Headspace VOC analysis of in vitro, ex vivo and biofluid samples

Discovering and validating biomarkers for disease is a key stage in developing valuable and effective diagnostic tests for clinical applications. Do you have a detailed understanding of the biology of your disease of interest? Headspace sampling underpins the biological understanding of disease, supporting the translation of clinically useful biomarkers.

Our headspace sampling pipeline, supported by our Breath Biopsy^® Services, can help you to identify volatile organic compounds (VOCs) produced by your laboratory samples that have the potential to be effective biomarkers for non-invasive clinical breath testing. We provide an optimized detection process supported by expert VOC analysis and identification that provides high confidence candidate biomarker discovery for clinical translation.

Our approach is designed to bridge the gap in biomarker research. Some existing studies have investigated metabolic processes involved in disease but have not identified clinically viable biomarkers. While others have taken an untargeted approach to discovering clinical biomarkers without insight from the underlying biology.

Many studies show that VOCs on breath have the potential to be valuable biomarkers for a range of diseases. The majority of these have hypothesized biological origins for biomarkers but relatively few have directly investigated the relationship between disease biology and biomarker abundance.

Analyzing VOCs produced by in vitro models of disease or drug response can demonstrate mechanistic links between disease biology and biomarker candidates. These can subsequently be verified in clinical studies sampling the same VOCs on breath, providing vital evidence that could bring diagnostic breath tests into clinical practice.

Key features:

• VOC collection using automated HiSorb probes dynamic sampling

• Direct from sample collection to HRAM GC-MS

• Suitable for study of cell cultures, ex vivo tissues, fecal headspace, biofluids and/or macromolecular isolations

• Compare disease site VOCs to breath samples on the same platform

Contact us to discuss integrating Breath Biopsy into your research:

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Aldo-keto reductases (AKRs) are upregulated in some cancers as an adaptation to oxidative stress and lipid peroxidation. We used in vitro cell cultures of lung cancer cell lines to try and target AKRs in these cells and monitor the metabolic processes they are involved in on breath. To do that we modulated the catalytic activity of AKRs using small compounds and also developed knock-out (KO) cells for target AKR genes.

We treated our cell cultures with a range of substrates then monitored substrate and product levels using the same GC-MS workflow used for the detection of VOCs in clinical breath samples (OML Breath Biopsy® workflow). This allowed us to demonstrate that the target enzymes were responsible for the metabolization of the substrates and appeared to confirm that in vitro cell cultures, coupled with VOC analysis can return information about genomic manipulations and drug treatments.

View the lung cancer results

Liver disease

We have used in vitro sampling to identify alterations of metabolic pathways induced by a chronic liver disease. By treating cell cultures of human primary hepatocytes with substrates we were able to use VOC analysis to find alterations in the bioproduct generation of healthy hepatocytes compared to hepatocytes in which a non-alcoholic steatohepatitis (NASH) phenotype was induced.

Using pre-clinical models, like this one, to evaluate the relationship between substrates and metabolism alterations represents a step-forward for the identification and assessment of prospective biomarkers for breath diagnosis of disease.

Read our scientific poster

Inflammatory disease

Lipid peroxidation is a biological mechanism associated with many diseases as well as inflammatory immune responses.

We performed in vitro analysis of lipid peroxidation of various polyunsaturated fatty acids to understand the VOCs produced by each lipid species, finding that each fatty acid produces a different complement of VOCs which can be detected and quantified using the Breath Biopsy Platform.

We have also investigated the VOCs associated with bronchial inflammation.

Using headspace sampling of the air liquid interface assay of in vitro bronchial mucosa samples, VOCs were collected from cell media headspace and bronchial mucus headspace. Inflammation was induced via bleomycin or lipopolysaccharide (LPS) treatment.

Both bleomycin and LPS were shown to produce distinct changes in VOC profiles across two days in both cell media and bronchial mucus. While some of these changes were shared across each treatment there are also differences, likely indicative of differences in inflammation mechanisms.

Analysis of cell medium yielded more detectable VOCs, with a greater distinction between groups and more consistent results across repeats. Significant detected VOCs such as alkanes and aldehydes appeared to relate to well established inflammation responses such as lipid peroxidation.

Further work could verify VOC identities and confirm their origins.

About Lipid Peroxidation

Gastrointestinal (Fecal Headspace Analysis)

The analysis of VOCs collected from fecal headspace is already a well-established practice in gastrointestinal disease research. Conducting analysis of fecal headspace VOCs in parallel to investigations into novel breath-based VOC biomarkers for gastrointestinal disease can be illuminating. Performing cross-matrix data comparisons could help suggest key VOCs that are worth focusing efforts on. Identifying prospective biomarkers that can be strongly corroborated across both matrices can help build confidence for further study.

Fecal headspace VOCs can be collected on sorbent tubes in the same way as breath VOCs are collected and then analyzed using our Breath Biopsy Workflow.

If you’re interested in conducting an in vitro study as part of your breath research, we can help.

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