over the past 30 years there has been a dramatic increase in the prevalence in obesity in the us , from 18% in the 80s to approximately 35% in 2010 [ 1 , 2 ] . in parallel , there has been an increase in the prevalence of asthma from 30.7 to 53.8 per thousand population between 1980 and 1994 . a prospective epidemiologic study demonstrated a positive independent association between obesity and the incidence of adult onset asthma . obesity is associated with a dose - dependent increase in the odds of incident asthma [ 8 , 9 ] . leukotrienes ( lt ) are potent lipid mediators of inflammation derived from arachidonic acid known to play a critical role in the pathogenesis of asthma . arachidonic acid is acted upon by the enzyme 5-lipoxygenase to synthesize ltb4 and cysteinyl leukotrienes ( cys - lt c4 , d4 , and e4 ) . both cys - lt and ltb4 are released by neutrophils , eosinophils , mast cells , peripheral blood monocytes , and macrophages and are involved in the pathogenesis of obstructive lung disease . lt synthesis has been shown to be increased in peripheral blood leukocytes [ 11 , 12 ] , airway macrophages , and eosinophils   from asthmatics compared to cells from healthy controls . interestingly , obese subjects that had reduced responsiveness to inhaled corticosteroids with increasing body mass index ( bmi ) demonstrated a stable response to treatment with the lt modifier , montelukast . therefore , lts may play a greater role in the pathogenesis of asthma in obese subjects compared to nonobese patients . obesity is a proinflammatory state associated with increased systemic cytokine levels such as tumor necrosis factor  , interleukin-1 , and interleukin-6 and other classical mediators produced by adipocytes termed adipokines . one such adipokine is leptin , a 16  kd protein synthesized by adipocytes and involved in the regulation of food intake and energy balance . levels of plasma leptin correlate with total body fat stores , being elevated in obesity and reduced in weight loss . leptin has been shown to enhance proinflammatory cytokine expression , specifically to upregulate th1 cytokines   and the eicosanoid , lt [ 19 , 20 ] . in contrast to the proinflammatory effects of leptin , adiponectin is predominantly an anti - inflammatory mediator , mainly involved in glucose control and fatty acid metabolism , that is synthesized in adipose tissue and plasma levels of adiponectin are decreased in obesity . a role for leptin in promoting pulmonary inflammation and bronchoconstriction has been demonstrated in murine models of obesity and asthma [ 21 , 22 ] . however , data supporting a role for leptin in the prevalence or severity of asthma in adult humans , independent of bmi , are inconsistent . adiponectin has also been shown to play a role in animal models of asthma but , in contrast to leptin , this adipokine attenuates pulmonary inflammation [ 24 , 25 ] . the impact of reduced serum adiponectin levels in humans with asthma is , like leptin , not clearly defined . preliminary data from our laboratory demonstrate increased plasma leptin levels in obese asthmatics compared to obese nonasthmatics . leptin levels also tended to increase in asthmatics compared to nonasthmatics whether they were obese or nonobese . in view of the positive association between leptin and lt level , we examined cys - lt levels in the exhaled breath condensate ( ebc ) and peripheral blood leukocytes of asthmatics compared to nonasthmatics and obese compared to nonobese subjects . all subjects were 1865  years old with or without asthma , male and female , with ethnicity and race distribution of the community 's population seen at the university of michigan health centers . the nonasthmatic subjects were healthy volunteers without a history of lung disease . fully informed consent was obtained from each subject before entry into the study . bmi calculations were determined by dividing the weight by the square of the height ( kg / m ) . asthmatic and nonasthmatic subjects were studied based on bmi , ( 1 ) normal bmi ( 2024.9  kg / m ) and ( 2 ) obese bmi ( > 30  kg / m ) to maximize potential differences on cys - lt synthesis . diagnosis of asthma was based on history , physical exam , and pulmonary function testing including peak expiratory flow rate ( pefr ) ( l / min ) ( asthmacheck , respironics , inc . approximately 50% of the asthmatics had spirometry , with forced expiratory volume in one - second ( fev1 ) measurements . peak flow meter and bmi values were determined in all subjects on the day of the study . an asthma questionnaire was administered which includes frequency of day and night time asthma symptoms . exclusion criteria included current smokers within the past 6 months and