Hydroperoxides from oxidation of linoleic and linolenic acids by soybean lipoxygenase: Proof of thetrans-11 double bond

Hydroperoxides produced by oxidation of linoleic and linolenic acids with soybean lipoxygenase were analyzed by nuclear magnetic resonance. The isomerized double bond α,β to the hydro-peroxide group at carbon-13 was determined to betrans. The complete structures of the major products proved to be 13-hydroperoxy-cis-9,trans-11-octa-decadienoic acid from linoleic acid and 13-hydroperoxy-cis-9,trans-11,cis-15-octadecatrienoic acid from linolenic acid. The configuration of the double bonds indicates that oxidation took place through a free radical mechanism as proposed previously by others. N. Market. Nutr. Res. Div., ARS, USDA.     

Purification of lipoxygenase from Chlorella: production of 9- and 13-hydroperoxide derivatives of linoleic acid

Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9-and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30–45 and 45–80% saturated (NH₄)₂SO₄ precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH₄)₂SO₄ precipitation (35–80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorilla is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.     

Method to produce 9(S)-hydroperoxides of linoleic and linolenic acids by maize lipoxygenase

Seed from maize (corn) Zea mays provides a ready source of 9-lipoxygenase that oxidizes linoleic acid and linolenic acid into 9(S)-hydroperoxy-10(F), 12(Z)-octadecadienoic acid and 9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, respectively. Corn seed has a very active hydro-peroxide-decomposing enzyme, allene oxide synthase (AOS), which must be removed prior to oxidizing the fatty acid. A simple pH 4.5 treatment followed by centrifugation removes most of the AOS activity. Subsequent purification by ammonium sulfate fractional precipitation results in negligible improvement in 9-hydroperoxide formation. This facile alternative method of preparing 9-hydroperoxides has advantages over other commonly used plant lipoxygenases.     

Gamma‐linolenic and stearidonic acids from Moroccan Boraginaceae

Seeds from 20 species belonging to Boraginaceae, subfamilies Boraginoideae and Heliotropioideae, were surveyed in a search for new sources of γ‐linolenic acid (GLA) and stearidonic acid (SDA). Seed oil content ranged from 7.5% in Echium humile ssp. pycnanthum to 28.8% in Anchusa undulata. GLA ranged from 0.2% of total fatty acids in Heliotropium undulatum to 20.2% in Lithodora maroccana. This last species may be considered as new source of GLA. GLA content was also tested in other Lithodora species from the south east of Spain, to compare GLA percentages among related taxa. GLA amounts in all Echium species reached approximately 12%, in good agreement with previous findings in other European Echium species. SDA ranged from an absence in several Cynoglossum species to 16.2% in Echium humile ssp. pycnanthum, which may be considered as a new source of this fatty acid.     

Dietary trans α‐linolenic acid does not inhibit Δ5‐ and Δ6‐desaturation of linoleic acid in man

