even small changes in glucose metabolism may contribute to the onset of cardiovascular disease and endothelial dysfunction due to peripheral insulin resistance .
acute myocardial infarction affects a large proportion of patients with dm2 ; thus , the evidence of subclinical atherosclerosis would be helpful for preventive strategies in these individuals [ 1 , 2 ] . even knowing that hyperglycemia is one of the ways that can lead to the development of atherosclerosis , the mechanisms underlying coronary artery disease ( cad ) in individuals with type ii diabetes are not known for sure . thus , besides serum glucose , other markers related to the pathophysiology of cardiovascular diseases might also be evaluated .
recent prominent candidates for prediction of cad in dm2 patients include thiols ( cysteine , homocysteine , methionine oxidized glutathione , and reduced glutathione ) , n - acetyl--d - glucosaminidase ( nagase ) , endogenous nitric oxide synthase inhibitors ( adma ) , nitrate + nitrite ( nox ) , nitrotyrosine , and s - nitrosothiols ( rsno ) .
hence , the purpose of this study was to identify important biochemical alterations that could distinguish dm2 individuals with and without cad .
in this study , 96 volunteers were enrolled and classified into the following groups : type 2 diabetes mellitus ( dm2 , n = 21 ) and type 2 diabetes mellitus with coronary artery disease ( dm2 + cad , n = 75 ) ( table 1 ) . volunteers of both genders ( aged  21 years ) were screened at the following brazilian institutions : instituto dante pazzanese de cardiologia , irmandade da santa casa de misericrdia de so paulo , and faculdade de cincias mdicas da universidade metodista de santos .
all subjects were fully informed about the details of the study and protocol and provided written informed consent .
dm2 was defined according to the american diabetes society criteria , whereas participants with coronary artery disease were defined as patients who had acute myocardial infarctions confirmed by ecg , laboratory exams , and clinical symptoms .
exclusion criteria used were pregnancy , psychiatric disorders , renal and/or hepatic diseases , smoking , alcoholism , cancer , or other pathological conditions that could interfere with the study .
venous blood was collected in tubes with and without edta after 12 hours of fasting .
the concentrations of fructosamine , glycated hemoglobin ( hba1c ) , glucose , total cholesterol , triglyceride , high - density lipoprotein- ( hdl- ) cholesterol , and very low density lipoprotein- ( vldl- ) cholesterol were estimated by enzymatic methods using kits ( biosystems sa , barcelona , spain ) .
the friedewald equation was used to calculate the low density lipoprotein- ( ldl- ) cholesterol .
n - acetyl--glucosaminidase activity was determined based on the methodology described by reglero and cabezas ( 1976 ) .
100  l of plasma was added to 1.25  ml citrate buffer ( 0.1  m , ph 4.4 ) and incubated at 37c with 0.25  ml 0.01 
m p - nitrophenyl - n - acetyl - glucosaminide in citrate buffer for 15 minutes .
the reaction was interrupted with 1.5  ml of sodium carbonate ( 0.2  m , ph 10.4 ) and the final product was measured by spectrophotometer at 405  nm .
one unit of enzyme was defined as the amount released 1 mol p - nitrophenol / min .
we used the nitric oxide analyzer ( noa , sievers , usa ) based on the chemiluminescence reaction between nitric oxide and ozone .
the reduction of nitrate and nitrite ( nox ) with vanadium chloride was used to convert nox to oxide nitric .
calibration curves with multiple levels were performed with an external standard ( sodium nitrate ) using the bag program software ( version 2.2 , sievers , usa ) .
the samples were extracted with cold ethanol ( 0.5  ml sample and 1.0  ml of ethanol at 0c ) .
after vortexing , the solution was stored on ice during 30 minutes and then centrifuged at 9000  g for fifteen minutes .
the standard s - nitroso - albumin ( sno - alb ) was used to quantify total s - ntrosothiol serum .
the synthesis was obtained by the reaction of nitrite with human albumin in 0.1  m hcl .
this was incubated in the dark for 2 hours and then the absorbance was measured at 336  mm (  = 3874  mcm ) .
the calibration curve was made with 10 , 50 , 100 , 250 , and 500  nm sno - alb .
