Gunji Yamashita

A Combination Therapy With Simvastatin and Ursodeoxycholic Acid Is More Effective for Cholesterol Gallstone Dissolution Than Is Ursodeoxycholic Acid Monotherapy

Inhibitors of 3-hydroxy,3-methylglutaryl coenzyme A (HMG-CoA) reductase have been reported to decrease the cholesterol saturation index (CSI) in duodenal bile in humans and to prevent formation of cholesterol gallstones in animal studies. We performed a prospective study to evaluate the role of HMG-CoA reductase inhibitors as gallstone-dissolving agents. Fifty patients with radiolucent gallstones in a gallbladder opacifying at drip infusion cholecystography were treated with either 10 mg/day simvastatin plus 600 mg/day ursodeoxycholic acid (group 1, n=26) or 600 mg/day ursodeoxycholic acid alone (group 2, n=24) for 12 months. The ratio of solitary to multiple gallstone cases was 21:29. Plasma lipid levels were assessed and ultrasonographic examination of the gallbladder was performed at baseline and at 3-month intervals during treatment. Duodenal bile sampling was performed in five patients in each group at baseline and after 12 months of treatment. Plasma cholesterol decreased significantly in group 1 but not in group 2. In solitary gallstone cases, no significant difference in dissolution rates was observed between groups 1 (3 of 9, 33%) and 2 (4 of 12, 33%). In contrast, the dissolution rate in multiple gallstone cases was significantly higher in group 1 (12 of 17, 71%) than in group 2 (3 of 12, 25%) (p < 0.01). Bile cholesterol saturation index was significantly decreased (p < 0.01) but did not significantly differ between the two groups. These results suggest that combination therapy with simvastatin and ursodeoxycholic acid is more effective for cholesterol gallstone dissolution than ursodeoxycholic acid monotherapy in patients with multiple gallstones.

Simultaneous microanalysis of biliary cholesterol, bile acids and fatty acids in lecithin using capillary column gas chromatography : an advantage to assess bile lithogenecity

Simultaneous determination of biliary lipids was performed by alkaline hydrolysis, the formation of the methyl ester derivatives of fatty acids that are constituents of phospholipids and of the acetylated methyl ester derivatives of bile acids, and subsequent analysis by capillary column gas chromatography. Complete separation and satisfactory recovery of cholesterol, bile acids, and fatty acids were achieved. Also, the accuracy of the calculation of the bile cholesterol saturation index was enhanced by computation. Since the degree of acyl chain unsaturation affects the cholesterol-holding capacity in vesicles, this method provides a unique insight into bile metastability by the quantitative assessment of fatty acids in lecithin.

Inhibitory Effects of Pravastatin, a Competitive Inhibitor of Hydroxymethylglutaryl Coenzyme A Reductase, on Cholesterol Gallstone Formation in Prairie Dogs

The effects of pravastatin on cholesterol gallstone formation were determined in prairie dogs. We fed 10 prairie dogs 1% cholesterol with or without 0.05% (w/w) pravastatin (n = 5, each) for 4 weeks. In addition, another 5 prairie dogs were fed a standard rodent chow as a control. Only the animals fed 1% cholesterol without pravastatin treatment formed cholesterol gallstones. Gallbladder bile from cholesterol-fed animals contained cholesterol monohydrate crystals, whereas those treated with pravastatin contained no crystal. Furthermore, marked increases in tissue cholesterol levels (serum, liver and bile), and in biliary mucous glycoprotein levels were evident in cholesterol-fed animals, whereas pravastatin treatment normalized these levels. These findings raise the possibility that such inhibitors might have a future role to play in the prevention of cholesterol gallstone formation and/or recurrence.

PARTIAL REPLACEMENT OF BILE SALTS CAUSES MARKED CHANGES OF CHOLESTEROL CRYSTALLIZATION IN SUPERSATURATED MODEL BILE SYSTEMS

Cholesterol crystallization is a key step in gallstone formation and is influenced by numerous factors. Human bile contains various bile salts having different hydrophobicity and micelle-forming capacities, but the importance of lipid composition to bile metastability remains unclear. This study investigated the effect of bile salts on cholesterol crystallization in model bile (MB) systems. Supersaturated MB systems were prepared with an identical composition on a molar basis (taurocholate/phosphatidylcholine/cholesterol, 152 mM:38 mM: 24 mM), except for partial replacement of taurocholate (10, 20, and 30%) with various taurine-conjugated bile salts. Cholesterol crystallization was quantitatively estimated by spectrophotometrically measuring crystal-related turbidity and morphologically scanned by video-enhanced microscopy. After partial replacement of taurocholate with hydrophobic bile salts, cholesterol crystallization increased dose-dependently without changing the size of vesicles or crystal morphology and the rank order of crystallization was deoxycholate>chenodeoxycholate>cholate (control MB). All of the hydrophilic bile salts (ursodeoxycholate, ursocholate and beta-muricholate) inhibited cholesterol precipitation by forming a stable liquid-crystal phase, and there were no significant differences among the hydrophilic bile-salt species. Cholesterol crystallization was markedly altered by partial replacement of bile salts with a different hydrophobicity. Thus minimal changes in bile-salt composition may dramatically alter bile lipid metastability.

