the study participants from the usa are members of a colorectal cancer prognosis study 
 ( colocare ) , with sites at the fred hutchinson cancer research center , seattle ( wa , usa ) , 
 h. lee moffitt cancer center and research institute , tampa ( fl , usa ) , and the national 
 center for tumor diseases , german cancer research center , heidelberg ( germany ) . the 
 objective of our consortium is to identify the factors that determine short - term survival 
 in a prospective cohort of colorectal cancer patients . these include inherited genetic and 
 tumour characteristics that affect early detection , treatment response and prognosis as 
 well as health behaviours that patients adopt that may have health outcomes . german 
 samples included in this pilot study were obtained from participants of the darmkrebs 
 chancen der verhtung durch screening ( dachs ) study at the german cancer research center 
 in heidelberg ( germany ) 
 . samples included from tampa were collected as part of moffitt 's total 
 cancer care program 
 . eligible for the study were colorectal cancer patients , at least 18 years of age , with a 
 first diagnosis of invasive colon or rectal cancer ( stages i  iv ) receiving care at any of 
 the colocare consortium / dachs sites . this pilot study involved 149 colorectal cancer cases 
 and ninety - one age- and sex - matched controls ( heidelberg , forty cases and forty controls ; 
 seattle , ninety - one cases and thirty - three frequency - matched controls ; tampa , eighteen 
 cases and eighteen controls ) , recruited between the years 2006 and 2010 and whose 
 biospecimens were obtained within the same time frame with identical blood - draw protocols 
 at each of the sites . blood samples were obtained at the time of recruitment into the 
 study . for the cases , the samples were pre - treatment samples obtained at the time of 
 surgery . in the dachs study , control subjects were community - based and randomly selected 
 from population registries . they were frequency - matched to cases on age , sex and county of 
 residence . we excluded controls with a history of colorectal cancer ; otherwise eligibility 
 criteria were the same as for the cases . controls were contacted through mail and 
 follow - up calls , and interviews were conducted at their homes . the participation rate 
 among eligible control participants was a little higher than 50  % 
 . the controls in moffitt , tampa were frequency matched to the cases on 
 age ( 5-year intervals ) , sex and race ( white non - hispanic ) . controls in seattle were drawn 
 from participants in a screening and surveillance colonoscopy programme who had no 
 evidence of pathology within the colon and were matched to the cases for age , sex and 
 race . this study was conducted according to the guidelines laid down by the declaration of 
 helsinki and all procedures involving human subjects were approved by the institutional 
 review boards of the german cancer research center ( dkfz ) , the fred hutchinson cancer 
 research center , seattle ( wa , usa ) and h. lee moffitt cancer center and research 
 institute , tampa ( fl , usa ) . plasma 25(oh)d3 was measured by reverse - phase hplc in the biochemistry and 
 biomarkers laboratory of the division of preventive oncology , german cancer research 
 center , dkfz ( dr owen ) . the intra - assay and inter - assay cv of 25(oh)d3 at a 
 concentration of 50  ng / ml were 23 and 31  % , respectively . snb 
 57co / i radioassay kit ( mp biomedicals ) in the biomarker laboratory ( dr 
 song ) at the fred hutchinson cancer research center with intra - assay cv of 31 and 35  % 
 and inter - assay cv of 70 and 51  % for folate and vitamin b12 , 
 respectively . to account for skewed distributions , wilcoxon or kruskal  wallis tests were used to test 
 for the differences in biomarker concentration by age group ( 1859 , 6069 and 7080 
 years ) , case status , tumour stage ( i  iv ) , study site ( heidelberg , seattle and tampa ) , and 
 season of blood draw ( spring : march  may ; summer : june  august ; autumn : september  november ; 
 winter : december  february ) . because the samples were unequally distributed across seasons 
 at the three study sites , we computed season - corrected levels for the descriptive analyses 
 of 25(oh)d3 in order to give equal weight to all four seasons . linear 
 regression analysis ( using both forward stepwise and enter methods ) with log - transformed 
 outcome parameters was used for multivariate analyses . all statistical analyses were 
 conducted using sas ( v9.1.2 , sas institute inc . ) . statistical significance was defined as 
