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Background: Oxylipins are products derived from polyunsaturated fatty acids (PUFAs) that play a role in cardiovascular disease and aging. Fish oil-derived n-3 PUFAs promote the formation of anti-inflammatory and vasodilatory oxylipins; however, there are little data on oxylipins derived from α-linolenic acid (C18:3n-3), the primary plant-derived n-3 PUFA. Walnuts are a source of C18:3n-3., Objectives: To investigate the effect on serum oxylipins of a diet enriched with walnuts at 15% energy (30-60 g/d; 2.6-5.2 g C18:3n-3/d) for 2 y compared to a control diet (abstention from walnuts) in healthy older males and females (63-79 y)., Methods: The red blood cell proportion of α-linolenic acid was determined by gas chromatography as a measure of compliance. Ultra-performance liquid chromatography-tandem mass spectrometry was used to measure serum concentrations of 53 oxylipins in participants randomly assigned to receive the walnut diet (n = 64) or the control diet (n = 51). Two-year concentration changes (final minus baseline) were log-transformed (base log-10) and standardized (mean-centered and divided by the standard deviation of each variable). Volcano plots were then generated (fold change ≥1.5; false discovery rate ≤0.1). For each oxylipin delta surviving multiple testing, we further assessed between-intervention group differences by analysis of covariance adjusting for age, sex, BMI, and the baseline concentration of the oxylipin., Results: The 2-y change in red blood cell C18:3n-3 in the walnut group was significantly higher than that in the control group (P < 0.001). Compared to the control diet, the walnut diet resulted in statistically significantly greater increases in 3 C18:3n-3-derived oxylipins (9-HOTrE, 13-HOTrE, and 12,13-EpODE) and in the C20:5n-3 derived 14,15-diHETE, and greater reductions of the C20:4n-6-derived 5-HETE, 19-HETE, and 5,6-diHETrE., Conclusions: Long-term walnut consumption changes the serum oxylipin profile in healthy older persons. Our results add novel mechanistic evidence on the cardioprotective effects of walnuts., Trial Registration: Clinicaltrials.gov Identifier: NCT01634841., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Impaired skin wound healing is a significant global health issue, especially among the elderly. Wound healing is a well-orchestrated process involving the sequential phases of inflammation, proliferation, and tissue remodeling. Although wound healing is a highly dynamic and energy-requiring process, the role of metabolism remains largely unexplored. By combining transcriptomics and metabolomics of human skin biopsy samples, we mapped the core bioenergetic and metabolic changes in normal acute as well as chronic wounds in elderly subjects. We found upregulation of glycolysis, the tricarboxylic acid cycle, glutaminolysis, and β-oxidation in the later stages of acute wound healing and in chronic wounds. To ascertain the role of these metabolic pathways on wound healing, we targeted each pathway in a wound healing assay as well as in a human skin explant model using metabolic inhibitors and stimulants. Enhancement or inhibition of glycolysis and, to a lesser extent, glutaminolysis had a far greater impact on wound healing than similar manipulations of oxidative phosphorylation and fatty acid β-oxidation. These findings increase the understanding of wound metabolism and identify glycolysis and glutaminolysis as potential targets for therapeutic intervention., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Although estimates of suboptimal adherence to oral corticosteroids in asthma range from 30% to 50%, no ideal method for measurement exists; the impact of poor adherence in severe asthma is likely to be particularly high., Research Questions: What is the prevalence of suboptimal adherence detected by self-reporting and direct measures? Is suboptimal adherence associated with disease activity?, Study Design and Methods: Data were included from individuals with severe asthma taking part in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study and prescribed daily oral corticosteroids. Participants completed the Medication Adherence Report Scale, a five-item questionnaire used to grade adherence on a scale from 1 to 5, and provided a urine sample for analysis of prednisolone and metabolites by liquid chromatography-mass spectrometry., Results: Data from 166 participants were included in this study: mean (SD) age, 54.2 (± 11.9) years; FEV 1 , 65.1% (± 20.5%) predicted; female, 58%; 37% completing the Medication Adherence Report Scale reported suboptimal adherence; and 43% with urinary corticosteroid data did not have detectable prednisolone or metabolites in their urine. Good adherence by both methods was detected in 49 of the 142 (35%) of participants in whom both methods were performed; adherence detection did not match between methods in 53%. Self-reported high adherers had better asthma control and quality of