, and low-Na diet. It became clear that the amount of dietary Ca necessary for the gonadal development of rats increases when rats are kept under constant darkness as a model of disturbed daily rhythm compared with the normal lighting condition.

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DNA vaccination is a simple method to induce antigen (Ag)-specific immunoresponse and has many potential advantages over other vaccines. Although people who need to receive vaccines often suffer undernutrition, there has been no study on a how nutritional status affects the immunoresponse induced by DNA vaccination. The aim of this study was to determine the relationship between protein deficiency and DNA vaccine-induced immunoresponses. C57BL/6 mice were fed a 5% or 20% casein diet for 30 d. The mice were immunized with an ovalubumin (OVA)-expression plasmid by the gene gun-based method three times at 10-d intervals. Body weight and serum albumin concentration in protein-deficient mice were significantly lower than those in mice fed the 20% casein diet (p<0.01, p<0.05). The percentage of OVA-specific CD8⁺ T cells was significantly decreased in the 5% casein group compared to that in the 20% casein group (p<0.05). Furthermore, CD4⁺ T cells from mice fed the low-protein diet showed lower interleukin (IL)-2 production than did those from the 20% group. In contrast to the T-cell function, protein deficiency did not affect OVA-specific Ab responses (p>0.05). These results suggest that protein deficiency impairs the induction of Ag-specific T-cell but not B-cell response in DNA-immunized mice. Our observation indicates that, in addition to development of an effective of DNA vaccine, the management of nutritional state is important for the prevention of infectious disease by DNA vaccination.

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We performed a double-blind, placebo-controlled trial to clinically evaluate the protective and ameliorative effects of ellagic acid-rich pomegranate extract on pigmentation in the skin after ultraviolet ray (UV) irradiation, using female subjects in their 20s to 40s. Thirteen healthy volunteers per group were randomly assigned to three groups; namely, high dose (200 mg/d ellagic acid), low dose (100 mg/d ellagic acid) and control (0 mg/d ellagic acid: placebo). Each group received the respective test foods for 4 wk. Each subject received a 1.5 MED (minimum erythema dose) of UV irradiation on an inside region of the right upper arm, based on the MED value measured on the previous day. Luminance (L), melanin and erythema values were measured before the start of the test food intake, and after 1, 2, 3 and 4 wk following the start of the test food intake. Further, questionnaires were conducted regarding the condition of the skin before the start of the test food intake and at the termination of the test food intake. As a result, decreasing rates of L values from the baseline in the low- and high-dose groups were inhibited by 1.35% and 1.73% respectively, as compared to the control group. Further, a stratified analysis using subjects with a slight sunburn revealed an inhibited decrease of L values compared with the control group at 1, 2 (p<0.01, respectively) and 4 wk (p<0.05) after the start of the test food intake in the low-dose group, and at 2 and 3 wk (p<0.05) in the high-dose group. Furthermore, the results of questionnaires showed ameliorating tendencies due to the test food, in some items such as “brightness of the face” and “stains and freckles.” Based on the above-mentioned results, it is suggested that ellagic acid-rich pomegranate extract, ingested orally, has an inhibitory effect on a slight pigmentation in the human skin caused by UV irradiation.

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Prunes are the dried fruits of certain cultivars of Prunus domestica L., and are recognized as a health food. The separated ethanol fraction from concentrated prune juice by DIAION HP-20 (PE) was investigated for cytotoxic effects on two different cancer cell lines in vitro. PE dose-dependently reduced the viable cell number of Caco-2, KATO III, but does not reduce the viable cell number of human normal colon fibroblast cells (CCD-18Co) used as a normal cell model. PE treatment for 24 h led to apoptotic changes in Caco-2 such as cell shrinkage and blebbed surfaces due to the convolutions of nuclear and plasma membranes and chromatin condensation, but this was not observed in CCD-18Co. PE induced nucleosomal DNA fragmentation typical of apoptosis in Caco-2 after 24 h of treatment. These results show that PE induced apoptosis in Caco-2. Furthermore, by Caco-2 treatment with H₂O₂ chelator catalase and Ca²⁺-chelator BAPTA/AM, the PE-induced cytotoxic pathway was completely blocked, and the viable cell number of Caco-2 was not affected.

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