Study of serum leptin level in obese and nonobese asthmatic patients

Aim: The aim of the study was to investigate serum leptin levels in obese and nonobese asthmatic patients and its change during acute attack and in remission, as well as its relation to the changes in pulmonary functions. Methods: The study was carried out on 55 participants (40 asthmatic patients and 15 controls) who were divided according to BMI into obese [(BMI >30 kg/m 2 ), 20 asthmatic patients and eight controls] and nonobese [(BMI <25 kg/m 2 ), 20 asthmatic patients and seven controls]. All participants were subjected to calculation of BMI, pulmonary function tests, and morning serum leptin level estimation (after at least 8 h of fasting). Results: Serum leptin levels (mean in ng/ml) in obese controls (64 ng/ml) and obese asthmatic patients (80.4 ng/ml during remission and 92.9 during exacerbation) were significantly higher than that in nonobese controls (6.3 ng/ml) and nonobese asthmatic patients (33.8 ng/ml during remission and 48.8 during exacerbation). There was a significant (r = −0.456 and P ≤ 0.05) negative correlation between the change in serum leptin (ng/ml) and the change in forced vital capacity (FVC) (% Predicted) and forced expiratory volume in first second (FEV 1 ) (% Predicted) in obese asthmatic patients, but not in nonobese asthmatic patients. There was a significant positive correlation between BMI (kg/m 2 ) and serum leptin levels (ng/ml) in obese (r = 0.712 and P ≤ 0.05) and nonobese (r = 0.747 and P ≤ 0.05) controls and a higher significant positive correlation in obese (r = 0.94 during exacerbation and r = 0.833 during remission, P ≤ 0.001) and nonobese (r = 0.687 during exacerbation, P ≤ 0.001 and r = 0.488 during remission, P ≤ 0.05) asthmatic patients. Conclusion: Serum leptin levels were higher in all asthmatic patients (more during exacerbation) compared with controls and the values were higher in obese than in nonobese asthmatic patients with a significant negative correlation between the change in serum leptin and the change in FEV 1 and FVC in obese asthmatic patients. These findings indicate that leptin is involved in asthma inflammation.


Introduction
Asthma is 'a chronic infl ammatory disorder of the airways associated with airway hyper-responsiveness that leads to recurrent episodes of widespread, and often reversible, airfl ow obstruction within the lung' [1].
Obesity has been signifi cantly associated with nonatopic asthma rather than with atopic asthma in women and children [2]. Some studies indicate that obesity may increase asthma severity and may reduce the effi cacy of standard asthma medications [3,4].
Growing evidence suggests that the proinfl ammatory eff ects of leptin may contribute to the higher incidence of asthma in the obese population [5,6]. erythrocyte sedimentation rate, liver function tests, kidney function tests, fasting and postprandial blood sugar), and pulmonary function tests (FEV 1 %, FVC%, FEF25-75%, and FEV 1 /FVC were measured for each patient before and 10 min after inhalation of 200-400 μg salbutamol). Body weight (kg) and height (meters) were measured for each patient for calculation of BMI. Serum leptin levels were determined (morning sample after at least 8 h of fasting) during acute exacerbation and after control of the attack and also in the control group by taking blood samples, subjecting them to centrifugation, and then measuring using an ELISA kit (DRG Diagnostics, Marburg, Germany).

Statistical analysis
Results are given as mean ± SD. Diff erences between groups were statistically analyzed using an unpaired Student t-test. For patients with leptin values below the detection limit (0.25 ng/ml) the value 0.25 ng/ml was used in the analysis. After curve estimation, linear, exponential, or logarithmic Pearson product moment correlation was calculated. After the simple correlations, a regression model was fi tted to the data to select the variables that contributed to the explained variation in plasma leptin concentration. Signifi cance was determined at the 5% level. Data were analyzed using statistical package for the social sciences, version 14.0 for Windows (SPSS Inc., Chicago, Illinois, USA).

