Mass spectrometry in the clinical laboratory. A short journey through the contribution to the scientific literature by CCLM.

Mass spectrometry (MS) has been a gold standard in the clinical laboratory for decades. Although historically refined to limited areas of study such as neonatal screening and steroid analysis, technological advancements in the field have resulted in MS becoming more powerful, versatile, and user-friendly than ever before. As such, the potential for the technique in clinical chemistry has exploded. The past two decades have seen advancements in biomarker detection for disease diagnostics, new methods for protein measurement, improved methodologies for reliable therapeutic drug monitoring, and novel technologies for automation and high throughput. Throughout this time, Clinical Chemistry and Laboratory Medicine has embraced the rapidly developing field of mass spectrometry, endeavoring to highlight the latest techniques and applications that have the potential to revolutionize clinical testing. This mini review will highlight a selection of these critical contributions to the field.

The Impact of a Graded Maximal Exercise Protocol on Exhaled Volatile Organic Compounds: A Pilot Study.

Exhaled volatile organic compounds (VOCs) are of interest due to their minimally invasive sampling procedure. Previous studies have investigated the impact of exercise, with evidence suggesting that breath VOCs reflect exercise-induced metabolic activity. However, these studies have yet to investigate the impact of maximal exercise to exhaustion on breath VOCs, which was the main aim of this study. Two-litre breath samples were collected onto thermal desorption tubes using a portable breath collection unit. Samples were collected pre-exercise, and at 10 and 60 min following a maximal exercise test (VO2MAX). Breath VOCs were analysed by thermal desorption-gas chromatography-mass spectrometry using a non-targeted approach. Data showed a tendency for reduced isoprene in samples at 10 min post-exercise, with a return to baseline by 60 min. However, inter-individual variation meant differences between baseline and 10 min could not be confirmed, although the 10 and 60 min timepoints were different (p = 0.041). In addition, baseline samples showed a tendency for both acetone and isoprene to be reduced in those with higher absolute VO2MAX scores (mL(O2)/min), although with restricted statistical power. Baseline samples could not differentiate between relative VO2MAX scores (mL(O2)/kg/min). In conclusion, these data support that isoprene levels are dynamic in response to exercise.

Association of gut-related metabolites with outcome in acute heart failure.

BACKGROUND: Trimethylamine N-oxide (TMAO), a gut-related metabolite, is associated with heart failure (HF) outcomes. However, TMAO is the final product of a complex metabolic pathway (ie, choline/carnitine) that has never been entirely investigated in HF. The present study investigates a panel of metabolites involved in the TMAO-choline/carnitine metabolic pathway for their associations with outcome in acute HF patients. METHODS: In total, 806 plasma samples from acute HF patients were analyzed for TMAO, trimethyllysine, L-carnitine, acetyl-L-carnitine, γ-butyrobetaine, crotonobetaine, trimethylamine, betaine aldehyde, choline, and betaine using a developed liquid chromatography-tandem mass spectrometry method. Associations with outcome of all-cause mortality (death) and a composite of all-cause mortality and/or rehospitalization caused by HF (death/HF) at 30 days and 1 year were investigated. RESULTS: TMAO, trimethyllysine, L-carnitine, acetyl-L-carnitine, and γ-butyrobetaine were associated with death and death/HF at 30 days (short term; hazard ratio 1.30-1.49, P≤ .021) and at 1 year (long term; hazard ratio 1.15-1.25, P≤ .026) when adjusted for cardiac risk factors. L-carnitine and acetyl-L-carnitine were superior for short-term outcomes whereas TMAO was the superior metabolite for association with long-term outcomes. Furthermore, acetyl-L-carnitine and L-carnitine were superior for in-hospital mortality and improved risk stratification when combined with current clinical risk scores (ie, Acute Decompensated HEart Failure National REgistry, Organized Program To Initiate Lifesaving Treatment In Hospitalized Patients With Heart Failure, and Get With The Guidelines-Heart Failure; odds ratio (OR) ≥ 1.52, P≤ .020). CONCLUSIONS: Carnitine-related metabolites show associations with adverse outcomes in acute HF, in particular L-carnitine and acetyl-L-carnitine for short-term outcomes, and TMAO for long-term outcomes. Further studies are warranted to investigate the role and implications of carnitine metabolites including intervention in the pathogenesis of HF.

