Enantiomeric separation of hydroxy and hydroperoxy eicosanoids by chiral column chromatography.

We describe high-performance liquid chromatography (HPLC) methods for the enantiomeric resolution of hydroxy and hydroperoxy fatty acids/eicosanoids using a Chiralpak AD or AD-RH chiral stationary phase. These columns achieve baseline resolution of all six positional/conjugated isomers of the hydroxy as well as of the hydroperoxy derivatives of arachidonic acid in chromatographic runs of less than 20 min. Hydro(pero)xy derivatives of linoleic and linolenic acids can be resolved with similar efficiencies. The individual hydroperoxy isomers are best resolved using the reversed-phase Chiralpak AD-RH column. For the synthesis of milligram quantities of enantiomerically pure hydro(pero)xy arachidonic acids, a simple scheme is presented starting with the autoxidation of the fatty acid methyl ester in the presence of 10% alpha-tocopherol followed by chromatographic purification of the positional isomers using a combination of reversed- and straight-phase HPLC columns. Mild alkaline hydrolysis of the methyl ester derivatives affords the free acids suitable for biological testing. The Chiralpak AD column appears to be efficient for the chiral resolution of prostaglandins and isoprostanes although a comprehensive evaluation is yet to be reported. For chiral analysis of endogenous hydroxy eicosanoids the availability of novel microflow Chiralpak capillary columns (0.3 mm i.d.) will be of great advantage, because sample sizes of a few nanograms can be analyzed using simple UV detection.

The hepoxilin connection in the epidermis.

The recent convergence of genetic and biochemical evidence on the activities of lipoxygenase (LOX) enzymes has implicated the production of hepoxilin derivatives (fatty acid epoxyalcohols) in the pathways leading to formation of the water-impermeable barrier of the outer epidermis. The enzymes 12R-LOX and eLOX3 are mutated in a rare form of congenital ichthyosis, and, in vitro, the two enzymes function together to convert arachidonic acid to a specific hepoxilin. Taken together, these lines of evidence suggest an involvement of these enzymes and their products in skin barrier function in all normal subjects. The natural occurrence of the specific hepoxilin products, and their biological role, whether structural or signaling, remain to be defined.

Epidermal lipoxygenase products of the hepoxilin pathway selectively activate the nuclear receptor PPARalpha.

Arachidonic acid can be transformed into a specific epoxyalcohol product via the sequential action of two epidermal lipoxygenases, 12R-LOX and eLOX3. Functional impairment of either lipoxygenase gene (ALOX12B or ALOXE3) results in ichthyosis, suggesting a role for the common epoxyalcohol product or its metabolites in the differentiation of normal human skin. Here we tested the ability of products derived from the epidermal LOX pathway to activate the peroxisome proliferator-activated receptors PPARalpha, gamma, and delta, which have been implicated in epidermal differentiation. Using a dual luciferase reporter assay in PC3 cells, the 12R-LOX/eLOX3-derived epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, activated PPARalpha with similar in potency to the known natural ligand, 8S-hydroxyeicosatetraenoic acid (8S-HETE) (both at 10 microM concentration). In contrast, the PPARgamma and PPARdelta receptor isoforms were not activated by the epoxyalcohol. Activation of PPARalpha was also observed using the trihydroxy hydrolysis products (trioxilins) of the unstable epoxyalcohol. Of the four trioxilins isolated and characterized, the highest activation was observed with the isomer that is also formed by enzymatic hydrolysis of the epoxyalcohol. Formation of a ligand for the nuclear receptor PPARalpha may be one possibility by which 12R-LOX and eLOX3 contribute to epidermal differentiation.

Human and mouse eLOX3 have distinct substrate specificities: implications for their linkage with lipoxygenases in skin.

