The effect of simvastatin and bezafibrate on bile composition and gall-bladder emptying in female non-insulin-dependent diabetics.

Female non-insulin-dependent diabetics have a high prevalence of gallstones. Treatment of hyperlipidaemia in these patients may modify the risk. Seventeen female non-insulin-dependent diabetics (age 35-65) were treated with simvastatin (n = 10) or bezafibrate (n = 7) and had the cholesterol saturation index (CSI) of bile and gall-bladder emptying measured before and after 3 months therapy. In both groups, there was a significant reduction in serum cholesterol following treatment. The mean pretreatment cholesterol saturation indices of bile did not differ between the two groups but, after 3 months therapy, there was a highly significant difference in CSI between the bezafibrate group (2.0 +/- 0.33) and the simvastatin group (1.1 +/- 0.14) P < 0.002. Whereas the increase in the CSI (42%) observed with bezafibrate therapy was significant, the decrease in the simvastatin group (14%) was only significant in those whose pretreatment cholesterol saturation indices were elevated. Despite the differences in CSI observed between the two treatment groups, no changes in gall-bladder emptying were detected.

Effect of phospholipase C on cholesterol solubilization in model bile. A concanavalin A-binding nucleation-promoting factor from human gallbladder bile.

Human bile contains a phospholipase C activity. To examine its pathophysiological importance, the effect of phospholipase C on the dynamics of lipid solubilization and nucleation (cholesterol crystal formation) were investigated in model bile. Phospholipase C from gallbladder bile from patients with gallstones was partially purified by competitively eluting from a concanavalin A (con A)-Sepharose (Sigma, St. Louis, MO) column and incubating with Pronase (Calbiochem, Behring Diagnostics, La Jolla, CA). Phospholipase C activity was resistant to Pronase digestion. When this fraction (concentrated to half the original volume) was mixed with model bile (1:1, vol/vol), a transfer of cholesterol and phospholipid from the micellar to the vesicular phase and an accelerate nucleation time were found concomitant with phospholipid hydrolysis. These effects were prevented by inhibiting the phospholipase C activity with ethylenediaminetetraacetic acid. To confirm that the results were caused by phospholipase C activity and not some other nucleation-promoting factor within the biliary con A preparation, model bile was incubated with bacterial phospholipase C. An identical cascade of events to that found with the partially purified biliary enzyme was observed. Further purification of the con A-bound proteins on DEAE-Sephadex (Pharmacia, Uppsala, Sweden) did not resolve any separate nucleation-promoting activity to that associated with phospholipase C activity. In conclusion, this study has identified phospholipase C as a/the con A nucleation-promoting activity in human gallbladder bile and has characterized a possible molecular mechanism by which cholesterol nucleation is stimulated by this fraction.

Comparative analysis of cholesterol transport in bile from patients with and without cholesterol gallstones.

Aggregation of cholesterol-phospholipid vesicles in supersaturated biles precedes cholesterol crystal formation. In this study we examined the relationship between the percentage of cholesterol carried by vesicles and/or their composition and the propensity to form cholesterol crystals (nucleation time). Bile (common bile duct, gallbladder and T-tube) was obtained from patients with and without gallstones. Gel filtration chromatography resolved three peaks, a void volume vesicle, a smaller vesicle (identified by electron microscopy and of distinct composition compared to the larger void volume vesicle), and the mixed micelle. The void volume vesicle was present in 11 of 28 abnormal gallbladder biles, but in none of the 10 normal gallbladder biles. Despite this difference, no correlation between the nucleation time of whole bile with either the percentage of cholesterol carried by or cholesterol/phospholipid ratio of the void volume vesicle was found. Nucleation time was, however, found to correlate with the composition of the small-vesicular transport form. No significant difference in the composition or percentage of the small-vesicular form or the combined vesicular forms was found between normal and abnormal gallbladder biles, although the latter nucleated significantly more rapidly. Our results confirm the importance of vesicles in the nucleation process but suggest that other factors, not yet identified, appear to be responsible for the more rapid nucleation seen in abnormal gallbladder biles.

Nucleation of cholesterol crystals from native bile and the effect of protein hydrolysis.

