In vitro and in vivo effects of exogenous lipids on the enzymatic hydrolysis of rat bile phospholipids

The addition of total phospholipids, phosphatidylcholines, triglycerides, cholesterol or glycerol to incubation media containing rat pancreatic juice and bile labeled with [9,10³H₂] oleic acid (90% of the radioactivity present as phospholipids) had no effect on the hydrolysis of bile endogenous phospholipids. The introduction of 2 or 10 mg of phosphatidylcholines and 0.5 ml of bile (≈ 1.5 mg of phospholipids)into the rat upper duodenum decreased the rate of absorption of rative bile phospholipids. It was not followed by an increase of free fatty acids released from biliary phospholipids in the intestinal lumen. The introduction of bile (0.5 ml) and small amounts of triolein (1.4–3.5 mg) into the duodenum had little effect on the rate of hydrolysis and absorption of native bile phospholipids, but caused a reduced absorption of the free fatty acids released or those coming from initial nonphosphorus biliary lipids. The introduction of bile (0.5 ml) and large amounts of triolein (30 mg) into the duodenum increased the rates of hydrolysis and absorption of endogenous bile phospholipids. These observations suggest that luminal lipid components can modify the organization of luminal micelles and, consequently, the action of the pancreatic phospholipase A₂ and the absorption of bile lipids.     

Protective effect of biliary lipids on rat pancreatic lipase and colipase

The effect of biliary components on the inactivation of rat pancreatic lipase and colipase was studied. In vitro incubations of these proteins were performed at 37 C in the presence or absence of trypsin, under various conditions. The influences of bile lipoprotein complex, bile salts below or above the critical micellar concentration (CMC), or albumin were investigated. The results showed that albumin and bile salts below the CMC had no protective effect on the inactivation rate of lipase or colipase. Under both denaturating conditions, bile salts above the CMC had a very slight effect, whereas the presence of the bile lipoprotein complex maintained lipase and colipase activity. The magnitude of this effect was related to the biliary phospholipid concentration. By means of gel filtration, the protective effect of bile was found to be due to associations of bile lipoprotein complex with these proteins in presence of bile salts. A correlation between the amount of colipase and the protection of lipase in the presence of biliary phospholipids was observed. Intestinal content of rats with normal and diverted bile secretion was submitted to the same in vitro incubation, and the enzyme was more stable in the segments containing biliary phospholipids. This suggests that the interaction between the bile lipoprotein complex, colipase and lipase in the presence of bile salts could have an important role in the intestinal lumen by retaining the enzyme activity.     

Formation of lymph chylomicron phosphatidylcholines in the rat during the absorption of safflower oil or triolein

The chylomicron phosphatidylcholines from rats fed safflower oil or triolein were isolated and separated into four different fractions according to the degree of unsaturation. Fraction 1, which was rich in palmitic, stearic and oleic acid, was a minor fraction (7.6–11.6 mole%) during the absorption of safflower oil, but was quantitatively important (27–51 mole%) after triolein feeding when significant amounts of dioleoylphosphatidylcholine were present. Fraction 2, which was a major fraction in all the experiments, contained linoleic acid in combination with a saturated or monounsaturated fatty acid. The third fraction contained mainly linoleic acid and was present only after safflower oil feeding. This indicates that dilinoleoyl-phosphatidylcholine is formed in the intestine after ingestion of linoleic acid. Fraction 4, which was rich in arachidonic acid and saturated fatty acids, accounted for 15–20 mole% of the chylomicron phosphatidylcholines with both kinds of fat meals. Incorporation of³H-choline indicated that the dilinoleoyl- and dioleoyl-phosphatidylcholines were formed by synthesis de novo while the majority of the rem aining phosphatidylcholines originated partly from acylated biliary lysolecithins and partly from the existing pool of mucosal phospholipids not formed during active fat absorption.     

