Role of phospholipase A2 in cholesterol gallstone formation is associated with biliary phospholipid species selection at the site of hepatic excretion: indirect evidence.

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.

Bilirubin overload modulates bile canalicular membrane fluidity in rats: association with disproportionate reduction of biliary lipid secretion.

We recently demonstrated that several organic anions cause dissociation of biliary lipid secretion from that of bile acids; namely, the "uncoupling phenomenon," in association with changes in the phospholipid molecular species in the canalicular membrane lipid bilayer. Because of the uncoupling phenomenon, transcytotic vesicles are retained inside cells, resulting in the accumulation of substances normally excreted in the bile. In the present study, bilirubin ditaurate (BDT; synthetic bilirubin) was used to investigate the effect of bilirubin overload on biliary lipid secretion and the lipid composition of hepatic subcellular fractions, as well as canalicular membrane packing density and fluidity. Male Sprague-Dawley rats underwent cannulation of the bile duct and femoral vein. Sodium taurocholate was infused intravenously at 100 nmol/min per 100 g body weight. Then BDT (50 nmol/min per 100 g body weight) was infused concomitantly, followed by periodic bile collection for analysis of lipids. Bile acid secretion was not significantly affected by the infusion of BDT. In contrast, the secretion of cholesterol and phospholipids was decreased by 56.7% and 49.2%, respectively, compared with control. The phosphatidylcholine hydrophobicity of canalicular membrane vesicles, estimated by the molar ratio of saturated to unsaturated fatty acids (S/U ratio) was decreased, but not significantly by BDT infusion. With BDT infusions, the biliary cholesterol/phospholipid (C/P) ratio was increased by 19%; canalicular membrane vesicle fluidity was decreased by 5.8%, whereas P-glycoprotein expression was unchanged. As P-glycoprotein expression was not altered, our findings suggested that the reduced canalicular membrane vesicle fluidity was a crucial regulator of canalicular membrane transporter function.

Influence of cholesterol crystallization effector proteins on vesicle fusion in supersaturated model bile.

BACKGROUND: In lithogenic bile, cholesterol-rich vesicles rapidly aggregate and fuse to eventually form cholesterol crystals. This process is modulated by cholesterol crystallization effector substances. In this study, we developed a method for quantitative assessment of vesicle fusion and used it to partly characterize the mechanisms of action of cholesterol crystallization effector proteins. METHODS: Cholesterol:phospholipid (1:1) liposomes were prepared and labelled with octadecyl rhodamine B chloride (R18). Fusion of these liposomes was detected by the increase of R18 fluorescence after incubation with various proteins, such as albumin, concanavalin-A bound glycoprotein, immunoglobulins, apolipoprotein A-I and apolipoprotein B (all at 100 microg/mL). RESULTS: Fusion of cholesterol/phospholipid liposomes was increased by 16 and 14% in the presence of concanavalin-A bound glycoprotein and immunoglobulins, respectively, and decreased by 21 and 9% after addition of apolipoprotein A-I and apolipoprotein B, respectively. The effect of each protein on vesicle fusion was correlated with its hydrophobicity. CONCLUSIONS: These results suggest that nucleation effector proteins modulate the stability of vesicles and, thus, affect cholesterol crystallization. Such modulation is based upon protein-vesicle association, which defines the physico-chemical metastability of vesicular cholesterol.

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. METHODS: Cannulation of the bile duct and femoral vein was done in male Sprague-Dawley rats. Sodium taurocholate was infused intravenously at a constant rate of 100 nmol/min per 100 g bodyweight. Bilirubin ditaurate (50 nmol/min per 100 g bodyweight) was infused concomitantly, followed by periodical bile collection for analysis of lipids, total protein and immunoglobulin A. RESULTS: Biliary bile acid secretion was not changed significantly by infusion of bilirubin ditaurate. In contrast, the secretion of cholesterol, phospholipids and immunoglobulin A was decreased by 57.3, 48.7 and 44.8%, respectively. The biliary cholesterol:phospholipid ratio was increased by 19%. Uncoupling was caused by bilirubin ditaurate and biliary immunoglobulin A secretion was decreased. CONCLUSIONS: As immunoglobulin A is a major protein carried by intrahepatic transcytotic vesicles, uncoupling may involve impairment of intrahepatic vesicular transport. Also, a reduction of immunoglobulin A secretion into bile by organic anion-induced uncoupling may weaken biliary immunity.

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.

