Peak Area Integration Research Articles

SLIM-Based High-Resolution Ion Mobility Reveals New Structural Insights into Isomeric Vitamin D Metabolites and their Isotopologues.

Testing for vitamin D deficiency remains a high-volume clinical assay, much of which is done using mass spectrometry-based methods to alleviate challenges in selectivity associated with immunoassays. Ion mobility-mass spectrometry (IM-MS) has been proposed as a rapid alternative to traditional LC-MS/MS methods, but understanding the structural ensemble that contributes to the ion mobility behavior of this molecular class is critical. Herein we demonstrate the first application of high-resolution Structures for Lossless Ion Manipulations (SLIM) IM separations of several groups of isomeric vitamin D metabolites. Despite previous IM studies of these molecules, the high resolving power of SLIM (Rp ∼ 200) has revealed additional conformations for several of the compounds. The highly similar collision cross sections (CCS), some differing by as little as 0.7%, precluded adequate characterization with low-resolution IM techniques where, in some cases, wider than expected peak widths and/or subtle shoulders may have hinted at their presence. Importantly, these newly resolved peaks often provided a unique mobility that could be used to separate isomers and provides potential for their use in quantification. Lastly, the contribution of isotopic labeling to arrival time distribution for commonly used 13C- and deuterium-labeled internal standards was explored. Minor shifts of ∼0.2-0.3% were observed, and in some instances these shifts were specific to the conformer being measured (i.e., "closed" vs "open"). Accounting for these shifts is important during raw data extraction to ensure reproducible peak area integration, which will be a critical consideration in future quantitative applications.

Structural equation model was used to evaluate the effects of soil chemical environment, fertility and enzyme activity on eucalyptus biomass.

This paper aims to reveal the effects of multi-generational succession of eucalyptus on soil fertility, organic structure and biological properties. Soil samples were collected from eucalyptus plantations of different stand ages (5, 11, 17 and 21 years old) in a typical area in south Asia, soil organic fraction structure and content characteristics were investigated using Fourier transform infrared (FTIR), and structural equation modelling (SEM) was used to explore influences of soil fertility, enzyme activity and organic fraction on stand biomass. FTIR analysis showed that 11 infrared absorption peaks existed in the soils of this study area, attributed to silicates, aromatics, carbonate ions, sugars, esters, polysaccharides, aliphatic hydrocarbons and phenolic alcohols. Combined with the results of peak area integration, the content of esters, aromatics and phenolic alcohols was significantly higher in 17- and 21-year-old stand soils than in control soils. The results of SEM showed that organic components were negatively related (p < 0.01) to enzyme activity and biomass, with standardized coefficients of 0.53 and 0.49, respectively. In summary, multi-generation succession of eucalyptus trees can change the structure of soil organic functional group composition and promote the enrichment of aromatic and phenolic alcohol functional groups. Such changes can directly inhibit the increase in eucalyptus biomass and indirectly negatively affect biomass by inhibiting enzyme activity.

Comprehensive Size Distribution and Composition Analysis of Adeno-Associated Virus Vector by Multiwavelength Sedimentation Velocity Analytical Ultracentrifugation.

During the manufacturing of recombinant adeno-associated virus vectors, it is generally difficult to purify out vectors that lack nucleic acids (empty particles, EPs), contain incomplete nucleic acids (intermediate particles, IPs) or aggregates. These impurities may cause side effects and therefore it is essential to both quantify and reduce them; however, comprehensive identification of the size distribution and components of virus vectors have been lagging. We developed multiwavelength sedimentation velocity analytical ultracentrifugation to characterize EPs, full particles, IPs, and aggregates in adeno-associated virus vector samples. The wavelength-dependent ultraviolet (UV) absorption of capsid protein and encapsulated single-stranded DNA could be deduced from the multiwavelength detection followed by size distribution analysis and peak area integration. Subsequently, a spectral deconvolution analysis using the wavelength-dependent UV absorption data enabled the identification of the protein-nucleic acid ratio of all species. A comprehensive approach for quantifying the viral vector particles and related impurities was established.