In vitro studies on interaction of rat pancreatic lipase and colipase with biliary lipids

Lipase and colipase were prepared separately from rat pancreatic juice, and their respective interaction with biliary lipids was investigated by gel filtration on agarose in the presence of a micellar solution of sodium taurocholate. It was found that the cofactor can associate with the biliary lipids, whereas the enzyme forms a high mol wt complex only in the presence of colipase. It is suggested that biliary phospholipids might participate in the in vivo formation of the enzyme-cofactor substrate complex at the triglyceride-water interface in the presence of bile salts.     

Effect of lecithin on jejunal absorption of micellar lipids in man and on their monomer activity in vitro

The effect of lecithin on jejunal absorption of fatty acids and octadecenoylglycerol was studied in healthy volunteers with a jejunal perfusion system which excluded pancreatic and biliary secretions from the test segment. Lecithin significantly reduced the absorption of oleic acid (P<0.05) and octadecenoylglycerol (P<0.01), while it had no effect on the absorption of ricinoleic acid. In vitro, lecithin reduced monomer activities of all three lipids; the changes were greater for oleic acid and octadecenoylglycerol than for ricinoleic acid (P<0.02). From these data it is concluded that lecithin reduces monomer activity of fatty acids in mixed micellar solutions and that it can thereby reduce the absorption rates of micellar lipids. Intact lecithin is not absorbed under these conditions. Maldigestion of lecithin in pancreatic insufficiency may, therefore, aggravate the steatorrhea observed in this condition.     

Lecithin inhibits fatty acid and bile salt absorption from rat small intestine in vivo

During digestion of a fatty meal, long chain free fatty acids (FFA) and lecithin are among the lipids solubilized in intestinal contents as mixed micelles with bile salts. We hypothesized that if lecithin were not hydrolyzed, the mixed micelles would be abnormal, and absorption of FFA and bile salts would be depressed. To test this hypothesis, isolated segments of rat small intestine were infused in vivo with micellar solutions of 2 mMolar linoleic acid and 10 mMolar taurocholate to which was added 3 mMolar 1-palmitoyl, 2-oleoyl lecithin (a common lecithin in bile and food), or 1-palmitoyl lysolecithin (the hydrolytic product of lecithin). Absorption of FFA and bile salt was measured under steady state conditions using a single-pass technique. Lecithin depressed the rate of FFA absorption by 40% (p<0.025) in jejunal and ileal segments whereas lysolecithin was associated with normal rates of FFA absorption. Lecithin also reduced taurocholate absorption from the ileum by 30% (p<0.05). These data support the idea that lecithin may depress FFA and bile salt absorption from the small intestine in pancreatic insufficiency. The following trivial names are used: lecithin (1,2-diacyl-sn-glycero-3-phosphorylcholine); lysolecithin (1-acyl-sn-glycero-3-phosphorylcholine).     

Role of luminal lecithin in intestinal fat absorption

The effects of biliary lecithin on fat absorption were studied in 1 day bile fistula rats fed micellar solutions of bile salt, monoglyceride and radioactive free fatty acids. By electron microscopy and measurement of uptake of radioactivity into liver and adipose tissue, it was shown that in the absence of bile lecithin there was significant impairment of fat release from mucosa. Fat clearance was effected by the feeding of phosphatidyl choline or choline, but not phosphatidyl ethanolamine, inositol or cholesterol. In the absence of luminal choline there was a decrease in incorporation of radioactive leucine into mucosal protein. It is concluded that biliary and dietary lecithin or choline play an important role in triglyceride transport out of intestinal mucosa, by providing surfactant lecithin for the chylomicron envelope and by supporting mucosal protein biosynthesis.     

