Recent Advances Digestion 14: 342-356 (1976)
The Role of the Digestive Tract in Cholesterol Metabolism C. Lutton Laboratoire de Physiologic de la Nutrition, Orsay
Key Words. Bile acids and salts • Cholesterol • Diet • Feces - Intestinal absorption • Intestinal mucosa • Isotopic equilibrium • Lymph • Sterols Abstract. New studies have permit to reevaluate the importance of the digestive tract in each of the mechanisms regulating cholesterol turnover. Particularly, the role of the diges tive tract in the rat cholesterol synthesis was underestimated. Is there a similar situation in man?
While the major aspects of absorption, synthesis or elimination of choles terol are now well known , the mechanisms of regulation of cholesterol metab olism in intact animals are not entirely clear. One reason that the regulatory processes have not been fully established is that generally only one process or at most two are studied in each experiment. Thus, the development in 1963 of a physiological method which permitted the simultaneous measurement of choles terol turnover rates in intact animals was promising. The theoretical and practical bases of this technique, the so-called isotopic equilibrium method, have been well described and shown by Chevallier (10). During the last decade the method has been applied extensively by several laboratories both in man and in animals (2, 35, 37, 40, 42, 53, 54, 82, 84). Recent studies have emphasized the importance of the digestive tract in the synthesis and excretion as well as in the absorption of cholesterol. The purpose of this review is to discuss some of the recent developments of intestinal secretion, excretion and absorption of choles terol. Since most of the experimental work has been done in animals, this review will emphasize the role of the digestive tract (particularly the intestine) in these functions in the rat. Some of the discrepancies between the rat and man will be pointed out. Several other reviews have been published on various aspects of cholesterol metabolism (4, 12, 25, 28, 32,43, 56, 78, 79, 88).
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Received: March 8. 1976: accepted: March 18, 1976.
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Since definitions concerning cholesterol dynamics differ among authors to avoid confusion 1 shall redefine the terminology used by Chevallier and his coworkers and compare it to the terminology commonly used in the United States. Much information can be obtain from whole body cholesterol turnover in respect to its dynamic equilibrium as previously pointed out by Chevallier (6, 7). For an animal in a steady state, the dynamic equilibrium of cholesterol in the transfer space is defined by the relationship m A + mIS = mT + mE (F+ U )’
(1)
m is the rate (mg/day) of absorption of cholesterol (A) internal secretion (IS), transformation into bile acids (T) and excretion (E) in feces (F) plus urine1 (U). Absorption is defined as the input of dietary cholesterol into the transfer space. The transfer space corresponds to the amount of cholesterol in equilibrium with plasma as it is determined at isotopic equilibrium (12). Thus, the cholesterol transfer space does not correspond exactly to the mobile body cholesterol since it does take into account the ‘cholesterol synthesis space’ (14). However, we know that the size of ‘synthesis cholesterol space’ is very small compared to that of cholesterol transfer space (2 -3 and 7 0-75 % of total body cholesterol, respectively). Excretion is defined as the transfer of cholesterol molecules from inside to the outside of the transfer space, for example by loss into the intestinal lumen or urinary tubules or by epithelial sloughing and sebaceous secretions (12). Secretion is synthesis by tissues and transfer into (internal secretion) or out of (external secretion) the transfer space (12). It is important to note that these definitions do not imply a mechanism and are simply related to the origin of the molecules. For a rat fed a cholesterol containing diet, fecal elimination of choles terol (m F) is the sum of unabsorbed dietary cholesterol (mUA), excreted fecal cholesterol (mEF) and externally secreted fecal cholesterol (mESF) (17) m F = m U A + mF.F + m E S F '
In the literature the term ‘endogenous sterol excretion’ corresponds gen erally to the sum of mEF + mESF, since very few authors are able to distinguish between the two types of sterols. However, it is important to make this distinc tion because fecal external secretion is not part of the output of the transfer space, as seen from equation (1). In man, ‘endogenous sterol excretion’ is probably only due to excreted cholesterol (58). This point will be discussed again later.
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1 In man. urinary elimination of cholesterol and its related compounds is quantita tively negligible compared to fecal loss of cholesterol and bile acids. In the rat it is not always slight (33, 46).
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Excretion and External Secretion
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In a rat which is fed a sterol-free diet and in which coprophagy and fur licking are prevented, the intestinal sterol contents are cholesterol (80—85 %) and some of its biosynthetic precursors (A7-cholesterol and methostenol) (48, 56). These sterols undergo bacterial modifications in the cecum and colon. For example, a part of the cholesterol is converted to coprosterol by reduction of the double bond at carbon 5 (48, 64, 70). In this article the term ‘fecal choles terol' will refer to the sum (cholesterol + coprosterol). Feces from rats fed a diet containing cholesterol (0.015 %) as the only sterol contain 0.5 mg/day of unabsorbed dietary cholesterol, 3 mg of excreted fecal cholesterol and 3 mg of cholesterol synthesized by the digestive tract and secreted into the lumen (fecal externally secreted cholesterol) (17). The mecha nisms by which endogenous cholesterol (excreted and secreted) enter the small intestine are not yet clear. It had been assumed that bile and sloughed epithelial cells were the only two sources of intestinal cholesterol and that opinion seems to be widely held especially for humans (55). However, several studies have shown that cholesterol synthesized by the intestine appears very rapidly in the lumen after the administration of labeled acetate. This appearance can hardly be explained on the basis of epithelial cell sloughing. Moreover, the biliary choles terol is only excreted cholesterol (13, 60). Though the bile contribution is difficult to measure accurately it approximates 2 -3 mg/day in the rat. Since a major part of biliary cholesterol (60—80 %) would be reabsorbed, it is reasonable to assume that bile does not account for more than one third of the fecal cholesterol excretion (19, 66). This conclusion is supported by other data. In rats fed a 2 % cholestyramine or 2 % cholesterol diet the observed fecal choles terol excretion is three and four times higher than that of controls, but the daily cholesterol output in bile undergoes only a slight increase or is unchanged (9, 50). Shortly after the subcutaneous or parenteral administration of 14C-cholesterol, radioactivity was found in the intestinal lumen even when the bile was diverted and replaced by unlabeled bile from a donor rat (22, 27). In a series of experiments, Chevallier and Lutton (19) estimated the daily flux of endogenous cholesterol entering the lumen to be 11 mg/day. If we assume that the villi accounts for some 20 % of the whole intestinal cholesterol and that epithelial cell turnover time is 30 h (44), the endogenous contribution from sloughed mucosal epithelium may be 3 mg/day. Thus, apparently at least 50% of the flux of endogenous cholesterol entering the lumen could be due to active exchange of cholesterol between epithelial cells and lumen in the rat. Experiments on anesthetized, fasted rats, by Cotton (26) concluded that the results obtained for lipid values (phospholipid, cholesterol and triglyceride) could be explained on the basis of exfoliated cells. Additional experiments will be necessary to resolve this important question.
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It is not likely that only a limited portion of the intestine is able to excrete or secrete cholesterol, since the ratio excreted cholesterol/secreted cholesterol in the intestinal lumen remained almost the same (about 1/1) from the duodenum to the ileum (19). Cholesterol synthesis takes place mainly or exclusively in the crypt area of the intestinal mucosa (31). It is not yet known whether the exchange of cholesterol between intestinal cells and plasma and between intes tinal cells and the lumen occurs in every cell of the villus or only in that part of it which has been shown to be involved in cholesterol absorption (76). Since synthesis occurs in the crypts and if exchange of cholesterol takes place along the entire villus, it can be expected that a progressive increase would occur in the ratio excreted cholesterol/secreted cholesterol in the absorptive cell from the crypt area to the top of the villus. In man ‘endogenous cholesterol excretion’ appears responsible solely by excreted fecal cholesterol; that is, there is no contribution from externally secreted fecal cholesterol (71). This raises interesting questions on the mecha nisms of cholesterol transfer in the intestinal mucosa. Some authors have found that sterols can be degraded by human bacterial flora whereas no degradation occurs in the rat (40,45). However, this degradation is probably greatly affected by the diet (29). It has been also observed that sterols are transformed more extensively by human flora than by rat flora. As an example, large proportions of sterols were found as stanones (coprostatone ...) in human feces but only in negligible amounts in the rat feces (48).
