Soy Isoflavone Intake Inhibits Bone Resorption and Stimulates Bone Formation in Menopausal Women: Meta-Analysis of Randomized Controlled Trials

Home , Isoflavone, Rimostil

ORIGINAL ARTICLE Soy isoflavone intake inhibits bone resorption and stimulates bone formation in menopausal women: meta-analysis of randomized controlled trials

D-F Ma1,2, L-Q Qin3, P-Y Wang1 and R Katoh2

1Department of Social Medicine and Health Education, School of Public Health, Peking University, Beijing, China; 2Department of Human Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan and 3Department of Nutrition and Food Hygiene, School of Radiation Medicine and Public Health, Soochow University, Suzhou, China

Objective: To clarify the effects of isoflavone intake on bone resorption and bone formation. Methods: We identified randomized controlled trials related to urinary deoxypyridinoline (Dpyr, a bone resorption marker) and serum bone-specific alkaline phosphatase (BAP, a bone formation marker) listed on MEDLINE (January 1966–April 2006), the Cochrane Controlled Trials Register, EMBASE (1985–January 2006), Science Citation Index and PUBMED (updated till April 2006). Results: Nine studies with a total of 432 subjects were selected for meta-analysis. The urinary Dpyr concentration in subjects who consumed isoflavones decreased significantly by À2.08 nmol/mmol (95% confidence interval (CI): À3.82 to À0.34 nmol/ mmol) in comparison with that in subjects who did not consume isoflavones. Isoflavone intake vs placebo intake significantly increased serum BAP by 1.48 mg/l (95% CI: 0.22–2.75 mg/l). Decreases in the urinary Dpyr concentration with isoflavone intake of o90 mg/day and with treatment lasting less than 12 weeks were À2.34 nmol/mmol (95% CI: À4.46 to À0.22 nmol/mmol) and À2.03 nmol/mmol (95% CI: À3.20 to À0.85 nmol/mmol), respectively. Conclusions: Isoflavone intervention significantly inhibits bone resorption and stimulates bone formation. These favorable effects occur even if o90 mg/day of isoflavones are consumed or the intervention lasts less than 12 weeks. European Journal of Clinical Nutrition (2008) 62, 155–161; doi:10.1038/sj.ejcn.1602748; published online 28 March 2007

Keywords: soy; isoflavone; osteoporosis; bone metabolism; deoxypyridinoline; bone-specific alkaline phosphatase

Introduction risk of hip fracture (Cauley et al., 2001; Rossouw et al., 2002). Furthermore, HRT has some negative side effects such as an With increases in life expectancy, osteoporosis has become a increased risk of cardiovascular disease and breast cancer common disease in post-menopausal women. Although (Recker, 1993). Thus, new bone protection options are hormone replacement therapy (HRT) is the first choice of needed. treatment for hormone-related osteoporosis, some results In recent years, isoflavones have received much attention of randomized controlled trials (RCTs) of this treatment in the medical and scientific literature. It is well known that showed no bone protection or a significant reduction in the the incidence of osteoporosis-related fracture is significantly lower in Southern and Eastern Asian women than in Western women (Ho et al., 1993; Tham et al., 1998). One possible Correspondence: Professor P-Y Wang, Department of Social Medicine and reason for this difference is high intake of phytoestrogens; Health Education, School of Public Health, Peking University, Xueyuan Road Asian people consume soy 10–20 times more than Western 38, Haidian District, Beijing, China. E-mail: [email protected] people (Kimira et al., 1998; Ho et al., 2003). Soy isoflavones Contributors: P-YW and RK contributed to the design of this article. D-FM and comprise mainly genistein, daidzein and glycitein, which L-QQ contributed to the collection of data and statistical analysis, and have have structures similar to that of 17b-estradiol, a potential also contributed equally to the work. All authors read and approved this alternative to HRT (Knight and Eden, 1996). However, the article. Received 25 October 2006; revised 14 February 2007; accepted 19 February effects of isoflavones on bone metabolism appear incon- 2007; published online 28 March 2007 sistent in RCTs. Thus, a statistical method of combining Effects of isoflavone on bone resorption and formation D-F Ma et al 156 these diverse data is needed to evaluate the usefulness of effects model considers both intra- and inter-study varia- isoflavone therapy. Meta-analysis combines or integrates the tions, it is more conservative and hence more appropriate results of several studies to provide increased statistical than an estimate from the fixed-effects model for an analysis power for the quantitative identification of trends (Brockwell such as this (Zhuo et al., 2004). Thus, we report results from and Gordon, 2001). In the RCTs, urinary deoxypyridinoline the random-effects model. To assess the heterogeneity (Dpyr) was generally used as a bone resorption marker, and (apparent diversity in weighted mean differences across serum bone-specific alkaline phosphatase (BAP) was the studies), we conducted a test based on w2 distribution. The most commonly used index of bone formation. Therefore, funnel plot was performed to detect publication bias. we identified all reported RCTs related to the effects of isoflavones on Dpyr or BAP and analyzed the effects of isoflavones on bone metabolism quantitatively. Results and discussion

