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Bone Biology Group, Department of Clinical Research, University of Bern, Murtenstrasse 35, CH-3010, Bern, Switzerland
2To whom correspondence should be addressed. E-mail: roman.muhlbauer{at}dkf5.unibe.ch.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bone resorption • vegetables • urinary excretion • acid • osteoporosis
Bone mass in adult humans decreases with age, leading to an increased risk of fractures (1). Osteoporotic fractures, besides causing suffering to the patient, are a major burden to health care resources; the direct annual expenditure for osteoporosis and associated fractures in the United States is 
$17 billion (2). From a medical and economic viewpoint, it would therefore be desirable to prevent loss of bone mass. A nutritional approach would be an inexpensive means of achieving this goal. However, the effects of the nutritional strategies recommended today are rather modest. Indeed, even the beneficial effect of calcium in milk on the relative risk of hip fracture seems to be restricted to the 10% of the female population with the lowest intake of calcium (3). Thus, research into novel nutritional strategies for preventing bone loss is needed.
Osteoporosis occurs most frequently in postmenopausal women following the decrease in estrogen levels. Hormone-replacement therapy (HRT)2 is effective in preventing bone loss. However, compliance with HRT therapy is low because of side effects. This has stimulated research into alternatives to the classical HRT therapy, i.e., the use of phytoestrogens to prevent bone loss.
It has been suggested that the high consumption of soy products in the traditional Japanese diet, providing 30 to 60 mg/d of the estrogenic isoflavones genistein and daidzein, may contribute to the low prevalence of postmenopausal osteoporosis in Japan (4). Indeed, treatment with soy protein containing isoflavones inhibits bone loss in an animal model for postmenopausal osteoporosis, ovariectomized rats (5). Treatment of perimenopausal and postmenopausal women with 40 g/d of a soy protein isolate providing 80 or 90 mg/d of isoflavones, respectively, attenuated the loss of bone mineral density (BMD) in the spine but not at other sites; lower doses were ineffective (6,7). A randomized double-blind placebo-controlled study recently found that a dose of 54 mg/d of genistein was as effective as HRT in preventing bone loss in the spine and in the femoral neck in early postmenopausal women (8). Thus, genistein appears to be a promising pharmacologically active agent. However, the doses required for this effect are beyond the possibilities of nutritional supplementation. Indeed, achieving an intake of 54 mg/d of genistein from soy products, for example, would require the consumption of 200 g/d of tofu (9). Such a strategy would imply a fundamental modification of Western nutritional habits, which is hardly feasible.
We have, therefore, chosen a different approach—we have investigated whether components of the Western diet display bone-modulating activities. We have previously shown that 14 common vegetables, salads and herbs that are part of the normal Western diet—arugula, broccoli, cucumbers, Chinese cabbage, red cabbage, dill, garlic, wild garlic, leeks, lettuce, onions, Italian parsley, common parsley and tomatoes—significantly inhibit bone resorption in rats when administered at a dose of 1 g/d (10). Others have shown that the consumption of fruit and vegetables is associated with greater bone mineral density in humans (11–13), an effect that is claimed to be caused by the base excess of fruits and vegetables buffering noncarbonic metabolic acids that would otherwise be buffered by bone mineral, leading to bone dissolution (11,12). We demonstrated, however, that at least in rats the effect on bone resorption of the 14 foodstuffs outlined above is not mediated by their base excess but possibly by pharmacologically active compounds (14). Because some vegetables, such as potatoes, carrots and soy, consumed at the same dosage, did not significantly inhibit bone resorption (10), it cannot be generally stated that the consumption of vegetables is beneficial to bone density. This prompted us to perform the present survey.
Here we demonstrate that individual foods shown to inhibit bone resorption are widely distributed among vegetable components of the human diet.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar Hanlbm rats were kept in standard animal facilities that comply with the Swiss and U.S. National Institutes of Health guidelines for care and use of experimental animals. The experiments performed were approved by the State Committee for the Control of Animal Experimentation. At completion of the experiment all rats were killed with carbon dioxide.
