Optimisation of vitamin D status in global populations.

Vitamin D is important for musculoskeletal health. Concentrations of 25-hydroxyvitamin D, the most commonly measured metabolite, vary markedly around the world and are influenced by many factors including sun exposure, skin pigmentation, covering, season and supplement use. Whilst overt vitamin D deficiency with biochemical consequences presents an increased risk of severe sequelae such as rickets, osteomalacia or cardiomyopathy and usually warrants prompt replacement treatment, the role of vitamin D supplementation in the population presents a different set of considerations. Here the issue is to keep, on average, the population at a level whereby the risk of adverse health outcomes in the population is minimised. This position paper, which complements recently published work from the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases, addresses key considerations regarding vitamin D assessment and intervention from the population perspective. This position paper, on behalf of the International Osteoporosis Foundation Vitamin D Working Group, summarises the burden and possible amelioration of vitamin D deficiency in global populations. It addresses key issues including screening, supplementation and food fortification.

Consensus statement from 2nd International Conference on Controversies in Vitamin D.

The 2nd International Conference on Controversies in Vitamin D was held in Monteriggioni (Siena), Italy, September 11-14, 2018. The aim of this meeting was to address ongoing controversies and timely topics in vitamin D research, to review available data related to these topics and controversies, to promote discussion to help resolve lingering issues and ultimately to suggest a research agenda to clarify areas of uncertainty. Several issues from the first conference, held in 2017, were revisited, such as assays used to determine serum 25-hydroxyvitamin D [25(OH)D] concentration, which remains a critical and controversial issue for defining vitamin D status. Definitions of vitamin D nutritional status (i.e. sufficiency, insufficiency and deficiency) were also revisited. New areas were reviewed, including vitamin D threshold values and how they should be defined in the context of specific diseases, sources of vitamin D and risk factors associated with vitamin D deficiency. Non-skeletal aspects related to vitamin D were also discussed, including the reproductive system, neurology, chronic kidney disease and falls. The therapeutic role of vitamin D and findings from recent clinical trials were also addressed. The topics were considered by 3 focus groups and divided into three main areas: 1) "Laboratory": assays and threshold values to define vitamin D status; 2) "Clinical": sources of vitamin D and risk factors and role of vitamin D in non-skeletal disease and 3) "Therapeutics": controversial issues on observational studies and recent randomized controlled trials. In this report, we present a summary of our findings.

Biotin supplementation causes erroneous elevations of results in some commercial serum 25-hydroxyvitamin d (25OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants' performance of specific methods for 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D). DEQAS occasionally circulates samples containing high levels of substances found in certain clinical situations e.g. 25-OHD2, 24,25-(OH)2D3, hypertriglyceridemia. The increased availability and use of health supplements containing biotin has led to case reports of assay interference in methods utilizing a biotin-streptavidin detection system. In October 2018, DEQAS included a serum sample (545) containing exogenous biotin (concentration =586 µg/L) which was analyzed by a total of 683 laboratories using 35 different methods. The same serum sample (544) without exogenous biotin was also included in the 5-sample set. All methods (760 laboratories) performed satisfactorily on sample 544 giving an All-Laboratory Trimmed Mean = 50.2 ± 6.5 nmol/L (±SD, CV = 12.9 %). The target value for this sample 544 (& 555) was 47.4 nmol/L as determined by Centers for Disease Control and Prevention (CDC) Atlanta, Georgia using their LC-MS/MS reference method. In contrast, #545 containing the exogenous biotin was reported by only 683 laboratories and gave an All-Laboratory Trimmed Mean = 66.8 ± 37.6 nmol/L (±SD, CV = 56.3 %). As expected, LC-MS/MS methods (143 labs) reported similar results for both 544 = 48.9 ± 4.4 nmol/L (±SD) and 545 = 48.3 ± 4.5 nmol/L (±SD) showing that assays involving chromatographic steps are unaffected by the presence of biotin. Several of the antibody-based assays including Abbott Architect, DiaSorin Liaison, Beckman Unicel and Siemens Centaur are also unaffected by the addition of biotin. Two assays, IDS-iSYS and Roche Total 25OHD, both of which use biotin-streptavidin, exhibit biotin interference yielding values with a significant positive bias for 545 of 102.6 nmol/L ± 78.7 nmol/L (±SD) and 517.8 nmol/L ± 209.8 nmol/L (±SD) respectively. Interestingly, the failure to report sample 545 data from 77 laboratories is due solely to those running Roche Total 25OHD or Roche Vitamin D Total II assays. Given the prevalence of the adversely affected assays (25 % of DEQAS users) and the high volume of 25OHD testing, clinicians using these assays should, where possible, only measure 25OHD when patients are off biotin.