The trial flow chart is illustrated in Figure 1. Our literature Materials and methods search identified 31 RCTs. Twenty-two studies were excluded because of lack of indices of interest (Agnusdei et al., MEDLINE (January 1966–April 2006), the Cochrane Con- 1997a, b; Gambacciani et al., 1997a, b; Potter et al., 1998; trolled Trials Register, EMBASE (1985–January 2006), Science Alekel et al., 2000; Wangen et al., 2000; Clifton-Bligh et al., Citation Index and PUBMED (updated till April 2006) were 2001; Katase et al., 2001; Anderson et al., 2002; Chiechi used to search articles that described RCTs investigating the et al., 2002; Lucas et al., 2002; Chen et al., 2003, 2004; Jones effect of isoflavones on bone metabolism. Titles, abstracts et al., 2003; Schult et al., 2003; Atkinson et al., 2004; and subject headings in the databases were searched with Harkness et al., 2004; Olsen et al., 2004; Mori et al., the help of the following keywords: bone, osteoporosis, bone 2004a, b), non-randomization (Dalais et al., 1998), lack of a metabolism, phytoestrogens, soy, isoflavones, genistein, control group (Agnusdei et al., 1997a, b), insufficient original daidzein, Dpyr or BAP. We also examined all references of data or baseline values (Gambacciani et al., 1997a, b; Khalil related reviews and papers identified by the search. Addi- et al., 2002). Thus, nine studies with a total of 432 subjects tionally, we contacted the experts for the obtaining of were included in this meta-analysis, in which five studies unpublished data. Studies were selected for analysis if they had quality score of five; three studies had quality score of met all of the following criteria: (1) subjects were limited to four and one study had quality score of three (Morabito et al., female; (2) subjects ingested soy products or isoflavones for 2002; Uesugi et al., 2002; Yamori et al., 2002; Arjmandi at least 4 weeks; (3) the RCT included a parallel control et al., 2003, 2005; Dalais et al., 2003; Brooks et al., 2004; Mori group; and (4) Dpyr or BAP was used as an index of bone et al., 2004a, b; Nikander et al., 2004) (Table 1). In five of metabolism. If the study sample was found to overlap with these studies, isolated soy protein that contained mainly that in another article or if two articles described aspects of isoflavones was used, and isoflavone tablets were used in the same study, only the publication with the largest sample other studies. Isoflavone intake varied from 37.3–118 mg/day group was used. in the various treatment groups. The duration of treatment Two researchers extracted data independently. A data also varied widely, ranging from 4 to 48 weeks including collection form was designed, and data were entered into three studies with a duration exceeding 12 weeks. Six studies the form twice to reduce input errors. The items entered in were performed in Caucasian women and three studies in the form included participant characteristics, treatment Asian women. Seven of these nine studies were carried out in duration, interventional dosage, and values of relevant indices before and after isoflavone or placebo treatments. Jadad scores were used to measure the quality of the RCTs Potentially relevant articles identified and screened for retrieval (n = 673) (Jadad et al., 1996). Two reviewers rated study quality independently, there was (90%) agreement on Jadad scores. Articles excluded because not RCTs (n = 642) If the reviews disagreed, a final score was reached by RCTs retrieved for more detailed evaluation (n = 31) discussion. In this meta-analysis, we obtained the mean differences RCTs excluded: did not meet the inclusion criteria (n = 22) from the post-randomization baseline and post-treatment RCTs included in meta-analysis (n = 9) values for each trial, and calculated the pooled standard

deviation of the mean differences according to the method RCTs withdrawn because did not report: of Yeung and Yu (2003). Weighted mean difference was Deoxypyridinoline (n = 0) Serum bone-specific alkaline phosphatase (n = 4) calculated by subtracting the mean difference of the control

group from that of the treatment group. The inverse variance RCTs with usable information on: method was used to pool the weighted mean difference with Deoxypyridinoline (n = 9) Serum bone-specific alkaline phosphatase (n = 5) Review Manager 4.2 software (Nordic Cochrane Center, Oxford, England). Because the estimate from the random- Figure 1 Results of search for eligible studies.

