NMR Metabolomic Profiling Research Articles

NMR-Metabolomic Profiling and Genome Mining Drive the Discovery of Cyclic Decapeptides from a Marine Streptomyces.

The integration of NMR-metabolomic and genomic analyses can provide enhanced identification of structural properties as well as key biosynthetic information, thus achieving the targeted discovery of new natural products. For this purpose, NMR-based metabolomic profiling of the marine-derived Streptomyces sp. S063 (CGMCC 14582) was performed, by which N-methylated peptides possessing unusual negative 1H NMR chemical shift values were tracked. Meanwhile, genome mining of this strain revealed the presence of an unknown NRPS gene cluster (len) with piperazic-acid-encoding genes (lenE and lenF). Under the guidance of the combined information, two cyclic decapeptides, lenziamides D1 (1) and B1 (2), were isolated from Streptomyces sp. S063, which contains piperazic acids with negative 1H NMR values. The structures of 1 and 2 were determined by extensive spectroscopic analysis combined with Marfey's method and ECD calculations. Furthermore, we provided a detailed model of lenziamide (1 and 2) biosynthesis in Streptomyces sp. S063. In the cytotoxicity evaluation, 1 and 2 showed moderate growth inhibition against the human cancer cells HEL, H1975, H1299, and drug-resistant A549-taxol with IC50 values of 8-24 μM.

The role of NMR-based circulating metabolic biomarkers in development and risk prediction of new onset type 2 diabetes

Associations of circulating metabolic biomarkers with type 2 diabetes (T2D) and their added value for risk prediction are uncertain among Chinese adults. A case-cohort study included 882 T2D cases diagnosed during 8-years’ follow-up and a subcohort of 789 participants. NMR-metabolomic profiling quantified 225 plasma biomarkers in stored samples taken at recruitment into the study. Cox regression yielded adjusted hazard ratios (HRs) for T2D associated with individual biomarkers, with a set of biomarkers incorporated into an established T2D risk prediction model to assess improvement in discriminatory ability. Mean baseline BMI (SD) was higher in T2D cases than in the subcohort (25.7 [3.6] vs. 23.9 [3.6] kg/m2). Overall, 163 biomarkers were significantly and independently associated with T2D at false discovery rate (FDR) controlled p < 0.05, and 138 at FDR-controlled p < 0.01. Branched chain amino acids (BCAA), apolipoprotein B/apolipoprotein A1, triglycerides in VLDL and medium and small HDL particles, and VLDL particle size were strongly positively associated with T2D (HRs 1.74–2.36 per 1 SD, p < 0.001). HDL particle size, cholesterol concentration in larger HDL particles and docosahexaenoic acid levels were strongly inversely associated with T2D (HRs 0.43–0.48, p < 0.001). With additional adjustment for plasma glucose, most associations (n = 147 and n = 129 at p < 0.05 and p < 0.01, respectively) remained significant. HRs appeared more extreme among more centrally adipose participants for apolipoprotein B/apolipoprotein A1, BCAA, HDL particle size and docosahexaenoic acid (p for heterogeneity ≤ 0.05). Addition of 31 selected biomarkers to an established T2D risk prediction model modestly, but significantly, improved risk discrimination (c-statistic 0.86 to 0.91, p < 0.001). In relatively lean Chinese adults, diverse metabolic biomarkers are associated with future risk of T2D and can help improve established risk prediction models.

O-258 Nuclear magnetic resonance profiling of the vaginal metabolome in adverse early pregnancy outcomes