Estimating uncertainty of target values for DEQAS serum materials.

The External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) distributes human serum samples to laboratories across the world to assess their performance in measuring serum total 25-hydroxyvitamin D [25(OH)D], i.e. the sum of the concentrations of serum 25(OH)D2 and 25(OH)D3. In 2013 DEQAS, in collaboration with the Vitamin D Standardization Program (VDSP), became an accuracy-based EQAS when the National Institute for Standards and Technology (NIST) began assigning 25(OH)D target values to DEQAS serum samples using their Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved reference measurement procedure (RMP). Historically, NIST has performed 4 determinations of 25-OHD2 and 25-OHD3 on each sample and used the mean values to calculate a single 'target value' for Total 25-OHD against which performance was judged. By definition the target values cannot be exact and each is associated with a level of uncertainty. The total uncertainty (UNIST) has two components, one from the 25(OH)D2, and 25(OH)D3 measurements and the other associated with the calibration procedure. The total combined uncertainty is calculated by adding up these uncertainties. In future, uncertainties will be attached to the target value in each DEQAS serum sample, starting with the next distribution cycle in 2019. Confidence intervals obtained using these uncertainties will allow DEQAS participants to determine if their result agrees with the NIST assigned target value. Furthermore, if the value falls within the confidence interval the laboratory's assay would be regarded as traceable, i.e. standardized, to the NIST RMP.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Hydroxyvitamin D assays: An historical perspective from DEQAS.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.

Quality assessment of vitamin D metabolite assays used by clinical and research laboratories.

The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH)2D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH)2D) was introduced. The 25-OHD scheme is accuracy - based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD2 and 25-OHD3. A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH)2D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

Vitamin D measurement standardization: The way out of the chaos.

Substantial variability is associated with laboratory measurement of serum total 25-hydroxyvitamin D [25(OH)D]. The resulting chaos impedes development of consensus 25(OH)D values to define stages of vitamin D status. As resolving this situation requires standardized measurement of 25(OH)D, the Vitamin D Standardization Program (VDSP) developed methodology to standardize 25(OH)D measurement to the gold standard reference measurement procedures of NIST, Ghent University and CDC. Importantly, VDSP developed protocols for standardizing 25(OH)D values from prior research based on availability of stored serum samples. The effect of such retrospective standardization on prevalence of "low" vitamin D status in national studies reported here for The Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) and the German Health Interview and Examination Survey for Children and Adolescents (KIGGS, 2003-2006) was such that in NHANES III 25(OH)D values were lower than original values while higher in KIGGS. In NHANES III the percentage with values below 30, 50 and 75 nmol/L increased from 4% to 6%, 22% to 31% and 55% to 71%, respectively. Whereas in KIGGS after standardization the percentage below 30, 50, and 70 nmol/L decreased from 28% to 13%, 64% to 47% and 87% to 85% respectively. Moreover, in a hypothetical example, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the 25(OH)D concentration which maximally suppressed PTH could vary from 20 to 35ng/mL. These examples underscore the challenges (perhaps impossibility) of developing vitamin D guidelines using unstandardized 25(OH)D data. Retrospective 25(OH)D standardization can be applied to old studies where stored serum samples exist. As a way forward, we suggest an international effort to identify key prior studies with stored samples for re-analysis and standardization initially to define the 25(OH)D level associated with vitamin D deficiency (rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, examples reported here highlight the importance of suspending publication of meta-analyses based on unstandardized 25(OH)D results.

