bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2019‒10‒27
thirty-nine papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Pharm Biomed Anal. 2019 Aug 15. pii: S0731-7085(19)31107-0. [Epub ahead of print]178 112815
      The carboxyl group is the functional group in both bile acids (BAs) and fatty acids (FAs) (BAFAs). Considering the functional correlation and the structural similarity of these compounds, a sensitive and efficient method was developed here for the first time to simultaneously profile BAFAs based on ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Through optimization of the chromatographic conditions, all BAFAs can be efficiently separated and quantified in 19 min with excellent peak shape. For comprehensive profiling of endogenous FAs without standards, a strategy was established to predict the retention times (RTs) of all theoretically possible FAs on the basis of the good multiple linear regression relationship between RT and FA carbon chain length and double bond number. High-resolution mass spectrometry was employed for the final confirmation of these predicted FAs. Twenty-eight FAs in rat serum were newly identified using this strategy. Though the regulation of collision energies (CEs) for highly abundant compounds, the problems of their poor quantification linearity and accuracy caused by MS signal saturation were solved, facilitating the simultaneous quantification of both high- and low-abundance BAFAs with good linearity and accuracy. The established UPLC-MS/MS method was further used to quantify BAFAs in rat serum and to explore the disturbance of BAFA metabolism in the Tripterygium glycoside-induced liver injury rat model. A total of 25 BAs and 55 FAs in rat serum were identified and quantified. Several BAFAs, including nordeoxycholic acid, taurodeoxycholic acid and some unsaturated FAs, were found to differ significantly in the control and model groups. These BAFAs are very promising biomarkers for the evaluation of Tripterygium glycoside-induced liver injury.
    Keywords:  Bile acids; Drug-induced liver injury; Fatty acids; UPLC-MS/MS
  2. Metabolites. 2019 Oct 24. pii: E247. [Epub ahead of print]9(11):
      Metabolomics, understood as the science that manages the study of compounds from the metabolism, is an essential tool for deciphering metabolic changes in disease. The experiments rely on the use of high-throughput analytical techniques such as liquid chromatography coupled to mass spectrometry (LC-ToF MS). This hyphenation has brought positive aspects such as higher sensitivity, specificity and the extension of the metabolome coverage in a single run. The analysis of a high number of samples in a single batch is currently not always feasible due to technical and practical issues (i.e., a drop of the MS signal) which result in the MS stopping during the experiment obtaining more than a single sample batch. In this situation, careful data treatment is required to enable an accurate joint analysis of multi-batch data sets. This paper summarizes the analytical strategies in large-scale metabolomic experiments; special attention has been given to QC preparation troubleshooting and data treatment. Moreover, labeled internal standards analysis and their aim in data treatment, and data normalization procedures (intra- and inter-batch) are described. These concepts are exemplified using a cohort of 165 patients from a study in asthma.
    Keywords:  LC-QToF-MS; asthma; large-scale; metabolomics; normalization
  3. Bioinformatics. 2019 Oct 23. pii: btz798. [Epub ahead of print]
      MOTIVATION: When metabolites are analyzed by electrospray ionization-mass spectrometry, they are usually detected as multiple ion species due to the presence of isotopes, adducts, and in-source fragments. The signals generated by these degenerate features (along with contaminants and other chemical noise) obscure meaningful patterns in MS data, complicating both compound identification and downstream statistical analysis. To address this problem, we developed Binner, a new tool for the discovery and elimination of many degenerate feature signals typically present in untargeted ESI-LC-MS metabolomics data.RESULTS: Binner generates feature annotations and provides tools to help users visualize informative feature relationships that can further elucidate the underlying structure of the data. To demonstrate the utility of Binner and to evaluate its performance, we analyzed data from RPLC-MS and HILIC platforms and demonstrated the accuracy of selected annotations using MS/MS. When we compared Binner annotations of 75 compounds previously identified in human plasma samples with annotations generated by three similar tools, we found that Binner achieves superior performance in the number and accuracy of annotations while simultaneously minimizing the number of incorrectly annotated principal ions. Data reduction and pattern exploration with Binner have allowed us to catalogue a number of previously unrecognized complex adducts and neutral losses generated during the ionization of molecules in LC-MS. In summary, Binner allows users to explore patterns in their data and to efficiently and accurately eliminate a significant number of the degenerate features typically found in various LC-MS modalities.
    AVAILABILITY AND IMPLEMENTATION: Binner is written in Java and is freely available from
    SUPPLEMENTARY INFORMATION: Available at Bioinformatics online.
  4. Metabolites. 2019 Oct 21. pii: E241. [Epub ahead of print]9(10):
      Illuminating the comprehensive lipid profiles after dietary supplementation of polyunsaturated fatty acids (PUFAs) is crucial to revealing the tissue distribution of PUFAs in living organisms, as well as to providing novel insights into lipid metabolism. Here, we performed lipidomic analyses on mouse plasma and nine tissues, including the liver, kidney, brain, white adipose, heart, lung, small intestine, skeletal muscle, and spleen, with the dietary intake conditions of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) as the ethyl ester form. We incorporated targeted and untargeted approaches for profiling oxylipins and complex lipids such as glycerol (phospho) lipids, sphingolipids, and sterols, respectively, which led to the characterization of 1026 lipid molecules from the mouse tissues. The lipidomic analysis indicated that the intake of PUFAs strongly impacted the lipid profiles of metabolic organs such as the liver and kidney, while causing less impact on the brain. Moreover, we revealed a unique lipid modulation in most tissues, where phospholipids containing linoleic acid were significantly decreased in mice on the ARA-supplemented diet, and bis(monoacylglycero)phosphate (BMP) selectively incorporated DHA over ARA and EPA. We comprehensively studied the lipid profiles after dietary intake of PUFAs, which gives insight into lipid metabolism and nutrition research on PUFA supplementation.
