Quantitative detection of formaldehyde using solid phase microextraction gas chromatography-mass spectrometry coupled to cysteamine scavenging.

Formaldehyde (HCHO) is a toxic and carcinogenic pollutant and human metabolite that reacts with biomolecules under physiological conditions. Quantifying HCHO is essential for ongoing biological and biomedical research on HCHO; however, its reactivity, small size and volatility make this challenging. Here, we report a novel HCHO detection/quantification method that couples cysteamine-mediated HCHO scavenging with SPME GC-MS analysis. Our NMR studies confirm cysteamine as an efficient and selective HCHO scavenger that out-competes O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, the most commonly used scavenger, and forms a stable thiazolidine amenable to GC-MS quantification. Validation of our GC-MS method using FDA and EMA guidelines revealed detection and quantification limits in the nanomolar and micromolar ranges respectively, while analysis of bacterial cell lysate confirmed its applicability in biological samples. Overall, our studies confirm that cysteamine scavenging coupled to SPME GC-MS analysis provides a sensitive and chemically robust method to quantify HCHO in biological samples.

Association of gut-related metabolites with respiratory symptoms in COVID-19: A proof-of-concept study.

Gut-related metabolites have been linked with respiratory disease. The crosstalk between the gut and lungs suggests that gut health may be compromised in COVID-19. The aims of the present study were to analyze a panel of gut-related metabolites (acetyl-L-carnitine, betaine, choline, L-carnitine, trimethylamine, and trimethylamine N-oxide) in patients with COVID-19, matched with healthy individuals and patients with non-COVID-19 respiratory symptoms. As results, metabolites from this panel were impaired in patients with COVID-19 and were associated with the symptoms of breathlessness and temperature, and it was possible to differentiate between COVID-19 and asthma. Preliminary results showed that lower levels of betaine appeared to be associated with poor outcomes in patients with COVID-19, suggesting betaine as a marker of gut microbiome health.

Household Air Pollution and Respiratory Symptoms a Month Before and During the Stringent COVID-19 Lockdown Levels 5 and 4 in South Africa.

Background: Household air pollution (HAP) is associated with adverse human health impacts. During COVID-19 Lockdown Levels 5 and 4 (the most stringent levels), South Africans remained at home, potentially increasing their exposure to HAP. Objectives: To investigate changes in fuel use behaviours/patterns of use affecting HAP exposure and associated HAP-related respiratory health outcomes during COVID-19 Lockdown Levels 5 and 4. Methods: This was a cross-sectional online and telephonic survey of participants from an existing database. Logistic regression and McNemar's test were used to analyse household-level data. Results: Among 2 505 participants, while electricity was the main energy source for cooking and heating the month before and during Lockdown Levels 5 and 4, some households used less electricity during Lockdown Levels 5 and 4 or switched to "dirty fuels." One third of participants reported presence of environmental tobacco smoke in the home, a source of HAP associated with respiratory illnesses. Prevalence of HAP-related respiratory health outcomes were <10% (except dry cough). Majority of households reported cooking more, cleaning more and spending more time indoors during Lockdown Levels 5 and 4 - potentially exposed to HAP. Conclusion: Should South Africa return to Lockdown Levels 5 or 4, awareness raising about the risks associated with HAP as well as messaging information for prevention of exposure to HAP, including environmental tobacco smoke, and associated adverse health impacts will be necessary.

Diagnosis of COVID-19 by exhaled breath analysis using gas chromatography-mass spectrometry.

BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic has claimed over two and a half million lives worldwide so far. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is perceived to be seasonally recurrent, and a rapid noninvasive biomarker to accurately diagnose patients early on in their disease course will be necessary to meet the operational demands for COVID-19 control in the coming years. OBJECTIVE: The aim of this study was to evaluate the role of exhaled breath volatile biomarkers in identifying patients with suspected or confirmed COVID-19 infection, based on their underlying PCR status and clinical probability. METHODS: A prospective, real-world, observational study was carried out, recruiting adult patients with suspected or confirmed COVID-19 infection. Breath samples were collected using a standard breath collection bag, modified with appropriate filters to comply with local infection control recommendations, and samples were analysed using gas chromatography-mass spectrometry (TD-GC-MS). RESULTS: 81 patients were recruited between April 29 and July 10, 2020, of whom 52 out of 81 (64%) tested positive for COVID-19 by reverse transcription-polymerase chain reaction (RT-PCR). A regression analysis identified a set of seven exhaled breath features (benzaldehyde, 1-propanol, 3,6-methylundecane, camphene, beta-cubebene, iodobenzene and an unidentified compound) that separated PCR-positive patients with an area under the curve (AUC): 0.836, sensitivity: 68%, specificity: 85%. CONCLUSIONS: GC-MS-detected exhaled breath biomarkers were able to identify PCR-positive COVID-19 patients. External replication of these compounds is warranted to validate these results.

Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

Automating and Extending Comprehensive Two-Dimensional Gas Chromatography Data Processing by Interfacing Open-Source and Commercial Software.

Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful analytical tool for both nontargeted and targeted analyses. However, there is a need for more integrated workflows for processing and managing the resultant high-complexity datasets. End-to-end workflows for processing GC×GC data are challenging and often require multiple tools or software to process a single dataset. We describe a new approach, which uses an existing underutilized interface within commercial software to integrate free and open-source/external scripts and tools, tailoring the workflow to the needs of the individual researcher within a single software environment. To demonstrate the concept, the interface was successfully used to complete a first-pass alignment on a large-scale GC×GC metabolomics dataset. The analysis was performed by interfacing bespoke and published external algorithms within a commercial software environment to automatically correct the variation in retention times captured by a routine reference standard. Variation in 1tR and 2tR was reduced on average from 8 and 16% CV prealignment to less than 1 and 2% post alignment, respectively. The interface enables automation and creation of new functions and increases the interconnectivity between chemometric tools, providing a window for integrating data-processing software with larger informatics-based data management platforms.

Breath analysis by two-dimensional gas chromatography with dual flame ionisation and mass spectrometric detection - Method optimisation and integration within a large-scale clinical study.

Precision medicine has spurred new innovations in molecular pathology leading to recent advances in the analysis of exhaled breath as a non-invasive diagnostic tool. Volatile organic compounds (VOCs) detected in exhaled breath have the potential to reveal a wealth of chemical and metabolomic information. This study describes the development of a method for the analysis of breath, based on automated thermal desorption (TD) combined with flow modulated comprehensive two-dimensional gas chromatography (GC×GC) with dual flame ionisation and quadrupole mass spectrometric detection (FID and qMS). The constrained optimisation and analytical protocol was designed to meet the practical demands of a large-scale multi-site clinical study, while maintaining analytical rigour to produce high fidelity data. The results demonstrate a comprehensive method optimisation for the collection and analysis of breath VOCs by GC×GC, integral to the standardisation and integration of breath analysis within large clinical studies.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Erratum: Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

This corrects the article DOI: 10.1038/nature23481.

Real-time multi-marker measurement of organic compounds in human breath: towards fingerprinting breath.

The prospects for exploiting proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) in medical diagnostics are illustrated through a series of case studies. Measurements of acetone levels in the breath of 68 healthy people are presented along with a longitudinal study of a single person over a period of 1 month. The median acetone concentration across the population was 484 ppbV with a geometric standard deviation (GSD) of 1.6, whilst the average GSD during the single subject longtitudinal study was 1.5. An additional case study is presented which highlights the potential of PTR-ToF-MS in pharmacokinetic studies, based upon the analysis of online breath samples of a person following the consumption of ethanol. PTR-ToF-MS comes into its own when information across a wide mass range is required, particularly when such information must be gathered in a short time during a breathing cycle. To illustrate this property, multicomponent breath analysis in a small study of cystic fibrosis patients is detailed, which provides tentative evidence that online PTR-ToF-MS analysis of tidal breath can distinguish between active infection and non-infected patients.

Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage.

The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G(2)/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G(2)/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G(2)/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53(-)/(-), HCT116p21(-)/(-), HT-29 and SW480). Flow cytometry confirmed that these lines underwent G(2)/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5-10 µM). In all eight lines, the majority of this arrest occurred at the G(2)/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G(2)/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage.

Detection of acetaldehyde derived N(2)-ethyl-2'-deoxyguanosine in human leukocyte DNA following alcohol consumption.

Epidemiological studies have shown an association between alcohol (ethanol) consumption and increased cancer risk. The effect of alcohol consumption on the levels and persistence of N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) formed by acetaldehyde, the oxidative metabolite of ethanol, in human leukocyte DNA was investigated. DNA was isolated from venous blood samples obtained from 30 male non-smoking individuals before consumption of alcohol (0h) and subsequently at 3-5h following the consumption of 150mL of vodka (containing 42% pure ethanol). Additional samples were collected 24h and 48h post-alcohol consumption. The levels of N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) in the DNA were determined following reduction of N(2)-ethylidene-dG with sodium cyanoborohydride using a liquid chromatography-tandem mass spectrometry selected reaction monitoring method. A slight time-dependent trend showing an increase and decrease in the levels of N(2)-ethyl-dG was observed following consumption of alcohol compared to time 0h, however, the differences were not statistically significant. The average levels of N(2)-ethyl-dG observed at 0h, 3-5h, 24h and 48h time points following ingestion of alcohol were 34.6±21.9, 35.1±21.0, 36.8±20.7 and 35.6±21.1 per 10(8) 2'-deoxynucleosides, respectively. In conclusion, alcohol consumption that could be encountered under social drinking conditions, does not significantly alter the levels of the acetaldehyde derived DNA adduct, N(2)-ethyl-dG in human leukocyte DNA from healthy individuals.

Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column-switching liquid chromatography/tandem mass spectrometry.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software-based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH-modified DNA samples were obtained by reaction of the anti-dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2'-deoxynucleosides. Positive LC/electrospray ionisation (ESI)-MS/MS collision-induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2'-deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2'-deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH-adducted 2'-deoxynucleosides was achieved using online column-switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H](+) to [M+H-116](+) transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H-116](+) transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods.

A novel mechanism of vasoregulation: ADP-induced relaxation of the porcine isolated coronary artery is mediated via adenosine release.

In this study, we have investigated the mechanism of ADP-induced relaxation of porcine coronary artery (PCA) rings. The P2Y receptor agonists ADP and ADPbetaS produced concentration-dependent relaxation of endothelium-denuded PCA smooth muscle with pD2 values of 5.3 and 4.9, respectively. RT-polymerase chain reaction (RT-PCR) and immunoblotting demonstrated mRNA and protein expression of P2Y1 and A2A adenosine receptors in the PCA. The nonselective P2 antagonist PPADS or the P2Y1-selective antagonist MRS2179 failed to alter ADP- or ADPbetaS-induced relaxations. Relaxations to ADP were, however, blocked by the A2A adenosine receptor-selective antagonists ZM241385 and SCH58261 (apparent pK(B) values of 9.2 and 8.9, respectively). We excluded roles for direct occupancy of A2A adenosine receptors by ADP or ADPbetaS as well as metabolism to adenosine as mechanisms for ADP-evoked relaxations. However, ADP responses were significantly enhanced in the presence of the ENT1 nucleoside transporter inhibitors dipyridamole and NBTI and were significantly inhibited by adenosine deaminase, indicating a role for extracellular adenosine. Suprafusion of [3H]-adenine-labeled PCA segments showed that ADP induced the release of a number of purines, including adenosine. These data suggest that ADP mediates relaxation of the PCA via a novel mechanism that involves adenine nucleotide-evoked adenosine release and the subsequent activation of A2A receptors.