Reaction of histidine with methyl linoleate: Characterization of the histidine degradation products

Efforts have been made to characterize the products that result from interactions between L-histidine (free base) and peroxidizing methyl linoleate (ML) in a model system consisting of reactants dispersed on a filter paper. Imidazole lactic acid and imidazole acetic acid are identified as breakdown products when histidine is incubated with ML, methyl linoleate hydroperoxide (MLHPO), or n-hexanal over a period of 3 weeks. Two other reaction products are found to give back histidine upon acid hydrolysis. These products are though to be Schiff's base compounds which result from the condensation of the histidyl α-amino group and carbonyl groups of reactive aldehydes formed during ML peroxidation. Most of the detectable reaction products have the imidazole moiety intact indicating the high relative reactivity of the functional groups, especially the amino group, associated with the α-carbon. Such high reactivity provides an explanation for the low concentrations of ninhydrin-positive free amino compounds that are, at best, barely detectable on thin layer chromatography. Presented at the AOCS Meeting, Chicago, September 1976.     

Hydrolysis of linoleate geometric isomers byGeotrichum candidum lipase

Lipase (EC 3.1.1.3) from the microorganismGeotrichum candidum preferentially hydrolyzescis-9 18∶1 andcis,cis-9,12 18∶2 from triacylglycerols, largely ignoring all other positional isomers ofcis 18∶1 as well astrans-9 18∶1. To obtain additional information about the specificity of the enzyme, two triacylglycerols were prepared and utilized as substrates. The lipase hydrolyzed 85%cis,cis-9,12 18∶2 and 15%trans,trans-9,12 18∶2 from the triacylglycerol, containing ca. 50% of each acid. From the triacylglycerol containing 46.3%cis,trans-9,12 18∶2 and 53.7%trans,cis-9,12 18∶2, 44.8 and 55.2% of the two acids were hydrolyzed. Therefore the enzyme discriminated against thetrans,trans isomer but not between thecis,trans andtrans,cis isomers. Scientific contribution No. 535, Agricultural Experiment Station, University of Connecticut. ARS, USDA.     

Studies on kinetics of catalytic isomerization of methyl linoleate

Kinetics of isomerization of methyl linoleate are studied on ruthenium (5%) on carbon in the temperature range 200–270 C with different solvents. Some equilibrium experiments also are carried out with rhodium and ruthenium catalysts. The reactions taking place are isomerization, hydrogenation and polymerization. The activities and the selectivities are dependent on the nature of the solvent used. Highly protic solvents like methanol or isopropyl alcohol exhibited very high activity and selectivity for hydrogenation, whereas aprotic solvents like hexane or cyclohexane showed very high selectivities for isomerization reaction. The reaction kinetics were found to be further complicated by polymer formation at low solvent concentrations. The effects of temperature, solvent concentration, catalyst quantity and time of reaction also were investigated.     

Analysis of the geometric isomers of methyl linoleate by gas chromatography

The four geometric isomers of methyl linoleate have been quantitatively determined by gas chromatography on Apiezon L and DEGS polyester capillary columns. Three peaks were eluted from the Apiezon L column: (a) the 9-cis, 12-cis isomer; (b) the 9-cis, 12-trans isomer; and (c) the 9-trans, 12-cis and 9-trans, 12-trans isomers combined. The DEGS polyester column also resolved three peaks: (a) the 9-trans, 12-trans isomer; (b) the 9-cis, 12-cis and 9-cis, 12-trans isomers combined; and (c) the 9-trans, 12-cis isomer. Since the separation of isomers was different on each column, the content of each of the four isomers could be determined from the combined results. Quantitative results agreed closely with the per centtrans bonds as determined by infrared analysis. Presented at the fall meeting of the American Oil Chemists' Society, Chicago, Illinois, October 30-November 1, 1961. Supported in part by a grant from Cargill, Ineorporated.     

Positional isomers formed during the hydrogenation of methyl linoleate under various conditions

Journal of the American Oil Chemists' Society -     

Alkali isomerization—Gas chromatography with the microreactor apparatus

A simple and rapid method of simultaneously conjugating polyunsaturation and forming methyl esters from triglyceride oils, other esters and fatty acids has been developed by using the microreactor apparatus (MRA). These reactions occur while a mixture of tetramethylammonium hydroxide and sample of fat is being injected into a gas chromatograph from the MRA reaction chamber. The speed of this microanalysis is limited only by peak elution and peak integration times. Analyses of soybean oil and six partially hydrogenated fats have been compared with those previously obtained by the alkali isomerization spectrometric method. The amount of conjugation that occurs during the reaction is a measure of the linoleic acid in the oil. This procedure should prove useful for rapid microscale analysis of conjugatable dienes in a variety of samples, including edible fat products. Presented at the Fall ISF-AOCS Meeting, Chicago, September 1970. No. Market. Nutr. Res. Div., ARS, USDA.     

