Identification of oxidized phospholipids in bronchoalveolar lavage exposed to low ozone levels using multivariate analysis

Abstract

Chemical reactions with unsaturated phospholipids in the respiratory tract lining fluid have been identified as one of the first important steps in the mechanisms mediating environmental ozone toxicity. As a consequence of these reactions, complex mixtures of oxidized lipids are generated in the presence of mixtures of non-oxidized naturally occurring phospholipid molecular species, which challenge methods of analysis. Untargeted mass spectrometry and statistical methods were employed to approach these complex spectra. Human bronchoalveolar lavage (BAL) was exposed to low levels of ozone, and samples with and without derivatization of aldehydes were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry. Data processing was carried out using principal component analysis (PCA). Resulting PCA scores plots indicated an ozone dose-dependent increase, with apparent separation between BAL samples exposed to 60 ppb ozone and non-exposed BAL samples as well as a clear separation between ozonized samples before and after derivatization. Corresponding loadings plots revealed that more than 30 phosphatidylcholine (PC) species decreased due to ozonation. A total of 13 PC and 6 phosphatidylglycerol oxidation products were identified, with the majority being structurally characterized as chain-shortened aldehyde products. This method exemplifies an approach for comprehensive detection of low-abundance, yet important, components in complex lipid samples.

Keywords: Lung; Mass spectrometry; Oxidized lipids; Ozone; Principal component analysis; Pulmonary surfactant.

Figure 1

Elution profiles of m/z 732 in an untreated (blue trace) and m/z 650 in an ozonized (red trace) BAL sample in positive mode. The ozonized sample was exposed to 300 ppb ozone for 60 min. then subjected to LC-MS analysis (positive electrospray ionization) and these ion traces extracted from the LC-MS data.

Figure 2

Principal component analysis of ozonized samples (duplicate samples, n=16) with and without derivatization analyzed by LC-MS/MS (precursors of m/z 184). A) Score plot for samples analyzed in full scan positive ion mode. White squares represent non-ozonized samples, circles represent samples exposed to 50 ppb ozone, diamonds 150 ppb and filled in squares 300 ppb, respectively. Symbols in red represent samples treated with methoxylamine. Here, the first principal component (PC1) separated the samples according to ozone exposure and the second principal component (PC2) separated the untreated samples and samples treated with methoxylamine. PC1 explained 65.8% and PC2 20.1% of the variation in the data. B) Loadings plot showing all variables explaining the separation of samples in the score plot. Principal component variable grouping (PCVG) groups the variables that display the same behavior across samples and was here based on the first 3 principal components, together explaining 91.4 % of the variation in the data. Labeled variables show the m/z and retention time.

Figure 3

Analysis of a novel product observed in human surfactant after exposure to 300 ppb ozone for 60 min. (A) The elution profile of m/z 808.8 obtained in LC-MS/MS analysis revealed two closely eluting components at 27.2 and 28.2 min. (B) Collison induced decomposition of m/z 808 [M+H]⁺ from the component eluting at 28.2 min yielded an abundant product ion corresponding to phosphocholine (m/z 184) as well as product ions consistent with cleavage of an ozonide of 16:0_18:1-ozonide PC where the double bond is at carbon-9. The earlier eluting component had an identical product ion spectrum.