Nine-week-old rats prelabeled with tritiated tetracycline (see below) were used to measure bone resorption. The experiments were planned as a block design with a final number of 10 rats for each treatment group. The number of rats per block was 26—6, 5, 5, 5 and 5 for the untreated control group fed the plain semipurified diet, the positive group control fed onions and three groups fed one of the newly investigated items, respectively. New blocks were performed until each item was investigated in two independent experiments. The results for each item were pooled (n = 2 x 5 = 10) and compared to the untreated control (n = 2 x 6 = 12) investigated simultaneously. The positive control group fed onions (if not stated otherwise, n = 2 x 5 = 10), also investigated simultaneously, served to evaluate the performance of each experiment. In four experiments, the n for onions was only 5 (spinach, celeriac, Brazil nuts and instant coffee). In three experiments, the positive control group fed onions was shared by two newly investigated items (prunes and oranges, peanuts and French beans and shiitake and yellow boletus, respectively). Finally, black tea was not monitored by the positive control group.
Feeding and diets.
From the time the rats were placed in the metabolic cages, they were provided with demineralized water to drink and the diets were presented in a stainless steel crucible as wet food to minimize spillage in the cage; deionized water was added to batches of the food powder to give a dough-like consistency easily formed into food balls.
During the 10-d acclimatization period in the metabolic cages and the 10-d baseline urine collection, the rats were fed the standardized normal diet 2134 (Kliba-Mühlen, Kaiseraugst, Switzerland) with high Ca and P concentrations (11 g Ca/kg and 12 g P/kg) similar to those in the semipurified diet described below. During the acclimatization period the rats were trained to consume 23 g/d of wet food (13.1 g/d of dry matter); rats that repeatedly did not eat the whole daily amount were eliminated during this period.
During the 10 d of treatment the rats were fed the semipurified diet 2160 (Kliba-Mühlen) in order to mimic a typical Western diet with large proportions of refined components as well as to avoid any interference with bone resorption by the natural components of a normal diet (15). In order to make the rats insensitive to Ca and P in the food items to be tested, a semipurified diet with high concentrations of Ca and P (11 and 12 g/kg, respectively) was used (Table 1).
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The calcium and phosphate concentrations of the diets were verified with triplicate ashed samples dissolved in 1 mol/L HCl. Calcium was determined by atomic absorption spectrophotometry and phosphate by photometry (16,17). The values given by the manufacturer were confirmed.
Processing of foodstuffs and beverages for the various additions to the diets.
Processing of foodstuffs was similar to that previously described (18). Briefly, fennel, celeriac and red pepper were purchased locally, carefully washed with tap water, minced, air-dried at about 50°C and ground to a fine powder. Ready-to-use frozen spinach was freeze-dried before grinding. Plums, oranges, bananas and apples were carefully washed, stoned (plums) and peeled (oranges and bananas), cut and freeze-dried before grinding. Dried commercially available farmed shiitake and field agaric mushrooms (Lentinus edodes and Agaricus hortensis, respectively) and locally harvested yellow boletus wild mushrooms (Boletus edulis) were air-dried at about 50°C and stocked dry, then dried further by adsorption over silica gel before grinding. Roasted peanuts were shelled before grinding. Flax seed and kernels of Brazil nuts were purchased from a local retailer and ground without prior treatment. French beans and dry kidney beans were cooked in water, mashed in a blender together with the water and freeze-dried. Parboiled steamed rice was freeze-dried, and dark bread without crust, made from wheat flour type 1050, was air-dried at about 50°C before grinding. A mixture of 15.1 parts glucose, 12.9 parts fructose and 17.2 parts sucrose ("sugars"), similar to the proportions contained in dried onion (19), was prepared from analytical grade sugars.
Regular instant coffee and soluble cocoa powder were used as purchased, whereas black and green tea were ground prior to use. Normal cola was freeze-dried because a pilot experiment revealed that rats drinking cola in liquid form had a much higher liquid intake after a few days than control rats drinking demineralized water (fluid intake increased from 28 ± 5 mL/d in control rats to 88 ± 3 mL/d in rats drinking cola). Because the rats drinking cola also consumed all the food, this led to a gain in body weight of 43 ± 7 g compared with 7 ± 1 g in the control rats during the same time period. To overcome these difficulties the dry residue from cola was added to the diet. To avoid a similar problem with beer and red wine, the alcohol was removed under reduced pressure at 60°C (Rotavapor R110, Büchi, Flawil, Switzerland) and the remnant was freeze-dried. All these items were packed in polyethylene bags from which the air was evacuated before sealing, and they were stored at 4°C until used.
