Quantitative Metabolite Research Articles

P0565 Spatial Metabolic Profiles in Ulcerative colitis patients determine response to therapy

Abstract Background Ulcerative Colitis (UC) is an heterogenous debilitating disease without a cure. The step-up therapy approach aims at controlling and alleviating intestinal inflammation by achieving and maintaining clinical and endoscopic remission. However, the rate of primary non-response or loss of response to therapies is still high. Predictive markers that accurately guide decisions about the best therapeutic choice are an unmet need. This study aims to determine the intestinal metabolic profiles of UC patients and explore differences in response groups of given treatments, thus helping a better patient stratification. Methods Mucosal biopsies collected from active UC patients (N=26) at week 0 and 14 after treatment (vedolizumab, tofacitinib and filgotinib) were sectioned at 10 µm thickness. Patients were classified as non-responders (NR), responders (R) and super responders (SR) based on both endoscopic and partial Mayo scores. Sections were processed by matrix-assisted laser desorption/ionization (MALDI) coupled with Orbitrap Exploris™ 120 Mass Spectrometer. Raw spectral data was processed with MSConverter and MSIpixel for metabolite quantification and annotation. Comprehensive statistical analyses including quantitative metabolite distributions, Moran’s I spatial correlations and pathway enrichment analysis were performed via an in-house developed computational pipeline. Results Spatial metabolomic analysis revealed different metabolite patterns between R and NR across all drugs. Notably, all non-responders exhibited a dysregulated lipid metabolism. In the Vedolizumab group, a fragmented pattern distribution of metabolites was found in NR characterized by an increase of canavalmine, N1-acetylspermidine and heptanenitrile at week 0, whereas SR showed a significant tissue reorganization environment, with increased number of metabolites such as LysoPA, LysoPE and 5alpha-Cholesta-7,24dien-3beta-ol, involved in anti-inflammatory pathways, as well as glutathione and purine metabolism. Additionally, SR showed histamine- and IgE-related pathways supporting cellular regeneration. Conclusion Overall, these data depicted the alteration of mucosal lipid metabolism as a hallmark of non-response to therapies, and evidenced that the effectiveness of a specific treatment in UC is impacted by the initial mucosal metabolic state conditioned by diet and medications. The validation of these results in a larger sample size will aid the prediction of therapy response. References ImmUniverse project: this project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No. 853995. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. The content provided in this abstract reflects only the author's view and neither the IMI JU nor the European Commission are responsible for any use that may be made of the information it contains.

Metabolomics in juvenile idiopathic arthritis: a distinct profile in patients under methotrexate

The objective of the present study was to evaluate biochemical quantitative metabolites in peripheral blood serum samples of Juvenile Idiopathic Arthritis (JIA) patients and healthy controls. A cross-sectional study included 33 post-pubertal JIA (21 without and 12 with Methotrexate (MTX) women and 28 age-matched healthy controls. Metabolomic analyses based on targeted electrospray ionization tandem mass spectrometry were used to identify possible biochemical pathway modifications in serum from JIA patients. The mean current age (p = 0.065) was similar in JIA patients and healthy controls. Current MTX use in all subtypes of JIA patients was associated with an increase in concentrations of free carnitine [21.74 (12.7‒35.2) vs. 27.49 (14.5‒41.3) µM/L, p = 0.02], suggesting an enhanced mitochondrial metabolism and intestinal absorptive function. In contrast, a decreased mitochondrial metabolism was observed in polyarticular and systemic JIA subtypes, with a decrease of several acylcarnitines' concentrations (p < 0.05). In conclusion, the present study identified a distinctive pattern of serum metabolic signatures in JIA patients under MTX therapy. Our findings indicate that MTX use is associated with a more efficient mitochondrial function.

Effects of Ganoderma lucidum extract on the antioxidant status of pork patties during storage and gastro-intestinal digestion

This study proposed determining the effects of Ganoderma lucidum hydroalcoholic extract (GHE at 0.5 and 1%) incorporation, storage, and gastro-intestinal digestion (GID) on the antioxidant status of raw/cooked pork patties. GHE was subjected to qualitative and quantitative metabolites assays and antioxidant assays. Raw/cooked pork samples were subjected to pH, color, lipid oxidation, metabolite content, and antioxidant assays during storage time (2 °C for nine days); before and after GID, cooked samples were subjected to metabolite and antioxidant assays. Results demonstrated (p < 0.05) that GHE presented metabolites and antioxidant activity. GHE incorporation in raw/cooked samples increased storage stability, regarding pH, color, and lipid oxidation values. Also, GHE incorporation in digested cooked meat samples increased phenolic components and antioxidant values. GHE is a promising ingredient for increasing the antioxidant status of meat products.

