Lipidomics Reveals Myocardial Lipid Composition in a Murine Model of Insulin Resistance Induced by a High-Fat Diet.

Ectopic fat accumulation in non-adipose tissues is closely related to diabetes-related myocardial dysfunction. Nevertheless, the complete picture of the lipid metabolites involved in the metabolic-related myocardial alterations is not fully characterized. The aim of this study was to characterize the specific lipid profile in hearts in an animal model of obesity/insulin resistance induced by a high-fat diet (HFD). The cardiac lipidome profiles were assessed via liquid chromatography-mass spectrometry (LC-MS)/MS-MS and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging in hearts from C57BL/6J mice fed with an HFD or standard-diet (STD) for 12 weeks. Targeted lipidome analysis identified a total of 63 lipids (i.e., 48 triacylglycerols (TG), 5 diacylglycerols (DG), 1 sphingomyelin (SM), 3 phosphatidylcholines (PC), 1 DihydroPC, and 5 carnitines) modified in hearts from HFD-fed mice compared to animals fed with STD. Whereas most of the TG were up-regulated in hearts from animals fed with an HFD, most of the carnitines were down-regulated, thereby suggesting a reduction in the mitochondrial ß-oxidation. Roughly 30% of the identified metabolites were oxidated, pointing to an increase in lipid peroxidation. Cardiac lipidome was associated with a specific biochemical profile and a specific liver TG pattern. Overall, our study reveals a specific cardiac lipid fingerprint associated with metabolic alterations induced by HFD.

Urinary Metabolomics Study on the Protective Role of Cocoa in Zucker Diabetic Rats via 1H-NMR-Based Approach.

Cocoa constitutes one of the richest sources of dietary flavonoids with demonstrated anti-diabetic potential. However, the metabolic impact of cocoa intake in a diabetic context remains unexplored. In this study, metabolomics tools have been used to investigate the potential metabolic changes induced by cocoa in type 2 diabetes (T2D). To this end, male Zucker diabetic fatty rats were fed on standard (ZDF) or 10% cocoa-rich diet (ZDF-C) from week 10 to 20 of life. Cocoa supplementation clearly decreased serum glucose levels, improved glucose metabolism and produced significant changes in the urine metabolome of ZDF animals. Fourteen differential urinary metabolites were identified, with eight of them significantly modified by cocoa. An analysis of pathways revealed that butanoate metabolism and the synthesis and degradation of branched-chain amino acids and ketone bodies are involved in the beneficial impact of cocoa on diabetes. Moreover, correlation analysis indicated major associations between some of these urine metabolites (mainly valine, leucine, and isoleucine) and body weight, glycemia, insulin sensitivity, and glycated hemoglobin levels. Overall, this untargeted metabolomics approach provides a clear metabolic fingerprint associated to chronic cocoa intake that can be used as a marker for the improvement of glucose homeostasis in a diabetic context.

Remission of obesity and insulin resistance is not sufficient to restore mitochondrial homeostasis in visceral adipose tissue.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

Effects of Lifestyle Intervention in Tissue-Specific Lipidomic Profile of Formerly Obese Mice.

Lipids are highly diverse in their composition, properties and distribution in different biological entities. We aim to establish the lipidomes of several insulin-sensitive tissues and to test their plasticity when divergent feeding regimens and lifestyles are imposed. Here, we report a proton nuclear magnetic resonance (1H-NMR) study of lipid abundance across 4 tissues of C57Bl6J male mice that includes the changes in the lipid profile after every lifestyle intervention. Every tissue analysed presented a specific lipid profile irrespective of interventions. Glycerolipids and fatty acids were most abundant in epididymal white adipose tissue (eWAT) followed by liver, whereas sterol lipids and phosphoglycerolipids were highly enriched in hypothalamus, and gastrocnemius had the lowest content in all lipid species compared to the other tissues. Both when subjected to a high-fat diet (HFD) and after a subsequent lifestyle intervention (INT), the lipidome of hypothalamus showed no changes. Gastrocnemius and liver revealed a pattern of increase in content in many lipid species after HFD followed by a regression to basal levels after INT, while eWAT lipidome was affected mainly by the fat composition of the administered diets and not their caloric density. Thus, the present study demonstrates a unique lipidome for each tissue modulated by caloric intake and dietary composition.

Unravelling the metabolic alterations of liver damage induced by thirdhand smoke.

