Biomedical subjects
Publications and source records attributed to N Domingo.
Explore the source record for details and available documents.
BACKGROUND/AIMS: Gallbladder bile from patients with cholesterol or mixed gallstones frequently contains biliary "sludge", a suspension of cholesterol monohydrate crystals and pigment granules embedded in mucin and proteins. The composition of biliary "sludge" and the preferential localization of mucin and proteins could be an indicator for its potential role in gallstone formation. METHODS: Ultracentrifugation (100000 g/l h) was used to precipitate "sludge" from bile, and the concentration difference of its main components between native bile and ultracentrifuged bile samples was calculated. After purification of the sediment, immunolocalization was performed for the detection of mucin, IgA, albumin, aminopeptidase, and anionic polypeptide fraction using polyclonal and monoclonal antibodies. RESULTS: The amount of sludge in gallbladder bile was 4.26 mg/ml-0.78 (mean+/-SEM) in patients with cholesterol and 2.51 mg/ml+/-0.39 in patients with mixed stones and cholesterol was the main component (48.9+/-4.6% and 44.4+/-7.1%). The sediment appeared as a mixture of vesicular aggregates and pigment particles which were linked by a gel matrix of mucin containing cholesterol crystals. While anionic polypeptide fraction and aminopeptidase were associated to pigments, IgA was uniformly spread in the crystalline parts of "core-like" structures, and albumin, when it was present, appeared as randomly located small spots. CONCLUSIONS: Our study demonstrates that the cholesterol content and the distribution pattern of mucin and different proteins is similar in the sediments of biliary "sludge" to that previously shown in cholesterol and mixed gallstones. This suggests that biliary "sludge" represents an early stage of gallstone formation in these patients.
The beneficial metabolic effects of dietary soybean lecithin on lipid metabolism are now more clearly established. The intestinal absorption of cholesterol is decreased by soybean phosphatidylcholine-enriched diet and results in a cholesterol-lowering effect. There is an enhancement of the cholesterol efflux by endothelial cells incubated with soybean phosphatidylcholines, and a stimulation of the reverse cholesterol transport by high density lipoprotein-phosphatidylcholines. As a result of all these processes, phosphatidylcholines provided by the soybean lecithin metabolism appear to be key molecules controlling the biodynamic exchanges of lipids. They regulate homeostasis of cholesterol and fatty acids by decreasing their synthesis and promoting cholesterol oxidation into bile salts. Finally, the outcome is the increase in bile secretion of these lipids and/or their metabolite forms. Such findings constitute promising goals in the field of nutritional effects of soybean lecithin in the treatment or prevention of hyperlipidemia and related atherosclerosis.
Liver fatty acid-binding protein (L-FABP) is a small cytoplasmic molecule highly expressed in the liver. Since L-FABP exhibits affinities for several biliary components, its presence in bile was explored by Western blotting and competitive ELISA in various mammalian species. A L-FABP-like immunoreactivity was consistently found in both hepatic and gallbladder bile. A close molecular identity between this 14 kDa biliary protein and the purified L-FABP was assessed by immunological analyses and high performance capillary electrophoresis. Pharmacological induction of hepatic L-FABP biosynthesis led to a similar increase in biliary L-FABP levels showing a close relationships between the cytosolic and biliary contents of this protein. Finally, a correlation between the presence of L-FABP in bile and both bile flow and bile acid release was found. These data suggest an output of L-FABP in bile in normal conditions which might be coupled with the physiological release of biliary components.
