Comprehensive Metabolite Profiling Research Articles

Abstract 5709: Disruption of mitochondrial metabolism underlies the efficacy of devimistat in acute myeloid leukemia

Abstract Background: Acute myeloid leukemia (AML) remains an agressive malignancy with poor clinical outcomes, particularly in older patients. Dysregulated metabolism has long presented a promising target for cancer therapeutics; however, the development of clinical-stage therapies targeting cancer cell metabolism has been limited and faces challenges to achieve both efficacy and acceptable toxicity. As an exception, the lipoic acid analogue devimistat has shown positive clinical outcomes in numerous early-phase clinical trials including for relapsed and refractory AML patients. While inhibition of lipoic acid-dependent ketoacid dehydrogenase (KADH) enzymes has been proposed as the mechanism of these antineoplastic effects, the mechanisms underlying the efficacy devimistat and its possible relation to metabolism in patients has not been established. Methods: Samples of bone marrow and blood plasma from patients with relapsed or refractory AML before and after treatment with devimistat were collected. The samples were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) methods to profile ~400 polar metabolites. Kinetic flux profiling in cultured cancer cell lines treated with devimistat was also performed. Results: Analysis of blood plasma samples collected following devimistat treatment revealed detectable levels of drug, consistent with clinically therapeutic concentrations. Comprehensive metabolite profiling of pretreatment and posttreatment blood plasma samples revealed distinct metabolic alterations induced by devimistat administration. Devimistat is thought to inhibit ketoacid dehydrogenase (KADH) enzyme, and consistent with this proposed mechanism of action, accumulations of KADH substrates were observed in the posttreatment samples. Local metabolism in the tumor microenvironment was also altered by devimistat therapy, with robust reductions of TCA intermediates measured in bone marrow plasma samples collected after treatment. Interestingly, quantification of these metabolites revealed an association between overall clinical outcomes and changes in concentrations of bone marrow metabolites. Kinetic flux tracing demonstrated an expected reduction of mitochondrial metabolic flux in cultured tumor cells following treatment with devimistat. Conclusions: Comprehensive metabolite profiling reveals that devimistat targets KADH enzymes and reduces mitochondrial metabolism in humans. Furthermore, these metabolic changes are quantitatively associated with clinical outcomes, posing a potential opportunity for biomarker development and precision metabolic therapy. Citation Format: Michael A. Reid, Shree Bose, Zhengtao Xiao, Peter G. Mikhael, Juan Liu, Jason W. Locasale, Timothy S. Pardee. Disruption of mitochondrial metabolism underlies the efficacy of devimistat in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5709.

Metabolite Profiling and Chemometric Study for the Discrimination Analyses of Geographic Origin of Perilla (Perilla frutescens) and Sesame (Sesamum indicum) Seeds.

Perilla and sesame are traditional sources of edible oils in Asian and African countries. In addition, perilla and sesame seeds are rich sources of health-promoting compounds, such as fatty acids, tocopherols, phytosterols and policosanols. Thus, developing a method to determine the geographic origin of these seeds is important for ensuring authenticity, safety and traceability and to prevent cheating. We aimed to develop a discriminatory predictive model for determining the geographic origin of perilla and sesame seeds using comprehensive metabolite profiling coupled with chemometrics. The orthogonal partial least squares-discriminant analysis models were well established with good validation values (Q2 = 0.761 to 0.799). Perilla and sesame seed samples used in this study showed a clear separation between Korea and China as geographic origins in our predictive models. We found that glycolic acid could be a potential biomarker for perilla seeds and proline and glycine for sesame seeds. Our findings provide a comprehensive quality assessment of perilla and sesame seeds. We believe that our models can be used for regional authentication of perilla and sesame seeds cultivated in diverse geographic regions.

