the wellgen study design has been described in detail elsewhere ( 21 ) . in brief , type 2 diabetic patients attending the diabetes clinic of the king edward memorial hospital in pune were enrolled since 2005 , and the study is currently ongoing . patients suffering from acute intercurrent illnesses ( n = 4 ) were rescheduled for a blood sample after 4 weeks . usually , it takes 712 days for crp to return to baseline after an acute illness ( 22 ) . for the current study , we included patients who were enrolled between march 2005 and may 2007 , and who were residing within the pune municipal corporation boundary . the study was approved by the institutional ethics committee , and all patients gave a written informed consent . in addition , all patients answered a standard questionnaire that gave information about their residential address , age , sex , smoking history , alcohol consumption , and medical history , including current drug treatment . a fasting blood sample was drawn to examine various biochemical measurements , including crp , glucose , hba1c , hemoglobin , and other hematological parameters . blood samples were stored for an average period of 20 months ( 432 months ) at 80c before the measurement of crp . high - sensitivity crp concentrations were measured with an elisa kit ( diagnostic biochem canada ) , and coefficient of variability ( cv ) for the assay was < 16% . air pollution data for the conventional pollutants sulfur dioxide ( so2 ) , oxides of nitrogen ( nox ) , and particulate matter with an aerodynamic profile 10 m ( pm10 ) have been measured in pune under the national air quality monitoring program since 2004 and are available online in a public domain ( http://mpcb.gov.in/ ) . these published data were collected from three locations ( swargate , nal stop , and karve road ) situated around the city center ( admissible as per air pollution and health : a european approach [ aphea ] protocol ) ( 23 ) , and the data were accessed in january 2010 . the monitoring of pollutants was carried out for 24 h ( 4-h sampling for gaseous pollutants and 8-h for particulate matter ) on 2 consecutive days per week ( other than weekends ) in swargate and nal stop and 6 days a week in karve road ( supplementary fig . 1 ) . the average concentrations of so2 , nox , and pm10 from these three sites were used as urban background levels . there was no significant difference in the average 24-h readings from different monitors on a given day . missing air pollution data were imputed by linear interpolation technique ( 24 ) . ambient air pollutants and meteorological variables for each person s day of visit ( lag0 ) , and up to 7 days before ( lag17 ) as a short - term exposure , were then computed . furthermore , cumulative exposures for more extended periods of time ( average of 7 , 14 , and 30 days before blood collection ) were also computed . meteorological data for the city of pune during the period of the study were obtained from the pune office of the india meteorological department , a national data center . apparent temperature value was calculated as 2.653 + ( 0.994  dbt ) + ( 0.0153  dpt ) , where dbt and dpt are dry bulb temperature and dew point temperature , respectively ( 25 ) . data are presented as mean ( sd ) when normally distributed and median ( 25th75th percentile ) when not normally distributed . crp and fasting plasma glucose ( fpg ) concentrations were skewed and were therefore normalized using log10 transformation . characteristics of the study population were stratified by season ( monsoon [ june  october ] , winter [ november  february ] , and summer [ march  may ] ) . the differences of variables across season were tested by anova ( for normally distributed variables ) , kruskal - wallis ( for skewed variables ) , and  tests ( for categorical variables ) . the differences of crp concentration between two groups ( i.e. , men vs. women ) were tested using mann - whitney test . associations were tested using spearman ( rs ) and pearson ( r ) correlation coefficients . we used multiple linear regression to examine the association between ambient air pollution levels and crp concentration . the final multiple linear regression model was adjusted for a priori chosen confounders ( known or plausible ) including age , sex , fpg , hemoglobin , bmi , treatment with anti - inflammatory agents ( i.e. , statin , aspirin , and thiazolidinedione [ tzd ] ) , season , and meteorological variables ( relative humidity and air temperature ) . model selection was based on minimum values of akaike s information criterion ( 26 ) to obtain best fit with the minimum number of parameters in the model . the ambient air pollution and corresponding meteorological parameters ( relative humidity and air temperature ) were entered in the model to capture exposure on the day of the study ( lag0 ) or up to 7 days before ( lag17 ) and also as moving averages up to the last 30 days . the autocorrelation plot of the residual for the fully adjusted model was investigated for periodicity in order to avoid bias in the regression coefficient . the results are given as percent changes in geometric mean ( gm ) of crp concentration for an sd increment in air pollutant ( % change in gm = [ 10  1 ]  100 ) , where b is the regression coefficient . additionally , we checked the effect modification by bmi ( below vs. above the median , 25.60 kg / m ) , hba1c ( 8.80% ) , fpg ( 153 mg / dl ) , current smoking ( yes vs. no ) , intake of anti - inflammatory agent ( yes vs. no ) , and the season ( monsoon , winter , and summer).the significance threshold of p = 0.05 was used in all analyses . all statistical analyses were performed using stata version 11.1 software ( stata corporation , college station , tx ) . to explore the robustness of our results , alternative ways of modeling were also performed : 1 ) possible influence of additional adjustments for meteorological variables , including barometric pressure and apparent temperature , and 2 ) multipollutant models . the wellgen study design has been described in detail elsewhere ( 21 ) . in brief , type 2 diabetic patients attending the diabetes clinic of the king edward memorial hospital in pune were enrolled since 2005 , and the study is currently ongoing . patients suffering from acute intercurrent illnesses ( n = 4 ) were rescheduled for a blood sample after 4 weeks . usually , it takes 712 days for crp to return to baseline after an acute illness ( 22 ) . for the current study , we included patients who were enrolled between march 2005 and may 2007 , and who were residing within the pune municipal corporation boundary . the study was approved by the institutional ethics committee , and all patients gave a written informed consent . , all patients answered a standard questionnaire that gave information about their residential address , age , sex , smoking history , alcohol consumption , and medical history , including current drug treatment . a fasting blood sample was drawn to examine various biochemical measurements , including crp , glucose , hba1c , hemoglobin , and other hematological parameters . blood samples were stored for an average period of 20 months ( 432 months ) at 80c before the measurement of crp . high - sensitivity crp concentrations were measured with an elisa kit ( diagnostic biochem canada ) , and coefficient of variability ( cv ) for the assay was < 16% . air pollution data for the conventional pollutants sulfur dioxide ( so2 ) , oxides of nitrogen ( nox ) , and particulate matter with an aerodynamic profile 10 m ( pm10 ) have been measured in pune under the national air quality monitoring program since 2004 and are available online in a public domain ( http://mpcb.gov.in/ ) . these published data were collected from three locations ( swargate , nal stop , and karve road ) situated around the city center ( admissible as per air pollution and health : a european approach [ aphea ] protocol ) ( 23 ) , and the data were accessed in january 2010 . the monitoring of pollutants was carried out for 24 h ( 4-h sampling for gaseous pollutants and 8-h for particulate matter ) on 2 consecutive days per week ( other than weekends ) in swargate and nal stop and 6 days a week in karve road ( supplementary fig . the average concentrations of so2 , nox , and pm10 from these three sites were used as urban background levels . there was no significant difference in the average 24-h readings from different monitors on a given day . missing air pollution data were imputed by linear interpolation technique ( 24 ) . ambient air pollutants and meteorological variables for each person s day of visit ( lag0 ) , and up to 7 days before ( lag17 ) as a short - term exposure , were then computed . furthermore , cumulative exposures for more extended periods of time ( average of 7 , 14 , and 30 days before blood collection ) were also computed . meteorological data for the city of pune during the period of the study were obtained from the pune office of the india meteorological department , a national data center . apparent temperature value was calculated as 2.653 + ( 0.994  dbt ) + ( 0.0153  dpt ) , where dbt and dpt are dry bulb temperature and dew point temperature , respectively ( 25 ) . data are presented as mean ( sd ) when normally distributed and median ( 25th75th percentile ) when not normally distributed . crp and fasting plasma glucose ( fpg ) concentrations were skewed and were therefore normalized using log10 transformation . characteristics of the study population were stratified by season ( monsoon [ june  october ] , winter [ november  february ] , and summer [ march  may ] ) . the differences of variables across season