Philadelphia Convention Center, Philadelphia, Pennsylvania, USA
Abstract Content
More than 15,000 people died due to influenza during winter 2017/18 in the United States. Recognition of virus presence and early diagnosis of infection is not possible with existing methods. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive detection of diseases. Lung infections can be expected to impact strongly onto exhaled VOC profiles. Breath samples were taken from 3 control animals and 10 animals infected intranasally with Influenza A over 30 days. For breath sampling under high safety conditions, a special sampling device and protocol was developed. Samples were taken with a glass syringe in the alveolar phase of expiration after placing the pigs in a canvas sling. VOCs were preconcentrated onto polymer needle traps. After thermal desorption, VOCs were separated and identified by means of gas chromatography mass spectrometry. White blood cells, temperature, lung pathology, and clinical scores were determined additionally. Six compounds increased significantly in pigs' breath on day 4, when Influenza A virus was detected in pigs' nasal cavity. On this day, virus positive animals showed no clinical symptoms or hematological changes. After pigs' recovery, exhaled VOCs went back to pre-infection levels. Using these six VOCs, infected and non-infected animals and virus positive and negative days in the infected animals could be separated clearly from each other by means of principal component analysis. Reproducible breath sampling from spontaneously breathing pigs was realized under high safety conditions. VOCs mirrored virus presence in pigs' nasal cavity despite lack of clinical symptoms. Thus, non-invasive breath analysis could enable early detection of the virus and could be used for large-scale screening purposes for human medicine, animal health control or border protection.
Oral Sessions
Author Name:
Ann-Christin Bischoff
Session Title:
Expanding Areas of Interest in Mass Spectrometry
Event Type:
Oral Sessions
Event Title:
The Volatile Fingerprint of Stem Cell Differentiation
Philadelphia Convention Center, Philadelphia, Pennsylvania, USA
Abstract Content
Analysis of volatile organic compounds (VOCs) in the headspace of cell cultures has the potential to provide additional information on cell status beyond classical biochemical methods. Especially the metabolic and morphological changes of stem cells during differentiation might influence and change VOC fingerprints.
VOC profiles were determined by means of needle trap micro extraction (NTME) coupled with gas chromatography and mass spectrometry (GC-MS) and GCxGC-ToF-MS. Neuronal stem cells isolated from 19 day old rat embryos were grown on petri dishes to a semi confluent status in proliferation media. Then, medium was changed to differentiation stimulating media. Cells were differentiated for 3 weeks and headspace samples were taken in triplicates two times per week. Headspace analysis was performed in a hermetically closed system made out of Teflon®. A negative control with pure cell culture medium was measured in parallel.
Variance of the analytical method could be reduced below 10% with the new sampling system. 30 different substances were detected by means of GC-MS and about 200 substances could be detected by means of GCxGC-ToF-MS. Concentrations of certain substances changed significantly during the differentiation process.
These changes could be linked to the specific metabolic activities of the cells, since the number of mitochondria increased and metabolism changed to higher respiratory chain activity during the differentiation. Substances emitted from stem cell cultures have the potential to mirror metabolism and differentiation status in a non-destructive way.
Poster Sessions and Flash Talks
Author Name:
Julia Bartels
Session Title:
Biomedical Methods
Event Type:
Poster Sessions
Event Title:
Volatile Profiles Emitted From Proliferating and Differentiating Human Mesenchymal Stem Cells
Philadelphia Convention Center, Philadelphia, Pennsylvania, USA
Abstract Content
Stem cells have the potential to differentiate into various specialized cell types. The cellular differentiation requires adaptation of biosynthetic pathways and changes of metabolic processes. Volatile organic compounds (VOCs) in headspace over cell cultures have been described to mirror adjustments of cell metabolism. VOC patterns may, therefore, help to recognize changes in the metabolic pathways of stem cells in a non-destructive way. This study was intended to investigate VOC profiles from human adipose tissue-derived mesenchymal stem/stromal cells (adMSC) during proliferation and differentiation.
Human adMSC were proliferated and cultured with adipogenic stimulation over a period of 3 weeks. VOC profiles were determined by means of needle trap micro extraction (NTME) coupled with gas chromatography and mass spectrometry. Samples were taken in triplicate from the headspace over cultures on day 1, 7, 14 and 21 of proliferation and differentiation. A hermetically closed Teflon® system was used to perform the headspace analysis over in vitro cultures. Pure medium samples were analyzed in parallel as a negative control.
