Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 1: NMR spectral characterization

Methyl esters of gamma-linolenic acid, alpha-linolenic acid and stearidonic acid were epoxidised using m-chloroperbenzoic acid to achieve nine cis-monoepoxy-C18 fatty acid methyl esters (FAMEs), including novel methyl cis-monoepoxy derivatives of stearidonic acid and a cis-6,7-epoxy derivative of gamma-linolenic acid. These nine monoepoxy FAMEs were purified by normal-phase HPLC, identified by LC-MS, 1H and 13C NMR, and characterized by mass spectrometry and NMR spectroscopy. This study is focused on structural characterization of these C18 monoepoxy FAMEs using techniques in NMR spectroscopy including 1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear correlation (HETCOR). The proton and carbon NMR chemical shifts of the epoxide, the double bonds, and the interrupted methylenes are assigned. Also discussed is an interpretation of the 1H and 13C NMR spectra of these monoepoxides including the changes in the 13C resonance of the olefinic carbons on the neighboring double bonds resulting from epoxide formation.     

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.     

Triterpenoid saponins and acylated prosapogenins from Harpullia austro-caledonica

Three new triterpenoid saponins have been isolated from the stem bark of Harpullia austro-caledonica and identified as 24-O-[alpha-L-rhamnopyranosyl-(1->2)-beta-D-glucopyranosyl]-28-O-[beta-D-glucopyranosyl-(1->2)-beta-D-glucopyranosyl]-protoaescigenin, 24-O-[alpha-L-rhamnopyranosyl-(1->2)-beta-D-glucopyranosyl]-28-O-[beta-D-glucopyranosyl-(1->2)-beta-D-glucopyranosyl]-16-desoxyprotoaescigenin, 24-O-[alpha-L-rhamnopyranosyl-(1->2)-beta-D-glucopyranosyl]-28-O-[beta-D-glucopyranosyl-(1->2)-beta-D-glucopyranosyl]-24-oxo-camelliagenin D. The 21,22-di-O-angeloate esters of protoaescigenin and barringtogenol C were isolated in the acid hydrolysate of the saponin extract together with a new prosapogenin identified as 21beta,22alpha-di-O-angeloyl camelliagenin D. The structures were established using one- and two- dimensional NMR and mass spectrometry.     

Oxygenation of linolenic and linoleic acid to novel vicinal dihydroxy acids by hepatic microsomes of the rabbit

[14C] Linolenic acid (18: omega 3) and [14C] linoleic acid (18:2 omega 6) were incubated with hepatic microsomes of the rabbit in the presence of NADPH (1 Mm) for 15 min at 37 degrees C. The products were extracted and purified by high performance liquid chromatography. The major metabolites of linolenic and linoleic acid were identified by capillary gas chromatography mass spectrometry as 15,16-dihydroxy-9,12-octadecadienoic acid, 12,13-dihydroxy-9,15-octadecadienoic acid and 9,10-dihydroxy-12,15-octadecadienoic acid and as 12,13-dihydroxy-9-octadecaenoic acid and 9,10-dihydroxy-12-octadecaenoic acid, respectively. The results were confirmed by comparison with mass spectra of the authentic compounds. These metabolites are presumably formed by cytochrome P-450 catalyzed epoxidation of the omega 3, omega 6 and omega 9 double bonds, followed by enzymatic hydrolysis to 1,2-diols. The ratio of omega 3, omega 6 and omega 9 oxygenated metabolites of linolenic acid was approximately 2:1:1 and the ratio of the omega 6 and omega 9 metabolites of linoleic acid was 2:1, indicating that the double bond closest the omega end is most easily oxygenated.     

The stereochemistry of desaturations of long-chain fatty acids in Chlorella vulgaris

1. A study was made of the stereospecificity of hydrogen removal in the sequential desaturations performed by intact cells of Chlorella vulgaris in the biosynthesis of oleic acid, linoleic acid and alpha-linolenic acid. 2. By use of erythro- and threo-9,10-(2)H(2)-, -12,13-(2)H(2)- and -15,16-(2)H(2)-labelled precursors, it was demonstrated that the pair of hydrogen atoms removed from each of these positions had the cis relative configuration. 3. That the hydrogen atoms removed in oleic acid and linoleic acid formation were of the d absolute configuration was proved by use of d- and l-9-(3)H-and -12-(3)H-labelled precursors. 4. The presence of a substantial kinetic isotope effect of deuterium at both positions of the putative double bond was indicated, suggesting that the mechanism of desaturation involves simultaneous concerted removal of the pair of hydrogen atoms.     

