The role of mdr2 in manganese-bilirubin induced cholestasis in mice.

The pathogenesis of manganese-bilirubin (Mn-BR) induced cholestasis has only been studied in rats and is associated with alteration in the hepatic homeostasis of cholesterol and phospholipids. Multidrug resistance-2 (mdr2) transporter, which mediates excretion of these lipids, is suggested to be involved in this phenomenon. The present study was undertaken to examine if Mn-BR induced cholestasis is reproducible in mice, then to clarify the role of mdr2 in its pathogenesis, using mice with disrupted mdr2 gene (mdr2 (-/-)). Results showed that Mn-BR combination decreased bile flow in mice. This reduction in bile flow was similar in mdr2 (-/-) and the wild type mdr2 (+/+). Furthermore, the change in biliary lipid excretion was comparable in both genotypes. These data indicate that Mn-BR induced cholestasis is reproducible in mice and provide evidence that mdr2 alteration is not a primary event in this form of cholestasis.

Synergistic role of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase in the pathogenesis of manganese-bilirubin-induced cholestasis in rats.

Manganese (Mn) and bilirubin (BR) induce intrahepatic cholestasis when injected sequentially. It was suggested that accumulation of newly synthesized cholesterol in the canalicular membrane is an initial step in the development of cholestasis. To clarify the involvement of cholesterol in the pathogenesis of Mn-BR-induced cholestasis, we examined biliary secretion and liver subcellular distribution of lipids in the cholestatic conditions (Mn-BR combination). We also examined hepatic metabolism of cholesterol under cholestatic and noncholestatic (Mn or BR given alone) conditions. The Mn-BR combination reduced bile flow by 50%, and bile acid, phospholipids, and cholesterol output by 42, 75, and 90%, respectively. There was a dramatic impairment of cholate excretion but not chenodeoxycholate excretion. Phosphatidylcholine species secreted into bile were unchanged, and microsomal total phospholipid content was significantly increased. Although there was no changes in liver membrane phospholipid content, the cholesterol/phospholipid ratio was increased by more than 50% in the canalicular fraction. Despite the increased concentration of cholesterol in canalicular membrane the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, key enzyme in cholesterol synthesis, and cholesterol 7alpha-hydroxylase, key enzyme in cholesterol conversion to bile acids, were significantly reduced. However, the injection of Mn alone significantly increased both enzymes, while BR alone inhibited cholesterol 7alpha-hydroxylase by 62%, without affecting HMG-CoA reductase. In conclusion, the critical cholestatic events in Mn-Br-induced cholestasis appear to be mediated through the synergistic effects of Mn and BR acting on synthesis and degradation of cholesterol.

Simultaneous evaluation of HMG-CoA reductase and cholesterol 7alpha-hydroxylase activities by electrospray tandem MS.

Simultaneous evaluation of HMG-CoA reductase and cholesterol 7alpha-hydroxylase activities by electrospray tandem mass spectrometry (ES-MS-MS) was performed. The assay was based on the measurement of mevalonolactone (MVL) and 7alpha-hydroxycholesterol (7alpha-OHC) produced by the incubation of HMG-CoA with hepatic microsomes in the presence of NADPH and glucose-6-phosphate dehydrogenase. Following extraction and purification using a cyanopropyl cartridge, MVL and 7alpha-OHC were analyzed, without derivatization, by ES-MS-MS. The analysis was achieved in 5 min. Calibration curves were made for MVL and 7alpha-OHC, and were linear from 0 to 100 microg. The recovery was >97%. The procedure was validated under similar calibration and recovery experiments, by measuring the above mentioned products as dimethylethylsilyl ether derivatives using the classical technique of GC-MS. Data obtained by ES-MS-MS and GC-MS showed a good correlation, with no significant differences. ES-MS-MS is a simple and reliable method for the evaluation of HMG-CoA reductase and cholesterol 7alpha-hydroxylase activities in liver microsomal preparations.