    Keywords:  arachidonic acid; dietary fat; fatty acid metabolism; lipidomics; mass spectrometry; omega-3 fatty acids
  5. Se Pu. 2019 Aug 08. 37(8): 815-823
      Phthalates (PAEs) are a class of endocrine-disrupting chemicals. In recent years, the harmful effects of PAEs on human health, in particular their toxicity toward the reproductive system and development, have received significant attention because of their increasing production and usage. PAEs are ubiquitous in the environment and food products, resulting in unavoidable and long-term exposure in humans. Therefore, exposure and risk assessments of PAEs in humans are necessary. Screening and quantification of phthalate metabolites in urine is an important method for evaluating PAEs exposure, and establishing accurate analytical methods for this purpose has become a high priority. Phthalate monoesters and secondary metabolites are the most commonly targeted biomarkers of exposure to short-and long-chain PAEs, respectively. The combination of off-line or on-line solid phase extraction (SPE) with high performance liquid chromatography-tandem mass spectrometry has become the preferred method for the determination of phthalate metabolites. This paper reviews the analytical methods available for the detection of urinary phthalate metabolites and discusses the advantages, limitations, and challenges presented by each method in practical applications.
    Keywords:  biomarkers; gas chromatography-mass spectrometry (GC-MS); liquid chromatography-tandem mass spectrometry (LC-MS/MS); phthalate exposure; review; solid phase extraction (SPE)
  6. Metabolites. 2019 Oct 18. pii: E236. [Epub ahead of print]9(10):
      Metabolomics studies aiming to find biomarkers frequently make use of historical or multicenter cohorts. These samples often have different pre-analytical conditions that potentially affect metabolite concentrations. We studied the effect of different storage conditions on the stability of small-molecule metabolites in cerebrospinal fluid to aid a reliable interpretation of metabolomics data. Three cerebrospinal fluid pools were prepared from surplus samples from the Amsterdam Dementia Cohort biobank. Aliquoted pools were exposed to different storage conditions to assess the temperature and freeze/thaw stability before final storage at -80 °C: storage up to four months at -20 °C and up to one week at either 5-8 °C or 18-22 °C and exposure to up to seven freeze/thaw cycles. Direct-infusion high-resolution mass spectrometry was performed, resulting in the identification of 1852 m/z peaks. To test the storage stability, principal component analyses, repeated measures analysis of variance, Kruskal‒Wallis tests, and fold change analyses were performed, all demonstrating that small-molecule metabolites in the cerebrospinal fluid (CSF) are relatively unaffected by 1‒3 freeze/thaw cycles, by storage at -20 °C up to two months, by storage at 5-8 °C for up to 72 h, or by storage at 18-22 °C for up to 8 h. This suggests that these differences do not affect the interpretation of potential small-molecule biomarkers in multicenter or historical cohorts and implies that these cohorts are suitable for biomarker studies.
    Keywords:  DIMS; biomarker stability; cerebrospinal fluid; direct-infusion mass spectrometry; metabolomics; neurometabolic diagnostics; pre-analytical storage conditions; small-molecule metabolites
  7. Se Pu. 2019 Nov 08. 37(11): 1142-1156
      An ultra-performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry (UPLC-Qtrap MS) method was developed for the determination of 84 toxic plant constiuents in plasma and urine. Plasma was precipitated by acetonitrile to remove proteins and then passed through a Prime HLB SPE column to remove phospholipids, while urine was diluted with methanol. Chromatographic separation of the analytes was achieved on an Acquity BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution using the mobile phase of 0.1% (v/v) formic acid and 2 mmol/L ammonium formate both in 97% (v/v) acetonitrile aqueous solution and water. Electrospray ionization mass spectrometry was carried out in the positive ion mode with multiple reaction monitoring-information dependent acquisition-enhanced product ion scan mode (MRM-IDA-EPI). The 84 analytes were quantified by the matrix working standard curve internal standard method, and a good linear relationship was observed, with correlation coefficients of ≥ 0.9911. The limits of detection (LODs) in plasma and urine were 0.01-1 μg/L and 0.03-2 μg/L, respectively. The intra- and inter-day precisions of these analytes were 0.7%-18.4% and 1.1%-18.5%, and the accuracy of all analytes ranged from 70.6% to 124.5%. This method is simple, sensitive, and accurate for the measurement of these analytes in plasma and urine for both clinical and forensic applications.
    Keywords:  plasma; toxic plant constituents; ultra-performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry (UPLC-Qtrap MS); urine
  8. Anal Bioanal Chem. 2019 Oct 19.
      Testosterone in human serum is commonly tested in clinical laboratories using immunoassay methods as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. To standardize and ensure the accuracy of the measurement results, reference procedures with higher metrological order are required. A simple measurement procedure based on one-step liquid-liquid extraction (LLE) and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) was developed for total testosterone in human serum. The procedure involved serum spiked with 13C3-testosterone, equilibration for 2 h, and extraction with an organic solvent. Testosterone certified reference material (CRM) was used as the calibration standard to ensure the traceability to the International System of Units (SI). Testosterone in serum CRMs from the National Institute for Standards and Technology (NIST) and LGC were used to validate the accuracy of the newly developed method. The deviations of the obtained values from the NIST and LGC certified values ranged from -0.55% to 0.45%. Similarly, the coefficient of variations (CVs) of the replicate measurements were in the range of 0.55% and 0.78%, respectively. The relative expanded uncertainties were comparable with those of the certified materials. The newly developed LC-IDMS/MS procedure demonstrated adequate trueness and precision, and was simple to perform. The method can be used for value assignment of testosterone in external quality assessment (EQA) materials as well as certification of CRMs in the future. Graphical abstract.