Thermal decomposition of individual positional isomers of methyl linoleate hydroperoxide: Evidence of carbon-oxygen bond scission

The methyl ester of individual positional isomers of linoleate hydroperoxide were prepared by an enzymic oxidation of linoleate. On injection onto a gas chromatographic column they were thermally decomposed and the resulting volatile components analyzed. The major (67–80% yield on a molar basis) cleavage products were found to be hexanal, methyl octanoate, 2,4-decadienal isomers, and methyl 9-oxononanoate. Both the 9 and 13 isomers of linoleate hydroperoxide gave rise to these same four compounds, an observation suggesting carbon-oxygen scission in their decomposition. This was confirmed by using very pure individual isomers obtained by high performance liquid chromatography. The involvement of an isomerization reaction of the hydroperoxides is discussed.     

Photosensitized oxidation of methyl linoleate: Secondary and volatile thermal decomposition products

Studies of photosensitized oxidation of methyl linoleate show that the greater relative concentration of 9- and 13-hydroperoxides than 10- and 12-hydroperoxides is characteristic of singlet oxygenation and not due to either simultaneous autoxidation or type 1 photosensitized oxidation. Cyclization of the internal 10- and 12-hydroperoxides accounts for their lower relative concentrations. Secondary products separated by silicic acid and high pressure liquid chromatography were characterized spectrally (IR, UV,¹H-NMR,¹³C-NMR, GC-MS). Major secondary products included diastereomeric pairs of 13-hydroperoxy-10,12-epidioxy-trans-8-octadecenote (I and III) and 9-hydroperoxy-10,12-epidioxy-trans-13-octadecenoate (II and IV); minor secondary products included hydroperoxy oxy genated and epoxy esters. Thermal decomposition of the hydroperoxy cyclic peroxides produced hexanal and methyl 10-oxo-8-decenoate as major volatiles from I and III and methyl 9-oxo-nonanoate and 2-heptenal from II and IV. Hydroperoxy cyclic peroxides may be important sources of volatile decomposition products of photooxidized fats. Presented at 72nd Annual Meeting of the American Oil Chemists Society, New Orleans, LA, May 1981.     

Characterization of some dicarbonyls from autoxidized methyl linoleate

The 2, 4-dinitrophenylosazones of dicarbonyl compounds isolated from oxidized methyl linoleate were resolved by means of adsorption and partition chromatography. Individual components were characterized by means of ultraviolet absorption, thin layer chromatographic techniques for class determination and for separation of homologous series, column co-chromatography of authentics and the unknowns, IR spectroscopy and melting point analysis. Evidence was obtained for the presence of glyoxal, methyl glyoxal, but-2-en-1, 4-dial, α-keto hexanal, α-keto heptanal, α-keto octanal, and α-keto nonanal. Technical Paper No. 1864, Oregon Agricultural Experiment Station.     

Preparation of deuterium-labelled methyl linoleate and its geometric isomers from natural seed oils

Multi-gram quantities of deuterium-labelled methyl linoleate (methyl cis-9,cis-12-octadecadienoate) and its geometric isomers are readily synthesized fromCrépis alpina (70–80% cis-9-octadecen-12-ynoic acid) andVernonia galamensis (70–80% 12,13-epoxy-cis-9-octadecenoic acid) seed oils. Methylcis- 9,cis- 12- andtrans- 9,cis- – octadecadienoate-12,13-d₂ were prepared by the Lindlar-catalyzed reduction (with D₂ gas) of methylcis- 9- and trans-9-octadecen-12-ynoates, respectively. Methyltrans- 9- octadecen-12-ynoate was synthesized by thep-toluene-sulfinic acid-catalyzed isomerization of the corresponding cis isomer. Methylcis- 9fiis- 12., trans- 9fiis- 12;cis- 9,trans- 12- andtrans- 9, frans-12-octadecadienoate-d₂, d₄ and d₆ were prepared by the Wittig coupling (with stereochemical control) of the appropriate d₂-, d₄- or de-alkyltriphenyl-phosphonium salt with methyl 12-oxo-cis-9- ortrans- 9- dodecenoate (prepared by the para-periodic acid cleavage of methyl 12,13-dihydroxy-cis-9- or trans-9-octadecenoate). Thecis dihydroxy ester was synthesized fromVernonia galamensis seed oil by acetolysis, saponification and then esterification. Thecis dihydroxy ester was isomerized by nitric acid/sodium nitrite to thetrans form and purified by silver resin chromatography. Isotopic purities ranged from 88% (for the d₆ isomers) to 99% (for the d₂ isomers). The mention of firm names or trade products does not imply that they are endorsed or recommended over other firms or similar products not mentioned.     

