Association between serum concentrations of 25-hydroxyvitamin D3 and periodontal disease in the US population

¹ From the Department of Periodontology and the Department of Oral Surgery and Oral Radiology, Charité, Humboldt University of Berlin, Berlin (TD); the Department of Epidemiology, German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany (TD); the Department of Oral Health Policy & Epidemiology, Harvard School of Dental Medicine, Boston (KJJ); the Department of Epidemiology, Harvard School of Public Health, Boston (KJJ); the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston (BD-H); the Division of Aging, Brigham and Women's Hospital, Boston (HAB-F); and the Division of Aging, Harvard Medical School, Boston (HAB-F).

² Supported by the Harvard Hartford Foundation, the Kirkland Scholar Award (to HAB-F), and the Charles A King Trust. TD was supported by the German Academic Exchange Service.

³ Reprints not available. Address correspondence to T Dietrich, Department of Health Policy and Health Services Research, Goldman School of Dental Medicine, Boston University, 715 Albany Street, 560 3rd floor, Boston, MA 02118. E-mail: tdietric{at}bu.edu.

ABSTRACT  
Background: Periodontal disease (PD) is a common chronic inflammatory disease and an important risk factor for tooth loss. Vitamin D might affect periodontal disease risk via an effect on bone mineral density (BMD) or via immunomodulatory effects.

Objective: The objective was to evaluate whether serum 25-hydroxyvitamin D₃ [25(OH)D₃] concentrations are associated with PD in the third National Health and Nutrition Examination Survey.

Design: We analyzed data on periodontal attachment loss (AL) and serum 25(OH)D₃ concentrations from 11 202 subjects aged 20 y. Mean AL was modeled in a multiple linear regression with quintile of serum 25(OH)D₃ concentration as an independent variable. The model was stratified by age and sex and was adjusted for age within age groups, race or ethnicity, smoking, diabetes, poverty income ratio, body mass index, estrogen use, and gingival bleeding.

Results: 25(OH)D₃ concentrations were significantly and inversely associated with AL in men and women aged 50 y. Compared with men in the highest 25(OH)D₃ quintile, those in the lowest quintile had a mean AL that was 0.39 mm (95% CI: 0.17, 0.60 mm) higher; in women, the difference in AL between the lowest and highest quintiles was 0.26 mm (0.09, 0.43 mm). In men and women younger than 50 y, there was no significant association between 25(OH)D₃ and AL. The BMD of the total femoral region was not associated with AL and did not mediate the association between 25(OH)D₃ and AL.

Conclusions: Low serum 25(OH)D₃ concentrations may be associated with PD independently of BMD. Given the high prevalence of PD and vitamin D deficiency, these findings may have important public health implications.

Key Words: Vitamin D • periodontal disease • periodontal attachment loss • prevention and control • risk factors • National Health and Nutrition Examination Survey • NHANES

INTRODUCTION  
Periodontal disease is a widespread chronic inflammatory disease that is characterized by the loss of periodontal attachment, ie, periodontal ligament and alveolar bone. It is a primary cause of tooth loss, particularly in the elderly (1-4). Periodontal disease and associated tooth loss affect the quality of life, dietary quality, and nutrient intake (5-7). In fact, recent reports found associations between periodontal disease and increased risks of cardiovascular diseases (8-10). Possible mechanisms involved in these associations are decreased dietary quality and chronic inflammation (5, 11, 12).

The periodontal tissue destruction observed in periodontal disease is widely accepted as being host-mediated through the release of proinflammatory cytokines by local tissues and immune cells in response to the bacteria of dental plaque and their products and metabolites (13). These cytokines have the potential to stimulate bone resorption in a receptive host, which may be most pronounced in persons with low bone density. In fact, several studies reported positive associations between osteoporosis or low bone density and alveolar bone and tooth loss, which indicates that poor bone quality may be a risk factor for periodontal disease (14-20).

Vitamin D might affect periodontal disease both through an effect on bone mineral density (BMD) and through immunomodulatory effects. Vitamin D is well established as being essential for bone growth and preservation. In the elderly, supplementation with vitamin D and calcium is effective in preventing nonvertebral fractures (21, 22). A potential antiinflammatory effect of vitamin D is supported by an increasing amount of literature. The active metabolite of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, has been found to inhibit cytokine production and cell proliferation (23, 24).

One study suggested that supplementation with vitamin D and calcium may significantly reduce tooth loss in the elderly over a period of 3 y (25). However, periodontal disease was not evaluated as an outcome in this study. Hence, whether this benefit was entirely due to a reduction in tooth loss caused by periodontal disease is unclear. Whether vitamin D, calcium, or both are beneficial is also unclear, as is whether the effect is mediated by the BMD or the BMD-independent effects of vitamin D.

