Obesity affects the functional capability of adipose-derived stem cells (ASCs) and their effective use in regenerative medicine through mechanisms that are still poorly understood. In the present study we used a multiplatform [LC/MS, GC/MS and capillary electrophoresis/MS (CE/MS)], metabolomics, untargeted approach to investigate the metabolic alteration underlying the inequalities observed in obesity-derived ASCs. The metabolic fingerprint (metabolites within the cells) and footprint (metabolites secreted in the culture medium), from obesity- and non-obesity-derived ASCs of humans or mice, were characterized to provide valuable information. Metabolites associated with glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and the polyol pathway were increased in the footprint of obesity-derived human ASCs, indicating alterations in carbohydrate metabolism, whereas, from the murine model, deep differences in lipid and amino acid catabolism were highlighted. Therefore, new insights on the ASCs’ metabolome were provided that enhance our understanding of the processes underlying ASCs’ stemness capacity and its relationship with obesity, in different cell models.

INTRODUCTION

Adipose tissue is a complex, dynamic and multifunctional organ capable of influencing and coordinating a variety of biological processes in the whole body, including energy metabolism, neuroendocrine function, and inflammatory and immune responses [13]. The relevance of the tissue is further emphasized by its function as the biggest adult stem cell reservoir in the body [4,5].

Adipose-derived stem cells (ASCs) are a type of mesenchymal stem cell (MSC) with extensive self-renewal capacity and a great ability to differentiate along multiple lineages (i.e. adipogenic, osteogenic, chondrogenic, neuronal, endothelial) [610]. In addition, their availability in large quantities, the minimal invasive harvesting procedure, their easy isolation and expansion in vitro and, most importantly, their immunocompatibility have meant that ASCs have been successfully used for regenerative medicine, tissue engineering and autologous cell therapy, as reported over the last few years [1115]. However, despite their wide therapeutic use, little is known about their metabolism and the mechanism of self-renewal. Previous studies pointed out that sex, age, metabolic environment and the donor's depot localization affect the functional capability of ASCs [1618].

Furthermore, it has been demonstrated that an obese environment exerts a detrimental influence on cells, leading to impaired migration and invasion abilities of the ASCs, together with reduced angiogenic potential and growth behaviour [1921]. Although this environment has been included as a variable in consideration of the therapeutic use of ASCs in regenerative medicine, characterization of the mechanisms underpinning the alteration that occurs is still lacking.

Metabolomics is an attractive strategy to delve into the stem cell's fate and physiology [22]. Indeed, it capitalizes on a small amount of material by providing relevant information on thousands of compounds that reflect the cellular phenotype under different conditions, thus facilitating the global comprehension of stem cell properties and functions.

Cell metabolomics can provide valuable information through the characterization of the cellular exo- and endo-metabolome. Indeed, complementary information is obtained from the study of the metabolites both within the cell (metabolic fingerprints), and depleted from or secreted into the culture media (metabolic footprints); these are linked by a mutual and direct relationship, by allowing monitoring of cell metabolic activity that is tightly associated with their self-renewal ability [23].

In addition, the metabolic footprint of ASCs has shown therapeutic potential for regenerative medicine through modulation of the host response via a paracrine effect mediated by soluble factors, such as cytokines, chemokines and growth factors (secretome); these factors are secreted by the transplanted cells and stimulate the recruitment of endogenous stem cells to the site, promoting their differentiation along the required lineage pathway [2426]. In the present study we used a multiplatform untargeted metabolomics approach to shed light on the complex and poorly understood model of the ASCs by combining the information on the metabolic footprint and fingerprint of murine and human ASCs derived from obese and non-obese individuals.

Previous results of the study of obesity-derived ASCs from mice and humans have recently been published [27]. Through a range of functional assays, a general collapse in the homoeostasis regulatory network of obesity-derived ASCs has been highlighted due to a reduced proliferative ability, altered telomerase activity and DNA telomerase length, and impaired mitochondrial content and function.

In the present study, we delve into the alteration of metabolic processes responsible for the inequalities, observed in obesity-derived ASCs, to lead to new breakthroughs in our understanding of the cell metabolome, and moreover to suggest new metabolic targets to revert cell physiology towards normal conditions.

EXPERIMENTAL

Cell culture

Cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS (Sigma), penicillin–streptomycin, L-glutamine and Hepes (all from Lonza) in a humidified atmosphere at 37°C, 5% CO2 and 95% room air.

Culture of mouse and human adipose stem cells

Adipose stem cells were obtained from non-obese or obese mice and humans as described previously [21,28]. Murine tissues were collected from adult C57BL6 mice as the non-obese individuals (non-obesity-derived ASCs) and from 4-month-old mice with diet-induced obesity as the obese individuals (obesity-derived ASCs). Mice were maintained and used in accordance with the National Institutes of Health Animal Care and Use Committee guidelines. DIO, rodent-purified, high-fat diet (formula 58Y1) was obtained from TestDiet (IPS Product Supplies Ltd). The human cells were obtained from non-obese patients [body mass index (BMI) <25 kg/m2] and obese patients (BMI 25–30 kg/m2). Human adipose samples were obtained from patients after bariatric surgery (women aged between 35 and 45 years), which was performed at the Princesa Hospital of Madrid, with a total of five non-obese and five obese patients. Informed consent was obtained from all participants and the sample collection conformed to the principles set out in the World Medical Association's Declaration of Helsinki and the National Institutes of Health's Belmont Report.

Adipose stem cells were isolated from mouse and human tissues, sorted and expanded as described [29]. Small pieces of subcutaneous adipose tissue were collected and placed on Matrigel-coated plates. Cells emerging from the explants were selected, cloned by limiting dilution and grown to obtain ASCs. Cells were incubated and maintained under the same conditions and characterized by flow cytometry for specific surface antigens to define this population: CD44+, CD34+, Sca1+and CD45−.

Sample preparation

Equal numbers of each cell line were used to obtain extracts (five biological replicates). For each replicate, 106 cells were collected by gentle scraping with a rubber cell scraper, suspended in cold methanol extraction solvent on dry ice and vortexed for 1 min. Samples were subjected to three freeze–thaw cycles for complete cell disruption, allowed to sit for 10 min at 4°C, placed in liquid nitrogen for 10 min and thawed in an ice bath for 10 min with brief vortexing. The supernatant was recovered by centrifugation at 5725 g for 5 min at 4°C and the pellet was re-extracted twice.

Afterwards, the extracted cells and the culture media were prepared for the metabolomics analysis as previously described [27,30]. Briefly, protein precipitation was achieved by adding cold organic solvent–a mixture of methanol and ethanol (1:1, v/v) for LC/MS, methanol for capillary electrophoresis/MS (CE/MS) and acetonitrile for GC/MS–to an aliquot of the medium sample in a ratio 3:1. The samples were then centrifuged at 15700 g for 20 min at 4°C to collect the supernatant. Afterwards, for LC/MS analysis, the extracted cell and medium samples were filtered through a 0.22-μm nylon filter; for CE/MS analysis, they were evaporated in a nitrogen stream and then resuspended in ultra-pure water with 0.1 M formic acid and, for GC/MS, they were evaporated and derivatized with O-methoxyamine hydrochloride (15 mg/ml) in pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide in 1% trimethylchlorosilane.

Internal standards, such as methionine sulfone in ultra-pure water (CE/MS) and methyl stearate in heptane (GC/MS), were added to monitor sample injection and correct the intra-batch fluctuation of the migration or retention time (MT or RT) of the metabolites.

Quality control samples

Quality control (QC) samples (n=10 for each analytical technique, where n=5 from cell samples and n=5 from medium samples) were prepared by pooling equal volumes of sample from all samples. QC samples were prepared simultaneously with the rest of the samples by following the aforementioned protocols. They were injected at the beginning, after every five samples, and at the end, to assess the reproducibility of both sample preparation and analysis.

LC/MS analysis

Samples were analysed using a 1290 Infinity series UHPLC system coupled through an ESI source with Jet Stream technology to a 6550 iFunnel quadrupole time-of-flight (QTOF)/MS system (Agilent Technologies) as described previously [31] (see Supplementary Experimental Procedures for full details).

CE/MS analysis

The analysis was carried out using a 7100 CE system coupled through an ESI source to a 6224 TOF/MS system (Agilent Technologies) by optimizing a previously reported method [32] (see Supplementary Experimental Procedures for full details).

GC/MS analysis

Samples were analysed using a 7890A GC system interfaced through an electron ionization source to a 5975C Q/MS (Agilent Technologies) as reported previously [33,34] (see Supplementary Experimental Procedures for full details).

LC/MS and CE/MS data processing

Data were processed using MassHunter Qualitative Analysis (MH Qual B.06.00) and Mass Profiler Professional (MPP B.02.00, both from Agilent Technologies) software as reported [32]. The resulting data matrix was then filtered through the MPP software by retaining the features present in 100% of QC samples with a coefficient of variation (CV or relative standard deviation, RSD) below 30%, either present or absent in 80% (LC/MS data) or 100% (CE/MS data) of the groups under study (see Supplementary Experimental Procedures and Supplementary Table S1 for details).

Compound identification: LC/MS and CE/MS analyses

The statistically significant compounds were putatively identified by searching for their accurate mass against public databases, i.e. METLIN (see http://www.metlin.scripps.edu), KEGG (see http://www.genome.jp/kegg), LIPIDMAPS [see http://www.lipidmaps.org, all simultaneously accessed by a developed search engine in house, CEU MassMediator (see http://ceumass.eps.uspceu.es/mediator)] and the Human Metabolome Database (HMDB, see http://www.hmdb.ca). In addition, to increase the reliability of metabolite identification, the match score between the distribution of the experimental isotopic pattern and the compound formula was computed using MH Qual software. For compounds included in our in-house libraries RTs/MTs were also compared.

Data processing and compound identification: GC/MS analysis

To deconvolute and simultaneously identify the eluted compounds, the Automated Mass Spectrometry Deconvolution and Identification System (AMDIS version 2.71, see http://chemdata.nist.gov/mass-spc/amdis/downloads) was employed as described previously [34]. Then, MPP software was used to align the data from all samples, and the resulting data matrix was filtered by retaining the features present in 100% of the QC samples with a CV below 40%, and the features either present or absent in 80% of the groups under study (see Supplementary Experimental Procedures and Supplementary Table S1 for details).

Statistical data analysis

Statistical analysis was carried out using univariate [UVA, MATLAB R2015 software (Mathworks, Inc.)] and multivariate [MVA, SIMCA P+12.0.1 software (Umetrics)] analyses. For the UVA, the non-parametric Mann−Whitney U-test with Benjamini–Hochberg post-hoc correction (level q=0.05) was applied. For the MVA, log-transformed and Pareto-scaled data, or just Pareto-scaled data, were used to create multivariate models. Afterwards, unsupervised (principal components analysis, PCA) and supervised (orthogonal partial least squares discriminant analysis, OPLS-DA) analyses were applied to check trends and outliers, and to select the variables responsible for the separation shown by the models through the S-plot and jackknife interval. Then, the models were statistically validated using the cross-validation tool by the leave-a-third-out approach to exclude model overfitting. Finally, the percentage change and fold change (FC) for the significant variables resulting from both UVA and MVA were calculated as follows: percentage change=[(average value in the tested group − average value in reference group)/(average value in reference group)]×100; FC=[(average value in the tested group)/(average value in reference group)].

Data quality assurance

Data quality was assured by using the QC samples as reported [35,36]. Briefly, raw data from all samples (participant and QC samples) were processed as described above for each analytical technique. Then, data were filtered by using the QC group as a reference: features present in 100% of the QC samples with a CV below 50% were retained. The final data matrix underwent PCA in SIMCA software to test the analytical reproducibility of the data [37].

RESULTS

In the present study, a naive approach has been used to evaluate the broader possible range of differences between ASCs from obese and non-obese individuals, both murine and human. The final goal of the study was to achieve useful information that allowed evaluation of the ASCs’ metabolic capacity according to the individual's obesity, as well as the suitability of the obese mouse model as a system for study of the metabolic changes in different conditions.

