data from the coronary artery risk development in young adults ( cardia ) study and its ancillary study , young adult longitudinal trends in antioxidants ( yalta ) , were used to examine the association between biomarkers of oxidative stress and insulin resistance . briefly , cardia is a longitudinal study aiming to investigate lifestyle and other factors that influence the evolution of cardiovascular disease in young adults . this study began in 1985 with a cohort of 5,115 healthy black and white men and women , aged 1830 years , who were free - living individuals residing in four u.s . cities ( birmingham , al ; chicago , il ; minneapolis , mn ; and oakland , ca ) . the yalta ancillary study analyzes blood and urine biomarkers of oxidative stress , endothelial dysfunction , and related concepts and interprets those data in conjunction with the other data that have been collected by cardia . follow - up examinations were conducted during 19871988 ( year 2 ) , 19901991 ( year 5 ) , 19921993 ( year 7 ) , 19951996 ( year 10 ) , 20002001 ( year 15 ) , and 20052006 ( year 20 ) . the percentages of the surviving cohort who returned for these examinations were 90 , 86 , 81 , 79 , 74 , and 72% , respectively . in this analysis , we included all participants with f2isop or oxldl measurements at year 15 and fasting insulin / fasting glucose measurements at year 20 . among them , we excluded those who had been diagnosed with diabetes , defined as a fasting glucose 126 mg / dl ( 7 mmol / l ) or who were receiving antidiabetes medication between years 0 and 20 in our analysis . self - reported demographic ( age , sex , race , and educational level ) and behavioral ( smoking , alcohol consumption , and physical activity ) information was obtained across cardia examination visits . self - reported alcohol beverages were quantified as average milliliters of alcohol consumed per day , and smoking status was classified as never , former , or current smoker . an interviewer - administered questionnaire queried participation spent in leisure , occupational , and household physical activities over the course of a year , weighting frequency and intensity in order to obtain a total activity score . body height was recorded to the nearest 0.5 cm , and bmi was calculated as weight in kilograms divided by the square of height in meters , using the average of the two measurements . waist circumference ( cm ) was measured with each participant 's weight distributed equally on both feet . the measurement was made midway between the iliac crest and the lowest lateral portion of the ribcage and anteriorly midway between the xyphoid process of the sternum and the umbilicus , keeping the tape ( vinyl anthropometric centimeter tape ) horizontal . since the adiposity measurements , total fat mass , and percent fat were obtained only from a subsample ( n = 1,072 ) of the study participants , race- and sex - specific predictive equations for percent body fatness ( % fat ) were computed using the year 10 dual - energy x - ray absorptiometry ( dxa ) examination ( 14 ) ; these estimates enabled study of total body fatness of each participant . year 10 dxa was obtained only from a subsample ( n = 1,072 ) , in birmingham and oakland , of the study participants using the qdr 2000 ( hologic , bedford , ma ) with software version 11.1 . these race-/sex - specific predictive equations included age , bmi , waist circumference ( referred to as wc ) , and squared terms for waist circumference and bmi ( wc and bmi ) : for black men : % fat = 55.5371 to 0.0578  age + 0.1585  bmi + 1.1741  wc  0.00365  wc ; for black women : % fat = 52.0734 + 0.0901  age + 1.9772  bmi + 0.9664  wc  0.015  bmi  0.0051  wc ; for white men : % fat = 82.6174 + 0.1015  age + 0.5448  bmi + 1.5883  wc  0.0065  wc ; and for white women : % fat = 75.2741 + 0.1698  age + 1.2678  bmi + 1.7154  wc  0.00972  wc . these equations had higher correlation coefficients and smaller mean square errors and predictive errors compared with the widely used general predictive equations for body composition , such as the durnin and womersley equations ( 15 ) . the correlations of observed year 10%fat with predicted values were r = 0.66 for black men , 0.76 for black women , 0.60 for white men , 0.74 for white women , and 0.82 for sex and race combined , compared with r = 0.75 for the durnin and womersley equations that relied solely on skinfolds . these formulae closely predicted dxa measured fat and lean mass at year 20 . before drawing blood , all of the