Role of bile salt hydrophobicity in distribution of phospholipid species to carriers in supersaturated model bile solutions.

BACKGROUND: Phospholipid species modulate cholesterol-holding capacity and, therefore, regulate bile metastability. METHODS: In this study, we investigated the effect of bile salt hydrophobicity on the distribution of phospholipids among lipid particles in supersaturated model bile solutions (total lipid concentration, 9 g/dL; taurocholate/phospholipid ratio 3.0, cholesterol saturation index 1.3), by using gel permeation chromatography. RESULTS: With an increase of bile salt hydrophobicity in the elution buffer, the uptake of cholesterol and phospholipids into bile salt micelles was increased, associated with an increased cholesterol/phospholipid molar ratio of the vesicles. In contrast, there was an inverse correlation between the hydrophobicity of the phospholipid species in the vesicles and that of bile salts in the elution buffer, suggesting that hydrophobic bile salts induced preferential uptake of hydrophobic phospholipids into bile salt micelles, while less hydrophobic phospholipids, with a relatively low cholesterol-holding capacity, remained in the vesicles. CONCLUSIONS: These data indicate that bile salt hydrophobicity regulates vesicular cholesterol metastability by modulating the hydrophobicity of phospholipids in vesicles, as well as the lipid distribution among various biliary lipid particles.

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 of phosphatidylcholine species secreted into bile and thereby modulate bile metastability. The aim of this study was to determine whether bile salts alter phosphatidylcholine species of the canalicular membrane, and if they do, to clarify whether the cytoprotective action of hydrophilic bile salts is associated with modulation of phosphatidylcholine composition in cell membrane bilayers. Bile salt-pool-depleted rats were infused intravenously with sodium taurocholate at a constant rate (200 nmol/min/100 g body wt) for 2 hr, followed by infusion of either sodium tauroursodeoxycholate, sodium tauroalphamuricholate, or sodium taurobetamuricholate (200 nmol/min/100 g) for 2 hr. Biliary outputs of cholesterol and phosphatidylcholine and phosphatidylcholine hydrophobicity in bile and subcellular fractions were determined. The cytoprotective action of hydrophilic bile salts was determined by the release of canalicular membrane-localizing enzymes (alkaline phosphatase, leucine aminopeptidase) into bile. Tauroursodeoxycholate, taurobetamuricholate, and tauroalphamuricholate decreased the release of these enzymes when compared to values under taurocholate infusion. Bile phosphatidylcholine hydrophobicity was also decreased by the bile salts, whereas the cholesterol/phosphatidylcholine ratio was increased. In contrast, phosphatidylcholine hydrophobicity in the canalicular membrane was increased by these three bile salts. In conclusion, hydrophilic bile salts promote biliary secretion of relatively hydrophilic phosphatidylcholine secretion into bile, and consequently phosphatidylcholine hydrophobicity in canalicular membranes increased. Such an alteration in phosphatidylcholine species within canalicular membrane enhances its lateral packing density with less fluidity, and this may account, in part, for the cytoprotective action of hydrophilic bile salts against hydrophobic bile salts.

The comparative potency of cholesterol crystallization-effector proteins in supersaturated model bile systems: association with vesicle transformation.

Various proteins which affect cholesterol crystallization are known to be present in bile, although the relative potency of their action is yet to be established. In this study, we evaluated the comparative potency of nucleating-effector proteins using a recently developed method for quantitative assessment of vesicle transformation in supersaturated model bile systems, to partially characterize mechanisms of their action. Concanavalin A-bound glycoproteins isolated from human gall-bladder bile shortened cholesterol crystallization time by 40% and increased cholesterol growth rate and final crystal mass by 161 and 19%, respectively, when compared to the control. In addition, immunoglobulins isolated from human gall-bladder bile increased cholesterol growth rate by 9%, but showed no significant effect on cholesterol crystallization time and final crystal mass. In contrast, human serum apolipoproteins A-I and B reduced cholesterol growth rate by 26 and 31% and reduced final crystal mass by 12 and 21%, but did not affect cholesterol crystallization time. Gel permeation chromatography revealed that proteins were distributed to both vesicles and bile salt micelles, but that no marked redistribution of lipids was caused by addition of these proteins. Furthermore, no significant difference in crystal structure was observed by video-enhanced contrast microscopy. These results indicate that nucleating-effector substances tested in this study may modulate vesicular cholesterol-holding capacity, thus affecting cholesterol crystallization. Such modulation is based upon the protein-vesicle association which defines the physico-chemical metastability of vesicular cholesterol.

