Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.     

Effect of Free Radical Scavengers and Metal Ion Chelators on Hydrogen Peroxide and Phenylhydrazine Induced Red Blood Cell Lipid Peroxidation

Desferoxamine a well-known iron chelator was found to decrease hydrogen peroxide and phenylhy-dra/ine induced lipid peroxidation of red blood cell membranes assessed by hydrocarbon gas release and loss of polyunsaturated fatty acids. Hydroxyl radical scavengers like mannitol and thiourea and proteins like albumin were unable to reduce peroxidative reactions to our system. Addition of uric acid (in an unphysiological concentration of 5mM) to the incubation medium resulted in a slight reduction in H,0₂/phenyIhydrazine mediated break-down of arachidonic (20:4) and docosahexaenoic acid (22:6) in the erythrocyte membrane and consequently in a decreased alkane release and haemolysis.     

The lipoxygenase pathways are involved in LH-stimulated progesterone production in bovine corpus luteum.

We have examined the effects of endogenous lipoxygenase products on basal progesterone (P4) production by cultured bovine mid-luteal cells. The involvement of lipoxygenase products in the stimulatory effect of LH on luteal cAMP accumulation and P4 production was also examined. Bovine luteal cells from mid-cycle corpora lutea (CL) were exposed for 16 h to a lipoxygenase inhibitor (nordihydroguaiaretic acid: NDGA; 0.33-33 microM). For the last 4 h of incubation, the cells were exposed to LH and/or three different lipoxygenase products, 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE). NDGA inhibited P4 production by the cells in a dose-dependent manner (P < 0.05). NDGA-reduced P4 production was reversed by the addition of 12-HETE, but not 5- or 15-HETE, whereas 5-, 12- and 15-HETE alone showed no significant effect on P4 production in the intact cells. Furthermore, NDGA (33 microM) blocked the stimulatory action of LH on P4 production (P < 0.05), without changing cAMP accumulation (P > 0.1). When the cells were exposed to 5-, 12- or 15-HETE with LH and NDGA, only 15-HETE maintained the stimulatory effect of LH on P4 production in the cells (P < 0.05). These results suggest that endogenous lipoxygenase products play important roles in P4 production by bovine CL, i.e. basal P4 production is supported by 12-HETE, and LH-stimulated P4 production is partially mediated via the activation of lipoxygenase and subsequent 15-HETE formation downstream of the LH-activated cAMP-PKA-phosphorylation pathway.     

Synthesis of two hydroxy fatty acids from 7,10,13,16,19-docosapentaenoic acid by human platelets

Platelets metabolize 7,10,13,16,19-docosapentaenoic acid (22:5(n-3] into 11-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-docosapentaenoic acid via an indomethacin-insensitive pathway. Time-dependent studies with 20 microM substrate show a lag in the synthesis of both the 11- and 14-isomers which was not observed for the synthesis of thromboxane B2 (TXB2), 5,8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from arachidonic acid. When platelets were incubated with increasing concentrations of 22:5(n-3), the 11- and 14-isomers were not produced until the substrate concentration exceeded 5 microM unless arachidonic acid was also added to the incubations. The stimulatory effect of arachidonic acid was not blocked by indomethacin thus suggesting that 12-hydroperoxyeicosatetraenoic acid or 12-HETE derived from arachidonic acid may activate the platelet lipoxygenase(s) which metabolize 22:5(n-3). Incubations containing 20 microM 22:5(n-3) and increasing levels of [1-14C]arachidonic acid show that the (n-3) acid inhibits the synthesis of both 5,8,10-heptadecatrienoic acid and TXB2 from arachidonic acid. At the same time, 12-HETE synthesis increased due to substrate shunting to the lipoxygenase pathway.     