Analysis of 40 samples, including stem cells and their culture media, from humans and mice, either obese or lean, was performed through a multiplatform metabolomics approach. After data alignment, performed separately according to sample type (cell or culture medium samples), the analysis provided a huge amount of data: 101965 variables, for cell samples, and 130909 variables for culture medium samples. After data filtration, the resulting set was employed for statistical data analysis. PCA models, in Figure 1, show clustering of the QC samples, pointing out the good quality of the data in the models under study. The comparisons evaluated by MVA (OPLS-DA) and UVA were obese (o) versus control (c) for both media (M) and cells (ASC), from human (h) and murine (m) models (hoM vs hcM, hoASC vs hcASC, moM vs mcM, moASC vs mcASC), for the three platforms. The OPLS-DA models, displayed in Figures 2 and 3, show clear separation of samples in all analytical techniques for all comparisons. The high quality of the OPLS-DA models is described by the values of explained variance, R2, and the predicted variance, Q2 (Figures 2 and 3). Furthermore, the models were successfully cross-validated as mentioned earlier.

Unsupervised multivariate analysis results

Figure 1
Unsupervised multivariate analysis results

PCA plots generated from all samples (□) and QC samples (◆). (AD) The fingerprinting study, (EH) the footprint study. The supervised models are built from: (A) LC/MS ESI+data (R2=0.69, Q2=0.28), (B) LC/MS ESI− data (R2=0.89, Q2=0.23), (C) CE/MS data (R2=0.63, Q2=0.53), (D) GC/MS data (R2=0.65, Q2=0.24), (E) LC/MS ESI+data (R2=0.49, Q2=0.22), (F) LC/MS ESI− data (R2=0.73, Q2=0.55), (G) CE/MS data (R2=0.60, Q2=0.47) and (H) GC/MS data (R2=0.69, Q2=0.47).

Figure 1
Unsupervised multivariate analysis results

PCA plots generated from all samples (□) and QC samples (◆). (AD) The fingerprinting study, (EH) the footprint study. The supervised models are built from: (A) LC/MS ESI+data (R2=0.69, Q2=0.28), (B) LC/MS ESI− data (R2=0.89, Q2=0.23), (C) CE/MS data (R2=0.63, Q2=0.53), (D) GC/MS data (R2=0.65, Q2=0.24), (E) LC/MS ESI+data (R2=0.49, Q2=0.22), (F) LC/MS ESI− data (R2=0.73, Q2=0.55), (G) CE/MS data (R2=0.60, Q2=0.47) and (H) GC/MS data (R2=0.69, Q2=0.47).

Multivariate analysis results (supervised models, OPLS-DA) for the comparison between obese and non-obese ASCs

Figure 2
Multivariate analysis results (supervised models, OPLS-DA) for the comparison between obese and non-obese ASCs

(AD) Human model [obese (■) vs non-obese (□)]. The supervised models are built from: (A) LC/MS ESI+data (R2=0.99, Q2=0.93), (B) LC/MS ESI− data (R2=1, Q2=0.69), (C) CE/MS data (R2=0.80, Q2=0.97) and (D) GC/MS data (R2=1, Q2=0.97). (EH) Murine model [obese (▲) vs non-obese (∆)]. The supervised models are built from: (E) LC/MS ESI+data (R2=0.99, Q2=0.94), (F) LC/MS ESI− data (R2=1, Q2=0.66), (G) CE/MS data (R2=0.99, Q2=0.99) and (H) GC/MS data (R2=0.94, Q2=0.84).

Figure 2
Multivariate analysis results (supervised models, OPLS-DA) for the comparison between obese and non-obese ASCs

(AD) Human model [obese (■) vs non-obese (□)]. The supervised models are built from: (A) LC/MS ESI+data (R2=0.99, Q2=0.93), (B) LC/MS ESI− data (R2=1, Q2=0.69), (C) CE/MS data (R2=0.80, Q2=0.97) and (D) GC/MS data (R2=1, Q2=0.97). (EH) Murine model [obese (▲) vs non-obese (∆)]. The supervised models are built from: (E) LC/MS ESI+data (R2=0.99, Q2=0.94), (F) LC/MS ESI− data (R2=1, Q2=0.66), (G) CE/MS data (R2=0.99, Q2=0.99) and (H) GC/MS data (R2=0.94, Q2=0.84).

Multivariate analysis results (supervised models, OPLS-DA) for the comparison between culture media harvested from obese and non-obese ASCs

Figure 3
Multivariate analysis results (supervised models, OPLS-DA) for the comparison between culture media harvested from obese and non-obese ASCs

(AD) Human model [obese (□) vs non-obese (■)]. The supervised models are built from: (A) LC/MS ESI+data (R2=1, Q2=0.57), (B) LC/MS ESI− data (R2=1, Q2=0.83), (C) CE/MS data (R2=1, Q2=0.79) and (D) GC/MS data (R2=0.95, Q2=0.90). (EH) Murine model [obese (∆) vs non-obese (▲)]. The supervised models are built from: (E) LC/MS ESI+data (R2=0.99, Q2=0.90), (F) LC/MS ESI− data (R2=0.97, Q2=0.92), (G) CE/MS data (R2=0.98, Q2=0.89) and (H) GC/MS data (R2=0.99, Q2=0.96).

Figure 3
Multivariate analysis results (supervised models, OPLS-DA) for the comparison between culture media harvested from obese and non-obese ASCs

(AD) Human model [obese (□) vs non-obese (■)]. The supervised models are built from: (A) LC/MS ESI+data (R2=1, Q2=0.57), (B) LC/MS ESI− data (R2=1, Q2=0.83), (C) CE/MS data (R2=1, Q2=0.79) and (D) GC/MS data (R2=0.95, Q2=0.90). (EH) Murine model [obese (∆) vs non-obese (▲)]. The supervised models are built from: (E) LC/MS ESI+data (R2=0.99, Q2=0.90), (F) LC/MS ESI− data (R2=0.97, Q2=0.92), (G) CE/MS data (R2=0.98, Q2=0.89) and (H) GC/MS data (R2=0.99, Q2=0.96).

For all analytical techniques, the mean classification scores for the cross-validated cell models were 91% (hoASC vs hcASC) and 90% (moASC vs mcASC), whereas for the cross-validated culture medium models they were 96% (hoM vs hcM) and 98% (hoASC vs hcASC). Thus, all models showed good classification accuracy and were reliably utilized to select the variables responsible for the separation shown by the models. The results from multivariate analysis highlighted 76 (hoASC vs hcASC) and 109 (moASC vs mcASC) variables as significant contributors to the variance, in the cell models, whereas 97 (hoM vs hcM) and 152 (moM vs mcM) were significant contributors in the culture medium models. In addition, P values were computed for the four comparisons under study. A total of 56 (hoASC vs hcASC) and 122 (moASC vs mcASC) compounds were statistically significant (P < 0.05) from the cell models, whereas a total of 118 (hoM vs hcM) and 243 (moM vs mcM) were significant from the culture media models. After a Benjamini–Hochberg post-hoc correction, the number of compounds was reduced to 24 (hoASC vs hcASC) and 64 (moASC vs mcASC), for the cell models, and to 66 (hoM vs hcM) and 87 (moM vs mcM), for the culture medium models. The results of the whole study are summarized in Tables 1 (human cells and medium) and 2 (murine cells and medium). A broad coverage of the metabolites that differed significantly for the comparisons under study was obtained. It has been possible to identify compounds from m/z 88.0160 (i.e. pyruvate) to m/z 940.6041, i.e. glycerophosphoinositol(42:5), with high polarity such as arginine or low polarity such as triacylglycerols, with different functional groups that are carboxylated (i.e. citrate) or aminated (i.e. ornithine), and, from a metabolic point of view, sugars (i.e. fructose), sugar phosphates (i.e. erythrose phosphate), glycolysis products (i.e. GA3P), the TCA cycle (i.e. succinate), amino acids (i.e. tryptophan) and their metabolites (i.e. kynurenine), as well as fatty acids (i.e. palmitic acid) and acyls (i.e. pentadecanal), glycerolipids (i.e. 2-monopalmitin), glycerophospholipids [i.e. glycerophosphoserine(36:2)], sphingolipids [i.e. sphingomyelin(d34:0)], bile acids (i.e. oxocholadienoic acid) and vitamin D3 metabolites. The information held in Tables 1 and 2 is summarized in Figure 4 (metabolite differences), whereas a summary of some of the processes that are altered in the comparison of obese and lean individuals is depicted in Figure 5 (metabolic differences).

Summary of metabolites found to be significant in the footprint and fingerprint study of (A) human and (B) murine ex vivo adipose stem cells from either obese or lean individuals

Figure 4
Summary of metabolites found to be significant in the footprint and fingerprint study of (A) human and (B) murine ex vivo adipose stem cells from either obese or lean individuals

Metabolites with concentrations that were increased in obese individuals are highlighted in yellow and those that were decreased in green. Metabolites found to be common between the two models are pictured within bold boxes. Coloured metabolites were detected using GC, LC, CE/MS (see Tables 1 and 2 for abbreviations of the depicted metabolites).

Figure 4
Summary of metabolites found to be significant in the footprint and fingerprint study of (A) human and (B) murine ex vivo adipose stem cells from either obese or lean individuals

Metabolites with concentrations that were increased in obese individuals are highlighted in yellow and those that were decreased in green. Metabolites found to be common between the two models are pictured within bold boxes. Coloured metabolites were detected using GC, LC, CE/MS (see Tables 1 and 2 for abbreviations of the depicted metabolites).

Overview of metabolic pathways found to be altered in ex vivo adipose stem cells from humans or mice, when obese and non-obese individuals are compared

Figure 5
Overview of metabolic pathways found to be altered in ex vivo adipose stem cells from humans or mice, when obese and non-obese individuals are compared

The main processes and metabolites for which involvement was uncovered by the present study are depicted in bold hexagonal and square boxes, respectively. The biological processes are highlighted in bold italics and enzymes in bold. Three symbols (i.e. ↑, ↓ and −) account for the changes observed. The blue symbol (on the left) refers to the human model, and the magenta symbol (on the right) to the murine model (see text for a description of the depicted pathways and Tables 1 and 2 for abbreviations of the depicted metabolites). AA, amino acids; BA, bile acids; CAR, carnitines; CoA, coenzyme A; DAG, diacylglycerols; DHAP, dihydroxyacetone phosphate; FFA, free fatty acids; LysoPLC, lysophospholipase C; MAG, monoacylglycerols; NSM, neutral sphingomyelinase; PA, phosphatidic acid; PL, phospholipids; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D; TAG, triacylglycerols.

Figure 5
Overview of metabolic pathways found to be altered in ex vivo adipose stem cells from humans or mice, when obese and non-obese individuals are compared

The main processes and metabolites for which involvement was uncovered by the present study are depicted in bold hexagonal and square boxes, respectively. The biological processes are highlighted in bold italics and enzymes in bold. Three symbols (i.e. ↑, ↓ and −) account for the changes observed. The blue symbol (on the left) refers to the human model, and the magenta symbol (on the right) to the murine model (see text for a description of the depicted pathways and Tables 1 and 2 for abbreviations of the depicted metabolites). AA, amino acids; BA, bile acids; CAR, carnitines; CoA, coenzyme A; DAG, diacylglycerols; DHAP, dihydroxyacetone phosphate; FFA, free fatty acids; LysoPLC, lysophospholipase C; MAG, monoacylglycerols; NSM, neutral sphingomyelinase; PA, phosphatidic acid; PL, phospholipids; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D; TAG, triacylglycerols.

Table 1
Metabolites identified in the study of exo- and endo-metabolome of human ASCs, which were statistically significant after adjustment for multiple testing (P<0.05), in the comparison between obese ASCs and control ASCs (hoASCs vs hcASCs, and hoM vs hcM)

Rows with bold text refer to ASC results; rows with Roman (non-bold) text refer to culture media results. Category and biochemical subclass were assigned according to the LIPIDMAPS consortium. The entity code was generated according to the analytical technique (G: GC/MS data; C: CE/MS data; LP: LC/(ESI+)/MS data; LN: LC/(ESI−)/MS data), compartment (M: culture medium, cells elsewhere) and consecutive numbers.

