Relationship between tooth loss and mortality in 80-year-old Japanese community-dwelling subjects

  • Toshihiro Ansai1Email author,

    Affiliated with

    • Yutaka Takata2,

      Affiliated with

      • Inho Soh1,

        Affiliated with

        • Shuji Awano1,

          Affiliated with

          • Akihiro Yoshida1,

            Affiliated with

            • Kazuo Sonoki2,

              Affiliated with

              • Tomoko Hamasaki3,

                Affiliated with

                • Takehiro Torisu2,

                  Affiliated with

                  • Akira Sogame4,

                    Affiliated with

                    • Naoko Shimada5 and

                      Affiliated with

                      • Tadamichi Takehara1

                        Affiliated with

                        BMC Public Health201010:386

                        DOI: 10.1186/1471-2458-10-386

                        Received: 18 August 2009

                        Accepted: 1 July 2010

                        Published: 1 July 2010

                        Abstract

                        Background

                        Findings from several studies suggest associations between tooth loss and health outcomes, including malnutrition, poor quality of life, and mortality, in older individuals. However, limited information is available regarding whether those associations remain true in very elderly subjects after adequately considering confounding factors such as sex and smoking status. Herein, we determined whether the number of teeth in 80-year-old subjects is an independent predictor of mortality.

                        Methods

                        We initially contacted 1282 80-year-old community-dwelling individuals born in 1917, of whom 697 responded and participated in a baseline study, with follow-up examinations conducted 4 and 5.5 years later. Data from interviews and medical and oral examinations were obtained, and oral health was determined according to the number of teeth remaining in the oral cavity.

                        Results

                        A total of 108 and 157 subjects died in 4 years and 5.5 years, respectively, after the baseline study. Tooth loss was significantly associated with mortality at age 85.5, but not at age 84, after adjusting for potential confounders. When the analysis was stratified by sex, we found a stronger association in females in follow-up examinations conducted at both 4- and 5.5 years. On the other hand, the effect of tooth loss on mortality was not significantly different between smokers and non-smokers.

                        Conclusion

                        Tooth loss is a significant predictor of mortality independent of health factors, socio-economic status, and lifestyle in octogenarians, with a stronger association in females.

                        Background

                        Associations between tooth loss and mortality have been reported, though issues related to important confounding factors such as age, gender, and smoking status, which may be related to oral health and are also risk factors for mortality, remain to be clarified. In a number of studies, the age range of the subjects is broad [13], while there is a limited number of reports regarding the association of tooth loss and mortality in subjects who are the same age. An important merit of limiting a study population to the same age is that there is no need to consider the effects of age on changes that may potentially confound factors related to systemic condition. Thus, results obtained with such a study design would be expected to reveal a much higher level of scientific evidence. In a search of published studies regarding the association between tooth loss and mortality in a single age group, we found 3 reports: a 10-year cohort study of 80-year-old subjects by Hämalainen et al., in which the hazard ratio (HR) for number of missing teeth was 1.026 [4], an 18-year cohort study of 1803 70-year-old subjects by Österberg et al., in which the HRs for numbers of present teeth in males and females were 0.98 and 0.79, respectively [5], and an investigation by Holm-Pedersen et al. of 573 70-year-old Danish individuals examined every 5 years for 20 years, in which the HR for mortality 21 years after beginning the study in subjects who were edentulous at the age 70 was 1.26 [6].

                        Smoking is a major risk factor for oral diseases, including periodontitis and systemic diseases, as well as cardiovascular disease and aspiration pneumonia. However, there is concern regarding the effects of smoking status, as previously published reports included inadequate measurement and control for smoking status, and confounding factors and effect modification have become increasingly important. Furthermore, another important issue is the source of study subjects, as nearly all published studies investigated subjects in Western countries. To the best of our knowledge, only 1 report focused on a single-age non-Western population, in which a Japanese population-based cohort was investigated [7]. In that study, the subjects were elderly (mean age approximately 82 years old) and the age range was narrow, though the sample size was small (59 males, 59 females) and smoking status was based on information obtained at the age of 60.

                        In the present cohort study, we analyzed the association between number of teeth at the age of 80-years old and mortality after 4 and 5.5 years, and then evaluated the effects of sex and smoking status on that association.

