Patent Application: US-88262707-A

Abstract:
a method for predicting onset or risk of atrial fibrillation in a subject includes determining the presence of an oxidative stress marker , such as glutathione , cysteine , and / or a derivative of a reactive oxidative metabolite .

Description:
this cross - sectional , case - control study recruited patients in af from outpatient clinics at the atlanta veterans affairs medical center ( vamc ) from may through july of 2005 under a protocol approved by the emory university institutional review board ( www . clinicaltrials . gov : nct00142194 ). eligible patients were over 18 years of age and in persistent or permanent af at the time of enrollment . ineligibility criteria included systemic inflammatory diseases , malignant neoplasm , severe stenotic or regurgitant valvular heart disease , new york heart association class iv heart failure , hyperthyroidism , uncontrolled hypertension (& gt ; 180 / 100 at rest ), an illness that may have resulted in death within one year , implanted devices designed for the active management of atrial arrhythmias by pacing or defibrillation , and current illicit drug or alcohol abuse . control patients were identified from outpatient clinic visits at the atlanta vamc over the same period of time . the same eligibility and ineligibility criteria applied with the exception that control patients were free of current af and any history of af as documented by an electrocardiogram ( ecg ) performed at the time of enrollment , history , and review of the medical record . cases and control subjects were matched for factors known to affect the oxidative markers used , age in decades , smoking , and diabetes status ( references 25 , 44 , 48 , 58 , 63 and 65 ). all patients enrolled gave written consent . data was collected from subject interviews , review of va hospital and clinic charts , telemetry recordings , and ecgs . the presence or absence of af was confirmed on the basis of an ecg done at the time of enrollment . demographic data obtained included : age , race , body mass index , new york heart association ( nyha ) functional class , and a history of previous myocardial infarction , hypertension , diabetes , smoking , or alcohol use . additionally , all medications being taken at the time of enrollment were recorded . a single blood draw was performed at the time of enrollment and analyzed for markers of oxidative stress and inflammation in the emory biomarkers core laboratory . markers used to measure oxidative stress were : ratios of oxidized to reduced glutathione ( e h gsh ) and cysteine ( e h cysh ) in plasma ( thiol ratios ) ( references 41 - 44 ) and derivatives of reactive oxygen species ( droms ) ( also known as the free oxygen radicals test — fort ) ( references 1 , 12 , 18 and 38 ). detailed methods to prevent rapid oxidation of samples have been delineated previously ( reference 43 ). briefly , blood was collected from an antecubital vein transferred immediately to a microcentrifuge tube containing 0 . 5 ml of a preservation solution of 100 mm serine - borate ( ph 8 . 5 ) containing ( per ml ) 0 . 5 mg sodium heparin , 1 mg bathophenanthroline disulfonate sodium salt , and 2 mg iodoacetic acid . use of this procedure minimizes autoxidation and hemolysis ( reference 43 ). following centrifugation to remove blood cells , aliquots ( 200 μl ) were transferred to tubes containing 200 μl of 10 % ( w / v ) perchloric acid containing 0 . 2 m boric acid and 10 μm γ - glu - glu as internal standard . samples were stored at − 80 ° c . (& lt ; 2 months ) prior to further processing to form n - dansyl derivatives and analysis by hplc with fluorescence detection . previous data have shown stable measurements with this length of storage ( reference 43 ). metabolites were identified by co - elution with standards , and quantification was obtained by integration relative to the internal standard . samples from control and af patients were treated identically . the redox states ( e h ) of the thiol / disulfide pools were calculated with the nernst equation , e h = e o + rt / nf in [ disulfide ]/[ thiol ] 2 , where e o is the standard potential for the redox couple , r is the gas constant , t is the absolute temperature , n is 2 for the number of electrons transferred , and f is faraday &# 39 ; s constant ( references 64 and 66 ). the standard potential e o used for the glutathione and cysteine redox couples was − 264 mv and − 250 mv , respectively ( reference 43 ). less negative e h numbers imply a more oxidized state . droms were measured in carr units with higher values indicating higher levels of oxidative stress . droms ( diacron international , grosseto , italy ) and inflammatory markers , high sensitivity c - reactive protein ( hscrp ; life diagnostics , west chester , pa . ), interleukin - 1 - β ( il - 1β ; r & amp ; d systems , minneapolis , minn . ), interleukin - 6 ( il - 6 ; r & amp ; d systems ), and tumor necrosis factorα ( tnfα ; r & amp ; d systems ), were measured using commercially available kits . intraassay cvs were & lt ; 1 % at − 156 and & lt ; 1 % at − 120 mv for e h gsh , 5 . 