Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.
Objective
To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.
Methods
Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.
Results
Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.
Conclusion
Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.
Data processing is one of the biggest problems in metabolomics, given the high number of samples analyzed and the need of multiple software packages for each step of the processing workflow.
Objectives
Merge in the same platform the steps required for metabolomics data processing.
Methods
KniMet is a workflow for the processing of mass spectrometry-metabolomics data based on the KNIME Analytics platform.
Results
The approach includes key steps to follow in metabolomics data processing: feature filtering, missing value imputation, normalization, batch correction and annotation.
Conclusion
KniMet provides the user with a local, modular and customizable workflow for the processing of both GC–MS and LC–MS open profiling data.
Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.
Objectives
To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.
Methods
The extracted compounds were analyzed by LC/MS and GC/MS.
Results
The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.
Conclusion
From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.
Central arterial stiffness is an accepted risk factor for cardiovascular disease. While aerobic activity is associated with reduced stiffness the influence of practicing yoga is unknown. The aims of this study were to: 1) evaluate arterial stiffness in middle-aged adults who regularly practiced yoga, performed regular exercise, or were inactive, 2) evaluate the reproducibility of arterial stiffness measured in the left and right carotid artery and by pulse wave velocity (PWV).
Methods
Twenty six healthy subjects (male and female, 40–65 yrs old) were tested on two separate days. Carotid artery distensibility (DC) was measured with ultrasound. Physical activity was determined by questionnaire.
Results
Yoga and aerobic subjects had similar physical activity levels. Yoga and aerobic groups were not different in either DC (p = 0.26) or PWV (p = 0.21). The sedentary group had lower DC and higher PWV compared to the aerobic and yoga groups (both, p < 0.001). Stiffness measures were reliable day to day (coefficients of variation ~2.5%) and similar between left and right arteries (CV = 2.2%).
Conclusion
Physical activity was a strong predictor of both measures of arterial stiffness, although other factors such as nutritional status need to be accounted for. An independent effect of practicing yoga could not be detected. Stiffness measures were reproducible and left and right sides were consistent with each other.
It is currently unclear whether reductions in adiposity mediate the improvements in vascular health that occur with aerobic exercise. The purpose of this longitudinal study of 13 healthy women (33 ± 4 years old) was to determine whether 14 weeks of aerobic exercise would alter functional measures of vascular health, namely resting aortic pulse wave velocity (aPWV, an index of arterial stiffness), femoral artery diameter (DFA), and femoral artery blood flow (BFFA) independent of changes in adiposity.
Methods
Aerobic fitness was assessed as VO2peak normalized to fat-free mass, and adiposity (percent body fat) was determined by dual energy x-ray absorptiometry. Serum concentrations of proteins associated with risk for cardiovascular disease, including C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), and leptin, were also measured. Subjects cycled for 50 minutes, 3 times per week.
Results
Aerobic fitness normalized to fat-free mass increased 6% (P = 0.03) whereas adiposity did not change. Resting DFA increased 12% (P < 0.001) and resting shear rate decreased 28% (P = 0.007). Aortic PWV, and serum sICAM-1, CRP and leptin did not change with training.
Conclusion
Significant reductions in adiposity were not necessary for aerobic exercise training to bring about improvements in aerobic fitness and arterial remodeling. Peripheral arterial remodeling occurred without changes in central arterial stiffness or markers of inflammation.
Dental calculus is a mineralized microbial dental plaque biofilm that forms throughout life by precipitation of salivary calcium salts. Successive cycles of dental plaque growth and calcification make it an unusually well-preserved, long-term record of host-microbial interaction in the archaeological record. Recent studies have confirmed the survival of authentic ancient DNA and proteins within historic and prehistoric dental calculus, making it a promising substrate for investigating oral microbiome evolution via direct measurement and comparison of modern and ancient specimens.
Objective
We present the first comprehensive characterization of the human dental calculus metabolome using a multi-platform approach.
Methods
Ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) quantified 285 metabolites in modern and historic (200 years old) dental calculus, including metabolites of drug and dietary origin. A subset of historic samples was additionally analyzed by high-resolution gas chromatography–MS (GC–MS) and UPLC–MS/MS for further characterization of metabolites and lipids. Metabolite profiles of modern and historic calculus were compared to identify patterns of persistence and loss.
