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Introduction

Recent studies have shown that endogenous hydrogen sulfide (H2S) is produced in the human body. Together with nitric oxide (NO) and carbon monoxide (CO), H2S is a gaseous signaling molecule with regulatory functions (1). Indeed, H2S relaxes vascular smooth muscle, inhibits vascular smooth muscle cell proliferation and reduces oxidative stress to play an important role in the structure and function of pulmonary circulation (2,3). Notably, endogenous H2S may be involved in the pathogenesis of airflow obstruction in chronic obstructive pulmonary disease (COPD) (4). In addition, epidemiological studies have shown that exogenous H2S exposure increases the incidence of asthma (5). However, whether endogenous H2S is involved in the pathogenesis of asthma and the precise effects of H2S on pulmonary function remain unclear. While a previous study indicated that serum H2S levels are lower in patients with asthma (6), serum H2S has yet to be correlated with pulmonary function in these individuals. In the present study, we analyzed H2S concentration in the serum in comparison to lung function parameters in 64 children with bronchial asthma and 60 healthy children to determine the effects of H2S on pulmonary function in children with bronchial asthma.

Materials and methods

Study population

Between June 2009 and June 2011, 64 children admitted to the Affiliated Nanjing Children’s Hospital, Nanjing Medical University (China), with acute bronchial asthma were recruited to the study. The study population included 36 males and 28 females between 6 and 12 years of age (mean 9.03±1.84). Acute disease lasted between 6 and 25 days. Eighteen individuals (28.1%) experienced fever, with body temperatures between 37.5 and 38.4°C. Asthma diagnosis followed the WHO criteria (7): all children had cough, asthma, obvious wheeze, rhonchus and moist rale in the lung, and other clinical manifestations; X-ray examination also showed increased lung markings or lung hyperinflation and infection. None of the children used asthma-related drugs prior to diagnosis. Children with combined respiratory and heart failure or other complications, or with congenital heart disease, tuberculosis infection and foreign body in the bronchus, were excluded. Sixty healthy children who had received physical examinations in our hospital during the same period were selected as controls. Control population included 31 males and 29 females between 6 and 12 years of age (mean 9.22±1.80). Mean age and gender distribution between the two groups of children were not statistically different. The study was approved by the Nanjing Health Bureau.

Detection of H2S concentration in the serum

Venous blood (3 ml) was collected from children in the morning following a minimum 10-h fast. Samples were centrifuged at 3,000 rpm for 10 min to separate sera and were stored at −70°C until testing for H2S. H2S was measured as described by Geng et al (8). A sensitive sulfur electrode [PXS-270 ion meter (Leici Company, Shanghai, China)] was used to determine H2S content in the plasma. Briefly, standard sulfion and antioxidant solutions were prepared. The electrode was activated for at least 2 h in deionized water prior to use. The ion meter was adjusted to mV and rake ratio to 100. Sensitive sulfur electrode and reference electrode were immersed together in the sample until the reading stabilized. Standard curves were determined with standard sulfion solution before each test.

Detection of pulmonary function

MINAT (Japan) AS-407 pulmonary function instrument was used by specialized technicians to determine pulmonary function in all participants. Main indicators included forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), peak expiratory flow (PEF), forced midexpiratory flow rate (FEF25–75), midexpiratory flow rate at 50% vital capacity (MEF50) and midexpiratory flow rate at 75% vital capacity (MEF25). All indicators are expressed as the percentage of the normal predicted value (based on the actual age, height and weight of children at sample collection).

Statistical analysis

SPSS13.0 statistical software was used for analyses. Data are expressed as the means ± standard deviation (χ̄ ± SD). Independent samples t-test was used to compare H2S concentrations in the serum as well as differences in pulmonary function parameters between groups. Pearson’s correlation was used to detect and analyze the correlation between H2S concentration and each pulmonary function parameter in children with bronchial asthma. Analyses were performed with two-sided tests at α level 0.05. p<0.05 denoted statistically significant differences.

Results

H2S serum concentration and pulmonary function differ in children with asthma

In children with bronchial asthma, H2S concentration in the serum and indicators of pulmonary function (FVC, FEV1, PEF, FEF25–75, MEF50 and MEF25) were all decreased in comparison to the healthy children. Each of these decreases were statistically significant between groups (p<0.05; Tables I and II).

Table I Mean measures of H2S, FVC, FEV1 and PEF in children with or without asthma (χ̄ ± s).

Table I

Mean measures of H2S, FVC, FEV1 and PEF in children with or without asthma (χ̄ ± s).

Study population	n	H2S (μM)	FVC (%)	FEV1 (%)	PEF (%)
Healthy control	60	52.60±5.56	104.51±8.20	99.02±7.81	100.59±7.66
Bronchial asthma	64	44.17±10.95	78.79±11.98	69.79±11.41	60.48±12.63
t		5.353	13.865	16.537	21.201
p-value		0.001	0.001	0.005	0.001

[i] H₂S, hydrogen sulfide; FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 sec; PEF, peak expiratory flow.

Table II Mean measures of FEF25–75, MEF50 and MEF25 in children with or without asthma (χ̄ ± s).

Table II

Mean measures of FEF25–75, MEF50 and MEF25 in children with or without asthma (χ̄ ± s).