                        Methods

                        Study population

                        A total of 1282 80-year-old candidates from 3 cities (Buzen, Yukuhashi, and Munakata), 4 towns (Katsuyama, Tsuiki, Toyotsu, and Kanda), 1 village (Shinyoshitomi), and 1 ward (Tobata of Kitakyushu City) in Fukuoka Prefecture on Kyushu Island in southern Japan were invited to participate. Those 9 locations were selected randomly from urban, suburban, and rural communities to achieve a balance of living environments in terms of socio-demographic backgrounds, dietary habits, health behaviors, and available medical care. This study was designed as an investigation of a representative population of individuals residing in the eastern area of Kyushu Island who were born in 1917. The percentage of 80-year-old individuals living in the study locations was approximately 0.62% of all residents, which was similar to the percentage (0.64%) of 80-year-old individuals residing in all of Kyushu. Of those 1282 individuals, 697 (54.4%) (277 males, 420 females) agreed to participate in the present study and completed a questionnaire regarding lifestyle, and oral and systemic health, and also underwent physical, laboratory blood, and oral examinations. The Human Investigations Committee of Kyushu Dental College approved the survey and all subjects gave written informed consent prior to participation.

                        Baseline data

                        The baseline survey was performed in March 1998, during which time the participants took part in a face-to-face interview, and answered a questionnaire containing 37 questions about oral and systemic health status, use of medical (or dental) services, personal hygiene, healthcare practices (including smoking habit), and medical conditions [8]. Dental examinations were also performed using WHO criteria [9] by 3 trained dentists assisted by a dental nurse. In this study, only the number of teeth and denture status (fixed prostheses, partial or full dentures) were used for analysis. Remaining roots were not counted as retained teeth.

                        Follow-up survey

                        This study utilized data obtained from 1998 to 2003 and was designed to analyze the results of follow-up examinations of all subjects in our previous study at the ages of 84 and 85.5 years old. Information on the survival of the subjects was collected from the registers at the Public Health Centers of each district included in the study. Of all subjects who took part in the baseline survey, there were no losses during the follow-up period, as even subjects who had moved away from Fukuoka Prefecture after the initial survey were successfully traced.

                        Statistical analyses

                        Power analysis was performed using the software package G-power. The statistical power of this study was found to be 87%, with sample sizes of 540 for n1 and 157 for n2, an effect size of 0.25, and an α value of 0.05 set (two tailed t-test with accuracy mode), which demonstrated reasonable power. The main characteristics of participants who died versus those who remained alive during the follow-up period are reported as mean values (standard deviation, SD) or percentages. Differences between groups were tested using ANOVA for continuous variables and a Pearson chi-square test for categorical variables. Covariates determined at the baseline were sex, marital status, place of residence, medical history, laboratory blood test results (serum total cholesterol, fasting serum glucose, serum albumin), systolic and diastolic blood pressures, body mass index, regular physical activity, regular checkup by family doctor, self-rated general health, smoking habit, and daily alcohol consumption. Place of residence was included as a proxy for socioeconomic status.

                        The association between number of teeth and mortality was analyzed using multivariate logistic analysis. Preliminary analyses included univariate analyses of the association between number of teeth and mortality, covariates and number of teeth, and covariates and mortality. Multivariate analyses included variables that were related to number of teeth and mortality (P < 0.05) in bivariate analyses, which included gender, smoking status, serum total cholesterol, fasting serum glucose, serum albumin, place of residence, marital status, and body mass index. We sequentially tested the effect modification of tooth loss-mortality associations by adding interaction terms between tooth loss and sex, and smoking status according to likelihood ratio tests.

                        All statistical analyses were performed using SPSS 14.0 for Windows (SPSS, Chicago, Illinois, USA).

                        Results

                        After 4 years, 108 of the subjects (58 males, 50 females) had died, while 157 (81 males, 76 females) had died by 5.5 years. Socio-demographic and medical characteristics of the study participants at the age of 80 are shown in Table 1. The deceased individuals had lower total cholesterol, higher glucose concentration, and lower serum albumin values as compared to those who remained alive, while BMI was higher in the survivors. Men and smokers were more likely to die during the follow-up period. The mortality rate was higher in males than in females at both 4 and 5.5 years, namely, 20.9% vs. 11.9%, and 29.2% vs. 18.1%, respectively. The dental characteristics of the subjects are shown in Table 2. Among the 697 subjects, the prevalence of edentulism was 33.9% (n = 236), and the proportion with 20 teeth or more was 15.2% (n = 106). As compared to edentulous males, edentulous females had a 1.3-fold higher rate of mortality in the 4-year follow-up examinations and a 1.2-fold higher rate of mortality in the 5.5-year follow-up examinations. The number of teeth (or number of missing teeth) and health-related behaviors such as brushing habit and regular check-ups by dentists differed between the survivors and deceased subjects.
                        Table 1

