Regiospecific analysis of diricinoleoylacylglycerols in castor (Ricinus communis L.) oil by electrospray ionization-mass spectrometry

HPLC fractions of diricinoleoylacylglycerols containing one non-ricinoleoyl chain from castor oil were used to identify the regiospecific location of this non-ricinoleoyl chain on the glycerol backbone using electrospray ionization-MS3 of lithium adducts. The regiospecific ions used were from the loss of alpha,beta-unsaturated fatty acid specific at the sn-2 position. The content of 1,3-diricinoleoyl-2-oleoyl-sn-glycerols (ROR) among the three stereospecific isomers, RRO, ROR and ORR, was about 91%. The contents of other 1,3-diricinoleoyl-2-acyl-glycerols among the three stereospecific isomers were as follows: 1,3-diricinoleoyl-2-linoleoyl-sn-glycerol, 95%; 1,3-diricinoleoyl-2-linolenoyl-sn-glycerol, 96%; 1,3-diricinoleoyl-2-stearoyl-sn-glycerol, 96%; 1,3-diricinoleoyl-2-palmitoyl-sn-glycerol, 78%; and 1,3-diricinoleoyl-2-lesqueroloyl-sn-glycerol, 31%. These non-hydroxyl fatty acids were mostly at the sn-2 position of triacylglycerols in castor oil. These results suggest that phospholipase A2 hydrolysis of phosphatidylcholine (PC) containing non-hydroxyl fatty acid at the sn-2 position is either blocked or partially blocked in vivo. Phospholipase A2 hydrolysis of 2-lesqueroloyl-PC is not blocked and is similar to that of 2-ricinoleoyl-PC. Transgenic inhibition of phospholipase C hydrolysis of PC might be used to block the incorporation of non-hydroxyl fatty acids into triacylglycerols, thus increasing the content of ricinoleate in seed oil.     

(13)C NMR characterization of triacylglycerols of Moringa oleifera seed oil: an "oleic-vaccenic acid" oil

The composition of acyl chains and their positions in the triacylglycerols of the oil extracted from seeds of Moringa oleifera were studied by (13)C NMR spectroscopy. The unsaturated chains of M. oleifera seed oil were found to comprise only mono-unsaturated fatty acids and, in particular, two omega-9 mono-unsaturated acids, (cis-9-octadecenoic (oleic acid) and cis-11-eicosenoic acids) and one omega-7 mono-unsaturated acid (cis-11-octadecenoic acid (vaccenic acid)). The mono-unsaturated fatty acids were detected as separated resonances in the spectral regions where the carbonyl and olefinic carbons resonate according to the 1,3- and 2-positions on the glycerol backbone. The unambiguous detection of vaccenic acid was also achieved through the resonance of the omega-3 carbon. The (13)C NMR methodology enabled the simultaneous detection of oleate, vaccenate, and eicosenoate chains according to their positions on the glycerol backbone (1,3- and 2-positions) through the carboxyl, olefinic, and methylene envelope carbons of the triacylglycerol acyl chains.     

Synthesis of structured lipids via acidolysis of docosahexaenoic acid single cell oil (DHASCO) with capric acid

Screening of five commercially available lipases for the incorporation of capric acid (CA) into docosahexaenoic acid single cell oil (DHASCO) indicated that lipase PS-30 from Pseudomonas sp. was most effective. Of the various reaction parameters examined, namely, the mole ratio of substrates, enzyme amount, time of incubation, reaction temperature, and amount of added water, for CA incorporation into DHASCO, the optimum conditions were a mole ratio of 1:3 (DHASCO/CA) at a temperature of 45 degrees C, and a reaction time of 24 h in the presence of 4% enzyme and 2% water content. Examination of the positional distribution of fatty acids on the glycerol backbone of the modified DHASCO with CA showed that CA was present mainly in the sn-1,3 positions of the triacylglycerol (TAG) molecules. Meanwhile, DHA was favorably present in the sn-2 position, but also located in the sn-1 and sn-3 positions. The oxidative stability of the modified DHASCO in comparison with the original DHASCO, as indicated in the conjugated diene values, showed that the unmodified oil remained relatively unchanged during storage for 72 h, but DHASCO-based structured lipid was oxidized to a much higher level than the original oil. The modified oil also attained a considerably higher thiobarbituric acid reactive substances value than the original oil over the entire storage period. However, when the oil was subjected to the same process steps in the absence of any enzyme, there was no significant difference (p > 0.05) in its oxidative stability when compared with enzymatically modified DHASCO. Therefore, removal of antioxidants during the process is primarily responsible for the compromised stability of the modified oil.     