previous smokers > 20 pack years , intercurrent infection , and treatment with lt - modifier drugs , aspirin , nonsteroidal anti - inflammatory agents , and oral steroids . aspirin sensitive asthmatics were excluded because of their tendency to display excess cys - lt synthesis at baseline . exhaled breath condensate ( ebc ) was collected with rtube to determine airway lining fluid levels of cys - lt levels [ 29 , 30 ] . the exhaled air cools below the dew point by transfer of heat to a chilled condenser surface . cys - lt were determined by enzyme immunoassay ( eia ) ( cayman chemicals , ann arbor , mi ) . samples were run in duplicate , neat , and diluted , using a competitive assay based on a high - affinity monoclonal antibody measuring cys - lt . the lower range detection limit ( 80%  b / b0 ) was 34  pg / ml . significant numbers of both male and female patients were studied because of possible greater levels of leptin   and lt in women than in men . a volume of 5  ml of heparinized whole blood was layered over 3.5  ml of a mixture of sodium metrizoate and ficoll ( 1-step polymorphs , accurate chemical & scientific corporation , westbury , ny ) in a 15  ml centrifuge tube . the sample was centrifuged at 450  g for 30  min in a swing - out rotor at 22c . adipokines , leptin , and adiponectin were measured in the plasma by enzyme immunoassay ( eia ) according to the protocol described . plasma leptin levels were determined spectrophotometrically using commercially available colorimetric eia kits ( millipore , ma usa ) and run according to the manufacturer 's instructions . the coefficient of variation for duplicate samples was 3.7% and the lower limit of detection was 0.5  ng / ml . similarly , plasma high molecular weight adiponectin ( linco research , a subsidiary of millipore , st . charles , mo ) was run in duplicate according to the manufacturer 's directions ( lld : 8.1% , 0.5  ng / ml . ) . blood samples were drawn from patients at the same time of the day , in the morning , and when patients were in the fasting state . total ige levels were determined in all subjects ( clinical pathology laboratory , university of michigan ) . we measured lt levels in pbm from subjects obese and nonobese with and without asthma . pbm were isolated from whole blood by ficoll ( one step , accurate chemical & scientific corp , westbury , ny ) centrifugation and adherence , as previously described . cells were > 90% pbm by differential staining and viability > 95% by trypan blue exclusion . isolated pbm were resuspended in lipopolysaccharide - free dulbecco 's modified eagle medium at 0.5  10/ml and adhered for 1  h at 37c in a humidified atmosphere of 5% co2/95% o2 for eia . the cells were stimulated ex vivo with 1  m calcium ionophore a23187 ( calbiochem - behring corp . final concentrations of dimethyl sulfoxide ( 0.05% ) added to cultures had no effect on either cell viability or eicosanoid synthesis . this compound liberates intracellular calcium from intact cells and releases arachidonic acid from cell membranes . the eicosanoid products were measured by eia . our study was designed to have 80% power with a type i error 5% to detect significant differences between asthmatics and normal subjects , as well as between obese and nonobese asthmatics . from the preliminary data , we estimate the median log ltb4 levels in exhaled breath condensate for nonobese asthmatics as 50  pg / ml , corresponding to a median log level of 3.90 . the study is powered to detect a doubling in the median lt levels for the obese compared to the nonobese groups . based on preliminary data and to achieve the above differences 20 subjects were needed in each group , that is , 80 total for the four groups : obese asthma , nonobese nonasthma , asthma nonobese , and obese nonasthma . mean and standard deviations for normally distributed data were calculated and differences between groups determined by student 's t - test . for skewed data mann - whitney tests correlations and partial correlations were performed using pearson 's coefficient . for the evaluation of possible associations between each study variable ( dependent ) and bmi ( independent ) , linear regression was performed . multivariable regression analysis was performed for leptin , adiponectin , and ebc cys - lt levels , adjusting for age , gender , and asthma status . we evaluated the association with bmi categories as well as bmi as a continuous variable . comparison of study variables among the four groups was performed with one - way analysis of variance ( anova ) . we set out to study four groups of subjects : obese asthmatics , nonobese asthmatics , obese nonasthmatics , and nonobese nonasthmatics . the bmi of the control and obese