Improved enantioselective analysis of polyunsaturated hydroxy fatty acids in psoriatic skin scales using high-performance liquid chromatography

Enantioselective analysis is used as a valuable tool for determining the biological origin of chiral derivatives of arachidonic, 11,14-eicosadienoic and linoleic acid in psoriatic skin scales and for clarifying their role in pathogenesis. This paper reports on a simple and rapid enantioselective determination (without any derivatization) of the fatty acid derivatives 13(R,S)-hydroxyoctadecadienoic acid [13(R,S)-HODE], 9(R,S)-hydroxyoctadecadienoic acid [9(R,S)-HODE] and 12(R,S)-hydroxyeicosatetraenoic acid [12(R,S)-HETE], using high-performance liquid chromatography (HPLC) with Chiralpak AD as the chiral selector and electrospray ionisation mass spectrometry (ESI-MS). The enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE in psoriatic skin scales of untreated patients (untreated during the last 4 weeks before sampling) was evaluated in comparison to psoriatic skin scales of patients underlying systemic treatment. The enantiomeric distribution of 12-HETE and 9-HODE showed no remarkable differences, whilst samples of patients under systemic treatment exhibited a lower predominance of 13(S)-HODE than samples of untreated patients. Furthermore, the effect of UVB phototherapy on the enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE was studied and a semiquantitation of these compounds in psoriatic skin scales performed. The detected amounts of 9-HODE in samples of untreated patients were remarkably lower than those in samples of patients underlying systemic treatment. In the case of UVB phototherapy, no influence on the enantiomeric distribution could be observed.     

Free and esterified 13(R,S)-hydroxyoctadecadienoic acids: principal oxygenase products in psoriatic skin scales

Characterization of the chemical form and stereo-specificity of the fatty acid derivatives of arachidonic and linoleic acid in psoriatic epidermis is needed to define the enzymatic origin of these compounds and their possible role in pathogenesis. In an analysis of psoriatic skin scales, both free and esterified 13-hydroxyoctadecadienoic acids were the principal fatty acid derivatives, present in mean concentrations of 115 and 17 ng/mg scales, respectively. The analysis included reversed-phase high performance liquid chromatography of the free acid and of its methyl ester, gas chromatography, gas-liquid chromatography-mass spectrometry of the methyl ester derivatives, and chiral separation. The free and esterified 13-hydroxyoctadecadienoic acids isolated from the psoriatic scales contained a mixture of the S/R stereoisomers, averaging 1.9:1 for free 13- hydroxyoctadecadienoic acid. These findings are not compatible with the strict S-stereospecificity for oxygen insertion exhibited by mammalian lipoxygenase but rather could point to the action of a cyclooxygenase. The demonstration that a hydroxylated fatty acid derivative is esterified in vivo in psoriatic keratinocytes suggests that the physiology of these cells may be altered early in the process of keratinization.     

In vivo activation of an omega-6 oxygenase in human skin

To test the hypothesis that an epidermal fatty acid oxygenase is activated in vivo under physiologic conditions, surface lipids from normal human skin were analyzed for oxygenase products. With high-performance liquid chromatography on reversed-phase and straight-phase chiral columns and gas-liquid chromatography/mass spectrometry, these lipids were found to contain free 13-hydroxyoctadeca-9Z,11E-dienoic acid and 9-hydroxyoctadeca-10E,12Z-dienoic acid. The 13-hydroxyoctadecadienoic acid was present as a stereoisomeric mixture, with an average S/R ratio of 2.2, and exceeded the concentration of 9-hydroxyoctadecadienoic acid by a factor of 2. These observations and others indicate that the 13-hydroxyoctadecadienoic acid was derived mostly from an omega-6 oxygenase (probably 15-lipoxygenase) which is activated in vivo in normal skin.     

Toxoplasma gondii stimulates the release of 13- and 9-hydroxyoctadecadienoic acids by human platelets.

We have recently demonstrated a novel cytotoxic effect of human platelets against Toxoplasma gondii and a role for thromboxane (TX) in this process (Yong et al., 1991). We now report on the spectrum of lipid mediators released by human platelets after interaction with T. gondii. In addition to TXB2, human platelets after incubation with T. gondii for 90 min released 12-hydroxyheptadecatrienoic acid (12-HHT), 12-hydroxyeicosatetraenoic acid (12-HETE), and an unidentified peak (UVmax 234 nm) as determined by reverse-phase high-performance liquid chromatography. Thermospray-liquid chromatography/mass spectrometry analysis and straight-phase HPLC identified the unknown peak as a mixture of 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE. Radiolabeling studies with [14C]linoleic acid indicated that the platelets were the cellular source of the octadecanoids with 13-HODE (87.7%) greater than 9-HODE (12.3%). Inhibitor studies with indomethacin indicated that 13-HODE was a lipoxygenase product and 9-HODE was a cyclooxygenase product of linoleic acid. Thus, Toxoplasma-stimulated platelets release oxygenated products of both arachidonic acid and linoleic acid which may be important in the host response to T. gondii infection.     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Conversion of linoleic acid and arachidonic acid by skin epidermal lipoxygenases