were tested by anova ( for normally distributed variables ) , kruskal - wallis ( for skewed variables ) , and  tests ( for categorical variables ) . the differences of crp concentration between two groups ( i.e. , men vs. women ) were tested using mann - whitney test . associations were tested using spearman ( rs ) and pearson ( r ) correlation coefficients . we used multiple linear regression to examine the association between ambient air pollution levels and crp concentration . the final multiple linear regression model was adjusted for a priori chosen confounders ( known or plausible ) including age , sex , fpg , hemoglobin , bmi , treatment with anti - inflammatory agents ( i.e. , statin , aspirin , and thiazolidinedione [ tzd ] ) , season , and meteorological variables ( relative humidity and air temperature ) . model selection was based on minimum values of akaike s information criterion ( 26 ) to obtain best fit with the minimum number of parameters in the model . the ambient air pollution and corresponding meteorological parameters ( relative humidity and air temperature ) were entered in the model to capture exposure on the day of the study ( lag0 ) or up to 7 days before ( lag17 ) and also as moving averages up to the last 30 days . the autocorrelation plot of the residual for the fully adjusted model was investigated for periodicity in order to avoid bias in the regression coefficient . the results are given as percent changes in geometric mean ( gm ) of crp concentration for an sd increment in air pollutant ( % change in gm = [ 10  1 ]  100 ) , where b is the regression coefficient . additionally , we checked the effect modification by bmi ( below vs. above the median , 25.60 kg / m ) , hba1c ( 8.80% ) , fpg ( 153 mg / dl ) , current smoking ( yes vs. no ) , intake of anti - inflammatory agent ( yes vs. no ) , and the season ( monsoon , winter , and summer).the significance threshold of p = 0.05 was used in all analyses . all statistical analyses were performed using stata version 11.1 software ( stata corporation , college station , tx ) . to explore the robustness of our results , alternative ways of modeling were also performed : 1 ) possible influence of additional adjustments for meteorological variables , including barometric pressure and apparent temperature , and 2 ) multipollutant models . of the 1,700 patients enrolled in the wellgen study between 2005 and 2007 , 1,392 who lived in pune city and had a measurement of crp were included in this study ( table 1 ) . none of the patients were clinically diagnosed as having a chronic infective or inflammatory condition and none were on a steroid treatment . characteristics of type 2 diabetic patients and local levels of environmental variables ( air pollutants and meteorological measurement ) the median crp concentration was 3.49 mg / l ( 1.787.58 mg / l ) . fifty - seven percent of patients had crp concentrations in the high coronary risk zone i.e. , > 3 mg / l , and 19% of patients had abnormally raised crp concentrations ( > 10 mg / l ) . crp concentration was weakly associated with age ( r = 0.06 , p = 0.02 ) , and women had a higher concentration compared with men ( 5.17 mg / l for women vs. 2.76 mg / l for men ; p < 0.001 ) , crp was found to be positively associated with white blood cell count ( r = 0.07 , p = 0.03 ) , fpg ( r = 0.12 , p < 0.001 ) , hba1c ( r = 0.09 , p = 0.005 ) , and bmi ( r = 0.27 , p < 0.001 ) but inversely associated with air temperature ( r = 0.10 , p < 0.001 ) and apparent temperature ( r = 0.10 , p < 0.001 ) . patients on statins , aspirin , and tzd treatment had lower crp concentrations compared with those not on these treatments ( median crp for subjects treated with all three agents , 2.30 mg / l ; any one agent , 3.45 mg / l ; no agent , 3.94 mg / l ) . crp concentrations were not associated with hemoglobin concentration , duration of diabetes , smoking , alcohol usage , and relative humidity . air pollution data were available for 647 of the 822 days of the study ; data were imputed for the remaining days . the air pollutant levels were highest in winter and lowest in monsoon . during the study period , the concentrations of so2 and nox never crossed the daily national ambient air quality standard ( naaqs ) of 80 g / m ( 27 ) . for 598 days ( 62% ) , the 24-h pm10 levels were found to be above the daily naaqs of 100 g / m . the daily concentrations of ambient so2 , nox , and pm10 were interrelated in all seasons ; rs ranged from 0.27 to 0.75 ( p < 0.001 ) , with a lower correlation observed during the winter ( supplementary fig . air temperature was inversely correlated with air pollutant concentration ; rs ranged from 0.13 to 0.22 ( p < 0.001 ) but was positive only with pm10 during monsoon ( rs = 0.12 , p = 0.002 ) . relative humidity showed negative correlations with ambient