The metabolism of leukotrienes in blood plasma studied by high-performance liquid chromatography.

The metabolism of leukotrienes (B4, C4, D4, and E4) within human plasma was studied and a simple sample preparation is presented. It was demonstrated that leukotriene E4 and leukotriene B4 were stable during incubation at 37 degrees C using the in vitro system. In contrast, leukotriene C4 was metabolized by gamma-glutamyl transpeptidase activities into leukotriene D4 which was further metabolized by dipeptidase activities of plasma into leukotriene E4. The transition state inhibitor of gamma-glutamyl transpeptidase L-serine-borate decreased the metabolism of leukotriene C4 in plasma. Dilution of plasma demonstrated that the dipeptidase was more active compared to the gamma-glutamyl transpeptidase. The metabolizing activities of plasma were functionally characterized by fractionating the plasma proteins.

Functional characteristics of leukotriene C4- and D4-metabolizing enzymes (gamma-glutamyl transpeptidase, dipeptidase) within human plasma.

Several properties of the leukotriene C4- and leukotriene D4-metabolizing enzymes within human plasma were studied after fractionation of the plasma proteins using ammonium sulfate precipitation. Leukotriene D4-metabolizing enzymes were widely distributed among the fractions obtained. They showed different pH optima (pH 6.5, pH 7.0 and pH greater than or equal to 8.5) and revealed a different degree of thermal stability. The results indicate the presence of more than one enzyme in plasma which interacts with leukotriene D4. EDTA and L-cysteine inhibited the metabolism of leukotriene D4. Two leukotriene C4-metabolizing activities (gamma-glutamyl transpeptidases) differing in their molecular weights were detected after gel filtration. Their molecular weights were estimated to be Mr greater than or equal to 150 000 and Mr between 55 000 and 100 000.

Metabolism of leukotriene B4 via reduction into dihydro-leukotriene B4 in human monocytes, alveolar macrophages, and U-937 cells.

The biological effects of leukotriene B4 (LTB4) within the microenvironment are controlled by rapid inactivation. In this regard human granulocytes convert LTB4 into omega-oxidated products (20-OH-LTB4 and 20-COOH-LTB4); moreover, we recently described the formation of unpolar metabolites of LTB4 in human tonsillar and lung macrophages. By means of high performance liquid chromatography (HPLC) we identified the main metabolite of LTB4 as dihydro-LTB4 (5,12-dihydroxyeicosatrienoic acid). Studies on a lymphocyte (74-78%), monocyte (19-22%), and basophil (less than 4%) containing cell fraction isolated from peripheral blood as well as peripheral monocytes purified by elutriation centrifugation revealed evidence that these cells metabolize LTB4 to a very low degree if incubated immediately after isolation. However, after culture for 24-72 h these cells showed a strongly increased capacity to metabolize LTB4. The pattern of metabolites in this cell fraction was identical to bronchoalveolar macrophages (purity greater than 95%). Similarly, the LTB4-reductase was expressed in differentiated human monocytic U-937 cells almost 5-7 h after the addition of dimethylsulfoxide (1.3%) or phorbol-myristate-acetate (16 nM). The expression of this pathway was blocked in the presence of cycloheximide (10 micrograms/ml) whereas actinomycin (3.8 micrograms/ml) had no effects. Dihydro-LTB4 was further metabolized by granulocytes probably via omega-oxidation; therefore, several metabolites could be detected by radioactive high performance liquid chromatography (HPLC) after incubation of bronchoalveolar cells consisting of macrophages and granulocytes with 3H-LTB4. Our data provide evidence for a unique role of macrophages to control the level of LTB4 by generation as well as metabolism into dihydro-LTB4.

Pharmacokinetic/pharmacodynamic evaluation of systemic effects of flunisolide after inhalation.

