Comprehensive polar metabolomics and lipidomics profiling discriminates the transformed from the non-transformed state in colon tissue and cell lines.

Colorectal cancer (CRC) is the fourth most lethal disease worldwide. Despite an urgent need for therapeutic advance, selective target identification in a preclinical phase is hampered by molecular and metabolic variations between cellular models. To foster optimal model selection from a translational perspective, we performed untargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry-based polar metabolomics and lipidomics to non-transformed (CCD841-CON and FHC) and transformed (HCT116, HT29, Caco2, SW480 and SW948) colon cell lines as well as tissue samples from ten colorectal cancer patients. This unveiled metabolic signatures discriminating the transformed from the non-transformed state. Metabolites involved in glutaminolysis, tryptophan catabolism, pyrimidine, lipid and carnitine synthesis were elevated in transformed cells and cancerous tissue, whereas those involved in the glycerol-3-phosphate shuttle, urea cycle and redox reactions were lowered. The degree of glutaminolysis and lipid synthesis was specific to the colon cancer cell line at hand. Thus, our study exposed pathways that are specifically associated with the transformation state and revealed differences between colon cancer cell lines that should be considered when targeting cancer-associated pathways.

Untargeted Metabolomics Reveals Elevated L-Carnitine Metabolism in Pig and Rat Colon Tissue Following Red Versus White Meat Intake.

SCOPE: The consumption of red and processed meat, and not white meat, associates with the development of various Western diseases such as colorectal cancer and type 2 diabetes. This work aims at unraveling novel meat-associated mechanisms that are involved in disease development. METHODS AND RESULTS: A non-hypothesis driven strategy of untargeted metabolomics is applied to assess colon tissue from rats (fed a high dose of beef vs. white meat) and from pigs (fed red/processed meat vs. white meat), receiving a realistic human background diet. An increased carnitine metabolism is observed, which is reflected by higher levels of acylcarnitines and 3-dehydroxycarnitine (rats and pigs) and trimethylamine-N-oxide (rats). While 3-dehydroxycarnitine is higher in HT29 cells, incubated with colonic beef digests, acylcarnitine levels are reduced. This suggests an altered response from colon cancer cell line towards meat-induced oxidative stress. Moreover, metabolic differences between rat and pigs are observed in N-glycolylneuraminic acid incorporation, prostaglandin, and fatty acid synthesis. CONCLUSION: This study demonstrates elevated (acyl)carnitine metabolism in colon tissue of animals that follow a red meat-based diet, providing mechanistic insights that may aid in explaining the nutritional-physiological correlation between red/processed meat and Western diseases.

Untargeted Metabolomics to Reveal Red versus White Meat-Associated Gut Metabolites in a Prudent and Western Dietary Context.

SCOPE: To improve understanding of the epidemiological link between red and processed meat consumption and chronic diseases, more insight into the formation of metabolites during meat digestion is warranted. METHODS AND RESULTS: Untargeted mass-spectrometry-based metabolomics is applied to explore the impact of red and processed meat consumption (compared to chicken), combined with a prudent or Western dietary pattern. A pig feeding study (n = 32), as a sentinel for humans, is conducted in a 2 × 2 factorial design for 4 weeks. The luminal content of the small intestine and colon are collected to determine their metabolic fingerprints. Seventy-six metabolites (38 in the small intestine, 32 in the colon, and 6 in both intestinal compartments) contributing to the distinct gut metabolic profiles of pigs fed either chicken or red and processed meat are (tentatively) identified. Consumption of red and processed meat results in higher levels of short- and medium-chain acylcarnitines and 3-dehydroxycarnitine, irrespective of dietary context, whereas long-chain acylcarnitines and monoacylglycerols are associated with the red and processed Western diet. CONCLUSION: The identification of red and processed meat-associated gut metabolites in this study contributes to the understanding of meat digestion in a complex but controlled dietary context and its potential health effects.

Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis.