The main steps of cholesterol absorption will be described from a ‘dynamic’ point of view. Figure 1 summarizes the movement of cholesterol from the lumen into the intestinal cell and then into the extracellular space and lymphatic duct. Before entering the duodenal lumen, dietary cholesterol mixes with excreted and secreted endogenous cholesterol from salivary and gastric fluids (78). Chevallier and Lutton (19) recently estimated the flux of endogenous cholesterol which mixes with dietary cholesterol in the stomach content to be 1.5 mg/day in the rat. Cholesterol is present in the intestinal contents mainly as free cholesterol because of the hydrolyzing action of pancreatic cholesterolesterase (48, 78). Cholesterol concentration increases greatly from the pyloric to the cecal valves (19, 57). Cholesterol in the intestinal lumen is a mixture of cholesterol originating from the stomach contents and endogenous cholesterol from sources other than the stomach contents. In contrast to a previous interpretation (78), a recent study has shown that the mixing of these two ‘types’ of cholesterol is not homogeneous in the rat (19). In fact, two cholesterol compartments seem to be present in the lumen, i.e. an ‘intermediary’ compartment containing endogenous
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Absorption
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Luiton
Com partm ents _______ l_______
Fig. i. Schematic representation of various processes responsible for lymph and chylo microns cholesterol. Adapted from Chevallier and Vyas (22) and Chevallier and Luiton (19).
mg Id
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Fig. 2. Flux of excreted and secreted cholesterol (mg/day) through walls of the upper part of the digestive tract and intestine in a control rat. From Chevallier and Lutton (19).
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cholesterol which exchanges with cholesterol of the intestinal cells, and an ‘axial’ compartment, containing a mixture of intestinal cholesterol and cholesterol from the stomach contents (19). Thus, three different sources of cholesterol are available for absorption: dietary, excreted and secreted cholesterol. Because of the partial mixing of these three sources one could have a different absorption coefficient (percentage absorption) for cholesterol from each source. Also the mixing of externally secreted with dietary cholesterol accounts for the contri bution of the digestive tract to the internal secretion (undirect internal secretion, fig. 2). In the same study, the total of cholesterol synthesized by the digestive tract and reabsorbed was estimated to be 4 mg/day, i.e. almost one third of the total internal secretion in the rat. Following centrifugation of the small intestinal contents, free sterols are found to be mainly in a micellar phase (clear aqueous supernatant) and in the sediment (57; unpubl. observations). The interrelationships between these two ‘types’ of cholesterol and the two compartments previously described remain to be established. The mechanisms of the mucosal uptake of free cholesterol from micelles, as well as the mode of transport of cholesterol across the mucosal cells are not yet fully understood. Migration of cholesterol across the cell seems to be associated with that of long chain fatty acids. These compounds migrate to the endoplasmic reticulum tubules in droplets of 250-650 A diameter which could represent chylomicrons being formed (80). Triglyceride synthesis and chylo micron formation probably take place in the endoplasmic reticulum located above the nucleus (41, 81). Inside the intestinal cell, cholesterol exchanges between chylomicra and the subcellular structures (arrows 6 and 7, fig. 1) (22). Part of the cholesterol in chylomicrons is esterified before the particles are secreted across the basal membrane (23, 41). In the extracellular space chylo microns adsorb free cholesterol and probably also esterified (arrows 12 and 13, fig. 1) (21, 22). Cholesterol exchange also takes place between intestinal cells and the lumen or intestinal cells and extracellular spaces (arrows 4, 5,9 and 10, fig. 1) (15). It is known that there is cholesterol transfer from plasma to extra cellular fluid (arrows 14, 15) (8, 22). It is probable that an opposite transfer exists (arrow 16) since even HDL proteins can appear in the perfusate of a lymph cannulated isolated rat intestine (87). The mechanisms associated with transfer of cholesterol to lymph constitute the routes by which the intestine directly secretes its synthesized cholesterol into the plasma (direct internal secretion; fig. 2); but the quantifying of this mechanism is very difficult. Regulation o f Absorption
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Each step previously described in the overall process of cholesterol absorp tion is potentially a rate-limiting step. The mechanisms which regulate these processes are not well established.
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The percentage of cholesterol which is absorbed varies according to species and dose. Numerous factors which modify cholesterol absorption have been described, e.g., age and sex (46), nature of dietary fatty acids (32), percentage of casein in the diet (47), bile flow (32), etc. It has been shown repeatedly that the size of the bile acid pool has a great effect on the rate of cholesterol absorption. Numerous studies support this view: cholestyramine lowers and a bile fistula or bile ligation completely inhibit absorption of cholesterol (47, 67, 79) while addition of taurocholate to the diet increases it (50, 79). This effect on the extent of cholesterol absorption has been attributed to tire role of bile acids in promoting micellar solubilization of cholesterol in the intestinal lumen and also to their effect on its transfer into the intestinal lymph (32, 85). If the dietary concentration of taurocholate was increased to 4 %, the absorption coefficient of cholesterol was decreased (50). This observation and others show that more than a simple mechanism is needed to explain tire role of bile acids in regulating cholesterol absorption. Some authors have claimed that the movement from the intestinal cell into the lymph is the limiting step in cholesterol absorption (75). If this is the case the production of the compounds necessary for the chylo micron coat (i.e. proteins and phospholipids) could be the key steps in the regulation of cholesterol absorption. Contradictory results have been published as to whether the mucosal synthesis of the protein coat is essential to chylo micron formation (63, 65). The recent observation that an active phospholipid exchange protein is present in mucosal cells also may be pertinent (51). How ever, this phospholipid exchange activity is not influenced by high lipid and cholesterol content in the diet suggesting that phospholipid exchange proteins are not rate limiting in the process of cholesterol absorption.
Excretion Fecal excretion of cholesterol in the rat is relatively low compared to that of bile acids, but in man cholesterol elimination in the form of fecal neutral steroids is equal to or greater than that of bile acids (32, 39, 58). Several authors have claimed that the fall in serum cholesterol that occurs when polyunsaturated fats are substituted for saturated fat in the diet may result from an increase in the excretion o f fecal cholesterol (1, 24, 59, 86). Fecal excretion of cholesterol is influenced by various factors such as cerebrosides or cellulose content of the diet, germfree status, etc. (32). Numerous isotopic equilibrium experiments in the rat have recently shown that in most of the nutritional conditions described by the authors, the total fecal excretion of cholesterol was inversely propor tional to the absorption coefficient of cholesterol (fig. 3 a) (49). The value for the fecal cholesterol excretion extrapolated to a theoretical absorption coeffi-
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Regulation o f Fecal Excretion and External Secretion
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A b s o rp tio n , %
Fig. 3. a Mass of excreted cholesterol found daily in the feces as a function of the absorption coefficient of cholesterol, b Mass of secreted cholesterol found daily in the feces as a function of the absorption coefficient of cholesterol, bach point represents an isotopic equilibrium experiment (4 rats) in a given experimental condition (17, 46, 47, 50).
