The activating effect of dietary protein on linoleic acid desaturation

The desaturation of¹⁴C-1-linoleic acid to γ-linolenic acid and their incorporation into the microsomal lipids of rats fed on a balanced diet and a protein diet were measured in vitro. It was shown that a protein diet does not change significantly the distribution of the radioactivity among the different lipidic fractions compared to the animals fed on a balanced diet. However the microsomal desaturation of linoleic acid to γ-linolenic acid increased in the rats maintained on a protein diet. Besides, the amount and composition of the free fatty acids present in the microsomes of the animals fed on both diets were similar enough to discard the hypothesis that they may modify the desaturation of linoleic acid produced by the diet. The enzymic activity of the linoleyl desaturase of liver microsomes of animals fed on a protein diet, measured in substrate saturating conditions, is greater than in animals with balanced diet. Consequently the results support the hypothesis that a protein diet increases specifically the desaturating activity of the microsomes.     

Comparative effect of a protein diet on the desaturation,elongation and simultaneous desaturation and elongation of linoleic acid

The effect of a protein diet on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied by incubation of rat liver microsomes with labeled linoleic acid. The incubation was performed in desaturating, elongating and desaturating-elongating conditions. In desaturating conditions, linoleic acid was converted to γ-linolenic acid, whereas in elongating conditions it was converted to 20∶2, 22∶2 and 24∶2. In desaturating-elongating conditions, labeling was found in γ 18∶3, 20∶2, 20∶3, 20∶4 and 22∶2. A protein diet increased the oxidative desaturation of linoleic acid to γ-linolenic and arachidonic acid biosynthesis, whereas the elongating reaction was not enhanced in the experimental conditions tested. It is suggested that the main controllable step in the linoleic acid family is the oxidative desaturation of linoleic acid to γ-linolenic acid.     

Effect of catecholamines and β-blockers on linoleic acid desaturation activity

The effect of catecholamines and adrenergic blocking agents on the oxidative desaturation of linoleic acid in rat liver microsomes was studied. Epinephrine (1 mg/kg/body weight) produced a significant decrease on the conversion of [1-¹⁴C]linoleic acid to γ-linolenic acid. The effect of epinephrine was blocked by single injections of the β blockers propranolol (10 mg/kg body weight) or dichloroisoproterenol 30 min before the hormone treatment. Isoproterenol (100 μg/kg body weight) produced a significant decrease on the activity of the linoleyl-CoA desaturase. The effect of the catecholamines was postulated to be mediated through β receptors by an enhancement of the intracellular levels of cyclic AMP.     

In vitro desaturation of 1-14C linoleic acid in Novikoff hepatoma

The lipid fatty acid pattern of normal liver, host liver, and Novikoff hepatoma was determined by gas liquid chromatography, and Δ6-desaturase activity for linoleic acid was measured in the microsomal fractions. The results showed that, in Novikoff hepatoma, there is a correlation between the low content of arachidonic acid and the low activity of Δ6-desaturase, a key enzyme in the biosynthetic pathway of this acid.     

Effects of conjugated linoleic acid isomers on the hepatic microsomal desaturation activities in vitro

The influence of individual conjugated linoleic acid (CLA) isomers on the Δ6 desaturation of linoleic and α-linolenic acids and on the Δ9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The Δ6 desaturation of 18∶2n−6 was decreased from 23 to 38% when the ratio of 9cis,11trans-18∶2 to 18∶2n−6 increased from 0.5 to 2. The compound 10trans,12cis-18∶2 exhibited a similar effect only at the highest concentration. The Δ6 desaturation of α-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the Δ9 desaturation of stearic acid was 10trans,12cis-18∶2.     

Effects of dietary proteins on linoleic acid desaturation and membrane fluidity in rat liver microsomes

The effect of dietary protein, casein (CAS) and soybean protein (SOY), on linoleic acid desaturation in liver microsomes was studied in rats. The activity of Δ6 desaturase in total and rough endoplasmic reticula (ER and RER) was significantly higher in the CAS group than in the SOY group. In ER and smooth endoplasmic reticulum, the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, when incorporated into the membrane, was decreased in the SOY group and accompanied by a reduction in the cholesterol/phospholipid (CHOL/PL) ratio, consistent with an increase in membrane fluidity. In a separate study, the effect of varying dietary proteins, CAS, milk whey protein, egg albumin, SOY, potato protein and wheat gluten, on the relationship between the Δ6 desaturase activity and microsomal membrane fluidity was also examined. The results indicated that the dietary protein-dependent change in the liver microsomal CHOL/PL ratio affected membrane fluidity, and subsequently the activity of Δ6 desaturase in liver microsomes. However, since dietary protein influenced the Δ6 desaturase activity in RER without influencing membrane fluidity, it is possible that some regulation might have taken place at the level of enzyme synthesis.     

