Newer pathogenetic concepts in cholesterol gallstone formation: a unitary hypothesis.
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Animals↗
Biomedical subjects
R T Holzbach
Publications and source records attributed to R T Holzbach.
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.
Adult↗
[Role of biliary proteins in pathogenesis of cholesterol gallstones].
Cholelithiasis is a frequent disease in developed countries with significant economical implications. While the multifactorial pathogenesis of cholesterol gallstones has been widely accepted, the relative importance of the various contributing factors is not yet clear. One focus of research in recent years has been the identification and functional analysis of biliary proteins. Few proteins are synthesized in the biliary tract itself, the majority reflect the composition of serum proteins. Many biliary proteins modify cholesterol crystallization, the initial step of cholesterol gallstone formation. In cholelithiasis, biliary concentration of many pronucleating proteins are increased. However, this may be a consequence rather than the cause of the disease, because a majority of these proteins are acute-phase reactants. They may be secreted into bile at increased concentrations because of--often asymptomatic--cholecystitis due to gallstone disease. A number of protein-lipid interactions have been observed in search of a mechanism of action of biliary effector proteins. Only recently the potential modification of lipid removal from gallbladder bile by Apo A-I was described in addition to its direct antinucleating effect. In conclusion, biliary proteins directly modify cholesterol crystallization by protein-lipid interactions and may influence lipid absorption from the gallbladder.
Apolipoprotein A-I↗
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.
Amino Acid Sequence↗
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.
Apolipoprotein A-I↗
Correlation between biliary alpha 1-acid glycoprotein concentration and cholesterol crystal nucleation time in gallstone disease.
A biliary form of the alpha 1-acid glycoprotein (AAG) promotes cholesterol crystallization in the lower-molecular-weight, concanavalin A-bound fraction of gallbladder bile. In addition, bile AAG concentration is higher in cholesterol gallstone patients with multiple stones than in control patients without gallstone disease. In this study we sought to determine whether the increased biliary concentration of AAG in cholesterol gallstone patients is accompanied by a more rapid nucleation time in patients with multiple stones. AAG concentration in native biles was measured by ELISA. Nucleation time was measured using a standard microscopy method. The concentration of biliary AAG was then related to nucleation time in biles from the same patients. Nucleation times were significantly shorter (< or = 5 days) in cholesterol gallstone patients with raised AAG concentrations (P < 0.03). There was a significant (P = 0.004) negative correlation (r = -0.53) between nucleation time and the AAG concentration in cholesterol gallstone patients with multiple stones. The concentration of biliary AAG appears to exert an important influence on the speed of cholesterol nucleation in bile in many patients with cholesterol gallstone disease.
Bile↗
Biliary alpha 1-acid glycoprotein concentrations in gallstone-free controls and in patients with multiple or solitary cholesterol gallstones.
We recently identified a promoting glycoprotein in the concanavalin A-bound fraction of gallbladder bile as a biliary form of alpha 1-glycoprotein (AAG). The concentration of biliary AAG appears to exert an important promoting effect on the speed of cholesterol nucleation in many patients with cholesterol gallstone disease. In the current study, we provide information about the biliary concentration of AAG as well as the amount and comparative potency of its subfractions in patients with and without cholesterol gallstone disease. The amount of total biliary AAG and the amounts of its different isoforms separated by concanavalin A affinity chromatography were measured by ELISA. Estimates of absolute concentrations of AAG for each sample were normalized to the sample total protein content to give relative AAG values. The promoting activity (potency) of immunopurified biliary AAG from gallstone patients and gallstone-free controls on cholesterol crystallization was compared by a crystal growth assay. The mean absolute concentration of AAG in gallstone-free controls was not significantly different from multiple stone patients. The relative concentration of AAG (micrograms per milligram total protein) was significantly increased in patients with multiple stones when compared to controls (P < 0.05), and both the absolute and relative concentrations of AAG (micrograms per milligram bile), were three- and to five fold higher in a number of these patients. The functional activity and distribution of AAG in different subfractions was similar in gallstone patients and gallstone-free controls. The relative concentration of biliary AAG is significantly greater in cholesterol gallstone patients with multiple stones than in gallstone-free controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Bile↗
Cholesterol nucleation in bile.
