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Characterisation, synthesis and functional investigation of epidermal lipoxygenase products in inflammation

Final Report Summary - LOXEPI (Characterisation, synthesis and functional investigation of epidermal lipoxygenase products in inflammation)

The aim of this project is to identify and characterize novel lipids, termed esterified lipoxygenase (LOX) products in the epidermis of both mouse and human skin. Lipoxygenases are a family of non-heme enzymes that catalyze oxygen insertion into a polyunsaturated fatty acid, yielding a hydroperoxide. Specific isoforms of the LOX enzymes reside in the skin although their range of products and function are currently unknown. Characterization of these lipids will deepen our understanding of how the barrier function of the epidermis is both formed and maintained.

Three specific objectives were detailed in the proposal;
(1) Structural characterization of esterified LOX products in skin
(2) Chemical synthesis of esterified eicosanoid standards, setting up quantitative assay and their application to murine skin samples from LOX-deficient and GsdmA3Dfl/+ skin
(3) Studies of bioactivity of esterified LOX products in healthy skin and in GsdmA3Dfl/+ mice, and backcrossing to determine role of these lipids in skin inflammation.
Progress made towards each section, including results obtained and future experiments, is outlined below.

Year 1

(1) Structural characterization of esterified LOX products in skin

Using a combination of HPLC-UV, HPLC-mass spectrometry and GC–mass spectrometry, several classes of novel linoleoyl-ω-hydroxyceramide and their eicosanoid derivatives have been identified in human and porcine epidermis. Porcine epidermis was initially used, as this is representative in terms of lipid content, of human skin. These products form via the consecutive action of 12R-LOX and eLOX3 on esterified linoleic acid (LA), and include an epoxy-alcohol derivative and its subsequent tri-hydroxy hydrolysis product. A recent publication from the Brash laboratory proposed that the oxidation of LA in these ceramides is required for freeing the ω-hydroxyceramide for covalent binding to protein, thus forming the micro-architecture of the human skin. The first year of the project involved the development of extraction and HPLC methods that allow the ceramide lipids to be accurately profiled with the use of mass spectrometry. Using these methods, I identified discrete sets of ceramides including novel LOX oxidized products and glucosylated ceramides. Linoleoyl-ω-hydroxyceramide (m/z 994-1078) and glucosyl analogues (m/z 1142-1238) represented ~10% and ~26% of total esterified ω-hydroxyceramide respectively. Less prominent peaks containing the epoxy-alcohol linoleate derivative (m/z 1040-1126) corresponded to ~3% of total esterified ω-hydroxyceramide in human skin. The putative trihydroxy-linoleate ceramide esters (m/z 1036-1100) with acyl chain lengths of C30:0-C34:0 were detected at ~2% of total. Structural characterization of the oxidized LA is now underway focusing on the stereochemistry of products obtained.

During the investigations into the formation of these ceramides, it became clear that cleavage of the oxidized LA is an important step in barrier formation. As a result, and given the laboratory’s expertise in protein expression, investigations into the enzyme that facilitates this cleavage were carried out. In pursuit of the esterase/hydrolase that specifically cleaves the oxidized linoleate from ceramide, a candidate hydrolase, secretory phospholipase A2 (sPLA2-IIF) was expressed in E.coli and its activity characterized with dilauroyl-phosphocholine liposomes containing oxidized stearoyl-linoleoyl-phosphoethanolamine (SLPE). It was found that sPLA2-IIF selectively hydrolyzes the oxidation products of SLPE, preferentially cleaving ~15% of available 9R- HPODE vs. ~3.5% linoleate, a selectivity relevant to processing of oxidized linoleate among polar lipids in the epidermis. Further experiments involving the incorporation of oxidized ceramides will be conducted to determine the role of this epidermal specific hydrolase.

