lone atrial fibrillation ( af ) is a term commonly used to denote af occurring in a small subset ( ~3% ) of patients without identifiable cardiovascular and extracardiac comorbidities or triggering factors [ 1 , 2 ] . with growing understanding of af pathophysiology , the existence of truly lone af has been challenged , since emerging risk factors and evidence of subclinical vascular disease have been found in some apparently lone af patients [ 24 ] . in particular , latent or masked arterial hypertension has been implicated as a possible  hidden  cause of af , while clinically overt hypertension has been the most frequently diagnosed vascular disease in cohorts of initially lone af patients during follow - up [ 5 , 6 ] . however , determinants of future development of vascular disease in previously lone af patients have not been broadly investigated . current evidence indicates that chronic low - grade inflammation in association with oxidative stress could represent a link between af and ( subclinical ) vascular disease [ 710 ] . increased plasma levels of inflammatory ( e.g. , c - reactive protein  crp )   and oxidative mediators ( e.g. , oxidized low density lipoprotein  oxldl ) have been reported in subjects with lone af compared to healthy individuals in sinus rhythm [ 11 , 12 ] . increased inflammatory and oxidative burden has been associated with af recurrence and perpetuation , but the association of markers of inflammation and oxidative stress with the development of vascular disease in lone af remains unknown . the aim of the present study was to evaluate the association of plasma biomarkers of inflammation ( crp ) and oxidative stress ( oxldl ) with the development of clinically evident vascular disease ( arterial hypertension , cerebrovascular disease , coronary / peripheral arterial disease , and pulmonary embolism ) in lone af patients . we hypothesized that if there was a relation between lone af , inflammation , and oxidative burden , ( i ) baseline plasma levels of crp and oxldl would be higher in lone af patients compared to healthy controls in sinus rhythm and ( ii ) crp and oxldl could be associated with increased risk for the development of overt vascular disorders in a group of lone af patients . of 854 consecutive af patients referred to the outpatient unit of the cardiology clinic , clinical center of serbia , between may 2010 and august 2011 , we prospectively enrolled 50 subjects ( 5.8% ) with carefully characterized lone af . we also included a control group of 44 healthy individuals in sinus rhythm , recruited among the hospital staff and acquaintances of af patients . none of the participants had any evidence of underlying cardiovascular disorders ( including hypertension ) or extracardiac comorbidities . the study included patients with prior history of af ( paroxysmal , persistent , or permanent ) , as well as patients with newly diagnosed af . thorough evaluation of medical records , physical examination , office and home blood pressure ( bp ) measurements , 12-lead electrocardiogram ( ecg ) , laboratory analyses , transthoracic echocardiographic examination , and chest radiography were performed in both af patients and the controls at inclusion , while additional diagnostic assessments were performed when indicated . subjects with prior cardiovascular disorders , stroke , transient ischemic attack ( tia ) , diabetes ( fasting plasma glucose 7.0  mmol / l or 2 h postload plasma glucose 11  mmol / l ) , hepatic , renal , or thyroid dysfunction , systemic inflammatory disorders , malignancy , or obesity ( body mass index [ bmi ] 30  kg / m ) were excluded . all participants were required to have normal findings on physical examination , including normal office ( systolic bp < 140  mmhg and diastolic bp < 90  mmhg ) and 7-day home bp measurements ( systolic bp < 135  mmhg and diastolic bp 85  mmhg ) prior to the institution of any medications . subjects with high - normal office bp ( systolic bp 130139  mmhg and/or diastolic bp 8589  mmhg ) were included if masked arterial hypertension was excluded by further ambulatory bp monitoring . arterial hypertension was diagnosed if office bp measurement 140/90  mmhg , and/or home bp measurement 135/85  mmhg was found ( and those subjects were excluded ) . normal 12-lead ecg and transthoracic echocardiographic examination , including normal indexes of left atrial volume and left ventricular mass , without evidence of diastolic left ventricular dysfunction or valvular heart disease , were required for all participants . subjects with symptoms suggestive of obstructive sleep apnea or chronic pulmonary disease were referred for further respiratory function examination . subjects with symptoms indicative of myocardial