Nucleation time represents the terminal step in in vitro studies examining bile lithogenicity. Because of the concern that residual microcrystals, left after ultracentrifugation, may be responsible for the rapid nucleation time of gallbladder bile from patients with cholesterol gallstones, we have included a final filtration step. However, we found this procedure to considerably lengthen the nucleation time of abnormal biles. In view of the central importance of the nucleation assay we compared the effect of three commonly used gallbladder bile pre-treatment regimes (designed to remove endogenous crystals) on nucleation time. They were: a) immediate filtration of bile (0.22 micron filter); b) ultracentrifugation; and c) ultracentrifugation followed by filtration. The respective nucleation times were: a) 9.3 +/- 3.7 days, n = 6; b) 2.9 +/- 0.4 days, n = 10; c) 12.8 +/- 2.3 days, n = 11. To determine whether the dramatic change in nucleation time was due to the removal of components other than seed crystals, we examined the mucus content, the total lipid composition of bile, and that of its cholesterol transport components following the different pre-treatments. No significant difference in total lipid, percentage cholesterol carried by the transport components, or their cholesterol/phospholipid ratio were found. Ultracentrifugation alone was sufficient to removal all detectable large molecular weight mucus glycoprotein. Although nucleation time of the abnormal gallbladder samples was extended in the ultracentrifuged/filtered biles, it was still significantly different (P less than 0.01) from that of normal gallbladder biles, confirming an intrinsic difference between abnormal and normal biles, in cholesterol metastability. We also examined the effect of protein digestion on the nucleation time of native biles.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase C and diacylglycerol lipase in human gallbladder and hepatic bile.

A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho [N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol.h-1.mg-1 of delipidated protein) were found in common bile duct (11.25 +/- 14.23) and gallbladder bile (19.07 +/- 22.24), although per milliliter of bile, the mean gallbaldder levels were 6.4 times greater than those found in common duct bile. With the tow substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio similar to that found with sn-2[3H]DPPC. Finally, no radiolabeled lysolecithin was identified with [3H]choline-DPPC or [14C]DPPC as substrate indicating the free fatty acid was the product of a diacylglycerol-lipase rather than a phospholipase A2. Phospholipase C and diacyl-glycerol-lipase activities were significantly correlated (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein localization in the human bile duct and gallbladder.

Apolipoproteins AI, AII and B were identified in the normal and pathological human bile duct and the gallbladder epithelium using an avidin-biotin immunoperoxidase technique. Small intestine and stomach sections served as positive and negative controls respectively. Staining was focal for apolipoproteins AI and AII, and continuous for apolipoprotein B. In addition to homogenous and granular cytoplasmic staining, foamy cytoplasmic staining, particularly for apolipoproteins AI and AII, was observed around lipid droplets in cells containing much lipid. No correlation between a particular pathological condition of the gallbladder (acute cholecystitis, mucocele, chronic cholecystitis, cholesterolosis) and staining pattern or intensity of staining was found for any of the apolipoproteins, although both apolipoproteins AI and AII stained more intensely than apolipoprotein B in each group. Positive staining was also found for all apolipoproteins in epithelial cells which had invaded the underlying connective tissue (gallbladder carcinoma), suggesting that the epithelial cells are capable of synthesizing apolipoproteins de novo. In this latter case, apolipoprotein B stained more intensely than for either AI or AII, and significantly (p less than 0.05) more strongly than that found in the other pathological groups. The identification of apolipoproteins in the gallbladder epithelium raises the interesting question of their origin and functional role.

The effect of ethanol on phospholipid metabolism in rat pancreas.