Synthesis and NMR characterization of the methyl esters of eicosapentaenoic acid monoepoxides

The activities of cytochrome P450-derived epoxide metabolites of omega-6 polyunsaturated fatty acids (PUFAs) in cellular homeostasis have generated considerable topical interest, but there is less information on the effects of omega-3 PUFA epoxides. Mass spectroscopic data on the epoxides of the omega-3 PUFA eicosapentaenoic acid (EPA) have been reported but the absence of corresponding NMR data currently hinders their biological assessment. In the present study five monoepoxy derivatives of EPA methyl ester were synthesized by treating EPA methyl ester with m-chloroperbenzoic acid. The individual regioisomers were purified by normal-phase chromatography and characterized by LC-MS/MS and a combination of NMR approaches including ¹H-, ¹³C-, ¹H-¹H-COSY, ¹H-¹³C-HSQC, and ¹H-¹³C-HMBC. The chromatographic properties for these monoepoxides were studied in normal-phase and reversephase-HPLC systems and the MS/MS fragmentation patterns using electrospray ionization were established. This paper also focuses on the NMR characterization of epoxide, olefinic and methylenic moieties and the complete assignments of the isomers.     

△6-脂肪酸脱氢酶的分子生物学研究进展

包括γ-亚麻酸在内的多不饱和脂肪酸由于在人类健康中的重要作用而成为有价值的产品,目前市场上对γ-亚麻酸的需求持续增长,然而当前来源难以满足市场的需求,寻找合适的替代来源将有助于解决这一问题。△^6-脂肪酸脱氢酶是多不饱和脂肪酸合成途径中的限速酶,这里从△^6-脂肪酸脱氢酶的基因克隆、结构和功能的研究、系统进化和基因工程应用等方面探讨了△^6-脂肪酸脱氢酶的研究进展。     

Application of liquid chromatography-mass spectrometry to the investigation of poisoning by Oenanthe crocata

Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.     

Metabonomic investigation of liver profiles of nonpolar metabolites obtained from alcohol-dosed rats and mice using high mass accuracy MSn analysis

Alcoholism is a complex disorder that, in man, appears to be genetically influenced, although the underlying genes and molecular pathways are not completely known. Here, the intragastric alcohol feeding model in rodents was used together with high mass accuracy LC-MS(n) analysis to assess the metabonomic changes in nonpolar metabolite profiles for livers from control and alcohol-treated rats and mice. Ion signals with a peak area variance of less than 30% (based on repeat analysis of a pooled quality control sample analyzed throughout the batch) were submitted to multivariate statistical analysis (using principal components analysis, PCA). PCA revealed robust differences between profiles from control and alcohol-treated animals from both species. The major metabolites seen to differ between control and alcohol-treated animals were identified using high accuracy MS(n) data and verified using external search engines ( http://www.lipidmaps.org ; http://www.hmdb.ca; http://www.genome.jp/kegg/ ) and authentic standards. The main metabolite classes to show major changes in the alcoholic liver-derived samples were fatty acyls, fatty acid ethyl esters, glycerolipids, and phosphatidylethanol homologues. Significant metabolites that were up-regulated by alcohol treatment in both rat and mouse livers included fatty acyls, metabolites such as octadecatrienoic acid and eicosapentaenoic acid, a number of fatty acid ethyl esters such as ethyl arachidonate, ethyl docosahexaenoic acid, ethyl linoleate, and ethyl oleate and phosphatidylethanol (PEth) homologues (predominantly PEth 18:0/18:2 and PEth 16:0/18:2; PEth homologues are currently considered as potential biomarkers for harmful and prolonged alcohol consumption in man). A number of glycerophospholipids resulted in both up-regulation (m/z 903.7436 [M + H](+) corresponding to a triglyceride) and down-regulation (m/z 667.5296 [M + H](+) corresponding to a diglyceride). Metabolite profiles were broadly similar in both mouse and rat models. However, there were a number of significant differences in the alcohol-treated group particularly in the marked down-regulation of retinol and free cholesterol in the mouse compared to the rat. Unique markers for alcohol treatment included ethyl docosahexaenoic acid. Metabolites were identified with high confidence using predominantly negative ion MS(n) data for the fatty acyl components to match to www.lipidmaps.org MS and MS/MS databases; interpreting positive ion data needed to take into account possible adduct ions which may confound the identification of other lipid classes. The observed changes in lipid profiles were consistent with alcohol-induced liver injury in humans.     

