Oxylipin profiling in endothelial cells in vitro - Effects of DHA and hydrocortisone upon an inflammatory challenge.

Omega-3 poly-unsaturated fatty acids have been shown to have beneficial effects on several inflammatory-driven endpoints such as cardiovascular diseases. The anti-inflammatory effects of docosahexaenoic acid (DHA) are largely mediated through various oxylipins. Yet, mechanistic insights are limited. Here, we measured 53 oxylipins using LC-MS/MS in an in vitro model of endothelial cell inflammation, and compared the changes induced by DHA to hydrocortisone, a well-established anti-inflammatory drug. DHA modified several oxylipins derived from different precursors such as DHA, AA, LA and EPA. In response to a TNFα and IL-1-ß challenge, DHA clearly reduced many COX-derived pro-inflammatory oxylipins, yet to a minor extent when compared to hydrocortisone. DHA also upregulated metabolites from the CYP and LOX pathways as opposed to hydrocortisone. Thus, DHA reduced pro-inflammation and enhanced pro-resolution, while hydrocortisone blunted both the pro- and anti-inflammatory pathways. Our results may fuel further research on the mitigation of corticosteroids adverse side-effects.

High-Resolution Mass Spectrometry Quantification: Impact of Differences in Data Processing of Centroid and Continuum Data.

High-resolution mass spectrometry (HRMS) in full scan mode acquires all ions present in the sample of interest offering a lot of qualitative information. This, in combination with the improved performance of the new generation HRMS systems, triggers more (bio) analysts to switch from triple quad MS systems to HRMS for quantitative analysis. Quantitative processing of HRMS data is performed based on narrow mass extraction windows rather than on nominal mass product ion chromatograms (SRM or MRM). Optimal processing of HRMS data requires different considerations and software tools and can have an impact on data processing and final results. The selection of centroid versus continuum/profile data for processing, selection of the optimal narrow mass extraction window, using theoretical versus measured accurate mass for the extraction of the ion chromatograms as well as differences in calculations and data handling residing in the different vendor software packages are tackled in the presented manuscript. These differences are illustrated on HRMS data acquired for the same plasma samples on three different platforms, i.e., a Sciex QToF, a Waters QToF, and a Thermo Orbitrap system, and processed in four different software packages, i.e., Sciex Analyst® TF, Waters Masslynx, Waters Unifi, and Thermo Xcalibur. The impact of these differences on quantitative HRMS performance was evaluated on calibration curves of eight small molecule compounds in plasma using four different ways of processing. Simple guidelines are provided for the selection of an optimal mass extraction window for continuum and centroided data. Graphical Abstract.

New software and breast boundary landmarks to calculate breast volumes from 3D surface images.

BACKGROUND: A method to accurately calculate breast volumes helps achieving a better breast surgery outcome. 3D surface imaging potentially allows these calculations in a harmless, quick, and practicable way. The calculated volume from a 3D surface image is dependent on the determined breast boundary and the method of chest wall simulation by software. Currently, there is no consensus on a robust set of breast boundary landmarks and validation studies on breast volume calculation software are scarce. The purposes of this study were to determine the robustness of newly introduced breast boundary landmarks and introduce and validate a new method to simulate a chest wall. METHODS: Sixteen subjects who underwent a unilateral simple mastectomy were included. In addition to the natural skin fold of the breast, the sternomanubrial joint, the transition of the pectoral muscle curve into the breast curvature, and the midaxillary line were used as landmarks to indicate the breast boundary. The intra- and interrater variability of these landmarks was tested. Furthermore, new chest wall simulation software was validated on the breastless chest side of the subjects. RESULTS: The intra- and interrater variability of the three breast boundary markers was small (mean 3.5-6.7 mm), and no significant difference was found between the intra- and interrater variability (p = 0.08, p = 0.06, and p = 0.10). The mean volume error of the most accurately simulated chest wall was 4.6 ± 37 ml. CONCLUSION: The newly introduced landmarks showed to be robust and our new chest wall simulation algorithm showed accurate results.Level of Evidence: Level IV, diagnostic study.

