Occupational asthma follow-up--which markers are elevated in exhaled breath condensate and plasma?

OBJECTIVES: To search for optimal markers in the exhaled breath condensate (EBC), plasma and urine that would reflect the activity/severity of occupational asthma (OA) after the withdrawal from the exposure to the allergen. MATERIAL AND METHODS: Markers of oxidative stress: 8-iso-prostaglandin F2α (8-isoprostane, 8-ISO), malondialdehyde (MDA), 4-hydroxy-trans-2-nonenale (HNE), cysteinyl leukotrienes (LT) and LTB4 were determined using liquid chromatography and mass spectrometry in 43 subjects with immunological OA (49.3 ± 11.8 years), removed from the exposure to the sensitizing agent 10.5 ± 6.5 years ago; and in 20 healthy subjects (49.0 ± 14.9 years). EBC was harvested both before and after the methacholine challenge test. In parallel, identical markers were collected in plasma and urine. The results were analyzed together with forced expiratory volume in one second (FEV1), blood eosinophils, immunoglobulin E (IgE) and eosinophilic cationic protein (ECP) and statistically evaluated (Spearman rank correlation rS, two- or one-sample t tests and alternatively Kruskal Wallis or pair Wilcoxon tests). RESULTS: Several parameters of lung functions were lower in the patients (FEV1% predicted, MEF25% and MEF50%, Rtot%, p < 0.001). Shorter time interval since the removal from the allergen exposure correlated with higher ECP (rS = 0.375) and lower FEV1%, MEF25% and MEF50% after methacholine challenge (rS = -0.404, -0.425 and -0.532, respectively). In the patients, IgE (p < 0.001) and ECP (p = 0.009) was increased compared to controls. In EBC, 8-ISO and cysteinyl LTs were elevated in the asthmatics initially and after the challenge. Initial 8-ISO in plasma correlated negatively with FEV1 (rS = -0.409) and with methacholine PD20 (rS = -0.474). 8-ISO in plasma after the challenge correlated with IgE (rS = 0.396). CONCLUSIONS: The improvement in OA is very slow and objective impairments persist years after removal from the exposure. Cysteinyl LTs and 8-ISO in EBC and 8-ISO in plasma might enrich the spectrum of useful objective tests for the follow-up of OA.

Leukotrienes B4, C4, D4 and E4 in the exhaled breath condensate (EBC), blood and urine in patients with pneumoconiosis.

Leukotrienes (LTs) are involved in the pathogenesis of lung fibrosis and were increased in exhaled breath condensate (EBC) of the patients with pneumoconiosis. However the possible influence of extra-pulmonary disorders on the EBC markers is not known. Therefore in parallel with EBC, LTs' levels in the plasma and urine were measured in patients with pneumoconiosis (45 × asbestos exposure, 37 × silica exposure) and in 27 controls. Individual LTs B4, C4, D4 and E4 were measured by liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). In EBC, LT D4 and LT E4 were increased in both groups of patients (p<0.001 and p<0.05), comparing with the controls. Both LT B4 and cysteinyl LTs were elevated in asbestos-exposed subjects (p<0.05). Asbestosis with more severe radiological signs (s1/s2-t3/u2) and lung functions impairment has shown higher cysteinyl LTs and LT C4 in the EBC (p<0.05) than mild asbestosis (s1/s0-s1/s1). In addition, in the subjects with asbestosis, cysteinyl LTs in EBC correlated with TLC (-0.313, p<0.05) and TLCO/Hb (-0.307, p<0.05), and LT C4 with TLC (-0.358, p<0.05). In pneumoconioses, EBC appears the most useful from the 3 fluids studied.

Determination of cysteinyl leukotrienes in exhaled breath condensate: method combining immunoseparation with LC-ESI-MS/MS.

A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.

Occupational hypersensitivity pneumonitis reported to the Czech National Registry Of Occupational Diseases in the period 1992-2005.

Between 1992 and 2005, 72 cases of occupational hypersensitive pneumonitis were reported to the Czech National Registry of Occupational Diseases. This represented 0.24% of all occupational diseases reported in the Czech Republic during that period. The greatest number of cases occurred in 1997 (10 cases), of which men constituted 58.3% (42 cases) and women 41.7% (30 cases). The most prevalent cases were farmer's lung (50 cases), malt worker's lung (7 cases) and chemical worker's lung (6 cases). Agriculture was the most common economic activity (total 48 cases), with cattleman and dairyman (total 26 cases) the most frequent occupations; less common were tractor driver (8 cases) and maltster (7 cases). Typical case reports after different exposures are presented. A peak in frequency was observed in the age groups of 45-49 and 50-54 yr (20.8% and 19.4% of cases, respectively) and within the first four years of employment (22.2% of cases). Median age was 48 yr and median exposure 12.5 yr. Incidence was in the range of 0.00-0.20 per 100,000 workers, which appears rather low. Due to the difficult diagnostics of hypersensitive pneumonitis, the actual number of cases is undoubtedly higher, and this disease is probably under-reported.

