Regiospecific and enantioselective metabolism of 8,9-epoxyeicosatrienoic acid by cyclooxygenase.

8(S),9(R)-epoxyeicosatrienoic acid, a major product of the renal cortex, was found to be a substrate for cyclooxygenase from human platelets and ram seminal vesicles. 11(R)-hydroxy-8(S),9(R)-epoxyeicosatrienoic acid was the sole metabolic product. The 8(R),9(S)-enantiomer formed both C-11 and C-15 hydroxylated metabolites. These novel findings suggest that the cyclooxygenase-dependent renal vasoconstrictor activity of 8(S),9(R)-epoxyeicosatrienoic acid may be due to the 11(R)-hydroxy metabolite.     

Lipoxygenase metabolism of polyunsaturated fatty acids in oocytes of the frog Xenopus laevis

Recently, oocytes or eggs of two marine invertebrates have been found to metabolize arachidonic acid to specific monohydroxy products. These studies have prompted our examination of the oocytes of higher organisms. In the present study, oocytes of an amphibian, Xenopus laevis, were examined for their capacity to biosynthesize hydroxyeicosatetraenoic acids (HETEs) and related hydroxy fatty acids. Two hydroxyeicosanoids were formed during incubations of oocyte homogenates with [14C]arachidonic acid; their structures and stereochemistry were determined by high-pressure liquid chromatography, uv spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as 15(S)- and 12(S)-hydroxyeicosatetraenoic acids. The synthesis of the two HETEs was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), or by prior exposure of the oocyte homogenates to carbon monoxide, an inhibitor of cytochrome P450. Furthermore, 12(S)- and 15(S)-hydroperoxyeicosatetraenoic acids were isolated from brief incubations of gel-filtered ammonium sulfate fraction of frog oocyte homogenates; isolation of the hydroperoxide is further support for the existence of 12(S)- and 15(S)-lipoxygenase activities in the oocytes of X. laevis. Other polyunsaturated acids, including C18.2, C18.3, C20.3, C20.5, and C22.6 were also substrates for the lipoxygenase, and in each case the major product was formed by omega 6 oxygenation.     

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.     

Formation of pentachlorophenol as the major product of microsomal oxidation of hexachlorobenzene

On incubation of [14C]-hexachlorobenzene with microsomes from livers of rats induced with hexachlorobenzene, the major product (80-90%) was pentachlorophenol. The only other detectable metabolite, tetrachlorohydroquinone (4-15%), was presumably formed from pentachlorophenol. A considerable amount of radioactivity (5-10% of the amount of extracted metabolites) was covalently bound to protein. Microsomes derived from male hexachlorobenzene--induced rats gave by far the highest conversion (approx. 1% of substrate). Microsomes from female hexachlorobenzene--induced rats were 3 times less efficient. Microsomes from untreated and 3-methyl-cholanthrene--treated animals gave less than 5% of the amount of pentachlorophenol formed by microsomes from hexachlorobenzene--induced male rats, while phenobarbital and aroclor 1254-induction resulted in formation of 51% and 34% respectively.     

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4- and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.     

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.     

In vitro metabolism of bladder carcinogenic nitrosamines by rat liver and urothelial cells.

