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PubMed1997

Structural Effects on the Intrinsic Basicities of alpha,beta-Unsaturated Lactones and Ketones.

The proton affinities of 2(5H)-furanone, 1 (836 kJ/mol), 5,6-dihydro-2H-pyran-2-one, 2 (862 kJ/mol), cyclopentenone, 3 (857 kJ/mol), and cyclohexenone, 4 (863 kJ/mol), have been measured by Fourier transform ion cyclotron resonance techniques. A comparison is made with (reexamined) data concerning saturated cyclic and unsaturated aliphatic analogs. Three general observations are made. First, the basicity is found to increase with the size of the ring. Second, unsaturated lactones are more basic than their corresponding aliphatic unsaturated esters. Third, unsaturated and saturated lactones have almost identical gas-phase basicities, while unsaturated and saturated lactones have almost identical gas-phase basicities, while unsaturated cyclic ketones are more basic than their saturated analogs. All these experimental findings have been rationalized by means of ab initio calculations up to the G2(MP2,SVP) level. The basicity trends along the series are the result of two main factors: the different hybridization pattern of the carbonyl carbon as the size of the ring changes and, in the case of lactones, the nonbonding interaction between the proton attached to the carbonyl group and the ether-like oxygen which contributes to the enhanced stability of the protonated form. For unsaturated ketones the C=C double bond participates fully in the change in charge distribution induced by the protonation, while for unsaturated lactones the existence of an oxygen atom within the ring impedes this shift of the electron density.

Journal Article↗
PubMed2001

Excretion into gastrointestinal tract of irinotecan lactone and carboxylate forms and their pharmacodynamics in rodents.

PURPOSE: To investigate the excretion of irinotecan hydrochloride (CPT-11) and its active metabolite, SN-38, into the gastrointestinal lumen via the biliary and/or intestinal membrane route after dosing with lactone and carboxylate forms of CPT-11, and to evaluate the toxic and antitumor effects of the two forms. METHODS: The excretions of CPT-11 and SN-38 were investigated by the in situ perfusion technique using rats. The incidence of delayed diarrhea was evaluated after i.v. dosing (60 mg/kg) with CPT-11 lactone and carboxylate forms for 4 days. Antitumor activity and changes in body weight were investigated in mice with Meth A tumors. RESULTS: The excretion of CPT-11 into bile was greater in dosing with CPT-11 carboxylate than that with its lactone form, whereas the exsorption across intestinal membrane was greater in dosing with CPT-11 lactone than that with its carboxylate form. Dosing with CPT-11 lactone dose-dependently inhibited the increase in tumor weights in Meth A tumor mice, whereas the dosing with its carboxylate form reduced the antitumor effect. CONCLUSIONS: The decreased antitumor effect caused by dosing with the CPT-11 carboxylate form could be due to less accumulation in the tissue including tumor cells resulting from the rapid elimination of the form in the body.

Animals↗
PubMed1999

The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction.

The Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages. We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels. OdDHL (1-30 microM) caused a concentration-dependent inhibition of U46619-induced contractions of the coronary artery through a largely endothelium-independent mechanism, but was markedly less effective in the pulmonary artery. Quantitively similar effects to those produced by OdDHL were observed with N-(3-oxododecanoyl)-L-homocysteine thiolactone, a thiolactone derivative, while N-3-oxododecanamide, a lactone-free acyl analogue, possessed 1/3rd the potency as a vasorelaxant. Neither N-butanoyl-L-homoserine lactone nor L-homoserine lactone (up to 30 microM) were active. Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig. The vasorelaxant action of OdDHL appears to be primarily determined by the N-acyl chain length, with a minor contribution by the homoserine lactone moiety.

4-Butyrolactone↗
PubMed1991

The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies.

Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.

Amino Acid Sequence↗
PubMed1995

Synthesis of 25-hydroxyvitamin D3-26,23-lactone but not 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 in opossum kidney cells treated with 1 alpha, 25-dihydroxyvitamin D3.

