Formation of 6,13-dihydroxyoctadecatrienoic acid isomers from gamma-linolenic acid

Incubation of gamma-linolenic acid with soybean lipoxygenase initially at pH 9.3 and subsequently at pH 7.9 gave rise to the conjugated triene dioxygenation product (lambda max = 267 nm, greater than 50% yield), which was reduced to form 9-cis isomer of 6,13-dihydroxyoctadecatrienoic acid (6,13-diHOT) accompanied by minor isomers. Meanwhile, hemoglobin converted 13-hydroperoxyoctadecatrienoic acid into two major 9-trans isomers of 6,13-diHOT and two 9-cis isomers as minor products. The four isomers of 6,13-diHOT methyl ester were separated from each other on SP-HPLC, and characterized by chromatographic, spectrometric and cis----trans isomerization analyses.     

The possible role of 9(S)-hydroperoxyoctadecatrienoic acid as a suicide substrate of soybean lipoxygenase

While incubation of soybean lipoxygenase with alpha-linolenic acid resulted in the gradual decrease of lipoxygenase activity, the incubation with linoleic acid had no change. The inactivation of soybean lipoxygenase during incubation with alpha-linolenic acid was markedly observed at pH 6.5, but not at pH 9.0. Among the lipoxygenation products of alpha-linolenic acid, only 9(S)-hydroperoxyoctadecatrienoic acid caused the inactivation of lipoxygenase. 9(S)-Hydroxyoctadecatrienoic acid, 13(S)-hydroperoxyoctadecatrienoic acid or 9,16-dihydroperoxy conjugated trienoic acid was without effect. Accordingly, it is suggested that the epoxide intermediate, one conversion product of 9(S)-hydroperoxyoctadecatrienoic acid, might be involved in the direct inactivation of lipoxygenase.     

Synthesis of four isomers of parinaric acid

A simple and reliable method for synthesizing four isomers of parinaric acid from alpha-linolenic acid (ALA) in high yields is described. The methylene-interrupted, cis triene system (1,4,7-octatriene) of ALA and common to other naturally occurring polyunsaturated fatty acids was transformed to a conjugated tetraene system (1,3,5,7-octatetraene). The synthesis involves bromination of ALA using 0.l M Br(2) in a saturated solution of NaBr in methanol, esterification of the fatty acid dibromides, double dehydrobromination by 1,8-diazabicyclo[5.4.0]undec-7-ene and saponification of the conjugated esters to a mixture of free conjugated acids. Addition of one molecule of bromine to the 12,13-double bond of ALA and subsequent dehydrobromination produces alpha-parinaric acid (9Z,11E,13E,15Z-octadecatetraenoic acid); addition of Br(2) to the 9,10-double bond or 15,16-double bond and then dehydrobromination and rearrangement yields 9E,11E,13E,15Z-octadecatetraenoic or 9E,11E,13E,15Z-octadecatetraenoic acids, respectively. The mixture of parinaric acid isomers is obtained in 65% yield, and the isomers can be purified by preparative HPLC; alternatively, the isomers can be converted by base catalyzed cis-trans isomerization (or by treatment with I(2)) to exclusively beta-parinaric acid (9E,11E,13E,15E-octadecatetraenoic acid). The various parinaric acid isomers were characterized by (1)H NMR, (13)C NMR, UV, GLC, HPLC and mass spectrometry.     

Soybean lipoxygenase-catalyzed formation of lipoxin A and lipoxin B isomers from arachidonic acid via 5,15-dihydroperoxyeicosatetraenoic acid

Soybean lipoxygenase converted arachidonic acid to a group of polar products (lambda max, 300-301 nm), which were increasingly formed during the continued incubation at 20 degrees C after the initial incubation (2 hrs, at 4 degrees C). These products were identified as lipoxin A and B isomers, based on the chromatographic and spectrometric analyses. In further chromatographic analyses, the lipoxin A and B isomers were separated into at least three isomers, respectively. The exposure of 5,15-dihydroperoxyeicosatetraenoic acid to the soybean lipoxygenase produced the identical product profile of chromatography, substantiating the intermediacy of 5,15-dihydroperoxyeicosatetraenoic acid in the soybean lipoxygenase-catalyzed formation of lipoxins. Based on these results, it is proposed that the conversion of arachidonic acid into lipoxins by soybean lipoxygenase may bear a mechanistic resemblance to the formation of lipoxins in the human leukocytes.     