Monitoring of bone resorption.
The urinary excretion of tritium-labeled tetracycline ([3H]-Tc) from chronically prelabeled rats, an extensively validated method, was used to monitor bone resorption continually (10,14,16,20–22) .
For each experiment three Wistar Hanlbm dams with 12 3-d-old male pups each were purchased (RCC Ltd., Füllinsdorf, Switzerland). From the first week of life the 36 pups were injected twice a week for 6 wk with increasing amounts of [3H]-Tc (20). The [3H]-Tc is deposited into bone and is released when bone is resorbed (20). After discontinuation of labeling, the rats were transferred to metabolic cages. After 10 d of acclimatization baseline bone resorption was monitored by measuring the daily urinary [3H] excretion. After 10 d of baseline measurement the 10-d dietary intervention was begun in rats that were homogeneously assigned to the groups; that is, the baseline [3H] urinary excretion of all rats was ranked and one animal with a similar rank was assigned to each treatment group until the complete number of animals per group was assigned (n = 6 per control group; n = 5 per treatment group). Using this protocol the mean [3H] excretion was similar for all groups at the start of the dietary intervention.
Concentration of 3H in urine was determined by liquid scintillation counting. Aliquots of 1 mL of urine were counted in 10 mL of Irga-Safe Plus scintillator (Packard International, Zürich, Switzerland) and the result (Bq) was multiplied by the 24-h urine volume.
Statistical methods.
Where appropriate, the 95% CI of the pertinent control groups was calculated by multiplying the SEM by 1.96 (shaded box in Fig. 1). Means of groups outside the 95% CI are significantly different from those of the control group (P < 0.05) (23). The correlation between the dose of vegetables and inhibition of bone resorption was analyzed by linear regression, and the significance of the slope was tested by ANOVA using GraphPad InStat version 3.05 statistical software (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Using this animal model, we found that vegetables such as fennel and celeriac but not spinach and red pepper significantly inhibited bone resorption. Fruits with a significant effect were prunes and oranges, but not banana and apple. From the beans, nuts and seeds group, only French beans displayed significant activity, but not Brazil nuts, peanuts, kidney beans and flax seed. Farmed and wild mushrooms (yellow boletus and field agaric) displayed a significant activity, but Chinese mushrooms (shiitake) were devoid of activity. The three carbohydrate sources, rice, dark bread and sugars, also showed no effect on bone resorption. Finally, of the beverages tested, only the residue from red wine significantly inhibited bone resorption, but not beer, green tea, instant coffee, cola or cocoa. A pilot experiment showed that green tea administered at a dosage of 1 g/d was not well tolerated; it reduced food intake and body weight. Therefore, green and black tea were administered at a dosage of 0.25 g/d. As green tea showed no effect in both experiments, a second independent experiment for black tea was not done. Bone resorption in rats fed 250 mg/d of black tea in the single experiment, expressed as treated/control, was 1.02 ± 0.03 (n = 5), and the 95% CI of the untreated control was ±7.7% (n = 6). Thus, black tea was also devoid of bone resorption inhibitory activity. In order to make sure that freeze-dried cola corresponds to native cola in terms of acid concentration, we compared the two items. For this, freeze-dried cola was reconstituted to its original volume with demineralized water, and the pH and the titratable acid concentration was measured as done previously (14). The pH of reconstituted cola was 2.55, compared with 2.52 for the control cola. The consumption of 0.01 mol/L NaOH to titrate 1 mL of reconstituted cola to pH 7.4 was 0.88 ± 0.1 mL, whereas the control cola required 1.10 ± 0.1 mL. Thus, reconstituted cola appears to have a 20% lower titratable acid concentration, compared with native cola. Nevertheless, rats fed 1 g/d of cola powder still received a conspicuous load of noncarbonic acid. Despite this, bone resorption was not significantly different from that of the control group.