Abstract Study question Is the vaginal metabolome predictive of early pregnancy outcomes? Summary answer The vaginal metabolome is moderately predictive of live normally-sited pregnancies when compared to a pregnancy with an adverse outcome. What is known already Approximately 15% of all pregnancies are lost in the first trimester and a further 1-2% are ectopic. To date, no non-invasive and clinically useful tests have been developed to differentiate live normally-sited pregnancies from non-viable and ectopic pregnancies. Metabolomics is the comprehensive analysis of small molecules in biological samples used to identify disease biomarkers. The vaginal metabolome has previously been shown to be predictive of later adverse pregnancy outcomes including preterm birth. In this study, we aimed to identify biomarkers of live normally-sited pregnancies when compared to non-viable intrauterine and ectopic pregnancies using NMR metabolomic profiling of vaginal swabs (LVS). Study design, size, duration This was a prospective cohort study (EXPEDITE). 222 pregnant women &amp;lt;10 weeks’ gestation with pain and/or bleeding were recruited over a period of 36 months at the Liverpool Women’s Hospital, a tertiary referral centre. Pregnancy outcomes were categorised according to ESHRE guidelines as live normally-sited pregnancy, miscarriage, tubal ectopic pregnancy or pregnancy of unknown location (PUL). Participants/materials, setting, methods Participants consented to self-collecting a LVS, which was extracted in acetonitrile and analysis by NMR spectroscopy; 1D 1H and 2D 1H-13C. Spectra were acquired at 25 °C on a 700 MHz spectrometer. Relative metabolite abundances underwent univariate and multivariate statistical analysis using MetaboAnalyst 5.0 and custom-built R scripts (p-value false discovery rate adjusted). Area under receiver operating characteristic curve (AUC) values were used to evaluate predictive models. Main results and the role of chance Our cohort of 222 women were categorised in to 100 (45 %) live normally-sited pregnancies, 29 (13 %) tubal ectopic pregnancies, 52 (23 %) miscarriages and 31 (14%) pregnancies of unknown location (PUL). Ten spectra from rare or uncertain pregnancy outcomes (e.g. molar pregnancy) were excluded. Univariate statistical analysis of all four groups identified isoleucine levels as significantly higher (p &amp;lt; 0.05) in live normally-sited pregnancies compared with all groups with adverse pregnancy outcome groups. Multivariate partial least squares discriminant analysis (PLS-DA) models of all groups failed to accurately predict pregnancy outcomes. To enhance the predictive models, miscarriage, tubal ectopic pregnancy and PUL groups were combined and compared to live normally-sited pregnancies. Univariate analysis revealed levels of several metabolites (including isoleucine, tyrosine and maltose) as significantly altered in live normally-sited pregnancies compared with all other outcomes. PLS-DA models demonstrated modest discrimination between live normally-sited pregnancies versus other outcomes (AUC = 0.75). In summary, this study has identified a vaginal metabolite profile and potential biomarkers associated with live normally-sited pregnancy in the first trimester. Limitations, reasons for caution The number of tubal ectopic pregnancies and PULs were relatively low compared to other outcomes. Additionally, several factors that may influence the vaginal metabolome, including infection and the vaginal microbiome, were not characterised in this study. Wider implications of the findings These findings may be useful in the development of diagnostic tests to confirm an intrauterine pregnancy and exclude potential life-threatening pregnancies. It is well established that the microbiome contributes to the vaginal metabolome. As such, our findings will inform future correlative studies of vaginal microbiome and metabolome in early pregnancy. Trial registration number NA

Critical assessment of metabolism and related growth and quality traits in trout fed spirulina-supplemented plant-based diets

