Isomers of leukotriene B4 possess different biological potencies

Rat elicited polymorphonuclear leucocytes (PMNs), when exposed to the ionophore A23187, release three isomers of leukotriene B4. The three isomers have been purified and tested for their ability to induce the chemokinesis of human PMNs in vitro, the aggregation of rat PMNs in vitro and changes in vascular permeability in rabbit skin in vivo in the presence of PGE2. The results demonstrate that all three isomers are biologically active and that the enzymatically produced isomer, in which the conjugated triene contains one cis and two trans double bonds, is more potent than the two diastereoisomers of LTB4 which contain all trans double bonds in the conjugated triene and which are produced by non-enzymatic hydrolysis.     

Syntheses and NMR Analyses of Eight Geometrical Isomers of 10,12,14-Hexadecatrienyl Acetate,Sex Pheromone Candidates of the Mulberry Pyralid

Eight geometrical isomers of 10,12,14-hexadecatrienyl acetate, female sex pheromone candidates of the mulberry pyralid (Glyphodes pyloalis Walker), were synthesized by six routes. Each route consisted of reactions by which two of three double bonds were introduced stereospecifically and another rather nonspecifically, giving mainly two geometrical isomers, which were analyzed by 2D-NMR after separating by reversed-phase HPLC or by a reaction with tetracyanoethylene. The signals of the olefinic protons and carbons of each geometrical isomer were assigned by COSY spectra and by C-H COSY spectra, respectively, in addition to the signals of the allylic protons and carbons, and its chemical structure was revealed by the values of their chemical shifts and coupling constants. Furthermore, a new empirical rule concerning the chemical shift changes of these carbons by converting the configuration of the conjugated triene system is suggested.     

Structural requirement for the action of leukotriene B4 on the guinea-pig lung:importance of double bond geometry in the 6, 8, 10-triene unit

The action of four synthetic 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acids has been compared to the action of natural leukotriene B₄ (LTB₄) in perfused guinea-pig lung and in the parenchymal strip preparations. Synthetic LTB₄ (Fig. 1) having the 6-$\text{cis}$, 8, 10-$\text{trans}$ triene unit was found to be as powerful as natural LTB₄ both for contracting the parenchymal strip and for releasing prostaglandins and thromboxanes from the perfused lung while three other isomers were inactive. The results indicate that the action of LTB₄ on the lung is highly dependent on the geometry of the conjugated triene.     

Triplet state spectra and dynamics of peridinin analogs having different extents of π-electron conjugation

The Peridinin-Chlorophyll a-Protein (PCP) complex has both an exceptionally efficient light-harvesting ability and a highly effective protective capacity against photodynamic reactions involving singlet oxygen. These functions can be attributed to presence of a substantial amount of the highly-substituted and complex carotenoid, peridinin, in the protein and the facts that the low-lying singlet states of peridinin are higher in energy than those of chlorophyll (Chl) a, but the lowest-lying triplet state of peridinin is below that of Chl a. Thus, singlet energy can be transferred from peridinin to Chl a, but the Chl a triplet state is quenched before it can sensitize the formation of singlet oxygen. The present investigation takes advantage of Chl a as an effective triplet state donor to peridinin and explores the triplet state spectra and dynamics of a systematic series of peridinin analogs having different numbers of conjugated carbon–carbon double bonds. The carotenoids investigated are peridinin, which has a C₃₇ carbon skeleton and eight conjugated carbon–carbon double bonds, and three synthetic analogs: C₃₃-peridinin, having two less double bonds than peridinin, C₃₅-peridinin which has one less double bond than peridinin, and C₃₉-peridinin which has one more double bond than peridinin. In this study, the behavior of the triplet state spectra and kinetics exhibited by these molecules has been investigated in polar and nonpolar solvents and reveals a substantial effect of both π-electron conjugated chain length and solvent environment on the spectral lineshapes. However, only a small dependence of these factors is observed on the kinetics of triplet energy transfer from Chl a and on carotenoid triplet state deactivation to the ground state.     