Despite intensive research and constant medical progress, sepsis remains one of the most urgent unmet medical needs of today. Most studies have been focused on the inflammatory component of the disease; however, recent advances support the notion that sepsis is accompanied by extensive metabolic perturbations. During times of limited caloric intake and high energy needs, the liver acts as the central metabolic hub in which PPARα is crucial to coordinate the breakdown of fatty acids. The role of hepatic PPARα in liver dysfunction during sepsis has hardly been explored. We demonstrate that sepsis leads to a starvation response that is hindered by the rapid decline of hepatic PPARα levels, causing excess free fatty acids, leading to lipotoxicity, and glycerol. In addition, treatment of mice with the PPARα agonist pemafibrate protects against bacterial sepsis by improving hepatic PPARα function, reducing lipotoxicity and tissue damage. Since lipolysis is also increased in sepsis patients and pemafibrate protects after the onset of sepsis, these findings may point toward new therapeutic leads in sepsis.

A Metabolomics Approach to Unravel Cricket Paralysis Virus Infection in Silkworm Bm5 Cells.

How a host metabolism responds to infection with insect viruses and how it relates to pathogenesis, is little investigated. Our previous study observed that Cricket paralysis virus (CrPV, Dicistroviridae) causes short term persistence in silkworm Bm5 cells before proceeding to acute infection. In this study, a metabolomics approach based on high resolution mass spectrometry was applied to investigate how a host metabolism is altered during the course of CrPV infection in Bm5 cells and which changes are characteristic for the transition from persistence to pathogenicity. We observed that CrPV infection led to significant and stage-specific metabolic changes in Bm5 cells. Differential metabolites abundance and pathway analysis further identified specific metabolic features at different stages in the viral life cycle. Notably, both glucose and glutamine levels significantly increased during CrPV persistent infection followed by a steep decrease during the pathogenic stages, suggesting that the central carbon metabolism was significantly modified during CrPV infection in Bm5 cells. In addition, dynamic changes in levels of polyamines were detected. Taken together, this study characterized for the first time the metabolic dynamics of CrPV infection in insect cells, proposing a central role for the regulation of both amino acid and carbohydrate metabolism during the period of persistent infection of CrPV in Bm5 cells.

Metabolomics-based biomarker discovery for bee health monitoring: A proof of concept study concerning nutritional stress in Bombus terrestris.

Bee pollinators are exposed to multiple natural and anthropogenic stressors. Understanding the effects of a single stressor in the complex environmental context of antagonistic/synergistic interactions is critical to pollinator monitoring and may serve as early warning system before a pollination crisis. This study aimed to methodically improve the diagnosis of bee stressors using a simultaneous untargeted and targeted metabolomics-based approach. Analysis of 84 Bombus terrestris hemolymph samples found 8 metabolites retained as potential biomarkers that showed excellent discrimination for nutritional stress. In parallel, 8 significantly altered metabolites, as revealed by targeted profiling, were also assigned as candidate biomarkers. Furthermore, machine learning algorithms were applied to the above-described two biomarker sets, whereby the untargeted eight components showed the best classification performance with sensitivity and specificity up to 99% and 100%, respectively. Based on pathway and biochemistry analysis, we propose that gluconeogenesis contributed significantly to blood sugar stability in bumblebees maintained on a low carbohydrate diet. Taken together, this study demonstrates that metabolomics-based biomarker discovery holds promising potential for improving bee health monitoring and to identify stressor related to energy intake and other environmental stressors.

Validated comprehensive metabolomics and lipidomics analysis of colon tissue and cell lines.

