O'Don n ell, Vale rie B. ORCID: h t t p s://o rcid.o r g/000 0-0 0 0 3-4 0 8 9-8 4 6 0, Fi tzGe r ald, Ga r r e t A., M u r p hy, Rob e r t C., Liebisch, Ge r h a r d , De n nis, E dw a r d A., Qu e h e n b e r g er, Os w ald, S u b r a m a nia m, S h a nk a r a n d Wakela m, Mich a el J.O. 2 0 2 0. S t e p s Tow a r d Mini m al Re po r ting S t a n d a r d s for Lipido mics M a s s S p e c t ro m e t ry in Biom e dic al Res e a rc h P u blica tions. Circ ula tion: Ge no mic a n d P r e cision M e dicin e 1 3 (6) , e 0 0 3 0 1 9. 1 0.11 6 1/CIRCGEN.120.0 03 0 1 9 file

Supplementary Methods. Supplementary Mass spectrometric methodology

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA 3 ). Herein, we demonstrate that signi fi cant amounts of DXA 3 are rapidly attached to phosphatidylethanolamine (PE) forming four esteri fi ed eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA 3 -PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA 3 generated by platelets, are formed in ng amounts (24.3 ±6.1 ng/2×10 8 ) and remain membrane bound. Pharmacological studies revealed DXA 3 -PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A 2 (cPLA 2 ), phospholipase C and intracellular calcium. They are generated primarily via esteri fi cation of newly formed DXA 3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA 3 -PEs were detected in human clots. Puri fi ed platelet DXA 3 -PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

Table S1 shows the composition of diets fed to F344 rats.

Rat body weights in the groups fed different EPA:omega-6 fatty acid ratios.

Table S2 shows the molar % of fatty acids in the colon at the fed EPA:AA ratios.

<p>Table Supplementary S-I. Glycerophosphocholine species generated in response to etoposide treatment of B16F10 cells</p>

Table S3 shows the eicosanoid concentrations in the colon at the fed EPA:AA ratios.

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx). The precise structure and localization of LOX-oxidized EOx in the human epidermis is unknown, as is their regulation in diseases such as psoriasis, one of the most common inflammatory diseases affecting the skin. Here, using precursor LC/MS/MS, we characterized multiple intermediates of EOx, including 9-HODE, 9,10-epoxy-13-HOME, and 9,10,13-TriHOME, in healthy human epidermis likely to be formed via the epidermal LOX pathways. The top layers of the skin contained more LA, 9-HODE, and 9,10,13-TriHOME EOSs, whereas 9,10-epoxy-13-HOME EOS was more prevalent deeper in the stratum corneum. In psoriatic lesions, levels of native EOx and free HODEs and HOMEs were significantly elevated, whereas oxidized species were generally reduced. A transcriptional network analysis of human psoriatic lesions identified significantly elevated expression of the entire biosynthetic/metabolic pathway for oxygenated ceramides, suggesting a regulatory function for EOx lipids in reconstituting epidermal integrity. The role of these new lipids in progression or resolution of psoriasis is currently unknown. We also discovered the central coordinated role of the zinc finger protein transcription factor, ZIC1, in driving the phenotype of this disease. In summary, long-chain oxygenated ceramide metabolism is dysregulated at the lipidomic level in psoriasis, likely driven by the transcriptional differences also observed, and we identified ZIC1 as a potential regulatory target for future therapeutic interventions.

Mass spectrometry has played a critical role in the identification and quantitation of lipids present in biological extracts. Various strategies have emerged in order to carry out lipidomic studies. These include both shotgun approaches as well as those engaging liquid chromatographic separation of lipid species prior to mass spectrometric analysis. Nonetheless challenges remain at every level of the lipidomic experiment, including extraction of lipids, identification of specific species, and quantitation of the vast array of lipids present in the sample extract. New strategies have emerged to address some of these issues; however, precise quantitation remains a significant challenge. The use of the ratio of the abundance of the molecular ion species to that of an internal standard enables quite accurate assessment of fold changes within complex lipid species without the need for exact quantitation. Challenges continue to remain in terms of availability of reference standard material as well as relevant internal standards.

Having a preterm infant is challenging for parents and families, with increased risk of psychological stress. Being separated from an infant, and dealing with the disruption to family life can impact on the development of parent – infant interaction and early bonding. These early interactive experiences are important in the development of communication skills. The antecedents for receptive and expressive language development can be supported and shaped in the neonatal unit. As preterm infants are at risk of speech, language and communication difficulties, providing parents with information about language development and strategies to promote communication are essential to integrate into neonatal care. This systematic review identified only five papers which investigated parent – infant interaction and the specific attributes associated with providing a good communication environment on the NICU. Due to the small number and differing outcome measures, a full meta –analysis was not possible. The authors recommend a clearer distinction between language and communication literature alongside investigations which have studied bonding and improving mental health for carers post pre-term birth.