Characterization and biological effects of di-hydroxylated compounds deriving from the lipoxygenation of ALA

We have recently described a di-hydroxylated compound called protectin DX (PDX) which derives from docosahexaenoic acid (DHA) by double lipoxygenation. PDX exhibits anti-aggregatory and anti-inflammatory properties, that are also exhibited by similar molecules, called poxytrins, which possess the same E,Z,E conjugated triene geometry, and are synthesized from other polyunsaturated fatty acids with 22 or 20 carbons. Here we present new biological activities of di-hydroxylated metabolites deriving from α-linolenic acid (18:3n-3) treated by soybean 15-lipoxygenase (sLOX). We show that 18:3n-3 is converted by sLOX into mainly 13(S)-OH-18:3 after reduction of the hydroperoxide product. But surprisingly, and in contrast to DHA which is metabolized into only one di-hydroxylated compound, 18:3n-3 leads to four di-hydroxylated fatty acid isomers. We report here the complete characterization of these compounds using high field NMR and GC-MS techniques, and some of their biological activities. These compounds are: 9(R),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic acid, 9(S),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic acid, 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid, and 9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid. They can also be synthesized by the human recombinant 15-lipoxygenase (type 2). Their inhibitory effect on blood platelet and anti-inflammatory properties were compared with those already reported for PDX.     

Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.

The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.     

Identification of nucleic acid adducts from trans-4-acetylaminostilbene

It has been proposed that trans-4-acetylaminostilbene (AAS) is an initiator for tumor formation in rat liver and that the metabolically formed hydroxamic acid ester ultimately reacts with nucleic acids in vivo. We have now studied the generation of a major adduct in vitro. trans-4-N-Acetoxy-N-acetylaminostilbene (N-acetoxy-AAS) was reacted with guanosine at pH 7.5 and reaction products were separated by chromatography on Sephadex LH-20 and RP18 HPLC. The major adduct isolated consists of four isomers which have been tentatively identified by mass- and 1H-NMR spectroscopy as (S,S)- and (R,R)-guanosine-N2,beta-N3,alpha-N-acetylaminobibenzyl and the respective regio isomers guanosine-N2,alpha-N3,beta-N-acetylaminobibenzyl. These adducts are formed in a ratio of 9:9:1:1. Under acidic conditions (pH 2) the ribose moiety is removed and two regio isomeric base adducts are formed in the ratio 9:1. Results to be published indicate that the adducts are also formed in vivo in rat liver RNA and DNA.     

Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9S)-hydroperoxide by lipoxygenase-1, but not the (13S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9S)- and (13S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest and active-site model for soybean lipoxygenase-1.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Conjugated linoleic acid metabolism

Conjugated linoleic acid (CLA) is a naturally occurring fatty acid that is produced by a bio-hydrogenation process in the rumen, and thus is present in dairy products and ruminant meat. In this case the predominant isomer formed is 9cis,11trans. However, CLA includes 28 positional and geometrical isomers, of which only 9cis,11trans and 10trans,12cis have thus far been proven to possess biological activities. Both of these CLA isomers have been shown to undergo elongation and desaturation processes similar to those that occur with linoleic acid, maintaining the conjugated diene structure. There are evidences supporting the hypothesis that CLA metabolism may interfere with eicosanoid formation. Other metabolites with 16 carbon atoms (conjugated 16:2 and 16:3, which are probably derived from peroxisomal beta-oxidation of CLA and its metabolites, respectively) have been detected. This suggests an efficient metabolism of CLA and its metabolites in peroxisomes, which might be linked to their capacity to activate peroxisome proliferator-activated receptors.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

植物乳杆菌ZS2058在磷酸盐缓冲液体系中生物转化共轭亚油酸

植物乳杆菌ZS2058是从泡菜中筛选到一株具有转化共轭亚油酸能力的乳酸菌。该菌株在MRS培养基中经0.5mg/mL的亚油酸诱导培养后,所获得的菌体细胞具有较强的转化能力。文中就植物乳杆菌ZS2058水洗细胞在磷酸盐缓冲液体系中生物转化共轭亚油酸进行了深入研究。在非厌氧条件下,植物乳杆菌ZS2058在亚油酸浓度为1mg/mL,湿细胞质量浓度约为150mg/mL,120r/min、37℃的条件下反应24h后,能将亚油酸转化为共轭亚油酸和羟基脂肪酸,其中c9,t11-CLA占所产生的CLA总量的96.4%,产量可高达312.4μg/mL,说明该菌株有很强的专一性。随着反应进一步进行,反应至36h时,c9,t11-CLA含量逐渐减少,伴随着大量羟基脂肪酸的产生;并且,以CLA(c9,t11-CLA和t10,c12-CLA的混合样品)为底物进行反应时,c9,t11-CLA被转化为羟基脂肪酸。由此可知,c9,t11-CLA可能是该菌株生物转化LA过程中的一个中间产物。     

Evidence for a novel pathway of leukotriene formation in human platelets

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.     