Category Biochemical subclass Entity Compound Acronym FC Change (%) CV QC P value TI (GC) or mass (LC, CE) Mass error (ppm) RT (GC, LC), MT (CE) (min) Score
(%) 
Formula 
Fatty acyls Fatty acids and conjugates C001 Acetylcarnitine† C2-CAR 1.64 64 7 4.50×10−2 203.1158 0 14.27 78 C9H17NO4 
Fatty acyls Fatty acids and conjugates GM004 Tetradecanoic acid
(myristic acid) 
MYR 0.58 −42 21 3.81×10−2 343 – 21.97 92 C14H28O2 
Fatty acyls Fatty acids and conjugates G001 Hexadecanoic acid
(palmitic acid) 
PAL 1.91 91 36 2.99×10−2 117  18.77 75 C16H32O2 
Fatty acyls Fatty acids and conjugates LP003 Heptadecanoic acid
(margaric acid)*† 
MAR 0 Con 6  270.2559 0 2.99 100 C17H34O2 
Fatty acyls Fatty acids and conjugates LP007 Nonadecanoic acid
(nonadecylic acid)*† 
NON 0 Con 9  298.2872 0 4.18 100 C19H38O2 
Fatty acyls Fatty acids and conjugates LP005 Hydroxy-heptadecanoic acid*† OHMAR 0 Con 4  286.2508 0 2.75 98 C17H34O3 
Fatty acyls Fatty acids and conjugates LP006 Hydroxy-nonadecanoic acid* OHNON 0 Con 5  314.2821 0 3.89 97 C19H38O3 
Fatty acyls Fatty acyls and conjugates LNM002 Glucosyl-octaconsanetriol* GLC-OTC 100 3.31×10−2 604.4841 12 8.11 – C34H68O8 
Glycerolipids Monoacylglycerols LP009 MG(14:0)* MG(14:0) 0 Con 16  302.2454 1 3.94 96 C17H34O4 
Glycerolipids Monoacylglycerols LPM003 MG(14:0)* MG(14:0) 0.62 −38 2.78×10−2 302.246 4.10 82 C17H34O4 
Glycerolipids Monoacylglycerols G002 MG(16:0)_2*
(2-monopalmitin) 
MG(16:0) 0 Con 15  371  23.36 97 C19H38O4 
Glycerolipids Monoacylglycerols GM005 MG(18:0)_1* MG(18:0) 0.59 −41 20 3.81×10−2 399 – 24.95 88 C21H42O4 
Glycerolipids Monoacylglycerols LPM006 MG(20:0)_1* MG(20:0) 0.44 −56 14 2.78×10−2 386.3395 6.35 92 C23H46O4 
Glycerolipids Diacylglycerols LPM008 DG(32:0) DG(32:0) 2.05 105 2.78×10−2 568.5067 8.10 38 C35H68O5 
Glycerolipids Diacylglycerols LPM009 DG(36:0)* DG(36:0) 1.99 99 15 2.78×10−2 624.5693 8.51 96 C39H76O5 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LNM004 PC(O-33:6)/PE(O-36:0)* PC/E(O-33/36:0) 1.24 24 4.67×10−2 719.6192 8.36 36 C41H86NO6
Glycerophospholipids Glycerophosphoglycerols LN002 PG(40:5) PG(40:5) 1.45 45 10 4.17×10−2 824.5617 5 0.28 92 C46H81O10P 
Glycerophospholipids Glycerophosphatidic acids LN001 PA(30:2) PA(30:2) 1.67 67 6 4.17×10−2 616.4104 8 0.27  C33H61O8P 
Glycerophospholipids Glycerophosphatidic acids LNM003 PA(44:10) PA(44:10) 1.2 20 11 4.67×10−2 796.5022 7.29 – C47H73O8
Glycerophospholipids Glycerophosphatidic acids LPM014 LysoPA(O-18:0)* LPA(O-18:0) 0.44 −56 13 2.78×10−2 424.2952 6.34 75 C21H45O6
Sphingolipids Sphingolipids LP002 Heptadecasphinganine*† C17-SPN 0 Con 6  287.2824 0 2.56 100 C17H37NO2 
Sterol lipids Bile acids, alcohols and derivatives LNM008 Taurodeoxycholic acid TDC 39 4.67×10−2 499.2962 1.38 84 C26H45NO6
Carbohydrates Monosaccharide GM007 Fructose* FRU 1.89 89 19 3.81×10−2 103 – 17.08 94 C6H12O6 
Carbohydrates Monosaccharide GM008 Glucose* GLU 2.37 137 26 2.38×10−2 319 – 17.34 99 C6H12O6 
Carbohydrates Monosaccharide GM009 Ribose RIB 1.65 65 18 2.38×10−2 73 – 15.05 88 C5H10O5 
Carbohydrates Sugar alcohols GM010 Sorbitol* SOR 2.23 123 22 2.38×10−2 73 – 17.87 77 C6H14O6 
Carbohydrates Sugar alcohols GM011 Threitol* THE 1.67 67 18 2.38×10−2 73 – 13.02 99 C4H10O4 
Carbohydrates Sugar alcohols GM012 Xylitol XYL 1.97 97 16 2.38×10−2 73 – 15.55 96 C5H12O5 
Carbohydrates Monosaccharide/phosphate of G014 Glycerol 1-phosphate* G1P 2 Obe   299  15.98 74 C3H9O6P 
Carbohydrates Monosaccharide/phosphate of C005 Glyceraldehyde 3-phosphate GA3P 0.63 −37 14 4.50×10−2 169.998 23 9.03  C3H7O6P 
Carbohydrates Nucleotides LNM011 dTDP-glucose d TDP-GLC 1.74 74 22 3.31×10−2 564.0758 0.24 – C16H26N2O16P2 
Organic acids Organic acid and derivatives GM013 3-Hydroxybutyric acid OHBUT 3.14 214 27 2.38×10−2 117 – 8.27 90 C4H8O3 
Organic acids Organic acid and derivatives GM014 Acetoacetic acid ACAC 1.69 69 20 2.38×10−2 73 – 7.86 81 C4H8O3 
Organic acids Organic acid and derivatives GM020 Pyruvic acid* PYR 1.89 89 19 2.38×10−2 73 – 6.65 100 C3H4O3 
Organic acids Organic acid and derivatives GM016 Citric acid CIT 3.36 236 12 3.81×10−2 273 – 16.57 91 C6H8O7 
Organic acids Organic acid and derivatives G004 Citric acid* CIT 2.41 141 12 8.06×10−4 273  16.53 97 C6H8O7 
Organic acids Organic acid and derivatives GM021 Succinic acid* SUC 300 29 3.81×10−2 147 – 10.44 98 C4H6O4 
Organic acids Organic acid and derivatives GM018 Malic acid MAL 3.54 254 28 2.38×10−2 73 – 12.73 95 C4H6O5 
Organic acids Organic acid and derivatives G006 Malic acid* MAL 1.33 33 18 2.38×10−2 73  12.75 97 C4H6O5 
Organic acids Organic acid and derivatives GM015 Aminomalonic acid AMA 2.59 159 28 2.38×10−2 218 – 12.53 97 C3H5NO4 
Organic acids Organic acid and derivatives GM017 Glyceric acid GLYC 3.38 238 29 2.38×10−2 73 – 10.68 92 C3H6O4 
Organic acids Organic acid and derivatives GM019 Oxalic acid* OXA 2.76 176 32 2.38×10−2 73 – 7.97 70 C2H2O4 
Amino acids Amino acids and derivatives G008 Glycine* GLY 1.63 63 4 1.11×10−3 174  10.31 98 C2H5NO2 
Amino acids Amino acids and derivatives GM023 Glycine* GLY 2.89 189 32 2.38×10−2 174 – 10.40 99 C2H5NO2 
Amino acids Amino acids and derivatives G007 Alanine* ALA 2.53 153 61 1.83×10−2 116  7.49 97 C3H7NO2 
Amino acids Amino acids and derivatives GM022 Alanine*a ALA 2.58 158 31 2.38×10−2 116 – 7.44 97 C3H7NO2 
Amino acids Amino acids and derivatives G013 Valine* VAL 1.96 96 27 1.11×10−2 144  9.06 99 C5H11NO2 
Amino acids Amino acids and derivatives GM033 Valine* VAL 2.06 106 18 2.38×10−2 144 – 9.06 99 C5H11NO2 
Amino acids Amino acids and derivatives GM025 Leucine* LEU 1.66 66 20 2.38×10−2 158 – 9.85 96 C6H13NO2 
Amino acids Amino acids and derivatives G009 Isoleucine*a ILE 2.32 132 7 2.69×10−2 86  8.63 97 C6H13NO2 
Amino acids Amino acids and derivatives GM024 Isoleucine* ILE 1.68 68 13 2.38×10−2 86 – 8.63 98 C6H13NO2 
Amino acids Amino acids and derivatives G010 Phenylalanine*a PHE 1.86 86 25 2.99×10−2 120  13.52 96 C9H11NO2 
Amino acids Amino acids and derivatives GM027 Phenylalanine* PHE 1.68 68 21 2.38×10−2 120 – 13.52 87 C9H11NO2 
Amino acids Amino acids and derivatives G012 Tyrosine*a TYR 3.58 258 16 1.11×10−3 179  17.25 90 C9H11NO3 
Amino acids Amino acids and derivatives GM032 Tyrosine* TYR 2.8 180 30 2.38×10−2 179 – 17.25 97 C9H11NO3 
Amino acids Amino acids and derivatives GM026 Methionine* MET 2.07 107 17 2.38×10−2 176 – 13.11 91 C5H11NO2
Amino acids Amino acids and derivatives GM028 Proline* PRO 2.28 128 27 2.38×10−2 142 – 10.22 98 C5H9NO2 
Amino acids Amino acids and derivatives GM029 Glutamic acid*† GLU 2.41 141 20 2.38×10−2 156 – 13.15 97 C5H9NO4 
Amino acids Amino acids and derivatives GM030 Serine* SER 2.65 165 27 3.81×10−2 204 – 11.08 99 C3H7NO3 
Amino acids Amino acids and derivatives GM031 Threonine* THR 2.66 166 24 2.38×10−2 73 – 11.40 98 C4H9NO3 
Amino acids Amino acids and derivatives CM005 Homocysteine* HCYS 0.66 −34 11 3.25×10−2 268.0551 15.76 74 C8H16N2O4S2 
Amino acids Amino acids and derivatives G011 Putrescine* PUT 2.71 171  1.29×10−3 174  15.69 70 C4H12N2 
Amino acids Amino acids and derivatives CM003 Acetyl-histidine ACHIS 1.25 25 3.25×10−2 197.0804 13.34 70 C8H11N3O3 
Amino acids Amino acids and derivatives CM004 Fructose-lysine* FRC-LYS 2.34 134 3.25×10−2 308.1583 12.75 98 C12H24N2O7 
Vitamins and cofactors Secosteroids LNM009 Vitamin D3 metabolite* D3_2 0.79 −21 3.31×10−2 394.2851 5.76 73 C27H38O2 
Vitamins and cofactors Secosteroids LNM010 Vitamin D3 metabolite* D3_3 0.78 −22 3.31×10−2 394.2849 5.62 73 C27H38O2 
Vitamins and cofactors Secosteroids LN003 Vitamin D3 metabolite* D3_5 0.39 −61 8 4.17×10−2 394.284 9 5.63  C27H38O2 
Vitamins and cofactors Water-soluble vitamins GM006 1-Methyl nicotinamide MB3 2.48 148 2.38×10−2 179 – 12.74 83 C7H9N2
Category Biochemical subclass Entity Compound Acronym FC Change (%) CV QC P value TI (GC) or mass (LC, CE) Mass error (ppm) RT (GC, LC), MT (CE) (min) Score
(%) 
Formula 
Fatty acyls Fatty acids and conjugates C001 Acetylcarnitine† C2-CAR 1.64 64 7 4.50×10−2 203.1158 0 14.27 78 C9H17NO4 
Fatty acyls Fatty acids and conjugates GM004 Tetradecanoic acid
(myristic acid) 
MYR 0.58 −42 21 3.81×10−2 343 – 21.97 92 C14H28O2 
Fatty acyls Fatty acids and conjugates G001 Hexadecanoic acid
(palmitic acid) 
PAL 1.91 91 36 2.99×10−2 117  18.77 75 C16H32O2 
Fatty acyls Fatty acids and conjugates LP003 Heptadecanoic acid
(margaric acid)*† 
MAR 0 Con 6  270.2559 0 2.99 100 C17H34O2 
Fatty acyls Fatty acids and conjugates LP007 Nonadecanoic acid
(nonadecylic acid)*† 
NON 0 Con 9  298.2872 0 4.18 100 C19H38O2 
Fatty acyls Fatty acids and conjugates LP005 Hydroxy-heptadecanoic acid*† OHMAR 0 Con 4  286.2508 0 2.75 98 C17H34O3 
Fatty acyls Fatty acids and conjugates LP006 Hydroxy-nonadecanoic acid* OHNON 0 Con 5  314.2821 0 3.89 97 C19H38O3 
Fatty acyls Fatty acyls and conjugates LNM002 Glucosyl-octaconsanetriol* GLC-OTC 100 3.31×10−2 604.4841 12 8.11 – C34H68O8 
Glycerolipids Monoacylglycerols LP009 MG(14:0)* MG(14:0) 0 Con 16  302.2454 1 3.94 96 C17H34O4 
Glycerolipids Monoacylglycerols LPM003 MG(14:0)* MG(14:0) 0.62 −38 2.78×10−2 302.246 4.10 82 C17H34O4 
Glycerolipids Monoacylglycerols G002 MG(16:0)_2*
(2-monopalmitin) 
MG(16:0) 0 Con 15  371  23.36 97 C19H38O4 
Glycerolipids Monoacylglycerols GM005 MG(18:0)_1* MG(18:0) 0.59 −41 20 3.81×10−2 399 – 24.95 88 C21H42O4 
Glycerolipids Monoacylglycerols LPM006 MG(20:0)_1* MG(20:0) 0.44 −56 14 2.78×10−2 386.3395 6.35 92 C23H46O4 
Glycerolipids Diacylglycerols LPM008 DG(32:0) DG(32:0) 2.05 105 2.78×10−2 568.5067 8.10 38 C35H68O5 
Glycerolipids Diacylglycerols LPM009 DG(36:0)* DG(36:0) 1.99 99 15 2.78×10−2 624.5693 8.51 96 C39H76O5 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LNM004 PC(O-33:6)/PE(O-36:0)* PC/E(O-33/36:0) 1.24 24 4.67×10−2 719.6192 8.36 36 C41H86NO6
Glycerophospholipids Glycerophosphoglycerols LN002 PG(40:5) PG(40:5) 1.45 45 10 4.17×10−2 824.5617 5 0.28 92 C46H81O10P 
Glycerophospholipids Glycerophosphatidic acids LN001 PA(30:2) PA(30:2) 1.67 67 6 4.17×10−2 616.4104 8 0.27  C33H61O8P 
Glycerophospholipids Glycerophosphatidic acids LNM003 PA(44:10) PA(44:10) 1.2 20 11 4.67×10−2 796.5022 7.29 – C47H73O8
Glycerophospholipids Glycerophosphatidic acids LPM014 LysoPA(O-18:0)* LPA(O-18:0) 0.44 −56 13 2.78×10−2 424.2952 6.34 75 C21H45O6
Sphingolipids Sphingolipids LP002 Heptadecasphinganine*† C17-SPN 0 Con 6  287.2824 0 2.56 100 C17H37NO2 
Sterol lipids Bile acids, alcohols and derivatives LNM008 Taurodeoxycholic acid TDC 39 4.67×10−2 499.2962 1.38 84 C26H45NO6
Carbohydrates Monosaccharide GM007 Fructose* FRU 1.89 89 19 3.81×10−2 103 – 17.08 94 C6H12O6 
Carbohydrates Monosaccharide GM008 Glucose* GLU 2.37 137 26 2.38×10−2 319 – 17.34 99 C6H12O6 
Carbohydrates Monosaccharide GM009 Ribose RIB 1.65 65 18 2.38×10−2 73 – 15.05 88 C5H10O5 
Carbohydrates Sugar alcohols GM010 Sorbitol* SOR 2.23 123 22 2.38×10−2 73 – 17.87 77 C6H14O6 
Carbohydrates Sugar alcohols GM011 Threitol* THE 1.67 67 18 2.38×10−2 73 – 13.02 99 C4H10O4 
Carbohydrates Sugar alcohols GM012 Xylitol XYL 1.97 97 16 2.38×10−2 73 – 15.55 96 C5H12O5 
Carbohydrates Monosaccharide/phosphate of G014 Glycerol 1-phosphate* G1P 2 Obe   299  15.98 74 C3H9O6P 
Carbohydrates Monosaccharide/phosphate of C005 Glyceraldehyde 3-phosphate GA3P 0.63 −37 14 4.50×10−2 169.998 23 9.03  C3H7O6P 
Carbohydrates Nucleotides LNM011 dTDP-glucose d TDP-GLC 1.74 74 22 3.31×10−2 564.0758 0.24 – C16H26N2O16P2 
Organic acids Organic acid and derivatives GM013 3-Hydroxybutyric acid OHBUT 3.14 214 27 2.38×10−2 117 – 8.27 90 C4H8O3 
Organic acids Organic acid and derivatives GM014 Acetoacetic acid ACAC 1.69 69 20 2.38×10−2 73 – 7.86 81 C4H8O3 
Organic acids Organic acid and derivatives GM020 Pyruvic acid* PYR 1.89 89 19 2.38×10−2 73 – 6.65 100 C3H4O3 
Organic acids Organic acid and derivatives GM016 Citric acid CIT 3.36 236 12 3.81×10−2 273 – 16.57 91 C6H8O7 
Organic acids Organic acid and derivatives G004 Citric acid* CIT 2.41 141 12 8.06×10−4 273  16.53 97 C6H8O7 
Organic acids Organic acid and derivatives GM021 Succinic acid* SUC 300 29 3.81×10−2 147 – 10.44 98 C4H6O4 
Organic acids Organic acid and derivatives GM018 Malic acid MAL 3.54 254 28 2.38×10−2 73 – 12.73 95 C4H6O5 
Organic acids Organic acid and derivatives G006 Malic acid* MAL 1.33 33 18 2.38×10−2 73  12.75 97 C4H6O5 
Organic acids Organic acid and derivatives GM015 Aminomalonic acid AMA 2.59 159 28 2.38×10−2 218 – 12.53 97 C3H5NO4 
Organic acids Organic acid and derivatives GM017 Glyceric acid GLYC 3.38 238 29 2.38×10−2 73 – 10.68 92 C3H6O4 
Organic acids Organic acid and derivatives GM019 Oxalic acid* OXA 2.76 176 32 2.38×10−2 73 – 7.97 70 C2H2O4 
Amino acids Amino acids and derivatives G008 Glycine* GLY 1.63 63 4 1.11×10−3 174  10.31 98 C2H5NO2 
Amino acids Amino acids and derivatives GM023 Glycine* GLY 2.89 189 32 2.38×10−2 174 – 10.40 99 C2H5NO2 
Amino acids Amino acids and derivatives G007 Alanine* ALA 2.53 153 61 1.83×10−2 116  7.49 97 C3H7NO2 
Amino acids Amino acids and derivatives GM022 Alanine*a ALA 2.58 158 31 2.38×10−2 116 – 7.44 97 C3H7NO2 
Amino acids Amino acids and derivatives G013 Valine* VAL 1.96 96 27 1.11×10−2 144  9.06 99 C5H11NO2 
Amino acids Amino acids and derivatives GM033 Valine* VAL 2.06 106 18 2.38×10−2 144 – 9.06 99 C5H11NO2 
Amino acids Amino acids and derivatives GM025 Leucine* LEU 1.66 66 20 2.38×10−2 158 – 9.85 96 C6H13NO2 
Amino acids Amino acids and derivatives G009 Isoleucine*a ILE 2.32 132 7 2.69×10−2 86  8.63 97 C6H13NO2 
Amino acids Amino acids and derivatives GM024 Isoleucine* ILE 1.68 68 13 2.38×10−2 86 – 8.63 98 C6H13NO2 
Amino acids Amino acids and derivatives G010 Phenylalanine*a PHE 1.86 86 25 2.99×10−2 120  13.52 96 C9H11NO2 
Amino acids Amino acids and derivatives GM027 Phenylalanine* PHE 1.68 68 21 2.38×10−2 120 – 13.52 87 C9H11NO2 
Amino acids Amino acids and derivatives G012 Tyrosine*a TYR 3.58 258 16 1.11×10−3 179  17.25 90 C9H11NO3 
Amino acids Amino acids and derivatives GM032 Tyrosine* TYR 2.8 180 30 2.38×10−2 179 – 17.25 97 C9H11NO3 
Amino acids Amino acids and derivatives GM026 Methionine* MET 2.07 107 17 2.38×10−2 176 – 13.11 91 C5H11NO2
Amino acids Amino acids and derivatives GM028 Proline* PRO 2.28 128 27 2.38×10−2 142 – 10.22 98 C5H9NO2 
Amino acids Amino acids and derivatives GM029 Glutamic acid*† GLU 2.41 141 20 2.38×10−2 156 – 13.15 97 C5H9NO4 
Amino acids Amino acids and derivatives GM030 Serine* SER 2.65 165 27 3.81×10−2 204 – 11.08 99 C3H7NO3 
Amino acids Amino acids and derivatives GM031 Threonine* THR 2.66 166 24 2.38×10−2 73 – 11.40 98 C4H9NO3 
Amino acids Amino acids and derivatives CM005 Homocysteine* HCYS 0.66 −34 11 3.25×10−2 268.0551 15.76 74 C8H16N2O4S2 
Amino acids Amino acids and derivatives G011 Putrescine* PUT 2.71 171  1.29×10−3 174  15.69 70 C4H12N2 
Amino acids Amino acids and derivatives CM003 Acetyl-histidine ACHIS 1.25 25 3.25×10−2 197.0804 13.34 70 C8H11N3O3 
Amino acids Amino acids and derivatives CM004 Fructose-lysine* FRC-LYS 2.34 134 3.25×10−2 308.1583 12.75 98 C12H24N2O7 
Vitamins and cofactors Secosteroids LNM009 Vitamin D3 metabolite* D3_2 0.79 −21 3.31×10−2 394.2851 5.76 73 C27H38O2 
Vitamins and cofactors Secosteroids LNM010 Vitamin D3 metabolite* D3_3 0.78 −22 3.31×10−2 394.2849 5.62 73 C27H38O2 
Vitamins and cofactors Secosteroids LN003 Vitamin D3 metabolite* D3_5 0.39 −61 8 4.17×10−2 394.284 9 5.63  C27H38O2 
Vitamins and cofactors Water-soluble vitamins GM006 1-Methyl nicotinamide MB3 2.48 148 2.38×10−2 179 – 12.74 83 C7H9N2