Of the 25 newly investigated nutritional items, 8 significantly inhibited bone resorption in rats when tested at a dosage of 1 g/d (Fig. 1). Seven items were clearly negative (mean ± SEM within the 95% CI of the controls), whereas 10 items were located at the lower limit of the 95% CI of the controls. No conclusions could be drawn for red pepper because the discriminative performance was poor in this set of experiments. For the other items, the question of whether a significant effect could be obtained with larger doses remains open. The daily dose of 4 g dry foodstuff/kg body weight corresponded to 8% of the daily intake of dry matter and was, therefore, already a high dose of an individual food item. The daily doses of coffee (400 mg of coffee extract/kg body weight) and of green tea (1 g of tea leaves/kg body weight) correspond to about 1 L/d if extrapolated to a 70 kg human; similarly, 1 g of cola powder corresponds to 2.2 L for a 70-kg human if the 20% loss of titratable acid is taken into account.
Twenty-four food items and the red wine residue were shown to significantly inhibit bone resorption when administered at a dosage of 1 g dry weight in this study and in the previous work (10,24) (Fig. 2). To allow a comparison of the activity of the food items as they are consumed in real life, the results were converted to fresh weight and ranked according to activity. A 10-fold difference appears between the most active fresh food item (garlic) and the least active (cucumber). Furthermore, herbs and spices are exclusively to the left of onion, indicating a higher activity than onion, while the activity of the 17 other nutritional items is lower than that of onion.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We found that bread made of dark wheat flour containing 5.2% dietary fiber had no inhibitory effect (19). Thus, it appears likely that no effect on bone resorption is to be expected from flours with a lower fiber concentration commonly used to make white bread and many kinds of pasta. Other processed carbohydrate sources, parboiled rice and sugars, were devoid of effect, and because potato also displayed no significant inhibitory effect (10), the major carbohydrate sources in the human diet are not BRIFI. Foodstuffs of animal origin were also devoid of inhibitory effect in this animal model (10). Therefore, BRIFI seem to be restricted to the categories of vegetables, salads, herbs, mushrooms, fruits and red wine residue.
Our results in rats can possibly confirm and explain the effects of fruits and vegetables on bone mineral density observed in human studies (11–13) and expand BRIFI to the categories of salads, herbs, mushrooms and red wine. Our interpretation of the effect, however, contrasts with the interpretation from the human studies, where it has been claimed that the base excess of fruits and vegetables buffers noncarbonic metabolic acids, generated from cystine- and methionine-rich dietary proteins, that would otherwise be buffered by bone mineral, leading to bone dissolution (11,12). If this hypothesis were correct, we should have found foodstuffs that stimulate bone resorption. In addition, it has been shown that the inhibitory effect of foodstuffs of vegetable origin on bone resorption is not mediated by their base excess but possibly by pharmacologically active compound(s) (14). Thus, based on the results from our dietary intervention studies in rats, we conclude that components of the human diet are either BRIFI or inert. As a consequence, a model that implies a high rate of bone resorption that can be opposed by BRIFI can be envisaged.
There is only scant information concerning the nature of the compounds in BRIFI that cause their inhibitory activity.
To date we have identified nine monoterpenes contained in the essential oils of sage, rosemary, thyme and other plants as inhibitors of bone resorption (24). In our previous work, we also showed that the active component in onion is extractable with water or with ethanol/water, inhibits resorption in vivo and in vitro and prevents bone loss in an osteoporosis model. The solubility of this active component is, therefore, different from that of the highly lipophilic monoterpenes, suggesting at least two different classes of active molecules.
Others have shown that rutin, a flavonoid abundant in onion, inhibits bone resorption in rats. Unfortunately, however, a single pharmacological dose was used (25) which was much higher than that contained in the 1 g/d dose of vegetables we used (10). It is therefore open whether rutin can partly explain the activity of BRIFI (26,27). Likewise, hesperidin, a citrus flavonoid, has been shown to inhibit bone loss in mice at a pharmacological dosage (28).
Beer could possibly be expected to have a significant effect on bone, considering the carryover of the estrogenic humulone from the hops used in brewing (29). Because intact male rats were used, the lack of effect might be due not only to an insufficient dosage, but possibly also to the fact that the model we used is not tailored to be exquisitely sensitive to phytoestrogens. Accordingly, our previous studies also found no significant effect with soy at a dosage of 1 g/d in rats (10); for an effect larger dosages of 2.5 g/d were necessary (unpublished observation). We chose this model, which is sensitive to inhibitors of bone resorption in general (30), because bone loss occurs in old age in both sexes.