Current untargeted approaches for metabolic fingerprinting of colon tissue and cell lines lack validation of reproducibility and/or focus on a selection of metabolites as opposed to the entire metabolome. Yet, both are critical to ensure reliable results and pursue a fully holistic analysis. Therefore, we have optimized and validated a platform for analyzing the polar metabolome and lipidome of colon-derived cell and tissue samples based on a consecutive extraction of polar and apolar components. Peak areas of selected targeted analytes and the number of untargeted components were assessed. Analysis was performed using ultra-high performance liquid-chromatography (UHPLC) coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (HRMS). This resulted in an optimized extraction protocol using 50% methanol/ultrapure water to obtain the polar fraction followed by a dichloromethane-based lipid extraction. Using this comprehensive approach, we have detected more than 15,000 components with CV < 30% in internal quality control (IQC) samples and were able to discriminate the non-transformed (NT) and transformed (T) state in human colon tissue and cell lines based on validated OPLS-DA models (R2Y > 0.719 and Q2 > 0.674). To conclude, our validated polar metabolomics and lipidomics fingerprinting approach could be of great value to reveal gastrointestinal disease-associated biomarkers and mechanisms.

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies.

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state-of-the-art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

Biomarkers of meat and seafood intake: an extensive literature review.

Meat, including fish and shellfish, represents a valuable constituent of most balanced diets. Consumption of different types of meat and fish has been associated with both beneficial and adverse health effects. While white meats and fish are generally associated with positive health outcomes, red and especially processed meats have been associated with colorectal cancer and other diseases. The contribution of these foods to the development or prevention of chronic diseases is still not fully elucidated. One of the main problems is the difficulty in properly evaluating meat intake, as the existing self-reporting tools for dietary assessment may be imprecise and therefore affected by systematic and random errors. Dietary biomarkers measured in biological fluids have been proposed as possible objective measurements of the actual intake of specific foods and as a support for classical assessment methods. Good biomarkers for meat intake should reflect total dietary intake of meat, independent of source or processing and should be able to differentiate meat consumption from that of other protein-rich foods; alternatively, meat intake biomarkers should be specific to each of the different meat sources (e.g., red vs. white; fish, bird, or mammal) and/or cooking methods. In this paper, we present a systematic investigation of the scientific literature while providing a comprehensive overview of the possible biomarker(s) for the intake of different types of meat, including fish and shellfish, and processed and heated meats according to published guidelines for biomarker reviews (BFIrev). The most promising biomarkers are further validated for their usefulness for dietary assessment by published validation criteria.

A validated multi-matrix platform for metabolomic fingerprinting of human urine, feces and plasma using ultra-high performance liquid-chromatography coupled to hybrid orbitrap high-resolution mass spectrometry.

In recent years, metabolomics has surfaced as an innovative research strategy in human metabolism, whereby selection of the biological matrix and its inherent metabolome is of crucial importance. However, focusing on a single matrix may imply that relevant molecules of complementary physiological pathways, covered by other matrices, are missed. To address this problem, this study presents a unique multi-matrix platform for polar metabolic fingerprinting of feces, plasma and urine, applying ultra-high performance liquid-chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry, that is able to achieve a significantly higher coverage of the system's metabolome and reveal more significant results and interesting correlations in comparison with single-matrix analyses. All three fingerprinting approaches were proven 'fit-for-purpose' through extensive validation in which a number of endogenous metabolites were measured in representative quality control samples. For targeted and untargeted validation of all three matrices, excellent linearity (coefficients of determination R2 ≥ 0.99 or 0.90 respectively), recovery and precision (coefficients of variance ≤ 15% or 30% respectively) were observed. The potential of the platform was demonstrated by subjecting fecal, urine and plasma samples (collected within one day) from ten healthy volunteers to metabolic fingerprinting, yielding respectively 9 672, 9 647, and 6122 components. Orthogonal partial least-squares discriminant analysis provided similar results for feces and plasma to discriminate according to gender (p-value, R2(X), R2(Y) and Q2(Y)), suggesting feces as an excellent alternative biofluid to plasma. Moreover, combining the different matrices improved the model's predictivity, indicating the superiority of multi-matrix platforms for research purposes in biomarker detection or pathway elucidation and in the selection of the most optimal matrix for future clinical purposes.

DNA adduct profiling of in vitro colonic meat digests to map red vs. white meat genotoxicity.

The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O6-carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N4-etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk.

Aminobacter sp. MSH1 invades sand filter community biofilms while retaining 2,6-dichlorobenzamide degradation functionality under C- and N-limiting conditions.