Oxidation products of α-tocopherol formed in autoxidizing methyl linoleate

The oxidation products of¹⁴C-α-tocopherol formed by heating with methyl linoleate in an air atmosphere at 60 C or 100 C were investigated. The products included a dimer, trimer and dihydroxy dimer, α-tocopherol quinone and unidentified degradation compounds. The dimer and trimer constituted the major products present after heating for 70 hr at 60 C. After 70 hr at 100 C most of the¹⁴C-α-tocopherol had been converted to degradation products; part of the¹⁴C originallt present in the 5-methyl group was recovered as¹⁴CO₂ and¹⁴CH₃OH.     

Nutritive value of methyl linoleate and its thermal decomposition products

Methyl linoleate was heated for 10 hrs. at 300°C, in the absence of air and fractionated by alembic distillation and urea adduct-formation. Intestinal absorptions of the urea adduct-forming monomeric nonadduct-forming monomeric, and dimeric fractions were determined. It was found that dimers were half as well absorbed as the monomers. When fed to rats, dimers were better accepted and exhibited some toxicity symptoms different from the non-adduct-forming monomers. The dimers caused diarrhea, irritability, and loss of hair during the early period of administration. The nonadduct-forming monomers were lethal and produced an increase in liver weight. Both fractions depressed growth. With the technical assistance of Oscar giacomantone and Perla Mordujovich.     

The migration of double bonds during the isomerization of methyl linoleate with palladium on carbon catalyst

Summary The isomerization of methyl linoleate by heating with palladium on carbon catalyst gave an initial rapid rise in the degree of conjugation which reached a maximum and then gradually decreased. The maximum conjugation obtained was about 24% after 4 hrs. of heating at 200±5°C. with 25% by weight of catalyst. Ultraviolet absorption spectra did not indicate the presence of any cyclized aromatic components even after 46 hrs. of heating at 200°C. but showed the formation of aromatics at 250°C. The limit of conjugation obtained is a result of random migration of the olefinic bonds rather than dimerization or polymerization reactions. One of the laboratories of the Southern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Effect of browning reaction products of phospholipids on autoxidation of methyl linoleate

Methyl linoleate was allowed to autoxidize in bulk phase at 50 C in the presence of either synthetic phospholipids consisting of saturated fatty acids or egg yolk phospholipids to estimate the effect of base group and fatty acid moiety of phospholipids on oil autoxidation. Dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) exhibited poor antioxidant activity at 50 C and showed no synergistic effect with α-tocopherol. The addition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of egg yolk accelerated the oxidation of methyl linoleate. However, egg yolk PC and PE collected at various stages of heating at 180 C inhibited hydroperoxide formation at the initial stage of oxidation. This effect could be attributed to the browning products formed during heating reaction. Thus, browning color products formed from unsaturated phospholipids at high temperatures may influence oil stability, although the base group of phospholipids did not exert any significant effect.     

Gas chromatographic study on high-temperature thermal degradation products of methyl linoleate hydroperoxides

Chromatographic techniques were used to separate secondary products generated by thermal degradation of methyl linoleate hydroperoxides (MLHP). The MLHP were obtained by oxidation, selected, and concentrated by solid-phase extraction (SPE) and thin-layer chromatography (TLC). The purified MLHP were then thermo-degraded in the gas-chromatographic glass liner and analyzed on-line by gas chromatography-mass spectrometry (GC-MS). The MLHP were also thermodegraded and collected in a short silicic acid-packed column, eluted, separated by TLC, and then analyzed by GC. By considering the elution in TLC, the GC retention times and the GC-MS analyses, it was possible to characterize the mono- and the dioxygenated secondary products, particularly those having a boiling point higher than methyl linoleate. The peaks that corresponded to the mono-oxygenated products (epoxy, hydroxy, and keto) were identified, and, on the basis of their MS spectra, molecular structures were proposed. A specific elution order was suggested for keto derivatives: 9-keto,Δ^10,12- and 13-keto,Δ^9,11-octadecadienoate. The hydroxy derivatives, which show the typical fragmentations of 9-hydroxy,Δ^10,12- and 13-hydroxy,Δ^9,11-octadecadienoate, were also identified. On the other hand, identification of the di-oxygenated compounds was more difficult, and, therefore, it was not possible to indicate each positional isomer; however, their elution order could be epoxy-hydroxy and epoxy-keto derivatives.     