ischemia were evaluated by stress - echo exercise testing , supplemented by coronary angiography if indicated . participants suspected of having lower or upper extremities ischemia or cerebral hypoperfusion were referred for doppler echosonographic examination to confirm peripheral arterial disease or carotid artery disease , respectively . patients with a sudden - onset focal neurological deficit suspected of having a stroke or tia were referred for neurological examination and imaging diagnostics . peripheral arterial thromboembolism was defined as thromboembolic events outside the brain , retina , heart , or lungs . pulmonary embolism was suspected in the presence of clinical symptoms , ecg , and laboratory findings and confirmed by computed tomography - pulmonary angiogram . following centrifugation ( 2,200  rpm , 20 minutes , 4c ) , ethylene - diamine - tetra - acetic acid - plasma samples were stored in multiple aliquots at 20c until further analysis . plasma oxldl ( mercodia , uppsala , sweden ) was determined by commercially available enzyme - linked immunosorbent assay ( elisa ) . determination of crp was performed by immunonephelometric technique ( dade behring bnii nephelopmeter , marburg , germany ) with detection limit of 0.2  mg / l and a coefficient of variation of 4.7% . follow - up visits were performed by cardiologists at 3-month intervals or more frequently if required , and data on incident vascular disease were collected . a composite study end - point was defined as the first occurrence of any clinically manifest vascular disease ( arterial hypertension , coronary / peripheral artery disease , significant carotid artery stenosis , pulmonary embolism , stroke , or tia ) in a patient with initially lone af or in a control group subject . diagnostic criteria and medical procedures used to confirm the diagnosis of vascular diseases during follow - up were the same as at study inclusion and included assessments of patients ' medical records during follow - up , physical exam , 12-lead ecg , and office and 7-day home bp measurements at each visit , supplemented by additional diagnostic tests if indicated ( e.g. , stress - echo exercise testing , coronary angiography , peripheral / carotid artery doppler echosonography , ct pulmonary angiography , and endocranial ct scan ) . vascular disease was confirmed when standard diagnostic criteria were satisfied . in patients with high - normal office bp at follow - up ( systolic bp 130139  mmhg and/or diastolic bp 8589  mmhg ) , ambulatory bp monitoring was performed . hypertension was diagnosed when either office bp 140/90  mmhg and/or home bp 135/85  mmhg was found or when 24-hour ambulatory bp of 130/80  mmhg was recorded . control laboratory and echocardiographic exams were performed at 12-month intervals in all participants . before the development of vascular disease , none of the participants received ace inhibitors or dihydropyridine ca channel blockers , while statins were prescribed in subjects with elevated plasma cholesterol . in af patients , vitamin k antagonists were used for the preparation for elective cardioversion . once vascular disease was diagnosed , patients ' treatment was reassessed in accordance with practice guidelines . the study was approved by the local ethics committee and all participants gave their informed consent . data are presented as mean values and standard deviations for normally distributed continuous variables , medians , and interquartile ranges ( 25th75th ) for skewed variables and counts ( n ) with percentages ( % ) for categorical variables . the differences between variables were tested by the two - tailed t - test , mann - whitney test ,  test , and fisher test , respectively . correlation between normally distributed or log - transformed variables was analyzed by pearson 's correlation . univariate cox proportional hazard analyses were performed to examine the relationships of clinical , echocardiographic , and biochemistry variables from table 1 with the risk for incident vascular disease . variables significantly associated with the risk for incident vascular disease were subsequently entered in the multivariate cox proportional hazard models with plasma biomarkers . the association of plasma biomarkers ( crp and oxldl ) with incident vascular disease was tested in univariate and multivariate cox proportional hazard models with biomarkers first entered as continuous ( oxldl ) or log - transformed ( crp ) predictor variables and then as predictor variables categorized per quartile increase . time to the first occurrence of vascular disease or time to the last follow - up ( for those participants who did not develop vascular disease ) was used in all cox proportional hazard analyses . the