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* JPOS Study Group, Department of Public Health, Kinki University School of Medicine, 377-2 Oono-Higashi, Osaka-Sayama, Osaka, Japan; Department of Public Health and Home Nursing, Nagoya University School of Health Sciences, Nagoya, Aichi, Japan; ** Department of Welfare Promotion and Epidemiology, Faculty of Medicine, University of Toyama, Toyama, Japan; Kagawa Nutrition University, Tokyo, Japan; and Syuuwa Sougoh Hospital, Kasukabe, Japan
2 To whom correspondence should be addressed. E-mail: yuyu{at}med.kindai.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • fermented soybean • menaquinone • isoflavone • bone density • Japanese women
Osteoporosis is one of the most important diseases affecting elderly people worldwide (1). Hip fractures resulting from osteoporosis cause many elderly people to become bedridden, and osteoporosis is a great burden on society today. In the prevention of osteoporosis, nutrition plays a key role in addition to other factors such as genetics, physical activity, cigarette smoking, and alcohol intake (1–4). Many studies have assessed the role of calcium, vitamin D, magnesium, and other micronutrients, as well as macronutrients such as protein, in the prevention of osteoporosis (3–6). There is emerging evidence, however, that vitamin K may also play a protective role against age-related bone loss (7–9).
Vitamin K is a cofactor of 
-carboxylase, which mediates the conversion of undercarboxylated osteocalcin to carboxylated osteocalcin by transforming the glutamyl residues of osteocalcin into carboxyglutamic acid residues; the last-mentioned have a high affinity for calcium ions in hydroxyapatite and regulate the growth of these crystals (4). Vitamin K is classified into 2 groups: phylloquinone, which is the major form and exists in plants, and menaquinone, which is synthesized by bacteria and exists in dairy products.
Natto is a kind of fermented soybean that is consumed very widely and frequently in Japan. This food is rich in menaquinone-7 and contains >100 times more menaquinone-7 than various kinds of cheese (10).
The incidence of hip fracture in Japan is lower than in European countries and the United States. The reason for this difference is not known, but it is thought to be due to the differences in dietary (11), bone-geometric (12), and lifestyle factors (13) between these populations. Natto is one of the candidates that may account for this difference in hip fracture rate. Furthermore, there is some evidence suggesting that natto is effective in maintaining bone stiffness (10), increasing serum levels of menaquinone-7 and -carboxylated osteocalcin (14), and maintaining the bone mineral density (BMD) of middle-aged women (15). However, these studies were conducted among subjects with a limited age distribution and selected from limited areas (14,15); the sample sizes were small (14) and the studies were performed cross sectionally (10,14,15).
The purpose of the present study was to assess whether habitual natto intake has a positive association with BMD and with change in BMD over time in healthy Japanese women of various ages who participated in a large representative cohort study.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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57 y old are reported to be postmenopausal (17). The remaining subjects were classified as perimenopausal and were excluded from further analysis. In total, 336 women were excluded from further analysis; the remaining 944 women served as the representative sample of Japanese women free from apparent abnormalities affecting bone mass. We obtained written informed consent for all study procedures from each subject. The study protocol was approved by the Ethical Committee of the Kinki University School of Medicine.
Bone mass. The BMD was measured at baseline and 3 y thereafter by dual-energy X-ray absorptiometry at the lumber spine (L2–4), the right hip (QDR 4500A, Hologic), and at the distal third of the radius (pDXA, Norland/Stratec). The short-term CV of the BMD measurement in vivo, as calculated on the basis of 5 measurements on different days for each of 5 volunteers, were 1.2, 1.6, 1.2, and 1.2% for the spine, femoral neck, total hip, and distal third of the radius, respectively (16). The BMD of the spine and forearm phantoms were measured every day for quality assurance; they did not drift significantly during the study period.
Body size. The height (cm) and weight (kg) of the subjects were measured with an automatic scale (TK-11868h, Takei Kagaku). The BMI (kg/m2) was calculated.
