Exhaled breath analysis in exercise and health

Research in the field of exhaled breath analysis is developing rapidly and is currently focussed on disease diagnosis and prognosis. The ability to identify early onset of life-threatening diseases, by a subtle change in exhaled profile that is picked up through a non-invasive measure, is of clinical interest. However, implementation of exhaled breath analysis can extend further beyond disease diagnosis and/or management. Using a non-invasive and rapid sample collection with high sensitivity, breath analysis may be seen to have potential benefit to the wider community. This research describes preliminary investigations into exhaled breath in exercise-based scenarios that aims to translate current breath analysis methodologies into a sport and exercise medicine context. An adaptive absorbent-based breath sampling methodology was used to collect a total of 220 breath samples from 54 participants over 3 studies. Breath volatiles were analysed using thermal desorption-gas chromatography-mass spectrometry. Data were analysed with targeted, and multivariate metabolomics-based approaches. Potential health impacts to high performance and recreational swimmers exposed to chlorinated water was studied. Following preliminary and scoping studies, 19 participants were sampled before a 30 min swim, and a further 5 times for 10 hrs after swimming. Environmental and control samples were also collected. Concentrations of chlorine-based disinfection by-products were observed to increase by up to a median of 121-fold, and take up to 8.5 hrs to return to pre-swimming levels. Metabolomic profiling identified the monoterpene geranylacetone to be a discriminant variable in samples taken 10 hrs after swimming. Geranylacetone is associated with membranes and extracellular fluids and an upregulated trend was observed across the five sampling time points post-swimming. Further research with an appropriately stratified and powered cohort (n=38) was recommended. The effects of intense exercise on breath profiles was explored for the possible use of breath analysis for exercise science with elite performance-based medicine. Twenty-nine participants provided exhaled breath samples before undergoing a maximal oxygen uptake (fitness) test and then provided 2 additional samples over the following 1 hr period. High and low fitness groupings, deemed by oxygen uptake values, were compared for exhaled metabolites. Lower exhaled acetone and isoprene were observed in participants with greater absolute oxygen uptake leading to a hypothesis for a non-invasive breath based fitness test. Finally, an interface for breath-by-breath analysis using a transportable mass spectrometer was developed. A controlled change in exhaled profiles was achieved through the ingestion of a peppermint oil capsule. Menthone was measured on-line and monitored for up to 10 hrs post-administration. Sixteen participants enabled the system to be demonstrated as exhaled menthone was at elevated concentrations for at least 6 hrs. Validation against thermal desorption-gas chromatography-mass spectrometry confirmed the system to be detecting metabolites at the sub-µg L-1 range

Clinical mass spectrometry in heart disease

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Translation of exhaled breath volatile analyses to sport and exercise applications

Exhaled breath gases are becomingly increasingly investigated for use as non-invasive measurements for clinical diagnosis, prognosis and therapeutic monitoring. Exhaled volatile organic compounds (VOCs) in the breath, which make up the exhaled volatilome, offer a rich sample medium that provides both information to external exposures as well as endogenous metabolism. For these reasons, exhaled breath analyses can be extended further beyond disease-based investigations, and used for wider biomarker measurement purposes. The use of a rapid, non-invasive (and potentially non-physically demanding) test in an exercise and/or sporting situation may provide additional information for translation to performance sport, recreational exercise/fitness and clinical exercise health. This review intends to provide an overview into the initial exploration of exhaled VOC measurements in sport and exercise science, and understand the current limitations in knowledge and instrumentation that have restricted these methodologies in becoming common practice

In reply: The emerging value of molecular forms of B-type natriuretic peptide in heart failure

We would like to commend Drs Yang and Wang for their forward-thinking perspectives and positive implications of the clinical measurements of molecular forms of B-type natriuretic peptide (BNP) (1). We share the view that these processed forms of BNP can provide beneficial clinical information for cardiovascular disease prognoses and therapeutic monitoring with an emphasis on personalized medicine. We have previously demonstrated the prognostic capabilities of BNP molecular forms in the acute setting through measurement in acute heart failure (HF) patients (2), and support the further investigation of these forms across cardiovascular conditions

Proteomic Biomarkers of Heart Failure

Heart failure (HF) is associated with significant morbidity and mortality. Biomarkers are used to assist clinicians with timely diagnosis, prognosis, and risk prediction of patients for personalized treatment. Using modern proteomic methods such as mass spectrometry, an increasing number of novel biomarkers have been identified that further aid clinicians in the early diagnosis and outcome prediction of HF. This article focuses on the array of common and novel protein-based biomarkers that provide diagnostic and prognostic information in HF

Mass spectrometry in medicine: a technology for the future?