 p  <  005 and all statistical tests were two - sided . cases and controls were , on average , aged 600 ( sd 133 ) and 609 ( sd 
 131 ) years , respectively . the majority of tumours were of stages iii ( 396  % ) and iv 
 ( 262  % ) . among the cases , the mean 25(oh)d3 , folate and vitamin b12 
 concentrations were 256 ( sd 135 )  ng / ml , 216 ( sd 140 )  ng / ml , and 638 
 ( sd 597 )  pg / ml , respectively , whereas among the controls , they were 259 
 ( sd 140 )  ng / ml , 180 ( sd 137 )  ng / ml , and 515 ( sd 241 )  pg / ml 
 ( table 1 ) . table 1.baseline characteristics of colorectal cancer cases and controlscases ( n 149)controls ( n 91)characteristic 
 n 
 % 
 n 
 % study siteheidelberg4026840440seattle9161133363tampa1812118198age ( years)mean6061 
 sd 
 1313185964433640606947323235709038262325sexmale80544853female69464347racewhite , non - hispanic137928492non - white or other12878tumour stagei ( i , ia , ib and ic)20134ii ( ii , iia , iib and iic)30201iii ( iii , iiia , iiib and iiic)59396iv39262unknown107season of blood drawspring ( march  may)2718123253summer ( june  august)3221525275autumn ( september  november)4429523253winter ( december  february)4630920220nutritional biomarker25(oh)d3 ( ng / ml)mean256259 
 sd 
 135140range4680040830folate ( ng / ml)mean216180 
 sd 
 140137range0370314625vitamin b12 ( pg / ml)mean638515 
 sd 
 597241range96375094135225(oh)d3 , 25-hydroxyvitamin d3 . baseline characteristics of colorectal cancer cases and controls 25(oh)d3 , 25-hydroxyvitamin d3 . in multivariate modelling , significant predictors for the biomarkers included season for 
 25(oh)d3 , study site for 25(oh)d3 , folate , vitamin b12 , 
 and tumour stage for vitamin b12 ( table 
 2 ) . probably because of the limited sample size of this pilot study , case status was 
 not statistically significant as a predictor ; hence , cases and controls were combined in 
 subsequent analyses . also , the results from models excluding case - status were identical to 
 those including case - status in the model . the association from controls only was similar , 
 yet did not reach statistical significance . plasma 25(oh)d3 concentrations were , 
 on average , higher in heidelberg ( 317  ng / ml ; range 110830 ng / ml ) than in seattle 
 ( 233  ng / ml ; range 40800 ng / ml ) and tampa ( 211  ng / ml ; range 46516 ng / ml ) ( table 3 ) . hypovitaminosis d ( plasma 
 25(oh)d3 < 20  ng / ml ) was more prevalent in tampa ( 54  % ) and seattle ( 50  % ) 
 than in heidelberg ( 23  % ) ( data not shown ) . on the other hand , plasma folate concentrations 
 were , on average , lower in heidelberg ( 111  ng / ml ; range 03482 ng / ml ) than in seattle 
 ( 253 ng / ml ; range 35703 ng / ml ) and tampa ( 238 ng / ml ; range 68697 ng / ml ) . no 
 participants from seattle and tampa had folate deficiency ( folate <3  ng / ml ) , while 
 10  % of those from heidelberg had folate deficiency . plasma vitamin b12 
 concentrations followed the same pattern as folate with lower average values recorded in 
 heidelberg ( 395  pg / ml ; range 961641 pg / ml ) than in seattle ( 740  pg / ml ; range 1833750 
 pg / ml ) and tampa ( 522  pg / ml ; range 941085 pg / ml ) . table 2.predictors of plasma 25-hydroxyvitamin d3 ( 25(oh)d3 ) , vitamin 
 b12 and folate among colorectal cancer cases and controlsbiomarkerpredictordirectionsignificance25(oh)d3heidelberg+**summer season+**autumn season+*vitamin b12heidelberg**stage iv+**folateheidelberg**age 6069 years+ * * p  <  010 , * * p  <  005.cases and controls were combined because case status was not statistically 
 significant . table 3.nutritional biomarker concentrations among colorectal cancer cases and controls in 
 the three study locations(mean values and ranges)heidelbergseattletampameanrangemeanrangemeanrange25(oh)d3 ( ng / ml)317110 , 83023340 , 80021146 , 516folate ( ng / ml)11103 , 48225335 , 70323868 , 697vitamin b12 ( pg / ml)39596 , 1641740183 , 375052294 , 108525(oh)d3 , 25-hydroxyvitamin d3 . predictors of plasma 25-hydroxyvitamin d3 ( 25(oh)d3 ) , vitamin 
 b12 and folate among colorectal cancer cases and controls * p  <  010 , * * p  <  005 . cases and controls were combined because case status was not statistically 
 significant . nutritional biomarker concentrations among colorectal cancer cases and controls in 
 the three study locations ( mean values and ranges ) 25(oh)d3 , 25-hydroxyvitamin d3 . seasonal differences in plasma 25(oh)d3 concentrations were evident in 
 heidelberg , where the highest plasma 25(oh)d3 concentrations were observed in 
 summer ( 370  ng / ml ) and the lowest in spring ( 213  ng / ml ) , but no such seasonal variation 