Nowadays in aquaculture, the nutrition of carnivorous fish species requires the development of sustainable plant-based diets. Supplementation with microorganisms such as spirulina could offer an alternative to full plant-based diets that are inappropriate for carnivorous species such as rainbow trout. However, the benefits and drawbacks of spirulina supplementation on fish performance and metabolism need to be assessed to substantiate spirulina as an appropriate ingredient. The objective of this work was to determine the metabolic utilization of a plant-based diet supplemented with two levels of spirulina (5% and 15%) and to compare it to a full plant-based (PB) and a commercial-like (COM) diet for trout. Spirulina supplementation induced both a decrease in feed intake and an increase in feed efficiency while maintaining growth and a putative reorientation of lipid deposition from viscera to muscle. Fatty acid changes, including an increase in saturated fatty acids, a decrease in monounsaturated fatty acids, and color changes with an increase in b* index (yellow), suggested a slight alteration of the nutritional and visual quality of fish fed a spirulina diet. 1H NMR metabolomics profiling was used to measure metabolites in experimental feeds and in fish plasma, liver, and muscle. Two specific compounds of the spirulina ingredient were identified and determined to have opposed metabolic fates: i) a quinone-like compound was found to accumulate in liver and ii) β-hydroxybutyrate was found to be completely metabolized. The main metabolites modulated by spirulina in plasma and tissues were involved in energy and amino acid metabolisms. Spirulina supplementation almost suppressed the accumulation of glucose and essential amino acids in fish plasma and muscle induced by the PB diet. However, these compounds, as well as betaine, were found to accumulate in the liver of fish fed spirulina-supplemented diets. An alteration of liver metabolism due to the specific fatty acid profile and quinone-like compound of spirulina is suspected. A correlative approach of fish performance, quality traits, and metabolomic data in plasma and liver suggests that the maintenance of growth rate and feed efficiency using spirulina supplementation of a PB diet requires not only the supply of a well-balanced protein but also the redirection of lipid metabolism. However, observed changes in fatty acid composition and color suggest further alterations of the phospholipid metabolism. This work thus provides a contrasted fate of spirulina supplementation of a PB diet with both expected positive effects and unexpected negative effects that must be investigated further.

Untargeted metabolomics-based response analysis of temperature and insecticide exposure in Aedes aegypti

In this study, we utilized an untargeted NMR metabolomics approach to identify the vector response in terms of metabolic profiling after temperature and insecticide exposure in comparison with the control. Clearly, temperature and insecticide exposure cause changes in the underlying metabolism, and the NMR metabolomic profile enables a direct examination of the immediate response of the vector to cope up with these changes. The present study was designed in four parts: A-Aedes aegypti were exposed to 40 °C for one-hour, DDT-4%, malathion-5%, and deltamethrin-0.05% separately and, part B-D; one-hour exposure at 35 °C and 40 °C temperatures followed by one-hour exposure to insecticide. The resultant metabolite profiles were compared with the control. In response to temperature and insecticide exposure, several metabolites and altered pathways were identified. Citrate, maltose, lipids, Nicotinate, Choline, Pyruvate and β-hydroxybutyrate were found as important components of major biological pathways such as tri-carboxylic acid cycle, branched amino acid degradation, glycolysis/gluconeogenesis, amino acid metabolism, lipid and carbohydrate metabolism, nucleotide PRPP pathway, and phospholipid metabolism. Furthermore, the results also suggest that the changes imposed by exposure to temperature and insecticides individually, are reversed with combined exposure, thus negating the impact of each other and posing a threat to the control of Aedes-borne diseases such as dengue, chikungunya, Zika and yellow fever.

Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome.

Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.

Bean cultivars (Phaseolus vulgaris L.) under the spotlight of NMR metabolomics

The seeds of Phaseolus vulgaris are a rich source of protein consumed around the world and are considered as the most important source of proteins and antioxidants in the Mexican diet. This work reports on the 1H NMR metabolomics profiling of the cultivars Peruano (FPe), Pinto (FPi), Flor de mayo (FM), Negro (FN) and Flor de junio (FJ). Total phenolics, total flavonoids and total protein contents were determined to complement the nutritional facts in seeds and leaves. According to our results, the metabolomics fingerprint of beans seeds and leaves were very similar, showing the presence of 52 metabolites, 46 in seeds and 48 in leaves, including 8 sugars, 17 amino acids, 15 organic acids, 5 nucleosides and 7 miscellaneous compounds. In seeds, free amino acids were detected in higher concentrations than in the leaves, whereas organic acids were more abundant in leaves than in seeds. With multivariate and cluster analysis it was possible to rank the cultivars according to their nutritional properties according to NMR profiling, then a machine learning algorithm was used to reveal the most important differential metabolites which are the key for correct classification. The results coincide in highlighting the FN seeds and FPe leaves for the best nutritional facts. Finally, in terms of cultivars, FN and FM present the best nutritional properties, with high protein and flavonoids content, as well as, a high concentration of amino acids and nucleosides.