c - statistic , a measure of the area under the receiver - operator characteristic ( roc ) curve , was used to assess the validity of the unadjusted and adjusted models of plasma biomarkers to predict the development of vascular disease during follow - up . pairwise comparison of the roc curves was also performed using delong approach ( the z - statistic ) . both groups included subjects of similar mean age with male predominance , and there were no differences regarding demographic , clinical , or routine laboratory findings , except for higher bmi and higher levels of fibrinogen in af patients ( table 1 ) . in af patients , newly diagnosed af was noted in 50.0% of subjects . echocardiographic parameters , including left atrial dimensions and volume index , were similar between af patients and healthy controls ( table 1 ) . medical treatment at baseline in af patients comprised of antiarrhythmic medications and ventricular rate controlling drugs , while none of the healthy controls received any medications ( table 1 ) . comparison of baseline plasma levels of the investigated biomarkers revealed that both median crp level and mean oxldl level were significantly higher in lone af patients than in healthy controls ( table 1 ) . in af patients , there was a significant positive correlation between plasma levels of crp and oxldl ( r = 0.343 , p = 0.040 ) , as well as a positive correlation between both biomarkers and patients ' age ( r = 0.520 and r = 0.568 , resp . ) . oxldl was also correlated with total- and ldl - cholesterol concentrations in af patients ( r = 0.432 and r = 0.460 , resp . ) , all p < 0.05 . positive correlations between crp and oxldl were also noted in the control group ( r = 0.334 , p = 0.028 ) . in the course of a median 36-month follow - up , incident vascular disease was diagnosed in 14 ( 28.0% ) lone af patients and in 5 ( 11.4% ) healthy controls ( table 2 ) . none of the other overt vascular disorders ( coronary / peripheral artery disease , significant carotid artery stenosis , pulmonary embolism , stroke , or tia ) occurred in lone af patients . in the control group , none of the study participants died or were lost to follow - up . the median time to incident vascular disease was similar in both groups , but af patients developed vascular disease at a younger age compared to healthy controls ( table 2 ) . during follow - up , af patients were treated with antiarrhythmic medications , beta - blockers , and non - dihydropyridine ca channel blockers , while vitamin k antagonists were prescribed for elective cardioversion ( table 2 ) . the kaplan - meier curve of the cumulative risk for vascular disease development in lone af patients and healthy controls is presented in figure 1 . in lone af patient group , subjects with incident hypertension were older , had a longer history of af , and higher baseline systolic and diastolic bp , bmi , fibrinogen , and total- and ldl - cholesterol plasma levels ( table 3 ) . all these characteristics were significantly associated with the risk of developing hypertension ( all p < 0.05 ) . there were no differences regarding other clinical , laboratory , or echocardiographic characteristics , including af type , smoking habits , alcohol intake , or medications used during follow - up ( not presented ) . on univariate cox proportional hazard analyses , both crp and oxldl were associated with increased risk for incident hypertension in lone af patients , both as continuous variables ( not presented ) and per quartile increase ( table 4 ) . both biomarkers demonstrated high sensitivity and specificity for incident hypertension ( figure 2 ) and their predictive ability did not significantly differ between biomarkers when pairwise comparison was performed ( table 4 ) . on multivariate cox proportional hazard analysis , investigated biomarkers maintained their predictive significance for subsequent occurrence of hypertension in lone af patients , with high discriminatory ability that was similar between models in pairwise comparison ( table 5 ) . multivariate hazard ratios ( hr ) with 95% confidence intervals ( ci ) for crp and oxldl were 2.54 ( 1.265.12 ) , p = 0.009 , and 2.24 ( 1.144.40 ) , p = 0.019 , respectively . in the control group , predictors of vascular disease occurrence were older age , higher systolic and diastolic bp , and higher plasma total- and ldl cholesterol levels ( table 3 ) . on univariate cox regression analysis , there was no statistically significant association of either crp ( hr 1.64 , 95% ci , 1.137.34 , p = 0.068 ) or oxldl ( hr , 1.23 , 95% ci , 0.846.78 , p = 0.223 ) with the risk for incident vascular disease in healthy controls . in the present study , we have demonstrated increased plasma levels of