Synthesis of 11,12-leukotriene A4, 11S,12S-oxido-5Z,7E,9E,14Z-eicosatetraenoic acid, a novel leukotriene of the 12-lipoxygenase pathway

A simple and efficient method for preparing 11,12-leukotriene A4 has been established by the stereospecific biomimetic route from arachidonic acid. 12S-Hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid was synthesized using a partially purified 12-lipoxygenase of porcine leukocytes. The methyl ester of the compound was then chemically converted to two labile epoxides with a conjugated triene structure. These compounds were identified by proton NMR and mass spectrometry to be 11S,12S-oxido-5Z,7E,9E,14Z-eicosatetraenoic acid (11,12-leukotriene A4) and its geometric isomer.     

Specificity of the effect of lipoxygenase metabolites of arachidonic acid on calcium homeostasis in neutrophils. Correlation with functional activity

The ability of the major neutrophil-derived lipoxygenase metabolites of arachidonic acid to increase the rate of 45Ca influx in rabbit neutrophils was examined. The results obtained demonstrate that (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid (leukotriene B4) is the most active of the arachidonic acid metabolites. The activity of leukotriene B4 is highly stereospecific in that its three nonenzymatically derived isomers are essentially inactive. The omega-hydroxylation of leukotriene B4 results in a compound that is nearly as active as leukotriene B4 as far as its ability to stimulate calcium influx and neutrophil aggregation while being a much weaker secretagogue. The further conversion of leukotriene B4 into a dicarboxylic acid removes all detectable biological activity. 5,6-Oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) methyl ester was also found to increase the rate of calcium influx, while the degradation products of native leukotriene A4 were essentially inactive. These results demonstrate that a close correlation exists between the ability of the various lipoxygenase products to alter calcium homeostasis in rabbit neutrophils and their biological activities.     

Effect of pH on conjugated linoleic acid (CLA) formation of linolenic acid biohydrogenation by ruminal microorganisms

Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of ¹³C-LNA with different pH conditions (5.5 and 6.5). The ¹³C enrichment was calculated as a ¹³C/¹²C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from ¹³C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.     

The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells

The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated (14)C-eicosanoids and (b) the incorporation of (14)C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of (14)C-prostaglandin F(2a) by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated (14)C-prostaglandins was observed with the CLA mixture (IC(50) 100 microM). The cis9,trans11 and trans10,cis12 (50 microM) isomers individually inhibited the overall production of stimulated (14)C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 microM), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of (14)C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of (14)C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of (14)C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.     

Bioconversion of linoleic acid into conjugated linoleic acid by immobilized Lactobacillus reuteri

Lactobacillus reuteri was immobilized on silica gel to evaluate the bioconversion of linoleic acid (LA) into conjugated linoleic acid (CLA), consisting of cis-9,trans-11 and trans-10,cis-12 isomers. The amount of cell to carrier, the reaction time, and the substrate concentration, pH, and temperature for CLA production were optimized at 10 mg of cells/(g of carrier), 1 h, 500 mg/L LA, 10.5, and 55 degrees C, respectively. In the presence of 1.0 mM Cu(2+), CLA production increased by 110%. Under the optimal conditions, the immobilized cells produced 175 mg/L CLA from 500 mg/L LA for 1 h with a productivity of 175 mg/(L.h) and accumulated 5.5 times more CLA than that obtained from bioconversion by free washed cells. The CLA-producing ability of reused cells was investigated over five reuse reactions and was maximal at pH 7.5, 25 degrees C, and 1.0 mM Cu(2+). The total amount of CLA by the combined five reuse reactions was 344 mg of CLA/L reaction volume. This was 8.6 times higher than the amount obtained from reuse reactions by free washed cells.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

Isomers of leukotriene B4 possess different biological potencies

Rat elicited polymorphonuclear leucocytes (PMNs), when exposed to the ionophore A23187, release three isomers of leukotriene B₄. The three isomers have been purified and tested for their ability to induce the chemokinesis of human PMNs in vitro, the aggregation of rat PMNs in vitro and changes in vascular permeability in rabbit skin in vivo in the presence of PGE₂. The results demonstrate that all three isomers are biologically active and that the enzymatically produced isomer, in which the conjugated triene contains one and two double bonds, is more potent than the two diastereoisomers of LTB₄ which contain all double bonds in the conjugated triene and which are produced by non-enzymatic hydrolysis.