Production of 10(S)-hydroxy-8(E)-octadecenoic acid mono-estolides by lipases in non-aqueous media

In this study, Novozym 435, a lipase B from Candida antarctica, was used for fatty acid polymerization. For the first time, an apolar reaction media, n-hexane, was used to synthesize in vitro estolides from trans-hydroxy-fatty acids derived from the biotransformation of oleic acid by Pseudomonas aeruginosa 42A2 NCIMB 40045. We studied the effects of the substrate, the enzyme ratio, the enzyme stability and the reusability of the biocatalyst. To determine the structure of the oligomers formed, both liquid chromatography mass spectrometry and MALDI-TOF mass spectrometry, with a DHB matrix neutralized with lithium hydroxide, were used to obtain simpler mass spectra. Estolides composed of two units of (10S)-HOME were synthesized with a reaction yield of 30%. Finally, various lipases were screened, and another apolar organic solvent, iso-octane, was assayed to try to increase the reaction yield.     

Production of 10,12-dihydroxy-8(E)-octadecenoic acid, an intermediate in the conversion of ricinoleic acid to 7,10,12-trihydroxy-8(E)-octadecenoic acid by Pseudomonas aeruginosa PR3

A bacterial isolate, Pseudomonas aeruginosa (PR3), has been reported to produce a new compound, 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD), from ricinoleic acid (Kuo TM, LK Manthey and CT Hou. 1998. J Am Oil Chem Soc 75: 875–879). The reaction is unique in that it involves an introduction of two additional hydroxyl groups at carbon 7 and 10 and a rearrangement of the double bond from carbon 9–10 (cis) to 8–9 (trans). In an effort to elucidate the metabolic pathway involved in the formation of TOD from ricinoleic acid by PR3, we have isolated another compound from the reaction mixture using HPLC. The structure of the new compound was determined to be 10, 12-dihydroxy-8(E)-octadecenoic acid (DHOD) by GC/MS, FTIR, and NMR. The structural similarity between DHOD and TOD and the results from the time course study of the above two compounds strongly suggested that DHOD was an intermediate in the bioconversion of ricinoleic acid to TOD by PR3. The optimum pH and temperature for the production of DHOD from ricinoleic acid by PR3 was 6.5 and 25°C, respectively. This is the first report on the production of 10,12-dihydroxy-8(E)-octadecenoic acid from ricinoleic acid by PR3. Journal of Industrial Microbiology & Biotechnology (2000) 24, 167–172. Received 28 July 1999/ Accepted in revised form 18 November 1999     

Structure-activity relationships of trans-substituted-propenoic acid derivatives on the nicotinic acid receptor HCA2 (GPR109A)

Nicotinic acid (niacin) has been used for decades as an antidyslipidemic drug in man. Its main target is the hydroxy-carboxylic acid receptor HCA2 (GPR109A), a G protein-coupled receptor. Other acids and esters such as methyl fumarate also interact with the receptor, which constituted the basis for the current study. We synthesized a novel series of substituted propenoic acids, such as fumaric acid esters, fumaric acid amides and cinnamic acid derivatives, and determined their affinities for the HCA2 receptor. We observed a rather restricted binding pocket on the receptor with trans-cinnamic acid being the largest planar ligand in our series with appreciable affinity for the receptor. Molecular modeling and analysis of the structure-activity relationships in the series suggest a planar trans-propenoic acid pharmacophore with a maximum length of 8 Å and out-of-plane orientation of the larger substituents.     

Biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and the determination of the absolute configuration of all isomers

2-Hydroxy-2-(ethoxyphenylphosphinyl)acetic acid, a new type of organophosphorus compound possessing two stereogenic centers, was investigated. Racemic 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid was synthesized and hydrolyzed using four bacterial species as biocatalysts. In all cases the reaction was more or less stereoselective and isomers bearing a phosphorus atom with an (S_P)-configuration were hydrolyzed preferentially. The observed ¹H and ³¹P NMR chemical shifts of Mosher esters of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid were correlated with the configurations of both stereogenic centers of all four stereoisomers.     

