Roles of the indole ring of Trp396 covalently bound with the imidazole ring of His398 coordinated to type I copper in bilirubin oxidase.

Bilirubin oxidase has a post-translationally formed covalent-bond between the imidazole ring of His398 coordinated to type I copper and the indole ring of Trp396 located in the outer-coordination sphere. We performed point mutations at Trp396 with Ala, Thr, Phe, and Tyr with the aim of elucidating the role of the imidazole-indole moiety found only in bilirubin oxidase. The result showed shifts in the redox potential of type I copper towards negative direction by > 100 mV and decreases in cathodic current in electrochemistry, whereas optical and magnetic properties of type I copper were not affected or sparingly affected. Along with the conspicuous changes in redox properties enzymatic activities of the Trp396 mutants were prominently decreased. Further, chemical modification of the Trp residues with N-bromosuccinimide and photo-induced formylations of bilirubin oxidase exerted more pronounced effects on both redox properties and enzymatic activities compared to the Trp396 mutants. All these results unequivocally indicate that the covalent-bond formed between Trp396 and His398 plays a crucial role to enhance enzymatic activities of bilirubin oxidase by shifting the redox potential of type I Cu towards positive direction and also by functioning as the effective pathway of electron transport.

Threshold value of serum 25-hydroxyvitamin D concentration in relation to elevated serum parathyroid hormone concentrations in elderly Japanese women.

This study was designed to determine the threshold value for 25-hydroxyvitamin D [25(OH)D] concentration in relation to elevated serum parathyroid hormone (PTH) concentrations in elderly Japanese women. The subjects were 582 noninstitutionalized, ambulant women who lived in a community in Japan. Serum 25(OH)D concentrations were determined using the Nichols Advantage chemiluminescent assay, and serum intact PTH concentrations were determined with a two-site immunoradiometric assay. Demographic characteristics, calcium intake, and serum 1,25(OH)(2)D levels were also determined. The average age, body mass index (BMI), and calcium intake of the subjects were 74.5 years (SD 4.5), 23.3 kg/m(2) (SD 3.4), and 579 mg/day (SD 248), respectively. The serum log-transformed intact PTH concentration was significantly predicted by the serum 25(OH)D concentration (r = -0.147, P = 0.0004), but not by age, BMI, the serum log-transformed 1,25(OH)(2)D concentration, or the log-transformed calcium intake. Analysis of variance with Dunnett's multiple comparisons showed that mean serum intact PTH concentrations with serum 25(OH)D concentrations less than 30 nmol/l (mean intact PTH = 5.89 pmol/l, P < 0.0001) and in the range 30-39 nmol/l (mean intact PTH = 4.54 pmol/l, P = 0.0067) were significantly higher than mean intact PTH concentrations for serum 25(OH)D concentrations greater than 50 nmol/l (mean intact PTH = 3.65 pmol/l, the baseline level), but the mean serum intact PTH concentration for 25(OH)D concentrations in the range 40-49 nmol/l (mean intact PTH = 3.70 pmol/l, P = 0.9975) was not. We conclude that serum 25(OH)D for ambulant elderly Japanese women should be maintained at 40 nmol/l or higher.

Nutrition, mild hyperparathyroidism, and bone mineral density in young Japanese women.

BACKGROUND: The adverse effects of poor nutrition on the bones of young Asian women have not been fully elucidated. OBJECTIVE: The purpose of this study was to investigate possible associations of vitamin D nutrition, calcium intake, and other nutrients with bone metabolism and bone mass in young Japanese women. DESIGN: The subjects were 108 female college students aged 19-25 y. Dietary nutrients were measured by using the duplicate sampling method on 3 weekdays. Serum 25-hydroxyvitamin D [25(OH)D], intact parathyroid hormone, and bone turnover markers were also measured. Bone mineral density (BMD) of the spine and femur was measured by dual-energy X-ray absorptiometry. RESULTS: The proportions of the subjects with low 25(OH)D (< 30 nmol/L) and high intact parathyroid hormone (> or = 6.9 pmol/L) concentrations were 32.4% and 15.7%, respectively. Serum 25(OH)D concentrations (P = 0.0265) and calcium intake (P = 0.0103) were inversely associated with serum intact parathyroid hormone. In addition to weight and physical activity, the presence of mild hyperparathyroidism was associated with a low BMD of the lumbar spine (P = 0.0062) and the femoral neck (P = 0.0250), and a low calcium intake was associated with a low BMD of the femoral neck (P = 0.0044). CONCLUSIONS: Low calcium intake (based on low BMD of the femoral neck only) and mild hyperparathyroidism (based on low BMD of both the femoral neck and lumbar spine), partly explained by low vitamin D nutrition and a low calcium intake, are important predictors of low BMD in young Japanese women. Effects of poor nutrition and mild hyperparathyroidism on bone peak bone mass in young women should be further investigated in longitudinal studies.