HPLC separation and NMR structural elucidation of sn-1,2-, 2,3-, and 1,3-diacylglycerols from olive oil as naphthylethylurethane derivatives

In this study, sn-1,2-, sn-2,3-, and sn-1,3-diacylglycerols were isolated from olive oil, and their urethane derivatives (urethanes) were prepared. Normal-phase high-performance liquid chromatography (NP-HPLC) separation of the urethane isomers was performed and the separate classes were studied by nuclear magnetic resonance (NMR). The use of 1H NMR and homo- and heteronuclear 2D techniques provided a great amount of information in a very short time, particularly when a high-field NMR instrument (700 MHz) was used. Particularly diagnostic for this kind of compound was the glyceridic moiety that presents typical chemical shifts both for carbon and hydrogen. These studies show the usefulness of NMR spectroscopy to recognize clearly the sn-1,3- and, moreover, sn-1,2- with respect to sn-2,3-diacylglycerols, although very minor differences occur between them.     

Analysis of regiospecific triacylglycerols by electrospray ionization-mass spectrometry(3) of lithiated adducts

A method of regiospecific analysis of triacylglycerols (TAGs) in vegetable oils and animal fats is reported here using the electrospray ionization-mass spectrometry (MS(3)) of TAG-lithiated adducts. The fragment ions of the MS(3) from the loss of fatty acids at the sn-2 position as alpha,beta-unsaturated fatty acids were used for regiospecific identification and quantification. The ratio of the regiospecific TAGs, ABA and AAB, in an oil sample usually fraction collected by high-performance liquid chromatography can be determined by the abundance of the fragment ions of [ABA + Li-ACOOH-B'CH=CHCOOH]+ and [AAB + Li-ACOOH-A'CH=CHCOOH]+. The method was used to analyze regiospecific TAGs in extra virgin olive oil. The results showed that the saturated fatty acids, palmitic and stearic acids, were mostly located at the sn-1,3 positions and unsaturated fatty acids, oleic and linoleic acids, were mostly located at the sn-2 position.     

Analysis of a model reaction system containing cysteine and (E)-2-methyl-2-butenal, (E)-2-hexenal, or mesityl oxide

Cysteine conjugates, resulting from the addition of cysteine to alpha,beta-unsaturated carbonyl compounds, are important precursors of odorant sulfur compounds in food flavors. The aim of this work was to better understand this chemistry in the light of the unexpected double addition of cysteine to two unsaturated aldehydes. These reactions were studied as a function of pH. When (E)-2-methyl-2-butenal (tiglic aldehyde, 4) was treated with cysteine in water at pH 8, the major product formed was the new compound (4R)-2-(2-[[(2R)-2-amino-2-carboxyethyl]thio]methylpropyl)-1,3-thiazolidine-4-carboxylic acid (6). Under acidic conditions (pH 1), we also observed a double addition, but the second cysteine was linked by a vinylic sulfide bond to form the previously unreported major product, (2R,2'R,E)-S,S'-(2,3-dimethyl-1-propene-1,3-diyl)bis-cysteine (7). When (E)-2-hexenal (12) was treated with cysteine under acidic conditions, the major product was the novel (4R,2' 'R)-2-[2'-(2' '-amino-2' '-carboxyethylthio)pentyl]-1,3-thiazolidine-4-carboxylic acid (13), and the formation of an vinylic sulfide compound analogous to 7 was not observed. Reduction of the acidic crude reaction mixture with NaBH(4) afforded 13 and the cysteine derivative (R)-S-[1-(2-hydroxyethyl)butyl]cysteine (14) in 14% yield. Treating (E)-2-hexenal with cysteine at pH 8 followed by NaBH(4) reduction yielded the new product (3R)-7-propylhexahydro-1,4-thiazepine-3-carboxylic acid (15). Addition of cysteine to mesityl oxide (16), at pH 8, followed by reduction with NaBH(4) furnished (R)-S-(3-hydroxy-1,1-dimethylbutyl)cysteine (3) and the new compound (3R)-hexahydro-5,7,7-trimethyl-1,4-thiazepine-3-carboxylic acid (18).     

Metabolism and distribution of [2,3-(14)C]acrolein in laying hens

The metabolism and distribution of [2,3-(14)C]-acrolein were studied in 10 laying hens orally administered 1.09 mg/kg of body weight/day for 5 days. Eggs, excreta, and expired air were collected. The hens were killed 12-14 h after the last dose and edible tissues collected. The nature of radioactive residues was determined in tissues and eggs. All of the identified metabolites were the result of the incorporation of acrolein-derived radioactivity into normal natural products of intermediary metabolism in the hen except for 1,3-propanediol, which is a known degradation product of glycerol in bacteria.     