participants were asked to fast for 12 h and to abstain from smoking for 2 h before their examinations . after venous blood samples were drawn , plasma was separated by centrifugation , transferred into airtight vials , stored at 70c , and shipped on dry ice . fasting glucose and insulin were measured at years 0 , 7 , 10 , 15 , and 20 . glucose was measured in stored blood samples using the hexokinase ultraviolet method on a cobas mira plus chemistry analyzer . the insulin measurements were performed by using a radioimmunoassay with an overnight , equilibrium - incubation format . based on reassays of glucose in december 2007 in 200 samples stored since year 7 and 100 samples stored since year 15 recalibrated glucose values were 6.98 + 0.94  year 7 glucose concentration , 7.15 + 0.96  year 10 glucose concentration , 6.99 + 1.01  year 15 glucose concentration , and 4.06 + 0.97  year 20 glucose concentration . oxldl concentrations were measured by the mercodia oxidized ldl elisa ( mercodia , uppsala , sweden ) in year 15 samples that had been stored at 70c for several years . the antibody ( mab-4e6 ) is directed against a conformational epitope in the apolipoprotein b-100 moiety of ldl , as a consequence of substitution of 60 lysine residues of apolipoprotein b-100 with aldehydes , and these substituting aldehydes can be produced by peroxidation of lipids of ldl , leading to the generation of oxldl . 11 ) , and showed the similar analytical performance and clinical applicability ( 11 ) . plasma levels of ascorbic acid were measured using high - performance liquid chromatography ( hplc ) at year 15 . levels of carotenoids ( including lycopene , -carotene , -carotene , -cryptoxanthin , and zeaxanthin plus lutein ) and tocopherols ( -tocopherol and -tocopherol ) were obtained by hplc at year 15 . bmi and waist circumference were set to missing at each examination at which a woman was pregnant . we investigated patterns of repeated measurements for individuals with large within - person sds from year 0 to year 20 by visual inspection of the raw data to detect if there were any substantial departures ( e.g. , outliers ) from patterns . six outliers of waist circumference at year 15 , one outlier of bmi at year 20 , one outlier of f2isop at year 15 , and one outlier of homa - ir at year 7 were replaced with missing values . insulin resistance was assessed by homa - ir , which was calculated as fasting plasma insulin ( mu / l )  fasting plasma glucose ( mmol / l)/22.5 ( 16 ) . homa - ir , f2isop , oxldl , and circulating antioxidant ( ascorbic acid , lycopene , -carotene , -carotene , -cryptoxanthin , zeaxanthin plus lutein , -tocopherol , and -tocopherol ) estimates were logarithmically transformed , since their distributions were skewed to the right . the subjects were classified into quartiles , based on their levels of oxidative stress markers . partial correlation coefficients between body fat measurements / antioxidants and oxidative stress markers were calculated by sex , adjusting for age and race . multivariable generalized linear models were used to test associations of oxidative stress markers with evolving homa - ir , using as dependent variable homa - ir at year 20 and adjusting for demographic and lifestyle factors ( e.g. , age , sex , race , study center , smoking status , physical activity , alcohol consumption , and education ) and the previous measure of homa - ir at year 15 . in addition , the models were adjusted for adiposity measurements ( e.g. , bmi , waist circumference , and equation - driven estimates of percent fat ) to examine if the association between oxidative stress and insulin resistance was independent of obesity . body height was recorded to the nearest 0.5 cm , and bmi was calculated as weight in kilograms divided by the square of height in meters , using the average of the two measurements . waist circumference ( cm ) was measured with each participant 's weight distributed equally on both feet . the measurement was made midway between the iliac crest and the lowest lateral portion of the ribcage and anteriorly midway between the xyphoid process of the sternum and the umbilicus , keeping the tape ( vinyl anthropometric centimeter tape ) horizontal . since the adiposity measurements , total fat mass , and percent fat were obtained only from a subsample ( n = 1,072 ) of the study participants , race- and sex - specific predictive equations for percent body fatness ( % fat ) were computed using the year 10 dual - energy x - ray absorptiometry ( dxa ) examination ( 14 ) ; these estimates enabled study of total body fatness of each participant . year 10 dxa was obtained only from a subsample ( n = 1,072 ) , in birmingham and oakland , of the study participants using the qdr 2000 ( hologic , bedford , ma ) with software version 11.1 . these race-/sex - specific predictive equations included age , bmi , waist circumference ( referred to as wc ) , and squared terms for waist circumference and bmi ( wc and bmi ) : for black men : % fat = 55.5371 to 0.0578  age + 0.1585  bmi + 1.1741  wc  0.00365  wc ; for black women : % fat = 52.0734 + 0.0901  age + 1.9772  bmi + 0.9664  wc  0.015  bmi  0.0051  wc ; for white men : % fat = 82.6174 + 0.1015  age + 0.5448  bmi + 1.5883  wc  0.0065  wc ; and for white women : % fat = 75.2741 + 0.1698  age + 1.2678  bmi + 1.7154  wc  0.00972  wc . these equations had higher correlation coefficients and smaller mean square errors and predictive errors compared with the widely used general predictive equations for body composition , such as the durnin and womersley equations ( 15 ) . the correlations of observed year 10%fat with predicted values were r = 0.66 for black men , 0.76 for black women , 0.60 for white men , 0.74 for white women , and 0.82 for sex and race combined , compared with r = 0.75 for the durnin and womersley equations that relied solely on skinfolds . before drawing blood , all of the participants were asked to fast for 12 h and to abstain from smoking for 2 h before their examinations . after venous blood samples were drawn , plasma was separated by centrifugation , transferred into airtight vials , stored at 70c , and shipped on dry ice . fasting glucose and insulin were measured at years 0 , 7 , 10 , 15 , and 20 . glucose was measured in stored blood samples using the hexokinase ultraviolet method on a cobas mira plus chemistry analyzer . the insulin measurements were performed by using a radioimmunoassay with an overnight , equilibrium - incubation format . based on reassays of glucose in december 2007 in 200 samples stored since year 7 and 100 samples stored since year 15 recalibrated glucose values were 6.98 + 0.94  year 7 glucose concentration , 7.15 + 0.96  year 10 glucose concentration , 6.99 + 1.01  year 15 glucose concentration , and 4.06 + 0.97  year 20 glucose concentration . oxldl concentrations were measured by the mercodia oxidized ldl elisa ( mercodia , uppsala , sweden ) in year 15 samples that had been stored at 70c for several years . the antibody ( mab-4e6 ) is directed against a conformational epitope in the apolipoprotein b-100 moiety of ldl , as a consequence of substitution of 60 lysine residues of apolipoprotein b-100 with aldehydes , and these substituting aldehydes can be produced by peroxidation of lipids of ldl , leading to the generation of oxldl . ( 11 ) , and showed the similar analytical performance and clinical applicability ( 11 ) . plasma levels of ascorbic acid were measured using high - performance liquid chromatography ( hplc ) at year 15 . levels of carotenoids ( including lycopene , -carotene , -carotene , -cryptoxanthin , and zeaxanthin plus lutein ) and tocopherols ( -tocopherol and -tocopherol ) were obtained by hplc at year 15 . bmi and waist circumference were set to missing at each examination at which a woman was pregnant . we investigated patterns of repeated measurements for individuals with large within - person sds from year 0 to year 20 by visual inspection of the raw data to detect if there were any substantial departures ( e.g. , outliers ) from patterns . six outliers of waist circumference at year 15 , one outlier of bmi at year 20 , one outlier of f2isop at year 15 , and one outlier of homa - ir at year 7 were replaced with missing values . insulin resistance was assessed by homa - ir , which was calculated as fasting plasma insulin ( mu / l )  fasting plasma glucose ( mmol / l)/22.5 ( 16 ) . homa - ir , f2isop , oxldl , and circulating antioxidant ( ascorbic acid , lycopene , -carotene , -carotene , -cryptoxanthin , zeaxanthin plus lutein , -tocopherol , and -tocopherol ) estimates were logarithmically transformed , since their distributions were skewed to the right . the subjects were classified into quartiles , based on their levels of oxidative stress markers . partial correlation coefficients between body fat measurements / antioxidants and oxidative stress markers were calculated by sex , adjusting for age and race . multivariable generalized linear models were used to test associations of oxidative stress