Study group	n	FEF25–75 (%)	MEF50 (%)	MEF25 (%)
Healthy control	60	92.30±7.08	95.93±8.24	86.39±7.56
Bronchial asthma	64	54.78±12.19	57.84±12.22	50.67±11.70
t		20.779	20.212	20.049
p-value		0.001	0.002	0.001

[i] FEF_25–75, forced midexpiratory flow rate; MEF₅₀, midexpiratory flow rate at 50% vital capacity; MEF₂₅, midexpiratory flow rate at 75% vital capacity.

Correlation between H2S concentration in the serum and lung function in bronchial asthma

To establish a connection between the decreases in endogenous H2S and the altered lung function in asthma patients, we assessed these results with Pearson’s correlation. The H2S concentration in the serum of children with bronchial asthma was positively correlated with each of the lung function parameters (FVC, FEV1, PEF, FEF25–75, MEF50 and MEF25) (p<0.05; Table III); i.e., for increasing H2S concentrations, lung function (FVC, FEV1, PEF, FEF25–75, MEF50 and MEF25) also significantly increased (Fig. 1).

Figure 1 Relationship between serum H2S concentration and pulmonary function parameters in children with asthma. FVC, forced vital capacity; FEV1, forced expiratory volume in 1 sec; PEF, peak expiratory flow; FEF25–75, forced midexpiratory flow rate; MEF50, midexpiratory flow rate at 50% vital capacity; MEF25, midexpiratory flow rate at 75% vital capacity.

Table III Correlation between H2S concentration and pulmonary function parameters in children with asthma.

Table III

Correlation between H2S concentration and pulmonary function parameters in children with asthma.

Statistics	FVC	FEV1	PEF	FEF25–75	MEF50	MEF25
r	0.550	0.554	0.555	0.543	0.540	0.567
p-value	0.001	0.001	0.001	0.001	0.001	0.001

[i] FVC, forced vital capacity; FEV_1, forced expiratory volume in 1 sec; PEF, peak expiratory flow; FEF_25–75, forced midexpiratory flow rate; MEF₅₀, midexpiratory flow rate at 50% vital capacity; MEF₂₅, midexpiratory flow rate at 75% vital capacity.

Discussion

H2S exists in the human blood in two forms: gas and an ion, accounting for 18.5 and 81.5% of H2S, respectively, constituting a dynamic balance (9). Metabolism of H2S in the body is not fully understood. Two main metabolic pathways have been proposed (10): i) H2S is rapidly oxidized into trisulfide in mitochondria and is further converted to sulfite and sulfate, as occurs in the colon; ii) H2S is methylated in the cytoplasm by sulfo-S-methyltransferase and combined with metahemoglobin to form sulfhemoglobin. Despite such uncertainties, the functions of H2S as a signaling molecule within the body are becoming increasingly evident. For example, H2S relaxes vascular smooth muscle, which, as reported by Zhao et al (11), occurs via direct regulation on vascular smooth muscle tone. H2S also inhibits proliferation of vascular smooth muscle cells (12,13). Additionally, conflicting evidence suggests both anti- and pro-inflammatory properties of this compound (14,15), making it of increasing interest for its potential involvement in promoting or preventing disease.

One disease in which H2S may play a preventative role is bronchial asthma. A chronic inflammatory disease of the airway, bronchial asthma involves changes in various cell types, eosinophils, mastocytes, T lymphocytes, neutrophils and airway epithelial cells, and cellular components (15). Of note, vascular smooth muscle changes are involved in the airway remodeling noted in asthma and may suggest a role of H2S in this disease (16,17). In the past 20 years, the incidence of asthma has significantly increased and it has become one of the most common chronic diseases worldwide (18). The pathogenesis of asthma is very complex and has not yet been fully elucidated. Uncovering a role of H2S in asthma pathogenesis may lead to new therapeutic or preventative approaches. Here, we confirmed that H2S concentration in the serum is lower in children with bronchial asthma than in healthy children, which indicates that endogenous H2S may play an anti-inflammatory role in the lung. Endogenous H2S may therefore be useful as a non-invasive indicator for monitoring bronchial asthma.

Pulmonary function is impaired in individuals with bronchial asthma (15,16). Lung function testing is commonly employed in the diagnosis and prognosis of this disease. Our findings of reduced lung function in children with bronchial asthma are not surprising. However, this is the first report of a correlation between indicators of lung function and concentration of endogenous H2S in the serum of patients with bronchial asthma. Increasing serum H2S concentrations corresponded to improved lung function. This result indicates that H2S concentration may predict disease severity. It is possible that endogenous H2S causes bronchoconstriction via effects on smooth muscle of bronchi to influence pulmonary function of both large and small airways. Indeed, a study in a rat model of asthma indicated that endogenous H2S reduced airway remodeling (19). Taken together, these findings suggest that further investigation of endogenous H2S in the context of asthma is warranted.

In conclusion, few studies have focused on the biological effects of endogenous H2S on airway and function in respiratory diseases. Our findings of a correlation between endogenous H2S and lung function indicate that further investigation of this compound in the context of airway disease is required, possibly leading to improvements in the diagnosis and treatment of such diseases.

Acknowledgements

This study was supported by the Nanjing Health Bureau (no. YKK10050).

References