our study sample comprised all patients newly diagnosed with type 1 diabetes consecutively admitted to the steno diabetes center between 1 september 1979 and 31 august 1984 .
nine patients were excluded ; seven because of serious mental illness and two because of microalbuminuria at the onset of diabetes .
plasma 25(oh)d3 was analyzed in samples from approximately 3 years after diagnosis or the subsequently closest available sample ( median [ range ] years after diagnose = 3.0 [ 2.39.1 ] ) and before any of the patients developed microalbuminuria .
the patients attended the outpatient clinic every 3 or 4 months as part of routine follow - up .
they were treated by diabetologists and nurses according to previously described guidelines ( 12 ) .
twenty - four hour urinary albumin excretion rate ( uaer ) was measured in each patient at least once a year ( 12 ) .
development of persistent microalbuminuria and persistent macroalbuminuria were defined as uaer between 30 and 300 mg/24 h and > 300 mg/24 h , respectively , in at least two of three consecutive samples , with an increase of at least 30% above the baseline level ( 13,14 ) .
plasma 25(oh)d3 is found to be stable when tested after more than 10 years of storage ( 15 ) , making long - term epidemiologic studies of circulating plasma 25(oh)d3 possible . before initiation of the current study
, we tested the stability of plasma 25(oh)d3 in samples taken from this exact cohort .
we analyzed plasma 25(oh)d3 in samples taken from the same subjects at the same time of year and stored for 5 , 10 , 15 , 20 , and 25 years .
no statistical significant difference between mean levels of plasma 25(oh)d3 was shown ( data not shown ) . plasma levels of 25(oh)d3 were determined by high performance liquid chromatography / tandem mass spectrometry ( department of clinical biochemistry , lilleblt hospital , vejle , denmark ) . a plasma 25(oh)d3 value equal to or below the 10% percentile ( 15.5
nmol / l ) was considered severe vitamin d deficiency in accordance with our previous study on vitamin d and mortality in type 2 diabetes ( 3 ) .
patients were classified as smokers if they smoked more than one cigarette a day ; smoking history was elicited by questionnaire .
retinopathy was assessed through dilated pupils and graded as absent , background , or proliferative ( 12 ) .
baseline data are from first assessment 6 months after the onset of type 1 diabetes , after initial glycemic stabilization .
variables with skewed distribution are expressed as medians ( interquartile range ) ; values for all other variables are means ( sd ) . for nonnormally distributed variables
, comparisons between groups were performed using the mann - whitney u test , and unpaired student t tests were used for normally distributed variables .
whether low vitamin d levels predicted all - cause mortality in an explanatory model , a cox proportional hazards regression analysis was performed .
initially , a univariate analysis was performed using baseline variables ( sex , age , plasma total cholesterol , systolic blood pressure , uaer , smoking ) , all which have previously been shown to be associated with increased all - cause mortality .
all time - to - event variables were analyzed with a log - rank test and displayed as kaplan - meier plots according to levels of vitamin d either above or below the lower 10% percentile .
all statistical calculations were performed using spss for windows , version 14.0 ( spss , chicago , il )
baseline data are from first assessment 6 months after the onset of type 1 diabetes , after initial glycemic stabilization .
variables with skewed distribution are expressed as medians ( interquartile range ) ; values for all other variables are means ( sd ) . for nonnormally distributed variables ,
comparisons between groups were performed using the mann - whitney u test , and unpaired student t tests were used for normally distributed variables .
whether low vitamin d levels predicted all - cause mortality in an explanatory model , a cox proportional hazards regression analysis was performed .
initially , a univariate analysis was performed using baseline variables ( sex , age , plasma total cholesterol , systolic blood pressure , uaer , smoking ) , all which have previously been shown to be associated with increased all - cause mortality .
all time - to - event variables were analyzed with a log - rank test and displayed as kaplan - meier plots according to levels of vitamin d either above or below the lower 10% percentile .
all statistical calculations were performed using spss for windows , version 14.0 ( spss , chicago , il )
we followed 220 patients newly diagnosed with type 1 diabetes for a median ( range ) of 26.0 ( 1.029.0 ) years .
the median ( range ) vitamin d concentration was 44.6 ( 1.7161.7 ) nmol / l .
baseline clinical and laboratory characteristics of 220 type 1 diabetic patients in accordance with levels of 25(oh)d3 data are n , means ( sd ) , or medians ( interquartile range ) .
the patients were divided into two subgroups based on their vitamin d level being either equal to and below or above the value of the lower 10% percentile .
nmol / l in both men and women , and 22 ( 10% ) of the 220 included patients with a vitamin d value below this level . vitamin d level was not associated with age , sex , uaer , and blood pressure , but a weak negative association was found with hba1c .