†Multiple putative identification. See Supplementary Table S2 for alternative identifications of the same entity. *Significant in both MVA and UVA. aMetabolite found to be significant in more than one analytical technique. Change (%) refers to obesity-derived ASCs or M compared with non-obesity-derived ASCs or M. Scored as isotopic pattern distribution score (LC/MS and CE/MS data) or quality match against the in-house library (GC/MS data). Obe: metabolite present only in the obese group; Con: metabolite present only in the control group. Acronym, arbitrary metabolite acronym; TI, target ion (m/z).

Table 2
Metabolites identified in the study of exo- and endo-metabolome of murine ASCs, which were statistically significant after adjustment for multiple testing (P<0.05), in the comparison between obese and control ASCs (moASCs vs mcASCs and moM vs mcM)

Rows with bold text refer to ASC results; rows with Roman (non-bold) text refer to culture media results. Category and biochemical subclass were assigned according to the LIPIDMAPS consortium. Entity code was generated according to the analytical technique (G: GC/MS data; C: CE/MS data; LP: LC/(ESI+)/MS data; LN: LC/(ESI−)/MS data), compartment (M: culture medium, cells elsewhere) and consecutive numbers.

Category Biochemical subclass Entity Compound Acronym FC Change (%) CV QC P TI (GC) or mass (LC, CE) Mass error (ppm) RT (GC, LC), MT (CE) (min) Score (%) Formula 
Fatty acyls Fatty acids and conjugates C001 Acetylcarnitine*† C2-CAR 0.39 −61 11 1.17×10−2 203.1158 0 14.26 79 C9H17NO4 
Fatty acyls Fatty acids and conjugates LP001 Butyrylcarnitine C4-CAR 0.53 −47 4 8.48×10−3 231.1474 1 0.26 98 C11H21NO4 
Fatty acyls Fatty acids and conjugates GM004 Tetradecanoic acid
(myristic acid) 
MYR Con 21 − 343 − 21.97 92 C14H28O2 
Fatty acyls Fatty acids and conjugates LP002 Heptadecanoic acid
(margaric acid)*† 
MAR 2 Obe 6  270.2559 0 2.56 100 C17H34O2 
Fatty acyls Fatty acids and conjugates LP008 Nonadecanoic acid
(nonadecylic acid)*† 
NON 2 Obe 4  298.2872 0 3.72 100 C19H38O2 
Fatty acyls Fatty acids and conjugates LPM001 Nonadecanoic acid
(nonadecylic acid)*† 
NON 0.33 −67 1.22×10−2 298.2880 5.75 77 C19H38O2 
Fatty acyls Fatty acids and conjugates LP004 Epoxy-eicosatetraenoic acid
(leukotriene A4)† 
LTA4 0.65 −35 6 5.06×10−3 318.2195 0 4.11 81 C20H30O3 
Fatty acyls Fatty acids and conjugates LP005 Hydroxy-heptadecanoic acid*† OHMAR 2 Obe 4  286.2508 0 2.75 98 C17H34O3 
Fatty acyls Fatty acids and conjugates LP006 Hydroxy-nonadecanoic acid* OHNON 2 Obe 5  314.2821 0 3.89 97 C19H38O3 
Fatty acyls Fatty acyls and conjugates LNM002 Glucosyl-octaconsanetriol* GLC-OTC 1.31 31 1.47×10−2 604.4841 12 8.11 − C34H68O8 
Fatty acyls Fatty acyls and conjugates LP017 Pentadecanenol* OHC25 2 Obe 8  226.2297 0 1.92 100 C15H30O 
Fatty acyls Fatty acyls and conjugates LPM002 Linoleoyl ethanolamide* LEAM 1.98 98 1.22×10−2 323.2824 4.76 64 C20H37NO2 
Glycerolipids Monoacylglycerols LP009 MG(14:0) MG(14:0) 0.63 −37 16 8.10×10−3 302.2459 0 4.11 98 C17H34O4 
Glycerolipids Monoacylglycerols LPM003 MG(14:0)* MG(14:0) 0.39 −61 1.22×10−2 302.2460 4.10 91 C17H34O4 
Glycerolipids Monoacylglycerols LNM001 MG(15:0)*† MG(15:0) 1.83 83 1.47×10−2 316.2612 2.44 97 C18H36O4 
Glycerolipids Monoacylglycerols GM001 MG(16:0)_2*
(2-monopalmitin) 
MG(16:0) Con 12 − 371 − 23.51 94 C19H38O4 
Glycerolipids Monoacylglycerols GM002 MG(16:0)_1
(1-monopalmitin) 
MG(16:0) Con 16 − 129 − 23.25 92 C19H38O4 
Glycerolipids Monoacylglycerols LPM004 MG(16:0)* MG(16:0) 0.54 −46 1.22×10−2 330.2773 5.03 100 C19H38O4 
Glycerolipids Monoacylglycerols GM003 MG(18:0)_2* MG(18:0) Con 20 – 129 – 24.69 88 C21H42O4 
Glycerolipids Monoacylglycerols LPM005 MG(18:0)* MG(18:0) 0.37 −63 1.22×10−2 358.3085 5.76 100 C21H42O4 
Glycerolipids Monoacylglycerols LPM006 MG(20:0)_1* MG(20:0) 0.17 −83 15 1.22×10−2 386.3395 6.34 97 C23H46O4 
Glycerolipids Monoacylglycerols LPM007 MG(20:0)_2 MG(20:0) 0.52 −48 12 1.22×10−2 386.3395 5.76 95 C23H46O4 
Glycerolipids Diacylglycerols LPM010 DG(41:5) DG(41:5) 0.45 −55 1.22×10−2 684.5693 5.03 87 C44H76O5 
Glycerolipids Diacylglycerols LPM011 DG(44:6) DG(44:6) 0.3 −70 15 1.22×10−2 724.6006 5.76 90 C47H80O5 
Glycerolipids Triacylglycerols LPM012 TG(46:2) TG(46:2) 1.43 43 12 1.22×10−2 774.6737 12 7.83 − C49H90O6 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM022 PC(20:1)/PE(23:1) PC/E(20/23:1) 0.57 −43 13 1.22×10−2 703.5143 7.12 − C38H74NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LP013 PC(32:3)/PE(35:3) PC/E(32/35:3) 0.52 −48 6 4.94×10−2 727.5143 1 7.11 66 C47H74NO8P 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM023 PC(38:0)/PE(41:0) PC/E(38/41:0) 0.57 −43 17 1.22×10−2 677.4968 7.02 − C36H72NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM024 PC(38:6)/PE(41:6)* PC/E(38/41:6) 1.5 50 3.81×10−2 805.5615 7.49 − C46H80NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM025 PC(41:6)/PE(44:6) PC/E(41/44:6) 1.4 40 12 3.81×10−2 847.6076 7.86 − C49H86NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM026 PC(O-42:6)/PE(O-45:6) PC/E(O-42/45:6) 1.4 40 3.81×10−2 847.6437 8.12 − C50H90NO7
Glycerophospholipids Glycerophosphoserine LP014 PS(36:2)* PS(36:2) 0.36 −64 14 5.77×10−3 773.5572 0 6.94  C42H80NO9P 
Glycerophospholipids Glycerophosphoserine LNM006 PS(42:7) PS(42:7) 1.38 38 11 3.26×10−2 861.5520 11 7.94 − C48H80NO10
Glycerophospholipids Glycerophosphoserine LP015 PS(P-34:0)* PS(P-34:0) 0.34 −66 11 8.10×10−3 747.5401 1 6.85 93 C40H78NO9P 
Glycerophospholipids Glycerophosphoinositol LNM005 PI(42:5) PI(42:5) 1.48 48 2.30×10−2 940.6041 11 8.11 − C51H89O13
Glycerophospholipids Glycerophosphoinositol C003 PIP(34:1)* PIP(34:1) 0.79 −21 4 2.14×10−2 888.4833 7 8.72  C41H78O16P2 
Glycerophospholipids Glycerophosphatidic acid LNM003 PA(44:10) PA(44:10) 1.38 38 25 3.97×10−2 796.5022 7.29 − C47H73O8
Glycerophospholipids Glycerophosphatidic acid LPM015 PA(O-42:6)* PA(O-42:6) 0.35 −65 14 1.22×10−2 762.5584 5.75 96 C45H79O7
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM017 LysoPC(16:0)/LysoPE(19:0) LPC/E(16/19:0) 1.38 38 10 2.21×10−2 495.3321 4.25 38 C24H50NO7
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM018 LysoPC(18:0)/LysoPE(21:0) LPC/E(18/21:0) 1.55 55 13 1.22×10−2 523.3633 5.10 96 C26H54NO7
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LP012 LysoPC(O-16:0)/LysoPE(O-19:0)* LPC/E(O-16/19:0) 0.35 −65 4 3.52×10−3 481.3530 0 4.73 93 C24H52NO6P 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM019 LysoPC(O-22:0)/LysoPE(O-25:0) LPC/E(O-22/25:0) 1.48 48 2.21×10−2 565.4468 6.62 − C30H64NO6
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM020 LysoPC(O-32:0)/LysoPE(O-35:0)* LPC/E(O-32/35:0) 0.23 −77 1.22×10−2 705.6036 5.75 − C40H84NO6
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM021 LysoPC(34:0)/LysoPE(37:0) LPC/E(34/37:0) 1.51 51 1.22×10−2 761.5928 7.89 − C42H84NO8
Glycerophospholipids Glycerophosphatidic acid LPM013 LysoPA(O-16:0)* LPA(O-16:0) 0.38 −62 1.22×10−2 396.2645 5.76 85 C19H41O6
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM016 Glycerophosphocholine* GPC 5.73 473 13 1.22×10−2 257.1028 0.24 99 C8H20NO6
Sphingolipids Sphingoid bases LP003 Heptadecasphinganine*† C17-SPN 2 Obe 8  287.2824 0 2.99 100 C17H37NO2 
Sphingolipids Ceramide LP010 Dihydroceramide C2* CER-DHC2 2 Obe 11  343.3080 1 4.62 85 C20H41NO3 
Sphingolipids Phosphosphingolipids LPM027 Cer(34:0) CER 0.56 −44 15 2.21×10−2 571.5157 7.94 − C34H69NO5 
Sphingolipids Phosphosphingolipids LPM029 SM(d34:0) SM(d34:0) 1.4 40 1.22×10−2 704.5820 7.42 94 C39H81N2O6
Sphingolipids Phosphosphingolipids LPM030 SM(d34:1)* SM(d34:1) 1.38 38 1.22×10−2 702.5677 7.29 100 C39H79N2O6
Sphingolipids Phosphosphingolipids LPM031 SM(d39:1) SM(d39:1) 1.42 42 1.22×10−2 772.6444 7.97 − C44H89N2O6
Sphingolipids Phosphosphingolipids LPM032 SM(d40:1)* SM(d40:1) 1.38 38 1.22×10−2 786.6610 8.07 96 C45H91N2O6
Sphingolipids Phosphosphingolipids LPM033 SM(d41:1) SM(d41:1) 1.36 36 1.22×10−2 800.6760 8.18 − C46H93N2O6
Sphingolipids Phosphosphingolipids LPM034 SM(d42:2)* SM(d42:2) 1.57 57 1.22×10−2 812.6766 8.10 98 C47H93N2O6
Sphingolipids Neutral glycosphingolipids LPM028 GlcCer(d38:1)/GalCer(d38:1) GCER 1.59 59 16 3.81×10−2 755.6268 8.20 − C44H85NO8 
