13-Hydroxyoctadecadienoic acid is the vessel wall chemorepellant factor, LOX

We have previously reported that endothelial cells synthesize a cytosol-associated, lipoxygenase-derived metabolite, LOX, which acts as a chemorepellant and, in so doing, maintains the vessel wall thromboresistance. In this study we demonstrate that LOX is a 13-hydroxylinoleic acid (13-OH-18:2) derived from linoleic acid and identical to 13-hydroxy-9-cis,11-trans-octadecadienoic acid, as measured by both reverse phase high pressure liquid chromatography and gas chromatography/mass spectrometry. In addition, we demonstrate that 13-OH-18:2 is produced in significantly greater quantities by endothelial cells than by smooth muscle cels or by fibroblasts. Furthermore, we demonstrate that 13-OH-18:2 is produced in microgram amounts under basal conditions and is decreased by thrombin, calcium ionophore, and trypsin stimulation. And finally, we demonstrate that endothelial cells do not synthesize any significant amounts of lipoxygenase-derived arachidonic acid metabolites either under basal or stimulated conditions unless exogenous arachidonic acid is added. These observations indicate that the major lipoxygenase-derived, chemorepellant metabolite produced by the endothelial is 13-hydroxy-9-cis,11-trans-octadecadienoic acid.     

Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts

Epoxyeicosatrienoic acids (EETs), the eicosanoid biomediators synthesized from arachidonic acid by cytochrome P450 epoxygenases, are inactivated in many tissues by conversion to dihydroxyeicosatrienoic acids (DHETs). However, we find that human skin fibroblasts convert EETs mostly to chain-shortened epoxy-fatty acids and produce only small amounts of DHETs. Comparative studies with [5,6,8,9,11,12,14,15-(3)H]11,12-EET ([(3)H]11,12-EET) and [1-(14)C]11,12-EET demonstrated that chain-shortened metabolites are formed by removal of carbons from the carboxyl end of the EET. These metabolites accumulated primarily in the medium, but small amounts also were incorporated into the cell lipids. The most abundant 11, 12-EET product was 7,8-epoxyhexadecadienoic acid (7,8-epoxy-16:2), and two of the others that were identified are 9, 10-epoxyoctadecadienoic acid (9,10-epoxy-18:2) and 5, 6-epoxytetradecaenoic acid (5,6-epoxy-14:1). The main epoxy-fatty acid produced from 14,15-EET was 10,11-epoxyhexadecadienoic acid (10, 11-epoxy-16:2). [(3)H]8,9-EET was converted to a single metabolite with the chromatographic properties of a 16-carbon epoxy-fatty acid, but we were not able to identify this compound. Large amounts of the chain-shortened 11,12-EET metabolites were produced by long-chain acyl CoA dehydrogenase-deficient fibroblasts but not by Zellweger syndrome and acyl CoA oxidase-deficient fibroblasts. We conclude that the chain-shortened epoxy-fatty acids are produced primarily by peroxisomal beta-oxidation. This may serve as an alternate mechanism for EET inactivation and removal from the tissues. However, it is possible that the epoxy-fatty acid products may have metabolic or functional effects and that the purpose of the beta-oxidation pathway is to generate these products.     

13-Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets

The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.     

Cyclic AMP regulation of endothelial cell triacylglycerol turnover, 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and endothelial cell thrombogenicity

The 15-omega-lipoxygenase enzyme in endothelial cells metabolizes endogenous linoleic acid (18:2) into 13-hydroxyoctadecadienoic acid (13-HODE) under basal conditions, i.e., in unstimulated endothelial cells. 13-HODE is thought to regulate the non-adhesivity of the endothelium, contributing to vessel wall/blood cell biocompatibility. We performed experiments, therefore, to determine the relationship between basal levels of cAMP, 13-HODE synthesis, and platelet/endothelial cell adhesion. We found that 13-HODE synthesis increased with elevated cAMP levels and that the elevated 13-HODE levels correlated with increased 18:2 turnover in the triacylglycerol pool. In contrast, neither 18:2 nor arachidonic acid (20:4) turnover in the phospholipid nor prostacyclin (PGI2) production were changed with elevated cAMP levels. Platelet/endothelial cell adhesion was inversely proportional to 13-HODE synthesis. We conclude that intracellular 13-HODE influences platelet/vessel wall interactions, is synthesized from 18:2 released from the endogenous triacylglycerol pool, and that this pathway is modulated by intracellular cAMP levels.     