We explored the possibility that the biliary protein fraction may support part of the variation in the nucleating activity previously measured in gallbladder biles of pigs. Eighteen gallbladder aspirates freshly obtained from three dietary groups (0, 5, or 10% beta-cyclodextrin) of six pigs were chromatographed to purify their total protein fraction. Proteins were quantified, and analysed through electrophoresis and immunoblotting or enzyme-linked immunosorbent assay for albumin, and five putative effectors of cholesterol crystallisation, mucins, immunoglobulin A, 130 kDa, apolipoprotein A-I, and anionic polypeptide fraction. Each total protein fraction was also assayed for its ability to influence cholesterol precipitation, when added to supersaturated model bile. The current data provided evidence that the cholesterol crystallisation-promoting activity of biliary proteins in model biles increased with the beta-cyclodextrin dietary content. This occurred without any significant change in the total biliary protein content, but was associated with a significant decrease in the concentration of albumin and apolipoprotein A-I, resulting in changes in the overall balance of proteins in bile. Comparison of these results with the crystallisation figures previously obtained from the corresponding native biles led us to conclude that biliary proteins might influence the outcome of the crystallisation process, namely the final crystal concentration at equilibrium, but would not systematically represent a major driving force for determining the velocity of crystal formation in native bile of pigs.
Anionic polypeptide fraction (APF) is a phospholipid- and calcium-binding apoprotein present in animal and human bile, predominantly associated with cholesterol-phospholipid vesicles. In bile, the protein may play a physiological role in preventing precipitation of calcium salts. APF has also been suggested to be of regulatory importance in the process of biliary lipid secretion. The aim of the present study was to investigate whether the secretion rates of APF and that of biliary lipids are coupled, which would support a physiological role of APF in biliary lipid secretion. Biliary secretion rates of bile acids, phospholipids, and cholesterol were experimentally modulated in three different rat models. Secretion rates of APF were compared with that of bile acids, lipids, and with that of two other biliary proteins, the lysosomal protein beta-glucuronidase and apolipoprotein A-I (apo A-I). Model 1: diurnal variation in bile formation during chronic bile diversion; model 2: specific inhibition of biliary phospholipid and cholesterol, but not of bile acid secretion by infusion of the organic anion, sulfated lithocholyltaurine; model 3: acute interruption of the enterohepatic circulation in unanesthetized rats. The diurnal variation in bile formation involved a parallel increase of the biliary secretion rates of bile acids (+56 +/- 7%, mean +/- SD), phospholipids (+53 +/- 29%), cholesterol (+73 +/- 54%), and APF (+72 +/- 86%) during the night phase of the cycle. Infusion of sulfated lithocholyltaurine inhibited biliary phospholipid and cholesterol secretion (-78 +/- 15%, and -54 +/- 25%, respectively), but did not affect biliary bile acid or APF secretion rate (-19 +/- 14%, and +12 +/- 107%, respectively). Within 4 hours after interruption of the enterohepatic circulation, bile secretion rates for bile acids (-92 +/- 3%), phospholipids (-74 +/- 13%), cholesterol (-64 +/- 8%), and APF (-58 +/- 24%) rapidly declined to a new steady-state level. Correlation analysis using the data from the three experimental models indicated that the biliary secretion rate of APF was independent from that of phospholipids, cholesterol, beta-glucuronidase, and, presumably, apolipoprotein A-I, and positively correlated to bile acid secretion rate and bile flow. The data from three experimental models indicate that the biliary secretion rates of APF and of phospholipids/cholesterol are not coupled and, therefore, do not support a direct physiological role of APF secretion in biliary lipid secretion. APF secretion into bile may, at least partially, be controlled by biliary bile acid secretion.