    Keywords:  Human serum; Isotope dilution mass spectrometry; Liquid chromatography-tandem mass spectrometry; Liquid-liquid extraction; Reference measurement procedure; Testosterone
  9. J Pharm Biomed Anal. 2019 Aug 19. pii: S0731-7085(19)30606-5. [Epub ahead of print]178 112812
      Drug induced liver injury (DILI) is a diverse set of liver injury occurring after exposure to any manufactured or naturally occurring chemical compounds. DILI has already become the leading cause of acute liver failure in developed countries. Early diagnosis is of great significance for the prevention and treatment of DILI, which can effectively avoid its progress to acute liver failure. In this paper, a novel targeted metabolomics method based on isobaric tagging reagent iTRAQ®-LC-MS/MS was developed for the exploring of 42 common amino acids and related amines alterations in rats treated with hydrazine, aiming to discover the potential biomarkers for early diagnosis of DILI. Forty-two amino acids and related amines were covered in this new method. Through derivatization by iTRAQ reagent, all derivatized amino acids and related amines can be separated and quantified in 16 min with excellent peak shape and good separation efficiency. Fragments related to reporter group (m/z 113 or 121) of iTRAQ reagents could be generated from all derivatized amino acids and related amines under general multiple reaction monitoring (MRM) parameters. Isotope dilution method was established for the quantification of amino acid, which significantly reduced the interference of matrix effect on quantitative accuracy. Using this iTRAQ®-LC-MS/MS method, the changes of amino acid metabolism were comprehensively investigated in rat serum and urine samples after DILI modeling by hydrazine. More significant changes of amino acids were observed in serum and urine samples with fold changes ranging from 0.5 to 193.7. Six significantly increased amino acids in serum, including L-citrulline, L-α-amino-adipic acid, L-tyrosine, L-glutamic acid, glycine and L-lysine, and ten amino acids and related amines in urine including L-citrulline, L-α-amino-adipic acid, L-tyrosine, taurine, β-alanine, ethanolamine, argininosuccinic acid, D,L-β-amino-isobutyric acid, γ-amino-n-butyric acid and L-glutamine, 3 of which were detected in both serum and urine. Except for L-lysine all these significantly increased amino acids and related amines possessed 92.5% to 100% specificity and sensitivity calculated at best cut-off points of their ROC curves in distinguishing control and DILI model group.
    Keywords:  Amino acid and related amines; Drug induced liver injury; Hydrazine; LC-MS/MS; iTRAQ
  10. J Appl Lab Med. 2019 May;3(6): 974-992
      BACKGROUND: Monitoring of medication compliance and drug abuse is used by clinicians to increase patient prescription drug compliance and reduce illicit drug abuse and diversion. Despite available immunoassays, chromatography-mass spectrometry-based methods are considered the gold standard for urine drug monitoring owing to higher sensitivities and specificities. Herein, we report a fast, convenient ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect or quantify 37 clinically relevant prescription drugs, drugs of abuse, and related glucuronides and other metabolites in human urine by single diluted sample injection.METHODS: Analytes consisted of prescription and illicit opioids, benzodiazepines, and drugs of abuse, including parent compounds and glucuronidated and nonglucuronidated metabolites. Urine samples were diluted with water and supplemented with deuterated internal standards without enzymatic hydrolysis, analyte extraction, or sample purification. Analytes were separated by reversed-phase UPLC and quantified by positive-mode electrospray ionization and collision-induced dissociation MS. Assay validation followed Food and Drug Administration bioanalytical guidelines.
    RESULTS: Total analytical run time was 5.5 min. All analytes demonstrated acceptable inter- and intraassay accuracy, imprecision, and linearity throughout clinically relevant analytical ranges (1-2000 ng/mL, depending on analyte). All analytes demonstrated acceptable selectivity, stability, matrix effects, carryover, and performance compared to national reference laboratory or previously validated in-house methods. A total of 23 and 14 analytes were validated for quantitative and qualitative testing, respectively.
    CONCLUSIONS: A convenient UPLC-MS/MS assay for simultaneously monitoring 37 analytes in human urine was validated for use in pain management testing. Advantages of this multiplex assay include facile sample preparation and higher-throughput definitive detection including glucuronide metabolite quantification.
  11. Clin Chem Lab Med. 2019 Oct 22. pii: /j/cclm.ahead-of-print/cclm-2019-0354/cclm-2019-0354.xml. [Epub ahead of print]
      Background A method for bile acid profiling measuring 21 primary and secondary bile acids in serum samples was developed and validated with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample preparation included spiking with internal standards followed by protein precipitation, centrifugation, drying under nitrogen gas and reconstitution. Extracted samples were injected onto a Phenomenex Kinetex C18 column (150 × 4.60 mm, 2.6 μm). Methods Data was collected with LC-MS/MS operated in negative ion mode with multiple reaction monitoring (MRM) and single reaction monitoring (SRM). The analytical run time was 12 min. Results The method showed excellent linearity with high regression coefficients (>0.99) over a range of 0.05 and 25 μM for all analytes tested. The method also showed acceptable intra-day and inter-day accuracy and precision. As a proof of concept, the analytical method was applied to patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), biliary atresia (BA), and necrotizing enterocolitis (NEC), and distinct bile acids profiles were demonstrated. Conclusions The method could be poised to identify possible biomarkers for non-invasive early diagnosis of these disorders.
    Keywords:  bile acids profiling; biliary atresia (BA); liquid chromatography-tandem mass spectrometry (LC-MS/MS); necrotizing enterocolitis (NEC); neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD)
  12. Front Cell Dev Biol. 2019 ;7 210
      Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C16 fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d3 and L-serine-d3. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B1). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates - naturally occurring or chemically tailored - as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.
    Keywords:  ceramides; mass spectrometry; serine palmitoyltransferase; sphingolipid de novo synthesis; stable-isotope labeling
  13. J Chromatogr Sci. 2019 Oct 23. pii: bmz068. [Epub ahead of print]
      Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5-100 μg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 μg/mL for PTX and 0.4419 and 1.3389 μg/mL for SFN, respectively. A total of 98-101% of the injected samples was recovered with RSD of 0.06-0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.