The phospholipid effect involves agonist-induced breakdown of phosphatidyl inositol (or polyinositides) generating second messengers followed by increased incorporation of 32P during the resynthetic phase of the cycle. Ethanol, an aetiological factor in pancreatitis, has been shown to have various effects on pancreatic secretion. In this study ethanol decreased the incorporation of 32P into phosphatidyl inositol but had no effect on the stimulated breakdown of prelabelled phosphatidyl inositol. However, in addition to recycling of phosphatidyl inositol stimulation of pancreatic tissue results in increased incorporation of precursors into other phospholipids. Cholecystokinin increased the incorporation of both [U-14C] glucose and 32P into phosphatidyl ethanolamine 3-fold but had no effect on 32P incorporation into phosphatidyl choline. As well as increased incorporation of 32P into phosphatidyl inositol (8-fold) cholecystokinin also increased the incorporation of [U-14C] glucose into phosphatidyl inositol (4-5-fold) implying significant de novo synthesis of 1,2 diacyl glycerol in addition to the currently accepted recycling of the 1,2 diacyl glycerol back to phosphatidyl inositol. Ethanol caused an inhibition of 32P incorporation into total phospholipid of rat pancreas during basal and stimulated conditions. When individual phospholipids were separated ethanol was found to decrease the incorporation of 32P into phosphatidyl choline under basal conditions and into all phospholipids during cholecystokinin stimulation. With [U-14C] glucose as the precursor, ethanol inhibited its incorporation into phosphatidyl choline only. Ethanol did not alter the total 32P radioactivity in the aqueous phase of the pancreatic extract nor the percent incorporated into nucleotides. This excluded decreased uptake of 32P and incorporation into nucleotides as a mechanism for the differential inhibition of 32P versus [U-14C] glucose incorporation into phospholipids other than phosphatidyl choline under stimulated conditions.

Identification of a phosphatidylcholine active phospholipase C in human gallbladder bile.

This study describes the identification of a phospholipase C activity against phosphatidylcholine in delipidated human gallbladder bile. All biles were obtained from cholesterol gallstone patients and were negative on bacterial culture. The biliary enzyme was inhibited by EDTA and had a pH optimum of between 7-8. All of the 15 gallbladders examined contained significant phospholipase C activity (32.85 +/- 8.37 nmol/h/mg delipidated protein). The finding of a phospholipase C in gallbladder bile of patients with cholesterol gallstones may be one of the factors responsible for or related to the rapid in vitro nucleation seen in these biles.

The effect of ethanol on enzyme synthesis and secretion in isolated rat pancreatic lobules.

This study investigates the effect of ethanol on enzyme synthesis and secretion in rat pancreatic lobules. Ethanol caused a dose-dependent inhibition of 3H-leucine incorporation into total protein. Examination of the time dependence showed that ethanol inhibited protein synthesis at each time point. Removal of ethanol partially reversed this inhibition. An autoradiograph of the newly synthesized proteins separated on SDS-PAGE showed that ethanol inhibited synthesis of all proteins. 14C-cycloleucine uptake was not altered by ethanol, excluding inhibition of amino acid uptake as the mechanism for the decreased protein synthesis induced by ethanol. Electron microscopy revealed no ultrastructural damage. Ethanol had no effect on the stimulated release of (i) amylase from zymogen granules nor (ii) newly synthesized pulse labelled enzymes. Acetaldehyde had no inhibitory effect on enzyme synthesis or secretion indicating that ethanol per se and not its metabolite is inhibitory. The decreased synthesis after acute exposure to ethanol with preservation of exocytosis would limit the autodigestive potential of pancreatic tissue. This may explain why isolated toxic doses of ethanol are rarely if ever associated with pancreatitis.

Biliary secretion of cefoperazone and its inter-relationship with bile acid transport in the rat.

Cefoperazone is a third generation cephalosporin which is secreted predominantly in bile. This study set out to examine the effect of stimulating bile choleresis on the biliary secretion of cefoperazone. Stimulation of both bile acid-dependent and independent bile flow (phenobarbitone pretreatment) hastened the peak appearance of a pulse of cefoperazone into bile. Although the biliary secretion rate of cefoperazone was enhanced by bile acid infusion, the % recovery and maximal biliary concentration were reduced. The reciprocal effect of continuous cefoperazone infusion on the rate of biliary transport of a pulse of bile acid was examined. Cefoperazone infusion hastened the biliary transport of glycocholate. Net recovery of glycocholate was unaffected.

Inhibition of biliary phospholipid and cholesterol secretion by cefoperazone.