A divergent synthesis of [1-14C]-mono-E isomers of fatty acids

A convenient synthesis of [1-14C]-mono-trans fatty acid using olefin inversion as a key-step is described. This methodology allows for a facile synthesis of [1-14C]-labelled mono-trans analogues of oleic, linoleic and linolenic acids. As an example, only eleven steps were necessary to obtain the [1-14C]-mono-E isomers of linolenic acid from its commercial all-Z form. In the first step, Barton's decarboxylation procedure yielded a bromo intermediate. Epoxidation of this compound resulted in the formation of three monoepoxides, which could be separated by HPLC. After identification by 1H NMR and MS, the pure monoepoxides were then subjected to inversion consisting of a stereospecific deoxygenation followed by a beta-elimination step. Finally, the labelling was introduced by substitution of the bromine by a [14C]-cyano group followed by hydrolysis.     

A peroxygenase pathway involved in the biosynthesis of epoxy fatty acids in oat

While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation.     

On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.     

Leaf lipids of Ribes nigrum: a plant containing 16:3, alpha-18:3, gamma-18:3 and 18:4 fatty acids

The glycerolipid composition of Ribes nigrum (blackcurrant) leaves was determined. The total fatty acid composition was unusual in that alpha-linolenic acid (alpha-18:3) occurred together with cis-7,10,13-hexadecatrienoic acid (16:3) and lower amounts of stearidonic acid (18:4) and gamma-linolenic acid (gamma-18:3). Monogalactosyldiacylglycerol contained the highest proportion of 16:3 with less in digalactosyldiacylglycerol. gamma-18:3 and 18:4 were present in all lipids and 18:4 was always greater than gamma-18:3. The highest percentages of gamma-18:3 and 18:4 were in phosphatidylcholine, but phosphatidylglycerol was particularly low in these acids. In summary, the lipid composition was largely typical of 16:3 plants but there was a minor contribution typical of 18:4 plants. The possibility of three pathways for glycolipid biosynthesis is discussed.     

Effect of dietary alpha-linolenic acid on the conversion of linoleic and gamma-linolenic acids (1-14C) into arachidonates in rats in vivo]

The effects of alpha-linolenic acid (9-12-15 octadecadienoic) upon the conversion in vivo of [1-14C] linoleic acid and of [1-14C] gamma-linolenic acid into arachidonate have been studied in adult rats. The two tracers have been administered by stomach tubing and the amounts of [14C]-radioactivity incorporated into arachidonate in the liver, kidneys and whole rat have been measured 48 h later. Three experiments have been carried out on rats fed on alpha-linolenic acid containing diets prior to the radioactive tubing. In these diets, alpha-linolenic acid was brought either as ethyl ester or in the form of Primor oil (erucic acid free rapeseed oil). In all of them, the ratio alpha-linolenic acid: linoleic acid did not exceed 0.45. Control animals were fed, in the same conditions, ethyl oleate or peanut oil respectively. Comparing the alpha-linolenic acid fed-rats to the control animals, we were able to observe the following results: (1) The exogenous supplies of alpha-linolenic acid used in the diets have not brought about any significant alteration in the amounts (weights) of arachidonic acid present in the liver, kidneys and whole animal. (2) Using [1-14C] linoleic acid as a precursor, the amounts of [14C]-radioactivity incorporated into arachidonate in the same organs as well as in the whole rat have been significantly lowered by dietary alpha-linolenate. (3) alpha-Linolenate, on the contrary, had no significant effect upon the amounts of radioactivity incorporated into hepatic, renal and whole body arachidonate following the administration of [1-14C] gamma-linolenic acid. These results lead to the conclusion that alpha-linolenic acid, when present in the diet of rats at a limited, phyisological level, partly inhibits the desaturation of linoleic acid in vivo but does not affect the subsequent reactions in the biosynthesis of arachidonic acid.     