 was noted in seattle and tampa ( fig . 1.(a ) plasma vitamin d ( 25-hydroxyvitamin d ; 25(oh)d ) concentration by study site . the horizontal bars represent medians and the circles 
 represent potential outliers in the data . ( d ) seasonal plasma 25(oh)d by study site : 
 , spring ; , 
 summer ; , autumn ; , 
 winter . values are means . ( a ) plasma vitamin d ( 25-hydroxyvitamin d ; 25(oh)d ) concentration by study site . the horizontal bars represent medians and the circles 
 represent potential outliers in the data . ( d ) seasonal plasma 25(oh)d by study site : 
 , spring ; , 
 summer ; , autumn ; , 
 winter . to the best of our knowledge , this is the first study to compare a range of nutritional 
 biomarkers ( 25(oh)d3 , folate and vitamin b12 ) among colorectal cancer 
 patients and controls in different geographical regions . our international pilot study 
 revealed differences in biomarker concentrations between the us sites and germany . because dietary sources of vitamin d are very limited , cutaneous synthesis of vitamin d 
 ( within the uvb wavelength of 290315  nm ) is a very important source of plasma 
 25(oh)d3 
 . the uv radiation required for the synthesis of vitamin d in the skin is 
 more abundant and more functional during summer than during winter 

 . hence , among people who do not use adequate vitamin d supplements or 
 consume vitamin d - fortified foods , season is one of the most important determinants of 
 plasma 25(oh)d3 concentrations . thus , the seasonal variations in plasma 25(oh)d3 observed in heidelberg are in 
 line with expectations . somewhat surprising is the lack of seasonal variation and the low 
 plasma 25(oh)d3 concentrations observed in seattle and tampa . of the three sites , 
 tampa has the highest daytime temperatures during summer but the participants from tampa had 
 the lowest plasma 25(oh)d3 concentrations during summer . the differences in 
 plasma 25(oh)d3 concentration during summer among participants in our study could 
 reflect the extent of outdoor activities and possible sun - avoidance behaviours ( including 
 sunscreen use ) in the three cities . in heidelberg , people are more likely to commute with 
 bicycles or walk than use cars , whereas people are more likely to practise sun - avoidance in 
 florida 
 . few studies have examined the impact of season on 25(oh)d3 concentrations in 
 lower altitude regions in the usa . although a previous study in south florida reported a 
 seasonal variation in serum 25(oh)d3 concentrations 
 , the magnitude ( 14  % ) is much smaller than what is observed ( 38  % ) among 
 residents of northern latitudes in the usa 

 . compared with the previous study from south florida , the winter 
 25(oh)d3 concentrations among participants from tampa in our study are 
 identical ( 233 v. 231  ng / ml , respectively ) but the summer concentrations 
 were lower ( 177 v. 268  ng / ml ) . thus , the low summer 25(oh)d3 
 concentrations in tampa could be a chance finding due to the low number of participants from 
 tampa in our study . studies have revealed that current food fortification practices in the usa may not be 
 adequate to prevent vitamin d deficiency among vulnerable people during 
 winter 

 . likewise , it has been shown that although supplement use increases 
 serum 25(oh)d3 concentrations , it does not make up for insufficient sun 
 exposure 
 , and current supplement recommendations are not adequate to prevent 
 vitamin d deficiency 
 . as vitamin d supplementation is discussed in the context of improving 
 survival in colorectal cancer patients 

 , our study shows that high plasma vitamin d concentrations can be 
 achieved in populations with substantial outdoor activity during summer ( such as 
 heidelberg ) . nevertheless , vitamin d fortification in the usa did not prevent low levels of 
 serum 25(oh)d3 in tampa or seattle , cities that practise sun - avoidance or reduced 
 sun exposure . the lower plasma folate concentrations among participants from heidelberg compared with 
 those from the usa are not unexpected considering the fact that mandatory fortification of 
 staple foods with folic acid commenced in the usa in 1998 
  while fortification is not practised in germany 
 . the mandatory fortification programme has resulted in a substantial 
 increase in plasma folate concentrations in the usa , where the prevalence estimates for low 
 serum folate concentrations ( <3  ng / ml ) declined from 206  % in the period 19881994 
 to 06  % in 2004 
 . however , we also observed large intra - site differences in plasma folate and vitamin 
 b12 concentrations at each study site . this may be due mostly to the use of 