( r = 0.14 , p = 0.04 ) . during follow - up , 44 ( 20% ) of
seven of the 22 ( 32% ) patients with vitamin d levels 15.5 nmol / l died compared with 37 ( 19% ) of the patients with vitamin d levels > 15.5 nmol / l ( p = 0.06 ) .
figure 1 shows a kaplan - meier curve for mortality according to a vitamin d level equal to and below or above the lower 10% percentile . in a cox proportional hazards model ,
the unadjusted hr ( 95% ci ) for mortality in subjects with vitamin d levels equal to or below the lower 10% percentile was 2.0 ( 0.94.4 ) , p = 0.1 .
the association persisted and became statistically significant after adjustment for uaer , hba1c , and conventional cardiovascular risk factors ( age , sex , blood pressure , cholesterol , smoking ) ( covariate adjusted hr 2.7 [ 1.16.7 ] , p = 0.03 ) .
an analysis of vitamin d as a continuous variable in the cox model could not demonstrate a significant relation to all - cause mortality , suggesting the relationship is not linear over the range of vitamin d values .
kaplan - meier curves of all - cause mortality in 220 type 1 diabetic patients in accordance with a vitamin d level equal to and below or above the lower 10% percentile , 25(oh)d3 = 15.5
nmol / l ; dashed black line , 25(oh)d3 15.5 nmol / l . during follow - up , 81 ( 34% )
of these patients , 6 ( 27% ) had a vitamin d level equal to or below the lower 10% percentile .
of the 81 patients who developed microalbuminuria , 27 ( 12% ) progressed to persistent macroalbuminuria , but only 2 of the patients with vitamin d levels equal to or below the lower 10% percentile progressed , corresponding to 9% .
a cox proportional hazards model showed that low vitamin d levels at baseline did not predict the development of micro- or macroalbuminuria ( unadjusted hr 1.1 [ 0.52.4 ] , p = 0.8 and hr 1.3 [ 0.35.4 ] , p = 0.7 , respectively ) .
a total of 192 ( 80% ) patients developed background retinopathy , and 18 ( 82% ) of the patients had vitamin d levels equal to or below the lower 10% percentile .
of the 192 patients , 34 ( 15% ) patients progressed to proliferative retinopathy , and one of these patients had low vitamin d level at baseline , corresponding to 5% .
vitamin d levels equal to or below the lower 10% percentile at baseline did not predict development of background retinopathy or proliferative retinopathy ( hr 1.1 [ 0.71.7 ] , p = 0.8 and hr 3.1 [ 0.422.9 ] , p = 0.3 , respectively ) .
after adjusting for progression promoters , the associations between severe vitamin d deficiency at baseline and development or progression of microvascular complications weakened further .
of these patients , 6 ( 27% ) had a vitamin d level equal to or below the lower 10% percentile .
of the 81 patients who developed microalbuminuria , 27 ( 12% ) progressed to persistent macroalbuminuria , but only 2 of the patients with vitamin d levels equal to or below the lower 10% percentile progressed , corresponding to 9% .
a cox proportional hazards model showed that low vitamin d levels at baseline did not predict the development of micro- or macroalbuminuria ( unadjusted hr 1.1 [ 0.52.4 ] , p = 0.8 and hr 1.3 [ 0.35.4 ] , p = 0.7 , respectively ) .
a total of 192 ( 80% ) patients developed background retinopathy , and 18 ( 82% ) of the patients had vitamin d levels equal to or below the lower 10% percentile .
of the 192 patients , 34 ( 15% ) patients progressed to proliferative retinopathy , and one of these patients had low vitamin d level at baseline , corresponding to 5% .
vitamin d levels equal to or below the lower 10% percentile at baseline did not predict development of background retinopathy or proliferative retinopathy ( hr 1.1 [ 0.71.7 ] , p = 0.8 and hr 3.1 [ 0.422.9 ] , p = 0.3 , respectively ) . after adjusting for progression promoters , the associations between severe vitamin d deficiency at baseline and development or progression of microvascular complications weakened further .
in this 26-year prospective observational follow - up study , we found very low levels of plasma 25(oh)d3 ( equal to or below the 10% percentile ) after onset of diabetes to be a strong and independent predictor of all - cause mortality in type 1 diabetic patients .
this association was not only independent of glycemic control and conventional cardiovascular risk factors , but also independent of uaer .
severe vitamin d deficiency at baseline did not predict development or progression of microvascular complications in the eye and kidney .
our finding of an association between severe vitamin d deficiency and increased risk of mortality complement recent data from epidemiological studies suggesting similar associations in other populations .
for the general population , a cross - sectional study of 13,331 participants from the national health and nutrition examination survey ( nhanes ) iii ( 2 ) showed low vitamin d levels to be associated with all - cause mortality .