Screening of Volatile Compounds in Mate (Ilex paraguariensis) Tea—Brazilian Chimarrão Type—By HS-SPDE and Hydrodistillation Coupled to GC-MS

The volatile fraction of mate (Ilex paraguariensis) tea—specifically Brazilian chimarrão type, which has an odor profile comprising distinctive fresh, green, grass, and herbal notes—was investigated. Hydrodistillation in a Clevenger apparatus was employed in order to extract volatiles from the tea matrix. Headspace–solid-phase dynamic extraction (HS-SPDE) was employed to extract the volatiles from two types of infusions of this tea—a simple single infusion and a traditional preparation of consecutive infusions. Volatiles were analyzed by gas chromatography–flame ionization detection/mass spectrometry (GC-FID/MS). In total, 85 compounds were either identified or tentatively identified and semi-quantified. Semi-quantification comprised peak area integration of all the peaks (including the unidentified ones) in the chromatogram. Results obtained by hydrodistillation and by HS-SPDE were distinct, covering mostly different ranges of volatility and showing only 15 compounds in common. The identified compounds had their respective average and minimum odor thresholds and odor characteristics compiled from the literature. Several major compounds considered as key odorants in other mate tea products were not detected or only present at low levels in the samples of this research. Approximately half of the odorants identified in these samples were commonly reported in different mate tea types; the remaining 41 molecules—predominantly terpenoids (isoprenoids)—could be listed as specific to the Brazilian chimarrão type and are suggested to underlie its typical freshness.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Dynamics of Collisions and Adsorption in the Stochastic Electrochemistry of Emulsion Microdroplets.

Current-time recordings of emulsified toluene microdroplets containing 20 mM Ferrocene (Fc), show electrochemical oxidation peaks from individual adsorption events on disk microelectrodes (5 μm diameter). The average droplet diameter (∼0.7 μm) determined from peak area integration was close to Dynamic Light Scattering measurements (∼1 μm). Random walk simulations were performed deriving equations for droplet electrolysis using the diffusion and thermal velocity expressions from Einstein. The simulations show that multiple droplet-electrode collisions, lasting ∼0.11 μs each, occur before a droplet wanders away. Updating the Fc-concentration at every collision shows that a droplet only oxidizes ∼0.58% of its content in one collisional journey. In fact, it would take ∼5.45 × 106 collisions and ∼1.26 h to electrolyze the Fc in one droplet with the collision frequency derived from the thermal velocity (∼0.52 cm/s) of a 1 μm-droplet. To simulate adsorption, the droplet was immobilized at first contact with the electrode while the electrolysis current was computed. This approach along with modeling of instrumental filtering, produced the best match of experimental peaks, which were attributed to electrolysis from single adsorption events instead of multiple consecutive collisions. These results point to a heightened sensitivity and speed when relying on adsorption instead of collisions. The electrochemical current for the former is limited by the probability of adsorption per collision, whereas for the latter, the current depends on the collision frequency and the probability of electron transfer per collision (J. Am. Chem. Soc. 2017, 139, 16923-16931).

Quantification of uranium, plutonium, neodymium and gadolinium for the characterization of spent nuclear fuel using isotope dilution HPIC-SF-ICP-MS

A method was developed for the determination of the nuclide-specific concentrations of U, Pu, Nd and Gd in two types of spent nuclear fuel (UOx and Gd-enriched). High-performance ion chromatography (HPIC) was used to separate the target elements from one another while sector-field inductively coupled plasma-mass spectrometry (SF-ICP-MS) was used for their determination relying on isotope dilution for calibration. In order to obtain the best possible precision for these isotope ratios extracted from the transient HPIC-SF-ICP-MS signals, the SF-ICP-MS data acquisition parameters were optimized and the most suitable method for calculating the isotope ratios from the transient signals was identified. The point-by-point (PbP), linear regression slope (LRS) and peak area integration (PAI) approaches were compared in the latter context. It was found that data acquisition in the flat centre of the spectral flat top peak using a mass window of 25%, a dwell time of 10 ms and 20 samples per peak, while using PAI for isotope ratio calculation, gave the best precision on the isotope ratios extracted from the HPIC-SF-ICP-MS transient signals. These parameters were used in the determination of the nuclide-specific mass fractions of Pu, Nd and Gd in two types of spent nuclear fuel using isotope dilution HPIC-SF-ICP-MS. For U, which was present at a higher concentration, the element fraction was collected and analyzed off-line after dilution. For the other target elements, an online approach was used. An uncertainty budget estimation was made using the bottom-up approach for the resulting mass fractions, and the accuracy and precision obtained when using isotope dilution HPIC-SF-ICP-MS were compared with those obtained with the routinely used techniques, isotope dilution TIMS & alpha spectrometry (an ISO 17025 accredited method).