Metabolomic analysis reveals reliance on secondary plant metabolites to facilitate carnivory in the Cape sundew, Drosera capensis.

BACKGROUND AND AIMS: Secondary metabolites are integral to multiple key plant processes (growth regulation, pollinator attraction and interactions with conspecifics, competitors and symbionts) yet their role in plant adaptation remains an underexplored area of research. Carnivorous plants use secondary metabolites to acquire nutrients from prey, but the extent of the role of secondary metabolites in plant carnivory is not known. We aimed to determine the extent of the role of secondary metabolites in facilitating carnivory of the Cape sundew, Drosera capensis. METHODS: We conducted metabolomic analysis of 72 plants in a time-series experiment before and after simulated prey capture. We used ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and the retention time index to identify compounds in the leaf trap tissue that changed up to 72 h following simulated prey capture. We identified associated metabolic pathways, and cross-compared these compounds with metabolites previously known to be involved in carnivorous plants across taxa. KEY RESULTS: For the first time in a carnivorous plant, we have profiled the whole-leaf metabolome response to prey capture. Reliance on secondary plant metabolites was higher than previously thought - 2383 out of 3257 compounds in fed leaves had statistically significant concentration changes in comparison with unfed controls. Of these, ~34 compounds are also associated with carnivory in other species; 11 are unique to Nepenthales. At least 20 compounds had 10-fold changes in concentration, 12 of which had 30-fold changes and are typically associated with defence or attraction in non-carnivorous plants. CONCLUSIONS: Secondary plant metabolites are utilized in plant carnivory to an extent greater than previously thought - we found a whole-metabolome response to prey capture. Plant carnivory, at the metabolic level, likely evolved from at least two distinct functions: attraction and defence. Findings of this study support the hypothesis that secondary metabolites play an important role in plant diversification and adaptation to new environments.

The Athlete and Gut Microbiome: Short-chain Fatty Acids as Potential Ergogenic Aids for Exercise and Training.

Short-chain fatty acids (SCFAs) are metabolites produced in the gut via microbial fermentation of dietary fibers referred to as microbiota-accessible carbohydrates (MACs). Acetate, propionate, and butyrate have been observed to regulate host dietary nutrient metabolism, energy balance, and local and systemic immune functions. In vitro and in vivo experiments have shown links between the presence of bacteria-derived SCFAs and host health through the blunting of inflammatory processes, as well as purported protection from the development of illness associated with respiratory infections. This bank of evidence suggests that SCFAs could be beneficial to enhance the athlete's immunity, as well as act to improve exercise recovery via anti-inflammatory activity and to provide additional energy substrates for exercise performance. However, the mechanistic basis and applied evidence for these relationships in humans have yet to be fully established. In this narrative review, we explore the existing knowledge of SCFA synthesis and the functional importance of the gut microbiome composition to induce SCFA production. Further, changes in gut microbiota associated with exercise and various dietary MACs are described. Finally, we provide suggestions for future research and practical applications, including how these metabolites could be manipulated through dietary fiber intake to optimize immunity and energy metabolism.

Applying mass spectrometry-based assays to explore gut microbial metabolism and associations with disease.

The workings of the gut microbiome have gained increasing interest in recent years through the mounting evidence that the microbiota plays an influential role in human health and disease. A principal focus of this research seeks to further understand the production of metabolic by-products produced by bacteria resident in the gut, and the subsequent interaction of these metabolites on host physiology and pathophysiology of disease. Gut bacterial metabolites of interest are predominately formed via metabolic breakdown of dietary compounds including choline and ʟ-carnitine (trimethylamine N-oxide), amino acids (phenol- and indole-containing uremic toxins) and non-digestible dietary fibers (short-chain fatty acids). Investigations have been accelerated through the application of mass spectrometry (MS)-based assays to quantitatively assess the concentration of these metabolites in laboratory- and animal-based experiments, as well as for direct circulating measurements in clinical research populations. This review seeks to explore the impact of these metabolites on disease, as well as to introduce the application of MS for those less accustomed to its use as a clinical tool, highlighting pertinent research related to its use for measurements of gut bacteria-mediated metabolites to further understand their associations with disease.

