Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs ( )

Two fractions with prostaglandin E-like activity were isolated from onion ( ) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 μg of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E₂.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

Direct cardiovascular actions of two metabolites of linoleic acid

Two newly discovered oxidation products of linoleic acid (i.e., 9,10-epoxy-12-octadecenoate termed Leukotoxin A, and 12,13-epoxy-9-octadecenoate termed Leukotoxin B) are produced by neutrophils in a variety of species. These substances appear to combat bacterial infection although they also have detrimental effects on normal organ function. Administration of Leukotoxin A or B to isolated cat papillary muscles decreased developed force, an index of myocardial contractility, in a concentration-dependent manner. Leukotoxin B was more active in decreasing the developed force than Leukotoxin A at high concentrations. Leukotoxin A or B, when added to isolated perfused cat carotid arteries, produced a significant vasoconstriction which in vivo would result in an increased vascular resistance. Thus, leukotoxins exert significant direct effects on the cardiovascular system in cats. Leukotoxins A and B are both cardiodepressant and vasoactive independent of release of other blood borne mediators.     

Stimulation of progesterone and prostaglandin E2 production by lipoxygenase metabolites of arachidonic acid

The role of several lipoxygenase metabolites of arachidonic acid in the action of luteinizing hormone-releasing hormone (LHRH) on ovarian hormone production was investigated. Like LHRH, treatment of rat granulosa cells with 5-HETE, 5-HPETE, 12-HETE, 15-HETE or 15-HPETE stimulated progesterone (P) and prostaglandin E2 (PGE2) production. 12-HEPE was most potent and stimulated P and PGE2 equally well. By contrast, 5-HETE stimulated P better than PGE2, while 15-HETE was a potent stimulator of PGE2 but not of P. Stimulation of P and PGE2 by LHRH or 12-O-tetradecanoylphorbol 13-acetate (TPA) was further augmented by several HETEs and HPETEs. Like protein kinase C, arachidonic acid metabolites appear to mediate the multiple actions of LHRH in the ovary.     

Mass spectral characterization of a protein-nucleic acid photocrosslink

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.     

Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion ($\text{Allium cepa}$) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 μg of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E₂.     

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.     

Spin-state exchange in fusarium lipoxygenase on binding of linoleic acid.

The electron paramagnetic resonance(EPR) signals of Fusarium lipoxygenase were measured at liquid nitrogen temperature in the presence or absence of substrate, linoleic acid. The spin-state exchange of heme iron in Fusarium lipoxygenase from a low to high spin-state by the addition of linoleic acid was observed. The addition of linoleic acid to the enzyme at pH 9.0 gave rise to the appearance of EPR lines at g=5.92 and 3.58, while at pH 12.0, lines at g=6.12 and 3.41 were newly appeared. At the same time, the resonance at g=4.31 was increased both at pH 9.0 and 12.0 in the presence of linoleic acid.     

Mass spectral characterization of 6-oxo-OGF

The mass spectra of eleven derivatives are presented to provide structural support for the recently discovered prostaglandin, 6-oxo-PGF1alpha, which we have isolated from incubations of arachidonic acid with ram seminal vesicles or released during isolated perfusions of sensitized guinea pig lungs.     

Metabolic profile of linoleic acid in porcine leukocytes through the lipoxygenase pathway

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.     

Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B4, C4, D4 and E4 and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone resorption in vitro. Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E2 (PGE2) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE and 12-HETE. These results indicate that lipoxygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.     

Preparation of oxygen-18-labeled lipoxygenase metabolites of arachidonic acid

Plasma pseudocholinesterase and porcine liver esterase were used to catalyse the incorporation of the stable isotope oxygen-18 into the carboxyl moiety of lipoxygenase metabolites of arachidonic acid. This simple method produces eicosanoid products containing two oxygen-18 atoms; but the enzymes studied were found to display large substrate specificity in the efficiencies at which oxygen-18 could be incorporated into the lipoxygenase metabolites. Furthermore, [18O2]LTB4 was found not to back exchange during in vitro incubation with human neutrophils. The methods involved for stable isotope incorporation are simple, efficient and produce highly enriched species in a short time. By varying the type of esterase, the amount of esterase or the length of incubation highly enriched species of all eicosanoids tested could be prepared.     

Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B₄, C₄, D₄ and E₄ and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone respiration . Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E₂ (PGE₂) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE nd 12-HETE. These results indicate that liposygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.     

Specificity of the effect of lipoxygenase metabolites of arachidonic acid on calcium homeostasis in neutrophils. Correlation with functional activity

The ability of the major neutrophil-derived lipoxygenase metabolites of arachidonic acid to increase the rate of 45Ca influx in rabbit neutrophils was examined. The results obtained demonstrate that (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid (leukotriene B4) is the most active of the arachidonic acid metabolites. The activity of leukotriene B4 is highly stereospecific in that its three nonenzymatically derived isomers are essentially inactive. The omega-hydroxylation of leukotriene B4 results in a compound that is nearly as active as leukotriene B4 as far as its ability to stimulate calcium influx and neutrophil aggregation while being a much weaker secretagogue. The further conversion of leukotriene B4 into a dicarboxylic acid removes all detectable biological activity. 5,6-Oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) methyl ester was also found to increase the rate of calcium influx, while the degradation products of native leukotriene A4 were essentially inactive. These results demonstrate that a close correlation exists between the ability of the various lipoxygenase products to alter calcium homeostasis in rabbit neutrophils and their biological activities.     

Hydroperoxide isomers and ketohydroxy product from oxidation of linoleic acid by eggplant lipoxygenase

Hydroperoxides produced by oxidation of linoleic acid with purified eggplant lipoxygenase were separated by TLC and analysed by IR spectroscopy. The methyl hydroxystearates from the enzymatically produced hydroperoxides were analysed by MS and GLC. Both analyses indicated that the eggplant enzyme converted linoleic acid almost exclusively (96%) into the 13-hydroperoxy isomer whereas the 9-hydroperoxy isomer was only a minor product (4%). HPLC of the methyl ester of the isolated hydroperoxides showed three components. Each component was collected, reduced to methyl hydroxystearate and characterized by GLC, MS and IR analysis. The components were identified as 13-hydroperoxy cis-trans isomer (92.8%), 13-hydroperoxy trans-trans isomer (2.6%) and 9-hydroperoxy cis-trans isomer (4.6%). A polar by-product present in the reaction mixture was identified by IR, ¹H NMR, and MS (of the toluene-p-sulphonyl derivative) as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid.     

Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases

Arachidonic acids (AA) and linoleic acids (LAs) are metabolized, in several tissues, to hydroxylated metabolites that are important mediators of many physiological and pathophysiological processes. The conjugation of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 13-hydroxyoctadecadienoic acid (HODE) by the human UDP-glucuronosyltransferase (UGT) enzymes was investigated. All substrates tested were efficiently conjugated by human liver microsomes to polar derivatives containing the glucuronyl moiety as assessed by mass spectrometry. The screening analyses with stably expressed UGT enzymes in HK293 showed that glucuronidation of LTB4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7, whereas UGT1A1, UGT1A3, UGT1A4, and UGT1A9 also conjugated most of the HETEs and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7 that conjugate all HETE and 13-HODE. Interestingly, UGT2B10 and UGT2B11, which are considered as orphan enzymes since no conjugation activity has so far been demonstrated with these enzymes, conjugated 12-HETE, 15-HETE, and 13-HODE. In summary, our data showed that several members of UGT1A and UGT2B families are capable of converting LA and AA metabolites into glucuronide derivatives, which is considered an irreversible step to inactivation and elimination of endogenous substances from the body.