                        Sociodemographic and medical characteristics of 80-year-old subjects based on survival during 5.5-year follow-up period

                        Characteristic

                        Alive

                        Died

                        P value

                         

                        (n = 540)

                        (n = 157)

                         

                        Sex

                           

                           Female

                        344 (81.9%)

                        76 (18.1%)

                        0.001

                           Male

                        196 (70.8%)

                        81 (29.2%)

                         

                        Marital statusa

                           

                           Currently married

                        253 (74.2%)

                        88 (25.8%)

                        0.022

                           Currently unmarried

                        268 (80.9%)

                        63 (19.1%)

                         

                        Place of residenceb

                           

                           Ward

                        149 (85.6%)

                        25 (14.4%)

                        0.008

                           City

                        215 (76.2%)

                        67 (23.8%)

                         

                           Town/village

                        170 (73.3%)

                        62 (26.7%)

                         

                        Medical history

                           

                           CVD

                        90 (76.3%)

                        28 (23.7%)

                        0.358

                           Cancer

                        2 (66.7%)

                        1 (33.3%)

                        0.528

                           Pneumonia

                        13 (72.2%)

                        5 (27.7%)

                        0.364

                        Medical examinations

                           

                           Serum total cholesterol (mg/dL)

                        208.1 (37.4)

                        196.8 (40.1)

                        0.002

                           Fasting serum glucose (mg/dL)

                        116.5 (44.5)

                        138.9 (74.6)

                        < 0.001

                           Serum albumin (g/dL)

                        4.3 (0.29)

                        4.1 (0.35)

                        < 0.001

                        Systolic blood pressure (mmHg)

                        150.8 (23.6)

                        149.2 (21.8)

                        0.463

                           Diastolic blood pressure (mmHg)

                        79.3 (12.5)

                        77.5 (11.7)

                        0.117

                        Disease risk factors

                           

                           Body mass index

                        22.9 (3.3)

                        21.9 (3.2)

                        0.002

                           Regular physical activityc

                           

                        Yes

                        300 (77.9%)

                        85 (22.1%)

                        0.614

                        No

                        212 (76.3%)

                        66 (23.7%)

                         

                           Regular checkup by family doctord

                           

                        Yes

                        430 (77.5%)

                        125 (22.5%)

                        0.688

                        No

                        81 (75.7%)

                        26 (24.3%)

                         

                           Self-rated general healthe

                           

                        Very good

                        224 (79.2%)

                        59 (20.8%)

                        0.140

                        Good

                        245 (78.3%)

                        68 (21.7%)

                         

                        Moderate

                        61 (69.3%)

                        27 (30.7%)

                         

                           Smoking habitc

                           

                        Smoker

                        173 (68.4%)

                        80 (31.6%)

                        < 0.001

                        Non-smoker

                        358 (82.9%)

                        74 (17.1%)

                         

                           Daily alcohol consumptionf

                           

                        Non-drinker

                        221 (75.4%)

                        72 (24.6%)

                         

                        Drinker

                        294 (79.0%)

                        78 (21.0%)

                        0.156

                        Abbreviations: CVD, cardiovascular disease.

                        Data indicate the number of subjects (%) or mean (SD).

                        Data available for a672, b688, c663, d662, e684, and f665 people, respectively.

                        Differences between groups were tested using a Pearson chi-square test for categorical variables and an ANOVA for continuous variables.