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.

Effects of cerivastatin sodium, a new HMG-CoA reductase inhibitor, on biliary lipid metabolism in patients with hypercholesterolemia.

The use of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has become common in the treatment of hypercholesterolemia. The present uncontrolled study was undertaken to determine the effect of cerivastatin sodium (BAY w 6228), a new HMG-CoA reductase inhibitor, on biliary lipid levels in patients with hypercholesterolemia. Twenty-one hypercholesterolemic patients (World Health Organization type IIa = 16 patients; type IIb = 5 patients) received placebo during a 4- to 6-week observation period, after which they received cerivastatin sodium 0.2 mg/d for 12 weeks. Fasting blood samples were drawn for the measurement of serum lipid levels early in the morning before the start of treatment and once a month for each of the 12 weeks of cerivastatin sodium treatment. Gallbladder bile samples were aspirated with a duodenal tube by cerulein stimulation to assess bile lithogenicity. Serum total cholesterol levels decreased markedly after 12 weeks. However, no significant difference was found in the molar percentage composition of biliary lipids (e.g., cholesterol, phospholipids, and total bile acids) or in individual biliary bile acids. Consequently, no significant change in bile cholesterol saturation index was found. The index values before and after 12 weeks of treatment were 0.81 +/- 0.38 and 0.80 +/- 0.47, respectively, whereas when patients were grouped by type of hypercholesterolemia, there was a tendency toward decreased lithogenicity in patients with type IIb but not type IIa hypercholesterolemia. We concluded that cerivastatin sodium was an effective cholesterol-lowering drug that did not appear to worsen biliary lipid metabolism and that may decrease lithogenicity in patients with type IIb hypercholesterolemia.

Quantitative assessment of comparative potencies of cholesterol-crystal-promoting factors: relation to mechanistic characterization.

The crystallization of cholesterol is affected by various factors in bile. The present study evaluated the relative importance of cholesterol-nucleation-promoting factors and partially characterized the mechanisms of their action. Model biles with an identical relative composition of cholesterol, egg-yolk phosphatidylcholine and taurocholate, except for replacing phosphatidylcholine (5-20%) with dilinoleoyl-phosphatidylcholine or taurocholate (10-30%) with taurodeoxycholate. Cholesterol crystallization was quantitatively assessed spectrophotometrically and morphologically estimated by the laser-scattering diffraction analyser and video-enhanced microscopy in the absence and presence of concanavalin A-binding glycoprotein isolated from human bile. In a series of experiments, lipid distribution among particulate species was determined after isolation by FPLC. In all experiments, cholesterol crystallization was dose-dependently enhanced with a rank order of: concanavalin A-binding glycoprotein > dilinoleoyl - phosphatidyl choline> taurodeoxycholate. No morphological alteration was evident for vesicles and crystals, but the cholesterol/phospholipid ratio in vesicles was increased significantly by replacement with dilinoleoyl-phosphatidylcholine and excess cholesterol. A high proportion of relatively hydrophilic phosphatidylcholine species such as dilinoleoyl-phosphatidylcholine and excess cholesterol in bile cause a redistribution of cholesterol to increase a vesicular cholesterol/phospholipid ratio, eventually promoting cholesterol crystallization, whereas concanavalin A-binding glycoprotein acts via differing mechanisms.

Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease.

Gallbladder mucosal absorption of fluid during fasting is a well-known process. Indirect in vivo and recent in vitro evidence for physiologically relevant gallbladder absorption of cholesterol and phospholipids from bile has been observed in humans. The present study explored and compared by indirect means the relative efficiences of human gallbladder mucosal absorption of fluid and lipids in health and disease. Biliary lipids and pigment content were measured in fasting gallbladder bile samples obtained from gallstone-free controls and from four study groups: multiple and solitary cholesterol gallstone patients, and morbidly obese subjects with and without gallstones. Bile salts and pigment content were significantly greater in gallstone-free controls than in all other disease study groups. This was interpreted as evidence of more effective gallbladder mucosal fluid absorption in nonobese gallstone-free controls compared to that in all other groups. Correlation plot analyses of biliary lipids showed lower concentrations of phospholipids than expected from the index bile salt concentrations. The same was found for cholesterol concentrations but only in supersaturated samples. These findings were much more pronounced in gallstone free-controls and were accordingly interpreted as evidence of more efficient gallbladder absorption of both phospholipids and cholesterol in controls compared with that found in each of the disease study groups. Moreover, impaired gallbladder mucosal function, while invariably associated with cholesterol gallstone disease, was not found to be a necessary consequence of the physical presence of stones. It is concluded that efficient gallbladder mucosal absorption of both fluid and apolar lipids from bile is a normal physiological process that is often seriously impaired in the presence of either cholesterol gallstone disease or at least one of its precursor forms.