Selectivity of celite-immobilized patatin (lipid acyl hydrolase) from potato (Solanum tuberosum L.) tubers in esterification reactions As influenced by water activity and glycerol analogues as alcohol acceptors

Lipid acyl hydrolase (LAH; patatin) was purified from potato tubers by ammonium sulfate fractionation followed by anion-exchange and affinity chromatography. The major protein band of 40-43 kDa on SDS-PAGE appeared to be patatin, and it stained positive for lipase activity on native PAGE. Selectivity of a Celite-immobilized potato LAH in esterification reactions with n-acyl fatty acids (FA; C4, C6, C8, C10, C12, C14, C16, and C18) and alcohol acceptors (n-propanol, 2-propanol, 1,3-propanediol, and glycerol; 1,2-propanediol was not sufficiently reactive) was studied in isooctane. Immobilized LAH was highly selective for medium chain FAs (C8/C10) with a secondary optimum for chain lengths of C14/16. Water activity (a(w)) influenced activity and FA selectivity of the enzyme. Initial rates of ester synthesis were greatest at a(w) of 0.90 for all alcohol acceptors except for glycerol, where greatest initial rates were observed at a(w) of 0.19. Immobilized LAH preparations exhibited a bell-shape pH profile with optimum activity at pH 6-7 for ester synthesis, and no effect of pH on FA selectivity was observed.     

Bioactive compounds and 1,3-Di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9, 12-octadecadienoyl]glycerol from Apium graveolens L. seeds

Bioassay-directed isolation and purification of the hexane extract of Apium graveolens L. seeds led to the characterization of three compounds: beta-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2) and 5-allyl-2-methoxyphenol (3). The structures of these compounds were established by using (1)H and (13)C NMR spectral methods. Compounds, 1-3 demonstrated 100% mortality on fourth-instar Aedes aegyptii larvae at 50, 25, and 200 microg mL(-)(1), respectively, in 24 h. Also, 2 inhibited the growth of Candida albicans and Candida kruseii at 100 microg mL(-)(1). It inhibited both topoisomerase-I and -II enzyme activities at 100 microg mL(-)(1). Compound 2 displayed 100% mortality at 12.5 and 50 microg mL(-)(1), respectively, when tested on nematodes, Panagrellus redivivus and Caenorhabditis elegans. The triglyceride, 1,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9, 12-octadecadienoyl]glycerol (4) and 3 were isolated for the first time from A. graveolens seeds, although 4 was not biologically active.     

Regioisomerism of triacylglycerols in lard, tallow, yolk, chicken skin, palm oil, palm olein, palm stearin, and a transesterified blend of palm stearin and coconut oil analyzed by tandem mass spectrometry.

Triacylglycerols (TAG) of lard, tallow, egg yolk, chicken skin, palm oil, palm olein, palm stearin, and a transesterified blend of palm stearin and coconut oil (82:18) were investigated by chemical ionization and collision-induced dissociation tandem mass spectrometry. Accurate molecular level information of the regioisomeric structures of individual TAGs was achieved. When existing in a TAG molecule of lard, palmitic acid occupied 90-100% of the sn-2 position. Within the major fatty acid combinations in tallow TAGs, the secondary position sn-2 was preferentially occupied in the decreasing order by oleoyl > palmitoyl > stearoyl residues, the order in saturated TAGs being myristoyl > stearoyl = palmitoyl. TAGs in egg yolk were more asymmetric than in chicken skin, with linoleic acid highly specifically attached in the yolk sn-2 carbon. Nearly 50% of yolk TAGs contained 52 carbon atoms with two or three double bonds. Linoleic, oleic, and palmitic acids were in the sn-2 location in decreasing quantities in palm oil and its fractions. Triacylglycerols of equal molecular weight behaved similarly in the fractionation process. Randomization of the parent oil TAGs was seen in the transesterified oil. The tandem mass spectrometric analysis applied provided detailed information of the distribution of fatty acids in individual combinations in TAGs.     