1.0  ml of plasma was added to 10  l of n - ethylmaleimide ( 500  mm ) .
after homogenizing , 100  l of sulfanilamide ( 1% ) was added and homogenized again .
we used the equipment nitric oxide analyzer ( noa , sievers , usa ) and injected 500  l of sample and 500  l of standard .
the reaction solution was composed of 8  ml of glacial acetic acid , 2  ml of ki ( 50  mg / ml ) , 300  l of decanol , and 200  l of cuso4 ( 200  mm ) at 70c .
plasma concentrations of adma , sdma , and l - arginine were determined by the technique of capillary electrophoresis ( biofocus 2000 , bio - rad laboratories , inc . ) .
the blood was collected with edta and centrifuged at 1,000  g for 10 minutes at 4c to obtain plasma .
the calibration curve was performed by adding the standard of adma , sdma , and l - arginine in pooled plasma .
samples and standard were precipitated with ethanol , centrifuged at 9.000  g for 15 minutes at 4c , and then derivatized with fluorescein 5-isothiocyanate .
the injection was done under pressure ( 1  psi.sec ) and the race was performed in a fused silica capillary ( 85  cm length and 50 internal diameter m ) at 20  kv . running buffer consisted of 50  mm boric acid and 20  mm 3-cyclohexylamino-1-propanesulfonic adjusted to ph 10.8 . the detector laser - induced fluorescence ( biofocus lif , bio - rad laboratories , inc . )
operated at 488  nm ( excitation ) and 520  nm ( emission ) ( causs et al .
the blood was collected with edta and centrifuged at 1000  g for 10 minutes at 4c to obtain plasma . in 200  l in plasma 2  l internal standard ( 1  mm n-(2-mercaptopropionyl ) glycine ) and 20  l tri - n - butylphosphine 10% ( v / v dimethylformamide ) were added .
then , 200  l 10% trichloroacetic acid containing 1  mm edta was added .
after homogenizing samples , they were centrifuged at 13.000  g for 15 minutes at 4c . in 100 
l of the supernatant was added to 100  l 0.5  m phosphate buffer and adjusted to ph 7.5 with 1  m na3po4 .
the derivatizer ( 5-bromomethylfluorescein ) was added to the samples at a molar ratio of 5 to 10 times excess and the preparations are incubated for 15 minutes at 60c .
after derivatization , the sample was diluted at 1  :  10 with phosphate buffer 0.25  m ph 7.6 and injected with a pressure of 0.5  psi for 1.5 seconds on capillary ( 70  cm length and 50  m internal diameter ) . the capillary electrophoresis instrument ( biofocus 2000 , bio - rad laboratories , inc . )
the detector laser - induced fluorescence ( biofocus lif , bio - rad laboratories , inc . ) operated in 488  nm ( excitation ) and 520  nm ( emission ) . for each run
we used the polyclonal anti - nitrotyrosine antibody ( upstate , catalog number : 06 - 284 ) .
the standard used was nitrated bovine albumin ( nitro - albumin ) prepared by alkaline addition of 1  mm peroxynitrite and 1  mm albumin .
the concentration of the nitro - albumin was determined using the molar extinction coefficient of 4300  mcm at 438  nm and ph 9.0 .
the plate was sensitized with 0.05  ug nitro - albumin per well and then washed , blocked with milk protein , and rinsed again .
the following was added to the plate anti - nitrotyrosine antibody and sample / standard .
after incubation , the plate was washed and added to the peroxidase - conjugated antibody ( stressgen biotechnologies corp . ) .
after incubation and washing , 2.3  mm luminol and 0.9  mm p - iodophenol ( 200  ul / well ) and 3.9  mm hydrogen peroxide ( 50  ul / well ) were added .
the reading of the chemiluminescence produced was immediately performed ( lumicount , packard , meriden , usa ) .
the present nitrotyrosine concentrations in the sample were estimated by using the calibration curve and nitrated albumin was expressed as equivalent nitro - albumin .
data were expressed as means  standard deviation ( sd ) , median ( interquartile range ) , or counts ( percentage ) when appropriate . for univariate analyses ,
groups were compared by the t - test for approximately normally distributed variables or by means of the mann - whitney u - test for variables with skewed distribution .
in addition , we constructed multiple logistic regression models using the backward stepwise selection procedure to ascertain predictors for cad in dm2 patients ( coding scheme : dm2 + cad = 1 , dm2 = 0 ) .