Intakes of calcium and other nutrients related to bone health in Japanese female college students: a study using the duplicate portion sampling method.

The purposes of this study were to determine intakes of nutrients related to bone health, such as calcium (Ca), phosphorus (P), sodium (Na), potassium (K), and protein, in Japanese female college students, using the duplicate portion sampling method, and to identify possible lifestyle factors explaining their calcium intakes. Subjects were 106 Japanese female college students aged 19-23 years. All foods in the duplicate portions, as eaten by the subjects during a three-weekday period, were collected. The minerals and protein in the food samples were analyzed. Life-style information was obtained by interview. Levels of cognitive eating restraint (CER) were assessed by the Three-Factor Eating Inventory. Average intakes of dietary Ca, P, Na, K, and protein were 380 (SD 209) mg/day, 649 (SD 212) mg/day, 2,535 (SD 847) mg/day, 1,108 (SD 429) mg/day, and 41.7 (SD 12.6) g/day, respectively. Ca intake was significantly associated with the frequency of milk intake (R2 = 0.278, p < 0.001), intake of other dairy products (R2 = 0.338, p < 0.001), and meal skipping (R2 = 0.161, p < 0.001), but not with CER score (p = 0.378). Female college students are at high risk for poor Ca nutrition. Low intake of nutrients relevant to bone health is considered to adversely affect bone metabolism in young women.

Weight gain in childhood and bone mass in female college students.

The attainment of maximal peak bone mass early on in life is one of the most important strategies for the prevention of osteoporosis in women. The aim of this study was to clarify the correlation between gains in body size in all growth phases in childhood and adult bone mass in women. The subjects were 86 female first-year university students, aged 18-21 years. We measured the subjects' bone mineral content (BMC) and bone mineral density (BMD) at the lumbar spine and the left hip, including the femoral neck, with dual energy X-ray absorptiometry. Each subject was measured for current height and weight. Height and weight at birth, and at 1.5, and 3 years were obtained from each maternity record book, and those between 6 and 18 years were obtained from their school health records. Other information, including physical activity and calcium intake, was obtained through an interview. Bivariate analysis showed that weight gains during the periods from birth to 1.5 years and from 9 to 12 years significantly correlated with both BMC and BMD values at any site. The stepwise method of multiple regression analysis showed that a weight gain during the period from birth to 1.5 years was significantly associated with BMC at the lumbar spine (P = 0.0001) and at the femoral neck (P = 0.0290) and with BMD at the lumbar spine (P = 0.0387). Birth weight was significantly associated with BMC at the lumbar spine (P = 0.0474) and the total hip (P = 0.0352), and weight gain during the period from 9 to 12 years was significantly associated with BMC at the femoral neck (P = 0.0376). In conclusion, birth weight and weight gain in infancy are important determinants of bone mass in young women. Additionally, a girl's prepubertal growth spurt is likely to be a key phase for the acquisition of bone mass in relation to body weight. Our findings suggest that osteoporosis prevention programs may need to start very early in the life cycle.

Dietary calcium, sodium, phosphorus, and protein and bone metabolism in elderly Japanese women: a pilot study using the duplicate portion sampling method.

OBJECTIVES: Associations between dietary factors and bone metabolism in Asians have not been thoroughly investigated. The purpose of this study was to determine the effect of dietary intake of calcium (Ca), phosphorus (P), sodium (Na), and protein on bone metabolism in elderly Japanese women by the duplicate portion sampling method. METHODS: The subjects were 43 healthy female volunteers (mean age, 68.3 y; standard deviation, 6.8). Dietary nutrients were directly determined by using a 24-h duplicate meal portion. Serum osteocalcin and bone alkaline phosphatase and urinary deoxypyridinoline (DPD) and type I collagen cross-linked N-telopeptides (NTX-I) were measured as markers of bone turnover. Hormones related to bone metabolism, including serum 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), and intact parathyroid hormone also were determined. Urinary parameters were corrected for urinary creatinine concentration. RESULTS: The mean (standard deviation) daily dietary intakes of Ca, P, Na, and protein were 660 (195) mg, 996 (208) mg, 4080 (1142) mg, and 63.9 (15.5) g, respectively. Dietary Ca was negatively correlated with urinary DPD (r = -0.417, P = 0.005) and NTX-I (r = -0.324, P = 0.034), and dietary P was negatively correlated with urinary DPD (r = -0.307, P = 0.045). Multiple regression analysis showed that only dietary Ca was associated with urinary DPD and NTX-I. Dietary Ca intake was significantly associated with bone resorption markers, but no other dietary factors were associated with any of the biochemical markers. CONCLUSION: The major factor having an adverse effect on the bone health of the elderly Japanese women is low Ca intake and not other dietary minerals or protein.