  14. J Mass Spectrom. 2019 Oct 26.
      The eye is an elegant organ consisting of a number of tissues and fluids with specialised functions that together allow it to effectively transmit and transduce light input to the brain for visual perception. One key determinant of this integrated function is the spatial relationship of ocular tissues. Biomolecular distributions within the main ocular tissues cornea, lens and retina have been studied extensively in isolation, yet the potential for metabolic communication between ocular tissues via the ocular humours has been difficult to visualise. To address this limitation, the current study presents a method to map spatial distributions of metabolites and small molecules in whole eyes, including ocular humours. Using a tape-transfer system and freeze-drying, the spatial distribution of ocular small molecules was investigated in mouse, rat, fish (black bream) and rabbit eyes using negative ion mode MALDI imaging mass spectrometry. Full-scan imaging was used for discovery experiments, while MS/MS imaging for identification and localisation was also demonstrated. In all eyes, metabolites such as glutathione and phospholipids were localised in the main ocular tissues. In addition, in rodent eyes, major metabolites were distributed relatively uniformly in ocular humours. In contrast, both uniform and spatially-defined ocular metabolite distributions were observed in the black bream eye. Tissue and ocular humour distributions were reproducible, as demonstrated by the three dimensional analysis of a mouse eye, and able to be captured with high spatial resolution analysis. The presented method could be used to further investigate the role of inter-tissue metabolism in ocular health, and to support the development of therapeutics to treat major ocular diseases.
    Keywords:  MALDI imaging mass spectrometry; eye; humours; lens; metabolites; retina
  15. Contemp Clin Trials Commun. 2019 Dec;16 100445
      Background: Lung cancer is a major cause of global morbidity and mortality. Current low dose CT screening is invasive and its role remains contentious. There are no known biomarkers to monitor treatment response, detect disease recurrence and patient selection for adjuvant treatment after curative surgical resection. Hence there is an urgent need to explore non-conventional and non-invasive tools to develop novel biomarkers to improve the outcome of this lethal cancer.Methods: This is an ongoing exploratory and translational study involving collection of bio fluids from 50 patients with early stage non-small cell lung cancer before and after surgical resection. The primary objective is to identify cancer specific metabolome in body fluids - sputum, exhaled breath condensate, blood and urine of the patients with early stage non-small cell lung cancer using Magnetic Resonance Spectroscopy and Mass Spectroscopy.
    Conclusion: The trajectory of change in metabolic profile of body fluids before and after surgical resection may have potential clinical applications in lung cancer screening, as biomarkers for disease recurrence and exploration of novel targets for therapeutic intervention.
    Keywords:  Biomarkers; EBC, exhaled breath condensate; LC, liquid chromatography; LC-QTOF-MS, liquid chromatography quadrupole time-of-flight mass spectrometry; Lung cancer; MRS, magnetic resonance spectroscopy; MS, mass spectrometry; Metabolomics; NMR, nuclear magnetic resonance; NSCLC, non-small cell lung cancer
  16. Biol Pharm Bull. 2019 Oct 24.
      To improve the efficiency of drug-discovery research on pyrrole-imidazole polyamides (PIs), a more rapid method for quantitative and qualitative measurement of PI in rat plasma samples was developed here using ultra-fast liquid chromatography-ultraviolet spectrometry (UFLC-UV) in order to shorten the measurement time. A measurement method of PIs by HPLC developed until now takes 45 min for one sample measurement. This method was inefficient to investigate extraction conditions from biological samples and measurement of animal experimental samples. In the developed method of this study, PI and phenacetin (internal standard, IS) were separated with an ACQUITY UPLC HSS T3 (1.8 mm, 2.1 × 50 mm; Nihon Waters K.K., Japan) column using a mobile phase of 0.1% acetic acid (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min with a linear gradient. The detection wavelength was 310 nm. The calibration curve was linear in the range of 0.225-4.5 μg/mL (correlation coefficients ≥ 0.9995, n = 5). The intra- and inter-day accuracies were in the range of -6.04% to 12.2%, and the precision was less than 2.99%. The measurement time of this method (7 min per injection) was markedly shortened to about one-sixth of the previous measurement time (45 min per injection). This is the first report describing the quantitative and qualitative measurement of PI in plasma using UFLC-UV. The present method will be very useful for the drug-discovery research of PIs.
    Keywords:  drug-discovery research; pyrrole-imidazole polyamide; ultra-fast liquid chromatography
  17. Anal Chem. 2019 Oct 23.
      We report a high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to quantify without derivatizaton dehyroepiandrosterone (DHEA), 17β-estradiol (E2), testosterone (T), and their sulfates in serum and tissues. This assay functions well with multiple adipose depots, a previously unattained analysis. To delipidate and facilitate recovery, tissues were homogenized in acetonitrile and the homogenate was frozen. The supernatant was evaporated, resuspended in an aqueous acetate buffer, and extracted with hexane to separate free (unconjugated) from sulfated steroids. Sulfated steroids in the aqueous medium were then hydrolyzed with sulfatase and extracted with hexane. Each extract was analyzed separately. HPLC resolution combined with the sensitivity and specificity of MS/MS allowed quantification of DHEA, E2, and T with 10, 10 and 5 fmol lower limits of quantification and linear ranges to 1 pmol. Application of the method to mouse serum and tissues reveals ranges of DHEA, E2, and T and their sulfates, and tissue-specific differences in steroid profile, especially white vs brown adipose. In addition, marginal decreases of T in all tissues and considerable increases in DHEA in male iWAT and eWAT in response to high-fat diet further strengthen the inference regarding role of steroid metabolism in adipogenesis. This assay permits detailed studies of interactions between adiposity and sex steroids in serum and tissues, including adipose.
  18. Biomed Chromatogr. 2019 Oct 21. e4717
      A sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established to analyze furanodienone in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for furanodienone and patchouli alcohol (internal standard, IS) were m/z 231.1→83.2 and m/z 205.1→95.1, respectively. Great linearity of furanodienone in plasma samples was found in the corresponding concentration range (r > 0.995). Intra- and inter-day precisions (RSD%) were less than 11.3% in plasma, and the accuracy (RE%) was within ±10.7%. This method was used to the furanodienone study on rat pharmacokinetics after a single oral dose of 10 mg/kg of furanodiene. The results indicated that the maximum observed plasma concentration (Cmax ) was 52.4±19.1 ng/mL at 1.2±0.7 h (time to reaching Cmax ) with the elimination half-life (t1/2 ) of 2.2±0.7 h. The obtained data indicated that furanodienone could be moderately distributed and eliminated.