BACKGROUND: Thirdhand smoke (THS) is the accumulation of tobacco smoke gases and particles that become embedded in materials. Previous studies concluded that THS exposure induces oxidative stress and hepatic steatosis in liver. Despite the knowledge of the increasing danger of THS exposure, the metabolic disorders caused in liver are still not well defined. OBJECTIVES: The aim of this study is to investigate the metabolic disorders caused by THS exposure in liver of male mice and to evaluate the effects of an antioxidant treatment in the exposed mice. METHODS: We investigated liver from three mice groups: non-exposed mice, exposed to THS in conditions that mimic human exposure and THS-exposed treated with antioxidants. Liver samples were analyzed using a multiplatform untargeted metabolomics approach including nuclear magnetic resonance (1H NMR), liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and laser desorption/ionization mass spectrometry imaging (MSI), able to map lipids in liver tissues. RESULTS: Our multiplatform approach allowed the annotation of eighty-eight metabolites altered by THS exposure, including amino acids, nucleotides and several types of lipids. The main dysregulated pathways by THS exposure were D-glutamine and D-glutamate metabolism, glycerophospholipid metabolism and oxidative phosphorylation and glutathione metabolism, being the last two related to oxidative stress. THS-exposed mice also presented higher lipid accumulation and decrease of metabolites involved in the phosphocholine synthesis, as well as choline deficiency, which is related to Non-Alcoholic Fatty Liver Disease and steatohepatitis. Interestingly, the antioxidant treatment of THS-exposed mice reduced the accumulation of some lipids, but could not revert all the metabolic alterations, including some related to the impairment of the mitochondrial function. CONCLUSIONS: THS alters liver function at a molecular level, dysregulating many metabolic pathways. The molecular evidences provided here confirm that THS is a new factor for liver steatosis and provide the basis for future research in this respect.

Identification of metabolic changes leading to cancer susceptibility in Fanconi anemia cells.

Fanconi anemia (FA) is a chromosomal instability disorder of bone marrow associated with aplastic anemia, congenital abnormalities and a high risk of malignancies. The identification of more than two dozen FA genes has revealed a plethora of interacting proteins that are mainly involved in repair of DNA interstrand crosslinks (ICLs). Other important findings associated with FA are inflammation, oxidative stress response, mitochondrial dysfunction and mitophagy. In this work, we performed quantitative proteomic and metabolomic analyses on defective FA cells and identified a number of metabolic abnormalities associated with cancer. In particular, an increased de novo purine biosynthesis, a high concentration of fumarate, and an accumulation of purinosomal clusters were found. This was in parallel with decreased OXPHOS and altered glycolysis. On the whole, our results indicate an association between the need for nitrogenous bases upon impaired DDR in FA cells with a subsequent increase in purine metabolism and a potential role in oncogenesis.

Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics.

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC's superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.

Hepatic Lipidomics and Molecular Imaging in a Murine Non-Alcoholic Fatty Liver Disease Model: Insights into Molecular Mechanisms.

An imbalance between hepatic fatty acid uptake and removal results in ectopic fat accumulation, which leads to non-alcoholic fatty liver disease (NAFLD). The amount and type of accumulated triglycerides seem to play roles in NAFLD progression; however, a complete understanding of how triglycerides contribute to NAFLD evolution is lacking. Our aim was to evaluate triglyceride accumulation in NAFLD in a murine model and its associations with molecular mechanisms involved in liver damage and adipose tissue-liver cross talk by employing lipidomic and molecular imaging techniques. C57BL/6J mice fed a high-fat diet (HFD) for 12 weeks were used as a NAFLD model. Standard-diet (STD)-fed animals were used as controls. Standard liver pathology was assessed using conventional techniques. The liver lipidome was analyzed by liquid chromatography-mass spectrometry (LC-MS) and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging. Liver triglycerides were identified by MS/MS. The transcriptome of genes involved in intracellular lipid metabolism and inflammation was assessed by RT-PCR. Plasma leptin, resistin, adiponectin, and FABP4 levels were determined using commercial kits. HFD-fed mice displayed increased liver lipid content. LC-MS analyses identified 14 triglyceride types that were upregulated in livers from HFD-fed animals. Among these 14 types, 10 were identified in liver cross sections by LDI-MS tissue imaging. The accumulation of these triglycerides was associated with the upregulation of lipogenesis and inflammatory genes and the downregulation of ß-oxidation genes. Interestingly, the levels of plasma FABP4, but not of other adipokines, were positively associated with 8 of these triglycerides in HFD-fed mice but not in STD-fed mice. Our findings suggest a putative role of FABP4 in the liver-adipose tissue cross talk in NAFLD.

Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.

2D Diffusion-Ordered 1 H-NMR Spectroscopy Lipidomic Profiling after Oral Single Macronutrient Loads: Influence of Obesity, Sex, and Female Androgen Excess.