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cient of zero (14.6 mg) could represent a maximal flux of cholesterol from the transfer space to the lumen. In the control rat where the absorption coefficient reached 75 %, the net flux for excreted cholesterol has been estimated to be 5.5 mg/day by another method (19). Thus it is probably that this net flux increases when the absorption coefficient diminishes. Another interesting observation is that the extrapolation of the regression line to 100 % absorption gives a positive value for fecal excretion, suggesting that the absorption coeffi cient of excreted cholesterol is lower than that of dietary cholesterol since when the dietary absorption of cholesterol is 100 % fecal cholesterol excretion should be zero (19. 49). The interrelationship between fecal excretion of cholesterol and the absorption coefficient could be of general significance. It is noteworthy that the relative quantitative importance of fecal excretion of cholesterol com pared to conversion to bile acids increases from rabbit to rat to man, species in which the absorption coefficient for cholesterol is very high, high, and low, respectively.
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External Secretion Like fecal excretion the daily mass of fecal external secretion of cholesterol increases when the absorption coefficient decreases in the rat (fig. 3 b). The similar response of fecal excretion and fecal external secretion to changes in the absorption coefficient suggests that the transfer of plasma cholesterol and synthesized cholesterol is regulated by a similar mechanism in the ‘intermediary’ and ‘axial’ compartments. However, the slope of the regression line for fecal external secretion versus absorption coefficient is lower than that for excretion, and the extrapolated value for a theoretical absorption of 0 % is only slightly higher (7.7 mg/day) than the estimated net flux of cholesterol secreted into the lumen of a control rat (5.5 mg/day) (19). This observation could indicate that the flux of secreted cholesterol from intestine to lumen is fairly constant and independant of nutritional conditions in these rats.
Although is has been known since 1950 that the intestine synthesizes cholesterol, its relative contribution to the internal secretion is still questionable (61, 72). Based on in vitro incorporation of various labeled precursors, the liver has the highest rate of sterol synthesis (31, 56). Although it is difficult to extrapolate such in vitro information to the intact animal, the liver has been generally assumed to be the main source of synthesized cholesterol. However, several new studies do not agree with this point of view. It is well established that hepatic cholesterol synthesis varies greatly with the type of diet and is inhibited by fasting or cholesterol feeding (68, 77). However, the internal secre tion of cholesterol as measured by the isotopic equilibrium method undergoes only slight changes under numerous dietary conditions and does not change when cholesterol is added to the diet (11). The rate of cholesterol synthesis determined by the same method is lower in female than in male rats, although incorporation of labeled precursors into liver homogenates or slices is higher in the female (34, 46). It has been recently estimated that more than 4 mg of the cholesterol synthesized by the digestive tract and secreted into the lumen could be reabsorbed daily with dietary cholesterol (19). This represents almost one third of total internal secretion in the rat. Thus it is likely that tire role played by the intestine in cholesterol synthesis will be reevaluated in the near future. Dietschy and Siperstein (31) have shown that incorporation of labeled acetate into cholesterol in intestinal slices occurs predominantly in the cells of the crypts. This fact agrees with a general hypothesis correlating cholesterol synthesis and formation of cells (18). Whether or not such a mechanism can entirely explain the production of synthesized cholesterol is unknown. In contrast with Dietschy's experiment, Mak and Trier (52) found a higher in corporation of labeled mevalonate into cholesterol on tire brush border of cells
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Regulation o f Synthesis
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in the upper part of the villus than in crypts cells from everted ileal sacs. In this latter experiment, however, the high amount of label observed on the upper part of the villi could be due to labeled cholesterol previously secreted by the crypts cells and reabsorbed by the absorptive cells. Dietschy (30) has claimed that the luminal concentration of bile acid controls cholesterogenesis in the small intestine based upon the observations that in vitro incorporation of acetate into jejunal cholesterol was greatly enhanced by diversion of bile or by ligating the bile duct and was depressed by infusion of solutions of whole bile or pure bile acids. In contrast to the situation in the liver he did not find any feedback inhibition by cholesterol itself. This finding has been questioned by several other investigators who observed a moderate inhibitory effect of cholesterol on intestinal cholesterogenesis in the baboon and guinea pig (74. 83). When rats were fed tomatine, an agent which precipitates cholesterol without interferring with bile acids, Cayen (5) observed an increase in small intestinal cholesterogenesis. Thus, cholesterol synthesis in the small intestine may be controlled by several mechanisms. The analysis of 21 isotopic equilibrium experiments has recently shown that there exists an inverse relation ship between total cholesterol synthesis and the absorption coefficient for cholesterol in the rat. Since a correlation also exists between external fecal secretion and absorption coefficient, it has been suggested that intestinal choles terogenesis could be inversely correlated with the absorption coefficient (20). It is obvious that the absorption coefficient for cholesterol depends on several factors such as the concentration of bile acids and cholesterol in the lumen; the rate of cell turnover in the intestinal mucosa which probably modifies intestinal cholesterol synthesis could also play a role. It is known that, in regard to the villus, cholesterol absorption occurs in the apical area (76). This functional particularity could be due to a progressive development of enzymes by absorp tive cells during their migration (73). When the renewal of crypts cells is increased, the area o f ‘mature’ cells could be diminished. In some of the isotopic equilibrium experiments cited above, total choles terol synthesis did not follow the relation previously described. It is remarkable that these particular cases in which there was an unusual elevated synthesis could always be correlated with a hyperfunction of an extradigestive source of choles terol synthesis such as placenta and mammary glands in pregnant and lactating females, respectively, or liver (dietary cholestyramine or bile duct ligation) (20, 49). Thus, under normal conditions the importance by the liver in cholesterol synthesis has probably been overestimated.
This discussion has only been concerned with describing processes involving the movements of cholesterol in the digestive tract. However, an important
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Elimination as Bile Salts and Conclusion
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mechanism of cholesterol turnover is by hepatic transformation of cholesterol into bile acids (3, 69). The isotopic equilibrium method is a good technique for measuring the rate of this process (16, 17,45). In a rat fed a sterol-free diet this daily rate is 13 mg, i.e. 4 times more than the daily excretion of cholesterol into the feces. The rate varies when the rates of excretion (fecal or urinary) internal secretion or absorption change or even when the transfer space of cholesterol increases (45). So this transformation rate seems to be an adaptative process for maintaining a dynamic equilibrium of cholesterol in the rat (50). In man the rate of cholesterol excretion is quantitatively as large as bile acid production (24, 36, 84). Moreover, the conversion of cholesterol into bile acids does not seem to be greatly affected by large amounts of cholesterol in the diet (62). The ‘unadaptability’ of this process may explain the frequency of dysfunc tion of the dynamic equilibrium in man. Finally, interrelationships between absorption and synthesis have been studied but the results are conflicting (38, 62). Additional difficulties occurring in human experimentation explain the slower advance of knowledge in this important area. New methods developed during the last decade have solved numerous problems concerned with cholesterol turnover but several questions are still not adequately answered. An important problem is whether the mechanisms regu lating cholesterol turnover in man are similar to those in the rat. The methods presented permit the simultaneous measure of input-output rates of cholesterol turnover in man may give the information needed to fully understand this important area of metabolism.
Acknowledgments I would like to thank Prof. F. Clievallier for his continuous interest and stimulation. I am indebted to J. Balmer and Dr. M. Green for their help in correcting the text and to Prof. J.L. Rabinowitz for his helpful suggestions.