The pharmacokinetics and pharmacodynamics of flunisolide were studied in healthy volunteers after inhalation. In the morning on the day the study began, volunteers inhaled 0.5 mg of flunisolide with and without oral administration of charcoal, or 1 mg, 2 mg, and 3 mg of flunisolide with concomitant administration of charcoal. A placebo group was used to assess the endogenous cortisol, granulocyte, and lymphocyte baseline levels. Flunisolide plasma levels were determined by high-performance liquid chromatography using a tandem mass spectrometer as detector (HPLC/MS/MS). Cortisol plasma levels and differential white blood cell counts were obtained over 12 hours. An integrated pharmacokinetic/pharmacodynamic (PK/PD) model was applied to link the flunisolide plasma concentrations with the effects on lymphocytes, granulocytes, and cortisol. Maximum concentration levels of 3 to 9 ng/mL of flunisolide were observed after 0.2 to 0.3 hours for all of the investigated doses. The terminal half-life ranged from 1.3 to 1.7 hours. There was no statistical difference between treatments in the presence or absence of orally administered charcoal. The pharmacokinetic/pharmacodynamic (PK/PD) models satisfactorily described the time-courses of the effects on granulocytes, lymphocytes, and cortisol suppression. The resulting E50-values (concentrations to induce 50% of the maximum effect) concurred with the reported values of in vitro receptor binding affinities. The duration of the systemic effects were short because of the short half-life of the drug. Cumulative cortisol suppression increased with dose administration and ranged from 20% to 36%. The PK/PD simulations resulted in a smaller degree of cortisol suppression for the drug administered at 10 PM. The cumulative change from baseline was slightly smaller for the effects on granulocytes and lymphocytes than those on cortisol. This information promotes the comparison with other inhaled glucocorticoids.

Decreased expression of leukotriene B4 receptor sites on polymorphonuclear granulocytes of severely burned patients.

Polymorphonuclear granulocytes were isolated from patients with burn injury and the specific binding of (3H)leukotriene B4 was assessed. We observed a decreased receptor expression as compared to healthy donor cells, which may be the result of receptor downregulation as a consequence of cellular preactivation. In addition, leukotriene B4-synthesis was also reduced and differential cell counts demonstrated a shift from segmented neutrophils to immature cells. In survivors the values returned to normal parameters whereas nonsurvivors who succumbed in the course of generalized sepsis showed depressed cellular functions up to their death.

Modulation of leukotriene formation by cellular composition and exogenous leukotriene A4.

We investigated the interactions of exogenous leukotriene A4 (LTA4) with isolated cells in the presence or absence of cellular stimuli. The majority of isolated cells are able to transform exogenous LTA4 into LTB4 as well as LTC4. In eosinophils, LTA4 induced 15-hydroxy-eicosatetranoic acid formation and was converted into LTB4. The Ca-ionophore-induced generation of LTB4 from polymorphonuclear leukocytes or from the cell fraction containing lymphocytes, monocytes and basophils was significantly suppressed with LTA4 while the formation of LTC4 was increased. Conversely, the Na-fluoride- and fMLP-induced generation of LTB4 was significantly increased. Our results suggest that the stimulus and the cellular composition determine the pattern of the generated inflammatory mediators.

Determination of aspoxicillin (TA-058) by high-performance liquid chromatography. Stability at different temperatures.

A rapid and sensitive HPLC-method has been developed for the determination of serum concentrations of aspoxicillin (TA-058), a new semisynthetic beta-lactam antibiotic. Aspoxicillin was chromatographed with a phosphate buffer/methanol (92:8 v/v) mobile phase and a C-18 reversed phase column and was detected at a wavelength of 220 nm. The stability of aspoxicillin in serum and buffer at different temperatures was studied over a time period of 3 months. Furthermore, the degradation of aspoxicillin versus piperacillin was determined in serum and buffer at 37 degrees C. Aspoxicillin remains stable only at -70 degrees C whereas degradation has been observed at -20 degrees C and 4 degrees C. At 37 degrees C, 20% of aspoxicillin is degraded in serum within 24 h whereas piperacillin is completely degraded under the same conditions.

Degradation of acylaminopenicillins with regard to their pH dependency.

Determination of antibiotic concentration is performed in many biological fluids and tissues which all have different pH values. Therefore, we investigated the in vitro stability of three acylaminopenicillins (azlocillin, mezlocillin and piperacillin) in borate buffer by the HLPC technique with regard to pH dependency. HPLC allows the detection of all three substances together with their metabolites, penicilloate and penilloate, within 15 min. Decomposition was monitored at 37 degrees C during a 24 h incubation period (pH values ranged between pH 3.0 and 10.0). The highest degradation rates were observed with a buffer solution of pH = 10.0: 50% of the azlocillin and 83% of the mezlocillin were decomposed after 8 h while under the same conditions, piperacillin was completely decomposed already after 1 h. The highest stability was detected in borate buffer at pH values of 4.0, 5.0, and 6.0. At pH = 3.0, degradation was determined as follows: 31% of the piperacillin, 39% of the mezlocillin, and 45% of the azlocillin were decomposed after 24 h. Penilloic acid was identified as the main metabolite in contrast to buffer solutions with higher pH values which only revealed negligible amounts of this compound.

Mezlocillin in pleural effusions--analysis by HPLC.