0 % at − 100 and 4 . 5 % at − 60 mv for e h cysh , 0 . 2 % at 300 and 2 . 3 % at 550 carr units for droms ; 10 . 1 % at 0 . 2 and 5 . 2 % at 10 ng / l for il - 1β , 5 . 1 % at 1 and 3 . 6 % at 8 mg / l for hscrp , 20 . 9 % at 3 . 2 and 6 . 2 % at 50 ng / l for il - 6 , and , 11 . 9 % at 2 and 7 . 3 at 50 ng / l for tnfα . statistical analysis was performed using sas software 9 . 1 ( sas institute , cary , n . c ., usa ). baseline characteristics of af patients and their matched controls were compared using a paired t - test for continuous variables ( expressed as mean ± sd ), and fisher &# 39 ; s exact test for categorical variables . all statistical tests were two - tailed , and significance was assumed at p ≦ 0 . 05 . correlations between markers of inflammation and oxidative stress were assessed using spearman rank - order correlation coefficients . all oxidative and inflammatory markers were examined as predictors of af occurrence in single - variate conditional logistic regression models ; age category , smoking and diabetes status were accounted for by matching . variables exhibiting borderline normality were also examined after logarithmic transformation . parameter estimates for each oxidative and inflammatory marker were scaled so that reported odds ratios correspond to an approximate inter - quartile range increase . using the parameter estimate from the e h gsh model , odds ratios for af were computed and plotted as a function of increase in e h gsh level . multivariate conditional logistic regression models were used to examine the association between each oxidative marker and the presence of af while controlling for inflammatory markers . statins were assessed as predictors of af presence and in linear regression models as predictors of e h gsh . twenty subjects with persistent or permanent af along with 20 individuals free of af were compared in the study . control subjects were matched to cases by age , sex , smoking and diabetes status because these variables are known to affect the oxidative stress measures used ( references 25 , 44 , 48 , 58 , 63 and 65 ) table 1 ( below ) compares the demographics of cases and their controls . the af subjects had ages ranging from 58 - 86 with a mean age of 74 . 8 years . five subjects ( 25 %) were diabetic . all af subjects were male and non - smokers . the mean length of af was 10 . 1 years with a median of 6 . 4 ± 13 . 3 years . in non - matched variables , hypertension and heart failure were slightly more common in the af group , consistent with these conditions predisposing to the arrhythmia ( references 5 , 16 , 31 and 45 ). in all other parameters , the populations were statistically similar ( p & gt ; 0 . 05 ). all measures of oxidative stress were significantly increased in af patients compared with controls . thiol ratios in the af group were significantly more oxidized ( i . e ., negative ) than in the controls ( p & lt ; 0 . 001 ; fig1 a ). the af group showed more oxidation , with a mean ( sd ) e h gsh of − 133 ( 21 ) mv ( median − 143 mv , interassay cv , 15 . 8 %) and e h cysh of − 68 ( 6 ) mv ( median − 67 mv , cv = 8 . 8 %) compared to the control group , which had a mean ( sd ) e h gsh of − 154 ( 12 ) mv ( median − 156 mv , cv 7 . 8 %) and e h cysh of − 77 ( 6 ) mv ( median − 76 , cv 7 . 8 %). consistent with the thiol results , the droms also showed more oxidation in the af group [ 388 ( 54 ) carr units , median 370 carr units , cv 13 . 9 %] than the controls [ 310 ( 44 ) carr units , median 308 carr units , cv 14 . 2 %] ( p = 0 . 001 ; fig1 b ). the inflammatory markers il - 1β , il - 6 , tnfα , and hscrp were mildly , but insignificantly increased in the af group compared to controls . mean ( sd ) values for il - 1β , il - 6 , tnfα , and hscrp in the af group were 0 . 5 ( 0 . 8 ) ng / l ( median 0 . 3 ), 5 . 5 ( 3 . 9 ) ng / l ( median 4 . 2 ), 6 . 5 ( 8 . 1 ) ng / l ( median 3 . 8 ), and 5 . 1 ( 3 . 8 ) mg / l ( median 4 . 5 ) compared to 0 . 4 ( 0 . 4 ) ng / l ( median 0 . 3 ), 3 . 9 ( 1 . 6 ) ng / l ( median 3 . 6 ), 5 . 5 ( 3 . 4 ) ng / l ( median 4 . 7 ), and 3 . 6 ( 3 . 1 ) μg / ml ( median 2 . 