life, whereas directly measured high adherers had lower blood eosinophil levels., Interpretation: Low adherence is a common problem in severe asthma, whether measured directly or self-reported. We report poor agreement between the two methods, suggesting some disassociation between self-assessment of medication adherence and regular oral corticosteroid use, which suggests that each approach may provide complementary information in clinical practice., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Background: Exposure to polycyclic aromatic hydrocarbons (PAHs) has been linked to adverse pulmonary effects. However, the impact of low-level environmental PAH exposure on lung function in early adulthood remains uncertain., Objectives: To evaluate the associations between urinary PAH metabolites and lung function parameters in young adults., Methods: Urinary metabolites of pyrene, phenanthrene, and fluorene were analysed in 1000 young adults from Sweden (age 22-25 years) using LC-MS/MS. Lung function and eosinophilic airway inflammation were measured by spirometry and exhaled nitric oxide fraction (FeNO), respectively. Linear regression analysis was used to evaluate associations between PAH metabolites and the outcomes., Results: Median urinary concentrations of 1-OH-pyrene, ∑OH-phenanthrene, and ∑OH-fluorene were 0.066, 0.36, 0.22 μg/L, respectively. We found inverse associations of ∑OH-phenanthrene and ∑OH-fluorene with FEV1 and FVC, as well as between 1-OH-pyrene and FEV1/FVC ratio (adjusted P < 0.05; all participants). An increase of 1% in ∑OH-fluorene was associated with a decrease of 73 mL in FEV1 and 59 mL in FVC. In addition, ∑OH-phenanthrene concentrations were, in a dose-response manner, inversely associated with FEV1 (B from -109 to -48 compared with the lowest quartile of ∑OH-phenanthrene; p trend 0.004) and FVC (B from -159 to -102 compared with lowest quartile; p-trend <0.001). Similar dose-response associations were also observed between ∑OH-fluorene and FEV1 and FVC, as well as between 1-OH-pyrene and FEV1/FVC (p-trend <0.05). There was no association between PAH exposure and FeNO, nor was there an interaction with smoking, sex, or asthma., Conclusion: Low-level PAH exposure was, in a dose-response manner, associated with reduced lung function in young adults. Our findings have public health implications due to i) the widespread occurrence of PAHs in the environment and ii) the clinical relevance of lung function in predicting all-cause and cardiovascular disease mortality., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined., (Copyright © 2021. Published by Elsevier B.V.)

Background & Aims: Acute-on-chronic liver failure (ACLF) is a newly described syndrome, which develops in patients with acute decompensation of cirrhosis, and is characterized by intense systemic inflammation, multiple organ failures and high short-term mortality. The profile of circulating lipid mediators, which are endogenous signaling molecules that play a major role in inflammation and immunity, is poorly characterized in ACLF., Methods: In the current study, we assessed the profile of lipid mediators by liquid chromatography coupled to tandem mass spectrometry in plasma from patients with acute decompensation of cirrhosis, with (n = 119) and without (n = 127) ACLF, and from healthy controls (n = 18). Measurements were prospectively repeated in 191 patients with acute decompensation of cirrhosis during a 28-day follow-up period., Results: Fifty-nine lipid mediators (out of 100) were detected in plasma from cirrhotic patients, of which 16 were significantly associated with disease status. Among these, 11 lipid mediators distinguished patients at any stage from healthy controls, whereas 2 lipid mediators (LTE 4 and 12-HHT, both derived from arachidonic acid) shaped a minimal plasma fingerprint that discriminated patients with ACLF from those without. Levels of LTE 4 distinguished ACLF grade 3 from ACLF grades 1 and 2, followed the clinical course of the disease (increased with worsening and decreased with improvement) and positively correlated with markers of inflammation and non-apoptotic cell death. Moreover, LTE 4 together with LXA 5 (derived from eicosapentaenoic acid) and EKODE (derived from linoleic acid) were associated with short-term mortality. LXA 5 and EKODE formed a signature associated with coagulation and liver failures., Conclusion: Taken together, these findings uncover specific lipid mediator profiles associated with disease severity and prognosis in patients with acute decompensation of cirrhosis., Lay Summary: Acute-on-chronic liver failure (ACLF) is characterized by intense systemic inflammation, multiple organ failures and high short-term mortality. In the current study, we assessed the plasma lipid profile of 100 bioactive lipid mediators in healthy controls, patients with decompensated cirrhosis, and those who had developed ACLF. We identified lipid mediator signatures associated with inflammation and non-apoptotic cell