1145Obesity-related changes in metabolomic profiles in youth

Abstract Background Obesity during childhood and adolescence is linked to subclinical atherosclerosis, metabolic dysfunction and later cardiovascular disease. Adults with obesity have metabolomic profiles that predict cardiovascular disease and mortality. Analogous data from children with obesity are sparse. Here, we aimed to investigate i) the relationship between the severity of obesity (determined by the percentage above the 95th BMI-centile (%&amp;gt;95th BMI-centile)) and metabolomic profiles, ii) the effect of changes in the severity of obesity on the metabolomic profile and iii) the relationship between obesity-related metabolites and subclinical atherosclerosis outcomes. Methods Participants from the Childhood Overweight Biorepository of Australia (COBRA) cohort had %&amp;gt;95th BMI-centile and NMR metabolomic profile (Nightingale, Finland) from fasted blood analysed at two time-points (mean interval of 5.5 years). At the follow-up visit, subclinical atherosclerosis phenotypes (carotid elasticity, carotid intima-media thickness, and pulse-wave velocity) were assessed. Results There were 98 participants who attended both visit 1 (mean %&amp;gt;95th BMI-centile 134.6±19.0) and visit 2 (mean %&amp;gt;95th BMI-centile 130.7±26.2). Higher absolute, and increasing severity, of obesity between visits were associated with increased phenylalanine, tyrosine, GlycA (a marker of chronic inflammation), and pyruvate, in both sexes (estimated increases of 0.14-0.18 standard deviations per 10% BMI-centile at visit 2, and 0.15-0.25 per 10% increase in BMI-centile between visits). There was modest evidence for a relationship between lower alanine and higher carotid elasticity. Conclusions In children with obesity, the overall severity of obesity and changes in obesity severity were associated with a metabolomic pattern that in adults is predictive of cardiovascular disease. In children, these metabolites were not related subclinical atherosclerosis; these relationships may become manifest with increasing age. Key messages There is evidence for an early effect of severe obesity on metabolomic profiles in childhood and adolescence.

O-234 Using serum metabolomics to identify biomarkers of viable early, intrauterine pregnancy: an untargeted 1H NMR-based approach

Abstract Study question Which metabolites are associated with a viable intrauterine pregnancy (VIUP) when compared to other early pregnancy outcomes (failed intrauterine and ectopic pregnancies)? Summary answer Serum levels of four metabolites (phenylalanine, alanine, glutamate and glutamine) were significantly altered in VIUPs compared to other early pregnancy outcomes. What is known already Around 10% of all intrauterine pregnancies are lost in the first trimester. A further 1-2% of pregnancies are located outside the endometrial cavity; these ectopic pregnancies are the leading cause of maternal mortality in the first trimester of gestation. Early miscarriages may also cause significant morbidity when bleeding or infection occurs. The symptoms of miscarriages and ectopic pregnancy are often similar (pain and bleeding), however, such symptoms are also common in VIUPs. To date, no biomarkers have been identified to differentiate VIUPs from non-viable and ectopic pregnancies. Study design, size, duration This is a prospective cohort study that included 332 pregnant women at less than ten weeks of gestation, who attended the early pregnancy assessment unit (EPAU) at Liverpool Women’s Hospital with pain and/or bleeding. Participants/materials, setting, methods Blood samples were collected from the 332 pregnant women prior to final clinical diagnosis of pregnancy outcome. Serum samples were subjected to NMR metabolomics profiling (14 spectra that did not meet the recommended minimum reporting standards were removed from subsequent analysis). 1D 1H-NMR spectra were acquired at 37 °C on a 700 MHz spectrometer. Relative metabolite abundances underwent statistical analysis using MetaboAnalyst 5.0 (p-value FDR adjusted). Main results and the role of chance Final pregnancy outcomes were as follows: one hydatidiform mole (0.3%), 48 ectopic pregnancies (14.4%), three pregnancies of unknown location (PULs, 0.9%), 78 failed pregnancies of unknown location (FPULs, 23.4%), 47 miscarriages (14.1%), two vanishing twin pregnancies (0.6%) and 153 VIUPs (45.8%). Due to small sample numbers, the hydatidiform mole, PULs and vanishing twin pregnancies were excluded from further analysis. To compare VIUPs to other pregnancy outcomes, ectopic pregnancies, FPULs and miscarriages were grouped together. Univariate analysis of serum metabolite concentrations identified four metabolites (phenylalanine, alanine, glutamate and glutamine) as significantly different in VIUPs compared to other pregnancy outcomes. Multivariate partial least squared discriminant analysis provided only weak correlation between the serum metabolome and pregnancy outcome. In summary, we have identified differences in the metabolome of women with VIUPs compared to other common pregnancy outcomes, which may provide diagnostic utility. Limitations, reasons for caution In this study, women with VIUPs presented with pain and/or bleeding. The presence of symptoms may influence the metabolome of this group versus VIUPs without symptoms, thus limiting the translation of our findings. Furthermore, environmental factors were not controlled (e.g. fasting status), making it likely that cohort heterogeneity was enhanced. Wider implications of the findings This study identifies a metabolite profile associated with VIUPs. These findings may be useful in the development of a diagnostic test to confirm VIUPs and thus exclude potentially life-threatening pregnancy outcomes. Such a test would be invaluable in clinical emergencies. Trial registration number NA