In order to establish the importance of the target organ in the activation of bladder carcinogens, we compared rat liver and urothelial cell alpha-hydroxylation activities using as substrates N-nitrosobutyl(4-hydroxybutyl)amine and its metabolite N-nitrosobutyl(3-carboxypropyl)amine, two potent urinary bladder carcinogens in animals. Previous studies have shown that the production of molecular nitrogen can serve as an indicator of nitrosamine alpha-hydroxylation. The use of doubly 15N-labelled nitrosamines and the gas chromatography-mass spectrometric detection of 15N2 formed gives a measurement of the extent of this metabolic step. Various amounts of 15N-labelled substrates were incubated for 60 min at 37 degrees C with rat liver S9 preparations or urothelial cell homogenates in the presence of a NADPH generating system. Both enzyme sources metabolized 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine through the alpha-hydroxylation pathway. Using hepatic S9 fractions, 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine increased from 1.69 +/- 0.02 nmol/h per mg protein (mean +/- S.E.) to 5.78 +/- 0.5 with substrate concentrations ranging between 0.55 and 5.55 mM. 15N2 produced by urothelial cell homogenates was about 40-50% that of the liver S9. 15N-labelled N-nitrosobutyl(3-carboxypropyl)amine was also metabolized through the alpha-hydroxylation pathway both by hepatic S9 and urothelial cell homogenates, though to a lesser extent. 15N2 production was about 10-times less than from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine, but again urothelial cell 15N2 production was about 40-50% that of the liver. Treatment with phenobarbital resulted in a 2.7-fold increase in the 15N2 produced from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9. No effect was observed with urothelial cell homogenates. Acetone treatment had no effect on 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9, but raised 15N2 production by urothelial cell homogenates 1.8 times. Although the liver has a greater capacity than the bladder for activating the 15N-labelled nitrosamines studied, the target organ can metabolize bladder carcinogens, thus increasing the possibility of a local toxic effect. Moreover, the distribution of P-450 isozymes might be different in the bladder and this could affect the metabolism of nitrosamines reportedly formed in the human bladder in some pathological conditions.     

Xanthobacter sp. C20 contains a novel bioconversion pathway for limonene

Xanthobacter sp. C20 was isolated from sediment of the river Rhine using cyclohexane as sole source of carbon and energy. Xanthobacter sp. C20 converted both enantiomers of limonene quantitatively into limonene-8,9-epoxide, a not previously described bioconversion product of limonene. With (4R)-limonene, (4R,8R)-limonene-8, 9-epoxide was formed as the only reaction product, while (4S)-limonene was converted into a (78:22) mixture of (4S,8R)- and (4S,8S)-limonene-8,9-epoxide. Cytochrome P-450 was shown to be induced concomitantly with limonene bioconversion activity following growth of Xanthobacter sp. C20 on cyclohexane. Maximal limonene bioconversion rate was observed at an initial substrate concentration of 12 mM. The amount of limonene-8,9-epoxide formed, up to 0.8 g l(-1), was limited by a strong product inhibition.     

12(R)-hydroxyeicosatrienoic acid: a vasodilator cytochrome P-450-dependent arachidonate metabolite from the bovine corneal epithelium

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, two biologically active metabolites of arachidonic acid were formed. The structure of one of the metabolites, compound C, was previously reported to be 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid and was found to be a potent inhibitor of the Na+/K+-ATPase in the cornea. The second metabolite, compound D, was found to be a potent vasodilator as well as having the property of stimulating protein influx into the aqueous humor of the eye. Following purification of compound D by thin layer chromatography and high pressure liquid chromatography, it was found to lack a UV chromophore in contrast to the previously reported cytochrome P-450-dependent metabolite. Mass spectrometric analysis using positive and negative ionization modes was carried out on derivatized compound D that had been synthesized from a mixture of labeled [( 5,6,8,9,11,12,14,15-2H8]) and unlabeled arachidonic acid incubated with corneal microsomes. The novel arachidonate metabolite had abundant fragment ions consistent with compound D being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon 12 of the eicosanoid backbone; only seven deuterium atoms from [2H8]arachidonate were retained in the structure. Oxidative ozonolysis yielded a product indicating that the double bonds in metabolite D resided between carbons at positions 8 and 9 and positions 14 and 15 of the 20-carbon chain. Compound D was therefore characterized as 12-hydroxy-5,8,14-eicosatrienoic acid. Model compounds were synthesized from dimethyl malate with the hydroxy at the 12 position with both the R and S absolute configuration and with all double bonds of the cis configuration. Only the 12(R) isomer was found to be a potent vasodilator and to increase aqueous humor protein concentration, suggesting that the biologically active compound D was 12(R)-hydroxy-5,8,14-(Z,Z,Z)-eicosatrienoic acid. As this compound possesses proinflammatory properties, it may play a role in the wound-healing processes of corneal injury.     