Prunes have been shown to inhibit bone loss in rats at a very high dosage (25% of daily intake), the rationale for the study with prunes being their abundant polyphenol content, thought to protect bone by scavenging free radicals (31). We found prunes to have a significant effect when fed at a dosage corresponding to 8% of the daily intake. If polyphenols in general were responsible for the effect observed, other foodstuffs containing large amounts of polyphenols could also be inhibitory. However, among the beverages tested, only red wine inhibited bone resorption, despite the fact that some beverages have a large concentration of polyphenols. The polyphenol concentration provided per rat with the additions we used, calculated from published data (32), was within a range of 2.4 to 3.9 mg/d for beer, 40 to 161 mg/d for red wine, 55 to 82 mg/d for black tea, 50 to 87 mg/d for green tea and 120 to 180 mg/d for cocoa. Thus, it appears unlikely that polyphenols in general can explain BRIFI.
Coffee, tea, cola and cocoa are rich in caffeine. Caffeine intake is a risk factor for bone loss in humans because caffeine increases the urinary excretion of calcium. However, it appears that the risk can be offset by moderate milk consumption (33). We used a high calcium diet throughout these studies to make the rats insensitive to the calcium concentration of the food items tested, which makes it plausible that any stimulation of bone resorption by caffeine-rich food items might have been masked.
Finally, concerning cola, we have shown previously that young rats with intact renal function can excrete large amounts of acid into the urine so that buffering by bone is not required (14); this might well explain why cola did not stimulate bone resorption in our animal model.
To minimize stimulation of bone resorption by calcium depletion, the recommended daily intake of calcium for women and men over 65 y of age is 1500 mg/d (34). The preferred source of calcium is through calcium-rich foods such as dairy products (34). However, the effect of this strategy might be rather modest (3). As the effect of BRIFI occurred in our rat model despite a high calcium diet, our findings suggest that an abundant consumption of BRIFI, together with a sufficient mineral supply, might offer an additional benefit in humans.
The minimum effective dosage of BRIFI was 170 mg/d in rats as assessed with a mixture of 14 active vegetables. This dose corresponds to 6.2 g fresh BRIFI/kg body weight, indicating that the amount of BRIFI consumed by way of a standard Western diet might not be sufficient to elicit a protective effect in humans. However, other compounds occurring in plant-derived foodstuffs, such as vitamins K and C and the phytoestrogen genistein, have been identified as potentially important for bone health because they support bone formation (8,35,36). Our methodology does not allow us to make conclusions as to the effect of foodstuffs on bone formation. Notwithstanding this limitation, it can be summarized that the evidence available today suggests that foodstuffs of vegetable origin offer a variety of active compounds (possibly secondary plant products; the nature of many, however, is still unknown) that either stimulate bone formation or are BRIFI, which, when present in the human diet in sufficient amounts, in concerted action could slow down bone loss.
If the high incidence of osteoporosis in Western societies is taken as evidence that the amount of such active compounds consumed by way of the typical 2 to 3 servings of fruits and vegetables per day in the Western diet is not sufficient to elicit a protective effect, a strategy to increase the number of servings should be recommended. Although it is difficult to change nutritional habits in humans, the international "Take Five" campaign shows that it is possible to increase the daily number of servings, at least of fruits and vegetables, from 3 to 5 servings of 80 g each (37). The Take Five campaign was started as a result of the overwhelming epidemiological evidence that generous consumption of fruits and vegetables is associated with a reduced risk for certain forms of cancer (38). Higher consumption of fruits and vegetables seems to reduce the risk for another condition associated with age, low bone mass (11–13), the main risk factor for osteoporotic fractures. It will, therefore, be of interest to assess bone mass in persons who have increased their fruit and vegetable intake to 5 servings per day for cancer prevention.
Our survey shows that not all fruits and vegetables are effective and that BRIFI are widely distributed among vegetable components of the human diet. Thus, regular consumption of BRIFI could be an efficient alternative means of ingesting sufficient amounts of the active agents to protect against bone loss. However, before such a recommendation can be given, the amounts necessary to obtain an effect in humans must be established in a clinical intervention study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BMD, bone mineral density; BRIFI, bone resorption inhibitory food items; HRT, hormone replacement therapy; [3H]-Tc, tritium-labeled tetracycline.
Manuscript received 8 May 2003. Initial review completed 2 June 2003. Revision accepted 11 August 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Lindsay, R. & Cosman, F. (1992) Primary osteoporosis. Coe, F. L. Favus, M. J. eds. Disorders of Bone and Mineral Metabolism 1992:831-888 Raven Press New York, NY. .
