Brooks Wendy M, Lash Heath, Kettle Anthony J, Epton Michael J
Canterbury Respiratory Research Group, Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, New Zealand.
Redox Rep. 2006;11(2):78-84. doi: 10.1179/135100006X101011.
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.
To explore factors that affect accurate quantitation of H2O2 in EBC.
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.
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.
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.
呼出气冷凝物（EBC）分析已被提议作为评估气道病理的一种非侵入性方法。已在EBC中测量了多种物质，包括过氧化氢（H2O2），但关于验证和优化的详细公开信息不足。
探讨影响EBC中H2O2准确定量的因素。
使用4-羟基苯乙酸荧光法测量EBC样本中的H2O2。研究了一些可能改变定量的因素，包括pH和缓冲条件、试剂储存以及测定温度。
标准曲线斜率受pH显著影响，导致H2O2定量的潜在差异高达42%。通过增加反应混合物的缓冲能力解决了这些差异。添加到EBC中的H2O2在冰上储存时1小时内保持稳定。冷冻测定试剂对测定无影响。根据所用缓冲液的不同，H2O2的检测限在3.4 nM至8.8 nM之间。
该测定所需的试剂可储存数月，从而在纵向研究中实现有价值的一致性。EBC中H2O2的定量取决于pH，但增加测定缓冲可降低这种影响。EBC中H2O2的灵敏可重复定量需要严格优化。