Two different lipoxygenases have been identified in human and rat epidermis. One lipoxygenase has a (n-9)-specificity, converts arachidonic acid into 12-hydroxyeicosatetraenoic acid (12-HETE), and has been described by several investigators. Linoleic acid is not a substrate for this enzyme. The other lipoxygenase, with (n-6)-specificity, converts arachidonic acid into 15-HETE and linoleic acid into 13-hydroxyoctadecadienoic acid (13-HOD). Especially the latter lipoxygenase is thought to be involved in the regulation of the differentiation of the skin cells into a proper water-barrier layer. Linoleate is supposed to be the physiological substrate; this fatty acid is especially present in characteristic sphingolipids with unique structures.     

Demonstration of a 12-lipoxygenase activity in bovine polymorphonuclear leukocytes

In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.     

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.     

Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean

When linoleic acid was incubated with the purified potato lipoxygenase under O2 atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [+2] or [-2] lipoxygenases.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

The epithelium, endothelium, and stroma of the rabbit cornea generate (12S)-hydroxyeicosatetraenoic acid as the main lipoxygenase metabolite in response to injury

Previous work has shown that, shortly after rabbit corneas are injured, arachidonic acid metabolism is activated, and 12-hydroxyeicosatetraenoic acid (12-HETE) is one of the main products formed (Bazan, H. E. P., Birkle, D. L., Beuerman, R., and Bazan, N. G. (1985) Invest. Ophthalmol. & Visual Sci. 26, 474-480; Bazan, H. E. P. (1987) Invest. Ophthalmol. Visual Sci. 28, 314-319). In order to determine whether this metabolite is a lipoxygenase product, anesthetized rabbit corneas injured in vivo, either cryogenically or by 1 M NaOH, were subsequently incubated in vitro with [14C] arachidonic acid in the presence of indomethacin. 12-HETE was the main metabolite produced, as established by gas chromatography-mass spectrometry. The (R)- and (S)-enantiomers of novel naphthoyl-pentafluorobenzoyl derivatives of 12-HETE were resolved by chiral-phase high performance liquid chromatography. The radiolabeled 12-HETE from whole cornea and from isolated epithelium, endothelium, or stroma eluted as a single peak co-chromatographing with the (S)-enantiomer and was detected both by UV absorbance at 234 nm and by radioactivity. In noninjured corneas a smaller peak of radiolabeled (12S)-HETE was also eluted from the chiral column. The stereochemistry was additionally confirmed by liquid chromatography-mass spectrometry. These studies suggest that (12S)-lipoxygenase is activated in the injured rabbit cornea.     

Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry.

A method for determination of the lipoxygenase products of linoleic acid (9- and 13-hydroxyoctadecadienoic acid; 9-HODE, 13-HODE) and of arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid; 5-, 8-, 9-, 11-, 12-, and 15-HETE) is described. The method combines solid-phase extraction, derivatization to the corresponding fully hydrogenated methylester/trimethylsilylether derivatives and capillary gas chromatography coupled with electron impact mass spectrometry. Each regioisomeric HODE and HETE shows a unique pair of mass spectrometric fragment ions originating from fission of the fatty acid carbon chain at the hydroxylated position. The carboxyl-terminal fragment is used for quantification relative to a carboxyl-18O2-labeled analogue added as internal standard and the methyl-terminal fragment is monitored for confirmation. The assay can be extended for quantification of the complete hydroxylation profile of linoleic and arachidonic acid. Applications of this assay are demonstrated for the quantification of HODEs and HETEs in normal, hyperplastic, and neoplastic mouse epidermis. In mouse epidermis papilloma, the tissue levels of 8- and 12-HETE were found to be increased by one to two orders of magnitude compared to levels in normal epidermis.     

Lipid lymphocyte chemoattractants in psoriasis

In view of the evidence that lymphocyte infiltrates are a constant feature of the skin lesions of psoriasis and the demonstration that certain hydroxylated metabolites of arachidonic acid are present in lesional psoriatic skin and possess lymphocyte chemoattractant properties, lipid extracts of samples from lesional and normal skin were assayed to determine which are the predominant lipid lymphocyte chemoattractants in psoriasis. Dilution-related lymphocyte chemoattractant activity was found in lipid extracts of stratum corneum samples from psoriatic lesions, but not in similar extracts the samples from both sources contained equivalent amounts of this activity. Subsequent purification of lesional stratum corneum lipid extracts by straight and reversed phase high performance liquid chromatography (HPLC) revealed the presence of at least two different lipid chemoattractants, one major component being identified as 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12[R]-HETE) by its biological and chromatographic properties. These compounds may play a role in the pathogenesis of the lymphocyte infiltrates in psoriatic lesions.     

Role of peroxisomal oxidation in the conversion of arachidonic acid to eicosatrienoic acid in human skin fibroblasts.