Conversion of dihomo-gamma-linolenic acid to mono- and dihydroxy acids by potato lipoxygenase: evidence for the formation of 8,9-leukotriene A3

Evidence for the formation of a positional isomer of leukotriene (LT) C3 (8,9-LTC3) from dihomo-gamma-linolenic acid has been published (Hammarstr?m, S. J. Biol. Chem. 256, 7712-7714, 1981). This report describes the conversion of dihomo-gamma-linolenic acid to a postulated intermediate in former reaction, 8,9-LTA3, by purified lipoxygenase from potato tubers. 8(S)-Hydroperoxyeicosatrienoic acid (8(S)-HPETrE) was the most abundant dioxygenation product formed followed by 11-, 15-, and 12-HPETrEs (in decreasing order of abundance). In addition, 8(S),15(S)- plus 8(S), 15(R)-dihydroperoxyeicosatetraenoic acid (DiHPE-TrE) (EZE), and 8(S),15(S)- plus 8(S),15(R)-dihydroxy-eicosatetraenoic acid (DiHETrE) (EEE) were generated. Under anaerobic conditions only the latter two isomers of 8,15-DiHETrE (EEE) were obtained from 8-HPETrE. The results suggest that 8,9-LTA3 is synthesized by the sequential action of 8- and 11-lipoxygenase activities associated with the potato enzyme.     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.     

On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.     

Methene bridge carbon atom elimination in oxidative heme degradation catalyzed by heme oxygenase and NADPH-cytochrome P-450 reductase

Physiological heme degradation is mediated by the heme oxygenase system consisting of heme oxygenase and NADPH-cytochrome P-450 reductase. Biliverdin IX alpha is formed by elimination of one methene bridge carbon atom as CO. Purified NADPH-cytochrome P-450 reductase alone will also degrade heme but biliverdin is a minor product (15%). The enzymatic mechanisms of heme degradation in the presence and absence of heme oxygenase were compared by analyzing the recovery of 14CO from the degradation of [14C]heme. 14CO recovery from purified NADPH-cytochrome P-450 reductase-catalyzed degradation of [14C]methemalbumin was 15% of the predicted value for one molecule of CO liberated per mole of heme degraded. 14CO2 and [14C]formic acid were formed in amounts (18 and 98%, respectively), suggesting oxidative cleavage of more than one methene bridge per heme degraded, similar to heme degradation by hydrogen peroxide. The reaction was strongly inhibited by catalase, but superoxide dismutase had no effect. [14C]Heme degradation by the reconstituted heme oxygenase system yielded 33% 14CO. Near-stoichiometric recovery of 14CO was achieved after addition of catalase to eliminate side reactions. Near-quantitative recovery of 14CO was also achieved using spleen microsomal preparations. Heme degradation by purified NADPH-cytochrome P-450 reductase appeared to be mediated by hydrogen peroxide. The major products were not bile pigments, and only small amounts of CO were formed. The presence of heme oxygenase, and possibly an intact membrane structure, were essential for efficient heme degradation to bile pigments, possibly by protecting the heme from indiscriminate attack by active oxygen species.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

Identification of the major urinary metabolite of the highly reactive cyclopentenone isoprostane 15-A(2t)-isoprostane in vivo