Sterol lipids Bile acids, alcohols and derivatives LPM036 Hydroxy-oxocholadienoic acid OH-OCD 0.57 −43 1.22×10−2 368.2332 5.03 64 C24H32O3 
Sterol lipids Bile acids, alcohols and derivatives LNM007 Oxocholadienoic acid* OCD 1.78 78 1.47×10−2 370.2508 2.44 − C24H32O3 
Sterol lipids Bile acids, alcohols and derivatives C004 Trihydroxy-methyl-cholestanoic acid* TOHMC 0.6 −40 16 1.17×10−2 464.3466 7 17.10  C28H48O5 
Carbohydrates Monosaccharide/phosphate of CM002 Erythritol phosphate* ERYP 1.94 94 14 6.81×10−3 202.0242 9.98 − C4H11O7
Organic acids Organic acid and derivatives LNM012 (Homo)2-citrate* HCIT 1.67 67 1.47×10−2 220.0583 0.25 66 C8H12O7 
Organic acids Organic acid and derivatives LPM038 Dihydroxyphenyl-pyruvate* DOHPHPYR 1.37 37 10 1.22×10−2 196.0372 0.25 81 C9H8O5 
Organic acids Organic acid and derivatives G003 2-Ketoisocaproic acid* KIC 0.19 −81 32 3.79×10−3 73  9.04 93 C6H10O3 
Organic acids Organic acid and derivatives G005 Lactic acid* LAC 0.23 −77 13 4.05×10−2 73  6.89 100 C3H6O3 
Organic acids Organic acid and derivatives G006 Malic acid* MAL 0.22 −78 18 4.05×10−2 73  12.75 97 C4H6O5 
Amino acids Amino acids and derivatives LNM013 Arginine ARG 1.5 50 13 1.37×10−2 174.1115 0.26 82 C6H14N4O2 
Amino acids Amino acids and derivatives C011 Glutamine* GLN 0.53 −47 8 1.17×10−2 146.0693 1 16.89 77 C5H10N2O3 
Amino acids Amino acids and derivatives G008 Glycine* GLY 0.41 −59 4 4.91×10−2 174  10.31 99 C2H5NO2 
Amino acids Amino acids and derivatives C013 Histidine* HIS 0.42 −58 12 1.17×10−2 155.0694 0 12.15 98 C6H9N3O2 
Amino acids Amino acids and derivatives C014 Isoleucine* ILE 0.43 −57 8 1.17×10−2 131.0947 0 16.16 99 C6H13NO2 
Amino acids Amino acids and derivatives C016 Methionine* MET 0.52 −48 13 1.17×10−2 149.0509 1 16.78 97 C5H11NO2S 
Amino acids Amino acids and derivatives C021 Threonine* THR 0.42 −58 6 1.17×10−2 119.0579 2 16.54 47 C4H9NO3 
Amino acids Amino acids and derivatives C017 Phenylalanine* PHE 0.42 −58 7 1.17×0−2 165.0790 0 17.24 99 C9H11NO2 
Amino acids Amino acids and derivatives LNM014 Phenylalanine PHE 1.38 38 1.47×10−2 165.0790 0.26 78 C9H11NO2 
Amino acids Amino acids and derivatives C023 Tyrosine* TYR 0.41 −59 6 1.17×10−2 181.0738 0 17.59 99 C9H11NO3 
Amino acids Amino acids and derivatives C022 Tryptophan* TRP 0.81 −19 10 1.17×10−2 204.0895 1 17.07 91 C11H12N2O2 
Amino acids Amino acids and derivatives LNM015 Tryptophan TRP 1.48 48 1.47×10−2 204.0895 0.27 99 C11H12N2O2 
Amino acids Amino acids and derivatives C006 Aspartic acid* ASP 0.48 −52 8 1.17×10−2 133.0374 0 17.89 86 C4H7NO4 
Amino acids Amino acids and derivatives C010 Glutamic acid* GLU 0.57 −43 8 1.17×10−2 147.0537 3 17.09 97 C5H9NO4 
Amino acids Amino acids and derivatives C007 Creatine*a CRE 0.29 −71 9 1.17×10−2 131.0695 0 14.20 97 C4H9N3O2 
Amino acids Amino acids and derivatives C012 Glutathione* GSH 0.26 −74 16 1.17×10−2 307.0833 1 19.85 96 C10H17N3O6S 
Amino acids Amino acids and derivatives C018 Pyroglutamic acid* OPRO 0.56 −44 10 1.17×10−2 129.0425 0 16.93 87 C5H7NO3 
Amino acids Amino acids and derivatives C015 Kynurenine KYN 0 Con 15  208.0846 0 15.96 76 C10H12N2O3 
Amino acids Amino acids and derivatives CM006 Kynurenine* KYN Con − 208.0855 14.66 90 C10H12N2O3 
Amino acids Amino acids and derivatives C008 Formylkynurenine FKYN 0 Con 36 1.17×10−2 236.0794 1 18.32 98 C11H12N2O4 
Amino acids Amino acids and derivatives C009 Glucosyl-galactosyl hydroxylysine* GGOHLYS 0.61 −39 5 1.17×10−2 486.2079 3 15.45 80 C18H34N2O13 
Amino acids Amino acids and derivatives C019 S-Adenosylmethionine (SAM)* SAM 0.5 −50 9 1.17×10−2 398.1384 2 11.86 92 C15H22N6O5S 
Amino acids Amino acids and derivatives C020 Spermidine* SPE 0.62 −38 11 1.17×10−2 145.1576 2 7.79 99 C7H19N3 
Amino acids Amino acids and derivatives LPM039 Thiourocanic acid SUR 0.61 −39 11 1.22×10−2 170.0150 0.24 72 C6H6N2O2
Vitamins and cofactors Coenzyme M biosynthesis C024 O-Phospho-3-sulfolactate* PSLAC 0.65 −35 11 1.17×10−2 249.9548 0 9.65  C3H7O9PS 
Vitamins and cofactors Water-soluble vitamins CM001 1-Methylnicotinamide* MB3 0.39 −61 4.71×10−3 136.0633 15.21 79 C7H8N2
Vitamins and cofactors Secosteroids LPM037 Vitamin D3 metabolite D3_1 2.04 104 13 3.81×10−2 516.4178 0.53 – C33H56O4 
Vitamins and cofactors Secosteroids LNM009 Vitamin D3 metabolite* D3_2 1.29 29 1.47×10−2 394.2851 5.76 73 C27H38O2 
Vitamins and cofactors Secosteroids LNM010 Vitamin D3 metabolite* D3_3 2.65 165 1.47×10−2 394.2849 5.62 73 C27H38O2 
Vitamins and cofactors Secosteroids LPM035 Vitamin D3 metabolite*† D3_4 0.37 −63 1.22×10−2 400.3341 5.76 52 C27H44O2 
Others Quaternary ammonium LP011 Phosphocholine* PC 0.29 −71 25 3.64×10−5 183.0665 2 0.30 99 C5H14NO4P 
Others Purine base derivative LP016 5-Hydroxyisourate OHIUR 0.52 −48 34 3.52×10−3 184.0259 14 0.28 69 C5H4N4O4 
Category Biochemical subclass Entity Compound Acronym FC Change (%) CV QC P TI (GC) or mass (LC, CE) Mass error (ppm) RT (GC, LC), MT (CE) (min) Score (%) Formula 
Fatty acyls Fatty acids and conjugates C001 Acetylcarnitine*† C2-CAR 0.39 −61 11 1.17×10−2 203.1158 0 14.26 79 C9H17NO4 
Fatty acyls Fatty acids and conjugates LP001 Butyrylcarnitine C4-CAR 0.53 −47 4 8.48×10−3 231.1474 1 0.26 98 C11H21NO4 
Fatty acyls Fatty acids and conjugates GM004 Tetradecanoic acid
(myristic acid) 
MYR Con 21 − 343 − 21.97 92 C14H28O2 
Fatty acyls Fatty acids and conjugates LP002 Heptadecanoic acid
(margaric acid)*† 
MAR 2 Obe 6  270.2559 0 2.56 100 C17H34O2 
Fatty acyls Fatty acids and conjugates LP008 Nonadecanoic acid
(nonadecylic acid)*† 
NON 2 Obe 4  298.2872 0 3.72 100 C19H38O2 
Fatty acyls Fatty acids and conjugates LPM001 Nonadecanoic acid
(nonadecylic acid)*† 
NON 0.33 −67 1.22×10−2 298.2880 5.75 77 C19H38O2 
Fatty acyls Fatty acids and conjugates LP004 Epoxy-eicosatetraenoic acid
(leukotriene A4)† 
LTA4 0.65 −35 6 5.06×10−3 318.2195 0 4.11 81 C20H30O3 
Fatty acyls Fatty acids and conjugates LP005 Hydroxy-heptadecanoic acid*† OHMAR 2 Obe 4  286.2508 0 2.75 98 C17H34O3 
Fatty acyls Fatty acids and conjugates LP006 Hydroxy-nonadecanoic acid* OHNON 2 Obe 5  314.2821 0 3.89 97 C19H38O3 
Fatty acyls Fatty acyls and conjugates LNM002 Glucosyl-octaconsanetriol* GLC-OTC 1.31 31 1.47×10−2 604.4841 12 8.11 − C34H68O8 
Fatty acyls Fatty acyls and conjugates LP017 Pentadecanenol* OHC25 2 Obe 8  226.2297 0 1.92 100 C15H30O 
Fatty acyls Fatty acyls and conjugates LPM002 Linoleoyl ethanolamide* LEAM 1.98 98 1.22×10−2 323.2824 4.76 64 C20H37NO2 
Glycerolipids Monoacylglycerols LP009 MG(14:0) MG(14:0) 0.63 −37 16 8.10×10−3 302.2459 0 4.11 98 C17H34O4 
Glycerolipids Monoacylglycerols LPM003 MG(14:0)* MG(14:0) 0.39 −61 1.22×10−2 302.2460 4.10 91 C17H34O4 
Glycerolipids Monoacylglycerols LNM001 MG(15:0)*† MG(15:0) 1.83 83 1.47×10−2 316.2612 2.44 97 C18H36O4 
Glycerolipids Monoacylglycerols GM001 MG(16:0)_2*
(2-monopalmitin) 
MG(16:0) Con 12 − 371 − 23.51 94 C19H38O4 
Glycerolipids Monoacylglycerols GM002 MG(16:0)_1
(1-monopalmitin) 
MG(16:0) Con 16 − 129 − 23.25 92 C19H38O4 
Glycerolipids Monoacylglycerols LPM004 MG(16:0)* MG(16:0) 0.54 −46 1.22×10−2 330.2773 5.03 100 C19H38O4 
Glycerolipids Monoacylglycerols GM003 MG(18:0)_2* MG(18:0) Con 20 – 129 – 24.69 88 C21H42O4 
Glycerolipids Monoacylglycerols LPM005 MG(18:0)* MG(18:0) 0.37 −63 1.22×10−2 358.3085 5.76 100 C21H42O4 
Glycerolipids Monoacylglycerols LPM006 MG(20:0)_1* MG(20:0) 0.17 −83 15 1.22×10−2 386.3395 6.34 97 C23H46O4 
Glycerolipids Monoacylglycerols LPM007 MG(20:0)_2 MG(20:0) 0.52 −48 12 1.22×10−2 386.3395 5.76 95 C23H46O4 
Glycerolipids Diacylglycerols LPM010 DG(41:5) DG(41:5) 0.45 −55 1.22×10−2 684.5693 5.03 87 C44H76O5 
Glycerolipids Diacylglycerols LPM011 DG(44:6) DG(44:6) 0.3 −70 15 1.22×10−2 724.6006 5.76 90 C47H80O5 
Glycerolipids Triacylglycerols LPM012 TG(46:2) TG(46:2) 1.43 43 12 1.22×10−2 774.6737 12 7.83 − C49H90O6 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM022 PC(20:1)/PE(23:1) PC/E(20/23:1) 0.57 −43 13 1.22×10−2 703.5143 7.12 − C38H74NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LP013 PC(32:3)/PE(35:3) PC/E(32/35:3) 0.52 −48 6 4.94×10−2 727.5143 1 7.11 66 C47H74NO8P 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM023 PC(38:0)/PE(41:0) PC/E(38/41:0) 0.57 −43 17 1.22×10−2 677.4968 7.02 − C36H72NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM024 PC(38:6)/PE(41:6)* PC/E(38/41:6) 1.5 50 3.81×10−2 805.5615 7.49 − C46H80NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM025 PC(41:6)/PE(44:6) PC/E(41/44:6) 1.4 40 12 3.81×10−2 847.6076 7.86 − C49H86NO8