Alternate reversed-phase and hydrophilic interaction liquid chromatography coupled with mass spectrometry for broad coverage in metabolomics analysis

Because of the greatly different physicochemical properties of metabolites, comprehensive metabolite profiling analysis has always been a challenging task. Reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) have been used to the analysis of nonpolar metabolites and polar metabolites, respectively. In this work, an alternate HILIC/RPLC-mass spectrometry (MS) approach was developed for the comprehensive and high-throughput analysis of polar and nonpolar metabolites. HILIC and RPLC are respectively performed on two ultra-high performance LC (UHPLC) systems, and coupled to one mass spectrometer to acquire the data. When HILIC gradient elution is running RPLC is in a washing and equilibration state, and vice versa. As a result, the total analysis time was reduced by about one third to 25.4 min. Two hundred and eight representative standards including at least twelve types of commonly met metabolites, SRM 1950 plasma, serum, urine and liver tissue samples were used to test the established alternate HILIC/RPLC-MS method. The results demonstrated that the method possessed high metabolite coverage. The developed method was validated to have good linearity and repeatability. As an example of application, 61 significantly changed metabolites in the colon cancer tissues were defined by this established method.

Comprehensive lipid and metabolite profiling in healthy adults with low and high consumption of fatty fish: a cross-sectional study.

Fish consumption is associated with reduced risk of CVD, which may be partly mediated by alterations in plasma lipids, such as HDL-cholesterol. However, comprehensive analyses of associations between fatty fish consumption and lipoprotein subclass profile are limited and show inconsistent results. Therefore, the aim of the present exploratory study was to investigate the association between fatty fish consumption and lipoprotein subclass particle concentrations and composition, with an emphasis on HDL. We performed a comprehensive plasma metabolite profiling in 517 healthy adults, using a targeted high-throughput NMR spectroscopy platform. The participants were divided into tertiles based on consumption of fatty fish, reported through a validated FFQ. We compared the concentration of metabolites between the participants in the lowest and highest tertiles of fatty fish consumption. We show that high consumers of fatty fish (>223 g/week, median intake 294 g/week) had higher particle concentrations and content of total lipids, free cholesterol and phospholipids in large and extra-large HDL particles and higher content of total cholesterol, cholesteryl esters and TAG in large HDL particles than low consumers (<107 g/week, median intake 58 g/week). Using fatty fish consumption as a continuous variable, we found that fatty fish consumption was associated with lower levels of the inflammation marker glycoprotein acetyls. In conclusion, high consumers of fatty fish seem to have a more favourable HDL-cholesterol-related lipoprotein profile and anti-inflammatory phenotype than low consumers of fatty fish. Thus, these data support the current Norwegian dietary recommendations for fish consumption regarding CVD risk.

Linden tea from Serbia – an insight into the phenolic profile, radical scavenging and antimicrobial activities

Linden tea has been used in Serbian folk medicine for centuries to induce sweating for colds, relieve throat irritation and cough, reduce blood pressure, as well as a diuretic, spasmolytic, and as a sedative. The main aim of this study was to determine the comprehensive metabolite profile, free radical scavenging activity, and antimicrobial activities against 23 human and plant pathogens of both commercial and field-collected linden tea samples from Serbia. Developed high-performance thin-layer chromatography (HPTLC) method allowed identification of eight major metabolites in investigated samples, while forty-six metabolites were tentatively identified using high resolution mass spectrometry (HRMS): 14 phenolic acids and their derivatives, 15 glycosides, 9 flavonoids, and 8 procyanidins. Both commercial and field-collected linden tea samples showed similar total phenolic content and radical scavenging activity. Seven compounds such as p-hydroxybenzoic acid 4-O-hexoside, caffeic acid cinnamyl ester, pinocembrin, galangin, luteolin 7-O-glucuronide (and its isomer), isorhamnetin hexosyl hexoside were found in Tilia samples for the first time. Radical reagent 2,2-Diphenyl-1-picrylhydrazyl· (DPPH·) was used in DPPH-HPTLC assay which identified chlorogenic acid, caffeic acid, astragalin, quercitrin, tiliroside, and rutin as compounds that exhibit radical scavenging activity. Tilia samples showed inhibitory effect overwhelming only on Gram-positive bacteria, especially on Bacillus subtilis with the lowest MIC values observed, as well as towards Staphylococcus aureus and oral cavity isolates of Streptococcus pyogenes and Streptococcus mutans. Vaginal isolate of Candida glabrata showed more susceptibility than Candida albicans isolate. Generally, extract from Tilia cordata Miller (L4) showed the highest antimicrobial activity against the most of the tested pathogens, among all field-collected Tilia samples.