6 ) for the control group , respectively . the relationship of oxidative stress and inflammatory markers to af was analyzed in single exposure conditional logistical models . the odds ratios for af were computed based on an inter - quartile range increase for each single marker ; comparing the risk of af in subjects at the 25 th percentile to those at the 75 th percentile . single exposure model odds ratios were controlled for matching variables of age , sex , smoking , and diabetes status , but are otherwise unadjusted . oxidative stress markers , e h gsh , e h cysh , and droms , all predict af with odds ratios of 6 . 1 ( 95 % ci : 1 . 3 - 28 . 3 , p = 0 . 02 ), 13 . 6 ( 95 % ci : 2 . 5 - 74 . 1 , p = 0 . 01 ), 15 . 9 ( 95 % ci : 1 . 7 - 153 . 9 , p = 0 . 02 ) respectively ( table 2 below ). none of the odds ratios for any of the four inflammatory markers measured were statistically significant ( fig2 , p & gt ; 0 . 05 ). there is a complex relationship between oxidative stress and inflammation . to evaluate this further , we compared the correlations between markers in our study ( table 3 below ). consistent with the idea that thiol ratios best represent the redox states of the hydrophilic phase while droms more likely measure the redox state of a lipid phase , spearman correlation coefficients revealed a statistically significant correlation between e h gsh and e h cysh ( r = 0 . 66 ) while the relationship of thiol ratios to droms was weaker ( table 3 ). for the most part , oxidative stress markers were independent of inflammatory markers , except for the case of a statistically significant positive correlation between tnfα and droms ( r = 0 . 38 ) and a negative correlation between tnfα and e h cysh ( r =− 0 . 42 ). on the other hand , most inflammatory markers showed a significant degree of correlation between each other . because statins are postulated to have antioxidant activity and have been associated with a reduced incidence of af ( references 2 , 55 and 60 ), we analyzed the relationship between use of statins and af among patients in our study . statin use was negatively correlated with af with an or of 0 . 2 ( 95 % ci 0 . 05 - 0 . 99 , p = 0 . 05 ). moreover , linear regression analysis revealed that e h gsh and statins were associated with a 14 . 3 mv ( 95 % ci 0 . 8 - 27 . 8 ) decrease in the oxidative stress marker , e h gsh , suggesting that the statin effect of af may be related to a reduction in oxidative stress . in multivariate analyses , the association of af and more oxidized thiol ratios remained statistically significant when correcting for hypertension , congestive heart failure , and statin use . oxidative stress has been implicated in the pathogenesis of af . the purpose of this study was to compare the relative changes in oxidative stress markers between patients with and without persistent or permanent af . we found that oxidative stress markers differed between the two groups . inter - quartile range increases across all markers of oxidative stress strongly and significantly correlated with increased risk of af . this remained true even after correction for differences in hypertension , congestive heart failure , and statin use between the two groups . the strong correlation of af with oxidative stress markers may suggest novel measures to predict the onset and efficacy of treatment in af . fig3 shows the relationship of e h gsh to the odds ratio for af based on the parameters determined in the multivariate logistical regression analysis . a change in e h gsh of 15 mv implies a ˜ 4 fold increase in the odds ratio of af . this change in e h gsh corresponds to roughly that expected with either a decade increase in age or the presence of diabetes , hypertension , or smoking , all known risk factors for af ( references 44 , 58 and 65 ). the mechanisms whereby oxidative stress may contribute to af are unknown , but there is evidence that oxidants can affect ion channel activity ( references 4 , 30 and 61 ). also , oxidative stress is known to activate redox sensitive transcription factors such as nf - κb . recently , we have shown that the cardiac sodium channel ( scn5a ) promoter region contains an nf - κb response element that could lead to na + channel transcriptional regulation by a nfκb - dependent mechanism ( reference 67 ). in our study , we showed an inverse relationship between statin use and oxidative stress or the incidence of af . statins are thought to have anti - oxidant properties at least in part as a result of preventing nadph oxidase induced oxygen free radical production ( references 14 and 71 - 74 ). our findings are consistent with reports that statins prevent electrical remodeling in rapid pacing - induced af ( reference 69 ) and experimentally induced sterile pericarditis ( reference 51 ) in canine models and reducing af burden after cardiac ( reference 55 ) or non - cardiac surgeries ( reference 2 ). moreover , it is consistent with a recent report of statins preventing recurrence of