Linda L. Gallo

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.

Role of Pancreatic Cholesterol Esterase in the Uptake and Esterification of Cholesterol by Isolated Intestinal Cells

Summary Isolated intestinal mucosal cells from normal and common duct-cannu-lated rats have been prepared by collagen-ase dissociation. These cells displayed ultra-structural integrity and retained several metabolic characteristics typical of intact intestinal mucosa. These included galactose accumulation and its inhibition by dinitro-phenol, glucose oxidation, incorporation of labeled leucine into cellular protein, and the uptake and esterification of fatty acid. The uptake of cholesterol by cells from common duct-cannulated rats was comparable to that in cells from control animals; however, esterification was only about 25% of that in control cells. Preincubation of the “defective” cells for 30 min with the purified subunit or active form of pancreatic sterol ester hydrolase had no effect on cholesterol uptake but resulted in a fourfold to sixfold increase in the ability of the cells to esterify cholesterol. These studies provide additional evidence for the essential role of pancreatic sterol ester hydrolase in the mucosal esterification of absorbed cholesterol prior to lymphatic transport.

Dietary Fibers VI: Binding of Fatty Acids and Monolein from Mixed Micelles Containing Bile Salts and Lecithin

Abstract Mixed micelles were prepared containing sodium taurocholate, monolein dioleyl lecithin, cholesterol, and an equimolar mixture of palmitic, oleic, and linoleic acids. 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 two viscosity grades of guar gum. Binding of monolein and fatty acids was determined as the difference between the radioactivity of the added micellar component, and that recovered in the centrifugal supernatant after incubation. In general, the extent of monoglyceride or fatty acid sequestration was characteristic and reproducible for each binding agent. Cholestyramine and DEAE-Sephadex essentially quantitatively bound monoglycerides and all three fatty acids from micellar medium. Low- and high-viscosity grades of guar gum sequestered 15-23% of the monolein and 32-33% of the fatty acids, showing a significant preference for linoleic acid in each case. Alfalfa fiber removed about 18% of the micellar monoglyceride and mixed fatty acids, again showing some preference for the polyunsaturated acid. Lignin, the hydrophobic component of dietary fibers, sequestered about 13% of the available lipids and displayed an apparent preference for oleic acid. Wheat bran and cellulose showed little affinity for micellar lipids binding about 11 and 4.7%, respectively. These data on resin and fiber sequestration of micellar fatty acids and monoglycerides compare favorably with the binding of other micellar components including phospholipid, bile salt, and cholesterol.

Dietary fiber and lymphatic absorption of cholesterol in the rat.

The indirect effects of short-term (3-day) feeding of several types of dietary fiber and nonnutritive materials on the subsequent absorption of cholesterol has been investigated in thoracic duct cannulated rats. Absorption was studied at timed intervals over 24 hr after duodenal introduction of a tracer dose of cholesterol at least 20 hr after the last feeding. In animals fed for 3 days with diets containing cholestryamine, bran, or cellulose, cholesterol absorption was significantly less than in control animals maintained on rat chow. Rats fed for 3 days with an alfalfa-containing diet showed large variations in cholesterol absorption that were not significantly different from controls. However, after 5 weeks, rats on the alfalfa diet showed a marked reduction in lymphatic absorption of the tracer sterol. These indirect effects of cholestryamine and fibers on cholesterol absorption were not attributable to a common mechanism; i.e., differences in transit times that were not significant, or dirrect binding of bile acids and cholesterol by the test materials.

Immunological comparison of cholesterol esterases

Abstract Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.

LOCALIZATION OF CHOLESTEROL ESTERASE AND CHOLESTEROL IN MUCOSAL FRACTIONS OF RAT SMALL INTESTINE.

Summary A modified procedure for brush border preparation has been developed which gives 90–100% yields of invertase activity in the brush border. It was determined that the esterifying and hydrolytic activities of cholesterol esterase are concentrated in the supernatant fraction of the mucosa as opposed to the brush border. Further fractionation of the mucosal cells into nuclei and cell debris, mitochondria, microsomes, and soluble fraction, and assay of these fractions for esterase activity demonstrated that 60–66% of the esterase activity of the whole homogenate was in the soluble fraction. The remaining activity was distributed among the other fractions. The data suggest that esterification of cholesterol occurs in the mucosal cell rather than in the brush border. Cholesterol levels in rat intestinal wall, intestinal mucosa, and mucosal subcellular fractions are also reported.