The effect of cefoperazone, a third-generation cephalosporin, on biliary lipid secretion in rats was examined. Rats were anesthetized with ether and the mid-lumbar vein and common bile duct cannulated. Bile acid secretion was maintained by intravenous taurocholic acid infusion (28 mumol/hr). A 1-hr control period was followed by intravenous cefoperazone infusion at either submaximal (20 mumol/hr), or supramaximal (60 mumol/hr) concentrations. At the cefoperazone infusion rate of 20 mumol/hr (biliary secretion of 7.1 +/- 1.6 mumol/hr) phospholipid secretion fell 19% and cholesterol secretion fell 31%; at a cefoperazone infusion rate of 60 mumol/hr (biliary secretion rate of 27.1 +/- 5.1 mumol/hr) phospholipid and cholesterol secretion were further reduced 40% and 56%, respectively, of controls. All changes were significant (P less than 0.01). Inhibition of both cholesterol and phospholipid secretion paralleled each other, was dose-dependent, and reversible. Cefoperazone's inhibitory action was abolished at a bile acid infusion rate of 108 mumol/hr. Cefoperazone was not found to be associated with bile acid micelles or mixed micelles as determined by ultracentrifugation and gel filtration. Thus, the effect of cefoperazone on biliary lipid secretion is not due to the impairment of mixed micelle formation in the canalicular lumen but rather its inhibitory effect appears to be due to a presecretory event.

The incorporation of [myo-2-3H] inositol into phosphatidyl inositol of stimulated rat pancreas.

The 'phospholipid effect' involves agonist induced breakdown of phosphatidyl inositol (PI) or its phosphorylated derivates with increased incorporation of 32P or [myo-2-3H] inositol during resynthesis. In rat pancreas pancreozymin and bethanecol resulted in the standard dose dependent increased incorporation of 32P into PI which was paralleled by increased amylase secretion. By contrast the incorporation of [myo-2-3H] inositol into PI was significantly decreased by pancreozymin whereas bethanecol had no effect. However, pancreozymin caused a 30% decrease in labelled PI irrespective of whether it was prelabelled with 32P or [myo-2-3H] inositol. Thus in rat pancreas, pancreozymin resulted in the standard agonist induced breakdown of pre-labelled PI but inhibited the incorporation [2-3H-myo] inositol during the resynthetic phase.

Distribution of biliary cholesterol between mixed micelles and nonmicelles in relation to fasting and feeding in humans.

To further investigate the nonmicelle mode of cholesterol transport in human bile, we examined its levels in relation to fasting and feeding. T-tube bile samples were collected (for 30 min) every 4 h over a 24-h period. All patients (3 with cholesterol gallstones; 1 with pigment stone) had their T tube clamped for a minimum of 4 days before the study to allow the bile acid pool to replete. Biliary lipid concentrations in all patients increased with feeding and decreased with fasting. However, because of a greater decrease in bile acid concentration relative to cholesterol concentration during the fasting period, fasting bile was consistently more saturated than bile obtained while feeding. Associated with the increase in lithogenic index with fasting was a decrease in the micelle solubilized cholesterol and an increase in nonmicelle solubilized cholesterol. At low bile acid concentrations (fasting) most biliary cholesterol is therefore transported as a nonmicelle complex, whereas at high bile acid concentrations (feeding) most of the cholesterol is transported in the mixed micelle. No nucleation of the biliary samples was observed in any of the patients over a 10-day period of observation. Thus the biliary nonmicelle complex (presumably vesicles) has a major cholesterol transport function especially at low bile acid concentrations.

Solubilisation of cholesterol in human bile.

Two non-disruptive separation techniques (gel filtration, and CsCl density gradient ultracentrifugation) were used to characterise the mode of cholesterol transport in human bile. Both methods showed that biliary cholesterol is solubilised as a high--Mr non-micelle (lipoprotein) complex as well as the mixed micelle. The lipoprotein complex was separated between the densities 1.01 and 1.08, had a cholesterol to phospholipid ratio of 0.95 +/- 0.41 and contained protein (7-20% by wt). The mixed micelle on the other hand sedimented at d greater than or equal to 1.18, and had a cholesterol to phospholipid ratio of 0.41 +/- 0.13. Eighteen bile samples, obtained from either T-tube, gallbladder or endoscopy, were analysed, and without exception they all contained a fraction of biliary cholesterol in the non-micelle form. The results indicate that cholesterol is secreted, at least in part, as a lipoprotein complex independently of bile acids (mixed micelles).

