Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Erratum: Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

This corrects the article DOI: 10.1038/nature23481.

Advancements in Pulsed Stable Isotope-Resolved Metabolomics.

The understanding of biochemical processes of metabolism is gained through the measurement of the concentration of intermediates and the rate of metabolite conversion. However, the measurement of metabolite concentrations does not give a full representation of this dynamic system. To understand the kinetics of metabolism, the system must be described and quantified in terms of metabolite flow as a function of time. In order to measure the metabolite flow, or more precisely the metabolic flux through a biological system, substrates of the cell are labelled with stable isotopes. The usage of these substrates by the cell leads to the incorporation of the isotopes into downstream intermediates.The most important metabolic pathways are encompassed in the central carbon metabolism (CCM). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the central carbon metabolism "is the most basic aspect of life". It includes all metabolites and enzymatic reactions within: glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), amino acids and nucleotide metabolic pathways. Some molecules are at the crossroad of metabolic pathways, interconnecting diverse metabolic and therefore functional outcomes. Labelling these nodal metabolites and analysing their isotopic composition allows the precise determination of the metabolic flow within the biochemical networks that they are in.Application of stable isotope labelled substrates allows the measurement of metabolic flux through a biochemical pathway. The rapid turnover of metabolites in pathways requires pulse-feeding cells with a labelled substrate. This method allows for the determination of different cell states. For example, the action of a drug from immediate impact until the compensatory response of the metabolic system (cell, organs, organisms). Pulsed labelling is an elegant way to analyse the action of small molecules and drugs and enables the analysis of regulatory metabolic processes in short time scales.

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

BACKGROUND: Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known. METHODS: Mice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of Lrrc8b-e, or with inducible or cell-specific ablation of Lrrc8a, were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics. RESULTS: The kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms. CONCLUSIONS: VRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments despite their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.

Kinetic modelling of quantitative proteome data predicts metabolic reprogramming of liver cancer.

BACKGROUND: Metabolic alterations can serve as targets for diagnosis and cancer therapy. Due to the highly complex regulation of cellular metabolism, definite identification of metabolic pathway alterations remains challenging and requires sophisticated experimentation. METHODS: We applied a comprehensive kinetic model of the central carbon metabolism (CCM) to characterise metabolic reprogramming in murine liver cancer. RESULTS: We show that relative differences of protein abundances of metabolic enzymes obtained by mass spectrometry can be used to assess their maximal velocity values. Model simulations predicted tumour-specific alterations of various components of the CCM, a selected number of which were subsequently verified by in vitro and in vivo experiments. Furthermore, we demonstrate the ability of the kinetic model to identify metabolic pathways whose inhibition results in selective tumour cell killing. CONCLUSIONS: Our systems biology approach establishes that combining cellular experimentation with computer simulations of physiology-based metabolic models enables a comprehensive understanding of deregulated energetics in cancer. We propose that modelling proteomics data from human HCC with our approach will enable an individualised metabolic profiling of tumours and predictions of the efficacy of drug therapies targeting specific metabolic pathways.

L-Glyceraldehyde Inhibits Neuroblastoma Cell Growth via a Multi-Modal Mechanism on Metabolism and Signaling.

Glyceraldehyde (GA) is a three-carbon monosaccharide that can be present in cells as a by-product of fructose metabolism. Bruno Mendel and Otto Warburg showed that the application of GA to cancer cells inhibits glycolysis and their growth. However, the molecular mechanism by which this occurred was not clarified. We describe a novel multi-modal mechanism by which the L-isomer of GA (L-GA) inhibits neuroblastoma cell growth. L-GA induces significant changes in the metabolic profile, promotes oxidative stress and hinders nucleotide biosynthesis. GC-MS and 13C-labeling was employed to measure the flow of carbon through glycolytic intermediates under L-GA treatment. It was found that L-GA is a potent inhibitor of glycolysis due to its proposed targeting of NAD(H)-dependent reactions. This results in growth inhibition, apoptosis and a redox crisis in neuroblastoma cells. It was confirmed that the redox mechanisms were modulated via L-GA by proteomic analysis. Analysis of nucleotide pools in L-GA-treated cells depicted a previously unreported observation, in which nucleotide biosynthesis is significantly inhibited. The inhibitory action of L-GA was partially relieved with the co-application of the antioxidant N-acetyl-cysteine. We present novel evidence for a simple sugar that inhibits cancer cell proliferation via dysregulating its fragile homeostatic environment.

