The use of the colorimetric Jaffé method for the measurement of creatinine in mouse and rat plasma has been criticized as prior studies have shown a dramatic overestimation. We compared a colorimetric picric acid, an enzymatic, and a high-performance liquid chromatography (HPLC) method to assess their appropriateness for routine measurements of creatinine in plasma of healthy and diseased mice (n=61) and rats (n=56). For the colorimetric Jaffé method a pronounced overestimation is confirmed. Additionally the method showed interference with hemoglobin already in a very low, non-visible concentration range in rat plasma. The enzymatic measurement demonstrated a hemoglobin interference in mice, only when hemolysis was visible. The comparison between HPLC and the enzymatic measurement gave a good agreement between both methods in both species. Therefore the enzymatic method fulfills the requirements for a routine screening test for plasma creatinine in healthy as well as diseased mice and rats Kiover a broad concentration range.

The significance of cysteinyl leukotrienes was investigated in patients with liver diseases by measurements of leukotriene E4 and N-acetyl-leukotriene E4 in urine. A marked increase of renal cysteinyl leukotriene excretion was observed in patients with cirrhosis without and with ascites, intrahepatic cholestasis, and obstructive jaundice as compared with healthy subjects (leukotriene E4: means 82, 264, 221 and 142 versus 40 nmol/mol creatinine, respectively; N-acetyl-leukotriene E4: means 25, 64, 61 and 47 versus 13 nmol/mol creatinine, respectively). The urinary concentration of leukotriene E4 was positively correlated with the one of N-acetyl-leukotriene E4 (r = 0.81, p < 0.001). In patients with cirrhosis, the excretion of cysteinyl leukotrienes was strongly increased in patients in Child-Turcotte stage C as compared with those in Child-Turcotte stages A and B. In patients with intrahepatic cholestasis and in those with obstructive jaundice, the excretion of leukotriene E4 plus N-acetyl-leukotriene E4 was positively correlated with total serum bilirubin. In patients with cirrhosis and in those with obstructive jaundice, the cysteinyl leukotrienes in urine were negatively correlated with creatinine clearance. The elevated renal excretion of cysteinyl leukotrienes decreased after biliary drainage in patients with obstructive jaundice. These data support the concept that increased urinary excretion of cysteinyl leukotrienes in patients with cirrhosis is due to a reduced functional liver mass and that in patients with cholestasis it is mainly due to an impaired elimination into the biliary tract that results in a diversion to renal excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments on the metabolism and excretion of i.v. administered selectively labeled [3H8]1eukotriene C4 in bile duct-cannulated guinea pigs indicated predominantly biliary excretion of tritium. The major leukotriene metabolite in bile was identified as leukotriene D4. By monitoring leukotriene excretion radioimmunochromatographically, it was shown that guinea pigs suffering from anaphylactic shock produce leukotriene D4 endogenously. Immunological challenge of animals sensitized to ovalbumin was accompahied by an increase of biliary leukotriene D4 concentrations from 10 ± 1 to 86 ± 10 nM (mean ± SEM, n = 5, P < 0.001). When considering that bile flow was decreased to abobt half after challenge, the excretion rate of leukotriene D4 in bile increased from 0.88 ± 0.16 before to 3.18 ± 0.38 pmolmin-'-kg-' after challenge (mean ± SEM, n = 5, P < 0.002). It is concluded that systemic anaphylaxis in the guinea pig is associated with endogenous generation of leukotriene C4 (Up to 1 nmol/kg during a 30-min period after the challenge).Leukotriene C4 (LTC4) is a biologically active substance presumed to play major roles as a mediator of allergic and anaphylactic reactions (1, 2). It is formed by basophilic (3) and eosinophilic leukocytes (4), monocytes (5), macrophages (6), and mast cells (7). In cells having IgE receptors, bridging of these receptors by divalent anti-IgE receptor antibodies or by interaction between receptor-bound IgE and anti-IgE will induce LTC4 formation (5-7). Leukotriene formation has also been demonstrated in other in vitro models of immediate hypersensitivity (8-10). The biological actions of LTC4 comprise induction of airway obstruction, constriction of coronary arteries, hypotension, and plasma extravasation (11)(12)(13)(14). Leukotriene formation in vivo may mediate anaphylactic shock symptoms and cause the death of an animal. To prove the presumed mediator role of this substance in anaphylactic reactions, it is necessary to demonstrate its endogenous formation during anaphylaxis. Studies on LTC4 metabolism have revealed rapid catabolism by various transformations of the peptide substituent (15). Three metabolites have been demonstrated to be excreted as end-products: leukotriene E4 (LTE4) (16) in man and N-acetyl-LTE4 plus N-acetyl-11-trans-LTE4 (17) in the rat. By monitoring biliary N-acetyl-LTE4 levels, endogenous leukotriene formation in the rat was demonstrated in vivo after tissue trauma and endotoxin shock (18,19). We now wish to report evidence for endogenous LTC4 production during anaphylactic shock in guinea pigs.

Leukotriene C4 (LTC4) underwent rapid elimination from the circulating blood and was extensively converted to LTD4 within the vascular space of the guinea pig. To mimic the elimination and metabolism of endogenous LTC4 generated during anaphylaxis, 14,15-3H-labeled LTC4 was infused intravenously over a period of 15 min, leading to a recovery in bile of 85% of the infused LT radioactivity within 2 h. Corresponding to the tracer studies, LTD4 and, to a lesser extent, LTC4 were the predominant endogenous cysteinyl LTs in guinea pig bile. The biliary production rate of endogenous LTD4 increased from 0.3 +/- 0.1 to 6.2 +/- 1.8 pmol x min-1 x kg-1 (p less than 0.001) during anaphylactic shock induced by intravenous injection of OVA (0.2 mg/kg) into sensitized guinea pigs. A novel LT biosynthesis inhibitor (MK-886; 10 mg/kg, i.v., 15 min before antigen challenge) suppressed the antigen-induced cysteinyl LT production by greater than 92% (p less than 0.001). This inhibition of systemic LTC4 formation was associated with a complete protection against lethal anaphylactic shock in animals pretreated in addition with the H1 receptor antagonist pyrilamine. Pretreatment with either the inhibitor of LT synthesis or the histamine receptor antagonist reduced the lethality during anaphylactic shock from 100 to 60 and 78%, respectively. In artificially ventilated, pyrilamine-pretreated animals, the antigen-induced decrease in dynamic lung compliance and the rise in hematocrit were significantly reduced (p less than 0.05) by pretreatment with the inhibitor of LT synthesis. Dexamethasone at high doses (10 mg/kg, i.p., once daily for 7 d, or in a single dose of 10 mg/kg, i.v., 3.5 h before challenge) had no inhibitory effect on LT generation during anaphylaxis in vivo. However, in resident peritoneal macrophages, harvested from these dexamethasone-treated sensitized guinea pigs and stimulated with zymosan, both cysteinyl LT and 6-keto-PGF1 alpha formation were strongly suppressed. These studies indicate an important role of cysteinyl LTs in systemic anaphylaxis in vivo and demonstrate the blockade of anaphylactic LT generation by a novel inhibitor of LT biosynthesis (MK-886) but not by dexamethasone.