 folic acid - containing supplements . it has been shown that supplement and multivitamin use is 
 higher among cancer patients compared with the general population 
 . between 64 and 81  % of cancer patients use any vitamin or mineral 
 supplement , compared with 50  % in the general population 
 . in a large study in washington state , usa , colorectal cancer patients 
 were 33  % more likely to use folic acid supplements compared with cancer - free 
 controls 
  and such a supplement use is associated with higher plasma folate and 
 vitamin b12 concentrations 
 . therefore , the use of folic acid - containing supplements is an important 
 determinant of plasma folate concentrations in colorectal cancer patients . this may be of 
 potential concern considering the fact that very high plasma folate concentrations that can 
 be achieved with supplement use may accelerate the growth of already existing colorectal 
 cancers 
 . in a randomised controlled trial , folic acid given at a dosage of 
 1 mg / d was associated with a 17-fold increased risk of advanced colorectal 
 cancers 
 , thus excluding its utility and safety as a secondary or tertiary 
 prevention agent for colorectal cancer . however , this is an area of ongoing research and 
 further studies are needed to elucidate the relationship between folate and survival in 
 patients with colorectal cancer . a particular strength of our study is the comparison of biomarkers across international 
 study sites with identical laboratories for assays and consistent blood - draw protocols . however , our sample size as well as variable set was limited and we had no information on 
 supplement use ; hence we could not investigate the association of supplement use with 
 nutritional biomarker concentrations . in conclusion , we observed substantial differences in nutritional biomarker concentrations 
 across international study sites , probably reflecting dissimilarities in supplement use , 
 food fortification , and sun - seeking behaviour , among other health practices . intra - site differences at each study location were larger than between - location variability , 
 suggesting that individual health behaviours play a significant role . as we aim to 
 understand predictors of colorectal cancer prognosis , there is much to learn from 
 international cohorts , which offer a broad range of exposure and health behaviours that 
 can not be obtained at a single site .
vitamin d and folate are associated with decreased colorectal cancer risk and their 
 association with colorectal cancer prognosis is under investigation . 
 we assessed the 
 levels of plasma 25-hydroxyvitamin d3 ( 25(oh)d3 ) , folate and vitamin 
 b12 in an international pilot study in order to determine variability of 
 these biomarkers based on geographical location . 
 plasma 25(oh)d3 , folate and 
 vitamin b12 concentrations were measured in 149 invasive , newly diagnosed 
 colorectal cancer cases from heidelberg ( germany ) , seattle ( wa , usa ) , and tampa ( fl , usa ) 
 and in ninety - one age- and sex - matched controls . their associations with potential 
 predictors were assessed using multivariate linear regression analyses . 
 plasma 
 25(oh)d3 , folate and vitamin b12 concentrations differed by 
 location . 
 other predictors were season for 25(oh)d3 and tumour stage ( vitamin 
 b12 ) . 
 season - corrected average 25(oh)d3 concentrations were higher 
 in heidelberg ( 317  ng / ml ; range 110830 ng / ml ) than in seattle ( 233  ng / ml ; range 
 40800 ng / ml ) and tampa ( 211  ng / ml ; range 46516 ng / ml ) . in heidelberg , a strong 
 seasonal variation was observed . 
 folate ( 111  ng / ml ) and vitamin b12 
 ( 395  pg / ml ) concentrations in heidelberg were lower than those in seattle ( 253  ng / ml and 
 740  pg / ml , respectively ) and tampa ( 238  ng / ml and 522  pg / ml , respectively ) . 
 differences 
 in plasma 25(oh)d3 and folate concentrations between heidelberg and the us 
 sites were observed , probably reflecting variation in outdoor activities and sun - avoidance 
 behaviour during summer as well as in folic acid fortification and supplement use . 
 intra - site differences at each study location were greater than between - location 
 variability , suggesting that individual health behaviours play a significant role . 
 nevertheless , the intra - site differences we observed may be due to chance because of the 
 limited sample size . 
 our pilot study illustrates the value of an international cohort in 
 studying colorectal cancer prognosis to discern geographical differences in a broad range 
 of exposures .