biomarkers of inflammation ( crp ) and oxidative stress ( oxldl ) in carefully characterized lone af patients compared to healthy controls in sinus rhythm . we have also demonstrated that baseline plasma levels of crp and oxldl are associated with the risk for incident vascular disease ( hypertension ) during a median 3-year follow - up in lone af patients , but not in healthy controls . despite positive mutual correlations , both crp and oxldl were strong multivariate predictors for incident hypertension in our group of lone af patients . vascular disease occurred more frequently and at a younger age in lone af patients than in healthy controls . thus far , prognostic significance of biomarkers of inflammation and oxidative burden in lone af has been demonstrated for arrhythmia perpetuation following cardioversion or catheter ablation [ 11 , 13 , 14 ] . to our knowledge , this is the first study to report on the link between increasing levels of crp and oxldl and the development of arterial hypertension in patients with lone af . from the clinical perspective , development of any vascular disorder , including arterial hypertension , in lone af patients implies an increased risk of stroke , heart failure , and other complications [ 1 , 5 , 15 ] . therefore , efforts to establish contributors or associates of vascular disease development in lone af might aid to earlier recognition and adequate treatment of patients at risk , since it pertains to their prognosis . such af patients merit a more intensive follow - up , rigorous life - style changes , and possibly earlier institution of medical treatment to prevent vascular disorders and their complications , while appropriate af thromboprophylaxis should be commenced once vascular disease becomes apparent . although heterogeneous pathophysiological mechanisms may contribute to the development of lone af , there is a considerable interest in the role of inflammation and oxidative stress as underlying mechanisms . despite evidence of inflammatory atrial lesions   and increased levels of various biomarkers in lone af , there is no unequivocal link between inflammation , oxidative burden , af , and vascular disease . in the present study , we have demonstrated higher crp levels in af patients compared to healthy controls , which is in line with our previous findings of impaired endothelial function and increased crp concentrations in subjects with persistent lone af compared to healthy individuals . similarly , increased levels of crp   and other mediators of inflammation   have been reported in other studies of lone af patients , with a stepwise crp elevation associated with increased af burden . however , data on inflammatory mediators in lone af are still conflicting since some studies have failed to demonstrate higher inflammatory burden in lone af . we have also found higher oxldl levels in lone af patients compared to the controls . data on oxldl in lone af are sparse , but in a recent study , kim et al . also reported increased plasma concentrations of oxidized lipoproteins in females with paroxysmal lone the origin of the observed abnormalities in lone af remains elusive , but it is conceivable that they represent harbingers of a pathological process that predates an overt vascular disease and first becomes manifest as ( lone ) af . the results of the present study support the notion that in the background of some lone af cases there are inflammatory and oxidative mechanisms ( as reflected by increased plasma concentrations of crp and oxldl compared to healthy subjects ) that are associated with or possibly contributing to the development of vascular disease ( e.g. , arterial hypertension ) . in subjects without af , several epidemiological studies have established an association of crp with the occurrence of hypertension [ 21 , 22 ] while oxldl has been linked to the development of subclinical vascular disease . presently , we have not observed a significant relation of either crp or oxldl with a risk for incident vascular disease in healthy controls , which does not preclude an association in a larger group of subjects with more pronounced overall cardiovascular risk . additionally , the absence of an association of the investigated biomarkers with incident vascular disease in the control group further strengthens clinical significance of the association observed in lone af patients . considering other variables associated with hypertension occurrence in lone af patients in the present study , we have found that aging , longer af history , higher baseline systolic and diastolic bp , higher serum cholesterol and fibrinogen levels , and increased bmi are linked with incident hypertension . nevertheless , all of the listed variables were