af after cardioversion ( reference 60 ). since inflammation has been associated with af and oxidative stress , we also measured inflammatory markers between our two groups . we did not find an association in our study , however . this is consistent with findings of several other groups investigating the use of crp to predict post - operative af ( references 2 , 33 , 34 and 77 ). conway et al . found that crp predicted only initial but not long term cardioversion success , ( reference 17 ). conversely , other reports suggest a correlation of inflammatory markers with af . there is a well documented increase in af incidence after cardiac surgery , this increase in af correlates temporally with the peak elevation in crp levels ( reference 8 ). moreover , in two trials , patients with high crp levels were more likely to develop af ( references 3 and 53 ). a recent meta - analysis of 16 trials does suggest a relationship between inflammation and persistent or permanent af ( reference 6 ). in one trial , il - 6 but not crp or tnfα predicted post - operative af ( reference 39 ). the concomitant lack of elevation of il - 1β , il - 6 , and crp is consistent with the known roles of these interleukins as synergistic upstream stimuli for crp production ( reference 32 ). since our patients had persistent or permanent af , the association of af with oxidative stress but not inflammatory markers could represent a more prominent role for oxidative stress relative to inflammation in the maintenance of af . alternatively , differences in post - operative and non - operative af , a lack of sensitivity given our high baseline crp levels compared to other trials , or the limited power of the trial to detect a relationship could explain the findings . our levels of il - 6 and tnfα were comparable with baseline levels in a recent report , however , suggesting that our patients were not substantially different in inflammatory state from those in other trials ( reference 39 ). although our results do not directly address the role of oxidative stress in the initiation of af , they do not rule out a potential role for inflammation in the maintenance of af . interestingly , cardiac surgery has also been reported to increase oxidative stress as measured by thiol ratios in the plasma and myocardium , ( reference 19 ) and supplementing postoperative patients with ascorbate , a known anti - oxidant , cuts rates of af over 2 - fold ( reference 11 ). as noted above , af has been associated with cardiac oxidative stress , but a recent trial suggests that oxidative stress in af may be more widespread ( reference 35 ). therefore , it is possible that systemic oxidative stress contributes to af risk and , once af is established , local cardiac oxidative stress reinforces the risk ( references 23 and 47 ). interestingly , congestive heart failure and hypertension are associated with oxidative stress , perhaps contributing to their unequal distribution between the two groups ( references 13 , 37 and 70 ). a hypothesis potentially explaining the relationship of af and its risk factors to oxidative stress is presented in fig4 . in conclusion , persistent or permanent af is associated with increased blood markers of oxidative stress when compared to an age -, sex -, smoking -, and diabetes - matched control population . in this study , the use of statins was associated with a lower prevalence of af and with decreased oxidative stress levels . therefore , lower oxidative stress marker levels may predict a lower risk of af or efficacy of drugs in the prevention of af . * approximate increase in the number of measured units ( e . g . mv for thiol ratios , carr units for droms , pg / ml for il - 1β , il - 6 , and tnfα , and μg / ml for hscrp , respectively ) needed to move from the 25 th to 75 th percentile . while this invention has been described as having preferred sequences , ranges , steps , materials , structures , features , and / or designs , it is understood that it is capable of further modifications , uses and / or adaptations of the invention following in general the principle of the invention , and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains , and as may be applied to the central features hereinbefore set forth , and fall within the scope of the invention and of the limits of the appended claims . the following references , and those cited in the disclosure herein , are hereby incorporated herein in their entirety by reference . 1 . abramson , j . l . ; hooper , w . c . ; jones , d . p . ; ashfaq , s . ; rhodes , s . d . ; weintraub , w . s . ; harrison , d . g . ; quyyumi , a . a . ; vaccarino , v . association between novel oxidative stress markers and c - 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