The cyclopentenone isoprostanes (A(2)/J(2)-IsoPs) are formed in significant amounts in humans and rodents esterified in tissue phospholipids. Nonetheless, they have not been detected unesterified in the free form, presumably because of their marked reactivity. A(2)/J(2)-IsoPs, similar to other electrophilic lipids such as 15-deoxy-Delta(12,14)-prostaglandin J(2) and 4-hydroxynonenal, contain a highly reactive alpha,beta-unsaturated carbonyl, which allows these compounds to react with thiol-containing biomolecules to produce a range of biological effects. We sought to identify and characterize in rats the major urinary metabolite of 15-A(2t)-IsoP, one of the most abundant A(2)-IsoPs produced in vivo, in order to develop a specific biomarker that can be used to quantify the in vivo production of these molecules. Following intravenous administration of 15-A(2t)-IsoP containing small amounts of [(3)H(4)]15-A(2t)-IsoP, 80% of the radioactivity excreted in the urine remained in aqueous solution after extraction with organic solvents, indicating the formation of a polar conjugate(s). Using high pressure liquid chromatography/mass spectrometry, the major urinary metabolite of 15-A(2t)-IsoP was determined to be the mercapturic acid sulfoxide conjugate in which the carbonyl at C9 was reduced to an alcohol. The structure was confirmed by direct comparison to a synthesized standard and via various chemical derivatizations. In addition, this metabolite was found to be formed in significant quantities in urine from rats exposed to an oxidant stress. The identification of this metabolite combined with the finding that these metabolites are produced in in vivo settings of oxidant stress makes it possible to use this method to quantify, for the first time, the in vivo production of cyclopentenone prostanoids.     

Stereochemistry of leukotriene B4 metabolites formed by the reductase pathway in porcine polymorphonuclear leukocytes: inversion of stereochemistry of the 12-hydroxyl group

Leukotriene B4 (LTB4), a potent proinflammatory agent, is a major metabolite of arachidonic acid in polymorphonuclear leukocytes (PMNL). When porcine PMNL were incubated with LTB4 and the products purified by reversed-phase high-pressure liquid chromatography (HPLC), we previously identified two metabolites: 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 [Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369]. Further analysis of the reaction products by normal-phase HPLC has now revealed the presence of a third major metabolite of LTB4. This product is not formed in detectable amounts in the first 5 min of the reaction but accounts for about 20-30% of the reaction products after 60 min, when LTB4 has been completely metabolized. The mass spectrum and gas chromatographic properties of the new metabolite are identical with those of 10,11-dihydro-LTB4, suggesting that it is a stereoisomer of this compound. This product was identified as 10,11-dihydro-12-epi-LTB4 [i.e., 5(S),12(R)-dihydroxy-6,8,14-eicosatrienoic acid] by comparison of its chromatographic properties with those of the authentic chemically synthesized compound. Both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 were enzymatically converted to 10,11-dihydro-12-epi-LTB4 by porcine PMNL, the former compound being the better substrate. The reaction was reversible, since both 10,11-dihydro-12-epi-LTB4 and 10,11-dihydro-12-oxo-LTB4 could be converted to 10,11-dihydro-LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydroperoxy-arachidonic acid mediated n-hexanal and (Z)-3- and (E)-2-nonenal formation in Laminaria angustata

In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.     

Compartmentation of metabolism probed by [2-13C]alanine: improved 13C NMR sensitivity using a CryoProbe detects evidence of a glial metabolon

The labelling of metabolites with the NMR active nucleus 13C allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-13C, 15N]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using 13C NMR spectroscopy. The spectra were obtained using a CryoProbe optimised for 13C detection (dual CryoProbe [13C, 1H]) in which the receiver and transmitter coils are cooled to approximately 20K to reduce contributions to noise in the signal obtained. Compared with conventional inverse geometry probe, the signal-to-noise ratio (S/N) was increased by approximately 17-fold using this device. A large proportion of alanine was initially metabolised over the first 20 min by glial cells, as indicated by the relative importance of the glial, only enzyme pyruvate carboxylase to the labelling pattern of glutamate, with the ratio of pyruvate carboxylase to pyruvate dehydrogenase derived glutamate being 0.25, and exported [2-13C, 15N]aspartate.Using the increased sensitivity of the CryoProbe, [2-13C, 15N]aspartate was also detected in the extracts of cerebral tissue. This metabolite could only have been derived via the pyruvate carboxylase pathway, and given the large export of the metabolite into the superfusion buffer suggests the occurrence of a "metabolon" arrangement of enzymes within glial cells.