A comprehensive pharmacokinetic strategy for systematic evaluation of whole interaction of different constituents in Astragali Radix -Fructus Corni to improve diabetic kidney disease

Ethnopharmacological relevanceAstragali-Radix (the dried root of Astragalus mongholicus Bunge, AR) - Fructus Corni (the dried ripe fruit of Cornus officinalis Sieb. et Zucc., FC) has been used as a herb-pair remedy to treat diabetic kidney disease (DKD) for hundred years. Polysaccharides, saponins, and flavonoids in AR, and the iridoid glycosides in FC were deemed as the main bioactive constituents that can offer beneficial nephroprotective activities. A systematic evaluation of the nephroprotective effects of AR-FC herb pair, the main bioactive constituents extracted from the herb pair, and their combinations in different ratios was performed, CG6 (polysaccharides, flavonoids, saponins, and iridoid glycosides, in a ratio of 2:3:1:2) as the best compatibility proportion was screened out in our previous study. Aim of the studyThis study aimed to investigate the pharmacokinetic characteristics of AR-FC herb-pair in DKD rats, and explore the interactions between constituents from AR-FC and the rational compatibility of different constituents. Materials and methodsThe protective effect of AR-FC and CG6 on renal injury caused by DKD was first verified by histopathological examination. Then, an analytical method based on UHPLC-Q-TOF-MS and UHPLC-QqQ-MS/MS for qualitative and quantitative metabolites without reference standards was established and applied to pharmacokinetic (PK) studies in following different aspects: between single groups (polysaccharides, flavonoids, saponins and iridoid glycosides) and compatibility groups (AR-FC, CG6), in normal and DKD rats, in single-dose administration and long-term administration. ResultsPathological observations confirmed that AR-FC could improve renal injury in DKD rats. PK profiles of nine prototypes and four metabolites in various groups were obtained, revealing the compatibility of multiple constitutes, pathological states, and long-term administration could alter PK characteristics of the main components from AR-FC, and promoting the absorption of them (Cmax, AUC0-t, and AUC0-t increased). Notably, co-administration of iridoid glycosides could significantly increase the absorption of flavonoids and saponins in vivo. The pharmacokinetics based on homologous compounds revealed that saponins first acted, then its initial metabolites affected flavonoids, and ultimately the metabolites of flavonoids influenced iridoid glycosides. ConclusionThis study demonstrated the existence of interactions between constituents from AR-FC herb-pair and the importance of their rational compatibility. It provides experimental evidence for developing a therapeutic agent based on AR-FC (especially CG6) to treat DKD. It is also expected to provide a reference for the multi-component pharmacokinetic study of other herbal medicines.

Hypometric genetics: Improved power in genetic discovery by incorporating quality control flags.

Balancing the tradeoff between quantity and quality of phenotypic data is critical in omics studies. Measurements below the limit of quantification (BLQ) are often tagged in quality control fields, but these flags are currently underutilized in human genetics studies. Extreme phenotype sampling is advantageous for mapping rare variant effects. We hypothesize that genetic drivers, along with environmental and technical factors, contribute to the presence of BLQ flags. Here, we introduce "hypometric genetics" (hMG) analysis and uncover a genetic basis for BLQ flags, indicating an additional source of genetic signal for genetic discovery, especially from phenotypic extremes. Applying our hMG approach to n= 227,469UK Biobank individuals with metabolomic profiles, we reveal more than 5% heritability for BLQ flags and report biologically relevant associations, for example, at APOC3, APOA5, and PDE3B loci. For common variants, polygenic scores trained only for BLQ flags predict the corresponding quantitative traits with 91% accuracy, validating the genetic basis. For rare coding variant associations, we find an asymmetric 65.4% higher enrichment of metabolite-lowering associations for BLQ flags, highlighting the impact of putative loss-of-function variants with large effects on phenotypic extremes. Joint analysis of binarized BLQ flags and the corresponding quantitative metabolite measurements improves power in Bayesian rare variant aggregation tests, resulting in an average of 181% more prioritized genes. Our approach is broadly applicable to omics profiling. Overall, our results underscore the benefit of integrating quality control flags and quantitative measurements and highlight the advantage of joint analysis of population-based samples and phenotypic extremes in human genetics studies.