Results
Dipeptides, free amino acids, free nucleotides, and carbohydrates substantially decrease in abundance and ubiquity in archaeological samples, with some exceptions. Lipids generally persist, and saturated and mono-unsaturated medium and long chain fatty acids appear to be well-preserved, while metabolic derivatives related to oxidation and chemical degradation are found at higher levels in archaeological dental calculus than fresh samples.
Conclusions
The results of this study indicate that certain metabolite classes have higher potential for recovery over long time scales and may serve as appropriate targets for oral microbiome evolutionary studies.
To compare the degradation performance and biodiversity of a polyvinyl alcohol-degrading microbial community under aerobic and anaerobic conditions.
Results
An anaerobic–aerobic bioreactor was operated to degrade polyvinyl alcohol (PVA) in simulated wastewater. The degradation performance of the bioreactor during sludge cultivation and the microbial communities in each reactor were compared. Both anaerobic and aerobic bioreactors demonstrated high chemical oxygen demand removal efficiencies of 87.5 and 83.6 %, respectively. Results of 16S rDNA sequencing indicated that Proteobacteria dominated in both reactors and that the microbial community structures varied significantly under different operating conditions. Both reactors obviously differed in bacterial diversity from the phyla Planctomycetes, Chlamydiae, Bacteroidetes, and Chloroflexi. Betaproteobacteria and Alphaproteobacteria dominated, respectively, in the anaerobic and aerobic reactors.
Conclusions
The anaerobic–aerobic system is suitable for PVA wastewater treatment, and the microbial genetic analysis may serve as a reference for PVA biodegradation.
Natural products from culture collections have enormous impact in advancing discovery programs for metabolites of biotechnological importance. These discovery efforts rely on the metabolomic characterization of strain collections.
Objective
Many emerging approaches compare metabolomic profiles of such collections, but few enable the analysis and prioritization of thousands of samples from diverse organisms while delivering chemistry specific read outs.
Method
In this work we utilize untargeted LC–MS/MS based metabolomics together with molecular networking to inventory the chemistries associated with 1000 marine microorganisms.
Result
This approach annotated 76 molecular families (a spectral match rate of 28 %), including clinically and biotechnologically important molecules such as valinomycin, actinomycin D, and desferrioxamine E. Targeting a molecular family produced primarily by one microorganism led to the isolation and structure elucidation of two new molecules designated maridric acids A and B.
Conclusion
Molecular networking guided exploration of large culture collections allows for rapid dereplication of know molecules and can highlight producers of uniques metabolites. These methods, together with large culture collections and growing databases, allow for data driven strain prioritization with a focus on novel chemistries.
Raspberries are becoming increasingly popular due to their reported health beneficial properties. Despite the presence of only trace amounts of anthocyanins, yellow varieties seems to show similar or better effects in comparison to conventional raspberries.
Objectives
The aim of this work is to characterize the metabolic differences between red and yellow berries, focussing on the compounds showing a higher concentration in yellow varieties.
Methods
The metabolomic profile of 13 red and 12 yellow raspberries (of different varieties, locations and collection dates) was determined by UPLC–TOF-MS. A novel approach based on Pearson correlation on the extracted ion chromatograms was implemented to extract the pseudospectra of the most relevant biomarkers from high energy LC–MS runs. The raw data will be made publicly available on MetaboLights (MTBLS333).
Results
Among the metabolites showing higher concentration in yellow raspberries it was possible to identify a series of compounds showing a pseudospectrum similar to that of A-type procyanidin polymers. The annotation of this group of compounds was confirmed by specific MS/MS experiments and performing standard injections.
Conclusions
In berries lacking anthocyanins the polyphenol metabolism might be shifted to the formation of a novel class of A-type procyanidin polymers.
Cognitive impairment may be seen in as many as 43–70% of patients with multiple sclerosis (MS) and may be observed in all MS subtypes. The Brief International Cognitive Assessment in Multiple Sclerosis (BICAMS) battery may be used to evaluate cognition status. The purpose of the current study is to validate the BICAMS battery in Turkish.
Methods
Patients with MS attending our clinic between September 2014 and April 2015 were invited to participate. Healthy control participants were matched in terms of age, gender and years of education.
Results
One hundred seventy-three MS patients and 153 healthy control participants were enrolled in the study. MS patients performed significantly worse in all trials than the members of the healthy control group. In addition, cognitive dysfunction was identified in 78 of the 173 (45.1%) patients. In the MS with cognitive impairment group, 64 out of 151 (42.4%) subjects were RRMS patients, 12 out of 18 (66.7%) were secondary progressive MS patients, and 2 out of 4 (50%) were primer progressive MS patients.