  19. Biosci Rep. 2019 Oct 30. pii: BSR20192502. [Epub ahead of print]39(10):
      Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.
    Keywords:  PIP4K; lipid kinase; mass spectrometry; phosphatidylinositol; phosphatidylinositol 5 phosphate
  20. J Mass Spectrom. 2019 Oct 25.
      Supercritical fluid chromatography (SFC) has experienced a particular revival in recent years thanks to the development of robust and efficient commercial systems. Due to its physico-chemical properties, supercritical carbon dioxide (CO2 ) mixed with co-solvents and additives is particularly suitable for SFC to allow the elution of compounds of different polarity, and more particularly complex lipids. Hyphenation with mass spectrometry (MS) is increasingly described in the literature but still requires many further developments in order to be as user-friendly as coupling with liquid chromatography. The basic concepts of SFC and MS hyphenation will be first considered. Then a representative example of method development in lipidomics will be introduced. In conclusion, the challenges and future needs in this field of research will be discussed.
  21. Anal Chem. 2019 Oct 22.
      Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which can be then measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases, and by comparison to concentrations provided for commercial materials, or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop Spectrophotometry or established from droplet-digital PCR (ddPCR) techniques agree well to the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling SI-traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases, or mixtures.
  22. Metabolites. 2019 Oct 18. pii: E235. [Epub ahead of print]9(10):
      The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.
    Keywords:  biomarker; glutathione; homocysteine; mass spectrometry; methionine metabolism; targeted metabolic profiling; thiol
  23. Anal Chem. 2019 Oct 22.
      Analytical scientists working in many sectors have long expressed the need for increasingly fast chromatographic separations. The pharmaceutical industry, for example, has a growing need for high-throughput methodologies in order to accelerate the discovery and development of new chemical entities. Analytical toxicology labs require fast, robust methods for routine analysis of many clinical and forensic samples. As biomedical researchers increase the need for in-depth proteomic and metabolomic analysis of complex biological systems, rapid separations-based techniques will be central to advancing this field. Progress in these areas and others has advanced in parallel with improvements to LC column technology, especially as it relates smaller packing materials, over the past several decades. Arguably the most impactful result of the steady reduction in the size of chromatographic stationary phase supports is faster LC, run at higher mobile phase velocities without the significant losses in efficiency that are observed on columns packed with larger diameter particles. This improvement in chromatographic speed due to smaller particless necessitated the development of ultrahigh pressure liquid chromatography (UHPLC) instrumentation, with typical pressure limits of 1000-1500 bar, because both the use of smaller particles and higher mobile phase velocities require higher pressures than achievable with traditional HPLC instruments limited to 400 bar. It has been over twenty years since the first demonstration of capillary UHPLC and over fifteen years since the release of the first commercial UHPLC platform. Since that time, there has been a sharp increase in the use of UHPLC for analytical separations, with continuing trends toward higher speed and higher performance.
  24. J Chromatogr A. 2019 Oct 10. pii: S0021-9673(19)31018-0. [Epub ahead of print] 460613
      In this work, the use of design of experiments and posterior data modelling by artificial neural network (ANN) and least squares (LS) is presented as a suitable analytical tool for the performance optimization of a tandem mass spectrometric detector coupled to ultra-high performance liquid chromatography for the analysis of seventeen veterinary drugs. Firstly, a central composite design was built considering as factors the cone, capillary, extractor and radio frequency voltages of the mass spectrometer in order to obtain a proper combination to improve the sensitivity of the method. Secondly, a one factor design considering the collision voltage was built to define the adequate voltage for each daughter ion. The response surface methodology (RSM) was then applied, and the prediction capability of ANN and LS were compared. As conclusion, the ANN modelling provided better results than LS, both in terms of the ANOVA and predicted areas results. The accuracy of the model prediction was between 85 and 125%, confirming that the estimates of the model were correct, and endorsing the optimization procedure as a suitable way to gather excellent results. The suitability of the new approach and its implications on the simultaneous analysis of seventeen veterinary drugs by ultra-high liquid chromatography coupled to tandem mass spectrometry detection are discussed.
    Keywords:  Artificial neural networks (ANN); Desirability function; Response surface methodology (RSM); Ultra-high performance liquid chromatography coupled to tandem mass spectrometric detection (UHPLC–MS/MS); Veterinary drugs
  25. Biomed Chromatogr. 2019 Oct 21. e4713
      Metabolomes are small molecule metabolites (<1000 Da) produced by cellular processes. Metabolomes are close counterparts to the genome, transcriptome, and proteome. The aim of this study was to develop a method to detect and quantify candidate nucleoside metabolomes 1-methyl adenosine (1-MA), 1-methylguanosine (1-MG), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the urine of patients with breast cancer using gas chromatography-mass spectrometry (GC-MS). The method was applied to urine specimens from patients with breast cancer (n=56) and benign breast tumors (n=22), as well as from healthy females (n=20). The relative standard deviations of precision and repeatability analysis were <10%, and recoveries ranged from 88.5% to 105.6%. Limits of detection were 0.014, 0.012, and 0.018 mg/L for 1-MA, 1-MG, and 8-OHdG, respectively. The lower limits of quantitation were 0.056, 0.048, and 0.072 mg/L, respectively. There were significant differences in concentrations of candidate metabolomes between patients with cancer and the healthy individuals, especially for those in the early stages of the disease (p<0.001). No significant differences were observed between the benign and healthy groups. In conclusion, a reliable GC-MS method for detection and quantification of 1-MA, 1-MG, and 8-OHdG metabolomes in urine has been developed.