pollutants ; rs ranged from 0.28 to 0.45 ( p < 0.001 ) . we studied the contribution of ambient air pollution at lag07 and different averaging time periods ( 7 , 14 , and 30 days ) to the concentration of serum crp . we found that a 1 sd increment in city - wide so2 was associated with a significant increase in crp concentrations , ranging from 8.67 to 12.42% for lag periods up to 7 days and of 11.23 to 14.40% for multiday averaging periods of up to 30 days before blood collection . for nox , the increase was between 7.77 and 11.60% for lag up to 7 days and 12.80% for 7 days for pm10 , there were no significant associations with crp concentrations either at different lag days or during averaging times , except during the summer ( e.g. , for lag4 11.31% [ 95% ci 3.3619.88 ] ) ( fig . 1 ) . percent change in gm of crp per 1 sd increase in air pollutants . models are adjusted for age , sex , bmi , log10 fpg , hemoglobin , statin , aspirin , and tzd treatment , season , relative humidity , and air temperature . we present the effect modification only on the most influential lag days of so2 ( lag2 ) . enhanced associations between so2 and crp concentrations were observed among patients with a duration of diabetes < 7 years and patients who were not treated with statin and tzd medication , reflected in significant interaction terms ( fig . 2 ) . season did not modify the association between air pollution and crp concentrations significantly ( fig . effect modification ; the estimates are given as percent change in gm of crp per 1 sd increase of so2 ( lag2 ) . the estimates are given as percent change in the gm of crp per 1 sd increase of so2 ( lag2 ) , nox ( lag2 ) , and pm10 ( lag4 ) . additional adjustment for meteorological variables ( i.e. , barometric pressure and apparent temperature ) did not show any significant effect on the inflammatory response ( supplementary table 1 ) . multipollutant models indicated that so2 was the most important pollutant associated with serum crp concentration . of the 1,700 patients enrolled in the wellgen study between 2005 and 2007 , 1,392 who lived in pune city and had a measurement of crp were included in this study ( table 1 ) . none of the patients were clinically diagnosed as having a chronic infective or inflammatory condition and none were on a steroid treatment . characteristics of type 2 diabetic patients and local levels of environmental variables ( air pollutants and meteorological measurement ) the median crp concentration was 3.49 mg / l ( 1.787.58 mg / l ) . fifty - seven percent of patients had crp concentrations in the high coronary risk zone i.e. , > 3 mg / l , and 19% of patients had abnormally raised crp concentrations ( > 10 mg / l ) . crp concentration was weakly associated with age ( r = 0.06 , p = 0.02 ) , and women had a higher concentration compared with men ( 5.17 mg / l for women vs. 2.76 mg / l for men ; p < 0.001 ) . , crp was found to be positively associated with white blood cell count ( r = 0.07 , p = 0.03 ) , fpg ( r = 0.12 , p < 0.001 ) , hba1c ( r = 0.09 , p = 0.005 ) , and bmi ( r = 0.27 , p < 0.001 ) but inversely associated with air temperature ( r = 0.10 , p < 0.001 ) and apparent temperature ( r = 0.10 , p < 0.001 ) . patients on statins , aspirin , and tzd treatment had lower crp concentrations compared with those not on these treatments ( median crp for subjects treated with all three agents , 2.30 mg / l ; any one agent , 3.45 mg / l ; no agent , 3.94 mg / l ) . crp concentrations were not associated with hemoglobin concentration , duration of diabetes , smoking , alcohol usage , and relative humidity . air pollution data were available for 647 of the 822 days of the study ; data were imputed for the remaining days . the air pollutant levels were highest in winter and lowest in monsoon . during the study period , the concentrations of so2 and nox never crossed the daily national ambient air quality standard ( naaqs ) of 80 g / m ( 27 ) . for 598 days ( 62% ) , the 24-h pm10 levels were found to be above the daily naaqs of 100 g / m . the daily concentrations of ambient so2 , nox , and pm10 were interrelated in all seasons ; rs ranged from 0.27 to 0.75 ( p < 0.001 ) , with a lower correlation observed during the winter ( supplementary fig . air temperature was inversely correlated with air pollutant concentration ; rs ranged from 0.13 to 0.22 ( p < 0.001 ) but was positive only with pm10 during monsoon ( rs = 0.12 , p = 0.002 ) . relative humidity showed negative correlations with ambient pollutants ; rs ranged from 0.28 to 0.45 ( p < 0.001 ) . we studied the contribution of ambient air pollution at lag07 and different averaging time periods ( 7 , 14 , and 30 days ) to the concentration of serum crp . we found that a 1 sd increment in city - wide so2 was associated with a significant increase in crp concentrations , ranging from 8.67 to 12.42% for lag periods up to 7 days and of 11.23 to 14.40% for multiday averaging periods of up to 30 days before blood collection . for nox , the increase was between 7.77 and 11.60% for lag up to 7 days and 12.80% for 7 days average . for pm10 , there were no significant associations with crp concentrations either at different lag days or during averaging times , except during the summer ( e.g. , for lag4 11.31% [ 95% ci 3.3619.88 ] ) ( fig . 