Extracellular and intracellular regulation of biliary lecithin hydrophobicity.

Bromosulfophthalein and papaverine have been demonstrated to inhibit biliary lipid secretion without affecting secretion of bile salts in normal rats, so-called uncoupling. Bromosulfophthalein inhibits the capacity of intracanalicular bile salt micelles to induce biliary lipid secretion, and papaverine inhibits vesicular transport within the hepatocyte. We compared the effects of bromosulfophthalein and papaverine on biliary lipid secretion in normal Sprague-Dawley rats and Eizai hyperbilirubinuria rats. The fatty acyl chain saturation in biliary lecithin increased during bromosulfophthalein infusion and decreased during papaverine infusion in Sprague-Dawley rats. Bromosulfophthalein had no effect on biliary lipid secretion in Eizai rats, while papaverine induced uncoupling. The degree of fatty acyl chain saturation in biliary lecithin was unchanged during bromosulfophthalein infusion, but decreased with papaverine in Eizai rats. We deduce that selection of biliary lecithin species occurs at various points in the lipid transport pathway at intracellular and intracanalicular sites.

Variations in pigment and carbohydrate content of gallbladder bile affect accurate quantitation of total protein when using the fluorescamine method.

BACKGROUND: Despite solute dilution and reduced total lipid concentrations, an unexplained increase in protein concentration has been reported to occur in the gallbladder bile of cholesterol gallstone patients. METHODS: Solutes in gallbladder bile from gallstone-free controls and from four study groups were measured using standard methods. Total proteins were measured using amino acid analysis and a conventional fluorescamine method. RESULTS: Bile salts and pigment content were greater in gallstone-free controls than in all other study groups, including morbidly obese gallstone-free subjects. Total biliary protein concentration, as determined by amino acid analysis in the gallstone-free control group was higher than in non-obese gallstone patients with multiple stones and in morbidly obese gallstone-free subjects. Total biliary proteins as measured with fluorescamine, however, did not show intergroup differences. A major problem of the conventional fluorescamine assay is shown to be an artefact arising from the high pigment content of the more concentrated samples. CONCLUSIONS: Very dilute gallbladder bile samples are often found in the presence of gallstone disease. This also occurs in morbidly obese subjects, even in the absence of gallstones. Although the contribution of protein secretion/absorption by the gallbladder can also be relevant, especially in the presence of morbid obesity, the protein concentration in gallbladder bile, when accurately measured, generally parallels the concentrations of non-absorbed biliary solutes, reflecting the efficiency of fluid absorption. Measurement of biliary proteins by the conventional fluorescamine method is unreliable in clinical studies in which intergroup differences in pigment content are commonly present.

A quantitative assessment of serum chylomicron by light scattering intensity: application to the intestinal fat absorption test.

A novel fat absorption test to clarify the malabsorption syndrome was developed using a micronephelometric technique and compared with the classic conventional technique using 131I-triolein. An integrity of time-sequential light scattered from chylomicron-related turbidity in serum was determined between 0 and 300 min after butter fat load, being expressed in terms of the light scattering intensity (LSI). A good correlation was obtained between LSI and the serum level of chylomicron-triglyceride determined by an ultracentrifugation technique (r = 0.819, P < 0.001). The maximal LSI was consistently observed at 180 min after administration of a test meal in the normal group (n = 39), whereas the malabsorption syndrome group (n = 35) was distinctly different and could be further classified according to four patterns of LSI changes. In addition, an inverse correlation was found between this fat absorption test and the 131I-triolein absorption test. It was concluded that the micronephelometric technique which does not use a radionuclide is advantageous in its simple and safe evaluation of fat malabsorption syndrome.

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 was 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.

Comparison of haptoglobin and apolipoprotein A-I on biliary lipid particles involved in cholesterol crystallization.