References
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Antonis. A. and Bersohn, I.: The influence of diet on fecal lipids in South Africa white and Bantu prisoners. Am. J. clin. Nutr. 11: 142-146 (1962). Avigan, J. and Steinberg. D.: Sterol and bile acid excretion in man and the effects of dietary fats. J. clin. Invest. 44: 1845- 1855 (1965). Bloch. K.; Berg. B.. and Rittenberg, D.: The biological conversion of cholesterol to cholic acid. J. biol. Chcm. 149: 511-517 (1943). Borgstrom, B.: Fat digestion and absorption in biomembranes, vol. 4B, pp. 556 620 (Plenum Press, London 1974). Cayen. M.N.: Effect of dietary tomatine on cholesterol metabolism in the rat. J. Lipid Res.12: 482 490(1971). Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:04:34 AM
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Chevallier, F.: Le cholestérol: données chimiques et apport des méthodes isotopiques dans la connaissance de son métabolisme. Ann. Nutr. Aliment. 7: 305 308 (1953). 7 Chevallier, F.: L’espace cholestérol du rat. Archs Sci. physiol. 10: 249-275 (1956). 8 Chevallier, F.: Etude des origines des stérols fécaux du rat à l’aide d’indicateurs nuclé aires. IL Détermination des quantités de stérols excrétés et sécrétés, et de la fraction du cholestérol des parois digestives renouvelé par transfert. Bull. Soc. Chim. biol. 42: 633-641 (1960). 9 Chevallier, F.: Origine des stérols fécaux. III. Influence de la concentration du choles térol alimentaire sur les quantités de cholestérol excrété par la bile et par la paroi intestinale. Bull. Soc. Chim. biol. 42: 643 654 (1960). 10 Chevallier F : La méthode d’équilibre isotopique. Ann. Nutr. Aliment. 17: 51-70 (1963). 11 Chevallier, F.: Vitesses des processus de renouvellement du cholestérol contenu dans son espace de transfert, chez le rat. II. Influence de la concentration du cholestérol alimentaire dans le cas d’un régime semi-synthétique témoin. Bull. Soc. Chim. biol. 48: 715-729 (1966). 12 Chevallier, F.: Dynamics of cholestérol in rats studied by the isotopic equilibrium method. Adv. Lipid Res. 5: 209 239 (1967). 13 Chevallier. F. et Branco, R.J.: Synthèse de cholestérol hépatique à partir d’acétate 14C chez les rats normaux et porteurs de fistule biliaire. Origine du cholestérol biliaire. Revue Etud. clin. biol. 8: 903 909 (1963). 14 Chevallier, F.: D'Hollander, F. et Simonnet, F.: Renouvellement, par synthèse et par transfert du cholestérol libre et estérifié. Grandeur des compartiments chez le rat adulte et leur répartition tissulaire. Biochim. biophys. Acta 164: 339 356 (1968). 15 Chevallier, F.: D ’Hollander, F., and Vaughan, AL. Plasma cholestérol ester formation in situ and their transfer into the rat tissues in vivo. Biochim. biophys. Acta 248: 524 529 (1971). 16 Chevallier, F. et Lutton, C.: Cinétiques journalières et horaire de la transformation du cholestérol 26 laCen acides biliaires. Bull. Soc. Chim. biol. 48: 295-311 (1966). 17 Chevallier, F. et Lutton, C.: Vitesses des processus de renouvellement du cholestérol contenu dans son espace de transfert chez le rat. I. Méthodes et résultats obtenus dans le cas d’un régime semi-synthétique témoin. Bull. Soc. Chim. biol. 48: 507 523 (1966). 18 Chevallier, F. et Lutton, C.: Origines du cholestérol de sécrétion interne chez le rat. Théorie du renouvellement cellulaire. Revue eur. Etud. clin. biol. 16: 16 18 (1971 ). 19 Chevallier, F. et Lutton, C.: Mouvements des stérols dans le tube digestif d’un rat. Absorption du cholestérol de synthèse. Biochim. biophys. Acta 274: 3 8 2 411 (19 72). 20 Chevallier, F. and Lutton. C.: The intestine is the major site of cholestérol synthesis in the rat. Nature new Biol. 242: 61 62 (1973). 21 Chevallier, F. et Mathe, D.: Destinée du cholestérol des chylomicrons. III. Mouvements de cholestérol 4 ‘"C entre les chylomicrons et la lymphe ou le sérum in vivo. Bull. Soc. Chim. biol. 46: 509-527 (1964). 22 Chevallier, F. et Vyas, M.: Les origines du cholestérol du chyle. Mise en évidence à l’aide de la méthode des indicateurs nucléaires. Bull. Soc. Chim. biol. 44: 253-275 (1963). 23 Clemenl J.: Les lipides intestinaux et fécaux chez le rat soumis pendant de longues périodes à un régime lipidoprive. Archs Sci. physiol. 15: 345-361 (1961 ). 24 Connor, W.F.: Witiak, D.T.: Stone, D.B., and Armstrong, M.L.: Cholestérol balance and fecal neutral steroid and bile acid excrétion in normal men fed dietary fats of different fatty acid composition. J. clin, tnvest. 48: 1363-1375 (1969). Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:04:34 AM
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Cook, R.P.: Cholesterol chemistry, biochemistry and pathology (Academic Press, New York 1958). 26 Cotton. P.B.: Non-dietary lipid in the intestinal lumen. Gut 13: 675 681 (1972). 27 Danielsson, H.: On the origin of the neutral fecal sterols and their relation to choles terol metabolism in the rat: bile acids and steroids. Acta physiol, scand. 48: 364 372 (1960). 28 Danielsson, H.: Mechanisms of bile acid formation, bile salt metabolism (Thomas, Springfield 1969). 29 Denbesten, /..; Connor, IV./;’., Kent, T.H., and Un, D.: Effect of cellulose in the diet on the recovery of dietary plant sterols from the feces. J. Lipid Res. 11: 341-345 (1970). 30 Dietscliy, J.M.: The role of bile salts in controlling the rate of intestinal cholesterogenesis. J. clin. Invest. 47: 286 300 (1968). 31 Dietscliy, J.M. and Siperstein, M.D.: Cholesterol synthesis by the gastrointestinal tract: localization and mechanisms of control. J. clin. Invest. 44: 1311 1327 (1965). 32 Dietscliy, J.M. and Wilson, J.D.: Regulation of cholesterol metabolism. New Engl. J. Med. 282: 1128-1138, 1179-1183,1241-1249 (1970). 33 Dorfman, R.l. and Ungar, F.: in Metabolism of steroid hormones, pp. 123-213 (Aca demic Press, New York 1965). 34 Dupont. J.: Synthesis of cholesterol and total lipid by male and female rats fed beef tallow or corn oil. Lipids 1: 409-414 (1966). 35 Eggen, D.A. and Strong. J.P.: Cholesterol metabolism in baboon, rhesus and squirrel monkeys on a atherogenic diet. Circulation 40: 3 -7 (1969). 36 Gordon, H.: Lewis. B.: Bales, /.., and Brock, J.F.: Dietary fat and cholesterol metabo lism. Faecal elimination of bile acids and other lipids. Lancet 28: 1299-1306 (1957). 37 Grundy, S.M. and Ahrens, E.H.: Measurements of cholesterol turnover, synthesis and absorption in man, carried out by isotope kinetic and sterol balance methods. J. Lipid Res. 10: 91 107 (1969). 38 Grundy, S.M.: Ahrens, E.H., and Davignon, J.: The interaction of cholesterol absorp tion and cholesterol synthesis in man. J. Lipid Res. 10: 304-315 (1969). 39 Grundy, S.M.: Ahrens. E.H., and Miettinen, T.A.: Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J. Lipid Res. 6: 397 410 (1965). 40 Grundy, S.M.; Ahrens, E.H.,and Salen, G.: Dietary fi sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies. J. Lipid Res. 9: 374-387 (1968). 41 Hatch, F.T.: Aso, Y.: Hagopian, L.M., and Rubinstein, J.L.: Biosynthesis of lipoprotein by rat intestinal mucosa. J. biol. Chem. 241: 1655 1665 (1966). 42 Kaplan, J.A.: Cox. G.E., and Taylor. C.B.: Cholesterol metabolism in man. Studies on absorption. Arehs Path. 76: 359- 368 (1963). 43 Kritchevsky, D.: Cholesterol (Wiley. New York 1958). 44 Leblond, C.P. and Stevens. C.E.: The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100: 357-377 (1948). 45 Lutton. C. el Chevallier, F: Vitesses des processus de renouvellement du cholestérol contenu dans son espace de transfert, chez le rat. III. Modifications et étude critique de la méthode d’équilibre isotopique. Biochim. biophys. Acta 255: 762-779 (1972). 46 Lutton. C. et Chevallier. F.: Vitesses des processus de renouvellement du cholestérol contenu dans son espace de transfert, chez le rat. IV. Influence de l’âge, du poids, du sexe, de la gestation et de la lactation. Biochim. biophys. Acta 280: 116-130 (1972). 47 Lutton. C. et Chevallier. F.: Vitesses des processus de renouvellement du cholestérol contenu dans son espace de transfert, chez le rat. V. Influence des proportions de glucides, lipides et protides dans l’alimentation. Biochim. biophys. Acta 280: 131 -144 (1972). Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:04:34 AM