Metabolomic Disparities in Intraocular Fluid Across Varied Stages of Cataract Progression: Implications for the Analysis of Cataract Development.

Introduction: The lens's metabolic demands are met through a continuous circulation of aqueous humor, encompassing a spectrum of components such as organic and inorganic ions, carbohydrates, glutathione, urea, amino acids, proteins, oxygen, carbon dioxide, and water. Metabolomics is a pivotal tool, offering an initial insight into the complexities of integrated metabolism. In this investigative study, we systematically scrutinize the composition of intraocular fluid in individuals afflicted with cataracts. Methods: The investigation involved a comprehensive analysis of aqueous humor samples from a cohort comprising 192 patients. These individuals were stratified by utilizing the SPONCS classification system, delineating distinct groups characterized by the hardness of cataracts. The analytical approach employed targeted quantitative metabolite analysis using HILIC-based liquid chromatography coupled with high-resolution mass spectrometric detection. The metabolomics data analysis was performed with MetaboAnalyst 5.0. Results: The results of the enrichment analysis have facilitated the inference that the discerned disparities among groups arise from disruptions in taurine and hypotaurine metabolism, variations in tryptophan metabolism, and modifications in mitochondrial beta-oxidation of short-chain saturated fatty acids and pyrimidine metabolism. Conclusion: A decline in taurine concentration precipitates diminished glutathione activity, prompting an elevated requirement for NAD+ and instigating tryptophan metabolism along the kynurenine pathway. Activation of this pathway is additionally prompted by interferon-gamma and UV radiation, leading to the induction of IDO. Concurrently, heightened mitochondrial beta-oxidation signifies a distinctive scenario in translocating fatty acids into the mitochondria, enhancing energy production.

Therapeutic application of nicotinamide: As a potential target for inhibiting fibrotic scar formation following spinal cord injury.

We aimed to confirm the inhibitory effect of nicotinamide on fibrotic scar formation following spinal cord injury in mice using functional metabolomics. We proposed a novel functional metabolomics strategy to establish correlations between gene expression changes and metabolic phenotypes using integrated multi-omics analysis. Through the integration of quantitative metabolites analysis and assessments of differential gene expression, we identified nicotinamide as a functional metabolite capable of inhibiting fibrotic scar formation and confirmed the effect invivo using a mouse model of spinal cord injury. Furthermore, to mimic fibrosis models invitro, primary mouse embryonic fibroblasts and spinal cord fibroblasts were stimulated by TGFβ, and the influence of nicotinamide on TGFβ-induced fibrosis-associated genes and its underlying mechanism were examined. Administration of nicotinamide led to a reduction in fibrotic lesion area and promoted functional rehabilitation following spinal cord injury. Nicotinamide effectively downregulated the expression of fibrosis genes, including Col1α1, Vimentin, Col4α1, Col1α2, Fn1, and Acta2, by repressing the TGFβ/SMADs pathway. Our functional metabolomics strategy identified nicotinamide as a metabolite with the potential to inhibit fibrotic scar formation following SCI by suppressing the TGFβ/SMADs signaling. This finding provides new therapeutic strategies and new ideas for clinical treatment.

Qualitative and Quantitative Metabolite Comparison of Korean Traditional Alcoholic Beverages: Takju, Yakju, and Traditional-Soju.

With increasing interest in Korean foods and beverages, Korean traditional alcoholic beverages need to be studied. To characterize Korean traditional alcoholic beverages, we analyzed the metabolites of Takju, Yakju, and Traditional-Soju using 48 commercial products. We performed non-targeted metabolite profiling using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and identified 33 significantly discriminant metabolites, including nine organic acids, three amino acids, and seven fatty acids, in the three types of alcoholic beverage. Subsequently, we quantified the profiled metabolites in each product and compared their contents to identify alcoholic beverage type-specific metabolites. Thus, we figured out seven metabolites using receiver operating characteristic (ROC) curves. The results revealed that octadecanoic acid (limit of detection (LOD) to 168.72 mg/L), nonanoic acid (LOD to 112.54 mg/L), and octanoic acid (8.00 to 145.08 mg/L) in Takju; succinic acid (LOD to 1.90 mg/mL), heptanoic acid (LOD to 343.23 mg/L), and hexadecanoic acid (20.28 to 126.45 mg/L) in Yakju; and malonic acid (LOD to 19.13 mg/mL) in Traditional-Soju, with an area under the curve (AUC) > 0.7, are important metabolites that can distinguish the type of alcoholic beverage. Our results provide qualitative and quantitative metabolite information about Korean traditional alcoholic beverages that can be used by consumers and manufacturers.