A Metabolomics Workflow for Analyzing Complex Biological Samples Using a Combined Method of Untargeted and Target-List Based Approaches.

In the highly dynamic field of metabolomics, we have developed a method for the analysis of hydrophilic metabolites in various biological samples. Therefore, we used hydrophilic interaction chromatography (HILIC) for separation, combined with a high-resolution mass spectrometer (MS) with the aim of separating and analyzing a wide range of compounds. We used 41 reference standards with different chemical properties to develop an optimal chromatographic separation. MS analysis was performed with a set of pooled biological samples human cerebrospinal fluid (CSF), and human plasma. The raw data was processed in a first step with Compound Discoverer 3.1 (CD), a software tool for untargeted metabolomics with the aim to create a list of unknown compounds. In a second step, we combined the results obtained with our internally analyzed reference standard list to process the data along with the Lipid Data Analyzer 2.6 (LDA), a software tool for a targeted approach. In order to demonstrate the advantages of this combined target-list based and untargeted approach, we not only compared the relative standard deviation (%RSD) of the technical replicas of pooled plasma samples (n = 5) and pooled CSF samples (n = 3) with the results from CD, but also with XCMS Online, a well-known software tool for untargeted metabolomics studies. As a result of this study we could demonstrate with our HILIC-MS method that all standards could be either separated by chromatography, including isobaric leucine and isoleucine or with MS by different mass. We also showed that this combined approach benefits from improved precision compared to well-known metabolomics software tools such as CD and XCMS online. Within the pooled plasma samples processed by LDA 68% of the detected compounds had a %RSD of less than 25%, compared to CD and XCMS online (57% and 55%). The improvements of precision in the pooled CSF samples were even more pronounced, 83% had a %RSD of less than 25% compared to CD and XCMS online (28% and 8% compounds detected). Particularly for low concentration samples, this method showed a more precise peak area integration with its 3D algorithm and with the benefits of the LDAs graphical user interface for fast and easy manual curation of peak integration. The here-described method has the advantage that manual curation for larger batch measurements remains minimal due to the target list containing the information obtained by an untargeted approach.

Soil sample drying temperature affects specific organic mid-DRIFTS peaks and quality indices

Peak area (PA) integration of diffuse reflectance mid infrared Fourier transform spectroscopy (DRIFTS) spectra is useful to study soil organic matter (SOM) quality and as a potential modelling proxy. Residual water of soil samples after drying affects DRIFTS spectra, mainly in the region >2500 cm−1. Therefore, SOM quality related PA at different wavenumbers should be influenced to varying degrees. We studied how absolute, normalized and relative PA related to SOM functional groups (at 2930, 1620, 1530 and 1159 cm−1), as well as their ratios were influenced by oven drying of soil samples at increasing temperatures (from 32 °C to 105 °C). All organic and even mineral associated PA significantly increased with drying temperature. The PA of aliphatic CH stretching (2930 cm−1), located on the shoulder of the broad OH stretching PA, was influenced most strongly. Our results indicate that using 105 °C as drying temperature and storing samples in a desiccator is the best way to minimize water interference. These findings apply to relative PA and ratios of organic compounds, while no effect of drying temperatures on PA correlations to C or N were found.

Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform.

In the field of quantitative proteomics, the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In the present study, after peptide and protein identification, we compared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (ProteinPilot) to the alternative method based on the calculation of ratios of the sum of peak intensities (jTRAQx [Quant]) and we processed output data with the in-house Customizable iTRAQ Ratios Calculator (CiR-C) algorithm. Quantitation based on peak area ratios displayed no significant linear correlation with Western blot quantitation. In contrast, quantitation based on the sum of peak intensities displayed a significant linear association with Western blot quantitation (non-zero slope; Pearson correlation coefficient test, r = 0.296, P=0.010**) with an average bias of 0.087 ± 0.500 and 95% Limits of Agreement from −0.893 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing adjunct to the iTRAQ technology.