markers with evolving homa - ir , using as dependent variable homa - ir at year 20 and adjusting for demographic and lifestyle factors ( e.g. , age , sex , race , study center , smoking status , physical activity , alcohol consumption , and education ) and the previous measure of homa - ir at year 15 . in addition , the models were adjusted for adiposity measurements ( e.g. , bmi , waist circumference , and equation - driven estimates of percent fat ) to examine if the association between oxidative stress and insulin resistance was independent of obesity . the mean age of the participants during 20002001 ( year 15 ) was 40 years , and 45% were men among 2,774 nondiabetic study participants . participants who had higher levels of f2isop were more likely to be women ( p < 0.001 ) ( table 1 ) . an increased level of f2isop was positively associated with smoking ( p < 0.001 ) , whereas oxldl was not associated with this factor . physical activity was inversely associated with both markers , though the gradient was stronger over f2isop than over oxldl . means ( or proportions ) of demographic , lifestyle , and type 2 diabetes  related factors by quartile of oxidative stress markers * data are means  sd unless otherwise indicated . * adjusted for year 15 hdl cholesterol , ldl cholesterol , and triglycerides ( 25 ) . body fat measurements showed strong positive associations with both markers ( p < 0.01 ) , as shown in table 1 . f2isop showed much stronger correlations with adiposity markers in women than in men ( table 2 ) . correlations between f2isop and body fat measurements , including bmi , waist circumference , and estimated body percent fat , were 0.400.43 in women , whereas these correlations were only 0.150.16 in men , after adjustment for race and age . f2isop was negatively correlated with sum of five serum carotenoids , ascorbic acid , and -tocopherol but positively correlated with serum -tocopherol . interestingly , the two oxidative stress markers , isop and oxldl , were minimally correlated in our sample ( r = 0.09 , p = 0.0007 , in women and r = 0.003 , p = 0.9 , in men ) . sex - specific partial correlation coefficients of observed body fat components , estimated percent body fat , and antioxidant markers at year 15 with f2isop and oxldl * all models were adjusted for age and race . based on race-/sex - specific prediction from bmi , waist circumference , and age ; see research design and methods . additional adjustment for year 15 hdl cholesterol , ldl cholesterol , and triglycerides ( 25 ) . sum of five carotenoids : -carotene , -carotene , -cryptoxanthin , lutein / zeaxanthin , and lycopene . table 3 includes adjusted estimates for year 20 homa - ir , according to the quartiles of year 15 f2isop and oxldl . we observed a significant increase in the estimates of evolving homa - ir with increasing levels of oxldl ( p < 0.001 ) and a marginally significant association with f2isop ( p = 0.06 ) . after additional adjustment for the adiposity measurements , oxldl was still significantly associated with year 20 homa - ir , whereas the association between f2isop and homa - ir became substantially attenuated . further adjustments for hdl cholesterol , ldl cholesterol , and triglycerides did not affect the magnitude and significance of the association between oxldl and homa - ir . adiposity measurements remained significant , predicting homa - ir in the models adjusted for either marker of oxidative stress ( p < 0.0001 ) . relationship between year 15 oxidative stress markers and year 20 homa - ir -coefficient is a change in homa - ir [ ln ( mu l  mmol  l ) ] per one - unit increment of independent variables [ ln(f2isop ) ( ng / l ) or ln(oxldl ) ( units / l ) ] . * model 1 : adjusting for age , sex , race , study center , and year 15 homa - ir . model 2 : model 1 plus year 15 smoking status , physical activity , alcohol consumption , and education . model 3 : model 2 plus year 15 body fat measurements ( bmi , waist circumference , or estimated percent body fat ; adding each variable separately ) . in our analysis , we did not adjust for medication use ( e.g. , lipid- or blood pressure  lowering medications ) because such use could mediate or be directly correlated with mediators in the causal pathway for associations of oxidative stress on insulin resistance . omission of those taking these medications did not materially change our study results ( data not shown ) . there were no significant interactions between oxidative stress markers and other covariates , including