Quantitation of 13-hydroxyoctadecadienoic acid (13-HODE) by radioimmunoassay

Antibodies against 13-hydroxyoctadecadienoic acid (13-HODE) were produced in rabbits by immunizing the animal with 13-HODE-thyroglobulin conjugate. The antibodies appeared to be rather specific for 13-HODE since other hydroxy fatty acids showed minimal crossreaction. The radioimmunoassay was capable of detecting 50 pg per assay tube and was applied to the study of the biosynthesis of 13-HODE in platelets and leukocytes. In contrast to reported findings from endothelial cells, A-23187, thrombin and collagen stimulated synthesis and release of 13-HODE from platelets. However, insignificant synthesis of 13-HODE was found in leukocytes following A-23187 stimulation. Exogenous addition of linoleic acid stimulated the synthesis of 13-HODE from both platelets and leukocytes. The majority of 13-HODE synthesized was found in the medium. These studies suggest that both types of blood cells possess active (omega-6) lipoxygenase. Platelets may use endogenously released linoleic acid to synthesize 13-HODE, whereas leukocytes may utilize linoleic acid released from other cell types for 13-HODE synthesis.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

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.     

Binding of 13-HODE and 5-, 12- and 15-HETE to endothelial cells and subsequent platelet, neutrophil and tumor cell adhesion

Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.     

Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy

Peroxisomal disorders have been associated with malfunction of peroxisomal metabolic pathways, but the pathogenesis of these disorders is largely unknown. X-linked adrenoleukodystrophy (X-ALD) is associated with elevated levels of very-long-chain fatty acids (VLCFA; C(>22:0)) that have been attributed to reduced peroxisomal VLCFA beta-oxidation activity. Previously, our laboratory and others have reported elevated VLCFA levels and reduced peroxisomal VLCFA beta-oxidation in human and mouse X-ALD fibroblasts. In this study, we found normal levels of peroxisomal VLCFA beta-oxidation in tissues from ALD mice with elevated VLCFA levels. Treatment of ALD mice with pharmacological agents resulted in decreased VLCFA levels without a change in VLCFA beta-oxidation activity. These data indicate that ALDP does not determine the rate of VLCFA beta-oxidation and that VLCFA levels are not determined by the rate of VLCFA beta-oxidation. The rate of peroxisomal VLCFA beta-oxidation in human and mouse fibroblasts in vitro is affected by the rate of mitochondrial long-chain fatty acid beta-oxidation. We hypothesize that ALDP facilitates the interaction between peroxisomes and mitochondria, resulting, when ALDP is deficient in X-ALD, in increased VLCFA accumulation despite normal peroxisomal VLCFA beta-oxidation in ALD mouse tissues. In support of this hypothesis, mitochondrial structural abnormalities were observed in adrenal cortical cells of ALD mice.     

Dual metabolic pathways of 12-HETE in rat aortic smooth muscle cells.

12(S)-HETE, a major lipoxygenase-derived compound from arachidonic acid is incorporated and metabolized by vascular smooth muscle cells via beta-oxidation. We have now identified for the first time in this cell type 12(S)-HETE metabolites formed by a combination of reductase and oxidation pathways. HPLC and GC-MS analysis of time-course experiments allow us to characterize two different metabolic pathways: a direct peroxisomal beta-oxidation of 12(S)-HETE leading to the formation of 16:3 (8-OH) which accumulates first and a reduction of one of the conjugated double bonds of 12(S)-HETE giving the dihydro-intermediate 20:3(12-OH) that transiently accumulates before being converted itself by peroxisomal beta-oxidation to 16:2(8-OH). Taken together these results may suggest that the transient accumulation of 20:3(12-OH) through transcellular metabolism of 12(S)-HETE may represent a part of the modulatory effect of 12(S)-HETE on vascular function.     

Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in endothelial cells

Human umbilical vein endothelial cells convert linoleic acid to two monohydroxyoctadecadienoic (HODE) acids, 9- and 13-HODE. More 9-HODE than 13-HODE is formed under most conditions. The production of these metabolites is reduced substantially by acetylsalicylic acid, ibuprofen, or arachidonic acid, suggesting that cyclooxygenase may be involved in endothelial HODE synthesis. Incubations lasting up to 4 h indicate that the endothelial cells can convert [U-14C] linoleic acid into at least four additional products, some of which may be derived from the HODE that is formed initially. Radioactive 9- and 13-HODE are produced when the endothelial cells are labeled with linoleic acid and then exposed to thrombin, suggesting that these metabolites also may be formed when the endothelium is activated. If endothelial monolayers grown on micropore filters are incubated with linoleic acid, a substantial amount of the HODE formed accumulates in the basolateral fluid. This suggests that HODE may have extracellular effects, especially within the vascular wall. Furthermore, when 9- or 13-HODE are added, endothelial cultures produce less prostaglandin I2 and convert less 12-hydroxyeicosatetraenoic acid to its main metabolite, 8-hydroxyhexadecatrienoic acid. Therefore, in addition to extracellular actions, HODE also may have functional effects within the endothelium.     

Nonradiometric HPLC measurement of 13(S)-hydroxyoctadecadienoic acid from rat tissues

A major bioactive metabolite of linoleic acid formed by the action of 15-lipoxygenase-1 is 13(S)-hydroxy-cis-9, trans-11-octadecadienoic acid (13(S)-HODE). 13(S)-HODE is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Separation and quantification of 13(S)-HODE from biological materials has previously been achieved only by using radiolabeled linoleic acid as the substrate and two serially connected or two separate HPLC columns to achieve separation of 13(S)-HODE. In the current method, separation and quantification of 13(S)-HODE was achieved by use of a normal-phase HPLC and a solvent system containing hexane/isopropanol/acetonitrile/acetic acid (800/8/30/1, v/v) using isocratic elution with detection at 235 nm. With the currently described method, good separation from unreacted interfering compounds and quantification for 13(S)-HODE were achieved within 35 min with a minimum detection limit of 0.5 ng per injection.     

Very long chain fatty acid beta-oxidation by subcellular fractions of normal and Zellweger syndrome skin fibroblasts

Very long chain fatty acid (VLCFA) beta-oxidation was compared in homogenates and subcellular fractions of cultured skin fibroblasts from normal individuals and from Zellweger patients who show greatly reduced numbers of peroxisomes in their tissues. beta-Oxidation of lignoceric (C24:0) acid was greatly reduced compared to controls in the homogenates and the subcellular fractions of Zellweger fibroblasts. The specific activity of C24:0 acid beta-oxidation was highest in the crude peroxisomal pellets of control fibroblasts. Fractionation of the crude mitochondrial and the crude peroxisomal pellets on Percoll density gradients revealed that the C24:0 acid oxidation was carried out entirely by peroxisomes, and the peroxisomal beta-oxidation activity was missing in Zellweger fibroblasts. In contrast to the beta-oxidation of C24:0 acid, the beta-oxidation of C24:0 CoA was observed in both mitochondria and peroxisomes. We postulate that a very long chain fatty acyl CoA (VLCFA CoA) synthetase, which is different from long chain fatty acyl CoA synthetase, is required for the effective conversion of C24:0 acid to C24:0 CoA. The VLCFA CoA synthetase appears to be absent from the mitochondrial membrane but present in the peroxisomal membrane.     

13-HPODE and 13-HODE modulate cytokine-induced expression of endothelial cell adhesion molecules differently