H - nuclear magnetic ressonance (H-NMR) -based metabolomic profiling provides a specific type 2 diabetes mellitus associated profile in Spanish population beyond glucose: The Di@bet.es study

Background and Aims: The [email protected] Study, showed an overall DM2 prevalence of 13.8% in Spain. The primary objective of our study was to characterize the NMR metabolomic profile of a sub-group of 720 individuals, – men and women in a wide age range – 25% of them with DM2 and to evaluate 1H-NMR molecular panels to find a specific metabolomic profile associated with DM.

Effect of Very-Low-Calorie Ketogenic Diet on Psoriasis Patients: A Nuclear Magnetic Resonance-Based Metabolomic Study.

Psoriasis is an inflammatory disease of the epidermis based on an immunological mechanism involving Langerhans cells and T lymphocytes that produce pro-inflammatory cytokines. Genetic factors, environmental factors, and improper nutrition are considered triggers of the disease. Numerous studies have reported that in a high number of patients, psoriasis is associated with obesity. Excess adipose tissue, typical of obesity, causes a systemic inflammatory status coming from the inflammatory active adipose tissue; therefore, weight reduction is a strategy to fight this pro-inflammatory state. This study aimed to evaluate how a nutritional regimen based on a ketogenic diet influenced the clinical parameters, metabolic profile, and inflammatory state of psoriasis patients. To this end, 30 psoriasis patients were subjected to a ketogenic nutritional regimen and monitored for 4 weeks by evaluating the clinical data, biochemical and clinical parameters, NMR metabolomic profile, and IL-2, IL-1β, TNF-α, IFN-γ, and IL-4 concentrations before and after the nutritional regimen. Our data show that a low-calorie ketogenic diet can be considered a successful strategy and therapeutic option to gain an improvement in psoriasis-related dysmetabolism, with significant correction of the full metabolic and inflammatory status.

Metabolomics profiling of fermented cantaloupe juice and the potential application to extend the shelf life of fresh cantaloupe juice for six months at 8 °C