European Journal of Clinical Nutrition Effects of isoflavone on bone resorption and formation D-F Ma et al 157 Table 1 Characteristics of the nine selected randomized controlled trials of isoflavone intake in women

Study Length of treatment No. of subjects Menopausal Intervention Indices Quality score (weeks) status

Arjmandi et al. (2003) 12 42 Post ISP: soy protein 40 g/day, Iso 88.4 mg/day BAP, Dpyr 5 Con: Milk-based protein 40 g/day Arjmandi et al. (2005) 48 62 Post ISP: soy protein 25 g/day, Iso: 60 mg/day BAP, Dpyr 5 Con: placebo Brooks et al. (2004) 16 28 Post ISP: soy protein 25 g/day, Iso: 41.9 mg/day BAP, Dpyr 4a Con: placebo Dalais et al. (2003) 12 78 Post ISP: soy protein 40 g/day, Iso: 118 mg/day Dpyr 5 Con: placebo Morabito et al. (2002) 48 60 Post Iso: genistein 54 mg/day BAP, Dpyr 4b Con: placebo Mori et al. (2004) 4 43 Peri Iso: Iso 40 mg/day Dpyr 3a,b Con: placebo Nikander et al. (2004) 12 56 Post Iso: Iso 114 mg/day BAP, Dpyr 5 Con: placebo Uesugi et al. (2002) 4 23 Peri ISP: Iso 61.8 mg/day Dpyr 5 Con: placebo Yamori et al. (2002) 10 40 Post Iso: Iso 37.3 mg/day Dpyr 4b Con: placebo

Abbreviations: BAP, serum bone-specific alkaline phosphatase; Con, control group; Dpyr, deoxypyridinoline; Iso, isoflavone tablet; ISP, isolated soy protein; peri, peri-menopause; post, post-menopause. Highest total score is 5. aPoints were deleted from Quality score because there was no description of withdrawal and dropouts. bPoints were deleted from Quality score because the method of blinding was either not described or not appropriate. post-menopausal women. The age of the female being Favors treatment Favors control studied ranged from 51 to 62.4 years. In all RCTs, subjects Arjmandi 2003 were healthy and were not undergoing any other therapy for Arjmandi 2005 osteoporosis; common soy diets such as tofu and natto were Brooks 2004 restricted for the treatment duration; and the daily nutrient intake assessed by food-frequency questionnaire or 3-day Dalais 2003 dietary record for subjects in both treatment groups was Morabito 2002 similar before and after the treatment period. All studies Mori 2004 reported no significant differences regarding baseline char- Nikander 2004 acteristics such as age, body mass index, urinary isoflavones and Dpyr excretion or serum BAP between groups. There Uesugi 2002 were also no significant weight changes or negative side Yamori 2002 effects reported. Total Bone metabolism is a complex process involving bone -10 -5 0 5 10 remodeling and bone modeling. Dpyr is a small cross-linking Weighted mean difference, 95% CI peptide of the type I collagen molecule. During bone remodeling, Dpyr is released into the circulatory system Figure 2 Weighted mean difference of urinary Dpyr concentration and excreted in the urine. It is thought to be more specific after treatment with isoflavone. Lines correspond to 95% CI. The CI of the Morabito study is from À15.87 to À6.13 nmol/mmol. than other classical bone resorption markers (Demers, 1992). In the present analysis, nine studies reported values of urinary Dpyr before and after isoflavone or placebo treat- À2.08 nmol/mmol (95% confidence interval (CI) ¼À3.82 to ments. Three of nine studies did not find a significant effect À0.34 nmol/mmol) (Figure 2). Even when we excluded the of isoflavones on bone metabolism and the remainder found trial by Morabito et al. (2002), in which the change was that isoflavone intake significantly decreased urinary Dpyr. much larger than that in other trials, isoflavones were still This discrepancy may be explained by the different chemical associated with a significant decrease of À1.22 nmol/mmol forms of isoflavone, various ‘equol producer’ states of the (95% CI ¼À2.41 to À0.02 nmol/mmol). subjects and the influence of other foods eaten during the Further analysis of the effects of isoflavones on urinary study (Cassidy et al., 2006). Moreover, limited sample sizes Dpyr is shown in Table 2. It is nowadays a general viewpoint often prevent the detection of significant effects in indivi- that soy protein and isoflavones are needed together for dual studies. When we combined the nine studies, the beneficial effects. Unexpectedly, when the five studies in isoflavones significantly decreased urinary Dpyr by which isolated soy protein was used as an intervention were

European Journal of Clinical Nutrition Effects of isoflavone on bone resorption and formation D-F Ma et al 158 Table 2 Subgroup analysis of the effects of isoflavones on Dpyr