Reaction of hydroxy-bismuth polycations with montmorillonite.

Interlayer sodium ions of montmorillonite were exchanged with hydroxy-bismuth polycations which were prepared from bismuth perchlorate solutions by the addition of NaOH. Assuming the charge density of the silicate layer to be unchanged, the compositions of the polycations involved in the exchange can be estimated from the amount of bismuth taken up by the montmorillonite and from the ignition loss between 110° and 800°C. The derived compositions are near [Bi6(OH)16]2+ irrespective of the ratio of OH:Bi in the perchlorate solution. The basal spacing of the hydroxy-bismuth montmorillonites is about 16 Å at 110°C, which corresponds to that of hydroxy-chromium montmorillonite having a high surface area of about 250 m2/g. The surface areas of the hydroxy-bismuth montmorillonites, however, are less than 80 m2/g.РезюмеМежслойные натриевые ионы монтмориллонита были заменены поликатионами окси-висмута, которые приготовлялись из растворов висмутового перхлората добавлением ИаОН. Предполагая, что плотность зарядов силикатного слоя не должна изменяться, состав поликатионов, вовлеченных в обмен, можно определить по количеству висмута принятого монтмориллонитом и из потерь прокаливания при температурах от 110° до 800°C. Полученные составы близки [Bi6(OH)16]2+ независимо от отношения OH:Bi в перхлоратовых растворах. Основной промежуток оксивисму-товых монтмориллонитов равен примерно 16 Å при 110°С, который соответствует промежутку оксихромового монтмориллонита, имеющего большую поверхностную площадь, примерно 250 м2/г. Поверзностные площади висмутовых монтмориллонитов, однако, меньше 80 м2/г. [N.R.]ResümeeZwischenschichtnatrium von Montmorillonit wurde gegen Hydroxy-Wismut-Polykationen ausgetauscht, die durch die Zugabe von NaOH aus Wismutperchloratlösungen hergestellt wurden. Unter der Annahme einer unveränderten Ladungsdichte der Silikatlagen kann man die Zusammensetzung der am Austausch beteiligten Polykationen aus der Wismutmenge, die durch den Montmorillonit aufgenommen wird, und aus dem Glühverlust zwischen 110° und 800°C abschätzen. Die abgeleiteten Zusammensetzungen sind etwa [Bi6(OH)16]2+, unabhängig vom OH:Bi-Verhältnis in der Perchloratlösung. Der Basisabstand der Hydroxy-Wismut-Montmorillonite beträgt bei 110°C etwa 16 Å. Dies entspricht dem von Hydroxy-Chrom-Montmorillonit, der eine große Oberfläche von etwa 250 m2/g hat. Die Oberflächen der Hydroxy-Wismut-Montmorillonite sind dagegen unter 80 m2/g. [U.W.]RésuméDes ions de sodium interfeuillets de montmorillonite ont été échangés avec des polycations hydroxy-bismuth qui avaient été préparés à partir de solutions de perchlorate par addition de NaOH. Si l’on suppose que la densité de charge de la couche silicée demeure inchangée, les compositions des polycations concernés par l’échange peuvent être estimées par la quantité de bismuth prise par la montmorillonite et par la perte à l’ignition entre 110° et 800°C. Les compositions derivées sont près de [Bi6(OH)16]2+ de manière irrespective de la proportion de OH:Bi dans la solution de perchlorate. L’espacement basal des montmorillonites hydroxy-bismuth est à peu près 16 Å à 110°C, ce qui correspond à celui de la montmorillonite hydroxy-chromium ayant une aire de surface élevée d’approximativement 250 m2/g. Les aires de surface des montmorillonites hydroxy-bismuth sont cependant moins que 80 m2/g. [D.J.]

Human Gallbladder Mucosal Function (Effects on Intraluminal Fluid and Lipid Composition in Health and Disease)

Gallbladder mucosal absorption of fluid duringfasting is a well-known process. Indirect in vivo andrecent in vitro evidence for physiologically relevantgallbladder absorption of cholesterol and phospholipids from bile has been observed in humans. Thepresent study explored and compared by indirect meansthe relative efficiences of human gallbladder mucosalabsorption of fluid and lipids in health and disease. Biliary lipids and pigment content weremeasured in fasting gallbladder bile samples obtainedfrom gallstone-free controls and from four study groups:multiple and solitary cholesterol gallstone patients, and morbidly obese subjects with and withoutgallstones. Bile salts and pigment content weresignificantly greater in gallstone-free controls than inall other disease study groups. This was interpreted as evidence of more effective gallbladdermucosal fluid absorption in nonobese gallstone-freecontrols compared to that in all other groups.Correlation plot analyses of biliary lipids showed lowerconcentrations of phospholipids than expected from the indexbile salt concentrations. The same was found forcholesterol concentrations but only in supersaturatedsamples. These findings were much more pronounced in gallstone free-controls and were accordinglyinterpreted as evidence of more efficient gallbladderabsorption of both phospholipids and cholesterol incontrols compared with that found in each of the disease study groups. Moreover, impaired gallbladdermucosal function, while invariably associated withcholesterol gallstone disease, was not found to be anecessary consequence of the physical presence ofstones. It is concluded that efficient gallbladdermucosal absorption of both fluid and apolar lipids frombile is a normal physiological process that is oftenseriously impaired in the presence of either cholesterol gallstone disease or at least one of itsprecursor forms.

