Endotoxin is known to cause a dose-dependent impairment of hepatic bile secretion, organic anion excretion, and activity of Na+,K'-activated ATPase. Since it is possible that this impaired excretory function is a result of direct interaction of endotoxin with hepatocytes, we examined: (a) the excretion of endotoxin into bile, and (b) its association with an hepatocyte-enriched, Kupffer cell-depleted population of liver cells. Escherichia coli 0127:B8 endotoxin (17 to 54 pg per 100 g m body weight) was given i.v. to male Sprague Dawley rats and bile collected up to 48 hr. The expected significant decrease in bile secretion was seen, confirming the biological effectiveness of the endotoxin. Endotoxin was quantitated by a gas chromatography/mass spectroscopy method which detects the P-hydroxymyristic acid in the lipid A moiety of endotoxin. Approximately 7% of the administered dose of endotoxin was recovered as P-hydroxymyristic acid in bile in the 48 hr following injection. Approximately two thirds of the administered dose of endotoxin was recovered as P-hydroxymyristic acid in the whole liver. Further analysis of the /3-hydroxymyristic acid in bile and liver by Folch extraction and thin-layer chromatography was performed. The P-hydroxymyristic acid in bile was associated with polar and nonpolar metabolites of endotoxin. The P-hydroxymyristic acid recovered in the liver distributed similarly in Folch extraction to intact endotoxin added in vitro. Endotoxin quantitated as P-hydroxymyristic acid was measured in whole liver homogenate and hepatocyte-enriched preparations 3 hr following i.v. administration. The mass abundance by gas chromatography/mass spectrometry of P-hydroxymyristic acid per lo6 hepatocytes was similar in crude liver homogenate and the hepatocyte-enriched fractions indicating the presence of substantial amounts of P-hydroxymyristic acid in association with parenchymal liver cells. Thus, our studies indicate that the bulk of the P-hydroxymyristic acid of endotoxin given i.v. to rats is found in hepatocytes in a form suggesting minimal degradation of the endotoxin. A small proportion of the P-hydroxymyristic acid associated with the endotoxin in the liver is then released slowly into bile as a complex mixture of polar and nonpolar metabolites of endotoxin.
The liver plays the major role in clearing circulating endotoxin from the blood of all experimental animals studied. Using (51Cr) -labeled endotoxin, within 15 min after injection, the liver contained 65% of the administered radioactivity (1). In vzuo studies have shown subcellular localization of 14C-endotoxin in the nuclear and mitochondrial fractions of hepatocytes as well as Kupffer cells (2). In a recent study, (I2'I) radioactivity was present in the gallbladder bile of rabbits following i.v. injection of '251-lipopolysaccharide (LPS) suggesting the possibility that LPS might be processed by hepatocytes and released into the bile canalicular system (3). However, the same