subjects were recruited to obtain optimal separation of the groups ( table 1 ) . mean peak flow meter ( pfm ) readings of the asthmatics and nonasthmatics were also significantly different ( table 1 ) . there was no difference in pfm between obese and nonobese ( 82.5  21 versus 80.3  12.2 l / min , p = ns ) asthma subjects . there was also no significant difference in fev1 between obese and nonobese asthmatics ( 74.6  19.4 versus 91.5  12.5% predicted , p = 0.052 ) . the asthma control test ( act ) questionnaire was lower in obese asthma ( 19.8  11.9 ) compared to nonobese asthma ( 22.1  1.6 ) patients , p = 0.01 . total ige levels were different but quite variable in both asthmatics and nonasthmatics ( 326  519 versus 73  124  ku / l , p = 0.004 ) . there was no difference in ige levels between obese and nonobese asthmatics ( 315  448 versus 336  586  ku / l , p = 0.13 ) . the gender breakdown was 64% female ( 36% male ) , which was as close as we could get to equality in recruitment . males ( 35.2  12.2 years ) and females ( 35.1  12.7 years ) displayed no difference in age . no significant difference occurred in bmi between the genders , obese ( 36.5  5.7 versus 38.4  4.6 years male  :  female ) or nonobese ( 23.6  1.3 versus 22.5  1.7 ) . the recruitment age in the subjects , asthma versus nonasthma subjects , was not significantly different ( 32.7  12.3 versus 37  12.1 years , p = 0.31 ) . obese subjects were generally older , displaying an age - related increase in fat mass ( obese 41.8  11.6 versus nonobese 27.7  8.4 ,  p < 0.001 ) . obese asthmatics were on average older than nonobese asthmatics ( 40.6  12.8 versus 25.5  5.9 years , p < 0.001 ) . twelve of the obese subjects had a diagnosis of obstructive sleep apnea ( osa ) . gastroesophageal reflux disease ( gerd ) was more common in obese than nonobese ( 8/42 versus 3/40 subjects ) subjects . gerd was equally distributed between asthmatics and nonasthmatics ( 6/42 versus 5/40 subjects ) . the asthmatics studied had mild to moderate persistent disease with all of them on ics ( 42 subjects ) . none of the asthmatics were on leukotriene modifier therapy , since that was an exclusionary criterion for subject recruitment . as expected , fasting leptin levels were higher in obese subjects than nonobese subjects ( 32.7  20.2 versus 8.5  6.3  ng / ml , p = 0.0001 ) . there was no significant difference in leptin levels between asthmatics and nonasthmatics ( table 2 ) . mean leptin levels were higher in females than in males ( 26.4  21 versus 12.4  12.3  ng / ml , p < 0.05 ) . female asthmatics had higher leptin levels than males with asthma ( 31.6  24.1 compared to 13.1  11.9  ng / ml , p = 0.01 ) . leptin levels were higher in obese female asthmatics compared to obese male asthmatics ( 52.1  18.6 versus 20.8  11.6  ng / ml , p < 0.001 ) . leptin levels in female asthmatics were 31.6  24.1 compared to females without asthma 20.9  3.7  ng / ml , p = 0.03 . leptin levels in men with and without asthma were 13.1  11.9 and 11.7  8.5  ng / ml , respectively ( p = 0.29 ) . leptin 's correlation with weight is 0.77 which was significant ( p < 0.0001 ) . when it was adjusted for asthma , it is 0.78 ( p < 0.0001 ) . the correlation of leptin with asthma is 0.17 ( p = 0.119 ) . there was also a positive correlation between leptin and the female gender ( p = 0.001 ) . using multiple linear stepwise regression only weight and gender ( female ) had a positive correlation , when the variables age , gender , weight , and asthma were considered . using anova , leptin was significantly different between the four groups : obese asthmatics , nonasthma nonobese , asthma nonobese , and nonasthma obese . obese asthmatics had significantly higher levels than both nonobese asthmatics and nonobese nonasthmatics ( p < 0.0001 ) ( figure 1 ) . the only groups that were not statistically different were the obese asthmatics and obese nonasthmatics ( p = 0.27 ) . adiponectin was decreased overall in the obese versus nonobese subjects ( mean 9.3  4.7 versus 14.1  20  g / ml , p < 0.05 ) , as expected . there was no difference in adiponectin levels between asthmatics compared to nonasthmatics ( table 2 ) . as noted for leptin , mean adiponectin levels were higher in females than in males ( 14.1  5.5 versus 7.2  3.1  g / ml , p < 0.05 ) especially in nonobese . this was also true for obese females who had higher mean levels compared to obese males ( 11.3  4.8 versus 6.4  2.7  g / ml , p < 0.05 ) . males with asthma display lower mean levels of adiponectin than those seen in females with asthma ( 6.9  2.8 versus 13.3  5.6  g / ml p < 0.05 ) . adiponectin 's correlation with weight is 0.44 which was significant ( p < 0.0001 ) . the correlation of