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Background: Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H2O2), have been measured in EBC, without adequate published details of validation and optimisation., Objectives: To explore factors that affect accurate quantitation of H2O2 in EBC., Materials and Methods: H2O2 was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature., Results: Standard curve slope was significantly altered by pH, leading to a potential difference in H2O2 quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H2O2 added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H2O2 ranged from 3.4 nM to 8.8 nM depending on the buffer used., Conclusions: The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H2O2 in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H2O2 in EBC requires rigorous optimisation.

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1 x 10(6) M(-1)s(-1)), it reacts sluggishly with compound II (kII 7 M(-1)s(-1)). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 microM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 microM) unless superoxide dismutase was present (I50 5 microM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. This heme enzyme uses hydrogen peroxide and chloride to catalyze the production of hypochlorous acid, which is the major strong oxidant generated by neutrophils in appreciable amounts. In addition to chlorination, myeloperoxidase displays several other activities. It readily oxidizes thiocyanate to hypothiocyanite, converts a myriad of organic substrates to reactive free radicals, and hydroxylates aromatic compounds. Depending on the concentration of its competing substrates and the conditions of the local environment, myeloperoxidase could substantially affect oxidant production by neutrophils. Superoxide is undoubtedly a physiological substrate for myeloperoxidase. Its interactions with the enzyme are key factors in determining how neutrophils use superoxide to kill pathogens and promote inflammatory tissue damage. Superoxide modulates the chlorination and peroxidation activities of myeloperoxidase. It also reacts with the enzyme to form oxymyeloperoxidase which is catalytically active and hydroxylates phenolic substrates. Myeloperoxidase reacts rapidly with nitric oxide and peroxynitrite so that at sites of inflammation there is a strong possibility that these reactions will impact on oxidative damage caused by neutrophils. Under certain conditions, many substrates of myeloperoxidase act as inhibitors and regulate oxidant production by the enzyme. Given the numerous reactions of myeloperoxidase, all its activities should be considered when assessing the injurious oxidants produced by neutrophils.