Results
Th is study included 38 female (95%) and two male (5%) patients. Table 1 shows the age and BMI of obese (mean age ± SD = 34.5 ± 4.44 and mean BMI ± SD = 34.1 ± 1.2) and nonobese (mean age ± SD = 42.4 ± 7.35 and mean BMI ± SD = 23.7 ± 1) controls and obese (mean age ± SD = 42.65 ± 8.98 and mean BMI ± SD = 35.15 ± 3.32) and nonobese (mean age ± SD = 42.5 ± 8.88 and mean BMI ± SD = 23.15 ± 1.81) asthmatic patients.    Table 3 shows that serum leptin levels (ng/ml) in obese controls were signifi cantly higher than that in nonobese controls with a mean of 64 ng/ml in obese controls and 6.3 ng/ml in nonobese controls. Table 4 shows that serum leptin (ng/ml) was signifi cantly higher in obese asthmatic patients during an attack than in obese asthmatic patients during remission and in obese controls. Table 5 shows that serum leptin (ng/ml) was signifi cantly higher in nonobese asthmatic patients during an attack than in nonobese asthmatic patients during remission and in nonobese controls. Table 6 shows that serum leptin (ng/ml) was signifi cantly higher in obese than in nonobese asthmatic patients both during an attack and during remission. Table 7 shows that serum leptin (ng/ml) was signifi cantly increased in both obese and nonobese asthmatic patients during an attack than during remission. Table 8 shows the nonsignifi cant negative correlation between serum leptin (ng/ml), FVC (% Predicted), and FEF 25-75 (% Predicted) in obese controls, nonsignifi cant positive correlation between serum leptin (ng/ml), FEV 1 (% Predicted), and FEV 1 /FVC in obese controls, and nonsignifi cant positive correlation between serum leptin (ng/ml), FEV 1 (% Predicted), FVC (% Predicted), FEV 1 /FVC, and FEF25-75 (% Predicted) in nonobese controls. Table 9 shows the signifi cant negative correlation between change in serum leptin (ng/ml) and change in FVC (% Predicted) and FEV 1 (% Predicted) in obese asthmatic patients, the nonsignifi cant positive correlation between change in serum leptin (ng/ml) and change in FEV 1 /FVC and FEF25-75 (% Predicted) in obese asthmatic patients, the nonsignifi cant negative correlation between the change in serum leptin (ng/ ml) and the change in FVC (% Predicted) and FEV 1 (% Predicted) in nonobese asthmatic patients, and the nonsignifi cant positive correlation between change in serum leptin (ng/ml) and change in FEV 1 /FVC and FEF25-75 (% Predicted) in nonobese asthmatic patients. Table 10 shows that there was a nonsignifi cant negative correlation between age (years) and serum leptin (ng/ml) in obese and a signifi cant negative correlation between age (years) and serum leptin (ng/ml) in nonobese controls.
Th ere was a nonsignifi cant positive correlation between age (years) and serum leptin (ng/ml) in obese asthmatic patients during an attack and during remission Table 11. Th ere was signifi cant positive correlation between age (years) and serum leptin levels (ng/ml) in nonobese asthmatic patients during an attack and during remission.    Table 12 shows the signifi cant positive correlation between BMI (kg/m 2 ) and serum leptin levels (ng/ml) in obese and nonobese controls. Table 13 shows the highly signifi cant positive correlation between BMI (kg/m 2 ) and serum leptin (ng/ml) in obese asthmatic patients during an attack and during remission. Th ere was a highly signifi cant positive correlation between BMI (kg/m 2 ) and serum leptin levels (ng/ml) in nonobese asthmatic patients during an attack and a signifi cant positive correlation during remission.

Discussion
Obesity and bronchial asthma are both chronic, prevalent conditions that pose a signifi cant challenge to the clinician as well as to public health worldwide [8].
Th e aim of our study was to investigate serum leptin levels in obese and nonobese asthmatic patients and its change during acute attack and during remission as well as its relation to the changes in pulmonary functions.
Th is study included 38 female (95%) and two male (5%) patients, indicating a higher frequency and morbidity of bronchial asthma in women compared with men. In our study all asthmatic patients were admitted as inpatients in the chest department, Benha University Hospitals. Other studies showed much higher frequency of asthma and associated morbidity in women (regardless of their BMI) compared with men [9][10][11][12].
In our study serum leptin levels in all asthmatic patients were much higher than that in controls and this was statistically signifi cant and increased sharply during acute exacerbation and decreased after control of the attack; however, it was still higher than in the control group, and the level in obese asthmatic patients was higher than the level in nonobese asthmatic patients, which may indicate a role for leptin in asthma pathogenesis.
Our results agreed with data from the Nurses' Health Study [13], which evaluated the relationship between   body weight and the incidence of self-reported physician-diagnosed asthma in women. Th at study included 85 911 patients and was conducted over 4 years. As in the case-control studies, those investigators found a signifi cant relationship between BMI and the incidence of asthma, with obesity increasing the asthma risk by 2.7-3.8-fold and overweight increasing the risk by 50-70%.