The "olfactory fingerprint": can diagnostics be improved by combining canine and digital noses?

A sniffer (detecting) dog is conventionally defined as an animal trained to use its olfactory perceptions for detecting a vast array of substances, mostly volatile organic compounds (VOCs), including those exceptionally or exclusively generated in humans bearing specific pathologies. Such an extraordinary sniffing performance translates into the capability of detecting compounds close to the femtomolar level, with performance comparable to that of current mass spectrometry-based laboratory applications. Not only can dogs accurately detect "abnormal volatilomes" reflecting something wrong happening to their owners, but they can also perceive visual, vocal and behavioral signals, which altogether would contribute to raise their alertness. Although it seems reasonable to conclude that sniffer dogs could never be considered absolutely "diagnostic" for a given disorder, several lines of evidence attest that they may serve as efficient screening aids for many pathological conditions affecting their human companions. Favorable results have been obtained in trials on cancers, diabetes, seizures, narcolepsy and migraine, whilst interesting evidence is also emerging on the capability of early and accurately identifying patients with infectious diseases. This would lead the way to proposing an "olfactory fingerprint" loop, where evidence that dogs can identify the presence of human pathologies provides implicit proof of the existence of disease-specific volatilomes, which can be studied for developing laboratory techniques. Contextually, the evidence that specific pathologies are associated with abnormal VOC generation may serve as reliable basis for training dogs to detect these compounds, even (or especially) in patients at an asymptomatic phase.

Evidence for alternative exhaled elimination profiles of disinfection by-products and potential markers of airway responses to swimming in a chlorinated pool environment.

Chlorine-based disinfectants protect pool water from pathogen contamination but produce potentially harmful halogenated disinfection by-products (DBPs). This study characterized the bioaccumulation and elimination of exhaled DBPs post-swimming and investigated changes in exhaled breath profiles associated with chlorinated pool exposure. Nineteen participants provided alveolar-enriched breath samples prior to and 5, 90, 300, 510, and 600 minutes post-swimming. Known DBPs associated with chlorinated water were quantitated by thermal desorption-gas chromatography-mass spectrometry. Two distinct exhaled DBP elimination profiles were observed. Most participants (84%) reported peak concentrations immediately post-swimming that reduced exponentially. A sub-group exhibited a previously unobserved and delayed washout profile with peak levels at 90 minutes post-exposure. Metabolomic investigations tentatively identified two candidate biomarkers associated with swimming pool exposure, demonstrating an upregulation in the hours after exposure. These data demonstrated a hitherto undescribed exhaled DBP elimination profile in a small number of participants which contrasts previous findings of uniform accumulation and exponential elimination. This sub-group which exhibited delayed peak-exhaled concentrations suggests the uptake, processing, and immediate elimination of DBPs are not ubiquitous across individuals as previously understood. Additionally, non-targeted metabolomics highlighted extended buildup of compounds tentatively associated with swimming in a chlorinated pool environment that may indicate airway responses to DBP exposure.

Physical activity and lipidomics in a population at high risk of type 2 diabetes mellitus.

The aim was to investigate how measurements of the lipidome differ according to the level and intensity of physical activity in a population at high risk of type 2 diabetes (T2DM). A targeted metabolomics platform provided quantitative molecular data on lipid species. Linear regression examined the associations between plasma lipid concentrations, particle size and time spent in objectively measured physical activity intensity domains, in increments of 500 counts per minute (cpm) (up to >4500 cpm (~>5.6METs)). Results are presented as % difference in the concentration (lower/higher) or particle size (smaller/larger) per 10 min of activity within each intensity. Five hundred and nine participants were included. Time spent in the lowest physical activity intensity domain (<500 cpm) was unfavourably associated with VLDL (2%), HDL (-2%) and Apolipoprotein A-1 particle concentrations (-2%) and HDL diameter (-2%). Conversely, time spent in intensities ≥1000 cpm were favourably associated with HDL subclass concentrations; with stronger associations seen at moderate intensities (2000-3999 cpm (~4.5METs)). For Apolipoprotein-B concentration and VLDL particle concentration and size, a negative association was consistently observed at the highest physical activity intensity only. If these associations are causal, HDL subclasses appear sensitive to light-intensities whereas only the high category of physical activity intensity was consistently associated with VLDL subclasses.