Genetic and biochemical evidence suggests a functional link between human 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3) in normal differentiation of the epidermis; LOX-derived fatty acid hydroperoxide is isomerized by the atypical eLOX3 into a specific epoxyalcohol that is a potential mediator in the pathway. Mouse epidermis expresses a different complement of LOX enzymes, and therefore this metabolic linkage could differ. To test this concept, we compared the substrate specificities of recombinant mouse and human eLOX3 toward sixteen hydroperoxy stereoisomers of arachidonic and linoleic acids. Both enzymes metabolized R-hydroperoxides 2-3 times faster than the corresponding S enantiomers. Whereas 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) is the best substrate for human eLOX3 (2.4 s(-1); at 30 microM substrate), mouse eLOX3 shows the highest turnover with 8R-HPETE (2.9 s(-1)) followed by 8S-HPETE (1.3 s(-1)). Novel product structures were characterized from reactions of mouse eLOX3 with 5S-, 8R-, and 8S-HPETEs. 8S-HPETE is converted specifically to a single epoxyalcohol, identified as 10R-hydroxy-8S,9S-epoxyeicosa-5Z,11Z,14Z-trienoic acid. The substrate preference of mouse eLOX3 and the unique occurrence of an 8S-LOX enzyme in mouse skin point to a potential LOX pathway for the production of epoxyalcohol in murine epidermal differentiation.

Mutations associated with a congenital form of ichthyosis (NCIE) inactivate the epidermal lipoxygenases 12R-LOX and eLOX3.

Non-bullous congenital ichthyosiform erythroderma (NCIE) is one of the main clinical forms of ichthyosis. Genetic studies indicated that 12R-lipoxygenase (12R-LOX) or epidermal lipoxygenase-3 (eLOX3) was mutated in six families affected by NCIE [F. Jobard, C. Lefevre, A. Karaduman, C. Blanchet-Bardon, S. Emre, J. Weissenbach, M. Ozguc, M. Lathrop, J.F. Prud'homme, J. Fischer, Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1, Hum. Mol. Genet. 11 (2002) 107-113.], but the impact of these mutations on LOX function has not been defined. To explore this, we overexpressed the wild-type or mutated enzymes in E. coli and COS7 cells and then analyzed the essential catalytic properties. We showed recently that human eLOX3 is a hydroperoxide isomerase (hepoxilin synthase) that converts the product of 12R-LOX, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) to a specific epoxyalcohol. Using incubations with [(14)C]-labeled substrates and HPLC analyses, we found that the naturally occurring mutations totally eliminate the lipoxygenase activity of 12R-LOX and the hydroperoxide isomerase activity of eLOX3. We further demonstrate that the 12R-LOX/eLOX3-derived 8R-hydroxy-11R,12R-epoxide is converted by an epoxide hydrolase in COS7 cells and in human keratinocytes to a single isomer of 8,11,12-trihydroxyeicosa-5,9,14-trienoic acid. Taken together, the results support the hypothesis that 12R-LOX, eLOX3, and perhaps an epoxide hydrolase function together in the normal process of skin differentiation, and that the loss of function mutations are the basis of the LOX-dependent form of NCIE.

The lipoxygenase gene ALOXE3 implicated in skin differentiation encodes a hydroperoxide isomerase.

Lipoxygenase (LOX) enzymes form fatty acid hydroperoxides used in membrane remodeling and cell signaling. Mammalian epidermal LOX type 3 (eLOX3) is distinctive in totally lacking this typical oxygenase activity. Surprisingly, genetic evidence has linked mutations in eLOX3 or a colocalizing enzyme, 12R-LOX, to disruption of the normal permeability barrier of the skin [Jobard, F., Lefèvre, C., Karaduman, A., Blanchet-Bardon, C., Emre, S., Weissenbach, J., Ozgüc, M., Lathrop, M., Prud'homme, J. F. & Fischer, J. (2002) Hum. Mol. Genet. 11, 107-113]. Herein we identify a logical link of the biochemistry to the genetics. eLOX3 functions as a hydroperoxide isomerase (epoxyalcohol synthase) by using the product of 12R-LOX as the preferred substrate. 12R-Hydroperoxyeicosatetraenoic acid (12R-HPETE) is converted to 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, one of the isomers of hepoxilin A3, and to 12-ketoeicosatetraenoic acid in a 2:1 ratio. Other hydroperoxides, including 8R-HPETE, 12S-HPETE, and 15S-HPETE, as well as the 13S- and 13R-hydroperoxides of linoleic acid are converted less efficiently. Mass spectrometric analysis of the epoxyalcohol formed from [18O]15S-HPETE showed that both hydroperoxy oxygens are retained in the product. We propose that the ferrous form of eLOX3 initiates a redox cycle, unprecedented among LOX in being autocatalytic, in which the hydroperoxy substrate is isomerized to the epoxyalcohol or keto product. Our results provide strong biochemical evidence for a functional linkage of 12R-LOX and eLOX3 and clues into skin biochemistry and the etiology of ichthyosiform diseases in humans.