Dietary oxidized cholesterol modulates cholesterol metabolism and linoleic acid desaturation in rats fed high-cholesterol diets

The interactive effect of high dietary levels of oxidized cholesterol on exogenous cholerterol and linoleic acid metabolism was examined in male 4-wk-old Sprague-Dawley rats given high-cholesterol diets. The rats were pair-fed purified diets free of or containing either 0.5% cholesterol alone or both 0.5% cholesterol and 0.5% oxidized cholesterol mixture (containing 93% oxidized cholesterol) for 3 wk. Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity was reduced in rats given cholesterol alone or both cholesterol and oxidized cholesterol. However, hepatic cholesterol 7α-hydroxylase activity was lowered only when rats were given both cholesterol and oxidized cholesterol, although dietary cholesterol increased this activity. Reflecting this effect, acidic steroid excretion was lowest among the groups of rats given cholesterol and oxidized cholesterol. On the other hand, the activity of hepatic Δ6 desaturase, a key enzyme in the metabolism of linoleic acid to arachidonic acid, was increased in rats given both cholesterol and oxidized cholesterol, although dietary cholesterol alone lowered its activity. As a result, the Δ6 desaturation index, 20∶3n-6+20∶4n-6/18∶2n-6, in liver and serum phosphlipids tended to be higher in the group fed both cholesterol and oxidized cholesterol than in the one fed cholesterol alone. Thus, dietary oxidized cholesterol significantly modulated exogenous cholesterol metabolism and promoted linoleic acid desaturation even when it was given at high levels together with a high cholesterol diet.     

Comparative effects of docosa-4,7,10,13,16-pentaenoic acid and docosa-4,7,10,13,16,19-hexaenoic acid on the desaturation of linoleic acid and α-linolenic acid

Varying concentrations of free docosa-4,7,10,13,16-pentaenoic acid or its CoA ester were incubated with a given variable concentration of 1-¹⁴C-linoleate or 1-¹⁴C-α-linolenate as either the free fatty acid or the CoA ester, microsomal enzymes, and the appropriate cofactors for fatty acid desaturation. The results obtained were compared to the effects of docosa-4,7,10,13,16,19-hexaenoyl CoA when incubated in a similar manner in the presence of the labeled substrates. Both feedback and crossed inhibition effects were observed; these inhibition effects may play a role in the regulation of polyunsaturated fatty acid biosynthesis.     

Zinc deficiency increases the rate of Δ6 desaturation of linoleic acid in rat mammary tissue

The effect of zinc deficiency on the Δ⁶-desaturation of [1-¹⁴C] linoleic acid was studied in mammary tissue microsomes from lactating rats. The rats were maintained on zinc-adequate (20 ppm zinc) or zinc-deficient (10 ppm zinc changing to 0.5 ppm zinc during last trimester) diets throughout gestation and for the first 3 days of lactation. Mammary tissue microsomes were incubated with [1-¹⁴C] linoleic acid and other samples of mammary tissue, mammary milk and the milk in the stomachs of the pups were analyzed for total fatty acid composition. In mammary microsomes from zinc-deficient rats, Δ⁶-desaturation of linoleic acid was 3.4 times greater than in microsomes from zinc-adequate rats. This change in metabolism of linoleic acid was reflected by comparable changes in the relative tissue and milk composition of linoleic and arachidonic acids and in the ratios of palmitic to palmitoleic acid, stearic to oleic acid and linoleic and arachidonic acid.     

Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to γ-linolenic acid in rat liver microsomes

The effects of antibodies against microsomal electron-transport components on the in vitro activity of Δ⁶-desaturation of linoleic acid to γ-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the Δ⁶-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b₅ and NADPH-cytochrome c reductase, only the antibody against cytochrome b₅ demonstrated a marked ability to inhibit the Δ⁶-desaturase activity. This evidence supports a participation of cytochrome b₅ in the Δ⁶-desaturation of linoleic acid and suggests a pathway analogous to the Δ⁹-desaturation of stearyl-CoA.     