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS3), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA(3)). Here, we achieved enzymatic synthesis and H-1 NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-hydroperoxy-eicosatetraenoic acid (11-HpETE) and thereby identified a candidate sharing the precise reverse-phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by H-1 NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA(3). We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern and, using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and four isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.

Chronic inflammation and oxidative stress are critical components in the pathogenic cascade of early diabetic retinopathy, characterized by neuronal and vascular degeneration. We investigated pharmacologic inhibition of the proinflammatory leukotriene cascade for therapeutic benefit in early diabetic retinopathy. Using the streptozotocin-induced diabetes mouse model, we administered montelukast, a leukotriene receptor antagonist, and diabetes-related retinal pathology was assessed. Early biochemical and cellular function measures were evaluated at 3 months’ diabetes duration and included vascular permeability, superoxide production, leukotriene generation, leukocyte-induced microvascular endothelial cell death, and retinal function by electroretinography. Histopathology assessments at 9 months’ diabetes duration included capillary degeneration and retinal ganglion cell loss. Leukotriene receptor antagonism resulted in a significant reduction of early, diabetes-induced retinal capillary leakage, superoxide generation, leukocyte adherence, and leukotriene generation. After 9 months of diabetes, the retinal microvasculature from untreated diabetic mice demonstrated a nearly threefold increase in capillary degeneration compared with nondiabetic mice. Montelukast inhibited the diabetes-induced capillary and neuronal degeneration, whether administered as a prevention strategy, immediately after induction of diabetes, or as an intervention strategy starting at 4.5 months after confirmation of diabetes. Pharmacologic blockade of the leukotriene pathway holds potential as a novel therapy to prevent or slow the development of diabetic retinopathy.

Enzymatically oxidized phospholipids (eoxPLs) are formed through regulated processes by which eicosanoids or prostaglandins are attached to phospholipids (PLs) in immune cells. These eoxPLs comprise structurally diverse families of biomolecules with potent bioactivities, and they have important immunoregulatory roles in both health and disease. The formation of oxPLs through enzymatic pathways and their signaling capabilities are emerging concepts. This paradigm is changing our understanding of eicosanoid, prostaglandin, and PL biology in health and disease. eoxPLs have roles in cellular events such as ferroptosis, apoptosis, and blood clotting and diseases such as arthritis, diabetes, and cardiovascular disease. They are increasingly recognized as endogenous bioactive mediators and potential targets for drug development. This review will describe recent evidence that places eoxPLs and their biosynthetic pathways center stage in immunoregulation.

Ion mobility measurements of product ions were used to characterize the collisional cross section (CCS) of various complex lipid [M-H]- ions using traveling wave ion mobility mass spectrometry (TWIMS). TWIMS analysis of various product ions derived after collisional activation of mono- and dihydroxy arachidonate metabolites was found to be more complex than the analysis of intact molecular ions and provided some insight into molecular mechanisms involved in product ion formation. The CCS observed for the molecular ion [M-H]- and certain product ions were consistent with a folded ion structure, the latter predicted by the proposed mechanisms of product ion formation. Unexpectedly, product ions from [M-H-H2O-CO2]- and [M-H-H2O]- displayed complex ion mobility profiles suggesting multiple mechanisms of ion formation. The [M-H-H2O]- ion from LTB4 was studied in more detail using both nitrogen and helium as the drift gas in the ion mobility cell. One population of [M-H-H2O]- product ions from LTB4 was consistent with formation of covalent ring structures, while the ions displaying a higher CCS were consistent with a more open-chain structure. Using molecular dynamics and theoretical CCS calculations, energy minimized structures of those product ions with the open-chain structures were found to have a higher CCS than a folded molecular ion structure. The measurement of product ion mobility can be an additional and unique signature of eicosanoids measured by LC-MS/MS techniques. Graphical Abstract ᅟ.

Murine mastocytoma cells ated with um ionophore A.23187 prod a slow-reacting substan (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist 12. On the basis of isotope incorporation experiments, spectroscopy, and chemical degra" dation% the SRS was identified as a cysteine-containing derivative of 5"hydroxy-7,9,11,14-icosatetraenoie acid. This amino acid was attached in thioether linkage at C-6. The SRS is structurally related to previously identified epoxy and dihydroxy metabolites of arachidonic acid in leukocytes. A common feature is the presence of a conjugated triene, and the name "leakotrienc" has been introduced to designate these compounds. Leukotriene A (5,6-epoxy-7,9,11,14-icosatetraenoic acid) is an intermediate in the formation of leukotriene B (5,12-dihr droxy-6,8,10,14-icosatetraenoic acid) and is proposed to be a precursor also of leukotriene C, which is the SRS identified here.

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.