Obligatory role of bile for the intestinal absorption of vitamin E

Normal, white female rats subjected to cannulation of the abdominal thoracic duct have been utilized for a study on the essentiality of biliary and pancreatic secretions for the intestinal absorption of vitamin E. In all animals the thoracic duct lymph was collected. Some rats had the enterohepatic circulation undisturbed and in others bile or pancreatic juice or both were drained to the exterior by appropriate catheters in the common bile duct. On the first postoperative day, the animals received intragastrically an emulsion containing protein, carbohydrate, monoolein, 2 mg ofd,l-α-tocopheryl acetate plus 50 μC ofd,l-α-tocopheryl-1’,2’-³H-acetate. The appearance of radioactive α-tocopherol and its derivatives was determined in lymph, hourly, after emulsion administration. The obligatory role of bile in intestinal absorption ofd,l-α-tocopheryl-1’,2’-³H-acetate has been established. Pancreatic juice seems to be necessary for the hydrolysis of the vitamin E acetate ester. The simultaneous infusion of bile and pancreatic juice promotes absorption of about 10% of the administered dose into the lymph. A chromatographic separation of the radioactive vitamin E fractions revealed that most of the vitamin E, which is actively transfered from the intestinal lumen to the lymph, is nonesterified. An oxidation product of α-tocopherol, presumably itsp-quinone, appears in small amounts in the lymph, but almost no labeled α-tocopheryl acetate could be detected under these experimental conditions.     

Effect of orlistat on fat absorption in rats: A comparison of normal rats and rats with diverted bile and pancreatic juice

Orlistat is a specific inhibitor of pancreatic and gastric lipases leading to decreased absorption of fat. In the present study, we measured the effect of orlistat on lymphatic fat transport in rats following intake of oils very different in FA composition and TAG structure, and compared this with the transport in normal rats and rats with fat malabsorption. Rats were subjected to cannulation of the main mesenteric lymph duct, and a feeding catheter was inserted into the stomach. In addition, malabsorbing rats were cannulated in the common bile and pancreatic duct. Emulsified safflower, fish, and randomized oils were administered, and lymph was collected for 24 h and analyzed for FA composition. Administration of 25 mg orlistat together with the dietary oils resulted in very small changes from baseline lymphatic transport, indicating that inhibition of the fat absorption was almost complete and furthermore that the source of fat had no influence on the inhibitory effect of orlistat. Orlistat did not interfere with the absorption of the hydrolysis products, since high absorption of sn-2 MAG and FFA (oleic acid) mixed with orlistat was observed. The baseline lymphatic transport in the orlistat group was higher than in the malabsorbing group, but this was the result of generally lower transport of endogenous FA in the malabsorbing group, presumably caused by the absence of bile FA. The transport of FA in normal rats was several-fold higher than the transport after orlistat addition and in malabsorbing rats. Thus, this study showed that orlistat inhibited fat hydrolysis, and thereby lymphatic absorption, almost completely independently of the fat administered.     

Thyroid hormone is required for dietary fish oil to induce hypersecretion of biliary cholesterol in the rat

In the rat, both fish oil diet and thyroid hormone replacement are reported to augment bile cholesterol secretion out of proportion to bile flow or secretion of other bile lipids. We sought common mechanisms for these effects and evaluated the role of phospholipid fatty acid composition in the process. Methimazole-treated hypothyroid rats were fed low-fat chow or chow supplemented with 10% corn oil or fish oil, and were studied before and after thyroid hormone treatment. Serum, hepatic, and bile lipids were measured, phospholipid fatty acid composition determined, and hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity assayed. Fish oil diet stimulated cholesterol secretion into bile only after thyroid hormone was given, and this action was synergistic with that of thyroid hormone. Reduced serum cholesterol in fish oil-treated rats was associated with increased biliary cholesterol secretion and diminished hepatic cholesterol content. This suggests that augmented biliary cholesterol secretion may contribute to the fish oil-induced reduction of serum cholesterol. No definite relationship between hepatic or biliary phospholipid fatty acid composition and biliary secretion was apparent, although high bile cholesterol secretion was associated with a low percentage of hepatic and bile phospholipid linoleic acid.     

Solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats

Intestinal absorption of various plant sterols was investigated in thoracic duct-cannulated normal rats. Lymphatic recovery was the highest in campesterol, intermediate in brassicasterol and sitosterol, and the lowest in stigmasterol and sitostanol. Higher solubility in the bile salt micelle was observed in sitosterol, campesterol, and sitostanol than in brassicasterol and stigmasterol. The solubility of the latter two sterols was extremely low. When the affinity of plant sterols for the bile salt micelle was compared in an in vitro model system, which assessed sterol transfer from the micellar to the oil phase, the transfer rate was the highest in brassicasterol, intermediate in campesterol and stigmasterol, and lowest in sitosterol and sitostanol. Although no significant correlations between lymphatic recovery of plant sterols and their micellar solubility or transfer rate from the bile salt micelle were observed, highly positive correlation was obtained between the lymphatic recovery and the multiplication value of the micellar solubility and the transfer rate. These observations strongly suggest that both solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats.     