A Survey of Orbitrap All Ion Fragmentation Analysis Assessed by an R MetaboList Package to Study Small-Molecule Metabolites

Leukemia cell and melanoma tumor tissue extracts were studied for small (mostly m/z < 250) polar metabolites by LC-ESI-HRMSn analysis powered by a hybrid Quadrupole-Orbitrap. MS data were simultaneously acquired in fast polarity switching mode operating in MS1 and MS/MS (All Ion Fragmentation, AIF) full-scan analyses at high mass resolution. Positive metabolite assignments were achieved by AIF analysis considering at least two characteristic transitions. Targeted metabolite profiling was achieved by the relative quantification of 18 metabolites through spiking of their respective deuterated counterparts. Manual data processing of MS1 and AIF scans were compared for the accurate determination of natural metabolites and their deuterated analogs by chromatographic alignment and peak area integration. Evaluation of manual and automated (MetaboList R package) AIF data processing yielded comparable results. The versatility of AIF analysis also enabled the untargeted metabolite profiling of leukemia and melanoma samples in which 22 and 53 compounds were, respectively, identified outside those studied by labeling. The main limitation of this method was that low abundance metabolites with scan rates below 8 scans/peak could not be accurately quantified by AIF analysis. The combination of AIF analysis with MetaboList R package represents an opportunity to move towards automated, faster, and more global metabolomics approaches supported by an entirely flexible open source data processing platform freely available from Comprehensive R Archive Network (CRAN, https://CRAN.R-project.org/package=MetaboList ).

Prediction of peroxide value in omega-3 rich microalgae oil by ATR-FTIR spectroscopy combined with chemometrics.

Our work explored, for the first time, monitoring peroxide value (PV) of omega-3 rich algae oil using ATR-FTIR spectroscopic technique. The PV of the developed method was compared by that obtained by standard method of Association of Official Analytical Chemists (AOAC). In this study, peak area integration (PAI), Partial Least Squares Regression (PLSR), and Principal Component Regression (PCR) were used as the calibration techniques. PV obtained by the AOAC method and by FTIR-ATR technique were well correlated considering the peak area related to trans double bonds and chemometrics techniques of PLSR and PCR. Calibration model was established using the band with a peak point at 966cm-1 (990-940cm-1) related to CH out of plane deformation vibration of trans double bond. Algae oil oxidation could be successfully quantified using PAI, PLSR and PCR techniques. Additionally, hierarchical cluster analysis was performed and significant discrimination was observed coherently with oxidation process.

Influence of solvent chemistry on 1H NMR spectral and relaxation properties of a long-chain ionic surfactant in chloroform-d

1H NMR chemical shift, line width, indirect nuclear splitting value, peak area integration value, and spin–lattice and spin–spin relaxation times at 298 K are compared for low-concentration isotropic solutions of n-octylammonium n-octadecanoate prepared via different techniques and conditions using dried, distilled, and degassed deuterochloroform and the nontreated solvent containing tetramethylsilane. The nature of the variation of observed spectral parameters and relaxation/rotational behavior with chemical composition (presence of oxygen and other paramagnetic species, stabilizer, impurities, and degradation products) of the solvent, history of the solution, and sample containment are analyzed. Relaxation times are interpreted in terms of monomer structure and reorientation and internal rotation modes as a function of atomic position along the n-alkyl chains. Collectively, the relaxation behavior of the surfactant complies with the two-step model of fast picosecond internal rotations of different size segments containing methylene groups separated in timescale from slower large segment and overall molecular tumbling modes of the monomer. Fast motional phenomena do not appear to be appreciably influenced by the chemistry of the solvent in contrast to spectral parameters such as chemical shift and line width of the labile ammonium protons. A model is also presented to explain anomalous variation of the peak area integration value with chemical shift of the ammonium resonance peak.

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCl (110°C for 24h), 4.2M NaOH (105°C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60°C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25°C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25°C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal).