Protein factors, primarily glycoproteins, present in native human bile, have been found to modify, e.g., retard/accelerate, the process of de novo monohydrate crystal formation and, by inference, the rate of formation of cholesterol "nuclei" in such metastable cholesterol supersaturated systems. Neither the process of nucleation itself or how these modifiers work is yet well understood. From the health standpoint, an inhibitor type of protein has been found that may help explain why about 50% of the population have biliary cholesterol supersaturation; whereas, only about 10% actually form gallstones. To date, only one inhibitor glycoprotein has been isolated and characterized. Its role thus seems clear. This is in contrast to the situation with so-called promoter glycoproteins which accelerate nucleation and crystal formation. A number of these proteins have now been identified. An understanding of the comparative roles of each of these proteins has not yet been established. This is partly because of conflicting results, insufficient potencies or lack of information about physiologic concentrations.
Animals↗
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)
Antibody Specificity↗
Identification of human biliary alpha 1-acid glycoprotein as a cholesterol crystallization promoter.
BACKGROUND/AIMS: We have recently outlined the biochemical features of a human 42-kilodalton biliary glycoprotein that shows concentration-dependent cholesterol crystallization-promoting activity. The goal in this work was to establish its identity and to examine some aspects of its biochemical properties relative to its activity. METHODS: Internal amino acid sequencing following tryptic digestion was performed. Based upon this result, immunoreactivity against the 42-kilodalton glycoprotein was examined using a relevant antibody. With the same antibody, the 42-kilodalton glycoprotein was isolated from bile and assayed for activity. Sequential enzymatic deglycosylation of successive terminal glycans of the purified glycoprotein was performed, and the effects on both reductions in molecular radius (M(r)) and on comparative promoter activities were examined. RESULTS: Both amino acid sequence and immunochemical data identify the 42-kilodalton glycoprotein as a biliary form of alpha 1-acid glycoprotein. When purified by immunoaffinity chromatography, potent promoting activity shown was proportionately reduced by successive removal of terminal glycans that also reduced the M(r)s. CONCLUSIONS: The 42-kilodalton cholesterol crystallization-promoting glycoprotein is now identified as a biliary form of alpha 1-acid glycoprotein. Further, some aspects of the important role of glycans in this extensively glycosylated protein have been explored.
Bile↗
Isolation of a human biliary glycoprotein inhibitor of cholesterol crystallization.
BACKGROUND: About 50% of populations in developed countries have bile supersaturated with cholesterol, which is a major risk factor for cholesterol gallstone formation. Despite the prevalence of supersaturated bile, only about 10% of these populations develop gallstones. The existence of a biliary protein that inhibits cholesterol crystallization was hypothesized to explain this discrepancy. This report outlines the purification and characterization of such a human biliary glycoprotein. METHODS: Chromatographic methods were used for separation and characterization. Additional steps included activity analysis by crystal growth assay, electrophoresis, and deglycosylation. RESULTS: The glycoprotein consists of a heterodimer, M(r) of 120 kilodalton, with subunits of M(r) of 63 kilodalton and 58 kilodalton. Each of the subunits is characterized by an isoelectric point of 6.6 and shows comparable inhibitory activity. Deglycosylation of the subunits show that they share a similar polypeptide backbone (M(r) of 35 kilodalton) based upon a highly similar amino acid profile. This suggests that differential subunit glycosylation alone may account for the apparent heterodimeric structure. CONCLUSIONS: No other human biliary glycoprotein has been found thus far that shows cholesterol crystal growth-inhibiting activity. Thus, it may be of importance in preventing gallstone formation in healthy populations.
Amino Acids↗
Isolation and characterization of a cholesterol crystallization promoter from human bile.