death that discriminate disease severity and evolution, short-term mortality and organ failures., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Purpose: It is well established that end-stage renal disease (ESRD) is associated with increased cardiovascular morbidity and mortality both in the adult and pediatric population. Although the underlying molecular mechanisms are poorly understood, compromised nitric oxide (NO) bioactivity has been suggested as a contributing factor. With this in mind, we investigated the effects of hemodialysis on NO homeostasis and bioactivity in blood., Methods & Results: Plasma and dialysate samples were obtained before and after hemodialysis sessions from adults (n = 33) and pediatric patients (n = 10) with ESRD on chronic renal replacement therapy, and from critically ill adults with acute kidney injury (n = 12) at their first sustained low-efficiency dialysis session. Levels of nitrate, nitrite, cyclic guanosine monophosphate (cGMP) and amino acids relevant for NO homeostasis were analyzed. We consistently found that nitrate and cGMP levels in plasma were significantly reduced after hemodialysis, whereas post-dialysis nitrite and amino acids coupled to NO synthase activity (i.e., arginine and citrulline) were only significantly reduced in adults with ESRD. The amount of excreted nitrate and nitrite during dialysis were similar to daily endogenous levels that would be expected from endothelial NO synthase activity., Conclusions: Our results show that hemodialysis significantly reduces circulating levels of nitrate and cGMP, indicating that this medical procedure may impair NO synthesis and potentially NO signaling pathways., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Succinate is a Krebs cycle intermediate whose formation is enhanced under metabolic stress, and for which a selective sensor GPR91 has been identified on various cell types including platelets. Platelet-derived eicosanoids play pivotal roles in platelet activation/aggregation, which is key to thrombus formation and progression of atherothrombosis., Objectives: This study aims to decipher the molecular mechanism(s) and potential involvement of eicosanoids in succinate enhanced platelet activation/aggregation., Methods: We used liquid chromatography-mass spectrometry (LC-MS)/MS-based lipid mediator profiling to identify eicosanoids regulated by succinate. We ran light transmittance aggregometry and flow cytometry to assess platelet aggregation, P-selectin expression, and platelet-polymorphonuclear leukocyte (PMN) adherence. Various pharmacological tools were used to assess the contributions of GPR91 signalling and eicosanoids in platelet aggregation., Results: Succinate and two types of synthetic non-metabolite GPR91 agonists-cis-epoxysuccinate (cES) and Cmpd131-potentiated platelet aggregation, which was partially blocked by a selective GPR91 antagonist XT1. GPR91 activation increased production of 12-hydroxy-eicosatetraenoic acid (12-HETE), thromboxane (TX) A 2 , and 12-hydroxy-heptadecatrienoic acid (12-HHT) in human platelets, associated with phosphorylation of cytosolic phospholipase A 2 (cPLA 2 ), suggesting increased availability of free arachidonic acid. Blocking 12-HETE and TXA 2 synthesis, or antagonism of the TXA 2 receptor, significantly reduced platelet aggregation enhanced by GPR91 signalling. Moreover, platelet-PMN suspensions challenged with succinate exhibited enhanced transcellular biosynthesis of leukotriene C 4 (LTC 4 ), a powerful proinflammatory vascular spasmogen., Conclusion: Succinate signals through GPR91 to promote biosynthesis of eicosanoids, which contribute to platelet aggregation/activation and potentially vascular inflammation. Hence, GPR91 may be a suitable target for pharmacological intervention in atherothrombotic conditions., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived lipid mediators reported to be dysregulated in obstructive lung disease. In contrast to many other oxylipins, TriHOME biosynthesis in humans is still poorly understood. The association of TriHOMEs with inflammation prompted the current investigation into the ability of human granulocytes to synthesize the 16 different 9,10,13-TriHOME and 9,12,13-TriHOME isomers and of the TriHOME biosynthetic pathway. Following incubation with linoleic acid, eosinophils and (to a lesser extent) the mast cell line LAD2, but not neutrophils, formed TriHOMEs. Stereochemical analysis revealed that TriHOMEs produced by eosinophils predominantly evidenced the 13(S) configuration, suggesting 15-lipoxygenase (15-LOX)-mediated synthesis. TriHOME formation was blocked following incubation with the 15-LOX inhibitor BLX-3887 and was shown to be largely independent of soluble epoxide hydrolase and cytochrome P450 activities. TriHOME synthesis was abolished when linoleic acid was replaced with 13-HODE, but increased in incubations with 13-HpODE, indicating the intermediary role of epoxy alcohols in TriHOME formation. In contrast to eosinophils, LAD2 