The aim of this study was to evaluate the potential of adding fermented cantaloupe juice at different concentrations to extend the shelf life of fresh cantaloupe juice stored at 8 °C for six months. Cantaloupe juice was fermented with Lactobacillus plantarum FBS05 for 48 h at 37 °C. The antimicrobial activity of the fermented juice was determined using microtiter plate assays, and the bioactive metabolites were identified using 1H NMR metabolomics analysis. Fermented juice was added to fresh juice at different ratios including 10:90, 20:80 and 30:70 (v/v) to determine the effects on the shelf life and sensory properties. Fermented cantaloupe juice demonstrated strong antibacterial activity against Escherichia coli, Salmonella typhimurium, Aspergillus flavus and Penicillium spp. The 1H NMR metabolomics profiling identified 23 metabolites in fermented and non-fermented cantaloupe juice. The PCA model demonstrated significant discrimination level between the fermented and non-fermented cantaloupe juice. The metabolites responsible for the discrimination were lactic acid, GABA, acetoin, acetoacetate, cellobiose and alanine in the fermented cantaloupe juice, and sucrose, glucose, mannose and epicatechin in non-fermented cantaloupe juice. The ratio 20:80 of fermented cantaloupe juice to the fresh juice extended the shelf life for 6 months at 8 °C and sustained good sensory properties for cantaloupe juice. The findings of this study showed high potential for the fermented cantaloupe juice to be added to the fresh cantaloupe juice as natural preservative to extend the shelf life and improve the sensory properties.

NMR-based metabolomics reveals effect of Ganoderma boninense infection on oil palm leaf at 30 days post-infection

Basal stem rot is the major disease in oil palm industry that caused by a fungal named Ganoderma boninense (G. boninense) species. Infected palms are symptomless at the early stage of this disease which imposes difficulties in detecting the disease. Therefore, this study was carried out to obtain the 1H NMR metabolomic profiling of both non-infected and G. boninense infected oil palm leaf at 30 days post-infection (dpi). This combination has provided a rapid approach in investigating the changes in the compound variations of non-infected and G. boninense infected oil palm leaf. Non-infected and G. boninense infected oil palm leaf at 30 dpi was extracted using aqueous methanol (methanol: water, 80: 20 v/v). The crude extracts obtained were analyzed by 1H NMR-based metabolomics approach. Analysis of metabolomics data from 1H NMR was conducted by multivariate data analysis of principal component analysis (PCA). Significant differences were found between the two groups. Compared to the non-infected leaf, the G. boninense infected leaf had higher relative abundance of choline, asparagine, alanine, succinic acid, gallic acid, epicatechin, trimethylamine, N-acetylglucosamine, N-acetyltyrosine, β-sitosterol, 2,3-butanediol, lactic acid, caffeic acid, p-hydroxybenzoic acid, α-tocopherol, β-cryptoxanthin and kaempferol. The non-infected leaf showed higher level of sucrose, xylose, α-glucose, S-sulfocysteine and indole-3-acetic acid. NMR-based metabolomics applied in this study reveals that G. boninense alters a manifold of primary and secondary compounds in oil palm leaf.

1 H NMR metabolomic profiling elucidated attenuation of neurometabolic alterations by lercanidipine in MCAo model in rats.

Stroke is a leading cause of death and disability worldwide with limited therapeutic interventions. The current study explored proton nuclear magnetic resonance spectroscopy (1 H NMR)-based metabolomic approach to elucidate the effect of lercanidipine on neurometabolic alterations in transient model of ischaemic stroke in rats. In the present investigation, male Wistar rats were subjected to middle cerebral artery occlusion (MCAo) for 2h followed by reperfusion using intraluminal filament method. Rats were randomly divided into three groups as vehicle-treated sham control, vehicle-treated MCAo control and lercanidipine-treated MCAo. Vehicle or lercanidipine (0.5mg/kg, i.p.) was administered 120min post-reperfusion. The rat brain cortex tissues were isolated 24h post-MCAo and were investigated by 1 H NMR spectroscopy through perchloric extraction method. A total of 23 metabolites were altered significantly after cerebral ischaemic-reperfusion injury in MCAo control as compared to sham control rats. Lercanidipine significantly reduced the levels of valine, alanine, lactate, acetate and tyrosine, while N-acetylaspartate, glutamate, glutamine, aspartate, creatine/phosphocreatine, choline, glycerophosphorylcholine, taurine, myo-inositol and adenosine di-phosphate were elevated as compared to MCAo control. Present study illustrates effect of lercanidipine on neurometabolic alterations which might be mediated through its antioxidant, anti-inflammatory, vasodilatory and anti-apoptotic property in MCAo model of stroke.

Evaluation of sample preparation protocols for quantitative NMR-based metabolomics.