a study of mainly nondiabetic ckd patients ( 16 ) found vitamin d to independently predict all - cause mortality and increased risk of progression to dialysis . also among patients with nondiabetic end - stage renal disease ,
low levels of vitamin d have been associated with all - cause mortality ( 17 ) . in healthy subjects ,
vitamin d deficiency mainly results from inadequate sunlight exposure , skin color , and inadequate nutritional supply of vitamin d  containing foods .
seasonal variations in vitamin d levels occur depending on geographic latitude and sun exposure in particularly .
a study ( 18 ) done in the general population in a northern european country showed a seasonal variation of vitamin d insufficiency of 73% and 29% for winter and summer , respectively .
furthermore , several conditions such as obesity and absorption , liver , or kidney disorders pose an increased risk of developing vitamin d deficiency .
the mechanisms of action behind the increased mortality risk seen among patients with the lower levels of vitamin d at baseline are unclear .
a growing amount of evidence indicates that vitamin d , through activation of the vitamin d receptor , has clinically important noncalcemic pleiotropic effects .
activation of the vitamin d receptor is associated with suppression of the raas ( 19 ) , cardiac myocyte hypertrophy ( 20 ) , vascular calcification , and atherosclerosis lowering , anti - inflammatory ( 21 ) , and immunomodulatory actions . also , an inverse relationship is found between vitamin d and increased incident risk of certain cancers as well as a higher mortality from these cancers ( 1 ) .
diabetic patients have a higher rate of cardiovascular morbidity and mortality compared with the general population . in a 10 year follow - up study of a cohort of 593 normoalbuminuric patients with type 1 diabetes
, we have previously shown that more than one - third died of known cardiovascular causes and 25% died of unknown , potentially cardiovascular causes ( 4 ) , hence a large number of the observed deaths in the cohort of the current study are likely to be from cardiovascular causes . in a cross - sectional study of type 2 diabetic patients with mild kidney impairment ,
vitamin d deficiency was shown to be strongly associated with a higher prevalence of manifest cardiovascular disease when compared with normal vitamin d status ( 22 ) .
in a recent study of type 2 diabetic patients ( 23 ) , which investigated the mechanism by which vitamin d deficiency mediates increased risk of cardiovascular disease , investigators found a reduced vitamin d receptor signaling to be a potential mechanism underlying increased foam cell ( macrophages that ingested oxidized ldl ) formation and accelerated cardiovascular disease in diabetic compared with nondiabetic patients .
given the observational design , the current study does not elaborate further on underlying mechanisms but indirectly adds to the increasing amount of data suggesting that vitamin d substitution might be a potential therapeutic target to prevent vascular disease progression . however , it is not possible to rule out that low levels of vitamin d may result from a single yet unidentified factor that is at the same time responsible for the increased risk seen in this study .
the pathogenesis behind microvascular complications in diabetes is complex and thought to involve multiple pathways .
development of microalbuminuria in diabetic patients is closely associated with an increased risk of both renal and cardiovascular disease .
it has been suggested that microalbuminuria often regresses to normoalbuminuria , but in this cohort , spontaneous and permanent regression to normoalbuminuria occurred in only 16% of the patients progressing to microalbuminuria , whereas 34% progressed to persistent macroalbuminuria during follow - up ( 24 ) .
of particular importance to diabetic nephropathy is an activation of the intrarenal raas and the proinflammatory nuclear factor-b pathway , both promoting progressive renal damage . in a post
( 10 ) found that administration of a vdra compared with placebo caused reduction in albuminuria in nondiabetic ckd as measured by a dipstick method ( p = 0.004 ) .
importantly , the effects appeared to be independent of concomitant therapies to inhibit the raas . recently
the vital study , a randomized controlled clinical trial with 281 type 2 diabetic patients with diabetic nephropathy , has shown that administration of a vdra ( paricalcitol ) in addition to blockade of the raas causes sustained reduction in albuminuria and thereby potentially has clinically relevant renoprotective effects in patients with diabetic nephropathy ( 11 ) .
a cross - sectional study of 581 type 2 diabetic patients shows significant association between the existence of proliferative retinopathy and a decrease in 25(oh)d3 .
also , investigators found a decrease in 25(oh)d3 according to the number of microvascular complications present ( 25 ) .
a cross - sectional design does not contribute to clarification on causality and effect , but fuels speculation that low levels of vitamin d might be a risk marker of development or progression of both diabetic nephropathy and/or retinopathy . in this study , however , we did not find low 25(oh)d3 levels to significantly predict development or progression of microvascular complications .
this could be because of the lack of power based on a low number of events as the hr for both retinopathy and albuminuria was above 1 in patients with severe vitamin d deficiency .