SCOPE: Postprandial dysmetabolism plays a major role in the pathogenesis of metabolic disorders such as obesity and the polycystic ovary syndrome (PCOS). The aim is to characterize the circulating lipoprotein particle profiles in response to oral glucose, lipid, and protein challenges. METHODS AND RESULTS: 17 women with PCOS, 17 control women, and 19 healthy men selected to have similar age and body mass index are studied. Blood samples are collected following the ingestion of 300 kcal in the form of glucose, lipids, or proteins, and they are submitted to two-dimensional (2D) diffusion-ordered 1 H-NMR spectroscopy. Regardless of macronutrient administered, the number of very low-density (VLDL) particles increases whereas low density-lipoprotein (LDL) decreases. High density-lipoprotein (HDL) particles increase only after lipid ingestion. Obese subjects show an increase in the number of large VLDL particles and a decrease in large LDL particles, with a significant reduction in the average particle size of LDL. Patients with PCOS show a particularly unfavorably smaller LDL particle size response to oral lipid intake, regardless of obesity. CONCLUSIONS: Oral macronutrient challenges induce immediate class-specific postprandial changes in particle number and size of lipoproteins, with lipids inducing a more pro-atherogenic lipoprotein profile compared to glucose and proteins, particularly in obese subjects and women with PCOS.

Niveles plasmáticos de glucosa, triglicéridos, VLDL, leptina y resistina como potenciales biomarcadores de la grasa miocárdica en ratones / Plasma glucose, triglycerides, VLDL, leptin and resistin levels as potential biomarkers for myocardial fat in mice

Introducción: El incremento de grasa miocárdica ha sido propuesto como uno de los principales precursores de la disfunción miocárdica de etiología diabética independiente de la enfermedad arterial coronaria. Sin embargo, actualmente se carece de biomarcadores que reflejen el contenido de grasa miocárdica para la detección clínica de esta patología. Métodos: Las correlaciones entre el contenido de triglicéridos cardíacos y los niveles plasmáticos de las principales moléculas alteradas durante la diabetes y los niveles cardíacos de ARNm de genes implicados en el metabolismo cardíaco (Cd36 y Pdk4) han sido exploradas en un modelo murino de resistencia a la insulina inducida por una dieta con alto contenido en grasas. Resultados: En ratones resistentes a la insulina, la dieta grasa aumentó los niveles de triglicéridos del miocardio, en comparación con animales controles alimentados con una dieta estándar. El contenido de triglicéridos cardíacos se encontró directamente asociado con los niveles plasmáticos de glucosa, triglicéridos, VLDL, resistina y leptina. Además, se observó una correlación inversa entre el contenido de triglicéridos y los niveles cardíacos de ARNm de Cd36 y Pdk4. Conclusiones: Nuestros datos revelan que el contenido cardíaco de triglicéridos se encuentra asociado con un perfil bioquímico plasmático alterado y con una reprogramación de la expresión de genes dirigida a atenuar el impacto de la acumulación ectópica de lípidos en miocardio

Introduction: The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction due to diabetic aetiology, independently of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. Methods: Correlations between 4cardiac triglyceride content and plasma levels of major altered molecules during diabetes and cardiac mRNA levels of genes involved in cardiac metabolism (Cd36 and Pdk4) have been explored in a murine model of insulin resistance induced by a high-fat diet. Results: In insulin-resistant mice, the fatty diet increased myocardial triglyceride levels, compared to control animals fed with a standard diet. The content of cardiac triglycerides was directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin. In addition, an inverse correlation was observed between the content of cardiac triglycerides and the cardiac mRNA levels of Cd36 and Pdk4. Conclusions: Our data reveal that the cardiac triglyceride content is associated with altered plasma biochemical profile and reprogramming of gene expression aimed to mitigate the impact of ectopic lipid accumulation in the myocardium

Plasma glucose, triglycerides, VLDL, leptin and resistin levels as potential biomarkers for myocardial fat in mice. / Niveles plasmáticos de glucosa, triglicéridos, VLDL, leptina y resistina como potenciales biomarcadores de la grasa miocárdica en ratones.

INTRODUCTION: The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction due to diabetic aetiology, independently of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. METHODS: Correlations between cardiac triglyceride content and plasma levels of major altered molecules during diabetes and cardiac mRNA levels of genes involved in cardiac metabolism (Cd36 and Pdk4) have been explored in a murine model of insulin resistance induced by a high-fat diet. RESULTS: In insulin-resistant mice, the fatty diet increased myocardial triglyceride levels, compared to control animals fed with a standard diet. The content of cardiac triglycerides was directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin. In addition, an inverse correlation was observed between the content of cardiac triglycerides and the cardiac mRNA levels of Cd36 and Pdk4. CONCLUSIONS: Our data reveal that the cardiac triglyceride content is associated with altered plasma biochemical profile and reprogramming of gene expression aimed to mitigate the impact of ectopic lipid accumulation in the myocardium.