Dietary trans monoenes have been associated with an increased risk of heart disease in some studies and this has caused much concern. Trans polyenes are also present in the diet, for example, trans α‐linolenic acid is formed during the deodorisation of α‐linolenic acid‐rich oils such as rapeseed oil. One would expect the intake of trans α‐linolenic acid to be on the increase since the consumption of rapeseed oil in the western diet is increasing. There are no data on trans α‐linolenic acid consumption and its effects. We therefore carried out a comprehensive study to examine whether trans isomers of this polyunsaturated fatty acid increased the risk of coronary heart disease. Since inhibition of Δ6‐desaturase had also been linked to heart disease, the effect of trans α‐linolenic acid on the conversion of [U‐¹³C]‐labelled linoleic acid to dihomo‐γ‐linolenic and arachidonic acid was studied in 7 healthy men recruited from the staff and students of the University of Edinburgh. Thirty percent of the habitual fat was replaced using a trans ‘free’‐ or ‘high’ trans α‐linolenic acid fat. After at least 6 weeks on the experimental diets, the men received 3‐oleyl, 1,2‐[U‐¹³C]‐linoleyl glycerol (15 mg twice daily for ten days). The fatty acid composition of plasma phospholipids and the incorporation of ¹³C‐label into n‐6 fatty acids were determined at day 8, 9 and 10 and after a 6‐week washout period by gas chromatography‐combustion‐isotope ratio mass spectrometry. Trans α‐linolenic acid of plasma phospholipids increased from 0.04 ? 0.01 to 0.17 ? 0.02 and cis ? ‐linolenic acid decreased from 0.42 ? 0.07 to 0.29 ? 0.08 g/100 g of fatty acids on the high trans diet. The composition of the other plasma phospholipid fatty acids did not change. The enrichment of phosphatidyl ¹³C‐linoleic acid reached a plateau at day 10 and the average of the last 3 days did not differ between the low and high trans period. Both dihomo‐γ‐linolenic and arachidonic acid in phospholipids were enriched in ¹³C, both in absolute and relative terms (with respect to ¹³C‐linoleic acid). The enrichment was slightly and significantly higher during the high trans period (P<0.05). Our data suggest that a diet rich in trans α‐linolenic acid (0.6% of energy) does not inhibit the conversion of linoleic acid to dihomo‐γ‐linolenic and arachidonic acid in healthy middle‐aged men consuming a diet rich in linoleic acid.     

Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSⁿ mass spectra of the main metabolites of oleic, linoleic, α-linolenic and γ-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS³ analysis of hydroperoxides and MS² analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18∶2n−6, 18∶3n−3, and 18∶3n−6 in a ratio of ∼1∶1–3 at high substrate concentrations. 8-Hydroxy-9(10)expoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSⁿ is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.     

Studies on the chemistry of the fatty acids VIII the reaction of thiocyanogen with linoleic and linolenic acids

Summary The thiocyanogen reagent may be stabilized by storage at temperatures of 3° or less. The reaction of thiocyanogen with highly purified linoleic and linolenic acids has been studied under many variations of experimental conditions. Reaction rates of thiocyanogen with oleic, linoleic and linolenic acids at 3°, 16° and 25° and with variable excess of reagent are described. Oleic acid gives a maximum thiocyanogen absorption which is apparently equal to the iodine number, within 3–6 hours. The reaction with linoleic acid is rapid up to 3 hours and shows a slow constant increase thereafter. A modified procedure of analysis, similar to the official method, employs a 0.2 N thiocyanogen reagent, containing 10 percent carbon tetrachloride and a reaction temperature of 16°. Results of analysis of several specimens of acids by several preparations of reagent shows an average thiocyanogen value of 96.6 for linoleic acid and 166.3 for linolenic. Simultaneous equations, derived from these values, were used in the analysis of five mixtures of known composition with excellent results. Presented in part at the Cincinnati meeting of the American Chemical Society, 1940. Instructor in Biochemistry at Emory University, Emory University, Ga. Presented in partial fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University.     

Analysis of genetic effects and heritabilities for linoleic and α‐linolenic acid content of Brassica napus L. across Chinese environments

A genetic model for quantitative traits of seed in diploid plant was applied to analyze the main genetic effects and genotype × environment (GE) interaction effects for linoleic and α‐linolenic acid content of rapeseed (Brassica napus L.) by using two year experimental data with 8 parents and their F₁ and F₂. Results indicated that the performance of linoleic (C18:2) and α‐linolenic acid content (C18:3) of rapeseed were simultaneously controlled by diploid embryo nuclear genes, cytoplasmic genes and diploid maternal plant nuclear genes, and the GE interaction effects for these two seed quality traits were more important than the genetic main effects. The total narrow‐sense heritability was 69.5% and 41.8% for C18:2 and C18:3, respectively, and the GE interaction heritabilities were found to be larger than the general heritabilities for both quality traits. It was suggested by the predicted genetic effects that Huashuang 3 was better than other parents for improving offspring of C18:2 and Youcai 601, Zhongyou 821, Eyouchangjia and Zhong R‐888 for decreasing C18:3.     