                        Table 2

                        Dental characteristics of 80-year-old subjects based on survival during 5.5-year follow-up period

                        Characteristic

                        Alive

                        Died

                        P value

                         

                        (n = 540)

                        (n = 157)

                         

                        Number of teetha

                        8.4 (9.0)

                        6.7 (8.2)

                        0.031

                        Number of missing teetha

                        23.5 (9.0)

                        25.3 (8.2)

                        0.026

                        Number of edentulous subjectsa

                        178 (75.4%)

                        58 (24.6%)

                        0.196

                        Number of dentate subjects

                        353 (78.6%)

                        96 (21.4%)

                         

                        Number of teeth & denture statusb

                           

                           No denture

                        89 (83.9%)

                        17 (16.1%)

                        0.108

                           PDs

                        114 (80.9%)

                        27 (19.1%)

                         

                           Either PD or FD

                        142 (74.3%)

                        49 (25.7%)

                         

                           FDs

                        164 (73.9%)

                        58 (26.1%)

                         

                        Self-carec

                           

                           Yes

                        400 (78.7%)

                        108 (21.3%)

                        0.091

                           No

                        103 (72.1%)

                        40 (27.9%)

                         

                        Toothbrushingd

                           

                           ≥ 2 times/day

                        240 (83.6%)

                        47 (16.4%)

                        0.004

                           < 2 times/day

                        213 (73.7%)

                        76 (26.3%)

                         

                        Regular checkups by dentiste

                           

                           Yes

                        360 (79.6%)

                        92 (20.4%)

                        0.027

                           No

                        151 (71.9%)

                        59 (28.1%)

                         

                        Abbreviations: FD, full denture; PD, partial denture (including fixed prostheses).

                        Data indicate the number of subjects (%) or mean (SD).

                        Data available for a685, b660, c651, d576, and e662 people, respectively.

                        Differences between groups were tested using a Pearson chi-square test for categorical variables and an ANOVA for continuous variables.

                        Table 3 shows adjusted odds ratios (ORs) for number of teeth in regard to mortality at the 4-year follow-up examination. The overall OR was 0.980 [95% confidence interval (CI), 0.953-1.007]. We performed interaction tests to investigate whether sex and smoking status were associated with tooth loss, and found a significant association between the risk of tooth loss and sex (P = 0.006), while there was no such association between smoking status and tooth loss. Accordingly, it was suggested that sex should be treated as an effect modifier in the following analyses. When stratified by sex, only the OR for number of teeth in females was significant (0.937, 95% CI, 0.889-0.987), while there was not a significant association in males (OR, 1.004, 95% CI, 0.969-1.040).
                        Table 3

                        Odds ratios for effect of number of teeth on mortality at 4-year follow-up examination according to sex: Multivariate logistic regression analysis

                           

                        OR by sexc

                         

                        Adjusted OR (95% CI) a

                        P

                         

                        Cases

                        Adjusted OR (95% CI) b

                        P

                        Number of teeth

                        0.980 (0.953-1.007)

                        0.148

                        Males

                        277

                        1.004 (0.969-1.040)

                        0.841

                           

                        Females

                        420

                        0.937 (0.889-0.987)

                        0.014

                        OR: odds ratio, CI: confidence interval.

                        aAdjusted for sex, smoking habit, serum total cholesterol, fasting serum glucose, serum albumin, place of residence, marital status, and body mass index.

                        bAdjusted for smoking habit, serum total cholesterol, fasting serum glucose, serum albumin, place of residence, marital status, and body mass index.

                        cStratified by sex due to results of interaction tests, which indicated a statistically significant difference for risk related to tooth loss between males and females (P = 0.006) (see statistical analyses).

                        Table 4 shows adjusted ORs for number of teeth in regard to mortality at the 5.5-year follow-up examination. The overall OR for number of teeth was 0.972 (95% CI, 0.948-0.996). Furthermore, the adjusted OR for female gender was 0.946 (95% CI, 0.907-0.987), while that for male gender was 0.986 (95% CI, 0.955-1.019), which was not significantly different.
                        Table 4

                        Odds ratios for effect of number of teeth on mortality at 5.5-year follow-up examination according to sex: Multivariate logistic regression analysis

                           

                        OR by sexc

                         

                        Adjusted OR (95% CI) a

                        P

                         

                        Cases

                        Adjusted OR (95% CI) b

                        P

                        Number of teeth

                        0.972 (0.948-0.996)

                        0.023

                        Males

                        277

                        0.986 (0.955-1.019)

                        0.399

                           

                        Females

                        420

                        0.946 (0.907-0.987)

                        0.011

                        OR: odds ratio, CI: confidence interval.

                        aAdjusted for sex, smoking habit, serum total cholesterol, fasting serum glucose, serum albumin, place of residence, marital status, and body mass index.