Whether measures of periodontal disease are associated with vitamin D status as reflected by serum concentrations of 25-hydroxyvitamin D₃ [25(OH)D₃] is unclear. Therefore, we conducted an analysis to determine whether serum 25(OH)D₃ concentrations are associated with periodontal disease in a large, representative sample of the US population [third National Health and Nutrition Examination Survey (NHANES III)] and, if so, whether this association is mediated by total hip BMD.

SUBJECTS AND METHODS  
Data source
The NHANES III survey was conducted in 2 phases between 1988 and 1994 to study the health and nutritional status of the civilian, noninstitutionalized US population. The survey was designed as a complex, multistage, stratified, clustered sample survey. Interviews were conducted at respondents' homes. Standardized examinations were conducted in mobile examination centers and included extensive dental examinations with periodontal assessments, where appropriate. A detailed description of the survey can be found elsewhere (26). The oral health component of the survey was described in detail previously (27). Furthermore, detailed analyses of destructive periodontal disease, gingival recession, gingival bleeding, dental calculus, and smoking-attributable destructive periodontitis in NHANES III were also published previously (28-30).

Clinical oral examinations
Periodontal measurements were performed at the mesiobuccal and midbuccal sites of all fully erupted teeth except the third molars in 2 randomly selected quadrants, one maxillary and one mandibular. Clinical attachment levels were measured with a periodontal probe as the distance between the cemento-enamel junction and the bottom of the periodontal pocket. Gingival bleeding on gentle probing (a measure of oral hygiene) was scored as a dichotomous variable at each site (ie, bleeding present or bleeding absent).

Serum concentrations of 25-hydroxyvitamin D₃
Venous blood samples were taken in a standardized fashion in the mobile examination center. Serum 25(OH)D₃ concentrations were measured with the use of a radioimmunoassay kit (DiaSorin, Stillwater, MN). Details of the assay method were published previously (31). The reference range for the assay is 22.5–94 nmol/L and was established in healthy, predominantly white volunteers in the Midwestern United States in October, when 25(OH)D₃ concentrations are expected to be lower than those reported in the present study. NHANES III contains an inherent season and latitude structure insofar as data were collected in the northern latitudes during the summer and in the southern latitudes during the winter. This structure has to be taken into consideration when 25(OH)D₃ concentrations are analyzed, because they have been shown to be affected by seasonality and latitude. Exact latitudes are not given in the public use data file; however, county codes are given for a subset of counties with >500 000 inhabitants, and the month of blood sampling is given for all participants.

Data on other covariates
Smoking status and diabetes mellitus were assessed during the household interview. Respondents were classified as never smokers (if they had smoked <100 cigarettes in their lifetime), former smokers (100 lifetime cigarettes, not currently smoking), and current smokers (100 lifetime cigarettes, currently smoking). Current smokers were further stratified by the number of cigarettes smoked per day (10, 11–20, 21–30, or >30 cigarettes/d). The self-reported smoking measures used in NHANES III were validated previously with serum cotinine measurements (32).

The poverty income ratio was computed as the ratio of family income to the poverty threshold produced annually by the Census Bureau. These threshold values adjust for changes caused by inflation between calendar years. Hence, the higher the family income relative to the poverty threshold, the higher the poverty income ratio is.

Body mass index (in kg/m²) was computed from measurements of weight and standing height. For female respondents, a variable for estrogen use (never, former, or current) was created from answers to questions on the use and duration of use of "birth control pills" and "estrogen or female hormone pills." Calcium intake (mg/d) was calculated from a 24-h dietary recall and did not include calcium intake from supplements. BMD measurements of the total proximal femoral regions were available for a subset of the subjects. The inclusion and exclusion criteria for BMD assessment and details about the methodology and results were published previously (33, 34).

Statistical analyses
All statistical analyses were performed with STATA 7.0 (Stata Corp, College Station, TX). The mean periodontal attachment loss (AL) per subject and the proportion of bleeding sites per subject were calculated. To reduce the chance of misclassification of periodontal disease due to gingival recession, only mesiobuccal sites were included. Serum 25(OH)D₃ concentrations were categorized in quintiles. Body mass index was also categorized (<20, 20 to <25, 25 to <30, 30). Univariate comparisons were made by using the Student's t test or chi-square test as appropriate.