sex , race , smoking , adiposity measurements , and physical activity . for example , although the association between f2isop and homa - ir was positive in whites and negative in blacks , race - specific coefficients were small and p values for interactions were not significant . increase in f2isop was not predicted by homa - ir in longitudinal analysis , controlling for body fat measurements ( bmi , waist circumference , or estimated body percent fat ) , age , sex , race , study center , smoking status , physical activity , alcohol consumption , education , and f2isop , all of which were measured in the same year as the independent variable ( bmi - adjusted regression coefficient = 0.004 , p = 0.9 ; waist circumference  adjusted regression coefficient = 0.009 , p = 0.7 ; body fat percent  adjusted regression coefficient = 0.008 , p = 0.7 ) . we observed positive associations between oxidative stress markers and insulin resistance in a nondiabetic adult population . oxldl was positively associated with insulin resistance , even after accounting for various adiposity measurements , a finding akin to a recent report ( 12 ) in these data that oxldl predicted incident metabolic syndrome . although the association between f2isop and insulin resistance was also positive , it was explained by additional adjustment for adiposity measurements . the levels of year 20 homa - ir predicted by each oxidative stress marker were not significantly different by sex . recently , meigs et al . ( 9 ) showed in a cross - sectional design that the association between homa - ir and oxidative stress , measured by urinary isoprostanes ( 8-epi - pgf 2 /creatinine ) , was significant , independent of bmi , but association between insulin resistance prevalence and oxidative stress was attenuated by additional adjustment for bmi ( p = 0.06 ) in individuals without type 2 diabetes in the framingham offspring cohort study ( 9 ) . our study substantially extends what is known about the impact of oxidative stress on insulin resistance in people without diabetes . in our longitudinal analysis , the results show that plasma f2isop was marginally related with elevated levels of homa - ir , but a substantial part of this association was explained by anthropometric measurements ( bmi and waist circumference ) . furthermore , we examined this association by using an alternate marker , oxldl , that showed a stronger association with insulin resistance , independent of body fatness , compared with f2isop . oxidative stress results from an imbalanced condition in which the generation of free radicals is greater than the capacity of the antioxidant defense system to detoxify them ( 17 ) . when oxidative stress is chronic , it is thought to result in damage to dna , lipids , proteins , and other molecules , which may contribute to the development and progression of chronic disease , including cardiovascular disease and cancer ( 13,18 ) . recently , various indirect markers of oxidative stress have been used in epidemiological studies to measure oxidation damage due to feasibility and cost issues , instead of direct measures of free radicals and reactive oxygen species , which include electron resonance or spin trapping , capturing free radical reactions in real time ( 19 ) . among them , oxldl and f2isop have been known as robust reflectors of oxidative stress in humans and have been widely used in biology , medicine , and epidemiological research ( 20,21 ) . each marker may capture different stages of the oxidative process or reflect different pathophysiological pathways ( 22 ) . the apparent compartmentalization of oxidative stress is illustrated in the differing associations of f2isop , oxldl , and different carotenoids with each other and with homa - ir . there was little correlation between the two measures of oxidative stress , though f2isop was well correlated with the sum of serum carotenoids and other serum antioxidant vitamins ( e.g. , ascorbic acid and -tocopherol ) , lower values of which in part indicate reduced carotenoid intake and in part oxidative stress . in contrast , correlations between oxldl and these antioxidant nutrients were substantially weaker . in longitudinal analysis using cardia / yalta , hozawa et al . ( 23 ) reported that serum carotenoid concentrations were inversely associated with incident diabetes and homa - ir in nonsmokers , concluding that oxidative stress may be involved in the development of type 2 diabetes in nonsmokers . it has been reported that serum carotenoids were