Nutritional covariates of dietary calcium in elderly Japanese women: results of a study using the duplicate portion sampling method.

OBJECTIVES: The purposes of this study were to determine the intake of nutrients related to bone health, including calcium (Ca), phosphorus (P), sodium (Na), potassium (K), protein, and vitamin D, in elderly Japanese women and to examine possible intercorrelations between Ca intake and other nutrients. METHODS: Fifty-three elderly women in a rural community in Japan participated by supplying duplicate meal samples of everything they ate during a 24-h period. Dietary Ca, P, Na, K, protein, and vitamin D intakes were examined. The mean (standard deviation) age of the 53 women was 68.2 y (6.4 y). RESULTS: The mean (standard deviation) intakes of dietary Ca, P, Na, K, protein, and vitamin D were 670 (219) mg/d, 1019 (267) mg/d, 4203 (1341) mg/d, 2752 (844) mg/d, 65.2 (18.3) g/d, and 9.90 (8,89) microg/d, respectively. Dietary Ca was significantly correlated with dietary P (r = 0.732, P < 0.0001), Na (r = 0.336, P = 0.0140), K (r = 0.571, P < 0.0001), and protein (r = 0.563, P < 0.0001), but not with vitamin D. After adjusting the data to reflect the total dry weight, dietary Ca was still correlated with P (r = 0.696, P < 0.0001), K (r = 0.423, P = 0.0018), and protein (r = 0.405, P = 0.0029). CONCLUSIONS: The intake of dietary nutrients relevant to bone health in ambulatory Japanese elderly women was examined and documented. The potential confounding effects of these nutrients, especially P, K, and protein, should be taken into account when evaluating the effects of dietary Ca on bone health in observational studies.

Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot.

Our previous studies identified the extract of Beta vulgaris (beetroot), commercially also known as betanin, as a potent cancer chemopreventive agent in both in vitro Epstein-Barr early antigen activation assay and in an in vivo two-stage mouse lung and skin carcinogenesis. To explore this issue further, we have now investigated its cancer chemopreventive potentials in three different chemical carcinogen initiation-promotion experimental tumor models in mice. Following tumor initiation with 390 nmol of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 microl of acetone, the mouse skin tumor promotion with 3430 J/m(2) of ultraviolet light-B (UV-B) as well as splenomegaly was significantly inhibited by oral administration of 0.0025% betanin. At the same dose, betanin also afforded significant protection in the mouse skin cancer model following the topical application of 390 nmol of (+/-)-(E)-4-methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) in 100 microl of acetone and promoted by topical administration of 1.7 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). In the two-stage model of hepatocarcinogenesis in mice with N-nitrosodiethylamine (DEN, 30 mg/kg) as the initiator and phenobarbital as the promoter, oral administration of 0.0025% betanin also showed a very significant inhibition of both the incidence and multiplicity of the liver tumors. These findings along with our initial reports suggest that betanin which is a regularly consumed natural product colorant is an effective cancer chemopreventive agent in mice. The most interesting observation is that the cancer chemopreventive effect was exhibited at a very low dose used in the study and thus indicating that beetroot warrants more attention for possible human applications in the control of malignancy.

Fish as a major source of vitamin D in the Japanese diet.

OBJECTIVES: We investigated the amount and sources of vitamin D in the Japanese diet by analyzing diet records collected over a 4-mo period. METHODS: Dietary data for this study were provided by a nursing home in Niigata, Japan. Diet records, written by the nursing home's dietitian, for 122 consecutive days between September and December 1999, were used. The amount of food for an individual was weighed before cooking and recorded on the diet record. Vitamin D-containing foods, including fish, eggs, meat, and mushrooms, were selected from the diet records, and their vitamin D (vitamin D2 plus D3) per day was calculated by referring to the Standard Tables of Food Composition in Japan. RESULTS: The overall average vitamin D intake per day was 7.10 microg (284 IU), which is about 70% of the recommended dietary allowance of 10 microg (400 IU). There were no significant differences in vitamin D values over the 4 mo (P = 0.822). Overall, the contribution of vitamin D from fish to total vitamin D intake was 90.7%, followed by mushrooms (4.4%), eggs (3.2%), and meat (1.7%). CONCLUSIONS: Frequent fish intake appears to be an advisable health practice in terms of preventing vitamin D insufficiency in the elderly.