In vivo studies on the metabolism of the monoterpene pulegone in humans using the metabolism of ingestion-correlated amounts (MICA) approach: explanation for the toxicity differences between (S)-(-)- and (R)-(+)-pulegone

The major in vivo metabolites of (S)-(-)-pulegone in humans using a metabolism of ingestion-correlated amounts (MICA) experiment were newly identified as 2-(2-hydroxy-1-methylethyl)-5-methylcyclohexanone (8-hydroxymenthone, M1), 3-hydroxy-3-methyl-6-(1-methylethyl)cyclohexanone (1-hydroxymenthone, M2), 3-methyl-6-(1-methylethyl)cyclohexanol (menthol), and E-2-(2-hydroxy-1-methylethylidene)-5-methylcyclohexanone (10-hydroxypulegone, M4) on the basis of mass spectrometric analysis in combination with syntheses and NMR experiments. Minor metabolites were be identified as 3-methyl-6-(1-methylethyl)-2-cyclohexenone (piperitone, M5) and alpha,alpha,4-trimethyl-1-cyclohexene-1-methanol (3-p-menthen-8-ol, M6). Menthofuran was not a major metabolite of pulegone and is most probably an artifact formed during workup from known (M4) and/or unknown precursors. The differences in toxicity between (S)-(-)- and (R)-(+)-pulegone can be explained by the strongly diminished ability for enzymatic reduction of the double bond in (R)-(+)-pulegone. This might lead to further oxidative metabolism of 10-hydroxypulegone (M4) and the formation of further currently undetected metabolites that might account for the observed hepatotoxic and pneumotoxic activity in humans.     

Production of human milk fat analogue containing docosahexaenoic and arachidonic acids

Human milk fat (HMF) analogue containing docosahexaenoic acid (DHA) and arachidonic acid (ARA) at sn-1,3 positions and palmitic acid (PA) at sn-2 position was produced. Novozym 435 lipase was used to produce palmitic acid-enriched hazelnut oil (EHO). EHO was then used to produce the final structured lipid (SL) through interesterification reactions using Lipozyme RM IM. Reaction variables for 3 h reactions were temperature, substrate mole ratio, and ARASCO/DHASCO (A:D) ratio. After statistical analysis of DHA, ARA, total PA, and PA content at sn-2 position, a large-scale production was performed at 60 °C, 3:2 A:D ratio, and 1:0.1 substrate mole ratio. For the SL, those results were determined as 57.3 ± 0.4%, 2.7 ± 0.0%, 2.4 ± 0.1%, and 66.1 ± 2.2%, respectively. Tocopherol contents were 84, 19, 85, and 23 μg/g oil for α-, β-, γ-, and δ-tocopherol. Melting range of SL was narrower than that of EHO. Oxidative stability index (OSI) value of SL (0.80 h) was similar to that of EHO (0.88 h). This SL can be used in infant formulas to provide the benefits of ARA and DHA.     

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482, flumioxazin) in the rat: II. Identification of reduced metabolites

On single oral administration of (14)C-S-53482 [7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one, Flumioxazin] labeled at the 1- and 2-positions of tetrahydrophthaloyl group to rats at 1 (low dose) or 100 (high dose) mg/kg, the radiocarbon was almost completely eliminated within 7 days after administration in both groups with generally very low residual (14)C tissue levels. The predominant excretion route was via the feces. The major fecal and urinary metabolites involved reduction or sulfonic acid addition reactions at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety and hydroxylation of the cyclohexene or cyclohexane ring. One urinary and four fecal metabolites were identified using chromatographic techniques and spectroanalyses (NMR and MS). Three of five identified metabolites were unique forms, reduced at the 1,2-double bond of the 3,4,5, 6-tetrahydrophthalimide moiety. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed. To specify tissues forming reduced metabolites, an in vitro study was conducted. Reduction was found to take place in red blood cells.     

(E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene: a potent grape-derived odorant in wine

(E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene (TPB) was identified as a potent odorant in acid hydrolysates of crude glycoconjugate fractions isolated from grapes and grape vine leaves. TPB was also identified in a Semillon wine, using gas chromatography/mass spectrometry, by co-injection with an authentic sample. TPB had an aroma detection threshold of 40 ng/L in a neutral white wine and the concentration of TPB in four out of five white wines analyzed ranged from 50 to 210 ng/L.     

Metabolism of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and its isomer, N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), in rats. 1. Identification of metabolites in feces and urine

Rats were orally dosed with a 1:1 diastereomixture of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), both labeled with 14C, at 200 mg/kg/day for 5 consecutive days, and 16 metabolites in urine and feces were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Several of them were unique decyanated and/or cyclic compounds (lactone, imide, cyclic amide, cyclic imino ether forms). Major biotransformation reactions of the mixture of S-2900 and S-2900S in rats are proposed on the basis of the metabolites identified in this study.     