Subgroup outcome No. of references No. of subjects Treatment effect on Dpyr (nmol/mmol)a Heterogeneity P-value

Form of intervention Isoflavone tablet 19, 20, 21, 23 199 À4.59 [À8.35, À0.83]b o0.05 Isolated soy protein 15, 16, 17, 18, 22 233 À0.23 [À1.02, 0.57] 0.45

Isoflavone intake (mg/day) p90 15, 16, 17, 19, 20, 22, 23 298 À2.34 [À4.46, À0.22]b o0.05 490 18, 21 134 À1.20 [À3.76, 1.36] 0.49

Treatment length (weeks) p12 15, 18, 20, 21, 22, 23 282 À2.03 [À3.20, À0.85]b 0.71 412 16, 17, 19 150 À3.36 [À8.72, 1.99] o0.05

Race Asian 20, 22, 23 106 À2.79 [À4.55, À1.02]b 0.56 Western 15, 16, 17, 18, 19, 21 326 À2.03 [À4.27, 0.22] o0.05

Menopausal status Peri-menopause 20, 22 66 À1.50 [À4.45, 1.45] 0.88 Post-menopause 15, 16, 17, 18, 19, 21, 23 366 À2.47 [À4.31, À0.52]b o0.05

Abbreviations: CI, confidence interval; Dpyr, deoxypyridinoline. a95% CI in square bracket. bStatistically significant.

analyzed collectively, there was no significant effect on resorption (Uesugi et al., 2002; Yamori et al., 2002). In the urinary Dpyr. This suggests that the components of soy present analysis, we could not judge whether the effects protein are complex and include both favorable and adverse become more obvious when isoflavones are consumed for factors (Setchell et al., 1984). Further researches are required more than 12 weeks because of the large range of CIs. to determine the effective components and their mutual Interestingly, we found that isoflavone intake had sig- reaction with isoflavones. Branca (2003) ever reviewed the nificant effects on urinary Dpyr in Asian women but not available literature and recommended that 90 mg isoflavones Caucasian women after sub-group analysis. Isoflavone is per day is required to achieve bone health benefits. In the converted into its active metabolite equol and inactive present analysis, significant reduction in urinary Dpyr did metabolite p-ethylphenol in intestinal flora, respectively. not change when studies with isoflavone intake of more Equol is associated with an increased benefit of isoflavones than 90 mg/day were not included. Potter et al. (1998) on bone metabolism (Setchell et al., 2002). Approximately showed that 90 mg isoflavones was able to attenuate bone one-third of Caucasian women can metabolize isoflavones loss; however, Dalais et al. (2003) reported that soy protein into equol, whereas more than half of Asian women possess supplementation with the highest dosage of 118 mg/day this capacity (Setchell et al., 1984; Watanabe et al., 1998). isoflavones did not appear to have estrogenic effects on Because the hormone levels of peri-menopausal women are markers of bone resorption. This suggests that isoflavones quite diverse, we pooled results of all trials involving post- exert biphasic dose-dependent effects on bone metabolism, menopausal women who have stable lower endogenous stimulating osteogenesis at low concentrations and inhibit- estrogen levels. The pooled result from seven trials showed ing osteogenesis at high concentrations (Dang and Lowik, that isoflavones have a slightly stronger favorable effect in 2005). Animal studies further indicated that low-dose post-menopausal women than in peri-menopausal women. genistein, a common kind of isoflavone, increased rat femur In a crossover study, Mei et al. (2001) found that isoflavone bone retention, whereas high doses were less effective intake of 53.3 mg/day was associated with increased bone (Anderson et al., 1998). An intake of 90 mg/day soy mineral density in post-menopausal but not in pre-meno- isoflavones can be attained by daily consumption of two pausal women. Together with the findings of other studies, packs (92 g) of natto, or 249 g miso, amounts that are this suggests that isoflavones may act as an estrogen agonist consumed habitually by the population of countries such as under estrogen-depletion circumstances to provide beneficial China and Japan (Arai et al., 2000). When the intervention effects on bone (Xu et al., 1998). duration was shortened to less than 12 weeks, the effect of During bone modeling, osteoblasts produce BAP, a non- isoflavones was still significant. Some studies also indicated collagenic protein that is the biochemical marker of bone that treatment lasting less than 12 weeks may be adequate formation, which is frequently used (Gomez et al., 1995). In to produce the effect of isoflavones on indices of bone five studies that examined serum BAP values, we found a

European Journal of Clinical Nutrition Effects of isoflavone on bone resorption and formation D-F Ma et al 159 Favors control Favors treatment 1.2

