Different Fatty Acids Compete with Arachidonic Acid for Binding to the Allosteric or Catalytic Subunits of Cyclooxygenases to Regulate Prostanoid Synthesis.

Prostaglandin endoperoxide H synthases (PGHSs), also called cyclooxygenases (COXs), convert arachidonic acid (AA) to PGH2. PGHS-1 and PGHS-2 are conformational heterodimers, each composed of an (Eallo) and a catalytic (Ecat) monomer. Previous studies suggested that the binding to Eallo of saturated or monounsaturated fatty acids (FAs) that are not COX substrates differentially regulate PGHS-1 versus PGHS-2. Here, we substantiate and expand this concept to include polyunsaturated FAs known to modulate COX activities. Non-substrate FAs like palmitic acid bind Eallo of PGHSs stimulating human (hu) PGHS-2 but inhibiting huPGHS-1. We find the maximal effects of non-substrate FAs on both huPGHSs occurring at the same physiologically relevant FA/AA ratio of ∼20. This inverse allosteric regulation likely underlies the ability of PGHS-2 to operate at low AA concentrations, when PGHS-1 is effectively latent. Unlike FAs tested previously, we observe that C-22 FAs, including ω-3 fish oil FAs, have higher affinities for Ecat than Eallo subunits of PGHSs. Curiously, C-20 ω-3 eicosapentaenoate preferentially binds Ecat of huPGHS-1 but Eallo of huPGHS-2. PGE2 production decreases 50% when fish oil consumption produces tissue EPA/AA ratios of ≥0.2. However, 50% inhibition of huPGHS-1 itself is only seen with ω-3 FA/AA ratios of ≥5.0. This suggests that fish oil-enriched diets disfavor AA oxygenation by altering the composition of the FA pool in which PGHS-1 functions. The distinctive binding specificities of PGHS subunits permit different combinations of non-esterified FAs, which can be manipulated dietarily, to regulate AA binding to Eallo and/or Ecat thereby controlling COX activities.

Major urinary metabolites of 6-keto-prostaglandin F2α in mice.

Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF2α urinary metabolites included dinor-6-keto-PGF2α (~10%) and dinor-13,14-dihydro-6,15-diketo-PGF1α (~10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI3 by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases.

Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids.

Recombinant human prostaglandin endoperoxide H synthase-1 (huPGHS-1) was characterized. huPGHS-1 has a single high-affinity heme binding site per dimer and exhibits maximal cyclooxygenase (COX) activity with one heme per dimer. Thus, huPGHS-1 functions as a conformational heterodimer having a catalytic monomer (E(cat)) with a bound heme and an allosteric monomer (E(allo)) lacking heme. The enzyme is modestly inhibited by common FAs including palmitic, stearic, and oleic acids that are not COX substrates. Studies of arachidonic acid (AA) substrate turnover at high enzyme-to-substrate ratios indicate that nonsubstrate FAs bind the COX site of E(allo) to modulate the properties of E(cat). Nonsubstrate FAs slightly inhibit huPGHS-1 but stimulate huPGHS-2, thereby augmenting AA oxygenation by PGHS-2 relative to PGHS-1. Nonsubstrate FAs potentiate the inhibition of huPGHS-1 activity by time-dependent COX inhibitors, including aspirin, all of which bind E(cat). Surprisingly, preincubating huPGHS-1 with nonsubstrate FAs in combination with ibuprofen, which by itself is a time-independent inhibitor, causes a short-lived, time-dependent inhibition of huPGHS-1. Thus, in general, having a FA bound to E(allo) stabilizes time-dependently inhibited conformations of E(cat). We speculate that having an FA bound to E(allo) also stabilizes E(cat) conformers during catalysis, enabling half of sites of COX activity.

Fluorescent n-3 and n-6 very long chain polyunsaturated fatty acids: three-photon imaging in living cells expressing liver fatty acid-binding protein.