Biocatalysis of linoleic acid to conjugated linoleic acid

CLA refers to a group of geometrical and positional isomers of linoleic acid (LA) with conjugated double bonds. CLA has been reported to have diverse health benefits and biological properties. Traditional organic synthesis is highly capital-intensive and results in an isomeric mixture of CLA isomers. Biotechnology presents new alternatives to traditional lipid manufacturing methods. The objective of this study was to examine the effect of protein isolation procedures on linoleate isomerase (LAI) recovery from microbial cells and biocatalysis of LA to CLA. Protein isolation experiments were carried out using Lactobacillus acidophilus L1 and two strains of Lactobacillus reuteri (ATCC 23272 and ATCC 55739). Under the same assay conditions, ATCC 55739 had the highest LAI activity among the microbial cultures examined in this study. Efficiency of cell lysis methods, which included various combinations of lysozyme and mutanolysin treatments in combination with sonication and osmotic rupture of cells with liquid nitrogen, was very low. Although treatment of cell material with a detergent (octylthioglucoyranoside) freed a significant amount of LAI activity into the solution, it was not sufficient to recover all the LAI activity from the residual cells. Crude LAI preparations produced mainly the cis-9,trans-11 CLA isomer. Time and substrate/protein ratio had a significant effect on biocatalysis of LA to CLA. It appears that the mechanism and kinetics of enzymatic conversion of LA to CLA are quite complex and requires further research using pure LAI preparations. Published with approval of the Director, Oklahoma Agricultural Experiment Station.     

Reciprocal interactions in the desaturation of linoleic acid into γ-linolenic and eicosa-8,11,14-trienoic into arachidonic

Variable concentrations of [I¹⁴C] linoleic acid and [I¹⁴C] eicosa-8,11,14-trienoic acid were incubated with liver microsomes in a medium containing the necessary cofactors for fatty acid desaturation. The conversion of linoleic into γ-linolenic acid and eicosatrienoic into arachidonic acid were mutually inhibited and the inhibition depended on the concentration of the fatty acids incubated.     

Reinvestigation of the antioxidant properties of conjugated linoleic acid

Despite repeated suggestions that antioxidant activity of conjugated linoleic acid (CLA), a collective of conjugated dienoic isomers of linoleic acid, underlies its reported anticarcinogenic and antiatherosclerotic effects, the antioxidant properties of CLA remain ill-defined. Therefore, this study was undertaken to gain more insight into the mechanism of potential CLA antioxidant activity. It was tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or-independent oxidative stress. Progress of oxidation was determined by direct spectrophotometric measurement of conjugated diene formation and by gas chromatographic/mass spectrometric analysis of fatty acids. The oxidative susceptibility of CLA was higher than that of linoleic acid, and comparable to arachidonic acid. When oxidation of PLPC (1.0 mM) was initiated using the lipid-soluble 2,2′-azobis(2,4-dimethylvaleronitrile) or the water-soluble 2,2′-azobis(2-amidinopropane) hydrochloride, the radical scavengers vitamin E and butylated hydroxytoluene (BHT) at 0.75 μM efficiently inhibited PLPC oxidation, as evident from a clear lagphase. In contrast, 0.75 μM CLA did not have any significant effect on PLPC oxidation. Inhibition of PLPC oxidation by higher concentrations of CLA appeared due to competition, not to an antioxidant effect. When oxidation of PLPC was initiated by hydrogen peroxide/Fe²⁺ (500 μM/0.05–20 μM), both vitamin E (1 μM) and ethylene glycol-bis(aminoethyl ether) tetraacetic acid (50 μM) efficiently inhibited PLPC oxidation. However, CLA (1–50 μM) did not show a clear protective effect under any of the conditions tested. We conclude that CLA, under these test conditions, does not act as an efficient radical scavenger in any way comparable to vitamin E or BHT. CLA also does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress, as had previously been suggested. On the basis of our observations, a role for CLA as an antioxidant does not seem plausible.     