Arjmandi 2003 1

Arjmandi 2005 0.8 Brooks 2004 0.6 Morabito 2002 1/SE 0.4 Nikander 2004 TotalTotal 0.2

-10 -5 0 5 10 0 -12 -8 -4 0 4 Weighted mean difference, 95% CI Weighted mean difference Figure 3 Weighted mean difference of serum BAP concentration after treatment with isoflavone. Lines correspond to 95% CI. Figure 4 Heterogeneity of difference in Dpyr after isoflavone treatment. Funnel plot showing no significant publication bias. significant increase of 1.48 mg/l (95% CI ¼ 0.22–2.75 mg/l) in weighted mean difference. Even if 30 of these null results serum BAP, suggesting that isoflavones have a favorable were added, the pooled results changed little. This suggested effect on bone formation (Figure 3). Because of the low that unpublished studies like published studies with non- number of studies, subgroup analysis was not appropriate. significant results do not seem to influence this combined- After isoflavone administration, Dpyr and BAP parameters effect estimate over a large range. can change quickly; however, bone mineral density and Although the relatively low number of studies limited the content change slowly. Ravn et al. (1996) observed 979 power of our meta-analysis, the results clearly suggested women and found that high bone turnover is associated with that isoflavones contribute significantly to inhibiting bone a significantly lower bone mass in menopausal women. Then resorption and stimulating bone formation, especially in they further indicated that high bone turnover is a risk factor post-menopausal women. These favorable effects are ob- for osteoporosis (Ravn et al., 1997). Harkness et al. (2004) served even if isoflavones are consumed o90 mg/day or for found that soy isoflavones significantly decreased bone less than 12 weeks. Further RCTs are required to provide resorption and significantly increased bone mineral density more comprehensive information about the effective within 24 weeks. Recently, some RCTs reported that amount and duration of isoflavone intake. isoflavone intake less than 6 months significantly increased bone mineral density or bone mineral content (Alekel et al., 2000; Mori et al., 2004a, b). Thus, the change of Dpyr and Acknowledgements BAP is relevant to bone mineral density and content. In our meta-analysis, we found that isoflavone supplementation This research was supported in part by a grant from National significantly decreased urinary Dpyr and increased serum Natural Science Foundation of China (No. 30471448) to Dr BAP. Cassidy et al. (2006) concluded that isoflavone intake Pei-Yu Wang. may be beneficial for bone in post-menopausal women in a review. The mechanism of the Dpyr-lowering and BAP- increasing effects of isoflavones is not well understood. References Likely mechanisms include prevention of urinary calcium Agnusdei D, Bufalino L (1997a). Efficacy of ipriflavone in established loss, beneficial effects on osteoblasts and influences on the osteoporosis and long-term safety. Calcif Tissue Int 61, S23–S27. secretion of calcitonin, which suppresses bone resorption Agnusdei D, Crepaldi G, Isaia G, Mazzuoli G, Ortikabu S, Passeri M (Kurzer and Xu, 1997). Isoflavones, as phytoestrogens, may et al. (1997b). A double blind placebo-controlled trial of ipri- stimulate estrogen receptors. Because estrogen receptors flavone for prevention of postmenopausal spinal bone loss. Calcif Tissue Int 61, 142–147. have been found in osteoblasts, isoflavones may cause an Alekel DL, Germain AS, Peterson CT, Hanson KB, Stewart JW, Toda T alteration in the production of some proteins in bone via the (2000). Isoflavone-rich soy protein isolate attenuates bone loss in estrogen-receptor pathway (Dang and Lowik, 2005). the lumbar spine of perimenopausal women. Am J Clin Nutr 72, The potential for publication bias was examined by 844–852. construction of a ‘funnel plot’ of the relation between the Anderson JJ, Ambrose WW, Garner SC (1998). Biphasic effects of genistein on bone tissue in the ovariectomized, lactating rat reciprocal of standard error and the weighted mean differ- model. Proc Soc Exp Biol Med 217, 345–350. ence. It did not provide strong evidence of publication bias Anderson JJ, Chen X, Boass A, Symons M, Kohlmeier M, Renner JB for either index (Figure 4). Generally, studies with significant et al. (2002). Soy isoflavones: no effects on bone mineral content and bone mineral density in healthy, menstruating young adult results were easier to publish than those with nonsignificant women after one year. J Am Coll Nutr 215, 388–393. results (Qin et al., 2004). Thus, we randomly added three Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, Kinae N null results appearing in the analysis and calculated a new (2000). Dietary intakes of flavonols, flavones and isoflavones by

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