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles

Ethnopharmacological relevanceEthnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. Aim of the studyA pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. Materials and methodsThe roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. ResultsThe RME showed 80% inhibition of PTZ-induced locomotor activity (300 μg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 μM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 μM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35–37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. ConclusionsThis study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.

Comparative metabolic profiling of cultivated and wild black soybeans reveals distinct metabolic alterations associated with their domestication

Generally, cultivated black soybean (CBS) has been used as a major source of various nutrients for humans and animals. To assess the metabolic alterations induced by domestication in soybean, we performed a comprehensive metabolite profiling of 56 soybean varieties, including 28 CBS and 28 wild black soybean (WBS) varieties. A total of 48 metabolites were characterized, including 45 primary and 3 secondary metabolites, from CBS and WBS. The results of principal component analysis and hierarchical cluster analysis (HCA) revealed significant metabolic differences between CBS and WBS that were closely related to metabolic pathways. The results indicate that flavonoids correlated positively with phenylalanine, a precursor for phenylpropanoid biosynthesis; the contents of flavonoids and phenylpropanoids were higher in WBS. Pathway analysis revealed that CBS contained large amounts of TCA cycle intermediates, amino acids, and fatty acids as a result of increased energy metabolism, amino acid metabolism, and seed filling. The projection to latent structure method, using the partial least squares method, was applied to predict the flavonoid content in soybean seed, which indicated that sucrose, threonic acid, citric acid, and fatty acids are important in predicting the antioxidant content of samples. This work will provide important information for designing new soybean cultivars with enhanced nutritional and agricultural traits.

Differential annotation of converted metabolites (DAC-Met): Exploration of Maoto (Ma-huang-tang)-derived metabolites in plasma using high-resolution mass spectrometry

IntroductionTraditional herbal medicine (THM) contains a vast number of natural compounds with varying degrees of pharmacological activity. To elucidate the mode of action, comprehensive metabolite profiling in the plasma before and after administration of THM is essential.ObjectiveThe aim of this study was to explore and identify/annotate converted metabolites after administration of THM in humans.MethodsWe performed untargeted metabolome analysis of human plasma collected before and after administration of maoto (ma-huang-tang), a traditional Japanese Kampo medicine. Maoto-derived metabolites were then selected and annotated following the DAC-Met strategy, which is an annotation method that uses mass differences of major metabolic reactions among the detected peaks and a differential network analysis.ResultsAbout 80% of maoto-derived components were found to be converted forms. Following DAC-Met, the structures of 15 previously unidentified metabolites were determined, and five of these were later confirmed with authentic standards. Using published literature, we also reconstructed the metabolic pathway of maoto components in humans. A kinetic time-course analysis revealed their diverse kinetic profiles.ConclusionThe results demonstrated that time-resolved comprehensive metabolite profiling in plasma using the DAC-Met strategy is highly useful for elucidating the complex nature of THM.

Untargeted and Targeted Metabolomic Profiling of Australian Indigenous Fruits.

Selected Australian native fruits such as Davidson’s plum, finger lime and native pepperberry have been reported to demonstrate potent antioxidant activity. However, comprehensive metabolite profiling of these fruits is limited, therefore the compounds responsible are unknown, and further, the compounds of nutritional value in these native fruits are yet to be described. In this study, untargeted and targeted metabolomics were conducted using the three fruits, together with assays to determine their antioxidant activities. The results demonstrate that targeted free and hydrolysed protein amino acids exhibited high amounts of essential amino acids. Similarly, important minerals like potassium were detected in the fruit samples. In antioxidant activity, Davidson’s plum reported the highest activity in ferric reducing power (FRAP), finger lime in antioxidant capacity (ABTS), and native pepperberry in free radical scavenging (DPPH) and phosphomolybdenum assay. The compounds responsible for the antioxidant activity were tentatively identified using untargeted GC×GC-TOFMS and UHPLC-QqQ-TOF-MS/MS metabolomics. A clear discrimination into three clusters of fruits was observed using principal component analysis (PCA) and partial least squares (PLS) analysis. The correlation study identified a number of compounds that provide the antioxidant activities. GC×GC-TOFMS detected potent aroma compounds of limonene, furfural, and 1-R-α-pinene. Based on the untargeted and targeted metabolomics, and antioxidant assays, the nutritional potential of these Australian bush fruits is considerable and supports these indigenous fruits in the nutraceutical industry as well as functional ingredients for the food industry, with such outcomes benefiting Indigenous Australian communities.