Interviews. Detailed interviews were conducted during the survey by trained nurses both at baseline and at follow-up. The interviews were conducted to confirm the information given on a questionnaire that was delivered to the subjects by mail and completed beforehand. The questionnaire included points on menstrual history and on the past history and present involvement of gynecological and other diseases or medications that may affect bone metabolism.
Information on the habitual intake of natto, bean curd (tofu), and other soybean products was obtained through an interview conducted by trained dietitians. We determined the portion size and weekly frequency of intake using a FFQ that was developed and validated previously to estimate dietary calcium intake (18). The FFQ was given to the subjects during the baseline and follow-up surveys. Natto is sold in a pack that usually contains 40 g of natto, a quantity considered to be suitable for 1 meal in Japan. One pack of natto contains 35 mg of calcium and 350 µg menaquinone-7 (19). Natto intake can be determined quite precisely by counting the number of natto packs consumed by the subject over a period of 1 wk. The usual portion size of tofu or other soybean products was determined through the interview by comparison with the portion size of the reference food samples. The total calcium intake, which included calcium intake from mineral supplements, was estimated on the basis of the same FFQ.
Statistical data analysis. The subjects were divided into premenopausal and postmenopausal groups; each of these 2 groups was then classified into 3 subgroups according to their mean habitual intake of natto at baseline or the mean intakes at baseline and at follow-up. Because the premenopausal women did not consume large amounts of natto compared with the postmenopausal women, we used different intervals to classify the weekly natto intake for these 2 groups: no intake, 1 or 2 packs, and <2 packs for the premenopausal women, and no intake, 1–4 packs, and >4 packs for the postmenopausal women.
The rate of BMD change was calculated as follows:
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The results of the indices are expressed as means and SD or SEM. Student's t test was used to test the significance of the difference in mean BMD and other continuous variables between 2 groups. For comparisons among 3 groups with different habitual natto intake or among 3 cohorts, ANOVA was performed and, if significant, pairwise comparisons were done using t tests. The effects of confounding variables, such as age, height, weight, smoking habits, physical activity, years since menopause, and intake of calcium, tofu, and other soybean products, were adjusted using analysis of covariance (ANCOVA) where necessary. The linear regression analysis for BMD or its rate of change was performed after introducing the ordinal variable for natto intake, which was coded as 0, 1 or 2 according to the amount of habitual natto intake. The significance for the regression coefficient for the ordinal variable for natto intake was interpreted as the significance of trend of BMD or its rate of change with the increase in natto intake. The 
2 test was used to compare the prevalence rates of lifestyle factors, such as smoking, exercise, or milk consumption. Differences were considered significant at P < 0.05. Scheffé's multiple comparison procedure was applied to adjust the level of statistical significance to 5% when 3 groups were compared. All statistical analyses were performed using the SAS® system for personal computers (release 8.2, SAS Institute).
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We also tested whether there was a relation between the intake of tofu or other soybean products and the rate of BMD change after adjusting for the effects of the covariates, but there were no associations (Table 4).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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350 µg of menaquinone-7, a remarkably high amount for a food. Menaquinone-7 may have mediated the association found in the present study. Two epidemiologic studies derived from the Framingham cohort examined the association between phylloquinone intake and BMD (20,21). In the Framingham Offspring Study (20), greater dietary phylloquinone intake was associated with higher BMD at the hip and spine in women aged 29–83 y (mean age: 58.9 y). However, there was no association between dietary phylloquinone intake and femoral-neck BMD or its change during the 4 y of follow-up in postmenopausal women aged 68–94 y (mean age: 75.3 y) in the Framingham osteoporosis study (21). The authors of these studies suspected that the lack of association in the latter study was due to the relatively small age-related bone loss because the subjects were older. The present findings support this hypothesis because the significant association was present both at the femoral neck and the distal third of the radius in subjects in their 60s but only at the femoral neck in subjects in their 70s. The mean annual BMD change at the femoral neck in the subjects in their 70s, 1.4%, was greater than that of women in their 60s, –1.2%. At the distal third of the radius, however, the mean annual change in BMD in the subjects in their 70s was –0.9%, which was smaller than that of women in their 60s, –1.1%; this change was so much less than the measurement error that the statistical power for detecting a significant association in this age group was reduced. A similar mechanism may have operated in the Framingham osteoporosis study.