cells formed TriHOMEs having predominantly the 13(R) configuration, demonstrating that there are multiple synthetic routes for TriHOME formation. These findings provide for the first-time insight into the synthetic route of TriHOMEs in humans and expand our understanding of their formation in inflammatory diseases., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The eicosanoids are a family of lipid mediators of pain and inflammation involved in multiple pathologies, including asthma, hypertension, cancer, atherosclerosis, and neurodegenerative diseases. These signaling mediators act locally, but are rapidly metabolized and transported to the systemic circulation as a mixture of primary and secondary metabolites. Accordingly, urine has become a useful readily accessible biofluid for monitoring the endogenous synthesis of these molecules. Herein, we present the validation of a rapid, repeatable, and precise method for the extraction and quantification of 32 eicosanoid urinary metabolites by LC-MS/MS. For 12 out of 17 deconjugated glucuronide eicosanoids, there was no improvement in recovered signal. These metabolites cover the major synthetic pathways, including prostaglandins, leukotrienes, and isoprostanes. The method linearity was >0.99 for all metabolites analyzed, the limit of detection ranged from 0.05-5 ng/ml, and the average extraction recoveries were >90%. All analytes were stable for at least three freeze/thaw cycles. The method was formatted for large-scale analysis of clinical cohorts, and the long-term repeatability was demonstrated over 15 months of acquisition, evidencing high precision (CV <15%, except for tetranorPGEM and 2,3-dinor-11β-PGF 2α , which were <30%). The presented method is suitable for focused mechanistic studies as well as large-scale clinical and epidemiological studies that require repeatable methods capable of producing data that can be concatenated across multiple cohorts., (Copyright © 2019 Gómez et al.)

Accurate annotation is vital for data interpretation; however, metabolite identification is a major bottleneck in untargeted metabolomics. Although community guidelines for metabolite identification were published over a decade ago, adaptation of the recommended standards has been limited. The complexity of LC-MS data due to combinations of various chromatographic and mass spectrometric acquisition methods has resulted in the advent of diverse workflows, which often involve non-standardized manual curation. Herein, we review the parameters involved in metabolite reporting and provide a workflow to estimate the level of confidence in reported metabolite annotation. The future of metabolite identification will be heavily based upon the use of metabolome data repositories and associated data analysis tools, which will enable data to be shared, re-analyzed and re-annotated in an automated fashion., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The health benefits of substituting dietary polyunsaturated fatty acids (PUFAs) for saturated fatty acids are well known. However, limited information exists on how the response to dietary intake of linoleic acid (LA; 18:2n-6) is modified by polymorphisms in the fatty acid desaturase (FADS) gene cluster., Objectives: The aim of the current study was to test the hypothesis that the FADS1 rs174550 genotype modifies the effect of dietary LA intake on the fatty acid composition of plasma lipids, fasting glucose, and high-sensitivity C-reactive protein (hsCRP)., Methods: Associations were investigated between genotype, plasma PUFAs, fasting glucose, and hsCRP concentrations in the cross-sectional, population-based Metabolic Syndrome in Men cohort (n = 1337). In addition, 62 healthy men from the cohort who were homozygotes for the TT or CC genotype of the FADS1 rs174550 were recruited to a 4-wk intervention (FADSDIET) with an LA-enriched diet. The fatty acid composition of plasma PUFAs and concentrations of plasma fasting glucose, serum hsCRP, and plasma lipid mediators (eicosanoids and related analogs) were measured at the beginning and end of the 4-wk intervention period., Results: In the FADSDIET trial, the plasma LA proportion increased in both genotype groups in response to an LA-enriched diet. Responses in concentrations of serum hsCRP and plasma fasting glucose and the proportion of arachidonic acid (20:4n-6) in plasma phospholipids and cholesteryl esters differed between genotype groups (interaction of diet × genotype, P < 0.05). In TT homozygous subjects, plasma eicosanoid concentrations correlated with the arachidonic acid proportion in plasma and with hsCRP (r = 0.4-0.7, P < 0.05), whereas in the CC genotype there were no correlations., Conclusions: Our findings show that the FADS1 genotype modifies metabolic responses to dietary LA. The emerging concept that personalized dietary counseling should be modified by the FADS1 genotype needs to be tested in larger randomized trials. The study was registered at clinicaltrials.gov as NCT02543216.

