Current Metabolomics (v.2, #3)

Metabolite Identification in NMR-based Metabolomics by Santosh Kumar Bharti, Raja Roy (163-173).
To achieve goals in metabolomics investigations, it is necessary to produce a comprehensive metabolic profiling from biological samples. Identification of metabolites is one of the important steps in metabolomics studies and the conclusion drawn from such studies depends on how exactly the metabolites are identified. NMR is one of the most selective analytical techniques which offers structural information of molecules. But, due to complex biological sample matrix, metabolic identification step needs application of advanced NMR techniques and analytical strategies for better accuracy. This review covers the analytical methods and strategies used for identification of metabolites in NMR-based metabolomics. The specific problems and troubleshoots associated with identification of metabolites in biological samples are discussed in details.

A Comparison of the Metabolome of Male and Female Drosophila melanogaster by Rong Zhang, Tong Zhang, Dominika Korzekwa, Shadi Al-Johani, Julian A.T. Dow, David G. Watson (174-183).
Metabolite profiles of ten batches of male and female Drosophila melanogaster were compared using three different chromatographic methods interfaced to an Orbitrap Exactive mass spectrometer. Several thousand features were observed and were reduced after data extraction and careful checking of around 390 metabolites excluding lipids. Chromatographic traces for these metabolites are presented as supplementary information. There were many significant differences between male and female flies. Female flies contained much higher levels of methylated lysines and methylated arginine suggesting differences in histone metabolism. In addition, there were differences in the methylation of nucleosides, and Sadenosylmethionine metabolism. Differences in the methylome may relate to the requirement of compensation for the single X chromosome present in males since methylated histones inhibit gene transcription. Nucleoside phosphate levels were elevated in female flies which may relate to increased requirement for DNA biosynthesis for egg production. A series of acylated amino acids previously observed in Drosophila was further characterised and these metabolites were present to a much greater extent in female flies and may be associated with the microbiome.

Metabolomics Approach Reveals Altered Plasma Amino Acid and Sphingolipid Profiles Associated with Patholological State in Transition Dairy Cows by Dagnachew Hailemariam, Rupasri Mandal, Fozia Saleem, Suzanna M. Dunn, David S. Wishart, Burim N. Ametaj (184-195).
Every other cow in a dairy herd is affected by one or multiple diseases during the periparturient period; however, the reason for this high incidence rate of diseases is not clear yet. The objective of this study was to evaluate whether diseased cows show alterations in the amino acid and sphingolipid profiles around calving. Transition dairy cows affected by multiple periparturient diseases (n=6) and healthy controls (n=6) were selected to quantify amino acid and sphingolipid components in the plasma at 4 time points (-4, -1, +1, and +4 wks) around parturition. Samples were sorted retrospectively and analyzed by DI-MS/MS. Univariate and multivariate data analyses were performed at each time point to elucidate the relationship between metabolite alterations and disease progression. Results from the univariate ananlysis indicated increased concentrations of multiple plasma amino acids in the sick cows as compared to healthy controls at -4 wk before parturition. At -1 wk before parturition marked elevations of sphingolipids occured in the sick cows. During the week of parturition very minor differences in amino acid and sphingolipid profiles were observed between healthy and sick cows. However, at +4 wk after parturion a marked decrease in various amino acid and sphingolipid compounds were observed in cows with health problems. Multivariate analysis also classified the sick and healthy cows on the basis of plasma amino acid profiles at -4 wk before calving. In conclusion, altered profiles of plasma amino acids and sphingolipids in the sick cows during the transition period suggest an association among disease state and the dynamics of amino acid and sphingolipid metabolism giving insight on the etiopathology of the disease state. Moreover, this suggests utilization of selected amino acids as biomarkers for disease or deviations from normal state of health.

