Stability of plasma metabolomics over 10 years among women

Background: In epidemiological studies, samples are often collected long before disease onset or outcome assessment. Understanding the long-term stability of biomarkers measured in these samples is crucial. We estimated within-person stability over 10 years of metabolites and metabolite features (N=5938) in the Nurses' Health Study (NHS): The primary dataset included 1880 women with 1184 repeated samples donated 10 years apart while the secondary dataset included 1456 women with 488 repeated samples donated 10 years apart. Methods: We quantified plasma metabolomics using two liquid chromatography mass spectrometry platforms (lipids and polar metabolites) at the Broad Institute (Cambridge, MA). Intra-class correlations were used to estimate long-term stability (10 years) of metabolites and were calculated as the proportion of the total variability (within-person + between-person) attributable to between-person variability. Within-person variability was estimated among participants who donated two blood samples approximately 10 years apart while between-person variability was estimated among all participants. Results: In the primary dataset, the median ICC was 0.43 (1st quartile [Q1]: 0.36; 3rd quartile [Q3]: 0.50) among known metabolites and 0.41 (Q1: 0.34; Q3: 0.48) among unknown metabolite features. The most stable (median ICCs: 0.54-0.57) metabolite classes were nucleosides, nucleotides and analogues, phosphatidylcholine plasmalogens, diglycerides, and cholesteryl esters. The least stable (median ICCs: 0.26-0.36) metabolite classes were lysophosphatidylethanolamines, lysophosphatidylcholines and steroid and steroid derivatives. Results in the secondary dataset were similar (Spearman correlation=0.87) to corresponding results in the primary dataset. Conclusion: Within-person stability over 10 years is reasonable for lipid, lipid-related, and polar metabolites, and varies by metabolite class. Additional studies are required to estimate within-person stability over 10 years of other metabolites groups.

Metabolomics Coupled with Pathway Analysis Provides Insights into Sarco-Osteoporosis Metabolic Alterations and Estrogen Therapeutic Effects in Mice

Postmenopausal osteoporosis (PMOP) and sarcopenia are common diseases that predominantly affect postmenopausal women. In the occurrence and development of these two diseases, they are potentially pathologically connected with each other at various molecular levels. However, the application of metabolomics in sarco-osteoporosis and the metabolic rewiring happening throughout the estrogen loss-replenish process have not been reported. To investigate the metabolic alteration of sarco-osteoporosis and the possible therapeutical effects of estradiol, 24 mice were randomly divided into sham surgery, ovariectomy (OVX), and estradiol-treated groups. Three-dimensional reconstructions and histopathology examination showed significant bone loss after ovariectomy. Estrogen can well protect against OVX-induced bone loss deterioration. UHPLC-Q-TOF/MS was preformed to profile semi- polar metabolites of skeletal muscle samples from all groups. Metabolomics analysis revealed metabolic rewiring occurred in OVX group, most of which can be reversed by estrogen supplementation. In total, 65 differential metabolites were identified, and pathway analysis revealed that sarco-osteoporosis was related to the alterations in purine metabolism, glycerophospholipid metabolism, arginine biosynthesis, tryptophan metabolism, histidine metabolism, oxidative phosphorylation, and thermogenesis, which provided possible explanations for the metabolic mechanism of sarco-osteoporosis. This study indicates that an UHPLC-Q-TOF/MS-based metabolomics approach can elucidate the metabolic reprogramming mechanisms of sarco-osteoporosis and provide biological evidence of the therapeutical effects of estrogen on sarco-osteoporosis.

Changes in Polar Metabolites Content during Natural and Methyl-Jasmonate-Promoted Senescence of Ginkgo biloba Leaves

The present study clarified changes in the contents of polar metabolites (amino acids, organic acids, saccharides, cyclitols, and phosphoric acid) in leaf senescence in Ginkgo biloba with or without the application of methyl jasmonate (JA-Me) in comparison with those in naturally senescent leaf blades and petioles. The contents of most amino acids and citric and malic acids were significantly higher in abaxially, and that of myo-inositol was lower in abaxially JA-Me-treated leaves than in adaxially JA-Me-treated and naturally senescent leaves. The levels of succinic and fumaric acids in leaves treated adaxially substantially high, but not in naturally senescent leaves. In contrast, sucrose, glucose, and fructose contents were much lower in leaf blades and petioles treated abaxially with JA-Me than those treated adaxially. The levels of these saccharides were also lower compared with those in naturally senescent leaves. Shikimic acid and quinic acid were present at high levels in leaf blades and petioles of G. biloba. In leaves naturally senescent, their levels were higher compared to green leaves. The shikimic acid content was also higher in the organs of naturally yellow leaves than in those treated with JA-Me. These results strongly suggest that JA-Me applied abaxially significantly enhanced processes of primary metabolism during senescence of G. biloba compared with those applied adaxially. The changes in polar metabolites in relation to natural senescence were also discussed.

