Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15alpha-hydroxylithocholic acid

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15alpha-hydroxylithocholic acid (3alpha,15alpha-dihydroxy-5beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta14 intermediate. The synthesis of its C-15 epimer, 15beta-hydroxylithocholic acid (3alpha,15beta-dihydroxy-5beta-cholan-24-oic acid), is also reported. The taurine conjugate of 15alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15alpha-hydroxylation of lithocholic acid that was formed by bacterial 7alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.     

Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S), 12(S)-dihydroxyeicosatetraenoic acid and 14(R), 15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5 - and 15 -hydroxyeicosatetraenoic acids, and 5,15 -dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)- dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R), 15(S)- dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 microm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)- dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 x 10(5) for 15 -HETE, 1.4 x 10(5) for 12-HETE, and 0.9 x 10(5) for 5 -HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.     

Arachidonic acid 15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Partial purification and properties

Arachidonic acid 15-lipoxygenase was purified from rabbit peritoneal polymorphonuclear leukocytes. The enzyme was recovered in the cytosol fraction after sonication and purified about 250-fold by acetone precipitation, column chromatography on CM52, Sephadex G-150, and hydroxyapatite. The enzyme catalyzed the conversion of arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which then decomposed to a mixture of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), 15-keto-5,8,11,13-eicosatetraenoic acid, 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid. The enzyme was specific for oxygenation at carbon 15 of arachidonic acid. The apparent molecular weight of the enzyme was about 61,000 as measured by Sephadex G-150 gel filtration chromatography. The enzyme was sensitive to sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid. The enzyme activity was inhibited by eicosatetraynoic acid (ETYA) or 3-amino-1-(m-(trifluoromethyl)-phenyl)2-pyrazoline (BW755C), but not by indomethacin up to 200 micrograms/ml.     

FXR agonists and FGF15 reduce fecal bile acid excretion in a mouse model of bile acid malabsorption

Bile acid malabsorption, which in patients leads to excessive fecal bile acid excretion and diarrhea, is characterized by a vicious cycle in which the feedback regulation of bile acid synthesis is interrupted, resulting in additional bile acid production. Feedback regulation of bile acid synthesis is under the control of an endocrine pathway wherein activation of the nuclear bile acid receptor, farnesoid X receptor (FXR), induces enteric expression of the hormone, fibroblast growth factor 15 (FGF15). In liver, FGF15 acts together with FXR-mediated expression of small heterodimer partner to repress bile acid synthesis. Here, we show that the FXR-FGF15 pathway is disrupted in mice lacking apical ileal bile acid transporter, a model of bile acid malabsorption. Treatment of Asbt-/- mice with either a synthetic FXR agonist or FGF15 downregulates hepatic cholesterol 7alpha-hydroxylase mRNA levels, decreases bile acid pool size, and reduces fecal bile acid excretion. These findings suggest that FXR agonists or FGF15 could be used therapeutically to interrupt the cycle of excessive bile acid production in patients with bile acid malabsorption.     

Novel biological transformations of 15-Ls-hydroperoxy-5,8,11,13-eicosatetraenoic acid

[1-14C]Arachidonic acid was incubated with homogenates of the fungus, Saprolegnia parasitica. The products consisted of comparable amounts of two epoxy alcohols, 15-Ls-hydroxy-11,12-epoxy-5cis,8cis,13trans- eicosatrienoic acid and 15-hydroxy-13,14-epoxy-5cis,8cis,11cis-eicosatrienoic acid. Results of incubations carried out in the presence of nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, p-hydroxymercuribenzoate as well as glutathione peroxidase plus reduced glutathione demonstrated that transformation of arachidonic acid into epoxy alcohols occurred with the formation of 15-Ls-hydroperoxy-5cis,8cis,11cis,13trans- eicosatetraenoic acid (15-HPETE) as an intermediate. The pathway involved a lipoxygenase catalyzing the oxygenation of arachidonic acid at the 15L position to produce 15-HPETE, and a hydroperoxide isomerase activity which catalyzed conversion of 15-HPETE into the two epoxy alcohols. Studies with 15-[18O2]HPETE demonstrated that both oxygens of 15-HPETE were retained in the epoxy alcohols. Furthermore, experiments with mixtures of 15-[18O2]-and 15-[16O2]HPETE showed that conversion of 15-HPETE into epoxy alcohols occurred by an intramolecular transfer of hydroperoxide oxygen.     

Labdane diterpenes from Cistus symphytifolius

The investigation of the aerial part of Cistus symphytifolius afforded, in addition to sitosterol, trimethoxykaempferol, cativic acid, labdenic acid, labdanolic acid and labdan-8α,15-diol, three new bicyclic diterpenes: cistadienic acid, cistenolic acid and labd-13(E)- ene-8α,15-diol. The structures of these were determined by spectral studies and correlations. CD spectral studies showed that cistenolic acid and salvic acid are enantiomeric compounds, so the stereochemistry of salvic acid (7α-hydroxy-labd-8(17)-ene-15-oic acid) should be changed to 7β-hydroxy-eperu-8(17)-ene-15-oic acid (7β-hydroxy-ent-labd-8(17)-ene-15-oic acid).     