25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites.

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3 (52.4nmol/L), 24R,25(OH)2D3 (14.4nmol/L) and 24S,25(OH)2D3 (57.9nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC-MS/MS methods were able to resolve 3-epi-25-OHD3 from 25-OHD3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3 (24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC-MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.

Developing vitamin D dietary guidelines and the lack of 25-hydroxyvitamin D assay standardization: The ever-present past.

Unstandardized laboratory measurement of 25-hydroxyvitamin D (25(OH)D) confounds efforts to develop clinical and public health vitamin D guidelines. The Vitamin D Standardization Program (VDSP), an international collaborative effort, was founded in 2010 to correct this problem. Nearly all published vitamin D research is based on unstandardized laboratory 25(OH)D measurements. While it is impossible to standardize all old data, it may be possible to identify a small subset of prior studies critical to guidelines development. Once identified it may be possible to calibrate their 25(OH)D values to the NIST and Ghent University reference measurement procedures using VDSP methods thereby permitting future guidelines to be based on standardized results. We simulated the calibration of a small set of ten clinical trials of vitamin D supplementation on achieved 25(OH)D under minimal sun exposure. These studies were selected because they played a prominent role in setting the 2010 vitamin D dietary reference intakes (DRI). Using random-effects meta-regression analysis, Vitamin D External Quality Assessment (DEQAS) data on assay bias was used to simulate the potential bias due to the lack of assay standardization by calibrating the achieved 25(OH)D levels from those 10 studies to: (1) the largest negative, and (2) the largest positive bias from the DEQAS all laboratory trimmed mean (ALTM) for the appropriate assay and year of analysis. For a usual vitamin D intake of 600IU/day the difference in mean achieved 25(OH)D values for those two options was 20nmol/L. However, without re-calibration of 25(OH)D values it is impossible to know the degree to which any of the current guidelines may have been biased. This approach may help stimulate the search for and standardization of that small subset of key studies and, in the cases where standardization is impossible, to identify areas of urgently needed vitamin D research.

Antioxidants, oxidative stress, and pulmonary function in individuals diagnosed with asthma or COPD.

OBJECTIVE: The objective of this study was to investigate the association between antioxidant nutrients and markers of oxidative stress with pulmonary function in persons with chronic airflow limitation. DESIGN: Cross-sectional study exploring the association of antioxidant nutrients and markers of oxidative stress with forced expiratory volume in the first second (FEV1%) and forced vital capacity (FVC%). SETTING/SUBJECTS: The study data included 218 persons with chronic airflow limitation recruited randomly from the general population of Erie and Niagara counties, New York State, USA. RESULTS: After adjustment for covariates, multiple linear regression analysis showed that serum beta-cryptoxanthin, lutein/zeaxanthin, and retinol, and dietary beta-carotene, beta-cryptoxanthin, lutein/zeaxanthin, vitamin C, and lycopene were positively associated with FEV1% (P < 0.05, all associations). Serum vitamins beta-cryptoxanthin, lutein/zeaxanthin, and lycopene, and dietary beta-cryptoxanthin, beta-carotene, vitamin C, and lutein/zeaxanthin were positively associated with FVC% (P < 0.05, all associations). Erythrocytic glutathione was negatively associated with FEV1%, while plasma thiobarbituric acid-reactive substances (TBARS) were negatively associated with FVC% (P < 0.05). CONCLUSION: These results support the hypothesis that an imbalance in antioxidant/oxidant status is associated with chronic airflow limitation, and that dietary habits and/or oxidative stress play contributing roles.