2. Melton, L. J. & Heaney, R. P. (2003) Too much medicine? Or. too. little?. Bone 32:327-331.[Medline]
3. Kanis, J. A. (1999) The use of calcium in the management of osteoporosis. Bone 24:279-290.[Medline]
4. Fujita, T. (1991) Osteoporosis—East and West. Calcif. Tissue. Int. 48:151-152.
5. Harrison, E., Adjei, A., Ameho, C., Yamamoto, S. & Kono, S. (1998) The effect of soybean protein on bone loss in a rat model of postmenopausal osteoporosis. J. Nutr. Sci. Vitaminol. (Tokyo). 44:257-268.[Medline]
6. Alekel, D. L., Germain, A. S., Peterson, C. T., Hanson, K. B., Stewart, J. W. & Toda, T. (2000) Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar spine of perimenopausal women. Am. J. Clin. Nutr. 72:844-852.
7. Potter, S. M., Baum, J. A., Teng, H., Stillman, R. J., Shay, N. F. & Erdman, J. W. Jr (1998) Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am. J. Clin. Nutr. 68(suppl.):1375S-1379S.[Abstract]
8. Morabito, N., Crisafulli, A., Vergara, C., Gaudio, A., Lasco, A., Frisina, N., D’Anna, R., Corrado, F., Pizzoleo, M. A., Cincotta, M., Altavilla, D., Ientile, R. & Squadrito, F. (2002) Effects of genistein and hormone-replacement therapy on bone loss in early postmenopausal women: a randomized double-blind placebo-controlled study. J. Bone Miner. Res. 17:1904-1912.[Medline]
9. Coward, L., Barnes, N. C., Setchell, K. D. R. & Barnes, S. (1993) Genistein, daidzein, and their ß-glycoside conjugates: Antitumor isoflavones in soybean foods from American and Asian diets. J. Agric. Food. Chem. 41:1961-1967.
10. Mühlbauer, R. C. & Li, F. (1999) Effect of vegetables on bone metabolism. Nature 401:343-344.[Medline]
11. Tucker, K. L., Hannan, M. T., Chen, H., Cupples, L. A., Wilson, P. W. & Kiel, D. P. (1999) Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women. Am. J. Clin. Nutr. 69:727-736.
12. New, S. A., Robins, S. P., Campbell, M. K., Martin, J. C., Garton, M. J., Bolton-Smith, C., Grubb, D. A., Lee, S. J. & Reid, D. M. (2000) Dietary influences on bone mass and bone metabolism: further evidence of a positive link between fruit and vegetable consumption and bone health?. Am. J. Clin. Nutr. 71:142-151.
13. Tucker, K. L., Chen, H., Hannan, M. T., Cupples, L. A., Wilson, P. W., Felson, D. & Kiel, D. P. (2002) Bone mineral density and dietary patterns in older adults: the Framingham Osteoporosis Study. Am. J. Clin. Nutr. 76:245-252.
14. Mühlbauer, R. C., Lozano, A. & Reinli, A. (2002) Onion and a mixture of vegetables, salads and herbs affect bone resorption in the rat by a mechanism independent of their base excess. J. Bone Miner. Res. 17:1230-1236.[Medline]
15. Brown, N. M. & Setchell, K.D.R. (2001) Animal models impacted by phytoestrogens in commercial chow: implications for pathways influenced by hormones. Lab. Invest. 81:735-747.[Medline]
16. Mühlbauer, R. C. & Fleisch, H. (1995) The diurnal rhythm of bone resorption in the rat: effect of feeding habits and pharmacological inhibitors. J. Clin. Invest. 95:1933-1940.
17. Chen, P. S., Jr, Toribara, T. Y. & Warner, H. (1956) Microdetermination of phosphorus. Anal. Chem. 28:1756-1758.
18. Mühlbauer, R. C., Li, F., Lozano, A., Reinli, A. & Tschudi, I. (2000) Some vegetables (commonly consumed by humans) efficiently modulate bone metabolism. J. Musculoskel. Neuron. Interact. 1:137-140.
19. Souci, S. W., Fachmann, W. & Kraut, H. (1989) Food Composition and Nutrition Tables 1989/90. 1989 Wissenschaftliche Verlagsgesellschaft mbH Stuttgart, Germany.
