Consequences of Lipid Remodeling of Adipocyte Membranes Being Functionally Distinct from Lipid Storage in Obesity.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (ob/ob), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.

Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid, free fatty acids and amino acid levels.

BACKGROUND: Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn's disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression. OBJECTIVES: The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples. METHODS: A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis. RESULTS: Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease. CONCLUSIONS: Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.

Metabonomics of newborn screening dried blood spot samples: a novel approach in the screening and diagnostics of inborn errors of metabolism.

A novel, single stage high resolution mass spectrometry-based method is presented for the population level screening of inborn errors of metabolism. The approach proposed here extends traditional electrospray tandem mass spectrometry screening by introducing nanospray ionization and high resolution mass spectrometry, allowing the selective detection of more than 400 individual metabolic constituents of blood including acylcarnitines, amino acids, organic acids, fatty acids, carbohydrates, bile acids, and complex lipids. Dried blood spots were extracted using a methanolic solution of isotope labeled internal standards, and filtered extracts were electrosprayed using a fully automated chip-based nanospray ion source in both positive and negative ion mode. Ions were analyzed using an Orbitrap Fourier transformation mass spectrometer at nominal mass resolution of 100,000. Individual metabolic constituents were quantified using standard isotope dilution methods. Concentration threshold (cutoff) level-based analysis allows the identification of newborns with metabolic diseases, similarly to the traditional electrospray tandem mass spectrometry (ESI-MS/MS) method; however, the detection of additional known biomarkers (e.g., organic acids) results in improved sensitivity and selectivity. The broad range of detected analytes allowed the untargeted multivariate statistical analysis of spectra and identification of additional diseased states, therapeutic artifacts, and damaged samples, besides the metabolic disease panel.

Analysis of colorectal adenocarcinoma tissue by desorption electrospray ionization mass spectrometric imaging.

Negative ion desorption electrospray ionization (DESI) was used for the analysis of an ex vivo tissue sample set comprising primary colorectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples. Frozen sections (12 µm thick) were analyzed by means of DESI imaging mass spectrometry (IMS) with spatial resolution of 100 µm using a computer-controlled DESI imaging stage mounted on a high resolution Orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ceramides, sphingolipids, and sulfatides among others. Molecular constituents were identified based on their exact mass and MS/MS fragmentation spectra. An identified set of molecules was found to be in good agreement with previously reported DESI imaging data. Different histological tissue types were found to yield characteristic mass spectrometric data in each individual section. Histological features were identified by comparison to hematoxylin-eosin stained neighboring sections. Ions specific to certain histological tissue types (connective tissue, smooth muscle, healthy mucosa, healthy liver parenchyma, and adenocarcinoma) were identified by semi-automated screening of data. While each section featured a number of tissue-specific species, no potential global biomarker was found in the full sample set for any of the tissue types. As an alternative approach, data were analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) which resulted in efficient separation of data points based on their histological types. A pixel-by-pixel tissue identification method was developed, featuring the PCA/LDA analysis of authentic data set, and localization of unknowns in the resulting 60D, histologically assigned LDA space. Novel approach was found to yield results which are in 95% agreement with the results of classical histology. KRAS mutation status was determined for each sample by standard molecular biology methods and a similar PCA/LDA approach was developed to assess the feasibility of the determination of this important parameter using solely DESI imaging data. Results showed that the mutant and wild-type samples fully separated. DESI-MS and molecular biology results were in agreement in 90% of the cases.

Electrospray post-ionization mass spectrometry of electrosurgical aerosols.

The feasibility of electrospray (ES) ionization of aerosols generated by electrosurgical disintegration methods was investigated. Although electrosurgery itself was demonstrated to produce gaseous ions, post-ionization methods were implemented to enhance the ion yield, especially in those cases when the ion current produced by the applied electrosurgical method is not sufficient for MS analysis. Post-ionization was implemented by mounting an ES emitter onto a Venturi pump, which is used for ion transfer. The effect of various parameters including geometry, high voltage setting, flow parameters, and solvent composition was investigated in detail. Experimental setups were optimized accordingly. ES post-ionization was found to yield spectra similar to those obtained by the REIMS technique, featuring predominantly lipid-type species. Signal enhancement was 20- to 50-fold compared with electrosurgical disintegration in positive mode, while no improvement was observed in negative mode. ES post-ionization was also demonstrated to allow the detection of non-lipid type species in the electrosurgical aerosol, including drug molecules. Since the tissue specificity of the MS data was preserved in the ES post-ionization setup, feasibility of tissue identification was demonstrated using different electrosurgical methods.

Real time analysis of brain tissue by direct combination of ultrasonic surgical aspiration and sonic spray mass spectrometry.

