Kuniharu Nakai

Phospholipid alterations in hepatocyte membranes and transporter protein changes in cholestatic rat model.

Biliary components are transported by hepatic adenosine triphosphate-binding cassette (ABC) transporters that are located in canalicular membranes. Physiological transporter function is related to membrane fluidity, which is modulated by the phospholipid composition of the lipid bilayer. We hypothesized that cholestasis may alter transporter function by modifying phospholipid species to protect the cell from cholestatic damage. Therefore, we examined the expression of ABC transport proteins and their mRNA levels in canalicular membrane vesicles isolated from rat liver 6 hr or three days after bile duct ligation. Membrane lipid composition and membrane fluidity of both sinusoidal and canalicular membrane vesicles were also examined. By 6 hr after bile duct ligation, we found a clear increase of mdr2 and bsep mRNA. These changes were associated with an increase of mdr-Pgp and with a clear decrease of mrp2 protein, and small decrease of bsep protein. In addition, mdr1b mRNA showed a strong increase by three days after bile duct ligation. Canalicular membrane fluidity decreased in a marked time-dependent manner, whereas sinusoidal membranes showed biphasic changes: increased fluidity at 6 hr and a decrease at three days. These changes were closely related to the changes of membrane lipid constitution; the saturated/unsaturated fatty acid ratio increased for phosphatidylcholine in canalicular membrane and the reverse occurred in sinusoidal membrane, and those for sphingomyelin showed the opposite pattern. We conclude that cholestasis causes modulation of ABC transporters as well as that of the lipid constitution in lipid bilayer. These may confer cytoprotective resistance to hepatocytes against cholestatic stress.

Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation.

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by beta-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of beta-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without beta-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with beta-glucuronidase-free bile, (1) beta-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96+/-17.0 vs. 114+/-20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with beta-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without beta-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with beta-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of beta-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.

Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influences of bilirubin on bile lithogenicity.

Bilirubin is found in the center of cholesterol gallstones, but its pathogenic role in their formation is unknown. Bilirubin causes a disproportionate reduction of biliary lipid secretion without affecting bile salt secretion in association with a change of biliary lecithin species, which modulates the cholesterol crystallization process. Therefore, the present study investigated whether bilirubin can influence the cholesterol crystallization procedure, and the mechanism(s) of any such action. Supersaturated model bile was prepared (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 9.0 g/dl, and cholesterol saturation index of 1.8), and cholesterol crystallization was monitored over time using a spectrophotometer and video-enhanced differential contrast microscopy in the absence or presence of bilirubin (at a final concentration of 10 microM, 20 microM, 40 microM, and 100 microM). Bilirubin enhanced the onset of cholesterol crystallization by 50%, whereas the crystal growth rate and final crystal mass were reduced at a high concentration of bilirubin. Taken together, these results suggest that bilirubin influences the cholesterol crystallization process, by either a direct interaction with biliary lipids that alters metastability, an indirect alteration of the bile salt-micellar lipid holding capacity, or both. Thus, bilirubin may play a role in the pathogenesis of both cholesterol and pigment gallstones.

Factors affecting gallstone recurrence after successful extracorporeal shock wave lithotripsy.

Ninety-six patients treated successively for symptomatic cholelithiasis with extracorporeal shock wave lithotripsy (ESWL) and oral bile acid therapy consisting of ursodeoxycholic acid in daily dosages of 600 mg were prospectively followed for gallstone recurrence for a median of 13 months. Ultrasonography was performed to detect stone recurrence at 3, 6, and 12 months, and then yearly after the termination of therapy. Recurrent stones were found in 17 patients (18%). The cumulative probability of gallstone recurrence was 15.8% at 12 months, 26.1% at 24 months, and 30.7% at 36 months. The probability of stone recurrence over the entire period of observation was not dependent on stone number, whereas the median interval to detection of recurrence was significantly shorter in the patients with multiple stones (2 months) than in those with solitary stones (8 months) (p < 0.05). The rate of impaired gallbladder contractility was higher in patients with recurrence (8/15, 53.3%) when compared with those with no recurrence (15/72, 20.8%) (p < 0.01). Neither age, gender, or stone characteristics predicted stone recurrence. Only one patient with a recurrence reported biliary pain. Of the 15 patients with recurrent stones who opted for further nonsurgical treatment, complete stone disappearance was achieved in 10. Impaired gallbladder function may predict gallstone recurrence after ESWL.