A 15N NMR study on D-lysine metabolism in Neurospora crassa

The metabolic relationship of D-lysine, L-lysine, and L-pipecolic acid has been investigated in Neurospora crassa. Kinetic experiments show that radioactivity from D-lysine is efficiently incorporated into L-pipecolic acid and that this metabolite is converted to L-lysine. The alpha-amino group from D-[alpha-15N]lysine is lost in the course of its conversion to L-pipecolic acid and is trapped by pyruvate and alpha-keto glutarate to give L-[alpha-15N]alanine and L-[alpha-15N]glutamic acid. These amino acids are devoid of any label, however, when D-[epsilon-15N]lysine is applied to the fungus. As determined by mass and 15N NMR spectrometry the label from D-[epsilon-15N]lysine migrate via L-pipecolic acid into the alpha position of L-lysine, i.e. D-[epsilon-15N]lysine is converted to L-[alpha-15N]lysine. L-Pipecolic acid functions as an intermediate in this conversion.     

Transformation of arachidonic acid by 5- and 15-lipoxygenase pathways in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with isolated bovine adrenal fasciculata cells for 15 min at 37gC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), leukotriene B4 and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid (11,14,15-THET). Addition of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculata cells resulted in the formation of 11,14,15-THET. The formation of 11,14,15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome P-450 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculata cells and that 15-HPETE is further metabolized to 11,14,15-THET by adrenal microsomal cytochrome P-450.     

Significance of lipoxygenase-derived monohydroxy fatty acids in cutaneous biology

The skin displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA), an 18-carbon (n-6) PUFA, results in characteristic scaly skin disorder and excessive epidermal water loss. Although arachidonic acid (AA), a 20-carbon (n-6) PUFA, is metabolized via cyclooxygenase pathway into predominantly prostaglandin E2 (PGE2) and PGF2alpha. The 15-lipoygenase is very active in this tissue and catalyzes the transformation of 20-carbon AA into predominantly 15-hydroxyeicosatetraenoic acid (15-HETE). Similarly, the epidermal 15-lipoxygenase also catalyzes the transformation of 18-carbon LA and 20-carbon dihomo-gamma-linolenic acid (DGLA) to 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatrienoic acid (15-HETrE), respectively. The monohydroxy fatty acids are incorporated in phospholipids which undergo catalysis to yield substituted-diacylglycerols (13-HODE-DAG) and 15-HETrE-DAG) which exert anti-inflammatory/antiproliferative effects on the skin.     

Conditions for the formation of the oxo derivatives of arachidonic acid from platelet 12-lipoxygenase and soybean 15-lipoxygenase

Three carbonyl compounds derived from arachidonic acid have recently been characterized in human platelets, namely, 12-ketoeicosatetraenoic acid and two isomeric 12-oxododecatrienoic acids. The conditions for the synthesis of these compounds and for the synthesis of analogous products from soybean lipoxygenase, i.e., 15-ketoeicosatetraenoic acid and 15-oxopentadecatetraenoic acids, were compared with regard to the role of oxygen and fatty acid availability, and heme catalysis. Using platelet homogenates or soybean lipoxygenase and arachidonic acid as a substrate, it was found that the establishment of anaerobic conditions during the incubation was mandatory only for the synthesis of 15-oxopentadecatetraenoic acids. Anaerobic conditions, however, greatly increased the formation of 15-ketoeicosatetraenoic acid and, to a lesser extent, of 12-oxododecatrienoic acids. On the other hand, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was transformed into 12-ketoeicosatetraenoic acid and 12-oxododecatrienoic acids by platelet homogenates or soybean lipoxygenase. This transformation was increased when the incubation was performed in anaerobic conditions and in the presence of a fatty acid substrate of the enzyme. These data suggest that oxygen deprivation and excess fatty acid could play a stimulatory role in the synthesis of 12-oxo compounds by platelets. Finally, we have compared the heme-catalyzed generation of the 12-oxo and 15-oxo derivatives from their hydroperoxide precursors: whereas 12-oxododecatrienoic acids and 12-ketoeicosatetraenoic acid were formed in the proportion of 8.5: 1.5 from 12-HPETE incubated with hematin (150 nM), 15-ketoeicosatetraenoic acid was the only carbonyl compound generated from 15-HPETE in the same conditions, emphasizing the unique reactivity of the 12-HPETE.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14, 15-leukotriene A4