Accuracy and reproducibility of a newly developed tool for volume measurements of the arm using 3D stereophotogrammetry.

BACKGROUND: Lymph edema of the arm is a common complication after breast cancer treatment. To evaluate lymph edema volume and treatment outcome, an easy to use, objective quantification method of arm volume is necessary. Most often, water displacement is used to measure arm volume, as it is an easy and robust method that can be performed at any place with a simple equipment. However, when using water displacement, no exact localization of volume difference can be seen. To accurately measure hand and forearm volumes separately, an in-house-developed device was constructed for an accurate landmark placement. METHOD: An in-house developed measurement tool was used for placing artificial landmarks proximal to the wrist crease on ten healthy subjects. Three-dimensional (3D) images of the hand and forearm were acquired, and volume measurements of the hand and forearm were performed. Measurements were repeated to investigate the intra- and inter-rater variability caused by the landmark placement. RESULTS: Measuring volume of the hand and forearm while using artificially placed landmarks turned out to be a highly reproducible, quick, and easy procedure. Both intra- and inter-rater variability showed high reproducibility for hand (ICC = 0.96 and 0.98, respectively) and forearm (ICC = 0.99 and 0.99, respectively) volumes. CONCLUSION: Measuring volumes of the hand and forearm while using artificially placed landmarks was found to be a highly reproducible, quick, and easy procedure. The device enables to analyze the localization of lymph edema in greater details.

The effect of aging on the three-dimensional aspect of the hand: A pilot study.

BACKGROUND: With the increasing popularity of rejuvenation treatments of the skin, there has been an increase in patient inquiry for rejuvenation of the hand. Thus far, no imaging tool exists to analyze the aging process of the hand. Three-dimensional (3D) stereophotogrammetry is a reliable technique, which is used for other purposes in soft tissue analysis of the upper extremity. This pilot study aimed to investigate the possibility of visualizing the hand aging process by 3D stereophotogrammetry. METHODS: A total of 64 healthy volunteers were divided into four groups based on age and sex, and a 3D photograph of both hands was captured. Differences in the aspect of the dorsum of the hands were quantified and visualized using two methods. The first method quantified the smoothness of the old and young dorsa. The second method visualized the differences between an average young and old hand by creating a color-coded distance map. RESULTS: The first method showed that the young hands were smoother than the old hands; however, this difference was not statistically significant (p = 0.30). The distance map resulting from the second method showed a relative volume loss in the intermetacarpal spaces of the average old hand. These differences were not present when male hands were compared with female hands. CONCLUSIONS: This pilot study shows that 3D stereophotogrammetry can be used to visualize the exact areas of volume loss on the dorsum of the aging hand. On the basis of this finding, specific treatment areas can be identified, and the results of different esthetic hand surgery procedures can be objectively analyzed and compared.

Comprehensive metabolomics to evaluate the impact of industrial processing on the phytochemical composition of vegetable purees.

The effects of conventional industrial processing steps on global phytochemical composition of broccoli, tomato and carrot purees were investigated by using a range of complementary targeted and untargeted metabolomics approaches including LC-PDA for vitamins, (1)H NMR for polar metabolites, accurate mass LC-QTOF MS for semi-polar metabolites, LC-MRM for oxylipins, and headspace GC-MS for volatile compounds. An initial exploratory experiment indicated that the order of blending and thermal treatments had the highest impact on the phytochemicals in the purees. This blending-heating order effect was investigated in more depth by performing alternate blending-heating sequences in triplicate on the same batches of broccoli, tomato and carrot. For each vegetable and particularly in broccoli, a large proportion of the metabolites detected in the purees was significantly influenced by the blending-heating order, amongst which were potential health-related phytochemicals and flavour compounds like vitamins C and E, carotenoids, flavonoids, glucosinolates and oxylipins. Our metabolomics data indicates that during processing the activity of a series of endogenous plant enzymes, such as lipoxygenases, peroxidases and glycosidases, including myrosinase in broccoli, is key to the final metabolite composition and related quality of the purees.

Pharmacometabolomics reveals that serotonin is implicated in aspirin response variability.

