calcimycin and 5-12-20-trihydroxy-6-8-10-14-eicosatetraenoic-acid

Human polymorphonuclear granulocytes (PMNs) synthesize leukotriene B4 (LTB4) as a response of cell activation. Inactivation of the potent inflammatory mediator proceeds via omega-oxidation, resulting in the formation of 20-hydroxy- and 20-carboxy-LTB4. The main metabolite after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is 20-carboxy-LTB4, and after stimulation with the calcium ionophore A23187 is 20-hydroxy-LTB4. Differences in the LTB4 inactivation pathway were also observed when the catabolism of exogenously added LTB4 was analysed. In contrast to resting cells or cells preactivated with FMLP, prestimulation with the ionophore or with phorbol esters resulted in the inhibition of 20-carboxy-LTB4-generation. This decrease correlated with the reduction in specific [3H] LTB4-receptor expression. Studies with the non-penetrating diazonium salt of sulphanilic acid, which is known to interact with ectoenzymes, revealed that LTB4 is metabolized via receptor-mediated uptake. Our data suggest that the reduction in the amount of LTB4-receptor sites inhibits the conversion of 20-OH-LTB4 into 20-COOH-LTB4.

Topics: Calcimycin; Cell-Free System; Diazonium Compounds; Humans; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Immunologic; Receptors, Leukotriene B4; Sulfanilic Acids; Tetradecanoylphorbol Acetate; Thermolysin

The metabolism of endogenous arachidonic acid P(AA) was investigated in activated neutrophils from 20 patients with Crohn's disease, 20 with ulcerative colitis, and 25 healthy volunteers. 1-14C-P(AA) was incorporated into intracellular pools of phospholipids prior to activation of the cells with ionophore A23187 and analyses of released arachidonic acid metabolites by thin layer chromatography. Total release of radioactivity expressing the release of arachidonic acid and its metabolites, was equal in the experimental and control groups, which suggests a normal substrate availability. In contrast, there was a marked increase in the relative release of leucotriene B4 (LTB4) and its omega-oxidation products, 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4), with LTB4 values exceeding the reference interval in seven of 20 patients with Crohn's disease, median 8.7%, and in six of 20 patients with ulcerative colitis, median 7.7%, as compared with a median of 5.3% in healthy volunteers. Furthermore, a decreased release of unmetabolised arachidonic acid, correlating inversely with the release of LTB4 in all experimental and control groups, and normal values for the production of other metabolites of arachidonic acid--for example, 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyheptadecatrienoic acid (HHT), point to an enzymatic abnormality such as increased activity of leucotriene B synthetase. An increased capacity for release of LTB4, the major pro-inflammatory metabolite of arachidonic acid lipoxygenation by polymorphonuclear leucocytes, may contribute to perpetuation of the inflammation and to tissue destruction in chronic inflammatory bowel disease. Our findings agree with previous reports of an increased release of LTB4 by the colonic mucosa in this condition.

Topics: Adolescent; Adult; Arachidonic Acid; Arachidonic Acids; Calcimycin; Colitis, Ulcerative; Crohn Disease; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Middle Aged; Neutrophils

Leukotriene B4 release from polymorphonuclear granulocytes of severely burned patients was reduced as compared to healthy donor cells. This decrease is due to an enhanced conversion of LTB4 into the 20-hydroxy- and 20-carboxy-metabolites and further to a decreased LTB4-synthesis. In addition, studies on the exogenous LTB4-conversion revealed an unidentified compound which was derived from LTB4. Our data suggest a modulation of the enzymatic activities involved in omega-oxidation of LTB4 (isoenzymes of cytochrome P-450).

Topics: Adult; Burns; Calcimycin; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Middle Aged; Neutrophils

The generation and metabolism of leukotrienes (LTs) from polymorphonuclear granulocytes of four polytraumatic patients on stimulation with the Ca-Ionophore A 23187 were studied by high performance liquid chromatography. In contrast to healthy donors the concentration of LTB4 within the supernatant of the stimulated granulocytes from these patients is reduced. The ratio of LTB4 versus the combined amounts of the biologically inactive 6-trans and 12-epi-6-trans-isomers is significantly decreased. In one patient who suffered from an Adult Respiratory Distress Syndrome (ARDS) a pronounced enhancement of LTC4 synthesis was observed.

