Intestinal cholesterol uptake: Comparison between mixed micelles containing lecithin or lysolecithin

The aim of our study was to define the mechanism by which cholesterol uptake is inhibited by lecithin but not by lysolecithin. The work compared the cholesterol uptake by everted rat jejunal sacs from bile salt-lecithin-cholesterol or bile salt-lysolecithin-cholesterol micelles. The micellar size and the cholesterol saturation were measured. The size or molecular weight increases when the lecithin concentration rises, and the cholesterol uptake decreases and leads to zero when the micelles contain more than 30% lecithin. The size of bile salt-lysolecithin-cholesterol micelles is smaller than that of lecithin micelles in comparable molar ratios. Consistent with this result is the fact that, for a given phospholipid concentration, cholesterol uptake is greater in the presence of lysolecithin than in the presence of lecithin. The diffusion rate of the micelles through the unstirred water, layer decreases when micellar size increases. However, the comparison of uptakes from lecithin or lysolecithin micelles similar in size and in cholesterol saturation showed that the cholesterol uptake is still lower for lecithin micelles. This shows that with larger micelles some factor other than micellar size and cholesterol content of the micelles is important. We observe that lysolecithin absorption is 15-fold greater, than lecithin absorption. We suggest that lysolecithin absorption results in a rapid supersaturation with cholesterol leading to cholesterol absorption.     

Dietary fibers: V. Binding of bile salts,phospholipids and cholesterol from mixed micelles by bile acid sequestrants and dietary fibers

Mixed micelles were prepared containing combinations of either taurocholate or taurochenodeoxycholate, monoolein, oleic acid, dioleylphosphatidylcholine (lecithin) and cholesterol. These were incubated with commercial bile-acid-sequestering resins, cholestyramine and DEAE-Sephadex, or various dietary fibers and fiber components including wheat bran, cellulose, alfalfa, lignin and 2 viscosity grades of guar gum. Binding was determined as the difference between the radioactivity of each micellar component added and that recovered in the centrifugal supernatant after incubation. In general, the extent of bile salt sequestration was characteristic and reproducible for each bile salt, and was largely unaffected by the presence of one or more additional components of the micellar mixture, including the other bile salt. Cholestyramine bound 81–92% of the bile salts and 86–99% of the phospholipid and cholesterol present in micelles. DEAE-Sephadex sequestered only 49% of the taurocholate and 84% of the taurochenodeoxycholate, but completely removed all of the phospholipid and cholesterol from micelles containing either bile salt. Among the dietary fibers, guar gum of either viscosity bound between 20–38% of each micellar component, whereas lignin, alfalfa, wheat bran and cellulose were progressively less effective in sequestratin of individual components of mixed micelles. The extent of sequestration of micellar components by these resins and fibers is reasonably correlated with the effects of these same materials on lymphatic absorption of lipids and to their suggested hypocholesteremic properties.     

The effect of lipids on taurocholate absorption from intestinal loops in the rat

The rates of uptake and serosal trans-fer of [¹⁴C]-labelled taurocholate (7.77 mM in bicarbonate buffer, pH 6.5) were determined in situ in ligated segments of rat intestine in the presence of lipids. Oleic acid, monoolein, lecithin, and lysolecithin enhanced taurocholate uptake and transfer in the jejunum, each lipid exhibiting and optimal concentration at which the bile acid fluxes were maximal. The maximal rates of bile acid uptake observed with the various lipids were close to four times the uptake rates found with the lipid-free taurocholate medium, whereas serosal transfer rates under optimal conditions were enhanced about six-fold. The optimal concentrations differed widely among the various lipids, being inversely related to the lipids’ polarity. Simultaneous measurement of taurocholate and [³H]-labelled oleic acid showed that under optimal conditions, when the molar concentration of oleic acid was about equal to that of the bile acid, the fatty aicd and bile acid also exhibited closely similar rates of absorption. At other fatty acid concentrations, the fractional rate of absorption of the bile acid was much lower than that of the fatty acid. The rates of uptake and serosal transfer of pure taurocholate by the ileum exceeded those of the jejunum by factors of about 7 and 15, respectively, but in the presence of lipids this difference in absorptive capacity for bile acid between the distal and proximal segment largely disappeared.     

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.     

Lecithins and lysolecithins of wheat flour

Lecithin and lysolecithin from the bound lipid of Thatcher wheat endosperm were separated and purified by column and thin-layer silicic acid chromatography. Lecithin was hydrolyzed with phospholipaseA (Crotalus adamanteus) and the products isolated and purified by silicic acid chromatography. The fatty acid composition of the original lecithin and lysolecithin and of the hydrolysis products was determined by gas-liquid chromatography. The fatty acids in the beta position of the lecithin were found to be almost entirely unsaturated, whereas those in the alpha position were saturated and unsaturated in nearly equal amounts. The differences between the fatty acid composition of the original lysolecithin and that of the lysolecithin obtained by hydrolysis of lecithin with phospholipaseA suggested the presence of both alpha and beta acyl species in the naturally occurring lysolecithin. Contribution No. 48, Food Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, Canada.     

