Novel transformation of arachidonic acid in Saprolegnia parasitica.
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Arachidonate Lipoxygenases↗
Biomedical subjects
M Hamberg
Publications and source records attributed to M Hamberg.
Characterization of prostaglandin E2 20-hydroxylase of sheep vesicular glands.
The microsomal fraction of homogenates of the sheep vesicular glands, supplemented with 1 mM NADPH, metabolized 0.2 mM prostaglandin E2 to 20-hydroxyprostaglandin E2 at a rate of 76 +/- 9 pmol/min per mg of protein (with a Km of about 0.1 mM and a Vmax of about 0.1 nmol/min per mg of protein). Prostaglandin E1 was metabolized at a rate of only 8.5% of that of prostaglandin E2. The metabolism of prostaglandin E2 was decreased by 66% using 1 mM NADH instead of NADPH. alpha-Naphthoflavone (50 microM) and carbon monoxide inhibited the 20-hydroxylase by more than 60%, while 1 mM beta-diethylaminoethyl-2,2-diphenyl-pentanoate and 1 mM metyrapone inhibited it by less than 50%. The enzyme catalyzed the incorporation of atmospheric oxygen into the substrate. The findings suggest that the 20-hydroxylase could be a cytochrome P-450. The 20-hydroxylase could not be detected in vesicular glands of five rams 3 weeks after castration. The function of the enzyme is presumably to create the high level of 20-hydroxyprostaglandin E compounds in ram semen.
Animals↗
Identification of the major urinary metabolite of prostaglandin E3 in the rat.
[11,12-3H2]Prostaglandin E3 was administered subcutaneously into male Sprague-Dawley rats in doses of 0.4 microgram-10 mg/kg body weight. 40-60% of the administered radioactivity was excreted in the urine. The major metabolite was isolated by solid phase extraction followed by three steps of high-performance liquid chromatography. The structure of the major metabolite (5-11% of the administered radioactivity) was 7 alpha,11 alpha-dihydroxy-5-ketotetranorprosta-9,13-dienoic acid as shown by gas-liquid chromatography-mass spectrometry and by its conversion into 11 alpha-hydroxy-5-ketotetranorprosta-4(8),9, 13-trienoic acid.
Alprostadil↗
Novel biological transformations of 15-Ls-hydroperoxy-5,8,11,13-eicosatetraenoic acid.
[1-14C]Arachidonic acid was incubated with homogenates of the fungus, Saprolegnia parasitica. The products consisted of comparable amounts of two epoxy alcohols, 15-Ls-hydroxy-11,12-epoxy-5cis,8cis,13trans- eicosatrienoic acid and 15-hydroxy-13,14-epoxy-5cis,8cis,11cis-eicosatrienoic acid. Results of incubations carried out in the presence of nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, p-hydroxymercuribenzoate as well as glutathione peroxidase plus reduced glutathione demonstrated that transformation of arachidonic acid into epoxy alcohols occurred with the formation of 15-Ls-hydroperoxy-5cis,8cis,11cis,13trans- eicosatetraenoic acid (15-HPETE) as an intermediate. The pathway involved a lipoxygenase catalyzing the oxygenation of arachidonic acid at the 15L position to produce 15-HPETE, and a hydroperoxide isomerase activity which catalyzed conversion of 15-HPETE into the two epoxy alcohols. Studies with 15-[18O2]HPETE demonstrated that both oxygens of 15-HPETE were retained in the epoxy alcohols. Furthermore, experiments with mixtures of 15-[18O2]-and 15-[16O2]HPETE showed that conversion of 15-HPETE into epoxy alcohols occurred by an intramolecular transfer of hydroperoxide oxygen.
Arachidonic Acid↗
Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid.
Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.
Alprostadil↗
Isolation of two novel E prostaglandins in human seminal fluid.
cis-8,11,14,17-[1-14C]Eicosatetraenoic acid was incubated with microsomes of ram seminal vesicles and 1 mM glutathione for 3 min at 37 degrees C. The main metabolite was identified as 17,18-dehydroprostaglandin E1 by capillary column gas chromatography-mass spectrometry. Human seminal fluid was analyzed for the presence of 17,18-dehydroprostaglandin E1 and prostaglandin E3. Whereas prostaglandin E3 could be demonstrated by capillary gas chromatography-mass spectrometry, 17,18-dehydroprostaglandin E1 could not be found under these conditions. However, human seminal fluid contained two compounds with a similar polarity on reversed phase high performance liquid chromatography as 17,18-dehydroprostaglandin E1 and prostaglandin E3. The two compounds were identified as 18,19-dehydroprostaglandin E1 and 18,19-dehydroprostaglandin E2 by gas chromatography-mass spectrometry, by UV analysis after conversion to the corresponding prostaglandin B compounds, and by ozonolysis. The amount of each of the two prostaglandins in human seminal fluid seemed to be in the same order of magnitude as the amount of prostaglandin E3.
5,8,11,14-Eicosatetraynoic Acid↗
On the stereochemistry and biosynthesis of lipoxin B.
Lipoxin B (LXB) was prepared by incubation of (15S)-15-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with human leukocytes. Comparison with a number of trihydroxyicosatetraenes prepared by total synthesis showed that biologically derived LXB is (5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-icosatetraenoi c acid. Two isomers of LXB were identified by using an improved isolation procedure. These compounds were shown to be (5S,14R,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid (8-trans-LXB) and (5S,14S,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid [(14S)-8-trans-LXB]. Experiments with 18O2 showed that formation of LXB and its two isomers occurred with incorporation of molecular oxygen at C-5 but not at C-14. These results together with the finding that (15S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) is a precursor of LXB compounds in activated leukocytes suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-icosatetraenoic acid or its equivalent is a common intermediate in the biosynthesis of LXB and its two isomers.
Arachidonic Acids↗
Biosynthesis of a novel prostaglandin, delta 17-PGE1, in the ram.
The prostaglandin, delta 17-PGE1, was purified from extracts of ram seminal fluid by reversed phase high performance liquid chromatography (HPLC) and identified after conversion to delta 17-PGB1 by UV-analysis and by capillary column gas chromatography-mass spectrometry (GC-MS). It was also formed by incubation of a homogenate of ram vesicular glands. The amount of delta 17-PGE1 in the seminal fluid and in the homogenate averaged 12% and 25% of the amount of PGE2, respectively. The results show that cis-8,11,14,17-eicosatetraenoic acid can be metabolized to prostaglandins in vivo in the ram.
Alprostadil↗
External fixation as a test for instability after spinal fusion L 4-S 1. A case report.
A case presented with severe backache after fusion of the L 4-S 1 levels; the patient became immediately painfree after external transpedicular fixation between L 4 and the sacrum. The device was kept in place for 10 weeks. After an additional 4 weeks the patient was able to return to his work after several years of sick-leave. The case indicates instability as a cause of backache. Painful nonunion of a fusion can be present in spite of signs of healing on radiographs and CT-scan. External transpedicular fixation may be a good tool in assessing instability of the lower lumbar spine.
Adult↗
Spontaneous effect of increased stability of the lower lumbar spine in cases of severe chronic back pain. The answer of an external transpeduncular fixation test.
Eighteen patients with severe low-back pain of long duration were externally stabilized over selected segments of the lumbar spine to evaluate the pain relieving effect of increased stability. Five millimeter Schantz screws were driven into the vertebral body transpeduncularly by a closed technique using an image intensifier. A modified Hoffmann fixation device with possibilities to compress and distract was used for external stabilization. The results were recorded by means of pain area sketches and pain lines. All but one patient experienced remarkable relief of low-back pain and often of pain radiating into the lower extremities. No serious complications were seen. Of eight patients with residual severe pain after fusions, five were considered healed using radiologic techniques and three were improved by external stabilization. This test could be used to identify candidates, select levels for lumbar fusion, and evaluate the stability of previous fusions.
Adult↗
Lipoxins: stereochemistry, biosynthesis and biological activities.
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Animals↗
15-Lipoxygenase in human platelets.
The metabolism of arachidonic acid by washed human platelets was investigated. [1-14C]Arachidonic acid was extensively converted to [1-14C]12-hydroxyeicosatetraenoic acid. In addition, several minor labeled products were formed with a considerably lower specific activity, indicating their preferential formation from endogenous substrate. These were dihydroxy metabolites of arachidonic acid with conjugated triene structures and were identified as 14,15-dihydroxyeicosatetraenoic acid (three isomers) and 8,15-dihydroxyeicosatetraenoic acid (three isomers). The identification was based on comparison with reference compounds with respect to chromatographic properties, characteristic UV spectra, and mass spectrometry of several derivatives. Bradykinin (10(-8)-10(-5) M) was found to enhance the formation of all these compounds. In addition, the monohydroxy acid fraction was found to contain 15-hydroxyeicosatetraenoic acid. The present investigation thus demonstrates the occurrence of a 15-lipoxygenase in human platelets in addition to the previously known 12-lipoxygenase.
Arachidonate Lipoxygenases↗
Formation of 15-HETE as a major hydroxyeicosatetraenoic acid in the atherosclerotic vessel wall.
Atherosclerosis was induced in New Zealand White rabbits through cholesterol feeding. Aortae were taken out from treated animals and incubated with arachidonic acid. Aortae from cholesterol-fed animals converted arachidonic acid into 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). This conversion was not seen in aortae from control animals. The immediate precursor of 15-HETE, 15-HPETE, is an inhibitor of prostacyclin synthetase and might hamper prostacyclin production.
Animals↗
Circulating hydroxy fatty acids in familial Mediterranean fever.
Episodes of fever, serositis, and arthritis in familial Mediterranean fever (FMF) suggested circulating mediators of acute inflammation (e.g., neutrophil activation). The mean serum neutrophil-aggregating activity of 51 FMF patients was 2.5 +/- 0.2 cm2/min, compared to 1.0 +/- 0.1 cm2/min in 20 normal controls (P less than 0.0002). Lipid extracts of FMF sera retained neutrophil-aggregating activity and had UV absorbance peaks at 269 and 279 nm, indicating the presence of lipids with a conjugated triene structure. Chromatography of extracts yielded peaks that were coeluted with reference dihydroxyicosatetraenoic acids, had UV absorbance peaks at 259, 269, and 279 nm, and possessed neutrophil-aggregating activity. The presence of leukotriene B4 was excluded by chromatography following methyl-esterification. Monohydroxy compounds identified in FMF extracts by gas chromatography/mass spectrometry included 5-hydroxyicosatetraenoic acid, and 9- and 13-hydroxyoctadecadienoic acids. Hydroxy acids were present in 19 of 31 FMF sera and absent in extracts of sera from 8 patients with active systemic lupus erythematosus, 7 with fever from infection, and 12 normal controls. The finding of circulating mono- and dihydroxy fatty acids in FMF suggests that defects in the formation or elimination of these compounds might play a role in the pathogenesis of FMF.
Cell Aggregation↗
Biosynthesis and biological activities of lipoxins.
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Animals↗
Structural determination of leukotrienes and lipoxins.
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Animals↗
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.
Arachidonic Acid↗
Isotope effects in enzymatic and non-enzymatic oxygenation of 6,9,12-octadecatrienoic acid.
Synthesis of intramolecularly labeled 6,9,12-[11,11-2H2,1-14C]octadecatrienoic acid is described. The doubly labeled acid was added to 6,9,12-[9,10-3H2]octadecatrienoic acid and the mixture was incubated with soybean and human platelet lipoxygenases and was subjected to autoxidation. Methyl 13-hydroxyoctadecanoate obtained in the three sets of experiments had a considerably reduced 14C/3H ratio compared to that of the starting material, i.e., 20-24% (autoxidation) and 12-15% (lipoxygenase oxygenations). This indicates that subtraction of a hydrogen atom from the methylene group of the cis,cis-pentadiene structure is the rate-limiting step in autoxidation as well as in the enzymatic oxygenations.
Blood Platelets↗