Two very similar small anionic, amphipathic proteins, a phospholipid-binding apoprotein (anionic polypeptide fraction [APF]) and a calcium-binding polypeptide (CBP), are found abundantly in bile and all types of gallstones. The often disparate properties among various preparations of APF/CBP could reflect different sources and separation procedures, leading to partly degraded and/or denatured protein and varied association of bile salts, lipids, bile pigments, and detergents. The present study presents new methods for isolation and purification of APF/CBP, and characterizes the preparations thus obtained. It was found that isolation by selective precipitation of proteins from fresh T-tube bile by added calcium chloride, followed by demineralization with ethylenediaminetetraacetic acid (EDTA), removal of salts, lipids, and some pigment by Sephadex LH-20, and serial ultrafiltration yields the purest preparations. Though free of lipids, bile salts, detergents, and most pigments, these new preparations all show the same 7-kd and 12-kd bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the same major peaks on hydrophobic high-performance liquid chromatography (HPLC), and retain the self-associative, lipid- and calcium-binding functions, typical of older preparations obtained by potentially denaturative procedures. The varied properties among APF/CBP preparations are thus apparently related mainly to their content of different proportions of two major components, lipid-binding APF and calcium-binding CBP. Immunologic cross-reactions indicate common epitopes, and amino acid analyses are also similar, suggesting that APF and CBP may have the same polypeptide backbone, but differ because of posttranslational modification(s). Sufficiently pure APF and CBP have now been obtained to permit possible structural identification by sequencing and molecular biological techniques, though such attempts have thus far been unsuccessful.
BACKGROUND/AIMS: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstone pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. METHODS: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. RESULTS: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. CONCLUSIONS: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.
BACKGROUND/METHODS: In this study, pigs fed for 3 weeks a well-balanced semi-purified diet enriched with 0.3% cholesterol and 0, 5 or 10% beta-cyclodextrin were proposed as new animal donors of gallbladder bile exhibiting different rates of cholesterol crystallization, in order to gain insight into the early mechanisms underlying cholesterol precipitation in vivo. The appearance and growth of cholesterol crystals were monitored in the incubated freshly collected gallbladder biles through light microscopy and concomitant time-sequential determination of crystallized cholesterol concentration, and interpreted in terms of the composition of the bile. RESULTS: Although the concentration of total lipids and proteins and the relative proportions of bile acids, phospholipids, and cholesterol remained unchanged under beta-cyclodextrin, the cholesterol crystallization increased in the following order: 0<<10<5% beta-cyclodextrin. Concomitantly, the proportion of chenodeoxycholic acid in bile, and the hydrophobicity index of the biliary bile acid mixture increased in the following order: 0<5<10% beta-cyclodextrin (the same as reported elsewhere for the decrease in the antinucleating ApoA1), while sn-2 arachidonoyl biliary lecithins were specifically increased with 5% beta-cyclodextrin in the diet. CONCLUSIONS: We hypothesized that lecithin molecular species may be the determinant factor in modulating high cholesterol crystallization rates in biles otherwise enriched with hydrophobic bile acids.
BACKGROUND/AIMS: Cholesterol gallstones consist of cholesterol crystals and smaller amounts of pigments and calcium salts, arrayed on a mucin plus protein matrix. The localization of the various biliary proteins in the stones has not been characterized. We aimed to localize several biliary proteins in gallstones in order to determine their possible role in stone formation and growth. METHODS: The distribution of several matrix proteins and their relationships to the minerals were determined using immunostaining and EDAX microanalysis on hemisected cholesterol gallstones. RESULTS: Pigment areas were rich in calcium and contained Cu, P and S. These elements were absent in cholesterol regions. Mucin was identified in a three-dimensional network intercalated between cholesterol crystals and as septa between deposits of pigments and cholesterol; APF/CBP and ApN coated only the pigment deposits. No specific topographical localization was found for albumin or IgA. CONCLUSIONS: This suggests a role for mucin, APF/ CBP and ApN in the formation of cholesterol gallstones. We propose that cholesterol crystals bind directly to mucin, whereas calcium salts and pigments deposit on APF/CBP and ApN bind to the mucin.
We present here a computational method based on the analysis of amino acid composition for performing comparisons between proteins. This user-friendly and reliable test is aimed at rapidly identifying, from data-base subsets, sequences--if necessary, partial sequences--which share similar amino acid compositions to the input composition (deduced from experimental results). Apparent molecular weight (as determined by SDS-PAGE) and artefactual modifications due to the experimental determination of the amino acid composition are taken into account to perform the comparison. This program thus constitutes a useful tool in searching for the probable identification of either non-sequenced proteins or peptides from hydrolysed proteins.