The pathway from 25-hydroxyvitamin D3(25[OH]D3) to 25-hydroxyvitamin D3-26,23-lactone (25[OH]D3-26,23-lactone) is important in the renal degradation of vitamin D metabolites. Our objective was to investigate the regulation of 25(OH)D3 metabolism in the opossum kidney (OK) cell line which has been used for studying the actions of the calcium-regulating hormones. While the untreated OK cells did not produce 25(OH)D3 metabolites, the synthesis of the lactone was increased dose-dependently by 20h pretreatment with 1 alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3), peaked at 10(-7) M 1,25(OH)2D3, and decreased with higher concentrations. Little production of 24,25-dihydroxyvitamin D3 (24,25[OH]2D3) was observed after any dose of 1,25(OH)2D3. The increase in the production of lactone induced by 10(-7) M 1,25(OH)2D3 was detectable at 2 h, peaked between 4 and 24 h, and decreased gradually until 48 h. Its induction was inhibited by actinomycin D and cycloheximide, suggesting that de novo protein synthesis is required for such induction. After treatment with 10(-7) M 1,25(OH)2D3 for 20 h, 24-hydroxylase (24-OHase) mRNA was not detected in OK cells, but was found evident in COS-7 cells (a renal cell line). Moreover, the COS-7 cells treated with 10(-7) M 1,25(OH)2D3 metabolized 25(OH)D3 to 24,25(OH)2D3. These results indicate that 1,25(OH)2D3 stimulates the synthesis of 25(OH)D3-26,23-lactone in a dose- and time-dependent manner, but does not induce the gene expression or activity of 24-OHase in OK cells.

24,25-Dihydroxyvitamin D 3↗
PubMed1976

Immunologic evidence that the gene for L-gulono-gamma-lactone oxidase is not expressed in animals subject to scurvy.

L-Gulono-gamma-lactone oxidase (L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8) is the enzyme that catalyzes the terminal step of L-ascorbic acid biosynthesis in mammalian liver. The absence of the oxidase activity in primates and guinea pigs is the reason why these animals are subject to scurvy, which must be considered an inborn error of metabolism. Attempts were made to determine if a protein immunologically crossreactive with L-gulono-gamma-lactone oxidase is present in these animals. Detergent-solubilized microsomal preparations from guinea pig and African green monkey liver did not precipitate the antisera directed to either rat or goat enzyme, nor did any of the other cell fractions obtained from guinea pig liver react with either antiserum. No crossreactive protein was detectable in guinea pig microsomes even with the sensitive procedure or micro-complement fixation. On the other hand, extracts of all 10 other mammalian (4 orders) liver microsomes tested were shown to contain L-gulono-gamma-lactone oxidase activity that did crossreact with antibodies to the rat and goat enzymes. One explanation of these findings is that, in the guinea pig, and perhaps in primates too, the structural gene for L-gulono-gamma-lactone oxidase is not expressed.

Alcohol Oxidoreductases↗
PubMed2000

Intracellular Ca(2+) mobilization and kinase activity during acylated homoserine lactone-dependent quorum sensing in Serratia liquefaciens.

Quorum sensing in Gram-negative bacteria involves acylated homoserine lactones (AHLs) and a transcription factor, activated by the AHLs. In this study, a possible involvement of intracellular Ca(2+) as second messenger and/or protein kinase activity during signal transduction is analyzed. When N-hexanoyl-l-homoserine lactone was added to a suspension of Fura-2-loaded Serratia liquefaciens, there was a decline in [Ca(2+)](i), measured as a decrease in the Fura-2 fluorescence ratio. As controls, the addition of the signal molecule N-3-oxohexanoyl-l-homoserine lactone, which is not produced by S. liquefaciens, did not induce changes in [Ca(2+)](i). Using a protein kinase activity assay on AHL-stimulated cells, an increase in kinase activity after N-butanoyl-l-homoserine lactone stimulation of S. liquefaciens cells was detected, whereas the kinase activity induced by N-3-oxohexanoyl-l-homoserine lactone was not statistically significant. The conclusion from this study is that changes in [Ca(2+)](i) are involved in quorum sensing signal transduction in the Gram-negative bacteria S. liquefaciens. We also conclude that kinase activity is induced in S. liquefaciens upon AHL stimulation. We suggest that the transient intracellular [Ca(2+)] changes and kinase activity, activated by the AHL signal, are critical for the quorum-sensing signal transduction.

4-Butyrolactone↗
PubMed1998

L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis.

L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.

Amaryllidaceae Alkaloids↗
PubMed1970

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii.

Fixation of (14)CO(2) by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of (14)C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total (14)C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-(14)C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With (14)C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of (14)CO(2) fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.

Journal Article↗
PubMed1990

Induction of mouse anti-melanoma cytotoxic and suppressor T cells in vitro by an artificial antigen, GM3-lactone.

We investigated the ability of GM3-lactone liposomes to induce anti-melanoma T cell responses in mice. GM3-lactone liposomes, like murine B16 melanoma cells, induced anti-melanoma cytotoxic T cells (CTL) and also suppressor T cells (Ts). A small dose of GM3-lactone (0.0003 micrograms/ml) was enough to generate CTL in the in vitro primary response, whereas relatively large amounts of the antigen (0.03-0.3 microgram/ml) were required for anti-melanoma Ts induction. As the epitope for anti-melanoma Ts is NeuAc but not NeuGc residue on GM3, and anti-melanoma CTL are effectively induced by either GM3(NeuAc) or GM3(NeuGc)-lactone liposomes, GM3(NeuGc)-lactone or GM3(NeuGc) liposomes have potent activity as an artificial melanoma antigen to induce anti-melanoma CTL in vitro.

Animals↗
PubMed1992

Purification and characterization of a novel FMN-dependent enzyme. Membrane-bound L-(+)-pantoyl lactone dehydrogenase from Nocardia asteroides.

Membrane-bound L-(+)-pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L-(+)-pantoyl lactone, was solubilized with Brij 35 and purified 78-fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE-Sephacel, Sepharose CL-6B and Cellulofine GC-700-m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high-performance gel-permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L-(+)-pantoyl lactone as a substrate (Km = 26.8 mM; Vmax = 4.22 mumol.min-1.mg protein-1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L-(+)-pantoyl lactone dehydrogenase was identified as noncovalently bound FMN.

Alcohol Oxidoreductases↗
PubMed1999

Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells.

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.

4-Butyrolactone↗
PubMed2001

Multiple N-acyl homoserine lactone signals of Rhizobium leguminosarum are synthesized in a distinct temporal pattern.

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R. leguminosarum cells with and without the symbiosis plasmid pRL1JI. Cells containing pRL1JI were found to produce three predominant signals. In decreasing order of abundance, these were N-(3-oxo)octanoyl homoserine lactone [(3-O)C(8)HSL], N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone. Cells without pRL1JI produced only two major signals, N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone [(3-OH)C(14:1)HSL] and (3-O)C(8)HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C(8)HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C(14:1)HSL did activate synthesis of the three AHLs made by cells containing pRL1JI. These results indicate that the synthesis of multiple AHLs in R. leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C(14:1)HSL can supersede these controls in pRL1JI-containing cells. This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.

4-Butyrolactone↗
PubMed1975

Beta-ketoadipate enol-lactone hydrolases I and II from Acinetobacter calcoaceticus.

Beta-Ketoadipate enol-lactone hydrolase catalyzes a common step in the utilization of protocatechuate and cis,cis-muconate by bacteria. Either of the two compounds elicits the synthesize of an enol-lactone hydrolase in Acinetobacter. The enol-lactone hydrolase that is induced by each compound was purified, and the properties of the proteins were compared. Both enzymes appear to be dimers with molecular weights of approximately 25,000. The amino acid compositions of the enzymes differ, and the two proteins do not cross-react serologically. The NH2-terminal amino acid residue of the protocatechuate-induced enol-lactone hydrolase (ELH I) is methionine and the NH2-terminal amino acid residue of the cis,cis-muconate-induced enol-lactone hydrolase (ELH II) is proline. Therefore, ELH I and ELH II appear to be the products of different structural genes. The serological specificity of ELH I and ELH II made it possible to demonstrate the mutually independent regulation of their synthesis in wild type cells and in constitutive mutant strains. The synthesis of ELH I is not impaired in mutant strains that cannot synthesize ELH II. The rapid characterization of mutant strains that produce ELH I or ELH II constitutively was made possible by the development of pH indicator enzyme assays that were performed with toluenized cells. cis,trans-Muconate, which does not support the growth of Acinetobacter, elicits the synthesis of the enzymes that normally are induced by cis,cis-muconate to 20% of fully induced levels.

Acinetobacter↗
PubMed1988

An IgG3 monoclonal antibody established after immunization with GM3 lactone: immunochemical specificity and inhibition of melanoma cell growth in vitro and in vivo.

In previous studies, an IgM monoclonal antibody (M2590), established after immunization of C57BL/6 mice with syngeneic B16 melanoma cells, was found to react with melanoma cells, but not with various normal cells and tissues (Taniguchi, M., and Wakabayashi, S., Jpn. J. Cancer Res., 75:418-426, 1984). The structure defined by this antibody was identified as GM3 (Hirabayashi, Y., et al., J. Biol. Chem., 260:13328-13333, 1985) organized in membranes at high density, although the real immunogen was suggested to be GM3 lactone (Nores, G. A., et al., J. Immunol., 139:3171-3176, 1987). Since GM3 lactone was found to be highly immunogenic, we subsequently immunized C57BL/6 mice with GM3 lactone coated on Salmonella minnesotae and established hybridoma DH2, secreting an IgG3 antibody showing preferential reactivity with GM3 lactone over GM3 under certain conditions. The reactivity of the DH2 antibody was competitively inhibited by M2590, and it showed a preferential reactivity with melanoma cells and displayed various immunochemical and immunobiological properties similar to those of M2590. However, DH2 antibody inhibited melanoma cell growth in vivo, induced antibody-dependent cytotoxicity in vitro, and showed a preferential accumulation in melanoma growth in vivo. These properties are characteristic of the IgG3 subclass, in striking contrast to IgM antibody M2590, which does not inhibit cell growth in vivo or in vitro and does not induce antibody-dependent cytotoxicity. Thus, immunization with lactone forms of tumor-associated ganglioside antigens might be useful in the production of antibodies and prevention of tumor cell growth in vivo (antitumor vaccines).

Animals↗
PubMed1980

Evolutionary divergence of co-selected beta-ketoadipate enol-lactone hydrolases in Acinetobacter calcoaceticus.

Muconolactone isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive catabolic steps in bacteria. Separately inducible beta-ketoadipate enol-lactone hydrolases I and II are formed in representatives of Acinetobacter calcoaceticus. When subjected to DEAE-cellulose chromatography, Acinetobacter enol-lactone hydrolase I displays heterogeneous behavior which, in whole or in part, appears to be due to modifications of sulfhydryl groups in the protein; the enzyme is unusual in that its NH2-terminal amino acid is cysteine. Comparison of the NH2-terminal amino acid sequence of Acinetobacter enollactone hydrolase I, reported here, with the corresponding amino acid sequences of Acinetobacter enollactone hydrolase II and Pseudomonas enol-lactone hydrolase indicates that all three proteins have diverged widely from a common evolutionary origin. Sequence comparisons suggest that divergence of the Acinetobacter enol-lactone hydrolase structural genes was achieved by substitution with DNA derived from an ancestral muconolactone isomerase structural gene.

Acinetobacter↗
PubMed1995

In vitro and in vivo biotransformations of the naphthalenic lignan lactone 5-lipoxygenase inhibitor, L-702,539.

It has been reported previously that the tetrahydropyranyl naphthtalenic lignan lactone L-702,539 is a potent nonredox, 5-lipoxygenase inhibitor that has the advantage that it can be dosed either as the lactone or as the corresponding nonactive hydroxy acid L-702,618 (opened lactone). Studies with hepatic microsomes from the rat, rhesus monkey, and human were undertaken in a phosphate buffer and suggested that the closure of the hydroxy acid L-702,618 to the lactone L-702,539 was an enzymatic process. The incubation of L-702,539 under oxidative conditions with these specific hepatic microsomes resulted in the formation of three significant metabolites (> 0.4 nmol/mg protein/hr) as determined by HPLC with UV detection. These metabolites were isolated from large microsomal incubations and were characterized by MS and NMR spectroscopy. Data showed that the lactone and tetrahydropyran portions of the molecule were both susceptible to hydroxylation, and the hydroxylated tetrahydropyran was further oxidized to the hydroxy acid. Analysis of plasma samples obtained from rat and rhesus monkeys following L-702,618 administration indicated that the in vivo metabolic pathway was similar to the one observed in vitro using hepatic microsomes. Studies conducted with microsomes from genetically engineered human cell lines expressing individual cytochrome P450s indicated that the isozyme responsible for the metabolism at the tetrahydropyran ring, was P4503A4. These findings were supported by studies conducted in human microsomes using an inhibitory P4503A4 antibody and troleandomycin, which is a potent P4503A inhibitor.

Animals↗
PubMed1998

A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol.

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

8,11,14-Eicosatrienoic Acid↗