C. R. Treadwell

Dynamic Aspects of Cholesterol Metabolism in Different Areas of the Aorta and Other Tissues in Man and Their Relationship to Atherosclerosis

Tracer doses of cholesterol-4-C14 were administered to each of 8 patients with limited life expectancies. Chemical determinations of free and esterified cholesterol and determinations of their C14-specific activities were made in serially collected sera, in tissues, and in the different layers of the abdominal aorta, the intimas of which were separated where possible, into normal-appearing intima, thickened intima, and free-lying lipid material. The turnover times of the free and esterified cholesterol fractions of the various tissues and the portions of those fractions derived from the plasma and by synthesis were calculated from these data. The plasma and liver free cholesterol fractions were in equilibrium at the earliest time, 2.5 days. The free cholesterol fractions of other tissues were in equilibrium with the plasma free cholesterol at later dates. The esterified cholesterol fractions of the various tissues, including liver, were in equilibrium with that of the plasma at later dates than were their free fractions. The findings indicate that a large portion of the cholesterol ester of the plasma is not derived from the liver. The greater part of both the free and ester cholesterols of other tissues was derived from the plasma, although a significant portion was derived by synthesis, except for both fractions of skeletal muscle. The adrenal glands were the most active in the synthesis of free cholesterol. The data indicate that a major portion of adrenal cortical hormones is synthesized directly from acetate, without cholesterol as an intermediary product. A major portion of the cholesterol of the intima was derived from plasma through interchange but a considerable amount of cholesterol was synthesized in the intima. The free-lying lipids in the intima appeared to be inert. Data concerning the changes of the lipid contents of the intima with the development of atherosclerosis have been presented. One patient, who died of a second myocardial infarction, differed from the others in having a longer turnover time of the cholesterol of his intima.

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.

Function of specific bile acids in cholesterol esterase activity in vitro

Abstract Substrates for synthetic and hydrolytic pancreatic juice cholesterol esterase (sterol ester hydrolase, EC 3.1.1.13), activities were solubuized in micelles of phosphatidylcholine alone or mixed micelles of phosphatidylcholine and a bile acid. The comparative effectiveness of various bile acids in solubilizing the substrates, cholesterol and oleic acid, or cholesterol oleate, was measured turbidimetrically, and the effects of the same bile acids on enzymatic activity were quantitatively determined. In esterification studies, dihydroxy bile acids solubuized substrates as effectively as trihydroxy bile acids. However, only with the latter group, cholic acid and its conjugates, was there significant cholesterol esterification. Comparable results were obtained in hydrolysis studies; essentially no splitting of cholesterol oleate occurred in the absence of trihydroxycholanic acids. Even when cholesterol oleate was effectively solubuized in mixed micelles of glycodeoxycholate and phospholipid, no enzymatic hydrolysis of the substrate occurred until taurocholate was added. Thus, cholic acid and its conjugates appear to be cofactors for pancreatic juice cholesterol esterase. Data obtained by microtitration of fatty acids and thin-layer silicic acid chromatography of the enzyme digests indicated that the bile acid did not complex with either the fatty acid or cholesterol substrates. The presence of taurocholate effectively prevented tryptic (trypsin, EC 3.4.4.4) and chymotryptic (chymotrypsin, EC 3.4.4.5) inactivation of cholesterol esterase, even though general proteolysis of pancreatic proteins was not effected. In the absence of taurocholate, trypsin addition resulted in complete loss of cholesterol esterase activity within 10 min. It appears, therefore, that the specific requirement of trihydroxy bile acids for cholesterol esterase activity, and the protective effect of taurocholate against proteolytic inactivation of this enzyme, are due to the formation of a specific bile acid-enzyme complex.

Portal absorption of fatty acids in lymph- and portal vein-cannulated rats

Abstract The route and the rate of the intestinal absorption of [1- 14 C]oleic, [1- 14 C]-caprylic, and 2-[ 14 C]ethyl- n -caproic acids have been studied using lymph- and portal vein-cannulated rats. It was found that 85% of absorbed oleic acid was transported via lymphatic system. However, as much as 15% of absorbed oleic acid was transported directly via the portal system. With short-chain fatty acids, between 94–98% of the absorbed acids were transported via the portal system. Studies on the rate of the intestinal absorption of these acids indicated that 2-ethyl- n -caproic acid was absorbed less completely and subsequently metabolized less effectively than the corresponding straight-chain fatty acid, caprylic acid. Studies on the distribution of radioactivity in lymph lipids showed that most of the radioactivity (85%) was present as triglycerides when [ 14 C]oleic acid was administered to lymph and portal vein fistula rats. However, when the 14 C-labeled short-chain fatty acids were given, 96–102% of the radioactivity was present as free fatty acids. Studies on the distribution of radioactivity in lipid fractions of portal vein blood showed that 50 and 98–100% of the radioactivity present were in the form of free fatty acids when [ 14 C]oleic acid and 14 C-labeled short-chain fatty acids, respectively, were administered to lymph and portal vein fistula rats.

