Total and free endotoxin release in time from cultures of Escherichia coli by different antibiotics was studied in vitro for 4 h in relation to the antibiotic effect on viable counts and morphological features of the test cultures. The most rapid fall in viable counts was seen after treatment with imipenem or the combination of imipenem with tobramycin, accompanied by an early, but minimal increase (1.8-fold) of the total (free plus cell-bound) endotoxin level at 1 h. Total endotoxin levels increased approximately 5-fold upon incubation with ceftazidime, tobramycin or the combination of tobramycin with cefuroxime, whereas incubation with cefuroxime or aztreonam alone caused a late 22-and 49-fold increase in total endotoxin, respectively, at 4 h. In chloramphenicol treated cultures there was still an increase in viable counts during therapy, resulting in an ultimately 78-fold increase of mean levels of total endotoxin. Free endotoxin levels increased approximately 6-fold within 1 h upon treatment with imipenem, alone or in combination with tobramycin, or ceftazidime as the result of rapid lysis of bacteria. Treatment with cefuroxime or aztreonam induced a relatively late but much higher release of free endotoxin (118-and 222-fold, respectively), which was due to the formation of long filamentous structures during the first 2 h of incubation and eventually cell lysis. Both tobramycin and the combination of tobramycin with cefuroxime caused a more gradual rise in free endotoxin, with a +/- 15-fold increase in free endotoxin at 4 h. In chloramphenicol treated cultures, as in the control cultures, the level of free endotoxin remained proportional to the amount of viable organisms. We also studied plasma endotoxin levels in 20 patients with septic shock. 10 out of these 20 patients had a detectable endotoxemia (greater than 5 ng/l) on admission. We describe the patterns of plasma endotoxin in these patients during the first 24 h of antibiotic treatment. We conclude that, in the in-vitro study, values of total endotoxin, free endotoxin, and the rate of release of endotoxin varies with the antibiotic used. We also demonstrate that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics.
In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.
Increased ALA intakes decrease the estimated IHD risk to an extent similar to that found with increased LA intakes. Group nutritional education can effectively increase fish intake.
SummaryWe investigated sensitivity to activated protein C (APC) in 43 men, 42 women not using oral contraceptives (OC) and 38 women using OC containing 30.0-37.5 μg ethinyl estradiol. A commercially available kit was used. Men were more sensitive to APC as compared with women not using OC (p <0.005), but this difference seems not to be of clinical importance. Women using OC showed to be significantly less sensitive to APC, reflected by a lower APC-ratio, as compared with men (p <0.005) and women not using OC (p <0.05).So, in normal individuals the APC-sensitivity differs according to sex and estrogen intake. This should be taken into account when interpreting APC-ratios.
SummaryProtein S, an important cofactor of activated protein C, and C4b-binding protein were purified from human plasma. Specific antibodies against the purified proteins were raised in rabbits and used for the development of immunologic assays for these proteins in plasma: an immunoradiometric assay for protein S (which measures both free protein S and protein S complexed with C4b-binding protein) and an electroimmunoassay for C4b- binding protein. Ranges for the concentrations of these proteins were established in healthy volunteers and patients using oral anticoagulant therapy. A slight decrease in protein S antigen was observed in patients with liver disease (0.78 ± 0.25 U/ml); no significant decrease in protein S was observed in patients with DIC (0.95 ± 0.25 U/ml).Criteria were developed for the laboratory diagnosis of an isolated protein S deficiency
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