More than 20 substances were identified in the culture samples. Variance among the samples analyzed in triplicate was below 10%. VOC profiles changed with ongoing differentiation. The degree of differentiation was qualitatively verified by means of immune histological staining.
NTD-GC-MS analysis in combination with an adapted inert sampling system allows trace analysis of VOCs of human cultures with minimal interference of the investigated in vitro system. Changing VOC concentrations could be correlated to the differentiation process of human adMSC. VOC analysis from stem cells has great potential to provide additional information on the metabolic changes during cell differentiation in a non-invasive and destruction-free way.
Poster Sessions and Flash Talks
Author Name:
Radost Saß
Session Title:
Biomedical Methods
Event Type:
Poster Sessions
Event Title:
Trace VOC Profiles Emitted from Human Cells Change after (Co-)Infection with Virus and Bacteria
Philadelphia Convention Center, Philadelphia, Pennsylvania, USA
Abstract Content
Co-infections with viruses and bacteria represent a common problem in clinical medicine. E.g., a considerable part of mortality from influenza is due to bacterial co-infection. Non-invasive methods to recognize (co-)infections could promote understanding of pathomechanisms and help to improve diagnostics and therapy control. Bacteria and cells are known to emit volatile organic compounds (VOCs). This study was intended to gather basic knowledge on volatile emissions from (co-)infected cell cultures.
Human cells were stored in a custom made inert sampling box and infected with a virus followed by bacterial inoculation. Headspace above these cells was analyzed at different time points after inoculation. VOCs in the samples were pre-concentrated by means of triple bed needle trap devices (NTDs). Substances were thermally desorbed from the NTDs and analyzed by means of GC-MS. Media and pure cell samples, incubated under identical conditions as co-infected cells, were taken as controls.
The NTD-GC-MS assay provided reliable quantitation of VOCs down to the pptV level. Concentrations of several compounds in the headspace over human cells changed significantly during (co-)infection with virus and bacteria when compared to pure cell cultures. One substance increased when cells were infected with virus. Concentrations of two compounds increased after bacterial inoculation.
NTD-GC-MS analysis in combination with an adapted inert sampling system allows trace analysis of VOC's of human cell cultures with minimal interference of investigated in vitro system. Co-infection with virus and bacteria had distinct effects onto concentrations of volatile organic compounds in the headspace over infected human cells. In a perspective, VOC analysis may be used for the characterization of viral-bacterial co-infections in living organisms.
Poster Sessions and Flash Talks
Author Name:
Giovanni Pugliese
Session Title:
Mass Spectrometric Methods
Event Type:
Poster Sessions
Event Title:
Real-Time Analysis of Nitrogen Containing Compounds Under Varying Nutrition
Co-Authors
Brock, Beate, Miekisch, Wolfram, Phillip, Trefz, Schubert, Jochen K
Affiliation:
Rostock University Medical Center
Date:
Wednesday, March 20, 2019
Start Time:
10:00 am
Location:
Philadelphia Convention Center, Philadelphia, Pennsylvania, USA
Abstract Content
Nitrogen containing molecules represent an important class of volatile compounds in breath which can be related to disease, metabolic processes and bacterial activity. Real time analysis of these compound is hampered through their high reactivity. As they can react with any surface of sampling or calibration devices reliable detection and calibration are difficult.
We developed and optimized a PTR-Tof-MS based analytical method for real-time determination of N-containing compounds.
Gaseous standard solutions were prepared using a liquid calibration unit (LCU). PTR-Tof-MS conditions were optimized in terms of inlet flow, temperature, drift temperature, drift pressure and electric field. We carried out experiments at different temperatures and pH and tested different materials for LCU and PTR inlets.
Using inert materials, high temperature and pH 7.4 compounds reactivity on the surfaces of the analytical system was considerably reduced. Under these conditions, calibrations showed good linearity with correlation coefficients > 0.99. LODs ranged from 0.15 ppbV to 63 ppbV and LOQs from 0.24 ppbV to 1.94 ppbV.
In a proof of concept study, we applied this method for breath-resolved determination of nitrogen containing compounds in 10 healthy subjects after consuming a protein meal. For each volunteer, we analyzed breath every 30 minutes for 5 hours.
Methylamine, acetone and indole concentrations increased significantly after protein intake.
The newly developed method reliably enables real-time determination of nitrogen containing compounds in human breath. Breath VOC concentration changes may provide a non-invasive insight into protein metabolism.