Glomeruli cooperate with macrophages in converting arachidonic acid to prostaglandins and hydroxyeicosatetraenoic acids

The interaction of proliferating macrophages with the glomerulus may play an important role in certain forms of glomerulonephritis. This interaction could involve metabolites of arachidonic acid (C20:4) such as prostaglandins (PG) and lipoxygenase products. We therefore investigated the conversion of exogenous |³H| C20:4 into hydroxyeicosatetraenoic acids (HETE) and PG by isolated glomeruli and macrophages from rats, alone and in combination. As demonstrated by HPLC, glomeruli converted C20:4 predominantly to lipoxygenase products -mainly 12-HETE- and, to a lesser extent, to PG. Resident macrophages converted C20:4 to equivalent amounts of HETE and PG, mainly 12-HETE and 6 keto-PGF_1α. When macrophages and glomeruli were studied in combination, a striking interaction was detected in both pathways of C20:4 metabolism. Production of 6 keto-PGF_1α was stimulated and considerable amounts of TXB₂, PGD₂ and hydroxyheptadecatrienoic acid (HHT) were also produced Total 12-HETE production was unchanged. When a lipid extract of glomeruli, containing oxygenated metabolites of C20:4, was added to macrophages, stimulation of 12-HETE occurred without any change in HHT or PG formation. When, on the contrary, a lipid extract from macrophages was added to glomeruli, 12-HETE production by the glomeruli was nearly completely abolished. Thus the unchanged total 12-HETE production by coincubated glomeruli and macrophages resulted from its increased production by macrophages and its decreased production by glomeruli. These data suggest that interaction between glomeruli and macrophages results in activation of C20:4 metabolism by macrophages followed by inhibition of C20:4 metabolism by glomeruli. Such a regulatory process could play a role in the inflammatory response to immunological injuries during macrophage-dependent human and experimental glomerulonephritis.     

Arachidonate metabolism in the mouse thyroid implication of the lipoxygenase pathway in thyrotropin action

The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.     

Relation of fatty acid composition in lead-exposed mallards to fat mobilization,lipid peroxidation and alkaline phosphatase activity

The increase of n-6 polyunsaturated fatty acids (PUFA) in animal tissues has been proposed as a mechanism of lead (Pb) poisoning through lipid peroxidation or altered eicosanoids metabolism. We have studied fatty acid (FA) composition in liver and brain of mallards (Anas platyrhynchos) feeding for 3 weeks on diets containing combinations of low or high levels of vitamin E (20 or 200 UI/kg) and Pb (0 or 2 g/kg). Saturated FA, n-6 PUFA and total concentrations of FA were higher in livers of Pb-exposed mallards, but not in their brains. The percentage of n-6 PUFA in liver and brain was slightly higher in Pb-exposed mallards. The increase of n-6 PUFA in liver was associated with decreased triglycerides and increased cholesterol in plasma, thus could be in part attributed to feed refusal and fat mobilization. The hepatic ratios between adrenic acid (22:4 n-6) and arachidonic acid (20:4 n-6) or between adrenic acid and linoleic acid (18:2 n-6) were higher in Pb exposed birds, supporting the existing hypothesis of increased fatty acid elongation by Pb. Among the possible consequences of increased n-6 PUFA concentration in tissues, we found increased lipid peroxidation in liver without important histopathological changes, and decreased plasma alkaline phosphatase activity that may reflect altered bone metabolism in birds.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

The function of very long chain polyunsaturated fatty acids in the pineal gland

The mammalian pineal gland is a prominent secretory organ with a high metabolic activity. Melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. Approximately 25% of the total fatty acids present in the rat pineal lipids are represented by arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These very long chain polyunsaturated fatty acids play important roles in the pineal gland. In addition to the production of melatonin, the mammalian pineal gland is able of convert these polyunsaturated fatty acids into bioactive lipid mediators. Lipoxygenation is the principal lipoxygenase (LOX) activity observed in the rat pineal gland. Lipoxygenation in the pineal gland is exceptional because no other brain regions express significant LOX activities under normal physiological conditions. The rat pineal gland expresses both 12- and 15-lipoxygenase (LOX) activities, producing 12- and 15-hydroperoxyeicosatetraenoic acid (12- and 15-HpETE) from arachidonic acid and 14- and 17-hydroxydocosahexaenoic acid (14- and 17-HdoHE) from docosahexaenoic acid, respectively. The rat pineal also produces hepoxilins via LOX pathways. The hepoxilins are bioactive epoxy-hydroxy products of the arachidonic acid metabolism via the 12S-lipoxygenase (12S-LOX) pathway. The two key pineal biochemical functions, lipoxygenation and melatonin synthesis, may be synergistically regulated by the status of n-3 essential fatty acids.     

Glucose induces lipid peroxidation and inactivation of membrane-associated ion-transport enzymes in human erythrocytes in vivo and in vitro.

Erythrocytes of diabetic subjects (non-insulin dependent) were found to have eight- to ten-fold higher levels of endogenously formed thiobarbituric acid reactive malonyldialdehyde (MDA), thirteen-fold higher levels of phospholipid-MDA adduct, 15-20% reduced Na(+)-K(+)-ATPase activity with unchanged Ca+2-ATPase activity, as compared with the erythrocytes from normal healthy individuals. Incubation of normal erythrocytes with elevated concentrations (15-35 mM) of glucose, similar to that present in diabetic plasma, led to the increased lipid peroxidation, phospholipid-MDA adduct formation, reduction of Na(+)-K(+)-ATPase (25-50%) and Ca+2-ATPase (50%) activities. 2-doxy-glucose was 80% as effective as glucose in the lipid peroxidation and lipid adduct formation. However, other sugars, such as fructose, galactose, mannose, fucose, glucosamine and 3-O-methylmannoside, and sucrose, tested at a concentration of 35 mM, resulted in reduced (20-30%) lipid peroxidation without the formation of lipid-MDA adduct. Kinetic studies show that reductions in Na(+)-K(+)-ATPase and Ca+2-ATPase activities precede the lipid peroxidation as the enzyme inactivation occur within 30 min of incubation of erythrocytes with high concentration (15-35 mM) of glucose, while lipid peroxidation product, MDA appears at 4 hr and lipid-MDA adducts at 8 hr. The lipoxygenase pathway inhibitors, 5,8,11-eicosatriynoic acid and Baicalein (5,6,7-trihydroxyflavone), reduced the glucose-induced lipid peroxidation by 30% and MDA-lipid adduct formation by 26%. Indomethacin, a cyclooxygenase pathway inhibitor, had no discernible effect on the lipid peroxidation in erythrocytes. However, the inhibitors of lipid peroxidation, 3-phenylpyrazolidone, metyrapone, and the inhibitors of lipoxygenase pathways did not ablate the glucose-induced reduction of Na(+)-K(+)-ATPase and Ca+2-ATPase activities in erythrocytes. Erythrocytes produce 15-HETE (15-hydroxy-eicosatetraenoic acid), which is augmented by glucose. These results suggest that the formation of lipoxygenase metabolites potentiate the glucose-induced lipid peroxidation and that the inactivation of Na(+)-K(+)-ATPase and Ca+2-ATPase occurs as a result of non-covalent interaction of glucose with these enzymes.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

Influence of N-phenyllinoleamide from toxic oil samples on the lipoxygenase metabolism of exogenous arachidonic acid in mouse peritoneal macrophages.

N-phenyllinoleamide (NPLA) is a useful marker for adulterated oil samples associated with cases of toxic oil syndrome (TOS). To date, NPLA has not reproduced the human poisoning episode in experimental animal models and, thus, its pathological role in the syndrome remains controversial. The present report describes the effect of NPLA on the lipoxygenase metabolism of exogenous arachidonic acid (AA) in mouse peritoneal macrophages (MPM). Results show that MPM cells exposed to 1mM NPLA for 2 h, when subsequently incubated with exogenous 3H-AA, undergo a significant increase in the biosynthesis of 3H-12-hydroxyeicosatetraenoic acid (3H-12-HETE) whereas levels of 3H-15-HETE are relatively stable. These data indicate that NPLA selectively potentiates the lipoxygenase metabolism of exogenous AA, supporting the possible implication of lipid peroxidative processes in the ethiopathology of TOS, although the relatively high NPLA concentration required 'in vitro' makes it unlikely that this xenobiotic could be directly related to human toxicity.     

