Studies on the Influence Ofdietary Cholesterolon Cholesterol
Total Page：16
File Type：pdf, Size：1020Kb
Journal of Clinical Investigation Vol. 44, No. 11, 1965 Studies on the Influence of Dietary Cholesterol on Cholesterol Metabolism in the Isotopic Steady State in Man * JEAN D. WILSON t AND CHARLES A. LINDSEY, JR. (From the Department of Internal Medicine, The University of Texas Southwestern Medical School, Dallas, Texas) The relation between dietary cholesterol and it can quantitatively account only for a degree of the concentration of cholesterol in serum has been protection equal to that amount ordinarily syn- the subject of intense interest since the original thesized in the liver, whereas the serum choles- demonstration that both hypercholesterolemia and terol remains virtually stable despite the in- atherosclerosis could be induced in the rabbit by gestion of quantities of cholesterol many times the feeding of diets rich in cholesterol (2). On this value. Second, the ingestion of cholesterol the other hand, in man virtually all studies have could cause an increase in the rate of degradation demonstrated that rather wide variations in the of cholesterol to bile acids. This phenomenon cholesterol intake result in less marked effects has been clearly documented in the rat (12, 13); on the serum cholesterol (3-8). Even when the in this species increased bile acid production oc- intake of dietary cholesterol greatly exceeds the curring with very effective cholesterol absorption normal turnover rate, the concentration of choles- is the major means by which serum cholesterol terol in the serum almost never rises more than is stabilized. Finally, it is obvious that any effect 100 mg per 100 ml (8), and indeed, in careful of exogenous cholesterol on cholesterol metabo- balance studies it is necessary to compare cho- lism is dependent on its rate of absorption. Be- lesterol diets with diets totally devoid of choles- cause cholesterol is both absorbed and secreted terol before any significant changes can be across the wall of the intestine, the best means detected (4-7). This ability of the serum choles- of quantifying cholesterol absorption in the intact terol of man to stabilize at only moderately animal involves the use of a complicated double elevated levels despite enormous intake resembles isotopic steady state technique (13), and, conse- the situation in such animals as the rat (9) and quently, very little is known about net cholesterol is in marked contrast to the profound degrees of absorption in various species. hypercholesterolemia produced in the rabbit by To evaluate in the human the possible roles of cholesterol feeding (2). these three factors-the inhibition of cholesterol At least three compensatory mechanisms could synthesis, increased bile acid production, and a account for this phenomenon. First, dietary cho- limited ability to absorb dietary cholesterol-five lesterol suppresses the hepatic synthesis of cho- balance studies have been performed in two nor- lesterol in the human (10) as well as in other mal men who were fed varying quantities and species (11) via a negative feedback system. types of cholesterol-4-14C until an isotopic steady However, the suppression of hepatic synthesis state was attained. The results clearly demon- the compensatory mechanism since cannot be only strate that dietary cholesterol, whether fed in the * Submitted for publication April 29, 1965; accepted form of eggs or in a purified state, has little July 15, 1965. effect on the total rate of endogenous cholesterol Supported by grants from the U. S. Public Health to Service and the American Heart Association. synthesis or of cholesterol degradation bile The results have been published in abstract form (1). acids. We have concluded, therefore, that in tWork performed during tenure as an Established In- man the major mechanism that stabilizes the vestigator of the American Heart Association. serum cholesterol during large variations in the Address requests for reprints to Dr. Jean D. Wilson, is a limited ca- Dept. of Internal Medicine, The University of Texas intake of exogenous cholesterol 'Southwestern Medical School, Dallas, Texas 75235. pacity for cholesterol absorption. 