Intestinal absorption of retinol and retinyl palmitate in the rat. Effects of tetrahydrolipstatin

The aim of the present study was to characterize the intestinal absorption of retinol and retinyl palmitate in thoracic duct and bile duct fistulated rats and to investigate the effect of a simultaneously administered lipase inhibitor, tetrahydrolipstatin (THL). Absorption was determined as lymphatic recovery over a 24-hr period, including an initial 12-hr continuous intraduodenal infusion of either [11,12-³H]retinol or [11,12-³H]retinyl palmitate given in emulsified glyceryl trioleate or in mixed micellar solution of monoolein and oleic acid. From micellar dispersion, labeled retinol and retinyl palmitate were recovered in the lymph to 50–60% and both to the same extent. Administered in emulsified form, labeled retinol from fed retinyl palmitate was recovered to 47%, but retinol from fed retinol to only 18%. THL (10⁻⁴ M) in the infusate had no significant effect on the recovery of¹⁴C-labeled oleic acid. The recovery of label from emulsified glyceryl tri[1-¹⁴C]oleate was significantly decreased at this concentration of THL (76.5% vs 19.6% recovery). When administered in emulsified form, retinol absorption was not significantly affected by THL at 10⁻⁴ M, while retinyl palmitate absorption was very significantly decreased (5.0% compared to 47.8%). In the presence of THL, retinol absorption from retinyl palmitate in micellar solution was decreased (from 58% to 17%). Most of the retinol in the lymph extracts (72.2 to 91.3) was present as retinyl ester, regardless of the chemical and physical form of administration. Furthermore, THL did not induce any change in this pattern.     

Bile acids LVII. Bile acids and colorectal cancer

Significant correlations have been reported by epidemiologists between the mortality from colorectal cancer in various populations and the consumption of meat or lipids by these populations. These have directed considerable attention to possible relationships between diet and the occurrence of this neoplasm. We have carried out studies of the composition of bile from rats as influenced by diets of varying lipid content. Two cannulas were surgically implanted to form an externalized bile duct through which bile was drained from the common duct and returned to the duodenum. Small aliquots were analyzed for total bile acids by enzymatic assay and for individual bile acids by high-pressure liquid chromatography, gas chromatography and gas chromatography-mass spectrometry. Animals consuming diets highest in lipid content provided bile with the greatest amounts of bile acids. The primary bile acids, taurocholic, taurochenodeoxycholic, and tauro α- and β-muricholic acids made up more than 99% of the 3α-hydroxy bile acids and were found in approximate molar ratio of 2∶1∶1. Either complete drainage of bile without return to the duodenum, or biliary tract obstruction had pronounced influence on the rate of secretion of bile and its composition.     

Effect of cholestyramine on bile acid metabolism in conventional rats

Effects of cholestyramine on biliary secretion of cholesterol, phospholipids and bile acids and fecal excretion of sterols and bile acids were examined in Wistar male rats. Six rats were fed a basal diet, and the other six were fed a basal diet supplemented with 5% cholestyramine for eight days. Bile flow and biliary secretion of bile acids and phospholipids (per hour per rat) decreased with cholestyramine treatment, while biliary cholesterol secretion (per hour per rat) remained unchanged. In the biliary bile acid composition, a marked increase of chenodeoxycholic acid with a concomitant decrease of β-muricholic acid was observed in cholestyramine-treated rats. Fecal excretion of total sterols and bile acids increased about three-and four-fold, respectively, after cholestyramine treatment. The increase of fecal bile acids derived from cholic acid was more predominant than that derived from chenodeoxylcholic acid, resulting in an increase of the cholic acid group/chenodeoxycholic acid group ratio.     