Rat Intestinal Acyl Coenzyme A: Cholesterol Acyl Transferase Properties and Localization

Abstract Microsomal acyl coenzyme A:cholesterol acyltransferase activity of rat intestinal mucosa was measured as the incorporation of [1-14C]oleic acid into cholesterol esters. Exogenous cholesterol and sterol carrier protein singly and together extended the time over which the reaction rate was linear. Cholesterol esterification was suppressed by 2-monooleoyl glyceryl ether a 2-monoglyceride analog and potential substrate, but was unaltered by α-glycerophosphate or lysolecithin. Esterifying activity was lowest in microsomes from the intestinal segment 0–15 cm distal to the pylorus and highest in microsomes from the 30- to 45-cm segment. The total and specific activities of acyl coenzyme A:cholesterol acyltransferase were highest in cells isolated from the crypt zones. The location and level of activity were unaffected by diversion of either pancreatic juice or pancreatic juice and bile from the intestinal lumen. These findings question the physiological significance of acyl coenzyme A:cholesterol acyl transferase in the regulation of the absorption of exogenous cholesterol.

Intestinal mucins and cholesterol uptake in vitro

A mucus material, secreted by intestinal segments, with a high affinity for cholesterol, has been isolated and chemically characterized. The mucin contained 11% carbohydrate, largely as glucosamine, galactose and N-acetylneuraminic acid, and 19% lipid, of which 86% was unesterified fatty acid. The isolated material readily bound cholesterol in a stoichiometric manner. Conditions known to enhance cholesterol absorption in vivo also decreased mucin complexing to cholesterol in vitro. This association of cholesterol and intestinal surface mucin also occurred during incubations of intestinal segments with dispersed cholesterol, resulting in a high level of intestinal adsorption, with little or no cellular absorption of the sterol. However, when cholesterol was solubilized in simple or complex micelles containing bile salts, surface adsorption of cholesterol was reduced and net absorption was increased. The results suggest that surface mucin binding of cholesterol may represent at least one major diffusion limitation to cholesterol absorption in the intestine.

The 1- and 2-Octadecyl Glyceryl Ethers as Model Compounds for Study of Triglyceride Resynthesis in Cell Fractions of Intestinal Mucosa

Summary The 1- and 2-octadecyl-(1-14C) glyceryl ethers have been used as model compounds to study triglyceride synthesis. The data suggest that: (a) The rat mucosa utilizes 2-monoglyceride for the formation of triglyceride; (b) the 1,2-diglyceride is the intermediate which is acylated to form triglyceride in the monoglyceride pathway; (c) and that utilization of the 1,3-diglyceride in triglyceride synthesis probably involves preliminary isomerization to the 1,2-diglyceride.

DIRECT EVIDENCE FOR STEROL CARRIER PROTEIN-2 (SCP-2) PARTICIPATION IN ACTH STIMULATED STEROIDOGENESIS IN ISOLATED ADRENAL CELLS

Intact, dispersed adrenal fasiculata cells were fused with liposomal entrapped anti-sterol carrier protein-2 IgG, washed and subsequently exposed to adrenocorticotropic hormone (ACTH). The steroidogenic response (measured as corticosterone production) of these cells was inhibited by 45–60%, compared to cells fused with liposomally entrapped non-immune IgG or buffer. Furthermore, the degree of inhibition was shown to be dependent on the amount of antibody utilized. Fusion of cells with liposomally entrapped antibody to fatty acid binding protein (FABP) had no effect on ACTH-induced steroidogenesis. The incorporation of liposomal SCP-2 into adrenal fasiculata cells pre-treated with affinity purified anti-SCP-2 IgG resulted in a concentration dependent release of the inhibition of ACTH-induced steroidogenesis caused by the antibody. It was also demonstrated indirectly that the fusion of liposomal anti-SCP-2 IgG had no effect on ACTH binding to adrenal cells. Finally, cells treated with liposomal anti-SCP-2 IgG and subsequently exposed to ACTH in the presence of aminoglutethimide, accumulated unesterified cholesterol in their cytoplasmic lipid inclusion droplets. These results, taken together, establish that an important physiological function for SCP-2 in adrenal cells is the transfer of unesterified cholesterol from the cytoplasmic lipid inclusion droplets to mitochondria. This translocation is generally considered to be the rate-limiting step in steroid hormone biosynthesis.