Human skin fibroblasts converted [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]20:4) to eicosatrienoic acid (20:3), but appreciable amounts of radiolabeled 20:3 were not detected in corresponding incubations with [1-(14)C]20:4. This indicates that the main pathway for synthesizing 20:3 from arachidonic acid in the fibroblast involves oxidative removal of the carboxyl group of arachidonic acid. Fibroblasts deficient in long-chain acyl coenzyme A dehydrogenase (LCAD) converted [3H]20:4 to [3H]20:3. However, Zellweger fibroblasts that are deficient in peroxisomal fatty acid oxidation did not, indicating that the oxidative removal of the carboxyl group occurs in the peroxisomes. [3H]Hexadecatrienoic acid (16:3) was the main product that accumulated when [3H]20:4 was incubated with normal, LCAD deficient, and very long-chain acyl coenzyme A dehydrogenase (VLCAD) deficient fibroblasts, but Zellweger fibroblasts did not form this product. Normal fibroblasts converted [3H]16:3 to radiolabeled 20:3 and arachidonic acid. These findings suggest that some of the 16:3 produced from arachidonic acid by peroxisomal beta-oxidation can be recycled and that this recycling process constitutes a novel pathway for the conversion of arachidonic acid to 20:3 in human fibroblasts.     

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.     

Inhibition of fatty acid transport and proliferative activity in tissue-isolated human squamous cell cancer xenografts perfused in situ with melatonin or eicosapentaenoic or conjugated linoleic acids

Melatonin and eicosapentaenoic and 10t,12c-conjugated linoleic acids suppress the growth-stimulating effects of linoleic acid (LA) and its metabolism to the mitogenic agent 13-(S)-hydroxyoctadecadienoic acid (13-(S)-HODE) in established rodent tumors and human cancer xenografts. Here we compared the effects of these 3 inhibitory agents on growth and LA uptake and metabolism in human FaDu squamous cell carcinoma xenografts perfused in situ in male nude rats. Results demonstrated that these agents caused rapid inhibition of LA uptake, tumor cAMP content, 13-(S)-HODE formation, extracellular signal-regulated kinase p44/ p42 (ERK 1/2) activity, mitogen-activated protein kinase kinase (MEK) activity, and [3H]thymidine incorporation into tumor DNA. Melatonin's inhibitory effects were reversible with either the melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-bromoadenosine-cAMP, suggesting that its growth-inhibitory effect occurs in vivo via a receptor-mediated, pertussis-toxin-sensitive pathway.     

Synthesis and applications of stereospecifically (3)H-labeled arachidonic acids as mechanistic probes for lipoxygenase and cyclooxygenase catalysis

Stereospecifically (3)H-labeled substrates are useful tools in studying the mechanism of hydrogen abstractions involved in the oxygenation of polyunsaturated fatty acids. Here, we describe modified methods for the synthesis of arachidonic acids labeled with a single chiral tritium on the methylene groups at carbons 10 or 13. The appropriate starting material is a ketooctadecanoic acid which is prepared from an unsaturated C18 fatty acid precursor or by total synthesis. The (3)H label is introduced by NaB(3)H(4) reduction and the resulting tritiated hydroxy fatty acid then is tosylated, separated into the enantiomers by chiral phase HPLC, and subsequently transformed into stearic acids. A variety of stereospecifically labeled unsaturated fatty acids are obtained using literature methods of microbial transformation with the fungus Saprolegnia parasitica. Two applications are described: (i) In incubations of [10S-(3)H]- and [10R-(3)H]arachidonic acids in human psoriatic scales we show that a 12R-lipoxygenase accounts not only for synthesis of the major product 12R-HETE, but it contributes also, through subsequent isomerization, to the minor amounts of 12S-HETE. (ii) The [10R-(3)H]- and [10S-(3)H]arachidonic acids were also used to demonstrate that prostaglandin ring formation by cyclooxygenases does not involve carbocation formation at C-10 of arachidonic acid as was hypothesized recently.     

Conversion of linoleic acid into arachidonic acid by cultured murine and human keratinocytes

The origin of arachidonic acid (AA) found in the epidermis is not known. Two possibilities exist: either de novo synthesis within the epidermal keratinocyte, or transport of AA formed at distant tissue sites. The current study examined the ability of cultured murine and human keratinocytes to metabolize exogenously added linoleic acid (LA). Conversion of radiolabeled substrate (14C-LA) into 18:3(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) (AA) was noted. The conversion of non-radiolabeled 18:3(n-6) or 20:2(n-6) was also examined and the pattern of metabolites synthesized suggests that the preferred metabolic pathway for conversion of linoleic acid into arachidonic acid is via the classically described pathway in which a delta 6 desaturase constitutes the initial reaction. Although cultured skin fibroblasts are known to convert linoleic acid into arachidonic acid, the current study demonstrates that cultured epidermal keratinocytes can also avidly metabolize exogenous linoleic acid. The ability of cultured keratinocytes, and not of whole epidermis in vivo, to convert linoleic acid into arachidonic acid suggests that specific enzymatic activities may be induced by the tissue culture system itself. Hence, findings of metabolic capabilities in cultured cells may not necessarily be extrapolated to the in vivo situation.