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. 2024 Feb 12;12(1):14.
doi: 10.1186/s40635-024-00600-3.

HS-GC-MS analysis of volatile organic compounds after hyperoxia-induced oxidative stress: a validation study

Thijs A Lilien  1   2 , Dominic W Fenn  3   4 , Paul Brinkman  4 , Laura A Hagens  5 , Marry R Smit  5 , Nanon F L Heijnen  6 , Job B M van Woensel  7 , Lieuwe D J Bos  3   5 , Reinout A Bem  7   8 ; DARTS study group
Collaborators, Affiliations

HS-GC-MS analysis of volatile organic compounds after hyperoxia-induced oxidative stress: a validation study

Thijs A Lilien et al. Intensive Care Med Exp. .

Abstract

Background: Exhaled volatile organic compounds (VOCs), particularly hydrocarbons from oxidative stress-induced lipid peroxidation, are associated with hyperoxia exposure. However, important heterogeneity amongst identified VOCs and concerns about their precise pathophysiological origins warrant translational studies assessing their validity as a marker of hyperoxia-induced oxidative stress. Therefore, this study sought to examine changes in VOCs previously associated with the oxidative stress response in hyperoxia-exposed lung epithelial cells.

Methods: A549 alveolar epithelial cells were exposed to hyperoxia for 24 h, or to room air as normoxia controls, or hydrogen peroxide as oxidative-stress positive controls. VOCs were sampled from the headspace, analysed by gas chromatography coupled with mass spectrometry and compared by targeted and untargeted analyses. A secondary analysis of breath samples from a large cohort of critically ill adult patients assessed the association of identified VOCs with clinical oxygen exposure.

Results: Following cellular hyperoxia exposure, none of the targeted VOCs, previously proposed as breath markers of oxidative stress, were increased, and decane was significantly decreased. Untargeted analysis did not reveal novel identifiable hyperoxia-associated VOCs. Within the clinical cohort, three previously proposed breath markers of oxidative stress, hexane, octane, and decane had no real diagnostic value in discriminating patients exposed to hyperoxia.

Conclusions: Hyperoxia exposure of alveolar epithelial cells did not result in an increase in identifiable VOCs, whilst VOCs previously linked to oxidative stress were not associated with oxygen exposure in a cohort of critically ill patients. These findings suggest that the pathophysiological origin of previously proposed breath markers of oxidative stress is more complex than just oxidative stress from hyperoxia at the lung epithelial cellular level.

Keywords: Headspace gas chromatography–mass spectrometry; Hyperoxia; Intensive care unit; Mechanical ventilation; Oxidative stress; Volatile organic compounds.

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Conflict of interest statement

PB reports grants from Amsterdam UMC (Innovation Impulse grant), Vertex (Vertex Innovation Award), Stichting Astma Bestrijding (SAB grant), Boehringer Ingelheim Grant, Eurostars (Public–Private Partnership grant), Horizon Europe Framework Programme (HORIZON grant) outside the submitted work.

LDJ reports grants from the Dutch lung foundation (Young investigator grant), grants from the Dutch lung foundation and Health Holland (Public–Private Partnership grant), grants from the Dutch lung foundation (Dirkje Postma Award), grants from IMI COVID19 initiative, grants from Amsterdam UMC fellowship, grants from ZonMW (VIDI) outside the submitted work. He has also served in advisory capacity for Sobi NL, Impentri, Novartis, AstraZeneca, CSL Behring and Scailyte with money paid to his institution.

All other authors have no competing interests to disclose.

Figures

Fig. 1
Fig. 1
Hyperoxia-induced cytotoxicity of A549 cells. Relative fold change of interleukin-8 (A) and lactate dehydrogenase (B) supernatant concentrations of control, hyperoxia-exposed and H2O2-exposed cells in comparison to the levels before experimental exposure are shown. Differences were tested by Dunn’s test with Holm’s correction for multiple comparisons
Fig. 2
Fig. 2
Targeted analysis of volatile organic compounds (VOCs) associated with oxidative stress. Volcano plot with the relative change of the median intensity of VOCs from the headspace of hyperoxia-exposed cells compared to controls is shown on the x-axis and the false discovery rate-adjusted P value on the y-axis. VOCs previously associated with oxidative stress that could be identified by gas standard are labelled
Fig. 3
Fig. 3
Target volatile organic compounds (VOCs) associated with oxidative stress. Intensities of identified target VOCs previously associated with oxidative stress per experimental condition, differences were tested by Wilcoxon rank-sum test with adjustment for false discovery rate
Fig. 4
Fig. 4
Untargeted analysis of volatile organic compounds (VOCs). Volcano plot (A) with the relative change of the median intensity of VOCs from the headspace of hyperoxia-exposed cells compared to controls is shown on the x-axis and the false discovery rate-adjusted P value on the y-axis. VOCs with at least a twofold increase of the median and an adjusted P value < 0.05 are labelled and their change per group is shown (B–F)
Fig. 5
Fig. 5
Correlation of volatile organic compounds (VOCs) and clinical oxygen exposure. Spearman’s correlation (ρ) of hexane (A), octane (B), and decane (C) with oxygen exposure in patients on the first measurement day is shown. Quantile regression was used to estimate the median (orange line) VOC intensity and interquartile range (grey lines) as a function of PaO2. Within subject correlation of VOCs and PaO2 over repeated measures is shown in D, 1 represents a strong positive correlation (red) and − 1 a strong negative correlation (blue). **, P < 0.01; ***, P < 0.001
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