Lesional skin of seborrheic dermatitis patients is characterized by skin barrier dysfunction and correlating alterations in the stratum corneum ceramide composition.

Seborrheic dermatitis (SD) is a chronic inflammatory skin disease characterized by erythematous papulosquamous lesions in sebum rich areas such as the face and scalp. Its pathogenesis appears multifactorial with a disbalanced immune system, Malassezia driven microbial involvement and skin barrier perturbations. Microbial involvement has been well described in SD, but skin barrier involvement remains to be properly elucidated. To determine whether barrier impairment is a critical factor of inflammation in SD alongside microbial dysbiosis, a cross-sectional study was performed in 37 patients with mild-to-moderate facial SD. Their lesional and non-lesional skin was comprehensively and non-invasively assessed with standardized 2D-photography, optical coherence tomography (OCT), microbial profiling including Malassezia species identification, functional skin barrier assessments and ceramide profiling. The presence of inflammation was established through significant increases in erythema, epidermal thickness, vascularization and superficial roughness in lesional skin compared to non-lesional skin. Lesional skin showed a perturbed skin barrier with an underlying skewed ceramide subclass composition, impaired chain elongation and increased chain unsaturation. Changes in ceramide composition correlated with barrier impairment indicating interdependency of the functional barrier and ceramide composition. Lesional skin showed significantly increased Staphylococcus and decreased Cutibacterium abundances but similar Malassezia abundances and mycobial composition compared to non-lesional skin. Principal component analysis highlighted barrier properties as main discriminating features. To conclude, SD is associated with skin barrier dysfunction and changes in the ceramide composition. No significant differences in the abundance of Malassezia were observed. Restoring the cutaneous barrier might be a valid therapeutic approach in the treatment of facial SD.

High-resolution ion mobility spectrometry-mass spectrometry for isomeric separation of prostanoids after Girard's reagent T derivatization.

RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial for understanding the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution ion mobility spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry. METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques, namely linear cyclic IMS, linear trapped IMS, and nonlinear high-field asymmetric waveform IMS, were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue. RESULTS: Direct infusion of GT-derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS are discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research. CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

Quantitative Mass Spectrometry Imaging to Study Drug Distribution in the Intestine Following Oral Dosing.

Local delivery to the lower gut to treat diseases of the colon has become a topic of special attention. Tissue exposure of locally acting agents is not represented by plasma concentrations. Therefore, reliable methods to measure tissue uptake at the primary site of action (e.g., epithelial layer or lamina propria) are vital. This work investigates the suitability of mass spectrometry imaging (MSI) in quantitatively visualizing intestinal transmural drug distribution. Tofacitinib (Tofa), a drug approved for the treatment of several autoimmune diseases, including ulcerative colitis, was selected as a tool compound for feasibility studies. One- and 7-h postdose sections of the ileum, proximal- and distal-colon from rats that received an oral solution of Tofa were subjected to matrix-assisted laser desorption ionization (MALDI)-MSI. A dilution series of individual concentrations sprayed over an entire tissue section allowed for tissue type-specific quantitation. At 1 h (systemic Tmax), the signal was highest in the ileum, whereas at 7 h, the signal was highest in the colon, when the unabsorbed fraction of the compound reached the colon. A combination of three-dimensional (3D) intensity plots and hematoxylin and eosin (H&E) stains showed a visually observable gradual decrease in Tofa concentration from the lumen toward the muscular layer of the proximal colon. The high luminal concentration of Tofa indicated that flushing of the intestines with saline does not result in complete removal of the drug material from the lumen. This could cause an overestimation of drug concentration in gut tissue homogenates by conventional liquid chromatography-mass spectrometry (LC-MS) methods. This study demonstrates the utility of MSI to differentiate between the lumen and intestinal wall layers and enables proper interpretation of tissue distribution data.

Quantitative mass spectrometry imaging of drugs and metabolites: a multiplatform comparison.