1. The objective of this study was to compare in cultured human hepatocytes or Hep G2 cells, changes in the fate of unesterified low density lipoprotein (LDL)-cholesterol induced by crilvastatin, a new cholesterol lowering drug and a reference statin, simvastatin. 2. The experiments were carried out for 20 h, each well contained 4.2 x 10(5)/cm2 Hep G2 cells or 0.5 x 10(5)/Cm2 human hepatocytes, 130 microM ursodeoxycholate, 0.68 microCi or 1.59 microCi unesterified human [14C]-LDL-cholesterol, crilvastatin or simvastatin at 0 or 50 microM (both cell types) or 300 microM (Hep-G2 cells). Incubation with the two drugs resulted in increased amounts of unesterified [14C]-LDL-cholesterol taken by the two cell types, compared to control. 3. Crilvastatin 50 microM led to significantly higher quantities of [14C]-glyco-tauro-conjugated bile salts, compared to simvastatin. Statins reduced the apo B100 level secreted by the two cell types (simvastatin) or human hepatocytes (crilvastatin). Crilvastatin enhanced both the level of apo A1 secreted by the Hep G2 cells and the level of APF, a high density lipoprotein (HDL) and biliary apoprotein. 4. Crilvastatin not only acts by stimulating LDL-cholesterol uptake by hepatocytes, but also by enhancing the catabolism of LDL-cholesterol in bile salts and probably by stimulating HDL and/or bile component secretion. Such a mechanism was not previously described for HMG CoA reductase inhibitors. Our results on APF show that this apoprotein could be considered also as an indicator of changes in bile and/or HDL compartments. 5. The human hepatocyte model appeared to be a suitable and relevant model in the pharmacological-metabolic experiments carried out in this study. It led to more consistent data than those obtained with Hep G2 cells.
A calcium-binding protein of 68 kDa was isolated from human bile that precipitates upon addition of 5 mM CaCl2. This protein was recognized by an immunoaffinity purified anti-annexin VI antibody and it had a similar aminoacid composition as annexin VI. Phospholipase A2 activity was inhibited in vitro in a dose-dependent manner as reported for annexin VI. It is the first time that an annexin secretion is demonstrated in bile.
We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl-, phosphate and Ca2+ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na+,K+ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs.(ABSTRACT TRUNCATED AT 250 WORDS)
The small (7 kD) biliary phospholipid and calcium binding polypeptide (anionic polypeptide fraction/calcium binding protein) has been found in higher concentrations in the bile of patients with pigment stones than in controls. In different model systems it was variously found to promote or retard cholesteral crystalization. In the present study we investigated its distribution between cholesterol carriers in bile and its effect on cholesterol crystalization in native and model biles. On gel chromatography anionic polypeptide fraction/calcium binding protein was found predominantly in three areas: in the vesicular fraction, in the non-vesicular lipid fraction and in another fraction unassociated with biliary lipids. It was much more concentrated in the vesicular than in the non-vesicular fraction, the mean anionic polypeptide fraction/phospholipid molar ratio being 219 +/- 181 vs. 30.4 +/- 16, respectively. Anionic polypeptide fraction/calcium binding protein was added at three dose levels, 0.14, 0.28, 0.42 mg/ml (representing approximately 18%-55% of the physiologic biliary concentration), to 19 human and five model biles. This did not produce any significant changes in the nucleation time. The addition of anionic polypeptide fraction/calcium binding protein at a dose level of 0.42 mg/ml to 13 different human biles did not induce changes in the distribution of cholesterol among its carriers. The present experiments do not support a role for anionic polypeptide fraction/calcium binding protein in the process of cholesterol nucleation in bile. Qualitative changes in the protein molecule, as demonstrated in other human secretions, cannot be excluded.