Correction of Dyssynergic Defecation, but Not Fiber Supplementation, Reduces Symptoms of Functional Dyspepsia in Patients With Constipation in a Randomized Trial.

BACKGROUND & AIMS: Patients with functional dyspepsia are believed to have increased sensitivity of the gastrointestinal tract, and some also have functional constipation. We investigated whether in patients with functional dyspepsia, correction of dyssynergic defecation can reduce postprandial fullness. METHODS: We performed a parallel trial at 2 referral centers in Spain, from June 2016 through January 2018 of 50 patients who fulfilled the Rome IV criteria for functional dyspepsia with postprandial distress syndrome and functional constipation and dyssynergic defecation. After a 2-week pretreatment phase, the patients were randomly assigned to groups that learned to correct dyssynergic defecation (2-3 sessions of biofeedback combined with instructions for daily exercise; n = 25) or received dietary fiber supplementation (3.5 g plantago ovata per day; n = 25) for 4 weeks. The primary outcome was change in postprandial abdominal fullness, measured daily on a scale of 0-10, during the last 7 days treatment phase vs the last 7 days of the pretreatment phase. Anal gas evacuations were measured (by an event marker) during the last 2 days of the pretreatment vs treatment phases. RESULTS: Biofeedback treatment corrected dyssynergic defecation in 19/25 patients; corrected dyssynergic defection reduced postprandial fullness by 22%±1% in these patients (P < .001), and reduced the number of anal evacuations by 21%±8% (P = .009). Fiber supplementation did not reduce postprandial fullness or anal evacuations (P ≤ .023 between groups for both parameters in the intent to treat analysis). CONCLUSIONS: Diagnosis and correction of dyssynergic defecation reduces dyspeptic symptoms by more than 20% in patients with functional dyspepsia and associated constipation. Dietary fiber supplementation does not reduce symptoms in these patients. ClinicalTrials.gov no: NCT02956187.

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells.

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.

Lipid Profiling Using 1H NMR Spectroscopy.

Lipid profiling, which includes fatty acids, phospholipids, glycerides, and cholesterols is extremely important because of the essential role lipids play in the regulation of metabolism in animals. 1H-NMR-based protocols for high-throughput lipid analysis in complex mixtures have been developed and applied to biological systems. Many classes of lipids can be quantitatively analyzed in many sample matrices including serum, cells, and tissues using a simple 1H NMR experiment. In this chapter, we provide protocols for NMR-based lipid profiling including sample preparation, NMR experiments, and quantification using the LipSpin software tool.

Fatty acid binding protein 4 (FABP4) as a potential biomarker reflecting myocardial lipid storage in type 2 diabetes.

OBJECTIVE: Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone involved in the crosstalk between adipose and peripheral tissues, and it contributes to widespread insulin resistance in cells, including cardiac cells. However, the role of this adipokine in regulating cardiac metabolism and myocardial neutral lipid content in patients with type 2 diabetes has not been elucidated. METHODS: The impact of circulating FABP4 on the cardiac neutral lipid content was measured by proton magnetic resonance spectroscopy (1H-MRS) in patients with type 2 diabetes. Additionally, circulating FABP4 and the cardiac triglyceride content were analysed in high-fat diet (HFD)-fed mice, and the impact of the exogenous FABP4 was explored in HL-1 cardiac cells. RESULTS: Serum FABP4 levels were higher in type 2 diabetic patients compared to healthy individuals. Circulating FABP4 levels were associated with myocardial neutral lipid content in type 2 diabetic patients. In HFD-fed mice, both serum FABP4 and myocardial triglyceride content were increased. In FABP4-challenged HL-1 cells, extracellular FABP4 increased intracellular lipid accumulation, which led to impairment of the insulin-signalling pathway and reduced insulin-stimulated glucose uptake. However, these effects were partially reversed by FABP4 inhibition with BMS309403, which attenuated the intracellular lipid content and improved insulin signalling and insulin-stimulated glucose uptake. CONCLUSIONS: Taken together, our results identify FABP4 as a molecule involved in diabetic/lipid-induced cardiomyopathy and indicate that this molecule may be an emerging biomarker for diabetic cardiomyopathy-related disturbances, such as myocardial neutral lipid accumulation. Additionally, FABP4 inhibition may be a potential therapeutic target for metabolic-related cardiac dysfunctions.