variables were sequentially removed from the full model ( all regression terms included ) when the correspondent p value was higher than 0.10 . in order to assess the model performance ,
we did not employ k - fold partitioning due to the relatively low number of subjects .
instead , we used the resubstitution approach , in which the same data are used for both training and testing . for these analyses
, each participant has an estimated probability of being dm2 + cad , which is calculated from the logistic model - derived equation based on the participant 's own variables .
if the calculated probability was 50% , the subject was assigned as dm2 + cad or dm2 only , otherwise .
we quantified model performance by using two measures of classification accuracy that are not susceptible to class imbalance : balanced accuracy ( ba ) and normalized mutual information ( nmi ) .
ba is an accuracy measure that takes into account both sensitivity and specificity of the models and is calculated as the average of sensitivity and specificity .
nmi ranges from 0 to 100% and is interpreted as the amount by which the examined model reduces one 's uncertainty about the true state of the participants ( e.g. , 0% means that the status is independent of the studied explanatory variables , while 100% suggests that the model fully predicts the status for each individual ) .
permutation was used to construct empirical null distributions ( 2,000 shuffles ) in order to compute the statistical significance of both ba and mni measures .
the hypothesis of a significantly better fit for the model with biochemical biomarkers plus classical variables for cad ( full model ) compared to the simpler models ( nested models ) was tested by means of a likelihood - ratio test .
this test assumes the form  = 2(l0  l1 ) , where l1 and l0 are the natural log - likelihoods under the full and nested models , respectively .
we used permutation to test whether the full model fits the data significantly better than simpler models by comparing the observed test statistic  to those obtained in 2,000 randomly generated shuffles .
all data analyses were performed using the stata package ( version 11.0 , stata corp . , college station , tx , usa ) .
all calibration curves were linear with a coefficient of determination ( r )  0.98 .
the quantification limit ( 10  :  1 ; signal  :  noise ) by capillary electrophoresis ( adma , sdma , and l - arginine : 25  nm ; thiols : 50  nm ) and nitric oxide analyzer ( nox and rsno : 10  nm ) were suitable for detection of the analytes in the samples .
the elisa also showed a quantification limit adequate for detection of nitrotyrosine ( 30  nm ) .
the main demographical , clinical , and biochemical characteristics of the studied participants are shown in table 1 .
twenty - one ( 22% ) out of 96 participants with dm2 were classified as dm2 + cad .
age , body mass index ( bmi ) , and gender proportion were comparable between dm2 + cad and dm2 groups .
however , in an exploratory ( unadjusted ) analysis , levels of methionine , ldl / hdl ratio , n - acetyl--glucosaminidase , and nitrite plus nitrate ( nox ) were significantly higher in dm2 + cad .
in addition , levels of l - arginine  :  adma ratio , fructosamine , and reduced glutathione were significantly lower in dm2 + cad patients compared to their dm2 counterparts . after a holm - bonferroni correction for 25 tests , both nox and fructosamine levels remained significantly higher in dm2 + cad participants compared to the dm2 group ( p = 0.02 and p = 0.006 , resp . ) . to determine putative predictor variables for cad in dm2 , we built a multiple logistic regression model with backward elimination .
of the 25 variables tested , eight remained in the final model : ldl / hdl ratio , methionine , reduced glutathione ( gsh ) , l - arginine  :  adma , nox ( nitrate plus nitrite ) , n - acetyl--glucosaminidase ( nagase ) , gender , and bmi ( table 2 ) .
on the basis of this model ( hereafter named full model ) , we constructed an equation that was applied to the complete dataset to predict the status of the 96 studied participants .
the overall classification accuracy of this model ( resubstitution method ) is presented in table 3 , along with other two competing models including either only classical variables for cad ( i.e. , ldl / hdl ratio , gender , and bmi ; model 2 ) or emerging biochemical markers only ( i.e. , l - arginine  :  adma ratio , methionine , gsh , nagase , and nox ; model 3 ) .
all of the three models significantly predicted cad more often than can be expected by chance , although it can be verified from table 3 that the performance of the studied models ranges from small to moderate .
we sought next to assess whether there is an increase in accuracy for prediction models when emerging biochemical markers are used in combination with classical risk factors for cad .
in other words , we tested whether simpler models ( models 2 and 3 ) are equally capable of predicting cad in dm2 patients compared to the full model .
this is of paramount importance , since there is a potential of overfitting as the number of variables included in the model increases .