Despite the considerable beneficial effects of n-3 and n-6 very long chain polyunsaturated fatty acids (VLC-PUFAs), very little is known about the factors that regulate their uptake and intracellular distribution in living cells. This issue was addressed in cells expressing liver-type fatty acid-binding protein (L-FABP) by real time multiphoton laser scanning microscopy of novel fluorescent VLC-PUFAs containing a conjugated tetraene fluorophore near the carboxyl group and natural methylene-interrupted n-3 or n-6 grouping. The fluorescent VLC-PUFAs mimicked many properties of their native nonfluorescent counterparts, including uptake, distribution, and metabolism in living cells. The unesterified fluorescent VLC-PUFAs distributed either equally in nuclei versus cytoplasm (22-carbon n-3 VLC-PUFA) or preferentially to cytoplasm (20-carbon n-3 and n-6 VLC-PUFAs). L-FABP bound fluorescent VLC-PUFA with affinity and specificity similar to their nonfluorescent natural counterparts. Regarding n-3 and n-6 VLC-PUFA, L-FABP expression enhanced uptake into the cell and cytoplasm, selectively altered the pattern of fluorescent n-6 and n-3 VLC-PUFA distribution in cytoplasm versus nuclei, and preferentially distributed fluorescent VLC-PUFA into nucleoplasm versus nuclear envelope, especially for the 22-carbon n-3 VLC-PUFA, correlating with its high binding by L-FABP. Multiphoton laser scanning microscopy data showed for the first time VLC-PUFA in nuclei of living cells and suggested a model, whereby L-FABP facilitated VLC-PUFA targeting to nuclei by enhancing VLC-PUFA uptake and distribution into the cytoplasm and nucleoplasm.

Chemical C2-elongation of polyunsaturated fatty acids.

Three fatty acids were synthesized from commercially available alpha-linolenic, stearidonic and eicosapentaenoic acids by C2-elongation using a four step preparative technique. The parent fatty acid methyl esters were reduced to alcohols with LiAlH(4), converted to bromides by treatment with triphenylphosphine dibromide, coupled with a lithiated C2-elongation block--2,4,4-trimethyl-2-oxazoline--to form the corresponding 2,2-dimethyloxazolines of C2-elongated fatty acids, and finally, converted to the target polyunsaturated fatty acids by acidic alcoholysis. Yields of more than 60% were achieved on a gram scale. The resulting 11Z,14Z,17Z-eicosatrienoic, 8Z,11Z,14Z,17Z-eicosatetraenoic and 7Z,10Z,13Z,16Z,19Z-docosapentaenoic acids were obtained as colorless oils with >98% purity and could be used for biochemical investigations without additional purification. The elongated fatty acids were free of byproducts that could result from Z-E isomerization or migration of double bonds.

Synthesis of four isomers of parinaric acid.

A simple and reliable method for synthesizing four isomers of parinaric acid from alpha-linolenic acid (ALA) in high yields is described. The methylene-interrupted, cis triene system (1,4,7-octatriene) of ALA and common to other naturally occurring polyunsaturated fatty acids was transformed to a conjugated tetraene system (1,3,5,7-octatetraene). The synthesis involves bromination of ALA using 0.l M Br(2) in a saturated solution of NaBr in methanol, esterification of the fatty acid dibromides, double dehydrobromination by 1,8-diazabicyclo[5.4.0]undec-7-ene and saponification of the conjugated esters to a mixture of free conjugated acids. Addition of one molecule of bromine to the 12,13-double bond of ALA and subsequent dehydrobromination produces alpha-parinaric acid (9Z,11E,13E,15Z-octadecatetraenoic acid); addition of Br(2) to the 9,10-double bond or 15,16-double bond and then dehydrobromination and rearrangement yields 9E,11E,13E,15Z-octadecatetraenoic or 9E,11E,13E,15Z-octadecatetraenoic acids, respectively. The mixture of parinaric acid isomers is obtained in 65% yield, and the isomers can be purified by preparative HPLC; alternatively, the isomers can be converted by base catalyzed cis-trans isomerization (or by treatment with I(2)) to exclusively beta-parinaric acid (9E,11E,13E,15E-octadecatetraenoic acid). The various parinaric acid isomers were characterized by (1)H NMR, (13)C NMR, UV, GLC, HPLC and mass spectrometry.

Synthesis of long chain n-3 and n-6 fatty acids having a photoactive conjugated tetraene group.