Year 2

To determine the exact molecular species including the structural isomers, a series of oxidized LA fatty acids were synthesized chemically and using a novel enzyme found in beetroot. The non esterified trihydroxy acids were generated using a hydroperoxide precursor, and through reaction with hematin and subsequent hydrolysis, 4 structural isomers were generated and characterized in detail using a combination of 1H NMR, gas chromatography–mass spectrometry and circular dichroism. Thus, a set of analytical standards was generated, for later use during skin product analysis. The 1H NMR used to determine the chirality of the products formed needed development as an existing method was not available. The development of these assays and analysis of an enzymatically formed trihydroxy product from beetroot formed the subject of a manuscript due to be submitted to Chemistry and Physics of Lipids. This analysis method will be used to identify the structure of the trihydroxy products formed within the skin, and will enable confirmation of the hypothesis that these products are formed enzymatically during barrier formation.

The generation of the trihydroxy products in-vivo is likely to involve soluble epoxide hydrolase (sEH). As part of the Winter Eicosanoid Conference held in Baltimore on the 11th March 2012, I presented a poster on the work described here. Through this I discussed my work with Dr. Anette Cronin from the University of Zurich who studies these sEH enzymes. Collaboration was initiated and recombinant enzymes were kindly sent to me to test the activity with the oxidized LA products, with this area of research currently ongoing.

Another collaboration with Dr. Erin Seeley in the Vanderbilt Mass Spectrometry Tissue Profiling and Imaging Core allowed initial studies examining the location within the epidermal tissue of specific lipids. This yielded very interesting results concerning localization of the ceramide species tentatively assigned within the tissue. It would appear that different ceramides localize in particular regions of the epidermis. Work is needed to confirm structures of these ceramides and in particular examine localization of oxidized ceramides in different regions within the epidermis.


(2) Chemical synthesis of esterified eicosanoid standards, setting up quantitative assay and its application to murine skin samples from LOX-deficient and GsdmA3Dfl/+ skin

Biochemical synthesis and characterization of products are currently ongoing employing the use of recombinant lipoxygenase enzymes and a variety of analytical approaches. These include, chiral HPLC, mass spectrometry and 1H NMR analysis.

The quantitative assay was being developed in Vanderbilt just before the return phase of the grant began. This involved monitoring one particular ceramide of each species using mass spectrometry i.e. one particular un-oxidized linoleoyl-ω-hydroxyceramide with a specific mass, one particular epoxy-hydroxy ceramide with a specific mass etc. The ceramides followed represented the most abundant of each species. Through separation of the stratum corneum (the outer most skin layer) and the remaining epidermis and following the specific ceramides it was possible to show that the ceramides of interest in this project i.e. the LOX products were confined to the stratum corneum. This discovery has implications concerning the action of these lipids in the barrier formation of the skin. These methods must now be developed further to enable the use of the mass spectrometry instruments at Cardiff.

Chemical synthesis is an on-going project as starting materials must first be isolated in sufficient amounts for further use.

(3) Studies of bioactivity of esterified LOX products in healthy skin and in GsdmA3Dfl/+ mice, and backcrossing to determine role of LOX in skin inflammation

This objective is scheduled for investigation in year 3 of the fellowship in Cardiff as stated in Research Methodology.


Year 3

Since returning to Cardiff University my focus has been on quantitation of tri-hydroxy compounds in human skin utilizing the available mass spectrometry in the Cardiff Lipidomic Group. Summarized below is the overall progress towards the specific objectives as detailed above in this reporting period.