While aspirin is generally effective for prevention of cardiovascular disease, considerable variation in drug response exists, resulting in some individuals displaying high on-treatment platelet reactivity. We used pharmacometabolomics to define pathways implicated in variation of response to treatment. We profiled serum samples from healthy subjects pre- and postaspirin (14 days, 81 mg/day) using mass spectrometry. We established a strong signature of aspirin exposure independent of response (15/34 metabolites changed). In our discovery (N = 80) and replication (N = 125) cohorts, higher serotonin levels pre- and postaspirin correlated with high, postaspirin, collagen-induced platelet aggregation. In a third cohort, platelets from subjects with the highest levels of serotonin preaspirin retained higher reactivity after incubation with aspirin than platelets from subjects with the lowest serotonin levels preaspirin (72 ± 8 vs. 61 ± 11%, P = 0.02, N = 20). Finally, ex vivo, serotonin strongly increased platelet reactivity after platelet incubation with aspirin (+20%, P = 4.9 × 10(-4), N = 12). These results suggest that serotonin is implicated in aspirin response variability.

Integrating metabolomics profiling measurements across multiple biobanks.

To optimize the quality of large scale mass-spectrometry based metabolomics data obtained from semiquantitative profiling measurements, it is important to use a strategy in which dedicated measurement designs are combined with a strict statistical quality control regime. This assures consistently high-quality results across measurements from individual studies, but semiquantitative data have been so far only comparable for samples measured within the same study. To enable comparability and integration of semiquantitative profiling data from different large scale studies over the time course of years, the measurement and quality control strategy has to be extended. We introduce a strategy to allow the integration of semiquantitative profiling data from different studies. We demonstrate that lipidomics data generated in samples from three different large biobanks acquired in the time course of 3 years can be effectively combined when using an appropriate measurement design and transfer model. This strategy paves the way toward an integrative usage of semiquantitative metabolomics data sets of multiple studies to validate biological findings in another study and/or to increase the statistical power for discovery of biomarkers or pathways by combining studies.

Transient inflammatory-like state and microbial dysbiosis are pivotal in establishment of mucosal homeostasis during colonisation of germ-free mice.

The gut microbiota is increasingly recognised as a key-player in defining the health status of the gastrointestinal tract. Recently, we demonstrated that colonisation of healthy germfree mice with a conventional microbiota (conventionalisation) elicits temporal and region specific host-microbe communication responses that lead to the establishment of a microbiota-accommodating homeostatic state within 30 days. Here, the microbiota composition profiles, mucosal transcriptomes and plasma-analytes in germ-free and conventionalised C57/BL 6 J mice were assessed to decipher the features of the distinctive and pivotal events occurring four days after initiation of the conventionalisation process. The dominance of the microbial genera Helicobacter, Sphingomonas and Mucispirillum in the gut microbiota coincided with the transient mounting of proinflammatory responses in the mucosa and the transiently elevated levels of specific (inflammatory) cytokines and amines in plasma. The overrepresented microbes have previously been associated with the potential to cause disease under certain conditions, illustrating that conventionalisation proceeds through a transient state that resembles situations associated with dysbiosis. However, no overt mucosal inflammation was observed, suggesting a pivotal role of the overrepresented bacterial groups in priming and maturation of the immune system during the process of conventionalisation. These findings imply that the transiently elevated relative overgrowth of particular microbial genera functions as pivotal adjuvants to elicit the corresponding proinflammatory cascades, which precede the full maturation of the different arms of the immune system following these events and is required to achieve a microbiota-accommodating homeostasis in healthy animals.

Three-dimensional stereophotogrammetrical analysis of peristomal fixation of adhesive base plates during hands-free speech among laryngectomised patients related to tracheostoma volumes.