Primary Metabolite Responses to Oxidative Stress in Early-Senescing and Paraquat Resistant Arabidopsis thaliana rcd1 (Radical-Induced Cell Death1).

Rcd1 (radical-induced cell death1) is an Arabidopsis thaliana mutant, which exhibits high tolerance to paraquat [methyl viologen (MV)], herbicide that interrupts photosynthetic electron transport chain causing the formation of superoxide and inhibiting NADPH production in the chloroplast. To understand the biochemical mechanisms of MV-resistance and the role of RCD1 in oxidative stress responses, we performed metabolite profiling of wild type (Col-0) and rcd1 plants in light, after MV exposure and after prolonged darkness. The function of RCD1 has been extensively studied at transcriptomic and biochemical level, but comprehensive metabolite profiling of rcd1 mutant has not been conducted until now. The mutant plants exhibited very different metabolic features from the wild type under light conditions implying enhanced glycolytic activity, altered nitrogen and nucleotide metabolism. In light conditions, superoxide production was elevated in rcd1, but no metabolic markers of oxidative stress were detected. Elevated senescence-associated metabolite marker levels in rcd1 at early developmental stage were in line with its early-senescing phenotype and possible mitochondrial dysfunction. After MV exposure, a marked decline in the levels of glycolytic and TCA cycle intermediates in Col-0 suggested severe plastidic oxidative stress and inhibition of photosynthesis and respiration, whereas in rcd1 the results indicated sustained photosynthesis and respiration and induction of energy salvaging pathways. The accumulation of oxidative stress markers in both plant lines indicated that MV-resistance in rcd1 derived from the altered regulation of cellular metabolism and not from the restricted delivery of MV into the cells or chloroplasts. Considering the evidence from metabolomic, transcriptomic and biochemical studies, we propose that RCD1 has a negative effect on reductive metabolism and rerouting of the energy production pathways. Thus, the altered, highly active reductive metabolism, energy salvaging pathways and redox transfer between cellular compartments in rcd1 could be sufficient to avoid the negative effects of MV-induced toxicity.

Cortisol Measurements in Cushing's Syndrome: Immunoassay or Mass Spectrometry?

Determination of cortisol levels in the urine (24 hours urine free cortisol), saliva (late-night), or serum (total cortisol after dexamethasone suppression) is recommended to screen for Cushing's syndrome (CS). This review focuses on the differences between the frequently used cortisol-antibody immunoassay-based methods and the highly specific mass-spectrometry-based methods that are progressively being employed in clinical laboratories for CS screening. The particular characteristics of cortisol metabolism and the lack of specificity of the immunoassays cause marked differences between both methods that are in turn highly dependent on the biological matrix, in which the cortisol is measured. Understanding the origin of these differences is essential for the interpretation of these results. Although cross-reactivity with endogenous steroids leads to grossly inaccurate results of immunoassay measurements of cortisol in the saliva and urine, preliminary evidence suggests that the clinical sensitivity of CS screening using immunoassays may be similar to CS screening using mass spectrometry. However, mass spectrometry offers more accurate results and considerably reduced variation across laboratories, while avoiding false-positive results. Moreover, mass spectrometry can overcome some common diagnostic challenges, such as identification of exogenous corticosteroids or simultaneous assessment of appropriate dexamethasone levels in suppression tests. Further, comprehensive mass spectrometry-based profiling of several steroid metabolites may be useful for discriminating among different subtypes of CS. Finally, this review discusses the main preanalytical factors that could cause variations in cortisol measurements and their influence on the reliability of the results.

Metabolite and Phytohormone Profiling Illustrates Metabolic Reprogramming as an Escape Strategy of Deepwater Rice during Partially Submerged Stress.