    Keywords:  Breast cancer; Gas chromatography; Mass spectrometry; Metabolome; Metabolomics
  26. Metabolites. 2019 Oct 21. pii: E242. [Epub ahead of print]9(10):
      Inborn errors of metabolism (IEMs) are a group of inherited diseases with variable incidences. IEMs are caused by disrupting enzyme activities in specific metabolic pathways by genetic mutations, either directly or indirectly by cofactor deficiencies, causing altered levels of compounds associated with these pathways. While IEMs may present with multiple overlapping symptoms and metabolites, early and accurate diagnosis of IEMs is critical for the long-term health of affected subjects. The prevalence of IEMs differs between countries, likely because different IEM classifications and IEM screening methods are used. Currently, newborn screening programs exclusively use targeted metabolic assays that focus on limited panels of compounds for selected IEM diseases. Such targeted approaches face the problem of false negative and false positive diagnoses that could be overcome if metabolic screening adopted analyses of a broader range of analytes. Hence, we here review the prospects of using untargeted metabolomics for IEM screening. Untargeted metabolomics and lipidomics do not rely on predefined target lists and can detect as many metabolites as possible in a sample, allowing to screen for many metabolic pathways simultaneously. Examples are given for nontargeted analyses of IEMs, and prospects and limitations of different metabolomics methods are discussed. We conclude that dedicated studies are needed to compare accuracy and robustness of targeted and untargeted methods with respect to widening the scope of IEM diagnostics.
    Keywords:  LC-MS; aminoacidemia.; lysosomal storage disease; mass spectrometry; mitochondrial disorders; organic aciduria; phenylketonuria
  27. Anal Bioanal Chem. 2019 Oct 22.
      In this work, a straightforward analytical approach based on headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was developed for the analysis of salivary volatile organic compounds without any prior derivatization step. With a sample volume of 500 μL, optimal conditions were achieved by allowing the sample to equilibrate for 10 min at 50 °C and then extracting the samples for 10 min at the same temperature, using a carboxen/polydimethylsiloxane fibre. The method allowed the simultaneous identification and quantification of 20 compounds in sample headspace, including short-chain fatty acids and their derivatives which are commonly analysed after analyte derivatization. The proof of applicability of the methodology was performed with a case study regarding the analysis of the dynamics of volatile metabolites in saliva of a single subject undergoing 5-day treatment with rifaximin antibiotic. Non-stimulated saliva samples were collected over 3 weeks from a nominally healthy volunteer before, during, and after antibiotic treatment. The variations of some metabolites, known to be produced by the microbiota and by bacteria that are susceptible to antibiotics, suggest that the study of the dynamics of salivary metabolites can be an excellent indirect method for analysing the gut microbiota. This approach is novel from an analytical standpoint, and it encourages further studies combining saliva metabolite profiles and gut microbiota dynamics. Graphical abstract.
    Keywords:  HS-SPME-GC-MS method; Microbiota; Saliva; Volatile organic compounds
  28. Food Chem. 2019 Oct 01. pii: S0308-8146(19)31650-4. [Epub ahead of print]307 125531
      In the present study, submicrometer flow-through silica microspheres (Sub-FTSiO2) was for the first time obtained via a suspension polymerization method coupled with sol-gel transition and phase separation. The Sub-FTSiO2 was characteristic of rich mesopores, penetrable macropores and small particle size, which would be beneficial to fast mass transfer, low column backpressure and high column efficiency. It was directly used as the hydrophilic interaction liquid chromatographic (HILIC) stationary phase, and the fast separation of seven water-soluble vitamins in 2.2 min was realized. The proposed method was successfully applied to the determination of water-soluble vitamins in two functional beverages on the market. The prepared Sub-FTSiO2 was well demonstrated for fast HILIC, and would be potential as the stationary phase matrix for fast liquid chromatography in diverse separation modes.
    Keywords:  Beverage; Fast separation; Flow-through silica microspheres; Hydrophilic interaction liquid chromatography; Water-soluble vitamins
  29. Metabolites. 2019 Oct 24. pii: E250. [Epub ahead of print]9(11):
      Metabolomics generates massive and complex data. Redundant different analytical species and the high degree of correlation in datasets is a constraint for the use of data mining/statistical methods and interpretation. In this context, we developed a new tool to detect analytical correlation into datasets without confounding them with biological correlations. Based on several parameters, such as a similarity measure, retention time, and mass information from known isotopes, adducts, or fragments, the algorithm principle is used to group features coming from the same analyte, and to propose one single representative per group. To illustrate the functionalities and added-value of this tool, it was applied to published datasets and compared to one of the most commonly used free packages proposing a grouping method for metabolomics data: 'CAMERA'. This tool was developed to be included in Galaxy and will be available in Workflow4Metabolomics ( Source code is freely available for download under CeCILL 2.1 license at /tool-acf and implement in Perl.
    Keywords:  data filtration; high-resolution mass spectrometry; metabolomics
  30. Wiley Interdiscip Rev Syst Biol Med. 2019 Oct 23. e1466
      Lipids are essential for all facets of life. They play three major roles: energy metabolism, structural, and signaling. They are dynamic molecules strongly influenced by endogenous and exogenous factors including genetics, diet, age, lifestyle, drugs, disease and inflammation. As precision medicine starts to become mainstream, there is a huge burgeoning interest in lipids and their potential to act as unique biomarkers or prognostic indicators. Lipids comprise a large component of all metabolites (around one-third), and our expanding knowledge about their dynamic behavior is fueling the hope that mapping their regulatory biochemical pathways on a systems level will revolutionize our ability to prevent, diagnose, and stratify major human diseases. Up to now, clinical lipid measurements have consisted primarily of total cholesterol or triglycerides, as a measure for cardiovascular risk and response to lipid lowering drugs. Nowadays, we are able to measure thousands of individual lipids that make up the lipidome. nuclear magnetic resonance spectrometry (NMR) metabolomics is also being increasingly used in large cohort studies where it can report on total levels of selected lipid classes, and relative levels of fatty acid saturation. To support the application of lipidomics research, LIPID MAPS was established in 2003, and since then has gone on to become the go-to resource for several lipid databases, lipid drawing tools, data deposition, and more recently lipidomics informatics tools, and a lipid biochemistry encyclopedia, LipidWeb. Alongside this, the recently established Lipidomics Standards Initiative plays a key role in standardization of lipidomics methodologies. This article is categorized under: Laboratory Methods and Technologies > Metabolomics Analytical and Computational Methods > Analytical Methods.
