The kinetics of the autoxidation of polyunsaturated fatty acids

The kinetics of the autoxidation of a series of polyunsaturated fatty acids (PUFA) with increasing degrees of unsaturation and the mono-, di-and triglycerides of linoleate have been studied in homogeneous chlorobenzene solution at 37 C under 760 torr of oxygen. The autoxidations were initiated by thermal decomposition of azo initiators and followed by measuring the rate of oxygen uptake. The rate of chain initiation was determined by the induction period method using α-tocopherol as the chainbreaking antioxidant. The measured oxidizabilities of the PUFA are linearly dependent on the number of doubly allylic positions present in the molecule. Thus, the oxidizability of linoleate is 2.03×10⁻² M^−1/2 sec^−1/2, and the value for docosahexaenoate is five times greater, 10.15×10⁻² M^−1/2 sec^−1/2. The rate of autoxidation for all PUFA studied and for the mono- and diglyceride is proportional to the substrate concentration and to the square root of the rate of chain initiation, implying that the autoxidation of these compounds follows the usual kinetic rate law. The autoxidation of the triglyceride is more complex and does not appear to follow the same rate law at all substrate concentrations. This deviation from the usual kinetic rate expression may be due to lipid aggregation at low concentrations of the triglyceride.     

The biosynthesis of polyunsaturated fatty acids in plants

This paper is a review of some of the work being done at the author's laboratory. The phospholipids and glycolipids of the alga,Chlorella vulgaris, have been implicated in fatty acid transformations such as chain elongation and desaturation. Labeling studies with [¹⁴C] acetate have shown that newly synthesized galactosyl glycerides have mainly saturated fatty acids. Subsequent to de novo synthesis, a series of alterations of fatty acid structure takes place within the same glycolipid molecules. The specific incorporation of [¹⁴C] oleic acid intoChlorella phosphatidyl choline provides a convenient model system for studying the lipid dependent desaturation of oleic to linoleic acid. The inhibitor of fatty acid desaturation, sterculic acid, only inhibits the conversion of oleate into linoleate if added before the precursor fatty acid has been incorporated into a complex lipid. Studies with isomeric monoenoic fatty acids have suggested that there are two enzymes which catalyze the formation of linoleic from oleic acid. One measures the position of the second double bond from the carboxyl group, the other, from the methyl end of the chain. The latter enzyme probably requires the complex lipid substrate.     

Lipase-assisted concentration of n-3 polyunsaturated fatty acids in acylglycerols from marine oils

Preparation of n-3 polyunsaturated fatty acid (PUFA) concentrates from seal blubber oil (SBO) and menhaden oil (MHO) in the form of acylglycerols was carried out by hydrolysis with a number of commercial microbial lipases. The lipases tested were Aspergillus niger, Candida cylindracea (CC), Chromobacterium viscosum, Geotrichum candidum, Mucor miehei, Pseudomonas sp., Rhizopus oryzae, and Rhizopus niveus. After lipase-assisted hydrolysis of oils, free fatty acids were removed, and fatty acid composition of the mixture containing mono-, di-, and triacylglycerols was determined. All lipases were effective in increasing the n-3 PUFA content of the remaining acylglycerols of both SBO and MHO. The highest concentration of n-3 PUFA was provided by CC lipase; 43.5% in SBO [9.75% eicosapentaenoic acid (EPA), 8.61% docosapentaenoic acid (DPA), and 24.0% docosahexaenoic acid (DHA)] and 44.1% in MHO (18.5% EPA, 3.62% DPA, and 17.3% DHA) after 40 h of hydrolysis. Thus, CC lipase appears to be most suitable for preparation of n-3 PUFA in the acylglycerol form from marine oils.     

Component fatty acids in body fats of some marine fishes

Summary The component fatty acids of the body fats of three typical species of Bombay marine fish were investigated. The saturated acid contents of pala and black pomfret oil were about the same. The black pomfret oil was abnormally high in palmitoleic acid. Linoleic acid was found to be remarkably low in pala and black pomfret oils. In white pomfret the amount of it present was about the same as that of linolenic. Polyunsaturated acids were present in black and white pomfret oils but only present in traces in pala oil.     