20. Mühlbauer, R. C. & Fleisch, H. (1990) A method for continual monitoring of bone resorption in rats: evidence for a diurnal rhythm. Am. J. Physiol. 259:R679-R689.
21. Egger, C. D., Mühlbauer, R. C., Felix, R., Delmas, P. D., Marks, S. C. & Fleisch, H. (1994) Evaluation of urinary pyridinium crosslink excretion as a marker of bone resorption in the rat. J. Bone Miner. Res. 9:1211-1219.[Medline]
22. Antic, V. N., Fleisch, H. & Mühlbauer, R. C. (1996) Effect of bisphosphonates on the increase in bone resorption induced by a low calcium diet. Calcif. Tissue Int. 58:443-448.[Medline]
23. Sachs, L. (1974) Der Weg zum statistischen Test. Statistische Sicherheit. Angewandte Statistik 1974:90-91 Springer-Verlag Berlin, Germany; Heidelberg, Germany; New York, NY.
24. Mühlbauer, R. C., Lozano, A., Palacio, S., Reinli, A. & Felix, R. (2003) Common herbs, essential oils, and monoterpenes potently modulate bone metabolism. Bone 32:372-380.[Medline]
25. Horcajada-Molteni, M. N., Crespy, V., Coxam, V., Davicco, M. J., Remesy, C. & Barlet, J. P. (2000) Rutin inhibits ovariectomy-induced osteopenia in rats. J. Bone Miner. Res. 15:2251-2258.[Medline]
26. Mühlbauer, R. C. (2001) Rutin cannot explain the effect of vegetables on bone metabolism. (Letter to the Editor). J. Bone Miner. Res. 16:970.[Medline]
27. Barlet, J. P. (2001) A possible rut(in) the road. (Reply). J. Bone Miner. Res. 16:971.
28. Chiba, H., Uehara, M., Wu, J., Wang, X., Masuyama, R., Suzuki, K., Kanazawa, K. & Ishimi, Y. (2003) Hesperidin, a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice. J. Nutr. 133:1892-1897.
29. Tobe, H., Muraki, Y., Kitamura, K., Komiyama, O., Sato, Y., Sugioka, T., Maruyama, H. B., Matsuda, E. & Nagai, M. (1997) Bone resorption inhibitors from hop extract. Biosci. Biotechnol. Biochem. 61:158-159.[Medline]
30. Mühlbauer, R. C. (1994) Biochemical assessment of bone metabolism in rodents. Ziegler, R. Pfeilschifter, J. Bräutigam, M. eds. Sex Steroids and Bone 1994:191-202 Springer Berlin, Germany; Heidelberg, Germany; New York, NY. .
31. Arjmandi, B. H., Lucas, E. A., Juma, S., Soliman, A., Stoecker, B. J., Khalil, D. A., Smith, B. J. & Wang, C. (2001) Dried plums prevent ovariectomy-induced bone loss in rats. JANA 4:50-56.
32. Bravo, L. (1998) Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56:317-333.[Medline]
33. Barrett-Connor, E., Chang, J. C. & Edelstein, S. L. (1994) Coffee-associated osteoporosis offset by daily milk consumption. The Rancho Bernardo Study. J. Am. Med. Assoc. 271:280-283.[Abstract]
34. NIH Consensus conference. (1994) Optimal calcium intake. NIH Consensus. Development Panel on Optimal Calcium Intake. J. Am. Med. Assoc. 272:1942-1948.[Medline]
35. Feskanich, D., Weber, P., Willett, W. C., Rockett, H., Booth, S. L. & Colditz, G. A. (1999) Vitamin K intake and hip fractures in women: a prospective study. Am. J. Clin. Nutr. 69:74-79.
36. Heaney, R. P. (1996) Nutrition and Risk for Osteoporosis. Osteoporosis 1996:483-509 Academic Press New York, NY.
37. Cox, D. N., Anderson, A. S., Reynolds, J., McKellar, S., Lean, M. E. & Mela, D. J. (1998) Take Five, a nutrition education intervention to increase fruit and vegetable intakes: impact on consumer choice and nutrient intakes. Br. J. Nutr. 80:123-131.[Medline]
38. Willett, W. C. (1994) Diet and health: what should we eat?. Science 264:532-537.
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