Mezlocillin concentrations in the pleural fluid of six patients (42-76 years of age, suffering from cytologically confirmed malignant pleural effusions) were determined after intravenous infusion of 10 g mezlocillin. Serum and pleural fluid samples were withdrawn 15, 30, 45, 60 min, 2, 4, and 8 h post infusion. Detection of mezlocillin and its metabolites penicilloic acid and penilloic acid was carried out by means of high performance liquid chromatography (HPLC). Mezlocillin concentrations in serum increased up to 778 +/- 270 micrograms/ml after 15 min, steadily decreasing to 55 +/- 50 micrograms/ml (8 hours post infusion) comparable to the known pharmacokinetic behaviour of mezlocillin; in the pleural effusions mezlocillin levels increased up to 100 +/- 38 micrograms/ml after 1 h. This concentration was maintained throughout the following 7 h. Penicilloic levels ranged about 2-4% within serum, whereas levels below 1% were measured in the pleural fluid.

Determination of mezlocillin and its penicilloate and penilloate by high-performance liquid chromatography and stability of mezlocillin at different temperatures.

A method is described for the determination of mezlocillin and its metabolites penicilloic acid and penilloic acid in biological fluids by high-performance liquid chromatography. The stability of mezlocillin in serum and buffer was studied at different temperatures (4, -20, -70, and -196 degrees C) over a period of 6 months. Mezlocillin remained stable at -70 and -196 degrees C, whereas degradation was observed at -20 and 4 degrees C in serum and buffer.

Effects of cefaclor, cefetamet and Ro 40-6890 on inflammatory responses of human granulocytes.

The effect of three cephalosporins (cefetamet, cefaclor and Ro 40-6890) upon human granulocytes and their ability to modulate the chemiluminescence response, phagocytose, kill bacteria and generate leukotrienes was studied. In the presence of the cephalosporins there was a significant increase in phagocytosis of Escherichia coli. The bactericidal activity of human granulocytes for several other bacteria was also enhanced. Cefetamet and cefaclor increased the chemiluminescence response of human neutrophils to Pseudomonas aeruginosa and Proteus mirabilis in contrast to Ro 40-68790, which decreased the chemiluminescence response. The cephalosporins decreased the synthesis of leukotrienes from human neutrophils after stimulation with Escherichia coli and Staphylococcus aureus. These data emphasize the immunomodulatory functions of various cephalosporins on cells involved in host defence.

The role of lipid mediators in asthma (current views and perspectives).

Among the lipid mediators, leukotrienes and the platelet activating factor (PAF) have been attributed various roles in the immunopathology of allergic diseases. PAF has been shown to be a chemotactic factor for eosinophils, which conversely release leukotriene C4 on activation. PAF also may induce airway hyperreactivity. Leukotrienes (LTB4) are also powerful chemotactic factors and predominantly attract neutrophils, which stimulates dependent release lipid mediators (hydroxyeicosatetraenoic acids--HETES, leukotrienes, PAF) and thus amplify the inflammatory process. Leukotrienes (LTC4, D4, E4) induce bronchoconstriction, and mucus production. Lipid mediators are released by immunological as well as nonimmunological processes, e.g. by inflammatory stimuli. While analytical methods for leukotriene determinations have been established, the analysis of PAF is mainly carried out by biological studies. Further investigations with antagonists may be helpful to clarify the role of the lipid mediators during allergic and inflammatory processes.

Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity.

Influence of ciprofloxacin on leukotriene generation from various cells in vitro.

The effect of ciprofloxacin on leukotriene generation from human polymorphonuclear leucocytes (PMN) and the respective lymphocyte, monocyte and basophil (LMB) containing cell fraction has been studied. Furthermore, the influence on the LTB4-receptor expression of PMNL, as well as on the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets, was analysed. The antibiotic concentrations ranged from 0.5 to 4 micrograms/10(7) cells. Analysis of the generated leukotrienes was performed by high performance liquid chromatography (HPLC). The calcium-ionophore A23187 induced leukotriene generation from PMNs and LMBs was significantly suppressed by preincubation with ciprofloxacin in a dose-dependent manner. Incubation of PMNs with the same concentrations of ciprofloxacin induced an augmentation of the LTB4-receptor expression. Preincubation of human platelets led to a suppression of the calcium-ionophore induced 12-HETE generation at high concentrations (8 and 4 micrograms/10(8) cells) and to an increased synthesis of 12-HETE at lower concentrations (0.5, 1, and 2 micrograms ciprofloxacin/1 x 10(8) platelets).