Identification of carotenoid pigments and their fatty acid esters in an avian integument combining HPLC-DAD and LC-MS analyses

Yellow-orange-red ornaments present in the integuments (feathers, bare parts) of birds are often produced by carotenoid pigments and may serve to signal the quality of the bearer. Although carotenoid esterification in tissues is a common phenomenon, most of the work on avian carotenoids has been focused on the identification of free forms or have been done after sample saponification. Here we determined free and esterified carotenoid composition in a bird species with red ornaments: the red-legged partridge (Alectoris rufa). Carotenoids from leg integument were extracted and processed by TLC to separate three major carotenoid groups (free form, mono- and diesters with fatty acids), whereas saponified extracts gave only free forms of carotenoids. TLC fractions were then analyzed by HPLC-DAD with C18 phase column for a preliminary identification of carotenoid groups. The final characterization of free carotenoids and its esters with fatty acids was performed with direct extracts analyzed by LC-MS and LC-MS/MS with a C30 phase, always with a system coupled to DAD. The main carotenoid (λ(max) 478 nm and [M+H](+) at m/z 597.2) was identified as astaxanthin by comparison with standards. A second carotenoid (λ(max) between 440 and 480 nm and [M+H](+) at m/z 581.3) was not identified among any of the commercially available carotenoid standards, although it could correspond to pectenolone according to its fragmentation pattern. Both the unidentified carotenoid and astaxanthin formed monoesters with fatty acids, but only astaxanthin was in its diesterified form. Monoesters were mainly formed with palmitic, stearic, oleic and linoleic acids. Complementary analyses of fatty acid composition in partridge integument by GC-MS revealed high amounts of these and other fatty acids, such as myristic, arachidic and docosanoic acids. The combination of HPLC-DAD and LC-MS/MS spectra was especially useful to identify the carotenoids present in the esterified forms and the probable masses of the fatty acids included in them, respectively.     

Chemical C2-elongation of polyunsaturated fatty acids

Three fatty acids were synthesized from commercially available alpha-linolenic, stearidonic and eicosapentaenoic acids by C2-elongation using a four step preparative technique. The parent fatty acid methyl esters were reduced to alcohols with LiAlH(4), converted to bromides by treatment with triphenylphosphine dibromide, coupled with a lithiated C2-elongation block--2,4,4-trimethyl-2-oxazoline--to form the corresponding 2,2-dimethyloxazolines of C2-elongated fatty acids, and finally, converted to the target polyunsaturated fatty acids by acidic alcoholysis. Yields of more than 60% were achieved on a gram scale. The resulting 11Z,14Z,17Z-eicosatrienoic, 8Z,11Z,14Z,17Z-eicosatetraenoic and 7Z,10Z,13Z,16Z,19Z-docosapentaenoic acids were obtained as colorless oils with >98% purity and could be used for biochemical investigations without additional purification. The elongated fatty acids were free of byproducts that could result from Z-E isomerization or migration of double bonds.     

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). II. Quantitative determination of cis-EODA in human plasma

Cytochrome P450 dependent epoxidation and non-enzymic lipid peroxidation of oleic acid (cis-9-octadecenoic acid) result in the formation of cis-9,10-epoxyoctadecanoic acid (cis-EODA). This oleic acid oxide has been identified indirectly in blood and urine of humans. Reliable concentrations of circulating cis-EODA have not been reported thus far. In the present article, we report on the first GC-tandem MS method for the accurate quantitative determination in human plasma of authentic cis-EODA as its pentafluorobenzyl (PFB) ester. cis-[9,10-2H2]-EODA (cis-d2-EODA) was synthesized by chemical epoxidation of commercially available cis-[9,10-2H2]-9-octadecenoic acid and used as an internal standard for quantification. Endogenous cis-EODA and externally added cis-[9,10-2H2]-EODA were isolated from acidified plasma samples (1 ml; pH 4.5) by solvent or solid-phase extraction, converted into their PFB esters, isolated by HPLC and quantified by selected reaction monitoring. The parent ions [M-PFB]- at mass-to-charge ratio (m/z) 297 for cis-EODA and m/z 299 for (cis-d2-EODA) were subjected to collisionally-activated dissociation and the corresponding characteristic product ions at m/z 171 and 172 were monitored. In plasma of nine healthy humans (5 females, 4 males), cis-EODA was found to be present at 47.6+/-7.4 nM (mean+/-S.D.). Plasma cis-EODA levels were statistically insignificantly different (P=0.10403, t-test) in females (51.1+/-3.4 nM) and males (43.1+/-2.2 nM). cis-EODA was identified as a considerable contamination in laboratory plastic ware and found to contribute to endogenous cis-EODA by approximately 2 nM. The present GC-tandem MS method should be useful in investigating the physiological role(s) of cis-EODA in humans.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (S_Ni) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.