A marker of hydroxyl radical damage to DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG); 8-isoprostane, a marker of lipid peroxidation; and 3-nitrotyrosine (3-NT), a product of tyrosine nitration mediated by peroxynitrite, were measured in sputum supernatants using commercially available enzyme immunoassays (Cayman Chemical Co) according to the manufacturer’s instructions. Sputum nitrite levels were measured using the Griess assay. The Meso Scale Discovery platform (Meso Scale Discovery LLC) was used to measure inflammatory mediators in sputum and BAL supernatants. The mediators measured were the proinflammatory cytokines IL-1β, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF); the neutrophil chemokine CXCL8/IL-8; and the protease matrix metalloprotease-9 (MMP-9). Neutrophil elastase was measured using an enzyme-linked immunosorbent assay according to the manufacturers’ instructions (Immunodiagnostik AG). Further details are provided in the e-Appendix .
Protocol detail
Oxidative Stress Markers in Sputum
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3 nitrotyrosine
8 isoprostane
8 ohdg
Assay
Cayman
Cytokines
Enzyme immunoassays
Enzyme linked immunosorbent assay
Granulocyte macrophage colony stimulating factor
Hydroxyl radical
Il 1β
Inflammatory mediators
Lipid peroxidation
Matrix metalloprotease 9
Neutrophil
Neutrophil elastase
Nitration
Nitrite
Peroxynitrite
Protease
Sputum
Tumor necrosis factor α
Tyrosine
Corresponding Organization : Imperial College London
Other organizations : National University Health System
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Variable analysis
independent variables
- None explicitly mentioned
- 8-hydroxy-2'-deoxyguanosine (8-OHdG) (a marker of hydroxyl radical damage to DNA)
- 8-isoprostane (a marker of lipid peroxidation)
- 3-nitrotyrosine (3-NT) (a product of tyrosine nitration mediated by peroxynitrite)
- Sputum nitrite levels
- Proinflammatory cytokines IL-1β, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF)
- Neutrophil chemokine CXCL8/IL-8
- Protease matrix metalloprotease-9 (MMP-9)
- Neutrophil elastase
- None explicitly mentioned
- Positive and negative controls not explicitly mentioned
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