1 ) . percent change in gm of crp per 1 sd increase in air pollutants . models are adjusted for age , sex , bmi , log10 fpg , hemoglobin , statin , aspirin , and tzd treatment , season , relative humidity , and air temperature . we present the effect modification only on the most influential lag days of so2 ( lag2 ) . enhanced associations between so2 and crp concentrations were observed among patients with a duration of diabetes < 7 years and patients who were not treated with statin and tzd medication , reflected in significant interaction terms ( fig . 2 ) . season did not modify the association between air pollution and crp concentrations significantly ( fig . effect modification ; the estimates are given as percent change in gm of crp per 1 sd increase of so2 ( lag2 ) . the estimates are given as percent change in the gm of crp per 1 sd increase of so2 ( lag2 ) , nox ( lag2 ) , and pm10 ( lag4 ) . additional adjustment for meteorological variables ( i.e. , barometric pressure and apparent temperature ) did not show any significant effect on the inflammatory response ( supplementary table 1 ) . multipollutant models indicated that so2 was the most important pollutant associated with serum crp concentration . in this study , we report that type 2 diabetic patients residing in pune , india , show high concentrations of serum crp concentrations that correlate positively and significantly with ambient levels of so2 and nox , even at levels below the naaqs . although ambient pm10 levels exceeded naaqs on 62% of study days , it did not show significant correlation with crp concentrations . among the gaseous pollutants , so2 showed a stronger association compared with nox . moreover , the association was particularly stronger in patients with a shorter ( < 7 years ) duration of time since their diabetes diagnosis , and weaker in those who were receiving statins , aspirin , and tzd , drugs with known anti - inflammatory properties . to the best of our knowledge , this is the first study in this region of the world that has shown a significant association between ambient air pollutants and crp concentrations in type 2 diabetic patients . inflammation is a powerful risk factor for cvd in type 2 diabetic patients ( 28 ) , and our results suggest a possible environmental and preventable contribution to this risk . temporal characteristics of the associations indicate that circulating crp concentrations are relatively rapidly affected by exposure to so2 and nox ( within 1 day ) ( fig . the peak association was by 2-day lag , similar to a previous study in patients with coronary heart disease ( 12 ) , and it wore off progressively thereafter . multiday averaging analysis suggested an effect up to 30 days for so2 and 7 days for nox . for pm10 , the effect of air pollution on crp was first demonstrated in 112 elderly subjects in the u.k . , in whom crp was significantly associated with city mean concentration of pm10 over the previous 3 days ( 10 ) . a number of other studies made similar observations in different groups of subjects ( 1119 ) , although some studies have failed to show an association ( 29,30 ) . the difference in the findings in these studies may be attributable to differences in the composition of pollution , underlying medical conditions , and concomitant medication and dietary diversity ( 31).there is only one previous study in diabetic patients in belgium ( n = 233 ) ( 14 ) that found a significant association between crp concentrations and exposure to pm10 up to 1 week before blood collection ; there was no measurement of so2 and nox in this study . in other studies , subgroup analysis reported for diabetic patients suggests that diabetes enhances the effect of pollutants on crp concentrations ( 13,16 ) . crp is an established risk factor for coronary heart disease ( 9 ) and type 2 diabetes ( 8) . it is produced exclusively in the liver during systemic inflammation , and it is synthesized within 2448 h by hepatocytes ( 32 ) in response to cytokines released into circulation by activated leukocytes . its half - life is 19 h ( 33 ) , and plasma concentrations of crp reflect its synthesis rate . inhaled pollutants stimulate the alveolar macrophages and airway epithelial cells to secrete cytokines into circulation , which release leukocytes from bone marrow ( 34 ) . preexisting inflammation and oxidative stress