J Nutr Sci Vitaminol, 50,114-120, 2004 Promotion of Bone Formation by Fermented Soybean (Natto) Intake in Premenopausal Women Hironobu KATSUYAMA1,Seiji IDEGUCHI2,Masao FUKUNAGA3, Tatsushige FUKUNAGA4, Kiyofumi SAIJOHSand Shigeo SUNAMII Departments of 1Public Health, 2Health Care Medicine arid 3NuclearMedicine , Kawasaki Medical School,Kurashiki 701-0192, Japan 4Departrnent of Forensic Medicine and Sciences , Mie University School of Medicine, Tsu 514-8507, Japan 5Department of Hygiene , Kanazawa University School of Medicine and Graduate School of Medical Sciences, Kanazawa 920-8640, Japan (Received July 24, 2003) Summary A therapeutic agent of vitamin K2 is approved for the treatment of osteoporosis in Japan. However, little is known about the efficacy of dietary intake of vitamin K2for bone health. We compared the effects of various levels of fermented soybeans (Natto) intake , which contains plenty of vitamin K2, on bone stiffness and bone turnover markers in healthy premenopausal women, Seventy-three healthy premenopausal women were ran domly divided into four groups matched for age and parity categories . Natto was supplied as follows: Group 1(no intake), Group 2 (once per month), Group 3 (once per week) and Group 4 (three times per week). Subjects took Natto at a lunch for 1 y, and the stiffness index by quantitative ultrasound and bone turnover markers were assessed at baseline, 6 mo and 1 y There was no statistical difference in the stiffness index during the 1 y observation . How ever, bone specific alkaline phosphatase (BAP) in Group 4 was higher than that in Group 3 at 1 y and undercarboxylated osteocalcin (Glu) in Group 4 was significantly lower than those in Groups 1, 2 and 3 at 6 mo. Logistic regression analysis showed that the risk of reduction of bone formation markers declined to 0.07 in Group 4 based on that in Group 1. In premenopausal women who had to keep the stiffness index as high as possible before menopause, Natto intake may have contributed to the promotion of bone formation . Key Words premenopausal women , Natto intake, stiffness index, bone formation marker, vitamin K2 With the rapidly increasing population of the elderly high as possible in adolescence but also to maintain in Japan, the incidence of osteoporosis and the number high BMD until menopause. of elderly patients suffering from bone fractures are It is well known that nutritional and pharmacologi increasing (1). Although osteoporosis results in inade cal factors are necessary to prevent age-related bone quate skeletal strength predisposing its sufferers to frac loss. Recently, it has been suggested that vitamin K2 ture, most osteoporotic patients are diagnosed after the may play an important role in both aspects . From the occurrence of fractures since osteoporosis itself lacks pharmacological perspective, one of vitamin K2analog, specific symptoms. Moreover, L.S. Prevention Services menatetrenone or menaquinone-4 (MK-4), is approved Task Force (USPSTF), and 2002 clinical practice guide as a therapeutic agent of osteoporosis in Japan . Since lines for the diagnosis and management of osteoporosis vitamin K2 activates osteocalcin through y-carboxyla in Canada do not recommend for routine osteoporosis tion, its deficiency may increase the circulating level of screening in postmenopausal women who are younger undercarboxylated osteocalcin (Glu) and the risk of hip than 60 or in women aged 60 to 64 who are not at fracture (5). Moreover, subjects with osteoporotic pa increased risk for osteoporotic fractures (2, 3). Even tients treated with menatetrenone for 48 wk shows the though anti-resorptive therapies have been shown to significant increase in carboxylated osteocalcin (Gla) reduce fracture risk and increase bone mineral density and decrease in Glu (6). Not only vitamin D3 adminis (BMD)(3), it is difficult to avoid the risk of fracture since tration alone but also the combined administration of it takes significant time to recover BMD except for treat vitamin D3 and vitamin K2 significantly increase BMD ment. In females, BMD reaches its maximal level in ado in postmenopausal women (7). On the other hand , lescence, and gradually decreases until menopause , from the nutritional perspective, the prospective cohort after which it deceases dramatically (4). In order to pre study including both premenopausal and postmeno vent the incidence of involutional osteoporosis, it seems pausal women shows that dietary intake of vitamin K important not only to make a maximal level of BMD as from vegetables, namely vitamin Kl intake , reduces the risk of femoral neck fracture (8). Soybeans fermented by E-mail:[email protected] Bacillus Natto (Natto), a traditional Japanese food, con 114 romotion of Bone Formation by Natto 115 taro more than 100 times more vitamin K2 (mainly 5.7 timeslmo, respectively, and therefore this study was MK-7) than various cheeses (9). It has been reported not blinded for previous