Conclusions
The BICAMS has been proposed for assessing cognitive impairment in MS patients. This study shows that the battery is suitable for use in Turkey.
Climate change is a major concern for the scientific community, demanding novel information about the effects of environmental stressors on living organisms. Metabolic profiling is required for achieving the most extensive possible range of compounds and their concentration changes on stressed conditions.
Objectives
Individuals of the crustacean species Daphnia magna were exposed to three different abiotic factors linked to global climate change: high salinity, high temperature levels and hypoxia. Advanced chemometric tools were used to characterize the metabolites affected by the exposure.
Method
An exploratory analysis of gas chromatography-mass spectrometry (GC–MS) data was performed to discriminate between control and exposed daphnid samples. Due to the complexity of these GC–MS data sets, a comprehensive untargeted analysis of the full scan data was performed using multivariate curve resolution-alternating least squares (MCR-ALS) method. This approach enabled to resolve most of the metabolite signals from interference peaks caused by derivatization reactions. Metabolites with significant changes in their peak areas were tentatively identified and the involved metabolic pathways explored.
Results
D. magna metabolic biomarkers are proposed for the considered physical factors. Metabolites related with energy metabolic pathways including some amino acids, carbohydrates, organic acids and nucleosides were identified as potential biomarkers of the investigated treatments.
Conclusions
The proposed untargeted GC–MS metabolomics strategy and multivariate data analysis tools were useful to investigate D. magna metabolome under environmental stressed conditions.
Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes.
Objectives
We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform.
Methods
Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models.
Results
The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs?≥?0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68–0.93).
Conclusion
Most metabolites measured by the UPLC–MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.
Traumatic brain injury (TBI) is physical injury to brain tissue that temporarily or permanently impairs brain function.
Objectives
Evaluate the use of metabolomics for the development of biomarkers of TBI for the diagnosis and timing of injury onset.
Methods
A validated model of closed injury TBI was employed using 10 TBI mice and 8 sham operated controls. Quantitative LC–MS/MS metabolomic analysis was performed on the serum.
Results
Thirty-six (24.0 %) of 150 metabolites were altered with TBI. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) analyses revealed clear segregation between TBI versus control sera. The combination of methionine sulfoxide and the lipid PC aa C34:4 accurately diagnosed TBI, AUC (95 % CI) 0.85 (0.644–1.0). A combination of metabolite markers were highly accurate in distinguishing early (4 h post TBI) from late (24 h) TBI: AUC (95 % CI) 1.0 (1.0–1.0). Spermidine, which is known to have an antioxidant effect and which is known to be metabolically disrupted in TBI, was the most discriminating biomarker based on the variable importance ranking in projection (VIP) plot. Several important metabolic pathways were found to be disrupted including: pathways for arginine, proline, glutathione, cysteine, and sphingolipid metabolism.
Conclusion
Using serum metabolomic analysis we were able to identify novel putative serum biomarkers of TBI. They were accurate for detecting and determining the timing of TBI. In addition, pathway analysis provided important insights into the biochemical mechanisms of brain injury. Potential clinical implications for diagnosis, timing, and monitoring brain injury are discussed.
The prevalence of metabolic syndrome (MS) increased in recent years in both adolescents and children groups. The aim of the study is evaluating the relationship between insulin and uric acid (UA) level in MS in adolescents
Materials and Methods
we studied 120 adolescence aged 10 to 19 in two groups: control group without metabolic syndrome and case group with metabolic syndrome. The Criteria of ATP III was considered as a diagnosis factor for metabolic syndrome.
Discussion
Various studies have been conducted in various populations to evaluate the relationship between UA level and MS in adolescents. Abdominal obesity, low HDL, hypertriglyceridemia and hypertension are associated with high UA level. In their analysis, the MS OR in UA level?4.9, 4.9-5.8 and ?5.8 mg/dl was 1, 2.53 and 9.03, respectively, which were higher than our findings in current study. Hyperinsulinemia caused by insulin resistance is one of the complications associated with MS, which puts individuals at risk of diabetes and cardiovascular events.
Results
Uric acid level in the Case group was significantly higher than the control group (p = 0.0001, 43.8±1.4 vs. 4.1±1 mg/dl, respectively). Insulin level was significantly higher in the case group in compare to the control group (p = 0.008, 9.8± 5.3 vs. 12.2±6 μU/ml, respectively).