Patterns of infant fecal metabolite concentrations and social behavioral development in toddlers.

Gut-derived metabolites, products of microbial and host co-metabolism, may inform mechanisms underlying children's neurodevelopment. We investigated whether infant fecal metabolites were related to toddler social behavior. Stool samples collected from 6-week-olds (n = 86) and 1-year-olds (n = 209) in the New Hampshire Birth Cohort Study (NHBCS) were analyzed using nuclear magnetic resonance spectroscopy metabolomics. Autism-related behavior in 3-year-olds was assessed by caregivers using the Social Responsiveness Scale (SRS-2). To assess the association between metabolites and SRS-2 scores, we used a traditional single-metabolite approach, quantitative metabolite set enrichment (QEA), and self-organizing maps (SOMs). Using a single-metabolite approach and QEA, no individual fecal metabolite or metabolite set at either age was associated with SRS-2 scores. Using the SOM method, fecal metabolites of six-week-olds organized into four profiles, which were unrelated to SRS-2 scores. In 1-year-olds, one of twelve fecal metabolite profiles was associated with fewer autism-related behaviors, with SRS-2 scores 3.4 (95%CI: -7, 0.2) points lower than the referent group. This profile had higher concentrations of lactate and lower concentrations of short chain fatty acids than the reference. We uncovered metabolic profiles in infant stool associated with subsequent social behavior, highlighting one potential mechanism by which gut bacteria may influence neurobehavior. Differences in host and microbial metabolism may explain variability in neurobehavioral phenotypes, but prior studies do not have consistent results. We applied three statistical techniques to explore fecal metabolite differences related to social behavior, including self-organizing maps (SOMs), a novel machine learning algorithm. A 1-year-old fecal metabolite pattern characterized by high lactate and low short-chain fatty acid concentrations, identified using SOMs, was associated with social behavior less indicative of autism spectrum disorder. Our findings suggest that social behavior may be related to metabolite profiles and that future studies may uncover novel findings by applying the SOM algorithm.

Efficient Quantification of Milk Metabolites from 1H NMR Spectra Using the Signature Mapping (SigMa) Approach: Chemical Shift Library Development for Cows' Milk and Colostrum.

Cow milk contains essential nutrients for humans, and its bulk composition is usually analyzed using Fourier transform infrared spectroscopy. The higher sensitivity of nuclear magnetic resonance (NMR) spectroscopy can augment the extractible qualitative and quantitative information from milk to nearly 60 compounds, enabling us to monitor the health of cows and milk quality. Proton (1H) NMR spectroscopy produces complex spectra that require expert knowledge for identifying and quantifying metabolites. Therefore, an efficient and reproducible methodology is required to transform complex milk 1H NMR spectra into annotated and quantified milk metabolome data. In this study, standard operating procedures for screening the milk metabolome using 1H NMR spectra are developed. A chemical shift library of 63 milk metabolites was established and implemented in the open-access Signature Mapping (SigMa) software. SigMa is a spectral analysis tool that transforms 1H NMR spectra into a quantitative metabolite table. The applicability of the proposed methodology to whole milk, skim milk, and ultrafiltered milk is demonstrated, and the method is tested on ultrafiltered colostrum samples from dairy cows (n = 88) to evaluate whether metabolic changes in colostrum may reflect the metabolic status of cows.