10-Hydroxy-8(Z)-octadecenoic acid,an intermediate in the bioconversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid

Summary Previously, we reported the discovery of a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) which was produced from oleic acid by a new bacterial isolate PR3 [6,7]. The reaction is unique in that it involves a hydroxylation at two positions and a rearrangement of the double bond of the substrate molecule. Now, we have isolated another compound from the reaction mixture determined by GC/MS to be 10-hydroxy-8-octadecenoic acid (HOD). NMR and IR data indicate that the unsaturation is probablycis. The optimum pH and temperature for the production of HOD by strain PR3 were 6.5 and 30°C, about the same as those for DOD. However, the amount of HOD detected remained small throughout an 48-h reaction period during which the amount of DOD increased sharply. At 48 h of reaction, the ratio between HODDOD was 110. HOD may be an intermediate in the biosynthesis of DOD from oleic acid.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Determination of abscisic acid in Eucalyptus haemastoma leaves using gas chromatography/mass spectrometry and deuterated internal standards

Abscisic acid and 2-trans-abscisic acid each with three deuterium atoms in the C-3 methyl group, have been synthesized chemically and used as internal standards in selected ion monitoring experiments to establish the endogeneous concentrations of these compounds and their conjugates in turgid and wilted Eucalyptus haemastoma leaves. The analytical procedure used GC/CIMS(methane) to detect the methyl esters of abscisic acid, 2-trans-abscisic acid and their deuterated internal standards. A three-fold increase in the concentration of abscisic acid occurred on wilting and the amounts of 2-trans-abscisic acid and conjugates of both compounds were determined.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.     

Perennial peanut (Arachis glabrata Benth.) leaves contain hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase activity and accumulate hydroxycinnamoyl-tartaric acid esters

Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg^?1 crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.     

A facile reactor process for producing 7,10-dihydroxy-8(E)-octadecenoic acid from oleic acid conversion by Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). The bioconversion was scaled up in a 7-l bench-top, stirred-batch reactor to produce DOD for testing of potential industrial uses. Aeration was supplied continuously from the top through two ports on the headplate and periodically through a bottom sparger, in conjunction with the use of marine impellers for agitation. This unique aeration arrangement maintained the dissolved O₂ concentration in the 40–60% range during the period of maximal bioconversion and it also avoided excessive medium foaming during the reaction. Furthermore, the level of dissolved O₂ in the first 24 h of reaction played an important role in the initial rate of DOD production. DOD production reached a plateau after 72 h with a yield up to 100 g (or 50% recovery) from a total of 9 l medium from two reactors run simultaneously. The final culture broth was processed using newly adapted procedures in the pilot plant that included crystallization of DOD from ethyl acetate solution at –15°C. The newly developed bioprocess will serve as a platform for the scale-up production of other value-added products derived from vegetable oils and their component fatty acids.     

Metabolism and biological activity of all-trans 4,4-difluororetinyl acetate

All-trans [11-³H]4,4-difluororetinyl acetate was synthesized by treating methyl all-trans [11-³H]4-oxoretinoate with diethylaminosulfurtrifluoride, followed by reduction and acetylation of the product. After oral administration of the radioactive difluoro analog in oil to rats, difluororetinol, difluororetinyl palmitate and related esters, 4-oxoretinol, 4-oxoretinoic acid and polar conjugated derivatives were identified in the intestine, liver, kidney and / or blood. The major metabolic products were difluororetinyl palmitate and related esters, which were stored in the liver. The presence of the difluoro analog in liver oil from treated rats was confirmed by ¹⁹F-NMR spectroscopy. Neither retinol nor retinyl esters were detected as products of the metabolism of the difluoro analog. Nonetheless, all-trans difluororetinyl acetate showed 26 ± 12% of the biological activity of all-trans retinyl acetate in the rat growth assay. Presumably, the difluoro analog is active per se in growth rather than by conversion to retinol or to one of its known growth-promoting metabolites. In general, however, the difluoro analog was metabolized in a manner very similar to vitamin A. The vitamin A moiety of administered difluororetinyl acetate and retinyl acetate was poorly stored (1.8–3.3%) in the liver of vitamin A-depleted rats, confirming and extending past reports that the liver storage mechanism is severely impaired when initial liver stores are very low.     

Chiral enamines derived from 2-(2′-pyrido)acetophenone and 2-(2′-quinolino)acetophenone as ligands in copper(I) catalyzed enantioselective cyclopropanations

Optically active enamines of 2-(2′-pyrido)acetophenone or 2-(2′-quinolino)acetophenone with (R)-1-phenylethylamine, (R)-1-(1-naphthyl)ethylamine, (R)-cyclohexylethylamine, and (R)-phenylglycinol were prepared and their copper(I) complexes used in the enantioselective cyclopropanation of styrene with ethyl- and menthyldiazoacetate. Enantioselectivities of up to 42% enantiomeric excess were obtained for cis/trans 2-phenylcyclopropan-1-carboxylic acid ethyl esters, as determined by gas-liquid chromatography (GLC) on chiral chromatographic columns. © 1995 Wiley-Liss, Inc.     