Lithocholate detoxification and biliary secretion in the rat.

To define the efficiency of hepatic detoxification, 14C lithocholate in combination with taurocholate was continuously infused intravenously into rats until steady-state. Quantitation of hepatic radiolabelled bile acid under these conditions showed only 11% of total liver bile acid was unmetabolised, indicating very efficient detoxification of lithocholate, in its most hepatotoxic state. Interestingly we found the rate for each bile acid to reach steady-state differed. To test whether the difference was due to glutathione S-transferase binding, as proposed by Strange et al (8), glutathione S-transferase levels were induced by phenobarbitone treatment. An increase in cytosol glutathione S-transferase levels had no effect on the time it took for each bile acid to reach steady-state.

Isolation of two cholic acid-binding proteins from rat liver cytosol using affinity chromatography.

Using an affinity matrix coupled with cholic acid, two proteins that recognise bile acids were isolated from rat liver cytosol. One protein of molecular weight 68 000 was immunologically identical to rat albumin. The other protein was of molecular weight 46 000. On discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis the 46 000 molecular weight protein dissociated to a single band with an RF value identical to the Yb subunit of the bromosulphophthalein-binding fraction (Y-fraction) of whole liver cytosol. The monomers of purified ligandin under these conditions resolved into two bands which corresponded to the Ya and Yc subunits of liver cytosol Y-fraction. Anti-serum to the purified ligandin reacted monospecifically with purified ligandin and whole liver cytosol, but did not cross-react with the Yb dimer eluted from the affinity column. The Yb dimer was shown to possess glutathione-S-transferase activity with a substrate specificity distinct from ligandin but similar to glutathione-S-transferase C. Cholic acid inhibited the catalytic activity of the transferase.

Comparison of three methods to estimate steatorrhoea.

Faecal fat excretion was estimated in 24 patients using three methods. Quantitative estimations of fat excretion were calculated from both a three day faecal fat collection and a twenty-four hour faecal collection corrected for excretion of a cuprous thiocyanate marker. Breath 14CO2 excretion was measured after ingestion of a liquid meal containing 2.5 microCi of 14C-triolein in an arachis oil emulsion. Peak concentrations of 14CO2 in breath were used as an estimate of the degree of fat absorption. Correlation between the two quantitative measures of faecal fat was good (r = 0.87), 17 patients having steatorrhoea of more than 7 g fat per day by both estimations. Results of the breath test were disappointing. With the standard meal containing 20 g arachis oil the lowest peak 14CO2 excretion rate seen in subjects without steatorrhoea, 3 percent of the dose per hour, was taken as the lower limit of normal. Seven subjects with steatorrhoea as shown by faecal collections excreted normal amounts of 14CO2. When the size of the fat meal was increased to 1.0 g arachis oil per kg body weight in 16 of the subjects previously studied, all patients with proven steatorrhoea excreted less than 3 percent of the dose per hour but three of the subjects without steatorrhoea gave abnormal breath excretion results. It is concluded that the collection of a 24 hour faecal specimen using a cuprous thiocyanate marker provides a more reliable estimate of faecal fat excretion than the 14C-triolein breath test.

Formation of cyclic AMP from exogenous ATP by isolated hepatocytes and adipocytes.

Suspensions of isolated rat hepatocytes and adipocytes converted exogenous ATP to cyclic AMP at a rate which was about 30--50% of that observed with homogenates of isolated cells. Formation of cyclic AMP was stimulated by hormones (isoprenaline in the case of adipose tissue and glucagon in the case of liver) and sodium fluoride. Experiments with [alpha-32P]ATP indicated that the conversion of exogenous ATP to cyclic AMP did not occur within the cells. It is proposed that in isolated hepatocytes ad adipocytes some catalytic units of adenylate cyclase are exposed on the outer surface of the cell membrane.