Lifestyle Intervention Decreases Urine Trimethylamine N-Oxide Levels in Prepubertal Children with Obesity.

OBJECTIVE: Early lifestyle interventions in children with obesity decrease risk of obesity and metabolic disorders during adulthood. This study aimed to identify metabolic signatures associated with lifestyle intervention in urine samples from prepubertal children with obesity. METHODS: Thirty-four prepubertal children with obesity were studied before and after a 6-month lifestyle intervention program, and anthropometric, metabolic, and nutritional variables were collected. A nuclear magnetic resonance approach was applied to obtain the metabolomic profile from urine samples. Partial least squares-discriminant analysis (PLS-DA) was used to achieve group classification and variable importance on projection (VIP) for biomarker selection. RESULTS: The intervention reduced caloric intake by 10% (P < 0.05) and BMI standard deviation score by 0.47 SD (P < 0.001). PLS-DA identified trimethylamine N-oxide (TMAO, VIP = 2.21) as the metabolite with the highest discrimination properties between groups. Urine TMAO levels were reduced after the intervention (P < 0.05). TMAO is a biomarker of cardiovascular disease risk and is a product of gut microbiota-dependent metabolism of certain dietary compounds, including choline. Notably, changes in TMAO levels after the intervention did not correlate to differences in choline intake but were inversely associated with fiber intake (P < 0.05). CONCLUSIONS: These results indicate that lifestyle intervention decreases TMAO levels in children with obesity.

Metabolomics reveals novel blood plasma biomarkers associated to the BRCA1-mutated phenotype of human breast cancer.

Hereditary breast and ovarian cancer syndrome (HBOC) is partly due to the presence of mutations in the BRCA genes. Triple-negative (TN) breast cancer (BC) shares histological characteristics with germline BRCA1 mutation-associated tumours. We have investigated the metabolic profiles of human breast cancer (BC) cell lines carrying BRCA1 pathogenic mutations by non-targeted liquid chromatography coupled to mass spectrometry technology. Based on our in vitro results, we performed a targeted metabolomic analysis of plasma samples from TN HBOC patients taking into account their BRCA1 genotype. BRCA1 promoter hypermethylation and the BRCAness phenotype of BC cell lines were also studied. The purpose of this study was to determine the metabolic signature of HBOC syndrome and TNBC patients and to evaluate the potential contribution of the metabolites identified to the genetic diagnosis of breast cancer. The present results show the existence of a differential metabolic signature for BC cells based on the BRCA1 functionality. None of the studied BC cell lines presented hypermethylation of the BRCA1 promoter region. We provide evidence of the existence of free methylated nucleotides capable of distinguishing plasma samples from HBOC patients as BRCA1-mutated and BRCA1 non-mutated, suggesting that they might be considered as BRCA1-like biomarkers for TNBC and HBOC syndrome.

eRah: A Computational Tool Integrating Spectral Deconvolution and Alignment with Quantification and Identification of Metabolites in GC/MS-Based Metabolomics.

Gas chromatography coupled to mass spectrometry (GC/MS) has been a long-standing approach used for identifying small molecules due to the highly reproducible ionization process of electron impact ionization (EI). However, the use of GC-EI MS in untargeted metabolomics produces large and complex data sets characterized by coeluting compounds and extensive fragmentation of molecular ions caused by the hard electron ionization. In order to identify and extract quantitative information on metabolites across multiple biological samples, integrated computational workflows for data processing are needed. Here we introduce eRah, a free computational tool written in the open language R composed of five core functions: (i) noise filtering and baseline removal of GC/MS chromatograms, (ii) an innovative compound deconvolution process using multivariate analysis techniques based on compound match by local covariance (CMLC) and orthogonal signal deconvolution (OSD), (iii) alignment of mass spectra across samples, (iv) missing compound recovery, and (v) identification of metabolites by spectral library matching using publicly available mass spectra. eRah outputs a table with compound names, matching scores and the integrated area of compounds for each sample. The automated capabilities of eRah are demonstrated by the analysis of GC-time-of-flight (TOF) MS data from plasma samples of adolescents with hyperinsulinaemic androgen excess and healthy controls. The quantitative results of eRah are compared to centWave, the peak-picking algorithm implemented in the widely used XCMS package, MetAlign, and ChromaTOF software. Significantly dysregulated metabolites are further validated using pure standards and targeted analysis by GC-triple quadrupole (QqQ) MS, LC-QqQ, and NMR. eRah is freely available at http://CRAN.R-project.org/package=erah .