The association between serum 25(OH)D₃ concentrations and periodontal disease was analyzed by using multiple linear regression with mean AL as a continuous outcome. Robust SEs based on the Huber-White-sandwich estimator of variance were used for tests of significance (35). Variables stayed in the model if they were either significant predictors of periodontal disease or confounders of the association between 25(OH)D₃ and periodontal disease when a criterion of a 10% change in estimate was used. Race or ethnicity, sex, age, and calcium intake were considered a priori to be potentially important effect-measure modifiers, and the significance of the respective interactions was evaluated by using partial F tests. Finally, BMD was entered in the model to assess to what extent the association between serum 25(OH)D₃ concentrations and periodontal disease was explained by BMD.

Estimates were calculated to account for stratification and clustering (36). We did not use sampling weights because our stratified models were adjusted for age, race or ethnicity, and poverty income ratio, and this adjustment is regarded as a good compromise between efficiency and bias (36, 37).

RESULTS  
A total of 12 976 subjects aged 20 y had periodontal attachment level measurements performed, and among those subjects, 12 449 had their serum 25(OH)D₃ concentrations measured. The final sample for the analysis consisted of 11 202 subjects for whom complete assessment of all covariates was performed.

Age modified the association between serum 25(OH)D₃ concentrations and periodontal disease (20–49 compared with 50 y; P < 0.001). The interaction with sex was marginally significant (P = 0.09). No effect modification by race or ethnicity (P = 0.55) or calcium intake (P = 0.52) was found. Thus, the regression models were stratified for age group and sex.

The distribution of the covariates within the analytic subgroups is given in Table 1. In the men and the women, the mean AL and the percentage of sites bleeding was significantly higher in the older age group than in the younger age group, whereas mean serum 25(OH)D₃ concentrations were similar between age groups and higher in the men than in the women. Dietary calcium intake was significantly higher in the younger age group in both the men and the women, whereas the prevalence of diabetes was significantly higher in the older age group. The percentage of women who had ever used estrogen was significantly higher in the younger women than in the older women.

View this table:
TABLE 1. Distribution of sample characteristics in young and old men and women

 
ß coefficients and 95% CIs for mean AL by quintile of serum 25(OH)D₃ concentration in the men and the women are given in Tables 2 and 3, respectively. The final linear regression model was adjusted for age, sex, race or ethnicity, smoking, diabetes, body mass index, estrogen use among the women, poverty income ratio, gingival bleeding, survey phase, and dental examiner. Calcium intake was not significantly associated with periodontal disease and did not affect the association between 25(OH)D₃ and periodontal disease. Likewise, the month of examination and the month of blood sampling did not confound the exposure-disease association and were therefore not included in the final model. Furthermore, in a subgroup of subjects with available county codes (ie, proxy for latitude), adjustment for county code did not change the main effect estimates. There was no association between serum 25(OH)D₃ concentrations and periodontal disease in the younger men and women.

View this table:
TABLE 2. ß Coefficients and 95% CIs for mean periodontal attachment loss by quintile of serum 25-hydroxyvitamin D₃ [25(OH)D₃] concentration in men

 

View this table:
TABLE 3. ß coefficients and 95% CIs for mean periodontal attachment loss by quintile of serum 25-hydroxyvitamin D₃ [25(OH)D₃] concentration in women

 
In both the men and the women aged 50 y, lower serum 25(OH)D₃ concentrations were associated with higher AL (P for trend = 0.001 for the men and 0.008 for the women). On average, the men aged 50 y who were in the lowest quintile of serum 25(OH)D₃ concentration lost 0.39 mm (95% CI: 0.17, 0.60 mm) more periodontal attachment than did those who were in the highest quintile. Among the women, the corresponding value was 0.26 mm (95% CI: 0.09, 0.43 mm). Expressed as percentages, these differences correspond to 27% AL (95% CI: 12%, 42%) and 23% AL (95% CI: 8%, 38%) in the men and the women, respectively.

In the subgroup of subjects with available BMD measurements (n = 10 175), BMD was not significantly associated with AL. Furthermore, adjustment for BMD did not change the ß coefficients for the association between serum 25(OH)D₃ concentrations and periodontal disease.

DISCUSSION  
In the present study, we found a significant inverse association between serum 25(OH)D₃ concentrations and periodontal disease in both the men and the women aged 50 y. Compared with the subjects of the same sex in the highest quintile of serum 25(OH)D₃ concentration in this age group, the men and the women in the lowest quintile lost 27% (95% CI: 12%, 42%) and 23% (95% CI: 8%, 38%) more attachment. This association was independent of race or ethnicity, socioeconomic status, estrogen use among the women, smoking, and gingival bleeding.