Synthesis and evaluation of (E)-2-(acrylamido)cyclohex-1-enecarboxylic acid derivatives as HCA1, HCA2, and HCA3 receptor agonists

2-(3-(Naphthalen-2-yl)propanamido)cyclohex-1-enecarboxylic acid and its 6-hydroxynaphthalen-2-yl analogue are well-known hydroxyl-carboxylic acid (HCA) receptor HCA2 agonists. A series of novel aryl derivatives of 2-amidocyclohex-1-ene carboxylic acid that contained rigidity elements, such as an E-double bond, triple bond, and trans or cis-substituted cyclopropane rings, instead of the saturated ethane linker in the amide part of the molecules were designed and synthesized, and the derivatives’ potency for the activation of HCA1, HCA2, and HCA3 receptors by 3′–5′-cyclic adenosine monophosphate (cAMP) assay were evaluated. The SAR studies revealed that the rigidifying of appropriate molecules enabled modulation of the potency and selectivity of the HCA2 receptor activation.     

Activity and metabolism of C-(+/-)-abscisic Acid derivatives

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-¹⁴C-abscisic acid and the cis- and trans-1′,4′-diols of 2-¹⁴C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.     

Synthesis of 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranoses

Five 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranoses (lipophilic, muramoyl dipeptide analogs) were synthesized from benzyl 2-(benzyloxycarbonylamino)-3-O-(d-1-carboxyethyl)-2-deoxy-5,6-O-isopropylidene-β-dglucopyranoside (1). Methanesulfonylation of 3, derived from the methyl ester of 1 by O-deisopropylidenation, gave the 6-methanesulfonate (4). (Tetrahydropyran-2-yl)ation of 4 gave benzyl 2-(benzyloxycarbonylamino)-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-6-O-(methylsulfonyl)-5-O-(tetrahydropyran-2-yl)-β-d- glucofuranoside, which was treated with sodium azide to give the corresponding 6-azido derivative (6). Condensation of benzyl 6-amino-2-(benzyloxycarbonyl-amino)-2,6-dideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-5-O-(tetrahydropyran-2-yl)-β-d-glucofuranoside, derived from 6 by reduction, with the activated esters of octanoic, hexadecanoic, and eicosanoic acid gave the corresponding 6-N-fatty acyl derivatives (8–10). Coupling of the 2-amino derivatives, obtained from compounds 8, 9, and 10 by catalytic reduction, with the activated esters of the fatty acids, gave the 2,6-(diacylamino)-2,6-dideoxy derivatives (11–15). Condensation of the acids, formed from 11–15 by de-esterification, with the benzyl ester of l-alanyl-d-isoglutamine, and subsequent hydrolysis, afforded benzyl 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine benzyl ester)-β-d-glucofuranosides. Hydrogenation of the dipeptide derivatives thus obtained gave the five lipophilic analogs of 6-amino-6-deoxymuramoyl dipeptide, respectively, in good yields.     

Cobalt(III) complexes with racemic polyamine ligands 1. Isomers formed using 5,5,7(R,S),12,12,12,14(R,S)-hexamethyl-1,4,8,11-tetraazacyclotetradecane (tetb)

cis-[Co(ox)(tetb]ClO₄ is readily isolated from the reaction between tetb (tetb = rac-Me₆-cyclam = 5,5,7(R,S),12,12,14(R,S)-hexamethyl-1,4,8,11-tetraazacyclotetradecane) and K₃Co(ox)₃·3H₂O in aqueous solution. Removal of the coordinated oxalato ligand by acid hydrolysis (with HCl/HClO₄) results in the formation of α-trans-(RRRR,SSSS), [CoCl₂{(SSeq,RReq)-tetb}]ClO₄ with both six-membered rings in the twist conformation. The (RR)-(+)-tartrate (−2) ion coordinates enantioselectively with this isomer to give (–)-cis-[Co₂{μ-(RR)-(+)-(tart)}{(RR)-tetb}₂](ClO₄)₂ and this, in turn, reacts with HCl/HClO₄ to give (–)-α-trans-(1S,4S,8S,11S)-[CoCl₁₂{7Req,14Req)-tetb}]ClO₄. The absolute configuration of the tetraamine obtained in the resolution procedure was established by synthesizing the α-trans-dichloro isomer using tetb of known absolute configuration.     

