14,15-Dihydroxy-5,8,10,12-eicosatetraenoic acid. Enzymatic formation from 14,15-leukotriene A4

When 14C-labeled (14S, 15S)-14,15-trans-oxido-5,8-cis-10,12-trans-eicosatetraenoic acid (14,15-leukotriene A4) was incubated with cytosolic epoxide hydrolase purified from mouse liver, one major radiolabeled product appeared. The structure was assigned as (14R, 15S)-14,15-dihydroxy-5,8-cis-10,12-trans-eicosatetraenoic acid (14,15-DHETE), based on analytical data as well as enzyme mechanistic considerations. The formation of this compound was dependent on time and enzyme concentration and was abolished after heat treatment of the enzyme. The apparent Km and Vmax values at 37 degrees C were 11 microM and 900 nmol X mg-1 X min-1 respectively. This enzymatic hydrolysis of 14,15-leukotriene A4 represents an additional mode of formation for 14,15-DHETE, a compound previously found to modulate functions of human leukocytes.     

Stereochemistry, total synthesis, and biological activity of 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid

The stereochemistry of the major isomer of 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid formed from 15-hydroperoxyeicosatetraenoic acid in human leukocytes was determined. The structure (erythro-14(R),15(S]-14,15-dihydroxy-5,8-cis-10,12-trans-eicosatetraenoi c acid) was assigned based on sodium arsenite thin-layer chromatography, NMR spectroscopy, and comparison with material prepared by total synthesis. This compound was found to inhibit leukotriene B4-induced superoxide anion generation in human neutrophils (IC50 = 10(-8)-10(-7) M). Superoxide anion generation induced by either formylmethionyl-leucyl-phenylalanine or arachidonic acid was not affected.     

Transformation of arachidonic acid in the rat anterior pituitary

Rat anterior pituitaries were incubated with [1-¹⁴C]-arachidonic acid. The metabolites were purified by reversed-phase high pressure liquid chromatography. Conclusive identification of the compounds was performed by gas chromatography-mass spectrometry. The major metabolite of arachidonic acid was the 12-hydroxy-5,8,10,14-icosatetraenoic acid (0.1% of added radioactivity). Smaller amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid and of 15-hydroxy-5,8,11,13-icosatetraenoic acid (0.01% of added radio-activity) were also isolated. Trace amounts of prostaglandins E₂, D₂ and F₂α were detected.     

Metabolism of (2Z,4E)-γ-Ionylideneethanol and (2Z,4E)-γ-Ionylideneacetic Acid in Cercospora cruenta

[2–¹⁴C]-(2Z,4E)-γ-Ionylideneethanol and [2–¹⁴C]-(2Z,4E)-γ-ionylideneacetic acid were converted by Cercospora cruenta to [2–¹⁴C]-(2Z,4E)-1′,4′-dihydroxy-γ-ionylideneacetic acid and [2-¹⁴C]-(2Z,4E)-4′-hydroxy-γ-ionylideneacetic acid, which are intermediates of ABA biosynthesis in C. cruenta.     

Investigation of the chemical conversion of hydroperoxyeicosatetraenoate to leukotriene epoxide using stereospecifically labeled arachidonic acid. Comparison with the enzymatic reaction

A series of stereospecifically labeled polyunsaturated fatty acids were prepared by biosynthesis from [8-DR-3H]- and [8-LS-3H]stearic acids. The labeled stearic acids were synthesized by a novel scheme employing readily available alkyne and aldehyde starting materials. The stereochemical purity of the prochiral tritium labels was judged to be greater than 99%, as determined by analysis of the octadec-1-yn-8(R)- and 8(S)-ol intermediates in the synthesis. Previously, the labeled arachidonic acids were used to investigate the stereoselectivity of hydrogen abstraction in the biosynthesis of leukotriene epoxides. We have now investigated the selectivity of hydrogen abstraction in a chemical synthesis of 14,15-leukotriene (LT) A4 from mixtures of [3-14C]- and either [10-DR-3H]- or [10-LS-3H]15(S)-HPETE methyl esters. Reaction with either chirally labeled precursor led to 70-95% retention of 3H relative to 14C in the 14,15-LTA4 and 10-Z-14,15-LTA4 products after purification by high performance liquid chromatography. The 15-dienone obtained from this reaction was consistently enriched in 3H relative to 14C after isolation and purification. Evidence was obtained to indicate that the majority of the 3H in the products was retained in its original location and configuration. These results indicate that the biomimetic chemical reaction is stereo-random with respect to hydrogen loss from carbon 10 and that, in contrast to the reaction as it occurs in leukocytes and platelets, in the chemical model the reaction begins by decomposition of the hydroperoxide group, with hydrogen loss from carbon 10 occurring as a late or final step.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

Mechanisms of leukotriene formation: hemoglobin-catalyzed transformation of 15-HPETE into 8,15-DiHETE and 14,15-DiHETE isomers

Four isomers of 8,15-diHETE as well as 14,15-diHETEs are isolated and characterized after exposure of 15-HPETE to hemoglobin. It is found that 83% of the C-8 oxygen atoms in 8(R), 15(S)-diHETE and 8(S), 15(S)-diHETE, and 41% of the C-8 oxygen atoms in 8(R), 15(S)-11Z-diHETE and 8(S), 15(S)-11Z-diHETE are derived from H2(18)O. These results suggest that hemoglobin catalyzes the transformation of 15-HPETE into these products via a free radical process, possibly involving the intermediacy of 14,15-LTA. Intact human leukocytes contain a distinct enzyme system for catalyzing the conversion of 15-HPETE into 14,15-LTA. This enzyme activity is inhibited by ETYA and is rapidly denatured upon homogenization of the intact leukocytes.     

Mechanistic studies of the dioxygenase and leukotriene synthase activities of the porcine leukocyte 12S-lipoxygenase

Lipoxygenases react with hydroperoxy fatty acids and catalyze dioxygenase or dehydrase (leukotriene A4 (LTA4) synthase) types of reactions. In the present investigation we studied the mechanism of reaction of the purified porcine leukocyte 12S-lipoxygenase with 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE). Oxygen-18 labeling experiments with GC-MS analysis were used to distinguish dioxygenase and leukotriene synthase activities of the enzyme; 8S,15S-DiHPETE and 14R,15S-DiHPETE were formed by oxygenation, and a series of 8,15- and 14,15-diols were formed via enzymatic synthesis of 14,15-LTA4 and nonenzymatic hydrolysis of the epoxide. 10D-3H- and 10L-3H-labeled substrates were used to study the stereospecificity of the C-10 hydrogen abstraction in the synthesis of these products. Formation of 14,15-DiHPETE and 14,15-LTA4 was associated with stereoselective abstraction of hydrogen from the 10L position of 15S-HPETE. The same type of measurements on the 8S,15S-DiHPETE product indicated a variable (50-250%) retention of the 10L-3H label, and a consistent 90% retention of the 10D-3H. In contrast, the synthesis of 8S,15S-DiHPETE by the soybean lipoxygenase was associated with the expected stereoselective abstraction of the 10D hydrogen. It appears that the porcine leukocyte 12S-lipoxygenase synthesizes 8S,15S-DiHPETE by a different mechanism.     

A novel leukotriene formed by transpeptidation of leukotriene E

A new leukotriene 5(S)-hydroxy-6(R)-$\text{S}$-γ-glutamylcysteine-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid (leukotriene F₄) was isolated after incubating leukotriene E₄ with γ-glutamyltranspeptidase and glutathione. Leukotriene F₄ induced contractions of the isolated quinea pig ileum and was less potent in this respect than leukotriene E₄.     

Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro

One juvenile hormone was isolated from culture medium containing isolated corpora allata of the grasshopper $\text{Schistocerca vaga}$ (Orthoptera: Acrididae) and was shown by microchemical methods to be methyl (2$\text{E}$, 6$\text{E}$) - (10$\text{R}$) - 10, 11-epoxy-3, 7, 11-trimethyldodeca-2, 6-dienoate. This compound (JH III), which occurs in a sphingid moth $\text{Manduca sexta}$, is the first juvenile hormone identified in an insect order other than the Lepidoptera. Grasshopper organs incorporate both [2?¹⁴C] acetate and [methyl-¹⁴C] methionine into JH III showing $\text{de novo}$ biosynthesis, but no indication of the synthesis of JH I or JH II was seen.     

Isolation and identification of a naturally occurring 7, 8-didemethyl-8-hydroxy-5-deazariboflavin derivative from Mycobacterium avium

A yellow pigment (C₂₄H₂₆N₄PO₁₅Na₃) was isolated from Mycobacterium avium. On the acid hydrolysis the pigment (1 mol) gave L-glutamic acid (1 mol), lactic acid (1 mol) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5′-phosphoric acid. The structure of $\text{N-[O-[[5-(8-}\text{hydroxypyrimidol[4,5-b]}\text{quinoline}\text{-10-}\text{yl}\text{-2,4(3H, 10H)-}\text{dione}\text{)-2,3,4-}\text{trihydroxypentyloxy]hydroxyphosphoryl]-}\text{L}\text{-lactyl]-}\text{L}\text{-glutamic acid}$ was proposed for this compound. The compound isolated functions presumably as a new cofactor in a redox system of the bacteria.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Studies on the biosynthesis of N-(γ-L-glutamyl)-4-hydroxyaniline] in Agaricus bisporus: Identification of the position in shikimic acid at which the amination occurs

Labelled shikimic acid was efficiently incorporated into the aniline moiety of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$, a characteristic aromatic compound of the common mushroom, Agaricus bisporus. Incubations with [3-³H]- and [1,6-¹⁴C]shikimic acid clearly proved that the amination of shikimic acid occurs at its 4-position during the biosynthesis of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$.     

A study on the stability of an estrogen-protein conjugate in vivo and under in vitro conditions

[³ H] Estradiol-17β-succinyl bovine serum albumin conjugate ([³H]-E₂-BSA) was synthesized with a specific activity of 1.92 × 10⁷ cts/min/mg. The conjugate was administered iv to ovariectomized rats and the quantity of free [³H] steroid in the uterus was determined. Radioactive material was detected in all of the subcellular fractions of the uterus and identified as estradlol-17β. Similar subcellular distribution of the radioactivity was observed when [³H]E₂-BSA was added $\text{in vitro}$ to uterine homogenate. Free estradlo1-17β was released when the conjugate was Incubated with rat uterine homogenate or with serum. The results of the present study suggest that E₂-BSA is hydrolyzed $\text{in vivo}$ and under $\text{in vitro}$ conditions. It is recommended that the stability of a hormone-protein conjugate be established before use.     

11-Trans leukotriene C: A naturally occurring slow reacting substance

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9,11-$\text{trans}\text{-14-}\text{cis}\text{-eicosatetraenoic acid (11-}\text{trans}$ leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts in vitro

The effects of endometrium on metabolism of [³H]-arachidonic acid ([³H]-AA) by bovine blastocysts recovered on day 19 postmating were studied $\text{in vitro}$. Blastocysts (n = 12) and endometrial slices were assigned to four incubation groups. In group 1, blastocysts were incubated alone; group 2, endometrial slices were incubated alone; group 3, blastocysts were incubated with endometrial slices; group 4, blastocysts were incubated in 7.5 ml fresh incubation medium plus 7.5 ml frozen-thawed medium from endometrial incubations. In all groups, tissues were incubated in 15 ml modified minimum essential medium (MEM) containing 5 μCi of [³H]-AA and 200 μg radioinert arachidonic acid for 24 h at 37°C in an atmosphere of 50% N₂:45% O₂:5% CO₂. For incubation controls, 5 μCi of [³H]-AA were added to 15 ml MEM and incubated at the same time as tissues from each cow. To evaluate metabolism of [³H]-AA, [³H]-AA and its metabolites were extracted from aliquots of MEM and separated on columns of Sephadex LH-20. Most (78.3 ± 3.2%) of the radioactivity (dpm) in the incubation controls was recovered as [³H]-AA, indicating that there was little breakdown of [³H]-AA in the absence of tissue. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α ([²H]-PGFM), [³H]-PGE₂ and [³H]-PGF_2^α. Endometrial slices metabolized very little of the [³H]-AA. Data from groups 1 and 4 were combined (group $\text{1}\text{4}$) for analysis because the distribution of dpm did not differ between the two groups. In group 3, blastocysts and endometrial slices incubated together tended(P<.10) to produced more [³H]-PGE₂ than did group $\text{1}\text{4}$, there tended to be less (P<.10)_[³H]-PGF_2^α, and there was more (P<.05) [³H]-PGFM than in group $\text{1}\text{4}$. Neither endometrial secretions nor endometrial slices altered the proportion of [³H]-AA metabolized by blastocysts. Endometrial slices appear capable of metabolizing [³H]-PGF_2^α synthesized by blastocysts, and capable of directing blastocyst metabolism of [³H]-AA away from synthesis of [³H]-PGF_2^α and toward synthesis of [³H]-PGE₂. It is postulated that the endometrium has an important role in regulating the amounts and ratios of prostaglandins in th uterine lumen during early prenancy in cows.     

Prostaglandins and congeners V. (1) Synthesis of d1-prostaglandin E1, d1-11-deoxyprostaglandin E1, and d1-15-hydroxy-9-oxo-13-cis-prostenoic acid via conjugate addition of 3-trityloxy-1-octenylmagnesium bromides

d1-Prostaglandin E₁ and d1-11-deoxyprostaglandin E₁ are conveniently synthesized via the copper (I) catalyzed conjugate addition of the Grignard reagent prepared from 3-trityloxy-$\text{trans}$-1-octenyl bromide to the appropriate cyclopentenone precursor. The Grignard reagent also afforded the synthesis of a novel structure, d1-15-hydroxy-9-oxo-13-$\text{cis}$-prostenoic acid.