Enzymatic formation of 9,16-dihydro(pero)xyoctadecatrienoic acid isomers from alpha-linolenic acid

Incubation of alpha-linolenic acid with soybean lipoxygenase at pH 6.5 led to formation of conjugated triene oxidation products exhibiting maximum uv absorption at 267 nm, which were converted into four 9,16-dihydroxyoctadecatrienoic acid isomers. In the precursor-substrate study, it seems that 9,16-dihydroxy acid isomers are derived from the doubly oxygenated products and the epoxide intermediate, which are both produced from hydrogen removal at C-14 of 9(S)-hydroperoxyoctadecatrienoic acid. Optimum pH and Km values for soybean lipoxygenase-1-catalyzed conversion of 9(S)-hydroperoxyoctadecatrienoic acid into the conjugated triene products were 8.5 and 80 microM, respectively.     

Synthesis and Characterization of Hexadecadienyl Compounds with a Conjugated Diene System,Sex Pheromone of the Persimmon Fruit Moth and Related Compounds

Hexadecadien-1-ol and the derivatives (acetate and aldehyde) with a conjugated diene system have recently been identified from a pheromone gland extract of the persimmon fruit moth (Stathmopoda masinissa), a pest insect of persimmon fruits distributed in East Asia. The alcohol and acetate showed their base peaks at m/z 79 in a GC-MS analysis by electron impact ionization, but the aldehyde produced a unique base peak at m/z 84, suggesting a 4,6-diene structure. To confirm this inference, four geometrical isomers of each 4,6-hexadecadienyl compound were synthesized by two different routes in which one of two double bonds was furnished in a highly stereoselective manner. Separation of the two isomers synthesized together by each route was facilely accomplished by preparative HPLC. Their mass spectra coincided well with those of natural components, indicating that they were available for use as authentic standards for determining the configuration of the natural pheromone. Furthermore, other hexadecadienyl compounds, including the conjugated diene system between the 3- and 10-positions, were synthesized to accumulate the spectral data of pheromone candidates. 5,7-Hexadecadienal interestingly showed the base peak at m/z 80; meanwhile, the base peaks of its alcohol and acetate were detected at m/z 79 like the corresponding 4,6-dienes. The base peaks of all 6,8-, 7,9-, and 8,10-dienes universally appeared at m/z 67 like 9,11-, 10,12-, and 13,15-dienes, the spectra of which have already been published. Although 3,5-hexadecadienal was not prepared, base peaks at m/z 67 and 79 were recorded for the alcohol and acetate, respectively.     

Slow Absorption of Conjugated Linoleic Acid in Rat Intestines,and Similar Absorption Rates of 9c,11t-Conjugated Linoleic Acid and 10t,12c-Conjugated Linoleic Acid

We have previously shown that the 9c,11t-conjugated linoleic acid (CLA) concentration was always significantly higher than the 10t,12c-CLA concentration following the administration of these compounds to mice and rats, and considered that structural differences between the conjugated double bonds in these isomers affected absorption in the small intestine. This study investigates the absorption of CLA in the rat intestine by a lipid absorption assay of lymph from the thoracic duct. In Study 1, we used safflower oil and a triacylglycerol form of CLA (CLA-TG), while in Study 2, we used 9c,11t-CLA and 10t,12c-CLA. The cumulative recovery of CLA was lower than that of linoleic acid until two hours after sample administration. There was no difference in the extent of lymphatic recovery of 9c,11t-CLA and 10t,12c-CLA after the administration of CLA-TG, 9c,11t-CLA, and 10t,12c-CLA to the rats, suggesting that geometrical and positional isomerism of the conjugated double bonds did not influence the absorption.     

A facile E,Z-isomerization of α,β-unsaturated terpenoid lactones and its effect on plant growth activity

Various synthetic C-16 lactones prepared in earlier work are the Z-isomers. These have been isomerized chemically to the corresponding E-isomers. It is observed that these isomers have different root initiating properties, which reflect the significance of the geometry of double bonds in conjugated γ-lactones which act as plant growth regulators.     