Alkali isomarization of methyl 9, 12, 15-octadecatrienoate geometrical isomers

Geometrical isomers of methyl 9,12,15-octadecatrienoate were subjected to alkali isomerization according to the official AOCS method. Thec,t,t andt,t,c isomers gave higher specific extinction coefficients at 268 mμ than the all-cis isomer. The all-trans isomer was not completely conjugated. These results are in conformity with previous predictions based on theoretical and experimental evidence.     

Epoxidation of methyl linoleate. II. The two isomers of methyl 9, 10: 12, 13-diepoxystearate

Epoxidation of methyl linoleate results in the formation of two stereoisomeric methyl 9,10:12,13-diepoxystearates. The previously well-known isomer, mp 32C, represents only 60% of the total diepoxides formed. The newly isolated form is liquid at room temperature and is measured incompletely by standard titrimetric oxirane analysis. The isomers differ by the relative positions of their oxirane oxygen atoms. The oxygens of the solid are located on opposite sides and those of the liquid are on the same side of the molecular plane. E. Utiliz. Res. Devel. Div., ARS, USDA.     

Chemical lonization-mass spectrometry of secondary oxidation products from methyl linoleate and linolenate

Chemical ionization-mass spectrometry (CI-MS) with a direct exposure probe was used to analyze a series of hydroperoxy cyclic peroxides and dihydroperoxides obtained from methyl linoleate and linolenate by either autoxidation or photosensitized oxidation. The mass spectra obtained with isobutane and ammonia as reacting gases showed fragmentation patterns similar to those ditions of gas chromatography (GC)-electron impact (EI)-MS. Because the fragmentation patterns obtained under either CI-MS with a direct exposure probe or GC-(EI)-MS conditions are sufficiently predictable, these techniques are powerful analytical tools for the structural characterization of lipid oxidation products. These techniques are also useful to elucidate the cleavage pathways to volatile lipid oxidation products of flavor and biological significance.     

Effect of water activity on secondary products formation in autoxidizing methyl linoleate

The role of water activity on the formation of peroxides and carbonyl compounds during lipid oxidation is important to know because there could be either beneficial or detrimental effects of water activity on lipid oxidation in stored foods. Therefore, methyl linoleate was chosen as a model lipid and was autoxidized to 1% at water activity ranging from 0.02 to 0.79 at 37°C. Oxygen uptake was monitored manometrically. The peroxide and carbonyl contents were determined upon termination of the autoxidation studies. Methyl linoleate autoxidation was characterized by three phases: i) an initial induction period of no oxygen absorption; ii) a slow rate of oxygen absorption, up to 0.15% oxidation; and iii) a relatively faster rate of oxygen absorption beyond 0.15% up to 1% oxidation. Water activity had considerable influence during the first phase. There was no induction period at or below water activity 0.22. The induction period begins at water activity 0.32 and could be extended to a limit with increase in water activity. Once the induction period was passed water activity had no influence on the rate of oxidation. However, during the second and third phases water activity becomes important in the stabilization of peroxides/hydroperoxides and decides the course of secondary reactions that follow peroxide decomposition. Higher water activity values, particularly water activity 0.67, tended to stabilize peroxides. Water activity had considerable influence on the formation of secondary products of autoxidation as evidenced by the variation in the type and quantity of carbonyl compounds at different water activity values.     

Singlet oxygen oxidation of methyl linoleate: Isolation and characterization of the NaBH4-reduced products

The mixture of diene hydroperoxides from methylene blue-sensitized oxidation of methyl linoleate was reduced with NaBH₄ and the resulting alcohols were separated by high pressure liquid chromatography (HPLC). Four diene alcohols were isolated in approximately equal yields from adsorption and reversed phase HPLC; the isomers were identified as methyl esters of 9-hydroxy-10,12-, 10-hydroxy-8,12-, 12-hydroxy-9,13- and 13-hydroxy-9,11-octadecadienoate. Formation of equal yields of both conjugated and nonconjugated diene alcohols from methyl linoleate is characteristic of singlet oxygen oxidations. The detection of the easily separated nonconjugated isomer methyl 10-hydroxy-trans-8,cis-12-octadecadienoate from methyl linoleate is proposed as a test to probe the involvement of singlet oxygen in biological oxidations. A preliminary report of these results was presented at the 177th meeting of the American Chemical Society, Honolulu, HI, April 1–6, 1979; see abstracts of papers, paper No. ORGN-375.