Synthesis of copolymers of a linoleic acid derivative and properties of the copolymer films

Addition copolymers of maleic anhydride and a commercially available conjugated fatty acid, Pamolyn 380, were synthesized via thermal initiation. The copolymers had moderately high molecular weights and were obtained in reasonable yields. The copolymers were characterized by nuclear magnetic resonance and infrared spectroscopy and by elemental analysis. Based on this analytical data and examples from the literature, the copolymers were assigned regularly alternating chain morphology. Coatings were formulated with the copolymers, and films over a metal substrate were evaluated. The films were found to have excellent solvent resistance, high hardness, and good gloss. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 261–267, 2001     

Epoxidation of a conjugated linoleic acid isomer

Epoxidation of methyl (9Z, 11E)‐octadecadienoate ( 1 ) with various epoxidizing agents viz. m‐chloroperoxybenzoic acid, dimethyl dioxirane, methyltrioxorhenium/hydrogen peroxide, potassium peroxomonosulfate (Oxone, 2KHSO₅ · KHSO₄ · K₂SO₄)/tetrahydrothiopyran‐4‐one, and Novozyme 435/hydrogen peroxide is described. The reactions furnished the corresponding mono‐epoxy [methyl (11, 12E)‐epoxy‐(9Z)‐octadecenoate ( 2 ) and methyl (9, 10Z)‐epoxy‐(11E)‐octadecenoate ( 3 )] and a mixture of diastereomers of syn‐ and anti‐diepoxy‐stearate [methyl (9, 10Z;11, 12E)‐diepoxystearate ( 4a‐4d )], which were identified by a combination of spectroscopic and spectrometric analyses.     

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.     

Free radical polymerization and lipid binding of lysozyme reacted with peroxidizing linoleic acid

Insolubilization and polymerization of proteins exposed to peroxidizing lipids may be due either to cross-linking with incorporation of fragments of the lipid oxidation products, or to free radical transfer from lipid to protein and subsequent free radical polymerization of protein. The second mechanism which has been proposed was inferred from measurements of electron spin resonance signals in proteins. In this study, uniformly labeled linoleic acid, [¹⁴C(U)] LA, was reacted with lysozyme. Volatile oxidation products of LA were also used in some experiments. Incubation was done in the absence of water. Oligomers of lysozyme, as well as the monomer, were isolated after incubation, and the [¹⁴C] label incorporated into each fraction was determined. The results show that the dominant mechanism of protein polymerization after exposure to peroxidizing linoleic acid is the transfer of free radical from lipid to protein, and subsequent free radical polymerization.     

Buttonweed seed oil a source of linoleic acid

Summary Buttonweed seeds (Abutilon theophrasti) contain 15 to 17% oil. The oil contains about 58% linoleic acid which may be concentrated by simple fractionation into fractions containing as high as 83% of the acid. This suggests the possible use of the oil as a source of linoleic acid. The seed also has an appreciable sterol content.     

The linoleic acid content of seed fats and the isomerism of linoleic acid

The linoleic acid content of a series of seed fats was determined by the thiocyanometric and the tetrabromide-precipitation methods in a search for isomeric linoleic acids. The results indicated the presence of only one form of linoleic acid. The tetrabromide number was shown to be affected by the pronounced solubility of alpha tetrabromostearic acid in the other bromides and in the solid acids, its determination therefore being of only limited value. The thiocyanogen numbers of pure linoleic and linolenic acids were found to be empirical values differing markedly from the theoretical constants, requiring a revision of the accepted equations for the calculation of the per cent concentration of the unsaturated acids in oils and mixed fatty acids. Aided by grants from the National Livestock and Meat Board and the Graduate School of the University. Presented before the 13th convention of the American Oil Chemists’ Society in Chicago, October 6, 1939. Assistance in the preparation of these materials was furnished by the personnel of Work Projects Administration, Official Project No. 65-1-71-140, Sub-project No. 325.     

The performic acid oxidation of linoleic acid

The performic acid oxidation of linoleic acid has been shown to form 9,12-dihydroxy-trans-10,11-methylene-heptadecanoic acid (I) after hydrolysis of the formate ester. A sequence of reactions led to the identification of dimethyl-trans-1,2-cyclopropanedicarboxylate by gas liquid chromatography. Spectroscopic evidence is presented for thetrans geometry in I. Failure of the monoepoxide of linoleic acid to give the formate ester of I suggests the alternative that a homoallylic carbonium ion is formed directly upon attack of the peroxide reagent.