The application of metabolomics to investigating the cell secretome has garnered popularity owing to the method's large-scale data output, biochemical insight, and prospects for novel target compound discovery. However, there are no standardized protocols for the use of cell growth media, a factor that can exert profound effects upon the detected metabolites, and thus in the interpretability of the resulting data. Herein, we applied a liquid chromatography-high resolution mass spectrometry-based metabolomics approach to examine the influence of 5 different media combinations upon the obtained secretome of two phenotypically different cell lines: human embryonic kidney cells (HEK293) and L6 rat muscle cells. These media combinations were, M1: Medium 199, M2: Medium 199 + 2% fetal bovine serum (FBS), M3: Dulbecco's Modified Eagle's Medium (DMEM), M4: DMEM + 2% FBS and M5: Krebs-Henseleit Modified Buffer (KHB). The effect of incubation (37 °C) vs. refrigeration (4 °C) on DMEM medium over a 24 h period was also investigated. Results were validated for a selected panel of 5 metabolites measured from an additional cell culture experiment. Metabolomics identified a total of 53 polar metabolites that exhibited differential patterns on a cell type- and medium-specific basis. We observed that choice of media was the primary contributor to the secreted metabolite profile detected. The addition of FBS resulted in unique detected metabolites, compared to media-only controls (M199 and DMEM alone). Glutamine and pyroglutamate were more abundant in incubated relative to refrigerated DMEM medium. The overall metabolic pattern of the metabolites from the targeted approach matched with that exhibited across M1-M5 of the metabolomics experiment, and aided in further identifying the presence of compounds that were below the limit of detection in metabolomics. Based upon these findings, we highlight the following considerations in designing a cell secretome-based metabolite profiling experiment: (1) multiple media combinations (with and without FBS) should be tested for each cell line to be investigated; (2) cell-free media combinations should be plated separately, and incubated/treated in the same experimental conditions as the cells; and (3) a compromise between cell death and metabolite detection should be identified in order to avoid batch-specific contributions from FBS supplementation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuro-active pharmaceuticals have been reported to act as endocrine disruptors enhancing reproduction in the model crustacean Daphnia manga at environmental concentrations of ng/L. Oxylipins and more specifically eicosanoids, which are lipid mediators formed from polyunsaturated fatty acids (PUFAs), are known to regulate reproduction together with other physiological processes in insects. In D. magna, the biosynthesis of eicosanoids and their putative role in the regulation of reproduction has been studied using transcriptomics, genomics and exposures to cyclooxygenase inhibitors. Quantification of eicosanoids and oxylipins derived from PUFAs upon exposure to pharmaceuticals is therefore crucial for a better understanding of the mode of action of neuro-active pharmaceuticals on aquatic invertebrates. The aim of this study was to investigate shifts in the oxylipin profile in D. magna adults upon exposure to environmental concentrations of the three psychiatric drugs, fluoxetine, diazepam and carbamazepine, with known effects of enhancing offspring production. Oxylipin profiles were determined in whole organism tissues using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Up to 28 different oxylipins belonging to arachidonic (AA), linoleic acid (LA), α-linoleic acid (α-LA) and eicosapentaenoic acid (EPA) pathways were detected and quantified in D. magna adults. Exposure to the selected psychiatric drugs showed that fluoxetine enhanced the accumulation of the cyclooxygenase (COX) product 12-hydroxyheptadecatrienoic acid (12-HHTrE), whereas diazepam increased the concentration of eicosanoids belonging to the lipoxygenase (LOX) and cytochrome P450 (CYP) pathways (HETEs, EpOMEs, HODEs, HOTrEs and HEPEs) from the AA, LA, αLA and EPA pathways. Carbamazepine had little effect and only one LA-derived compound from the LOX pathway (13-HODE) increased significantly. This means that despite having different modes of action in humans, fluoxetine and diazepam up-regulated eicosanoid pathways in D. magna, closely related to known biologically active products that regulate reproduction in insects., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human blood is a self-regenerating lipid-rich biological