the ludwigshafen risk and cardiovascular health ( luric ) study is a prospective cohort study designed to evaluate determinants of cardiovascular health ( 1517 ) . between july 1997 and january 2000
, 3,316 caucasian subjects referred for coronary angiography were recruited at the herzzentrum ( cardiac center ) ludwigshafen in southwest germany .
exclusion criteria were any acute illness other than acute coronary syndrome , any predominant noncardiac chronic disease , and a history of malignant neoplasm(s ) within the past 5 years .
written informed consent was obtained from each participant , and the study was approved by the institutional review board at the rztekammer rheinland - pfalz ( medical association of rheinland - pfalz ) .
a detailed questionnaire was used to collect a range of demographic characteristics , including lifestyle factors such as alcohol consumption , smoking , and physical activity .
daily physical activity was recorded using a nonvalidated 11-point scale ranging from bedridden to extremely active .
key points on the scale were 1 , bed rest ; 2 , mostly supine , 3 , not very active , 6 , usual office work ; 9 , heavy work or sports ; and 11 , extremely sportive .
a fasting venous blood sample was obtained in the morning before coronary angiography from supine subjects .
a summary of analytic methods relevant to this study has been previously reported ( 15 ) .
serum levels of 25(oh)d were assayed weekly using a radioimmunoassay ( diasorin sa , antony , france ) with intra - assay and interassay coefficients of variation of 8.6% and 9.2% , respectively ( 15,16 ) .
we have also validated this radioimmune assay using liquid chromatography coupled to tandem mass spectrometry , which correlated significantly ( r = 0.88 , p < 0.001 ) , with intra - assay and interassay coefficients of variation of less than 10% .
nmol / l ) , or severe deficiency ( < 25 nmol / l ) , as recommended by the endocrine society ( 18,19 ) . for reference , other organizations may use other criteria , such as the institute of medicine , that recommend levels > 50 nmol / l ( 20 ) .
metabolic syndrome in adults was defined using the consensus statement from the international diabetes federation task force on epidemiology and prevention ; the national heart , lung , and blood institute ; the american heart association ; the world heart federation ; the international atherosclerosis society ; and the international association for the study of obesity ( 21 ) .
specifically , it was identified in those having 3 or more of the following ( 1 ) : blood pressure 130/85 mmhg or receiving antihypertensive medication ( 2 ) ; fasting glucose 5.6
mmol / l or receiving drug treatment for diabetes ( 3 ) ; triglycerides 1.7 mmol / l or receiving specific drug treatment ( 4 ) ; hdl cholesterol < 1.03 mmol / l in men or < 1.30 mmol / l in women or receiving specific drug treatment ( 5 ) ; or waist circumference > 102 cm in men or > 88 cm in women ( 21 ) .
follow - up procedures ( median 7.7 years ) have been described in detail elsewhere ( 1517 ) .
we used death certificates to classify the deceased into those who died of cardiovascular versus noncardiovascular causes .
this classification was done independently by two experienced clinicians who were blinded to the study participants , except for information that was required to classify the cause of death . in the event of disagreement or uncertainty ,
a classification decision was made by one of the luric study principal investigators ( w.m . ) .
subjects were stratified according to widely used cutoffs for the definition of vitamin d status based on 25(oh)d levels ( 18 ) .
baseline demographics of the subjects categorized by vitamin d status are described as percentages for categoric data and as , depending on their distribution , continuous data are presented as means  sd or as geometric means with 95% cis .
comparisons between groups were performed using the  test for categoric data and anova for continuous data .
kaplan - meier curves , followed by the log - rank test , were used to evaluate differences in overall and cardiovascular mortality for the 25(oh)d groups .
hazard ratios ( hr ) with 95% cis for the mortality categories were calculated using cox proportional hazards regression models , which enabled adjustment for potential confounding parameters . in these analyses ,
model 1 describes the crude association ; model 2 was adjusted for age , sex , smoking , alcohol and physical activity ; and model 3 was further adjusted for bmi , waist circumference , diastolic blood pressure , type 2 diabetes , total cholesterol , cystatin c , c - reactive protein , the new york heart association ( nyha ) functional classification , cardiovascular medication , and month of blood sampling ( seasonality ) .
hrs for 25(oh)d categories were calculated using the severe vitamin d deficiency group as the reference .
assumptions underlying the cox proportional hazards regression model were evaluated by log minus log survival and partial ( schnfeld ) residuals versus survival time plots , and were found valid .
all statistical tests were two - sided , and statistical significance was defined as p < 0.05 .