Bile lipids are secreted in association with a newly identified major apoprotein called anionic polypeptide fraction-calcium binding protein (APF-CBP), which is synthesized in the hepatocytes and has been detected in both bile and plasma and characterized. The secretion of the lipids in bile depends both on the concentration and the hydrophobicity of the bile salts (BS) secreted. The present study was undertaken to determine whether the synthesis and the secretion of APF-CBP are similarly regulated by BS, using two methods. The synthesis and secretion of labelled, newly synthesized APF-CBP by isolated rat hepatocytes were monitored by solid-phase immunoassay. For this purpose, hepatocytes were incubated with either glycodeoxycholate (GDC) or taurocholate (TC). The synthesis and secretion of labelled, newly synthesized APF-CBP by perfused rat liver were measured by immunological enzyme-linked assay (ELISA) upon perfusing the liver with either GDC or TC. We found that (i) the synthesis and the secretion of APF-CBP were increased during either TC or GDC perfusion, but the increase was more pronounced with TC; (ii) in GDC perfusion the APF-CBP levels measured were more closely related to the levels of bile salts and not to phospholipid levels, (iii) when the two bile salts were perfused in reverse order, i.e., first GDC and then TC, the secretion of APF-CBP in bile decreased when GDC was perfused, but increased when TC was perfused. Similar results were obtained in experiments with isolated hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
With the demonstration of pronucleating and antinucleating proteins, the role of biliary proteins became of considerable research interest. Anionic polypeptide fraction is the third most abundant biliary protein; it is found in association with biliary lipids, has antinucleating properties for calcium and is found in gallstones. Its levels in various human biles have not been studied as of this writing. In this investigation the concentration of anionic polypeptide fraction in gallbladder bile was measured in 16 subjects without gallstones, 19 subjects with cholesterol stones and 15 subjects with pigment stones in Tel Aviv. Anionic polypeptide fraction concentrations in bile (mean +/- S.D.) were 0.76 +/- 0.09 gm/L in controls and 0.81 +/- 0.25 gm/L (which was not significant) in patients with cholesterol gallstones. They were significantly higher 1.03 +/- 0.23 (p < 0.05) in patients with pigment gallstones. The anionic polypeptide fraction/phospholipid ratio and the anionic polypeptide fraction/total lipid ratio were significantly higher in patients with pigment gallstones (p < 0.005 and 0.05, respectively). The anionic polypeptide fraction lipid ratios were insignificantly elevated in biles of patients with cholesterol stones compared with the ratios in biles of controls. Only the anionic polypeptide fraction/phospholipid ratio was significantly higher in biles of patients with pigment stones compared with those with cholesterol gallstones. The values were similar although higher in a small group of gallstone patients from Marseilles. The role of anionic polypeptide fraction in the pathogenesis of gallstones, particularly pigment gallstones, requires further study.
This work was undertaken to study the effect of intravenously infused dehydrocholate (DHCA) and cholate (CA) on lipid and anionic polypeptide fraction (APF) secretion in bile. APF is a small acidic amphipathic apoprotein closely associated with biliary lipids and bilirubin and involved in the control of bile-destined cholesterol. Rats were infused with increasing doses of DHCA (2 and 3 mumols/min/100 g b.w.) and then CA (1, 2, and 3 mumols/min/100 g b.w.). Each dose was infused for 30 min. As expected, intravenous DHCA inhibited biliary phospholipid (PL) and cholesterol secretion, and CA restored it. When DHCA was infused, the level of APF increased fourfold compared with controls. The APF/PL ratio also increased, but biliary albumin remained stable. When bile secretion was stimulated by infusion of CA, biliary APF returned to normal. These data indicate that biliary secretion of APF depends on the nature and the amount of bile salts returning to the liver, and consequently, APF can be considered a marker of bile secretion disorders.