Alcoholic beverage preference and characteristics of drinkers and nondrinkers in western New York (United States).

BACKGROUND AND AIM: Dietary and lifestyle characteristics may differ for drinkers of specific alcoholic beverages and nondrinkers which would have important implications for studies of alcohol and disease. Our aim in this study was to describe differences in dietary and lifestyle characteristics associated with alcoholic beverage preference in a population-based sample of healthy study participants. METHODS AND RESULTS: Data were collected as part of a series of case-control studies of alcohol use, myocardial infarction, and lung, breast and prostate cancer in western New York from 1846 men and 1910 women aged 35 to 79, randomly selected from the general population of Erie and Niagara Counties. Beverage preference was defined for noncurrent vs current drinkers, and drinkers of beer, wine, liquor, and mixed beverages. Generalized linear models for continuous variables and Cochran-Mantel-Haenszel statistics for categorical variables were computed for the entire sample and stratified by gender. Participant characteristics differed by alcoholic beverage preference and drinking status. In general, wine drinkers had higher education and household incomes, lower prevalence of current smoking, higher intakes of dietary fiber, potassium, vitamin E, and total carotenoids, lower total fat intakes and higher amounts of fruits, vegetables, and grain products than consumers of other beverages. Conversely, beer and liquor drinkers had somewhat lower education and household incomes, higher rates of current smoking, higher energy and total fat intakes and consumed lower amounts of fruits, vegetables, and grain products. Finally, current nondrinkers were more likely to be older, less educated, have lower household incomes, and consume diets less consistent with dietary guidelines than current drinkers. CONCLUSIONS: These results suggest that usual beverage preference may encompass other health-related behaviors and underline the importance of accurate exposure measurement and use of statistical methods to accommodate these interrelationships.

Alcohol and cardiovascular disease--more than one paradox to consider. Average volume of alcohol consumption, patterns of drinking and risk of coronary heart disease--a review.

The effect of average volume of alcohol on coronary heart disease (CHD) is J-shaped in established market economies. Light to moderate drinkers have less risk than abstainers, with heavy drinkers displaying the highest level of risk. This relationship between average volume of alcohol consumption and CHD is modified by different patterns of drinking. Heavy drinking occasions as well as drinking outside meals are related to increased CHD risk, independently of volume of drinking. Beverage type does not seem to have much impact, even though there are some indications that wine is more protective than other forms of alcohol. Physiological mechanisms have been identified to explain this complex relationship between alcohol and CHD. Since patterns of drinking are important in determining CHD risk, they should be included in future epidemiologic studies, together with biomarkers further to test hypotheses about pathways.

Is allergen skin test reactivity a predictor of mortality? Findings from a national cohort.

BACKGROUND: The importance of atopy on subsequent mortality is controversial. A clearer understanding is important as atopy is increasing worldwide. OBJECTIVE: To determine the influence of allergen skin test reactivity on observed mortality of a national cohort. METHODS: Baseline health status and atopic status (allergen skin testing) was measured as part of the second National Health and Nutrition Examination Survey (NHANES II), a representative sample of the US population, during the years 1976-80. Vital status and cause of death were assessed through December 31, 1992 for all examinees 30 years of age or older at baseline (n = 9252) as part of the NHANES II Mortality Study (NH2MS). The analytic sample contained 8179 men and women after excluding missing data. Allergen skin test reactivity was defined as weal >/= 3 mm to one of eight 1 : 20 (w/v), 50% glycerinated ('No US Standard of Potency') allergens licensed by the FDA: house dust, cat, dog, Alternaria, mixed giant/short ragweed, oak, perennial rye grass, and Bermuda grass. Survival analyses were conducted using multivariate adjusted Cox regression models to evaluate the association between atopy and all-cause, cardiovascular, and cancer mortality. RESULTS: There was no association between allergen skin test reactivity and all cause mortality: 30-44 years RR = 1.07 (95% CI 0.63-1.84); 45-59 years RR = 1.10 (0.78-1.55); 60-75 years RR = 1.07 (0.91-1.25). Results were unchanged when cancer or heart disease mortality were examined separately. The presence or absence of allergic symptoms, using the flare to define skin test reactivity, eliminating deaths in the first 5 years of follow-up, or eliminating individuals with pre-existing conditions did not alter the findings. CONCLUSIONS: Atopy, defined by allergen skin test reactivity, with or without symptoms, is not a predictor of subsequent mortality.