(1) Structural characterization of esterified LOX products in skin

A collaboration initiated with Prof. James Birchall at the School of Pharmacy and Pharmaceutical Sciences at Cardiff has allowed human skin samples to be obtained and analyzed for the presence of LOX metabolites and continuing the work carried out at Vanderbilt. Use of a Thermo Orbitrap Elite mass spectrometer at Cardiff has allowed detailed characterization of free tri-hydroxy fatty acids with accurate mass determination up to 4 decimal places. This enables exact molecular and fragment structures to be determined and therefore structures to be defined. Using a combination of the mass accuracy of the Orbitrap Elite system and the sensitivity of the ABSciex 4000 Q-Trap, I showed that skin contains 2 isomers of tri-hydroxy linoleate derivatives, namely the 9,10,13 and the 9,12,13 tri-hydroxy linoleate. These are likely derived from a single epoxy-hydroxy linoleate, 9,10-epoxy, 13-hydroxy. The results suggest a non-enzymatic hydrolysis of the epoxide moiety but this is in the process of being confirmed with the soluble epoxide hydrolase enzymes as discussed above. Both the 9,10,13 and the 9,12,13 tri-hydroxy have now been quantified with levels in the high ng – μg region and therefore highly likely to be involved in the physiology of the skin. Further experiments would determine their role in inflammation and immunity by study of possible receptors.
Due to the difference in the mass spectrometers available at Vanderbilt and Cardiff the methods employed to study the ceramide and free fatty acid tri-hydroxy linoleates needed development. In the case of the free acid moieties this has been completed and has allowed quantitation. The ceramide analysis method is in the final stages of completion and methods are being employed to quantitate the ceramide tri-hydroxy species.
Ethical approval from the Cardiff University School of Medicine Research Ethics Committee allowed tape-stripping experiments to begin which involves the analysis of individual layers of human skin removed by small sections of adhesive tape. Analysis of the depth profile of the LOX products in the skin of a range of volunteers will allow an overview of the levels of these products and their distribution within normal healthy skin.

(2) Chemical synthesis of esterified eicosanoid standards, setting up quantitative assay and its application to murine skin samples from LOX-deficient and GsdmA3Dfl/+ skin

During this project several methods were investigated using recombinant enzyme systems in an attempt to synthesis enough oxidized material to be used as standards and in formulations to add to the skin. These are summarized below.

1. Isolation of linoleoyl-ω-hydroxyceramide was performed from large-scale porcine skin extraction and subsequent HPLC purification. The crudely purified substrate was then incubated with 12R-LOX to generate hydroperoxy intermediates that were then incubated with eLOX3 to generate the epoxy-hydroxy derivative. These experiments experienced several procedural difficulties, as the solubility of the large, very long chain ceramides was restricted to organic solvents unsuitable for the enzymes. To overcome this several liposome models were employed to package the ceramides as they would be expected in the epidermis. Unfortunately very low quantities of oxidized ceramides were generated in this manner.
2. Isolation of ω-hydroxyceramide was performed from large-scale porcine skin extraction and subsequent HPLC purification. This substrate was used in a previously described rat liver microsome system designed to couple a fatty acid to a free hydroxyl moiety. Again these experiments were unsuccessful in generating the required quantities of product.
3. The isolated linoleoyl-ω-hydroxyceramide was used in an auto-oxidation system where a free radical initiator is used to generate a radical reaction in an oxygen rich atmosphere. This would generate a range of oxidized linoleate species that could be purified by HPLC. These experiments provided very little product for further experiments.

The methods detailed above did not yield sufficient quantities of oxidized ceramide product to add to the LOX-deficient and GsdmA3Dfl/+ skin. In this respect this specific objective could not be completed.

(3) Studies of bioactivity of esterified LOX products in healthy skin and in GsdmA3Dfl/+ mice, and backcrossing to determine role of LOX in skin inflammation

Upon return to Cardiff in the final year of this project it was proposed to initiate crossing experiments with LOX deficient and GsdmA3Dfl/+ mice. The project on which the GsdmA3Dfl/+ mice had been kept unfortunately was not continued and so the mice could not be maintained at Cardiff. Embryos from the mice were frozen but the cost of regenerating this line was not within budget of this project. This objective could not be completed.


Main results
The main results achieved so far in the project are as follows:
1. LOX products have been detected for the first time in human skin in several forms including free acid products and ceramides.
2. Novel analysis methods for structural identification have been developed and published.
3. LOX product lipid structures have been characterized and quantified for the first time.
4. A novel mass spectrometry imaging system has revealed that particular ceramides localize in discrete regions within the epidermis.
5. Small quantities of oxidized lipid standards have been generated.
6. A PLA2 enzyme implicated in the generation of free acid LOX products in the epidermis has been cloned and expressed. Use of the enzyme has demonstrated a preferential activity towards oxidized linoleate lipids.

Expected final results and impact
It is expected that the oxidized ceramide content of human skin will be established. The discovery of these lipids and their free acid counterparts opens a new paradigm in epidermal research. These previously un-described products represent a novel enzyme function within the skin and yields exciting opportunities to further explore the bioactivity of these products and their role with skin function.