    Keywords:  biomarkers; lipid; lipidomics; mass spectrometry; precision medicine
  31. Int J Oncol. 2019 Oct 14.
      The purpose of the present study was to compare metabolites from formalin‑fixed and paraffin‑embedded (FFPE) pancreatic tissue blocks with those identified in optimal cutting temperature (OCT)‑embedded pancreatic tissue blocks. Thus, ultra‑performance liquid chromatograph‑mass spectrometry/mass spectrometry‑based metabolic profiling was performed in paired frozen (n=13) and FFPE (n=13) human pancreatic adenocarcinoma tissue samples, in addition to their benign counterparts. A total of 206 metabolites were identified in both OCT‑embedded and FFPE tissue samples. The method feasibility was confirmed through reproducibility and a consistency assessment. Partial least‑squares discriminant analysis and heatmap analysis reliably distinguished tumor and normal tissue phenotypes. The expression of 10 compounds, including N‑acetylaspartate and creatinine, was significantly different in both OCT‑embedded and FFPE tumor samples. These ten compounds may be viable candidate biomarkers of malignant pancreatic tissues. The super‑categories to which they belonged exhibited no significant differences between FFPE and OCT‑embedded samples. Furthermore, purine, arginine and proline, and pyrimidine metabolism used a shared pathway found in both OCT‑embedded and FFPE tissue samples. These results supported the notion that metabolomic data acquired from FFPE pancreatic cancer specimens are reliable for use in retrospective and clinical studies.
  32. J Sep Sci. 2019 Oct 20.
      Adopting a stationary phase convention circumvents problematic definition of the boundary between the stationary and the mobile phase in the LC, resulting in thermodynamically consistent and reproducible chromatographic data. Three stationary phase definition conventions provide different retention data, but equal selectivity: 1. the complete solid phase moiety; 2. the solid porous part carrying the active interaction centers; 3. the volume of the inner column pores. The selective uptake of water from the bulk aqueous-organic mobile phase affects significantly the volume and the properties of polar stationary phases. Some polar stationary phases provide dual-mode retention mechanism in aqueous-organic mobile phases, reversed-phase in the water-rich range and normal-phase at high concentrations of the organic solvent in water. The Linear Solvation Energy Relationship model characterizes the structural contributions of the non-selective and selective polar interactions both in the water-rich and in the organic solvent-rich mobile phases. The inner-pore convention provides a single hold-up volume value for the retention prediction on the dual-mode columns over the full mobile phase range. Using the dual-mode monolithic polymethacrylate zwitterionic micro-columns alternatively in each mode in the first dimension of two-dimensional LC, in combination with a short reversed-phase column in the second dimension, provides enhanced sample information This article is protected by copyright. All rights reserved.
    Keywords:  dual retention mechanism; hydrophilic interaction liquid chromatography; mixed-mode columns; phase volume convention; two-dimensional separation
  33. Clin Chem Lab Med. 2019 Oct 22. pii: /j/cclm.ahead-of-print/cclm-2019-0974/cclm-2019-0974.xml. [Epub ahead of print]
      The workings of the gut microbiome have gained increasing interest in recent years through the mounting evidence that the microbiota plays an influential role in human health and disease. A principal focus of this research seeks to further understand the production of metabolic by-products produced by bacteria resident in the gut, and the subsequent interaction of these metabolites on host physiology and pathophysiology of disease. Gut bacterial metabolites of interest are predominately formed via metabolic breakdown of dietary compounds including choline and ʟ-carnitine (trimethylamine N-oxide), amino acids (phenol- and indole-containing uremic toxins) and non-digestible dietary fibers (short-chain fatty acids). Investigations have been accelerated through the application of mass spectrometry (MS)-based assays to quantitatively assess the concentration of these metabolites in laboratory- and animal-based experiments, as well as for direct circulating measurements in clinical research populations. This review seeks to explore the impact of these metabolites on disease, as well as to introduce the application of MS for those less accustomed to its use as a clinical tool, highlighting pertinent research related to its use for measurements of gut bacteria-mediated metabolites to further understand their associations with disease.
    Keywords:  TMAO; biomarker; gut microbiome; mass spectrometry; short-chain fatty acids; uremic toxins
  34. J Appl Lab Med. 2019 Jul;4(1): 101-107
      BACKGROUND: Assessing vitamin A status in populations remains a high public health priority for low- and middle-income countries. However, analytical difficulties with serum retinol measurements persist in international laboratories. Nearly all participants in a Centers for Disease Control and Prevention external quality assessment program use HPLC to measure serum retinol, but round-to-round results failing to meet acceptable criteria suggest the need to provide a straightforward stable HPLC ultraviolet (UV) method that can be adopted by these laboratories to improve performance. We present a protein precipitation HPLC-UV method that measures serum retinol below the deficiency cutoff value (<0.7 μmol/L or 20 μg/dL) that is suitable for low- and middle-income countries and uses commercially available materials.METHODS: Serum (25 μL) added to retinyl acetate was precipitated with acetonitrile (125 μL) to extract retinol. Solvent-based calibration solutions required no extraction. Calibration used either single-point (50 μg/dL) or multipoint solutions (0.52-100 μg/dL). C18 column (4.6 × 100 mm) and acetonitrile with 0.1% triethylamine/water (83/17, v/v) as isocratic mobile phase (1.1 mL/min), achieved baseline separation (7 minutes).