all data were analyzed using spss 18.0 software ( spss inc , chicago , il ) .
the ludwigshafen risk and cardiovascular health ( luric ) study is a prospective cohort study designed to evaluate determinants of cardiovascular health ( 1517 ) . between july 1997 and january 2000
, 3,316 caucasian subjects referred for coronary angiography were recruited at the herzzentrum ( cardiac center ) ludwigshafen in southwest germany .
exclusion criteria were any acute illness other than acute coronary syndrome , any predominant noncardiac chronic disease , and a history of malignant neoplasm(s ) within the past 5 years .
written informed consent was obtained from each participant , and the study was approved by the institutional review board at the rztekammer rheinland - pfalz ( medical association of rheinland - pfalz ) .
a detailed questionnaire was used to collect a range of demographic characteristics , including lifestyle factors such as alcohol consumption , smoking , and physical activity .
daily physical activity was recorded using a nonvalidated 11-point scale ranging from bedridden to extremely active .
key points on the scale were 1 , bed rest ; 2 , mostly supine , 3 , not very active , 6 , usual office work ; 9 , heavy work or sports ; and 11 , extremely sportive .
a fasting venous blood sample was obtained in the morning before coronary angiography from supine subjects .
a summary of analytic methods relevant to this study has been previously reported ( 15 ) .
serum levels of 25(oh)d were assayed weekly using a radioimmunoassay ( diasorin sa , antony , france ) with intra - assay and interassay coefficients of variation of 8.6% and 9.2% , respectively ( 15,16 ) .
we have also validated this radioimmune assay using liquid chromatography coupled to tandem mass spectrometry , which correlated significantly ( r = 0.88 , p < 0.001 ) , with intra - assay and interassay coefficients of variation of less than 10% .
serum vitamin d status was categorized based on 25(oh)d levels as being optimal ( 75 nmol / l ) , or having insufficiency ( 5074.99 nmol / l ) , moderate deficiency ( 2549.99
nmol / l ) , or severe deficiency ( < 25 nmol / l ) , as recommended by the endocrine society ( 18,19 ) . for reference
, other organizations may use other criteria , such as the institute of medicine , that recommend levels > 50 nmol / l ( 20 ) .
metabolic syndrome in adults was defined using the consensus statement from the international diabetes federation task force on epidemiology and prevention ; the national heart , lung , and blood institute ; the american heart association ; the world heart federation ; the international atherosclerosis society ; and the international association for the study of obesity ( 21 ) .
specifically , it was identified in those having 3 or more of the following ( 1 ) : blood pressure 130/85 mmhg or receiving antihypertensive medication ( 2 ) ; fasting glucose 5.6 mmol / l or receiving drug treatment for diabetes ( 3 ) ; triglycerides 1.7 mmol / l or receiving specific drug treatment ( 4 ) ; hdl cholesterol < 1.03 mmol / l in men or < 1.30 mmol / l in women or receiving specific drug treatment ( 5 ) ; or waist circumference > 102 cm in men or > 88 cm in women ( 21 ) .
follow - up procedures ( median 7.7 years ) have been described in detail elsewhere ( 1517 ) .
we used death certificates to classify the deceased into those who died of cardiovascular versus noncardiovascular causes .
this classification was done independently by two experienced clinicians who were blinded to the study participants , except for information that was required to classify the cause of death . in the event of disagreement or uncertainty ,
a classification decision was made by one of the luric study principal investigators ( w.m . ) .
subjects were stratified according to widely used cutoffs for the definition of vitamin d status based on 25(oh)d levels ( 18 ) .
baseline demographics of the subjects categorized by vitamin d status are described as percentages for categoric data and as , depending on their distribution , continuous data are presented as means  sd or as geometric means with 95% cis .
comparisons between groups were performed using the  test for categoric data and anova for continuous data .
kaplan - meier curves , followed by the log - rank test , were used to evaluate differences in overall and cardiovascular mortality for the 25(oh)d groups .
hazard ratios ( hr ) with 95% cis for the mortality categories were calculated using cox proportional hazards regression models , which enabled adjustment for potential confounding parameters . in these analyses ,
model 1 describes the crude association ; model 2 was adjusted for age , sex , smoking , alcohol and physical activity ; and model 3 was further adjusted for bmi , waist circumference , diastolic blood pressure , type 2 diabetes , total cholesterol , cystatin c , c - reactive protein , the new york heart association ( nyha ) functional classification , cardiovascular medication , and month of blood sampling ( seasonality ) .