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l.utton, C. el Chevallier, F.: Analyse des stérols des contenus digestifs et des fèces du rat Biochim. biophys. Acta 260: 133 145 (1972). l.utton, C. et Chevallier, F.: Coefficient d’absorption du cholestérol alimentaire, paramètre fondamental de l’équilibre dynamique du cholestérol chez le rat. Bioméde cine 2 (in press). l.utton, C.; Mathe, D. et Chevallier, F.: Vitesses des processus de renouvellement du cholestérol contenu dans son espace de transfert chez le rat. VI. Influence de la ligature du cholédoque et de l’ingestion d’acides biliaires ou de cholestyramine. Biochim. biophys. Acta 306: 483- 496 (1973). Lutton, C. and Zilversmit, D.B.: Presence of phospholipid exchange protein in rat intestine. Lipids//.' 16-20 (1976). Mak, K.M. and Trier, J.S.: Radioautographic and chemical evidence for (53H) mcvalonate incorporation into cholesterol by rat villous absorptive cells. Biochim. biophys. Acta 280: 316 328(1972). Mann, G. V.: Cystine deficiency and cholesterol metabolism in primates. Circulation 28: 205 212 (1966). Manning. P.J.: Clarkson, T.B., and l.ofland, H.B.: Cholesterol absorption, turnover and excretion rates in hypercholestérolémie rhesus monkeys. Expl molec. Path. 14: 75 -89 (1971). McIntyre, N.: Sterol absorption from lumen to liver. Gut / 2: 411 -416 (1971). McIntyre, N. and Isselhacher, K.J.: Role of the small intestine in cholesterol metabo lism. J. clin. Nutr. 26: 647-656 (1973). McIntyre, N.; Kirsch, K.: Orr, J.C., and Isselhacher, K.I„: Sterols in the smalt intestine of the rat, guinea pig and rabbit. J. Lipid Res. 12: 336 346 (1971 ). Miettinen, T.A.: Ahrens, F.H., and Grundy, S.M.: Quantitative isolation and gas liquid chromatographic analysis of total dietary and fecal neutral steroids. J. Lipid Res. 6: 411-424 (1965). Moore, R.B.: Anderson, J.T.: Taylor, H.L: Keys, A., and Frantz, I.D.: Effect of dietary fat on the fecal excretion of cholesterol and its degradation products in man. J. clin. Invest. 47: 1517-1534 (1968). Ogura, M.: Shiga, J., and Yamasaki, K.: Studies on the cholesterol pool as the precursor of bile acids in the rat. Biochcm. J. 70: 967 - 9 7 2 (1971). Popjak, G. and Beckmans, M .L: Extrahepatic lipid synthesis. Biochem. J. 47: 233 238 (1950). Quintaô, E.; Grundy, S.M., and Ahrens, E.H.: Effects of dietary cholesterol on the regulation of total body cholesterol in man. J. Lipid Res. 12: 233 -247 (1971). Redgrave, T.G.: Inhibition of protein synthesis and absorption of lipid into thoracic duct lymph of rats. Proc. Soc. exp. Biol. Med. 130: 776-780 (1969). Rosenfeld, R.S.: Fukushima, D.K.: Heilman, !... and Gallagher, T.F.: The transforma tion of cholesterol to coprostanol. J. biol. Chem. 211: 301- 311 (1954). Sabesin, S.M. and Isselhacher, K.J.: Protein synthesis inhibition. Mechanism for the production of impaired fat absorption. Science, N.Y. 147: 1149 1151 (1965). Shi-Kaung Peng: Kang-Jey Ho, and Taylor, C.B.: The role of the intestinal mucosa in cholesterol metabolism. Its relation to plasma and luminal cholesterol. Expl molec. Path. 21: 138 153 (1974). Siperstein, M.D.: Cliaikoff 1.1.., and Reinhard, W.O.: |4C-cholesterol. V. Obligatory function of bile in intestinal absorption of cholesterol. J. biol. Chem. 198: 111 114 (1952). Siperstein, M.D. and Fagan. V.M.: Feedback control of mcvalonate synthesis by di etary cholesterol. J. biol. Chem. 241: 602 609 (1966). Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:04:34 AM
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Siperstein. M.D.; Jayko, M.E.; Chaikoff, I.L., and Dauben, W.G.: Nature of the meta bolic products of l4C cholesterol excreted in bile and feces. Proc. Soc. exp. Biol. Med. 81: 720 724 (1952). Snog-Kjaer, A.: Praune. /.. and Dam, H.: Conversion of cholesterol into coprostanol by bacteria in vitro. 1. gen. Microbiol. 14: 256-260 (1956). Spritz. N.: Ahrens. E.H., and Grundy. S.: Sterol balance in man as plasma cholesterol concentrations are altered by exchanges of dietary fats. J. clin. Invest. 44: 1482 1493 ( 1965).
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Stere. P.A.; Cltaikoff, I.L.; Treitman, S.S., and Burstein, L.S.: The extrahepatic syn thesis of cholesterol. J. biol. Chem. 182: 629- 634 (1950). Strauss, F.W.: Handbook of physiology, vol. 3, pp. 1377 1406 (Am. Physiol. Soc., Washington 1968). Swann, A. and Siperstein, M.D.: Distribution of cholesterol feedback control in the guinea pig. J. clin. Invest. 51: 95 (1972). Sylven, C. and Borgstrôm, ¡1: Absorption and lymphatic transport of cholesterol in the rat. J. Lipid Res. 9: 596-601 (1968). Sylven. C. and Nordstrom, C: The site of absorption of cholesterol and sitosterol in the rat small intestine. Scand. J. Gastroenterol. 5: 58- 63 (1970). Tomkins, G.M. and Chaikoff. 1. 1,.: Cholesterol synthesis by liver. I. Influence of fasting and of diet. J. biol. Chem. 196: 569 573 (1952). Treadwell. C.R. and Vahouny. G. V.: Cholesterol absorption. Handbook of physiology, vol. 3, pp. 1407 1438 (Am. Physiol. Soc., Washington 1968). Van Belle. I f: Cholesterol, bile acids and atherosclerosis (North-Holland, Amsterdam 1965). Vodovar, N.: Flamy, J. et François, A.C.: Pénétration et acheminement des graisses dans la cellule épithéliale absorbante de l'intestin grêle du porc. C.r. hebd. Scanc. Acad. Sci., Paris 262/ 812 815 (1966). Vodovar, N.; Flamy, J., and François, A.C.: The chylomicrons of the intestinal ab sorptive cell: site of formation, fine structure and function in the mechanism of long chain fatty acid absorption. Ann. biol. Animal 9: 219 232 (1969). Wilson, J.D.: The quantification of cholesterol excretion and degradation in the iso topic steady state in the rat: the influence of dietary cholesterol. J. Lipid Res. 5: 409- 417 (1964). Wilson. J.D.: The relation between cholesterol absorption and cholesterol synthesis in the baboon. J. clin. Invest..?/. 1450 1458 (1972). Wilson, J.Ü. and Lindsey, C.A.: Studies on the influence of dietary cholesterol on cholesterol metabolism in the isotopic steady state in man. J. clin. Invest. 44: 1805 1814 (1965). Wilson, .1.1). and Reinke, R.T.: Transfer of locally synthesized cholesterol from intes tinal wall to intestinal lymph. J. Lipid Res. 9: 85 92 (1968). Wilson. J.D. and Siperstein. M.D.: liffect of saturated and unsaturated fed on fecal excretion of end products of cholesterol 4 ,4C metabolism in the rat. Am. J. Physiol. 196: 596 602 (1959). Windmueller, H.G. and Spaeth, A.F.: l-'at transport and lymph and plasma lipoproteins biosynthesis by isolated intestine. J. Lipid Res. 13: 92- 105 (1972). /.ilversmil. D.B.: Chylomicrons in structural and functional aspects of lipoproteins in living systems (Academic Press, London 1969).