Mass Spectrometry in Carbohydrate Chemistry

In this study, the physiological effects of fatty acids with conjugated double bonds were widely examined in vitro and in vivo. Initially, a method for determination of conjugated fatty acids in food and biological samples was established. I then clarified that the oxidative stability of conjugated fatty acids was improved by the form of triacylglycerol and addition of an antioxidant, and the influence of this effect on the metabolism and pharmacokinetics of conjugated fatty acids was clarified in vivo. In addition, antitumor, anti-angiogenesis, and antiobesity effects of conjugated fatty acids were found for the first time, thus demonstrating the usefulness of conjugated fatty acids. This communication mainly outlines the data obtained for conjugated linolenic acid. In addition, this review summarizes my research on conjugated fatty acid.     

A C-NMR study of 10,12-tricosadiynoic acid and the corresponding phospholipid and phospholipid polymer

Diacetylene phospholipids are presently being studied because of their potential to polymerise in vesicles, multilayers and natural biomembranes. ¹³C-NMR spectra and spin-lattice relaxation times have now been obtained of a diacetylene phospholipid present in a sonicated dispersion in water. Similar data have been obtained of a monoacetylene phospholipid and a saturated phospholipid. For further comparison the spectrum of a diacetylenic fatty acid in benzene-d₆ was also examined and relaxation data obtained. A comparison of the various relaxation data provides an indication of the restricted motion associated with the two conjugated triple bonds of the diacetylene phospholipid within the lipid bilayer structure. A proximity interaction between diacetylene groups occurs and a conformation for the diacetylene part of the lipid in the bilayer is deduced. The ¹³C-NMR spectrum of a soluble phospholipid polymer in C²HCl₃, obtained by ultraviolet irradiation of the diacetylene phospholipid, shows that the two conjugated triple bonds of the monomer is replaced in the polymer by an alternating double and triple bonded conjugated structure.     

RE‐EXAMINATION OF THE DOUBLE BOND POSITIONS IN ALKENONES AND DERIVATIVES: BIOSYNTHETIC IMPLICATIONS1

By derivatizing with OsO₄, we determined the double bond positions of all the diunsaturated alkenones present in different haptophytes and demonstrated that the positions in C₃₇–C₄₀ homologs occur at a fixed carbon number from the carbonyl group, a finding contrary to earlier speculation. These data have allowed us to recognize three “families” of alkenones, for which we propose different biosynthetic pathways. The proposed biosynthesis of classical C₃₇–C₄₀ alkenones starts with acetyl‐ or propionyl‐SCoA and involves classical chain elongation steps with malonyl‐ and methylmalonyl‐SCoA affording alkanoyl‐ACP intermediates, which undergo subsequent Δ^{14, 21}‐desaturation and decarboxylation reactions. Unusual diunsaturated shorter‐chain (C₃₅ and C₃₆) ketones now being produced for yet unknown physiological reasons by our specimen of Emiliania huxleyi (Lohm). Hay et Mohler strain CCMP1742. These compounds exhibit ω15,22 and ω16,23 double bonds, which are at a shorter distance (two carbon atoms) from the carbonyl group than the higher homologs, implying that their biosynthesis must involve an additional chain‐shortening step after Δ^{14, 21}‐desaturation of alkanoyl‐ACP. Alkenones exhibiting a very unusual double bond spacing (three methylene groups instead of five) were detected in Holocene Black Sea sediments, in particulate matter collected in the Ligurian Sea and in Gephyrocapsa oceanica strain JB‐02. The formation of these compounds seems to be linked to the biosynthesis of monounsaturated alkenones previously detected in several haptophytes. Our work demonstrates the value of determining the double bond locations of alkenones whenever possible.     

Metabolism of leukotriene B4 to dihydro and dihydro-oxo products by porcine leukocytes

Porcine leukocytes contain a novel pathway for the metabolism of leukotriene B4 (LTB4) which results in reduction of the conjugated triene chromophore to a conjugated diene. These cells converted LTB4 to two major metabolites, both of which exhibited maximal absorbance at 230 nm in their UV spectra. These products were purified by high pressure liquid chromatography and identified as 10, 11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 on the basis of the mass spectra of various derivatives. The position of the double bond of LTB4 which had been reduced was established by cleaving the remaining double bonds of 10, 11-dihydro-LTB4 with ozone followed by oxidation or reduction of the resulting ozonide and analysis of the products by mass spectrometry. Experiments with deuterium-labeled substrate indicated that LTB4 could be directly converted to 10, 11-dihydro-LTB4 without the prior oxidation of either of its hydroxyl groups, as is required for the formation of dihydro metabolites of prostaglandins. Incubation of porcine leukocytes with 10, 11-dihydro-LTB4 and 10, 11-dihydro-12-oxo-LTB4 indicated that these two products can be interconverted and are in equilibrium with one another. The dihydro-oxo metabolite can therefore be formed from 10, 11-dihydro-LTB4, although we have not ruled out the possibility that it is also produced via 12-oxo-LTB4, which could be a transitory intermediate. These results indicate that porcine leukocytes contain a novel reductase/dehydrogenase pathway distinct from the pathway responsible for the metabolism of prostaglandins. This pathway is also different from the pathway in human polymorphonuclear leukocytes which converts 6-trans-isomers of LTB4 to dihydro products, since the latter pathway involves 5-oxo intermediates and results in a shift in the positions of the remaining double bonds.     

Conjugated double bonds in lipid bilayers: a molecular dynamics simulation study

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is attributed to have the anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be responsible for the anti-obesity effects. Since dietary CLA are incorporated into membrane phospholipids, we have used Molecular Dynamics (MD) simulations to investigate the comparative effects of the two isomers on lipid bilayer structure. Specifically, simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed. Force field parameters for the torsional potential of double bonds were obtained from ab initio calculations. From the MD trajectories we calculated and compared structural properties of the two lipid bilayers, including areas per molecule, density profiles, thickness of bilayers, tilt angle of tail chains, order parameters profiles, radial distribution function (RDF) and lateral pressure profiles. The main differences found between bilayers of the two CLA isomers, are (1) the order parameter profile for C9T11 has a dip in the middle of sn-2 chain while the profile for T10C12 has a deeper dip close to terminal of sn-2 chain, and (2) the lateral pressure profiles show differences between the two isomers. Our simulation results reveal localized physical structural differences between bilayers of the two CLA isomers that may contribute to different biological effects through differential interactions with membrane proteins or cholesterol.     

Double dioxygenation of arachidonic acid by soybean lipoxygenase--1.

Soybean lipoxygenase-1, has been found to catalyze the incorporation of two oxygen molecules into arachidonic acid. The product appears to be 8,15 dihydroperoxy-5,9,11,13-eicosatetraenoic acid. This is apparently the first report of the enzymatic production of a conjugated aliphatic triene $\text{in vitro}$.     

Biodegradation of commercial linear alkyl benzenes byNocardia amarae

Laboratory degradation studies of two indigeneously produced linear alkyl benzenes byNocardia amarae MB-11 isolated from soil showed an overall degradation of linear alkyl benzenes isomers to the extent of 57–70%. Degradation of 2-phenyl isomers of linear alkyl benzenes was complete and faster than that of other phenyl position (C3–C7) isomers which were degraded to the extent of 40–72% only. Length of alkyl side chains (C₁₀–C₁₄) had little or no impact on the degradation pattern. Major metabolities detected were 2-, 3-and 4-phenyl butyric acids, phenyl acetic acid and cis, cis-muconic acid. Minor metabolites weretrans-cinnamic acid, 4-phenyl 3-butenoic acid and 3-phenyl pentanoic acid along with two unidentified hydroxy acids. On the basis of the formation pattern of these metabolities, three catabolic pathways of linear alkyl benzenes isomers inNocardia amarae MB-11 were postulated. All the phenyl position (C2–C7) isomers of C₁₀, C₁₂, and C₁₄ linear alkyl benzenes along with 3-phenyl and 5-phenyl isomers of C₁₁ and C₁₃ linear alkyl benzenes were degraded viacis,cis-muconic acid pathway. Other phenyl position isomers of C₁₁ and C₁₃ linear alkyl benzenes with phenyl substitution at even number carbon atoms were principally degraded via phenyl acetic acid pathway whiletrans-cinnamic acid formation provided a minor pathway     

Anti-Angiogenic Effects of Conjugated Docosahexaenoic Acid in Vitro and in Vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK,OK-432 or N-CWS

Peritoneal polymorponuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 µg)/head of OK-432 or 200 µg/head ofNocardia rubra cell wall skeleton (N-CWS). Withoutin vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in⁵¹Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 µg/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H₂O₂ production of PSK-induced PMNs was increased by thein vitro addition of 25 µg/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.     

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization

We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain α-alkyl-β-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M−H]⁻ ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing α-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths of the meromycolate chain and the α-branch. This study revealed that the mycolic acids isolated from pathogenic Rhodococcus equi 103 contained a series of homologous ions having C₃₀ to C₅₀ chain with 0–2 double bonds. The α-branch ranged from C₁₀ to C₁₈ with 0 to 1 double bond, in which 16:0 and 14:0 are the most prominent, whereas the meromycolate chain ranged from C₁₄ to C₃₄ with 0 to 2 double bonds. The major molecular species consisted of more than 3 isomers that differ by the lengths of the α-branch or meromycolate chain, and up to 10 isobaric isomers were identified for some minor ions. We also employed tandem quadrupole mass spectrometry with precursor ion and neutral loss scans for profiling mycolic acid with specific structure in mixtures. The tandem spectra obtained from precursor ion scans of m/z 255 (16:0-carboxylate anion) and m/z 227 (14:0-carboxylate anion) may provide a simple specific means for classification of Rhodococci species, whereas tandem spectra from neutral loss of meroaldehyde residue scans provided a simple approach to reveal the mycolic acid molecules with specific meromycolate chain in mixtures.     

cis–trans isomerization of carbon double bonds in monounsaturated triacylglycerols via generation of free radicals

We investigated the heat-induced cis/trans isomerization of double bonds in monounsaturated lipids. When triolein (9-cis, 18:1) was heated around 180 °C, small amounts of isomerization products were obtained depending on the heating period. The heat-induced isomerization of triolein was considerably suppressed by the addition of different antioxidants or under nitrogen stream, and these additives simultaneously inhibited the thermal oxidation of double bonds in triolein. Therefore, an intermediate of the thermal oxidation reaction might be responsible for the heat-induced isomerization of the double bonds in triolein. The thermodynamics of the heat-induced isomerization of triolein (9-cis, 18:1) and trielaidin (9-trans, 18:1) were investigated using Arrhenius plot. The Arrhenius activation energies of cis double bonds in triolein and trans double bonds in trielaidin were 106 kJ/mol and 137 kJ/mol, respectively. The calculated internal rotational barrier heights of these double bonds were similar to those of the double bond of 2-butene radical and significantly lower than those of non-radicalized double bonds in 2-butene. These results suggest that heat-induced cis/trans isomerization of triolein and trielaidin occurs mainly through the formation of radical species, which are the intermediates produced during thermal oxidation. The activation energy difference between the two forms suggests that trans trielaidin radicals are more stable than cis triolein radicals. The high thermodynamic stability of the trans double bonds in lipid radicals would influence the population of cis and trans isomers in edible oils and contribute to slight accumulation of trans-18:1 isomers during heating or industrial processing.     

Novel leukotrienes: Products formed by initial oxygenation of arachidonic acid at C-15

A preparation of human leukocytes was incubated with arachidonic acid. Two new dihydroxy acids with conjugated triene structures, were isolated and characterized as 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-leukotriene B₄) and 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene B₄).