Micellar-solubilized substrates and cholesterol esterase activity in vitro☆

Abstract Substrates for synthetic and hydrolytic pancreatic juice cholesterol esterase activity have been solubilized in phospholipid-bile salt micelles and prepared as albumin-stabilized emulsions. For esterification studies, the micellar preparation did not provide an adequate substrate system since it appeared that the sterol and fatty acid were not equally available to the enzyme. Disrupting the micellar medium by addition of Mg ++ caused increased esterification of cholesterol. For hydrolysis studies, the micelle was an excellent vehicle for the sterol ester and gave hydrolytic rates higher than previously noted. By using various esters in micellar media, it was found that all esters tested, from cholesterol propionate to cholesterol linoleate, are hydrolyzed equally effectively. Thus, it is shown that there is no fatty acid specificity for hydrolytic cholesterol esterase. Enzymic hydrolysis or esterification was not evident in the absence of the bile salt.

Pancreatic juice cholesterol esterase: Studies on molecular weight and bile salt-induced polymerization☆

Abstract The molecular weight of pancreatic juice cholesterol esterase (sterol ester hydrolase, EC 3.1.1.13) has been determined to be 65–69,000 by Sephadex G-200 gelfiltration chromatography and sodium dodecylsulfate-polyacrylamide disc-gel electrophoresis. The amino acid composition resembles that of classical pancreatic lipase (EC 3.1.1.3) but is unique in the virtual absence of tyrosine in the molecule. Evidence for a direct molecular interaction between specific bile salts, i.e., cholic acid or its conjugates, and the pancreatic juice enzyme was obtained by Sephadex G-50 filtration studies. This interaction results in a polymerization of the enzyme protein to give an apparent molecular weight of approximately 400,000 as determined by standardized gel-filtration chromatography. Studies with phenylmethanesulfonylfluoride, a serine hydroxyl group reagent, indicate that the polymerized protein is the active enzyme involved in the reversible synthesis and hydrolysis of sterol esters.

Correlation of arachidonic acid of serum cholesterol esters in different species with susceptibility to atherosclerosis.

Summary Serum CEFA composition of 8 different species has been determined by gas-liquid chromatography. Different CEFA spectrums were found for each species. The outstanding variation was the proportions of arachidonic acid in CEFA of the serums. That of the rat had 50% of arachidonic acid and that of the dog had 17%. All species also had a large proportion of linoleic acid in the CEFA of their serums. The rat and the dog, with high arachidonic acid levels in CEFA of their serums, are known to be highly resistant to development of atherosclerosis. Those species having small proportions of arachidonic acid in the CEFA of their serums are susceptible to atherosclerosis and develop the disease spontaneously. This study provides evidence, by association, that level of arachidonic acid in the CEFA may be related to susceptibility to atherosclerosis. It remains open to investigation whether the proportion of arachidonic acid in the CEFA is a characteristic of the species or whether it, and the susceptibility to atherosclerosis, can be altered by dietary measures.

A rapid, quantitative determination of total and free cholesterol with anthrone reagent.

Abstract A simple, rapid microcolorimetric determination for total and free 3β-hydroxy sterols in serum and tissues has been described. Following extraction of total sterols and saponification of the esterified fraction by established procedures, the sterol is precipitated in 15 min at 45°C as the digitonide using 10% aluminum chloride. The isolated sterol digitonide is purified by twice recrystallizing from methanol-10% aluminum chloride (1 : 1), each step requiring 20 min at 45°C. The precipitate is washed with acetone and suspended in glacial acetic acid, and the digitonin moiety is determined with a stable anthrone reagent. Multiple analyses of sterol concentrations as low as 15–20 μg are routinely determined with an accuracy comparable to the method of Sperry and Webb, and with a greater sensitivity and precision than obtained with the latter method.

Effect of hypocholesterolemic agents on intestinal cholesterol absorption.

Summary 1. The direct effect of several blood cholesterol-lowering agents on intestinal absorption of cholesterol-4-C14 was studied in lymph-fistula rats. 2. MK-135 (cholestyramine) and pectin caused significant reductions in lymph total cholesterol and absorption of cholesterol-4-C14. 3. With MK-135, the larger of 2 doses studied also reduced absorption of endogenous cholesterol from the intestine. 4. Cholesterol trimethyl-acetate, nicotinuric acid and pyridine-3-acetic acid had no effect on cholesterol absorption or lymph cholesterol levels. 5. In all groups where significant cholesterol absorption occurred, the percentage esterification of absorbed cholesterol was constant (88–93%), irrespective of the extent of absorption.

Sterol specificity of pancreatic cholesterol esterase.

Summary Pancreatic cholesterol esterase catalyzes the esterincation and hydrolysis of a number of different sterols and sterol esters. The order of activity observed in the esterifying system with oleic acid was as follows: dihydrocholesterol † cholesterol † β-sitosterol † sitosterol † stigmasterol † ergosterol. Of these sterols, only dihydrocholesterol and cholesterol were esterified with butyric acid. In the hydrolytic system all of the sterol butyrates tested were split very rapidly, with the exception of ergosterol butyrate. the order of splitting of the oleates in the first 4 hours was: cholesterol oleate † sitosterol oleate † stigmasterol oleate † ergosterol oleate. The relation of enzyme activity to sterol structure is discussed.