Preservation of exhaled breath samples for analysis by off-line SESI-HRMS: proof-of-concept study.

Secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) is an established technique in the field of breath analysis characterized by its short analysis time, as well as high levels of sensitivity and selectivity. Traditionally, SESI-HRMS has been used for real-time breath analysis, which requires subjects to be at the location of the analytical platform. Therefore, it limits the possibilities for an introduction of this methodology in day-to-day clinical practice. However, recent methodological developments have shown feasibility on the remote sampling of exhaled breath in Nalophan® bags prior to measurement using SESI-HRMS. To further explore the range of applications of this method, we conducted a proof-of-concept study to assess the impact of the storage time of exhaled breath in Nalophan® bags at different temperatures (room temperature and dry ice) on the relative intensities of the compounds. In addition, we performed a detailed study of the storage effect of 27 aldehydes related to oxidative stress. After 2 h of storage, the mean of intensity of allm/zsignals relative to the samples analyzed without prior storage remained above 80% at both room temperature and dry ice. For the 27 aldehydes, the mean relative intensity losses were lower than 20% at 24 h of storage, remaining practically stable since the first hour of storage following sample collection. Furthermore, the mean relative intensity of most aldehydes in samples stored at room temperature was higher than those stored in dry ice, which could be related to water vapor condensation issues. These findings indicate that the exhaled breath samples could be preserved for hours with a low percentage of mean relative intensity loss, thereby allowing more flexibility in the logistics of off-line SESI-HRMS studies.

Real-Time Volatile Metabolomics Analysis of Dendritic Cells.

Dendritic cells (DCs) actively sample and present antigen to cells of the adaptive immune system and are thus vital for successful immune control and memory formation. Immune cell metabolism and function are tightly interlinked, and a better understanding of this interaction offers potential to develop immunomodulatory strategies. However, current approaches for assessing the immune cell metabolome are often limited by end-point measurements, may involve laborious sample preparation, and may lack unbiased, temporal resolution of the metabolome. In this study, we present a novel setup coupled to a secondary electrospray ionization-high resolution mass spectrometric (SESI-HRMS) platform allowing headspace analysis of immature and activated DCs in real-time with minimal sample preparation and intervention, with high technical reproducibility and potential for automation. Distinct metabolic signatures of DCs treated with different supernatants (SNs) of bacterial cultures were detected during real-time analyses over 6 h compared to their respective controls (SN only). Furthermore, the technique allowed for the detection of 13C-incorporation into volatile metabolites, opening the possibility for real-time tracing of metabolic pathways in DCs. Moreover, differences in the metabolic profile of naïve and activated DCs were discovered, and pathway-enrichment analysis revealed three significantly altered pathways, including the TCA cycle, α-linolenic acid metabolism, and valine, leucine, and isoleucine degradation.

Rapid detection of Staphylococcus aureus and Streptococcus pneumoniae by real-time analysis of volatile metabolites.

Early detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment of infections. In this study, we show that secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) can be used as a diagnostic tool for rapid detection of bacterial infections as a supportive system for current state-of-the-art diagnostics. Volatile organic compounds (VOCs) produced by growing S. aureus or S. pneumoniae cultures on blood agar plates were detected within minutes and allowed for the distinction of these two bacteria on a species and even strain level within hours. Furthermore, we obtained a fingerprint of clinical patient samples within minutes of measurement and predominantly observed a separation of samples containing live bacteria compared to samples with no bacterial growth. Further development of this technique may reduce the time required for microbiological diagnosis and should help to improve patient's tailored treatment.

Suitability evaluation of new endogenous biomarkers for the identification of nitrite-based urine adulteration in mass spectrometry methods.

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5-hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO2 ). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO2 concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and - 20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after biomarker quantification (n = 100 per group). Blinded measurements (n = 50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH 4-5). Storage at -20°C ensured sufficient stability for all oxidative markers over one month. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO2 and are ready for implementation into routine MS procedures.

A new metabolomics-based strategy for identification of endogenous markers of urine adulteration attempts exemplified for potassium nitrite.

Urine adulteration to circumvent positive drug testing represents a problem for toxicological laboratories. While creatinine is a suitable marker for dilution, detection of chemicals is often performed by dipstick tests associated with high rates of false positives. Several methods would be necessary to check for all possible adulterants. Untargeted mass spectrometry (MS) methods used in metabolomics should theoretically allow detecting concentration changes of any endogenous urinary metabolite or presence of new biomarkers produced by chemical adulteration. As a proof of concept study, urine samples from 10 volunteers were treated with KNO2 and analyzed by high-resolution MS. For statistical data evaluation, XCMSplus and MetaboAnalyst were used. Compound identification was performed by database searches using an in-house database, Chemspider, METLIN, HMDB, and NIST. Principle component analysis revealed clear separation between treated and untreated urine samples. In detail, 307 features showed significant concentration changes with fold changes greater than 2 (79 decreased; 228 increased). Mainly amino acids (e.g., histidine, methylhistidine, di- and trimethyllysine) and purines (uric acid) were detected in lower amounts. 5-HO-isourate was found to be formed as a new compound from uric acid and, e.g., imidazole lactate concentrations increased due to the breakdown of histidine. This metabolomics-based strategy allowed for a broad identification range of markers of urinary adulteration. More studies will be needed to investigate routine applicability of identified potential markers exploring urinary conditions of their formation and stability. Selected markers might then be integrated into routine MS screening procedures allowing for detection of adulteration within routine MS analysis. Graphical Abstract ᅟ.

Parelaphostrongylus tenuis-associated meningoencephalitis in a sika deer (Cervus nippon).

An adult, female, free-ranging, sika deer (Cervus nippon yakushimae) from Wicomico County, Maryland, USA, was found circling and having no fear of humans. The animal was euthanized and submitted for a postmortem exam. There were no gross lesions and the deer was negative for rabies. Microscopic examination revealed lymphoplasmacytic, neutrophilic, and eosinophilic meningoencephalitis with intralesional adult nematodes, larvae, and eggs consistent with nematodes in the family Protostrongylidae. Parelaphostrongylus tenuis was identified by polymerase chain reaction and DNA sequencing. To our knowledge, this is the first report of P. tenuis-associated encephalitis in a sika deer.