HEK293 producing the extracellular domain HER1: Full datasets of continuous fermentation process and metabolites analysis.

The data for provide evidences of the multi steady state of the human cell line HEK 293 was obtained from 2 L bioreactor continuous culture. A HEK 293 cell line transfected to produce soluble HER1 receptor was used. The bioreactor was operated at three different dilution rates in sequential manner. Daily samples of culture broth were collected, a total of 85 samples were processed. Viable cell concentration and culture viability was addressing by trypan blue exclusion method using a hemocytometer. Heterologous HER1 supernatant concentration was quantified by a specific ELISA and the metabolites by mass spectrometry coupled to a liquid chromatography. The primary data were collected in excel files, where it was calculated the kinetic and other variables by using mass balance and mathematical principles. It was compared the steady states behavior each other's to find out the existence of steady states' multiplicity, taking into account the stationary phase with respect to the cell density (which means its coefficient of variation is less than 20 %). From the metabolic measurements by using Liquid Chromatography coupled to mass spectrometry (LC-MS), it was also built the data matrix with the specific rates of the 76 metabolites obtained. The data were processed and analyzed, using multivariate data asssnalysis (MVDA) to reduce the complexity and to find the main patterns present in the data. We describe also the full data of the metabolites not only for steady states but also in the time evolution, which could help others in terms of modeling and deep understanding of HEK293 metabolism, especially under different culture conditions.

Mitochondrial injury during normothermic regional perfusion (NRP) and hypothermic oxygenated perfusion (HOPE) in a rodent model of DCD liver transplantation.

BACKGROUND: Normothermic regional perfusion (NRP) and hypothermic-oxygenated-perfusion (HOPE), were both shown to improve outcomes after liver transplantation from donors after circulatory death (DCD). Comparative clinical and mechanistical studies are however lacking. METHODS: A rodent model of NRP and HOPE, both in the donor, was developed. Following asystolic donor warm ischemia time (DWIT), the abdominal compartment was perfused either with a donor-blood-based-perfusate at 37 °C (NRP) or with oxygenated Belzer-MPS at 10 °C (donor-HOPE) for 2 h. Livers were then procured and underwent 5 h static cold storage (CS), followed by transplantation. Un-perfused and HOPE-treated DCD-livers (after CS) and healthy livers (DBD) with direct implantation after NRP served as controls. Endpoints included the entire spectrum of ischemia-reperfusion-injury. FINDINGS: Healthy control livers (DBD) showed minimal signs of inflammation during 2 h NRP and achieved 100% posttransplant recipient survival. In contrast, DCD livers with 30 and 60 min DWIT suffered from greater mitochondrial injury and inflammation as measured by increased perfusate Lactate, FMN- and HMGB-1-levels with subsequent Toll-like-receptor activation during NRP. In contrast, donor-HOPE (instead of NRP) led to significantly less mitochondrial-complex-I-injury and inflammation. Results after donor-HOPE were comparable to ex-situ HOPE after CS. Most DCD-liver recipients survived when treated with one HOPE-technique (86%), compared to only 40% after NRP (p = 0.0053). Following a reduction of DWIT (15 min), DCD liver recipients achieved comparable survivals with NRP (80%). INTERPRETATION: High-risk DCD livers benefit more from HOPE-treatment, either immediately in the donor or after cold storage. Comparative prospective clinical studies are required to translate the results. FUNDING: Funding was provided by the Swiss National Science Foundation (grant no: 32003B-140776/1, 3200B-153012/1, 320030-189055/1, and 31IC30-166909) and supported by University Careggi (grant no 32003B-140776/1) and the OTT (grant No.: DRGT641/2019, cod.prog. 19CT03) and the Max Planck Society. Work in the A.G. laboratory was partially supported by the NIH R01NS112381 and R21NS125466 grants.

Proteome dynamics and early salt stress response of the photosynthetic organism Chlamydomonas reinhardtii.