The thiocyanogen values of the methyl esters of oleic,linoleic, and linolenic acids

Summary Thiocyanogen values were determined on the methyl esters of oleic, linoleic and linolenic acids and on six different mixtures of these esters, using 0.1 and 0.2 normal thiocyanogen solutions. The values determined with 0.1 N solutions showed less variation than those determined with 0.2 N. The composition of the mixtures calculated from equations based on the found thiocyanogen values of the esters agreed with the known composition within reasonable limits. Comparisons were made with the composition calculated with the Kaufmann-theory values. It is suggested that the F.A.C. consider adopting tentatively the values 89.4 for oleic acid, 93.9 for linoleic acid, and 162.0 for linolenic acid when 0.1 N thiocyanogen solutions are used; the values 89.4, 96.8, and 167.5 when 0.2 N solutions are employed. These represent the average of the values for these acids which have been reported in the literature. Agricultural Chemical Research Division Contribution No. 15     

13‐Phenyltridec‐9‐enoic and 15‐phenylpentadec‐9‐enoic acids in Arum maculatum seed oil

The seed oil of Arum maculatum has been found to contain 13‐phenyltridec‐9‐enoic (0.4%) and 15‐phenyl‐pentadec‐9‐enoic (1%) acids, detected by gas chromatographymass spectrometry of the picolinyl ester and related derivatives.     

Lipase‐mediated hydrolysis of flax seed oil for selective enrichment of α‐linolenic acid

Polyunsaturated fatty acids (PUFA) are important ingredients of human diet because of their prominent role in the function of human brain, eye and kidney. α‐Linolenic acid (ALA), a C18, n‐3 PUFA is a precursor of long chain PUFA in humans. Commercial lipases of Candida rugosa, Pseudomonas cepacea, Pseudomonas fluorescens, and Rhizomucor miehei were used for hydrolysis of flax seed oil. Reversed phase high performance liquid chromatography followed by gas chromatography showed that the purified oil contained 12 triacylglycerols (TAGs) with differences in fatty acid compositions. Flax seed oil TAGs contained α‐linolenic acid (50%) as a major fatty acid while palmitic, oleic, linoleic made up rest of the portion. Among the four commercial lipases C. rugosa has preference for ALA, and that ALA was enriched in free fatty acids. C. rugosa lipase mediated hydrolysis of the TAGs resulted in a fatty acid mixture that was enriched in α‐linolenic to about 72% yield that could be further enriched to 80% yield by selective removal of saturated fatty acids by urea complexation. Such purified ALA can be used for preparation of ALA‐enriched glycerides. Practical applications : This methodology allows purifying ALA from fatty acid mixture obtained from flax seed oil by urea complexation.     

Quasi‐alternating polyesteramides from ε‐caprolactone and α‐amino acids

Glycine‐ɛ‐caprolactone‐based and α‐alanine‐ɛ‐caprolactone‐based polyesteramides with a strong tendency to form alternating sequences (degree of randomness = 1.64 and 1.31) were synthesized by melt polycondensation of intermediate hydroxy‐ and ethyl ester‐terminated amides. These intermediates were synthesized by the reaction of equimolar amounts of ɛ‐caprolactone and glycine or L‐α‐alanine ethyl esters in mild conditions. The structure and microstructure of these polyesteramides are discussed on the basis of an in‐depth nuclear magnetic resonance study. Both polyesteramides are semi‐crystalline, but the glycine‐based one presents the highest melting enthalpy. This polyesteramide also exhibits higher Young's modulus and stress at break than its α‐ and β‐alanine counterparts. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 44220.     