Mass spectrometry imaging (MSI) provides insight into the molecular distribution of a broad range of compounds and, therefore, is frequently applied in the pharmaceutical industry. Pharmacokinetic and toxicological studies deploy MSI to localize potential drugs and their metabolites in biological tissues but currently require other analytical tools to quantify these pharmaceutical compounds in the same tissues. Quantitative mass spectrometry imaging (Q-MSI) is a field with challenges due to the high biological variability in samples combined with the limited sample cleanup and separation strategies available prior to MSI. In consequence, more selectivity in MSI instruments is required. This can be provided by multiple reaction monitoring (MRM) which uses specific precursor ion-product ion transitions. This targeted approach is in particular suitable for pharmaceutical compounds because their molecular identity is known prior to analysis. In this work, we compared different analytical platforms to assess the performance of MRM detection compared to other MS instruments/MS modes used in a Q-MSI workflow for two drug candidates (A and B). Limit of detection (LOD), linearity, and precision and accuracy of high and low quality control (QC) samples were compared between MS instruments/modes. MRM mode on a triple quadrupole mass spectrometer (QqQ) provided the best overall performance with the following results for compounds A and B: LOD 35.5 and 2.5 µg/g tissue, R2 0.97 and 0.98 linearity, relative standard deviation QC <13.6%, and 97-112% accuracy. Other MS modes resulted in LOD 6.7-569.4 and 2.6-119.1 µg/g tissue, R2 0.86-0.98 and 0.86-0.98 linearity, relative standard deviation QC < 19.4 and < 37.5%, and 70-356% and 64-398% accuracy for drug candidates A and B, respectively. In addition, we propose an optimized 3D printed mimetic tissue model to increase the overall analytical throughput of our approach for large animal studies. The MRM imaging platform was applied as proof-of-principle for quantitative detection of drug candidates A and B in four dog livers and compared to LC-MS. The Q-MSI concentrations differed <3.5 times with the concentrations observed by LC-MS. Our presented MRM-based Q-MSI approach provides a more selective and high-throughput analytical platform due to MRM specificity combined with an optimized 3D printed mimetic tissue model.

Enhanced Sensitivity Using MALDI Imaging Coupled with Laser Postionization (MALDI-2) for Pharmaceutical Research.

Visualizing the distributions of drugs and their metabolites is one of the key emerging application areas of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) within pharmaceutical research. The success of a given MALDI-MSI experiment is ultimately determined by the ionization efficiency of the compounds of interest, which in many cases are too low to enable detection at relevant concentrations. In this work we have taken steps to address this challenge via the first application of laser-postionisation coupled with MALDI (so-called MALDI-2) to the analysis and imaging of pharmaceutical compounds. We demonstrate that MALDI-2 increased the signal intensities for 7 out of the 10 drug compounds analyzed by up to 2 orders of magnitude compared to conventional MALDI analysis. This gain in sensitivity enabled the distributions of drug compounds in both human cartilage and dog liver tissue to be visualized using MALDI-2, whereas little-to-no signal from tissue was obtained using conventional MALDI. This work demonstrates the vast potential of MALDI-2-MSI in pharmaceutical research and drug development and provides a valuable tool to broaden the application areas of MSI. Finally, in an effort to understand the ionization mechanism, we provide the first evidence that the preferential formation of [M + H]+ ions with MALDI-2 has no obvious correlation with the gas-phase proton affinity values of the analyte molecules, suggesting, as with MALDI, the occurrence of complex and yet to be elucidated ionization phenomena.

Targeted Drug and Metabolite Imaging: Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

Mass spectrometry imaging (MSI) has proven to be a valuable tool for drug and metabolite imaging in pharmaceutical toxicology studies and can reveal, for example, accumulation of drug candidates in early drug development. However, the lack of sample cleanup and chromatographic separation can hamper the analysis due to isobaric interferences. Multiple reaction monitoring (MRM) uses unique precursor ion-product ion transitions to add specificity which leads to higher selectivity. Here, we present a targeted imaging platform where desorption electrospray ionization is combined with a triple quadrupole (QqQ) system to perform MRM imaging. The platform was applied to visualize (i) lipids in mouse brain tissue sections and (ii) a drug candidate and metabolite in canine liver tissue. All QqQ modes were investigated to show the increased detection time provided by MRM as well as the possibility to perform dual polarity imaging. This is very beneficial for lipid imaging because some phospholipid classes ionize in opposite polarity (e.g., phosphatidylcholine/sphingomyelin in positive ion mode and phosphatidylserine/phosphatidylethanolamine in negative ion mode). Drug and metabolite images were obtained to show its strength in drug distribution studies. Multiple MRM transitions were used to confirm the local presence and selective detection of pharmaceutical compounds.