Quantitative estimation of metabolites and antifungal efficacy of leaf extracts &lt;i&gt;Aspilia africana&lt;/i&gt; on cucumber and pawpaw fruit spoilage fungi

Several reports have shown that about 25% of harvested fruits globally are lost to spoilage by microorganisms. The study determined the quantitative metabolites and bioactivities of Aspilia africana extracts on fungi from spoiled Cucumis sativus and Carica papaya fruits. The fungi from spoiled C. sativus and C. papaya fruits were obtained using mycological techniques. The quantitative metabolites and bioactivities of the aqueous (ALEAA) and ethanol (ELEAA) extracts of A. africana were determined using the standard protocol and disc diffusion technique, respectively. The fungal genera obtained were Aspergillus, Penicillium, Mucor, Fusarium, and Rhizopus. There were variations in percentage yields, physical appearances, and pH of the extracts. The results of quantitative estimation of metabolites showed that ALEAA had mean protein, carbohydrate, and lipid contents of 15.36 ± 0.32%, 60.97 ± 1.14%, and 6.66 ± 0.04%, respectively. Alkaloids showed a positive correlation with protein (r = 0.2028) and carbohydrate (r = 0.421), and a negative correlation with lipid (r = -0.6556) at p &lt; 0.05. The ELEAA exhibited more inhibitory effects on test fungal isolates, with mean zones of inhibition (IZs) ranging from 9.3 ± 0.1 to 18.8 ± 0.3 mm, than the ALEAA, with mean IZs ranging between 9.4 ± 0.4 and 16.0 ± 1.0 mm. The R coefficients of the extracts and IZs as exhibited by the fungi ranged from 0.5985 to 0.9936. The results have revealed quantitative metabolites and antifungal activities of the extract and also provided rationale for its utilization as a preservative for fruits against spoilage by fungi.

The Hepatic Antioxidant System Damage Induced with the Cafeteria (CAF) Diet Is Largely Counteracted Using SCD Probiotics during Development of Male Wistar Rats.

The cafeteria (CAF) diet, reflective of predominant Western dietary behaviors, is implicated in hastening weight gain, subsequently resulting in health complications such as obesity, diabetes, and cancer. To this end, it is vital to notice the deleterious consequences of the CAF regimen prior to the onset of complications, which is fundamental for early intervention in the context of numerous diseases. Probiotic-derived postbiotic metabolites have gained attention for their antioxidative properties, offering a potential countermeasure against oxidative stress. This research sought to discern the protective efficacy of SCD Probiotics against liver glutathione system damage arising from the CAF diet during developmental phases. Male Wistar rats, from weaning on day 21 to day 56, were categorized into four groups: a control on a conventional diet; a group on a standard diet enriched with SCD Probiotics; a mixed-diet group comprising both CAF and standard feed; and a combination diet group supplemented with SCD Probiotics. Through the application of real-time PCR, enzyme activity assessments, and quantitative metabolite analyses, our findings highlight the CAF diet's adverse influence on the liver's antioxidant defenses via shifts in gene expression. Yet, the inclusion of SCD Probiotics mostly ameliorated these harmful effects. Remarkably, the positive regulatory influence of SCD Probiotics on the liver's antioxidant system was consistently observed, independent of the CAF diet's presence.

In Vivo Metabolite Profiles of an N-Acetylgalactosamine-Conjugated Antisense Oligonucleotide AZD8233 Using Liquid Chromatography High-Resolution Mass Spectrometry: A Cross-Species Comparison in Animals and Humans.