                        bAdjusted for smoking habit, serum total cholesterol, fasting serum glucose, serum albumin, place of residence, marital status, and body mass index.

                        cStratified by sex due to results of interaction tests, which indicated a statistically significant difference for risk related to tooth loss between males and females (P = 0.006) (see statistical analyses).

                        Discussion

                        In the present longitudinal study, we investigated associations between tooth loss and mortality in 80-year-old Japanese subjects, and obtained the following important findings: (1) tooth loss was associated with higher mortality in our 80-year-old subjects; (2) the associations differed due to sex, with female gender significantly associated with mortality; and (3) smoking status did not affect the association between tooth loss and mortality. Abnet et al. [1] reported a difference between genders for the association between tooth loss and mortality, as that association was stronger in males, though the difference between females and males was only slight. Also, 2 different reports presented by Japanese researchers found an association between the number of teeth and mortality in elderly males [2, 7]. Their findings did not coincide with ours, though the reasons for the differences remain unclear. They might be partly explained by the different age range and life-style related factors of the study subjects.

                        There are a number of reports regarding the association between number of teeth and mortality. However, some of those analyzed subjects with a broad age range, used a small number of subjects, studied mixed genders, and/or investigated subjects with smoking habit information lacking. In a literature search, we found only 3 reports of a cohort study of the association between number of teeth and mortality in a single age elderly population [46]. The baseline ages of the subjects in those reports were either 80 or 70 years old, and they presented similar conclusions stating that tooth loss is independently associated with mortality and independent of other potential confounding factors. On the other hand, treatment of smoking habit varied among those 3 reports, as Hämalainen et al. [4] did not provide adequate information regarding smoking status, while the others indicated smoking status, but did not show the effects of smoking [5, 6]. An explanation for our result regarding the role of smoking status may be related to survivor effects. In our subjects who survived to the age of 85.5, the effect of smoking may have been reduced.

                        The mechanism of the association between tooth loss and mortality has been hypothesized to consist of 2 pathways; an infection and inflammation pathway, and a nutritional pathway, as reported by Janket et al. [10]. Tooth loss is known to mainly be caused by periodontal disease, the most common oral infectious disease, indicating an inflammatory burden from a past infection, and periodontitis is associated with a steady increase in circulatory levels of pro-inflammatory cytokines [11, 12]. In addition, inadequate dentition due to tooth loss may affect eating behavior, including mastication and food choice, causing individuals to substantially reduce their intake of fruits, vegetables, and other key nutrients [13], which have been shown to be associated with increased survival and lower cardiovascular mortality [14]. Furthermore, adults who have no natural teeth and wear complete replacement dentures tend to ingest a high fat, low fiber diet [15]. However, we have no direct evidence to support any of those speculations at the present time.

                        A limitation of our study is that the sample consisted largely of generally healthy elderly subjects, who might have been more eager and/or able to participate. Thus, our findings may indicate the association in generally healthy elderly subjects. The response rate to participate in the study was 54%, which was related to location and health-oriented behavior, as individuals who resided in towns, and those who regularly attended check-up examinations by a family doctor or dentist had higher response rates. On the other hand, several findings have been presented regarding the effect of inadequate dental status on mortality of institutionalized elderly individuals [16], in which poor dentition was found to be associated with high overall mortality, thus systematic attention to dental status is recommended.

                        Conclusions

                        In this study, our results showed a significant relationship between tooth loss and mortality in 80-year-old individuals, even after extensive adjustment for potential confounding variables. Since a stronger association was found in females, we recommend that researchers include gender when performing related statistical analyses. Our findings also have significant implications in terms of public health strategy, because they indicate that improving oral health and keeping more natural teeth will increase the health status of the population as well as longevity.

                        Declarations

                        Acknowledgements

                        The authors extend their gratitude to the subjects who participated in this investigation. This investigation was supported by a research grant from the 8020 Promotion Foundation, and by Grants-in-Aid (B) 15390655 and (B) 17390566 from the Ministry of Education, Culture, Sports, and Technology of Japan.