Effect of Castration and hormonal supplementation on cholesterol cholelithiasis in the male hamster

This study examined the effect of castration and dietary hormonal supplementation on cholesterol cholelithiasis in male hamsters. Animals fed a standard lithogenic diet developed cholesterol gallstones (17%) after 6 wk, while castrated hamsters did not form any stones. Addition of a synthetic androgen, methyltestosterone, to the lithogenic diet induced cholelithiasis in castrated animals (50%). The biles of normal and castrated-hormone supplemented hamsters had cholesterol saturation indices of 1.0 and 1.1, respectively, while the bile of the castrated animals remained unsaturated (0.6). The ratio of cholic acid/chenodeoxycholic acid in bile increased after castration, but returned to normal levels following hormonal supplementation. Biliary cholesterol carriers were separated by ultracentrifugation. Animals in the stone-forming groups (normal and castrated-hormone treated) had a significant proportion of their biliary cholesterol in vesicles (44 and 46%, respectively); castrated hamsters had less cholesterol in vesicle form (9%). The molar ratio of cholesterol/phospholipid in vesicles was reduced after castration (0.93 vs. 0.42) and increased by hormonal supplementation (1.89). In conclusion, when compared to normal male hamsters fed a standard lithogenic diet, castration reduced the cholesterol saturation of bile, lowered the vesicular/micellar ratio in bile, and inhibited cholesterol cholelithiasis. Dietary androgen supplementation increased the lithogenicity of bile, resulting in stone formation in castrated animals.     

Equilibrium of taurodeoxycholate between mixed micellar and aqueous phases: Effect of amphiphile

The equilibration of taurodeoxycholate between mixed micellar and aqueous phases has been studied by equilibrium dialysis. The presence of amphiphiles in the form of lecithin, long chain monoglyceride, and fatty acid in the bile salt solution will greatly decrease the bile salt concentration in the aqueous (intermicellar) phase. At high amphiphile concentration relative to bile salt, the concentration of bile salt in the aqueous phase will be below the critical micellar concentration (CMC) of the pure bile salt solution. Under these conditions, few simple micelles will be present and no binding of bile salts to protein takes place as indicated by experiments with colipase. The lowering of the concentration of bile salt in the aqueous phase by the presence of amphiphile may be a physiological mechanism to regulate bile salt absorption during the digestive phase of fat absorption.     

Effect of biliary obstruction on canine plasma and biliary lipids

The common bile duct was obstructed in 17 dogs. Reciprocal changes were noted for the plasma and biliary lipid concentrations of each after obstruction. As the plasma lecithin and free cholesterol concentrations increased, the biliary lipid concentrations declined. After biliary obstruction the reflux of biliary lecithin into the plasma of these animals was demonstrated with both labeled and unlabeled lecithin. The plasma lipid abnormalities seen after acute biliary obstruction were closely simulated by the reflux of lecithin alone from the biliary tree. The isolated reflux of biliary tract taurocholate produced a distinct lowering of plasma phospholipid and cholesterol concentrations, quite different from the plasma lipid alteration noted with acute biliary obstruction. Similar to observations in human obstruction, some of the plasma lipid was in mesophase form after these animals were obstructed.     