The formation of phosphatidylinositol by acylation of 2-acyl-sn-glycero-3-phosphorylinositol in rat liver microsomes

The conversion of 2-acyl-sn-glycero-3-phosphorylinositol into phosphatidylinositol via acyl-CoA: 2-acyl-sn-glycero-3-phosphorylinositol acyltransferase activity was found to occur in rat liver microsomes. Over a wide range of conditions, stearic acid was preferred over palmitate by the acyltransferase when these acids were presented in mixtures as acyl-CoA derivatives. The potential importance of this enzyme activity for the entry of stearic acid into the 1-position of hepatic phosphatidylinositol is further supported by its greater preference for stearate relative to the acyl-CoA: 2-acyl-sn-glycero-3-phosphorylcholine acyltransferase under certain assay conditions.     

The importance of the steric configuration of lysophosphatidylcholine in the lymphatic transport of fat

The importance of the steric configuration of lysophosphatidylcholine in the lymphatic transport of fat was investigated in bile fistula rats. It was found that the feeding of 1-palmitoyl-sn-glycero-3-phosphocholine increased the lymphatic output of phosphatidyl choline and triacylglycerol, while the feeding of 3-palmitoyl-sn-glycero-1-phosphocholine had no effect. In intestinal microsomes of the bile fistula rats, it was found that the lysophosphatidylcholine acyltransferase was stereospecific in acylating the 1-acyl-sn-glycero-3-phosphocholine enantiomer. The significance of these findings is briefly discussed.     

Solubilization of carotenoids from carrot juice and spinach in lipid phases: II. Modeling the duodenal environment

We have been investigating the factors determining the bioavailability of carotenoids from vegetables. The previous paper [Rich, G.T., Bailey, A.L., Faulks, R.M., Parker, M.L., Wickham, M.S.J., and Fillery-Travis, A. (2003) Solubilization of Carotenoids from Carrot Juice and Spinach in Lipid Phases: I. Modeling the Gastric Lumen, Lipids 38, 933–945] modeled the gastric lumen and studied the solubilization pathway of carotenes and lutein from carrot juice and homogenized spinach to oil. Using the same vegetable preparations, we have extended our investigations to solubilization pathways potentially available in the duodenum and looked at the ease of solubilization of carotenes and lutein within simplified lipid micellar and oil phases present within the duodenum during digestion. Micellar solubility of raw spinach carotenoids was low and was enhanced by freezing, which involved a blanching step. The efficiency of solubilization of carotenoids in glycodeoxycholate micelles decreased in the order lutein_carrot>lutein_{blanched-frozen spinach}>carotene_{blanched-frozen spinach}>carotene_carrot. Frozen spinach carotenoids were less soluble in simple micelles of taurocholate than of glycodeoxycholate. The results comparing the solubility of the carotenoids in mixed micelles (bile salt with lecithin) with simple bile salt micelles are explained by the relative stability of the carotenoid in the organelle compared to that in the micelle. The latter is largely determined by the polarity of the micelle. Below their critical micelle concentration (CMC), bile salts inhibit transfer of carotenoids from tissue to a lipid oil phase. Above their CMC, the bile salts that solubilize a carotenoid can provide an additional route to the oil from the tissue for that carotenoid by virtue of the equilibrium between micellar phases and the interfacial pathway. Mixed micellar phases inhibit transfer of both carotenoids from the tissue to the oil phase, thereby minimizing this futile pathway.     

Two synthetic phosphonate analogs of lecithin

The diether-phosphonate lecithin analog DL-2-hexadecoxy-3-octadecoxypropyl-phosphonylcholine was prepared in 55% overall yield by reaction of 2-hexadecoxy-3-octadecoxypropylphosphonic acid with choline iodide andp-toluenesulfonyl chloride, and purified chromatographically. The diester-phosphonate lecithin analog 1,2-dipalmitoyl-sn-glycero-3-[2′-trimethyl-ammonium) ethylphosphonate) was prepared fromsn-glycerol-1,2-dipalmitate and 2-(trimethylammonium) ethylphosphonyl dichloride, and the reaction mixture purified chromatographically. The lecithin analog was obtained in an anhydrous form.     

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver

The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.     