Rapid and easy method for monitoring oxidative stress markers in body fluids of patients with asbestos or silica-induced lung diseases.

Sensitive assay method was developed for a parallel, rapid and precise determination of the most prominent oxidative stress biomarkers: 8-iso-prostaglandin F(2alpha), malondialdehyde and 4-hydroxynonenal. The method consisted of a pre-treatment part a solid-phase extraction, for rapid and effective isolation of biomarkers from body fluids (exhaled breath condensate, plasma and urine) and the detection method LC-ESI-MS/MS, where the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the imprecision was below 14.3%, the mean inaccuracy was determined to be lower than 13.1%. The method was tested on samples obtained from patients diagnosed with asbestosis, pleural hyalinosis or silicosis, i.e. occupational lung diseases caused by fibrogenic dusts, inducing oxidative stress in the respiratory system, and then compared to samples from healthy subjects. The difference in concentration levels of biomarkers between the two groups was perceptible in all the body fluids (the difference observed in an exhaled breath condensate was statistically most significant).

Increased 8-isoprostane, a marker of oxidative stress in exhaled breath condensate in subjects with asbestos exposure.

Asbestosis and pleural plaques exhibit unpredictable but progressive development, and there are no markers routinely available to measure their prognosis. Asbestos exposure induces the generation of reactive oxygen species, and 8-isoprostane is involved in experimental asbestos-related lung toxicity. This oxidative stress marker was measured in exhaled breath condensate (EBC) in 92 former asbestos workers with mean age 68.8+/-1.7 yr and mean duration of asbestos exposure 24.1+/-2.0 yr. The control group had 46 subjects with mean age 65.2+/-3.3 yr. The mean level of 8-isoprostane, analyzed by liquid chromatography-electrospray ionization-mass spectrometry, was higher in asbestos-exposed subjects (69.5+/-6.6 pg/ml, p=0.0001) compared with the control group, where the concentration was 47.0+/-7.8 pg/ml. The results presented support the hypothesis that oxidative stress due to asbestos is the main cause of increased 8-isoprostane in EBC. Measurement of 8-isoprostane in EBC is a promising non-invasive means for assessing the activity of asbestos-induced diseases.

Lung function impairment in relation to asbestos-induced pleural lesions with reference to the extent of the lesions and the initial parenchymal fibrosis.

OBJECTIVES: The study focused on the effect of pleural lesions on the lung function of asbestos-exposed workers. METHODS: A clinical check-up, chest radiography, high-resolution computed tomography (HRCT), and lung function testing were performed on 162 asbestos-exposed workers without any sign of parenchymal fibrosis on their chest radiographs. According to the HRCT scans, two subgroups were delineated, 97 subjects with pleural lesions and 65 referents without pleural lesions. Four categories of pleural lesions were specified according to the extent. Parenchymal changes, if identified on the HRCT scans, were recorded. RESULTS: The radiographic sensitivity and specificity for pleural lesion detection when compared with that of HRCT were 64.9% and 98.5%, respectively. The HRCT scans showed parenchymal abnormalities in 46.3% of the participants, more frequently in those with pleural lesions (67.0% versus 15.4%, P<0.0001). After the effect of parenchymal fibrosis was taken into account, pleural lesions were found to have a significant effect on the decrease in total lung capacity, forced vital capacity, and forced expiratory volume in 1 second, if being classified into category 2 or higher. CONCLUSIONS: Pleural lesions proved to have a negative effect on lung function, depending on their extent. The effect of the initial parenchymal fibrosis detectable in the HRCT scans only was also significant. A normal chest radiograph does not exclude the presence of pleural lesions or initial parenchymal fibrosis, with a possible negative effect on lung function.

Asbestos exposure and antineutrophil cytoplasmic Antibody (ANCA) positivity.

The authors used indirect immunofluorescence to examine the association of antineutrophil cytoplasmic antibodies (ANCAs) with exposure to asbestos among 61 asbestos-exposed patients (mean exposure = 24.6 yr) and 39 nonexposed controls. ANCA positivity was detected significantly more frequently (p = 0.034) in the asbestos-exposed group (21.3%) than in the control group (5.1%). ANCA-associated diseases did not occur more frequently among subjects exposed previously to asbestos than among unexposed controls. These findings confirmed that exposure to asbestos is another occupational factor, as is silica exposure, that is associated with ANCA positivity. The influence of asbestos appears stronger than that of silica because ANCA positivity was found among subjects who had histories of exposure to asbestos but who did not exhibit typical radiographic signs of asbestosis on their chest x-rays. Additional stimuli may be necessary to induce systemic vasculitis in asbestos-exposed persons.