J. Knöller

Serum CD14 levels in poly traumatized and severely burned patients

Recently it has been demonstrated that the CD14 molecule which is expressed on monocytes and macrophages serves as a receptor for lipopolysaccharide (LPS) bound to LPS‐binding protein (LBP) and thus mediates LPS‐induced tumour necrosis factor (TNF) production. Here we report that CD14 is found as a soluble (s) molecule in serum. In healthy volunteers sCD14 levels (mean ± s.e.m.) were 3.7±0.05 μg/ml (n= 30, 25–50 years of age) as determined by ELISA (detection limit 20 μg/ml serum) using two monoclonal antibodies in a sandwich technique. In polytraumatized patients (n= 16) significantly decreased levels (1.7 ± 0.3) were detected immediately after the trauma, which increased to 4.9±0.3 μg/ml within the first 6 days post trauma. sCD14 remained elevated during the first 14 days post trauma in patients with the most severe injuries (injury severity score > 45 points), whereas a return to normal levels was observed in patients with an injury score of < 45 points. In addition, the levels of the high‐density lipoproteins that partially inactivate free endotoxin are significantly decreased post trauma. No correlation between parameters of inflammation (C3a and neopterin levels, leucocyte counts, amount of band cells), liver function and sCD14 levels was established. Comparable to polytraumatized patients, increased sCD14 serum levels were observed in five patients with burn trauma (burned area > 35%) within the second week post trauma when clinical signs of septicaemia were evident.

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.

Application of high-performance liquid chromatography of some antibiotics in clinical microbiology

During recent years high-performance liquid chromatography has become an excellent tool for the determination of antibiotics in biological fluids. Compared with biological assays, the major benefits of this method are specificity and rapidity. In particular, the determination of biologically inactive metabolites emphasizes that this technique plays an outstanding role for the analysis of antibiotics. This paper describes how the method can be used in the analysis of several antibiotics and demonstrates the efficacy of this method for clinical microbiology. Methods for the determination in biological fluids of acylaminopenicillins (azlocillin, mezlocillin, piperacillin and aspoxicillin), quinolones (ciprofloxacine, norfloxacine and ofloxacine), a penem (imipenem) and a cephalosporin (cefixime) are summarized. Furthermore, their application to in vitro studies and their trial in clinical studies are described.

Generation and Metabolism of Leukotrienes in Granulocytes of Patients with Cystic Fibrosis

We studied the generation and metabolism of lipoxygenase products in peripheral granulocytes from children suffering from cystic fibrosis (CF). Peripheral granulocytes were stimulated at different times (days) before and during anti-infectious treatment with the Ca ionophore (7.5 microM, 5 and 20 min), opsonized zymosan (2 mg) and arachidonic acid (50 microM); the amount of lipoxygenase products in the cell supernatants was determined by high performance liquid chromatography. Granulocytes from patients with CF, compared to an age-matched control group, showed an increased omega-oxidation of the synthesized leukotriene (LTB4) into 20-OH- and 20-COOH-LTB4 after stimulation with the Ca ionophore (ratio of LTB4 versus omega-oxidated products in patients with CF: 0.77 +/- 0.07, mean +/- SEM, n = 11; control group: 1.07 +/- 0.1, n = 11, p less than 0.01) whereas the combined amounts of LTB4 and its omega-oxidated products did not differ significantly. A comparable profile was observed with opsonized zymosan. Stimulation of the cells with the Ca ionophore combined with arachidonic acid led to a significantly increased formation of lipoxygenase products in the patient group, whereas only a slight enhancement was observed in the control group. During the 14-day anti-infectious treatment a normalization of the altered pattern was observed. 12-Hydroxyeicosatetraenoic acid (12-HETE) production from platelets within the granulocyte fraction was significantly depressed in the CF group compared to the controls (38.5 +/- 12.5 versus 339 +/- 93 ng/5 +/- 10(6) cells, p less than 0.005). Within the CF group a strong correlation between the release of LTB4 and its metabolites, the production of 12-HETE and clinical (e.g. pO2, FEV1) and laboratory findings (e.g. IgE and IgG levels, C-reactive protein) was established. Our data suggest that the inflammatory process in patients with CF is associated with an alteration of the lipoxygenase pathway of granulocytes which correlates with the clinical signs of inflammation.

Functional characteristics of leukotriene C4- and D4 -metabolizing enzymes (γ-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.

Arachidonic acid metabolites from polymorphonuclear leukocytes of healthy donors, severely burned patients and children with cystic fibrosis--routine monitoring by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for the determination of arachidonic acid metabolites was developed. This method provides a clear and simple separation of the omega-oxidation products as well as of leukotriene B4 and 5S,12S-dihydroxy-6-cis-8-trans-10-trans-14-cis-eicosatetraenoic acid. Furthermore, a solvent switch enables the detection of the monohydroxyeicosatetraenoic acids together with the cysteinyl-leukotrienes as well as leukotriene B4 and the dihydroxyeicosatetraenoic acids within 70 min (including column equilibration for 10 min). The efficiency of the method for the routine monitoring of leukotrienes was performed with human polymorphonuclear leukocytes of healthy donors, severely burned patients and of children with cystic fibrosis. The advantages of this method include high sensitivity and easy handling, which are important for routine analyses of the arachidonic acid metabolites.

Leukotriene generation in patients with multiple injuries

We determined the generation and metabolism of lipoxygenase products in isolated granulocyte fractions of patients with multiple trauma (n = 9) and compared the results with those of healthy volunteers (n = 8). The supernatants of stimulated cells were analyzed by high-performance liquid chromatography. During the first week after injury a significantly reduced capacity to generate LTB4 and an increased metabolism of LTB4 into omega-oxidated products (20-OH-LTB4 and 20-COOH-LTB4) were observed after stimulation of the granulocytes with Ca ionophore. The depressed leukotriene production could be partly abrogated by the addition of arachidonic acid. These findings are comparable with alterations previously described in severely burned patients with postburn sepsis. Additionally, an elevated production of LTC4 by peripheral granulocyte fractions was observed in two patients suffering from adult respiratory distress syndrome (ARDS) as well as an increased number of eosinophils during the time of lung dysfunction. Analysis of bronchoalveolar lavage in patients with multiple trauma (11 patients with ARDS and 11 patients without ARDS) by a specific radio-immunoassay confirmed an elevated production of cysteinyl-leukotrienes.(ABSTRACT TRUNCATED AT 250 WORDS)

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 ω‐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 (<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 >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 μg/ml) whereas actinomycin (3.8 μg/ml) had no effects. Dihydro‐LTB4 was further metabolized by granulocytes probably via ω‐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.

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.

Altered arachidonic acid metabolism in granulocytes of polytraumatized patients

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.