AZD8233, a liver-targeting antisense oligonucleotide (ASO), inhibits subtilisin/kexin type 9 protein synthesis. It is a phosphorothioated 3-10-3 gapmer with a central DNA sequence flanked by constrained 2'-O-ethyl 2',4'-bridged nucleic acid (cEt-BNA) wings and conjugated to a triantennary N-acetylgalactosamine (GalNAc) ligand at the 5'-end. Herein we report the biotransformation of AZD8233, as given by liver, kidney, plasma and urine samples, after repeated subcutaneous administration to humans, mice, rats, rabbits, and monkeys. Metabolite profiles were characterized using liquid chromatography high-resolution mass spectrometry. Metabolite formation was consistent across species, mainly comprising hydrolysis of GalNAc sugars, phosphodiester-linker hydrolysis releasing the full-length ASO, and endonuclease-mediated hydrolysis within the central DNA gap followed by exonuclease-mediated 5'- or 3'-degradation. All metabolites contained the 5'- or 3'-cEt-BNA terminus. Most shortmer metabolites had the free terminal alcohol at 5'- and 3'-positions of ribose, although six were found retaining the terminal 5'-phosphorothioate group. GalNAc conjugated shortmer metabolites were also observed in urine. Synthesized metabolite standards were applied for (semi)quantitative metabolite assessment. Intact AZD8233 was the major component in plasma, whereas the unconjugated full-length ASO was predominant in tissues. In plasma, most metabolites were shortmers retaining the 3'-cEt-BNA terminus, whereas metabolites containing the 5'- or 3'-cEt-BNA terminus were detected in both tissues and urine. All metabolites in human plasma were also detected in all nonclinical species, and all human urine metabolites were detected in monkey urine. In general, metabolite profiles in animal species were qualitatively similar and quantitatively exceeded the exposures of the circulating metabolites in humans at the doses studied. SIGNIFICANCE STATEMENT: This study presents metabolite identification and profiling of AZD8233, an N-acetylgalactosamine-conjugated antisense oligonucleotide (ASO), across species. A biotransformation strategy for ASOs was established by utilizing biologic samples collected from toxicology and/or clinical studies and liquid chromatography high-resolution mass spectrometry analysis without conducting bespoke radiolabeled absorption, distribution, metabolism, and excretion studies. The generated biotransformation package was considered adequate by health authorities to progress AZD8233 into a phase 3 program, proving its applicability to future metabolism studies of ASOs in drug development.

A systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions.

Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation, and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for HeLa carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol/chloroform/water [1:1:1 v/v/v], 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent HeLa carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids (AAs), adenosine nucleotides, and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33nmol per million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile were found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect AA metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to two-dimensional (2D) cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.

Biomarker discovery for tuberculosis using metabolomics.

Tuberculosis (TB) is the leading cause of death among infectious diseases, and the ratio of cases in which its pathogen Mycobacterium tuberculosis (Mtb) is drug resistant has been increasing worldwide, whereas latent tuberculosis infection (LTBI) may develop into active TB. Thus it is important to understand the mechanism of drug resistance, find new drugs, and find biomarkers for TB diagnosis. The rapid progress of metabolomics has enabled quantitative metabolite profiling of both the host and the pathogen. In this context, we provide recent progress in the application of metabolomics toward biomarker discovery for tuberculosis. In particular, we first focus on biomarkers based on blood or other body fluids for diagnosing active TB, identifying LTBI and predicting the risk of developing active TB, as well as monitoring the effectiveness of anti-TB drugs. Then we discuss the pathogen-based biomarker research for identifying drug resistant TB. While there have been many reports of potential candidate biomarkers, validations and clinical testing as well as improved bioinformatics analysis are needed to further substantiate and select key biomarkers before they can be made clinically applicable.

Differential Metabolomic Fingerprinting of the Crude Extracts of Three Asteraceae Species with Assessment of Their In Vitro Antioxidant and Enzyme-Inhibitory Activities Supported by In Silico Investigations

The Asteraceae is a large family, rich in ornamental, economical, and medicinally valuable plants. The current study involves the analytical and pharmacological assessment of the methanolic extracts of three less investigated Asteraceae plants, namely Echinops ritro, Centaurea deflexa, and Tripleurospermum decipiens, obtained by three different extraction methodologies viz. maceration (MAC), ultrasound-assisted extraction (UAE), and homogenizer-assisted extraction (HAE). LC-MS-MS analysis of E. ritro, C. deflexa, and T. decipiens extracts led to the identification of ca. 29, 20, and 33 metabolites, respectively, belonging to flavonoids, phenolic acids, and fatty acids/amides. Although there were significant differences in the quantitative metabolite profiles in the extracts of E. ritro and T. decipiens based on the used extraction method, no significant variation was observed in the extracts of C. deflexa in the three implemented extraction techniques. The antioxidant activities of the nine extracts were assessed in vitro using six different assays viz. DPPH, ABTS, CUPRAC, FRAP, PDA, and metal chelation assay (MCA). The HAE/UAE extracts of E. ritro and the UAE/ MAC extracts of C. deflexa displayed the highest antioxidant activity in the DPPH assay, while the UAE extract of T. decipiens showed the strongest antioxidant activity in both the CUPRAC and MCA assays. The enzyme inhibitory activities of the nine extracts were studied in vitro on five different enzymes viz. tyrosinase, α-amylase, α-glucosidase, acetylcholinesterase (AChE), and butyrylcholinestrase (BChE), affecting various pathological diseases. Concerning C. deflexa, its MAC /UAE extracts showed the strongest inhibition on α-amylase, while its UAE/HAE extracts displayed strong inhibitory power on AChE. However, no significant difference was observed on their effects on tyrosinase or BChE. For T. decipiens, its UAE/HAE showed potent inhibition to α-glucosidase, MAC/ HAE significantly inhibited AChE and BChE, while UAE could strongly inhibit tyrosinase enzyme. For E. ritro, all extracts equally inhibited α-amylase and α-glucosidase, MAC/HAE strongly affected tyrosinase, HAE/MAC best inhibited BChE, while HAE inhibited AChE to a greater extent. Chemometric analysis using PCA plot was able to discriminate between the plant samples and between the implemented extraction modes. The in vitro enzyme inhibitory activities of the extracts were supported by in silico data, where metabolites, such as the lignan arctiin and the flavonoid vicenin-2, dominating the extract of C. deflexa, displayed strong binding to AChE. Similarly, chlorogenic and dicaffeoyl quinic acids, which are some of the major metabolites in the extracts of E. ritro and T. decipiens, bound with high affinity to α-glucosidase.