Non-targeted metabolomics in sport and exercise science.

Metabolomics incorporates the study of metabolites that are produced and released through physiological processes at both the systemic and cellular levels. Biological compounds at the metabolite level are of paramount interest in the sport and exercise sciences, although research in this field has rarely been referred to with the global 'omics terminology. Commonly studied metabolites in exercise science are notably within cellular pathways for adenosine triphosphate production such as glycolysis (e.g., pyruvate and lactate), ß-oxidation of free fatty acids (e.g., palmitate) and ketone bodies (e.g., ß-hydroxybutyrate). Non-targeted metabolomic technologies are able to simultaneously analyse the large numbers of metabolites present in human biological samples such as plasma, urine and saliva. These analytical technologies predominately employ nuclear magnetic resonance spectroscopy and chromatography coupled to mass spectrometry. Performing experiments based on non-targeted methods allows for systemic metabolite changes to be analysed and compared to a particular physiological state (e.g., pre-/post-exercise) and provides an opportunity to prospect for metabolite signatures that offer beneficial information for translation into an exercise science context, for both elite performance and public health monitoring. This narrative review provides an introduction to non-targeted metabolomic technologies and discusses current and potential applications in sport and exercise science.

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.

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.

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 µmol/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.

Prognostic Role of Molecular Forms of B-Type Natriuretic Peptide in Acute Heart Failure.

BACKGROUND: B-type natriuretic peptide (BNP) molecular forms 5-32, 4-32, and 3-32 are known to be present in the circulation of heart failure (HF) patients. This study investigated the prognostic role of circulating BNP molecular forms on risk prediction for patients with acute HF. METHODS: BNP molecular forms were measured in plasma using an immunocapture MALDI-TOF-mass spectrometry (MS) method. Associations of molecular BNP forms with adverse outcome of all-cause mortality (death) and a composite of all-cause mortality and rehospitalization due to HF (death/HF) at 6 months and 1 year were investigated. RESULTS: BNP molecular forms 5-32, 4-32, and 3-32 were detected in 838 out of 904 patient samples. BNP molecular forms were all able to independently predict death and death/HF at 6 months and 1 year. BNP 5-32 was the superior form with strongest predictive qualities for death at 6 months [adjusted hazard ratio (HR) 1.31, P = 0.005] and 1 year (adjusted HR 1.29, P = 0.002) and death/HF at 1 year (adjusted HR 1.18, P = 0.011). BNP 5-32, 4-32, and 3-32 showed decreased survival rates across increasing tertiles of circulating concentrations (P ≤ 0.004). BNP molecular forms showed prognostic ability comparable with conventional BNP measurements across all end points (P = 0.002-0.032 vs P = 0.014-0.039, respectively) and reduced associations with renal dysfunction (blood urea; Spearman correlation rs = 0.187-0.246 vs rs = 0.369, respectively). CONCLUSIONS: BNP molecular forms, notably BNP 5-32, showed association with poor prognosis at 6 months and 1 year in patients with acute HF. This is the first study reporting the prognostic ability of molecular BNP forms in HF patients and demonstrated comparable qualities to conventional BNP measurements.

Advances in quadrupole and time-of-flight mass spectrometry for peptide MRM based translational research analysis.

The application of unit resolution tandem quadrupole and high-resolution orthogonal acceleration ToF mass spectrometers for the quantitation and translational analysis of proteolytic peptides is described. The MS platforms were contrasted in terms of sensitivity and linear response. Moreover, the selectivity of the platforms was investigated and the effect on quantitative precision studied. Chromatographic LC conditions, including gradient length and configuration, were investigated with respect to speed/throughput, while minimizing isobaric interferences, thereby providing information with regard to practical sample cohort size limitations of LC-MS for large cohort experiments. In addition to these fundamental analytical performance metrics, precision and linear dynamic ranges were also studied. An LC-MS configuration that encompasses the best combination of throughput and analytical accuracy for translational studies was chosen, despite the MS platforms giving similar quantitative performance, and instances were identified where alternative combinations were found to be beneficial. This configuration was utilized to demonstrate that proteolytically digested nondepleted samples from heart failure patients could be classified with good discriminative power using a subset of proteins previously suggested as candidate biomarkers for cardiovascular diseases.

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.