The reasons for improved survival following minimally invasive surgery remain elusive. Circulating mediators link surgical trauma, vascular and tissue homeostasis. Acute phase reactants, leukocytes and leukocyte Reactive Oxygen Species (ROS) are affected differentially by minimally invasive video-assisted thoracic surgery (VATS). Also, immunoglobulins, complement, TNF receptor and P-selectin changes have been observed, but the influence of minimally invasive surgery on these opsonins is less well defined. In this prospective randomised trial, 41 patients were randomly assigned to minimally invasive or open thoracic surgery, and immunoglobulins and vascular endothelial damage biomarkers were analysed. Humoral mediators (blood IgG, IgM, IgA; complement fragments C3, C4, and complement haemolytic index of activation CH50; TNF receptors I, II and P-selectin) were analysed before and 2, 5 and 7 days after surgery. Post-surgical changes in individual patients were determined. ; Substantial immunoglobulin decreases followed minimally invasive and open surgery. Decreased IgG, IgM and IgE were detected 2 days after surgery, and IgG and IgM after 7 days. These changes were greater than haemodilution, reaching greater significance in open surgery patients. Immunoglobulin decreases followed lymphocyte decreases. In contrast, increased complement and inflammatory endothelial cell signals (C3 and C4, soluble TNFR-II) were detected 7 days after surgery. In both groups, increased C3 and TNFR-II followed early acute phase reactants CRP, IL-6 and ROS. Acute phase reactants and CD4/CD8 lymphocytes were factors most attenuated in patients undergoing minimally invasive thoracic surgery (VATS). This study suggests local trauma mediators are better biomarkers than circulating opsonins in defining the response to minimally invasive surgery, and a systems approach, comparing individual metabolic responses, is effective in small patient groups.

NMR-based Metabolite Profiling of Pancreatic Cancer by Kwadwo Owusu-Sarfo, Vincent M. Asiago, Lingli Deng, Haiwei Gu, Siwei Wei, Narasimhamurthy Shanaiah, G. A. Nagana Gowda, Bowei Xi, Elena G. Chiorean, Daniel Raftery (204-212).
Metabolite profiles of serum from pancreatic cancer (PC) patients (n=51) and non-disease controls (n=47) were measured using 1H nuclear magnetic resonance (NMR) spectroscopy with a focus on the metabolic changes associated with PC pathology and the development and external validation of the statistical models developed using the metabolite data. Univariate statistical analysis indicated 42 metabolite features showing significant differences between PC and controls (p<0.05). Based on multivariate regression analysis of the data from 38 PC patients and 32 controls, twelve distinguishing metabolites (alanine, choline, citrate, creatinine, glucose, glutamine, glutamic acid, 3- hydroxybutyrate, lactate, lipids, methionine and valine) were determined based on their ranked importance in a partial least square discriminant analysis (PLS-DA) model. A cross-validated regression PLS-DA model built using these metabolites differentiated the cancer and control groups with high accuracy and an area under the receiver operating characteristic curve (AUROC) of 0.95. Notably, external validation of this model using the NMR data from a second, distinct set of samples (13 PC patients and 15 controls) collected approximately 1 year later showed an AUROC of 0.86, which represents very good performance compared to the current approaches for identifying pancreatic cancer patients. Metabolic changes in pancreatic cancer patients compared to healthy controls as shown in this study demonstrate the potential for the development of regression models based on blood metabolites to identify patients with pancreatic cancer.

Non-target Metabolomic Profiling of the Marine Microalgae Dunaliella tertiolecta After Exposure to Diuron using Complementary High- Resolution Analytical Techniques by Petra Booij, Marja H. Lamoree, Sascha B. Sjollema, Pim de Voogt, Jennifer E. Schollee, A. Dick Vethaak, Pim E.G. Leonards (213-222).
Traditionally, bioassays are used to assess the toxicity of chemicals. Bioassays often focus on one specific mode of action or end point and their responses offer a limited understanding of the health status and underlying pathways of the species under consideration. Metabolomics can be used to detect hundreds of metabolites in which each metabolite, or set of metabolites, represents short term and long term changes, indicating the status of the organism. The effects of the herbicide diuron, one of the compounds of concern for European water bodies, on the marine microalgae Dunaliella tertiolecta were investigated through non-target metabolomic profiling and bioassay testing. The pulse amplitude modulation (PAM) fluorometry bioassay was employed to measure the effective photosystem II efficiency (?PSII), while non-target metabolomic profiling using complementary analytical techniques characterized the metabolomic response in the algae during diuron exposure. The use of complementary analytical techniques was necessary to identify a broad range of metabolites. Twenty-eight compounds were identified as metabolites affected by diuron exposure, including several amino acids, adenosine, lactic acid, and citric acid. Collectively, these metabolites indicated that diuron negatively affects energy processes in the algae both at the citric acid cycle pathway as well as on the amino acid metabolism at realistic environmental concentrations. In addition, dose-response relationships were found between a number of affected metabolites and the inhibition of the ?PSII of D. tertiolecta. Non-target metabolomic profiling using complementary analytical techniques proved to have additional and complementary benefits to traditional toxicology tests.