    RESULTS: With only 25 μL of serum, the limit of detection was 0.52 μg/dL. Single- and multipoint calibration generated equivalent results. Over several years, between-run imprecision was ≤7.1% in multiple quality-control materials. Overall mean (CV) method bias for NIST-certified reference materials (e-series) was -0.2% (5.8%). Maximally, 180 samples were processed within 24 h.
    CONCLUSIONS: This method was robust and stable over years and accurately measured serum retinol with low-volume samples. Thus, it may be of interest to low- and middle-income countries and to pediatric and finger stick applications.
  35. J Circ Biomark. 2019 Jan-Dec;8:8 1849454419879848
      There is a wide variety of extracellular vesicles (EVs) that differ in size and cargo composition. EVs isolated from human plasma or serum carry lipid, protein, and RNA cargo that provides insights to the regulation of normal physiological processes, and to pathological states. Specific populations of EVs have been proposed to contain protein and RNA cargo that are biomarkers for neurologic and systemic diseases. Although there is a considerable amount of evidence that circulating lipids are biomarkers for multiple disease states, it not clear if these lipid biomarkers are enriched in EVs, or if specific populations of EVs are enriched for particular classes of lipid. A highly reproducible workflow for the analysis of lipid content in EVs isolated from human plasma or serum would facilitate this area of research. Here we optimized an MS/MSALL workflow for the untargeted analysis of the lipid content in EVs isolated from human serum. A simple sequential ultracentrifugation protocol isolated three distinct types of serum EVs that were identified based on size, targeted protein, and untargeted lipidomic analyses. EVs in the upper and middle fractions were approximately 140 nm in diameter, while EVs in the pellet were approximately 110 nm in diameter. EVs in the upper most buoyant fractions contained the highest concentration of lipids, were enriched with phospholipids, and immunopositive for the cytoskeletal markers actin, α-actinin, and the mitochondrial protein mitofillin, but negative for the typical EV markers CD63, TSG101, and flotillin. A central fraction of EVs was devoid of cytoskeletal and mitochondrial markers, and positive for CD63, and TSG101, but negative for flotillin. The EV pellet contained no cytoskeletal or mitochondrial markers, but was positive for CD63, TSG101, and flotillin. The EV pellet contained the lowest concentration of most lipids, but was enriched with ceramide. These results provided new insights into the lipid composition of EVs isolated from serum using a simple ultracentrifugation isolation method suitable for lipidomic analysis by mass spectrometry.
    Keywords:  Extracellular vesicle; blood; exosome; lipid; lipidomics; serum
  36. Bull Exp Biol Med. 2019 Oct 19.
      The metabolomics of urinary steroids was studied by gas chromatography-mass spectrometry in 25 patients with Cushing's syndrome without malignant potential and in 12 patients with malignant potential of adrenal neoplasms (Weiss score 1-3). Patients with adrenocortical adenoma (N=24) constituted the control group. In patients with Cushing's syndrome and malignant potential, increased urinary excretion of 16-oxo-androstendiol, tetrahydro-11-deoxycortisol, and 16-hydroxypregnendiol, which had 100% specificity and sensitivity >90% for the diagnosis of malignant potential. Additionally, non-classical 5-ene-pregnenes (16-OHpregnenolone, 21-OH-pregnenolone, 3β,16,20-pregnentriol, and 3β,17,20-pregnentriol) were identified. The revealed changes in the metabolomics of steroids can be early signs of malignant potential in patients with Cushing's syndrome. In patients with malignant potential, three signs of reduced activity of 11β-hydroxysteroid dehydrogenase type 2 were detected and in patients without malignant potential, one sign was found. In patients with and without malignant potential, three signs increased activity of 5β-reductase were found.
    Keywords:  Cushing’s syndrome; gas chromatography-mass spectrometry; malignant potential; steroid profile in urine
  37. Front Oncol. 2019 ;9 1010
      Magnetic resonance spectroscopic imaging (SI) is a unique imaging technique that provides biochemical information from in vivo tissues. The 1H spectra acquired from several spatial regions are quantified to yield metabolite concentrations reflective of tissue metabolism. However, since these metabolites are found in tissues at very low concentrations, SI is often acquired with limited spatial resolution. In this work, we test the hypothesis that deep learning is able to upscale low resolution SI, together with the T1-weighted (T1w) image, to reconstruct high resolution SI. We report on a novel densely connected UNet (D-UNet) architecture capable of producing super-resolution spectroscopic images. The inputs for the D-UNet are the T1w image and the low resolution SI image while the output is the high resolution SI. The results of the D-UNet are compared both qualitatively and quantitatively to simulated and in vivo high resolution SI. It is found that this deep learning approach can produce high quality spectroscopic images and reconstruct entire 1H spectra from low resolution acquisitions, which can greatly advance the current SI workflow.
    Keywords:  artificial intelligence; deep learning (DL); magnetic resonance spectroscopic imaging (SI); magnetic resonance spectroscopy (1H MRS); super-resolution
  38. Biochim Biophys Acta Gene Regul Mech. 2019 Oct 19. pii: S1874-9399(19)30224-X. [Epub ahead of print] 194418
      Gaussian Graphical Models (GGMs) are tools to infer dependencies between biological variables. Popular applications are the reconstruction of gene, protein, and metabolite association networks. GGMs are an exploratory research tool that can be useful to discover interesting relations between genes (functional clusters) or to identify therapeutically interesting genes, but do not necessarily infer a network in the mechanistic sense. Although GGMs are well investigated from a theoretical and applied perspective, important extensions are not well known within the biological community. GGMs assume, for instance, multivariate normal distributed data. If this assumption is violated Mixed Graphical Models (MGMs) can be the better choice. In this review we provide the theoretical foundations of GGMs, present extensions such as MGMs or multi-class GGMs, and illustrate how those methods can provide insight in biological mechanisms. We summarize several applications and present user-friendly estimation software.
    Keywords:  (Multi-)omics; Gaussian Graphical Model; Gene regulatory network; Mixed Graphical Model