A sensitive and precise method for simultaneous quantification of cysteinyl leukotrienes (=cys LTs) - leukotriene C4 (=LTC4), leukotriene D4 (=LTD4) and leukotriene E4 (=LTE4) - essential biomarkers of bronchial asthma present in exhaled breath condensate (=EBC) was developed. An immunomagnetic molecular probe was prepared by anchoring cysteinyl leukotrienes antibody on the surface of functionalized monodispersed magnetic particles and used to selectively isolate cys LTs from biological matrices - EBC, plasma and urine. Immobilization and the immunoaffinity capture procedures were optimized to maximize the amount of separated cys LTs, which were detected "off-beads" after acidic elution by UHPLC-ESI-MS/MS operated in a multiple reaction monitoring mode. The developed method was characterized with high precision ≤13.6% (intra-day precision determined as RSD) and ≤14.5% (inter-day precision determined as RSD), acceptable accuracy ≤18.5% (determined as RE), and high recovery of immunoseparation (≥93.1%) in aforementioned biological matrices. The applicability of the method was demonstrated on EBC, plasma and urine clinical samples of patients with various subtypes of bronchial asthma (occupational, steroid-resistant, moderate with and without corticosteroids therapy) and healthy subjects where reasonable differences in cys LTs concentration levels were found. Combining extremely selective immunomagnetic separation with highly sensitive and precise detection step, the developed method was used to aid diagnosis, predict the most effective therapy, and monitor the response to treatment. The detection of elevated inflammatory mediators (cys LTs) in EBC of subjects with relatively asymptomatic asthma and normal pulmonary function tests could offer a novel way for monitoring the lung inflammation and perhaps initiating treatment in an earlier stage.
- MeSH
- bronchiální astma diagnóza farmakoterapie patofyziologie MeSH
- dechové testy MeSH
- glukokortikoidy terapeutické užití MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- imunomagnetická separace metody MeSH
- leukotrien C4 analýza MeSH
- leukotrien D4 analýza MeSH
- leukotrien E4 analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- studie případů a kontrol MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky kontrolované MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
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.
- MeSH
- azbestóza metabolismus radiografie MeSH
- dechové testy * MeSH
- leukotrien B4 analýza krev moč MeSH
- leukotrien C4 analýza krev moč MeSH
- leukotrien D4 analýza krev moč MeSH
- leukotrien E4 analýza krev moč MeSH
- leukotrieny analýza krev moč MeSH
- lidé středního věku MeSH
- lidé MeSH
- respirační funkční testy MeSH
- senioři MeSH
- silikóza metabolismus radiografie MeSH
- stupeň závažnosti nemoci MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- antiflogistika nesteroidní škodlivé účinky terapeutické užití MeSH
- Aspirin škodlivé účinky terapeutické užití MeSH
- bronchiální astma chemicky indukované imunologie patofyziologie MeSH
- kyselina arachidonová metabolismus MeSH
- leukotrien C4 imunologie MeSH
- rinitida chemicky indukované imunologie patofyziologie MeSH