Natto intake. The subjects were that intake of Natto increases serum level of vitamin K2, not always fond of Natto and some of them never ate especially MK-7, and Gla in normal individuals (10). Natto because of its peculiar smell. Nonetheless, they Higher incidence of hip fracture is found in women liv were allocated randomly to each group. Since 30g of ing in western Japan where people seldom eat Natto as Natto, containing approximately 9ƒÊg/g of vitamin K2 opposed to eastern Japan where Natto is very popular (9), was taken at lunchtime in the hospital for 1 y, sub (11, 12). Recently, it has been demonstrated that vita jects in Group 2, Group 3 and Group 4 took 270ƒÊg per min K2 (MK-7) can directly stimulate calcification in month, 270ƒÊg per week and 810ƒÊg per week of vita the femoral metaphyseal tissues obtained from normal min K2, respectively. Although 37.9% of subjects took rats in vitro (13). Although both vitamin K1 and MK-7 Natto more than three times per week prior to study, are reportedly converted to MK-4 (14), serum concen they were forced to ingest Natto only in the study proto tration of MK-4 is not measured (15). Since bioavail col during the observation period. It is not necessary to ability from food and their divergent metabolism in vivo take into account for serum levels of vitamin K2 due to are yet unknown, the efficacy of continuous intake of the short half-life (approximately 2.5 d) (15). No statis neither vitamminK2 nor vitamin K1 in the development tical differences were observed in BMI, dietary intakes and maintenance of bone has not been fully clarified. such as energy, protein, calcium and vitamin D, physi Since Natto is recognized to be a healthy food, it cal activity, and stiffness index among subjects divided seems noteworthy to examine its efficacy for mainte by the frequencies of Natto intake prior to study. nance of bone health. Thus, we investigated the effects In addition that Natto contains plenty of vitamin K2 of various levels of Natto intake on bone stiffness and compared to other fermented foods such as cheese, but bone turnover markers in healthy premenopausal ter, etc., the subjects were fond of Japanese foods rather women with consideration of their lifestyle. than western foods. Thus, vitamin K2 intake from other sources was negligible in this study. SUBJECTS AND METHODS None of the subjects had an unbalanced diet for vita Subjects and lifestyle factors. One hundred and one min K1 rich food, e.g. green vegetables and fermented healthy female workers at a local hospital in Japan were foods, indicating that there was no difference in vitamin recruited with their written, informed consent. Twenty K1 intake among each group. The classic role of vitamin eight workers were excluded because of dropping out, K1 involves the synthesis of several blood coagulation menopause, pregnancy, cardiovascular diseases, bilat factors (18, 19), and the maintenance of plasma pro eral ovariectomy, use of anticoagulant therapy, or thrombin concentrations is the basis for the recom neglection of protocol, so that 73 premenopausal work mended dietary allowance of 1ƒÊg/kg/d (20). Since sub ers (mean age 33,5•}10.0 y-old) were included in the jects took vitamin K1 from vegetables around recom present study. This study was aimed for the mainte mended dietary allowance levels, the difference of vita nance of bone health, not for treatment, hence all sub min K2 intake could be assessed. jects were considered to be free from low bone mineral When stiffness index, bone turnover markers, and density or bone disease. other health examinations were performed at baseline, Usual dietary intake was assessed using a food fre 6 mo and 1y, we also confirmed that the subjects were quency questionnaire as previously reported (16). This keeping to the protocol and examined dietary intake, questionnaire had been designed and validated previ physical activity and stature of body. ously against a 7-d frequent food intake, and daily Stiffness index. The speed of sound (SOS; m/s) and dietary intakes were calculated from this 7-d weighed the broadband ultrasound attenuation (BUA; dB/MHz) food intake. Physical activity level was assessed by the in the calcaneus were measured using an Achilles number of steps taken per day as measured by a pedom ultrasound bone densitometer (A-1000, Lunar Corpo eter. The height and weight were measured with light ration, Madison, WI). SOS was recognized to reflect clothes on, and their body mass index (BMI) was calcu density and elastic properties of bone, and BUA was lated. thought to relate to the structure and density of bone Among subjects, 20% and 50% had smoking and (21). The mathematical index, designated as 'stiffness drinking habits, respectively. However, those who had a index' was automatically calculated according to the drinking habit drank as little as drink I (17) per day on formula: 'stiffness index'=0.67•~BUA+0.28•~SOS average.