Conclusion
The findings of this case-control study showed that adolescents with metabolic syndrome have a higher uric acid and insulin level in compare to normal subjects. We hypothesis that increase in serum insulin and uric acid level can be a risk factor in the development of metabolic syndrome.
Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC–MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters.
Objective
Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using design of experiments (DoE).
Methods
We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term Comprehensive optimization of LC–MS metabolomics methods using design of experiments (COLMeD). Multivariate statistical analysis guided our decision process in the method optimizations.
Results
LC–MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5 % (p < 0.0001) over initial conditions with a 13.3 % increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8 and 57.3 %, with median metabolite response increases of 106.1 and 10.3 % (p < 0.0001 and p < 0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8 % response increase (p < 0.0001) over initial conditions.
Conclusions
The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.
Polyploidy is a widespread phenomenon in nature and is thought to play a major role in the evolution of flowering plants. Additionally, polyploidization produces novel phenotypes that through plant breeding have enhanced the production of biomass and improved the stress tolerance of major economic crops. However, the effect of polyploidization on plant metabolism is still unclear.
Objectives
In order to test whether there are common metabolic responses following genome doubling, we performed a comparative metabolomic analysis of mature leaves from doubled diploids and the corresponding diploids of red tangerine (Citrus reticulata Blanco), trifoliate orange (Poncirus trifoliata L. Raf.) and precocious trifoliate orange (P. trifoliata).
Methods
Non-targeted and targeted metabolic profiling of mature leaves from three doubled diploids and their diploid controls were performed by using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS).
Results
About 11–34% of the detected metabolic features differentially accumulated in the doubled diploids, mostly by less than fivefold. The levels of primary metabolites tended to increase in the doubled diploids. Concentrations of tricarboxylic acid cycle intermediates—citric acid, malic acid, fumaric acid and succinic acid, enhanced in all of the doubled diploids. The levels of secondary metabolites, including phenylpropanoids and terpenoids, tended to decrease in the doubled diploids. This is consistent with the lower C/N ratios in the doubled diploids.
Conclusions
Polyploidization had a significant but relatively limited influence on the accumulation of metabolites in these citrus species. We conclude that primary metabolism takes priority over secondary metabolism in doubled diploid plants to relieve the “genomic stress” encountered during the early stages of genome doubling, probably to promote vitality and growth.
Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.
Objectives
To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS.
Methods
Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst.
Results
It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation.
Conclusion
We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.
The benefits of physical activity in persons with Multiple Sclerosis (MS) are considerable. Knowledge about factors that correlate to physical activity is helpful in order to develop successful strategies to increase physical activity in persons with MS. Previous studies have focused on correlates to physical activity in MS, however falls self-efficacy, social support and enjoyment of physical activity are not much studied, as well as if the correlates differ with regard to disease severity. The aim of the study was to examine associations between physical activity and age, gender, employment, having children living at home, education, disease type, disease severity, fatigue, self-efficacy for physical activity, falls self-efficacy, social support and enjoyment of physical activity in a sample of persons with MS and in subgroups with regard to disease severity.
Methods
This is a cross-sectional survey study including Swedish community living adults with MS, 287 persons, response rate 58.2%. The survey included standardized self-reported scales measuring physical activity, disease severity, fatigue, self-efficacy for physical activity, falls self-efficacy, and social support. Physical activity was measured by the Physical Activity Disability Survey – Revised.
Results
Multiple regression analyzes showed that 59% (F(6,3)?=?64.9, p?=?0.000) of the variation in physical activity was explained by having less severe disease (β?=??0.30), being employed (β?=?0.26), having high falls self-efficacy (β?=?0.20), having high self-efficacy for physical activity (β?=?0.17), and enjoying physical activity (β?=?0.11). In persons with moderate/severe MS, self-efficacy for physical activity explained physical activity.
Conclusions
Consistent with previous research in persons with MS in other countries this study shows that disease severity, employment and self-efficacy for physical activity are important for physical activity. Additional important factors were falls self-efficacy and enjoyment. More research is needed to confirm this and the subgroup differences.
The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis.
Objectives
We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples.
Method
Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS).
Results
The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol.
Conclusion
Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders).
Graphical Abstract
Urine desalting and preconcentration in bursting bubbles.