Rice varieties that can survive under submergence conditions respond to flooding either by enhancing internode elongation or by quiescence of shoot elongation. Despite extensive efforts to identify key metabolites triggered by complete submergence of rice possessing SUBMERGENCE 1 (SUB1) locus, metabolic responses of internode elongation of deepwater rice governed by the SNORKEL 1 and 2 genes remain elusive. This study investigated specific metabolomic responses under partial submergence (PS) to deepwater- (C9285) and non-deepwater rice cultivars (Taichung 65 (T65)). In addition, we examined the response in a near-isogenic line (NIL-12) that has a C9285 genomic fragment on chromosome 12 introgressed into the genetic background of T65. Under short-term submergence (0–24 h), metabolite profiles of C9285, NIL-12, and T65 were compared to extract significantly changed metabolites in deepwater rice under PS conditions. Comprehensive metabolite and phytohormone profiling revealed increases in metabolite levels in the glycolysis pathway in NIL-12 plants. Under long-term submergence (0–288 h), we found decreased amino acid levels. These metabolomic changes were opposite when compared to those in flood-tolerant rice with SUB1 locus. Auxin conjugate levels related to stress response decreased in NIL-12 lines relative to T65. Our analysis helped clarify the complex metabolic reprogramming in deepwater rice as an escape strategy.

Characterization of tea leaf metabolites dependent on tea (Camellia sinensis) plant age through 1H NMR-based metabolomics

The chemical or metabolic compositions of tea (Camellia sinensis) varies according to numerous factors, such as geographical origin, cultivar, climate, plucking position, and horticultural practices. However, how the age of tea plants affects the metabolite compositions of tea leaves has not been reported yet. Therefore, we extended the metabolomic approach to the investigation of the age-related differences of tea leaf metabolites in the fresh leaves collected from tea plants aged 8 and 25 years. Multivariate statistical analysis with comprehensive metabolite profiles analyzed by 1H NMR spectroscopy showed the clear metabolic differentiation between the fresh tea leaves from different ages of the tea plants. Of the various tea leaf metabolites varied according to the age of the tea plants, theanine, glutamine, catechin, and gallocatechin were uniquely dependent on the age of tea plants, demonstrating a difference of theanine metabolism between young and old tea plants. These results suggest that leaves from 25-year-old tea plants would still be worthy as a functional ingredient for the production in the food or cosmetic industry rather than quality-enhanced tea infusions for human consumption.

Emerging Insights into the Metabolic Alterations in Aging Using Metabolomics.

Metabolomics is the latest ‘omics’ technology and systems biology science that allows for comprehensive profiling of small-molecule metabolites in biological systems at a specific time and condition. Metabolites are cellular intermediate products of metabolic reactions, which reflect the ultimate response to genomic, transcriptomic, proteomic, or environmental changes in a biological system. Aging is a complex biological process that is characterized by a gradual and progressive decline in molecular, cellular, tissue, organ, and organismal functions, and it is influenced by a combination of genetic, environmental, diet, and lifestyle factors. The precise biological mechanisms of aging remain unknown. Metabolomics has emerged as a powerful tool to characterize the organism phenotypes, identify altered metabolites, pathways, novel biomarkers in aging and disease, and offers wide clinical applications. Here, I will provide a comprehensive overview of our current knowledge on metabolomics led studies in aging with particular emphasis on studies leading to biomarker discovery. Based on the data obtained from model organisms and humans, it is evident that metabolites associated with amino acids, lipids, carbohydrate, and redox metabolism may serve as biomarkers of aging and/or longevity. Current challenges and key questions that should be addressed in the future to advance our understanding of the biological mechanisms of aging are discussed.