fluid that is routinely collected in hospital settings. The inventory of lipid molecules found in blood plasma (plasma lipidome) offers insights into individual metabolism and physiology in health and disease. Disturbances in the plasma lipidome also occur in conditions that are not directly linked to lipid metabolism; therefore, plasma lipidomics based on MS is an emerging tool in an array of clinical diagnostics and disease management. However, challenges exist in the translation of such lipidomic data to clinical applications. These relate to the reproducibility, accuracy, and precision of lipid quantitation, study design, sample handling, and data sharing. This position paper emerged from a workshop that initiated a community-led process to elaborate and define a set of generally accepted guidelines for quantitative MS-based lipidomics of blood plasma or serum, with harmonization of data acquired on different instrumentation platforms across independent laboratories as an ultimate goal. We hope that other fields may benefit from and follow such a precedent., (Copyright © 2018 Burla et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived oxylipins with potential physiological relevance in inflammatory processes as well as in maintaining an intact skin barrier. Due to the high number of possible TriHOME isomers with only subtle differences in their physicochemical properties, the stereochemical analysis is challenging and usually involves a series of laborious analytical procedures. We herein report a straightforward analytical workflow that includes reversed-phase ultra-HPLC-MS/MS for rapid quantification of 9,10,13- and 9,12,13-TriHOME diastereomers and a chiral LC-MS method capable of resolving all sixteen 9,10,13-TriHOME and 9,12,13-TriHOME regio- and stereoisomers. We characterized the workflow (accuracy, 98-120%; precision, coefficient of variation ≤6.1%; limit of detection, 90-98 fg on column; linearity, R 2 = 0.998) and used it for stereochemical profiling of TriHOMEs in bronchoalveolar lavage fluid (BALF) of individuals with chronic obstructive pulmonary disease (COPD). All TriHOME isomers were increased in the BALF of COPD patients relative to that of smokers ( P ≤ 0.06). In both COPD patients and smokers with normal lung function, TriHOMEs with the 13(S) configuration were enantiomerically enriched relative to the corresponding 13(R) isomers, suggesting at least partial enzymatic control of TriHOME synthesis. This method will be useful for understanding the synthetic sources of these compounds and for elucidating disease mechanisms., (Copyright © 2018 Fuchs et al.)

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Background & Purpose: Infants on chronic peritoneal dialysis (PD) have an increased risk of developing neurological morbidities; however, the underlying biological mechanisms are poorly understood. In this clinical study, we investigated whether PD-mediated impairment of nitric oxide (NO) bioavailability and signaling, in patients with persistently low systolic blood pressure (SBP), can explain the occurrence of cerebral ischemia., Methods & Results: Repeated blood pressure measurements, serial neuroimaging studies, and investigations of systemic nitrate and nitrite levels, as well as NO signaling, were performed in ten pediatric patients on PD. We consistently observed the loss of both inorganic nitrate (-17 ± 3%, P < 0.05) and nitrite (-34 ± 4%, P < 0.05) during PD, which may result in impairment of the nitrate-nitrite-NO pathway. Indeed, PD was associated with significant reduction of cyclic guanosine monophosphate levels (-59.4 ± 15%, P < 0.05). This reduction in NO signaling was partly prevented by using a commercially available PD solution supplemented with l-arginine. Although PD compromised nitrate-nitrite-NO signaling in all cases, only infants with persistently low SBP developed ischemic cerebral complications., Conclusions: Our data suggests that PD impairs NO homeostasis and predisposes infants with persistently low SBP to cerebral ischemia. These findings improve current understanding of the pathogenesis of infantile cerebral ischemia induced by PD and may lead to the new treatment strategies to reduce neurological morbidities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Omega-3 fatty acid supplements have been reported to inhibit exercise-induced bronchoconstriction (EIB). It has not been determined whether omega-3 supplements inhibit airway sensitivity to inhaled mannitol, a test for bronchial hyperresponsiveness (BHR) and model for EIB in people with mild to moderate asthma., Methods: In a double-blind, crossover trial, subjects with asthma who had BHR to