Effect of 1-substituted imidazole derivatives for the curing process of epoxy- -isocyanate composition

Abstract The kinetics of the curing process of isocyanate-epoxy materials hardened in the presence of 1- substituted imidazole derivatives was studied by the Coast-Redfern method. The extent of a conversion parameter of the curing process in two ways was calculated: DSC (peak area integration) and rheology (viscosity changes). The activation energy values were determined for epoxy-isocyanate cured in the presence of 0.5; 1.0 and 2.0 phr 1-substituted imidazole derivatives respectively. Increasing of accelerators amount results in decreasing the activation energy and other kinetic parameters.

Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress

A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into proteome changes that underlie the remodeling of tilapia gill epithelium in response to environmental salinity change.

Software Solution for Post-Column Isotope Dilution Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry

The application of isotope dilution in high performance liquid chromatography (HPLC) and inductively-coupled plasma mass spectrometry (ICP-MS) represents an important tool for elemental speciation analysis. Currently commercially available software deals only with one type of HPLC-ID-ICP-MS measurement comprising an addition of isotope-enriched species into the sample before HPLC separation, that is, species-specific isotope dilution. However, there is another possibility: species-unspecific isotope dilution, which is, unfortunately, not easily employed by common software. In this method, the isotope enriched solution containing common inorganic compound of the analyzed element is added to the sample after HPLC separation. Here a novel software system addresses this problem. The most important features of the computer program include easy operation, data adjustment by smoothing, detector dead-time correction, mass discrimination correction, background correction, integration of peak area and ID enumeration, processing of multi-elemental analyses, a choice of various isotopes, and a database of isotope abundances. The reliability of the software was demonstrated by processing of artificial chromatographic data and real chromatograms of standard reference materials containing mercury species and mixtures of selenium species.

Quantitative analysis of 5-HMF produced from fructose

Quantitative analysis of 5-hydroxymethylfufural (5-HMF) conversion from fructose by dehydration and rearrangement was investigated by <TEX>$^1H$</TEX>-NMR spectroscopic method. Fructose was converted to 5-HMF in dimethylsulfoxide (DMSO)-<TEX>$d^6$</TEX> or acidic deuterium hydroxide at controlled reaction temperature and time. With addition of internal standards (biphenyl for DMSO-<TEX>$d^6$</TEX> solvent, and 2,5-dihydroxybenzoic acid for deuterium oxide solvent), conversion from fructose to 5-HMF was analyzed by <TEX>$^1H$</TEX>-NMR spectroscopy. Quantitative analysis was run by comparison with peak area integration between of 5-HMF and internal standard. In DMSO solvent, 5-HMF was stable end product but part of 5-HMF was converted to formic and levulinic acid at acidic aqueous medium.

비알콜성 지방간환자 평가를 위한 3.0T 고자기장 자기공명분광법을 이용한 PRESS 기법의 유용성

3.0T 고자기장 자기공명분광법에서 비알콜성 지방간 환자 평가를 위해 PRESS 기법과 Biopsy결과의 상관관계를 토대로 PRESS 기법의 임상적 유용성을 알아보았다. 본 실험은 3.0T 고자지장 자기공명영상 장비를 이용하여 실험을 하였고 분광스펙트럼을 적분(peak area integration) 방법으로 면적을 구하여 간지방량에 상대비율에 따른 공명피크의 면적을 이용하여 계산하였다. 비알콜성 지방간환자에서 MR 스펙트럼 피크가 모두 0.9 - 1.6 ppm에서 나타났으며 비알콜성 환자에 대하여 MRS와 Biopsy 결과 민감도 94%와 특이도 80%로 높은 값을 얻었다(p=0.000). 정상(r=0.79, p=0.04) 및 비알콜성 지방간질환 대한 결과( r=0.89, p=0.002) 역시 높은 상관관계를 보여주었다. 따라서 3.0T 고자장 MRS을 적용했을 때 PRESS 기법은 비알콜성 지방간 환자를 평가하는데 있어서 정상부분과 지방간부분의 구별을 가능하게 하였으며 생검검사와 MRS의 상관관계 분석에서 간지방량 비율(%)은 r값이 0.7 이상의 높은 Lipid를 제공하여 PRESS 기법의 임상적 유용성을 권고 할 만하였다. This study was to evaluate the clinical usefulness of the PRESS technique based on the correlation between PRESS technique and biopsy results by applying 3.0T high magnetic field MRS technique for evaluation of non-alcoholic fatty liver disease patients. This experiment were carried out using a 3.0T magnetic resonance imaging equipment. The part data of each spectrum is taken by peak area integration. The part data of resonance peak was used to calculate relative ratio. MR spectral peak in patients with non-alcoholic fatty liver disease is from 0.9 to 1.6 ppm. According to MRS method study result, Patients with NAFLD were obtained with 94% sensitivity and 80% specificity(p=0.000). When compared to normal based on MRS and Biopsy results was valid correlation(r=0.79, p=0.04). Results for NAFLD(r=0.89, p=0.002) also showed a correlation. Therefore, PRESS technique to evaluate patients with non-alcoholic fatty liver disease, the distribution difference between normal liver and fatty liver part is significantly distinguished. Biopsy and MRS fatty liver peak ratio(%) proves high lipid over grade(r = 0.7).

