1. A technique is described for the continuous collection of bile, for long periods, from unrestrained conscious rats housed in standard glass metabolism cages. 2. Bile is collected in cooled vessels outside the cage through a cannula which is exteriorized at the back of the neck and is protected from damage by an outer covering. 3. A minimum recovery period of three days is allowed after the operation, by which time liver function and intestinal motility are normal. 4. An extension of the technique can be used to assess enterohepatic circulation.
The considerable interest in the Group II secreted phospholipase A, (PLA,; EC 3.1.1.4) as a possible mediator of the inflammatory response is because of the association of elevated levels of this enzyme with inflammatory disorders as in septic shock patients.An important question in this area of inflammation research is the origin of this serum PLA,. In a previous communication, we have highlighted the liver as a possible source of this enzyme particularly as the activity of this enzyme was elevated in liver after pretreatment of animals with endotoxin (1).In a separate study by Besler and Grimble [2], an enhanced inflammatory response to endotoxin challenged in rats fed for 28 days on a 10% corn oil (polyunsaturated) diet was observed as compared to animals on an olive oil (monounsaturated) diet and in rats fed an equivalent saturated diet consisting of butter fat.In this communication, we have investigated the effect of endotoxin challenge on the activity of the liver Group II PLA, enzyme which is normally associated with the mitochondrial fraction from a rat liver homogenate [3] and, in particular, how this endotoxin challenge might be modulated by diet.Male Wistar rats (1 50 -2009 body wt), were used throughout the study. In the acute endotoxin response investigation, the rats were injected intraperitoneally with a dose of 10 mglkg of pure LPS from Escherichia coli K-235. After a 4 hour incubation, the rats were sacrificed. The liver was removed and homogenised using the same method as Hatch eta/ [l] to obtain the mitochondrial fraction. A celldebris and nuclei free supernatant was centrifuged at 105,000 g for 60 min to isolate a total membrane fraction. The pellets were resuspended in buffer B (0.25 M sucrose, 20 mM Tris/HCI, pH 7.4, 1 mM EDTA, 1 M KCI). The samples were assayed for PLA, [4]. These samples were also sonicated and centrifuged at 350.000 g for 15 min in a Beckman Optima TLX ultracentrifuge. The supernatants were then assayed for PLA, activity [4].In the 28 day diet study, rats were fed on a standard chow diet and nine experimental diets which varied in the type and level of fat. Corn oil, olive oil and butter were included in the diet at different concentrations -5%, 10% and 20% by weight. A single dose of 0.8 mg/kg of Escherichia coli endotoxin (DIFCO strain 055:B9) was administered interperitonally to half the rats. A similar dose of sterile saline was given to the control rats. After 24 hours the rats were sacrificed. The livers were removed and processed using the procedure essentially described above. One possible explanation for the endotoxin effect on enhancing PLA, activity [l] might be a redistribution and/or increased releasibility of PLA, activity. In order to address this problem the Table 1. The effect of endotoxin on PLA, activity in rat liver.Sample was assayed as a crude fraction without the final centrifugation step at 350.000 g for 15 min to remove the membrane Darticles after KCI treatment and sonication.
1. Following single intramuscular doses of [14C]fluprostenol (0.5--2.4 micrograms/kg) to three female horses and to three gelded male horses, radioactivity was present in the plasma within 5 min; peak concn. (0.32--1.30 ng/ml fluprostenol equiv.) occurred 5 to 90 min after injection. Radioactivity was still present in the plasma of the females after three days. About 88% of fluprostenol is bound to plasma proteins. 2. Radioactivity was present in the parotid saliva of the gelded male horses within 10 min. Peak concn. (45--91 pg/ml fluprostenol equiv.) occurred from 5 min to 1 h after injection. Saliva : plasma concn. ratios varied inversely with saliva flow rate and limiting ratios were 0.33 and 0.41 for the combined results of two experiments on each of two male horses; the calculated value is 0.46 Chromatography indicated that the majority of plasma and saliva radioactivity was [14C]fluprostenol. 3. Excretion of radioactivity in the urine was rapid and virtually complete 12 h after dosing. The total radioactivity excreted in urine by the female horses was 45% of the dose (96 h) and by the gelded male horses 53% (30 h). About 30% of the radioactivity present in the urines was unchanged fluprostenol. 4. Faecal excretion, which was substantially complete after 2 days, accounted for 32% of the radioactivity administered to the female horses. 5. Tissue conc. of radioactivity in the female horses at four days were below the limits of detection (90 pg/g), but 0.2--0.9% of the dose was detected at the site of injection.
1. Disposition of the 3R,4S(+) and 3S,4R(-) enantiomers of the racemic antihypertensive drug cromakalim has been studied in rats and cynomolgus monkeys using the 14C-drug labelled in either the 3R,4S(+) or the 3S,4R(-) enantiomer. 2. After oral administration to rat, blood concentrations of the 3R,4S(+) enantiomer were up to fourfold higher than those of the 3S,4R(-) enantiomer. Metabolism of the former was not as extensive as that of the latter and consequently plasma and urinary radiometabolite patterns were quantitatively different. 3. In contrast to rat, there were much greater differences in the disposition of the two enantiomers following oral administration of cromakalim to the cynomolgus monkey. Plasma concentrations of the 3R,4S(+) enantiomer were approximately 100 x those of the 3S,4R(-) enantiomer and the rate of urinary 14C elimination for the 3R,4S(+) enantiomer was much faster than that for the 3S,4R(-) enantiomer. Plasma and urinary radiometabolite patterns were very different for the two isomers. Metabolic end products of the 3R,4S(+) enantiomer were predominantly phase I metabolites whereas the 3S,4R(-) enantiomer was almost entirely metabolized by glucuronidation. 4. A study of the racemic drug alone would have led to a misunderstanding of the fate of the compound in these species.
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