A comparison of serum total cholesterol levels and their determinants between the Federal Republic of Germany and the United States.

Mean serum total cholesterol levels appear to be higher in the Federal Republic of Germany (FRG) than in the United States (US) while coronary heart disease death rates are lower. The study examined possible factors for the difference including possible differences in laboratory methodology. Cross-sectional data from the first two waves of the German National Health Surveys (1984-1986 and 1987-1989; n = 9709) and from the Second National Health and Nutrition Examination Survey (1976-1980; n = 7832) were compared for men and women 25-69 years of age. The influence of age, body mass index, diet, cigarette smoking, education, income, use of oral contraceptives or antihypertensive agents, alcohol consumption and potential differences in laboratory measurement were explored using multiple regression techniques separately for men and women for ages 25-39, 40-59 and 60-69 years of age. Overall ages, unadjusted mean total cholesterol levels were higher in German than US men (6.02 vs. 5.64 mmol/l) and in German than US women (6.04 vs. 5.80 mmol/l) as were HDL cholesterol levels (men: 1.30 vs 1.14 mmol/l; women: 1.65 vs. 1.38 mmol/l). Adjusting for lifestyle factors explained, on the average, 40% of the differences in mean total cholesterol of which half or 20% was accounted for by adjusting for alcohol intake. Adjusting for possible laboratory differences explained, on the average, an additional 30% of the differences. Frequency of alcohol intake was the most important factor in explaining differences in mean HDL cholesterol levels. Adjustment for differences in alcohol intake had negligible effects on reducing the differences in mean non-HDL cholesterol.

Periodontal disease and risk of cerebrovascular disease: the first national health and nutrition examination survey and its follow-up study.

BACKGROUND: Periodontal disease has been found to be a potential risk factor for coronary heart disease. However, its association with cerebrovascular accidents (CVAs) is much less studied. METHODS: This study examines the association between periodontal disease and CVA. The study cohort comprises 9962 adults aged 25 to 74 years who participated in the First National Health and Nutrition Examination Survey and its follow-up study. Baseline periodontal status was categorized into (1) no periodontal disease, (2) gingivitis, (3) periodontitis, and (4) edentulousness. All CVAs (International Classification of Diseases, Ninth Revision [ICD-9], codes 430-438) were ascertained by hospital records for nonfatal events and death certificates for fatal events. The first CVA, nonfatal or fatal, was used to define incidence. Relative risks were estimated by hazard ratios from the Cox proportional hazard model with adjustment for several demographic variables and well-established cardiovascular risk factors. Weights were used to generate risk estimates. RESULTS: Periodontitis is a significant risk factor for total CVA and, in particular, nonhemorrhagic stroke (ICD-9, 433-434 and 436-438). Compared with no periodontal disease, the relative risks (95% confidence intervals) for incident nonhemorrhagic stroke were 1.24 (0.74-2.08) for gingivitis, 2.11 (1.30-3.42) for periodontitis, and 1.41 (0.96-2.06) for edentulousness. For total CVA, the results were 1.02 (0.70-1.48) for gingivitis, 1.66 (1.15-2.39) for periodontitis, and 1.23 (0.91-1.66) for edentulousness. Increased relative risks for total CVA and nonhemorrhagic stroke associated with periodontitis were also seen in white men, white women, and African Americans. Similar results were found for fatal CVA. CONCLUSION: Periodontal disease is an important risk factor for total CVA and, in particular, nonhemorrhagic stroke.