Table 9 Correlations between changes in serum leptin (ng/ml) and changes in pulmonary function test scores in obese and nonobese asthmatic patients during attack and remission
Haynes et al. [14] concluded that leptin could predispose to asthma through its eff ect on immune function [tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6)] and its eff ect on the sympathetic nervous system; in addition, Guler et al. [15] found that the median serum leptin concentrations of children (especially boys) were signifi cantly higher in those with asthma than in healthy controls (3.53 vs. 2.26 ng/ml, P = 0.01), even though there was no diff erence in BMI levels.
Our study also agreed with that by Mai et al. [16] involving children born with very low birth weight who subsequently became overweight. Th e study showed that current asthmatic patients had median leptin concentrations twice as high as that in children without current asthma (30.8 vs. 14.3 ng/ml, P = 0.14), but this was not the case in nonoverweight children. Taken together, our study and prior studies suggest that leptin may potentially play an important role in the pathophysiology of asthma.
In a study [17] conducted on 5876 individuals after exclusion of those who were either pregnant or had missing values for covariates it was found that adults with the highest quartile of leptin concentrations had an odds ratio (OR) of 1.56 (95% confi dence interval 0.96-2.53) for current asthma after adjustment in a multivariable logistic regression analysis for age, sex, race/ethnicity, educational status, smoking status, concentration of cotinine, physical activity, and atopy. Th is association was stronger in women (OR 1.85) than in men (OR 1.27). In women, adjustment for triceps skin fold thickness strengthened the association between serum leptin concentrations and asthma, whereas adjustment for BMI weakened this association.
Kilic et al. [18] found that the median leptin level was higher in patients with uncontrolled asthma than in those with controlled asthma, but the diff erence was not signifi cant. In the obese group, a nonsignifi cant negative correlation (r = -0.138, P = 0.390) was found between leptin levels and asthma control test scores.
In the nonobese group, mean unadjusted leptin concentrations were higher in participants who had current asthma than in those who had never had asthma.
Shore et al. [5] found that leptin concentrations are increased acutely during infl ammation and, in turn, promote infl ammation. Other experiments showed a prompt dose-dependent increase in serum leptin levels and leptin mRNA expression in the adipose tissue of mice following administration of proinfl ammatory cytokines such as TNF-a and IL-1 [19,20] as well as demonstration of increased serum TNF-α, IL-6, and IL-12 levels and increased phagocytosis by macrophages on exogenous administration of leptin [21]. Infl ammatory mediators such as TNF-α also promote the expression and release of leptin from the adipose tissue, formulating a positive feedback mechanism [22].
Vernooy et al. also reported that leptin had an eff ect on infl ammation through enhancement of production of TNF-α and IL-6 from endotoxin-treated macrophages and lymphocytes [23].
In our study there was a signifi cant negative correlation between serum leptin and both FVC and FEV 1 in obese asthmatic patients but not in nonobese asthmatic patients.
Our fi ndings agreed with a population-based study [24] in which individuals with impaired lung function had raised serum leptin levels; King et al. [25] also reported declines in airway conductance in obese compared with nonobese individuals, which could refl ect the proinfl ammatory role of leptin. In our study there was a signifi cant positive correlation between BMI (kg/m 2 ) and serum leptin levels (ng/ml) in obese and nonobese control and asthmatic individuals.
Several studies have identifi ed an association between asthma and obesity in women [9,13,18]. BMI was positively associated with asthma, wheezing, asthma treatment, atopy, and immunoglobulin E, and inversely with the FEV 1 /FVC ratio in women. One would expect that most obese individuals would become short of breath much more quickly, especially if some of them had exercise-induced asthma. It remains unclear whether this association is due to asthma itself or due to symptoms caused by overweight or adipokines such as leptin.
Hancox et al. [26] reported that a raised BMI was associated with asthma and atopy in women than in men, and population attributable fraction calculations estimated that 28% (95% confi dence interval 7-45) of asthma cases in women after age 9 are due to overweight.
Guler et al. [15] noted that, even after controlling the BMI, serum leptin is higher in male asthmatic patients compared with nonasthmatic individuals.
Some studies have suggested that leptin may play an important role in asthma pathophysiology through its ability to activate the sympathetic nerves. Leptin was found to increase the activity of sympathetic nerves in various organs, but its eff ects on the lung sympathetic nerves are unknown [14,27,28]. In animal studies, leptin-treated mice were found to exhibit augmented responses to metacholine and increased levels of IgE, following ovalbumin challenge, when compared with saline-infused mice.
Leptin release from adipose tissue or the lung may be induced by disease-related infl ammation, which further increases the airway infl ammation and hyperreactivity [17,29,30]. Leptin has been identifi ed to have proinfl ammatory properties through the stimulation of TNF-α and IL-6 from the adipose tissue [31], and through the modulation of immunity to promote Th 1 immune responses with increased production of interferon-c (IFN-γ) [32]. In asthmatic children, IFNγ-producing CD4+ T cells are inversely correlated with blood eosinophilia but positively correlated with airway hyper-responsiveness, suggesting a possible involvement of IFN-γ in nonatopic asthma [33].
In our study there was a signifi cant negative correlation between change in serum leptin (ng/ml) and change in both FVC (% Predicted) and FEV 1 (% Predicted) in obese asthmatic patients but not in nonobese asthmatic patients. Th is fi nding may indicate that leptin has an eff ect on airway response in obese asthmatic patients during exacerbation.

Conclusion
Serum leptin levels were higher in all asthmatic patients (more during exacerbation) than in controls and the values were higher in obese than in nonobese asthmatic patients with a signifi cant negative correlation between the change in serum leptin and the change in FEV 1 and FVC in obese asthmatic patients. Th ese fi ndings indicate that leptin is involved in asthma infl ammation.