inhaled mannitol (n = 23; 14 men; mean age, 28 years; one-half taking regular inhaled corticosteroids) were randomized to omega-3 supplements (4.0 g/d eicosapentaenoic acid and 2.0 g/d docosahexaenoic acid) or matching placebo for 3 weeks separated by a 3-week washout. The primary outcome was the provoking dose of mannitol (mg) to cause a 15% fall in FEV1 (PD15). Secondary outcomes were sputum eosinophil count, spirometry, Asthma Control Questionnaire (ACQ) score, serum triacylglyceride level, and lipid mediator profile in urine and serum., Results: PD15 (geometric mean, 95% CI) to mannitol following supplementation with omega-3s (78 mg, 51-119 mg) was not different from placebo (88 mg, 56-139 mg, P = .5). There were no changes in sputum eosinophils (mean ± SD) in a subgroup of 11 subjects (omega-3, 8.4% ± 8.2%; placebo, 7.8% ± 11.8%; P = .9). At the end of each treatment period, there were no differences in FEV1 % predicted (omega-3, 85% ± 13%; placebo, 84% ± 11%; P = .9) or ACQ score (omega-3, 1.1% ± 0.5%; placebo, 1.1% ± 0.5%; P = .9) (n = 23). Omega-3s caused significant lowering of blood triglyceride levels and expected shifts in serum fatty acids and eicosanoid metabolites, confirming adherence to the supplements; however, no changes were observed in urinary mast cell mediators., Conclusions: Three weeks of omega-3 supplements does not improve BHR to mannitol, decrease sputum eosinophil counts, or inhibit urinary excretion of mast cell mediators in people with mild to moderate asthma, indicating that dietary omega-3 supplementation is not useful in the short-term treatment of asthma., Trial Registry: ClinicalTrials.gov; No.: NCT00526357; URL: www.clinicaltrials.gov.

Background: Nitrate, which is an inorganic anion abundant in vegetables, increases the efficiency of isolated human mitochondria. Such an effect might be reflected in changes in the resting metabolic rate (RMR) and formation of reactive oxygen species. The bioactivation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species., Objective: We studied effects of inorganic nitrate, in amounts that represented a diet rich in vegetables, on the RMR in healthy volunteers., Design: In a randomized, double-blind, crossover study, we measured the RMR by using indirect calorimetry in 13 healthy volunteers after a 3-d dietary intervention with sodium nitrate (NaNO₃) or a placebo (NaCl). The nitrate dose (0.1 mmol · kg⁻¹ · d⁻¹) corresponded to the amount in 200-300 g spinach, beetroot, lettuce, or other vegetable that was rich in nitrate. Effects of direct nitrite exposure on cell respiration were studied in cultured human primary myotubes., Results: The RMR was 4.2% lower after nitrate compared with placebo administration, and the change correlated strongly to the degree of nitrate accumulation in saliva (r² = 0.71). The thyroid hormone status, insulin sensitivity, glucose uptake, plasma concentration of isoprostanes, and total antioxidant capacity were unaffected by nitrate. The administration of nitrite to human primary myotubes acutely inhibited respiration., Conclusions: Dietary inorganic nitrate reduces the RMR. This effect may have implications for the regulation of metabolic function in health and disease.

Mast cells are inflammatory cells that play key roles in health and disease. They are distributed in all tissues and appear in two main phenotypes, connective tissue and mucosal mast cells, with differing capacities to release inflammatory mediators. A metabolic profiling approach was used to obtain a more comprehensive understanding of the ability of mast cell phenotypes to produce eicosanoids and other lipid mediators. A total of 90 lipid mediators (oxylipins) were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), representing the cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) metabolic pathways. In vitro-derived murine mucosal-like mast cells (MLMC) and connective tissue-like mast cells (CTLMC) exhibited distinct mRNA expression patterns of enzymes involved in oxylipin biosynthesis. Oxylipins produced by 5-LO and COX pathways were the predominant species in both phenotypes, with 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs. Multivariate analyses demonstrated that CTLMCs and MLMCs secrete differing oxylipin profiles at baseline and following calcium ionophore stimulation, evidencing specificity in both a time- and biosynthetic pathway-dependent manner. In addition to the COX-regulated prostaglandin PGD(2) and 5-LO-regulated cysteinyl-leukotrienes (e.g., LTC(4)), several other mediators evidenced phenotype-specificity, which may have biological implications in mast cell-mediated regulation of inflammatory responses.