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Dr. C. Lutton. Laboratoire de Physiologie de la Nutrition. Bâtiment 447, F-91405 Orsay (France)
The Role of the Digestive Tract in Cholesterol Metabolism C. Lutton Laboratoire de Physiologic de la Nutrition, Orsay
Key Words. Bile acids and salts • Cholesterol • Diet • Feces - Intestinal absorption • Intestinal mucosa • Isotopic equilibrium • Lymph • Sterols Abstract. New studies have permit to reevaluate the importance of the digestive tract in each of the mechanisms regulating cholesterol turnover. Particularly, the role of the diges tive tract in the rat cholesterol synthesis was underestimated. Is there a similar situation in man?
While the major aspects of absorption, synthesis or elimination of choles terol are now well known , the mechanisms of regulation of cholesterol metab olism in intact animals are not entirely clear. One reason that the regulatory processes have not been fully established is that generally only one process or at most two are studied in each experiment. Thus, the development in 1963 of a physiological method which permitted the simultaneous measurement of choles terol turnover rates in intact animals was promising. The theoretical and practical bases of this technique, the so-called isotopic equilibrium method, have been well described and shown by Chevallier (10). During the last decade the method has been applied extensively by several laboratories both in man and in animals (2, 35, 37, 40, 42, 53, 54, 82, 84). Recent studies have emphasized the importance of the digestive tract in the synthesis and excretion as well as in the absorption of cholesterol. The purpose of this review is to discuss some of the recent developments of intestinal secretion, excretion and absorption of choles terol. Since most of the experimental work has been done in animals, this review will emphasize the role of the digestive tract (particularly the intestine) in these functions in the rat. Some of the discrepancies between the rat and man will be pointed out. Several other reviews have been published on various aspects of cholesterol metabolism (4, 12, 25, 28, 32,43, 56, 78, 79, 88).
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Received: March 8. 1976: accepted: March 18, 1976.
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Since definitions concerning cholesterol dynamics differ among authors to avoid confusion 1 shall redefine the terminology used by Chevallier and his coworkers and compare it to the terminology commonly used in the United States. Much information can be obtain from whole body cholesterol turnover in respect to its dynamic equilibrium as previously pointed out by Chevallier (6, 7). For an animal in a steady state, the dynamic equilibrium of cholesterol in the transfer space is defined by the relationship m A + mIS = mT + mE (F+ U )’
(1)
m is the rate (mg/day) of absorption of cholesterol (A) internal secretion (IS), transformation into bile acids (T) and excretion (E) in feces (F) plus urine1 (U). Absorption is defined as the input of dietary cholesterol into the transfer space. The transfer space corresponds to the amount of cholesterol in equilibrium with plasma as it is determined at isotopic equilibrium (12). Thus, the cholesterol transfer space does not correspond exactly to the mobile body cholesterol since it does take into account the ‘cholesterol synthesis space’ (14). However, we know that the size of ‘synthesis cholesterol space’ is very small compared to that of cholesterol transfer space (2 -3 and 7 0-75 % of total body cholesterol, respectively). Excretion is defined as the transfer of cholesterol molecules from inside to the outside of the transfer space, for example by loss into the intestinal lumen or urinary tubules or by epithelial sloughing and sebaceous secretions (12). Secretion is synthesis by tissues and transfer into (internal secretion) or out of (external secretion) the transfer space (12). It is important to note that these definitions do not imply a mechanism and are simply related to the origin of the molecules. For a rat fed a cholesterol containing diet, fecal elimination of choles terol (m F) is the sum of unabsorbed dietary cholesterol (mUA), excreted fecal cholesterol (mEF) and externally secreted fecal cholesterol (mESF) (17) m F = m U A + mF.F + m E S F '
In the literature the term ‘endogenous sterol excretion’ corresponds gen erally to the sum of mEF + mESF, since very few authors are able to distinguish between the two types of sterols. However, it is important to make this distinc tion because fecal external secretion is not part of the output of the transfer space, as seen from equation (1). In man, ‘endogenous sterol excretion’ is probably only due to excreted cholesterol (58). This point will be discussed again later.
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1 In man. urinary elimination of cholesterol and its related compounds is quantita tively negligible compared to fecal loss of cholesterol and bile acids. In the rat it is not always slight (33, 46).
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Excretion and External Secretion
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In a rat which is fed a sterol-free diet and in which coprophagy and fur licking are prevented, the intestinal sterol contents are cholesterol (80—85 %) and some of its biosynthetic precursors (A7-cholesterol and methostenol) (48, 56). These sterols undergo bacterial modifications in the cecum and colon. For example, a part of the cholesterol is converted to coprosterol by reduction of the double bond at carbon 5 (48, 64, 70). In this article the term ‘fecal choles terol' will refer to the sum (cholesterol + coprosterol). Feces from rats fed a diet containing cholesterol (0.015 %) as the only sterol contain 0.5 mg/day of unabsorbed dietary cholesterol, 3 mg of excreted fecal cholesterol and 3 mg of cholesterol synthesized by the digestive tract and secreted into the lumen (fecal externally secreted cholesterol) (17). The mecha nisms by which endogenous cholesterol (excreted and secreted) enter the small intestine are not yet clear. It had been assumed that bile and sloughed epithelial cells were the only two sources of intestinal cholesterol and that opinion seems to be widely held especially for humans (55). However, several studies have shown that cholesterol synthesized by the intestine appears very rapidly in the lumen after the administration of labeled acetate. This appearance can hardly be explained on the basis of epithelial cell sloughing. Moreover, the biliary choles terol is only excreted cholesterol (13, 60). Though the bile contribution is difficult to measure accurately it approximates 2 -3 mg/day in the rat. Since a major part of biliary cholesterol (60—80 %) would be reabsorbed, it is reasonable to assume that bile does not account for more than one third of the fecal cholesterol excretion (19, 66). This conclusion is supported by other data. In rats fed a 2 % cholestyramine or 2 % cholesterol diet the observed fecal choles terol excretion is three and four times higher than that of controls, but the daily cholesterol output in bile undergoes only a slight increase or is unchanged (9, 50). Shortly after the subcutaneous or parenteral administration of 14C-cholesterol, radioactivity was found in the intestinal lumen even when the bile was diverted and replaced by unlabeled bile from a donor rat (22, 27). In a series of experiments, Chevallier and Lutton (19) estimated the daily flux of endogenous cholesterol entering the lumen to be 11 mg/day. If we assume that the villi accounts for some 20 % of the whole intestinal cholesterol and that epithelial cell turnover time is 30 h (44), the endogenous contribution from sloughed mucosal epithelium may be 3 mg/day. Thus, apparently at least 50% of the flux of endogenous cholesterol entering the lumen could be due to active exchange of cholesterol between epithelial cells and lumen in the rat. Experiments on anesthetized, fasted rats, by Cotton (26) concluded that the results obtained for lipid values (phospholipid, cholesterol and triglyceride) could be explained on the basis of exfoliated cells. Additional experiments will be necessary to resolve this important question.