we observed that the full model fits significantly better the data compared to either model 2 ( p < 0.001 ) or model 3 ( p = 0.004 ) , indicating that there is a statistically significant gain in accuracy when both emerging biochemical markers and classical risk factors are added to the model for cad prediction . for comparison purposes , less conservative measures of diagnostic accuracy ( e.g. , classification error )
also favor the full model over models 2 and 3 ( data not shown ) .
we found that individuals with dm2 + cad have several significant metabolic changes related to endothelial dysfunction and oxidative stress compared to dm2 without cad . in our sample , we observed significant alterations in the following variables : hdl - cholesterol , fructosamine , gsh , sdma , l - arginine / adma , nox , and nagase .
in addition , we showed that the prediction accuracy for cad in dm2 patients is significantly increased when six emerging biochemical markers are incorporated into the model along with classical risk factors for cad .
first , our investigation should be regarded as a  predictor finding study  only . although we have developed a multivariable prediction model , we did not aim at the development of a model for use in clinical practice to guide patient management . instead
, our study should be viewed as a hypothesis - generating study that warrants further confirmation in larger , well - powered investigations with external validation .
second , criticism might be directed at the fact that our definition of cad might lack specificity . because cad was classified conservatively
, silent cad might be present among a few patients in the dm2 + cad group .
however , in our study , misclassification of the cad status is likely to lead to downwardly biased estimates , ultimately leading to less impressive model predictions ( i.e. , more conservative models ) .
third , our sampling scheme was based on volunteers and was not designed to be representative of the population . with that said , our results are prone to a survival bias , since there is a reduced likelihood of enrolling patients who died acutely or who are experiencing severe consequences of myocardial infarction .
again , these results are likely to lead to more conservative model predictions , since more prominent metabolic alterations are plausible in advanced cad patients . on the other hand , even though we employed permutation to compute statistical significance , our small sample size is prone to overoptimistic results .
indeed , there is a clear potential for overfitting and overestimation , because our models were constructed with a large number of biomarkers relative to the number of participants . in another study conducted by our research group  
we showed that hyperglycemia correlated with increased production of nitric oxide based on the evaluation of concentrations of nitrate and nitrite .
endothelial dysfunction is generally defined as impaired endothelium - dependent vasodilation , related to lower no production or bioavailability .
reactive oxygen and nitrogen species ( rons ) , such as h2o2 , o2 , no , and onoo , can be produced in blood vessels by certain drugs and pathological conditions and accentuate or induce endothelial dysfunction .
rons may reduce no bioactivity by formation of peroxynitrite or by decrease of enzyme or transporter activities by oxidation of their thiols groups .
endothelial dysfunction can contribute to the initiation , progression , and clinical manifestations of cad .
the increase in s - nitrosothiols may be a consequence of glycation of proteins , which facilitates the nitrosation of protein thiols by nitric oxide .
the glycated proteins can act as a sink for nitric oxide because s - nitrosation reduces their competence to release nitric oxide which reflects directly on the pathogenesis of endothelial dysfunction . nitric oxide can react with superoxide anion , which leads to the formation of a potent oxidant , peroxynitrite , and in sequence generation of hydroxyl radicals and nitrogen dioxide [ 18 , 21 , 22 ]
. the tyrosine , free or protein - bind , may react with nitrogen dioxide generating nitrotyrosine , which is considered a biomarker of the formation of peroxynitrite . in this present study ,
only the increase of nox was demonstrated with no differences for nitrotyrosine , even though reduced glutathione and reduced glutathione / oxidized glutathione ratio concentrations were observed .
the endogenous nitric oxide synthase inhibitors , represented by symmetric dimethylarginine ( sdma ) and asymmetric dimethylarginine ( adma ) , also play an important role in endothelial dysfunction . in the present study ,
reduced concentration of l - arginine / adma in dm2 + cad group in comparison with dm2 without cad reinforces the fact that endothelial nitric oxide synthase inhibition occurs along the atherosclerosis process .
accordingly , it has been shown that l - arginine / adma levels diminished parallel to the impairment of vasodilatation in dm2 patients after lipid ingestion .
moreover , it was previously reported that low l - arginine / adma ratio was not related to either the presence of macroangiopathy or the time of disease manifestation in dm2 patients , but rather to the metabolic control of the diabetes .