N‐3 fatty acids and conjugated linoleic acids of longissimus muscle in beef cattle

The objective of the experiment with cattle was to produce high quality beef under different feeding conditions and to increase the concentration of essential fatty acids in muscle. In total 10 German Simmental (GS) bulls and 9 German Holstein (GH) steers were kept either on pasture (grass feeding) or in stable (concentrate feeding). Despite biohydrogenation in the rumen, linolenic acid (C18:3n‐3) contained in grass was absorbed and deposited into the lipids of muscle. This led to a significantly (p ≤ 0.05) higher content of n‐3 fatty acids in the muscle lipids of grazing cattle. The relative amount of total n‐3 fatty acids increased from 1.4 g/100 g fatty acid methyl ester (%FAME) in the intensively fed Simmental bulls to 5.5 %FAME in grass fed cattle. The n‐6/n‐3 ratio of pasture grazing GS bulls was 1.3 in contrast to 13.7 of the animals kept in the byre. The total n‐3 fatty acid concentration in beef muscle increased from 24.6 mg (concentrate) to 108.6 mg/100 g wet weight (grazing). In GH steers the total n‐3 fatty acid concentration was significantly (p ≤ 0.05) increased up to 86.3 mg/100 g wet weight in pasture grazing steers compared to 28.8 mg/100 g wet weight in animals fed the concentrate. The relative content (%FAME) of CLAcis‐9, trans‐11 (0.6 vs 0.56 %FAME in GS; 0.55 vs 0.52 %FAME in GH) in muscle was not significantly increased by grazing on pasture in comparison to concentrate feeding neither in GS bulls nor in GH steers, respectively.     

Biodegradation of 2‐, 3‐ and 4‐iodobenzoic acids

The biodegradation of 2‐iodobenzoic acid (2IBA), 3‐iodobenzoic acid (3IBA) and 4‐iodobenzoic acid (4IBA) was investigated. Substrate disappearance was monitored in conjunction with product formation (ie iodide production) in order to verify complete mineralisation. Samples from SmithKline Beecham (SB) Wastewater Treatment Plants (WWTP) were employed to test their ability to degrade the iodinated aromatics listed above. Complete mineralisation of 3IBA was observed and the initial pathway(s) of biotransformation (via hydroxybenzoic acid formation) was proposed based on the comparison with standards of metabolites previously elucidated for similar halogenated benzoic acids. Evidence for co‐metabolism of 4IBA was observed since biodegradation only occurred in the presence of 3IBA. No evidence of biodegradation of 2IBA was observed over a period of 20 days, neither when fed individually nor as a mixture of substrates. © 1999 Society of Chemical Industry     

Regio- and stereochemical analysis of trihydroxyoctadecenoic acids derived from linoleic acid 9- and 13-hydroperoxides

The methyl esters of 9S,10S,13R-trihydroxy-11E-octadecenoic acid, 9S,10R,13S-trihydroxy-11E-octadecenoic acid, and 9S,10R,13R-trihydroxy-11E-octadecenoic acid were prepared from 9S-hydroperoxy-10E,12Z-octadecadienoic acidvia the epoxy alcohols methyl 10R,11R-epoxy-9S-hydroxy-12Z-octadecenoate and methyl 10S,11S-epoxy-9S-hydroxy-12Z-octadecenoate. The trihydroxyesters, as well as four stereoisomeric methyl 9,12,13-trihydroxy-10E-octadecenoates earlier prepared [Hamberg, M.,Chem. Phys. Lipids 43, 55–67 (1987)], were characterized by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and by chemical degradation. Availability of these chemically defined trihydroxyoctadecenoates made it possible to design a method for regio- and stereochemical analysis of 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids obtained from various sources. Application of the method revealed that the mixture of 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids formed during autoxidation of linoleic acid in aqueous medium contained comparable amounts of the sixteen possible regio- and stereoisomers. Furthermore, hydrolysis of the allylic epoxy alcohol, methyl 9S,10R-epoxy-13S-hydroxy-11E-octadecenoate, yielded a major trihydroxyoctadecenoate,i.e., methyl 9S,10S,13S-trihydroxyl-11E-octadecenoate, together with smaller amounts of methyl 9S,10R,13S-trihydroxy-11E-octadecenoate, methyl 9S,12R,13S-trihydroxy-10E-octadecenoate, and methyl 9S,12S,13S-trihydroxy-10E-octadecenoate.