Fluorescence spectroscopy of 9-anthroyloxy fatty acids in solvents

A series of anthroyloxy fatty acid (AF) fluorescent probes, with the anthroyloxy group covalently linked at various positions along the alkyl chain, were studied in solvents exhibiting a wide range of polarity and hydrogen-bond donor (H_d) and acceptor (H_a) ability. These probes were sensitive to the solvent polarity as reflected by the Stokes' shift observed in steady state fluorescence. As determined by multi-linear regression analysis of the observed Stokes' shift and solvent parameters, such as orientation polarizability (Δf), H_d and H_a of the solvents, all the probes were sensitive to the H_d of solvents but were not affected by the H_a of solvents except the 2-AF. Due to the proximity of the polar headgroup to the fluorophore, it appears that some intramolecular hydrogen-bonding is present in 2-AF, an interaction that is sensitive to the pH of the solvent, but is less sensitive to the H_d and H_a of the solvents. Fluorescence lifetimes measured by the multi-frequency phase-modulation technique in mixtures of hexane and ethanol reflect a modified Stern-Volmer behavior suggesting the second solvent, ethanol, specifically interacts with the probe, in part through collisional quenching. Also, the lifetime data were sensitive to very low concentrations of the second solvent (0–0.1%, by vol.). The results from this study provide insight into the intrinsic differences between the different AF positions that must be taken into consideration while investigating the dynamics of lipid bilayer systems. Moreover, this study illustrates the utility and resolving power of lifetime based measurements needed for the interpretation of heterogeneous biophysical environments.     

Localization of cyanine dye binding to brush-border membranes by quenching of n-(9-anthroyloxy) fatty acid probes

The location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush-border membrane vesicles was examined from the quenching of n-(9-anthroyloxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 greater than 12 much much greater than 6 greater than 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90 degrees in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-15 s.     

The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers

A set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12) have been used to examine gradients in fluorescence polarization, lifetime (tau F), relative quantum yield (phi rel) and positions of emission maxima (lambda max) through bilayers composed of synthetic phospholipids. The fluorophores of these probes report the environment at a graded series of depths from the surface to the centre of the bilayer structure. 1. Polarizations decrease as the fluorophore is moved deeper into the bilayer indicating greater rotational motion of the fluorophore in the hydrocarbon core of the bilayer. 2. The different responses of the probe diphenylhexatriene and the anthroyloxy fatty acids to the action of cholesterol on lipid bilayers are discussed in terms of the orientation of these probes in the bilayer and the types of anisotropic rotational motions which result in depolarization of fluorescence. 3. Stearic acid derivatives which have the fluorophore in the 6-, 9- and 12-positions along the acyl chain have a similar response to solvent polarity as measured by values of lambda max and phi rel in a variety of organic solvents. 4. The position of the emission maximum has little dependence on solvent viscosity, but viscosity does change the degree of vibrational structure seen in the emission spectrum. The vibrational structure itself may be used as an indication of the 'mciroviscosity' gradient in the transverse plane of the bilayer. 5. Values of lambda max, tau F and phi rel indicate that a gradient of polarity exists from the surface to the centre of the bilayer. For dipalmitoyl phosphatidylcholine in the crystalline phase, cholesterol acts to make this polarity gradient shallower.     

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.     

Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of (n-9) and (n-7) cis-monounsaturated fatty acids in seeds of higher plants

The relative abundance of (n-9) and (n-7) isomers in the monounsaturated fatty acids of seed lipids has been determined for selected plants in order to assess the biosynthetic reactions involved in their formation. 9 Desaturation of stearic acid to (n-9) octadecenoic acid is almost exclusively operative in the formation of monounsaturated fatty acids in the seeds of Helianthus annuus, Glycine max and Brassica napus, cv. Quinta and Erglu, in which chain elongation of monounsaturated fatty acids terminates at the level of an 18 carbon chain. 9 Desaturation of palmitic acid is a minor yet significant pathway in the seeds of Sinapis alba and Brassica napus, cv. Rapol and Tira, in which chain elongation of monounsaturated fatty acids occurs extensively beyond the 18 carbon chain. In each of these seeds, both (n-9) and (n-7) octadecenoic acids formed are subsequently elongated to icosenoic acids. However, elongation of the (n-7) isomer is terminated at the level of a 20 carbon chain, whereas the (n-9) icosenoic acid is selectively elongated to docosenoic acid and even up to tetracosenoic acid in Sinapis alba. 9 Desaturation of palmitic acid followed by elongation to (n-7) octadecenoic acid occurs to a minor extent in the seeds of Tropaeolum majus. Only the (n-9) octadecenoic acid, and not its (n-7) isomer, is elongated to icosenoic and docosenoic acids.     