Dietary fat alters biliary lipid secretion in the hamster

Dietary fat has been found to alter the incidence of cholesterol gallstones in hamsters: butterfat intensifies while safflower oil reduces lithiasis. We now report how dietary fat affects bile flow and biliary lipid secretion in this model. Male hamsters were fed one of three experimental diets: a control diet (containing 0.3% cholesterol); control diet +4.0% butterfat; or control diet +4.0% safflower oil. After three weeks, bile samples were collected via an external biliary fistula. The endogenous bile acid pool was depleted for 120 min followed by increasing rates of taurocholate infusion for 160 min. Basal secretion of biliary lipids was measured during the bile acid depletion period. Basal bile flow and bile acid output were not significantly different in the three groups. Dietary butterfat increased basal cholesterol output compared to the control diet (0.037 vs. 0.025 μmol/min·kg, respectively); safflower oil did not change cholesterol output (0.027 μmol/min·kg). Hamsters fed butterfat or safflower oil secreted more phospholipid (0.171 and 0.178 μmol/min·kg, respectively) than controls (0.131 μmol/min·kg). The cholesterol/phospholipid output ratio of the butterfat group was higher than the safflower oil group (0.220 vs. 0.153, respectively). Effects of dietary fat on several relationships between bile flow and biliary lipid secretion were analyzed by linear regression using the data for the entire bile collection period (bile acid depletion and taurocholate infusion). Butterfat and safflower oil did not change either bile acid dependent or bile acid independent bile flow. Hamsters fed butterfat had a higher linkage coefficient (slope) of cholesterol vs. bile acid output than the safflower oil group (0.023 vs. 0.009, respectively). The linkage coefficient of phospholipid vs. bile acid output of the butterfat group was higher than the controls (0.278 vs. 0.185, respectively). In summary, butterfat induced a high cholesterol and phospholipid secretion with a high cholesterol/phospholipid output ratio; safflower oil induced a high phospholipid secretion with a low cholesterol/phospholipid output ratio. Butterfat and safflower oil have different effects on biliary lipid secretion. These differences in biliary lipid secretion may explain, in part, how butterfat and safflower oil differ in affecting gallstone formation in hamsters.     

Oxygenated fatty acid constituents of soybean phosphatidylcholines

Bitter-tasting phosphatidylcholines from hexane-defatted soybean flakes were chromatographically separable from ordinary soy phosphatidylcholines (SPC). The bitter-tasting SPC contain 32% oxygenated fatty acids in addition to palmitic, stearic, oleic, linoleic, and linolenic acids. Identification of these oxygenated acids was based on infrared, ultraviolet, proton nuclear magnetic resonance, and mass spectral characteristics of methyl ester derivatives which were separated and purified by column and thin layer chromatography. The fatty acid methyl esters identified were (a) 15, 16-epoxy-9, 12-octadecadienoate, (b) 12, 13-epoxy-9-octadecenoate, both with double bonds and epoxide groups predominantly ofcis configuration; (c) 13-oxo-9,11-and 9-oxo-10, 12-octadecadienoates; (d) 13-hydroxy-9, 11- and 9-hydroxy-10, 12-octadecadienoates; (e) 9, 10, 13-trihydroxy-11- and 9,12,13-trihydroxy-10-octadecenoates. In addition, trace amounts of (f) 11-hydroxy-9,10-epoxy-12-and 11-hydroxy-12,13-epoxy-9-octadecenoates; (g) 13-oxo-9-hydroxy-10-and 9-oxo-13-hydroxy-11-octadecenoates; (h) 9,10-dihydroxy-12- and 12, 13-dihydroxy-9-octadecenoates; and (i) 9,12,13-dihydroxyethoxy-10- and 9,10,13-dihydroxyethoxy-11-octadecenoates were indicated by mass spectrometry. Dihydroxyethoxy compounds (i) were possibly formed upon extraction of the SPC from flakes by 80% ethanol. Except for the first two epoxy compounds, labelled a and b, the oxygenated fatty acids are similar to the products formed by homolytic decomposition of linoleic acid hydroperoxide. The first two compounds with predominantlycis configuration may occur by action of fatty acid hydroperoxides on an unsaturated fatty acid. Presented in part at the 13th World Congress of the International Society for Fat Research, Marseille, France, August 31–September 4, 1976.     

Hydrolysis of intralipid by pancreatic lipase and phospholipase A2-gel filtration studies

Intralipid was incubated with pancreatic lipase (EC 3.1.1.3) and/or phospholipase A₂ (EC 3.1.1.4) at two bile salts/phosphatidylcholine molar ratios and at two different triglyceride hydrolysis rates using various amounts of lipase. Incubations were studied by gel filtration. Results show: (i) During lipase action, three phases of lipids coexist: an emulsified phase, a micellar phase and an intermediate heavy phase sized between the two others. The equilibrium between each phase is dependent upon the bile salts concentration. (ii) Under these conditions, pancreatic lipase was at 60% bound to the emulsified phase, whereas pancreatic phospholipase A₂ was bound at 94% to the micellar phase.