OBJECTIVES: This study evaluates the relation between stoma volumes by means of objective three-dimensional measurements of laryngectomised individuals and peristomal fixation of adhesive baseplates during hands-free speech. DESIGN: A three-dimensional stereophotogrammetrical image was captured of the tracheostoma and its surrounding tissue for each participant. The placement of a baseplate adhesive was virtually simulated. Individual stoma volumes were calculated and related to the duration of lifetime of adhesive baseplates. SETTING: Prospective trial in a university hospital setting. PARTICIPANTS: Twenty-four laryngectomised patients. MAIN OUTCOME MEASURES: Stoma volume in cm(3) and adhesive baseplate adherence in minutes. RESULTS: For the 24 three-dimensional images captured, the mean volume of the area under the adhesive (stoma volume) was 9.5 cm(3) (range 3.5-22.5). After relating the different volumes of all patients to their individual fixation score, a significant decrease in duration of adhesive lifetime was found with increasing volumes of the stoma (P = 0.001). An increase of 1 cm(3) in tracheostoma volume resulted in a decrease of 21% in median adhesive lifetime (95% confidence interval 10-31%). CONCLUSIONS: Longer lifetime of the adhesive baseplate in patients with smaller volume outcomes suggests that a more accurate fit between baseplate and stoma leads to better fixation. This should be taken into account when shaping a stoma during laryngectomy. On the other hand, a more custom-made patch that has a more accurate fit could increase the lifetime of fixation. We believe that our three-dimensional volumetric data can contribute to the development of such an adhesive.

Determination of polar organophosphorus pesticides in aqueous samples by direct injection using liquid chromatography-tandem mass spectrometry.

It was demonstrated that four out of six of the very polar organophosphorus pesticides (OPs), i.e. acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl and vamidothion, could not be extracted from water using commonly available SPE cartridges. In addition, GC analysis on all six compounds was found to be troublesome due to their polar and thermolabile character. This initiated the development of an alternative highly sensitive and selective method for the determination of the above mentioned very polar OPs in water, based on LC-MS. Large volume (1 ml) water samples were directly injected onto an RP18 HPLC column with a polar endcapping. The latter was essential for obtaining retention and maintaining column performance under 100% aqueous conditions during the sampling. The compounds were ionized using atmospheric pressure chemical ionization and detected on a tandem mass spectrometer operated in multiple reaction-monitoring mode. The detection limits were in the range of 0.01-0.03 microg/l. Compared to conventional GC methods, the developed LC-MS procedure is very straightforward, fast and more reliable. This application demonstrates the applicability of LC-MS for analysis of polar OPs in surface, ground and drinking water, as a more favourable alternative to GC.

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides.

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.

Determination of chlormequat in pears by liquid chromotagraphy/mass spectrometry.

A straightforward and reliable method was developed for the determination of chlormequat in pears by liquid chromatography/mass spectrometry (LC/MS). Water and methanol were compared as extraction solvents. Because no significant differences in extraction efficiency or repeatability were found, water was chosen as the extraction solvent. The extracts were analyzed without cleanup by either an ion-trap liquid chromatograph/mass spectrometer in the single MS mode or a triple-quadrupole instrument in the MS/MS mode, using electrospray ionization. Both instruments were equally suitable for quantitation and confirmation of identity. Recoveries were 76-103%, and reproducibility was < or = 12%. The lowest detection limit (0.007 mg/kg) was obtained with the triple-quadrupole instrument in the MS/MS mode.

Determination of daminozide in apples and apple leaves by liquid chromatography--mass spectrometry.

A straightforward and efficient method was developed for the determination of intact daminozide in apples and apple leaves. After extraction with methanol and a clean-up step using a graphitized carbon cartridge, the extract was analysed by ion-trap liquid chromatography-tandem mass spectrometry (LC-MS-MS) using atmospheric pressure chemical ionisation in the positive ion mode. Recoveries for apple were 98-102% with a R.S.D. < or = 11% (n = 6) and for leaves were 112-116% with a R.S.D. < or = 18% (n = 6). The limits of detection were 0.008 and 0.02 mg/kg for apples and leaves, respectively.

Rapid target analysis of microcontaminants in water by on-line single-short-column liquid chromatography combined with atmospheric pressure chemical ionization tandem mass spectrometry.