                        Authors’ Affiliations

                        (1)
                        Division of Community Oral Health Science, Department of Health Promotion, Kyushu Dental College
                        (2)
                        Division of General Internal Medicine, Department of Health Promotion, Kyushu Dental College
                        (3)
                        Division of Nutrition, Department of Home Economics, Kyushu Women's University
                        (4)
                        Kurate Office for Health, Human Services and Environmental Issues
                        (5)
                        Kitakyushu Public Health and Welfare Bureau, Kyushu Dental College

                        References

                        1. Abnet CC, Qiao YL, Dawsey SM, Dong ZW, Taylor PR, Mark SD: Tooth loss is associated with increased risk of total death and death from upper gastrointestinal cancer, heart disease, and stroke in a Chinese population-based cohort. Int J Epidemiol 2005, 34:467–74.PubMedView Article
                        2. Fukai K, Takiguchi T, Ando Y, Aoyama H, Miyakawa Y, Ito G, Inoue M, Sasaki H: Mortality rates of community-residing adults with and without dentures. Geriatr Gerontol Int 2008, 8:152–9.PubMedView Article
                        3. Heitmann BL, Gamborg M: Remaining teeth, cardiovascular morbidity and death among adult Danes. Prev Med 2008, 47:156–60.PubMedView Article
                        4. Hämalainen P, Meurman JH, Keskinen M, Heikkinen E: Relationship between dental health and 10-year mortality in a cohort of community-dwelling elderly people. Eur J Oral Sci 2003, 111:291–6.PubMedView Article
                        5. Österberg T, Carlsson GE, Sundh V, Mellstrom D: Number of teeth--a predictor of mortality in 70-year-old subjects. Community Dent Oral Epidemiol 2008, 36:258–68.PubMedView Article
                        6. Holm-Pedersen P, Schultz-Larsen K, Christiansen N, Avlund K: Tooth loss and subsequent disability and mortality in old age. J Am Geriatr Soc 2008, 56:429–35.PubMedView Article
                        7. Morita I, Nakagaki H, Kato K, Murakami T, Tsuboi S, Hayashizaki J, Toyama A, Hashimoto M, Simozato T, Morishita N, et al.: Relationship between survival rates and numbers of natural teeth in an elderly Japanese population. Gerodontology 2006, 23:214–8.PubMedView Article
                        8. Ansai T, Takata Y, Soh I, Akifusa S, Sogame A, Shimada N, Yoshida A, Hamasaki T, Awano S, Fukuhara M, et al.: Relationship between chewing ability and 4-year mortality in a cohort of 80-year-old Japanese people. Oral Dis 2007, 13:214–9.PubMedView Article
                        9. WHO: Oral Health Surveys. 3rd edition. Geneva: WHO; 1987:22–44.
                        10. Janket SJ, Qvarnstrom M, Meurman JH, Baird AE, Nuutinen P, Jones JA: Asymptotic dental score and prevalent coronary heart disease. Circulation 2004, 109:1095–100.PubMedView Article
                        11. D'Aiuto F, Parker M, Andreou G, et al.: Periodontitis and systemic inflammation: control of the local infection is associated with a reduction in serum inflammatory markers. J Dent Res 2004, 83:156–160.PubMedView Article
                        12. Montebugnoli L, Servidio D, Miaton RA, Prati C, Tricoci P, Melloni C: Poor oral health is associated with coronary heart disease and elevated systemic inflammatory and haemostatic factors. J Clin Periodontol 2004, 31:25–29.PubMedView Article
                        13. Sheiham A, Steele J: Does the condition of the mouth and teeth affect the ability to eat certain foods, nutrient and dietary intake and nutritional status amongst older people? Public Health Nutr 2001, 4:797–803.PubMedView Article
                        14. Tucker KL, Hallfrisch J, Qiao N, Muller D, Andres R, Fleg JL: The combination of high fruit and vegetable and low saturated fat intakes is more protective against mortality in aging men than is either alone: the Baltimore Longituginal Study of Aging. J Nutr 2005, 135:556–561.PubMed
                        15. Allen PF: Association between diet, social resources and oral health related quality of life in edentulous patients. J Oral Rehabil 2005, 32:623–628.PubMedView Article
                        16. Ohrui T, Matsui T, Yoshida M, Yoneyama T, Adachi M, Akagawa Y, He M, Yamaga M, Arai H, Sasaki H, et al.: Dental status and mortality in institutionalized elderly people. Geriatr Gerontol Int 2006, 6:101–108.View Article
                        17. Pre-publication history

                          1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2458/​10/​386/​prepub

                        Copyright

                        © Ansai et al. 2010

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.