Several proteins are known to modulate cholesterol crystallization. We recently demonstrated that haptoglobin has cholesterol crystallization promoting activity. However, this effect is still not well understood mechanistically. The current study examined the distribution of haptoglobin compared to apolipoprotein A-I (apo A-I) to micelles, vesicles and crystals as an initial step in providing a focus for further studies of the mechanism of cholesterol crystallization activity. Specific protein purification was accomplished by immunoaffinity chromatography. The crystallization-promoting activity of biliary haptoglobin, albumin and commercial apo A-I was measured by a photometric crystal growth assay. The distribution of micelles, vesicles and proteins in model bile was determined by Sepharose CL-6B column chromatography. Detection of the presence of test proteins in cholesterol crystals was determined using specific 125I-radiolabelled proteins. Haptoglobin (20 micrograms/mL) showed a significant crystallization promoting-activity, whereas apo A-I (30 micrograms/mL) only tended to show a slight inhibitory activity. The cholesterol crystal-bound protein in each case was found to be less than 1% of the total concentration of that protein that had been added to the model bile system. The elution profile of commercial apo A-I from a Sepharose CL-6B column was strikingly altered when it was added to model bile prior to elution. In contrast, the column elution profiles for both haptoglobin and albumin were unchanged when model bile was similarly added to the sample. Haptoglobin increased the amount of cholesterol found in the vesicular fraction when compared to apo A-I. Haptoglobin does not bind tightly to either biliary lipid particles or to cholesterol crystals but does increase the amount of cholesterol in vesicles by inducing a shift from micellar cholesterol (P = 0.046). This shift appears to explain in part its promoting effect on cholesterol crystallization.

Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile.

Crystallization-inhibiting proteins can explain longer nucleation times associated with bile from gallstone-free subjects as compared with bile from patients with cholesterol gallstones. We partially characterized and examined the crystallization inhibitory potency of a newly purified 15 kd human biliary protein. Gallbladder bile was passed through an anti-apolipoprotein A-I (apo A-I) immunoaffinity column to extract lipid-associated proteins. The bound fraction was separated by 30 kd ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) was performed under nonreducing and reducing conditions. Cholesterol crystallization activity was tested in a photometric cholesterol crystal growth assay. Isoelectric focusing was performed by using a standard gel. The purified 15 kd protein was subjected to N-terminal amino acid sequencing. Although the whole apo A-I-bound fraction contained a variety of proteins and lipids, its 30 kd filtrate yielded a nearly pure 15 kd protein with only minor contamination from apo A-1. Amino acid sequencing showed that the protein was unique. Enzymatic deglycosylation revealed no evidence for glycosylation. At a protein concentration of 10 micrograms/ml, crystallization time was delayed as compared with control and apo A-I, and final crystal mass was reduced to 75% of control. Its isoelectric point was 6.1 without isoforms. Under nonreducing conditions, the protein formed a 30 kd dimer and a 60 kd tetramer. We conclude that this protein is a novel potent biliary crystallization inhibitor protein.

Biliary haptoglobin, a potent promoter of cholesterol crystallization at physiological concentrations.

BACKGROUND/AIMS: Several proteins present in human bile have been reported to promote cholesterol crystallization and thus are potentially important in the formation of cholesterol crystals as the initial stage in gallstone pathogenesis. To be physiologically relevant, such proteins must either be present in high concentration in bile or have a potent promoting activity. The current study explored several of the more abundant but unexamined biliary proteins based upon their also having sufficiently high serum concentrations that antibodies were available for both their isolation and quantitation. METHODS: Protein purification was accomplished by immunoaffinity chromatography of bile followed by delipidation. Con A affinity chromatography of bile was used to obtain the bound fraction, a portion of which was delipidated. Crystallization-promoting activity of both the purified proteins and Con A-bound glycoprotein fractions (CABG) was measured by a photometric crystal growth assay. A competitive antibody-capture ELISA assay was developed to measure concentrations of alpha 1-antitrypsin, transferrin, and haptoglobin in native bile. RESULTS: At their relevant physiological concentrations, biliary haptoglobin (15 micrograms/ml) had a crystallization-promoting activity twice that of the biliary IgM (75 micrograms/ml) used as a reference standard (P < 0.05). Biliary transferrin (20 micrograms/ml) had only modest promoting activity (P < 0.05). Biliary alpha 1-antitrypsin (50 micrograms/ml), by contrast, showed no promoting activity. Delipidation of the CABG fraction decreased its promoting activity by 75%. Biliary haptoglobin accounts for about 30% of delipidated total CABG-promoting activity. CONCLUSIONS: Biliary haptoglobin at its physiological concentration has a highly potent crystallization-promoting activity and thus becomes a candidate for major attention in understanding gallstone pathogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)