The applicability of trace enrichment and separation of microcontaminants on a 10 mm x 2 mm I.D. high-pressure packed (8 microns C18 bonded silica or 10-15 microns PLRP-S) column combined on-line with an atmospheric pressure chemical ionization MS-MS system is demonstrated for the target analysis of herbicides in river water. Tailor-made procedures are obtained for a limited number of analytes by tuning the chromatographic efficiency of the short LC column and the specificity of tandem MS, in order to minimize the analysis time. With the on-line short-column LC-MS-MS method, good linearity is obtained for the herbicides in the range of 0.1-10 micrograms/l. The relative standard deviations of peak areas are less than 5% and, with only 4-ml samples, detection limits of 0.01-0.1 microgram/l can be achieved. The total analysis time is 10-15 min. The 10 mm x 2 mm I.D. LC columns packed with 8 microns particles show good stability and can be used for at least 40 analyses. Target compound analyses of river water allowed the confirmation of the presence of herbicides such as diuron, simazine, atrazine and terbutylazine at sub-microgram/l levels.

On-line post-column Diels-Alder derivatization for the determination of vitamin D3 and its metabolites by liquid chromatography/thermospray mass spectrometry.

Liquid chromatography/thermospray mass spectrometry (LC/TSP MS) has been used for the determination of vitamin D3 and some of its metabolites, i.e. 1 alpha(OH) vitamin D3, 25(OH) vitamin D3, 1 alpha,25(OH)2 vitamin D3 and 24,25(OH)2 vitamin D3, using positive and negative ion detection. Using these two modes positional isomers can be identified. Detection in the negative ion mode was preferred because of the slightly higher sensitivity. The limits of detection, using multiple ion detection, are 50-100 nM (6-12 pmol injected). On-line post-column derivatization based on [4 + 2] cyclo-addition (Diels-Alder reaction) proceeds within 1 min at room temperature. If this step is included in LC/TSP MS, the detection limits of the analytes can be improved 7-70-fold depending on the analyte tested. The best results (detection limits down to 1 nM, i.e. 0.12 pmol injected) are obtained with discharge ionization in the negative ion mode.

Evaluation of eluents in thermospray liquid chromatography-mass spectrometry for identification and determination of pesticides in environmental samples.

The influence of different eluents in positive and negative ion mode thermospray liquid chromatography-mass spectrometry was studied with several groups of pesticides, including carbamates, chlorotriazines, phenylureas, phenoxy acids and organophosphorus and quaternary ammonium compounds, and the corresponding degradation products. Using the positive ion mode in combination with reversed-phase eluents the base peaks generally corresponded either to [M + H]+ for the chlorotriazines and their hydroxy metabolites or to [M + NH4]+ for the carbamates, the phenylureas, the organophosphorus pesticides and their oxygen analogues. In the negative ion mode different processes such as (dissociative) electron-capture and anion attachment mechanisms occurred. Fragment ions such as [M - CONHCH3]- for the carbamates, [M - H]- for the chlorotriazines, phenylureas and chlorinated phenoxy acids and [M].-, [M - R]- (R being a methyl or ethyl group) for organophosphorus pesticides were usually formed. Depending on the eluent additive used (ammonium acetate, ammonium formate and/or chloroacetonitrile), three different adduct ions were formed: [M + CH3COO]-, [M + HCOO]- and [M + Cl]-. Normal-phase eluents with cyclohexane, n-hexane and/or dichloromethane provided more structural information and enhanced the response of several compounds. The positive ion mode was useful for the detection of chlorinated phenoxy acids and chlorophenols which could not be detected in the positive ion mode using reversed-phase systems. The base peaks generally corresponded to [M].+, [M + H]+ or [M - Cl]+. For the characterization of difenzoquat, a quaternary ammonium pesticide of which trace level analysis is troublesome, a post-column ion-pair extraction system was used. An aqueous mobile phase with a sulphonate-type counter ion was applied and an extraction solvent containing cyclohexane-dichloromethane-n-butanol (45:45:10) was used in thermospray liquid chromatography-mass spectrometry. Illustrative examples of the determination of residue levels of pesticides in soil matrices are shown.