A two steps enzymatic procedure to obtain sterol esters,tocopherols and fatty acid ethyl esters from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) was enzymatically modified to obtain a product mixture comprised mainly of sterol esters, tocopherols, and fatty acid ethyl esters. Firstly, the original SODD was mixed with oleic acid to reduce its melting point from 65–70 to 30–35 °C and also to produce a reaction mixture with a ratio of free fatty acids (FFA) to sterols close to 2 to improve the progress of sterols esterification. Two enzymatic steps were used in order to separate sterols esterification and ethyl esterification in time and space. The first enzymatic step (in the presence of Candida rugosa lipase) allowed to efficiently transform more than 90% of the original sterols in a short period of time (5 h). The second enzymatic step (in the presence of Novozym 435) converted more than 95% of the FFA in less than 3 h. In addition, the stability of both biocatalysts has been evaluated and both bioprocesses have been scaled-up reutilizing the same batch of lipase up to 8 and 3 times for the first and the second enzymatic step, respectively. The final product obtained is intended to be used as starting material for the purification of sterol esters, tocopherols, and fatty acid ethyl esters via supercritical fluid extraction.     

Sporotrichum thermophile type C feruloyl esterase (StFaeC): purification,characterization, and its use for phenolic acid (sugar) ester synthesis

A feruloyl esterase (StFaeC) produced by Sporotrichum thermophile was purified to homogeneity. The native StFaeC was homodimer with a subunit of M_r 23,000 and pI 3.1. The enzyme activity was optimal at pH 6.0 and 55 °C. The esterase displayed remarkable stability at pH 10.0 and retained 50% of its activity after 133 and 55 min at 55 and 60 °C, respectively. Determination of k_cat/K_m revealed that the enzyme had a broad spectrum of activity against the (hydroxyl) cinnamate esters indicating a type C feruloyl esterase. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and hydrolysed 4-nitrophenyl-5-O-trans-feruloyl-α-l-arabinofuranoside three times more efficiently than 4-nitrophenyl-2-O-trans-feruloyl-α-l-arabinofuranoside. Ferulic acid was efficiently released from wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 41% total ferulic acid released after 1 h incubation). StFaeC by itself could release FA but at a level almost 10-fold lower than that obtained in the presence of xylanase. The potential of StFaeC for the synthesis of various phenolic acid esters was examined using as a reaction system a ternary water–organic mixture consisting of n-hexane, 1-butanol and water. Also StFaeC catalyzed the transfer of the feruloyl group to l-arabinose in a similar system using t-butanol, with about a 40% conversion of l-arabinose to feruloylated derivative was achieved. This work is the first example of enzymatic feruloylation of a carbohydrate.     

Cyclic fatty esters: Stereochemistry of monounsaturated products from the hydrogenation and reduction of 9-(6-propyl-3-cyclohenxenyl)-8-nonenoic acid or its ester

Diunsaturated, C-18 cyclic fatty acid methyl esters (CFAME) were previously synthesized as model derivatives for characterization and biological evaluation of cyclic fatty acids (CFA) formed in heat-abused vegetable oils. The propyl substituted, diunsaturated CFMAE (I) was selectively reduced to prepare two monounsaturated, positional isomers with the double bond located either in the ester substituent (alkene isomer II) or in the ring (cyclohexene isomer III). The stereochemistry of these monounsaturated products was investigated by capillary GLC and NMR. Capillary GLC showed that each positional isomer was a mixture of two ‘ring’ isomers (i.e. a mixture of two isomers with side chains either cis or trans). The ring double bond in diene I was readily hydrogenated with various metal catalysts, and no cyclohexene isomer III was detected in the product. Platinum oxide poisoned with Ph₃P was the most selective catalyst examined to convert diene I to monoene II. Diimide reduction was the only method foud to reduce selectively the double bond in the ester side chain of diene I. This diimide reduction was facilitated when the Z-double bond in the side chain was isomerized to E-double bond with p-toluenesulfinic acid. Cyclohexene isomer III and alkene isomer II were separated by argentation HPLC. These two isomeric monoenes were characterized by GC-MS, capillary GLC, micro-ozonolysis, IR and NMR. Catalytic hydrogenation with Ph₃P-poisoned PtO₂ and diimide reduction of the diunsaturated cyclic ester may provide useful methods to synthesize and label monounsaturated cyclic fatty esters.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC–MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal–glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione–alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d₁₁-4-hydroxy-2-(E)-nonenal (d₁₁-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d₁₁-4-HNE-perfused rat livers, the amount of d₁₁-(S)-4-HNE–GSH released from the rat liver was higher than that of d₁₁-(R)-4-HNE–GSH, and more d₁₁-(R)-4-HNE–GSH than d₁₁-(S)-4-HNE–GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.