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It is not likely that only a limited portion of the intestine is able to excrete or secrete cholesterol, since the ratio excreted cholesterol/secreted cholesterol in the intestinal lumen remained almost the same (about 1/1) from the duodenum to the ileum (19). Cholesterol synthesis takes place mainly or exclusively in the crypt area of the intestinal mucosa (31). It is not yet known whether the exchange of cholesterol between intestinal cells and plasma and between intes tinal cells and the lumen occurs in every cell of the villus or only in that part of it which has been shown to be involved in cholesterol absorption (76). Since synthesis occurs in the crypts and if exchange of cholesterol takes place along the entire villus, it can be expected that a progressive increase would occur in the ratio excreted cholesterol/secreted cholesterol in the absorptive cell from the crypt area to the top of the villus. In man ‘endogenous cholesterol excretion’ appears responsible solely by excreted fecal cholesterol; that is, there is no contribution from externally secreted fecal cholesterol (71). This raises interesting questions on the mecha nisms of cholesterol transfer in the intestinal mucosa. Some authors have found that sterols can be degraded by human bacterial flora whereas no degradation occurs in the rat (40,45). However, this degradation is probably greatly affected by the diet (29). It has been also observed that sterols are transformed more extensively by human flora than by rat flora. As an example, large proportions of sterols were found as stanones (coprostatone ...) in human feces but only in negligible amounts in the rat feces (48).
The main steps of cholesterol absorption will be described from a ‘dynamic’ point of view. Figure 1 summarizes the movement of cholesterol from the lumen into the intestinal cell and then into the extracellular space and lymphatic duct. Before entering the duodenal lumen, dietary cholesterol mixes with excreted and secreted endogenous cholesterol from salivary and gastric fluids (78). Chevallier and Lutton (19) recently estimated the flux of endogenous cholesterol which mixes with dietary cholesterol in the stomach content to be 1.5 mg/day in the rat. Cholesterol is present in the intestinal contents mainly as free cholesterol because of the hydrolyzing action of pancreatic cholesterolesterase (48, 78). Cholesterol concentration increases greatly from the pyloric to the cecal valves (19, 57). Cholesterol in the intestinal lumen is a mixture of cholesterol originating from the stomach contents and endogenous cholesterol from sources other than the stomach contents. In contrast to a previous interpretation (78), a recent study has shown that the mixing of these two ‘types’ of cholesterol is not homogeneous in the rat (19). In fact, two cholesterol compartments seem to be present in the lumen, i.e. an ‘intermediary’ compartment containing endogenous
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Absorption
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Luiton
Com partm ents _______ l_______
Fig. i. Schematic representation of various processes responsible for lymph and chylo microns cholesterol. Adapted from Chevallier and Vyas (22) and Chevallier and Luiton (19).
mg Id
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Fig. 2. Flux of excreted and secreted cholesterol (mg/day) through walls of the upper part of the digestive tract and intestine in a control rat. From Chevallier and Lutton (19).
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cholesterol which exchanges with cholesterol of the intestinal cells, and an ‘axial’ compartment, containing a mixture of intestinal cholesterol and cholesterol from the stomach contents (19). Thus, three different sources of cholesterol are available for absorption: dietary, excreted and secreted cholesterol. Because of the partial mixing of these three sources one could have a different absorption coefficient (percentage absorption) for cholesterol from each source. Also the mixing of externally secreted with dietary cholesterol accounts for the contri bution of the digestive tract to the internal secretion (undirect internal secretion, fig. 2). In the same study, the total of cholesterol synthesized by the digestive tract and reabsorbed was estimated to be 4 mg/day, i.e. almost one third of the total internal secretion in the rat. Following centrifugation of the small intestinal contents, free sterols are found to be mainly in a micellar phase (clear aqueous supernatant) and in the sediment (57; unpubl. observations). The interrelationships between these two ‘types’ of cholesterol and the two compartments previously described remain to be established. The mechanisms of the mucosal uptake of free cholesterol from micelles, as well as the mode of transport of cholesterol across the mucosal cells are not yet fully understood. Migration of cholesterol across the cell seems to be associated with that of long chain fatty acids. These compounds migrate to the endoplasmic reticulum tubules in droplets of 250-650 A diameter which could represent chylomicrons being formed (80). Triglyceride synthesis and chylo micron formation probably take place in the endoplasmic reticulum located above the nucleus (41, 81). Inside the intestinal cell, cholesterol exchanges between chylomicra and the subcellular structures (arrows 6 and 7, fig. 1) (22). Part of the cholesterol in chylomicrons is esterified before the particles are secreted across the basal membrane (23, 41). In the extracellular space chylo microns adsorb free cholesterol and probably also esterified (arrows 12 and 13, fig. 1) (21, 22). Cholesterol exchange also takes place between intestinal cells and the lumen or intestinal cells and extracellular spaces (arrows 4, 5,9 and 10, fig. 1) (15). It is known that there is cholesterol transfer from plasma to extra cellular fluid (arrows 14, 15) (8, 22). It is probable that an opposite transfer exists (arrow 16) since even HDL proteins can appear in the perfusate of a lymph cannulated isolated rat intestine (87). The mechanisms associated with transfer of cholesterol to lymph constitute the routes by which the intestine directly secretes its synthesized cholesterol into the plasma (direct internal secretion; fig. 2); but the quantifying of this mechanism is very difficult. Regulation o f Absorption
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Each step previously described in the overall process of cholesterol absorp tion is potentially a rate-limiting step. The mechanisms which regulate these processes are not well established.
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The percentage of cholesterol which is absorbed varies according to species and dose. Numerous factors which modify cholesterol absorption have been described, e.g., age and sex (46), nature of dietary fatty acids (32), percentage of casein in the diet (47), bile flow (32), etc. It has been shown repeatedly that the size of the bile acid pool has a great effect on the rate of cholesterol absorption. Numerous studies support this view: cholestyramine lowers and a bile fistula or bile ligation completely inhibit absorption of cholesterol (47, 67, 79) while addition of taurocholate to the diet increases it (50, 79). This effect on the extent of cholesterol absorption has been attributed to tire role of bile acids in promoting micellar solubilization of cholesterol in the intestinal lumen and also to their effect on its transfer into the intestinal lymph (32, 85). If the dietary concentration of taurocholate was increased to 4 %, the absorption coefficient of cholesterol was decreased (50). This observation and others show that more than a simple mechanism is needed to explain tire role of bile acids in regulating cholesterol absorption. Some authors have claimed that the movement from the intestinal cell into the lymph is the limiting step in cholesterol absorption (75). If this is the case the production of the compounds necessary for the chylo micron coat (i.e. proteins and phospholipids) could be the key steps in the regulation of cholesterol absorption. Contradictory results have been published as to whether the mucosal synthesis of the protein coat is essential to chylo micron formation (63, 65). The recent observation that an active phospholipid exchange protein is present in mucosal cells also may be pertinent (51). How ever, this phospholipid exchange activity is not influenced by high lipid and cholesterol content in the diet suggesting that phospholipid exchange proteins are not rate limiting in the process of cholesterol absorption.
Excretion Fecal excretion of cholesterol in the rat is relatively low compared to that of bile acids, but in man cholesterol elimination in the form of fecal neutral steroids is equal to or greater than that of bile acids (32, 39, 58). Several authors have claimed that the fall in serum cholesterol that occurs when polyunsaturated fats are substituted for saturated fat in the diet may result from an increase in the excretion o f fecal cholesterol (1, 24, 59, 86). Fecal excretion of cholesterol is influenced by various factors such as cerebrosides or cellulose content of the diet, germfree status, etc. (32). Numerous isotopic equilibrium experiments in the rat have recently shown that in most of the nutritional conditions described by the authors, the total fecal excretion of cholesterol was inversely propor tional to the absorption coefficient of cholesterol (fig. 3 a) (49). The value for the fecal cholesterol excretion extrapolated to a theoretical absorption coeffi-
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Regulation o f Fecal Excretion and External Secretion
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A b s o rp tio n , %
Fig. 3. a Mass of excreted cholesterol found daily in the feces as a function of the absorption coefficient of cholesterol, b Mass of secreted cholesterol found daily in the feces as a function of the absorption coefficient of cholesterol, bach point represents an isotopic equilibrium experiment (4 rats) in a given experimental condition (17, 46, 47, 50).