hrs for 25(oh)d categories were calculated using the severe vitamin d deficiency group as the reference .
assumptions underlying the cox proportional hazards regression model were evaluated by log minus log survival and partial ( schnfeld ) residuals versus survival time plots , and were found valid .
all statistical tests were two - sided , and statistical significance was defined as p < 0.05 .
all data were analyzed using spss 18.0 software ( spss inc , chicago , il ) .
the metabolic syndrome was identified in 1,811 subjects ( 54.6% ) referred for coronary angiography , for whom serum levels of 25(oh)d were available in 1,801 ( 99.4% ) .
in the 1,801 subjects with the metabolic syndrome and complete 25(oh)d data , 92% had suboptimal ( < 75
nmol / l ) , and 22.2% severe deficiency ( < 25 nmol / l ) .
as reported in table 1 , 25(oh)d levels had an inverse relationship with fasting glucose and the prevalence of diabetes , whereas hdl cholesterol levels increased with increasing 25(oh)d levels .
surprisingly , there was also a small positive association with diastolic blood pressure , but no association was observed with systolic blood pressure or prevalence of hypertension , although pulse pressure did decrease with increasing 25(oh)d levels .
the decrease in the proportion of subjects at nyha functional classes iii and iv suggested lower rates of heart failure in those with increased 25(oh)d levels ( table 1 ) .
a number of lifestyle factors were positively associated with 25(oh)d levels , including alcohol consumption and physical activity ( table 1 ) .
baseline characteristics in those with the metabolic syndrome according to 25(oh)d concentrations across the 12,514 person - years of follow - up , 462 deaths were recorded , of which 267 ( 57.8% ) were cardiovascular in origin ( table 2 ) .
figure 1 presents the kaplan - meier plots for all - cause and cardiovascular mortality according to the four 25(oh)d groups .
there was a clear dose - dependent reduction in all - cause mortality ( p < 0.001 for trend ; table 2 ) , showing a 75% reduction ( hr 0.25 [ 95% ci 0.130.46 ] ) in those with optimal 25(oh)d levels , relative to those with severe deficiency , after full adjustment . for cardiovascular disease , a comparable reduction in mortality
hrs for all - cause and cardiovascular mortality in those with the metabolic syndrome according to 25(oh)d concentrations kaplan - meier plots for all - cause ( left ) and cardiovascular mortality ( right ) according to 25(oh)d groups in those with the metabolic syndrome .
log - rank analysis indicated a significant difference between all 25(oh)d groups ( p < 0.001 ) .
diabetes is associated with a strong reduction in 25(oh)d levels and is a major predictor of mortality .
however , the removal of 965 subjects with type 2 diabetes from this analysis only modestly reduced the observed effect sizes .
for example , after removal of those with type 2 diabetes , we found a 64% reduction for all - cause mortality in subjects with optimal 25(oh)d levels ( hr 0.36 [ 95% ci 0.200.64 ) , relative to those with severe deficiency ( p < 0.001 for trend ) .
we also performed a sensitivity analysis to rule out that 25(oh)d is a marker of end - stage disease in this population by excluding subjects who died within 2 years of follow - up , leaving 1,561 subjects .
the results remained essentially unaltered : comparing optimal versus severe deficiency yielded hrs of 0.16 ( 95% ci 0.060.39 ) for all - cause mortality and 0.24 ( 0.090.67 ) for cardiovascular disease  specific mortality .
we further removed 268 subjects with a glomerular filtration rate of < 60 ml / min/1.73 m , which represents a loss of more than half the adult level of normal kidney function ( 22 ) .
likewise , we found the current study results remained essentially unchanged ( data not shown ) .
a total of 35 subjects ( 2.0% ) reported regularly taking vitamin supplements , which usually contained b complex vitamins or vitamin d3 . because mean 25(oh)d levels were only modestly higher in users of vitamin d preparations ( 51.4  31.2 nmol / l ) compared with the remaining cohort ( 40.7  24.7 nmol / l ; p = 0.048 ) ,
while 1,25-dihydroxyvitamin d levels , as well age and parathyroid hormone levels , did not differ significantly ( data not shown ) , these subjects were included in the present analysis .
sensitivity analyses excluding those subjects did not affect the risk estimates ( data not shown ) . despite a limited number of subjects available for analyses of cause - specific mortality
, we observed a 85% reduced sudden death in those with optimal 25(oh)d levels ( hr 0.15 [ 95% ci 0.040.63 ] ) contrasted with severe deficiency ( p = 0.004 for trend , table 2 ) .