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Antigenic variation is a common mechanism employed by many pathogenic organisms to avoid recognition of surface proteins by the host immune system. The malaria parasite, Plasmodium falciparum, among many others, exploits this mechanism and manages to survive in an otherwise hostile environment. Although similarities in the mechanisms used among different species to generate antigenic variation are broadly recognized, there is a lack of studies using cross-species data. The varDB project (http://www.vardb.org) was created to study antigenic variation at a range of different levels, both within and among species. The project aims to serve as a resource to increase our understanding of antigenic variation by providing a framework for comparative studies. In this review we describe the varDB project, its construction, and the overall organization of information with the intent of increasing the utility of varDB to the research community. The current version of varDB supports 27 species involved in 19 different diseases affecting humans as well as other species. These data include 42 gene families that are represented by over 67,000 sequences. The varDB project is still in its infancy but is expected to continue to grow with the addition of new organisms and gene families as well as input from the general research community., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Eicosanoids are a family of oxygenated metabolites of arachidonic acid, including the prostaglandins, thromboxanes, leukotrienes and lipoxins. These lipid mediators play essential roles in normal cellular homeostasis as well as in a number of disease states. This review will focus on recent advances in the field of eicosanoids and highlight specific discoveries and achievements. Emphasis will be placed on structure and receptor biology, which are of significant pharmacological and clinical relevance., (2010 Elsevier Inc. All rights reserved.)

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.

Endogenous 2-arachidonoylglycerol (2-AG) inhibits invasion of androgen-independent prostate cancer cells. Blocking cellular hydrolysis of 2-AG to increase its endogenous concentration results in a decrease in cell invasion. A series of compounds containing a trifluoromethyl ketone (TFK) moiety or the methyl analog (known to inhibit carboxylesterases) were investigated for their ability to inhibit 2-AG hydrolysis and prostate cancer cell invasion. Compounds containing a thioether beta to a TFK moiety inhibited 2-AG hydrolysis as well as cell invasion in a concentration-dependent manner. Inhibition of 2-AG hydrolysis increased concomitantly with inhibitor alkyl chain length from 4- to 12-carbons while inhibition of cell invasion exhibited a maximum at 8- to 10-carbons of the compounds. These results demonstrate a new series of 2-AG hydrolysis inhibitors as a potential therapeutic approach for prostate cancer.

Carboxylesterases are enzymes that catalyze the hydrolysis of ester and amide moieties. These enzymes have an active site that is composed of a nucleophile (Ser), a base (His), and an acid (Glu) that is commonly known as a catalytic triad. It has previously been observed that the majority of carboxylesterases and lipases contain a second conserved serine in their active site [Proteins, 34 (1999) 184]. To investigate whether this second serine is also involved in the catalytic mechanism, it was mutated to an alanine, a glycine or a cysteine. Site-directed mutagenesis of this conserved serine resulted in a loss of specific activity, in both the S247G and S247A mutants (5- to 15-fold), which was due to a decrease in the rate of catalysis (kcat). Due to the instability of the S247C mutant no reliable data could be attained. A carbamate inhibitor, carbaryl, was then employed to investigate whether this decrease in the kcat was due to the rate of formation of the acyl-enzyme intermediate (k2) or the rate of deacylation (k3). The S247A mutant was found only to alter k2 (2.5-fold decrease), with no effect on k3. Together with information inferred from a human carboxylesterase crystal structure, it was concluded that this serine provides an important structural support for the spatial orientation of the glutamic acid, stabilizing the catalytic triad so that it can perform the hydrolysis.

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of alpha-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against alpha-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity., (Copyright 2003 Published by Elsevier Science (USA))