it is also possible that a potentially damaging effect of low vitamin d is offset by optimized patient treatment aiming at improving blood glucose and lowering blood pressure inclusive of raas blockade . in our study ,
international consensus is lacking in regard to definitions of what vitamin d levels are to be regarded as normal , insufficiency , and deficiency .
the limits for a physiological optimal vitamin d level are still a matter of debate in the literature .
although 25(oh)d3 is shown to be stable in stored samples ( 15 ) , storing could affect absolute concentrations because of evaporation , thus we arbitrarily chose the lower 10% percentile rather than an absolute value .
we have previously used the same method to define the cutoff in a follow - up study in type 2 diabetic patients ( 3 ) .
furthermore , we have recently analyzed plasma 25(oh)d3 in fresh samples from 200 type 2 diabetic patients and we found the lower 10% percentile in these patients to deviate only 0.1 nmol / l from the danish national definition of severe vitamin d deficiency existing at the time .
the stability of plasma 25(oh)d3 levels in our samples was tested before analysis as previously described .
no statistically significant difference in levels was found when compared according to years of storage .
one element of methodological strength is the prospective design and long follow - up period as well as completeness of follow - up .
given the observational design , it is not possible to infer causality from the associations described .
further limitations of our study are related to possible changes in the level of vitamin d throughout the year .
we did not adjust for seasonal change . nor did we have baseline data on physical activity , which could be related to outdoor activity , sun exposure and thereby levels of vitamin d. vitamin d levels are also influenced by nutritional status . unfortunately no nutritional parameters were available except bmi , which was not related to vitamin d levels ( data not shown ) .
we were therefore not able to adjust for any of these possible cofounders , but in general , the patients were young and healthy apart from newly diagnosed type 1 diabetes .
more observational studies are needed to confirm this finding , but as mentioned , we have performed the same analysis in a cohort consisting of type 2 diabetic patients and found similar results in regards to all - cause mortality and development / progression of diabetic nephropathy .
furthermore , in the study comprising type 2 diabetic patients , very low levels of 25(oh)d3 were also able to predict an increased risk of cardiovascular disease .
another limitation to the current study is that we do not have access to data on the cause of death . in conclusion , with the present prospective follow - up study we are now able to show that baseline levels of 25(oh)d3 equal to or below the 10% percentile predict increased risk of all - cause mortality in type 1 diabetic patients , as already shown for the general population , in patients with nondiabetic ckd and for patients with type 2 diabetes ( 3 ) .
we were not able to demonstrate an association between baseline levels of 25(oh)d3 equal to or below the 10% percentile and development of microvascular complications in type 1 diabetic patients .
randomized controlled clinical trials administrating vdra are necessary in order to prove causality between vitamin d status and survival prognosis in diabetic patients .
 objectiveto evaluate vitamin d as a predictor of all - cause mortality , progression from normoalbuminuria to micro- or macroalbuminuria , and the development of background or proliferative retinopathy in patients with type 1 diabetes.research design and methodsa prospective observational follow - up study in which an inception cohort of type 1 diabetic patients was followed from onset of diabetes diagnosed between 1979 and 1984 . 
 plasma vitamin d [ 25(oh)d3 ] levels were determined by high performance liquid chromatography / tandem mass spectrometry in 227 patients before the patients developed microalbuminuria . 
 values equal to or below the 10% percentile ( 15.5 
 nmol / l ) were considered severe vitamin d deficiency.resultsmedian ( range ) vitamin d was 44.6 ( 1.7161.7 ) nmol / l . 
 vitamin d level was not associated with age , sex , urinary albumin excretion rate ( uaer ) , or blood pressure . during follow - up , 44 ( 18% ) 
 patients died . in a cox proportional hazards model , 
 the hazard ratio for mortality in subjects with severe vitamin d deficiency was 2.7 ( 1.16.7 ) , p = 0.03 , after adjustment for uaer , hba1c , and conventional cardiovascular risk factors ( age , sex , blood pressure , cholesterol , smoking ) . 
 of the 220 patients , 81 ( 37% ) developed microalbuminuria and 27 ( 12% ) of these progressed to macroalbuminuria . 
 furthermore , 192 ( 87% ) patients developed background retinopathy , whereas 34 ( 15% ) progressed to proliferative retinopathy . 
 severe vitamin d deficiency at baseline did not predict the development of these microvascular complications.conclusionsin patients with type 1 diabetes , severe vitamin d deficiency independently predicts all - cause mortality but not development of microvascular complications in the eye and kidney . 
 whether vitamin d substitution in type 1 diabetic patients can improve the prognosis remains to be investigated . 
RESEARCH DESIGN AND METHODS
Statistical analysis
RESULTS
Microvascular complications
CONCLUSIONS