Lysophosphatidylcholine acyltransferases activity during experimental cholelithiasis

The accumulation of (1-palmitoyl)lysophosphatidylcholine, lysolecithin, in gallbladder bile was observed during the first week of cholesterol-induced experimentals cholelithiasis using the prairie dog model for cholesterol gallstone formation. Gallbladder fluid transport function decreased as bile lysolecithin concentration increased. These observations suggest that lysolecithin play an important, early role in the etiology of gallstone disease. Furthermore, the relative activities of hepatic and gallbladder mucosa lysophosphatidylcholine acyltransferase and acylcoenzyme A hydrolases may be responsible for the turnover of gallbladder bile lysolecithin.     

Relevance of interactions between sphingomyelin and cholesterol in biliary and intestinal tract

Phosphatidylcholine and sphingomyelin are the major phospholipids of the hepatocytic canalicular membrane outer leaflet. Sphingomyelin may preferentially reside with cholesterol in liquid‐ordered domains. In contrast, phosphatidylcholine is the exclusive phospholipid secreted in rat bile (enriched in hydrophilic species compared to the canalicular membrane), subsequently incorporated into bile salt‐cholesterol micelles. We determined the bile lipid composition in 95 vertebrate species (Moschetta et al., J Lipid Res. 2005, 46 , 2221–2232). Phospholipid was often virtually absent in bile of cartilaginous fish and reptiles, occurred in low relative amounts (compared to bile salts) in bony fish or birds and in high relative amounts in most mammals. Biles with low relative amounts of phospholipid often contained high proportions of sphingomyelin. Phosphatidylcholine was the predominant phospholipid in biles with high phospholipid contents. We then compared, in CaCo2 cells (without appreciable phospholipase A₂ activity), the effects of incorporating sphingomyelin, egg yolk phosphatidylcholine or lyso‐phosphatidylcholine in apical bile salt‐cholesterol micelles. Egg yolk phosphatidylcholine and (more pronounced) sphingomyelin inhibited cholesterol absorption with decreased ABC‐A1 and ‐G1 expression. Lyso‐phosphatidylcholine enhanced cholesterol absorption with increased basolateral HDL‐dependent cholesterol efflux and high expression of ABC‐A1 and ‐G1. In conclusion, sphingomyelin plays a pivotal role in protecting hepatocytes against detergent bile salts. Dietary sphingomyelin may inhibit intestinal cholesterol absorption.     

Activity of phospholipid-synthesizing enzymes in rat liver plasma membranes and the source of biliary lecithin

The potential for the synthesis of phosphatidylcholine by the bile canalicular membrane of the liver cell was assessed by measuring the activity of a number of phospholipid synthesizing enzymes in isolated bile canalicular membrane fractions from rat liver. The activity of these various enzymes was compared to that present in noncanalicular liver cell plasma membranes and in microsomes. The CDP-choline: 1,2-diacyl-sn-glycerol-cholinephosphotransferase was virtually absent from the bile canalicular membranes but the specific activities of S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase and acyl-CoA:1-acyl-sn-glycero-3-phosphoryl-choline acyltransferase were 11–15% of those found in the microsomes. The bile canalicular membranes also contained detectable acyl-CoA:sn-glycero-3-phosphate acyltransferase activity and the ability to potentiate the Ca⁺⁺-stimulated exchange of bases between different phospholipids. These findings indicate that the bile canalicular membranes have a very limited capacity for the formation of phosphatidylcholine under the assay conditions employed. A preliminary report of this paper was given at the AOCS Spring Meeting, Dallas, April 1975.     

Interaction of bile salts with gastrointestinal mucins

The properties of three mucins were examined to identify the structural features responsible for their functional differences. Bovine submaxillary mucin (BSM), porcine gastric mucin (PGM), and rat intestinal mucin (RIM) were each characterized, and high carbohydrate contents were found for RIM and PGM. The amino acid compositions were typical of mucin glycoproteins, with over half comprising small, neutral amino acids. Thereafter, each mucin was equilibrated with three different series of concentrations of the bile salts sodium taurocholate, sodium taurodeoxycholate, and sodium taurochenodeoxycholate. Following multiple centrifugations, the supernatant and mucin pellet concentrations of the bile salts were measured. The bile salt pellet concentration was plotted as a function of supernatant concentration, and from the slopes, the excluded volumes were calculated as 25, 29–44, and 28–55 mL/g for BSM, RIM, and PGM, respectively. The intercepts were 8–10, 2–3, and 1–3 mM for BSM, RIM, and PGM, respectively, which represents an estimate of the bound concentration of bile salt. Differences among the bile salts were observed in the excluded volume and amount bound, but no trends were evident. The bile salts may interact as aggregates with the hydrophobic areas and carbohydrate side chains of the mucins, providing favorable sites for association. The binding at low concentrations with exclusion at high concentrations is significant for modulating the absorption of lipid aggregates from the intestine. Finally, the differences among the mucins reflect the unique structure-function relationship of these gastrointestinal mucins.     