Cross-Species Molecular Imaging of Bile Salts and Lipids in Liver: Identification of Molecular Structural Markers in Health and Disease.

The liver is the primary organ involved in handling of bile salts, a class of amphipathic molecules with signaling activities as well as desired and detrimental detergent actions. To allow in-depth investigation of functions of bile salts in healthy and diseased liver, the spatial distribution of bile salt species within the liver needs to be studied. Therefore, the aim of our study was to determine hepatic bile salt distribution and identify specific lipid markers that define the structural elements of the liver. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to monitor the spatial distribution of bile salts and lipids in liver sections of rat, dog, and patients with unaffected and cholestatic parenchyma. MALDI-MSI in negative ion mode showed the local presence of a variety of bile salts, predominantly taurine-conjugates, as localized patches of varying sizes (representing the bile ducts) throughout the liver tissue. Specific molecular markers were identified for the connective tissue (phosphatidic acids, e.g., [PA (18:0_18:1)-H]-), the liver parenchyma (phosphatidylinositols, e.g., [PI (18:0_20:4)-H]-), and the bile ducts (hydroxylated-sulfatides, e.g., [ST-OH (18:1_24:0)-H]-). One of these sulfatides (at m/ z 906.6339) was found to be uniquely localized in a thin lining on the inside of the bile duct, colocalized with cytokeratins, and encased luminal bile salts. A similar distribution of the aforementioned sulfatide was observed, albeit in constricted ductular structures, in the liver of a patient with a mild clinical phenotype of primary sclerosing cholangitis (PSC). In contrast, sulfatides were virtually absent in the liver of patients with PSC and a severe clinical phenotype, with (atypical) cholanoids (e.g., the bile alcohol 5-cyprinolsulfate) abundant in the extra-ductular space and glyco(cheno)deoxycholic acid-3-sulfate localized to fibrotic connective tissue. The latter two molecular species were able to discriminate between healthy liver tissue ( n = 3) and tissue from PSC patients with a severe clinical phenotype ( n = 3). In conclusion, the distinct structural elements of the mammalian liver are characterized by specific classes of lipids. We propose that (hydroxylated-)sulfatides are specific molecular markers of the bile duct.

Development and application of a UHPLC-MS/MS metabolomics based comprehensive systemic and tissue-specific screening method for inflammatory, oxidative and nitrosative stress.

Oxidative stress and inflammation are underlying pathogenic mechanisms associated with the progression of several pathological conditions and immunological responses. Elucidating the role of signalling lipid classes, which include, among others, the isoprostanes, nitro fatty acids, prostanoids, sphingoid bases and lysophosphatidic acids, will create a snapshot of the cause and effect of inflammation and oxidative stress at the metabolic level. Here we describe a fast, sensitive, and targeted ultra-high-performance liquid chromatography-tandem mass spectrometry metabolomics method that allows the quantitative measurement and biological elucidation of 17 isoprostanes as well as their respective isomeric prostanoid mediators, three nitro fatty acids, four sphingoid mediators, and 24 lysophosphatidic acid species from serum as well as organ tissues, including liver, lung, heart, spleen, kidney and brain. Application of this method to paired mouse serum and tissue samples revealed tissue- and serum-specific stress and inflammatory readouts. Little correlation was found between localized (tissue) metabolite levels compared with the systemic (serum) circulation in a homeostatic model. The application of this method in future studies will enable us to explore the role of signalling lipids in the metabolic pathogenicity of stress and inflammation during health and disease.

Fetal Metabolic Stress Disrupts Immune Homeostasis and Induces Proinflammatory Responses in Human Immunodeficiency Virus Type 1- and Combination Antiretroviral Therapy-Exposed Infants.