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM026 PC(O-42:6)/PE(O-45:6) PC/E(O-42/45:6) 1.4 40 3.81×10−2 847.6437 8.12 − C50H90NO7
Glycerophospholipids Glycerophosphoserine LP014 PS(36:2)* PS(36:2) 0.36 −64 14 5.77×10−3 773.5572 0 6.94  C42H80NO9P 
Glycerophospholipids Glycerophosphoserine LNM006 PS(42:7) PS(42:7) 1.38 38 11 3.26×10−2 861.5520 11 7.94 − C48H80NO10
Glycerophospholipids Glycerophosphoserine LP015 PS(P-34:0)* PS(P-34:0) 0.34 −66 11 8.10×10−3 747.5401 1 6.85 93 C40H78NO9P 
Glycerophospholipids Glycerophosphoinositol LNM005 PI(42:5) PI(42:5) 1.48 48 2.30×10−2 940.6041 11 8.11 − C51H89O13
Glycerophospholipids Glycerophosphoinositol C003 PIP(34:1)* PIP(34:1) 0.79 −21 4 2.14×10−2 888.4833 7 8.72  C41H78O16P2 
Glycerophospholipids Glycerophosphatidic acid LNM003 PA(44:10) PA(44:10) 1.38 38 25 3.97×10−2 796.5022 7.29 − C47H73O8
Glycerophospholipids Glycerophosphatidic acid LPM015 PA(O-42:6)* PA(O-42:6) 0.35 −65 14 1.22×10−2 762.5584 5.75 96 C45H79O7
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM017 LysoPC(16:0)/LysoPE(19:0) LPC/E(16/19:0) 1.38 38 10 2.21×10−2 495.3321 4.25 38 C24H50NO7
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM018 LysoPC(18:0)/LysoPE(21:0) LPC/E(18/21:0) 1.55 55 13 1.22×10−2 523.3633 5.10 96 C26H54NO7
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LP012 LysoPC(O-16:0)/LysoPE(O-19:0)* LPC/E(O-16/19:0) 0.35 −65 4 3.52×10−3 481.3530 0 4.73 93 C24H52NO6P 
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM019 LysoPC(O-22:0)/LysoPE(O-25:0) LPC/E(O-22/25:0) 1.48 48 2.21×10−2 565.4468 6.62 − C30H64NO6
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM020 LysoPC(O-32:0)/LysoPE(O-35:0)* LPC/E(O-32/35:0) 0.23 −77 1.22×10−2 705.6036 5.75 − C40H84NO6
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM021 LysoPC(34:0)/LysoPE(37:0) LPC/E(34/37:0) 1.51 51 1.22×10−2 761.5928 7.89 − C42H84NO8
Glycerophospholipids Glycerophosphatidic acid LPM013 LysoPA(O-16:0)* LPA(O-16:0) 0.38 −62 1.22×10−2 396.2645 5.76 85 C19H41O6
Glycerophospholipids Glycerophosphocholines/glycerophosphoethanolamines LPM016 Glycerophosphocholine* GPC 5.73 473 13 1.22×10−2 257.1028 0.24 99 C8H20NO6
Sphingolipids Sphingoid bases LP003 Heptadecasphinganine*† C17-SPN 2 Obe 8  287.2824 0 2.99 100 C17H37NO2 
Sphingolipids Ceramide LP010 Dihydroceramide C2* CER-DHC2 2 Obe 11  343.3080 1 4.62 85 C20H41NO3 
Sphingolipids Phosphosphingolipids LPM027 Cer(34:0) CER 0.56 −44 15 2.21×10−2 571.5157 7.94 − C34H69NO5 
Sphingolipids Phosphosphingolipids LPM029 SM(d34:0) SM(d34:0) 1.4 40 1.22×10−2 704.5820 7.42 94 C39H81N2O6
Sphingolipids Phosphosphingolipids LPM030 SM(d34:1)* SM(d34:1) 1.38 38 1.22×10−2 702.5677 7.29 100 C39H79N2O6
Sphingolipids Phosphosphingolipids LPM031 SM(d39:1) SM(d39:1) 1.42 42 1.22×10−2 772.6444 7.97 − C44H89N2O6
Sphingolipids Phosphosphingolipids LPM032 SM(d40:1)* SM(d40:1) 1.38 38 1.22×10−2 786.6610 8.07 96 C45H91N2O6
Sphingolipids Phosphosphingolipids LPM033 SM(d41:1) SM(d41:1) 1.36 36 1.22×10−2 800.6760 8.18 − C46H93N2O6
Sphingolipids Phosphosphingolipids LPM034 SM(d42:2)* SM(d42:2) 1.57 57 1.22×10−2 812.6766 8.10 98 C47H93N2O6
Sphingolipids Neutral glycosphingolipids LPM028 GlcCer(d38:1)/GalCer(d38:1) GCER 1.59 59 16 3.81×10−2 755.6268 8.20 − C44H85NO8 
Sterol lipids Bile acids, alcohols and derivatives LPM036 Hydroxy-oxocholadienoic acid OH-OCD 0.57 −43 1.22×10−2 368.2332 5.03 64 C24H32O3 
Sterol lipids Bile acids, alcohols and derivatives LNM007 Oxocholadienoic acid* OCD 1.78 78 1.47×10−2 370.2508 2.44 − C24H32O3 
Sterol lipids Bile acids, alcohols and derivatives C004 Trihydroxy-methyl-cholestanoic acid* TOHMC 0.6 −40 16 1.17×10−2 464.3466 7 17.10  C28H48O5 
Carbohydrates Monosaccharide/phosphate of CM002 Erythritol phosphate* ERYP 1.94 94 14 6.81×10−3 202.0242 9.98 − C4H11O7
Organic acids Organic acid and derivatives LNM012 (Homo)2-citrate* HCIT 1.67 67 1.47×10−2 220.0583 0.25 66 C8H12O7 
Organic acids Organic acid and derivatives LPM038 Dihydroxyphenyl-pyruvate* DOHPHPYR 1.37 37 10 1.22×10−2 196.0372 0.25 81 C9H8O5 
Organic acids Organic acid and derivatives G003 2-Ketoisocaproic acid* KIC 0.19 −81 32 3.79×10−3 73  9.04 93 C6H10O3 
Organic acids Organic acid and derivatives G005 Lactic acid* LAC 0.23 −77 13 4.05×10−2 73  6.89 100 C3H6O3 
Organic acids Organic acid and derivatives G006 Malic acid* MAL 0.22 −78 18 4.05×10−2 73  12.75 97 C4H6O5 
Amino acids Amino acids and derivatives LNM013 Arginine ARG 1.5 50 13 1.37×10−2 174.1115 0.26 82 C6H14N4O2 
Amino acids Amino acids and derivatives C011 Glutamine* GLN 0.53 −47 8 1.17×10−2 146.0693 1 16.89 77 C5H10N2O3 
Amino acids Amino acids and derivatives G008 Glycine* GLY 0.41 −59 4 4.91×10−2 174  10.31 99 C2H5NO2 
Amino acids Amino acids and derivatives C013 Histidine* HIS 0.42 −58 12 1.17×10−2 155.0694 0 12.15 98 C6H9N3O2 
Amino acids Amino acids and derivatives C014 Isoleucine* ILE 0.43 −57 8 1.17×10−2 131.0947 0 16.16 99 C6H13NO2 
Amino acids Amino acids and derivatives C016 Methionine* MET 0.52 −48 13 1.17×10−2 149.0509 1 16.78 97 C5H11NO2S 
Amino acids Amino acids and derivatives C021 Threonine* THR 0.42 −58 6 1.17×10−2 119.0579 2 16.54 47 C4H9NO3 
Amino acids Amino acids and derivatives C017 Phenylalanine* PHE 0.42 −58 7 1.17×0−2 165.0790 0 17.24 99 C9H11NO2 
Amino acids Amino acids and derivatives LNM014 Phenylalanine PHE 1.38 38 1.47×10−2 165.0790 0.26 78 C9H11NO2 
Amino acids Amino acids and derivatives C023 Tyrosine* TYR 0.41 −59 6 1.17×10−2 181.0738 0 17.59 99 C9H11NO3 
Amino acids Amino acids and derivatives C022 Tryptophan* TRP 0.81 −19 10 1.17×10−2 204.0895 1 17.07 91 C11H12N2O2 
Amino acids Amino acids and derivatives LNM015 Tryptophan TRP 1.48 48 1.47×10−2 204.0895 0.27 99 C11H12N2O2 
Amino acids Amino acids and derivatives C006 Aspartic acid* ASP 0.48 −52 8 1.17×10−2 133.0374 0 17.89 86 C4H7NO4 
Amino acids Amino acids and derivatives C010 Glutamic acid* GLU 0.57 −43 8 1.17×10−2 147.0537 3 17.09 97 C5H9NO4 
Amino acids Amino acids and derivatives C007 Creatine*a CRE 0.29 −71 9 1.17×10−2 131.0695 0 14.20 97 C4H9N3O2 
Amino acids Amino acids and derivatives C012 Glutathione* GSH 0.26 −74 16 1.17×10−2 307.0833 1 19.85 96 C10H17N3O6S 
Amino acids Amino acids and derivatives C018 Pyroglutamic acid* OPRO 0.56 −44 10 1.17×10−2 129.0425 0 16.93 87 C5H7NO3 
Amino acids Amino acids and derivatives C015 Kynurenine KYN 0 Con 15  208.0846 0 15.96 76 C10H12N2O3 
Amino acids Amino acids and derivatives CM006 Kynurenine* KYN Con − 208.0855 14.66 90 C10H12N2O3 
Amino acids Amino acids and derivatives C008 Formylkynurenine FKYN 0 Con 36 1.17×10−2 236.0794 1 18.32 98 C11H12N2O4 
Amino acids Amino acids and derivatives C009 Glucosyl-galactosyl hydroxylysine* GGOHLYS 0.61 −39 5 1.17×10−2 486.2079 3 15.45 80 C18H34N2O13 
Amino acids Amino acids and derivatives C019 S-Adenosylmethionine (SAM)* SAM 0.5 −50 9 1.17×10−2 398.1384 2 11.86 92 C15H22N6O5S 
Amino acids Amino acids and derivatives C020 Spermidine* SPE 0.62 −38 11 1.17×10−2 145.1576 2 7.79 99 C7H19N3 
Amino acids Amino acids and derivatives LPM039 Thiourocanic acid SUR 0.61 −39 11 1.22×10−2 170.0150 0.24 72 C6H6N2O2
Vitamins and cofactors Coenzyme M biosynthesis C024 O-Phospho-3-sulfolactate* PSLAC 0.65 −35 11 1.17×10−2 249.9548 0 9.65  C3H7O9PS 
Vitamins and cofactors Water-soluble vitamins CM001 1-Methylnicotinamide* MB3 0.39 −61 4.71×10−3 136.0633 15.21 79 C7H8N2
Vitamins and cofactors Secosteroids LPM037 Vitamin D3 metabolite D3_1 2.04 104 13 3.81×10−2 516.4178 0.53 – C33H56O4 
Vitamins and cofactors Secosteroids LNM009 Vitamin D3 metabolite* D3_2 1.29 29 1.47×10−2 394.2851 5.76 73 C27H38O2 
Vitamins and cofactors Secosteroids LNM010 Vitamin D3 metabolite* D3_3 2.65 165 1.47×10−2 394.2849 5.62 73 C27H38O2 
Vitamins and cofactors Secosteroids LPM035 Vitamin D3 metabolite*† D3_4 0.37 −63 1.22×10−2 400.3341 5.76 52 C27H44O2 
Others Quaternary ammonium LP011 Phosphocholine* PC 0.29 −71 25 3.64×10−5 183.0665 2 0.30 99 C5H14NO4P 
Others Purine base derivative LP016 5-Hydroxyisourate OHIUR 0.52 −48 34 3.52×10−3 184.0259 14 0.28 69 C5H4N4O4 