mortality due to congestive heart failure similarly showed a significant trend ( p = 0.001 ) .
risk of fatal strokes was also not significantly related to 25(oh)d levels , but interpretation of this analysis is limited by the very low number of events ( n = 25 ) . because exposure to sunlight is a major potential confounder , although all analyses were adjusted for seasonality , we also performed a sensitivity analysis stratifying the study into those measured during the  summer  ( may to october ) and  winter 
the associations were similar to those from the overall analysis , with hrs in the  summer  of 0.62 ( 95% ci 0.450.87 )
, 0.62 ( 0.410.93 ) , and 0.32 ( 0.150.68 ) for moderate deficiency , insufficient , and optimal , respectively , compared with those with severe deficiency . likewise , for the  winter 
period , the hrs were 0.63 ( 0.460.87 ) , 0.56 ( 0.340.91 ) , and 0.17 ( 0.040.69 ) , respectively .
the observations also remained similar for all - cardiovascular disease mortality ( data not shown ) .
to the best of our knowledge , this is the first study to show that optimal levels of 25(oh)d substantially reduce the risk of all - cause and cardiovascular mortality in subjects with the metabolic syndrome , a population well - known to exhibit excess mortality ( 14 ) .
we observed a 75% and 69% reduction in all - cause and cardiovascular disease mortality , respectively , in those with optimal levels compared with those with severe 25(oh)d deficiency .
the analyses further showed that these associations were not driven by diabetes , a factor known to be associated with reduced 25(oh)d levels .
the findings presented here strengthen a limited longitudinal literature describing the association between vitamin d and mortality ( 1012,16 ) and extend our prior observations derived from the luric cohort ( 17 ) .
although answers regarding the causal role of vitamin d need to come from randomized controlled trials , it is promising to note that most criteria usually taken as support for causality were met : these include temporality , strength , consistency , biologic gradient , coherence , and plausibility .
indeed , numerous mechanisms have been identified by which vitamin d metabolites may affect metabolic homeostasis and atherogenesis .
for example , vitamin d effects include regulation of glycemic homeostasis , as observed in the current study ; whereby reduced vitamin d levels are associated with lower glucose levels and/or reduced insulin secretion and increased insulin resistance ( 18,2326 ) .
not all studies have identified the association with vitamin d and insulin action , including in subjects with the metabolic syndrome ( 27 ) .
however , vitamin d can also influence the effects of glycemia independently of these mechanisms by blunting the effect of advanced glycation end products on endothelial cells ( 28 ) , which may contribute to the increased arterial stiffness and endothelial dysfunction observed in individuals deficient in vitamin d ( 29 ) .
vitamin d can also exert protective effects on the vessel walls by inhibition of macrophage to foam cell formation ( 30 ) and via its anti - inflammatory effects ( 31,32 ) .
the latter is likewise consistent with the current data , which showed an inverse association of 25(oh)d levels with c - reactive protein .
vitamin d suppresses atherosclerotic disease and attenuates thrombogenesis ( 32,33 ) . in the current study
, however , we did not find an association with acute myocardial infarction or stroke ; the latter might have resulted from the limited number of events .
vitamin d sufficiency has been associated with an downregulation of the renin - angiotensin - aldosterone system , thus potentially attenuating the development of hypertension ( 18 ) , although only pulse pressure was inversely associated with 25(oh)d levels in the current study .
the action of vitamin d on the renin - angiotensin - aldosterone system ( 18 ) , along with its suppression of cardiac hypertrophy ( 34 ) and hypercontractility ( 35,36 ) , likely contributes to the reduction in sudden cardiac death and heart failure mortality .
surprisingly , 25(oh)d levels were not associated with the measures of adiposity , namely waist circumference and bmi ( 37 ) . however
, this may be an artifact of selecting those with the metabolic syndrome , for which central adiposity is a component and who are thus overall generally more obese .
likewise , in these more ill subjects ( e.g. , those with heart failure ) , issues of wasting may also potentially be present that interfere with the association .
in the overall population of 3,316 subjects , when including those with and without the metabolic syndrome , we did observe a significant inverse association between bmi and 25(oh)d levels ( 16 ) .
short - term studies have suggested that alcohol intake does not affect vitamin d metabolism ( 38 ) ; however , consumption at moderate levels might have assisted in the reduction in cardiovascular disease mortality ( 39 ) .
overall alcohol consumption per se is usually associated with an overall excess of deaths ( 39 ) , so is unlikely to be a significant determinant of the apparent protective effect on mortality associated with the 25(oh)d levels , particularly given that we adjusted for alcohol consumption in the analyses .
