EIA and GC/MS analysis of 8-isoprostane in EBC of children with problematic asthma

Abstract

Asthmatic airways are characterised by enhanced oxidative stress, which can be studied by measuring biomarkers, such as 8-isoprostane. The aims of the present study were: 1) to measure the concentrations of 8-isoprostane in exhaled breath condensate (EBC) and urine of children with problematic and well-controlled asthma; 2) to compare the concentrations of 8-isoprostane measured by gas chromatographic/negative ion chemical ionisation mass spectrometry (GC/NICI-MS) and by an enzymatic immunoassay (EIA).We recruited 20 asthmatic allergic children, 13 with well-controlled asthma and seven with problematic asthma. They underwent exhaled nitric oxide measurements and spirometry, and both EBC and urine samples were collected. 8-isoprostane was measured in EBC by GC/NICI-MS and EIA.8-isoprostane concentrations in EBC were significantly higher in children with problematic asthma than in children with well-controlled asthma (p50.01). An acceptable reproducibility emerged between GC/NICI-MS and EIA (coefficient of reproducibility 11.5 pg?mL -1 ). 8-isoprostane levels measured in urine did not correlate with those measured in EBC.We showed that 8-isoprostane in EBC was significantly increased in children with problematic asthma, suggesting a role for oxidative stress in this asthma phenotype. In addition we found an acceptable reproducibility of EIA compared to GC/NICI-MS, even if the latter method had higher accuracy.

Evidence of unexpected oxidative stress in airways of adolescents born very pre-term

“…The lowest detection limit was 3.7 pg?mL -1 . For samples below the lowest detection limit, data were arbitrarily expressed as half the detection limit [23]. This assay was previously validated using a reference analytical method (gas chromatographic/negative ion chemical ionisation mass spectrometry) [23].…”

“…For samples below the lowest detection limit, data were arbitrarily expressed as half the detection limit [23]. This assay was previously validated using a reference analytical method (gas chromatographic/negative ion chemical ionisation mass spectrometry) [23]. The repeatability of the EIA 8-isoprostane measurements in EBC on two different days had previously been tested at our laboratory, showing a coefficient of repeatability of 3.2 pg?mL -1 .…”

An update on paediatric asthma

“…Leukotriene B 4 , prostaglandin E2 and 8-isoprostane in breath condensate from the nose and the mouth were analysed in an attempt to study the relationship between persistent allergic rhinitis and asthma; possible correlations were reported [94]. In two other paediatric studies, eoxin and eotaxin were measured in EBC along with 8-isoprostane, all three were significantly increased in children with problematic severe asthma [95,96]. Finally, a new and advanced approach to utilising EBC was reported by BLOEMEN et al [97].…”

Assessment of problematic severe asthma in children: Figure 1–

“…Few, if any, EBC biomarkers have been validated for clinical use, and considerable methodological challenges remain [74]. A recent study [75] demonstrated an increased level of EBC 8-isoprostane in children with problematic asthma suggesting a role for oxidative stress in this asthma phenotype.…”