Genome-wide Dissection of Co-selected UV-B Responsive Pathways in the UV-B Adaptation of Qingke

Qingke (Tibetan hulless barley) has long been cultivated and exposed to long-term and strong UV-B radiation on the Tibetan Plateau, which renders it an ideal species for elucidating novel UV-B responsive mechanisms in plants. Here we report a comprehensive metabolite profiling and metabolite-based genome-wide association study (mGWAS) using 196 diverse qingke and barley accessions. Our results demonstrated both constitutive and induced accumulation, and common genetic regulation, of metabolites from different branches of the phenylpropanoid pathway that are involved in UV-B protection. A total of 90 significant mGWAS loci for these metabolites were identified in barley-qingke differentiation regions, and a number of high-level metabolite trait alleles were found to be significantly enriched in qingke, suggesting co-selection of various phenylpropanoids. Upon dissecting the entire phenylpropanoid pathway, we identified some key determinants controlling natural variation of phenylpropanoid content, including three novel proteins, a flavone C-pentosyltransferase, a tyramine hydroxycinnamoyl acyltransferase, and a MYB transcription factor. Our study, furthermore, demonstrated co-selection of both constitutive and induced phenylpropanoids for UV-B protection in qingke.

The Origin and Evolution of Plant Flavonoid Metabolism.

During their evolution, plants have acquired the ability to produce a huge variety of compounds. Unlike the specialized metabolites that accumulate in limited numbers of species, flavonoids are widely distributed in the plant kingdom. Therefore, a detailed analysis of flavonoid metabolism in genomics and metabolomics is an ideal way to investigate how plants have developed their unique metabolic pathways during the process of evolution. More comprehensive and precise metabolite profiling integrated with genomic information are helpful to emerge unexpected gene functions and/or pathways. The distribution of flavonoids and their biosynthetic genes in the plant kingdom suggests that flavonoid biosynthetic pathways evolved through a series of steps. The enzymes that form the flavonoid scaffold structures probably first appeared by recruitment of enzymes from primary metabolic pathways, and later, enzymes that belong to superfamilies such as 2-oxoglutarate-dependent dioxygenase, cytochrome P450, and short-chain dehydrogenase/reductase modified and varied the structures. It is widely accepted that the first two enzymes in flavonoid biosynthesis, chalcone synthase, and chalcone isomerase, were derived from common ancestors with enzymes in lipid metabolism. Later enzymes acquired their function by gene duplication and the subsequent acquisition of new functions. In this review, we describe the recent progress in metabolomics technologies for flavonoids and the evolution of flavonoid skeleton biosynthetic enzymes to understand the complicate evolutionary traits of flavonoid metabolism in plant kingdom.

Ancient Danish Apple Cultivars-A Comprehensive Metabolite and Sensory Profiling of Apple Juices.

In recent decades, intensive selective breeding programs have allowed the development of disease-resistant and flavorsome apple cultivars while leading to a gradual decline of a large number of ancient varieties in many countries. However, the re-evaluation of such cultivars could lead to the production new apple-based products with health beneficial properties and/or unique flavor qualities. Herein, we report the comprehensive characterization of juices obtained from 86 old, mostly Danish, apple cultivars, by employing traditional analysis (ion chromatography, °Brix, headspace gas chromatography/mass spectrometry (GC–MS), and panel test evaluation) as well as an innovative nuclear magnetic resonance (NMR)-based screening method developed by Bruker for fruit juices, known as Spin Generated Fingerprint (SGF) Profiling™. Principal component analysis showed large differences in aroma components and sensory characteristics, including odd peculiar odors and flavors such as apricot and peach, and very different levels of phenolic compounds, acids and sugars among the analyzed juices. Moreover, we observed a tendency for late-season juices to be characterized by higher °Brix values, sugar content and they were perceived to be sweeter and more flavor intense than early-season juices. Our findings are useful for the production of specialty vintage-cultivar apple juices or mixed juices to obtain final products that are characterized both by healthy properties and peculiar sensory attributes.

Metabolomics Analysis of Skeletal Muscles from FKRP-Deficient Mice Indicates Improvement After Gene Replacement Therapy