enhanced susceptibility of diabetic patients to inhaled particles is likely to be attributed to a heightened state of inflammation , oxidative stress , and the excess adiposity characteristic of this condition ( 35 ) . it is interesting that in our study , we found that those on statin and tzd treatments ( which have anti - inflammatory action ) ( 36 ) had a lower crp concentration and a smaller rise in crp concentrations in relation to ambient pollution compared with those who were not on this treatment . higher inflammatory response in newly diagnosed diabetic patients could be attributed to their younger age and therefore possibly more exposure to outdoor air pollution , and because many were not yet started on medication that lowers inflammation . unlike gaseous pollutants , we did not find an association between pm10 exposure and crp concentration , even though the average pm10 levels were above naaqs ( 27 ) . the isolated positive association only during summer may be related to the composition of pm10 and meteorological factors ( i.e. , temperature ) , which can play an important role in pm toxicity ( 37,38 ) . this is a large - scale study reporting an association between air pollution and crp in urban indian diabetic patients . the study was possible because we could link the phenotypic data from the wellgen study with air pollutant data available in the public domain . the availability of extensive information on diabetic patients and meteorological variables allowed adjustments for individual as well as extraneous confounders . our models and results are robust to additional adjustment to barometric pressure and apparent temperature , and no indication of seasonality was seen in residuals . we believe that the estimated air pollution effect on crp concentrations in this study represents an underestimate because we used centrally measured air pollution as a surrogate for personal exposure and we did not account for the effect of indoor pollution and time spent outdoors . in addition , we were not able to account for a dietary pattern that may be associated with crp concentration ( 39 ) . diets rich in saturated fat and refined carbohydrates induce oxidative and inflammatory stress , whereas diets rich in fiber and fruits do not have such an effect ( 40 ) . in our future research , we will focus on the improvement of the exposure data considering emission inventory , meteorological data , traffic density , time activity , and diet . in summary , we found that exposure to traffic - related air pollutants is associated with a rapid increase in systemic inflammation ( i.e. , crp ) in diabetic patients . given the strong evidence that crp concentrations are associated with the development and complications of the metabolic syndrome ( 41 ) , our findings may have implications for enhanced risk of cvd in urban indian diabetic patients . our results should promote studies on the effect of air pollution on the risk of noncommunicable disease in india .
objectiveto study the association between ambient air pollutants and serum c - reactive protein ( crp ) concentration in 1,392 type 2 diabetic patients in pune , india.research design and methodsa cross - sectional study was conducted that linked daily time series of ambient air pollution data ( obtained from central monitoring sites ) and plasma crp concentration in type 2 diabetic patients from the wellcome trust genetic ( wellgen ) study , recruited between march 2005 and may 2007 . 
 air pollution effects on crp concentration were investigated with delays ( lags ) of 07 days and multiday averaging spans of 7 , 14 , and 30 days before blood collection adjusted for age , sex , bmi , hemoglobin , fasting plasma glucose , treatment with agents with anti - inflammatory action , season , air temperature , and relative humidity.resultsmedian crp concentration was 3.49 mg / l . for 1 sd increase in so2 and oxides of nitrogen ( nox ) concentrations in ambient air , a day before blood collection ( lag1 ) , we observed a significant increase in crp ( 9.34 and 7.77% , respectively ) . 
 the effect was higher with lag2 ( 12.42% for so2 and 11.60% for nox ) and wore off progressively thereafter . 
 we also found a significant association with multiday averaging times of up to 30 and 7 days for so2 and nox , respectively . 
 no significant associations were found between particulate matter with an aerodynamic profile 10 m ( pm10 ) and crp concentration except in summer . 
 the association was significantly higher among patients with a shorter duration of diabetes , and in those not on statin and thiazolidinedione treatment.conclusionswe demonstrate , for the first time , a possible contribution of ambient air pollution to systemic inflammation in indian type 2 diabetic patients . 
 this may have implications for vascular complications of diabetes .