In vivo studies on the metabolism of the monoterpenes S-(+)- and R-(-)-carvone in humans using the metabolism of ingestion-correlated amounts (MICA) approach

The major in vivo metabolites of S-(+)- and R-(-)-carvone in a metabolism of ingestion correlated amounts (MICA) experiment were newly identified as alpha,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), alpha-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone) on the basis of mass spectral analysis in combination with syntheses and NMR experiments. Minor metabolites were identified as reduction products of carvone, namely, the alcohols carveol and dihydrocarveol. The previously identified major in vivo metabolite in rabbits, 10-hydroxycarvone, could not be detected, indicating either concentration effects or interspecies differences. Metabolic pathways for carvone in humans including oxidation of the double bond in the side chain and, to a minor extent 1,2- and 1,4 + 1,2-reduction of carvone, are discussed. No differences in metabolism between S-(+)- and R-(-)-carvone were detected.     

Improved synthesis and deployment of (2S,3R)-2-(2Z,5Z-octadienyl)-3-nonyloxirane, a pheromone of the pink moth, Lymantria mathura

We report two new syntheses of (2S,3R)-2-(2Z,5Z-octadienyl)-3-nonyloxirane, the main sex pheromone component of the pink moth, Lymantria mathura. The key step in the first route was the construction of (Z,Z)-1-bromo-1,4-heptadiene (6), which was coupled in the final step with 2-iodomethyl-3-nonyloxirane 4 via a Grignard reaction. The second approach employed alkylation of 1,4-heptadiynyllithium with epoxy triflates 7 in ether/hexane and provided the pheromone in >/=37% overall yield from alcohol 2. The 4:1 ratio of pheromone enantiomers, reportedly the most attractive to pink moth males, can be directly crafted from appropriately selected Sharpless asymmetric epoxidation conditions.     

Phenolic derivatives from soy flour ethanol extract are potent in vitro quinone reductase (QR) inducing agents

The fractionation of soy flour directed by a cellular bioassay for induction of phase 2 detoxification enzymes was used to identify quinone reductase (QR) inducing agents. A phospholipid-depleted, 80% methanol-partitioned isolate from a crude ethanol extract of soy flour was resolved using normal phase medium-pressure liquid chromatography (MPLC). Early eluting fractions were found to be the most potent QR inducing agents among the separated fractions. Fraction 2 was the most potent, doubling QR at <2 mug/mL. Further fractionation of this isolate led to the identification of several constituents. Fatty acids and sn-1 and sn-2 monoacylglycerols were identified, but were not highly potent QR inducers. Benzofuran-3-carbaldehyde, 4-hydroxybenzaldeyde, 4-ethoxybenzoic acid, 4-ethoxycinnamic acid, benzofuran-2-carboxylic ethyl ester, and ferulic acid ethyl ester (FAEE) were also identified as QR inducing constituents of this fraction. FAEE was the most potent of the identified constituents, doubling QR specific activity at 3.2 muM in the cellular bioassay.     

Comparison of the incorporation of oleate and ricinoleate into phosphatidylcholines and acylglycerols in soybean microsomes

The incorporations of oleate (endogenous) and ricinoleate (nonendogenous) into phosphatidylcholine (PC) and acylglycerol (AG) in immature soybean microsomes were compared. [(14)C]Oleate and [(14)C]ricinoleate were incubated individually with soybean microsomal preparations for up to 4 h, and molecular species of PC and AG incorporated were identified and quantified by HPLC. The activities of acyl CoA:lysoPC acyltransferase and phospholipase A(2) are in general not affected by the fatty acid (FA) chain at the sn-1 position. However, comparison between oleate and ricinoleate revealed that different FA incorporated at sn-2 of PC showed some different selection of the molecular species of lysoPC. The incorporation of [(14)C]ricinoleate into triacylglycerols (TAG) was slightly better than that of [(14)C]oleate and indicated that soybean was capable of incorporating ricinoleate into TAG when ricinoleate can be produced endogenously in a transgenic soybean. The incorporation of FA into TAG in soybean microsomes was much slower than that in castor microsomes.     

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482) in rat. 1. Identification of a sulfonic acid type conjugate

To examine the metabolic fate of 7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one (S-53482), rats were given a single oral dose of [phenyl-(14)C]-S-53482 at 1 (low) or 100 (high) mg/kg. The radiocarbon was almost completely eliminated within 7 days after administration in both groups. (14)C recoveries (expressed as percentages relative to the dosed (14)C) in feces and urine were 56-72 and 31-43%, respectively, for the low dose and 78-85 and 13-23%, respectively, for the high dose. S-53482 and seven metabolites were identified in urine and feces. Six of them were purified by several chromatographic techniques and identified by spectroanalyses (NMR and MS). Alcohol derivatives and an acetoanilide derivative were isolated from urine. Three sulfonic acid conjugates having a sulfonic acid group incorporated into the double bond of the 3,4,5,6-tetrahydrophthalimide moiety were isolated from feces. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed.