Reduction of fatty acid hydroperoxides by human parotid saliva

Arachidonic acid hydroperoxide (15-hydroperoxyeicosatetraenoic acid; 15-HPETE) was introduced into human parotid saliva and incubated at 37°C. Straight phase high-performance liquid chromatography analysis of the reaction mixture showed that 15-HPETE was detoxified to its reduced form, 15-hydroxyeicosatetraenoic acid, in the presence of glutathione. Therefore, it is concluded that human parotid saliva possesses fatty acid hydroperoxide-reducing ability. However, its effectiveness was found to be lower than that of blood plasma.     

Decomposition of unsaturated fatty acid hydroperoxides by hemoglobin: Structures of major products of 13L-hydroperoxy-9,11-octadecadienoic acid

13L-hydroperoxy-9, 11-octadecadienoic acid was decomposed rapidly in the presence of hemoglobin. The product consisted of five major compounds, i.e. 13-keto-9, 11-octadecadienoic acid, 13L-hydroxy-9, 11-octadecadienoic acid, erythro-11-hydroxy-12, 13-epoxy-9-octadecenoic acid, threo-11-hydroxy-12, 13-epoxy-9-octadecenoic acid, and 9 DL-hydroxy-12, 13-epoxy-10-octadecenoic acid.     

Oxidative activation of guanylate cyclase by prostaglandin endoperoxides and fatty acid hydroperoxides

Purified prostaglandin endoperoxides (PGG₂ and PGH₂) and hydroperoxides (15-OOH-PGE₂) as well as fatty acid hydroperoxides (12-OOH-20∶4, 15-OOH-20∶4, and 13-OOH-18∶2) were examined as effectors of soluble splenic cell guanylate cyclase activity. The procedures employed for the preparation and purification of these components circumvented the use of diethyl ether which obscured effects of lipid effectors because of contaminants presumed to be ether peroxides which were stimulatory to the cyclase. Addition of prostaglandin endoperoxides or fatty acid hydroperoxides to the reaction mixture led to a time-dependent activation of guanylate cyclase activity; 2.5-to 5-fold stimulation was seen during the first 6 min. The degree of stimulation and rate of activation were dependent on the concentration of the fatty acid effector; when initial velocities (6 min) were assessed, half maximal stimulation was achieved in the range of 2 to 3 μM. However, by extending the incubation time to 90 min, similar maximal increases in specific activity could be achieved with 3 or 10 μM PGG₂ or PGH₂. Activation of guanylate cyclase upon addition of prostaglandin endoperoxides or fatty acid hydroperoxides was prevented or reversed by the thiol reductants dithiothreitol (3 to 5 mM) or gluthathione (10 to 15 mM). Na₂S₂O₄, not known as an effective reducing agent of disulfieds, prevented but was relatively ineffective in reversing activation after it had been induced by PGG₂. Pretreatment of the enzyme preparation with increasing concentrations of N-ethyl-maleimide in the range of 0.01 to 1.0 mM prevented activation by PGG₂ without effecting basal guanylate cyclase activity. These observations indicate that fatty acid hydroperoxides and prostaglandin endoperoxides promote activation of the cyclase by oxidation of enzyme-related thiol functions. In contrast, PGE₂, PGF_2α, hydroxy fatty acids (13-OH-18∶2, 12-OH-20∶4) as well as saturated (18∶0), monoenoic (18∶1), dienoic (18∶2), and tetraenoic (20∶4) fatty acids were ineffective in promoting cyclase activation in the range of 1 to 10 μM. Studies to identify the species of the rapidly metabolized prostaglandin endoperoxides that serve as effectors of the cyclase indicated that PGG₂ but not 15-OOH-PGE₂ (the major buffer-rearrangement product of PGG₂) is most likely an activator. In the case of PGH₂ a rapidly generated (30 sec) metabolite of PGH₂ was found which contained a hydroperoxy or endoperoxy functional group and was equally as effective as PGH₂ as an apparent activator of the enzyme. The combined effects of PGG₂ and dehydroascorbic acid, another class of activator, exhibited additivity with respect to the rate at which the time-dependent activation was induced. These results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryls that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.     

Reduction of polyunsaturated fatty acid hydroperoxides by human brain glutathione peroxidase

Glutathione peroxidase (GSHPx) activity in the normal human brain was investigated using lipid hydroperoxides as substrates. Samples were obtained from autopsied frontal gray matter of 5 normal human males with no known central nervous system (CNS) disease. Aliquots were homogenized in 0.9% NaCl-0.5% Triton X-100, and the supernatant solution, obtained after centrifugation at 105,000 × g, was used for GSHPx assay. Glutathione peroxidase was measured by following the oxidation of NADPH at 340 nm. Hydroperoxides of linoleic, linolenic, gamma linolenic, 11,14 eicosodienoic, homo gamma linolenic, arachidonic, docosotetraenoic and docosohexaenoic acids were prepared and used as substrates. All these hydroperoxides were reduced by the brain GSHPx system, but at different rates. Gamma linolenic and docosotetraenoic hydroperoxides were reduced rapidly, whereas the peroxides of docosohexaenoic and 11,14 eicosodienoic were reduced at the lowest rate. Arachidonic hydroperoxide had the highest affinity for the enzyme and linolenic the lowest. Our results suggest that the brain GSHPx system is capable of reducing hydroperoxides of polyunsaturated fatty acids.     

Analyses of lipids and oxidation products by partition chromatography. Fatty acid hydroperoxides

A liquid partition chromatographic method was developed to isolated and determine hydroperoxides in autoxidized fatty acids or their methyl esters. By the use of benzene containing 2 to 4% methanol as the mobile solvent, the hydroperoxides were separated from unoxidized fatty acids or methyl esters and from secondary and polymeric decomposition products. In the analyses of oxidized fatty acids, diethyl ether was necessary to elute the secondary decomposition products. Saponification of autoxidized fatty esters destroyed the peroxides as determined iodometrically, but the resulting acids contained a fraction which was eluted in the same position as hydroperoxide acids. Evidence showed that this fraction is a monomeric hydroxy fatty acid containing conjugated cis-traux and trans-trans unsaturation. Fatty ester hydroperoxides were isolated chromatographically in yields and purity comparable to those reported in the literature by countercurrent distribution. The concentrations of methyl linoleate hydroperoxide determined chromatographically were smaller than indicated by the peroxide value and diene conjugation of the autoxidized methyl linoleate. This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Analysis of lipoxygenase-derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

A rapid method is described for the identification of fatty acid hydroperoxides by electrospray ionization-tandem mass spectrometry coupled to liquid chromatography without any derivatization required prior to analysis. Localization of fatty acid hydroperoxides in complex mixtures was achieved by monitoring the loss of hydrogen peroxide using constant neutral loss scanning. In the presence of 5 mM NH₄OAc in methanol-water, adductions [M+NH₄]⁺ were formed almost exclusively, directly revealing the molecular mass of the thermolabile hydroperoxides. In addition, low energy collision-induced dissociation of precursor ions [M+NH₄]⁺ led to characteristic product ions from both the 9- and 13-regioisomers. Thus, electrospray ionization-tandem mass spectrometry provides a straightforward approach for the study of the regioselectivity of lipoxygenase catalysis without any derivatization step required prior to analysis.     

Immobilized lipoxygenase in continuous production of fatty acid hydroperoxides

Soybean lipoxygenase-1 was covalently coupled to agarose with 75% recovery of catalytic activity. Because evidence was obtained that the immobilization resulted in improved operational stability of the enzyme, a lipoxygenase-reactor and a continuous process for the synthesis of 13-hydroperoxy-linoleic acid and 15-hydroperoxyarachidonic acid were developed. A procedure based on spectrophotometric hydroperoxide assay and constant oxygraphic monitoring of the effluent is presented for the calibration of the reactor to operate at the highest conversion efficiency when oxygenating quantitatively the substrate. Under these conditions, the reactor was capable of producing about 0.6 mg of hydroperoxy fatty acid/1.0 ml of wet gel/hr. The covalently coupled enzyme has been stable during six months of storage at 3 C in 0.2 M Na-borate buffer, pH 9.0, and during the same period, its operational stability in the column has been unaltered under the conditions used.     

Mass spectrometry of isomeric fatty acid hydroperoxides by chemical ionization via direct exposure probe

Intact isomeric methyl 9- and 13-hydroperoxy linoleates are analyzed by chemical ionization (CI) mass spectrometry using a direct exposure probe. Both isobutane and ammonia CI spectra were obtained. With isobutane CI, ions are observed for protonated fragments that are indicative of an acid catalysis mechanism similar to that observed in solution, whereas ammonia CI yields primarily adduct ions. The collisionally activated decomposition daughters of the high mass ions can be used to identify the position of the hydroperoxy group in the isomers studied.     

Cis-Unsaturated fatty acid products by hydrogenation with chromium hexacarbonyl

The use of Cr(CO)₆ was investigated to convert polyunsaturated fats intocis unsaturated products. With methyl sorbate, the same order of selectivity for the formation ofcis-3-hexenoate was demonstrated for Cr(CO)₆ as for the arene-Cr(CO)₃ complexes. With conjugated fatty esters, the stereoselectivity of Cr(CO)₆ toward thetrans, trans diene system was particularly high in acetone. However, this solvent was not suitable at elevated temperatures required to hydrogenatecis, trans- andcis, cis-conjugated dienes (175 C) and nonconjugated soybean oil (200 C). Reaction parameters were analyzed statistically to optimize hydrogenation of methyl sorbate and soybean oil. To achieve acceptable oxidative stability, it is necessary to reduce the linolenate constituent of soybean oil below 1–3%. When this is done commercially with conventional heterogenous catalysts, the hydrogenated products contain more than 15%trans unsaturation. By hydrogenating soybean oil with Cr(CO)₆ (200 C, 500 psi H₂, 1% catalyst in hexane solution), the product contains less than 3% each of linolenate andtrans unsaturation. Recycling of Cr(CO)₆ catalyst by sublimation was carried through three hydrogenations of soybean oil, but, about 10% of the chromium was lost in each cycle by decomposition. The hydrogenation mechanism of Cr(CO)₆ is compared with that of arene-Cr(CO)₃ complexes. Presented in part at Seventh Conference on Catalysis in Organic Syntheses, Chicago, Illinois, June 5–7, 1978.     

Influence of native antioxidants on the formation of fatty acid hydroperoxides in model systems

The effect of alpha‐tocopherol (alpha‐T) and quercetin on the formation of hydroperoxides of linoleic and linolenic acids during autoxidation at 60 ± 1 °C was investigated. Three isomers of hydroperoxides were detected using HPLC. Of isomers of linoleic acid hydroperoxides, 13‐hydroperoxy‐octadecadienoic acid trans‐trans (13‐HPODE t‐t), 9‐HPODE cis‐trans (9‐HPODE c‐t) and 9‐HPODE trans‐trans (9‐HPODE t‐t) were identified, constituting 64, 19 and 17% of the total amount, respectively. For linolenic acid, the components 13‐hydroperoxy‐octadecatrienoic acid trans‐trans (13‐HPOTE t‐t), 9‐HPOTE c‐t and 9‐HPOTE t‐t contributed 7, 33 and 60% to the total, respectively. The different dominant hydroperoxide isomers detected in linoleic and linolenic acids during oxidation are related to their chemical structure and the microenvironment of emulsion droplets. The ratios between specific isomers for both fatty acid hydroperoxides did not change during oxidation with or without antioxidants. Alpha‐T effectively inhibited the oxidation of fatty acids and reduced the formation of hydroperoxides. The total amount of the hydroperoxides decreased along with the increase in the concentration of alpha‐T, 1–40 µM. Quercetin inhibited the oxidation of both fatty acids at similar efficiency only at 40 µM concentration. A synergistic antioxidant effect of quercetin with alpha‐T in a binary system on both fatty acids was observed.     

脂肪酸淀粉酯的合成与结构表征

脂肪酸淀粉酯是一种新型的非离子型表面活性剂,具有广泛的应用价值.以天然的淀粉和大豆油为原料,通过化学法进行酯化改性,制得混合脂肪酸淀粉酯.研究了基本的合成路线,并用FT-IR和UV对其结构进行表征.产物乳化性能比较优越.     

Nuclear magnetic resonance characterization of reaction products of interesterification of peracetylated α-d-glucopyranose and fatty acid methyl esters

Mono- and diesters of fatty acids of peracetylated α-d-glucopyranose were prepared by chemical interesterification. Substituent-induced chemical shift effects on the carbonyl carbons rather than the ring carbons and proton atoms unambiguosly show the fatty acyl substituents to be at C1 in the monosubstituted, and at C1 and C6 in the disubstituted products. ¹H nuclear magnetic resonance (NMR) integration data before and after interesterification complemented ¹³C chemical shift data in verifying the molecular structures. Empirical data from classical ¹H and ¹³C NMR experiments thus provide a simple self-contained method for determining the number and position of fatty acyl substituents, and the anomeric compositions of peracetylated glucose fatty esters.     

强酸性阳离子树脂催化棕榈油副产物合成脂肪酸甲酯

以强酸性阳离子交换树脂为催化剂,经过自制的固定床反应器,使棕榈油脱臭馏出物(PODD)中的脂肪酸与甲醇起酯化反应,合成脂肪酸甲酯。结果表明,用固定床可从PODD连续制备脂肪酸甲酯,酯化反应的最佳条件为:n(甲醇)/n(PODD)=17.3;反应温度在甲醇正常沸点以下时,温度越高,转化率越大;转化率随催化反应时间增大而增大,但增大速度逐渐趋缓。当在常压下,64℃反应56 min时,游离脂肪酸的转化率可达87%左右。     

Utilization of acid oils in making valuable fatty products by microbial lipase technology

Microbial lipase-catalyzed hydrolysis, esterification, and alcoholysis reactions were carried out on acid oils of commerce such as coconut, soybean, mustard, sunflower, and rice bran for the purpose of making fatty acids and various monohydric alcohol esters of fatty acids of the acid oils. Neutral glycerides of the acid oils were hydrolyzed byCanadida cylindracea lipase almost completely within 48 h. Acid oils were converted into fatty acid esters of short- and long-chain alcohols like C₄, C₈, C₁₀, C₁₂, C₁₆, and C₁₈ in high yields by simultaneous esterification and alcoholysis reactions withMucor miehei lipase as catalyst. Acid oils of commerce can be utilized as raw materials in making fatty acids and fatty acid esters using lipase-catalyzed methodologies.     

Homolytic decomposition of linoleic acid hydroperoxide: Identification of fatty acid products

An isomeric mixture of linoleic acid hydroperoxides, 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (79%) and 9-hydroperoxy-cis-12,trans-10-octadecadienoic acid (21%), was decomposed homolytically by Fe(II) in an ethanol-water solution. In one series of experiments, the hydroperoxides were decomposed by catalytic concentrations of Fe(II). The 10⁻⁵ M Fe(III) used to initiate the decomposition was kept reduced as Fe(II) by a high concentration of cysteine added to the reaction in molar excess of the hydroperoxides. Nine different monomeric (no detectable dimeric) fatty acids were identified from the reaction. Analyses of these fatty acids revealed that they were mixtures of positional isomers identified as follows: (I) 13-oxo-trans,trans-(andcis,trans-) 9,11-octadecadienoic and 9-oxo-trans,trans- (andcis,trans-) 10,12-octadecadienoic acids; (II) 13-oxo-trans-9,10-epoxy-trans-11-octadecenoic and 9-oxo-trans-12, 13-epoxy-trans-10-octadecenoic acids; (III) 13-oxo-cis-9,10-epoxy-trans-11-octadecenoic and 9-oxo-cis-12, 13-epoxy-trans-10-octadecenoic acids; (IV) 13-hydroxy-9,11-octadecadienoic and 9-hydroxy-10,12-octadecadienoic acids; (V) 11-hydroxy-trans-12, 13-epoxy-cis-9-octadecenoic and 11-hydroxy-trans-9, 10-epoxy-cis-12-octadecenoic acids; (VI) 11-hydroxy-trans-12, 13-epoxy-trans-9-octadecenoic and 11-hydroxy-trans-9,10-epoxy-trans-12-octadecenoic acids; (VII) 13-oxo-9-hydroxy-trans-10-octadecenoic acids; (VIII) isomeric mixtures of 9, 12, 13-dihydroxyethoxy-trans-10-octadecenoic and 9, 10, 13-dihydroxyethoxy-trans-11-octadecenoic acids; and (IX) 9, 12, 13-trihydroxy-trans-10-octadecenoic and 9, 10, 13-trihydroxy-trans-11-octadecenoic acids. In another experiment, equimolar amounts of Fe(II) and hydroperoxide were reacted in the absence of cysteine. A large proportion of dimeric fatty acids and a smaller amount of monomeric fatty acids resulted. The monomeric fatty acids were examined by gas liquid chromatography-mass spectroscopy. Spectra indicated that the monomers were largely similar to those produced by the Fe(III)-cysteine reaction. Presented in part at the American Chemical Society Meeting, Los Angeles, March 1974. ARS, USDA.     

Cyclization of linolenic acid by alkali isomerization

Summary Methyl linolenate was isomerized by heating in an alkaline ethylene glycol solution for 7 hrs. at 200°. Methyl esters of the isomerized acids were treated with urea in methanol, and the nonurea-adduct-forming fraction was distilled. The isomerized esters consisted of 81.8% of nonurea-adduct-forming monomeric distillate (NAM), 13.7% nonurea-adduct-forming polymeric nondistillable material, and 4.5% of urea adduct-former The NAM has a maximum absorption in the ultraviolet region at 238 mμ. Ultraviolet absorption extends into the region associated with triene conjugation, but the characteristic structure of triene absorption is absent. Infrared absorption bands in the 10 μ region ordinarily found withtrans,trans orcis,trans are absent. The NAM is shown to contain a cyclic monomer by treatment with N-bromosuc-cinimide, followed by dehydrobromination with dimethylaniline. The resultant product was oxidized to phthalic acid with permanganate. The NAM was separated both by countercurrent distribution and by gas chromatography into fractions having different ultraviolet absorption spectra. However complete fractionation of the mixture into its pure components has not yet been achieved. Presented at fall meeting, American Oil Chemists' Society, Chicago, Ill., October 20–22, 1958. This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Synthesis and characterization of maltose fatty acid monoesters as biosurfactants

Maltose long-chain fatty acid esters (MFAE), esterified at the 6 and 6′ position, were synthesized with stearic, palmitic, myristic, and oleic groups. Synthesis yields were 15–20% based on initial maltose present, and structural confirmation was obtained using plasma desorption mass spectrometry and nuclear magnetic resonance spectroscopy. These surfactants have surface tensions in the range of 34–36 dyn/cm at their critical micelle concentrations (CMC) of approximately 10⁻⁵–10⁻⁶ mol/L. The increased chain lengths have a marked effect, reducing CMC values for MFAE by approximately three orders of magnitude over similar carbohydrate-based dodecyl chain sources. Within chain lengths between 14 and 18 carbons, the rate of change in CMC is significant and decreases with increasing chain length for MFAE. The melting points of MFAE are approximately 40°C, and the heat capacities range from 1.6 to 1.9 J/g·K. These numbers are comparable to those of sucrose esters, indicating their applicability in similar uses. However, because MFAE, unlike sucrose, possess an anomeric carbohydrate carbon position, these surfactants maintain their reducing nature and are susceptible to further derivatization. They are also synthesized from renewable, economical carbohydrates and lipids and may provide an excellent alternative to pertrochemical-derived products.     

Synthesis and characterization of telechelic macromers containing fatty acid derivatives

Telechelic macromers end-capped with (meth)acrylic functionalities are the most commonly used materials in rigid, dental formulations. In order to provide higher flexibility to the final product (not necessarily for dental applications), long chain aliphatic fatty acid derivatives may be chosen. Thus, new telechelic macromers comprising methacrylic functionalities and dimer fatty acid derivatives have been synthesized for the first time, and their chemical structure is discussed in detail. Six different systems comprising ester, anhydride and urethane bonds were synthesized from non-toxic raw materials. FTIR spectroscopy and NMR analysis were used to evaluate chemical structure of new systems. Their molecular masses were estimated from GPC measurements and from an analytical method based on iodine value determination. The latter one proved to be very accurate in determining molecular masses of methacrylated telechelics according to a new method developed in this work. We demonstrated that, via simple organic chemistry, different architectures of telechelic macromers comprising commercially available, long chain derivatives of fatty acid, mainly linoleic acid, with α,ω-dihydroxy, α,ω-dicarboxy or α,ω-diamino functionalities were successfully synthesized. These macromers facilitate the development of new reactive (preferably, photocurable) flexible systems for potential biomedical applications.     

A new reaction of unsaturated fatty acid hydroperoxides: Formation of 11-hydroxy-12,13-epoxy-9-octadecenoic acid from 13-hydroperoxy-9,11-octadecadienoic acid

One of the main compounds formed from 13L-hydroperoxy-9cis,11trans-octadecadienoic acid anaerobically at 100 C in aqueous ethanol was found to bethreo-11-hydroxy-12,13-epoxy-9-octadecenoic acid. The major part (ca. 90%) of this compound was formed from the fatty acid hydroperoxide in a reaction involvingcis-addition to the Δ¹¹ double bond of the proximally linked hydroperoxide oxygen and hydroxyl ion or hydroxyl radical from the solvent. A small part (ca. 10%) was formed bycis-addition of the two hydroperoxide oxygens to the Δ¹¹ double bond. 11-Hydroxy-12,13-epoxy-9-octadecenoic acid and its isomer, tentatively identified as 11-hydroxy-9,10-epoxy-12-octadecenoic acid, also were isolated from a sample of autoxidized linoleic acid.     

Inhibition of bacterial urease by autoxidation of furan C-18 fatty acid methyl ester products

Autoxidation products of synthetic methyl 9,12-epoxy-octadeca-9,11-dienoate (MEFA) were investigated by gas chromatography-mass spectrometry analysis and tested for bacterial urease inhibition. A suspension of oxidized MEFA in 10% Tween 80 was an effective inhibitor for bacterial urease extract fromHelicobacter pylori (I₅₀=1.3 mM) and for commercial urease fromBacillus pasteurii (I₅₀=0.06 mM). The urease inhibitory effect was cancelled by adding cysteine to the reaction mixture. The total content of biologically active oxidized products in the mixture was found to be 6.2%. Dioxo-ene derivatives of MEFA on the thin-layer chromatography plate surface were converted into more stable compounds, whose formation in the mixture reduced inhibition ofB. pasteurii to about 2% of the former level. The mechanism of urease inhibition is supposed to involve the interaction of the thiol groups of the enzyme’s active center with the inhibitor molecules.