we assessed 25(oh)d only at one time point , and this may not reflect long - term vitamin d status . however , despite significant seasonal variation , 25(oh)d levels are reported to track over time in a comparable manner similar to other established risk factors such as blood pressure and lipids ( 40 ) .
however , we adjusted the analyses for seasonality by incorporating the timing of the sampling , which should partly address this issue .
we also performed sensitivity analyses stratified by season , for which the results consistently showed a clear protective effect for all - cause and all - cardiovascular mortality .
significantly , momentary 25(oh)d was found to be a powerful long - term predictor : the kaplan - meier plots showed a consistent linear relationship with mortality , continuing for more than 8 years after a single assessment of 25(oh)d .
inclusion of only caucasian subjects limits the generalizability of the study but does help to minimize potential confounding resulting from the use of an ethnically diverse population ; for example , skin pigmentation and cultural aspects , such as style of dress , could differentially influence the magnitude of photosynthesis at different times of the year ( 8) . likewise , because these subjects with the metabolic syndrome had undergone elective angiography due to cardiovascular symptoms or events , they are thus not representative of the general population with the metabolic syndrome and are likely to display an elevated mortality risk . although , we performed multivariable adjustments , we can not rule out that unmeasured or unknown confounders could have influenced our results .
a possible alternative explanation of our results is that those with the highest risk for mortality had limited sunlight exposure due to existing health - related issues , thereby confounding the association between increased risk and reduced vitamin d levels .
related , those with greater physical activity levels would likely be healthier , and if the activities were performed outdoors , would also have increased ultraviolet b exposure .
moreover , increased physical activity may also be associated with other beneficial lifestyle practices that reduce morbidity and mortality .
we addressed these potential confounding issues in several ways : we adjusted our cox regression analyses for parameters that are related to outdoor activities and sun light exposure , including nyha functional class , physical activity , cardiovascular medication , renal function , and month of blood sampling .
sensitivity analyses that excluded those who died within the first 2 years of follow - up only minimally attenuated the observed effects . considering the magnitude of the observed benefit of higher 25(oh)d levels and the very modest effect - size attenuation seen after comprehensive statistical and sample adjustments , it seems fair to conclude that residual confounding is a unlikely hypothesis . in summary ,
25(oh)d levels were dose - dependently associated with a robust reduction in all - cause and cardiovascular mortality in subjects with the metabolic syndrome .
we hope these findings will spur interventional randomized , controlled trials to confirm the effects of vitamin d on mortality and , if positive , help establish recommendations for supplementation in these subjects .
 objectiveoptimal vitamin d levels are associated with reduced cardiovascular and all - cause mortality . 
 we investigated whether optimal 25-hydroxyvitamin d ( 25[oh]d ) is protective in individuals with the metabolic syndrome.research design and methodsthe ludwigshafen risk and cardiovascular health ( luric ) study is a cohort study of subjects referred for coronary angiography between 1997 and 2000 , from which 1,801 with the metabolic syndrome were investigated . 
 mortality was tracked for a median of 7.7 years . 
 multivariable survival analysis was used to estimate the association between 25(oh)d levels and mortality.resultsmost subjects ( 92% ) had suboptimal levels of 25(oh)d ( < 75 nmol / l ) , with 22.2% being severely deficient ( < 25 nmol / l ) . during follow - up , 
 462 deaths were recorded , 267 ( 57.8% ) of which were cardiovascular in origin . 
 after full adjustment , including the metabolic syndrome components , those with optimal 25(oh)d levels showed a substantial reduction in all - cause ( hazard ratio [ hr ] 0.25 [ 95% ci 0.130.46 ] ) and cardiovascular disease mortality ( 0.33 [ 0.160.66 ] ) compared with those with severe vitamin d deficiency . for specific cardiovascular disease mortality , 
 there was a strong reduction for sudden death ( 0.15 [ 0.040.63 ] ) and congestive heart failure ( 0.24 [ 0.061.04 ] ) , but not for myocardial infarction . 
 the reduction in mortality was dose - dependent for each of these causes.conclusionsoptimal 25(oh)d levels substantially lowered all - cause and cardiovascular disease mortality in subjects with the metabolic syndrome . 
 these observations call for interventional studies that test whether vitamin d supplementation provides a useful adjunct in reducing mortality in these subjects . 
RESEARCH DESIGN AND METHODS
Study population
Laboratory analyses
Diagnoses
Follow-up
Statistical analysis
RESULTS
CONCLUSIONS