The significance of polyunsaturated fatty acids in cutaneous biology

The skin epidermis displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA) and 18-carbon (n-6) PUFA results in characteristic scaly skin disorder and excessive epidermal water loss. Arachidonic acid, a 20-carbon (n-6) PUFA is metabolized via the cyclooxygenase pathway into predominantly prostaglandin E₂ (PGE₂) PGF_2α′ and PGD₂ and via the lipoxygenase pathway into predominantly 15-hydroxyeicosatetraenoic acid (15-HETE). The prostaglandins modulate normal skin physiological processes at low concentrations and inflammatory reactions at high concentrations. Similarly, the very active epidermal 15-lipoxygenase transforms dihomogammalinolenic acid (DGLA) into 15-hydroxy eicosatrienoic acid (15-HETrE), eicosapentaenoic acid (EPA) into 15-hydroxyeicosapentaenoic acid (15-HEPE) and docosahexaenoic acid (DHA) into 17-hydroxydocosahexaenoic acid (17-HDoHE), respectively. These monohydroxy acids exhibit anti-inflammatory properties. In contrast, the 18-carbon (n-6) PUFA is transformed into 13-hydroxy-9,11-octadecadienoic acid (13-HODE), which exerts antiproliferative properties in the tissue. Thus the supplementation of diets with appropriate purified vegetable oils and/or fish oil may generate local cutaneous anti-inflammatory metabolites which could serve as a less toxicin vivo monotherapy or as adjuncts to standard therapeutic regimens for the management of skin inflammaory disorders.     

Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis

When whale oil triglycerides were subjected to pancreatic lipase hydrolysis, eicosapentaenoic and docosahexaenoic acids were found mainly in the di- and triglyceride products, suggesting that they are in the 1,3-positions but resistant to the action of the lipase. Their presence in the 1,3-positions was confirmed. Their resistance to pancreatic lipase hydrolysis was demonstrated by analysis of the products of the enzyme action on: (a) a concentrate of highly unsaturated whale oil triglycerides; (b) the latter after randomization; and (c) synthetic 1,2-di-octadecenoyl-3-eicosapentaenoyl glycerol. Docosapentaenoic acid was also shown to be present in the 1,3-position of whale oil triglycerides but was not lipase resistant. It is postulated that the presence of a double bond near the carboxyl group exercises an inhibitory effect, or that the location of the double bonds in the resistant acids places their terminal methyl groups close to the carboxyl, producing a steric hindrance effect.     

The desaturation step in the animal biosynthesis of polyunsaturated fatty acids

The aerobic desaturation of unsaturated fatty acids in the microsomes has been systematically explored and some of the relevant experimental works have been assembled. The contribution of the microsomal electron transport chain and lipoproteic structure of the microsomes is analyzed. Evidences of linoleyl-CoA and α-linolenyl-CoA being desaturated by the same enzyme are presented. The linoleic acid desaturation is shown to be different in different tissues and to decrease with aging. The effect of competitive reactions with acids of the same or different series, the competition of desaturation and transesterification, and dietary and hormonal contributions to unsaturated fatty acids desaturation are summarized. Alltrans linoleic acid and elaidic acid were not desaturated in our experimental conditions by rat liver microsomes. From the bulk of data collected, a hypothetical model of 6-olefinase is drawn. The main features of the model are: The existence of both binding and desaturating sites; the binding of acyl-CoA and enzyme through hydrophobic forces of the Van der Waals type and weak polar attractions due to double bonds; the orienting binding characteristics of double bond proximate to the place where olefination will take place; and the importance of enzyme conformation that requires that the orienting double bond must have acis structure.     

Isomers of monoethylenic fatty acids in some partially hydrogenated marine oils

An analytical study of the monoethylenic isomers in commercial samples of partially hydrogenated herring, whale and seal oils is presented. The results show that with hydrogenated herring oil there is a slight decline in monoenetrans content from 37% in C₁₆ through to 32% in C₂₂. With both whale and seal oils, monoenetrans contents were constant at 54% and 59%, respectively, throughout all chain lengths. In general thecis andtrans positional isomers from hydrogenated whale and seal oils were more scattered than those from hydrogenated herring oil; however in each oil the majorcis isomers of each chainlength were indicative of originalcis fatty acid isomers in the raw oils.     

Industrial chemical uses of polyunsaturated fatty acids

Production of vegetable, animal and marine oils containing more than about 40% unsaturated fatty acids totaled 15,000 million pounds in 1968, almost on the scale of petrochemical production. The greater share (64%) of this nonfossil oil production was directed toward food uses, the remainder toward industrial and animal feed uses. The variety of chemical reactions carried out on these unsaturated fatty acid products include hydrogenation, interesterification, dimerization, sulfation, formation of nitrogen compounds, epoxidation, alkaline cleavage and oxidative ozonolysis. Some of these reactions have been developed at Utilization Research and Development Divisions of the Agricultural Research Service, U.S. Department of Agriculture. Research is continuing in developing new reactions for potential industrial application. An example is reductive ozonolysis of unsaturated fatty esters to produce monofunctional aldehydes and bifunctional aldehyde esters. Presented at the ISF-AOCS World Congress, Chicago, September 1970. No. Market. Nutr. Res. Div., ARS, USDA.     

Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids

Lipase hydrolysis was evaluated as a means of selectively enriching long-chain ω3 fatty acids in fish oil. Several lipases were screened for their ability to enrich total ω-3 acids or selectively enrich either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). The effect of enzyme concentration, degree of hydrolysis, and fatty acid composition of the feed oil was studied. Because the materials that were enriched in long-chain ω3 acids were either partial glycerides or free fatty acids, enzymatic reesterification of these materials to triglycerides by lipase catalysis was also investigated. Hydrolysis of fish oil by eitherCandida rugosa orGeotrichum candidum lipases resulted in an increase in the content of total ω3 acids from about 30% in the feed oil to 45% in the partial glycerides. The lipase fromC. rugosa was effective in selectively enriching either DHA or EPA, resulting in a change of either the DHA/EPA ratio or the EPA/DHA ratio from approximately 1:1 to 5:1. Nonselective reesterification of free fatty acids or partial glycerides that contained ω3 fatty acids could be achieved at high efficiency (approximately 95% triglycerides in the product) by using immobilizedRhizomucor miehei lipase with continuous removal of water.     

Influence of moderate amounts of trans fatty acids on the formation of polyunsaturated fatty acids

The effect of trans fatty acids from partially hydrogenated soybean oil and butterfat on the formation of polyunsaturated fatty acids was investigated. Five groups of rats were fed diets that contained 20 wt% fat. The content of linoleic acid was adjusted to 10 wt% of the dietary fats in all diets, whereas the amount of trans fatty acids from partially hydrogenated soybean oil (PHSBO) was varied from 4.5 to 15 wt% in three of the five diets. The fourth group received trans fatty acids from butterfat (BF), while the control group was fed palm oil without trans fatty acids. Trans fatty acids in the diet were portionally reflected in rat liver and heart phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol, and phosphatidylserine. Incorporation in the sn-1 position was compensated by a decrease in saturated fatty acids. Trans fatty acids were not detected in diphosphatidylglycerol. Compared to the presence in the dietary fats, 8t- and 10t-18:1 were discriminated against in the incorporation in PE and PC from liver and heart, whereas 9t- and 12t-18:1 were preferred. The formation of 20:4n-6 was not influenced by 4.5 wt% trans fatty acids (from PHSBO) but apparently was by 10 wt% in liver. In contrast, even a content of 2.5 wt% trans fatty acids from BF reduced the formation of 20:4n-6. The inhibitory effect of trans isomers on linoleic acid conversion was reflected less in heart than in liver and less for PE than for PC. Groups with trans fatty acids showed increased 22:6n-3 and 22:5n-3 deposition in liver and heart PE and PC.     

The phospholipid fatty acids of the marine spongeXestospongia muta

The rare phospholipid fatty acids 3,7,11-trimethyldodecanoic (1), 5,9-hexadecadienoic (2) and 12-methyl-hexadecanoic (3) were identified in the marine spongeXestospongia muta. Branched fatty acids inX. muta accounted for 35% of the total fatty acid mixture. It was observed that the occurrence of the 5,9-hexadecadienoic acid (2) coincides with the complete absence of the very long chain fatty acid 5,9-hexacosadienoic. The acid 5,9,19-octacosatrienoic seems to be found in mostXestospongia species.     

Effect of polyunsaturated fatty acids in obese mice

Genetically obese (ob/ob) mice display a variety of metabolic differences from lean litter mates. In the obese state, fatty acid desaturation-elongation in brown adipose tissue mitochondria is apparently altered, resulting in differences in membrane fatty acid composition. This change in membrane lipid environment appears to influence GDP binding and there-fore the activity of the proton conductance pathway associated with regulation of energy expenditure in these animals. In liver, binding of insulin to the nuclear membrane is increased by feeding a high polyunsaturated/saturated (P/S) diet fat. Consumption of a high P/S diet decreased mRNA levels for fatty acid synthase, acetyl-CoA carboxylase, malic enzyme, and pyruvate kinase in obese and lean animals. Expression of mRNA for these lipogenic enzymes was higher in obese animals and suggests that obese mice may be resistant to polyunsaturated fatty acid feedback control of gene expression.     