BACKGROUND: The cellular proteome and metabolome are underlying dynamic regulation allowing rapid adaptation to changes in the environment. System-wide analysis of these dynamics will provide novel insights into mechanisms of stress adaptation for higher photosynthetic organisms. We applied pulsed-SILAC labeling to a photosynthetic organism for the first time and we established a method to study proteome dynamics in the green alga Chlamydomonas reinhardtii, an emerging model system for plant biology. In addition, we combined the analysis of protein synthesis with metabolic profiling to study the dynamic changes of metabolism and proteome turnover under salt stress conditions. RESULTS: To study de novo protein synthesis an arginine auxotroph Chlamydomonas strain was cultivated in presence of stable isotope-labeled arginine for 24 hours. From the time course experiment in 3 salt concentrations we could identify more than 2500 proteins and their H/L ratio in at least one experimental condition; for 998 protiens at least 3 ratio counts were detected in the 24 h time point (0 mM NaCl). After fractionation we could identify 3115 proteins and for 1765 of them we determined their de novo synthesis rate. Consistently with previous findings we showed that RuBisCO is among the most prominent proteins in the cell; and similar abundance and turnover for the small and large RuBisCO subunit could be calculated. The D1 protein was identified among proteins with a high synthesis rates. A global median half-life of 45 h was calculated for Chlamydomonas proteins under the chosen conditions. CONCLUSION: To investigate the temporal co-regulation of the proteome and metabolome, we applied salt stress to Chlamydomonas and studied the time dependent regulation of protein expression and changes in the metabolome. The main metabolic response to salt stress was observed within the amino acid metabolism. In particular, proline was up-regulated manifold and according to that an increased carbon flow within the proline biosynthetic pathway could be measured. In parallel the analysis of abundance and de novo synthesis of the corresponding enzymes revealed that metabolic rearrangements precede adjustments of protein abundance.

Investigating the role of GLUL as a survival factor in cellular adaptation to glutamine depletion via targeted stable isotope resolved metabolomics.

Cellular glutamine synthesis is thought to be an important resistance factor in protecting cells from nutrient deprivation and may also contribute to drug resistance. The application of ?targeted stable isotope resolved metabolomics" allowed to directly measure the activity of glutamine synthetase in the cell. With the help of this method, the fate of glutamine derived nitrogen within the biochemical network of the cells was traced. The application of stable isotope labelled substrates and analyses of isotope enrichment in metabolic intermediates allows the determination of metabolic activity and flux in biological systems. In our study we used stable isotope labelled substrates of glutamine synthetase to demonstrate its role in the starvation response of cancer cells. We applied 13C labelled glutamate and 15N labelled ammonium and determined the enrichment of both isotopes in glutamine and nucleotide species. Our results show that the metabolic compensatory pathways to overcome glutamine depletion depend on the ability to synthesise glutamine via glutamine synthetase. We demonstrate that the application of dual-isotope tracing can be used to address specific reactions within the biochemical network directly. Our study highlights the potential of concurrent isotope tracing methods in medical research.

Metformin Is a Pyridoxal-5'-phosphate (PLP)-Competitive Inhibitor of SHMT2.

The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5'-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

Metabolite AutoPlotter - an application to process and visualise metabolite data in the web browser.

BACKGROUND: Metabolomics is gaining popularity as a standard tool for the investigation of biological systems. Yet, parsing metabolomics data in the absence of in-house computational scientists can be overwhelming and time-consuming. As a consequence of manual data processing, the results are often not analysed in full depth, so potential novel findings might get lost. METHODS: To tackle this problem, we developed Metabolite AutoPlotter, a tool to process and visualise quantified metabolite data. Other than with bulk data visualisations, such as heat maps, the aim of the tool is to generate single plots for each metabolite. For this purpose, it reads as input pre-processed metabolite-intensity tables and accepts different experimental designs, with respect to the number of metabolites, conditions and replicates. The code was written in the R-scripting language and wrapped into a shiny application that can be run online in a web browser on https://mpietzke.shinyapps.io/autoplotter. RESULTS: We demonstrate the main features and the ease of use with two different metabolite datasets, for quantitative experiments and for stable isotope tracing experiments. We show how the plots generated by the tool can be interactively modified with respect to plot type, colours, text labels and the shown statistics. We also demonstrate the application towards 13C-tracing experiments and the seamless integration of natural abundance correction, which facilitates the better interpretation of stable isotope tracing experiments. The output of the tool is a zip-file containing one single plot for each metabolite as well as restructured tables that can be used for further analysis. CONCLUSION: With the help of Metabolite AutoPlotter, it is now possible to simplify data processing and visualisation for a wide audience. High-quality plots from complex data can be generated in a short time by pressing a few buttons. This offers dramatic improvements over manual analysis. It is significantly faster and allows researchers to spend more time interpreting the results or to perform follow-up experiments. Further, this eliminates potential copy-and-paste errors or tedious repetitions when things need to be changed. We are sure that this tool will help to improve and speed up scientific discoveries.

Towards a More Reliable Identification of Isomeric Metabolites Using Pattern Guided Retention Validation.