Pharmacometabolomic profiles in type 2 diabetic subjects treated with liraglutide or glimepiride

Background Treatment with glucagon-like peptide-1 receptor agonists (GLP-1 RAs) leads to multiple metabolic changes, reduction in glucose levels and body weight are well established. In people with type 2 diabetes, GLP-1 RAs reduce the risk of cardiovascular (CV) disease and may also potentially represent a treatment for fatty liver disease. The mechanisms behind these effects are still not fully elucidated. The aim of the study was to investigate whether treatment with liraglutide is associated with favourable metabolic changes in cases of both CV disease and fatty liver disease. Methods In a prespecified post-hoc analysis of a double-blind, placebo-controlled trial in 62 individuals with type 2 diabetes (GLP-1 RA liraglutide or glimepiride, both in combination with metformin), we evaluated the changes in plasma molecular lipids and polar metabolites after 18 weeks of treatment. The lipids and polar metabolites were measured by using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). Results In total, 340 lipids and other metabolites were identified, covering 14 lipid classes, bile acids, free fatty acids, amino acids and other polar metabolites. We observed more significant changes in the metabolome following liraglutide treatment compared to with glimepiride, particularly as regards decreased levels of cholesterol esters hexocyl-ceramides, lysophosphatidylcholines, sphingolipids and phosphatidylcholines with alkyl ether structure. In the liraglutide-treated group, lipids were reduced by approximately 15% from baseline, compared to a 10% decrease in the glimepiride group. At the pathway level, the liraglutide treatment was associated with lipid, bile acid as well as glucose metabolism, while glimepiride treatment was associated with tryptophan metabolism, carbohydrate metabolism, and glycerophospholipid metabolism. Conclusions Compared with glimepiride, liraglutide treatment led to greater changes in the circulating metabolome, particularly regarding lipid metabolism involving sphingolipids, including ceramides. Our findings are hypothesis-generating and shed light on the underlying biological mechanisms of the CV benefits observed with GLP-1 RAs in outcome studies. Further studies investigating the role of GLP-1 RAs on ceramides and CV disease including fatty liver disease are warranted. Trial registration: NCT01425580

Cysteinolic Acid Is a Widely Distributed Compatible Solute of Marine Microalgae

Phytoplankton rely on bioactive zwitterionic and highly polar small metabolites with osmoregulatory properties to compensate changes in the salinity of the surrounding seawater. Dimethylsulfoniopropionate (DMSP) is a main representative of this class of metabolites. Salinity-dependent DMSP biosynthesis and turnover contribute significantly to the global sulfur cycle. Using advanced chromatographic and mass spectrometric techniques that enable the detection of highly polar metabolites, we identified cysteinolic acid as an additional widely distributed polar metabolite in phytoplankton. Cysteinolic acid belongs to the class of marine sulfonates, metabolites that are commonly produced by algae and consumed by bacteria. It was detected in all dinoflagellates, haptophytes, diatoms and prymnesiophytes that were surveyed. We quantified the metabolite in different phytoplankton taxa and revealed that the cellular content can reach even higher concentrations than the ubiquitous DMSP. The cysteinolic acid concentration in the cells of the diatom Thalassiosira weissflogii increases significantly when grown in a medium with elevated salinity. In contrast to the compatible solute ectoine, cysteinolic acid is also found in high concentrations in axenic algae, indicating biosynthesis by the algae and not the associated bacteria. Therefore, we add this metabolite to the family of highly polar metabolites with osmoregulatory characteristics produced by phytoplankton.

Metabolism of Selected 2-Arylbenzofurans in a Colon In Vitro Model System

2-arylbenzofurans represent a small group of bioactive compounds found in the plant family Moraceae. As it has not been investigated whether these substances are stable during passage through the gastrointestinal tract, their biological effects may be altered by the metabolism of intestinal microbiota or cells. The aim of the present study was to investigate and compare mulberrofuran Y (1), moracin C (2), and mulberrofuran G (3) in an in vitro model of human intestinal bacterial fermentation and in an epithelial model using the Caco-2 cell line. The analysis of compounds by LC-MS-Q-TOF showed sufficient stability in the fermentation model, with no bacterial metabolites detected. However, great differences in the quantity of permeation were observed in the permeability assay. Moreover, mulberrofuran Y (1) and moracin C (2) were observed to be transformed into polar metabolites by conjugation. Among the test compounds, mulberrofuran Y (1) was mostly stable and accumulated in endothelial cells (85.3%) compared with mulberrofuran G (3) and moracin C (2) (14% and 8.2%, respectively). Thus, only a small amount of mulberrofuran Y (1) was conjugated. Moracin C (2) and mulberrofuran G (3) were metabolized almost completely, with only traces of the unchanged molecule being found on the apical and cellular sides of the system. Only conjugates of mulberrofuran Y (1) and moracin C (2) were able to reach the basolateral side. Our results provide the basic description of bioavailability of these three compounds, which is a necessary characteristic for final evaluation of bio-efficacy.

Ion-pairing chromatography and amine derivatization provide complementary approaches for the targeted UPLC-MS analysis of the polar metabolome

Liquid chromatography coupled to mass spectrometry is a key metabolomics technology. Reversed-phase liquid chromatog-raphy (RPLC) is very widely used as a separation step, possessing excellent characteristics with respect to reproducibility and reliability, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alter-native methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. We detected 132 metabolites for real biological samples (liver extracts) with good reproducibility (based on coefficients of variation in pooled biological quality control samples). Finally, we tested the combination of methods with real-world samples by analyzing a set of liver extracts from aged male and female mice that had been treated with the poly-phenol compound ampelopsin. Furthermore, we also compared the results of these LC-MS methods to 1H NMR spectrosco-py as an orthogonal method (also termed statistical heterospectroscopy (SHY)), and found a strong correlation between the results of these different analytical approaches. By these means, not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the di-rection of metabolite change was opposite for males and females.