The biological consequences of feeding polyunsaturated fatty acids to antioxidant-deficient animals

The addition of polyunsaturated fatty acids (PUFA) to diets deficient in vitamin E and other effective antioxidants results in a variety of symptoms in animals. For example, the feeding of such diets to rats results in muscular dystrophy, testis degeneration, dental depigmentation, brown discoloration of the fat and and uterus and creatinuria Similar diets fed to rabbits and ruminants results in muscular dystrophy. In chickens the symptoms observed are encephalomalacia, lowered egg production, and poor hatchability. The addition of PUFA to diets is known to result in the destruction of vitamin E in the diet or in the tissues of animals as a result of free radicals produced during the autooxidation of the PUFA. However, in several studies, this possible explanation for the development of vitamin E deficiency symptoms has been made untenable. In such studies the more likely explanation for development of symptoms is thein vivo peroxidation of PUFA in the tissues of animals following incorporation of large amounts of PUFA in lipid structures and depletion from the tissues of vitamin E and other biologically effective antioxidants.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

2-Hydroxy fatty acids from marine sponges 2. The phospholipid fatty acids of the caribbean spongesVerongula gigantea andAplysina archeri

The α-hydroxy fatty acids 2-hydroxy-eicosanoic (1) acid, 2-hydroxyheneicosanoic (2) acid, 2-hydroxydocosanoic (3) acid, 2-hydroxytetracosanoic (4) acid, 2-hydroxy-23-methyl-tetracosanoic acid and 2-hydroxypentacosanoic (5) acid were isolated from the Caribbean spongesVerongula gigantea andAplysina archeri. The very long chain fatty acids 5,9-nonacosadienoic acid (29∶2) and 5,9,23-tricontatrienoic acid (30∶3) were also identified together with theiso-prenoid fatty acid 3,7,11,15-tetramethylhexadecanoic (phytanic) acid that seems to be common in the Aplysinidae.A. archeri contained an extremely long chain fatty acid tentatively characterized as dotricontaenoic (32∶1) acid. These acids were found to occur in phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and traces of phosphatidylglycerol.     

Unsaturated fatty acids. I. Low temperature urea complexes of polyunsaturated acids from bovine tissue

Summary A mixture of fatty acids obtained from autolyzed saline extracts of beef testicular tissue was fractionated by crystallization of the urea complexes at 5°, −20°, and −75°C. Fractions rich in docosahexaenoic and in arachidonic acids were obtained as solid complexes. The filtrate remaining after precipitation of the solid urea complexes contained a high percentage of hexaenoic acid of shorter chain length than docosahexaenoic, probably eicosahexaenoic acid. Presented at the fall meeting of the American Oil Chemists' Society, Minneapolis, Oct. 11–13, 1954. Presented in part before the Lipoid Section, IId International Congress of Biochemistry, Paris, France, Sept. 21, 1952.     

The intestinal mucosa as a target for dietary polyunsaturated fatty acids

Several studies have reported beneficial effects of dietary polyunsaturated fatty acids (PUFA) on various aspects of both human and animal health, and particular reference has been made to their effects on systemic immune responses. Both immune stimulation and immune suppression have been reported, with the outcome dependent on the type of PUFA, the target cell, as well as the immune competence of the cells before exposure. The systemic and the mucosal immune systems are discrete entities, which have evolved specific approaches in the defense of the host. The latter comprises several interconnected tissues, which communicate with one another through the action of soluble mediators and the trafficking of cellular components. After the oral mucosa, the intestinal epithelium and its associated gutassociated lymphoid tissue are the primary targets of dietary components. Absorption of dietary PUFA and its incorporation into intestinal tissues has been well studied, but the consequences of these events in relation to local immune responses have received little attention. This article describes some of the immune mechanisms operating at this barrier and, where possible, pinpoints areas for which a modulatory role for PUFA has already been demonstrated. Although not an exhaustive treatise of the subject, it is hoped that this review will foster research into the specific interaction between dietary PUFA and cell populations comprising the intestinal barrier.     

Enzymatic synthesis of steryl esters of polyunsaturated fatty acids

Steryl esters of long-chain fatty acids have water-holding properties, and polyunsaturated fatty acids (PUFA) have various physiological functions. Because steryl ester of PUFA can be expected to have both features, we attempted to synthesize steryl esters of PUFA by enzymatic methods. Among lipases used, Pseudomonas lipase was the most effective for the synthesis of cholesteryl docosahexaenoate. When a mixture of cholesterol/docosahexaenoic acid (3:1, mol/mol), 30% water, and 3000 units/g of lipase was stirred at 40°C for 24 h, the esterification extent attained 89.5%. Under the same reaction conditions, cholesterol, cholestanol, and sitosterol were also esterified efficiently with docosahexaenoic, eicosapentaenoic, arachidonic, and γ-linolenic acids.