The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis

The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The results demonstrated the protective effect of alpha-tocopherol on lipid peroxidation in microsomes and mitochondria from rat testis.     

Fatty acid composition of phospholipids and neutral lipids from human diabetic small arteries and veins by a new TLC method

It has been suggested that lipid peroxidation of polyunsaturated fatty acids (PUFA) may play a role in the pathogenesis of diabetic complications. To test this hypothesis, we aimed to compare PUFA composition of small arteries and veins (< 500 microm diameter) obtained from diabetic or non-diabetic Guadeloupean patients undergoing arterio-venous shunt surgery before renal dialysis. Small forearm subcutaneous vessels were analysed by a new TLC method which involved inclusion of vascular biopies directly in alveoles made in the TLC gel and lyophilization onto the plate. The TLC plate was then chromatographed and lipids were both extracted and eluted during this step. Fatty acid composition of phospholipid and neutral lipid fractions were determined. Similar fatty acid composition was obtained for arteries and veins from diabetic or non-diabetic subjects. In phospholipids from diabetic vessels, major changes consisted of a 20% decrease of arachidonic acid (20:4 n-6), a 40% decrease of its elongation product 22:4 n-6 and 30% increase of 18:2 n-6. In neutral lipids, 20:4 n-6 was also diminished by 60% whereas oleic acid increased by 15%. This loss of arachidonic acid in small diabetic vessels suggests impaired delta6-desaturase forming 20:4 n-6 or alternatively increased peroxide formation, in the vascular wall of small vessels in diabetic patients.     

Lipoxygenation in rat brain?

It has been previously claimed that rodent brain possesses lipoxygenase activity, based upon the structure of products which were formed from arachidonic acid and the inhibition of this activity by "lipoxygenase inhibitors." Our studies confirm that various positional isomers of hydroxyeicosatetraenoic acids (HETE) are formed (e.g., 15-, 12-, 11-, 9-, 8- and 5-HETE) by brain homogenate and that their production is inhibited by certain lipoxygenase inhibitors, such as nordihydroguaiaretic acid (NDGA) but not by cyclooxygenase or cytochrome P-450 inhibitors. However, stereochemical analysis indicated racemic distributions of these products suggesting that they were not formed by a lipoxygenase enzyme but rather by a peroxidative process. It should also be noted that the presence of 12(S)-lipoxygenase activity could be demonstrated by stereochemical analysis only when the brain was not perfused properly, indicating this activity was due to blood cell contamination. It is known that many lipoxygenase inhibitors are also capable of inhibiting peroxidative reactions apparently due to their free radical scavenging properties. For these reasons, it is essential that the stereochemical purity of purported lipoxygenase products be determined and that previous claims of lipoxygenase activity in mammalian brain be reexamined.     

Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.     

Localization of 12-hydroxyeicosatetraenoic acid in endothelial cells

Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage.     

Mechanism of biosynthesis of 11R-and 12R-hydroxyeicosatetraenoic acids by eggs of the sea urchin Strongylocentrotus purpuratus

11(R)-Hydroxyeicosatetraenoic acid [11(R)-HETE] and 12(R)-HETE are biosynthesized by eggs of the sea urchin S. purpuratus. We report here the isolation of the 11(4)- and 12(R)-hydroperoxy-eicosanoids from incubations of the desalted 30-50%(NH4)2SO4 fraction of the egg homogenate; biosynthesis required the addition of calcium but not NADPH. Egg 11- and 12-HETE were formed from octadeuterated arachidonic acid without loss of geminal 2H from C11 or C12, thus revealing that 11- or 12-keto intermediates are not involved in the biosynthesis. The results support the conclusion that egg 11(R)- and 12(R)-HETE are synthesized by a lipoxygenase and not by an NADPH-dependent cytochrome P450 monooxygenase mechanism.