†Multiple putative identification. See Supplementary Table S3 for alternative identifications of the same entity. *Significant in both MVA and UVA. aMetabolite found to be significant in more than one analytical technique. Change (%) refers to obesity-derived ASCs or M compared with non-obesity-derived ASCs or M. Scored as isotopic pattern distribution score (LC/MS and CE/MS data) or quality match against the in-house library (GC/MS data). Obe: metabolite present only in the obese group; Con: metabolite present only in the control group. Acronym, arbitrary metabolite acronym; TI, target ion (m/z).

DISCUSSION

In the present study, a comprehensive, untargeted, metabolic approach was used to investigate the underlying biology of ASCs through the study of two different and complementary compartments: the endo-metabolome, which allowed us to discover changes in the metabolites associated with the cell, and the exo-metabolome, which gave us the opportunity to find differences in the metabolites excreted or absorbed by the cells.

Human

Among the list of significantly different metabolites in Table 1, it is noteworthy that most of the proteinogenic amino acids were higher in both culture media and obesity-derived ASCs (see Figure 1). It has to be taken into account that the culture medium contained these amino acids, so the difference between non-obese and obese metabolomes may be related more to their lack of uptake (and usage) in the case of obesity-derived ASCs. Few differences between hcASCs and hoASCs were found in lipid metabolism (Figures 4A and 5), in either lipolysis-related metabolites (fatty acids, lysophospholipids) or fatty acid oxidation-related (acylcarnitine) metabolites.