Expression of cellular adhesion molecules (CAMs) at endothelial surfaces represents a physiological response to vascular damage and mediates the initiation of inflammation and possibly of atherogenesis. The cytokines TNF alpha and IL-1 are potent inducers of CAMs in endothelial cells. Reactive oxygen species comprising lipid oxidation products have been implicated in the signaling pathways of both TNF alpha and IL-1 and accordingly could modulate atherogenic events. We, therefore, investigated the potential role of the lipoxygenase product, 13-hydroperoxyoctadecadienoic acid (13-HPODE), which has also been identified in oxidized low density lipoproteins on CAM expression in HUVEC. 13-HPODE induced the expression of ICAM-1 in a concentration dependent manner up to 75 microM. Higher concentrations were toxic. Similar effects were observed with H2O2 and phosphatidylcholine hydroperoxide. VCAM-1 and E-selectin were not induced by 13-HPODE. 13-HPODE administered simultaneously with IL-1 or TNF alpha induced ICAM-1 additively, suggesting that hydroperoxides and cytokines act on the same signaling pathways. In contrast, pretreatment of cells with 50 microM 13-HPODE for 1 hour rather inhibited subsequent cytokine-induced ICAM-1 and E-selectin expression. Surprisingly, the reduction product of 13-HPODE, 13-hydroxyoctadecadienoic acid (13-HODE) proved to be an even better inducer of ICAM-1 than 13-HPODE. Pretreatment with 13-HODE did not show any inhibitory effect on ICAM-1 expression. Our data show that lipoxygenase products differentially affect CAM expression. 13-HPODE is stimulatory by itself and can positively or negatively affect cytokine signaling depending on time of exposure. 13-HODE induces CAM expression by itself but does not inhibit cytokine signaling. Thus, the interplay of lipoxygenase products with proinflammatory cytokines can not simply be explained by an oxidant-mediated facilitation of cytokine signaling.     

Coordination of peroxisomal beta-oxidation and fatty acid elongation in HepG2 cells

A major product of mitochondrial and peroxisomal beta-oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal beta-oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of beta-oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-(13)C(18)]stearate or [U-(13)C(16)] palmitate. Using mass isotopomer analysis we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the tricarboxylic acid cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mm [U-(13)C(18)]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mm [U-(13)C(16)]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that 1) chain elongation activity is a useful and novel probe for peroxisomal beta-oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.     

9- and 13-hydroxy-linoleic acid possess chemotactic activity for bovine and human polymorphonuclear leukocytes

The presence of polymorphonuclear leukocytes (PMNs) within the airways is a characteristic feature of a variety of lung diseases. Pulmonary alveolar macrophages (PAMs) and epithelial cells release many different factors which contribute to the recruitment of inflammatory cells into infected airways. PAMs and tracheal epithelial cells are able to produce linoleic acid metabolites (9-HODE and 13-HODE) besides arachidonic acid metabolites. The objective of the present study was to determine whether 9-HODE and 13-HODE possess chemotactic activity for isolated PMNs. It was found that 9-HODE and 13-HODE induced a chemotactic response of both human and bovine PMNs in vitro. The HODEs evoked chemotaxis with a linear dose response from 10(-10) to 10(-6) M to the same extent as the arachidonic acid metabolite 15-HETE. At 10(-8) M, 9-HODE and 13-HODE were approximately half as potent in inducing chemotaxis as compared to LTB4.     

Incorporation of arachidonic and linoleic acid hydroperoxides into cultured human umbilical vein endothelial cells

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.     

Peroxisomal retroconversion of docosahexaenoic acid (22:6(n-3)) to eicosapentaenoic acid (20:5(n-3)) studied in isolated rat liver cells.

Retroconversion of docosahexaenoic acid (DHA, 22:6(n-3)) to eicosapentaenoic acid (EPA, 20:5(n-3)) was studied in isolated rat liver cells. 20% of the substrate was retroconverted to EPA in control cells by one cycle of beta-oxidation probably with delta 4 enoyl CoA reductase and delta 3, delta 2 enoyl CoA isomerase as auxiliary enzymes. This conversion was not stimulated by (-)-carnitine and was not inhibited by the addition of (+)-decanoylcarnitine. In hepatocytes from fasted rats little EPA was formed from DHA. These results strongly suggest that the retroconversion of DHA to EPA is a peroxisomal function. Retroconverted EPA, produced from DHA was rapidly incorporated in triacylglycerol, the phosphatidylcholine and phosphatidyletanolamine fractions. During longer incubation time EPA was partly removed from the phospholipid fractions, chain-elongated to 22:5(n-3) and incorporated in the triacylglycerol fraction.     

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions.

We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.