Determining the presence of asthma-related molecules and salivary contamination in exhaled breath condensate.
Determining the presence of asthma-related molecules and salivary contamination in exhaled breath condensate.
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DOI：
10.1186/s12931-017-0538-5
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发表时间：
2017-04-12
影响因子：
5.8
通讯作者：
Reisdorph N
中科院分区：
文献类型：
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作者：
Cruickshank-Quinn C;Armstrong M;Powell R;Gomez J;Elie M;Reisdorph N
研究哮喘等肺部疾病的研究人员质疑先前报道的呼出气冷凝物 (EBC) 中的某些化合物是否源自唾液污染。此外，尽管 EBC 在“组学分析研究”中的使用越来越多，但 EBC 的成分在很大程度上仍然没有被表征。本研究旨在通过使用靶向和非靶向质谱法比较 EBC、唾液和唾液污染的 EBC，以及 EBC 样品收集管吸附造成的代谢物损失的可能性，来确定 EBC 在研究肺部疾病中的有用性。使用液相色谱质谱法 (LC-MS) 分析了来自三个不同队列的 133 名个体的样本。使用靶向 LC-MS 测量氨基酸和类二十烷酸（之前在 EBC 和唾液中报道的两类分子）的水平。第 1 组用于检查唾液对 EBC 的污染。第 1 组的样本包括干净的 EBC、唾液污染的 EBC 以及来自 13 名健康志愿者的干净唾液；使用非靶向 LC-MS 分析样品。第 2 组用于比较匹配的 EBC 和从 107 名哮喘受试者收集的唾液中的类二十烷酸水平。使用靶向和非靶向 LC-MS 分析样品。第 3 组样本由从 13 名受试者（包括吸烟者和非吸烟者）收集的清洁 EBC 组成，用于独立确认研究结果；使用靶向 LC-MS、非靶向 LC-MS 和蛋白质组学对样品进行分析。除了人体样本外，还使用内部开发的雾化系统来确定 EBC 样本被唾液污染的可能性。在 EBC 和唾液中检测到的 400 种代谢物中，77 种是 EBC 特有的；然而，EBC 样品浓缩了 20 倍才能达到这一水平的灵敏度。氨基酸浓度范围为 196 pg/mL – 4 µg/mL（干净的 EBC）、1.98 ng/mL – 6 µg/mL（唾液污染的 EBC）和 13.84 ng/mL – 1256 mg/mL（唾液）。类二十烷酸浓度范围降低了一个数量级； 10 pg/mL – 76.5 ng/mL（干净的 EBC）、10 pg/mL – 898 ng/mL（唾液污染的 EBC）和 2.54 ng/mL – 272.9 mg/mL（唾液）。尽管复制队列（队列 3）的样本量不允许进行统计比较，但在吸烟者与非吸烟者 clean-EBC 中检测到了两种蛋白质和 19 种类二十烷酸。我们得出的结论是，EBC 中存在代谢物，并且可以使用 LC-MS 检测到代谢物；然而，需要大量的起始样品体积。本文的在线版本 (doi:10.1186/s12931-017-0538-5) 包含补充材料，可供授权用户使用。
Researchers investigating lung diseases, such as asthma, have questioned whether certain compounds previously reported in exhaled breath condensate (EBC) originate from saliva contamination. Moreover, despite its increasing use in ‘omics profiling studies, the constituents of EBC remain largely uncharacterized. The present study aims to define the usefulness of EBC in investigating lung disease by comparing EBC, saliva, and saliva-contaminated EBC using targeted and untargeted mass spectrometry and the potential of metabolite loss from adsorption to EBC sample collection tubes. Liquid chromatography mass spectrometry (LC-MS) was used to analyze samples from 133 individuals from three different cohorts. Levels of amino acids and eicosanoids, two classes of molecules previously reported in EBC and saliva, were measured using targeted LC-MS. Cohort 1 was used to examine contamination of EBC by saliva. Samples from Cohort 1 consisted of clean EBC, saliva-contaminated EBC, and clean saliva from 13 healthy volunteers; samples were analyzed using untargeted LC-MS. Cohort 2 was used to compare eicosanoid levels from matched EBC and saliva collected from 107 asthmatic subjects. Samples were analyzed using both targeted and untargeted LC-MS. Cohort 3 samples consisted of clean-EBC collected from 13 subjects, including smokers and non-smokers, and were used to independently confirm findings; samples were analyzed using targeted LC-MS, untargeted LC-MS, and proteomics. In addition to human samples, an in-house developed nebulizing system was used to determine the potential for EBC samples to be contaminated by saliva. Out of the 400 metabolites detected in both EBC and saliva, 77 were specific to EBC; however, EBC samples were concentrated 20-fold to achieve this level of sensitivity. Amino acid concentrations ranged from 196 pg/mL – 4 μg/mL (clean EBC), 1.98 ng/mL – 6 μg/mL (saliva-contaminated EBC), and 13.84 ng/mL – 1256 mg/mL (saliva). Eicosanoid concentration ranges were an order of magnitude lower; 10 pg/mL – 76.5 ng/mL (clean EBC), 10 pg/mL – 898 ng/mL (saliva-contaminated EBC), and 2.54 ng/mL – 272.9 mg/mL (saliva). Although the sample size of the replication cohort (Cohort 3) did not allow for statistical comparisons, two proteins and 19 eicosanoids were detected in smoker vs. non-smoker clean-EBC. We conclude that metabolites are present and detectable in EBC using LC-MS; however, a large starting volume of sample is required. The online version of this article (doi:10.1186/s12931-017-0538-5) contains supplementary material, which is available to authorized users.