467 QUANTITATIVE PROTEOMIC ANALYSIS OF EXTRACELLULAR MICROVESICLES IN AN IN VITRO MODEL OF URINARY TRACT OBSTRUCTION

You have accessJournal of UrologyPediatrics: Congenital Anomalies - Kidney & Ureter1 Apr 2012467 QUANTITATIVE PROTEOMIC ANALYSIS OF EXTRACELLULAR MICROVESICLES IN AN IN VITRO MODEL OF URINARY TRACT OBSTRUCTION Dennis Orton, Alan Doucette, Geoffrey Maksym, and Dawn MacLellan Dennis OrtonDennis Orton Halifax, Canada More articles by this author , Alan DoucetteAlan Doucette Halifax, Canada More articles by this author , Geoffrey MaksymGeoffrey Maksym Halifax, Canada More articles by this author , and Dawn MacLellanDawn MacLellan Halifax, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.535AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Congenital hydronephrosis affects up to 1% of children. An in vitro model of urinary traction obstruction (UTO) using renal proximal tubule cells (NRK-52E) subjected to mechanical stretch has helped to elucidate the molecular pathology of UTO (Nguyen, et al., Nephron. Exp. Nephrol., 2006). Renal proximal tubule cells have been suggested to release protein-containing extracellular microvesicles (exosomes) into the urinary space that reflect the proteomic profile of these cells in vivo. The goal of this study was to confirm that proximal tubule cells generate extracellular microvesicles in vitro, and characterize specific changes in protein abundance in these microvesicles that result from mechanical stretch using quantitative proteomics. METHODS NRK-52E cells were grown on BioFlex plates and subjected to 24 hours of stretch-relaxation, with a maximum of 20% maximal biaxial stretch on a FX-4000 Flexercell Strain Unit (Flexcell, Hillsborough, NC, USA). The efficacy of UTO simulation is measured by cell death ELISA (Roche, Indianapolis, IN, USA). Microvesicles are isolated by ultracentrifugation from the cell culture media. Relative protein abundance is then quantitatively measured by spectral counting following mass spectrometry using a ThermoFisher LTQ linear ion trap mass spectrometer with 5 replicates. RESULTS Microvesicle isolation was confirmed by electron microscopy and immunoblotting for a known exosomal protein, Annexin 1. On average, stretched cells showed a 3 fold increase in cell death. Quantitative analysis reveals 25 proteins with altered abundance as a result of stretch. Histone proteins H4, H2B-291, H2b-61, H3 and H1 had the most significant changes in abundance, while other proteins, gelsolin, nebulin, haptoglobin, an integral membrane serine 2 protease, and polyubiquitin were also noted to have significantly altered abundances in stretch cell extracellular microvesicles (p-value <0.05 by student’s t-test). Fibronectin 1 (FN1) abundance was investigated by peak area integration, and found to be slightly increased in stretched cell microvesicles. CONCLUSIONS Renal proximal tubule cells generate extracellular microvesicles whose protein profile is altered in an in vitro model of UTO. Quantitative proteomic analysis of these extracellular microvesicles demonstrates proteins important in apoptosis, the cytoskeleton, and cellular response to oxidative stress are altered in abundance after mechanical stretch, providing insight into the pathophysiology of UTO. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e191 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Dennis Orton Halifax, Canada More articles by this author Alan Doucette Halifax, Canada More articles by this author Geoffrey Maksym Halifax, Canada More articles by this author Dawn MacLellan Halifax, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...