Quantification of metabolites in biological fluids and tissues by NMR spectroscopy is challenged by the presence of abundant macromolecules and lipoproteins in samples, which give broad signals in the NMR spectra. To improve the quality of NMR spectra the different protocols for protein and lipid removal from the sample are used. This work is aimed at the evaluation of the effectiveness of various methods of purification of blood serum from proteins and lipids for 1H NMR metabolomic profiling. The advantages and limitations of different methods of the sample preparation for NMR-based quantitative metabolomics have been compared, including ultrafiltration, methanol and ethanol extractions with and without additional lipid removal, and methanol-chloroform extraction. The concentrations of 30 abundant metabolites extracted from human blood serum have been measured. It is found that ultrafiltration provides the best lipid removal, but causes significant and inhomogeneous metabolite losses. Ethanol and methanol extractions demonstrate similar performance with the minimal metabolite losses, and are ideal for fluids and tissues with low lipid content. The additional purification of alcohol extracts from lipids allows for the significant improving of NMR spectra, but causes additional metabolite losses. The methanol-chloroform extraction seems to be an optimal method for tissues with the high lipid content, providing a satisfactory lipid removal and low metabolite losses. The ultrafiltration leads to large losses of metabolites (up to 60%) and for this reason is not suitable for quantitative analysis.

NMR-Based Tissular and Developmental Metabolomics of Tomato Fruit.

Fruit is a complex organ containing seeds and several interconnected tissues with dedicated roles. However, most biochemical or molecular studies about fleshy fruit development concern the entire fruit, the fruit without seeds, or pericarp only. We studied tomato (Solanum lycopersicum) fruit at four stages of development (12, 20, 35, and 45 days post-anthesis). We separated the seeds and the other tissues, exocarp, mesocarp, columella with placenta and locular tissue, and analyzed them individually using proton NMR metabolomic profiling for the quantification of major polar metabolites, enzymatic analysis of starch, and LC-DAD analysis of isoprenoids. Pericarp tissue represented about half of the entire fruit mass only. The composition of each fruit tissue changed during fruit development. An ANOVA-PCA highlighted common, and specific metabolite trends between tissues e.g., higher contents of chlorogenate in locular tissue and of starch in columella. Euclidian distances based on compositional data showed proximities within and between tissues. Several metabolic regulations differed between tissues as revealed by the comparison of metabolite networks based on correlations between compounds. This work stressed the role of specific tissues less studied than pericarp but that impact fruit organoleptic quality including its shape and taste, and fruit processing quality.

Metabolomics and Their Ability to Distinguish Thyroid Disorders: A Retrospective Pilot Study.

Early diagnosis of thyroid disorders is key to further treatment. We assessed the ability of a high-throughput proton NMR metabolomic profile to distinguish disease type amongst of Graves' disease (n=87), Hashimoto's thyroiditis (n=17), toxic goiter (n=11), and autoimmune thyroiditis [i. e., subacute thyroiditis (n=4), postpartum thyroiditis (n=1)]. This observational study was conducted investigating patients presenting with a thyroid disorder at a Swiss hospital endocrine referral center and an associated endocrine outpatient clinic. The main outcome was diagnosis of thyroid disorder based on classical parameters. Blood draws took place as close as possible to treatment initiation. We performed one-way ANOVA and partial least squares discriminant analysis (PLS-DA) as multivariate classification and feature ranking method. One-way ANOVA analysis yielded following significantly different metabolites, triglycerides in small VLDL, triglycerides in very small VLDL, and triglycerides in large LDL (FDR=0.04). There was no distinct separation of any of the 4 diagnoses by PLS-DA. We did not find a metabolomic biomarker combination capable of predicting diagnosis. Preanalytical issues might have influenced our results. We strongly suggest replicating our work in another cohort.