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cient of zero (14.6 mg) could represent a maximal flux of cholesterol from the transfer space to the lumen. In the control rat where the absorption coefficient reached 75 %, the net flux for excreted cholesterol has been estimated to be 5.5 mg/day by another method (19). Thus it is probably that this net flux increases when the absorption coefficient diminishes. Another interesting observation is that the extrapolation of the regression line to 100 % absorption gives a positive value for fecal excretion, suggesting that the absorption coeffi cient of excreted cholesterol is lower than that of dietary cholesterol since when the dietary absorption of cholesterol is 100 % fecal cholesterol excretion should be zero (19. 49). The interrelationship between fecal excretion of cholesterol and the absorption coefficient could be of general significance. It is noteworthy that the relative quantitative importance of fecal excretion of cholesterol com pared to conversion to bile acids increases from rabbit to rat to man, species in which the absorption coefficient for cholesterol is very high, high, and low, respectively.
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External Secretion Like fecal excretion the daily mass of fecal external secretion of cholesterol increases when the absorption coefficient decreases in the rat (fig. 3 b). The similar response of fecal excretion and fecal external secretion to changes in the absorption coefficient suggests that the transfer of plasma cholesterol and synthesized cholesterol is regulated by a similar mechanism in the ‘intermediary’ and ‘axial’ compartments. However, the slope of the regression line for fecal external secretion versus absorption coefficient is lower than that for excretion, and the extrapolated value for a theoretical absorption of 0 % is only slightly higher (7.7 mg/day) than the estimated net flux of cholesterol secreted into the lumen of a control rat (5.5 mg/day) (19). This observation could indicate that the flux of secreted cholesterol from intestine to lumen is fairly constant and independant of nutritional conditions in these rats.
Although is has been known since 1950 that the intestine synthesizes cholesterol, its relative contribution to the internal secretion is still questionable (61, 72). Based on in vitro incorporation of various labeled precursors, the liver has the highest rate of sterol synthesis (31, 56). Although it is difficult to extrapolate such in vitro information to the intact animal, the liver has been generally assumed to be the main source of synthesized cholesterol. However, several new studies do not agree with this point of view. It is well established that hepatic cholesterol synthesis varies greatly with the type of diet and is inhibited by fasting or cholesterol feeding (68, 77). However, the internal secre tion of cholesterol as measured by the isotopic equilibrium method undergoes only slight changes under numerous dietary conditions and does not change when cholesterol is added to the diet (11). The rate of cholesterol synthesis determined by the same method is lower in female than in male rats, although incorporation of labeled precursors into liver homogenates or slices is higher in the female (34, 46). It has been recently estimated that more than 4 mg of the cholesterol synthesized by the digestive tract and secreted into the lumen could be reabsorbed daily with dietary cholesterol (19). This represents almost one third of total internal secretion in the rat. Thus it is likely that tire role played by the intestine in cholesterol synthesis will be reevaluated in the near future. Dietschy and Siperstein (31) have shown that incorporation of labeled acetate into cholesterol in intestinal slices occurs predominantly in the cells of the crypts. This fact agrees with a general hypothesis correlating cholesterol synthesis and formation of cells (18). Whether or not such a mechanism can entirely explain the production of synthesized cholesterol is unknown. In contrast with Dietschy's experiment, Mak and Trier (52) found a higher in corporation of labeled mevalonate into cholesterol on tire brush border of cells
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Regulation o f Synthesis
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in the upper part of the villus than in crypts cells from everted ileal sacs. In this latter experiment, however, the high amount of label observed on the upper part of the villi could be due to labeled cholesterol previously secreted by the crypts cells and reabsorbed by the absorptive cells. Dietschy (30) has claimed that the luminal concentration of bile acid controls cholesterogenesis in the small intestine based upon the observations that in vitro incorporation of acetate into jejunal cholesterol was greatly enhanced by diversion of bile or by ligating the bile duct and was depressed by infusion of solutions of whole bile or pure bile acids. In contrast to the situation in the liver he did not find any feedback inhibition by cholesterol itself. This finding has been questioned by several other investigators who observed a moderate inhibitory effect of cholesterol on intestinal cholesterogenesis in the baboon and guinea pig (74. 83). When rats were fed tomatine, an agent which precipitates cholesterol without interferring with bile acids, Cayen (5) observed an increase in small intestinal cholesterogenesis. Thus, cholesterol synthesis in the small intestine may be controlled by several mechanisms. The analysis of 21 isotopic equilibrium experiments has recently shown that there exists an inverse relation ship between total cholesterol synthesis and the absorption coefficient for cholesterol in the rat. Since a correlation also exists between external fecal secretion and absorption coefficient, it has been suggested that intestinal choles terogenesis could be inversely correlated with the absorption coefficient (20). It is obvious that the absorption coefficient for cholesterol depends on several factors such as the concentration of bile acids and cholesterol in the lumen; the rate of cell turnover in the intestinal mucosa which probably modifies intestinal cholesterol synthesis could also play a role. It is known that, in regard to the villus, cholesterol absorption occurs in the apical area (76). This functional particularity could be due to a progressive development of enzymes by absorp tive cells during their migration (73). When the renewal of crypts cells is increased, the area o f ‘mature’ cells could be diminished. In some of the isotopic equilibrium experiments cited above, total choles terol synthesis did not follow the relation previously described. It is remarkable that these particular cases in which there was an unusual elevated synthesis could always be correlated with a hyperfunction of an extradigestive source of choles terol synthesis such as placenta and mammary glands in pregnant and lactating females, respectively, or liver (dietary cholestyramine or bile duct ligation) (20, 49). Thus, under normal conditions the importance by the liver in cholesterol synthesis has probably been overestimated.
This discussion has only been concerned with describing processes involving the movements of cholesterol in the digestive tract. However, an important
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mechanism of cholesterol turnover is by hepatic transformation of cholesterol into bile acids (3, 69). The isotopic equilibrium method is a good technique for measuring the rate of this process (16, 17,45). In a rat fed a sterol-free diet this daily rate is 13 mg, i.e. 4 times more than the daily excretion of cholesterol into the feces. The rate varies when the rates of excretion (fecal or urinary) internal secretion or absorption change or even when the transfer space of cholesterol increases (45). So this transformation rate seems to be an adaptative process for maintaining a dynamic equilibrium of cholesterol in the rat (50). In man the rate of cholesterol excretion is quantitatively as large as bile acid production (24, 36, 84). Moreover, the conversion of cholesterol into bile acids does not seem to be greatly affected by large amounts of cholesterol in the diet (62). The ‘unadaptability’ of this process may explain the frequency of dysfunc tion of the dynamic equilibrium in man. Finally, interrelationships between absorption and synthesis have been studied but the results are conflicting (38, 62). Additional difficulties occurring in human experimentation explain the slower advance of knowledge in this important area. New methods developed during the last decade have solved numerous problems concerned with cholesterol turnover but several questions are still not adequately answered. An important problem is whether the mechanisms regu lating cholesterol turnover in man are similar to those in the rat. The methods presented permit the simultaneous measure of input-output rates of cholesterol turnover in man may give the information needed to fully understand this important area of metabolism.
Acknowledgments I would like to thank Prof. F. Clievallier for his continuous interest and stimulation. I am indebted to J. Balmer and Dr. M. Green for their help in correcting the text and to Prof. J.L. Rabinowitz for his helpful suggestions.
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Dr. C. Lutton. Laboratoire de Physiologie de la Nutrition. Bâtiment 447, F-91405 Orsay (France)