adiponectin with asthma was 0.10 but was not significant ( p = 0.36 ) . there was a positive correlation between adiponectin and act ( 0.35 ) which did reach significance ( p = 0.002 ) . when adjusted for asthma it was a positive correlation ( 0.36 ) and was significant ( p = 0.001 ) . linear regression demonstrated a negative correlation between adiponectin and obesity . using multiple linear stepwise regression , there was a negative correlation with weight and a positive correlation with gender when the variables age , gender , weight , and asthma were considered . anova demonstrated that adiponectin was significantly different between the four groups : obese asthmatics , nonasthma nonobese , asthma nonobese , and nonasthma obese . obese asthmatics had lower adiponectin levels than nonobese nonasthmatics ( p < 0.008 ) ( figure 2 ) . obese nonasthmatics also had lower adiponectin than nonobese nonasthmatics ( p = 0.017 ) . since both leptin and adiponectin are increased in women compared to men , we next examined the leptin / adiponectin ratio as a method to better compare genders with regard to the role of adipokines in the pathogenesis of asthma in obesity . the mean leptin / adiponectin ratio was 2.8  3.6 in women compared to 2.1  1.9 in men ( p = 0.15 ) . obese subjects had a significantly higher mean ratio than nonobese subjects ( 4.3  3.5 versus 0.78  0.8 , p < 0.01 ) . asthmatics did not have a significantly higher mean ratio compared to nonasthmatics ( table 2 ) . although the mean leptin / adiponectin ratio trended to be higher in patients with worse airway obstruction ( pfr < 80% predicted ) , it did not reach statistical significance ( p = 0.26 ) . linear regression demonstrated a positive correlation between leptin / adiponectin ratio and weight ( p = 0.0001 ) , as well as age ( p = 0.0001 ) . using multiple linear stepwise regression , there was a correlation with weight ( p = 0.0001 ) . anova demonstrated that leptin / adiponectin ratio was significantly different between the four groups : obese asthmatics , nonasthma nonobese , asthma nonobese , and nonasthma obese . obese asthmatics had higher leptin / adiponectin ratios than nonobese nonasthmatics ( 5.3  4.3 versus 0.75  0.9 , p < 0.0001 ) and nonobese asthmatics ( p < 0.0001 ) ( figure 3 ) , but not obese nonasthmatics ( p = 0.16 ) . this was mainly accounted for by female obese asthmatics ( 6.3  4.9 versus 3.3  1.7 for males , p = 0.03 ) . exhaled breath concentrate ( ebc ) cys - lt levels were higher in all asthmatics compared to nonasthmatics ( table 2 ) . there was no difference in ebc cys - lt levels between obese and nonobese subjects ( 66.1  29.00 versus 62.8  29.1  g / ml , p = 0.3 ) . obese asthmatics had higher ebc cys - lt levels than nonobese nonasthmatic subjects ( 73  29.7 versus 57  28.1  g / ml , p = 0.05 ) . the correlation of ebc with obesity was 0.11 ( p = 0.34 ) . ebc correlation with asthma was 0.23 which is significant ( p = 0.04 ) . when this is adjusted for bmi it was 0.24 ( p = 0.03 ) . multiple regression analysis revealed a significant correlation between ebc cys - lt and asthma ( p = 0.04 ) . there was no correlation with weight , age , gender , ics , or osa . anova demonstrated no difference between four groups , likely due to the variability of the samples . the greatest difference was between obese asthmatics and nonobese nonasthmatics but only reached a p value of 0.3 ( figure 4 ) . pbm cys - lt production was higher in females than males ( 222  132 versus 142  116  g / ml , p < 0.05 ) . pbm cys - lt levels were comparable in the asthmatic versus nonasthma subjects ( 197  148 versus 190  115  g / ml , p = 0.4 ) . however , in view of the increased variability of the pbm cys - lt levels , there were no significant increased values in obese asthmatics compared to nonobese nonasthmatics . the main findings of this study are the following : ( 1 ) plasma leptin levels are increased in obese asthmatics compared to obese nonasthmatics . this was mainly accounted for by higher leptin levels in females , and especially obese female asthmatics . ( 3 ) the leptin / adiponectin ratio was significantly higher in obese asthmatics compared to nonobese nonasthmatics . ( 4 ) ebc cys - lt levels were higher in asthmatics than in nonasthmatics . ( 5 ) pbm cys - lt levels were higher in women than in men , irrespective of asthma or obesity . an important finding in our study was the association between leptin and asthma , and specifically in obese asthmatics . leptin levels are thought to be higher in females because of increased percentage body fat in women   as well as increased secretion of leptin from adipose tissue . the latter may be related to increased estradiol levels and its