Increased morbidity and fetal growth restriction are reported in uninfected children born to human immunodeficiency virus type 1 (HIV-1)-infected women treated with antiretroviral (ARV) therapy. Viruses and/or pharmacological interventions such as ARVs can induce metabolic stress, skewing the cell's immune response and restricting (cell) growth. Novel metabolomic techniques provided the opportunity to investigate the impact of fetal HIV-1 and combination ARV therapy (cART) exposure on the infants' immune metabolome. Peroxidized lipids, generated by reactive oxygen species, were increased in cART/HIV-1-exposed infants, indicating altered mitochondrial functioning. The lipid metabolism was further dysregulated with increased triglyceride species and a subsequent decrease in phospholipids in cART/HIV-1-exposed infants compared to control infants. Proinflammatory immune mediators, lysophospholipids as well as cytokines such as CXCL10 and CCL3, were increased whereas anti-inflammatory metabolites from the cytochrome P450 pathway were reduced in cART/HIV-1-exposed infants. Taken together, these data demonstrate that the fetal metabolism is impacted by maternal factors (cART and HIV-1) and skews physiological immune responses toward inflammation in the newborn infant.

Identification of a Polyketide Synthase Involved in Sorbicillin Biosynthesis by Penicillium chrysogenum.

UNLABELLED: Secondary metabolism in Penicillium chrysogenum was intensively subjected to classical strain improvement (CSI), the resulting industrial strains producing high levels of ß-lactams. During this process, the production of yellow pigments, including sorbicillinoids, was eliminated as part of a strategy to enable the rapid purification of ß-lactams. Here we report the identification of the polyketide synthase (PKS) gene essential for sorbicillinoid biosynthesis in P. chrysogenum We demonstrate that the production of polyketide precursors like sorbicillinol and dihydrosorbicillinol as well as their derivatives bisorbicillinoids requires the function of a highly reducing PKS encoded by the gene Pc21g05080 (pks13). This gene belongs to the cluster that was mutated and transcriptionally silenced during the strain improvement program. Using an improved ß-lactam-producing strain, repair of the mutation in pks13 led to the restoration of sorbicillinoid production. This now enables genetic studies on the mechanism of sorbicillinoid biosynthesis in P. chrysogenum and opens new perspectives for pathway engineering. IMPORTANCE: Sorbicillinoids are secondary metabolites with antiviral, anti-inflammatory, and antimicrobial activities produced by filamentous fungi. This study identified the gene cluster responsible for sorbicillinoid formation in Penicillium chrysogenum, which now allows engineering of this diverse group of compounds.

Metabolomics profiling of the free and total oxidised lipids in urine by LC-MS/MS: application in patients with rheumatoid arthritis.

Oxidised lipids, covering enzymatic and auto-oxidation-synthesised mediators, are important signalling metabolites in inflammation while also providing a readout for oxidative stress, both of which are prominent physiological processes in a plethora of diseases. Excretion of these metabolites via urine is enhanced through the phase-II conjugation with glucuronic acid, resulting in increased hydrophilicity of these lipid mediators. Here, we developed a bovine liver-ß-glucuronidase hydrolysing sample preparation method, using liquid chromatography coupled to tandem mass spectrometry to analyse the total urinary oxidised lipid profile including the prostaglandins, isoprostanes, dihydroxy-fatty acids, hydroxy-fatty acids and the nitro-fatty acids. Our method detected more than 70 oxidised lipids biosynthesised from two non-enzymatic and three enzymatic pathways in urine samples. The total oxidised lipid profiling method was developed and validated for human urine and was demonstrated for urine samples from patients with rheumatoid arthritis. Pro-inflammatory mediators PGF2α and PGF3α and oxidative stress markers iPF2α- IV, 11-HETE and 14-HDoHE were positively associated with improvement of disease activity score. Furthermore, the anti-inflammatory nitro-fatty acids were negatively associated with baseline disease activity. In conclusion, the developed methodology expands the current metabolic profiling of oxidised lipids in urine, and its application will enhance our understanding of the role these bioactive metabolites play in health and disease.

Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes.

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10µl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, 'high-throughput' profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.

Genomic mutational analysis of the impact of the classical strain improvement program on ß-lactam producing Penicillium chrysogenum.