Characterizing alternative feeds for rainbow trout (O. mykiss) by 1H NMR metabolomics

IntroductionFish feed formulations are constantly evolving to improve the quality of diets for farmed fish and to ensure the sustainability of the aquaculture sector. Nowadays, insect, microalgae and yeast are feedstuff candidates for new feeds. However, the characterization of aquafeed is still based on proximate and targeted analyses which may not be sufficient to assess feed quality.ObjectivesOur aim was to highlight the soluble compounds that specifically differ between selected plant-based feeds complemented with alternative feedstuffs and discuss their origin and potential for fish nutrition.MethodsA growth trial was carried out to evaluate growth performances and feed conversion ratios of fish fed plant-based, commercial, insect, spirulina and yeast feeds. 1H NMR metabolomics profiling of each feed was performed using a CPMG sequence on polar extracts. Spectra were processed, and data were analyzed using multivariate and univariate analyses to compare alternative feeds to a plant-based feed.ResultsFish fed insect or yeast feed showed the best growth performances associated with the lowest feed conversion ratios compared to plant-based feed. Soluble compound 1H NMR profiles of insect and spirulina alternative feeds differed significantly from the plant-based one that clustered with yeast feed. In insect and spirulina feeds, specific differences compared to plant-based feed concerned glycerol and 3-hydroxybutyrate, respectively.ConclusionThis strategy based on compositional differences between plant-based and alternative feeds can be useful for detecting compounds unsuspected until now that could impact fish metabolism.

1H NMR metabolomics profiling unveils the compositional changes of hydro-cooled rockmelon (Cucumis melo L. reticulatus cv glamour) during storage related to in vitro antioxidant activity

From field harvest to cold room storage, rockmelon accumulates heat from the high ambient temperature which accelerates the metabolic changes and shortens the market life of the fruit. Hydro-cooling treatment has been used to reduce the temperature of fresh produce after harvest in order to maintain quality during postharvest storage. The purpose of this study was to elucidate the influence of hydro-cooling on phytochemical content and metabolite changes in the rockmelon. In this study, hydro-cooling application has effectively preserved the beta-carotene of rockmelon pulp. The results also suggested that high phenolic content in rind and pulp of rockmelon was highly maintained by using 1/2 cooling time (CT). A discriminatory analysis on the metabolites responsible for the variation between weeks of storage (WS) at each CT of rockmelon was successfully achieved by using 1H NMR-based metabolomics. The principal component analysis scores showed a noticeable and clear separation into four clusters representing the WS 0, 1, 2 and 3 for 0 and 3/4 CT rind extracts. Various assigned 1H NMR chemical shifts referring to the metabolites responsible for the variation were ascribed. Among the identified metabolites, the aromatic amino acids such as phenylalanine, tryptophan and tyrosine were not changed significantly during WS 0, 1 and 2 as they were grouped into the same cluster in 1/2 CT hydro-cooled rind extracts.

Metabolomics for Prediction of Relapse in Graves' Disease: Observational Pilot Study.

Background: There is a lack of biochemical markers for early prediction of relapse in patients with Graves' disease [GD], which may help to direct treatment decisions. We assessed the prognostic ability of a high-throughput proton NMR metabolomic profile to predict relapse in a well characterized cohort of GD patients.Methods: Observational study investigating patients presenting with GD at a Swiss hospital endocrine referral center and an associated endocrine outpatient clinic. We measured 227 metabolic markers in the blood of patients before treatment initiation. Main outcome was relapse of hyperthyroidism within 18 months of stopping anti-thyroid drugs. We used ROC analysis with AUC to assess discrimination.Results: Of 69 included patients 18 (26%) patients had a relapse of disease. The clinical GREAT score had an AUC of 0.68 (95% CI 0.63–0.70) to predict relapse. When looking at the metabolomic markers, univariate analysis revealed pyruvate and triglycerides in medium VLDL as predictors with AUCs of 0.73 (95% CI 0.58–0.84) and 0.67 (95% CI 0.53–0.80), respectively. All other metabolomic markers had lower AUCs.Conclusion: Overall, metabolomic markers in our pilot study had low to moderate prognostic potential for prediction of relapse of GD, with pyruvate and triglycerides being candidates with acceptable discriminatory abilities. Our data need validation in future larger trials.