inversely associated with f2isop in the same data ( 24 ) . our study shows that a substantial portion of the association between f2isop and homa - ir was explained by body fat measurements , and f2isop was strongly correlated with adiposity markers , especially in women . in a previous report using cardia / yalta , gross et al . ( 10 ) reported that the level of plasma f2isop was higher in women , especially for those with a bmi > 25 kg / m , than men . since f2isop is a product of lipid peroxidation , it is closely related to adipose tissue mass , especially for women . in contrast , correlations between oxldl and body fat measurements did not differ by sex . furthermore , oxldl was a strong precursor of the risk of insulin resistance , independent of obesity . therefore , we suggest that each oxidative stress marker may capture a different oxidative stress pathway and that their interaction with adiposity differs between men and women . nevertheless , in our study the estimated levels of evolving homa - ir with increasing levels of the oxidative stress markers did not differ significantly by sex . studies examining the longitudinal associations between oxidative stress and insulin resistance are rare , especially with plasma oxidative stress markers in healthy young people in large , community - based studies , while most previous epidemiological studies have used urinary markers . also , cardia has maintained a high participation rate and high - quality data collection through rigorous quality - control procedures throughout the study . furthermore , we could test prediction in the reverse direction and added to the specificity of our finding by showing that homa - ir did not predict future oxidative stress , measured by f2isop , although we only measured oxldl once and so were not able to measure the reverse direction with it . among potential limitations , first , oxidative stress is a highly complex phenomenon and our measures may not be sufficiently detailed to fully understand the role of oxidative stress and adipose tissue in development of insulin resistance . second , the study was conducted only among black and white individuals , with no representation of hispanic , asian , or other individuals . as a result finally , there could be residual confounding due to this study 's observational nature . in summary , the independent association of oxldl with insulin resistance supports the hypothesis that oxidative stress leads to insulin resistance , independent of obesity . f2isop is closely correlated with adiposity , especially for women , and it is possible that f2isop is intermediate in a pathway that links insulin resistance with total body fat . further research is needed to elucidate the underlying etiological relationships between adiposity and oxidative stress in the genesis and progression of insulin resistance by various biomarkers of oxidative stress and body fat measurements in diverse populations .
objectivealthough cumulative evidence suggests that increased oxidative stress may lead to insulin resistance in vivo or in vitro , community - based studies are scarce . 
 this study examined the longitudinal relationships of oxidative stress biomarkers with the development of insulin resistance and whether these relationships were independent of obesity in nondiabetic young adults.research design and methodsbiomarkers of oxidative stress ( f2-isoprostanes [ f2isop ] and oxidized ldl [ oxldl ] ) , insulin resistance ( the homeostasis model assessment of insulin resistance [ homa - ir ] ) , and various fatness measures ( bmi , waist circumference , and estimated percent fat ) were obtained in a population - based observational study ( coronary artery risk development in young adults ) and its ancillary study ( young adult longitudinal trends in antioxidants ) during 20002006.resultsthere were substantial increases in estimated mean homa - ir over time . 
 oxldl and f2isop showed little association with each other . 
 mean evolving homa - ir increased with increasing levels of oxidative stress markers ( p < 0.001 for oxldl and p = 0.06 for f2isop ) , measured in 20002001 . 
 after additional adjustment for adiposity , a positive association between oxldl and homa - ir was strongly evident , whereas the association between f2isop and homa - ir was not.conclusionswe observed positive associations between each of two oxidative stress markers and insulin resistance . 
 the association with oxidized ldl was independent of obesity , but that with f2isop was not .