On the other hand, metabolites from glycolysis and the tricarboxylic acid (TCA) cycle did not show adequate turnover: indeed, they were accumulated (citrate and malate levels were higher in the cell) or excreted further (pyruvate, citrate, malate and succinate levels were higher in the medium), as shown in Table 1 and Figure 4(A). Glycolysis and the TCA cycle together constitute the main oxidative process for obtaining energy from carbohydrates. But in hoASCs the mitochondrial function was not working as efficiently as in hcASCs, even though the numbers of mitochondria were no different in the two [27]. The higher levels of pyruvate observed could be associated, not with a higher rate of glycolysis, but with a lower activity of the mitochondrial pyruvate dehydrogenase, probably due to higher levels of NADH from the oxidation of sorbitol to fructose (see below).

Moreover, incorporation of acetyl-coenzyme A into the cycle was probably lower in hoASCs, because higher levels of acetylcarnitine were found within the cells; ketogenesis was therefore increased, and the overproduced acetoacetate and 3-hydroxybutyrate were secreted into the medium. In addition, it must be mentioned that succinic acid was found to be the compound with the highest percentage of change (300% increment) in the medium from obese humans (hoM) compared with the control (hcM, see Table 1). It has been mentioned previously that reduced activity of the mitochondrial enzymes of the TCA cycle may result in an accumulation of the compounds of this pathway, but it has also been published that, in macrophages activated by lipopolysaccharide, succinate (from glutamine anaplerosis) is an inflammatory signal that induces interleukin (IL)-1β [38].

The release of such acid metabolites (from glycolysis and the TCA cycle, as well as ketone bodies) could explain the already observed reduction in the medium's pH [27]. Release of excess acid may be a protective strategy, because it has been known for a long time that maintenance of intracellular pH is very important for several processes such as the activity of either the glycolysis rate-limiting enzyme (phosphofructokinase), which is inhibited by pH decrease, or the cell cycle, because low intracellular pH is associated with the cellular resting state (see, for example, Madshus [39]).

Obesity is strongly related to insulin resistance [40,41]. In fact, the obese state is characterized by what has been called low-grade systemic inflammation [42]. It is consistent with previous findings on the same cell model that have shown lower adipokine production and higher tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) from obesity-derived ASCs (either human or murine) [21].

Another process related to both inflammation and obesity is oxidative stress, which can be induced by the obese state [43] and associated with some of the characteristic complications of obesity, such as microvascular and cardiovascular complications [44]. Unused glucose can undergo the so-called ‘polyol pathway’, which is a source for reactive oxygen species (ROS), and therefore responsible for higher oxidative stress [45]. In this process, aldohexoses can be reduced to sorbitol with a concomitant decrease in the availability of NADPH, necessary for glutathione synthesis. Sorbitol can then be converted into fructose with NADH being produced in the reaction. Moreover, it must be taken into account that unbalanced NADH/NAD will reduce the glycolysis rate for the oxidation of glyceraldehyde 3-phosphate (GA3P) to 1,3-bisphosphoglycerate.

In addition to the aforementioned mechanism associated with the generation of ROS, it is remarkable that increased levels of aminomalonate were found in the human exo-metabolome. Knowledge about such a metabolite is scarce. Its presence in atherosclerotic plaques had already been described in 1984 [46], and subsequent biodistribution studies of antibodies against aminomalonate injected into normal and hyperlipidaemic rabbits showed that atheromatous aortas had a significantly higher staining for aminomalonate than normal aortas [47]. The origin of aminomalonate has been attributed to oxidation of glycine mediated by radicals [48], and recently it was found to be decreased in the plasma of patients with acute coronary syndrome [49], as well as those with an abdominal aortic aneurysm [50].

One mechanism that could be strongly involved in all processes that have been mentioned is the activation/inhibition of the serine/threonine kinase mammalian target of rapamycin (mTOR) [5155]. It would be a further connection with the glycolytic flux mediated by GA3P, because it has been suggested that this glycolytic intermediate acts as a messenger to regulate mTORC1 signalling through the destabilization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Ras family kinase Rheb [56]. The linkage between stemness and mTOR has been demonstrated in several stem cell models [57,58]; furthermore, the delivery of cytosol from control ASCs into obesity-derived ASCs has been described as restoring insulin sensitivity and reversing the proinflammatory cytokine profile, due in part to the restoration of Lin28 protein levels; this coincides with previous studies about the metabolic rescue of these cells [21].

Mice

In the case of the murine ASCs’ exo-metabolome (Table 2 and see Figure 4B), the results are not comparable with those seen in humans, neither in the significantly different number of metabolites that resulted, nor in the identification (few common metabolites) or direction of changes (increase/decrease) when the same metabolite was found in both models.

In addition, no differences were found for the metabolites involved in glycolysis, the polyol pathway or the pentose phosphate pathway, pointing out that carbohydrate metabolism was similar in both conditions. Furthermore, ketone bodies and TCA cycle metabolites also did not differ between the two conditions under study. The similarity between the carbohydrate metabolic rate in obesity- and non-obesity-derived cells must be interpreted, taking into account the previous demonstration that their mitochondrial numbers were higher in moASCs and their oxygen consumption was higher when the energy source was free fatty acids. Under these conditions, the production of ROS was higher [27] through a contribution to the insulin resistance observed in this cell model, similar to what was observed in adipocytes [59].

In the moASC exo-metabolome, three amino acids were found to be higher when compared to the mcASC exo-metabolome, whereas 10 were lower in the endometabolome of moASC compared to the mcASC endometabolome. This suggests that the amino acid uptake was similar in both conditions, but their utilization differed. Together with the reduction in amino acids, lower levels of amino acid metabolites (kynurenine, formylkynurenine, 5-oxoproline, ketoisocaproic acid) were observed. The most probable usage of amino acids by the moASCs is not for protein synthesis but for their catabolism, because they serve as an alternative energy source to carbohydrates as usually described for the insulin-resistant condition [21]. Alterations in plasma amino acids levels have been associated with insulin secretion in obesity since 1969 [60] and, more recently, using metabolomic approaches, obesity-related alterations in the amino acid profile were shown in plasma and white adipose tissue [61,62].

In the murine model, we found large differences in lipid metabolism-associated compounds that were not related to the lipolysis from neutral glycerides, because none of the five most common fatty acids (palmitic, stearic, oleic, linoleic and arachidonic acids) differed. This is also supported by the fact that, of the 11 neutral glycerides found to be different, all but 2 had lower levels in the medium from obesity-derived ASCs, which was probably related to the activity of phospholipases. Lysophospholipids (LysoPLs) would be the result of the activity of phospholipases A1 and A2 (phospholipase B) and, if both fatty acids are cleaved, the result is glycerophosphocholine, the metabolite that showed the biggest variation in the present study (+473% in moM, see Table 2). LysoPLs are not merely by-products of phospholipase A activity, but are strongly involved not only in stem cell regulation [63,64] but also in their chemotaxis [65].

Phosphocholine is the result of the activity of phospholipase C, which was decreased in mouse obesity-derived ASCs (see Table 2). Together with the increase in lysophospholipids already mentioned, and the decrease in monoacylglycerols in the medium (see Table 2 and Figure 4B), it is possible that there was in fact a decrease in the activity of lysophospholipase C, which has been proposed as a supplier of phosphocholine in the brain [66]. This is also supported by the accumulation of sphingomyelins and the reduction of ceramides in the exo-metabolome, because lysophospholipase C shows the activity of neutral sphingomyelinase [67]. In obesity-derived ASCs there can be a combination of higher sphingomyelin release and lower activity of sphingomyelinase, although this can be specific to the stem cells, because expression of enzymes involved in ceramide generation was elevated in obese adipose tissues from ob/ob mice, as well as sphingomyelin, ceramide, sphingosine and sphingosine 1-phosphate [68].

It is noticeable that, in the exo-metabolome from both types of ASCs (from humans and mice), significant differences in bile acids were found (taurodeoxycholic acid in humans, and oxocholadienoic acid and hydroxyoxocholadienoic acid in mice). Liver is the only organ that synthesizes primary bile acids, and its presence in the medium can be due to two sources: either the cells absorbed bile acids in vivo and released them ex vivo, or there was a significant supply from the culture medium. However, the intriguing facts are that we can identify differences between obesity- and non-obesity-derived ASCs in bile acid-related metabolites, and furthermore that in humans and rodents different derivatives were involved, and finally that in the case of mice oxocholadienoic acid was increased whereas hydroxyoxocholadienoic acid was decreased. This opens an interesting line of research to elucidate the possible role of bile acids in stem cell regulation.

CONCLUSIONS

In the present study we observed, in obesity-derived human ASCs, an increased release of the metabolites associated with glycolysis, the TCA cycle, the pentose phosphate pathway and the polyol pathway, together with a lack of amino acid and lipid catabolism. Those changes, concerning both the type of energy-related metabolites and their abundance within the cells and the culture medium, occur together with the worsening of the ability of obesity-derived ASCs to proliferate, migrate and differentiate. However, further studies are needed to investigate the actual role of those metabolites and their possible involvement in the regulation of key enzymes of the metabolic routes responsible for energy homoeostasis in the realm of the stemness process.

Furthermore, in the present study we corroborated the problematic feasibility of the diet-induced obesity of the ob/ob mouse as a useful model for the study of stemness capacity in human ASCs. Indeed, it has been unveiled that the metabolic conditions differed between the two models (human and murine). Lower levels of amino acids were found in obesity-derived murine ASCs and, together with unbalanced levels of phospho- versus lysophospho-lipids and sphingomyelins versus ceramide, were a hallmark of alterations in amino acid and lipid catabolism, whereas no differences were highlighted for the carbohydrate metabolism-related compounds.

Thus, the footprint/fingerprint, multiplatform, metabolomics approach has provided a huge amount of information that can be readily used for deeper understanding of the biochemical processes underlying ASCs’ stemness capacity, and their relationship with obesity and/or the selected cell model (murine/human).

Finally, the present study opens new avenues for future investigations aimed at delving into the role of enzymes such as mTOR, GAPDH, phospholipase A and sphingomyelinase (SMase), among others, in the alterations described, and testing whether their external modulation may enhance the obesity-derived ASCs’ stemness capacity and/or make the murine model comparable to the human model.

AUTHOR CONTRIBUTION

All authors participated in writing and revising the manuscript. L.M. Pérez worked on the cell isolation and culture, under the supervision of B.G. Gálvez. A. Mastrangelo was in charge of the sample treatment, instrumental analysis and data processing, under the direct supervision of A. García and C. Barbas. M.I. Panadero, A. Mastrangelo and F.J. Rupérez worked together for the interpretation and presentation of the results. F.J. Rupérez was in charge of coordinating all the information.

The authors wish to thank Maria Bove for her help in sample preparation and data processing.

FUNDING

A. Mastrangelo receives a PhD grant from the Spanish Ministry of Economy and Competitiveness [AP-2012-1385]. The authors wish to express their gratitude for the financial support received by B.G. Gálvez from the Spanish Ministry of Economy and Competitiveness MINECO [CTQ2014-55279-R] and from MINECO/FEDER [SAF2015-67911].

Abbreviations

     
  • ASC

    adipose-derived stem cell

  •  
  • BMI

    body mass index

  •  
  • CE

    capillary electrophoresis

  •  
  • CV

    coefficient of variation

  •  
  • FC

    fold change

  •  
  • GA3P

    glyceraldehyde 3-phosphate

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • LysoPL

    lysophospholipid

  •  
  • MSC

    mesenchymal stem cell

  •  
  • MT

    migration time

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • MVA

    multivariate analysis

  •  
  • OPLS-DA

    orthogonal partial least squares discriminant analysis

  •  
  • PCA

    principal components analysis

  •  
  • QC

    quality control

  •  
  • QTOF

    quadrupole time-of-flight

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    retention time

  •  
  • TOF

    time-of-flight

  •  
  • UVA

    univariate analysis

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Supplementary data