Fatty acids of the n-3 and n-6 families containing a photoactive conjugated tetraene group near the carboxylate were prepared from several naturally occurring fatty acids by sequential iodolactonization and treatment with excess 1,8-diazabicyclo[5.4.0]undec-7-ene. The new conjugated fatty acids include 5E,7E,9E,11Z,14Z- and 5E,7E,9E,11E,14Z-eicosapentaenoic acids derived from arachidonic acid; 5E,7E,9E,11Z,14Z,17Z- and 5E,7E,9E,11E,14Z,17Z-eicosahexaenoic acids from eicosapentaenoic acid; and 4E,6E,8E,10Z,13Z,16Z,19Z- and 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acids from docosahexaenoic acid. All of the newly synthesized fatty acids were characterized by UV, 1H NMR and mass spectroscopy. These new products represent the first examples of directed conjugation of methylene interrupted double bond systems. The products can be synthesized in gram quantities and in high yields (>50%). Interestingly, it did not prove possible to synthesize fatty acids having a triene group near the carboxyl group even using mild conditions and different synthetic approaches. Once initiated, the isomerization always continued until a tetraene group was formed. Because of the sensitivity of the tetraene group to light, these fatty acids have the potential for being used in tracking fatty acid movements in cells employing fluorescence techniques and in UV light-induced cross linking to membrane proteins.

Epoxy acetylenic lipids: their analogues and derivatives.

Currently, approximately 250 natural acetylenic epoxy structures are known. The present review describes research concerning biologically active epoxy acetylenic lipids and related compounds isolated from different sources. Intensive searches for new classes of pharmacologically potent agents produced by living organisms have resulted in the discovery of dozens of such compounds that possess high anticancer, cytotoxic, antibacterial, antiviral, and other activities. Acetylenic epoxides primarily belong to a class of molecules containing triple bond(s) and epoxy group(s) belonging to different lipid classes and/or other groups. This review emphasises natural and synthetic acetylenic epoxides and other related compounds as important sources of leads for drug discovery. The present review is the first article devoted to natural acetylenic epoxides.

Bioactive acetylenic metabolites.

This article focuses on anticancer, and other biological activities of acetylenic metabolites obtained from plants and fungi. Acetylenic compounds belong to a class of molecules containing triple bond(s). Naturally occurring acetylenics are of particular interest since many of them display important biological activities and possess antitumor, antibacterial, antimicrobial, antifungal, and immunosuppressive properties. There are of great interest for medicine, pharmacology, medicinal chemistry, and pharmaceutical industries. This review presents structures and describes cytotoxic activities of more than 100 acetylenic metabolites, including fatty alcohols, ketones, and acids, acetylenic cyclohexanoids, spiroketal enol ethers, and carotenoids isolated from fungi and plants.

Cyclooxygenase Allosterism, Fatty Acid-mediated Cross-talk between Monomers of Cyclooxygenase Homodimers.

Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2, also known as cyclooxygenases (COXs), catalyze the oxygenation of arachidonic acid (AA) in the committed step in prostaglandin (PG) biosynthesis. PGHSs are homodimers that display half of sites COX activity with AA; thus, PGHSs function as conformational heterodimers. Here we show that, during catalysis, fatty acids (FAs) are bound at both COX sites of a PGHS-2 dimer. Initially, an FA binds with high affinity to one COX site of an unoccupied homodimer. This monomer becomes an allosteric monomer, and it causes the partner monomer to become the catalytic monomer that oxygenates AA. A variety of FAs can bind with high affinity to the COX site of the monomer that becomes the allosteric monomer. Importantly, the efficiency of AA oxygenation is determined by the nature of the FA bound to the allosteric monomer. When tested with low concentrations of saturated and monounsaturated FAs (e.g. oleic acid), the rates of AA oxygenation are typically 1.5-2 times higher with PGHS-2 than with PGHS-1. These different kinetic behaviors of PGHSs may account for the ability of PGHS-2 but not PGHS-1 to efficiently oxygenate AA in intact cells when AA is a small fraction of the FA pool such as during "late phase" PG synthesis.

A procedure for preparing oxazolines of highly unsaturated fatty acids to determine double bond positions by mass spectrometry.

A convenient, mild, reliable method has been developed for preparing oxazolines of fatty acids and for using these derivatives to determine double bond locations in long-chain polyunsaturated and polyconjugated fatty acids. Fatty acyl mixed anhydrides are prepared using isobutylchloroformate and then converted to their ethanolamides by treatment with ethanolamine. Ethanolamides are subsequently cyclized to the corresponding oxazolines in >or=85% yields by treatment with trifluoroacetic anhydride under mild conditions (>50 degrees for 30-60 min). This general protocol can also be used to synthesize 4,4-dimethyloxazoline and benzoxazole derivatives of fatty acids. Gas chromatography-mass spectrometry of oxazoline derivatives of fatty acids yields prominent ions diagnostic of the structures of the parent fatty acids and, in the case of unsaturated fatty acids, indicating the positions of the double bonds. The utility of the method is illustrated with several fatty acids, including the conjugated 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acid.