Quantitative T1-relaxation corrected metabolite mapping of 12 metabolites in the human brain at 9.4 T

Magnetic resonance spectroscopic imaging (MRSI) is a non-invasive imaging modality that enables observation of metabolites. Applications of MRSI for neuroimaging have shown promise for monitoring and detecting various diseases. This study builds off previously developed techniques of short TR, 1H FID MRSI by correcting for T1-weighting of the metabolites and utilizing an internal water reference to produce quantitative (mmol kg−1) metabolite maps. This work reports and shows quantitative metabolite maps for 12 metabolites for a single slice. Voxel-specific T1-corrections for water are common in MRSI studies; however, most studies use either averaged T1-relaxation times to correct for T1-weighting of metabolites or omit this correction step entirely. This work employs the use of voxel-specific T1-corrections for metabolites in addition to water. Utilizing averaged T1-relaxation times for metabolites can bias metabolite maps for metabolites that have strong differences between T1-relaxation for GM and WM (i.e. Glu). This work systematically compares quantitative metabolite maps to single voxel quantitative results and qualitatively compares metabolite maps to previous works.

A multimodal pipeline using NMR spectroscopy and MALDI-TOF mass spectrometry imaging from the same tissue sample.

NMR spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple-negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several phospholipid metabolite species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy-MALDI imaging to advance metabolomic and lipidomic studies.

Comprehensive metabolite quantitative assay based on alternate metabolomics and lipidomics analyses

Metabolomics-based precision medicine is facing several obstacles including cross-platform data comparison issue and the lack of metabolome benchmark values of healthy population, one of main reasons is the shortage of comprehensive metabolome quantitation methods. Here, we developed an alternate reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) method to quantitatively determine metabolites and lipids. Assisted by a wide set of reference standards and real samples, up to 397 multiple reaction monitoring (MRM) transitions (239 for positive and 158 for negative ion modes) and 1080 MRM transitions (607 for positive and 473 for negative ion modes) were defined respectively in the metabolomic and lipidomic analyses with more than 1000 metabolites and lipids being quantified. Among them, 144 analytes including amines, amino acids, benzenoids, peptides, nucleobases and related, bile acids, carboxylic acids, fatty acids, hormones, indoles and others were absolutely quantified, while carnitines, lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, free fatty acids, sphingomyelins, phosphatidylcholines (PCs), alkyl and alkenyl substituted PCs, phosphatidylethanolamines (PEs), alkyl and alkenyl substituted PEs and triacylglycerols were semiquantified. The developed method was validated to have good analytical characteristics. Analytical results of standard reference material 1950 human plasma had a good agreement with literature data. As a proof of application, this method was used to study serum metabolic pattern changes of patients with hyperuricemia and nonalcoholic fatty liver. This alternate RPLC-MS method for quantitative metabolites and lipids analysis can further be used to provide technology and large-scale data support for precision medicine and life sciences.

Development and Characterization of Indole-Responsive Whole-Cell Biosensor Based on the Inducible Gene Expression System from Pseudomonas putida KT2440

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/PPP_RS00425 from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/PPP_RS00425 is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization.