Biomedical subjects
Publications and source records attributed to M Hamberg.
Incubation of arachidonic acid or 12-Ls-hydroxy-5,8,10,14-eicosatetraenoic acid with suspensions of human polymorphonuclear leukocytes led to the formation of 12-Ls,20-dihydroxy-5,8,10,14-eicosatetraenoic acid. The structure of the new metabolite was by physical methods and by chemical degradation.
Addition of 15L-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) to human leukocytes led to the formation of a novel series of compounds containing four conjugated double bonds. The yield of tetraenes was increased approx. 100-fold when ionophore A23187 (5 microM) was added simultaneously with 15-HPETE. The structure of the major tetraene was established by physical methods as well as by chemical degradation and found to be 5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid.
Mechanistic aspects of lipoxygenase reactions are briefly reviewed. Results of studies on the degradation of an unsaturated fatty acid hydroperoxide, i.e. 13-LS-hydroperoxy-9,11-octadecadienoic acid, are presented.
Trihydroxytetraenes, a novel series of oxygenated derivatives formed from arachidonic acid in human leukocytes, were recently isolated [Serhan, C. N., Hamberg, M. & Samuelsson, B. (1984) Biochem. Biophys. Res. Commun. 118, 943-949]. The structure of the major compound was established--i.e., 5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid. The present study reports the structure of a second member of the trihydroxytetraene series of compounds--i.e., 5D,14,15L-trihydroxy-6,8,10,12-icosatetraenoic acid. When added to human neutrophils, 5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid stimulated superoxide anion generation and degranulation at submicromolar concentrations without provoking a substantial aggregation response. With respect to superoxide anion generation, 5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid proved to be as potent as leukotriene B4. In contrast, the compound was approximately 2 orders of magnitude less potent than either leukotriene B4 or fMet-Leu-Phe at provoking degranulation. The results indicate that interaction(s) between the 5- and 15-lipoxygenase pathways of human leukocytes leads to formation of a new series of oxygenated derivatives of arachidonic acid that may be involved in regulating specific cellular responses. The trivial names lipoxin A (5,6,15L-trihydroxy-7,9,11,13-icosatetraenoic acid) and lipoxin B (5D,14,15L-trihydroxy-6,8,10,12-icosatetraenoic acid) are proposed for the new compounds.
[1-14C]6, 9, 12-Octadecatrienoic acid was incubated with suspensions of human platelets. Three monohydroxy acids were isolated, i.e. 10LS-hydroxy-6, 8-pentadecadienoic acid, 10LS-hydroxy-6, 8, 12-octadecatrienoic acid, and 13LS-hydroxy-6, 9, 11-octadecatrienoic acid. Aspirin (0.5 mM) and indomethacin (10 microM) completely inhibited formation of the first mentioned compound whereas 5, 8, 11, 14-eicosatetraynoic acid (34 microM) inhibited formation of all three compounds. Isolation of 13LS-hydroxy-6, 9, 11-octadecatrienoic acid demonstrates that human platelets possess a lipoxygenase activity catalyzing omega 6-oxygenation of suitable poly-unsaturated fatty acids.
13-LS-Hydroperoxy-9,11-octadecadienoic acid (13-HPOD) was incubated with human hemoglobin. Apart from monoketo-, monohydroxy- as well as a number of hydroxy-epoxy acid derivatives earlier identified (Hamberg, M. (1975) Lipids 10, 87-92) a small conversion (1-2%) of 13-HPOD into 8-LR, 13-LS- and 8-DS, 13-LS-dihydroxy-9trans,11trans-octadecadienoic acids was demonstrated. An unstable intermediate, i.e., 12,13-epoxy-8,10-octadecadienoic acid, was detected in the conversion of 13-HPOD into the two dihydroxy-octadecadienoates by trapping experiments. It was concluded that compounds structurally related to the leukotrienes may be formed nonenzymatically from 13-HPOD.
[10D-3H; 3-14C]- and [10L-3H; 3-14C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B4 and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the 3H/14C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8-cis,11cis,14cis-eicosatetraenoic acid into leukotriene A4 whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.
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5,8,11,14,17-[1-14C]Eicosapentaenoic acid was prepared biosynthetically from [1-14C]arachidonic acid using the fungus, Saprolegnia parasitica. Incubation of 5,8,11,14,17-[1-14C]eicosapentaenoic acid with suspensions of human platelets led to the formation of three labeled compounds which were identified as thromboxane B3 (2-5% yield), 12-hydroxy-5,8,10,14-heptadecatetraenoic acid (2-5% yield), and 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid (7-59% yield).
9-Hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid are formed from linoleic acid upon incubation with the microsomal fraction of homogenates of the sheep vesicular gland (Hamberg, M. and Samuelsson, B. (1967) J. Biol. Chem. 242, 5344-5354. This communication is concerned with the stereochemical aspects of the conversion. The ratio between the 9- and 13-hydroxy isomers was 77:23. Steric analysis of the individual isomers showed that the hydroxyl group of both isomers had mainly the L configuration, i.e. 9L:9D, 79:21 and 13L:13D, 9- and 13-hydroxyoctadecadienoates which had largely lost the tritium label (6% and 7% retention of tritium relative to precursor, respectively) showing that the hydrogen which is removed from C-11 during the conversion has the L (pro-S) configuration.
The spontaneous degradation of 15-keto-13,14-dihydro-PGE2 was studied under various conditions. In aqueous media, dehydration rapidly occurred, particularly at high or very low pH. The acid catalyzed dehydration product was identified as 15-keto-13,14-dihydro-PGA2. This compound was also formed at alkaline pH, however, at higher pH a bicyclic compound, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE2, was formed in addition (cf. Fig. 1). The amounts of the latter compound increased with time and with increasing pH. In samples containing albumin, 15-keto-dihydro-PGE2 was degraded even more rapidly than in buffer of the same pH. In addition to the formation of the dehydration products, a considerable part of the metabolite was bound to albumin to yield water soluble adducts. A similar binding occurred to a low molecular weight fraction of plasma. The compound that was bound was 15-keto-dihydro-PGA2, whereas both 15-keto-dihydro-PGE2 and the bicyclic product were relatively inert in this respect. Due to its chemical stability, the bicyclic degradation product, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE2, is suggested as a suitable target for measurements instead of the labile parent compound, 15-keto-dihydro-PGE2, or the reactive dehydration product, 15-keto-dihydro-PGA2.
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The transformation of [1-14C]arachidonic acid by homogenates of human umbilical arteries was studied. The major compound formed was the stable end product of PGI2, i.e. 6-keto-PGF1 alpha (lactol form) as analyzed by gas-liquid chromatography-mass spectrometry. PGI2 was generated by incubating PGH2 with a lyophilized pig aorta microsome preparation. PGI2 concentrations around 10 ng/ml relaxed the human umbilical artery preparation significantly. Formation of PGI2 by umbilical arteries during pregnancy might be a mechanism for regulation of blood flow to the fetus.
The main urine metabolite of prostaglandin F2 alpha (5 alpha, 7 alpha-dihydroxy-11-ketotetranor-phostane-1, 16-dioic acid, PGF-metabolite) was determined by mass spectrometry (MS) and radioimmunoassay (RIA) in burned patients treated under routine conditions. The amount of the PGF-metabolite as determined by MS was 130 and 67 microgram/24 h (normal value 24 +/- 17 microgram/24 h) on days 3 and 5 respectively in one patient. In serial determinations using RIA the urine level of the PGF-metabolite was within normal values during the first days and rose to a broad peak 1-4 weeks after the injury. Thromboxane B2 (TXB2) was identified and quantitated in burn blister fluid. The amount of TXB2 was 1.7 ng/ml.
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