Aminobacter sp. MSH1 is of interest for bioaugmentation of biofiltration units in drinking water treatment plants (DWTPs) due to its ability to degrade the groundwater micropollutant 2,6-dichlorobenzamide (BAM). Using a continuous flow chamber biofilm model, MSH1 was previously shown to colonize surfaces and degrade BAM at trace concentrations as low as 1 µg/L under the oligotrophic conditions found in DWTPs. In DWTP filtration units, MSH1 has to compete with the resident biofilm microbiota for space and nutrients. Using the same model, we examined how a sand filter community (SFC) affects MSH1's BAM-degrading activity and biofilm formation under C- and N-limiting conditions when fed with trace concentrations of BAM. MSH1 was inoculated simultaneously with the SFC (co-colonization mode) or after the SFC formed a biofilm (invasion mode). MSH1 successfully established in the SFC biofilm showing growth and activity. In co-colonization mode, MSH1 decreased in number in the presence of the SFC and formed isolated colonies, while specific BAM-degradation activity increased. In the invasion mode, MSH1 also decreased in numbers in the presence of the SFC but formed mixed colonies, while specific BAM degradation was unaffected. Our results show that MSH1 invades and performs successfully in an SFC biofilm under the oligotrophic conditions of DWTPs.

Genetic (In)stability of 2,6-Dichlorobenzamide Catabolism in Aminobacter sp. Strain MSH1 Biofilms under Carbon Starvation Conditions.

Aminobacter sp. strain MSH1 grows on and mineralizes the groundwater micropollutant 2,6-dichlorobenzamide (BAM) and is of interest for BAM removal in drinking water treatment plants (DWTPs). The BAM-catabolic genes in MSH1 are located on plasmid pBAM1, carrying bbdA, which encodes the conversion of BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) (BbdA+ phenotype), and plasmid pBAM2, carrying gene clusters encoding the conversion of 2,6-DCBA to tricarboxylic acid (TCA) cycle intermediates (Dcba+ phenotype). There are indications that MSH1 easily loses its BAM-catabolic phenotype. We obtained evidence that MSH1 rapidly develops a population that lacks the ability to mineralize BAM when grown on nonselective (R2B medium) and semiselective (R2B medium with BAM) media. Lack of mineralization was explained by loss of the Dcba+ phenotype and corresponding genes. The ecological significance of this instability for the use of MSH1 for BAM removal in the oligotrophic environment of DWTPs was explored in lab and pilot systems. A higher incidence of BbdA+ Dcba- MSH1 cells was also observed when MSH1 was grown as a biofilm in flow chambers under C and N starvation conditions due to growth on nonselective residual assimilable organic carbon. Similar observations were made in experiments with a pilot sand filter reactor bioaugmented with MSH1. BAM conversion to 2,6-DCBA was not affected by loss of the DCBA-catabolic genes. Our results show that MSH1 is prone to BAM-catabolic instability under the conditions occurring in a DWTP. While conversion of BAM to 2,6-DCBA remains unaffected, BAM mineralization activity is at risk, and monitoring of metabolites is warranted.IMPORTANCE Bioaugmentation of dedicated biofiltration units with bacterial strains that grow on and mineralize micropollutants was suggested as an alternative for treating micropollutant-contaminated water in drinking water treatment plants (DWTPs). Organic-pollutant-catabolic genes in bacteria are often easily lost, especially under nonselective conditions, which affects the bioaugmentation success. In this study, we provide evidence that Aminobacter sp. strain MSH1, which uses the common groundwater micropollutant 2,6-dichlorobenzamide (BAM) as a C source, shows a high frequency of loss of its BAM-mineralizing phenotype due to the loss of genes that convert 2,6-DCBA to Krebs cycle intermediates when nonselective conditions occur. Moreover, we show that catabolic-gene loss also occurs in the oligotrophic environment of DWTPs, where growth of MSH1 depends mainly on the high fluxes of low concentrations of assimilable organic carbon, and hence show the ecological relevance of catabolic instability for using strain MSH1 for BAM removal in DWTPs.