effect of increased leptin secretion from adipose tissue . leptin has been noted to be a proinflammatory adipokine driving the immune system towards a predominant th1 phenotype . leptin receptors have been described in the airway and investigators have shown a correlation between plasma and bal levels . leptin deficient mice display reduced lt synthesis , and exogenous leptin augments lt production in macrophages from these animals . obesity has also been associated with an alteration in adipokines that predispose to neutrophilic inflammation . however , other investigators have demonstrated decreased inflammation in the airways of obese asthmatics , which is reversed by weight loss . recently , it has also been shown that leptin may also affect the airway diameter through noninflammatory pathways by inhibiting the cholinergic pathway . although we have shown an association between leptin and asthma , population studies by sutherland et al . have shown no such association . sampling of nonfasting subjects , some of whom smoked , can affect leptin levels and may explain the differences in conclusions of these studies . in contrast to leptin , adiponectin levels are decreased in obesity and tend to increase in starvation . it has a number of metabolic actions including increasing insulin sensitivity , diminishing atherosclerosis , and being generally anti - inflammatory . although adiponectin receptors have been detected in the lungs , serum levels do not consistently correlate with bal fluid levels . this finding may be explained by the fact that active transport of adiponectin , which is a large molecule ( full length form ) , may be necessary for penetration into the airways . higher adiponectin levels have been associated with lower asthma prevalence rates in women . in our population , obese asthmatics had lower adiponectin levels than nonobese nonasthmatics . however , the higher levels of adiponectin in women compared to men , as was noted for leptin , may be the confounding variable , which detracts from the association of lower adiponectin levels in obese asthmatics compared to other groups . cross - sectional analysis of an asthmatic population , who were nonfasting and smokers , did not demonstrate an association between adiponectin and asthma . an increased leptin / adiponectin ratio is associated with asthma compared to control subjects without asthma . the increased leptin / adiponectin ratio is also associated with severe asthma compared to mild to moderate asthma . in our study , however , in our study the leptin / adiponectin ratio was not associated with asthma or increased airway obstruction . the reduced responsiveness to inhaled corticosteroids with increasing bmi , with a relatively stable response to montelukast , has been previously described . sutherland et al . also examined the comparative effects of bmi in response to asthma controller medication . by contrast , they demonstrated that inhaled corticosteroids were superior to lt modifiers at all bmi levels , in patients who were underweight compared to those with morbid obesity . in our study we demonstrated that ebc cys - lt levels correlated with asthma , being higher in asthmatics than in nonasthmatics . other investigators have demonstrated an association between bmi and urine cys - lt in obese asthmatics . obese patients had significantly higher values of lte4/creatinine in urine compared to subjects who were preobese and in the normal range . in a linear regression model using the same model , log leptin and log adiponectin presented positive and negative associations , respectively , with lte4/creatinine in urine . this was because of the increased variability noted with ebc samples , which may also explain the marginal increase in ebc cys - lt levels in obese asthmatics in our study . the other problem is the inability to adequately standardize dilution factors for ebc across subjects . an alternative explanation is that alveolar cys - lt synthesis may not be as important as airway levels . a recent study demonstrated elevated submucosal but not sputum eosinophil levels in obese subjects with severe asthma [ 47 , 48 ] . this may explain the elevated urine cys - lt levels in obese subjects compared to ebc measurements . in our cohort subjects with the older obese women phenotype trended to have increased leptin and ebc cys - lt levels with worse pulmonary function . recently , investigators have demonstrated that bmi was associated with the incidence of asthma in women but not in men . furthermore , adult onset nonatopic asthma has become the most common type of asthma in women . investigators have demonstrated that both testosterone ( negative ) and estrogen ( positive ) were associated