This editorial looks to highlight the major advantages of MS (Mass Spectrometry) in the clinical laboratory, explain the major hurdles in place and provide an insight into the potential applications for clinical biomarker analysis

B-type natriuretic peptide molecular forms for risk stratification and prediction of outcome after acute myocardial infarction

Background: B-type natriuretic peptide (BNP) is known to be a risk marker following acute myocardial infarction (MI). More recently, truncated molecular forms of the BNP molecule have been identified, with the association of these forms and outcome in acute MI not known. The present study investigated their use as risk stratifying biomarkers of this condition. Methods: BNP molecular forms (BNP 5–32, BNP 4–32 and BNP 3–32) were measured in plasma from 1078 acute MI patients using immunocapture followed by MALDI-ToF-mass spectrometry. Associations of molecular forms with short-term and long-term adverse outcomes were assessed. Results: BNP molecular forms were independent predictors of mortality/reinfarction, mortality/rehospitalization due to heart failure, and a composite of all events at 6 months, 1 year and 2 years and showed prognostic ability comparable with conventional BNP measurements (P < .001–0.026 vs. N-terminal [NT]-proBNP P < .001–0.020, respectively). Reclassification analyses showed BNP molecular forms successfully reclassified patient risk when used in addition to the GRACE clinical risk score (P ≤ .005). BNP 5–32 showed utility as a secondary risk stratification biomarker when used in combination with the GRACE score and NT-proBNP by successful down-classification of high-risk patients. Conclusions: BNP molecular forms were associated with poor prognosis at 6 months, 1 year and at 2 years in patients with acute MI. BNP 5–32 showed successful utility as a secondary marker in combination with NT-proBNP after GRACE scoring. This study suggests a potential role for BNP molecular forms in prognosis and risk stratification after acute MI

Trimethylamine N-oxide and Risk Stratification after Acute Myocardial Infarction

Background: Risk stratification in acute myocardial infarction (MI) remains a clinical challenge. Trimethylamine N-oxide (TMAO), a gut-derived metabolite, was investigated for its ability to assist in risk stratification for acute MI hospitalizations. Methods: TMAO was analyzed in 1079 acute MI patients. Associations with adverse outcome of all-cause mortality or reinfarction (death/MI) for shorter (6-month) and longer (2-year) terms were assessed and compared to other cohort-specific biomarkers. Added value in risk stratification by combined use with the Global Registry of Acute Coronary Events (GRACE) score was also investigated. Results: TMAO independently predicted death/MI at 2 years [292 events, hazard ratio 1.21 (95% CI, 1.03–1.43), P = 0.023], but was not able to predict death/MI at 6 months (161 events, P = 0.119). For death/MI at 2 years, TMAO retained independent prediction of risk (P = 0.034) and improved stratification even after addition of multiple alternative and contemporary biomarkers previously shown to provide added prognostic value in this cohort. From these contemporary biomarkers, TMAO remained the only significant predictor of outcome. Further, TMAO improved risk stratification for death/MI at 6 months by down-classifying risk in patients with GRACE score >119 and plasma TMAO concentration ≤3.7 μ mol/L. Conclusions: TMAO levels showed association with poor prognosis (death/MI) at 2 years and superiority over contemporary biomarkers for patients hospitalized due to acute MI. Furthermore, when used with the GRACE score for calculating risk at 6 months, TMAO reidentified patients at lower risk after initial categorization into a higher-risk group and showed usefulness as a secondary risk stratification biomarker

In Reply

Response to Letter to the Editor ‘Predictive Value of NT-proBNP in Patients with Acute Myocardial Infarction’; Regarding Article ‘Trimethylamine N-oxide and Risk Stratification after Acute Myocardial Infarction

High mass accuracy assay for trimethylamine N-oxide using stable-isotope dilution with liquid chromatography coupled to orthogonal acceleration time of flight mass spectrometry with multiple reaction monitoring

Trimethylamine N-oxide (TMAO) has attracted interest as circulating levels have reported prognostic value in patients with cardiovascular conditions, such as heart failure. With continual advances in accurate mass measurements, robust methods that can employ the capabilities of time of flight mass spectrometers would offer additional utility in the analysis of complex clinical samples. A Waters Acquity UPLC was coupled to a Waters Synapt G2-S high-resolution mass spectrometer. TMAO was measured in plasma by stable-isotope dilution-hydrophilic interaction liquid chromatography-time of flight mass spectrometry with multiple reaction monitoring (LC-ToF-MRM). Two transitions were monitored: m/z 76.1 to 58.066/59.073 and m/z 85.1 to 66.116/68.130. The method was assessed for linearity, lower limits of detection and quantitation, and reproducibility. A selected cohort of patients with systolic heart failure (SHF; n = 43) and healthy controls (n = 42) were measured to verify the assay is suitable for the analysis of clinical samples. Quantitative analysis of TMAO using LC-ToF-MRM enabled linearity to be established between 0.1 and 75 μmol/L, with a lower limit of detection of 0.05 μmol/L. Relative standard deviations reported an inter-day variation of ≤20.8 % and an intra-day variation of ≤11.4 % with an intra-study quality control variation of 2.7 %. Run times were 2.5 min. Clinical application of the method reported that TMAO in SHF was elevated compared to that of healthy controls (p < 0.0005). LC-ToF-MRM offers a highly selective method for accurate mass measurement of TMAO with rapid and reproducible results. Applicability of the method was shown in a selected cohort of patient samples