To date, 2 studies have addressed a potential association between vitamin D and periodontal disease or tooth loss. One longitudinal study of 550 predominantly white, middle-aged, elderly men found no association between vitamin D intake from foods and supplements and the number of teeth with progression of periodontal bone loss over a 4-y period (38). Progression of bone loss was defined as a change from <20% alveolar bone loss to >20% alveolar bone loss. However, intake of vitamin D alone may be a poor indicator of vitamin D status because exposure to sunshine is another important determinant of serum vitamin D concentrations, and this fact might account for the negative findings in this study (39, 40). The second study was a secondary analysis of a randomized, double-blind, placebo-controlled trial including 145 healthy ambulatory men and women. Over a 3-y period, the subjects received either supplementation with calcium (500 mg/d) and vitamin D (700 IU cholecalciferol/d) or placebo. The subjects who received supplementation with vitamin D and calcium had a 60% lower risk of losing at least one tooth (OR: 0.4; 95% CI: 0.2, 0.9) than did those who received the placebo. This study gave a first indication that vitamin D may be important for preventing tooth loss and potentially periodontal disease. However, because the study medication was a combination of calcium and vitamin D, the relative effect of vitamin D remains unclear (25).

Vitamin D could affect periodontal disease in different ways, including BMD-mediated effects and BMD-independent antiinflammatory effects. Vitamin D has an important role in bone formation and preservation (41, 42) and successfully increased BMD and reduced nonvertebral fractures in several randomized clinical trials (22, 43, 44). Because recent findings suggest that alveolar bone loss in periodontal disease may be more pronounced in osteoporotic bone (45), the pathway involving BMD-mediated effects may be an important one. However, we did not find an association between AL and BMD in this national survey, and BMD did not attenuate the association between serum 25(OH)D₃ concentrations and AL. Therefore, we suggest that the inverse association between serum 25(OH)D₃ concentrations and AL that was observed in the men and the women aged 50 y in this survey is likely to be explained by BMD-independent factors, such as the antiinflammatory effects of vitamin D. Vitamin D has been found to inhibit cytokine production and cell proliferation in various tissues (23) and could thus affect the inflammatory resorption of alveolar bone (23, 24). In a recent clinical study, supplementation with vitamin D reduced serum concentrations of C-reactive protein and interleukin 6 in critically ill patients (46). In addition, vitamin D receptors are ubiquitous (23), and associations between vitamin D receptor polymorphisms and periodontal disease have been described (47-50).

We can only speculate why the association between serum 25(OH)D₃ concentrations and AL was limited to the older subjects of both sexes. One reason may be the higher prevalence and extent of AL in older subjects than in younger subjects. In this way, older subjects may be more susceptible to a potential benefit of vitamin D.

The strength of the present analysis of the NHANES III database was its population-based design. The large sample size provided enough power to perform stratification by sex and age group. The consistency of the association between 25(OH)D₃ concentrations and periodontal disease in both sexes in the older age group, in whom attachment loss is most prevalent, lends credibility to the main findings. Serum 25(OH)D₃ concentrations are considered to be a suitable biomarker of vitamin D status that reflects both exposure to sunlight and vitamin D intake from food and supplements. The major limitation of the present analysis was its cross-sectional design. AL is a cumulative measure of periodontal disease; as such, it reflects life-long disease experience. A causal association between vitamin D and periodontal disease, therefore, cannot be established solely on the basis of the present analysis. Furthermore, the analysis was prone to nondifferential bias to the extent that serum 25(OH)D₃ concentrations measured at a single point in time do not reflect average life-long vitamin D status, which probably led to a bias toward the null hypothesis. Therefore, the true association may be stronger than the association reported here.

Both hypovitaminosis D and periodontal disease are highly prevalent in the United States and various European countries (51-53). Therefore, our findings might have important public health implications, especially because beneficial effects of vitamin D have been shown for other disabling and chronic diseases and conditions, such as osteoporosis (54), risk of falls in the elderly (55), osteoarthritis (56, 57), and certain cancers (58, 59).

In conclusion, our results suggest that higher serum 25(OH)D₃ concentrations are associated with less AL in subjects aged 50 y. This association appears to be independent of BMD. Future studies are needed to prospectively assess the beneficial effect of vitamin D on periodontal disease. Confirmation of our results would lend further support to the public health benefit of vitamin D and suggest that oral health is an important new target.

ACKNOWLEDGMENTS  
The analysis was planned by TD and HAB-F. TD conducted the analysis with contributions by KJJ and HAB-F. All authors evaluated the results and contributed to their interpretation. TD wrote the manuscript with input from all other authors. None of the authors had a conflict of interest to report.

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