Role of Phospholipase A2 in Cholesterol Gallstone Formation Is Associated with Biliary Phospholipid Species Selection at the Site of Hepatic Excretion

Phospholipase A2 plays a role in cholesterol gallstone development by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. Lysolecithin and polyunsaturated free fatty acids are known to stimulate the synthesis and/or secretion of gallbladder mucin via a prostanoid pathway, leading to enhancing cholesterol crystal nucleation and growth, and therefore, the action of phospholipase A2 is associated, in part, with bile phospholipid fatty acid. To clarify this hypothesis, we evaluated the effect on bile lipid metastability in vitro of replacing phospholipids with lysolecithin and various free fatty acids. Supersaturated model biles were created with an identical composition (cholesterol saturation index, 1.8; egg yolk lecithin, 34 mM; taurocholate, 120 mM; cholesterol, 25 mM) except for 5%, 10%, or 20% replacement of egg yolk lecithin with a combination of palmitoyl–lysolecithin and a free fatty acid (palmitate, stearate, oleate, linoleate, or arachidonate), followed by time-sequentially monitoring of vesicles and cholesterol crystals using spectrophotometer and video-enhanced differential contrast microscopy. Replacement with hydrophilic fatty acids (linoleate and arachidonate) reduced vesicle formation and promoted cholesterol crystallization, whereas an enhanced cholesterol-holding capacity was evident after replacement with hydrophobic fatty acids (palmitate and stearate). These results indicate that the effect of phospholipase A2 on bile lithogenecity is modulated by the fatty acid species in bile phospholipids, and therefore, that the role of phospholipase A2 in cholesterol gallstone formation is dependent, in part, on biliary phospholipid species selection at the site of hepatic excretion.

Impaired gallbladder mucosal function in aged gallstone patients suppresses gallstone recurrence after successful extracorporeal shockwave lithotripsy.

Background:  Absorption of water, as well as emptying of bile, are important functions of the gallbladder. We studied the changes of gallbladder function with age in gallstone patients and their influence on the outcome of extracorporeal shockwave lithotripsy (ESWL).

Dose-Dependent Conjugation of Sulfobromophthalein and Hepatic Transit Time in Bile Fistula Rats

Sulfobromophthalein (BSP) is selectively taken up by the liver and secreted into the bile as unconjugated and conjugated forms. Our previous study demonstrated that unconjugated BSP, but not conjugated BSP, caused the dissociation of biliary lipid secretion from that of bile acids, suggesting that the hepatic BSP conjugation rate partly regulated biliary lipid secretion. To evaluate the mechanisms through which biliary lipid secretion is regulated by exogenous organic anions, we intravenously administered BSP to male Sprague–Dawley rats at various doses either continuously or as a bolus. Then the relationship of the dose of BSP to its conjugation rate, hepatic transit time, and biliary lipid secretion was determined. BSP decreased biliary secretion of cholesterol and phospholipids in a dose-dependent manner without affecting bile acid secretion. In contrast, the proportion of conjugated BSP in bile was associated with the dose. Although the serum clearance of BSP after bolus infusion was constant regardless of the dose administered (50 or 200 nmol/100 g), BSP secretion was delayed with increasing doses: unconjugated BSP was secreted predominantly in the early phase (0–15 min after bolus injection), and conjugated BSP was the predominant form in the late phase (15–30 min). Pretreatment with colchicine reduced the conjugation rate and hepatic transit time of BSP, suggesting that the microtubule-dependent vesicle pathway plays a role in biliary excretion and conjugation of BSP. We conclude that biliary lipid secretion is influenced by organic anions with an affinity for bile acids such as BSP and that this effect is dependent upon the hepatic metabolic rate, i.e., conjugation rate. The hepatic transit time also plays a key role in this process by influencing metabolism.

Choleretic action of diosgenin is based upon the increases in canalicurar membrane fluidity and transporter activity mediating bile acid independent bile flow

It is known that diosgenin alters the lipid composition of hepatic plasma membranes as well as bile lipids. We recently reported that changes in the lipid composition of the canalicular membrane bilayer were associated with alterations of membrane fluidity. Therefore, the present study was performed to determine the effect of diosgenin on bile secretion, focusing on canalicular membrane composition, membrane fluidity, transporter expression, and transporter activity. METHODS: SD rats were fed a diet with or without diosgenin. Bile duct cannulation was done and the bile acid pool was depleted, followed by infusion of sodium taurocholate for 120 min to collect bile samples. Bile flow and the biliary output of cholesterol (Ch), phospholipids, bile salt, and total glutathione (GSH+GSSG) were estimated. Liver canalicular membrane vesicles (CMV) were prepared for the assessment of lipid composition, ATP-dependent transporters, and membrane fluidity. RESULTS: Bile flow, especially bile acid-independent fraction, was increased significantly by diosgenin. Biliary output of Ch and total glutathione were significantly increased by diosgenin. CMV fluidity and Mrp2 expression were increased by diosgenin. CONCLUSION: The fact that diosgenin increased bile flow and biliary cholesterol and glutathione output indicates a choleretic action along with enhancement of solute secretion. The increase of CMV fluidity and Mrp2 suggests that the choleretic action of diosgenin was based upon both the direct stimulation of transporter expression and indirect transporter activation by an increase of membrane fluidity.