within reference limits acceptable for the healthy population and comparable to the healthy controls , except for higher fibrinogen levels and bmi in af patients . longer overall history of af has been already linked to the risk for vascular disease development in lone af , possibly due to longer exposition to as yet unrecognized causal or confounding factors . earlier studies have reported occurrence of arterial hypertension in lone af between 7.5% ( after 7 years )   and 44% ( after 2 years ) . in a recent trial , weijs et al . reported increased incidence , younger age at onset , and more severe characteristics of vascular disease in lone af patients compared to healthy subjects . during a 5-year follow - up , incident hypertension occurred in 30% of lone af patients , while other comorbidities ( coronary artery disease , heart failure and stroke ) occurred in 49% of patients . , we have also demonstrated that lone af patients developed vascular disease ( i.e. , hypertension ) more frequently and at a younger age compared to sinus rhythm controls , but despite careful follow - up we have not documented occurrence of other vascular disorders . however , it is not precluded that over time other comorbidities and vascular complications would become apparent with a possible relation to inflammation and oxidative burden . the most important limitation to the present study is a relatively small number of highly selected participants in both study groups , originating from a single ethnic background and from one referral center . therefore , our results do not necessarily apply to all lone af patients and should be reproduced in other groups of lone af subjects before any general conclusions could be drawn . biomarker determination has been conducted at a single time point at study inclusion and we have no information on possible changes in the levels of investigated biomarkers during the follow - up or whether these changes ( if occurred ) were pertinent to vascular disease development . furthermore , only 2 highly correlated biomarkers have been evaluated , and adjustments have been conducted for the routine biochemistry parameters and clinical characteristics of study participants , while other possible confounding biochemical variables or risk factors might have been missed . despite our meticulous efforts to include strictly lone af patients , some cases with occult comorbidities , particularly latent arterial hypertension , might have been missed . considering that we have not demonstrated an association of the investigated biomarkers with vascular disease development in the control group , no comparisons of their predictive abilities could have been made between lone af patients and healthy controls . the present study showed that levels of circulating biomarkers of inflammation , crp , and oxidative stress , oxldl , are higher in lone af patients compared to healthy individuals and that both biomarkers were associated with increased risk for incident hypertension in lone af . further research should better define pathophysiological role of these biomarkers in the development of af and vascular disease and refine their clinical relevance in lone af patients , particularly concerning risk stratification and therapeutic implications . meanwhile , a regular clinical follow - up of lone af patients should be routinely conducted in daily clinical practice .
we compared plasma levels of biomarkers of inflammation ( crp ) and oxidation ( oxldl ) , determined at study inclusion in lone atrial fibrillation ( laf ) patients ( 48.6  11.5 years ; 74.0% men ) and sinus rhythm controls ( 49.7  9.3 years ; 72.7% men , p > 0.05 ) , and investigated the association of baseline crp and oxldl levels with the risk for vascular disease ( vd ) development ( hypertension , cerebrovascular disease , coronary / peripheral artery disease , and pulmonary embolism ) during prospective follow - up . 
 baseline crp ( 1.2 [ 0.71.9 ] mg / l versus 1.1 [ 0.71.6 ] mg / l ) and oxldl levels ( 66.3  21.2 
 u / l versus 57.1  14.6 
 u / l ) were higher in laf patients 
 ( both p < 0.05 ) . following a median of 36 months , incident vd occurred in 14 ( 28.0% ) laf patients , all of whom developed arterial hypertension , and in 5 ( 11.4% ) controls ( hypertension , n = 4 ; coronary artery disease , n = 1 ) , p < 0.05 . 
 laf patients developed vd more frequently and at a younger age . both crp ( hr , 2.54 ; 95% ci , 1.265.12 ; p = 0.009 ) and 
 oxldl ( hr , 2.24 ; 95% ci , 1.144.40 ; p = 0.019 ) were multivariate predictors of incident hypertension in laf patients , but not in the controls . 
 further research should clarify clinical relevance of investigated biomarkers for risk stratification and treatment of laf patients .