the penn diabetes heart study ( pdhs ) is an ongoing , cross - sectional study of individuals with type 2 diabetes ( aged 3575 years ) without clinical evidence of cvd ( myocardial infarction , coronary revascularization or angiographic disease , positive stress test , clinical peripheral arterial disease or peripheral arterial revascularization , stroke , or transient ischemic attack ) .
the university of pennsylvania institutional review board approved the study protocol , and all subjects gave written informed consent ( 4 ) . the ankle - brachial index ( abi )
was calculated by dividing the average of the ankle pressures by the highest brachial pressure .
participants with an abi > 1.4 ( n = 13 ) reflecting noncompressible arteries were excluded ( 5 ) .
each subject had the estimated glomerular filtration rate ( egfr ) calculated based on the abbreviated modification of diet in renal disease study equation ( 6 ) .
plasma levels of fetuin - a ( 4.3 and 10.2% , respectively , intra - assay and inter - assay coefficient of variation ) were measured by enzyme - linked immunosorbent assay ( biovendor laboratory medicine , modrice , czech republic ) .
high - sensitivity c - reactive protein ( hscrp ) was measured by immunoturbidimetric assay ( wako , osaka , japan ) .
student 's t test was used for normal continuous variables and the kruskal - wallis for nonnormal distributed variables .
the association between a 1-sd change in fetuin - a and pad was examined using unadjusted and multivariable adjusted logistic regression models as described in table 1 .
analyses were performed on stata 10.0 software ( stata , college station , tx ) .
association between fetuin - a and pad data are odds ratios ( 95% ci ) and were obtained by logistic regression model .
model 2 : model 1 + smoking , hypertension , hypercholesterolemia , metabolic syndrome , a1c , framingham risk score ( % ) , and medications ( ace inhibitors , aspirin , statins , insulin , metformin , sulfonylureas , and thiazolidinediones ) .
the ankle - brachial index ( abi ) was calculated by dividing the average of the ankle pressures by the highest brachial pressure .
participants with an abi > 1.4 ( n = 13 ) reflecting noncompressible arteries were excluded ( 5 ) .
each subject had the estimated glomerular filtration rate ( egfr ) calculated based on the abbreviated modification of diet in renal disease study equation ( 6 ) .
plasma levels of fetuin - a ( 4.3 and 10.2% , respectively , intra - assay and inter - assay coefficient of variation ) were measured by enzyme - linked immunosorbent assay ( biovendor laboratory medicine , modrice , czech republic ) . high - sensitivity c - reactive protein ( hscrp )
student 's t test was used for normal continuous variables and the kruskal - wallis for nonnormal distributed variables .
the association between a 1-sd change in fetuin - a and pad was examined using unadjusted and multivariable adjusted logistic regression models as described in table 1 .
analyses were performed on stata 10.0 software ( stata , college station , tx ) .
association between fetuin - a and pad data are odds ratios ( 95% ci ) and were obtained by logistic regression model .
model 2 : model 1 + smoking , hypertension , hypercholesterolemia , metabolic syndrome , a1c , framingham risk score ( % ) , and medications ( ace inhibitors , aspirin , statins , insulin , metformin , sulfonylureas , and thiazolidinediones ) .
among 738 subjects in the study sample , pad prevalence was 5.1% , the mean age was 58.7  9.3 years , 37.1% were female , 63.2% were caucasians , and 32.1% were african americans .
the median fetuin - a level was 292.4 ng / ml ( interquartile range 115.5 ) . individuals with pad had a significantly lower fetuin - a level compared with individuals without pad ( 269.3 vs. 293.4 , p < 0.001 ) .
no significant differences were noted in other cardiovascular risk factors , metabolic syndrome , kidney function , inflammation , bmi , serum albumin ( the only surrogate marker of liver function collected on the pdhs ) , or medication use .
fetuin - a levels decreased consistently ( p for trend < 0.02 ) across abi clinically relevant cut points ( < 0.7 , 0.70.9 , 0.91.1 , and 1.11.4 ) ( 7 ) ( see supplemental fig . 1 in the online appendix [ available at http://care.diabetesjournals.org/cgi/content/full/dc09-1541/dc1 ] ) . the odds of pad were significantly increased for each sd decrease in fetuin - a ( odds ratio 1.6 [ 95% ci 1.12.3 ] , p = 0.001 ) and the association persisted in incremental models that adjusted fully for age , sex , race , kidney function , cardiovascular risk factors , medication use , and hscrp ( 1.6 [ 1.0582.5 ] , p = 0.03 ) ( table 1 ) .
a similar trend was noted among subjects with egfr > 80 ml  min  1.73 m ( 1.9 [ 1.03.4 ] , p = 0.05 ) , suggesting that our findings were not confounded by the presence of moderate kidney disease .
finally , in a fully adjusted subgroup analysis , participants with hscrp levels <3 mg / dl ( n = 460 ) had higher odds of pad ( 2.6 [ 1.45.0 ] , p = 0.002 ) , whereas subjects with high hscrp levels ( 3 mg / dl ; n = 234 ) did not ( 0.82 [ 0.41.6 ] , p = 0.59 ) .
low levels of fetuin - a have been linked to medial arterial calcification and flow limiting aortic stenosis in humans ( 8,9 ) . in this study , we demonstrate that lower levels of fetuin - a are associated with pad in subjects with type 2 diabetes . to our knowledge , this is the first study to report such a relationship in the absence of advanced kidney disease or prevalent cvd .
notably , in analysis stratified by the centers for disease control and prevention / american heart association  defined hscrp risk strata ( 10 ) , fetuin - a conferred increased odds of pad in subjects with hscrp <3 mg / dl and also in participants with interleukin ( il)-6 levels below the median ( data not shown ) .
this is consistent with studies reporting an increased cvd risk mortality with fetuin - a deficiency independent of hscrp and il-6 ( 11 ) and points to a unique role for this negative acute - phase protein as a biomarker of pad beyond traditional and novel cardiovascular risk factors . remarkably , animal models appear to track with our clinical observations . in mice lacking the fetuin - a gene ,
the aorta was found to be spared of calcification and fibrosis , whereas peripheral vessels in the skin and kidney showed evidence of extensive calcification ( 2 ) , and the small artery involvement preceded the renal impairment ( 3 ) . however , in the absence of direct imaging data , we are unable to define the type of vascular phenotype ( intimal calcification or medial calcification ) that account for the observed association .
an abi of < 0.9 is 95% sensitive and 99% specific for a stenotic lesion ( > 50% ) ( 12 ) .
therefore , we assume that some degree of eccentric atherosclerotic calcification contributes to the association observed while acknowledging that multiple types of vascular calcification may coexist in type 2 diabetes ( 13 ) . nonalcoholic liver disease and other phenotypes related to insulin resistance , including type 2 diabetes ( 14 ) , are associated with higher levels of fetuin - a ( 15 ) .
we controlled for most potential confounding factors and found no attenuation of the inverse association of fetuin - a with pad .
in particular , because of this inverse relationship , a significant confounding effect was not expected by any condition associated with high fetuin - a levels .
finally , our study is limited by cross - sectional design , which limits causal inferences . in summary ,
low plasma fetuin - a levels are associated with pad in type 2 diabetes independent of traditional and contemporary risk factors .
our findings suggest a unique role for fetuin - a deficiency as a biomarker of pad in the setting of type 2 diabetes .
 objectivefetuin - a is an inhibitor of vascular calcification and a mediator of insulin resistance . 
 this study evaluated the association of plasma fetuin - a and peripheral arterial disease ( pad).research design and methodsa total of 738 individuals with type 2 diabetes ( mean age 58.7 years , 37.1% female ) without known cardiovascular or kidney disease were included in this cross - sectional analysis.resultssubjects with pad had a significantly lower fetuin - a ( 264.3 vs. 293.4 ng / dl , p < 0.001 ) . in multivariable analysis , a 1-sd decrease in fetuin - a increased the odds of pad ( odds ratio 1.6 , p = 0.02 ) . 
 subgroup analysis revealed an increased odds even in subjects with glomerular filtration rate > 80 ( odds ratio 1.9 , p = 0.05 ) or high - sensitivity c - reactive protein <3 mg / dl ( odds ratio 2.7 , p = 0.002).conclusionslower circulating fetuin - a is associated with pad in type 2 diabetes beyond traditional and novel cardiovascular risk factors . 
 our findings suggest a potentially unique role for fetuin - a deficiency as a biomarker of pad in patients with type 2 diabetes . 
RESEARCH DESIGN AND METHODS
Evaluated parameters
Statistical analysis
RESULTS
CONCLUSIONS
Supplementary Material