with alterations in adipokines , specifically leptin levels . we detected no significant difference between premenopausal obese women with asthma compared to nonobese patients . there was no decline in leptin levels in asthma with age , in postmenopausal compared to premenopausal subjects matched for bmi . the increase in bmi with age may have offset the effect of any change in estrogen / testosterone levels on adipokine levels as subjects increased in years . we did document that pbm from female subjects had higher cys - lt levels than that of male subjects . this has been noted by other investigators and may in part be due to suppression of 5-lo enzymatic activity by testosterone . although pbm cys - lt levels were trending higher in asthmatics than nonasthmatics , this did not reach significance . increased plasma leptin levels in women may be associated with the elevated pbm cys - lt production noted in females . poorly controlled osa has been shown to worsen asthma control , in part through increased release of proinflammatory mediators during episodes of hypoxia and disrupted sleep . nocturnal gerd may exacerbate asthma with aspiration of acid , during inhalation against a closed airway , following episodes of upper airway obstruction . like osa , the incidence of gerd is also increased in obese subjects and may exacerbate airway disease after eating . gerd can result in reflex bronchospasm from irritation of the esophagus or directly spilling into the airway with gross reflux . although both osa and gerd were more common in obese than nonobese subjects in our study , the incidence was equally distributed in the asthmatic and nonasthmatic groups . it is a relatively small study number - wise , examining the role of obesity in the pathogenesis of asthma . although the diagnosis of asthma was made by a physician with history , physical examination , and pulmonary function testing , spirometry or methacholine challenge testing was not performed . in addition , a number of variables to assess the role of ebc cys - lt readings limited the interpretation of results . inhaled corticosteroids can result in ~18% reduction in ebc cys - lt levels , but we wanted to include asthmatics that were with mild to moderate severity . although adipokines can be affected by the gender and the menstrual cycle , we did not determine the reproductive hormone concentrations in our premenstrual patients . despite these issues , there was good characterization and separation of subjects between the control group and obese subject group . in addition , the asthmatics were well characterized compared to the nonasthmatics with peak flow rates and asthma control test questionnaires . furthermore , the significance of the study was improved since we studied predominantly moderate persistent asthmatics all on inhaled corticosteroid therapy . none of the asthmatics were on lt modifier medications . in summary , we have demonstrated that leptin levels and the leptin / adiponectin ratio are increased in obese asthmatics compared to obese nonasthmatics . proinflammatory mediators , cys - lt , were increased in obese asthmatics compared to nonobese nonasthmatics . adipokines , especially in obese female asthmatics , may promote an asthma phenotype that has become more prevalent and results in difficult to control airway disease .
background . 
 the prevalence of obesity has increased dramatically over the last decades , and its association with asthma is being increasingly recognized . 
 aims . 
 our hypothesis is that increased leptin and decreased adiponectin levels in obese subjects play a direct role in regulating inflammation in asthmatics . 
 we wanted to examine the hypothesis that cysteinyl leukotrienes ( cys - lt ) , inflammatory mediators that are regulated by adipokines , are involved in the pathogenesis of asthma . methods . we studied a population of asthmatics and nonasthmatics , who in turn were divided into obese and nonobese categories . 
 we examined leptin and its ratio to adiponectin , in asthmatics and nonasthmatics , with and without obesity . 
 in addition , we measured cys - lt levels in exhaled breath condensate ( ebc ) and in peripheral blood monocytes ( pbm ) in these groups . results . 
 leptin levels were increased in obese asthmatics compared to obese nonasthmatics . the leptin / adiponectin ( l / a ) ratio was higher in obese asthmatics compared to obese nonasthmatics . 
 ebc cys - lt levels were elevated in asthmatics compared to nonasthmatics . 
 discussion . 
 proinflammatory adipokines , released from adipose tissue , may promote an asthma phenotype through enhanced cys - lt production that may result in more prevalent and difficult to control airway disease .