Direct combination of cavitron ultrasonic surgical aspirator (CUSA) and sonic spray ionization mass spectrometry is presented. A commercially available ultrasonic surgical device was coupled to a Venturi easy ambient sonic-spray ionization (V-EASI) source by directly introducing liquified tissue debris into the Venturi air jet pump. The Venturi air jet pump was found to efficiently nebulize the suspended tissue material for gas phase ion production. The ionization mechanism involving solely pneumatic spraying was associated with that of sonic spray ionization. Positive and negative ionization spectra were obtained from brain and liver samples reflecting the primary application areas of the surgical device. Mass spectra were found to feature predominantly complex lipid-type constituents of tissues in both ion polarity modes. Multiply charged peptide anions were also detected. The influence of instrumental settings was characterized in detail. Venturi pump geometry and flow parameters were found to be critically important in ionization efficiency. Standard solutions of phospholipids and peptides were analyzed in order to test the dynamic range, sensitivity, and suppression effects. The spectra of the intact tissue specimens were found to be highly specific to the histological tissue type. The principal component analysis (PCA) and linear discriminant analysis (LDA) based data analysis method was developed for real-time tissue identification in a surgical environment. The method has been successfully tested on post-mortem and ex vivo human samples including astrocytomas, meningeomas, metastatic brain tumors, and healthy brain tissue.

In situ, real-time identification of biological tissues by ultraviolet and infrared laser desorption ionization mass spectrometry.

Laser desorption ionization-mass spectrometric (LDI-MS) analysis of vital biological tissues and native, ex vivo tissue specimens is described. It was found that LDI-MS analysis yields tissue specific data using lasers both in the ultraviolet and far-infrared wavelength regimes, while visible and near IR lasers did not produce informative MS data. LDI mass spectra feature predominantly phospholipid-type molecular ions both in positive and negative ion modes, similar to other desorption ionization methods. Spectra were practically identical to rapid evaporative ionization MS (REIMS) spectra of corresponding tissues, indicating a similar ion formation mechanism. LDI-MS analysis of intact tissues was characterized in detail. The effect of laser fluence on the spectral characteristics (intensity and pattern) was investigated in the case of both continuous wave and pulsed lasers at various wavelengths. Since lasers are utilized in various fields of surgery, a surgical laser system was combined with a mass spectrometer in order to develop an intraoperative tissue identification device. A surgical CO(2) laser was found to yield sufficiently high ion current during normal use. The principal component analysis-based real-time data analysis method was developed for the quasi real-time identification of mass spectra. Performance of the system was demonstrated in the case of various malignant tumors of the gastrointestinal tract.

Intact skin analysis by desorption electrospray ionization mass spectrometry.

In vivo skin analysis by Desorption Electrospray Ionization was characterized on healthy human volunteers by directing pneumatically assisted electrospray directly onto their fingertips. In order to eliminate the risk of electric shock, a high ohmic resistor was built into the system. Positive ion DESI-MS analysis yields low intensity spectra, while negative ion spectra feature a number of various biogenic carboxylic acids. Compounds of external origin and excreted molecules were found to have different analysis kinetics, with the exception of highly hydrophobic species. The difference was demonstrated in the case of nicotine and cotinine. Pharmacokinetic studies were performed using a rat animal model. The kinetics of the anesthetic ketamine was followed by DESI, and results were in agreement with off-line HPLC-MS blood analysis. Using a similar approach for N,N'-dimethylthiourea (DMTU), a novel method was developed for the real-time quantification of oxidative stress. DMTU was administered to the animals, and the ratio of the molecule and its oxidized form was monitored from the skin surface. The ratio was found to be highly sensitive to experimentally induced diabetes mellitus type I and angiotensin-induced chronic oxidative stress. It was concluded that the method has a number of potential applications in the fields of forensics, pharmacology and clinical chemistry.

Identification of biological tissues by rapid evaporative ionization mass spectrometry.

The newly developed rapid evaporative ionization mass spectrometry (REIMS) provides the possibility of in vivo, in situ mass spectrometric tissue analysis. The experimental setup for REIMS is characterized in detail for the first time, and the description and testing of an equipment capable of in vivo analysis is presented. The spectra obtained by various standard surgical equipments were compared and found highly specific to the histological type of the tissues. The tissue analysis is based on their different phospholipid distribution; the identification algorithm uses a combination of principal component analysis (PCA) and linear discriminant analysis (LDA). The characterized method was proven to be sensitive for any perturbation such as age or diet in rats, but it was still perfectly suitable for tissue identification. Tissue identification accuracy higher than 97% was achieved with the PCA/LDA algorithm using a spectral database collected from various tissue species. In vivo, ex vivo, and post mortem REIMS studies were performed, and the method was found to be applicable for histological tissue analysis during surgical interventions, endoscopy, or after surgery in pathology.

Analysis of biological fluids by direct combination of solid phase extraction and desorption electrospray ionization mass spectrometry.

A novel, solid phase extraction (SPE)-based sample preparation method was developed for desorption electrospray ionization (DESI) mass spectrometry. Conventional SPE sample preparation was followed by a custom elution procedure. The eluate was evaporated from the closing frit of the cartridge using a gas jet. Thus the analyte was concentrated on the surface of the frit, which is ideal for DESI analysis. Application of the above SPE protocol allowed the concentration of the analyte content of up to 1 L liquid sample into a 1 mm diameter circular spot. The sample preparation procedure can improve the overall sensitivity of the method by up to 6 orders of magnitude if the sample volume is sufficient. The device has been tested using aqueous solutions of Rhodamine 116; the limit of detection was comparable to the LOD of electrospray analysis. Methodology was tested for drug monitoring applications in human serum. Levels of Cyclosporine A were determined using a 0.1 mL serum sample. Dynamic range of the method exceeded 3 orders of magnitude; the detection limit was below the therapeutic serum concentration of the drug.