BACKGROUND: Penicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of ß-lactams. The high ß-lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved ß-lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown. RESULTS: To examine the impact of CSI on secondary metabolism, a comparative genomic analysis of ß-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to ß-lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum. CONCLUSIONS: The classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of ß-lactams.

Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

Matrix Effect Compensation in Small-Molecule Profiling for an LC-TOF Platform Using Multicomponent Postcolumn Infusion.

The possible presence of matrix effect is one of the main concerns in liquid chromatography-mass spectrometry (LC-MS)-driven bioanalysis due to its impact on the reliability of the obtained quantitative results. Here we propose an approach to correct for the matrix effect in LC-MS with electrospray ionization using postcolumn infusion of eight internal standards (PCI-IS). We applied this approach to a generic ultraperformance liquid chromatography-time-of-flight (UHPLC-TOF) platform developed for small-molecule profiling with a main focus on drugs. Different urine samples were spiked with 19 drugs with different physicochemical properties and analyzed in order to study matrix effect (in absolute and relative terms). Furthermore, calibration curves for each analyte were constructed and quality control samples at different concentration levels were analyzed to check the applicability of this approach in quantitative analysis. The matrix effect profiles of the PCI-ISs were different: this confirms that the matrix effect is compound-dependent, and therefore the most suitable PCI-IS has to be chosen for each analyte. Chromatograms were reconstructed using analyte and PCI-IS responses, which were used to develop an optimized method which compensates for variation in ionization efficiency. The approach presented here improved the results in terms of matrix effect dramatically. Furthermore, calibration curves of higher quality are obtained, dynamic range is enhanced, and accuracy and precision of QC samples is increased. The use of PCI-ISs is a very promising step toward an analytical platform free of matrix effect, which can make LC-MS analysis even more successful, adding a higher reliability in quantification to its intrinsic high sensitivity and selectivity.

A mutasynthesis approach with a Penicillium chrysogenum ΔroqA strain yields new roquefortine D analogues.

Penicillium chrysogenum, which lacks the roqA gene, processes synthetic, exogenously added histidyltryptophanyldiketopiperazine (HTD) to yield a set of roquefortine-based secondary metabolites also produced by the wild-type strain. Feeding a number of synthetic HTD analogues to the ΔroqA strain gives rise to the biosynthesis of a number of new roquefortine D derivatives, depending on the nature of the synthetic HTD added. Besides delivering semisynthetic roquefortine analogues, the mutasynthesis studies presented here also shed light on the substrate preferences and molecular mechanisms employed by the roquefortine C/D biosynthesis gene cluster, knowledge that may be tapped for the future development of more complex semisynthetic roquefortine-based secondary metabolites.

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements.

Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.

Collagen Induced Arthritis in DBA/1J Mice Associates with Oxylipin Changes in Plasma.

Oxylipins play important roles in various biological processes and are considered as mediators of inflammation for a wide range of diseases such as rheumatoid arthritis (RA). The purpose of this research was to study differences in oxylipin levels between a widely used collagen induced arthritis (CIA) mice model and healthy control (Ctrl) mice. DBA/1J male mice (age: 6-7 weeks) were selected and randomly divided into two groups, namely, a CIA and a Ctrl group. The CIA mice were injected intraperitoneally (i.p.) with the joint cartilage component collagen type II (CII) and an adjuvant injection of lipopolysaccharide (LPS). Oxylipin metabolites were extracted from plasma for each individual sample using solid phase extraction (SPE) and were detected with high performance liquid chromatography/tandem mass spectrometry (HPLC-ESI-MS/MS), using dynamic multiple reaction monitoring (dMRM). Both univariate and multivariate statistical analyses were applied. The results in univariate Student's t-test revealed 10 significantly up- or downregulated oxylipins in CIA mice, which were supplemented by another 6 additional oxylipins, contributing to group clustering upon multivariate analysis. The dysregulation of these oxylipins revealed the presence of ROS-generated oxylipins and an increase of inflammation in CIA mice. The results also suggested that the collagen induced arthritis might associate with dysregulation of apoptosis, possibly inhibited by activated NF-κB because of insufficient PPAR-γ ligands.

Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection.

An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a flow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofluids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipids in biological samples of different matrices.

Metabolic phenotyping reveals a lipid mediator response to ionizing radiation.

Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.