1805 1806 JEAN D. WILSON AND CHARLES A. LINDSEY, JR. Methods position of the tip of the tube as evidenced by a mercury marker was checked by fluoroscopy just before the col- Two normal men, ages 31 and 23, were studied during lection of the samples, and consequently, although tele- five different feeding periods varying from 21 to 40 days scoping of bowel around the tubes can occur, the position- over an 8-month interval. During the periods of study ing of the tubes was thought to be reliable. the subjects were ambulatory outpatients who ate only a Serum cholesterol analysis. The serum cholesterol constant weighed quantity of formula diet, vitamins, specific activity and content were measured as follows: Hawk-Oser salt mix, and clear liquids. After a steady 2 ml of serum was mixed with 0.4 ml of 10 N KOH and state had been approached, stools were collected into re- saponified in an autoclave for 30 minutes at 15 pounds frigerated, tared aluminum containers for periods of time pressure. An equal volume of ethanol was then added, that varied from 4 to 7 days; the stool samples. were and the solution was brought to a boil on a steam bath. pooled and treated as a single sample for the purpose of Neutral sterols were then extracted into a tenfold excess the subsequent analyses. of ethyl ether, and after backwashing the ether extracts Two different formula diets were utilized in these were dried. After dissolving the residue in acetone-alco- studies. For the periods in which crystalline cholesterol hol (1:1) cholesterol digitonides were then formed, was added to the diets the basic formulas consisted of washed, and dried by the method of Sperry and Webb triolein,l vitamin-free casein,1 gelatin, sucrose, vanilla ex- (16). The cholesterol digitonides were dissolved in tract, egg white, and methyl cellulose; the analyzed sterol methanol; one aliquot was added to 0.4% diphenyloxazole content of this basic formula was less than 5 mg of digi- in toluene and assayed for 14C in a Packard liquid scintil- tonin precipitable material per day. Varying amounts of lation spectrometer, and one aliquot was assayed for cho- crystalline cholesterol-4-1C were added to this basic diet lesterol content (16). Each determination was performed by dissolving the cholesterol in a portion of the triolein in duplicate. Specific activity determinations performed and feeding it directly in either two or three portions. per in this way varied less than 5% when rechecked on differ- day. The cholesterol-4-14C used in these studies was ob- ent days. tained by adding cholesterol-4-14C 2 that had been purified Bile acid and neutral sterol content of feces. To the by chromatography on silica gel in benzene: ethyl acetate weighed feces samples was added 2 vol of water, and the (4:1) (14) to cholesterol that had been regenerated from mixture was shaken on a paint can shaker for 5 minutes the dibromide and then recrystallized five times (15). and then transferred to a Waring blendor and homogenized This mixture was then recrystallized three times from for 5 minutes. One- to 2-g samples of the liquid mixture ethanol. were then carefully weighed in triplicate, mixed with 100 For the formula diets in which the cholesterol source ml chloroform: methanol (2: 1), and refluxed for 5 min- was egg yolk, the eggs were obtained as follows: eight utes. The chloroform: methanol extracts were filtered by laying hens were each injected with 1.2 X 108 cpm of washing into 250-ml Erlenmeyer flasks, dried under ni- cholesterol-4-j4C dissolved in ethanol, and all the eggs trogen by boiling, mixed with 3.5 ml of 10 N NaOH and were collected for 1 month. The pooled, frozen yolks (6 10 ml H20, and autoclaved for 3 hours at 15 pounds dozen) were then diluted with 140 dozen fresh egg yolks. pressure and 1300 C. Two vol of ethanol was then added, This mixture was stirred repeatedly in two large batches and the samples were refluxed on a hot plate for 5 min- and frozen in weighed aliquots. The formula for this diet utes. Neutral sterols were extracted three times from consisted of sucrose, vanilla extract, gelatin, and egg the mixture by shaking with pentane; the pentane extracts yolk that were whipped together and then frozen. The were combined, backwashed with 10 ml of ethanol: water total daily caloric intakes for each of the study periods (1: 1), and dried under nitrogen. After dissolving the and the calculated distribution of the calories are given residue in methanol one aliquot was assayed for 14C, and in Table I. In each instance the cholesterol content and one was saved for thin-layer chromatography. The wa- the cholesterol specific activity were measured directly ter layer remaining after the pentane extraction was acidi- by analysis of the entire diet. fied to pH 2 to 3 by titrating with concentrated H2SO4, At the end of the first feeding period a study of the and the bile acid fraction was extracted into ethyl ether. change in specific activity of the dietary cholesterol at The ethyl ether extracts were dried under nitrogen and various levels in the gastrointestinal tract was performed dissolved in 50 ml acetone. The acetone extract was then in each of the subjects. A Levine tube was allowed to passed through a Florisil column (1 X 10 cm), and the pass overnight into the duodenum (95 cm from the bile acids were eluted with 50 ml of acetone: methanol: mouth), and then a second tube was passed into the stom- acetic acid (80: 20: 1) (17). The eluate was. then dried ach (65 cm from the mouth).