BACKGROUND: Recent studies on the pathogenesis of cholesterol gallstone disease have focused on the potential importance of an imbalance between biliary proteins having either inhibitory or promoting activities on nucleation and/or growth of cholesterol crystals as the initial stage in stone formation. The current study describes the purification and partial characterization of a 42-kilodalton biliary glycoprotein that shows concentration-dependent cholesterol crystallization-promoting activity. METHODS: Chromatographic methods were used for separation and purification. Characterization steps included electrophoresis, deglycosylation, amino acid and carbohydrate analysis, and activity analysis by crystal growth assay. RESULTS: The 42-kilodalton purified glycoprotein is an extensively glycosylated (37%) monomer with an acidic isoelectric point (pl < 4.1) that is probably based on the sialic acid content of the carbohydrate moiety. Enzymatic N-deglycosylation removes the carbohydrate moiety and inactivates the promoting activity. Furthermore, enzymatic proteolysis results in both its complete structural degradation and functional inactivation. Although the glycoprotein was isolated from normal human gallbladder biles, its presence in gallstone-associated samples is clearly shown. CONCLUSIONS: This report outlines biochemical features of a human biliary glycoprotein that may be of major pathophysiological significance in gallstone disease.
Amino Acids↗
Cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, alpha 1-acid glycoprotein, phospholipase C, and aminopeptidase N1.
Concanavalin A (Con A)-binding glycoproteins accelerate the rate of cholesterol crystal formation as a prelude to gallstone formation. Immunoglobulins (IgM, IgA, and IgG), aminopeptidase N (APN), phospholipase C (pcPLC), and alpha 1-acid glycoprotein from this Con A fraction have all been proposed as candidate promoters. We immunopurified each of the six putative promoters and examined their comparative effects by adding equal amounts to a cholesterol crystal growth assay. The effects of immunoabsorptive removal of each of the specific candidate promoters from native bile were also compared. In additional studies, the potency of these proteins was in the following order: IgM > IgA = AAG > IgG. APN and pcPLC showed no effect on cholesterol crystal growth at their apparent physiological concentrations. In subtractive experiments, only a minor loss (< 10%) of net promoting activity from that of the whole Con A-bound fraction was observed after immunoabsorptive removal of pcPLC, APN, or immunoglobulins. Total removal of AAG, however, showed a far greater loss (/33%) of the net promoting activity. These data indicate that AAG accounts for the greatest portion of net biliary Con A-bound promoting activity derived from currently defined and well-identified glycoproteins. However, more than 60% of total Con A-binding promoting activity remains unaccounted for, indicating the presence of other important and still unidentified promoters in human bile.
Aminopeptidases↗
Transcellular transport of organic anions in the isolated perfused rat liver: the differential effects of monensin and colchicine.
Nonbile salt cholephiles and bile salts are two classes of organic anions that are efficiently taken up and excreted by the liver. Recent evidence suggests that a microtubular system-dependent, colchicine-sensitive transcellular pathway may transport both classes of these ligands. The relationship of this pathway to flux rates, however, remains unclear. Some structural evidence suggests an important role for a Golgi-associated vesicular system. Monensin, like colchicine, is a perturbing agent that is believed to target primarily Golgi and related organelles. The effects of a minimal effective dose of both colchicine (0.06 mg to 0.12 mg/100 gm body wt) and monensin (0.6 mg/100 gm body wt) were examined in the isolated perfused rat liver in a single-pass mode. The nonbile salt cholephile, phenol red, was studied at two doses: 1 nmol and 5 mumol. Sodium taurocholate was studied at three doses: 2 nmol, 1 mumol and 5 mumol. Colchicine affected the transcellular transport for both classes of organic anions equally. Partially inhibitory effects on both anions occurred only at high ligand flux rates. In contrast, monensin greatly impaired the transport of nonbile salt cholephiles but had no influence on transcellular bile salt flux. We conclude that the monensin effect appears to define a distinct transcellular transport pathway for each of the two classes of organic anions.