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.     

Interaction of differently oriented lipids in monolayer: mixed monolayers of 16-(9-anthroyloxy)palmitic acid with phosphatidylcholine and cholesterol

16-(9-Anthroyloxy)palmitic acid (16-AP) is a bifunctional molecule with carboxyl and 9-anthroyloxy groups attached at both ends of the hydrocarbon chain. At the air-water interface, in a monolayer, the 16-AP molecule has horizontal and vertical orientations, depending on the surface pressure of the monolayer. The miscibilities of 16-AP with dimyristoylphosphatidylcholine (DMPC), cholesterol (CH), and fatty acids in mixed monolayers were evaluated in investigations of monolayer phase transitions. Lipid molecules with flexible hydrocarbon chains, i.e., DMPC and fatty acids, formed homogeneous mixed monolayers with horizontally oriented 16-AP. On the other hand, the rigid molecule, CH, could not accommodate the horizontally oriented 16-AP in a monolayer, and there was a phase separation from 16-AP. In biological and reconstituted membranes, preferential binding of phospholipid to the integral protein and exclusion of cholesterol in close vicinity of the membrane protein have been recognized. On the basis of this work, it can be expected that flexible lipids readily accommodate the rough hydrophobic surface of integral proteins and stabilize the structure of the protein, while rigid lipids such as cholesterol are removed from the immediate environment of the membrane protein, if the protein does not interact specifically with the rigid lipids.     

n-3 and n-6 polyunsaturated fatty acids have different effects on acyl-CoA:cholesterol acyltransferase in J774 macrophages.

The effects of incubating J774 mouse macrophages with different fatty acids on cholesterol esterification were investigated. In cells incubated with n-3 polyunsaturated fatty acids, the rate of cholesterol esterification was significantly reduced compared with cells incubated with n-6 polyunsaturated fatty acids or with oleic acid. This change in cholesterol esterification appears to be the result of reductions in the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum of the macrophages incubated with the n-3 polyunsaturated fatty acids. No differences in microsomal cholesterol were observed among cells incubated with different fatty acids. However, cellular cholesterol levels were lower in cells incubated with n-3 polyunsaturated fatty acids. In microsomes from cells incubated with n-3 polyunsaturated fatty acids, both the Km and the Vmax of ACAT were lower than in microsomes from cells incubated with n-6 fatty acids or oleic acid. These findings may explain some of the reduction in atherosclerotic lesions that are observed with dietary fish oils that contain high levels of n-3 polyunsaturated fatty acids.     

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.     

An assessment of the fluidity gradient of the lipid bilayer as determined by a set of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12, 16).

The rotational behavior of a set of n-(9-anthroyloxy) fatty acid fluorescent probes is examined in two liquid paraffins and in liposomes composed of dipalmitoyl phosphatidylcholine. As has been observed with other membrane fluorescent probes (Hare, F., and Lussan, C. (1977) Biochim. Biophys. Acta 467, 262-272), the degree of fluorescence depolarization for a given solvent viscosity is dependent on the solvent standard employed. In addition, when the anthroyloxy group is in the terminal position of the acyl chain, it has more rotational freedom than when it is conjugated to positions 6, 9, or 12 where the rotational motion of the fluorophore is similar. When incorporated into lipid bilayers, values of fluorescence polarization reflect the gradient of "fluidity" which extends from the surface to the center of the membrane. The nature of this polarization gradient is discussed in relation to the intrinsic differences between the probes and the anisotropic rotations responsible for depolarization.     

A comment on the localization of cyanine dye binding to brush-border membranes by the fluorescence quenching of n-(9-anthroyloxy) fatty acid probes

This work comments on the location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) in renal brush-border membrane vesicles (RBBMV) (Cabrini, G. and Verkman, A.S. (1986) Biochim. Biophys. Acta 862, 285-293) evaluated from collisional quenching of n-(9-anthroyloxy)stearic acid (n-AS) fluorescence. At variance with these authors, it is concluded that the quenching is due to resonance energy transfer. It is also shown that the fluorescence data are not clear evidence for the reported monomer and dimer locations.     

Steady-state fluorescence polarization in planar lipid membranes. Experimental and theoretical analysis of the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-Diphenyl-1,3,5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids

In continuation of earlier work, the steady-state fluorescence polarization in a globally oriented system of planar lipid membranes was analyzed experimentally and theoretically for the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-diphenyl-1,3, 5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids. The theoretical analyses of experiments were mainly done in terms of the mean orientation of transition moments with respect to the membrane normal, an angle describing the region of hindered rotational diffusion and the coefficients of rotational diffusion perpendicular to the membrane and around the membrane normal. The nonvanishing angle between the moments of absorption and emission was taken into account. In the case of n-(9-anthroyloxy) fatty acids it was found that the orientational disorder increases significantly with the depth of the fluorophore within the membrane. In order to compare with recent results from time-dependent fluorescent polarization in globally isotropic membrane suspensions and with 2H-NMR experiments, the second moment ('order parameter') of the steady-state orientational distribution of absorption dipoles was calculated. For all fluorophores the theoretical analysis indicates a preferred orientation of absorption moments within the membrane plane.     

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.     

(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

Metabolism of polyunsaturated (n-3) fatty acids by monkey seminal vesicles: isolation and biosynthesis of omega-3 epoxides.

Monooxygenases of monkey seminal vesicles can metabolize arachidonic acid (20:4(n-6)) by w3-hydroxylation to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and eicosapentaenoic acid (20:5(n-3)) to 17,18-dihydroxyeicosatetraenoic acid (Oliw, E.H. (1989) J. Biol. Chem. 264, 17845-17853). The present study aimed to further characterize the oxygenation of (n-3) polyunsaturated fatty acids. 14C-Labelled 22:6(n-3), 20:5(n-3), 20:4-(n-3) and 18:3(n-3) were incubated with microsomes of seminal vesicles of the cynomolgus monkey, NADPH and a cyclooxygenase inhibitor, diclofenac, and the main metabolites were identified by capillary gas chromatography-mass spectrometry. 22:6(n-3) was slowly metabolized to 19,20-dihydroxy-4,7,10,13,16-docosapentaenoic acid, while 20:5(n-3), 20:4(n-3) and 18:3(n-3) were metabolized more efficiently to the corresponding w4,w3-diols. The w3 epoxides, which were obtained from 20:5(n-3) and 18:3(n-3), were isolated in the presence of an epoxide hydrolase inhibitor, 1(2)epoxy-3,3,3-trichloropropane, and the geometry of the epoxides was determined to be 17S, 18R and 15S, 16R, respectively. While 20:5(n-3) was metabolized almost exclusively to the epoxide and diol pair of metabolites, 18:3(n-3) was metabolized not only to the w3 epoxide and the corresponding diol, but also to the w2 alcohol, 17(R)-hydroxy-9,12,15-octadecatrienoic acid. 22:6(n-3) and 5,8,11,14-eicosatetraynoic acid inhibited the biosynthesis of 18(R)-HETE from arachidonic acid (IC50 0.16 and 0.14 mM, respectively). In comparison with 20:4 or 18:3(n-3), 18:1(n-9) and 22:5(n-6) appeared to be slowly metabolized by seminal monooxygenases, while 18:2(n-6) was converted to the w3 alcohol and to smaller amounts of the w2 alcohol (4:1). Together, the results indicate that the w3-hydroxylase and w3-epoxygenase enzyme(s) metabolize 20:4(n-6) and 20:5(n-3) almost exclusively to the w3(R) alcohol and the w3(R, S) epoxide, respectively, while longer and shorter fatty acids either are poor substrates or metabolized with a lesser degree of position specificity.     

Short-term effect of dietary cholesterol on tissue n-6 fatty acids in fat-deficient rats

Weanling female rats raised on a fat-free diet for 8 weeks were then given the same diet supplemented with 0, 0.25, 0.5, or 1% by weight of cholesterol in addition to 10% of safflower oil for 3 days. Fatty acid compositions of cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) in liver and plasma were examined. Cholesterol feeding increased plasma and liver cholesterol contents and also affected the patterns of n-6 polyunsaturated fatty acids. There were no consistent changes in either plasma and liver TG which contained little 20:3n-6 and 20:4n-6. The levels of 20:3n-6 increased in plasma and liver PL, while proportions of 20:4n-6 decreased in liver and plasma CE. However, the absolute amount of 20:4n-6 in cholesteryl esters increased because of a threefold rise in cholesteryl ester levels. The changes might be attributable to an increased utilization of 20:4n-6 for cholesterol transport and/or an inhibition of delta 5-desaturation of n-6 fatty acids by cholesterol feeding.