The purified lipoxygenase of rabbit reticulocytes converts arachidonic acid at 0 degrees C to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) via reactions which involve hydrogen abstraction at C-13 and C-10, respectively. At 37 degrees C the enzyme converts arachidonic acid to additional products which were identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, 8,15-dihydroperoxy-5,9,11,13- and 5,15-dihydroperoxy-6, 6,8,11,13-eicosatetraenoic acids (8,15-diHPETE and 5,15-HPETE, respectively) and diastereoisomers of 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-diHPETEs). The 8,15- and 5,15-diHPETEs were formed by double lipoxygenation since each incorporated 2 molecules of 18O2 and since their synthesis from 15-HPETE was blocked under anaerobic conditions. The 8,15-diHETEs each incorporated 18O from 18O2 at C-15 and were found to arise from nonenzymatic hydrolysis of an epoxytriene which was identified as 14,15-leukotriene A4 by trapping in acidic methanol. This compound was a major product of 15-HPETE in anaerobic incubations. The conversion of 15-HPETE to 14,15-leukotriene A4 was inhibited by the lipoxygenase inhibitors nordihydroguairetic acid and 5,8,11,14-eicosatetraynoic acid. The 14,15-leukotriene A4 synthase and 15-lipoxygenase activities were inhibited by 5,8,11,14-eicosatetraynoic acid in a similar time-dependent manner. The results support a mechanism whereby 14,15-leukotriene A4 is synthesized from 15-HPETE by a further enzymatic step carried out by the reticulocyte 15-lipoxygenase via hydrogen abstraction at C-10 and a redox cycle of the non-heme iron atom of the enzyme.     

Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis

L-[15N]Glutamic acid was prepared in high yields via a fermentative process. Brevibacterium lactofermentum, growing on a medium containing 97% enriched 15NH4Cl as a sole isotopic precursor, excreted mostly L-[15N]glutamic acid. The L-[15N]glutamic acid was purified and identified. Gas chromatography-mass spectrometry analysis was performed to demonstrate its usefulness in clinical studies.     

Inhibition of human leukocyte 5-lipoxygenase by 15-HPETE and related eicosanoids

The inhibition of human leukocyte 5-lipoxygenase by 15-hydroperoxyeicosatetraenoic acid and its chemical or enzymatic rearrangement products was investigated. 15-Hydroperoxyeicosatetraenoic acid was the most potent inhibitor tested. The inhibition was found to be time dependent and is not due to chemical or enzymatic decomposition products nor metabolism of 15-hydroperoxyeicosatetraenoic acid to 5,15-dihydroperoxyeicosatetraenoic acid.     

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.     

Modulation of Fcγ receptors on T cells and monocytes by 15 hydroperoxyeicosatetranoic acid

We investigated the effects of the 15-lipoxygenase products, 15 hydroxyeicosatetranoic acid (15 HPETE) and 15 hydroxyeicosatetric acid (15 HETE) on Fcγ receptor expression on human T coils and monocytes. Incubation of these cells with 15 HPETE but not 15 HETE results in a shift to decreased density of Fcγ receptors on the cell surface.     

15S-Lipoxygenase-2 mediates arachidonic acid-stimulated adhesion of human breast carcinoma cells through the activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.     

Formation of 15-HETE as a major hydroxyeicosatetraenoic acid in the atherosclerotic vessel wall

Atherosclerosis was induced in New Zealand White rabbits through cholesterol feeding. Aortae were taken out from treated animals and incubated with arachidonic acid. Aortae from cholesterol-fed animals converted arachidonic acid into 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). This conversion was not seen in aortae from control animals. The immediate precursor of 15-HETE, 15-HPETE, is an inhibitor of prostacyclin synthetase and might hamper prostacyclin production.     

Inhibition of thrombin-induced platelet arachidonic acid release by 15-hydroperoxy-arachidonic acid

Measuring platelet thromboxane B₂ biosynthesis by gas-liquid chromatography with capillary column, we found that 15-hydroperoxy-arachidonic acid (15-HPETE) abolished the above biosynthesis under thrombin stimulation but not from exogenous arachidonic acid. This fact indicates that 15-HPETE inhibits the release of arachidonic acid from platelet phospholipids. However, the hydroxy-derivative of 15-HPETE, called 15-HETE does not have this activity. In addition, 15-HPETE has no inhibiting effect on platelet phospholipase A₂ activity. Since, it has been previously published that 15-HPETE inhibits platelet diglyceride lipase, we conclude that the phosphatidylinositol specific phospholipase C-diglyceride lipase pathway could be essential in providing arachidonic acid from phospholipids, at least under low doses of thrombin.     

Inhibition of leukotriene biosynthesis by acetylenic analogs

The monoacetylenic acid, 5,6-dehydroarachidonic acid (5,6-DHA), inhibits the 5-lipoxygenase in RBL-1 extracts in a time-dependent irreversible manner. In intact cell systems, 5,6-DHA is not as effective as ETYA or 15(S)-HEYA in inhibiting the 5-lipoxygenase activities, because 5,6-DHA is metabolized into triglycerides, phospholipids and hydroxylated products. While lipoxygenation of arachidonic acid at C-5 and C-12 is inhibited by 15-HETE, the transformation of arachidonic acid into 5,15-diHETE via 15-HPETE in human leukocytes is relatively insensitive to 15-HETE.