Synthesis and polymerization of novel methacrylate having glycerophosphorylcholine

A novel vinyl monomer, 1-(2-methacryloyloxyethyl) carbamoyl-sn-glycero-3-phosphorylcholine (MCGPC), was synthesized from sn-3-glycero-phosphorylcholine (GPC) and 2-methacryloyloxyethyl isocyanate (MOI) in the presence of di-n-butyltin dilaurate (BTL) as a catalyst, and its critical micelle concentration (CMC) was determined to be 2.7 × 10⁻³ mol/l. MCGPC was copolymerized with n-butyl methacrylate (BMA) in the presence of a radical initiator in ethanol for 24 hr and 48 hr at 60 C. Bovine serum albumin (BSA) was adsorbed onto membranes prepared from copolymers of MCGPC with BMA to estimate the blood compatibility of those copolymers. The BSA adsorption was reduced with an increase of the composition of MCGPC in copolymers.     

Acylation of lyso platelet-activating factor by splenocytes of the rainbow trout,Oncorhyncus mykiss

In mammalian systems, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) is rapidly inactivated by a deacetylation/reacylation system that produces 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine which is highly enriched in arachidonic acid. There is some evidence that n−3 fatty acids may have an impact on this system in humans but the nature of this impact is unclear. In rainbow trout, n−3 fatty acids are known to be essential dietary components which are derived through the food chain. Substantial quantities of n−3 fatty acids are found in trout membrane phospholipids. We show here that in sharp contrast to mammalian cells, trout cells acylate lyso platelet-activating factor, alkyl-GPC, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, (lyso-PAF) with a high degree of specificity for n−3 fatty acids. When [³H]lysoPAF was incubated with these cells, only three molecular species of alkylacylglycerophosphocholine were produced, and 92% contained n−3 fatty acids. Since isolated membranes yielded similar results, it appears that the acylation proceedsvia a coenzyme A-independent transacylase as found in mammalian systems.     

Synthesis of a monobrominated analog of dipalmitoyl lecithin

The synthesis of 1-palmitoyl-2-monobromopalmitoyl lecithin, where the β-chain is brominated in either the 9- or 10-position, is reported, Monobromopalmitic acid was prepared by addition of HBr to palmitoleic acid, and the anhydride of the fatty acid conjugated with 1-palmitoyl lysolecithin. The resulting lecithin was isolated by preparative thin layer chromatography and its structure confirmed by chemical and enzymatic analysis.     

Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [³H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [³H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 2-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[³H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[³H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1′-enyl-2-[³H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.     

Mechanism of formation of chloropropanols present in protein hydrolysates

Chloropropanols are formed in protein hydrolysates by the reaction of hydrochloric acid with residual lipids associated with the proteinaceous materials used in their production. The products formed from glycerol, triolein, 1,2-diacyl-sn-glycero-3-phosphorylcholine and soya meal have been analyzed by thin-layer and gas chromatography. The yields and isomer ratios of the chloropropandiols and dichloropropanols formed are interpreted in terms of reaction mechanisms for their formation, which involve preferential nucleophilic substitution by the chloride anion at positions activated by neighboring ester groups. These provide anchimeric assistance and govern regioselectivity through steric and electronic effects.     

The incorporation of phosphorylethanolamine into the phospholipids of brain microsomes in vitro

Synthesis of ethanolamine phosphoglycerides (EPG) from labeled¹⁴C-phosphorylethanolamine (PE) and cytidine triphosphate (CTP) has been studied in vitro in particles and in soluble fractions of chicken brain. The microsomal membranes can carry out this conversion, but supplementing the microsomes with an enzymic fraction derived from the particle-free supernatant results in a noticeable increase of the rate of EPG synthesis. Mitochondria are almost inactive, in this connection. The conversion of PE to lipid is very low, in no case exceeding 0.5–0.6%, even in the presence of diacyl glycerols or 1-alkenyl 2-acyl glycerol. No stimulation occurs by supplementing the incubation system with natural lipid acceptors, intermediates of lipid synthesis, energy-producing cofactors or monoacylsn-glycero-3-phosphorylethanolamine (GPE), monoacylsn-glycero-3-phosphorylcholine (GPC) and other lipid material. From these and other results, the conclusion is made that the PE:CTP cytidylyltransferase (E.C. 2.7.7.14) must display very low activity in vitro, thus limiting the overall rate of synthesis from PE. Diacyl GPE is the only lipid which has been found labeled after incubation with PE of the brain preparations. A small synthesis of alkenyl acyl GPE takes place, only when PE is incubated with suitable concentrations of a plasmalogenic diglyceride.