For Asian populations, several clinical trials were conducted to investigate the effect of menaquinone in pharmacologic doses on BMD (22,23) and fracture risk (24–26). Shiraki, et al. (24) reported that 45 mg/d of menatetrenone maintained BMD at the spine more effectively than the control regimen (–0.4% vs. –2.6% for 24 mo, respectively) and reduced the incidence of fracture in patients with osteoporosis. That study showed that menaquinone had a preventive effect on bone loss and supports the findings of the present study. The menaquinone-7 content in an ordinary pack of natto is <1% of the pharmacologic dose. Therefore, the preventive effects of dietary natto intake on bone loss may be small compared with that of menaquinone medication. The preventive effect of phylloquinone or menaquinones on fractures was also previously described (14,21,27). These studies support our speculation that the habitual intake of phylloquinone or menaquinones attenuates bone loss at the femoral neck, and may lead to reduction in the incidence of hip fractures in postmenopausal women. We should follow the present subjects further to assess whether natto intake reduces the risk of hip fracture by maintaining the BMD at the femoral neck.
Isoflavones also may be effective components of natto. Natto contains large amounts of isoflavones, which were reported to reduce bone resorption through estrogenic mechanisms (28–30). One portion of tofu also contains an amount of isoflavones similar to that in 1 pack of natto. However, tofu intake was not associated with bone loss prevention in our subjects. This may be due to the difference in the types of isoflavones present in natto and tofu. Isoflavone aglycones are absorbed faster in humans and are more bioavailable than isoflavone glycosides (31). Fermented soy products contain larger amounts of aglycones than other soy products (32–34). Morabito et al. (35) conducted a randomized placebo-controlled trial to evaluate the effect of genistein, one of the aglycones, on BMD in women aged 47–57 y. The administration of 54 mg/d of genistein significantly increased BMD at the femoral neck (genistein: 3.6 ± 3.0% vs. placebo: –0.7 ± 0.1%) after 1 y of treatment. In the present study, only natto decreased bone loss. We assume, therefore, that this may be due to the greater amount of isoflavone aglycone contained in natto in addition to menaquinone-7.
The present study had several advantages over previous studies with respect to study design. It used a larger representative sample of the general population, the ages of the subjects varied widely, and the follow-up rate was acceptable. However, its limitations should be addressed. First, our FFQ listed only calcium-rich foods. Therefore, we could not adjust the results for potential confounding due to energy intake and protein intake. Subjects who consumed a large amount of natto may also have consumed large amounts of energy and protein, which may have resulted in part in the protective effect on bone loss in the present study. This possible confounding could be controlled for through the adjustment for height and weight in the present analysis, but may still have been present. Second, natto contains large amounts of menaquinone-7, but we did not examine the effects of any other food containing menaquinone-7. Therefore, the results should be interpreted carefully and the conclusion should be restricted to the effect of natto. Third, the information on natto intake was obtained through interviews based on a questionnaire, and there may have been individual misclassifications of natto intake. Fourth, we did not observe any association between tofu or other soybean products and BMD in this study. One reason for this is that natto intake could be determined easily and precisely through the FFQ (because natto is sold in packs containing a similar amount of natto). This information may yield a more accurate estimate of the consumption of this soybean product compared with any others. Fifth, the 3 study areas were not randomly selected from all municipalities in Japan; thus, the subjects might not be representative of the Japanese female population. However, there were no differences in body size or the BMD at the skeletal sites between the present subjects and the Japanese female population. It is not likely, therefore, that the present findings originated from a sampling bias.
The present representative cohort study suggests that natto intake may decrease the loss of bone mass at the femoral neck and possibly at the distal third of the radius in postmenopausal women. Further studies on this topic should be designed to allow for possible confounding effects due to protein and energy intakes and to address whether the effect of menaquinone-7 or of the isoflavones is the primary mechanism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 12 August 2005. Initial review completed 16 September 2005. Revision accepted 8 February 2006.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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