Animals↗
A novel transcellular transport pathway for non-bile salt cholephilic organic anions.
Non-bile salt cholephilic organic anions comprise a single class of nonhomologous ligands having a range of hydrophobicity. Hydrophobicity enhances the hepatic extraction of cholephiles as well as their partitioning into secreted biliary lipid particles. When hydrophobicity is correlated with patterns of biliary excretion for studying transcellular transport, however, the more hydrophobic probes are unsuitable. Specifically, with the isolated perfused rat liver technique, the excretory times for sulfobromophthalein and rose bengal were significantly longer compared with that for the much more hydrophilic analogue phenol red (PR), which showed only a single, nearly symmetrical excretory peak at 10 min. Colchicine affected the apparently well-defined PR pathway only at a saturation dose (10,000 times the tracer dose). In contrast, the effect of a different perturbant, monensin, was striking at a tracer dose of PR, but was less evident at a saturation dose. The combined administration of colchicine and monensin had no additive inhibitory effect on PR excretion at tracer doses. At a saturation dose of PR, where monensin is less inhibiting, however, a significant additive inhibitory effect was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Inhibition and promotion of cholesterol crystallization by protein fractions from normal human gallbladder bile.
Pooled, normal human gallbladder biles were initially separated on a molecular sieving chromatography column to remove soluble mucin glycoproteins as well as high molecular weight proteins (greater than 200,000). The remaining lower molecular weight proteins and other bile components were then examined by lectin affinity chromatography with four different types of lectin. The separated bound fractions were compared for inhibiting and promoting activities with a newly devised sensitive cholesterol crystal growth assay and for differences in electrophoretic patterns on SDS-gels. Protein factors (presumably glycoproteins) were found to have both inhibiting and promoting activities, even in the absence of cholesterol gallstone disease. The promoting effect was indicated by shortened crystal detection times and increases in crystal growth rate; whereas the inhibiting effect was indicated by decreases in crystal growth rate and reductions in the final crystal concentration as determined by the growth assay. Affinity chromatography mitigated the major problems of removing both lipids and pigment from the glycoproteins. In addition, partial purification of bound fractions with potent cholesterol crystal nucleation-altering activity can be obtained by this technique.
Anticholesteremic Agents↗
Hepatic extraction of organic anions in the rat depends on ligand hydrophobicity.
Non-bile-salt cholephilic organic anions are efficiently taken up by the liver. Recent work from our group has suggested the possible importance of relative hydrophobicity among various organic anions in hepatic uptake. To further validate and clarify this, we studied hepatic extraction of five different cholephilic dyes using the isolated perfused rat liver in single-pass mode. Albumin binding affinities and capacities for each of the ligands were measured in vitro to permit evaluation of in vivo interactions for each of them over a spectrum of unbound ligand concentrations. As expected, a strong positive correlation was found between ligand hydrophobicity and the relative degree of albumin binding affinity and capacity. Using appropriate experimental conditions, we also found a strong positive correlation between hepatic extraction efficiency for a given ligand and both its hydrophobicity and its unbound concentration. These data indicate that where the unbound ligand concentration is significant, the greater the ligand hydrophobicity, the greater is its efficiency of hepatic extraction. We conclude that hepatic extraction efficiency for non-bile-salt cholephilic organic anions depends on a combination of ligand hydrophilic/hydrophobic balance and the availability of the unbound ligand for uptake.
Animals↗
Nucleation and growth of cholesterol crystals. Kinetic determinants in supersaturated native bile.
Among the factors affecting the stability of supersaturated bile that culminates in the nucleation and growth of cholesterol crystals are biliary lipids. The abundance and composition of these lipids affect the composition of biliary vesicles that, if rich in cholesterol, are themselves unstable and that constitute precursor particles from which cholesterol crystals somehow arise. Kinetic factors can affect stability in either of two ways: They can inhibit cholesterol crystallization, thus stabilizing the system, or they can promote the process of cholesterol crystal nucleation, thus stabilizing the system.
Bile↗