Reliable analyte identification is critical in metabolomics experiments to ensure proper interpretation of data. Due to chemical similarity of metabolites (as isobars and isomers) identification by mass spectrometry or chromatography alone can be difficult. Here we show that isomeric compounds are quite common in the metabolic space as given in common metabolite databases. Further, we show that retention information can shift dramatically between different experiments decreasing the value of external or even in-house compound databases. As a consequence the retention information in compound databases should be updated regularly, to allow a reliable identification. To do so we present a feasible and budget conscious method to guarantee updates of retention information on a regular basis using well designed compound mixtures. For this we combine compounds in "Ident-Mixes", showing a way to distinctly identify chemically similar compounds through combinatorics and principle of exclusion. We illustrate the feasibility of this approach by comparing Gas chromatography (GC)-columns with identical properties from three different vendors and by creating a compound database from measuring these mixtures by Liquid chromatography-mass spectrometry (LC-MS). The results show the high influence of used materials on retention behavior and the ability of our approach to generate high quality identifications in a short time.

Formate metabolism in health and disease.

BACKGROUND: Formate is a one-carbon molecule at the crossroad between cellular and whole body metabolism, between host and microbiome metabolism, and between nutrition and toxicology. This centrality confers formate with a key role in human physiology and disease that is currently unappreciated. SCOPE OF REVIEW: Here we review the scientific literature on formate metabolism, highlighting cellular pathways, whole body metabolism, and interactions with the diet and the gut microbiome. We will discuss the relevance of formate metabolism in the context of embryonic development, cancer, obesity, immunometabolism, and neurodegeneration. MAJOR CONCLUSIONS: We will conclude with an outlook of some open questions bringing formate metabolism into the spotlight.

Amino acid dependent formaldehyde metabolism in mammals.

Aldehyde dehydrogenase class 3, encoded by ADH5 in humans, catalyzes the glutathione dependent detoxification of formaldehyde. Here we show that ADH5 deficient cells turn over formaldehyde using alternative pathways starting from the reaction of formaldehyde with free amino acids. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine - timonacic and spinacine - are increased. These reactions take place spontaneously and the formation of timonacic is reversible. The levels of timonacic are higher in the plasma of Adh5-/- mice relative to controls and they are further increased upon administration of methanol. We conclude that mammals possess pathways of cysteine and histidine dependent formaldehyde metabolism and that timonacic is a formaldehyde reservoir.

Impact of Formate Supplementation on Body Weight and Plasma Amino Acids.

Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here, we investigated the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM of sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology analyser. At the end of the study, blood and tissues were collected for metabolomics analysis and immune cell profiling. Formate supplementation had no significant physiological effect on male mice, except for a small decrease in body weight. Formate supplementation had no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice, however, the body weight and spleen wet weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of bifidobacteria, a probiotic bacterium, in the stools of female mice. We conclude that formate supplementation induces physiological changes in a gender-specific manner.

Formate induces a metabolic switch in nucleotide and energy metabolism.

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.

An automated GCxGC-TOF-MS protocol for batch-wise extraction and alignment of mass isotopomer matrixes from differential 13C-labelling experiments: a case study for photoautotrophic-mixotrophic grown Chlamydomonas reinhardtii cells.

Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of (13)C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC-TOF-MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2-reference spectra and new search algorithms. Using those techniques we analysed the dynamics of (13)CO(2) and (13)C-acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions.

Stratification of cancer and diabetes based on circulating levels of formate and glucose.

BACKGROUND: Serum and urine metabolites have been investigated for their use as cancer biomarkers. The specificity of candidate metabolites can be limited by the impact of other disorders on metabolite levels. In particular, the increasing incidence of obesity could become a significant confounding factor. METHODS: Here we developed a multinomial classifier for the stratification of cancer, obesity and healthy phenotypes based on circulating glucose and formate levels. We quantified the classifier performance from the retrospective analysis of samples from breast cancer, lung cancer, obese individuals and healthy controls. RESULTS: We discovered that circulating formate levels are significantly lower in breast and lung cancer patients than in healthy controls. However, the performance of a cancer classifier based on formate levels alone is limited because obese patients also have low serum formate levels. By introducing a multinomial classifier based on circulating glucose and formate levels, we were able to improve the classifier performance, reaching a true positive rate of 79% with a false positive rate of 8%. CONCLUSIONS: Circulating formate is reduced in HER2+ breast cancer, non-small cell lung cancer and highly obese patients relative to healthy controls. Further studies are required to determine the relevance of these observations in other cancer types and diseases.