Muscular dystrophy-dystroglycanopathies comprise a heterogeneous and complex group of disorders caused by loss-of-function mutations in a multitude of genes that disrupt the glycobiology of α-dystroglycan, thereby affecting its ability to function as a receptor for extracellular matrix proteins. Of the various genes involved, FKRP codes for a protein that plays a critical role in the maturation of a novel glycan found only on α-dystroglycan. Yet despite knowing the genetic cause of FKRP-related dystroglycanopathies, the molecular pathogenesis of disease and metabolic response to therapeutic intervention has not been fully elucidated. To address these challenges, we utilized mass spectrometry-based metabolomics to generate comprehensive metabolite profiles of skeletal muscle across diseased, treated, and normal states. Notably, FKRP-deficient mice elicit diverse metabolic abnormalities in biomarkers of extracellular matrix remodeling and/or aging, pentoses/pentitols, glycolytic intermediates, and lipid metabolism. More importantly, the restoration of FKRP protein activity following AAV-mediated gene therapy induced a substantial correction of these metabolic impairments. While interconnections of the affected molecular mechanisms remain unclear, our datasets support the notion that global metabolic profiling can be valuable for determining the involvement of previously unsuspected regulatory or pathological pathways as well as identifying potential targets for drug discovery and diagnostics.

Comparison of the Phytochemical Composition of Serenoa repens Extracts by a Multiplexed Metabolomic Approach.

Phytochemical extracts are highly complex chemical mixtures. In the context of an increasing demand for phytopharmaceuticals, assessment of the phytochemical equivalence of extraction procedures is of utmost importance. Compared to routine analytical methods, comprehensive metabolite profiling has pushed forward the concept of phytochemical equivalence. In this study, an untargeted metabolomic approach was used to cross-compare four marketed extracts from Serenoa repens obtained with three different extraction processes: ethanolic, hexanic and sCO2 (supercritical carbon dioxide). Our approach involved a biphasic extraction of native compounds followed by liquid chromatography coupled to a high-resolution mass spectrometry based metabolomic workflow. Our results showed significant differences in the contents of major and minor compounds according to the extraction solvent used. The analyses showed that ethanolic extracts were supplemented in phosphoglycerides and polyphenols, hexanic extracts had higher amounts of free fatty acids and minor compounds, and sCO2 samples contained more glycerides. The discriminant model in this study could predict the extraction solvent used in commercial samples and highlighted the specific biomarkers of each process. This metabolomic survey allowed the authors to assess the phytochemical content of extracts and finished products of S. repens and unequivocally established that sCO2, hexanic and ethanolic extracts are not chemically equivalent and are therefore unlikely to be pharmacologically equivalent.

Untargeted Metabolomics and Cognitive Function in the Boston Puerto Rican Health Study (OR32-03-19)

ObjectivesWe sought to determine the metabolic signature associated with poor vs good cognition in a cohort of Boston-area Puerto Ricans. MethodsWe studied 638 participants the Boston Puerto Rican Health Study (BRPHS), a prospective Boston-area cohort study of Puerto Ricans, with comprehensive metabolite profiling (>700 metabolites) at baseline and a concurrent assessment of global cognitive function. Cognition was assessed at baseline using a comprehensive cognitive battery from which a global cognitive score was derived.Our analysis focused on 638 annotated metabolites that had reported values in ≥80% of the participants. We performed univariate linear regression analyses to identify metabolites significantly associated with global cognitive score. To examine whether these metabolites could then discriminate between the low from (<=1SD below the mean, N = 85) the high (>=1SD above the mean, N = 99) cognition groups, and to identify metabolites that could best discriminate between low and high cognition groups, we conducted partial least square discrimination analysis (PLS-DA). We used separation distance as the test statistic and plotted the scores of the first two partial least square components to inspect the separation of cases from controls. We also identified metabolites contributing most strongly to the separation (VIP > 1). Results113 metabolites were significantly associated with cognitive function in univariate analyses. In PLS-DA analyses using these 113 metabolites. when plotting the first two PLS components, there was a clear separation between the low and high cognition groups and controls (Figure 1) (permutation test based on separation distance p-value 0.012) (Figure 1). 23 metabolites had a Variable Importance in Projection (VIP) scores > 1. The metabolites with the highest VIP scores and elevated in the low relative to the high cognition group included cystathionine (amino acid and downstream metabolite of homocysteine), quinate (xenobiotic/food component, component medication used to treat malaria) and p-cresol-glucuronide (amino acid). (Table 1). ConclusionsIn this cross sectional study of Boston Area Puerto Ricans, we observed a separation between high and low cognitive function groups in the Boston Puerto Rican study based on metabolomic profiles. Funding SourcesThis work was supported by the National Institutes of Health. Supporting Tables, Images and/or Graphs▪▪