Topics: Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; SRS-A; Stereoisomerism; Wounds and Injuries

Leukotriene B4 [LTB4; (5S,12R)-5,12-dihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid] and its 20-hydroxy derivative [20-OH-LTB4; (5S,12R)-5,12,20-trihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid] are principal metabolites produced when human neutrophils (hPMNs) are stimulated by the calcium ionophore A23187. These compounds were purified to homogeneity by Nucleosil C18 and silicic acid HPLC and identified by UV absorption and gas chromatographic/mass spectral analyses. 20-OH-LTB4 is considerably more polar than LTB4 and interacts weakly with the hydrophobic Nucleosil C18 resin, whereas LTB4 interacts strongly, reflecting the hydrophobic C13-C20 domain in LTB4. Specific binding of highly purified [3H]LTB4 and [3H]20-OH-LTB4 to hPMNs was assessed. Binding of [3H]20-OH-LTB4 could be largely displaced by an excess of nonlabeled LTB4 or 20-OH-LTB4 but not by 15-hydroxyicosatetraenoic acid (15-HETE), (5S,12S)-5,12-dihydroxy-6,10-trans-8,14-cis-icosatetraenoic acid [(5S,12S)-diHETE], or the 6-trans stereoisomer of LTB4 at 1 microM. In contrast, [3H]LTB4 displays a high level of nonspecific binding to human PMNs, which makes assessment of the Kd for LTB4 binding unobtainable. Binding measurements for [3H]LTB4 were performed in a buffer containing bovine serum albumin, and under these conditions significantly less nonspecific binding was observed. The apparent Kd for high-affinity binding sites on human PMNs at 0 degrees C was 31.3 X 10(-9) M for LTB4 and 14.3 X 10(-9) M for 20-OH-LTB4. In addition, we observed a saturable low-affinity receptor for 20-OH-LTB4 with a Kd of approximately 100 X 10(-9) M and 2 X 10(5) receptors per cell. The data from this study suggest that omega oxidation represents a major pathway for metabolism of LTB4 as well as other arachidonate metabolites. LTB4 and 20-OH-LTB4 express similar functional activities and share common binding properties to hPMNs but differ significantly in their physical properties. It is the unique physical characteristics of 20-OH-LTB4 that suggest that arachidonate metabolites oxidized at the omega position may be more important agents in inflammation than LTB4.

Topics: Arachidonic Acids; Calcimycin; Cell Membrane; Humans; Kinetics; Leukotriene B4; Lipoxygenase; Neutrophils; Oxidation-Reduction; Receptors, Immunologic; Receptors, Leukotriene B4

Suspensions of human peripheral blood leukocytes were incubated with arachidonic acid and calcium ionophore A23187. Acidic lipid extracts were purified by silicic acid chromatography and reversed phase high performance liquid chromatography. A polar metabolite with an ultraviolet absorption spectrum identical to that of leukotriene B4 was identified by gas chromatography and mass spectrometry as a 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid. The data indicated that this compound was the 20-hydroxy metabolite of leukotriene B4. The compound showed chemokinetic activity towards human polymorphonuclear leukocytes and aggregating activity towards rat peritoneal polymorphonuclear leukocytes. Its activity was greater than that of 5-hydroxy-6,8,11,14-eicosatetraenoic acid but less than that of leukotriene B4. It is concluded that the 15,12,20-trihydroxyeicosatetraenoic acid may play a role as a mediator of inflammatory reactions.

Topics: Calcimycin; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Leukocytes; Leukotriene B4; Neutrophils

Topics: Arachidonic Acids; Calcimycin; Cells, Cultured; Humans; Leukotriene B4; Mass Spectrometry; Neutrophils

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chemical Phenomena; Chemistry; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; Hydroxylation; Leukocytes; Leukotriene B4; Lung; Mass Spectrometry; Muscle Contraction