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.

Partial characterization of mechanisms of cytoprotective action of hydrophilic bile salts against hydrophobic bile salts in rats: Relation to canalicular membrane fluidity and packing density

Bile salts regulate the subselection ofphosphatidylcholine species secreted into bile andthereby modulate bile metastability. The aim of thisstudy was to determine whether bile salts alterphosphatidylcholine species of the canalicular membrane, and ifthey do, to clarify whether the cytoprotective action ofhydrophilic bile salts is associated with modulation ofphosphatidylcholine composition in cell membrane bilayers. Bile salt-pool-depleted rats wereinfused intravenously with sodium taurocholate at aconstant rate (200 nmol/min/100 g body wt) for 2 hr,followed by infusion of either sodiumtauroursodeoxycholate, sodium tauroalphamuricholate, or sodiumtaurobetamuricholate (200 nmol/min/100 g) for 2 hr.Biliary outputs of cholesterol and phosphatidylcholineand phosphatidylcholine hydrophobicity in bile andsubcellular fractions were determined. The cytoprotectiveaction of hydrophilic bile salts was determined by therelease of canalicular membrane-localizing enzymes(alkaline phosphatase, leucine aminopeptidase) into bile. Tauroursodeoxycholate,taurobetamuricholate, and tauroalphamuricholatedecreased the release of these enzymes when compared tovalues under taurocholate infusion. Bilephosphatidylcholine hydrophobicity was also decreased by the bile salts, whereasthe cholesterol/phosphatidylcholine ratio was increased.In contrast, phosphatidylcholine hydrophobicity in thecanalicular membrane was increased by these three bile salts. In conclusion, hydrophilicbile salts promote biliary secretion of relativelyhydrophilic phosphatidylcholine secretion into bile, andconsequently phosphatidylcholine hydrophobicity in canalicular membranes increased. Such analteration in phosphatidylcholine species withincanalicular membrane enhances its lateral packingdensity with less fluidity, and this may account, inpart, for the cytoprotective action of hydrophilic bilesalts against hydrophobic bile salts.

Effect of cholestasis induced by organic anion on the lipid composition of hepatic membrane subfractions and bile in rats

Several organic anions inhibit the secretion of cholesterol and phospholipid into bile without affecting total bile acid secretion (uncoupling). The uncoupling induced by sulphobromophthalein (BSP) alters the fatty acid composition of biliary lecithin. The purpose of this study was to investigate the relationship between the lipid composition of bile and of liver subcellular membrane fractions during BSP‐induced uncoupling. After depletion of the bile salt pool, rats fitted with a bile duct cannulus were infused with sodium taurocholate given either alone or with BSP. Bile was collected and liver microsomes and canalicular membranes were isolated for analysis of lipid composition. In bile, uncoupling increased the cholesterol/phospholipid ratio (C/P ratio) and the saturated/unsaturated fatty acid ratio (S/U ratio) in phosphatidylcholine. The C/P ratio was increased in the canalicular membrane, but the membrane phosphatidylcholine S/U ratio decreased during uncoupling. In microsomes, the S/U ratio of membrane phosphatidylcholine was slightly increased, but the C/P ratio was unaffected during uncoupling. These results support the hypothesis that an increased secretion of hydrophobic phosphatidylcholine species from the canalicular membrane into bile reduces the proportion of hydrophobic phosphatidylcholine species in the canalicular membrane during uncoupling. The decreased contribution of hydrophobic phosphatidylcholine species may ameliorate the decrease in membrane fluidity resulting from the accumulation of cholesterol in the canalicular membrane and stimulate the synthesis of hydrophobic phosphatidylcholine species in the microsomes.

Effects of bilirubin ditaurate on biliary secretion of proteins and lipids: Influence on the hepatic vesicle transport system

Background : Several organic anions cause dissociation of biliary lipid secretion from bile acid secretion (uncoupling). As bile lipids originate from liver microsomes and are transported by carrier proteins and/or transcytotic vesicles, such a reduction of biliary lipid secretion may lead to cytosolic accumulation of vesicles. This study investigated whether bilirubin conjugate, a physiologically important organic anion, caused uncoupling and whether hepatic retention of compounds carried by transcytotic vesicles occurred subsequently, using bilirubin ditaurate, a synthetic commercially available compound.