Organic metabolites in exhaled human breath—A multivariate approach for identification of biomarkers in lung disorders

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Abstract

The gas chromatographic profiles of exhaled air from lung cancer patients have been investigated. The breath from healthy volunteers, smokers and non-smokers, and lung cancer patients without treatment and under radio and/or chemotherapy, was collected using Tedlar bags. Different profiles for healthy people and cancer patients could be recognized by multivariate analysis and significant diagnostic compounds could be established. Target compounds showed to be linear and branched hydrocarbons between C14 and C24. Solid phase microextraction (SPME) coupled to gas chromatography mass spectrometry GC–(TOF)-MS was used. The method showed good precision (RSD below 26%) and limit of detection ranged from 0.04 to 8.0 ppb. These findings show a high potential for establishment of laboratorial screening methods. Validation studies in a larger number of patients are being done.

Introduction

Metabolomics is a discipline dedicated to the global study of metabolites, their composition, and responses to interventions or to changes in their environment, in cells, tissues and biofluids [1]. In general terms, biomarkers are indicators of a biological process or perturbation in a complex system as a result of a disease or its progression [2]. Biomarker discovery procedures use, among others, different molecular profiling technologies such as profiles of endogenous metabolites [2]. Some studies suggested that characteristic metabolite patterns could be obtained from patients suffering from different diseases. The ultimate goal is to identify a biomarker or a set of biomarkers with the greatest possibility for the risk assessment (preventive medicine), early diagnosis of a disease, and prognosis [2]. Appropriate biomarkers will also be expected to provide valuable information for selection of a medical treatment or prediction and measures of outcome.

Volatile organic compounds (VOCs) in human breath were identified as early as 1970 [3] as a non-invasive indicator of health. Composition and amount of some of the VOCs in human breath has been related to different diseases [4]. Despite the obvious advantages, only a few breath tests are used in the routine applications: the nitric oxide test to recognize asthma, the evaluation of ethanol concentrations in expiratory gas after alcohol consumption and the 13C-urea breath test for the diagnosis of Helicobacter pylori infection [5], [6], [7]. The most used methods for identifying diseases are computer tomography (CT), magnetic resonance imaging (MRI), endoscopy and ultrasonography together with clinical analysis. However, these methods are time-consuming, unpleasant for patients, require skilled medical staff and expensive devices. There are increasing interests in human breath analysis for clinical diagnostic purposes and therapeutic monitoring [8]. Since 1988, groups of researchers have reported some volatile organic compounds as markers of human metabolic processes, especially lung cancer [8]. A recent study used canine scent detection for early- and late-stage lung and breast cancer and melanoma identification [9]. This work, once more, demonstrated the presence in expired breath of target compounds, potential biomarkers for cancer detection, especially lung cancer, which is a major cause of death among adult and its incidence is increasing globally. A literature survey [8], [10], [11], [12], [13], [14] revealed very different target compounds detected in cancer patients, such as aliphatic and aromatic hydrocarbons, alcohols, aldehydes, acetone, and amines which were detected using different sampling and analyzing methodologies. Therefore, further studies are needed to confirm the usefulness of breath VOCs for detecting lung cancer in general population.

The present work reports on a systematic study of gas chromatographic profiles of metabolites in exhaled human breath by pattern recognition methods. Linear and branched C14–C24 hydrocarbons from exhaled air of lung cancer patients, smokers and non-smokers, were used as profile defining variables. These potential biomarkers of lung cancer were analyzed by headspace solid phase microextraction followed by gas chromatography (time of flight) mass spectrometry (HS-SPME–GC–(TOF)-MS).

Section snippets

Instrumentation and chromatographic conditions

The fiber used in this study, coated with 100 μm of polydimethylsiloxane (PDMS), was purchased from Supelco (Bellefonte, PA, USA). Fibers were initially conditioned according to the manufacturer's instructions (each one was conditioned by inserting it into the GC injector during 30 min at 250 °C) in order to remove contaminants and to stabilize the polymeric phase. For the SPME procedure, standard solutions were placed in 1 L erlenmeyer (Pyrex, France), sealed with PTFE-faced silicone septum

Separation of hydrocarbons

Headspace SPME was used. In order to develop the HS-SPME method for the extraction of target C8–C24 hydrocarbons from exhaled air, the following variables were taken into account: SPME coating, effect of extraction temperature, extraction time, desorption conditions and humidity.

As far as selection of the SPME fiber concerns, the nature of the analytes influences the SPME fiber selection. In this study, two SPME fibers were tested in order to obtain the best extraction efficiency of target

Conclusions

VOCs are exhaled in picomolar concentrations revealing the necessity of collecting and concentrating them before assay. HS-SPME(PDMS)–GC–(TOF)-MS seems to be an adequate method, capable of monitoring profiles and detecting target VOCs, linear and branched hydrocarbons between C14 and C24, with potential to distinguish healthy volunteers from lung cancer patients. Profiles of exhaled air of lung cancer patients indicate an increased lipid peroxidation and oxidative stress.

The results show that

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