Untargeted metabolomics of colonic digests reveals kynurenine pathway metabolites, dityrosine and 3-dehydroxycarnitine as red versus white meat discriminating metabolites.

Epidemiological research has demonstrated that the consumption of red meat is an important risk factor for the development of colorectal cancer (CRC), diabetes mellitus and cardiovascular diseases. However, there is no holistic insight in the (by-) products of meat digestion that may contribute to disease development. To address this hiatus, an untargeted mass spectrometry (MS)-based metabolomics approach was used to create red versus white meat associated metabolic fingerprints following in vitro colonic digestion using the fecal inocula of ten healthy volunteers. Twenty-two metabolites were unequivocally associated with simulated colonic digestion of red meat. Several of these metabolites could mechanistically be linked to red meat-associated pathways including N'-formylkynurenine, kynurenine and kynurenic acid (all involved in tryptophan metabolism), the oxidative stress marker dityrosine, and 3-dehydroxycarnitine. In conclusion, the used MS-based metabolomics platform proved to be a powerful platform for detection of specific metabolites that improve the understanding of the causal relationship between red meat consumption and associated diseases.

Reducing Compounds Equivocally Influence Oxidation during Digestion of a High-Fat Beef Product, which Promotes Cytotoxicity in Colorectal Carcinoma Cell Lines.

We studied the formation of malondialdehyde, 4-hydroxy-nonenal, and hexanal (lipid oxidation products, LOP) during in vitro digestion of a cooked low-fat and high-fat beef product in response to the addition of reducing compounds. We also investigated whether higher LOP in the digests resulted in a higher cyto- and genotoxicity in Caco-2, HT-29 and HCT-116 cell lines. High-fat compared to low-fat beef digests contained approximately 10-fold higher LOP concentrations (all P < 0.001), and induced higher cytotoxicity (P < 0.001). During digestion of the high-fat product, phenolic acids (gallic, ferulic, chlorogenic, and caffeic acid) displayed either pro-oxidant or antioxidant behavior at lower and higher doses respectively, whereas ascorbic acid was pro-oxidant at all doses, and the lipophilic reducing compounds (α-tocopherol, quercetin, and silibinin) all exerted a clear antioxidant effect. During digestion of the low-fat product, the hydrophilic compounds and quercetin were antioxidant. Decreases or increases in LOP concentrations amounted to 100% change versus controls.

In vitro DNA adduct profiling to mechanistically link red meat consumption to colon cancer promotion.

Colorectal cancer (CRC) is the third most common cancer type in the world. Epidemiological research has demonstrated that both red and processed meat consumption significantly contribute to CRC risk. In this study, red meat toxicity was investigated by means of simulated gastrointestinal conditions, malondialdehyde (MDA) analysis and UHPLC-(HR)MS(/MS) based DNA adductomics. Since dairy products with high calcium content are associated with a decreased CRC-risk, the possible CRC-protective effects of calcium were assessed as well. The obtained results confirmed the earlier reported finding that heme-rich meat stimulates lipid peroxidation and O6-carboxymethylguanine (O6-CMG) DNA adduct formation during digestion. Calcium carbonate (CaCO3) supplementation resulted in both toxic and anti-toxic effects; i.e. stimulation of O6-CMG production, but reduction of MDA formation. DNA adductome mapping of meat digests revealed a significant interindividual variability. The observed DNA adduct profile also differed according to the digested meat type, uncovering different putative DNA adducts that seem to be associated with digestion of beef or chicken with or without supplemented CaCO3. Formamidopyrimidine-adenine was found to be discriminative for meat digests without added CaCO3, carboxyethylcytosine was significantly higher in beef digests and methoxymethylcytosine (or its hydroxyethylcytosine isomer) was found to be lower in meat digests supplemented with CaCO3. These results demonstrate that DNA adduct formation may be involved in the pathway that links red meat digestion to CRC promotion. In addition, the possible CRC-protective attributes of calcium through anti-oxidant actions could be documented.