thus , in the present study , hypercholesterolemia observed in dm2 + cad group could be related to adma increase , occasioning a lower production of  no and leading to endothelial dysfunction .
however , nitric oxide low production could not be confirmed in this study based only on the increase of nitrite plus nitrate concentration generation .
studies have shown a reduction of endothelial dysfunction by rising l - arginine / adma ratio .
lipid peroxidation causes peroxidative damage to tissue proteins and may raise the rate of proteolysis . alternatively , there may be a dysfunction or downregulation of di - methylarginine dimethylaminohydrolase , the enzyme that converts adma to l - citrulline .
hypercholesterolemia may alter the regulation or function of di - methylarginine dimethylaminohydrolase , thereby resulting in intracellular accumulation of adma .
hypercholesterolemia plus hyperglycemia is a factor that contributes to oxidative stress due to autoxidation of glucose and nonenzymatic protein glycation .
thiols play an important role on the oxidative processes and therefore may directly or indirectly contribute in endothelial dysfunction .
glutathione is a major thiol used as a biomarker of oxidative stress and is found in the forms of gsh ( reduced glutathione ) and gssg ( oxidized glutathione ) . in the present study ,
the dm2 + cad group showed lower gsh / gssg and gsh concentration , compared with dm2 without cad group .
this decrease of reduced glutathione may be due to activation of the polyol pathway in which glucose is reduced to sorbitol by the nadph - dependent aldose reductase [ 30 , 31 ] .
this enzyme presents a high michaelis - menten constant for glucose and therefore this metabolic via is only quantitatively significant in hyperglycemia .
thus , whenever the nadph / nadp ratio is decreased in hyperglycemic state , it will result in prejudice in the regeneration of reduced glutathione . also as a consequence
, the synthesis of nitric oxide by nitric oxide synthase will be impaired due to the nadph dependence [ 31 , 32 ] . apparently , the decrease in gsh concentration has a relation to the presence of atherosclerotic inflammation process in patients with cad .
the activity of n - acetyl--d - glucosaminidase ( nagase ) in plasma has been used as a biomarker of endothelial dysfunction . in the present study ,
nagase activity was significantly higher in dm2 + cad group in comparison with the dm2 without cad group .
nagase is a lysosomal enzyme produced by many cells , including not only endothelial cells , but also smooth muscle and kidney .
showed correlation between measurements of brachial artery diameter and blood flow via doppler ultrasonography with nagase activity in patients with dac . additionally , nagase activity showed positively correlated with insulin resistance in patients with cad . according to komosiska - vassev et al
, the increase of nagase activity can be considered an indicator of the intensity of endothelial cell dysfunction .
several studies also demonstrated that nagase is increased in individuals with dm2 , when compared to healthy individuals and that nagase activity is high in individuals with dm2 and previous history of cardiovascular disease ( myocardial infarction ) in comparison with individuals with dm2 without previous coronary events [ 34 , 35 , 37 ] .
thus , our data reinforce previous data showing that the increase of nagase activity in individuals with dm2 can be associated with the presence of cad .
 we investigated the potential of a panel of 22 biomarkers to predict the presence of coronary artery disease ( cad ) in type 2 diabetes mellitus ( dm2 ) patients . 
 the study enrolled 96 dm2 patients with ( n = 75 ) and without ( n = 21 ) evidence of cad . 
 we assessed a biochemical profile that included 22 biomarkers : total cholesterol , ldl , hdl , ldl / hdl , triglycerides , glucose , glycated hemoglobin , fructosamine , homocysteine , cysteine , methionine , reduced glutathione , oxidized glutathione , reduced glutathione / oxidized glutathione , l - arginine , asymmetric dimethyl - l - arginine , symmetric dimethyl - l - arginine , asymmetric dimethyl - l - arginine / l - arginine , nitrate plus nitrite , s - nitrosothiols , nitrotyrosine , and n - acetyl--glucosaminidase . 
 prediction models were built using logistic regression models . 
 we found that eight biomarkers ( methionine , nitratate plus nitrite , n - acetyl--glucosaminidase , bmi , ldl , hdl , reduced glutathione , and l - arginine / asymmetric dimethyl - l - arginine ) along with gender and bmi were significantly associated with the odds of cad in dm2 . 
 these preliminary findings support the notion that emerging biochemical markers might be used for cad prediction in patients with dm2 . 
 our findings warrant further investigation with large , well - designed studies . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion